JP2002540813A - Novel antisense-oligo with better stability and antisense effect - Google Patents

Abstract

(57) SUMMARY The present invention relates to a novel antisense (AS) oligo comprising one or more antisense sequences for an mRNA region having less secondary structure. In particular, the invention relates to covalently closed multiple antisense (CMAS) -oligos, which are constructed to form closed forms by ligation with complementary primers, and sequences complementary at both 5 'ends. The present invention relates to a ribbon-type antisense (RiAS) -oligo composed of two loops containing a multiple antisense sequence constructed by ligation using, and one stem connecting these two loops. Because the novel AS-oligos of the present invention are very stable to exonuclease activity and show significant inhibition of tumor cell growth, pharmaceutical compositions comprising the novel types of AS-oligos of the present invention may It is effective in treating immune diseases, infectious diseases, and other human diseases caused by abnormal gene expression.

Description

DETAILED DESCRIPTION OF THE INVENTION

FIELD OF THE INVENTION [0001] The present invention relates to novel antisense oligos comprising one or more antisense sequences to an mRNA region with less secondary structure to improve the specificity of the target sequence and stability against nuclease activity. Things.

In particular, the present invention relates to multiple target antisense sequences for mRNAs of various proto-oncogenes such as c-myb, c-myc and k-ras.
Covalently closed multiple antisense (CMAS) containing ntisense sequence)
(covalently-closed multiple antisense)-pertains to oligos. This C
MAS-oligos are constructed to form a closed form by ligation with complementary primers.

Further, the present invention provides a multiple target antisense sequence (mRNA) for mRNA of various proto-oncogenes such as c-myb, c-myc and k-ras.
ribbon-type antisense (RiAS) including antisense sequence
antisense)-for oligos. The RiAS-oligo was ligated with complementary sequences at both major ends, resulting in a stem-loop.
) Constructed to form the structure.

[0004] The present invention relates to a drug comprising a novel type of AS-oligo for the treatment of cancers, immune diseases, infectious diseases, and other human diseases caused by abnormal gene expression. It relates to a composition.

BACKGROUND [0005] Antisense oligonucleotides (hereinafter referred to as "A")
(Referred to as "S-oligo") is important for studying the function of genes by repressing gene expression in a sequence-specific manner (Tompson, CB, et al., Nature, 314, 363-36).
6, 1985). Much effort has also been made to develop molecular antisense agents that eliminate abnormal expression of genes involved in tumor development and progression (Chavany,
C., et al., Mol. Pharm., 48, 738-746, 1995).

[0006] Synthetic AS-oligos have been widely used to facilitate design and synthesis, as well as because of their potential specificity for disease-causing genes. Short-length (13-30 nucleotides) AS-oligos show Watson-Crick base pairing
(Watson-Crick base pairing) and is designed to bind to complementary sequences, thereby providing specificity and affinity. Inhibition of gene expression is thought to be achieved through steric hindrance of RNase H activity or ribosomal complex binding after formation of the DNA-mRNA duplex (Dolnick, BJ, Cancer Inv., 9, 185). -194,1991). The AS-oligo also inhibits gene expression by binding to genomic DNA to form a triple-helix or using a duplex oligo-decoy, which is the primary objective, or by competing with the promoter region of genomic DNA. Efforts have been made
(Young, SL, et al., Proc. Natl. Acad. Sci., USA, 88, 10023-10026, 1991
).

The efficacy of AS-oligos has been confirmed through several animal models and recent clinical studies on human disease. Phosphorothioate (hereinafter, phosphorothioate)
Hepatitis B virus DNA disappeared from duck liver after 10 days of intravenous injection of AS-oligo (designated "PS") (Offenserger, WB, et al., EMBO J., 12, 12).
57-1262, 1993). AS-oligos against angiotensinogen were found to be effective in lowering blood pressure when injected into hypertensive inbred rats (T
omita, N., et al., Hypertension, 26, 131-136, 1995). Phosphorothioate AS for the RIα subunit of protein kinase A subcutaneously in nude mice
-Application of oligos arrested tumor growth (Nesterova, M., et al., Nat.
ed., 1, 528-533, 1995). Also, various clinical trials using AS-oligos for different genes that cause various diseases are producing results in ovarian cancer and Crohn's disease (Roush, W., Science, 276, 1192-1193, 1997). .

However, because the results from many investigators are not always clear and sometimes conflicting, the high expectation of AS-oligos that has the advantage of its sequence specificity for genes and hence the potential for disease is: Often disappointed. AS
-A significant problem with oligos was instability to nucleases and inefficient cell uptake.

[0009] The stability of AS-oligo is determined by the fact that PS- and methylphosphonate (hereinafter "MP") are used to increase the stability to nucleases.
To some extent by using modified oligos such as oligos. However, each of the modified nucleotides has its own problems of lack of sequence specificity and insensitivity to RNaseH. In addition, there has long been a concern that unwanted mutations may be introduced upon recyclization of the hydrolyzed nucleotide.

[0010] AS-oligos bind to complementary target sequences that are effective. Not all sequences of the mRNA show the same accessibility to the AS-oligo. AS-
The unequal binding of oligos can be explained, at least in part, by the secondary and / or tertiary structure of the target mRNA (Gryaznov, S., et al., Nucleic Ac
ids Res., 24, 1508-1514, 1996). Thus, regions with less secondary structure are likely to be easily targeted by AS-oligos.

[0011] To increase the stability of AS-oligos, we needed to construct an AS-oligo with multiple antisense sequences that had a stem-loop structure or covalently closed circles. A rational way to find better target sites using computer simulation to predict the next structure was devised.

[0012] AS-oligos against the c-myb gene can be used to inhibit tumor cell growth.

[0013] The Myb protein, encoded by the c-myb proto-oncogene, is located primarily in the nucleus and functions as a transcriptional regulator in the G1 / S phase transition between cell cycles. The proto-oncogene c-myb plays an important role in the proliferation and differentiation of hematopoietic cells. Hematopoietic cells show differential expression of c-myb and show little expression at the last stage of differentiation (Melani, C., et al., Cancer Res., 51, 2897).
-2901, 1991). In leukemia cells, c-myb was often found to be overexpressed.

