Li L, Yang L, Scudiero D A, Miller S A, Yu Z-X, Stukenberg P T, Shoemaker R H, Kotin R M
Laboratory of Biochemical Genetics, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Gene Ther. 2007 May;14(10):814-27. doi: 10.1038/sj.gt.3302933. Epub 2007 Mar 1.
Transcript depletion using small interfering RNA (siRNA) technology represents a potentially valuable technique for the treatment of cancer. However, delivering therapeutic quantities of siRNA into solid tumors by chemical transfection is not feasible, whereas viral vectors efficiently transduce many human tumor cell lines. Yet producing sufficient quantities of viral vectors that elicit acute and selective cytotoxicity remains a major obstacle for preclinical and clinical trials. Using the invertebrate Spodoptera frugiperda (Sf9) cell line, we were able to produce high titer stocks of cytotoxic recombinant adeno-associated virus (rAAV) that express short hairpin RNA (shRNA) and that efficiently deplete Hec1 (highly expressed in cancer 1), or Kntc2 (kinetochore-associated protein 2), a kinetochore protein directly involved in kinetochore microtubule interactions, chromosome congression and spindle checkpoint signaling. Depletion of Hec1 protein results in persistent spindle checkpoint activation followed by cell death. Because Hec1 expression and activity are only present in mitotic cells, non-dividing cells were not affected by rAAV treatment. On the basis of the results of screening 56 human tumor cell lines with three different serotype vectors, we used a tumor xenograft model to test the effects in vivo. The effects of the shHec1 vector were evident in sectioned and stained tumors. The experiments with rAAV-shRNA vectors demonstrate the utility of producing vectors in invertebrate cells to obtain sufficient concentrations and quantities for solid tumor therapy. This addresses an important requirement for cancer gene therapy, to produce cytotoxic vectors in sufficient quantities and concentrations to enable quantitative transduction and selective killing of solid tumor cells.
使用小干扰RNA（siRNA）技术进行转录本消减是一种潜在的有价值的癌症治疗技术。然而，通过化学转染将治疗剂量的siRNA递送至实体瘤中并不可行，而病毒载体可有效转导许多人类肿瘤细胞系。然而，生产足够量的能引发急性和选择性细胞毒性的病毒载体仍然是临床前和临床试验的主要障碍。利用无脊椎动物草地贪夜蛾（Sf9）细胞系，我们能够生产出高滴度的细胞毒性重组腺相关病毒（rAAV），其表达短发夹RNA（shRNA），并能有效消减Hec1（在癌症中高表达1）或Kntc2（动粒相关蛋白2），Kntc2是一种直接参与动粒微管相互作用、染色体排列和纺锤体检查点信号传导的动粒蛋白。Hec1蛋白的消减会导致纺锤体检查点持续激活，随后细胞死亡。由于Hec1的表达和活性仅存在于有丝分裂细胞中，非分裂细胞不受rAAV处理的影响。基于用三种不同血清型载体筛选56种人类肿瘤细胞系的结果，我们使用肿瘤异种移植模型来测试体内效果。shHec1载体的效果在切片和染色的肿瘤中很明显。rAAV-shRNA载体的实验证明了在无脊椎动物细胞中生产载体以获得足够浓度和数量用于实体瘤治疗的实用性。这满足了癌症基因治疗的一项重要要求，即生产足够数量和浓度的细胞毒性载体，以实现实体瘤细胞的定量转导和选择性杀伤。
