AU2003227281B2 - Packaging systems for human recombinant adenoviruses to be used in gene therapy - Google Patents

Description

I

Regulation 3.2

AUSTRALIA

Patents Act 1990 DIVISIONAL APPLICATION Name of Applicant: Crucell Holland B.V. AND Rijksuniversiteit Leiden Actual Inventor(s): Frits Jacobus FALLAUX, Robert Cornelis HOEHEN, Abraham BOUT, Domenico VALERIO and Alex Jan VAN DER EB.

Address for Service: DAVIES COLLISON CAVE, Patent Attorneys, Level 3, 303 Coronation Drive, Milton, Queensland, 4064, Australia Invention Title: "Packaging systems for human recombinant adenoviruses to be used in gene therapy." Details of Parent Application No: 19705/01 The following statement is a full description of this invention, including the best method of performing it known to me/us: Q \Oper\VpalJulyUu 2003\2177242 PO div crucell et al.205.doc 24/7/03 1 Title: Packaging systems for human recombinant adenovirus to be used in gene therapy.

The invention relates to the field of recombinant DNA technology, more in particular to the field of gene therapy. In particular the invention relates to gene therapy using materials derived from adenovirus, in particular human recombinant adenovirus. It especially relates to novel virus derived vectors and novel packaging cell lines for vectors based on adenoviruses.

Gene therapy is a recently developed concept for which a wide range of applications can be and have been envisaced.

In gene therapy a molecule carrying genetic information is introduced into some or all cells of a host, as a result of which the genetic information is added tc the host in a functional format.

The genetic information added may be a gene or a derivative of a gene, such as a cDNA, which encodes a protein. In this case the functional format means that the protein can be expressed by the machinery of the host cell.

The generic information can also be a seauence of nucleotides complementary to a sequence of nucleotides (be it DNA zr RNA) present in the host cell. The functional format in this case is that the added DNA (nucleic acid) molecule or copies made thereof in situ are capable of base pairing with the complementary sequence present in the host cell Appiications include the treatment of genetic disorders by suppiementing a protein or other substance which is, through said genetic disorder, nct present or at least present in insufficient amounts in the host, the treatment of tumors and (other) acquired diseases such as (auto)immune diseases or infections, etc.

As may be clear from the above, there are basically three different approaches in gene therapy, one directed towards compensating a deficiency present in a (mammalian) host; the second directed towards the removal or elimination of unwanted substances (organisms or cells) and the third towards application of a recombinant vaccine (tumors or foreign micro-organisms).

For the purpose of gene therapy, adenoviruses carrying deletions have been proposed as suitable-.-ehicles.

Adenoviruses are non-enveloped DNA viruses. Gene-transfer vectors derived from adenoviruses (so-called adenoviral vectors) have a number of features that make them particularly useful for gene transfer for such purposes. Eg.

the biology of the adenoviruses is characterized in detail, the adenovirus is not associated with severe human pathology, the virus is extremely efficient in introducing its DNA into the host cell, the virus can infect a wide variety of cells and has a broad host-range, the virus can be produced in large quantities with relative ease, and the virus can be rendered replication defective by deletions in the earlyregion 1 (Zl) of the viral genome.

The adenovirus genome is a linear double-stranded DNA molecule of anproximately 36000 base pairs with the terminal crotein covalentiv bound to the 5'terminus of each strand. The Ad DNA contains identical Inverted Terminal Repeats (ZTR) of about 100 base pairs with the exact length depending on the serotype- The viral origins of replication are located within the ITRs exactly at the genome ends. DNA synthesis occurs in two stages. First, the replication proceeds cy strand displacement, generating a daughter duplex molecule and a parental displaced strand. The displaced strand is sinale stranded and can form a so-called "pannandle" intermediate, which allows reolication initiation and seneration of a daughter duplex molecule. Alternatively.

replication may proceed from both ends of the genome simultaneously, obviating the requirement to form the panhandle structure. The replication is summar-zed in Figure 14 adapted from (Lechner and Kelly, 197-).

During the productive infection cycle, the viral genes are expressed in two phases: the early phase, which is the period upto viral DNA replication, and the late phase, which coincides with the initiation of iral DNA replication. During the early phase only the early gene products, encoded by regions El, E2, E3 and are expressed, which carry out a number of functisns that prepare the cell for synthesis of viral structural proteins (Berk, 1986). During the late phase the late viral gene products are expressed in addition to the early gene products and host cell DNA and protein synthesis are shut off. Consequently, the cell becomes dedicated to the production of viral DNA and of viral structural proteins (Tooze, 1981).

The El region of adenovirus is the first region of adenovirus expressed after infection of the target cell.

This region consists of two transcriptional units, the ElA and ElB genes, which both are required for oncogenic transformation of primary (embryonal) rodent cultures. The main functions of the EIA gene products are: i) to induce quiescent cells to enter the cell cycle and resume cellular DNA synthesis, and il) to transcriptionally activate the E1B cene and the other early regions (E2, E3, E4). Transfecticn of primary cells with the E1A gene alone can induce unlimited proliferation (immortalization), but does not result in complete transformation. However, expression cf E1A in most cases results in induction of programmed cell death (apoptosis), and only occasionally immortalization is obtained (Jochemsen et al. 1987). Co-expression of the E13 aene is required to prevent induction of apoptosis and for complete morphological transformation to occur. In established immortal cell lines, high level exoression of ElA can cause complete transformation in the absence of EB1 (Roberts et al., 1985).

The E1B encoded proteins assist EIA in redirecting the cellular functions to allow viral replication. The E1B kD and E4 33kD proteins, which form a complex that is essentially localized in the nucleus, function in inhibiting the synthesis of host proteins and in facilitating the expression of viral genes. Their main influence is to establish selective transport of viral mRNAs from the nucleus to the cytoplasm, concomittantiv with the onset of the late phase of infection. The E1B 21 kD protein is important for correct temporal control of the productive infection cycle, thereby preventing premature death of the host cell before the virus life cycle has been completed. Mutant viruses incapable of expressing the EIB 21 kD gene-product exhibit a shortened infection cycle that is accompanied by excessive degradation of host cell chromosomal DNA (deg-phenorype) and in an enhanced cytopathic effect (cyc-phenotype) (Telling et al., 1994). The deg and cyt phenotypes are suppressed when in addition the E1A gene is mutated, indicating that these phenotypes are a function of E1A (White et al., 1988). Furthermore, the E1B 21 kDa protein slows down the rate by which E1A switches on the other viral genes. It is not yet known through which mechanisms) E1B 21 kD quenches these E1A dependent functions.

25 Vectors derived from human adenoviruses, in which at least the El region has been deleted and replaced by a gene of interest, have been used extensively for gene therapy experiments in the pre-clinical and clinical phase.

As stated before all adenovirus vectors currentlused in gene therapy have a deletion in the El region, where novel genetic information can be introduced. The El deletion renders the recombinant virus replication defective (Stratford-Perricauder and Perricaudet, 1991) We have demonstrated that recombinant adenoviruses are able to efficiently transfer recombinant genes to the rat liver and airway epithelium of rhesus monkeys (Bout et al., 1994b; Bout et al., 1994a). In addition, we (Vincent et al., 1996a; Vincent et al., 1996b) and others (see e.g.

Haddada et al 1993) have observed a very eff cient Tvivo adenovirus mediated gene transfer to a variety of tumor cells in vitro and to solid tumors in animals models (lung tumors, glioma) and human xenografts in immunodeficient mice (lung) in vivo (reviewed by Blaese et al., 1995).

In contrast to for instance retroviruses, adenoviruses a) do not integrate into the host cell genome; b) are able to infect non-dividing cells and c) are able to efficiently transfer recombinant genes in vivo (Brodv and Crystal, 1994). Those features make adenoviruses attractive candidates for in vivo gene transfer of, for instance, suicide or cytokine genes into tumor cells.

However, a problem associated with current recombinant adenovirus technology is the possibility of unwanted generation of replication competent adenovirus (RCA) during the production of recombinant adenovirus (LochmUller et ai. 1994; Imler et al., 1996). This is caused by homologous recombination between overlapping sequences from the recombinant vector and the adenovirus constructs present in the complementing cell line, such as the 293 cells (Graham et al., 1977). RCA in batches to be used in clinical trials is unwanted because RCA i) will replicate in an uncontrolled fashion; ii) can complement replication defective recombinant adenovirus, causing uncontrolled multiplication of the recombinant adenovirus and iii) batches containing RCA induce significant tissue damage and hence strong pathological side effects (Lochmuller et al.. 1994). Therefore, batches to be used in clinical trials should be proven free of RCA (Ostrove, 1994). In one aspect of the invention this problem in virus production is solved in thar we have developed packaging cells that have no overlapping sequences with a new basic vector and thus are suited for safe large scale production of recombinant adenoviruses one of the additional problems associated with the use of recombinant adenovirus vectors is the host-defence reaction against treatment with adenovirus.

Briefly', recombinant adenoviruses are deleted for the El region (see above). The adenovirus El products trigger the transcription of the other early genes (E2, E3, E4), which consequently activate expression of the late virus genes. Therefore, it was generally thought that El deleted vectors would not express any other adenovirus genes.

However, recently it has been demonstrated that some cell types are able to express adenovirus genes in the absence of El sequences. This indicates, that some cell types possess the machinery to drive transcription of adenovirus genes. In particular, it was demonstrated that such cells synthesize E2A and late adenovirus proteins.

In a gene therapy setting, this means that transfer of the therapeutic recombinant gene to somatic cells not only results in expression of the therapeutic protein but may also result in the synthesis of viral proteins. Cells that express adenoviral proteins are recognized and killed by Cytotoxic T Lymphocytes, which thus a) eradicates the transduced cells and b) causes inflammations (Bout et al.

1994a: Engelhardt et al., 1993; Simon et al., 1993).As this adverse reaction is hampering gene theraov, several solutions to this problem have been suggested, such as a) using immunosuppressive agents after treatment; b) retainment of the adenovirus E3 region in the recombinant vector (see patent application EP 95202213) and c) and using ts mutants of human adenovirus, .hich have a point mutation in the E2A region (patent WO/29938) However, these strategies to circumvent the immune response have their limitations.

