Ex Vivo/In vivo Gene Editing in Hepatocytes Using All-in-One CRISPR-Adeno-Associated Virus Vectors with a Self-Linearizing Repair Template

Adeno-assodated virus (AAV)-based vectors are considered efficient and safe gene delivery systems in gene therapy. We combined two guide RNA genes, Cas9, and a self-linearizing repair template in one vector (AIO-SL) to correct fumarylacetoacetate hydrolase (FAH) deficiency in mice. The vector genome of 5.73 kb was packaged into VP2-depleted AAV particles (AAV2/8(Delta V)(P2)), which, however, did not improve cargo capacity. Reprogrammed hepatocytes were treated with AIO-SL.AAV2(Delta V)(P2) and subsequently transplanted, resulting in large clusters of FAH-positive hepatocytes. Direct injection of AIO-SL.AAV8(Delta V)(P2) likewise led to FAH expression and long-term survival. The AIO-SL vector achieved an similar to 6-fold higher degree of template integration than vectors without template self-linearization. Subsequent analysis revealed that AAV8 particles, in contrast to AAV2, incorporate oversized genomes distinctly greater than 5.2 kb. Finally, our AAV8-based vector represents a promising tool for gene editing strategies to correct monogenic liver diseases requiring (large) fragment removal and/or simultaneous sequence replacement.