Luận án: Tối ưu hóa Liệu pháp Gene Lentiviral cho FHL3 - Sarah Takushi, Emory University

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Signature: ______________________________ ______________________________ Sarah Takushi Date 2 Optimization and Validation of Lentiviral Based Gene Therapy for FHL3 By Sarah Takushi Doctor of Philosophy Graduate Division of Biological and Biomedical Sciences Immunology and Molecular Pathogenesis Program __________________________________ H. Trent Spencer, PhD Advisor __________________________________ __________________________________ Christopher B. Doering, PhD David Archer, PhD Committee Member Committee Member __________________________________ __________________________________ Mandy Ford, PhD Sean Stowell, M./PhD Committee Member Committee Member Accepted: __________________________________ __________________________________ Lisa A. Tedesco, PhD Date Dean of the James T.

Laney School of Graduate Studies 3 Optimization and Validation of Lentiviral Based Gene Therapy for FHL3 By Sarah Takushi B., Illinois Wesleyan University, 2013 Advisor: H. Trent Spencer, PhD An abstract of a dissertation submitted to the Faculty of the James T. Laney School of Graduate Studies of Emory University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate Division of Biological and Biomedical Science, Immunology and Molecular Pathogenesis Program 2020 4 Abstract Optimization and Validation of Lentiviral Based Gene Therapy for FHL3 “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.

5 Optimization and Validation of Lentiviral Based Gene Therapy for FHL3 By Sarah Takushi B., Illinois Wesleyan University, 2013 Advisor: H. Trent Spencer, PhD A dissertation submitted to the Faculty of the James T. Laney School of Graduate Studies of Emory University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate Division of Biological and Biomedical Science, Immunology and Molecular Pathogenesis. 2020 6 Acknowledgments It is with deep and sincere appreciation that I would like to thank Dr.

Trent Spencer for supporting my development as a scientist, for believing in me as a person, and for helping me find my way through the winding and ever-surprising journey that is graduate school. Thank you for adopting me when I was a wayward third-year graduate student, and giving me the tools to forge my own way. Thank you for the pre-dawn pow-wows, the after-midnight check-ins, and your willingness to offer guidance and support during all the hours in between. As a pillar of the Emory Gene Therapy program and an advisor on my committee, Dr.

Chris Doering has also helped guide every step of this project, sometimes even pointing out detours and alternative routes that I had not known even existed. Thank you for helping me untangle experimental problems, puzzle through inscrutable datasets, and never failing to bring the infectious, creative energy that makes biology both an art and a science. Though not formally an advisor to me, the HLH gene therapy program at Emory University would not exist without Dr. Shanmuganathan “Shan” Chandrakasan.

Shan combines clinical expertise with a deep understanding of immunology that has been inspiring for an immunology graduate student such as myself. Thank you for saving me years of my life that would have otherwise been spent trouble- shooting and head-scratching. None of this would be possible without you. Thank you as well to the other members of my committee: Dr.

David Archer, Dr. Mandy Ford, and Dr. I sincerely appreciate how each one of you helped me refine and develop my project. Thank you for keeping me focused on what would be the best use of my time.

Thank you for helping foster connection and mentorship—not just with me, but throughout the Emory community. Similarly, I would like to thank Dr. Nael McCarty and Ms. Tamara Hutto for their work with the Graduate Division of Biology and Biological Sciences, as well as through the Atlanta BEST program.

Both Nael and Tammy have an unshakable dedication to graduate students and helping them achieve self- actualization as well as professional success. The lessons they taught me about personal fulfillment and finding my own way are ones that I will never forget. Lastly and most important to me, I would like to thank my friends and family. Thank you, dear lab friends, for shouldering daunting workloads, pepping me up after setbacks, feeding me delicious food during viciously hangry moments, and helping me troubleshoot equipment in the wee hours of the morning.

Thank you to all the friends with whom I have spent time outside of lab for helping me find psychic pressure safety valves through which I can let off steam and find my center again. Finally, thank you to my family for being emotionally close despite being geographically distant. Though many of you have at one point or other confessed that “I don’t really get what you do,” it has meant everything to me that you have still believed that whatever it is, I can do it. 7 Table of Contents Abstract.