The use of ts mutant recombinant adenovirus diminishes the immune response to some extent, but was less effective in preventing pathological responses in the lungs (Enaelhardt et al., 1994a) The E2A protein may induce an immune response by itself and it plays a pivotal role in the s:.-itch to the synthesis of late adenovirus proteins. Therefore, it is attractive to make recombinant adenoviruses which are mutated in the E2 region, rendering it temperature sensitive as has been claimed in patenr application WO/28938.

A major drawback of this system is the fact that, although the E2 protein is unstable at the non-permissive temperature, the immunogenic protein is still being synthesized. In addition, it is to be expected that the unstable protein does activate late gene expression, albeit to a low extent. tsl25 mutant recombinant adenoviruses have been rested, and prolonged recombinant gene expression was reported (Yang et al., 1994b: Engeihardt et al.. 1994a; Engelhardt ez al.. 1994b; Yang et al., 1995). However, pathology in the lungs of cotton rats was still high (Engelhardt et al., 1994a), indicating that the use of ts mutants results in only a partial improvement in recombinant adenovirus technology. Others (Fang et al., 1996) did not observe prolonged gene expression in mice and dogs using ts125 reccmbinant adenovirus- An additional difficulty associated with the use of ts125 mutant adenoviruses is that a hiah freouencof reversion is observed. These revertants are either real revertants or the result of second site mutations (Kruijer et ai., 1983; Nicolas et al., 1981). Both t-v.es of revertants have an E2A protein that functions at normal temperature and have therefore similar toxicity as the wild-type virus.

In another aspect of the present invention we therefore delete E2A coding sequences from the recombinant aderovirus genome and transfeco these E2A sequences into the (packaging) cell lines containing El sequences to complement recombinant adenovirus vectors.

Major hurdles in this approach are a) that E2A should be expressed to very high levels and b) that E2A protein is very toxic to cells.

The current invention in yet another aspect therefore discloses use of the tsl25 mutant E2A gene, which produces a protein that is not able to bind DNA sequences at the non permissive temperature. High levels of this protein may be maintained in the cells (because it is not toxic at this temperature) until the switch to the permissive temperature is made. This can be combined with placing the mutant E2A gene under the direction of an inducible promoter, such as for instance tet, methallothionein, steroid inducible promoter, retinoic acid P-receotor or other inducible systems. However in yet another asoect of the invention, the use of an inducible promoter to control the moment of production of toxic wild-type E2A is disclosed.

Two salient additional advantages of E2A-deleted recombinant adenovirus are the increased capacity to harbor heterologous sequences and the permanent selection for cells that express the mutant E2A. This second advantage relates to the high frequency of reversion of mutation: when reversion occurs in a cell line narboring tsl25 E2A, this will be lethal to the cell i Therefore, there is a permanent selection for those cells that express the ts125 mutant E2A protein. In addition, as we in one aspect of the invention generate E2A-deleted recombinant adenovirus, we will not have the problem of reversion in our adenoviruses.

In vet another aspect of the invention as a further improvement the use of non-human cell lines as packaging cell .lines is disclosed.

For GMP production cf clinical batches of recombinant viruses it is desirable =z use a ceil line that has been Sused widely for production of other biotechnology products. Most of the latter cell lines are from monkey origin, which have been used to produce e.g. vaccines.

These cells can not be used directly for the production of recombinant human adenovirus, as human adenovirus can not or only to low levels replicate in cells of monkey origin.

A block in the switch of early to late phase of adenovirus lytic cycle is underlying defective replication. However, host range mutations in the human adenovirus genome are described (hr400 404) which allow replication of human viruses in monkey cells. These mutations reside in the gene encoding E2A protein (Klessig and Grodzicker, 1979; Klessig et al., 1984; Rice and Klessig, 1985)(Klessig et al., 1984). Moreover, mutant viruses have been described that harbor both the hr and temperature-sensitive ts125 phenotype (Brough et al., 1985; Rice and Klessig, 1985).

We therefore generate packaging cell lines of monkey origin VERO. CVi) that harbor: a. El sequences, to allow replication of E1/E2 defective adenoviruses, and b. E2A sequences, containing the hr mutation and the ts 125 mutation, named ts400 (Brough et al., 1985: Rice and Klessig, 1985) to prevent cell death by E2A overexpression, and/or c. E2A sequences, just containing the hr mutation, under the control of an inducible promoter, and/or d. E2A sequences, containing the hr mutation and the ts 125 muration (ts400), under the control of an inducible promoter Furthermore we disclose the construction of novel and improved combinations of (novel and improved) packaging cell lines and (novel and improved) recombinant adenovirus vectors. We orovide: .a novel packaging cell line derived from diploid human embryonic retinoblasts (HER) that harbors n. 80 5788 of the Ad5 senome.This cell line, named 911, deposited under no 95062101 at the ECACC, has many characteristics that make it superior to the commonly used 293 cells (Fallaux et al., 1996).

2. novel packaging cell lines that express just E1A genes and not E1B genes.

Established cell lines (and not human diploid cells of which 293 and 911 cells are derived) are able to express ELA to high levels without undergoing apoptotic cell death, as occurs in human diploid cells that express EIA in the absence of E1B.

Such cell lines are able to trans-complement E1Bdefeczve recombinant adenoviruses, because viruses mutated for E1B 21 kD protein are able to complete viral 'replication even faster than wild-type adenoviruses (Telling et al., 1994). The constructs are described in detail below, and graphically represented in Figures 1-5. The constructs are transfecred into the different established cell lines and are selected for high expression of EIA. This is done by operatively linking a selectable marker gene NEO gene) directly to the E1B promoter. The E1B promoter is transcriptionally activated by the E1A gene product and therefore resistance to the selective agent G418 in the case NEO is used as the selection marker) results in direct selection for desirec expression of tne E1A gene 3 Packagina constructs that are mutated or deleted for

E

1B 2 1 kD, but just express the 55 kD protein.

4. Packaging constructs to be used for generation of compiementing packaging ceil lines from diploid cells (not exclusively of human origin) without the need of selectrin with marker genes. These cells are immortalized by expression of EIA. However, in this particular case expression of E13 is essential to orevent apoptosis induced by E1A proteins.

Selection of El expressing cells is achieved by selecEtin for focus formation (immortalization), as described for 293 cells (Graham e 1977) and 911 cels (Fallaux et al, 1996), that are El-transformed human embryonic kidney (HEK) cells and h-man embryonic retinoblasts (HER), respectively.

After transfection of HER cells with construct pIG.E1B (Fig. seven independent cell lines cculd be established. These cell lines were desicnated PER.C1, PER.C3, PER.C4, PER.C5, PER.C6, PER.C8 and PER.C9. PER denotes PGK-El-Retinoblasts. These cell lines express E1A and E1B proteins, are stable PER.C6 for more than 57 passages) and complement El defective adenovirus vectors. Yields of recombinant adenovirus obtained on PER cells are a little higher than obtained on 293 cells. One of these cell lines (PER.C6) has been deposited at the ECACC ander number 96022940.

6. New adenovirus vectors with extended El deletions (deletion nt. 459 3510). Those viral vectors lack sequences homologous to El sequences in said packaging cell lines. These adenoviral vectors contain pIX promoter sequences and the pIX gene, as pIX (from its natural promoter sequences) can only b expressed from the vector and not by packaging cells (1.arsui et al 1986, Hoeben and Fallaux, pers.comm.; Imrler et al., 1996).

E2A expressing packaging cell lines preferably based on either E1A expressing established cell lines or E1A E1B expressing diploid cells (see under 2 E2A expression is either under the control cf an inducible promoter or the E2A tsl25 mutant is driven by either an inducible or a constitutive promoter.

S. Recombinant adenovirus vectors as described before (see 6) but carrying an additional deletion of E2A sequences.

Adenovirus packaging cells from monkey crigin that are able to trans-complement El-defective recombinant adenoviruses. They are preferably co-transfected with pIG.ElAElB and pIG.NEO, and selected for NEO resistance. Such cells expressing E1A and ELB are able to transcomplement El defective recombinant human adenoviruses, but will do so inefficiently because of a block of the synthesis of late adenovirus proteins in cells of monkey origin (Klessig and Grodzicker.

3 1979). To overcome this problem, we generate recombinant adenoviruses that harbor a host-range mutation in the E2A gene, allowing human adenoviruses to replicate in monkey cells. Such viruses are generated as described in Figure 12,-except DNA from a hr-mutant is used for homologous recombination.

Adenovirus packaging cells from monkey origin as described under 9, except that they will also be co-transfected with E2A sequences harborina the hr mutation. This allows replication of human adenoviruses lacking El and E2A (see under E2A in these cell lines is either under the control of an inducible promoter or the tsE2A mutant is used. In the latter case, the E2A gene will thus carry both the ts mutation and the hr mutation (derived from ts400).

Replication competent human adenoviruses have been described that harbor both mutatibns (Brough et al., 1985; Rice and Klessig, 1985).

A further aspect of the invention provides otherwise improved adenovirus rectors, as well as novel stratecies ror generation and application of such vectors and a method for the intracellular amplification of linear DNA fragments in mammalian cells.

The so-called "minimal" adenovirus vectors according 3C to the present invention retain at least a portion of the -iral genome that is required for encapsidation of the genome into virus particles (the encapsidation signal:,. as well as at least one copy of at least a functional oart or a derivative of the Inverted Terminal Repeat !ITR), rhat 3 _is DNA sequences derived from the termini of the linear adenovirus genome. The vectors according to the present invention will also contain a transgene linked to a promoter sequence to govern expression of the transgene.

Packaging of the so-called minimal adenovirus vector can be achieved by co-infection with a helper virus or, alternatively, with a packaging deficient replicating helper system as described below.

