Testing the Therapeutic Potential of iPS Cells for Inherited Skin Diseases

描述(申请人提供)：大疱性表皮松解症(EB)是一组罕见的遗传性皮肤起泡疾病，导致严重的水泡和结痂。EB的一些变种判处那些遭受严重疼痛和残疾甚至过早死亡的人的生活。尽管导致这些毁灭性疾病的基因缺陷已经知道近20年了，但目前EB的治疗仅限于伤口护理。由于表皮由增殖基底层的干细胞不断更新，EB的永久矫正疗法必须针对干细胞群体。到目前为止，还没有人报道成功地使用同源重组技术来纠正从EB患者中分离的人角质形成干细胞中的缺陷基因。唯一成功的遗传性皮肤起泡疾病临床试验利用逆转录病毒载体恢复缺失蛋白的表达，由于使用逆转录病毒载体的安全性问题，该试验被搁置。为此，我们认为必须探索替代战略。因此，我们建议使用来自同一EB患者皮肤细胞的自体诱导多能干细胞(IPSC)来开发基于干细胞的EB治疗方法。针对患者的IPSC的产生不仅有可能避免免疫排斥的并发症，而且还提供了恢复活力的成人干细胞的来源，这些干细胞很可能是由于修复水泡组织的失败尝试而耗尽的。在测试基于IPSC的干细胞疗法治疗EB之前，最好利用临床前的动物模型来确定这些方法的安全性和有效性。我们之前已经建立了一个可诱导的小鼠模型，在基因水平上模拟最严重的大疱性表皮松解症-道林-米拉(EBS-DM)。使用这个模型，我们已经获得了非常令人信服的数据，证明与野生型角质形成细胞干细胞相比，EBS角质形成干细胞显示出生长劣势。这些结果表明，EBS是一种遗传性皮肤病的例子，如果将经过基因矫正的IPSC来源的角质形成细胞种植到容易起泡的区域，就会对它们进行“自然选择”。因此，由于EBS是EB的主要形式，而且患者的免疫系统不会排斥经过基因纠正的IPSC来源的角质形成细胞，我们认为EBS是生成支持使用IPSC治疗EB的“概念证明”数据的理想模型。EBS-DM患者会出现皮肤和口腔粘膜上皮病变；因此，需要局部和全身治疗来治疗这些患者。Jakub Tole博士是第一个使用系统交付的异基因骨髓(BM)来源的细胞治疗RDEB患者的临床试验，因此，他被招募为这一多PI应用的PI。尽管托勒博士最初的临床试验非常有希望，但异基因移植也存在安全问题，例如对移植前进行调理所需的化疗的毒性，以及由于必须进行免疫抑制治疗以防止异基因移植排斥反应而易受感染。不幸的是，最初试验中的7名RDEB儿童中有2名死于这些并发症。为了避免异基因移植的并发症，我们建议从EBS-DM患者中产生IPSC，并使用锌指核酸酶(ZFN)介导的基因组编辑来灭活突变的角蛋白14(KRT14)等位基因，这在大多数EBS-DM患者中是有缺陷的。我们建议使用这些矫正的IPSC来生成角质形成细胞，通过移植修复皮肤，并使用间充质细胞来系统修复口腔中的损伤。在克服IPSC能够安全应用于临床之前所需的一些障碍方面，我们已经取得了实质性的进展，例如开发了一种非病毒的重新编程方法，以及有效的将IPSC分化为角质形成细胞和间充质细胞的方案，但其他安全问题仍然存在。在这项应用中，我们建议使用新的模型来解决IPSC来源的细胞的组织相容性、致瘤性和遗传稳定性的剩余问题。我们还设计了一种ZFN介导的策略，以灭活导致约70%的EBS-DM病例的“热点突变”。如果本申请中概述的研究成功，这些临床前数据将为批准基于IPSC的临床试验铺平道路，不仅适用于其他形式的EB，也适用于其他遗传性皮肤病。

DESCRIPTION (provided by applicant): Epidermolysis bullosa (EB) is a group of rare inherited skin blistering diseases that result in severe blistering and scaring. Some of the variants of EB sentence those afflicted to a life of severe pain and disability and even early death. Although the genetic defects that cause these devastating diseases have been known for almost 2 decades, current therapy for EB is limited to wound care. Since the epidermis is continuously renewed by stem cells in the proliferative basal layer, a permanent corrective therapy for EB must target the stem cell population. To date, no one has reported the successful use of homologous recombination technology to correct a defective gene in human keratinocyte stem cells isolated from an EB patient. The only successful clinical trial for an inherited skin blistering disease utilized a retroviral vector to restore expression of the missing protein, and that trial was placed on hold because of safety concerns with the use of retroviral vectors. For this reason, we believe that alternative strategies must be explored. Therefore, we are proposing to develop stem-cell based therapies for EB using autologous induced pluripotent stem cells (iPSC) derived from skin cells harvested from the same EB patient. The generation of patient-specific iPSC would not only potentially avoid the complication of immune rejection, but also provide a source of rejuvenated adult stem cells that are most likely exhausted as a result of unsuccessful attempts to repair blistered tissues. Prior to testing an iPSC-based stem cell therapy for EB in humans, it is desirable