RNA interference in respiratory epitheial cells
View Patent ↗The present invention is directed to small interfering RNA molecules targeted against a gene of interest in respiratory epithelial cells, and methods of using these RNA molecules.
1. A precursor RNAi molecule capable of inhibiting the expression of a respiratory syncytial virus (RSV)-specific messenger RNA (mRNA) to which it corresponds comprising:
a first oligonucleotide strand that is 22-30 nucleotides in length; and
a second oligonucleotide strand that is 22-30 nucleotides in length and comprises a nucleotide sequence that is sufficiently complementary to a sequence of an RNA of a target gene to direct target-specific RNAi and wherein the second oligonucleotide strand anneals to the first oligonucleotide strand under biological conditions,
wherein the first strand of RNA comprises SEQ ID NO:279 and the second strand of RNA comprises SEQ ID NO:280, and wherein the precursor RNAi molecule is a substrate for a Dicer.
2. The precursor RNAi molecule of claim 1 , wherein the first and second oligonucleotide strands have a different length and wherein one of the first and second oligonucleotide strands has a 3′ overhang that enhances Dicer processing of the precursor RNAi molecule.
3. The precursor RNAi molecule of claim 2 , wherein the overhang is one to three nucleotides.
4. The precursor RNAi molecule of claim 2 , wherein the overhang is two nucleotides.
5. The precursor RiNAi molecule of claim 1 , wherein at least one of the first and second oligonucleotide strands is modified on the 3′ end to direct Dicer processing of the RNAi molecule.
6. The precursor RNAi molecule of claim 5 , wherein the modification is a deoxyribonucleotide, dideoxyribonucleotide, acyclonucleotide, 3 ′-deoxyadeno sine (cordycepin), 3′-azido-3′-deoxythymidine (AZT), 2′,3′-dideoxyinosine (ddI), 2′,3-dideoxy-3′-thiacytidine (3TC), 2′,3′-didehydro-2′,3′-dideoxythymidine (d4T), a monophosphate nucleotide of 3′-azido-3′-deoxythymidine (AZT), 2′,3′-dideoxy-3′-thiacytidine (3TC) or a monophosphate nucleotide of 2′,3′-didehydro-2′,3′-dideoxythymidine (d4T).
7. The precursor RNAi molecule of claim 5 , wherein the modification is 1-3 deoxynucleotides substituted for ribonucleotides at the 3′ end of the first oligonucleotide strand.
8. The precursor RNAi molecule of claim 1 , wherein the first and second oligonucleotide strands are linked by a linker, wherein the strands anneal under biological conditions.
9. The precursor RNAi molecule of claim 8 , wherein the linker is an oligonucleotide.
10. The precursor RNAi molecule of claim 9 , wherein the linker contains 4 to 10 nucleotides.
11. The precursor RiNAi molecule of claim 9 , wherein the linker contains 4, 5 or 6 nucleotides.
12. The precursor RNAi molecule of claim 1 , wherein a ribonucleotide of the first or second strand is modified.
13. The precursor RNAi molecule of claim 1 , wherein a ribonucleotide is substituted by a locked nucleic acid (LNA).
14. The precursor RNAi molecule of claim 13 , wherein the LNA is in the 5 ′region of the first strand.
15. An isolated or purified expression cassette comprising
a nucleic acid encoding a first oligonucleotide strand that is 22-30 nucleotides in length, wherein the first strand of RNA comprises SEQ ID NO:279;
a second oligonucleotide strand that is 22-30 nucleotides in length, wherein the second strand of RNA comprises SEQ ID NO:280 and comprises a nucleotide sequence that is sufficiently complementary to a seciuence of an RNA of a target gene to direct target-specific RNAi;
an oligonucleotide linker positioned between the first oligonucleotide strand and the second oligonucleotide strand; and
a promoter operably linked to the first oligonucleotide strand.
16. The isolated or purified expression cassette of claim 15 , wherein the promoter is a regulatable promoter.
17. The isolated or purified expression cassette of claim 15 , wherein the promoter is a constitutive promoter.
18. The isolated or purified expression cassette of claim 17 , wherein the promoter is a CMV, RSV, pol II or pol III promoter.
19. The isolated or purified expression cassette of claim 15 , wherein the expression cassette further comprises a polyadenylation signal.
20. The isolated or purified expression cassette of claim 15 , further comprising a marker gene.
21. An isolated cell comprising the isolated or purified expression cassette of claim 15 .
22. The cell of claim 21 , wherein the cell is a mammalian cell.
23. A vector comprising the isolated or purified expression cassette of claim 15 .
24. The vector of claim 23 , wherein the vector is an adenoviral, lentiviral, adeno-associated viral (AAV), poliovirus, Herpes Simplex Virus (HSV), or murine Moloney-based viral vector.