Adenovirus-derived DNA fragments that can replicate in suitable cell lines and that may serve as a packaging deficient replicating helper system are generated as follows. These DNA fragments retain at least a portion of the transcribed region of the "late" transcription unit of the adenovirus genome and carry deletions in at least a portion of the El region and deletions in at least a portion of the encapsidation sianal. In addition, these DNA fragments con-ain at least one copy of an inverted terminal repeat (ITR). At one terminus of the transfected DNA molecule an ITR is located. The other end may contain an ITR, or alternatively, a DNA sequence that is complementary to a portion of the same strand of the DNA molecule other than the ITR. If, in the latter case, the two complementary sequences anneal, the free 3'-hydroxyl group of the 3' terminal nucleotide of the hairpinstructure can serve as a primer for DNA synthesis by cellular and/or adenovirus-encoded DNA polymerases, resulting in conversion into a double-stranded form of at least a portion cf the DNA molecule. Further reolication initiating at the ITR will result in a linear doublestranded DNA molecule, that is flanked by two ITR's, and is larger than the original transfected DNA molecule (see Fig: 13). This molecule can replicate itself in the transfected cell by virtue or the adenovirus proteins encoded by the DNA molecule and the adenoviral and cellular proteins encoded by genes in the host-cell genome. This DNA molecule can not be encapsidaied due to its large size (greater than 39000 base pairs; or due to the absence of a functional encapsidation signal. This DNA molecule is intended to serve as a helper for the production of defective adenovirus vectors in suitable cell lines.

The invention also comprises a method for the amplification of linear DNA fragments of variable size in suitable mammalian cells. These DNA fragments contain at least one copy of the ITR at one of the termini of the fragment. The other end may contain an ITR, or alternatively, a DNA sequence that is complementary to a portion of the same strand of the DNA molecule other than the ITR. in the latter case, the t-.o complementary sequences anneal, the free 3'-hydroxyi group of the 3' terminal nucleotide of the hairpin-structure can serve as a primer :or DNA synthesis by cellular and/or adenovirus-encoded DNA poiymerases, resultina in conversion cf the displaced stand into a double stranded form of at least a portion of the DNA moiecule. Further replication initiating at the ITR will result in a linear double-stranded DNA molecule, that is flanked by two ITR's, which is larger than the original transfected

DNA

molecule. A DNA molecule that contains ITR sequences at both ends can replicate itself in transfected cells by virtue of :he presence of at least the adenovirus E2 proteins the DNA-binding protein ;DBP), the adenovirus DNA polymerase (Ad-pol), and the preterminal protein The required proteins may be e-cressed from adenovirus genes on the DNA molecule itself, from adenovirus E2 genes integrated in the host-cell genome, or from a repicating helper fragment as described above.

Several groups have shown that the presence of ITR sequences a- the end of DNA molecules are sufficient to generate adenovirus minichromosomes that can replicate, if the adenc-:-rus-proteins required for replication are provided :rans e.g. by infection w~i, a heiper--irus (Hu et al., 1992;: (Wang and Pearson, 1985 (Hay en ai., 1984). Hu ea al., (1992) observed the cresence and replicaticn of symmetrical adenovirus -inichromosomedimers after :ransfection of plasmids containing a single ITR. The authors were able to demonstrate that these dimeric minichromosomes arize after tail-to-rail ligation of the single ITR DNA molecules. In DNA extracted from defective adenovirus type 2 particles, dimeric molecules of various sizes have also been observed usina electronmicroscopy (Daniell, 1976). It was suggested that the incomplete genomes were formed by illegitimate reccmbination between different molecules and that variations in the position of the sequence at which the illegitimate base pairing occurred were resonsible for the heterogeneous nature of the incomplete genomes. Based on this mechanism it was speculated that, in theory, defective molecules with.a total length of uc to two times the normal genome could be generated. Such molecules couic contain duplicated sequences from either end zf the genome. However, no DNA molecules larger than the fulllength virus were found packaged in the defective parricles (Daniell, 1976). This can be explained by the size-limitations that apply to the packaging. In addition, it was observed that in the virus particles DNA-molecules with a duplicated left-end predominated over those containing the right-end terminus (Daniel,. 1976). This is fully explained by the presence of the encapsidation signal near that left-end of the genome (Grable and Hearing, 1990; Grhble and Hearing, 1992: Hearinc et al..

198-; The major problems associated with the current adencvirus-derived vectors are: A) The strong immunogenicit of the virus particle 3) The expression of adenovirus genes that reside in the adenoviral vectors, resulting in a C-totoxic T-celrespcnse against the transduced cells.

2) The low amount of heerologous sequences that can be accommodated in the current vectors (Up to maximally approx. 8000 bp. of heteroloaous DNA).

Ad A) The strong immunogenicity of the adenovirus particle results in an immunological response of the host, even after a single administration of the adenovirai vector. As a result of the development of neutralizing 3 antibodies, a subsequent administration of the virus will be less effective or even completely ineffective. However, a prolonged or persistent expression of the transferred genes will reduce the number of administrations required and may bypass the problem.

Ad B) Experiments performed by Wilson and collaborators have demonstrated that after adenovirusmediated gene transfer into immunocompetent animals, the expression of the transgene gradually decreases and disappears approximately 1 4 weeks post-infection (Yana Set al., 1994a; Yang et al., 1994b). This is caused bv the development of a Cytotoxic T-Cell (CTL) response against the transduced cells. The CTLs were directed against adenovirus proteins expressed by the viral vectors. In the transduced cells synthesis of the adenovirus DNA-binding.

protein (the E2A-gene product), penton and fiber proteins (late-gene products) could be established. These adenovirus proteins, encoded by the viral vector, were expressed despite deletion of the El region. This demonstrates that deletion of the El region is not Ssuificient to completely prevent expression of the viral genes (Engelhardt et al., 1994a).

Ad C) Studies by Graham and collaborators have cemonstrated that adenoviruses are capable of encapsidating DNA of up to 105% of the normal genome size ;Bett et al., 1993). Larger genomes tend to be instable resulting in loss of DNA sequences during propagation of the virus. Combining deletions in the El and E3 regions of tne virual genomes increases the maximmum size of the foreign that can be encapsidated to approx. 8.3 kb. In Saddition, some sequences of the E4 region appear to be dispensable for virus growth (adding another 1.8 kb to the maximum encapsidation capacity). Also the E2A region can be deleted from the vector, when the E2A gene product is provided in trans in the encapsidation cell line, adding another 1.6 kb. It is, however, unlikely that the maximum capacity of foreign DNA can be significantly increased further than 12 kb.

We developed a new strategy for the generation and production of helperfree-stocks of recombinant adenovirus vectors that can accomodate up to 38 kb of foreign DNA.

Only two functional ITR sequences, and sequences that can function as an encapsidation signal need to be part of the vector genome. Such vectors are called minimal adenovectors. The helper functions for the minimal adenovectors are provided in trans by encapsidation defective-replication competent DNA molecules that contain all the viral genes encoding the required gene products, with the exception of those genes that are present in the host-cell genome, or genes that reside in the vector genome.

The applications of the disclosed inventions are outlined below and will be illustrated in the experimental part, which is only intended for said purpose, and should not be used to reduce the scope of the present invention as understood by the person skilled in the art.

Use of the IG packaging constructs Diploid cells.

The constructs, in particular pIG.ElA.ElB, will be used to transfecr diploid human cells, such as Human Embryonic Retinoblass (HER), Human Embryonic Kidney cells (HEK;, and Human Embryonic Lung cells (HEL). Transfected cells will be selected for transformed phenotype (focus formation) and tested for their ability to support propagation of El-deleted recombinant adenovirus, such as IG.Ad.MLPI.TK. Such cell lines will be used for the generation and (large-scale) production of El-deleted recombinant adenoviruses. Such cells, infected with recombinant adenovirus are also intended to be used in vivo as a local producer of recombinant adenovirus, e.g. for the treatment of solid tumors.

911 cells are used for the titration, generation and production of recombinant adenovirus vectors (Fallaux et al., 1996).

HER cells transfected with pIG.EIA.ElB has resulted in 7 independent clones (called PER cells). These clones are used for the production of El deleted (including nonoverlapping adenovirus vectors) or El defective recombinant adenovirus vectors and provide the basis for introduction of e.g. E2B or E2A constructs tsl25E2A, see below), E4 etc., that will allow propagation of adenovirus vectors that have mutations in e.a. E2A or E4.

In addition, diploid cells of other species that are permissive fur human adenovirus, such as the cotton rat (Siamodon .ispidus) (Pacini et al., 1964), Syrian hamster (Morin et al., 1987) or chimpanzee. (Levrero er al., 1991), will be immortalized with these constructs. Such cells, infected with recombinant adenovirus, are also intended to be used in vivo for the local production of recombinant adenovirus, e.g. for the treatment of solid tumors.

Established cells.

The constructs, in particular pIG.1A.NEO; can be used to transfecr established cells, e.g. A549 (human bronchial carcinoma), KB (oral carcinoma), MRC-5 (human diploid lung cell line) or GLC cell lines (small cell lung cancer) (de Leij et al., 1985; Postmus en al.. 1988) and selected for NEO resistance. Individual colonies of resistant cells are isolated and tested for their caoaci-,to support propagation of El-deleted recombinant adenovirus, such as IG.Ad.MLPI.TK. When propagation of El deleted viruses on E1A containing cells is possible, such cells can be used for the generation and production of El-deleted recombinant adenovirus. The: are also be used for the propagation of ElA deleted/ElB retained recombinant adenovirus.

Established cells can also be co-transfected with pIG.ElA.ElB and pIG.NEO (or another NEO containing expression vector). Clones resistant to G418 are tested for their ability no support propagation of El deleted recombinant adenovirus, such as IG.Ad.MLPI.: and used for the generation and production of El deleted recombinant adenovirus and will be applied in vivo for Iccal production of recombinant virus, as described for the diploid cells (see above).

All cell lines, including transformed diploid cell lines or NEO-resistant established lines, can be used as the basis for the aeneration of 'next aenerazzon' packaging cells lines, that support propagatl:n of El-defective recombinant adenoviruses, that also carry deletions in other genes, such as E2A and E4. Moreover, they will provide the basis for the generation of minimal adenovirus vectors as disclosed herein.

E2 expressing cell lines Packaging cells expressing E2A sequences are and will be used for the generation and 'large scale) :roduction of E2A-deleted recombinant adenovirus.

The newly generated human adenovirus packaging cell lines or cell lines derived from species permissive for human adenovirus (E2A or tsl25E2A: E1A E2A; ElA ElB E2A; ElA E2A/tsl25; ElA E1B E2A/ts125) or nonpermissive cell lines such as monkey cells (hrE2A or hr tsl:5E2A; E1A hrE2A; ElA EiS hrE2A; E1A hrE2A/tsl25; ElA ElB hrE2A/ts125) are and will be used ror e generation and (large scale) production of E2A delernd recombinant adenovirus vectors. In addition, thev will be applied in vivo for local production of recc=oinant virus, as described for the diploid cells (see above.

