Correction of a CADASIL point mutation using adenine base editors in hiPSCs and blood vessel organoids

a.
MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China
b.
Key Laboratory of Reproductive Medicine of Guangdong Province, the First Affiliated Hospital and School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China
c.
Center of Reproductive Medicine, Children’s Hospital of Shanxi and Women Health Center of Shanxi, Taiyuan, Shanxi 030013, China

Funds: This study was funded by the National Natural Science Foundation of China (31971365), the Guangdong Basic and Applied Basic Research Foundation (2020B1515120090), the Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program (2019BT02Y276).

摘要

Abstract: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a monogenic small vessel disease caused by mutations in the NOTCH3 gene. However, the pathogenesis of CADASIL remains unclear, and patients have limited treatment options. Here, we use human induced pluripotent stem cells (hiPSCs) generated from the peripheral blood mononuclear cells (PBMCs) of a patient with CADASIL carrying a heterozygous NOTCH3 mutation (c.1261C>T, p.R421C) to develop a disease model. The correction efficiency of different adenine base editors (ABEs) is tested using the HEK293T-NOTCH3 reporter cell line. ABEmax is selected based on its higher efficiency and minimization of predicted off-target effects. Vascular smooth muscle cells (VSMCs) differentiated from CADASIL hiPSCs show NOTCH3 deposition and abnormal actin cytoskeleton structure, and the abnormalities are recovered in corrected hiPSC-derived VSMCs. Furthermore, CADASIL blood vessel organoids generated for in vivo modeling show altered expression of genes related to disease phenotypes, including the downregulation of cell adhesion, extracellular matrix organization, and vessel development. The dual adeno-associated virus (AAV) split-ABEmax system is applied to the genome editing of vascular organoids with an average editing efficiency of 8.82%. Collectively, we present potential genetic therapeutic strategies for patients with CADASIL using blood vessel organoids and the dual AAV split-ABEmax system.

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参考文献(45)

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