Stanford University

NIH Research Projects · FY 2024 · 2024-07

PROJECT SUMMARY/ABSTRACT Chimeric antigen receptor (CAR) T cells have emerged as breakthrough treatments for patients with hematologic malignancies, earning 12 approvals from the U.S. Food and Drug Administration (FDA) since 2017. Experimental CAR T cell therapies have also demonstrated complete remissions in solid tumors, and the FDA is projecting to grant 10-15 approvals per year by 2025, highlighting the potential of these ‘living therapies’. Despite this, current CAR T cell designs have not yet mediated sustained efficacy in solid tumors, and only 30-50% of B cell leukemia and lymphoma patients experience long-term disease control. To develop safe and potent next-generation CAR T cell therapies, it is critical to understand why existing CAR T cells succeed or fail in patients. As a scientist trained in both experimental and computational immuno-oncology, I have chosen to focus my career on using a systems biology approach to uncover the molecular mechanisms governing efficacy of engineered T cell immunotherapies. This proposal outlines a structured 2-year training plan and a comprehensive 5-year career development program to complete my training and launch an independent research career. My specific research goals are: (1) to define the most therapeutically relevant CAR T cell subsets in patients with large B cell lymphoma (LBCL), and (2) to overcome an immune suppression mechanism of resistance to CAR T cell therapy for LBCL. First, I will follow individual CAR T cell clones through time in patients treated for LBCL using matched single-cell sequencing of transcriptome, a panel of surface proteins, and endogenous T cell receptors (Aim 1). This approach, termed reverse fate mapping, will pinpoint T cell clones in the pre-manufacture apheresis and infusion products with sought-after properties, including abilities to expand, persist, and home to the tumor. In Aim 2, I will apply reverse fate mapping and methylation analyses to identify the origin of circulating CAR T regulatory (Treg) cells that I recently linked to limited CAR T cell efficacy in LBCL. In Aim 3, I will mechanistically dissect the interplay between Treg and non-Treg CAR T cells to design a potent ‘Treg-free’ CAR T cell therapy for clinical evaluation. My work will generate a comprehensive CAR T cell atlas and insights, leading to promising avenues for engineering the next-generation CAR T cell therapies. The results of my proposed research will positively impact public health, as they will gather sufficient preliminary data for testing a ‘Treg-free’ CD19-CAR T cell therapy for LBCL in a clinical trial and will deliver fundamental insights into CAR Treg biology that may generalize to other diseases, including solid tumors, where engineered T cell therapies have not manifested similarly potent effects as in LBCL. To build upon my skills, I have assembled a mentorship team, including my primary mentor, Dr. Crystal Mackall, a pioneer in CAR T cell immunotherapies; co-mentor, Dr. Sylvia Plevritis, a leader in cancer systems biology; and an advisory committee with extensive expertise relevant to all aspects of this proposal. The completion of this K99/R00 program will prepare me to compete for R01 funding and to launch an independent research career focused on improving immunotherapies for patient with cancer.

NIH Research Projects · FY 2026 · 2024-07

Project Summary/Abstract Proximity-based protein modulators such as proteolysis targeting chimeras (PROTACs) and molecular glues represent a new therapeutic modality because they can, through proximity-induced protein depletion, ablate all functions of their targets. Because of their ability to target non-enzymatic proteins, these molecules have been heralded as key to eliminating the “undruggable” human proteome. However, the transformational potential of this technology is limited by the necessity of drug-like ligands for target proteins, which are sometimes intrinsically disordered or do not possess small molecule binding pockets. One of these target proteins is BCL11A, the transcriptional repressor of fetal hemoglobin and a validated target for the treatment of sickle cell disease and beta-thalassemia. This proposal focuses on the development of modulators for BCL11A to spur new therapeutics for these disorders. Sickle cell disease and beta-thalassemia are the most common genetic hematologic disorders, affecting millions of people worldwide. In these diseases, oxygen transport to metabolizing tissues is impaired. This leads to anemia, chronic pain, cardiac and pulmonary issues, decreased liver and spleen functions, etc. Many patients require costly lifelong care. Extensive research has firmly established that fetal hemoglobin, an alternate form that is predominantly expressed during development but is silenced in adults, offers protection to patients. Yet, few therapies exist to reactivate fetal hemoglobin. There is one FDA-approved drug, that although widely prescribed in the US, does not work in many patients. More recently, gene therapy targeting BCL11A, has successfully increased fetal hemoglobin in patients by 3 to 4-fold. While exciting, this therapy is costly, complicated, and has limited access, resulting in only hundreds of patients undergoing these procedures globally. Because it also requires autologous transplantation of genetically modified cells following myeloablative conditioning, it is restricted to severely ill patients who have access to advanced clinical care. Clearly, alternative therapies are needed. The research described here aims to leverage chemical biology advances to develop tools for proximity- based depletion of BCL11A. In proof-of-principle studies, we recently reported first-in-class degraders for BCL11A (Shen et al., 2022; Yin et al., 2023) that deplete up to ~ 70% of cellular BCL11A. BCL11A loss led to a significant induction of fetal hemoglobin to levels that, if achieved in patients, will be curative for sickle cell disease and beta-thalassemia. The proposed work will build on this finding to further advance these degraders into viable therapeutic leads. Such advancement will require extensive ligand optimization and the development of cell- and organ-specific delivery vehicles to negate hematopoietic stem cell mobilization and ex vivo treatment. Beyond sickle cell disease and beta-thalassemia, the proposed strategy can be used to similarly modulate other intractable but disease relevant protein targets.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY/ABSTRACT Hair cells (HCs) are the specialized sensory cells of the inner ear that play a crucial role in transmitting environmental information, such as sound and motion, to the brain. In adult mammals, HCs cannot regenerate naturally. Therefore, a large effort has been made to understand the normal development of auditory and vestibular HCs, so that effective regenerative therapies can be established. Technological advancements in studying transcriptional control have provided valuable insights into the roles of different transcription factors (TFs) such as ATOH1, POU4F3, GFI1, INSM1, TBX2, and IKZF2 in the development of HCs. However, the application of certain tools, such as CRISPR-mediated gene activation and inhibition, for studying HC development has been limited by the complicated use of animal models. Recently, a highly scalable protocol was developed to guide mouse embryonic stem cells (mESCs) towards inner ear sensory cells in vitro. Over a span of 2-3 weeks, these inner ear organoids produce sensory epithelia-like structures complete with mechanosensitive HCs and underlying supporting cells. The development of this inner ear organoid model facilitates the progress of cell-based assays for studying HC-specific development and testing potential treatments for sensory cell loss. In this proposal, I aim to utilize the inner ear organoid model to investigate the transcriptional control of HC development in vitro. To achieve this, I plan to: I. Define the TFs involved in in vitro inner ear organoid HC development compared to in vivo HC development. II. Generate a versatile toolkit of mESC lines designed for inducible and reversible CRISPR-mediated gene activation and inhibition. III. Utilize this toolkit to systematically investigate the roles of candidate TFs in organoid HCs through CRISPR-mediated knockdown and activation, followed by multi-omic analyses. Successful completion of this project will not only uncover additional TFs essential for HC development but will also provide a valuable resource for fellow inner ear researchers seeking to manipulate the expression of genes of interest within inner ear organoid cell types.

