IN VIVO CUTANEOUS GENE THERAPY

本研究的长期目标是开发遗传性皮肤病的皮肤基因治疗。目前，最受欢迎的方法是离体基因转移，然后将基因修饰的细胞移植回供体。该手术成本高，耗时长，并且需要在移植部位进行全层切除。此外，这种手术会导致疤痕和挛缩，并最终限制可以治疗的区域。优选的替代方案是体内基因转移，其中新的遗传物质被直接引入表皮。作为一名博士后研究助理，我成功地开发了一种使用逆转录病毒载体进行皮肤体内转导的小鼠模型。磨皮后，直接用高滴度逆转录病毒转导再上皮化表面。在免疫缺陷小鼠和耐受转基因产物（β-gal）的转基因小鼠中，注意到长期表达（40周）。然而，在正常小鼠中，转导后三周表达丧失。初步研究表明，转基因特异性免疫反应的存在和转基因表达的持续时间之间的相关性。从培养细胞中获得的其他证据表明，逆转录病毒介导的转基因表达可受细胞因子（如干扰素）的调节。该提案提出了一个计划，以确定是否免疫介导的转基因表达的损失是由于表达抑制或转导细胞的细胞溶解;表征负责任的免疫应答;并最终使用这些知识来设计载体以规避对转基因的免疫应答。提出的策略包括除了转基因之外还表达与β 2-微球蛋白互补的反义RNA以抑制MHC I类表达或编码免疫抑制性细胞因子基因（例如IL-10）的载体。开发一种能够避免对转基因产生免疫反应的载体将克服基因治疗临床应用的主要障碍。

The long-term objective of this research is to develop cutaneous gene therapy for inherited dermatological disorders. Currently, the most favored approach is ex vivo gene transfer followed by transplantation of the gene-modified cells back to the donor. This procedure is costly, time consuming and will require full thickness excision at the graft site. In addition, this surgery will result in scarring and contracture and ultimately limit the area that can be treated. A preferred alternative would be in vivo gene transfer whereby new genetic material is introduced directly into the epidermis. As a postdoctoral research associate I succeeded in developing a mouse model for in vivo transduction of skin using retroviral vectors. Following dermabrasion, the re-epithelializing surface was transduced directly with high titer retroviruses. In immunodeficient mice and transgenic mice tolerant to the transgene product (beta-gal), long term expression was noted (40 weeks). However, in normal mice, expression was lost by three weeks post-transduction. Preliminary studies showed a correlation between presence of transgene-specific immunological responses and duration of transgene expression. Additional evidence obtained with cells in culture suggested that retrovirus-directed transgene expression could be modulated by cytokines such as interferons. This proposal sets forth a plan to determine whether immune-mediated loss of transgene expression results from inhibition of expression or cytolysis of transduced cells; to characterize the responsible immune responses; and finally use this knowledge to design vectors to circumvent the immune responses to the transgene. Strategies proposed include the vectors that in addition to the transgene, express an anti-sense RNA complementary to beta2-microglobulin to inhibit MHC class I expression or encode immunosuppressive cytokines gene (e.g. IL-10). Development of a vector that circumvents the immune responses to the transgene would overcome a major obstacle to clinical application of gene therapy.