Genomic editing of a pathogenic mutation in ACTA2 rescues multisystemic smooth muscle dysfunction syndrome
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ABSTRACT:
PROVIDER: PRJNA1228008 | ENA |
REPOSITORIES: ENA
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Project description:BACKGROUND: Vascular smooth muscle cells (vSMCs), the predominant cell type in the aortic wall, play a crucial role in maintaining aortic integrity, blood pressure, and cardiovascular function. vSMC contractility and function depend on smooth muscle alpha-actin 2 (ACTA2). The pathogenic variant ACTA2 c.536G>A (p. R179H) causes multisystemic smooth muscle dysfunction syndrome (MSMDS), a severe disorder marked by widespread smooth muscle abnormalities, resulting in life-threatening aortic disease and high risk early mortality from aneurysms or stroke. No effective treatments exist for MSMDS. METHODS: To develop a comprehensive therapy for MSMDS, we utilized CRISPR-Cas9 adenine base editing to correct the ACTA2 R179H mutation. We generated isogenic human induced pluripotent stem cell (iPSC) lines and humanized mice carrying this pathogenic missense mutation. iPSC-SMCs were evaluated for key functional characteristics, including proliferation, migration, and contractility. The adenine base editor (ABE) ABE8e-SpCas9-VRQR under control of either a SMC-specific promoter or a CMV promoter, and an optimized single guide RNA (sgRNA) under control of U6 promoter were delivered intravenously to humanized R179H mice using adeno-associated virus serotype 9 (AAV9) and phenotypic outcomes were evaluated. RESULTS: The R179H mutation causes a dramatic phenotypic switch in human iPSC-SMCs from a contractile to a synthetic state, a transition associated with aneurysm formation. Base editing prevented this pathogenic phenotypic switch and restored normal SMC function. In humanized mice, the ACTA2R179H/+ mutation caused widespread smooth muscle dysfunction, manifesting as decreased blood pressure, aortic dilation and dissection, bladder enlargement, gut dilation, and hydronephrosis. In vivo base editing rescued these pathological abnormalities, normalizing smooth muscle function. CONCLUSIONS: This study demonstrates the effectiveness of adenine base editing to treat MSMDS and restore aortic smooth muscle function. By correcting the ACTA2 R179H mutation, the pathogenic phenotypic shift in SMCs was prevented, key aortic smooth muscle functions were restored, and life-threatening aortic dilation and dissection were mitigated in humanized mice. These findings underscore the promise of gene-editing therapies in addressing the underlying genetic causes of smooth muscle disorders and offer a potential transformative treatment for patients facing severe vascular complications.
2025-07-09 | GSE290426 | GEO
Project description:Multisystem smooth muscle dysfunction syndrome (MSMDS) is a smooth muscle myopathy with major dysfunction in multiple organ systems. Described cases have been exclusively associated with recurrent missense variants at amino acid position 179 in the ACTA2 gene. We describe a patient with multiple major manifestations of MSMDS including steno-occlusive cerebrovascular arteriopathy, prune belly syndrome, gastrointestinal dysmotility, bladder dysfunction, and pupillary hyporeactivity without variation in ACTA2. Whole genome sequencing revealed a single nucleotide variant contained within the seed sequence of MIR145, which encodes the smooth muscle associated microRNA; hsa-miR-145-5p. RNA expression analysis on patient-derived fibroblasts was consistent with actin cytoskeletal dysfunction, and mutant miR-145-5p was unable to mediate cytoskeletal modulation of smooth muscle cells. MSMDS can be caused by a loss-of-function mutation in the smooth muscle differentiation factor, miR-145-5p, the first described non-coding genetic vasculopathy.
