Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, February, 2021 / Cataloged from the official PDF version of thesis. / Includes bibliographical references (pages 185-197). / Forward genetic screens are powerful tools for the unbiased discovery and functional characterization of specific genetic elements associated with a phenotype of interest. By perturbing thousands of genes simultaneously and selecting for a desired phenotype, genetic features can be systematically mapped to phenotypic changes. Recently, CRISPR-Cas9 has emerged as a powerful genetic perturbation technology, opening up new opportunities for forward genetic screens. In this thesis, I present work to advance this approach and demonstrate its application in a range of contexts relevant to human health. We first established a detailed CRISPR-Cas9 screening protocol that outlines experimental design considerations. We then applied this methodology to develop a CRISPR toolkit for screening and characterizing long non-coding RNAs in the human genome, many of which remain uncharacterized. We identified the EMICERI locus as a regulator of four neighboring genes, one of which conferred resistance to a melanoma therapeutic. We next sought to use CRISPR activation screening to gain insight into the cellular processes that govern tumor resistance to immunotherapy. We identified four candidate genes in our screen, which we validated in diverse cancer cell types and explored through mechanistic studies, leading to the discovery of novel immunotherapy resistance pathways. Finally, we developed a pooled transcription factor (TF) screening platform that provides a generalizable approach for studying cellular programming. We created a comprehensive human TF library and applied it to identify TFs that can drive differentiation of embryonic stem cells toward neural cell fates. We discovered that one TF, RFX4, leads to differentiation of neural progenitors that produced inhibitory neurons, providing an efficient method for generating this important cell type. During the COVID-19 pandemic, we paused the screening work and developed a streamlined SARS-CoV-2 detection assay, STOPCovid, suited for low-complexity settings. STOPCovid combines viral RNA concentration with isothermal amplification and CRISPR-mediated detection. STOPCovid achieved a sensitivity and specificity of 93.1% and 98.5%, respectively, on patient samples. Together, our applications of forward genetic screens address diverse problems in human health and broadly demonstrate the potential of this approach for systematically interrogating genetic elements. / by Julia Joung. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2019 / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 186-197). / Hands-on demonstrations greatly enhance the teaching of STEM concepts and foster engagement and exploration in the sciences. While numerous chemistry and physics classroom demonstrations exist, few biology demonstrations are practical and accessible due to the challenges and concerns of growing living cells in classrooms. Here I introduce a platform to develop hands-on molecular and synthetic biology educational activities based on easy-to-use, shelf-stable, freeze-dried, cell-free (FD-CF) reactions, which are simply activated by water. By using fluorescent proteins as a visual output, I created a variety of engaging modules using this platform that can teach the central dogma of biology, how certain cellular functions work, and other basic molecular biology topics that are otherwise difficult to easily teach in a hands-on manner. By expanding the platform to other non-visual outputs (such as smell or touch), as well as further incorporating components, such as RNA switches, I also developed modules that can teach more advanced biology topics, such as biochemistry, biomaterials, and synthetic biology, as well as basic laboratory skills such as pipetting, experimental design, and the scientific method. Pilot testing of a prototype kit based on these elements were tested in classrooms across the country and initial results suggest that the activities are accessible, easy to use, educational, and engaging for high school students. Overall, the platform introduces low-cost, user-friendly, and hands-on activities that can be used in classrooms to improve the quality of biology education and open the door for student-driven, independent explorations in the life sciences. / by Ally Huang. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2019 / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 154-176). / Lack of medication adherence is a worldwide problem. As many as 50-70% of patients have trouble following treatment recommendations. Whereas adherence is driven by many factors including the socioeconomic status of a patient and the quality of the health care team, drug regimen complexity also affects treatment outcomes. For example, adherence decreases as the number of pills per dose and the number of doses per day increases. For diseases where potent medications are available, depot formulations provide sustained drug release to simplify dosing. For diseases lacking potent compounds for treatment, there remains an unmet need for depot systems that could transform medication adherence. Tuberculosis (TB) is one such disease with a high pill burden, where poor patient adherence to the treatment regimen is a major cause of treatment failure and contributes to the emergence of drug-resistant TB strains. / For example, an average 60-kg patient with TB needs to take 3.3 g of antibiotics per day, which is a dose that exceeds the largest swallowable capsule and current depot systems. According to the World Health Organization (WHO), 10 million people developed TB in 2017 with a global economic burden amounting to $12 billion annually. This thesis presents a solution to the challenge of prolonged dosing for regimens such as TB that require multigram drug dosing. First, a gastric resident system (GRS) compatible with transesophageal administration was designed using biocompatible materials. The GRS consists of a series of drug pills on a coiled superelastic nitinol wire; the ends are protected with a retainer and tubing. Safe administration, gastric retention for 1 month, and retrieval of the GRS were demonstrated in a swine model. Next, sustained release formulations for 6 TB antibiotics were formulated into drug-polymer pills, and first-order drug release kinetics were achieved in vitro. / Then, the GRS was demonstrated to be capable of safely encapsulating and releasing 10 grams of an antibiotic over the period of weeks in a swine model. Lastly, end-user assessment was evaluated with a field questionnaire in India and an economic model to estimate the impact of the GRS on the health care system. There are multiple applications of the GRS in the field of infectious diseases, as well as for other indications where multigram depots could impart meaningful benefits to patients, helping maximize adherence to their medication. / "Funding and Resources: -- Bill and Melinda Gates Foundation -- National Institutes of Health -- National Science Foundation Graduate Research Fellowship -- MIT Tata Center and leadership team for believing in and guiding our project" / by Malvika Verma. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2019 / Cataloged from PDF version of thesis. / Includes bibliographical references. / Fireflies (Lampyridae) and certain other families of beetles including the American railroad worms (Phengodidae), Asian starworms (Rhagophthalmidae), and American click-beetles (Elateridae), produce light in a process known as bioluminescence. The bioluminescent systems of beetles, natively used for the purposes of mating communication and/or an aposematic warning signal, are now well understood and have been widely applied in biotechnology and biomedical research. There have been considerable advancements in the engineering of the luciferin substrate, and the luciferase enzyme, for beneficial characteristics such as altered emission wavelength, improved thermostability, and improved catalytic parameters, but despite this substantial effort focused on the biotechnological applications of beetle bioluminescence, major questions remain regarding its natural biochemistry and evolutionary origins. / Four major questions that were unanswered at the beginning of this PhD study were: (1) Do fireflies possess a storage form of their luciferin? (2) What is the evolutionary relationship of bioluminescence amongst the bioluminescent beetles families, and has this trait independently evolved multiple times? (3) How is firefly luciferin biosynthesized? And (4) Are there accessory genes from the bioluminescent beetles which act in bioluminescent metabolism, and might these genes be useful for biotechnological applications? Here I describe the discovery and characterization of the presumed storage form of luciferin in fireflies, sulfoluciferin, and the enzyme which produces it, luciferin-sulfotransferase. / Furthermore, I describe the sequencing, assembly, and characterization of the genome of the North American "Big Dipper" firefly Photinus pyralis, along with the Japanese "heike" firefly Aquatica lateralis genome, and the genome of the Puerto Rican bioluminescent click beetle or "cucubano" Ignelater luminosus. Genomic comparisons amongst these three species support the hypothesis that firefly and click beetle luciferase evolved independently, suggesting an independent evolutionary origin of the bioluminescent systems between these fireflies and click beetles. I also describe stable isotope tracing experiments in live fireflies, establishing that adult and larval fireflies likely do not de novo biosynthesize firefly luciferin, and may instead rely on a "recycling" pathway to re-synthesize luciferin from the luminescence product oxyluciferin. Lastly, I discuss the future directions resulting from this thesis, and the yet unanswered questions. / by Timothy Robert Fallon. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2019 / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 139-148). / Synthetic mRNA is an emerging therapeutic modality for gene and cell therapy. Unlike their synthetic DNA counterparts, synthetic mRNA has an increased safety profile due to its transient gene expression and ability to express outside of the nucleus. Furthermore, it can be more easily delivered to cells via entry only into the cytoplasm. While synthetic biology as a field has existed for over two decades, the main area of research and development has focused on DNA interfaces, building on the mechanisms of transcription factors with small molecule interfaces to create multi-input/multi-output genetic circuitry. Until recently, the field had not developed sufficient synthetic circuit control devices at the translational level due to 1) lack of perceived need and 2) deficiency of available natural systems for adaptation. In this thesis, I present the construction of a diverse synthetic biology toolbox for RNA-only synthetic biology. The creation of new synthetic biology frameworks can be broken down into three modules: Build, Control, and Apply. In the Build phase, I demonstrate how the current toolbox of mRNA binding and recognition proteins can be utilized to form diverse and orthogonal gene regulatory networks. In Control, I construct regulatory networks capable of responding to exogenous signals and utilize advanced circuit design to motivate dynamic control for novel behaviors. When I transition to Apply, I illustrate that these next-generation circuits can be layered into biologically active modalities that are therapeutically relevant. Taken as a whole, the work presented here represents a merging of the fields of synthetic biology and mRNA therapeutics, and serves as a foundational proof-of-principle for future efforts to expand synthetic biology across novel modalities. / by Jacob Robert Becraft. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2019 / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 118-128). / A variety of cancers are marked by the over-expression and over-activity of the EGF receptor (EGFR), rendering this protein an attractive therapeutic target. Anti-EGFR therapeutics are a mainstay of clinical practice for the treatment of colorectal, lung and head and neck cancers but efficacy is limited and response rates low. Opportunities for improving EGFR antagonism include higher potency inhibition of ligand binding, inducing receptor downregulation, or creating synergistic therapeutic combinations. The Wittrup lab has previously made significant advances in EGFR antagonism by demonstrating the therapeutic potential of inducing receptor downregulation through multi-epitopic targeting. The lab has also pioneered the use of a novel protein scaffold, called Sso7d, for yeast surface display-based libraries and selections. In the first part of this work I show that a combination of traditional yeast display techniques with simple but novel in silico approaches can be applied to derive a panel of Sso7d binders against EGFR with diverse paratopes. I demonstrate the superior EGFR inhibition of antibody-Sso7d fusions in vitro, and discuss the lessons learned from applying these proteins in vivo. In the second part of this work I use a structure-guided yeast display approach to create a novel research tool, a minimally modified verstion of cetuximab called "mCetux", which essentially enables in vivo experiments of cetuximab. I apply this antibody tool in vitro and in vivo in a new and highly relevant model system for colorectal cancer and subsequently discuss future opportunities for its use. / Funded by NIH/NIGMS Biotechnology Training Grant / by Alison W. Tisdale. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2018 / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 184-197). / Most malignant cancers are treated with chemotherapeutic agents that target and damage cellular DNA While genotoxic chemotherapies have proven to be highly effective agents for cancer therapy, it is well known that intrinsic and acquired cancer drug resistance is a problem that severely limits the successful elimination of a wide range of malignancies. This point is particularly important in the context of genotoxic chemotherapies, because the DNA damaging agents used in cancer treatment induce a diverse spectrum of toxic lesions that are recognized by a variety of DNA damage response (DDR) mechanisms. In this thesis I used CRISPR-Cas9 gene-editing and other molecular biology and biochemical techniques to examine the functional relevance of that Rev7, a multi-functional translesion synthesis (TLS) DNA damage tolerance protein in drug-resistant cancers. / In particular, I employed CRISPR-Cas9 gene-editing technologies to generate Rev7 knockout (KO) drug-resistant lung cancers cell to use as a tool to investigate the impact that Rev7 loss may have on chemotherapeutic efficacy. Excitingly, this work reveals that Rev7 loss sensitizes intrinsically drug-resistant lung tumors to cisplatin and drastically enhances the overall survival of syngeneic mice transplanted with drug-resistant lung tumors. Additionally, in this thesis I conducted immunoprecipitation and mass spectrometry to better elucidate the Rev7's functional relevance. Mass spectrometry findings in this thesis reveal that when Rev7 is immunoprecipitated under different cellular conditions (i.e. G2/M arrested or DNA damaged cells), Rev7 interacts with novel and diverse Rev7 protein interactors. Intriguingly, my mass spectrometry findings also reveal that Rev7 forms protein-protein interactions with many proteins that play a role in regulating double-strand break (DSB) repair. / Given these findings we conducted DSB repair studies investigating if Rev7 plays a role regulating DSB repair in drug-resistant lung cancer. Notably, these studies suggest that Rev7 loss results in a decrease in DSB repair capacity and increases in DSBs in drug-resistant lung cancer cells. Altogether, this thesis demonstrates that Rev7 has functional relevance in modulating chemotherapeutic response in drug-resistant lung cancer. Further, this thesis presents findings that strongly argue that the development of small molecule inhibitors targeting Rev7 may provide a new way to enhance chemotherapeutic efficacy in drug-resistant tumors in the clinic. / by Faye-Marie Vassel. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2018 / Cataloged from PDF version of thesis. / Includes bibliographical references. / The humble red blood cell (RBC) is the most abundant cell in the human body. Every second, a normal adult generates some 2.5 million RBCs, which subsequently circulate through the blood vessels for a lifespan of 50 or 120 days in mouse and human, respectively. RBCs are also unique in that they are completely enucleated once fully mature. These two characteristics exist as distinct assets for cellular therapy applications utilizing RBCs as a platform, enabling long-lasting availability in vivo and the ability to genetically modify precursor cells without worry of the terminally differentiated progeny carrying any foreign genetic material. The first part of this thesis is devoted to the establishment of methodologies that allow for the covalent attachment of both natural and synthetic cargoes to the surface of red blood cells without compromising its biological properties. This system employs genetic engineering and sortase A, a bacterial transpeptidases. / We show that this strategy is able to efficiently engineer both mature mouse and human RBCs in a site-specific and covalent manner. The next portion of this work describes how these established methodologies can be mixed and matched according to the diverse needs of engineered RBC applications. We provide a proof of concept that utilizes engineered RBCs to prolong prophylactic protection against deadly toxins. By expressing chimeric proteins of single domain antibodies (VHHs) against botulinum neurotoxin A (BoNT/A) with RBC-specific proteins, we demonstrated that mouse RBCs expressing anti-BoNT/A VHHs can provide resistance up to 10,000 times the lethal dose (LD₅₀) of BoNT/A. We validate this finding by repeating our results in a human RBC culture system that we have improved to achieve 90% enucleation, illustrating the broad translatability of our strategy for therapeutic applications. / Finally, drawing upon knowledge that the body clears 2.5 millions RBCs every second to maintain homeostasis, we use sortase to attach disease-associated autoantigens to genetically engineered and to unmodified red blood cells (RBCs). Such modified RBCs masquerade with these autoantigens as their own, and hijack the non-inflammatory nature of the RBC clearance pathway to promote tolerance to their carried payload. We show that this blunts the immune contribution of major subsets of immune effector cells (B cells, CD4+ and CD8+ T cells) in an antigen-specific manner. Transfusion of RBCs expressing self-antigen epitopes alleviates and even prevents signs of disease in an experimental system for autoimmune encephalomyelitis, and also maintains normoglycemia in a mouse model of type 1 diabetes, highlighting the potential of engineered RBCs for treating autoimmune diseases. / Taken together, the results of applying our engineered RBCs in areas of both acute infectious and toxic agents, as well as for longer-term chronic and autoimmune diseases, hint at the tremendous potential of this system, and we have only begun to scratch the surface. / by Novalia Pishesha. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2019 / Cataloged from PDF version of thesis. "June 2019." / Includes bibliographical references. / The ability to map the molecular organization of cells and tissues with nanoscale precision would open the door to understanding their biological functions as well as the mechanisms that lead to pathologies. Though recent technological advances have expanded the repertoire of biological tools, this crucial ability remains an unmet need. Expansion Microscopy (ExM) enables the 3D, nanoscale imaging of biological structures by physically magnifying cells and tissues. Specimens, embedded in a swellable hydrogel, undergo uniform expansion as covalently anchored labels and tags are isotropically separated. ExM thereby allows for the inexpensive nanoscale imaging of biological samples on conventional light microscopes. In this thesis, I describe the development of a method called Expansion FISH (ExFISH) that uses ExM to enable the nanoscale imaging of RNA throughout cells and tissues. A novel chemical approach covalently retains endogenous RNA molecules in the ExM hydrogel. After expansion, RNA molecules can be interrogated with in situ hybridization. ExFISH opens the door for the investigation of the nanoscale organization of RNA molecules in various contexts. Applied to the brain, ExFISH allows for the precise localization of RNA in nanoscale neuronal compartments such as dendrites and spines. Furthermore, the optical homogeneity of expanded samples enables the imaging of RNA in thick tissue-sections. ExFISH also supports multiplexed imaging of RNA as well as signal amplification techniques. Finally, this thesis describes strategies for the multiplexed characterization of biological specimens. Taken together, these approaches will find applications in developing an integrative understanding of cellular and tissue biology. / by Asmamaw T. Wassie. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2017 / Cataloged from PDF version of thesis. "February 2017." / Includes bibliographical references (pages 157-170). / The local cytokine milieu shapes the nature and function of immune cells and by extension the overall course of tissue-specific immune responses. In the context of cancer and autoimmunity, two opposing immune responses, the local immune environment can lead to dysfunctional T cell states. Understanding the mechanisms that distinguish these two states is key to identifying unique pathways that could be induced in autoimmunity and relieved without causing autoimmunity in cancer. We sought to characterize unique and shared T cell states in the context of multiple sclerosis (MS) and glioblastoma (GBM) using a combination of immunophenotyping and functional approaches, including ultra low-input transcriptional profiling. First we use a series of unbiased approaches to identify functional differences between auto-reactive T cells derived from MS or healthy donors. / We found that MS-derived CCR6⁺ T cells produce pathogenic inflammatory cytokines IFN-[upsilon], IL-17, and GM-CSF, while healthy controls produce IL-10 in response to myelin antigen. We also identified a transcriptional signature that characterizes these cells from MS donors and highlights the role of CTLA-4 signaling in autoreactive T cells derived from healthy donors. Next, we sought to better understand the role of another co-inhibitory receptor, PD-1, specifically in the context of Treg and CD4⁺ T effector function in GBM, a central nervous system cancer. We identify unique signatures of dysfunction in both the CD4⁺ T cell and Treg compartment correlated with PD-1 expression. Finally, adverse development of autoimmunity in the context of treatment with blocking CTLA-4 antibodies suggests that studies directly comparing T cells in the context of autoimmunity and cancer can yield valuable insight into mechanisms of T cell dysfunction. / Through a transcriptional comparison of isolated T cell subsets from the spinal fluid of MS patients, tumors of GBM patients, and matched blood we identify common biological mechanisms of CNS T cells and identify unique signatures of CNS exhaustion. Taken together, our data suggests that the CNS may be enriched for pathogenic cells in the context of both MS and GBM. / by Brittany A. Goods. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering