Methods for Cancer Gene Therapy

Yok‐Lam Kwong1, Shu‐Hsia Chen1, Savio L.C. Woo1

1 Baylor College of Medicine, Houston, Texas
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 13.5
DOI:  10.1002/0471142905.hg1305s12
Online Posting Date:  May, 2001
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Abstract

First, the testing of therapeutic gene vectors in vitro is described. This is followed by a discussion of the administration of therapeutic vectors in vivo. Two methods for assessing the development of anti‐tumor immunity after cytokine gene therapy are provided. In addition, two methods for the generation of murine tumor models in syngeneic hostsone subcutaneous and one orthotopicare also included.

     
 
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Table of Contents

  • Basic Protocol 1: In Vitro Functional Characterization of ADV.β‐Gal Reporter Vector
  • Basic Protocol 2: In Vitro Characterization of ADV.tk Suicide Gene Vector by Biochemical Assay
  • Alternate Protocol 1: In Vitro Functional Characterization of ADV.tk Suicide Gene Vector by Cytotoxicity Assay
  • Alternate Protocol 2: In Vitro Functional Characterization of ADV.tk Suicide Gene Vector by Assaying Bystander Effect
  • Basic Protocol 3: In Vitro Functional Characterization of ADV.IL‐2 Cytokine Gene Vector
  • Basic Protocol 4: In Vivo Administration of Therapeutic Vectors
  • Basic Protocol 5: Demonstration of Systemic Anti‐Tumor Immunity After Cytokine Gene Therapy by Second‐Site Rechallenge Protection
  • Basic Protocol 6: Demonstration of Systemic Anti‐Tumor Immunity After Cytokine Gene Therapy by Cytotoxic T Cell Assay
  • Support Protocol 1: Generation of Subcutaneous Tumors to Establish Tumor Models for In Vivo Gene Therapy
  • Support Protocol 2: Generation of Hepatic Metastases for In Vivo Gene Therapy
  • Reagents and Solution
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: In Vitro Functional Characterization of ADV.β‐Gal Reporter Vector

  Materials
  • Culture medium appropriate to the cell line used, with 1% FBS and with the normal amount of FBS used for growth (see appendix 3G)
  • Tumor cell line
  • Adenoviral vector: ADV.β‐Gal, pfu determined by plaque assay (unit 12.4); store at –70°C
  • Phosphate‐buffered saline (PBS; appendix 2D), ice‐cold
  • 0.5% (w/v) glutaraldehyde in PBS, ice‐cold
  • Xgal staining solution (see recipe)
  • 6‐well tissue culture plates (Costar)
  • Inverted microscope
  • Additional reagents and equipment for plaque assay of adenoviral vectors (unit 12.4) and growing cells in tissue culture ( appendix 3G)
NOTE: All culture incubations are performed in a humidifed 37°C, 5% CO 2 incubator unless otherwise specified.NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique must be used accordingly.

Basic Protocol 2: In Vitro Characterization of ADV.tk Suicide Gene Vector by Biochemical Assay

  Materials
  • Tumor cell line
  • Adenoviral vectors: ADV.tk with ADV.β‐Gal as control, pfu determined by plaque assay (unit 12.4); store at –70°C
  • Culture medium appropriate to the cell line used, with 1% FBS and with the normal amount of FBS used for growth, 37°C
  • Phosphate‐buffered saline (PBS; appendix 2D), 4°C
  • Buffer A (see recipe), 4°C
  • Buffer B (see recipe)
  • 65 µCi/ml 3H‐labeled acyclovir (DuPont NEN; 35 Ci/mmol)
  • Double‐distilled H 2O
  • Ganciclovir wash solution (see recipe)
  • 95% ethanol
  • 6‐well tissue culture plates (Costar)
  • 15‐ml Corning centrifuge tubes
  • Refrigerated centrifuge
  • DE81 filter paper (Whatman), cut into 1.7 × 1.7–cm square pieces
  • 200‐ml flasks
  • Additional reagents and equipment for growing cells in tissue culture counting, and trypsinizing cells ( appendix 3G)
NOTE: All culture incubations are performed in a humidifed 37°C, 5% CO 2 incubator unless otherwise specified.NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique must be used accordingly.

