Fusion protein delivery system and uses thereof

• US 20030157690A1
• Filed: 11/27/2002
• Published: 08/21/2003
• Est. Priority Date: 04/14/1995
• Status: Abandoned Application

• Current Assignee: UAB Research Foundation (University of Alabama System)
• Original Assignee: UAB Research Foundation (University of Alabama System)
• Inventors: Kappes, John C., Wu, Xiaoyun
• Application Number:
  US10/306,885
• Publication Number:
  US 20030157690A1
• Time in Patent Office: Days
• US Class Current: 435/235.1
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 47/645 Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 2740/16322 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Abstract

The present invention provides a composition of matter, comprising: DNA encoding a viral Vpx protein fused to DNA encoding a protein. In another embodiment of the present invention, there is provided a composition of matter, comprising: DNA encoding a viral Vpr protein fused to DNA encoding a protein. The present invention further provides DNA, vectors and methods for expressing a lentiviral pol gene in trans, independent of the lentiviral gag-pol. A gene transduction element is optionally delivered to a lentiviral vector according to the present invention. Also provided are various methods of delivering a virus inhibitory molecule to a target in an animal. Further provided is a pharmaceutical composition.

Claims

1. A viral vector system comprising:

a) at least a first nucleic acid segment comprising a nucleotide sequence encoding at least a functional portion of a Gag polypeptide, and said first nucleic acid segment does not encode at least one of a functional Reverse Transcriptase polypeptide and a functional Integrase polypeptide; and

,

b) at least a second nucleic acid segment comprising at least one nucleotide sequence encoding a polypeptide selected from the group consisting of;

i) a functional portion of a Reverse Transcriptase polypeptide; and

,

ii) a functional portion of an Integrase polypeptide;

wherein said second nucleic acid segment does not encode a functional Gag polypeptide; and

,

c) at least a third nucleic acid segment comprising a nucleic acid sequence encoding a functional portion of an envelope polypeptide, wherein said third nucleic acid segment does not encode a functional Gag-Pol precursor polypeptide;

wherein said viral vector system produces an infectious viral particle.

2. The viral vector system of claim 1, wherein

said first nucleic acid segment does not encode the functional Reverse Transcriptase and the functional Integrase polypeptide;

said second nucleic acid segment comprises a nucleotide sequence encoding the functional portions of the Reverse Transcriptase polypeptide fused in frame to the functional portions of the Integrase polypeptide.

3. The viral vector system of claim 1, wherein

said first nucleic acid segment does not encode the functional Reverse Transcriptase and the functional Integrase polypeptide;

said second nucleic acid segment comprises a nucleotide sequence encoding a functional portion of the Reverse Transcriptase polypeptide; and

,

said viral vector system further comprises a fourth nucleic acid segment comprising a nucleotide sequence encoding a functional portion of the Integrase polypeptide, wherein said fourth nucleic acid segment does not encode the functional Gag polypeptide.

4. The viral vector system of claim 1, wherein said first nucleic acid segment further comprises a Protease polypeptide.

5. The viral vector system of claim 1 further comprising at least a fourth nucleic acid segment comprising a nucleic acid sequence of interest, wherein said fourth nucleic acid segment does not encode a functional Gag-Pol precursor polypeptide wherein said viral vector system produces an infectious viral particle capable of transducing a target cell.

6. The viral vector system of claim 3 further comprising at least a fifth nucleic acid segment comprising a nucleic acid sequence of interest, wherein said fifth nucleic acid segment does not encode a functional Gag-Pol precursor polypeptide wherein said viral vector system produces an infectious viral particle capable of transducing a target cell.

7. The viral vector system of claim 5, wherein said nucleotide sequence of interest encodes a polypeptide.

8. The viral vector system of claim 7, wherein said polypeptide is a viral inhibitory polypeptide.

9. The viral vector system of claim 5, wherein said nucleotide sequence of interest is operably linked to a promoter active in a target cell.

10. The viral vector system of claim 1, wherein said functional portions of said Gag polypeptide, said Reverse Transcriptase polypeptide, and said Integrase polypeptide are from a retrovirus.

11. The viral vector system of claim 10, wherein said retrovirus is a lentivirus.

12. The viral vector system of claim 11, wherein said lentivirus is a human immunodeficiency virus or a simian immunodeficiency virus.

13. The viral vector system of claim 12, wherein said human immunodeficiency virus is HIV-1 or HIV-2.

14. The viral vector system of claim 5, wherein said fourth nucleic acid segment further comprises a gene encoding a marker protein selected from the group consisting of β

-gal, fluorescence proteins, and luciferase.

15. The viral vector system of claim 1 further comprising promoters operatively linked to at least one of said first, said second, or said third nucleic acid segments.

