Toxicology and Biodistribution Studies of a Recombinant Adeno-Associated Virus 2 (rAAV2) Alpha-1 Antitrypsin (AAT) Vector

A series of safety and biodistribution studies were undertaken to support the development of a muscle-based gene therapy system for AAT deficiency. AAT is the primary circulating serum proteinase inhibitor, and its deficiency can result in emphysema. The test article that was used in this toxicology study was rAAV serotype 2 with the CMV/Beta Actin promoter driving expression of the human AAT gene. There were 4 dosing arms in the primary toxicology study (PTS) with sacrifice time points being at 21 days and 90 days post vector administration. In the biodistribution study (BDS), there were 5 dosing arms with duplicate doses given intramuscularly and intravenously. The sacrifice time point for this study was at 60 days post vector administration. The PTS was performed in 48-C57/Bl6 mice (24M/24F) with the primary safety endpoint being the histopathologic examination of organs collected at necropsy. CBC and serum chemistry analyses were also performed. Gene transfer efficiency was evaluated from gDNA isolated from organs and whole blood by real-time PCR. The BDS was performed in 36-C57/Bl6 mice (18M/18F) to determine the potential for vector dissemination following test article administration IV and IM. In the PTS, no abnormalities were noted in the CBC and serum chemistry data among test article and control article study arms. Histopathology results were essentially normal with no vector related findings. Real Time PCR indicated that vector DNA was present in the peripheral blood 24 hours post administration, but decreased to near baseline levels at the time of sacrifice. Vector DNA was not detectable in gonad tissue. Real Time PCR results in the BDS also indicated vector DNA present in peripheral blood at 24 hours post vector administration. At sacrifice, IV injected mice vector levels were higher than IM injected mice. Vector DNA was found in skeletal muscle, lung, heart, liver, diaphragm, kidney and pancreas at the time of necropsy via IM administration at dose dependent levels. No vector DNA was detected in the skeletal muscle following IV administration, but substantial amounts were found in other organs. Gonads were negative for vector DNA with both IM and IV administration indicating that the risk of germline transmission with our gene therapy vector would be relatively low.