THE JOURNAL OF GENE MEDICINE RESEARCH ARTICLE J Gene Med 2004; 6: 555–564. Published online 25 February 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jgm.542 Identification of a replication-defective herpes simplex virus for recombinant adeno-associated virus type 2 (rAAV2) particle assembly using stable producer cell lines Estelle Toublanc 1 Abdellatif Benraiss 2 Delphine Bonnin 2 V´ eronique Blouin 1 Nicole Brument 1 Nathalie Cartier 2 Alberto L. Epstein 3 Philippe Moullier 1 Anna Salvetti 1 * 1 INSERM U649, CHU Hotel-Dieu, 30 Avenue Jean Monnet, 44035, Nantes, France 2 INSERMU561, Hˆopital Saint Vincent de Paul, 82 Avenue Denfert Rochereau, 75014 Paris, France 3 CNRS UMR5534, Universit´ e Claude Bernard Lyon I, 43 Boulevard du 11 Novembre 1918, 69622 Villeurbanne, France *Correspondence to: Anna Salvetti, INSERM U649, CHU Hotel-Dieu, 30 Avenue Jean Monnet, 44035, Nantes, France. E-mail: salvetti@sante.univ-nantes.fr Received: 29 August 2003 Revised: 20 November 2003 Accepted: 11 December 2003 Abstract Background The development of stable producer cell lines for recombinant adeno-associated virus (rAAV) assembly is a strategy followed by many groups to develop scalable production methods suitable for good manufacturing practice (GMP) requirements. The major drawback of this method lies in the requirement for replicating adenovirus (Ad) for rAAV assembly. In the present study, we analyzed the ability of several replication-defective herpes simplex type 1 (HSV-1) helper viruses to induce rAAV2 particle production from stable producer cell lines. Methods Several stable rAAV producer cell clones were infected with wild- type and replication-defective HSV strains and analyzed for rep-cap gene amplification, viral protein synthesis and rAAV titers achieved. In vivo analysis following rAAV injection in the murine brain was also conducted to evaluate the toxicity and biopotency of the rAAV stocks. Results We demonstrated that an HSV strain mutated in the UL30 polymerase gene could efficiently be used in this context, resulting in rAAV titers similar to those measured with wild-type HSV or Ad. Importantly, with respect to clinical developments, the use of this mutant resulted in rAAV stocks which were consistently devoid of contaminating HSV particles and fully active in vivo in the murine central nervous system with no detectable toxicity. Conclusions This study, together with our previous report describing a rAAV chromatography-based purification process, contributes to the definition of an entirely scalable process for the generation of rAAV particles. Copyright  2004 John Wiley & Sons, Ltd. Keywords AAV production; stable producer cell lines; replication-defective HSV Introduction The adeno-associated virus type 2 (AAV-2) is a human parvovirus which requires the presence of a helper virus such as adenovirus (Ad) or herpes simplex virus (HSV) to accomplish a complete and productive replication cycle. The AAV-2 genome is composed of two open-reading frames, rep and cap, encoding for the regulatory and structural proteins, respectively, Copyright  2004 John Wiley & Sons, Ltd.