Grants

OD - NIH Office of the Director

Abstract. This HEAL project will employ computational, machine learning (ML) and Artificial Intelligence (AI) tools to accelerate the discovery of vaccines and antibodies against highly toxic ultrapotent synthetic opioids (UPSO). United States, Canada, and Europe have registered dramatic increases in fatal drug poisoning due to the widespread availability of fentanyl, fentanyl analogs, emerging compounds of the nitazene class, and their mixtures. Because of their potency, ease of illicit synthesis, and widespread availability, UPSO are fueling the ongoing overdose crisis. Beyond their impact on individuals with Opioid Use Disorder (OUD), these compounds could be involved in mass casualty incidents (MCI) or deliberately deployed in chemical attacks. Current FDA- approved countermeasures consist of opioid receptor antagonists, which may not always be sufficient to reverse overdoses involving UPSO or drug mixtures containing UPSO. To address this public health threat, our team has developed vaccines and monoclonal antibodies (mAb) against a series of UPSO. Vaccine-elicited antibodies and mAbs selectively bind the target drug in serum, reduce distribution of the unbound (free) drug to the brain, and prevent or reverse drug-induced effects. Based on their pre-clinical profile, anti-UPSO vaccines and mAbs have the potential to counteract overdose toxicity and poisoning in both civilian and defense scenarios. Due to their selectivity, vaccines and mAbs could be combined with existing treatments to increase survival. Because of the speed with which new and emerging UPSO can enter the drug supply, new tools to optimize and streamline the process of vaccine and antibody discovery are needed to rapidly address these threats by accelerating their translation into the product development space. To address this challenge, this project will employ state-of-the- art structure- and computational-guided platforms and AI/ML tools to accelerate discovery of vaccines and mAb against emerging UPSOs such as nitazenes. Specifically, AIM1 will focus on discovery of novel conjugate vaccines against nitazenes paired with computational methods to identify correlates of vaccine efficacy, AIM2 will focus on isolation of mAb against nitazenes and other UPSO accelerated by structure-informed design, and AIM3 will focus on evaluation of vaccines and mAb in vivo in rats challenged with nitazenes, fentanyl analogs, and their mixtures. Completion of this research proposal will lead to development and validation of computational methods to rapidly isolate vaccines and mAb against novel and rising UPSO, and other chemicals of concern.

NCI - National Cancer Institute

Project Summary/Abstract Resistance to chemotherapy is a major contributor to cancer relapse and treatment failure. Of many molecular mechanisms of cancer therapeutic resistance, increased drug efflux, mediated by ATP-binding cassette (ABC) transporters, results in multidrug resistance (MDR) and poses a huge barrier to effective cancer therapy. Small molecule inhibitors of efflux could be used to sensitize cancer cells toward chemotherapeutic treatment and improve treatment quality. Significantly, we have identified a highly potent lead compound, a deazaflavin analog which at single digit nanomolar concentrations drastically increased the intracellular concentration of etoposide (ETP), and to a lesser extent, that of teniposide and pemetrexed, all are important cancer chemotherapeutics. Our prior studies revealed that it is a potent inhibitor of MRP1. Notably, it displayed excellent selectivity over other major ABC transporters, including P-gp, BCRP, MRP2 and MRP3, and fully reversed the MRP1-mediated drug resistance to ETP and doxorubicin in H69AR small cell lung cancer cells. In addition, it exhibited desirable PK in preliminary mice testing without altering distribution of co-administered ETP to normal tissues in healthy mice, and hence may have limited toxicological consequences. These preliminary findings strongly suggest this lead and its analogs could be a highly valuable lead series for developing combination treatments with clinical TOP2 poisons or other anticancer drugs. The objectives of this grant application are to examine a library of deazaflavin analogs for MRP1 inhibiting activity and to further evaluate their potential for cancer chemotherapy sensitization in in vitro and in vivo models, by pursuing these specific aims: Aim 1. to screen and characterize deazaflavin analogs for MRP1 inhibition using membrane vesicle-based and cell-based drug transport assays. In this aim, we will screen an in-house deazaflavin analog library (~30 synthetic analogs) using vesicular transport assay and cell-based calcein retention assay for MRP1 inhibitory activity and characterize their potency, selectivity as well as examine the structure-activity relationship (SAR). We will also perform site-directed mutagenesis on MRP1 to identify residues that may be critical for the inhibitory effect of the analogs. Aim 2. to evaluate the deazaflavin analogs for cancer chemo- sensitization in in vitro and in vivo models. In this aim, we will first evaluate advanced analogs for chemo- sensitization in a panel of lung cancer and neuroblastoma cells toward selected chemotherapeutics and establish correlations to the MRP1 protein expression and inhibition. We will then profile the in vivo toxicity of our top analogs in mice, and further evaluate the anti-tumor effect and toxicological consequence of the analogs in combination with TOP2 poison ETP in a xenograft mouse model of multidrug resistant lung cancer. At the conclusion of the studies outlined in this proposal, a better understanding of the mechanism of action and the chemotherapeutic sensitizing potential of our lead compounds will be gained, which will lay a solid foundation for future preclinical studies.

NHLBI - National Heart Lung and Blood Institute

Heart disease continues to be a leading cause of disability and death worldwide. Recent research has refined our understanding of the many causes of heart disease and provided many new therapies to treat heart disease. Coronary microvascular and vasomotor dysfunction {CMVD) is a condition that results from aging, diabetes, obesity, hypertension, smoking, and other cardiovascular risk factors and is a common cause of chest pain. Many studies have established that CMVD is associated with adverse cardiac outcomes, including death. Indeed, CMVD is likely to be a causal contributor to epicardial coronary artery disease and vulnerable atherosclerotic plaques through decreased wall shear stress. Despite this, standard testing modalities are unable to consistenUy diagnose CMVD. Generally, CMVD is diagnosed with either expensive, specialized noninvasive stress imaging modalities or even more costly invasive angiographic measurements performed with intracoronary probes. As a result most CMVD diagnoses are presumptive-made after ruling out all other possibilities. This is a cosUy approach with uncertainty for both patients and treating clinicians. Furthermore, difficulty in establishing confirmed diagnoses of CMVD has been a critical impediment to the development of novel therapeutic approaches to CMVD. Consequently, there is a major unmet clinical need for a simple, readily available, and inexpensive diagnostic test for CMVD. The primary aims of this project will be to develop and validate artificial intelligence methods and computer software tools for noninvasive diagnosis and characterization of CMVD. Artificial intelligence methods are able to identify subtle features in complex data that are sometimes difficult or time-consuming for physicians to see. The long-term objective is to develop and commercialize such tools to facilitate improved management of patients with myocardial ischemia without obstructive coronary artery disease, to guide and monitor treatment of such patients, and to provide tools to enable the development of novel therapeutic approaches for CMVD.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

