Massachusetts Institute Of Technology

NIH Research Projects · FY 2025 · 2021-09

We propose to develop a probe technology for monitoring human brain function with molecular precision; in conjunction with magnetic resonance imaging (MRI) or other imaging modalities, the probes will provide a combination of sensitivity and resolution that could permit unprecedented noninvasive studies of dynamic neu- rophysiological processes in people. Our strategy is based on a fundamentally new type of chemical imaging probe designed to produce neuroimaging readouts by purposefully manipulating endogenous hemodynamic contrast in the brain—repurposing the blood oxygen level dependent (BOLD) effect that underlies conventional functional MRI (fMRI). This new “vasoprobe” concept offers three key advantages: First, by providing time-de- pendent sensitivity to dilute molecular species such as neurotransmitters, the probes can enable well-defined neurobiological phenomena to be mapped dynamically across the entire brain, dramatically surpassing existing nonspecific fMRI approaches. Second, because of the endogenous contrast source they influence, the probes are detectable on a variety of spatiotemporal scales by noninvasive imaging modalities complementary to fMRI, such as diffuse optical or ultrasound-based methods. Third, by circumventing limitations of established optical, magnetic, and radioactive probe designs, vasoprobes combine exquisite sensitivity approaching that of positron emission tomography (PET) with the resolution and versatility of MRI. In this project, we will build on our recent proof-of-concept work with vasoprobes to establish noninvasive brain-wide delivery strategies and to develop robust neurochemical sensors that function in primates. The technology we establish will address multiple goals in basic and applied neuroscience, and we expect it to yield molecular probes that will be appropriate for clinical evaluation in human subjects by the end of the project period. In Aim 1, we will create vasoprobe variants that can be delivered to the brain via intravenous injection and spontaneous permeation through the blood-brain barrier (BBB). We will form conjugates of vasoprobe-based sensors with “brain shuttle” antibodies that have previously been shown to enable brain import via receptor- mediated transcytosis. Demonstration of brain-permeable vasoprobes will establish a clinically viable path for facile, noninvasive applications of vasoprobes throughout the brain. In Aim 2, we will optimize vasoprobes to sense the key neurotransmitters dopamine and glutamate; we will then apply them on a brain-wide scale for molecular-level fMRI in rodent brains. These experiments, in conjunction with outcome of Aim 1, will set the stage for applications of neurotransmitter-sensitive vasoprobes and related sensors in primate brains. Accord- ingly, in Aim 3, we will adapt neurotransmitter-sensitive vasoprobe technology for functional molecular neuroim- aging in marmosets, a tractable primate species with which we have previous experience. Successful completion of validation experiments in marmosets will therefore establish groundbreaking imaging agents suitable for trans- lation to humans, as well as for adaptation to many further neurophysiological targets.

NIH Research Projects · FY 2025 · 2021-09

Project Summary ​ ​ RFA-AI-19-037 The rising incidence of Lyme disease demands new strategies for prevention. Existing methods such as acaricides, deer reduction, landscaping, and personal protective clothing, are inherently short-term and must be regularly re-applied, maintained and worn. ​The Mice Against Ticks project seeks to develop a durable one-time intervention to disrupt the ecological cycle of Lyme disease transmission for many decades​. The causative agent of Lyme disease ​B. burgdorferi ​is passed back and forth between ticks and their small animal hosts, which serve as zoonotic reservoirs of disease. The white-footed mouse ​P. leucopus​ is widely considered to be the most important reservoir because it is both ubiquitous and extremely efficient at acquiring and transmitting pathogens via ticks. Our overarching goal for this proposed project is to heritably immunize white-footed mice against Lyme by encoding protective ​P​. ​leucopus​ antibodies targeting ​B. burgdorferi ​outer surface protein A (OspA) in the mouse germline. According to our calculations, combining at least four such antibodies should prevent evolutionary escape by ​B. burgdorferi ​because too many simultaneous OspA mutations would be needed. Crucially, even if these mice are less important in some areas than currently thought, immunization will reduce the number of infected ticks, which in turn will infect fewer secondary reservoirs, which will infect fewer ticks, reinforcing a negative feedback spiral anticipated to greatly reduce the local burden of Lyme disease. We have already successfully isolated anti-OspA antibodies from OspA-immunized ​P. leucopus​, derived putative ​P. leucopus embryonic stem cells, and shown that the albumin locus appears suitable for antibody secretion from the liver. We now seek to (1) identify antibodies that bind to at least four different OspA epitopes, (2) establish a stable embryonic stem cell line and perform germline editing, and (3) generate heritably resistant mice that express antibodies from a cisgenic cassette linked to a reciprocal chromosomal translocation, a naturally occurring form of high-threshold gene drive that would enable the reversible and tightly localized engineering of wild ​P. leucopus populations. Our open and community-guided approach has met with apparent enthusiasm by residents of Nantucket and Martha’s Vineyard, indicating that local communities suffering from tick-borne disease throughout the Northeast and Upper Midwest may wish to immunize their own wild mouse populations in order to help prevent Lyme disease for many decades.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY Although genetic tools have dramatically advanced our understanding of brain function, they have largely been confined to mice. While mice are essential models for many areas of neuroscience, there are also many aspects of higher brain function that cannot be adequately modeled in rodents. Similarly, many brain disorders affect higher cognitive functions that have no clear parallels in rodents. Furthermore, recent large- scale single cell transcriptomic analyses have revealed many neuron types, connections and gene expression patterns that are unique to primates. Thus, there is an urgent need for new genetic models that have brain structure and function closer to humans. Non-human primates (NHP) are much more closely related to humans than are rodents, and this is reflected in their brain development, structure and physiology. Hence, it is increasingly recognized that they provide an attractive model to study higher brain function and brain disorders. A promising emerging NHP model is the common marmoset, a small new world primate that has many advantages for neuroscience and genetic research. However, lack of tools with cell type specificity has been a major obstacle in advancing structural and functional studies in NHP. With the combined single cell RNA-seq and single cell ATAC-seq, it is now possible to nominate short cell type-specific enhancer sequences. If validated, these enhancers will provide an effective tool to map connectivity and interrogate function using virus mediated expression. The difficulty lies in the identification of functional enhancers from the hundreds or thousands of nominated potential enhancer sequences in NHP. Here we propose (1) to use a novel high throughput in vivo approach to identify functional enhancers, and (2) to establish a whole-brain circuit mapping pipeline for use striatal circuitry to validate our approach for cell type-specific connectivity mapping in marmosets. When completed, these studies will provide much needed essential tools, methods and computational pipelines for cell type-specific mapping and functional interrogation of the marmoset brain in healthy and disease models.

