Lanuti M, Gao G P, Force S D, Chang M Y, El Kouri C, Amin K M, Hughes J V, Wilson J M, Kaiser L R, Albelda S M
Department of Surgery, University of Pennsylvania Medical Center, Philadelphia 19104, USA.
Hum Gene Ther. 1999 Feb 10;10(3):463-75. doi: 10.1089/10430349950018904.
Studies with first-generation adenoviral vectors have uncovered limitations that include finite transgene persistence, potential hepatotoxicity, and contamination with replication-competent adenovirus (RCA). To address these limitations within the context of cancer suicide gene therapy, a new adenoviral vector was developed containing the herpes simplex virus type 1 thymidine kinase (HSV tk) gene inserted in the E1 region of a recombinant vector containing deletions in the E1 and E4 regions of the Ad5 genome. The HSV tk minigene was placed under transcriptional control of a Rous sarcoma virus (RSV) promoter. This new E1E4-deleted vector was compared with the first-generation E1E3-deleted Ad.RSVtk vector. Generation of replication-competent adenovirus during production was eliminated. Using semiquantitative immunoblotting, the two vectors produced equivalent amounts of the expected 44-kDa tk-encoded protein in three different cell lines tested. The ability of the E1E4-deleted vector to sensitize tumor cells to ganciclovir (GCV) using in vitro assays and mixing studies was comparable to that of the E1E3-deleted vector. In vivo bystander effects were investigated using mixing studies in a syngeneic flank tumor model and demonstrated no difference between vectors in either immunocompetent or immunodeficient mice. To test the efficiency of these vectors in treating tumors in clinically relevant models, virus was injected intraperitoneally into tumor-bearing SCID mice and intrapleurally in a syngeneic rat mesothelioma model. After treatment of animals with ganciclovir, both vectors were roughly equivalent in their ability to increase mean survival (from approximately 40 to approximately 70 days) and markedly reduce tumor burden. Finally, formal toxicology studies were performed and showed similar amounts of local inflammation without systemic toxicity. In summary, this series of in vitro and in vivo experiments indicates that the performance of the recombinant E1E4-deleted adenoviral vector was virtually identical to that of the E1E3-deleted vector. Since the E1E4 vector has a much lower rate of recombination during production and has been shown to be less hepatotoxic in animal models, this new vector should prove superior to the first-generation Ad.HSVtk vectors in clinical cancer gene therapy trials.
对第一代腺病毒载体的研究揭示了其局限性，包括转基因持久性有限、潜在的肝毒性以及与复制型腺病毒（RCA）的污染。为了在癌症自杀基因治疗的背景下解决这些局限性，开发了一种新的腺病毒载体，其在Ad5基因组E1和E4区域缺失的重组载体的E1区域插入了单纯疱疹病毒1型胸苷激酶（HSV tk）基因。HSV tk小基因置于劳氏肉瘤病毒（RSV）启动子的转录控制之下。将这种新的E1E4缺失载体与第一代E1E3缺失的Ad.RSVtk载体进行了比较。生产过程中复制型腺病毒的产生被消除。使用半定量免疫印迹法，在三种测试的不同细胞系中，这两种载体产生的预期44 kDa tk编码蛋白量相当。通过体外试验和混合研究，E1E4缺失载体使肿瘤细胞对更昔洛韦（GCV）敏感的能力与E1E3缺失载体相当。在同基因侧翼肿瘤模型中使用混合研究对体内旁观者效应进行了研究，结果表明在免疫健全或免疫缺陷小鼠中，两种载体之间没有差异。为了在临床相关模型中测试这些载体治疗肿瘤的效率，将病毒腹腔内注射到荷瘤SCID小鼠体内，并胸膜内注射到同基因大鼠间皮瘤模型中。在用更昔洛韦治疗动物后，两种载体在提高平均生存期（从约40天延长至约70天）和显著减轻肿瘤负荷的能力方面大致相当。最后，进行了正式的毒理学研究，结果显示局部炎症程度相似且无全身毒性。总之，这一系列体外和体内实验表明，重组E1E4缺失腺病毒载体的性能与E1E3缺失载体几乎相同。由于E1E4载体在生产过程中的重组率低得多，并且在动物模型中已显示出较低的肝毒性，这种新载体在临床癌症基因治疗试验中应证明优于第一代Ad.HSVtk载体。
