利用突变基因修复的人iPSC-MSC细胞改善成骨不全症小鼠异常成骨的研究

Over 90% cases of osteogenesis imperfecta (OI) are caused by dominant mutations in the COL1A1 and COL1A2 genes. A variety of investigations have demonstrated that allogeneic mesenchymal stem cells (MSC) transplantation is able to greatly improve the abnormal bone production. However, to date, there has been no report on the effect of the transplantation of autologous gene-corrected stem cells on bone phenotype in murine OI models. In the preliminary experiments, we confirmed the feasibility of using CRISPR/Cas9-mediated homologous recombination in induced pluripotent stem cells (iPSC). In this proposal, we intent to create human induced pluripotent stem cells (iPSC) carrying COL1A1 gene mutation using the CRISPR/Cas9 genome editing system to model OI. Then, we will use the CRISPR/Cas9 system to correct the mutant COL1A1 gene by homologous recombination. After gene-corrected iPSC differentiate into MSC (iPSC-MSC), they will be transplanted into the murine OI models. The mechanism of iPSC-MSC migration and engraftment and the improvement of bone production will be investigated. Finally, this technique will be used to correct the mutant COL1A1 gene in OI patient-specific iPSC to confirm its feasibility.This investigation may lay the foundation for the utilization of autologous gene-corrected stem cells for the treatment of OI in the future.

90%以上的成骨不全症(OI)是由于I型胶原编码基因COL1A1/ COL1A2突变引起的常染色体显性遗传。众多研究表明，同种异体间充质干细胞(MSC)移植可明显改善OI的异常成骨，但自体突变基因修复干细胞移植治疗OI至今未有报道。在前期工作中，我们已验证所构建的CRISPR/Cas9系统介导的同源重组在诱导性多能干细胞(iPSC)中的可行性。本课题组拟利用CRISPR/Cas9技术构建COL1A1基因突变的人OI模拟iPSC细胞系，并用该技术介导的同源重组修复COL1A1基因的突变，将突变基因修复的iPSC诱导分化为MSC(iPSC-MSC)后移植入OI小鼠体内，对iPSC-MSC示踪分析，研究其体内迁移、定植和改善异常成骨的机制，最后利用该技术修复OI患者iPSC中COL1A1基因的突变，进一步验证其可行性，从而为患者自体干细胞移植治疗OI奠定基础。

成骨不全症(OI)主要是由于I型胶原编码基因COL1A1或COL1A2发生突变而引起的常染色体显性遗传病，目前尚无治愈的手段。众多研究表明，同种异体间充质干细胞(MSCs)移植可明显改善OI的异常成骨，但异体MSCs来源有限、携带病毒/细菌风险及受体需放化疗处理等缺陷限制其发展。因此，本研究主要探索自体突变基因修复的MSCs移植治疗OI的可行性，从而规避上述风险。本课题组成功利用CRISPR/Cas9技术建立了COL1A1基因突变的OI模拟细胞系及OI小鼠模型，并利用同源重组技术修复了OI模拟细胞系突变的COL1A1基因，细胞重新表达结构和功能正常的I型胶原，突变基因修复的自体iPSC-MSCs经尾静脉移植入OI小鼠体内后发现小鼠骨量显著增加，异常成骨明显改善。本项目不仅成功构建了OI模拟细胞系及小鼠模型，为今后OI的病理机制、药物筛选及细胞治疗研究等提供有效的细胞和动物模型，也为利用突变基因修复自体干细胞移植治疗OI的可行性提供了依据，从而为OI干细胞治疗提供一种新思路。本项目研究成果在Biosci Rep及Biochim Biophys Acta Mol Basis Dis等期刊发表论文2篇。