Novel adenovirus vectors.

The newly developed adenovirus vectors harborin- an El deletion of nt. 459-3510 will be used for gene transfer purposes. These vectors are also the basis for the development of further deleted adenovirus vectors thar are mutated for e.g. E2A, E2B or E4. Such vectors will -e generated e.g. on the newly developed packaging cell lines described above (see 1-3] Minimal adenovirus packaging system We disclose adenovirus packaging constructs (to be used for the packaging of minimal adenovirus -ectors: may 1 nave the following cnaracteristics: a. the packaging construct replicates b. the packaging construct can not be packaged because the packaging signal is deleted c. the packaging construct contains an internal hairpin- 2C forming sequence (see section 'Experimental: suggested hairpin' see Fig. d. because of the internal hairpin structure, the packaging construct is duplicated, that ,s the DNA of the packaging construct becomes twice as Icna as "2 it was before transfection into the packagina tell (in our sample -i duplicates from 35 kb to 70 kb) This duplication also prevents packaging. Note that this duplicated DNA molecule has ITR's at both termini (see e.g. Fig. 13) e. this duplicated packaging molecule is able to replicate like a 'normal adenovirus' DNA molecule f. the duplication of the genome is a prerecuisiz for the production of sufficient levels of adeno--_rus proteins, required to package the minimal adencvirus vector g. the packaging construct has no overlapping sequences with the minimal vector or cellular sequences that may lead to generation of RCA by homologous recombination.

This packaging system will be used to produce minimal adenovirus vectors. The advantages of minimal adenovirus vectors e.g. for gene therapy of vaccination purposes, are well known (accommodation of up to 38 kb, gutting of all potentially toxic and immunogenic adenovirus genes) Adenovirus vectors containing mutations in essential genes (including minimal adenovirus vectors) can also be propagated using this system.

Use of intracellular E2 expressing vectors.

Minimal adenovirus vectors are generated using the helper functions provided in trans by packaging-deficient replicating helper molecules. The adenovirus-derived ITR sequences serve as origins of DNA replication in the presence of at least the E2-gene products. When the E2 gene products are expressed from genes in the vector genome the gene(s) must be driven by an Elindependent promoter the vector genome can replicate in the targer cells. This will allow an significantiv increased number of template molecules in the targer cells, and, as a result an increased expression of the genes of interest encoded by the vector. This is of particular interest for approaches of gene therapy in cancer.

Applications of intracellular amplification of linear DNA fragments.

A similar aoproach could also be taken if amplification of linear DNA fragments is desired. DNA fragments of known cr unknown sequence could be amplified in cells containing the E2-gene products if at least one ITR sequence is located near or at its terminus. There are no apparent constraints on the size of the fragment. Even fragments much larger than the adenovirus genome (36 kb) should be amplified using this approach. It is thus possible to clone large fragments in mammalian cells without either shuttling the fragment into bacteria (such as E.coli) or use the polymerase chain reaction At the end stage of an productive adenovirus infection a single cell can contain over 100,000 copies of the viral genome. In the optimal situation, the linear DNA fragments can be amplified to similar levels. Thus, one should be able to extract more than 5 gg of DNA fragment per million cells (for a 35-kbp fragment;. This system can be used to express heterologous proteins equivalent to the Simian Virus 40-based COS-cell system) for research or for therapeutic purposes. In addition, the system can be used to identify genes in large fragments of DNA. Random DNA fragments may be amplified (after addition of ITRs) and expressed during intracellular amplification. Election or,selection of those cells with the desired phenotype can be used to enrich the fragment of interest and to isolate the gene.

EXPERIMENTAL

Generation of cell lines able to transcomplement El defective recombinant adenovirus vectors.

1. 911 cell line We have aenerated a cell line that harbors El sequences cf adenovirus type 5, able t3 trans-complement El deleted recombinant adenovirus (Fallaux et al., 1996) This cell line was obtained by transfection of human diploid human embryonic retinoblasts (HER) with pAdSXhoIC.

that contains nt. 80 5788 of Ad5; one of the resulting iransformants was designated 911. This cell line has been shown to be very useful in the propagation of El defective recombinant adenovirus. It was found to be superior to the 293 cells. Unlike 293 cells, 911 cells lack a fully transformed phenotype, which most likely is the cause of performing better as adenovirus packaging line: plaque assays can be performed faster (4 5 days instead of 8-14 days on 293) monolayers of 911 cells survive better under agar overlay as required for plaque assays higher amplification of El-deleted vectors In addition, unlike 293 cells that were :ransfected with sheared adenoviral DNA, 911 cells were cransfected usinc a defined construct. Transfection efficiencies of 911 cells are comparable to those of 293.

New packaging constructs.

Source of adenovirus sequences.

Adenovirus sequences are derived either from pAd5.SalB, containing nt. 80 9460 of human adenovirus type 5 (Bernards et al., 1983) or from wiid-t-:pe Ad5 DNA.

pAdS.SalB was digested with Sail and Xhco and the large fragment was religated and this new clone was named The oTN construct (constructed by Dr. Vogels, IntrcGene. The Netherlands) was used as a source for the human PGK promoter and the NEO gene.

Human PGK promoter and NEO R gene.

Transcription of E1A sequences in the new packaging constructs is driven by the human PGK promoter (Michelson et 1983; Singer-Sam et al., 1984), derived from plasmid pTN (gift of R. Vogels), which uses ?UC119 (Vieira and Messing, 1987) as a backbone. This plasmid was also used as a source for NEO gene fused to the Hepatitis B Virus (HBV) poly-adenylation signal.

Fusion of PGK promoter to El genes (Fig. 1) In order to replace the El sequences of Ad5

TR,

origin of replication and packaging signal) bn heterologous sequences we have amplified El sequences (nt.459 to nt. 960) of Ad5 by PCR, using primers Eal and Ea2 (see Table The resulting PCR product was digested with Clal and ligated into Bluescript (Stratagene predigested with Clal and EcoRV, resulting in construct pBS.PCRI.

Vector pTN was digested with restriction enz-mes EcoRI (partially) and Scal, and the DNA fragment containina the PGK promoter sequences was liated into PBS.PCRI digested with Scal and EcoRI. The resulting Ii construct PBS.PGK.PCRI contains the human PGK promoter operatively linked to Ad5 1E sequences from n.459 to nr.

916.

Construction of pIG.E1A.E1B.X (Fig. 2) PIG.ElA.ElB.X was made by replacing the ScaI-BspEI :ragment of pAT-X/S by the corresponding fragment from 3 BS-PGK.PCRI (containing the PGK promoter linked to E1A sequences).

PIG.EIAElB.X contains the E1A and E1B codino sequences under the direction of the PGK promoter.

As Ad5 sequences from nt.459 to nt. 5788 are present in this construct, also pIX protein of adenovirus is encoded by this piasmid.

Construction of pIG.ElA.NEO (Fig. 3) In order to introduce :he complete EIB promoter and c^ ruse this promoter in such a way that the AUG codon of SE1B 21 kD exact!-. functions as the AUG codon of NEOR, we amolified the EIB promoter using primers Ea3 and Eo2, wnere primer Ep2 introduces an Ncol site in the PCR fragment. The resulting PCR fragment, named PCRII, was digested with HpaI and NcoI and ligated into pAT-X/S, which was predigested with HpaI and with NcoI. The resulting plasmid was designated pAT-X/S-PCR2. The NcoI StuI fragment of pTN, containing the NEO gene and part of the Hepatitis B Virus (HBV) poly-adenylation signal, was cloned into pAT-X/S-PCR2 (digested with Ncol and Nrul).

The resulting construct: pAT-PCR2-NEO. The polyadenylation signal was completed by replacing the Scal- Sall fragment of pAT-PCR2-NEO by the corresponding fragment of pTN (resulting in pAT.PCR2.NEO.p(A)). The Scal XbaI of pAT.PCR2.NEO.p was replaced by the corresponding fragment of pIG.E1A.EB-X, containing the PGK promoter linked to E1A genes.

The resulting construct was named pIG.ElA.NEO, and thus contains Ad5 El sequences (nt.459 to nt 1713) under the control of the human PGK promoter.

Construction of pIG.EIA.EIB (Fig. 4) pIG.E1A.E!B was made by amplifying the sequences encoding the N-terminal amino acids of E1B 55kd using primers Ebl and Eb2 (introduces a XhoI site). The resulting PCR fragment was digested with BglII and cloned into BlII/Nrul of pAT-X/S, thereby obtaining DAT-PCR3.

pIG.ElA.ElB was constructed by introducing the HBV poly(A) sequences of pIG.ElA.NEO downstream of E1B sequences of pAT-PCR3 by exchange of XbaI Sall fragment of pIg.E1A.NEO and the XbaI XhoI fragment of pAT.PCR3.

pIG.EIA.E13 contains nt, 459 to nt. 3510 of Ad5, that encode the E1A and E1B proteins. The E1B sequences are terminated at the splice acceptor at nt.3511. No pIX sequences are present in this construct.

Construction of pIG.NEO (Fig. pIG.NEO was generated by cloning the HpaI Scal fragment of plG.E1A.NEO, containing the NEO gene under the control of the Ad.5 E1B promoter, into pBS digested with EcoRV and ScaI.

This construct is of use when established cells are transfected with E1A.ElB constructs and NEO selection is required. Because NEO expression is directed by the E1B promoter, NEO resistant cells are expected to co-express E1A, which also is advantageous for maintaining high levels of expression of E1A durina long-term culture of the cells.

13 Testing of constructs.

The intearity of the constructs pIG.I!A.NEO, pIG.E1A.E1B.X and pIG.E1A.ElB was assessed by restriction enzyme mapping; furthermore, parts of the constructs that were obtained by PCR analysis were confirmed by sequence analysis. No changes in the nucleotide sequence were found.

The constructs were transfected into primary

BRK

(Baby Rat Kidney) cells and tested for their ability to immortali'e (zIG.ElA.NEO) or fully transform G.ElA.E1B.X and pIG.EIA.EiB) these cells.

