Modification of the specificity of non-coding RNA molecules for silencing gene expression in eukaryotic cells

Abstract

This invention provides a method for modifying genes that encode or are processed by non-coding RNA molecules that do not possess RNA silencing activity in eukaryotic cells. [Solution] The method is not for eukaryotic cells but for plant cells, and the method includes the step of introducing a DNA editing agent that confers silencing specificity of a non-coding RNA molecule to a target RNA of interest into the eukaryotic cells, and provides a method for modifying a gene that codes for an RNA silencing molecule against a target RNA or is processed by it in eukaryotic cells.

Claims

1. A method for modifying a gene that codes for or processes a non-coding RNA molecule that does not have RNA silencing activity in a eukaryotic cell, wherein the eukaryotic cell is not a plant cell, and the method comprises the steps of introducing a DNA editing agent into the eukaryotic cell that confers silencing specificity of the non-coding RNA molecule to a target RNA of interest, thereby modifying the gene that codes for or processes the non-coding RNA molecule.

2. A method for modifying a gene encoding or being processed by an RNA silencing molecule for a target RNA in a eukaryotic cell, wherein the eukaryotic cell is not a plant cell, and the method comprises the step of introducing a DNA editing agent into the eukaryotic cell that redirects the silencing specificity of the RNA silencing molecule toward a second target RNA, thereby modifying the gene encoding the RNA silencing molecule, wherein the target RNA and the second target RNA are different.

3. The method according to claim 1, wherein the gene encoding or being processed by the non-coding RNA molecule is endogenous to the eukaryotic cell.

4. The method according to claim 2, wherein the gene encoding the RNA silencing molecule is endogenous to the eukaryotic cell.

5. The method according to claim 1 or 3, wherein modifying the gene that encodes or processes the non-coding RNA molecule confers at least 45% complementarity to the target RNA of the purpose to the non-coding RNA molecule.

6. The method according to claim 2 or 4, wherein modifying the gene encoding the RNA silencing molecule confers at least 45% complementarity to the RNA silencing molecule with respect to the second target RNA.

7. The method according to claim 1, claim 3, or claim 5, wherein the silencing specificity of the non-coding RNA molecule is determined by measuring the RNA level or protein level of the target RNA of the purpose.

8. The method according to claim 2, claim 4, or claim 6, wherein the silencing specificity of the RNA silencing molecule is determined by measuring the RNA level or protein level of the second target RNA.

9. The method according to any one of claims 1 to 8, wherein the silencing specificity of the non-coding RNA molecule or the RNA silencing molecule is phenotypically determined.

10. The method according to claim 9, wherein the phenotypic determination is brought about by determining at least one phenotype selected from the group consisting of cell size, growth rate / inhibition, cell shape, cell membrane integrity, tumor size, tumor shape, pigmentation of the organism, infection parameters, and inflammation parameters.

11. The method according to any one of claims 1 to 10, wherein the silencing specificity of the non-coding RNA molecule or the RNA silencing molecule is determined genotypeically.

12. The method according to claim 11, wherein the phenotype is determined before the genotype.

13. The method according to claim 11, wherein the genotype is determined before the phenotype.

14. The method according to any one of claims 1 to 13, which is processed from the non-coding RNA molecule or the RNA silencing molecule precursor.

15. The method according to any one of claims 1 to 14, wherein the non-coding RNA molecule or the RNA silencing molecule is an RNA interference (RNAi) molecule.

16. The method according to claim 15, wherein the RNAi molecule is selected from the group consisting of small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), Piwi-interacting RNA (piRNA), and trans-acting siRNA (tasiRNA).

17. The method according to any one of claims 1, 3, 5, 7 and 9 to 14, wherein the non-coding RNA molecule is selected from the group consisting of nuclear small RNA (snRNA), nucleolar small RNA (snoRNA), long non-coding RNA (lncRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), repeat-derived RNA, and transfer factor RNA.

18. The method according to claim 15 or claim 16, wherein the RNAi molecule is modified to preserve structural originality and to be recognized by a cellular RNAi factor.

19. The method according to any one of claims 1 to 18, wherein the modification of the gene is performed by a modification selected from the group consisting of deletions, insertions, point mutations and combinations thereof.

20. The method according to claim 19, wherein the modification is in the stem region of the non-coding RNA molecule or the RNA silencing molecule.

21. The method according to claim 19, wherein the modification is in the loop region of the non-coding RNA molecule or the RNA silencing molecule.

22. The method according to claim 19, wherein the modification is in the unstructured region of the non-coding RNA molecule or the RNA silencing molecule.

23. The method according to claim 19, wherein the modification is in the stem region and loop region of the non-coding RNA molecule or the RNA silencing molecule.

24. The method according to claim 19, wherein the modification is in the stem region, loop region, and unstructured region of the non-coding RNA molecule or the RNA silencing molecule.