NIH Research Projects · FY 2025 · 2021-09

Primate brains contain cortical areas that exhibit selective engagement in high-level sensory or behavioral operations. The functional specialization of these regions is thought to be central to primate-specific cognitive faculties and to associated disorders. Deciphering the origins of functional specialization in primate brain regions has been an enormously challenging task, however, due in large part to the absence of suitable experimental tools. To address this problem, we will develop a method for measuring the activity of inputs to specialized areas from throughout the brain, permitting systematic analyses of information flow in the multiregional neural circuitry that gives rise to high-level functions. Our method will employ a conceptually new family of genetically encoded imaging probes called NOSTICs, which transduce the calcium signaling of NOSTIC-expressing neurons into localized hemodynamic signals that can be dynamically monitored using brain-wide measurement techniques like functional magnetic resonance imaging (fMRI). When delivered using retrogradely transported viral vectors, NOSTICs can permit targeted fMRI-based recording of neural activity in distributed cell populations that provide monosynaptic input to any injection target in the brain. In our preliminary work, we have created first-generation NOSTIC probes and used them to demonstrate genetically targeted functional imaging in rodents. In Aim 1 of this project, we will take two steps that adapt this tool for use in nonhuman primates. We will create second- generation NOSTICs that display improved performance for circuit-specific functional imaging, while also devel- oping viral vectors that allow expression of these probes to be tracked longitudinally in primate brains. We will also adapt the NOSTIC probes for incorporating into adeno-associated viruses, which provide extended capa- bility compared with the herpes viruses we currently use. In Aim 2, we will perform pilot experiments to investigate whether NOSTICs can provide circuit-specific readouts in nonhuman primates. These tests will already be pos- sible using our currently available probes and vectors, and new variants from Aim 1 will also be tested when available. Successful demonstration of NOSTIC functionality for circuit imaging in marmosets constitutes our proposed go/no-go criterion for entry into the UH3 stage of this project. Then in Aim 3 (UH3 stage), we will validate NOSTIC probes in two paradigms that explore their performance across brain regions, experimental contexts, and primate species. In the first paradigm, we will apply NOSTICs to examine origins of functional specialization in face-selective regions of the marmoset brain. In the second paradigm, we will apply NOSTICs to investigate brain-wide contributions to object selective responses in the ventral stream of the macaque visual cortex. These experiments will be performed as multi-laboratory collaborations that both harness and dissemi- nate the NOSTIC technology; this work will therefore establish a broadly applicable transformative approach for mechanistic analysis of primate brain function.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY / ABSTRACT Eukaryotic cells are defined by their organelles, membrane-enclosed compartments in which specific cellular processes are carried out. The nucleus is the largest organelle, contains all genetic material, and enables separation of gene transcription from protein translation. As the nuclear envelope (NE) serves as a tight barrier enclosing the nucleus, the cell requires machinery to establish and control nucleo-cytoplasmic communication. There are two principally different components to this machinery. On one hand, nuclear pore complexes (NPCs) serve as the main conduit for molecular exchange across the NE. On the other hand, universally conserved linker of nucleo- and cytoskeleton (LINC) complexes serve as physical tethers across the NE, which are necessary for positioning the nucleus and for mechano-sensing in a diverse set of circumstances. Dysfunction of the machinery is at the core of important human diseases, including skeletal and cardiac myopathies, premature aging, and cancer. Our goal is to understand the structure of the protein complexes involved in nucleo-cytoplasmic communication at high (atomic) resolution. Such information helps to identify and separate the myriad functions this machinery carries out and that we are still only beginning to fully grasp. High resolution information further provides the basis for structure-guided drug design to interfere with the salient human diseases, such as Emery-Dreifuss Muscular Dystrophy (EDMD) and Primary Dystonia, which are still not cured. The structural characterization of the NPC and the LINC complex are challenging, because of the size and complexity of these multi-MDa assemblies. Over the past 15 years, we have made significant advances on both problems. For the NPC, we have chosen a highly productive bottom-up approach, in which we characterized multi-subunit complexes predominantly by X-ray crystallography, the building blocks of the massive, 40-100 MDa NPC. Those structures have now been used in combination with cryo-electron tomographic (cryo-ET) maps of assembled NPCs to generate composite structures that attempt to position the roughly 500 individual proteins within one NPC. For the LINC complex, we solved the universally conserved core component and have started to untangle the diverse network of its components, the Sad1/UNC-84 (SUN) and Klarsicht/ANC1/Syne-Homology (KASH) proteins. Going forward, the challenge is the structural characterization of large and dynamic assemblies, which is true for both, the NPC and the LINC complex, for the latter particularly when including the connection to the nucleo- and cytoskeletal components. The dramatic advances in cryo-electron microscopy (cryo-EM) over the recent past make this technology particularly important for our studies. We anticipate combining X-ray crystallography and cryo-EM for studying the most relevant structures going forward. The success of this will depend upon innovative, tailored methods to address the particular challenges that come with each project. We have repeatedly shown over the past decade how to successfully approach such challenges and have devised methods to meet them.

