文章基本信息

标题：Ex Vivo/ In vivo Gene Editing in Hepatocytes Using “All-in-One” CRISPR-Adeno-Associated Virus Vectors with a Self-Linearizing Repair Template
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作者：Simon Alexander Krooss ; Zhen Dai ; Florian Schmidt 等
期刊名称：iScience
印刷版ISSN：2589-0042
出版年度：2020
卷号：23
期号：1
页码：1-30
DOI：10.1016/j.isci.2019.100764
语种：English
出版社：Elsevier
摘要：SummaryAdeno-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.AAV2ΔVP2and subsequently transplanted, resulting in large clusters of FAH-positive hepatocytes. Direct injection of AIO-SL.AAV8ΔVP2likewise 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.Graphical AbstractDisplay OmittedHighlights•Single AAV vector mediates efficient large fragment replacementin vivoandex 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 genomeGenetics; Techniques in Genetics; Genetic Engineering