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AU772630B2 - Adenovirus vectors, packaging cell lines, compositions, and methods for preparation and use - Google Patents
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AU772630B2 - Adenovirus vectors, packaging cell lines, compositions, and methods for preparation and use - Google Patents

Adenovirus vectors, packaging cell lines, compositions, and methods for preparation and use Download PDF

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AU772630B2
AU772630B2 AU24372/00A AU2437200A AU772630B2 AU 772630 B2 AU772630 B2 AU 772630B2 AU 24372/00 A AU24372/00 A AU 24372/00A AU 2437200 A AU2437200 A AU 2437200A AU 772630 B2 AU772630 B2 AU 772630B2
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fiber
adenovirus
protein
gene
cells
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AU2437200A (en
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Paul L Hallenbeck
Glen Robert Nemerow
Yelena Skripchenko
Susan C. Stevenson
Daniel J. Von Seggern
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Novartis AG
Scripps Research Institute
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Novartis AG
Scripps Research Institute
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    • C03B37/018—Manufacture of preforms for drawing fibres or filaments made entirely or partially by chemical means, e.g. vapour phase deposition of bulk porous glass either by outside vapour deposition [OVD], or by outside vapour phase oxidation [OVPO] or by vapour axial deposition [VAD] by glass deposition on a glass substrate, e.g. by inside-, modified-, plasma- or plasma modified- chemical vapour deposition [ICVD, MCVD, PCVD, PMCVD], i.e. by thin layer coating on the inside or outside of a glass tube or on a glass rod
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    • C03B37/018—Manufacture of preforms for drawing fibres or filaments made entirely or partially by chemical means, e.g. vapour phase deposition of bulk porous glass either by outside vapour deposition [OVD], or by outside vapour phase oxidation [OVPO] or by vapour axial deposition [VAD] by glass deposition on a glass substrate, e.g. by inside-, modified-, plasma- or plasma modified- chemical vapour deposition [ICVD, MCVD, PCVD, PMCVD], i.e. by thin layer coating on the inside or outside of a glass tube or on a glass rod
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Description

Adenovirus Vectors, Packaging Cell Lines, Compositions, and Methods for Preparation and Use This invention was made with U.S. government support under NIH Grant No.
HL 54352. The government has certain rights in the invention.
The present invention relates to gene therapy, especially to adenovirus-based gene therapy. In particular, novel packaging cell lines are disclosed, for use in facilitating the development of high-capacity and targeted vectors. High-capacity adenovirus vectors are also disclosed herein, as are related compositions, kits, and methods of preparation and use of the disclosed vectors, cell lines and kits.
Enhanced transfer of DNA conjugates into cells has been achieved with adenovirus, a human DNA virus which readily infects epithelial cells (Horwitz, "Adenoviridae and Their Replication", in Virology, Fields and Knipe, eds., Raven Press, NY (1990) pp. 1679-1740).
There is a need in the art to obtain Adenovirus vectors capable of incorporating large segments of foreign DNA and capable of being targeted to specific cells, as well as to obtain cell lines which can package such adenovirus- S gene deficient vectors or targeted vectors. These needs, as well as others, are S met by the invention.
This invention utilizes recombinant adenovirus constructs which duplicate the cell receptor binding and DNA delivery properties of intact adenovirus virions and thus represents an improved method for gene therapy and cell targeting as well as for antisense-based antiviral therapy.
In contrast to the disadvantages of using intact adenovirus, modified S adenovirus vectors requiring a helper plasmid or virus, or so-called replicationdeficient adenovirus in the art, the use of recombinant adenovirus-derived vectors according to one aspect of the present invention provides certain advantages for gene delivery. First, the Ad-derived vectors of the present invention possess all of the functional properties required for gene therapy including binding to epithelial cell receptors and penetration of endocytic vesicles. Therapeutic viral vectors of the present invention may also be engineered to target the receptors of and WO 00/42208 PCT/EP00/00265 -2achieve penetration of non-epithelial cells; means of engineering viral vectors to accomplish these ends are described in detail herein below.
Second, the vectors of the present invention have deletions of substantial portions of the Ad genome, which not only limits the ability of the Ad-derived vectors to "spread" to other host cells or tissues, but allows significant amounts of "foreign" (or non-native) nucleic acids to be incorporated into the viral genome without interfering with the reproduction and packaging of the viral genome.
Therefore, the vectors of the present invention are ideal for use in a wide variety of therapeutic applications.
Third, while the vectors disclosed herein are safe for use as therapeutic agents in the treatment of a variety of human afflictions, some of these vectors do not require the presence of any "helpers" for propagation and packaging, largely because of the novel cell lines in which they are reproduced. Such cell lines referred to herein as packaging cell lines comprise yet another aspect of the invention.
To reduce the frequency of contamination with wild-type adenovirus, it is desirable to improve either the viral vector or the cell line to reduce the probability of recombination. For example, an adenovirus from a group with less homology to the group C viruses may be used to engineer recombinant viruses with little propensity for recombination with the Ad5 sequence contained in the packaging lines. The invention describes the preparation of packaging cells lines which stably expresses adenovirus proteins or polypeptides. These cell lines are useful for complementing viral vectors bearing deletions of regulatory and/or structural genes, irrespective of the serotype from which such a vector was derived.
It is also contemplated that the constructs and methods of the present invention will support the design and engineering of chimeric viral vectors which express amino acid residue sequences derived from two or more Ad serotypes.
Thus, unlike methods and constructs available prior to the advent of the present disclosure, this invention allows the greatest possible flexibility in the design and preparation of useful viral vectors and cell lines which support their construction and propagation all with a decreased risk of recombining with wild-type Ad to produce potentially-harmful recombinants.
In part, the present invention discloses a simpler, alternative means of reducing the recombination between viral and cellular sequences than those discussed in the art. One such means is to increase the size of the deletion in the recombinant virus and thereby reduce the extent of shared sequences between 11-03-'04 09:45 FROM-DCC +61392542770 T-227 P05/26 U-250 -3that virus and any Ad genes present in a packaging cell line the AdS genes in 293 cells, or the various Ad genes in the novel cell lines of the present invention.
Deletions of all or portions of structural genes of the adenovirs have been considered undesirable because of the anticipated deleterious effects such deletions would have on viral reproduction and packaging. Indeed, the use of "helper" viruses or plasmids has often been recommended when using Ad-derived vectors containing large deletions in structural protein sequences precisely for this reason.
Contrary to what has been suggested in the art, however, this invention discloses the preparation, propagation and use of recombinant Ad-derived vectors having deletions of all or part of various gene sequences encoding Ad structural proteins, both as a way of reducing the risk of wild-type adenovirus contamination in virus preparations, as a way of allowing foreign DNA to be packaged in such vectors for a variety of diagnostic and therapeutic applications and as a way of targeting an adenovirus vector to a specific cell type.
The invention further discloses a wide variety of nucleic acid sequences and viral vectors. Thus, in one embodiment, the invention discloses a nucleic acid sequence encoding any one of the adenovirus fiber proteins mentioned in the specification, polypeptides or fragments thereof including, without limitation, those that include 20 deletions or other mutations; those that are chimeric; and those that have linkers, foreign amino acid residues, or other molecules attached for various purposes as disclosed herein.
Nucleic acid sequences encoding various other adenovirus structural and/or regulatory "proteins or polypeptides are also within the scope of the present invention.
In various embodiments, the adenovirus is a Group C adenovirus selected from serotypes 1, 2, 5 or 6; while in other embodiments, adenovirus selected from other serotypes, such as for example Ad37 (subgroup D) are useful as disclosed herein.
The invention is also directed to an isolated nucleic acid molecule comprising: a sequence of nucleotides encoding an adenovirus tripartite leader (TPL), wherein said TPL 9. 9.
nucleotide sequence consists essentially of at least two different TPL exons selected from complete exons 1, 2 and 3 and optionally also includes intron 1. A preferable embodiment 30 of the invention may further comprise an intron operatively linked to the TPL, wherein 9.9999o COMS ID No: SMBI-00658976 Received by IP Australia: Time 09:33 Date 2004-03-11 11-03-'04 09:46 FROM-DCC +61392542770 T-227 P06/26 U-250 <f.E\flha2472mj s2.dat lX3lJ -4said intron also contains requisite processing signals for the intron's removal. Another preferable embodiment of the invention is directed to the isolated nucleic acid molecule wherein said TPL nucleotide sequence consists essentially of complete TPL exon 1 operatively linked to complete TPL exon 2 operatively linked to complete TPL exon 3. A related embodiment may further include an intron and appropriate processing signals.
Additional embodiments of the invention are directed to nucleic acid molecules contained in plasmids selected from the group consisting of pDV60 (ATCC Accession No. PTA- 1144), pDV67 (ATCC Accession No. PTA-1145), pDV69 (ATCC Accession No. PTA- 1146), pDV80 (ATCC Accession No. PTA-1147) and pDV90 (ATCC Accession No.
PTA-1148). Packaging cell lines and adenovirus particles containing the nucleic acids described above are also included in the invention.
The invention is further directed to methods for producing an adenovirus vector particle containing a helper-independent fiberless recombiant adenovirus vector genome comprising providing a) a packaging cell line which complements replication and packaging of said genome and b) a helper-independent fiberless recombinant adenovirus vector genome which is deficient in expressing sufficient functional fiber protein to support assembly of fiber-containing particles. The genome is introduced into the cell line.
Additional embodiments of the invention may also include the following steps; a) growing the cell line produced under conditions for producing particles; and/or b) harvesting an 20 adenovirus vector particle containing said helper-independent fiberless recombinant adenovirus vector genome. The method may also include a cell line that expresses a fiber protein and complements a fiber mutation in the vector.
The invention is also directed to an adenovirus vector packaging cell line comprising a stably integrated nucleic acid molecule as described above, an operativelylinked promoter and a nucleic acid sequence which encodes an adenovirus structural protein. Another embodiment of the invention comprises adenovirus structural protein, such as adenovirus fiber protein or a chimeric protein which includes an adenovirus fiber protein tail domain.
The invention is further directed to a recombinant adenovirus particle comprising a 30 recombinant adenovirus vector genome wherein said genome: does not encode or does *0o0oo COMS ID No: SMBI-00658976 Received by IP Australia: Time 09:33 Date 2004-03-11 11-03-'04 09:46 FROM-DCC +61392542770 T-227 P07/26 U-250 P.\JPErmrcm read-lMW/ not express sufficient adenovirus fiber protein to support packaging of a fiber-containing adenovirus particle without complementation of said fiber gene, and encodes an adenovirus packaging signal and inverted terminal repeats containing adenovirus origin of replication. The invention is also directed to a helper-independent fiberless recombinant adenovirus vector genome comprising genes which encode all adenovirus structural gene products but do not express sufficient adenovirus fiber protein to package a fibercontaining adenovirus particle without complementation of said fiber gene or said genome lacks at least the fibre gene and encodes an exogenous protein. Either of the above embodiments may substitute a helper-dependent for a helper-independent recombinant adenovirus vector genome. In a preferable embodiment, no fiber protein is expressed. In yet another embodiment of the invention, the recombinant adenovirus particle fails to express sufficient fiber protein to allow fiber incorporation into the particle such that the particle can use the fiber pathway for infection.
The invention is further directed to a method for producing an adenovirus vector particle containing a helper-independent fiberless recombinant adenovirus vector genome, said method comprising providing a packaging cell line which complements replication and packaging of said genome and a helper-independent fiberless recombinant adenovirus Svector genome which is deficient in expressing sufficient functional fiber protein to support assembly of fiber-containing particles, wherein said packaging cell comprises an 20 isolated nucleic acid molecule comprising a sequence of nucleotides encoding an adenovirus tripartite leader (TPL), wherein said TPL nucleotide sequence consists essentially of at least two different TPL exons selected from complete exons 1, 2 and 3 and optionally also includes intron 1, and harvesting said adenovirus particles produced by said cell line. In a preferable embodiment the adenovirus particle further comprises an exogenous protein or a modified fiber protein. The method may also comprise a step of coating a particle providing fiber protein in any way) with an adenovirus fiber protein.
*9 9 COMS ID No: SMBI-00658976 Received by IP Australia: Time 09:33 Date 2004-03-11 II- 111 WO 00/42208 PCT/EP00/00265 -6- Another aspect of the invention is directed to a method for pseudotyping recombinant viral vectors comprising complementing a missing fiber gene of a helper-independent fiberless recombinant adenovirus vector genome by expressing in packaging cells a fiber gene from a different adenoviral serotype than said recombinant adenovirus vector, thereby pseudotyping said vector. An additional embodiment of the invention is directed to the method for pseudotyping recombinant viral vectors comprising: a) providing a packaging cell line for propagating a fiber gene deleted recombinant adenovirus vector, b) introducing into said cell line a helper-independent fiberless recombinant adenovirus vector genome, and c) complementing the missing fiber gene by expression in the cells of a fiber gene from a different adenoviral serotype thereby pseudotyping the vector.
The invention is further directed to a method for specifically targeting an adenovirus vector to a cell of choice comprising introducing a helper-independent or helper-dependent fiberless recombinant adenovirus vector genome into a packaging cell line for producing a fiber gene-deleted adenovirus vector and providing, wherein said gene for a missing fiber protein is complemented with a gene for a desired modification for targeting the vector to a cell of choice The invention is further directed to a method for producing a modified adenovirus comprising providing in vitro an exogenous fiber protein to a fiberless adenovirus. Additional embodiments of the invention may provide any combination of all of the following steps such that the invention be directed to a method for producing a modified adenovirus comprising: a) providing a packaging cell line for producing a fiberless adenovirus vector, b) introducing into said cell line a helperindependent fiberless or helper-dependent fiberless recombinant adenovirus vector genome, c) growing and harvesting a fiberless adenovirus, d) maintaining the fiberless adenovirus in any suitable buffer, and e) providing exogenous fiber, wherein said fiber may be a modified fiber, to the fiberless adenovirus by adding conditioned media or a soluble fiber preparation or a fiber in any suitable buffer to a virus preparation thereby producing the modified adenovirus.
The invention is further directed to a method for producing a modified adenovirus comprising providing a packaging cell line for producing a helperdependent fiberless adenovirus vector genome and providing a helper virus vector, wherein said cell line complements at least a deficient fiber protein gene, thereby producing the modified adenovirus. Another aspect of the invention is directed to a method for producing a modified adenovirus comprising: a) providing a packaging cell line for producing a fiberless adenovirus vector, b) introducing into said cell line a helper dependent fiberless recombinant adenovirus vector genome I WO 00/42208 PCT/EPOO/00265 -7and a fiberless helper virus vector, c) growing and harvesting a fiberless adenovirus, and d)maintaining the fiberless adenovirus in infectious media, and e) providing exogenous fiber to the fiberless adenovirus by adding conditioned media or a soluble fiber preparation to a virus preparation thereby producing the modified adenovirus.
Additional aspects of the invention are directed to hybrid Ad/AAV vectors and to new helper-dependent vectors used with fiberless adenovirus vectors.
The invention is also directed to a method for delivering a heterologous gene to an EBV-infected B cells comprising infecting said B cells with a pseudotyped F particle or other fiber-deleted adenovirus particle, said particle having a chimeric fiber with the receptor-binding knob domain of the adenovirus type 3 fiber.
The invention is also directed to an isolated nucleic acid comprising a posttranscriptional regulatory element (PRE) and a TPL. Preferably the PRE is the woodchuck hepatitis virus PRE (WPRE).
The invention is further directed to a composition for preparing a therapeutic vector, said composition comprising a plasmid comprising an adenovirus genome lacking a nucleotide sequence encoding a fiber protein or a genome that is incapable of expressing sufficient fiber to result in packaging.
Another aspect of the invention is directed to a method of delivering a heterologous gene to a human or any animal comprising providing an exogenous gene to a target cell comprising contacting said cell in vivo or ex vivo with an amount of a recombinant adenovirus particle sufficient to infect said cell.
The invention is also directed to A method for producing a gutless adenoviral vector particle comprising: a) delivering a helper adenovirus vector genome to an adenovirus vector packaging cell, wherein said helper adenovirus vector genome lacks any gene encoding adenovirus fiber protein or lacks the ability to encode sufficient adenovirus fiber protein to produce an adenoviral vector comprising fiber protein in the absence of complemetation by said packing cell and wherein said packaging cell comprises the nucleic acid molecule of claim 2 operably linked to a promoter and to an adenoviral fiber protein or to a chimeric protein that includes an adenovirus fiber protein tail domain; delivering a gutless adenovirus vector genome to said packaging cell; and recovering the gutless adenoviral vector particle produced by said cell.
Another aspect of the invention is directed to a helper adenovirus particle comprising an adenovirus vector genome that does not encode or does not express sufficient adenovirus fiber protein to support packaging of a fiber- WO 00/42208 PCT/EP00/00265 -8containing adenovirus particle without complementation of said fiber gene, wherein said genome has a mutation in its packaging sequence that renders said genome substantially incapable of being packaged. Packaging sequence are those sequences are those sequences involved in packaging the viral particle.
The invention is further directed to a helper adenovirus particle comprising an adenovirus vector genome with recombinase sites flanking its packaging sequence, wherein said vector genome does not encode or does not express sufficient adenovirus fiber protein to support packaging of a fiber-containing adenovirus particle without complementation of said fiber gene.
The invention is also directed to an adenovirus particle comprising a gutless adenoviral vector genome and a fiberless capsid, as well as an adenovirus particle comprising a gutless adenoviral vector genome and a capsid comprising a modified fiber protein.
Another aspect of the invention is directed to a packaging cell for the production of a fiberless or fiber-modified gutless adenovirus particle comprising an adenovirus vector complementing plasmid and a nucleotide sequence encoding a recombinase, wherein said complementing plasmid comprises the nucleic acid molecule of claim 2 operably linked to a promoter and to a nucleotide sequence encoding an adenoviral fiber protein or a chimeric adenoviral fiber protein.
Preferably the cell line may comprise a recombinase. In an embodiment of the invention the recombinase may be Cre.
In another embodiment of the invention, the fiber-deleted adenovirus vectors of the invention and the fiber-complementing adenovirus packaging cells of the invention are used to produce a gutless adenovirus vector particle. Such particle comprises a gutless adenoviral vector genome in an adenoviral capsid. The fiber proteins of the capsid may be wild-type fiber, or the modified fiber proteins disclosed herein. Alternatively, such particle may have a fiberless capsid as disclosed herein. Preferably, the gutless genome contains at least one heterologous gene as described herein. As used herein, the term "gutless adenoviral vector genome" means an adenoviral vector genome from which all of the viral genes have been deleted..
The invention also discloses systems or kits for use in any of the aforementioned methods. The systems or kits may contain any appropriate combination of the within-described vectors, plasmids, cell lines, virus particles and additional therapeutic agents as disclosed. Preferably, each such kit or system includes a quantity of the appropriate therapeutic substance or sequence sufficient for at least one administration, and instructions for administration and use. Thus, WO 00/42208 PCT/EP00/00265 -9one system further comprises an effective amount of a therapeutic agent which enhances the therapeutic effect of the therapeutic viral vector-containing composition. Another variation discloses that the composition and the therapeutic agent are each included in a separate receptacle or container.
It will also be appreciated that any combination of the preceding elements may also be efficacious as described herein, and that all related methods are also within the scope of the present invention.
Figure 1 is a schematic diagram of the entire adenoviral E4 transcriptional unit with the open reading frames (ORF) indicated by blocked segments along with the promoter and terminator sequences. The location of primers for amplifying specific portions of E4 are also indicated as further described in Example 1A.
Figure 2 is a schematic map of plasmid pE4/Hygro as further described in Example 1B.
Figure 3 is a schematic map of plasmid pCDNA3/Fiber as further described in Example 1B.
Figure 4 is a schematic map of plasmid pCLF as further described in Example 1B.
Figure 5 is a photograph of a Southern blot showing the presence of intact adenovirus E4 3.1 kilobase (kb) insert in the 211 cell line as further described in Example 1C.
Figure 6 is an autoradiograph showing labeled fiber protein immunoprecipitated from cells and electrophoresed under native and denaturing electrophoresis conditions as described in Example 1C. The 293 cells lack fiber while the sublines 211A, 211B and 211R contain fiber protein detectable in functional trimerized form and denatured monomeric form.
Figure 7 is a schematic map of plasmid pDEX/E1 as further described in Example 1 D.
Figure 8 is a schematic map of plasmid pE1/Fiber as further described in Example 1F1.
Figure 9 is a schematic map of plasmid pE4/Fiber as further described in Example 1 F2).
Figure 10 is a schematic illustration of linearized pD E1Bb gal delivery plasmids for use in cotransfection and recombination to form a recombinant adenoviral vector having multiple adenoviral gene deletions. The plasmids and recombination event are more fully described in Example 2A.
WO 00/42208 PCT/EP00/00265 Figure 11 is a schematic of plasmid p11.3 as further described in Example 2A used in the construction of pDV44 delivery plasmid.
Figure 12 is a schematic of plasmid 8.2.
Figure 13 shows the trimeric structure of the recombinant fiber. 293, 211A, 211B, or 211R cells as indicated were metabolically labeled with 35 S]methionine, soluble protein extracts prepared, and fiber was immunoprecipitated. A portion of the precipitated protein was electrophoresed on an 8% SDS-PAGE gel under either semi-native or denaturing conditions. The positions of trimeric and monomeric fiber are indicated. As a control for electrophoretic conditions, recombinant Ad2 fiber produced in baculovirus-infected cells was run under identical conditions and stained with Coomassie blue. Figure 14 shows the complementation of a fiber mutant adenovirus by fiber-producing cells. The cell lines indicated (2x10 6 cells per sample) were infected with the temperature-sensitive fiber mutant adenovirus H5ts142 at 10 PFU/cell and incubated at either the permissive (32.5 0
C,
stippled bars) or the restrictive (39.5°C, solid bars) temperature. 48 hours postinfection, virus was isolated by freeze-thaw lysis and yields determined by fluorescent focus assay on SW480 cells. Each value represents the mean of duplicate samples, and the data shown is representative of multiple experiments.
Figure 15 shows the incorporation of the recombinant Ad5 fiber into Ad3 particles. In Figure 15A, the alignment of the N-terminal (penton base-binding) domains of fiber proteins from several different adenovirus serotypes is shown.
From top to bottom, the five different serotypes are listed as SEQ ID NOs 21-25. In Figure 15B, type 3 adenovirus was propagated in 293, 211B, or 211R cells as indicated and purified by two sequential CsCI centrifugations. 10 pg of the purified viral particles was then electrophoresed under denaturing conditions and transferred to a PVDF membrane. Ad5 fiber was detected with a polyclonal rabbit antibody raised against recombinant Ad2 fiber. As a positive control for detection, 400 ng of wild-type Ad2 was run in the lane marked "Ad2". Under these conditions, the mobilities of the Ad2 and Ad5 fibers are indistinguishable and the antibody reacts with both proteins.
Figure 16 shows the fiber deletion in pDV44 and the genomic structures of the Ad5.pgal.AF and Ad5.pgal.wt vectors: Figure 16A shows pDV44 that was constructed by removing the fiber gene and residual E3 sequences (nt 30819:32743 of AD5) from pBHG10. Figures 16B shows viruses constructed by cotransfection of either pBHG10 or pDV44 with pAE1Bgal. Both are E1/E3 deleted Ad5 vectors, and Ad5.pgal.AF has the additional fiber (L5) deletion as in pDV44.
WO 00/42208 PCT/EP00/00265 -11- Figure 17 shows the analysis of the viral chromosomes. Figure 17A shows the predicted EcoRI restriction maps of Ad5.pgal.wt and Ad5.pgal.AF. The 5.9 kb fragment at the right end of the Ad5.pgal.wt genome is reduced to 4.0 kb by the deletion of fiber sequences in Ad5.pgal.AF. Figure 17B shows an ethidium bromide-stained gel of EcoRI-digested viral DNA. Figure 17C shows a Southern blot of the gel as described in Example 2 probed either with labeled fiber or E4 sequences.
Figure 18 shows the analysis of vertex proteins in the viral particles. 293 (non-fiber expressing) or 211B (fiber-expressing) cells were infected with or with Ad5.pgal.AF('AF') and the resulting viral particles were purified on CsCI gradients. 10 pg of purified virions was then electrophoresed on 5-16% gradient gels and Western blotted. Proteins were detected with polyclonal anti-fiber or anti-penton base antibodies.
Figure 19 shows the infectivity of Ad particles on THP-1 monocytic cells.
Figure 19A shows THP-1 cells that were infected with Ad5.pgal.wt or with fiberless at 100,000 particles/cell. Forty-eight hours after infection, cells were fixed and stained with X-gal and the fraction of infected cells was determined by light microscopy. Figure 19B shows cells that were infected with 1000 particles per cell of Ad5.pgal.wt or with 100,000 particles/cell of Ad5.pgal.AF. As indicated, cells were pretreated with 100 pg/ml of recombinant penton base or with 20 pg/ml of recombinant Ad2 fiber.
Figure 20 shows a schematic of improved fiber-complementing cell lines, 633 and 644 as further described in the Examples.
Figures 21 and 22 illustrates pseudotyping of fiberless particles with fiber proteins and infectivity data as further described in the Examples.
Figure 23 shows the Clal to Bglll fragment of Figure 24 shows the plasmid pGRE5-2/EBV Figure 25 shows the plasmid pGRE5-E1.
Figure 26 shows the plasmid pSE280-E2 BamHI-Smal.
Figure 27. The fiber-deleted adenovirus vector Ad5.bgal.DF was grown in cells expressing either no fiber (293; 'Ad5.bgal.DF/0'), the Ad5 fiber (633; or the Ad37 fiber with modifications as described in the text (705; 'Ad5.bgal.DF/37F') and CsCI-purified. 10 pg of the purified particles were electrophoresed and transferred to a nylon membrane. As controls, 10 pg of wildtype Ad37 or the fiber gene-containing vector Ad5.bgal.wt or a sample of purified recombinant Ad37 fiber knob were also run. The blot was probed with polyclonal WO 00/42208 PCT/EP00/00265 -12antisera against recombinant Ad37 fiber or Ad2 fiber proteins. As a loading control, the same filter was reprobed with an antibody against the Ad2 penton base (the anti-Ad2 sera cross-recognized the very similar Ad5 fiber and Ad5 penton base proteins).
Figure 28 shows PCR analysis for fiber presence.
Figure 29 shows the transduction efficiency for fiberless virus with and without soluble fiber.
Figure 30 shows the transduction efficiency of AD5BgF on HDF cell line with the presence of different amounts of 633 conditioned media.
To reduce the frequency of contamination with wild-type adenovirus, it is considered desirable to improve either the viral vector or the cell line to reduce the probability of recombination. For example, an adenovirus from a group with less homology to the group C viruses may be used to engineer recombinant viruses with little propensity for recombination with the Ad5 sequence in 293 cells.
Similarly, an epithelial cell line e.g. the cell line known as 293 may be used or further modified according to within-disclosed methods which stably expresses adenovirus proteins or polypeptides from Ad3 and/or proteins or polypeptides from another non-group-C or group C serotype; such a cell line would be useful to support adenovirus-derived viral vectors bearing deletions of regulatory and/or structural genes, irrespective of the serotype from which such a vector was derived.
It is also contemplated that the constructs and methods of the present invention will support the design and engineering of chimeric viral vectors which express amino acid residue sequences derived from two or more Ad serotypes.
Thus, unlike methods and constructs available prior to the advent of the present disclosure, this invention allows the greatest possible flexibility in the design and preparation of useful viral vectors and cell lines which support their construction and propagation all with a decreased risk of recombining with wild-type Ad to produce potentially-harmful recombinants.
In part, the present invention discloses a simpler, alternative means of reducing the recombination between viral and cellular sequences than those discussed in the art. One such means is to increase the size of the deletion in the recombinant virus and thereby reduce the extent of shared sequences between that virus and any Ad genes present in a packaging cell line the Ad5 genes in 293 cells, or the various Ad genes in the novel cell lines of the present invention.
WO 00/42208 PCT/EPOO/00265 -13- Therefore, the present invention makes it feasible to engineer and produce novel viral vectors that are able to package and deliver significantly larger foreign nucleic acid sequences for efficacious use in a variety of therapeutic applications, without endangering the subject to whom they are administered, due to their impaired ability to self-replicate in non-complementing cell lines. Due to the fact that "helper" viruses or plasmids need not be used in conjunction with many of the viral vectors of the present invention, those vectors of the present invention are also simpler to use than those previously described in the art.
In order to provide a clearer understanding of the specification and claims, the following definitions are provided.
Adenoviral Vector or Ad-Derived Vector Any adenovirus-derived plasmid, genome or virus into which a foreign DNA may be inserted or expressed. This term may also be used interchangeably with "viral vector." This "type" of vector may be utilized to carry nucleotide sequences encoding therapeutic proteins or polypeptides to specific cells or cell types in a subject in need of treatment, as described further herein below.
Amino Acid Residue: An amino acid formed upon chemical digestion (hydrolysis) of a polypeptide at its peptide linkages. The amino acid residues described herein are preferably in the isomeric form. However, residues in the isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property is retained by the polypeptide. NH 2 refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxyl terminus of a polypeptide. Standard polypeptide nomenclature described in J. Biol. Chem., 243:3552-59 (1969) and adopted at 37 C.F.R. 1.821 1.822 is used.
It should be noted that all amino acid residue sequences represented herein by formulae have a left to right orientation in the conventional direction of amino-terminus to carboxyl-terminus. In addition, the phrase "amino acid residue" is broadly defined to include the amino acids listed in the Table of Correspondence and modified and unusual amino acids, such as those referred to in 37 C.F.R. 1.821-1.822, and incorporated herein by reference. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino acid residues or to an amino-terminal group such as NH 2 or to a carboxyl-terminal group such as
COOH.
WO 00/42208 PCT/EPOO/00265 -14- Complementing Plasmid: This term is generally used herein to describe plasmid vectors used to deliver particular nucleotide sequences into a packaging cell line, with the intent of having said sequences stably integrate into the cellular genome.
Delivery Plasmid: This term is generally used herein to describe a plasmid vector that carries or delivers nucleotide sequences in or into a cell line a packaging cell line) for the purpose of propagating therapeutic viral vectors of the present invention.
DNA Homolog: A nucleic acid having a preselected conserved nucleotide sequence and a sequence encoding a preferred polypeptide according to the present invention, where the nucleic acid is substantial homologous to a named preferred embodiment. By the term "substantially homologous" is meant having at least 80%, preferably at least 90%, most preferably at least 95% homology therewith.
The terms "homology" and "identity" are often used interchangeably. In this regard, percent homology or identity may be determined, for example, by comparing sequence information using a GAP computer program. The GAP program utilizes the alignment method of Needleman and Wunsch Mol. Biol.
48:443 (1970), as revised by Smith and Waterman (Adv. Appl. Math. 2:482 (1981).
Briefly, the GAP program defines similarity as the number of aligned symbols nucleotides or amino acids) which are similar, divided by the total number of symbols in the shorter of the two sequences. The preferred default parameters for the GAP program may include: a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) and the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745 (1986), as described by Schwartz and Dayhoff, eds., ATLAS OF PROTEIN SEQUENCE AND STRUCTURE, National Biomedical Research Foundation, pp. 353-358 (1979); a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and no penalty for end gaps.
Whether any two nucleic acid molecules have nucleotide sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% "identical" can be determined using known computer algorithms such as the "FAST A" program, using for example, the default parameters as in Pearson and Lipman, Proc. Natl. Acad.
Sc. USA 85:2444 (1988). Alternatively the BLAST function of the National Center for Biotechnology Information database may be used to determine identity In general, sequences are aligned so that the highest order match is obtained. "Identity" per se has an art-recognized meaning and can be calculated WO 00/42208 PCT/EP00/00265 using published techniques. (See, Computational Molecular Biology, Lesk, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, and Griffin, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, eds., M Stockton Press, New York, 1991). While there exist a number of methods to measure identity between two polynucleotide or polypeptide sequences, the term "identity" is well known to skilled artisans (Carillo, H. Lipton, SIAM J Applied Math 48:1073 (1988)). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H. Lipton, SIAM J Applied Math 48:1073 (1988). Methods to determine identity and similarity are codified in computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCG program package (Devereux, et al., Nucleic Acids Research 12(1):387 (1984)), BLASTP, BLASTN, FASTA (Atschul, et al., J Molec Biol 215:403 (1990)).
Therefore, as used herein, the term "identity" represents a comparison between a test and a reference polypeptide or polynucleotide. For example, a test polypeptide may be defined as any polypeptide that is 90% or more identical to a reference polypeptide. As used herein, the term at least "90% identical to" refers to percent identities from 90 to 99.99 relative to the reference polypeptides.
Identity at a level of 90% or more is indicative of the fact that, assuming for exemplification purposes a test and reference polynucleotide length of 100 amino acids are compared. No more than 10% 10 out of 100) amino acids in the test polypeptide differs from that of the reference polypeptides. Similar comparisons may be made between a test and reference polynucleotides. Such differences may be represented as point mutations randomly distributed over the entire length of an amino acid sequence or they may be clustered in one or more locations of varying length up to the maximum allowable, e.g. 10/100 amino acid difference (approximately 90% identity). Differences are defined as nucleic acid or amino acid substitutions, or deletions.
An embodiment of the invention may use polynucleotides at least 90% or identical to those encoding the TPL nucleic acid sequences. A further embodiment of the invention may include those polynucleotides that encode a WO 00/42208 PCT/EP00/00265 -16polypeptide of interest that are at least 95% identical when the variation in such a polynucleotide is due to more than merely degenerate changes.
Expression or Delivery Vector: Any plasmid or virus into which a foreign DNA may be inserted for expression in a suitable host cell the protein or polypeptide encoded by the DNA is synthesized in the host cell's system. Vectors capable of directing the expression of DNA segments (genes) encoding one or more proteins are referred to herein as "expression vectors." Also included are vectors which allow cloning of cDNA (complementary DNA) from mRNAs produced using reverse transcriptase.
Foreign Gene: This term is used to identify a DNA molecule not present in the exact orientation and position as the counterpart DNA molecule found in wildtype adenovirus. It may also refer to a DNA molecule from another organism or species exogenous) or from another Ad serotype.
Gene: A nucleic acid whose nucleotide sequence encodes an RNA or polypeptide. A gene can be either RNA or DNA. Genes may include regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).
Isolated: This term is used to indicate a nucleic acid or polypeptide sequence separated from the genetic environment from which the sequences were obtained. It may also mean altered from the natural state. For example, a polynucleotide or a polypeptide naturally present in a living animal is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein. Thus, a polypeptide or polynucleotide produced and/or contained within a recombinant host cell is considered isolated for purposes of the present invention. Also intended as an "isolated polypeptide" or an "isolated polynucleotide" are polypeptides or polynucleotides that have been purified, partially or substantially, from a recombinant host cell or from a native source. For example, a recombinantly produced version of a compounds can be substantially purified by the one-step method described in Smith and Johnson, Gene 67.31-40 (1988). The terms isolated and purified are sometimes used interchangeably.
By "isolated" is meant that the DNA is free of the coding sequences of those genes that, in the naturally-occurring genome of the organism (if any) from which the DNA of the invention is derived, immediately flank the gene encoding the DNA of the invention. The isolated DNA may be single-stranded or double-stranded, and may be genomic DNA, cDNA, recombinant hybrid DNA, or synthetic DNA. It WO 00/42208 PCT/EPOO/00265 -17may be identical to a native DNA sequence, or may differ from such sequence by the deletion, addition, or substitution of one or more nucleotides.
Isolated or purified as it refers to preparations made from biological cells or hosts should be understood to mean any cell extract containing the indicated DNA or protein including a crude extract of the DNA or protein of interest. For example, in the case of a protein, a purified preparation can be obtained following an individual technique or a series of preparative or biochemical techniques and the DNA or protein of interest can be present at various degrees of purity in these preparations. The procedures may include for example, but are not limited to, ammonium sulfate fractionation, gel filtration, ion exchange change chromatography, affinity chromatography, density gradient centrifugation and electrophoresis.
A preparation of DNA or protein that is "pure" or "isolated" should be understood to mean a preparation free from naturally occurring materials with which such DNA or protein is normally associated in nature. "Essentially pure" should be understood to mean a "highly" purified preparation that contains at least of the DNA or protein of interest.
A cell extract that contains the DNA or protein of interest should be understood to mean a homogenate preparation or cell-free preparation obtained from cells that express the protein or contain the DNA of interest. The term "cell extract" is intended to include culture media, especially spent culture media from which the cells have been removed.
Packaging Cell line: A packaging cell line is a cell line that provides a missing gene product or its equivalent.
Particle: The adenovirus (Ad) particle is relatively complex and may be resolved into various substructures. The particle is the minimal structural or functional unit of a virus. A virus can refer to a single particle, a stock of particles or a viral genome.
Penton: The terms "penton" or "penton complex" are preferentially used herein to designate a complex of penton base and fiber. The term "penton" may also be used to indicate penton base, as well as penton complex. The meaning of the term "penton" alone should be clear from the context within which it is used.
Plasmid: An autonomous self-replicating extrachromosomal circular DNA Post-transcriptional Regulatory Element (PRE) is a regulatory element found in viral or cellular messenger RNA that is not spliced, i.e. intronless messages.
Examples include, but are not limited to, human hepatitis virus, woodchuck WO 00/42208 PCT/EPOO/00265 -18hepatitis virus, the TK gene and mouse histone gene. The PRE may be placed before a polyA sequence and after a heterologous DNA sequence.
Pseudotyping: This term as generally used herein describes the production of adenoviral vectors having modified capsid protein or capsid proteins from a different serotype than the serotype of the vector itself. One example, is the production of an adenovirus 5 vector particle containing a chimeric Ad3/Ad5 fiber protein. This may be accomplished by producing the adenoviral vector in packaging cell lines expressing different fiber proteins.
Promoter: Useful promoters according to the present invention may be inducible or constitutive. Inducible promoters will initiate transcription only in the presence of an additional molecule; constitutive promoters, on the other hand, do not require the presence of any additional molecule to regulate gene expression.
A regulatable or inducible promoter may also be described as a promoter wherein the rate of RNA polymerase binding and initiation is modulated by external stimuli.
Such stimuli include various compounds or compositions, light, heat, stress, chemical energy sources, and the like. Inducible, suppressible and repressible promoters are considered regulatable promoters.
Receptor: Receptor is a term used herein to indicate a biologically active molecule that specifically binds to (or with) other molecules. The term "receptor protein" may be used to more specifically indicate the proteinaceous nature of a specific receptor.
Recombinant As used herein, the term is intended to refer to any progeny formed as the result of genetic engineering. This may also be used to describe a virus formed by recombination of plasmids in a packaging cell.
Transgene or Therapeutic Nucleotide Sequence: As described and claimed herein, such a sequence includes DNA and RNA sequences encoding an RNA or polypeptide. Such sequences may be "native" or naturally-derived sequences; they may also be "non-native" or "foreign" sequences which are naturally- or recombinantly-derived. The term "transgene," which may be used interchangeably herein with the term "therapeutic nucleotide sequence," is often used to describe a heterologous or foreign (exogenous) gene that is carried by a viral vector and transduced into a host cell.
Therefore, therapeutic nucleotide sequences may also include antisense sequences or nucleotide sequences which may be transcribed into antisense sequences. Therapeutic nucleotide sequences (or transgenes) further comprise sequences which function to produce a desired effect in the cell or cell nucleus into which said therapeutic sequences are delivered. For example, a therapeutic WO 00/42208 PCT/EP00/00265 -19nucleotide sequence may encode a functional protein intended for delivery into a cell which is unable to produce that functional protein.
Adenovirus Fiber plays a crucial role in adenovirus infection by attaching the virus to a specific receptor on the cell surface. The fiber is an elongated protein which exists as a trimer of three identical polypeptides (polypeptide IV) of 582 amino acids in length. An adenovirus fiber consists of three domains: an N-terminal tail domain that interacts with penton base; a shaft composed of variable numbers of repeats of a 15-amino-acid segment that forms beta-sheet and beta-bends; and a knob at the C-terminus ("head domain") that contains the type-specific antigen and is responsible for binding to the cell surface receptor. The gene encoding the fiber protein from Ad2 has been expressed in human cells and has been shown to be correctly assembled into trimers, glycosylated and transported to the nucleus.
(See, Hong and Engler, Virology 185: 758-761, 1991). Thus, alteration of the fiber in recombinant Ad vectors can lead to alteration in gene delivery. This has great utility for a variety of gene therapy applications and is one of the objects of the present invention.
Hexon, penton and fiber capsomeres are the major components on the surface of the virion. Their constituent polypeptides, nos. II, III and IV, contain tyrosine residues that are exposed on the surface of the virion and can be labeled by iodination of intact particles.
The 35,000+ base pair (bp) genome of adenovirus type 2 has been sequenced and the predicted amino acid sequences of the major coat proteins (hexon, fiber and penton base) have been described. (See, Neumann et al., Gene 69: 153-157 (1988); Herisse et al., Nuc. Acids Res. 9: 4023-4041 (1981); Roberts et al., J. Biol. Chem. 259: 13968-13975 (1984); Kinloch et al., J. Biol.
Chem. 259: 6431-6436 (1984); and Chroboczek et al., Virol. 161: 549-554, 1987).
The sequence of Ad5 DNA was completed more recently; its sequence includes a total of 35,935 bp. Portions of many other adenovirus genomes have also been sequenced. It is presently understood that the upper packaging limit for adenovirus virions is about 105% of the wild-type genome length. (See, Bett, et al., J. Virol. 67(10): 5911-21, 1993). Thus, for Ad2 and Ad5, this would be an upper packaging limit of about 38kb of DNA.
Adenovirus DNA also includes inverted terminal repeat sequences (ITRs) ranging in size from about 100 to 150 bp, depending on the serotype. The inverted repeats enable single strands of viral DNA to circularize by base-pairing WO 00/42208 PCT/EP00/00265 of their terminal sequences, and the resulting base-paired "panhandle" structures required for replication of the viral DNA.
For efficient packaging, the ITRs and the packaging signal (a few hundred bp in length) comprise the "minimum requirement" for replication and packaging of a genomic nucleic acid into an adenovirus particle. Helper-dependent vectors lacking all viral ORFs but including these essential cis elements (the ITRs and contiguous packaging sequence) have been constructed, but the virions package less efficiently that the helper and package as multimers part of the time, which suggests that the virus may "want" to package a fuller DNA complement (see, e.g., Fisher, etaL, Virology 217: 11-22, 1996).
The viral vectors of the present invention may retain their ability to express the genome packaged within they may retain their "infectivity" they do not act as infectious agents, however, to the extent that they cause disease in the subjects to whom they are administered for therapeutic purposes.
It is to be appreciated that Ad vectors have several distinct advantages over other viral vectors in the art. For example, recombination of such vectors is rare; there are no known associations of human malignancies with adenoviral infections despite common human infection with adenoviruses; the genome may be manipulated to accommodate foreign genes of a fairly substantial size; and host proliferation is not required for expression of adenoviral proteins.
An extension of this invention is that the Ad-derived viral vectors disclosed herein may be used to target and deliver genes into specific cells by incorporating the attachment sequence for other receptors (such as CD4) onto the fiber protein by recombinant DNA techniques, thus producing a chimeric molecule. This should result in the ability to target and deliver genes into a wide range of cell types with the advantage of evading recognition by the host's immune system. The withindisclosed delivery systems thus provide for increased flexibility in gene design to enable gene delivery into proliferating and nonproliferating cell types.
For example, U.S. Patent Nos. 5,756,086 and 5,543,328 as well as, W095/26412 and WO 98/44121 and Krasnykh, et al. Virol. 70: 6839-46, 1996) describe modifications that may be made to the adenovirus fiber protein. Such modifications are useful in altering the targeting mechanism and specificity of adenovirus and could readily be utilized in conjunction with the constructs of the present invention to target the novel viral vectors disclosed herein to different receptors and different cells. Moreover, modifications to fiber protein which alter its tropism may permit greater control over the localization of viral vectors in therapeutic applications.
WO 00/42208 PCT/EP00/00265 -21- Similarly, incorporation of various structural proteins into cell lines of the present invention, whether or not those proteins are modified, is also contemplated by the present invention. Thus, for example, modified penton base polypeptides such as those described in Wickham, et al. Virol. 70' 6831-8, 1996) may have therapeutic utility when used according to the within-disclosed methods.
While some of the Examples appearing below specifically recite fiber proteins, polypeptides, and fragments thereof, it is expressly provided herein that other structural and non-structural Ad proteins and polypeptides regulatory proteins and polypeptides) may be used as components of the various disclosed vectors and cell lines. Moreover, chimeric molecules comprised of proteins, polypeptides, and/or fragments thereof which are derived from different Ad serotypes may be used in any of the within-disclosed methods, constructs and compositions. Similarly, recombinant DNA sequences of the present invention may be prepared using nucleic acid sequences derived from different Ad serotypes, in order to design useful constructs with broad applicability, as disclosed and claimed herein.
It should also be appreciated that, while the members of Group C or Group D adenovirus Ad serotypes 1, 2, 5, 6 or 37 are specifically recited in various examples herein, the present invention is in no way limited to those serotypes alone. In view of the fact that the adenovirus serotypes are all closelyrelated in structure and functionality, therapeutic viral vectors, packaging cell lines, and plasmids of the present invention may be constructed from components of any and all Ad serotypes and the within-disclosed methods of making and using the various constructs and cell lines of the present invention apply to all of said serotypes.
The family of Adenoviridae includes many members with at least 47 known serotypes of human adenovirus (Ad1-Ad47) (Shenk, Virology, Chapter 67, in Fields et al., eds. Lippincott-Raven, Philadelphia, 1996,) as well as members of the genus Mastadenovirus including human, simian, bovine, equine, porcine, ovine, canine and opossum viruses, and members of the Aviadenovirus genus, including bird viruses, e.g. CELO. Thus it is contemplated that the disclosed inventions can be applied to any adenovirus species, and the invention need not be so limited. One of skill in the art would have knowledge of the different adenoviruses as evidenced by (Shenk, Virology, Chapter 67, in Fields et al., eds.
Lippincott-Raven, Philadelphia, 1996,) which is herein incorporated by reference.
WO 00/42208 PCT/EP00/00265 -22- Packaging Cell Lines A. Adenovirus Complementation Genetics The first generation of recombinant adenoviral vectors currently available typically have a deletion in the first viral early gene region which is generally referred to as El, which comprises the Ela and Elb regions. (These regions span genetic map units 1.30 to 9.24.) Figure 3 in chapter 67 of Fields Virology, 3d Ed. (Fields et al. eds, Lippincott-Raven Publ., Philadelphia, 1996, p. 2116) illustrates a transcription and translation map of adenovirus type 2 (Ad2).
Deletion of the viral El region renders the recombinant adenovirus defective for replication and incapable of producing infectious viral particles in subsequently-infected target cells. Thus, to generate El-deleted adenovirus genome replication and to produce virus particles requires a system of complementation which provides the missing El gene product. El complementation is typically provided by a cell line expressing El, such as the human embryonic kidney packaging cell line, i.e. an epithelial cell line, called 293.
Cell line 293 contains the El region of adenovirus, which provides El gene region products to "support" the growth of El-deleted virus in the cell line (see, e.g., Graham et al., J. Gen. Virol. 36: 59-71, 1977). Additionally, cell lines that may be usable for production of defective adenovirus having a portion of the adenovirus E4 region have been reported (WO 96/22378). Multiply deficient adenoviral vectors and complementing cell lines have also been described (WO 95/34671, U.S. Patent No. 5,994,106). Nevertheless, inherent problems exist concerning first-generation recombinant adenoviruses.
B. Adenovirus Particle Packaging Cell Lines Packaging cell lines disclosed herein support viral vectors with deletions of major portions of the viral genome, without the need for helper viruses.
Additionally, the invention provides novel cell lines and helper viruses for use with helper-dependent vectors.
Thus, in one embodiment of the present invention, a packaging cell line is disclosed having DNA sequences stably integrated into the cellular genome wherein the DNA sequences encode one or more adenovirus regulatory and/or structural polypeptides which complement the genes deleted or mutated in the adenovirus vector genome to be replicated and packaged.
In another embodiment, the packaging cell line expresses one or more adenovirus structural proteins, polypeptides, or fragments thereof, wherein said WO 00/42208 PCTIEP00/00265 -23structural protein is selected from the group consisting of penton base, hexon, fiber, polypeptide Ilia, polypeptide V, polypeptide VI, polypeptide VII, polypeptide VIII, and biologically active fragments thereof.
In one variation, the sequences are constitutively expressed; in another, one or more sequences is under the control of a regulatable promoter. In a preferred embodiment expression is constitutive. In various preferred embodiments, the polypeptides expressed by the DNA sequences are biologically active.
In a further and preferred embodiment the packaging cell line of the present invention supports the production of a viral vector. In a preferred embodiment the viral vector is a therapeutic vector.
The present invention also discloses a packaging cell line which complements a viral vector having a deletion or mutation of a DNA sequence encoding an adenovirus structural protein, regulatory polypeptides E1A and E1B, and/or one or more of the following regulatory proteins or polypeptides: E2A, E2B, E3, E4, L4, or fragments thereof.
Various useful packaging cells are contemplated which complement adenovirus. In one aspect of the present invention, each DNA sequence is introduced into the genome of the within-disclosed cell lines via a separate complementing plasmid. In other embodiments, two or more DNA sequences were introduced into the genome via a single complementing plasmid. In one variation, the complementing plasmid comprises a DNA sequence encoding adenovirus fiber protein, polypeptide or fragment thereof. An example of a useful complementing plasmid according to the present invention is a plasmid having the characteristics of pCLF (for deposit details, see Example 3) One embodiment discloses a packaging cell useful in the preparation of recombinant adenovirus viral vectors comprising a delivery plasmid comprising an adenovirus genome lacking a nucleotide sequence encoding fiber. In one variation, the delivery plasmid further comprises a nucleotide sequence encoding a foreign polypeptide. A preferred delivery plasmid is pDV44, pE1B gal, or pElsplB. In another variation, the cell further comprises a complementing plasmid containing a nucleotide sequence encoding fiber, the plasmid being stably integrated into the cellular genome of the cell.
In one embodiment, a composition comprises a cell containing first and second delivery plasmids wherein a first delivery plasmid comprises an adenovirus genome lacking a nucleotide sequence encoding fiber and incapable of directing the packaging of new viral particles in the absence of a second WO 00/42208 PCT/EP00/00265 -24delivery plasmid, and a second delivery plasmid comprises an adenoviral genome capable of directing the packaging of new viral particles in the presence of the first delivery plasmid.
In another variation, the first and second delivery plasmids interact within the cell to produce a therapeutic viral vector. In yet another variation, the cell further comprises a complementing plasmid containing a nucleotide sequence encoding fiber, the plasmid being stably integrated into the cellular genome of the cell. In still another, the first or second delivery plasmid further comprises a nucleotide sequence encoding a foreign polypeptide. In various embodiments, the polypeptide is a therapeutic molecule.
Another embodiment discloses a composition as before, wherein the first delivery plasmid lacks adenovirus packaging signal sequences. In another aspect, the second delivery plasmid contains a LacZ reporter construct. In another variation, the second delivery plasmid further lacks a nucleotide sequence encoding an adenovirus regulatory protein. In one variation, the regulatory protein is El. In one embodiment of the above-noted compositions, the complementing plasmid has the characteristics of pCLF.
In another embodiment, a composition is disclosed wherein the first delivery plasmid lacks a nucleotide sequence encoding an adenovirus structural protein and the second delivery plasmid lacks a nucleotide sequence encoding adenovirus El protein. In another, the first delivery plasmid lacks a nucleotide sequence encoding adenovirus E4 protein and the second delivery plasmid lacks a nucleotide sequence encoding adenovirus El protein. In still another, the cell contains at least one complementing plasmid encoding an adenoviral regulatory protein and a structural protein.
In one preferred variation of the present invention, a packaging cell line expresses fiber protein. In one embodiment, the fiber protein has been modified to include a non-native amino acid residue sequence which targets a specific receptor, but which does not disrupt trimer formation or transport of fiber into the nucleus. In another variation, the non-native amino acid residue sequence alters the binding specificity of the fiber for a targeted cell type. In still another embodiment, the structural protein is fiber comprising amino acid residue sequences from more than one adenovirus serotype. As disclosed herein, the nucleotide sequences encoding fiber protein or polypeptide need not be modified solely at one or both termini; fiber protein and indeed, any of the adenovirus structural proteins, as taught herein may be modified "internally" as well as at the termini.
WO 00/42208 PCTIEPOO/00265 In one variation, the non-native amino acid residue sequence is coupled to the carboxyl terminus of the fiber. In yet another, the non-native amino acid residue sequence further includes a linker sequence. Alternatively, the fiber protein further comprises a ligand coupled to the linker. A suitable ligand may be selected from the group consisting of ligands that specifically bind to a cell surface receptor and ligands that can be used to couple other proteins or nucleic acid molecules. Typically, any of the packaging cell lines of this invention may have a DNA sequence encoding all or part of a fiber protein including modified or chimeric proteins stably integrated into the genome.
In various aspects of the present invention, a packaging cell line of the present invention is derived from a procaryotic cell line; in another, it is derived from a eucaryotic cell line. While various embodiments suggest the use of mammalian cells, and more particularly, epithelial cell lines, a variety of other, non-epithelial cell lines are used in various embodiments. Thus, while various embodiments disclose the use of a cell line selected from the group consisting of 293, A549, W162, HeLa, Vero, 211, and 211A cell lines, it is understood that various other cell lines are likewise contemplated for use as disclosed herein.
Therapeutic Viral Vectors and Related Systems A. Nucleic Acid Segments A therapeutic viral vector or composition of the present invention comprises a nucleotide sequence, nucleic acid molecule or segment as described herein.
Typically, the nucleic acid molecule or molecule encodes a protein or polypeptide molecule or a biologically active fragment thereof which may be used for therapeutic applications. A nucleotide sequence may further comprise an enhancer element or a promoter located 3' or 5' to and controlling the expression of such a therapeutic nucleotide sequence or gene.
A subject nucleotide sequence consists of a nucleic acid molecule that comprises at least 2 different operatively linked DNA segments. The DNA can be manipulated and amplified by PCR as described herein and by using standard techniques, such as those described in Molecular Cloning: A Laboratory Manual, 2nd Ed., Sambrook et al., eds., Cold Spring Harbor, New York (1989). Typically, to produce a nucleotide sequence of the present invention, the sequence encoding the selected polypeptide and the promoter or enhancer are operatively linked to a DNA molecule capable of autonomous replication in a cell either in vivo or in vitro.
WO 00/42208 PCT/EP00/00265 -26- By operatively linking the enhancer element or promoter and the nucleotide sequence to the vector, the attached segments are replicated along with the vector sequences.
Thus, a recombinant DNA molecule (rDNA) of the present invention is a hybrid DNA molecule comprising at least 2 nucleotide sequences not normally found together in nature. In various preferred embodiments, one of the sequences is a sequence encoding an Ad-derived polypeptide, protein, or fragment thereof.
Stated another way, a nucleotide sequence of the present invention is one that encodes an expressible protein, polypeptide or fragment thereof, and it may further include an active constitutive or regulatable inducible) promoter sequence.
A nucleotide sequence of the present invention is optimally from about base pairs to about 40,000 base pairs in length. Preferably the nucleotide sequence is from about 50 bp to about 38,000 bp in length. In various embodiments, the nucleotide sequence is of sufficient length to encode one or more adenovirus proteins or functional polypeptide portions thereof. Since individual Ad polypeptides vary in length from about 19 amino acid residues to about 967 amino acid residues, corresponding nucleotide sequences will range from about 50 bp up to about 3000 bp, depending on the number and size of individual polypeptide-encoding sequences that are "replaced" in the viral vectors by therapeutic nucleotide sequences of the present invention.
1. Tripartite Leader (TPL) Nucleic Acid Sequences In one aspect of the invention, it has been discovered that expression of adenovirus late proteins such as the structural proteins in a packaging cell line according to the present invention is substantially improved when the expression cassette present on the complementing plasmid or in the packaging cell line's genome contains an adenovirus tripartite leader (TPL) nucleic acid sequence.
Thus, the invention contemplates a nucleic acid molecule comprising a TPL nucleotide sequence. Preferably, the TPL nucleotide sequence may be operatively linked to an intron containing RNA processing signals (such as for example, splice donor or splice acceptor sites) suitable for expression in the packaging cell line.
Most preferably, the intron contains a splice donor site and a splice acceptor site.
Alternatively, the TPL nucleotide sequence may not comprise an intron.
In one embodiment, a subject nucleic acid molecule of this invention is isolated, separated from the genetic environment from which the component sequences were obtained. Thus, molecular cloning of fragments of a gene will WO 00/42208 PCT/EP00/00265 -27produce an isolated nucleic acid, as will the chemical synthesis of an oligonucleotide to build a nucleic acid molecule.
The intron useful in the present invention is any nucleotide sequence which functions in the packaging cell line to provide RNA processing signals, as are well known in the art, including splicing signals. Introns have been well characterized from a large number of structural genes, and therefor the invention should not be considered as limited. Well characterized and preferred introns include a native intron 1 from adenovirus, such as Ad5's TPL intron 1; others include the SV40 VP intron; the rabbit beta-globin intron, and synthetic intron constructs. See, for example, Petitclerc et al., J. Biothechnol., 40:169, 1995, and Choi et al., Mol. Cell.
Biol., 11:3070, 1991.
The TPL nucleotide sequence comprises either first and second TPL exons or first, second and third TPL exons, where each TPL exon in the sequence is selected from the group consisting of complete TPL exon 1, partial TPL exon 1, complete TPL exon 2 and complete TPL exon 3. A complete exon is one which contains the complete nucleic acid sequence based on the sequence found in the wild type viral genome. Preferably the TPL exons are from Ad2, Ad3, Ad7 and the like, however, they may come from any Ad serotype, as described herein. A preferred partial TPL exon 1 is described in the Examples.
The use of a TPL with a partial exon 1 has been reported (WO98/13499).
The intron and the TPL exons can be operatively linked in a variety of configurations to provide a functional TPL nucleotide sequence, although in some embodiments of the invention, an intron may not be a part of the construct. For example, the intron can be positioned between any of TPL exons 1, 2 or 3, and the exons can be in any order of first and second, or first/second/third. The intron can also be placed preceding the first TPL exon or following the last TPL exon. In a preferred embodiment, complete TPL exon 1 is operatively linked to complete TPL exon 2 operatively linked to complete TPL exon 3. In a preferred variation, adenovirus TPL intron 1 is positioned between complete TPL exon 1 and complete TPL exon 2. It may also be possible to use analogous translational regulators from other viral systems such as rabiesvirus.
A preferred "complete" TPL nucleic acid molecule containing complete TPL exons 1, 2 and 3 with adenovirus intron 1 inserted between exons 1 and 2 has a nucleotide sequence shown in SEQ ID NO: 32. A preferred "partial" TPL nucleic acid molecule containing partial TPL exon 1 and complete TPL exons 2 and 3 in that order has a nucleotide sequence shown in SEQ ID NO: 26. The construction of these preferred TPL nucleotide sequences is described in the Examples.
WO 00/42208 PCT/EP00/00265 -28- Thus, preferred expression cassettes and complementing plasmids for expressing adenovirus structural genes, particularly fiber protein, contain an adenovirus TPL nucleotide sequence as described herein. Preferred packaging cell lines containing the subject nucleic acid molecules also contain a TPL nucleotide sequence of the invention.
2. Complementing Plasmids The invention describes in a related embodiment nucleic acid molecules and nucleotide sequences, typically in the form of DNA plasmid vectors, which are capable of expression of an adenovirus structural protein or regulatory protein.
Because these expression plasmids are used to complement the defective genes of a recombinant adenovirus vector genome of this invention, the plasmids are referred to as complementing or complementation plasmids.
The complementing plasmid contains an expression cassette, a nucleotide sequence capable of expressing a protein product off the gene encoded by the nucleotide sequence. Expression cassettes are described in more detail herein, but typically contain a promoter and a structural gene operatively linked to the promoter and whose expression is controlled by the promoter. A preferred complementing plasmid further includes a TPL nucleotide sequence described herein to enhance expression of the structural gene product when used in the context of adenovirus genome replication and packaging.
In one embodiment, a complementing plasmid comprises a promoter nucleotide sequence operatively linked to a nucleotide sequence encoding an adenovirus structural polypeptide. The adenovirus structural polypeptide is selected from the group consisting of penton base; hexon; fiber; polypeptide Ilia; polypeptide V; polypeptide VI; polypeptide VII; polypeptide VIII; and biologically active fragments thereof. In another variation, a complementing plasmid further comprises a nucleotide sequence encoding a first adenovirus regulatory polypeptide, a nucleotide sequence encoding a second regulatory polypeptide, a nucleotide sequence encoding a third regulatory polypeptide; or any combination of the foregoing.
The present invention also discloses a complementing plasmid comprising a promoter nucleotide sequence operatively linked to a nucleotide sequence encoding an adenovirus structural protein, polypeptide or fragment thereof and a nucleotide sequence encoding an adenovirus regulatory protein, polypeptide or fragment thereof. In one variation, the early region polypeptide is E4; in another, WO 00/42208 PCT/EP00/00265 -29the plasmid comprises pE4/Hygro. In still another variation, the early region polypeptides are El and E4.
In another aspect of the present invention, the complementing plasmid used to transform a cell line of the present invention further comprises a DNA sequence encoding an adenovirus regulatory protein, polypeptide or fragment thereof. In one variation, the regulatory protein is selected from the group consisting of E1A, E1B, E2A, E2B, E3, E4 and L4 (also referred to as "the 100K protein"); an exemplary complementing plasmid has the characteristics of is pE4/Hygro (for deposit details, see the Examples). In another aspect, the complementing plasmid used to transform a cell line of the present invention further comprises a DNA sequence encoding two or more of the above mentioned adenovirus regulatory proteins, polypeptides or fragments thereof.
Preferred complementing plasmids include pCLF, pDV60, pDV61, pDV67, pDV69, pDV80, pDV90 and the like plasmids described in the Examples. The nucleic acid sequence of particularly preferred complementing plasmids are shown in SEQ ID NO: 43 for pDV60, SEQ ID NO: 44 for pDV67, SEQ ID NO: 47 for pDV69, SEQ ID NO: 64 for pDV80 and SEQ ID NO: 65 for 3. Nucleic Acid Molecule Synthesis A nucleic acid molecule comprising synthetic oligonucleotide sequences of the present invention can be prepared using any suitable method, such as, the phosphotriester or phosphodiester methods. See Narang et al., Meth. Enzymol., 68:90, (1979); U.S. Patent No. 4,356,270; and Brown et al., Meth. Enzymol., 68:109, (1979).
For oligonucleotide sequences in which a family of variants is preferred, the synthesis of the family members can be conducted simultaneously in a single reaction vessel, or can be synthesized independently and later admixed in preselected molar ratios. For simultaneous synthesis, the nucleotide residues that are conserved at preselected positions of the sequence of the family member can be introduced in a chemical synthesis protocol simultaneously to the variants by the addition of a single preselected nucleotide precursor to the solid phase oligonucleotide reaction admixture when that position number of the oligonucleotide is being chemically added to the growing oligonucleotide polymer.
The addition of nucleotide residues to those positions in the sequence that vary can be introduced simultaneously by the addition of amounts, preferably equimolar amounts, of multiple preselected nucleotide precursors to the solid phase WO 00/42208 PCT/EP00/00265 oligonucleotide reaction admixture during chemical synthesis. For example, where all four possible natural nucleotides (A,T,G and C) are to be added at a preselected position, their precursors are added to the oligonucleotide synthesis reaction at that step to simultaneously form four variants.
This manner of simultaneous synthesis of a family of related oligonucleotides has been previously described for the preparation of "Degenerate Oligonucleotides" by Ausubel et al. (Current Protocols in Molecular Biology, Suppl.
8. p.2.11.7, John Wiley Sons, Inc., New York ,1991), and can readily be applied to the preparation of the therapeutic oligonucleotide compositions described herein.
Nucleotide bases other than the common four nucleotides (A,T,G or or the RNA equivalent nucleotide uracil can also be used in the present invention. For example, it is well known that inosine is capable of hybridizing with A, T and G, but not C. Examples of other useful nucleotide analogs are known in the art and may be found referred to in 37 C.F.R. §1.822.
Thus, where all four common nucleotides are to occupy a single position of a family of oligonucleotides, that is, where the preselected nucleotide sequence is designed to contain oligonucleotides that can hybridize to four sequences that vary at one position, several different oligonucleotide structures are contemplated. The composition can contain four members, where a preselected position contains A,T,G or C. Alternatively, a composition can contain two nucleotide sequence members, where a preselected position contains I or C, and has the capacity the hybridize at that position to all four possible common nucleotides. Finally, other nucleotides may be included at the preselected position that have the capacity to hybridize in a non-destabilizing manner with more than one of the common nucleotides in a manner similar to inosine.
Similarly, larger nucleic acid molecules can be constructed in synthetic oligonucleotide pieces, and assembled by complementary hybridization and ligation, as is well known.
B. Adenovirus Expression Vector Systems One key component of the present invention for producing gene therapy reagents comprised of recombinant adenovirus particles is the recombinant adenovirus vector genome which is encapsulated in the virus particle and which expresses exogenous genes in a gene therapy setting. Thus, the components of an recombinant adenovirus vector genome include the ability to express selected
~I
WO 00/42208 PCT/EP00/00265 -31adenovirus structural genes, to express a desired exogenous protein, and to contain sufficient replication and packaging signals that the genome is packaged into a gene delivery vector particle. The preferred replication signal is an adenovirus inverted terminal repeat containing an adenovirus origin of replication, as is well known and described herein.
According to the present invention, a preferred recombinant adenovirus vector genome is "helper independent" which means the genome can replicate and be packaged without the help of a second, complementing helper virus. Instead, the complementation is provided by a packaging cell line of the present invention.
Additional embodiments of the invention, however, are drawn to a vector genome referred to as "gutless" which is "helper dependent." In a preferred embodiment, the adenovirus vector genome does not encode a functional adenovirus fiber protein. A non-functional fiber gene refers to a deletion, mutation or other modification to the adenovirus fiber gene such that the gene does not express any or insufficient adenovirus fiber protein to package a fiber-containing adenovirus particle without complementation of the fiber gene by a complementing plasmid or packaging cell line. Such a genome is referred to as a "fiberless" genome, not to be confused with a fiberless particle. Alternatively, a fiber protein may be encoded but is insufficiently expressed to result in a fiber containing particle.
Thus, the invention describes a helper-independent fiberless recombinant adenovirus vector genome comprising genes which express all adenovirus structural gene products but express insufficient adenovirus fiber protein to package a fiber-containing adenovirus particle without complementation of said fiber gene, express an exogenous protein, and contains an adenovirus packaging signal and inverted terminal repeats containing adenovirus origin of replication.