PROJECT SUMMARY/ABSTRACT Obesity, affecting over 40% of U.S. adults, is a major health concern that contributes to the development of metabolic disorders such as type 2 diabetes and insulin resistance. Current FDA-approved hunger suppressant drugs can induce weight loss but do not directly impact adipose tissue, leading to quick weight regain once treatment is stopped. White adipose tissue (WAT), which stores excess fat, plays a central role in obesity, while brown adipose tissue (BAT) helps burn fat through fat oxidation, converting fatty acids into energy and increasing thermogenesis. This project aims to create a drug delivery system that is retained in the adipose tissue and promotes fat browning—the conversion of WAT to a more metabolically active state similar to BAT—to enhance systemic metabolism. The overarching goal is to develop a locally injected adipose tissue nano-reservoir for continuous delivery of forskolin, a cyclic AMP activator molecule known to stimulate fat thermogenesis in adipose tissue. The project comprises three specific aims: Aim 1 focuses on establishing a correlation between the physicochemical properties of engineered hybrid lipid-coated mesoporous silica nanoparticles (LCMSN) and the cell uptake in the adipose tissue as well as the in vivo distribution of LCMSN and forskolin, assessing how shell configurations impact cell retention, forskolin release and efficacy. Aim 2 aims to identify the optimal properties and dosing strategies for the LCMSN to effectively induce thermogenesis in WAT, evaluating its impact on systemic metabolism, energy expenditure, and weight management in mice on a high-sucrose high-fat diet. Aim 3 will determine the efficacy of this nanomedicine in promoting weight loss in a diet-induced obesity model, correlating treatment outcomes with changes in food intake, body composition, and metabolic markers. Through preclinical studies, this project seeks to assess the impact of optimized forskolin delivery on weight management and metabolic health, ultimately creating a novel targeted tool for long-term obesity management and improved patient care.

NIAMS - National Institute of Arthritis and Musculoskeletal and Skin Diseases

ABSTRACT Osteoarthritis (OA) is a painful and debilitating joint disease and a leading cause of disability in the US. It is preliminarily characterized by cartilage degeneration. Previous drug discovery efforts predominantly focused on monotherapy (the use of a single therapeutic drug) targeting a single joint tissue (predominantly cartilage). However, rapid clearance of these potential drugs from the joint space requires repeated administrations and high doses, which increase the risk of treatment-related toxicity, escalate healthcare expenditures, and diminish patient quality of life. To date, there are no disease-modifying drugs available to halt disease progression or reverse its course. In healthy joints, articular cartilage is maintained through a fine balance between anabolic and catabolic activities of chondrocytes. In OA joints, this balance is tipped toward increased catabolic activity and diminished anabolic activity, leading to cell death and matrix degradation. We recently developed two innovative nanoparticle (NP)-based therapeutics, transforming growth factor alpha ( TGFα) -conjugated polymeric micellar nanoparticles (TGFα-NPs) and superoxide dismutase (SOD)-loaded porous polymersome nanoparticles (SOD-NPs), with independent intervention mechanisms for OA treatment. Our preliminary in vitro and in vivo data demonstrated that TGFα-NPs stimulate the anabolic activity via enhancing epidermal growth factor receptor (EGFR) signaling in chondroprogenitors, and SOD-NPs block the inflammation-mediated catabolic activity via reducing oxidative stress in the synovium. Despite encouraging and proof-of-principle results in a mouse model of OA induced by destabilization of the medial meniscus (DMM), neither of them restored the joints to a fully healthy state, and the injection frequency used in the pilot studies (once every 2-3 weeks) falls short of clinical practicality. We hypothesize that a combination treatment, using an advanced drug delivery system that targets distinct joint tissues to simultaneously enhance anabolic activity and reduce catabolic activity, can more effectively restore cartilage integrity and achieve better therapeutic outcomes than single treatments. The overall goal of this proposal is to engineer and optimize dual-action, nanoparticle-loaded microparticles (NMPs) for OA treatment with clinically relevant injection frequencies, using both injury- and age-induced animal OA models. The specific aims for the proposal are 1) Synthesize and characterize physical-chemical properties of NMPs; 2) Evaluate the efficacy of combination therapy in a mouse OA injury model; and 3) Evaluate the efficacy of MRI- guided combination therapy in a spontaneous OA model. In the short-term, this proposal will demonstrate for the first time that a two-pronged approach using advanced drug delivery system is feasible for OA treatment. In the long-term, data from this proposal will pave the foundation for future clinical trials. Thus, the success of this project will greatly benefit patients with disabilities caused by OA as well as other forms of degenerative joint disease.

National Park Service

United State Department of the Interior, National Park Service (NPS) Notice of Intent to Award. This is not a request for applications. This funding opportunity is to provide public notice of NPS's intention to fund the following project activities without full and open competition. Task Agreement # P15AC00108 under Cooperative Agreement P10AC00570 with the Joshua Tree National Park Association, for project titled "Development of Educational Digital Technology Tools"

DEEP

Development of engineering and cost information for municipal sewer and water improvements

