Optimizing Normal Collagen Replacement in Osteogenesis Imperfecta

描述（由申请人提供）：成骨不全（OI）是一种遗传性疾病，其中骨骼非常脆，非常容易骨折。大多数OI病例是由I型胶原基因突变引起的，其导致正常胶原的量减少或三螺旋结构缺陷，导致异常原纤维形成和/或组装。OI的疾病谱从伴有宫内骨折/围产期致死性的严重形式到不伴有骨折的轻度形式不等。目前治疗OI的标准是双膦酸盐治疗。然而，最近对这些药物抑制骨重建和损害骨折愈合的潜力的担忧，以及对儿童双膦酸盐治疗的长期后果缺乏了解，为寻找替代性OI治疗带来了新的紧迫性。我们的实验室最近产生了一个“绿色胶原小鼠”，其中胶原蛋白pro 2（I）链与绿色荧光蛋白（GFP）融合，并在3.6kb COL 1A 1启动子的控制下表达，用于在骨中的成骨细胞中表达。我们的初步数据验证了这种新的<$2（I）-GFP-胶原融合蛋白，表明它的行为与野生型形式相似，在加入抗坏血酸后分泌，与胶原<$1共沉淀， 链，并组装成带状胶原纤维阵列。表达GFP-胶原构建体的小鼠在骨基质、肌腱、椎间盘和皮肤中显示绿色荧光胶原，并且表现出正常的表型。这些小鼠提供了一种新的和强大的新的研究工具，探索全骨髓移植，干细胞或诱导多能干细胞（iPS）作为潜在的治疗方法，用于修复OI中的异常胶原蛋白。在这个探索性的R21中，我们计划将GFP胶原表达细胞和骨祖细胞联合骨髓移植到代表中度严重和轻度形式的OI的两种小鼠模型中。这些包括oim小鼠，它不产生功能性的pro 2（I）胶原蛋白链和G610 C小鼠，它在<$2（I）链中携带半胱氨酸突变。表达GFP-胶原的移植细胞提供了强有力的体内读数，我们不仅可以评估供体细胞的植入，还可以评估供体（GFP-阳性）胶原替代突变（宿主）骨胶原的程度。这些创新研究将确定移植细胞中的胶原蛋白在多大程度上可以取代异常胶原蛋白作为时间的函数，并探索通过骨合成代谢和抗吸收剂治疗来提高胶原蛋白替代程度和数量的潜在方法。这些研究有可能为OI患者带来新的或改进的治疗方法。

DESCRIPTION (provided by applicant): Osteogenesis imperfecta (OI) is a genetic disorder in which the bones are extremely brittle and highly susceptible to fracture. Most cases of OI are caused by mutations in type I collagen genes that result in reduced amounts of normal collagen or structural defects of the triple helix, leading to abnormal fibril formation and/or assembly. Th disease spectrum in OI varies from severe forms with intrauterine fractures/perinatal lethality to mild forms without fractures. The current standard of care for OI is bisphosphonate treatment. However, recent concerns over the potential of these drugs to inhibit bone remodeling and impair fracture healing, as well as the lack of knowledge about the long term consequences of bisphosphonate treatment in children bring new urgency to the search for alternative OI therapies. Our laboratory has recently generated a "green collagen mouse" in which the collagen pro¿2(I) chain is fused with green fluorescent protein (GFP) and is expressed under control of the 3.6kb COL1A1 promoter for expression in osteoblasts in bone. Our preliminary data validating this novel ¿2(I)-GFP-collagen fusion protein have shown that it behaves similarly to the wild type form, is secreted upon addition of ascorbic acid, co-precipitates with collagen ¿1 chains, and is assembled into banded collagen fibril arrays. Mice expressing the GFP-collagen construct show green fluorescent collagen in the bone matrix, tendon, intervertebral discs and skin and appear phenotypically normal. These mice provide a novel and powerful new research tool with which to explore the utility of transplantation of whole marrow, stem cells or induced pluripotent stem cells (iPS) as potential therapies for repair of abnormal collagen in OI. In this exploratory R21, we plan to perform combined bone marrow transplantation of GFPcollagen expressing cells and osteoprogenitors into two mouse models of OI representing moderately severe and mild forms of OI. These include the oim mouse, which does not produce functional pro¿2(I) collagen chains and the G610C mouse, which carries a cysteine mutation in the ¿2(I) chain. The GFP-collagen expressing transplanted cells provide a powerful in vivo readout with which we can assess not only engraftment of donor cells, but also the degree of replacement of mutant (host) bone collagen with donor (GFP-positive) collagen. These innovative studies will determine the extent to which abnormal collagen can be replaced by the collagen from the transplanted cells as a function of time and explore potential approaches to enhance the extent and amount of collagen replacement through treatment with bone anabolic and antiresorptive agents. These studies have the potential to lead to novel or improved treatments for patients with OI.