Massachusetts Institute Of Technology

NIH Research Projects · FY 2026 · 2022-05

Abstract The most common neurodegenerative diseases, including Alzheimer’s, Parkinson’s, and Huntington’s diseases, all display distinct clinical presentations. The basis of these distinct clinical presentations is the enhanced vulnerability of specific neuronal cell types to death or dysfunction in each disease, despite widespread expression of disease-associated genes. My work uses innovative approaches to address these long-standing questions of enhanced vulnerability, which have remained open questions in the field for decades. I was centrally involved in developing a widely used cell type-specific profiling methodology known as translating ribosome affinity purification (TRAP) that allows cell type-specific RNA profiling. My lab has recently developed a new genetic in vivo screening platform for the CNS, a powerful new approach for brain studies as it allows for systematically testing the causal effect (versus correlation) of each gene in the genome for disease phenotypes, rather than more standard approaches that test a single gene per animal. Additionally, using single cell sequencing approaches, we have also conducted the largest single cell studies of Huntington’s disease patient tissue conducted to date. These studies have collectively revealed the scope of transcriptional dysregulation in Huntington’s disease and Huntington’s disease model tissue, and also have implicated neuronal innate immune activation as a likely key driver of cell type-specific vulnerability in Huntington’s disease. My long-term research goal, starting with Huntington’s disease as a model disorder, is to elucidate the basis of enhanced vulnerability in neurodegenerative disease, not only as a window for discovering valuable insights into the cell biology of disease-relevant neuronal cell types, but also identifying new therapeutic targets.

NIH Research Projects · FY 2026 · 2022-05

ABSTRACT/PROJECT SUMMARY Continuous monitoring of physiological state (oxygenation, breathing, circulation) is a standard practice for all patients receiving general anesthesia and sedation. Anesthetics produce their primary effects of unconsciousness and antinociception by acting on molecular targets and neural circuits in the brain and central nervous system. Nevertheless, continuous monitoring of brain function is not a practice requirement. It is no surprise that brain dysfunction following general anesthesia is highly prevalent, particularly among the elderly. Similarly, COVID 19 patients who can be anesthetized for weeks in the intensive care unit, are often left with profound brain dysfunction following termination of ventilatory support. Many years of research have shown that the level of unconsciousness of a patient receiving general anesthesia can be reliably tracked using real-time processing of electroencephalogram (EEG) recordings. In recent years, dramatic advances have been made in sensors, actuators, artificial intelligence and control theory algorithms. A highly plausible solution is the development of closed loop anesthesia delivery (CLAD) systems that determine in real time from the EEG the patient’s level of unconsciousness and precisely control an anesthetic infusion to maintain the level at an appropriate target. The Federal Drug Administration (FDA) readily acknowledges the significant enhancement to patient care that CLAD systems can provide. To date, no system has been approved for human use due to a lack of appropriate animal models to test adequately the reliability and robustness of these systems. Therefore the research design of this project will be to conduct in non-human primates neurophysiological recordings (EEG, local field potentials and neural spiking activity) while simultaneously administering anesthetics using a computer-controlled syringe pump as the animals execute a behavior task to characterize level of unconsciousness. The data will be analyzed by combining pharmacokinetics and pharmacodynamic modeling, modern control theory and statistical signal processing approaches to develop and test real-time CLAD systems. The specific aims of this research project are to develop and test in a non-human primate model, CLAD systems for real-time control of unconsciousness using the anesthetics: propofol, dexmedetomidine, and propofol and dexmedetomidine administered simultaneously. The broad long-term objectives are to: establish a non-human primate model paradigm for development and testing of CLAD systems; and make the use of CLAD systems a standard for intelligent brain state monitoring and precise second-to-second drug dosing in anesthesiology. The health relatedness impact of the research will be a new paradigm for computer-assisted vigilance of brain state and computer-assisted dosing of anesthetic agents. Such systems should enhance patient safety by reducing provider errors and by fostering significant decreases in anesthesia-associate brain dysfunction as well as other anesthesia-related morbidities (inadequate pain control, hypotension, nausea) commonly experienced by the millions of patients who each year receive anesthesia care in operating rooms and intensive care units.

NIH Research Projects · FY 2025 · 2022-05

Cell-based therapies, where naturally or artificially engineered cells secreting therapeutic proteins are grafted onto the body to act as biological drug factories, are an attractive approach for long-term treatment of chronic diseases such as hemophilia, diabetes and liver disorders. However, ‘off the shelf’ therapeutic cells are immunogenic to the host and must be protected from the host immune system. Cell-encapsulation has emerged as an attractive strategy to transplant these cells without chronic immunosuppression. Here, cells are placed in an immune-isolating device which physically separates the cells from the components of the immune system while providing access to oxygen and nutrients. Retrievable macroscale cell- encapsulation devices (macrodevice), are attractive in this context as they provide a safer path to clinical translation. Unfortunately, a standalone macrodevice that remains functional in humans over long-periods (>6 months) is yet to be realized due to two core challenges: 1) a foreign-body reaction to the implanted device causing inflammation and fibrosis, and 2) inadequate supply of oxygen and nutrients to the encapsulated cells. Here, we propose to build on several promising recent advances in biomaterials design, microfabrication, bioelectronics and cell engineering from our team to develop an advanced “smart” macrodevice platform with integrated electronic components which overcomes the major limitations of current device designs. First, we will develop an engineered cell line which is amenable to long term encapsulation and suitable for clinical translation. Landing pads within these cells will ensure stable transgene expression, allowing for broad control of therapeutic protein secretion (Aim 1). Separately, we will develop a bioelectronic macrodevice as a platform for long- term transplant of these cells in vivo. Our device will incorporate novel membranes with uniform/controlled pore-sizes and enhanced oxygen transport properties. In parallel, we will develop new surface coating techniques to minimize fibrosis and ensure long- term graft survival. We will integrate proton exchange membranes and optoelectronic components to allow a) in-situ oxygen generation, and b) optical gene activation to allow for triggerable control of protein production by the encapsulated cells (Aim 2). Finally, we will test the device in B6 mice using a model protein (SEAP) to test for long term survival of cells and external control of protein delivery. We will develop the device as a platform to delivery of Factor VIII for the treatment of Hemophilia A (Aim 3) as a model disease. If successful, the platform will represent a qualitative technological advancement in the field of cell therapy.

