AAV transduction vectors

• US 5,139,941 A
• Filed: 10/25/1991
• Issued: 08/18/1992
• Est. Priority Date: 10/31/1985
• Status: Expired due to Term

• Current Assignee: University of Florida Research Foundation, Incorporated (State University System of Florida)
• Original Assignee: University of Florida Research Foundation, Incorporated (State University System of Florida)
• Inventors: Hermonat, Paul L., Muzyczka, Nicholas, Samulski, Richard J., Berns, Kenneth I.
• Primary Examiner(s): Martinell, James
• Application Number:
  US07/785,224
• Time in Patent Office: 298 Days
• Field of Search: 435/69.1, 435/70.1, 435/70.3, 435/172.1, 435/172.3, 435/320.1, 435/240.1, 435/240.2, 435/235, 435/236, 536/27, 935/32, 935/34, 935/57, 935/70, 935/71
• US Class Current: 435/456
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2750/14143 viral genome or elements thereof as genetic vector

Abstract

A hybrid gene vector suitable for introducing foreign DNA into a mammalian cell comprising the foreign DNA ligated to an AAV genome; a method of constructing the hybrid gene vector; a method of transducing foreign DNA into mammalian cells comprising infecting the cells with the above hybrid gene vector and a method of rescuing foreign DNA from mammalian cells utilizing helper virus.

Claims

1. A hybrid gene vector comprising foreign DNA ligated into an AAV genome in place of or in addition to the cap, lip or rep coding sequence thereof or in place of or in addition to an AAV DNA sequence excluding the first and last 145 base pairs, said vector being capable of transducing data foreign DNA into a mammalian cell in the presence of a promoter other than an AAV transcription promoter, and said AAV genome being cloned in a prokaryotic vector plasmid or bacteriophage vector capable of growth in prokaryotic cells or yeast plasmid vector capable of growth in fungal cells and isolating a recombinant AAV hybrid gene vector by transfection of the ligated vector into a mammalian cell, said AAV genome being the entire AAV viral DNA or a deletion mutant thereof, capable of replication upon infection of said mammalian cell when complemented in trans by an AAV gene.

2. The vector of claim 1 wherein said AAV genome is AAV-type 2.

3. The vector of claim 2 wherein said AAV genome is the deletion mutant dl52-91 or dl3-94.

4. A method of constructing a hybrid gene vector comprising ligating foreign DNA into an AAV genome in place of or in addition to the cap, lip or rep coding sequence thereof or in place of or in addition to an AAV DNA sequence excluding the first and last 145 base pairs, said vector being capable of transducing said foreign DNA into a mammalian cell in the presence of a promoter other than an AAV transcription promoter, and said AAV genome being cloned in a prokaryotic vector plasmid or bacteriophage vector capable of growth in prokaryotic cells or yeast plasmid vector capable of growth in fungal cells and isolating a recombinant AAV hybrid gene vector by transfection of the ligated vector into a mammalian cell, said AAV genome being the entire AAV viral DNA or a deletion mutant thereof, capable of replication upon infection of said mammalian cell when complemented in trans by an AAV gene.

5. The method of claim 4 wherein said AAV genome is an AAV-type 2 genome.

6. The method of claim 5 wherein said AAV genome is the deletion mutant dl52-91 or dl13-94.

7. The method of claim 4 wherein said AAV genome is cloned in the prokaryotic plasmid vector pBR322.

8. The method of claim 4 wherein said transfection is conducted in the presence of a helper virus and/or a helper AAV plasmid ins 96/λ

-M each of which contain genes necessary for the replication of AAV genome or the production of AAV virus.

9. A method of transducing foreign DNA into mammalian cells comprising infecting said cells with a hybrid gene vector comprising foreign DNA ligated into an AAV genome in place of or in addition to the cap, lip or rep coding sequence thereof or in place of or in addition to an AAV DNA sequence excluding the first and last 145 base pairs, said vector being capable of transducing said foreign DNA into a mammalian cell in the presence of a promoter other than an AAV transcription promoter, and said AAV genome being cloned in a prokaryotic vector plasmid or bacteriophage vector capable of growth in prokaryotic cells or yeast plasmid vector capable of growth in fungal cells and isolating a recombinant AAV hybrid gene vector by transfection of the ligated vector into a mammalian cell, said AAV genome being the entire AAV viral DNA or a deletion mutant thereof, capable of replication upon infection of said mammalian cell when complemented in trans by an AAV gene.

10. A method of rescuing foreign DNA from mammalian cells transduced according to a method of claim 9 comprising infecting said transduced mammalian cells with helper virus containing genes necessary for the replication of the AAV genome or the production of AAV virus.