NIH

The current proposal aims to develop a for at-home-use neuroprosthesis for restoring brain-controlled functional hand grasping to Veterans and others living with chronic tetraplegia due to spinal cord injury (SCI). This system leverages and advances two established technologies – functional-electrical stimulation (FES) systems for re-animating paralyzed limbs, and intracortical brain-machine interfaces (BMIs) for recording and discerning motor intent. Our previous work successfully demonstrated a Veteran with chronic tetraplegia using a simple proof-of-concept FES+BMI system, composed of temporarily implanted intramuscular wires for movement restoration and an implanted brain electrode for deciphering movement intent, to regain brain- controlled movements of basic arm and hand movements to perform limited but meaningful activities of daily living (ADLs), such as self-feeding. We have more recently demonstrated a more advanced system that uses composite flat-interface-nerve-electrode (C-FINE) cuffs to provide for more selective activation of peripheral musclesfor FES, and a multi-network BMI system for recording from multiple nodes in the cortical grasp network (primary motor, premotor, and parietal cortices) to provide for more intuitive brain control of dexterous hand function. These demonstrations have been limited in that research participants could only use them in the laboratory. The proposal, leveraging existing and to be enrolled study participants of the ReHAB (Re-connecting the Hand and Arm to the Brain) clinical trial (NCT03898804) who already have implanted C-FINE and BMI electrodes, will result in developing a C-FINE based FES system, able to be commanded by multiple modalities (voice, EMG, and a portable BMI system), that will be demonstrated for use in a home environment. A major consideration for development of at-home FES+BMI use is that factors unforeseen in the laboratory may become evident that result in variable system performance. Specifically, it is well known that the behaviors of individual neurons exhibit nonstationarities that could negative impact decoding the movement command. Additionally changes in a user's internal state (e.g. attention, stress, fatigue) has been shown to affect non-invasively recorded brain signals. As a result, neural decoders are recalibrated on a daily basis (and sometime more often) in regular laboratory use. It is unclear the time of these changes, how these changes affect neural decoding, and how to best mitigate these potentially deleterious changes during continual FES+BMI use. The present proposal aims to understand how neural variability (Aim 1), and changes in a user's internal state (measurable through correlated physiological biomarkers) (Aim 2) can be accounted for in development of an FES+BMI system for at-home use. Examples of these physiological biomarkers include: 1) heart rate variability (decreased by stress, increased by attention, decreased by fatigue), 2) blood pressure (increased by stress and attention), 3) pupil size (dilated under stress or intense attention), 4) blink rate (decreased with intense attention), and 5) saccade velocity (increased with increased attention and error state). Finally, the proposal will compare FES movement restoration through use of a portable BMI with a more standard EMG command option (Aim 3). Participants will use each during the at-home portion of the study, and will report out measures of satisfaction, perceived ease of use, and personal independence. The study will also use clinically validated tests of upper extremity function, during regular in-laboratory assessments, to compare performance of these command options for movement restoration through the FES arm and hand neuroprosthesis. The success of this proposal will result in the first FES+BMI system suitable for at-home use. The time is appropriate for this work, and the need is great, and the investigative team is the best in the world to pursue the aims of the proposal.

NCI - National Cancer Institute

PROJECT SUMMARY Ovarian cancer remains the most lethal gynecologic cancer. Treatment strategies developed over the past four decades, however, have only achieved limited success. The current standard of care is surgical debulking followed by paclitaxel/platinum-based chemotherapy. Although patients respond initially, almost all will relapse due to rapid development of chemotherapy resistance, the mechanism of which is still largely unknown. Given the fact that protein kinases are critical in regulating pro-survival signaling, we conducted a kinome- wide shRNA screening, which identified AP2 associated kinase 1 (AAK1) as a “synthetic lethal” partner of paclitaxel. AAK1 is known for its role in regulating clathrin-dependent endocytosis. However, its role in cancer remains unexplored. TCGA datamining revealed that high AAK1 levels are associated with poor overall and progression-free survival in taxane-treated ovarian cancer patients, and AAK1 levels are higher in taxane non- responders than in responders. Consistently, silencing AAK1 sensitized paclitaxel to ovarian cancer cell lines and organoids, both in vitro and in vivo, demonstrating the critical role of AAK1 in promoting paclitaxel resistance. To investigate how AAK1 regulates paclitaxel sensitivity, we first discovered that caspase 7, activated by paclitaxel, cleaves AAK1 at D767, creating AAK1 (1-767) truncation form. We further identified CCR4-NOT complex as the key downstream effector for AAK1 (1-767) to promote paclitaxel resistance. Collectively, these findings position AAK1 as a promising target for enhancing paclitaxel efficacy in ovarian cancer. However, AAK1 inhibitors, despite their high potency in inhibiting cellular AAK1 kinase activity, require high concentrations to sensitize ovarian cancer cells to paclitaxel, indicating limited clinical translation potential for AAK1 inhibitors in ovarian cancer treatment. We hypothesize that the proteolysis targeting chimera (PROTAC) approach can more effectively target AAK1 for ovarian cancer treatment. Indeed, HC-332, a lead AAK1-targeting PROTAC, induces potent, selective, deep, and long-lasting degradation of AAK1 in ovarian cancer cells, in vitro and in vivo. HC-332 also sensitizes paclitaxel treatment in various ovarian cancer patient-derived organoids, primary cancer cells, and cell lines, including paclitaxel-resistant sublines, indicating its potential to improve paclitaxel response in both naive and recurred patients. Despite suboptimal PK properties, in vivo studies further demonstrated that HC-332 greatly improves paclitaxel response in ovarian cancer cell line and PDXs without obvious systemic toxicity. Collectively, our data supports the potential of AAK1 PROTACs in treating ovarian cancer. Thus, we plan to develop AAK1 PROTACS with high selectivity, potency, and druglike properties; and evaluate of the efficacy of AAK1 PROTAC/paclitaxel combination in ovarian cancer and elucidate the mechanism of action of PROTACs.