25. The vector of claim 24 , wherein the vector is an adenoviral vector.
26. The vector of claim 23 , wherein the vector is a plasmid vector.
27. A composition comprising a polymer or excipient and the precursor RNAi molecule of claim 1 .
28. The composition of claim 27 , wherein the polymer or excipient is bioadhesive, mucoadhesive or viscoelastic.
29. The composition of claim 27 , wherein the polymer or excipient is a gel.
30. The composition of claim 29 , wherein the gel is a viscoelastic gel.
31. The composition of claim 30 , wherein the viscoelastic gel is methylcellulose, carboxymethylcellulose or a poloxamer polymer.
32. The composition of claim 30 , wherein the viscoelastic gel is methylcellulose or carboxymethylcellulose.
33. The composition of claim 27 , further comprising a calcium chelator.
34. The composition of claim 33 , wherein the calcium chelator is ethylene glycol -bis-(2-aminoethyl)-N,N,N′, N′-tetraacetic acid (EGTA), ethylenedioxy-diethylene -dinitrilo-tetraacetic acid (EDTA) or 1,2-bis(aminophenoxy)ethane-N,N,N′, N′-tetra-acetic acid (BAPTA).
35. The composition of claim 33 , wherein the calcium chelator is ethylene glycol -bis-(2-aminoethyl)-N,N,N′, N′-tetraacetic acid (EGTA).
36. A vector comprising two expression cassettes,
the first expression cassette comprising a first promoter operably linked to a nucleic acid encoding a first oligonucleotide strand that is 22-30 nucleotides in length, wherein the first oligonucleotide strand comprises SEQ ID NO:279 operably linked to a promoter; and
the second expression cassette comprising a second promoter operably linked to a second oligonucleotide strand that is 22-30 nucleotides in length, wherein the second oligonucleotide strand comprises SEQ ID NO:280 and comprises a nucleotide sequence that is sufficiently complementary to a sequence of an RNA of a target gene to direct target-specific RNAi.
37. The vector of claim 36 , wherein the vector is an adenoviral, lentiviral, adeno-associated viral (AAV), poliovirus, HSV, or murine Moloney-based viral vector.
38. The vector of claim 37 , wherein the vector is an adenoviral vector.
39. The vector of claim 36 , wherein the vector is a plasmid vector.
40. A composition comprising a polymer or excipient and the vector of claim 23 .
41. The composition of claim 40 , wherein the polymer or excipient is bioadhesive, mucoadhesive or viscoelastic.
42. The composition of claim 40 , wherein the polymer or excipient is a gel.
43. The composition of claim 42 , wherein the gel is a viscoelastic gel.
44. The composition of claim 43 , wherein the viscoelastic gel is methylcellulose, carboxymethylcellulose or a poloxamer polymer.
45. The composition of claim 43 , wherein the viscoelastic gel is methylcellulose or carboxymethylcellulose.
46. The composition of claim 40 , further comprising a calcium chelator.
47. The composition of claim 46 , wherein the calcium chelator is ethylene glycol-bis -(2-aminoethyl)-N,N,N′, N′-tetraacetic acid (EGTA), ethylenedioxy-diethylene-dinitrilo -tetraacetic acid (EDTA) or 1,2-bis(aminophenoxy)ethane-N,N,N′, N′-tetra-acetic acid (BAPTA).
48. The composition of claim 47 , wherein the calcium chelator is ethylene glycol-bis -(2-aminoethyl)-N,N,N′, N′-tetraacetic acid (EGTA).
49. A composition comprising a polymer or excipient and the vector of claim 36 .
50. The composition of claim 49 , wherein the polymer or excipient is bioadhesive, mucoadhesive or viscoelastic.
51. The composition of claim 49 , wherein the polymer or excipient is a gel.
52. The composition of claim 51 , wherein the gel is a viscoelastic gel.
53. The composition of claim 51 , wherein the viscoelastic gel is methylcellulose, carboxymethylcellulose or a poloxamer polymer.
54. The composition of claim 51 , wherein the viscoelastic gel is methylcellulose or carboxymethylcellulose.
55. The composition of claim 49 , further comprising a calcium chelator.
56. The composition of claim 55 , wherein the calcium chelator is ethylene glycol-bis -(2-aminoethyl)-N,N,N′, N′-tetraacetic acid (EGTA), ethylenedioxy-diethylene-dinitrilo -tetraacetic acid (EDTA) or 1 ,2-bis(aminophenoxy)ethane-N,N,N′, N′-tetra-acetic acid (BAPTA).
57. The composition of claim 55 , wherein the calcium chelator is ethylene glycol-bis -(2-aminoethyl)-N,N,N′, N′-tetraacetic acid (EGTA).