[0014] Blockage of c-myb expression by AS-oligos is associated with promyelocytic cancer.
cer) cell line HL-60 and chronic myelogenous leukemia cell line K562 are reported to inhibit growth (Kimura, S., et al., Cancer
Res., 55, 1379-1384, 1995). However, the c-myb used in the above experiment
AS-oligos are shown to have a partial effect. The c-myb AS-oligo used in the above experiment is a phosphodiester (hereinafter referred to as “PO”)-
Oligo or PS capped-oligo (Anfossi, G., e
Natl. Acad. Sci. USA, 86, 3379-3383, 1989). These oligo molecules, especially PO-oligos, are not stable and may account for a partial antisense effect.

AS-oligos with improved stability, selected by rational target site search, have been used to completely remove c-myb mRNA, thereby better improving leukemic cell growth. Can be suppressed. Recently, molecular therapy based on AS-oligo strategy (malignancies) for human malignancies (malignancies)
There is great interest in the development of molecular therapeutics). Therefore, it is desirable to find improved c-myb antisense molecules that completely block leukemic cell growth.

Thus, in order to develop AS-oligos with novel structures with better stability and antisense effects, we in a preferred embodiment analyzed the c-myb mRNA from secondary structure analysis in a preferred embodiment. Eight sites were selected and the selected c-
Myb antisense sequences are combined to create new large antisense molecules, namely covalently-closed multiple antisense molecules.
ense, hereinafter referred to as “CMAS” —Ribbon-type antisense having oligo and loop and stem structures (hereinafter “RiAS”)
-Designed oligos. Therefore, the present inventors have developed these novel AS-
The oligos were shown to be stable to nucleases and show significant specificity to suppress gene expression.

SUMMARY OF THE INVENTION It is an object of the present invention to provide a novel AS-oligo comprising one or more antisense sequences for an mRNA region having less secondary structure to improve specificity for the target sequence and stability against nuclease activity. It is to provide.

According to the present invention, the above objects and advantages are easily obtained.

In this aspect of the invention, the invention provides an antisense sequence selected from a c-myb, c-myc, or k-ras mRNA region having less secondary structure.

The present invention provides covalently closed multiple antisense (CMAS) -oligos comprising multiple antisense sequences to c-myb mRNA. This CMA
The S-oligo is constructed to form a closed form by ligation with complementary primers.

The present invention also provides a ribbon-type antisense (RiAS) -oligo comprising multiple antisense sequences to c-myb mRNA. The RiAS-oligo is composed of two loops containing multiple antisense sequences and a stem linking the two loops constructed by ligation using complementary sequences at both 5'-ends. In addition, the present invention relates to c-myc mRNA or k-ras mRN.
A RiAS-oligo comprising multiple antisense sequences to A is provided.

The present invention further provides a pharmaceutical composition comprising a novel type of AS-oligo for the treatment of cancer, immune diseases, infectious diseases, and other human diseases caused by abnormal gene expression. I do.

[0023] Further objects and advantages of the present invention will be set forth below.

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

FIG. 2 shows the migration pattern of electrophoresis of CMAS-oligo.

A is an oligo analyzed on a 5% Metaphor agarose gel,
At this time, lane 1; size marker, lane 2; 14-mer ligation primer, lane 3; linear 60-mer oligo, and lane 4; CMAS-
Oligo.

B shows the stability of linear and covalently closed oligos in denaturing polyacrylamide gels, lanes 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 c-myb RiAS-oligo.

FIG. 4 shows a migration pattern of electrophoresis of RiAS-oligo.

A is an oligo analyzed on a 15% denaturing agarose gel, where lane 1 is a 58-mer MIJ-78 molecule and lane 2 is a 116-mer Ri
AS-oligo.

B shows MIJ-78 and RiAS- when treated with exonuclease III
FIG. 3 shows the stability test of oligos, in which lanes 1 and 3; no treatment with exonuclease III, and lane 2
And 4; treatment with exonuclease III.

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

A shows the stability test of the linear AS-oligo, where lane 1; serum untreated (negative control), lane 2; 50% live serum (r
aw serum), lane 3; FBS treatment, and lane 4; CS, 24 hours each.

B shows the stability test of CMAS-oligo, in which lane 1; serum untreated (negative control), lane 2; 50% live serum (r
aw serum), lane 3; FBS treatment, and lane 4; CS, 24 hours each.

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

A shows the stability test of the MIJ-78 molecule, in which lane 1; serum untreated (negative control), lane 2; 50% live serum (r
aw serum), lane 3; FBS treatment, and lane 4; CS, 24 hours each.

B shows the stability test of RiAS-oligo, in which lane 1; serum untreated (negative control), lane 2; 50% live serum (r
aw serum), lane 3; FBS treatment, and lane 4; CS, 24 hours each.

FIG. 7 shows c-myb CMA for c-myb expression in HL-60 cells.
It shows the effect of S-oligo.

A shows RT-P performed using total RNA and two c-myb primers.
This shows CR, in which lane 1; 0.3 μg of 60-mer CMAS-oligo + lipofectin (lipofec
1 μg of tin), lane 2; 1 μg of 60-mer CMAS-oligo + 1 μg of lipofectin, lane 3: 1 μg of scrambled AS-oligo + 1 μg of lipofectin.

B: RT-P performed using total RNA and two c-myb primers
5 shows CR, where the upper row: hybridized RT-PCR band of c-myb mRNA, and the lower row; hybridized RT-PCR band of β-actin mRNA.

FIG. 8 shows the cm for expression of c-myb mRNA in HL-60 cells.
It shows the effect of yb RiAS-oligo.

A shows RT-PC performed on total RNA using two c-myb primers.
R, where lane 1; RiAS-oligo 0.1 μg + lipofectin 0
. 8 μg, lane 2; RiAS-oligo 0.2 μg + Lipofectin 0.8
μg and lane 3: SC-oligo 0.2 μg + lipofectin 0.8 μg
.

B: RT-P performed using total RNA and two c-myb primers
5 shows CR, where the upper row: hybridized RT-PCR band of c-myb mRNA, and the lower row; hybridized RT-PCR band of β-actin mRNA.

FIG. 9 shows the effect of 60-mer CMAS or linear AS-oligo on the proliferation of HL-60 cells, where-◆-; CMAS-oligo (1),-■-; CMAS- Oligo (2),-●-;
AS-oligo,-▲-; S-MIJ-7, ●: lipofectin alone, Δ; untreated control, and (1) or (2); number of AS-oligo treatments.

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

A shows the MTT assay for c-myb RiAS-oligo.

