Massachusetts Institute Of Technology

NSF Awards · FY 2024 · 2024-07

This project will study the turbulence in a stratified layer at the air-water interface, as caused by waves and wind. The study will carry out simulations with laboratory experiments and with computer models. Simulations will test the hypothesis that to represent the deepening of a surface layer reliably, it is necessary to couple currents and waves. Simulations will also test a parametrization of turbulence related to waves (Craik-Leibovich parametrization), and the results of combining a couple of parametrizations. For broader impacts, the project could improve the reliability in representing the ocean surface boundary layer of Earth-system models. Moreover, the PIs would produce educational materials to be used at their home institutions. In addition to supporting 4 PIs, this effort would fund one postdoctoral scholar and one graduate student. The proposed study seeks to advance understanding of wind-driven and wave-driven near-surface turbulence in a stratified surface boundary layer. This goal would be pursued with a combination of a) controlled laboratory experiments of stratified turbulent mixing under the influence of surface waves in the surface boundary layer, and b) state-of-the-art (Large-Eddy Simulations) numerical experiments. Laboratory and numerical simulations will test the hypothesis that the coupling between waves and wind-driven currents is necessary to reliably represent the surface mixed-layer deepening. The comparison between lab measurements and numerical simulations would seek to i) assess the reliability of LES (Craik-Leibovich) simulations in representing lab observations of turbulent mixing beneath horizontally heterogenous surface waves, and (ii) determine the effects of combining coarser grids in LES simulations with a turbulence closure. As broader impacts, the project could potentially improve the accuracy of Earth-system numerical simulations of the ocean surface boundary layer. Moreover, the PIs would produce educational materials to be used at their home institutions. In addition to supporting 4 PIs, this effort would fund one postdoctoral scholar and one graduate student. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NSF Awards · FY 2024 · 2024-06

Energy-constrained Cyber-Physical Systems (CPS), ranging from smartphones and lightweight augmented reality (AR)/ virtual reality (VR) headsets to insect-size flying robots and pill-sized medical micro-robots, could transform a diverse set of applications in consumer electronics, targeted medication delivery, search and rescue missions, and space exploration. All these applications place severe constraints on the size, weight and power of on-board computers and sensors. Yet, to safely operate in unknown complex environments, each CPS should perform several fundamental tasks including: (i) localization: determining its location, typically without any strong external aids such as GPS (Global Positioning System) and (ii) mapping: creating a compact representation of obstacles in the environment. Existing algorithms that enable these fundamental tasks often require large memory overhead for storing temporary variables during computation and are typically executed on general-purpose computers which are too energy hungry. Thus, enabling autonomy on energy-constrained robots requires not only the design of robust and efficient algorithms for localization and mapping, but also their specialized energy-efficient computing hardware. The project will also support the development of a new graduate course that is at the intersection of computer architecture, integrated circuits and robotics, and an outreach program for high school students. This project explores the co-design of algorithms and hardware for localization and mapping that is efficient, robust, and accurate all at the same time. To achieve energy efficiency, the approach is to develop new algorithms and their computing hardware such that: (i) the number of memory accesses do not dominate the algorithm, and (ii) the amount of memory required during computation remains small enough to be on-the-chip. The team will demonstrate the new methodologies by designing and fabricating a new chip to execute effective localization and mapping tasks in a fraction of the size, weight, and power of the state of the art. The innovation here is the focus on minimizing memory utilization and data flow, as opposed to optimizing the number of computing operations. If successful, the research will impact miniature and/or extremely-long-endurance mobile CPS application in agriculture, environmental quality, healthcare and personalized medicine, and manufacturing as well as consumer applications, robotics, and sustainability. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2026 · 2024-06

Project Summary/Abstract Here, we propose to study cancer etiology in the context of DNA damage caused by a methylating agent, N- nitrosodimethylamine (NDMA), a probable human carcinogen that has been found in water, food, and drugs. NDMA is both mutagenic and toxic, and our overriding hypothesis is that its biological consequences are shaped interactions among three repair pathways: direct reversal, mismatch repair (MMR), and homologous recombination (HR). One of the key DNA lesions created by NDMA is O6MeG, which mispairs readily with thymine. The direct reversal protein O6-methylguanine DNA methyltransferase (MGMT) removes the offending methyl group, restoring the structure of guanine. Interestingly, O6MeG can become toxic when acted upon by Mismatch Repair (MMR), although the underlying mechanism remains to be fully elucidated in vivo. Normally, MMR recognizes mismatches behind the replication fork and removes the newly synthesized strand to give the cell another chance at accurate replication. In the case of O6MeG, it has been posited that MMR removes the strand opposite the lesion forming a single strand gap that upon the subsequent replication cycle becomes a broken fork (i.e., a double strand break [DSB]) that requires HR for repair. Despite the prevalence of this model in the literature, these predicted processes have been largely untested in vivo, a key gap in the literature that will be addressed in part by using an in-house genetically engineered mouse model, namely RaDR, for which HR yields a fluorescent signal. An advantage of RaDR is that it can also be used for lineage tracing, which makes it possible to monitor clonal expansion. Using the RaDR mice, we made the remarkable discovery that MGMT strongly suppresses clonal expansion. Our overriding hypothesis is that MMR promotes toxicity and inflammation, providing selective pressure for clonal expansion. Specific Aim 1 is to test the MMR model by measuring the replication dependence of DSBs and HR to elucidate possible dependence on two cycles of replication, which would be consistent with gap-driven fork breakdown. Specific Aim 2 is to quantify clonal expansion, to leverage 2-photon microscopy for whole-organ 3D imaging of clonal outgrowths, and to peer into clonal outgrowths via spatial transcriptomics to reveal underlying mechanisms of clonal expansion. Specific Aim 3 is to quantify the impact of MGMT, MMR, and their interaction on NDMA-induced inflammation and cancer in the liver. The proposed work will not only elucidate the ways by which interactions among DNA repair pathways modulate susceptibility to DNA damage, but it will also be one of the first studies of how DNA repair shapes the risk of clonal expansion, a fundamentally important step in carcinogenesis. Together, these studies will have a significant and lasting impact by advancing our understanding of the molecular forces that shape disease with important implications to public health and the clinic.

