Development of epidermal progenitor cell-based therapy for regenerative medicine

项目摘要 体细胞基因治疗为治疗各种其他疾病提供了一种有前途的治疗方法。 晚期或严重致残的疾病。基因组编辑技术的最新发展，包括 CRISPR（成簇的规则间隔短回文重复序列）系统，已经使得执行 精确的细胞基因工程然而，CRISPR技术在人类患者中的临床应用， 由于使用常规递送方法的体内功效不足，因此具有挑战性。照经上所 迫切需要开发一种离体平台，该平台可以将体外精确基因组编辑与体外精确基因组编辑联合收割机结合起来， 工程细胞在体内的有效应用。 皮肤的表皮祖细胞具有几个独特的优点，使其特别适合于 离体基因治疗。人体皮肤是人体内最大、最容易接触的器官，因此很容易隔离 皮肤表皮祖细胞，并监测组织的潜在有害并发症。从解剖学上讲，皮肤 表皮通过基底膜与脉管系统分离，这防止了 基因修饰的细胞在体内，使潜在的治疗组织特异性和安全。最后，潜力 皮肤基因治疗的适用性是广泛的，因为已经有充分的文献证明， 在皮肤中，表皮细胞可以穿过表皮/真皮屏障并到达循环以达到治疗效果 以系统的方式。此外，皮肤表皮细胞中代谢酶的异位表达可 将工程皮肤转化为“代谢槽”，用于纠正各种代谢紊乱。然而，在这方面， 尽管具有潜在的临床重要性，但基于表皮祖细胞的治疗（皮肤基因治疗）的研究 治疗）由于缺乏合适的小鼠模型而受到极大阻碍。虽然老鼠或人的皮肤 可以移植到免疫缺陷的小鼠身上，但由于缺乏完整的免疫系统， 治疗可能在体内引起的潜在结果和并发症。我们现在已经解决了 技术障碍，并建立了一个独特的小鼠对小鼠皮肤移植模型，可以稳定地引入 将基因组编辑的表皮祖细胞植入免疫活性小鼠。在这个提议中，我们将利用 并探讨皮肤基因治疗治疗的可行性和临床潜力， 遗传性疾病，包括苯丙酮尿症（PKU）和血友病A。我们的研究将建立一个 目前基因组编辑技术的皮肤基因治疗的独特和强大的模型，揭示了 表皮祖细胞体细胞基因治疗的治疗潜力。

Project Summary Somatic gene therapy provides a promising therapeutic approach for treatment of a variety of otherwise terminal or severely disabling diseases. The recent development of genome editing technology, including CRISPR (clustered regularly-interspaced short palindromic repeats) system, has made it possible to perform precise genetic engineering in cells. However, clinical application of CRISPR technology to human patients has been challenging due to the inadequate efficacy in vivo using conventional delivery approach. Thus, it is urgently needed to develop an ex vivo platform that can combine both precise genome editing in vitro with effective application of engineered cells in vivo. The epidermal progenitor cells of skin have several unique advantages, making it particularly suited for ex vivo gene therapy. Human skin is the largest and most accessible organ in the body, making it easy to isolate skin epidermal progenitor cells and monitor the tissue for potential detrimental complications. Anatomically, skin epidermis is separated from vasculature by the basement membrane, which prevents potential dissemination of genetically modified cell in vivo, making the potential therapy tissue specific and safe. Lastly, the potential applicability of cutaneous gene therapy is broad because it has been well documented that proteins expressed in skin epidermal cells can cross the epidermal/dermal barrier and reach circulation to achieve therapeutic effect in a systematic manner. In addition, ectopic expression of metabolic enzymes in skin epidermal cells can transform the engineered skin into a “metabolic sink” for correction of various metabolic disorders. However, despite the potential clinical importance, research in epidermal progenitor cell-based therapy (cutaneous gene therapy) has been greatly hindered due to lack of an appropriate mouse model. Although mouse or human skin can be transplanted to immunodeficient mice, lack of an intact immune system makes it impossible to examine the potential outcomes and complications that the therapy may elicit in vivo. We have now resolved the technical hurdle and established a unique mouse-to-mouse skin transplantation model that can stably introduce genome-edited epidermal progenitor cells into immunocompetent mice. In this proposal, we will take advantage of this novel platform and explore the feasibility and clinical potential of cutaneous gene therapy for treatment of genetic diseases, including phenylketonuria (PKU) and hemophilia A. Together, our studies will establish a unique and powerful model for cutaneous gene therapy with current genome editing technology, revealing the therapeutic potential for somatic gene therapy with epidermal progenitor cells.