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，会导致严重起泡和疤痕。EB的一些变体判处那些患有严重疼痛和残疾甚至过早死亡的人。虽然导致这些毁灭性疾病的遗传缺陷已经被发现了近20年，但目前EB的治疗仅限于伤口护理。由于表皮不断更新的干细胞在增殖基底层，永久性的EB矫正治疗必须针对干细胞群体。迄今为止，还没有人报道成功使用同源重组技术来纠正从EB患者分离的人角质形成细胞干细胞中的缺陷基因。唯一成功的遗传性皮肤起泡病临床试验利用逆转录病毒载体恢复缺失蛋白质的表达，由于使用逆转录病毒载体的安全性问题，该试验被搁置。因此，我们认为必须探索其他战略。因此，我们建议开发基于干细胞的EB治疗方法，使用来自同一EB患者的皮肤细胞的自体诱导多能干细胞（iPSC）。患者特异性iPSC的产生不仅可能避免免疫排斥的并发症，而且还提供了再生成体干细胞的来源，这些干细胞很可能由于修复受损组织的尝试失败而耗尽。在测试用于人类EB的基于iPSC的干细胞疗法之前，期望利用临床前动物模型来确定这些方法的安全性和功效。我们以前已经产生了一个诱导型小鼠模型，模仿最严重的形式单纯性大疱性表皮病，道林-米拉（EBS-DM），在遗传水平。使用该模型，我们已经获得了非常令人信服的数据，证明EBS角质形成干细胞与野生型角质形成干细胞相比表现出生长劣势。这些结果表明，EBS是遗传性皮肤病的一个例子，其中如果将遗传校正的iPSC衍生的角质形成细胞接种到易于起泡的区域中，则会对它们进行"自然选择"。由于这个原因，以及基因校正的iPSC衍生的角质形成细胞不会被患者的免疫系统排斥的事实，因为EBS是EB的主要形式，我们认为EBS是用于生成支持使用iPSC治疗EB的"概念验证"数据的理想模型。患有EBS-DM的患者在皮肤和口腔粘膜上皮中发生病变;因此，需要局部和全身治疗来治疗这些患者。Jakub Tolar博士是第一个使用全身递送的同种异体骨髓（BM）衍生细胞治疗RDEB患者进行临床试验的人，因此，他被招募为这项多PI申请的PI。虽然Tolar博士的初步临床试验非常有希望，但同种异体移植存在安全性问题，例如移植前预处理所需的化疗毒性，以及由于需要免疫抑制治疗以防止同种异体移植排斥而对感染的易感性。不幸的是，初步试验中纳入的7名RDEB儿童中有2名因这些并发症而死亡。为了避免同种异体移植的并发症，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消除突变角蛋白14（KRT14）等位基因，该等位基因在大多数EBS-DM患者中存在缺陷。我们建议使用这些矫正的iPSC来产生角质形成细胞，以通过移植修复皮肤，并产生间充质细胞来全身修复口腔中的病变。我们已经在克服iPSC在临床上安全使用之前所需的一些障碍方面取得了实质性进展，例如开发用于重编程的非病毒方法和将iPSC分化为角质形成细胞和间充质细胞的有效方案，但其他安全问题仍然存在。在本申请中，我们建议使用新型模型来解决iPSC衍生细胞的组织相容性、致瘤性和遗传稳定性等剩余问题。我们还设计了一种ZFN介导的策略来灭活导致~70% 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，还适用于其他遗传性皮肤病。 公共卫生相关性：大疱性表皮病（EB）是一组罕见的遗传性皮肤起泡疾病，导致严重的起泡和疤痕，在某些情况下，早期死亡。目前EB的治疗仅限于伤口护理。我们建议开发基于干细胞的EB治疗方法，使用来自同一EB患者的皮肤细胞的自体诱导多能干细胞（iPSC）。如果本申请中概述的研究取得成功，这些临床前数据将为批准EB患者的iPSC临床试验铺平道路。

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. PUBLIC HEALTH RELEVANCE: Epidermolysis bullosa (EB) is a group of rare inherited skin blistering diseases that result in severe blistering and scaring, and in some cases, early death. Current therapy for EB is limited to wound care. 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. 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 EB patients.