IN VIVO CUTANEOUS GENE THERAPY

这项研究的长期目标是开发遗传性皮肤病的皮肤基因疗法。目前，最受欢迎的方法是体外基因转移，然后将基因修饰的细胞移植回捐赠者。这一手术费用昂贵，耗时长，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痉挛，并最终限制可治疗的区域。一种首选的替代方案是体内基因转移，即将新的遗传物质直接引入表皮。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，将高滴度逆转录病毒直接导入再上皮化表面。在免疫缺陷小鼠和对转基因产物(β-GAL)耐受的转基因小鼠中，注意到长期表达(40周)。然而，在正常小鼠中，基因转导后三周就失去了表达。初步研究表明，转基因特异性免疫反应的出现与转基因表达持续时间之间存在相关性。在培养细胞中获得的更多证据表明，逆转录病毒导向的转基因表达可以受到干扰素等细胞因子的调节。这项建议提出了一项计划，以确定免疫介导的转基因表达丧失是由于转导细胞的表达抑制或细胞溶解所致，并表征负责的免疫反应，最后利用这一知识设计载体以绕过对转基因的免疫反应。建议的策略包括除转基因外，还表达与β2-微球蛋白互补的反义RNA以抑制MHC I类基因的表达或编码免疫抑制细胞因子基因(如IL-10)。开发一种绕过转基因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.