to utilize a pre-clinical animal model to determine the safety and efficacy of these approaches. We have previously generated an inducible mouse model that mimics the most severe form of epidermolysis bullosa simplex, Dowling-Meara (EBS-DM), at the genetic level. Using this model, we have obtained very compelling data documenting that EBS keratinocyte stem cells exhibit a growth disadvantage compared to wild-type keratinocyte stem cells. These results suggest that EBS is an example of an inherited skin disease where there would be a "natural selection" for genetically corrected iPSC-derived keratinocytes if they were seeded into areas prone to blistering. For this reason, and the fact that genetically corrected iPSC-derived keratinocytes would not be rejected by the patient's immune system, since EBS is a dominant form of EB, we believe that EBS is an ideal model for generating "proof of concept" data supporting the use of iPSC for the treatment of EB. Patients with EBS-DM develop lesions in the skin and the oral mucosal epithelia; therefore, both local and systemic therapy will be required to treat these patients. Dr. Jakub Tolar was the first to perform clinical trials using systemically delivered allogeneic bone marrow (BM)-derived cells in the treatment of RDEB patients, and for that reason, he was recruited as a PI on this multi-PI application. Although Dr. Tolar's initial clinical trials were very promising, there are safety concerns with allogeneic transplants, such as toxicity to chemotherapy required for conditioning before transplantation, and susceptibility to infections due to the necessity for immunosuppressive therapy to prevent rejection of allogeneic transplants. Unfortunately, 2 of the 7 RDEB children included in this initial trial died due to these complications. To avoid the complications of allogeneic transplants, we are proposing to generate iPSC from EBS-DM patients, and use zinc-finger nuclease (ZFN)-mediated genome editing to inactivate the mutant keratin 14 (KRT14) allele, which is defective in the majority of EBS-DM patients. We are proposing to use these corrected iPSC to generate keratinocytes to repair the skin by grafting, and mesenchymal cells to systemically repair lesions in the oral cavity. We have made substantial progress in overcoming some of the obstacles that will be required before iPSC can be used safely in the clinic, such as developing a non-viral method for reprogramming and efficient protocols for differentiating iPSC into keratinocytes and mesenchymal cells, but other safety issues remain. In this application, we are proposing to use novel models to address the remaining issues of histocompatibility, tumorigenicity, and genetic stability of iPSC-derived cells We have also designed a ZFN-mediated strategy to inactivate a 'hot spot mutation" responsible for ~70% of the EBS-DM cases. If the studies outlined in this application are successful, these pre-clinical data will pave the way for approval of iPSC-based clinical trials for not only other forms of EB, but also other inherited skin disorders.