NSF Awards · FY 2024 · 2024-07

Cities across the US have experienced a significant increase in people experiencing homelessness, especially since the beginning of the COVID pandemic. Timely and early intervention that improves the well-being of those who are experiencing homelessness significantly improves their outcomes, reduces time spent in homelessness, and prevents persistent homelessness. However, because of the dynamic movements of unhoused persons (due to clearing of encampments, weather, safety, etc.) coupled with a reluctance to provide information to the authorities, it is difficult for existing programs to determine the magnitude and location of service needs and to ensure that well-intentioned programs do not inadvertently reduce overall wellbeing. The project will support research that will measure neighborhood conditions and factors that impact the wellbeing of homeless populations through cameras, noise, and environmental sensors mounted on cars driving throughout the city of San Jose. This data will help determine neighborhood conditions at a granular level and the localized need of the homeless population and to optimize the services they receive (e.g., meal delivery, trash and waste removal, and toilets) through our partners including the City of San Jose, Loaves & Fishes, and Feed My Lamb. The project has four main technical research steps to achieve the goal of understanding neighborhood wellbeing and the local needs of the homeless population: (1) developing a community-driven vehicular and mobile crowdsensing system to measure neighborhood conditions, (2) designing clustered federated learning algorithms to reconstruct city-wide maps of neighborhood environments and service needs, (3) modeling the causal relationships between neighborhood environments and wellbeing across different communities, and (4) developing methods to optimize services to improve and reduce inequality in wellbeing. The research project involves three types of community partners: local food pantries, local residents, and the city government of San Jose. Through collaboration with these partners, the project will have immediate impact to provide localized actionable needs relating to food, trash, and toilets, and to improve the wellbeing of vulnerable populations in San Jose, CA. The methods and models developed in the project will be generally applicable to other cities and areas with diverse neighborhoods. 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 · 2024-07

Neural networks have revolutionized science and engineering in recent years, but their theoretical properties are still poorly understood. The proposed projects aim to gain a deeper understanding of these theoretical properties, especially the statistical ones. It is a matter of intense debate whether neural networks can "think" like humans do, by recognizing logical patterns. The project aims to take a small step towards showing that under ideal conditions, perhaps they can. If successful, this will have impact in a vast range of applications of neural networks. This award includes support and mentoring for graduate students. In one direction, it is proposed to study features of deep neural networks that distinguish them from classical statistical parametric models. Preliminary results suggest that the lack of identifiability is the differentiating factor. Secondly, it is proposed to investigate the extent to which neural networks may be seen as algorithm approximators, going beyond the classical literature on universal function approximation for neural networks. This perspective may shed light on recent empirical phenomena in neural networks, including the surprising emergent behavior of transformers and large language models. 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 · 2024-07

Project Summary/Abstract Our project will address a major gap in existing neuroscience research tools, through the development of intersectional genetic tools for drug-controlled manipulation of specific synaptic connections between two genetically- or anatomically-defined neuronal populations. The tools we have engineered build from established cell manipulation tools – DREADDs, PSAMs, and tetanus toxin – but critically, introduce trans-synaptic gating. Thus, the tools become activatable only at user-selected, genetically-defined cell-cell contact sites. We propose to develop three tool platforms in Aims 1-3, which enable drug-controlled synapse manipulation by three different mechanisms and on three different timescales – ranging from seconds to minutes for antigen-gated PSAM ion channels to hours for antigen-gated trans-tetanus toxin. We have designed the antigen-gating of our tools to be both modular and programmable, so that either exogenous (e.g., surface GFP) or endogenous (e.g., tumor marker GD2) trans-synaptic triggers can be used. To develop and optimize these tools, we will rely on the extensive protein engineering and directed evolution expertise of PI Alice Ting, who has previously developed proximity labeling enzymes and calcium integrators that are widely used in the neuroscience community. We will carefully validate the tools in two distinct mouse brain regions in the labs of PIs Xiaoke Chen and Ivan Soltesz. Chen will evaluate the specificity and sensitivity of the proposed tools by assessing their ability to thoroughly and selectively silence connections from the paraventricular thalamus and prefrontal cortex onto two types of nucleus accumbens medium spiny neurons. We will use a combination of electrophysiological recording and optogenetic pathway stimulation and calcium imaging on ex vivo brain slices to read out drug-gated inhibition of these circuits. We will then utilize these tools in vivo to study the circuit mechanisms of opioid withdrawal and cocaine-induced behavioral sensitization. In the Soltesz lab, we plan to apply the tools to answer previously untestable questions about the roles of cell-type specific projections in regulating hippocampal function across scales from local microcircuits to long-range inputs. Our hippocampal experiments will use single cell electrophysiology, optogenetics, in vivo imaging of behavior-associated neuronal activity, local field potential recordings and behavioral memory tests. Constant feedback between these ex vivo and in vivo studies and the tool engineering efforts in Aims 1-3 will ensure that our tools are optimized for maximal efficacy, specificity, and robustness. Our team is highly interdisciplinary and diverse, combining the chemical biology and protein engineering expertise of Alice Ting with the systems and molecular neuroscience expertise of Xiaoke Chen and Ivan Soltesz. Soltesz is a pioneer in exploring the roles of hippocampal neuronal subpopulations in normal and pathological circuit function, while Chen has uncovered novel circuit mchanisms underlying pain and addiction. Though our proposed aims are highly ambitious, they are feasible given the track record, expertise, and synergy of the team, as well as the strong preliminary data presented.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY Primary cilia play a pivotal role in human health, acting as signaling hubs to sense extracellular cues such as odorants, metabolites, light, neurotransmitters, and more. Defects or failure of these signaling hubs to function leads to developmental disorders, immune dysfunction, diabetes, obesity, Parkinson’s disease, cancer, and other pathologies. An early focus of this grant is the generation of better tools to dissect primary cilia function and mechanism, such as a protocol for synchronized ciliogenesis in human retinal pigemented epithelial (RPE) cells coupled with microscopy and shotgun/phosphoproteomic mass spectrometry, to dissect signaling events temporally and spatially. While mapping ciliogenesis via immunofluorescence with known markers of the stages, one protein that emerged as a strong candidate for regulating interpathway communication was TULP3, a 50kDa ciliary protein whose primary known function is in driving the import of ciliary GPCRs through unclear mechanisms. A combination of synchronization and classical cell biological approaches were used to uncover novel phenotypes revealing temporal and spatial timing of TULP3’s function in GPCR traffic as well as new functions in ciliogenesis, downstream of TTBK2 recruitment but before axoneme protrusion. Patient mutations were identified from the use of GWAS databases to probe for links between protein function and human health. Constitutive expression of these TULP3 mutants in TULP3 KO background generated new tools to perturb select functions for TULP3, which is especially powerful for probing function-specific binding domains and partners. This proposal tests the following hypotheses: TULP3 (i) regulates receptor traffic prior to cargo arrival at the basal body and coupling to IFT machinery to mediate receptor entry into cilia and (ii) licenses axonemogenesis. Furthermore, primary cilia perform two different functions in lymphoid tissues: regulating fate change of hematopoietic stem cells into lymphocytes in the bone marrow, and in the lymph node facilitating lymphocyte maturation. These hypotheses will be tested in the following aims: Aim 1- Determine the mechanism by which TULP3 mediates membrane receptor transport into cilia. Aim 2- Identify the function and mechanism of TULP3 in ciliogenesis. Aim 3- Uncover the function of primary cilia and TULP3 in the generation of white blood cells. These aims will be addressed using cell culture and mouse model systems to probe TULP3 function and its role in immunity in both in vitro and in vivo contexts. The success of any and all of these aims will provide novel insight into key mechanisms driving ciliary function and reveal cellular contexts for how disrupted immune function arises. The study of primary cilia in the lymphatic system is largely uncharted territory. As a result, these studies are expected to open new fields of investigation into mechanisms of immune regulation.

