Stanford University

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY Genome-wide association studies have now discovered tens of thousands of noncoding variants associated with human diseases and traits. It has proven challenging to interpret these associations. A majority of causal variants lie in the noncoding genome and appear to affect DNA cis-regulatory elements, which control the logic of gene expression and could point us to new cell types, genes, and pathways for disease. However, we have lacked the tools needed to systematically characterize how these cis-regulatory variants and elements impact genome function and phenotype. Our team at Stanford University has now developed innovative single-cell, CRISPR mapping, and computational technologies that will enable identifying and functionally characterizing many thousands of elements and variants directly in the human genome. These tools include single-cell ATAC-seq to identify candidate elements in cells and tissues; sensitive CRISPR tiling methods to connect thousands of elements and variants to effects on gene expression and cellular phenotypes; and the ABC and BPNet models to predict how disease variants regulate gene expression. Together, these technologies suggest a new strategy to systematically connect DNA variants and elements to function and phenotype. Here we will apply these new technologies in collaboration with the NHGRI Impact of Genomic Variation on Function Consortium. We will use four cardiovascular cell types derived from human pluripotent stem cells as model systems. First, we will leverage single-cell maps of cardiac differentiation and development to select elements and risk variants for adult and children’s heart diseases likely to control cardiovascular cell function. Second, we will apply single-cell CRISPR tools to measure the effects of thousands of unbiased elements and variants on gene expression, and connect prioritized disease variants to target genes, cellular phenotypes, and tissue phenotypes. Third, we will leverage these experimental datasets to calibrate and refine computational models to build a variant-element-phenotype catalog across many human cell types and diseases. Fourth, we will enable future studies by sharing data, protocols, and software, and by conducting systematic evaluations of CRISPR technologies and computational models to connect variants to phenotypes. Together, these studies will advance our understanding of how DNA variants and elements impact genome function and demonstrate a novel strategy to leverage high-throughput genomic tools to understand biological mechanisms of human diseases.

NIH Research Projects · FY 2025 · 2021-09

Electronic cigarettes (e-cigarettes, e-cigs, vapes, ENDS) are the most widely used tobacco product among adolescents, aged 12-18. Despite clear connections between e-cigarette use, nicotine addiction, and physical and mental health outcomes, adolescents continue to harbor misperceptions about e-cigarettes, perpetuated by exposure to marketing and flavors, and a lack of understanding about their harms and addictive properties. While schools have historically provided a key venue in which to implement tobacco prevention programs, most school-based tobacco prevention programs focus on conventional cigarette smoking only, have had mixed results, and have several gaps in their educational content on e-cigarettes. Further, studies have rarely determined if there are specific groups for whom e-cigarette prevention and cessation programs are, and are not, helping. Using a community-based participatory research approach, we developed the “Be Vape-Free” Curriculum, a free 5-session school-based education, prevention, and reduction (de-escalation) program for middle and high school students. This curriculum is part of and includes the most effective components of the Tobacco Prevention Toolkit, a free online comprehensive tobacco prevention program used by thousands of schools and educators across the United States, having reached more than 1.7 million middle and high schools students. Aligning with the NIH Stage Model for Behavioral Intervention Development, we have addressed 3 of the 6 stages needed to adequately develop, evaluate, refine, and fully implement and disseminate our Be Vape-Free Curriculum. Thus, the Specific Aims of this project are to: (1) Determine whether the Be Vape-Free Curriculum is effective in increasing middle and high school students’ knowledge of e-cigarettes and resistance to using, and decreasing their positive attitudes towards and intentions to use e- cigarettes; (2) Determine whether the Be Vape-Free Curriculum is effective in changing middle and high school students’ actual use of e-cigarettes (including preventing initiation, continuation, escalation; encouraging decreased use and cessation; and use of e-cigarettes with other tobacco and marijuana products); and (3) Examine the heterogenous treatment effects (HTE) of the intervention, identifying both those who benefit the most and those who do not benefit from the curriculum. We will employ a cluster-randomized trial, stepped- wedge design, with 60 middle and 60 high schools in California (n=10,800 students). Schools will be randomized to either the treatment (Be Vape-Free Curriculum) or delay-in-treatment arm (i.e., standard health education first year and Be Vape-Free curriculum in second year), with all students followed for another 12 months. The timing of this proposed research is extremely important as we have outstanding momentum, have established an extensive team of school and community partners and stakeholders, have numerous schools already interested in using the Be Vape-Free Curriculum, and have garnered tremendous support from the California Department of Education and schools.

NIH Research Projects · FY 2025 · 2021-09

ABSTRACT A central challenge in human genomics is to interpret the regulatory functions of the noncoding genome, and to identify and interpret variants with regulatory functions. In this project we plan to leverage recent advances in experimental functional genomics (including single cell methods and high throughput perturbation methods) alongside recent progress in deep learning models of gene regulation, to make fundamental progress on these problems. We have assembled a team of investigators with diverse and complementary expertise – in deep learning, single-cell genomics, cellular QTLs and GWAS, and high throughput validations – to build, test, and implement predictive models for interpreting disease associations. Specifically, we aim to (1) Develop interpretable base-resolution deep-learning models for regulatory sequences; (2) Predict and validate cell type- specific effects of regulatory variants on molecular phenotypes and disease; (3) Collaborate with the IGVF Consortium to build nucleotide-level regulatory maps. Our ultimate goal in this project will be to create a nucleotide-resolution cis-regulatory map of the human genome to connect disease variants to functions and phenotypes, in diverse cell types, states, and spatial contexts.