NIH Research Projects · FY 2026 · 2024-06

PROJECT SUMMARY Humans with normal hearing excel at deriving information about the world from sound. Our auditory abilities represent stunning computational feats that only recently have been replicated to any extent in machine systems. And yet our auditory abilities are highly vulnerable, being greatly compromised in listeners with hearing impairment, cochlear implants, and auditory neurodevelopmental disorders, particularly in the presence of noise. Difficulties in recognition often lead to frustration and social isolation, and are not adequately addressed by current hearing aids, implants, and remediation strategies. The long-term goal of the proposed research is to build models that replicate our abilities to recognize and localize sounds, and to use these models to facilitate improved prosthetic devices and therapeutic interventions. The development of more effective devices and therapies is currently limited by an incomplete understanding of the link between peripheral auditory processing and auditory behavior. We propose to leverage machine learning to build models that instantiate this link by performing realistic tasks given simulated peripheral auditory input, and to evaluate the models with large-scale behavioral assays. Aim 1 will build models of recognition and localization of real-world sounds, and compare them to human recognition and localization judgments. Aim 2 will augment these models with selective attention, and compare them to human performance on recognition and localization tasks that require selective attention. Aim 3 will introduce different types of simulated hearing loss to these models and attempt to isolate distinct behavioral signatures of different types of hearing loss. The research will generate models that can predict human auditory behavior along with new behavioral benchmarks with which to evaluate these and future models. The results will clarify the role of peripheral auditory processing in real-world auditory abilities, setting the stage for new strategies for remediation.

NIH Research Projects · FY 2025 · 2024-06

PROJECT SUMMARY / ABSTRACT Preliminary work has shown that two different environmental hepatocarcinogens, aflatoxin B1 (AFB1) and N-nitrosodimethylamine (NDMA), produce high-resolution mutational spectra (HRMS) that occur shortly after carcinogen exposure, are distinct from one another, and are mechanistically in accord with the established mutational properties of the DNA adducts these agents form. Extending that work, the goal of the proposed project is to develop a “blood-based” analytical tool enabling rapid detection of the mutational profiles of these agents. The hypothesis to be tested is that mechanistically informative mutational fingerprints of environmental toxicants are present in circulating cell-free DNA (cfDNA) obtained from blood of exposed individuals. This project will address a gap in knowledge connecting environmental exposures to cancer risk. Sequencing of cancer genomes has revealed ~100 mutational patterns termed “signatures,” with some signatures showing similarity to mutational spectra produced by known environmental carcinogens (e.g., UV light, AFB1, benzo(a)pyrene). Currently, there is no facile way to measure the genetic consequences of prior genotoxic exposures because of: (1) the invasive procedures required to obtain tissue samples, and (2) the insensitivity of “typical” DNA sequencing methods. This proposal will take advantage of recent advances to overcome these limitations. Consisting of fragments ~170 bp long, cfDNA originates from normal cell turnover as well as from apoptotic and necrotic cells following exposure to toxins. Conventional NextGen DNA sequencing tools cannot reliably identify the low levels of mutations that are present in cfDNA. To overcome this sensitivity limitation, we shall use Duplex Consensus Sequencing, which we have shown affords up to 104-fold higher accuracy/sensitivity over conventional sequencing. The Specific Aims will determine if HRMS previously observed in mouse liver genomic DNA following treatment with AFB1 and NDMA can be identified in cfDNA in blood. Aim 1 will quantify the levels of cfDNA in blood after treatment. Circulating cfDNA will be assayed at several times after treatment to determine the temporal relationship between exposure, hepatotoxicity and the levels of cfDNA in blood. Aim 2 will determine compound-specific mutational spectra in cfDNA and compare them to spectra obtained from genomic DNA from target tissue (liver) as well as white blood cells from blood, lymph nodes and thymus. Aim 3 will examine mutational spectra in liver-specific cfDNA generated by challenging the animals with mild hepatotoxicants (e.g., ethanol, acetaminophen), which trigger increased cell turnover. The success of these studies will provide an innovative approach to mutational fingerprinting of environmental exposures that could, in turn, lead to predictive biomarkers that would trigger interventions to limit future exposures and reduce healthcare needs by preventing the development of advanced disease.

NIH Research Projects · FY 2026 · 2024-06

PROJECT SUMMARY: An organism's capacity to develop and grow is heavily dependent on its ability to organize various aspects of structure, ranging from G-actin to F-actin, cross-linked filaments, F-actin networks, and multicellular networks—in short, coordinate structure at multiple levels of order. Despite advances in characterizing proteins that affect actin at different levels of the structural hierarchy, we have limited knowledge of how regulation of actin is integrated across these hierarchical levels. Bitesize, the only Synaptotagmin-like protein in flies, is a membrane-bound protein that has recently been demonstrated to also directly bundle filamentous actin; preliminary data has further demonstrated that Bitesize promotes F-actin assembly. However, the mechanisms driving these actin-organizing behaviors of Bitesize have not been determined; moreover, the manner in which such mechanisms combine to produce a net effect in vivo is unknown. The long-term goal of this work is to elucidate how mechanisms that control different hierarchical levels of actin organization are integrated to facilitate morphogenesis. The overall objective of this proposal is to determine the mechanisms underlying Bitesize's organization of actin and their combined effect on Drosophila embryo development. My rationale for the proposed work is that Bitesize demonstrates direct interaction with actin, and therefore offers a unique opportunity to study the direct regulation of actin's structural hierarchy at multiples levels through the probing of a single protein. My hypothesis is that Bitesize integrates the regulation of actin assembly and actin bundling via distinct domains along its length. My aims are to: 1) determine the mechanism used by Bitesize to bundle actin, and 2) determine whether Bitesize separately regulates assembly and nucleation of actin. This approach is innovative in its coupling of structure-function analysis of Bitesize’s interaction partners with the in vivo study of btsz gene function in an animal tissue using precise gene edits. My proposed research is significant because it will determine means of coordinating hierarchical structures of actin, and because it is expected to demonstrate new mechanisms of regulating actin, such as stimulation of Dia via formin elongation effector domains, that have not been demonstrated in animals in vivo. This research will be accompanied by training in biochemistry as well as Drosophila embryology and genetics, which will take place in the Martin lab and Schwartz lab in the Department of Biology and Cryo-EM Facility at MIT, as well as in the Goode lab at Brandeis University. Together, this research and training will prepare me to establish an independent lab to investigate fundamental questions related to the formation of functional, hierarchical structures in cells and tissues.