NIH Research Projects · FY 2026 · 2024-06

Project Summary Over 4,000 patients receive life-saving lung transplants every year. Unfortunately, chronic respiratory infection frequently complicates these cases and undermines the long-term benefits of this procedure. However, it can be difficult to identify infections early or to distinguish them from rejection. Unfortunately, the current diagnostic gold standard, bronchoalveolar lavage (BAL) fluid culture, is invasive and impractical. We need better, non-invasive ways to diagnose lung infections. One novel but currently insufficient technology that could be developed for this purpose is Next Generation Sequencing of circulating free DNA (cfDNA). These degraded DNA fragments are released by human cells, and mirobes into blood plasma. High-throughput sequencing of cfDNA from blood samples is easy, non-invasive, and has already transformed diagnostics in many areas of medicine. There is great interest in using microbial cfDNA to diagnose infections. Unfortunately, microbial cfDNA protocols cannot distinguish between pathogens and closely related organisms that frequently colonize the lungs of lung transplant recipients. Consequently, microbial cfDNA is not widely used to diagnose infections. We have identified a novel approach for identifying microbial pathogens using phages – viral parasites of bacteria, fungi, and mycobacteria. Because phages are exquisitely specific to their particular host species and strain, they can provide insights into microbial population dynamics at the strain level. In particular, we find that when a particular microbial strain causes infection, phages unique to that strain explode in abundance in peripheral blood. These data suggest that phages can be used to identify their bacterial hosts. Further, we find that the circulating phages are a window into the lung. In paired samples of plasma and bronchoalveolar lavage (BAL) fluid, circulating phages closely mirror the composition of respiratory flora. We envision that phages in plasma could be used to monitor patients for infections. It may also be possible to use phages to distinguish between infection and allograft rejection. Donor-derived cell-free DNA fragments (dd-cfDNA), released by damaged allograft tissues, are a sensitive but not specific biomarker for rejection. We postulate that phages might add to this specificity by helping to rule out infection. Our hypothesis is that circulating phages reflect microbial strain dynamics in the lung and can be used to identify infections and rejection in lung transplant recipients. To test this, in Aim 1 we will develop protocols for identifying phages associated with respiratory pathogens and AMR strains. Then, in Aim 2 we will define the circulating phageome and its relationship to respiratory flora in lung transplant recipients. Finally, in Aim 3 we will determine whether phages can retrospectively diagnose infections and distinguish these from rejection. Together, these bold studies will provide unprecedented insights into the bacterial flora of the lung as well as help fulfill the unrealized potential of cfDNA in infectious diseases.

NIH Research Projects · FY 2026 · 2024-06

Project Summary My laboratory combines mechanistic cell biology with synthetic biology, focusing on understanding cellular behaviors like recognition and communication. We aim to advance the applications of engineered multicellular systems, particularly in engineered T cells targeting solid tumors. Our research has a foundation on two main questions. 1) How can we engineer highly specific cellular recognition? We seek to understand the limits and synergies of strategies for enhancing cellular specificity. Through a comparative analysis of synthetic circuits, we will explore mechanisms such multi-step signaling, and molecular titration to optimize T cell discrimination of tumors from bystander tissue based on antigen density. We also aim to uncover general principles for engineering gene expression systems and its relationship with genome organization. This may lead to general rules to engineer robust circuit behavior that could help in therapeutics. 2) how does multicellular organization affect cellular recognition? In addressing the complexity and heterogeneity of tumors, we aim for a quantitative understanding of how tissue architecture impacts T cell activity. Using an engineered spheroid platform, our research will focus into how variation in antigen density in solid tumors influence the antigen density sensing T cell activity. We anticipate extending this research to include factors like tumor inhibitory signals, inter T cell communication and the role of chemokine secretion, on T cell trafficking and tumor infiltration. The fundamental idea in this project is to control the composition and spatial organization of a spheroid and to study how these properties affect the activity of engineered T cells. In summary, our group will apply principles of molecular recognition and novel methods in cell and tissue engineering to understand and control cellular behavior. By systematically deconstructing the problem of how T cells recognize tumors from bystander tissue based on antigen density and studying the influence of tumor organization to the immune response, we aim to layout fundamental rules to engineering recognition at the cellular level and improve therapeutic cells.

NSF Awards · FY 2024 · 2024-06

Data visualization literacy plays a pivotal role in effectively communicating patterns in quantitative data, making it a cornerstone of STEM education. However, the landscape of test-based measures for assessing these skills is fragmented, without clear agreement on how to measure the key components of data visualization literacy. Furthermore, there might also be important aspects of data visualization literacy that are not well captured by existing measures. This project seeks to overcome these limitations by thoroughly characterizing existing measures and leveraging the resulting insights to develop improved and unified measures of data visualization literacy. The research plan is organized into three objectives. The first will focus on conducting controlled studies to determine the internal reliability and convergent validity of existing measures of data visualization literacy in demographically diverse samples. The second objective will focus on the development of reproducible procedures for generating new measures that span the full spectrum of tasks and data visualizations used in existing measures, while also overcoming some of their most important limitations, including uneven estimation precision across different components of data visualization literacy. The third objective will administer multiple variants of newly developed measures to multiple diverse samples, providing a strong test of their reliability. Taken together, these activities will generate a suite of well validated and comprehensive measures for assessing data visualization literacy skills. In the future, these new measures can then be used to understand how well core data literacy skills are being learned in real-world educational settings. This project is supported by NSF's EDU Core Research (ECR) program. The ECR program emphasizes fundamental STEM education research that generates foundational knowledge in the field. Investments are made in critical areas that are essential, broad and enduring: STEM learning and STEM learning environments, broadening participation in STEM, and STEM workforce development. 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 2025 · 2024-06