NIH Research Projects · FY 2025 · 2021-09

Our goal is to develop towards an IND a novel class of small molecule inhibitors of phosphoinositide (PI) 4 kinase IIIb (PI4KIIIb) with potent dual activity against both SARS-CoV-2 and the excess cytokine release associated with COVID-19 disease. Entry of SARS-CoV has been shown to depend on PI4KIIIb, and strong inhibition of entry was achieved following knockdown of PI4KIIIb via siRNA, and SARS-CoV-2 is believed to enter cells via a similar mechanism. This likely reflects a requirement for enrichment of phosphorylated isoforms of PI, such as PI-4, in the lipid organelle required for viral fusion upon entry. We have developed potent and specific small molecule inhibitors of PI4KIIIb, and optimized them for high oral bioavailability. Our lead inhibitor, STF-1019 has nanomolar efficacy against enteroviruses (EV) which are also dependent on PI4KIIIb, and is the only molecule to have demonstrated in vivo efficacy in the animal model of EV-71, and without toxicity. We have now shown that STF-1019’s EC50 against SARS-CoV-2 is 210 nM, with a CC50 of >100 microM, reflecting a therapeutic index (TI) of ~500. Finally, likely due to PI4KIIIb’s role in Golgi-mediated secretion, we have also recently shown that STF-1019 can potently inhibit the LPS-induced secretion of IL-6 from human PBMC. STF-1019’s metabolic stability, however, is suboptimal, requiring co-administration with an inhibitor (i.e. ritonavir) of its metabolism by CYP3A4 for optimal sustained tissue exposure. We hypothesize that: 1) STF-1019’s SAR and major metabolites indicates that our lead PI4KIIIb inhibitor can be further optimized to increase its activity and metabolic stability to achieve an optimal exposure profile; 2) modifications that further increase PI4KIIIb inhibition can provide a buffer for modifications that may increase metabolic stability at the expense of efficacy; 3) the optimized inhibitor will inhibit SARS-CoV-2 in vitro, and in vivo; 4) the optimized inhibitor will have a high barrier to the development of resistance; 5) because of its orthogonal mechanism of action, our PI4KIIIb inhibitor can be used in combination with other agents to maximize efficacy; 6) STF-1019’s inhibition of IL-6 reflects an ability to modulate the release of other cytokines, and this non- antiviral activity can be of great additional benefit in addressing the cytokine storm associated with severe COVID-19 infection; 7) determination of key pharmacokinetic, in vitro ADME-Tox parameters and initial preclinical in vivo toxicity assessment of our optimized lead can advance its translational development, and form the basis of a future IND package. We propose the test these hypotheses by: 1) Identifying the STF-1019 analog (and back-up compound) with greatest in vivo trough:EC90 ratios; 2) determining the in vivo activity of the optimized PI4KIIIb inhibitors against SARS-CoV-2 and their effect on cytokine production; 3) determining the relative barrier to resistance, and potential for synergy with other agents; and 4) nominating a PI4KIIIb inhibitor IND candidate by subjecting the optimized lead to initial in vitro ADME-tox and IND-enabling preclinical animal safety studies.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY/ABSTRACT To combat the US opioid epidemic, massive efforts have been focused on expanding access to medications for opioid use disorder (MOUD). While there are indications of improved reach and adoption, an ironic gap persists—only about one-third of specialty addiction treatment organizations offer MOUD. This proposal, Stagewise Implementation-To-Target – Medications for Addiction Treatment (SITT-MAT), not only advances the science of implementation, but advances our empirical understanding of how to best respond to a substance-related epidemic. This is a revised application in response to PAR-19-274: Dissemination and Implementation Research in Health” and aligned with the National Institute on Drug Abuse Strategic Plan “ensuring the effective translation and implementation of scientific research findings to improve the prevention and treatment of substance use disorders.” Within an adaptive implementation strategy trial design, using an innovative stagewise implementation-to-target approach, 72 community addiction treatment programs will participate. The stagewise implementation-to-target, stepped “care” type approach, deploys increasingly intensive strategies only if needed. The sequence of implementation strategies are: 1) Enhanced Monitoring and Feedback; 2) “NIATx/MAT Academy,” a 2-day workshop on MOUD and NIATx (Network for Improvement of Addiction Treatment)—an evidence-based process improvement strategy; 3) Randomization to either NIATx Internal Facilitation or NIATx External Facilitation; and, 4) If outcome targets are not achieved in the NIATx Internal Facilitation arm, assignment to NIATx External Facilitation. We evaluate the relative impact of 5 possible paths of implementation strategies on RE-AIM target outcomes: reach, effectiveness, adoption, and implementation quality. Maintenance of outcomes is evaluated for sustainment. Measures of multi-level contextual determinants are rigorous and systematic. In opening the “black box” of implementation strategies, we detail procedures, fidelity, participation and costs using standardized measures. The collective expertise of the research team, the established partnership with a state system of care and addiction treatment organizations, forecasts successful project execution. As we submit this application, the US is still coping with the COVID19 pandemic. The global health situation may rebound to relative normalcy in the months from December 2020. Meanwhile, the CDC, SAMHSA, CMS, and the State of Washington Health Care Authority have all made accommodations to continue the initiation and management of MOUD for patients receiving addiction treatment services. Therefore, even if the current quarantine restrictions persist, we do not anticipate major modifications to the study protocol, except in-person implementation support activities would be transitioned to videoconference formats. During this pandemic, it is even more critical for patients with opioid use disorders to access pharmacological interventions that require minimal physical contact with providers. This study has the potential to shift the paradigm in public health and implementation research.

NIH Research Projects · FY 2025 · 2021-09

Gold-standard quantitative imaging studies are often difficult to implement, limited by financial and logistical issues, or expose the patient to unnecessary risks. Deep learning has shown great promise in recent years for many medical applications; one use is to synthesize improved images. Such image transformation methods offer the potential to improve the quality, value, and accessibility of medical imaging. The goal of this project is to develop deep convolutional neural network approaches to FDG PET imaging, the most commonly performed clinical brain-focused PET study in the USA. Using simultaneous PET/MRI, we will train networks to produce diagnostic PET images from ultra-low dose PET and MR images. We will explore the three reimbursed clinical indications for this imaging modality (tumor recurrence, dementia, and epilepsy) using both quantitative metrics and repeated reader studies to assess equivalence and evaluate model generalization related to simultaneity, scanner type, age, sex, and disease prevalence. Next, we will evaluate whether we can move beyond ultra-low dose and remove the radiation dose altogether, synthesizing FDG brain PET images from MR inputs only, relying on the information in multi-modal functional MRI. Finally, we will assess whether we can use deep networks to combine imaging and non-imaging data such as clinical and genetic information to further improve image transformation and predict future images and image-based biomarkers. Significantly reducing or even eliminating the need for radiation to produce brain FDG PET images would be truly transformative while the ability to predict the future will enable personalized radiology and enhance our ability to perform clinical trials.

