AU773641B2 - The novel antisense-oligos with better stability and antisense effect - Google Patents

Description

UU429f/31 I SUBSTITUTE PAGE THE NOVEL ANTISENSE-OLIGOS WITH BETTER STABILITY AND ANTISENSE EFFECT FIELD OF THE INVENTION The present invention relates to novel covalently-closed antisense deoxynucleotides (ASoligos) containing one or more antisense sequence to mRNA region with a less secondary structure "toimprove their antisense activity and specificity and stability to target mRNA, to nuclease activities.

Particularly, the present invention relates to covalently-closed multiple antisense (CMAS)-oligos containing multiple target antisense sequences to various protooncogene mRNAs including c-myb, c-myc, or k-ras. The CMAS-oligos are constructed to form a closed type by ligation using complementary ligation primers.

In addition, the present invention relates to ribbon-type antisense (RiAS)-oligos containing multiple target antisense sequences to various protooncogene mRNAs including c-myb, c-myc, or k-ras.

25 The RiAS-oligos are constructed to form a stem-loop structure by ligation using complementary sequences at both 5 prime ends.

The present invention relates to pharmaceutical 004296731 composition containing the novel types of AS-oligos for treatment cancer, immune diseases, infectious diseases, and other human diseases caused by aberrant gene expression.

BACKGROUND

Antisense oligonucleotides (hereinafter, referred to as 'AS-oligos') have been valuable in the functional study of a gene by reducing expression of the gene in a sequence specific manner (Thompson, C.

B. et al., Nature, 314,363-366,1985). Intense efforts have also been made to develop molecular antisense agents by ablating aberrant expression of genes involved in tumor initiation and progression (Chavany, C. et al., Mol. Pharm., 48,738-746,1995).

Synthetic AS-oligos have been widely utilized for the ease of design and synthesis as well as for 20 potential specificity to genes causing diseases. ASoligos with short length (13 30 nucleotides) have been designed to bind a complementary sequences by forming Watson-Crick base paring, providing specificity and affinity. Inhibition of gene 25 expression is believed to be achieved through either RNaseH activity following formation of DNA-mRNA duplex or sterical hindrance of binding of ribosomal ooo 004296731 complex (Dolnick, B. Cancer Inv., 9,185-194, 1991). There also have been some effort to inhibit gene expression by employing triple helix formation or duplex oligo-decoy mainly aiming at or competing with the promoter region of genomic DNA (Young, S. L.

et al., Proc. Natl. Acad. Sci. USA, 88,10023-10026, 1991).

Efficacy of AS-oligos has been validated in some animal models as well as in some of recent clinical studies for human diseases. Intravenous injection of phosphorothioate (hereinafter, referred to as 'PS') AS-oligos for 10 days have eliminated virus DNA of hepatitis B from the duck liver (Offenserger, W. B.

et al., EMBO 12,1257-1262,1993). AS-oligos to angiotensin Ogen has been found effective to lower blood pressure when injected in spontaneously Shypertensive inbred rats (Tomita, N. et al., :Hypertension, 26,131-136,1995). A subcutaneous application of phosphorothioate AS-oligo against RIa 20 subunit of protein kinase A in nude mice has stopped tumor growth (Nesterova, M. et al., Nat. Med., 1, 528-533,1995). Several clinical trials using ASoligos to different genes causing various diseases are also in progress with some results in ovarian 25 cancer and Crohn's disease (Roush, Science, 276, *ee* 1192-1193,1997) o 004296731 However, high expectation for an AS-oligo taking advantages of its sequence specificity for a gene and thus potentially for a disease have frequently met with disappointments as results from many researchers have not always unambiguous and they were at times contradicting. Salient problems for an AS-oligo were instability to nucleases and inefficient cellular uptake.

Stability of an AS-oligo has been improved to a certain extent by either using modified oligos such as PS-and methylphosphonate (hereinafter, referred to as 'MP')-oligos that are utilized to augment stability against nucleases. However, each of the modified nucleotides exposed problems of its own.

They are lack of sequence specificity and insensitivity to RNaseH. Further, there is lingering apprehension for introduction of unwanted mutations upon recycling of the hydrolyzed nucleotides.

S:.i AS-oligos bind to complementary target sequences. All sequences in mRNA have not been found S* to be equally accessible to AS-oligos. Unequal binding of an AS-oligo could be explained, at least in part, by secondary and/or tertiary structures of target mRNA (Gryaznov, S. et al., Nucleic Acids Res., 25 24,1508-1514,1996). Thus, it is conceivable that a region with a less secondary structure could be o Ooooo UU4296731 targeted readily for an AS-oligo.

In an effort to enhance stability of AS-oligos, the present inventors have devised a rational way of searching better target sites using computer simulation by which secondary structures of mRNA are predicted, so they construct AS-oligos with a stemloop structure or covalently-closed multiple antisense sequences.

AS-oligos to c-myb gene could be used for inhibition of tumor cell growth.

The Myb protein, encoded by the c-myb protooncogene, is located mainly inside the nucleus and functions as a transcriptional regulator for Gl/S phase transition during the cell cycle. Protooncogene c-myb plays an important role in proliferation and e differentiation of hematopoietic cells. Hematopoietic cells exhibit differential expression of c-myb and :show little expression of the gene when 20 differentiated to term (Melani, C. et al., Cancer Res., 51,2897-2901, 1991). C-myb has often been found to be overexpressed in leukemic cells.

It is reported that blockage of c-myb expression by AS-oligos inhibits growth of a promyelocytic S. 25 cancer cell line HL-60 and a chronic myelogenous leukemia cell line K562 (Kimura, S. et al., Cancer Res., *e 004296731 1379-1384,1995). However, the c-myb AS-oligo used in the above experiments was demonstrated to be partially effective. The c-myb AS-oligo employed for the above experiments was either a phosphodiester (hereinafter, referred to as 'PO')-oligo or a PS capped-oligo (Anfossi, G. et al., Proc. Natl. Acad.

Sci. USA, 86,3379-3383,1989). These oligo molecules are not stable, especially for PO-oligo, possibly explaining the partial antisense effect.

AS-oligos selected by rational target site search combined with improved stability would be employed for complete ablation of c-myb mRNA, leading to better inhibition of leukemic cell growth.

Recently, a great deal of interest has been focused on developing molecular therapeutics based on ASoligo strategies against human malignancies. Thus, it is desired to find an improved c-myb antisense molecule that could block leukemic cell growth to Scompletion.

20 Therefore, to develop AS-oligo of a novel structure with better stability and antisense effect, the present inventors selected 8 sites along c-myb mRNA from secondary structure analysis in the preferred embodiment and combined antisense sequences 25 of the selected c-myb to construct novel large antisense molecules, a covalently-closed multiple e 004296731 antisense (hereinafter, referred to as 'CMAS')-oligo and a ribbon type antisense (hereinafter, referred to as 'RiAS')-oligo, with loops and a stem structure.

