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

Cornel Fraefel¹

¹Institute of Virology, Zurich, Switzerland

Publication Name:

Current Protocols in Human Genetics

Unit Number:

Unit 12.12

DOI:

10.1002/0471142905.hg1212s33

Online Posting Date:

August, 2002

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Abstract

Herpes simplex virus type 1 (HSV-1)-based amplicon vectors contain only ~1% of the viral genome. Consequently, replication and packaging of amplicons depend on helper functions provided either by replication-defective mutants of HSV-1 (helper viruses) 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. 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. Resulting stocks of packaged amplicon vectors are free of contaminating helper virus. The basic protocol describes the cotransfection of amplicon and cosmid DNA into 2-2 cells and the harvesting of packaged vector particles. Support protocols describe preparing cosmid DNA and methods for determining the titers of amplicon stocks.

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Unit Introduction
Basic Protocol: Preparation of Helper Virus–Free Amplicon Stocks
Support Protocol 1: Preparation of HSV-1 Cosmid DNA for Transfection
Support Protocol 2: 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)
0.25% trypsin/0.02% EDTA (Life Technologies)
Opti-MEM I reduced-serum medium (Life Technologies)
PacI-digested cosmid DNA of set C6a48a (see Support Protocol 1)
HSV-1 amplicon DNA (maxiprep DNA isolated from E. coli)
LipofectAMINE reagent (Life Technologies)
10%, 30% and 60% (w/v) sucrose in PBS
Phosphate-buffered saline (PBS; appendix 2D)

75-cm² tissue culture flasks
Humidified 37°C, 5% CO₂ incubator
60-mm-diameter tissue culture dishes
15-ml conical centrifuge tubes
Dry ice/ethanol bath
Probe sonicator
0.45-µm syringe-tip filters (use Sarstedt polyethersulfone membrane filters)
20-ml disposable syringes
30-ml centrifuge tubes (Beckman Ultra-Clear 25 × 89 mm and 14 × 95 mm)
Sorvall SS-34 rotor
Fiber-optic illuminator
Ultracentrifuge with Beckman SW28 and SW40 rotors

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

Materials

E. coli clones of HSV-1 cosmid set C6a48a,which includes cos6a, cos14, cos28, cos48a, and cos56 (see Fig. 12.12.1)
SOB medium containing 50 µg/ml ampicillin (SOB/amp; see recipe)
Dimethyl sulfoxide (DMSO)
Plasmid Maxi Kit (Qiagen), which 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
TE buffer, pH 7.5 (appendix 2D)
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 3C)
24:1 (v/v) chloroform/isoamyl alcohol
100% ethanol
3 M sodium acetate, pH 5.5 (appendix 2D)
17 × 100–mm graduated snap-cap tubes (e.g., Falcon 2059), sterile
Sorvall GSA and SS-34 rotors
65° and 37°C water baths
250-ml polypropylene centrifuge tubes
30-ml centrifuge tubes

Support Protocol 2: 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)
PBS (appendix 2D)
Samples collected from vector stocks (see Basic Protocol, steps , , or )
4% (w/v) paraformaldehyde solution, pH 7.0 (see recipe)
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
Inverted light microscope

NOTE: The titers expressed as transducing units per milliliter (t.u./ml) are relative and do not necessarily reflect numbers of infectious vector particles per milliliter. Factors influencing relative transduction efficiencies include: (1) the cells used for titration, (2) the promoter regulating the expression of the transgene, (3) the transgene, and (4) the sensitivity of the detection method.

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

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Figures

Figure 12.12.1

Helper virus–free packaging of HSV-1 amplicons into HSV-1 particles. (A) The HSV-1 genome (152 kbp) is composed of unique long (U_L) and unique short (U_S) segments (horizontal lines), which are flanked by inverted repeats (open rectangles):IR_S, internal repeat of the short segment; TRM_S, terminal repeat of the short segment;IR_L, internal repeat of the long segment;TR_L, terminal repeat of the long segment. The origins of DNA replication, ori_S (solid circle) and ori_L (open circle), and the DNA cleavage/packaging signals,pac (solid rectangles), are also shown. (B) Schematic diagramof HSV-1 cosmid set C6a48a with deleted pac signals (X) (Fraefel et al., 1996; Cunningham and Davison, 1993), which includes cos6a, cos14, cos28, cos48a, and cos56. (C) 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 DNA from cosmid set C6a48a. In the absence of the pac signals, this cosmid set 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.