NIH Research Projects · FY 2025 · 2021-09

PROJECT ABSTRACT Appropriate functioning of the nervous system is tremendously important to our quality of life. The nerves within our body branch from the spinal column and can extend up to one meter away. Peripheral nerve injuries are a common occurrence and can readily heal in most cases. However, end-organs beyond ~18 inches away from the nerve body, are highly susceptible to poor reinnervation outcomes and long-term detriments. A main reason for this being the slow growth rate of the axons (1-2mm/day or 1 inch/month). Some tissues, such as skeletal muscle, can remain ready for reinnervation for 18-24 months. However, current intervention strategies are limited, and most injuries are left to resolve naturally. This application aims to develop a strategy capable of promoting quicker neurite outgrowth and preserving the muscle in a healthy state until reinnervation can occur. This application focuses on tissue-nanotransfection mediated delivery of non-viral gene cargo to induce molecular changes and to reprogram cells. The ability to deliver cargos locally to the site of injury opens avenues beyond nerve injury for gene and cell-based therapeutics with a precision medicine approach. Completion of the F99 phase sets a strong intellectual, technical and professional foundation for the postdoctoral (K00) phase of this award. During the K00 phase, training in understanding how cell and gene- based therapies impact neurodevelopmental and neurodegenerative conditions will develop knowledge, expertise, and skills essential to becoming an independent investigator.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY/ABSTRACT Alzheimer’s disease (AD) is a common, progressive, and ultimately fatal brain disease. Currently approved treatments provide only minimal symptomatic benefits and do not stop the disease from progressing. The field is in dire need of novel drug targets which could lead to disease-modifying therapies. The most common genetic risk factor for AD is the ε4 variant of the apolipoprotein E gene (APOE4). The effect of APOE4 varies greatly between people of African ancestry and people of European ancestry. The current study—Illuminating the APOE Locus with Long-Read Sequencing and Targeted Genomics—will apply a new genome sequencing technology (long-read sequencing) to the study of APOE and several other AD-relevant genes including ABCA7. Long-read sequencing will be performed on DNA from roughly 2000 African-Americans with AD and 2000 healthy older African-American control subjects as well as DNA from roughly 5000 European-American AD patients and 5000 European-American controls. A subset of these patients will also have long-read sequencing of these genes’ RNA derived from white blood cells, fibroblasts, or brain tissue. These analyses will help us understand how local genetic variants near the APOE4 variant can alter the type or amount of the APOE4 protein and how this affects risk of AD. Similar analyses will be done on ABCA7 and another 15-20 targeted genes that will be selected just before sequencing begins and following an up-to-date review of the AD genetics literature. In addition to understanding the local variants regulating a gene and the protein it produces, long-read sequencing will be useful in detecting large, damaging genetic mutations that are easily missed with standard whole-genome sequencing. The results will allow for more specific estimates of AD risk in individuals of diverse ancestral backgrounds and will provide novel targets for drug development.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY/ABSTRACT A comprehensive genome-wide map of DNA regulatory elements and gene expression in human cells is of critical importance for understanding how genomic variation impacts human health and disease. Since regulatory DNA elements are exceptionally cell type-, tissue-, and disease state-specific, a comprehensive catalog of these elements has been difficult to achieve. The overall mission of this IGVF Mapping Center is to create a high- quality, open-access, and single cell-resolution reference map of human regulatory elements and gene expression in immune cells during human development, across organ systems in healthy adults, and in tissues from diverse immune-related diseases. Our Mapping Center will leverage: (i) our recent advances in developing scalable and cost-efficient single-cell epigenome and multi-omic technologies to simultaneously map open chromatin sites, gene expression, intracellular and cell surface proteins, and clonal lineage tracing in each tissue sample, (ii) our prior technical improvements and application of these methods to primary tissues from humans, and (iii) our pre-existing human tissue biobank consisting of samples from more than 500 human individuals, 20 organ systems, and 15 disease conditions, consented for unrestricted access, genomic sequencing, and data sharing. In Specific Aim 1, we will work closely with the IGVF Consortium to establish cross-center plans for data generation, analyses, and effective coordination of sample access and sharing. In Specific Aim 2, we will generate a single-cell multi-omic atlas of immune cell types (and non-immune cells types, as determined with the IGVF) during development in early life, healthy aging, and across human organ systems. In Specific Aim 3, we will generate a single-cell multi-omic atlas in immune cell types from primary tissues in patients with autoimmunity, cancer, neurodegenerative disease, and infection. In Specific Aim 4, we will analyze regulatory sites and gene expression in the context of clonal differentiation trajectories inferred from mitochondrial lineage tracing and develop and maintain an integrated reference map of each datatype and tissue sample for the research community. Our Mapping Center, composed of 7 new investigators with extensive experience in single- cell genomic technologies and human disease analysis, will work closely with the IGVF to share technologies, resources, data, and tissue samples towards the shared goal of developing a comprehensive single-cell atlas of cell types, functional regulatory elements, and gene expression in humans.

NIH Research Projects · FY 2024 · 2021-09

PROJECT SUMMARY/ABSTRACT Meta-analyses critically shape clinical recommendations and policy, but their credibility may be undermined by both within-study biases (e.g., confounding in observational studies) and across-study biases (e.g., filtering by publication bias). These biases can produce meta-analysis estimates that are too large, too small, or in the wrong direction. Scientists, clinicians, and health policymakers are increasingly concerned about these biases given recent empirical evidence that meta-analyses on the same topic can disagree with one another and with the results of systematic replication studies, which are designed to minimize publication bias by having independent investigators repeat published studies. This eroding confidence in the published literature and in meta-analyses represents an epistemic turning point. This proposal develops and empirically validates an innovative, domain-independent statistical framework for quantitatively synthesizing studies subject to within- and across-study biases. Aim 1 will thus develop novel, domain-independent statistical sensitivity analyses will quantify how results of meta-analysis estimates might be shifted by within- and across-study biases, that allow bias-corrected synthesis of studies with these two forms of bias, and that forecast the likely range of results of new studies and the impact of adding them to an existing meta-analysis. The methods will be made broadly accessible via user-friendly websites and R software, and their use will be illustrated in meta-analyses on pediatric obesity. Aim 2 will illustrate the methods' real-world impact and compare their performance to that of existing methods by using the methods to characterize the credibility of Cochrane database meta-analyses. Aim 3 will involve a collaboration with “ManyBabies'', an innovative initiative to conduct conceptual replications of landmark results in developmental psychology. The results of these planned replications will be forecasted using the new methods of Aim 1 as well as existing methods; the forecasts will be compared studies' results after they are conducted, providing real performance benchmarks. Aims 2-3 will also provide online “dashboards” allowing intuitive exploration of the results. The immediate-term goal is to develop methods and software that, unlike existing statistical methods, assess the robustness of a given meta-analysis to the joint effects of within- and across-study biases; that synthesize and compare results of meta-analyses with those of studies subject to less publication bias (e.g., replication studies); and that use potentially biased meta-analyses to plan the optimal design of new studies. The long-term goal is to calibrate confidence in meta-analyses to more swiftly inform scientifically robust conclusions that will improve practice and health policy.

