OSTEOBLASTIC STEM CELLS AS GENE THERAPY

描述(改编自申请人的摘要)：兴趣正在形成 认为体细胞基因治疗是治疗人类遗传性疾病的一种方法 成骨细胞如成骨不全细胞(OI)。重大问题 可能是骨骼和其他结缔组织细胞所特有的，需要严重 在制定一个有说服力的战略之前，要先考虑一下。具体地说，它 需要确定是否有骨干细胞可以 靶向，将产生分化的成骨细胞的祖细胞池 表达纠正基因。五个基本问题将被问到 本应用利用携带标记基因的转基因供体小鼠 可以指示来源和分化状态的细胞 人工操纵的干细胞：(1)更新的成骨细胞是否自然产生于 循环干细胞，组织驻留干细胞，还是两者兼而有之？ 老年人骨强度明显正常的分子和细胞学基础 哪些患者是导致OI的突变的体细胞嵌合体？对于干细胞来说 要想治疗成功，工程细胞需要进入自然池 循环细胞充填OI骨，取代OI的功能 细胞和减轻疾病的严重程度。这些问题将通过以下方式得到解答 来自不同转基因胚胎供者的异种小鼠；(3) 即使干细胞不能正常循环，干细胞也能 植入骨骼并产生分化的成骨细胞？MSC将在 并用于异位和髓内骨形成的实验 和干细胞数量。一只携带TK基因的小鼠，由 COL1A1启动子，将用于评估移植的MSC的能力 植入骨并参与骨形成；(4)和(5)间充质干细胞能否 用逆转录病毒载体操纵，逆转录病毒载体包含无处不在的 COL1A1启动子是否仍能保持其归巢、造骨的能力？ 这个项目代表了三个主要调查人员的融合，带来了 不同的，但相互作用的学科来处理复杂的 骨髓间充质干细胞作为细胞成分的可行性 骨骼的体细胞基因治疗。

DESCRIPTION (Adapted from the Applicant's Abstract): Interest is building to consider somatic gene therapy as an approach to heritable diseases of the osteoblast such as osteogenesis imperfecta (OI). Significant problems which may be unique to bone and other connective tissue cells need serious consideration before a cogent strategy can be formulated. Specifically, it needs to be determined whether there is a bone stem cell that can be targeted which will yield a progenitor pool of differentiated osteoblasts expressing the correcting gene. Five fundamental questions will be asked by this application, utilizing transgenic donor mice bearing marker genes which can indicate the source and the differentiation state of cells derived from a manipulated stem cell: (1) do renewing osteoblasts naturally arise from a circulating stem cell, a tissue resident stem cell, or both?; (2) what is the molecular and cellular basis of apparently normal bone strength in patients who are somatic mosaics for a mutation causing OI? For stem cell therapy to be successful, engineered cells will need to enter a natural pool of circulating cells to populate OI bone, replace the function of the OI cells and ameliorate disease severity. These issues will be answered with allophenic mice derived from embryo donors bearing different transgenes; (3) even if stem cells do not normally circulate, can stem cells be made to populate bone and yield differentiated osteoblasts? MSC will be expanded in vitro and used in a heterotopic and intramedullary assay of bone formation and stem cell number. A mouse engineered with the TK gene, controlled by the COL1A1 promoter, will be used to assess the ability of transplanted MSC to engraft bone and participate in bone formation; (4) and (5) can MSC be manipulated with retroviral vectors containing either ubiquitous, or a COL1A1 promoter and still maintain its ability to home to, and make bone? This project represents a fusion of three principal investigators, bringing different, but interacting disciplines to the complex question of feasibility of MSCs as the cellular ingredient in an overall strategy of somatic gene therapy of bone.