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

Simon Alexander Krooss; Zhen Dai; Florian Schmidt; Alice Rovai; Julia Fakhiri; Akshay Dhingra; Qinggong Yuan; Taihua Yang; Asha Balakrishnan; Lars Steinbrück; Sangar Srivaratharajan; Michael Peter Manns; Axel Schambach; Dirk Grimm; Jens Bohne; Amar Deep Sharma; Hildegard Büning; Michael Ott

doi:10.1016/j.isci.2019.100764

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

iScience. 2020 Jan 24;23(1):100764. doi: 10.1016/j.isci.2019.100764. Epub 2019 Dec 12.

Authors

Simon Alexander Krooss¹, Zhen Dai², Florian Schmidt³, Alice Rovai⁴, Julia Fakhiri⁵, Akshay Dhingra⁶, Qinggong Yuan⁴, Taihua Yang⁷, Asha Balakrishnan⁴, Lars Steinbrück⁶, Sangar Srivaratharajan⁶, Michael Peter Manns⁸, Axel Schambach⁹, Dirk Grimm¹⁰, Jens Bohne⁶, Amar Deep Sharma², Hildegard Büning¹¹, Michael Ott¹²

Affiliations

¹ Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany; Twincore Centre for Experimental and Clinical Infection Research, Hannover, Germany; Institute for Virology, Hannover Medical School, Hannover, Germany.
² Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany; Junior Research Group MicroRNA in Liver Regeneration, Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, Germany.
³ Bioquant, University of Heidelberg, Heidelberg, Germany; German Center for Infection Research (DZIF), and German Center for Cardiovascular Research (DZHK), Partner Site Heidelberg, Hannover, Germany.
⁴ Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany; Twincore Centre for Experimental and Clinical Infection Research, Hannover, Germany.
⁵ Bioquant, University of Heidelberg, Heidelberg, Germany; Center for Infectious Diseases/Virology, Cluster of Excellence Cell Networks, Heidelberg University Hospital, Heidelberg, Germany.
⁶ Institute for Virology, Hannover Medical School, Hannover, Germany.
⁷ Junior Research Group MicroRNA in Liver Regeneration, Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, Germany.
⁸ Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany.
⁹ Institute for Experimental Hematology, Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, Germany.
¹⁰ Bioquant, University of Heidelberg, Heidelberg, Germany; German Center for Infection Research (DZIF), and German Center for Cardiovascular Research (DZHK), Partner Site Heidelberg, Hannover, Germany; Center for Infectious Diseases/Virology, Cluster of Excellence Cell Networks, Heidelberg University Hospital, Heidelberg, Germany.
¹¹ Institute for Experimental Hematology, Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, Germany; German Center for Infection Research (DZIF), Partner Site Hannover-Braunschweig, Hannover, Germany.
¹² Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany; Twincore Centre for Experimental and Clinical Infection Research, Hannover, Germany. Electronic address: ott.michael@mh-hannover.de.

Abstract

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 (AAV2/8^ΔVP2), which, however, did not improve cargo capacity. Reprogrammed hepatocytes were treated with AIO-SL.AAV2^ΔVP2 and subsequently transplanted, resulting in large clusters of FAH-positive hepatocytes. Direct injection of AIO-SL.AAV8^ΔVP2 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.

Keywords: Genetic Engineering; Genetics; Techniques in Genetics.