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

• Published in: iScience Vol. 23; no. 1; p. 100764
• Main Authors: Krooss, Simon Alexander, Dai, Zhen, Schmidt, Florian, Rovai, Alice, Fakhiri, Julia, Dhingra, Akshay, Yuan, Qinggong, Yang, Taihua, Balakrishnan, Asha, Steinbrück, Lars, Srivaratharajan, Sangar, Manns, Michael Peter, Schambach, Axel, Grimm, Dirk, Bohne, Jens, Sharma, Amar Deep, Büning, Hildegard, Ott, Michael
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 24.01.2020; Elsevier
• Subjects: Genetic Engineering; Genetics; Techniques in Genetics; Genetics; Techniques in Genetics; Genetic Engineering
• ISSN: 2589-0042; 2589-0042
• DOI: 10.1016/j.isci.2019.100764

Adeno-associated 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 (AAV2/8ΔVP2), which, however, did not improve cargo capacity. Reprogrammed hepatocytes were treated with AIO-SL.AAV2ΔVP2 and subsequently transplanted, resulting in large clusters of FAH-positive hepatocytes. Direct injection of AIO-SL.AAV8ΔVP2 likewise led to FAH expression and long-term survival. The AIO-SL vector achieved an ∼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. [Display omitted] •Single AAV vector mediates efficient large fragment replacement in vivo and ex vivo•Fah-corrected iHeps repopulate the liver of recipient mice•Self-linearizing donor template enhances integration rate•AAV2 and AAV8 reveal differences in packaging the oversized AIO-SL vector genome Genetics; Techniques in Genetics; Genetic Engineering