Production and Titration of Lentiviral Vectors

Patrick Salmon1, Didier Trono2

1 Department of Neuroscience, Faculty of Medicine, University of Geneva, Geneva, null, 2 School of Life Sciences, École Polytechnique Fédérale de, Lausanne and “Frontiers in Genetics,” National Center for Competence in Research, Lausanne, null
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 12.10
DOI:  10.1002/0471142905.hg1210s54
Online Posting Date:  July, 2007
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Abstract

Lentiviral vectors have emerged over the last decade as powerful, reliable and safe tools for stable gene transfer in a wide variety of mammalian cells. Unlike other vectors derived from oncoretroviruses, they allow for stable gene delivery into most nondividing primary cells. This is why LVs are becoming useful and promising tools for future gene and cell therapy approaches. Lentivectors (LVs) derived from HIV-1 have gradually evolved to display many desirable features aimed at increasing both their safety and their versatility. These latest designs are reviewed in this unit. This unit also describes protocols for production and titration of LVs that can be implemented in a research laboratory setting, with an emphasis on standardization to improve transposability of results between laboratories Curr. Protoc. Hum. Genet. 54:12.10.1-12.10.24. © 2007 by John Wiley & Sons, Inc.

Keywords: Lentiviral vectors; gene therapy; gene delivery

     
 
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Table of Contents

  • Introduction
  • Basic Protocol: Production of High-Titer HIV-1-Based Vector Stocks by Transient Transfection of 293T Cells
  • Support Protocol 1: Determination of Total Vector Concentration Using Anti-p24 Immunoassay
  • Support Protocol 2: Biological Titration of Lentivectors Using Flow Cytometry
  • Support Protocol 3: Biological Titration of Lentivectors by Quantitative PCR (QPCR)
  • Support Protocol 4: RCR Assay for LVS: Real-Time Quantitative PCR (QPCR) Detection of Replication-Competent Recombinants
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol: Production of High-Titer HIV-1-Based Vector Stocks by Transient Transfection of 293T Cells

 Materials
  • 293T/17 cells (ATCC cat. no. SD-3515)
  • Dulbecco's modified Eagle medium/10% FBS (DMEM-10; appendix 2D)
  • 0.05% trypsin/EDTA (e.g., Invitrogen)
  • Plasmids (available from the Trono lab, http://tronolab.epfl.ch):
    • pMD2G (encoding the VSV G envelope protein)
    • pWPT-GFP (second-generation transfer vector, abbreviated “pWPT”)
    • pRRL-cPPT-PGK-GFP-W-SIN (third-generation transfer vector, abbreviated “pRRL”)
    • psPAX2 (encoding HIV-1 Gag, Pol, Tat and Rev proteins)
    • pMDLgag/polRRE (encoding the HIV-1 Gag and Pol proteins)
    • pRSVrev (encoding the HIV-1 Rev protein)
  • TE buffer, pH 8.0 (appendix 2A)
  • Buffered water (see recipe)
  • 0.5 M CaCl2 (see recipe)
  • 2× HeBS (see recipe)
  • Phosphate-buffered saline (PBS; appendix 2D), 37°C
  • 75% (v/v) ethanol in spray bottle
  • 20% (w/v) sucrose (SigmaUltra from Sigma), filter-sterilized through 0.22-µm filter (store at 4°C)
  • CellGro Stem Cell Growth Medium (CellGenix GmbH, http://www.cellgenix.com/; optional, if subsequent experiments require absence of serum)
  • Phosphate-buffered saline containing Ca2+ and Mg2+ (e.g., Invitrogen; optional, if subsequent experiments require absence of protein)
  • 10-cm tissue culture dishes
  • 15- and 50-ml conical centrifuge tubes, sterile
  • 50-ml syringes and 0.45-µm pore size PVDF filters
  • 30-ml Beckman Konical ultracentrifuge tubes (Beckman Coulter)
  • Ultracentrifuge with SW 28 rotor (Beckman Coulter) or equivalent
  • Additional reagents and equipment for tissue culture (appendix 3G) and quantitation of DNA by absorption spectroscopy (appendix 3D)