2023-12-14 | GSE220038 | GEO
Project description:Smooth muscle cells exist in many different locations within the body, including blood vessels and airways. The heterogeneity of smooth muscle cells has been related to their embryological origins and could have implications in many diseases, including atherosclerosis, pulmonary hypertension, and asthma. Here we aimed to study the heterogeneity of Acta2+ cells in the mouse lung and to identify specific markers for Acta2+ sub-populations. We used a mouse line containing an Acta2hrGFP/Cspg4dsRed reporter and performed single cell RNA sequencing of Acta2+ cells isolated from adult mouse lungs. We identified 3 main distinct clusters, corresponding to airway smooth muscle (ASM), vascular smooth muscle (VSM) and mesenchymal alveolar niche cells (MANCs), and extracted specific gene signatures for each of these cell types. Our dataset and gene signatures help address the heterogeneity of Acta2+ lineages in the lung and will be of use for future studies focusing on smooth muscle cells in the lung and in other organs.
2023-05-08 | GSE203243 | GEO
Project description:Single-cell RNA datasets for aortic tissue from 8-week-old inducible smooth muscle cell-specific knock-in of Acta2 R179C mutation vs. littermate controls and aortic aneurysm tissue from a human patient carrying an ACTA2 R179H variant vs. a pediatric organ donor healthy control.
2023-01-01 | GSE201091 | GEO
Project description:This experiment was performed in order to investigate the difference in the gene expression pattern between differentiated airway/vascular smooth muscle cells and progenitors for alveolar myofibroblasts in embryonic lung. To answer this question, we genetically labeled Smooth muscle actin-expressing cells using Sma-Cre-ERT2 crossed with Tomato Cre-reporter line. This line allows to label both airway/vascular smooth muscle cells (high SMA expression) and progenitors for alveolar myofibroblasts (low SMA expression). Mice were treated with tamoxifen at embryonic day E15.5 and cells from embryonic lungs were collected at E18.5. Mice used in this study were offspring from RjOrl:SWISS (CD1) [mother] x STOCK Tg (Acta2-cre/ERT2)12Pcn Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/sbel (Acta2-CreERT2; tdTomatoflox) [father]
2017-10-04 | GSE87683 | GEO
Project description:Mutations in RNA binding motif protein 20 (RBM20) are a common cause of dilated cardiomyopathy (DCM). Many RBM20 mutations cluster within an arginine/serine rich (RS-rich) domain, resulting in mis-localization of RBM20 to ribonucleoprotein granules within the cytoplasm, abnormal splicing of cardiac genes, and cardiomyocyte dysfunction. We used adenine base editing (ABE) and prime editing to correct pathogenic p.R634Q and p.R636S mutations in the RS-rich domain in human isogenic induced pluripotent stem cell-derived cardiomyocytes. We also created humanized Rbm20R636Q mutant mice, which succumbed to severe cardiac dysfunction, heart failure and premature death. Systemic delivery of ABE components by adeno-associated virus in these mice restored cardiac function and extended life span. These findings demonstrate the potential of precise correction of genetic mutations as a promising therapeutic approach for DCM.
2022-08-14 | GSE211005 | GEO
Project description:Mutations in RNA binding motif protein 20 (RBM20) are a common cause of dilated cardiomyopathy (DCM). Many RBM20 mutations cluster within an arginine/serine rich (RS-rich) domain, resulting in mis-localization of RBM20 to ribonucleoprotein granules within the cytoplasm, abnormal splicing of cardiac genes, and cardiomyocyte dysfunction. We used adenine base editing (ABE) and prime editing to correct pathogenic p.R634Q and p.R636S mutations in the RS-rich domain in human isogenic induced pluripotent stem cell-derived cardiomyocytes. We also created humanized Rbm20R636Q mutant mice, which succumbed to severe cardiac dysfunction, heart failure and premature death. Systemic delivery of ABE components by adeno-associated virus in these mice restored cardiac function and extended life span. These findings demonstrate the potential of precise correction of genetic mutations as a promising therapeutic approach for DCM.
