Development of Molecular Genetic Interventions for HIV Infection

Clive Woffendin1, Udaykumar Ranga1, Gary J. Nabel1

1 University of Michigan Medical Center, Ann Arbor, Michigan
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 13.6
DOI:  10.1002/0471142905.hg1306s12
Online Posting Date:  May, 2001
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Abstract

This unit includes a set of protocols for the ex vivo transfer of genes into CD4+ T cells, to be used in the initial evaluation of genes protecting against HIV infection in gene therapy protocols. The describes isolating and expanding CD4+ T cells from the patient. The cells are then transduced by either retroviral transduction or particle‚Äźmediated gene transfer and reinfused into the patient. To monitor the effectiveness of gene transfer, genomic DNA is prepared from the patient's cells. Detection of vector DNA by PCR analysis of the patient's genomic DNA following gene transfer is also described in detail.

     
 
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Table of Contents

  • Basic Protocol 1: Ex Vivo Gene Transfer into CD4+–Enriched T Cells
  • Support Protocol 1: Retroviral Transduction of CD4+ Cells
  • Support Protocol 2: Particle‐Mediated Gene Transfer to CD4+ Cells
  • Support Protocol 3: Small‐Scale Preparation of Genomic DNA from Peripheral Blood Lymphocytes
  • Support Protocol 4: Detection of the Vector DNA by PCR Following Ex Vivo Gene Transfer to Peripheral Blood Lymphocytes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Ex Vivo Gene Transfer into CD4+–Enriched T Cells

  Materials
  • Human patient
  • Histopaque 1077 (Sigma), room temperature
  • D‐PBS (Dulbecco's phosphate‐buffered saline without Mg2+ and Ca2+; Life Technologies)
  • 1.33 µg/ml OKT 3 monoclonal antibody (Ortho) in HBSS ( appendix 2D) or borate buffer, pH 8.6 (see recipe)
  • Hanks' balanced salt solution (HBSS) without Ca2+ or Mg2+ (Life Technologies)
  • X‐Vivo 15 medium containing gentamycin and phenol red (Bio‐Whittaker), 37°C
  • Delavirdine (Upjohn)
  • IL‐2: aldesleukin (Proleukin from Chiron Therapeutics); reconstitute 2. 2 × 107 IU stock vial with 1.2 ml H 2O (1.8 × 107 IU/ml final; store at –20°C)
  • p24 ELISA kit (Coulter)
  • 25% human albumin (from hospital pharmacy)
  • Physiological saline (from hospital pharmacy)
  • Branched DNA analysis kit (Chiron Therapeutics)
  • Beckman GPR centrifuge with GH‐3.7 rotor (or equivalent) and aerosol containment device
  • 50‐ml polypropylene centrifuge tubes
  • 250‐ml plug‐seal centrifuge tubes
  • CD4+ cell selection devices (e.g., CELLector CD8 150‐cell culture flasks, Applied ImmuneSciences)
  • 225‐cm2 tissue culture flasks
  • 600‐ml Fenwal transfer pack (Baxter)
  • 10‐ml heparinized blood‐collection tubes
  • 10‐ml EDTA blood‐collection tubes
  • Additional reagents and equipment for growing cells in tissue culture and counting cells ( appendix 3G) retroviral transduction of cells (see protocol 2) or particle‐mediated gene transfer (see protocol 3), preparation of genomic DNA (see protocol 4), and detection of vector DNA by PCR (see protocol 5)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.CAUTION: All procedures should be performed following standard Biosafety Level 2/Biosafety Level 3 procedures as described in Biosafety in Microbiology and Biomedical Labs (CDC/NIH.HHS publication no. CDC39‐8395).

Support Protocol 1: Retroviral Transduction of CD4+ Cells

  Materials
  • Retrovirus‐containing supernatants, test and control (store at −70°C; thaw and warm to 37°C immediately prior to transduction)
  • 1 × 106 cell/ml suspension of activated CD4+ lymphocytes (see protocol 1, steps to ) and conditioned medium
  • 10 mg/ml protamine sulfate stock solution (must be FDA‐approved, e.g., Elkins‐Sinn)
  • IL‐2: aldesleukin (Proleukin from Chiron Therapeutics); reconstitute 2. 2 × 107 IU stock vial with 1.2 ml H 2O (1.8 × 107 IU/ml final; store at –20°C)
  • D‐PBS (Dulbecco's phosphate‐buffered saline without Mg2+ and Ca2+; Life Technologies)
  • X‐Vivo 15 medium containing gentamycin and phenol red (Bio‐Whittaker)
  • Delavirdine (Upjohn)
  • Beckman GPR centrifuge with GH‐3.7 rotor (or equivalent)
  • 3000‐ml Fenwal Lifecell tissue culture flasks (Baxter) or equivalent
  • Additional reagents and equipment for growing cells in tissue culture ( appendix 3G)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 2: Particle‐Mediated Gene Transfer to CD4+ Cells