B shows the incorporation of [ ³ H] -thymidine in the c-myb RiAS-oligo.

FIG. 11 shows a photomicrograph of the inhibition of HL-60 cell growth by c-myb RiAS-oligo.

A is a c-myb RiAS-oligo, B is an SC-oligo, and C is lipofectin alone.

FIG. 12 shows photomicrographs of the inhibition of HT-29 cell growth by c-myb RiAS-oligo.

A is a c-myb RiAS-oligo, B is an SC-oligo, and C is lipofectin alone.

FIG. 13 shows photomicrographs of the inhibition of HT-29 cell growth by c-myc RiAS-oligo.

A is a c-myc RiAS-oligo, B is an SC-oligo, and C is lipofectin alone.

FIG. 14 shows a micrograph of the inhibition of HT-29 cell growth by k-ras RiAS-oligo.

A is a k-ras RiAS-oligo, B is an SC-oligo, and C is lipofectin alone.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail.

According to one aspect, the present invention provides novel AS-oligos comprising one or more antisense sequences for regions having less secondary structure.

In a particularly preferred embodiment, eight different regions of the c-myb mRNA, one of the proto-oncogenes, were selected as target sites for the antisense oligo. The rational target site search for AS-oligos is used to improve the chance of predicting the native secondary structure. The eight antisense sequences are:
Complementary to the sequence of the selected target site. Of the eight selected target sites for the AS-oligo, four sites (MIJ-1, MIJ-2, MIJ-3, and 4) to form a minimal intramolecular secondary structure (see Table 1) MIJ-
4) is finally selected in combination when forming a CMAS molecule, and the three sites (MIJ-3, MIJ-4, and MIJ-17) are combined to form a RiAS molecule.

An AS-oligo having a phosphodiester backbone is
Lack of stability which is essential for good antisense application. Modified oligos, such as PS-oligos or MP-oligos, exhibited improved stability,
The increase in stability was only partial and involved a potential dangerous misincorporation of hydrolyzed modified nucleotides during DNA replication or repair.
It has been previously reported that stem-loop oligos complexing with cationic liposome also show a partial increase in stability. However, AS-
For oligos, stability is still an important concern. For this reason, the present inventors
An attempt was made to develop improved AS-oligos with better stability.

Thus, the present invention provides covalently closed multiple antisense (covalently-
closed multiple antisense (CMAS) -provides an oligo.

In particular, the intracellular secondary structure of the AS-oligo was determined by the AS-oligo and target mR
Built without duplex formation between NAs. In a preferred embodiment, a CMAS (covalently linked) comprising four antisense sequences (MIJ-1, MIJ-2, MIJ-3, and MIJ-4) described by SEQ ID NOs: 1, 2, 3, and 4 in a loop These AS-oligos, referred to as multiple antisense) -oligo forms closed in tandem, are placed in tandem to increase the length of the CMAS-oligo (see FIG. 1). The CMAS-oligo is about 10% slower than the linear precursor on a 15% denaturing PAGE gel,
y pattern) (see FIG. 2A). The CMAS-oligo is, as expected, resistant to exonuclease III and shows multiple bands on denaturing PAGE gels, where the monomer (60 mer) is the most abundant and the dimer (120 mer) And a trimmer (180mer). In contrast to CMAS-oligos, linear oligos are completely degraded after 2 hours incubation with exonuclease III (see FIG. 2B).

The present invention also provides a ribbon type antisense (RiAS) -oligo.

[0063] CMAS-oligos are very stable, but require primers for intramolecular ligation that must be subsequently removed. Thus, in order to avoid the use of ligation primers and to obtain a homogenous population of AS-oligos, we have established that the RiAS-
A ribbon-shaped closed molecule called an oligo is formed.

The RiAS-oligo (116mer) is composed of two loops and one stem connecting the two loops (see FIG. 3). In a preferred embodiment,
Three antisense sequences (MIJ-3, MIJ-4 and MIJ-17) described by SEQ ID NOs: 3, 4 and 5 in the loop are placed in tandem to increase the length of the CMAS-oligo. As a result, two of the three different antisense sequences
Copies (all six antisense sequences) are on the RiAS-oligo. Such extended length loops in the RiAS-oligo relieve the torsional stress caused by forming a duplex with the target mRNA sequence. RiAS
-The oligo is found to be significantly slower than its linear precursor (MIJ-78) on denaturing PAGE gels (see Fig. 4A). RiAS-oligo, as expected,
It is resistant to exonuclease III and is shown on the PAGE gel as a major band (116 mer). Contrary to RiAS-oligo, MIJ-78 is
Completely degraded after 2 hours incubation with exonuclease III (
(See FIG. 4B).

In order to demonstrate the enhanced stability of the CMAS-oligos and RiAS-oligos of the invention to nuclease activity, the CMAS-oligos and RiAS-oligos with heat-inactivated serum to maintain nuclease activity. Incubated.

As a result, a linear 60-mer oligo (precursor of CMAS-oligo, see FIG. 5A) and a linear 59-mer oligo (precursor of RiAS-oligo, FIG. 6)
A) is completely digested after 24 hours incubation in the presence of serum. However, CMAS-oligos and RiAS-oligos remain almost intact after 24 hours of incubation with raw human serum, FBS, and bovine serum, thus significantly improving stability compared to the linear one for nuclease activity. (See FIG. 5B and FIG. 6B).

In addition, it is shown that CMAS-oligos function to better eliminate sequence-specific target mRNAs.

In particular, CMAS-oligo was delivered intracellularly in combination with Lipofectin. Lipofectin is used because it has been shown to be less toxic to cells and produce reproducible results. MIJ-5, a CM for human c-myb
AS-oligo can reduce 95% or more of c-myb mRNA compared to control SC-oligo. On the other hand, MIJ-5A, a linear counterpart of MIJ-5, reduces about 37% of c-myb mRNA (FIG. 7).
A). These results indicate that the CMAS-oligos of the invention are superior to the linear ones for excision of target mRNA, even when used in lower amounts.

The function of RiAS-oligo to remove target mRNA is that CMAS-
Indicated by a similar method as in the oligo.

