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Development of recombinant adeno-associated virus vectors carrying small interfering RNA (shHec1)-mediated depletion of kinetochore Hec1 protein in tumor cells

Gene Therapy volume 14, pages 814–827 (2007)Cite this article

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.

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Abbreviations

BEV:

baculovirus expression vector

CGNS:

cerebella granule neurons

CNS:

central nervous system

dsRNA:

double-strand RNA

eGFP:

enhanced green fluorescence protein

Hec1:

highly expressed in cancer 1

hrGFP:

humanized Renilla green fluorescence protein

HSPG:

heparan sulfate proteogylcans

kmts:

kinetochore microtubles

Kntc2:

kinetochore-associated protein 2

MOI:

multiplicity of infection (encapsidated vector genome particles per cell)

rAAV:

recombinant adeno-associated virus

RNAi:

RNA interference

Sf:

Spodoptera frugiperda

shRNA:

short hairpin RNA

siRNA:

small interfering RNA

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Acknowledgements

We thank Scott Q Harper in Beverly Davidson's Laboratory at the University of Iowa and Wenqing Li at LMI of NCI, NIH for advise on shRNA design; Tracy Laabs in Herbert Geller's laboratory at NHLBI, NIH for providing primary CGNS cells; and Shigui Zhu at NHGRI, NIH for DNA sequencing. We acknowledge the professional skills and advice of NHLBI core facility members Philip McCoy Jr and Ann Williams at Flow Cytometry core; Fillipina Giacometti at Pathology Core; Christian A Combs and Daniela Malide at Light Microscopy Core regarding FACS-, pathology-, and microscopy-related experiments performed in this paper. We appreciate the help from Andres Mendez in our laboratory.

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Authors and Affiliations

  1. Laboratory of Biochemical Genetics, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA

    L Li, L Yang & R M Kotin

  2. Division of Cancer Treatment and Diagnosis, Screening Technologies Branch, National Cancer Institute, National Institutes of Health, Frederick, MD, USA

    D A Scudiero & R H Shoemaker

  3. Department of Biochemistry and Molecular Genetics, University of Virginia Medical School, Charlottesville, VA, USA

    S A Miller & P T Stukenberg

  4. Pathology Core Facility, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA

    Z-X Yu

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  1. L Li
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Correspondence to R M Kotin.

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Li, L., Yang, L., Scudiero, D. et al. Development of recombinant adeno-associated virus vectors carrying small interfering RNA (shHec1)-mediated depletion of kinetochore Hec1 protein in tumor cells. Gene Ther 14, 814–827 (2007). https://doi.org/10.1038/sj.gt.3302933

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