Modeling correction of severe urea cycle defects in the growing murine liver using a hybrid recombinant adeno‐associated virus/piggyBac transposase gene delivery system

Abstract

Liver-targeted gene therapy based on recombinant adeno-associated viral vectors (rAAV) shows promising therapeutic efficacy in animal models and adult-focused clinical trials. This promise, however, is not directly translatable to the growing liver, where high rates of hepatocellular proliferation are accompanied by loss of episomal rAAV genomes and subsequently a loss in therapeutic efficacy. We have developed a hybrid rAAV/piggyBac transposon vector system combining the highly efficient liver-targeting properties of rAAV with stable piggyBac-mediated transposition of the transgene into the hepatocyte genome. Transposition efficiency was first tested using an enhanced green fluorescent protein expression cassette following delivery to newborn wild-type mice, with a 20-fold increase in stably gene-modified hepatocytes observed 4 weeks posttreatment compared to traditional rAAV gene delivery. We next modeled the therapeutic potential of the system in the context of severe urea cycle defects. A single treatment in the perinatal period was sufficient to confer robust and stable phenotype correction in the ornithine transcarbamylase-deficient Spf ash mouse and the neonatal lethal argininosuccinate synthetase knockout mouse. Finally, transposon integration patterns were analyzed, revealing 127,386 unique integration sites which conformed to previously published piggyBac data. Conclusion: Using a hybrid rAAV/piggyBac transposon vector system, we achieved stable therapeutic protection in two urea cycle defect mouse models; a clinically conceivable early application of this technology in the management of severe urea cycle defects could be as a bridging therapy while awaiting liver transplantation; further improvement of the system will result from the development of highly human liver-tropic capsids, the use of alternative strategies to achieve transient transposase expression, and engineered refinements in the safety profile of piggyBac transposase-mediated integration. (HEPATOLOGY 2015;62:417-428)

Prevention of Cholestatic Liver Disease and Reduced Tumorigenicity in a Murine Model of PFIC Type 3 Using Hybrid AAV‐piggyBac Gene Therapy

“…To overcome rapid loss of transgene expression following neonatal vector delivery, our previously reported hybrid rAAV‐ piggyBac transposon vector strategy was used with the intent of stabilizing transgene expression by transposition of the expression cassette out of AAV vector episomes and into the host hepatocyte genome before episome loss occurs . Although earlier experiments confirmed the functionality of hABCB4 splice variant B, the phosphatidylcholine floppase activity relative to the two other known hABCB4 splice variants (A and C) had not been previously studied .…”

“…For therapeutic evaluation of the hybrid rAAV‐ piggyBac transposon vector system, a previously reported rAAV construct encoding piggyBac transposase under the transcriptional control of a liver‐specific promoter (pAAV2‐LSP1.PBase) was used to mediate integration of rAAV‐delivered transposons into hepatocyte genomes in vivo . The dose and ratio between transposon and transposase rAAV vectors were based on previously reported work …”

Recombinant Adeno-Associated Viral Integration and Genotoxicity: Insights from Animal Models

“…A recent publication describing a hybrid AAV-piggyBac transposase delivery method might also add important information about whether AAV integration events can cause HCC. 55 Another area of future experimental need surrounds the precise characterization of rAAV integration events. A particularly relevant observation is that both sides of the integration junction have never been captured in any rAAV HCC study.…”

Gene therapy for monogenic liver diseases: clinical successes, current challenges and future prospects

“…Other genome editing tools have been successfully tested with AAV vectors in a neonatal OTC deficiency mouse model. In these experiments, two AAV vectors were injected simultaneously, one of which encoded the transgene and the other the enzymatic system for integration or site specific cutting by Piggybac transposase (Cunningham et al 2015) and CRISPR-Cas9 (Yang et al 2016), respectively.…”