Production of research and clinical‐grade recombinant adeno‐associated virus vectors

Abstract

Recombinant adeno-associated virus (rAAV) vectors are constructed by replacing the rep and cap genes with the transgene of interest while retaining the flanking ITR sequences. Construction of rAAV vectors is possible because the AAV inverted terminal repeats (ITRs) supply all of the cis acting sequences for vector production and transduction. Recombinant AAV vector constructs are flanked by AAV ITRs and other cis-sequences control transgene expression including constitutive or regulated promoters, enhancers, and intron sequences. In general, there are two different approaches for packaging rAAV vectors: ‘‘isotype’’ and ‘‘pseudotyped’’ vectors. The major improvements in production and purification of rAAV vectors include enhanced output of the number of DNase resistant particles (drp) per cell and the emergence of scaleable systems. Large-scale purification by column chromatography is made possible by two changes in the preparations of crude lysates. Having reliable, specific, and sensitive assays is crucial during development of production and purification methods, and to show lot consistency. Once made and purified, the rAAV vector stocks are characterized using a variety of techniques to ensure they are safe, pure, potent, and stable.