AAV vectors for gene therapy

A method for producing recombinant adeno-associated virus (AAV) with reduced levels of wild-type helper adenovirus compared to co-infection of cells with AAV vector and wild-type helper adenovirus, said method comprising the steps of:

• (1) culturing a composition comprising cells which have been transiently transfected with:
• (a) an AAV helper plasmid comprising AAV rep and cap nucleic acid sequences encoding AAV rep and cap proteins;
• (b) an adenoviral helper plasmid comprising essential adenovirus helper genes selected from the group consisting of E1A, E1B, E2A, E4, E4ORF6, E4ORF6/7, VA and combinations of the above;
• and
• (c) an AAV vector comprising first and second AAV ITRs or portions thereof, wherein said first and second AAV ITRs flank a DNA sequence encoding a polypeptide of interest, said DNA sequence being operably linked to a promoter DNA sequence;
• in the absence of adenovirus particles and under conditions suitable for the production of recombinant AAV;
• and
• (2) purifying recombinant AAV produced therefrom.

A method for producing recombinant adeno-associated virus (AAV) with reduced levels of wild-type helper adenovirus to co-infection of cells with AAV and wild-type helper adenovirus, said method comprising the steps of:

• (1) culturing a composition comprising cells which have been transiently transfected with:
• (a) an AAV helper plasmid comprising AAV rep and cap nucleic acid sequences encoding AAV rep and cap proteins;
• (b) an adenoviral helper plasmid consisting essentially of adenovirus helper genes E2A, E4 ORF6 and VA RNA, wherein the adenoviral helper plasmid comprises AAV ITR sequences flanking the adenovirus helper genes, and said AAV ITR sequences allow the plasmid to replicate;
• and
• (c) an AAV vector comprising first and second AAV ITRs or portions thereof, wherein said first and second AAV ITRs flank a DNA sequence encoding a polypeptide of interest, said DNA sequence being operably linked to a promoter DNA sequence;
• in the absence or reduced presence of adenovirus particles and under conditions suitable for the production of recombinant AAV;
• and
• (2) purifying recombinant AAV produced therefrom.

A method for producing recombinant adeno-associated virus (AAV) with reduced levels of wild-type helper adenovirus compared to co-infection of cells with AAV and wild-type helper adenovirus, said method comprising the steps of:

• (1) culturing a composition comprising cells which have been transiently transfected with:
• (a) an AAV helper plasmid comprising AAV rep and cap nucleic acid sequences encoding AAV rep and cap proteins;
• (b) an adenoviral helper plasmid consisting essentially of adenovirus helper genes E2A, E4 ORF6 and VA RNA, wherein the adenoviral helper plasmid comprises AAV ITR sequences flanking the adenovirus helper genes, and said AAV ITR sequences allow the plasmid to replicate;
• and
• (c) an AAV vector comprising first and second AAV ITRs or portions thereof, wherein said first and second AAV ITRs flank a DNA sequence encoding a polypeptide of interest, said DNA sequence being operably linked to a promoter DNA sequence;
• in the absence or reduced presence of adenovirus particles and under conditions suitable for the production of recombinant AAV;
• and
• (2) purifying recombinant AAV produced therefrom.