NIH Research Projects · FY 2025 · 2022-04

Overall: Summary From the moment of fertilization to birth, the maternal immune system evolves, adapts, and supports the growth of a fetal allograft that ultimately perpetuates the human race. Immunological changes throughout a pregnancy play a key deterministic role in the success of the pregnancy. While pregnancy was historically regarded as a simple shift towards tolerance, emerging immunological data point to remarkable dynamic changes during pregnancy. The pregnancy immunome must protect the fetus from a maternal attack while at the same time it must afford the maternal-fetal dyad protection from invading pathogens. The health of the mother and the fetus requires that these two opposing immunological tasks work in concert. Thus collectively, pregnancy marks a whirlwind of immune adaptations that render the pregnant immune system a truly unique immunologic marvel. Despite our growing appreciation for these highly controlled dynamic shifts, the precise mechanisms that lead to optimal pregnancy health, profoundly impacting both mother and fetus, are incompletely understood, delaying the development of targeted therapies for this population. Capitalizing on this unique moment in vaccine history, with the introduction of several novel-vaccine platforms for SARS-CoV-2, the consortium will build a Pregnancy Immune Atlas via the application of high-density immunological profiling technologies to deeply and comprehensively dissect the overall changes that occur across pregnancy and how the immune system, as a collective, responds to in vivo perturbations with vaccines. Using both de novo vaccine induced immune responses and booster vaccination, the consortium will capture overall changes in the pregnant ImmunOME as well as shifts in the pregnant AdaptOME to fully capture the immunological mechanisms that govern the balanced growth of the fetus and battle of the maternal:fetal dyad against invading pathogens. Thus, together the Maternal ‘Omics to Maximize Immunity (MOMi) consortium seeks to build the foundational data to advance our knowledge of natural tolerance, fertility, shifts in immunity during pregnancy to better understand this evolutionary marvel required for the perpetuation of the human species.

NIH Research Projects · FY 2025 · 2022-04

Project Summary This is a new MPI R01 proposal bringing together protein engineering for immunotherapy (Wittrup, MIT) with comparative oncology/veterinary medicine (Fan, UIUC) to test clinical strategies for combining radiotherapy with intratumoral cytokine administration/retention in pet dogs with melanoma, at the UIUC veterinary clinic. We have developed a strategy for retaining injected cytokines (IL-2 and IL-12 in particular) in situ by expressing them as fusions to natural collagen-binding domains. This approach has been found to be safely curative in challenging murine transplant and GEM tumor models, and will now be advanced into a more faithful model for human cancer: spontaneous canine melanoma. These tumors arise spontaneously in outbred populations, and undergo a natural progression of immunoediting prior to clinical presentation. Canine melanoma exhibits pathophysiology similar to human melanoma, including the presence of immune infiltrated, excluded, and desert subtypes. In Aim 1, we will exploit the more-realistic anatomy of these tumors to optimize the micropharmacokinetics of intratumoral administration, establishing foundational principles with respect to injectable volume fractions, needle types, and numbers of sites. In Aim 2, we will test the therapeutic hypothesis that precisely temporally programmed intense localized cytokine stimulation can be optimally combined with radiation therapy so as to prime a strong T cell vaccinal response with consequent systemic impact on efficacy. In Aim 3, we will perform a clinical trial in canine melanoma to rigorously compare alternative dose scheduling for intratumoral cytokine therapy following irradiation. We hypothesize that the time delay prior to cytokine injection will have a critical, all-or-nothing effect on outcomes. This intradisciplinary collaboration has commenced, and exciting preliminary treatment data is presented herein. The overarching objective of this project is to develop improved human cancer immunotherapy protocols that combine intratumoral immunotherapy with local radiation. Multiple previous clinical trials in this area have yet to realize the full promise of this approach, but by performing rapid design-build-test-learn cycles in spontaneous canine melanoma, we hope to converge more efficiently to efficacious strategies.

NIH Research Projects · FY 2025 · 2022-04

Project Summary Rare and unnatural carbohydrates play an essential role in the potency and selectivity of hundreds of bioactive natural products and pharmaceutical compounds. These scaffolds often feature unusual relative/absolute stereochemistry, pyranose/furanose ring branching, heteroatom substitutions, and varying degrees of deoxygenation. Despite the biological significance of rare and unnatural sugars, synthetic challenges limit access to these important molecules. Due to their functional group density and stereochemical complexity, current syntheses of rare and unnatural sugars require multistep chemical synthesis, and commonly rely on protecting group manipulations to achieve selective reaction outcomes. New, selective methods are needed for the expedient synthesis of pyranose and furanose sugars. This proposal describes the development of selective radical reactions to transform unprotected and minimally protected carbohydrates into diversely functionalized monosaccharides and glycans. We specifically target epimerization reactions and radical rearrangements to achieve broad synthetic access to rare isomeric and deoxygenated sugars, respectively. Using state-of-the-art synthetic, mechanistic and theoretical tools, our approach involves the identification of new catalytic strategies to control bond breaking, bond forming, and radical reorganization steps within the context of complex glycan molecular frameworks. The successful development of this proposed research is anticipated to transform carbohydrate synthesis, dramatically reducing the time and resources necessary to access these complex pharmacophores. Fundamental mechanistic findings revealed en route to this goal are further anticipated to contribute significantly to our understanding of carbohydrate reactivity patterns and to lay the groundwork for catalytic approaches to selective radical functionalization reactions, more broadly.

