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 的突变会导致显性成骨不全症，最近，已发现许多不同基因的突变会导致隐性成骨不全症。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物，并共同修饰胶原蛋白肽。 LEPRE1 突变已被确定会导致隐性成骨不全，其中一种突变 IVS5 1G>T 在非裔美国人中被发现，其携带频率高达百分之一到三百分之一。成骨不全的治疗选择有限，移植研究的结果虽然令人鼓舞，但只能实现非常低的植入率。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，并评估基因纠正的干细胞在我们的条件性敲入 OI 小鼠模型中的体内生长潜力。该项目将研究两个独立的目标。目标 1：使用 AAV 基因靶向载体纠正多能干细胞中的 LEPRE1 突变。内源基因的修饰允许其调节受到内部元件的控制，确保适当的基因表达。我们将使用腺相关病毒载体来改变患者特异性细胞中的 LEPRE1 基因座，并使用泡沫病毒载体将目标细胞重新编程为诱导多能干细胞 (iPSC)。这些 iPSC 可用作未来移植实验的可再生干细胞来源。目标 2：开发再生医学 OI 小鼠模型以评估移植参数。人们对干细胞移植和植入骨骼知之甚少。过去的骨移植努力并未取得长期、高水平的移植效果。通过开发我们的条件敲入 Lepre1 OI 小鼠模型，我们将能够确定正常胶原蛋白表达细胞的生长优势、要输送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.