¹Applied Genetic Technologies Corporation, Alachua, FL, USA

Correspondence: Dr DR Knop, Applied Genetic Technologies Corporation, 11801 Research Drive, Suite D, Alachua, FL 32615, USA. E-mail: dknop@agtc.com

²Current address: Florida Biologix, 13706 Innovation Drive, Alachua, FL 32615, USA.

³Current address: Monsanto Company, 800 N. Lindbergh Blvd., St Louis, MO 63167, USA.

Received 10 June 2008; Revised 9 September 2008; Accepted 10 September 2008; Published online 16 October 2008.

Abstract

Recombinant herpes simplex virus type 1 (rHSV)-assisted recombinant adeno-associated virus (rAAV) vector production provides a highly efficient and scalable method for manufacture of clinical grade rAAV vectors. Here, we present an rHSV co-infection system for rAAV production, which uses two ICP27-deficient rHSV constructs, one bearing the rep2 and cap (1, 2 or 9) genes of rAAV, and the second bearing an AAV2 ITR-gene of interest (GOI) cassette. The optimum rAAV production parameters were defined by producing rAAV2/GFP in HEK293 cells, yielding greater than 9000 infectious particles per cell with a 14:1 DNase resistance particle to infectious particle (DRP/ip) ratio. The optimized co-infection parameters were then used to generate large-scale stocks of rAAV1/AAT, which encode the human -1-antitrypsin (hAAT) protein, and purified by column chromatography. The purified vector was extensively characterized by rAAV- and rHSV-specific assays and compared to transfection-made vector for in vivo efficacy in mice through intramuscular injection. The co-infection method was also used to produce rAAV9/AAT for comparison to rAAV1/AAT in vivo. Intramuscular administration of 1 10¹¹ DRP per animal of rHSV-produced rAAV1/AAT and rAAV9/AAT resulted in hAAT protein expression of 5.4 10⁴ and 9.4 10⁵ ng ml^-1 serum respectively, the latter being clinically relevant.