CAUTION: P2 practices require that open tubes always be handled in the laminar flow hood. Tubes can be taken out of the laminar flow only when they are closed, and that they be sprayed with 75% ethanol. All solid waste and plasticware must be discarded in a trash bin in the laminar flow hoods and all liquids must be aspirated into a liquid waste bottle containing fresh concentrated bleach. Refill the liquid waste bottle with fresh bleach when the color of the liquid is no longer yellow. When full, bags are closed inside the laminar flow hood, then autoclaved. When full, and at least 15 min after neutralization with fresh bleach, the liquid waste bottle can be emptied into a regular sink. In case of a major spill of vector-containing liquid, absorb liquid with paper towels and neutralize with fresh concentrated bleach prior to disposal. In case there is a leak in the SW 28 buckets, remove the tubes in the hood, fill the buckets with 75% ethanol, and invert them several times. Leave under the hood for ³20 min. Discard the 75% ethanol and remove the conical adapters under the hood. Spray the adapters with 75% ethanol and leave them under the hood for >20 min.

NOTE: All solutions and equipment coming into contact with living cells must be sterile, and proper aseptic technique should be used accordingly.

NOTE: All culture incubations should be performed in a humidified, 37°C, 5% CO2 incubator unless otherwise specified.

Support Protocol 1: Determination of Total Vector Concentration Using Anti-p24 Immunoassay

 Materials
  • Lentiviral vector sample for titration (see Basic Protocol) and positive control (provided with p24 ELISA kit)
  • Phosphate-buffered saline (PBS; appendix 2D)
  • 5% (v/v) Triton X-100 (store at room temperature)
  • p24 ELISA kit (Perkin-Elmer cat. no. NEK05000 1KT) including:
    • Anti-p24-coated ELISA 96-well plates (or well strips)
    • Adhesive plate covers
    • p24 wash buffer
    • Biotinylated anti-p24 polyclonal antibody for primary reaction
    • Streptavidin–horseradish peroxidase (HRP) for secondary reaction
    • Substrate: o-phenylenediamine HCl (OPD) tablets
    • Substrate diluent
    • Stop solution: 4 N sulfuric acid
  • ELISA plate reader with 492 nm filter

Support Protocol 2: Biological Titration of Lentivectors Using Flow Cytometry

 Materials
  • HeLa cells (ATCC cat. no. CCL-2)
  • Dulbecco's modified Eagle medium/10% FBS (DMEM-10; appendix 2D)
  • Lentiviral vector sample for titration, carrying GFP transgene (see Basic Protocol) and positive control (lentiviral supernatant already titered)
  • Phosphate-buffered saline (PBS), pH 7.4 (appendix 2D)
  • 0.05% trypsin/EDTA (e.g., Invitrogen)
  • 1% (w/v) formaldehyde: dilute 1 ml of 37% formaldehyde (Sigma) in 36 ml PBS (appendix 2D); store at 4°C
  • 6-well tissue culture plates (e.g., BD Biosciences)
  • Fluorescence-activated cell sorter (FACS, Becton Dickinson; with 488 nm excitation laser and green filter) or equivalent flow cytometer, and appropriate tubes

NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.

NOTE: All culture incubations should be performed in a humidified, 37°C, 5% CO2 incubator unless otherwise specified.

Support Protocol 3: Biological Titration of Lentivectors by Quantitative PCR (QPCR)

 Materials
  • HeLa cells (ATCC cat. no. CCL-2)
  • Lentiviral vector (LV) sample for titration (see Basic Protocol) and DNA from positive and negative control cells (see annotation to step 1)
  • DNAeasy Genomic DNA Extraction Kit (Qiagen)
  • Kit for preparing QPCR master mix (RT-QP2X-03; Eurogentec), including 2× reaction buffer
  • 10× TaqMan GAG set (see recipe)
  • 10× TaqMan HB2 set (see recipe)
  • MicroAmp 96-well optical reaction plate (Applied Biosystems)
  • Optical caps (Applied Biosystems)
  • Centrifuge with microtiter plate carrier
  • Real-time PCR machine (7700 Sequence Detector, Applied Biosystems)
  • Computer running ABI Prism software (Applied Biosystems) and Microsoft Excel
  • Additional reagents and equipment for transducing HeLa cells with lentivectors (Support Protocol 2)

