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

Abstract

Valoctocogene roxaparvovec (AAV5-hFVIII-SQ) is an adenoassociated 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.

Interindividual variability in transgene mRNA and protein production following adeno-associated virus gene therapy for hemophilia A

“…We also looked for any safety concerns at the tissue level and explored potential mechanisms mediating interindividual variability. Alongside similar investigations in mice and NHPs, reported separately 17 , the results also further our knowledge of interspecies translatability in gene therapy.…”

“…Circular vector genomes were primarily in H–T configuration (Extended Data Fig. 3f ), as reported when AAV5-hFVIII-SQ was administered to mice and NHPs 17 .…”

“…Studies in lung, liver and muscle transduced by AAV gene therapy with normal-sized vectors (<4.7 kb 7 ) in mice and nonhuman primates (NHPs) have shown that circularized monomeric and concatemeric episomes are the major DNA species associated with long-term, persistent expression of the gene product in the target cell 8 – 16 . More recently, we have similarly demonstrated that long-term FVIII expression from AAV5-hFVIII-SQ transduction in mice and NHPs is associated with formation of full-length circular episomes in the liver 17 . Whether this is the case in human liver has not been previously determined.…”

“…Multiple preclinical studies demonstrating lung, liver and muscle transduction by AAV gene therapy in mice and NHPs have shown that circularized monomeric and concatemeric episomes are the major DNA species associated with long-term, persistent expression of the gene product in the target cell 8 – 16 . In nonclinical studies of AAV5-hFVIII-SQ 17 , circularized full-length episomes were evident in NHP and mouse livers up to 6 months after vector administration and correlated significantly with hFVIII-SQ RNA. In both humans and animals, there were dose-dependent trends in the numbers of hepatocytes transduced with vector genomes, in quantities of circular full-length/ITR-fused vector genomes, and in expression of FVIII-SQ RNA.…”

“…3 (ref. 17 ). FVIII activity following valoctocogene roxaparvovec administration in the phase 1/2 study generally increased over the first 20–28 weeks leading to peak FVIII levels in the first year, and declined in subsequent years 5 with a shallower rate of decline beyond year 2.…”