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 A O'Neill; Erno Pungor; Stuart Bunting; Sylvia Fong

doi:10.1016/j.omtm.2021.12.004

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

Mol Ther Methods Clin Dev. 2021 Dec 21:24:142-153. doi: 10.1016/j.omtm.2021.12.004. eCollection 2022 Mar 10.

Authors

Affiliations

¹ Biology Research, BioMarin Pharmaceutical, Inc., Novato, CA, USA.
² Bioanalytical Sciences, BioMarin Pharmaceutical, Inc., Novato, CA, USA.
³ Pharmacological Sciences, BioMarin Pharmaceutical, Inc., Novato, CA, USA.
⁴ Analytical Chemistry, BioMarin Pharmaceutical, Inc., Novato, CA, USA.

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.

Keywords: AAV genome processing; AAV vectors; AAV5-hFVIII-SQ; circular episomes; hepatocyte-directed AAV gene transfer; oversized AAV vectors; valoctocogene roxaparvovec.