Thus, the present inventors have demonstrated that the novel AS-oligos are stable to nuclease activities and show a significant specificity to repress gene expression.

SUMMARY OF THE INVENTION The present invention relates to novel AS-oligos containing one or more antisense sequences to mRNA regions with a less secondary structure to improve target sequence specificity and stability to nuclease activities.

In such aspects of this invention, the present 20 invention provides antisense sequences selected from mRNA region of c-myb, c-myc, or k-ras with a less secondary structure.

The present invention provides a covalently- 25 closed multiple antisense (CMAS)-oligo containing multiple antisense sequences to c-myb UU42aD(d1 mRNA. The CMAS-oligo is constructed to form a closed type by ligation using complementary primer.

The present invention also provides a ribbontype antisense (RiAS)-oligo containing multiple antisense sequences to c-myb mRNA. The RiAS-oligo is composed of two loops containing multiple antisense sequences and a stem connecting the two loops that is constructed by ligation using complementary sequences at both 5 prime ends. In addition, the present invention provides the RiAS-oligos containing multiple antisense sequences to c-myc mRNA or k-ras mRNA.

The present invention further provides pharmaceutical composition containing the novel types of AS-oligos for treatment cancer, immune diseases, infectious diseases, and other human diseases caused by aberrant gene expression.

The present invention further provides a method 20 for preparing covalently-closed antisense oligodeoxynucleotides (AS-oligos) which have improved antisense activity and specificity to a target gene or a target mRNA, and which have improved stability to nuclease, comprising the following steps: 25 selecting mRNA target regions wherein less secondary structures are formed, by analysing putative secondary structures of target mRNA which are predicted from the base sequences of a target gene or target mRNA; 004296731 synthesizing single-stranded linear ASoligos which contain sequences complementary to one or more mRNA target regions selected in the above step and ligating the linear AS-oligo synthesized in the above step by using either a ligation primer as a ligation template or a linear AS-oligo molecule having a cohesive end as a ligation template.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a scheme for the construction of a c-myb CMAS-oligo.

FIG. 2 shows electrophoretic mobility patterns of a CMAS-oligo.

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*o *o o o oo oe goo ooo UU4ZaOIJI A is oligos analyzed by 5% Metaphor agarose gel, where lane 1; size marker, lane 2; 14 mer ligation primer, lane 3; linear 60 mer oligo, and lane 4; CMAS-oligo.

B shows stability of linear and covalently closed oligos on denaturing polyacrylamide gel, where lane 1 and 3; no treatment with exonuclease III, and lane 2 and 4; treatment with exonuclease III.

FIG. 3 shows a scheme for the construction of a c-myb RiAS-oligo.

FIG. 4 shows electrophoretic mobility patterns of a RiAS-oligo.

A is oligos analyzed by a 15% denaturing polyacrylamide gel, where lane 1; 58 mer MIJ-78 molecule, and lane 2; 116 mer RiAS-oligo.

B shows stability test of MIJ-78 and a RiASoligo upon treatment with exonuclease III, where lane 1 and 3; no treatment with exonuclease III, and 20 lane 1 and 3; no treatment with exonuclease III, and lane 2 and 4; treatment with exonuclease

III.

FIG. 5 shows degradation patterns of linear and CMAS-oligos in the presence of serum.

A shows stability test of linear AS-oligo, where 25 lane 1; no treatment with serum (negative control), lane 2; treatment with 50% raw serum, lane oo 004296/31 3; FBS, and lane 4; CS for 24hr respectively.

B shows stability test of CMAS-oligos, where lane 1; no treatment with serum (negative control), lane 2; treatment with 50% raw serum, lane 3; FBS, and lane 4; CS for 24hr respectively.

FIG. 6 shows degradation patterns of linear and RiAS-oligos in the presence of serum.

A shows stability test of MIJ-78 molecules, where lane 1; no treatment with serum (negative control), lane 2; treatment with 50% raw serum, lane 3; FBS, and lane 4; CS for 24hr respectively.

B shows stability test of RiAS-oligos, where lane 1; no treatment with serum (negative control), lane 2; treatment with 50% raw serum, lane 3; FBS, and lane 4; CS for 24hr respectively.

FIG. 7 shows an effect of c-myb CMAS-oligo on cmyb expression in HL-60 cells.

A shows RT-PCR which is performed with total RNA and two c-myb primers, where 20 lane 1; 60 mer CMAS-oligo 0.3 ug Lipofectin 1 ug, lane 2; 60 mer CMAS-oligo 1 ug Lipofectin 1 ug, and lane 3; scrambled AS-oligo 1 ug Lipofectin 1 o* ug.

B shows RT-PCR which is performed with total RNA 25 and two c-myb primers, where upper panel; the hybridized RT-PCR bands of cmyb mRNA, and lower panel; the hybridized RT-PCR bands of C 0 oo 004296731 P-actin mRNA.

FIG. 8 shows an effect of c-myb RiAS-oligo on the c-myb mRNA expression in HL-60 cells.

A shows RT-PCR which is performed with total RNA using two c-myb primers, where lane 1; RiAS-oligo 0.1 ug Lipofectin 0.8 ug, lane 2; RiAS-oligo 0.2 ug Lipofectin 0.8 ug, and lane 3; SC-oligo 0.2 ug Lipofectin 0.8 ug.

B shows RT-PCR which is performed with total RNA and two c-myb primers, where upper panel; the hybridized RT-PCR bands of cmyb mRNA, and lower panel; the hybridized RT-PCR bands of P-actin mRNA.

FIG. 9 shows an effect of 60 mer CMAS or linear AS-oligo on proliferation of HL-60 cells, where CMAS-oligos U CMAS-oligos AS-oligos, S-MIJ-7, S Lipofectin alone, "0 O; untreated control, and or times of *g* treatment with AS-oligos.

20 FIG. 10 shows an effect of c-myb RiAS-oligo on proliferation of HL-60 cells.

A shows MTT assay of a c-myb RiAS-oligo.

B shows 3 H] thymidine incorporation of a c-myb RiAS-oligo.

25 FIG. 11 shows a photomicrograph for inhibition of HL-60 cells with c-myb RiAS-oligo.

*o*oo *ooooo 004296731 A is c-myb RiAS-oligo, B is SC-oligo, Lipofectin alone.

FIG. 12 shows a photomicrograph for of HT-29 cells with c-myb RiAS-oligo.

A is c-myb RiAS-oligo, B is SC-oligo, Lipofectin alone.

FIG. 13 shows a photomicrograph for of HT-29 cells with c-myc RiAS-oligo.

A is c-myc RiAS-oligo, B is SC-oligo, Lipofectin alone.

FIG. 14 shows a photomicrograph for of HT-29 cells with k-ras RiAS-oligo.

A is k-ras RiAS-oligo, B is SC-oligo, Lipofectin alone.

and C is inhibition and C is inhibition and C is inhibition and C is DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Hereinafter, the present invention is described in detail.

In one aspect, the present invention provides novel AS-oligos containing one or more antisense sequence to regions which form a less secondary structure within a target mRNA.