NIH Research Projects · FY 2026 · 2024-06

Project Summary Proper regulation of gene expression is required for cellular function. Conversely, dysregulation of gene expression is a major cause of disease. Furthermore, expression of two genes is frequently coupled. Coupled expression is expected if two genes respond to the same signal or are expressed from a bidirectional promoter (BDP). About one in ten human genes are controlled by a BDP, defined as two genes with less than 1 kilobase between their transcription start sites. However, coupled expression in general and BDPs in particular remain very poorly understood. The premise of this proposal is that our current lack of understanding of coupled expression is due to a lack of tools. The goal of this proposal therefore is to develop an integrated experimental and computational toolkit for real-time analysis of coupled nascent transcription. For example, we currently do not understand if transcription of two BDP-controlled genes is synergistic, antagonistic, or independent in quantity and in time. More generally, understanding coupled expression in both quantity and time requires real-time single-molecule imaging of nascent transcription integrated with computational modeling. Here we propose to develop an experimental platform that allows direct visualization of nascent RNA of two genes (Aim 1). Specifically, we will adapt RNA hairpin systems from bacteriophage to allow real-time visualization and counting of single nascent RNAs in live cells. We will evaluate multiple microscope modalities and optimize a microscopy platform for long-term gentle live-cell imaging at high spatiotemporal resolution. Next, we will develop computational methods for quantification of nascent BDP transcription imaging data (Aim 2). We will also develop Bayesian Inference methods for inference of two-gene coupled nascent transcription data, and identify the minimal mathematical models required to understand BDP transcription. Next, we will apply these new experimental and computational tools for an initial exploration of BDPs (Aim 3). Specifically, we will take a two-pronged approach exploring multiple BDPs at a safe-harbor locus as well as dedicated studies of the endogenous NIPBL BDP. This will serve as proof-of-concept for our integrated toolkit. In summary, we propose to establish an integrated experimental and computational toolkit for real-time analysis of coupled nascent transcription. We will devote significant efforts to disseminate the experimental and computational tools and anticipate this being useful beyond BDP studies. We anticipate this toolkit will allow future mechanistic studies to better understand BDPs, will make it possible to establish BDPs as powerful tools for synthetic biology, and empower studies of coupled nascent transcription beyond BDPs.

NIH Research Projects · FY 2026 · 2024-05

Abstract A long-term goal of my research is to develop a comprehensive understanding of the computational principles, anatomical substrates, and neural mechanisms of sensorimotor function. Our research is guided by the concept of internal models, which provides a unified computational framework for investigating the neural basis of sensorimotor coordination, integration, and learning. Currently, we do not have a comprehensive understanding of how the brain instantiates internal models. Our broad research objective in to elucidate the functional role of the ascending cerebello-thalamocortical (Cb-Th-Ctx) and basal ganglia-thalamocortical (Bg-Th-Ctx) pathways in forming, updating, and using internal models. We will tackle this problem in the nonhuman primate model using a multidisciplinary and cutting-edge approach that leverages (1) a sensorimotor task with suitable variants for investigating the neural basis of sensorimotor coordination, integration, and learning, (2) large-scale multi- region electrophysiology techniques to characterize neural signals with high spatiotemporal resolution across entire subcortico-cortical pathways, and (3) a mathematically rigorous framework to integrate knowledge across scales (cells, circuits, areas) and disciplines (systems, computational), and link computational concepts (e.g., internal models) to directly measurable neurobiological variables (e.g., firing rates). In the next decade, we will use this integrative approach to gain a mechanistic understanding of how Cb-Th-Ctx and Bg-Th-Ctx pathways establish internal models and support adaptive sensorimotor behaviors.

NIH Research Projects · FY 2026 · 2024-05

Alzheimer's disease (AD) is a fatal neurodegenerative disease characterized by progressive cognitive decline and brain pathologies including amyloid-β (Aβ) peptide deposition, hyperphosphorylated tau accumulation, inflammation, and synaptic and neuronal loss. In addition to protein pathology, the abnormal accumulation of lipids in AD brains – originally described by Alzheimer himself in his 1907 publication – has recently been rediscovered as an important factor in AD. Many recent discoveries in Alzheimer’s disease raise questions of how lipid-rich microdomains within cell membranes known as lipid rafts might play roles in Alzheimer’s pathology; why contact points between mitochondria and the endoplasmic reticulum (ER), known as mitochondria-associated ER membranes or MAMs, increase in AD and are impacted by the presence of APOE4, the strongest genetic risk factor for late-onset AD; how intraneuronal aggregation of tau and toxic Aβ trigger ER stress in early stages of AD; why changes in mitochondrial morphology such as fragmentation and elongation occur in AD; as well as countless other recent discoveries. One recent example from our teams was a demonstration of accumulation of neutral lipid droplets and cholesterol in glial cells harboring the APOE4 allele in the gene coding for apolipoprotein E (ApoE), one of the most significant genetic risk factors for developing AD. The presence of APOE4 led to LD accumulation in astrocytes, microglia, and oligodendrocytes. While these findings clearly suggest that subcellular organelle organization and morphologies play important roles in the development of AD, the mechanisms mediating these roles remain unclear, largely due to the lack of analytical tools that allow the unambiguous characterization of intracellular structures in brain tissue from animal models of AD and from human AD patients. The standard method for investigating morphological characteristics of organelles is electron microscopy (EM) which struggles to identify specific biomolecules amidst organelle architecture. Our recent invention of expansion microscopy (ExM) allows nanoscale imaging of biological specimens, including molecular contrast, with conventional microscopes, and has been employed in studies on the Golgi apparatus, the ER, mitochondria, and myelin. Here we propose to extend the ExM toolbox to confront, head-on, the key needs of Alzheimer’s lipid research. Specifically, we will (Aim 1) optimize, and validate, a form of ExM that combines lipid preservation and staining, multiplexed antibody staining, and expansion microscopy – which we call multiplexed ultrastructure expansion microscopy (multiplex-umExM); (Aim 2) optimize, and validate, multiplex-umExM for human brain tissue; (Aim 3) perform a comprehensive characterization of lipid accumulation and organelles by brain cell type and AD risk genotype in mouse and human tissue. The net result of this grant will be a toolbox that anyone in biology can use to characterize lipid and organelle properties, with nanoscale precision and molecular contrast, in diseases such as Alzheimer’s disease, as well as the validation data to show its utility.

NIH Research Projects · FY 2026 · 2024-05

Abstract Understanding how T cell receptors (TCRs) see tumor antigens presented by MHCs is necessary to fully understand how the immune system recognizes tumor antigens, and to reap the full potential of antigen-specific immunotherapy. To achieve this goal, a quantum leap forward is required in which the revolutionary advances in machine learning are combined with a large volume of structure, function, data on matched TCR-pMHC pairs. The development of accurate predictors of TCR-antigen recognition will be dependent on the creation and integration sequencing-based datasets with high-throughput structural and functional insights. Our proposal, submitted as a CRUK/NCI Grand Challenge team (MATCHMAKERS) will combine researchers with expertise in immunology, methods development, structural biology, and computation to enable generalized prediction and design of TCR recognition. This work will be spread across four Work Packages (WPs): WP1: Large-scale generation of TCR-pMHC pairs from naturally occurring sources. We will build datasets of naturally occurring TCR-pMHC pairs. Our team will use an array of approaches to collect these datasets, from humans and from mouse models, and in the context of both cancer and immunity more generally. WP2: Ultra-high throughput TCR-pMHC matching using molecular engineering. Efforts to create general models will require a broader array of data than feasible to collect from natural TCR systems. We will use an array of synthetic approaches developed by our team to comprehensively match TCRs with pMHCs to train computational models. WP3: Large-scale structural and biochemical analyses of TCR-pMHC interactions. A key to our team’s vision is to match interaction datasets with high throughput structural and functional insights. A deep understanding of how the TCR contacts with MHC helices control function and orientation will be essential for training and testing computational models. WP4 AI-based prediction and design of TCR-pMHC interactions. We will integrate our data to train next- generation algorithms capable of generally predicting and designing TCR-pMHC interactions. These predictions will proceed through a reiterative testing and feedback circuits for further model optimization.

NIH Research Projects · FY 2026 · 2024-05

ABSTRACT Our vision is to unravel and ultimately reverse the intricate network of causal factors throughout the life course that disrupts biological homeostasis to promote colorectal cancer (CRC) among individuals younger than age 50 years. Uniting leading scientific minds in early-onset colorectal cancer (EOCRC) research and complementary fields, we have embraced disruptive, transdisciplinary approaches spanning cells to individuals to populations to address the core Grand Challenge to “Determine why the incidence of early-onset cancers is rising globally”. We will address specific questions of “the mechanisms linking lifetime exposures with cancer initiation and promotion” by focusing on EOCRC as an ideal model for early-onset cancer due to the availability of well- characterized animal models and a well-established and prevalent precursor lesion, the adenomatous polyp (adenoma), offering a unique opportunity for interception and prevention. Our work will transform the field by directly addressing our overarching goal to “identify and understand the processes through which different biological and environmental factors cause early-onset cancers”, and reverse the burden in a timely, effective, and feasible fashion. Our team, both working independently and in collaboration, has uncovered several risk factors that are likely to be drivers for the rising incidence for EOCRC. We are now uniquely positioned to translate etiologic understanding to actionable prevention by identifying novel factors, including environmental determinants, and deepening our understanding into overlooked dimensions of exposure throughout the life course. The unprecedented scope and scale of our proposal can only be supported through Cancer Grand Challenges since our “high-risk” disruptive approach requires deep interactions between work packages (WP)s led by leaders in distinct disciplines. This will enable incorporation of fresh perspectives to move beyond traditional risk-factor epidemiology toward an integrated, mechanistically-informed model with population scale and cellular resolution of the multiple and cumulative “hits” that promote EOCRC to inform the development of actionable prevention. Our innovations intersect epidemiology, small molecule discovery, genomics, stem cell biology, immunology, and computational biology with these key features: 1) harmonization of cohorts with data and biospecimens collected across the lifecourse; 2) innovative and reliable analysis of small molecules to detect novel exposures; 3) high-resolution technologies for analysis of target tissues; 4) model systems capable of interrogating accumulating exposures across the lifespan and their impact on the cellular ecosystem; 5) prevention through risk assessment and pharmacologic/lifestyle interventions. Collectively, our work will serve as an exemplar for transforming research into other early-onset cancers.

NIH Research Projects · FY 2025 · 2024-02

PROJECT SUMMARY/ABSTRACT This proposal will elucidate the various steric and electronic parameters of a P-catalyst necessary for enantioinduction in tandem amidation/amide functionalizations through deoxygenative P(III)/P(V) catalysis; an important transformation for the synthesis of biologically active compounds. This proposal will first examine P- catalyzed enantioselective amidations, followed by P-catalyzed enantioretentive amide functionalizations. Finally, these catalysts will be combined for a one-pot synthesis of enantioenriched secondary nitriles from racemic carboxylic acids, a moiety important to the pharmaceutical community. Overall, during this fellowship period I seek to gain expertise in inorganic main-group chemistry, including computational analyses and modern inorganic synthetic techniques. Such skills will complement my existing skillset, creating a strong foundation in catalyst design for my future career goals.

NIH Research Projects · FY 2025 · 2024-01

Project Summary Title: Systematic identification of novel anti-phage defense mechanisms in the E. coli pangenome There is an urgent need for new therapies and approaches for treating antibiotic-resistant bacterial infections. One promising, but underdeveloped approach called phage therapy aims to use the viruses that infect bacteria, called bacteriophages (or just phages). Although there have been a handful of case studies reporting success, including for the treatment of ESKAPE pathogens of most dire concern, the long-term efficacy and prospects for wide-spread use of phage therapy remains highly uncertain. A key challenge is that bacteria often harbor potent anti-phage defense mechanisms that enable them to resist or overcome viral infection. These anti-phage mechanisms have emerged from the long-standing, fierce coevolutionary battle between bacteria and phages, with a molecular 'arms race' leading bacteria to evolve diverse mechanisms for defending themselves and phages, in turn, evolving counter-defense strategies. The anti-phage arsenal of bacteria includes restriction- modification (RM) and CRISPR-Cas systems. In recent years computational studies have identified dozens of additional systems, but these studies have critical limitations, and our own experimental studies have indicated that there are dozens, and likely hundreds, of additional systems still to be discovered. By developing and applying a powerful, high-throughput functional selection procedure, we aim to identify the anti-phage systems present in a diverse collection of 1,500+ strains of E. coli, including a range of pathogenic strains. We will screen for defense against a panel of 10 different coliphages. Bioinformatic analyses, particularly homology detection and structural predictions, will be done to assess the conservation, genomic context, and predicted biochemical functions of the newly identified systems. Thus, our work will lay the foundation for detailed molecular studies of the diverse new systems identified. As with prior studies of anti-phage defense, we anticipate that the new systems will drive the discovery of new molecular mechanisms, which may, in turn, form the foundation of a new generation of precision molecular tools. It has also become clear in recent years that many cell autonomous components of eukaryotic innate immunity have distant homologs in bacteria. As such, our work may also reveal evolutionarily conserved facets of immunity across the kingdoms of life. Finally, the methodology developed will be broadly applicable to virtually any bacterial pathogen, work that we anticipate will inform ongoing and future efforts to develop phages as therapeutic agents.