Characterizing biopsychological mismatch during cognitive training in mild cognitive impairment as a means of improving transfer ABSTRACT Cognitive training is a scalable, well-tolerated intervention for slowing cognitive decline and preventing Alzheimer’s Disease and Alzheimer’s Disease related dementias (AD/ADRD) with minimal side effects. Despite much promise, there have been mixed findings on strong, reliable transfer of cognitive training to non-trained domains in older adults at risk for AD/ADRD, including those with mild cognitive impairment (MCI), a critical pre-clinical stage for intervention. Transfer is hypothesized to occur when a mismatch between cognitive resources and task demands leads to increases in the efficiency with which existing cognitive resources can be applied to untrained tasks. However, few studies have attempted to quantify mismatch. The overall objective of this K01 award is to develop a new conceptual and operational framework for biopsychological mismatch in cognitive training in MCI, quantified as high mental energy and task absorption (using experience sampling) and autonomic adaptation (from electrocardiogram/ECG) both within and across training sessions. Using an existing speed of processing training (SOPT) dataset (Aim 1) with weekly measures of mental energy and autonomic adaptation we hypothesize that higher biopsychological mismatch across sessions will be associated with greater far transfer to executive function and episodic memory in MCI. We will also compare biopsychological mismatch in MCI with healthy controls. In a small pilot experiment (Aim 2) we will collect measures of mental energy, autonomic adaptation, and task absorption during SOPT sessions in a local community representative sample of Asian, Hispanic/Latino, and Non-Hispanic White older adults with MCI, and hypothesize that higher biopsychological mismatch will be associated with increased near transfer from pre- to post-session. We will also explore differences in biopsychological mismatch across racial/ethnic groups. This K01 award application will enhance the career of Dr. Adam Turnbull, a cognitive neuroscientist and young investigator with a strong research record in experience sampling (method) and cognitive aging (content), allowing him to become a lead investigator in slowing and preventing AD/ADRD by developing personalized, scalable non-pharmacological interventions (NPIs: e.g., cognitive training). The candidate will gain research skills in: 1) behavioral intervention trial design and analysis applying principles from the Science of Behavior Change and the NIH Stage Model, 2) novel biobehavioral measures and relevant signal processing, data harmonization, and computational modeling; and 3) intersections on biobehavioral and sociocultural research using a multi-dimensional framework. Dr. Vankee Lin, who was the candidate’s postdoc mentor with a strong track record and lab infrastructure for NPIs in AD/ADRD, will guide the candidate in establishing his independent research program at Stanford University. The candidate will also be mentored by Dr. Booil Jo (clinical trial design), Dr. Ehsan Adeli (machine learning), Dr. James Gross (psychophysiology/affect science), and Dr. Michelle Odden (aging and population health research).

NIH Research Projects · FY 2026 · 2024-06

Regenerative medicine cell transplantation strategies are limited by poor transplanted cell survival, retention, and integration. To overcome this, I propose development of an engineered biomaterial that addresses two major causes of transplanted cell death: (i) acute membrane damage during injection and (ii) long-term exposure to a toxic microenvironment, by providing (i) cell encapsulation within an injectable hydrogel and (ii) slow-release of growth factors. To evaluate the preclinical effectiveness of this biomaterial, I will target cervical spinal cord injury (SCI), which results in permanent sensorimotor dysfunction and has no clinically available regenerative therapies. My data demonstrates that transplanting human induced pluripotent stem cell-derived neurons (hiPSC-neurons) significantly improves anatomical and functional outcomes in rat models of cervical SCI; however, this cell type suffers from poor transplanted cell viability, reducing therapeutic efficacy. I have already shown that use of an injectable hydrogel significantly improves the acute viability of encapsulated cells by providing mechanical shielding during injection, which resulted in statistically improved neurite outgrowth and forelimb function. I now hypothesize that tuning the temporal growth factor-release properties of this designer, injectable hydrogel will significantly improve long-term survival of transplanted hiPSC-neurons, leading to significantly improved anatomical and functional outcomes. Specifically, in Aim 1, I will optimize a growth factorrelease system in vitro to assist hiPSC-neurons in surviving oxidative stress and excitotoxicity that are present at the injury site. In Aim 2, I perform functional evaluation of this biomaterial/cell therapy in a subacute model of SCI in comparison to standard delivery vehicles (saline and fibrinogen) and appropriate controls. My career goal is to lead a translational laboratory that leverages expertise in biomaterials and stem cell biology to develop regenerative therapies for central nervous system (CNS) injury. This Career Development Award would enable me to enhance my strong background in CNS neurodegeneration and stem cell biology with new expertise in biomaterial design and translational bioengineering. My career development plan includes (1) formal coursework in materials science and bioengineering, (2) technical training in recombinant biomaterials synthesis and characterization, (3) close mentorship by an outstanding bioengineer with a strong track-record of successful training and collaboration, and (4) career guidance by an Advisory Committee to prepare for my transition to independence. My training plan leverages the outstanding resources available within Stanford University and national and international events to strengthen my scientific network, build on my extensive mentorship and grantsmanship skills, and polish my scientific communication. This plan will position me to submit innovative and interdisciplinary application packets for tenure-track Assistant Professorships in my last year of this award as I embark on an independent career at the intersection of biomaterials science and CNS regeneration.

NIH Research Projects · FY 2024 · 2024-06

The Gordon Research Conference (GRC) on Signaling by Adhesion Receptors was established in 2000 and has become the premier meeting on cellular adhesion biology, mechanobiology, and their relevance to development, homeostasis, and disease. The 2024 meeting, subtitled “Adhesion and Growth Factor Receptors in Health and Disease,” will be held from July 14 to July 19, 2024 at the Southern New Hampshire University in Manchester, NH. The meeting Chairs will be Dr. Alexander Dunn, Dr. Patrick Derksen, and Dr. Maddy Parsons. These chairs are leading investigators in the fields of cellular adhesion and cancer biology and provide complimentary expertise in these fields. A prominent, new goal of this meeting will be to couple fundamental discovery science with clinical cancer diagnosis and treatment. As in previous meetings, we aim to disseminate the latest discoveries concerning mechanical and biochemical crosstalk between adhesion and growth factor receptors, as well as how these two receptor classes interact in the context of multiple diseases. Additionally, we will highlight the ongoing development of novel tools and techniques that promise to accelerate progress in our field. We likewise look forward to showcasing near-physiological modeling to better understand development and disease, and clinical applications of adhesion and growth factor receptor modulation. Lastly, we will emphasize emerging topics in the field. This interdisciplinary meeting plays an essential role in instigating and fostering collaborations among scientists with diverse backgrounds in bioimaging, bioengineering, and cancer and developmental biology. These interdisciplinary interactions provide a powerful means of uncovering commonalities in how disruptions in signaling at cellular adhesion complexes contribute to diseases that are typically studied by separate intellectual communities. Speakers will include established leaders in the field, as well as young, promising investigators. In addition to plenary talks, the meeting will feature informal and interactive poster sessions. We have also planned afternoons that facilitate informal engagement among trainees and investigators. We have initiated a concept called ‘walk with a PI’ (introduced in the 2022 meeting; Chairs were Drs. Ann Miller and Patrick Derksen), in which junior scientists are given the opportunity to engage senior leaders in one-on-one sessions. Importantly, a total of 10 junior scientists (trainees or newly appointed young investigators) will be invited to give oral presentations, a policy that contributes to the mentoring culture of the meeting. As an important feature of this meeting, the main conference will be preceded by a two-day Gordon Research Seminar (GRS) planned by and for predoctoral and postdoctoral trainees. The GRS (Chairs: Drs. Timo Kohler and Fernando Valencia) will provide a forum for future leaders in the field to present their work through oral and poster presentations in a collegial and interactive environment. In addition, interactions with leaders from academia, industry, and government will provide GRS participants with mentoring aimed at advancing the careers of these exceptionally promising young scientists.