6 Table of Contents. 7 List of Tables. 14 List of Figures. 14 List of Abbreviations .1 A History of Gene Therapy and Retroviral Biology .1 What is Gene Therapy? .2 Discovery of Viral Mediated Gene Transfer .3 Nascent Gene Transfer Studies .4 Early Gene Therapy Successes (1970s – 1990s) .5 Gene Therapy Setbacks (1999 – early 2000s) .6 Recovery of Gene Therapy Research and the Beginning of Commercialization (2000s – present) .7 Retroviruses and Applications towards Ex Vivo Gene Transfer .2 An Introduction to HLH .2 Clinical Manifestations of HLH .4 Primary vs Secondary HLH .5 Treatment of HLH .6 Landmarks in HLH Research .1 Expression of the UNC13D gene .2 Regulation of the UNC13D gene.3 Structure of Munc13-4 Protein .4 Regulation of the Munc13-4 protein .5 Functions of Munc13-4 Protein .6 The FHL3 Mouse Model .7 Gene therapies with regards to FHL3.3 MATERIALS AND METHODS.

104 Lentiviral Vector Packaging and Titering. 104 9 Viral Copy Number (VCN) Analysis. 108 T-Cell Degranulation Assay. 108 Neutrophil degranulation assay.

110 Primary Cell and Cell Lines .01 Optimization of the lentiviral construct .02 Optimization of HSPC isolation .03 Design and validation of the codon-optimized-tCD271 lentiviral construct .04 The effect of transfection plasmid ratios on LV producer cells and transduced target cells .05 A comparison of different lentiviral vector purification methods .06 Primary human T cells do not maintain their CD271 expression over time .07 Microfluidics based transduction .08 The impact of Munc13-4 expression on transduction efficiency .09 A comparison of different transduction enhancers.10 Optimization of the T-cell degranulation assay .11 Neutrophil degranulation assay .01 Optimization of the lentiviral construct.02 Optimization of HSPC isolation .03 Design and validation of the codon-optimized-tCD271 lentiviral construct .04 The effect of transfection plasmid ratios on LV producer cells and transduced target cells .05 A comparison of different lentiviral vector purification methods .06 Primary human T cells do not maintain their CD271 expression over time .07 Microfluidics based transduction .08 The impact of Munc13-4 expression on transduction efficiency .09 A comparison of different transduction enhancers .10 Optimization of T-cell degranulation assay .11 Neutrophil degranulation assay .01 Stable expression of Munc13-4 in healthy donor cells .02 Transduction of FHL3 patient T cells .03 Identification of potentially therapeutic Munc13-4 expression levels .04 Gene transfer into the FHL3 disease mouse model .01 Stable expression of Munc13-4 in healthy donor cells .02 Transduction of FHL3 patient T cells .03 Identification of potentially therapeutic Munc13-4 expression levels .04 Gene transfer into the FHL3 disease mouse model .5 MATERIALS AND METHODS. 204 Lentiviral Vector Packaging and Titering. 206 Viral Copy Number (VCN) Analysis. 208 Primary Cell and Cell Lines .1 SUMMARY OF RESULTS .2 IMPLICATIONS OF FINDINGS .3 LIMITATIONS AND FUTURE DIRECTIONS.

229 14 List of Tables Table 1 Overview of the sub-categories of primary HLH and associated diseases. 46 Table 2 Prognosis of clinical HLH studies. 58 Table 3 Different clinical trials use different methods for calculating the quantity of lentiviral vector modified cells which will constitute a single dose or treatment. 100 Table 4 Transduction enhancers and their mechanism of action.

103 Table 5 Variations in TC degranulation protocols. 163 Table 6 Alternative flow gating strategies for neutrophils. We gated our neutrophils as being Ly6G+, but different murine neutrophils gating strategies exist. 169 Table 7 A comparison of in vitro murine neutrophil degranulation assay protocols.

169 List of Figures Figure 1. 211 17 List of Abbreviations 2B4 CD244 A-HLH Autoimmune hemophagocytic lymphohistiocytosis AAV Adeno-associated viruses ADA Adenosine deaminase ADA-SCID Adenosine deaminase severe combined immunodeficiency ADP Adenosine diphosphate AIDS Acquired immunodeficiency ALC Absolute lymphocyte counts ALL Acute lymphoblastic leukemia ALPS Autoimmune lymphoproliferative syndrome ANA Antinuclear antibody ANOVA ANalysis Of VAriance APC Antigen presenting cell ASGCT American Society of Gene and Cell Therapy AT Antithrombin ATTC American Type Culture Collection ATG Anti-thymocyte globulin Atg9b Autophagy Related 9B ATP Adenosine triphosphate B-LCL B cell large cell lymphoma Ba Barium BLAST Basic local alignment search tool.