Factors Influencing Recombinant Adeno-Associated Virus Production

Abstract

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 Française contre les Myopathies/Généthon. 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.

Chromatographic purification of recombinant adenoviral and adeno-associated viral vectors: methods and implications

“…Alternatively, the minimal viral genes required for the helper function can be supplemented on a separate plasmid. 16,32,33 In the latter case, the rAAV production system requires simultaneous transfection with three plasmids. Since helper virus is not necessary for rAAV packaging via triple plasmid transfection, this system offers significant safety advantages for clinical use.…”

Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield

“…14,15 rAAV is most often generated by cotransfection of rAAV vector plasmid and wild-type (wt) AAV helper plasmid into Ad-infected 293 cells. 16 Recent improvements in AAV helper design 17 as well as construction of non-infectious mini-Ad plasmid helper [18][19][20] have eliminated the need for Ad infection and improved the yield of rAAV per transfected cell in a crude lysate. Scalable methods of rAAV production that do not rely on DNA transfection have also been developed.…”

Efficient gene transfer into human cord blood CD34+ cells and the CD34+CD38− subset using highly purified recombinant adeno-associated viral vector preparations that are free of helper virus and wild-type AAV

“…Preparations entirely free of adenovirus can be obtained by substituting a plasmid, which encodes the required adenoviral gene products for the helper virus. 16,[29][30][31] A reduction in the risk for generation of wild-type AAV (wt-AAV) has been achieved by rearranging the coding sequences for AAV proteins in the packaging plasmid. 32 Column chromatography has been substituted for cesium chloride gradient centrifugation as a purification step, improving the ratio of biologically active to physical particles in the final preparations.…”