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Published on: September 25, 2019
Correction of a CADASIL point mutation using adenine base editors in hiPSCs and blood vessel organoids
Jingwen Wang1, Lei Zhang2, Guanglan Wu3
1MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong 510275, China; Key Laboratory of Reproductive Medicine of Guangdong Province, The First Affiliated Hospital and School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong 510275, China.
Insights
Researchers developed a cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) model using patient stem cells. They demonstrated gene editing in blood vessel organoids, offering potential genetic therapies for this rare brain disorder.
Area of Science:
- Neuroscience
- Genetics
- Stem Cell Biology
Background:
- Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a genetic small vessel disease linked to NOTCH3 gene mutations.
- The exact disease mechanisms of CADASIL are not fully understood, and effective treatments are limited.
Purpose of the Study:
- To establish a human induced pluripotent stem cell (hiPSC)-based disease model for CADASIL.
- To investigate potential genetic therapeutic strategies for CADASIL using advanced gene editing techniques.
Main Methods:
- Generated hiPSCs from a CADASIL patient with a specific NOTCH3 mutation.
- Differentiated hiPSCs into vascular smooth muscle cells (VSMCs) and created blood vessel organoids.
- Tested and optimized adenine base editors (ABEs), selecting ABEmax for its efficiency and safety.
- Applied a dual adeno-associated virus (AAV) split-ABEmax system for genome editing in vascular organoids.
Main Results:
- CADASIL hiPSC-derived VSMCs exhibited NOTCH3 deposition and abnormal actin cytoskeleton, which were corrected post-editing.
- CADASIL blood vessel organoids showed altered gene expression related to vessel development and extracellular matrix organization.
- The dual AAV split-ABEmax system achieved an average editing efficiency of 8.82% in vascular organoids.
Conclusions:
- hiPSC-derived vascular cells and organoids serve as a valuable model for studying CADASIL pathogenesis.
- The dual AAV split-ABEmax system shows promise for in vivo genome editing in CADASIL models.
- This study presents viable genetic therapeutic strategies for CADASIL patients.
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 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.
