Methods in Molecular Biology
Part I: Immunogene Therapy
1 Aptamer Targeting the ERBB2 Receptor Tyrosine Kinase for Applications in Tumor Therapy
1.1 Molecular Targeted Therapies of Cancer
1.3 The ERBB Family of Growth Factor Receptors
1.4 Aptamers to ERBB Receptors
2.1 DNA Library and Beads for SELEX Selection
2.6 Animals, Other Reagents and Materials
3.2 Cloning, Sequencing, and Synthesis of Selected Aptamer Sequences
3.3 Heterogeneous, Noncompetitive Direct ELISA to Test ERBBưSpecific Aptamer Binding
3.4 Dot Blot to Show Aptamer Specificity for ERBB2
3.10 In Vivo Mouse Experiments
3.7 Immunofluorescence Analysis
3.8 Fluorescence-Activated Cell Sorting (FACS) Analysis
3.9 Cell Proliferation Assays
2 Gene Gun Her2/neu DNA Vaccination: Evaluation of Vaccine Efficacy in a Syngeneic Her2/neu Mouse Tumor Model
2.1 In Vivo DNA Vaccination
2.3 In Vitro Analysis of T Cell Responses
2.3.3 Cytokine Detection Assays
2.3.4 Cytometric Bead Array
2.4 Analysis of Humoral Immune Responses
3.1 Preparation of the DNA Vaccine for In Vivo Experiments
3.2 Gene Gun Immunization in Mice
3.3.1 Preparation of Tumor Cells for Tumor Challenge
3.3.2 Tumor Challenge and Measurement of Tumor Growth
3.4 T Cell Responses In Vitro
3.4.1 Preparation of Lymphocytes for Pentamer Staining
3.4.3 Preparation of Splenocytes from Mice Vaccinated with the Her2/neu DNA Vaccine
3.4.4 IFNg and IL-4 ELISpot
3.4.5 IFNg and IL-4 Secretion Assay
3.4.6 ⁵ạChromium (Cr⁵ạ) Release Assay
3.4.7 Cytometric Bead Array
3.5 Anti-Her2/neu Humoral Immune Response
3.5.1 Collection of Serum
3.5.2 Preparation of SK-BR3 Cells
3.5.3 Preparation of Serum for Flow Cytometric Detection of Anti-Her2/neu Antibodies
3.5.4 Preparation of the Antibody Solutions
3 MIDGE Technology for the Production of a Fourfold GeneưModified, Allogenic Cell-Based Vaccine for Cancer Therapy
2.1 MIDGE Vector Solutions
3.2 Preparation of Dishes with Low Attachment Surface
3.3 Preparation of Cell Suspension for Electroporation
3.5 Analysis of Efficacy of Gene Modification
3.6 Analysis of Expression of Surface Proteins by Flow Cytometry
3.7 Analysis of Expression of Cytokines by ELISA
Part II: Suicide Gene Therapy
4 Evaluation of Bystander Cell Killing Effects in Suicide Gene Therapy of Cancer: Engineered Thymidylate Kinase (TMPK)/AZT Enzyme-Prodrug Axis
2.1 Cell Culture Reagents
2.2 Transfection and Transduction Reagents
2.3 SDS-PAGE and Western Blotting Reagents
2.6 Consumables and Equipment
3.3 Production of Recombinant LV/TMPK
3.4 Transduction of PC-3 Cells and Analysis of Transgene Expression
3.5 Evaluation of AZT-Induced Cell Death In Vitro by a Colorimetric Proliferation Assay
3.6 Evaluation of AZT-Induced Apoptosis In Vitro
3.7 Evaluation of AZT-Induced Apoptosis in Bystander Cells In Vitro
3.8 Evaluation of AZT-Induced Bystander Cell Killing In Vivo in Mixed Tumors
3.9 Evaluation of AZT-Induced Bystander Cell Killing Effect In Vivo by Intratumoral Injection of TMPK Lentivirus
5 Oncoleaking: Use of the Pore-Forming Clostridium perfringens Enterotoxin (CPE) for Suicide Gene Therapy
2.3 Transfection with CPEưExpressing Vector
2.4 Quantitative RealưTime RT-PCR
2.4.2 Reverse Transcription
2.6 Protein Isolation and Western Blot
2.6.1 Lysis and Quantification
2.8 MTT Cytotoxicity Assay
3.1 Generation of Clostridium perfringens Enterotoxin (CPE) Expressing Vectors
3.3 Transfection of Human Tumor Cell Lines with CPEưExpressing Vectors