OD - NIH Office of the Director

The evolving geopolitical landscape has heightened the risk of radiation-induced skin injuries due to large- scale radiological incidents, including state or terrorist-improvised nuclear devices. Additionally, radiation induced skin injuries are a significant adverse effect of radiotherapy in cancer patients. However, effective countermeasures remain limited, primarily due to the lack of physiologically and anatomically relevant human models for studying radiation-induced injuries.This proposal aims to address this critical gap by developing an extracorporeal human skin perfusion model to investigate radiation injury mechanisms and facilitate the development of effective therapeutics. The long-term goal is to create a fully automated, immune- competent human skin perfusion system that mimics in vivo conditions, enabling precise studies on radiation injury dynamics and therapeutic interventions. The overall objectives of this proposal are to (i) automate perfusion bioreactor and establish parameters to enable immune capability of the skin perfusion system, (ii) determine the utilization of human skin perfusion system to study mechanism(s) of radiation injury and (iii) as a proof-of-principle demonstrate the utilization of human skin perfusion model for developing radiation countermeasure using metformin lotion as an example. Our central hypothesis is that an automated full- thickness human skin perfusion model will provide a robust ex vivo platform for studying radiation-induced skin injuries and developing countermeasures. This hypothesis will be tested through the following three specific aims: 1) Automate perfusion bioreactor and optamize immune capabilities of human skin perfusion model – we will develop a fully automated perfusion bioreactor in collaboration with Daedalus Inc. to improve reproducibility, operational efficiency, and real-time monitoring. Additionally, integrate immune components to enhance the physiological relevance of the model; 2) elucidate the mechanism(s) of radiation induced injuries in human skin - investigate molecular and cellular responses to radiation exposure across diverse human donor samples (sex, age, race) using histological, genomic, and proteomic analyses; and 3) evaluate the use of metformin lotion as a mitigator of radiation induced skin injury – proof -of-principle for employing human skin perfusion model for therapeutics development - as a proof-of-concept, assess metformin lotion efficacy in mitigating radiation- induced skin injuries using the developed perfusion model, demonstrating its potential for therapeutic screening. This project is highly innovative as it will deliver the first-of-its-kind, fully automated, immune-competent human skin perfusion platform, transforming the study of radiation-induced injuries and enabling broader applications for skin-related research. The proposed research is highly significant because it will: i) Provide strong scientific justification for using human skin-based models in radiation injury research; ii) Reduce reliance on animal models, aligning with ethical and regulatory priorities; and iii) accelerate the development and FDA approval process for radiation countermeasures and other skin therapeutics. By successfully achieving these aims, this project will establish a new paradigm in preclinical skin research, bridging the gap between ex vivo modeling and clinical translation to improve radiation injury management and therapeutic development.

NEI - National Eye Institute

PROJECT SUMMARY Glaucoma is a neurodegenerative disease defined by the injury of retinal ganglion cell (RGC) axons at the optic nerve head, leading to axon degeneration, cell death and irreversible vision loss. There has been a longstanding need to develop neuroprotective therapies that could complement intraocular pressure-lowering therapies as well as biomarkers to assess disease activity and the need for escalated treatment. Sterile alpha and TIR motif containing 1 (SARM1) is a key mediator of the active genetic program underlying axon degeneration in glaucoma and other neurodegenerative disease. This makes SARM1 a potential neuroprotective drug target as well as an indicator of disease activity. While small molecule inhibitors and sensors are being developed for SARM1, use in the eye will require highly optimized formulations and pharmacokinetics and will not have systemic availability. In contrast, genetically-encoded SARM1 inhibitors and biosensors can be robust, readily delivered with adeno- associated virus (AAV) and used across a wide range of animal models. Moreover, they themselves can be the basis for future clinical diagnostics and therapeutics. To develop genetically-encoded SARM1 inhibitors and biosensors, we propose to use nanobodies (Nbs). These small, single chain antibodies from camelids can have high antigen affinity and, because of a protruding complementarity determining region 3 (CDR3), are particularly suited to inhibit the active sites of enzymes like SARM1. Moreover, they are modular, allowing one to add protein domains that can degrade or image target antigens. We have developed a novel bacterial display platform capable of identifying high affinity Nbs and shown that we can find Nbs against purified human SARM1 protein. In Specific Aim 1, we use molecular evolution, including a novel approach based on genetic algorithms, and structural determination to optimize Nb binding. Then, in Specific Aim 2, we use a novel mammalian cell platform in which cell survival is coupled to SARM1 activity in order to evolve Nbs with intrinsic neutralizing ability. As a complementary approach, we will functionalize the Nbs without neutralizing ability using the substrate recognition module of E3 ligases in order to retarget them to degrade SARM1. We will then validate the strategy in human stem cell-derived RGCs as well as a rat model of glaucoma. Finally, in Specific Aim 3, we will take advantage of the in vitro nature of the Nb screen in order to develop a conformational biosensor of SARM1 activity. This will be tested in a rodent model of glaucoma. We expect that the aims proposed here will culminate in the development of robust genetically-encoded inhibitors and biosensors of SARM1 activity. In order to successfully achieve this in a five-year timeframe, we have brought together two PIs with expertise in glaucoma neuroprotection, nanobodies and molecular evolution and included additional expertise in AAV production, retinal organoids and live animal imaging.

NINDS - National Institute of Neurological Disorders and Stroke

Summary Neocortical layer 1 (L1), the outermost layer of the cortex and the “crowning mystery” of David Hubel, is a major target of cortico-cortical and thalamocortical projections that carry “top-down” information (behavioral saliency, expectations, predictions, and memories) to be integrated with the “bottom-up” sensory information received by pyramidal cells, the output neurons of the cortex, in order to produce percepts and context-dependent behavior. Understanding how top-down and bottom-up information streams are integrated is necessary to understand perception and behavior, and is of medical significance since disturbances in these processes are likely at the root of neurocognitive diseases such as autism and schizophrenia. L1 is unique among neocortical layers; it is the only layer that lacks excitatory neurons but instead contains the distal (tuft) dendrites of the pyramidal cells (PCs) located in L2-5. These apical dendrites receive the diverse long-range projections arriving in L1 and mediate the integration of these top-down inputs with the feedforward sensory input arriving at the basal dendrites of the PCs. L1 also contains a specialized population of GABAergic interneurons (INs) that sculpt how top-down information is delivered to the PC distal dendrites, but the mechanism by which these neurons regulate top-down processing is poorly understood. L1 INs are distinct from those in other layers that have been the subject of intense research in recent years. Our understanding of the cellular and circuit mechanisms of top- down signaling has lagged behind our knowledge of bottom-up sensory processing, largely due to the lack of molecular genetic tools that have helped elucidate IN and PC circuitry in other layers. We and others have recently discovered that both in mice and humans, the majority of L1 INs specifically express the marker neuron- derived neurotrophic factor (NDNF). Characterization of the properties of NDNF INs in the mouse revealed that there are two main subtypes: L1 neurogliaform cells (NGFCs) and an IN subtype that is unique to L1, the canopy cells. The evolutionary conservation of these two L1 NDNF IN subtypes has recently been confirmed in humans. The availability of genetic reagents to specifically label and manipulate these IN populations would provide unprecedented opportunities to advance our understanding of the circuit mechanisms of top-down processing. Thus, the goal of this exploratory R21 application is to develop and validate novel genetic approaches for the study of L1 NDNF INs. In Aim 1, we will utilize electrophysiology and neuronal reconstructions to assess the specificity and efficacy of a novel intersectional genetic targeting strategy (NDNF/Cxcl14) for the selective labeling and manipulation of canopy cells (Aim 1A). In Aim 1B, we will utilize our NDNF/Cxcl14 intersectional genetics to study the efferent connectivity of canopy cells using optogenetics. In Aim 2, we will leverage a recently developed intersectional inhibitory DREADD reporter mouse line with our intersectional NDNF/Cxcl14 genetic strategy to suppress the output of canopy cells. These experiments will allow us to generate hypotheses about the role of these neurons in regulating the integration of top-down information in the cerebral cortex.

