Micro-RNA reprogrammed human CD34 stem cells for cardiovascular disease therapy

Micro-RNA重编程人类CD34干细胞用于心血管疾病治疗

• 批准号: 8105927
• 负责人: KEITH A WEBSTER
• 金额: $ 39.4万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8105927
• 关键词: Acute myocardial infarction; Age; Animal Model; Apoptosis; Atherosclerosis; Autologous; Back; Biological Assay; Biomedical Engineering; Blood; Blood Vessels; Bone Marrow; Bone Marrow Cells; CD34 gene; Cell Therapy; Cell physiology; Cells; Clinical Trials; Coronary Arteriosclerosis; Cyclin D1; Cytokine Gene; Defect; Dermal; Diabetes Mellitus; Disease; Down-Regulation; Engineered Gene; Engineering; Environment; Event; Failure; Gene Expression; Gene Targeting; Genes; Genetic Databases; Genetic Engineering; Genetic Transcription; Growth; Growth Factor; Human; Hypoxia; In Vitro; Infection; Inflammatory; Integrins; Ischemia; Limb structure; Mediating; MicroRNAs; Modeling; Molecular; Molecular Genetics; Molecular Profiling; Mus; Muscle; Nature; Non-Insulin-Dependent Diabetes Mellitus; Oxygen measurement, partial pressure, arterial; Pathway interactions; Patients; Performance; Peripheral; Peripheral arterial disease; Phosphotransferases; Physiological; Population; Proteins; Protocols documentation; Publishing; RNA; Recommendation; Regulation; Reperfusion Therapy; Reporting; Role; Secondary to; Series; Serum; Skin; Stem Cell Factor; Stem cells; Stimulus; Surgical Flaps; Switch Genes; TNF gene; Testing; Tissue Engineering; Tissues; Transcriptional Silencer Elements; Transfection; Vascular Endothelial Growth Factors; adeno-associated viral vector; age effect; aged; angiogenesis; bone; cardiovascular disorder therapy; cdc Genes; cell motility; cytokine; healthy volunteer; improved; in vivo; inhibitor/antagonist; migration; mouse model; neointima formation; non-diabetic; progenitor; promoter; repaired; research study; response; self-renewal; skeletal; stem cell therapy; success; vector

DESCRIPTION (provided by applicant): Defective endothelial progenitor cells (EPCs) may contribute to the progression of coronary artery disease. Defective EPCs and a reduced responsiveness of aged/diseased tissues may also be reasons for the limited success of clinical trials of autologous CD34+ bone marow cels for the treatment of acute myocardial infarction and peripheral artery disease (PAD). In the Preliminary Results section of the proposal we present the first micro!array and micro!RNA (miR) profiles of human CD34+/Lin! EPCs, comparing healthy volunteers with coronary artery disease (CAD) and age!matched non!CAD patients. The results reveal down!regulation of multiple angiogenic, growth and survival factors including VEGF, integrin!1V, Akt, and eNOS in the CAD group, and >3!fold increased expression of miR!92a, a global anti!angiogenesis miR that targets integrin!1V and Akt, miR!16, that targets VEGF and cell cycle genes CCDN!1 and 2, and miR!21 that targets cell migration factors and stem cell self renewal and has also been associated with neointimal formation. Here we will test the hypothesis that ischemia can be effectively and safely resolved on multiple disease backgrounds by tissue engineering with hypoxia!regulated AAV9 vectors expressing pro!angiogenic growth factors, combined with EPC therapy after engineering the EPCs with selective micro!RNAs (premirs) and micro!RNA inhibitors (antagomirs). We have created and tested a series of unique AAV vectors that express pro!angiogenic genes under the direction of hypoxia!regulated, conditionally silenced promoters. When expressing VEGF, these vectors support directional vessel growth, arteriogenesis and stable reperfusion of ischemic hind limbs, and are protective against AMI in a mouse model. The vectors are highly regulated due to the presence of silencer elements that switch off gene expression under physiological oxygen tension. We propose to profile miR expression in different CD34+ and CD133+ cell populations from CAD patients with and without type 2 diabetes and investigate the role of serum factors in regulating miRs expression, and whether normal regulation and function can be recovered pharmacologically. We will determine whether EPC functions can be recovered by molecular genetic engineering of EPCs, and test engineered EPC therapy in multiple models of ischemia on backgrounds of age and atherosclerosis. In Aims 1 and 2 we will profile miRs, their targets and functions in EPCs from CAD patients 1 T2D and investigate pathways to regain function. In Aim 3 we will characterize the cellular and molecular events that promote revascularization in response to optimal gene and engineered cell therapy in each model of ischemia and each age/disease background. PUBLIC HEALTH RELEVANCE: Studies are proposed to determine why bone marrow cells from patients with coronary artery disease and peripheral artery disease have defective functions and do not provide efficient therapy when they are collected from a patient's bone marrow, purified and delivered back to the same patient for stem cell therapy. Experiments are proposed to correct the defects in these cells and optimize a cellular bioengineering protocol that will improve stem cell therapy for patients with peripheral and coronary artery disease.

