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Ex Vivo/In vivo Gene Editing in Hepatocytes Using "All-in-One" CRISPR-Adeno-Associated Virus Vectors with a Self-Linearizing Repair Template.
Krooss, Simon Alexander Dai, Zhen Schmidt, Florian Rovai, Alice Fakhiri, Julia Dhingra, Akshay Yuan, Qinggong Yang, Taihua Balakrishnan, Asha Steinbrück, Lars
Krooss, Simon Alexander
Dai, Zhen
Schmidt, Florian
Rovai, Alice
Fakhiri, Julia
Dhingra, Akshay
Yuan, Qinggong
Yang, Taihua
Balakrishnan, Asha
Steinbrück, Lars
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2020-01-24
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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 (AAV2/8ΔVP2), 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.
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iScience. 2020 Jan 24;23(1):100764. doi: 10.1016/j.isci.2019.100764. Epub 2019 Dec 12.
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en
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2589-0042
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Attribution-NonCommercial-ShareAlike 4.0 International