CELL AND GENE THERAPY IN A MOUSE MODEL OF HUMAN OSTEOGEN

成骨不全(OI)是一组遗传性结缔组织疾病，其共同特征是骨脆性。大多数形式的OI是编码I型胶原前α1和前α2多肽链的基因突变的结果，I型胶原是骨骼的主要蛋白质。本建议的长期目标是测试使用细胞疗法或基因疗法治疗某些形式的OI和其他与骨骼相关的疾病的可行性。该方案的主要重点是利用人OI III型小鼠模型，该模型具有前α2(I)链的合成缺陷，以评估通过骨髓基质细胞移植或利用骨髓基质细胞向骨传递治疗性基因来逆转OI缺陷的可行性。本研究的目的是：(1)评价正常供体小鼠骨髓基质细胞移植到同种异体OI小鼠体内合成和沉积正常I型胶原的能力；(2)证实转导胶原基因的骨髓基质细胞能在骨中定位和表达，从而验证基因治疗的可行性。作为前奏，正常小鼠的骨髓将通过冲洗股骨和胫骨来建立骨髓基质细胞。建立的骨髓基质细胞将被含有LacZ和neo基因的逆转录病毒载体(BAG-LacZ Neo)转导，然后将它们移植到受体小鼠身上，以帮助细胞跟踪。从正常小鼠建立的骨髓基质细胞将被注射到辐射或非辐射OI小鼠的股骨中，并将用小鼠α2(I)抗血清和溴化氰消化受体小鼠的骨胶原通过免疫荧光定位来确定前α2(I)链的表达。为了检测骨髓基质细胞在骨中胶原基因的表达情况，将转小鼠proalpha2(I)基因的骨髓基质细胞注射到OIM小鼠的股骨中。受体小鼠体内的转导细胞的存在将通过聚合酶链式反应进行评估，基因的表达将通过骨基质中α2(I)链的免疫荧光定位来确定。未来的计划将包括确定移植细胞在受体小鼠骨骼中产生的胶原蛋白的数量，并通过对受体小鼠骨骼的放射学、组织学和生物力学分析来评估骨骼质量。

Osteogenesis imperfecta (OI) 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 present proposal is to test the feasibility of using cell therapy or gene therapy for some forms of OI and other bone related diseases. The major focus of the proposal is to utilize a mouse model of human OI type III that has defective synthesis of proalpha2(I) chains to evaluate the feasibility of reversing OI defects by bone marrow stromal cell transplantation or by use of bone marrow stromal cells to deliver therapeutic 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 synthesize and deposit normal type I collagen in bone matrix of the recipient mice and (2), to test the feasibility of gene therapy by demonstrating that bone marrow stromal cells transduced with collagen genes will home to bone 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) anti-serum and cyanogen bromide digestion of the bone collagen of the recipient mice. To test for the gene expression of collagen genes in bone by bone marrow stromal cells, bone marrow stromal cells transduced with the mouse proalpha2(I) cDNA will be injected in the femurs of the oim mice. The presence of the transduced cells in the recipient mice will be assessed by PCR and gene expression will be determined by immunofluorescence localization of the alpha2(I) chain in bone matrix. Future plans will involve determining 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.