Particularly, in the preferred embodiment, 8 different regions of c-myb mRNA, one of protooncogenes, for target sites of antisense oligos UU4Z~ti31 were selected. The rational target site search for an AS-oligo was employed to improve the chance to predict a natural secondary structure. The above 8 antisense sequences are complementary to the selected target sites. Among the 8 selected target sites for AS-oligos, 4 sites (MIJ-1, MIJ-2, MIJ-3, and MIJ-4) were finally chosen in a combination upon forming a CMAS molecule and a 3 sites (MIJ-3, MIJ-4, and MIJ- 17) in a combination forming a RiAS molecule as they form minimal intramolecular secondary structure (see Table 1).

AS-oligos having phosphodiester backbone lacked stability which was essential for successful antisense application. Modified oligos, such as PSoligo or MP-oligo, exhibited improved stability, but the gain in stability was only partial and bore potential hazard of modified nucleotide S. misincorporation during DNA replication or repair.

20 It was previously reported that stem-loop oligos complexed with cationic liposomes also showed partial S. improvement of stability. However, stability still remains a major concerns for AS-oligos. So, these inventors tried to develop an improved AS-oligo 25 having better stability to nucleases.

Therefore, the present invention provides a *o* go *www UULzI 31 covalently-closed multiple antisense (CMAS)-oligo which is characterized by having covalently closed end.

Particularly, CMAS-oligos are designed and synthesized not to form intermolecular duplex formation, especially when they are delivered into cells. In the preferred embodiment, these AS-oligos are designated as CMAS (covalently-closed multiple antisense)-oligo containing four antisense sequences (MIJ-1, MIJ-2, MIJ-3, and MIJ-4) which are described by SEQ ID NO 1, NO 2, NO 3, and NO 4 and which are placed in a loop in tandem to increase the length of CMAS-oligos (see FIG. The CMAS-oligos showed electrophoretic mobility patterns in that their mobility were slowed by about 10% than its linear precursor on a 15% denaturing PAGE gel (see A of FIG. The CMAS-oligos were, due to covalentlyclosed ends, resistant to exonuclease III and are shown in multiple bands on a denaturing PAGE gel, 20 with monomer (60 mer) being the most abundant, dimers (120 mer) and trimers (180 mer). In contrast to CMAS-oligo, linear oligos are completely degraded after 2 hr incubation with exonuclease III (see B of FIG. 2).

S 25 The present invention also provides a ribbontype antisense (RiAS)-oligo.

UU4Z~b(J1 The CMAS-oligo, although very stable, needs a primer f or intramolecular ligation that must be eliminated afterward. So, to avoid using a ligation *14a UUL4o oJI primer and obtain a homologous population of ASoligo, these inventors made two AS-oligos enzymatically ligated to form a ribbon-type closed molecule termed as RiAS-oligo (see FIG.3).

The RiAS-oligo (116 mer) consists of two loops and one stem connecting the two loops (see FIG. 3).

In the preferred embodiment, three antisense sequences (MIJ-3, MIJ-4, and MIJ-17) which are described by SEQ IN NO 3, NO 4, and NO 5 are placed in a loop region in tandem to increase the length of RiAS-oligo. Consequently, two copies of three different antisense sequences (total 6 antisense sequences) are placed in the RiAS-oligo.

This enlarged length of the loop in RiAS-oligo is beneficial to accommodate torsional stress caused by forming a duplex with the target mRNA sequences. The RiAS-oligo is found to be slowed markedly than its linear precursor (MIJ-78) on a denaturing PAGE gel (see A of FIG. The RiAS-oligo is, due to 20 covalently-closed ends, resistant to exonuclease III and is shown in a major band (116 mer) on a PAGE gel.

In contrast to the RiAS-oligo, MIJ-78 (linear ASoligos) is completely degraded after 2 hr incubation with exonuclease III (see B of FIG. 4).

To demonstrate the enhanced stability of the CMAS-oligo and the RiAS-oligo of this invention r" against nuclease activities, the CMAS-oligo and the a a UU429f/31 RiAS-oligo are incubated with different ser that are not heat inactivated to retain nuclease activities.

As a result, linear 60 mer AS-oligo (precursor of the CMAS-oligo, see A of FIG. 5) and linear 59 mer AS-oligo (precursor of the RiAS-oligo, see A of FIG.

6) are completely digested after 24 hr incubation in the presence of serum. The CMAS-oligo and the RiASoligo, however, are remained mostly intact after 24 hr incubation with raw human serum, FBS, and calf serum, exhibiting significantly improved stability than does the linear one against nucleases activities (see B of FIG. 5 and B of FIG. 6).

In addition, it is demonstrated that the CMAS-oligo functions well eliminating target mRNA in a sequence specific manner.

Particularly,, the CMAS-oligo was combined with Lipofectin to deliver into cells. Lipofectin is employed as it is found to be less toxic to cells and yield consistent results. MIJ-5, the CMAS-oligo to *o 20 human c-myb, is able to reduce more than 95% of c-myb mRNA when compared to a control SC-oligo. Meanwhile, the linear counterpart of MIJ-5, MIJ-5A, decreases some 37% of c-myb mRNA (see A of FIG. These S* results indicate that the CMAS-oligo of this invention is superior to linear one in ablating target mRNA even when used in a smaller amount.

The function of RiAS-oligo that eliminates target mRNA was demonstrated by the same method in the CMAS-oligo.

cells were tested with RiAS-oligos, scrambled (SC)-oligos as well as Lipofectin alone.

The RiAS-oligo is delivered into cells after forming a complex with Lipofectin. Consequently, the RiASoligo is able to ablate c-myb mRNA completely. In contrast, SC-oligo exhibits a mild reduction of c-myb mRNA by about 30% when compared to Lipofectin treatment alone (see A of FIG. These results indicate that the RiAS-oligo of this invention is excellent in ablating target mRNA even when used in a small amount.

The present inventors also examined antisense effect of the CMAS-oligo and the RiAS-oligo by Southern blotting of the PCR product.

20 In case of the CMAS-oligo, when c-myb mRNA is 0* amplified with RT-PCR, more than 90% of the mRNA is found to be reduced with treatment of MIJ-5 (see B of FIG. 7).

0* In RiAS-oligo, c-myb PCR product amplified by RT-PCR is detected with a labelled internal hybridization oligo (30 mer) (B of FIG. The result confirms that the amplified PCR product is indeed c-myb derived.

The present invention also provides pharmaceutical composition containing the novel types of AS-oligos for treatment cancer, immune diseases, infectious diseases, and other human diseases caused by aberrant gene expression.

It is demonstrated that the c-myb CMAS-oligo or the c-myb RiAS-oligo inhibits leukemic cell growth.

Particularly, growth inhibition of the c-myb CMASoligo and the c-myb RiAS-oligo to leukemic cells was measured by three methods, MTT assay, [3H] thymidine incorporation or colony formation on soft agarose.