Kidneys of 6-day old WAG-Rij rats were isolated, homogenized and trypsinized. Subconfluent dishes (diameter cm) of the BRK cell cultures were transfected with 1 or 5 Ag of DIG.NEO, pIG.EIA.NEO, pIG.EIA. B. pIG.E1A.E1B.X, or with pIG.ElA.NEO together with PDC26 (Van der Elsen et al., 1983), carrying the AdS.ElB aene under control of the SV40 early promoter. Three weeks post-transrec-ion, when foci were 7isibie, the dishes were fixed, Giemsa stained and the foci counted.

An over-.-'iew of the generated adenovirus packaging constructs, and their ability to transform BRK, is presented in Fig. 6. The results indicate that the constructs pIG.ElA.ElB and pIG.EIA.E1B.X are able to transform BRK cells in a dose-dependent manner. The efficiency of transformation is similar fcr both constructs and is comparable to what was found with the construct that was used to make 911 cells, namely As expected, pIG.EA.NEO was hardly able to immortalize BRK. However, co-transfectioo of an E1B expression construct (PDC26) did result in a significant increase of the number of transformants (IS versus 1), indicatinu that E1A encoded by pIG.lIA.NEO is functional.

We conclude therefore, that the newiv aenerated packaging constructs are suited for the generation of new adenc--irus packaging lines.

Generation of cell lines with new packaging constructs Cell lines and cell culture Human A549 bronchial carcinoma cells 'Shapiro et ai., 1978). human embryonic retinoblasts (HER), transfor-med human embryonic kidney (HEK) cells (293; Graham er al., 1977) cells and AdS-iransfo~red HER cells (911: Fallaux et ai, 1996)) and PER cells .ere grown in Dulbecco's Modified Eagle Medium :DMEM) suzpcemented with Fetal Calf Serum (FCS) and antibiotics _n a 5% C02 atmosphere at 370C. Cell culture media, reaaents and sera were purchased from Gibco Laboratories (Grand Island, NY) Culture plastics were purchased from Greiner (Nurtingen, Germany) and Corning (Corning, NY' Viruses and virus techniques The construction of adenoviral vectors :G.d..nlsac GAd.MLP.s.acZ IGAdMLP.uc. IG.Ad.ML?.TK and IG.Ad.CMV.TK is described in detail in patent application EP 95202213.

The recombinant adenoviral vector IG.Ad.MLP.nlslacZ contains the E.coli lacZ gene, encoding B-galactosidase, under control of the Ad2 major late promoter (MLP).IG.Ad.MLP.luc contains the firefly luciferase gene driven by the Ad2 MLP. Adenoviral vectors IG.Ad.MLP.TK and IG.Ad.CMV.TK contain the Herpes Simplex Virus thvmidine kinase (TK) gene under the control of the Ad2 MLP and the Cytomegalovirus (CMV) enhancer/promoter, respectively.

Transfections All transfections were performed by calcium-phosphate precipitation DNA (Graham and Van der Eb, 1973) with the GIBCO Calcium Phospnaze Transfecrion System (GIECO 3RL Life Technologies Inc., Gaithersburg, USA), according to the manufacturers protocol.

Western blotting Subconfluent cultures of exponentially growina 293,911 and Ad5-El-transformed A549 and PER cells were washed with PBS and scraped in Fos-RIPA buffer (10 mM Tris 'pH 150 mM NaCI. 1% NP40,01% sodium dodec-! sulphate 'SDS), 1% NA-DOC, 0,5 mM phenvl methyl sulphon-l fluoride :PMSF), 0,5 mM trypsin inhibitor, 50 mM NaF and I mM sodium vanadate). After 10 min. at room temperature, Iysates were cleared by centrifugation. Protein concentrations were measured with the Biorad protein assay kit, and 25 4g total cellular protein was loaded on a 12.5% SDS-PAA gel. After electrophoresis, proteins were transferred to nitrocellulose (lh at 300 mA). Prestained standards (Sigma, USA) were run in parallel. Filters were blocked with 1% bovine serum albumin (BSA) in TBST (10 mM Tris, pH 8, 15 mM NaC1, and 0.05% Tween-20) for 1 hour.

First antibodies were the mouse monoclonal antibody A1C6 (Zantema et ai. unpublished), the rat monoclonal anti-Ad5-E1B-221-kDa antibody C1G11

I

(Zantema et al., 1985). The second antibody was a horseradish peroxidase-labeled goat anti-mouse antibody (Promega). Signals were visualized by enhanced chemoluminescence (Amersham Corp, UK).

Southern blot analysis High molecular weight DNA was isolated and 10 Mg was digested to completion and fractionated on a 0.7% agarose gel. Southern blot transfer to Hybond N+ (Amersham, UK) was performed with a 0.4 M NAOH, 0.6 M NaC1 transfer solution (Church and Gilbert, 1984). Hybridization was performed with a 2463-nt SspI-HindIII fragment from (Bernards et al., 19831. This fragment consists of Ad5 bp. 342-2805. The fragment was radiolabeled with a-2P-dCTP with the use of random hexanucleotide primers and Klenow DNA polymerase. The southern blots were exposed to a Kodak XAR-5 .film at -80°C and to a Phospho-Imager screen which was analyzed by B&L systems Molecular Dynamics software.

A549 A549 human bronchial carcinoma cell lines were generated by transfecnion with pIG.ElA.NEO and selection for G418 resistance. Thirty-one G418 resistant clones were established. Co-transfection of pIG.ElA.ElB with pIG.NEO yielded seven G418 resistant cell lines.

PER

human embryonic retina (HER) cells were generated by transfection of primery HER cells with piasmid pIG.ElA.ElB. Transformed cell lines were established from well-separated foci. We were able to

I

establish seven clonal cell lines, which we called PER.C1, PER.C3, PER.C4, PER.C5, PER.C6, PER.CS and PER.C9.

One of the PER clones, namely PER.C6, has been deposited at the ECACC under number 96022940.

Expression of Ad5 EIA and E1B genes in transformed A549 and PER cells Expression of the Ad5 E1A and the 55-kDa and 21 kDa EIB proteins in the established A549 and PER cells was studied by means of Western blotting, with the use of monoclonal antibodies (mAb). Mab M73 recognizes the E1A products, whereas. Mabls AIC6 and CIGII are directed against the 55-kDa and 21 kDa E1B proteins, respecrively.

The antibodies did not recognize proteins in extracts from the parental A549 or the primary HER cells (data not shown). None of the A549 clones that were generated by co-transfection of pIG.NEO and pIG.EIA.EIB expressed detectable levels of ElA or E1B proteins (not shown). Some of the A549 clones that were generated by cransfection with pIG.EIA.NEO expressed the AdS E1A proteins (Fig. 7), but the levels were much lower than those detected in protein iysares from 293 cells. The steady state E1A levels detected in protein extracts from PER cells were much higher zhan those detected in extracts from A549derived cells. All PER cell lines expressed similar levels of E1A proteins (Fig. The expression of the ElB proteins, parzicularly in the case of ElB 55 kDa, was mote variable. ompared to 911 and 293, the majority of the PER clones express high levels of EIB 55 kDa and 21 kDa. The steadv sar7e level of E1B 21 kDa was the highest in PER.C3. None of the PER clones lost expression of the El genes zpon serial passage of the cells (not shown) ie found -Iat the level of El expression in PER cells remained stable for at least 100 population doublings.

We decided t= characterize the PER clones in more detail Southern analysis of PER clones To study the arrangement of the AdS-El encoding sequences in the PER clones we performed Southern analyses. Cellular DNA was extracted from all PER clones, and from 293 and 911 cells. The DNA was diaested with HindII, which cuts once in the Ad5 El regicn. Southern hybridization on HindIII-digested DNA, usinc a radiolabeled Ad5-El-specific probe revealed the presence of several integrated copies of pIG.EIA.E1B in the genome of the PER clones. Figure 8 shows the distribution pattern of El sequences in the high molecular weight DNA of the different PER cell lines. The copies are concentrated in a single band, which suggests that they are in-egrated as tande.m repeats. In the case of PER.C3, C5, Z5 and C9 we found additional hybridizing bands of low molecular weight that indicate the presence of truncated copies of pIG.E1A.E1B. The number of copies was determined with the use of a Phospho-Imager. We estimated that ?ER.C1, C3, C4, C5, C6. C8 and C9 contain 2, 88. 5.4, 5, 5 and 3 copies of the Ad5 El coding region, respec=ively, and that 911 and 293 cells contain I and 4 copies of the Ad5 El sequences, respectLveyv.

Transfection efficiency Recombinant adenovectors are generated by cotransfecrion of adaptor plasmids and the larce Clal fragment of Ad5 into 293 cells (gee patent apolication EP 95202213). The recombinant virus DNA is formed bv homolocous recombination between the homolocous viral sequences that are present in the plasmid and the adeno--rus DNA. The efficacy of this method, as well as that cf alternative strategies, is highly deoendent on the transfecrability of the helper cells. Therefore, we compared the transfection efficiencies of some of the PER clones with 911 cells, using the E.coli 0-galactosidase-encoding lacZ gene as a reporter (Fig. 9).

Production of recombinant adenovirus Yields of recombinant adenovirus obtained after inoculation of 293, 911, PER.C3, PER.C5 and PER.C6 with different adenovirus vectors are presented in Table II.

The results indicate that the yields obtained on PER cells are at least as high as those obtained on the existing cell lines.

In addition, the yields of the novel adenovirus vector IG.Ad.MLPI.TK are similar or higher than the yields obtained for the other viral vectors on all cell lines tested.

Generation of new adenovirus vectors (Fig. The used recombinant adenovirus vectors (see patent application on EP 95202213) are deleted for El sequences from 459 to nt. 3328.

As construct pElA.E1B contains Ad5 sequences 459 Lo nt. 3510 there is a sequence overlap of 183 nr. between E1B sequences in the. ackaging construct pIG.ElA.ElB and recombinant adenoviruses, such as e.g. IG.Ad.MLP.TK. The overlapping sequences were deleted from the new adenovirus vectors. In addition, non-coding sequences derived from lacZ, that are present in the original contructs, were deleted as well. This was achieved (see Fig. 10) by PCR amplification of the SV40 poly(A) sequences from oMLP.TK using primers SV40-1 (intrcduces a BamHI site) and SV40-2 (introduces a BglII site). -n addition, Ad5 sequences present in this construct were amplified from nt 2496 introduces a BglII site) ro nt. 2779 (Ad5-2). Both PCR fragments were digested wi-ih BglII and were ligated.

