Molecular analysis of AAV5-hFVIII-SQ vector-genome-processing kinetics in transduced mouse and nonhuman primate livers

• Published in: Molecular therapy. Methods & clinical development Vol. 24; pp. 142 - 153
• Main Authors: Sihn, Choong-Ryoul, Handyside, Britta, Liu, Su, Zhang, Lening, Murphy, Ryan, Yates, Bridget, Xie, Lin, Torres, Richard, Russell, Chris B., O'Neill, Charles A., Pungor, Erno, Bunting, Stuart, Fong, Sylvia
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 10-03-2022; American Society of Gene & Cell Therapy; Elsevier
• Subjects: AAV genome processing; AAV vectors; AAV5-hFVIII-SQ; circular episomes; hepatocyte-directed AAV gene transfer; Original; oversized AAV vectors; valoctocogene roxaparvovec; circular episomes; hepatocyte-directed AAV gene transfer; oversized AAV vectors; AAV vectors; valoctocogene roxaparvovec; AAV genome processing; AAV5-hFVIII-SQ
• ISSN: 2329-0501; 2329-0501
• DOI: 10.1016/j.omtm.2021.12.004

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.
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AAV vectors are a well-established transgene delivery method, but the steps that transform oversized vector genomes into stable structures capable of transgene expression remain uncertain. Here, we demonstrate the formation of durable, circular, full-length episomes in hepatocytes of mice and non-human primates dosed with valoctocogene roxaparvovec (AAV5-hFVIII-SQ).