EP1175430A4 - Oligonucleotides antisens a stabilite amelioree et a effet antisens - Google Patents

Oligonucleotides antisens a stabilite amelioree et a effet antisens

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Publication number
EP1175430A4
EP1175430A4 EP00913157A EP00913157A EP1175430A4 EP 1175430 A4 EP1175430 A4 EP 1175430A4 EP 00913157 A EP00913157 A EP 00913157A EP 00913157 A EP00913157 A EP 00913157A EP 1175430 A4 EP1175430 A4 EP 1175430A4
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Prior art keywords
oligo
rias
cmas
oligos
antisense
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EP1175430A1 (fr
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Jong-Gu Park
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed

Definitions

  • the present invention relates to novel antisense oligos containing one or more antisense sequence to
  • the present invention relates to covalently-closed multiple antisense (CMAS) -oligos containing multiple target antisense sequences to various protooncogene mRNAs including c-myb, c-myc, or k-ras.
  • CMAS-oligos are constructed to form a closed type by ligation using complementary primers.
  • the present invention relates to ribbon-type antisnese (RiAS) -oligos containing multiple target antisense sequences to various protooncogene mRNAs including c-myb, c-myc, or k-ras.
  • the RiAS-oligos are constructed to form a stem- loop structure by ligation using complementary sequences at both 5 prime ends .
  • the present invention relates to pharmaceutical composition containing the novel types of AS-oligos for treatment cancer, immune diseases, infectious diseases, and other human diseases caused by aberrant gene expression.
  • Antisense oligonucleotides have been valuable in the functional study of a gene by reducing expression of the gene in a sequence specific manner (Thompson, C. B. et al., Nature, 314, 363-366, 1985). Intense efforts have also been made to develop molecular antisense agents by ablating aberrant expression of genes involved in tumor initiation and progression (Chavany, C. et al., Mol. Pharm. , 48, 738-746, 1995).
  • AS-oligos have been widely utilized for the ease of design and synthesis as well as for potential specificity to genes causing diseases.
  • AS-oligos with short length 13 ⁇ 30 nucleotides
  • AS-oligos with short length 13 ⁇ 30 nucleotides
  • Inhibition of gene expression is believed to be achived through either RNaseH activity following formation to DNA-mRNA duplex or sterical hindrance of binding of ribosomal complex (Dolnick, B. J. , Cancer Inv., 9, 185-194, 1991) .
  • AS-oligos Efficacy of AS-oligos has been validated in some animal models as well as in some of recent clinical studies for human diseases. Intravenous injection of phosphorothioate (hereinafter , referred to as 'PS') AS-oligos for 10 days has eliminated virus DNA of hepatitis B from the duck liver (Offenserger, W. B. et al . , EMBO J., 12, 1257-1262, 1993). AS-oligos to angiotensiongen has been found effective to lower blood pressure when injected in spontaneously hypertensive inbred rats(Tomita, N. et al . , Hypertension, 26, 131-136, 1995) .
  • 'PS' phosphorothioate
  • AS-oligos bind to complementary target sequences to be effective. All sequences in mRNA haves not been found to be equally accessible to AS-oligos. Unequal binding of an AS-oligo could be explained, at least in part, by secondary and/or tertiary structures of target mRNA (Gryaznov, S. et al . , Necleic Acids Res., 24, 1508-1514, 1996). Thus, it is conceivables that a region with a less secondary structure could be targeted readily for an AS-oligo.
  • AS-oligos In an effort to enhance stability of AS-oligos, the present inventors have devised a rational way of searching better target sites using computer simulation by which secondary structures of mRNA are predicted, so they construct AS-oligos with a stem-loop structure or covalently-closed multiple antisense sequences.
  • the AS-oligos to c-myb gene could be used for inhibition of tumor cell growth.
  • the Myb protein encoded by the c-myb protooncogene, -is located mainly inside the nucleus and functions as a transcriptional regulator for Gl/S phase transition during the cell cycle.
  • Protooncogene c-myb plays an important role in proliferation and differentiation of hematopoietic cells. Hematopoietic cells exhibit differential expression of c-myb and show little expression of the gene when differentiated to term(Melani, C. et al . , Cancer Res., 51, 2897-2901, 1991) . C-myb has often been found to be overexpressed in leukemic cells.
  • c-myb AS-oligos blockage of c-myb expression by AS-oligos inhibits growth of a promyelocytic cancer cell line HL-60 and a chronic myelogenous leukemia cell line K562 (Kimura, S. et al . , Cancer Res., 55, 1379-1384, 1995) .