NIH Research Projects · FY 2024 · 2022-04

Research Strategy Summary of Parent Award: Enzymes with complex metallocofactors in their active sites catalyze myriad transformations relevant to human health and disease. Understanding their reaction mechanisms requires molecular-level characterization of their resting states and intermediate states, and metal-specific spectroscopic techniques are especially useful in this endeavor. However, the high nuclearity of many metallocofactors can limit the usefulness of such techniques; the signals arising from multiple metal sites can be challenging to resolve, especially in mixtures of reaction intermediates. Moreover, it is often impossible to map the rich spectroscopic information onto the geometric structure, and this severely limits our understanding of the chemical bonding—and therefore the reactivity—of complex metallocofactors. We propose to address these challenges by developing methods for modifying the isotopic and elemental compositions of complex metallocofactors, in particular the nitrogenase catalytic cofactors. Nitrogenases are responsible for supplying a significant portion of the fixed nitrogen on the planet, and they therefore play an important role in maintaining a healthy and growing human population. Their catalytic cofactors are among the most complex in Nature, and as a result their reaction mechanisms have been especially difficult to characterize. To overcome these challenges and gain new insights into the mechanism of biological nitrogen fixation, we will develop chemical methods for precisely altering the isotopic and elemental composition of nitrogenase cofactors. Our approach is to discover mild protocols for removing specific Fe sites in nitrogenase cofactors and subsequently replacing them with 57Fe. The site-selectivity of the label will allow for the electronic structure (as elucidated spectroscopically) to be connected to the geometric structure (as defined crystallographically), and will thereby provide unprecedented insights into the chemical bonding and reactivity of nitrogenase cofactors. Studies of these cofactors in both their resting states and intermediate states comprise the heart of the proposal. We will also extend the site- selective 57Fe labeling protocol to incorporating different metals into specific sites of nitrogenase cofactors. This will yield artificial metalloenzymes that will serve as mechanistic probes with potentially unique properties and/or reactivity. Completion of this project will provide unprecedented mechanistic insights into biological nitrogen fixation and will articulate concepts and protocols for rendering complex metallocofactors as mechanistically tractable as mononuclear active sites. Justification for proposed equipment: The proposed modern ultracentrifuge would replace our current ultracentrifuge (a decades-old Beckman Coulter L70, heretofore referred to as the “Old Ultracentrifuge”), which was listed in the Equipment section of our grant proposal submitted in 2021. We first describe the scientific relevance of an ultracentrifuge with respect to this project, and then the reasons for replacing the Old Ultracentrifuge with a modern ultracentrifuge. Scientific necessity for a functioning ultracentrifuge. The project is absolutely dependent on large-scale overproduction of nitrogenase proteins from Azotobacter vinelandii (Av), and this can only be accomplished by conducting large-scale cell growths and protein purifications. The extraction of nitrogenase proteins from Av cells (50-150 g) requires cell lysis and clarification of the cell lysate in an ultracentrifuge; without this step, particles in the lysate would clog the chromatography resins, rendering protein purification nearly impossible. Thus, an ultracentrifuge is critical for cell lysis. Indeed, when the Old Ultracentrifuge is broken, the project grinds to a halt because every experiment requires access to nitrogenase proteins (e.g., the Mo nitrogenase) and/or nitrogenase-protein-derived cofactors (e.g., FeMo- co, the catalytic cofactor of the Mo nitrogenase, which is extracted from the Mo nitrogenase). The specific aims that would be affected without an ultracentrifuge are: 1. Aim 1. This Aim cannot be undertaken because it relies on the large-scale isolation of nitrogenase cofactors. 2. Aim 2. This Aim cannot be undertaken because it relies on the large-scale isolation of nitrogenase cofactors as well as the apo-proteins in which they are reinserted. 3. Aim 3. This Aim cannot be undertaken for the same reasons as articulated for Aim 2. Simply put: an ultracentrifuge is one of a few critical pieces of equipment, without which the project cannot be pursued. Rational for purchasing a modern ultracentrifuge. My lab inherited the Old Ultracentrifuge from Prof. Stephen Lippard, who purchased this equipment many years ago. Because of its age and because we use it frequently, it breaks several times per year. This has not significantly slowed down our research progress because it is typically repaired within a few days by a talented technician from Beckman Coulter. However, Beckman Coulter recently informed us that they have stopped making replacement parts and we will therefore no longer be able to source them. As such, Beckman Coulter is not guaranteeing the repairability of the Old Centrifuge, which means that it is essentially obsolete; soon (probably within a year), the Old Centrifuge will break and will remain permanently broken. This will be catastrophic for our research project for the reasons described above. Replacing the Old Centrifuge with a modern centrifuge would solve this problem since a modern centrifuge would be serviceable now and for the remainder of the project. Additionally, it would likely break far less frequently, and thus would result in less down-time in our research efforts. Note that we are proposing to purchase a modern ultracentrifuge (Beckman Optima XE) that has essentially the same features as the Old Ultracentrifuge. The Optima XE is not only their best-selling model, it’s also their most economical ultracentrifuge, and as such, our proposal to purchase this piece of equipment is financially conservative. Future costs. There will be no additional costs associated with the new ultracentrifuge as any repairs will be covered under a service contract.

NIH Research Projects · FY 2026 · 2022-04

PROJECT SUMMARY/ABSTRACT Tools to determine and analyze the structures of molecular machines in motion Single particle cryo-electron microscopy (cryo-EM) has transformed our ability to rapidly determine high resolution structures of static, structurally homogeneous macromolecular complexes. However, we have not realized cryo-EM’s potential to uncover the full ensemble of heterogeneous structures these molecules adopt as they function. The overall objective of this work is to develop novel cryo-EM image processing tools to: 1) determine the complete ensemble of structural states adopted by imaged complexes; 2) quantify the relative abundance of these states; 3) monitor how the distribution of these states changes as the machine functions; and 4) use this information to understand the molecular mechanism of how these machines assemble and function. This objective is important as visualizing structural ensembles can be vital in developing and testing hypotheses for how these machines function, and in developing therapeutics to modulate their activity. Here, we specifically aim to develop two tools to facilitate achieving these overall objectives. First, we will generate ‘benchmark’ datasets that will be distributed to the methods development community to aid in building and quantitatively assessing of the fidelity of different approaches to reconstruct 3D density maps from single particle cryo-EM data. These benchmark datasets will include macromolecular complexes bearing elements of structural heterogeneity we have specifically designed for this purpose, and that we have biochemically assembled and imaged. Additionally, it will design, implement, and validate a machine learning-based computational tool that more realistically simulates the imaging process than existent software, thereby enabling users to rapidly construct custom synthetic benchmark datasets to test specific aspects of their own algorithms. Recently, as a proof-of-concept, we published the first method using deep neural networks to perform 3D reconstruction from single particle data, and this approach was particularly efficacious is revealing heterogeneous structures. Thus, our second aim is to develop this approach into a complete software package enabling users to readily reconstruct hundreds-to-thousands of density maps from a single dataset; to implement tools to focus the analysis on specific structural regions; and to deploy methods guiding the interpretation of the density maps and the construction of ensembles of associated atomic models. This work in innovative in its objective to analyze heterogeneous structural ensembles as opposed to static structures at high resolution; in our approach to model model conformational changes as originating from a continuous distribution of structures as opposed to isolated, discrete states; and in our application of deep learning methods to both the generation of benchmark datasets and in the reconstruction process itself. As a proof-of-concept, our reconstruction approach has proven significant as evidenced by its recent application in multiple structural studies, and we expect the tools we propose to develop here will be broadly impactful on a wide-array of NIH-funded research programs that rely on single particle cryo-EM.

