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: 8527943
• 负责人: KEITH A WEBSTER
• 金额: $ 6.66万
• 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/8527943
• 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.

描述（由申请人提供）：有缺陷的内皮祖细胞（EPC）可能会导致冠状动脉疾病的进展。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+ 骨髓细胞治疗急性心肌梗死和外周动脉疾病 (PAD) 临床试验成功有限的原因。在该提案的初步结果部分，我们展示了人类 CD34+/Lin 的第一个微阵列和 microRNA (miR) 图谱！ EPC，将患有冠状动脉疾病 (CAD) 的健康志愿者与年龄匹配的非 CAD 患者进行比较。结果揭示了 CAD 组中多种血管生成、生长和存活因子的下调，包括 VEGF、整合素！1V、Akt 和 eNOS，并且 miR!92a 的表达增加了 3 倍以上，miR!92a 是一种靶向整合素!1V 和 Akt 的全局抗血管生成 miR，靶向 VEGF 和细胞周期基因 CCDN!1 和 2 的 miR!16，以及靶向细胞迁移因子和干细胞的 miR!21 自我更新也与新内膜形成有关。在这里，我们将测试以下假设：通过使用表达促血管生成生长因子的低氧调节 AAV9 载体进行组织工程，并在用选择性 microRNA（premirs）和 microRNA 抑制剂（antagomirs）工程化 EPC 后结合 EPC 治疗，可以在多种疾病背景下有效且安全地解决缺血。我们已经创建并测试了一系列独特的AAV载体，其在缺氧调节、条件性沉默启动子的指导下表达促血管生成基因。当表达 VEGF 时，这些载体支持定向血管生长、动脉生成和缺血后肢的稳定再灌注，并且在小鼠模型中具有针对 AMI 的保护作用。由于沉默元件的存在，这些载体在生理氧张力下关闭基因表达，因此受到高度调控。我们建议对患有和不患有2型糖尿病的CAD患者的不同CD34+和CD133+细胞群中的miR表达进行分析，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学恢复正常的调节和功能。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，并在年龄和动脉粥样硬化背景下的多种缺血模型中测试工程化的 EPC 疗法。在目标 1 和 2 中，我们将分析 CAD 患者 1 T2D 的 EPC 中的 miR、它们的靶点和功能，并研究恢复功能的途径。在目标 3 中，我们将描述在每种缺血模型和每种年龄/疾病背景中响应最佳基因和工程细胞治疗而促进血运重建的细胞和分子事件。