  • the c-myb AS-oligo used in the above experiments is demonstrated to be partially effective.
  • the c-myb AS-oligo employed for the above experiments is either a phosphodiester (hereinafter , referred to as 'P0')-oligo or a PS capped-oligo (Anfossi , G. et al . , Proc . Natl. Acad. Sci.
  • the present inventors selected 8 sites along c-myb mRNA from secondary structure analysis in the preferred embodiment and combined antisense sequences of the selected c-myb to construct novel large antisense molecules, a covalently-closed multiple antisense (hereinafter, referred to as ' CMAS ') -oligo and a ribbon type antisense (hereinafter, referred to as ' RiAS ') -oligo, with loops and a stem structure.
  • ' CMAS ' covalently-closed multiple antisense
  • ' RiAS ' ribbon type antisense
  • the present invention provides antisense sequences selected from mRNA region of c-myb, c-myc, or k-ras with a less secondary structure.
  • the present invention provides a covalently-closed multiple antisense (CMAS) -oligo containing multiple antisense sequences to c-myb mRNA.
  • the CMAS-oligo is constructed to form a closed type by ligation using complementary primer.
  • the present invention also provides a ribbon-type antisense (RiAS) -oligo containinj multiple antisense sequences to c-myb mRNA.
  • the RiAS-oligo is composed of two loops containing multiple antisense sequences and a stem connecting the two loops that is constructed by ligation using complementary sequences at both 5 prime ends.
  • the present invention provides the RiAS-oligos containing multiple antisense sequences to c-myc mRNA or k-ras mRNA.
  • the present invention further provides pharmaceutical composition containing the novel types of AS-oligos for treatment cancer, immune diseases, infectious diseases, and other human diseases caused by aberrant gene expression.
  • FIG. 1 shows a scheme for the construction of a c-myb CMAS-oligo.
  • FIG. 2 shows electrophoretic mobility patterns of a CMAS-oligo.
  • A is oligos analyzed by 5% Metaphor agarose gel, where lane 1; size marker, lane 2; 14 mer ligation primer, lane 3; liner 60 mer oligo, and lane 4; CMAS-oligo.
  • FIG. 3 shows a scheme for the construction of a c-myb RiAS -oligo.
  • FIG. 4 shows elextrophoretic mobility patterns of a RiAS-oligo.
  • A is oligos analyzed by a 15% denaturing poilyacrylamide gel, where lane 1; 58 mer MIJ-78 molecule, and lane 2; 116 mer RiAS-oligo.
  • B shows stability test of MIJ-78 and a RiAS-oligo upon treatment with exonuclease III, where lane 1 and 3; no treatment with exonuclease III, and lane 2 and 4; treatment with exonuclease III.
  • FIG. 5 shows degradation patterens of linear and CMAS-oligos in the presence of serum.
  • A shows stability test of linear AS-oligo, where lane 1; no treatment with serum (negative control) , lane 2 ; treatment with 50% raw serum, lane 3 ; FBS, and lane 4 ; CS for 24hr respectively.
  • B shows stability test of CMAS-oligos, where lane 1; no treatment with serum (negative control) , lane 2 ,- treatment with 50% raw serum, lane 3 ; FBS, and lane 4 ; CS for 24hr respectively.
  • FIG. 6 shows degradation patterens of linear and RiAS-oligos in the presence of serum.
  • A shows stability test of MIJ-78 molecules, where lane 1; no treatment with serum (negative control) , lane 2 ; treatment with 50% raw serum, lane 3 ; FBS, and lane 4 ; CS for 24hr respectively.
  • B shows stability test of RiAS-oligos, where lane 1; no treatment with serum (negative control) , lane 2 ; treatment with 50% raw serum, lane 3 ; FBS, and lane 4 ; CS for 24hr respectively.
  • FIG. 7 shows an effect of c-myb CMAS-oligo on c-myb expression in HL-60 cells.
  • A shows RT-PCR which is performed with total RNA and two c-myb primers, where lane 1; 60 mer CMAS-oligo 0.3 ug + Lipofectin 1 ug, lane 2; 60 mer CMAS-oligo 1 ug + Lipofectin 1 ug, and lane 3; scrambled AS-oligo 1 ug + Lipofectin 1 ug.
  • B shows RT-PCR which is performed with total RNA and two c-myb primers, where upper panel; the hybridized RT-PCR bands of c-myb mRNA, and lower panel; the hybridized RT-PCR bands of ⁇ -actin mRNA.
  • FIG. 8 shows an effect of c-myb RiAS-oligo on the c-myb mRNA expression in HL-60 cells.