As a result of MTT assay, cell number is reduced progressively when treated with increasing amounts of the CMAS-oligo to human c-myb. Inhibition of cell growth is more pronounced when cells are treated twice with MIJ-5. More than 80% of growth inhibition of HL-60 cells is observed even at a low concentration (see FIG. Meanwhile, the linear mer AS-oligo, MIJ-5A, and linear sense oligo does not 20 bring about any significant inhibition of cell growth when compared with a sham control. These results indicate that the c-myb CMAS-oligo of this invention is an effective antisense agent and is efficacious against tumor growth in a concentration dependent manner.

addition, cell growth is observed to be *oooo oo* inhibited by 91% with the RiAS-oligo (see A of FIG.

Meanwhile, the SC-oligo and Lipofectin alone do not significantly inhibit cell growth when compared to that of the untreated control. These results indicate that the c-myb RiAS-oligo of this invention is also an effective antisense agent for the inhibition of leukemic cell growth.

In colony formation on soft agarose, MIJ-5, the CMAS-oligo to human c-myb, reduces the number of colonies formed by more than 90% (see Table MIJalso reduces colonies formed but less effective for growth inhibition, about 70% reduction of colonies. On the other hand, a sense oligo and a SColigo exhibits marginal reduction of colonies, by about 11% and 32% respectively.

Also, the c-myb RiAS-oligo transfected into cells is able to reduce the number of colonies formed by about 92% (see Table 3) when compared to an 20 untreated control. Meanwhile, a SC-oligo and Lipofectin alone exhibits marginal reduction of colonies, by about 7.9% and 7.1% respectively.

:In addition, it is observed growth inhibition of the c-myb RiAS-oligo to leukemic cells by [3H] thymidine incorporation. Particularly, the RiAS-oligo inhibits growth of HL-60 cells by 93%. Meanwhile, the Iaisob VVuuQOIJI SC-oligo and Lipofectin alone exhibit mild inhibition of cell growth, by about 16.8% and 15.4k respectively (see B of FIG. 10). On a microscopic observation, after treatment with the c-myb RiAS-oligo, growth of HL-60 cells are markedly inhibited when compared with cells treated with scrambled oligo and Lipofectin alone (see FIG. 11).

Encouraged by the remarkable inhibition activity of c-myb RiAS-oligo in this .invention, these inventors construct other RiAS-oligos against two different protooncogenes, c-myc and k-ras, and examine if the c-myc RiAS-oligo and the k-ras RiASoligo functions well in inhibiting cell growth.

As a result of microscopic observations, growth of HT-29 cells is markedly inhibited by all RiASoligos, c-myb RiAS-oligo, c-myc RiAS-oligo, and k-ras RiAS-oligo, when compared with cells treated with scrambled oligos and Lipofectin alone (see FIG. 12, FIG. 13, and FIG. 14) Therefore, the novel RiAS-oligos of the present invention show effective inhibition of tumor cell growth to various target sequences as well as enhanced stability to nuclease activity. So, the novel RiAS-oligos of this invention may be effectively employed for developing molecular antisense oligos to treat various human diseases that are caused by abnormal expression of certain genes.

p* e VU I I

EXAMPLES

Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.

However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

Example 1: Selection of target sites for an AS-oligo Target site selection of an AS-oligo had been found to be critical to achieve antisense effect, reduction or ablation of target mRNA. However, the approach for the target site selection had been rather arbitrary. So, these inventors scanned the entire sequence of human c-myb mRNA for putative secondary structures to search a rational target site in the preferred embodiment.

Particularly, simulation of secondary structures was carried out with the DNAsis program (Hitach Software, Japan). Entire c-myb sequence was scanned sequentially for secondary structure formation in contiguous frames of 100 bases. Then, frames for the simulation of secondary structure were staggered down by 30 bases, resulting in an overlap of 60 bases on *oo o* *o*eo *ooo uuaLuoDII the 5 prime end. This process was repeated again such that any given sequence was scanned for its potential secondary structure in three different frames.

As a result, eight sequences which had minimal secondary structures in three different frames were selected from the c-myb mRNA sequence (Table The rational target site search for an AS-oligo was employed to improve the chance to predict a natural secondary structure.

TABLE 1. Eight target sequences for antisense oligos selected from the c-myb mRNA sequences Name Complementary Type Size Sequence site (mer) MIJ-1 253-267 Antisense 15 SEQ ID NO: 1 MIJ-2 401-415 Antisense 15 SEQ ID NO: 2 MIJ-3 613-627 Antisense 15 SEQ ID NO: 3 MIJ-4 1545-1559 Antisense 15 SEQ ID NO: 4 MIJ-6 253-267 Antisense 15 SEQ ID NO: 9 MIJ- 585-602 Antisense 18 SEQ ID NO: 16 MIJ- 961-978 Antisense 18 SEQ ID No: 17 MIJ- 97-114 Sense 8 SEQ ID NO:11 19 As illustrated in the table 1, the above 8 antisense sequences were complementary to the selected target sites. Among the 8 selected target sites for AS-oligos, 4 sites (MIJ-1, MIJ-2, MIJ-3, and MIJ-4) were finally chosen in a combination upon forming a CMAS molecule and a 3 sites (MIJ-3, MIJ-4, and MIJ-17) in a combination forming a RiAS-oligo as they form minimal intramolecular secondary structure.

Example 2: Construction of a covalently-closed multiple antisense (CMAS)-oligo These inventors tried to develop an improved AS-oligo containing better stability.

4 different AS-oligos (MIJ-1, MIJ-2, MIJ-3, and MIJ-4) obtained by Example 1 was used to construct a CMAS-oligo. To bind to target sites more readily, one CMAS-oligo was constructed to harbor 4 different antisense sequences in a combination with the least secondary structure. AS-oligos were phosphorylated during synthesis at the 5 prime end to follow intra- 20 or intermolecular covalent ligations (FIG. 1) The sequence of the 60 mer AS-oligo containing 4 different antisense sequences was described by SEQ.

ID NO 7. Both ends of the AS-oligo was joined with a ligation primer which has complementary sequences in its both halves to the both extreme end sequences (7 bases on each side) of the 60 mer AS-oligo. The sequence of *oo* UU4ZaU/J1 the 14 mer ligation primer was described by SEQ ID NO 6. Ligation primer was mixed with AS-oligo and was heated at 85 0 C for 2 min followed by gradual cooling to ambient temperature. One unit of T4 ligase was added and incubated at 16 0 C for 16 hr to generate a covalently-closed molecule. The CMAS-oligo was electrophoresed on a 5k Metaphor TM agarose gel (FMC, USA) or on 12% denaturing PAGE and identified for its resistance to exonuclease III as well as for slight gel retardation compared with the linear 60 mer oligos. Ligation primer was degraded with exonuclease III or detached from the CMAS-oligo by running on a denaturing gel after heating the oligos at 90 0

C.