NIMH - National Institute of Mental Health

ABSTRACT/SUMMARY Understanding the role of sex steroids like estrogens and androgens within the brain is crucial for elucidating their impact on various brain functions, including mood, cognition, and reproductive behavior. However, our current understanding of how these steroids modulate neuronal physiology and behavior in vivo remains limited, largely due to the absence of technologies capable of monitoring these hormones in real time within the brain. The goal of this project is to bridge this gap by developing a suite of genetically encoded fluorescent biosensors for steroid hormones. These biosensors will enable cell-specific monitoring of steroid hormone levels in awake and behaving animals, providing unprecedented insights into the dynamic roles of neuroactive steroids derived from endocrine tissues or synthesized within the brain. Our central goal is to engineer green fluorescent biosensors for estrogens and androgens based on their nuclear receptors. By incorporating fluorescent reporters into receptor regions that change conformation upon ligand binding, we aim to create sensors with enhanced ligand affinity and signal strength through structure-guided engineering and high-throughput screening. Additionally, we plan to develop far-red shifted sensors for multiplexed applications, extending our innovative approach to a broader spectrum of steroid hormones. Our project is innovative because it employs structure-guided design and a high-throughput engineering platform to rapidly screen thousands of protein variants. This approach significantly accelerates the development of biosensors capable of real-time steroid hormone monitoring in vivo. Furthermore, by applying these sensors to study the rapid effects of steroid signaling on neural activity in the hypothalamus of behaving mice, we aim to dissect the temporal dynamics of steroid action beyond their genomic effects. This research is significant because it addresses a critical gap in our ability to study steroid hormones in live animals. By providing tools for real-time monitoring of steroid hormone signaling within genetically specified brain cell types, we pave the way for a deeper understanding of how steroids influence brain circuit function and behavior. The successful completion of this project will not only advance our knowledge of steroid hormone dynamics but also offer new avenues for therapeutic intervention in steroid-related disorders.

NIAID - National Institute of Allergy and Infectious Diseases

Background: Lutzomyia sandflies are the primary vectors of Leishmania in the neotropics. Leishmania infection manifests as multiple diseases, some of which are lethal. During the past 20 years, the distribution of Leishmania-carrying sandflies has spread, with significant increases in the number of reported cases of leishmaniasis disease (1–6). Given the contemporary geographic range of Lutzomyia sandflies, over 350 million people are at risk of leishmaniasis. Nearly 1.5 million cases are reported annually (1, 3, 5, 7, 8), in what is likely an underestimate of infection burden (7, 9–14). Understanding the processes that lead to genetic and phenotypic variation in sandflies influences the ability to monitor and predict vector spread and control. Aims and Approach: In this proposal, we leverage our expertise to advance sandfly management using integrative analyses of insecticide resistance (IR). We have the necessary permits and facilities to collect and work with sandflies, enabling us to rapidly initiate and efficiently complete the proposed work. Aim 1 will generate pangenome graphs with highly contiguous reference genomes for five vector species. This will enable the detection of loci under selection across sandfly species and establish a sandfly community genomic resource. Aim 2 will build on our preliminary data and expertise ecological and evolutionary genetics to sample natural variation and map alleles associated with pyrethroid resistance – one of the main strategies for controlling sandflies – in Lutzomyia evansi. This work will provide insights into the genetic basis of IR in a genetically understudied disease vector. Long-term goal: Our research program focuses on evolutionary and population genetics in insects. We are now applying our expertise to systems relevant to human health. This proposal launches an integrative research program combining population genomics, lab experimentation, and field sampling to generate genomic resources and foundational knowledge for Lutzomyia sandfly vectors. The outcomes—quantifying IR and identifying its genetic basis—will improve understanding of Lutzomyia adaptation to control strategies. Our preliminary data, necessary permits, and expertise in sampling and analyzing natural variation enable us to efficiently complete the proposed work on time. Health-relatedness: Leishmania disease burden is substantial, with estimates that over 12 million individuals are currently infected (8, 15). This disease burden is projected to grow as global trade, and travel facilitate range expansion of vectoral Lutzomyia species. Identifying genomic regions under selection – and specifically those involved in IR – using innovative approaches contributes significantly toward understanding and ultimately projecting leishmaniasis risk in contemporary and in novel geographical areas as sandfly vector populations continue their expansion toward the US.

