Targeted gene therapy for malignant melanoma

通过整合素靶向腺病毒载体向人黑色素瘤进行有效基因转移。重组腺病毒载体（Adv）用于基因治疗的效用受到其低转导效率和缺乏靶细胞特异性的限制。低转导效率通常被认为是由于初级腺病毒受体（柯萨奇病毒-腺病毒受体（CAR））的缺陷。本文通过对人黑色素瘤细胞系CAR水平的研究证实，转导效率低与腺病毒受体缺陷密切相关。为了通过Adv实现CAR非依赖性基因转移，我们通过5型腺病毒（Ad 5）纤维蛋白的遗传改变来修饰病毒嗜性。在纤维结结构域的HI环中插入含有Arg-Gly-Asp（RGD）的肽，允许病毒在细胞进入过程中使用替代受体整联蛋白受体。在表达由RGD肽基序识别的α（v）β（3）、α（v）β（5）类整联蛋白的五种人黑素瘤细胞中，相对于含有野生型纤维的载体（Adv-F/wt），用这种修饰的载体（Adv-F/RGD）转导增加了5- 96倍。相反，在293细胞中观察到Adv-F/RGD和Adv-F/wt之间的转导效率没有显著差异，其显示CAR的高水平表达。本研究尝试将Adv-F/RGD应用于恶性黑色素瘤的基因治疗。在相同的感染复数下，用携带人白细胞介素2的Adv-F/RGD（AxCAhIL 2-F/RGD）感染的黑素瘤细胞比用AxCAhIL 2-F/wt感染的细胞产生更高水平的细胞因子。在黑素瘤异种移植模型中，通过肿瘤内注射AxCAhIL 2-F/RGD的治疗比肿瘤内注射AxCAhIL 2-F/wt在使肿瘤消退方面更有效。这些数据表明，整合素靶向腺病毒载体可能是CAR缺陷型黑色素瘤基因治疗的有力工具。

Effective gene transfer to human melanomas via integrin-targeted adenoviral vectors. The utility of recombinant adenoviral vectors (Adv) for gene therapy is limited by their low transduction efficiency and lack of specificity for target cells. The low transduction efficiency is often recognized as due to deficiency of the primary adenoviral receptor, the coxsackievirus-adenovirus receptor (CAR). In this paper, studies of CAR levels on human melanoma cell lines confirmed that low transduction efficiency was closely related to deficiency of the adenoviral receptor. To achieve CAR-independent gene transfer via Adv, we modified viral tropism via genetic alteration of the adenovirus type 5 (Ad5) fiber protein. Insertion of an Arg-Gly-Asp (RGD)-containing peptide in the HI loop of the fiber knob domain allowed the virus to use an alternative receptor, the integrin receptor, during the cell entry process. With this modified vector (Adv-F/RGD) transduction was increased 5- to 96-fold relative to a vector containing wild-type fiber (Adv-F/wt) in five human melanoma cells expressing integrins of the alpha(v)beta(3), alpha(v)beta(5) class, which are recognized by the RGD peptide motif. In contrast, no significant difference in transduction efficiency between Adv-F/RGD and Adv-F/wt was observed in 293 cells, which show high-level expression of CAR. In this study, we attempted to apply Adv-F/RGD for gene therapy for malignant melanoma. At the same multiplicity of infection, melanoma cells infected with Adv-F/RGD carrying human interleukin 2 (AxCAhIL2-F/RGD) produced a higher level of cytokine than cells infected with AxCAhIL2-F/wt. Treatment by intratumoral injection of AxCAhIL2-F/RGD was more effective than intratumoral injection of AxCAhIL2-F/wt in regressing tumors in a melanoma xenograft model. These data suggest that integrin-targeted adenoviral vectors may be a powerful tool in gene therapy for CAR-deficient melanomas.