3.4 Quantitative Real-Time RT-PCR for Analysis of Claudin and CPE Expression Analysis
3.5 Preparation of Protein Lysates and Western Blot
3.6 Immunocytochemistry for Claudin-3 and -4 Detection
3.7 Determination of Biological Activity of CPE by MTT Cytotoxicity Assay
3.8 CPE-Specific Enzyme-Linked Immunosorbent Assay
3.9 Lactate Dehydrogenase Release Assay
6 iCaspase 9 Suicide Gene System
1.1 Development of Cellular Safety Switches
1.2 Development of Inducible Caspase 9 Safety Switches
1.3 Current Clinical Applications of iC9 Safety Switch
2.2 Common Materials and Reagents
2.6 In Vitro Evaluation of Genetically Modified T Cells
3.2 Co-culture Donor PBMC with Recipient LCL (Day 0)
3.3 Proliferation Assay (Day 0)
3.4 Treatment with Immunotoxin (Day 3)
3.5 Coat Flasks with Anti-CD3 Antibody (OKT3) (Day 3)
3.6 Wash Off RFT5-dgA After 15–18 h (Day 4)
3.7 Sampling for Proliferation Assay and FACS Analysis (Day 4)
3.10 Pre-coat T75 Flasks with Retronectin (Day 5)
3.11 Transduction with SFG.iC9.2A.dCD19 Retrovirus (Day 6)
3.8 Activation of Allodepleted Cells with OKT3 (Day 4)
3.9 Feed OKT3ưActivated T Cells with 100 U/mL IL-2 (Day 5)
3.12 Transfer Cells into Tissue Culture-Treated Flasks (Day 7)
3.13 Feed Non-transduced Cells (Day 7)
3.14 Split Cells (Optional) (Day 9)
3.15 Preparation of CD19 Selection on CliniMACS (Day 10)
3.16 CliniMACS Selection (Day 10)
3.17 Feed Non-transduced Cells (Day 10)
3.18 Killing Assay (Day 11)
3.19 iC9 Activity Evaluated by Flow Cytometry (Day 12)
3.20 Release Criteria for Clinical Product
Part III: Gene Replacement Therapies
7 p53-Encoding pDNA Purification by Affinity Chromatography for Cancer Therapy
2.1 Plasmid and Bacterial Growth
2.2 Alkaline Lysis and Sample Clarification
2.3 Preparative Chromatography
2.4 Agarose Gel Electrophoresis
2.5 Analytical Chromatography
2.6 p53 Protein Expression and Western Blot
3.1 Plasmid Amplification by Bacterial Growth
3.2 Alkaline Lysis and Plasmid Sample Clarification
3.3 Arginine Affinity Chromatography
3.4 Agarose Gel Electrophoresis
3.5 Assessment of sc pDNA Purity and Yield
3.6 p53 Protein Expression
8 A qRT-PCR Method for Determining the Biodistribution Profile of a miR-34a Mimic
2.1 Reverse Transcription (RT) Components
3.1 Reverse Transcription (RT), 384-Well Plate Format
3.2 qPCR, 384-Well Plate Format
Part IV: Gene Suppression and Signaling Modulation Therapy
9 Design and Selection of Antisense Oligonucleotides Targeting Transforming Growth Factor Beta (TGF-β) Isoform mRNAs for the Treatment of Solid Tumors
2.1 Human TGF-β Isoform mRNA Sequences
2.2 Cell Culture of Human Panc-1 Pancreatic Carcinoma Cells
2.3 Detection of TGF-β Isoform mRNAs in Cultured Cell Lysates
2.4 Detection of TGF-β Proteins in Cell CultureưConditioned Medium
2.6 Cell Culture of Human 786-O Renal Cell Carcinoma Cells
3.1 Oligonucleotide Sequence Design and Nucleotide Chemical Modification Pattern
3.2 Gymnotic Delivery of Antisense Oligonucleotides in Tumor Cells
3.3 Detection of TGF-β Protein Isoforms in Cell Supernatants by ELISA
3.4 Detection of TGF-β Isoform mRNAs in Cell Lysates Using the bDNA Assay
3.5 Acute Liver Toxicity Evaluation in Mice
3.6 Cell Culture of Human 786-O Renal Cell Carcinoma Cells
3.7 Evaluation of Target Downregulation (Target Engagement) In Vivo
10 RNA Interference for Antimetastatic Therapy
2.2 Plasmids and Plasmid DNA Purification