NIH Research Projects · FY 2025 · 2022-04

Alzheimer's disease (AD) is an incurable brain disorder, with a staggering human and financial cost in a rising aging population. The complexity of its pathophysiology has proven to be a daunting challenge in the development of effective pharmacological interventions. 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, cerebral vasculature, as well as reduced accumulation of amyloid and hyperphosphorylated tau in respective amyloid and tauopathy mouse models. However, the mechanisms by which GENUS impacts cell-cell interactions, in particular, interactions between neurons and microglia, are not clear. To this end, we will utilize a combination of AD mouse models and innovative iPSC 3D co-culture system to assess the role of microglia in modifying the GENUS response, focusing on the contributions of microglial AD risk genes, APOE and TREM2, in modulating the neuroimmune interactions in response to GENUS.

NIH Research Projects · FY 2025 · 2022-03

Project Summary: Center for Engineering Endometriosis Care (CEEC) The high prevalence, diversity of morphological and symptomatic presentations, array of potential etiological explanations, and variable response to existing interventions suggest that different subgroups of endometriosis patients with mechanistic bases of disease may exist. These factors, combined with the weak links to genetic predisposition, make the entire spectrum of the human condition challenging to model in animals. The majority of endometriosis research approaches questions as "diseased" or "control", with stratification among clinical status of patients according to ASRM stages. The overarching goal the CEEC is to reframe the way the clinical and basic science researchers together approach the complex landscape of endometriosis: first, by creating a new framework for defining clinical cohorts, based on presumed distinct biological mechanisms among different patient groups, for corresponding basic science studies; and second, by developing and implementing new computational systems biology, tissue engineering, and organ-on-chip models designed to address specific scientific questions arising from the mechanistic groupings of patients. The average age of patients in published studies on endometriosis is above 30 - more than twice the age of onset for many patients. Endocrine, metabolic, and immune systems are, on average, very different in 16 and 32 year olds; the physiology of lesions very likely is, also. We know little about the interplay between systemic host factors and the drugs we now use to treat lesions on the physiology of the lesions. Why is the disease invasive in some patients, and not others? Here, we propose to classify patient cohorts into 4 distinct subgroups that differ by systemic physiology {ages 16-21 and ages 32-42) and lesion physiology {superficial only, persistent; or invasive +/- superficial). This scheme allows us to construct an engineering landscape of in vitro lesion microenvironments, according to the features of the lesion physical microenvironment and systemic microenvironment, and a corresponding parameter space in which the magnitudes of cues are varied. Three projects allow us to develop correlations between patient clinical phenotypes and in vitro models:: Project 1: Parsing Effects of Donor Source and Lesion Microenvironment on Lesion Phenotypes in Vitro Project 2: Dissecting macrophage signal integration and function in endometriosis Project 3: Correlates of a holistic in vivo cellular and molecular signature with clinical phenotypes These projects will draw from a Biospecimen Coordinating Core. At the completion of this work, we will have new tools, new insights into how existing hormone therapies work in patients, and hopefully a new language for communication between clinicians and basic scientists in the trenches of endometriosis research. We will also have a substantial impact on education of the next generation of endometriosis researchers, and patient awareness of research efforts, through the activities of the education and outreach program.

NIH Research Projects · FY 2026 · 2022-02

Gene editing is a promising strategy for treating or even permanently curing genetic diseases. In particular, a new technique called prime editing has the potential to make small targeted insertions, deletions, and substitutions with very high potential coverage of known disease- causing mutations, and while minimizing dangerous double-stranded breaks in DNA. In order to realize this potential, robust delivery strategies must be developed to deliver prime editing tools efficiently to disease-relevant organs. One such delivery strategy is lipid nanoparticle delivery of RNA and/or protein-based prime editing components. LNPs are nonviral, nontoxic, and clinically validated delivery tools. However, there is an extremely diverse space of possible LNPs, with tens of thousands of potential lipid structures that may be useful for LNP delivery. Selecting the best possible LNP for a prime editing application, therefore, is challenging because in vitro testing is often unreliable and in vivo testing of one LNP at a time is extremely low throughput. Here, we propose to combine two scalable techniques to generate and test safe, potent LNP formulations for performing prime editing. First, we will employ combinatorial chemistry techniques to generate large libraries of biodegradable lipids for inclusion into LNPs. Second, we will introduce a new technique which we term pegRNA barcoding to screen dozens to hundreds of LNPs for successful prime editing in a single mouse. We will employ this technique to identify the best biodegradable LNPs for editing of multiple organs, including in particular the lung and the liver. Having identified the top candidates, we will proceed to use our LNPs to apply prime editing to treat mouse models of two different inherited genetic diseases: hereditary tyrosinemia type I (HTI), a liver disease, and cystic fibrosis (CF), primarily a lung disease. We will evaluate the efficiency of prime editing, the levels of undesired editing events, and phenotypic correction of these mice. The results may identify promising preclinical candidates for the treatment of HTI, CF, and many other lung and liver diseases.

NIH Research Projects · FY 2026 · 2022-02

Hemodynamic neuroimaging methods like functional magnetic resonance imaging (fMRI) have revolution- ized neuroscience by allowing researchers to characterize spatiotemporal features of brain-wide activity in hu- mans and animals. A major disadvantage of such approaches, however, is their lack of specificity for well-defined cellular and molecular sources; this limits their ability to yield explanatory insights into neural function. To address this problem, we recently developed an unprecedented family of genetically encodable molecular probes, called NOSTICs, that transduce intracellular calcium activity into artificial hemodynamic responses, permitting spatially comprehensive neuroimaging of genetically targeted cells and circuit elements. Hemogenetic signals arising from the NOSTICs may be differentiated from endogenous blood flow changes by pharmacological means and can be detected by any hemodynamic imaging modality. Our preliminary experiments indicate that cell-specific activity of even sparse neuronal populations can be identified using hemogenetic fMRI. These capabilities will enable hemogenetic imaging to confront some of the most outstanding problems in neuroscience, such as de- scribing functional properties of discrete cell populations on a brain-wide scale, defining input-output relation- ships among interacting brain regions and neural circuit components, and relating behavior and activity to plas- ticity and gene expression changes that occur throughout the brain. In this project, we will use hemogenetic imaging to address each of these broad problems in the context of sensory function in rodents, while at the same time refining the technology and laying a foundation for its wider application to many research topics and model systems in neuroscience. In Aim 1, we propose to use the technology for investigation of network-level processing in the somatosen- sory system. Anticipated results will inform a first-of-its-kind model of multiregional stimulus processing that con- stitutes a data-driven alternative to traditional correlative functional connectivity measures. We will use this model to examine the importance of feedback relationships and to help explain the phenomena of sensory adaptation and salience encoding at the network level. In Aim 2, we will exploit this capability by applying NOSTIC probes for genetically targeted fMRI of excitatory and inhibitory neural subtypes during forepaw stimulation and resting state dynamics in rats, addressing hypotheses about the functional roles of the different cell types. In addition, we will apply ultrahigh resolution fMRI to examine the relationships between single vessel-level hemodynamics and the cell type-specific distributions of NOSTIC expression, enabling a rich analysis that simultaneously in- forms interpretation of conventional fMRI results and rigorously characterizes performance of the hemogenetic technique itself. In Aim 3, we propose to improve the NOSTIC reporters themselves. Improvements we plan will enhance the detectability of hemogenetic signals and give rise to hemogenetic gene reporters that will be useful for mapping neural connectivity and plasticity in future applications.

