Schmitz M, Graf C, Gut T, Sirena D, Peter I, Dummer R, Greber U F, Hemmi S
Institute of Molecular Biology, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
Gene Ther. 2006 Jun;13(11):893-905. doi: 10.1038/sj.gt.3302739.
Replicating adenovirus (Ad) vectors with tumour tissue specificity hold great promise for treatment of cancer. We have recently constructed a conditionally replicating Ad5 AdDeltaEP-TETP inducing tumour regression in a xenograft mouse model. For further improvement of this vector, we introduced four genetic modifications and analysed the viral cytotoxicity in a large panel of melanoma cell lines and patient-derived melanoma cells. (1) The antiapoptotic gene E1B-19 kDa (Delta19 mutant) was deleted increasing the cytolytic activity in 18 of 21 melanoma cells. (2) Introduction of the E1A 122-129 deletion (Delta24 mutant), suggested to attenuate viral replication in cell cycle-arrested cells, did not abrogate this activity and increased the cytolytic activity in two of 21 melanoma cells. (3) We inserted an RGD sequence into the fiber to extend viral tropism to alphav integrin-expressing cells, and (4) swapped the fiber with the Ad35 fiber (F35) enhancing the tropism to malignant melanoma cells expressing CD46. The RGD-fiber modification strongly increased cytolysis in all of the 11 CAR-low melanoma cells. The F35 fiber-chimeric vector boosted the cytotoxicity in nine of 11 cells. Our results show that rational engineering additively enhances the cytolytic potential of Ad vectors, a prerequisite for the development of patient-customized viral therapies.
具有肿瘤组织特异性的复制型腺病毒（Ad）载体在癌症治疗方面具有巨大潜力。我们最近构建了一种条件性复制的Ad5 AdDeltaEP-TETP，在异种移植小鼠模型中可诱导肿瘤消退。为了进一步改进该载体，我们进行了四项基因改造，并在大量黑色素瘤细胞系和患者来源的黑色素瘤细胞中分析了病毒的细胞毒性。（1）删除抗凋亡基因E1B-19 kDa（Delta19突变体），可增强21种黑色素瘤细胞中18种的溶细胞活性。（2）引入E1A 122-129缺失（Delta24突变体），据推测可减弱病毒在细胞周期停滞细胞中的复制，但并未消除这种活性，反而增强了21种黑色素瘤细胞中2种的溶细胞活性。（3）我们在纤维中插入了一个RGD序列，以将病毒嗜性扩展到表达αv整合素的细胞，并且（4）将纤维与Ad35纤维（F35）进行交换，增强对表达CD46的恶性黑色素瘤细胞的嗜性。RGD纤维修饰显著增强了所有11种CAR低表达黑色素瘤细胞的细胞溶解作用。F35纤维嵌合载体增强了11种细胞中9种的细胞毒性。我们的结果表明，合理的工程改造可累加增强Ad载体的溶细胞潜力，这是开发针对患者的病毒疗法的先决条件。