NIAMS - National Institute of Arthritis and Musculoskeletal and Skin Diseases

Abstract Rheumatoid arthritis (RA) is a disease with no cure that affects approximately 1% of the world's population. It is characterized by inflammation, cartilage/bone destruction, and disorders of the cardiovascular, pulmonary, and skeletal systems. Disease-modifying antirheumatic drugs such as methotrexate (MTX) effectively manage the symptoms. Clinically, methotrexate is administered in a high dose once a week due both to the rapid renal clearance and toxic side effects. Studies show that patients undergoing treatment experience a twenty-six percent reduction in the number of inflamed joints. While the MTX is highly effective, some patients experience articulations that do not respond to systemic treatment with the drug. To address this issue, we will create nanocarriers to deliver MTX to specific joints that do not respond well to its systemic delivery. The conjugation chemistry will be developed to bind MTX to gold nanoparticles through environmentally responsive linkers, which will serve as a slow-release nanocarrier system when injected into the targeted joints. Colloidal gold has demonstrated anti-inflammatory properties when used to treat RA. The gold nanocarriers, along with MTX and linkers sensitive to environmental cues like reactive oxygen species and the acidic conditions of the synovial fluid, are expected to have a synergistic and pronounced effect in reducing inflammation. The acid and reactive oxygen species release mechanisms of the nanocarriers will be studied along with their cytotoxicity. Next, the efficacy of the gold nanocarriers will be determined by analyzing the levels of reactive oxygen species, tumor necrosis factor (TNF-), interleukin 6 (IL-6) and interleukin 1 (IL-1) produced by RAW 264.7 macrophages upon in vitro inflammatory stimulation. RAW 264.7 macrophages are used as a model cell line to study inflammation responses of other gold nanoparticle formulations. It is expected that the inflammatory variables evaluated will be reduced in vitro. This research will lay the groundwork for testing in animal models and may eventually lead to changes in the administration of methotrexate to patients.

Environmental Protection Agency

The U.S. Environmental Protection Agency (EPA) is planning a new funding opportunity under the Clean School Bus (CSB) Program to encourage broad participation and fleet turnover by providing school districts with greater choice in school bus technology while strengthening oversight and compliance. To support the development of this new funding opportunity, the EPA invites public comments to inform the Agency's understanding of the availability of alternative fuels and associated technologies in the medium- and heavy-duty school bus sector. The EPA is committed to ensuring that all technologies covered by the definition of "clean school bus" in 42 U.S.C. 16091(a)(3) are included in a new funding opportunity later this year. In addition to seeking information on available alternative fuels and buses for the upcoming funding opportunity, the EPA is also seeking information from the public on additional ways the Agency can further safeguard taxpayer dollars. The EPA conducted a programmatic review of the previous CSB funding rounds and identified areas for enhanced controls. The EPA is interested in identifying additional ways to prevent waste, fraud, and abuse within the CSB Program and is requesting comment to ensure that the Agency has the most comprehensive information available regarding robust oversight and compliance.

National Park Service

The objectives of this task agreement is to support the development of Groundwork Cincinnati - Mill Creek (GW CMC) to support projects and programs that facilitate community led conservation, youth engagement, brownfield remediation and green space improvements and the development and stewardship of parkland, recreation areas, nature reserves, and trail systems in Cincinnati, OH.

NIGMS - National Institute of General Medical Sciences

ABSTRACT Halogenation is a chemical reaction that is responsible for the introduction of a halogen atom including chlorine, bromine, iodine and fluorine (Cl, Br, I, F) into an organic compound. It has emerged as one of the most important reaction types for the selective synthesis of pharmaceuticals and biologically active compounds and for late- stage functionalization of drugs to enhance their therapeutic properties. Despite the importance of halogenation in drug development, current methods are largely reliant on reagents that are air- and moisture-senstive and/or unselective in the halogenation event, leading to the production of undesired halogenated byproducts. Enzymes are an attractive catalyst option for performing halogenation and halogenation-mediated reactions because of their unmatched selectivity and sustainability parameters. The overarching goal of my research group is to discover and develop halogenating enzymes for perfoming selective halogenation or halogenation-mediated reactions for chemical synthesis. Among the broad range of halogenase enzymes in nature, the vanadium- dependent haloperoxidase (VHPO) class of enzymes have been recognized as an attractive option for halogenation in chemical synthesis. These fascinating enzymes perform halogenation using inert halide salts and hydrogen peroxide as the terminal oxidant. Some additional notable features of VHPOs are their ease of access, tolerance to a broad range of solvents and temperatures and their ability to act on a wide range of substrate types. Over the next 5 years, the central goal of our research team is to expand the synthetic capabilities of this enzyme class in both halogenation-mediated and halogenation reactions for downstream application in the synthesis of biologically active compounds. In our first approach (Thrust A), we will use a concept referred to as enzymatic halide recycling to perform new biocatalytic oxidation and bond forming reactions. In this approach, a catalytic quantity halide salt is repeatedly oxidized by the enzyme to faciliate new bond formation using hydrogen peroxide as the terminal oxidant. In our second approach (Thrust B), we will develop a series of defunctionalative halogenation (replacing a functional group with a halogen) and halooxidation (addition of a halogen and an oxygen atom) reactions. Collectively, these approaches will provide a sustainable and selective catalyst platform for accessing intermediate and target compounds that are relevant to drug discovery and also dramatically expand the synthetic repertoire of halogenase enzymes for chemical synthesis.

