Optimization and Validation of Lentiviral Based Gene Therapy for FHL3 Público

Abstract

“Gene therapy” is the introduction, removal, or alteration of a person’s genetic code in order to treat or prevent disease. Many different sub-types of gene therapy exist. Some therapies can modify specific cell types for transient expression of foreign DNA, and others integrate permanently into the genomes of stem cells and create lasting change across all the subsequently generated daughter cells. While originally envisioned to correct diseases that were caused by a single defective gene, researchers are now developing gene therapy products to combat complex diseases and disorders such as cancer, arthritis, and HIV. Compared to other disease interventions such as drug based therapies or vaccines, gene therapy is still in the early phases of development. The first successful report of human gene therapy was made less than thirty years ago, and the field has experienced distinct periods of rapid growth, setback, and re-acceleration. Excitingly, within the last three years the FDA approved the first gene therapy for use in the USA, signaling that the translational potential of this field will be actualized. 

This dissertation focuses on the creation, validation, and optimization of a lentiviral based gene therapy for treating the primary immune disorder familial hemophagocytic lymphohistiocytosis Type III (FHL3). This disease, which is characterized by fever, hepatosplenomegaly, cytopenias, and, if left untreated, organ failure and death, is caused by a mutation to the UNC13D gene which renders cytotoxic cells incapable of degranulating cytolytic vesicles. Here we evaluated two lentiviral vector gene therapy approaches. The first approach seeks to transduce hematopoietic stem and progenitor cells (HSPCs) for autologous hematopoietic stem cell transplant (HSCT). The second approach seeks to resolve the hyperinflammation common to most FHL3 patients by transducing the patient’s own T cells and re-infusing them back into the patient such that the underlying cause of inflammation could be resolved. Furthermore, in order to maximize the chances of this gene therapy making it to clinical trial, we have sought to optimize transduction efficiency by altering our protocols for lentiviral vector production, isolation of target cells, and transduction. Our hope is that this work provides insight into optimization strategies broadly used throughout the field of lentiviral gene therapy, and that our work pertaining specifically to FHL3 gene therapy might one day benefit patients.