2.4 Cell Lines and Animals
2.5 Cell Line Transplantation, Tail Vein Injection, Blood Sample Collection, and Bioluminescence Imaging
2.6 Quantitative Real-Time Reverse Transcriptase (RT) PCR
3.1 Large-Scale Plasmid Preparation from Bacteria
3.2 Cell Culture, Passaging, and Plasmid Transfection
3.3 Luciferase Activity Assay
3.4 Generating Xenograft Mice by Intrasplenic Transplantation
3.5 Tail Vein Injection of Plasmid DNA
3.6 Monitoring Plasmid Concentration in Mouse Blood
3.7 In Vivo Imaging of Tumor Growth and Metastasis Formation
3.8 Sacrifice of Mice and Organ Storage
3.9 Evaluation of Tumor Transfection and S100A4 Expression in Tumor Tissue by qPCR
3.9.1 Nucleic Acid Extraction
3.9.2 Reverse Transcription
3.9.3 Quantitative Polymerase Chain Reaction
11 STAT3 Decoy ODN Therapy for Cancer
2.1 STAT3 Decoy/Scramble ODN
2.3 The Fluorescence-Conjugated Antibodies
2.10 Cytokines and Blocking Antibodies
2.9 The Dual-Glo Luciferase Assay System
2.12 Reagents Used in Western Blotting
2.13 MTT (3- (4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) Solution
2.14 Immunohistochemistry
3.2 Assessment of ODN Transfection Efficiency
3.3 Luciferase Reporter Gene Assay
3.4 Cell Proliferation Assay (See Note 10)
3.6 Apoptosis Determination
3.7 NK Cells Treated with the Supernatant from Tumor Cells (See Note 14)
3.8 NK Cell Cytotoxicity Assay
3.10 Quantitative RealưTime RT-PCR
3.11 Western Blot Analysis
3.12 Enzyme-Linked Immunosorbent Assay (ELISA) (See Note 20)
3.13 Human Tumor-Bearing Nude Mouse Model (See Note 21)
3.14 Terminal Deoxynucleotidyl Transferase-Mediated Nick End Labeling (TUNEL) Assay (See Note 22)
3.15 Immunohistochemistry (See Note 23)
3.16 Antitumor Immunity Analysis (See Note 24)
Part V: Oncolytic Virotherapy and Bacterial Vectors for Cancer Treatment
12 Oncolytic Viral Therapy Using Reovirus
2 Prototypical Strains of Reovirus
3 Reovirus and Human Disease
4 Molecular Basis of Reovirus Oncolysis
5 Mechanisms of Reovirus-Mediated Cell Death
6 Preclinical Studies of Reovirus as a Cancer Therapeutic
7 Reovirus Induction of Immunogenic Cell Death and Immune Modulation with Reovirus
7.1 Inflammatory Storm Generated by Reovirus Leads to the Activation of the Innate and Adaptive Immune Responses
7.2 Mechanisms of Immunogenic Tumor Cell Death and Cytokine Release in ViralưInfected Tumor Microenvironment
7.3 Reovirus Effects on Dendritic Cells
7.4 Reovirus Modulates Innate Immune Responses
7.5 Innate Immune Response Manipulation Leads to Enhancement of Reoviral Therapy
7.6 Reovirus Modulates the Adaptive Immune System with Enhancement of Tumor Lysis
7.7 Myeloid-Derived Suppressor Cells and Their Effects on Reovirus Therapy
7.8 Gemcitabine and Reovirus Therapy
7.9 Immune Check Point Inhibitors and Reovirus
7.10 Immune Modulation by Reovirus in Clinical Trials
8 Reolysin Clinical Trials
8.1 Reovirus as Monotherapy
8.1.1 Reovirus Intratumoral Therapy
8.1.2 Intravenous Monotherapy of Reovirus
8.2 Reovirus in Combination with Radiation Therapy
8.3 Reovirus in Combination with Chemotherapy
13 Use of GLV-1h68 for Vaccinia Virotherapy and Monitoring
2.4 Animals and Equipments
2.5 Buffers and Plasticware
3.1 A549 or DU-145 Tumor Cell Preparation and Implantation
3.2 GLV-1h68 Injection (Retro-Orbital Sinus Vein Injection)
3.4 Optical Imaging of Renilla Luciferase [17, 18] (Coelenterazine Injection, Anesthesia, Overlay, Quantification, See Note 19)
