BONE MARROW TOTIPOTENT STEM CELLS & ADENOVIRAL VECTORS

骨髓全能干细胞

• 批准号: 6668355
• 负责人: Shahin Rafii
• 金额: $ 19.94万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6668355
• 关键词: Adenoviridae; angiogenesis factor; biotechnology; cooperative study; cytokine; enzyme activity; enzyme induction /repression; gene therapy; hematopoietic stem cells; laboratory mouse; metalloendopeptidases; stem cell transplantation; stem cells; transfection /expression vector

Hematopoietic stem cells (HSC) and endothelial precursor cells (EPC) provide an invaluable source of cells for autologous and allogeneic transplantation as well as gene therapy for congenital hematological and vascular disorders. The focus of this proposal is the challenge to identify and deliver factors that will allow for efficient in vivo expansion and mobilization for adequate pluripotent HSC and EPC that could be used for transplantation and gene therapy. The strategy is to capitalize on the robust, albeit transient expression mediated by adenovirus (Ad) gene transfer to express stem-cell active chemokines and angiogenic factors that promote extramedullary mobilization of both HSC and EPC. The preliminary data shows that Ad vector mediated in vivo expression of stem cell active cytokines and angiogenic factors with chemotactic potential such as vascular endothelial growth factor (VEGF), stromal derived factor-1 (SDF-1) and Angiopoietin-1 in the peripheral circulation can induce mobilization of HSCs and EPCs. Based on these studies, we hypothesize that regional and temporal expression of secreted and membrane bound angiogenic factors and stem cell active chemocytokines by Ad gene delivery will promote in vivo expansion and mobilization of marrow derived EPCs and HSCs to the peripheral circulation. These mobilized puripotent stem cells may be used for autologous or allogeneic transplantation or gene therapy. On the basis, this project seeks this strategy in the context of moving in to human application. First, we plan to determine whether regional delivery of Ad vectors expressing stem active chemocytokines induced in vivo expansion and mobilization of HSC and EPCs. Studies will be carried out to: 1) investigate whether sufficient, soluble and membrane bound Kit-ligand (Skl, Mkl) AND Flk- 2, alone or in combination can be delivered to marrow using Ad vectors to promote expansion and mobilization of HSCs; and 2) to determine whether sufficient angiogenic factors including soluble VEGF/121, VEGF/165, and matrix bound VEGF/189, placental growth factor (PLGF), Angiopoietin-1 and Angiopoietin-2 could be delivered and produced by Ad vectors to induce proliferation and mobilization of EPCs. Second, we plan to define the mechanism whereby chemocytokines induce mobilization of HSC and EPCs. The studies are planned to 1) evaluate the significance of chemokine-induced metalloproteinase (MMP) activation in the mobilization of stem cells by Ad vectors expressing SDF-1, VEGF isoforms in MMP (MMP-9) knock out mice; and 2) examine the role of endothelial specific adhesion molecules in the regulation of stem cell mobilization by Ad vectors expressing SDF-1, VEGF and angiopoietins in ICAM1, E-selectin and P-selectin knock out mice; and 3) assess the role of chemocytokine modulation of stem cell cycle in the mobilization of HSC and EPCs. Third, assess the efficacy of a novel approach of transplantation ex vivo AD vector transduced hematopoietic cells over-expressing chemocytokines, in mobilization HSC and EPCs. To evaluate this, studies have vector transduced hematopoietic cells over-expressing chemocytokines in mobilization HSC and EPCs. To evaluate this, studies have been designed to: 1) examine whether sufficient hematopoietic cells over-expressing Mkl, Flk-2, SDF- 1, VEGF/165 and VEGF/189 can be transplanted and delivered to the marrow to induce expansion of HSC and EPCs; and 2) assess whether sufficient chemokines can be delivered to the marrow environment by transduced hematopoietic cells to induce mobilization of HSC and EPCs.

造血干细胞（HSC）和内皮前体细胞（EPC）为自体和同种异体移植以及先天性血液和血管疾病的基因治疗提供了宝贵的细胞来源。该提案的重点是识别和递送因子的挑战，这些因子将允许有效的体内扩增和动员足够的多能HSC和EPC，这些HSC和EPC可用于移植和基因治疗。该策略是利用腺病毒（Ad）基因转移介导的强大的，虽然短暂的表达，以表达干细胞活性趋化因子和血管生成因子，促进HSC和EPC的髓外动员。初步数据显示，Ad载体介导外周循环中干细胞活性细胞因子和具有趋化潜力的血管生成因子（如血管内皮生长因子（VEGF）、基质衍生因子-1（SDF-1）和血管生成素-1）的体内表达，可以诱导HSC和EPCs的动员。基于这些研究，我们假设，区域和时间的表达分泌和膜结合的血管生成因子和干细胞活性的趋化细胞因子的Ad基因传递将促进在体内扩增和动员骨髓来源的EPCs和HSC的外周循环。这些动员的纯能干细胞可用于自体或同种异体移植或基因治疗。在此基础上，该项目寻求在人类应用的背景下实现这一战略。首先，我们计划确定表达干细胞活性趋化因子的Ad载体的区域递送是否诱导HSC和EPCs的体内扩增和动员。将进行研究以：1）研究是否存在足够的、可溶的和膜结合的Kit-配体，（Skl，Mkl）和Flk- 2单独或组合可使用Ad载体递送至骨髓以促进HSC的扩增和动员;和2）确定是否有足够的血管生成因子，包括可溶性VEGF/121、VEGF/165和基质结合的VEGF/189，胎盘生长因子（PLGF），腺病毒载体可介导血管生成素-1和血管生成素-2诱导内皮祖细胞的增殖和动员。其次，我们计划确定的机制，使化学细胞因子诱导动员的HSC和EPCs。本研究的目的是：1）在MMP-9基因敲除小鼠中，评价趋化因子诱导的MMP激活在表达SDF-1、VEGF亚型的Ad载体动员干细胞中的意义;和2）检测内皮特异性粘附分子在通过表达ICAM 1中的SDF-1、VEGF和血管生成素的Ad载体调节干细胞动员中的作用，E-选择素和P-选择素敲除小鼠;和3）评估干细胞周期的化学细胞因子调节在HSC和EPCs动员中的作用。第三，评估体外移植AD载体转导的过表达趋化细胞因子的造血细胞的新方法在动员HSC和EPCs中的功效。为了评估这一点，研究了在动员HSC和EPCs中过表达趋化细胞因子的载体转导的造血细胞。为了评估这一点，已经设计了研究以：1）检查是否可以将足够的过表达Mkl、Flk-2、SDF- 1、VEGF/165和VEGF/189的造血细胞移植并递送至骨髓以诱导HSC和EPC的扩增;和2）评估是否可以通过转导的造血细胞将足够的趋化因子递送至骨髓环境以诱导HSC和EPC的动员。