25. The method according to any one of claims 19 to 24, wherein the modification comprises a modification of up to 200 nucleotides.

26. The method according to any one of claims 19 to 25, further comprising introducing a donor oligonucleotide into the eukaryotic cells.

27. The method according to any one of claims 1 to 26, wherein the DNA editing agent comprises at least one gRNA operably linked to a plant expression promoter.

28. The method according to any one of claims 1 to 27, wherein the DNA editing agent does not contain an endonuclease.

29. The method according to any one of claims 1 to 27, wherein the DNA editing agent comprises an endonuclease.

30. The method according to any one of claims 1 to 29, wherein the DNA editing agent is selected from the group consisting of meganuclease, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and CRISPR.

31. The method according to claim 29 or claim 30, wherein the endonuclease comprises Cas9.

32. The method according to any one of claims 1 to 31, wherein the DNA editing agent is applied to the cells as DNA, RNA, or RNP.

33. The method according to any one of claims 1 to 32, wherein the DNA editing agent is linked to a reporter for monitoring expression in eukaryotic cells.

34. The method according to claim 33, wherein the reporter is a fluorescent protein.

35. The method according to any one of claims 1 to 34, wherein the target RNA or the second target RNA is endogenous to the eukaryotic cell.

36. The method according to claim 35, wherein the target RNA for the purpose or the second target RNA is related to cancer.

37. The method according to any one of claims 1 to 34, wherein the target RNA for the purpose or the second target RNA is exogenous to the eukaryotic cell.

38. The method according to claim 37, wherein the target RNA for the purpose or the second target RNA is related to an infectious disease.

39. The method according to any one of claims 1 to 38, wherein the eukaryotic cells are obtained from eukaryotes selected from the group consisting of mammals, insects, nematodes, birds, reptiles, fish, crustaceans, fungi, and algae.

40. The method according to any one of claims 1 to 39, wherein the eukaryotic cell is a mammalian cell.

41. The method according to claim 40, wherein the mammalian cells include human cells.

42. The method according to any one of claims 1 to 41, wherein the eukaryotic cell is a totipotent stem cell.

43. A method for treating an infectious disease in a subject requiring treatment, comprising modifying a gene that encodes or is processed by a non-coding RNA molecule or an RNA silencing molecule or is processed by an RNA silencing molecule, in accordance with the method of any one of claims 1 to 42, thereby treating the infectious disease in the subject. A method comprising the above-mentioned target RNA, wherein the target RNA is associated with the onset or progression of the infectious disease.

44. A method for treating a monogene recessive genetic disorder in a subject of interest, comprising the step of modifying a gene that encodes or is processed by a non-coding RNA molecule or encodes or is processed by an RNA silencing molecule, according to the method of any one of claims 1 to 42, thereby treating the monogene recessive genetic disorder in the subject, wherein the target RNA of interest is associated with the monogene recessive genetic disorder.

45. A method for treating an autoimmune disease in a subject of interest, comprising the step of modifying a gene that encodes or is processed by a non-coding RNA molecule or encodes or is processed by an RNA silencing molecule, in accordance with the method of any one of claims 1 to 42, thereby treating the autoimmune disease in the subject, wherein the target RNA of interest is associated with the autoimmune disease.

46. A method for treating a cancerous disease in a subject of interest, comprising the step of modifying a gene that encodes or is processed by a non-coding RNA molecule or encodes or is processed by an RNA silencing molecule, in accordance with the method of any one of claims 1 to 42, thereby treating the cancerous disease in the subject, wherein the target RNA of interest is associated with the cancerous disease.

47. A method for enhancing the efficacy and / or specificity of a chemotherapeutic agent in a target subject, comprising the step of modifying a gene that encodes or is processed by a non-coding RNA molecule or encodes or is processed by an RNA silencing molecule, in accordance with the method of any one of claims 1 to 42, thereby enhancing the efficacy and / or specificity of the chemotherapeutic agent in the target subject, wherein the target RNA of the object is related to the enhancement of the efficacy and / or specificity of the chemotherapeutic agent.

48. A method for inducing cellular apoptosis in a target subject, comprising the step of modifying a gene that encodes or is processed by a non-coding RNA molecule or encodes or is processed by an RNA silencing molecule, according to the method of any one of claims 1 to 42, thereby inducing cellular apoptosis in the target subject, wherein the target RNA of the purpose is associated with the apoptosis.

49. A method for generating a eukaryotic non-human organism, wherein the organism is not a plant, and at least some of the cells of the organism contain a modified gene that encodes or processes a non-coding RNA molecule having silencing specificity for a target RNA of interest, the method comprising the step of modifying a gene according to the method of any one of claims 1 to 42 to generate the eukaryotic non-human organism.

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