NIH Research Projects · FY 2025 · 2021-09

Major advancements in stem cell biology have paved the way for innovation in organoid engineering. Organoids are 3D tissues derived from human induced pluripotent stem cells (hiPSCs) generated by reprogramming patient- specific adult cells, such as fibroblasts. While organoids show great promise as testbeds for investigating devel- opmental biology, current methods for organoid production are limited by their reliance on external inputs, such as growth factors and small molecules, which affect cells imprecisely and give rise to immature organoids that do not faithfully recapitulate in vivo physiology and functionality. The resulting organoids are size-constrained, lim- ited to a small set of cell types, and do not generally develop mature tissue that exhibits the functionality of fully developed organs. While we have previously demonstrated genetic programs that enable organoids to generate all requisite cell types in liver, variability in cell ratios remains an open challenge for achieving reproducible, high quality organoids. Further, progress is blocked by the inability to reliably guide multi-lineage specification, the lack of precise timing of multistep differentiation, and the inability to make robust bifurcation decisions that determine the ratios of the resulting cell types. To overcome these obstacles, we will combine synthetic biology, developmental biology, and control theory to design novel open and closed loop genetic controllers that individually guide differentiation from within each cell to form unique new 3D tissue: vascularized pancreatic organoids with defined ratios of endocrine and exocrine cells. We will demonstrate how these new organoids can serve as more sophisticated and comprehensive models for investigating developmental biology principles. This work will spearhead a transformation in organoid synthesis by shifting the field from manual addition of inductive chemical signals to cell type conditional, self-timed ectopic expression of transcription factors that induce differentiation. Building upon the premise that 1) gene sensors can detect cell types specific to differentiation stages, and 2) at least in certain important cases, regulated expression of lineage-specifying transcription factors can guide differentiation to the next stage, our main hypothesis is that feedback regulation of cell lineage bifurcation decisions can lead to more robust and reproducible sub-population ratios in organoids in comparison to open loop approaches. Our organoids will contain synthetic developmental programs that are self-timed and globally-orchestrated, with cells working together to generate the requisite ratios. We will create a platform for programmed bifurcation decisions that can be used for other differentiation steps in the pancreas, and more broadly to other organoid and tissue types. We will use this platform to perform novel developmental studies to systematically vary the ratio of endocrine to exocrine cells and measure the consequences on exocrine/endocrine cells and their differentiation and function. The ability to precisely vary the ratio while studying gene expression profiles, the organoid secretome, and its affects on target cells will provide invaluable benefit in the investigation of pancreas development and dysfunction.

NIH Research Projects · FY 2026 · 2021-09

Project Summary/Abstract Adoptive cell therapy (ACT) is a promising therapeutic approach for the treatment of cancer. However, the initial success of ACT has been limited to chimeric antigen receptor (CAR)-T cell therapies for hematological malignancies. Applying this cell therapy to solid tumors is challenged by the lack of targetable tumor antigens, the severe systemic toxicity and the suppressive tumor microenvironment. T cell receptor (TCR) gene therapy can overcome some of these challenges because it enables targeting of intracellular proteins presented as peptide antigens on the human leukocyte antigen (HLA) complex. However, the majority of naturally occurring TCRs are of low-affinity to their peptide-HLA targets. Engineering these TCRs via phage display or yeast display for higher affinity is complicated by the introduction of unwanted cross-reactivity and the poor association between affinity and function. This project seeks to tackle each of the major challenges of ACT in order to effectively reprogram the immune system to combat solid tumors. The F99 phase is focused on a TCR engineering platform for the creation, modification, and profiling of TCRs that can target tumor-associated self-proteins with minimal toxicity profiles. In this approach, I first raise T cells from the natural repertoire that recognize a related ‘foreign’ peptide that differs by one amino acid from the self-peptide. Then, I modulate the fine specificity of the TCR by directed evolution of the peptide binding region to switch its specificity towards the tumor self-antigen of interest. I demonstrate the value of this approach by the creation of libraries of viral-specific TCRs and the subsequent in vitro selection of TCRs that switched specificity to a closely related epitope. The engineered TCRs showed robust T-cell activation after ligand recognition and are of equal or higher efficiency than the parental receptor. Importantly, the engineered TCRs displayed no additional promiscuity or off-target specificities as compared to the parental TCRs. The goal for the remainder of my dissertation project is to apply this approach to the generation of cancer reactive TCRs. By controlling the fine specificity of TCRs, this approach will overcome two of the major challenges of ACT, namely increasing the breadth of antigens that can be used for ACT while also minimizing cross-reactivities. For the K00 phase, I will shift my focus to addressing the suppressive tumor microenvironment that surrounds solid tumors by developing novel synthetic receptors and testing them in mouse models. I plan to build upon my synthetic biology background to implement novel high-throughput screens, learn new statistical analysis methods, and gain experience working with in vivo mouse models of cancer. These new approaches, coupled with my already strong background in genetics, molecular biology and biochemistry will allow me to address the most pressing and challenging issues facing targeted immunotherapies. With the aid of this award, I intend to continue my research contribution to become a leader in the field of cancer immunotherapy.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY/ABSTRACT Spotted Fever Group (SFG) Rickettsia species are obligate intracellular bacterial pathogens that cause mild-to- life-threatening vascular diseases in humans. To promote widespread disease, SFG Rickettsia species have evolved dynamic strategies to invade host cells, escape into and thrive within the cytosol, and spread from cell to cell. We hypothesize that SFG Rickettsia coordinate their complex life cycle by delivering an arsenal of secreted bacterial proteins (i.e., effectors) into cells to reprogram cellular processes, but the identity and targets of these secreted effectors have remained largely unknown. Furthermore, how these effectors function in the vector are a mystery. To overcome these barriers, we have used a forward genetic screen and cell-selective proteomics to identify and study secreted effectors. We discovered that the effector Sca4 promotes cell-to-cell spread in mammalian cells in contrast to its critical role regulating bacterial growth in tick cells. Each of these activities have been linked to distinct adhesion and endocytic machinery, respectively, highlighting the different contributions effectors play across environments. Then, using a bioorthogonal non-canonical amino acid tagging strategy for the first time in the Rickettsia genus, we also discovered seven new effectors we called Secreted rickettsia factors (Srfs). Most of these Srfs are annotated as hypothetical proteins and localize to distinct subcellular compartments in host cells. This proposal will leverage these key discoveries alongside our multidisciplinary approaches in cell biology, biochemistry, and genetics to examine how secreted effectors promote different aspects of the R. parkeri infectious life cycle. In Aim 1, we will investigate the functions of Sca4 across cellular environments to elucidate how Sca4 specifically targets the subcellular pool of a host cell-cell adhesion factor during R. parkeri cell-to-cell spread (Aim 1.1) and how Sca4 promotes bacterial growth in tick cells (Aim 1.2). In Aim 1.3 we will ask if these functions are also required in in vivo models of infection, highlighting critical functions of Sca4 in mammalian and tick colonization and pathogenicity. Then in Aim 2, we will use high- throughput phenotypic and interactome screens to begin to dissect the functions of the novel Srfs. Collectively, the proposed research will dramatically improve our fundamental understanding of rickettsia biology and rickettsia-host/vector interactions, which is essential to combat the growing threat of tickborne diseases.

