Identification of a replication-defective herpes simplex virus for recombinant adeno-associated virus type 2 (rAAV2) particle assembly using stable producer cell lines.
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.
Toublanc
Estelle
E
INSERM U649, CHU Hotel-Dieu, 30 Avenue Jean Monnet, 44035, Nantes, France.
Benraiss
Abdellatif
A
Bonnin
Delphine
D
Blouin
Véronique
V
Brument
Nicole
N
Cartier
Nathalie
N
Epstein
Alberto L
AL
Moullier
Philippe
P
Salvetti
Anna
A
D000818:Animals; D002522:Chlorocebus aethiops; D000229:Dependovirus / Q000235:genetics / Q000502:physiology*; D005260:Female; D005822:Genetic Vectors; D006367:HeLa Cells; D018259:Herpesvirus 1, Human / Q000235:genetics / Q000502:physiology*; D006801:Humans; D051379:Mice; D009154:Mutation; D011995:Recombination, Genetic; D014709:Vero Cells; D019065:Virus Assembly; D014779:Virus Replication