Vectors for regulating gene expression

• US 20060239971A1
• Filed: 02/23/2004
• Published: 10/26/2006
• Est. Priority Date: 02/21/2003
• Status: Active Grant

• Current Assignee: University of South Florida (State University System of Florida)
• Original Assignee: University of South Florida (State University System of Florida)
• Inventors: Mohapatra, Shyam S.
• Granted Patent: US 8,796,235 B2
• Time in Patent Office: Days
• US Class Current: 424/93.200
• CPC Class Codes: A61K 48/005 characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
• CPC Class Codes: A61K 48/0058 Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/1131 against viruses
• CPC Class Codes: C12N 15/86 Viral vectors
• 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 2310/53 partially self-complementary or closed
• CPC Class Codes: C12N 2330/30 chemically synthesised
• CPC Class Codes: C12N 2750/14143 viral genome or elements thereof as genetic vector
• 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 pertains to vectors for regulating gene expression having at least one gene expressing cassette and at least one gene suppressing cassette, wherein the gene expression cassette encodes a polypeptide of interest, and wherein the gene suppressing cassette encodes a short interfering RNA (siRNA) molecule that reduces expression of a target gene by RNA interference. The present invention further includes vectors that contain suppressor cassettes in conjunction with cassettes upregulating gene expression regulated by either a constitutive promoter, such as a general CMV promoter, or a tissue specific promoter. The present invention further includes vectors that contain Dengue virus gene suppression cassettes. The present invention further includes pharmaceutical compositions containing such vectors, methods of modulating the expression of genes in a host using such vectors, and method of producing such vectors.

Claims

1. A vector comprising a plurality of expression cassettes, wherein said plurality of expression cassettes comprise:

(a) at least one gene promoting cassette comprising a first polynucleotide operably linked to a first promoter sequence; and

(b) at least one gene suppressing cassette comprising a second polynucleotide operably-linked to a second promoter sequence, wherein said second polynucleotide encodes a short interfering RNA (siRNA) molecule that reduces expression of a target gene by RNA interference.

7. The vector of claim 1, wherein the vector comprises a plurality of said gene promoting cassettes.

8. The vector of claim 1, wherein said vector comprises a plurality of said gene suppressing cassettes.

10. The vector of claim 1, wherein said first promoter sequence, or said second promoter sequence, or both said first and second promoter sequences are inducible.

11. The vector of claim 1, wherein said first promoter sequence, or said second promoter sequence, or both said first and second promoter sequences are tissue-specific.

16. The vector of claim 1, wherein said first polynucleotide sequence encodes a polypeptide.

17. The vector of claim 16, wherein said polypeptide comprises a protein selected from the group consisting of an enzyme, cytokine, growth factor, hormone, receptor, and receptor ligand.

22. The vector of claim 1, wherein the target gene is a Dengue virus gene encoding a structural protein.

23. The vector of claim 1, wherein the target gene is a Dengue virus gene encoding a non-structural protein.

24. The vector of claim 1, wherein said second polynucleotide comprises at least one nucleic acid sequence selected from the group consisting of SEQ ID NO:

1, SEQ ID NO;

2, SEQ ID NO;

3, and SEQ ID NO;

4.

34. A method of modulating the expression of multiple genes within a host, said method comprising administering the vector of claim 1 to the host, wherein the first polynucleotide sequence is expressed in the host, wherein the second polynucleotide is transcribed to produce the siRNA molecule, and wherein the siRNA molecule is capable of reducing expression of a target gene within the host by RNA interference.

36. The method of claim 34, wherein the target gene is an endogenous gene of the host.

37. The method of claim 34, wherein the target gene is exogenous to the host.

48. A method for producing a vector for modulating the expression of multiple genes, said method comprising combining at least one gene promoting cassette with at least one gene suppressing cassette to form the vector of claim 1.

2-6. -6. (canceled)

9. (canceled)

12-15. -15. (canceled)

18-21. -21. (canceled)

25. A vector comprising at least one gene suppressing cassette, wherein said gene suppressing cassette comprises a polynucleotide operably-linked to a promoter sequence, wherein said polynucleotide encodes a short interfering RNA (siRNA) molecule that reduces expression of a target Dengue virus gene by RNA interference.

26. The vector of claim 25, wherein said vector comprises a plurality of gene suppressing cassettes.

27. The vector of claim 25, wherein said target gene encodes a structural protein.

28. The vector of claim 25, wherein said target gene encodes a non-structural protein.

29. The vector of claim 25, wherein said target gene is at least one gene encoding a protein selected from the group consisting of C, prM, E, NS1, NS2a, NS3, NS4a, NS4b, and NS5.

30. The vector claim 25, wherein said polynucleotide comprises the nucleotide sequence of SEQ ID NO:

3 or SEQ ID NO;

4.

42. A method for inhibiting the expression of Dengue virus genes within a host, said method comprising administering the vector of claim 25 to the host, wherein the polynucleotide sequence is transcribed to produce the siRNA molecule, and wherein the siRNA molecule is capable of reducing expression of a target Dengue virus gene within the host by RNA interference.

45. The method of claim 42, wherein the host is suffering from a Dengue virus infection.

46. The method of claim 42, wherein the host is not suffering from a Dengue virus infection, and wherein the vector is administered prophylactically.

49. A method for producing a vector for inhibiting the expression of Dengue virus genes within a host, said method comprising combining at least one polynucleotide with an operably linked promoter sequence to form the vector of claim 25.

31-33. -33. (canceled)

35. (canceled)

38-41. -41. (canceled)

43-44. -44. (canceled)

47. (canceled)

50. A pharmaceutical composition comprising:

(a) a vector and a pharmaceutically acceptable carrier, said vector comprising;

at least one gene promoting cassette comprising a first polynucleotide operably linked to a first promoter sequence; and

at least one gene suppressing cassette comprising a second polynucleotide operably linked to a second promoter sequence, wherein said second polynucleotide encodes a short interfering RNA (siRNA) molecule that reduces expression of a target gene by RNA interference;

or

(b) a vector and a pharmaceutically acceptable carrier, said vector comprising;

at least one gene suppressing cassette, wherein said gene suppressing cassette comprises a polynucleotide operably linked to a promoter sequence, wherein said polynucleotide encodes a short interfering RNA (siRNA) molecule that reduces expression of a target Dengue virus gene by RNA interference.