NIH Research Projects · FY 2025 · 2024-01

PROJECT SUMMARY Colorectal cancer (CRC) metastasis is a major cause of mortality, yet there is a distinct lack of therapies targeting metastasis. Among the major modifiable external factors known to affect CRC risk are diet and obesity; however, unlike cancer initiation, how pro-obesity high fat diets (HFD) can impact cancer metastasis remains an essential question. Our understanding of the biology of metastatic cells is significantly impeded by a lack of in-vivo CRC models that recapitulate metastatic disease. This proposal leverages our orthotopic transplantation model of CRC, where genetically-engineered CRC organoids harboring common mutations found in human CRC are transplanted via colonoscopy into recipient host colons, forming tumors that later metastasize to the liver. Using this system, we have begun to study how CRC with different driver mutations such as APC, KRAS and p53, metastasize and adapt to the liver microenvironment in diet induced obesity. We have observed that diet induced obesity increases CRC metastasis to the liver. Our initial single cell RNA-Sequencing analyses of the primary colon and liver metastatic tumors have identified tumor intrinsic and extrinsic changes in response to an obesogenic HFD. Tumor intrinsic responses to HFD include increased YAP1 signaling and tumor-specific “revival stem cell” or revSC like populations. In our AIM1 of mentored K99 phase, using CRISPR mediated knock out models and untargeted metabolite profiling, we will identify the role of Hippo/YAP1 signaling in tumor metastasis and metabolic adaptations in pro-obesity HFD. In our AIM2 (K99 phase), leveraging reporter mouse models and single cell RNA-Sequencing, we will identify the contribution of revSCs in metastatic seeding, and the role of YAP1 regenerative signaling in mediating revSC like phenotypes in obesity. In our AIM3 of independent R00 phase, we will identify tumor extrinsic niche factors that contribute to increased metastasis in HFD. In our preliminary single cell profiling of the tumor niche in HFD, we observe evolution of specific Spp1 and Cxcl1 expressing cancer associated fibroblasts (CAFs) in the colon and liver metastatic niche. AIM3 will specifically characterize the origin, role and interactions of these CAF populations. The K99 training will mainly focus on cancer stem cell niche and modeling, single cell analyses, and tumor metabolism. This will be accomplished by training with the primary mentor, Dr Omer Yilmaz, with expertise in intestinal stem cells and their niche, colorectal cancer and gastrointestinal pathology, and mentoring team Dr Alex Shalek (single cell and computational analysis), Drs Alpaslan Tasdogan and Matthew Vander Heiden (tumor metabolism) and Dr Jacqueline Lees (cancer stem cells). This will be supplemented with conference, coursework and workshops in Metabolomics, Metastasis and Bioinformatics, and mentorship and teaching exercises to prepare for future academic career in the independent R00 phase. Successful completion of this study will identify mechanisms integral to initiate and maintain metastasis, revealing targetable vulnerabilities as well as provide insights to guide dietary interventions.

NIH Research Projects · FY 2025 · 2024-01

PROJECT SUMMARY/ABSTRACT The stereochemistry of a pharmaceutical drug is of paramount importance as alternative stereoisomers can lead to vastly completely different outcomes in its efficacy, pharmacokinetic properties, and side-effects. Therefore, site-selective control to access the desired stereochemistry of a molecule (stereochemical editing), especially at a late-stage, has a direct impact on drug discovery and is at the forefront of innovation in synthetic organic chemistry. The objective of this proposed research is to develop a mild and efficient stereochemical editing strategy for quaternary stereocenters guided by enantioselective recombination of C–C bonds. Traditionally, stereochemical editing relies on the homolytic cleavage of a C–H bond via photoredox catalysis in the presence of a hydrogen bond donor and a hydrogen bond abstractor. Nevertheless, this system is simply unapplicable to quaternary stereocenters because they lack the required hydrogen bond. To overcome this challenge, the proposed research engages an innovative application of photoredox catalysis and asymmetric recombination of a C–C bond. The first approach will establish a dual asymmetric photoredox/nickel catalysis strategy. This system will be applied to effect a mesolytic cleavage of a target C–C bond, followed by asymmetric induction of a chiral nickel catalyst to promote an recombination of the C–C bond via intramolecular sp3–sp3 cross-coupling. If successful, this dual catalytic system will provide a fully stereocontrolled means to access to the quaternary stereocenters under mild conditions. The second approach will deploy a chiral counteranion of the photocatalyst to induce asymmetric ion-pairing with carbocationic intermediates. This method will utilize exceptionally simple, yet underexplored conditions to recombine C–C bonds to manipulate the quaternary stereocenters orthogonally to the previous approach. These studies are expected to enable a novel mode of action toward stereochemical editing of quaternary stereocenters and have applications, such as epimerization, racemization, and deracemization, in the discovery of pharmaceuticals, natural product synthesis, as well as derivatization of the existing drugs. The proposed research aligns well with my future development plan to broaden my expertise in modern methodology by ensuring exposure to the development of new techniques in photoredox and transition metal catalysis with mechanistic and kinetic studies. The Wendlandt lab’s extensive experience and expertise in photoredox catalysis, coupled with the state-of-the-art resources and facilities at MIT, provides the optimal environment to pursue and successfully execute the proposed research.