The HL-60 cells were transformed with RiAS-oligo, scrambled (S
C) -oligos and also lipofectin alone were tested. RiAS-oligo,
After forming a complex with lipofectin, it is delivered into cells. As a result, R
iAS-oligos can completely excise c-myb mRNA. In contrast, SC-oligo was about 30 times less than that treated with lipofectin alone.
% Of c-myb mRNA (see FIG. 8A). These results indicate that the RiAS-oligos of the present invention show that the target mR
It shows that it is excellent for excision of NA.

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

In the case of CMAS-oligo, it can be seen that when the c-myb message is amplified by RT-PCR, more than 90% of the message is reduced by the MIJ-5 treatment (see FIG. 7B). ).

In the RiAS-oligo, the c-myb message amplified by RT-PCR is detected together with a labeled internal hybridization oligo (30mer) (see FIG. 8B). From the above results, the amplified message is truly c-my
b.

The present invention also provides a pharmaceutical composition comprising a novel type of AS-oligo for the treatment of cancer, immune diseases, infectious diseases, and other human diseases caused by abnormal gene expression. Is what you do.

It is shown that c-myb CMAS-oligo or c-myb RiAS-oligo inhibits leukemic cell growth. In particular, c-myb against leukemia cells
Growth inhibition of CMAS-oligo and c-myb RiAS-oligo was measured by ^three methods, MTT assay, [ ³ H] thymidine incorporation or colony formation on soft agarose.

As a result of the MTT assay, the cell number gradually decreases with increasing amounts of MIJ-5, a CMAS-oligo against human c-myb. Inhibition of cell growth is more pronounced when cells are treated twice with MIJ-5. Inhibition of growth of more than 80% of HL-60 cells is also observed at low concentrations (see FIG. 9). On the other hand, the linear 60-mer AS-oligo, MIJ-5A, and the linear sense oligo, do not result in significant inhibition of cell growth as compared to the sham control.
These results indicate that the c-myb CMAS-oligo of the present invention is a useful antisense agent and is effective for tumor growth depending on the concentration.

In addition, it is observed that cell growth is inhibited by more than 91% by RiAS-oligo (see FIG. 9A). On the other hand, SC-oligo and lipofectin alone,
Does not significantly inhibit cell growth as compared to untreated controls. These results indicate that the c-myb RiAS-oligo of the present invention is also an effective antisense agent for inhibiting the growth of leukemic cells.

For colony formation on soft agarose, MIJ-5 reduces the number of colonies formed by more than 90% (see Table 2). MIJ-5A also reduces the number of colonies formed, although less effective at inhibiting growth, reducing about 70% of the colonies. In contrast, sense oligo and SC-oligo were about 11% and 3%, respectively.
It shows a slight colony reduction of 2%.

In addition, c-myb RiAS-oligo transfected into cells can reduce the number of colonies formed by about 92% (see Table 3) as compared to the untreated control. On the other hand, SC-oligo and Lipofectin alone show a slight colony reduction of about 7.9% and 7.1%, respectively.

In addition, inhibition of growth of c-myb RiAS-oligo on leukemia cells [ ^Three H] Observed by thymidine incorporation. In particular, RiAS-oligos are HL-oligos.
Inhibits the growth of 60 cells by 93%. On the other hand, SC-oligo and Lipofectin alone
Shows a moderate inhibition of cell growth of about 16.8% and 15.4%, respectively.
(See FIG. 10B). Microscopic observation revealed that c-myb RiAS-oligo
After treatment with HL-60 cells, the growth of HL-60 cells was scrambled.
It is significantly inhibited compared to cells treated with rigo and lipofectin alone (FIG. 1).
1).

Inspired by the remarkable inhibitory activity of c-myb RiAS-oligos in the present invention, we constructed other RiAS-oligos for two different proto-oncogenes, c-myc and k-ras. And c-myc RiAS-oligo and k
It was investigated whether -ras RiAS-oligo functions well in inhibiting cell growth.

According to the results of microscopic observation, the growth of HT-29 cells was scrambled (scram
bled) All RiA compared to cells treated with oligo and lipofectin alone
It is significantly inhibited by S-oligo, c-myb RiAS-oligo, c-myc RiAS-oligo and k-ras RiAS-oligo (see FIGS. 12, 13 and 14).

Thus, the novel RiAS-oligos of the present invention exhibit effective growth inhibition of tumor cells against various target sequences, as well as enhanced stability against nuclease activity. For this reason, the novel RiAS-oligos of the present invention are usefully used for the development of molecular antisense oligos for treating various human diseases.

EXAMPLES The actual and presently preferred embodiments of the present invention are described in detail, as set forth in the following examples.

However, it is conceivable that those skilled in the art, in light of this disclosure, will make modifications and improvements within the spirit and concept of the invention.

Example 1 Selection of Target Site for AS-Oligo The selection of the target site for AS-oligo was based on
It was found to be important in achieving NA reduction or ablation. However, the method of selecting the target site was rather arbitrary. For this reason, we scanned the complete sequence of human c-myb mRNA for putative secondary structure and searched for a reasonable target site in the preferred embodiment.

In particular, the simulation of the secondary structure is performed by the DNAsis program (Hitachi So
ftware, Japan). The complete c-myb sequence was scanned sequentially for the formation of secondary structure in a continuous frame of 100 bases. Next, the frame for the simulation of the secondary structure was shifted by 30 bases at a time to obtain an overlap of 60 bases on the 5 ′ side. This process was repeated again to scan a given sequence for potential secondary structures in three different frames.

As a result, eight sequences having the minimum secondary structure in three different frames were selected from the c-myb mRNA sequence (Table 1). Using a rational target site search for AS-oligos has improved the chance of predicting the native secondary structure.

[0089]

[Table 1]

As shown in Table 1, the eight antisense sequences were complementary to the selected target sites. Of the eight selected target sequences for the AS-oligo, four sites (MI
J-1, MIJ-2, MIJ-3, and MIJ-4) are selected in combination when forming a CMAS molecule, and three sites (MIJ-3, MIJ-4, and MIJ) are selected.
-17) was selected in combination to form RiAS-oligos.

Example 2: Construction of multiple antisense (CMAS) -oligos covalently closed We tried to develop improved AS-oligos with better stability.

