Compositions and methods for helper-free production of recombinant adeno-associated viruses

A method for producing recombinant adeno-associated virus (rAAV), comprising the step of:

• culturing a host cell containing:
• (a) an AAV rep sequence and an AAV cap sequence under the control of regulatory sequences directing expression thereof;
• and
• (b) an adenovirus/AAV hybrid virus comprising a recombinant adeno-associated viral (rAAV) vector and nucleic acid sequences encoding adenovirus E1a and adenovirus E1b are under the control of regulatory sequences directing expression of the E1a and E1b gene products, wherein said hybrid virus contains sufficient adenoviral sequences to permit replication of said hybrid virus in a host cell;
• wherein said host cell is cultured under conditions which inhibit hybrid virus replication, resulting in enhanced rAAV production.

A method for producing recombinant adeno-associated virus (rAAV) comprising the step of:

• culturing a host cell comprising:
• (a) an AAV rep sequence and an AAV cap sequence under the control of regulatory sequences directing expression thereof;
• and
• (b) an adenovirus/AAV hybrid virus comprising:
• (i) adenovirus 5′ cis-elements necessary for replication and packaging;
• (ii) a deletion of adenoviral sequences in the native adenoviral E1a and E1b region;
• (iii) a recombinant adeno-associated viral (rAAV) vector;
• (iv) a deletion of adenoviral sequences from the E3 region;
• (v) nucleic acid sequences encoding adenovirus E1a and adenovirus E1b under the control of regulatory sequences directing expression of the E1a and E1b gene products, wherein said E1a and E1b nucleic acid sequences are located in the site of the E3 region;
• and
• (vi) adenovirus 3′ cis-elements necessary for replication and packaging.