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

描述(申请人提供)：有缺陷的内皮祖细胞(EPC)可能导致冠状动脉疾病的进展。自体CD34+骨髓干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也可能是EPC缺陷和老年/患病组织的反应性降低。在该提案的初步结果部分，我们展示了人类CD34+/Lin！的第一个微阵列和微RNA(MiR)图谱。将健康志愿者与冠状动脉疾病(CAD)患者和年龄匹配的非CAD患者进行比较。结果显示，在CAD组中，多种血管生成、生长和生存因子的表达下调，包括血管内皮生长因子、整合素1V、Akt和eNOS，以及以整合素1V和Akt为靶点的全球抗血管生成miR miR的表达增加3倍，以VEGF和细胞周期基因CCDN！1和2为靶标的miR！16和以细胞迁移因子和干细胞自我更新为靶点的miR！21的表达也与新生内膜形成有关。在这里，我们将检验这一假设，即通过组织工程和低氧调控的表达促血管生成因子的AAV9载体，结合EPC治疗，可以在多种疾病背景下有效和安全地解决缺血问题。我们已经创建并测试了一系列独特的AAV载体，它们在低氧调节的、条件沉默的启动子的指导下表达促血管生成基因。当表达血管内皮生长因子时，这些载体支持缺血后肢的定向血管生长、动脉生成和稳定的再灌注，并对小鼠模型的急性心肌梗死具有保护作用。由于存在在生理氧分压下关闭基因表达的沉默元件，这些载体受到高度调控。我们建议分析合并和不合并2型糖尿病的冠心病患者不同CD34+和CD133+细胞群中miR的表达，并探讨血清因素在调节miR表达中的作用，以及正常的调节和功能是否可以从药物上恢复。我们将确定是否可以通过内皮祖细胞的分子基因工程恢复内皮祖细胞的功能，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内皮祖细胞治疗。在AIMS 1和AIMS 2中，我们将描述MIR、它们的靶点和在CAD患者1 T2D内皮祖细胞中的功能，并探索恢复功能的途径。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，促进血管重建的细胞和分子事件，以响应最优的基因和工程细胞治疗。

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.