  • A shows RT-PCR which is performed with total RNA using two c-myb primers, where lane 1; RiAS-oligo 0.1 ug + Lipofectin 0.8 ug, lane 2; RiAS-oligo 0.2 ug + Lipofectin 0.8 ug, and lane 3; SC-oligo 0.2 ug + Lipofectin 0.8 ug .
  • B shows RT-PCR which is performed with total RNA and two c-myb primers, where upper panel; the hybridized RT-PCR bands of c-myb mRNA, and lower panel; the hybridized RT-PCR bands of ⁇ -actin mRNA.
  • FIG. 9 shows an effect of 60 mer CMAS or linear AS-oligo on proliferation of HL-60 cells, where
  • FIG. 10 shows an effect of c-myb RiAS-oligo on proliperation of HL-60 cells.
  • A shows MTT assay of a c-myb RiAS-oligo.
  • FIG. 11 shows a photomicrograph for inhibition of
  • HL-60 cells with c-myb RiAS-oligo A is c-myb RiAS-oligo, B is SC-oligo, and C is Lipofectin alone.
  • FIG. 12 shows a photomicrograph for inhibition of HT-29 cells with c-myb RiAS-oligo.
  • A is c-myb RiAS-oligo
  • B is SC-oligo
  • C is
  • FIG. 13 shows a photomicrograph for inhibition of HT-29 cells with c-myc RiAS-oligo.
  • A is c-myc RiAS-oligo
  • B is SC-oligo
  • C is Lipofectin alone.
  • FIG. 14 shows a photomicrograph for inhibition of HT-29 cells with k-ras RiAS-oligo.
  • A is k-ras RiAS-oligo
  • B is SC-oligo
  • C is Lipofectin alone.
  • the present invention provides novel AS-oligos containing one or more antisense sequence to regions with a less secondary structure.
  • 8 different regions of c-myb mRNA, one of protooncogenes, for target sites of antisense oligos were selected.
  • the rational taregt site search for an AS-oligo is employed to improve the chance to predict a natural secondary structure.
  • the above 8 antisense sequences are complementary to the selected target sites.
  • 4 sites (MIJ-1, MIJ-2, MIJ-3, and MIJ-4) are finally chosen in a combination upon forming a CMAS molecule and a 3 sites (MIJ-3, MIJ-4, and MIJ-17) in a combination forming a RiAS molecule as they form minimal intramolecular secondary structure (see Table 1) .
  • AS-oligos having phosphodiester backbone lacked stability which was essential for successful antisense application.
  • Modified oligos such as PS-oligo or MP-oligo, exhibited improved stability, but the gain in stability was only partial and beared potential harzard misincorporation of the hydrolyzed odified-nucleotides during DNA replication or repair. It was previously reported that stem-loop oligos complexed with cationic liposomes also showed partial improvement of stability. However, stability still remains a major concerns for AS-oligos. So, these inventors tried to develop an improved AS-oligo containing better stability.
  • the present invention provides a covalently-closed multiple antisense (CMAS) -oligo .
  • CMAS covalently-closed multiple antisense
  • AS-oligos intracellular secondary structure of AS-oligos was constructed without duplex formation between an AS-oligo and target mRNA.
  • these AS-oligos are designated a form of a CMAS (covalently-closed multiple antisense) -oligo containing four antisense sequences (MIJ-1 , MIJ-2, MIJ-3, and MIJ-4) which is described by SEQ ID NO : 1, NO : 2, NO : 3, and NO : 4 in a loop are placed in tandem to increase the length of CMAS-oligos (see FIG.l).
  • the CMAS-oligos show electrophoretic mobility patterns that it is slowed by about 10% than its linear presusor on a 15% denaturing PAGE gel (see A of FIG. 2) .
  • the CMAS-oligos are, as expected, resistant to exonuclease III and are shown in multiple bands on a denaturing PAGE gel, with monomer (60 mer) being the most abunbant, dinners (120 mer) and trimers(180 mer). In contrast to a CMAS-oligo, linear oligos are completely degraded after 2 hr incubation with exonuclease III (see B of FIG. 2).
  • the present invention also provides a ribbon-type antisense (RiAS) -oligo .
  • the CMAS-oligo although very stable, needs a primer for intramolecular ligation that must be eliminated afterward. So, to avoid using a ligation primer and obtain a homologus population of AS-oligo, these inventors makes two AS-oligos enzymatically ligated to form a ribbon-type closed molecule termed a RiAS-oligo.
  • the RiAS-oligo (116 mer) consists of two loops and one stem connecting two loops (see FIG. 3) .
