EVALUATION OF CELLULAR GENE THERAPY FOR OI

描述（取自应用程序）： 个体发育障碍（Ontogenesis prostituta，0 I）是一组遗传性结缔组织疾病， 其共同特征是骨骼脆弱的组织。大多数形式的OI都是 编码前α 1和前α 2多肽的基因中的突变 I型胶原蛋白链是骨骼的主要蛋白质。的长期目标 该建议是开发使用细胞疗法或基因疗法的策略， 治疗某些形式的骨质疏松症和其他骨相关疾病。的焦点 这项研究计划是利用人类OI（oim）的小鼠模型， proalpha 2（I）链的合成缺陷，以评估 通过骨髓或骨髓移植逆转OI缺陷和其他骨相关疾病 基质细胞移植或将正常胶原基因递送至骨。的 目的：（1）评价骨髓基质细胞的潜能， 将正常供体小鼠移植到同基因OI小鼠体内， 并在受体小鼠的骨基质中存款正常的I型胶原， (2)，通过评估基因治疗的潜力， 用胶原蛋白转导骨髓基质并递送和表达 骨骼中的基因作为前奏，骨髓基质细胞将 通过从股骨和胫骨冲洗骨髓从正常小鼠中建立。 建立的骨髓基质细胞将用逆转录病毒转导， 在移植前含有LacZ和neo基因的载体（BAG-LacZ neo） 将它们植入受体小鼠以帮助细胞追踪。骨髓基质 从正常小鼠建立的细胞将被注射到小鼠的股骨中。 照射或未照射的OI小鼠和proalpha 2（I）的表达 链将通过使用小鼠的免疫荧光定位来确定 α 2（I）抗血清和溴化氰消化的骨胶原蛋白， 受体小鼠检测骨髓基质细胞胶原基因的表达， 细胞，将用含有小鼠的腺病毒转导细胞。 前α 2（I）胶原蛋白基因，并且将转导的细胞注射到 o=s小鼠的股骨。α 2（I）胶原蛋白表达将通过以下方法测定： 使用小鼠proalpha 2（I）和氰的免疫荧光定位 组织的溴化物消化。未来的计划将包括确定 移植细胞在接受者骨骼中产生的胶原蛋白量 通过放射学、组织学和 受体小鼠骨骼的生物力学分析。

DESCRIPTION (Taken from the application): Ontogenesis imperfecta (0I) is a group of heritable disorders of connective tissue whose common feature is bone fragility. Most forms of OI are the result of mutations in the genes that encode proalpha1 and proalpha2 polypeptide chains of type I collagen the major protein of bone. The long-term objective of the proposal is to develop strategies using cell therapy or gene therapy for the treatment of some forms of OI and other bone related diseases. The focus of this research proposal is to utilize a mouse model of human OI (oim) that has defective synthesis of proalpha2(I) chains to evaluate the feasibility of reversing OI defects and other bone related disease by either bone marrow stromal cell transplantation or delivery of normal collagen genes to bone. The aims are: (1) to evaluate the potential of bone marrow stromal cells from normal donor mice transplanted into syngeneic OI mice to engraft, synthesize and deposit normal type I collagen in bone matrix of the recipient mice and (2), to test the feasibility of gene therapy by evaluating the potential of bone marrow stromal to be transduced with collagens and to deliver and express the genes in bone. As a prelude to this, bone marrow stromal cells will be established from the normal mice by flushing the marrow from femurs and tibias. The established bone marrow stromal cells will be transduced with a retroviral vector containing LacZ and neo~ genes (BAG-LacZ neo) prior to transplanting them to the recipient mice to aid in cell tracking. The bone marrow stromal cells established from normal mice will be injected in the femurs of the irradiated or non-irradiated OI mice and the expression of the proalpha2(I) chains will be determined by immunofluorescence localization using a mouse alpha2(I) antiserum and cyanogen bromide digestion of the bone collagen of the recipient mice. To test for the collagen gene expression by bone marrow stromal cells, the cells will be transduced with an adenovirus containing the mouse proalpha2(I) collagen gene and the transduced cells will be injected in the femurs of the o=s mice. The alpha2(I) collagen expression will be determined by immunofluorescence localization using the mouse proalpha2(I) and the cyanogen bromide digestion of the tissue. Future plans will involve determination of the amount of collagen made by the transplanted cells in the bones of the recipient mice and the assessment of the bone quality by radiographic, histological and biomechanical analysis of the bones of the recipient mice.