UNIVERSITY OF CALIFORNIA Los Angeles Gene Editing of Bruton’s Tyrosine Kinase for Treatment of X-Linked Agammaglobulinemia A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Molecular Biology by David Gray 2020 © Copyright by David Gray 2020 ABSTRACT OF THE DISSERTATION Gene Editing of Bruton’s Tyrosine Kinase for Treatment of X-Linked Agammaglobulinemia by David Gray Doctor of Philosophy in Molecular Biology University of California, Los Angeles, 2020 Professor Donald Barry Kohn, Chair X-Linked Agammaglobulinemia (XLA) is a primary immunodeficiency characterized by a lack of mature B lymphocytes and antibody production. Patients with XLA have loss of function mutations in the Bruton’s Tyrosine Kinase (BTK) gene. The standard of care for XLA is immunoglobulin supplementation, which has a profound effect on patient wellbeing and life expectancies. However, immunoglobulin supplementation requires frequent, expensive injections throughout a patient’s life and patients remain susceptible to certain recurring illnesses. The only permanent cure for XLA is an allogeneic hematopoietic stem cell (HSC) transplant, though it is rarely performed due to the associated risks. Gene therapy-based methods to replace or repair the BTK gene in autologous HSCs provide an alternative with the benefits of a permanent cure for XLA while circumventing much of the risk. While previous efforts to deliver a functional copy of the BTK gene using viral vector mediated gene transfer have shown promise, these vectors carry a risk of insertional oncogenesis ii which may not be tolerated for treatment of XLA. Instead, this dissertation lays a foundation for targeted integration of a functional copy of the BTK gene into HSCs using Cas9 endonuclease mediated gene editing to drastically reduce those risks. Initial work identified and optimized a target site for integration into both cell lines and primary cells. However, integration of the BTK sequence alone generated insufficient transgene expression. We identified three modifications that improved integration and expression of the construct: mutation of the protospacer adjacent motif, re-addition of the BTK terminal intron, and addition of the woodchuck hepatitis virus posttranscriptional regulatory elemental. Together, these modifications generated expression of transgenic BTK nearing wildtype levels in cell lines with seamless DNA integration. Finally, an unbiased comparison of three different methods of targeted integration (homology directed repair, homology independent targeted integration, and precise integration into target chromosome) was performed to identify the optimal donor design for treatment of XLA. This work has produced a novel treatment for XLA that is ready to progress into in vivo models and towards the clinic. The new, effective donor modifications may be invaluable for similar treatments in development for other genetic disorders. iii The dissertation of David Gray is approved. Gay Crooks Kenneth Dorshkind Owen Witte Dinesh Rao Donald Barry Kohn, Committee Chair University of California, Los Angeles 2020 iv DEDICATION This dissertation is dedicated to my family. Steve Gray, Terri Gray, Christie Gray, Pat Thomas, Dave Thomas, Gerry Gray, Harry Gray, and Meztli España and a remaining list too long to name. Their constant support, compassion, and love has made this possible. v TABLE OF CONTENTS List of Figures and Tables vii Acknowledgements ix VITA xiii CHAPTER 1 1 Introduction: Hematopoietic Stem Cell Gene Therapy – Progress and Lessons Learned CHAPTER 2 51 Optimizing Integration and Expression of Transgenic Bruton’s Tyrosine Kinase for CRISPR/Cas9 Mediated Gene Editing of X-Linked Agammaglobulinemia CHAPTER 3 93 A Comparison of DNA Repair Pathways to Achieve Site-Specific Correction in Bruton’s Tyrosine Kinase CHAPTER 4 130 Discussion and Closing Remarks: Major Steps Towards a Cure for XLA vi LIST OF FIGURES AND TABLES CHAPTER 1 Figure 1-1. Overview of Targets for Gene Therapy 5 Figure 1-2. Autologous hematopoietic stem cell transplantation combined with gene 6 addition or editing Table 1-1. Genetic Diseases of Blood Cells and the Transplantation Modalities that 15 Have Been Applied Clinically as Therapies or Are in Pre-Clinical Development Figure 1-3. Summary of Gene Editing Pathways 19 CHAPTER 2 Figure 2-1. Targeted integration of BTK cDNA into intron 1 of the gene leads to 80 sub-physiological levels of mRNA and protein expression Figure 2-2. Addition of a terminal intron of the woodchuck hepatitis virus post- 82 transcriptional regulatory element (WPRE) to donor templates increases transgenic BTK expression Figure 2-3. Combining successful donor modifications leads to an additive effect on 84 transgene expression Figure 2-4. Integration of WPRE containing donors led to elevated BTK expression 86 in non-BTK expressing Jurkat T lymphocytes Figure 2-5. Chemical modification of sgRNA and Cas9 