NIH Research Projects · FY 2025 · 2021-08

Humans make rich inferences about the relationships between entities in the world from scarce information. For example, we can find a novel destination after seeing a few street numbers, or find a page in a dictionary by glancing at a few words in other pages. Theoretical considerations suggest that the brain makes such inferences by constructing "internal models" of the relationships in the environment (relationships between actions and states of the world), and by mentally simulating those models. However, the neural substrates and mechanisms of mental simulation are not understood. Our overarching goal is to integrate insights from theory and modeling with behavior and electrophysiology in awake, behaving monkeys to understand how mental simulation of internal models support relational inference. We will develop a behavioral task for monkeys in which they have “navigate” mentally from one stimulus to another along a one-dimensional abstract space of discrete stimuli (i.e., a sequence of images). We will assess whether animals’ behavioral characteristics exhibit hallmarks of mental simulation. We will then create a large library of neural network models to generate hypotheses for alternative computational strategies (including mental simulations) that the brain might employ for navigating abstract spaces. Next, we will record from candidate brain areas in the parietal and entorhinal cortex of monkeys, and analyze the data at single cell and population levels looking for signatures of mental simulation. Finally, we will adopt an iterative approach involving model-based data analyses and data-driven model revision with the ultimate goal of creating models that simultaneously succeed in performing task-relevant computations (i.e., behavior) and account for observed neural responses. Finally, we will validate our framework by evaluating the predictions of our models for both behavior and electrophysiology in new behavioral tasks.

NIH Research Projects · FY 2025 · 2021-08

Protein synthesis underpins a cell’s decision to growth, proliferate and/or differentiate.2,6,11–14,14,15,18 Understanding how protein synthesis allow cells to perform these fundamental activities is a major challenge in biology. Therefore, there is a critical need to elucidate the mechanisms determining protein synthesis rates and whether these mechanisms operate in a cell type-specific manner to impart a new layer of regulation in the control of gene expression. To explore these questions, two orthogonal, but complementary, research programs, namely Program 1 and 2, have been designed. Program 1 investigates new factors and mechanisms involved in the regulated assembly of ribosomes in stem cells. Program 1 is built upon recent studies from my lab and others demonstrating that stem cells relies on ribosome assembly to ensure adequate protein synthesis rates and the transition from self-renewal to differentiation.2,3,6,11–14,14,15,18 My lab has characterized the composition of the small subunit (SSU) processome in human cells, and identified DNA-dependent protein kinase (DNA-PK) as an RNA-dependent regulator of ribosome assembly and proteins synthesis in hematopoietic stem cells.6 Thus, the immediate goal of Program 1 is to establish the mechanisms by which DNA-PK regulates ribosome biogenesis in stem cells. Program 2 explores how customizing ribosome assembly and function contributes to protein synthesis and selective mRNA translation during embryogenesis. Program 2 is underscored by recent findings suggesting that ribosomes composition and activity are dynamically regulated in a cell type- and tissue- specific manner, allowing protein expression to be regulated with exquisite temporal and spatial precision.8,12 The immediate goal of Program 2 is to generate in vivo model systems to understand how the cell creates and regulates ribosome heterogeneity and the importance of this form of regulation for proper cellular function and organismal development. To address these, we have generated transgenic zebrafish in which two compositionally distinct and developmentally regulated ribosomes have been genetically labeled, a unique and powerful tool to study functional aspects of the ribosome in an in vivo developmental model system. Over the next five years, we expect Program 1 and 2 to uncover new mechanisms regulating ribosomes assembly and function in stem cells and vertebrate development and to provide powerful insights into ribosomopathies, tissue- specific disorders linked to defects in ribosome biogenesis and function.

NIH Research Projects · FY 2026 · 2021-08

PROJECT SUMMARY HIV and influenza pose major health burdens in the US and worldwide, claiming hundreds of thousands of lives annually, with the latter additionally posing a significant pandemic threat. Protein subunit vaccines formulated as nanoparticles with multivalent display of pathogenic immunogens are a proven, successful strategy to safely and effectively induce long lasting humoral, antibody-based immunity. Indeed, this strategy may be particularly important for challenging-to-neutralize pathogens such as HIV, which still does not have an approved preventative vaccine, and for designing future-proofed vaccines against the rapidly evolving pathogen influenza that poses an ongoing pandemic threat. However, to date it remains unclear what the optimal characteristics of nanoparticulate vaccines are. In addition, because most particulate subunit vaccines are composed of protein virus-like particles (P-VLPs) that multivalently display pathogenic antigens that are the target of broadly neutralizing antibody (bnAb) responses, it remains unclear whether the protein scaffolds used to formulate these VLPs are in themselves eliciting humoral immune responses that distract the immune system away from the pathogenic antigens of interest. In this project, we previously discovered that DNA-based VLPs (D-VLPs) can be used to spatially organize antigens multivalently in a manner similar to P-VLPs to elicit humoral antibody responses across multiple pathogens, yet without the protein-based scaffold VLP and resulting off-target antibody response. Here, we investigate how D-VLPs can be used to optimize priming, shepherding, and polishing phases of HIV vaccination using sequential introduction of engineered immunogens to drive the generation of target bnAbs. Specifically, we test the relative roles of antigen copy number, spacing, VLP size and geometry, glycosylation, and passivation, on lymph node targeting, germinal center formation, and on-target B cell activation. We investigate how these principles can be used together with controlled presentation of T cell helper epitopes and cytokine co-delivery to promote robust, long-lived GCs that result in bnAbs for HIV. Finally, we explore how the D-VLP platform generalizes to the distinct, challenging-to-neutralize pathogen influenza, towards identifying future-proofed vaccines to avert pandemic threats. Taken together, these results will help elucidate optimal VLP design rules for HIV and influenza, and may eventually also offer a clinically relevant new vaccine platform in future work.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY While mice are essential models for many areas of neuroscience, there are also many aspects of higher brain function and dysfunction that cannot be adequately modeled in rodents. Thus, there is a need for new genetic models that have brain structure and function closer to humans. For these reasons, non-human primates (NHP) provide an attractive model to study higher brain function and brain disorders. A promising emerging NHP model is the common marmoset, a small New World primate that has many advantages as a genetic model. Although the adaptation of bacterial CRISPR/Cas systems for targeted genome engineering and model creation has revolutionized modern biology, editing of the marmoset genome is still in its infancy. Given the significant time and money required for marmoset genome editing, new methods to increase editing efficiency, decrease mosaicism, and identify correctly-edited embryos prior to transfer to recipient females are critical. Additionally, new methods for controlling the zygosity of founder animals are necessary to enable analysis of homozygous F0 animals and to avoid homozygous editing when targeting essential genes that cause embryonic lethality upon biallelic disruption. To these ends, we propose a research program that will significantly enhance our ability to introduce multiple types of edits into the marmoset genome, reduce mosaicism, control the zygosity of edits, and identify successfully edited embryos through prenatal genetic testing. We will disseminate these technologies and models through direct resource sharing, in-person trainings, deposition to NIH-supported Marmoset Coordination Center. Together, the proposed advances will significantly reduce the time, effort, costs and animal numbers necessary to marmoset genetic models and will unlock the true potential of marmosets for basic and translational neuroscience research.

