EVALUATION OF CELLULAR GENE THERAPY FOR OI

成骨不全症细胞基因疗法的评估

• 批准号: 6375351
• 负责人: CHRISTOPHER NIYIBIZI
• 金额: $ 7.26万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-18 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6375351
• 关键词: bone marrow; cell transplantation; collagen; gene mutation; gene therapy; immunofluorescence technique; laboratory mouse; musculoskeletal imaging /visualization /scanning; nonhuman therapy evaluation; osteogenesis imperfecta; protein biosynthesis; protein localization

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.

描述（摘自应用程序）： 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。大多数成骨不全症的结果都是 编码 proalpha1 和 proalpha2 多肽的基因突变 I 型胶原蛋白链是骨的主要蛋白质。长期目标 该提案是制定使用细胞疗法或基因疗法的策略 治疗某些形式的成骨不全症和其他骨骼相关疾病。重点是 这项研究计划是利用人类成骨不全症 (oim) 小鼠模型，该模型具有 有缺陷的 proalpha2(I) 链合成以评估可行性 通过任一骨髓逆转成骨不全缺陷和其他骨相关疾病 基质细胞移植或将正常胶原蛋白基因递送至骨骼。这 目的是：（1）评估骨髓基质细胞的潜力 将正常供体小鼠移植到同基因 OI 小鼠中进行植入、合成 并在受体小鼠的骨基质中沉积正常的 I 型胶原蛋白， (2)、通过评估基因治疗的潜力来测试基因治疗的可行性 骨髓基质被胶原蛋白转导并输送和表达 骨骼中的基因。作为这一切的前奏，骨髓基质细胞将被 通过冲洗股骨和胫骨的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 移植前含有 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追踪。骨髓基质 将正常小鼠建立的细胞注射到小鼠的股骨中 受辐射或未受辐射的 OI 小鼠以及 proalpha2(I) 的表达 将使用小鼠通过免疫荧光定位来确定链 α2(I)抗血清和溴化氰消化骨胶原 受体小鼠。检测骨髓基质中胶原蛋白基因的表达 细胞，细胞将被含有小鼠的腺病毒转导 proalpha2(I) 胶原蛋白基因和转导细胞将被注射到 o=s 小鼠的股骨。 α2(I) 胶原表达将通过以下方式确定 使用小鼠 proalpha2(I) 和氰进行免疫荧光定位 组织的溴化物消化。未来的计划将涉及确定 受者骨骼中移植细胞产生的胶原蛋白量 小鼠的骨质量通过放射学、组织学和 对受体小鼠的骨骼进行生物力学分析。