NIH Research Projects · FY 2026 · 2024-06

Cell membrane (CellMem) coating has emerged as a biomimetic strategy to modify polymeric nanoparticles for drug delivery applications. Compared to using individual ligands, CellMem coating has unique advantages in biomimicry by borrowing the entire ligand repertoire contained in the cell membrane. Our lab has recently explored combining CellMem coating with macroporous scaffolds for enhancing tissue regeneration via immunomodulation. We demonstrated that coating of macroporous gelatin microribbon (µRB) scaffolds with CellMem from primed mesenchymal stem cells (pMSCs) induced regenerative immune response and enhanced bone healing in a mouse disease model. While our previous study demonstrates the promise of CellMem-coated µRB scaffolds for regeneration, it was limited to using cells from young donors and in a young disease model, yet patients needing therapies are mostly aged population. Aging is known to be associated with excessive inflammation and delayed healing. As such, the goal of this proposal is to determine optimal CellMem coating for enhancing bone regeneration in aging via targeting immunomodulation and to elucidate how varying CellMem coating modulates MSC/immune cell crosstalk. We propose to use allogeneic CellMem isolated from young donors due to their more abundant supply and more potent functionality than CellMem from aged donors. We choose CellMem from pMSCs and M1 Mφ due to their immunomodulatory functions and ability to scavenge inflammatory cytokines, respectively. We hypothesize that varying the ratio of MSC/ Mφ CellMem coating will modulate Mφ polarization and MSC osteogenesis in vitro. We further hypothesize that cells from young and aged donors would require a different optimal ratio of CellMem coating. To test these hypotheses, we will assess the effects of varying ratios of CellMem coating on individual cell types and MSC/immune cell crosstalk in vitro using cells from young and aged donors. We devised a tri- culture model (MSC/ Mφ/T cells) to better mimic the complex cellular crosstalk in vivo. Lead formulations that result in robust bone formation in the tri-culture model will be validated in vivo using young and aged mouse critical-sized bone defect models. Our team has a long track record of productive collaborations and this project will integrate our complementary expertise in biomaterials, immunology, bone biology, aged animal models, tissue engineering, mass cytometry, and single-cell sequencing. This project will pioneer the translation of CellMem- coated scaffolds for enhanced regeneration in aging and elucidate the underlying mechanisms through the use of a tri-culture model and high-dimensional assays.

NIH Research Projects · FY 2026 · 2024-06

Summary Stanford University and the SLAC National Accelerator Laboratory propose to continue the Stanford-SLAC CryoEM Center (S2C2) as a National Center for Cryogenic Electron Microscopy to meet the growing demand for atomic-resolution structural biology. S2C2 will 1) upgrade and maintain six cutting-edge cryo-electron microscopes, 2) record and archive image data with comprehensive metadata, 3) assist users to overcome technical challenges in preparing vitrified samples suitable for high-resolution cryoEM structure determination and in data acquisition, 4) provide real-time data quality assessment, 5) ensure public accessibility of our resources regardless of user’s geographical location, 6) select user proposals based on scientific impact and readiness as judged by external experts’ evaluations, 7) disseminate cryoEM best practices through seminars, workshops, and user meetings, 8) train independent cryoEM researchers, 9) integrate and evaluate user feedback for facility improvement, 10) implement and validate new instrument and technology as developed , and 11) optimize center operational efficiency. Led by Dr. Wah Chiu, Dr. Michael Schmid and Dr. Britt Hedman, supported by an Advisory Committee comprising external expert scientists and user representatives, and leveraging SLAC's many decades of expertise in serving a wide range of scientific users, including those from IDeA states and under-resourced institutions, S2C2 will foster high-throughput cryoEM data generation, optimization of vitrified sample preparation, and cross-disciplinary training for novice and advanced users. Our collective efforts will shape the future of cryoEM structural biology, enabling unprecedented discoveries crucial to fundamental biomedical sciences.

NIH Research Projects · FY 2024 · 2024-06

Otitis Media (OM) is one of the most common conditions of early childhood, accounting for a very high proportion of all pediatric physician office visits annually with national health care cost estimated to be greater than $2 billion. Despite the overuse and emergence of resistant pathogens, antibiotics remain the primary medical form of OM management. In fact, antibiotics for acute otitis media (AOM) are the most common prescriptions for children. In turn, tympanostomy tube placement to treat chronic otitis media (COM) is the most common pediatric surgical procedure requiring anesthesia in the United States. The most reliable and commonly used method for the diagnosis of OM in clinical practice is pneumotoscopy, which provides visible light illumination and mobility assessment of the tympanic membrane (TM). However, pediatricians and otolaryngologists correctly identify middle ear effusions at a rate of 51% and 73%, respectively highlighting the need for new diagnostic modalities in the primary care setting. To address the inability to diagnose otitis media adequately, we have developed three innovative methods leveraging novel optical modalities and molecular probes to target bacteria and cysteine proteases. For our first aim, we developed a shortwave infrared (SWIR) fluorescence otoscopy capable of imaging bacterial infections using a maltotriose probe that targets gram-positive and gram-negative bacteria via an ATP-binding cassette (ABC) transporter unique to prokaryotic and not eukaryotic cells. Identifying a bacterial otitis media infection can help reduce unnecessary antibiotic prescriptions, which contribute to multi-resistant bacteria. In our second aim, we have designed a Spatial Offset Raman Spectroscopy (SORS) probe able to be used with current ear speculums to provide molecular information regarding changes in the eardrum and middle ear fluid.. Understanding the molecular changes in the tympanic membrane can provide prognostic information regarding the progression or resolution of otitis media. Clinically we rely on otoscopy and significant visible changes to assess the middle ear. Understanding the optical signatures Raman spectroscopy provides can help us improve how we monitor otitis media. We will also collect middle ear samples to test our previously described algorithm to differentiate serous from mucoid middle ear effusions. Lastly, to address our goal of clinical translation, for our third aim, we have developed a shortwave infrared otoscope to diagnose middle ear effusion based on the increased absorption of light by water in the shortwave infrared wavelengths at around 1450nm. We will perform a pilot clinical trial in two institutions on the ability of shortwave infrared otoscopy to diagnose middle ear effusions. The proposed application will build on novel imaging modalities to refine non-invasive diagnostic imaging strategies for preclinical and clinical translation applications for otitis media.