View Image
Figure 12.12.2

Analytical agarose gel showing restriction endonuclease patterns of clones from cosmid set C6a48a. Cosmids were either individually digested with DraI (lanes 3-7) and KpnI (lanes 8-12), 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, cos6a; lanes 4 and 9, cos14; lanes 5 and 10, cos28; lanes 6 and 11, cos48a; 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).

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Figure 12.12.3

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 C6a48a and amplicon DNA were cotransfected into 2-2 cells, and the cultures were examined after (A) 24 hr and (B) 60 hr.

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Figure 12.12.4

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 (amp^r) 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

Literature Cited

Literature Cited
	Aboody-Guterman, K.S., Pechan, P.A., Rainov, N.G., Sena-Esteves, M., Jacobs, A., Snyder, E.Y., Wild, P., Schraner, E., Tobler, K., Breakefield, X.O., and Fraefel, C. 1997. Green fluorescent protein as a reporter for retrovirus and helper virus–free HSV-1 amplicon vector-mediated gene transfer into neural cells in culture and in vivo. Neuroreport 8:3801-3808.
	Cunningham, C. and Davison, A.J. 1993. A cosmid-based system for constructing mutants of herpes simplex virus type 1. Virology 197:116-124.
	Fraefel, C., Song, S., Lim, F., Lang, P., Yu, L., Wang, Y., Wild, P., and Geller, A.I. 1996. Helper virus-free transfer of herpes simplex virus type 1 plasmid vectors into neural cells. J. Virol. 70:7190-7197.
	Fraefel, C., Jacoby, D.R., Lage, C., Hilderbrand, H., Chou, J.Y., Alt, F.W., Breakefield, X.O. and, Majzoub, J.A. 1997. Gene transfer into hepatocytes mediated by helper virus-free HSV/AAV hybrid vectors. Mol. Med. 3:813-825.
	Fraefel, C., Breakefield, X.O., and Jacoby, D.R. 1998. HSV-1 amplicon. In Gene Therapy for Neurological Disorders and Brain Tumors (E.A. Chiocca and X.O. Breakefield, eds.) pp. 63-82. Humana Press, Totowa, N.J.
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	Kwong, A.D. and Frenkel, N. 1985. The herpes simplex virus amplicon. IV.Efficient expression of chimeric chicken ovalbumin gene amplified within defective virus genomes. Virology 142:421-425.
	Lim, F., Hartley, D., Starr, P., Lang, P., Song, S., Yu, L., Wang, Y., and Geller, A. I. 1996. Generation of high-titer defective HSV-1 vectors using an IE2 deletion mutant and quantitative study of expression in cultured cortical cells. BioTechniques 20:458-469.
	Pechan, P.A., Fotaki, M., Thompson, R.L., Dunn, R.J., Chase, M., Chiocca, E.A., and Breakefield, X.O. 1996. A novel “piggyback” packaging system for herpes simplex virus amplicon vectors. Hum. Gene Ther. 7:2003-2013.
	Smith, I.L., Hardwicke, M.A., and Sandri-Goldin, R.M. 1992. Evidence that the herpes simplex virus immediate-early protein ICP27 acts post-transcriptionally during infection to regulate gene expression. Virology 186:74-86.
	Spaete, R.R. and Frenkel, N. 1982. The herpes simplex virus amplicon: A new eucaryotic defective-virus cloning-amplifying vector. Cell 30:295-304.
	Wang, S. and Vos, J. 1996. A hybrid herpesvirus infectious vector based on Epstein-Barr virus and herpes simplex virus type 1 for gene transfer into human cells in vitro and in vivo. J. Virol. 70:8422-8430.