Micro-RNA Reprogrammed Human CD34 Stem Cells for Cardiovascular Disease Therapy

描述（由申请人提供）：有缺陷的内皮祖细胞（EPCs）可能与冠状动脉疾病的进展有关。有缺陷的EPCs和衰老/病变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）的临床试验有限成功的原因。在提案的初步结果部分，我们提出了第一个微！阵列和微！人CD34+/Lin！EPCs：比较冠心病（CAD）和年龄的健康志愿者！匹配非!CAD患者。结果揭晓下来！调节多种血管生成、生长和生存因子，包括VEGF、整合素！1V, Akt，和eNOS在CAD组，>3！miR的表达增加1倍。92a，全球反！靶向整合素的血管生成miR ！1V和Akt， miR！16、靶向VEGF和细胞周期基因CCDN！1和2，还有miR！21，靶向细胞迁移因子和干细胞自我更新，也与内膜形成有关。在这里，我们将验证缺氧组织工程可以有效和安全地解决多种疾病背景下的缺血的假设！表达pro！血管生成生长因子，结合EPC治疗后工程的EPCs选择性微！rna（先导物）和微rna ！RNA抑制剂（安塔戈米）。我们已经创建并测试了一系列独特的AAV载体，表达亲！缺氧指导下的血管生成基因！受管制的、有条件地沉默的发起人。当表达VEGF时，这些载体支持血管定向生长、动脉发生和缺血后肢的稳定再灌注，并在小鼠模型中对AMI具有保护作用。由于存在沉默元件，该载体在生理氧张力下关闭基因表达，因此受到高度调节。我们拟分析伴有和不伴有2型糖尿病的CAD患者不同CD34+和CD133+细胞群中miR的表达，并研究血清因子在调节miR表达中的作用，以及是否可以从药理学上恢复正常的调节和功能。我们将确定EPCs的分子基因工程是否可以恢复EPCs的功能，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EPC治疗。在目标1和目标2中，我们将分析冠心病患者1 T2D EPCs中的miRs、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/疾病背景下，促进血管重建的最佳基因和工程细胞治疗的细胞和分子事件。

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.