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US 2013/0171719 A1Published application
Recombinant aav production in mammalian cells
OwnerKyu-Kye Hwang, David Knopinventors
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Abstract
The present invention includes methods and compositions for the production of high titer recombinant Adeno-Associated Virus (rAAV) in a variety of mammalian cells. The disclosed rAAV are useful in gene therapy applications. Disclosed methods based on co-infection of cells with two or more replication-defective recombinant herpes virus (rHSV) vectors are suitable for high-titer, large-scale production of infectious rAAV.
Claims30
A method for producing recombinant Adeno-Associated Virus (rAAV), comprising:
- (a) simultaneously infecting a mammalian cell, wherein the mammalian cell is selected from the group consisting of a Cos-7 cell and a HT1080 cell, with:
- (i) a first replication-defective recombinant herpes simplex virus (rHSV) comprising a nucleic acid sequence operably linked to a promoter wherein the nucleic acid comprises an AAV rep gene and an AAV cap gene;
- and
- (ii) a second replication-defective rHSV comprising a nucleic acid sequence including AAV inverted terminal repeat sequences (ITRs) and a gene of interest, said gene of interest being operably linked to a promoter;
- (b) incubating the infected mammalian cell;
- and
- (c) obtaining rAAV from the cell of step (b), wherein the titer of rAAV produced by the cell is between about 1000 and about 9000 infectious particles (i.p.) per cell.
4 more elements
A method for producing recombinant rAAV in a mammalian cell, comprising:
- (a) simultaneously infecting the mammalian cell, wherein the mammalian cell is selected from the group consisting of a BHK cell and a 293 cell, with:
- (i) a first replication-defective rHSV comprising a nucleic acid sequence operably linked to a promoter wherein the nucleic acid comprises an AAV rep gene and an AAV cap gene, wherein the AAV rep and cap genes are integrated into the tk gene of the first rHSV-1;
- and
- (ii) a second replication-defective rHSV comprising a nucleic acid sequence including AAV ITRs and a gene of interest, said gene of interest being operably linked to a promoter;
- (b) incubating the infected mammalian cell;
- and
- (c) obtaining rAAV from the cell of step (b), wherein the titer of rAAV produced by the cell is between about 1000 and about 9000 infectious particles (i.p.) per cell.
4 more elements
Dependencies are inferred from the claim text.