NIH Research Projects · FY 2025 · 2021-08

Project summary Synthetic biology aims to harness the power of biological systems to dynamically access information in the cell, enabling synthetic biomedical tasks such as tumor surveillance, pathogen identification, or cell-fate reprogramming. Such tasks in cellular engineering rely on robust mechanisms to regulate transgenes for the delivery of enzymes, genetic corrections, and cellular therapies. To unleash its full potential, mammalian synthetic biology requires foundational tools for implementing reliable control of gene expression in primary cells. For example, transgene silencing (e.g. loss of expression) remains a common challenge to effectively engineering primary cells. Layers of regulation across a range of length- and time-scales coordinate events from molecular binding to cell signaling regulate gene expression and thus cell fate. Multiscale approaches are needed to integrate the diverse processes that control cell-fate transitions. Cell-fate transitions represent pivotal events requiring coordination of multiple processes from epigenetic and cytoskeletal remodeling to proliferation and transcription. Understanding these transitions may illuminate how oncogenes coopt these processes to drive cellular transformation. Here, we propose a multiscale approach for understanding and engineering cell-fate transitions (e.g. reprogramming, differentiation). From our previous work to identify principles of cell-fate transitions, we identified systems-level constraints that limited reprogramming and developed a cocktail that increased reprograming 100-fold in mouse cells. Comparing the human and mouse response to reprogramming, we identified species-specific differences in proliferation, signaling, and the innate immune response during reprogramming that may contribute to lower reprogramming rates for human cells. We propose to examine these molecular correlates to determine how each impacts the reprogramming process and outcomes. We will use these insights to design genetic controllers to guide cells through reprogramming. Already we have identified a strategy to optimize reprogramming by inducing a transient “erase” phase followed by a “write” phase to establish the new cell fate. We propose to develop controllers capable of autonomously guiding cells through these competing objectives to enhance the efficiency of reprogramming. Genetic controllers are composed from synthetic gene circuits connected to native gene regulatory networks. While significant efforts have been devoted to the logical design of enhanced synthetic circuitry (e.g. circuits for synchronized quorum sensing, edge-detection), less is understood regarding how cellular hardware and the emergent three-dimensional structure of genetic elements affect circuits. Here, we propose to improve our understanding how transcription reshapes DNA and how it impacts the performance of gene circuits. Defining the role of chromatin structure in cellular identity will guide molecular engineering efforts to build genetic controllers capable of regulating behavior. We anticipate that the principles and tools we develop will be broadly applicable across cellular engineering applications as well as for investigating cell-fate transitions.

NIH Research Projects · FY 2025 · 2021-07

Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. This proposal is for support of the pre-doctoral program in Cellular, Biochemical and Molecular Science at the Massachusetts Institute of Technology (MIT). Our mission is to train the next generation of biological/biomedical scientists, many of whom will be innovators and leaders in research, education, and other fields. This proposal builds on an outstanding training record developed over a 46-year partnership with NIGMS. Our aims are to: educate students to understand the fundamental and underlying principles of molecular, biochemical, and cellular biology, train students to be critical and creative thinkers, prepare students to be ethical decision makers, teach and provide practice in written and oral communication, provide experience with teaching and mentoring younger students, mentor students to become effective and rigorous researchers, guide students through completion and publication of research projects, advise students as they determine best-fit careers based on their individual interests and skills, provide a comprehensive learning and research environment, and ensure participation in biomedical research careers. Trainees admitted to our program have outstanding academic records and strong motivation and aptitude to pursue research. A key feature of our program is an intensive, focused curriculum required of all first-year students. Students work together in lecture and discussion-style courses taught by dedicated faculty to master a fundamental set of approaches that underpin molecular biological science. The training program exposes students to the research interests of all faculty members in the Biology Department prior to a sequence of rotations that, in combination with first-semester courses, support an informed choice of a thesis advisor and topic. Students gain experience in scientific communication and in teaching and mentoring junior students. Responsible conduct of research is taught in both classroom and laboratory settings, including an intense mini-course for 2nd year students. Students have many opportunities to learn about career paths open to them following doctoral training. Students’ progress through the program is monitored in regular thesis committee meetings with faculty members, with oversight by the graduate committee. We seek to build and maintain a welcoming and supportive learning community. This training grant would constitute a critical source of support for ~50% of training-grant eligible graduate students in our program during their first two years. Our students perform research of outstanding quality, and most trainees go on to careers in biomedical research. Many of our former trainees are now leaders in their chosen fields and bring to their positions the knowledge, rigor, and thoughtful perspectives that we emphasize at MIT Biology. We anticipate exciting futures for our alumni as they help transform the US biomedical landscape.