The four different AS-oligos obtained according to Example 1 ((MIJ-1, MIJ
-2, MIJ-3, and MIJ-4) were used to construct CMAS-oligos. One CMAS-oligo was constructed to accommodate a combination of four different antisense sequences that minimizes secondary structure so as to more easily bind to the target site. AS-oligos are phosphorylated during synthesis at the 5 'end, followed by intramolecular or intermolecular covalent ligation (Figure 1). The sequence of a 60-mer AS-oligo containing four different antisense sequences is set forth by SEQ ID NO: 7. Both ends of the AS-oligo are ligated with ligation primers having a sequence complementary to both ends of the 60-mer AS-oligo at both ends (7 bases on each side). The sequence of the 14mer ligation primer is set forth by SEQ ID NO: 6. The ligation primer was mixed with the AS-oligo, heated at 85 ° C. for 2 minutes, and then gradually cooled to ambient temperature. One unit of T4 ligase was added and incubated at 16 ° C. for 16 hours to produce a covalently closed molecule. The CMAS- oligo, 5% Metaphor A Gallo - Sugeru ^{(Metaphor T M agarose gel) (} FMC, USA) or were electrophoresed on 12% denaturing PAGE, and compared to oligo resistance and linear 60mer to exonuclease III Identified by some gel delay. The ligation primer was exonuclease I
After being digested by II or heating the oligo at 90 ° C., it was detached from the CMAS-oligo by running on a denaturing gel.

As a result, the intracellular secondary structure of the AS-oligo was
Constructed without duplex formation of RNA. This AS-oligo was used for CMAS
(Multiple antisense covalently closed) -oligo. The CMAS-oligo showed an electrophoretic migration pattern that was about 10% slower than the linear precursor on a 15% denaturing PAGE gel (FIG. 2A). The CMAS-oligo is, as expected, resistant to exonuclease III, showing multiple bands on denaturing PAGE gels, where the monomer (60 mer) is the most abundant, followed by the dimer (120 mer) and It was a trimmer (180mer) (FIG. 2B). C
Contrary to the MAS-oligo, the linear oligo is exonuclease III
After a period of incubation, it was completely degraded.

Example 3 Construction of Ribbon-Type Antisense (RiAS) -Oligo We enzymatically link two AS-oligos to form a ribbon-type closed ring molecule called RiAS-oligo. did.

In particular, the RiAS-oligo is composed of two loops and one stem connecting the two loops. Each loop has three different antisenses (MIJ-3, MIJ-4, and MIJ-1) described by SEQ ID NOs: 3, 4, and 5.
7) Included sequence. A combination of three antisense sequences with as few secondary structures as possible was chosen for the AS-oligo to bind more easily to the target site. C-myb AS-oligo (MIJ-78) was phosphorylated at the 5 'end. The sequence of the 58mer MIJ-78 is set forth by SEQ ID NO: 8. MIJ-78 forms a stem-loop structure. The stem was formed by the complementary sequences at both ends of each oligo. The 5 'end of the stem is 5'-(
p) Had 4 bases of a single standard sequence of GATC-3 '. Two MIJs
-78 molecules were linked by complementary 4 base sequences at both 5 'ends. MIJ-78 molecules were mixed, heated at 85 ° C. for 2 minutes, and then gradually cooled to ambient temperature. One unit of T4 DNA ligase was added and incubated at 16 ° C. for 24 hours to produce a diad-symmetric covalently linked molecule (FIG. 3). RiAS-oligos were electrophoresed on a 15% denaturing polyacrylamide gel to determine resistance to exonuclease III and gel delay.

As a result, a RiAS-oligo (116 mer) composed of two loops and one stem connecting the two loops was constructed. The three antisense sequences in the loop were placed in tandem to increase the length of the RiAS-oligo. As a result, two copies of three different antisense sequences (all six antisense sequences)
Was in the RiAS-oligo. Such extended length loops in the RiAS-oligo relieved the torsional stress created by forming a duplex with the target mRNA sequence. RiAS-oligo was found to be significantly slower than its linear precursor (MIJ-78) on denaturing PAGE gels (FIG. 4A).
). The RiAS-oligo was, as expected, resistant to exonuclease III and showed a major band (116 mer) on the PAGE gel (FIG. 4B).
). Contrary to RiAS-oligo, MIJ-78 is an exonuclease III
Was completely degraded after incubation for 2 hours.

Example 4: Enhanced safety of CMAS-oligos and RiAS-oligos on nuclease activity To test the stability of the CMAS-oligos and RiAS-oligos of the invention on nuclease activity, CMAS-oligos and RiAS-oligos -Oligos were incubated with serum that was not heat inactivated to maintain nuclease activity.

In particular, non-specific control-phosphodiester oligos (linear 60 me
1 μg of each of r) and CMAS-oligo were incubated with raw human serum, FBS and bovine serum (inactivated without heating; HyClone, Logan, Utah, USA) or exonuclease III. 15% serum was added to each 100
Added to μl reaction volume of AS-oligo and incubated at 37 ° C. for 24 hours. Next, the AS-oligo was extracted with phenol and chloroform and examined on a 15% denaturing PAGE gel. 160 U / μg oligo exonuclease III (T
akara, Japan) to the linear and CMAS-oligos at 37 ° C. for 2 hours,
Incubated. AS-oligo treated with exonuclease III was also extracted and electrophoresed in the same manner as described above.

As a result of the CMAS-oligo, the linear 60-mer oligo was 2
After 4 hours incubation, it was completely digested (FIG. 5A). However, the CMAS-oligos of the present invention were used with live human serum, FBS, and bovine serum for
Even after 4 hours of incubation, most remain intact,
It showed significantly improved stability to nucleases than the linear ones (FIG. 5).
B).

In the case of RiAS-oligo, the linear 58mer was completely hydrolyzed after 24 hours incubation in the presence of each different serum (FIG. 6A). However, the RiAS-oligos of the invention showed significantly improved stability over the nuclease-linear one, since most remained intact even after 24 h incubation with live serum ( FIG. 5B).

Example 5: c-myb mRN with CMAS-oligo and RiAS-oligo
Specific Reduction of A Inspired by the remarkable stability of CMAS-oligos and RiAS-oligos in the present invention, we have found that AS-oligos function well to sequence-specifically remove target mRNAs. I checked whether to do it.

<5-1> Cell line and tissue culture A leukemia cell line, HL-60 (promyelocytic leu
kemia cell line) and K562 (chronic myelogenous cell line)
leukemia cell line) and 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 / streptomycin. Cells were maintained at 37 ° C. in a CO ₂ incubator. Routine cell culture practices were rigorously implemented to maintain the proper cell density and thaw the stock vials and culture the cells no more than 5 generations. The culture solution is AS
-Changed one day before treating with oligo.