14 Back to the Future: Are Tumor-Targeting Bacteria the Next-ưGeneration Cancer Therapy?
3.1 GFP Labeling of S. typhimurium (See Note 1)
3.2 Isolation of High-Tumor Virulence Strain, S. typhimurium A1-R (See Notes 2–4, 8, and 9)
3.3 Adherence and Invasion Assay Comparison S. typhimurium A1 and A1-R
3.4 Invasiveness of Dual-Color Cancer Cells by S. typhimurium A1-R (See Notes 5 and 6)
3.5 Surgical Orthotopic Implantation of Breast Tumors
3.6 Bacterial Targeting of Experimental Lung Metastasis (See Note 15)
3.7 Orthotopic Osteosarcoma Model in Nude Mice
3.8 S. typhimurium A1-R Therapy of Experimental Pancreatic Cancer Lymph Node Metastasis (See Notes 13 and 14)
3.10 Intra-tumoral Bacterial Therapy for Pancreatic Cancer (See Notes 10 and 11)
3.11 Selection of Highly Aggressive Subpopulations of XPA-1 RFP Human Pancreatic Cancer Cells (See Notes 11 and 12)
3.9 S. typhimurium A1-R Therapy of Spontaneous Lymph Node Metastasis
3.12 Intrasplenic Injection of Pancreatic Cancer Cells
3.13 Dose Response of S. typhimurium A1-R Treatment of Metastatic Pancreatic Cancer
3.14 Orthotopic Transplantation of Red Fluorescent ProteinưExpressing U87 Human Glioma Cells
3.15 S. typhimurium A1-R Therapy of the Orthotopic IMSCT Model (See Notes 16 and 17)
3.16 Functional Evaluation of Hind Limbs to Determine Degree of Paralysis
3.17 RFP-Expressing Murine Cutaneous Lung Cancer Model (See Note 18)
3.18 S. typhimurium A1-R Treatment of Tumor
3.19 S. typhimurium A1-R Targeting of Tumor Vascularity
3.20 Experimental Lewis Lung Carcinoma Metastasis in the Lungs of C57 Mice
3.21 Bacterial Dosing (See Note 7)
3.22 Craniotomy Open Window Model for Brain Cancer Treatment and Imaging
3.23 Stereotactic Injection of Cancer Cells in the Brain
3.24 Bacterial Therapy in the Brain Tumor Model (See Note 19)
3.25 Mammary Fat Pad Injection of MDA-MBư435-RFP Cells
3.26 Targeting Breast Cancer Cells by S. typhimurium A1-R In Vitro
3.27 Antitumor Efficacy of S. typhimurium A1-R Administered by Three Different Routes (See Note 7)
3.28 Reversible Skin Flap
3.29 Primer-Dose S. typhimurium A1-R Therapy (See Note 20)
3.30 Effect of S. typhimurium A1-R on TNF-α
3.31 Orthotopic Pancreatic Cancer Implantation
3.32 S. typhimurium A1-R Therapy and Chemotherapy of Pancreatic Cancer Stem Cells (See Notes 21–24)
3.33 Establishment of Patient-Derived Orthotopic Xenograph (PDOX) Model
3.34 Establishment of fPDOX Model
3.35 Whole-Body Imaging Equipment (See Notes 26
28)