NIH Research Projects · FY 2026 · 2024-06

Adult survivors of childhood cancer face a lifetime of health risks due to past cancer treatments, with an estimated 80% experiencing at least one severe or life-threatening chronic health condition by age 45. To receive the recommended lifelong survivorship care, young adult childhood cancer survivors (YA-CCS) need to develop skills to manage their own care as they age. Triadic communication among YA-CCS, parents/caregivers, and clinicians is essential for YA-CCS to learn about their cancer history and develop these skills; however, communicating about survivorship topics is complex and even more challenging for families with different language preferences. Language and other social determinants of health (income, education, rurality) contribute to adverse health outcomes among childhood cancer survivors, yet very few interventions have addressed this. Community health workers help connect communities with services such as cancer screening but have not been widely employed in cancer survivorship settings. New strategies are critically needed to reduce communication gaps and improve survivorship care among YA-CCS at risk for poor health. To address these gaps, the applicant Dr. Smith will leverage her partnership with a community organization serving all families of children with cancer in a rural, low-income region in California. The proposed study will develop, pilot test, and refine a tailored family-centered communication intervention for YA-CCS and parents/caregivers. Specifically, the study aims to 1) Evaluate survivorship-related communication gaps and preferences among YA-CCS and parents through focus groups; 2) Develop a community health worker-led intervention to facilitate communication among YA-CCS, parents, and clinicians, applying principles of community-based participatory research to involve cancer survivors, parents, and community members in an iterative intervention design process; and 3) Pilot test the communication intervention to evaluate feasibility and acceptability among 18 YA-CCS–parent dyads and refine the intervention based on feedback. Through leading this study, Dr. Smith will develop advanced skills in community-based participatory research, family-centered communication, and intervention science. She will do so through structured training with support from a strong mentorship team with expertise in community-based participatory research, communication, behavioral interventions, and cancer survivorship. Dr. Smith’s mentorship and training plan, combined with Stanford’s robust institutional support for research, are anticipated to launch Dr. Smith’s independent career leading patient-centered, community-based clinical research studies to improve health outcomes among childhood cancer survivors. This research fills an important gap by involving a novel population and addressing a novel intervention target (triadic communication).

NIH Research Projects · FY 2026 · 2024-06

PROJECT SUMMARY Schistosomiasis is a neglected tropical parasitic disease that infects over 150 million people in low- and middle-income countries. WHO has targeted schistosomiasis for elimination as a public health problem by 2030 but is not on track due to geographically focal areas of persistent endemicity, especially hot spots of high Schistosoma prevalence. After decades of the longstanding public health strategy of mass drug administration (MDA), endemic areas and hot spots are often geographically focal and missed by the standard district-level prevalence surveys that inform MDA decisions. More granular prevalence mapping to inform targeted MDA is limited by: (1) inefficient diagnostics (i.e., stool microscopy) that are slow, insensitive, and prohibitively resource intensive; and (2) lack of understanding of the optimal geographic scale (i.e., district, sub-district, community) for mapping and MDA implementation. Our broad, long-range goal is to evaluate whether more geographically precise mapping with newer diagnostics for targeted MDA, including more intensive efforts in hot spots, can improve schistosomiasis control while being cost-effective. Our proposal will evaluate a novel ‘rapid mapping strategy’ that will compare two rapid, sensitive lateral flow urine antigen tests: (1) WHO endorsed semi-quantitative POC-CCA; and (2) fully quantitative UCP-LF-CAA, in an innovative pooled sampling strategy, which is supported by our preliminary data. Our application will leverage a decade-long, productive collaboration in Côte d'Ivoire and includes a multi-disciplinary team that merges expertise across epidemiology, diagnostics, mathematical modeling, cost-effectiveness analysis, clinical medicine, and policy. In Aim 1, we will evaluate rapid urine antigen tests with pooled sampling for identification of endemic and hot spot locations of Schistosoma mansoni prevalence for improved surveillance. We will test pooled urine samples with two rapid antigen tests (qualitative POC-CCA, pool size 4-8; highly sensitive and quantitative UCP-LF- CAA, pool size 5-10), using a one-stage (pooling only) and two-stage system, in comparison to WHO- recommended thresholds for being an endemic or hot spot location. In Aim 2, we will apply a geospatial model of S. mansoni prevalence and project the impact and cost-effectiveness of implementing surveillance, including the rapid mapping strategies, and MDA against S. mansoni at more granular geographic scales (sub-district, community-level), compared to current standard district-level decisions. We will develop an open-source modeling tool to guide optimal mapping for schistosomiasis, which may be used by national MDA programs across endemic countries. This proposal’s aim to evaluate new rapid mapping strategies to guide precise intervention against schistosomiasis will support the 2030 WHO goal of eliminating schistosomiasis as a public health problem.

NIH Research Projects · FY 2026 · 2024-06

Project Summary To grow and survive, bacteria rely on a multitude of physiological processes occurring along and across the cell envelope, including nutrient uptake, respiration, and the secretion of waste products. These processes are sustained by a dense arrangement of proteins located within and along the cell envelope. To better predict bacterial growth behavior in different environments it is thus essential to better understand how bacterial cells control their envelope proteome depending on their cell-physiological state and the specific characteristics of the environment they encounter. This research integrates experiments and mathematical modeling to promote such a systems-level understanding of the cell envelope in rod-shaped Gram-negative bacteria. Envelope proteins in Gram-negative bacteria are distributed across two membranes and the periplasmic space enclosed by these membranes. As the dimensions of these envelope layers are inherently linked to the size of the cell, this study tightly integrates cell-size control to investigate how envelope protein masses and envelope size are regulated by the cell. Experimentally, microscopy, genetic engineering, and a novel biochemical assay are combined to quantify changes in cell size and the envelope proteome across bacterial species and for a broad range of physiologically distinct growth conditions. Mathematically, Bayesian inference and resource allocation models are integrated to dismantle the interdependence of measured quantities and specifically probe the role of two hypothesized constraints of envelope composition and cell size control: macromolecular density and aspect-ratio maintenance. The three specific aims are: (i) Revealing the fundamental growth laws of envelope composition and cell size in different Gram-negative species. (ii) Establishing a dynamical resource allocation model to predict adjustment of cell size and envelope composition over the cell cycle. (iii) Probing the molecular regulation of envelope-dependent aspect-ratio control. Major pathogenic bacteria belong to the group of rod-shaped Gram-negatives considered in this study, including five of the seven ESKAPEE pathogens known for their aggressive acquisition of multiple antibiotics resistance. As such, this systematic study of envelope and size control builds an important physiological foundation for the targeted development of novel prevention and treatment strategies against an increasing global health threat.