<5-2> CMAS-oligo and R Formed in Complex with Cationic Liposomes
Transfection of iAS-oligo 0.3 μg CMAS-oligo and 0.8 μg Lipofectin ^™ (Gibco BRL, USA)
Alternatively, add 0.2 μg of RiAS-oligo and 0.8 μg of Lipofectin ^™ to 20 μl
Of OPTI-MEM ^™ each and incubated for 40 minutes at ambient temperature. Each component was mixed to form a complex at ambient temperature for 15 minutes. One day before the addition of the oligos, the cells were washed with a fresh culture medium (RP
MI 1640 + 10% FBS) and washed twice with OPTI-MEM before conducting the experiment. The cell density was adjusted to 5 × 10 ⁵ cells / ml, and 100 μl was dispensed into a 48-well plate (Falcon, USA). 40 μl of the liposome-oligo complex was added to the cells twice, once on day 0 and once on day 1. The oligo treated cells were incubated for 4 hours at 37 ° C., 5% CO ₂ , followed by 10%
100 μl of OPTI-MEM containing FBS was added. The next day, 100 μl of the supernatant was carefully removed and fresh OPTI-M containing oligo-liposome complexes was removed.
Replaced with 20 μl of EM. After 4 hours, the cells were treated with 100 μl of complete medium containing antibiotics.
1 was added and incubated another day at 37 ° C. before the assay.

<5-3> Isolation of Total RNA and RT-PCR According to the method recommended by the manufacturer, Tripure ^™ isolation reagent (Tripu
total RNA was isolated using re ^™ Isolation Reagent) (Boehringer Manhein, Germany). Briefly, 0.4 ml of Tripure reagent, 10 μl was added to the collected cells.
g of glycogen and 80 μl of chloroform were added to obtain total RNA. R
T-PCR was performed in one reaction tube using the Access ^™ RT-PCR kit (Promega, USA). RNA, PCR primer,
AMV reverse transcriptase (5 U / μl), Tfl DNA polymerase (5 U / μl)
, DNTPs (10 mM, 1 μl) and MgSO ₄ (25 mM, 2.5 μl). First strand cDNA synthesis was performed at 48 ° C for 45 minutes.
Performed on a termal cycler (Hybaid, USA). Next, PCR amplification was performed for 25 cycles according to the method recommended by the manufacturer. The amplified PCR product was confirmed on a 1% agarose gel and quantified by a gel documentation program (Bio-Rad, USA).

<5-4> Southern Hybridization of RT-PCR Fragment The RT-PCR product was electrophoresed on a 1% agarose gel. 0.4M DNA
Transferred onto a nylon membrane (New England Biolab, USA) for 4 hours in NaOH. The membrane was hybridized with a 30 mer internal primer labeled with ECL 3'-end oligolabeling and a detection system (Amersham Life Science, England). The sequence of the 30 mer internal primer is set forth by SEQ ID NO: 9. Hybridization was performed at 62 ° C. for 60 minutes in 6 ml of a buffer containing 5 × SSC and 0.02% SDS. The membrane was washed twice with 5 × SSC containing 0.1% SDS and twice with 1 × SSC containing 0.1% SDS for 15 minutes.
After blocking the membrane with the blocking solution, 30 minutes before autoradiography,
Treated with HRP (horseradish peroxidase) anti-fluorescein conjugated antibody.

The CMAS-oligos of the present invention have been shown to work well to remove target mRNA specifically to the sequence.

In particular, CMAS-oligos were complexed with lipofectin and delivered intracellularly. Lipofectin was used because it has been shown to be less toxic to cells and to give reproducible results. As a result, a complex was formed with 1 μg of lipofectin for transfection of HL-60 cells with 0.3 μg of MIJ-5 and CMAS-oligo against human c-myb. MIJ-5 is
The c-myb mRNA could be reduced by more than 95% compared to the control SC-oligo. On the other hand, MIJ-5's linear counterpart, MIJ
-5A reduced c-myb mRNA by about 37% (FIG. 7A). These results indicate that the CMAS-oligos of the invention are superior to the linear ones for excision of target mRNA, even when used in lower amounts.

It has also been shown that the RiAS-oligos of the present invention function well to remove target mRNA specifically to the sequence.

HL-60 cells were transfected with RiAS-oligo, SC-oligo, and lipofectin alone. The RiAS-oligo was delivered into cells after forming a complex with Lipofectin. RiA against human c-myb
S-oligo (0.1 μg or 0.2 μg) was complexed with 0.8 μg lipofectin for transfection into HL-60 cells. As a result, 0.2 μg of RiAS-oligo (40 nM) was able to completely excise c-myb mRNA. On the other hand, 0.1 μg of RiAS-oligo was c-my
b Reduced about 70% of mRNA (FIG. 8A). In contrast, SC-oligo had about 30% c-myb mRN compared to that treated with lipofectin alone.
A showed a slight decrease. However, the expression of β-actin shown at the bottom was not affected by the treatment of RiAS-oligo and further by other treatment conditions.
These results indicated that RiAS-oligo was excellent at excision of target mRNA even when used in small amounts.

We also investigated the antisense effect of CMAS-oligo and RiAS-oligo by Southern blotting with PCR products. HL-60 cells in M
Transfected with IJ-5 and oligos such as control oligos, these cells were used to isolate total DNA.

In the case of CMAS-oligo, it was found that when the c-myb message was subjected to RT-PCR, more than 90% of the messages were reduced by MIJ-5 treatment (
FIG. 7B). However, the expression of β-actin shown at the bottom shows that MIJ-5
Was not affected by the treatment.

In the case of RiAS-oligo, C-myb message amplified by RT-PCR was detected with a labeled internal hybridization oligo (30mer) (FIG. 8B). From these results, the amplified message is indeed cm-m
It was derived from yb, and it was confirmed that the message could be completely removed by treating with 0.2 μg of c-myb RiAS-oligo.

Example 6 Inhibition of Effective Growth of Leukemia Cells by c-myb CMAS-Oligo and c-myb RiAS-Oligo It was reported that c-myb plays a key role in leukocyte proliferation. c
The effect of AS-oligo on -myb was also reported to selectively block leukemic cell growth. For this reason, we tested the c-myb CMAS-oligo and c-myb RiAS-oligo of the invention for inhibition of leukemic cell growth.