The introduction of exogenous DNA into eucaryotic cells has become one of the most powerful tools of the molecular biologist. The term "exogenous" encompasses any therapeutic composition of this invention which is administered by the therapeutic methods of this invention. Thus, "exogenous" may also be referred to herein as "foreign," "non-native," and the like. The methods of this invention preferably require efficient delivery of the DNA into the nucleus of the recipient cell and subsequent identification of cells that are expressing the foreign
DNA.
The adenovirus vector genome is propagated in the laboratory in the form of rDNA plasmids containing the genome, and upon introduction into an appropriate WO 00/42208 PCT/EP00/00265 -32host, the viral genetic elements provide for viral genome replication and packaging rather than plasmid-based propagation. Exemplary methods for preparing an Advector genome are described in the Examples.
A widely-used plasmid is pBR322, a vector whose nucleotide sequence and endonuclease cleavage sites are well known. Various other useful plasmid vectors are described in the Examples that follow.
A nucleic acid vector of the present invention comprises a nucleic acid (preferably DNA) molecule capable of autonomous replication in a cell and to which a DNA segment, a gene or polynucleotide, can be operatively linked so as to bring about replication of the attached segment. In the present invention, one of the nucleotide segments to be operatively linked to vector sequences encodes at least a portion of a therapeutic nucleic acid molecule in effect, a nucleic acid sequence that encodes one or more therapeutic proteins or polypeptides, or fragments thereof.
As one of skill in the art will note, in various embodiments of the present invention, different "types" of vectors are disclosed. For example, one "type" of vector is used to deliver particular nucleotide sequences into a packaging cell line, with the intent of having said sequences stably integrate into the cellular genome; these "types" of vectors are generally identified herein as complementing plasmids.
A further "type" of vector described herein carries or delivers nucleotide sequences in or into a cell line a packaging cell line) for the purpose of propagating therapeutic viral vectors of the present invention; hence, these vectors are generally referred to herein as delivery plasmids. A third "type" of vector described herein is utilized to carry nucleotide sequences encoding therapeutic proteins or polypeptides to specific cells or cell types in a subject in need of treatment; these vectors are generally identified herein as therapeutic viral vectors or Ad-derived vectors and are in the form of a virus particle encapsulating a viral nucleic acid containing an expression cassette nucleic acid sequence for expressing the therapeutic gene.
1. Nucleic Acid Gene Expression Cassettes In various embodiments, a peptide-coding sequence of the therapeutic gene is inserted into an expression vector and expressed; however, it is also feasible to construct an expression vector which also includes some non-coding sequences as well. Preferably, however, non-coding sequences are excluded. Alternatively, a nucleotide sequence for a soluble form of a polypeptide may be utilized. Another preferred therapeutic viral vector includes a nucleotide sequence encoding at least WO 00/42208 PCT/EP00/00265 -33a portion of a therapeutic nucleotide sequence operatively linked to the expression vector for expression of the coding sequence in the therapeutic nucleotide sequence.
As used herein with regard to DNA sequences or segments, the phrase "operatively linked" generally means the sequences or segments have been covalently joined into one piece of DNA, whether in single or double stranded form.
The choice of viral vector into which a therapeutic nucleotide sequence of this invention is operatively linked depends directly, as is well known in the art, on the functional properties desired, vector replication and protein expression, and the host cell to be transformed these being limitations inherent in the art of constructing recombinant DNA molecules. Although certain adenovirus serotypes are recited herein in the form of specific examples, it should be understood that the present invention contemplates the use of any adenovirus serotype, including hybrids and derivatives thereof. As one will observe, it is not unusual or outside the scope of the present invention to utilize nucleotide and/or amino acid residue sequences of two or more serotypes in constructs, compositions and methods of the invention.
A translatable nucleotide sequence is a linear series of nucleotides that provide an uninterrupted series of at least 8 codons that encode a polypeptide in one reading frame. Preferably, the nucleotide sequence is a DNA sequence. The vector itself may be of any suitable type, such as a viral vector (RNA or DNA), naked straight-chain or circular DNA, or a vesicle or envelope containing the nucleic acid material and any polypeptides that are to be inserted into the cell.
2. Promoters As noted elsewhere herein, an expression nucleic acid in an Ad-derived vector of the present invention may also include a promoter sequence.
In general, promoters are DNA segments that contain a DNA sequence that controls the expression of a gene located 3' or downstream of the promoter. The promoter is the DNA sequence to which RNA polymerase specifically binds and initiates RNA synthesis (transcription) of that gene, typically located 3' of the promoter. A promoter also includes DNA sequences which direct the initiation of transcription, including those to which RNA polymerase specifically binds. If more than one nucleic acid sequence encoding a particular polypeptide or protein is included in a therapeutic viral vector or nucleotide sequence, more than one promoter or enhancer element may be included, particularly if that would enhance WO 00/42208 PCT/EP00/00265 -34efficiency of expression. For purposes of the present invention, regulatable (inducible) as well as constitutive promoters may be used, either on separate vectors or on the same vector.
Both constitutive and regulatable (often called "inducible") promoters are useful in constructs and methods of the present invention. For example, some useful regulatable promoters are those of the CREB-regulated gene family and include inhibin, gonadotropin, cytochrome c, glucagon, and the like. (See, e.g., published International App. No. W096/14061.
A regulatable or inducible promoter may be described as a promoter wherein the rate of RNA polymerase binding and initiation is modulated by external stimuli. (See U.S. Patent Nos. 5,750,396 and 5,998,205). Such stimuli include various compounds or compositions, light, heat, stress, chemical energy sources, and the like. Inducible, suppressible and repressible promoters are considered regulatable promoters.
Regulatable promoters may also include tissue-specific promoters. Tissuespecific promoters direct the expression of the gene to which they are operably linked to a specific cell type. Tissue-specific promoters cause the gene located 3' of it to be expressed predominantly, if not exclusively, in the specific cells where the promoter expressed its endogenous gene. Typically, it appears that if a tissue-specific promoter expresses the gene located 3' of it at all, then it is expressed appropriately in the correct cell types (see, Palmiter et al., Ann.
Rev. Genet. 20: 465-499, 1986).
When a tissue-specific promoter controls the expression of a gene, that gene will be expressed in a small number of tissues or cell types rather than in substantially all tissues and cell types. Examples of tissue-specific promoters include the immunoglobulin promoter described by Brinster et al., Nature 306: 332-336 (1983) and Storb et al., Nature 310- 238-231 (1984); the elastase-l promoter described by Swift et al., Cell 38: 639-646 (1984); the globin promoter described by Townes et al., Mol. Cell. Biol. 5: 1977-1983 (1985), and Magram et al., Mol. Cell. Biol. 9: 4581-4584 (1989), the insulin promoter described by Bucchini et PNAS USA, 83: 2511-2515 (1986) and Edwards et al., Cell 58: 161 (1989); the immunoglobulin promoter described by Ruscon et al., Nature 314: 330-334 (1985) and Grosscheld et al., Cell 38: 647-658 (1984); the alpha actin promoter described by Shani, Mol. Cell. Biol. 6: 2624-2631 (1986); the alpha crystalline promoter described by Overbeek et al., PNAS USA 82: 7815-7819 (1985); the prolactin promoter described by Crenshaw et al., Genes and Development 3: 959-972 (1989); the propiomelanocortin promoter described by Tremblay et al., WO 00/42208 PCT/EP00/00265 PNAS USA 85: 8890-8894 (1988); the beta-thyroid stimulating hormone (BTSH) promoter described by Tatsumi et al., Nippon Rinsho 47: 2213-2220 (1989); the mouse mammary tumor virus (MMTV) promoter described by Muller et al., Cell 54: 105 (1988); the albumin promoter described by Palmiter et al., Ann. Rev. Genet.
465-499 (1986); the keratin promoter described by Vassar et al., PNAS USA 86: 8565-8569 (1989); the osteonectin promoter described by McVey et al., J. Biol.
Chem. 263: 11,111-11,116 (1988); the prostate-specific promoter described by Allison et al., Mol. Cell. Biol. 9 2254-2257 (1989); the opsin promoter described by Nathans et al., PNAS USA 81: 4851-4855 (1984); the olfactory marker protein promoter described by Danciger et al., PNAS USA 86: 8565-8569 (1989); the neuron-specific enolase (NSE) promoter described by Forss-Pelter et al., J.
Neurosci. Res. 16: 141-151 (1986); the L-7 promoter described by Sutcliffe, Trends in Genetics 3: 73-76 (1987) and the protamine 1 promoter described Peschon et al., Ann. New York Acad. Sci. 564: 186-197 (1989) and Braun et al., Genes and Development 3: 793-802 (1989).
3. Adenovirus Vectors Although adenovirus consists of many proteins, not all adenovirus proteins are required for assembly of a recombinant adenovirus particle (vector) of this invention. Thus, deletion of the appropriate genes from a recombinant Ad vector as taught herein will thus allow the vector to accommodate even larger "foreign" DNA segments. Thus, if the sequences encoding one or more adenovirus polypeptides or proteins are supplanted by a recombinant nucleotide sequence of the present invention, the length of the recombinant sequence can conceivably extend nearly to the packaging limit of the relevant adenovirus-derived vector.
In view of the fact that preferred embodiments disclosed herein are helperindependent Ad-derived vectors, the entire wild-type Ad genome cannot be completely supplanted by recombinant nucleic acid molecules without transforming such a vector into a vector requiring "help" of some kind. However, most of the Adderived vectors of the present invention do not depend on a helper virus; instead, the vectors of the present invention are propagated in cell lines stably expressing proteins or polypeptides that have been removed from said vectors to allow the addition of "foreign" DNA into the vectors. In various disclosed embodiments, specific early region and structural polypeptides are deleted from the vectors of the present invention, thereby enabling the vectors to accommodate recombinant nucleic acid sequences (or cassettes) of various lengths. For example, Ad-derived WO 00/42208 PCT/EP00/00265 -36vectors of the present invention may easily include 12 kb or more of foreign (or "therapeutic") DNA sequences.
Thus, adenovirus viral vectors are also disclosed which comprise nucleotide sequences encoding a packaging signal and a foreign protein or polypeptide, wherein the nucleotide sequence encoding an adenovirus structural protein has been deleted.
In one variation, the nucleotide sequence encoding the foreign protein or polypeptide is a DNA molecule up to about 3 kb in length; in another, the nucleotide sequence encoding the foreign protein or polypeptide is a DNA molecule up to about 9.5 kb in length; in still another, the nucleotide sequence encoding the foreign protein or polypeptide is a DNA molecule up to about 12.5 kb in length. Nucleotide sequences of intermediate lengths are also contemplated by the present invention, as are sequences in excess of 12.5 kb.
The invention also discloses viral vectors wherein the sequence encoding a foreign protein or polypeptide is a sequence encoding an anti-tumor agent, a tumor suppressor protein, a suicide protein, or a fragment or functional equivalent thereof. In one variation, nucleotide sequences encoding one or more regulatory proteins have also been deleted from the vector. In another, the regulatory proteins are selected from the group consisting of ElA, E1B, E2A, E2B, E3, E4, and L4 (100K protein).
A wide variety of therapeutic viral vectors are also embodiments of the present invention. In one embodiment, a therapeutic viral vector is disclosed which lacks a DNA sequence encoding fiber protein, or a portion thereof. In another variation, a therapeutic viral vector may further or alternatively comprise deletion of a DNA sequence encoding one or more regulatory proteins, polypeptides, or fragments thereof. In various embodiments, foreign DNA sequences are inserted in place of the DNA sequence encoding fiber protein in the viral vectors of the present invention. In other embodiments, the therapeutic viral vectors further comprise foreign DNA sequences inserted in place of the DNA sequences encoding one or more regulatory proteins, polypeptides, or fragments thereof, and/or one or more structural proteins, polypeptides, or fragments thereof.
The present invention further discloses a number of viral vectors. In one variation, a viral vector comprises a deletion or mutation of a DNA sequence encoding an adenovirus structural protein, polypeptide, or fragment thereof. A vector may further comprise deletion or mutation of the DNA sequences encoding regulatory polypeptides ElA and ElB; and it may still further comprise deletion or mutation of the DNA sequence encoding one or more of the following regulatory I WO 00/42208 PCT/EPOO/00265 -37proteins or polypeptides: E2A, E2B, E3, E4, L4, or fragments thereof. In another variation, in a viral vector of the present invention, the structural protein comprises fiber. Any combination of the foregoing is also contemplated by the present invention. The viral vectors of the present invention are suitable for the preparation of pharmaceutical compositions comprising any of the therapeutic viral vectors disclosed herein including combinations thereof are also disclosed herein. A further use of the viral vectors of the present invention is for targeting specific cells in a cell population comprising different cell types.
Yet another variation discloses that a foreign DNA sequence encoding one or more foreign proteins, polypeptides or fragments thereof has been inserted in place of any of the deletions in the therapeutic viral vector. In one embodiment, the foreign DNA encodes a tumor-suppressor protein or a biologically active fragment thereof. In another embodiment, the foreign DNA encodes a suicide protein or a biologically active fragment thereof.
The invention further contemplates that a viral vector comprises a foreign DNA sequence encoding one or more foreign proteins, polypeptides or fragments thereof wherein said DNA sequence has been inserted in place of any structural and/or regulatory proteins (or portions thereof) that have been deleted. Thus, in one embodiment, the foreign DNA encodes a therapeutic molecule such as a tumor-suppressor protein; a suicide protein; a cystic fibrosis transmembrane conductance regulator (CFTR) protein; or a biologically active fragment of any of them.
The therapeutic (or foreign) nucleotide sequence can be a gene or gene fragment that encodes a protein or polypeptide or a biologically active fragment thereof. (See, Crystal, et al., Nature Genetics 8:42-51 (1994); Zabner, et aL., Cell 75: 207-216 (1993); Knowles, et al., NEJM 333(13): 823-831 (1995); and Rosenfeld, et al., Cell 68:143-155 (1992).
It is further contemplated that a therapeutic protein or polypeptide expressed by a therapeutic viral vector of the present invention may be used in conjunction with another therapeutic agent when appropriate a thymidine kinase metabolite may be used in conjunction with the gene encoding thymidine kinase and its gene product in order to be even more effective.
Alternatively, a therapeutic viral vector can include a DNA or RNA oligonucleotide sequence that exhibits therapeutic activity without needing to be translated into a polypeptide product before exerting a therapeutic effect.
Examples of the latter include antisense oligonucleotides that will inhibit the transcription of deleterious genes or ribozymes that act as site-specific
-I
WO 00/42208 PCT/EP00/00265 -38ribonucleases for cleaving selected mutated gene sequences. In another variation, a therapeutic nucleotide sequence of the present invention may comprise a DNA construct capable of generating therapeutic nucleotide molecules, including ribozymes and antisense DNA, in high copy numbers in target cells, as described in published PCT application No. WO 92/06693 (the disclosure of which is incorporated herein by reference). Other preferred therapeutic nucleotide sequences according to the present invention are capable of delivering HIV antisense oligonucleotides to latently-infected T cells via CD4. Similarly, delivery of Epstein-Barr Virus (EBV) EBNa-1 antisense oligonucleotides to B cells via CR2 is capable of effecting therapeutic results.
A preferred recombinant adenovirus vector genome is based on the vector described in the Examples and designated Ad5.3gal.AF. This vector is a helper independent, fiberless vector genome which can host, upon insertion, an exogenous gene for expression of an exogenous or therapeutic protein. The genome of Ad5.pgal.AF has a nucleotide sequence shown in SEQ ID NO: 27. A virus particle containing Ad5.pgal.AF vector genome has been prepared as described in the Examples and is deposited with the ATCC as Accession No. VR- 2636 The Ad5.pgal.AF genome nucleic acid can be manipulated to contain any exogenous gene in place of the beta-galactosidase gene present in the construct, as described herein.
Construction of Therapeutic Viral Vectors for Gene Delivery A. Adenovirus Particles Various methods of making and using the vectors, plasmids, cell lines and other compositions and constructs of the present invention are also disclosed herein. The following methods are considered exemplary and not limiting.
Thus, in one variation, the invention discloses a method of constructing therapeutic viral vectors, comprising introducing a delivery plasmid into an Ad fiberexpressing complementing cell line, wherein the DNA sequence encoding Ad fiber protein has been deleted from the delivery plasmid. In one variation, the delivery plasmid further includes a DNA sequence encoding a foreign protein, polypeptide, or fragment thereof. In other embodiments, a combination of pDV44 and pAEIBIgal or a similar construct such as, for example, that found in pDV44, pAE1 Bpgal or the equivalent.
WO 00/42208 PCT/EP00/00265 -39- A recombinant adenovirus particle may be produced with a fiber protein, or it may be produced without a fiber protein ("fiberless particle") according to the present invention. Where the particle is made without fiber, such as by passaging the fiberless viral vector genome, Ad5.pgal.AF in the 293 cells, a fiberless genome is replicated and packaged in a fiberless particle. In contrast, where the fiberless genome Ad5.pgal.AF is passaged in the 211B or other fiber expressing cells, a fiberless genome is replicated and packaged into a fiber-containing particle.
Recombinant adenovirus particles may be made such that they include no fiber proteins, modified fiber proteins or other exogenous proteins. They may also be produced in systems using either helper-independent or helper-dependent adenovirus recombinant genomes, i.e. with or without helper viruses.
B. Targeting of Particles to Tissues Virus Trophism A preferred viral vector particle in which therapeutic nucleotide compositions of this invention are present is derived from adenovirus As taught herein, viral vector particles of this invention may be designed and constructed in such a way that they specifically target a preselected recipient cell type, depending on the nature of therapy one seeks to administer. Methods of making and using therapeutic viral vectors that target specific cells are further described in the Examples that follow.
Novel vectors, viral particles or compositions may also be designed and prepared to preferentially target cells that might not otherwise be targeted by wildtype adenovirus virions. For example, in order to target non-epithelial cells, one following the teachings of the present. specification may be able to prepare a therapeutic vector particle including a nucleotide sequence encoding a foreign protein, polypeptide or other ligand directed to a non-epithelial cell or to a different receptor than that generally targeted by a particular adenovirus. Examples of useful ligands directed to specific receptors (identified in parentheses) include the V3 loop of HIV gp120 (CD4); transferring (transferrin receptor); LDL, apolipoprotein B100, apolipoprotein E (LDL receptors); and deglycosylated proteins (asialoglycoprotein receptor). Various useful ligands which may be added to adenovirus fiber and methods for preparing and attaching same are set forth in U.S. Patent Nos. 5,756,086 and 5,543,328.
In yet another embodiment, the non-native amino acid residue sequence is incorporated into the fiber amino acid residue sequence at a location other than WO 00/42208 PCT/EP00/00265 one of the fiber termini. Alternatively, the non-native amino acid residue sequence alters the binding specificity of the fiber for a targeted cell type. In other embodiments, the linker sequence alters the binding specificity of the fiber for a targeted cell type. The expressed fiber may, in various embodiments, bind to a specific targeted cell type not usually targeted by adenovirus and/or may comprise amino acid residue sequences from more than one adenovirus serotype.
Useful ligands may be encoded by a foreign nucleotide sequence contained within a viral vector of the present invention, or may be linked to proteins or polypeptides, include antibodies and attachment sequences, as well as receptors themselves. For example, antibodies to cell receptor molecules such as integrins and the like, MHC Class I and Class II, asialoglycoprotein receptor, transferrin receptors, LDL receptors, CD4, and CR2 are but a few which are useful according to the present invention. It is also understood that the ligands typically bound by receptors, as well as analogs to those ligands, may be used as cellular targeting agents, as disclosed herein.
Therapeutic Methods The recombinant adenovirus vectors of the present invention, typically in the form of an adenovirus particle encapsulating a recombinant adenovirus vector genome containing an expression cassette for expressing a therapeutic gene, are particularly suited for gene therapy. Thus, various therapeutic methods are contemplated by the present invention.
For example, it has now been discovered that Ad-derived viral vectors are capable of delivering a therapeutic nucleotide sequence to a specific cell or tissue, based on the tissue tropism of the particle, thereby expanding and enhancing treatment options available in numerous conditions in which more conventional therapies are of limited efficacy. Accordingly, methods of gene therapy utilizing a recombinant adenovirus particle containing a modified fiber or chimeric fiber which targets a preselected tissue, as described herein, is within the scope of the invention. Vector particles are typically purified and then an effective amount is administered in vivo or ex vivo (in vitro)into the subject.
For in vitro or ex vivo gene transfer, administration is often accomplished by first isolating a selected cell population from a patient such as lung epithelial cells, lymphocytes and the like followed by in vitro or ex vivo gene transfer of the therapeutic compositions of this invention and the replacement of the cells into the patient. In vivo therapy is also contemplated, via the administration of WO 00/42208 PCT/EP00/00265 -41therapeutic compositions of this invention by various delivery means. For example, aerosol administration and administration via subcutaneous, intravenous, intraperitoneal, intramuscular, ocular means and the like are also within the scope of the present invention.
Other gene-delivery methods are also useful in conjunction with the methods, compositions and constructs of the present invention; see, e.g., published International Application No. WO 95/11984.
The present invention also contemplates various methods of targeting specific cells cells in a subject in need of diagnosis and/or treatment. As discussed herein, the present invention contemplates that the viral vectors and compositions of the present invention may be directed to specific receptors or cells, for the ultimate purpose of delivering those vectors and compositions to specific cells or cell types. The viral vectors and constructs of the present invention are particularly useful in this regard.
In general, adenovirus attachment and uptake into cells are separate but cooperative events that result from the interaction of distinct viral coat proteins with a receptor for attachment and av integrin receptors for internalization. Adenovirus attachment to the cell surface via the fiber coat proteins has been discovered to be dissociable and distinct from the subsequent step of internalization, and the present invention is able to take advantage of and function in conjunction with these differing receptors.
The invention also discloses methods of transforming a pathologic hyperproliferative mammalian cell comprising contacting the cell with any of the vectors described herein. In another embodiment, methods of infecting a mammalian target cell with a viral vector containing a preselected foreign nucleotide sequence are disclosed. One such variation comprises the following steps: infecting the target cell with a viral vector of the present invention, the viral vector carrying a preselected foreign nucleotide sequence; and expressing the foreign nucleotide sequence in the targeted cell.
The invention also encompasses mammalian target cells infected with a preselected foreign nucleotide sequence produced by the methods disclosed herein. In one variation, the target cells are selected from the group consisting of replicating, slow-replicating and non-replicating human cells.
Methods of treating an acquired or hereditary disease are also disclosed.
One method comprises administering a pharmaceutically acceptable dose of a viral vector to a target cell, wherein the vector comprises a preselected therapeutic nucleotide sequence; and expressing the therapeutic sequence in the target WO 00/42208 PCT/EP00/00265 -42cell for a time period sufficient to ameliorate the acquired or hereditary disease in the cell. Method of gene therapy comprising administering to a subject an effective amount of a therapeutic viral vector produced by a packaging cell line of the present invention are also disclosed.
Also contemplated by the present invention are various methods of inhibiting the proliferation of a tumor in a subject comprising administering an effective amount of a therapeutic viral vector of the present invention under suitable conditions to the subject. In one variation, the gene encodes an anti-tumor agent.
In another variation, the agent is a tumor-suppressor gene. In still another embodiment, the agent is a suicide gene or a functional equivalent thereof. In another variation, the vector is administered via intra-tumoral injection.
A composition of this invention may be used prophylactically or therapeutically in vivo to disrupt HIV infection and mechanisms of action by inhibiting gene expression or activation, via delivery of antisense HIV sequences or ribozymes to T cells or monocytes. Using methods of the present invention, one may target therapeutic viral vectors as disclosed herein to specific cells and tissues, including hematopoietic cells, as infection of such cells appears to be mediated by distinct integrins to which viral vectors of the present invention may readily be targeted. (See, Huang, etal., J. Virol. 70 4502-8, 1996).
Other useful therapeutic nucleotide sequences include antisense nucleotide sequences complementary to EBV EBNa-1 gene. Use of such therapeutic sequences may remediate or prevent latent infection of B cells with EBV. As discussed herein and in the Examples below, targeting and delivery may be accomplished via the use of various ligands, receptors, and other appropriate targeting agents.
Thus, in one embodiment, a therapeutic method of the present invention comprises contacting the cells of a subject infected with EBV or HIV with a therapeutically effective amount of a pharmaceutically acceptable composition comprising a therapeutic nucleotide sequence of this invention. In a related embodiment, the contacting involves introducing the therapeutic nucleotide sequence composition into cells having an EBV or HIV-mediated infection.
Methods of gene therapy are well known in the art (see, Larrick and Burck, Gene Therapy: Application of Molecular Biology, Elsevier Science Publ.
Co., Inc., New York, NY (1991); Kriegler, Gene Transfer and Expression: A Laboratory Manual, W. H. Freeman and Company, New York, 1990). The term "subject" should be understood to include any animal particularly mammalian WO 00/42208 PCT/EP00/00265 -43patient, such as any murine, rat, bovine, porcine, canine, feline, equine, ursine, or human patient.
When the foreign gene carried in the vector encodes a tumor suppressor gene or another anti-tumor protein, the vector is useful to treat or reduce hyperproliferative cells in a subject, to inhibit tumor proliferation in a subject or to ameliorate a particular, related pathology.
The present invention also contemplates methods of depleting suitable samples of pathologic mammalian hyperproliferative cells contaminating hematopoietic precursors during bone marrow reconstitution via the introduction of a wild-type tumor suppressor gene into the cell preparation using a vector of this invention. As used herein, a suitable sample is defined as a heterogeneous cell preparation obtained from a patient, a mixed population of cells containing both phenotypically normal and pathogenic cells.
Administration includes but is not limited to the introduction of therapeutic agents of the present invention into a cell or subject via various means, including direct injection, intravenously, intraperitoneally, via intra-tumor injection, via aerosols, or topically. Therapeutic agents as disclosed herein may also be combined for administration of an effective amount of the agents with a pharmaceutically-acceptable carrier, as described herein.
As used herein, "effective amount" generally means the amount of vector particle (or proteins produced/released thereby) which achieves a positive outcome in the subject to whom the vector is administered. The total volume administered will necessarily vary depending on the mode of administration, as those of skill in the relevant art will appreciate, and dosages may vary as well.
The dose of a biologic vector (particle) is somewhat complex and may be described in terms of the concentration (in plaque-forming units per milliliter (pfu/ml)), the total dose (in pfus), or the estimated number of particles administered per cell (the estimated multiplicity of infection or MOI). Thus, if a vector is administered via infusion say, across nasal epithelium at a constant total volume, the respective concentration, etc. may be described as follows: WO 00/42208 PCT/EP00/00265 -44- In general, when recombinant adenoviral vector particles are administered via infusion across the nasal epithelium an area of nasal epithelium containing 2 x 107 cells,) administered amounts producing an estimated MOI (multiplicity of infection) of about 10 or greater are much more effective than lower Concentration Volume Dose Estimated (pfu/ml) (ml) (Dfu) MOI 107 2 2 x 10 7 1 108 2 2 x 10 8 109 2 2 x 10 9 100 1010 2 2 x 10 1 0 1000 Table 2 dosages. (See, Knowles, et al., New Eng. J. Med. 333: 823-831, 1995).
Similarly, when direct injection is the preferred treatment modality direct injection of a viral vector into a tumor doses of 1 x 109 pfu or greater are generally preferred. (See, published International App. No. W095/11984.) Thus, depending on the mode of administration, an effective amount administered in a single dose preferably contains from about 106 to about 1015 infectious units. A typical course of treatment would be one such dose per day over a period of five days. As those of skill in the art will appreciate, an effective amount may vary depending on the pathology or other condition to be treated, the status and sensitivity of the patient, and various other factors well known to those of skill in the art, such as the patient's tolerance to other courses of treatment that may have been applied previously. Thus, those of skill in the art may easily and precisely determine effective amounts of the agents/vectors of the present invention which may be administered to a particular patient, based on their understanding of and evaluation of such factors.
The present invention also contemplates methods of ameliorating pathologies characterized by hyperproliferative cells or genetic defects in a subject, by administering to the subject an effective amount of a vector as described herein.
Such vectors preferably contain a foreign gene encoding a gene product (e.g.
polypeptide or protein) having the ability to ameliorate the pathology, under suitable conditions. As used herein, the term "genetic defect" means any disease, WO 00/42208 PCT/EP00/00265 condition or abnormality which results from inherited factors, e.g. Huntington's Disease, Tay-Sachs Disease, or Sickle Cell Disease.
The present invention further provides methods for reducing the proliferation of tumor cells in a subject by introducing into the tumor mass an effective amount of an adenoviral expression vector containing an anti-tumor gene other than a tumor suppressor gene. The anti-tumor gene can encode, for example, thymidine kinase An effective amount of a therapeutic agent is then administered to the subject; the therapeutic agent, in the presence of the anti-tumor gene, is toxic to the cell.
Using thymidine kinase as exemplary, the therapeutic agent is a thymidine kinase metabolite such as ganciclovir (GCV), 6-methoxypurine arabinonucleoside (araM), or a functional equivalent thereof. Both the thymidine kinase gene and the thymidine kinase substrate must be used concurrently in order to exert a toxic effect on the host cell. In the presence of the TK gene, GCV is phosphorylated and becomes a potent inhibitor of DNA synthesis, whereas araM is converted to the cytotoxic anabolite araATP. Thus, the precise method of action or synergism is not relevant to therapeutic efficacy; what is relevant is the fact that the concurrent use of appropriate genes and therapeutic agents may effectively ameliorate a specific disease condition.
Another useful example contemplates use of a vector of the present invention which expresses the enzyme cytosine deaminase. Such a vector could be used in conjunction with administration of the drug 5-fluorouracil (Austin and Huber, Mol. Pharm. 43: 380-387, 1993) or the recently-described E. coli Deo gene in combination with 6-methyl-purine-2'-deoxyribonucleoside (Sorscher et al., Gene Therapy 1: 233-238, 1994).
As with the use of the tumor suppressor genes described previously, the use of other anti-tumor genes, either alone or in combination with the appropriate therapeutic agent, provides a treatment for the uncontrolled cell growth or proliferation characteristic of tumors and malignancies. Thus, the present invention provides therapies to halt the uncontrolled cellular growth in a patient, thereby alleviating the symptoms or the disease or cachexia present in the patient. The effect of this treatment includes, but is not limited to, prolonged survival time of the patient, reduction in tumor mass or burden, apoptosis of tumor cells, or the reduction in the number of circulating tumor cells. Means of quantifying the beneficial effects of this therapy are well known to those of skill in the art.
The present invention provides a recombinant adenovirus expression vector characterized by the partial or total deletion of one or more adenoviral structural WO 00/42208 PCT/EP00/00265 -46protein genes, such as the gene encoding fiber, which allows the vector to accommodate a therapeutic, foreign nucleic acid sequence encoding a functional foreign polypeptide, protein, or biologically active fragment thereof. A therapeutic gene sequence may be introduced into a tumor mass by combining the adenoviral expression vector with a suitable pharmaceutically acceptable carrier. Introduction can be accomplished, for example, via direct injection of the recombinant Ad vector into the tumor mass.
A method of tumor-specific delivery of a tumor-suppressor gene is accomplished by contacting target tissue in a subject with an effective amount of a recombinant Ad-derived vector of this invention. In the case of anti-tumor therapy, the gene is intended to encode an anti-tumor agent, such as a functional tumor suppressor gene product or suicide gene product. The term "contacting" is intended to encompass any delivery method for the efficient transfer of the vector, such as via intra-tumoral injection.
In another example, adenovirus vectors of the present invention can be used to transfer genes to central nervous system (CNS) tumors in vivo.
The present invention also contemplates methods for determining the efficacy of the within-disclosed therapeutic compositions and methods. One such method for confirming efficacy utilizes the human/SCID (severe combined immunodeficient) mouse model of EBV-induced LPD (lymphoproliferative disease) to ascertain whether EBV-antisense therapeutic nucleotide sequences block tumor formation. (See, Pisa, et al., Blood 79:173-179 (1992); Rowe, et al., Curr.
Top. Microbiol. Immunol. 166 325 (1990); and Cannon, et al., J. Clin. Invest. 1333-1337 (1990).
Finally, the use of Ad vectors of the present invention to prepare medicaments for the treatment, therapy and/or diagnosis of various diseases is also contemplated by this invention. Moreover, other anti-tumor genes may be used in combination with the corresponding therapeutic agent to reduce the proliferation of tumor cells. Such other gene-and-therapeutic-agent combinations are known to those of skill in the art and may be applied as taught herein.
A. Therapeutic Compositions In various alternative embodiments of the present invention, therapeutic sequences and compositions useful for practicing the therapeutic methods described herein are contemplated. Therapeutic compositions of the present invention may contain a physiologically tolerable carrier together with one or more WO 00/42208 PCT/EP00/00265 -47therapeutic nucleotide sequences of this invention, dissolved or dispersed therein as an active ingredient. In a preferred embodiment, the composition is not immunogenic or otherwise able to cause undesirable side effects when administered to a subject for therapeutic purposes.
As used herein, the terms "pharmaceutically acceptable," "physiologically tolerable" and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a subject a mammal without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like.
For example, the present invention comprises therapeutic compositions useful in the specific targeting of epithelial or non-epithelial cells as well as in delivering a therapeutic nucleotide sequence to those cells. Therapeutic compositions designed to preferentially target to epithelial cells may comprise a recombinant adenovirus-derived vector particle including a therapeutic nucleotide sequence. As described herein, a number of adenovirus-derived moieties are described, including particles lacking fiber, particles that contain wild type adenovirus fiber, and particles that contain modified or chimeric fiber, each type providing a different tissue tropism to the particle.
The preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art. Typically such compositions are prepared as injectables either as liquid solutions or suspensions however, solid forms suitable for solution or suspension in liquid prior to use can also be prepared. A preparation can also be emulsified, or formulated into suppositories, ointments, creams, dermal patches, or the like, depending on the desired route of administration.
Physiologically tolerable carriers are well known in the art. Exemplary of liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes.
Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions.
WO 00/42208 PCT/EP00/00265 -48- A therapeutic composition typically contains an amount of a therapeutic material, a nucleotide sequence or adenovirus vector particle of the present invention, sufficient to deliver a therapeutically effective amount to the target tissue, typically an amount of at least 0.1 weight percent to about 90 weight percent of therapeutic material per weight of total therapeutic composition. A weight percent is a ratio by weight of therapeutic material, a nucleotide sequence, to total composition. Thus, for example, 0.1 weight percent is 0.1 grams of DNA segment per 100 grams of total composition.
Other Applications The cell lines, viral vectors and methods of the present invention may also be used for purposes other than the direct administration of therapeutic nucleotide sequences. In one such application, the production of large quantities of biologically active proteins or polypeptides in cells transfected with the withindisclosed viral vectors is contemplated herein. For example, human lymphoblastoid cells may be transfected with a viral vector of the present invention carrying a human hematopoietic growth factor such as the gene for erythropoietin (EPO); cells so transfected are thus able to produce biologically active EPO. (See, Lopez et al., Gene 148: 285-91, 1994).
Various other applications and uses of the within-described methods, cell lines, plasmids, vectors, and compositions of the present invention shall become apparent upon closer examination of the Examples that follow.
The following examples are intended to illustrate, but not limit, the present invention. As such, the following description provides details of the manner in which particular embodiments of the present invention may be made and used.
This description, while exemplary of the present invention, is not to be construed as specifically limiting the invention. Variations and equivalents, now known or later developed, which would be within the understanding and technical competence of one skilled in this art are to be considered as falling within the scope of this invention.
Example 1 Preparation of Adenovirus Packaging Cell Lines WO 00/42208 PCT/EP00/00265 -49- Cell lines that are commonly used for growing adenovirus are useful as host cells for the preparation of adenovirus packaging cell lines. Preferred cells include 293 cells, an adenovirus-transformed human embryonic kidney cell line obtained from the ATCC, having Accession Number CRL 1573; HeLa, a human epithelial carcinoma cell line (ATCC Accession Number CCL-2); A549, a human lung carcinoma cell line (ATCC Accession Number CCL 1889); and the like epithelialderived cell lines. As a result of the adenovirus transformation, the 293 cells contain the El early region regulatory gene. All cells were maintained in complete DMEM 10% fetal calf serum unless otherwise noted.
The cell lines of this invention allow for the production and propagation of novel adenovirus-based gene delivery vectors having deletions in preselected gene regions, that are obtained by cellular complementation of adenoviral genes.
To provide the desired complementation of such deleted adenoviral genomes in order to generate a novel viral vector of the present invention, plasmid vectors that contain preselected functional units were designed as described herein. Such units include but are not limited to El early region, E4 and the viral fiber gene. The preparation of plasmids providing such complementation, thereby being "complementary plasmids or constructs," that are stably inserted into host cell chromosomes are described below.
A. Preparation of an E4-Expressing Plasmid for Complementation of E4-Gene-Deleted Adenoviruses The viral E4 regulatory region contains a single transcription unit which is alternately spliced to produce several different mRNAs. The E4-expressing plasmid prepared as described herein and used to transfect the 293 cell line contains the entire E4 transcriptional unit as shown in Figure 1. A DNA fragment extending from 175 nucleotides upstream of the E4 transcriptional start site including the natural E4 promoter to 153 nucleotides downstream of the E4 polyadenylation signal including the natural E4 terminator signal, corresponding to nucleotides 32667-35780 of the adenovirus type 5 (hereinafter referred to as genome as described in Chroboczek et al. (Virol., 186:280-285 (1992), GenBank Accession Number M73260), was amplified from Ad5 genomic DNA, obtained from the ATCC, via the polymerase chain reaction (PCR). Sequences of the primers used were 5'CGGTACACAGAATTCAGGAGACACAACTCC3' (forward or 5' primer referred to as E4L) (SEQ ID NO: 1) and 5'GCCTGGATCCGGGAAGTTACGTAACGTGGGAAAAC3' (SEQ ID NO: 2) WO 00/42208 PCT/EP00/00265 (backward or 3' primer referred to as E4R). To facilitate cloning of the PCR fragment, these oligonucleotides were designed to create novel sites for the restriction enzymes EcoRI and BamHI, respectively, as indicated with underlined nucleotides. DNA was amplified via PCR using 30 cycles of 92 C for 1 minute, C for 1 minute, and 72 C for 3 minutes resulting in amplified full-length E4 gene products.
The amplified DNA E4 products were then digested with EcoRI and BamHI for cloning into the compatible sites of pBluescript/SK+ by standard techniques to create the plasmid pBS/E4. A 2603 base pair (bp) cassette including the herpes simplex virus thymidine kinase promoter, the hygromycin resistance gene, and the thymidine kinase polyadenylation signal was excised from the plasmid pMEP4 (Invitrogen, San Diego, CA) by digestion with Fspl followed by addition of BamHI linkers (5'CGCGGATCCGCG3') (SEQ ID NO: 3) for subsequent digestion with BamHI to isolate the hygromycin-containing fragment. The isolated BamHImodified fragment was then cloned into the BamHI site of pBS/E4 containing the E4 region to create the plasmid pE4/Hygro containing 8710 bp (Figure The pE4/Hygro plasmid has been deposited with the ATCC as described in Example 3.
The complete nucleotide sequence of pE4/Hygro is listed in SEQ ID NO: 4.
Position number 1 of the linearized vector corresponds to approximately the middle portion of the pBS/SK+ backbone as shown in Figure 2 as a thin line between the 3' BamHI site in the hygromycin insert and the 3' EcoRI site in the E4 insert. The and 3' ends of the E4 gene are located at respective nucleotide positions 3820 and 707 of SEQ ID NO: 4 while the 5' and 3' ends of the hygromycin insert are located at respective nucleotide positions 3830 and 6470. In the clone that was selected for use, the E4 and hygromycin resistance genes were divergently transcribed.
WO 00/42208 PCT/EP00/00265 -51- B. Preparation of a Fiber-Expressing Plasmid for Complementation of Fiber-Gene-Deleted Adenoviruses To prepare a fiber-encoding construct, primers were designed to amplify the fiber coding region from Ad5 genomic DNA with the addition of unique BamHI and Notl sites at the 5' and 3' ends of the fragment, respectively. The Ad5 nucleotide sequence is available with the GenBank Accession Number M18369. The 5' and 3' primers had the respective nucleotide sequences of 5'ATGGGATCCAAGATGAAGCGCGCAAGACCG3' (SEQ ID NO: 5) and 5'CATAACGCGGCCGCTTCTTTATTCTTGGGC3' (SEQ ID NO: where the inserted BamHI and NotI sites are indicated by underlining. The 5' primer also contained a nucleotide substitution 3 nucleotides 5' of the second ATG codon (C to A) that is the initiation site. The nucleotide substitution was included so as to improve the consensus for initiation of fiber protein translation.
The amplified DNA fragment was inserted into the BamHI and Notl sites of pcDNA3 (Invitrogen) to create the plasmid designated pCDNA3/Fiber having 7148 bp, the plasmid map of which is shown in Figure 3. The parent plasmid contained the CMV promoter, the bovine growth hormone (BHG) terminator and the gene for conferring neomycin resistance. The viral sequence included in this construct corresponds to nucleotides 31040-32791 of the Ad5 genome.
The complete nucleotide sequence of pCDNA3/Fiber is listed in SEQ ID NO: 7 where the nucleotide position 1 corresponds to approximately the middle of the pcDNA3 vector sequence. The 5' and 3' ends of the fiber gene are located at respective nucleotide positions 916 with ATG and 2661 with TAA.
To enhance expression of fiber protein by the constitutive CMV promoter provided by the pcDNA vector, a Bglll fragment containing the tripartite leader (TPL) of adenovirus type 5 was excised from pRD112a (Sheay et al., BioTechniques, 15:856-862 (1993) and inserted into the BamHI site of pCDNA3/Fiber to create the plasmid pCLF having 7469 bp, the plasmid map of which is shown in Figure 4. The adenovirus tripartite leader sequence, present at the 5' end of all major late adenoviral mRNAs as described by Logan et al., Proc.
Natl. Acad. Sci., USA, 81:3655-3659 (1984) and Berkner, BioTechniques, 6:616- 629 (1988), also referred to as a "partial TPL" since it contains a partial exon 1, shows correspondence with the Ad5 leader sequence having three spatially separated exons corresponding to nucleotide positions 6081-6089 (the 3' end of the first leader segment), 7111-7182 (the entire second leader segment), and WO 00/42208 PCT/EP00/00265 -52- 9644-9845 (the third leader segment and sequence downstream of that segment).
The corresponding cDNA sequence of the partial tripartite leader sequence present in pCLF is listed in SEQ ID NO: 8 bordered by BamHI/Bglll 5' and 3' sites at respective nucleotide positions 907-912 to 1228-1233. The nucleotide sequence of an isolated partial TPL of the present invention is also listed separately as SEQ ID NO: 26 with the noted 5' and 3' restriction sites and with the following nucleotide regions identified: 1-6 nt Bglll site; 1-18 nt polylinker; 19-27 nt last 9 nt of the first leader segment (exon 28-99 nt second leader segment (exon 100-187 nt third leader segment (exon 188-301 nt contains the nt sequence immediately following the third leader in the genome with an unknown function; and 322-327 nt Bglll site.
The pCLF plasmid has been deposited with the ATCC as described in Example 3. The complete nucleotide sequence of pCLF is listed in SEQ ID NO: 8 where the nucleotide position 1 corresponds to approximately the middle of the pcDNA3 parent vector sequence. The 5' and 3 ends of the Ad5 fiber gene are located at respective nucleotide positions 1237-1239 with ATG and 2980-2982 with TAA. The rest of the vector construct has been previously described above.
C. Generation of an Adenovirus Packaging Cell Line Carrying Plasmids Encoding Functional E4 and Fiber Proteins The 293 cell line was selected for preparing the first adenovirus packaging line as it already contains the El gene as prepared by Graham et al., J. Gen. Virol., 36:59-74 (1977) and as further characterized by Spector, Virol., 130:533-538 (1983). Before electroporation, 293 cells were grown in RPMI medium 10% fetal calf serum. Four x 106 cells were electroporated with 20 pg each of pE4/Hygro DNA and pCLF DNA using a BioRad GenePulser and settings of 300 V, 25 pF.
DNA for electroporation was prepared using the Qiagen system according to the manufacturer's instructions (Bio-Rad, Richmond, CA).
Following electroporation, cells were split into fresh complete DMEM fetal calf serum containing 200 pg/ml Hygromycin B (Sigma, St. Louis, MO).
From expanded colonies, genomic DNA was isolated using the "MICROTURBOGEN" system (Invitrogen) according to manufacturer's instructions.
The presence of integrated E4 DNA was assessed by PCR using the primer pair E4R and ORF6L (5'TGCTTAAGCGGCCGCGAAGGAGA AGTCC3') (SEQ ID NO: the latter of which is a 5' forward primer near adenovirus 5 open reading frame 6. Refer to Figure 1 for position of the primers relative to the E4 genes.
WO 00/42208 PCT/EP00/00265 -53- One clone, designated 211, was selected exhibiting altered growth properties relative to that seen in parent cell line 293. The 211 clone contained the expected product, indicating the presence of inserted DNA corresponding to most, if not all, of the E4 fragment contained in the pE4/Hygro plasmid. The 211 cell line has been deposited with the ATCC as described in Example 3. This line was further evaluated by amplification using the primer pair E4LE4R described above, and a product corresponding to the full-length E4 insert was detected. Genomic Southern blotting was performed on DNA restricted with EcoRI and BamHI. The E4 fragment was then detected at approximately one copy/genome compared to standards with the EcoRI/BamHI E4 fragment as cloned into pBS/E4 for use as a labeled probe with the Genius system according to manufacturer's instructions (Boehringer Mannheim, Indianapolis, IN). In DNA from the 211 cell line, the expected labeled internal fragment pE4/Hygro hybridized with the isolated E4 sequences. In addition, the probe hybridized to a larger fragment which may be the result of a second insertion event (Figure Although the 211 cell line was not selected by neomycin resistance, thus indicating the absence of fiber gene, to confirm the lack of fiber gene, the 211 cell line was analyzed for expression of fiber protein by indirect immunofluorescence with an anti-fiber polyclonal antibody and a FITC-labeled anti-rabbit IgG (KPL) as secondary. No immunoreactivity was detected. Therefore, to generate 211 clones containing recombinant fiber genes, the 211 clone was expanded by growing in RPMI medium and subjected to additional electroporation with the fiber-encoding pCLF plasmid as described above.
Following electroporation, cells were plated in DMEM 10% fetal calf serum and colonies were selected with 200 pg/ml G418 (Gibco, Gaithersburg, MD).
Positive cell lines remained hygromycin resistant. These candidate sublines of 211 were then screened for fiber protein expression by indirect immunofluorescence as described above. The three sublines screened, 211A, 211B and 211R, along with a number of other sublines, all exhibited nuclear staining qualitatively comparable to the positive control of 293 cells infected with AdRSVpgal (1 pfu/cell) and stained 24 hours post-infection.
Lines positive for nuclear staining in this assay were then subjected to Western blot analysis under denaturing conditions using the same antibody.
Several lines in which the antibody detected a protein of the expected molecular weight (62 kd for the Ad5 fiber protein) were selected for further study including 211A, 211B and 211R. The 211A cell line has been deposited with ATCC as described in Example 3.
WO 00/42208 PCT/EP00/00265 -54- Immunoprecipitation analysis using soluble nuclear extracts from these three cell lines and a seminative electrophoresis system demonstrated that the fiber protein expressed is in the functional trimeric form characteristic of the native fiber protein as shown in Figure 6. The predicted molecular weight of a trimerized fiber is 186 kd. The lane marked 293 lacks fiber while the sublines contain detectable fiber. Under denaturing conditions, the trimeric form was destroyed resulting in detectable fiber monomers as shown in Figure 6. Those clones containing endogenous El, newly expressed recombinant E4 and fiber proteins were selected for use in complementing adenovirus gene delivery vectors having the corresponding adenoviral genes deleted as described in Example 2.
D. Preparation of an El-Expressing Plasmid for Complementation of El-Gene-Deleted Adenoviruses In order to prepare adenoviral packaging cell lines other than those based on the El-gene containing 293 cell line as described in Example 1C above, plasmid vectors containing El alone or in various combinations with E4 and fiber genes are constructed as described below.
The region of the adenovirus genome containing the Ela and Elb gene is amplified from viral genomic DNA by PCR as previously described. The primers used are E1L, the 5' or forward primer, and E1R, the 3' or backward primer, having the respective nucleotide sequences 5'CCG AGCTAGC GACTGAAAATGAG3' (SEQ ID NO: 10) and 5'CCTCTCGAG AGACAGC AAGACAC3' (SEQ ID NO: 11).
The E1L and E1R primers include the respective restriction sites Nhel and Xhol as indicated by the underlines. The sites are used to clone the amplified El gene fragment into the Nhel/Xhol sites in pMAM commercially available from Clontech (Palo Alto, CA) to form the plasmid pDEX/E1 having 11152 bp, the plasmid map of which is shown in Figure 7.
The complete nucleotide sequence of pDEX/E1 is listed in SEQ ID NO: 12 where the nucleotide position 1 corresponds to approximately 1454 nucleotides from the 3' end of the pMAM backbone vector sequence. The pDEX/E1 plasmid includes nucleotides 552 to 4090 of the adenovirus genome positioned downstream (beginning at nucleotide position 1460 and ending at 4998 in the pDEX/E1 plasmid) of the glucocorticoid-inducible mouse mammary tumor virus (MMTV) promoter of pMAM. The pMAM vector contains the E. coli gpt gene that allows stable transfectants to be isolated using ~1 WO 00/42208 PCT/EP00/00265 hypoxanthine/aminopterin/thymidine (HAT) selection. The pMAM backbone occupies nucleotide positions 1-1454 and 5005-11152 of SEQ ID NO: 12.
E. Generation of an Adenovirus Packaging Cell Line Carrying Plasmids Encoding Functional El, and Fiber Proteins To create separate adenovirus packaging cell lines equivalent to that of the 211 sublines, 211A, 211B and 211R, as described in Example 1C, alternative cell lines lacking adenoviral genomes are selected for transfection with the plasmid constructs as described below. Acceptable host cells include A549, Hela, Vero and the like cell lines as described in Example 1. The selected cell line is transfected with the separate plasmids, pDEX/EI and pCLF, respectively for expressing El, and fiber complementary proteins. Following transfection procedures as previously described, clones containing stable insertions of the two plasmids are isolated by selection with neomycin and HAT. Integration of fulllength copy of the El gene is assessed by PCR amplification from genomic DNA using the primer set E1UE1R as described above. Functional insertion of the fiber gene is assayed by staining with the anti-fiber antibody as previously described.
The resultant stably integrated cell line is then used as a packaging cell system to complement adenoviral gene delivery vectors having the corresponding adenoviral gene deletions as described in Example 2.
F. Preparation of a Plasmid Containing Two or More Adenoviral Genes for Complementing Gene-Deleted Adenoviruses The methods described in the preceding Examples rely on the use of two plasmids, pE4/Hygro and pCLF, or, pCLF and pDEX/E1 for generating adenoviral cell packaging systems. In alternative embodiments contemplated for use with the methods of this invention, complementing plasmids containing two or more adenoviral genes for expressing of encoded proteins in various combinations are also prepared as described below. The resultant plasmids are then used in various cell systems with delivery plasmids having the corresponding adenoviral gene deletions. The selection of packaging cell, content of the delivery plasmids and WO 00/42208 PCT/EP00/00265 -56content of the complementing plasmids for use in generating recombinant adenovirus viral vectors of this invention thus depends on whether other adenoviral genes are deleted along with the adenoviral fiber gene, and, if so, which ones.
1. Preparation of a Complementing Plasmid Containing Fiber and El Adenoviral Genes A DNA fragment containing sequences for the CMV promoter, adenovirus tripartite leader, fiber gene and bovine growth hormone terminator is amplified from pCLF prepared in Example 1B using the forward primer 5'GACGGATCGGGAGATCTCC3' (SEQ ID NO: 13), that anneals to the nucleotides 1-19 of the pCDNA3 vector backbone in pCLF, and the backward primer 5'CCGCCTCAGAAGCCATAGAGCC3' (SEQ ID NO: 14) that anneals to nucleotides 1278-1257 of the pCDNA3 vector backbone. The fragment is amplified as previously described and then cloned into the pDEX/E1 plasmid, prepared in Example 1D. For cloning in the DNA fragment, the pDEX/E1 vector is first digested with Ndel, that cuts at a unique site in the pMAM vector backbone in pDEX/E1, then the ends are repaired by treatment with bacteriophage T4 polymerase and dNTPs.
The resulting plasmid containing El and fiber genes, designated pE1/Fiber, provides both dexamethasone-inducible El function as described for DEX/E1 and expression of Ad5 fiber protein as described above. A schematic plasmid map of pE1/Fiber, having 14455 bp, is shown in Figure 8.
The complete nucleotide sequence of pE1/Fiber is listed in SEQ ID NO: where the nucleotide position 1 corresponds to approximately to 1459 nucleotides from the 3' end of the parent vector pMAM sequence. The 5' and 3 ends of the El gene are located at respective nucleotide positions 1460 and 4998 followed by pMAM backbone and then separated from the Ad5 fiber from pCLF by the filled-in blunt ended Ndel site. The 5' and 3' ends of the pCLF fiber gene fragment are located at respective nucleotide positions 10922-14223 containing elements as previously described for pCLF.
The resultant pE1/Fiber plasmid is then used to complement one or more delivery plasmids expressing El and fiber.
The pE1/Fiber construct is then used to transfect a selected host cell as described in Example 1E to generate stable chromosomal insertions preformed as previously described followed by selection on HAT medium. The stable cells are then used as packaging cells as described in Example 2.
WO 00/42208 PCT/EPOO/00265 -57- 2. Preparation of a Complementing Plasmid Containing E4 and Fiber Adenoviral Genes pCLF prepared as described in Example 1B is partially digested with Bglll to cut only at the site in the pCDNA3 backbone. The pE4/Hygro plasmid prepared in Example 1A is digested with BamHI to produce a fragment containing E4. The E4 fragment is then inserted into the BamHI site of pCLF to form plasmid pE4/Fiber.
The resultant plasmid provides expression of the fiber gene as described for pCLF and E4 function as described for pE4/Hygro.
A schematic plasmid map of pE4/Fiber, having 10610 bp, is shown in Figure 9. The complete nucleotide sequence of pE4/Fiber is listed in SEQ ID NO: 16 where the nucleotide position 1 corresponds to approximately 14 bp from the 3' end of the parent vector pCDNA3 backbone sequence. The 5' and 3 ends of the E4 gene are located at respective nucleotide positions 21 and 3149 followed by fused Bglll/BamHI sites and pCDNA3 backbone including the CMV promoter again followed by Bglll/BamHI sites. The adenovirus leader sequence begins at nucleotide position 4051 and extends to 4366 followed by fused BamHI/Bglll sites and the 5' and 3' ends of the fiber gene located at respective nucleotide positions 4372 and 6124.
Stable chromosonal insertions of pE4/Fiber in host cells are obtained as described above.
Example 2 Preparation of Adenoviral Gene Delivery Vectors Using Adenoviral Packaging Cell Lines Adenoviral delivery vectors of this invention are prepared to separately lack the combinations of El/fiber and E4/fiber. Such vectors are more replicationdefective than those previously in use due to the absence of multiple viral genes.
A preferred adenoviral delivery vector of this invention that is replication competent but only via a non-fiber means is one that only lacks the fiber gene but contains the remaining functional adenoviral regulatory and structural genes. Furthermore, the adenovirus delivery vectors of this invention have a higher capacity for insertion of foreign DNA.
WO 00/42208 PCT/EP00/00265 -58- A. Preparation of Adenoviral Gene Delivery Vectors Having Specific Gene Deletions and Methods of Use To construct the El/ fiber deleted viral vector containing the LacZ reporter gene construct, two new plasmids were constructed. The plasmid pA E1Bpgal was constructed as follows. A DNA fragment containing the SV40 regulatory sequences and E. coli P-galactosidase gene was isolated from pSVpgal (Promega) by digesting with Vspl, filling the overhanging ends by treatment with Klenow fragment of DNA polymerase I in the presence of dNTP's and digesting with Barn H1. The resulting fragment was cloned into the EcoRV and BamHI sites in the polylinker of pA ElsplB (Microbix Biosystems, Hamilton, Ontario) to form pAE1 B3gal that therefore contained the left end of the adenovirus genome with the Ela region replaced by the LacZ cassette (nucleotides 6690 to 4151) of pSVpgal.
Plasmid DNA may be prepared by the alkaline lysis method as described by Birnboim and Doly, Nuc. Acids Res., 7:1513-1523 (1978) or by the Quiagen method according to the manufacturer's instruction, from transformed cells used to expand the plasmid DNA was then purified by CsCI-ethidium bromide density gradient centrifugation. Alternatively, plasmid DNAs may be purified from E. co/i by standard methods known in the art see Sambrook et al.) The second plasmid (pDV44), prepared as described herein, is derived from a vector prepared as described by Bett et al., Proc. Natl. Acad. Sci., USA, 91:8802-8806 (1994), now described in International Application Publication Number WO 9500655, with methodology well known to one of ordinary skill in the art and also is commercially available from Microbix, which contains an genome with the packaging signals at the left end deleted and the E3 region (nucleotides 28133:30818) replaced by a linker with a unique site for the restriction enzyme Pad. An 11.9 kb BamHI fragment, which contains the right end of the adenovirus genome, is isolated from pBHG10 and cloned into the BamHI site of pBS/SK(+) to create plasmid p11.3 having approximately 14,658 bp. A schematic of the plasmid map is shown in Figure 13. The p11.3 plasmid was then digested with Pad and Sail to remove the fiber, E4, and inverted terminal repeat (ITR) sequences.
This fragment was replaced with a 3,4 kb fragment containing the ITR segments and the E4 gene which was generated by PCR amplification from using the following oligonucleotide sequences TGTACACCG GATCCGGCGCACACC3' SEQ ID NO: 17) and WO 00/42208 PCT/EP00/00265 -59- AATTAATTAATTGCCACATCCTC3' SEQ ID NO: 18). These primers incorporated sites for Pad and BamHI. Cloning this fragment into the Pad and blunt ended Sail sites of the p11.3 backbone resulted in a substitution of the fused ITRs, E4 region and fiber gene present in pBHG10, by the ITRs and E4 region alone. The resultant p11.3 plasmid containing the ITR and E4 regions, now called plasmid pDV43a, was then digested with BamHI. This BamHI fragment was then used to replace a BamHI fragment in pBHG10 thereby creating pDV44 in a pBHG10 backbone.
In an alternative approach to preparing pDV44 with an additional subcloning step to facilitate the incorporation of restriction cloning sites, the following cloning procedure was performed. pDV44 as above was constructed by removing the fiber gene and some of the residual E3 sequences from pBHG10 (Microbix Biosystems).
As above, to simplify manipulations, the 11.9 kb BamHI fragment including the rightmost part of the Ad5 genome was removed from pBHG10 and inserted into pBS/SK. The resulting plasmid was termed p11.3. The 3.4 kb DNA fragment corresponding to the E4 region and both ITRs of adenovirus type 5 was amplified as described above from pBHG10 using the oligonucleotides listed above and subcloned into the vector pCR2.1 (Invitrogen) to create pDV42. This step is the additional cloning step to facilitate the incorporation of a Sail restriction site.
pDV42 was then digested with Pad, which cuts at a unique site (bold type) in one of the PCR primers, and with Sail, which cuts at a unique site in the pCR2.1 polylinker. This fragment was used to replace the corresponding Pacl/Xhol fragment of p11.3 (the pBS polylinker adjacent to the Ad DNA fragment contains a unique Xhol site), creating pDV43. Finally, pDV44 was constructed by replacing the 11.9 kb BamHI fragment of pBHG10 by the analogous BamHI fragment of pDV43. As generated in the first procedure, pDV44 therefore differs from by the deletion of Ad5 nucleotides 30819:32743 (residual E3 sequences and all but the 3'-most 41 nucleotides of the fiber open reading frame).
Thus, to summarize, the cloning procedures described above result in the production of a fiber-deleted Ad5 genomic plasmid (pDV44) that was constructed by removing the fiber gene and some of the residual E3 sequences from (Figure 16A). pDV44 contains a wild-type E4 region, but only the last 41 nucleotides of the fiber ORF (this sequence was retained to avoid affecting expression of the adjacent E4 transcription unit). Both pBHG10 and pDV44 contain unpackageable Ad5 genomes, and must be rescued by cotransfection and subsequent homologous recombination with DNA carrying functional packaging signals. In order to generate vectors marked with a reporter gene, either pDV44 or was cotransfected with pAE1BBgal, which contains the left end of the WO 00/42208 PCT/EPOO/00265 genome with an SV40-driven 1-galactosidase reporter gene inserted in place of the El region.
In general, and as described below, the method for virus production by recombination of plasmids followed by complementation in cell culture involves the isolation of recombinant viruses by cotransfection of any one of the adenovirus packaging cell systems prepared in Example 1, namely 211A, 211B, 211R, A549, Vero cells, and the like, with plasmids carrying sequences corresponding to viral gene delivery vectors.
A selected cell line is plated in dishes and cotransfected with pDV44 and pAE1BBgal using the calcium phosphate method as described by Bett et al., Proc.
Natl. Acad. Sci., USA, 91:8802-8806 (1994). Recombination between the overlapping adenovirus sequences in the two plasmids leads to the creation of a full-length viral chromosome where pDV44 and pAE1BIgal recombine to form a recombinant adenovirus vector having multiple deletions. The deletion of El and of the fiber gene from the viral chromosome is compensated for by the sequences integrated into the packaging cell genome, and infectious virus particles are produced. The plaques thus generated are isolated and stocks of the recombinant virus are produced by standard methods.
A pDV44-derived virus is expected to be replication-defective due to the fiber deletion, so that the cells in which it is grown must complement this defect.
The 211B cell line (a derivative of 293 cells which expresses the wild-type (wt) fiber and is equivalent to 211A on deposit with ATCC as described in Example 3) was used for rescue and propagation of the virus described here. pDV44 and pAE1BBgal were cotransfected into 211B cells, and the monolayers were observed for evidence of cytopathic effect (CPE). Briefly, for virus construction, cells were transfected with the indicated plasmids using the Gibco Calcium Phosphate Transfection system according to the manufacturer's instructions and observed daily for evidence of CPE.
One of a total of 58 transfected dishes showed evidence of spreading cell death at day 15. A crude freeze-thaw lysate was prepared from these cells and the resulting virus (termed Ad5.Bgal.AF) was plaque purified twice and then expanded.
To prepare purified viral preparations, cells were infected with the indicated Ad and observed for completion of CPE. Briefly, at day zero, 211B cells were plated in DMEM plus 10% fetal calf serum at approximately 1 X 10 7 cells/150 cm 2 flask or equivalent density. At day one, the medium was replaced with one half the original volume of fresh DMEM containing the indicated Ad, in this case Ad5.Bgal.AF, at approximately 100 particles/cell. At day two, an equal volume of medium was WO 00/42208 PCT/EPOO/00265 -61added to each flask and the cells were observed for CPE. Two to five days after infection, cells were collected and virus isolated by lysis via four rapid freeze-thaw cycles. Virus was then purified by centrifugation on preformed 15-40% CsCI gradients (111,000 x g for three hours at The bands were harvested, dialyzed into storage buffer (10 mM Tris-pH 8.1, 0.9% NaCI, and 10% glycerol), aliquoted and stored at 700C. Purified Ad5.Bgal.AF virus particles containing human adenovirus Ad5.Bgal. AFgenome (described further below) have been deposited with the ATCC on January 15, 1999 as further described in Example 3.
For viral titering, as necessary in the below Examples, Ad preparations were titered by plaque assay on 211B cells. Cells were plated on polylysine-coated 6 well plates at 1.5 x 106 cells/well. Duplicate dilutions of virus stock were added to the plates in 1 ml/well of complete DMEM. After a five hour incubation at 370C, virus was removed and the wells overlaid with 2 ml of 0.6% low-melting agarose in Medium 199 (Gibco). An additional 1 ml of overlay was added at five day intervals.
As a control, the first-generation virus Ad5.B gal.wt, which is identical to AF except for the fiber deletion, was constructed by cotransfection of and pAE1BBgal (Figure 16B). In contrast to the low efficiency of recovery of the fiberless genome (1/58 dishes), all of 9 dishes cotransfected with pAE1BBgal and pBHG10 produced virus.
In a preferred embodiment of this invention as more fully described herein and below, a delivery plasmid is prepared that does not require the abovedescribed recombination events to prepare a viral vector having a fiber gene deletion. In one embodiment, a single delivery plasmid containing all the adenoviral genome necessary for packaging but lacking the fiber gene is prepared from plasmid pFG140 containing full-length Ad5 that is commercially available from Microbix. The resultant delivery plasmid referred to as pFG140-f is then used with pCLF stably integrated cells as described above to prepare a viral vector lacking fiber. In a preferred aspect of this invention, the fiber gene is replaced with a therapeutic gene of interest for preparing a therapeutic delivery adenoviral vector.
Other embodiments including production of fiberless vector with a complete TPL are described in Example Vectors for the delivery of any desired gene and preferably a therapeutic gene are prepared by cloning the gene of interest into the multiple cloning sites in the polylinker of commercially available pAElsplB (Microbix Biosystems), in an analogous manner as performed for preparing p E1BIgal as described above. The same cotransfection and recombination procedure is then followed as described herein to obtain viral gene delivery vectors as further discussed in later Examples.
WO 00/42208 PCT/EPOO/00265 -62- 1. Characterization of the Ad5.pgal.AF Genome To confirm that the vector genomes had the expected structures and that the fiber gene was absent from the Ad5.Bgal.0)F chromosome, the DNA isolated from viral particles was analyzed. Briefly, purified viral DNA was obtained by adding pl of 10 mg/ml proteinase K, 40 pl of 0.5 M EDTA and 50 pl of 10% SDS to 800 pl of adenovirus-containing culture supernatant. The suspension was then incubated at 55C for 60 minutes. The solution was then extracted once with 400 pl of a 24:1 mixture of chloroform:isoamyl alcohol. The aqueous phase was then removed and precipitated with sodium acetate/ethanol. The pellet was washed once with 70% ethanol and lightly dried. The pellet was then suspended in pl of 10 mM Tris-HCI, pH 8.0, 1 mM EDTA. Genomic DNA from both and Ad5.pgal.AF produced the expected restriction patterns (Figure 17A) following digestion with either EcoRI (Figure 17B) or with Ndel (data not shown). Southern blotting, performed with standard methods, with labeled fiber DNA as a probe demonstrated the presence of fiber sequence in Ad5.Bgal.wt but not in Ad5.Bgal. AF DNA (Figure 17C). As a positive control, the blot was stripped and reprobed with labeled E4 sequence. Fiber and E4 sequences were detected by using labeled inserts from pCLF and pE4/Hygro, respectively. As expected, E4 signal was readily detectable in both genomes at equal intensities (Figure 17C).
The complete nucleotide sequence of Ad5.pgal.AF is presented in SEQ ID NO: 27 and is contained in the virus particle on deposit with ATCC.