NIH Research Projects · FY 2024 · 2021-09

PROJECT SUMMARY WNT signaling is crucial for embryonic development and adult tissue homeostasis, with aberrant signaling resulting in developmental disorders and disease, including cancer. Although much is known, a deeper mechanistic understanding of this signaling cascade will improve our understanding of cancer formation, progression and metastasis, allowing for the development of more effective therapeutics. WNT/b-catenin signaling is driven by the stabilization of the transcriptional co-activator, b-catenin. In the absence of WNT ligand, a cytosolic destruction complex phosphorylates, ubiquitylates and degrades b-catenin. In the presence of WNT ligand, the WNT receptors, Frizzled and LRP6, and intracellular proteins form an alternative complex called the WNT signalosome. This results in b-catenin accumulation and activation of b-catenin target genes. Recent data demonstrate that upon WNT ligand engagement, the signalosome is endocytosed. Although conflicting data exist within the literature, a consensus is beginning to emerge that clathrin-dependent endocytosis of the signalosome results in signalosome degradation. This training proposal and my thesis project is devoted to elucidating the molecular events and dynamics of signalosome formation, stabilization and endocytosis in normal cells and in cancer, with an emphasis on kinases. In the first half of my graduate training, I utilized a gain-of- function screen of the kinome to identify AAK1 as a negative regulator of WNT signaling. I demonstrated that AAK1 activates a transcription independent negative feedback loop to promote LRP6 internalization, resulting in WNT signaling downregulation. In the course of these studies, we demonstrated that AAK1 promotes the phosphorylation of a clathrin adapter protein, AP2M1, 8-10 hrs post-WNT3A and that AAK1 and AP2M1 interact with the tumor suppressor, WTX. My lab previously discovered the WTX tumor suppressor as a component of the signalosome and b-catenin destruction complex. Therefore, I will define comprehensive WNT3A and WTX- dependent changes to the phosphoproteome by quantitative mass spectrometry and test whether WTX regulates signalosome endocytosis via AAK1. Additionally, CSNK1g is known to regulate phosphorylation of LRP6, an essential step for signalosome formation. CSNK1g has 3 isoforms, CSNK1g1/2/3, all identified as understudied kinases. My preliminary data suggest each isoform functions differently to activate WNT signaling and promote LRP6 internalization. A main focus for the remainder of my graduate work will be to functionally characterize the role of each CSNK1g isoform in regulating WNT signaling, define comprehensive protein-protein interaction networks and evaluate isoform specific changes to the WNT-driven phosphoproteome. Because this work is descriptive in nature, I expect it to be submitted for publication in 14 months. To summarize, the precise role of endocytosis in WNT signaling remains unclear, with numerous questions surrounding the mechanism(s) and components of endocytosis and its effects on signaling. This work, and my future postdoctoral work, will provide me training in and experience in the mechanisms of WNT signaling and feedback attenuation.

NIH Research Projects · FY 2025 · 2021-09

Project Summary/Abstract The goals of the IGVF Data Administrative and Coordinating Center (DACC) are to support the IGVF Consortium by defining and establishing a strategy that connects all participants to the project’s science. By creating avenues of access that distribute these data to the greater biological research community, the DACC provides a critical connection between scientific producers and consumers. The IGVF Consortium brings together laboratories that generate complex data types via novel experimental assays, often focusing at the single-cell level of gene expression. This work is extended and regularized by laboratories that integrate these unique data using computational analyses to discover the associations and networks between human variation, chromosomal elements and molecular phenotypes for the purpose of elucidating their complex relationship in human cells and tissues. The DACC’s participation enhances the data created by the consortium through the creation of structured procedures for the verification and validation of all submitted data and providing processes for the documentation of metadata that describe each biological sample and assay method. To facilitate access to all the data created, the DACC will construct a state of the art data warehouse, design and develop robust software to enable data submission, and harden unified data processing pipelines. All experimental and computational results will be made available via the IGVF Portal, developed by the DACC. The Portal will integrate these data resources and provide enhanced search and browsing capabilities, along with powerful web services. The DACC will develop tools for semantically-enhanced graph-based searches of experiment metadata, individual genomic elements, variation and phenotype, and will implement methods to distribute these results in matrices suitable for machine learning. Beyond computational infrastructure to house and distribute consortium data, the DACC will also function as the administrative hub of the IGVF. Consortium science thrives on clear and forthright communication between its component parts, and it is the DACC’s responsibility to manage this relationship. This effort will be facilitated by management of consortium working groups, organization of scientific results and publications, and providing regular reporting and feedback to the Steering committee. To fully support the community, the DACC will act as a service organization, allowing biomedical research to take full advantage of the results from the IGVF. To this end, the DACC will organize and host consortium- focused and user-focused meetings, and will provide documentation via many media including written documentation, video tutorials, webinars, and meeting presentations. The various component projects of the IGVF (DACC, mapping, systematic characterization, genetic network regulation, modeling of genomic variation centers and groups) will be tightly woven together to create the IGVF Consortium.

NIH Research Projects · FY 2025 · 2021-09

Project Summary / Abstract Absence seizures occur in pediatric generalized epilepsy and involve excessive synchrony of the thalamocortical neural network. An unexplored possibility is that aberrant activity-dependent myelination contributes to absence seizure progression by promoting network synchrony. A recent discovery is that neuronal activity drives myelin plasticity (changes in myelin structure) in vivo. Myelination, in turn, is a critical determinant of neuronal network synchrony and function. Activity-regulated formation of new myelin requires Brain Derived Neurotrophic Factor (BDNF) signaling through its receptor, TrkB, on oligodendrocyte precursor cells (OPCs). Pathological seizure activity may also induce changes in myelin structure, which in turn could contribute to network dysfunction. This proposal investigates the relationship between absence seizures and activity-dependent myelin plasticity. Preliminary data indicate that absence seizures are associated with abnormally increased myelination in two rodent models with spontaneous absence seizures: Wag/Rij rats (a widely used inbred rat strain) and Scn8a+/mut mice. These mice have a loss of function mutation in SCN8A, similar to children with generalized epilepsy due to loss of function in SCN8A. Both models exhibit increased OPCs and myelin sheath thickness in the anterior corpus callosum. Preventing seizures with ethosuximide prevented the increased callosal myelination, indicating that seizures are required. My hypothesis is that seizure-induced aberrant myelination facilitates excessive synchrony and contributes to seizure burden. In Aim 1, the nature and extent of abnormal myelination in the thalamocortical network will be investigated using magnetization transfer and diffusion-based magnetic resonance imaging of Scn8a+/mut mice. Measurements will be validated by the gold standard method of quantifying myelination, electron microscopy. Aim 2 will determine the role of activity-dependent myelination in thalamocortical hyper-synchrony underlying absence seizures. This will be accomplished by conditionally deleting the TrkB receptor from OPCs in Scn8a+/mut mice specifically during the period of seizure initiation and progression, using a novel mouse line (Scn8a+/mut; trkB fl/fl; PDGFR::Cre). Indices of network synchrony will be measured in acute thalamic slices from Scn8a+/mut mice with or without normal activity-dependent myelination. Aim 3 will determine whether myelin plasticity contributes to seizure burden, by genetically blocking activity-dependent myelination as in Aim 2, and quantifying seizures with EEG. Thus, the proposed studies will use innovative methods to elucidate a novel and potentially paradigm-shifting pathological mechanism in epilepsy, with implications for new therapeutic strategies.