Support Protocol 4: RCR Assay for LVS: Real-Time Quantitative PCR (QPCR) Detection of Replication-Competent Recombinants

 Materials
  • HeLa cells (ATCC cat. no. CCL-2)
  • Lentiviral vector (LV) sample(s) and standards for titration (see Basic Protocol; also see last annotation to step 1 below)
  • 8E5 cells (ATCC cat. no. CRL-8993)
  • DNAeasy Genomic DNA Extraction Kit (Qiagen)
  • Kit for preparing QPCR master mix (RT-QP2X-03; Eurogentec), including 2× reaction buffer
  • 10× TaqMan GAG set (see recipe) or 10× TaqMan POL set (see recipe)
  • 10× TaqMan HB2 set (see recipe)
  • MicroAmp 96-well optical reaction plate (Applied Biosystems)
  • Optical caps (Applied Biosystems)
  • Centrifuge with microtiter plate carrier
  • Real-time PCR machine (7700 Sequence Detector, Applied Biosystems)
  • Computer running ABI Prism software (Applied Biosystems) and Microsoft Excel
  • Additional reagents and equipment for transducing HeLa cells with lentivectors (Support Protocol 2)

CAUTION: ATCC recommends that 8E5 cells be handled in a P2 laboratory. Indeed, although they contain a full copy of noninfectious HIV, they can form syncytia with uninfected CD4+ cells.
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Figures