2. Characterization of the Fiberless Adenovirus To verify that Ad5.Bgal.AF was fiber-defective, 293 cells (which are permissive for growth of El-deleted Ad vectors but do not express fiber) were infected with Ad5.Bgal. AF or with Ad5.Bgal.wt. Twenty-four hours post infection, the cells were stained with polyclonal antibodies directed either against fiber or against the penton base protein. Cells infected with either virus were stained by the anti-penton base antibody, while only cells infected with the Ad5.3gal.wt control virus reacted with the anti-fiber antibody. This confirms that the fiber-deleted Ad mutant does not direct the synthesis of fiber protein.
3. Growth of the Fiber-Deleted Ad5.pgal.AF Vector in Complementing Cells WO 00/42208 PCT/EP00/00265 -63was found to readily be propagated in 211B cells. As assayed by protein concentration, CsCI-purified stocks of either Ad5.pgal.AF or contained similar numbers of viral particles (Table and the particles appeared to band normally on CsCI gradients. However, infectivity of the Ad5.pgal.AF particles was lower than the Ad5.Bgal.wt control, as indicated by an increased particle/PFU ratio (Table This is likely due to a reduced amount of fiber protein incorporated into mutant particles during growth in the WO 00/42208 PCT/EP00/00265 -64- Virus CsCI- Cell Particles/ PFU/ml b Particle Fiber source purified line mla /PFU prepn ratio 1 211 7.4 x 10 7.5 x 10 Ad B 1010 chromosom e 2 211 3.0 x 10 5.0 x 10 60 Ad B chromosom e 3 211 7.7 x 10 1 3.5 x 10 2200 Packaging B cells 4 211 1.9 x101 2.3x10 9 808 Packaging B cells 293 4.5 x10 1 9.5 x10 6 47400 None 6 293 3.4 x 10 3.5 x 10 9700 None aCalculated from viral protein concentratic bAssayed by plaquing on 211B cells.
on (1ug of protein 4 x 10 9 particles).
Table 3 *Particle numbers and infectious titers of representative adenovirus preps. Each line represents a single CsCI-purified preparation of the indicated virus. Particle numbers were calculated from viral protein concentration (1 pg protein 4 x 10 9 particles). Pfu was assayed by plaquing on 211B cells (see above).
211B cells (see below). Ad5.pgal.AF was also found to plaque more slowly than the control virus. When plated on 211B cells, Ad5.Bgal.wt plaques appeared within 5-7 days, while plaques of Ad5.pgal.AF continued to appear until as much as 15-18 days post infection. Despite their slower formation, the morphology of plaques was essentially normal.
WO 00/42208 PCT/EP00/00265 4. Production of Fiberless Ad5.Bgal. AF Particles As Ad5.Pgal.AF represents a true fiber null mutation and its stocks are free of helper virus, the fiber mutant phenotype was readily investigated. A single round of growth in cells (such as 293) which do not produce fiber generating a homogeneous preparation of fiberless Ad allowed for the determination of whether such particles would be stable and/or infectious. Either Ad5.Bgal.wt or Ad5.pgal.AF was grown in 293 or 211B cells, and the resulting particles purified on CsCI gradients as previously described. Ad5.Pgal.AF particles were readily produced in 293 cells at approximately the same level as the control virus and behaved similarly on the gradients, indicating that there was not a gross defect in morphogenesis of fiberless capsids (Table 1).
As shown in Figure 18, particles of either virus contained similar amounts of penton base regardless of the cell type in which they were grown. This demonstrated that fiber is not required for assembly of the penton base complex into virions. However, as predicted, the Ad5.Pgal.AF particles produced in 293 cells did not contain fiber protein. 211B-grown Ad5.pgal.AF also contained less fiber than the Ad5.Bgal.wt control virus (Figure 18). Importantly, the infectivities of the different viral preparations on epithelial cells (Table 1) correlated with the amount of fiber protein present. The fiberless Ad particles were several thousand-fold less infectious than the first-generation vector control on a per-particle basis, while infectivity of 211B-grown Ad5.pgal.AF was only 50-100 fold less than that of These studies confirmed fiber's crucial role in infection of epithelial cells via CAR binding.
Composition and Structure of the Fiberless AF Particles The proteins contained in particles of 293-grown Ad5.pgal.AF were compared to those in Ad5.Bgal.wt, to determine whether proteolysis or particle assembly was defective in this fiber null mutant (data not shown). The overall pattern of proteins in the fiberless particles was observed to be quite similar to that of a first-generation vector, with the exception of reduced intensity of the composite band resulting from both proteins Ilia and IV (fiber) (data not shown). The fiberless particles also had a reduced level of protein VII. Although substantial amounts of uncleaved precursors WO 00/42208 PCT/EP00/00265 -66to proteins VI, VII, and VIII were not seen, it is possible that the low-molecular weight bands migrating ahead of protein VII represent either aberrantly cleaved viral proteins or their breakdown products.
Cryo-electron microscopy was used to more closely examine the structure of the 293 grown Ad5.pgal.AF and of Ad5Bgal.wt. The fiber, which consists of an extended stalk with a knob at the end, was faintly visible in favorable orientations of wild-type Ad5 particles, but not in images of the fiberless particles (data not shown).
Filamentous material likely corresponding to free viral DNA was seen in micrographs of fiberless particles. This material was also present in micrographs of the first-generation control virus, albeit at much lower levels.
Three-dimensional image reconstructions of fiberless and wild-type particles at -20 A resolution showed similar sizes and overall features, with the exception that fiberless particles lacked density corresponding to the fiber protein. The densities corresponding to other capsid proteins, including penton base and proteins Ilia, VI, and IX, were comparable in the two structures. This confirms that absence of fiber does not prevent assembly of these components into virions. The fiber was truncated in the wild-type structure as only the lower portion of its flexible shaft follows icosahedral symmetry. The RGD protrusions on the fiberless penton base were angled slightly inward relative to those of the wild-type structure. Another difference between the two penton base proteins was that there is a -30 A diameter depression in the fiberless penton base around the five-fold axis where the fiber would normally sit. The Ad5 reconstructions confirm that capsid assembly, including addition of penton base to the vertices, is able to proceed in the complete absence of fiber.
6. Integrin-Dependent Infectivity of Fiberless AdS.Bgal. AF Particles While attachment via the viral fiber protein is a critical step in the infection of epithelial cells, an alternative pathway for infection of certain hematopoietic cells has been described. In this case, penton base mediates both binding to the cells (via 32 integrins) and internalization (through interaction with av integrins). Particles lacking fiber might therefore be expected to be competent for infection of these cells, even though on a per-particle basis they are several thousand-fold less infectious than normal Ad vectors on epithelial cells.
WO 00/42208 PCT/EP00/00265 -67- To investigate this, THP-1 monocytic cells were infected with Ad5.Bgal.wt or with Ad5.pgal.AF grown in the absence of fiber. Infection of THP-1 cells was assayed by infecting 2 x 10 cells at the indicated m.o.i. in 0.5 ml of complete RPMI.
Forty-eight hours post-infection, the cells were fixed with glutaraldehyde and stained with X-gal, and the percentage of stained cells was determined by light microscopy.
The results of the infection assay showed that the fiberless particles were only a few-fold less infectious than first-generation Ad on THP-1 cells (Figure 19A).
In contrast to this, very large differences were seen in plaquing efficiency on epithelial (211B) cells (Table Infection of THP-1 cells by either Ad5.pgal.AF or was not blocked by an excess of soluble recombinant fiber protein, but could be inhibited by the addition of recombinant penton base (Figure 19B). These results indicate that the fiberless Ad particles use a fiber-independent pathway to infect these cells. Furthermore, the lack of fiber protein did not prevent from internalizing into the cells and delivering its genome to the nucleus, demonstrating that fiberless particles are properly assembled and are capable of uncoating.
The foregoing results with the recombinant viruses thus produced indicates that they can be used as gene delivery tools both in cultured cells and in vivo as described more fully in the Examples. For example, for studies of the effectiveness and relative immunogenicity of multiply-deleted vectors, virus particles are produced by growth in the packaging lines described in Example 1 and are purified by CsCI gradient centrifugation. Following titering, virus particles are administered to mice via systemic or local injection or by aerosol delivery to lung. The LacZ reporter gene allows the number and type of cells which are successfully transduced to be evaluated. The duration of transgene expression is evaluated in order to determine the long-term effectiveness of treatment with multiply-deleted recombinant adenoviruses relative to the standard technologies which have been used in clinical trials to date. The immune response to the improved vectors described here is determined by assessing parameters such as inflammation, production of cytotoxic T lymphocytes directed against the vector, and the nature and magnitude of the antibody response directed against viral proteins.
Versions of the vectors which contain therapeutic genes such as CFTR for treatment of cystic fibrosis or tumor suppressor genes for cancer treatment are evaluated in the animal system for safety and efficiency of gene transfer and expression. Following this evaluation, they are used as experimental therapeutic agents in human clinical trials.
WO 00/42208 PCT/EP00/00265 -68- B. Retargeting of Adenoviral Gene Delivery Vectors by Producing Viral Particles Containing Different or Altered Fiber Proteins As the specificity of adenovirus binding to target cells is largely determined by the fiber protein, viral particles that incorporate modified fiber proteins or fiber proteins from different adenoviral serotypes (pseudotyped vectors) have different specificities. Thus, the methods of expression of the native Ad5 fiber protein in adenovirus packaging cells as described above is also applicable to production of different fiber proteins.
In one aspect of invention, chimeric fiber proteins are produced according to the methods of Stevenson et al., J. Virol., 69:2850-2857 (1995). The authors showed that the determinants for fiber receptor binding activity are located in the head domain of the fiber and that isolated head domain is capable of trimerization and binding to cellular receptors. The head domains of adenovirus type 3 (Ad3) and were exchanged in order to produce chimeric fiber proteins. Similar constructs for encoding chimeric fiber proteins for use in the methods of this invention are contemplated. Thus, instead of the using the intact Ad5 fiber-encoding construct prepared in Example 1 as a complementing viral vector in adenoviral packaging cells, the constructs described herein are used to transfect cells along with E4 and/or El-encoding constructs.
Briefly, full-length Ad5 and Ad3 fiber genes were amplified from purified adenovirus genomic DNA as a template. The Ad5 and Ad3 nucleotides sequences are available with the respective GenBank Accession Numbers M18369 and M12411. Oligonucleotide primers are designed to amplify the entire coding sequence of the full-length fiber genes, starting from the start codon, ATG, and ending with the termination codon TAA. For cloning purposes, the 5' and 3' primers contain the respective restriction sites BamHI and Notl for cloning into pcDNA plasmid as described in Example 1A. PCR is performed as described above.
The resultant products are then used to construct chimeric fiber constructs by PCR gene overlap extension, as described by Horton et al., BioTechniques, 8:525- 535 (1990). The Ad5 fiber tail and shaft regions (5TS; the nucleotide region encoding amino acid residue positions 1 to 403) are connected to the Ad3 fiber head region (3H; the nucleotide region encoding amino acid residue positions 136 to 319) to form the 5TS3H fiber chimera. Conversely, the Ad3 fiber tail and shaft regions (3TS; the nucleotide region encoding amino acid residues positions 1 to 135) are WO 00/42208 PCT/EP00/00265 -69connected to the Ad5 fiber head region (5H; the nucleotide region encoding the amino acid residue positions 404 to 581) to form the 3TS5H fiber chimera. The fusions are made at the conserved TLWT (SEQ ID NO: 19) sequence at the fiber shaft-head junction.
The resultant chimeric fiber PCR products are then digested with BamHI and Notl for separate directional ligation into a similarly digested pcDNA 3.1. The TPL sequence is then subcloned into the BamHI as described in Example 1A for preparing an expression vector for subsequent transfection into 211 cells as described above or into the alternative packaging cell systems as previously described. The resultant chimeric fiber construct-containing adenoviral packaging cell lines are then used to complement adenoviral delivery vectors as previously described. Other fiber chimeric constructs are obtained using a similar approach with the various adenovirus serotypes known.
In an alternative embodiment, the methods of this invention contemplate the use of the modified proteins including novel epitopes as described by Michael et a., Gene Therapy, 2:660-668 (1995) and in International Publication WO 95/26412.
Both publications describe the construction of a cell-type specific therapeutic viral vector having a new binding specificity incorporated into the virus concurrent with the destruction of the endogenous viral binding specificity. In particular, the authors described the production of an adenoviral vector encoding a gastrin releasing peptide (GRP) at the 3' end of the coding sequence of the Ad5 fiber gene. The resulting fiber-GRP fusion protein was expressed and shown to assemble functional fiber trimers that were correctly transported to the nucleus of HeLa cells following synthesis.
Based on the teachings in the paper and International Publication, similar constructs are contemplated for use in the complementing adenoviral packaging cell systems of this invention for generating new adenoviral gene delivery vectors that are targetable, replication-deficient and less immunogenic. Heterologous ligands contemplated for use herein to redirect fiber specificity range from as few as amino acids in size to large globular structures, some of which necessitate the addition of a spacer region so as to reduce or preclude steric hindrance of the heterologous ligand with the fiber or prevent trimerization of the fiber protein. The ligands are inserted at the end or within the linker region. Preferred ligands include those that target specific cell receptors or those that are used for coupling to other moieties such as biotin and avidin.
WO 00/42208 PCT/EP00/00265 A preferred spacer includes a short 12 amino acid peptide linker composed of a series of serines and alanine flanked by a proline residue at each end. One of ordinary skill in the art is familiar with the preparation of linkers to accomplish sufficient protein presentation and for altering the binding specificity of the fiber protein without compromising the cellular events that follow viral internalization.
Moreover, within the context of this invention, preparation of modified fibers having ligands positioned internally within the fiber protein and at the carboxy terminus as described below are contemplated for use with the methods described herein.
The preparation of a fiber having a heterologous binding ligand is prepared essentially as described in the above-cited paper. Briefly, for the ligand of choice, site-directed mutagenesis is used to insert the coding sequence for a linker into the 3' end of the Ad5 fiber construct in pCLF as prepared in Example 1.
The 3' or antisense or mutagenic oligonucleotide encodes a preferred linker sequence of ProSerAlaSerAlaSerAlaSerAlaProGlySer (SEQ ID NO: 20) followed by a unique restriction site and two stop codons, respectively, to allow the insertion of a coding sequence for a selected heterologous ligand and to ensure proper translation termination. Flanking this linker sequence, the mutagenic oligonucloetide contains sequences that overlap with the vector sequence and allo its incorporation into the construct. Following mutagenesis of the pCLF sequence adding the linker and stop codon sequences, a nucleotide sequence encoding a preselected ligand is obtained, linkers corresponding to the unique restriction site in the modified construct are attached and then the sequence is cloned into linearized corresponding restriction site. The resultant fiber-ligand construct is then used to transfect 211 or the alternative cell packaging systems previously described to produce complementing viral vector packaging systems for use with the methods of this invention.
In a further embodiment, intact fiber genes from different Ad serotypes are expressed by 211 cells or an alternative packaging system as previously described.
A gene encoding the fiber protein of interest is first cloned to create a plasmid analogous to pCLF, and stable cell lines producing the fiber protein are generated as described above for Ad5 fiber. The adenovirus vector described which lacks the fiber gene is then propagated in the cell line producing the fiber protein relevant for the purpose at hand. As the only fiber gene present is the one in the packaging cells, the adenoviruses produced contain only the fiber protein of interest and therefore have the binding specificity conferred by the complementing protein. Such viral particles are used in studies such as those described above to determine their properties in experimental animal systems.
WO 00/42208 PCT/EP00/00265 -71 Example 3 Deposit of Materials The following cell lines and plasmids have been deposited on September 1996, with the American Type Culture Collection, 10801 University Blvd, Manassas, Virginia, USA (ATCC) under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations thereunder (Budapest Treaty):Plasmid pE4/Hygro (accession number 97739), Plasmid pCLF (accession number 97737), 211 Cell Line (accession number CRL-12193) and 211A Cell Line (accession number CRL-12194) The following virus, Ad5.pgal.AF deposit was deposited on January 1999, with the ATCC as listed above and provided with accession number VR2636.
Additionally, plasmids pDV60, pDV67, pDV69, pDV80 and pDV90 were also deposited at the ATCC on 5 January, 2000 and provided with accession numbers PTA-1144, PTA-1145, PTA-1146, PTA-1147 and PTA-1148 respectively.
Example 4 Complementation of Fiber-Defective and Fiber-Modified Virus The native fiber protein is a homotrimer (Henry L.J. et al., J. Virol.
68:5239-5246 (1994)), and trimerization is essential for assembly of the penton/fiber complex (Novelli A et al., J. Biol. Chem. 266.9299-9303 (1991)). To assess the multimeric structure of the recombinant fiber protein produced by the cell lines, cells were labeled with 50 pCi/ml 3 5 S] Translabel (ICN) for two hours at 37 0 C, lysed in RIPA buffer, and fiber protein was immunoprecipitated as described (Harlow E et a., Antibodies. Cold Spring Harbour Laboratory, Cold Spring Harbor (1988). Immune complexes were collected on Protein A-Sepharose beads (Pierce), extensively washed with RIPA buffer, and incubated at room temperature in 0.1 M triethylamine, pH 11.5 to release bound fiber protein. A portion of the precipitated fiber was electrophoresed on a 8% SDS-PAGE gel under denaturing SDS in loading buffer, samples boiled for 5 minutes) or semi-native SDS in loading buffer, samples not heated) conditions.
As seen in Fig. 13, lines 211A, 211B, and 211 R, but not the control 293 cells, expressed an immunologically reactive protein which migrated at the predicted molecular weight for trimer (186 kD) under seminative conditions and for monomer WO 00/42208 PCT/EPOO/00265 -72- (62 kD) under denaturing conditions. The behavior of the precipitated fiber was indistinguishable from that of purified baculovirus-produced recombinant Ad2 fiber (Wickham T et al., Cell 73:309-319 (1993)) (the 58 kD Ad2 and 62 kD Ad5 fibers have very similar mobilities under these conditions).
To determine whether the fiber-expressing lines could support the growth of a fiber-defective adenovirus, we performed one-step growth experiments using the temperature-sensitive fiber mutant Ad H5ts142 (the gift of Harold Ginsberg). At the restrictive temperature this mutant produces an underglycoslyated fiber protein which is not incorporated into mature virions (Chee-Sheung C. C et al., J.
Virol 42: 932-950 (1982)). This results in the accumulation of non-infectious viral particles. We asked whether the recombinant fiber protein expressed by our cell lines could complement the H5ts142 defect and rescue viral growth.
Cell lines 293, 211A, 211B and 211R (2 x 106 cells/sample) were infected with H5ts142 at 10 pfu/cell. 48 hours later, cells were detached with 25 mM EDTA and virus was harvested by four rapid freeze-thaw cycles. Debris was removed by a minute spin at 1500 x g, and viral titers determined by fluorescent focus assay (Thiel J.F et al., Proc. Soc. Exp. Biol. Med. 125:892-895 (1967)) on SW480 cells with a polyclonal anti-penton base Ab (Wickham T et al., Cell 73:309-3.19 (1993)). As shown in Fig. 14, the fiber mutant virus replicated to high titers in 293 cells at 32.5°C (the permissive temperature), but to a much lower extent at the restrictive temperature of 39.5°C. The fiber-producing packaging lines 211A, 211 B, or 211R supported virus production at 39°C to levels within two- to three-fold of those seen at the permissive temperature in 293 cells, indicating that these cells provided partial complementation of the fiber defect.
Interestingly, virus yields from the fiber-producing cell lines were also somewhat higher than those from 293 cells at 32.5°C (the 'permissive' temperature).
This suggests that fiber produced by the ts142 virus may be partially defective even at the permissive temperature. Alternatively, a non-specific increase in adenoviral titer could result when viruses are grown in the packaging cells, by a mechanism not involving fiber complementation. However, it was found that viruses with wild type fiber genes (such as Ad.RSVpgal) replicate to identical levels either in our packaging lines or in 293 cells (data not shown). Taken together, these results demonstrate that the observed increase in H5ts142 growth is due to specific complementation of the fiber mutation.
Even in the fiber-expressing cell lines, the fiber mutant grows to higher titers at 32°C than at 39.50C. This incomplete complementation may be due to the WO 00/42208 PCT/EP00/00265 -73packaging lines' expression of fiber at a level somewhat below that seen in a wildtype infection (data not shown). A recent study reported an E4-deleted vector which coincidentally reduced fiber protein expression, resulting in a large reduction in the titer of virus produced (Brough et al., J. Virol. 70.6497-6501 (1996)). Another possibility is that the defective ts142 fiber protein produced at the restrictive temperature might form complexes with some of the wild type protein produced by the cells and prevent its assembly into particles.
Although the fiber proteins of different Ad serotypes differ in the length of their shaft domains and in their receptor-binding knob domains, the N-terminal regions responsible for interaction with the viral penton base are highly conserved Arnberg N et Virology 227.239-244 (1997)) (Figure 15A). This suggests that fibers from many viral serotypes, with their different cell-binding specificities, may be amenable for use in producing gene delivery vectors.
In order to determine whether the recombinant Ad5 fiber produced by the packaging cells could be incorporated into particles of another adenovirus serotype, adenovirus type 3 was grown either in fiber-producing cell lines or in 293 cells. Viral particles were purified by two sequential centrifugations (3 h at 111,000 x g) on preformed 15-40% CsCI gradients to remove soluble cellular proteins and then dialyzed extensively against 10 mM Tris-HCI, pH 8.1, 150 mM NaCI, 10% glycerol.
fiber protein was detected by immunoblotting using the polyclonal anti-fiber serum, followed by detection with a horseradish peroxidase-conjugated goat antirabbit antibody (Kirkegaard and Perry Laboratories) and the ECL chemiluminescence substrate (Amersham). The purified Ad3 particles contained fiber protein after a single passage through a fiber-expressing cell line but not after passage through 293 cells (Figure 15B). Previous work has demonstrated that Ad2 fiber is capable of interacting in vitro with Ad3 penton base (Fender et al., Nature Biotech. 15:52-56 (1997)), and our result demonstrates that the type 5 fiber protein produced by the cells is capable of assembling into complete Ad3 particles.
A vector based on Ad5 but containing the gene for the Ad7 fiber protein has been described (Gall J. et al., J. Virol. 70:2116-2123 (1996)), as well as Ads containing chimeric fiber genes (Krasnykh et al., J. Virol. 70:6839-6846 (1996) and Stevenson et al., J. Virol. 69.2850-2857 (1995)). Chimeric Ad5/Ad3 vectors have also been reported (Stevenson, S. et J. Virol. 71:4782-4790, (1997). Addition of a short peptide linker to the fiber in order to confer binding to a different cellular protein has also been reported (Michael et al., Gene Therapy 2:660-668 (1995). By WO 00/42208 PCT/EP00/00265 -74using packaging technology such as that presented here, Ad vectors equipped with different fiber proteins may be produced simply by growth in cells expressing the fiber of interest, without the time-consuming step of generating a new vector genome for each application.
Replacing or modifying the fiber gene in the vector chromosome would also require that the new fiber protein bind a receptor on the surface of the cells it which it is to be grown. The packaging cell approach will allow the generation of Ad particles containing a fiber which can no longer bind to its host cells, by a single round of growth in cells expressing the desired fiber gene. This will greatly expand the repertoire of fiber proteins which can be incorporated into particles, as well as simplifying the process of retargeting gene delivery vectors.
Finally, a novel fiber-independent pathway of infection has recently been described in hematopoietic cells, in which penton base provides the initial virus-cell interaction by binding to integrin amb 2 (Huang S. et al., J. Virol 70:4502-4508 (1996)). This suggests that viral particles lacking fiber protein may be useful in targeting gene delivery to specific cell types via this pathway.
Example Preparation of Alternative TPLs The present invention contemplates the use of tripartite leader sequences (TPLs) that are useful in enhancing the expression of complementing adenoviral proteins, particularly fiber protein, for use in preparing an adenoviral gene delivery vector. One preferred TPL is the complete Ad5 tripartite leader contained in complementing vectors such as pDV67 and pDV69, both of which are prepared as described below. The complete Ad5 TPL was constructed by assembling PCR fragments. First, the third TPL exon (exon 3) (nt 9644-9731 of the Ad5 genome) was amplified from Ad5 genomic DNA using the synthetic oligonucleotide primers 5'CTCAACAATTGTGGATCCGTACTCC3'(SEQ ID NO: 28) and 5'GTGCTCAGCAGATCTTGCGACTGTG3' (SEQ ID NO: 29). The resulting product was cloned to the BamHI and Bglll sites of pAE1Spla (Microbix Biosystems) using novel sites in the primers (shown in bold) to create plasmid pDV52. A fragment corresponding to the first TPL exon (exon the natural first intron (intron and the second TPL exon (exon 2) (Ad5 nt 6049-7182) was then amplified using primers 5'GGCGCGTTCGGATCCACTCTCTTCC3' (SEQ ID NO:30) and CATGCTAGGCAGATCTCGTTCGGAG3' (SEQ ID NO: 31), and cloned into the WO 00/42208 PCT/EP00/00265 BamHI site of pDV52 (again using novel sites in the primers) to create This plasmid contains a 1.2 kb BamHI/Bglll fragment consisting of the first TPL exon, the natural first intron, and the fused second and third TPL exons. The nucleotide sequence of the complete TPL containing the noted 5' and 3' restriction sites is shown in SEQ ID NO: 32 with the following nucleotide regions identified: 1-6 nt BamHI site; 7-47 nt first leader segment (exon 48-1068 nt natural first intron (intron 1069-1140 nt second leader segment (exon 1141-1146 nt fused BamHI and Bglll sites; 1147-1234 nt third leader segment (exon and 1235-1240 nt Bglll site.
TPLs fragments containing two of the three exons, exons in non-native order, or containing either the first or second TPL intron are also constructed for use in preparing complementing plasmids for use in the methods of the present invention.
Briefly, DNA fragments containing any combination of 2 TPL exons can be constructed as follows: Exon 1 is amplified from genomic DNA as prepared above by using the oligonucleotides 5'GGCGCGTTCGGATCCACTCTCTTCC3'(SEQ ID NO: 33) and 5'GGGAGTAGATCTCCCAACAG3' (SEQ ID NO: 34). Exon 2 is similarly amplified from the same genomic DNA using oligonucleotides ITTTTTTTGGATCCCTCGCGG3' (SEQ ID NO: 35) and 5'CTACATGCTAGGCAGATCTCGTTCGGAG3' (SEQ ID NO: 36). Exon 3 is amplified using the oligonucleotides 5'CTCAACAATTGTTGGATCCGTACTCC3' (SEQ ID NO: 37) and 5'GTGCTCAGCAGATCTTGCGACTGTG3' (SEQ ID NO: 38).
The amplified exons are ligated together in any desired number and/or order by virtue of the unique BamHI and Bglll restriction sites (bold) in the primers for subsequent ligation into a construct analogous to pDV67, prepared as described below, for expression of viral structural genes.
Similarly, a fragment consisting of the first TPL exon (exon the native first intron (intron and the second TPL exon (exon 2) is produced by amplification from Ad5 genomic DNA with the oligonucleotide pair ACTCTCTTCC3' (SEQ ID NO: 39) and CGTTCGGAG3' (SEQ ID NO: 40). Finally, a fragment consisting of the second TPL exon (exon the native second intron (intron and the third TPL exon (exon 3) is produced by amplification using the oligonucleotides CTCGCGG3' (SEQ ID NO: 41) and 5'GTGCTCAGCAGATCTTGCGACTGTG3' (SEQ ID NO: 42). Either of the intron-containing fragments are used either alone or in combination with another TPL fragment(s) in constructs analogous to pDV67.
Introns in addition to adenoviral intron 1 used herein that have been shown to WO 00/42208 PCT/EP00/00265 -76increase the expression of recombinant proteins when included in expression constructs include SV40 VP1 intron, rabbit P-globin intron among others. The use of these alternative intron sequences are contemplated for use in preparing a TPL in the present invention.
Example 6 Preparation and Use of Adenoviral Packaging Cell Lines Containing Plasmids Containing Alternative TPLs Plasmids were first constructed as described below that contained TPLs are described above. The resultant plasmids containing different selectable markers such as neomycin or zeocin were then used to prepare stable cell lines for use as complementing vectors for preparing adenoviral vectors for use in the present invention. In a preferred embodiment, the resulting cell lines represent improvements over preexisting fiber-complementing cell lines in that fiber expression is enhanced with the use of alternative TPLs.
A. was constructed by inserting this TPL cassette of SEQ ID NO: 32 into the BamHI site upstream of the Ad5 fiber gene in pcDNA3/Fiber, a neomycin selectable plasmid, prepared as described in Example 1 and also as described by Von Seggern et al., J. Gen Virol, 79: 1461 (1998). The nucleotide sequence of is listed in SEQ ID NO: 43.
B. pDV61 To construct pDV61, an Asp718/Notl fragment containing the CMV promoter, partial Ad5 TPL, wildtype Ad5 fiber gene, and bovine growth hormone terminator was transferred from pCLF, prepared as described in Example 1 and also as described by Von Seggern et al., J. Gen Virol., 79: 1461 (1998), to a zeocin selectable cloning vector referred to as pCDNA3.1/Zeo (commercially available from Invitrogen and the sequence is also available).
C. pDV67 WO 00/42208 PCT/EP00/00265 -77- In an analogous process, pDV67 containing complete TPL was constructed by transferring an Asp 718/Xbal fragment from pDV60 to the pcDNA3.1/Zeo(+) backbone. The nucleotide sequence of pDV67 is listed in SEQ ID NO: 44.
D. pDV69 To prepare pDV69 containing a modified fiber protein, the chimeric fiber gene was amplified from pGEM5TS3H (Stevenson et al., J. Virol., 69: 2850-2857, 1995)) using the primers CAAGATGAAGCGCGCAAGACCG3' (SEQ ID NO: 45) and 5'CACTATAGCGGCCGCATTCTCAGTCATCTT3' (SEQ ID NO: 46), and cloned to the BamHI and Notl sites of pcDNA3.1/Zeo(+) via novel BamHI and Notl sites engineered into the primers to create pDV68. Finally, the complete TPL fragment described above was then added to the unique BamH1 site of pDV68 to create pDV69. The nucleotide sequence of pDV69 is listed in SEQ ID NO: 47.
E. Preparation of Stable Adenovirus Packaging Cell Lines E1-2a S8 cells are derivatives of the A549 lung carcinoma line (ATCC CCL 185) with chromosomal insertions of the plasmids pGRE5-2.E1 (also referred to as GRE5-E1-SV40-Hygro construct and listed in SEQ ID NO: 48) and pMNeoE2a-3.1 (also referred to as MMTV-E2a-SV40-Neo construct and listed in SEQ ID NO: 49), which provide complementation of the adenoviral El and E2a functions, respectively. This line and its derivatives were grown in Richter's modified medium (BioWhitaker) 10% FCS. E1-2a S8 cells were electroporated as previously described (Von Seggern et al., J. Gen Virol., 79: 1461 (1998)) with pDV61, pDV67, or with pDV69, and stable lines were selected with zeocin (600 pg/ml). The cell line generated with pDV61 is designated 601. The cell line generated with pDV67 is designated 633 while that generated with pDV69 is designated 644. Candidate clones were evaluated by immunofluorescent staining with a polyclonal antibody raised against the Ad2 fiber. Lines expressing the highest level of fiber protein were further characterized.
For the S8 cell complementing cell lines, to induce El expression, 0.3 pM of dexamethasone was added to cell cultures 16-24 hours prior to challenge with virus for optimal growth kinetics. For preparing viral plaques, 5 X 10 5 cells/well in 6 well plates are prepared and pre-induced with the same concentration of dexamethasone WO 00/42208 PCT/EPOO/00265 -78the day prior to infection with 0.5 pM included at a final concentration in the agar overlay after infection.
F. Development of Cell Lines for Complementation of E17E2a' Vectors This example shows the construction of S.8 cells The Adenovirus 5 genome was digested with Scal enzyme, separated on an agarose gel, and the 6,095 bp fragment comprising the left end of the virus genome was isolated. The complete Adenovirus 5 genome is registered as Genbank accession #M73260, incorporated herein by reference, and the virus is available from the American Type Culture Collection, Manassas, Virginia, under accession number VR-5. The Scal 6,095 bp fragment was digested further with Clal at bp 917 and Bglll at bp 3,328. The resulting 2,411 bp Clal to Bglll fragment was purified from an agarose gel and ligated into the superlinker shuttle plasmid pSE280 (Invitrogen, San Diego, CA), which was digested with Clal and Bglll, to form pSE280-E. (Figure 23).
Polymerase chain reaction (PCR) was performed to synthesize DNA encoding an Xhol and Sail restriction site contiguous with Adenovirus 5 DNA bp 552 through 924. The primers which were employed were as follows: end, Ad5 bp 552-585: C-3' (SEQ ID NO: 66) 3' end, Ad5 bp 922-891: 5'-CGAGATCGATCACCTCCGGTACAAGGTTTGGCATAG-3' (SEQ ID NO: 67) This amplified DNA fragment (sometimes hereinafter referred to as Fragment A) then was digested with Xhol and Clal, which cleaves at the native Clal site (bp 917), and ligated to the Xhol and Clal sites of pSE280-E, thus reconstituting the end of the El region beginning 8 bp upstream of the ATG codon.
PCR then was performed to amplify Adenovirus 5 DNA from bp 3,323 through 4,090 contiguous with an EcoRI restriction site. The primers which were employed were as follows: end, Ad5 bp 3323-3360: 5'-CATGAAGATCTGGAAGGTGCTGAGGTACGATGAGACC-3' (SEQ ID NO: 68) 3' end, Ad5 bp 4090-4060: WO 00/42208 PCT/EP00/00265 -79- 5'-GCGACTTAAGCAGTCAGCTG-AGACAGCAAGACACTTGCTTGATCCAAATCC-3 '(SEQ ID NO: 69) This amplified DNA fragment (sometimes hereinafter referred to as Fragment B) was digested with Bglll, thereby cutting at the Adenovirus 5 Bglll site (bp 3,382) and EcoRI, and ligated to the Bglll and EcoRI sites of pSE280-AE to reconstruct the complete Ela and Elb region from Adenovirus 5 bp 552 through 4,090. The resulting plasmid is referred to as pSE280-E1 (Figure 23).
A construct containing the intact Ela/b region under the control of the synthetic promoter GRE5 was prepared as follows. The intact Ela/b region was excised from pSE280-E1, which was modified previously to contain a BamHI site 3' to the El gene, by digesting with Xhol and BamHI. The Xhol to BamHI fragment containing the Ela/b fragment was cloned into the unique Xhol and BamHI sites of pGRE5-2/EBV (Figure 4, U.S. Biochemicals, Cleveland, Ohio) to form pGRE5-E1 (Figure 24).
Bacterial transformants containing the final construct were identified. Plasmid DNA was prepared and purified by banding in CsTFA prior to use for transfection of cells.
Construction of plasmid including Adenovirus 5 E2A sequence.
The Adenovirus 5 genome was digested with BamHI and Spel, which cut at bp 21,562 and 27,080, respectively. Fragments were separated on an agarose gel and the 5,518 bp BamHI to Spel fragment was isolated. The 5,518 bp BamHI to Spel fragment was digested further with Smal, which cuts at bp 23,912. The resulting 2,350 bp BamHI to Smal fragment was purified from an agarose gel, and ligated into the superlinker shuttle plasmid pSE280, and digested with BamHI and Smal to form pSE280-E2 BamHI-Smal (Figure 26).
PCR then was performed to amplify Adenovirus 5 DNA from the Smal site at bp 23,912 through 24,730 contiguous with Nhel and EcoRI restriction sites. The primers which were employed were as follows: end, Ad5 bp 24,732-24,708: 5'-CACGAATTCGTCAGCGCTTCTCGTCGCGTCCAAGACCC-3' (SEQ ID NO: 3' end, Ad5 bp 23,912-23,934: 5'-CACCCCGGGGAGGCGGCGGCGACGGGGACGGG-3' (SEQ ID NO: 71) This amplified DNA fragment was digested with Smal and EcoRI, and ligated to the Smal and EcoRI sites of pSE280-E2 Bam-Sma to reconstruct the complete E2a region from Ad5 bp 24,730 through 21,562. The resulting construct is pSE280-E2a.
(Figure 27.) WO 00/42208 PCT/EP00/00265 In order to convert the BamHI site at the 3' end of E2a to a Sail site, the E2a region was excised from pSE280-E2a by cutting with BamHI and Nhel, and recloned into the unique BamHI and Nhel sites of pSE280. (Figure Subsequently, the E2a region was excised from this construction with Nhel and Sail in order to clone into the Nhel and Sail sites of the pMAMneo (Clonetech, Palo Alto, CA) multiple cloning site in a 5' to 3' orientation, respectively. The resulting construct is pMAMneo E2a. (Figure 27).
Bacterial transformants containing the final pMAMneo-E2a were identified.
Plasmid DNA was prepared and purified by banding in CsTFA. Circular plasmid DNA was linearized at the Xmnl site within the ampicillin resistance gene of pMAMneo-E2a, and further purified by the phenol/chloroform extraction and ethanol precipitation prior to use for transfection of cells.
Transfection and selection of cells.
In general, this process involved the sequential introduction, by calcium phosphate precipitation, or other means of DNA delivery, of two plasmid constructions each with a different viral gene, into a single tissue culture cell. The cells were transfected with a first construct and selected for expression of the associated drug resistance gene to establish stable integrants. Individual cell clones were established and assayed for function of the introduced viral gene. Appropriate candidate clones then were transfected with a second construct including a second viral gene and a second selectable marker. Transfected cells then were selected to establish stable integrants of the second construct, and cell clones were established.
Cell clones were assayed for functional expression of both viral genes.
In order to determine the most suitable cell lines for the above-mentioned transfections, sequential transfections and selections were carried out with the following parental cell types: A549 (ATCC Accession No. CCL-185); Hep-2 (ATCC Accession No. CCL-23); or KB (ATCC Accession No. CCL-17).
Appropriate selection conditions were established for both G418 and hygromycin B for all three cell lines by standard kill curve determination.
Transfection of cell lines with plasmids including El and E2a regions.
pMAMNeo-E2a was linearized with Xmnl with the AmpR gene, introduced into cells by transfection, and cells were selected for stable integration of this plasmid by G418 selection until drug resistant colonies arose. The clones were isolated and screened for E2a expression by staining for E2a protein with a polyclonal antiserum, WO 00/42208 PCT/EPOO/00265 -81 and visualizing by immunofluorescence. E2a function was screened by complementation of the temperature-sensitive mutant Ad5ts125 virus which contains a temperature-sensitive mutation in the E2a gene. (Van Der Vliet, et al., J. Virology, Vol. 15, pgs. 348-354 (1975)). Positive clones expressing the E2a gene were identified and used for transfection with the 7 kb EcoRV to Xmnl fragment from pGRE5-E1 (Figure which contains the GRE5 promoted Ela/b region plus the hygromycin R gene. Cells were selected for hygromycin resistance and assayed for Ela/b expression by staining with a monoclonal antibody for the El protein (Oncogene Sciences, Uniondale, El function was assayed by ability to complement an El-deleted vector. At this point, expression and function of E2a was verified as described above, thus establishing the expression of both Ela/b and E2a in the positive cell clones.
One of the transfected A549 cell lines showed good Ela/b and E2a expression and was selected for further characterization. It was designated the S8 cell line.
G. Preparation of Adenoviral Vectors Containing Ad5.pgal.AF Genome in S8 Improved Fiber-Complementing Cell Lines To prepare adenoviral vectors containing Ad5.pgal.AF in S8 cells containing alternative forms of TPL for enhancing the expression of fiber proteins, the protocol as described in Example 2 for preparing Ad5.pgal.AF in 211B cells was followed with the exception of pretreatment with 0.3 pM dexamethasone for 24 hours as described above. Thus, viral particles with the wildtype Ad5 fiber protein on their surface and containing the fiberless Ad5.pgal.AF genome were produced in 633 cells. Particles produced in 644 cells also contained the fiberless genome, but had the chimeric 5T3H fiber protein, with the Ad3 fiber knob, on their surface.
The preparation of the cell lines and demonstration of stable nuclear expression of either wild-type Ad5 fiber protein or chimeric Ad5/Ad3 protein is shown in Figure 20. In the figure, schematic diagrams are presented of the constructs used to generate the cell lines as well as immunofluorescence results indicating the presence of expressed fiber protein in the nucleus of the cells. An indirect immunofluorescence assay of A549 based cell lines which stably express the different Ad fibers is shown. Line 633 expresses the native Ad5 fiber protein and line 644 expresses a chimeric fiber protein with the tail and shaft domains of the WO 00/42208 PCT/EP00/00265 -82protein and the knob domain of the Ad3 fiber. Previous work (Stevenson et al., 1996) showed that a virus containing this protein had the tropism expected for Ad3.
Thus, these viral preparations, prepared as described herein and in Example 2, are useful for targeting delivery of Ad5.pgal.AF fiberless genome with either wildtype or modified fibers, embodiments of which uses have been previously discussed and as further exemplified with the pseudotyping and infectivity results described in Example 7.
Example 7 Pseudotyping and Infectivity of Recombinant Adenoviral Vectors Produced with Improved Fiber-Complementing Cell Lines A. Pseudotyping of To verify that adenoviral vectors were produced had altered tropisms, viral particles were purified from either 633 (expressing wilt type Ad5 fiber) or 644 cells (expressing the chimeric Ad5/Ad3 fiber) 10 .g of the purified particles were Western blotted and probed with a polyclonal rabbit antibody against the Ad2 fiber (which detects both the Ad5 and chimeric 5T3H fiber proteins.). Equal amounts of purified Ad.pgal.wt or Av9LacZ which has the chimeric fiber gene in the viral chromosome) were run as controls. The results are shown in Figure 21 where both fiber proteins were detectable confirming pseudotyping.
B. Infectivity of Cells with 633 or 644 Generated Virus Particles The cell lines, 633 or 644, prepared as described above, were infected with the indicated number of particles/cell of Ad5.pgal.AF and virus particles produced.
Virus was then used to infect, as previously described, selected cell lines as shown in Figure 22, including 211B, MRC-5 human fibroblasts, A-10 rat aortic endothelial cells, and THP-1 human monocytic cells. Unbound virus was removed by washing the cells and the cells were further incubated at 37°C for 48 hours. Cells were then fixed with glutaraldehyde and stained with X-gal. The percentage of stained cells was then determined by light microscopy where all experiments were done in triplicate.
The results shown in Figure 22 indicate that adenoviral vectors could be retargeted by pseudotyping using packaging cell lines expressing different fiber WO 00/42208 PCT/EP00/00265 -83proteins. The data marked with "none" indicates virus grown in 293 cells and lacking fiber, while "Ad5" indicates virus prepared in 633 cells (containing the wild type fiber) and Ad3 indicates virus prepared in 644 cells (containing the chimeric 5T3H fiber.) Particles containing either fiber were equally infectious on 211B cells, while fibroblasts and THP-1 cells were more readily infected by virus containing the chimeric fiber. The A-10 rat endothelial cells were more readily infected by particles containing the wildtype Ad5 fiber protein.
Example 8 Targeted Gene Delivery Using Viral Vector Particles Lacking Fiber Protein An alternative mode of entry for adenoviral infection of hematopoietic cells has been described by Huang, et al., J. Virol., 69:2257-2263 (1995) which does not involve the fiber protein-host cell receptor interaction. As infection of most other cell types does require the presence of fiber protein, vector particles which lack fiber may preferentially infect hematopoietic cells, such as monocytes or macrophages.
To produce a fiber-free adenovirus vector particle, a vector lacking the fiber gene as described above in Example 2A but containing a gene of interest for delivery is amplified by growth in cells which do not produce a fiber protein, such as the 211 cells prepared in Example 1 or 293 or S8 cells as described herein, thereby producing large numbers of particles lacking fiber protein. The recovered fiber-free viral particles are then used to deliver the inserted gene of interest following the methods of this invention via targeting mechanisms provided by other regions of the adenoviral vector, via the native penton base.
A. Construction of an Adenovirus Vector Deleted for El, E3, and Fiber, and Carrying a Therapeutic Gene of Interest A general method of constructing a fiber-deleted Ad vector containing a therapeutic gene of interest (in this example, the Herpes Simplex Virus Thymidine Kinase (TK) gene) is described here. Linear viral DNA is isolated from a preparation of Ad5.pgal.AF particles. This DNA is digested with the restriction enzyme Clal, which removes the leftmost viral sequences including the left ITR, the WO 00/42208 PCT/EP00/00265 -84packaging signals, and part of the SV40-driven p-galactosidase gene. The large Clal fragment with the remainder of the fiber-deleted viral genome is then isolated by centrifugation on a sodium chloride or sucrose gradient. The plasmid pAdShuttleTK, which contains the left part of the Ad chromosome with an RSV-driven TK gene inserted in place of the El region, is linearized by digestion with Notl. The nucleotide sequence of the pAdShuttleTK is shown in SEQ ID NO: 50. The large Clal fragment of Ad5.pgal.AF and the linearized pAdShuttleTK are cotransfected into 211B cells, and an infectious adenovirus genome is generated by homologous recombination. A virus deleted for El, E3, and fiber that contains the TK cassette in the place of the El deletion is thus recovered. A virus containing any desired therapeutic gene of interest can be created in this manner by replacing the TK gene of the example with the gene of interest.
An alternative method of constructing a fiber-deleted genome containing a therapeutic gene (in this example the retinal degeneration-slow (RDS) gene driven by the CMV immediate early promoter) is described here. RDS is a protein expressed in photoreceptors, and essential for their proper development and functioning. RDS mutations have been implicated in retinal degenerative disorders, and transfer of the wildtype RDS gene by means of an Ad vector provides an avenue towards treating such disorders.
A plasmid (pDV50) analogous to pAE1B3gal but containing a CMV-driven RDS gene was constructed as follows. First, a fragment containing the CMV promoter and enhancer was excised from pCHaMIEP by digestion with Hindlll, filling the overhanging ends with the large fragment of E. coli DNA polymerase 1, ligation of BamHI linkers (5'CGCGGATCCCG3' SEQ ID NO: 51) to the blunt ends, and digesting with BamHI. The resulting fragment was then ligated into the BamHI site of pAElspla (Mikrobix) to create pDV45. A fragment containing the polyadenylation signal was amplified from pSVpgal (Promega) using the oligonucleotides 5'CTGACAAACTCAGATCTTGTTTATTG3' (SEQ ID NO: 51) and 5'GTCGACTCTAGAGGATCCAGA3' (SEQ ID NO: 52). This fragment was ligated into the Bglll site of pDV45 to create pDV46, using the unique BamHI and Bglll sites (bold type) in the primers. Finally, the human RDS open reading frame was amplified from the plasmid pRDS-T7 using the oligonucleotides AGATGGCAACCATGGCGCTAC3' (SEQ ID NO: 53) and GGGGAAGCTTGGCCCTCAGCCAGCCTCT3' (SEQ ID NO: 54). This fragment was inserted into the Hindlll and Xbal sites of pDV46, again using unique restriction sites in the primers, to create pDV50. pDV50 therefore contains a cassette WO 00/42208 PCT/EP00/00265 consisting of the CMV promoter, the RDS open reading frame, and the terminator sequences inserted in place of the Ad5 El region.
In a manner analogous to the construction of Ad5.pgal.AF pDV50 and pDV44 are then co-transfected into 211B cells, and an infectious Ad genome is recovered. A fiber-deleted Ad vector containing any desired gene to be expressed can be constructed by replacing the RDS gene of this example with the gene of interest.
Example 9 Transient Transcomplementation Human adenovirus type 5 (Ad5) is being developed as a vector for gene therapy. Its ability to deliver therapeutic genes to cells is mediated by the interaction of the adenoviral fiber protein with the coxsackievirus-adenoviral receptor (CAR).
Because a wide-range of cells express CAR, it can be difficult to use adenoviruses to deliver genes to specific cell types. One way to address this is to target the virus to a particular cell type by genetically altering the fiber. However, the genetic manipulations involved in cloning and production of the viruses with altered fibers can be time-consuming. Thus it would be a significant advancement in the field of adenoviral gene therapy to have a more streamlined system for testing modified fiber genes. An in vitro system has thus been developed that involves infection of tissue culture cells with a fiber-deleted Ad and transient co-transfection with a plasmid directing fiber expression. This system allows one to produce and evaluate such modified fibers in the context of a viral particle easily and quickly. In addition this system can be envisioned to actually produce therapeutic quantities of adenoviral vectors with modified fiber proteins, with such fibers having a new tropism added by insertion of a desired ligand into the fiber gene. These fibers may also have the natural tropism binding to CAR) ablated.
Plasmids used were pDV60 and pDV55, prepared as described herein.
is an pcDNA3.1-based expression plasmid that contains the CMV promoter, tripartite leader, an intron, and the Ad5 fiber gene sequence. pDV55 contains no fiber gene and serves as the negative control. Ad5.pgal.AF and 211B are described above. 293T cells are identical to 293 cells except they express an integrated SV40 large T antigen gene. HDF cells are human diploid fibroblasts.
293T cells express CAR and a, integrins; HDF cells express av integrins but no CAR. Transfections with fiber expression plasmids were performed with WO 00/42208 PCT/EP00/00265 -86- Lipofectamine (GIBCO-BRL) using 20mg DNA and 50ml Lipofectamine per dish. Cells were maintained in DMEM supplemented with 10% fetal bovine serum.
The fiber deletion mutation of Ad5.pgal.AF is complemented in trans by passaging virions through 211B, a cell line that stably expresses functional fiber. The present system was designed to complement Ad5.pgal.AF by modified fibers expressed from transfected episomal plasmids in 293T cells. The result is a simplified and rapid method to incorporate modified fibers on a viral particle containing the Ad5.pgal.AF genome that does not require propagation of the virus.
The feasibility of transcomplementation of Ad5.pgal.AF with episomal fiber-expressing plasmids was demonstrated in the following experiment. 293T cells were transfected with one of two plasmids: pDV55, which expresses no fiber or which expresses wildtype Ad5 fiber. Fiber expression persists for at least six days, suggesting that the plasmid is stable as an episome for this amount of time Twenty-four hours after transfection, these cells were infected at 2000 particles/cell with Ad5.pgal.AF passaged through 211B cells. Seventy-two hours later, a crude viral lysate (CVL) was generated by exposing the cells to five freeze-thaw cycles.
Viral particles were purified by cesium chloride gradient centrifugation. The resulting virions incorporated the fiber expressed from the episomal plasmid, as confirmed by Western blots performed with an antibody specific to the Ad5 fiber.
To demonstrate the functionality of these virions, the transduction efficiency was tested. The virions containing no fiber (pDV55) or wildtype fiber (pDV60) were applied to monolayers of 293T and HDF cells at different multiplicity of infection (MOl's). 293T cells express CAR and a a,'integrins; HDF cells express om integrins but no CAR. After 2 days, the cells were fixed and stained with X-gal to detect the Pgalactosidase reporter gene activity. The results showed low transduction efficiency for the pDV55-complemented virions in both cell lines. As expected, the virions transduced 293T cells to a high degree but did not transduce HDF cells, indicating that functional fiber proteins had been expressed from the episomal plasmids and incorporated into the virions. This transduction efficiency was comparable to or better than that of Ad5.pgal.AF virions passaged through the 211B cells.
Episomal plasmid transcomplementation system is suitable for quickly expressing and evaluating the properties of modified fibers in the context of a viral particle. Episomal plasmid transcomplementation will also be of great utility for quickly evaluating a bank of modified fibers for other binding properties, including novel tropism and the ablation of the native tropism. In addition to the rapid WO 00/42208 PCT/EP00/00265 -87generation and testing of large numbers of modified fibers, there are other advantages to the Ad5.pgal.AF transcomplementation system in terms of production and safety. Episomal plasmid transcomplementation has the inherent advantage over transcomplementation in that it is not necessary to make a stable cell line for every modified fiber with which you want to complement Ad5.pgal.AF Because the is deleted in El, E3 and fiber, there is an additional gene deletion compared to other first generation vectors. This makes Ad5.pgal.AF more replication defective and presumably safer. In addition, the presence of the fiber gene deletion decreases the opportunity to generate replication-competent virus via recombination in the packaging cells. In terms of production a single Ad vector prep could be retargeted to any number of different cell types simply by transfecting the cells with the appropriate fiber-expression construct.
Example Adenoviral Gene Delivery Vectors Containing the Ad37 Fiber Protein Adenovirus type 37 (subgroup D) has been associated with infections of the eye and genital tract, and may be useful for targeting these tissues or other mucous membranes, as well as other cell types. The tropism of Ad37 is due to the binding preference of its fiber protein, which binds to an as yet-unidentified receptor located on the surface of cells including Chang C, conjunctival epithelial cell line (Huang et al., J. Virology 73(4):2798-2802 (1999)). As this fiber directs viral infection to cell types different than those infected by Ad5, it is likely to provide a method for targeting gene delivery. This example describes construction of packaging cell lines expressing the Ad37 fiber protein, and their use in generating particles of a fiberdeleted Ad vector (such as Ad5.pgal.AF containing this fiber protein. The fiber protein is attached to the viral capsid by binding to the penton base protein through its N-terminus, and the Ad37 fiber was modified in order to make its N-terminal sequence more closely match that of the Ad5 protein to ensure that it would efficiently bind the Ad5 penton base in these vectors.
1. Construction of an Expression Plasmid for the Ad37 Fiber Protein (pDV8O) WO 00/42208 PCT/EP00/00265 -88- This plasmid uses the same regulatory elements as contained in pDV67, and pDV69 to express the Ad37 fiber in packaging lines, and was constructed in two steps. First, the Ad37 fiber open reading frame was amplified from Ad37 genomic DNA (obtained from the ATCC accession number VR- 929)using the synthetic oligonucleotides primers L37 TGT CTT GGA TCC AAG ATG AAG CGC GCC CGC CCC AGC GAA GAT GAC TTC (SEQ ID NO: 56) and 37FR AAA CAC GGC GGC CGC TCT TTC ATT CTT G (SEQ ID NO: 57). L37 contains nucleotides that differ from the Ad37 genomic sequence in order to add an unique Barn H1 site (bold in the above sequence) and create point mutations to make the N-terminal sequence of the fiber more closely match that of the Ad6 protein (underlined in the above sequence; the start codon is italicized). 37FR incorporates changes to create a unique Not 1 site (bold). The PCR product was inserted into the Bam H1 and Not 1 sites of pCDNA3.1zeo(+) (Invitrogen) to create pDV78. The correct sequence of the Ad37 fiber gene, including the predicted changes, was confirmed by sequencing.
Second, a 1.2 kb Bam H1/Bgl II fragment containing an adenovirus type tripartite leader was excised from pDV55 (DVS 1999) and inserted into the Bam H1 site of pDV78 to create pDV80 (SEQ ID NO:64) 2. Isolation of Cell Lines Expressing the Ad37 Fiber Protein DNA was purified using the Qiagen method and electroporated into the adenovirus-complementing cell line E1-2a S8 (Gorziglia et al., J. Virology 70(5):4173-4178 (1996)) as previously described (Von Seggern, et al., J. Gen. Virol.
79:1461-1418), and stable clones were selected with 600 pg/ml zeocin (Invitrogen).
Clones were expanded and screened for fiber expression by indirect immunofluorescence using a rabbit polyclonal antibody directed against the Ad37 fiber. Two clones (lines 705 and 731) that expressed the protein at a uniformly high level were selected for further study.
3. Production of Pseudotyped Ad Vector Particles To generate vector particles equipped ('pseudotyped') with the Ad37 fiber protein, the Ad37 fiber-expressing 705 cells were infected (approximately 1000 particles/cell) with Ad5.pgal.AF or with WO 00/42208 PCTIEPOO/00265 -89is prepared as previously described. Ad5.GFP.AF was constructed by recombination in bacteria using a modification of the method of (He, et al., PNAS 95:2509-2514 (1998)). First, a fiber-deleted genomic plasmid was constructed by removing the fiber gene from pAdEasyl (He, et al., PNAS 95:2509- 2514 (1998)). pDV43 (Von Seggern, et al., J. Virol. 73:1601-1608 (1999)) was digested with Pac 1, the ends blunted by treatment with the large fragment of E. coli DNA polymerase and dNTPs, and the product re-ligated. The resulting plasmid, pDV76, is identical to pDV43 except for loss of the Pac 1 site and contains the right end of the Ad5 genome with E3 and fiber deletions. A 4.2 kb fragment was amplified from pDV76 using the oligonucleotides primers 5' CGC GCT GAC TCT TA GGA CTA GTT TC 3' (SEQ ID NO: 58) (including the unique Spe 1 site in the genome, bold) and 5' GCG CTT AAT TAA CAT CAT CAA TAA TAT ACC TTA TTT T 3' (SEQ ID NO: 59) (including a novel Pac 1 site (bold) adjacent to the right ITR). This PCR fragment therefore contains nucleotides 27,082 to 35,935 of the genome with a deletion of nucleotides 28133 to 32743 (the E3 and fiber genes), and was used to replace the corresponding Spe 1/Pac 1 fragment of PAdEasyl to create pDV77.
E. coli strain BJ5183 was electroporated with a mixture of pDV77 and Pme 1linearized pAdTrack as described (He et al., 1998), and DNA was isolated from kanamycin-resistant colonies. The resulting plasmid, pDV83, contains a complete El-, E3-, and fiber-deleted Ad5 genome with a CMV-driven GFP reporter gene inserted at the site of the El deletion. The full-length Ad chromosome was isolated by Pac 1 digestion, and transfected to the El- and fiber-complementing 633 cells (Von Seggern et al., J. Virol January 2000). The recovered virus was then plaque purified by plating on 633 cells and stocks were prepared.
particles were generated by virus growth in 633 cells, which express the wild type Ad5 fiber protein. Viral particles were isolated and purified over CsCI gradients as previously described (Von Seggern et al., J. Virol 73:1601- 1608, 1999). For analysis of viral proteins, ten pg of the purified particles were electrophoresed on 8-16% gradient gels and the protein transferred to nylon membranes. The blot was then probed with rabbit polyclonal antibodies raised against recombinant Ad37 fiber or Ad5 fiber or penton base proteins expressed in baculovirus-infected cells (Figure 27).
Example 11 WO 00/42208 PCT/EP00/00265 Construction of a Fiber Expression Construct Containing a Post- Transcriptional Regulatory Element Previous studies have shown that mRNA transcribed from the woodchuck hepatitis virus (WHV) genome contains an element (the WHV post-transcriptional regulatory element, or WPRE) which can increase expression of a protein encoded by the mRNA via a post-transcriptional mechanism (Loeb et al., Human Gene Therapy 10:2295-2305 (1999)). The WPRE has also been shown to enhance expression of transgenes delivered by retroviral vectors. (Zufferey, R. et al., J. Virol.
73:2886-2892 (1999)). This example describes the construction of a fiber expression construct (pDV90) containing a WPRE as well as the promoter and TPL sequences as contained in pDV67.
A plasmid (pBS/WPRE) which contains the WPRE was obtained from Dr.
Thomas Hope, Salk Institute. Digestion of pBS/WPRE with C/a1 releases a 600 bp fragment containing the WPRE (nt 193-1684 of the WHV genome.) Following C/a1 digestion, the ends of this fragment were filled by treatment with the large fragment of E. coli DNA polymerase 1 in the presence of dNTPs to render them blunt. pDV67 DNA was digested with Xbal (which cuts at a unique site in the transcribed region downstream of the Ad5 fiber open reading frame) and the ends filled by the same treatment. The filled WPRE fragment was then ligated into the filled Xba 1 site of pDV67 to create pDV90 (SEQ ID NO: 65). The sequence is found at GenBank accession no. J04514 (entire genome) in Zufferey, R. et al., J. Virol. 73:2886-2892 (1999).
was electroporated into E1-2a S8 cells and stable clones expressing fiber isolated as described previously for Example 12 Construction of an Ad5 Fiber Protein with Heterologous Peptide Sequences Inserted in the HI Loop The receptor-binding knob domain of the Ad5 fiber protein contains several surface loops which are attractive candidates for the insertion of heterologous peptide sequence, as an additional ligand for vector targeting. This example describes the construction of a fiber gene which encodes a fiber protein containing a 6 amino acid peptide linker in the HI loop, and retains the ability to trimerize. The WO 00/42208 PCT/EP00/00265 -91 modified gene also contains a unique novel restriction site at the position of the linker insertion to facilitate addition of the targeting ligand into the HI loop.
The Ad5 fiber gene was amplified from Ad5 genomic DNA (ATCC accession number VR-5) using the primers Fiber ATG TGA AGC GCG CAA GAC CGT CTG AAG (SEQ ID NO: 60) and Fiber TAA CAT AAC ACT GCA GAT TCT TTA TTC TTG G (SEQ ID NO: 61), and cloned to the Ndel (filled with the large fragment of E. coli DNA polymerase 1 in the presence of dNTPs) and Pst 1 sites of pT7-7 using a unique Pst 1 site (bold) in the 'Fiber TAA' oligo. The resulting plasmid, pT7/fiber, was digested with Xba 1 and Pst 1 to excise the fiber gene, which was then cloned into the Pst 1 and Xba 1 sites of pUC119 to create pUC/fiber. This pUC-derived plasmid contains an origin for single-stranded DNA replication and can therefore be used to create template DNA for site-directed mutagenesis.
Site-directed mutagenesis was carried out according to the method of Kunkel Kunkel, PNAS 82:488-492 (1985)) using the oligonucleotide primer T542 GGT ACA CAG GAA ACA GGA GGT TCC GGA GGT GGA GGA GAC ACA ACT CC (SEQ ID NO: 62). This results in the addition of 18 new bases (underlined) encoding the sequence Gly Gly Ser Gly Gly Gly (SEQ ID NO: 63), with a novel BspE1 site (bold) for the addition of further sequences. The inserted sequence is between Thr542 and Gly543 of the Ad5 fiber protein, in the HI loop. The modified plasmid is termed pDV14.
Finally, the modified fiber gene was excised from pDV14 by digestion with Pst 1 and Xba 1 and cloned into the Pst 1 and Xba 1 sites of pGEM3Z (Promega) to create pDV18. In vitro transcription/translation experiments with pDV18 (using the
TNT
T kit, Promega) demonstrated that the modified fiber gene encoded a protein which was capable of trimerizing.
Alternatively an Ad5 fiber open reading frame (ORF) is amplified from genomic DNA (wildtype Ad5 was purchased from the ATCC) using the oligonucleotides 5' ATG GGA TCC AAG ATG AAG CGC GCA AGA CCG 3' (SEQ ID NO: 72) and 5' CAT AAC CTG CAG GAT TCT TTA TTC TTG GGC 3' (SEQ ID NO:,73) and inserted into the BamHI and Pst 1 sites of pGEM-3Zf(+) (Promega Inc., Madison, WI) via novel restriction sites (bold type) designed into the primers. The oligonucleotide also contains a G to A change 3 nucleotides 5' of the initial ATG codon (underlined), designed to improve the consensus for translation initiation.
Site-directed mutagenesis is performed by the method of Kunkel (Proc. Nat.
Acad. Sci. 82:488-492 (1985)), using the synthetic oligonucleotide 5' GGT ACA CAG GAA ACA GGA GGT TCC GGA GGT GGA GGA GAC ACA ACT CC 3' ((SEQ ID WO 00/42208 PCT/EP00/00265 -92- NO: 74). This operation introduced sequence (bold type) encoding 6 novel amino acids (Gly Gly Ser Gly Gly Gly) immediately following Threonine 542 of the fiber, and including a unique restriction site for the insertion of further heterologous sequences (underlined). The resulting plasmid (pDV18A) contains the modified fiber gene under the control of the T7 promoter in the parental pGEM-3Zf(+) and can be used for in vitro transcription/translation reactions to produce labeled fiber protein.
Example 13 Use of the Fiber Expression System to Retarget ('Pseudotype') Hybrid Ad/AAV Vectors Adenoviral vectors which lack essentially all Ad genes ('helper-dependent' or 'gutless' vectors) have recently been developed. In a modification of this idea, vectors ('hybrid' vectors) which contain an adeno-associated virus (AAV) or retroviral genome have been generated. As AAV and retroviral genomes integrate into the chromosome of the target cells, the hybrid Ad/AAV or Ad/retroviral vectors have the potential to provide very long-term gene expression.
Lieber et al., Virol. 73(11):9314-9324) describe an Ad vector (Ad.AAV1) which contains an AAV vector genome (a transgene insert flanked by the AAV inverted terminal repeats) inserted into the El region. When 293 cells are infected by Ad.AAV1, recombination between the AAV sequences generates a minimal Ad chromosome which carries the Ad inverted terminal repeats and packaging signal flanking the AAV vector genome. This chromosome cannot direct the synthesis of Ad proteins, but can be packaged into Ad vector particles. The remaining unrecombined Ad chromosomes provide the Ad structural proteins in trans, and both the full-length and minimal genomes are packaged into particles. The particles carrying the minimal Ad/AAV hybrid vector are then isolated by CsCI centrifugation.
These particles have the capsid structure of adenovirus, and infect cells using the efficient fiber- and penton base-mediated pathway used by Ad. Following infection, the hybrid genome is able to integrate into the cell's chromosomes by virtue of its AAV sequences. In this example, the AAV vector genome is inserted into the El region of a fiber-deleted vector, and the resulting vector is grown in packaging lines expressing either the Ad5 or Ad37 fiber proteins. The particles recovered therefore have the tropisms expected from the respective fiber proteins combined with the ability to integrate their AAV genome into target cells. Such WO 00/42208 PCT/EP00/00265 -93pseudotyping should be possible with any of a number of modified fiber proteins, as for the fiber-deleted vectors already described by us.
The Ad vector is constructed in a manner analogous to that described for by recombination between pAd.AAV1 (Lieber et al. J. Virol. 73.9314- 9324, 1999) and pDV44 (as described earlier in the specification.) pAd.AAV1 carries an MLV promoter-driven secreted alkaline phosphatase gene (SEAP) as a reporter, and an SV40-driven neomycin phosphotransferase (neo) gene to allow the selection of cells stable transduced by the AAV cassette. The resulting vector (Ad.AAV1.DF) has the AAV vector cassette of Ad.AAV1 inserted into the El region of a genome with the fiber deletion of Ad5.pgal.AF Growth of Ad.AAV1.AF in 633 cells results in particles carrying the AAV genome and the Ad5 fiber, and which have the tropism associated with Ad5. Growth of Ad.AAV1.0F in 705 cells produces particles bearing the Ad37 fiber and therefore having its associated different tropism.
Tropism is evaluated by infecting Chang C cells (which express the Ad37 receptor) and A549 cells which do not express this protein but do express the receptor (CAR). The extent of infection is monitored by assaying alkaline phosphatase expression, and the fraction of cells stable transduced is assayed by selection with neomycin. By using purified recombinant Ad5 or Ad37 fiber proteins as competitors during infection, the usage of the expected receptors by the pseudotyped particles is evaluated.
