EVALUATION OF CELLULAR GENE THERAPY FOR OI

描述(取自申请表)： 个体发育不全(0I)是一组遗传性结缔组织疾病 其共同特征是骨脆性的组织。大多数形式的OI都是 编码前α1和前α2多肽的基因突变 I型胶原链是骨骼的主要蛋白质。的长期目标是 该提议是开发使用细胞疗法或基因疗法的策略来治疗 治疗某些形式的OI和其他与骨相关的疾病。的关注点 本研究建议利用人类OI(OIM)的小鼠模型，该模型具有 缺陷合成前α2(I)链以评价其可行性 一种骨髓逆转OI缺陷症及其他骨相关疾病 基质细胞移植或将正常的胶原基因输送到骨中。这个 目的是：(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，合成 并在受体小鼠骨基质中沉积正常的I型胶原， (2)，通过评估基因治疗的潜力来测试基因治疗的可行性 胶原蛋白转导骨髓基质及其输送和表达 骨骼中的基因。作为前奏，骨髓基质细胞将 通过冲洗股骨和胫骨的骨髓从正常小鼠建立。 已建立的骨髓基质细胞将被逆转录病毒转导 移植前携带lacZ和neo~基因的载体(Bag-lacz neo) 将它们传递给受体小鼠，以帮助细胞跟踪。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 辐射与非辐射OI小鼠及前α2的表达(I) 链将通过使用小鼠的免疫荧光定位来确定 α2(I)抗血清和溴化氰消化小鼠骨胶原 受体小鼠。骨髓基质细胞胶原基因表达的检测 细胞，这些细胞将被含有小鼠的腺病毒转导 Proalpha2(I)胶原基因和转导细胞将被注射到 O=S小鼠的股骨。α2(I)胶原蛋白的表达将由 用小鼠前α2(I)和氰基进行免疫荧光定位 组织的溴化物消化。未来的计划将涉及确定 移植细胞在受者骨骼中制造的胶原量 小鼠与骨质量的放射学、组织学和骨质量评价 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.