  • three antisense sequences MIJ-3 , MIJ-4, and MIJ-17 which is described by SEQ IN NO : 3, NO : 4, and NO : 5 in a loop are placed in tandem to increase the length of RiAS-oligo.
  • the RiAS-oligo is found to be slowed markedly than its linear precusor (MIJ-78) on a denaturing PAGE gel (see A of FIG. 4) .
  • the RiAS-oligo is, as expected, resistant to exonuclaese III and is shown in a major band (116 mer) on a PAGE gel.
  • MIJ-78 is completely degraded after 2 hr incubation with exonuclease III (see B of FIG. 4) .
  • the CMAS-oligo and the RiAS-oligo of this invention are incubated with serums that are not heat inactivated to maintain nuclease activities.
  • linear 60 mer oligo (precusor of the CMAS-oligo, see A of FIG. 5) and linear 59 mer oligo (precusor of the RiAS-oligo, see A of FIG. 6) are completely digested after 24 hr incubation in the presence of serum.
  • the CMAS-oligo and the RiAS-oligo are remained mostly intact after 24 hr incubation with raw human serum, FBS, and calf serum, exhibiting significantly improved stability that does the linear one against nucleases activities (see B of FIG. 5 and B of FIG. 6) .
  • the CMAS-oligo was combined with Lipofectin to deliver into cells.
  • Lipofectin is employed as it is found to be less toxic to cells and yield consistent results.
  • MIJ-5 the CMAS-oligo to human c-myb, is able to reduce more than 95% of c-myb mRNA when compared to a control SC-oligo.
  • MIJ-5A the linear counterpart of MIJ-5, MIJ-5A, decreases some 37% of c-myb mRNA (see A of FIG. 7) .
  • the RiAS-oligo functions of eliminating target mRNA was demonstrated by the same method in the CMAS-oligo.
  • HL-60 cells were tested with RiAS-oligos, scrambled (SC) -oligos as well as Lipofectin alone.
  • the RiAS-oligo is delivered into cells after forming a complex with Lipofectin. Consequently, the RiAS-oligo is able to ablate c-myb mRNA to completion.
  • SC-oligo exhibits a mild reduction of c-myb mRNA by about 30% when compared to Lipofectin treatment alone (see A of FIG. 8) .
  • the present inventors also examines antisense effect of the CMAS-oilgo and the RiAS-oligo by Southern blotting of the PCR product.
  • c-myb meassage amplified by RT-PCR is detected with a labeled internal hybridization oligo (30 mer) (B of FIG. 8) .
  • the result confirms that the amplified meassage is indeed c-myb derived.
  • the present invention also provides pharmaceutical composition containing the novel types of AS-oligos for treatment cancer, immune diseases, infectious diseases, and other human diseases caused by aberrant gene expression.
  • c-myb CMAS-oligo or the c-myb RiAS-oligo inhibits leukemic cell growth.
  • growth inhibition of the c-myb CMAS-oligo and the c-myb RiAS-oligo to leukemic cells was measured by three methods, MTT assay, [ 3 H] thymidine incorporation or colony formation on soft agarose .
  • MIJ-5 reduces the number of colonies formed by more than 90% (see Table 2) . MIJ-5A also reduces colonies formed but less effective for growth inhibition, about 70% reduction of colonies. On the other hand, a sense oligo and a SC-oligo exhibits marginal reduction of colonies, by about 11% and 32% respectively.
  • the c-myb RiAS-oligo transfected into cells is able to reduce the number of colonies formed by about 92% (see Table 3) when compared to an untreated control. Meanwhile, a SC-oligo and Lipofectin alone exhibits marginal reduction of colonies, by about 7.9% and 7.1% respectively.
  • these inventors construct other RiAS-oligos against two different protooncogenes, c-myc and k-ras, and examine if the c-myc RiAS-oligo and the k-ras RiAS-oligo functiones well in inhibiting cell growth.
  • the novel RiAS-oligos of the present invention show effective growth inhibition of tumor cells to various target sequences as well as enhanced stability to nuclease activity. So, the novel RiAS-oligos of ' this invention may be effectively employed for developing molecular antisense oligos to treat various human diseases.
  • Example 1 Selection of target sites for an AS-oligo
  • Target site selection of an AS-oligo had been found to be critical to ' achive antisense effect, reduction or ablation of target mRNA. However, the approach for the target site selection had been rather arbitrary. So, these inventors scanned the entire sequence of human c-myb mRNA for putative secondary structures to search a rational target site in the preferred embodiment . Particulary, simulation of secondary structures
  • the above antisense sequences were complementary to the selected target sites.
