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Adeno-associated viral (AAV) capsid proteins, thought to be a rate-limiting step in the production of recombinant AAV (rAAV), are translated from spliced mRNAs. Improvement of the native AAV nonconsensus donor sequence increases splicing yet leaves the relative levels of VP1- and VP2/3-encoding mRNAs unchanged, and thus provides a means to increase delivery of correct ratios of AAV capsid proteins. This effect is independent of the AAV serotype used, and occurs whether the rep and cap genes supplied in trans are on the same or separate expression vectors. In the split-vector system, replacement of the more traditionally used cytomegalovirus promoter with that of the AAV5 P41 promoter allowed for even greater levels of splicing, and together with an improved intron donor, led to a 10- to 15-fold increase in the levels of splicing, rAAV production, and transduction compared with levels achieved by traditional cotransfection methods. Thus, the enhancement of splicing presents a useful method to enhance rAAV production via transient transfection.

Improved Splicing of Adeno-Associated Viral (AAV) Capsid Protein-Supplying Pre-mRNAs Leads to Increased Recombinant AAV Vector Production