Table of Contents

Abstract 4

Acknowledgments. 6

Table of Contents. 7

List of Tables. 14

List of Figures. 14

List of Abbreviations. 17

Chapter 1. 25

1.1     A History of Gene Therapy and Retroviral Biology. 26

1.1.1  What is Gene Therapy?. 26

1.1.2  Discovery of Viral Mediated Gene Transfer 27

1.1.3  Nascent Gene Transfer Studies. 28

1.1.4  Early Gene Therapy Successes (1970s – 1990s) 29

1.1.5  Gene Therapy Setbacks (1999 – early 2000s) 31

1.1.6  Recovery of Gene Therapy Research and the Beginning of Commercialization (2000s – present) 34

1.1.7  Retroviruses and Applications towards Ex Vivo Gene Transfer 35

1.2     An Introduction to HLH.. 39

1.2.1  Prevalence. 39

1.2.2  Clinical Manifestations of HLH.. 41

1.2.3  Diagnosing HLH.. 42

1.2.4  Primary vs Secondary HLH.. 45

1.2.5  Treatment of HLH.. 50

1.2.6  Landmarks in HLH Research. 61

1.3  FHL3. 62

1.3.1  Expression of the UNC13D gene. 62

1.3.2  Regulation of the UNC13D gene. 64

1.3.3  Structure of Munc13-4 Protein. 66

1.3.4  Regulation of the Munc13-4 protein. 70

1.3.5  Functions of Munc13-4 Protein. 72

1.3.6  The FHL3 Mouse Model 89

1.3.7  Gene therapies with regards to FHL3. 94

Chapter 2. 97

2.1  ABSTRACT.. 98

2.2  INTRODUCTION.. 98

2.3  MATERIALS AND METHODS. 104

Cloning. 104

Lentiviral Vector Packaging and Titering. 104

Viral Copy Number (VCN) Analysis. 105

Transduction. 106

Western Blot 107

CFU Assays. 107

Magnetic sorting. 108

T-Cell Degranulation Assay. 108

Neutrophil degranulation assay. 109

Mice. 110

LCMV Infection. 110

Primary Cell and Cell Lines. 110

Cell Culture. 111

Flow Cytometry. 111

Statistical Analysis. 112

2.4  RESULTS. 113

2.4.01  Optimization of the lentiviral construct 113

2.4.02  Optimization of HSPC isolation. 114

2.4.03  Design and validation of the codon-optimized-tCD271 lentiviral construct 120

2.4.04  The effect of transfection plasmid ratios on LV producer cells and transduced target cells. 125

2.4.05  A comparison of different lentiviral vector purification methods. 128

2.4.06  Primary human T cells do not maintain their CD271 expression over time. 129

2.4.07  Microfluidics based transduction. 130

2.4.08  The impact of Munc13-4 expression on transduction efficiency. 132

2.4.09  A comparison of different transduction enhancers. 133

2.4.10  Optimization of the T-cell degranulation assay. 137

2.4.11  Neutrophil degranulation assay. 144

2.5  DISCUSSION.. 149

2.5.01  Optimization of the lentiviral construct 149

2.5.02  Optimization of HSPC isolation. 151

2.5.03  Design and validation of the codon-optimized-tCD271 lentiviral construct 153

2.5.04  The effect of transfection plasmid ratios on LV producer cells and transduced target cells. 153

2.5.05  A comparison of different lentiviral vector purification methods. 155

2.5.06  Primary human T cells do not maintain their CD271 expression over time. 157

2.5.07  Microfluidics based transduction. 157

2.5.08  The impact of Munc13-4 expression on transduction efficiency. 158

2.5.09  A comparison of different transduction enhancers. 160

2.5.10  Optimization of T-cell degranulation assay. 162

2.5.11  Neutrophil degranulation assay. 166

2.6 CONCLUSIONS. 175

2.7  AKNOWLEDGEMENTS. 176

2.8  Supplemental Figures. 177

Chapter 3. 182

3.1  ABSTRACT.. 183

3.2  INTRODUCTION.. 184

3.3  RESULTS. 187

3.3.01  Stable expression of Munc13-4 in healthy donor cells. 187

3.3.02  Transduction of FHL3 patient T cells. 188

3.3.03  Identification of potentially therapeutic Munc13-4 expression levels. 191

3.3.04  Gene transfer into the FHL3 disease mouse model 195

3.4  DISCUSSION.. 199

3.3.01  Stable expression of Munc13-4 in healthy donor cells. 199

3.3.02  Transduction of FHL3 patient T cells. 200

3.3.03  Identification of potentially therapeutic Munc13-4 expression levels. 200

3.3.04  Gene transfer into the FHL3 disease mouse model 202

3.5  MATERIALS AND METHODS. 204

Cloning. 204

Lentiviral Vector Packaging and Titering. 204

Transduction. 205

Transplants. 206

Viral Copy Number (VCN) Analysis. 206

Infection. 207

CFU Assays. 207

Western Blot 207

Degranulation Assay. 207

Cytotoxicity Assay. 208

Primary Cell and Cell Lines. 209

Cell Culture. 209

Flow Cytometry. 209

Statistical Analysis. 210

3.6  SUPPLEMENTAL FIGURES. 211

3.7  ACKNOWLEDGMENTS. 212

Chapter 4. 213

4.1  SUMMARY OF RESULTS. 214

4.2  IMPLICATIONS OF FINDINGS. 216

4.3  LIMITATIONS AND FUTURE DIRECTIONS. 218

4.4  CONCLUSION.. 227

3.7  ACKNOWLEDGMENTS. 228

References 229

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Immunology and Molecular Pathogenesis
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, General Biology, Cell Biology, Molecular
Palavra-chave	Gene Therapy Munc13-4 Lentiviral vector Immunology UNC13D
Committee Chair / Thesis Advisor	Dr. Spencer, Emory University
Committee Members	Dr. Archer, Emory University Dr. Ford, Emory University Dr. Stowell, Emory University Dr. Doering , Emory University

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