FACTORS INFLUENCING RECOMBINANT ADENOASSOCIATED VIRUS PRODUCTION

Recombinant adeno-associated virus (rAAV) is produced by transfecting cells with two constructs: the rAAV vector plasmid and the rep-cap pla smid, After subsequent adenoviral infection, needed for rAAV replicati on and assembly, the virus is purified from total cell lysates through CsCl gradients, Because this is a long and complex procedure, the pre cise titration of rAAV stocks, as well as the measure of the level of contamination with adenovirus and rep-positive AAV, are essential to e valuate the transduction efficiency of these vectors in vitro and in v ivo., Our vector core is in charge of producing rAAV for outside inves tigators as part of a national network promoted by the Association Fra ncaise contre les Myopathies/Genethon. We report here the characteriza tion of 18 large-scale rAAV stocks produced during the past year, Thre e major improvements were introduced and combined in the rAAV producti on procedure: (i) the titration and characterization of rAAV stocks us ing a stable rep-cap HeLa cell line in a modified Replication Center A ssay (RCA); (ii) the use of different rep-cap constructs to provide AA V regulatory and structural proteins; (iii) the use of an adenoviral p lasmid to provide helper functions needed for rAAV replication and ass embly, Our results indicate that: (i) rAAV yields ranged between 10(11 ) to 5 x 10(12) total particles; (ii) the physical particle to infecti ous particle (measured by RCA) ratios were consistently below 50 when using a rep-cap plasmid harboring an ITR-deleted AAV genome; the physi cal particle to transducing particle ratios ranged between 400 and 600 ; (iii) the use of an adenoviral plasmid instead of an infectious viri on 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 determi ned by RCA; (iv) all the rAAV stocks were contaminated with rep-positi ve AAV as detected by RCA, In summary, this study describes a general method to titrate rAAV, independently of the transgene and its express ion, 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 w ith undetectable adenovirus contamination.