Targeted gene therapy for malignant melanoma

通过整合素靶向腺病毒载体对人黑色素瘤进行有效的基因转移。重组腺病毒载体(ADV)转导效率低，对靶细胞缺乏特异性，限制了其在基因治疗中的应用。转导效率低通常被认为是由于主要的腺病毒受体-柯萨奇病毒-腺病毒受体(CAR)的缺乏。本文通过对人黑色素瘤细胞株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实现非CAR基因转移，我们通过改变5型腺病毒(Ad5)纤维蛋白的基因改变了病毒的趋向性。将含有Arg-Gly-Asp(RGD)的多肽插入纤维结节结构域的HI环，使病毒在细胞进入过程中使用另一种受体-整合素受体。与含有野生型纤维(ADV-F/wt)的载体(ADV-F/wt)相比，该修饰载体(ADV-F/RGD)在5个人黑色素瘤细胞中的转导强度提高了5~96倍，该载体表达由RGD多肽基序识别的α(V)β(3)、α(V)β(5)类整合素。而在高表达CAR的293细胞中，ADV-F/RGD和ADV-F/wt的转导效率无显著差异。在本研究中，我们尝试将AdV-F/RGD用于恶性黑色素瘤的基因治疗。在相同的感染复数下，携带人白介素2的AdV-F/RGD(AxCAhIL2-F/RGD)感染的黑色素瘤细胞产生的细胞因子水平高于感染AxCAhIL2-F/wt的黑色素瘤细胞，瘤内注射AxCAhIL2-F/RGD比瘤内注射AxCAhIL2-F/wt更有效。这些数据表明，整合素靶向腺病毒载体可能是治疗汽车缺陷性黑色素瘤的有力工具。

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.