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In Vitro Human Tissue-Engineered Blood Vessel Disease Model of Progeria

早衰症体外人体组织工程血管疾病模型

基本信息

批准号：
10445145
负责人：
George A Truskey
金额：
$ 70.21万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-08-01 至 2026-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10445145
关键词：
20 year old Adenosine Adenovirus Vector Aging Alternative Splicing Apoptosis Architecture Arterial Fatty Streak Arteries Award Biological Assay Biomechanics Blood Vessels CCI-779 Cause of Death Cell Nucleus Cell physiology Cells Cellularity Child Chromatin Structure Clinical Trials Collaborations DNA DNA Sequence Alteration Development Disease Disease model Endothelial Cells Enrollment Epigenetic Process Exhibits Exons Exposure to FDA approved Farnesyl Transferase Inhibitor Fibroblasts Fibrosis First Independent Research Support and Transition Awards Gene Expression Genes Genetic Diseases Guide RNA Human Human Engineering In Vitro Individual Inflammation Inflammatory Lead Lentivirus Lipids Lonafarnib Measures Medial Mediating Mitochondria Mitosis Modeling Mus Mutation Myocardial Infarction NOS3 gene Nuclear Oxidative Stress Pathologic Pathology Patients Penetration Perfusion Periodicity Point Mutation Pre-Clinical Model Process Progeria Proteins RNA RNA Splicing Rare Diseases Recovery SDZ RAD Site Smooth Muscle Myocytes Stimulus Stretching Stroke Structure Syndrome System Testing Therapeutic Time Tissue Engineering Translations Variant Vascular Smooth Muscle Vascular calcification Vasodilation Virus arteriole base editing base editor calcification cell growth cellular engineering disease phenotype dosage effectiveness testing functional restoration genome editing histone methylation human tissue improved functioning in vivo induced pluripotent stem cell mouse model novel novel therapeutics prevent rare genetic disorder response shear stress single-cell RNA sequencing tool transcriptome sequencing

项目摘要

ABSTRACT Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare autosomal dominant disease of accelerated aging. Patients present with vascular stiffening, vascular calcification, and fibrous atherosclerotic plaque formation causing vessel occlusion, which causes death between 7 and 20 years of age due to heart attack or stroke. The disease arises from a point mutation (c.1824C>T) that produces the alternately spliced and farnesylated protein progerin that accumulates in the cell nucleus. Progerin alters gene expression, causing increased oxidative stress, apoptosis, and altered mitochondrial function. Pathological analysis of arteries of HGPS patients shows loss of medial vascular smooth muscle cells (SMCs) and progerin in the medial vascular SMCs, adventitial fibroblasts, and endothelial cells (ECs). While several potential therapeutics have been developed, progress is limited by the few HGPS individuals available to enroll in clinical trials. During the first award period, we developed an arteriole-scale tissue engineered blood vessel (TEBV) model using ECs and SMCs derived from induced pluripotent stem cells (iPSCs) obtained from individuals with HGPS. HGPS TEBVs exhibit the pathology observed in the disease including progerin expression, loss of SMCs, and calcification. HGPS ECs exhibit reduced expression of flow-mediated genes, are pro-inflammatory, and have reduced NOS3 gene expression that prevents TEBV vasodilation. HGPS TEBVs show improved function in response to the farnesyltransferase inhibitor Lonafarnib with or without the rapamycin analogue, Everolimus. In this competing renewal, in collaboration with Dr. David Liu and Dr. Kan Cao we will evaluate the hypotheses that (1) adenosine base editors (ABEs), precision genome editing tools that can directly correct the most common genetic mutation in HGPS, eliminate progerin accumulation in HGPS vascular iPSC-derived ECs (viECs) and SMCs (viSMCs), restoring normal function of individual cells and TEBVs; and (2) functional and genetic changes observed in ABE-treated TEBVs are observed in an HGPS mouse model treated with ABEs. We will examine the extent to which base editing of HGPS viECs and viSMCs restores function and gene expression after biomechanical stimulation. We will evaluate TEBVs made with edited cells for vasoactivity, stiffness, cellularity, EC function, progerin expression, and inflammation to establish if normal function is maintained over 5 weeks. To simulate in vivo conditions, we will perfuse HGPS TEBVs with adenovirus vectors containing guide RNAs and ABEs. We will establish dosage, percent transduction, and measure virus penetration into TEBVs to determine conditions needed to achieve effective correction of vascular pathology in HGPS. Using a mouse G608 HGPS model, we will treat with conditions identified in TEBV studies and compare cellularity, stiffness, and EC inflammation. Single cell RNA-Seq will be used to evaluate the impact of editing on the vascular cells in the mouse vessels and TEBVs after ABE treatment. Results of this study will provide important information to advance ABEs to clinical trials for HGPS and use of ABEs to correct genetic diseases.

摘要