24. Widespread and Efficient Gene Delivery to the Heart and Liver Using AAV Serotype 9: Implications for Cardiovascular Disease and Hemophilia

Heart disease remains one of the most prominent health challenges. Gene therapy may provide an alternative treatment to attain functional correction of the damaged heart. However, gene delivery into the heart has been particularly challenging which was least partly due to the inability to achieve widespread gene transfer following localized intra-cardiac delivery of gene transfer vectors, which typically remain confined to the injection site. The present study shows that a single intravenous injection of an adeno-associated viral vector derived from the human serotype 9 (AAV9) resulted in unprecedented and widespread transduction of the heart in adult mice. Almost the entire heart was GFP positive as up to 90% of the cardiomyocytes were predominantely transduced. Transduction efficiency was far greater than with any other gene delivery system described so far, including lentiviral and HC-Ad vectors, and obviated the use of neonatal recipients or the need for pharmacologic intervention to enhance vector penetration into the heart tissue. The use of AAV9 was not associated with any apparent side-effects, consistent with the lack of immune complications or pro- inflammatory cytokine induction (IL-6, TNF) following gene delivery. Moreover, AAV9 also resulted in highly efficient gene transfer into the liver paving the way towards improved gene therapeutics for liver disease and for production of therapeutically relevant proteins that are secreted into the circulation. As proof-of- concept, an AAV9 vector encoding human clotting factor IX (FIX) was generated and injected into hemophilia B mice. This resulted in stable therapeutic levels of factor IX (100 |[mu]|g/ml, i.e. 20-fold higher than normal levels) which far exceeded levels obtained using the prototypical AAV2 serotype that has been used previously in clinical trials. No neutralizing antibodies against transgene-endoded FIX could be detected. Hepatic FIX expression may provide an attractive alternative to ectopic FIX expression (e.g. in muscle), which we showed resulted in abnormal post-translational glycosylations and impaired propeptide cleavage of the transgene-encoded FIX protein . The present study identifies a highly efficient and safe means to deliver genes to the heart and liver using AAV9, which may overcome the current challenges that hamper progress to treat genetic or acquired heart disease and bleeding disorders. Moreover, the use of AAV9 opens up a new platform technology for screening or validating promising new therapeutic leads or for the rapid generation of animal models of human heart disorders that could be used to validate new pharmacologic agents.