  Materials
  • 1.6‐µm gold particles (Bio‐Rad)
  • 70% and 100% ethanol
  • 50% (v/v) glycerol
  • 1 mg/ml DNA to be transferred (test and control; linearized or supercoiled and gradient purified) in TE buffer or distilled H 2O
  • 2.5 M CaCl 2
  • 0.1 M spermidine (Sigma)
  • Activated CD4+ T lymphocytes (see protocol 1, steps to ) and conditioned medium
  • Biolistic PDS‐1000/He particle‐delivery system (Bio‐Rad)
  • Macrocarrier disks for Biolistic PDS‐100/He (Bio‐Rad)
  • Beckman GPR centrifuge with GH 3.7 rotor (or equivalent)
  • 35‐mm diameter petri dishes
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.

Support Protocol 3: Small‐Scale Preparation of Genomic DNA from Peripheral Blood Lymphocytes

  Materials
  • PBL from clinical samples (e.g., see protocol 1, steps and ) to be analyzed for the presence of transduced DNA
  • Untransduced PBL from normal HIV‐seronegative donor for background DNA
  • PBS ( appendix 2D), sterile
  • Filler cells: untransduced PBL or any transformed T cell lines—e.g., CEM cells or Jurkat cells
  • recipeQuick lysis solution A (see recipe)
  • recipeQuick lysis solution B (see recipe)
  • Light mineral oil (Sigma)
  • Aerosol‐barrier pipet tips
  • Heating block
  • Additional reagents and equipment for counting cells ( appendix 3G)

Support Protocol 4: Detection of the Vector DNA by PCR Following Ex Vivo Gene Transfer to Peripheral Blood Lymphocytes

  Materials
  • 10× reaction buffer without MgCl 2 (Promega; supplied with the enzyme)
  • 25 mM MgCl 2 (Promega; supplied with the enzyme)
  • recipe1 mM dNTP mix (see recipe)
  • recipe2.5 µM sense and antisense primer solutions (see recipe)
  • Paraffin wax (Aldrich)
  • 5 U/µl Taq DNA polymerase (Promega)
  • Template DNA: lysates of serial cell dilutions (see protocol 4) including positive DNA standards
  • 0.65‐ml thin‐walled PCR vials (Diagnostic Concepts)
  • Clean razor blade
  • 25‐ml glass beaker
  • Heating block accommodating 0.65‐ml PCR vials
  • Template Tamer PCR work station (Coy Laboratory Products)
  • Thermal cycler
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Figures

Videos

Literature Cited

Literature Cited
   Leavitt, M.C., Yu, M., Yamada, O., Kraus, G., Looney, G., Poeschla, E., Wong‐Staal, F. 1994. Transfer of an anti‐HIV‐1 ribozyme gene into primary human lymphocytes. Hum. Gene Ther. 5(9):1115‐1120.
   Levine, L.L., Mosca, J.D., Riley, J.L., Carroll, R.G., Vahey, M.T., Jagodzinski, L.L., Wagner, K.F., Mayers, D.L., Burke, D.S., Weislow, O.S., St. Louis, D.C., and June, C.H. 1996. Antiviral effect and ex vivo CD4 T cell proliferation in HIV positive patients as a result of CD28 costimulation. Science 272:1939‐1943.
   Nabel, G.J., Gene therapy approaches to AIDS. 1994. AIDS 8 (suppl. 1):S61‐69.
   Nabel, G.J., Fox, B.A., Post, L., Thompson, C.B., and Woffendin, C. 1994. A molecular genetic intervention for AIDS‐effects of a transdominant negative form of Rev. Hum. Gene Ther. 5:79‐92.
   Poli, G. and Fauci, A.S. 1993. General guidelines for experimenting with HIV. In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, and W. Strober, eds.) pp. 12.1.1‐12.1.7. John Wiley & Sons, New York.
   Sanford, J.C., Smith, F. D., and Russell, J.A. 1993. Optimizing the biolistic process for different biological applications. Methods Enzymol. 217:483‐509.
   Strober, W. 1996. Obtaining human peripheral blood cells. In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, and W. Strober, eds.) pp. A.3.13‐A.3.14. John Wiley & Sons, New York.
   Woffendin, C., Yang, Z.Y., Ranga, U., Xu, L., Yang, N.S., Sheehy, M.J., and Nabel, G.J. 1994. Nonviral and viral delivery of HIV protective gene into primary T cells. Proc. Natl. Acad. Sci. U.S.A. 91:11581‐11585.
   Woffendin, C., Ranga, U., Yang, Z.Y., Xu, L., and Nabel, G.J. 1996. Expression of a protective gene prolongs survival of T cells in HIV infected patients. Proc. Natl. Acad. Sci. U.S.A. 93:2889‐2894.
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