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Growing into their Own? Plant Molecular Farming and the Pursuit of Biotechnological Sovereignty for Lower and Middle Income Countries. Ana Christine Zeghibe Submitted in Partial Fulfillment of the Prerequisite for Honors In Biological Sciences Under the advisement of Martina Koniger, Ph.D. Wellesley College May 2021 © 2021 Ana Zeghibe Acknowledgements I am extraordinarily grateful for the support, kindness and guidance of my major and thesis advisor Professor Martina Koniger. The strong plant biology influence on an otherwise biotechnologically and medically oriented thesis was largely inspired by her own expertise in chloroplasts. When coming to this thesis, I knew I wanted to write something that was not simply a research paper, but a synthesis of ideas and observations about science and its interactions with the world beyond the laboratory that I had made over my 4 years at Wellesley. Thank you, as always, for allowing me to develop this unusual project, for keeping me sane through the many pitfalls and trials of writing, and for making me fall in love with science communication and plant biology. Thank you to my committee member Professor Natali Valdez for supporting and guiding my analyses and thoughts on the societal implications of plant molecular pharming. Professor Valdez's classes and work in Feminist Science and Technology Studies strongly influenced the later chapters of this thesis, helping me to think about the hopeful possibilities and critical realities for humanitarian applications of this unusual biotechnology. I am extremely thankful for her critiques of my argument and writing, her insight into the existing literature on biotechnological sovereignty and her passion for this project's potential. I am thankful for the guidance and critiques of my other committee members Professor Yui Suzuki and Professor Adam Matthews. Their suggestions for how my work might be able to fit in the extant literature and for the future of my thesis project have been essential to defining the scope of my thesis and how I chose to write it in the end. Many deep thanks to Professor Yue Hu for being my honors visitor. I will never forget the kindness you showed me throughout my final year. I am also deeply grateful for the aid and moral support of my loved ones and friends both in the US and abroad in the UK throughout this thesising process. Amidst truly extraordinary global circumstances, the love, cheer, pride, forgiveness and flexibility you have all shown me as I have worked on this project will always be treasured. Finally, I would like to thank the Department of Biological Sciences at Wellesley College for supporting me in exploring and writing this unconventional piece. I dedicate this thesis to everyone above. I would not have been able to complete this piece without all of your combined efforts to keep me sane, smiling and well fed. Table of Contents I. Introduction 1 II. Chapter 1- Methods of Transformation 8 III. Chapter 2- Plant Molecular Pharming and Platform Diversity 33 IV. Chapter 3- Plant Molecular Pharming and Biotechnological Sovereignty for LMICs 58 V. Chapter 4- Hurdles for Biotechnological Sovereignty Using Plant Molecular Pharming 83 VI. Conclusion 111 VII. References Cited 116 VIII. Appendix 179 Introduction For over 40 years, the biopharmaceutical industry has studied and used genetically modified organisms to produce desirable proteins for use in medicines, therapies and biological tests. Today, such proteins are dominantly produced by a combination of prokaryotes like Escherichia coli , simple eukaryotes like yeast, or complex eukaryotes like mammalian cell lines. Whilst the products of living organisms have been used in medical practice since the dawn of history, it was only at the beginning of the 1980s when genetically modified organisms were first used to produce commercially sold proteins and peptides, with early products including synthetic insulin pharmaceuticals produced in genetically modified E.coli.1,2 However, using bacteria as the first commercially established genetically modified platforms, also suffered from protein production limitations that stemmed from the organisms' prokaryotic biology. Prokaryotic cells do not have the organelles nor perform all the metabolic processes needed to create proteins that originate from eukaryotes. This means that if a prokaryotic cell is genetically modified to produce a eukaryotic protein product, it may not necessarily fold the protein into the correct shape for proper function, nor add important post-translational modifications that influence the protein's stability and activities.3 Furthermore, many bacteria collect eukaryotic protein products in aggregated clumps called inclusion bodies, which require difficult and time consuming processes to extract product from.4 As such, eukaryotic platforms had to be developed to expand the portfolio of proteins and peptides that genetically modified organisms could produce commercially. 1 Numerous types of eukaryotic platform organisms have since been developed to compensate for the drawbacks of prokaryotic platforms. These eukaryotes range from simple unicellular organisms like yeast, to cultures of tissue cells derived from multicellular animals, like Chinese Hamster Ovary (CHO) or insect ovary cell lines.5 Unlike prokaryotic platforms, these eukaryotic platforms have most of the molecular machinery and metabolic processes necessary for properly producing proteins from most eukaryotic and prokaryotic organisms. However, each type of eukaryotic platform faces its own series of challenges as well. Yeasts struggle with not being able to correctly replicate complex post-translational modifications, like glycan addition, for foreign eukaryotic proteins, which limits the products they are capable of producing.6 Cell lines derived from multicellular organisms, like CHOs and insect cells, also struggle with slower cell multiplication and the extended production time that results.7 Yet, for all of the innovation, improvement and platform diversification over 50 years of genetically modified organism research, all of the above prokaryotic and eukaryotic platforms are grown in fairly similar production facilities which have their own series of drawbacks. Most genetically modified organism production methods that dominate industry remain dependent on enclosed, sterile bioreactor facilities for growth of organisms in culture and product production. Such facilities are expensive to set up and maintain, demand highly skilled labour to run, require extensive sterility measures to protect the cultures from pathogens, and ultimately limit the number of productive cells to what can fit within the enclosed bioreactor.8,9,10 A relatively new eukaryotic cell based production platform is currently expanding the capabilities and production methods of using genetically modified organisms. Molecular 2 pharming, or plant molecular farming, is the production and recovery of protein or peptide products from a genetically modified platform derived from plants. Unlike a eukaryotic or prokaryotic cell culture, what a molecular pharming platform looks like is far more difficult to define. Molecular pharming platforms include production methods that rely on familiar cultures of plant tissue cells, but that also can prominently involve whole plant organisms grown like agricultural crops11,12. Further complicating molecular pharming's definition are the diversity of species, target tissues, growth environments and transformation methods that the biotechnology can employ, granting genetically modified plant-based production a unique flexibility that few other platforms can match.13-16 Molecular pharming platforms have a specific and unique set of advantages. These advantages include the ability to avoid the expenses and scale limitations of bioreactor facilities through whole-plant based processes, their inherent safety against many dangerous pathogens, and their retention of most benefits of complex eukaryote-based production platforms. Whole plant platforms, unlike enclosed bioreactor based platforms, only rely on well-established and affordable agricultural techniques for growth.17 Whole plants are also not necessarily limited by scale, with open field production easily scaled up to meet demand through sewing more seeds or planting more plants.18 Even within more enclosed growth environments, like greenhouses or underground vertical farms, meeting multi-tonne scale levels of protein production is entirely possible.19 Furthermore, the costs and maintenance efforts are typically not as extensive for whole plant platforms as those for eukaryotic mammalian cell platforms, their closest functional equivalent in protein production capability. 20-22 3 Plant platforms, whether whole or in cell culture, also require less maintenance than their animal or prokaryotic counterparts because they are less susceptible to pathogens that can harm humans or animals. Plants are not currently known to host human- or animal- infecting pathogens23. In a similar vein, plant-specific pathogens are also generally not harmful to the humans and animals that are exposed to them.24 This ultimately means that, during product extraction, the pathogen removal steps that are essential for prokaryotes, yeasts or animal cell platforms may not be necessary for plant-based platforms. A direct display of this advantage was seen in the production story of Elelyso, a therapeutic enzyme for Gaucher's disease produced using genetically modified carrot cells. When Elelyso was being

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