Alternate Protocol 1: In Vitro Functional Characterization of ADV.tk Suicide Gene Vector by Cytotoxicity Assay

  • 10 mg/ml ganciclovir (Hoffmann‐LaRoche) in double‐distilled water (sterile; store up to 6 months at –20°C)
  • Additional reagents and equipment for determining number of viable cells by trypan blue staining ( appendix 3G)
NOTE: All culture incubations are performed in a humidifed 37°C, 5% CO 2 incubator unless otherwise specified.NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique must be used accordingly.

Alternate Protocol 2: In Vitro Functional Characterization of ADV.tk Suicide Gene Vector by Assaying Bystander Effect

  • 10 mg/ml ganciclovir (Hoffman‐LaRoche) in double‐distilled water (sterile; store up to 6 months at –20°C)
  • 50‐ml conical polypropylene centrifuge tubes
NOTE: All culture incubations are performed in a humidifed 37°C, 5% CO 2 incubator unless otherwise specified.NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique must be used accordingly.

Basic Protocol 3: In Vitro Functional Characterization of ADV.IL‐2 Cytokine Gene Vector

  Materials
  • Tumor cell line
  • Culture medium appropriate to the cell line used, with 1% FBS and with the normal amount of FBS used for growth, 37°C
  • Adenoviral vector: ADV.IL‐2 with ADV.β‐Gal as control, pfu determined by plaque assay (unit 12.4); store at –70°C
  • ELISA kit for murine IL‐2 (Endogen)
  • CTLL‐2 cells (ATCC #TIB 214) growing in tissue culture
  • [3H]thymidine (e.g., DuPont NEN)
  • PBS ( appendix 2D)
  • IL‐2 solutions for standard curve
  • 6‐well tissue culture plates
  • 96‐well flat‐bottom microtiter plates (Costar)
  • Semiautomated cell harvester and filters
NOTE: All culture incubations are performed in a humidifed 37°C, 5% CO 2 incubator unless otherwise specified.NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique must be used accordingly.

Basic Protocol 4: In Vivo Administration of Therapeutic Vectors

  Materials
  • ADV.tk or other therapeutic viral vector and ADV.β‐Gal as control, pfu determined by plaque assay (unit 12.4); store at –70°C
  • Vector dilution buffer (see recipe), sterile
  • Mice with 5 × 5–mm tumors (see Support Protocols protocol 91 & protocol 102)
  • Avertin (see recipe), sterile
  • 10 mg/ml ganciclovir (Hoffman‐LaRoche) in double‐distilled water (sterile; store up to 6 months at –20°C)
  • PBS ( appendix 2D), sterile
  • Surgical instruments including electric cauterizer
  • Ear tags for animal identification
  • Hamilton syringe and 30‐G needle
  • Balance appropriate for weighing animals
  • Additional reagents and equipment for plaque assay of adenoviral vectors unit 12.4)
NOTE: The following procedures correspond to the animal experimentation protocols used at the authors' institution. For workers at other institutions, all procedures should be approved by the animal care and use committee of the individual institution, and should be in accordance with established guidelines of animal welfare. While performing animal surgery, workers should wear caps, face masks, gloves, and protective clothing. All survival surgery should be performed in a sterile cabinet using aseptic technique. Animals should be euthanized as soon as experimental objectives are reached. Euthanasia of animals must be carried out in accordance with institutional guidelines.

