KR20020013522A - The novel antisense-oligos with better stability and antisense effect - Google Patents

Abstract

PURPOSE: Provided are novel antisense oligos containing one or more antisense sequence to mRNA region with a less secondary structure to improve their target sequence specificity and stability to nuclease activities. CONSTITUTION: The novel antisense (AS) oligos contains one or more antisense sequence to mRNA region with a less secondary structure. In particular, they are a covalently-closed multiple antisense (CMAS)-oligo, which is constructed to form a closed type by ligation using complementary primer, and a ribbon-type antisense (RiAS)-oligo, which is composed of two loops containing multiple antisense sequences and a stem connecting the two loops that is constructed to by ligation using complementary sequences at both 5 prime ends. Since the novel AS-oligos are extremely stable to exonuclease activities, and show a significant growth inhibition of tumor cells, pharmaceutical compositions containing the novel types of AS-oligos are effectively used in treating cancer, immune diseases, infectious diseases, and other human diseases caused by aberrant gene expression.

Description

The novel antisense-oligos with better stability and antisense effect}

Antisense oligonucleotides (hereinafter referred to as "AS-oligo") are important for the study of gene function through the reduction of sequence specific gene expression (Tompson, CB, et al., Nature , 314, 363). -366, 1985 ). Much effort has been made to develop molecular antisense agents that eliminate abnormal expression of genes involved in the development and development of cancer (Chavany, C., et al., Mol. Pharm., 48, 738). -746, 1995).

Synthesized AS-oligo has been widely used because of its potential specificity for disease-causing genes and its ease of design and synthesis. Short-length (10-30 nucleotides) AS-oligoses designed to bind to the target gene sequence according to the Watson-Crick base pairing provide specificity and affinity. Inhibition of gene expression is achieved by the formation of DNA-mRNA duplexes by RNaseH activity or by disrupting the formation and progression of ribosomal complexes for protein translation (Dolnick, BJ, Cancer Inv ., 9). , 185-194, 1991 ). AS-oligo also inhibits gene transcription by binding to genomic DNA to form triple-helix structures (Young, SL, et al., PNAS, 88, 10023-10026, 1991 ). .

The efficacy of AS-oligo has been confirmed through clinical studies on several animal models and recent human diseases. After 10 days of intravenous injection of phosphorothioate (hereinafter referred to as "PS") AS-oligo, hepatitis B virus DNA disappeared from the duck's liver (Offenserger, WB, et al., EMBO) . J. , 12, 1257-1262, 1993 ). AS-oligo has also been shown to have an effect of lowering blood pressure when injected into hypertensive rats (Tomita, N., et al., Hypertension , 26, 131-136, 1995 ). Injecting phosphorothioate AS-oligo into the RI _a subunit of protein kinase A subcutaneously in hairless mice has been reported to stop cancer growth (Nesterova, M., et al., Nat. Med ., 1, 528-533, 1995 ). Clinical trials using AS-oligo against different disease-causing genes have been found to have significant effects on ovarian cancer and Crohn's disease (Roush, W., Science , 276, 1192-1193, 1997 ).

However, the effect of AS-oligo using sequence specificity for the target gene sequence is often not expected. A prominent problem with AS-oligo in this case is that instability to nucleases and inhalation into the cells are not effective.

The stability of the AS-oligo was somewhat improved by using modified oligos such as PS- and methylphosphonate (hereinafter referred to as "MP")-oligo, which are used to increase the stability to nucleases. However, each modified nucleotide has a problem of lacking sequence specificity or insensitivity to RNaseH.

AS-oligos bound to complementary target sequences are effective. Not all sequences of mRNA show the same access to AS-oligo. Binding to unequal AS-oligos can be explained at least in part by the secondary and / or tertiary structure of the target mRNA (Gryaznov, S., et al., Nucleic Acids Res ., 24, 1508 -1514, 1996 ). Therefore, it is important to select a region with less secondary structure as the target sequence for AS-oligo.

In order to increase the stability of AS-oligo, the inventors have devised a method of finding a better target sequence using a computer program that can predict the secondary structure of the mRNA, and thus the AS-oligo or shared with a stem-loop structure. AS-oligoes with closed multiple antisense sequences were constructed.

AS-lifts for the c-myb gene can be used to inhibit the growth of cancer cells.

The Myb protein, encoded by the c-myb primary cancer gene, is located primarily inside the nucleus and acts as a transcriptional regulator of G1 / S transition during the cell cycle. The primary oncogene c-myb plays an important role in the proliferation and differentiation of hematopoietic cells. The expression of c-myb in hematopoietic cells varies according to differentiation stages and is rarely expressed at the end of differentiation (Melani, C., et al., Cancer Res ., 51, 2897-2901, 1991 ). However, it has been discovered that c-myb is overexpressed in leukemic cells.

It has been reported that blocking the expression of c-myb using AS-oligo inhibits the growth of HL-60, a promyelocytic cancer cell line, and K562, a chronic myelogenous leukemia cell line (Kimura, 1999). S., et al., Cancer Res ., 55, 1379-1384, 1995 ). However, it was confirmed that the c-myb AS-oligo used in the experiment had only a partial effect. The c-myb AS-oligo used in the experiment was phosphodiester (hereinafter referred to as "PO")-oligo or PS-oligo (Anfossi, G., et al., Proc. Natl. Acad. Sci . USA). , 86, 3379-3383, 1989 ), these oligo molecules, especially PO-oligo, exhibit only partial antisense effects due to their poor stability.

AS-oligo with increased stability, selected by rational site search, can be used to completely remove c-myb mRNA, effectively inhibiting the growth of leukemia cells. Recently, the focus has been on the development of molecular therapeutics based on the use of AS-oligo for malignancies in humans. Therefore, it is desirable to find improved c-myb antisense molecules that can completely block the growth of leukemia cells.