16. The viral vector system of claim 3 further comprising promoters operatively linked to at least one of said first, said second, said third or said fourth nucleic acid segments.

17. A viral vector system comprising:

a) at least a first nucleic acid segment comprising a nucleotide sequence encoding at least a functional portion of a Gag polypeptide, and said first nucleic acid segment does not encode at least one of a functional Reverse Transcriptase polypeptide and a functional Integrase polypeptide; and

,

b) at least a second nucleic acid segment comprising at least one nucleotide sequence encoding a fusion protein selected from the group consisting of;

i) a functional portion of a Vpr or a Vpx polypeptide and a functional portion of a Reverse Transcriptase polypeptide; and

,

ii) a functional portion of a Vpr or Vpx polypeptide and a functional portion of an Integrase polypeptide;

wherein said functional portion of the Vpr or the Vpx polypeptide is capable of providing for the incorporation of said fusion protein into a viral particle and said second nucleic acid segment does not encode a functional Gag polypeptide; and

,

wherein said viral vector system produces an infectious viral particle.

18. The viral vector system of claim 17, wherein

said first nucleic acid segment does not encode the functional Reverse Transcriptase and the functional Integrase polypeptide;

said second nucleic acid segment comprises a nucleotide sequence encoding the fusion protein comprising the functional portion of the Vpr or the Vpx polypeptide and the functional Reverse Transcriptase polypeptide fused in frame to the functional Integrase polypeptide.

19. The viral vector system of claim 17, wherein

said first nucleic acid segment does not encode the functional Reverse Transcriptase polypeptide and the functional Integrase polypeptide;

said second nucleic acid segment comprises a nucleotide sequence encoding the fusion protein comprising the functional portion of the Vpr or the Vpx polypeptide and the functional portion of the Reverse Transcriptase polypeptide; and

,

said viral vector system further comprises a third nucleic acid segment comprising a nucleotide sequence encoding a second fusion protein comprising a second functional portion of the Vpr or the Vpx polypeptide and the functional portion of the Integrase polypeptide, wherein said third nucleic acid segment does not encode the functional Gag polypeptide.

20. The viral vector system of claim 17, further comprising at least a third nucleic acid segment comprising a nucleic acid sequence encoding a functional portion of an envelope polypeptide, wherein said third nucleic acid segment does not encode a functional Gag-Pol precursor polypeptide.

21. The viral vector system of claim 19, further comprising at least a fourth nucleic acid segment comprising a nucleic acid sequence encoding a functional portion of an envelope polypeptide, wherein said fourth nucleic acid segment does not encode a functional Gag-Pol precursor polypeptide.

22. The viral vector system of claim 17, wherein said first nucleic acid segment further comprises a Protease polypeptide.

23. The viral vector system of claim 20 further comprising at least a fourth nucleic acid segment comprising a nucleic acid sequence of interest, wherein said fourth nucleic acid segment does not encode a functional Gag-Pol precursor polypeptide wherein said viral vector system produces an infectious viral particle capable of transducing a target cell.

24. The viral vector system of claim 21 further comprising at least a fifth nucleic acid segment comprising a nucleic acid sequence of interest, wherein said fifth nucleic acid segment does not encode a functional Gag-Pol precursor polypeptide wherein said viral vector system produces an infectious viral particle capable of transducing a target cell.

25. The viral vector system of claim 23, wherein said nucleotide sequence of interest encodes a polypeptide.

26. The viral vector system of claim 25, wherein said polypeptide is a viral inhibitory polypeptide.

27. The viral vector system of claim 23, wherein said nucleotide sequence of interest is operably linked to a promoter active in a target cell.

28. The viral vector system of claim 17, wherein said functional portions of said Gag polypeptide, said Reverse Transcriptase polypeptide, and said Integrase polypeptide are from a retrovirus.

29. The viral vector system of claim 28, wherein said retrovirus is a lentivirus.

30. The viral vector system of claim 29, wherein said lentivirus is a human immunodeficiency virus or a simian immunodeficiency virus.

31. The viral vector system of claim 30, wherein said human immunodeficiency virus is HIV-1 or HIV-2.

32. The viral vector system of claim 23, wherein said fourth nucleic acid segment further comprises a gene encoding a marker protein selected from the group consisting of β

-gal, fluorescence proteins, and luciferase.

33. The viral vector system of claim 17 further comprising promoters operatively linked to at least one of said first or said second nucleic acid segments.

34. The viral vector system of claim 19 further comprising promoters operably linked to at least one of said first, said second, or said third nucleic acid segment.

35. The viral vector system of claim 23 further comprising promoters operatively linked to at least one of said first, said second, said third or said fourth nucleic acid segments.