Testing the Therapeutic Potential of iPS Cells for Inherited Skin Diseases

测试 iPS 细胞对遗传性皮肤病的治疗潜力

• 批准号: 8896426
• 负责人: Dennis Roop
• 金额: $ 34.59万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-17 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8896426
• 关键词: Address; Alleles; Allogenic; Animal Model; Area; Autologous; Autologous Transplantation; Biological Assay; Biopsy; Bone Marrow; Bulla; Caring; Cell Therapy; Cells; Cessation of life; Child; Clinic; Clinical Data; Clinical Trials; Codon Nucleotides; Collagen Type VII; Complication; Coupled; Data; Derivation procedure; Disadvantaged; Disease; Disease model; Epidermis; Epidermolysis Bullosa; Epidermolysis Bullosa Simplex; Epigenetic Process; Epithelium; Exhibits; Fibroblasts; Freezing; Gene Expression; Generations; Genes; Genetic; Genetically Engineered Mouse; Growth; Harvest; Histocompatibility; Hot Spot; Human; Immune; Immune response; Immune system; Immunocompromised Host; Infection; Inherited; Junctional Epidermolysis Bullosa; Keratin; Laminin; Lesion; Life; Local Therapy; Mediating; Mesenchymal; Methods; Modeling; Molecular Profiling; Mus; Mutation; Natural Selections; Oral cavity; Oral mucous membrane structure; PDGFRB gene; Pain; Patients; Predisposition; Process; Proteins; Protocols documentation; Recruitment Activity; Reporting; Retroviral Vector; Safety; Skin; Skin graft; Somatic Mutation; Source; Stem cells; Stratum Basale; Systemic Therapy; Technology; Testing; Therapeutic; Therapeutic immunosuppression; Tissues; Toxic effect; Transplantation; Tumorigenicity; Variant; Zinc Fingers; adult stem cell; base; chemotherapy; conditioning; design; disability; effective therapy; exhaust; exome sequencing; gene correction; genome editing; homologous recombination; induced pluripotent stem cell; injured; interest; keratin 5; keratinocyte; mouse model; mutant; novel; nuclease; pre-clinical; prevent; repaired; research study; skin disorder; stem cell population; stem cell therapy; transcriptome sequencing; wound

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.

描述（由申请人提供）：大疱性表皮松解症（EB）是一组罕见的遗传性皮肤水疱疾病，会导致严重的水疱和惊吓。 EB 的一些变体会导致患者终生遭受剧烈疼痛和残疾，甚至早逝。尽管导致这些毁灭性疾病的基因缺陷已为人所知近 20 年，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，因此 EB 的永久性纠正疗法必须针对干细胞群。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 患者分离的人类角质形成细胞干细胞中的缺陷基因。针对遗传性皮肤起泡疾病的唯一成功的临床试验利用逆转录病毒载体来恢复缺失蛋白质的表达，但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，我们认为必须探索替代策略。因此，我们建议使用源自同一 EB 患者皮肤细胞的自体诱导多能干细胞 (iPSC) 开发基于干细胞的 EB 疗法。患者特异性 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体干细胞的来源，这些干细胞很可能因修复起泡组织的不成功尝试而耗尽。在对人类测试基于 iPSC 的 EB 干细胞疗法之前，需要利用临床前动物模型来确定这些方法的安全性和有效性。我们之前已经生成了一种诱导型小鼠模型，该模型在基因水平上模拟了最严重形式的单纯性大疱性表皮松解症，Dowling-Meara (EBS-DM)。使用该模型，我们获得了非常令人信服的数据，证明 EBS 角质形成细胞干细胞与野生型角质形成细胞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，如果将经过基因校正的 iPSC 衍生的角质形成细胞接种到容易起水泡的区域，就会出现“自然选择”。因此，由于 EBS 是 EB 的主要形式，经过基因校正的 iPSC 衍生的角质形成细胞不会被患者的免疫系统排斥，因此我们相信 EBS 是生成支持“概念验证”数据的理想模型。使用 iPSC 治疗 EB。 EBS-DM 患者的皮肤和口腔粘膜上皮会出现病变；因此，需要局部和全身治疗来治疗这些患者。 Jakub Tolar 博士是第一个使用系统输送的同种异体骨髓 (BM) 衍生细胞治疗 RDEB 患者进行临床试验的人，因此，他被招募为该多 PI 应用的 PI。尽管托拉尔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，例如移植前调理所需的化疗的毒性，以及由于需要免疫抑制治疗来防止同种异体移植排斥而导致的感染易感性。不幸的是，最初试验中的 7 名 RDEB 儿童中有 2 名因这些并发症死亡。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灭活突变角蛋白 14 (KRT14) 等位基因，该基因在大多数情况下都有缺陷EBS-DM 患者。我们建议使用这些校正后的 iPSC 生成角质形成细胞，通过移植修复皮肤，并生成间充质细胞，系统性修复口腔病变。我们在克服 iPSC 在临床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以及将 iPSC 分化为角质形成细胞和间充质细胞的有效方案，但其他安全性问题仍然存在。在此应用中，我们建议使用新模型来解决 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，以灭活负责约 70 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。还有其他遗传性皮肤病。