In this regard, the present inventors selected eight sites from c-myb mRNA through secondary structure analysis and developed antisense sequences of selected c-myb in order to develop a novel AS-oligo with increased stability and antisense effect. One antisense molecule, ie, covalently-closed multiple antisense (hereinafter referred to as "CMAS")-ribbon-type antisense ("RiAS") with lift and loop and stem structure The present invention was completed by preparing oligos and confirming that these novel AS-oligoes specifically inhibit the expression of primary cancerous genes and show stability against nucleases.

Summary of the Invention

It is an object of the present invention to provide novel AS-oligomers comprising one or more antisense sequences for mRNA sites with less secondary structure in order to improve specificity for the desired sequence and stability for nuclease activity. To provide them.

The present invention also provides antisense sequences selected from the mRNA regions of c-myb, c-myc or k-ras with less secondary structure.

The present invention also provides a covalently-closed multiple antisense (CMAS) -oligo comprising a multiple antisense sequence for c-myb mRNA. CMAS-oligo was made closed by ligation with complementary primers.

The present invention also provides a ribon-type antisense (RIAS) -oligo comprising multiple antisense sequences for c-myb mRNA. RIAS oligos were prepared to have a stem-loop structure by ligation using complementary sequences on both 5'-ends. The invention also provides RIAS-oligos comprising multiple antisense sequences for c-myc mRNA or k-ras mRNA.

The present invention also provides a pharmaceutical composition comprising an AS-oligo effective for the treatment of cancer, immune diseases, infectious diseases and other human diseases caused by abnormal gene expression.

The present invention relates to novel antisense oligos comprising one or more antisense sequences for mRNA sites with fewer secondary structures in order to improve response specificity for the gene of interest and stability for nucleases. .

More specifically, the present invention relates to a CMAS (covalently- including multiple target antisense sequence) for mRNAs of various protooncogenes including c-myb, c-myc or k-ras. closed multiple antisense). CMAS-oligo was made closed by ligation with complementary primers.

In addition, the present invention provides a ribon-type antisense (RIAS) -oligoe comprising multiple target antisense sequences for mRNAs of various primary cancer genes, including c-myb, c-myc or k-ras. It is about. RIAS oligos were prepared to have a stem-loop structure by ligation using complementary sequences on both 5'-ends.

The invention also relates to a pharmaceutical composition comprising a novel AS-oligo effective for the treatment of cancer, immune diseases, infectious diseases and other human diseases caused by aberrant gene expression.

1 is a diagram showing the structure of c-myb closed multiple antisense oligo (CMAS- oligo),

2 is an electrophoresis picture showing the electrophoresis pattern of CMAS-oligo,

A: oligos analyzed with 5% Metaphor agarose gel,

Lane 1; Size marker, lane 2; 14 mer ligation primer,

Lane 3; Linear 60 mer oligos, lane 4; CMAS-Ups.

B: Electrophoresis picture showing the stability of linear and co-closed oligos on denaturing polyacrylamide gels.

Lane 1 and lane 3; Untreated exonuclease III,

Lane 2 and lane 4; Exonuclease III treatment.

3 is a diagram showing the structure of the c-myb ribbon antisense oligo (ribbon-type AS oligo, RiAS- oligo),

4 is an electrophoresis picture showing the electrophoresis pattern of RiAS-oligo,

A: oligo analyzed with 15% modified agarose gel,

Lane 1; 58 mer MIJ-78 molecule, lane 2; 116 Mer RIAS-Up.

B: Electrophoresis picture showing the stability of MIJ-78 and RiAS-oligo to exonuclease III.

Lane 1 and lane 3; Untreated exonuclease III,

Lane 2 and lane 4; Exonuclease III treatment.

FIG. 5 is an electrophoresis photograph showing the degradation pattern of linear and CMAS-oligo in the presence of serum,

A: electrophoresis photo showing the stability of the linear AS-lift,

Lane 1; Untreated serum (negative control), lane 2; 50% serum treatment,

Lane 3; FBS treatment, lane 4; Bovine serum (CS) treatment.

B: Electrophoresis picture showing stability of CMAS-oligo.

Lane 1; Untreated serum (negative control), lane 2; 50% serum treatment,

Lane 3; FBS treatment, lane 4; Bovine serum (CS) treatment.

6 is an electrophoresis picture showing the degradation pattern of linear and RiAS-oligo in the presence of serum,

A: electrophoresis photo showing the stability of the MIJ-78 molecule,

Lane 1; Untreated serum (negative control), lane 2; 50% serum treatment,

Lane 3; FBS treatment, lane 4; Bovine serum (CS) treatment.

B: Electrophoresis picture showing the stability of RiAS-oligo.

Lane 1; Untreated serum (negative control), lane 2; 50% serum treatment,

Lane 3; FBS treatment, lane 4; Bovine serum (CS) treatment.

7 is an electrophoretic photograph showing the effect of c-myb CMAS-oligo on c-myb expression in HL-60 cells,

A: Electrophoresis picture showing the results of RT-PCR performed using total RNA and two c-myb primers,

Lane 1; 60 μM CMAS-oligo 0.3 μg + lipofectin 1 μg,

Lane 2; 1 μg 60 mer CMAS-oligo + 1 μg lipofectin,

Lane 3; 1 μg mixed AS-oligo + 1 μg lipofectin.

B: Electrophoresis picture showing RT-PCR results performed using total RNA and two c-myb primers.

Stomach: hybridized RT-PCR band of c-myb mRNA,

Below: Hybridized RT-PCR band of β-actin mRNA.

8 is an electrophoretic image showing the effect of c-myb RIAS-oligo on the expression of c-myb mRNA in HL-60 cells,

A: Electrophoresis picture showing the results of RT-PCR performed using total RNA and two c-myb primers,

Lane 1; 0.1 μg RiAS-oligo + 0.8 μg lipofectin,

Lane 2; 0.1 μg RiAS-oligo + 0.8 μg lipofectin,

Lane 3; SC-oligo 0.2 μg + lipofectin 0.8 μg.

B: Electrophoresis picture showing RT-PCR results performed using total RNA and two c-myb primers.

Stomach: hybridized RT-PCR band of c-myb mRNA,

Below: Hybridized RT-PCR band of β-actin mRNA.

