Methods and compositions for reducing target gene expression using cocktails of siRNAs or constructs expressing siRNAs

• US 20040029275A1
• Filed: 11/15/2002
• Published: 02/12/2004
• Est. Priority Date: 08/10/2002
• Status: Abandoned Application

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Brown, David, Jarvis, Rich, Ford, Lance P.
• Application Number:
  US10/298,480
• Publication Number:
  US 20040029275A1
• Time in Patent Office: Days
• US Class Current: 435/375
• CPC Class Codes: A61K 31/713 Double-stranded nucleic acids or oligonucleotides
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/113 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 when used in plants C12N15/8218
• CPC Class Codes: C12N 15/1135 against oncogenes or tumor suppressor genes
• CPC Class Codes: C12N 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• CPC Class Codes: C12N 2310/111 spanning the whole gene, or a large part of it
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2330/31 Libraries, arrays
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The present invention concerns methods and compositions involving the production or generation of siRNA mixtures or pools capable of triggering RNA-mediated interference (RNAi) in a cell. Compositions of the invention include kits that include reagents for producing or generating siRNA pools. The present invention further concerns methods using polypeptides with RNase III activity for generating siRNA mixtures or pools that effect RNAi, including the generation of a number of RNA molecules to the same target gene.

Claims

1. A method for reducing or eliminating expression of at least one target gene in a cell comprising introducing a pool of siRNA molecules directed to at least one target gene into the cell.

2. The method of claim 1, wherein the pool of siRNA molecules is introduced into the cell by transfection.

3. The method of claim 1, wherein the pool of siRNA molecules is introduced by transfecting one or more expression constructs encoding siRNAs into the cell.

4. The method of claim 3, wherein the expression construct comprises one or more siRNA expression domains.

5. The method of claim 3, wherein the expression construct is a plasmid DNA expression construct, a linear DNA expression construct, or a viral expression construct.

6. The method of claim 1, wherein the pool of siRNA molecules is directed to at least two target genes.

7. The methods of claim 1, wherein the pool of siRNA molecules is directed to at least three target genes.

8. The method of claim 1, wherein the siRNA molecules are generated by transcribing in vitro one or more plasmids encoding the siRNA molecules to create RNA transcripts;

and hybridizing the RNA transcripts to allow hybridization within complementary regions of the RNA transcripts.

9. The method of claim 1, further comprising generating the pool of siRNA molecules to at least one target gene.

10. The method of claim 9, wherein the siRNA molecules are generated by hybridizing complementary oligonucleotides that were synthesized in vitro.

11. The method of claim 9, wherein the siRNA molecules are generated by hybridizing complementary ribonucleotides transcribed in vitro.

12. The method of claim 9, wherein the pool of siRNAs are generated in vitro.

13. The method of claim 12, wherein the pool of siRNA molecules comprises at least two siRNA molecules.

14. The method of claim 12, wherein the pool of siRNA molecules comprises at least three siRNA molecules.

15. The method of claim 12, wherein the siRNA molecules are isolated before or after combining the siRNA molecules with each other.

16. The method of claim 12, wherein the siRNA molecules are generated by a process comprising:

a) obtaining at least one dsRNA that corresponds to at least 15 contiguous basepairs of the first target gene;

b) incubating the at least one dsRNA with a nuclease under conditions to allow cleavage of the at least one dsRNA, wherein the at least one dsRNA is cleaved at least once to generate at least two candidate siRNA molecules that correspond to the first target gene.

17. The method of claim 1, wherein the pool of siRNA molecules comprises 2 to 20 different candidate siRNA molecules.

18. The method of claim 17, wherein the pool of siRNA molecules are directed to one target gene.

19. The method of claim 17, wherein the pool of siRNA molecules comprises 3 to 10 different candidate siRNA molecules.

20. The method of claim 19, wherein the pool of siRNA molecules are directed to one target gene.

21. The method of claim 1, further comprising reducing or eliminating the expression of at least a second target gene in a cell comprising introducing a pool of siRNA molecules directed to at least a second target gene into the cell.

