Gupta Vinay, Wang Weijun, Sosnowski Barbara A, Hofman Florence M, Chen Thomas C
Department of Pathology, K. Norris Jr. Comprehensive Cancer Center, University of Southern California, Los Angeles, California 90033, USA.
Neurosurg Focus. 2006 Apr 15;20(4):E26.
Adenovirus transduction in gene therapy is dependent on the expression of the coxsackie virus-adenovirus receptor (CAR) for initial binding and on the integrin receptors (avb3, avb5) for viral internalization. Low and variable expression of CAR may be responsible for the low transduction rates seen with native adenoviral vectors. The goal of this study was to demonstrate increased transduction efficiency by retargeting the adenovirus with a fibroblast growth factor (FGF) ligand, FGF-2.
The retargeted adenoviruses were used to transduce human glioblastoma multiforme (GBM)-derived ECs (tumor-associated brain endothelial cells [TuBECs]), in which there is minimal CAR expression but a high expression of FGF receptor (FGFR). The results demonstrate that the transduction efficiency of TuBECs can reach as high as 80% when one uses an FGF2-conjugated adenovirus containing green fluorescent protein (FGF2-AdGFP) yet be only 5% when one uses the native adenovirus (AdGFP). The TuBECs were transduced with either a native adenovirus (AdHSV-TK) or a retargeted adenovirus (FGF2-AdHSV-TK), both of which carry the suicide herpes simplex virus-thymidine kinase (HSV-TK) gene. Administered as a cytotoxic prodrug, ganciclovir induced a significant decline in the proliferation rate and increased apoptosis in TuBECs treated with the retargeted adenovirus, compared with its effect on TuBECs treated with the native adenovirus. Increased transduction efficiency was determined by performing GFP-based flow cytometry, and the expression of the TK protein by the retargeted adenovirus was assessed by performing an immunohistochemical analysis focused on HSV-TK. The mechanism of cytotoxicity was determined to be apoptosis by performing a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay.
Fibroblast growth factor-2-retargeted adenoviral vectors may be used to increase the transduction of GBM-derived endothelial cells, enabling a new and efficient antiangiogenesis strategy for the treatment of malignant gliomas.
基因治疗中的腺病毒转导依赖于柯萨奇病毒-腺病毒受体（CAR）的表达以进行初始结合，以及整合素受体（αvβ3、αvβ5）以实现病毒内化。CAR的低表达和可变表达可能是天然腺病毒载体转导率低的原因。本研究的目的是通过用成纤维细胞生长因子（FGF）配体FGF-2对腺病毒进行重新靶向，来证明转导效率的提高。
使用重新靶向的腺病毒转导人多形性胶质母细胞瘤（GBM）来源的内皮细胞（肿瘤相关脑内皮细胞[TuBECs]），其中CAR表达极少，但FGF受体（FGFR）表达高。结果表明，当使用含有绿色荧光蛋白的FGF2偶联腺病毒（FGF2-AdGFP）时，TuBECs的转导效率可高达80%，而使用天然腺病毒（AdGFP）时仅为5%。用携带自杀性单纯疱疹病毒胸苷激酶（HSV-TK）基因的天然腺病毒（AdHSV-TK）或重新靶向的腺病毒（FGF2-AdHSV-TK）转导TuBECs。作为细胞毒性前药给予更昔洛韦，与对用天然腺病毒处理的TuBECs的作用相比，其导致用重新靶向的腺病毒处理的TuBECs的增殖率显著下降且凋亡增加。通过基于GFP的流式细胞术确定转导效率的提高，并通过进行聚焦于HSV-TK的免疫组织化学分析来评估重新靶向的腺病毒对TK蛋白的表达。通过进行末端脱氧核苷酸转移酶介导的脱氧尿苷三磷酸缺口末端标记测定，确定细胞毒性机制为凋亡。
成纤维细胞生长因子-2重新靶向的腺病毒载体可用于提高GBM来源的内皮细胞的转导，为恶性胶质瘤的治疗提供一种新的高效抗血管生成策略。