Example 14 Use of the Fiber Expression System to Retarget ('Pseudotype') Helper-dependent Ad Vectors Gutted Ad vectors are those from which most or all viral genes have been deleted. They are grown by co-infection of the producing cells with a "helper" virus (such as using an El-deleted Ad vector). The helper virus trans-complements the missing Ad functions, including production of the viral structural proteins needed for particle assembly. In one embodiment of this invention, the helper virus is a fiberdeleted Ad (such as that described in Von Seggern et al., J. Virol. 73:1601-1608 (1999)). The vector is prepared in a fiber expressing cell line such as has been previously described by Von Seggern et al., J. Gen. Virol. 79:1461-1468 (1998), Von Seggern et al., J. Virol. 74:354-362 (2000). All the necessary Ad proteins except fiber are provided by the fiber-deleted helper virus, and the particles are equipped with the particular fiber expressed by the host cells. A concern with gutted vectors WO 00/42208 PCT/EP00/00265 -94has been contamination of a vector preparation with residual helper virus. As the helper virus in one aspect of this invention is deleted for both El and for fiber, it is more replication defective and therefore safer than those currently used.
A helper adenovirus vector genome and a gutless adenoviral vector genome are delivered to the packaging cells of the invention. The cells are maintained under standard cell maintenance or growth conditions, whereby the helper vector genome and the packaging cell together provide the complementing proteins for the packaging of the adenoviral vector particle. Such gutless adenoviral vector particles are recovered by standard techniques. The helper vector genome may be delivered in the form of a plasmid or similar construct by standard transfection techniques, or it may be delivered through infection by a viral particle containing the genome. Such viral particle is commonly called a helper virus. Similarly, the gutless adenoviral vector genome may be delivered to the cell by transfection or viral infection.
The helper virus genome is preferably the fiberless adenovirus vector genome as disclosed herein. Preferably, such genome also lacks the genes encoding the adenovirus E1A and E1B proteins. More preferably, the genome further lacks the adenovirus genes encoding the adenovirus E3 proteins. Alternatively, the genes encoding such proteins may be present but mutated so that they do not encode functional E1A, E1B and E3 proteins. Furthermore, such vector genome may not encode other functional early proteins, such as E2A, E2B, and E4 proteins.
Alternatively, the genes encoding such other early proteins may be present but mutated so that they do not encode functional proteins.
The helper virus genome is used in conjunction with the packaging cell of the invention. As disclosed elsewhere herein, the packaging cell also provides proteins necessary for the complementation of the gutless vector so that an adenovirus particle containing the gutless vector genome may be produced. Thus, the packaging cell can provide wild-type or modified fiber protein as described herein.
Alternatively, the cell could package a fiberless particle which could be used by itself or to which exogenously provided fiber could be added as described elsewhere herein.
In producing the gutless vectors, the helper virus genome is also packaged, thereby producing helper virus. In order the minimize the amount of helper virus produced and maximize the amount of gutless vector particles produced, it is preferable to delete or otherwise modify the packaging sequence in the helper virus genome, so that packaging of the genome is prevented or limited. Since the gutless vector genome will have a packaging sequence, it will be preferentially packaged.
WO 00/42208 PCT/EP00/00265 One way to do this is to mutate the packaging sequence by deleting one or more of the nucleotides comprising the sequence or otherwise mutating the sequence to inactivate or hamper the packaging function. An alternative approach is to engineer the helper genome so that recombinase target sites flank the packaging sequence and to provide a recombinase in the packaging cell. The action of recombinase on such sites results in the removal of the packaging sequence from the helper virus genome. Preferably, the recombinase is provided by a nucleotide sequence in the packaging cell that encodes the recombinase. Most preferably, such sequence is stably integrated into the genome of the packaging cell. Various kinds of recombinase are known by those skilled in the art. The preferred recombinase is Cre recombinase, which operates on so-called lox sites, which are engineered on either side of the packaging sequence as discussed above. Further information about the use of Cre-loxP recombination is found in U.S. Pat. No. 5,919,676 and Morsy and Caskey, Molecular Medicine Today, Jan. 1999, pgs. 18-24, both incorporated herein by reference.
This example demonstrates how the fiber-expressing packaging lines can be used to generate pseudotyped particles of helper-dependent or 'gutless' vectors with altered tropisms. As the gutless vectors lack many or all Ad genes, they must be grown as mixed cultures in the presence of a helper virus which can provide the missing functions. To date, such helper viruses have provided all Ad functions except El, and El is complemented by growth in 293 cells or the equivalent. The resulting virus particles are harvested, and the helper virus is typically removed by CsCI gradient centrifugation (the vector chromosome is generally shorter than the helper chromosome, resulting in a difference in buoyant density between the two particles).
An example of a gutless vector is pAdARSVDys (Haecker et aL, Human Gene Therapy 7:1907-1914 (1996)). This plasmid contains a full-length human dystrophin cDNA driven by the RSV promoter and flanked by Ad inverted terminal repeats and packaging signals. 293 cells are infected with a first-generation Ad which serves as a helper virus, and then transfected with purified pAdARSVDys DNA. Both the helper Ad genome and the pAdARSVDys DNA are replicated as Ad chromosomes, and packaged into particles using the viral proteins produced by the helper virus.
Particles are isolated and the pAdARSVDys-containing particles separated from the helper by virtue of their smaller genome size and therefore different density on CsCI gradients.
WO 00/42208 PCT/EPOO/00265 -96 To generate pseudotyped particles containing the pAdARSVDys genome, the vector is grown in either 633 or 705 cells and Ad5.Pgal.AF is used as a helper virus.
As in the published method, both the Ad5.pgal.AF and AdARSVDys genomes replicate and are packaged into particles. The Ad5.pgal.AF helper provides all the essential Ad proteins except fiber, and the fiber protein is that produced by the cells fiber in 633 cells and Ad37 fiber in the case of 705 cells). The particles containing AdARSVDys genomes are then isolated by centrifugation.
Tropism is evaluated by infecting Chang C cells (which express the Ad37 receptor) and A549 cells which do not express this protein but do express the natural receptor (CAR). The extent of infection is assessed by immunofluorescence staining of the infected cells with an anti-dystrophin antibody. By using purified recombinant Ad5 and Ad37 fiber proteins as competitors during infection, the usage of the expected receptors by the pseudotyped particles is evaluated.
Example Targeting EBV-Infected B Cells There are a number of cell types, such as EBV-transformed B-lymphocytes, that are involved in human disease which are not transducible using standard Ad vectors. To address this problem 'pseudotyped' Ad5.Bgal.AF particles containing either the wildtype Ad5 fiber protein or a chimeric fiber with the receptor-binding knob domain of the adenovirus type 3 (Ad3) fiber were generated. (Von Seggem et al., J. Virol. January, 2000). The strategy used for targeting the B-cells should be -broadly applicable for targeting gene delivery to other specific cell types.
Cells and Viruses. THP-1, MRC-5, FaDu, and A-10 cells were purchased from the ATCC. 211B is a 293-derived cell line that expresses the wild-type fiber protein (Von Seggem et al., J. Gen. Virol. 79:1461-1468 (1998)). El- 2a (Gorziglia et al., J. Virol. 70:4173-4178 (1996)) is an A549-derived cell line which complements adenoviral El and E2a functions. The JR, TO, and TL LCL lines were established as described (Huang et al., Proc. Natl. Acad. Sci. 94:8156-8161 (1997) WO 00/42208 PCT/EP00/00265 -97by EBV infection of lymphocytes from three normal donors. THP-1 and all LCL lines were maintained in RPMI 1640 medium (Gibco) 10% fetal calf serum (FCS) (Hyclone). 211B, MRC-5, and A-10 cells were grown in DMEM 10% FCS. E1-2a and its derivatives were grown in Richter's modified medium (BioWhitaker) FCS. Peripheral blood mononuclear cells were isolated from normal human blood (General Clinical Research Center, Scripps Clinic) by sedimentation on Ficoll-Paque (Pharmacia) per the manufacturer's instructions. Wild type Ad2 and Ad3 were purchased from the ATCC. Construction of Ad5.pgal.wt and Ad5.pgal.AF (Von Seggern et al., J. Virol. 73:1601-1608 (1999)) has been previously described.
AvlLacZ4 (Mitttereder et al., J. Virol. 70:7498-7509 (1996)) is a first-generation vector containing an RSV-driven p-galactosidase reporter gene. Av9LacZ4 (Stevenson et al., J. Virol. 71:4782-4790 (1997)) is identical to Av1LacZ4 except that the fiber gene in the vector chromosome was replaced by a recombinant gene encoding a chimeric fiber protein with the receptor-binding domain of the Ad3 fiber (Stevenson et al., J. virol. 69:2850-2857 (1995)). Accession numbers for the above are as follows. THP-1: TIB-202, MRC-5: CCL-171, FaDu: HTB-43, A-10: CRL-1476, Ad2: VR-846, Ad3: VR-3.
DNA constructs. The complete Ad5 tripartite leader contained in pDV67 and pDV69 was constructed by assembly of PCR fragments. pDV55 was constructed similar to Example 5. This plasmid contains a 1.2 kb Bam HI/Bgl II fragment consisting of the first TPL exon, the natural first intron, and the fused second and third TPL exons. Finally, pDV60 was constructed by inserting this TPL cassette into the Barn HI site upstream of the Ad5 fiber gene in pcDNA3/Fiber (Von Seggern et al., J. Gen. Virol. 79:1461-1468 (1998)). pDV61 and pDV67 were then constructed similar to example 6.
The chimeric Ad3/Ad5 fiber gene was amplified from pGEM5T3H (Stevenson et al., J.Virol. 69:2850-2857 (1995) using the primers 5' ATG GGA TCC AAG ATG AAG CGC GCA AGA CCG 3'(SEQ ID NO: 75) and 5' CAC TAT AGC GGC CGC ATT CTC AGT CAT CTT 3' (SEQ ID NO:76) and cloned to the Barn HI and Not I sites of pcDNA3.1/Zeo(+) via novel Bam HI and Not I sites (bold) engineered into the primers to create pDV68. Finally, the complete TPL fragment described above was then added to the unique Bam HI site of this plasmid to create pDV69.
Construction of Stable Cell Lines. E1-2a cells were electroporated as previously described (Von Seggern et al., J. Gen. Virol. 79:1461-1468 (1998)) with pDV61, pDV67, or pDV69, and stable lines were selected with 600 pg/ml Zeocin (Invitrogen). Candidate clones were evaluated by immunofluorescence (Von WO 00/42208 PCT/EP00/00265 -98- Seggern et al., J. Gen. Virol. 79:1461-1468 (1998)) using a polyclonal antibody generated against the Ad2 fiber (Wickham et al., Cell 73.309-319 (1993). Those lines expressing the highest level of nuclear fiber expression were further characterized. Line 601 and 633 were produced by transfection of pDV61 and pDV67, respectively, and therefore express the wildtype Ad5 fiber. Line 644 contains pDV69 and expresses the chimeric 5T3H fiber.
Virus Growth and Analysis. Adenovirus stocks were prepared in the indicated cell lines, and plaque-titered on 633 cells essentially as described (Von Seggern et al., J. Virol. 73:1601-1608 (1999)). E1-2a cells (Gorziglia et al., J. Virol. 70:4173- 4178 (1996). and their derivatives contain a dexamethasone-inducible construct for complementation of Ela. 601, 633, or 644 cells were therefore treated with 0.3 pM dexamethasone for 24 hours prior to infection, and 0.5 pM dexamethasone was included in the overlay for plaque assays. Protein concentration of viral preparations was determined using the BioRad Protein Assay (BioRad) with purified bovine serum albumin as a standard. Particle number was calculated using the formula 1 pg protein 4 x 10 9 viral particles. Western blotting was performed as described (Von Seggern et al., J. Gen. Virol. 79:1461-1468 (1998)) using polyclonal rabbit antibodies raised against either the Ad2 (Wickham et al., Cell 73:309-319 (1993) or Ad3 fibers (Stevenson et al., J.Virol. 71:4782-4790 (1997).
Determination of infection and binding to receptor was performed using methods known to those of skill in the art. 2 x 10 5 cells in a total volume of 200 pl were incubated with the indicated Ad preparation for three hours at 37 Cells were then washed twice with fresh medium, and returned to 37 Two days later, cells were fixed and stained with X-gal and counted by light microscopy as described (Von Seggern et al., J. Virol. 73:1601-1608 (1999)). For competition assays, cells were pre-incubated on ice for one hour with either recombinant Ad3 fiber (10 pg/ml) purified from baculovirus or with a crude baculovirus lysate (100 pg/ml) containing the recombinant Ad2 fiber protein (Wickham et al., Cell 73:309-319 (1997)).
Expression of oa integrins on cell surfaces was assayed by FACS assay using monoclonal antibodies (the gift of David Cheresh, TSRI) against either avPs (LM609) or ocvs (P1F6) as previously described (Huang et al., Proc. Natl. Acad. Sci USA 94:8156-8161 (1997)). For virus binding assays, CsCI-purified Ad2 or Ad3 was labeled with 1251 using lodogen tubes (Pierce). Free iodine was removed by filtration with a PD-10 Sephadex column (Pharmacia). Cells (1 x 106 cells in a volume of 200 pl either with or without a 100-fold excess of unlabeled virus) were rocked at 4 °C for WO 00/42208 PCT/EP00/00265 -99two hours with 1 x 106 cpm of the labeled virus, washed three times with PBS and counted.
Altered in vitro tropism and infection of B lymphoid cell lines.
Experiments with genetically modified viruses showed that a number of different cell types are more readily infected through interaction with the Ad3 receptor than by the CAR-dependent pathway used by Ad5 (Stevenson et al., J.
Virol. 71:4782-4790 (1997)). In order to further evaluate the pseudotyping system, the ability of Ad5.pgal.AF carrying either the Ad5 or chimeric 5T3H fibers to infect several cell lines was assayed: FaDu (a head and neck tumor line), THP-1 monocytic cells, and MRC-5 fibroblasts were assayed. Consistent with the previous studies (Stevenson et al., J. Virol. 71:4782-4790 (1997)), use of the chimeric Ad5/Ad3 fiber protein increased infection of all of these lines at equal particle/cell ratios. In contrast, the rat smooth muscle cell line A-10 was infected somewhat more readily by Ad5- than by Ad3-pseudotyped particles.
Gene delivery to EBV-infected B cells could allow the development of therapies for a variety of lymphoproliferative disorders. For example, ex vivo purging of donor marrow to eliminate infected cells could reduce the risk of EBV-associated lymphoproliferative disease, and EBV-induced malignancies such as AIDSassociated lymphoma are also potential targets. However, neither B cells nor EBVtransformed lymphoblastoid cell lines (LCLs) are efficiently infected by vectors. As the tropism of Ad3-pseudotyped particles appeared to be somewhat broader, it was asked whether EBV-infected LCLs could be infected using this system. The ability of Ad3-pseudotyped particles to infect LCLs generated by EBV infection of lymphocytes from three different normal human donors was tested. In agreement with previous reports, there was little or no infection of these by particles carrying the Ad5 fiber. In contrast, virus particles equipped with the chimeric fiber protein were able to efficiently infect all of these lines. At equal particle/cell ratios, all LCLs examined were at least 10-fold more infectible using the Ad3 receptor.
Further studies were performed to correlate the efficiency of infection with the level of attachment and internalization receptors expressed by the cells. The three LCL lines tested all bound very low levels of radiolabeled Ad2 particles, indicating that they expressed little or no CAR. In contrast, all three were able to specifically bind labeled Ad3 particles. This result suggested that fiber receptor distribution was largely responsible for the increased infection of these cells by Ad3-pseudotyped particles. Selective gene delivery to EBV-infected cells. The results above WO 00/42208 PCT/EP00/00265 -100suggested that the minority of EBV-infected B cells present in donor marrow or peripheral blood would be preferentially infected by vectors using the Ad3 receptor.
To test this hypothesis, a mixing experiment with normal uninfected peripheral blood mononuclear cells (PBMCs) and EBV-infected cells was performed. JR-LCL cells were mixed at varying ratios with PBMCs isolated from a normal human donor, and the mixture was then infected with Ad5.pgal.AF particles containing the 5T3H fiber protein. No infection of normal PBMCs alone was detected. Moreover, the percent of total cells infected increased with the fraction of JR cells added. These experiments indicate that EBV-infected cells can be selectively infected in vitro by relatively short (3 hours) exposure to a retargeted Ad vector.
Example 16 Production of Adenovirus Vectors by Addition of Exogenous Fiber The production of fiberless viruses by growth in a complementing cell line may result in a preparation that also contains contaminating fiber genome resulting from recombination in the complementing cell lines. This disadvantage is eliminated by addition of exogenous fiber to a fiberless adenovirus vector.
Production of fiberless virus by standard methods may include a two-step preparation protocol. This has been described in the earlier examples and is briefly described here again as follows: Step I amplification of fiber containing fiberless virus (Ad5/FV/F or fiberless, but there is fiber on the surface, not encoded in genome) on 211B cell line (which stably expresses fiber), followed by CsCI-purification and characterization.
Step II preparation of virus particles lacking fiber (Ad5F) by infection of S.8 cell line with Ad5/F/F', followed by CsCI purification and characterization. This produces a large stock of particles which do not contain fiber.
Step 1 is necessary because the infection efficiency of fiberless virus is extremely low, e.g. the dose of 20,000 particles/cell of Ad5/pg F gives only infected cells.
Contrary to the above, the production of fiberless virus by addition of exogenous fiber involves only a one-step protocol. The fiberless virus is amplified using the S.8 cell line with addition of exogenous fiber into infection media. The WO 00/42208 PCT/EP00/00265 -101 amount of exogenous fiber necessary for production is very low, no more than of purified fiber required per roller bottle. If desired the process may be followed by CsCI purification. As mentioned above, one advantage to this protocol is that it should provide no chance for recombination of adenovector during preparation.
A 10 roller bottle (RB) preparation of fiberless virus was made using the above two-step procedure. The yield of adenovector was 6.6x10 12 particles total Ad/pgalF'. A 1 RB preparation of fiberless adenovector was also made from the same initial material using a one-step procedure with exogenous fiber. The total yield was 2.5x1011 particles Ad5pgalF (one step procedure).
DNA was isolated from both preparations and a PCR assay for fiber contamination was performed. (Figure 28). The PCR assay was developed for detection of very low amounts of fiber contamination, as low as 10- 8 g. PCR assay showed much lower contamination for the preparation which was done by adding exogenous fiber (1015g one-step procedure) vs. 10- 8 two-step procedure).
Therefore, less contamination was obtained by simpler one-step approach.
Experiments were done using soluble purified fiber which does not have Histaq on the end (Ad5Fiber 5F) and with His-taq on the end (Ad5Fiber His These experiments showed that addition of Ad5Fiber can dramatically increase transduction efficiency of fiberless adenovector by simply adding it exogenously to a fiberless vector. The presence of the His tag on the Ad5FiberHis doesn't have any effect.
The results of these experiments suggest that the fiber is self-assembling with the fiberless vector. This self-assembled virus can then infect cell through the normal entry pathway. (Figure 29) Also, an experiment was done using conditioned media from 633 cell line, which can stably express fiber. A Western blot analysis for 633 condition media, showed that soluble fiber was present in the media during the period of cultivation of this cell line. Presence of soluble fiber in the media gives the possibility to increase transduction efficiency of fiberless adenovector on the HDF cell line. (Figure 30) Because the HDF cell line doesn't have a CAR-receptor, it is especially difficult to transduce this particular cell line, not only with fiberless vector, but also with regular fiber containing adenovector. Different amounts of 633 conditioned media (250pl, 500pl or 1000pl) were added to infectious media during the incubation period with fiberless adenovector.
P:\OPER\Kbn 24372-00 rml.doc-06/02/04 102- This experiment also showed a role of soluble fiber in the process of cell entry.
The conclusion is that by adding any fiber (wild-type, mutated, with ligand fusions) as long as one has the wild-type shaft (or region necessary to bind penton) one can retarget fiberless vector with any genome inside (gutless, oncolytic, expressing any transgene, etc.) to any cell type that your fiber is specific to. The advantage of this approach is that one does not have to make vectors with each new ligand. Just one fiberless vector need be made that can then be used to make different backbones by adding an exogenous "targetable" fiber off the shelf.
The foregoing specification, including the specific embodiments and examples, is intended to be illustrative of the present invention and is not to be taken as limiting.
Numerous other variations and modifications can be effected without departing from the true spirit and scope of the present invention. All publications, patents and patent applications cited herein are incorporated by reference in their entirety into the present disclosure.
"The reference to any prior art in this specification is not, and should not be taken S.as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and eo1 "comprising", will be understood to imply the inclusion of a stated integer or step or group S. of integers or steps but not the exclusion of any other integer or step or group of integers or oo 25 steps.
o o.
EDITORIAL NOTE APPLICATION NUMBER 24372/00 The following Sequence Listing pages 1 to 59 are part of the description. The claims pages follow on pages "103" to "109".
WO 00/42208 PCT/EP00/00265 -1- SEQUENCE LISTING <110> Novartis Ag The SCRIPPS RESEARCH INSTITUTE <120> ADENOVIRUS VECTORS, PACKAGING CELL LINES, COMPOSITIONS, AND METHODS FOR PREPARATION AND USE <130> 1294.0010001 <140> <141> <160> 76 <170> PatentIn Ver. 2.1 <210> 1 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 1 cggtacacag aattcaggag acacaactcc <210> 2 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 2 gcctggatcc gggaagttac gtaacgtggg aaaac <210> 3 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: linker <400> 3 cgcggatccg cg 12 <210> 4 <211> 8710 <212> DNA <213> Artificial Sequence WO 00/42208 WO 0042208PCT/EPOO/00265 -2- <220> <223> Description of Artificial Sequence: plasmid <400> 4 cacctaaatt ctcatttttt cgagataggg ctccaacgtc accctaatca gagcccccga gaaaqcgaaa caccacaccc gcgcaactgt agggggatgt ttgtaaaacg gccccccct c caagtgcata tatttgccac ttatgtttca agtatagccc accctagtat cqgctggcct cacacggttt tcacttaagt tqcttaacgg atcaggatag gtcctgcagg ataagqcgcc taactqcagc aagctcatg aagtggcgac ttcaccacct ctaaaccagc caatgacagt atgttggcac gttagaacca ggaagacctc ggatgatcct ctgtacggac acgccggacc gcgtctccgj caaagcatcc cctgataacz cgagtcacac aaagattatc cgtggtcaa cttccaaaac qaatctcctc accttctcai gct ccagag( ttcctcaca( taggtccctl cacttcccci agctatgct gcaaggtgc, catgctcat, ttctctcaa ttaaacatt tacggccat cagctcctc catcggtca gagacaaca gtaagcgtta aaccaatagg ttgagtgttg aaagggcgaa agttttttgg tttagagctt ggagcgggcg gccgcgctta tgggaagggc gctgcaaggc acggccagtg gaggtcgacg ctctatgtca atcctcttac acgtgtttat caccaccaCa tcaacctgcc taaaaagcat cctgtcgagc tcatgtcgct gcggcgaagq ggcggtggtg aatacaacat ttgtcctccg acaqcaccac cggggaccac ccctcataaa cccggtacca tggccaaaac ggagagccca aacacagqca tatcccagq *gcacgtaact ccagtatggt tgcgccgagz Itagtcatatt Itctcgccgct aggcgcccc( itccaccacc( -acgggagga( -caaaacctc ictctacagc( j gcaaacggc( tataaacatl -gccctccaci Sacctgtata, Scgcagggcc.
Sccaggaacc' a accaqcgta t gctcaaaaa g cagataaag a catgtctgc a gaagcctgt g ccggcgtga g gtcatgtcc g tgctaaaaa t tacagcccc atattttgtt ccgaaatcgg ttccagtttg aaaccgtcta gqtcgaggtg gacggggaaa ctagggcgct atgcgccgct gatcggtgcg qattaagttg aattgtaata gtatcgataa ttttcatggg actttttcat ttttcaattg tagcttatac acctccctcc catatcatgg caaacgctca gtccagctgc aqaagtccac ctgcagcagc ggcagtqgtc qgcacaqcag aatattgttc agaacccacg cacgctggac tataaacctc ctgcccgCcg ggactcgtaa *cacgtgcata aacaacccat *cacgttgtgc agcgcgqgtt icaaccgagat 1tcctg~agca tagatcgctc tggcttcggq caqaataagc I cgggaagagc a aaatgaagat -aaagaacagE ctcacgtccE ccagcacctt a agcaaatC :ttcagcctce a qattcaaaaC.
a gctgaacatU t tgacaaaagi g ccccgatgti a tcagqcaaa( g caggtaagcl g ggtttctgc c ttacaacagi c cgtaaaaaa, g gagtcataa g cgaccgaaa .c ataggaggt aaaattcgcg caaaatccct gaacaagagt tcagqgcgat ccgtaaagca gccggcgaac ggcaagtgta acagggcgcg ggcct cttcg ggt aacgcca cgactcacta gcttgatatc actggtctgg acattgccca cagaaaattt aqat caccgt caacacacag gtaacagaca tcagtgatat tgagccacag gcctacatgg qcgcgaataa tcctcagcga cgcaccctga aaaatcccac tggccatcat ataaacatta tgattaaaca gctatacact ccatggatca cacttcctca tcctgaatca attgtcaaag tctgtctcaa cgtgttggtc aaaccagqtg tgtgtagtag ttctatgtaa cacacccagc tggaagaacc ctattaagtc itaatggcatt iagtggacgta caaccatgcc gaatattaac iagcagcgaat j cggaacattz a atcgtgcag a acccacactc a agctttgtt(.
j cctcgcgcai t ccggaacca( ai taaacacaai g aaaaacaaci a ctggtcacc, t gtaagactci t agcccgggg' a taacaaaat ttaaattttt tataaatcaa ccactattaa ggcccactac ctaaatcgga gtggcgagaa gcggtcacgc tcccattcgc ctattacgcc gggttttccc tagggcgaat gaattcagga ccacaactac agaataaaga caagt'zattt accttaatca agtacacagt tattcttagg taataaactc gctgctgtc gqgtagagtc actgctgcq tgattcgcac tctcacttaa agtgcaaggc accacaagcq cctcttttgg tggcgccatc gcagqgaac tcatqctcgt ggattacaag gcgtaaatcc tgttacattc aagqaggtag gtagtgtcat cgggcgtgac ttgtagtata actccttcat caacctacac atgttttttt aacgcgctcc tgtaagatgt kaaggctaaac caaataattc tccggccatt catgattgca iacaaaaatac ;tctgcacgg2 ;attatgacac Scatggqcggc a aaaagaaagc :cacagaaaaz a ataaaataac =cttataagcz g tgattaaaai g gtaaacacal 9 aatacatac( t aataggaga( gttaaatcag aagaatagac 120 agaacgtgga 180 gtgaaccatc 240 accctaaaqg 300 aggaagggaa 360 tgcgcgtaac 420 cattcaggct 480 agctggcgaa 540 agtcacqacg 600 tgggtaccgg 660 gacacaactc 720 attaatgaaa 780 atcgtttgtg 840 ttcattcagt 900 aactcacaqa 960 cctttctccc 1020 tgttatattc 1080 cccgggcagc 1140 aacttqcggt 1200 ataatcqtgc 1260 ccqccgctcc 1320 cgcccgcagc 1380 atcagcacag 1440 gctgtatcca 1500 caqgtagatt 1560 catqttgtaa 1620 caccaccato 1680 gggactggaa 1740 catgatatca 1800 ctcctcccgc 1860 cacactqcaq 1920 gggcagcagc 1980 acgatcccta 2040 gccaaatqqa 2100 aaacagatct 2160 tccactctct 2220 gcgccgctgc 2280 attcgttctg 2340 ttttattcca 2400 cctccggtgg 2460 tgcacaatgg 2520 ccttcaqggt 2580 tcatctcgcc 2640 gtaaaaatct 2700 aaaattcagg 2760 cgcgatcccg 2820 iccagcgcggc 2880 qcatactcgg 2940 gatataaaat 3000 acatcgtagt 3060 igacaccattt 3120_ -aaaaaaacat 3180 itaaqacggac 3240 xgcaccaccga 3300 :caggttgatt 3360 -cgcaggcgta 3420 1 aaaaacacat 3480 WO 00/42208 WO 0042208PCTIEPOO/00265 aaacacctga acagcgcttc aaaaaacacc gcagagcgag ccagaaaacc caaatcgtca tctccgcaag ccggcttcca cagacaaggt gcggcataaa gcgagcgatc tgcaacgcgg cagcctcgcg ttcatcccc~g catgtcttta aacacgcaga cgtgtggcct ccgcaqatcc agtttctgat aatctcgtgc qcgccgatgg cgattccgga qccgtgcaca agccqgtcgc tcggcccatt cqattgctga ccgtcgcgca acct cgtgca cgqtcattga tcttctggag atccggagct aactctatca gcgacgcaat gcgcqgccgt ccagcactcg aggagacaat acgggtgttg qataccccac cacccccaa gccataca tcqtggqggt cagacccatc ccgggcgtct cgcccagtgc gccctgacgj gagctgcatc taatcagccE cctgaacctc taatggttac gcattctagi gttctagag( atttcgagcl acaattcca( gtgagctaa4 tcgtgccagi cgctcttcci gtatcagct, aagaacatg gcgtttttc aggtggcga gtgcgctct ggaagcgtg aaaaccctcc acagcggcag actcgacacg tatatatagg gcacgcgaac cttccgtttt aattgattgg ttcaggtcga atagggcggc tcgccgtgac cttgaagctg gcatcccgat tcgcgaacgc tggcccgttg gttctatgat tg'cagtcggg cgaacaccga cgggcaatga cgaaaagttc tttcagcttc tttctacaaa agtqcttgac gggtgtcacg ggaggccatg cggaccgcaa t cccc atgt g ggctctcgat cgcggatttc ctggagcgag gccgt ggttg tgcaggatcg gagcttggtt cgtccgatcc ctggaccgat tccgagggcz accggaaggz ggtcgtttgt *cgagaccccE *gttcgggtgz *ctggccccgt tattatttt( fgtttttggal 1 gtggctgcci ,cgtcgaccg( Igcttgtctgi jtgtcagaggl itaccacatti ;aaacataaa, -aaataaagc -tgtggtttg -ggccgccac :tggcgtaat acaacatac tcacattaa tgcattaat g cttcctcgc c actcaaagg t gagcaaaag ataggctcc a acccgacag ctgttccga g cgctttctc tgcctaggca cctaacagtc gcaccagctc actaaaaaat ctacgcccag cccacgttac ctccaattct ggtggcccgg gcctacaatc gatcagcggt tccctgatgg gccgccggaa cagcaagacg ctcgcgtttg gacacaaacc gcggcgcggt gcgaccctgc gatatgaaaa gacagcgtct gatgtaggag gatcgttatg attggggaat ttgcaagacc gatgcgatcg ggaatcggtc tatcactggc gagctgatgc ggctccaaca gcgatgttcg gcttgtatg Iccgcggctcc qacggcaatt ggagccggga ggctgtqtag iaaggaatagg tacccgcgcta tcataaacgc Ittggggccaa iaggcccagg -gggttagggz I ggcgttqcgt :ggcctgggcz a aacaccccc'( a tcatggctg( :tcccggcat( t gtagaggttl a tqaatgCaal a atagcatca( t ccaaactcal gcggtggag, atggtcata, g agccggaag, t tgcgttgcg g aatcggcca t cactgactc c ggtaatacg g9 ccagcaaaa g ccccCtga Ig actataaag .c cctgccgct :a tagctcacg aaatagcacc agccttacca aatcagtcac gacgtaacgg aaaCgaaagc gtaacttccc tggagtggtg ctccatgcac catgccaacc ccagtgatcg tcgtcatcta gcgagaagaa tagcccagcg ctggcggtgt ccgcccagcg cccaggtcca agcgacccgc agcctgaact ccqacctqat ggcgtggata tttatcggca tcagcgagag tgcctqaaac ctgcggccga aatacactac aaactgtqat tttgggccga atgtcctgac gqgattccca agcagcagac ggqcgtatat tcgatgatgc ctqtcgggCg aagtactcgc qgagatggg tgacggcaat ggggttcggt tacgcccgcg Ictcgcagcca Lcqgggtcccc -gggqtctggt itggaccgcat ;accccaaaa gccccgacac cgcttacaqz atcaccgaaE tact tgcttt tqttgttqtt aaatttcacz 1caatgtatct tccagctttt ataaagtgti tcaCtgccc( a cgcgcggggi g ctgCgCtCgi g ttatccacal g gccagqaac, gagcatcac a taccaggcg t accggataC c tgtaggtat ctccgctc gtaaaaaaga agtgtaaaaa ttaaagtcca caaaaaaccc ggatccgcgg aatccgttag cgcgacgcaa cgttccatgt aagttaggct cctqcctgga tcataatggg cgtcggCCgC ccccggaaqa tcttgtcatt cttcgcatat ttaacagcgt caccgcgacg gcagctctcg tgtcctgcgg ctttgcatcg cctqacctat cgaactqccc tcttagccag atggcgtgat ggacgacacc ggactgcccc ggacaatggc atacgaggtc gcgctacttc gctccgcatt agcttggqcg tacacaaatc cgatagtgga gaggctaact aaaaagacag cccagggctg tttcttcctt *acgtcggggc catggggaat *ccacgactgq *gtactggcqc accaccgcgc ccgccaacac tcaagctgtga icgcgcgaggc aaaaaacctc aacttgttta iaataaagcat :tatcatqtct :gcttcccttta I tgtgaaattg a aagcctgggg j ctttccagtc a gaggcggttt g tcgttcggct g aatcagggga gtaaaaaggc aaaatcgacc t ttccccctgi c tgtccgcctt tcagttcggt agaacaacat 3540 aaacctatta 3600 agggccaagt 3660 caaaaaacac 3720 acaacttcct 3780 cattcacagt 3840 cgaggtgccg 3900 cgcggggagg 3960 gctcgccgag 4020 ggtaagagcc 4080 cagcatggcc 4140 gaaggccatc 4200 catgccctgc 4260 aatatatttg 4320 ggcgaattcg 4380 taaggtgaCg 4440 caacagcgtg 4500 tctgtcgaga 4560 gagggcgaag 4620 gtaaatagct 4680 gccgcgctcc 4740 tgcatctccc 4800 gctgttctgc 4860 acgagcgggt 4920 ttcatatgcg 4980 gtcagtgcgt 5040 gaaqtccqgc 5100 cgcataacaq 5160 gccaacatct 5220 gagcggaggc 5280 ggtcttgacc 5340 cagggtcgat 5400 gcccgcagaa 5460 aaccgacgcc 5520 gaaacacqga 5580 aataaaacgc 5640 gcactctqtc 5700 ttccccaccc 5760 ggcaggccct 5820 ggtttatggt 5880 actgaqcaga 5940 qacacgaaca 6000 ggatttctgg 6060 ccgctgacgc 6120 ccqtctccgg 6180 aqccqgatca 6240 cccacctccc 6300 ttgcagctta 6360 ttttttcact 6420 ggatccacta 6480 gtgagggtta 6540 ttatccgctc 6600 tgcctaatga 6660 gggaaacctg 6720 qcgtattggg 6780 gcggcgagcg 6840 taacgcagga 6900 cgcgttgctg 6960 ictcaagtcag 7020 1aagctccctc 7080 tctcccttcg 7140 gtaggtcgtt 7200 WO 00/42208 WO 0042208PCT/EPOO/00265 cgctccaagc ggtaactatc actggtaaca tggcctaact gttaccttcg ggtggttttt cctttgatct ttggtcatga tttaaatcaa agtgaggcac gtcgtgtaga ccgcgagacc gccgagcgca cgggaagct a acaggcatcg cgatcaaggc cctccgatcg ctgcataatt tcaaccaagt atacgggata tcttcgqggc actcgtgcac aaaacaggaa ctcatactct ggatacatat cgaaaagtgc tgggctgtgt gtcttgagtc ggattagcag acggctacac gaaaaagagt ttgtttgcaa tttctacggg gattatcaaa tctaaagtat ctatctcagc taactacgat cacgctcacc gaagtggtcc gagtaagtag tggtgtcacg gagttacatg ttgtcagaag ctcttactgt cattctgaga ataccgcgcc gaaaactctc ccaactgatc ggcaaaatgc tcctttttca ttgaatgtat gcacgaaccc caacccggta agcgaggtat tagaaggaca.
tggtagctct gcagcagatt gtctgacgct aaggatcttc atatgagtaa gatctgtcta acgggagggc ggctccagat tqcaacttta ttcgccagtt ctcgtcgttt atcccccatg taagttggcc catqccatcc atagtgtatg acatagcaga aaggatctta ttcagcatct cgcaaaaaag atattattga ttagaaaaat cccgttcagc agacacgact gtaggcggtg gtatttggta tgatccggca acgcgcagaa cagtggaacg acctagatcc acttggtctg tttcgttcat ttaccatctg ttatcagcaa tccgcctcca aatagtttgc ggtatggctt ttgtgcaaaa gcagtgttat gtaagatgct cggcgaccga actttaaaag ccgctgttga tttactttca ggaataaggq agcatttatc aaacaaatag ccgaccgctg tatcgccact ctacagagtt tctgcgctCt aacaaaccac aaaaaggatc aaaactcacg ttttaaatta acagttacca ccatagttgc gccccagtgc taaaccaqcc tccagtctat gcaacgttgt cattcagctc aagcggttag cactcatggt tttctgtgac gttgctcttg tgctcatcat gatccaqttc ccagcqtttc cgacacggaa aggttattg gggttccgcg cgccttatcc 7260 ggcagcagcc 7320 cttqaagtgg 7380 gctgaagcca 7440 cgctgqtagc 7500 tcaagaagat 7560 ttaagggatt 7620 aaaatgaagt 7680 atgcttaatc 7740 ctgactcccc 7800 tgcaatgata 7860 agccggaagg 7920 taattgttgc 7980 tgccattgct 8040 cggttcccaa 8100 ctccttcggt 8160 tatggcagca 8220 tggtgagtac 8280 cccggcgtca 8340 tggaaaacgt 8400 gatgtaaccc 8460 igqgtgagca .8520 atgttgaata 8580 tctcatgagc 8640 cacatttccc 8700 8710 <210> <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 5 atgggatcca agatgaaqcg cgcaagaccg <210> 6 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequenlce: primer <400> 6 cataacgcgg ccgcttcttt attcttgggc <210> 7 <211> 7148 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: plasmid <400> 7 gacggatcgq gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg WO 00/42208 WO 0042208PCT/EPOO/00265 ccgcatagtt cgagcaaaat ttagggttag gattattgac tggagttccg cccgcccatt attgacgtca atcatatgcc atgcccagta tcgctattac actcacgggg aaaatcaacg gtagqcgtgt ctgcttactg gagctcggat ccatatgaca cccaatgggt gttacctcca ggcaacctta aacataaacc gccgccgcac accgtgcacg aaqctagccc actgcctcac atttatacac gacctaaaca caaactaaag gcaggaggac tttgatgctc gcccacaact tccaaaaagc atagccatta cccctcaaaa aaactaggaa aatgataagc gagaaagatg gtttcagttt catcttatta qaatattgqa ggatttatgc attgtcagtc ctaaacggta tgggactqgt tcatacattg ctatagtgtc cagccatctq actgtccttt attctggggq catgctggq agggggtatc cgcagcgtg2 tcctttctcc ggqttccgat tcacgtagtc ttctttaatz tcttttgatt taacaaaaat ccccaggct( aggtgtggai tagtcagcai tccgcccatl gcctctgccl aagccagtat ttaagctaca gcgttttgcg taqttattaa cgttacataa gacgtcaata atggqtggac aagtacgccc catgacctta catggtgatg atttzcaagt ggactttcca acggtgggag gcttatcgaa ccaagatgaa cggaaaccgg ttcaagagag atggcatgct cctcccaaaa tqgaaatatc ctctaatggt actccaaact tgcaaacatc cccctctaac aaaatgqaaa ctttgaccgt ttactgqagc taaggattga aaaaccaact tggatattaa ttgaggttaa atqcaggaga caaaaattgg ctggccttag taactttgtg ctaaactcac tggctgttaa taagatttga actttagaaa ctaacctatc aagtttactt cacaggaaac ctqgccacaa cccaagaata acctaaatgc ttgtttgCCC cctaataaaa Igtggggtgqgc Iatgcggtgg cccacgcgcc iccgctacact Iccacgttcqc ttaqtgcttt Iqgccatcgcc igtggactctt tataaqggat :ttaacgcgai cccaggcag( i agtccccagC iccatagtcc( -ctccgcccci ctgagctatl ctgctccctg acaaggcaag ctgcttcgCg tagtaatcaa cttacggtaa atgacgtatg tatttacggt cctattgacg tgggactttc cggttttggc ctccacccca aaatgtcgta gtct a tat aa attaatacga gcgcgcaaga tcctccaact tccccctggg tgcgctcaaa tgtaaccact tgcaccctc cgcgggcaac tagcattqcc aggcccctc tactgccact actaggacta agcaactgqt cttgggtttt ttctcaaaac aaatctaaga ctacaacaaa cctaagcact tgggcttgaa ccatggccta ttttgacagc gaccacacca tttgqtctta aggcagtttg cgaaaatgga tggagatctt agcttatcca aaacggagac aggagacaca ctacattaat aagaagcggc tagagctcgc ctcccccgtg tgaggaaatt fgcaggacagc Ictctatggct ctgtagcggc tgccagcgcc cgqctttccc acggcacctc ctgatagacg gttccaaact tttggggatt j cagaagtatc, j ctccccagca -gcccctaact a tggctgactE t ccagaagtac cttgtgtgtt gcttgaccga atgtacgggc t ta cgqggt c atggcccgcc ttcccatagt aaactgcca tcaatgacgg ctacttggca agtacatcaa ttgacgtcaa acaactccgc gcagagctct ctcactatag ccgtctgaag qtgccttttc gtactctctt atgggcaacg gtgagcccac acagttacct acactcacca acccaaggac accaccaccg ggtagcttgg aagtacqgggg ccaggtgtga gattcacaag agacgcctta ctaggacagg ggcctttact gccaaggggt tttggttcac gaatttgatt acaggtgcca gctccatctc acaaaatgtg gctccaatat gtgctactaa a ctgaaggca aaatctcacg aaaactaaac actccaagtg gaaatatttg cgctcgagca tgatcagcct ccttccttga gcatcgcatt aagggggagg tctgaggcgg gcattaagcg ctagcgccq cgtcaagctc gaccccaaaa *gtttttcgcc ggaacaacac tcggcctatt *ggaatgtgtg fcaaagcatgc ggcagaagta ccgcccatcc katttttttta Itgaggaggct ggaggtcgct caa ttgcatg cagatatacg attagttcat tggctgaccg aacgccaata cttggcagta taaatggccc gtacatctac tgggcgtgga tgggagtttg cccattgacg ctggctaact ggagacccaa ataccttcaa ttactcctcc tgcgcctatc gcctctctct ctctcaaaaa cagaagccct tgcaatcaca ccctcacagt ataqcagtac gcattgactt ctcctttqca ctattaataa gcaatatqca tacttgatgt gccctctttt tgtttacagc tgatgtttga ctaatgcacc caaacaaggc ttacagtagq ctaactgtag gcagtcaaat ctggaacagt acaattcctt cagcctatac gtaaaactgc ctgtaacact catactctat ccacatcctc tgcatctaga cgactgtgcc ccctggaagg gtctgagtag attgggaaga aaagaaccag cggcgggtgt ctcctttcgc taaatcgggg aacttgatta ctttgacgtt tcaaccctat ggttaaaaaa tcagttaggg atctcaatta tgcaaagcat cgcccctaac tttatgcaga tttttggagg gagtagtgcg 120 aagaatctgc 180 cgttgacatt 240 agcccatata 300 cccaacgacc 360 gggactttcc 420 catcaagtgt 480 gcctggcatt 540 gtattagtca 600 tagcggtttg 660 ttttggcacc 720 caaatgggcg 780 agagaaccca 840 gcttggtacc 900 ccccgtgtat 960 ctttgtatcc 1020 cgaacctcta 1080 ggacgaggcc 1140 aaccaagtca 1200 aactgtggct 1260 gqccccgcta 1320 gtcagaagga 1380 ccttactatc 1440 gaaaqagccc 1500 tqtaacagac 1560 tacttccttq 1620 acttaatgta 1680 tagttatccg 1740 tataaactca 1800 ttcaaacaat 1860 cqctacagcc 1920 aaacacaaat 1980 tatggttcct 2040 aaacaaaaat 2100 actaaatgca 2160 acttgctaca 2220 tcaaagtgct 2280 cctggaccca 2340 aaacgctgtt 2400 caaaagtaac 2460 aaccattaca 2520 gtcattttca 2580 ttacactttt 2640 gggccctatt 2700 ttctagttgc 2760 tgccactccc 2820 gtgtcattct 2880 caatagcagg 2940 ctggggctct 3000 ggtggttacg 3060* tttcttccct 3120 catcccttta 3180 gggtgatggt 3240 ggagtccacg 3300 ctcggtctat 3360 tgagctgatt 3420 tgtggaaagt 3480' gtcagcaacc 3540 gcatctcaat 3600 tccgcccagt 3660 ggccgaggcc 3720 cctaggcttt 3780 WO 00/42208 PCT/EPOO/00265 tgcaaaaagc ggatcgtttc gagaggctat ttccggctgt ctgaatqaac tgcgcagctg gtgCCggggC gctgatgcaa gcgaaacatc gatctggacg cgcatgcccg atggtggaaa cgctatcagq gctgaccgct tatcgccttd cgacgcccaa gcttcggaat tggagttctt atagcatcac ccaaactcat cgtaatcatq acatacgagc cattaattgc attaatgaat cctcgctcac caaaggcggt caaaaggcca qgctccgCCC cgacaggact ttccgaccct tttctcaatc gctgtgtgc ttgagtccaz ttagcagaqc gctacactaC, aaagagttg( tttgcaaqc ctacggggt( tatcaaaaal aaagtatat, tctcagcga ctacgatac gctcaccgg gtggtcctg taagtagtt tgtcacgct ttacatgat tcagaagta ttactgtca tctgagaat ccgcgccac aactctcaa actgatctt aaaatgccc tttttcaat aatgtattt ctgacgtc tcccgggagc gcatgattga tcggctatga cagcgcaggg tgcaggacga tgctcgacgt aggatctcct tgcggcggCt gcatcgagcg aagagcatca acggcgagga atggccgctt acatagcgtt tcctcqtgct ttgacgagtt cctgccatca cgttttCCgg cgcccacccc aaatttcaca caatgtatct gtcatagctg cgqaagcata gttgcgctca cggccaacgc tgactcqctg aatacggtta g caaaaggcc ccctgacgag *ataaagatac g ccgcttacc ctcacgctgt icgaacccccc icccggtaagE -gaggtatgtz Saaggacagt SgcagattacC.
-tqacgctcac, 9 gatcttcac( a tqaqtaaacl t ctgtctattl g ggagggctt c tccagattt, c aactttatc gccagttaa gtcqtttgq ccccatgtt a gttggccgc t gccatccgt a gtqtatgcg a tagcagaac q gatcttaCc :c agcatcttt ;c aaaaaaggg :a gaaaaataa ttgtatatcc acaagatgga ctgggcacaa gcgcccgqtt ggcagcgcgg tgtcactgaa gtcatctcac gcatacgctt agcacgtaCt ggggctcgcg tctcgtcgtg ttctggattc ggctacccgt ttacggtatc cttctgagcq cgagatttcg gacgccggct aacttgttta aataaagcat tatcatqtct tttcctgtgt aagtgtaaag ctqcccgctt qcggggagag cqctcggtog tccacagaat aggaaccgta catcacaaaa caggcgtttc ggatacctgt aggtatctcZ gttcagcccci cacgacttat ggcggtgCtE tttggtatct Itccggcaaa( j cgcagaaaai j tggaaogaai tagatccttl :tggtctgaci Scgttcatcc, a ccatctggc' a tcagcaata c gcctccatc t agtttgcgC t atggcttca g tqcaaaaaa a gtgttatca a agatgcttt g cgaccqagt t ttaaaagtg g ctgttgaga t actttcacc a ataagqggcc rc atttatcac La caaatagc.
attttcggat ttgcacgcag caqacaatcg ctttttgtca ctatcgtggc gcgggaaggg cttgctCCtg gatccggcta cggatggaag ccagccgaac acccatggcg atcgactqtg gatattgctq gccgctcccg ggactctgqg attccaccgc ggatgatcct ttgcagctta ttttttcact gtataccgtc gaaattqtta cctggggtgC tccagtcggg gcggtttgcg ttcggctgcg cagggqataa aaaaggccgc atcgacgctc cccctggaag ccgcctttct Lgttcggtgta jaccqctgcgc :cgccactggc icaqagttctt gcgctctgct aaaccaccgc a aaggatCtCE a actcacgttE :taaattaaaE agttaccaatc atagttgcctc c ccagtgctgc a accagccag( c aqtctattai a acgttgttg( t tcagctccgc g cggttagctl c tcatggttal t ctgtgactg, t gctcttgCC c tcatcattg t ccagttcga a gcgtttctg [a cacggaaat Ig gttattgtC Ig ttccgcgca ctgatcaaga gttCtCCggC gctgctctga agaccgacct tqgccacqac actggctgct ccgagaaagt cctgcccatt ccggtcttgt tgttcgccag atgcctgctt gccggctggg aagagcttgg attcgcagcg gttcgaaatg cgccttctat ccagcgcggg taatqgttac gcattctagt gacctctaqc tccgctcaca ctaatqagtg aaacctgtcg tattgggcgc gcgagcggta cgcaqgaaag gttgctqgg aagtcagagg ctccctcgtg cccttcggga ggtcqttcgc cttatccgqt agcaoccact g aaqtqgtgg *gaagccagtt tggtagcggt iagaagatcct kagggattttc iatgaatttt cttaatcagt actccccgtC aatgatacc! cggaagggcC cattgctac jttcccaacgi cttcggtCCi t ggcagcactC g tgaqtactc c ggcgtcaati g aaaacgttc' t gtaacccac g gtgagoaaa g ttgaatact t catgagcgg c atttccccg gacaggatga 3840 cgcttgggtg 3900 tgccgccgtg 3960 gtccggtgcc 4020 gggcgttcct 4080 attgggcgaa 4140 atccatcatg 4200 cgaccaccaa 4260 cgatcaggat 4320 gctcaaggcg 4380 gccgaatatc 4440 tgtggcggac 4500 cggcgaatgg 4560 catcgccttc 4620 accgaccaag 4680 gaaaggttgg 4740 gatctcatgc 4800 aaataaagca 4860 tgtgqtttgt 4920 tagaqcttgg 4980 attccacaca 5040 agctaactca 5100 tgccagctgc 5160 tcttccgctt 5220 tcagctcact 5280 aacatgtgaq 5340 tttttccata 5400 tqgcgaaacc 5460 cgctctcctg 5520 agcqtggcgc 5580 tccaagctgg 5640 aactatcgtc 5700 ggtaacagqa 5760 *cctaactacg 5820 accttcggaa 5880 ggtttttttq 5940 *ttgatctttt 6000 gtcatgaqat 6060 *aaatcaatct 6120 gaggcaccta 6180 qtgtagataa 6240 1cgagacccac 6300 gagcgcaqaa 6360 gaagctagag 6420 iggcatcgtgg 6480 a tcaaggcgaq 6540 :ccgatcgttg 6600 j cataattctc 6660 a accaagtcat 6720 a cgggataata 6780 t tcggggcgaa 6840 t cgtgcaccca 6900 a acaggaaggc 6960 c atactcttcc 7020 a tacatatttg 7080 a aaagtgccac 7140_ 7148 c210> 8 <z211> 7469 <212> DNA WO 00/42208 WO 0042208PCTIEPOO/00265 -7- <213> Artificial Sequence <220> <223> Description of Artificial Sequenlce: plasinid <400> 8 gacggatcgg ccgcatagtt cqagcaaaat ttaqggttag gattattgac tggagttccg cccgcccatt attgacgtca atcatatgc6 atgcccagta tcgctattac actcacqgggg aaaatcaacg gtaggCgjtgt ctgcttaCtc gaqctCggal gtctttcca( gggacctga agtcacagt tqtttCtgg tggtcgagg gataccttC cttactcct ttqcgCCta ggcctctCt cctctcaaa tcagaagCC atgcaatc~ cccctcaC gatagcag 1 ggcattga gctccttt actattaa ggcaatat atacttga ggccctct ttgtttac ttgatgtt cctaatg( tcaaacac attacaq 1 cctaact' ggcagtc t ctggaa aacaatt acagcct ggtaaaa cctgtaa gcatact gccacat atgcat( tcgact( accctg' tgtctg gattgg gaaaga gagatctcc gatc aagccagtat ctgc ttaagctaca acaa gcgttttgcg ctgC tagttattaa tagt cgttacataa cttE gacgtcaata atg~ atggqtggac tati aagtaCCC cct.
catgacctta tgg *catggtgatg cgg atttccaagt ctc qgactttcca aaa acggtgggag gtc gcttatCgaa att -ctgaattCga gct g cgaqtCcgc tc c gcaaqgtagg ct c ggaggtgctg ctc t gaggtgtggc ag( *a ccccgtgta tc C cctttgtatc cc' .t ccqaacctct ag *c tggacgaggc C9 *a aaaccaagtc aa taactgtggC tg ac aggccccgct aa ag tgtcagaagg aa t~a cccttaCtat ca ct tgaaagagCc ca gc atgtaaCaga cc ta atacttcctt gc qc aacttaatgt a( tt ttataaatc~ a ag cttcaaacaa t tg acgctacagC c .ac caaacacaaa t agg ctatggttCc t tag gaaacaaaaa t gta gactaaatgc a aaa tacttgctaC z cag tt~caaagtgCI cct tcctqqaccc ata caaacgctgt *ctg ccaaaagtaa ~cac taaccattac .cta tgtcattttC cct cttacacttt ,tag agggccctat gaag gtgccactcc agta ggtctcattc gaag acaatagcag acca gctggggctc ccctat ggtc :tccctg cttc ggczaag gctl ttcgcg atg 1 aatcaa tta ~cggtaa atg jcgtatg ttc :tacggt aaa attgacg tca gactttc cta ttttggc agt cacccca ttc tgtcgta aca tatataa gcz aataCga ct( .cgctgtt gg( 1 gaaaccc gt( 1 accggat cg' ;agCaccg tg latgatgt aa ;cttgaga tc catatgac ac ccaatggg tt ttaCCtCC aa gcaacCtt ac acataaac Ct ccgccgCa cc ccgtgcaC g9 *agCtaqCC ci ictgcctCa c( ~tttataca c ;acCtaaaC a -aaactaaa g gcaggagqa c tttgatgct c gcccZacaac t tccaaaaag c atagCCatt a cccctcaaa a ~aaactagga aatgataag Lgagaaagat ~gtttcagtt catcttatt igaatattgg tggatttatg cattgtcagt actaaacggt atgggactgg ttcatacatt tctatagtgt ccagccatct cactgtcctt tattctgggg gcatgctggg tagggggtat -gactct cagi Itgtgtt gga( tgaccga caal tacgggc cag cggggtc att gccgCC tgg ccatagt aac ctgccca ctt atgacgg taa cttggca gta .acatcaa tgg ~acgtcaa tgcj LactcgC ccc ~gagctCt ct' actatag gg9 3ctcgCgq tt~ cggcctcc ga gaaaacct ct gcgggcgg ca ttaaagta qg caagatqa ag ggaaaCCg gt tcaagaga gt *tggcatgC tt :ctcccaaa at :ggaaatat ct tctaatgg tc ctccaaac tt :gcaaaCat c~ cccctctaa ci aaaatggaa a~ ctttgaccg t ttactggag
C
taaggattg a aaaaccaaC t tggatatta a :ttgaggtta a atgcaggag a c~aaaaAttg 9 ~ctggccttac ctaactttgt gctaaactca ttggctgtta ataagatttg aactttagaa cctaacct at caagtttact acacaggaaa tctggccaca gcccaagaat cacctaaatg gttgtttgcc tcctaataaa ggtggggtgg gatgcggtgg ccccacgCgc :acaatc ggtcgct ttgcatg atatacg agttcat ctgaccg gccaata ggcagta atggCCC .catctac gcgtgga ;gagtttg :attgacg 3 gctaact agacccaa giaggacaz acggtact cgagaaa( gcgggtg( cggtctt cgcgcaa cctccaa ccccctg .gcgctca .gtaacca :gcacccc :qcgqgca :agcattci aggcCCCCC tactgcca actaggac agcaact( cttgggt' ttctcaa aaatcta ctacaac *cctaagc tgggCtt jccatggc ;ttttgac jgaccaca ctttggtc aagqcaqt acgaaaat atggagal cagcttal taaacgg caggaga actacat aaagaag ctagagc cctCCCC atgagga ggcagga gctctat cctgta tgctctgatg gagtagtgcg 120 aagaatctgc 180 cgttgacatt 240 agcccatata 300 cccaacgacc 360 gggactttcc 42C catcaagtgt 48C gcctggcatt 540 gtattaqtca tagcggtttq 66( ttttggcacC 72( caaatgggcg 781 agagaaccca 841 gcttggtacc 90' actcttcgcg 96 ccgccaccga 10 g gcgtCtaacc 10 g cggtCgg9cJt 11 g agacggCgg 12 g accgtCtgaa 12 c tqtgcctttt 13 g ggtactctct 13 a aatgggCaaC 14 C tgtgagCCca 15 .t cacagttacc 11 .a CaCactCaCC 14 jc cacccaagga 14 ~t caccaccacc 1 ic tggtagcttg 11 -t aaagtaCggg 1 gg tccaggtgtg 1 tt tgattcacaa 1 aa cagacgCCtt 2 ag actaqgacaq 2 aa aggcctttaC 2 ac tgccaagggg 2 ga atttggttca 2 ct agaatttgat 2 ag cacaggtgCC2 cc agCtccatct :tt ggIctccaata :gg agtgCtacta :ct tactgaaggc tcc aaaatctcac aga caaaactaaa cac aactccaagt taa tgaaatattt cgg ccgctcgagc tcg ctgatcagcc :cgt gccttCCttg aat tgcatcgcat icag caagggggag :ggc ttctgaggcg ;cgg cgcattaagC 0 0 00 ~00 620 680 740 800 860 920 980 040 100 160 .220 280 ~340 ~400 460 MO2 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 WO 00/42208 PCT/EP00100265 gcggcgggtg gctcctttcg ctaaatcggq aaacttgatt cctttgacgt ctcaacccta tggttaaaaa gtcagttagg catctcaatt atgcaaagca ccgcccctaa atttatgcag ttttttgqaq tctgatcaag ggttctccgg ggctgctctg aagaccgacc ctggccacga gactggctgc gccgagaaag acctqcccat gccggtcttg ctgttcqcca gatgcctgct ggccggctgg qaagagcttg gattcgcagc gqttcgaaat ccgccttcta tccagcgcqg ataatggtta tgcattctaq cgacctctag atccgctcac cctaatgagt gaaacctgtc gtattgggcg ggcgagcggt acqcaggaaa cgttgctggc c aaqtcagag gctccctcgt tcccttcggg aggtcgttcg ccttatccgg cagcagccac tgaagtqgtg tgaagccagt ctggtagcgg aaqaagatcc aagggatttt aatgaagttt gcttaatcag gact cccog t caatgatacc ccggaagggc attgttgccg ccattgctac gttcccaacg CCttcqgtcc tggcagcact gtgagtactc tggtgqttac ctttcttccc gcatcccttt agggtgatgg tggagtccac tctcggtcta atgagctgat gtgtggaaag agtcagcaac tgcatctcaa ctccgcccag aggccgaggc gcctaggctt agacaggatg ccgcttgggt atgccgccgt tqtccqgtgc cgggcgttcc tattgggcga tatccatcat t ogaccacca tcgatcagga gqctcaaggc tgccgaatat gtgtggcgga gcqgcgaatg gcat cgcctt gaccgaccaa tgaaaggttg ggatctcatg caaataaagc ttgtggtttg ctagagcttg aattccacac gaqctaactc gtgccagctg ctcttccgct atcagctcac gaacatgtga gtttttccat gtggcgaaac gcgctctcct aagcgtggcg ctccaagctg taactatcgt tggtaacagg gcctaactac taccttcgga tggttttttt tttgatcttt ggtcatgaga taaatcaatc tgaggcacct cgtgtagata gcgagaccca cgagcgcaga ggaagctaga aggcatcgtg atcaaggcga tccgatcgtt gcataattct aaccaagtca gcgcagcgtg ttcctttctc agggttccga ttcacgtagt gttctttaat ttcttttgat ttaacaaaaa tccccaggct caggtgtgga ttagtcagca ttccgcccat cgcctctgcc ttgcaaaaag aggatcgttt ggagaggcta gttccggctg cctgaatgaa ttgcgcagct agtgccgggg ggctgatgca agcgaaacat tgatctggac gcgcatgccc catggtggaa ccgctatcag ggctgaccgc ctatcgcctt qcgacgccca qgcttcggaa ctggagttct aatagcatca tccaaactca gcgtaatcat aacatacgag acattaattg cattaatgaa tcctcgctca tcaaaggcgg gcaaaagqcc aggctccgcc ccgacaggac gttccgaccc ctttctcaat ggctgtqtgc cttgaqtcca attagcagag ggctacacta aaaagagttg gtttgcaagc tctacggggt ttatcaaaaa taaagtatat atctcagcga actacgatac cgctcaccqg agtggtcctg gtaagtagtt gtgtcacgct gttacatgat gtcagaaqta cttactgtca ttctgagaat accgctacac gccacgttcg tttagtgctt gggccatcgc, agtggactct t tat aa ggga tttaacgqga ccccaggcag aagtccccag accatagtcc tctccgcccc tctgagctat ctcccgggag cgcatgattg ttcggctatg tcagcgcagg ctgcaggacg gtgctcgacg caggatctcc atgcggcggc cqcatcgaqc qaagagcatc gacggcgaqg aatggccqct gacatagcgt ttcctcgtgc cttgacgagt acctgccatc tcgttttccg tcgcccaccc caaatttcac tcaatgtatc1 ggtcatagct ccgqaagcat cgttgcgctc tcggccaacq ctgactcgct taatacgqtt agcaaaaggc cccctgacga tataaagata tgccgcttac gctcacgctgt acgaaccccc acccggtaag z cgaggtatgt z gaaggacagt z gtagctcttg z agcagattac ctgacgctca q ggatcttcac c atgagtaaac t tctgtctatt t gggagggctt a ctccagattt a caactttatc c cgccagttaa t zgtcgtttgg t Zccccatgtt g agttggccgc a tgccatccgt a agtqtatgcg g t tgccagcgc ccggctttcc tacggcacct cctgatagac tgttccaaac ttttggggat attaattctg gcagaagtat gctccccagc cgcccctaac atggctgact tccagaagta cttgtatatc aacaagatgg actgggcaca ggcgcccggt agqcagcgcg ttgtcactga tqtcatctca tgcatacgct gagcacgtac aggggctcgc atctcgtcgt tttctqgatt tggctacccg tttacgqtat tcttctgagc acgagatttc ggacgccggc caacttgttt aaataaagca1 ttatcatgtc1 gtttcctgtg1 aagtgtaaa actgcccgct cqcggggaga jcgctcggtc atccacagaa t zaggaaccgtE Icatcacaaa z ::caggcgtttc :ggatacctgt :aggtatctc E ,gttcagccc lcacgactta t ~ggcggtgct a itccggcaaa c ;cgcagaaaa a ~tggaacgaa a :tagatcctt t :tggtctgac a :cgttcatcc a iccatctggc c ttcagcaata a ~gcctccatc c :agtttgcgc a ~atggcttca t jtgcaaaaaa g gtgttatca c agatgcttt t jcgaccgagt t cctagcgccc 3420 ccgtcaagct 3480 cgaccccaaa 3540 ggtttttcgc 3600 tggaacaaca 3660 ttcggcctat 3720 tggaatgtgt 3780 gcaaagcatg 3840 aggcagaagt 3900 tccgcccatc 3960 aatttttttt 4020 gtgaggaggc 4080 cattttcgga 4140 attgcacgca 4200 acagacaatc 4260 tctttttgtc 4320 gctatcgtgg 4380 agcgggaagg 4440 ccttgctcct 4500 tgatccggct 4560 tcggatggaa 4620 qccagccgaa 4680 gacccatggc 4740 catcgactgt 4800 tgatattgct 4860 cgccgctccc 4920 gggactctgg 4980 gattccaccg 5040 tqgatgatcc 5100 attgcagctt 5160 tttttttcac 5220 tgtataccgt 5280 :qaaattgtt 5340 gcctggggtg 5400 ttccaqtcgg 5460 jgcggtttqc 5520 Ittcggctgc 5580 :caggggata 5640 ~aaaagqccg 5700 iatcgacgct 5760 ~cccctggaa 5820 :ccgcctttc 5880 gqttcggtgt 5940 ~accgctgcg 6000 :cgccactgq 6060 icagagttct 6120 :gcgctctgc 6180 :aaaccaccg 6240 Laaggatctc 6300 actcacgtt 6360 taaattaaa 6420 .gttaccaat 6480 .tagttgcct 6540 :ccagtgctq 6600 accagccag 6660 :agtctatta 6720 acgttgttg 6780 tcagctccg 6840 cggttagct 6900 tcatggtta 6960 ctgtgactg 7020 gctcttgcc 7080 WO 00/42208 WO 0042208PCT/EPOO/00265 cggcgtcaat gaaaacgttc tgtaacccac ggtgagcaaa gttgaatact tcatgagcgg catttccccg acgggataat ttcggggcga tcgtgcaccc aacaggaagg catactcttc atacatattt aaaagtgcca accgcgccac aaactctcaa aactgatctt caaaatgccg ctttttcaat gaatgtattt cctgacgtc atagcagaac ggatcttacc cagcatcttt caaaaaaggg attattgaag agaaaaataa tttaaaagtg gctgttgaga tactttcacc aataagggcg catttatcag acaaataggg ctcatcattg tccagttcga agcgtttctg acacggaaat ggttattgtc.
