title: Development of recombinant adeno-associated virus vectors carrying small interfering RNA (shHec1)-mediated depletion of kinetochore Hec1 protein in tumor cells.
abstract: 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.
authors.affiliation: Laboratory of Biochemical Genetics, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: L
authors.initials: L
authors.lastname: Li
authors.forename: L
authors.initials: L
authors.lastname: Yang
authors.forename: D A
authors.initials: DA
authors.lastname: Scudiero
authors.forename: S A
authors.initials: SA
authors.lastname: Miller
authors.forename: Z-X
authors.initials: ZX
authors.lastname: Yu
authors.forename: P T
authors.initials: PT
authors.lastname: Stukenberg
authors.forename: R H
authors.initials: RH
authors.lastname: Shoemaker
authors.forename: R M
authors.initials: RM
authors.lastname: Kotin
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D003598:Cytoskeletal Proteins; D000229:Dependovirus / Q000235:genetics*; D005434:Flow Cytometry; D017353:Gene Deletion; D005818:Genetic Engineering; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics / Q000627:therapeutic use*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D018386:Kinetochores / Q000378:metabolism; D051379:Mice; D008817:Mice, Mutant Strains; D008819:Mice, Nude; D008856:Microscopy, Fluorescence; D009369:Neoplasms / Q000378:metabolism / Q000628:therapy*; D009374:Neoplasms, Experimental; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000627:therapeutic use*; D014161:Transduction, Genetic / Q000379:methods; D014183:Transplantation, Heterologous
chemical_list: D003598:Cytoskeletal Proteins; C120529:NDC80 protein, human; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins