Gene Delivery Using Helper Virus‐Free HSV‐1 Amplicon Vectors

Andrea S. Laimbacher1, Cornel Fraefel1

1 University of Zurich, Zurich, Switzerland
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 4.14
DOI:  10.1002/0471142301.ns0414s60
Online Posting Date:  July, 2012
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Abstract

Herpes simplex virus type 1 (HSV‐1)‐based amplicon vectors contain only a very small percentage of the 152‐kbp viral genome. Consequently, replication and packaging of amplicons depend on helper functions that are provided either by replication‐defective mutants of HSV‐1 or by replication‐competent, but packaging‐defective, HSV‐1 genomes. Sets of cosmids that overlap and represent the entire HSV‐1 genome can form, via homologous recombination, circular replication‐competent viral genomes, which give rise to infectious virus progeny. However, if the DNA cleavage/packaging signals are deleted, reconstituted virus genomes are not packageable, but still provide all the helper functions required for the packaging of cotransfected amplicon DNA. The resulting stocks of packaged amplicon vectors are essentially free of contaminating helper virus. This unit describes the cotransfection of amplicon and cosmid or bacterial artificial chromosome (BAC) DNA into 2‐2 cells by cationic liposome‐mediated transfection and the harvesting of packaged vector particles. Support protocols provide methods for preparing cosmid and BAC DNA and determining the titers of amplicon stocks. Curr. Protoc. Neurosci. 60:4.14.1‐4.14.21. © 2012 by John Wiley & Sons, Inc.

Keywords: Herpes simplex virus; HSV‐1; amplicon; helper virus free; cotransfection; cosmid; BAC (bacterial artificial chromosome); gene delivery

     
 
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Table of Contents

  • Introduction
  • Basic Protocol: Preparation of Helper Virus–Free Amplicon Stocks
  • Support Protocol 1: Preparation of HSV‐1 Cosmid DNA for Transfection
  • Support Protocol 2: Preparation of HSV‐1 BAC DNA for Transfection
  • Support Protocol 3: Titration of Amplicon Stocks
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol: Preparation of Helper Virus–Free Amplicon Stocks

 Materials
  • 2‐2 cells (Smith et al., 1992)
  • Dulbecco's modified Eagle medium (Life Technologies) with 10% and 6% fetal bovine serum (DMEM/10% FBS and DMEM/6% FBS)
  • G418 (Geneticin; Life Technologies)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 0.25% trypsin/0.02% EDTA (Life Technologies)
  • Opti‐MEM I reduced‐serum medium (Life Technologies)
  • HSV‐1 amplicon DNA (maxiprep DNA isolated from E. coli)
  • PacI‐digested cosmid DNA of set C6Δa48Δa (see Support Protocol 1) or HSV‐1 BAC fHSVΔpacΔ27ΔKn (see Support Protocol 2)
  • pEBHICP27 plasmid DNA
  • Lipofectamine Reagent (Life Technologies)
  • Plus Reagent (Life Technologies)
  • Liquid nitrogen
  • 10%, 25 %, 30%, and 60% (w/v) sucrose in PBS
  • Humidified 37°C, 5% CO2 incubator
  • 75‐cm2 tissue culture flasks
  • Hemacytometer
  • 60‐mm diameter tissue culture dishes
  • 15‐ml conical centrifuge tubes
  • Rubber policeman
  • 37°C water bath
  • Probe sonicator
  • 0.45‐µm syringe‐tip polyethersulfone membrane filters (Sarstedt)
  • 20‐ml disposable syringes
  • 30‐ml centrifuge tube
  • Beckman Ultra‐Clear centrifuge tubes (25 × 89 mm and 14 × 95 mm)
  • Ultracentrifuge with Beckman SW28 and SW40 rotors
  • Fiber‐optic illuminator

NOTE: All cell culture incubations are performed in a humidified 37°C, 5% CO2 incubator unless otherwise stated.

