Assessment of Insertional Mutagenesis Risk Following AAV Vector-Mediated Factor IX Gene Transfer in Mice.
Authors: Li Hojun , Malani Nirav , Hamilton Shari R , Schlachterman Alexander , Zhang Jim Z , Bussadori Giulio , Edmonson Shyrie A , Mingozzi Federico , Wright J. Fraser , Arruda Valder R. , Bushman Frederic D , High Katherine A. 
Published: October 2019

Abstract
Abstract 2465
Poster Board II-442
Gene therapy for Hemophilia B is a promising alternative to recombinant protein therapy. Long-term expression of coagulation factor IX via stable gene transfer could reduce costs and risks associated with intravenous recombinant factor IX infusions. Liver-directed, adeno-associated virus (AAV) vector-mediated gene transfer of factor IX (FIX) is a gene therapy strategy currently used in two human clinical trials. Our group is performing one of these trials using an AAV vector (AAV-hFIX16) expressing human FIX from a liver-specific promoter. Hepatic artery delivery of AAV-hFIX16 has shown short-term efficacy and disease correction. A fundamental issue facing clinical gene transfer has to do with risk related to vector integration. Though AAV vector DNA is predominantly episomal in transduced cells, chromosomal integration can occur, and consequences of AAV genotoxicity at the molecular level in in vivo systems require further analysis. Here we present the results of a large-scale longitudinal study in which we followed 120 male, wild-type C57BL/6 mice for 18 months after portal vein injection of either 5e12 vg/kg of AAV-hFIX16 (60 mice), 1e14 vg/kg of AAV-hFIX16 (20 mice), 1e14 vc/kg of empty AAV capsid (20 mice), or saline (20 mice). At 18 months we found circulating hFIX levels of 12 ug/mL in mice receiving the lower AAV dose, and 25 ug/mL in mice receiving the higher AAV dose, with no detectable hFIX in mice receiving empty capsid or saline. To assess the risk of insertional mutagenesis in cells transduced by AAV vector, we analyzed the incidence of hepatocellular carcinoma (HCC). We found an HCC incidence rate of 9.1% in untreated mice, 0% in mice receiving saline, 0% in mice receiving empty capsid, 3.8% in mice receiving the lower AAV dose, and 12.5% in mice receiving the higher AAV dose, resulting in a p-value of 0.64 when comparing both AAV groups to controls. We quantified vector genome copy number by qPCR in the HCC's occurring in mice treated with AAV and compared them to vector genome copy number in normal tissue adjacent to the tumors and found no statistically significant difference (p=0.3). We then used LM-PCR to recover vector-chromosome integration junctions from tumor tissue and normal adjacent tissue. We were able to clone 249 unique integrants from tumor tissue and 862 unique integrants from normal adjacent tissue. This yields an average of 0.00018 unique integrants per diploid genome in tumor tissue and 0.00064 unique integrants per diploid genome in normal adjacent tissue. When determining integration profiles within the mouse genome, we found that integrants in both tumor tissue and normal adjacent tissue had a preference for integrating within genes, particularly within exons, as well as within CpG-rich regions. In addition, we found that integrants in both tumor tissue and normal adjacent tissue were more likely than not to be located within 50kb of the 5′ end of an oncogene. While this preference was stronger for integrants in normal adjacent tissue (p=6.3e-5) than integrants in tumor tissue (p=7.7e-2), it was even more likely for the HIV virus to integrate within 50kb of the 5′ end of an oncogene (p=4.1e-11). Functional consequences of these sites of AAV vector integration are not yet known, but are currently being investigated further. In conclusion, our large-scale prospective study of mice receiving AAV-hFIX16 did not demonstrate a statistically significant difference in HCC incidence between AAV-treated groups and control groups, although there was a trend of increasing HCC incidence with very high AAV doses. When analyzing tumors from mice treated with AAV, we found no relationship between vector genome copy number and tumor formation. When analyzing integrated vector in tumor tissue and normal adjacent tissue, we found that integrants in both normal adjacent tissue and tumor tissue had less likelihood for localizing within 50kb of the 5' end of oncogenes than HIV.

Disclosures:
Wright: Genzyme: Consultancy; Tacere: Consultancy.