Ancestral Sequence Reconstruction as a Lead Optimization Approach in Gene Therapy Drug Development Pubblico
Coyle, Christopher (Fall 2023)
Abstract
Gene therapy is revolutionizing 21st century medicine. Currently, 27 gene therapy products are approved by the U.S. Food and Drug Administration and/or the European Medicines Agency. These initial products are predominantly focused on two disease classes, monogenic disorders and cancer. Within monogenic disorders, approved products are now available for the severe bleeding disorders, hemophilia A and B. Historically, these diseases have been treated using lifelong protein replacement therapy, which is difficult to manage from both compliance and economic standpoints. Gene therapy has the potential to change the paradigm to a one-time treatment that is effective for years instead of days. However, as a new drug class, 1st generation gene therapies are hindered by critical limitations in manufacturing efficiency, optimal and durable efficacy, and dose-related toxicities. Unlike small molecule drug optimization, relatively few methods and technologies exist to optimize gene therapies. Common methods employed include nucleic acid ‘codon’ optimization, vector selection and tropism engineering, and transgene product (i.e., therapeutic protein) selection. The studies completed for the current dissertation were designed to test and further develop a unique strategy for gene therapy potency optimization through transgene product engineering. Improving gene therapy potency has the potential to address all three primary limitations currently hindering gene therapies. To accomplish this goal, we adopted a method historically used to study molecular evolution and applied it as a drug discovery platform.
Ancestral sequence reconstruction (ASR) utilizes bioinformatics to predict the protein sequences of ancient, extinct species, which have evolved over time to meet the ecological, physiological, and sociological demands of each species. While this approach has traditionally been applied to studies of protein and gene evolution, it also can be harnessed to facilitate the exploration and mapping of functional amino acid substitutions that confer pharmaceutically-inspired properties to protein drugs and gene therapies under development. In the current work, we present the results of ASR studies designed to identify enhanced variants of blood coagulation factors VIII (FVIII) and IX (FIX), which are associated with hemophilia A and B, respectively. As a validation of the ASR drug discovery approach, we identified ancestral variants of FVIII and FIX that display enhanced pharmaceutical properties such as specific activity, half-life and biosynthetic efficiency. Subsequently, reductionist screening strategies were successfully employed to map the amino acids responsible for the functional enhancements. In the case of FIX, an ancestral variant that displayed 10-fold higher specific activity than human FIX was identified during the initial ASR screen. Subsequently, the amino acid substitutions necessary and sufficient to confer 10-fold higher activity to human FIX were mapped and functionally characterized. Finally, by combining these 5 critical amino acid substitutions with an additional single amino acid substitution FIX variant identified in a family with elevated FIX activity levels, a final lead FIX candidate was identified and designated ET9. ET9 represents a 99% human FIX variant containing only six amino acid substitutions that confer 51-fold increased specific activity in clinical coagulation assays and 10-fold higher potency in vivo in hemophilia B mice compared to human FIX. ET9 is, to our knowledge, the most potent FIX variant described to date, and a premiere candidate for utilization in 2nd generation gene therapies for hemophilia B. These results further validate the ASR approach and justify its implementation in broader protein drug and gene therapy development as an effective discovery to lead optimization platform.
Table of Contents
Chapter 1: Introduction…………………………………………………………….……………….….1
1.1: Blood Coagulation Network………………………………………………………….………….….2
1.2 Hemophilia A and B……………………………………………………………………………...…..4
1.3 FVIII…………………………………………………………………………………………………....5
1.4 FIX……………………………………………………………………………………………………..8
1.5 Brief History of Gene Therapy……………………………………………………………………...9
1.6 Pharmacology and Optimization Strategies of AAV Gene Therapy…………………………..11
1.7 Gene Therapy of hemophilia A and B using AAV vectors……………………………………...17
1.8 Ancestral Sequence Reconstruction as a Platform for Protein Engineering…………………22
1.9 Hypothesis and objectives of this research……………………………………………………...24
Chapter 2: Molecular coevolution of coagulation factor VIII and von Willebrand factor……………………………………………………………………………………………………..25
2.1: Abstract……………………………………………………………………………………………..26
2.2: Introduction…………………………………………………………………………………………26
2.3: Materials and Methods………………………………………………………………….…………27
2.4: Results……………………………………………………………………………………………....33
2.5: Discussion…………………………………………………………………………………………..45
2.6: Acknowledgements………………………………………………………………………..………48
2.7: Supplementary Figures and Tables…………………………………………………………..…49
Chapter 3: Expression of Ancient Coagulation FVIII Molecules and Discovery of Relevant Domains…………………………………………………………………………………………..…….58
3.1: Abstract……………………………………………………………………………………………..59
3.2 Introduction………………………………………………………………………………………….60
3.3 Materials and Methods……………………………………………………………………….…….61
3.4 Results……………………………………………………………………………………………….61
3.5 Discussion…………………………………………………………………………………………...73
3.6 Acknowledgements…………………………………………………………………………………75
Chapter 4: Identification of coagulation factor IX variants with enhanced activity through ancestral sequence reconstruction………………………………………………………………..77
4.1: Abstract……………………………………………………………………………………………..78
4.2: Introduction…………………………………………………………………………………………78
4.3: Materials and Methods…………………………………………………….................................80
4.4: Results………………………………………………………………………………......................86
4.5: Discussion……………………………………………………………………………....................97
4.6: Acknowledgements……………………………………………………………………...............101
4.7: Supplementary Figures and Tables………………………………………………...................103
Chapter 5: Humanization and functional characterization of enhanced coagulation factor IX variants identified through ancestral sequence reconstruction…….............................108
5.1: Abstract…………………………………………………………………………………...............109
5.2: Introduction……………………………………………………………………………................110
5.3: Materials and Methods……………………………………………………………....................112
5.4: Results…………………………………………………………………………………................115
5.5: Discussion………………………………………………………………………….....................129
5.6: Acknowledgements…………………………………………………………………..................133
5.7: Supplementary Figures and Tables……………………………………………......................134
Chapter 6: General Discussion………………………………………………………...................141
6.1 Summary of Results…………………………………………………………………..................142
6.2 Implications of Findings……………………………………………………………....................145
6.3 Limitations and Future Directions……………………………………………….......................147
6.4 Conclusion…………………………………………………………………………......................149
References…………………………………………………………………………..........................150
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