NIH Research Projects · FY 2026 · 2024-01

Project Summary. Cancer is governed by evolutionary principles whereby sequential changes at the genetic and epigenetic level enable proliferation, immune evasion, drug resistance, and metastasis. An outstanding goal in cancer biology is to understand the spatiotemporal processes underpinning this evolution. To do so would greatly improve our ability to create more effective treatment strategies and forecast tumor development far into the future. However, this goal remains elusive due to our incomplete catalog of molecular processes driving evolution and lack of molecular and computational tools for holistically profiling tumors. One emerging driver is extrachromosomal DNA (ecDNA). Found in approximately half of cancers and strongly associated with poor survival, ecDNAs are a unique form of oncogene amplification: they reside outside of chromosomes and exhibit elevated copy-number, gene expression, and chromatin accessibility as compared to chromosomal amplifications. New evidence has underscored the importance of ecDNA dynamics and heterogeneity in driving tumor progression: first, ecDNAs asymmetrically segregate during mitosis, leading to accelerated copy-number gains and rapid adaptation to stressful conditions. Second, several varieties of ecDNAs can exist in single cells where they form cooperative, intermolecular “hubs”. Despite this appreciation, these features of ecDNA have remained elusive to study due to a scarcity of tools for profiling their vast heterogeneity and stochastic evolutionary dynamics. In this project, I will develop the requisite computational tools for profiling ecDNA variability and evolution in cancer and use these tools to more thoroughly investigate how ecDNA heterogeneity is created, maintained, and leveraged in response to targeted therapies. First, I will build on breakthroughs in long-read sequencing to develop tools that enable unbiased multi-omic profiling of ecDNA variability across biological conditions (Aim 1). Second, I will leverage new molecular techniques to infer the phylodynamic properties of ecDNA lineages and learn the molecular fitness landscape of ecDNA (Aim 2). Third, I will explore the co-evolutionary principles of ecDNA by combining evolutionary modeling and CRISPR- based screens (Aim 3). Together, these studies will illuminate properties of ecDNA evolution, nominate new therapeutic strategies, and provide innovative computational tools for the greater scientific community. This work will be performed in the excellent training environment of Stanford University under the mentorship of Dr. Howard Chang, an expert in epigenomics and ecDNA. An advisory committee of leaders in the fields of ecDNA, cancer biology, bioinformatics, and tumor evolution will provide additional expertise and mentorship. The first half of each aim will be completed predominantly during the K99 phase of the award, providing a solid foundation for the aims in the R00 phase and eventually an independent R01 application.

NIH Research Projects · FY 2025 · 2023-12

PROJECT SUMMARY Every human being is endowed with a finite number of neurons that must endure a lifetime of environmental stressors. This proposal focuses on studying a neuronal stress response and recovery mechanism with relevance to neuronal function and survival. These studies are directly applicable to human disease, aging, and external impacts of neuronal health. Briefly, we have found that neuronal cells respond to heat stress in a non- canonical way, by downregulating translation. This is rapidly recoverable when returned to baseline conditions within a finite window, beyond which point the cells die. We also discovered that stress response factors were being activated during recovery and are important for neuronal cell function. Based on these and other original findings during the predoctoral phase, our central hypothesis is that neurons endure heat stress in part through translational reprogramming to prepare the system for survival in the event that recovery is an option. Testing this hypothesis, we discovered neurons upregulate Hsp70 in order to survive this recovery phase. Intriguingly, upregulation of Hsp70 has been shown to be relevant to a variety of neurodegenerative disorders, including Alzheimer’s disease. The rationale for the proposed research during the F99 phase is that the molecular programs that mediate neuronal function and survival during heat stress are poorly understood and further understanding will provide critical insight into the underlying causes of stressors that impact the human nervous system in health and disease. Given the critical importance of neuronal stress response to human health, the long-range objective of the proposed research is to understand the role of dysfunctional stress response in neurodegeneration and how it contributes to decreased neuronal stress resilience and inability to recover from stress. We will do this through biochemical, molecular, cellular and physiological measurement to characterize stress response in neuronal cells and molecular mechanisms essential for recovery. Specifically: Aim 1. To investigate the mechanism of stress induced translation regulation and recovery, to test our hypothesis that heat stress induced translation reprogramming is vital to prepare the system for potential recovery and is a key feature in neuronal health and survival; Aim 2. To study disrupted proteostasis in purkinje neurons of a cerebellar ataxia mouse model, to test my hypothesis that mutations resulting in the loss of RREB1 alters protein degradation, which may participate in the observed neurodegeneration and make cells more susceptible to cellular stress. The completed predoctoral and proposed F99 studies create a rigorous program for studying neuronal stress response that will be applied to the proposed K00 studies. The proposed K00 studies will identify factors that lead to more rapid onset or progressive neurodegenerative disease, as well as factors that disallow neuronal recovery from stress. Findings from the K00 hold promise for generating therapeutic targets to extend cognitive well-span and promote neuronal recovery from stress.

NIH Research Projects · FY 2025 · 2023-09

Project Summary Membrane proteins regulate the cellular processes by which all organisms survive. Due to this crucial role, membrane proteins are 60% of drug targets. However, improvements to drug design are often impeded by open questions about their mechanisms. A fundamental function of membrane proteins is to transduce information across the membrane by encoding the presence of stimuli in their conformation. Therefore, knowledge of their conformations is required for the missing mechanistic understanding. However, high- resolution structural methods are often limited to individual domains and/or non-native conditions. In contrast, fluorescence-based single-molecule methods are amenable to physiological environments, yet can lack the spatial or temporal resolution required for key conformational changes. Our laboratory recently introduced new methods to improve the temporal resolution of single-molecule spectroscopy and, in the proposed work, will improve the spatial resolution. Investigations into transmembrane behaviors require the full-length protein structure, and thus its native membrane environment. Therefore, we have also developed robust protocols to solubilize membrane proteins from bacteria, plants, and mammals within discoidal lipid bilayers, known as nanodiscs. In initial studies, we used single-molecule spectroscopy and nanodiscs to reveal ligand-induced transmembrane conformational changes for two important receptors, the mammalian epidermal growth factor and the bacterial sugar chemoreceptor Tar. We are now primed to follow the propagation of ligand-induced conformational changes through the receptors and how these changes are controlled by the complex composition and organization of the plasma membrane. Altogether, this NIGMS MIRA application seeks to merge two of my laboratory's primary interests: (1) Developing and applying advanced single-molecule methods for molecular-level insight into protein machinery; and (2) Isolating and interrogating full-length membrane proteins in a near native environment using nanodiscs. Through this combination, we open a window into transmembrane conformational changes and the role of these conformations in cellular processes. Our contributions will impact fields ranging from single-molecule biophysics to cancer biology to microbial signaling.