NIH Research Projects · FY 2025 · 2021-06

Project Summary With the rise of high-throughput sequencing and multiplexed biotechnologies enabling single-cell multi-omics and massively parallel CRISPR experiments, the biomedical community is generating a monumental amount of data. These data promise to reveal new biology and drive personal and precision medicine. However, the sheer volume of genomic data is overwhelming current computational resources, requiring prohibitively high compute time, memory usage, and storage. My lab has been at the forefront of solving big data challenges in genomics, designing novel algorithms that enable efficient and secure analyses that were previously computationally infeasible, and that reveal novel structural, cellular, and systems biology. Drawing upon our expertise in developing scalable and insightful algorithms for analyzing genomic, transcriptomic, and proteomic data, we aim to tackle two key data-driven challenges facing the biological community: 1) efficient, accurate, and robust characterization of tissues at the single-cell level, and 2) translating high-throughput datasets into biological discoveries via machine learning-based prediction. To solve the first challenge, we will leverage our discovery that seemingly high-dimensional sequencing data often lies on low-dimensional manifolds that capture the underlying biological state of interest. We will design algorithms that generate these compact, meaningful manifold representations of single-cell omics datasets. This will enable a number of key applications including characterizing co-expression and gene-modules that define healthy and pathologic cell states; integrating multi-modal single-cell omics datasets to more richly characterize cellular diversity; and investigating the mechanisms underlying transcriptomic diversity across tissues and developmental states. To solve the second challenge, we will take a two-pronged approach. First, we will design novel machine learning frameworks that provide a measure of confidence when predicting in unfamiliar biological states, enabling prediction that is robust to “out-of-distribution” (unobserved) examples. We will then work with our experimental collaborators and CROs to rapidly perform experimental validation of model-based predictions. Finally, we will return the experimental results to the model to further improve performance. This will enable an “active learning” feedback loop to efficiently explore a complex biological space for outcomes of interest. We will use this uncertainty-powered active learning approach to explore several pressing biological concerns such as the identification of small molecule compounds with enzymatic or whole-cell growth inhibitory properties, efficient design of spatial- transcriptomic experiments, computationally guided CRISPR perturbation experiments, and identification of functional non-coding mutations. This project will result in 1) numerous software tools with wide utility that efficiently analyze massive biological datasets and guide complex experimentation, and 2) reveal biological insights, especially into biomolecular interactions and cellular heterogeneity.

NIH Research Projects · FY 2026 · 2021-06

Substance use disorder (SUD) is a debilitating condition characterized by compulsive use of a substance, in spite of the subjective recognition of the drawbacks associated with its use. Chronic use of the substance leads to the development of withdrawal and dependence, hindering intentional controls over its use. Among all substances, opioids are among the ones that pose the greatest negative impact on our society. Animal and human studies have implicated several brain regions involved in opioid use disorder (OUD), in particular various components of the dopamine system. Major dopamine-containing neurons are clustered in regions of the midbrain called the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA). Classically, their activities correlate with valence, e.g., promoting approach behavior to rewarding stimuli. Upstream of them, two major circuits are involved: one including the nucleus accumbens projecting onto the VTA, and the other including the dorsal striatum projecting onto the SNc. The scope of this study is thus to conduct in-depth transcriptomics across the cells of these two circuits to dissect genes, pathways, and cell types mediating opioid action in the mouse brain. We will use these data to predict driver genes, regulatory regions, pathways, and cell types involved in the emergence of opioid addiction behaviors. Finally, to test the causal role of these predicted drivers, we will use opioid self-administration, a classical paradigm used in the field of addiction. By combining a rich set of behavioral protocols with state-of-the-art transcriptomics, epigenomics, and computational data analysis, we aim to obtain multi-modal data with an unprecedented level of single-cell resolution for identifying genes, pathways, and cell types affected in mouse models of OUD.

NIH Research Projects · FY 2025 · 2021-05

Alzheimer's disease (AD) is an incurable brain disease, distinguished by the progressive accumulation of toxic amyloid and tau protein aggregates that are partly due to impaired waste clearance by the glymphatic and meningeal lymphatic systems. We have recently shown that noninvasive Gamma ENtrainment Using Sensory stimuli (GENUS) to induce neural oscillations in the gamma frequency range (30-90 Hz) could ameliorate pathology in various AD mouse models. Mice subjected to GENUS regime exhibited positive effects on microglia, astrocytes and the brain vasculature as well as reduced accumulation of amyloid and hyperphosphorylated tau in respective amyloid and tauopathy mouse models. However, the impact of GENUS on the glymphatic/lymphatic systems in the clearance of amyloid and tau accumulation is not clear. We will use amyloid and tauopathy mouse models to determine whether and identify the mechanisms by which GENUS enhances paravascular fluid movement and thereby promotes meningeal lymphatic drainage and glymphatic clearance of brain toxic metabolites including those associated with amyloid and tau.

NIH Research Projects · FY 2025 · 2021-05

PROJECT SUMMARY As the elderly population increases, the number of people with Alzheimer's disease (AD) is rising rapidly. There is, therefore, a growing interest in the development of in vitro human disease models to better understand and study the physiological and pathological mechanisms associated with AD. Emerging evidence has implicated an early breakdown of the BBB in AD patients, even before the cognitive decline and brain pathology. In addition, meningeal lymphatics are responsible for clearance of Aβ peptides from the brain parenchyma, and disruption of meningeal lymphatics in AD transgenic mice accelerates Aβ deposition in the meninges, aggravates parenchymal Aβ accumulation, and induces cognitive impairment. Yet, our understanding of the role of fluid transport across the BBB, through the brain parenchyma, and exiting via the lymphatic system is poorly understood. Therefore, the development of physiologically realistic human neural cell culture models of AD with a neurovascular unit and meningeal lymphatic vessels is urgently needed to dissect molecular mechanisms underlying the pathogenic cascade of AD and accelerate the discovery of new AD drugs. Building on our extensive set of preliminary and related studies, we propose to develop a three-dimensional (3D) in vitro model by integrating BBB, meningeal lymphatics, and stem-cell-derived AD cell culture to closely mimic the AD brain environment. With this model, we aim to identify AD-specific mechanisms underlying pathophysiological changes in the BBB, meningeal lymphatics, and Aβ clearance. Our 3D AD model will be useful for not only studying the mechanisms of AD progression but also for drug discovery in a human brain-like environment.

NIH Research Projects · FY 2025 · 2021-05

SUMMARY We propose developing a systems-biology approach to understand interactions between tumor and immune cells and their clinical implications. Our work will focus on medulloblastoma, a malignant pediatric brain tumor in which our team has extensive expertise. We and others have shown that medulloblastoma tumors are sites of immune activity despite the blood-brain barrier. However, the clinical consequences of these immune cells are unclear and there is little information that might guide development of therapeutics that modulate these immune cells. Our innovative strategy combines single-cell methods, including single-cell proteomics, with a sophisticated computational analysis. In Aim 1, we map the landscape of tumor-immune interactions using sequencing and imaging methods on human samples. Aim 2 builds a causal model of the molecular interactions that govern interactions among cell types in medulloblastoma, determines the clinical correlates of these cells, and identifies potential therapeutic targets. Aim 3 maps the tumor-immune environment in well-validated mouse models of the disease, and builds computational models for mice parallel to those for humans. Hypotheses from Aim 2 that are likely to translate well to the mouse models are then tested for their effects on tumor growth and survival. The mouse results are used to update the computational models and refine the therapeutic strategies. We expect that successful completion of this project will have a substantial impact on medulloblastoma therapeutics. Further, the methods we develop will catalyze research of interactions between immune cells and many other tumor types beyond medulloblastoma.

