Compositions and methods for generating adeno-associated viral vectors with undetectable capsid gene contamination

Abstract

In one aspect, the present invention provides an intron-modified capsid expression cassette useful for generating adeno-associated virus (AAV) vector particles. In another aspect, the present invention provides a method of reducing the immune response in a mammalian subject undergoing treatment with an AAV vector.

1 . A method of reducing the immune response in a mammalian subject undergoing treatment with an AAV vector comprising:
 administering to the mammalian subject a preparation of AAV vector particles comprising a nucleic acid sequence encoding a therapeutic molecule, wherein the preparation of AAV vector particles was produced with an intron-modified cap expression cassette, wherein the intron-modified cap expression cassette comprises:
 (a) a nucleic acid molecule comprising a nucleotide sequence encoding an AAV capsid protein operably linked to a promoter, wherein the encoded AAV capsid protein is capable of packaging AAV vectors in the presence of rep and adeno helper functions, and 
 (b) at least one heterologous intron sequence operably linked to the nucleotide sequence encoding the AAV capsid protein; 
 wherein the total length of the expression cassette is at least 5 kilobases; and 
   wherein the immune response in the mammalian subject to the AAV vector generated using the intron-modified cap expression cassette is significantly reduced or absent as compared to the immune response generated with a preparation of AAV vector particles generated using a standard cap expression cassette.   
     
     
         2 . The method of  claim 1 , wherein the AAV capsid protein is from an AAV serotype selected from the group consisting of AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, and AAV-13, or a variant or hybrid protein thereof. 
     
     
         3 . The method of  claim 1 , wherein the AAV capsid protein is from an AAV serotype selected from the group consisting of AAV-8 and AAV-9, or a variant or hybrid protein thereof. 
     
     
         4 . The method of  claim 1 , wherein the promoter is a heterologous promoter. 
     
     
         5 . The method of  claim 1 , wherein the at least one heterologous intron sequence is an excisable heterologous intron sequence. 
     
     
         6 . The method of  claim 1 , wherein the at least one heterologous intron sequence is positioned between the promoter and an open reading frame encoding said AAV capsid protein. 
     
     
         7 . The method of  claim 1 , wherein the at least one heterologous intron sequence comprises at least one splice donor and at least one splice acceptor site. 
     
     
         8 . The method of  claim 1 , wherein the intron-modified cap expression cassette further comprises an AAV intron sequence. 
     
     
         9 . The method of  claim 1 , wherein the intron-modified cap expression cassette further comprises a polyadenylation signal sequence. 
     
     
         10 . The method of  claim 1 , wherein the intron-modified cap expression cassette is incorporated into a plasmid. 
     
     
         11 . The method of  claim 1 , wherein the nucleic acid sequence encoding a therapeutic molecule is flanked by AAV terminal repeat sequences (ITRs). 
     
     
         12 . The method of  claim 1 , wherein the therapeutic molecule is a structural protein or an enzyme. 
     
     
         13 . The method of  claim 12 , wherein the therapeutic molecule is an enzyme. 
     
     
         14 . The method of  claim 1 , wherein the therapeutic molecule is useful to relieve a deficiency caused by a missing, defective, or sub-optimal level of a structural protein or enzyme in the mammalian subject. 
     
     
         15 . The method of  claim 1 , wherein the therapeutic molecule is an antisense molecule. 
     
     
         16 . The method of  claim 1 , wherein the mammalian subject is administered the preparation of AAV vector particles via a parenteral route of administration. 
     
     
         17 . The method of  claim 16 , wherein the parenteral route of administration is selected from an intranasal, intratracheal, intraarterial, intraocular, intracochlear, intravenous, intramuscular, subcutaneous, or intradermal route of administration. 
     
     
         18 . The method of  claim 17 , wherein the parenteral route of administration is an intravenous route of administration. 
     
     
         19 . The method of  claim 1 , wherein the mammalian subject is administered the preparation of AAV vector particles at a dose of about 10 9  to 10 16  genomes of AAV vector. 
     
     
         20 . The method of  claim 1 , wherein the mammalian subject is a human.