NIH Research Projects · FY 2026 · 2023-09

The endometrium -- the innermost mucosal lining of the uterus that provides the site of embryo implantation- is crucial for our species preservation and alterations on its function underlay infertility and disease. Developments in single-cell and spatial technologies have illuminated previously unknown subtypes of cells and their spatial arrangements in the endometrium, providing valuable insights into the signalling pathways involved in the different endometrial compartments and hinting at their roles in determining cell specification in the luminal or basal zones. While inferences can be made from these static pictures about physiological functions ranging from blastocyst implantation and invasion to heavy menstrual bleeding, without tractable in vitro models that capture the endometrium's dynamic spatial interactions, mechanistic hypotheses about endometrial function remain challenging to test. Here, we develop in vitro models of the endometrium by combining tissue-level spatial mapping approaches with in vitro tissue engineering and microfluidic approaches. We will refine existing approaches and develop/test new models, prioritizing robustness and reproducibility, and allowing dissemination and implementation by the broader community, as follows Aim 1: Design a robust and scalable microphysiological systems (MPS) model that replicates the spatial niches of the human endometrium in the early-to-mid secretory phase. We we will use a co-culture of epithelia and stroma in a microfluidic device, in which organoids undergo morphogenesis in a special hydrogel. We will inform the media composition and other metrics by querying the in vivo cellular atlases. Aim 2: Quantify the interplay between cell origin (“nature”) and spatial environment (“nurture”) in defining epithelial cell identity in the in vitro models built in Aim 1. We will use single-cell and spatial transcriptomics technologies to profile the devices, and we will use clonal tracing to map the origin of a cell type. By integrating vivo/in vitro datasets, we will conduct a systematic investigation into the various parameters that characterize the cell source (such as luminal / basalis), as well as the magnitudes of gradients in diffusible signaling molecules and nutrients. Aim 3: Investigate the functional consequences of introducing endothelial cells into the in vitro model from Aim 1, using a simple “monolayer” protocol. We will focus on the cell state transitions that are modulated by endothelial-stromal-epithelial crosstalk during decidualization, and use the model to investigate immune cell trafficking. Our ultimate goal is to obtain a systems biology view of the tissue, which will allow us to inform the development of new treatments for endometrial-related disorders. Our tools and knowledge have potential to bring preclinical models of the endometrium more firmly into the realm of humanised research. This work has significant implications for both academic and industrial research, leading towards a personalised medicine approach for gynaecology.

NIH Research Projects · FY 2025 · 2023-09

ABSTRACT – OVERALL The primary focus of the MIT/DFCI Center for Systems Biology of Glioblastoma is to understand the intersections between neurons, immune cells, and tumor cells in this deadly tumor. The lack of response to immunotherapy strategies despite prominent infiltrates of immune cells in many GBM highlights the immuno- suppressive nature of the GBM microenvironment and the importance of more clearly understanding the dynamic interactions at the tumor/immune interface. Similarly, interactions between tumor cells and neural cells in the tumor microenvironment have emerged as driving forces in tumor progression and invasion, with electrical signals from neurons providing growth and migration stimuli to tumor cells, while tumor cells lead to aberrant electrical signaling in local neurons. The central hypothesis of this proposal is that developing a systems-level understanding of the dynamic interactions between tumor cells, neurons and immune cells will provide unprecedented insights into glioma tumor biology and foster development of novel therapeutic strategies to abrogate tumor invasion, enhance the efficacy of cytotoxic therapies, and increase clearance of tumor burden by the innate and adaptive immune system. The planned analyses will enable building an integrated computational model of tumor-neural-immune interactions for GBM tumors. The model will be based on a foundation of in vitro, in vivo, and ex vivo model systems, and then validated in dozens of human patients. Image-registered biopsies from different tumor regions within each patient will be analyzed to test predictions of this model against the ‘ground truth’ of human tumors. The ultimate goal of the MIT/DFCI Center for Systems Biology of Glioblastoma is to improve patient care by using systems biology and computational modeling to identify therapeutic strategies to specifically disrupt critical tumor cell – microenvironment interactions.

NIH Research Projects · FY 2025 · 2023-09

Project Summary/Abstract With our expected lifespans increasing, the rapidly expanding aging population is bringing an increased prevalence of dementia, including Alzheimer’s disease (AD) and vascular cognitive impairment (VCI). However, there are still no neuroprotective medicines for treating patients with these conditions. AD and VCI are the most common types of dementia and impose a huge socioeconomic burden as well as devastating impacts on the lives of patients and their caregivers. Both of these forms of dementia are characterized by deterioration of the neurovascular unit that forms the blood-brain barrier (BBB), which in many cases even precedes the onset of cognitive deficits. Unfortunately, however, the mechanisms of BBB deterioration in AD and VCI are unclear. As a result, there are no therapies to protect the BBB. In my thesis work, I have established that the prostaglandin degrading enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is pathologically elevated in both human AD and VCI in human patients. I have also shown that the enzymatic activity of 15-PGDH in the brain is increased in the 5xFAD mouse model of AD, as well as normal aging. Importantly, I have established that pharmacologic and genetic inhibition of 15-PGDH in 5xFAD mice shows robust protection against BBB deterioration and other AD-related pathology, including cognitive deficits, impaired neurogenesis, and axon degeneration, independently of amyloid β pathology. I have also found that of the prostaglandins, prostaglandin D2 (PGD2) is most prominently elevated in the brain by 15-PGDH inhibition in 5xFAD mice. Therefore, I hypothesize that PGD2 is responsible for 15-PGDH inhibition-mediated protection of the BBB, and that this is related to improved endothelial cell barrier function. During the F99 portion of my proposal, I will evaluate whether 15-PGDH inhibition also protects from BBB deterioration in the high fat diet mouse model of VCI. I will utilize innovative 2-photon microscopy in vivo imaging and electron microscopy to determine the trajectory of BBB deterioration, as well as test the protective efficacy of 15-PGDH inhibition. I will also determine whether PGD2 mediates the protective effect both in vivo and in vitro, as previous literature suggests a role of PGD2 in increasing endothelial cell barrier function. During the K00 phase, I will expand my BBB research by investigating the interaction between perivascular macrophages (PVMs) and endothelial cells in the brain in Dr. Chenghua Gu’s laboratory. I will utilize an innovative cre-recombinase system to specifically target PVMs in the brain and investigate altered glucose metabolism in PVMs and transcriptomic profiling in both PVMs and endothelial cells, as a function of AD-related risk factors. Then, I will test how this altered metabolism in PVMs interacts with endothelial cells to initiate BBB deterioration. Successful completion of this study will provide new perspectives of how the BBB deteriorates with aging and dementia-related pathology, which will enable the discovery and development of new neuroprotective approaches for patients suffering from AD and VCI.