  •  FigureFigure 12.10.1 Representative FACS analysis of HeLa cells used for titration of GFP-coding LV. HeLa cells (105) were incubated with various volumes (5 µl, 50 µl, and 500 µl) of a supernatant containing an LV-expressing GFP under the control of the human PGK promoter (pRRL), as described above. After 5 days, cells were detached, fixed, and analyzed by FACS for GFP fluorescence (x axis, 4-decade log scale, FL1) versus number of cells (y axis, linear scale). The percentage of GFP-expressing cells was measured by placing a marker discriminating between GFP-negative (mean of fluorescence intensity 3 to 4) and GFP-positive cells (mean of fluorescence intensity 200).
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  •  FigureFigure 12.10.2 Representative QPCR analysis used for titration of HIV-1-based LVs. DNA from HeLa cells transduced with serial 10-fold dilutions of RRL-GFP vectors was subjected simultaneously to FACS titration analysis (Support Protocol 2) and QPCR analysis (Support Protocol 3). A sample of each dilution was submitted to QPCR amplification and monitoring using a Perkin-Elmer 7700 (Applied Biosystems) and sets of primers and probes specific for HIV gag sequences (GAG-FAM, panel A) or -actin sequences (HB2-VIC, panel B). Amplification plots were displayed and cycle threshold values (Ct) were set as described in text. Values of GAG Ct and HB2 Ct were exported in an Excel worksheet to calculate Ct values (x axis, linear scale) and plot them against copy number values (y axis, log scale); panel C). The sample giving 10% of GFP-positive cells was set as HeLa DNA containing 0.1 copy of HIV sequences per cell. The regression curve can then be used to calculate GAG copy numbers (y value) of unknown samples by applying the formula to Ct values (y values) of the sample. For the color version of this figure go to http://www.currentprotocols.com.
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  •  FigureFigure 12.10.3 Evolution in the design of HIV-1 based LV vectors. HIV-1-based LV vectors are derived from wild-type HIV-1 by dissociation of the trans-acting components (blue boxes, above HIV genome) coding for structural and accessory proteins, and the cis-acting sequences required for packaging and reverse transcription of the genomic RNA (gold boxes, below HIV genome). Sequences added between two vector versions are in red. Abbreviations: CMV, human cytomegalovirus immediate-early promoter; RRE, rev-responsive element; RSV, Rous sarcoma promoter; poly(A), polyadenylation site; U3-R-U5, HIV-1 LTR; SD, major splice donor; , HIV-1 packaging signal; cPPT, central polypurine tract; SA, splice acceptor; dPPT, distal (3¢) polypurine tract; Prom, promoter of the internal expression cassette; TG, transgene of the internal expression cassette; U3, self-inactivating deletion of the U3 part of the HIV-1 LTR; WPRE, Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element. For the color version of this figure go to http://www.currentprotocols.com.
    View Image
  •  FigureFigure 12.10.4 SIN-HLox vector design and predicted life cycle. (A) Schematic diagram of the SIN-HLox 3¢ LTR. The sizes of the LTR segments (U3, R, U5) and positions of major transcriptional elements are indicated. After removal of the –418 to –18 segment of the HIV-1 LTR, only 124 nt are left upstream of R, comprising 71 nt overlapping with nef, 35 nt of the proximal region of U3, and 18 nt of the distal end of U3. In this self-inactivating (SIN) LTR, the loxP sequence (58 nt) is inserted within the SIN deletion. (B) Schematic diagram of the SIN-HLox vector life cycle. The basic elements of HIV-based vectors, i.e., LTRs, SD, SA (splice donor and acceptor, respectively), (packaging signal), Ga (fragment of gag), RRE (Rev-responsive RNA), have been described (Naldini et al., 1996). Abbreviations: CMV, human cytomegalovirus immediate early promoter; EGFP, enhanced green fluorescent protein; IRES, internal ribosomal entry site of the encephalomyocarditis virus; HSV1-TK, thymidine kinase of herpes simplex virus type 1.
    View Image
  •  FigureFigure 12.10.5 Loaded LTRs. (A) Schematic diagram of the LTR present in pLVTHM vectors as described in Wiznerowicz and Trono (2003). This LTR is based on the SIN-Hlox LTR described above. U3 represents the deleted SIN U3, TetO is composed of seven Tet operator sequences, H1 is the polymerase-III H1-RNA gene promoter, and siRNA is where the small interfering RNA sequence is cloned. In the presence of tetracycline (depicted as the light-shaded circle under KRAB at the upper left of panel A), tTR-KRAB does not bind to the TetO sequences. In the absence of tetracycline, tTR-KRAB binds to the TetO sequences and represses transcription from the H1 promoter. (B) Schematic diagram of the LTR present in pRIX vectors. This LTR is based on the SIN-Hlox LTR described above. U3 represents the deleted SIN U3. Insulator is composed of a 242 bp sequence derived from the chicken -globin gene (Genbank locus number GGU78775, nt 26 to 245), which contains DNA elements with both chromatin-barrier and enhancer-blocking activities (Recillas-Targa et al., 2002).
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  •  FigureFigure 12.10.6 Multicistronic vector LTRs. Schematic diagram of current LV used for expression of multiple cistrons (shown as plasmids in producer cells). (A) Schematic diagram of the pRRL LV. Abbreviations: RSV, Rous Sarcoma Virus LTR U3 sequence; U3, SIN deletion of the U3 region of LTR; , packaging signal; cPPT, central HIV polypurine tract; hPGK, human PGK promoter; GFP, enhanced green fluorescent protein. (B) Schematic diagram of the pRRL-PGK-IRES-GFP LV. Abbreviations: GOI, gene of interest; IRES, internal ribosomal entry site of the encephalomyocarditis virus (see Fig. 12.10.4). (C) Schematic diagram of a bicistronic LV with a tissue-specific cassette in line with the ubiquitous PGK-GFP cassette. Abbreviations: Pro, tissue-specific promoter; Intron, intronic sequence containing regulatory elements; Enh, tissue-specific enhancer. (D) Schematic diagram of the pRPA LV. Same as in C but with an inverted cassette for the expression of the gene of interest, back-to-back with PGK-GFP ubiquitous cassette. Abbreviations: pA, polyadenylation signal from the herpes simplex 1 virus thymidine kinase gene (Genbank locus number HE1CG, nt 46565 to 46693). Transcription units (mRNAs) are represented as dotted arrows. Transcript 1 is the LV genome. It is synthesized only in producer cells from the active RSV-RU5 LTR. Transcripts 2 and 3 are synthesized from internal cassettes. They can be present in producer cells and in target cells.
    View Image
  •  FigureFigure 12.10.7 Troubleshooting diagram for lentiviral vector production and transduction.
    View Image

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Literature Cited

Literature Cited
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