NCI - National Cancer Institute

Project Summary/Abstract The technical and biologic expertise offered by Research Specialists provides a foundation of support that expedites the growth of research programs, shared resources, and cancer centers. Especially important in complex fields, such as the growing use of immunotherapeutics in personalized treatment of cancer, a comprehensive understanding of biologic responses requires cutting-edge technology and expertise that would be cost prohibitive to develop in individual laboratories. Monitoring immune responses in patients treated with immunotherapies is vital to understanding the mechanisms involved in these treatments and for the rational design of combinatorial clinical trials. This proposal demonstrates the need for support of the Research Specialist in the continued development of critical services provided by the Human Immune Monitoring Shared Resource (HIMSR), a University of Colorado Cancer Center (UCCC) Shared Resource at the University of Colorado Anschutz Medical Campus. The Research Specialist collaborates with UCCC members to support cancer-related investigator-initiated clinical trials with a full-service approach, from sample processing, to optimization and implementation of high parameter correlative assays for liquid and tissue biopsies, to primary data analyses. Continued support of the Research Specialist with this NCI R50 funding mechanism will allow time for the implementation of new technologies for spatial proteomic and transcriptomic imaging for use in the study of tumor microenvironments, development of novel computational approaches and improvements to existing computational tools for primary image analyses, and continued advancement of existing imaging technology with customized multiplex panels. Dr. Jordan's expertise and training in immunology and human clinical studies is a vital component of the Unit Director's Cancer Center Support Grant, many research programs of UCCC members, and to the completion of projects under the Head and Neck SPORE (NCI P50). As evidenced by rapidly expanding publications and funding opportunities that stem from its advanced techniques, the HIMSR has become an integral part of many research programs on campus and will be a vital component of the future growth of immunotherapies at CUAMC.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY Globally, respiratory syncytial virus (RSV) causes over 3 million hospitalizations of neonates/infants. While there are licensed vaccines in the U.S. for use in elderly populations, they are neither approved nor appropriate for use in neonates/infants who have immature immune systems. Palivizumab, a prophylactic monoclonal antibody (mAb) marketed by AstraZeneca and Sobi as Synagis™, has been available since 1998, but due to high cost and modest efficacy, its use has been restricted to high-risk infants. The recently approved single dose, long-acting mAb, nirsevimab (previously known as MEDI8897 and marketed as Beyfortus™ by Sanofi), has improved efficacy compared to palivizumab and was approved for use in all children up to 24 months of age. However, severe shortages and cost barriers have limited its availability in countries where it is currently approved (EU, US, Japan, Australia, Canada and China). In October 2023 the CDC issued Health Alert Network CDCHAN-00499 that recommended limiting the use of nirsevimab to higher risk children in the 2023- 2024 season due to the shortages, resulting in many eligible infants not receiving prophylaxis. In addition, nirsevimab is not currently available in any low- or middle-income countries (LMICs) and is unavailable in 8/10 of the most populous countries in the world. Mapp Biopharmaceutical, Inc. is developing a fully human mAb product, MBP002, that like nirsevimab, can be administered in a single dose per RSV season. Mapp’s objective is to dramatically lower the price and increase the global availability of RSV immunoprophylaxis. Further, unlike nirsevimab and Merck’s Phase 3 candidate, MK-1654, Mapp’s product is a two mAb cocktail, reducing the risk not only of clinical development of resistance, but also of a naturally occurring strain evading a single mAb. Indeed, this method of product failure occurred during a recent Phase 3 trial with Regeneron’s RSV mAb candidate and is the reason several mAb candidates lost their emergency use authorization for use against COVID-19. While nirsevimab resistant mutations were rare in a survey of 5675 RSV strains between 2015 and 2021, several mutations in the nirsevimab binding site have increased since 2020, including the resistant RSV-B variant K68N present in the U.S. and Australia. The impact of widespread use of nirsevimab on the frequency of resistant variants is under close post-marketing surveillance due to the potential for selection of escape mutants. Mapp has screened 70+ RSV clinical strains and found neutralization resistant strains to palivizumab, nirsevimab (MEDI8897) and Regeneron’s candidate, but no resistant strains to one of the MBP002 mAbs. In addition, the MBP002 mAbs are dramatically more potent in vitro and in vivo than palivizumab and have comparable neutralization and in vivo activity to nirsevimab. The requested SBIR support, together with significant in-kind contributions by Mapp, will allow for the advancement of MBP002 to IND submission (Investigational New Drug Application) and initiation of a Phase 1 clinical trial.

NIAID - National Institute of Allergy and Infectious Diseases

ABSTRACT Members of the IL-17 family (IL-17A-F) and its cognate receptors (IL-17RA-RE) mediate physiologically important immune responses, however, they can also drive inflammatory diseases, such as allergic asthma in case of IL-25. Despite its emerging clinical need, no drugs against IL-25 or its signaling pathway are currently clinically available. Our long-term goal is to identify small molecule inhibitors of the IL-25 pathway to probe and prevent IL-25-mediated diseases. The goal of this exploratory R21 application is to establish and validate a high throughput screening (HTS) system as well as relevant orthogonal assays that will enable successful IL-25 small molecule drug discovery using HTS-based hit identification and a defined hit advancement strategy. One major barrier for small molecule drug (SMD) development targeting IL-25 (and other members of the IL-17 family) is that the defined part of the signaling cascade relies on a series of hierarchically acting protein interactions that are inherently difficult for targeted drug development: IL-25 initiates signaling via binding and dimerization of IL-17 receptor RA- and RB-chains, which leads to recruitment/ dimerization of the common IL- 17R adaptor ACT1, followed by recruitment/ dimerization of TRAF6. IL-25 and IL-17RA/RB define the IL-25- specific signaling pathway, while ACT1 defines the common IL-17R family pathway. TRAF6, in turn, defines the boundary between various IL-17R family members and common, IL-17R-non-specific downstream pathways. Thus, signaling events up-stream of ACT1 represent a ‘drug target window’ that defines IL-25-specific inhibitors. Signaling events between ACT1 and TRAF6 represent a ‘drug target window’ that defines common inhibitors of the IL-17 family, including IL-17A as major driver of Th17 immune responses and inflammatory diseases. To overcome limitations related to targeted drug development, we developed a unique, cellular high throughput screening (HTS) platform that retains the advantages of phenotypic screening, i.e. the testing of complex responses in the natural environment of cells and, at the same time, eliminates non-specific compounds, thereby mitigating the major disadvantage of phenotypic screening. In this application, we propose (i) to validate already established reporter cell lines on robots in HTS/ 386 well- format and perform a small 3.6k pilot screen to test and calibrate our screening tool and (ii) to establish and validate a set of orthogonal assays that are suitable to advance IL-25-inhibitory compounds in a full-scale drug discovery campaign. We expect that establishment of these key components of HTS-based drug discovery (along with medicinal chemistry, pharmacology and clinical support by collaborators) will put us in an ideal position for future work to (i) identify specific IL-25-inhibitory small molecule compounds and (ii) develop them towards lead compounds with suitable drug-like properties to be used as probes and starting point for future drug development.

