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全（OI）。结构蛋白基因COL1A1和COL1A2的突变导致显性形式的成骨不全，最近，许多不同基因的突变已被确定导致隐性形式的成骨不全。其中一个基因LEPRE1编码一种蛋白质，这种蛋白质与另外两种蛋白质形成复合物，并共同修饰胶原蛋白肽。LEPRE1的突变已被确定可导致隐性OI，其中一种突变IVS5+1G>T已在非裔美国人群中被发现，其携带者频率高达1 / 100至1 / 300。成骨不全的治疗选择有限，移植研究的结果虽然令人鼓舞，但植入率非常低。在这里，我们开发了一种aav介导的基因靶向方法来纠正患者细胞系中突变的LEPRE1基因，并在我们的条件敲入OI小鼠模型中评估基因纠正干细胞的体内生长潜力。这个项目将研究两个独立的目标。目的1：利用AAV基因靶向载体纠正多能干细胞中的LEPRE1突变。内源性基因的修饰允许其调节由内部元件控制，确保适当的基因表达。我们将使用腺相关病毒载体改变患者特异性细胞中的LEPRE1位点，并使用泡沫病毒载体将靶细胞重编程为诱导多能干细胞（iPSCs）。这些多能干细胞可作为干细胞的再生来源，用于未来的移植实验。目的2：建立再生医学成骨不全小鼠模型来评估移植参数。干细胞移植和骨内植入尚不清楚。过去的骨移植工作并没有产生长期的、高水平的植入。通过开发我们的条件敲入Lepre1成骨不全小鼠模型，我们将能够确定正常胶原表达细胞的生长优势，传递的细胞类型，以及改善成骨不全症状所需的移植细胞的百分比。此外，我们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.