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

• US 7,846,729 B2
• Filed: 08/06/2007
• Issued: 12/07/2010
• Est. Priority Date: 08/09/1999
• Status: Expired due to Fees

• Current Assignee: Genzyme Corporation (Sanofi )
• Original Assignee: Genzyme Corporation (Sanofi )
• Inventors: Carter, Barrie J.
• Primary Examiner(s): Peng; Bo
• Application Number:
  US11/890,656
• Publication Number:
  US 20080206197A1
• Time in Patent Office: 1,219 Days
• Field of Search: None
• 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 preparing a recombinant adeno-associated virus (rAAV), the method comprising:

1) incubating a host cell under conditions that allow AAV replication and encapsidation, wherein said host cell comprises;

(a) a 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; and

2) purifying rAAV particles produced from the host cell, wherein the rAAV particles comprise a rAAV genome which forms intrastrand base pairs along its length, such that expression of a coding region of the heterologous sequence is enhanced relative to a rAAV vector that lacks sufficient intrastrand base pairing to enhance said expression.

2. The method of claim 1 wherein rep and cap functions are provided by a rep-cap cassette that is stably integrated in the host cell genome.

3. The method of claim 1 wherein rep and cap functions are provided by a plasmid.

4. The method of claim 1 wherein the rAAV vector is provided in a plasmid.

5. The method of claim 1, wherein the rAAV vector is stably integrated into the host cell genome.

6. The method of claim 1 wherein helper functions are provided by adenovirus infection.

7. A population of rAAV particles produced according to the method of claim 1.

8. A population of rAAV vectors produced according to the method of claim 1.

9. The method of claim 1, wherein the rAAV particles are purified to deplete helper virus.