Consequently, intramolecular secondary structure of an AS-oligo was designed and synthesized to form no intermolecular secondary structure to form without duplex formation. This AS-oligo was designated a CMAS (covalently-closed multiple antisense)-oligo. The CMAS-oligo showed electrophoretic mobility patterns that it was slowed by about 10% than its linear precursor on a 15% denaturing PAGE gel (A of FIG. 2) The CMAS-oligo was, as expected, resistant to exonuclease III and was shown in multiple bands on a denaturing PAGE gel, with monomer (60 mer) being the most abundant, then dimers (120 mer) and trimers (180 mer) (B of FIG In contrast to the CMAS-oligo, a linear oligo was

S

completely degraded after 2 hr incubation with exonuclease III.

Example 3: Construction of a ribbon-type antisense (RiAS)-oligo These inventors made two AS-oligos enzymatically ligated to form a ribbon-type closed molecule termed a RiAS-oligo.

Particularly, the RiAS-oligo consisted of two loops and one stem connecting the two loops. Each loop contained three different antisense (MIJ-3, MIJ- 4, and MIJ-17) sequences that were described by SEQ ID NO 3, NO 4, and NO 5. To bind to target sites more readily, a combination of three antisense sequences with a least possible secondary structure was chosen for the AS-oligo. C-myb AS-oligo (MIJ-78) was phosphorylated at the 5 prime end. Sequences of the 58 mer MIJ-78 was described by SEQ ID NO 8.

MIJ-78 was to form a stem-loop structure. The stem o 20 was formed by complementary sequences at both ends of each oligo. The 5 prime terminus of the stem had 4 bases of a single stranded sequence of GATC- S, Two MIJ-78 molecules were joined by the complementary 4 base sequences at both 5 prime ends.

MIJ-78 molecules were mixed and heated to 85 0 C for 2 min followed by gradual cooling to ambient temperature. One unit of e*eo

I

T4 DNA ligase was added and incubated at 16 0 C for 24 hr to generate a covalently ligated molecule with diad-symmetry (FIG. The RiAS-oligo was electrophoresed on 15% denaturing polyacrylamide gel and examined for its resistance to exonuclease III as well as for gel retardation.

As a result, the RiAS-oligo (116 mer) consisting of two loops and one stem connecting two loops was constructed. Three antisense sequences in a loop were placed in tandem to increase the length of the RiASoligo. Consequently, two copies of three different antisense sequences (total 6 antisense sequences) were in the RiAS-oligo. This enlarged length of the loop in the RiAS-oligo was to accommodate torsional stress caused by forming a duplex with the target mRNA sequences. The RiAS-oligo was found to be slowed markedly than its linear precursor (MIJ-78) on a denaturing PAGE gel (A of FIG. 4) The RiAS-oligo was, as expected, resistant to exonuclease III and 20 was shown in a major band (116 mer) on a PAGE gel (B of FIG. In contrast to RiAS-oligo, MIJ-78 was completely degraded after 2 hr incubation with exonuclease III.

25 Example 4: Enhanced stability of the CMAS-oligo and the RiAS-oligo to nuclease activities *oooo* 004296731 In order to test stability of the CMAS-oligo and the RiAS-oligo of this invention against nuclease activities, the CMAS-oligo and the RiAS-oligo were incubated with serums that were not heat inactivated to maintain nuclease activities.

Particularly, one microgram of each nonspecific control-phosphodiester oligo (liner 60 mer) and CMAS-oligo respectively was incubated with either raw human serum, FBS and calf serum (non-heat inactivated; HyClone, Logan, Utah, USA) or exonuclease III. 15% of each serum was added to ASoligos in an 100 ul reaction volume and incubated at 37 0 C for 24 hr. AS-oligos were then extracted with phenol and chloroform, and were examined on denaturing PAGE gel. Exonuclease III (Takara, Japan) at 160 U/ug oligo was added to linear and CMAS-oligos and incubated at 37 0 C for 2 hr. AS-oligos treated with exonuclease III were also extracted and electrophoresed in the same manner.

S 20 Compared to CMAS-oligo, linear 60 mer oligo was completely digested after 24 hr incubation in the presence of serum of FIG. The CMAS-oligo of this invention, however, was remained mostly intact after 24 hr incubation with raw human serum, FBS, and 25 calf serum, exhibiting significantly improved stability than the linear one against nucleases (B of FIG. WO 00/61595 PCT/KR00/00305 In the case of the RiAS-oligo, linear 58 mer was completely hydrolyzed after 24 hr incubation in the presence of each different serum(A of FIG. The RiAS-oligo of this invention, however, remained mostly intact after 24 hr incubation with the raw serums, exhibiting significantly improved stability than the linear one against nucleases(B of FIG. Example 5 Specific reduction of c-myb mRNA by the CMAS-oligo and the RiAS-olico Encouraged by the remarkable stability of the CMAS-oligo and the RiAS-oligo in this invention, these inventors examined if the AS-oligo functioned well in eliminating target mRNA in a sequence specific manner.

Cell lines and tissue culture Leukemic cell lines, HL-60(promyelocyte leukemic cell line) and K562(chronic myelogenous leukemic cell line), were obtained from ATCC(American Type Culture Collection, USA) and cultured in RPMI 1640(Gibco BRL, USA) supplemented with 10% heat-inactivated FBS(HyClone, USA) and 1% penicillin/streptomycine.

Cells were maintained in a CO 2 incubator at 37C.

Routine cell culture practices were strictly adhered SUBSTiTUTE PAGE to keep proper cell density and to avoid cells cultured more than 5 generations after thawing stock vials. Culture media were exchanged a day before treating with AS-oligos.

Transfection of the CMAS-oligo and the RiAS-oligo complexed with cationic liposomes 0.3 ug CMAS-oligo plus 0.8 ug Lipofectin m (Gibco BRL, USA) or 0.2 ug RiAS-oligo plus 0.8 ug Lipofectin were diluted in 20 ul OPTI-MEMTM separately and incubated at ambient temperature for 40 min. Each component was then mixed to form a complex at ambient temperature for 15 min. Cells were added with fresh culture media without antibiotics (RPMI 1640 FBS) 1 day prior to adding oligos and washed twice with OPTI-MEM before an experiment. Cell density was adjusted to 5 X 105 cells/ml and aliquoted in 100 ul each in a 48-well plate (Falcon, USA). 40 ul of liposome-oligo complex was added to cells twice, once 20 on day 0 and once on day 1. Cells treated with oligos were incubated at 37 0 C and 5% C02 for 4 hr and then added 100 ul of OPTI-MEM with 10% FBS. The next day, 100 ul of supernatant was carefully removed and replaced with 20 ul of fresh OPTI-MEM containing 25 oligo-liposome complex. Four hours later, cells were added with additional 100 ul of complete media with antibiotics and incubated at 37 0 C 1 more day before assay.

Isolation of total RNA and RT-PCR Total RNA was isolated with Tripure TM Isolation Reagent (Boehringer Manheim, Germany) according to the procedure recommended by the manufacturer.