  • 4 sites (MIJ-1, MIJ-2, MIJ-3, and MIJ-4) were finally chosen in a combination upon forming a CMAS molecule and a 3 sites (MIJ-3, MIJ-4, and MIJ-17) in a combination forming a RiAS-oligo as they form minimal intramolecular secondary structure .
  • CMAS multiple antisense
  • Example 1 4 different AS-oligos (MIJ-1, MIJ-2, MIJ-3, and MIJ-4) obatined by Example 1 was used to construct a CMAS-oligo. To bind to target sites more readily, one CMAS-oligo was constructed to harbor 4 different antisense sequences in a combination with the least secondary structure. AS-oligos were phosphorylated during synthesis at the 5 prime end to follow intra- or intermolecular covalent ligations (FIG. 1) . The sequence of the 60 mer AS-oligo containing 4 different antisense sequences was described by SEQ. ID NO : 7.
  • Both ends of the AS-oligo was joined with a ligation primer which has complementary sequences in its both halves to the both extreame end sequences (7 bases on each side) of the 60 mer AS-oligo.
  • the sequence of the 14 mer ligation primer was described by SEQ ID NO : 6.
  • Ligation primer was mixed with AS-oligo and was heated at 85°C for 2 min followed by gradual cooling to ambient temperature .
  • One unit of T4 ligase was added and incubated at 16°C for 16 hr to generate a covalently-closed molecule.
  • the CMAS-oligo was electropheoresed on a 5% MethphorTM agarose gel (FMC, USA) or on 12% denaturing PAGE and identified for its resistance to exonuclease III as well as for slight gel retardation compared with the liner 60 mer oligos.
  • Ligation primer was degraded with exonuclease III or detached from the CMAS-oligo by running on a denaturing gel after heating the oligos at 90°C.
  • This AS-oligo was designated a CMAS (covalently-closed multiple antisense) -oligo .
  • the CMAS-oligo showed electrophoretic mobility patterns that it was slowed by about 10% than its linear presusor on a 15% denaturing PAGE gel (A of FIG. 2) .
  • the CMAS-oligo was, as expected, resistant to exonuclease III and was shown in multiple bands on a denaturing PAGE gel, with monomer (60 mer) being the most abunbant , then dimers(120 mer) and trimers(180 mer) (B of FIG. 2).
  • a linear oligo was completely degraded after 2 hr incubation with exonuclease III.
  • the RiAS-oligo consisted of two loops and one stem connecting the two loops.
  • Each loop contained three different antisense (MIJ-3 , MIJ-4, and MIJ-17) sequences that were described by SEQ ID NO : 3 , NO : 4 , and NO : 5.
  • C-myb AS-oligo (MIJ-78) was phosphorylated at the 5 prime end. Sequences of the 58 mer MIJ-78 was described by SEQ ID NO : 8. MIJ-78 was to form a stem-loop structure. The stem was formed by complementary sequences at both ends of each oligo.
  • the 5 prime terminus of the stem had 4 bases of a single stranded sequense of 5 ' - (p) GATC-3 ' .
  • Two MIJ-78 molecules were joined by the complementary 4 base sequences at both 5 prime ends. MIJ-78 molecules were mixed and heated to 85°C for 2 min followed by gradual cooling to ambient temperature.
  • One unit of T4 DNA ligase was added and incubated at I60C for 24 hr to generate a covalently ligated molecule with diad-symmetry (FIG. 3) .
  • the RiAS-oligo was electrophoresed on 15% denaturing polyacrylamide gel and examined for its resistance to exonuclease III as well as for gel retardation.
  • the RiAS-oligo (116 mer) consisting of two loops and one stem connecting two loops was constructed. Three antisense sequences in a loop were placed in tandem to increase the length of the RiAS-oligo. Consequently, two copies of three different antisense sequences (total 6 antisense sequences) were in the RiAS-oligo. This enlarged length of the loop in the RiAS-oligo was to accommodate torsional stress caused by forming a duplex with the target mRNA sequences. The RiAS-oligo was found to be slowed markedly than its linear precusor (MIJ-78) on a denaturing PAGE gel (A of FIG. 4) .
  • MIJ-78 linear precusor
  • the RiAS-oligo was, as expected, resistant to exonuclaese III and was shown in a major band (116 mer) on a PAGE gel (B of FIG. 4). In contrast to RiAS-oligo, MIJ-78 was completely degraded after 2 hr incubation with exonuclease III.
