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Targeted gene therapy for malignant melanoma

恶性黑色素瘤的靶向基因治疗

基本信息

批准号：
12557071
负责人：
HAMADA Hirofumi
金额：
$ 8.32万
依托单位：
Sapporo Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12557071/
关键词：
Adenoviral vector Gene therapy integrin malignant melanoma receptor peptide ligand cancer fiber 悪性黒色癌

项目摘要

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.

通过整合素靶向腺病毒载体有效地将基因转移到人黑色素瘤。重组腺病毒载体（Adv）在基因治疗中的应用受到其低转导效率和对靶细胞缺乏特异性的限制。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）的缺乏。本文通过对人黑色素瘤细胞系CAR水平的研究，证实了低转导效率与腺病毒受体缺乏密切相关。为了通过Adv实现与car无关的基因转移，我们通过对腺病毒5型（Ad5）纤维蛋白的遗传改变来修饰病毒的趋向性。在纤维球形结构域的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，即整合素受体。与含有野生型纤维的载体（Adv-F/wt）相比，该修饰载体（Adv-F/RGD）在表达α (v) β(3)、α (v) β(5)类整合素的5个人类黑色素瘤细胞中的转导增加了5至96倍，这些整合素被RGD肽基序识别。相比之下，在CAR高表达的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。在相同的感染次数下，携带人白细胞介素2 （AxCAhIL2-F/RGD）的Adv-F/RGD感染的黑色素瘤细胞比AxCAhIL2-F/wt感染的细胞产生更高水平的细胞因子。瘤内注射AxCAhIL2-F/RGD比瘤内注射AxCAhIL2-F/wt治疗黑色素瘤异种移植模型中肿瘤消退更有效。这些数据表明，整合素靶向腺病毒载体可能是car缺陷黑色素瘤基因治疗的有力工具。