Identification of AAV Vector Integration Sites in Primary Human and Macaque Hepatocytes in vivo, and Analysis of Oncogenic Risk.

Abstract

Recombinant adeno-associated virus (rAAV) vectors are widely used for in vivo gene therapy, yet their potential to integrate into the host genome raises concerns about insertional mutagenesis and oncogenic risk, particularly in the liver where vector exposure is highest. To address this, we analyzed rAAV integration patterns in primary human hepatocytes xenografted into FRG mouse livers and in hepatocytes from cynomolgus macaques following systemic rAAV administration. High-resolution integration site mapping yielded approximately 1.5 million and 1.3 million unambiguously mapped sites in human and macaque genomes, respectively. Both datasets revealed a bias toward integration within transcriptionally active genes and regions of open chromatin, consistent with previous reports, but no particular preference for genes implicated in hepatocellular carcinoma (HCC) was observed. While numerous common integration sites (CIS) were identified, their distribution differed between species. Notably, a CIS was observed at the AAVS1 locus in human hepatocytes, raising the possibility of Rep-mediated integration. These findings highlight the need for continued monitoring of integration events in clinical settings. Overall, the data support a low oncogenic risk profile for the evaluated vector while reinforcing the value of direct human liver integration analyses to refine risk assessment and guide the development of safer gene therapy platforms.