The ligation product was PCR amplified using primers SV40-1 and Ad5-2. The PCR product obtained was cut with BamHI and AflII and was ligated into pMLP.TK predigested with the same enzymes. The resulting construct, named pMLPI.TK, contains a deletion in adenovirus El sequences from nt 459 to nt. 3510.

Packaging system The combination of the new packaging construct pIG.ElA.ElB and the recombinant adenovirus pMLPI.TK, which do not have any sequence overlap, are presented in Fig. 11. In this figure, also the original situation is presented, where the sequence overlap is indicated.

The absence of overlapping sequences between pIG.E1A.E1B and pMLFI.TK (Fig. ila) excludes the possibility of homologous recombination between packaging construct and recombinant virus, and is therefore a significant improvement for production of recombinant adenovirus as compared to the original situation.

In Fig. lib the situation is depicted for pIG.E1A.NEO and IG.Ad.MLPI.TK. pIG.EIA.NEO when transfected into established cells, is expected to be sufficient to support propagation of El-deleted recombinant adenovirus. This combination does not have any sequence overlap, preventing generation of RCA by homologous recombination. In addition, this convenient packaging system allows the propagation of recombinant adenoviruses that are deleted just for E1A sequences and not for E1B sequences.

Recombinant adenoviruses expressing E1B in the absence of E1A are attractive, as the EIB protein, in particular E1B 19kD, is able to prevent infected human cells from Ivsis by Tumor Necrosis Factor (TNF) (Gooding et al., 1991).

Generation of recombinant adenovirus derived from pMLPI.TK.

Recombinant adenovirus was generated by cotransfection of 293 cells with Sall linearized OMLPT.TK

I

DNA and ClaI linearized Ad5 wt DNA. The procedure is schematically represented in Fig. 12.

Outline of the strategy to generate packaging systems for minimal adenovirus vector Name convention of the plasmids used: P plasmid I ITR (Adenovirus Inverted Terminal Repeat) C Cytomegalovirus (CMV) Enhancer/Promoter Combination L Firefly Luciferase Coding Sequence hac,haw Potential hairpin that can be formed after digestion with restriction endonuclease Asp718 in its correct and in the.

reverse orientation, respectively (Fig. Eg. pICLhaw is a plasmid that contains the adenovirus ITR followed by the CMV-driven luciferase gene and the Asp718 hairpin in the reverse (non-functional) orientation.

1.1 Demonstration of the competence of a synthetic DNA sequence, that is capable of forming a hairpinstructure, to serve as a primer for reverse strand synthesis fcr the generation of double-stranded

DNA

molecules in cells that contain and express adenovirus genes.

Plasmids pICLhac, pICLhaw, pICLI and pICL were generated using standard techniques. The schematic representation of these plasmids is shown in Figs. 16-19.

Plasmid pICL is derived from the following plasmids: nt.1 457 pMLPO (Levrero et al., 1991) nt.458 1218 pCMVP (Clontech, EMBL Bank No. U02451; nt.1219 3016 pMLP.luc (IntroGene, unpublished) na.3017 5620 pBLCAT5 (Stein and Wheian, 1989) The plasmid has been constructed as follows: The tet gene of plasmid pMLPI0 has been inactivated by deletion of the BamHI-SalI fragment, to generate pMLP10ASB. Using primer set PCR/MLP1 and PCR/MLP3 a 210 bp fragment containing the Ad5-ITR, flanked by a synthetic Sall restriction site was amplified using pMLP10 DNA as the template. The PCR product was digested with the enzymes EcoRI and SgrAI to generate a 196 bD. fragment.

Plasmid pMLPIOASB was digested with EcoRI and SgrAI to remove the ITR. This fragment was replaced by the EcoRI- SgrAI-rreated PCR fragment to generate pMLP/SAL.

Plasmid pCMV-Luc was digested with PvuII to completion and recirculated to remove the SV40-derived pcio-adenvlation signal and Ad5 sequences with exception of the left-terminus. In the resulting piasmid, pCMV-lucAAd, the ITR was replaced by the Sal-site-flanked ITR from plasmid pMLP/SAL by exchanging the XmnI-SacII fragments.

The resulting piasmid, pCMV-lucAAd/SAL, the Ad5 left terminus and the CMV-driven luciferase gene were isolated as an Sall-Smal fragment and inserted in the Sall and Hpai digested plasmid pBLCATS, to form plasmid DICL. Plasmid pICL is represented in Fig 19; is sequence is presented in Fic. Plasmid opCL contains the following features: nt. 1-457 Ad5 left terminus (Seauence 1-457 of human adenovirus :'ope nr. 458-969 Human cytomegaiovirus enhancer and immediate earl.- promoter (Boshart et al., 1985)(from plasmid pCMVJ, Clontech, Palo Al-o, USA) nt. 97.-1204 SV40 19S exon and truncated 16/195 inrron (from plasmid pCMV|) nt. 1215-2987 Firefly iuciferase gene (from pMLP.luc) ni. 3018-3131 SV40 tandem poly-adenylation signals from late transcript, derived from plasmid nt. 3132-5620 pUC12 backbone (derived from piasmid nt. 4337-5191 0-lactamase gene (Amp-resistence gene, reverse orientation) Plasmid pICLhac and pICLhaw Plasmids plCLhac and pICLhaw were derived from plasmid pICL by digestion of the latter plasmid with the restriction enzyme Asp718. The linearized plasmid was treated with Calf-Intestine Alkaline Phosphatase zo remove the 51 phoshate groups. The partially complementary synthetic single-stranded oligonucleotide Hp/aspl en Hp/asp2 were annealed and phosphorylated on their using T 4 -polynucleotide kinase.

The phosporylared double-stranded oligomers were mixed with the dephosporylated pICL fragment and ligated. Clones containing a single copy of the synthetic oiigonucleotide inserted into the plasmid were isolated and characterized using restriction enzyme digests. Insertion of the ciigonucieotide into the AsD718 site will at one junction recreate an Asp718 recognition site, whereas at the other junction the recognitionsite will be disrupted. The crientation and the integrity of the inserted cligonucleotide was verified in selected clones by sequence analyses. A clone containing the oiigonucleotide in the correct orientation ,the Asp718 site close to the 3205 EcoRI site) was denoted pICLhac. A clone with the cligonucleotide in the reverse orientation (the Asp71S =si close to the SV40 derived poly signal) was designated -ICLhaw. Plasmids pICLhac and pICLhaw are represented in Ics. 16 and 17.

Plasmid cICLI was created from plasmid pICL bv Insertion or the SalI-SgrAI fragment from pICL, containina the Ad5-ITR into the Asp718 site of pICL. The 194 bp Sall- SgrAI fragment was isolated from pICL, and the cohesive ends were converted to blunt ends using E.coli DNA polymerase I (Klenow fragment) and dNTP's. The Asp71B cohesive ends were converted to blunt ends by treatment with mungbean nuclease. By ligation clones were generated that contain the ITR in the Asp718 site of plasmid pICL.

A clone that contained the ITR fragment in the correct orientation was designated pICLI (Fig. 18).

Generation of adenovirus Ad-CMV-hcTK. Recombinant adenovirus was constructed according to the method described in Patent application 95202213. Two components are required to generate a recombinant adenovirus. First, an adaptor-plasmid containing the left terminus of the adenovirus genome containing the ITR and the packaging signal, an expression cassette with the gene of interest, and a portion of the adenovirus genome which can be used for homologous recombination. In addition, adenovirus DNA is needed for recombination with the aforementioned adaptor plasmid. In the case of Ad-CMV-hcTK, the plasmid PCMV.TK was used as a basis. This plasmid contains nt. 1-455 of the adenovirus type 5 genome, nt. 456-1204 derived from pCMV (Clontech, the PstI-StuI fragment that contains the CMV enhancer promoter and the 16S/19S intron from Simian Virus 40), the Herpes Simplex Virus thvmidine kinase gene (described in Patent application 95202213), the SV40-derived polyadenylation signal (nt. 2533-2668 of the SV40 sequence), followed by the BglII-ScaI fragment of (nt. 3328-6092 of the Ad5 sequence). These fragments are present in a pMLPI0-derived (Levrero et al., 1991) backbone. To generate piasmid pAD-CMVhc-TK, plasmid pCMV.TK was digested with Clal (the unique ClaI-site is located just upstream of the TK open readingframe) and dephosphorylated with Calf-Intestine Alkaline Phosphate.

To generate a hairpin-structure, the synthetic oligonucleotides HP/cla2 and HP/cla2 were annealed and phopsphorylared on their 5'-OH arouos with T4polynucleotide kinase and ATP. The double-stranded oligonucleotide was ligated with the linearized vector fragment and used to transform E.coli strain "Sure".

Insetion of the oligonucleotide into the ClaI site will disrupt the Clal recognition sites. In the oligonucleotide contains a new Clal site near one of its termini. In selected clones, the orientation and the inegrity of the inserted oligonucleotide was verified by.sequence analyses. A clone containing the oligonucleotide in the correct orientation (the Clal site at the ITR side) was denoted pAd-CMV-hcTK. This plasmid was co-transfected with ClaI diaested wild-type Adenovirus-t-pe5 DNA into 911 cells. A recombinant adenovirus in which the CMV-hcTK expression cassette replaces the El sequences was isolated and propagated using standard procedures.

To study whether the hairpin can be used as a primer for reverse strand synthesis on the displaced strand after replication had started at the ITR, the plasmid pICLhac is introduced into 911 cells (human embryonic retinoblasts transformed with the adenovirus El region). The plasmid pICLhaw serves as a control, which contains the oligonucleotide pair HP/asp 1 and 2 in the reverse orientation but is further completely identical to plasmid pICLhac. Also included in these studies are plasmids pICLI and pICL. In the plasmid pICLI the hairpin is replaced by an adenovirus ITR. Plasmid pICL contains neither a hairpin nor an ITR sequence. These plasmids serve as controls to determine the efficiency of replication by virtue of the terminal-hairpin structure. To provide the viral products other than the El proteins (these are produced by the 911 cells) required for DNA replication the cultures are infected with the virus IG.Ad.MLPI.TK after transfection.

