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 rem...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 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.展开更多
More than 32,000 pathogenic single nucleotide polymorphisms(SNPs)have been identified in the human genome(Gaudelli et al.,2017).Genetically modified mice with pathogenic SNPs are good models for studies of disease pat...More than 32,000 pathogenic single nucleotide polymorphisms(SNPs)have been identified in the human genome(Gaudelli et al.,2017).Genetically modified mice with pathogenic SNPs are good models for studies of disease pathogenesis and the development of new therapeutics.Accordingly,an efficient,high-throughput method for the generation of mouse models with SNPs is needed.展开更多
基金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).
文摘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 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.
基金supported by the National Key R&D Program of China(2017YFC1001901,2017YFA0102801 and 2017YFC1001603)the National Natural Science Foundation of China(91640119,31671540,81330055 and 31601196)+3 种基金the Guangdong Special Support Program(2019BT02Y276)the Natural Science Foundation of Guangdong Province(2016A030310206 and 2014A030312011)the Science and Technology Planning Project of Guangdong Province(2015B020228002)the Guangzhou Science and Technology Project(201707010085 and 201803010020)。
文摘More than 32,000 pathogenic single nucleotide polymorphisms(SNPs)have been identified in the human genome(Gaudelli et al.,2017).Genetically modified mice with pathogenic SNPs are good models for studies of disease pathogenesis and the development of new therapeutics.Accordingly,an efficient,high-throughput method for the generation of mouse models with SNPs is needed.