Briefly, cells harvested were added with 0.4 ml Tripure reagent, 10 ug glycogen and 80 ul chloroform to obtain total RNA. RT-PCR was performed in a single reaction tube with AccessT" RT-PCR kit (Promrga, USA). In a PCR tube were added RNA, PCR primers, AMV reverse transcriptase (5 U/ul), Tfl DNA polymerase U/ul), dNTP (10 mM, 1 ul) and MgS04 (25 mM, 2.5 ul).

Synthesis of the first strand cDNA was done at 480C for 45 min in a DNA thermal cycler (Hybaid, USA). cycles of PCR amplification were subsequently carried out with the recommended condition by the manufacturer. Amplified PCR product was confirmed in 20 an 1% agarose gel and quantitation was done with a gel documentation program (Bio-Rad, USA).

Southern hybridization of RT-PCR fragments RT-PCR products were electrophoresed on an 1% agarose gel. DNA was transferred onto a nylon membrane (New England Biolab, USA) for 4 hr in 0.4 M NaOH. The membrane was hybridized with 30 mer internal primer labelled with ECL 3 prime end oligolabelling and detection system (Amersham Life Science, England) The sequence of 30 mer internal primer was described by SEQ ID NO 9. Hybridization was carried out at 62 0 C for 60 min in 6 ml buffer containing 5 X SSC, 0.02% SDS. The membrane was washed twice in 5 X SSC containing 0.1% SDS and washed twice with 1 X SSC containing 0.1% SDS at for 15 min. The membrane was blocked with a blocking solution and then treated with HRP (horse radish peroxidase) anti-fluorescein conjugated antibody for min before autoradiography.

It was demonstrated that the CMAS-oligo of this invention functioned well eliminating target mRNA in a sequence specific manner.

Particularly, the CMAS-oligo was complexed with Lipofectin to deliver into cells. Lipofectin was So. 20 employed as it was found to be less toxic to cells and yield consistent results. As a result, 0.3 ug MIJ-5, a CMAS-oligo to human c-myb, was complexed with 1 ug Lipofectin for transfection into cells. MJ-5 was able to reduce more than 95% of c-myb S 25 mRNA when compared to a control SC-oligo. Meanwhile, the linear counterpart of MIJ-5, decreased some 37% of c-myb mRNA (A of FIG. These results indicate that the CMAS-oligo of this invention was superior to linear one in ablating target mRNA even when used in a smaller amount.

It was also demonstrated that the RiAS-oligo of this invention functioned well in eliminating target mRNA in a sequence specific manner.

cells were transfected with the RiASoligos, SC-oligos as well as Lipofectin alone. The RiAS-oligo was delivered into cells after forming a complex with Lipofectin. The RiAS-oligo (0.1 ug or 0.2 ug) to human c-myb was complexed with 0.8 ug Lipofectin for transfection into HL-60 cells.

Consequently, 0.2 ug of the RiAS-oligo (40 nM) was able to ablate c-myb mRNA to completion. Meanwhile, 0.1 ug of the RiAS-oligo decreased about 70% of c-myb mRNA (A of FIG. In contrast, SC-oligo exhibited a mild reduction of c-myb mRNA by about 30% when compared to Lipofectin treatment alone. However, P- 20 actin expression shown in the bottom panel was not affected by the treatment of the RiAS-oligo as well as other treatment conditions. These results indicated that the RiAS-oligo was excellent in ablating target mRNA even when used in a small amount.

The present inventors also examined antisense effect of the CMAS-oligo and the RiAS-oligo by Southern blotting with PCR products. HL-60 cells were transfected with oligos including MIJ-5 and control oligos, and the cells were used to isolate total RNA.

In case of the CMAS-oligo, when c-myb mRNA was amplified with RT-PCR, more than 900 of the mRNA was found to be reduced with treatment of MIJ-5 (B of FIG. However, 3-actin expression shown in the bottom panel was not affected by the treatment of In RiAS-oligo, C-myb PCR product amplified by RT-PCR was detected with a labelled internal hybridization oligo (30 mer) (B of FIG. The results confirmed that the amplified PCR product was indeed c-myb derived, with total elimination of the mRNA by treatment with 0.2 ug of the c-myb RiASoligo.

Example 6: Effective growth inhibition of leukemic 20 cells by the c-myb CMAS-oligo and the c-myb RiASoligo It was reported that c-myb played an important role in proliferation of leukocytes. AS-oligos to cmyb were also reported to block leukemic cell growth 25 preferentially. So, these inventors tested the c-myb CMAS-oligo and the c-myb RiAS-oligo of this invention for inhibiting leukemic cell growth.

o* oo *e Particularly, growth inhibition of the c-myb CMAS-oligo and the c-myb RiAS-oligo to leukemic cells was measured by three methods, MTT assay, 3

H]

thymidine incorporation or colony formation on soft agarose.

MTT assay For MTT (3,-[4,5-Dimethythiazol-2-yi]2,5diphenyl-tetrazolium bromide, hereinafter, referred to as 'MTT') assay, HL-60 cells were washed twice with OPTI-MEM and aliquoted in a 96-well plate (5 X 103 cells/well) in a 50 ul volume. Cells were treated with performed complex between oligos in different amount (0.01-1 ug/15 ul in CMAS-oligo or 0.2 ug/15 ul in RiAS-oligo) and Lipofectin (0.2 ug/15 ul) for 5 hr and cultured for 5 days. Cells were then harvested in an 100 ul volume and added with 20 ul (100 ug) of an MTT reagent (5 mg/ml in PBS; Sigma, USA), followed by 4 hr incubation at 37 0 C. 100 ul of isopropanol 20 (containing 0.1 N HC1) was added to the cells and incubated for one more hour at the ambient temperature. Absorbance was measured at 570 nm with an ELISA reader to score the amount of cells survived.

In CMAS-oligo, cell number was reduced progressively when treated with increasing amounts of UU4Zb/31 Inhibition of cell growth was more pronounced when cells were treated twice with MIJ-5. More than of growth inhibition of HL-60 cells was observed even at a low concentration, 0.13 ug (total 0.24 ug) of the CMAS-oligo (FIG. Meanwhile, the linear mer AS-oligo, MIJ-5A, and linear sense oligo did not bring about any significant inhibition of cell growth when compared with a sham control. These results indicate that the c-myb CMAS-oligo was an effective antisense agent against tumor growth in a concentration dependent manner.

In RiAS-oligo, cell growth was also observed to be inhibited by 91% with the RiAS-oligo (A of FIG.

Meanwhile, the SC-oligo and Lipofectin alone did not significantly inhibit cell growth when compared to that of the untreated control. These results indicate that the c-myb RiAS-oligo was an effective antisense agent for inhibition of leukemic cell growth.

S*e* Colony formation on soft agarose Growth inhibition of the c-myb CMAS-oligo and the c-myb RiAS-oligo to leukemic cells was also measured by colony formation on soft agarose as 25 another way.

