Manufacturing and use of recombinant aav vectors

Abstract

Presented herein are technologies and methods for improved production of AAV vectors.

We claim: 
     
         1 . A Rep/Helper plasmid comprising a polynucleotide sequence of SEQ ID NO: 1, wherein the plasmid does not comprise a polynucleotide sequence encoding a cap gene. 
     
     
         2 . A Rep/Helper plasmid comprising a polynucleotide sequence of SEQ ID NO: 2, wherein the plasmid does not comprise a polynucleotide sequence encoding a cap gene. 
     
     
         3 . A composition comprising;
 the plasmid of any one of  claims 1  or  2 ; and   a Payload/Cap plasmid comprising a polynucleotide sequence of SEQ ID NO: 11; wherein the Payload/Cap plasmid does not comprise a polynucleotide sequence encoding a rep gene.   
     
     
         4 . The composition of  claim 3 , wherein the Payload/Cap plasmid comprises:
 a polynucleotide sequence comprising a sequence encoding a cap gene; and   a polynucleotide sequence encoding a payload.   
     
     
         5 . The composition of  claim 4 , wherein the cap gene is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVC11.01, AAVC11.02, AAVC11.03, AAVC11.04, AAVC11.05, AAVC11.06, AAVC11.07, AAVC11.08, AAVC11.09, AAVC11.10, AAVC11.11, AAVC11.12, AAVC11.13, AAVC11.14, AAVC11.15, AAVC11.16, AAVC11.17, AAVC11.18, AAVC11.19, AAV-DJ, AAV-LK03, AAV-LK19, AAVrh.74, AAVrh.10, AAVhu.37, AAVrh.K, AAVrh.39, AAV12, AAV 13, AAVrh.8, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, ovine AAV, or a hybrid AAV. 
     
     
         6 . The composition of any one of  claims 4  or  5 , wherein the polynucleotide sequence comprising a sequence encoding a cap gene is inserted before position 2025 of SEQ ID NO: 11. 
     
     
         7 . The composition of any one of  claims 4 - 6 , wherein the polynucleotide sequence encoding a payload comprises a polynucleotide sequence encoding a transgene. 
     
     
         8 . The composition of any one of  claims 4 - 7 , wherein the polynucleotide sequence encoding a payload is inserted after position 2663 of SEQ ID NO: 11. 
     
     
         9 . The composition of any one of  claims 7  or  8 , wherein the transgene is or comprises a gene listed in  FIG.  29   , or a variant thereof. 
     
     
         10 . The composition of any one of  claims 7  or  8 , wherein the transgene is or comprises one or more of Propionyl-CoA Carboxylase, ATP7B, Factor IX, methylmalonyl-CoA mutase (MUT), α1-antitrypsin (A1AT), UGT1A1, fumarylacetoacetate hydrolase (FAH), cystathionine beta synthase (CBS), or variants thereof. 
     
     
         11 . The composition of any one of  claims 7  or  8 , wherein the transgene is FAH or a variant thereof. 
     
     
         12 . The composition of any one of  claims 7  or  8 , wherein the transgene is MUT or a variant thereof. 
     
     
         13 . The composition of any one of  claims 7  or  8 , wherein the transgene is CBS or a variant thereof. 
     
     
         14 . The composition of any one of  claims 7  or  8 , wherein the transgene is ATP7B or a variant thereof. 
     
     
         15 . The composition of any one of  claims 7  or  8 , wherein the transgene is Factor IX or a variant thereof. 
     
     
         16 . The composition of any one of  claims 7  or  8 , wherein the transgene is UGT1A 1 or a variant thereof. 
     
     
         17 . The composition of any one of  claims 3 - 16 , wherein the composition comprises no more than two distinct plasmids. 
     
     
         18 . The composition of any one of  claims 3 - 17  wherein the plasmid ratio of the Rep/Helper plasmid to the Payload/Cap plasmid is greater than or equal to 1.5:1 up to 10:1. 
     
     
         19 . The composition of any one of the above claims, for use in producing an AAV vector. 
     
     
         20 . A method of manufacturing a packaged AAV vector, comprising delivering to a cell a composition of any one of  claims 3 - 18 . 
     
     
         21 . The method of  claim 20 , additionally comprising use of a chemical transfection reagent.