Several parameters are being studied to demonstrate proper replication of the transfected DNA molecules. First, DNA extracted from the cell cultures transfected with aforementioned piasmids and infected with IG.Ad.MLPI.TK virus is being analyzed by Southern blotting for the presence of the expected replication intermediates, as well as for the presence of the duplicated cenomes.

Furthermore, from the transfected and IG.Ad.MLPI.TK infected cell populations virus is isolated, that is capable to transfer and express a luciferase marker gene into luciferase negative cells.

Plasmid-DNA of plasmids pICLhac, pICLhaw, pICLI and pICL have been digested with restriction endonuclease Sall and treated with mungbean nuclease to remove the 4 nucleotide single-stranded extension of the resulting DNA fragment. in this manner a natural adenovirus terminus on the DNA fragment is created. Subsequently, both tne plCLhac and pICLhaw plasmids were diaested with restriction endonuclease Asp718 to generate the terminus capable of forming a hairpin structure. The digested plasmids are introduced into 911 cells, using the standard calcium phosphate co-precipitation technique, four dishes for each plasmid. During the transfection, for each plasmid two of the cultures are infected with the IG.Ad.MLPI.TK virus using 5 infectious IG.Ad.MLPI.TK particles per cell. At twenty-hours post-Transfection and fort hours post-transfection one Ad.tk-virus-infected and one uninfecred culture are used to isolate small molecular-weinht DNA using the procedure devised b- Hirt.

Aliquo-s cf isolated DNA are used for Southern analysis.

After digestion of the samples with restriction endonuclease EcoRI using the luciferase gene as a probe a hybridizing fragment of approx. 2.6kb is detected only in the samples from the adenovirus infected cells transfected with plasmid pICLhac. The size of this fragment is consiszent with the anticipated duplication of the luciferase marker gene. This supports the conclusions that the inserted hairpin is capable to serve as a primer for reverse strand synthesis. The hybridizing fragment is absent if the IG.Ad.MLPI.TK virus is omitted, or if the hairpin oliaonucleotide has been inserted in the reverse orientation.

The restriction endonuciease DpnI recognizes the tetranucleotide sequence 5'-GATC-3', but cleaves only methylated DNA, (that is, only (plasmid) DNA propagated in, and derived, from E.coli, not DNA that has been replicated in mammalian cells). The restriction endonuclease MboI recognizes the same sequences, but cleaves only unmethylated DNA (viz. DNA propagated in mammalian cells). DNA samples isolated from the transfected cells are incubated with MboI and DpnI and analysed with Southern blots. These results demonstrate that only in the cells transfected with the pICLhac and the pICLI plasmids large Dpnl-resistant fragments are present, that are absent in the Mbol treated samcies.

These data demonstrate that only after transfecrion of plasmids pICLI and pICLhac replication and duplication of the fragments occur.

These data demonstrate that in -adenovirus-infected cells linear DNA fragments that have on one terminus an adenovirus-derived inverted terminal repeat (ITR; and at the other terminus a nucleonide sequence that can anneal to sequences on the same strand, when present in singlestranded form thereby generate a hairpin structure, and will be converted to structures that have inverted terminal repeat sequences on both ends. The resuitino DNA ,olecuies will replicate by the same mechanism as the wild type adenovirus genomes.

1.2 Demonstration that the DNA molecules that contain a luciferase marker gene, .a single copy of the ITR, the encapsidation signal and a synthetic DNA sequence, that is capable of forming a hairpin structure, are sufficient to generate DNA molecules that can be encapsidated into virions.

To demonstrate that the above DNA molecules zontaninin two copies of the CMV-luc marker gene can be encapsidated into virions, virus is harvested from the remaininc two cultures via three cycles of freeze-thaw crushing and is used to infect murine fibroblasts. Fortyeight hours after infection the infected cells are assayed for luciferase activity. To exclude the possibility that the luciferase activity has been induced by transfer of free DNA, rather than via virus particles, virus stocks are treated with DNaseI to remove DNA contaminants.

Furthermore, as an additional control, aliquots of the virus stocks are incubated for 60 minutes at 56 0 C. The heat treatment will not affect the contaminating DNA, but will inactivate the viruses. Significant luciferase activity is only found in the cells after infection with the virus stocks derived from IG.Ad.MLPI.TK-infected cells transfected with the pICLhc and pICLI plasmids. Neither in the non-infected cells, nor in the infected cells transfecred with the pICLhw and pICL significant luciferase activity can be demonstrated. Heat inactivation, but not DNaseI treatment, completely eliminates luciferase expression, demonstrating that adenovirus particles, and not free (contaminating) DNA fragments are responsible for transfer of the luciferase reporter gene.

These results demonstrate that these small viral genomes can be encapsidated into adenovirus particles and suggest that the ITR and the encapsidation signal are Ssufficient for encapsidation of linear DNA fraaments into adenovirus particles. These adenovirus particles can be used for efficient gene transfer. When introduced into cells that contain and express at least part of the adenovirus genes (viz. El, E2, E4, and L, and VA), recombinant DNA molecules that consist of at least one ITR, at least part of the encapsidation signal as well as a synthetic DNA sequence, that is capable of forming a hairoin structure, have the intrinsic caoacitv to autonomously generate recombinant genomes which can be encapsidated into virions. Such genomes and vector system.

can be used for cene transfer.

1.3 Demonstration that DNA molecules which contain nucleotides 3510 35953 (viz. 9.7 100 map units) of the adenovirus type 5 genome (thus lack the El protein-coding regions, the right-hand ITR and the encapsidation sequences) and a terminal DNA sequence that is complementary to a portion of the same strand of the DNA molecule when present in single-stranded form other than the ITR, and as a result is capable of forming a hairpin structure, can replicate in 911 cells.

In order to develop a replicating DNA molecule that can provide the adenovirus products required to allow the above mentioned ICLhac vector genome and alike minimal adenovectors to be encapsidated into adenovirus particles by helper cells, the Ad-CMV-hcTK adenovirai vector has been developed. Between the CMV enhancer/promoter region and the thymidine kinase gene the annealed oligonucleotide pair HP/cla 1 and 2 is inserted. The vector Ad-CMV-hcTK can be propagated and produced in 911 cell using standard procedures- This vector is grown and propagated exclusively as a source of DNA used fcr cransfection.

DNA

of the adenovirus Ad-CMV-hcTK is isolated from virus particles that had been purified using CsCI densitygradient centrifugation by standard techniques. The virus DNA has been diaested with resrrictzon endonuclease Cla.

The digested DNA is size-fractionated on an 0.7% agarose gel and the large fragment is isolated and used for further experiments. Cultures of 911 cells are transfected large Clal-fragment of the Ad-CMV-hcTK DNA using the standard calcium phosphate co-precipitation technique.

Much like in the previous experiments with olasmid pICLhac. the AD-CMV-hc will replicate starring at the right-nand ITR. Once the 1-strand is displaced, a hairpin can be formed a: the left-hand :erminus cf the fragment.

This facilitates the DNA polymerase to elongate che chain towards the right-hand-side. The process will proceed until the displaced strand is completely converted to its doubie-stranded form. Finayiv, the right-hand ITR will be

I

recreated, and in this location the normal adenovirus replication-initiation and elongation will occur. Note that the polymerase will read through the hairpin, thereby duplicating the molecule. The input DNA molecule of 33250 bp, that had on one side an adenovirus ITR sequence and at the other side a DNA sequence that had the capacity to form a hairpin structure, has now been duplicated, in a way that both ends contain an ITR sequence. The resulting DNA molecule will consist of a palindromic structure of approximately 66500 bp.

This structure can be detected in low-molecular weigh: DNA extracted from the transfected cells using Southern analysis. The palindromic nature of the DNA fragment can be demonstrated by digestion of the lowmolecular weight DNA with suitable restri=tion endonucleases and Southern blotting with the HSV-TK gene as the crobe. This molecule can replicate itself in the transr eced cells by virtue of the adenovirus gene products that are present in the cells. -n part, the adenovirus genes are expressed from remplaces that are integrated in the genome of the target cells (viz. the E1 gene products), the other genes reside in the replicating DNA fragment itself. Note however, that nis linear DNA rragmen cannot be encapsidated into virions. Not oniv does i: lack all the DNA sequences reouired for encapsidation, but also is its size much too large to be endaosilated.

1.4 Demonstration that DNA molecules which contain nuclectides 3503 35953 (viz. 9.7 100 map units) of the adenc-.-irs type 5 genome (thus lack the l1 protein-codino regions. the right-hand ITR and the encacsidation sequences) and a terminal DNA seauence that is compcementary to a portion the same strand of the DNA molecule other than the ITR. and as a result is caoabla of forminc a hairpin structure, can repiicame in 911 cells and can orovide the helper functions required to encapsi-cte the pICLI and pICLhac derived DNA fragments.

The next series of experiments aim to demonstrate that the DNA molecule described in part 1.3 could be used to encapsidate the minimal adenovectors described in part 1.1 and 1.2.

In the experiments the large fragment isolated after endonuclease ClaI-digestion of Ad-CMV-hcTK DNA is introduced into 911 cells (conform the experiments described in part 1.3) together with endonuciease Sall, mungbean nuclease, endonuclease Asp718-treated plasmid pICLhac, or as a control similarly treated plasmid pICLhaw. After 48 hours virus is isolated by freeze-thaw crushing of the transfected cell population. The viruspreparation is treated with DNaseI to remove contaminating rree DNA. The virus is used subsequently to infect Rat2 fibroblasts. Forty-eight hours post infection the cells are assayed for luciferase activity. Only in the cells infected with virus isolated from the cells transfected with the pICLhac plasmid, and not with the pICLhaw plasmid, significant luciferase activity can be demonstrated. Heatinactivarion of the virus prior to infection completely abolishes the luciferase activity, indicating that the luciferase gene is transferred by a virai particle. Infection of 911 cell with the virus stock did not result in any cytopathological effects.

demonstrating that the piCLhac is produced without anv infectious helper virus that can be propagated on 911 cells. These results demonstrate that the proposed method can be used to produce stocks of minimal-adenoviral vectors, that are completely devoid of infectious heloer ruses that are able to replicate autonomousil on adenovirus-transformed human cells or on non-adenovirus transformed human cells.

