Amplifiable adeno-associated virus (AAV) packaging cassettes for the production of recombinant AAV vectors

Abstract

High-efficiency AAV packaging constructs and methods for their use are provided in the present invention. These high-efficiency packaging constructs comprise an activating element (such as the P1 sequence located within the AAV S1 integration site of human chromosome 19) amplifiably linked to one or more AAV packaging genes. The constructs may be either integrated into a mammalian cell genome or maintained episomally. Use of the high-efficiency AAV packaging vectors of the invention provides for controlled amplifiable production of rAAV vector constructs.

1 . A recombinant polynucleotide sequence encoding an adeno-associated virus (AAV) packaging cassette comprising at least one AAV packaging gene amplifiably linked to an activating element.  
     
     
         2 . The packaging cassette of  claim 1  wherein the AAV packaging cassette comprises an AAV rep gene and an AAV cap gene.  
     
     
         3 . The packaging cassette of  claim 1  wherein the activating element is a P1 element.  
     
     
         4 . The packaging cassette of  claim 1  wherein the activating element is an inducible replication origin.  
     
     
         5 . The packaging cassette of  claim 1  wherein the activating element is a replication origin that is activated by helper function.  
     
     
         6 . The packaging cassette of  claim 5  wherein the helper function is provided by adenovirus.  
     
     
         7 . The packaging cassette of  claim 1  wherein the activating element comprises a viral replication origin.  
     
     
         8 . The packaging cassette of  claim 7  wherein the viral replication origin is dependent on helper function.  
     
     
         9 . The packaging cassette of  claim 8  wherein the helper function is provided by adenovirus.  
     
     
         10 . The packaging cassette of  claim 1  wherein the activating element comprises a mammalian replication origin.  
     
     
         11 . The packaging cassette of  claim 2  wherein the activating element comprises a sequence having activity in a replication assay.  
     
     
         12 . The packaging cassette of  claim 11  wherein the replication activity is Rep-dependent.  
     
     
         13 . The packaging cassette of  claim 1 , wherein said activating element comprises a Rep Binding Motif and a Terminal Resolution site.  
     
     
         14 . A method for producing high-titer stocks of an rAAV vector containing a heterologous gene of interest, comprising co-expressing the rAAV vector containing a gene of interest in a mammalian cell along with an AAV packaging cassette, said AAV packaging cassette comprising at least one AAV rep and/or cap gene amplifiably linked to an activating element.  
     
     
         15 . The method of  claim 14  wherein the activating element is a P1 element.  
     
     
         16 . The method of  claim 14  wherein the activating element is a replication origin that is activated by helper function.  
     
     
         17 . The method of  claim 16  wherein the helper: function is provided by adenovirus.  
     
     
         18 . The method of  claim 14  wherein the activating element is an inducible replication origin.  
     
     
         19 . A method for generating a cell line capable of producing high-titer stocks of an rAAV vector containing a foreign gene of interest, by transfecting mammalian cells with an rAAV vector containing a gene of interest and with an AAV packaging cassette, said AAV packaging cassette comprising at least one AAV rep and/or cap gene amplifiably linked to an activating element.  
     
     
         20 . The method of  claim 19  wherein the activating element is a P1 element.  
     
     
         21 . The method of  claim 19  wherein the activating element is a replication origin that is activated by helper function.  
     
     
         22 . The method of  claim 21  wherein the helper function is provided by adenovirus.  
     
     
         23 . The method of  claim 19  wherein the activating element is an inducible replication origin.  
     
     
         24 . An AAV packaging cell line produced by the method of  claim 23 .  
     
     
         25 . An AAV virus containing an rAAV vector produced by the method of  claim 14.