Keratin Gene Targeting for the Treatment of Epidermolysis Bullosa

描述(申请人提供)：单纯性大疱性表皮松解症(EBS)是一种在没有有效治疗的情况下，以衰弱、显性遗传性皮肤起泡为特征的疾病。我们建议通过基因打靶的方法纠正EBS患者角质形成细胞的表型，并为自体移植做好准备。这种方法允许通过将载体序列定向插入突变的等位基因来扰乱产生有毒蛋白质的基因的转录。腺相关病毒(AAV)载体能有效地转导原代培养的细胞，其单链DNA基因组已被证明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可行的方法，因为表型校正的细胞具有生长优势。大疱性表皮松解症患者的逆转体细胞嵌合体的报道进一步支持了表达正常和异常角蛋白的细胞生长差异的概念，这表明即使是罕见的背部突变或第二位点“抑制”突变也可以起到治疗作用。这项建议中描述的实验将证明基因靶向作为治疗EBS的方法的可行性，并为未来的研究奠定基础，这些研究可能导致对各种衰弱皮肤病的临床试验。

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.