gttccgcgca 7140 7200 7260 7320 7380 7440 7469 <210> 9 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 9 tgcttaagcg gccgcgaagg agaagtcc <210> <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> ccgagctagc gactqaaaat gag <210> 11 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 11 cctctcgaga gacagcaaga cac <210> 12 <211> 11152 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: plasmid <400> 12 aagcttgggc aaggggttgt aaagacatat ggggaagttg gtgcaagatt cagccaactt cttgctaaaa atgttaagaa agaaatggtt ttcccaccaa tcattctctg cggttcgtgc acaatctaaa cctcttacaa attatatttt atgaatcatt gaactcccga ggacgacccg ctgcaaactt tcgcagggct caattcggag gccgcatcga taccaataag atcttttagt gagtgtccta tctgcgcaca ggcatagctc ctcacccttg aactcgacct ttttgtcctt accaatccaa actattttta cacctagggg aacggatgag tgctttgcct actcttttaa tcctcctgag cagaaataga taggtagatt ctcaaattca agaagcagcc cccatcagac ggggctattg tagctcttct gcaaggacca aataagaatg attagttact gaagttagaa 120 180 240 300 360 420 480 WO 00/42208 PCT/EPOO/00265 atgggaatag ttgaccacag aggtggtqgc ttaccatata aaagtqttat tgtatgttgt ctaggaacag ggactaatag ttggcccaac aacgaggatg gctctgagtg ttatgtaaac cattcacctc tcactttcca gctggcgccc tgtttgtctt ccatagggac accgaaqaaa cttccacctc acggcccccg ttgqcggtgc ccgcctcacc atgccaaacc agtgacgacg cacggttqca tcgctttgct gtgggtgata ggtttaaaqa agcccgagcc tcctgagacq actccgqtcc aaccagttgc ttaacgagcc acctgtgatt taaagggtga atataatgcg tggaagattt tttggaggtt acaagtggga tgggtcacca ggcgcgctgc cccatctqag tgagacacaa aggagcagca acccgagagc agaactgaga ggacggggg cagacaccgt tgatctgctg ggatgatttt gtacaagatc cgaggtggag gccgggggtg ttttagcggt tgggtttaac ttactgctgc cctctttgaa tgtggcctcc catggtatgt ctgtcacctj tgagcataac accttaccaa aaaatagaaa gcctagaagt aaccagggac caggaagata atagatcct ctcaagaaqa gaatqcactt aacattattc cttgcggttc tgagacaagt ttctattttc caagatataa ttgtgtqttt gaqqgt cccc gaacagggac gtattgtctc caagctagcg tggccgccag ctagccattt aagatccaa aggaaggqat tttcccqgca ttgtaccgga aggatqaaga ggtcttgtca atatgaggac gagtggtggg attttgtatt agaaccqgag cccgacatca ttctaacaca cgtgagagtt tgggcaacct gcgtgtgtgg gataatgttt ccgtgggcta ttctgctgtg tctgtggqqc atttgaagag ggcgcttttc ggctgctgtt cqgggggtac gaatcgcctg gcagcagcag cggcctggac cgcattttga gcttgtgagg cctgagtgta gcgcagaagt gaggaggcta agcaaacttg atagatacgg cttggcatgg acggttttcc *aatacctgtg *tggaaggggg aggtgtacct *gactgtggtt *ggcaactgcq ctgaagacca *atactgaccc Ltgcaatttgo gagacqctca aaaaaaggga ttatagggga tgacttaaat cccttttcgt aaaagacgac ttggggaaag tgcaaaaact ccagggctta ggtttcctqa ctatgttctt aagaqtgctg gtgtctgttc ccgcagaccc cctcggataa tttcttgtct actgaaaatg tcttttggac tgaaccacct cgaggaggcg tqacttactc gcccgagcag ggtgatcgat gggtgaggag ttatcaccgg ctgtqgcatg tttggtgtgg qtgatttttt cctgcaagac cctgtgtcta cctcctgaga ggtgggcgtc ttggacttga ttaacgcctt aacttgcatg atcttggtta cqtaacttgc tcatcccagg cttttgaaat caagagaagg gcttttttga ctqctggatt ctactgttgt gaggaagcca cctcgggaat caattacaga ctacagagga ttacttttca attccataga ttagggtata taaatatcag aggatagggt acqgggtggt tggccaatac tggaagcctg tggtgtgtcg tgggtatcct gcttcatgct Faggacagggc ttcacgtagc gctgttcctt igtcacactaa acctcaattg aaaaagagtg ccttacatct tgggataggt gaaagactcg atgaaacaac attttccata catggcatga agtaagtttt cttggtttgg ttggaattta attttttgag gccatcccgt cggcgaccct gtgacccttg ggctatcatc agacatatta cagctgatcg acccttcacg gtttcgcaga acttttccgc ccggagcaga cttacctgcc tttgtgttag aggaatacgg tttgtctaca taattttttt taaaaggtcc ctacccgccg gagaatgcaa tacacccggt gccaggctgt gctgtaaacg tgtttgctga gcgtgttaaa catctgacct tggaacagag caaagttagt cctgtggtga tcatcaagac gttttataaa ttctggccat cttccgtccg ggcggcggcg gaatgttgta ggatgggcag ggctaggaat acagatcaag gcagctgacc tgcaaaggtg gaattgttgc ggcctttaga tattatgaat caaccttatc gaccgatgta ccccaaaagc gtctgagggt agtgaaaagc ctctcagatg cagccactct gcatttgggt gatattgctt aagaacaggt tttttgtcaa acagaccaac gggttacagt ccagagctag aggtacatga ccaaggaggg gttattatga tggttacaaa tatcaaaggt tccaaatctt taaacttgca ctccgctcgt caggtcggcc tctctatttc acaagagcgg tctgccacgg aagaggtact aactgtatga tttttcccga cggcgcccgg gagccttggg acgaggctgg attatgtgga gggacccaga gtaagtgaaa tttaattttt tgtgtctgaa tcctaaaatg tagtaqtacg ggtcccgctg ggaatgtatc ccccaggcca atgagttgat tggggcgggg catggaggct ctctaacaqt ctgcaqaatt gctgtttgat tttggatttt ggataaatqg gcatctgtgg cccggcgata gcaggagcag caggtggctg gggctaaagg ctagctttta gataattgcg acttactggc gcacttaggc tacatttctg tgtagcatga gtaaggttta ctacacggtg agggttcggg agggcttcaa aactccaggg gtggctgtga ctgacctgct cgcaaggcct aacaggaggg gagcccgaga gcaaggacta aataggagac agatgccccc caatggctat acctccttgg ttatatttat gacagtggct atagccttta ctgttcttaa tctgatctga atgtaaatgc acaqtcctaa cacttatcct gactgcqgca tactatttgg aacgqactca aggtgttatt ggctgataat tttagacgtg ctctgtaatg ttctccggag tccggtttct ctttccaccc gcaccccggg tattatgtqt attatgggca acaqttttgt cctgagcctg gcgcctgcta gatagctgtg tqccccatta gaggacttgc taaggtgtaa gtaagtttaa cttaaaqggt tgggagtgtt acctcttggt aaggaggatt tctttgaatc tccacaccgg agcgaagaaa aqagcggttg ataccgacgg agcccatgga aactgtatcc gggtaaagag gcttaatgac ct aat gagct tgcagccagg cagattgcaa ggaacggggc taaatatgtg ctggccccaa taagcttcta gctgtgcctt ttaagaaatg tgcgccacaa ttaagcataa cggacggcaa ggccagtgtt gggtgttcct gcatgtccaa 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 WO 00/42208 PTEO/06 PCT/EPOO/00265 qgtgaacctg tgagacccgc tgtgatgctg cgctgagttt cttaagggtg agcagccqcc gacaacgcgc tggtcgcccc gccgttggag tgtgactgac ccgcgatgac tgtcgtttct ccctcccaat gcaagtqtct cataattgga qtgtataatg gaactgatga aagaaatgcc aaaagaagag gtcatqctgt aagctgcact ataacaqtta ctattaataa ataaqgaata gtagaggttt atgaatgcaa aatagcatca tccaaactca qggtgtggaa tagtcagcaa atgcatctca actccgccca gaggccgagg ggcctaggct accactttat actggttttt tttttaagcc ctgggacatg atggaaaggc tgaactgggt gcgcgagctt tgacctggtg ctttgtcacc tatcccgcaa aatctccggt caggcgggt t acctgagcga gccgctttaa ctcactggta aaatcctcga tatacqatac tttgtgaagg taaagctcta attgtttgtg gcctttaatg actgctgact gactttcctt gcttgctttg gaaaaatatt tttcttactc acctttagct actagagatc aacggggtgt accaggtgca gatgtgaccg ggctctagcg ggaaagaata gccgccatga atgcccccat gtcctgcccg actgcagcct tttgctttcc aagttgacgg cagcagctgt gcggtttaaa tgctgtctct caaactacct tgttaaacta atgggagcag atctagtgat aaaggtagaa gtttagtaat gctatacaag taatcataac ctatgctcaa tttgatgtat tacttgcttt ttqttgttgt caaatttcac tcaatgtatc aqtccccagg ccaggtgtgg attagtcagc qttccgccca ccgcctcggc tttgcaaaaa cccgcgtcag agtgcgccag gtagataaac ttgcagatc attattgccg attcgtcatg aaagtgctga gataccggtg atcttcgcaa gatacctgga cgctaatctt acaatagttt aaccctgttc tcacggcgca tcgcatgatt cqtccaggca ggtgattggc aaccttactt aggtaaatat tattttagat aggaaaacct ctcaacattc cagaattgct ctatttacac ctgtaacctt cacacaggca ttttaatttg ataatcagcc ttgacatgac gaccctgcga aggagctgag atgaagatac tataaggtgg gcaccaactc gggccggggt caa actct a c ccgccgccgc tgagcccgct ctcttttggc tggatctgcg acataaataa cgagggatct acagagattt ctgattctaa tggtggaatg gatgaggcta gaccccaagg agaactcttg aaaattatgg atactgtttt aaattgtgta agtgccttqa aaaaaacctc taacttgttt aaataaagca ttatcatgtc ctccccagca aaagtcccca aaccatagtc ttctccgccc ctctgagcta gcttggacac ggagaggcag atctctataa aggctgggac atgcacgtaa taagccgtgq tcgataccgt aacgcgcaga gtactgcggt aaccggctgg ttgaacagcc ttcaacgcct ccagtaagta aaaccccgct caaccgcctg aaccgtctga cgtattgtga taccgtggcg ctgtggtqtg aaaattttta tccaacctat gttttgctca tactcctcca aagttttttq cacaaaggaa tataagtagg tagagtgtct taaaggggtt ataccacatt catgaagatc gtgtggcggt gcccgatcac agat tgaggt gggtcttatg gtttgatgga gcgtcagaat taccttgacc ttcagccgct tgcaagcagt acaattggat ccagcaggtt a aaa ccaga c ttgtgaagga aaagctctaa ttgtttgtgt cctttaatga ctgctgactc actttccttc cttgctttgc aaaaatattc ttcttactcc cctttagctt ctagagatca ccacacctc attgcagctt tttttttcac tggatcqggc gqcagaagta ggctccccag ccgcccctaa catggctgac ttccagaagt aaqacaggct tqcgtaaaaa tctcgcqcaa acttcacatg actcgcaagc cqgtctqgta ttgtatttcc aggcgatggc tgcgattcgt tcgtccgctg gtgqgatatg ggcactgccg ttctggaggc ttaaacatcc tgcagtcggc tgtqgatctg tgagcgatgc gcaactggat acataattgg agtgtataat ggaactgatg gaagaaatgc aaaaagaaga agtcatgctg aaagctgcac cataacagtt gctattaata aataaggaat tgtagaggtt tggaaggtgc aaacatatta ttggtgctgg actgaaatgt tagttttgta agcattgtga gtgatgggct tacgagaccg gcagccaccg gcagcttccc tctttgaccc tctgccctga tctgtttgga accttacttc ggtaaatata attttagatt ggaaaacctg tcaacattct agaattgcta tatttacac tgtaaccttt acacaggcat tttaatttgt taatcagcca ccctgaacct ataatggtta tgcattctag tgtqgaatgt tgcaaagcat caggcagaag ctccgcccat taattttttt agtgaggagg tgcgagatat gjacgcggact cctattttcc agcgaaaaat cgactgatgc ccgggtgcgt agctacgatc gaaggcttca gaaatgtatc gttgatgact gjgcgtcgtat ggcgttgttc tgcatccatg tgaaacctcg ccttgatggt gcgcqgcatt cgaacgtacc ttatgagtgg acaaactacc gtgttaaact aatgggagca catctagtga gaaaggtaga tgtttagtaa tgctatacaa ataatcataa actatgctca atttgatgta ttacttgctt tgaqgtacga ggaaccagcc cctqcacccg gtgggcgtgg tctgttttgc gctcatattt ccagcattga tgtctggaac cccgcgggat gttcatccgc gggaacttaa aggcttcctc tttggatcaa tgtggtgtga aaatttttaa ccaacctatg ttttgctcag actcctccaa agttttttga acaaaqgaaa ataagtaggc gagtgtctg aaaggggtta taccacattt gaaacataaa caaataaagc ttgtggtttg gtgtcagtta qcatctcaat tatgcaaagc cccgccccta tatttatgca cttttttgga gtttqagaat cat gt qaa at cctcgaacac acatcgtcac cttctgaaca tactggcgcq acgacaacca tcgttattga caaaagcgca atgttgttga tcgtcccgcc tttttaactt acacaggcaa acqctagtcc aaaaccatcc gacccacgcq gacgatgatt gccccggatc tacagagatt actgattcta gtgqtqgaat tgatgaggct agaccccaag tagaactctt gaaaattatg catactgttt aaaattgtgt tagtgccttg taaaaaacct 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 5580 5640 5700 5760 5820 5880 5940 6000 6060 6120 6180 6240 6300 6360 6420 6480 6540 6600 6660 6720 6780 6840 6900 6960 7020 7080* 7140 7200 7260 7320 7380 7440 7500 7560 7620 7680 7740 7800 7860 7920 WO 00/42208 WO 0042208PCT/EPOO/00265 -12cccacacctc tattgcagct atttttttca ctggatcccc acattccaat acaaaaagga atctgggaag caacagcaga gaagcactgt tgtaggttcc ctggataagc caactgtagc acacaccctg gaatgggttt tagcagttac cacaggttaa gatacgccta cacttttcgg tatgtatccg gagtatgagt tcctgttttt tgcacgaqtg ccccgaagaa atcccgtgtt cttggttgaq attatgcagt gat cggagga ccttgatcgt gatgcctgca agcttcccgg qcgctcggcc gtctcgcggt ctacacgacg tgcctcactg tgatttaaaa catgaccaaa gatcaaagga aaaaccaccg qaaggtaact gttaggccac qttaccagtg atagttaccg cttggagcga cacgcttccc agagcgcacg tcgccacctc gaaaaacgcc catgttcttt agctgatacc ggaagagcgc atggtgcact ctatcgctac ccctgacggg agctgcatgt cccctqaacc tgaaacataa tataatggtt ctgcattcta agqaagctcc cataggctgc aattgggtag tcccttccac aacatacaag ggttgctgtg aaaatatcta attatcctta at tt t ttgg cagctccaaa tccagcacca cccaataacc gtcctcattt tttttatagg ggaaatgtgc ctcatgagac attcaacatt gctcacccag ggttacatcg cgttttccaa gacgccqqgc tactcaccag gctgccataa ccgaaggagc tgggaaccgg gcaatggcaa.
caacaattaa cttccggctg atcattgcag gggagtcagg attaagcatt cttcattttt atcccttaac tcttcttgag ctaccagcgg ggcttcagca cacttcaaqa gctgctgcca gataaggcgc acgacctaca gaagggagaa agggagcttc tgacttgagc agcaacgcgg cctgcgttat gctcgccgca ctgatgcggt ctcagtacaa gtgactgggt cttgtctgct gtcagaggtt acaaataaag gttgtggttt tctgtgtcct ccatccaccc gggtttttca.
tgctgtgttc ctgtcagctt ttagtaatgt gtgttttcat tccaaaacag gttacagttt ggttccccac ttttcatgag tcagttttaa aaattaggca ttaatgtcat gcggaacccc aataaccctg tccgtgtcgc aaacgctggt aactggatct tgatgagcac aagagcaact tcacagaaaa ccatgagtga taaccgcttt agctgaatga caacgttgcg tagactggat gctggtttat cactggqgcc caactatgga ggtaactgtc aatttaaaag gtgaqttttc atcctttttt tgqtttgttt gagcgcagat actctgtagc gtgqcgataa agcggtcggg ccgaactgag aggcggacag cagggggaaa gtcgattttt cctttttacg cccctgattc gccgaacgac attttecct tctgctctga catggctgcg cccggcatcc ttcaccgtca aatgaatgca caatagcatc gtccaaactc cataaaccct tctgtgtcct cagaccgctt cagaagtgtt tgcacaaggg gcaaaacagg ttttacttgg ccttgtggtc gagcaggata caacagcaa~a ttttttgtgt cagtaacagc aaggaattct gataataatg tatttgttta ataaatgctt ccttattccc gaaagtaaaa caacagcggt ttttaaagtt cggtcgccgc gcatcttacg taacactgcg tttgcacaac aqccatacca caaactatta ggaggcqgat tgctgataaa agat ggtaag tgaacgaaat agaccaagtt qatctaggtq gttccactga tctgcgcgta gccggatcaa accaaatact accgcctaca gtcgtgtctt ctqaacggqg atacctacag gtatccggta cgcctggtat gtgatgctcg gttcctggcc tgtggataac cgagcgcaqc tacgcatctg tgccgcatag ccccgacacc gcttacagac tcaccgaaac attgttgttg ttaacttgtt 7980 acaaatttca atcaatgtat aacctcctct cctgttaatt tctaagggta ggtaaacagc cccaacaccc aggcacattt atcaggaacc agtgttcatc tttggtcctg aaaAtgaaaa ccctgaatgc ttcccacatc tgaaqacgaa gtttcttaga tttttctaaa.
caataatatt ttttttgcgg gatgctgaag aagatccttg ctgctatgtg atacactatt qatggcatga gccaacttac atgqqggatc aacqacgagc actggcgaac aaagttgcag tctggagccg ccctcccgta agacagatcg tactcatata aagatccttt gcgtcagac atctgctgct gagctaccaa gtccttctag tacctcgctc accgggttqg ggttcgtgca cgtgagctat agcggcaggg ctttatagtc tcaggggggc ttttgctggc cgtattaccg gagtcagtga tgcggtattt ttaagccagt cgccaacacc aaqctgtgac gcgcgaqgca caaataaagc 8040 cttatcatgt 8100 acttgagagg 8160 aggtcactta 8220 attttaaaat 8280 ccacaaatgt 8340' tgctcatcaa 8400 tccccacctg 8460 cagcactcca 8520 tgctgactgt 8580 taqtttgcta 8640 tttgaccctt 8700 aagtttaaca 8760 aaaatatttc 8820 agggcctcgt 8880 cgtcaggtgg 8940 tacattcaaa 9000 gaaaaaggaa 9060 cattttgcct 9120 atcagttggg 9180 agagttttcg 9240 gcgcggtatt 9300 ctcagaatga 9360 cagtaagaga 9420 ttctgacaac 9480 atgtaactcg 9540 gtgacaccac 9600 tacttactct 9660 gaccacttct 9720 gtgagcgtgg 9780 tcgtagttat 9840 ctgagatagg 9900 tactttagat 9960 ttqataatct 10020 ccgtagaaaa 10080 tqcaaacaaa 10140 ctctttttcc 10200 tgtagccgta 10260 tgctaatcct 10320 actcaagacg 10380 cacagcccag 10440 gagaaagcgc 10500 tcggaacagg 10560 ctgtcgggtt 10620 ggagcctatg 10680 cttttgctca 10740 cctttgagtg 10800 qcqaggaagc 10860 cacaccqcat 10920 atacactccg 10980 cgctgacgcg 11040 cgtctccggg 11100 gc 11152 <210> 13 <211> 19 <212> DNA <213> Artificial Sequence <220> WO 00/42208 WO 0042208PCT/EPOO/00265 -13- <223> Description of Artificial Sequence: primer <400> 13 gacggatcgg gagatctcc <210> 14 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 14 ccgcctcaga agccatagag cc <210> <211> 14455 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: plasmid <400> 15 aagcttgggc agaaatggtt aaggggttgt ttcccaccaa aaagacatat tcattctctq ggggaagttg cggttcgtgc gtgcaagatt acaatctaaa cagccaactt cctcttacaa cttgctaaaa attatatttt atgttaagaa atgaatcatt atgggaataq aaaatagaaa ttqaccacag gcctagaaqt agqtggtggc aaccaggqac ttaccatata caggaagata aaagtqttat atagatccct tgtatgttgt ctcaagaaqa ctaggaacag gaatgcactt ggactaatag aacattattc ttggcccaac cttgcqgttc aacgaggatq tqagacaagt gctctgagtq ttctattttc ttatgtaaac caagatataa cattcacctc ttgtgtgttt tcactttcca gagggtcccc gctggcgccc gaacagggac tgtttgtctt gtattgtctC ccatagggac caagctaqcg accgaagaaa tggccgccag cttccacctc ctagccattt acggcccccg aagatcccaa ttggcggtgc aggaagggat ccgcctcacc tttcccggca atgccaaacc ttgtaccgga agtgacgacg aggatgaaga cacggttgca ggtcttgtca tcgctttgct atatgaggac qtgggtgata gagtggtggg ;aactcccga ;gacgacccg ftgcaaactt :cgcagggct :aattcggag jccgcatcga :accaataag atcttttagt gagacgctca aaaaaaggga Ltatagggga tgacttaaat cccttttcgt aaaagacgac ttggggaaag tgcaaaaact ccagggctta ggtttcctga ctatgttctt aagagtgctg gtgtctgttc ccgcagaccc cctcggataa tttcttgtct actgaaaatg tcttttggac tgaaccacct cgaggaqgcg tgacttactc gcccgagcag ggtgatcgat gggtgaggag ttatcaccgg ctgtggcatj tttggtgtgq gagtgtccta tctgcgcaca ggcataqctc ctcacccttg aactcgacct ttttgtcctt accaatccaa actattttta acctcaattg aaaaagagtg ccttacatct tgggataggt gaaagactcg atgaaacaac attttccata catggcatga agtaagtttt cttggtttgg ttggaattta attttttgag gccatcccgt cggcgaccct gtgacccttg ggctatcatc agacatatta cagctgatcg acccttcacg gtttcgcaga acttttccgc ccggagcaga cttacctgcc tttgtgttag aggaatacgg ftttgtctaca rtaattttttt cacctaqgg aacggatqag tgctttqcct actcttttaa tcctcctgag cagaaataga taqgtagatt ctcaaattca aagaacaggt tttttgtcaa acagaccaac qggttacagt ccagagctag aggtacatga ccaaggaggg gttattatga tggttacaaa tatcaaaggt tccaaatctt taaacttgca ctccgctcgt caggtcggcc tctctatttc acaagagcgg tctgccacgg aagaggtact aactgtatga tttttcccga cggcgcccgg gagccttggg acgaggctgg attatgtgga gggacccaga gtaagtgaaa tttaattttt agaagcagcc cccatcagac ggggctattg tagctcttct gcaaggacca aataagaatg autagttact gaaqttaqaa gcaaggacta aataggaqac agatgcccCC caatggctat acctccttgg ttatatttat gacagtggct atagccttta ctgttcttaa tctgatctga atgtaaatgc acagtcctaa cacttatcct gactqcqgca tactatttgg aacggactca aggtgttatt gcjctgataat tttagacqtg ctctgtaatg ttctccggag tccggtttct ctttccaccc gcaccccggq tattatgtgt attatgggca acagttttgt 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740_ 1800 1860 1920 1980 2040 2100 WO 00/42208 PCT/EPOO/00265 -14ggtttaaaga agcccgagcc tcctgagacg actccggtcc aaccagttgc ttaacgagcc acctgtgatt taaagggtga atataatgcg tggaagattt tttggaggtt acaagtggga tgggtcacca ggcgcgctqc cccatctgag tgagacacaa aggagcagca acccgagagc agaactgaga ggagcggggg cagacaccgt tgatctgctg ggatgatttt gtacaagatc cgaggtggag gccqqgggtg ttttagcggt tgggtttaac ttactgctgc cctctttgaa tgtggcctcc catggtatgt ctgtcacctg tgagcataac accttaccaa ggtgaacctq tgagacccgc tgtgatgctg cgctgagttt cttaagggtg.
agcagccgcc gacaacgcgc tggtcgcccc gccgttggag tgtgactgac ccgcgatgac tgtcgtttct ccctcccaat gcaagtgtct cataattgga gtgtataatg gaactgatga aagaaatgcc aaaagaagag gtcatgctgt aagctgcact ataacagtta ctattaataa ataaggaata gtagaggttt atgaatgcaa aatagcatca attttgtatt agaaccggaq cccgacatca ttctaacaca cgtgagagtt tgggcaacct gcgtgtgtgg gataatgttt ccgtgggcta ttctgctgtg t ctgt ggggc atttgaagag ggcgcttttc ggctgctgtt cggggggtac gaatcgcctg gcagcagcag cgqcctggac cgcattttga gcttgtgaqg cctgagtgta gcqcagaaqt gaggaggcta agcaaacttg atagatacgg cttggcatgg acggttttcc aatacctqtg tggaaggggg aggtgtacct gactqtggtt ggcaactgcg ctgaagacca atactgaccc tgcaatttqa aacggggtgt accaggtgca gatgtgaccg ggctctagcg ggaaagaata gccqccatqa atgcccccat gtcctgcccg actgcagcct tttgctttcc aagttgacgg cagcagctgt gcggtttaaa tgctqtct ct caaactacct tgttaaacta atgggagcag atctagtgat aaaggtagaa gtttagtaat gctatacaag taatcataac ctatgctcaa tttgatgtat tacttgcttt ttgttgttgt caaatttcac gtgatttttt cctgcaagac cctqtgtcta cctcctgaga ggtgggcgtc ttggacttga ttaacgcctt aacttgcatg atcttggtta cgtaacttgc tcatcccagg cttttgaaat caagagaagg qcttttttga, ctgctgqatt ctactgttgt gaggaagcca cctcgggaat caattacaga ctacagagga ttacttttca attccataga ttagggtata taaatatcag aggatagqqt acggggtggt tggccaatac tggaaqcctg tggtgtgtcg tgggtatcct gcttcatgct aggacagggc ttcacgtagc gctgttcctt qtcacactaa ttgacatgac qaccctgcga aggagctgag atgaagatac tataaggtgg gcaccaactc gggccggggt caaactctac ccgccgccgc tgagcccgct ctcttttggc tgqatctgcg acataaataa cgagggatct acagagattt ctgattctaa tgqtggaatg gatgaggcta gaccccaagg agaactcttg aaaattatgq atactgtttt aaattgtgta agtgccttga aaaaaacctc taacttgttt aaataaagca taaaaggtcc ctacccgccg gagaatqcaa tacacccggt gccaggctgt gctgtaaacg tgtttgctga gcgtgttaaa catctgacct tggaacagag caaagttagt cctgtggtga tcatcaagac gttttataaa ttctggccat cttccgtccg ggcggcggcg qaatgttgta ggatgqgcag ggctaggaat acaqatcaag gcagctgacc tgcaaaggtg gaattgttgc qgcctttaga tat tatqaat caaccttatc gaccgatqta ccccaaaagc gtctgagggt agtqaaaagc ctctcagatg caqccactct gcatttqggt gatattgctt catgaagatc qtqtggcggt gcccgatcac agattgaggt gqgtcttatg gtttgatgga gcgtcagaat taccttgacc ttcagccqct tgcaagcagt acaattggat ccaqcaggtt aaaaccagac ttgtgaagga aaagctctaa ttgtttqtgt cctttaatga ctgctgactc actttccttc cttgctttgc aaaaatattc ttcttactcc cctttaqctt ctagagatca ccacacctcc attqcagctt tttttttcac tgtgtctgaa tcctaaaatg tagtagtacg ggtcccgctg ggaatgtatc ccccaggcca atgagttgat tggggcgggg catggaggct ctctaacaqt ctgcagaatt gctgtttgat tttggatttt gqataaatgq gcatctgtgg cccggcgata gcaggagcag caggtggctq gggctaaagg ctagctttta gataattgcq acttactggc gcacttaggc tacatttctg tgtagcatga gtaaggttta ctacacgqtg agggttcggq agggcttcaa aactccagqg gtggctgtqa ctqacctgct cgcaaggcct aacaggaggg gagcccqaga tggaagqtgc aaacatatta ttggtgctgg actgaaatqt tagttttgta agcattgtqa gtgatgqgct tacgagaccg gcagccaccg gcagcttccc tctttqaccc tctqccctga tctgtttgga accttacttc ggtaaatata attttagatt ggaaaacctg tcaacattct agaattgcta tatttacacc tgtaaccttt acacaggoat tttaatttgt taatcagcca ccctgaacct ataatggtta tqcattctag cctgagcctq 2160 gcgcctgcta 2220 gatagctgtg 2280 tgccccatta 2340 gaggacttgc 2400 taaggtgtaa 2460 gtaagtttaa 2520 cttaaagggt 2580 tgggagtgtt 2640 acctcttggt 2700 aaggaggatt 2760 tctttgaatc 2820 tccacaccgg 2880 agcgaagaaa 2940 aqagcggttg 3000 ataccgacgg 3060 agcccatgga 3120 aactgtatcc 3180 gggtaaagag 3240 qcttaatgac 3300 ctaatgaqct 3360 tgcaqccagg 3420 cagattgcaa 3480 ggaacggggc 3540 taaatatgtg 3600 ctqgccccaa 3660 taaqcttcta 3720 qctgtgcctt 3780 ttaagaaatg 3840 tgcgccacaa 3900 ttaagcataa 3960 cggacggcaa 4020 ggccagtgtt 4080 gqgtgttcct 4140 qcatgtccaa 4200 tqaggtacga 4260 ggaaccagcc 4320 cctgcacccg 4380 gtgggcgtgg 4440 tctgttttgc 4500 gctcatattt 4560 ccagcattga 4620 tqtctqgaac 4680 cccgcgggat 4740 gttcatccgc 4800 gggaacttaa 4860 aggcttcctc 4920 tttggatcaa 4980 tgtggtgtga 5040 aaatttttaa 5100 ccaacctatg 5160 ttttgctcag 5220 actcctccaa 5280 agttttttga 5340 acaaaggaaa 5400 ataagtaggc 5460 agagtgtctg 5520 aaaggggtta 5580 taccacattt 5640 gaaacataaa 5700 caaataaagc 5760 ttgtggtttg 5820 WO 00/42208 WO 0042208PCT/EPOO/00265 tccaaactca qggtgtggaa tagtcagcaa atgcatctca .actccgccca gaggccgagg ggcctaggct accactttat actggttttt tttttaagcc ctggqacatg atggaaaggc tgaactgggt gcgcgagctt' tqacctggtg ctttgtcac tatcccgcaa aatctccqgt caggcgggtt acctgagcqa gccgctttaa ctcactggta aaatcctcga tatacgatac tttgtgaagg taaagctcta attgtttgtg gcctttaatg actgctgact gactttcctt gcttgctttg gaaaaatatt tttcttactc acctttagct actagagatc cccacacctc tattgcagct atttttttca ctggatcccc acattccaat acaaaaagga atctgggaag caacagcaga gaagcactgt tgtaggttcc ctggataagc caactqtagc acacaccctc gaatgggttt tagcagttac cacaggtta gatacgcct cacttttcg( tatgtat~cc gagtatgagi tcctgttttl tgcacqagti ccccgaagai atcccgtgt cttggttga' attatgcag gatcggagg tcaatgtatc agtccccagg ccaggtgtgg attagtcagc gttccgccca ccgcctcggc tttgcaaaaa cccgcgtcag agtgcgccag gtagataaac ttgcagatcc attattqccg attcgtcatg aaagtgctga gataccggtg atcttcgcaa gatacctgga cgctaatctt aoaatagttt aaccctgttc tcacggcgca tcgcatgatt cgtccaggca ggtgattggc aaccttactt aggtaaatat tattttagat aggaaaacct ctcaacattc cagaattgct ctatttacac ctgtaacctt cacacaggca ttttaatttg ataatcagcc cccctgaacc tataatgqtt ctgcattcta aggaagctcc cataggctgc aattgggtag tcccttccac Laacatacaac g gttgcttc *aaaatatctz *attatccttz -attttttggi Icagctccaaz *tccagcacc -cccaataacc igtcctcattt a tttttatagc j ggaaatgtg( I ctcatgaga( t attcaacatl t gctcaccca( g ggttacatc( a cgttttcca.
t gacgccggg, g tactcacca, t gctgccata a ccgaaggag ttatcatgtc ctccccagca aaagtcccca aaccatagtc ttctccgccc ctctgagcta gcttggacac ggagaggcag atctctataa aggctgggac atgcacgtaa taagccgtgg tcgataccgt aacgcgcaqa gtactgcggt aaccggctgg ttgaacagc ttcaacgcct ccagtaagta aaaccccgct caaccgcctg aaccgtctga cgtattgtga taccgtggcg ctgtggtgtg aaaattttta tccaacctat gttttgctca tactcctcca aagttttttg cacaaaggaa tataagtagg tagagtgtct taaaggggtt ataccacatt tgaaacataa acaaataaag gttgtggttt tctgtgtcct ccatccaccc gggtttttca tgctgtgttc ctgtcagctt Ittagtaat gt gtgttttcat tccaaaacac gttacagttt ggttccccac ttttcatac tcagttttaz aaattaggcz ;ttaatgtcat gcggaaccc( aataaccctc, tccgtgtCg( Saaacgctggl g aactggatcl a tgatgagcai c aagagcaac g tcacagaaa, a ccatgagtg.
taaccgctt tggatccggc ggcagaagta ggctccccag ccgcccctaa catggctgac ttccagaagt aagacaggct tgcgtaaaaa tctcgcgcaa acttcacatg actcgcaagc cggtctggta ttgtatttcc aqqcgatggc tgcgattcqt tcgtccgctg gtgggatatg ggcactgccg ttctggaggc ttaaacatcc tgcagtcggc tgtggatctg tgagcgatgc gcaactggat acataattgg agtgtataat ggaactgatg gaagaaatqc aaaaagaaga agtcatgctg aaagctgcac cataacagtt gctattaata aataaggaat tgtagaqgtt aatgaatgca caatagcatc gtccaaactc cataaaccct tctgtgtcct cagaccgctt cagaagtgtt *tgcacaaggg *gcaaaacagg ttttacttgg Iccttgtggtc -gagcaggata -caacagcaaa i cagtaacagc i aaggaattct -gataataatc -tatttgtttz ;ataaatgctt -ccttattccc t gaaagtaaaz t caacagcggt attttaaagtt t cggtcgCCg( a gcatcttac( a taacactgcc.
t tttgcacaac tgtggaatgt tgcaaagcat caggcagaag ctccgcccat taattttttt agtgaggagg tgcgagatat gacgcggact cctattttcc agcgaaaaat cgactgatgc ccgggtgcgt agctacgatc gaaqgcttca gaaatgtatc gttgatqact qgcgtcgtat ggcgttgttc tgcatccatg tgaaacctcg ccttgatggt gccgcatt cgaacgtacc ttatgagtgg acaaactacc gtqttaaact aatgggagca catctagtga gaaaggtaga tgtttagtaa tgctatacaa ataatcataa actatgctca atttgatgta ttacttgctt attgttgttg acaaatttca atcaatgtat aacctcctct cctgttaatt tctaagggta ggtaaacagc cccaacaCc aggcacattt atcaggaacc agtgttcatc tttggtcctg aaaatgaaaa *ccctgaatgc ttcccacatc *tgaagacgaa Igtttcttaga tttttctaaa caataatatt ttttttgcgq igatgctgaag aagatccttg ctgctatgtq atacactatt I gatggcatga I gccaacttac -atgggggatc gtgtcagtta 5880 gcatctcaat 5940 tatgcaaagc 6000 cccgccccta 6060 tatttatgca 6120 cttttttgga 6180 gtttgagaat 6240 catqtgaaat 6300 cctcgaacac 6360 acatcgtcac 6420 cttctgaaca 6480 tactqgcgcg 6540 acgacaacca 6600 tcgttattqa 6660 caaaagcgca 6720 atqttgttga 6780 tcgtcccgcc 6840 tttttaactt 6900 acacaggcaa 6960 acgctagtcc 7020 aaaaccatcc 7080 qacccacgcg 7140 gacgatqatt 7200 gccccqqatc 7260 tacaqaqatt 7320 actgattcta 7380 gtggtggaat 7440 tqatgaggct 7500 agaccccaag 7560 tagaactctt 7620 gaaaattatq 7680 catactgttt 7740 aaaattgtgt 7800 tagtgccttg 7860 taaaaaacct 7920 ttaacttgtt 7980 caaataaaqc 8040 cttatcatgt 8100 acttgagagg 8160 aggtcactta 8220 attttaaaat 8280 ccacaaatgt 8340 tqctcatcaa 8400 tccccacctg 8460 cagcactcca 8520 tgctgactqt 8580 tagtttgcta 8640 tttgaccctt 8700 aagtttaaca 8760 aaaatatttc 8820 agggcctcgt 8880 cgtcaggtgg 8940 tacattcaaa 9000 gaaaaaggaa 9060 cattttgcct 9120 atcagttggg 9180 agagttttcg 9240 gcgcggtatt 9300 *ctcagaatga 9360 icagtaagaga 9420 ttctgacaac 9480 atgtaactcg 9540 WO 00/42208 WO 0042208PCT/EPOO/00265 -16ccttgatcgt gatgcctgca agcttcccgg gcgCt cggcc gtctcgcggt ctacacgacg tgcctcactg tgatttaaaa catgaccaaa gatcaaagga aaaaccaccg gaaggtaact gttaqgccac gttaccagtg atagttaccg cttggagcga cacgcttcc agagcgcacg tcgccacctc qaaaaacqcc catgttcttt agctgatacc ggaagagcgc accgcctcaq tcctccccct caatgcgatg qtcaaggaag gaggctqatc atgctcgagc gcaaatattt gcacttggag ggtttagttt ccgtgagatt gtqccttcag tttagtagca gatattqgag ccacattttg qgagatqgag atggcacctg gaatcaaatt qqtqaaccaa aaccccttgg aagtaaaggc ccctgtccta ataaggcgtc ccttgtqaat gtcacacctg gccccgtact cccaagctac tcggtggtgg ggtccttggq atggtgagtg gaqgtaactg ggtgggctca ccgttgccca aaagagagta agaaaaggca tcttcagacg catccgccgt caaccccgac ctggttagac cctcggtggc tgggaaccgg gcaatggcaa caacaattaa cttccggctg atcattgcag gggagtcagg attaagcatt cttcattttt atcccttaac tcttcttgag ctaccagcgg ggcttcagca cacttcaaga gctgctgcca gataaggcgc acgacctaca gaagggagaa agggagcttc tqacttgagc agcaacgcgg cctgcgttat gctcgccgca ctgatgcggt aagccataga tgctgtcctg caatttcctc gcacggggga agcqagctct ggccgcttct cattaatgta ttgtgtctcc tgtctccgtt ttggataaqc taagatctcc ctccattttc ccaaactgcc ttaagaccaa ctggtgtggt tgctgtcaaa ctaggccatg attcaagccc cagtgcttag ctttgttgta gtcttagatt tgttttgaga caaaacccaa gaccagttgc ttagtcctag cagtggcagt tgagggggcc tggcaatgct tqttgcccgc tgaqgggtgc cagtggttac ttttgagcgc ccccaggggg cagttggagg gtcttgcgcg *ctcaagaccg *cgccacccgc -gcctttctcg -ggagtaccgt agctgaatga caacgttgcg tagactggat gctggtttat cactggggc caactatgga ggtaactgtc aatttaaaag gtgagttttc atcctttttt tggtttgttt gagcgcagat actctgtagc gtggcgataa agcggtcggg ccgaactgag aggcggacag cagggggaaa gtcgattttt cctttttacq cccctgattc gccgaacgac attttctcct gcccaccgca ccccacccca attttattag ggqgcaaaca agcatttagg ttattcttgg qttqtggcca tgtttcctgt taagtaaact tgataggtta atttctaaaq gtcaaatctt tttaacagcc agtgagttta ccacaaaqtt actaaggcca gccaattttt atctcctgca gttaacctca gttaatatcc tagttggt atcaatcctt ggctccagta tacggtcaaa ttttccattt aqttagaggg tgatgtttgc aagtttggag gaccattaga agatatttcc attttgggag aagcatgcca actctcttga accggtttcc cttcatcttg Fcctactttaa tgccgcccgc Iagaggttttc tcggaggccq agccatacca caaactatta ggaggcggat tgctgataaa agatggtaag tgaacgaaat agaccaagtt gatctaggtg gttccactga tctgcgcgta gccggatcaa accaaatact accgcctaca gtcgtgtctt ctgaacggqg atacctacag gtatccggta cgcctggtat gtgatgctcg gttcctggcc tgtggataac cqagcgcagc tacgcatctg tccccagcat ccc ccca ga a gaaaggacag acagatgqct tgacactata gcaatgtatg gaccagtcc gtaccgttta tgactgacaa ggcataaatc ttccaatatt ataataagat aaaactgaaa gcatctttct agcttatcat gttcctagtt gttttgaggg ttaatggcta agctttttgg aagttgtggg tgagcatcaa agtcctcctg actttagttt gtgtttaggt tgtgtataaa ggtgaggcag agggctagct tcgtgcacgg gqtgCggCgg aggtttatqt gtaaggttgc ttggaggtaa aacccattgg gtgtcatatg gatctcaaqc ttacatcatc cacggtgctc cgatccggtc acgggtttcc aacgacgagc actggcgaac aaagttgcag tctggagccg ccctcccgta agacagatcg tactcatata aagatccttt gcgtcagacc atctgctgct gagctaccaa gtccttctag tacctcgctc accgggttgg ggttcgtgca cgtgagctat agcggcaggg ctttatagtc tcaggggggc ttttqctggc cgtattaccq gagtcagtga tgcggtattt gcctgctatt tagaatgaca tgggaqtggc ggcaactaga qaatagggc aaaaagtgta atgaaaatga qtgtaatggt tgttactttt caacagcgtt ctgqgtccag gagcactttg ctgtagcaag ctgcatttaq tat t tt tgt t taggaaccat gatttgtgtt tggctgtagc aattgtttga ctgagtttat acqgataact ctacattaag gcaaggaagt cgtctgttac tgggctcttt tgatagtaag ttccttctga ttagcggggc cagccacagt ttgacttggt cggcctcgtc ctagaggttc gggatacaaa gatacacggg ctgccacacc agcagcacct agcctacctt gatgcggact gatccaagag gtgacaccac tacttactct gaccacttct gtgagcgtgg tcgtagttat ctgagatagg tactttagat ttgataatct ccgtagaaaa tgcaaacaaa ctctttttcc tgtagccgta tgctaatcct actcaagacg cacagcccag qagaaagcgc tcggaacagg ctgtcgggtt qgagcctatg cttttgctca cctttgagtq 5cgaggaagc cacaccgcat gtcttcccaa cctactcaga accttccaqg aggcacagtc ctctagatgc agaggatgtg catagagtat tagtgttaca ggcagtttta tgtatagqct qaaggaattg aactgttcca tatttgactg t cta cagt ta tcctactgta agccttgttt tggtgcatta gtcaaacatc agctgtaaac aaaaagaggg aacatcaagt ttgcatattg attattaata atgcaaagga caagtcaatg ggtactgcta cactgtgagg ctgtgattqc tagggcttct ttttttgaga cagagagagg ggataggcgc gggaggagta gttqaaggta tcacctcgac ccgccagaaa gcgactgtga cgctcaggtc tactggaaag 9600 9660 9720 9780 9840 9900 9960 10020 10080 10140 10200 10260 10320 10380 10440 10500 10560 10620 10680 10740 10800 10860 10920 10980 11040 11100 11160 11220 11280 11340 11400 11460 11520 11580 11640 11700 11760 11820 11880 11940 12000 12060 12120 12180 12240 12300 12360 12420 12480 12540 12600 12660 12720 12780 12840 12900 1296b 13020 13080 13140 13200 13260 WO 00/42208 WO 0042208PCTIEPOO/00265 accgcgaaga tcggtaccaa agtgggttct accgcccatt ttggtgccaa gtcaaaccgc gatgactaat ataatgccag atacacttga atggaaagtc ggggtcgttg catatatqgq tcaatgtcaa aagcagattc cgcgcactac ggcatcaqag tctatggtgc ccgctatcqc gcgccctqac gggagctgca gtttgtcctc gcttgggtct ctagttagcc tgcgtcaatg aacaaactcc tatccacc acgtaqatgt gcgggccatt tgtactgcca cctattggcg ggcggtcagc ctatgaacta cgcgtatatc ttcatgcaat tcagcgacct cagattgtac actctcagta tacgtgactg gggcttgtct.
tgtgtcagag aaccgcgagc ccctatagtg agagagct ct gggcggagtt cattgacgtc cattgatgta actgccaagt taccgtcatt agtgggcagt ttactatggg caggcgggcc atgaccccgt tggcccgtac tgtcggtcaa ccaacacaca tgagagtcga caatctqctc ggtcatggct gctcccggca gttttcaccg ccaacagcga agtcgtatta gcttatatag gttacgacat aatggggtgg ctgccaaaac aggaaagtcc gacgtcaata ttaccgtaaa aacatacgtc atttaccgta aattgattac atcgcgaagc gccttgcctt agcagggagc ccatagggqa tgatgccgca gcgccccgac tccgcttaca tcatcaccqa gctcgaattc atttcgataa acctcccacc tttgqaaagt agacttggaa cgcatcacca cataaggtca gggggcgtac tagtccaccc attattgacg agttatgtaa tattaataac agcgcaaaac gttgtagctt agatactggc tcgggagatc tagttaagcc acccgccaac gacaaqctgt aacgcgcgag aqatccqagc gccagtaagc gtacacgcct cccgttgatt atccccgtga tggtaatagc tgtactgggc ttggcatatg attgacgtca tcaatgggcg cqcggaactc taqtcaataa gcctaaccct aaattttgct ttaactatgc tcccgatccg agtatacact acccgctgac gaccgtctcc gcagc 13320 13380 13440 13500 13560 13620 13680 13740 13800 13860 13920 13980 14040 14100 14160 14220 14280 14340 14400 14455 <210> 16 <211> 10610 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: plasrnid <400> 16 gacggatcqg tctatgtcat tcctcttaca cgtgtttatt accaccacat caacctgcca aaaaagcatc ctgtcgaqcc catgtcgctg cggcgaagga gcggtggtgc atacaacatg tgtcctccqg cagcaccaca ggggaccaca cctcataaac ccggtaccat ggccaaaacc gagagcccag acacaggcac atcccaggga cacgtaactc cagtatggta gcgccgagac agtcatattt CtcgCCgCtt ggCgCCCCCt ccaccaccgc cgggaggagc aaaacctcaa tctacagcca gagatccgcg tttcatggga ctttttcata tttcaattgc agcttataca cctccctccc atatcatggg aaacgctcat tccagctgct gaaqtccacg tgcagcagcg gcagtggtct gcacagcagc atattgttca gaacccacgt acgctggaca.
ataaacctct tgcccgccgg gactcgtaac acgtgcatac acaacccatt acgttgtgca gcgcgggttt aaccgagatc cctgaagcaa agatcgctct ggcttcgggt agaataagcc gggaagagct aatgaagatc aagaacagat cggtacacag ctggtctggc cattgcccaa agaaaatttc gatcaccgta aacacacaga taacagacat cagtgatatt gagccacagg cctacatqggg cgcqaataaa cctcagcgat gcaccctgat aaatcccaca ggccatcata taaacattac gattaaacat ctatacactg catgqatcat acttcctcag cctgaatcag ttgtcaaagt ctgtctcaaa gtgttggtcg aaccaggtgc gtqtagtagt tctatgtaaa acacccagcc ggaagaacca tattaagtqa aatggcattt aattcaggaq cacaactaca gaataaagaa aagtcatttt ccttaatcaa gtacacagtc attcttaggt aataaactcc ctgctgtcca gqtagagtca ctgctgccgc gattcgcacc ctcacttaaa gtqcaaggcg ccacaagcgc ctcttttggc ggcgccatcc cagggaaccq catgctcgtc gattacaagc cgtaaatccc qttacattcg aggaggtaga tagtgtcatg gggcgtgaca tgtagtatat ctccttcatg aacctacaca tgtttttttt acgcgctccc gtaagatgtt acacaactcc ttaatqaaat tcgtttqtgt tcattcagta actcacagaa ctttctcccc qttatattcc ccqggcagct acttqcggtt taatcgtgca cgccgctccg gcccgcagca tcagcacagt ctgtatccaa aggtagatta atgttgtaat accaccatcc ggactggaac atgatatcaa tcctcccgcg acactgcag ggcagcagcg cqat ccctac ccaaatggaa aacagatctg ccactctctc cqccgctgcc ttcgttctgc tttattccaa ctccgqtggc gcacaatggC aagtgcatac atttgccaca tat gt t tca a gtatagcccc ccctagtatt ggctggcctt acacggtttc cacttaagtt gcttaacggg tcaggatagg tcctgcagga taaggcgcct aactgcagca aqctcatggc agtggcgacc tcaccacctc taaaccagct aatgacagtg tgttggcaca ttagaaccat gaagacctcg gatgatcctc tgtacggagt cgccggacgt cgtctccggt aaagcatcca.
Ctgataacat gagtcacaca aaqattatcc gtggtcaaac ttccaaaagg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 WO 00/42208 WO 0042208PCT/EPOO/00265 caaacggccc ataaacattc atatctctaa ccctccacct cctgtataag gcagggccag caggaacctt ccagcgtagc ctcaaaaaat agataaaggc atgtctgcgg aagcctgtct cggcgtgacc tcatgtccg gctaaaaagc acagccccca aaaccctcct cagcggcagc ctcgacacgq atatatagga cacgcgaacc ttccgttttC tcagtacaat tgqaggtcgc acaattgcat ccagatatac cattagttca ctggctgacc taacgccaat acttggcagt gtaaatggcc agtacatcta atgggcgtgg atgqgagttt ccccattgac tctggctaac gggagacccz gttgaggacE tcacgtccaa cagcaccttc gcaaatcccg tcagcctcaa attcaaaagc ctgaacataa gacaaaagaa cccgatgtaa caggcaaagc aggtaagctc gtttctgcat tacaacagqa gtaaaaaaaC agtcataatg gaccgaaata taggaggtat gcctaggcaa ctaacagtca caccagctca ctaaaaaatg tacgcccaga ccacgttacg ctgctctgat tgagtagtgc qaagaatctg gcgttgacat tagcccatat gcccaacqac agggactttc acatcaagtg cqcctggcat cgtattaqtc atagcgqttt gttttgqcac gcaaatgggc tagagaaccc Lagcttgqtac iaactcttcgc gtggacgtaa aaccatgccc aatattaagt gcagcgaatc ggaacattaa tcgtgcaggt cccacactga gctttgttgC ctcgcgcaaa cggaaccacc aaacacaaaa aaaacaaCCC tggtcaccgt taagactcgg gcccggggga aacaaaatta aatagcaccc gccttaccag atcagtcaca acgtaacgqt aacgaaagcc taacttcccg gccgcatagt gcgagcaaaa cttagggtta tgattattga atggagttcc ccccgcccat cattgacgtc tatcatatgc tatgcccagt atcgctatta gactcacggg caaaatcaac ggtaggcgtc actgcttact cgagctcggE qgtctttccz agqqacctgz cagtcacagt ;ttgtttctg( ;atggtcgagc a agatacctt( tcttactccl tttqcgccti a cggcctctcl :acctctcaa.
7ctcagaaqci :catgcaatc, g acccctcac c cgatagcag t gggcattga g ggctccttt t gactattaa a aggcaatat t tatacttga a gggccctct a cttgtttac q gttgatgtt c acctaatgc a ttcaaacaa [c cattacagt aggctaaacc aaataattct ccggccattg atgattgcaa caaaaatacc ctgcacggac ttatgacacg atgggcggcg aaagaaagca acagaaaaag taaaataaca ttataagcat gattaaaaag taaacacatc atacataccc ataggagaga tcccgctcca taaaaaagaa gtgtaaaaaa taaagtccac aaaaaaccca gatcct ctcc taagccagta tttaagctac ggcgttttgc ctagttatta gcgttacata tgacgtcaat aatgggtgga caagt acgcc acatgacctt ccatqgtgat gatttccaag gggactttcc Itacggtggga :ggcttatcga itctgaattcg igtactcttgg igcqagtccgc :cgcaaggtag j cggaggtgct I tgaggtgtgg aaccccgtgt :ccctttgtat a tccgaacctc t ctqgacgagq a aaaaccaagt c ctaactgtgj a caggccccgC a gtgtcagaa( t acccttacti c ttgaaagag( g catgtaaca( t aatacttCCI g caacttaati t gttagttat4 :a gcttcaaac, .t gacgctaca' !a ccaaacaca Lg gctatggtt a ggaaacaaa cttcagggtg a catctcgcca c taaaaatctg c aaattcaggt t gcgatcccgt a cagcgcggcc a catactcgga g atataaaatg c catcgtagtc a acaccatttt t aaaaaacatt t aagacggact a caccaccgac a aggttgattc a gcaggcgtag a aaaacacata a gaacaacata c aacctattaa z gggccaagtg c aaaaaacacc caacttcctcz cgat~ccccta tctgctccct aacaaggcaa gctgcttcgc atagtaatca acttacggta aatgacgtat ctatttacgg1 ccctattgac atgggacttt gcggttttgg tctccacccc aaaatgtcqt ggtctatata aattaatacg agctcgctgt atcggaaacc atcgaccgga gctgagcacc gctgatgatg caggcttgag *atccatatga *cccccaatgg tagttacctc Iccggcaacct caaacataaa ctgccgccgc taaccgtqca I gaaaqctagc i tcactgcctc ccatttatac Sacgacctaaa tgcaaactaa ;tagcaggagg cgtttgatgc t cagcccacaa a attccaaaaa g ccatagccat a atcccctcaa ctaaactagg a ataatgataa atctcctct 1920 cttctcaat 1980 'tccagagcg 2040 cctcacaga 2100 ggtcccttc 2160 ,cttccccgc 2220 rctatgctaa 2280 :aaggtgctg 2340 *tgctcatgc 2400 .ctctcaaac 2460 .aaacattag 2520 Lcggccatgc 2580 Lgctcctcgg 2640 Ltcggtcagt 2700 igacaacatt 2760 Lacacctgaa 2820 ~agcgcttca 2880 ~aaaacacca 2940 agagcgagt 3000 ~agaaaaccg 3060 ~aatcgtcac 3120 Lggtcgactc 3180 jcttgtgtgt 3240 jgcttgaccg 3300 jatgtacggg 3360 attacggggt 3420 aatggcccgc 3480 gttcccatag 3540 taaactgccc 3600 gtcaatgacg 3660 cctacttggc 3720 cagtacatca 3780 attgacgtca 3840 aacaactccg 3900 agcagagctc 3960 actcactata 4020 tgggctcgcg 4080 cgtcggcctc 4140 tcggaaaacc 4200 gtggcgggcg 4260 taattaaagt 4320 atccaagatg 4380 cacggaaacc 4440 gtttcaagag 4500 caatggcatg 4560 tacctcccaa 4620 cctggaaata 4680 acctctaatg 4740 cgactccaaa 4800 cctgcaaaca 4860 accccctcta 4920 acaaaatgga 4980 cactttgacc 5040 agttactgga 5100 actaaggatt 5160 tcaaaaccaa 5220 cttggatatt 5280 gcttgaggtt 5340 taatgcagga 5400 aacaaaaatt 5460 aactggcctt 5520 gctaactttg 5580 cgaacggtac tccgccacc tctcgagaaa ggcgtctaac gcagcgggtg aggcggtctt aagcgcgcaa ggtcctccaa agtccccctg cttgcgctca aatgtaacca tctgcacccc gtcgcgggca cttagcattg tcaggccccc actactgcca aaactaggac gtagcaactq gccttgggtt gattctcaaa ctaaatctaa aactacaaca aacctaagca gatgggcttg ggccatggcc agttttgaca gcggtcggg( gagacggcgs gaccgtctgz ctgtgccttt gggtactct( aaatgggcai ctgtgagcc( tcacagttal acacactca( ccacccaag, tcaccacca ctggtagct taaagtacg gtccaggtq ttgattcac acagacgcc gactaggac aaggccttt ctgccaagg aatttggtt tagaatttg gcacaggtq.
WO 60/42208 WO 0042208PCTIEPOO/00265 -19tggaccacac actttggtct aaaggcagtt gacgaaaatg aatggagatc tcagcttatc ttaaacggag acaggagaca aactacatta taaagaagcg gctaqagctC ccctcccccq aatgaggaaa gggcaqgaca ggctctatgg ccctqtagcg cttgccagcg gccggctttc ttacqgcacc ccctgataga ttgttccaaa attttgga aattaattct ggcagaagta ggctccccag ccgcccctaa catggctgac ttccagaagt gcttgtatat gaacaagatc gactgggcac gggCqCCcg qaggcagcgC gttgtcactc.
ctgtcatctc ctgcatacg( cgagcacgti caggggctc( gatctcgtc( ttttctggal ttggctacc, ctttacggt, ttcttctga cacgagatt gggacgccg ccaacttqt caaataaag cttatcatg tgt t tcct g taaagtgta cactgcccg gcqcgggga tgcgctcq tatccacac ccaggaacc agcatcacz accaggcgt ccggatacc gtaggtatc ccgttcag( gacacgact taggcggt( cagctccatc taacaaaatg tggctccaat gagtgctact ttactgaagg caaaatctca acaaaactaa caactccaag atgaaatatt gccgctcgag gctgatcagc tgccttcctt .ttgcatcgca gcaaggggga cttctgaggc gcgcattaag ccctagcgcc cccgtcaagc tcgaccccaa cggtttttcg ctggaacaac tttcggccta gtggaatgtg tgcaaaqcat caggcagaag ctccgcccat taattttttt *agtqaggagg *ccattttcgg Igattgcacgc aacagacaat ttctttttgt ggctatcgtc aagcgggaac accttgctcc ttgatccggC actcggatgg j cqccagccgz j tgacccatqc t tcatcgact( c gtgatattgc a tcgccgctc( g cgggactcti t cgattccac, g ctggatgat t tattqcagc c atttttttc t ctgtatacc t gtgaaattg a agcctgggg tttccagtc g aggcqgttt [t cgttcggct ra atcagggga :g taaaaaggc La aaatcgacc :t tccccctgq 't gtccgcctt -t cagttcggt -c cgaccgctc :t atcgccact Ic tacagagtt tcctaactgt tggcagtcaa atctqgaaca aaacaattcc cacagcctat cggtaaaact acctgtaaca tgcatactct tgccacatcc cat gca tct a ctcgactgtg gaccctggaa ttgtctgagt ggattgggaa ggaaagaacc cgcggcgggt cgctcctttc tctaaatcgg aaaacttgat ccctttgacg actcaaccct ttgqttaaaa tgtcagttag gcatctcaat tatgcaaagc cccgccccta tatttatgca *cttttttgga atctgatcaa aggttctccg *cggctgctct icaagaccgac Igqtggccacc Iggactggctc tqccgagaaE tacctgcccE i agccggtctt i. actgttcgc 3 cgatgcctg( I tggccggCt( :tgaagagctl 7cgattcgcai 3 gggttcgaai zgccgccttCl c ctccagcgcl t tataatggt' a ctgcattct.
g tcgacctct t tatccgctc t gcctaatga g qgaaacctg g cgtattggg g cggcgagcg t aacgcagga c gcgttgctg [c tcaagtcag la agctccctc :g taggtcgtt ic gccttatcc :g gcagcagcc :c ttgaagtgc agactaaatg atacttgcta gttcaaagtg ttcctggacc acaaacgctg gccaaaagta ctaaccatta atgtcatttt tcttacactt gagggcccta ccttctaqtt ggtgccactc aggtgtcatt gacaatagca agctggggct gtggtggtta gctttcttcc gqcatccctt tagggtgatg ttggaqtcca atctcggtct aatgagctga ggtqtggaaa tagtcagcaa atgcatctca actccgccca gaggccgagg ggcctaggct gagacaggat gccgcttggg *gatgCCgccg -ctgtccggtg facgggcgttc Ictattggg igtatccatca ittcgaccacc gtcgatcagg aggctcaagg -ttgccgaata 3 ggtgtggCg ggcggcgaat I cgcatcgcct a tgaccgaccz t atgaaagqtt 9 gggatctcat t acaaataaaC.
a gttgtggttt a gctagagcti a caattccaci g tgagctaacl t cgtgccagcl c gctcttccgl g tatcagctc a agaacatgt, 'g cgtttttcc a ggtggcgaa :g tgcgctctC Ig gaagcgtgg :c gctccaagc :g gtaactatc .a ctggtaaca It ggcctaact cagagaaaga t cagtttcagt t ctcatcttat t.
cagaatattg g.
ttggatttat g acattgtcag t cactaaacgg t catgggactg g tttcatacat t ttctatagtg t gccagccatc t ccactgtcct t ctattctggg g ggcatgctgg g ctagggggta t cgcgcagCgt g cttcctttct C tagggttCCg a gttcacgtag t cgttctttaa t attcttttga t tttaacaaaa gtccccaggc t ccaggtgtqg a attagtcagc a gttccgccca t ccqcctctgc c tttgcaaaaa g gaggatcgttt tggagaggctz tgttccqCt ccctgaatqa cttqcgcagc aagtgccgq tggctgatgC aagcgaaacaI atgatctgqa cgcgcatgcc tcatggtgga faccgctatca gggctgaccg tctatcgcct iagcgacgccc :gggcttcgga :gctggagttc j caataqcatc :gtccaaactC :ggcgtaatca a caacatacga cacattaatt L gjcattaatga a ttcctcgctc a ctcaaaggcg g agcaaaaggc a taggctccgc a cccgacagga tgttccgacc c gctttctcaa t gggctgtgtg g tCttgaqtCC g gattagcaga a cggctacact gctaaactc 5640 ttggctgtt 5700 ataagattt 5760 aactttaga 5820 cctaaccta 5880 caagtttac 5940 acacaggaa 6000 tctggccac 6060 gcccaagaa 6120 cacctaaat 6180 gttgtttgc 6240 tcctaataa 6300 ggtggggtg 6360 gatgcggtg 6420 ccccacgcg 6480 accgctaca 6540 gccacgttc 6600 tttagtgct 6660 gggccatcg 6720 agtggactc 6780 ttataaggg 6840 .tttaacgcg 6900 .ccccaqgca 6960 aagtcccca 7020 accatagtc 7080 ~tctccgccc 7140 :tctgagcta 7200 ~ctcccgqga 7260 :cgcatqatt 7320 ittcggctat 7380 ~tcagcgcag 7440 actgcaggac 7500 :gtgctcgac 7560 )caggatctc 7620 3atgcggcgg 7680 tcgcatcqag 7740 cqaagagcat 7800 cgacgqgcgag 7860 aaatggccgc 7920 gqacatagcg 7980 cttcctcgtg 8040 tcttgacgag 8100 aacctqccat 8160 atcgttttcc 8220 ttcgcccacc 8280 acaaatttca 8340 atcaatgtat 8400 tggtcatagc 8460 gccggaagca 8520 gcgttgcgct 8580 atcggccaac 8640 actgactcgc 8700 gtaatacggt 8760 cagcaaaagg 8820 ccccctgacg 8880 ctataaagat 8940_ ctgccgctta 9000 tgctcacgct 9060 cacgaacccc 9120 aacccggtaa 9180 gcgaggtatg 9240 agaaggacag 9300 WO 00/42208 WO 0042208PCT/EPOO/00265 tatttggtat gatccggcaa cgcgcagaaa agtggaacga cctagatcct cttggtctga ttcgttcatc taccatctgg tatcagcaat ccgcctccat atagtttgcg gtatggcttc tgtgcaaaaa caqtgttatc taagatgctt ggcgaccgag ctttaaaagt cgctgttgag ttactttcac gaataagggc gcatttatca aacaaatagg ctgcgctctg acaaaccacc aaaaggatct aaact cacgt tttaaattaa cagttaccaa catagttgcc ccccagtgct aaaccagcca ccagtctatt caacgttgtt attcagctcc agcggttagc actcatgqtt ttctgtgact ttgctcttgc gctcatcatt atccagttcg cagcgtttct gacacggaaa gggttattgt ggttccgcgc ctgaagccag gctggtagcg caagaagatc taagggattt aaatgaagtt tgcttaatca tgactccccg gcaatgatac gccggaaggg aattgttgcc gccattgcta ggttcccaac tccttcggtc atggcagcac ggtgagtact ccggcgt caa qgaaaacgtt atgtaaccca gggt gaqcaa tgttgaatac ctcatgagcg acatttcCc ttaccttcgg gtggtttttt ctttgatctt tggtcatgag ttaaatcaat gtgaggcacc tcgtgtagat cgcgagaccc ccgagcgcaq gggaagctaq caggcatcgt gatcaaqgcq ctccgatcqt tgcataattc caaccaagtc tacgggataa cttcggggcg ctcgtgcacc aaacaggaag tcatactctt qatacatatt gaaaagtgc aaaaagagtt tgtttgcaag ttctacgggg attatcaaaa ctaaagtata tatctcagcg aactacgata acgctcaccg aaqtggtcct agtaagtagt ggtgtcacgc agttacatga tgtcagaagt tcttactgtc attctgagaa taccgcgcca aaaactctca caactgatct gcaaaatgc cctttttcaa tgaatgtatt acctgacgtc ggtagctctt cagcagatta tctgacgctc aggatcttca tatgagtaaa atctgtctat cgggagggct gctccagatt gcaactttat tcgccagtta tcgtcgtttg tcccccatgt aagttggccg atgccatccg tagtgtatgc catagcagaa aggatcttac tcagcatctt qcaaaaaagg tat tattqaa taqaaaaata 9360 9420 9480 9540 9600 9660 9720 9780 9840 9900 9960 10020 10080 10140 10200 10260 10320 10380 10440 10500 10560 10610 <210> 1'7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 17 tgtacaccgg atccggcqca cacc <210> 18 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 18 cacaacgagc tcaattaatt aattgccaca tcctc <210> 19 <211> 4 <212> PRT <213> adenovirus <400> 19 Thr Leu Trp Thr 1 <210> <211> 12 <212> PRT <213> adenovirus WO 00/42208 PCT/EP00/00265 -21- <400> Pro Ser Ala Ser Ala Ser Ala Ser Ala Pro Gly Ser 1 5 <210> 21 <211> 44 <212> PRT <213> adenovirus <400> 21 Met Lys Arg Ala Arg Pro Ser Glu Asp Thr Phe Asn Pro Val Tyr Pro 1 5 10 Tyr Asp Thr Glu Thr Gly Pro Pro Thr Val Pro Phe Leu Thr Pro Pro 25 Phe Val Ser Pro Asn Gly Phe Gln Glu Ser Pro Pro <210> 22 <211> 43 <212> PRT <213> adenovirus <400> 22 Met Ala Lys Arg Ala Arg Leu Ser Thr Ser Phe Asn Pro Val Tyr Pro 1 5 10 Tyr Glu Asp Glu Ser Ser Ser Gln His Pro Phe Ile Asn Pro Gly Phe 25 Ile Ser Pro Asp Gly Phe Thr Gln Ser Pro Asn <210> 23 <211> 43 <212> PRT <213> adenovirus <400> 23 Met Ser Lys Arg Leu Arg Val Glu Asp Asp Phe Asn Pro Val Tyr Pro 1 5 10 Tyr Gly Tyr Ala Arg Asn Gln Asn Ile Pro Phe Leu Thr Pro Pro Phe 25 Val Ser Ser Asp Gly Phe Lys Asn Phe Pro Pro <210> 24 <211> 42 <212> PRT <213> adenovirus <400> 24 Met Lys Arg Ala Arg Phe Glu Asp Asp Phe Asn Pro Val Tyr Pro Tyr 1 5 10 WO 00/42208 WO 0042208PCT/EPOO/00265 -22- Giu His Tyr Asn Pro Leu Asp Ile Pro Phe Ile Thr Pro Pro Phe Ala 25 Ser Ser Asn Gly Leu Gin Glu Lys Pro Pro <210> <211> 42 <212> PRT <213> adenovirus <400> 25 Met Lys Arg Thr Arg Asp Thr Ser Ser Thr Ile Giu Asp Asp Phe 10 Asn Pro Val Tyr Pro Tyr Pro Phe Val Pro Ser Ile Pro 25 Tyr Val Ala Pro Ser Ser Asp Giy Leu Gin Giu Asn <210> 26 <211> 327 <212> DNA <213> adenovirus Pro Pro <400> 26 agatctgaat ccagtactct tgagcgagtc agtcgcaagg tggcggaggt aggtgaggtg tcgagctcgc tggatcggaa cgcatcgacc taggctgagc gctgctgatg tggcaggctt tgttgqgctc gcgqttgaqq acaaactctt acccgtcggc ctccgaacgg tactccgcca ggatcggaaa acctctcqag aaaggcgtct accgtggcgg gcgqcagcgg gtgqcggtcq atgtaattaa agtagqcggt cttgagacqg gagatct cqcggtcttt ccgagqgacc aaccaqtcac gggttgtttc cqgatggtcg <210> 27 <211> 32480 <212> DNA <213> adenoviruS <400> 27 catcatcaat ttgtgacgtg gatgttgcaa gtgtgcgccg taaatttggg agtgaaatct aagcttgaat tatgcttccg cagctatgac aggaacttgg agggtgtgga ttagtcagca catgcatctc aactccgccc agaggccgag aggcctaggc tcgaacactt atcgtcacct tctgaacaat aatatacctt gcgcggggCg gtgtggcgga.