NIH Research Projects · FY 2025 · 2022-01

The past half-century has seen an amazing trend. Linked advances in vascular biology, endovascular intervention and drug delivery have dropped morality from cardiovascular disease 4.5 fold. NIH support has blessed us with involvement in these endeavors and we are humbled by the accomplishments of the community. Yet, atherosclerotic disease is not eradicated, we do not fully grasp the vascular biology of obstructive vascular diseases, and interventional therapy is not at a standardized consensus. There is much to be learned in all areas especially for complex lesions where lesion modification is deemed indispensable. Increasingly sophisticated methods (e.g. orbital atherectomy, lithotripsy etc.) modify complex plaque before angioplasty or implantation of devices like stents, and yet modifications are still guided by operator personal experience. There are no criteria as to which technology to use and when, what constitutes sufficient modification and how to balance benefits and risks. Intravascular imaging can help visualize lesions peri-modification, but provides no functional feedback, forcing even experienced interventionalists to guide intricate procedures by sensation (touch, feel, even sound). What is needed and what our team of academic and industrial scientists, engineers and clinicians aims to develop are mechanistic insight into the biology of modification and tools for predicting function from imaging and validated criteria for treatment outcomes. We will relate alterations in lesion micro-morphology (calcium, lipid, fibrous, fibro-fatty content) to changes in spatial micro-mechanical (compliance, stress) and local drug delivery (uptake, retention) response, and correlate image-based quantification of lesion micro-morphology to interventional outcome, providing a framework to predict and optimize, therapy. Our aims are to (1) Quantify changes in clinical lesion micro-morphology of complex arterial disease as a function of lesion modification using deep-learning-based image analysis, and investigate how initial lesion state can predict micro-morphological alterations for different modifications. (2) Use image processing and lesion- specific inverse modelling to examine effects of lesion modification on micro-mechanics and local drug distribution in excised human lesions, and (3) compare predictions with clinical performance after angioplasty and stenting. Combining aims 1 and 2 with computational virtual stent implantation we will predict vascular responses after modification of vascular morphology, and compare these predictions to outcomes from clinical trials that have imaging and longitudinal follow-up. In whole we will distinguish clinical outcomes that arise from optimization of lumen dimensions, from optimization of micro-morphology, -mechanics and drug distribution. The significance of our work lies in providing a mechanistic framework to explore increasing use of lesion modification pre-intervention and a means to leverage such insight to guide and optimize effect. The novelty is in using imaging and computational methods developed with the past NIH support to achieve this understanding. We are honored that our science may have clinical impact in treating complex vascular disease.

NIH Research Projects · FY 2026 · 2022-01

Project Summary/Abstract Actomyosin-based force generation sculpts tissues into a remarkable array of shapes during development. Successful tissue sculpting requires that actomyosin is precisely regulated and that the resulting force patterns are transmitted across the tissue. Force transmission itself affects contractile signaling, resulting in emergent behaviors that result in tissue shape change. We have demonstrated the role of dynamic RhoA-GTPase cycling in generating actomyosin pulses and waves in Drosophila gastrulation and oogenesis, respectively. In each of these cases, we identified a Rho GTPase activating protein (RhoGAP) that is required for cycling behavior and demonstrates the functional importance for the cycling in morphogenesis. Our work has demonstrated the requirement of RhoGTPase cycling in tissue invagination and the completion of cytoplasmic transport from germline support cells to the oocyte. The mechanisms that initiate these dynamic behaviors and how they are influenced by force transmission in a tissue are still unknown. Patterns of force transmission in a tissue are complex and extremely dynamic. We have identified the importance of supracellular actomyosin meshworks in transmitting forces between hundreds of cells in a tissue, which forms chains of mechanically interconnected cells. Supracellular actomyosin meshworks within epithelia can exhibit biased connections, which influence tissue mechanics. But, how a cell determines which neighbors to link to is unknown and critical to understand tissue shape. Furthermore, the cell biological mechanisms that dissipate forces in response to morphogenetic movements and how they are coordinated with movement are poorly understood. We will undertake a multidisciplinary and multiscale approach to understand tissue shape emergence. Combining our ability to visualize and perturb dynamic signaling pathways we will investigate the interconnection between forces `felt' by cells and resulting single cell signaling patterns with the goal of bridging molecular and tissue scales. Members of my lab include biologists, physicists, and engineers. In addition, we have excellent collaborators in Mathematics to supplement our research capabilities. We are poised to make additional important contributions to our understanding of how collective cell behaviors contribute to morphogenesis.

NIH Research Projects · FY 2024 · 2021-09

Project Summary/Abstract Combination treatments aiming to stimulate synergistic immune pathways employing cytokines or immunomodulatory antibodies are generally more effective than monotherapies in preclinical models of cancer immunotherapy. However when given systemically, these combination treatments suffer from high toxicity from on-target off-tumor stimulation as well as low local concentrations at the tumor site due to poor tumor penetrance and high clearance rates. Local intratumoral therapy is a viable approach to bypass some of the challenges associated with systemic delivery, but requires optimization to promote retention of the therapeutic agent at the injection site and minimize leakage into the circulation. We have recently developed an approach to enhance vaccine efficacy by engineering the binding of immunogens to the commonly used adjuvant aluminum hydroxide (alum) via a site-specific phosphoserine (pSer) peptide tag. The pSer moieties undergo a ligand-exchange reaction with free hydroxyl groups on the surface of alum leading to stable anchoring of proteins on alum particles. We propose here to apply this alum-anchoring platform in the context of cancer to retain potent immune agonists within the tumor site, promoting a robust systemic immune response with minimal toxicity. Our preliminary results show that this simple approach can be used to load stimulatory cytokines onto alum for retention at the tumor site up to a month, stimulating a strong anti-tumor response from a single shot treatment. We plan to develop and optimize this translational strategy through the following specific aims: (1) use in-cell phosphorylation to produce phosphoserine-tagged cytokines and other candidate immune agonists for optimal alum binding, (2) determine optimal treatment regimens for these intratumoral alum-bound therapeutic agents in vivo in multiple tumor models, (3) define the mechanism of action through which this therapy elicits a response, (4) evaluate the systemic immune response and assess strategies to enhance abscopal effects by promoting the transfer of immunostimulatory payloads to motile lymphocytes for trafficking to distal untreated tumors. These studies will establish a robust technology platform capable of safely delivering treatments currently viewed as too toxic, by addressing key limitations in existing localized therapeutic strategies