NIH Research Projects · FY 2026 · 2021-05

Project Summary/Abstract The detailed study of the fundamental chemistry and biological properties of complex natural products provides critical insight toward understanding their mode of action and enables development of new approaches for treatment of various human ailments. This research program focuses on the development of efficient and concise strategies for the total chemical syntheses of structurally complex and biologically active natural products. The target compounds are selected based on novelty of molecular architecture, paucity of prior synthetic studies, abundance of opportunities for development of new synthetic strategies and methodologies, possession of significant biological activity, and the potential for future chemical and biological studies. The systematic discovery, development, and application of new synthetic strategies and methodologies explored in this program continue to provide synthetic samples of rare and precious compounds for structure validation and detailed examination of their chemistry and biology. This program focuses on synthetic studies of the rich families of complex natural products including cyclotryptamine, diketopiperazine, thiolated diketopiperazines, and monoterpene indole alkaloids. A central interest is the development of generalizable directed, regioselective, stereoselective, and efficient union of complex fragments providing late-stage couplings to secure challenging linkages, including complete stereochemical control at (multiple) quaternary stereogenic centers. Convergent and guided assembly of advanced fragments, often inspired by novel biosynthetic hypotheses advanced within this program, is complemented by development of new highly selective chemical transformations for rapid generation of molecular complexity. These transformations include new methods for azaheterocycle syntheses, cascade bond–forming reactions involving sensitive enamines and iminium ions, stereoretentive hydroxylation of complex substrates, and stereocontrolled sulfidation made possible through employing new reagents and conditions developed in this program. This program’s access to potently bioactive collection of families of complex alkaloids and related derivatives continues to enables exciting biochemical collaborative investigations. The array of synthetic molecules accessed through this program behold great promise as new bioactive compounds and mechanistic tools, and these compounds are continually evaluated and examined through multidisciplinary collaborations in pursuit of novel anticancer and antimicrobial compounds.

NIH Research Projects · FY 2025 · 2021-05

The mission of the NBETP is to train early-mid stage graduate students to become future leaders at the interface between neurobiology and engineering. This is accomplished via a blend of course requirements in neuroscience and engineering, including several dedicated neurotechnology classes, as well as through mentorship and community-associated events that encourage student leadership and career development. Students admitted into the program are selected based on intellectual caliber, leadership potential, commitment to neurobiological engineering research, willingness to take on risky and potentially game-changing projects, and disciplinary breadth. Students are engaged in innovation of emerging bioengineered technologies that enable fundamental biological discoveries with translational potential related to a broad spectrum of neurobiological diseases including neuropsychiatric and neurological conditions, as well as addiction. Goals of the program are thus very well aligned to the objectives of NIBIB-funded T32 grants and to the mission of NIBIB overall, and they also synergize with interests of ICs such as NIMH, NINDS, and NIDA. An administrative structure oversees the training program’s direction, student admission, and assessments. Preceptors associated with the NBETP include a set of faculty with multidisciplinary expertise encompassing multiple departments and intersection of multiple traditional disciplines such as bioengineering, electrical engineering, chemistry, and neurobiology. The program thus closely fits the profile of a “broad-based NRSA training program,” as described in NOT-EB- 07-005. Funding currently provides for four predoctoral students to participate in the program for two years each. In its first five years, the program has attracted a remarkable set of trainees and offered them important and enriching experiences during and after their funded periods. The program has also contributed to substantial research and leadership accomplishments by individual trainees, as well as to significant expansion of the footprint and visibility of neuroengineering research and education at MIT and beyond.

NIH Research Projects · FY 2025 · 2021-04

Many HIV vaccine candidates have failed clinical trials, as they were unable to elicit a potent and durable response to HIV viral challenge. Broadly neutralizing antibodies (bnAbs) have been identified in a number of HIV+ individuals with well-controlled viral levels, and these bnAbs target epitopes that contain residues that are relatively conserved across viral strains. It is thought bnAbs may have efficacy against various strains of HIV pathogen. It is therefore widely believed that systems which induce a potent immune response that includes the generation of broadly neutralising antibodies (bnAbs) in humans could be effective HIV vaccines, and help to mitigate the wide genetic diversity in envelope proteins and relatively high mutation rate of HIV. However, developing a vaccine which can elicit the production of these bnAbs in vivo has proven to be extremely challenging. This is likely due to the complex affinity maturation process that is required to produce bnAbs. Immunization protocols typically administer a single dose of antigen (prime dose), which is sometimes followed by a “boost” dose delivered several weeks later. In a traditional bolus immunization, the half-life of the antigen present in lymph nodes is generally shorter than the time scale over which germinal centres start producing higher affinity IgG antibodies relative to the initial IgM response (~18 hrs). In contrast, natural infections expose the immune system to escalating antigen and inflammation over days to weeks, resulting in the formation of a germinal centre with dynamic antigen presentation. This germinal centre niche also supports activation of antigen presenting cells, T follicular helper cells, and appropriate cytokine signalling to generate bnAbs. It is likely that to develop effective bnAbs, sophisticated vaccination techniques which can more closely mimic natural infections and natural bnAb formation may be required. We believe that to develop a successful HIV vaccine, researchers must aim to engineer more sophisticated and biomimetic vaccines. Bioengineered vaccines should therefore consider three key parameters in parallel; 1) delivery of an appropriately selected antigen, with 2) favourable kinetics of antigen expression, and 3) control of the immune response in the germinal centre. We believe lymph node targeted delivery of computationally designed mRNA antigens inside immunostimulatory lipid nanoparticles (mRNA LNPs) administered with computationally optimized immunization protocols will address these three aspects in a unique way. Additionally,Translate Bio will provide expertise in manufacturing considerations for mRNA therapeutics. As modifications to mRNA structure may impact the mRNA antigen translation, stability, and immunogenicity, the input of our translational partner (Translate Bio) will allow us to develop vaccines with a potential avenue for commercial development. This R61/R33 proposal combines our expertise in computational antigen design, HIV immunology, combinatorial chemistry, and the commercialisation of mRNA therapeutics to develop a new class of HIV mRNA vaccine candidates.