Particularly, K562 cells were transfected as S. described above in Example 6 and cultured at 37 0

C

*s

S

UU4LZUDI/ and 5% C02 for 24 hr. An equal volume mixture of 0.8% low melting agarose and 2X (RPMI 1640 containing FBS plus antibiotics were added to cells and seeded in a 6 well-plate to solidify. The 6-well plate was cooled to 40C for 5 min and incubated for 15 days.

Colonies containing more than 20 cells were scored as positive.

As a result, CMAS-oligo MIJ-5 reduced the number of colonies formed by more than 90% (Table also reduced colonies formed but less effective for growth inhibition, about 70% reduction of colonies.

On the other hand, a sense oligo and a SC-oligo exhibited marginal reduction of colonies, by about 11% and 32% respectively.

TABLE 2. Effects of c-myb oligos on colony formation of K562 cells O000 6r 0*6* Su 0@S 0 0

S.

S. 0 0S06

S

0000 00 *0 00 Oligos Number of Colonies Structure Size Type colony formed (mer) Linear 15 AS-MIJ-1 55 44.4 Linear 15 S-MIJ-3 110 88.7 Linear 15 SC-MIJ-1 84 67.7 Linear 60 AS-MIJ-5A 29 23.4 CMAS 60 AS-MIJ-5 9 7.2 Lipofectin 109 88.0 alone WO 00/61595 PCT/KR00/00305 Untreated 124 100.0 control On the other hand, cells transfected with the c-myb RiAS-oligo was able to reduce the number of colonies formed by about 92%(Table 3) when compared to an untreated control. Meanwhile, a SC-oligo and Lipofectin alone exhibited marginal reduction of colonies, by about 7.9% and 7.1% respectively.

TABLE 3. Effects of colony formation c-myb oligos on of K562 cells Oligos Size(mer) Number of Colonies colony formed RiAS-oligo 116 7.6 T 1.53 7.8 Scrambled 116 0.5 2.12 92.1 oligo Lipofectin 91.3 T 4.16 92.9 alone Untreated 98.3 4.04 100.0 control [3H] thymidine incorporation Growth inhibition of leukemic cells by the c-myb RiAS-oligo was also measured by 3 H] thymidine incorporation.

For 3 H] thymidine incorporation, HL-60 cells were treated with AS-oligo as described above. Cells

I

SUBSTITUTE

PAGE

were added with 0.5 uCi of [3H] thymidine Ci/mmol; Amersham, England) and incubated for 16 hr in triplicate. Cells were then harvested on a glass microfiber filter (Whatman GF/C, England). The filter was washed with in the order of cold PBS, 5% TCA and absolute ethanol. 3 H] thymidine incorporation was measured with the liquid scintillation counter in a cocktail solution containing toluene, Triton X-100, PPO and POPOP.

Consequently, the RiAS-oligo (0.2 ug) inhibited growth of HL-60 cells by 93% (B of FIG. Meanwhile, the SC-oligo and Lipofectin alone exhibited mild inhibition of cell growth, by about 16.8% and 15.4% respectively. On a microscopic observation, after treated with the c-myb RiAS-oligo, growth of HL-60 cells was markedly inhibited when compared with cells treated with scrambled oligo and Lipofectin alone (FIG. 11).

20 Example 8: Effective growth inhibition of the c-myc :RiAS-oligo and the k-ras RiAS-oligo Encouraged by the remarkable inhibition activity of c-myb RiAS-oligo in this invention, these inventors constructed other RiAS-oligos against two different protooncogenes, c-myc and k-ras, as the *m same method in Example 3.

And then, they examined if the c-myc RiAS-oligo and the k-ras RiAS-oligo functioned well in inhibiting cell growth.

Particularly, they used different cell line, colorectal adenocarcinoma cell line HT-29. Growth inhibition of the c-myc RiAS-oligo and k-ras RiASoligo to tumor cells was measured by 3 H] thymidine incorporation as the same method in Example HT-29 cells were treated with cationic liposome complexes of 0.2 ug c-myb RiAS-oligo plus 0.6 ug Lipofectin or 0.5 ug c-myc RiAS-oligo plus 1.5 ug Lipofectin or 0.5 ug k-ras RiAS-oligo plus 1.5 ug Lipofectin, respectively. After treated respective RiAS-oligos for 5 days, growth of HT-29 cells was observed using microscopy. Each photomicrograph exhibited the effect on growth inhibition after treatment with respective RiAS-oligos scrambled oligo and Lipofectin alone As a result of microscopic observations, growth 20 of HT-29 cells was markedly inhibited by all RiAS- :oligos, c-myb RiAS-oligo, c-myc RiAS-oligo, and k-ras RiAS-oligo, when compared with cells treated with scrambled oligos and Lipofectin alone (FIG. 12, FIG.

13, and FIG. 14) Therefore, the novel RiAS-oligos of the present invention showed effective growth inhibition of tumor WO 00/61595 PCT/KR00100305 cells to various target sequences as well as enhanced stability to nuclease activity. So, the novel RiAS-oligos of this invention might be effectively employed for developing molecular antisense oligos to treat various human diseases.

uu4zO/IJ1 SUBSTBTUTE PAGE INDUSTRIAL APPLICABILITY The present invention provides novel AS-oligos containing one or more antisense sequence complementary to mRNA target region with a less secondary structure and having better target sequence specificity and stability to nuclease activities.

Particularly, the present invention provides a covalently-closed multiple antisense (CMAS)-oligo containing multiple target antisense sequences to cmyb mRNA which is constructed to form a closed type by ligation using complementary primer. In addition, the present invention provides a ribbon-type antisense (RiAS)-oligo containing multiple target antisense sequences to c-myb mRNA which is constructed to form a stem-loop structure by ligation using complementary sequences at both 5 prime ends.

It is demonstrated that aberrant gene expression 20 is effectively ablated by the novel AS-oligos of this invention when human tumor cells are treated with the c-myb RiAS-oligo and the c-myb CMAS-oligo as well as c-myc RiAS-oligo and k-ras RiAS-oligo. Thus, it suggests that the novel AS-oligos of this invention may be employed for developing molecular antisense °o drugs to various genes causing diseases as well as for the functional study of a gene. Particularly, the UU4j(lJ1 novel AS-oligos of this invention may be used for developing pharmaceutical composition for treatment cancers, immune diseases, infectious diseases, or other human diseases caused by aberrant gene expression.

Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.

Reference to any prior art in the specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms "..part of the common general knowledge in Australia or any other country.

oooo 20 It will be understood that the term "comprises" Sor its grammatical variants as used in this specification and claims is equivalent to the term "includes" and is not to be taken as excluding the presence of other elements or features.

V00.0 i o o 60 0 0 EDITORIAL NOTE APPLICATION NUMBER 34640/00 The following Sequence Listing pages 1 to 3 are part of the description. The claims pages follow on pages 43 to 47.

