Factors influencing recombinant adeno-associated virus production

Recombinant adeno-associated virus (rAAV) is produced by transfecting cells with two constructs: the rAAV vector plasmid and the rep-cap plasmid, After subsequent adenoviral infection, needed for rAAV replication and assembly, the virus is purified from total cell lysates through CsCl gradients, Because this is a long and complex procedure, the precise titration of rAAV stocks, as well as the measure of the level of contamination with adenovirus and rep-positive AAV, are essential to evaluate the transduction efficiency of these vectors in vitro and in vivo., Our vector core is in charge of producing rAAV for outside investigators as part of a national network promoted by the Association Francaise contre les Myopathies/Genethon. We report here the characterization of 18 large-scale rAAV stocks produced during the past year, Three major improvements were introduced and combined in the rAAV production procedure: (i) the titration and characterization of rAAV stocks using a stable rep-cap HeLa cell line in a modified Replication Center Assay (RCA); (ii) the use of different rep-cap constructs to provide AAV regulatory and structural proteins; (iii) the use of an adenoviral plasmid to provide helper functions needed for rAAV replication and assembly, Our results indicate that: (i) rAAV yields ranged between 10(11) to 5 x 10(12) total particles; (ii) the physical particle to infectious particle (measured by RCA) ratios were consistently below 50 when using a rep-cap plasmid harboring an ITR-deleted AAV genome; the physical particle to transducing particle ratios ranged between 400 and 600; (iii) the use of an adenoviral plasmid instead of an infectious virion did not affect the particles or the infectious particles yields nor the above ratio, Most of large-scale rAAV stocks (7/9) produced using this plasmid were free of detectable infectious adenovirus as determined by RCA; (iv) all the rAAV stocks were contaminated with rep-positive AAV as detected by RCA, In summary, this study describes a general method to titrate rAAV, independently of the transgene and its expression, and to measure the level of contamination with adenovirus and rep-positive AAV, Furthermore, we report a new production procedure using adenoviral plasmids instead of virions and resulting in rAAV stocks with undetectable adenovirus contamination.