gtgtacacag cgtaaccgag gaataatttt tcgattaatg gctcgtatgt catgattacg ttaggtacct aagtccccag accaggtgtg aattagtcag agttccgccc gccgcctcgg ttttgcaaaa tttaagccgt gggacatgtt ggaaaggcat attttggatt tgggaacggg acacatgtaa gaagtgacaa.
taaqatttgg gtgttactca tgagttagct tgtgtggaat aattcggcgc tctgaggcgg gctccccagC gaaagtcccc caacczatagt attctccgcc cctctgagct agcttgggat agataaacag gcagatccat tattgccgta gaagccaata gcgqgtgacq gcgacggatg ttttcgcgcg ccattttcgc tagcqcgtaa cactcattag tgtgagcgga agcaccatgg aaagaaccag aggcagaagt a ggctcccc a cccgcccct a ccatggctga attccagaag ctctataatc gctgggacaC gca cgtaa ac agccgtqgg tgataatgag tagtaqtqtg tggcaaaagt gttttagg gggaaaactg tctctagcat gcaccccagg taacaatttc cctgaaataa ctgtggaatg atgcaaagca gcaggcagaa actccgccca ctaatttttt tagtgaqgag tcgcgcaac ttcacatgag tcgcaagccg gtctggtacc gggqtqgagt gcggaagtgt 120 gacgtttttg 180 gatqttgtaq 240 aataagagga 300 cgatgtcgac 360 ctttacactt 420 acacaggaaa 480 cctctgaaag 540 tgtgtcagtt 600 tgcatctcaa 660 gtatgcaaag 720 tcccgcccct 780 ttatttatgc 840 qcttttttgg 900 tattttcccc 960 cgaaaaatac 1020 actqatgcct 1080 ggtggqtgaa 1140 WO 00/42208 WO 0042208PCT/EPOO/00265 -23gaccagaaac ggcgagatcg cttaatcgcc accgatcgcc ccggcaccag gtcgtcgtcc acctatccca tcgctcacat gatggcgtta qacagtcgtt ctcgcqgtga cggatgagcg gatttccatg qttcaqatgt gaaacgcagg ggttatgccg gaaatcccga gaagcagaag ctgaacggca ggtcaggtca tttaacgccg cgctaccjgcc aatcgtctga gtgcagcgcg cacggcgcta gtqcagtatg gcgcgcgtgg ctttcgctac aacagtcttg ggcggcttcg ccgtggtcgg aacggtctgg cagcagtttt ttccgtcata gcaagcgqtg gaactaccgc aacgcgaccg qaaaacct ca gaaatggatt tttctttcac ttcacccgtg aacgcctggg cagtgcacgg catcagggqa atggcgatta ctgaactgcc gaaaactatc gacatgtata ttgaattatg caacagcaac ctgaatatcg tcggcggaat taataataac tatttaaaaa atgggagcct agtgatacgg tttggtctgc aaagcaatag gtttgtccaa agggtgggaa gccgccgccg ac-gcgcatgc agcacctcga atcccgtcgt ttgcagcaca cttcccaaca aagcggtgcc cctcaaactg ttacggtcaa ttaatgttga actcggcgtt tgccgtctga tggtgctgcg gcattttccg ttgccactcg gcgqcgagtt tcgccagcgg atcgcgtcac atctctatcg cctgcgatgt aqccgttgct tggatgagca tgcgctgttc t gtat gtggt ccgatgatcc atcgtaatca atcacgacgc aaggcggcgg at gaaga cc a ctggagagac gcgqtttcqc t ctgggactg cttacggcgg tctttgccga tccagttccg gcgataacga aagtgcctct agccggagag catggtcaga gtgtgacgct tttgcatcga aqatgtggat caccqctgga tcgaacgctg cagatacact aaaccttatt ccgttgatgt agctggcgca ccgaccgcct ccccgtacgt gcccacacca tgatggaaac acggtttcca tccagctgag cgggcaggcc acacaaactt acttcccgtt gtattatttt tttctgacaa catcacaaat actcatcaat agaatatata ccatgagcac ccccatgggc actgagccgc tttacaacgt tccccctttc gttgcgcagc ggaaagctgg gcagatgcac tccgccgttt tgaaagctgg tcatctgtgg atttgacctg ttggagtgac tgacqtctcg ctttaatgat gcgtgactac caccgcgcct actacgtctg tgcggtggtt cggtttccgc gattcgaggc gacgatggtg gcattatccg ggatgaagcc gcgctggcta cccgagtgtg gctgtatcgc agccgacacc gcccttcccg gcgcccgctg taaatactgg ggtggatcag tgattttggc ccgcacqccg tttatccgg qct cctgcac ggatqtcgct cgccgggcaa agccgggcac ccccgccgcg gctgggtaat tggcgataaa taacgacatt gaagqcgqg tgctqatgcg tatcaqccgg tgaagtggcg ggtagcaqag tactgccgcc cttcccgagc gtggcgCggC cagccatcgc tatggggatt cgccggtcgc atgtctgccc ttggatgttc tttcccgatt tgccgctatt actcggaact ttcacaaata gtatcttatc agqtggggqt caactcgttt cggggtgcgt gatattgccc cqtgactggg gccagctgqc ctgaatggcg ctggagtgcg ggttacgatg gttcccacgg ctacaggaag tgcaacgggc agcgcatttt ggcagttatc ttgctgcata gatttcagcc ctacgggtaa ttcggcggtg aacgtcgaaa gaactqcaca gaggtgcgga gttaaccqtc caggatatcc aaccatccgc aatattgaaa ccggcgatqa atcatctggt tggatcaaat acggccaccg gctgtgccga atcctttgcg caggcgtttc tcqctgatta qatacgccga catccagcgc caaaccatcg tggatggtgg ccacaaqgta ctctgqctca atcagcgcct tcccacgcca aagcgttggc aaacaactgc qgcgtaagtq ggccattacc gtgctqatta aaaacctacc agcgatacac cqggtaaact tgttttgacc gaaaacggtc gacttccagt catctgctgc ggtggcgacg taccattacc gtatttcgcg ggt ttatt ct tggctacatg tctctgttct tgtttattgc aagcattttt atgtctggat cttatgtagt gatggaagca cagaatgtga agcgtttcaa aaaaccctgg gtaatagcga aatggcgctt atcttcctga cgcccatcta agaatccgac gccagacgcg gctgggtcgg tacgcgccgg tggaagatca aaccgactac gcgctgtact cagtttcttt aaattatcga acccgaaact ccgccgacgg ttgaaaatgg acgagcatca tgctgatgaa tgtggtacac cccacggcat gcgaacgcgt cgctggggaa ctgtcgatcc atattatttg aatggtccat aatacgccca gtcagtatcc aatatgatga acgatcgcca tgacqgaagc aagtgaccag cgctggatgg aacagttgat cagtacgcgt ggcagcagtg tcccgcatct aatttaaccq tgacqccgct aagcgacccg aggccgaagc cgaccgctca ggattgatqg cgcatccggc ggctcggatt gctgggatct tgcgctgcgg tcaacatcag acgcggaaga actcctggag agttggtctg taaggaaatc ttttctttta acatcaacca cgctattatt agcttataat ttcactgcat ccagatctgg tttgtatctg ttgtgagctc tgggctccag cgcgctgtat cgttacccaa agaggcccgc tgcctggttt ggccgatact caccaacgta gggttgttac aattattttt ttacggccag agaaaaccgc ggatatgtgg acaaatcagc ggaggct gaa atggcagggt tgagcgtggt gtggagcgcc cacgctgatt tctgctgctg tcctctgcat qcagaacaac gctgtqcgac 5gtqccaatg aacgcgaatg tgaatcaggc ttcccgcccg cccqatgtac caaaaaatgg cgcgatgggt ccgtttacag aaacggcaac gttctgtatg aaaacaccag cgaatacctg taagccgctg tgaactgcct aqtgcaaccg gcgtctqgcg gaccaccagc ccagtcaqgc gcgcgatcag cattgaccct agcgttgttg cgcgtggcag tagtggtcaa gcggattggc agggccgcaa gccattgtca gacgcgcgaa ccgctacagt aqqcacatgg cccgtcagta gtgtcaaaaa cattatgtac cttttttatc tatcagcaaa ccaaccgctg ggttacaaat tctagttgtg gcgtggctta ttttgcagca atatttgaca cattgatggt 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 .3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4566 4620 4680 4740 4800 4860 WO 00/42208 WO 0042208PCTIEPOO/00265 -24cgccccgtcc ttggagactg actgactttg gatgacaagt gtttctcagc cccaatgcgg gtgtcttgct cggtcgttga agatacatgg tgcggggtgg atgtctttca cqgttaagct aggttggcta acagtgtatc aacttggaqa atgggcccac tgttccagga tgcggtataa cacgctttga tccggggtag caqccggtgg caqctgccgt ttttccctqa gaagcaaagt ccaagcagtt atatctcctc ccagacgggc tcacggtgaa tgctggtgct tggtgtcata aggcgccgca ccgattccgq gccaggtgag gtttcttacc tgtccccgta atagaaactc agtgggaggg acatgtcgcc cgggtgttcc ccgcatcgct tgacttctgc ccgcggtgat tgtcaagctt gcagggtttg attcgcgcgc gcacgcgcca gtaggcgctc ggtctagctc gcgcgtcgaa cggcaagcgc cggaggcgtE atgtagggtz agggagcga( tctgcctgai tggcgtctgt tgaccagct( tgtcatactl ggtctttcc tgtagaact( cctgcgcggi tgaggtacti ccgtgcgct tgcccgcaaa cagcctccgc ctttcctgag tgacggctct agctgttgga tttaaaacat gtctttattt gggtcctqtg gcataagccc tgttgtagat gtagcaagct gggatgggtg tgttcccagc cggtgcactt cgcccttqtg gggCqqCggC tgagatcgtc tggttccatc gttcagatgg gggagatcag gcccgtaaat catccctgag ccaaatccgc ttttcaacgg ccagcjcggtc gtttcgcggg cagggtcatg ggggtgcgct gaaqcgctgc gtccagcccc cgagggcgag ggagtaggca ctctggccgt tctggtttcc tacagacttg ggaccactct gtaqcggtcg ctcttcggca tqaagggggg gtctgcgagg gctaagattg gcctttqagg gqtggcaaac gtttttgtcg aacgcaccgc accgcggttg gttggtccaq Icgtctcgtcc kgtagtctatc :gcgctcgtat icatgccgcaz igcatcttccF jgaggtcgggE i gatggcatgt gagacctac( ggcggtgac atcctgtcc( igtactcttg( Sgttgacggc( cttccggag( g gtatttgaa( t tttggaacgi ctctactacc cgccgcttca cccgcttgca tttggcacaa tctgcgccag aaataaaaaa aggggttttg tattttttcc gtctctgggg gatccagtcg gattgccagg catacgtggg catatccctc gggaaatttg acctccaaga ctggqcgaag ataggccatt cggcccaggg qgggatcatg ctggqaagaa cacacctatt caggggggcc cagaagqgcg tttgagaccg ccacagctcq ttggggcggC tctttccacg ccgggctgqq cggtcttcgc tccgcggcqt tqcagacttt t ccgcgCCgC tcggggtcaa atgagccggt agaggcctgt qagacaaagg ttgtccacta tcaagqaagg ctataaaagg gccagctgtt tcagtttcca gtggccgcat gacccgtaga cgatcggcgc cattcgggaa tgcagggtga cagaggcggc ggggggtCtg ttgcatcctt gggttgagtq tatgtcgtaaa iccgcggatgc iccgaggttgc gagttggatc gcgtcacgce tgcacgtctz ttttttttcc I atcggaaac tggtaggcg( gaggtgtgg( j tcagtgtcgi :ggatttggc ttgacctacg gccgctgcag agcagtgcag ttggattctt caggtttctg ccagactctg cgcgcgcggt aggacgtggt tggaggtagc tagcaggagc ggcaggccct gatatgagat cggggattca tcatgtagct ttttccatgc atatttctgg tttacaaagc gcgtagttac tctacctgcg agcaggttcc accgggtgca acttcgttaa tcgccgccca tccgccgtag gtcacctgct tttcgctgta ggcgcagqgt cgctggccaq cctgcgcgtc ggcccttggc tgagggcgta aggccccgca aaaccagqtt gtccacgctc cctcgagcgg ctcgcgtcca gggggtccaC tgattggttt gggtggggqC ggggtqagta aaaacgagga ccatctggtc gggcgttgga gctccttggc aqa cggtggt caaggtcaac cgcccttgcq cgtccacggt gcaagtctag ggggacccca cgtagagggg *tgqcgcgcac *tacgggcggg Iatatggttgg kcqaaggaggc kgggcgcagta acagctcgcq cgtcggcctC agcatccctt j tgagcgcaaz cgcatccgcc i gggcgaaggt agaccgtgtc ccaccgcccg cttcccgttc tgacccggga ccctgaaggc tttggatttg aggcccggga aaaggtgact accactgcag gctgggcgtg tggtgtaagt gcatcttgga tgttgtgcag tagaaggaaa attcgtccat gatcactaac gcgggcggag cctcacagat gggcqatgaa tgagcagctg actggtagtt gcatgtccct gcgatagcag gcatgctttt ctacggcatc cggcagtagt cctcgtcagc ggtgcgcttg qgccaggtag gcgcagcttg gagcttgggc gacggtctcg tcccccatgc ggtgacgaaa tgttccgcgg ggccagcacg tcgctccaqg gtaggtgtag gcgttcgtcc ctccctctga ggatttqata agaaaagaca cagcaacttg cgcgatgttt gcgctcgtcg gctggtggct cgagcagaat aaagaccccg cgcctgctgc tggcatgggg ctctctgagt gtaatcgtat ctgctctgct acgctggaag gtaggagtcg gtccagggtt Igttgagqaca cgaacggtaa ttctacgggt ggtgtccctg ctgctcccag gacatcgttg tggaacgccg 4920 cgggattgtg 4980 atccgcccgc 5040 acttaatgtc 5100 ttcctcccct 5160 gatcaagcaa 5220 ccagcggtct 5280 ctggatgttc 5340 agcttcatgc 5400 gtgcctaaaa 5460 gtttacaaag 5520 ctgtattttt 5580 aaccaccagc 5640 tgcqtggaag 5700 aatgatqgca 5760 gtcatagttg 5820 ggtgccagac 5880 ttgcatttcc 5940 gaaaacggtt 6000 cgacttaccg 6060 aagagagctg 6120 gactcgcatg 6180 ttcttgcaaq 6240 gagcqtttga 6300 tcqatccagc 6360 cggtgctcgt 6420 gtagtctggg 6480 aggctggtcc 6540 catttgacca 6600 cccttggaqg 6660 gcgaqaaata 6720 cattccacga 6780 tttttqatgc 6840 agqctgtccq 6900 tcctcctcgt 6960 aaggaggcta 7020 qtgtgaaqac 7080 gccacgtgac 7140 tcactctctt 7200 aaagcgggca 7260 ttcacctggc 7320 atctttttgt 7380 gcgatgqagc 7440 agctqcacgt 7500 ggcaccaggt 7560 acctctccgc 7620 qgcggtagqg 7680 qgcagcaggc 7740 catgcqcggq 7800 tgggtgagcq 7860 attccaagat 7920 agttcgtgcg 7980 cggaaqacta 8040 acgttgaagc 8100 cgcagcttgt 8160 tccttgatga 8220 aactcttcgc 8280 gagcctagca 8340 agcgcgtatg 8400 accatgactt 8460 agcaaaaagt 8520 aagagtatct 8580 WO 00/42208 WO 0042208PCT/EPOO/00265 ttcccgcgcg tgttaattac tgtaaagttc aggtgagctc ggttggaagc gaaaggtcct gcgggtcttg ctagaggctc aggcccccat gatgcgagcc tgtggtgaaa gtgcgcagta cgcgcacaag cttctactt~c ggaccaccac tgacaacatc gcgggagctc gatacctaat cccgcggCgc atgcatctaa cgggagagqq taqgttgctg gaagacqacg gtcgttgacg gatctcgqcc cacggtggcg tccctcgttc cacctgcgcg aaagaggtag tcgcaacgtg gt ccacggcq aagacggatq ttcttcttct tgggggaggg gatcatctcc gcgcagttgg cggcagggat gagggacctg ccagtcacag gttgtttctg gatggtcgac catgccccag ttctaccggc ggcggcggcg gcccctcatc ctgcacctgc cgtgttgatq ctgcgagagc gcaagtccgc gggccagcgt tccgtagatg gtcgcggacg ctggccggtc agcgggcact gggttcgagc aacccaggtg ggcggctgct gaaagcgaaa agtcgcggga ctccccgtca ttgcttttcc agagcaagaq aggcataaag ctgggcggcg caagaagcgc ttcaggggag gacgaatgag aaactggcga ttcccagcgg atctccgccg ccaagtatag gatcgggaag gtagaagtc ctggcagcgg gaagcagagt ggctgcttgt gccgcgcgag gcgcagatgg ctgcaggttt ttccaggggc gactacggta aagcggtgac ggcaggggca gcgaacgcga ggcccggtga qcggcctggc atgaactgct gcqaggtcgt cagacgcggc agattgagct ttgagggtgg gattcgttga aagttgaaaa agctcggcga tcaatctcct ggqacacggc ccgcggcgac aagacgccgc acqgcgctaa agcgagtccq tcgcaaggta gcggaggtgc agaagcacca gcttcgtttt acttcttctt gagtttggcc ggctgaagca gtgaggqtag gtgtaagtgc tcqgtgtacc accaggtact aqggtggccg tacctgqaca cggttccaga aggcgcgcgc cttccgtggt cccgtatccg tgcgacgtca gcgctagctt gcattaagtg cccccggttc tgcaagaccc cagatgcatc cagcggcaga ttgcgtgtga agcacgatct gggatgccct ctgagcccgt ctccacaggt cctatggcca tcccatccaa aacttcatga gtctctacat aactggatct ctgcgacggg tgcacgggct gggaatttga ccttgaccgt cccaaagtcc gagctgtcca acctcqcata tqgttggtgg ccgcgcggcg gcgggcgagc cgtcggcgCC cgacgcggcg gcttgagcct gcaaaatctc cgatctcttc tggaaatgcg tgtagaccac ccacgtgccg tggcggtgtg tatcccccaa actgggagtt cagtqtcgcq cttccataag ggcgacgacg gqcgcatggt ccgtcatgtc cgatgcatct catcgaccgg ggctgagcac tgctgatgat tgtccttgqg gacatcggcg ctccttcctc gtaggtggcg gggctaggtc actggaagtc agttggccat tgagacgcga ggtatcccac gggctcCggg tccaggtgat tgttgcgcag aatcgttgac ctqqtggata gccgtccgcc gacaacgggg ttttggccac gctcgctccc gagtctcgga cgcttgcaaa cgqtgctgcg catgcagggc tgcggaaggg cgtcaaagcc tgatggaagg gctctgaaag cacgggccat ttttttctgg ggttcgcggc ccagcatgaa cgtaggtgac cccgccacca ccgaacactc gtacatcctg gcccctcgcc ctggctgctc agatgtccgc tggtctggag gacgggtcag cggcgtcgat ggcggtgggc ccccggaggt gcqcgcggqc gttgatctcc gaaagagagt ctqcacgtct ctcctggaga ggccatqagc gcccccttcg ggcgaaqacg ttctgccacg ggcctcaagg gcgcgccgac cacctcqcgc ggcctccct gcgcaccggg ctcqgtgacg ccggttatgg caacaattgt atcggaaaac cqtqgcqggc gtaattaaag tccggcctgc caggtctttg ttgtcctqca ccctcttcct ggcgacaacg atccatgtcc aacggaccag gtaagccctc caaaaaqtgc ggcgagatct gccggcqgcg cggcaaaaaq gctctagacc aattcgcaaq gtgatccatg gagtgctcct tgqccgcgcg tgtagccqga ccggccggac ttcctccgga gcagatgcgc accctcccct tcccggcacc gttgatgttg caatt t ttta ggcccagtct tagcatttgc ggtgatgcag taggtctcgc gggcacgagc aaagagacgc attggaggag gtgctggctt cacqaggttg tggcgggttt gaggggagtt gcgcgqcggt ctcccgcggc qgcgcgggct ggcttgcaag cgcgggggtg agggggggct aggagctggt tgaatctqgc tcgacagaat cctgagttgt tctccgcqtc tgcgagaagg gcatcgcggg gcgtagtttc aagaagtaca cgctccatgg acqgttaact tcaaaggcta tcttcttctt aqgcqgtcga gcgcggccgt gttggCgggg tgtgtaggta ctctcgagaa ggcagcgggc taggcggtct tgaatgcgca tagtagtctt tctcttgcat cccatgcgtq cqctcggcta acaaaqgt ttaacggtct gagtcaaata ggcggcggct tccaacataa gtggtggagg t gct ccatgg gtgcaaaagg ggtatcatgg cggttaccgc tttggcttcc cagcgtaagc gqgttatttt tgcggcgaac aacagggacg ccccctcctc cctcctaccg tcqgaacggt tggcccacaa agttcctcgt gcaagatgag aggtggtcgc tagaaggtaa gcggcagtca tgcttcccaa tcggtgcgag tggctattga ttgtaaaaac acctgacgac ggctggtggt acggtggatc cggagcttga gtcaggtcag aqatccaggt agqccgcatc tccttggatg ccggacccgc gctgcgcgCg occtctgcgt caatttcggt cttgataggc cggctcgctc cgttgaggcc cgcgcatgac gcaggcgctg taacccagcg cctcgtagaa cctcctccag caggggcctc ctggcggcgg caaagcgctc tctcgcgggg ggctgccatg ctccgccgcc aggcgtctaa ggcggtcggg tgagacggcg ggcggtcggc gcatgagcct ctatcgctgc tgaccccgaa atatggcctg ggtatgcgcc gqtgacccgg cgtaqtcgtt ggcggtagag ggcqatgata cgcgcggaaa tcgggacgct agagcctgta cqgacgaccg ccgcgtgtcg ttccaggcgc ggttaggctg ccaagggttg gggggtttgC agcccctttt agcagcggca cgtcaggagg 8640 8700 8760 8820 8880 8940 9000 9060 9120 9180 9240 9300 9360 9420 9480 9540 9600 9660 9720 9780 9840 9900 9960 10020 10080 10140 10200 10260 10320 10380 10440 10500 10560 10620 10680 10740 10800 10860 10920 10980 11040 11100 11160 11220 11280 11340 11400 11460 11520 11580 11640 11700 11760 11820 11880 11940 12000 12060 12120 12180 12240 12300 WO 00/42208 WO 0042208PCT/EPOO/00265 -26ggcqacatcc ccggcactac tgagcggtac gaacctgttt cgcagggcgc tgagcccgac ggtaaccgca ccacgtgcgt ctttgtaagc tatagtgcag gcccqaqggc gcgcaqcttg caagttttac gatcgagggg cgtttatcg6 cgaccgcgag agaggccgag cctggaggca cggcggcgtg agcggtgatg ctgcagagc atcatgtcgc ctctccgcaa ctggcgatcg gtctacgacg ctggaccggc cagggcaacc gtgccgcggg gagacaccgc caaggcctgc gtgcggqctc ctgttgctgc ctaggtcact actttccagj gaggcaaccc ttaaacagcc atqcgcgac( ggcatgtat( gccgccgtgi cctggtttcl gacatagaC( gagcaggcai ctaggcgct, cttaccagc.
tcgctgctg gagagccta ccaggcccg gaggacgat tttgcgcac aataaaaaa tqcggCgCg cggcgccag tgcctCCgC cacccctat ccctgaact acagcccgj gcgacctgz ataagtttz tgaaatacc tagacctti ttctggaai ccgtcact( gcggttgacg ctggacttgg ccaagggtgc cgcgaccgcg gagctgcggc gcgcgaaccg tacgagcaga acgcttgtgg gcgctggagc cacagcaggg cgctggCtgC aqcctggctg gcccgcaaga ttctacatgc aacgagcgca ctgatgcaca tcctactttg gctggggcCg gaggaatatg tttctgatca agccgtccqg tgactgcgcg ttctggaagc taaacgcgct cgctgcttCa tggtggggga tggqctccat gacaggagga aaagtgaggt -agaccgtaaa -ccacaggcga -tgctaatagc tgctgacact Iagattacaac taaactacct j agqaggagcc I gggtaacgc( ScctcaaaccC.
i accccgagti L acaccggggc.
j acagcgtgtl j aggcggcgcl g cggccccgCl ctcgcacca, agccqcagc, g tggacaaga c gcccgccca g actcggcag ttcgcccca tcaccaagg ggcgatgta t ggcggCggC !g gtacctgcg 't cgacaccac .a ccagaacga fg ggaggcaac La aaccatcct La ggcgcgggt la gtgggtggz it gaacaacg( ig cgacatcg( 4g tcttgtcai cggcagcaqa aggagggcga agctgaagcg agggagaqga atggcctgaa ggattagtCC cggtqaacca cgcgcqagga aaaacccaaa acaacgaggc tcgatttgat acaaggtggc tataccatac gcatggct tccacaaggc qcctgcaaag acgcgggcgc gacctgggct acgaggacga gatgatqcaa ccttaactcc caatcctqac ggtggtcccq ggccgaaaac gcgcgtggct tgtgcgcgag ggttgcacta ctacaccaac gtaccagtct *cctgagccag *ccgcgcgacc gcccttcacg gtaccgcgag Itgtcagccgc gctgaccaac Icattttgcgc cagcgtggcq j gccgtttatc i. tttcaccaat j attcqaggtc ttccccgcaE L gcgaaaggaz g gtcagatgct cgccC~gc g cgaaaaaaac t gagtagatg( c ccgtcgtcai a cgacagcag( g gctgqggagi c catggcacc, t gaggaaggt, g ctgggttCt g cctaccggg c cgtgtgtac .c cacagcaac fc acacagacc g cataccaac :g atggtgtcg Ig ttcacgctg ,g atcgtggag Ig gtaaagttt :g cctggggta tggtgattac gaacccCCgc gggcctggCg c tgatacgcgt g gcccgaggag a tcgcgagcgg t cgcgCgcgCa c ggagattaac t ggtggctata g tagcaagccg c attcagggat g aaacatcctg c cgccatcaac. t cccttacgtt c qaaggtgctt a cgtgagcgtg a ggccctggct g tgacctgcgc t ggcggtggca c tgagtacgag c gaCgcaacgg z acggacgact gcqttccggcz qgcgcqcgaa aggqccatcc cgttacaaca gccgtggcgc aacgccttCC tttgtgagCg gggccagact qctttcaeaaa gtqtctagct gacagtggca gccataggtc gcgctggggC cggcggcaga tacgtgcagc ctggacatga aaccgcctaa gccatcttga icccgagggta iccgcagaCc iagcttccgca agtagcccat ctgctgggcg ctgcctccgg j aagacgtacg a aggcacgacc :gtcctggatt j agcgttggtt z: ctcctccctc c ccttcgatgc g ggagaaacag c tggtggacaa t ttctgaccac a tcaatcttga a tgccaaatgt gcttgcctac 'C ccgagggcaa c actacttgaa .g acacccgcaa Lt atacaaacga aggcgtacg t tqcgggatc g tgctgcgcg a acgtggcgg c ttcaaaaaa g gactgatgc a tcatggcgc a rcgctgctaa a :agagcatag t *attccatgc t :ccatagaca a ccttgagcg a LgcCggcggc 9 ~gcacgggca g :gggccccaa q :cCgcgCqCg c ~cagaggacg LCCCggcggt ;gcgccaggt c ~gcagccgca ~ccccacgca lgcccqacga 1 cggcaacgt agcgtgagcg :gagtacaca cactgcggct attttttcca acttgcaggq tgctgacgcc gcgtgtcccg aggcgcatgt aggaggacac agatcccotc agagcgtgag ccgcgcqcaa tggactactt acccgcactq acgatggatt tgctagagtt ggccaagcag ttccaagctt aggaggagta catttcccaa cgcaggagca gtcagcgggg tgggagggag aaaaaaaaag ttcttgtatt ctacgagagt tcccctggac catccgttac caagtcaacg ggtcattcaa cgaccggtcg gaacgagttc taaggacaat ct act ccgag agtgggcaga cttcagactg agccttccat gcgccgggc 12360 qccctctcc 12420 gccgcggca 12480 aaagttCCa 12540 ggaggactt 12600 cgccgacct 12660 ctttaacaa 12720 tctgtggga 12780 .gctgttcct' 12840 .catagtaga 12900 *ggtgcagga 12960 tagcctggg 13020 ggaggtaaa 13080 cgacctggg 13140 ~cgaqctcag 13200 rcggcgatag 13260 ~ccgacgcgc 13320 :tggcaacqt 13380 ~cgaqtacta 13440 ;cgggcggcg 13500 ~atggaccgc 13560 ~gccaaccgq 13620 -qagaaggtg 13680 gccggcctg 13740 )tagaccaac 13800 ::gcqcagcag 13860 jcccqccaac 13920 aatggtgact 13980 gaccagtaga 14040 gctgtggggg 14100 caactcgcgc 14160 ggacacatac 14220 ggacgagcat 14280 gggcaqcctq 14340 gttgcacagt 14400 ccttaacctg 14460 catggaaccg 14520 gcatcgcgcg 14580 gotacogccc 14640 cctctgggac 14700 gcaacagcgC 14760 cttgtccgat 14820 gatagggtct 14880 cctaaacaac 14940 caacgggata 15000 cagggacgtq 15060 tctqgtgtgg 15120 tggcaacccg 15180 catgatgcaa 15240 ccccttagta 15300 gtggtgagcg 15360 ccgccgtttg 15420 tctgagttgg 15480 gatgtggcat 15540 aacaatqact 15600 cactggjggcg 15660 atgtttacca 15720 caggtggagc 15780 accatgacca 15840 cagaacqqgg 15900 gggtttgacc 15960 ccaqacatca 16020 WO 00/42208 WO 0042208PCTIEPOO/00265 -27ttttgctgcc tccgcaagcg gtaacattcc aacagggcgg ccaacgcggc gcgacacctt ctgccgcccc ccctgacaga cccagtaccg catggaccct tgccagacat cggtggtggg tctactccca agaaccagat ctgctctcat tgaccattac tctcgccgcg ccagcaataa agcgctccga acaaacgcgg aggcgcgcaa ccgtggtgcq gtcgccaccg qcqcacqtcg ttgtcactgt gtgctatqac tgcgcgtgcc actcgtactg aaatcaaaga aagagcagga atgatgaact agtggaaagg ccggtgagcg acctgcttga acatgctqgc tgcagcaggt ctggtgactt tcttggaaaa agcaqgtggc gcaccagtat cggtggcgga tgcaaacgga agtacggcgc cccccggcta ccaccactgg tgcgcagggt ccagcatcgt tccgtttccc acggcctgac gcatgcgcgg tgcccggaat tgtggaaaaa tgtagaatgg atgggaaact tcgctgtgga tggaacagca aaggtggtag gtgcaaaata gccgtggaga gaaactctgg ctgcccacca gtaacqctgg aggatgcggg gcaacccttc cgcactgttg ggqtggcgca agccgcggca tgccacacgg cgctgcgcaa ggacagcaag cagctggtac gctttgcact gatgcaagac cgccgagctg actcatccgc tttggcgcgc agatcacgqg tgacgccaga cgtcctatcg cacaggctgg ccaacaccca ccqcactgqg ctacacgccc cggaqcccgg ccgccgaccc caccggccqa gccccccagg tcaqggtcgc cgtgcgcacc ttgtatgtat agagatgctc ttacaagccc tgacgacgag tcgacgcgta ctccacccgc gcaggccaac gttgccgctg gctgcccgcg ggcacccacc aatgaccgtg gccgggactg tgccaccgcc tgccgcgqtg cccqtggatg cqccagcgcg tcgtggctac aacccgccgc ggctcgcgaa ttaaaagccg ggtgccggga gggcggcatg cggtatcctg tgcatccgtg tcaaaataaa aagacatcaa ggcaagatat gcggcattaa gcacaggcca atggcctgqc agattaacag cagtgtctcc tgacgcaaat cccgtcccat acctgcctcc gtggacttca caggagggct gatgtggacg ggcggcagca atgcagccgg gctgaggaga cccgaggtcg aaacgcagtt cttgcataca cctqacgtaa cccgtgacct ttgcccgtgc cagtttacct ccgccagccc acgctaccgc cgccgcacct agccgcactt ggcctgcgct gtgcgcgtgc cgcaccaccg acgccgccac cgctatgcta ggcactgccg cgggcggcca tccaggcgac aggggcaacg cgccccccgc ccagcggcgg caggtcatcg cgaaagctaa gtggaactgc aaacgtgttt acctacaagc gagcgcctcg gacgagggca cttgcaccgt gtgcagctga gaacctgggc ggcgtgcaga acagagggca caggcggtcg tttcqcgttt ctactgcccg acctaccgcc cgccgtcqcc ggaggcagga gtctttgtgg ttccgaggaa cqtcgtgcgc cccctcctta gccttgcagg aagtctggac ctttgcgtct cggcaccagc aaatttcggt gatgctgagg ctctgqcatt taagcttgat agaggggcgt agacgagcct cgcgcccatg ccccgccgac cccacagccg ttaggatcac cctaccaggc acagcagtgg tggaggacat agcgcgctga agaagcctca acaacctaat actacggcga cctgcggctc tccgctccac actccaagag ctctgaccca ccaccatcac tgcgcaacag gcccctacgt t tt gagcaag tcccaagcaa gcgggcacta tcgatqacgc cagtgtccac aaatgaagag cccaacgcgc tgcgggccgc gagcggccgc tgtattgggt gcaactagat cggcgcgcaa cgccqqagat agcgggtcaa tgcacgctac tgcgacccgq gcgtgtatga gggagtttgc acccaacacc ccgaagaaaa tggtacccaa tggagcccqa ccgtggacgt tggagacaca ctgcggccgc cagccccccq aatatgccct ccagaagacg gtcgccagcc ccctggtgct ttcttgcaga gaatgcaccq accaccggcg ttccactgat cgcagagaca tctcacgctc ctggccccgc aatatgagcg tccaccgtta gataagttga agcggggtgg ccc cgccctc ggcgaaaagc ccctcgtacg gctaccggag acccagcaga cctgagcaac ctacgatgat gagcttgaaa cagcggcgcg gaacgatcat ggccgaagca gaagaaaccg aagcaatgac ccctcagacc ggagcaggtc gcgccagatc cttctacaac cgtgttcaat caccgtcagt catcggagga ttacaaggcc catgtccatc gatgtttggc ccgcgcgccc catcgacgcg agtggacgcg acggcggagg ggcggcggcc tcgaaggctq cgcagcaqcc gcgcgactcg tgcaagaaaa cgaagctatg ctatggcccc aaagaaaaag cgcgcccagg caccaccgta tgaggtgtac ctacggaaag tagcctaaaq gcgcggccta gcgccagcga gqtccgcgtg tcagataccc aacgtccccg gtccaagacc gcgcccgcgc acatccttcc agcaactacc cgtgctggcc gccaacagcg tatggccctc taggaggggc gcggcgcgcg cgccgcggCg ctgattaaaa gcttggtcct gacacggct c gtggcgcct t agaactatgg aagagcaaaa tggacctggc ccgtagagga gtccgcgccc aggaggcact tgctgggcca aacctgtgct ttgttgggca 16080 ctggagggtg 16140 gatgacaccg 16200 gaagagaact 16260 gccattcgcg 16320 gcggccgaaq 16380 gtgatcaaac 16440 agcaccttca 16500 ggaatccgct 16560 tactggtcgt 16620 agcaactttc 16680 gaccaggccg 16740 cgctttcccg 16800 gaaaacgttc 16860 gtccagcgag 16920 ctgggcatag 16980 cttatatcgc 17040 gggqccaaga 17100 tggggcgcgc 17160 gtggtqgagg 17220 gccattcaga 17280 cgcgtagcac 17340 ctgcttaacc 17400 qccgcgggta 17460 gcggccatta 17520 gttagcggcc 17580 aactacttag 17640 tccaagcgca 17700 ccgaagaagq 17760 aaagatgatg 17820 cgacgggtac 17880 gtctttacgc 17940 ggcgacgagg 18000 cqgcataagg 18060 cccgtaacac 18120 aagcgcqagt 18180 ctggaagatg 18240 cggccaatca 18300 actaccagta 18360 qttgcctcaq 18420 tctacggagg 18480 ggttcgagga 18540 attqcgccta 18600 cgacgccgaa 18660 ccgatttccg 18720 cgctaccacc 18780 acctgccgcc 18840 atggccggcc 18900 tcgcaccgtc 18960 attggcgccq 19020 acaagttgca 19080 gtaactattt 19140 gcgcccgttc 19200 cagctqgggc 19260 cagcaaggcc 19320 tttccaacaa 19380 caaccaggca 19440 gcctccaccg 19500 cgacagggaa 19560 aaagcaaggc 19620 gcacacaccc 19680 gccaggcccg 19740 WO 00/42208 WO 0042208PCTIEPOO/00265 -28accgccgttg cgatcgttgc ctgggggtgC tgtcatgtat ccaagatggc acgcctcgga tcagcctgaa accggtccca cgtacaaggc cgtactttga ctgcctacaa ctactqctct agcaagctga ttacaaagga catttcaacC cagctgggag aacccacaaa aaagtcaagt acttgactcc atatttctta ctatgcccaa acaacagcac tagatttgca atagaaccag gaattattga gtgtgattaa gggaaaaaga ccatggaaat tgtatttgcc acacctacga accttqgagc gcaatgctgg acatccaggt acacctacga atgacctaag tcttccccat acgaccagtc acgctaccaa ccttcacgcg acacctactc agaaggtggc cccccaacga gtaacatgac agggcttcta agcccatgac tcctacaccz gacaggcctz ttacccagaz ttatgtccat acgcgctagz tgtttgaagt tgtacctgc( aacaacagct tgqttgtggc acacaagct( gatggccttl ttctgacca( cgccattgcl ggggcccaai ctggccccai ctccatgcti cagcttccti ttgtaacccg ggcccgtagc aatccctgaa gcgtccatgt taccccttcg gtacctgagc taacaagttt gcgtttgacg gcggttcacc catccgcggc cgccctggct tgaaataaac gcagcaaaaa gggtattcaa tgaacctcaa agtccttaaa tgaaaatgga ggaaatgcaa taaaqtggta catgcccact caggcctaat gggtaatatg agacaqaaac gtacttttct aaatcatgga tacagagact tgctacagaa caatctaaat cgacaagcta ctacatgaac acgctggtcc cctgcgctac gcctcagaag gtggaacttc ggttgacgga ggcccacaac ctttaacgac cgtgcccata ccttaagact tggctctata *cattaccttt *gtttgaaatt -caaagactgg itatcccagag ccgtcaggtg iacacaacaac kccctgctaac iaaagtttctt gggcgcactc icatgactttt ctttgacgtc Icacgcccttc -gccgccatg.
jccatattttt -gcctgcgccz gcctggaacC 3 cgactcaagc tcttccccc( tcggccgcct a actcccatg( aacagtccc g gagcgccacl tcctagccgc cagtggcaac gcgccgacga cgccgccaga atgatgccgc cccgggctgg agaaacccca ctgcggttca ctagctgtgg qtgctggaca cccaagggtg ctagaagaag actcacgtat ataggtgtcg ataggagaat aagactaccc gggcaaggca tttttctcaa ttqtacagtg attaagqaag tacattgctt ggtgttctgg acagagcttt atgtggaatc actgaagatg cttaccaagg ttttcagata qccaacctgt aagtacagtc aagcgagtgg cttqactata cgctcaatgt ttctttgcca aggaaggatg gccagcatta accgcctcca tatctctcq tccatcccct aaggaaaccc ccctacctag gactcttctg aagcgctcag ttcctggtac agctacaaqg Fgtggatgata tctggatttg ttcccctatc tgcgatcgca acagaczctgg gaggtggatc Igtccgtgtgc :tcggccggca Igctccagtga -tgggcaccta Itagtcaatac -cgcactcaaa -aggtttacca j accgctgtat gtggactatt ;atcacaaccc aggtacagcc cgccctactt gcgtccctgc tggcaaagca tgcttctgaa ggagctgctg agtggtctta tgcagtttgc cgqtggcgCC tccctgtgga gtgataaccg ggggccctac ccccaaatcc aggacgatga ttqgggcaggc aaggtcaaac ctcagtggta caatgaaacc ttcttgtaaa ctactgaggc aaqatgtaga qtaactcacq ttagggacaa cgggccaagc cataccagct aggctgttga aacttccaaa taaaacctaa aaaatgaaat ggagaaattt cttccaacgt tggctcccqg tggacaacgt tgctgggcaa ttaaaaacct ttaacatqgt agtttgataq cgcttgaggc ccgccaacat cccgcaactg catcactggg atggaacctt tcagctggcc ttgacgggga aaatgctagc accgcatgta ctaaatacaa ttggctacct cgcttataqg ccctttggcq gccaaaacct ccatggacqa accggccgca acgccacaac qcaggaactg tgacaagcgc qgccggtcgc aacatgctac *gtttgagtac *aacgctggaa ctgctgcatg caccatqaac caccctgcgt ccgcagccac gccgcgCcgc cactgaacag tagctaacgt agccgccgcg catgcacatc ccgcgccacc tacgcacgac ccgtgaggat tgtgctggac ttttaagccc ttgcgaatgg caacgaagac gccttattct acctaaatat cgaaactgaa atgttacggt gcaacaaaat gaccgcaggc tatagaaacc agaactaatg ttttattggt atcgcagttg tttgcttgat cagctatgat t tact qctt t aacaggtcag aagagttgga cctgtactcc aaaaatttct gttaqtggac caacccattt tggtcgctat ccttctcctg tctqcagagc catttqcctt catgcttaga gctctaccct ggcggctttc ctcgggctac ttacctcaac tggcaatgac gggttacaac taactacaac ctccttcttt ggactaccaa tgcccccaCC caagaccgca catcccattc tctctacgcc qcccaccctt ccgcggcgtc ataaagaagc aaagccattg tttccaggct gagactgggg ctctttgagc gagtcactcc aagtccaccc *tttctccacg cttattaccg cgcaaccagg agtgcgcaga cagcggtccg 19800 catcgtgggt 19860 gtcgtatgtg 19920 cgcccgcttt 19980 tcgggccagg 20040 gagacgtact 20100 gtgaccacag 20160 actgcgtact 20220 atggcttcca 20280 tactctggca 20340 gatgaagctg 20400 gaagtaqacg 20460 ggtataaata 20520 gccgataaaa 20580 attaatcatg 20640 tcatatgcaa 20700 ggaaagctag 20760 aatggtgata 20820 ccagacactc 20880 ggccaacaat 20940 ctaatgtatt 21000 aatgctgttg 21060 tccattggtg 21120 ccagatgtta 21180 ccactgggag 21240 gaaaatggat 21300 aataattttg 21360 aacatagcgc 21420 gataacccaa 21480 tgctacatta 21540 aaccaccacc 21600 gtgcccttcc 21660 ccgggctcat 21720 tccctaggaa 21780 tacgccacct 21840 aacgacacca 21900 atacccgcca 21960 cgcggctggg 22020 gacccttatt 22080 cacaccttta 22140 cgcctgctta 22200 gttgcccagt 22260 attggctacc 22320 agaaacttcc 22380 caggtqggca 22440 atgcgcgaag 22500 gttqacagca 22560 tccagtaact 22620 aactccgccc 22680 ctttatgttt 22740 atcgaaaccg 22800 aagcaacatc 22860 tcaaagatct 22920 ttgtttctcc 22980 gcgtacactg 23040 cctttggctt 23100 tgcgccgtag 23160 aaagcgtaca 23220 cctttgccaa 23280 gggtacccaa 23340 aacagctcta 23400 ttaggaqcgc 23460 WO 00/42208 WO 0042208PCT/EPOO/00265 -29cacttctttt aggcaaatgc cgccqtttaa gttgcgatac ggtgaagttt cgatatcttg gttgcagcac ggagatcaga tagctgcctt catcaaaagg gatctgctta gccggaaaac ggagatctgc ctccttcagc atttatcata cagccacaac caggtacgcc cagctgcaac cacttggtca catcagcgcg cgggttcatc ccgcatacca tttgccatqc ttctctttct agaagggcgc ccgcgqgctg ctcgatacgc cggggacgac ggtggtttcg gatcatggag cqcctccacc ggagqaggaa ctcagtacca agtcgggcgg gaagcatctq gcccctcgcc accccccaaa cgtatttgc acccctatcc cgctgtcata acgcgacgaq ctctggagtg catcgaggtc agtcatgagt agaacaaaca aacgcgcgag taccgtggag agaggaaaca caacgtggaq gcaaaacgtg cgtttactta ggaggagtgc gacggccttc cctgcttaaa ctttaggaac tagcgacttt ccttctgcag tgacqgtcta ttgcaattcg ctcgcctgac ggcttacctt agaccaatcc tgtcacttqa t ttt att tgt aaatcaaagg tggtgtttag tcactccaca aagtcgcagt tggaacacta tccgcgtcca cccaaaaagg tgaccgtgcc aaagccacct tgattggccg accacatttc gcgcgctgcc atgcttccgt gcgcagcccg tgcaggaatc ccgcqgtgct ggca gt agt t cgcgcagcct accgtaattt cgcgccactg ttgattagca tcctcqctgt ttctttttct gqtgtgcgcg cgcctcatcc acgtcctcca cgctgctcct tcagtcgaga gatgccqcca gtgattatcg acaqaggata ggggacgaaa caqcgccagt atagcggatg cgccaagaaa gtgccagagg tgccgtgcca cctgatatcg aagcgcgcgg ttggtggaac acccactttg gagctgatcg gagqagggcc cctgccgact cttgagtgca ttgcactaca ctctgcaacc cttcattcca tttctatgct aacctcaagg aacgagcgct accctgcaac tttatcctag gtgcccatta ctagccaact ctggagtgtc cagctgctta gaaaagtccg cgcaaatttg cgcccgccaa aaaacatgta acactctcgg ggttctgccg tgctccactt ggctgcgcac tggggcctcc tcagcgccgg ggtcctccgc gcgcgtgccc cggtctgggc gagcctttgc gacaggccgc ggccccaccg cgttttcgct gtaqacactt tgggctcgtg gccccat cat cctcgttcag tgaagttcgc ccatgccctt cactttccgc ggtcgtcttc ccggtgggtt ccacgattac tcttgggcgc gcaccagcgc gcttttttgg tggttggggg cttcccgact agaaggacag acgcgcctac agcaggacc aaaagcaaga qgcatggcga qcgccattat tcagccttgc acggcacatq tgcttgccac accgcagccq cctcgctcaa caaacgctct tcgagggtga cctacccggc tgcgccgtgc tacccgcagt tggaggagcg tgcagcggtt cctttcgaca tggtctccta cgct caaggq acacctggca agctgcagaa ccgtggccgc agggtctgcc agcgctcagg agtaccgcga accttgccta actgtcgctg acgaaagtca cggctccggg tacctgagga atgcggagct aaaataatgt gtgattattt cgcatcgcta aaactcaggc catcaccaac gccctgcgcg gtggtgcacg gttgctcagg aggctttgag .gttaggatac gccttcagag qtcgtgcacg qttcttcacg cgtcacatcc aagctcgcct atqcttgtag cgtcacaaag ccaggtcttg ctttagatcg c tccca cqca ttcgctgggc attcagccgc gctgaaaccc ctctggtgat aatggccaaa gtcttqtgat gggcgcCcgg acgtcgcgcc ggccatttcc cctaaccgcc caccttcccc aggttttgta ccaggacaac ctacctagat ctgcga cgcg ctacgaacgc cgagcccaac ctatcacatc agcqgacaag cgaagtgcca gcaacaggaa caacgcgcgc acttaaccta gcagcccctg tggcgacgag acgcaaacta ctttgctgac gggctacgta ccttggaatt cgaggcgcgc gacggccatg actgctaaag gcacctggcg agacttcacc aatcttgccc atgccctccg ccactctqac caacctatgc aattatcggt gttgaaactc ctaccacgcc taccgcctgc actagagaca acccccaccc tgcgccactg acaaccatcc gcgtttagca cgcgagttgc ctggccagca gcgaacggaq ttgcactcgc agcgcctgca aagaacatgc cagcaccttg atcttggcct atttcaatca tcgatctcag gtcacctctg gtcttgttgc catacgqccg ttatccacgt gacacgatcq tcttcctctt cgcactgtqc accatttgta ggcqggcgct tccgccgccg gagtcttcct ggaggcggcg gcaccgcgtc ttctcctata ccctctgagt gtcgaggcac agcgaagacg gcagaqgcaa gtgggagacg ttgcaaqagc cacctattct ccgcgcct ca tttttccaaa cagctggcct aaaatctttg aacagcgaaa ctagccgtac ccccccaagg gagagggatg cagctagcgc atgatggccg ccggagatgc cgccaggcct ttgcacgaaa cgcgactacg ggcgtttggc caaaacttga gacatcattt agtcaaagca gccacctgct ccgctttggg ataatggaag accccgcacc acctttgagc actccggggc cacgagatta gtcattaccc ctttcaataa ttgccgtctg gcagggacac gcggcagctc ggtcgggcgc gatacacagg cgctcttgtc tcaactttgg accgtagtgg taaaagcctt cgcaagactt cgtcggtgtt tgctagactg cgtgctcctt cgcagcggtg caaacgactg tggtgaaggt ccagagcttc ggta ct tgt c gcacactcag cctcttgcgt qc tt a cc tcc gcgccacatc cgggcttgqg aggtcgatgg cgtcctcgga gcgacgggga cgcgctcggg ggcagaaaaa tcgccaccac ccccgcttga acgaggaccg acgaggaaca acqtgctgtt gcagcqatgt caccgcgcgt acttctaccc actqcaagat tgcggcaggg agggtcttgg atgaaagtca taaaacgcaq tcatgagcac caaatttgca gctggcttca cagtgctcgt agcgcaagct gcaagatctc accgccttgg tccgcgactg agcagtgctt aggacctatg tccccgaacg tgttgcagaa gtgcacttcc gccactgcta acgtgagcgg gctccctggt tgcagggtcc tgtggacgtc ggttctacga agggccacat 23520 23580 23640 23700 23760 23820 23880 23940 24000 24060 24120 24180 24240 24300 24360 24420 24480 24540 24600 24660 24720 24780 24840 24900 24960 25020 25080 25140 25200 25260 25320 25380 25440 25500 25560 25620 25680 25740 25800 25860 25920 25980 26040 26100 26160 26220 26280 26340 26400 26460 26520 26580 26640 26700 26760 26820 26880 26940 27000 27060 27120 27180 WO 00/42208 PTEO/06 PCT/EPOO/00265 tcttggccaa gggggtttac gccctatcag agctgccgcc tggacgagga aggtcgaaga cccagaaatc cactgcccgt ccaagcagcc gcgggcacaa cccgccgctt accgtcatct gccacacaga gcggcggcag cgcgagctta caagaacaag tatcacaaaa aaatactgcg aaactacgtc agcaaggaaa ggagctgccc tcccqggt ca accaccacac gaaagtcccg actaactcag ggt at aact c tcctcqcttg ttcacgcctc ggcattggaa tcgggacctc tcggcggacg ctggtccact gaattgcccg qagcttgccc aggggaccct ttaattaatt ttgtqttatg tcagtagtat acaqaaccct ctccccggct tattccacac gcagctcact gcggttgctt cgtgcatcag gctccgtcct qcagcataag cacagtaact atccaaagct agattaagtg tgtaattcac ccatcctaaa tqgaacaatg tatcaatgtt cccgcgttag tgcagggaag gcagcggatg ccctactgta atggaacgcc gatctgcgtc tctct caaag gctgccctga ttctgcgagt ttgcaagcca ttggaccccc cagcagccgc gccacccacg ggaggaggac ggtgtcagac ggcaaccggt t cgccgaccc gccgccgtt a qaacgccata tcttctctac ctacagccca agcaaaggcg cagcaggagg gaaacaggat agctgaaaat gcgaagatca cgctgactct atctccagcg ttcccacqcc aaqactact c acggaatccg ctcgtaataa ctcccaccac gggcgcaqct acctgacaat gtctccgtcc gtcaggcaat ctctgcaatt ccggccacta qctacgactg gtcgccqcca aggatcatat gtagcctgat gtqttctcac gccacatcct tttcaacgtg agccccacca agtattcaac ggccttaaaa ggtttcctgt taagttcatg aacgggcgqc gatagggcgg gcaggaatac gcgccttgtc gcaqcacagc catggcgggg gcgacccctc cacctcccgg ccagctggcc acagtggaga ggcacaacac aaccatatcc acctcgcacg atcctccagt cggagtgcgc ggacgtagtc tccggtctcg catccaggcg taacatccac cacacacggg tcaacaaagc agtccggcga gggcccttgc gacgaggagg atgatggaag gaaacaccgt tccagcatgg aaccgtagat gcccaagagc gttgcttgct catcacggcg tactgcaccg accggatagc aggagcgctg ttttcccact aaaaaacagg gcttcggcgc taaggactag gccacacccg ctacatgtgg aacccgaata cgcccaccga ccttaatccc tgtggtactt tgcgggcggc cagagggcga ggacgggaca cctaactctq tattgaggag tccggatcaa aatgttaagt caagtgcttt cgagggcccg tcgggagttt tgtgatttgc cttacacttt tttatttttc ccacatagct ctqccacctc agcatcatat cgagccaaac tcgctgtcca gaaggagaag tggtgctgca aacatggcag ctccgggcac accacaatat accacagaac ataaacacgc taccatataa aaaacctgcc gcccaggact aggcacacgt cagggaacaa taactcacgt atggtagcgc cgagacaacc atatttcctg ccgcttagat ccccctggct caccgcagaa aggagcggga ccgccaagag ggagctcaac ttcccaggat aatactggga actgggagag caccctcggt ctacaacctc gggacaccac aacaacagcg tgcaagactg tggccttccc gcggcagcgg aagactctga cgt ctggcgc ctgtatgcta tctctgcgat acgctggaag tttcqcgccc gcgccagcac agttaccaqc aactacatga aaccgaattc cgtagttggc cccagagacg tttcgtcaca ggtattcagc tttcagatcg cagacctcgt tttqtgccat tttattccta ggagaggcaq gcccgcgact gcgcacggcg acccagcgcc aactgtccta ttcatacatt aattgcagaa tatacagatc cctcccaaca catgggtaac gctcatcagt gctgctgagc tccacgccta gcagcgcgcq tggtctcctc agcagcgcac tgttcaaaat ccacgtggcc tggacataaa acctctgatt cgccggctat cgtaaccatg gcatacactt cccattcctg tgtgcattgt gggtttctgt gagatcgtgt aagcaaaacc cgctctgtgt tcgggttcta taagccacac agagctggaa tttctgctac ccaatccccc ggcacccaaa cagtcaggca cctagacgag cgcattcccc cgctcctcag tggaaccagg ccaaggctac tgggggcaac ccgtaacatc cagcggcaqc caaagcccaa ccaacgaacc tatttcaaca ccctcacccq acgcggaggc tttctcaaat ctgtcgtcag cacaaatggg gcgcgggacc tcttggaaca ccgctgccct cccaqgccga gggtgcggtc tcaacgacga gcgqcgccgg cctctgagcc cggtctactt actttgacgc agcaactgcg ccggtgagtt tccggcttac ccctgctagt accttggatt qcccaagaat aatttcaaqt accgtacctt cacagagtac agacatattc gatattaata cacaggctgc catgggggta aataaactgc agcgatgatt cctgatctca cccacagtgc atcataccac cattacctct aaacatggcg acactgcaqg gatcatcatg cctcaggatt aatcaqcgta caaagtgtta ctcaaaagga tqgtcgtagt aggtgcgggc agtagttgta tgtaaactcc ccaqccaacc gaaccatgtt gaaagggacg 27240 cgccgccgca 27300 aagaagctgc 27360 gaggaggttt 27420 gaagcttccg 27480 tcgccggcgc 27540 gcgccgccgg 27600 gccggtaagt 27660 cgctcatggc 27720 atctccttcg 27780 ctgcattact 27840 aacagcagcg 27900 gaaatccaca 27960 cgtatcgacc 28020 gagcaggggc 28080 cagctgcctg 28140 tctcttcagt 28200 ttaagcqcga 28260 cgccattatg 28320 acttgcggct 28380 ccacatgata 28440 ggcggctatt 28500 dgtqtaccag 28560 agttcagatg 28620 gcccggqcag 28680 gtcggtgagc 28740 ccgtccttca 28800 gcgctctgga 28860 taaccccttc 28920 ggtaaaggac 28980 cctgaaacac 29040 ttgctacttt 29100 cgcccaqgga 29160 tgagcqggac 29220 acatcaagat 29280 aaagaatcgt 29340 catttttcat 29400 aatcaaactc 29460 acagtccttt 29520 ttaggtgtta 29580 aactccccgg 29640 tgtccaactt 29700 gagtcataat 29760 tgccgccgcc 29820 cgcaccgccc 29880 cttaaatcag 29940 aaggcgctgt 30000 aagcgcaggt 30060 tttgqcatgt 30120 ccatccacca 30180 gaaccgggac 30240 ctcgtcatga 30300 acaagctcct 30360 aatcccacac 30420 cattcgggca 30480 ggtagacgat 30540 gtcatgccaa 30600 qtgacaaaca 30660 gtatatccac 30720 ttcatgcgcc 30780 tacacattcg 30840 ttttttttta 30900 WO 00/42208 PCTIEPOO/00265 -31ttccaaaaga ggtggcgtgg aatggcttcc agggtgaatc tcgccacctt aatctgctcc tcaggttcct tcccgtaggt gcggccactt ctcggagcta aaaatgcaag gtagtcatgc catttttctc aacatttaaa cggactacgg accgacagct tgattcatcg' gcgtagagac cacataaaca aacatacagc tattaaaaaa caagtgcaga aacacccaga ttcctcaaat caattcccaa cgccccgcgc aaqgtatatt ttatccaaaa tcaaactcta aaaaggcaaa tcctctataa ctcaatatat agagcgccct cacagacctg cccttcgcag ccccgccagg tgctaaccag gtgctgctca tcatgcagat tcaaacatgt cattagaagc ccatgccggc cctcggtcat gtcagtgcta aacattacag cctgaaaaac gcttcacagc acaccactcg gcgagtatat aaaccgcacg cgtcacttcc cacatacaag cacgtcacaa attgatgatq cctcaaaatg aagatctatt Cagccaaaga acagataatg cggccctcac-gtccaagtgg acattccagc ctctaagcaa ccaccttcag tataagattc ggccagctga aaccttgaca cgtagccccg aaaaatcagg aaaggcaggt ctgcgggttt ctgtcttaca gtgaccgtaa gtccggagtc aaaagcgacc cccccatagg cctcctgcct ggcagcctaa acacggcacc ataggactaa cgaacctacg gttttcccac ttactccgcc actccacccc accttcaacc atcccgaata cctcaagcag aaaagcggaa acataatcgt aaagaaccca atgtaagctt caaagcctcg aagctccgga ctgcataaac acaggaaaaa aaaaactggt ataatgtaag gaaatagccc aggtataaca aggcaaaata cagtcagcct agctcaatca aaaatgacgt cccagaaacg gttacgtaac ctaaaaccta ctcattatca aaqtgaacgc gcatttgtaa acgtaaaggc atgcccaaat ttaagtccgg cgaatcatga cattaacaaa gcaggtctgc cactgattat tgttgcatgg cgcaaaaaag accaccacag acaaaataaa caacccttat caccgtgatt actcggtaaa gqgggaatac aaattaatag gcaccctccc taccagtaaa gtcacagtqt aacggttaaa aaagccaaaa ttcccatttt cgtcacccgc tat tggctt c gctcccctcc 30960 gatgttgcac 31020 taaacccttc 31080 aattctcatc 31140 ccattgtaaa 31200 ttgcaaaaat 31260 aataccgcga 31320 acggaccagc 31380 qacacgcata 31440 qcggcgatat 31500 aaagcacatc 31560 aaaaagacac 31620 ataacaaaaa 31680 aagcataaga 31740 aaaaagcacc 31800 cacatcaggt 31860 atacccqcag 31920 gaqagaaaaa 31980 gctccagaac 32040 aaaqaaaacc 32100 aaaaaagggc 32160 gtccacaaaa 32220 Aacccacaac 32280 aagaaaacta 32340 cccgttccca 32400 aatccaaaat 32460 32480 <210> 28 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 28 ctcaacaatt gtggatccgt actcc <210> 29 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 29 gtgctcagca gatcttgcga ctgtg <210> <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> WO 00/42208 PCTIEPOO/00265 ggcgcgttcg gatccactct cttcc <210> 31 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 31 ctacatgcta ggcagatctc gttcggag <210> 32 <211> 1240 <212> DNA <213> adenovirus <400> 32 ggatccactc ctgaaaagcg gatattcacc gacaatcttt cttgqcgatg gtttagctgc gtcgggcacc ggctacctct qaatggcggt cccgggcagc ctgccatgcg ggggtgggtg gagtattcca gtatagttcg tgctcggaaq gaagacgttg gtcgcgcagc ggtttccttg gacaaactct agatccgtac tctcgagaaa tcttccgcat ggcatgactt tggcccgcgg ttgttgtcaa qagcgcaggg acgtattcgc aggtgcacgc ccgcgt aqqc agggggtcta aggcgcgcgt cgggcggcaa agcgcggagg agatatqtag tqcgagggag actatctgcc aagctggcgt ttgttgacca atgatgtcat tcgcggtctt tccgccgccg ggcgtctaac cgctqtctgc ctgcgct aag tqatgccttt gcttggtggc tttggttttt gcgcaacgca gccaaccgcg gctcgttggt gctgcgtctc cgaagt agt c gcgcgcqctc cgtacatgcc ggtagcatct cgagqaggtc tgaagatqgc ctgtgagacc gctcqgcggt acttatcctg tccagtactc agggacctga cagtcacagt gaqggccagc attgtcaqtt gagggtggcc aaacgacccg qtcgcgatcg ccgccattcg gttgtgcagq ccagcaqagg gtccqgggg tatcttgcat gtatgggttg gcaaatgtcg tccaccgcgg gggaccgagg atgtgagttq taccgcgtca gacctgcacg tccctttttt ttggatcgga gcgagt ccgc cgcaagatct tgttggggtq tccaaaaacg gcatccatct tagagggcgt gcgcgCtcct ggaaagacqg gtgacaaggt cggccgccct tctgcqtcca ccttgcaagt agtgggggac taaacgtaga atqctqgcqc ttgctacggg gatgatatqg cgcacgaagg tctagqgcgc ttccacaqct aacccgtcqg atcgaccgga -agtactccct ggaqgattt ggtcagaaaa tggacagcaa tggccqcgat tggtgcgctc caacqctggt tgcqcgagca cggtaaagac ctagcgcctg cccatqgcat ggggctctct gcacgtaatc Cgggctgctc ttggacgctg aggcgtagga aqtagtccag cgcqgt tgag cctccgaacg tcggaaaacc 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1240 <210> 33 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 33 ggcgcgttcg gatccactct cttcc <210> 34 <211> <212> DNA <213> Artificial Sequence <220> WO 00/42208 PCT/EP00/00265 -33- <223> Description of Artificial Sequence: primer <400> 34 gggagtagat ctcccaacag <210> <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> cccttttttt tggatccctc gcgg 24 <210> 36 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 36 ctacatgcta ggcagatctc gttcggag 28 <210> 37 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 37 ctcaacaatt gttggatccg tactcc 26 <210> 38 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 38 gtgctcagca gatcttgcga ctgtg <210> 39 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer WO 00/42208 WO 0042208PCTIEPOO/00265 -34- <400> 39 ggcgcgttcg gatccactct cttcc <210> <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> ctacatgcta ggcagatctc gttcggag <210> 41 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 41 cccttttttt tqgatccctc gcgg <210> 42 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 42 gtgctcagca gatcttgcga ctgtq <210> 43 <211> 8383 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: plasmid <400> 43 gacggatcgg ccgcatagtt cgagcaaaat ttagggttag gattattgac tggagttccg cccgcccatt attgacgtca atcatatgcc atgcccagta tcgctattac actcacgggg aaaatcaacg gagatctccc aagccagtat ttaagctaca gcgttttgcg tagttattaa cgttacataa gacgtcaata atgggtggac aagtacgccc catgacctta catggtgatg atttccaagt ggactttcca gatcccctat ctgctccctg acaaggcaag ctgcttcgcg tagtaatcaa cttacggtaa atgacgtatg tatttacggt cctattgacg tgggactttc cggttttggc ctccacccca aaatgtcgta ggtcgactct cttgtgtqtt gcttgaccga atgtacgggc ttacggggtc atggcccgcc ttcccatagt aaactgccca tcaatgacgg ctacttggca agtacatcaa ttgacgtcaa acaactccgc cagtacaatc qgaggtcgct caattgcatg cagatatacg attagttcat tggctgaccg aacgccaata cttggcagta taaatggccc gtacatctac tgggcgtgga tqggagtttg cccattgacg tgctctgatg gagtagtgcg aagaatctgc cgttgacatt agcccatata cccaacgacc gggactttcc catcaagtgt gcctggcatt gtattagtca tagcggtttg ttttggcacc caaatgggcg 120 180 240 300 360 420 480 540 600 660 720 780 WO 60/42208 WO 0042208PCT/EPOO/00265 gtaggcgtgt ctgcttactg gagctcggat ctccctctga ggatttgata agaaaagaca cagcaacttg cgcgatgttt gcgctcgtcg gctggtggct cgagcagaat aaagaccccg cgcctgctgc tggcatgggg ctctctgaqt gtaatcgtat ctqctctgct acgctggaag gtaggagtcg gtccagggtt gttgaggaca cgaacgagat aaaacctctc aagaccgtct aactgtgcct tggqtactc caaaatqggc cactgtgagc cctcacagtt caacacactc tgccacccaa cctcaccacc cactqgtagc actaaagtac tggtccaggt ttttqattca aaacagacgc aagactagga caaaggcctt cactgccaac tqaatttggt cctagaattt cagcacaggt accagctccz cttaacaaa tttggctcc tggagtgcti tcttactga tccaaaatcl agacaaaacl cacaactcci taatgaaati gcggccgctl ctcgctgat, ccgtgcctt, aaattgcat acagcaagg tggcttctg gcggcgcat gcgccctag ttccccgtc acctcgacc acggtgqag gcttatcgaa ccactctctt aaagcgggca ttcacctggc atctttttqt gcgatggagc agctgcacgt ggcaccaggt acctctccgc ggcggtaggg ggcagcaggc catgcgcggg tgggtgagcg attccaagat agttcgtgcg cggaagacta acgttgaagc cgcagcttgt tccttgatga aactcttcgc ccgtactccg gagaaagqg gaagatacct tttcttactc tctttgcgcc aacggcctct ccacctctca acctcagaag accatgcaat ggacccctca accgatagca ttgggcattg ggggctcctt gtgactatta *caaggcaata cttatacttg *cagggccctc *tacttgttta fgqgttgatgt *tcacctaatq *gattcaaaca gccattacac itctcctaact itgtggcagtc iatatctggaz i ctaaacaatt a ggcacaqcct :cacggtaaaz aaacctgtaz a agtgcatact a tttgccacal :gagcatgcal "agcctcgacl cttgaccct( gcattgtct, g ggaggattq4 a ggcggaaag t aagcgcggc, gcccgctcc' agctctaaa c caaaaaact gtctatataa attaatacga ccgcatcgct tgacttctgc ccgcggtgat tgtcaagctt gcagggtttg attcgcgcgc gcacgcgcca qtaggcgctc ggtctagctg gcgcgtcgaa cgqcaaqgcg cggaggcgta atgtaqggta agggagcgaq tctgcctgaa tgqcgt ctgt tgaccagctc tgtcatactt qgtctttcca ccgccgaggg tctaaccagt tcaaccccgt ctccctttgt tatccgaacc ctctggacga aaaaaaccaa ccctaactgt cacaggcccc cagtgtcaga qtacccttac acttgaaaga tgcatqtaac ataatacttc tgcaacttaa atgttagtta *tttttataai: *cagcttcaaF ttgacgctac caccaaacaC aggctatggt Itaggaaacaz gtagactaai aaatacttg( icagttcaaa( ccttcctggi atacaaacg( ictgccaaaa( icactaaccal ctatgtcatl cctcttacal ctagaggC gtgccttct, j gaagqtgc j agtaggtgt g gaagacaat a accagctgg g ggtgtggtg t ttcgctttc t cggggcatc t gattagggt gcagagctct ctcactatag gtctgcgagg gctaagattg qcctttgagg ggtggcaaac gt ttt tgt cg aacgcaccgc accgcggttg gttggtccag cgtctcgtcc gtagtctatc gcqctcgtat catgccgcaa gcatcttcca gaggtcggga gatggcatqt gagacctacc ggcggtgacc atcctgtccc gtactcttgg acctgagcga cacagtcgca gtatccatat atcccccaat tctagttacc ggccggcaac gtcaaacata qgctgccgcc qctaaccqtg aggaaagcta tatcactqcc gcccatttat agacgaccta cttgcaaact tgtagcagga Ltccgtttgat ,ctcagcccac icaattccaaa -agccatagcc -aaatcccctc :tcctaaacta iaaataatgat itqcagagaaa tacagtttca I tgctcatctt a cccagaatat tgttggattt Staacattgtc t tacactaaac t ttcatgggac ctttttcatac c ctattctatz a gttgccagcc a ctcccactgl c attctattcl a gcaggcatg( g gctctaggg( g ttacgcgca( t tcccttcCtl c ctttaqggt g atggttcaci ctggctaact ggagacccaa gccaqctgtt tcagtttcca gtggccgcat gacccgtaga cgatcggcgc cattcgggaa tgcagggtga cagaggcggc gggqqgtCtg ttqcatcctt gggttgagtg atgtcgtaaa ccgcggatgc ccgaggttgc gagttgqatg gcgtcacgca tgcacgtcta ttttttttcc atcggaaacc gtccgcatcg agatccaaqa gacacgqaaa qggtttcaag tccaatqgca cttacctccc aacctgqaaa gcacctctaa cacgactcca qccctgcaaa tcacccctc acacaaaatg aacactttqa aaagttactg ggactaagga qctcaaaacc aacttggata aagcttgaqg attaatgcag aaaacaaaaa *ggaactggcc *aagctaactt *gatgctaaac gttttggctg *attataaqat tgqaacttta atgcctaacc agtcaagttt ggtacacagq tggtctggCC attgcccaac gtgtcacctz atctgttgtt cctttcctaE ggggggtgg! tqqqgatgcj Sgtatccccac j cgtgaccgct t tctcgccac, t ccqatttaql ci tagtgggcci agagaaccca 840 gcttggtacc 900 qgggtgagta 960 aaaacgagga 1020 ccatctggtc 1080 gggcqttgga 1140 gctccttggc 1200 agacggtggt 1260 caaggtcaac 1320 cgcccttgcg 1380 cgtccacggt 1440 gcaagtctag 1500 qgggacccca 1560 cgtagagggg 1620 tggcgcgcac 1680 tacgggcggg 1740 atatggttgg 1800 cgaaggaggc 1860 gggcgcagta 1920 acaqctcgcg 1980 cgtcggcctc 2040 accggatcgg 2100 tgaagcgcgc 2160 ccggtcctcc 2220 agagtccccc 2280 tgcttgcgct 2340 aaaatqtaac 2400 tatctgcacc 2460 tgqtcgcggq 2520 aacttagcat 2580 catcaggccc 2640 taactactqc 2700.
