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）可能导致冠状动脉疾病的进展。有缺陷的EPCs和老化/病变组织的反应性降低也可能是自体CD 34+骨髓基质细胞治疗急性心肌梗死和外周动脉疾病（PAD）的临床试验成功有限的原因。在提案的初步结果部分，我们提出了第一个微！阵列和微型！人CD 34 +/Lin！EPCs，比较健康志愿者与冠心病（CAD）和年龄！匹配非！冠心病患者。结果揭晓！调节多种血管生成、生长和存活因子，包括VEGF、整合素！1V，Akt，eNOS在CAD组，>3！增加一倍的miR！92 a，全球反！针对整合素的血管生成miR！1V和Akt、miR！16，即靶向VEGF和细胞周期基因CCDN！1和2，和miR！21靶向细胞迁移因子和干细胞自我更新，也与新生内膜形成有关。在这里，我们将测试的假设，缺血可以有效地和安全地解决多种疾病背景下的组织工程缺氧！表达pro！血管生长因子联合内皮祖细胞治疗。RNA（premir）和micro！RNA抑制剂（阿托莫西汀）。我们已经创建和测试了一系列独特的AAV载体，表达亲！血管生成基因的方向下缺氧！有条件沉默的启动子。当表达VEGF时，这些载体支持缺血后肢的定向血管生长、动脉生成和稳定再灌注，并且在小鼠模型中对AMI具有保护作用。由于在生理氧张力下关闭基因表达的沉默元件的存在，载体受到高度调节。我们建议分析来自CAD合并和不合并2型糖尿病患者的不同CD 34+和CD 133+细胞群体中的miR表达，并研究血清因子在调节miR表达中的作用，以及是否可以恢复正常的调节和功能。我们将确定是否可以通过EPCs的分子基因工程恢复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EPC治疗。在目的1和2中，我们将分析来自CAD患者1 T2 D的EPC中的miR、其靶标和功能，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/疾病背景中促进血管重建的细胞和分子事件，以响应最佳基因和工程细胞治疗。 公共卫生相关性： 提出研究以确定为什么来自冠状动脉疾病和外周动脉疾病患者的骨髓细胞具有缺陷的功能，并且当它们从患者的骨髓中收集、纯化并输送回同一患者用于干细胞治疗时不能提供有效的治疗。实验提出纠正这些细胞的缺陷，并优化细胞生物工程方案，这将改善外周和冠状动脉疾病患者的干细胞治疗。

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.