NIH Research Projects · FY 2024 · 2024-06

Next Generation T cell therapies for childhood cancers [NexTGen] Current treatments fail to cure many children with solid cancers. Recent advances in adult cancers such as checkpoint blockade and targeted small molecules have made little impact in childhood disease. Engineered T-cell therapies can achieve durable responses in refractory lymphoid cancers without long-term toxicity. These are precisely the characteristics required for new treatments for pediatric solid cancers. In contrast to hematologic malignancies, solid cancers are challenging due to a lack of targets, tumor heterogeneity, and hostile tumor microenvironment (TME). We posit that through advanced cellular engineering we can overcome these challenges. Our vision is that engineered T-cell therapy for childhood solid cancers will become routine within a decade. Our central hypothesis is that coupling of advanced cellular engineering along with progressive clinical development is the fastest route to developing effective T-cell therapies for pediatric solid tumors. In NexTGen, we combine detailed studies of primary tumors to discover new targets and understand how the TME subverts T- cell function. This, along with a closely coupled clinical development program will guide the progressive engineering of T-cells to result in transformative therapies. NexTGen is composed of 6 inter-connected work-packages (WPs) with work initially focused on pediatric sarcomas and brain tumors. AIMS: WP1: To identify suitable targets for engineered T-cells. WP2: To understand the TME in pediatric solid cancers. WP3: To develop receptors and other engineering components which target tumor cells and resist or modulate the TME. WP4: To evaluate the function of engineered T-cells developed in WP3. WP5: To translate approaches from WP4 and test them in clinical studies designed for maximal impact. Cancer Grand Challenges - Full Application - 2021 WP6: To promote data sharing across all WPs. METHODS: Target discovery (WP1) and TME studies (WP2) will utilize mass spectroscopy and chip cytometry respectively. Component engineering (WP3) will use protein engineering methods. To model engineered cell function, WP4 will mostly use intact tumor models such as immune PDXs. In WP5, clinical product generation will involve autologous closed system semi-automated manufacturing. WP6 uses standard and custom databases and data sharing platforms. USE OF RESULTS: Tumor target and TME data from WP1 and 2 will be uploaded to databases developed by WP6 for widespread distribution. Engineering components from WP3 and functional data from WP4 will be available for incorporation into therapeutic T-cell strategies by the entire community. Clinical study data from WP5 should lead to registration studies, improving cure rates and mitigation of long-term toxicity to realize our Vision.

NIH Research Projects · FY 2026 · 2024-06

ABSTRACT Over 20% of patients discharged from the Emergency Department (ED) have unplanned revisits within 30 days, often due to preventable causes. Upon revisit, the ED physician lacks vital data on the timeline of events and physiologic changes leading to the patient’s return, which can lead to delayed diagnosis and over-testing. Continuous monitoring of vital signs and activity can produce detailed information about a patient’s condition and stability, both in the hospital and after discharge. It is not known, however, which patients benefit most from post-discharge monitoring (PDM), which monitoring signals and strategies best predict quality of life and ED revisit risk for specific patient populations, and how PDM data can be made diagnostically useful when a patient returns to the hospital. To address these gaps, we aim to produce a framework for the integration of PDM and acute care to improve our understanding of ED patient trajectories, both after discharge and upon revisit. Specifically, we hypothesize that integrating hospital data and PDM can improve the predictability of ED revisits, identify potential targets for post-discharge interventions, and improve diagnosis and disposition of ED revisits that cannot be prevented. We will enroll a clinically and demographically diverse cohort of ED patients at high risk of revisit within 30 days, and configure noninvasive wearable monitors with an accompanying smartphone app to continuously track activity and physiology after discharge. We will develop interpretable deep learning models to predict revisits and changes in health-related quality of life, and characterize, for specific patient populations, the monitoring signals and measurement frequencies most relevant to predicting revisits and quality of life, and the prediction horizons in which preventive interventions could be delivered. Finally, we will combine in-hospital and PDM data to develop and evaluate an intervisit report for the ED physician treating a returning patient, summarizing the relevant trends in patient physiology, activity, and health-related quality of life between visits, and including a large language model-derived interpretation of the antecedents of the return visit. Better understanding how and for which patient populations PDM can predict ED revisits and quality of life can improve the integration of acute and ambulatory care, identify new clinical use cases for existing monitoring technologies, and inform the design and timing of preventive interventions. Analyzing intervisit trajectories can reveal the antecedents of acute presentations, and improve diagnosis and disposition upon ED revisit.

NIH Research Projects · FY 2026 · 2024-06

Project Summary Chimeric Antigen Receptor (CAR) T cell therapy has revolutionized treatment for B cell malignancies by targeting T cytotoxicity to the site of the tumor. Despite the success of CAR-T cells in B cell malignancies, more than half of patients receiving CAR-T cell treatment fail to achieve long term disease control. Therapeutic failure can be attributed to many causes including the inverse relationship between CAR-T cell manufacturing duration and the resulting anti-tumor potency. Additionally, CAR-T cell manufacturing poses barriers to access such as cost and difficulty meeting supply demand equilibrium. To engineer the next generation of CAR-T cells with enhanced anti-tumor efficacy and greater patient access, I will generate CD19.28z CAR-T cells in vivo using modified lentiviral particles engineered to express a T cell targeting antibody fragment, referred to in this proposal as the Programmable Antibody-mediated Cellular Knock-In of T cells (PACK-IT) system. The PACK-IT system will be used to explore my central hypothesis: engineering T cells in vivo is feasible and will deliver a more efficacious CAR-T cells (PACK-IT CAR-T cells) with distinct biologic features, reducing cost and increasing access. I have demonstrated feasibility of the PACK-IT system to generate functional CD19.28z CAR T cells in vitro and extended the use of the PACK-IT system to successfully transduce T cells in tumor bearing mice. Based on the proof-of-concept experiments, I propose to (i) optimize the PACK-IT system in terms of transduction efficiency, phenotype, and anti-tumor potency of resulting CAR-T cells, (ii) in vivo comparison of PACK-IT CAR-T cells and those made via conventional manufacturing, and (iii) assess the impact of armoring PACK-IT CAR-T cells with drug regulatable cytokine receptors on anti-tumor potency in immunocompetent hosts. Collectively, the proposed work will result in a method to produce CAR-T cells in vivo, allow rigorous characterization of the impact of eliminating the ex vivo manufacturing process, and develop PACK-IT CAR-T cells armed with regulatable cytokine receptors to boost T cell function in vivo. The proposed work will take place at Stanford University School of Medicine, a leading institution in immunology and immunotherapy and a setting that emphasizes innovation. Dr. Crystal Mackall is the ideal sponsor for this project due to her extensive track record of mentoring successful physician scientists and her expertise in T cell biology and translational therapeutics. In addition, I will be supported by a multidisciplinary team including mentorship from Drs. Howard Chang (genome wide sequencing, engineered lentiviral vectors), Christopher Barnes (structural virology), and Anusha Kalbasi (engineered cytokine receptors).