WO 00/61595 WO 0061595PCTIKROO/00305 <110> <120> <130> <150> <151> <160> <170> <210> <211> <212> <213> <400> tcagtttttc SEQUENCE LISTING PARK, Jong-Gu The novel antisense oligos with better stability and antisense effect Ofpo-02710 KR 99-122917 1999-04-08 11

KOF

1

DN.P

Horn 1 atcct ATIN 0 sapiens <210> 2 <211> <212> DNA <213> Homo sapiens <400> 2 tgatcttctt ctttg <210> 3 <211> <212> DNA <213> Homo sapiens <400> 3 gctttgcgat ttctg <210> 4 1- WO 00/61595 WO 0061595PCT/KROO/00305 <211> <212> <213>

DNA

Homo sapiens <400> 4 accgtattta atttc <210> <211> 18 <212> DNA <213> Homo sapiens <400> ggtcttcatc- attatagt <210> <211> <212> <213> <220> <223> <400> tcagtttttc 6

DNA

Artificial Sequence antisense oligonucleotides 6 atcctgcttt gcgacttctg tgatcttctt ctttgaccgt atttaatttc <210> <211> <212> <213> <220> <223> 7 58

DNA

Artificial Sequence antisense oligonucleotides <400> 7 gatccgcgct tcatcattat agtaccgtat ttaatttcgc tttgcgattt ctggcgcg -2 WO 00/61595 WO 0061595PCTIKROO/00305 <210> 8 <211> <212> DNA <213> Artificial Sequence <220> <223> internal primer <400> 8 tgtaacgcta cagggtatgg aacatgactg <210> 9 <211> <212> DNA <213> Homo sapiens <400> 9 tattcttctg ctcta <210> <211> 17 <212> DNA <213> Homo sapiens <400> cccagtctct tgtgtgc 17 <210> 11 <211> 18 <212> DNA <213> Homo sapiens <400> .11 tggcgcggcg ggcggcgg 18 -3

Claims (23)

1. A method for preparing covalently-closed antisense oligodeoxynucleotides (AS-oligos) which have improved antisense activity and spec..ficity to a target gene or a target mRNA, and which have improved stability to nuclease, comprising the following steps: selecting mRNA target regions within the target mRNA wherein less secondary structures are formed, by analysing putative secondary structures of target mRNA which are predicted from the base sequences of a target gene or target mRNA; synthesizing single-stranded linear AS- 15 oligos which contain sequences complementary to one or more mRNA target regions selected ir: the above step and ligating the linear AS-oligo syrthesized in the above step by using either a. ligation primer 20 as a ligation template or a linear AS-oligo molecule having a cohesive end as a ligation templa.te.

2. The method as set forth in claim 1, wherein the 25 target gene or the target mRNA is one that. is related 25 to cancers, immune diseases, infectious diseases, or genetic diseases, which are caused by aberrant gene expression. 43 COMS ID No: SMBI-00700286 Received by IP Australia: Time 14:41 Date 2004-04-07

3. The method as set forth in claim 1 or 2, wherein the target gene is selected from the protooncogenes consisting of c-myc, c-myb, and k-ras.

4. The method for preparing covalently-closed multiple AS-oligo (CMAS-oligo) as set forth in any one of claims 1 to 3, wherein the ligation step (3) in which the ligation primer is used as a ligation template is characterised by: i) using the ligation primer which contains base sequences of linear AS-oligo synthesized in the step of claim 1, as a ligation template; and ii) ligating the AS-oligo with ligation enzyme.

5. The method as set forth in claim 4, wherein the ligation primer used as a ligation template contains 5-10 base sequences complementary to both the 5' -end and the 3' end of linear AS-oligo synthesized in the step of claim 1.

6. The method as set forth in claim 4, wherein the ligation enzyme is T4 DNA ligase.

7. A CMAS-oligo prepared by the method of claim 4.

8. A CMAS-oligo prepared by the method of claim 4, which contains one or more antisense sequences represented by SEQ. ID. No 1, No 2, No 3 or No 4 selected by analysing putative secondary structures of c-myb, mRNA as a target mRNA.

9 The CMAS-oligo as set forth in claim 8, wherein the CMAS-oligo contains an antisense sequence represented by SEQ. ID. No 6.

10. The method for preparing ribbon-type AS-oligo (RiAS-oligo) as set forth in claim 1, wherein the ligation step in which a linear AS-oligo molecule having a cohesive end is used as a ligation template is characterised by: i) using two AS-oligo molecules which have cohesive end owing to their complementary sequence within the stem regions of linear AS-oligos which are designed to form intramolecular stem-loop structure, as a ligation template; and ii) ligating the AS-oligo with ligation enzyme.

11. The method as set forth in claim 10, wherein the ligation enzyme is T4 DNA ligase. i" 20

12. A RiAS-oligo prepared by the method of claim or 11, which consists of two loop regions and a stem region connecting the loop regions.

13. A RiAS-oligo prepared by the method of any one 25 of claims 1 to 12, wherein the RiAS-oligo is prepared by ligating two linear AS-oligos which have the stem- loop structure with the cohesive end, and which contain one or more antisense sequences represented by SEQ. ID No 3, No 4 or No 5 selected by analysing by SEQ. ID No 3, No 4 or No 5 selected by analysing -U u3 I o.I putative secondary structures of c-myb mRNA as a target mRNA.

14. The RiAS-oligo as set forth in claim 13, wherein the RiAS-oligo is prepared by ligating two linear AS- oligos which have the stem-loop structure with the cohesive end, and which contain the antisense sequence represented by SEQ. ID. No 7.

15. The RiAS-oligo as set forth in claim 13 or claim 14, wherein the base sequence of the cohesive end within the linear stem-loop AS-oligo is 5' GATC- 3'

16. An AS-oligo-liposome complex containing CMAS- oligo prepared by the method of claim 4 or RiAS-oligo prepared by the method of claim

17. The AS-oligo-liposome complex as set forth in 20 claim 16, wherein the liposome is a cationic liposome.

18. A pharmaceutical composition containing CMAS- olio prepared by the method of claim 4 or RiAS-oligo prepared by the method of claim 10, as an effective ingredient, which can be used for the treatment of cancers, immune diseases, infectious diseases, or other human diseases caused by aberrant gene expression. U IO J I

19. Use of a CMAS-olio prepared by the method of claim 4 or RiAS-oligo prepared by the method of claim in the preparation of a medicament for the treatment of cancers, immune diseases, infectious diseases, or other human diseases caused by aberrant gene expression.

A method of treating cancers, immune diseases, infectious diseases or other human diseases caused by aberrant gene expression comprising the administration of CMAS-olio prepared by the method of claim 4 or RiAS-oligo prepared by the method of claim to a patient in need so such treatment.

21. A method according to claim 1, substantially as hereinbefore described with reference to the o: examples. *00:

22. A method according to claim 4, substantially as o0 20 hereinbefore described with reference to the 000**** S: examples.

23. A method according to claim 10 substantially as 20* hereinbefore described with reference to the S 25 examples. Freehills Carter Smith Beadle 0 Patent Attorneys for the Applicant JONG-GU PARK JONG-GU PARK