NIH Research Projects · FY 2025 · 2021-09

Abstract Alzheimer’s disease (AD) is a devastating neurodegenerative disorder that leads to dramatic effects on the affected individuals and their families. While the characterization of the genetic contribution to AD and underlying molecular mechanisms have advanced the understanding of the disease in recent years, studies show that sex differences account for much of the observed differences in risk, progression, and severity across individuals. Here, we directly dissect the contribution of sex-specific variation down to the region- specific and cell-type-specific molecular basis by systematic profiling, computational integration, and experimental validation of the transcriptional, epigenomic, and genetic signatures across individuals, brain regions, and cell types. In Aim 1, we use genetic, epigenomic, and transcriptional profiles, generating millions of single-cell (sc) level maps using scRNA-seq and scATAC-seq across human and mouse samples of varying ages and genetic risk status. In Aim 2, we analyze the resulting datasets in the context of known AD genetic risk variation and underlying molecular mechanisms, enabling us to discover and converge variants, regulatory regions, genes, pathways, cell types, and brain regions to functional, causal mechanisms that drive sex-related differences. In Aim 3, we use our well-established mouse and iPSC models to test our predicted mechanisms with both high-throughput and cell-type specific assays. The resulting datasets, computational predictions, and experimentally-supported mechanisms will shed light on the sex-related differences of AD and will help deepen our understanding the disease in general as we develop more personalized therapeutic approaches in treating AD.

NIH Research Projects · FY 2025 · 2021-09

Abstract The information flow between the peripheral organs and the brain is increasingly recognized as bidirectional, with activity in peripheral circuits influencing high-level behaviors including mood, motivation, and stress. To establish mechanistic links between activity of peripheral neurons and brain circuits, we will develop a species- agnostic framework for targeting and remote modulation of specific cells within the peripheral organs and the brain during behavior. Our framework will combine the homing, modulation, and contrast properties of synthetic magnetic nanomaterials with the targeting specificity of viral vectors. Magnetic nanomaterials have recently emerged as versatile transducers of remotely applied weak magnetic fields into thermal, chemical, or mechanical stimuli perceived by ion channels. We will dramatically expand the palette of magnetic nanotransducers to enable receptor-specific remote magnetic modulation of neurons (or other electrogenic cells) anywhere in the body during free behavior. Moreover, we will leverage recent advances in adeno-associated viral vectors for targeting specific cells and tissues by creating an array of fusions of nanotransducers and viral capsids. This will allow for magnetic guidance and localization of the hybrid magnetic- viral fusions to the locations of interest following systemic delivery regardless of the model organism. We will apply our framework to elucidate circuits connecting the enteric (gut) nervous system to the midbrain structures. Recent work has drawn links between gastrointestinal dysfunction and social and mood disorders as well as demonstrated vagal transmission of the enteric signals to the brain. By applying receptor-specific modulation to the enteric neurons we intend to test the hypothesis that their activity influences midbrain pathways governing reward and motivation, and possibly motor behaviors. In addition to empowering studies of gut-brain circuits, our species-agnostic framework can be extended to investigate connections between any peripheral organ and the brain thus opening opportunities to develop peripheral organ interventions for neurological and mental conditions.

NIH Research Projects · FY 2024 · 2021-09

Project Summary Chiral natural products and man-made drugs play a pivotal role in the study and treatment of a diverse spectrum of human diseases. The presence of stereogenic centers in drug candidates has been shown to correlate with diminished off-target toxicity, reduced CYP450 inhibition, and an overall enhanced probability of regulatory approval as drugs. However, despite the established importance of three-dimensionality in bioactive chemical structures, the selective synthesis of complex organic molecules containing stereogenic centers remains a venerable challenge. The research described in this proposal describes a conceptually new approach to the selective formation and revision of stereogenic centers through the stereoconvergent epimerization of C– H bonds. In contrast to the vast majority of stereoselective transformations, which establish the absolute and relative configuration of a stereogenic center during a key bond-formation step, our approach introduces the opportunity to enrich or invert individual stereocenters after bond connectivity has been finalized. Our strategy thus decouples the strategies needed to establish bond connectivity from the strategies needed to establish the three-dimensional configuration of a complex molecule. By targeting the most ubiquitous functional group, C–H bonds, this tool has expansive potential. If successful, the research program proposed here is anticipated to transform chemical synthesis, dramatically reducing the time and resources necessary to synthesize complex, bioactive molecules. Fundamental mechanistic findings revealed en route to this goal are further anticipated to contribute significantly to our ability to understand reactivity and selectivity patterns in the context of complex molecular environments. .

NIH Research Projects · FY 2025 · 2021-09

Project Summary The goal of the proposed work is to study the typical development of the critical ability to recognize and navigate the local visual environment, or ”scene”, which constitutes the bedrock of a healthy, independent, and productive life. A rich behavioral literature in humans has shown that remarkable spatial and navigational abilities are already develop- ing within the first few years of life. Likewise, extensive neuroimaging work has uncovered a network of brain regions dedicated to scene perception and navigation in adulthood. However, there is key knowledge gap about how these regions develop the human brain, particularly during the first year of life. Here we propose to study the typical devel- opment of scene-selective cortex in the awake infant brain using magnetic resonance imaging (fMRI) and functional near-infrared spectroscopy (fNIRS). Across three aims, we develop and test two competing theoretical frameworks. One framework suggests that early responses to scenes are driven by low-level visual information inherited from earlier visual systems, and that higher-level scene responses are built on these foundations via cumulative passive exposure to visual scenes over time. By contrast, the second framework suggests that early responses to scenes al- ready reflect higher-level information about the navigation-relevant features of scenes, influenced by connectivity with regions beyond the visual system, and that representations of the structure of the scene are specifically enhanced as infants begin to make independent choices of where to go and how. In Aim 1, we will use fMRI in 2-9 month old infants to test when responses to scenes depicting navigational affordances first emerge in the infant cortex; and whether connections guiding the development of the network are primarily from earlier visual areas, or also from areas beyond the visual cortex. In Aim 2, we will use fNIRS and wide-angle immersive displays in 5-11 month old infants to study whether early-emerging functional responses in this system are driven by low-level features (e.g., peripheral visual stimulation) only, or also by higher-level information about the functional relevance of scenes (e.g., for navigation). Finally, in Aim 3, we will quantify infants' ecological passive visual experience with scenes, and how that changes with motor development, in order to ask whether the onset of independent navigation specifically shapes the neural devel- opment of scenes, over and above passive visual experience. The results of this work will yield basic insights into the typical development of cortical scene processing, and shed light on the fundamental debate in development over the relative roles of maturation and experience. This work will also inform clinically focused investigations into how the basic developmental processes studied here go awry in developmental disorders, and hopefully, novel interventions and rehabilitation strategies for individuals who lose these abilities as a result of stroke or neurodegeneration.