NIH Research Projects · FY 2025 · 2023-09

The brain of an awake, mature human (and other mammals) consciously and subconsciously “knows” the body’s configuration in 3D space on a moment-by-moment basis. This is often referred to as the “body schema” representation in the brain. Body schema is critical for self- awareness and for motor control. For example, we can effortlessly use our hand to touch our nose or swat a mosquito that has just landed on the back of the neck, regardless of the starting positions of the hand, because we have an intimate knowledge of the body’s position in 3D space at any given moment. Yet how the brain generates body schema representation remains largely unknown. Here we propose to systematically examining the neural circuits and mechanisms that compute the 3D positions of all body segments (individually or combined) with egocentric reference frames in the mouse brain. This is achieved by (1) developing an algorithm, BodySchemeJ, that generates fully parameterized moment-to-moment description of full body configurations in freely moving mice; (2) performing large-scale multi-electrode array recordings from multiple brain areas with concurrent tracking of body configurations in free- moving behaviors; (3) computational analysis of neural data and testing the hierarchical body schema representation hypothesis; and (4) delineating the presynaptic inputs and output targets of identified body schema cells. Finally, since body schema representation deficits are observed in many neurological diseases, we will test how body schema is abnormally encoded in a mouse model of autism. The pioneering knowledge gained from this research will help to establish a new conceptual framework to advance our understandings of quintessential neurobiological processes such as self/body awareness, localizing sensations to body parts in 3D space, action selection and motor control.

NIH Research Projects · FY 2025 · 2023-09

SUMMARY This ambitious proposal will establish an integrated experimental-computational platform to create the first comprehensive brain-wide mesoscale connectivity map in a non-human primate, the common marmoset (Callithrix jacchus),. It will do so by tracing axonal projections of RNA barcode-identified neurons brain-wide in the marmoset, utilizing a sequencing-based imaging method that also permits simultaneous transcriptomic cell typing of the identified neurons. This will help bridge the gap between brain-wide mesoscale connectivity data available for the mouse from a decade of mapping efforts using modern techniques and the absence of comparable data in humans and NHP. The proposal will bring together new viral barcode-based approaches with established tracer-injection based methods to collect an unprecedented data set which will permit comprehensive mapping of region-to-region axonal projections in the marmoset brain with cell-type specificity, apply this data to scientific questions regarding how brain connectivity differs between primates and rodents, address the relation between MRI-derived measures to ground-truth connectivity, and utilize web-based tools to engage the research community in annotating and using the resulting data. A diverse team of world-leading experts and pioneers in the problem domain who are already collaborating, spanning diverse institutions and disciplines, and equipped with outstanding facilities and resources, will come together to assemble the enabling platform. There are five specific aims to achieve stated goals. Aim 1 is to establish data acquisition platform for mesoscale projection mapping in marmoset using barcoded + fluorescent AAV/AAV-retro. Aim 2 is to develop brain-wide single-axon projection maps using systemic injections with simultaneous transcriptomic cell-typing. Aim 3 is to develop data management and analysis pipelines. Aim 4 is to perform comparative studies of the mesoscale connectivity in marmoset and mouse, comparing MRI-based connectivity with ground truth. Aim 5 is to develop platforms for data dissemination, community engagement through web portal, collaborative proofreading and annotations. Such a data set will have profound scientific significance, comparable to the first whole genome map of a primate. As genomes encode the blueprint for understanding an organism’s phenotype, so does the architecture of brain circuitry contain the blueprint for explaining an animal’s behavior and holds the key to many open questions.

NIH Research Projects · FY 2025 · 2023-09

SUMMARY Water is the medium in which biochemistry operates. The amount of water inside a cell defines the concentration of all biomolecules and the degree of molecular crowding. Consequently, water content is predicted to influence virtually all cell functions from immune surveillance to stem cell renewal in vivo. There are also severe genetic diseases caused by mutated water channels on cell membranes. Despite the fundamental role of cellular water content in cell physiology and diseases, there are no direct and non-invasive methods that measure how much water a single cell or a cluster of cells, such as an organoid, contains. This lack of water content measurements has prevented us from understanding cell physiology in normal and disease states, and from discovering drugs that can modulate cellular water content in diseases where water content is perturbed. Here, we propose to develop a new method that will directly, non-invasively and precisely measure the absolute and fractional (v/v) water content of a single cell or an organoid. This method will enable the long-term monitoring of the same cell or organoid during growth, differentiation or drug exposure. To achieve this, we will integrate total volume and dry volume measurements obtained using two independent approaches that we have previously developed. Our proposed method will enable water content measurements in complex and biomedically relevant samples, such as immune cells and organoids, with high throughput and in conjunction with detection of fluorescent markers. This method will enable novel basic and biomedical research that will increase our understanding of water content regulation in health and disease, and provide a new platform for drug discovery.

CIHR Grants and Awards · FY 202526 · 2023-09

Advanced high-grade serous ovarian cancer (HGSOC) is the leading cause of gynecological cancer death in the developed world, with 5-year survival rates of only 25-30% due to late-stage diagnosis and the limitations of chemotherapies; this survival rate has remained unimproved since the 1980s. The existing standard of care consists of tumor removal surgery followed by chemotherapy, which has systemic toxicity affecting both tumors and healthy organs. Patients often develop resistance to chemotherapies, leading to shortened disease-free intervals, increased risk of metastasis, and worsened prognosis. Given that many types of ovarian cancer (OC) are caused by genetic mutations, small interfering ribonucleic acid therapies (siRNAs) have emerged as promising solutions for OC by targeting the underlying mutations with high specificity and high efficiency. Using siRNAs to target multiple tumor proliferation or chemotherapy resistance pathways thus presents an exciting opportunity to reduce tumor viability without the toxicity of systemic chemotherapy combinations. However, bare siRNA is unstable and causes immune reactions, thus requiring a well-engineered delivery system. We propose the development of layer-by-layer nanoparticles (LbL NPs) for siRNA delivery to treat OC, in which a charged NP core is layered with alternating charged polymer layers via electrostatic attraction. By incorporating the charged polymers, LbL NPs better avoid liver clearance than existing NPs and selectively target tumors without affecting healthy cells. This is particularly suitable for delivering combination therapies for HGSOC, such as an siRNA-small molecule drug combination or multiple siRNAs. Ultimately, the development of an optimized drug delivery platform for nucleic acid delivery will be broadly impactful for therapeutic delivery in treating additional cancers and genetic diseases, reducing side effects, and significantly improving patient experience and outcomes. Keywords: DRUG DELIVERY; NANOMEDICINE; NUCLEIC ACID THERAPEUTICS; BIOMATERIALS; MATERIALS SCIENCE; NANOPARTICLES; RNA INTERFERENCE (RNAI) THERAPY; CANCER THERAPY; OVARIAN CANCER; SYSTEMS BIOLOGY