22. The method of claim 1 wherein the pool of siRNA molecules to at least one target gene is generated by methods comprising:

a) obtaining a first dsRNA that corresponds to at least 15 contiguous basepairs of the at least one target gene; and

b) incubating the first dsRNA with a nuclease under conditions to allow cleavage of the first dsRNA, wherein the first dsRNA is cleaved at least once to generate at least two candidate siRNA molecules that correspond to the at least one target gene.

23. The method claim 1, further comprising labeling the pool of siRNA molecules.

24. A method for generating a pool of candidate siRNA molecules to a target gene comprising:

a) obtaining a first dsRNA that corresponds to at least 15 contiguous basepairs of the target gene; and

b) incubating the first dsRNA with a nuclease under conditions to allow cleavage of the first dsRNA, wherein the first dsRNA is cleaved at least once to generate at least two candidate siRNA molecules that correspond to the target gene.

25. The method of claim 24, wherein the nuclease is an RNase III polypeptide.

26. The method of claim 25, wherein the RNase III polypeptide is DICER.

27. The method of claim 25, wherein the RNase III polypeptide is a prokaryotic RNase III.

28. The method of claim 27, wherein the RNase III polypeptide is an E. coli RNase III polypeptide.

29. The method of claim 25, wherein the RNase III polypeptide is recombinant.

30. The method of claim 24, wherein the first dsRNA and the nuclease are incubated at 30 to 40°

C.

31. The method of claim 24, further comprising isolating the candidate siRNA molecules.

32. The method of claim 31, further comprising transfecting the candidate siRNA molecules into a cell.

33. The method of claim 24, further comprising incubating the nuclease with a second dsRNA that corresponds to at least 15 contiguous basepairs of a first target gene or a second target gene.

34. The method of claim 33, wherein the second dsRNA corresponds to at least 15 contiguous basepairs of the first target gene.

35. The method of claim 34, wherein the second dsRNA and the first dsRNA overlap in sequence by fewer than 15 basepairs.

36. The method of claim 35, wherein the second dsRNA and the first dsRNA do not overlap in sequence.

37. The method of claim 33, wherein the second dsRNA corresponds to at least 15 contiguous basepairs of a second target gene.

38. The method of claim 33, further comprising incubating the nuclease with at least a third dsRNA that corresponds to at least 15 contiguous basepairs of a third target gene.

39. The method of claim 33, further comprising isolating the siRNA molecules.

40. The method of claim 24, further comprising labeling the pool of candidate siRNA molecules.

41. A pool of candidate siRNA molecules targeting one or more genes generated by the method of claim 24.

42. The pool of candidate siRNA molecules of claim 41, wherein at least two of the candidate siRNA molecules overlap in sequence by at least 3 basepairs.

43. The pool of candidate siRNA molecules of claim 41, wherein at least two of the candidate siRNA molecules are contiguous with respect to each other.

44. The pool of siRNA molecules of claim 41, wherein the candidate siRNA molecules correspond to at least three different targets.

45. The pool of candidate siRNA molecules of claim 41, wherein the genes include at least one of the following:

developmental genes, oncogenes, tumor suppressor genes, or enzymes.

46. The pool of candidate siRNA molecules of claim 41, wherein the pool of candidate siRNA molecules is labeled.

47. A composition for reducing or eliminating expression of at least a first target gene in a cell comprising a pool of siRNAs comprising at least two different siRNA molecules directed to the at least a first target gene.

48. The composition of claim 47, wherein the pool of siRNAs comprises 2 to 20 different siRNA molecules.

49. The composition of claim 48, wherein the pool of siRNAs comprises 3 to 10 different siRNA molecules.

50. The composition of claim 47, further comprising at least two different siRNA molecules directed to a second target gene.

51. The composition of claim 50, wherein the pool of siRNAs comprises 2 to 20 different siRNA molecules directed to each of the target genes.

52. The composition of claim 51, wherein the pool of siRNAs comprises 3 to 10 different siRNA molecules directed to each of the target genes.

53. The composition of claim 50, further comprising at least two different siRNA molecules directed to a third target gene.

54. The composition of claim 53, wherein the pool of siRNAs comprises 2 to 20 different siRNA molecules directed to each of the three target genes.

55. The composition of claim 54, wherein the pool of siRNAs comprises 3 to 10 different siRNA molecules directed to each of the three target genes.

56. The composition of claim 47, wherein the pool of siRNA molecules is labeled.