Classifications

• • 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

Definitions

• the present invention relates to modified oligonucleotides, especially 3'-0H monophosphorylated modified oligonucleotides to increase stability and reduce toxic side effects.
• the monophosphorylated modified oligonucleotides can be used as a treatment Drug. technical background
• Oligonucleotides include antisense oligonucleotides, anti-gene oligonucleotides (also known as triple helix-forming oligonucleotides), etc., which can be used to suppress gene expression, and are a new therapeutic method developed in recent years Wagner, RW, Nature, 1994, 372, 333-335; Crook, ST, Annu. Rev. Pharmacol. Toxica l., 1992, 32, 329-376; Helene, C, Eur. J. Cancer, 1994, 30A, 1721-1726; Crooke, ST, Ant i sense Nucle ic ac ids Drug Dev., 1996, 6, 141-147).
• Its mechanism of action includes blocking the replication, transcription, and translation of viral genes, as well as the transcription and translation of harmful genes in the human body, and the degradation of target RNA by the action of RNase H enzymes in the body. It is a class of highly specific and toxic side effects Small therapeutic drugs.
• Antisense oligonucleotides currently in clinical trials include anti-HIV GEM91 (Phase II), anti-inflammatory ISIS2302 (Phase II), anti-cancer ISIS3521 (Phase I) and ISIS5132 (Phase I), anti-AIDS patients CMV retinitis ISIS2922 (Phase III), LR-3001 (Phase I) for the treatment of chronic myelogenous leukemia (Genet ic Engineer ing News, 1996, 16, 29-34).
• the oligonucleotide fragments enter the human body, they are susceptible to degradation by the enzyme system in the human body, that is, the oligonucleotides entering the human body do not reach the target organ, and the target cells are degraded, so the expected therapeutic effect cannot be achieved (Hoke, GD, et a l. Nuc le ic Ac ids Res., 1991, 19, 5734-5748) 0 Therefore, it is very important to improve the stability of the oligonucleotide, especially its anti-enzymatic ability. In this way, not only can Increasing the efficacy and reducing the amount of medicine can also further reduce the cost of treatment and reduce side effects.
• the object of the present invention is to provide a 3'-monophosphorylated oligonucleotide to increase the stability of the oligonucleotide and reduce the toxic and side effects.
• 3'-phosphate solid phase column (3'-phosphate CPG Glen Research company product, full name 2_ [2- (4, 4, -dimethoxytriphenylmethoxy) ethanesulfonyl) ethyl] Succinyl long-chain alkylamine-microporous glass beads (2- [2- (4, 4,-Dimethoxy tri tyloxy) ethylsulfomyl] ethyl-succinoyl long chain alkylamino-CPG)), synthesized on ABI 391EP DNA synthesizer The 3'-monophosphorylated oligonucleotide was obtained by removing the protecting group through concentrated ammonia.
• snake venom phosphodiesterase is used to represent the 3'-5 'exonuclease in the body
• DNase I is used to represent the endonuclease in the body.
• 3'-phosphorylated oligonucleotide fragments are resistant to snake venom phosphodiesterase, and the stability in serum and cells is significantly higher than that of 3 'unmodified or y-partial phosphorothioate-modified Nucleotide, slightly higher than phosphorothioate-modified oligonucleotide. Moreover, the 3'-phosphorylation modification does not affect the rate at which the oligonucleotide enters the cell, which is superior to the phosphorothioate-modified oligonucleotide.
• 3'-monophosphorylated oligonucleotides can be degraded by 3'-5 'exonuclease only after the 3' phosphate is cut off by a phosphodiesterase.
• the 3'-monophosphorylated oligonucleotide has high stability in serum and intracellular cells, indicating that the activity of phosphodiesterase in serum and intracellular cells is not high.
• 3′-monophosphorylated oligonucleotide primers once bound to DNA, can also inhibit virus D replication or RNA reverse transcription.
• Most of the currently used antisense oligonucleotides are phosphorothioate-modified, 3 ', 0H, oligonucleotides, which do not inhibit viral DM replication or RNA reverse transcription.
• the 3'-monophosphorylated oligonucleotide has no effect on normal base pairing, and thus can ensure that the modified oligonucleotide is correctly and specifically paired with the target DNA or RM.