NIH Research Projects · FY 2024 · 2021-09

Project Summary / Abstract (30 line maximum) This research, in response to the PAR-19-253, “Focused Technology Research andDevelopment,” aims to pioneer new advances in biological optical microscopy. Methods such as the development of fluorescent proteins, single molecule fluorescence detection, single molecule fluorescence resonance energy transfer (smFRET) and super-resolution microscopy enabled molecular level study of in vitro and live cells of increasing complexity. The single molecule methods allowed researchers to observe kinetic pathways and transient states unobservable with bulk methods. Despite recent advances, the existing optical probes have limitations. Fluorescent proteins are comparable in size to the proteins they label and photobleach quickly. In situ labeling of cytosol proteins is possible, but in vitro labeling methods are much preferred and there are no reliable methods to introduce these proteins into cytosol of cells. This research will address these grand challenges by fundamentally expanding the toolbox of optical microscopy. Aim 1 will develop new methods to introduce proteins labeled in vitro with organic dyes directly into the cytosol of cells and the insertion of dye-labeled membrane proteins into cell membranes, thereby expanding the application of optical probes to new biological systems. These methods will be used to insert up-converting nanoparticle (UCNP) probes into live cells to allow the long- term tracking of specific individual proteins from minutes to months with nanometer spatial resolution. This technology will also allow the controllable transfection of cells with multiple genes. Aim 2 will fundamentally improve the temporal resolution of smFRET to ≤ 100𝜇𝑠 and develop smFRET methods that can span across cell membranes. Aim 3 will extend biological optical microscopy to access the temporal and spatial scales of molecular motion. Here, UCNPs will be used to measure the continuous transport of cargos by dynein in DRG neurons capable of resolving single molecular steps with one millisecond time resolution over a distance of 900 𝜇𝑚. Using plasmonic optical probes, this work aims to achieve ~ 100 𝑛𝑠 time resolution and < 1 𝑛𝑚 spatial resolution in live cells. By the end of the 4-year funding period, a device will be demonstrated that is able to introduce controllable numbers of nanoparticles, proteins, and multiple genes and promoters into 1000s of cells with high survival rates. The cells will be transferred onto microscope coverslips or microfluidic cells suitable for high-resolution optical microscopy. An instrument capable of 100𝜇𝑠 smFRET will have been used to study the dynamics of G-protein couped receptors (GPCRs). Another instrument will be built to improve the time resolution of sub-nanometer movement to by up to ~ 100 𝑛𝑠. With this instrument, the real-time visualization of the motion of molecular systems may be possible.

NIH Research Projects · FY 2025 · 2021-09

Summary / Abstract Diamond Blackfan Anemia (DBA) is a congenital bone marrow failure syndrome associated with physical malformations and defects in early erythroid progenitors. Over 80% of patients carry mutations in one of over twenty ribosomal genes, leading to haploinsufficiency and defective global ribosome biogenesis, but the mechanism by which this leads to erythropoiesis defects is poorly understood. I observed that Nemo-like Kinase (NLK) is activated in erythroid progenitors with ribosome-insufficiency, irrespective of the driving ribosomal mutation. Suppression of NLK improves erythroid expansion of hematopoietic stem and progenitor cells (HSPCs) from DBA patients and mouse models in vitro. The overarching goal of this proposal is to define the role of NLK in the pathogenesis of DBA and identify novel upstream regulators and downstream substrates of NLK. Through the acquisition of new skills in state-of-the-art technologies pioneered by my mentoring team, I foresee the successful resolution of the proposed research aims and the development of the skillset and preliminary data necessary to establish my own independent research program. In Aim 1, I will use CRISPR/Cas9 to knock out NLK in ribosome- insufficient donor HSPCs and examine engraftment after transplantation into recipient mice. As NLK is activated in DBA models irrespective of the driving mutation, this represents a gene therapy approach for autologous stem cell transplantation with the potential to cure the hematological impacts of the disease. In Aim 2, I will identify and characterize downstream substrates of NLK in DBA. In Aim 3, I will identify and characterize deregulated proteins upstream of NLK activation in DBA. The last two aims include characterization of preliminary candidates, complimented by genome wide screens to identify novel factors. Collectively, these studies have the potential to identify new therapeutic targets and improve outcomes for DBA patients. This proposed work will also provide me with the necessary tools and expertise to successfully transition to an independent career. Bench skills I will acquire include mouse stem cell transplantation, CRISPR/Cs9-mediated gene therapy of stem cells, ribosome and mitochondrial biogenesis, translational analysis and genome-wide kinome analysis. Coursework covering bench skills (e.g. RNA biology, applied computational tools, and bioinformatics) and career development (e.g. mentorship, personnel management and faculty transitioning) will compliment guidance from my mentoring team. Dr. Sakamoto has an exemplary track record of producing leaders and is committed to continued guidance as I take on more autonomy. Dr. Sakamoto and Stanford have demonstrated exceptional commitment to my professional development throughout my fellowship and instructor training and I have no doubt their continued support towards our shared goal of developing a world class independent research program dedicated to understanding the pathogenesis of nonmalignant hematological disorders such as DBA.

NIH Research Projects · FY 2024 · 2021-09

Project Summary A protein traffic control system that regulates left-right patterning and heart development Structural birth defects represent the leading cause of infant deaths. Congenital Heart Defects (CHDs) are the most common structural birth defects, affecting ~40,000 babies each year. Amongst CHDs, a disproportionate burden of mortality and morbidity is due to “severe” CHDs, defined as those that require surgery or a procedure before the first year of life. The molecular mechanisms that drive severe CHDs are incompletely understood, hampering preventative, diagnostic and therapeutic advances. Data from mouse studies and human birth registries have revealed a striking association between severe CHDs and heterotaxy, defects in left-right patterning of visceral organs. By integrating the expertise of three investigators in signal transduction, mouse development, human genetics and CHDs, we have identified a novel cell-surface ubiquitination pathway (the “MMM pathway”) that plays widespread roles in the patterning of tissues during development. Disruption of this pathway leads to a characteristic syndrome of heterotaxy with severe CHDs in embryonic mice, along with defects in other tissues such as the limb, skeleton and face. Three dimensional reconstructions of the intracardiac anatomy of MMM mutant embryos reveal the presence of severe CHDs also often seen in human patients, including double outlet right ventricle and transposition of the great arteries. The MMM pathway is anchored at the cell surface by a receptor-like ubiquitin ligase complex composed of MEGF8, a single-pass transmembrane protein, and MGRN1, a RING superfamily E3 ligase. This unique membrane-tethered ubiquitination machine attenuates signaling through the iconic Hedgehog (Hh) pathway. Mechanistically, the MMM components decrease the abundance of the Hh transducer Smoothened (SMO) by direct ubiquitination, thereby reducing the sensitivity of target cells to Hh ligands. We propose to test the hypothesis that the MMM pathway functions as a traffic control system for signaling receptors that regulate left-right patterning and cardiac development. Our first aim is focused on understanding the biochemical function and developmental roles of MOSMO, an uncharacterized tetraspan membrane protein that we identified as a third component of the MMM pathway. In the second aim, we test whether the heterotaxy and CHDs seen in MMM mutant embryos are caused by elevated Hh signaling strength at critical periods in development and also search for other signaling receptors regulated by the MMM pathway. Finally, we leverage our comprehensive biochemical and developmental assays for MMM proteins to test the functionality of rare coding variants in MMM genes seen in human patients with severe CHDs. Successful completion of this project will uncover trafficking and signaling mechanisms that underlie the long-observed link between left-right patterning and heart development and consequently advance our understanding of the molecular pathophysiology of severe CHDs.