NIH Research Projects · FY 2024 · 2021-09

Project Summary/Abstract The human genome measures nearly 2 meters in length and must be compacted to fit in the nucleus, which measures only a few microns in diameter. In addition to compaction, the DNA must be organized to maintain genome integrity but also remain accessible to the molecular machines that read the genome. How the genome is organized directly influences which genes are expressed in a particular cell. Decoupling of genome organization and gene expression has profound consequences for cells, leading to cancer, intellectual disability, and developmental delay. It is mechanistically unclear how genome organization and the first step of gene expression, transcription, are physically coupled. Determining how both processes are linked is critical for understanding how cell type function and specificity are achieved. The first level of eukaryotic genome compaction and organization is mediated by nucleosomes that are formed by wrapping DNA around histone proteins. Here I will investigate how DNA sequence and nucleosomes impact gene expression in promoter proximal regions of human genes. I will mechanistically decipher how the processes of transcription and genome compaction are intertwined by reconstituting transcription reactions on chromatin in vitro and observing them using (1) a novel high throughput biochemical assay and (2) time-resolved cryo-electron microscopy (EM). In the first part of this proposal, we will develop a high throughput, single molecule transcription assay using reconstituted transcription complexes on thousands of DNA sequences. We will assess how DNA sequence, chromatin state, and transcription factors directly regulate transcriptional activity during early transcription elongation. This assay will overcome major hurdles in the field by revealing directly how nucleosomes and protein factors affect transcription behavior on an unprecedented number of DNA sequence contexts. In the second part of this proposal, we will define how chromatin and DNA sequence influence transcription dynamics by capturing structural snapshots of transcription complexes as they transcribe through nucleosomes. We will use time-resolved cryo-EM to directly assess which states transcription complexes adopt during early transcription and identify stable online and labile offline states. Together, this ambitious proposal will address important questions regarding how nucleosomes and DNA sequence are used to directly influence transcriptional output. The methods and data acquired in this proposal will help fulfill the long-term vision of my lab to fully reconstitute mammalian transcription to determine how cell fate is regulated by the coupling of genome organization and gene expression and how disease mutations can perturb this coupling.

NIH Research Projects · FY 2025 · 2021-09

Immunotherapy treatments such as checkpoint blockade and chimeric antigen receptor T cell therapy have demonstrated the power of the immune system to eradicate metastatic cancer, but the efficacy of immunotherapies in solid tumors remains confined to a minority of patients. A series of interlinked events are needed for efficacy – including induction of immunogenic tumor cell death, recruitment of immune cells to the tumor bed, and reversion of immunosuppressive cues in the tumor microenvironment (TME). We have developed a therapeutic approach using intratumorally-administered synthetic lipid nanoparticles (LNPs) to deliver self-replicating (replicon) RNAs to tumors that activate innate immune signaling pathways and potently express therapeutic payloads. As shown in our recently published preliminary data, this approach elicited profound anti-tumor immune responses in several tumor models, and enabled tumor regression of both injected and distal non-injected tumors. Here we bring together a strong interdisciplinary team to build on these initial findings and apply a synthetic biology toolkit to create next-generation LNP-replicon therapeutics, which combine multiple features to increase the safety and efficacy of this approach, including: (1) cell classifier circuits that allow replicon expression only in target cancer cells or immune cells, (2) optimized multi- subgenomic promoter replicons that encode multiple payload genes expressed at tunable predefined expression levels, and (3) small molecule-regulated replicons that allow two-stage therapeutic programs to be implemented following a single intratumoral injection. These engineered RNAs will be combined with optimized LNP formulations that promote efficient transfection of desired target cell types in the TME. We will apply this technology to treat the leading cause of cancer death, lung cancer, and assess its impact using a syngeneic mouse model of local intratumoral therapy in orthotopic and autochthonous lung cancer models that recapitulate the TME of human lung cancers. Our specific aims are: (1) Develop formulations for cell type- specific expression in cancer cells and T cells, (2) Create small molecule-regulated RNA circuits for cancer cells and T cells for programmable immunogenic cancer cell death and specifically expression in T cells. (3) Therapeutic testing of optimized replicon circuits in orthotopic lung cancer models alone and in combination. Altogether, this proposal brings together a highly interdisciplinary team, marrying cutting edge concepts from synthetic biology and cancer immunotherapy to achieve a more effective, safe, and scalable form of immunotherapy.

NIH Research Projects · FY 2025 · 2021-09

Overall: PROJECT SUMMARY Metastatic disease is responsible for the vast majority of cancer mortality. Understanding of the fundamental mechanisms leading to metastatic cancer has been hampered by the need for models that replicate the step- wise metastatic process in vivo, yet are amenable to tight control and facilitate high-resolution, time-lapse imaging and quantitative analysis of cell behavior. Over the past decade, our team has developed in vivo and in vitro methods capable of simulating many steps of the metastatic cascade including tumor cell invasion, intravasation, trapping in the microcirculation or adhesion to the vessel walls, and extravasation into the surrounding extracellular matrix. In parallel, we have developed computational studies that provided detailed insights often not possible through experiments. This collective prior work has shed new light on central aspects of single-cell and collective cell behavior during metastasis, and identified mechanical adaptations and vulnerabilities of the tumor cell with promise for targeted interventions. The goal of our proposed U54 Center is to employ these developed assays and methods in combination with new measurement techniques to interrogate the full spectrum of stressors experienced by tumor cells in the metastatic niche during arrest and extravasation, and couple these with parallel studies of changes in chromatin structure and the transcriptome of tumor cells (Core B). These changes are critical to mechano-adaptation of the tumor cells towards an organ-preferential initiation of a metastatic colony or transition to dormancy. A hallmark of our proposed center is the use of state- of-the-art in vitro (Project 1) and in vivo (Project 2) experiments and computation (Core A) to uncover and probe the factors that ultimately determine tumor cell fate. We anticipate that such integrated studies will provide new insights into metastatic cancer, not possible by the use of any method alone, and enhance our ability to identify and screen for new therapies to inhibit the tendency for metastatic spread of disease.

