Metabolically activated recombinant viral vectors and methods for their preparation and use

• US 7,125,717 B2
• Filed: 04/25/2003
• Issued: 10/24/2006
• Est. Priority Date: 08/09/1999
• Status: Expired due to Term

• Current Assignee: Genzyme Corporation (Sanofi )
• Original Assignee: Targeted Genetics Corporation (Innoviva, Inc.)
• Inventors: Carter, Barrie J.
• Primary Examiner(s): Mosher, Mary E.
• Assistant Examiner(s): Peng, Bo
• Application Number:
  US10/423,507
• Publication Number:
  US 20030219735A1
• Time in Patent Office: 1,278 Days
• Field of Search: 424/93.1, 424/93.6, 435/456, 435/320.1, 435/69.1, 435/325, 435/457, 536/23.1, 536/23.73, 536/24.3
• US Class Current: 435/456
• CPC Class Codes: A61K 38/191 Tumor necrosis factors [TNF], e.g. lymphotoxin [LT], i.e. TNF-beta
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: C07K 14/70578 NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95 NGF-receptor C07K14/71, TNF-receptor C07K14/7151
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 2319/30 Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• CPC Class Codes: C07K 2319/33 fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2750/14143 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12P 21/06 produced by the hydrolysis of a peptide bond, e.g. hydrolysate products preparing foodstuffs by protein hydrolysis A23J3/00

Abstract

Recombinant viral vectors, especially parvovirus vectors such as adeno-associated virus (AAV) vectors, capable of enhanced expression of heterologous sequences, and methods for their construction and use, are provided. The vectors have a structure, or are capable of rapidly adopting a structure, which involves intrastrand base pairing of at least one region in a heterologous sequence.

Claims

1. A method for introducing a polynucleotide into a cell, comprising contacting the cell essentially in the absence of an AAV helper virus with a recombinant adeno-associated virus (rAAV) particle comprising an rAAV vector under conditions that allow uptake of the rAAV vector, whereby the rAAV vector is introduced into the cell, wherein the rAAV vector comprises a single-stranded heterologous nucleotide sequence comprising a coding region which forms intrastrand base pairs such that expression of the coding region of the heterologous sequence is enhanced relative to a second rAAV vector that lacks sufficient intrastrand base pairing to enhance said expression, wherein the rAAV vector comprises one or more inverted terminal repeat (ITR) sequence flanking said heterologous sequence.

3. The method of claim 1, wherein the sequence complexity of the heterologous sequence is about one-half of the heterologous sequence, and wherein the heterologous sequence forms a double-stranded structure along most or all of its length.

4. The method of claim 3, wherein the double-stranded structure is formed within the cell.

5. The method of claim 1, wherein the coding region forms a double-stranded structure along most or all of its length.

6. The method of claim 5, wherein the double-stranded structure is formed within the cell.

7. The method of claim 1, wherein the rAAV vector further comprises an ITR flanked on both sides by heterologous sequences.

8. The method of claim 1, wherein the heterologous sequence encodes a protein of therapeutic interest.

9. The method of claim 1, wherein the heterologous sequence encodes an RNA of therapeutic interest.

10. The method of claim 9, wherein the heterologous sequence encodes an antisense RNA or a ribozyme.

11. The method of claim 1, wherein the cell is a mammalian cell.

2. A method for expressing a polynucleotide coding region in a cell, comprising subjecting the cell to conditions which allow expression of the coding region, whereby the coding region is expressed, wherein the polynucleotide coding region is introduced into the cell by contacting the cell essentially in the absence of an AAV helper virus with an rAAV particle comprising an rAAV vector, wherein the rAAV vector comprises a single-stranded heterologous nucleotide sequence comprising the coding region which forms intrastrand base pairs such that expression of the coding region of the heterologous sequence is enhanced relative to a second rAAV vector that lacks sufficient intrastrand base pairing to enhance said expression, wherein the rAAV vector comprises one or more inverted terminal repeat (ITR) sequences flanking said heterologous sequence.

12. The method of claim 2, wherein the sequence complexity of the heterologous sequence is about one-half of the heterologous sequence, and wherein the heterologous sequence forms a double-stranded structure along most or all of its length.

13. The method of claim 12, wherein the double-stranded structure is formed within the cell.

14. The method of claim 2, wherein the coding region forms a double-stranded structure along most or all of its length.

15. The method of claim 14, wherein the double-stranded structure is formed within the cell.

16. The method of claim 2, wherein the rAAV vector further comprises an ITR flanked on both sides by heterologous sequences.

17. The method of claim 2, wherein the heterologous sequence encodes a protein of therapeutic interest.

18. The method of claim 2, wherein the heterologous sequence encodes an RNA of therapeutic interest.

19. The method of claim 18, wherein the heterologous sequence encodes an antisense RNA or a ribozyme.

20. The method of claim 2, wherein the cell is a mammalian cell.