Support Protocol 1: Preparation of HSV‐1 Cosmid DNA for Transfection

 Materials
  • E. coli clones of HSV‐1 cosmid set C6Δa48Δa: includes cos6Δa, cos14, cos28, cos48Δa, and cos56 (Cunningham and Davison, 1993; Fraefel et al., 1996; see Fig. 4.14.1)
  • SOB medium containing 50 µg/ml ampicillin (SOB/amp; see recipe)
  • Dimethyl sulfoxide (DMSO)
  • Plasmid Maxi Kit (Qiagen): includes Qiagen‐tip 500 columns and buffers P1, P2, P3, QBT, QC, and QF (prewarm buffer QF to 65°C)
  • Isopropanol
  • 70% (v/v) and 100% (v/v) ethanol
  • TE buffer, pH 7.5 (appendix 2A)
  • Restriction endonucleases DraI, KpnI, and PacI
  • High‐molecular‐weight DNA standard (Life Technologies)
  • 1‐kb DNA ladder (Life Technologies)
  • Electrophoresis‐grade agarose
  • TAE electrophoresis buffer (see recipe)
  • 1 mg/ml ethidium bromide in H2O
  • 25:24:1 (v/v) phenol/chloroform/isoamyl alcohol (appendix 2A)
  • 24:1 (v/v) chloroform/isoamyl alcohol (appendix 2A)
  • 3 M sodium acetate, pH 5.5 (appendix 2A)
  • 17 × 100–mm graduated snap‐cap tubes (e.g., Falcon 2059), sterile
  • 2‐liter flask
  • 37°C shaker incubator
  • 250‐ml polypropylene centrifuge tubes
  • Sorvall GSA and SS‐34 rotors (or equivalent)
  • 30‐ml polypropylene tubes
  • 65° and 37°C water baths
  • Additional reagents and equipment for quantitation of DNA by absorption spectroscopy (appendix 1K), restriction endonuclease digestion of DNA (appendix 1M), agarose gel electrophoresis (appendix 1N), and purification of DNA (appendix 1G)

Support Protocol 2: Preparation of HSV‐1 BAC DNA for Transfection

 Materials
  • LB medium containing 12.5 µg/ml chloramphenicol (appendix 2A)
  • E. coli clones of HSV‐1 BAC fHSVΔpacΔ27ΔKn and plasmid pEBHICP27 (Saeki et al., 2001)
  • Dimethyl sulfoxide (DMSO)
  • Plasmid Maxi Kit (Qiagen): includes Qiagen‐tip 500 columns and buffers P1, P2, P3, QBT, QC, and QF (prewarm buffer QF to 65°C)
  • Isopropanol
  • 70% (v/v) ethanol, chilled
  • TE buffer, pH 7.4 (appendix 2A)
  • CsCl (Sigma)
  • 10 mg/ml and 1mg/ml ethidium bromide in H2O
  • Paraffin oil
  • TE/CsCl solution: prepared by dissolving 3 g CsCl in 3 ml TE buffer, pH 7.4 (appendix 2A); store up to several months at room temperature
  • n‐butanol (Merck)
  • Restriction endonucleases HindIII and KpnI
  • High‐molecular‐weight DNA standard (Life Technologies)
  • 1‐kb DNA ladder (Life Technologies)
  • Electrophoresis‐grade agarose
  • TAE electrophoresis buffer (see recipe)
  • 17 × 100–mm graduated snap‐cap tubes (e.g., Falcon 2059), sterile
  • 37°C shaker incubator
  • 2‐liter flask
  • 250‐ml polypropylene centrifuge tubes
  • Sorvall GSA and SS‐34 rotors (or equivalent)
  • 30‐ml polypropylene centrifuge tube
  • 120‐mm diameter folded filters (Macherey‐Nagel)
  • 65° and 37°C water baths
  • 13 × 51–mm Ultra‐Clear centrifuge tubes (Beckman)
  • Sorvall TV 865 ultracentrifuge rotor
  • Dialysis cassettes (Slide‐A‐Lyzer, 10,000 MWCO; Pierce)
  • 1‐ml disposable syringes
  • 21‐G and 36‐G hypodermic needles
  • UV‐lamp (366 nm)
  • UV spectrophotometer
  • Additional reagents and equipment for quantitation of DNA by absorption spectroscopy (appendix 1K), restriction endonuclease digestion of DNA (appendix 1M), and agarose gel electrophoresis (appendix 1N)