NIH Research Projects · FY 2025 · 2021-09

Previous work from the Tsai lab (Canter et al 2019) identified the mamillary body (MB) as one of the first sites of amyloid deposition in 5XFAD model mice, a region that also correlates with dementia severity in human patients. Single cell RNA sequencing of the mouse MB identified 2 distinct neuronal populations within the MB, with segregated distribution, target projection, and unique electrophysiology. Analysis of these populations in the 5XFAD mice found that one of these populations, those found in the lateral MB (LM), are uniquely susceptible to hyperactivity and neurodegeneration, while the second population (medial MB, MM) is largely unaffected. The activity of the LM population also directly contributes to mouse performance in memory tasks. Additionally, using iterative direct-expansion microscopy (idExM) from the Boyden lab, we have identified intriguing patterns of amyloid associated with specific projections in the fornix, the white matter tract from the subiculum with axonal inputs to the MB. This grant proposes to investigate the links between amyloid and excitability changes in the MB and fornix, including development of the tools necessary to achieve this goal. The hypothesis to be tested in this application is that amyloid preferentially associates with the subiculum-LM projection and that these axons exhibit hyperexcitability. Aim 1 will map the connections between this new population of LM neurons and its upstream inputs from the hippocampus, using a newly developed in situ sequencing techniques, as well as exploring pathology in the white matter projection regions of this circuit in 5XFAD mice and human brain samples, using recently developed expansion microscopy. Aim 2 will characterize the source and location of hyperactivity found in the LM neurons through advanced voltage imaging, as well as expand this work to other mouse models of AD. Aim 3 will use optogenetics and pharmacological approaches to determine if specific aberrant circuit activity drives the pathology and behavioral changes seen in the AD model mice.

NIH Research Projects · FY 2025 · 2021-09

Project Summary/Abstract Comprised of six distinct layers, the cerebral cortex is the key brain structure for all of our cognitive abilities, ranging from sensation to decision making to movement. Each layer contains distinct cell types differing in their genes, biophysical properties, and connectivity with other parts of the nervous system. Yet how these diverse cortical layers and cell types are involved in any given behavior remains unresolved. Moreover, we currently lack insight into how aging impacts interactions between cortical layers, which severely limits our understanding of how aging alters cortical circuit function. At the most basic level, the cortex can be divided into deep and superficial layers, each of which receives a complete copy of sensory information from the thalamus. This suggests that the two sets of layers constitute different processing systems, which begs the question: what are the possible purposes of these parallel networks? Because these processing streams differ in input, output, intrinsic membrane, synaptic integration, and spike generation properties, I hypothesize that deep and superficial layers have unique, independent functions. This also raises the intriguing possibility that these pathways are differentially susceptible to aging. I hypothesize that aging leads to layer-specific changes that ultimately lead to unique age-related deficits in cortical circuit function. Investigating the functions and age-related changes in deep and superficial cortical networks requires a cortex-dependent task. In Dr. Bruno’s lab at Columbia University (F99), I developed a whisker-mediated texture discrimination task for head-fixed mice, demonstrated that this behavior requires the cortex, and revealed that both deep and superficial layers are involved in processing texture information (Aim 1.0, progress report). I propose to characterize the sensorimotor strategies required for this behavior (Aim 1.1) and how layer-specific manipulations alter texture representation in the deep and superficial layers (Aim 1.2). Understanding the computations performed by individual layers will not only expand our understanding of the complex cortical circuitry, but will also provide insight into how aging and neurodegeneration – which often involve dysfunction of specific cortical cell types, layers, and their pathways – may be mitigated through the development of targeted therapies. In Dr. Tsai’s lab at Massachusetts Institute of Technology (K00), I will develop a novel therapy that galvanizes the brain’s own mechanisms to noninvasively improve cognitive and behavioral health in aged and Alzheimer’s disease (AD) model mice. To do so, I will first identify how aging and AD alter learning, performance, sensorimotor strategies (Aim 2.1), and sensory processing across cortical circuits (Aim 2.2) on my texture discrimination task. These findings will inform the development of a noninvasive therapy that stimulates cortical circuits to protect sensory and motor functions from aging and AD pathology (Aim 2.3). This proposal will advance our limited understanding of how aging dynamically alters cortical circuits in vivo and may lead to an effective intervention to prevent age and disease-related functional impairments in human patients.

NIH Research Projects · FY 2026 · 2021-09

Diamond Rotors (5R01GM139055) Renewal Proposal Project Summary/Abstract Modern medicine rests on an understanding of molecular biology, and molecular biology rests on an understanding of molecular structure. Nuclear Magnetic Resonance (NMR) is uniquely suited to capture both atomic-level structure and molecular dynamics under near-native conditions. This advantage contrasts with cryo-Electron Microscopy (cryo-EM), which lacks information on dynamics, operates at cryogenic temperatures, and is typically limited to larger complexes (>60 kDa). The complementary insights provided by NMR and cryo-EM can bridge gaps in data, offering a fuller understanding of complex biological systems such as membrane protein drug targets and amyloid-beta (Aβ) plaques implicated in neurodegenerative diseases. Despite its strengths, NMR suffers from inherent insensitivity. For large biomolecules and biomolecular complexes, it also faces challenges related to spectral crowding, which impacts resolution. The limitation of sensitivity is exacerbated in the small sample volumes (0.4, 0.7, 1.3 mm) rotors used for achieving high resolution. This proposal seeks to advance magic angle spinning (MAS) rotors laser-machined from single-crystal diamond. The motivation is twofold: to enhance resolution by spinning at unprecedented high frequencies and to improve sensitivity by leveraging diamond rotors in Dynamic Nuclear Polarization (DNP) experiments. There are four compelling advantages to the use of diamond rotors in magic angle spinning (MAS) NMR experiments. First, higher spinning frequencies improve the resolution in MAS spectra. At 200 kHz MAS frequencies, ZrO2 rotors cannot withstand the centrifugal forces, and therefore rotors fabricated from a stronger material such as diamond are required. In particular, the flexural strength of ZrO2 is ~850 MPa vs. 2-5 GPa for diamond. Second, as we move dynamic nuclear polarization to higher frequencies ZrO2 becomes less transparent to microwaves and at 527 GHz (800 MHz 1H NMR) the transmission drops to ~10%. However, diamond is transparent and DNP enhancements should improve using diamond rotors. Third, diamond's superior thermal conductivity (x10 that of Cu) ensures that the temperature of the sample will be uniform. Finally, the toughness of diamond means these rotors can be repacked and reused, unlike existing rotors. 1