Method Article

High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

DOI:

10.3791/2538

⸱

March 22nd, 2011

 ,  ,  ,  ,  ,  ,  ,  , 
1Department of Pediatrics, Division of Cellular and Molecular Therapy, University of Florida

In This Article

Summary

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In this article, we describe the identification of the adeno-associated virus serotype 3 (AAV3) as the most efficient vector for targeting human liver cancer cells.

Abstract

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Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a number of gene therapy clinical trials. More recently, a number of additional AAV serotypes have also been isolated, which have been shown to exhibit selective tissue-tropism in various small and large animal models1. Of the 10 most commonly used AAV serotypes, AAV3 is by far the least efficient in transducing cells and tissues in vitro as well as in vivo.

However, in our recently published studies, we have documented that AAV3 vectors transduce human liver cancer - hepatoblastoma (HB) and hepatocellular carcinoma (HCC) - cell lines extremely efficiently because AAV3 utilizes human hepatocyte growth factor receptor as a cellular co-receptor for binding and entry in these cells2,3.

In this article, we describe the steps required to achieve high-efficiency transduction of human liver cancer cells by recombinant AAV3 vectors carrying a reporter gene. The use of recombinant AAV3 vectors carrying a therapeutic gene may eventually lead to the potential gene therapy of liver cancers in humans.

Protocol

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1. Packaging of Recombinant Adeno-associated Virus Serotype 3 (rAAV3) Vectors

  1. Go to the website: "http://www.geguchadze.com/calc" and calculate the amount of DNA.
  2. Pre-mix plasmids pHelper (Adenovirus helper), pACG2c3 (AAV helper) and pdsAAV-CB-EGFP (rAAV vector) in medium without FBS and antibiotics. Total volume should be 8,750 μL for ten 15 cm2 plates.
  3. Add directly 1,250 μL of PEI (0.1% m/v, Polysciences, Cat# 23966, pH 4.5) into DNA solution in Step 1.2. Vortex the DNA-PEI solution for a few seconds.
  4. Incubate DNA-PEI solution to form a complex for 5 min at room temperature (RT).
  5. Add 1 mL of DNA-PEI solution per 15 cm2 plate.

rAAV3 dot blot analysis; virus quantification results; varying sample volumes, concentrations noted.

Gene expression comparison via fluorescence microscopy, showing mock, scAAV2-EGFP, scAAV3-EGFP results.

Discussion

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Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus (AAV) have been developed, and are currently in use in a number of gene therapy clinical trials5. Previously, of the 10 most commonly used AAV serotypes, the transduction efficiency of AAV3 vectors in general has been reported to be particularly low, both in vitro and in vivo1. However, our recent observation that AAV3 vectors transduce human liver cancer cell lines exceedingly well, as determined quantitatively by flow cytometry2, and that AAV3 utilizes the human hepatocyte growth factor receptor (hHGFR) as a cellular co-receptor for binding and entry in these cells3, strongly suggest a selective tissue-tropism of AAV3 for human liver cells in general, and human liver cancer cells in particular. However, since AAV3 vectors also transduce normal human hepatocytes efficiently2, their potential use in cancer gene therapy application in vivo would be negatively impacted. One possible strategy to circumvent this potential problem involves transcriptional-targeting of cancer cells by identifying a gene product that is selectively produced by the tumor, but not by normal hepatocytes. For example, previous studies have shown that serum levels of α-fetoprotein (AFP) are used as a specific marker for tracking the presence, progression, and/or reoccurrence of certain types of liver cancers, since normal hepatocytes generally produce very small amounts of this protein. Indeed, we have used AAV3 vectors containing the AFP promoter to target transgene expression in liver cancer cells, but not in normal hepatocytes2, and studies are currently underway to test the efficacy of this approach in a murine xenograft model of liver cancer. In our additional studies, we have observed that the transduction efficiency of various AAV serotype vectors can be significantly augmented by site-directed mutagenesis of surface-exposed tyrosine residues in the viral capsids6-14. Since 6 of 7 surface-exposed tyrosines are also conserved in AAV3, we have performed site-directed mutagenesis of these residues and observed that the transduction efficiency of tyrosine-mutant AAV3 vectors is significantly enhanced in human liver cancer cells (unpublished data). Studies are also currently underway to evaluate the safety and efficacy of the tyrosine-mutant AAV3 vectors in mouse xenograft models for human hepatoblastoma and hepatocellular carcinoma, and if successful, the optimal tyrosine-mutant AAV3 serotype vectors may prove to be useful for targeting human liver cancers for the potential gene therapy.

Disclosures

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No conflicts of interest declared.

Acknowledgements

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We thank Drs. R. Jude Samulski and Xiao Xiao for their kind gifts of recombinant AAV3 and AAV-EGFP plasmids, respectively, and Dr. Gail Tomlinson for generously providing Hep293TT cells. This research was supported in part by Public Health Service grant grants R01 HL-076901, R01 HL-097088, and P01 DK-058327 (Project 1) from the National Institutes of Health (to AS).

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
DMEMCellgro10-0170CM
PEIPolysciences, Inc.23966
BenzonaseNovagen, EMD Millipore70664-3
HiTrap Q HP columnGE Healthcare17-1154-01
Salmon sperm DNAFisher ScientificNC9753983
Iodixanol gradientOptiPrep1114542
G-50 ColumnsGE Healthcare27-5330-01

References

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Tags

AAV3 VectorsTransduction EfficiencyPlasmid TransfectionVirus PurificationIodixanol GradientFluorescence MicroscopyEGFP ExpressionHepatoblastoma CellsHepatocellular Carcinoma

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