9 is a graph showing the effect of 60mer CMAS or linear AS-oligo on proliferation of HL-60 cells,

-◆-: CMAS-Up (1),-■-: CMAS-Up (2),-●-: AS-Up,

-▲-: S-MIJ-7, ●: lipofectin alone, ○: control group,

(1) or (2): number of times of AS-oligo treatment

10 is a graph showing the effect of c-myb RiAS-oligo on proliferation of HL-60 cells,

A: graph showing the results of MTT analysis of c-myb RiAS-oligo,

B: Graph showing [ ³ H] -thymidine insertion of c-myb RiAS-oligo.

11 is a micrograph showing that growth of HL-60 cells is inhibited by c-myb RiAS-oligo,

A: c-myb RiAS-oligo, B: SC-oligo, C: lipofectin alone.

12 is a micrograph showing that growth of HT-29 cells is inhibited by c-myb RiAS-oligo,

A: c-myb RiAS-oligo, B: SC-oligo, C: lipofectin alone.

FIG. 13 is a micrograph showing that growth of HT-29 cells is inhibited by c-myc RiAS-oligo,

A: c-myc RiAS-oligo, B: SC-oligo, C: lipofectin alone.

14 is a micrograph showing that growth of HT-29 cells is inhibited by k-ras RiAS-oligo.

A: k-ras RiAS-oligo, B: SC-oligo, C: lipofectin alone.

Hereinafter, the present invention will be described in detail.

The present invention provides novel AS-oligos comprising one or more antisense sequences for a region with less secondary structure.

In particular, as in the preferred embodiment, eight different c-myb mRNA regions and one primary cancerous gene were selected as target sites for antisense oligos. The eight antisense sequences are complementary to the sequences of the selected site of interest. CMAS molecules were prepared by finally selecting and combining four sites (MIJ-1, MIJ-2, MIJ-3 and MIJ-4) from eight selected target sites for AS-oligo, and three sites (MIJ- 3, MIJ-4 and MIJ-17) were combined to produce RiAS molecules, which form a minimal intramolecular secondary structure (see Table 1 ).

AS-oligos with a phosphodiester backbone lack the stability necessary for successful application of antisense. Modified oligos, such as PS-oligo or MP-oligo, have increased stability, but the increased stability implies the risk of only partial or hydrolyzed modified-nucleotides misincorporation during DNA replication or repair. Doing. It is known that stem-loop oligos associated with cationic liposomes also show an increase in partial stability. However, stability is still an important concern for AS-lifting. We therefore sought for the development of improved AS-oligoes with increased stability.

As a result, the present invention provides covalently-closed multiple antisense (CMAS) -oligos.

In particular, the intracellular secondary structure of AS-oligo was created without polymer formation between AS-oligo and the target mRNA. These AS-oligols are covalently comprised of four antisense sequences (MIJ-1, MIJ-2, MIJ-3 and MIJ-4) described by SEQ ID NO: 1 , SEQ ID NO: 2 , SEQ ID NO: 3 and SEQ ID NO: 4 -closed multiple antisense)-in the form of oligos (see Figure 1 ). CMAS-oligo showed an electrophoretic mobility pattern that was about 10% slower than linear on a 15% denaturing PAGE gel (see A in FIG. 2 ). CMAS-oligoes also showed resistance to exonuclease III as expected and showed multiple band-60mer monomers, 120mer dimers and 180mer trimers-on modified denaturation PAGE gels. . Unlike CMAS-oligo, linear oligos were completely degraded by exonuclease III in 2 hours (see B in FIG. 2 ).

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

CMAS-oligo is very stable but requires a primer for intramolecular ligation, which must be removed later. Therefore, the present inventors prepared a ribbon-type RiAS-oligo by enzymatically polymerizing two AS-oligoes to avoid the use of ligation primers.

The RiAS oligo (116mer) consists of two loops and one stem connected to these loops (see FIG. 3 ). Specifically, three antisense sequences (MIJ-3, MIJ-4 and MIJ-17) described by SEQ ID NO: 3 , SEQ ID NO: 4 and SEQ ID NO: 5 are connected in series in a loop. Thus, two copies of three different antisense sequences (6 antisense sequences in total) form one RiAS-oligo. This increased length of the RiAS-oligo serves to purify the torsional stress caused by forming the polymer with the desired mRNA sequence. RiAS-oligo shows significantly reduced electrophoretic locomotor behavior compared to linear oligo (MIJ-78) on denaturing PAGE gels (see A of FIG. 4 ). RiAS-oligoes also showed resistance to exonuclease III as expected and showed a major band of 116mer on denaturing PAGE gels. Unlike RiAS-oligo, linear oligo (MIJ-78) was completely degraded by exonuclease III in 2 hours (see B in FIG. 4 ).

In order to demonstrate the increased stability of the nuclease activity of CMAS-oligo and RiAS-oligo of the present invention, CMAS-oligo and RiAS-oligo were incubated with untreated heat serum to maintain nuclease activity. .

As a result, linear 60mer oligos (circle of CMAS-oligo, see A in FIG. 5 ) and linear 59mer oligos (circle of RiAS-oligo, see A in FIG. 6 ) are completely degraded after 24 hours of incubation with serum. It was confirmed. However, CMAS-oligo and RiAS-oligo were found to be almost completely present after 24 hours of incubation with human serum, FBS and calf serum, indicating that the CMAS-oligo and RiAS-oligonus of the present invention to show that the stability of the nuclease activity was markedly increased (Fig. 5 B of B and 6).

It has also been demonstrated that CMAS-oligo functions to remove target mRNAs in a sequence specific manner.

In particular, CMAS-oligo binds to Lipofectin and is delivered into cells. Lipofectin was chosen because of its low toxicity to cells and reproducible results. The binding of MIJ-5, or CMAS-oligo, to human c-myb can reduce more than 95% of c-myb mRNA compared to the control SC-oligo. MIJ-5A, the linear counterpart of MIJ-5, reduces about 37% of c-myb mRNA (see FIG. 7 ). The results indicate that the CMAS-oligo of the present invention is superior to linear even when a small amount is used to remove the target mRNA.