NIH Research Projects · FY 2026 · 2021-08

PROJECT SUMMARY In the present proposal, we aim to validate novel translational approaches (i.e. targeted local delivery into the kidney and microenvironment modulation of the kidney) and novel regenerative therapies (i.e. pulsed focused ultrasound (pFUS) primed mesenchymal stem cells (MSCs) and their secreted extracellular vesicles (MSC-EVs)) to treat acute kidney injury (AKI) resulting from Ischemia-Reperfusion Injury (IRI). IRI-AKI is a significant cause of morbidity following major cardiac and vascular surgery, kidney transplantation, sepsis or hemorrhagic shock, and has a complex and dynamic pathophysiology as it progresses through 3 phases: ischemia (which causes metabolic shifts due to mitochondrial dysfunction and ATP depletion), reperfusion (which causes reactive oxygen species (ROS) production) and inflammation (which can activate the immune system and, if left unchecked, can trigger fibrotic pathways). We have developed and characterized a reproducible IRI-AKI preclinical mouse model, that has molecular changes closely matching those seen in human patients. Given the translational potential of this model, we now propose to evaluate bone marrow derived MSCs (BM-MSCs) and EVs (BM-EVs) as therapies for IRI-AKI. Based on our characterization studies, these therapies have the optimal functional phenotype to address the activated pathways in IRI-AKI compared to other MSC sources, given their high expression of pro- angiogenic, metabolic, growth, immunomodulatory and anti-inflammatory factors. Interestingly, we have developed a novel approach to reproducibly stimulate BM-MSCs to produce primed therapies (i.e. pBM-MSCs and pBM-EVs) using pFUS, with therapies having an enhanced therapeutic phenotype, enriched metabolic cargo, reduced expression of factors that can activate the coagulation system, and increased expression of connexin-43 (Cx43) that can form gap junctions (GJs) with corresponding Cx43 proteins whose expression are also increased on cells in the injured kidney. Hence, we will fully optimize the generation pBM-MSCs (Aim 1) and pBM-EVs (Aim 2) and characterize their phenotype to ensure reproducibility among different donors from different sexes. Next, we will validate their therapeutic capabilities in vitro in a hypoxia-normoxia injury model with renal organoids, and then in vivo in an IRI-AKI animal model especially after intra-arterial (IA) delivery of these therapies directly into the injured kidney. To confirm that our primed therapies are working to restore metabolic shifts by transferring their cargo into injured cells via Cx43-GJs, we will next test these therapies in which Cx43 has been silenced. To determine how pFUS modulates the injured kidney microenvironment in IRI- AKI, we will assess how different sonication protocols can stimulate regenerative pathways, correct metabolic shifts and even help pBM-MSCs and pBM-EVs retention, thus facilitating their spatial co-localization next to injured cells for cargo transfer (Aim 3). Finally, we will validate our optimized therapeutic candidate and approach (i.e. dose, delivery route, timing, and pFUS priming protocol) following IRI-AKI -/- KO mouse.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY / ABSTRACT The overall goal of the Discovery Science Collaborative for CKDu Renal Science Core is to bring the best investigative methods and scientific technology to the problem of CKDu. We will work closely with Field Epidemiology Sites, the Scientific Data Coordinating Center, NIDDK Leadership, the External Oversight Committee, and HHEAR to design clinical phenotyping studies and perform discovery science experiments. We will then integrate this data to elucidate the etiology and progression factors of CKDu occurring in diverse agricultural communities. The Renal Science Core is a multi-institutional collaborative organized around six Core components: Genomics, Pathology, Transcriptomics, Biomarkers, Physiology, and Bioinformatics. The specific aims of the RSC are to: (1) characterize the clinical phenotypes and natural history of patients with CKDu and unaffected individuals in CKDu-endemic regions, comparing features across CKDu sites and determining the degree of similarities and differences across sites and regions; (2) introduce cutting-edge molecular technologies to compare the risk factors and pathophysiology of CKDu across and within study sites to understand both shared and unique aspects of the etiology of disease; (3) define the relationship between AKI and CKDu in endemic regions; and (4) build a flexible, adaptable infrastructure for both hypothesis- and non-hypothesis-based approaches to understanding etiology that will form the core of ongoing CKDu studies. The leadership team of the RSC has extensive experience working on CKDu, a history of collaboration on international working groups, and leadership roles on other major NIDDK consortia. This group brings together expertise in nephrology, genomics, nephropathology, machine learning algorithms for digital pathology, single- cell transcriptomics, biomarker development, proteomics, metabolomics, renal physiology, bioinformatics, and data integration. We have also enlisted several advisors with expertise in novel pathogen discovery, novel imaging technologies, and environmental sampling. These studies will result in a greater understanding of the underlying pathophysiology of CKDu.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY There is little debate that sugar-sweetened beverages (SSBs; drinks with added sugar) lack nutrition, are a major source of added sugar and calories, and promote obesity and poor cardiometabolic health, especially when consumed during early childhood. Nearly half of children aged 2-5 drink SSBs on a daily basis, with heavier consumption in low-income Latino children. After decades of research, it is clear that there is no single “magic bullet” for solving obesity. What we need are bundled interventions that combine incremental changes that transform food environments with targeted behavior changes that steer people towards those healthy options. Childcare centers, which serve 12.5 million children per year, provide an efficient way to intervene early by engaging childcare providers and parents to make resonant, mutually reinforcing changes in both the home and childcare environment. Interventions that promote water consumption in place of SSBs have shown promise for preventing childhood obesity in schoolchildren. Yet, no studies have examined whether interventions to promote intake of water instead of SSBs in childcare could prevent childhood obesity at an even earlier stage of development. The proposed cluster-randomized controlled trial will test the efficacy of an intervention called Healthy Drinks, Healthy Futures (Bebidas Saludables, Futuros Saludables) that is culturally adapted for Latino children and families. Following the Social-Ecological Model and Social Learning Theory, the intervention supports complementary changes in the childcare and home food environments that promote water consumption while reducing SSB availability. This is combined with education for childcare providers and children, and a one-on-one brief motivational counseling intervention with parents to reduce SSB intake and encourage water consumption in the home. Fourteen childcare centers serving low-income, predominately Latino children (n=420) will participate in this trial. The primary outcome is child BMI z-score (BMI standard deviation score). Key secondary outcomes are intake of water and beverage calories at centers and at home. Outcomes will be captured using anthropometrics (weight, height), plate waste measurements (water and caloric intake at centers), and Automated Self-Administered 24-hour dietary recalls (water and caloric intake at home) at baseline, 6, and 12 months post-intervention. Surveys of childcare providers and parents will allow us to explore possible mediators of the intervention effect. We hypothesize that the childcare-based healthy beverage intervention will increase intake of water and reduce beverage calories consumed at both childcare and at home. BMI z-score will also improve among children in intervention centers vs. control centers. If shown to be effective, the Healthy Drinks, Healthy Futures intervention will offer a strategy for intervening early to prevent obesity for millions of low-income children attending childcare centers. Findings from this study will contribute to the science of multilevel obesity prevention, and inform the implementation of state, federal, and local policies to promote healthy beverage intake in childcare centers.