3.36 Laser Scanning Microscope
3.37 Confocal Imaging of Cancer Cells Infected with S. typhimurium A1-R
Part VI: Clinical Application of Cancer Gene Therapy
15 Ethics of Cancer Gene Transfer Clinical Research
1 Preclinical Research Activities
1.1 Preclinical Research Design
1.2 Preclinical Research Reporting
1.3 Distinctive Issues in Cancer Gene Transfer
2.1.1 Distinctive Risk Challenges for Cancer Gene Transfer
Risk–Benefit in Early Phase Studies
Bystander and Germ-Line Risk
2.2.1 Access to Trials and Justice
2.3 Consent to Cancer GT Studies
2.3.1 Distinctive Challenges for Cancer Gene Transfer and Consent
2.3.2 Therapeutic Misconception
2.3.3 Therapeutic Misestimation
3.2 Managing Public Expectations
3.3 Sustainability of the Research Enterprise
4 Conclusions: The Same, but Different?
Box 1
Recommendations for Researchers Pursuing Cancer GT Research
16 Planning an Academic Clinical Trial
2 Regulatory Considerations
2.1 Definition of Gene Therapy Medicinal Products
2.2 Clinical Trials of Investigational Medicinal Products
2.3 Applying for Authorization to Conduct a CTIMP
2.4 Clinical Trial Registration
2.5 Licensable Activities and Notifications
2.6 From the Clinical Trials Directive to a Regulation
4 Feasibility Assessments
4.1 Feasibility of the Clinical Trial
4.2 Feasibility of Trial Sites
5 Writing a Trial Protocol
7 Budgeting for a Clinical Trial
17 Production of Plasmid DNA as Pharmaceutical
2.3 Cell Harvest and Lysis
3 Quality Control and Quality Assurance
4.2 High Quality Grade (HQ)
18 Minicircle: Next Generation DNA Vectors for Vaccination
Part VII: Protocols of Clinical Cancer Gene Therapy
19 A Phase 2, Open-Label, Randomized Study of Pexa-Vec (JX-594) Administered by Intratumoral Injection in Patients with Unresectable Primary Hepatocellular Carcinoma
2.1 Neutralizing Antibody Titers in Serum
2.2 Detection of Pexa-Vec Genome in Blood
2.3 Detection of GM-CSF Transgene in Plasma
3.1.1 Inclusion/Exclusion Criteria
3.1.2 Pretreatment Evaluation/Follow-Up Studies
3.2.1 Plasma Neutralizing Antibody Titers
3.2.2 Detection of Pexa-Vec Genome in Blood
3.2.3 Detection of GM-CSF Transgene in Plasma
20 Antiangiogenic Metargidin Peptide (AMEP) Gene Therapy in Disseminated Melanoma
2.2 Preparation of Plasmid DNA (Plasmid AMEP)
2.3 Electroporation Equipment
21 Clinical Evaluation of ErbB-Targeted CAR T-Cells, Following Intracavity Delivery in Patients with ErbB-Expressing Solid Tumors
1.1 Gene-Modified Cell Therapy: The Rationale for Regional Delivery
1.2 ErbB Targeting with T4 Immunotherapy
1.3 Intracavity T4 Therapy
1.4 Malignant Pleural Mesothelioma
1.5 High-Grade Serous Ovarian Cancer
2.1 Cell Product Manufacture
2.2 Product Certification
3.1 Phase IB Clinical Trial of Intracavitary T4 Immunotherapy
3.2 An Open Label Randomized Phase II Trial of T4 Immunotherapy in Malignant Pleural Mesothelioma
3.3 The Patient Population
3.5 Inclusion and Exclusion Criteria
3.6 An Open Label Randomized Phase II Trial of T4 Immunotherapy in High-Grade Serous Ovarian Carcinoma
3.7 The Patient Population
3.10 Administration of T4 Immunotherapy
3.9 Inclusion and Exclusion Criteria
3.11 Patient Monitoring for Toxicity
3.12 Analysis for In Vivo Persistence of T4 Immunotherapy
3.13 Monitoring for Immunogenicity
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