Engineering Pluripotent Stem Cells to Treat Osteogenesis Imperfecta

描述(由申请人提供)：遗传性骨病最常见的形式是个体发育不完善(OI)。结构蛋白基因COL1A1和COL1A2的突变导致OI的显性形式，最近，一些不同基因的突变被发现导致隐性形式的OI。其中一个基因LEPRE1编码一种蛋白质，该蛋白质与另外两种蛋白质形成复合体，并共同修饰胶原蛋白多肽。LEPRE1中的突变已被发现导致隐性OI，其中一种突变IVS5+1G&GT；T已在非洲裔美国人群体中被发现，其携带者频率高达1/100到1/300。OI的治疗选择有限，移植研究的结果虽然令人鼓舞，但植入率很低。在这里，我们开发了一种AAV介导的基因打靶方法来纠正患者细胞系中突变的LEPRE1基因，并在我们的条件性敲入OI小鼠模型中评估经基因纠正的干细胞在体内的生长潜力。在这个项目中将调查两个独立的目标。目的1：利用AAV基因打靶载体纠正多能干细胞中的LEPRE1突变。内源性基因的修饰使得它的调节可以由确保适当基因表达的内部因素来管理。我们将使用腺相关病毒载体改变患者特定细胞中的LEPRE1基因，并使用泡沫病毒载体将靶细胞重新编程为诱导多能干细胞(IPSCs)。这些IPSCs可以作为未来移植实验的可再生干细胞来源。目的2：建立再生医学OI小鼠模型，评估移植参数。干细胞移植和植入骨中的情况还知之甚少。过去的骨移植努力并没有产生长期的、高水平的植入。通过建立我们的条件性敲入Lepre1 OI小鼠模型，我们将能够确定正常胶原表达细胞的生长优势、要传递的细胞类型以及改善OI症状所需的移植细胞百分比。此外，我们还将研究转基因干细胞对骨形态的影响。

DESCRIPTION (provided by applicant): The most common form of genetic bone disease is ontogenesis imperfect (OI). Mutations in the structural protein genes COL1A1 and COL1A2 result in dominant forms of OI and recently, mutations in a number of different genes have been identified that result in recessive forms of OI. One of these genes, LEPRE1, encodes a protein that forms a complex with two other proteins and together they modify collagen peptides. Mutations in LEPRE1 have been identified that result in recessive OI, and one of these mutations, IVS5+1G>T, has been found in the African American population at a high carrier frequency of 1 in 100 to 1 in 300. Treatment options for OI are limited and results from transplantation studies, while encouraging, achieved only very low engraftment rates. Here we develop an AAV-mediated gene targeting approach to correct the mutated LEPRE1 gene in patient cell lines, and assess the in vivo growth potential of genetically-corrected stem cells in our conditional, knock-in OI mouse model. Two independent aims will be investigated in this project. Aim 1: To correct LEPRE1 mutations in pluripotent stem cells using AAV gene targeting vectors. The modification of an endogenous gene allows for its regulation to be governed by internal elements ensuring appropriate gene expression. We will use adeno-associated viral vectors to alter the LEPRE1 locus in patient- specific cells and foamy viral vectors to reprogram targeted cells into induced pluripotent stem cells (iPSCs). These iPSCs can be used as a renewable source of stem cells for future transplantation experiments. Aim 2: Develop a regenerative medicine OI mouse model to assess transplantation parameters. Stem cell transplantation and engraftment into bone is poorly understood. Past transplantation efforts into bone have not yielded long-term, high level engraftment. By developing our conditional knock-in Lepre1 OI mouse model, we will be able to determine the growth advantage of normal collagen expressing cells, type of cell to deliver, and percent of engrafted cells needed to improve symptoms of OI. In addition, we will study the effects of genetically modified stem cells on bone morphology.