NIH Research Projects · FY 2024 · 2021-08

PROJECT SUMMARY Despite the ubiquitous role of fibrosis in tissue dysfunction arising from aging and disease, no representative in vitro model of the fibrotic microenvironment exists. Fibrosis is characterized by excess extracellular matrix (ECM) deposition that stiffens the cellular microenvironment. Therefore, to model fibrosis in vitro, cell culture substrates that permit quantitative, dynamic tuning of matrix mechanics are necessary. However, existing dynamic hydrogel culture platforms generally rely on chemistries that may be toxic to cells or that simultaneously change multiple parameters, making it difficult to assign causal relationships between altered matrix properties and cell fate changes. Fibrotic stiffening occurs in a wide range of tissues, including the skeletal muscles, liver, lungs, and heart. Numerous genetic cardiomyopathies are characterized by progressive fibrotic stiffening that precedes heart failure. While fibrotic stiffening is known to impair the heart’s ability to pump blood, the impact of stiffening on the phenotype of individual cardiomyocytes remains poorly understood. The goal of this research proposal is to develop an in vitro model of tissue fibrosis based on dynamic hydrogel biomaterials that enables real time measurement of cellular dysfunction to determine how progressive fibrotic stiffening detrimentally impacts cell fate. As a model system, we will interrogate the effects of stiffening on human cardiomyocytes differentiated from induced pluripotent stem cells from Duchenne muscular dystrophy (DMD) patients. DMD is an ideal model system for studying outside-in mechanosignaling, as DMD arises from a lack of dystrophin, a structural protein linking the contractile cytoskeleton to the ECM. We will use the dynamic hydrogels developed during this research to assess contractile dysfunction, aberrant activation of mechanotransduction signaling, and novel molecular mechanisms of “mechanical memory” arising from fibrotic stiffening. In Aim 1, we will develop a synthetic hydrogel system that uses near-infrared light and bioorthogonal reactions to dynamically stiffen the gels, mimicking fibrosis. These hydrogels will be used to determine how contractile dysfunction arises from fibrotic stiffening. In Aim 2, we will determine how increased stiffness alters biochemical signaling in cardiomyocytes, focusing both on “canonical” mechanotransduction through Rho GTPases and YAP signaling and on a new mechanosensitive pathway in actively contracting cells that involves mechanical generation of reactive oxygen species (ROS), DNA damage, and impaired mitochondrial biogenesis. In Aim 3, we will investigate the first example of “mechanical memory” in cardiomyocytes. We will develop a hydrogel platform that is stiffened by one wavelength of light and subsequently softened by a second wavelength. This system will enable identification of molecular mechanisms by which exposure to a stiffened microenvironment causes persistent cellular dysfunction and strategies to reverse this memory. The engineered platforms developed will be broadly useful for studying fibrosis in progressive genetic diseases as well as aging.

NIH Research Projects · FY 2026 · 2021-08

Project Abstract Gene editing technologies have revolutionized biomedical research, yet significant challenges remain in achieving safe, efficient, and immunologically compatible integration of large DNA sequences. Current technologies, including CRISPR-Cas based transposase, base-editing, prime-editing, or phage-derived integrases and recombinases, are limited by their non-human origins, which can trigger toxic cellular responses and restrict editing capabilities. During our previous NIGMS funding period, we successfully developed a phage-derived recombineering system (REDIT) that achieves efficient kilobase-scale editing without DNA cutting, significantly reducing off-target effects compared to Cas9 editing. While successful, this system still faced challenges including variable efficiencies, large system size constraining delivery, and potential immunogenicity from non-human components when applied in primary cell or complex models like stem cell derived organoids. To overcome these limitations, we made a breakthrough discovery through structural homology mining: human mitochondrial Twinkle helicase possesses DNA unwinding-annealing capabilities with structural and functional similarity to phage SSAPs, despite low sequence homology. Our preliminary data show that engineered nuclear-targeted Twinkle facilitates homology-driven DNA insertion with double-digit efficiency for kilobase-sized sequences. Additionally, we confirm Twinkle editor could be guided by CRISPR system or function independently from CRISPR via homology annealing. This renewal application proposes to develop this human-derived mitochondrial enzyme into a versatile platform for proteome-wide functional screening. We will optimize Twinkle through protein and donor engineering, elucidate DNA repair mechanisms affecting editing efficiency, and validate the system through pooled knock-in screening in cancer and stem cell models. Our goal is to develop the first fully human gene-editing platform capable of efficient kilobase-scale insertion, enabling scalable genome engineering for both basic research and disease studies.