RiAS-oligo ability to remove target mRNA was demonstrated in the same way as CMAS-oligo.

HL-60 cells were tested with Lipofectin alone as well as with RiAS-oligo and scrambled (SC) -oligo. RiAS-oligo complexes with lipofectin and is then delivered into the cell. As a result, RiAS-oligo can completely remove c-myb mRNA. On the other hand, SC-oligo reduced c-myb mRNA by about 30% compared to lipofectin alone (see Figure 8 ). These results indicate that RiAS-oligo of the present invention is excellent for removing target mRNA even when used in small amounts.

We also investigated the antisense effects of CMAS-oligo and RiAS-oligo through Southern blots of PCR products.

In the case of CMAS-oligo, when c-myb was amplified by RT-PCR, more than 90% was reduced by treatment with MIJ-5 (see B in FIG. 7 ).

In the case of RiAS-oligo, c-myb amplified by RT-PCR is detected by labeled internal hybrid oligos 30mer (see B in FIG. 8 ). The results confirm that the amplified is from c-myb.

The invention also provides a pharmaceutical composition comprising a salivary silver type of AS-oligo for treating cancer, immune diseases, infectious diseases and other human diseases caused by erroneous gene expression.

c-myb CMAS-oligo or c-myb RiAS-oligo has been shown to interfere with leukemia cell growth.

In particular, growth inhibition of leukemia cells by c-myb CMAS-oligo and c-myb RiAS-oligo was measured by three methods: MTT analysis, [ ³ H] thymidine mixing or colony formation in soft agarose. do.

As a result of the MTT assay, the number of cells was rapidly reduced when the treatment was performed by increasing the amount of CMAS-oligo bound to MIJ-5, that is, human c-myb. More than 80% growth inhibition of HL-60 cells was observed even at low concentrations (see FIG. 9 ). On the other hand, the linear 60mer AS-oligos, ie MIJ-5A and linear sense oligos, were not disturbed in cell growth when compared to the control. The results indicate that the c-myb CMAS-oligo of the present invention is a useful antisense agent and is an effective cancer growth inhibitor in a concentration dependent manner.

In addition, cell growth was observed to be inhibited by 91% or more by treatment with RiAS-oligo (see A in FIG. 9 ). On the other hand, SC-oligo and lipofectin alone did not interfere with cell growth much as compared to the control treated with nothing. The results indicate that the c-myb RiAS-oligo of the present invention is also an effective antisense agent in inhibiting leukemia cell growth.

In colony formation assays on light agarose, MIJ-5 reduces the number of colony formation by more than 90% (see Table 2 ). MIJ-5A also reduces colony formation by about 70% but has no effect on growth inhibition. On the other hand, sense oligos and SC-oligoes show reduced colonies by 11% and 32%, respectively.

In addition, c-myb RiAS-oligo transformed into cells can reduce about 92% of the number of colonies formed compared to the control (see Table 3 ). On the other hand, SC-oligo and lipofectin alone reduce colonies by 7.9% and 7.1%, respectively.

In addition, growth inhibition of leukemia cells of c-myb RiAS-oligo was observed by [ ³ H] thymidine mixing. In particular, RiAS-oligo inhibits the growth of HL-60 cells by 93%. On the other hand, SC- oligonucleotide and lipoic pectin alone will prevent the cell growth respectively about 16.8%, and 15.4% (refer to B of FIG. 10). Microscopically, growth of HL-60 cells after treatment with c-myb RiAS-oligo was significantly inhibited compared to cells treated with mixed oligo and lipofectin alone (see FIG. 11 ).

Due to the significant interfering activity of c-myb RiAS-oligo in the present invention, we have prepared different RiAS-oligos against two different protocogenes c-myc and k-ras, and c-myc RiAS-oligo. Oligo and k-ras RiAS-oligo were observed to function well in the inhibition of cell growth.

Microscopic observations showed that the growth of HT-29 cells was increased in all RiAS- raises, ie c-myb RiAS- raise, c-myc RiAS- raise and k-ras RiAS-oligo, compared to cells treated with mixed oligo and lipofectin alone. Significantly inhibited (see FIGS. 12 , 13 and 14 ).

Thus, the new RiAS-oligo of the present invention has been shown to effectively inhibit tumor cell growth for various target sequences as well as increased stability for nuclease activity. Therefore, the new RiAS-oligo of the present invention can be usefully used for the development of antisense oligos for the treatment of various human diseases.

Hereinafter, the present invention will be described in detail by examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Screening of Target Position for AS-Uplift

Target site selection of AS-oligo is very important for antisense effects, i.e. reduction or elimination of target mRNA. However, the method for target location selection is somewhat arbitrary. Therefore, we specifically examined the entire sequence of human c-myb mRNA for the presumed secondary structure to find a suitable target position.

In particular, the simulation of the secondary structure was performed using the DNAsis program (Hitach Software, Japan). The entire c-myb sequence was examined sequentially for secondary structure formation in a contiguous frame of 100 bases. Next, the frame for the simulation of the secondary structure was advanced by 30 bases and 60 bases of 5 'were duplicated. This process was repeated to examine a given secondary structure for a given sequence in three different frames.

As a result, eight sequences with fewer duplexes in three different frames were selected from c-mybmRNA sequences ( Table 1 ).

8 target sequences for antisense oligos selected from c-myb mRNA sequences name Complementary position type Size (mer) order 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-16 585-602 Antisense 18 SEQ ID NO: 10 MIJ-17 961-978 Antisense 18 SEQ ID NO: 5 MIJ-19 97-114 sense 8 SEQ ID NO: 11

As indicated in Table 1 , the eight antisense sequences are complementary to the selected target positions. In order to minimize the secondary structure formation of the internal molecules, four positions (MIJ-1, MIJ-2, MIJ-3 and MIJ-4) out of eight selected target sequences for AS-oligo may form a CMAS molecule. The last selection in the combination was performed, and the three positions (MIJ-3, MIJ-4 and MIJ-17) were selected in the combination forming the RiAS-oligo.

<Example 2> Preparation of covalently-closed multiple antisense (CMAS) -oligo

The inventors have attempted to develop an improved AS-oligo that is more stable. Four different AS-oligos (MIJ-1, MIJ-2, MIJ-3 and MIJ-4) obtained in Example 1 were used to make the CMAS-oligo. To more easily bind to the target position One CMAS-oligo was constructed by concatenating four different antisense sequences in a combination to minimize secondary structure, AS-oligo phosphorylated at the 5 ′ end during synthesis for intramolecular or intermolecular covalent bonds ( FIG. 1 ). The sequence of a 60mer AS-oligo comprising four different antisense sequences is set forth in SEQ ID NO: 7 .

Both ends of the AS-oligo are conjugated with ligation primers having complementary sequences at both halves of both terminal sequences (7 bases on each side) of the 60-mer AS-oligo. The sequence of the 14mer linking primers is set forth in SEQ ID NO: 6 . The linking primer was mixed with AS-oligo, heated at 85 ° C. for 2 minutes and cooled slowly at room temperature. One unit of T4 ligase was added and reacted at 16 ° C. for 16 hours to produce a co-closed molecule. CMAS-oligoes were electrophoresed on 5% Metaphor agarose (FMC, USA) or 12% denaturation PAGE to degrade slightly for gel delay and exonuclease III compared to linear 60mer oligos. It was confirmed that it was not. The linking primers are cleaved by exonuclease III or separated from CMAS-oligo by electrophoresis on denaturing gels after heating the oligo at 90 ° C.

As a result, the intracellular secondary structure of AS-oligo was produced without the duplex formation of AS-oligo and target mRNA. This AS-oligo was named CMAS (covalently-closed multiple antisense) oligo. CMAS-oligo shows about 10% slower electrophoretic migration than linear precursors in a 15% modified PAGE gel ( A in FIG. 2 ). As expected, CMAS-oligo is not degraded by exonuclease III and appears as multiple bands of monomer (60 mer), dimer (120 mer) and trimer (180 mer) that occupy most of the modified PAGE gel ( 2 B ). Unlike CMAS-oligo, linear oligos were completely degraded after 2 hours of understanding in the treatment of exonuclease III.

Example 3 Fabrication of Ribbon Type AS (RiAS) -Oligo

We enzymatically linked two AS-oligoes to form a ribbon closed molecule and named RiAS-oligo.

In particular, the RiAS-oligo consists of two loops, with one stem connecting the two loops. Each loop comprises three different antisense (MIJ-3, MIJ-4 and MIJ-17) sequences set forth in SEQ ID NO: 3 , SEQ ID NO: 4 and SEQ ID NO: 5 . In order to bind more easily to the target position, a combination of three antisense sequences was chosen for AS-oligo, minimizing the possible secondary structure. C-myb AS-oligo (MIJ-78) was phosphorylated at the 5 'end. The sequence of 58mer MIJ-78 is set forth in SEQ ID NO: 8 . MIJ-78 forms a stem-loop structure. The stem was formed by the complementary sequence at both ends of each oligo. The 5 'end of the stem has 4 bases of the single stranded sequence of 5' (p) FATC-3 '. Two MIJ78 molecules are linked by complementary four base sequences at both 5 'ends. The MIJ-78 molecules were mixed, heated at 85 ° C. for 2 minutes and slowly cooled at room temperature. One unit of T4 DNA ligase was added and reacted at 16 ° C. for 24 hours to generate a covalently linked molecule having diad-symmetry ( FIG. 3 ). It was observed that the electrophoresis of RiAS-oligo on 15% modified polyacrylamide gels did not degrade with respect to retardation and exonuclease III in the gel.

As a result, a RiAS-oligo (116mer) was constructed that included two loops and one stem connecting the two loops. Three antisense sequences in a loop were placed in line to increase the length of the RiAS-oligo.

As a result, two copies of the three different antisense sequences (all six antisense sequences) are in RiAS-oligo. The extended length of the loop in RiAS-oligo is to control the torsional stress caused by forming a duplex with the target mRNA sequence. RiAS-oligo migrated significantly slower than linear precursors (MIJ-78) in denatured PAGE gels ( A in FIG. 4 ). As expected, RiAS-oligo was not degraded by exonuclease III and major bands (116mers) appeared in PAGE gels ( FIG . 4B ). Unlike RiAS-oligo, MIJ-78 was completely degraded after 2 hours of exonuclease treatment.

Example 4 Improved Safety of CMAS-oligo and RiAS-oligo Against Nuclease Activity

To test the stability of the nuclease activity of the CMAS-oligo and RiAS-oligo of the present invention, CMAS-oligo and RiAS-oligo were reacted with serum not thermally inactivated to maintain nuclease activity.

In particular, 1 μg of each non-specific normal-phosphodiester oligo (linear 60mer) and CMAS-oligo unprocessed human serum, FBS and bovine serum (heated and inactivated; HyClone, Logan, Utah , USA) or exonuclease III was added to react. 15% of each serum was added to 100 μl of the reaction solution to the AS-oligo and reacted at 37 ° C. for 24 hours. AS-oligo was extracted with phenol and chloroform and observed on a 15% modified PAGE gel. Exonuclease III (Takara, Japan) was added to linear and CMAS-oligo with 160 U / μg oligo and reacted at 37 ° C for 24 hours. AS-oligo treated with exonuclease III was extracted and electrophoresed in the same manner as above.

Results of CMAS- oligonucleotide, linear 60 Merced oligonucleotide was completely decomposed after 24 hours reaction in the presence of serum (A in Fig. 5). However, oligonucleotide CMAS- of the invention's most After reaction for 24 hours with serum, FBS and bovine serum raw remains as originally exhibited enhanced stability than linear for nuclease (B in Fig. 5).

RiAS- For oligonucleotide, linear dimmer 58 were each fully decomposed after the reaction for 24 hours in the presence of other serum (A in Fig. 6). However, oligonucleotide RiAS- of the invention was mostly left to its original shape after the reaction for 24 hours with raw serum which showed improved stability than the linear ones against nuclease (B in Fig. 5).

