Molecular analysis of AAV5-hFVIII-SQ vector-genome-processing kinetics in transduced mouse and nonhuman primate livers
Choong‐Ryoul Sihn, Britta Handyside, Su Liu, Lening Zhang, Ryan Murphy, Bridget Yates, Lin Xie, Richard Torres, Chris B. Russell, Charles O’Neill, Ernö Pungor, Stuart Bunting, Sylvia Fong
Abstract
Valoctocogene roxaparvovec (AAV5-hFVIII-SQ) is an adeno-associated virus serotype 5 (AAV5)-based gene therapy vector containing a B-domain-deleted human coagulation factor VIII (hFVIII) gene controlled by a liver-selective promoter. AAV5-hFVIII-SQ is currently under clinical investigation as a treatment for severe hemophilia A. The full-length AAV5-hFVIII-SQ is >4.9 kb, which is over the optimal packaging limit of AAV5. Following administration, the vector must undergo a number of genome-processing, assembly, and repair steps to form full-length circularized episomes that mediate long-term FVIII expression in target tissues. To understand the processing kinetics of the oversized AAV5-hFVIII-SQ vector genome into circular episomes, we characterized the various molecular forms of the AAV5-hFVIII-SQ genome at multiple time points up to 6 months postdose in the liver of murine and non-human primate models. Full-length circular episomes were detected in liver tissue beginning 1 week postdosing. Over 6 months, quantities of circular episomes (in a predominantly head-to-tail configuration) increased, while DNA species lacking inverted terminal repeats were preferentially degraded. Levels of duplex, circular, full-length genomes significantly correlated with levels of hFVIII-SQ RNA transcripts in mice and non-human primates dosed with AAV5-hFVIII-SQ. Altogether, we show that formation of full-length circular episomes in the liver following AAV5-hFVIII-SQ transduction was associated with long-term FVIII expression.