gaaaactagg 2760 ccgtagcaac 2820 qagccttggg 2880 ttqattctca 2940 aactaaatct 3000 ttaactacaa 3060 ttaacctaag 3120 qagatqggct 3180 ttggccatgg 3240 ttaqttttga 3300 tgtggaccac 3360 tcactttggt 3420 ttaaaggcag 3480 ttgacgaaaa 3540 gaaatggaga 3600 tatcagctta 3660 acttaaacgg 3720 1 aaacaggaqa 3780 acaactacat 3840 aataaaagaa 3900 iaatgctaqag 3960 tgcccctccc 4020 itaaaatgagg 4080 1gtggggcagg 4140 Igtgggctcta 4200 -gcgccctgta 4260 :acacttgcca 4320 3ttcgccggct 4380 :gctttacggc 4440 3, tcgccctgat 4500 WO 00/42208 PTEO/06 PCT/EPOO/00265 -36agacggtttt aaactggaac qgatttcggc tctgtggaat gtatgcaaag cagcaggcag taactccgcc gact aatt tt agtagtgagg tatccatttt atqgattgca cacaacagac cggttctttt cgcggctatc ctgaagcggg ctcaccttgc cgcttgatcc qtactcggat tcgcgccagc tcgtgaccca gattcatcga cccgtgatat gtatcgccgc gagcgggact t t tcgat tcc cggctqgatg gtttattgca agcatttttt tgtctgtata tgtgtgaaat taaaqcctgg cgctttccag gagaggcggt ggtcgttcgg aqaatcaggg ccgtaaaaag caaaaatcga gtttccccct cctgtccqcc tctcagttcg gcccgaccgc cttatcgcca tgctacagag tatctgcgct caaacaaacc a aa aa a agg a cgaaaactca ccttttaaat tgacagttac atccatagtt tggccccagt aataaaccag catccagtct gcgcaacgt t ttcattcagc aaaagcggtt atcactcatg cttttctgtg gagttgctct agtgctcatc gagatccagt caccagcgtt tcgccctttg aacactcaac ctattqgtta gtqtgtcagt catgcatctc aagtatgcaa catcccgccc ttttatttat aggctttttt cggatctgat cgcaggttct aatcggctgc tgtcaagac gtggctggcc aagggactgg tcctgccgag ggctacctgc ggaagccggt cgaactgttc tggcgatgdc ctgtggccgg tgctgaaqag tcccgattcg ctggggttcg accgccgcct atcctccagc gcttataatg tcactgcatt ccgtcgacct tgttatccgc gqtgcctaat tcgggaaacc ttgcgtattg ctgcggcgag gataacgcag gccgcgttgc cgctcaagtc ggaagctccc tttctccctt gtqtaggtcg tgcgccttat ctggcagcag ttcttgaagt ctgctgaagc accgctggta tctcaagaag cgttaaggga taaaaatgaa caatqcttaa gcctgactcc gctgcaatga ccagccggaa attaattgtt gttgccattg tccggttccc agctccttcg gttatggcag actggtgagt tgcccggcgt attggaaaac tcgatgtaac tctgggtgag acgttggagt cctatctcgg aaaaatgagc tagggtgtgg aattagtcag agcatgcatc ctaactccgc gcagaggccg ggaggcctag caagagacag ccggccgctt tctgatgccg gacctgtccg acgacgggcg ctgctattgg aaagtatcca ccattcgacc cttgtcgatc qccaggctca tgcttgccga ctgqqtgtgg cttggcggcg cagcgcatcg aaatgaccga tctatgaaag gcggggatct gttacaaata ctagttgtg ctagctagag tcacaattcc gagtgaqcta tqtcgtgcca ggcgctcttc cggtatcagc gaaaqaacat tggcgttttt agagqtggcg tcgtgcgctc cgggaaqcgt ttcgctccaa ccggtaacta ccactggtaa ggtggcctaa cagttacctt qcggtqgttt atcctttgat ttttggtcat gttttaaatc tcagtgaggc ccgtcgtgta t accgcgaga gggccgagcg gccgggaagc ct acaggcat aacgatcaag gtcctccgat cactgcataa actcaaccaa caatacggga gttcttcggg ccactcgtgc caaaaacagq ccacgttctt tctattcttt tqatttaaca aaagtcccca caaccaggtg tcaattagtc ccagttccgc aggccgcctc gcttttgcaa gatgaggatc gggtggagag ccgtgttccg qtgccctgaa ttccttgcgc gcgaagtgcc tcatggctga accaagcgaa aggatgatct aggcgcgcat atatcatggt cggaccgcta aatgggctga ccttctatcg ccaagcgacg gttgggcttc catgctggag aagcaatagc tttgtccaaa cttggcgtaa acacaacata actoacatta gctgcattaa cgcttcctcg tcactcaaag gtgAgcaaaa ccataggctc aaacccgaca tcctgttccg ggcgctttct gctgggctgt tcgtcttgag caggattagc ctacggctac cggaaaaaga ttttgtttgc cttttctacg gagattatca aatctaaagt acctatctca gataactacg cccacgctca cagaagtggt tagagtaagt cgtggtgtca gcgagttaca cgttgtcaga ttctcttact gtcattctga taataccgcg gcgaaaactc acccaactga aaggcaaaat taatagtgga tgatttataa aaaatttaac ggctccccag tggaaagtcc agcaaccata ccattctccg tgcctctgag aaagctcccg gtttcgcatg gctattcggc gctgtcagcg tgaactgcag agctgtgctc ggggcaggat tgcaatgcgg acatcgcatc ggacgaagag gcccgacggc ggaaaatggc tcaggacata ccgcttcctc ccttcttgac cccaacctgc ggaatcgttt ttcttcgccc atcacaaatt ctcatcaatq tcatggtcat cgagccggaa attgcgttgc tgaatcggcc ctcactgact gcggtaatac ggccagcaaa cgcccccctg ggactataaa accctgccqc caatgctcac gtgcacgaac tccaacccgg agagcgaggt actagaagga gttggtagct aagcagcaga gggtctgacg aaaaggatct atatatgagt gcgatctgtc atacgggagg ccggctccag cctgcaactt agttcgccag cqctcgtcgt tgatccccca agtaagttgg gtcatgccat gaatagtgta ccacatagca tcaaggatct tcttcagcat ctcttgttcc gggattttgg gcgaattaat gcaggcagaa ccaggctccc gtcccgcccc ccccatggct ctattccaga ggagcttgta attgaacaag tatgactggg caggggcgcc gacgaggcag gacgttgtca ctcctgtcat cggctgcata gagcgagcac catcaggggc gaggatctcg cqcttttctg gcgttggcta qtgctttacg gagttcttct catcacgaga tccgggacgc accccaactt tcacaaataa tatcttatca agctgtttcc gcataaagtg qctcactgcc aacgcgcggg cgctgcgct c qgttatccac aggccaggaa acgagcatca gataccaggo ttaccggata gctgtaggta cccccgttca taagacacga atgtaggcgg cagtatttgg cttgatccgg ttacqcgcag ctcagtggaa tcacctagat aaacttggtc tatttcgttc gcttaccatc atttatcagc tatccgcctc ttaatagttt ttggtatggc tgttgtgcaa ccgcagtgtt ccgtaagatg tgcggcgacc gaactttaaa taccgctgtt cttttacttt 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 5580 5640 5700 5760 5820 5880 5940 6000 6060 6120 6180 6240 6300 6360 6420 6480 6540 6600 6660 6720 6780 6840 6900 6960 7020 7080 7140 7200 7260 7320 7380 7440 7500 7560 7620 7680 7740 7800 7860 7920 7980 8040 8100 8160 8220 gccgcaaaaa agggaataag WO 00/42208 WO 0042208PCTIEPOO/00265 -37ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaaqcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtc <210> 44 <211> 7960 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: plasmid 8280 8340 8383 <400> 44 gacggatcgq ccgcatagtt cgagcaaaat ttagggttag gattattgac tggagttccg cccgcccatt attgacgtca atcatatgcc atgcccagta tcgctattac actcacgggg aaaatcaacg qtaggcgtgt ctgcttactg gtttaaactt gggccagctg tgtcagtttc gggtggccgc acgacccgta cgcgatcggc gccattcgq tgtgcagggt agcagaggcg ccggggggt c tcttgcatcc atgggttgag aaatgtcgta caccgcggat gaccgaggtt gtgagttgga ccgcgtcacg cctgcacqtc cctttttttt ggatcggaaa gagtccgcat caagatccaa atgacacgga atgggtttca cctccaatgg accttacctc taaacctgga ccgcacctct tgcacgactc tagccctgca cctcaccccc atacacaaaa taaacacttt gagatctccc aaqccagtat ttaagctaca qcgttttgcg tagttattaa cgttacataa gacgtcaata atgggtqgac aagtacgccc catgacctta catqgtgatg atttccaagt gqactttcca acggtgqqag gcttatcgaa aaqcttggta ttggggtgag caaaaacgag atccatctgg gaggqcgttg gcgctccttg aaagacggtg gacaaggtca gccgcccttg tgcqtccacg ttgcaagtct tgggggaccc aacgtagagg gctggcgcgc gctacgggcg tgatatggtt cacgaaggag taqggcqcag ccacagctcg cccgtcggcc cgaccggatc gatqaagcgc aaccggtcct agagagtccc catgcttgcg ccaaaatgta aatatctgca aatggtcgcg caaacttagc aacatcaggc tctaactact tggaaaacta gaccgtagca gatcccctat ctgctccctg acaaggcaag ctgcttcgcg tagtaatcaa cttacggtaa atgacgtatg tatttacqgt cctattgacg tgggactttc cggttttggc ctccacccca aaatgtcgta gtctatataa attaatacga ccgagctcgg tactccctct qaggatttga tcagaaaaga gacagcaact gccgcgatgt gtgcgctcgt acgctggtgg cgcgagcaga gtaaagaccc agcgcctgct catggcatgg ggctctctga acgtaatcgt ggctgctctg ggacgctgga gcgtaggagt tagtccaggg cggttgagga tccgaacgag gqaaaacct c gcaagaccgt ccaactgtgc cctggggtac ctcaaaatg accactgtga cccctcacag ggcaacacac attgccaccc cccctcacca gccactggta ggactaaagt actggtccag qgtcgactct cttgtgtgtt gcttgaccga atgtacgggc ttacgggqtc atggcccgcc ttcccatagt aaactgccca tcaatgacqg ctacttggca aqt acat caa ttgacgtcaa acaactccgc gcagagctct ctcactatag atccactctc gaaaagcgqg tattcacctg caatcttttt tggcgatgga ttagctgcac cgggcaccag ctacctctcc atggcggtag cgggcagcag gccatgcqg ggtgggtgag gtattccaag atagttcgtg ctcggaagac agacgttqaa cgcgcagct t tttccttgat caaactcttc atccgtactc tcgagaaagq ctgaagatac cttttcttac tctctttgcg gcaacggcct gcccacctct ttacctcaga tcaccatgca aaggacccct ccaccgatag gcttgggcat acggggctcc gtgtgactat caqtacaatc ggaggtcqct caattgcatg cagatatacg attagttcat tggctqaccg aacgccaata cttgqcagta taaatggccc gtacatctac tgggcgtgga tgggagtttq cccattgacg ctggctaact ggagacccaa ttccqcatcg catgacttct gcccgcqgtg gttgtcaagc gcgcagggtt gtattcgcgc gtgcacgcgc gcgtaggcgc ggggtctaqc gcqcgcgtcg ggcqqcaagc cgcggaggcg atatgtaggg cgagggagcg tatctgcctg qctggcgtct gttgaccagc gatgtcatac gcggtctttc cgccgccgag cgtctaacca cttcaacccc t cct ccct tt cctatccgaa ctctctggac caaaaaaacc agccctaact atcacaggcc cacagtgtca cagtaccctt tgacttgaaa tttgcatgta taataatact tgctctgatg gagtagtgcg aagaatctgc cgttgacatt agcccatata cccaacgacc gggactttcc catcaagtgt gcctggcatt gtattagtca tagcggtttg ttttggcacc caaatgggcg agagaaccca qctggctagc ctgtctgcga gcgctaagat atgcctttga ttggtggcaa tggtttttgt gcaacgcacc caaccgcggt tcgttggtcc tgcgtctcgt aagtagtcta gcqcgctcgt tacatgccgc tagcatcttc aggaqqtcgg aagatggcat gtgagaccta tcqgcggtga ttatcctgtc cagtactctt ggacctgagc gtcacagtcq qtgtatccat gtatccccca cctctagtta gaggccggca aagtcaaaca gtggctgccg ccgctaaccg gaaggaaagc actatcactg gagcccatt t acagacgacc tccttgcaaa 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 WO 00/42208 WO 0042208PCT/EPOO/00265 ctaaagttac gaggactaag atgctcaaaa acaacttgga aaaagcttga ccattaatgc tcaaaacaaa taggaactgg ataagctaac aagatgctaa cagttttggc ttattataag attggaactt ttatgcctaa tcagtcaagt acggtacaca actggtctgg acattgccca gatcaqcctc cttccttgac catcgcattg agggqgagga ctgaggcgga cattaagcgc tagcgcccgc gtcaaqctct accccaaaaa tttttcqccc gaacaacact cggcctattg gaatgtgtgt aaagcatgca gcagaagtat cgcccatccc ttttttttat gaggaggct t t tt tcggat c ataatacgac caccgcgcgc ggacttcgtq cgcggtccac ggacgagctc gcdggccatc qgccggcaac cgattccacc ctggatgatC tattgcagct atttttttcz ctgtatacc.
gtqaaattgt agcctggggi tttccagtc( aggcggttt( cgttcggct( atcaggggal taaaaaggci aaatcgacgi tccccctgg, gtccgcctt cagttcggt.
cgaccgctg atcgccact tggagccttg gattgattct ccaactaaat tattaactac ggttaaccta aggagatgg aattggccat ccttagtttt tttgtggacc actcactttg tgttaaaggc atttgacgaa tagaaatgga cctatcagct ttacttaaac ggaaacagqa ccacaactac agaataaaag gactgtgcct cctggaaggt tctgagtagg ttgggaagac aagaaccagc qgcgqgtgtg tcctttcgct aaatcggggc acttgattaq tttgacgttg caaccctatc gttaaaaaat cagttagggt tctcaattag gcaaagcatg gcccctaact ttatgcagag ttttqgagqc *tgatcagcac *aaqgtqagga *gacgtcgccg gaggacgact fgaccaqgtgg ftacgccgagt faccgagatcc -tgcgtgcact -gccgccttct -ctccagcgcc -tataatggtt ictgcattctz jtcgacctctz tatccgctcz gcctaatgaC.
I ggaaacctgt I cqtattggg' 3 cggcgagcgc t aacgcaggai gcgttgctg( -tcaagtcag a agctccctci t ctcccttcgl g taggtcgtt, c gccttatcC, g gcagcagcc ggttttgatt caaaacagac ctaagactag aacaaaggcc agcactgcca cttgaatttg ggcctagaat gacagoacag acaccagctc gtcttaaCaa agtttggcC aatggagtgc gatcttactg tatccaaaat ggagacaaaa -gacacaactc attaatgaaa aagcggccgc tctagttgcc gccactccca tqtcattcta aatagcagqc tggggctcta gtggttacqC ttcttccctt atccctttag ggtgatggtt gagtccacgt tcggtctatt gaqctgattt qtggaaagtc tcagcaacca catctcaatt ccgcccagtt qccgaggccg ctaggctttt gtgttgacaa actaaaccat gagcggtcga tcqccggtqt tgccggacaa *ggtcqgaggt fgcgagcagCC *tcgtggcCge atgaaaggtt Igggatctcat acaaataaac gttgtggttt igctagagctt i caattccaci j tgagctaact cgtgccagcl gctcttccg( ;tatcaqctci a agaacatgt( a ggtggcgaa.
g tgcgctctCl g gaagcgtgg, c gctccaagc g gtaactatc a ctggtaaca cacaaggCaa gccttataCt gacagggccc tttacttgtt aggggttgat gttcacctaa ttgattcaaa gtgccattaC catctcctaa aatgtggcag caatatctgg tactaaacaa aaggcacagc ctcacggtaa ctaaacctgt caagtgcata tatttgccac tcgagtctag agccatctgt ctgtcctttc ttctgggggg atgctqggga qggggtatcc gcagcgtgac cctttctc ggttccgatt cacgtagtgg t ctt ta atag cttttgattt aacaaaaatt cccaggctcc ggtqtqgaaa agtcagcaaC ccqcccattc cctctgcctc gcaaaaagct ttaatcatcg ggccaagttg *gttctggacc g gtccgggac caccctggcc cgtgtccacg gtgggggCgg iggagcagqac gggcttcgga gctggagttc Icaatagcatc gtccaaactc ggcgtaatca icaacatacga cacattaatt gcattaatga ttcctcgctc a ctcaaaggcc g agcaaaagqc a taggctccgc a cccgacagg :tgttccgac c gctttctca t gggctgtgt( g tcttgagtc( g gattagcagi tatgcaactt tgatgttagt tctttttata tacagcttca gtttgacgct tgcaccaaac caaggctatg agtaggaaac ctgtagacta tcaaatactt aacagttcaa ttccttCCtg ctatacaaac aactgccaaa aacactaacc ctctatgtca atcctcttac agggcccgtt tgtttgCCCC ctaataaaat tggggtgggg tqcggtgggc ccacgcgccc cgctacactt cacgttcgcc tagtgcttta qccatcqccc tgqactcttg ataaqggatt taacgcgaat ccaggcaggc gtccccaggc catagtcccg tccgccccat tgagctattc cccgggagct gcatagtata accaqtgccg gaccgqctcg gacgtgaccc tgggtgtggg aacttccqggg gagttcccc tgacacgtgc atcgttttcc ttcgcccacc acaaatttca atcaatgtat tggtcatagc gccggaagca gcgttgcgct iatcggccaac -actgactcgc Igtaatacggt :cagcaaaagg :ccccctgacg ictataaagat ctgccgctta I tgctcacgct j cacgaacccc 2aacccggtaa a gcgaggtatj aatgtagcag 2940 tatccgtttg 3000 aactcagccc 3060 aacaattcca 3120 acagccatag 3180 acaaatcccc 3240 gttcctaaac 3300 aaaaataatg 3360 aatgcagaga 3420 gctacagttt 3480 agtgctcatc 3540 gacccagaat 3600 gctgttggat 3660 agtaacattg 3720 attacactaa 3780 ttttcatggg 3840 actttttcat 3900 taaacccgct 3960 tcccccgtgc 4020 gaggaaattg 4080 caggacagca 4140 tctatggctt 4200 ~tgtagcggcg 4260 gccagcgccc 4320 ggctttccc 4380 cqgcacctcg 4440 tgatagacgg 4500 ttccaaactg 4560 ttggggattt 4620 taattctgtg 4680 agaagtatgc 4740 tccccagcag 4800 cccctaactc 4860 ggctgactaa 4920 cagaagtagt 4980 tgtatatcca 5040 tcggcatagt 5100 ttccggtgct 5160 ggttctcccg 5220 tgttcatcag 5280 tqcgcggcct 5340 acgcctccgq 5400 tgcgcgaccc 5460 tacgaqattt 5520 gqgacgccgg 5580 ccaacttgtt 5640 caaataaagc 5700 cttatcatgt 5760 tgtttcctgt 5820 taaagtgtaa 5880 cactgcccgc 5940 gcgcggggag 6000 tgcgctcggt 6060 tatccacaga 6120 ccaggaaccg 6180 agcatcacaa 6240 accaggcgtt 6300 ccggatacct 6360 qtaggtatct 6420 ccgttcagcc 6480 gacacgactt 6540 taggcggtgc 6600 WO 00/42208 WO 0042208PCT/EPOO/00265 tacagagttc ctgcgctctq acaaaCCaCC aaaaggatct aaactcacgt tttaaattaa cagttaccaa catagttgcc ccccagtgct aaaccagcca ccagtctatt caacgttgtt attcagctcc agcggttagg actcatggtt ttctgtqact ttgctcttgC gctcatcatt atccagttcg cagcgtttct gacacggaaa qggttattgt ggttccgcgC ttgaagtggt ctgaagccag gctggtagcg caagaagatc taagggattt aaatgaagtt tgcttaatca tgactccccg gcaatgatac gccggaaggg aattgttgcc gccattgcta ggttcccaac tccttcggtC atggcagcac ggtgagtact ccggcgtcaa qgaaaacqtt atgtaaccca gggtgagcaa tgttgaatac ctcatgaqg acatttCCCC ggcctaacta ttaccttCgg gtggtttttt ctttgatctt tggtcatgag ttaaatcaat gtgaggcacc tcgtgtagat cgcgagaccc ccgagcgcag gggaagctag caggcatcgt gatcaaggcq ctccgatcgt tgcataattc caaccaagtc tacgggataa cttcgqggcg ctcgtgcacC aaacaggaaq t catactctt qatacatatt gaaaagtgcc cggctacact aaaaagagtt tgtttgcaag ttctacgggg attatcaaaa ctaaagtata tatctcagcg aactacgata acgctcaccg aagtggtcct agtaagtagt ggtgtcacgc agttacatga tgtcagaagt tcttactgtc attctgagaa taccqcgcca aaaactctca caactgatct gcaaaatgcc cctttttcaa tgaatgtatt acctgacgtc agaaggacag ggtagctctt qagcagatta tctgacgctc aggatcttca tatgagtaaa atctgtctat cgggagggct gctccagatt gcaactttat tcgccagtta tcgtcgtttg tcccccatgt aagttggccg atgccatccg tagtgtatgc catagcagaa aggatcttac tcagcatctt gcaaaaaaqq tattattqaa tagaaaaata tatttggtat gatccggcaa cgcgcagaaa agtggaacga cctagatcct cttggtctga ttcgttcatc taccatctgg tatcagcaat ccgcctccat atagtttgcg gtatggcttc tgtgcaaaaa cagtgttatc taagatgctt ggcgaccag ctttaaaagt cgctgttgag ttactttcac gaataagggc gcatttatca aacaaatagg 6660 6720 6780 6840 6900 6960 7020 7080 7140 7200 7260 7320 7380 7440 7500 7560 7620 7680 7740 7800 7860 7920 7960 <210> <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 45 atgggatcca aqatgaaqcg cgcaagaccg <210> 46 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequenlce: primer <400> 46 cactatagcg qccqcattct cagtcatctt <210> 47 <211> 7989 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: plasmid <400> 47 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatq aagaatctgc 180 ttagggttag qcgttttqcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240 WO 00/42208 WO 0042208PCT/EPOO/00265 gattattgac tggagttccg cccgcccatt attgacgtca atcatatgcc atgcccagta tcgctattac actcacgggg aaaatcaacg gtaggcgtgt ctgcttactg gtttaaactt gggccagctg tgtcagtttc gggtggccgC acgacccgt"a cgcgatcggc gccattcggg tgtgcagggt agcagaggcg ccgggggtc tcttgcatcc atgggttgag aaatgtcgta caccgcggat gaccgaggtt gtgagttgga ccgcgtcacg cctgcacgtc cctttttttt ggatcggaaa gagtccgcat caagatccaa atgacacgga atgggtttca cctccaatg accttacctc taaacctgga ccgcacctct tgcacgactc tagccctgca cctcaccccc atacacaaaa taaacacttt ctaaagttac gaggactaag atqctcaaaa acaacttgga aaaagcttqa ccattaatgc tcaaaacaaa taggaactg ataagctaac aacaaaaccc gatatgtaac tctccattaa ctcttaaaac ttatgccaag attatatttt ttactgttat tatggtcctt catttacctt tagttattaa cgttacataa gacgtcaata atgggtggac aagtacgccc catgacctta catggtgatg atttccaagt ggactttcca acggt gggag gcttatcgaa aagcttggta ttggggtgag caaaaacgag atccatctgg gagggcgttg, gcgctccttg aaagacggtg gacaaggtca gccgcccttg tgcgtccacg ttqcaagtct tgggggaccc aacgtagagg gctggcgcgc gctacgggcg tgatatggtt cacgaaggag tagggcgcag ccacagctcg cccgtcggcc cgaccggatc gatgaagcgc aaccggtcct agagagtccc catgcttgcg ccaaaatgta aatatctgca aatggtcgcg caaacttagc aacatcaqgc tctaactact tggaaaacta gaccgtagca tggagccttg gattgattct ccaactaaat tattaactac ggttaaccta aggaqatqgg aattggccat ccttagtttt tttgtggacc agatagcaaa gctaatggga tgtagaacta agatctagaa tactacagcg tggtcaatgc gcttaataaa gaatgctggt ttcctatatt tagtaatcaa cttacggtaa atgacgtatg tatttacggt cctattgacg tgggactttc cggttttggc ctccacccca aaatgtcgta gtctatataa attaatacga ccgagctcgg tactccctct gaggatttga tcagaaaaga gccgcgatgt gtgcgctcgt acgctggtgg cgcgagcaga gtaaagaccc agcgcctgct catggcatgg ggctctctqa acgtaatcgt ggctgctctg ggacgctgga gcgtaggagt tagtccaggg cggttgagga tccgaacgag ggaaaacctc gcaaqaccgt ccaactgtgc cctggggtac ctcaaaatgg accactgtga cccctcacag ggcaacacac attgccaccc cccctcacca gccactggta ggactaaagt actggtccag ggttttgatt caaaacagac ctaagactag aacaaaqqcc agcactgcca cttgaatttq ggcctagaat gacagcacag ggtccaaaac ctaactttaa gcctcagact tactttgatg ctaaaataca tatccatttg tactacaaag cgcctgccag ctagctccag agaqaagatg ttacggggtc atggcccgcc ttcccatagt aaactgccca tcaatgacgg ctacttggca agtacatcaa ttgacgtcaa acaactccgc gcagagctct ctcactatag atccactctc gaaaagcggg tattcacctg caatcttttt tggcgatgga ttagctgcac cggqcaccaq ctacctctcc atggcggtag cgggcagcag gccatgcgcq gqtgggtgaq gtattccaaq atagttcgtg ctcggaagac aqacgttgaa cgcgcagct t tttccttgat caaactcttc atccgtactc tcgagaaagg ctgaagatac cttttcttac tctctttgcg gcaacggcct qcccacctct ttacctcaga tcaccatgca aaggacccct ccaccgatag gcttgggcat acggggctcc gtgtgactat cacaaggcaa gccttatact gacagggccc tttacttgtt aggggttgat gttcacctaa ttgattcaaa gtgccattac cagaagccaa tccttgtaaa acgttaacac ccactggtca agcaaaccgc tccttcctaa caagcgatgg atagtcgcac aaactactca actgattttt attagttcat tggctgaccg aacgccaata cttggcagta taaatggccc gtacatctac tgggcgtgga tgggagtttg cccattgacg ctggctaact ggagacccaa ttccgcatcg catgacttct gcccgcqgtg qttgtcaagc gcgcagggtt gtattcgcgc gtgcacgcgc gcgtaggcgc ggggtctaqc qcgcgcqtcg ggcggcaagc cgcggagqcg atatgtagqg cgagggaqcq tatctgcctg gctggcgtct gttgaccagc gatgtcatac gcggtctttc cgccqccgag cgtctaacca cttcaacccc tcctcccttt cctatccgaa ctctctgqac caaaaaaacc agccctaact atcacaggcc cacagtqtca cagtaccctt tqacttqaaa tttgcatgta taataatact tatqcaactt tgatgttagt tctttttata tacagcttca qtttgacgct tgcaccaaac caaggctatg agtaggaaac ctgcataatt aaatggagga cttatttaaa tatattacca tgactttagt tgcgggaaca tgcccttttt atcctatgtt ggcaaccctc aagaagcqgc agcccatata 300 cccaacgacc 360 gggactttcc 420 catcaagtgt 480 gcctggcatt 540 gtattagtca 600 tagcgqtttg 660 ttttggcacc 720 caaatgggcg 780 agagaaccca 840 gctggctagc 900 ctgtctgcga 960 gcgctaagat 1020 atgcctttga 1080 ttggtggcaa 1140 tggtttttgt 1200 gcaacgcacc 1260 caaccgcggt 1320 tcgttggtcc 1380 tgcgtctcgt 1440 aagtagtcta 1500 gcgcgctcqt 1560 tacatgccgc 1620 tagcatcttc 1680 aggaggtcgq 1740 aagatggcat 1800 gtgagaccta 1860 tcggcggtga 1920 ttatcctgtc 1980 cagtactctt 2040 qgacctgagc 2100 gtcacagtcg 2160 gtgtatccat 2220 qtatccccca 2280 cctctagtta 2340 gaggccggca 2400 aagtcaaaca 2460 gtggctgccg 2520 ccgctaaccg 2580 gaaggaaagc 2640 actatcactg 2700 gagcccattt 2760 acagacgacc 2820 tccttgcaaa 2880 aatgtagcag 2940 tatccgtttq 3000 aactcacjccc 3060 aacaattcca 3120 acagccatag'3180 acaaatcccc 3240 gttcctaaac 3300 aaaaataatg 3360 gaatacggga 3420 attgttaatg 3480 aacaaaaatg 3540 gactcatctt 3600 gcaagaggtt 3660 cataatgaaa 3720 ccgttggaag 3780 atgacttttt 3840 ataacctccc 3900 cgctcgagtc 3960 WO 00/42208 WO 0042208PCT/EPOO/00265 -41tagagggccc tgttqtttgc ttcctaataa gggtggggtg ggatgcggtg ggggtatccc cagcgtgacc ctttctcqcc gttccgattt acgtagtggg ctttaatagt ttttgattta acaaaaattt ccaggctccc gtgtggaaaq cgcccattct ctctgcctct caaaaagctc taatcatcgg gccaagttga ttctggaccq qtccgggacg accctggcct gtgtccacga tggggCggg gagcaggact ggcttcgqaa ctggagttct aatagcatca tccaaactca qcgtaatcat aacatacgag acattaattg cattaatgaa tcctcqctca tcaaaggcgg gcaaaaggcc agqctccqcc ccgacaggac gttccgaccc ctttctcaat ggctgtgtgc cttgagtcca attagcagag ggctacacta aaaagagttg gtttgcaagc tctacggqqt ttatcaaaaa taaagtatat atctcagcga actacgatac cgctcaccgg agtggtcctg gtaagtagtt gtgtcacgct gttacatgat gtcagaagta cttactgtca ttctgagaat accgcgccac gtttaaaCc ccctcccccg aatqaggaaa qqgcaggaca ggctctatgq cacgcqccct gctacacttg acgttcgccg agtgctttac ccatcgcct ggactcttgt taagggattt aacgcgaatt caggca ggca tccccaggct atagtcccgc ccgccccatg gagctattcc ccgggagctt catagtatat ccagtgccgt accggctcqg acgtgaccct gggtgtgqgt acttccggga agttcgccct gacacgtgct tcgttttccg tcgcccaccc caaatttcac tcaatgtatc ggtcatagct ccggaagcat cgttgcgctc tcggccaacg ctgactcgct taatacggtt agcaaaagqc cccctgacga tataaagata tgccgcttac gctcacgctg acgaaccccc acccggtaaq cgagqtatgt gaaggacagt qtagctcttq agcaqattac ctgacgctca ggatcttcac atgaqtaaac tctgtctatt gggagggctt ctccaqattt caactttatc cgccagttaa cgtcgtttgg cccccatgtt agttggccgc tgccatccgt agtgtatgcq atagcagaac gctgatcagc tgccttcctt ttgcatcgca qcaaggggga cttctgaggc gtagcggcgc ccagcgccct gctttccccg ggcacctcga gatagacggt tccaaactgg tggggatttc aattctgtgg gaagtatgca ccccagcagg ccctaactcc gctgactaat agaagtagtg gtatatccat cqgcatagta tccggtgctc gttctcccgg gttcatcagc qcgcggcctg cgcctccgqg gcqcqacccq acgagatttc ggacqccggc caacttgttt aaataaagca ttatcatgtc gtttcctgtg aaagtgtaaa actgcccgct cgcggggaga gcgctcqgtc atccacagaa caggaaccgt gcatcacaaa ccaggcgttt cgqatacctg taggtatctc cgttcagccc acacgactta aggcggtgct atttggtatc atccggcaaa gcgcagaaaa gtggaacgaa ctagatcctt ttggtctgac tcgttcatcc accatctggc atcagcaata cgcctccatc tagtttgcgc tatgqcttca gtqcaaaaaa agtgttatca aagatgcttt gcgaccgagt tttaaaagtg ctcgactgtg gaccctggaa ttgtctgagt ggattgggaa ggaaagaacc attaagcgcg agcgcccgct tcaagctcta ccccaaaaaa ttttcgccct aacaacactc ggcctattgg aatgtgtgtc aagcatgcat cagaagtatg gcccatcccg tttttttatt aggaggcttt tttcgqatct taatacgaca accgcgcgcg gacttcgtgg gcggtccagg gacgagctgt ccggccatga gccggcaact gattccaccg tgqatgatcc attgcagctt tttttttcac tgtataccgt tgaaattqtt gcctggggtq ttccagtcgg ggcggtttgc gttcggctqc tcagqggata aaaaaggccg aatcgacgct ccccctggaa tccgcctttc agttcggtgt gaccgctgcg tcgccactgg acagagttct tgcgctctgc caaaccaccg aaaggatctc aactcacgtt ttaaattaaa agttaccaat at agt tgcct cccagt gctg aaccagccag cagtctatta aacgttgttg ttcagctccg gcggttagct ctcatggtta tctgtgactg tgctcttgcc ctcatcattg ccttctagtt ggtgccactc aggtgtcatt gacaatagca snccntagct gcgggtgtgg cctttcgctt aatcggggca cttgattagg ttgacgttgg aaccctatct ttaaaaaatg agttagggtg ctcaattagt caaagcatgc cccctaactc tatgcagagg tttggaggcc gatcaqcacg aggtqaggaa acgtcgccgg aggacqactt accaggtggt acqccgaqtg ccqagatcgq gcgtgcactt ccqccttcta t ccagcgcgg ataatgqtta tqcattctag cgacctctaq atccgctcac cctaatgagt gaaacctgtc gtattggqcg qqcgagcgqt acgcaggaaa cgttgctggc caagtcagag gctcctcgt tcccttcgqg aggtcgttcg ccttatccgg cagcagccac tgaagtggtg tgaagccagt ctggtagcgg aagaagatcc aagqgatttt aatqaagttt gcttaatcag gactccccgt caatgatacc ccggaagggc attgttgccg ccattgctac gttcccaacg ccttcggtcc tggcagcact gtgagtactc cggcgtcaat gaaaacgttc gccagccatc ccactgtcct ctattctggg ggcatgctgg ggggctctag tggttacgcg tcttcccttc tccctttagg gtgatggttc agtccacgtt cggtctattc agctgattta tggaaagtcc cagcaaccag atctcaatta cgcccagttc ccgaggccgc taggcttttg tgttgacaat ctaaaccatg agcggtcgag cgccggtgtg gccqgacaac gtcggaggtc cgagcagccg cgtggccgag tgaaaggttq ggatctcatg caaataaagc ttgtggtttg ctagagcttg aattccacac gagctaactc gtgccagctg ctcttccgct atcagctcac gaacatgtga gtttttccat gtggcqaaac gcqctctcct aagcgtggcg ctccaagctg taactatcgt tggtaacagg gcctaactac taccttcgga tggttttttt tttgatcttt qqtcatgaga taaatcaatc tgaggcacct cgtgtagata gcqagaccca cgagcgcaga ggaagctaga aggcatcgtg atcaaggcga tccgatcgtt gcataattct aaccaagtca acgggataat ttcggggcga 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 5580 5640 5700 5760 5820 5880 5940 6000 6060 6120 6180 6240 6300 6360 6420 6480 6540 6600 6660 6720 6780 6840 6900 6960 7020 7080 7140 7200 7260 7320 7380 7440 7500 7560 7620 7680 WO 00/42208 WO 0042208PCT/EPOO/00265 -42aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact ctttttcaat attattqaag catttatcag ggttattgtc tcatgagcgg gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg cctgacgtc <210> 48 <211> 7607 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: plasmnid tcgtgcaccc aacaggaagg catactcttc atacatattt aaaagtgcca 7740 7800 7860 7920 7980 7989 <400> 48 tctagaagat ttctagctac gtacaggatg agatcgatct gcgcgttcgt aacttcaggg atgttatatg tatcaccatg ttgtctcctc ttgtaacgaa tcactttttt acaattgtta ggaaatattc tggttacaat ccctctgcta gttattgtgc actcggtcqa ggccqccaqt tagccatttt agatcccaac ggaagggatt ttcccggcag tgtaccggag ggatgaagag qtcttgtcat tatgagqacc agtggtgggt ttttgtattg gaaccggagc ccgacatcac tctaacacac qtgagagttg gggcaacctt cgtgtgtggt ataatgttta cgtgggctaa tctgctgtgc ctgtggggct tttgaagagc gcgcttttcc gctgctgttg ggggggtacc aatcgcctgc cagcagcagg ggcctggacc ccgctgtaca tttattagat ttctagctac cctggccgtt cctcactctc tgagtttggg gagggggcaa gaccctcatg ttattttctt tttttaaatt ttcaaggcaa taattaaatg ttattggtag gatatacact accatgttca tgtctcatca ctgaaaatga cttttgqacc gaaccaccta gaggaggcgg gacttactca cccgagcagc qtgatcgatc ggtgaggagt tatcaccgga tgtqgcatgt ttggtgtggt tqattttttt ctgcaagacc ctgtqtctag ctcctgagat gtgggcgtcg tggacttgag taacgccttt acttgcatgg tcttggttac gtaacttgct catcccaggc ttttgaaatc aagagaaggt cttttttgag tgctggattt tactgttgtc aggaagccag ctcgqgaatg ggatgttcta ccgctgtaca tttattagat cggggtcaaa ttccgcatcg gacccttgat agttttcagg ataattttgt ttcattttct cacttttgtt tcagggtata ataaggtaga aaacaactac gtttgagatg tgccttcttc ttttggcaaa gacatattat agctgatcga cccttcacga tttcgcagat cttttccgcc cggagcagag ttacctgcca ttgtgttaga ggaatacggg ttgtctacaq aatttttttt aaaaggtcct t acccgccgt agaatgcaat acacccggtg ccaggctgtg ctgtaaacgc qtttgctgaa cgtgttaaat atctgacctc ggaacagagc aaagttagtc ctgtggtgag catcaagact ttttataaag tctggccatg ttccgtccgc gcggcggcgg aatgttgtac gctactttat ggatgttcta ccgtgtacag aaccaggttt ctgtctgcga tgttctttCt gtgttgttta ttctttcact gtaacttttt tatttgtcag ttatattgta atatttctgc atcctqgtca aggataaaat tttttcctac gaattagatc ctgccacgga agaggtactg actgtatgat ttttcccqac qgcgcccggt agccttgggt cgaggctggc ttatgtggag ggacccagat taagtgaaaa ttaattttta gtgtctgaac cctaaaatgg agtagtacgg gtcccgctgt gaatgtatcg cccaggccat tgagttgatg ggggcggggC atggaggctt tctaacagta tgcagaatta ctgtttgatt ttggattttt gataaatgga catctgtgga ccggcgataa caggagcaga aggtggctga tagatccgct gctactttat gatgttctag ggctataaaa gggccaggat ttttcgctat gaatgggaag ttctactctg cgttaaactt attgtaagta cttcagcaca atataaattc tcatcctgcc actctgagtc agctcctggq taagcttctq ggtgttatta gctgataatc ttagacgtga tctgtaatgt tctccggagc ccggtttcta tttccaccca caccccgggc attatgtgtt ttatgggcag cagttttgtg ctgagcctga cgcctgctat atagctgtga gccccattaa aggacttgct aaggtgtaaa taagtttaat ttaaagggta gggagtgttt cctcttggtt aggaggatta ctttgaatct ccacaccggg gcgaagaaac gagcggttgt taccgacgga gcccatggaa actgtatcca gtacaggatg tagatccgct ctactttatt gggggtgggg cgatcctgag tgtaaaattc atgtcccttg ttgacaacca tagcttgcat ctttctctaa gttttagaga tggctggcgt tttctcttta caaaccgggc caacgtgctg cagctcgagg ccgaagaaat ttccacctcc cggcccccga tggcggtgca cgcctcacct tgccaaacct gtgacgacga acggttgcag cgctttgcta tgggtgatag gtttaaagaa gcccgagcca cctgagacgc ctccggtcct accagttgcc taacgagcct cctgtgattg aaagggtgag tataatgcgc ggaagatttt ttggaggttt caagtgggaa gggtcaccag gcgcgctgcg ccatctgagc gagacacaag ggagcagcag cccgagagcc gaactgagac 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 WO 60/42208 WO 0042208PCTIEPOO/00265 -43gcattttgac cttgtgaggc ctgagtgtat cgcagaagta aggaggctat gcaaacttgt tagatacgga ttggcatgqa cggttttcct atacctgtqt ggaagggggt ggtgtacctt actqtggttg gcaactgcga tgaaqaccat tactqacccg gcaatttgag acggggtgtt ccaggtgcag atgtgaccga gctctagcga gaaagaatat ccqccatqag tgcccccatg tcctgcccgc ctgcagcctc ttqctttcct aqttgacggc agcagctqtt cggt t taa aa gctgtctcag ttaaagcaga aatttcacaa aatgtatctt ctgcgctcgq ttatccacac gccaggaacc gagcatcacE taccaggcgt accggatac tgtaggtat< cccgttcagc agacacgacl gtaggcggtc gtatttggti tgatccggci acgcgcagai cagtggaac, acctagatc, acttggtct tttcgttca ttaccatct ttatcagca tccgcctcc aataqtttg ggtatggct aattacagag tacagaggag tacttttcaa ttccatagag tagggtatat aaatatcagg ggatagggtg cggggtggtt ggccaatacc ggaagcctgg ggtgtgtcgc gggtatcctg cttcatgcta ggacagggcc tcacgtagc ctgttccttg t ca.cacta a g tgacatgacc accctgcgaq ggagctgagg tgaagataca ataaggtggg caccaactcg ggccggggtg aaactctact cgccqccgct gagcccgctt tcttttggca ggatctgcgc cataaataaa ctgactgctt *acttgtttat ataaagcatt *atcatgtctq ftcgttcggct iaatcaggggz gtaaaaaggc iaaaatcgacc -ttccccctg -tgtccgcctt -tcagttcggt -ccgaccgct( tatcgccacl g ctacagagtl a tctgcgctCi a aacaaacca( a aaaaaggati g aaaactcaC4 c ttttaaatt g acagttacc t ccatagttg g gccccagtg a taaaccagc a tccagtcta gcaacgttg t cattcagct gatgggcagg gctaggaatc cagatcaagg cagctgacca gcaaaggtgg aattgttgct gcctttagat attatgaatg aaccttatcc accgatgtaa cccaaaagCa tctgagggta gtgaaaagcg tctcagatgC agccactctc catttgggta atattgcttg atgaagatCt tgtggcggta cccgatcact gattgaggta ggtcttatgt tttgatggaa cgtcagaatg accttgacct tcagccgctg gcaagcaqtg caattggatt cagcagqttt aaaccagaCt aaqtcgcaag tgcagcttat tttttcactg IgtcgactCta gcggcgagcq taacgCagg2a cgcqttgctc ctcaagtcac aagctccctc tctcccttci gtaggtcgtt j cgccttatcC ggcagcagc =cttgaagtg( gctgaagCC cgctggtag( tcaagaagal ;ttaagggatl a aaaatgaagl a atgcttaat4 ctgaCtccC4 c tgcaatgat, agccggaag, t taattgttg, t tgccattgC c cggttccca g ctccttcgg t tatggcagc c tggtgagta g cccggcgtc .t tggaaaacg .c gatgtaacc ggctaaaggg tagcttttag ataattgcgc cttactggct cacttaggcc acatttctgg gtagcatgat taaggtttac tacacggtgt gggttcgqggg gggcttcaat actccagggt tggctgtgat tgacctgctc gcaaggcctg acagg--aggg agcCCgagag ggaaggtgct aacatattag tggtgctggc ctgaaatgtg agttttgtat gcattqtgag tqatggqctc acgagacCgt cagccaccgc cagcttCCCg ctttgacccq ctgccctgaa ctgtttggat ccgaattgga aatggttaca cattctagtt gactcttccg FgtatcagCtc aagaacatgt gcgtttttCC faggtggcgaa :qtgcgctctc Iggaagcgtgg cgctccaagc :ggtaactatc -actggtaaca j tggcctaact Sgttaccttcg ggtggttttt cctttgatct ttqgtcatga tttaaatCaa agtgaggcac gtcgtgtaga accgcqagaCC g gccgagcgca c cgggaagctz t acaggcatcc a cgatcaaggC t cctccgatcc a ctgcataatt tcaaccaagt a atacgggati t tcttcgggq( .c actcgtgCa( ggtaaagagg cttaatgacc taatgagctt gcagcaggg agattgcaag gaacggggCC aaatatgtgg tggccccaat aagcttctat ctgtgccttt taagaaatgc gcgccacaat taagcataaC ggacqgcaac gccagtgttt ggtgttccta catgtccaag gaggtacgat gaaccagcct ctqcacccgc tgqgcqtgqC ctgttttgca ctcatatttg caqcattgat gtctggaacg ccgcgqatt ttcatccgcc gqaacttaat qgcttcctCC ttggatcaaq tccaattcgg aataaaqcaa gtggtttgtc cttcctcgct actcaaaggc gagCaaaagq ataggctccg acccgacaqg ctgttccgac cgctttctca tqggctgtgt gtcttgaqtc ggattagcag acggctaCac gaaaaagagt ttgtttgcaa tttctacggg gattatcaaa tctaaagtat ctatCtcagc taactacgat cacgctCacc gaagtggtcc Lgagtaagtac Itggtgtcaci -gagttaCatc Ittgtcagaac :ctcttactgt :cattctgagz i atacCgCgC( zgaaaactct( ccaactgjat( gagcgggggg 2760 agacaccgtc 2820 gatctgctgg 2880 gatqattttg 2940 tacaagatca 3000 gaggtggaga 3060 ccgggggtgc 3120 tttagcggta 3180 gggtttaaca 3240 tactgctgct 3300 ctctttgaaa 3360 gtggcctccg 3420 atggtatgtg 3480 tgtcacctgc 3540 gagcataaca 3600 ccttaccaat 3660 qtgaacctga .3720 gagacccgca 3780 gtqat.gctgg 3840 gctgagtttg 3900 ttaagggtgg 3960 gcaqccgccg 4020 acaacgcgca 4080 qgtcgccccg 4140 ccgttggaga 4200 qtgactgact 4260 cgcgatgaca 4320 gtcgtttctc 4380 cctcccaatg 4440 caaqtgtctt 4500 atcgatctta 4560 tagcatcaca 4620 caaactcatc 4680 cactgactcg 4740 qgtaatacgg 4800 ccagcaaaag 4860 cccccctgaC 4920 actataaaga 4980 cctqccgctt 5040 tagctcacgc 5100 gcacgaaccc 5160 caacccggta 5220 agcgaggtat 5280 tagaaggaca 5340 tggtagctct 5400 gcagcagatt 5460 gtctgacgct 5520 *aaggatcttc 5580 atatgagtaa 5640 gatctgtcta 5700 *acgggagggc 5760 ggctccagat 5820 tgcaacttta 5880 ttcqccagtt 5940 1ctcqtcgttt 6000 1atcccccatg 6060 Itaagttggcc 6120 -catgccatcc 6180 iatagtgtatg 6240 :acatagcaga 6300 -aaggatctta 6360 ttcagcatct 6420 ttgtgcaaaa aagcggtta gcagtgttat cactcatgg gtaagatgct tttctgtga cqgcgaccga gttgctctt actttaaaag tgctcatca ccgctgttga gatccagtt WO 00/42208 WO 0042208PCT/EPOO/00265 tttactttca qgaataaggg agcatttatc aaacaaatag attattatca gcgtttcggt ttgtctgtaa cgggtgtcgg tatgcggtgt agcgttaata caataggccg agtgttgttc gggcgaaaaa tttttggggt agagcttgacgcgggcgct a gcgcttaatg gaagggcgat gcaaggcgat gccagtgaat ccagcgtttc cgacacggaa agggttattg gggttccgcg tgacattaac gatgacggtg gcggatgccg ggctggctta gaaataccgc ttttgttaaa aaatcggcaa cagtttg gaa c cg t ctat ca cgaggtgccg qgggaaagcc gggcgctqgccgccgctaca cggtgcgggc taagttgggt tgtaatacga tgggtgagca atgttgaata tctcatgagc cacatttccc ctataaaaat aaaacctctg ggagcagaca actatgcggc acagatgcgt attcgcgtta aatcccttat caagagtcca gggcgatggc taaagcacta gqcgaacgtq aagtgtagcg gggcgcgtcc ctcttcgcta aa cqccaggg ctcactatag aaaacaggaa ctcatactct ggatacatat cgaaaagtgc aggcgtatca acacatgcag agcccgt cag atcagagcag aaggagaaaa aatttttgtt aaatcaaaag ctattaaaga ccactacgtg aatcggaacc gcgagaaagg gtcacgctgc cattcgccat ttacgccagc ttttcccagt ggcgaattaa ggcaaaatgc tcctttttca ttgaatgtat cacctqacgt cgaggcccct ctcccggaga ggcgcgtcag attgtactga taccgcatca aaatcagctc aatagaccga acgtggactc aaccatcacc ctaaagggag aagggaagaa gcgtaacca tcagqctgcg tggcgaaagg cacgacgttg ttcqgggq cqcaaaaaag atattattga ttagaaaaat ctaagaaacc ttcgtctcgc cggtcacagc cgggtgttgg gagtgcacca ggaaattgta attttttaac gatagggttg caacgtcaaa ctaatcaagt cccccgattt agcgaaagga cacacccgcc caactgttgg gggatgtgct taaaacgacg 6480 6540 6600 6660 6720 6780 6840 6900 6960 7020 7080 7140 7200 7260 7320 7380 7440 7500 7560 7607 <210> 49 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: plasmid <400> 49 gaattccgca tgaccattca gagtgtccta t ctgcgcaca ggcatagctc ctcacccttg aactcgacct ttttgtcctt accaatccaa actattttta acctcaattg aaaaagagtq ccttacatct tgggataggt gaaagactcg atgaaacaac attttccata catggcatga agtaagtttt cttggtttgg ttggaattta attttttgag gccatcccgt cggcgaccct gtgacccttg ggctatcatc cgtccaagac gcgccctgcc ggtatcttgc acggggtaga ggtacgcgcg ttgcagagat ccacattggt cacctagggg aacggatgag tqctttgcct.
actcttttaa tcctcctgaq cagaaataga taggtagatt ctcaaattca aagaacaggt tttttgtcaa acagaccaac gggttacagt ccagagctag aggtacatga ccaaggaggg gttattatga tggttacaaa tatcaaaggt tccaaatctt taaacttgca ctccgctcgt caggtcggcc tctctatttc acaagagcgg cctcaaagat gcaaggccag agttttggaa agttgaggcg gtgagaatag attgtattta gtgcacctcc agaagcagcc cccatcagac ggqgctattg tagctcttct gcaaggacca aataagaatg attagttact gaagttagaa gcaaggacta aataggagac agatgccccc caatggctat acctccttgg ttatatttat gacagtggct atagccttta ctgttcttaa tctgatctga atgtaaatgc acagtcctaa cacttatcct gactgcggca tactatttgg aacggactca ttttggcact ctgcttgtcc aaagatgtga cgggttgggc gtggcgttcg agtgcctagc aagcttggqc aaggggttgt aaagacatat ggggaagttg gtqcaagatt cagccaactt cttgctaaaa atgttaagaa atgggaatag ttgaccacag aggtggtggc t tac a tat a aaagtgttat tgtatgttqt ctaggaacag ggactaatag ttggcccaac aacgaggatg gctctgagtg ttatgtaaac cattcacctc tcactttcca gctggcgccc tgtttgtctt ccatagggac tcgttgagcg gctcggctgc taggtggcaa tcgcatgtgc taggcaaggc tcgatacaat agaaatggtt ttcccaccaa tcattctctg cggttcgtgc acaatctaaa cctcttacaa attatatttt atgaatcatt aaaatagaaa gcctagaagt aaccagggac c agga aga ta atagatcct ctcaagaaga gaatgcactt aacattattc cttgcggttc tgagacaagt ttctattttc caagatataa ttgtgtgttt gagggtcccc gaacagggac gtattgtctc caagctagcg aggcgatatc ggttggcacg gcacctctgg Cgttttcttg tgacatccgc aaacgccatt gaactcccqa ggacgacccg ctgcaaactt t cqcagggct caattcggag gccgcatcga taccaataaq atcttttagt gagacgctca aaaaaaggga ttatagggga tgacttaaat cccttttcgt aaaaqacgac ttggggaaag tqcaaaaact ccagggctta ggtttcctga ctatgttctt aagagtgctg qtgtctgttc ccgcagaccc cctcggataa t t tct tgt ct cttctcgtcg aggtatgaca gcaggatagg cacggcaaat gcgtttgggg tatggcgagg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 WO 00/42208 WO 0042208PCTJEPOO/00265 ggcacatcgc1 ttcaacagca acttqttcct gagcggtcct tcctcaagcg gcggtggtgg atctttttct acccccgagc ccgtccccgt gagtccgagg cggccatcqa tctcccaagc qaagatgtqg aaaggagqta cggacgcaag ccgctgaqtg atggacaagt gtggaagctc ctgaccttca ctgcagtcgt ctgcaccgct aataaggagc gagcagtct a tccaacaccg ggcaagtctt atcaaggctt atgccactac ctaccaaagt tccgacaaqa aaccctqtgt tcgqcgcccg accgagctgc aacgtgtccc gcgcagacgg cctttattqa gtggcgctcc ctgtagagct gagttgggta cagttggcaa ccctgtacgc gcgctacggc gaaaagccaa atccagtgta tgtggagaaa ccaagatctt gttgatgttg acacggtttc acaaaacata ttacctcagg aattggacaa tataatgtgt ctgatgaatg aaatgccatc agaagagaaa atgctgtgtt ctgcactgct acagttataa ttaataacta aggaatattt gaggttttac aatgcaattg agcatcacaa tgcgctcttg cgtcgtct~c cgtttgcctc cgtcgtcttc ggggtgcctc cgaactcaga gcctatagga gcggacgcgg cgccgccgcc acgaggaaga cct cggcggc ccgagcgccc cgctacaaat agcgcacagt aqgaagagga tgccga tcgt accacgtgga tggcggccgt ccagcaacaa ttqcagaggt gcgctgagat acgtgattga gcaaggccaa acgcaaqgtg gcggcatgtt ttatqcaggc ggtgcgagtg tgactccgtt gcgtgctggc atcgcaactc acctgctaaa cgcggatggt tgccagtggc caagggtg aagtgtctct taatctqcgc gttcctqgtt ccccggtaat aggcgtggag tttgggtgga gcaggagtga aqggctcaaa cgcccccagt caaagcctgg tgacaatggc cttgcttctt gatgacgccg aagaaqggtq tcgacctcga actacctaca taaactactg ggagcagtgg tagtgatgat ggtagaagac tagtaataga atacaagaaa tcataacata tgctcaaaaa gatgtataqt ttgctttaaa ttgttgttaa atttcacaaa caacgcgtcg cacatctagg tgcgttgtcc gcttacaaaa gacggggaag gggggcggtt gaaggaaatg tgcggcgcga tccccgggcg ctcatcacaa ggatttggcc gccatcacca ggtgggtttc gcggcggctg agagcccagc gtctgcgtgg taacgatcta atgcaaqacc gacctttgtg gacctacaag cgaaggcgag aatggatgtg gatcgtgaag ct gcqtgcac cttctctgaa gctgtatcct caactcaaag cgccctgagc cagcgtgcac gcgcgcgcag cgcqttggtg tgtgcctgag gcatagcgat gqtaaataat agtacattat actqtggctg gcgacgcagg aaggttcatg aaacatgcaq cttttccagc ctcgtactca gaggtagcat ctcqcgaccg aaagcqcttq tttcagttcc tatqttgtgg cggtgCggCC ggctcgtcca gggatctttg gaqatttaaa attctaattg tggaatgcct gaggctactg cccaaggact actcttgctt attatggaaa ctgttttttc ttqtgtacct gccttqacta aaacctccca cttgtttatt taaagcattt cagataatgg tagtcgccat tggtcttgct cctgggtcct gtggtaggcg aggctgtcct gccagtcggg cgtcccccaa cccccaa aaa gacgcgctgg attgcgccca gaggtaatcg agcaacccac aatgaagacg gaagcggaaa aaggcgaact tggctgaacg acgatgatqg catcacgagc cttaagtgtc acgagcgaaa aaccggtggg ga cgcggcct ggcgcaaagg aacgcccaga cctgggcacg aacqcggagg cacccggcqc ggcggaggcc atggtgcgca tttaagtgga gcgcggcaga cacccgagag ttttacatgt cgqaagtagg gtgggctqta gtgqggttqt cagaatagtc gttatacagc aactggtaaa gtttttgagt qccgtattga tcataggtgc tgctcactgg cgttgccggc qgtgcacacq tgggat ccat tgaaggaacc gctctaaggt tttgtgtatt ttaatgagga ctgactctca ttccttcaga gctttgctat aatattctgt ttactccaca ttagcttttt gagatcataa cacctccccC gcagcttata t tt tca ctqc cgcactggcg gcctttcgtc ttttatcctc gctcgataat cgttggCggC tcttctcgac aagaggagca ccatggagga agcggatgag tgccgcgcac agaagaaaaa tggacagcga cggtgctaat acccagtggc gtgaaattac tg- nintflf tcaaactact aggagcaccg ggcgattcct ccacgggctg tacacggaag acgggcagcg gccgaaatgt gtccggccaa ctcaggtggc ccgggcacgg cgcccttttt acctggacgc tgatagtgtt ccaactgcga g cctgtggag gcactaaaca acccctttcja tgtacaaata ttttcaagtg gcgagtggcg cctgqggact gatccatggq cacaggcggc gtcggggga cctgcttgag gcgggttcca ctatggcqca ccaaaaaata agcccatggc cgagaagggc gaccacgtCa atatagggcc ttacttctgt aaatataaaa ttagattcca aaacctgttt acattctact attgcztaagt ttacaccaca aacctttata caggcataga aatttgtaaa tcagccatac tgaacctgaa atggttacaa attctagttq ctgcagatgc 1920 cccccgcccg 1980 tgttggtact 2040 cacttcctcc 2100 atcggtggag 2160 tgactccatg 2220 gcgcgaaacc 2280 cgtgtcgtcc 2340 gcggcgtatc 2400 acccagcccg 2460 gaagcgccct 2520 ggaagaaaga 2580 caagcatggc 2640 gcgtggtatg 2700 ggtgatgaac 2760 gcncgcgctg 2820 gcctgaccaa 2880 cgggttgcag 2940 gcaggcgtac 3000.