2022-08-11 | GSE210783 | GEO
Project description:In response to myocardial infarction (MI), quiescent cardiac fibroblasts differentiate into myofibroblasts mediating tissue repair in the infarcted area. One of the most widely accepted markers of myofibroblast differentiation is the expression of Acta2 which encodes smooth muscle alpha-actin (SMαA) that is assembled into stress fibers. However, the requirement of Acta2/ SMαA in the myofibroblast differentiation of cardiac fibroblasts and its role in post-MI cardiac repair remained largely unknown. To answer these questions, we generated a tamoxifen-inducible cardiac fibroblast-specific Acta2 knockout mouse line. Surprisingly, mice that lacked Acta2 in cardiac fibroblasts had a normal survival rate after MI. Moreover, Acta2 deletion did not affect the function or overall histology of infarcted hearts. No difference was detected in the proliferation, migration, or contractility between WT and Acta2-null cardiac myofibroblasts. It was identified that Acta2-null cardiac myofibroblasts had a normal total filamentous actin level and total actin level. Acta2 deletion caused a significant compensatory increase in the transcription level of non- Acta2 actin isoforms, especially Actg2 and Acta1, 2 other muscle actin isoforms. Moreover, in myofibroblasts the transcription levels of cytoplasmic actin isoforms were significantly higher than those of muscle actin isoforms. In addition, we found that myocardin-related transcription factor-A is critical for myofibroblast differentiation but is not required for the compensatory effects of non-Acta2 isoforms. In conclusion, the deletion of Acta2 does not prevent the myofibroblast differentiation of cardiac fibroblasts or affect the post-MI cardiac repair, and the increased expression and stress fiber formation of non-SMαA actin isoforms and the functional redundancy between actin isoforms are able to compensate for the loss of Acta2 in cardiac myofibroblasts.
2022-01-10 | GSE193163 | GEO
Project description:Lung smooth muscle cells are including bronchiolar and vascular smooth muscle cells. In order to get adult lung smooth muscle cells, we use transgenic mouse line with smooth muscle actin creERT2, which is a transgenic cre recombinase in Acta2 (contractile smooth muscle cell gene). This mouse line also contains a CAG promoter-driven red fluorescent protein variant (tdTomato) - all inserted into the ROSA26 locus. This mouse line express robust tdTomato fluorescence following cre-mediated recombination after Tamoxifen injection.
2020-05-01 | GSE148008 | GEO
Project description:Pathogenic variants in ACTA2, encoding smooth muscle α-actin, predispose to thoracic aortic aneurysms and dissections. ACTA2 variants altering arginine 179 predispose to a more severe, multisystemic disease termed smooth muscle dysfunction syndrome (SMDS; OMIM 613834). Vascular complications of SMDS include patent ductus arteriosus (PDA) or aortopulmonary window, early-onset thoracic aortic disease (TAD), moyamoya-like cerebrovascular disease, and primary pulmonary hypertension. Patients also have dysfunction of other smooth muscle-dependent systems, including congenital mydriasis, hypotonic bladder, and gut hypoperistalsis. Here, we describe five patients with novel heterozygous ACTA2 missense variants, p.Arg179Gly, p.Met46Arg, p.Thr204Ile, p.Arg39Cys, and p.Ile66Asn, who have clinical complications that align or overlap with SMDS. Patients with the ACTA2 p.Arg179Gly and p.Thr204Ile variants display classic features of SMDS. The patient with the ACTA2 p.Met46Arg variant exhibits exclusively vascular complications of SMDS, including early-onset TAD, PDA, and moyamoya-like cerebrovascular disease. The patient with the ACTA2 p.Ile66Asn variant has an unusual vascular complication, a large fusiform internal carotid artery aneurysm. The patient with the ACTA2 p.Arg39Cys variant has pulmonary, gastrointestinal, and genitourinary complications of SMDS but no vascular manifestations. Identifying pathogenic ACTA2 variants associated with features of SMDS is critical for aggressive surveillance and management of vascular and nonvascular complications and delineating the molecular pathogenesis of SMDS.
| S-EPMC9283281 | biostudies-literature