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

• US 6,596,535 B1
• Filed: 08/08/2000
• Issued: 07/22/2003
• 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): Housel, James
• Assistant Examiner(s): JENKS, ULRIKE WINKLER
• Application Number:
  US09/634,126
• Time in Patent Office: 1,078 Days
• Field of Search: 424/93.2, 424/93.6, 435/69.1, 435/320.1, 435/325, 435/366, 435/371, 435/455, 435/456, 435/457, 435/462, 435/463, 536/23.72, 536/24.3, 536/23.1
• US Class Current: 435/320.1
• 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 recombinant adeno-associated virus (rAAV) vector comprising a single-stranded heterologous nucleotide sequence comprising a region which forms intrastrand base pairs such that expression of a 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 region which forms intrastrand base pairs is in a coding region.

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

3. The rAAV vector of claim 2, further comprising an ITR flanked on both sides by heterologous sequences.

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

5. The rAAV vector of claim 1, 2, 3, or 4, wherein said rAAV vector is capable of being packaged in an AAV particle.

6. A host cell comprising the rAAV vector of claim 1.

7. The host cell of claim 6, which is mammalian.

8. A host cell comprising the rAAV vector of claim 5.

9. The host cell of claim 8, which is mammalian.

10. A host cell comprising the rAAV vector of claims 2, 3, or 4.

11. The host cell of claim 10, which is mammalian.

12. A recombinant AAV particle comprising the rAAV vector of claim 1.

13. A recombinant AAV particle comprising the rAAV vector of claim 5.

14. A recombinant AAV particle comprising the rAAV vector of claim 2.

15. A recombinant AAV particle comprising the rAAV vector of claim 3 or 4.

16. A composition comprising the rAAV vector of claim 1.

17. The composition of claim 14, further comprising a pharmaceutical acceptable excipient.

18. A composition comprising the rAAV vector of claim 5.

19. The composition of claim 18, further comprising a pharmaceutically acceptable excipient.

20. A composition comprising the rAAV vector of claims 2, 3, or 4.

21. The composition of claim 20, further comprising a pharmaceutically acceptable excipient.

22. A method for preparing an rAAV vector according to claim 5 packaged in a particle, wherein the method comprises incubating a host cell under conditions that allow AAV replication and encapsidation, wherein said host cell comprises:

(a) an rAAV vector comprising a heterologous nucleotide sequence and one or more AAV inverted terminal repeat (ITR) sequences flanking said heterologous sequence, wherein the vector is less than about 2.5 kb, and

(b) AAV rep function, AAV cap function, and helper virus function for AAV.

23. The method of claim 22 wherein rep arid cap functions are provided by a rep-cap cassette that is stably integrated in the host cell genome.

24. The method of claim 22 wherein rep and cap functions are provided by a plasmid.

25. The method of claim 22 wherein the rAAV vector is provided in a plasmid.

26. The method of claim 22, wherein the rAAV vector is stably integrated into the host cell genome.

27. The method of claim 22 wherein helper functions are provided by adenovirus infection.

28. A population of rAAV particles produced according to the method of claim 22.

29. A population of rAAV vectors produced according to the method of claim 22.