In particular, c-myb CMAS-oligo and c-myb R against leukemic cells
Inhibition of iAS-oligo growth was measured in three different ways: MTT assay, [ ³ H].
It was determined by thymidine incorporation or colony formation on soft agarose.

<6-1> MTT Assay MTT (3,-[4,5-dimethylthiazol-2-yl] 2,5-diphenyltetrazolium bromide (3,-[4,5-Dimethylthiazol-2-yl] 2) , 5-diphenyl-tetra
zolium bromide) (hereinafter referred to as “MTT”).
60 cells were washed twice with OPTI-MEM, and 50 μl was added to a 96-well plate (
(5 × 10 ³ cells / well). Cells were harvested with different amounts of oligo (CMAS-
For oligos, 0.01-1 μg / 15 μl or for RiAS-oligos, 0.2-1 μg / 15 μl.
μg / 15 μl) and lipofectin (0.2 μg / 15 μl) were treated for 5 hours and cultured for 5 days. Next, the cells were collected in a volume of 100 μl,
20 μl (100 μg) of MTT reagent (5 mg / ml in PBS; Sigma, USA)
Was added and incubated at 37 ° C. for 4 hours. 100 μl of isopropanol (containing 0.1 N HCl) was added to the cells and incubated for another hour at ambient temperature. Absorbance was measured at 570 nm in an ELISA reader to determine the amount of living cells.

For CMAS-oligo, cell numbers progressively decreased with increasing amounts of MIJ-5. When cells were treated twice with MIJ-5, the inhibition of cell growth became even more pronounced. Greater than 80% growth inhibition of HL-60 cells is achieved at low concentrations, ie, 0.
Even 13 μg (0.24 μg total) of CMAS-oligo was observed (FIG. 9).
On the other hand, the linear 60-mer AS-oligo, MIJ-5A and the linear sense oligo did not result in significant inhibition of cell growth as compared to the sham control. These results indicated that c-myb CMAS-oligo was an effective antisense agent and, depending on concentration, an effective agent for tumor growth.

With RiAS-oligo, cell growth was also observed to be inhibited by 91% with RiAS-oligo (FIG. 10A). On the other hand, SC-oligo and lipofectin alone did not significantly inhibit cell growth when compared to untreated controls. These results indicated that c-myb RiAS-oligo was an effective antisense agent for inhibiting leukemic cell growth.

<6-2> Colony formation with soft agarose c-myb CMAS-oligo and c-myb RiAS for leukemia cells
-Oligo growth inhibition was also measured by colony formation on soft agarose as an alternative.

In particular, K562 cells were transfected as described in Example 6 and cultured at 37 ° C. with 5% CO ₂ for 24 hours. An equal volume of a mixture of 0.8% low melting point agarose and 2 (RPMI-1640 containing 20% FBS and antibiotics) was added to the cells and seeded into 6-well plates to solidify. 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 2). MIJ-5A also reduced the number of colonies formed, although less effective at inhibiting growth, and reduced about 70% of the colonies. In contrast, the sense oligo and SC-oligo showed a slight colony reduction of about 11% and 32%, respectively.

[0121]

[Table 2]

In contrast, cells transfected with c-myb RiAS-oligo had about 92% more colonies formed than untreated controls (Table 3).
) Could be reduced. On the other hand, SC-oligo and Lipofectin alone showed a slight colony reduction of about 7.9% and 7.1%, respectively.

[0123]

[Table 3]

<6-3> Incorporation of [ ³ H] thymidine Inhibition of leukemic cell growth by c-myb RiAS-oligo was also measured by [ ³ H] thymidine incorporation.

For incorporation of [ ³ H] thymidine, HL-60 cells were treated with AS-oligo as described above. Cells were treated with 0.5 μCi of [ ³ H] thymidine (2.0 C
i / mmol; Amersham, England) and incubated in triplicate for 16 hours. The cells were then collected on glass microfiber filters (Whatman GF / C, England). The filters were washed sequentially with cold PBS, 5% TCA and absolute ethanol. [ ³ H] thymidine incorporation was measured in toluene, Triton X-100, P
A cocktail solution containing PO and POPOP was measured with a liquid scintillation counter.

As a result, RiAS-oligo (0.2 μg) inhibited the growth of HL-60 cells by 93
% Inhibition (FIG. 10B). On the other hand, SC-oligo and lipofectin alone showed about 16.8% and 15.4% inhibition of light cell growth, respectively. When observed under a microscope, it was scrambled after treatment with c-myb RiAS-oligo.
(scrambled) when compared to cells treated with oligo and lipofectin alone,
L-60 growth was significantly inhibited (FIG. 11).

Example 8: Effective growth inhibition of c-myc RiAS-oligo and k-ras RiAS-oligo Inspired by the remarkable inhibitory activity of c-myb RiAS-oligo in the present invention,
We proceeded in a similar manner as in Example 3 with two different proto-oncogenes,
Other RiAS-oligos for c-myc and k-ras were constructed.

Next, it was examined whether c-myc RiAS-oligo and k-ras RiAS-oligo function well to inhibit cell growth.

In particular, we used a different cell line, the colorectal adenocarcinoma cell line HT-29. C-myc RiAS-oligo and k-ras R for tumor cells
Inhibition of iAS-oligo growth was measured by [ ³ H] thymidine incorporation in the same manner as in Example <6-3>. HT-29 cells each had 0
. Cations of 2 μg c-myb RiAS-oligo and 0.6 μg lipofectin or 0.5 μg c-myc RiAS-oligo and 1.5 μg lipofectin or 0.5 μg k-ras RiAS-oligo and 1.5 μg lipofectin Treated with a soluble liposome complex. After treatment with each RiAS-oligo for 5 days, the growth of HT-29 cells was observed using a microscope. Each micrograph showed the effect on growth inhibition after treatment with RiAS-oligo (A), scrambled oligo (B) and lipofectin alone (C).

Observation of the micrographs showed that the growth of HT-29 cells was comparable to that of all RiAS- cells compared to cells treated with scrambled oligo and lipofectin alone.
Oligo, c-myb RiAS-oligo, c-myc RiAS-oligo and k
Significantly inhibited by -ras RiAS-oligo (FIGS. 12, 13 and 14).

