AAV1-CMV-iCre

Gene therapy with adeno-associated virus (AAV) vectors has recently witnessed enormous clinical progress. Exiting improvements in the treatment of diverse genetic diseases including congenital blindness and hemophilia have been made possible by recombinant rAAV-mediated gene delivery. The increasing request for clinical application requires robust and scalable production methods for a growing range of AAV serotypes. Currently, 12 major AAV serotypes have been isolated from human and non-human primates, not counting the numerous engineered variants thereof. The AAV serotypes have distinct capsids, which interact with specific cell surface receptors. Whereas AAV2 or AAV1 have mostly been used in clinical trials so far, the extended cell- and tissue-specific transduction patterns of alternative AAV serotypes are being increasingly translated into clinical gene therapy.

Recombinant AAV vectors (rAAV) are constructed as gene-deleted AAVs comprising the gene of interest (GOI) flanked the AAV-ITRs, the only cis elements required for rAAV replication and packaging. The AAV gene rep codes for the regulatory proteins Rep78/68 and Rep52/40, while the cap codes for the capsid proteins VP1, VP2, and VP3. For rAAV production rep and cap have to be provided in trans together with the required helper virus genes. Plasmid cotransfection of the rAAV genome, AAVrep/cap, and adenovirus (Ad) helper genes

in 293 cells represents the most widely used protocol for rAAV production.