cgcgttgtgg 3060 cattatgata 3120 cgcgctgaag 3180 ggtgcagatc 3240 tcagttttcc 3300 ttttaagcag 3360 tcaccttttg 3420 gggaaggcag 3480 ggatctgatc 3540 ccagtqctgc 3600 cttcaagata 3660 tqaaaacttc 3720 ccagtatcgc 3780 tttttaaacg 3840 aaagcatt tg 3900 acaaaaagaa 3960 ctccaggaag 4020 qttqagcatg 4080 agtttggggc 4140 cgagttgggc 4200 aqaagcaatg 4260 tcgctggtca 4320 qgcaaaggcc 4380 ggcgagcttg 4440 tggcccacaa 4500 ggcagctqtt 4560 gtgcgcaggt 4620 aagacttcaa 4680 cgggttataa 4740 ggtgtgacat 4800 tttttaagtg 4860 acctatggaa 4920 tgctcagaag 4980 cctccaaaaa 5040 tttttgagtc 5100 aaggaaaaag 5160 agtaggcata 5220 gtgtctgcta 5280 ggggttaata 5340 cacatttgta 5400 acataaaatg 5460 ataaagcaat 5520 tggtttgtcc 5580 WO 00/42208 WO 0042208PCTIEPOO/00265 -46aaactcatca tgtggaaagt tcagcaacca catctcaatt ccgcccagtt gccgaggccg ctaggctttt ggaagcggaa gctactgggc gtgggcttac tgccagctgg tcttgccgcc gatcgtttcg agaggctatt tccggctgtc tgaatgaact gcgcagctgt tgccggggca ctqatgcaat cgaaacatcg atctggacga gcatgcccga tggtggaaaa gctatcagga ctgaccqctt atcgccttct gacgcccaac cttcggaatc ggagttcttc gctggacgac cagcggctat tttggtcccg tacctacaga aactactgat agcagtggtg gtgatgatga tagaagaccc gtaatagaac acaagaaaat ataacatact ctcaaaaatt tqtatagtgc gctttaaaaa gttgttaact ttcacaaata gtatcttatc ctctacttga aattaggtca ggtaatttta cagcccacaa accctgctca attttcccca aacccagcac catctgctga cctgtagttt aaaatttgac atgcaagttt catcaaaata cgaaagggcc tagacgtcag taaatacatt tattgaaaaa atgtatctta ccccaggctc ggtgtggaaa agtcagcaac ccgcccattc cctcggcctc gcaaaaagct cacgtagaaa tatctggaca atggcgatag ggcgccctct aaggatctga catgattgaa cggctatgac agcgcagggg gcaggacgag gctcgacgtt ggatctcctg .gcggcqgctg catcgagcga agagcatcag cggcgaggat tggccgcttt catagcgttg cctcgtgctt tgacgagttc ctgccatcac gttttccggg gcccaccccg ctcgcggagt ccqcgcatcc gatctttgtg gatttaaagc tctaattgtt gaatgccttt ggctactgct caaggacttt tcttgcttgc tatggaaaaa gttttttctt gtgtaccttt cttgactaga acctcccaca tgtttattgc aagcattttt atgtctggat gaggacattc cttaacaaaa aaatatctgg atgtcaacag tcaagaagca cctgtgtagg tccactggat ctgtcaactg gctaacacac ccttgaatgg aacatagcag tttccacagg tcgtgatacg gtggcacttt caaatatgta ggaagagtat tcatgtctgg cccagcaggc gtccccaggc catagtcccg tccgccccat tgagctattc tcacgctgcc gccagtccgc agggaaaacg ctagactggg ggtaaggttg tggcgcaggg caagatggat tgggcacaac cgcccggttc gcagcgcqqc gtcactgaaq tcatctcacc catacgcttg gcacgtactc gggctcgcqc ctcgtcgtga tctggattca gctacccgtq tacggtatcg ttctgagcgg gagatttcqa acgccggctg ggctcgatcc tctaccggca atgcccccga aaggaacctt tctaagqtaa tgtgtatttt aatgaggaaa gactctcaac ccttcagaat tttgctattt tattctqtaa actccacaca agctttttaa gatcataatc cctccccctg agcttataat ttcactgdat ccccaggaag caatcatagg aggaaattgg gaagtccctt cagaaacata ctgtggttgc ttccaaaata aagcattatc tagcattttt cctgcagctc gttttccagc ttaccccaat ttaagtcctc cctattttta tcggggaaat tccgctcatg gagtattcaa atccggctgt agaagtatgc tccccagcaq cccctaactc ggctgactaa cagaagtagt gcaagcactc agaaac'ggtg caagcgcaaa cggttttatg ggaagccctg gatcaagatc tgcacgcagg agacaatcgg tttttgtcaa tatcgtggct cggqaaggga ttgctcctgc atccggctac ggatggaagc cagccgaact cccatggcqa tcgactgtgg atattgctga ccgctcccqa gactctgggg ttccaccgcc gatgatcctc cctcgcgaqt gtgcaaatcc actgcaggag acttctgtgg atataaaatt agattccaac acctgttttg attctactcc tgctaagttt acaccacaaa cctttataaq ggcatagagt tttgtaaagg agccatacca aacctgaaac ggttacaaat tctagttgtg ctcctctgtg ctgcccatcc gtagggqttt ccactgctgt caagctgtca tgtgttagta tct agtgtt t cttatccaaa tggggttaca.
caaaggttcc accattttca aacctcagtt atttaaatta taggttaatg gtgcgcggaa agacaataac catttccgtg ggaatgtgtg aaagcatgca gcagaaqtat cgcccatccc ttttttttat gaggaggctt agggcgcaag ctgaccccgg gagaaagcag gacagcaagc caaaqtaaac tgatcaagag ttctccggcc ctgctctgat gaccgacctg ggccacgacg ctggctgcta cgagaaagta ctgcccattc cggtcttgtc gttcgccagg tgcctgcttg ccggctgggt agagct tgqc ttcgcagcgc ttcgaaatga gccttctatg cagcgcgggg t ggt t cagct qtcggcatcc tggggaggca tgtgacataa tttaagtqta ctatggaact ctcagaagaa tccaaaaaag tttgagtcat qgaaaaagct taggcataac gtctgctatt ggttaataag catttgtaga ataaaatgaa aaagcaatag gtttgtccaa tcctcataaa accctctgtg ttcacagacc gttccagaag gctttgcaca atgtgcaaaa tcatttttac acagccttgt gtttgagcag ccaccaacag tgagtttttt ttaacagtaa ggcaaaggaa tcatgataat cccctatttg cctgataaat tcgcccttat tcagttaggg tctcaattag qcaaaqcatg qcccctaact ttatgcagaq ttttggaggc ggctqctaaa atgaatgtca gtagcttgca gaaccggaat tggatggctt acaggatgag gcttgggtgg gccgccqtgt tccggtgccc ggcqttcctt ttggqcgaag tccatcatgg gaccaccaag ga tcaggat g ctcaaggcgc ccgaatatca qtggcqgacc ggcgaatggg atcgccttct ccgaccaaqc aaaggttggg atctcatgct gctgcctgag aggaaaccag cgatggccgc ttgqacaaac taatgtgtta gatgaatgqq atgccatcta aagagaaagg gctgtgttta gcactqctat aqttataatc aataactatg gaatatttga ggt t ttact t tgcaattgtt catcacaaat act cat caat ccctaacctc tcctcctgtt gctttctaag tgttggtaaa agqqcccaac caggaggcac ttggatcagg gqtcagtgtt gatatttggt caaaaaaatg gtgtccctga caqcttccca ttcttgaaga aatggtttct tttatttttc gcttcaataa tccctttttt 5640 5700 5760 5820 5880 5940 6000 6060 6120 6180 6240 6300 6360 6420 6480 6540 6600 6660 6720 6780 6840 6900 6960 7020 7080 7140 7200 7260 7320 7380 7440 7500 7560 7620 7680 7740 7800 7860 7920 7980 8040 8100 8160 8220 8280 8340 8400 8460 8520 8580 8640 8700 8760 8820 8880 8940 9000 9060 9120 9180 9240 9300 WO 00/42208 WO 0042208PCT/EPOO/00265 -47gcggcatttt gaagatcagt cttgagagtt tgtggcgcgg tattctcaga atgacagtaa ttacttctga gatcatgtaa gagcgtgaca gaactactta gcaggaccac gccggtqagc cgtatcgtag atcgctqaga tatatacttt gaccccgtag tgcttqcaaa ccaactcttt ctagtqtagc gctctgctaa ttggactcaa tqcacacagc ctatgagaaa agggtcgqaa agtcctqtcg gggcggaqcc tggccttttg accgcctttg gtgagcqagg atttcacacc cagtatctgc qctacaacaa tttgcgctgc tttaggcgaa tttcgctttt acatggtaac gattggtgga catggattgg gccttcctgt tgggtgcacg ttcgccccga tattatcccg atgacttqgt gagaattatq caacgatcgg ctcgccttga ccacgatgcc ctctagcttc ttctgcgctc gtgggtctcg ttatctacac taggtgcctc aqattgattt aaaagatcaa caaaaaaac ttccgaagqt cgtagttaqg tcctgttacc gacgatagtt ccagcttgga gcgccacgct caggagaqg gqtttcgcca tatggaaaaa ctcacatgtt agtgagctqa aagcgqaaqa gcatatggtg tccctgcttg ggcaaggctt ttcgcgatgt aagcggggct gcatagggag gatgagttag agtaaggtgq acgaaccact ttttgctcac agtgggttac agaacgtttt tgttgacgcc tgagtactca cagtgctgcc agqaccgaag tcgttgggaa tgcagcaatg ccggcaacaa ggcccttccg cggtatcatt gacggggagt actgattaag aaaacttcat C-aaaatccCt aggatcttct accgctacca aactggcttc ccaccacttc agtggctgct accqgataag gcqaacgacc tcccgaaggg cacgagggag cctctgactt cgccaqcaac ctttcctgCg taccgctcgc qcgcctgatq cactctcaqt tgtgttgqag gaccgacaat acgggccaqa tcgqttgtac ggggaaatgt caacatqcct tacgatcgtg ccagaaacgc atcgaactgg ccaatgatga ggqcaagagc ccagtcacag ataaccatga gagctaaccg ccggagctga gcaacaacgt ttaatagact gctggctggt gcagcactgg caggcaacta cattggtaac ttttaattta taacqtqaqt tqagatcctt qcggtggttt agcagagcgc aagaactctg gccagtgqcg gcgcagcggt tacaccgaac agaaaggcgg ct tccaggg gagcgtcgat gcggcctttt ttatcccctg cgcagccgaa cggtattttc acaatctgct gtcgctgagt tgcatgaaga tatacgcgta gcggttagga agtcttatgc tacaaqqaqa ccttattagg tggtgaaagt atctcaacag gcacttttaa aactcggtcg aaaagcatct gtgataacac cttttttgca atgaagccat tgcgcaaact ggatqgaggc ttattgctga ggccagatgq tggatgaacg tgtcagacca a a agga tct a tttcgttcca tttttctgcg gtttgccgga agataccaaa taqcaccgcc ataagtcgtg cgggctgaac tgagatacct a ca ggt at cc qaaacgcctg ttttgtgatg tacggttcct attctgtgga cgaccgagcg tccttacgca ctgatgccgc agtgcgcqag atctgcttag tctgagggga gtcccctcag aatacacttg gaaaaagcac aaggcaacag aaaagatgct cggtaagatc agttctgcta ccgcatacac tacggatggc tgcggccaac caacatgggg accaaacgac attaactggc ggataaagtt taaatctgga taagccctcc aaatagacag agtttactca ggtgaagatc ctgagcgtca cgtaatctgc tcaagagcta tactgtcctt tacatacctc tcttaccggg ggggggttcg acagcgtgag ggtaagcggc gtatctttat ctcgtcaggg ggccttttgc taaccgtatt cagcgagtca tctqtgcggt atagttaagc caaaatttaa ggttaggcgt ctagggtgtg gatatagtaq tagtcttgca cgtgcatgcc acqqgtctga 9360 9420 9480 9540 9600 9660 9720 9780 9840 9900 9960 10020 10080 10140 10200 10260 10320 10380 10440 10500 10560 10620 10680 10740 10800 10860 10920 10980 11040 11100 11160 11220 11280 11340 11400 11460 11520 11580 11600 <220> <223> Description of Artificial Sequence: plasmid <400> 49 <210> <211> 8238 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: plasmid <400> gcggccgcca tcatcaataa tataccttat tttggattga agccaatatg ataatgaggg ggtggagttt gtgacgtggc gcggggcgtg ggaacqgggc gggtgacqta gtagtqtggc 120 ggaagtgtga tgttgcaagt gtggcggaac acatgtaaqc gacggatgtg gcaaaagtga 180 Cgtttttggt gtgcgccggt qtacacagga agtgacaatt ttcgcgcgqt tttaggcgga 240 WO 00/42208 PTEO/06 PCT/EPOO/00265 tgttqtagta taagaggaag gccgcgggga ccagatatac tgt acqcggt aatgtagtct tgccttacaa tcgtgcctta ccgcattgca attcaccaca gcgggggcca cttgqatcgg tccgcatcga tagaactagt gcacctcttt gcgccctcgc actgcgqgtt ggtggccctg ggtgctgggg gggtgagata catqccttat ttcacatgcc cctgtgctac gttcgtggcc ggaggacaga ggctatgctg qcagqgcggc ccagggtgcc taccctgttt ctgggccttg cgaccaatcg gacccacgtc ccgggagatg ataccgtcga aaatttcaca caatgtatct gacccgcacc gatgctggat tgagtttggc aagggtggga agccgccgcc aacgcgcatg tcgccccgtc gttggagact gactgacttt cqatgacaag cgtttctcag tcccaatgcg agtgtcttgc tcqgtcgttg cagatacatg ctgcggggtg aatgtctttc gcggttaagc taggttggct cacagtgtat gaacttggag aatgggccca gtgttccagg ctgcggtata ccacgctttg aatttgggcg tgaaatctga ctttgaccgt gcgtatctga taggagtccc tatgcaatac ggagagaaaa ttaggaaggc gagatattgt ttggtqtgca gctgttgggc aaacccgtcg ccggatcgga ggatcccccq ggcaagcgcc ttcgaccaggcggcagcaag tatatagacg ggttcgcgcg gcttccgaga tcggccgggg qccgtgaccg ccqcccccgg ccggccgcgc ctcatcccgc cacatcgacc qccgcgattc gggtcgtggt gagccccaga cgggcccccg gacgtcttgg cccgccgqct accaccccag ggggaggcta aacttgttta aataaagcat tatcatgtct aggtgcagac gtgaccgagg tctagcgatg aagaatatat gccatgagca cccccatggg ctgcccgcaa gcagcctccg gctttcctga ttgacggctc cagctgttgg qtttaaaaca tqtctttatt agggtcctgt ggca ta aqcc gtgttgtaga agtagcaagc tgggatgggt atgttcccag ccggtgcact acgcccttgt cgggcggcgg atgagatcgt atggttccat agttcagatg taaccgagta ataattttgt ttacgtggag ggggactagg ctcaggatat tcttgtagtc agcaccgtgc aacagacggg atttaagtgc cctccggccc tcgcggttga gcctccgaac aaacctctcg ggctgcagga ttgtagaagc aagccacgga gtcctcacgg acgatatcgt caatcgcgaa acqcggcggt acqccgttct ccctcaccct gataccttat cgaccttgcc gcctggccaa gccgcqttta gggaggattg gcaacgcggg agttqctggc ccaaacgcct gccgggacgc gctccatacc actgactcga ttgcagctta ttttttcact ggatccqacc cctgcgaqtg agctqagqcc aaqatacaga aaggtggggg ccaactcgtt ccggggtgcg actctactac ccgccgcttc gcccgcttgc ttttggcaca atctgcgcca taaataaaaa taggggtttt gtattttttc cgtctctgg tgatccaqtc tgattgccag gcatacgtgg ccatat ccct tgggaaattt qacctccaaq cctgggcgaa cataggccat ccggcccagg gggggatcat agatttggcc gttactcata actcgcccag gtgtgtttag agtagtttcg ttgcaacatg atgccgattg tctgacatgg ctagctcgat atatggccac ggacaaactc gqtactccgc agaaaggcgt attcgatgat gcgtatggct t ~cnnccat agtccgcctg gatgggqaaa ctacgtaccc catctacacc ggtaatgaca ggctcctcat catcttcgac gggcagcatg cggcacaaac acgccagcgc cggqctqctt gggacagctt cccacgaccc ccccaacggc ccgtcccatg cctgctgcaa gacgatctgc gaagcttggg taatqgttac qcattctagt tcggatctgg tggcgqtaaa cqatcacttg ttgaggtact tcttatgtag tgatggaagc tcagaatgtg cttgacctac agccgctgca aagcagtgca attggattct gcaggtttct accagactct gcgcgcgcgg caggacgtgg gtggaggtag gtagcaggag gggcaggccc ggatatgaga ccggggattc gtcatgtagc attttccatg qatatttctg ttttacaaag ggcgtagtta gtctacctgc attttcgcgg gcgcgtaata ggcgcgcccc gcgaaaagcg cttttqcata gtaacgatga gtggaagtaa attggacgaa acaataaacg tctcttccgc ttcgcggtct cgccgaggga gtaaccagtc cttggtggcg tcgtacccct agcaacCgac gagcaqaaaa accaccacca gagccgatga acacaacacc agcgcccaga gtcggqggggg cqccatccca accccccagg atcgtgttgg cccggcgagc gccaatacgg tcqgggacgg catatcqgg gacctgtata cacgtcttta cttacctccg gacctggcgc cccatcgatc aaataaagca tgtggtttgt aaggtgctga catattagga qtgctqgcct gaaatgtgtg ttttgtatct attgtgagct atgggctcca gagaccgtgt gccaccgcc gcttcccgtt ttgacccggg gccctgaagg qtttggattt taggcccggg taaaggtgac caccactgca cgctgggcgt ttggtgtaag tgcatcttgg atgttgtgca ttagaaggaa cattcqtcca ggatcactaa cgcgggcgga ccctcacaga ggggcgatga gaaaactgaa tttgtctagg gatgtacggg gggcttcggt gggaggggga gttagcaaca ggtggtacga ccactgaatt ccatttgacc atcgctgtct ttccagtact cctgagcgag acagtcgctc tgaaactccc gccatcaaca gta cggcgtt tgcccacgct cgcaactgct cttactggca gcctcgacca taacaatggg aggctgggag tcgccgccct ccgtgctggc gggcccttcc ggcttgacct tgcggtatct ccgtgccgcc acacgttatt acgtgtttgc tcctggatta ggatggtcca gcacgtttgc aagcttatcg atagcatcac ccaaactcat ggtacgatga accagcctgt gcacccgcgc ggcgtggctt gttttgcagc catatttgac gcattgatgg ctggaacgcc gcgggattgt catccgcccg aacttaatgt cttcctcccc ggatcaagca accagcggtc tctggatgtt gagcttcatg ggtgcctaaa tgtttacaaa actgtatttt gaaccaccag atgcgtggaa taatgatggc cgtcatagtt gggtgccaga tttgcatttc agaaaacggt 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 WO 00/42208 WO 0042208PCT/EPOO/00265 -49ttccggggta gcagccggtg gcagctgccg gttttccctg ggaagcaaag accaagcagt catatctcct tccagacggg gt cacggtga ctgctggtgc atggtgtcat gaggcgccgc accgattccg agccaggtga cgtttcttac g+ rr r-nr.r,+ tatagaaact aagtgggagg cacatgtcgc ccgggtgttc tccgcatcgc atgacttctg cccgcggtqa ttgtcaagct tgcgcgcttg aattccacac gagctaactc gtqccagctg ctcttccgct atcagctcac gaacatgtqa gtttttccat gtggcgaaac gcgctctcct aagcqtggcg ctccaagctq taactatcgt tggtaacagg gcctaactac taccttcgga tggttttttt tttgatcttt ggtcatgaga taaatcaatc tgaggcacct cgtgtagata qcgagaccca cgagcgcaga ggaagctaga aggcatcgtg atcaaggcqa tccgatcgtt gcataattct aaccaagtca acgggataat ttcggggcga tcgtgcaccc aacaggaagg catactcttc atacatattt aaaagtgcca gcgcagcgtg ggggagatca ggcccgtaaa tcatccctga accaaatccg tttttcaacg tccaggcggt cgtttcgcgg ccagggtcat aggggtgcgc tgaagcgctg agtccagccc acgaggggca gggagtaggc gctctggccg ctctggtttc atcgat cggaccactc ggtagcggtc cctcttcggc ctgaaggggg tgtctgcgag cgctaagatt tgcctttgag tcgagggggg gcgtaatcat aacatacgag acattaattg cattaatgaa tcctcgctca tcaaaqgcgg gcaaaaggcc aqgctccgcc ccgacaggac gttccgaccc ctttctcata ggctgtgtgc cttgagtcca attagcagag ggctacacta aaaagaqttg gtttgcaaqc tctacggggt ttatcaaaaa taaagtatat atctcagcga actacgatac cgctcaccgg agtggtcctg gtaagtagtt gtqtcacgct gttacatgat gtcagaagta cttactgtca ttctgagaat accgcgccac aaactctcaa aactgatctt caaaatgccg ctttttcaat gaatgtattt cctgacgcgc accgctacac gctgggaaga t cacaccta t gcaggggggc ccagaaggcg gtttgagacc cccacagctc gttggggcgg gtctttccac tccgggctgc ccggtcttcg ctccgcggcg gtgcagactt atccgcgccg ttcggggtca catgagccgg nagagrT1,icla tgagacaaag gttgt ccact atcaaggaag gctataaaag ggccagctgt qtcagtttcc ggtggccgca gcccggtacc ggtcatagct ccggaagcat cgttgcgctC tcggccaacg ctgactcgct taatacggtt agcaaaaggc cccctgacga tataaagata tgccgcttac gctcacgctg acgaaccccc acccggtaag cgaggtatgt gaaggacagt gtagctcttg agcagattac ctgacgctca ggatcttcac atgagtaaac tctgtctatt gggagggct t ctccagattt caactttatc cgccagttaa cgtcgtttgg cccccatgtt agttggccgc tgccatccgt agtgtatgcg atagcagaac ggatcttacc cagcatcttt caaaaaaggq attattgaag agaaaaataa cctgtagcgg t tgccagcgc aagcaggttc taccggctgc cacttcgtta ctcgccgccc gtccgccgta ggtcacctgc ctttcgctgt gggcgcaggg gcgctggcca ccctgcgcgt tggcccttgg ttgagggcgt caggccccgc aaaaccaggt tgtccacgct tnlctcgagcg gctcgcgtcc agggggtcca gtgattggtt ggggtgggqg tggggtgagt aaaaacgagg tccatctggt cagcttttgt gtttcctgtq aaagtgtaaa actgcccgct cgcggggaga gcgctcggtc atccacagaa caggaaccgt gcatcacaaa ccaggcgttt cggatacctg taggtatctc cgttcagCc acacgactta aggcggtgct atttggtatc atccggcaaa gcgcagaaaa gtggaacgaa ctagatcctt ttggtctgac tcgttcatcc accatctggc atcagcaata cgcctccatc tagtttgcgc tatggcttca gtgcaaaaaa agtgttatca aagatgcttt gcgaccgagt tttaaaagtg gctgttgaga tactttcacc aataagggcg catttatcag acaaataggg cgcattaagc cctagcgccc ctgagcagct aactggtagt agcatgtccc agcgatagca ggcatgcttt tctacggcat acggcagtag tcctcgtcag gggtgcgctt cggccaggta cgcgcagctt agagcttggg agacggtctc ttcccccatg cggtgacgaa gtgttccgcg aggccagcac ctcgctccag tgtaggtgta cgcgttcgtc actccctctq agqatttgat cagaaaagac tccctttagt tgaaattgtt gcctggggtg ttccagtcgg ggcggtttgc gttcggctgc tcaggggata aaaaaggccg aatcgacgct ccccctggaa tccgcctttc agttcggtgt gaccgctgcg tcgccactqg acagagttct tgcgctctgc caaaccaccg aaaggatctc aactcacgtt ttaaattaaa agttaccaat atagttgcct cccagtgctg aaccagccag cagtctatta aacgttgttg ttcaqctccg gcggt tagct ctcatggtta tctgtgactg tgctcttgcc ctcatcattg tccagttcga agcgtttctg acacggaaat ggttattgtc gttccgcgca gcggcgggt g gctcct ttcg gcgacttacc taagagagct tgactcgcat gttcttgcaa tgagcgtttg ctcgatccag tcggtgctcg cgtagtctgg gaggctggtc gcatttgacc gcccttgqag cgcgagaaat gcattccacg ctttttgatg aaggctgtc gtcctcctCg gaaggaggct ggtgtgaaga ggccacgtga ctcactctct aaaagcgggc ittcacctqg aatctttttg gaggttaat atccgctcac cctaatgagt gaaacctgtc gtattgggcg ggcgaqcggt acgcaqgaaa cgttqctggc caagtcagag gctccctcgt tcccttcggg aggtcgttcg ccttatccgg cagcagccac tgaagtqgtg tgaagccagt ctqgtagcgg aagaagatc6 aagggatttt aatgaagttt gcttaatcag gactccccgt caatgatacc ccggaagggc attgttgccg ccattgctac gttcccaacg ccttcggtcc tggcagcact gtgagtactc cggcgtcaat gaaaacgttc tgtaacccac ggtgagcaaa gttgaatact tcatgagcgg catttccccg tggtggttac ctttcttccc 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 5580 5640 5700 5760 5820 5880 5940 6000 6060 6120 6180 6240 6300 6360 6420 6480 6540 6600 6660 6720 6780 6840 6900 6960 7020 7080 7140 7200 7260 7320 7380- 7440 7500 7560 7620 7680 WO 00/42208 WO 0042208PCTIEPOO/00265 ttcctttctc gccacgttcq agggttccga ttcacgtagt gttctttaat ttcttttgat taacaaaaat tcaggctgcg tggcqaaagg cacgacgttg tggagctcca tttagtgctt gggccatcgc agtggactct ttataaggga ttaacgcgaa caactgttgg qggatgtgct taaaacgacg ccgcggtg ccggctttcc tacggcacct cctgatagac tgttccaaac ttttgcgatt ttttaacaaa gaagggcgat gcaaggcgat gccagtgagc ccgtcaaqct cgaccccaaa ggtttttcgc tggaacaaca tcgqcctatt atattaacgc cggtgcgggc taagttggqt gcgcgtaata ctaaatcggg aaacttgatt cctttgacgt ctcaacccta ggttaaaaaa ttacaatttc ctcttcgcta aacgccaggg cgactcacta ggctcccttt agggtgatgg tggagtccac tctcggtcta tgagctgatt cattcgccat ttacgccagc ttttcccagt tagggcgaat 7740 7800 7860 7920 7980 8040 8100 8160 8220 8238 <210> 51 <211> 11 <212> DNA <213> adenovirus <400> 51 cgcggatccc g <210> 52 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 52 ctgacaaact cagatcttqt ttattg <210> 53 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 53 gtcgactcta gaggatccag a <210> 54 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 54 ccggactcta gatggcaacc atggcgctac <210> <211> 31 <212> DNA <213> Artificial Sequence- WO 00/42208 PCT/EPOO/00265 -51- <220> <223> Description of Artificial Sequence: primer <400> ggaggggaag cttggccctc agccagcctc t 31 <210> 56 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 56 tgtcttggat ccaagatgaa gcgcgcccgc cccagcgaag atgacttc 48 <210> 57 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 57 aaacacggcg gccgctcttt cattcttg 28 <210> 58 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 58 cgcgctgact cttaggacta gtttc <210> 59 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 59 gcgcttaatt aacatcatca ataatatacc ttatttt 37 <210> <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer WO 00/42208 WO 0042208PCT/EPOO/00265 -52- <400> tgaagcgcgc aagaccgtct gaag <210> 61 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 61 cataacactg cagattcttt attcttgg <210> 62 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 62 ggtacacagg aaacaggagg ttccggaggt ggaggagaca caactcc <210> 63 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic peptide <400> 63 Gly Gly Ser Gly Gly Gly 1 <210> 64 <211> 7231 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: plasmid <400> 64 ctgctccctg acaaggcaag ctgcttcgcg tagtaatcaa cttacggtaa atgacgtatg tatttacggt cctattgacg tgggactttc cggttttggc ctccacccca aaatgtcgta cttgtgtgtt gcttgaccga atgtacgggc ttacggggtc atggcccgcc ttcccatagt aaactgccca tcaatgacgg ctacttggca agtacatcaa ttgacgtcaa acaactccgc ggaggtcgct caattgcatg cagatatacg attagttcat tggctgaccg aacgccaata cttggcagta taaatggccc gtacatctac tgggcgtgga tgggagtttg cccattgacg gagtagtqg aagaatctgc cgttqacatt agcccatata cccaacgacc gggactttcc catcaagtgt gcctggcatt gtattagtca tagcggtttg ttttggcacc caaatgggcg cgagcaaaat ttagggttag gattattgac tggagttccg cccgcccatt attgacgtca atcatatgcc atqcccagta tcgctattac actcacgggg aaaatcaacg gtaggcgtgt ttaagctaca gcgttttgcg tagttattaa cgttacataa gacgtcaata atgggtggac aagtacgccc catgacctta catqgtgatg atttccaagt ggactttcca acggtgggag 120 180 240 300 360 420 480 540 600 660 7.20 WO 00/42208 PTEO/06 PCT/EPOO/00265 -53gtctatataa attaatacga ccgagctcgg tactccctct gaggatttga tcagaaaaga gacagcaact gccgcgatgt gtgcgctcgt acgctggtgg cgcgagcaga gtaaagaccc agcgcctgct catggcatgg ggctctctga acgtaatcgt ggctgCtctg ggacgctgga gcgtaggagt tagtccaggg cggttgagga tccgaacgag ggaaaacctc gcccgcccca aatatcccct ggqgtactgt aaggtgggag ctgcaagtta gctaataaac gcggggttaa gaaaatttag gaagggttga cgcacacttt tctaaactca attgtggtcg tttactatta aaaqcttatt attggtttta agagacatag aaaactacct tccaaaacct caagaatgaa cgactgtgcc ccctggaagg gtctgagtag attgggaaga aaagaaccag cggcgg gtgt ctcctttcgc taaatcgggg aacttgatta ctttgacgtt tcaaccctat ggttaaaaaa tcagttaggg atctcaatta tgcaaagcat cgcccctaac tttatgcaga tttttggagg ctgatcagca caaggtgagg gcagagctct ctcactatag atccactctc gaaaagcggg tattcacctg tggcgatgga ttagctgcac cgggcaccag ctacctctcc atggcggtag cgggcagcag gccatgcgcg ggtgggtgag gtattccaag atagttcatg ctcggaagac agacgttgaa cgcgcagctt tttccttgat caaactcttc atccgtactc tcgagaaaqg qcgaagatga tcctcactcc cactcaaact gtggtctcac atactqataa ttagtttaaa aagatttaat aaaatacaga catttgacaa ggacaacacc ctttggtact caggaaagta aactgctatt ggaactttag tgcctaattt tttatggaac ttaaccaaga atgaaaatgt agagcggccg ttctagttgc tgccactccc gtgtcattct caatagcagg ctggggctct ggtggttacg tttcttccct catcccttta gggtgatgqt ggagtccacg ctcggtctat tgagctgatt tgtggaaagt gtcagcaacc gcatctcaat tccgcccagt ggccgaggcc cctaggcttt cgtgttgaca aactaaacca ctggctaact ggagacccaa ttccgcatcg catgacttct gcccgcggtg gttgtcaagc gcgcagggtt gtattcgcgc gtgcacgcgc gcgtaggcgc ggggtctagc gcqcgcgtcg ggcggcaagc cgcggaggcg atatgtaggg caggaacg tatctgctg gctggcgtct gttgaccagc gatgtcatac gcggtctttc cgccgccgag cgtctaacca cttcaacccc cccctttgtc ggctgatcca tttgcaagat aaaacttgag agtaggacat tggcaaactt tggtagcagc tgatggatac agacacatct tacaaagtgt ccacatcata taataagaac aagtggaaat gqtagcgtat tatatatctt aactggatqt tgaatttgaa ctcgagtcta cagccatctg actgtccttt attctggggg catgctgggg agggggtatc cgcagcgtga tcctttctcg.
gggttccgat tcacgtagtg ttctttaata tcttttgatt taacaaaaat ccccaggctc agqtgtggaa tagtcagcaa tccgcccatt gcctctgcct tgcaaaaagc attaatcatc tggccaagtt agagaaccca gctggctagc ctgtctgcga gcgctaagat atgcctttqa ttggtggcaa tggtttttgt gcaacgcacc caaccgcggt tcgttggtcc tgcgtctcgt aagtagtcta gcgcgctcgt tacatgccgc t agcat ctt c agaggatcgg aagatggcat gtgagaccta tcggcggtqa ttatcctgtc cagtactctt ggacctgagc gtcacagtcg gtctacccct tcctccgatg atcaccatta ggaagcctaa cttgcatatg ggattaaaag gtggttttaa agaggaattg ttggtagcat ccaaactgca ggaagtcaaa aataataaga qgagtqcttt tccaatgttt ccaaaaccca ggtqgaaaac gaatactcta accacctctt gagggcccgt ttgtttgccc cctaataaaa gtggggtggg atgcggtggg cccacgcgcc ccgctacact ccacgttcgc ttagtgcttt ggccatcgcc gtggactctt tataagggat ttaacgcgaa cccaggcagg agtccccagg ccatagtccc ctccgcccca ctgagctatt tcccgggagc ggcatagtat gaccagtgcc ctgcttactg gtttaaactt gggccagctg tgtcagtttc gggtggccgc acgacccgta cgcgatcggc gccattcggq tgtgcagggt agcagaggcg ccggggggtc tcttgcatcc atgggttqag aaatgtcgta caccgcggat gaccagtt gtgagttgga ccgcgtcacg cctgcacgtc cctttttttt ggatcggaaa gagtccgcat ca aga tcca a atggctacgc gattcaaaaa ccaatgggga ctgtaaaccc ataatccatt tattagatga caggaaaagq gta ta a atgt ggaacccaaa caattgctca tattagctaa caaatccaaa tagacaactc cgacagctta qtaattctaa ctgatcaqcc tcacatttaa ttaccttctc ttaaacccgc ctcccccgtg tgaqgaaatt gcaggacagc ctctatggct ctgtagcggc tgccagcgcc cggctttccc acggcacctc ctgatagacg qttccaaact tttggggatt ttaattctgt cagaaqtatg ctccccagca gcccctaact tggctgacta ccagaagtaq ttgtatatcc atcggcatag qttccggtgc gcttatcgaa aagcttggta ttggggtgag caaaaacgag atccatctgg gagggcgttg gcgctccttg aaagacggtg gacaaggtca gccgcccttg tgcgtccacg ttgcaagtct tgggggaccc aacgtagagg gctgqcgcgc gctacqaacg tgatatggtt cacgaaggag tagggcgcag ccacagctcg cccgtcggcc :gaccggatc gatgaaqcgc gcggaatcag cttcccccct tgtatccctc taaggctcca tgaaagtagt aaaaagtgct aataggcact aagagcaaga gtatgacacg agataaggac tgtgtctttg aataaaaagt aaatcttgga tgaaaaagca aaaatatgca agcagtcatt ctttagttgg ctatattgcc tgatcagcct ccttccttga gcatcgcatt aagggggagg tctgaggcgg gcattaagcg ctagcgcccg cqtcaagctc gaccccaaaa gtttttcgcc ggaacaacac tcggcctatt ggaatgtgtg caaagcatgc ggcagaagta ccgcccatcc atttttttta tgaggaggct attttcggat tataatacga tcaccgcgcg 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 WO 00/42208 WO 0042208PCT/EPOO/00265 -54cgacgtcgcc ggaggacgac ggaccaggtg gtacgccgag gaccgagatc ctgcgtgcac cgccgccttc cctccagcgc ttataatggt actgcattct gtcgacctct ttatccgctc tgcctaatga gggaaacctg gcgtattggg taacgcagga cgcgttgCtg ctcaagtcag aagctccctc tctcccttcg gtaggtcgtt cgccttatcc ggcagcagcc cttgaagtgg gctgaagcca cgctggtagc tcaagaagat ttaagggatt aaaatgaagt atgcttaatc ctgact cccc tgcaatqata agccggaagg taattgttgc tgccattgct cqgttcccaa ctccttcggt tatggcagca tggtgagtac cccggcgtca tggaaaacgt gatgtaaccc tgggtgagca atgttgaata tctcatgagc cacatttccc ggagcggtcg ttcgCcggtg gtgccggaca tggtcggaqg ggcgagcagc ttcgtggccq tatgaaaggt ggggatctca tacaaataaa agttgtggtt agctagagct acaattccac gtgagctaac t cgt gccagc cgctcttccg ~l-t cag2ctc'-C aagaacatgt gcgtttttcc aggtggcgaa gtqcgctctc ggaagcgtgg cgctccaagc ggtaactatc actggtaaca tggcctaact qttaccttcg ggtggttttt cctttgatct ttggtcatga t t taa atca a agtgaggcac qtcqtgtaqa ccgcgagacc gccgagcgca cggqaagcta acaggcatcg cgatcaaggc cctccgatcg ctqcataatt tcaaccaagt atacgggata tcttcggggc actcgtgcac aaaacaggaa ctcatactct ggatacatat cgaaaagtgc agttctggac tggtccggga acaccctggc tcgtgtccac cgtgggggCg aggagcagga tgggcttcgg tgctggagtt gcaatagcat tqtccaaact tggcgtaatc acaacatacq tcacattaat tgcattaatg cttcctcgct ac'taag gagcaaaagg ataggctccg acccgacagq ctgttccgac cgctttctca tqqgctgtgt gtcttgagtc ggattagcaq acggctacac gaaaaagagt ttqtttqcaa tttctacggg gattatcaaa tctaaagtat ctatctcagc taactacgat cacgctcacc gaagtggtcc gagtaagtag tggtgtcacg gagttacatg ttgtcagaag ctcttactgt cattctqaga ataccgcgcc gaaaactctc ccaactgatc ggcaaaatgc tcctttttca ttgaatgtat cacctgacgt cgaccggctc cgacgtgacc ctgggtgtgg gaacttccgg ggagttcgcc ctgacacgtg aatcgttttc cttcgcccac cacaaatttc catcaatgta atggtcatag agccggaagc tgcgttgcgC aatcggccaa cactgactcg (TotaaFt3Ccac ccagcaaaag cccccctgac actataaaga cctqCcgctt atgctcacgc qcacqaaccc caacccggta agcgaggtat tagaaggaca tggtagctct gcagcagatt gtctgacgct aaggatcttc atatgagtaa gatctgtcta acgggagggc ggctccagat tqcaacttta ttcqccaqtt ctcgtcgttt atcccccatg taagttggcc catqccatcc atagtgtatg acatagcaga aaggatctta ttcagcatct cgcaaaaaag atattattga ttagaaaaat gggttctccc ctgttcatca gtgcgCggCC gacgcctccg ctgcgcgacc ctacgagatt cgggacgccg cccaacttgt acaaataaag tcttatcatg ctgtttcctg ataaagtgta tcactgcccg cgcgcgggga ctgcgCtcgq ttatcacaq gccaggaacc gagcatcaca taccaggcgt accggatac tqtaggtatc cccgttcagc agacacgact gtaggcggtg gtatttggta tgatccggca acgcqcagaa cagtggaacg acctagatcc acttggtctg tttcgttcat ttaccatctg ttatcagcaa tccgcctcca aatagtttgc ggtatggctt ttgtgcaaaa qcagtgttat gtaaqatgct cggcgaccga actttaaaag ccgctgttga tttactttca cjgaataaggg agcatttatc aaacaaatag gggacttcgt gcgcggtcca tggacgagct ggccggccat cggccggcaa tcgattccac gctggatgat ttattgcagc catttttttc tctgtatacc tgtgaaattg aaqcctqgggg ctttccagtc gaggcggttt tcgttcggct aatcagggga gtaaaaaggc aaaatcgacg ttccccctgg tgtcCgCctt tcagttcggt qcgaccgctg tatcgccact ctacagagtt tctgcgctct aacaaaccac aaaaaggatc aaaactcacg ttttaaatta acaqttacca ccatagttgc gccccagtgc taaaccagcc tccagtctat gcaacgttgt cattcagctc aagcggttag cactcatggt tttctgtgac gttqctcttg tqctcatcat gatccagttc ccagcgtttc cgacacggaa agqgttattg gggttccgCg 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 5580 5640 5700 5760 5820 5880 5940 6000 6060 6120 6180 6240 6300 6360 6420 6480 6540 6600 6660 6720 6780 6840 6900 6960 7020 7080 7140 7200 7231 <210> <211> 8484 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: plasmid <400> ctgctccctg cttgtgtgtt qgaggtcqct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg 120 ctgcttcgcg atgtacgggc cagatatacg cgttgacatt gattattgac tagttattaa 180 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa 240 WO 00/42208 WO 0042208PCT/EPOO/00265 cttacggtaa atgacgtatg tatttacggt cctattgacg tgggactttc cggttttggc ctccacccca aaatgtcgta gtctatataa attaatacga ccgagctcgg tactccctct gaggatttga tcagaaaagat gacagcaact gccgcgatgt gtgcgctcgt acgctggtgg cgcgaqcaga gtaaagaccc agcgcctgct catggcatgg ggctctctga acgtaatcgt ggctgctctg ggacgctgga gcgtaggagt tagtccaggg cggttgagga tccgaacgag ggaaaacctc gcaagaccgt ccaactgtgc cctggqtac ctcaaaatgg accactgtga cccctcacag ggcaacacac attgccaccc cccctcacca gccactggta ggactaaagt actggtccag ggttttgatt caaaacagac ctaagactag aacaaaggcc agcactgcca cttgaatttg ggcctagaat gacagcacag acaccagctc gtcttaacaa agtttggctc aatggagtgc gatcttactg tatccaaaat ggagacaaaa gacacaactc attaatgaaa aagcggccgc actggtattc atggcccgcc ttcccatagt aaactgccca tcaatgacgg ctacttggca agtacatcaa ttgacgtcaa acaactccgc gcagagctct ctcactatag atccactctc gaaaagcggg tattcacctg caatcttttt tggcgatgga ttagctgcac cgggcaccag ctacctctcc atggcggtag cgggcagcag gccatqcgcg ggtgggtgag gtattccaag atagttcqtg ctcggaagac agacgttgaa cgcgcagctt tttccttgat caaactcttc atccgtactc tcgagaaagg ctgaagatac cttttcttac tctctttgcg gcaacggcct gcccacctct ttacctcaga tcaccatgca aaggacccct ccaccgatag qcttgggcat acggggctcc gtgtgactat cacaaggcaa gccttatact gacagggccc tttacttgtt aggggttgat gttcacctaa ttgattcaaa gtgccattac catctcctaa aatgtggcag caatatctgg tactaaacaa aaggcacagc ctcacggtaa *ctaaacctgt caagtgcata tatttgccac *tcgagtctag *ttaactatgt tggctgaccg aacgccaata cttggcagta taaatggccc gtacatctac tgggcgtgga tgggagtttg cccattgacg ctggctaact ggagacccaa ttccgcatcg .catgacttct gCccgcggtg gttgtcaagc gcgcagggtt gtattcgcgc gtgcacgcgc gcgtaggcgc ggggtctagc gcgcgcgtcg ggcggcaagc cgcggaggcg atatgtaggg cgagggagcg tatctgcctg gctggcgtct gttgaccagc gatgtcatac gcggtctttc cgccgccgag cgtctaacca cttcaaccc tcctcccttt cctatccgaa ctctctggac caaaaaaacc agccctaact atcacaggc cacagtgtca cagtaccctt tgacttgaaa tttgcatgta taataatact tatgcaactt tgatgttagt tctttttata tacagcttca gtttgacqct tgcaccaaac caaggctatq agtaggaaac ctgtagacta tcaaatactt aacagttcaa ttccttcctg ctatacaaac aactgccaaa aacactaacc ctctatgtca atcctcttac cgataatcaa tgctcctttt cccaacgacc gggactttcc catcaagtgt gcctggcatt gtattagtca tagcggtttg ttttggcacc caaatgggcg agagaaccca gctggctagc ctgtctgcga gcgctaagat atgcctttga ttggtggcaa tggtttttgt ge aacgcacc caaccgcggt tcgttggtcc tgcgtctcgt aagtagtcta gcgcgctcgt tacatgccgc tagcatcttc aggaggtcgg aagatggcat gtgagaccta tcggcggtga ttatcctgtc cagtactctt ggacctgagc gtcacagtcg gtgtatccat gtatccccca cctctagtta gaggccggca aagtcaaaca gtggctgccg ccgctaaccg gaaggaaagc actatcactg gaqcccattt acagacgacc tccttgcaaa aatgtagcag tatccgtttg aactcagccc aacaattcca acagccatag acaaatcccc gttcctaaac aaaaataatg aatgcagaga gctacagttt agtgctcatc gacccagaat gctgttggat agtaacattg attacactaa ttttcatggg actttttcat cctctggatt acgctatgtg cccgcccatt attgacgtca atcatatgcc atgcccagta tcgctattac actcacgggg aaaatcaacg gtaggcgtgt ctgcttactg gtttaaactt gggccagctg tgtcaqtttc gggtggCCgC acgacccgta cgcgatcggc gccattcggg tgtgcagggt agcagaggcg ccgggqggtc tcttgcatcc atgggttgag aaatgtcgta caccgcggat gaccgaggtt gtgagttgga ccgcgtcacg cct qcacgtc cctttttttt ggatcggaaa gagtccgcat caagatccaa atgacacgga atgqgtttca cctccaatgg accttacctc taaacctgga ccgcacctct tgcacgactc tagccctgca cctcaccccc atacacaaaa taaacacttt ctaaagttac gaggactaag atgctcaaaa acaacttgga aaaagcttga ccattaatqc tcaaaacaaa taggaactqg ataagctaac aagatgctaa cagttttggc ttattataag attggaactt ttatgcctaa tcagtcaagt acggtacaca actggtctgg acattgccca acaaaatttg gatacgctgc gacgtcaata atgggtggac aagtacgccc catgacctta catggtgatg atttccaagt ggactttcca acggtgggag gcttatcgaa aagcttggta ttggggtgag caaaaacgag atccatctgg gagggcgttg gcgctccttg aaagacggtg gacaaggtca gccgcccttg tgcgtccacg ttgcaagtct tgggggaccc acgtagagg gctggcgcgc gctacgggcg tgatatggtt cacgaaggag tagggcgcag ccacagctcg cccgtcggcc cgaccggat c gatgaagcgc aaccggtcct agagagtccc catgcttgcg ccaaaatgta aatatctgca aatqgtcgcg caaacttagc aacatcagqc tctaactact tggaaaacta gaccgtagca tggaqccttg gattgattct dcaactaaat tattaactac ggttaaccta aggagatggg aattggccat ccttagtttt tttgtggacc actcactttg tgttaaaggc atttgacgaa tagaaatgga cctatcagct ttacttaaac ggaaacagga ccacaactac agaataaaag tgaaaqattg tttaatgcct 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660- 3720 3780 3840 3900 3960 WO 00/42208 WO 0042208PCTIEPOO/00265 -56ttgtatcatg ttgctgtctc gtgtttgctg gggactttcg cgctgctgga ctgacgtcct ttctgctacg gctctgcggc gccgcct ccc gccttctagt aggtgccact taggtgtcat agacaatagc cagctggggc tgtggtggtt U. L U U LU gggcatccct ttagggtgat gttggagtcc tatctcggtc aaatgagctg gggtgtggaa ttagtcagca catgcatctc aactccgccc agaggccgag aggcctaggc gcacgtgttg aggaactaaa gccggagcgg gacttcgccg gtggtgccqq gagtggtcgg atcggcgagc cacttcgtgg ttctatgaaa cgcggggatc ggttacaaat tctagttgtg tctagctaga ctcacaattc tqagtgagct ctgtcgtgcc gggcgctctt gcggtatcag gqaaaqaaca ctggcgtttt cagaggtggc ctcgtgcgct tcgggaagcg gttcgctcca tccggtaact gccactggta tgqtggccta ccagttacct agcggtggtt gatcctttga attttggtca agttttaaat atcagtgagg cccgtcgtgt ataccgcgag ctattgcttc tttatgagga acgcaacccc ctttccccct caggggctcg ttccatggct tcccttcggc ctcttccgcg cgcctgatcg tqccagccat cccactgtcc tctattctgg aggcatqctg tctaqggggt acgcgcagcg ttagggttcc ggttcacgta acgttcttta tattcttttg atttaacaaa aqtccccagg accaggtgtg aattagtcag agttccgccc gccgcctctg ttttgcaaaa acaattaatc ccatggccaa tcgagttctg gtgtggtccg acaacaccct aggtcgtgtc agccgtgggg ccgaggagca ggttgqgctt tcatgctgga aaaqcaatag gtttgtccaa gcttggcgta cacacaacat aactcacatt agctgcatta ccgcttcctc ctcactcaaa tqtgagcaaa tccataggct gaaacccgac ctcctgttcc tggcgctttc agctgggctg atcgtcttga acaggattag actacggcta tcggaaaaag tttttgtttg tcttttctac tgagattatc caatctaaag cacctatctc agataactac acccacgctc ccgtatggct gttgtggccc cactggttgg ccctattgcc gctgttgggc gctcgcctgt cctcaatcca tcttcgcctt ctagagggcc ctgttgtttg tttcctaata ggggtggggt gggatgcggt atccccacgc tgaccgctac LLgcacgtt U'.
gatttagtgc gtgggccatc atagtggact atttataagg aatttaacgc ctccccaggc gaaagtcccc caaccatagt attctccgcc cctctgagct agctcccggg atcggcatag gttgaccagt gaccgaccgg ggacgacgtq gqcctgggtg cacgaacttc gcgggagttc ggactgacac cggaatcgtt gttcttcgcc catcacaaat actcatcaat atcatggtca acgagccgga aattgcgttg atgaatcggc gctcactgac ggcggtaata aggccagcaa ccgcccccct aggactataa gaccctgccg tcaatgctca tgtgcacgaa gtccaacccg cagagcgagg cactagaagg agttggtagc caagcagcag ggggtctgac aaaaaggatc tatatatgag agcgatctgt gatacgggag accggctcca ttcattttct gttgtcaggc ggcattgcca acggcggaac actgacaatt gttgccacct gcggaccttc cgccctcaga cgtttaaacc cccctccccc aaatgaggaa ggggcaggac gggctctatg gccctgtagc acttgccagc cgccggcttt-* tttacggcac gccctgatag cttgttccaa gattttgggg gaattaattc aggcagaagt aggctcccca cccgccccta ccatggctga attccagaag agcttgtata tatatcggca gccgttccgg ctcgggttct accctgttca tgggtgcgcg cgggacgcct gccctgcgcg gtgctacgag ttccggqacg caccccaact ttcacaaata gtatcttatc tagctgtttc agcataaagt cgctcactgc caacgcgcgg tcgctgcgct cqgttatcca aaggccagga gacgagcatc agataccagg ottaccggat cgctgtaggt ccccccgtt c gtaagacacg tatgtaggcg acagtatttg tcttgatccg attacgcgca gctcagtgga ttcacctaga taaacttggt ctatttcgtt ggcttaccat gatttatcag cctccttgta aacgtggcgt ccacctgtca tcatcgccgc ccgtggtgtt ggattctgcg cttcccgcgg cgagtcggat cgctgatcag gtgccttcct attgcatcgc agcaaggggg gcttctgagg ggcgcattaa gccctagcgc ccctca ctcgacccca acggtttttc act ggaacaa att tcggcct tgtggaatgt atgcaaagca gcaggcagaa actccgccca ctaatttttt tagtgaggag t ccatt t tcg tagtataata tgctcaccgc cccgggactt tcagcgcggt gcctggacga ccgggccggc acccggccgg atttcgattc ccggctggat tgtttattgc aagcattttt atgtctgtat ctgtgtqaaa gtaaagcctg ccgctttcca ggagaggcgg cggtcgttcg cagaatcagg accgtaaaaa acaaaaatcg cgtttcccc acctgtccgc atctcagttc agcccgaccg acttatcgcc gtgctacaqa gtatctgcgc gcaaacaaac gaaaaaaagg acgaaaactc tccttttaaa ctgacagtta catccatagt ctggccccag caataaacca taaatcctgq ggtgtgcact gctcctttcc ctgccttgcc gtcggggaag cgggacgtcc cctgctgccg ctccctttgg cctcgactgt tgaccctgga attgtctgag aggattggga cggaaagaac gcgcggcggg ccgctccttt ctctl-aaat"cg aaaaacttga gccctttgac cactcaaccc attggttaaa gtgtcagtta gcatctcaa.
gtat gcaaag tcccgcccct ttatttatgc gcttttttgg gatctgatca cgacaaggtg qcgcgacgtc cgtggagqac ccaggaccag gctgtacgcc catgaccgag caactgcgtg caccgccgcc gatcctccag agcttataat ttcactgcat accgtcgacc ttgttatccg gggtgcctaa gtcgggaaac tttgcgtatt gctgcggcga ggataacgca ggccgcgttg acgctcaagt tggaagctcc ctttctccct ggtgtaggtc ctgcgcctta actgqcagca gttcttgaag tctgctgaag caccgctggt atctcaagaa acgttaaggg ttaaaaatga ccaatgctta tgcctgactc tgctgcaatg gccagccgga 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 A 012 0 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 5580 5640 5700 5760 5820 5880 -5940 6000 6060 6120 6180 6240* 6300 6360 6420 6480 6540 6600 6660 6720 6780 6840 6900 6960 7020 7080 7140 7200 7260 7320 7380 7440 7500 7560 7620 7680 WO 00/42208 PCT/EPOO/00265 -57agggccgagc tgccgggaag gctacaggca caacgatcaa ggtcctccga gcactgcata tactcaacca.
tcaatacggg cgttcttcgg cccactcgtg gcaaaaacag atactcatac agcggataca ccccgaaaag gcagaagtgg ctagagtaag tcgtggtgtc ggcgagttac tcgttgtcag attctcttac agtcattctg ataataccgc qgcgaaaact cacccaactg gaaggcaaaa tcttcctttt tatttgaatg tgccacctga tcctgcaact tagttcgcca acgctcgtcg atgatccccc aagtaagttg tgtcatgcca agaatagtgt gccacatagc ctcaaggatc atcttcagca tgccgcaaaa tcaatattat tatttagaaa cgtc ttatccgcct gttaatagtt tttggtatgg atgttgtgca gccgcagtgt tccgtaagat atgcggcgac agaactttaa ttaccgctgt tcttttactt aagggaataa tgaagcattt aataaacaaa ccatccagtc tgcgcaacgt cttcattcaq aaaaagcggt tatcactcat gcttttctgt cgagttgctc aagtgctcat tgagatccag tcaccagcgt gggcgacacg atcagggtta taggggttcc tattaattgt tgttgccatt ctccggttcc tagctccttc ggttatggca gactggtgag ttgcccggcg cattggaaaa ttcgatgtaa.
ttctgggtga.
gaaatgttga ttgtctcatg gcgcacattt 7740 7800 7860 7920 7980 8040 8100 8160 8220 8280 8340 8400 8460 8484 <210> 66 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 66 gtcactcgag gactcggtcg actgaaaatg agacatatta tctgccacgg acc <210> 67 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 67 cgagatcgat cacctccggt acaaggtttg gcatag <210> 68 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 68 catgaagatc tggaaggtgc tgaggtacga tgagacc <210> 69 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 69 gcgacttaag cagtcagctg agacagcaag acacttgctt gatccaaatc c WO 00/42208 PCT/EP00/00265 -58- <210> <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> cacgaattcg tcagcgcttc tcgtcgcgtc caagaccc 38 <210> 71 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 71 caccccgggg aggcggcggc gacggggacg gg 32 <210> 72 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: oligonucleotide <400> 72 atgggatcca agatgaagcg cgcaagaccg <210> 73 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: oligonucleotide <400> 73 cataacctgc aggattcttt attcttgggc <210> 74 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: oligonucleotide <400> 74 ggtacacagg aaacaggagg ttccggaggt ggaggagaca caactcc 47 <210> WO 00/42208 PCT/EP00/00265 -59- <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> atgggatcca agatgaagcg cgcaagaccg <210> 76 <211> <212> DNA.
<213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 76 cactatagcg gccgcattct cagtcatctt

Claims (32)

11-03-'04 09:46 FROM-DCC +61392542770 T-227 P08/26 U-250 ?:OERlKb«M372-0 ,q2dqAI1S4 103 THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 1. An isolated nucleic acid molecule comprising: a sequence of nucleotides encoding an adenovirus tripartite leader (TPL), wherein said TPL nucleotide sequence consists essentially of at least two different TPL exons selected from complete exons 1, 2 and 3 and optionally also includes intron 1. 2. The isolated nucleic acid molecule of claim 1 wherein said intron is native adenovirus intron 1. 3. The isolated nucleic acid molecule of claims 1 or 2 wherein said TPL nucleotide sequence is shown in SEQ ID NO: 32. 4. The isolated nucleic acid molecule of any one of claims 1 to 3 further comprising a promoter and a nucleic acid sequence which encodes an adenoviral structural protein, operatively linked to said promoter and said TPL sequence. The isolated nucleic acid molecule of claim 4 wherein said adenoviral structural 9 protein is a fiber protein or a chimeric protein which includes an adenovirus fiber protein 20 tail domain. 6. The isolated nucleic acid molecule of claim 5 wherein said chimeric protein comprises an Ad3 head domain and an Ad5 tail domain or an Ad5 head domain and an Ad3 tail domain. 7. The isolated nucleic acid molecule of claim 6 wherein said molecule is contained in a plasmid selected from the group consisting of plasmids pDV60 (ATCC Accession No. PTA-1144), pDV67 (ATCC Accession No. PTA-1145), pDV69 (ATCC Accession No. PTA-1146), pDV80 (ATCC Accession No. PTA-1147) and pDV90 (ATCC Accession No. 30 PTA-1148). COMS ID No: SMBI-00658976 Received by IP Australia: Time 09:33 Date 2004-03-11 11-03-'04 09:47 FROM-DCC +61392542770 T-227 P09/26 U-250 -104- 8. The isolated nucleic acid molecule of claim 7 wherein said molecule has a nucleotide sequence selected from the group consisting of sequences shown in SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 47, SEQ ID NO: 64 and SEQ ID NO: 9. An adenovirus vector complementing plasmid comprising an isolated nucleic acid molecule according to any one of claims 1 to 8. An adenovirus vector packaging cell line comprising a stably integrated nucleic acid molecule as claimed in any one of claims 1 to 9, an operatively-linked promoter and a nucleic acid sequence which encodes an adenovirus structural protein. 11. The cell line of claim 10 wherein said TPL molecule comprises complete TPL exon 1 having the nucleotide sequence of SEQ ID NO: 32.
12. The cell line of claim 10 wherein said promoter is an inducible promoter.
13. The cell line of claim 10 wherein said adenovirus structural protein is adenovirus fiber protein or a chimeric protein which includes an adenovirus fiber protein tail domain. 20 14. The cell line of claim 10 wherein said chimeric protein comprises an Ad3 head 9 9 S* domain and an AdS tail domain or an Ad5 head domain and an Ad3 tail domain.
15. The cell line of claim 10 wherein said nucleic acid molecule is selected from the group consisting of plasmids pDV60 (ATCC Accession No. PTA-1144), pDV67 (ATCC Accession No. PTA-1145), pDV69 (ATCC Accession No. PTA-1146), pDV80 (ATCC Accession No- PTA- 1147) and pDV90 (ATCC Accession No. PTA-1148). 9 16. The cell line of claim 15 wherein said nucleic acid molecule has a nucleotide sequence from the group consisting of sequences shown in SEQ ID NO: 43, SEQ ID NO: 30 44 and SEQ ID NO: 47. COMS ID No: SMBI-00658976 Received by IP Australia: Time 09:33 Date 2004-03-11 11-03-'04 09:47 FROM-DCC +61392542770 T-227 P10/26 U-250 -105-
17. The cell line of claim 10 wherein said cell line is an epithelial cell line.
18. The cell line of claim 17 wherein said cell line supports the production of a recombinant adenovirus vector genome by complementation of a deficient viral gene in said vector genome.
19. The cell line of claim 18 -wheein said cell line frther produces an adenovirus protein and thereby complements a deficient adenovirus gene in said vector genome, and wherein said cell line complements an adenovirus early protein gene and a fiber gene. The cell line of claim 19 wherein the deletion of said deficient adenovirus gene is complemented by the expression of said gene under the control of an inducible promoter.
21. A method for producing an adenovirus vector particle containing a helper- independent fiberless recombinant adenovirus vector genome, said method comprising providing a packaging cell line which complements replication and packaging of said genome and a helper-independent fiberless recombinant adenovirus vector genome which is deficient in expressing sufficient functional fiber protein to support assembly of fiber- containing particles, wherein said packaging cell comprises an isolated nucleic acid 20 molecule comprising: a sequence of nucleotides encoding an adenovirus tripartite leader (TPL), wherein said TPL nucleotide sequence consists essentially of at least two different TPL exons selected from complete exons 1, 2 and 3 and optionally also includes intron 1, and harvesting said particles produced by said cell line.
22. The method of claim 21 wherein said packaging cell line complements adenovirus fiber protein.
23. The method of claim 21 wherein said adenovirus vector genome comprises genes that: 30 express all adenovirus structural gene products but do not express sufficient adenovirus fiber protein to package a fiber-containing adenovirus particle without COMS ID No: SMBI-00658976 Received by IP Australia: Time 09:33 Date 2004-03-11 11-03-'04 09:47 FROM-DCC +61392542770 T-227 P11/26 U-250 106 complementation of said fiber gene or said genome lacks at least the fibre gene, and express an exogenous protein.
24. The method of claim 21 wherein said packaging cell line comprises a stably integrated first nucleic acid molecule alternatively operatively linked to a promoter, and said first nucleic acid is operatively linked to a second nucleic acid molecule encoding an adenovirus structural protein. The method of claim 21 wherein said helper-independent fiberless recombinant adenovirus vector genome is introduced by infecting said cell line with a virus particle conatining said genome.
26. The method of claim 25 wherein said particle is a particle comprising a helper- independent recombinant adenovirus vector genome comprising genes that: encode all adenovirus structural gene products but do not express sufficient adenovirus fiber protein to support packaging of a fiber-containing adenovirus particle without complementation of said fiber gene or said genome lacks at least the fibre gene, and S(b) encode an exogenous protein, wherein said particle comprises an adenovirus fiber protein or a chimeric protein that includes an adenovirus fiber protein tail domain.
27. The method of claim 21 wherein said helper-independent fiberless recombinant adenovirus vector genome is introduced into said cell line by transfecting said cell line with said helper-independent fiberless recombinant adenovirus vector genome,
28. The method of claim 27 wherein said adenovirus vector genome comprises genes which: encode all adenovirus structural gene products but do not express sufficient 30 adenovirus fiber protein to package a fiber-containing adenovirus particle without complementation of said fiber gene or said genome lacks at least the fibre gene, and COMS ID No: SMBI-00658976 Received by IP Australia: Time 09:33 Date 2004-03-11 c I 11-03-'04 09:47 FROM-DCC +61392542770 T-227 P12/26 U-250 107 encode an exogenous protein.
29. The method of claim 21 wherein said packaging cell line is transfected with a nucleic acid molecule encoding adenovirus. fiber protein. The method of claim 29 wherein said nucleic acid molecule is a nucleic acid molecule comprisig a sequence of nucleotides encoding an adenovirus tripartite leader J± &U LLta.JLALLV, dig LL GCUfltJVAS LLL}WJLILt, A;;LdUU (TPL), wherein said TPL nucleotide sequence consists essentially of at least two different TPL exons, wherein said TPL exons are selected from complete exons 1, 2 and 3 and optionally also includes intron 1.
31. The method of claim 22 wherein said adenovirus fiber protein is a modified fiber protein.
32. The method of claim 21 further comprising the step of coating said particle with adenovirus fiber protein.
33. The packaging cell line of claim 10 wherein said cell line is selected from the group consisting of 293, A549, W163, HeLa, Vero, 211, 211A and an epithelial cell line comprising the stably integrated nucleic acid molecule.
34. A method for producing a gutless adenoviral vector particle comprising: delivering a helper adenovirus vector genome to an adenovirus vector packaging cell, wherein said helper adenovirus vector genome lacks any gene encoding adenovirus fiber protein or lacks the ability to encode sufficient adenovirus fiber protein to produce an adenoviral vector comprising fiber protein in the absence of complementation by said packaging cell and wherein said packaging cell comprises the nucleic acid molecule of claim 1 operably linked to a promoter and to an adenoviral fiber protein or to a chimeric protein that includes an adenovirus fiber protein tail domain; 30 delivering a gutless adenovirus vector genome to said packaging cell; and recovering the gutless adenoviral vector particle produced by said cell. COMS ID No: SMBI-00658976 Received by IP Australia: Time 09:33 Date 2004-03-11 11-03-'04 09:48 FROM-DCC +61392542770 T-227 P13/26 U-250 PIAWM\VMWflrA- eaaw-l w13M 108 The method of claim delivered by viral infection.
36. The method of claim delivered by transfection. 34, wherein said helper adenovirus vector genome is 35, wherein said gutless adenovirus vector genome is 0* go 0 @0 0 0 0 0 0 0 *005 @005 0 S 0 0 0 *o o**oo
37. The method of claim 36, wherein said gutless adenovirus vector genome comprises an operable packaging sequence.
38. The method of claim 37, wherein said helper adenovirus vector genome has a mutation in its packaging sequence that renders said genome substantially incapable of being packaged as an adenoviral vector particle by said packaging cell.
39. The method of claim 37, wherein said helper adenovirus vector genome comprises recombinase sites flanking its packaging sequence and said packaging cell further comprises a nucleotide sequence encoding a recombinase.
40. The method of claim 39, wherein said recombinase site is a lox site and said 20 recombinase is Cie.
41. A packaging cell for the production of a fiberless or fiber-modified gutless adenovirus particle comprising an adenovirus vector complementing plasmid and a nucleotide sequence encoding a recombinase, wherein said complementing plasmid 25 comprises the nucleic acid molecule of claim 1 operably linked to a promoter and to a nucleotide sequence encoding an adenoviral fiber protein or a chimeric adenoviral fiber protein.
42. The packaging cell of claim 41, wherein said complementing plasmid and said nucleotide sequence encoding a recombinase are stably integrated into the genome of said cell. COMS ID No: SMBI-00658976 Received by IP Australia: Time 09:33 Date 2004-03-11 11-03-'04 09:48 FROM-DCC +61392542770 T-227 P14/26 U-250 ;:OPt f4372m4O. r ol ItQ4 109
43. The packaging cell of claim 41, firther comprising a helper adenovirus vector genome.
44. The packaging cell of claim 41, wherein said recombinase is Cre. An isolated .nucleic acid molecule of claim 1, substatially as .hereinbefre "9J. 'Ll zA atW u l L."M h. a 4 LM L WA %gA4ajl I p O L O LlAI4CI4A, 00 !wa, .LU /L described.
46. An adenovirus vector of claim 9, substantially as hereinbefore described.
47. An adenovirus vector packaging cell line of claim 10, substantially as hereinbefore described.
48. A method for producing an adenovirus vector particle containing a helper- independent fiberless recombinant adenovirus vector genome of claim 21, substantially as hereinbefore described.
49. A method for producing a gutless adenoviral vector particle of claim 34 substantially as hereinbefore described. A packaging cell of claim 41, substantially as hereinbefore described. 25 DATED this 1 th day of March, 2004 Novartis AG AND The Scripps Research Institute By DAVIES COLLISON CAVE 30 Patent Attorneys for the Applicants COMS ID No: SMBI-00658976 Received by IP Australia: Time 09:33 Date 2004-03-11
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