NIH Research Projects · FY 2025 · 2024-06

Abstract Pancreatic adenocarcinoma (PDAC) is the third leading cause of cancer mortality, reflecting the fact that only 9% of patients present with tumor localized to the primary site, so that the vast majority of patients with pancreatic cancer are unresectable at the time of diagnosis. We have created a program that spans the identification of new molecular targets by spatial transcriptomics, the identification of peptide/small molecule ligands, and the preclinical and eventual clinical evaluation of these therapies. Peptide targeted radionuclide therapy (PTRT) is a molecularly targeted treatment strategy that uses radiolabeled peptides as biological targeting vectors designed to deliver cytotoxic levels of radiation dose precisely to cancer cells that overexpress specific receptors. Peptides with high receptor affinity are conjugated with a chelator for diagnostic imaging using positron emission tomography (PET) for patient staging, selection of suitable candidates for PTRT, and subsequent treatment monitoring. In turn, peptides are then stably radiolabeled with the intermediate-energy β-emitter lutetium-177 (177Lu). Peptides are superior for delivery due to the ease of synthesis, comparable potential affinity and specificity, improved pharmacokinetic profiles, and low immunogenicity. We now have 40 spatially sequenced pancreatic cancer samples, each with 4000 locations sequenced at a depth of 18,000 genes. Across all of these samples, we have identified Claudin-4 (CLDN4) as a particularly promising target; CLDN4 is the top target from our analysis as it 1) is a tight junction protein that is not accessible in normal tissue, and 2) is overexpressed (~16x) in all patients and in 97% of the cancer cluster spatial regions we have sequenced. Peptides have been identified with nanomolar affinity for CLDN4 and we have demonstrated specific accumulation of ~20%IA/cc in tumor and peritoneal metastases in a PDAC model. Our spatial analysis of these samples indicates that if we can deliver a peptide carrying a beta-emitter to all CLDN4 expressing cells, all cancer cells could receive a therapeutic dose of radiation. Further, we have developed the first computational method to generate cyclic peptides to specific binding domains and have now generated peptides against the tight junction region of CLDN4. Clostridium perfringens enterotoxin (Cpe) is a natural ligand to CLDN4, and we evaluate mutated peptide fragments from this ligand and cyclic peptides designed by new machine learning tools. Our specific aims are to: Aim 1) Assess and optimize claudin-4 peptides in vitro. Aim 2) Evaluate 64Cu and 68Ga-labeled claudin-4 peptides in cell culture and mouse models of pancreatic cancer Aim 3) Evaluate claudin-4 peptides labeled with lutetium-177 in 3A) cell culture, 3B) xenograft, transgenic and PDX mouse models and 3C) combination therapy with immunotherapy.

NIH Research Projects · FY 2025 · 2024-06

PROJECT SUMMARY/ABSTRACT Myelin sheaths accelerate conduction velocity along axons, and its loss in neurological diseases, such as multiple sclerosis, leads to devastating disability. The importance of oligodendrocyte function and myelin for neuron health is also emerging in many neurological disorders, such as Alzheimer’s and Parkinson’s disease. Therefore, understanding how myelin is formed, remodeled, and regenerated may reveal new strategies with broad relevance to prevent or rescue neurological disorders. The majority of myelin generates during neurodevelopment, but recent discoveries demonstrate that new myelin forms following learning and sensory stimulation in humans and rodent models. Experiments in rodent models show that neuronal activity can directly stimulate new myelin formation and that new myelin formation is necessary for cognition, learning, and memory. This emerging form of neuroplasticity, termed activity-dependent myelination, can tune action potential timing and neural circuitry by adjusting myelination patterns along axons, changing the number, length, and thickness of myelin sheaths. Myelin sheaths form from oligodendrocytes that extend multiple processes and dramatically expand their cell surface to wrap axons in spiraling layers of membrane. How does neuronal activity regulate membrane expansion in oligodendrocytes? I recently discovered that exocytosis through VAMP2 and VAMP3 drive membrane expansion in oligodendrocytes during neurodevelopment. In many cell types, VAMP-mediated exocytosis can be stimulated by extracellular stimuli, but the cues that regulate oligodendrocyte exocytosis are unknown. I hypothesize that neuronal activity stimulates oligodendrocyte exocytosis to drive activity-dependent myelination within activated circuits. My preliminary data reveals that neuronal activity increases VAMP3 exocytosis in cultured primary oligodendrocytes by more than 2-fold. In my proposal, I will determine which VAMP proteins in primary oligodendrocytes are stimulated by neuronal activity (Aim 1.1) and which neuron-derived factors stimulate oligodendrocyte exocytosis (Aim 1.2). Then, I will obtain new training to investigate how human-derived neuron subtypes affect oligodendrocyte exocytosis in co-cultures (Aim 1.3). To determine the role of oligodendrocyte exocytosis in vivo, I will inhibit exocytosis specifically in oligodendrocytes of adult mice via AAVs (Aim 2.1) or Cre-inducible botulinum toxin (Aim 2.2) and use optogenetic stimulation to induce activity-dependent myelination. I will determine if oligodendrocyte exocytosis is necessary for activity-dependent myelin changes. Finally, with additional training, I will test if oligodendrocyte exocytosis is necessary for motor learning, a functional task that requires activity- dependent myelination (Aim 2.3). Altogether, my aims will uncover key cellular mechanisms that drive activity- dependent myelination and expand my scientific training to launch an exciting independent research laboratory. In the long term, my discoveries may provide mechanisms that can be harnessed to stimulate myelin regeneration or enhance neuroplasticity to change the course of neurological disorders.

NIH Research Projects · FY 2026 · 2024-06

Expression of activated forms of the multifunctional type II Ca2+/calmodulin-dependent protein kinase (CaMKII) were recently demonstrated to confer robust retinal ganglion cell (RGC) neuroprotection in mouse models of optic nerve injury and NMDA excitotoxicity, making CaMKII a leading target for therapeutic intervention in glaucoma. However, new preliminary data show that expression of constitutively active CaMKII (caCaMKII) not only promotes RGC survival after optic nerve crush (ONC) injury, but also suppresses axon regeneration. As axon regeneration will be a necessary feature of any potential treatment for glaucoma that would restore vision, a better understanding of the mechanisms how caCaMKII confers neuroprotection will be important for the successful development of CaMKII-directed therapeutics. We hypothesize that the phosphorylated effectors driving CaMKII-dependent neuroprotection and axon growth repression are different. In this project, we will derive strategies that promote RGC survival, but not prevent axon regeneration by investigating how differential compartmentation and activation confer CaMKII phosphorylation of relevant effectors and their respective cellular processes. In particular, we hypothesize that induction of CREB and/or NF-κB gene expression by nuclear CaMKII and potentially CaMKIV is required for RGC survival, while as an off-target effect, caCaMKII expression elsewhere in the cell suppresses axon growth. Specific Aim 1: The role of endogenous CaMKII in RGC neuroprotection and axon suppression. In this Aim, we will use knock-out mice to test whether endogenous CaMKII and CaMKIV expression is required for maintenance of basal RGC survival or for neuroprotection after ONC. In addition, we will test whether CaMKII knock-out will promote axon regeneration. By AAV-mediated expression of CaMKII-activating peptides, we will differentiate between alternative mechanisms for caCaMKII’s neuroprotective effects: replacement of insufficiently expressed CaMKII or activation of insufficiently active endogenous CaMKII. In parallel to assay for neuroprotection, we will study the activity of CaMKII in vivo by transpupillary imaging of a CaMKII fluorescent reporter. Specific Aim 2: Compartment-specific caCaMKII neuroprotection and suppression of axon regeneration. We will test whether exogenously expressed caCaMKIIα fusion proteins localized to different intracellular domains selectively promote neuroprotection or suppress axon regeneration after optic nerve injury. Conversely, we will test whether localized expression of CaMKII inhibitory peptides will rescue caCaMKII-suppressed axon regeneration without inhibiting neuroprotection. Specific Aim 3: CaMKII-dependent RGC gene expression conferring neuroprotection. We hypothesize that activation of CaMKII signaling confers neuroprotection by induction of CREB- and NF-κB-dependent gene expression. scRNA-seq and scATAC-Seq will be used to identify genes and regulatory elements associated with neuroprotection, while Creb1 and Rela RGC-specific knock-out mice will be studied for RGC survival and axon regeneration.