Keratin Gene Targeting for the Treatment of Epidermolysis Bullosa

描述（由申请人提供）：单纯性大疱性表皮病（EBS）是一种使人衰弱的显性遗传性皮肤起泡疾病，未经有效治疗。我们建议通过基因靶向方法纠正EBS患者角质形成细胞的表型，并为自体移植做好准备。该方法允许通过将载体序列靶向插入突变等位基因来破坏产生毒性蛋白的基因的转录。腺相关病毒（AAV）载体在培养物中有效地扩增原代细胞，并且它们的单链DNA基因组已显示与同源染色体序列重组。此外，表达异常角蛋白的角质形成细胞可能处于生长劣势，这使得基因靶向策略成为一种合理的方法，因为赋予表型校正的细胞的生长优势。表达正常和异常角蛋白的细胞的生长差异的概念进一步得到了大疱性表皮病患者中回复突变体嵌合现象的报告的支持，这表明即使是罕见的回复突变或第二位点“抑制”突变也可以是治疗性的。本提案中描述的实验将证明基因靶向作为EBS治疗方法的可行性，并为未来可能导致各种衰弱性皮肤病临床试验的研究奠定基础。 公共卫生相关性：单纯性大疱性表皮病（EBS）是一种极其衰弱的疾病，其特征是从出生开始并持续终生的复发性疼痛性皮肤水疱。这种水泡形成的医疗并发症包括感染、疤痕，甚至死亡。目前没有有效的治疗方法来预防这些水泡。该提案描述了旨在测试用于治疗单纯性大疱性表皮病的基因治疗策略的实验。

DESCRIPTION (provided by applicant): Epidermolysis Bullosa Simplex (EBS) is a debilitating, dominantly inherited skin blistering condition without an effective treatment. We propose to correct the phenotype of EBS-patient keratinocytes and prepare them for autologous transplantation by using a gene targeting approach. This method allows transcription from genes producing toxic proteins to be disrupted by targeted insertion of vector sequences into the mutant allele. Adeno-Associated Virus (AAV) vectors efficiently transduce primary cells in culture, and their single stranded DNA genomes have been shown to recombine with homologous chromosomal sequences. Furthermore, keratinocytes expressing abnormal keratins may be at a growth disadvantage making gene targeting strategies a plausible approach because of the growth advantage conferred to phenotypically corrected cells. The concept of growth differences of cells expressing normal and abnormal keratins is further supported by reports of revertant somatic mosaicism in patients with epidermolysis bullosa suggesting that even infrequent back mutations, or second site "suppressor" mutations can be therapeutic. Experiments described in this proposal will demonstrate the feasibility of gene targeting as a treatment approach for EBS and serve as a foundation for future studies that may lead to clinical trials for a variety of debilitating skin diseases. PUBLIC HEALTH RELEVANCE: Epidermolysis Bullosa Simplex (EBS) is an extremely debilitating disease characterized by recurrent painful skin blisters beginning at birth and continuing throughout life. Medical complications of this blister formation include infection, scarring, and even death. There is currently no effective therapy for preventing these blisters. This proposal describes experiments designed to test gene therapy strategies for the treatment of epidermolysis bullosa simplex.