TESTING GENE THERAPY FOR EPIDERMOLYSIS BULLOSA SIMPLEX

描述（由申请人提供）： 单纯性大疱性表皮病（EBS-DM）的Dowling-Meara变异型是一种以常染色体显性方式遗传的严重水疱性疾病。除了对症治疗外，没有有效的治疗方法可用于EBS。因此，基因治疗是这些患者永久性矫正治疗的唯一选择。在测试用于人类EBS的基因治疗方法之前，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。我们最近产生了一个转基因小鼠模型，在遗传水平上模仿EBS-DM。该小鼠模型与人类疾病的不同之处在于突变型K14等位基因的表达，该突变型K14等位基因含有与大多数EBS-DM患者中发现的Arg 125 Cys突变等同的Arg 131 Cys突变，可以限制在皮肤的小区域。这种小鼠模型为缺乏镶嵌形式的EBS提供了解释。患者的皮肤大多正常，但有斑块的病变皮肤被称为马赛克。马赛克患者已被描述为几种皮肤病，但不是EBS。通过局部应用诱导剂局部激活突变型K14基因导致水疱形成。然而，几周后，水泡愈合，再也没有出现。我们已经证明，突变K14基因在表皮干细胞中被激活。然而，有缺陷的EBS干细胞被从水疱周围未处理区域迁移的正常表皮干细胞所取代。这种小鼠模型预测，如果在胚胎发育过程中形成了EBS皮肤的马赛克补丁，这些有缺陷的EBS表皮干细胞将无法存活，而是被正常的干细胞所取代。这解释了EBS的马赛克形式的缺乏。这一观察结果对EBS的基因治疗方法也有重要意义，因为它表明，如果从患者体内取出EBS干细胞，进行遗传校正，然后返回到受影响的区域，它们将具有选择性生长优势，优于有缺陷的EBS干细胞。进一步感兴趣的是观察到以野生型K14的约50%的水平表达突变K14等位基因的小鼠不能表现出皮肤表型。这表明，只要野生型与突变型K14的比例高于阈值，可能低至2：1，皮肤将具有正常外观并具有完全功能。因此，成功的基因治疗方法可能不需要校正或完全抑制突变等位基因。该提案将使用从EBS-DM小鼠模型中分离的表皮干细胞来测试基于这些新发现的新基因治疗策略。

DESCRIPTION (provided by applicant): The Dowling-Meara variant of epidermolysis bullosa simplex (EBS-DM) is a severe blistering disease inherited in an autosomal-dominant fashion. Besides symptomatic care, no effective therapeutic treatment is available for EBS. Therefore, gene therapy is the only option for a permanent corrective therapy for these patients. Prior to testing gene therapy approaches for EBS in humans, it is desirable to utilize a pre-clinical animal model to determine the safety and efficacy of these approaches. We have recently generated a transgenic mouse model that mimics EBS-DM at the genetic level. This mouse model differs from the human disease in that expression of the mutant K14 allele, which contains an Arg 131 Cys mutation equivalent to the Arg 125 Cys mutation found in the majority of EBS-DM patients, can be restricted to a small area of the skin. This mouse model has provided an explanation for the lack of mosaic forms of EBS. Patients with mostly normal skin that have patches of diseased skin are referred to as mosaics. Mosaic patients have been described for several skin diseases, but not for EBS. Focal activation of the mutant K14 gene by topical application of an inducer results in blister formation. However, after a few weeks, the blister heals and never reappears. We have demonstrated that the mutant K14 gene was activated in epidermal stern cells. However, the defective EBS stem cells were replaced by normal epidermal stem cells that migrate in from the untreated area surrounding the blister. This mouse model predicts that if a mosaic patch of EBS skin formed during development of an embryo, these defective EBS epidermal stem cells would not survive, but be replaced by normal stem cells. This explains the absence of mosaic forms of EBS. This observation also has important implications for gene therapy approaches for EBS, since it suggests that if EBS stem cells were removed from a patient, genetically corrected and then returned to a blistered area, they would have a selective growth advantage over defective EBS stem cells. Of further interest was the observation that mice which express the mutant K14 allele at levels approximately 50% of wild type K14 fail to exhibit a skin phenotype. This suggests that as long as the ratio of wild type to mutant K14 is above a threshold, possibly as low as 2:1, the skin will have a normal appearance and be fully functional. Thus, successful gene therapy approaches may not require correction or complete suppression of the mutant allele. This proposal will use epidermal stem cells isolated from the EBS-DM mouse model to test new gene therapy strategies that are based on these novel findings.