delivered as mRNA produced 87 efficient allelic disruption and integration rates in human CD34+ mobilized peripheral blood hematopoietic stem and progenitor cells Figure 2-6. Targeted integration of a corrective cDNA donor into the BTK exon 2 target 88 vii site led to efficient integration and expression of the transgene Figure 2-S1. Production and verification of a BTK-deficient K562 cell sub-line 90 Figure 2-S2. sgRNA variants demonstrated improved cutting efficiency when delivered 92 as ribonucleoprotein to human CD34+ peripheral blood stem cells CHAPTER 3 Figure 3-1. Editing schema and methods of targeted DNA integration 118 Figure 3-2. Identifying integration events of plasmid donors in BTK deficient K562 cells 120 Figure 3-3. Synchronizing K562 cells in G1 increases corrective donor integration via HITI 122 while decreasing integration from HDR and PITCh Figure 3-4. Targeted integration rates in bulk and population-sorted primary human 123 Mobilized peripheral blood stem cells Figure 3-S1. Schematics of donor template and targeted integration pathways 124 Figure 3-S2. K562 cells treated with only donor plasmid or only endonuclease produced 126 no integration events Figure 3-S3. Hydroxyurea cell cycle synchronization efficacy in K562 cells 127 Figure 3-S4. Optimizing reagents for delivery into human CD34+ mobilized peripheral 128 blood stem and progenitor cells viii ACKNOWLEDGEMENTS There are many people I need to thank for their personal and professional support through my graduate studies. First and foremost are my mentors, Dr. Donald Kohn and Dr. Caroline Kuo. Dr. Kohn first accepted me into the lab as an undergraduate in 2011 with minimal prior research experience. All said and done, I have spent nearly a decade under Dr. Kohn’s tutelage and he really taught most of what I know about scientific research. I have been incredibly lucky to have such a brilliant, kind, patient, and thoughtful mentor. I have always admired Dr. Kohn. As a scientist, he is unbelievably accomplished and internationally renowned, and yet he is very down to earth and always has time for those who need it whether it is a graduate student, a patient, or anyone else. He is always eager to help teach and support every member of his lab. I could not have asked for a better environment to do my doctoral studies in. The other research mentor I want to thank is Dr. Caroline Kuo. Dr. Kuo has been a constant source of guidance and support. Nearly every experiment I have begun and every project I have undertaken was bounced off her first, and her insight has made me a much better scientist. Outside of the scientific help Dr. Kuo has always provided me with, working with her and being a part of her team has been wonderful and an absolute joy. Our casual conversations shared over chocolate about anything from recent scientific papers to the best hamburgers in Los Angeles, has made the lab feel like home and will be one of the things I miss the most. I need to thank the entire Kohn Lab, as well. The lab has such a friendly and collaborative atmosphere, in large part due to the leaders within it. Dr. Beatrice Campo Fernandez is an absolute wizard in her mastery of essentially everything in the lab, and her contributions have been invaluable to so many aspects of my work. Dr. Zulema Romero has been a constant help throughout my time in the lab and her input and questions have helped me deepen my ix understanding of the field and my own project. Dr. Roger Hollis has been another deep well of knowledge, and he often comes up with perfect solutions when things seem to be otherwise stuck. Joseph Long, a technician in the lab, has been a wonderful collaborator and a great friend throughout my graduate career. I also want to thank the students and technicians who have assisted me on the project and that I have had the opportunity to mentor. Ali Abele, Jasmine Santos, Alex Keir, Aayushi Kapadia, Simon Maddock, and Isaac Villegas have all been huge helps to me in different ways and mentoring them has been one of my favorite parts of my time in the lab. All of them showed their ingenuity, determination, and eagerness each day and I am grateful for all the work they have performed over the years. I would particularly like to thank the two that were with me the longest: Jasmine and Isaac. Jasmine worked in the lab tirelessly for most of her undergraduate career, starting with zero research experience and leaving as a seasoned contributor. Her growth was tremendous and all thanks to her hard work and dedication. Isaac, a technician in the lab, joined with a lot of experience and quickly became my partner and right hand on the project. His independence, work ethic, and creativity were a huge asset to the science at hand. The Broad Stem Cell Research Center’s flow cytometry core run by Felicia Codrea, Jessica Scholes, and Jeffrey Calimim, taught me most of what I know about flow cytometry.