NIH Research Projects · FY 2025 · 2021-04

The locus coeruleus (LC), a small brainstem nucleus, is the primary source of the neuromodulator norepinephrine (NE) in the brain. LC receives input from widespread brain regions and projects throughout the forebrain, brainstem, cerebellum, and spinal cord. LC neurons release NE tonically to regulate baseline arousal, and phasically in the context of a variety of sensory-motor and behavioral functions. However, despite its brain- wide effects, the conditions under which LC-NE neurons are phasically activated and the modes of NE action during behavior are poorly understood. One prevailing theory suggests that NE acts to control the gain of output circuits, thereby modulating task performance by enhancing or dampening responses to stimuli. However, another theory suggests that NE release in cortical output regions acts to reset network activity, enabling task- switching or learning of new rules. Neither of these theories adequately explains the many observed roles of the LC-NE system in learning and behavior. We propose a new hypothesis of LC function, that spatiotemporal dynamics and modular circuits enable dissociated roles for the LC in behavioral execution and reinforcement during learned behaviors. Here, we propose to examine multiple features of this hypothesis using innovative approaches combining optically-tagged recordings of specific neuronal populations, advanced 2-photon imaging of identified neurons and axons, optogenetic manipulation of LC neurons and subpopulations, and computational approaches to define encoding of task variables by neurons. In Aim 1, we will record and manipulate the activity of LC neurons in mice performing an instrumentally conditioned task in which they detect auditory tones of variable intensity, execute a response, and receive positive or negative reinforcement. Using targeted recordings from LC-NE neurons as well as newly discovered LC-GABA neurons, we will examine the hypothesis that subsets of LC-NE and LC-GABA neurons encode task execution signals or reinforcement signals. Using this information, we will use cell-type specific optogenetics to activate or inhibit LC-NE or LC-GABA neurons during specific task epochs while measuring the effects on behavior. In Aim 2, we will assess anatomical modularity of LC projections to motor cortex or the prefrontal cortex (PFC), and examine the hypothesis that neurons with execution or reinforcement responses project preferentially to motor cortex or PFC. Subsequently, we will modulate the activity of LC neurons projecting to these targets and measure the effects on execution and learning. In Aim 3, we will examine the hypothesis that differential integration of NE release in motor cortex and PFC facilitates task execution and learning, respectively. We will monitor the fast kinetics of NE release in motor cortex and PFC using a genetically encoded NE sensor, and measure the impact on behavior of silencing NE activity in these cortical targets using optogenetic silencing of LC-NE axons. These data will provide essential information for a computationally informed theory of the role of LC in cognition, and provide a mechanistic basis for understanding the role of LC-NE dysfunction in a range of neuropsychiatric disorders.

NIH Research Projects · FY 2025 · 2021-04

The neural architecture of language is the foundation for the highest form of human interaction. Prior work has delineated a network of frontal and temporal brain areas that selectively support language processing, but the precise computations that underlie our ability to extract meaning from sequences of words have remained out of reach. Recent breakthroughs in natural language processing and machine learning have led to the development or artificial neural network (ANN) models of language that perfrom remarkalbly well on previously intractable tasks, providing the first computationally precise models of how these functions might be solved by the brain. However, the standard approaches in human cognitive neuroscience lack the spatial and temporal resolution necessary for precise comparisons to computational models. Here we propose to collect large sets of neural responses to language stimuli from the human brain using the only method up to the task-human intracranial recording, and to bring the recent advances in computational neuroscience and machine learning to bear on the ultimate scientific quest of understanding uniquely human linguistic ability. In Aim 1, we build on a strong foundation of our earlier fMRI work to tackle a fundamental distinction between two key components of language-understanding word meanings (lexico-semantic processing) and connecting those meanings into phrases and sentences representations (syntactic processing). This distinction will be tested across four controlled paradigms. In Aim 2, we will extend the results from Aim 1 to rich naturalistic materials, testing for sensitivity to word meanings vs. hierarchical syntactic structure in predicting upcoming words. We will also supplement this hypothesis-driven approach with a data-driven search for structure in neural responses to language using innovative analytic techniques recently developed in the study of auditory cortex. Finally, in Aim 3, we will use human intracranial responses to language stimuli to test computational models of language understanding. In recent work, we tested a large number of ANN language models and found that the most powerful 'transformer' models accurately predict neural responses. Here we harness the precision of human intracranial recordings, a large set of diverse linguistic materials-selected and created to discriminate among candidate ANNs using novel state-of-the-art design optimization approaches-and controlled minimal-pair network comparisons to powerfully test computational models of language understanding. In line with current emphasis in the field on robust, replicable, and open science, the full dataset (1,500+ sentences in each of ~40 participants) will be made public, enabling many analyses beyond the ones proposed here, thus turbo charging the quest to understand the neural computations underlying language processing and moving the field forward for years to come. This work will set the stage for the most comprehensive picture of the neural architecture of language and synergize with parallel ongoing advances in network modeling of human cognition, making it possible for the first time to discover the neural codes and computations underlying human language.

NIH Research Projects · FY 2025 · 2021-03

Abstract Frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) are devastating and fatal neurodegenerative diseases that strike middle-aged adults just as they reach full familial, financial and career potential. Initially thought to be quite distinct, FTLD and ALS are now recognized to share many clinical, pathological, and genetic signatures, but the mechanistic basis of their shared and distinct circuitry remains unknown at the molecular level. Genome-wide association studies (GWAS) have uncovered multiple common weak-effect variants, but the vast majority are non-coding, making it difficult to identify their target genes and the cell types where they act. To address this challenge, in Aim 1, we systematically profile the transcriptional and epigenomic alterations of FTLD and ALS patients at single-cell resolution using post-mortem brain samples. In Aim 2, we integrate the resulting datasets to study the link between genetic, epigenomic, transcriptional, and cellular signatures of FTLD and ALS, and to study the common and distinct genes and pathways altered in each, to predict new therapeutic targets. In Aim 3, we validate the molecular and cellular effects of these targets using high-throughput directed perturbation experiments and both cell-autonomous and non-autonomous phenotypes guided by our predicted pathways, and we disseminate all our results to the community. The resulting datasets, analyses, and validated targets will provide an invaluable resource to understand the mechanisms of action of FTLD and ALS, and the common and unique circuitry towards new therapeutic targets.