Example 5 Specific Reduction of c-myb mRNA by CMAS-oligo and RiAS-oligo

Due to the marked stability of CMAS-oligo and RiAS-oligo in the present invention, the inventors observed whether AS-oligo functions to remove target mRNA in a sequence specific manner.

<5-1> Cell Line and Tissue Culture

The leukemia cell lines HL-60 (promyelocytic leukemia cell line) and K562 (chronic myelogenous leukemia cell line) were distributed by Daejeon, Korea Institute of Science and Technology Research Institute of Biotechnology Research, Gene Bank and 10% FBS in RPMI 1640 (Gibco BRL, USA) medium. (Gibco BRL, USA) and 1% penicillin / streptomycin (Gibco BRL, USA) were added to incubate in a 37 ° C., 5% CO ₂ incubator, and the cell concentration was properly maintained during the culture. Cells were incubated no more than five generations after maintaining the appropriate cell density and dissolving stock vials. Cultures were replaced one day prior to treatment with AS-oligo.

<5-2> Transformation of CMAS-oligo and RiAS-oligo complexed with cationic liposomes

0.3 μg of CMAS-oligo and 0.8 μg of lipofectin (Gibco BRL, USA) or 0.2 μg of RiAS-oligo and 0.8 μg of lipofectin were diluted in 20 μl of OPTI-MEM and allowed to react at room temperature for 40 minutes. Each component was mixed to form a complex for 15 minutes at room temperature. The cells were added to fresh culture without antibiotics (RPMI 1640 + 10% FBS) one day before the oligo addition and washed twice with OPTI-MEM before the experiment. Cell density was adjusted to ⁵ × 10 ⁵ cells / ml and 100 μl were aliquoted into 48-well plates (Falcon, USA). 40 μl of liposome-oligo complex was added to the cells twice for 2 days (day 0, day 1). Cells treated with oligos were reacted for 4 hours at 37 ° C., 5% CO ₂ incubator and 100 μl of OPTI-MEM with 10% FBS was added. The next day 100 μl of supernatant was carefully removed and replaced with 20 μl of OPTI-MEM containing oligo-liposomal complexes. After 4 hours, 100 μl of complete medium with antibiotics was added to the cells and incubated for another day at 37 ° C. before analysis.

<5-3> RNA isolation and RT-PCR

All RNA was isolated with Tripure ^™ separation reagent (Boehringer Manhein, Germany) according to the manufacturer's instructions. To isolate all RNA, 0.4 mL of Tripure reagent, 10 μg of glycogen and 80 μl of chloroform were added to the cell culture. RT-PCR was performed in one reaction tube with the Access ^™ RT-PCR kit (promega, USA). RNA, PCR primer, AMV reverse transcriptase (5 U / μl), Tfl DNA polymerase (5 U / μl), dNTP (10 mM, 1 μl) and MgSO ₄ (25 mM, 2.5 μl) were added to the PCR tube. . The synthesis of primary chain cDNA was performed with a DNA thermal cycler (Hybaid, USA) at 48 ° C. for 45 minutes. PCR amplification was performed continuously in 25 cycles according to the manufacturer's instructions. Amplified PCR products were identified on 1% agarose gel and quantitatively determined by gel documentation program (Bio-Rad, USA).

<5-4> Southern Hybridization of RT-PCR Sections

RT-PCR products were electrophoresed on 1% agarose gel. DNA was transferred to nylon membrane (New England Biolab, USA) for 4 hours in 0.4 M NaOH. Membranes were hybridized with ECL 3 ′ oligo labeled 30mer internal primers and detection system (Amersham Life Science, England). The sequence of the 30mer internal primer is set forth in SEQ ID NO: 9 . Hybridization was performed at 62 ° C. for 60 minutes in 6 ml of buffer containing 5 × SSC, 0.02% SDS. Membranes were washed twice in 5X SSC with 1% SDS and twice in 1X SSC with 0.1% SDS for 15 minutes. The membrane was treated with blocking solution and then treated with horseradish peroxidase (HRP) anti-fluorescent conjugated antibody for 30 minutes before radiographing.

The CMAS-oligo of the present invention has been shown to function to remove target mRNAs in a sequence specific manner.

In particular, CMAS-oligo complexes with lipofectin and is delivered into cells. Lipofectin was chosen because of its low toxicity to cells and reproducible results. As a result, 0.3 μg MIJ-5, that is, CMAS-oligo bound to human c-myb was complexed with 1 μg of lipofectin for transformation into HL-60 cells. MIJ-5 was able to reduce more than 95% of c-myb mRNA compared to the control SC-oligo. Meanwhile, MIJ-5A, the linear counterpart of MIJ-5, reduced about 37% of c-myb mRNA ( A in FIG. 7 ). These results indicate that the CMAS-oligo of the present invention is superior in removing target mRNA even if a small amount is applied than linear one.

The RiAS-oligo of the present invention has also been demonstrated to be able to remove target mRNAs well by sequence specific methods.

HL-60 cells were transformed with lipofectin alone, RiAS-oligo and SC-oligo. RiAS-oligo was delivered into cells after complexing with lipofectin. RiAS-oligo (0.1 μg or 0.2 μg) binding to human c-myb was complexed with 0.8 μg lipofectin for transformation into HL-60 cells. As a result, 0.2 μg of RiAS-oligo (40 nM) can completely remove c-myb mRNA ( A in FIG. 8 ). SC-oligo, on the other hand, can reduce c-myb mRNA by about 30% compared to lectopectin alone. However, α-actin expression was not affected by RiAS-oligo treatment as well as other treatment conditions. The results indicate that RiAS-oligo is superior in removing target mRNA even if a small amount is applied.

In addition, the inventors examined the antisense effects of CMAS-oligo and RiAS-oligo by Southern blot using PCR products. After infecting the HL-60 cell line with oligos comprising MIJ-5 and control oligos, the infected cells were used to isolate total DNA. In the case of CMAS-oligo, RT-PCR was used to amplify the message of c-myb, and it was confirmed that more than 90% of messages were reduced by the treatment of MIJ-5 ( FIG . 7B ). However, the expression of α-actin observed in the bottom panel was not affected by the treatment of MIJ-5. RiAS- oligonucleotide for there, a raised inner (internal) labeled hybridization was detected with the C-myb messages amplified by RT-PCR using the (30 monomers) (B in Fig. 8). From the above results, it was confirmed that the amplified message is actually induced by c-myb and the message can be completely eliminated by treating 0.2 μg of c-myb RiAS-oligo.

Example 6 Inhibition of Effective Leukemia Cell Growth by c-myb CMAS-oligo and c-myb RiAS-oligo.

It is known that c-myb plays an important role in the proliferation of white blood cells. The action of AS-oligo on c-myb has also been reported, which selectively inhibits the growth of leukemia cells. Therefore, the present inventors confirmed whether the c-myb CMAS-oligo and c-myb RiAS-oligo of the present invention can inhibit the growth of leukemia cells.

In particular, growth inhibition of c-myb CMAS-oligo and c-myb RiAS-oligo on leukemia cells was characterized by MTT analysis, [ ³ H] thymidine incorporation or colony formation in soft agarose. It was measured by the method.

<6-1> MTT analysis

Washing HL-60 cells twice with OPTI-MEM for MTT (3, -4 [4,5-Dimethylthiazol-2-yl] 2,5-diphenyl-tetrazolium bromide, hereinafter abbreviated as "MTT") analysis 50 μl was dispensed into 96-well plates. Cells were pretreated for 5 hours with a complex of prepared oligos (0.01-1 μg / 15 μl for CMAS-oligo, 0.2 μg / 15 μl for RiAS-oligo) and lipofectin (0.2 μg / 15 μl) And then incubated for 5 days. After harvesting cells in a volume of 100 μl, 20 μl (100 μg) of MTT reaction solution (5 mg / ml in PBS; Sigma, USA) dissolved in PBS was added and reacted at 37 ° C. for 4 hours. 100 μl of isopropanol containing 0.1 N HCl was added to the cells, followed by further reaction at ambient temperature for 1 hour. Absorbance at 570 nm was measured using an ELISA reader to count the amount of viable cells.

As a result, in the case of CMAS-oligo, the number of cells gradually decreased as the throughput of MIJ-5 increased. When cells were treated twice with MIJ-5, cell growth inhibition was more apparent. More than 80% inhibition of HL-60 cell growth was observed even when treated with very low concentrations of CMAS-oligo, such as 0.13 μg (0.24 μg total) ( FIG. 9 ). On the other hand, no significant cell growth inhibition was observed when the linear 60 monomer AS-oligo, MIJ-5A and linear sense oligo were used compared to the control. From the above results, it was confirmed that c-myb CMAS-oligo is an effective antisense substance and has an effect on tumor cells in a concentration-dependent manner.

In the case of RiAS-oligo, it was observed that 91% of cell growth was inhibited by the treatment of RiAS-oligo ( A in FIG. 10 ). On the other hand, SC-oligo and lipofectin itself did not significantly inhibit cell growth compared to the untreated control. From the above results, it was confirmed that c-myb RiAS-oligo is an effective antisense substance for inhibiting the growth of white blood cells.

<6-2> Colony Formation in Soft Agarose

Colony formation in soft agarose was measured as another method to identify leukocyte cell growth inhibition by c-myb CMAS-oligo and c-myb RiAS-oligo. Specifically, K562 cells were infected by the method described in Example 6 above, and cultured for 24 hours in the presence of 37 ° C and 5% CO ₂ . A mixture of 2x RPMI-1640 medium containing 0.8% low melting point agarose, 20% FBS and antibiotics in the same volume was added to the cells and then aliquoted into 6-well plates to solidify. The 6-well plates were cooled for 5 minutes at 4 ° C. and then incubated for 15 days. Colonies containing 20 or more cells were counted positive.

As a result, CMAS-oligo MIJ-5 reduced the formation of colonies by more than 90% ( Table 2 ). MIJ-5A also reduced colony formation by 70%, but growth inhibition was less effective. On the other hand, sense oligo and SC-oligo showed about 11% and 32% reduction in colony formation, respectively.

Effect of c-myb Oligo on Colony Formation in K562 Cells Raise Number of colonies % Of colonies formed rescue Size (mer) Type 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 alone 109 88.0 Untreated control 124 100.0

In contrast, cells infected with c-myb RiAS-oligo reduced the number of colonies formed by about 92% compared to the untreated control ( Table 3 ). On the other hand, SC-oligo and lipofectin alone showed a slight inhibition of colony formation of about 7.9% and 7.1%, respectively.

Effect of c-myb Oligo on Colony Formation in K562 Cells Raise Size (mer) Number of colonies % Of colonies formed RiAS-Ups 116 7.6-1.53 7.8 Mixed oligo 116 0.5-2.12 92.1 Lipofectin alone 91.3-4.16 92.9 Untreated control 98.3-4.04 100.0

<6-3> [ ³ H] thymidine inhalation

[ ³ H] thymidine inhalation was measured as another method to confirm growth inhibition of leukemia cells by c-myb RiAS-oligo. To measure [ ³ H] thymidine inhalation, HL-60 cells were treated with AS-oligo as above. Cells were incubated in triplicate for 16 hours with 0.5 μCi of [ ³ H] thymidine (2.0 Ci / mmol; Amersham, England) and harvested with glass microfiber filter paper. The filter paper was washed using chilled PBS, 5% TCA and absolute ethanol in this order. [ ³ H] thymidine inhalation was measured using a liquid scintillation counter in a cocktail solution comprising toluene, Triton X-100, PPO and POPOP.

As a result, RiAS-oligo (0.2 μg) inhibited the growth of HL-60 cells by 93% ( B in FIG. 10 ). On the other hand, SC-oligo and lipofectin alone inhibited mild cell growth by about 16.8% and 15.4%, respectively. Microscopic observation showed that treatment of c-myb RiAS-oligo noticeably inhibited the growth of HL-60 as compared to cells treated with scrambled oligo and lipofectin alone ( FIG. 11 ).

