IN VIVO TRANSDUCTION OF KERATINOCYTES

该提案的长期目标是制定有效的方法， 将转基因递送和表达到角质形成细胞中， 通过体细胞基因疗法治疗人类疾病。 离体 基因治疗包括从体内去除靶细胞， 在体外用病毒载体和随后移植修饰的 将自体细胞输回患者体内 这种方法需要外科手术 嫁接是耗时且昂贵的。 直接基因转移到 表皮中的角质形成细胞将避开这些困难。 的 这项研究的目的是开发一种方法， 基底角质形成细胞原位逆转录病毒载体，并实现 表皮角质形成细胞中持续存在转基因。 这将 通过1）诱导表皮增生以允许 逆转录病毒整合所需的基底角质形成细胞的复制， 2)基底细胞对病毒转导的可及性的测定 使用腺病毒作为标记，3）产生非常滴度的假型 逆转录病毒载体，4）将逆转录病毒载体引入基底细胞 并确定转基因在表皮中的持久性。

The long term goal of this proposal is to develop methods for efficient delivery and expression of transgenes into keratinocytes for possible treatment of human diseases by somatic gene-based therapies. Ex vivo gene therapy involves removal of the target cells from body, transduction in vitro with viral vector and subsequent transplantation of the modified autologous cells back to the patient. This method requires surgical grafting, is time consuming and costly. Direct gene transfer to keratinocytes in epidermis would circumvent these difficulties. The objective of this research proposal is to develop a method to transduce basal keratinocytes in situ with retroviral vectors and achieve persistant presence of transgene in epidermal keratinocytes. This will be achieved by 1)induction of hyperplasia in epidermis to allow replication of basal keratinocytes required for retroviral integration, 2)determination of accessibility of basal cells to viral transduction using adenoviruses as a marker, 3)generation of very titer pseudotype retroviral vectors, 4)introduction of retroviral vectors to basal cells and determining persistency of the transgene in epidermis.