NIH Research Projects · FY 2025 · 2021-08

Abstract For the past decade, our laboratory has been studying the role of cellular kinases in intracellular trafficking of RNA viruses and as targets for broad-spectrum antivirals. Furthermore, we have provided a proof of concept for the potential feasibility of the host-targeted broad-spectrum antiviral approach by demonstrating that the inhibition of two cellular kinases, AAK1 and GAK, by novel or the approved anticancer drugs, sunitinib and erlotinib, protects mice from dengue and Ebola viruses with a high barrier to resistance. Since the therapeutic index (TI) of this drug combination is narrower for SARS-CoV-2 infection, here, we focus on an independent class of compounds, the isothiazolo[4,3-b]pyridine-based RMC-113 series, that emerged from our prior work, but does not inhibit AAK1 or GAK. We showed that RMC-113 and 25 related analogs have potent broad- spectrum antiviral activity with a high barrier to resistance. Excitingly, RMC-113 reduces SARS-CoV-2 titer to undetectable levels at non-toxic concentrations and binds PIKFYVE, a cell kinase that regulates endosomal trafficking. We hypothesize that RMC-113 analogs inhibit both multiple distinct steps in the SARS-CoV-2 life cycle and the inflammatory response to this virus, in part by targeting PIKFYVE, thereby offering attractive and safe candidate inhibitors to combat SARS-CoV-2, other pandemic coronaviruses and other emerging viruses. In Aim 1, we will use a multi-dimensional medicinal chemistry approach to optimize the TI and PK profile of lead RMC-113 analogs and define their in vitro therapeutic potential as broad anticoronavirus inhibitors. Aim 2 will determine the effect of prioritized analogs and apilimod, a repurposed drug candidate for COVID-19 that inhibits PIKFYVE, on viral replication, cytokine response and tissue injury in organoids derived from excised normal lung tissue supplemented with PBMCs from 20 human donors and in two rodent models. Aim 3 will generate ADME-toxicity and safety pharmacology datasets to select pre-IND candidates. In Aim 4, we will probe the mechanism of antiviral action of RMC-113. We will validate PIKFYVE as a candidate target and use an unbiased CRISPRi screen to identify RMC-113’s target(s) and profile its chemical-genetic landscape. In parallel, we will design a clickable RMC-113 probe to confirm the molecular target via activity-based protein profiling and to monitor target engagement. Lastly, we will probe functional relevance and specific roles of PIKFYVE and other candidates emerging via these approaches in SARS-CoV-2 infection, and validate them as the molecular target(s) mediating the antiviral effect. The predicted immediate impact is that this project will provide insight into the therapeutic potential and MOA of apilimod, a repurposed drug candidate (beyond the reported effect on viral entry), and will establish a unique human lung organoid model for studying SARS-CoV- 2 pathogenesis and response to treatment under more natural conditions. In the longer term, successful completion of our study will deliver a drug-like small molecule candidate designed to protect against resurge of COVID-19 and to provide readiness for future outbreaks with coronaviruses and other emerging viruses.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY Brain tumors are the leading cause of cancer related death in children; among these, diffuse intrinsic pontine glioma (DIPG) and other histone-3 K27M (H3K27M) mutated diffuse midline gliomas (DMGs) are the most aggressive and are universally fatal with current standard therapies. Despite several decades of investigational trials testing dozens of therapeutic approaches, median overall survival for DIPG is 11 months. Chimeric antigen receptor (CAR)-expressing T-cells have mediated impressive clinical activity in B-cell malignancies, and recent preclinical and early clinical results suggest benefit in CNS malignancies. We discovered homogenous, high overexpression of the GD2 ganglioside on H3K27M DMGs and demonstrated impressive antitumor effects in xenograft models of H3K27M-mutant DIPG following treatment with GD2-CAR T cells (GD2-CART, Mount, Nat Med 2018). Significant clinical experience with GD2 targeting CAR T cells, available mostly from studies in neuroblastoma, demonstrate safety and some early signals of antitumor activity. Safe and effective translation of these findings to children with DMGs would transform the landscape for this universally lethal pediatric brain tumor. This bench-to-bedside-to-bench project will conduct three aims in parallel leveraging a recently launched single institution Phase I trial of GD2.BB.z.iCasp9-CAR T cells administered intravenously following a lymphodepleting preparative regimen in children and young adults with H3K27M DMGs. The first aim focuses on safety, integrating insights gleaned in our preclinical models into trial design to diminish the risk of tumor inflammation associated neurotoxicity (TIAN), to establish best practices and to develop improved grading and treatment algorithms for this novel toxicity. The second aim focuses on efficacy, assessing clinical activity of GD2-CART in DMG and identifying biomarkers and clinical features associated with response. We further address the limitations of standard radiographic imaging in these infiltrative tumors using a novel machine learning aided MRI radiomics approach to quantify textural changes within the tumor and assess whether such changes correlate with clinical outcome, and we assess whether GD2-CART induced changes in CSF cell free DNA can provide a rapid quantitative assessment of antitumor response. Our third aim is a discovery aim, focused on improving understanding of the biology associated with myeloid cell activation following GD2-CART therapy for DMGs, which we observe in preclinical models and we observed in the first patient treated. Here we undertake comprehensive single cell profiling of CSF myeloid cells emerging post-GD2-CART in patients enrolled on the study and in preclinical models, and bedside-to-bench translation using murine models to test the hypotheses that GD2-CART induced CNS myeloid cell expansion/activation limit the efficacy of GD2-CART, are modulated by corticosteroid therapy and that this obstacle can be overcome by engineering CD47 overexpression in the GD2-CART.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY Hispanic households in the United States disproportionately experience food insecurity (22%). Food insecurity, defined as the lack of access to safe, affordable, and health-promoting foods, presents a challenge for effective management of nutrition-sensitive chronic conditions such as prediabetes and diabetes. This is especially distressing for Hispanic adults with prediabetes and diabetes, given that food insecurity is associated with worse glycemic control. Food is Medicine, the integration of nutrition programs with healthcare, holds promise for addressing the dual challenge of food insecurity and diabetes management. The goal of ADELANTE (Addressing Diabetes by ELevating Access to Nutrition: A Trial of Effectiveness) is to determine whether a Food is Medicine intervention to improve household food insecurity and glycemic control is effective for Hispanic patients with prediabetes or diabetes. We will use a type 1 hybrid trial to assess the effectiveness of the Food is Medicine intervention on the primary outcome of glycemic control (HbA1c) at 6 months. Participants (n=355) will be randomized to either: 1) 12 weeks of household deliveries of fiber-rich foods (vegetables, beans/legumes, and whole grains) plus a 12-month remotely delivered, culturally-adapted lifestyle behavioral intervention called Vida Sana, or 2) a waitlist control arm, receiving the intervention after a 6-month delay. We will follow participants for 12 months to assess the primary outcome of HbA1c at 6 months, as well as key secondary outcomes, such as HbA1c at 12 months and diabetes-related stress at 6 and 12 months. Additionally, we will recruit up to 2 household members for each participant to assess household-level secondary outcomes, such as household food insecurity and dietary behaviors. To assess the future potential of implementation and dissemination of the multilevel intervention in primary care, we will use mixed methods including quantitative measures (e.g., intervention dose and fidelity) and qualitative interviews with participants and key stakeholders (e.g., providers, clinic leadership, and a community-supported agricultural group) according to the RE-AIM (Reach, Effectiveness, Adoption, Implementation, and Maintenance) framework. We will involve patients and our longstanding community partners in all phases of the trial. The trial will take place in Alameda County, California, which is home to a large Hispanic population, at La Clínica de La Raza, a community clinic with multiple locations throughout Alameda County. Successful completion of these aims will provide evidence of the effectiveness of a Food is Medicine program for improving household food insecurity and glycemic control among Hispanic adults with prediabetes or diabetes and their household members.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY Augmentative and alternative communication (AAC) technology for people with severe speech and motor impairment (SSMI) continues to improve, with recent advances being made in the neural control of communication devices. In prior NIDCD-supported research, our research team developed a high-performance intracortical brain-computer interface (iBCI) that decodes arm movement intentions directly from brain activity. This technology has allowed people with SSMI to control a computer cursor with sufficient speed and accuracy to type at up to 8 words/min and has enabled full control of unmodified consumer devices using only decoded motor cortical activity. In the proposed U01 clinical research, performed as part of the multi-site BrainGate consortium, we will build upon decades of experience in studying the motor system in humans and non-human primates, with the end goal of advancing iBCI technology. The goals of this project are to study how speech is prepared and produced at the level of ensembles of single neurons in speech-related motor areas of the brain in people with amyotrophic lateral sclerosis (ALS), and to create a speech prosthesis that will allow communication at rates approaching conversational speech (120-150 words per minute). We will approach these investigations with a suite of advanced methods, including (1) newly-developed dynamical systems computational approaches that have provided fundamental insights into the function of the motor system, and (2) machine learning algorithms for decoding of movement intention and language modeling that have formed the basis of the fastest communication prosthesis yet reported. Finally, we will continue to evaluate the safety profile of Utah-array based iBCIs through the ongoing BrainGate2 pilot clinical trial. Upon completion, this project will advance both the capabilities of iBCIs for communication and our understanding of the detailed neural mechanisms of speech production.