Example 8 Effective Growth Inhibition of c-myc RiAS-oligo and k-ras RiAS-oligo

Encouraged by the significant inhibitory activity of the c-myb RiAS-oligo of the present invention, the inventors described RiAS-oligo against two different protocogenes, c-myc and k-ras, in the same manner as in Example 3. It was prepared by. Next, we examined whether c-myc RiAS-oligo and k-ras RiAS-oligo inhibit cell growth well. In particular, the present inventors used another type of cell line, the colorectal adenocarcinoma cell line, HT-29. Growth inhibition of tumor cells by c-myc RiAS-oligo and k-ras RiAS-oligo was measured by [ ³ H] thymidine inhalation in the same manner as in Example <6-3> . HT-29 cells were cationic liposomes of 0.2 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, respectively. Treated with complex. After 5 days of treatment with each RiAS-oligo, the growth of HT-29 cells was observed using a microscope. Photomicrographs examined the growth inhibition effect after treatment with RiAS-oligo (A), mixed oligo (B) and lipofectamine alone (C), respectively.

Microscopic observations showed that the growth of HT-29 cells showed all RiAS-up, c-myb RiAS-up, c-myc RiAS-up, and k-ras RiAS-up compared to cells treated with mixed oligo and lipofectamine alone. Markedly inhibited by oligos ( FIGS. 12 , 13 and 14 ).

Therefore, the novel RiAS-oligo of the present invention not only increased the stability to nuclease activity but also effectively inhibited the growth of tumor cells having various target sequences. Thus, the novel RiAS-oligos of the present invention can be effectively used for the development of antisense oligo molecules against various human diseases.

The present invention provides novel AS-oligos comprising one or more antisense sequences that have a less secondary structure for mRNA sites and also have better target sequence specificity and stability to nuclease activity. do.

More specifically, the present invention provides a covalently-closed multiple antisense comprising multiple target antisense sequences for c-myb mRNA, prepared to ligation with complementary primers to form a closed form. (CMAS) -provides raise. In addition, the present invention provides a ribbon antisense (RiAS) comprising multiple target antisense sequences for c-myb mRNA, prepared to be joined to complementary sequences at both 5 'ends to form a stem-loop structure. -Provide raises.

Treatment of human tumor cells with c-myc RiAS-oligo and k-ras RiAS-oligo, as well as c-myb RiAS-oligo and c-myb CMAS-oligo, resulted in the inadequacy of genes by the novel AS-oligo aberrant expression is effectively eliminated. Thus, the novel AS-oligo of the present invention can be used to study the function of genes as well as to develop molecular antisense agents for genes that cause a variety of diseases. In particular, the novel AS-oligos of the present invention can be used in the development of pharmaceutical compositions for treating cancer, immune diseases, infectious diseases or other human diseases caused by inappropriate expression of genes.

Claims (23)

A novel antisense oligo comprising increased one or more antisense sequences having a small secondary structure relative to the mRNA site and having a closed form, thereby increasing stability to nuclease activity. The novel AS-oligo of claim 1, wherein the mRNA is transcribed from various genes that cause human disease. The novel AS-oligo according to claim 1, which is a covalently closed multiple antisense (CMAS) -oligo prepared by closing the AS-oligo using a ligation primer. 4. The new AS-oligo according to claim 3, wherein the AS-oligo is 60 monomeric AS-oligo as described in SEQ ID NO: 7. 5. The novel AS-oligo of claim 4, wherein SEQ ID NO: 7 consists of four antisense sequences described as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4. 6. 4. The novel AS-oligo of claim 3, wherein the conjugated primer consists of 14 monomeric nucleotides set forth in SEQ ID NO: 6. 4. The new AS-oligo according to claim 3, wherein CMAS-oligo effectively eliminates or reduces inappropriate expression of genes related to human disease. 8. The novel AS-oligo according to claim 7, wherein the gene is c-myc, c-myb or k-ras which is a protocogene. 4. The novel AS-oligo according to claim 3, wherein CMAS-oligo effectively inhibits the growth of tumor cells. 10. The novel cell of claim 9, wherein the tumor cells are HL-60, a promyuelotic leukemia cell line, K562, a chronic myelogenous leukemia cell line, or HT-29, a colorectal adenocarcinoma cell line. AS-Up. The novel AS-oligo of claim 1, wherein the AS-oligo is a ribbon antisense (RiAS) -oligo prepared by conjugating two AS-oligoes using a sequence complementary to each 5 'end. . 12. The novel AS-oligo according to claim 11, wherein the RiAS-oligo has a stem-loop structure consisting of two loops and one stem connecting them. 12. The new AS-oligo according to claim 11, wherein the RiAS-oligo is 58 monomeric AS-oligo as described in SEQ ID NO: 8. 14. The novel AS-oligo according to claim 13, wherein SEQ ID NO: 8 consists of three antisense sequences set forth in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5. 12. The novel AS-oligo of claim 11, wherein the complementary sequence is described as 5 '-(p) GATC-3'. 12. The novel AS-oligo of claim 11, wherein RiAS-oligo effectively eliminates or reduces inappropriate expression of genes involved in human disease. 12. The novel AS-oligo of claim 11, wherein the gene is c-myc, c-myb or k-ras which is a causative oncogene. 12. The novel AS-oligo of claim 11, wherein RiAS-oligo effectively inhibits the growth of tumor cells. 19. The novel AS-oligo of claim 18, wherein the tumor cells are HL-60, a pro-cytoblastic leukemia cell line, K562, a chronic hematopoietic leukemia cell line, or HT-29, a colorectal adenocarcinoma cell line. An AS-oligo-liposome complex comprising the CMAS-oligo of claim 3 or the RiAS-oligo of claim 11. 21. The AS-oligo-liposomes of claim 20, wherein the liposomes are cationic liposomes. A pharmaceutical composition comprising the CMAS-oligo of claim 3 or RiAS-oligo of claim 11 as an active ingredient. The pharmaceutical composition according to claim 22, which is used for the treatment of cancer, immune diseases, infectious diseases or other human diseases caused by inappropriate expression of genes.