描述（由申请人提供）：有缺陷的内皮祖细胞（EPC）可能有助于冠状动脉疾病的进展。 EPC有缺陷的EPC和衰老/患病组织的反应性降低也可能是自体CD34+骨Marow胶在治疗急性心肌梗塞和周围动脉疾病（PAD）临床试验中成功率有限的原因。在提案的“初步结果”部分中，我们介绍了人类CD34+/lin的第一个微型阵列和Micro！RNA（miR）曲线！ EPC比较健康的志愿者与冠状动脉疾病（CAD）和年龄相匹配的非CAD患者。 The results reveal down!regulation of multiple angiogenic, growth and survival factors including VEGF, integrin!1V, Akt, and eNOS in the CAD group, and >3!fold increased expression of miR!92a, a global anti!angiogenesis miR that targets integrin!1V and Akt, miR!16, that targets VEGF and cell cycle genes CCDN!1 and 2, and miR!21 that targets cell migration factors and stem cell自我更新，也与新的形成有关。在这里，我们将测试以下假设：缺血可以通过缺氧的组织工程在多种疾病背景上进行有效，安全地解决。我们已经创建并测试了一系列独特的AAV载体，这些量在缺氧的指导下表达Pro！pros！在表达VEGF时，这些载体支持方向血管的生长，动脉生成和缺血性后肢的稳定再灌注，并且在小鼠模型中对AMI具有保护性。由于存在在生理氧张力下关闭基因表达的消音器元件，因此向量受到了高度调节。我们建议在不同的CAD患者和患有2型糖尿病的CAD患者的不同CD34+和CD133+细胞群中介绍MIR表达，并研究血清因子在调节MIR表达中的作用，以及是否可以通过药理恢复正常调节和功能。我们将确定是否可以通过EPC的分子基因工程来恢复EPC功能，并在多种模型的缺血模型中测试EPC治疗的年龄和动脉粥样硬化。在AIMS 1和2中，我们将介绍MIR，它们的靶标和功能在CAD患者1 T2D的EPC中，并研究恢复功能的途径。在AIM 3中，我们将表征每个缺血模型和每个年龄/疾病背景的最佳基因和工程细胞疗法响应最佳基因和工程细胞疗法的细胞和分子事件。 公共卫生相关性： 提出了研究以确定为什么冠状动脉疾病和周围动脉疾病患者的骨髓细胞具有缺陷的功能，并且当从患者的骨髓中收集，纯化并将其送回同一患者进行干细胞疗法时，不提供有效的治疗。提出了实验来纠正这些细胞中的缺陷，并优化了一种细胞生物工程方案，该方案将改善外围和冠状动脉疾病患者的干细胞疗法。