Optimizing Normal Collagen Replacement in Osteogenesis Imperfecta

描述(申请人提供)：成骨不全(OI)是一种遗传性疾病，其骨骼极其脆弱，极易骨折。大多数OI病例是由I型胶原基因突变引起的，这些突变导致正常胶原蛋白数量减少或三螺旋结构缺陷，导致纤维形成和/或组装异常。OI的疾病谱各不相同，从有宫内骨折/围产儿死亡的严重形式到没有骨折的轻度形式。目前对OI的护理标准是双磷酸盐治疗。然而，最近对这些药物可能抑制骨骼重塑和损害骨折愈合的担忧，以及对双膦酸类药物在儿童中的长期后果缺乏了解，给寻找替代OI疗法带来了新的紧迫性。我们实验室最近培育了一只绿色胶原蛋白小鼠，它的胶原蛋白原2(I)链与绿色荧光蛋白(GFP)融合，并在3.6kb的COL1A1启动子控制下表达，可在成骨细胞中表达。我们的初步数据验证了这种新型的GFP-胶原融合蛋白，它的行为类似于野生型，在添加抗坏血酸的情况下分泌，与胶原共沉淀 链，并组装成带状胶原原纤维阵列。表达GFP-胶原结构的小鼠在骨基质、肌腱、椎间盘和皮肤中显示绿色荧光胶原，并表现正常。这些小鼠为探索全骨髓移植、干细胞移植或诱导多能干细胞(IPS)作为修复OI异常胶原的潜在治疗方法提供了一种新的、强大的新的研究工具。在这个探索性的R21中，我们计划将表达GFP胶原的细胞和骨祖细胞联合移植到代表中重度和轻度OI的两种OI小鼠模型中。其中包括OIM小鼠和G610C小鼠，前者不产生功能性Pro2(I)胶原链，后者在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一个强大的体内读数，不仅可以评估供体细胞的植入，还可以评估供体(GFP阳性)胶原对突变(宿主)骨胶原的替代程度。这些创新性的研究将确定移植细胞中的胶原蛋白随时间的变化可以在多大程度上替代异常的胶原蛋白，并探索通过骨合成代谢和抗吸收药物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。 与公共健康相关：这项研究与公共健康相关，因为它将探索治疗骨骼疾病--成骨不全的新的和/或改进的方法。这是一种遗传性疾病，由于编码骨胶原的基因突变，骨骼极其脆弱，容易骨折。这项研究将使用我们开发的一种新型转基因小鼠，在这种小鼠中，骨胶原被荧光标记为绿色。来自这些“绿色胶原蛋白小鼠”的骨髓和骨髓细胞将被移植到发生成骨不全突变的小鼠体内。我们将确定有多少患病的胶原被绿色胶原取代，并研究药物是否可以促进移植细胞中的绿色胶原取代患病的胶原。

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. PUBLIC HEALTH RELEVANCE: This research is relevant to public health as it will investigate approaches for novel and/or improved treatments for the skeletal disorder, osteogenesis imperfecta. This is an inherited disease in which the bones are extremely brittle and prone to fracture due to mutations in genes that code for bone collagens. The research will use a new transgenic mouse we have developed, in which the bone collagen is fluorescently labeled green. Marrow and bone cells from these "green collagen mice" will be transplanted into mice with osteogenesis imperfecta mutations. We will determine how much of the diseased collagen is replaced by green collagen and investigate whether pharmacological agents can enhance the replacement of diseased collagen with green collagen from the transplanted cells.