Basic Protocol 5: Demonstration of Systemic Anti‐Tumor Immunity After Cytokine Gene Therapy by Second‐Site Rechallenge Protection

  Materials
  • Naive animals (mice or rats for murine tumor cell lines)
  • Parental tumor cells used to create tumor model and syngeneic tumor or cell line of a different type
  • Animals that received therapeutic ADV.IL‐2 vector (see protocol 6)
  • Additional reagents and equipment for generation of subcutaneous tumors (see protocol 9)
NOTE: The following procedures correspond to the animal experimentation protocols used at the authors' institution. For workers at other institutions, all procedures should be approved by the animal care and use committee of the individual institution, and should be in accordance with established guidelines of animal welfare. While performing animal surgery, workers should wear caps, face masks, gloves, and protective clothing. All survival surgery should be performed in a sterile cabinet using aseptic technique. Animals should be euthanized as soon as experimental objectives are reached. Euthanasia of animals must be carried out in accordance with institutional guidelines.

Basic Protocol 6: Demonstration of Systemic Anti‐Tumor Immunity After Cytokine Gene Therapy by Cytotoxic T Cell Assay

  Materials
  • Animals that received therapeutic vector (see protocol 6) designed to produce anti‐tumor immunity
  • Animals that received control vector
  • DMEM medium (Life Technologies) containing 10% FBS
  • Red blood cell lysing buffer: 8.3 g/liter ammonium chloride/0.01 M Tris·Cl, sterile (available premixed from Sigma)
  • Fetal bovine serum (FBS)
  • T cell culture medium A (see recipe), 37°C
  • Tumor cell line against which cytotoxicity is directed
  • Control tumor cell line against which cytotoxicity is not directed
  • Recombinant interleukin 2 (rIL‐2; Pharmingen)
  • T cell culture medium B (see recipe)
  • 1 mCi/ml 51Cr (Amersham; 200 to 500 mCi/mg Cr)
  • 2% SDS solution
  • Sterile glass microscope slides
  • Refrigerated centrifuge
  • Column for filtering splenocytes: Pasteur pipets packed with cotton wool and autoclaved
  • 6‐well tissue culture plates
  • Gamma radiation source (e.g., Gammacell 1000, Nordion International)
  • 96‐well flat‐bottom tissue culture plates
  • Multiwell supernatant harvesting system (Skatron)
  • Additional reagents and equipment for determining number of viable cells by trypan blue staining ( appendix 3G)
NOTE: All culture incubations are performed in a humidifed 37°C, 5% CO 2 incubator unless otherwise specified.NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique must be used accordingly.

Support Protocol 1: Generation of Subcutaneous Tumors to Establish Tumor Models for In Vivo Gene Therapy

  Materials
  • Tumor cell line
  • Hanks' balanced salt solution (Life Technologies), sterile
  • Animals to receive tumor cell injection
  • Avertin (see recipe), sterile
  • 70% ethanol
  • Betadine solution (10% povidone iodine, Purdue‐Frederick)
  • Refrigerated centrifuge
  • Electric hair clipper
  • Hamilton syringe and 25‐G needle
  • Additional reagents and equipment for trypsinizing cells and determining number of viable cells by trypan blue staining ( appendix 3G)
NOTE: The following procedures correspond to the animal experimentation protocols used at the authors' institution. For workers at other institutions, all procedures should be approved by the animal care and use committee of the individual institution, and should be in accordance with established guidelines of animal welfare. While performing animal surgery, workers should wear caps, face masks, gloves, and protective clothing. All survival surgery should be performed in a sterile cabinet using aseptic technique. Animals should be euthanized as soon as experimental objectives are reached. Euthanasia of animals must be carried out in accordance with institutional guidelines.

Support Protocol 2: Generation of Hepatic Metastases for In Vivo Gene Therapy

  Materials
  • Surgical instruments including electric cauterizer and wound clips
  • Sterile gauze
  • Hamilton syringe and 30‐G needle
  • Additional reagents and equipment for generation of subcutaneous tumors (see protocol 9)
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Figures

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Literature Cited

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