Besides the system described in this application, another approach for the generation of minimal adenovirus -ectors has been disclosed in WO 94/12649. The method described in WO 94/12649 exploits the function of the crotein IX for the packaging of minimal adenovirus vectors (Pseudo Adenoviral Vectors (PAV) in the terminology of WO 94/12649). PAVs are produced by cloning an expression plasmid with the gene of interest between the left-hand (including the sequences required for encapsidation) and the right-hand adenoviral ITRs. The PAV is propagated in the presence of a helper virus. Encapsidation of the PAV is preferred compared the helper virus because the helper virus is partially defective for packaging. (Either by virtue of mutations in the packaging signal or by virtue of its size (virus genomes greater than 37.5 kb package inefficiently). In addition, the authors propose that in the absence of the protein IX gene the PAV will be preferentially packaged. However, neither of these mechanisms appear tc be sufficiently restrictive to allow packaging of only PAVs/minimal vectors- The mutations proposed in the packaging signal diminish packaging, but do not provide an absolute block as the same packagingactivity is required to propagate the helper virus. Also neither an increase in the size of the helper virus nor the mutation of the protein IX gene will ensure that PAV is packaged exclusively. Thus, the method described in WO 94/12649 is unlikely to be useful for the production of helper-free stocks of minimal adenovirus vectors/PAVs.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

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54 Table T Primers used for PCR amplification of DNA fragments used f or generation of constructs described in this patent application.

a Ed 2 Ea 3 L a-b5 CGTGTAGTGTAT7TATACCCZ TccTCAC:GGGTCCAAAGCCA TACC'-CCCCcTAAAATGC TGOACTTCAGCcTTAAAcGC ED- 2 GCCTZ=-T-AGGSTCAcATcT Ed- I _C7TGAG:A ,CATG~fr Lb __75_=A-CTCAATCrcTATCr- 5V4 2 GCcc Jr'GAACTGTTTATTGCAC 5V4 0-S GGGAntr-7rAGACATGATAAGATAC Ad-i GG 'CTAC7AAATTTGGGC Ad-: ;GAGG;CTGACw-_CrAAccGccc ITPI CGGGGAWTZ AAArcTc.TrCCs: ITR2 GGGCAGSACATCTAATAATATAC PCR orimers sets Zo be used to c sites <aIxtanosed zo the ITR seaw PCR/MLP'- ZOCZAA7::zr:ZAcAWA PcR/MLP7: G22GAAT:CGGTACcATC_.TcAATAA: PcR/xLP2, rZTGTACACZCScCCA PCI? amolif icarion AdS nt459 PcI? amolificarion Ad5 nrtYGO< nr1284-13Q4 of Ad5 genorne nt1514-1533 of AdS genome nt1711--L702 of AdS; nZrOaucticc Of NCO! site n7z326"3289 Ad5 genome ncJSOE8-3496 af AdS genome; :ntroacuction of Xhoi site :ntroduction BamHI site (nt2162-2199 of pMLP.TK) adaption Of ecombinant a denav i ruse s introduction 301.site :ntL2312-2297 2.z DMLP.TK) -fltrocuction 3Cr1:7 site nz249E-z5z ILP.TN', zfPZULP.7- 257bt-35757o d (Int-roductio of, 0 BanH-I size) nt35935-35919 ZDr Ad! Jinzroduction Df Xbai site) reat-e the SalT and Asc7iB EnceS.

ATACW ;Adb 5 Ji3 ATAc2 (Ad5 t.10-13) ('AdS nt.200-1a4) Synthetic oligonucleotjde pair used to generate a synthetic hairpin, recreates an AspYIB site at one of the termini if inserted in Asp7lB site: HP/aspi 51 STACAcTcACCTAGTGCCGCCCGcGCAAAGCCCG.GGCGATGTA HP/asp2 5' ACCTGACCTAuTcGCCGrG~CTTTGCCCfGCGGCACAGC Synthetic £ligonucjeotjde pair used to generate a synthetic n-airpin, contains the Clal recoanirxon site to be used fzr hairpin formation.

RP/clal 5 J:ACATTACCTATGCCGCCCGGCAAACZ2GCCCTAGCAC HP/cla2 5 TCTGTGCCATC-CCGC TCC;GGC CTc;TAAT TABLE 11 Cell Passagenumber IG.Ad.CNIVjlac IG.Ad.CM )q.3 (1.40 5. X I G. A d. IN-1 1, P I.TK (11313 Pro(hicer Mean I C .A d. NI [P I (113 1 3 Prodneer NIcaii 1 1 LR.C3 17.

25.8 6. 201 ITR.CG 36 10 22 58 Yields x j 1 phi!!' 175 flask.

Table 11.

Yields oldifFerenti recoiI arit adciioviirses "I1i" Ijd aiffer itiOctlilliori of ailenlovinis 1: 1 packaging cell liures 2'JH, 9 11, PIl1R.( 3, PF R and PER.C6. The yields are thie m1eanil of two dilfer rent experiments.

IG.A ([CMV. laecZ anld AdI.CMV'I'K are rieser bed inl pajctetappjicmljiOni Il 95 20) 22 1 3 The conS1lniil of 1&;Arl.MI*PI.1'IK is descr ibed iii Illis patent application., Yields of virus per 'ISO0 Ma~sk were deterini by plue assay on, 911 cells, ais described [lallatix, 1996 Hl 149.3)

Claims (24)

1. A cell comprising in its genome nucleic acid encoding adenovirus E1A and E1B 21 kDa and E1B 55 kDa gene 00 eC products, wherein said nucleic acid does not contain C- pIX sequences and wherein said cell is not part of a pg human body.

2. A cell according to claim 1, wherein said cell is derived from a retina cell.

3. A cell according to claim 1 or 2, wherein said cell is derived from a primary cell.

4. A cell according to any one of claims 1-3, wherein said cell is derived from an embryonal cell.

A cell according to any one of claims 1-4, wherein said cell is a human cell.

6. A cell according to any one of claims 1-5, wherein said said cell is derived from a human embryonic retina (HER) cell.

7. A cell according to any one of claims 1-6, wherein said nucleic acid encoding adenovirus E1A and E1B 21 kDa and E1B 55 kDa gene products consists of nucleotides 459-3510 of the adenovirus 5 genome.

8. A cell according to claim 1, wherein said cell is a PER.C6 cell, as deposited under no. 96022940 at the P:\Oper\Vpa\VPA Prosecution\12177242 amended claims- cmcell 237.doc o -58- SECACC, or a derivative thereof. C) o

9. A cell according to any one of claims 1-8, further harbouring nucleic acid encoding an adenovirus E2A gene product. c00 (C

10.A cell according to claim 9, wherein the adenovirus M E2A gene product has a temperature sensitive 125 o mutation.

11.Use of a cell according to any one of claims 1-10 for the generation of a recombinant adenovirus comprising a gene of interest.

12. A method for producing a recombinant adenovirus comprising a gene of interest, without the concomitant generation of replication competent adenovirus through homologous recombination, said method comprising: a) providing a cell according to any one of claims 1- b) transferring recombinant nucleic acid into said cell, said recombinant nucleic acid comprising: at least one encapsidation signal, and at least one functional Inverted Terminal Repeat, said recombinant nucleic acid further comprising a gene of interest and all sequences required for replication of said recombinant nucleic acid which are not provided by said cell; said recombinant nucleic acid lacking overlapping sequences with the cellular nucleic acid, which overlap could otherwise lead to homologous recombination resulting in the formation of P:\Oper\Vpa\VPA Proseculion\l2177242 amended claims crucell 237.doc 0 -59- 0 j replication competent adenovirus; C) c) culturing said cell; and o d) harvesting recombinant adenovirus produced from said cell. 00 ci

13. A method according to claim 12 wherein said c recombinant nucleic acid is DNA. Ce o

14. A method according to claim 12 or claim 13 wherein said recombinant nucleic acid is one nucleic acid molecule in linear form and comprises functional Inverted Terminal Repeats at or near both termini.

A method according to claim 12 or claim 13, wherein said recombinant nucleic acid comprises at least two nucleic acid molecules that upon homologous recombination in said cell are capable of forming said recombinant nucleic acid.

16. A batch of recombinant adenovirus particles comprising a gene of interest, wherein said batch is obtained by a method according to any one of claims 12-15 and is free of replication competent adenovirus.

17. A recombinant nucleic acid encoding adenovirus E1A and ElB 21 kDa and E1B 55 kDa gene products, but not containing pIX sequences.

18. A recombinant nucleic acid according to claim 17, wherein said nucleic acid contains nucleotides 459- P:\Oper\Vpa\VPA Prosecution\12177242 amended claims crucell 237.doc 3510 of the human adenovirus 5 genome. 0

19. A recombinant nucleic acid according to claim 17 or claim 18, wherein the sequence encoding said ElA gene product is under control of a human PGK promoter. c ci

20. Use of a recombinant nucleic acid according to any r one of claims 17-19 for the generation of a cell Oaccording to any one of claims 1-10.

21. A cell according to any one of claims 1-10, substantially as herein described with reference to the Examples and/or Figures.

22. A use according to claim 11, substantially as herein described with reference to the Examples and/or Figures.

23. A method according to any one of claims 12-15, substantially as herein described with reference to the Examples and/or Figures.

24. A batch of recombinant adenovirus particles comprising a gene of interest, substantially as herein described with reference to the Examples and/or Figures. A recombinant nucleic acid according to any one of claims 17-19, substantially as herein described with reference to the Examples and/or Figures. P:\Oper\Vpa\VPA Prosecution\1 2177242 amended claims cmcell 237.doc 0 -61- 0 j 26. A use according to claim 20, substantially as herein described with reference to the Examples and/or SFigures. 00 ci DATED this seventh day of September 2005 Crucell Holland B.V. o DAVIES COLLISON CAVE o Patent Attorneys for the applicant ci P:\Oper\Vpa\VPA Prosecution\12177242 amended claims crucell 237.doc