Abstract 13778: Disease Modelling and Gene Therapy of Hereditary Hemorrhagic Telangiectasia Combined With Pulmonary Artery Hypertension

Abstract

Introduction: Hereditary hemorrhagic telangiectasia combined with pulmonary arterial hypertension(HHT-PAH) caused by pathogenic variants of ACVRL1 gene progresses with poor prognosis. However, there is few treatments, thus developing novel models and innovative therapy is crucial. Combination of iPSCs with gene editing technologies may be a promising applications to illuminate the disease. Hypothesis: Gene editing is a promising therapy for HHT-PAH with ACVRL1 gene mutations. Methods: Patients with HHT-PAH who carrying ACVRL1 heterozygous c.1270C>G mutation were recruited. The reprogramming factors (Oct3/4, Sox2, c-Myc, and Klf4) were transferred into the PBMCs by using non-integrating Sendai virus. The obtained hiPSCs were passaged, individually isolated and expanded 3 weeks after reprogramming factors transduction. The mutated ACVRL1 gene was repaired by CRISPR Base Editing tools to generate ACVRL1 corrected -iPSCs line. Both the patient-specific iPSCs and ACVRL1 corrected -iPSCs were differentiated into induced endothelium. EC adhesion and survival were tested in the endothelial lines. Results: The patient-specific iPSCs showed typical morphology with high alkaline phosphatase activity and normal karyotype (46, XX). Pluripotency markers OCT4, NANOG, SOX2, and TRA-1-60 were confirmed by Immunofluorescence staining and qRT-PCR. Markers of three germ layers, TUBB3+ , αSMA+ and SOX17+ cells were found in the differentiated cell population as revealed by immunostaining in vitro . These two iPSCs cell lines formed teratomas composed of tissues derived from three germ layers in immunodeficient mice. Fluorescence-activated cell sorting analysis showed that most of patient-specific iPSCs and ACVRL1 corrected -iPSCs population expressed the mature EC surface marker CD144 or CD31, and none of cells expressed the early mesoderm marker KDR during the differentiation process. Cell adhesion and survival capacity were preserved in ACVRL1 corrected iPSC-ECs, which were similar to the control, whereas these functions were significantly impaired in patient-specific iPSC-ECs. Conclusions: This study presented an in vitro cell model for exploring the pathogenesis of HHT-PAH and gene editing therapy might be a novel strategy.