• a 3'-monophosphorylated oligonucleotide fragment designed based on a specific sequence of DNA or RNA of a harmful gene such as a virus or a tumor gene can specifically bind to target DNA or RM to block the replication and transcription of viral DNA And translation, can also suppress the transcription and translation of harmful genes in the human body, and can also use RNaseH in the body to be 3'-monophosphorylated Oligonucleotide-bound target RNA is degraded. Therefore, due to the above-mentioned mechanism of action and superior properties, 3'-monophosphorylated oligonucleotides have more biological functions.
• the 3'-monophosphorylated oligonucleotide provided by the present invention has the advantages of being more stable and easier to be taken up by cells than the phosphorothioate-modified oligonucleotide.
• the phosphate group is an inherent component in natural nucleic acids, the use of the phosphate group as a modification group does not introduce non-natural modification components, and its metabolic degradation products do not have any toxic and side effects. Therefore, compared with other chemical modification methods It is safer to use and its overall performance is better than the most widely used thiophosphate-substituted oligonucleotides.
• -Monophosphorylated oligonucleotides can specifically bind to target DM or RNA, and inhibit replication, transcription and translation of viral DNA. Therefore, 3'-monophosphorylated oligonucleotides are expected to become a clinically effective drug.
• Article 1 y is an OH group
• oligonucleotide 2 is a monophosphate group
• the third oligonucleotide is connected through a phosphorothioate bond between all the nucleotides, and is a 0H group.
• the third oligonucleotide of the fourth oligonucleotide has three phosphorophosphate diester bonds, and the rest are phosphorodiphosphates. Ester bond, 3 'is a 0H group.
• Article 2 3'-monophosphorylated oligonucleotide uses 0.2 ⁇ ⁇ 3' phosphate solid phase column (3'-phosphate CPG Glen Research company product, full name 2 ⁇ [2- (4,4, —Dimethyltriphenylmethoxy) ethanesulfonyl] ethyl-succinyl long-chain alkylamine-microporous glass beads (2- [2- (4,4'- Dimethoxy trityloxy) ethylsulfomyl] ethyl-succinoyl long chain alkylamino-CPG)) was synthesized on a ABI 391EP DM synthesizer using a 0.2 mole synthesis sequence.
• Oligonucleotides whose 3 ′ is 0H in Article 1 were synthesized using a 0.2 ⁇ dT solid-phase column (Glen Research product) using the same synthesis sequence.
• Articles 3 and 4 were synthesized using a dA solid-phase column (Glen Research product), which was synthesized in the same synthetic sequence, and the sulfuric acid reagent was used instead of the oxidizing reagent at the position modified by thiophosphoric acid (see Iyer, RT, et al. J. Org Chem. 1990, 55, 4693-4699), the rest are the same.
• the four oligonucleotide fragments (3'0H, 3'P, SP, 3SP) obtained in Example 1 were each using T4 Polynucleotide kinase (T4 ⁇ ) at its 5 'end with the 32 ⁇ , taking 50 pmol oligonucleotide fragment, add 50 ⁇ ⁇ [ ⁇ - 32 p ] -ATP, 2 units of T4 polynucleotide Kinase, 1 ⁇ 1 10-fold buffer, add double distilled water to 10 ⁇ 1, and incubate at 37 ° C for 1 hour.
• T4 Polynucleotide kinase T4 Polynucleotide kinase
• Example 2 the same sample labeled with 32 P to prepare four oligonucleotides, which were added in the non-labeled oligonucleotide to a final concentration of 5 ⁇ mol / L, DNase I was added to 100 U / ml, buffered The solution is 10-leg ol / L Tris-HCl, 5 mraol / L MgCl 2 , 0.1% BSA, pH 8. 0, 37 ° C, and maintained at 0, 0.5, 1, 1.5, 2, 4 Take 5 ⁇ l each at 1, 8, 12, and 24 hours. Add an equal volume of loading buffer and mix. Use 7 mol / L urea-20% polyacrylamide gel electrophoresis for identification.
• Example 2-four kinds of 32 P-labeled oligonucleotides were prepared, and unlabeled oligos were respectively added thereto.
• Nucleotide to a final concentration of 5 ⁇ / L add human serum to a final concentration of 40%, incubate at 37 ° C, and take 5 ⁇ l each at 0, 0.5, 1, 1.5, 2, 4, 8, 12, 24 hours Add an equal volume of loading buffer and mix with 7 mol / L urea-20% polyacrylamide gel electrophoresis for identification.
• Inoculate 2 x 10 5 HeLa cells in a 35mm petri dish add 1.5 ml of cell culture solution (DMEM contains 10% calf serum), and grow to 37%-60% of the area of the culture well at 37 ° C 5% C0 2 and aspirate.
• DMEM contains 10% calf serum

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• 1997
  • 1997-06-28 CN CN97106495A patent/CN1060177C/zh not_active Expired - Fee Related
• 1998
  • 1998-06-26 AU AU79051/98A patent/AU7905198A/en not_active Abandoned
  • 1998-06-26 WO PCT/CN1998/000102 patent/WO1999000401A1/zh active Application Filing
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