NSF

With the support of the Chemical Catalysis Program in the Division of Chemistry, Professor Pavel Nagorny of the University of Michigan is developing new recyclable catalysts that promote chemical reactions resulting in enantiomerically enriched (chiral) products. Access to molecules in their enantioenriched form is critically important for drug discovery, as it affects key molecular characteristics such as therapeutic potency, thereby underscoring the need for catalysts that can selectively form these molecules. The Nagorny research group has expertise in designing and using polymers that embed chiral acid catalysts. Professor Nagorny and his students will investigate how these catalysts work and will optimize them for the synthesis of molecular fragments commonly found in pharmaceuticals. It will generate new knowledge about organic molecule reactivity that advances the fields of catalysis, polymer chemistry, and asymmetric synthesis. The resulting techniques and catalysts will enable chemists to shorten synthetic sequences and develop chemical processes that are safer and more sustainable, thereby saving time, money, and natural resources. These studies will also be used to train graduate and undergraduate students who plan to pursue careers in the chemical and pharmaceutical industries, biomolecular science, and medicine. Professor Nagorny and his students will also participate in various training and outreach efforts focused on attracting K–12 students to STEM fields. With the support of the Chemical Catalysis Program in the Division of Chemistry, Professor Pavel Nagorny of the University of Michigan is developing new polystyrene-based chiral Brønsted acids and using them as recyclable catalysts for asymmetric organocatalysis in both batch and continuous flow setups. These catalysts will be synthesized using approaches designed to enable late-stage modifications of advanced intermediates, allowing the generation of multiple catalysts in a minimal number of steps, thereby simplifying the process of catalyst tuning. The project will target the immobilization of the best-performing classes of Brønsted acid catalysts, such as monomeric BINOL-based chiral phosphoric acids (CPAs) and dimeric, spatially confined imidodiphosphates (IDPs). The resulting polymeric catalysts are designed to be easily recovered and recycled, and will be employed in both new and established asymmetric transformations, such as transfer hydrogenation, allylation, acetal formation, and cycloaddition reactions. In addition, new strategies aimed at streamlining asymmetric reaction optimization with immobilized catalysts in continuous flow will be pursued to accelerate the discovery of optimal reaction conditions and reagents. 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.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

Abstract Ulcerative colitis (UC) is a chronic, idiopathic inflammatory disease that affects the colon, most commonly afflicting adults aged 30–40 years and resulting in disability. UC is characterized by relapsing and remitting mucosal inflammation, starting in the rectum and extending to proximal segments of the colon. Existing biological treatments have proved effective for some patients, yet up to one third of moderate-severe colitis patients are refractory to these therapies and may require colectomy. Novel biological targets are needed to modulate the dysregulation of the intestinal epithelial cells (IECs) as well as the activated intestinal immune population causing excessive host damage. RAB27A belongs to the RAS superfamily of monomeric G proteins and has been shown to localize to specific cellular compartments to regulate membrane trafficking in numerous cell types. Recent reports have shown its upregulation in UC patient colon samples, both in the intestinal epithelial cells and the surrounding immune population. In mouse models of UC, knock-down of RAB27A has been shown to improve the disease phenotype. RAB27A activity has also been shown critical to the toxic function of certain refractory immune cells including the neutrophil, NK-cells and dendritic cells . Notably, studies have suggested a potential role for neutrophil modulation in improving disease outcome. Both results from UC rodent models and descriptive clinical studies have shown that activated neutrophil toxicity is present and damaging to the colon, indicating that lowering its effect could substantially alleviate disease burden particularly in refractory patients. Nonetheless, currently there are no RAB27A or neutrophil targeting therapeutics in the clinic. To this end, Immunyx Pharma has developed a soluble small molecule RAB27A inhibitor to block the deleterious activities of the protein in ulcerative colitis. Our lead compound, IMYX-3 has already shown efficacy in rodent inflammatory models and has demonstrated positive PK and toxicity results. In this Phase II application, we will define, optimize and validate a treatment regimen and dosing for IMYX-3 in advanced models of mouse ulcerative colitis. Based on these murine validation and dosing studies, we will evaluate specific pharmacokinetic data for the therapeutic window. Next, we will prepare our drug formulation for GMP manufacturing and use this validated batch for our GLP toxicity studies in rodents. These key milestones will form the basis of FDA regulatory filings and prepare IMYX-3 for first in human studies.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary Human Metapneumovirus (MPV) is the second leading cause of lower respiratory tract infections (LRTI) in infants and young children, and a major cause of respiratory illness in the immunocompromised and the elderly. Unfortunately, there is currently no effective therapy, prophylaxis, or vaccine available for MPV, and only supportive care is available for those hospitalized with MPV LRTIs. MPV LRTIs can be lethal, with an observed hospital mortality rate among all MPV infections of ~10%, which approximates the ~9% mortality rate for influenza-infected adults admitted to the ICU. MPV shares much of the same pathophysiology as RSV, a closely related respiratory infection. RSV- induced LRTIs in infants can be effectively reduced by passive immunization, as illustrated by the clinical success of Synagis® (palivizumab), a monthly shot given to premature infants during the RSV season for at least 20 years, and the recently approved Beyfortus® (nirsevimab), a one-time shot indicated for all infants entering their first RSV season (not just high-risk infants). The clinical effectiveness of both Synagis® and Beyfortus®, which reduce RSV LRTIs by 70-80%, suggests that passive immunization should help similarly reduce risk for MPV-LRTIs. Inhalon is a clinical stage startup focused on advancing interventions against diverse respiratory infections, including MPV, RSV, SARS-CoV-2, and influenza. We are actively developing IN-003 to address the unmet clinical burden of MPV LRTIs. IN-003 is comprised of a pair of neutralizing mAbs against MPV-F proteins, originally identified from a library of human neutralizing mAbs against MPV, each of which bind and neutralize diverse MPV strains with picomolar affinities. Importantly, both mAbs exhibit strong resistance to viral escape. In this work, we plan to advance IN-003PI (i.e. IN-003 for passive immunization) against MPV LRTIs. We have shown in vivo that the individual IN-003 mAbs offer effective passive immunization against MPV in cotton rats and mice, and also prevent MPV infections ex vivo when dosed basally to well-differentiated human airway epithelial cultures prior to apical inoculation of MPV. To enable once-seasonal protection, the IN-003 mAbs will incorporate LS mutations in the to greatly increase affinity to FcRn and therefore prolong circulation. Herein, we will file a pre-IND meeting with the FDA, produce a GLP tox batch of IN-003PI, and conduct key IND-enabling studies, including GLP tox in non-human primates. The proposed work represents the critical path to advance IN-003PI into the clinic. Successful completion of this work will put us in a position to quickly file IND following production of GMP clinical trial materials for the first clinical study of an immunoprophylaxis against MPV.