Thus, the novel RiAS-oligos of the present invention showed effective growth inhibition of tumor cells against various target sequences, as well as enhanced stability against nuclease activity. For this reason, the novel RiAS-oligos of the present invention can be effectively used for the development of molecular antisense oligos against various human diseases.

INDUSTRIAL APPLICABILITY The present invention relates to a novel AS comprising one or more antisense sequences for mRNA regions having less secondary structure and having better target sequence specificity and stability against nuclease activity. Providing an oligo.

In particular, the present invention relates to covalently closed multiple antisense (CMAS) comprising multiple target antisense sequences to c-myb mRNA constructed to form a closed form by ligation with complementary primers. ) -Provides an oligo. In addition, the present invention relates to a cm that is constructed to form a stem-loop structure by ligation with sequences complementary at both 5 'ends.
The present invention provides a ribbon-type antisense (RiAS) -oligo containing multiple target antisense sequences for yb mRNA.

Human cell lines were analyzed for c-myb RiAS-oligo and c-myb CMA.
S-oligos, and also c-myc RiAS-oligos and k-ras RiASs
-Treatment with oligos shows that aberrant gene expression is effectively eliminated by the novel AS-oligos of the invention. Thus, it is suggested that the novel AS-oligos of the present invention will be used to develop molecular antisense agents against a variety of disease-causing genes, as well as for functional studies of genes. In particular,
The novel AS-oligos of the present invention are used in the development of pharmaceutical compositions for the treatment of cancer, immune diseases, infectious diseases, or other human diseases caused by abnormal gene expression.

Those skilled in the art will recognize that the concepts and specific embodiments disclosed in the foregoing description are readily available as a basis for modifying or designing different embodiments to accomplish the same purpose of the invention. Will think. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and concept of the invention as set forth in the appended claims.

[Sequence list]

[Brief description of the drawings]

FIG. 1 shows a scheme for the construction of c-myb CMAS-oligo.

FIG. 2 shows the migration pattern of CMAS-oligo electrophoresis.

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

FIG. 4 shows the migration pattern of RiAS-oligo electrophoresis.

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

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

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

FIG. 8: c-myb R for expression of c-myb mRNA in HL-60 cells
It shows the effect of iAS-oligo.

FIG. 9 shows the effect of 60mer CMAS or linear AS-oligo on proliferation of HL-60 cells.

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

FIG. 11 shows a micrograph of the inhibition of HL-60 cell growth by c-myb RiAS-oligo.

FIG. 12 shows a micrograph of the inhibition of HT-29 cell growth by c-myb RiAS-oligo.

FIG. 13 shows photomicrographs of the inhibition of HT-29 cell growth by c-myc RiAS-oligo.

FIG. 14 shows photomicrographs of the inhibition of HT-29 cell growth by k-ras RiAS-oligo.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 35/02 A61P 37/00 37/00 C12Q 1/68 Z C12Q 1/68 C12N 15/00 A (81 ) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF) , CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW) ), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP , KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW

Claims (23)

[Claims] 1. Improving the specificity of its target sequence by including one or more antisense sequences to mRNA regions with less secondary structure and improving its stability to nuclease activity by constructing a closed form New antisense oligo. 2. The novel AS-oligo according to claim 1, wherein said mRNA is transcribed from various genes that cause human diseases. 3. The method of claim 1, wherein the antisense is a multiple antisense (CMAS) -oligo covalently closed, wherein the CMAS-oligo is constructed in a closed form by ligation of the AS-oligo with a ligation primer. New AS-
Oligo. 4. The AS-oligo is a 60-mer described by SEQ ID NO: 7.
The CMAS-oligo according to claim 3, which is an AS-oligo of (1). 5. The CMAS-oligo according to claim 4, wherein SEQ ID NO: 7 consists of the four antisense sequences described by SEQ ID NOs: 1, 2, 3, and 4. 6. The CMAS-oligo according to claim 3, wherein the ligation primer consists of the 14-mer nucleotide set forth by SEQ ID NO: 6. 7. The CMAS-oligo according to claim 3, which is effective for eliminating or suppressing the expression of an abnormal gene associated with a human disease. 8. The gene may be a proto-oncogene, c-myc, c-myb or k
The CMAS-oligo according to claim 7, which is -ras. 9. The CM according to claim 3, which effectively inhibits the growth of tumor cells.
AS-oligo. 10. The CMAS-oligo according to claim 9, wherein the tumor cells are the promyelocytic leukemia cell line HL-60, the chronic myeloid leukemia cell line K562, or the colorectal adenocarcinoma cell line HT-29. . 11. An antisense (RiAS) -oligo of the ribbon type, wherein R
2. The novel AS-oligo according to claim 1, wherein the iAS-oligo is constructed in a ribbon form by ligation of two AS-oligos with sequences complementary at each 5 'end. 12. The RiAS-oligo according to claim 11, which has a stem-loop structure having two loops and one stem connecting the two loops. 13. The AS-oligo according to claim 58, wherein the AS-oligo is 58 m
12. The RiAS-oligo of claim 11, which is an AS-oligo of er. 14. The RiAS-oligo according to claim 13, wherein said SEQ ID NO: 8 consists of the three antisense sequences set forth by SEQ ID NOs: 3, 4, and 5. 15. The RiAS-oligo of claim 11, wherein said complementary sequence is described as 5 '-(p) GATC-3'. 16. The RiAS-oligo according to claim 11, which is effective for eliminating or suppressing the expression of an abnormal gene associated with a human disease. 17. The RiAS-oligo according to claim 11, wherein the gene is a proto-oncogene, c-myc, c-myb or k-ras. 18. The RiAS-oligo of claim 11, which effectively inhibits tumor cell growth. 19. The RiAS-oligo according to claim 18, wherein the tumor cells are the promyelocytic leukemia cell line HL-60, the chronic myeloid leukemia cell line K562, or the colorectal adenocarcinoma cell line HT-29. . 20. An AS-oligo-liposome complex comprising the CMAS-oligo according to claim 3 or the RiAS-oligo according to claim 11. 21. The liposome of claim 2, wherein the liposome is a cationic liposome.
0. The AS-oligo-liposome complex according to 0. 22. A pharmaceutical composition comprising the CMAS-oligo according to claim 3 or the RiAS-oligo according to claim 11 as an active ingredient. 23. The pharmaceutical composition according to claim 22, which is used for treating cancer, an immune disease, an infectious disease, or other human diseases caused by abnormal gene expression.