  • Example 4 Enhanced stability of the CMAS-oligo
  • the RiAS-oligo to nuclease activities
  • the CMAS-oligo and the RiAS-oligo was incubated with serums that were not heat inactivated to maintain nuclease activities.
  • each of the nonspecific control-phosphodiester oligo (liner 60 mer) and the CMAS-oligo were incubated with either raw human serum, FBS and calf serum (non-heat inactivated; HyClone, Logan, Utah, USA) or exonuclease III. 15% of each serum was added to AS-oligos in an 100 ul reaction volume and incubated at 37°C for 24 hr. AS-oligos were then extrected with phenol and chloroform, and were examined on 15% denaturing PAGE gel .
  • Exonuclease III (Takara, Japan) at 160 U/ug oligo was added to linear and CMAS-oligos and incubated at 37°C for 2 hr. AS-oligos treated with exonuclease III were also extracted and electrophoresed in the same manner.
  • Leukemic cell lines HL-60 (promyelocyte leukemic cell line) and K562 (chronic myelogenous leukemic cell line), were obtained from ATCC (American Type Culture Collection, USA) and cultured in RPMI 1640 (Gibco BRL, USA) supplemented with 10% heat-inactivated FBS (HyClone, USA) and 1% penicillin/streptomycine . Cells were maintained in a C0 2 incubator at 37°C. Routine cell culture practices were strictly adhered to keep proper cell density and to avoid cells cultured more than 5 generations after thawing stock vials. Culture media were exchanged a day before treating with AS-oligos.
  • 0.3 ug CMAS-oligo plus 0.8 ug LipofectinT (Gibco BRL, USA) or 0.2 ug RiAS-oligo plus 0.8 ug LipofectinTM were dilluted in 20 ul OPTI-MEM TM seperately and incubated at ambient temperature for 40 min. Each component was then mixed to form a complex at ambient temperature for 15 min. Cells were added with fresh culture media without antibiltics (RPMI 1640 + 10% FBS) 1 day prior to adding oligos and washed twice with OPTI-MEM before an experiment. Cell density was adjusted to 5 X 10 5 cells/ml and aliquoted in 100 ul each in a 48-well plate (Falcon, USA) .
  • RNA was added with 0.4 ml Tripure reagent, 10 ug glycogen and 80 ul chloroform to obtain total RNA.
  • RT-PCR was performed in a single reaction tube with AccessTM RT-PCR kit (Promrga, USA) .
  • RNA PCR primers
  • AMV reverse transcriptase 5 U/ul
  • Tfl DNA polymerase 5 U/ul
  • dNTP 10 mM, 1 ul
  • MgS0 4 25 M, 2.5 ul) .
  • Synthesis of the first strand cDNA was done at 48 °C for 45 min in a DNA termal cycler (Hybaid, USA) . 25 cycles of PCR amplification were subsequently carried out with the recommended condition by the manufacturer. Amplified PCR product was confirmed in an 1% agarose gel and quantitation was done with a gel documentation program (Bio-Rad, USA).
  • RT-PCR products were electrophoresed on an 1% agarose gel. DNA was transferred onto a nylon membrane (New England Biolab, USA) for 4 hr in 0.4 M NaOH. The membrane was hybridized with 30 mer internal primer labeled with ECL 3 prime end oligo-labeling and detection system (Amersham Life Science, England) . The sequence of 30 mer internal primer was described by SEQ ID NO : 9. Hybridization was carried out at 62 °C for 60 min in 6 ml buffer containing 5 X SSC, 0.02% SDS. The membrane was washed twice in 5 X SSC containing 0.1% SDS and washed twice with 1 X SSC containing 0.1% SDS at for 15 min. The membrane was blocked with a blocking solution and then treated with HRP (horse radish peroxidase) anti-fluorescein conjugated antibody for 30 min before autoradiography.
  • HRP horse radish peroxidase
  • the CMAS-oligo was complexed with Lipofectin to deliver into cells. Lipofectin was employed as it was found to be less toxic to cells and yield consistent results. As a result, 0.3 ug MIJ-5, a CMAS-oligo to human c-myb, was complexed with 1 ug Lipofectin for transfection into HL-60 cells. MJ-5 was able to reduce more than 95% of c-myb mRNA when compared to a control SC-oligo. Meanwhile, the linear counterpart of MIJ-5, MIJ-5A, decreased some 37% of c-myb mRNA (A of FIG. 7) .
  • HL-60 cells were transfected with the RiAS-oligos, SC-oligos as well as Lipofectin alone.
  • the RiAS-oligo was delivered into cells after forming a complex with Lipofectin.