Support Protocol 3: Titration of Amplicon Stocks

 Materials
  • Vero (clone 76; ECACC #85020205), BHK (clone 21; ECACC #85011433), or 293 (ATCC #1573) cells
  • DMEM (e.g., Life Technologies) supplemented with 10% and 2% FBS (DMEM/10% FBS and DMEM/2% FBS; appendix 2A)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Samples collected from vector stocks (see Basic Protocol, steps 17, 22a, or 24b)
  • 4% (w/v) paraformaldehyde solution, pH 7.0 (unit 4.13)
  • X‐gal staining solution (see recipe), GST solution (see recipe), or appropriate primary and secondary antibodies
  • 24‐well tissue culture plates
  • Humidified 37°C, 5% CO2 incubator
  • Inverted fluorescence microscope or inverted light microscope

NOTE: All cell culture incubations are performed in a humidified 37°C, 5% CO2 incubator unless otherwise stated.
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Figures

  •  FigureFigure 4.14.1 Helper virus‐free packaging of HSV‐1 amplicons into HSV‐1 particles. (A) The HSV‐1 genome (152 kbp) is composed of unique long (UL) and unique short (US) segments (horizontal lines), which are flanked by inverted repeats (open rectangles): IRS, internal repeat of the short segment; TRS, terminal repeat of the short segment; IRL, internal repeat of the long segment; TRL, terminal repeat of the long segment. The origins of DNA replication, oriS (solid circle) and oriL (open circle), and the DNA cleavage/packaging signals, pac (solid rectangles), are also shown. (B) Schematic diagram of HSV‐1 cosmid set C6Δa48Δa with deleted (X) pac signals (Fraefel et al., 1996; Cunningham and Davison, 1993), which includes cos6Δa, cos14, cos28, cos48Δa, and cos56. (C) Schematic diagram of HSV‐1 BAC fHSVΔpacΔ27ΔKn (Saeki et al., 2001) with deleted (X) pac signals and ICP27 gene. (D) For the helper virus‐free packaging of amplicons into HSV‐1 particles, cells that are susceptible for HSV‐1 replication are cotransfected with amplicon DNA and either DNA from cosmid set C6Δa48Δa or BAC DNA fHSVΔpacΔ27ΔKn and the HSV‐1 ICP27‐expressing plasmid pEBHICP27. In the absence of the pac signals, the HSV‐1 cosmid set or the HSV‐1 BAC cannot generate a packageable HSV‐1 genome, but can provide all the functions required for the replication and packaging of the cotransfected amplicon DNA, which contains a functional pac signal. The resulting vector stocks are essentially free of helper virus.
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  •  FigureFigure 4.14.2 Analytical agarose gel showing restriction endonuclease patterns of clones from cosmid set C6Δa48Δa. Cosmids were either individually digested with DraI (lanes 3 to 7) and KpnI (lanes 8 to 12), or pooled and digested with PacI (lane 15). The reaction mixtures were loaded on a 0.4% agarose gel, and the fragments were separated overnight at 40 V in TAE electrophoresis buffer and stained with ethidium bromide. Lanes 3 and 8, cos6Δa; lanes 4 and 9, cos14; lanes 5 and 10, cos28; lanes 6 and 11, cos48Δa; lanes 7 and 12, cos56; lanes 1 and 14, high‐molecular‐weight DNA standard (Life Technologies); lanes 2 and 13, 1‐kb DNA ladder (Life Technologies).
    View Image
  •  FigureFigure 4.14.3 Photomicrographs showing GFP‐fluorescent cells in culture. For packaging of amplicon pHSVGFP (expressing the GFP gene; Aboody‐Guterman et al., 1997) into HSV‐1 particles, DNA from HSV‐1 cosmid set C6Δa48Δa and amplicon DNA were cotransfected into 2‐2 cells, and the cultures were examined after (A) 24 hr and (B) 60 hr.
    View Image
  •  FigureFigure 4.14.4 Amplicon structures. (A) Standard HSV‐1 amplicons are composed of (1) sequences from bacteria, including an origin of DNA replication (colE1) and an antibiotic resistance gene (ampr) that allow propagation of plasmid DNA in E. coli; (2) sequences from HSV‐1, in particular an origin of DNA replication (ori) and a DNA cleavage/packaging signal (pac), which support replication of the amplicon DNA and subsequent packaging into HSV‐1 particles in mammalian cells in the presence of helper functions; and (3) a transgene cassette containing one or more genes of interest. (B) In addition to the standard amplicon elements, HSV/AAV hybrid amplicons contain the AAV rep gene and a transgene cassette that is flanked by adeno‐associated virus (AAV) inverted terminal repeats (ITR).
    View Image

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Literature Cited

Literature Cited
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