  • the RiAS-oligo (0.1 ug or 0.2 ug) to human c-myb was complexed with 0.8 ug Lipofectin for transfection into HL-60 cells. Consequently, 0.2 ug of the RiAS-oligo (40 nM) was able to ablate c-myb mRNA to completion. Meanwhile, 0.1 ug of the RiAS-oilgo decreased about 70% of c-myb mRNA (A of FIG. 8) .
  • SC-oligo exhibited a mild reduction of c-myb mRNA by about 30% when compared to Lipofectin treatment alone.
  • ⁇ -actin expression shown in the bottom panel was not affected by the treatment of the RiAS-oligo as well as other treatment conditions.
  • the present inventors also examined antisense effect of the CMAS-oilgo and the RiAS-oligo by Sourthern blotting with PCR products.
  • HL-60 cells were transfected with oligos including MIJ-5 and control oligos, and the cells were used to isolate total DNA.
  • c-myb played an important role in proliferation of leukocytes.
  • AS-oligos to c-myb were also reported to block leukemic cell growth preferentially. So, these inventors tested the c-myb CMAS-oligo and the c-myb RiAS-oligo of this invention for inhibiting leukemic cell growth. Particulary, growth inhibition of the c-myb CMAS-oligo and the c-myb RiAS-oligo to leukemic cells was measured by three methods, MTT assay, [ 3 H] thymidine incorporation or colony formation on soft agarose .
  • HL-60 cells were washed twice with OPTI-MEM and aliquoted in a 96-well plate (5 X 10 3 cells/well) in a 50 ul volume. Cells were treated with performed complex between oligos in different amount (0.01 - 1 ug/15 ul in CMAS-oligo or 0.2 ug/15 ul in RiAS-oligo) and Lipofectin (0.2 ug/15 ul) for 5 hr and cultured for 5 days .
  • Cells were then harvested in an 100 ul volume and added with 20 ul(100 ug) of an MTT reagent (5 mg/ml in PBS; Sigma, USA), followed by 4 hr incubation at 37°C. An 100 ul of isopropanol (containing 0.1 N HCl) was added to the cells and incubated for one more hour at the ambient temperature. Absorbance was measured at 570 nm with an ELISA reader to score the amount of cells survived.
  • MTT reagent 5 mg/ml in PBS; Sigma, USA
  • CMAS-oligo cell number was reduced progressively when treated with increasing amounts of MIJ-5. Inhibition of cell growth was more pronounced when cells were treated twice with MIJ-5. More than 80% of growth inhibition of HL-60 cells was observed even at a low concentration, 0.13 ug (total 0.24 ug) of the CMAS-oligo (FIG. 9). Meanwhile, the linear 60 mer AS-oligo, MIJ-5A, and linear sense oligo did not bring about any significant inhibition of cell growth when compared with a sham control . These results indicated that the c-myb CMAS-oligo was an effective antisense agent and was efficacious agent against tumor growth in a concentration dependent manner.
  • RiAS-oligo cell growth was also observed to be inhibited by 91% with the RiAS-oligo (A of FIG. 10) . Meanwhile, the SC-oligo and Lipofectin alone did not significantly inhibited cell growth when compared to that of the untreated control. These results indicated that the c-myb RiAS-oligo was an effective antisense agent for inhibition of leukemic cell growth .
  • CMAS-oligo MIJ-5 reduced the number of colonies formed by more than 90% (Table 2) .
  • MIJ-5A also reduced colonies formed but less effective for growth inhibition, about 70% reduction of colonies.
  • a sense oligo and a SC-oligo exhibited marginal reduction of colonies, by about 11% and 32% respectively.
  • HL-60 cells were treated with AS-oligo as described above. Cells were added with 0.5 uCi of [ 3 H] thymidine (2.0 Ci/mmol; Amersham, England) and incubated for 16 hr in triplicate. Cells were then harvested on a glass microfiber filter (Whatman GF/C, England). The filter was washed with in the order of cold PBS, 5% TCA and absolute ethanol . [ 3 H] thymidine incorporation was measured with the liquid scintillation counter in a cocktail solution containing toluene, Triton X-100, PPO and POPOP .
  • the RiAS-oligo (0.2 ug) inhibited growth of HL-60 cells by 93% (B of FIG. 10). Meanwhile, the SC-oligo and Lipofectin alone exhibited mild inhibition of cell growth, by about 16.8% and 15.4% respectiverly . On a microscopic observation, after treated with the c-myb RiAS-oligo, growth of HL-60 cells was markedly inhibited when compared with cells treated with scrambled oligo and Lipofectin alone (FIG. 11) .
  • RiAS-oligo and the k-ras RiAS-oligo
  • Example 3 Example 3. And then, they examined if the c-myc RiAS-oligo and the k-ras RiAS-oligo functioned well in inhibiting cell growth.
  • HT-29 used different cell line, colorectal adenocarcinoma cell line HT-29. Growth inhibition of the c-myc RiAS-oligo and k-ras RiAS-oligo to tumor cells was measured by [ 3 H] thymidine incorporation as the same method in Example ⁇ 6-3>.
  • HT-29 cells were treated with cationic liposome complexes of 0.2 ug c-myb RiAS-oligo plus 0.6 ug Lipofectin or 0.5 ug c-myc RiAS-oligo plus 1.5 ug Lipofectin or 0.5 ug k-ras RiAS-oligo plus 1.5 ug Lipofectin, respectively.
  • the novel RiAS-oligos of the present invention showed effective growth inhibition of tumor cells to various target sequences as well as enhanced stability to nuclease activity. So, the novel RiAS-oligos of this invention might be effectively employed for developing molecular antisense oligos to treat various human diseases .
  • the present invention provides novel AS-oligos containing one or more antisense sequence to mRNA region with a less secondary structure and having better target sequence specificity and stability to nuclease activities.
  • the present invention provides a covalently-closed multiple antisense (CMAS) -oligo containing multiple target antisense sequences to c-myb mRNA which is constructed to form a closed type by ligation using complementary primer.
  • CMAS covalently-closed multiple antisense
  • the present invention provides a ribbon-type antisnese (RiAS) -oligo containing multiple target antisense sequences to c-myb mRNA which is constructed to form a stem-loop structure by ligation using complementary sequences at both 5 prime ends .
  • novel AS-oligos of this invention may be employed for developing molecular antisense drugs to various genes causing diseases as well as for the functional study of a gene.
  • novel AS-oligos of this invention may be used for developing pharmaceutical composition for treatment cancer, immune diseases, infectious diseases, or other human diseases caused by aberrant gene expression.

Abstract

L'invention concerne de nouveaux oligonucléotides antisens (AS) contenant une ou plusieurs séquences antisens d'une région d'ARNm avec une structure secondaire moindre. L'invention concerne plus particulièrement un oligonucléotide antisens (CMAS) multiple fermé par covalence construit pour former un type fermé par liaison utilisant une amorce complémentaire et un oligonucléotide antisens (RiAS) de type bande se composant de deux boucles contenant de multiples séquences antisens et une tige reliant les deux boucles construite à l'aide des séquences complémentaires aux deux extrémités des amorces 5. Les nouveaux oligonucléotides AS sont extrêmement stables aux activités de l'exonucléase et font preuve d'une inhibition importante à la croissance de cellules tumorales. Les compositions pharmaceutiques contenant ces nouveaux types d'oligonucléotides AS sont efficaces pour le traitement du cancer, de maladies immunes, de maladies infectieuses et d'autres maladies affectant l'homme provoquées par l'expression génique aberrante.
EP00913157A 1999-04-08 2000-04-04 Oligonucleotides antisens a stabilite amelioree et a effet antisens Withdrawn EP1175430A4 (fr)

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KR1019990012297A KR20000065690A (ko) 1999-04-08 1999-04-08 반응 특이성 및 안정성을 개선시킨 안티센스 올리고 뉴클레오타이드, 안티센스 ｄｎａ 및 그 제조방법
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AU2004242533B2 (en) * 2001-03-08 2007-08-30 Welgene, Inc. Large circular target-specific antisense nucleic acid compounds
KR20030056538A (ko) * 2001-12-28 2003-07-04 주식회사 웰진 리본형 안티센스 올리고뉴클레오티드에 의한 형질전이성장 인자-β1의 효과적 저해제 개발
US20060009409A1 (en) 2002-02-01 2006-01-12 Woolf Tod M Double-stranded oligonucleotides
ATE529512T1 (de) 2002-02-01 2011-11-15 Life Technologies Corp Doppelsträngige oligonukleotide
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JP2015529469A (ja) 2012-09-14 2015-10-08 ラナ セラピューティクス インコーポレイテッド 多量体オリゴヌクレオチド化合物
WO2019195738A1 (fr) 2018-04-06 2019-10-10 Children's Medical Center Corporation Compositions et procédés de reprogrammation de cellules somatiques et de modulation d'impression
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CN100381455C (zh) 2008-04-16
KR20000065690A (ko) 2000-11-15
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