Grants

NSF

With the support of the Chemical Measurement and Imaging Program in the Division of Chemistry, Professor Hao Chen of the New Jersey Institute of Technology focuses on the development of a new mass spectrometric approach for detecting elusive intermediates involved in the degradation of per- and poly-fluoroalkyl substances (PFASs). PFASs, widely regarded as "forever chemicals", are extremely stable due to their high carbon-fluorine (C-F) bond energies. From a human health perspective, PFAS exposure has been linked to the generation of reactive oxygen species (ROS), oxidative stress, DNA damage, and an elevated risk of chronic conditions such as cancer and inflammatory disorders. The widespread presence and harmful effects of PFASs in the environment have made their degradation a critical research focus. However, PFAS degradation mechanisms are not fully understood, largely due to lack of information about their elusive degradation intermediates. The project aims to tackle this very challenging problem. Its success would greatly benefit the public by enabling researchers to understand PFAS degradation chemistry and to discover new and effective ways for PFAS removal. This project will engage undergraduates in the research and reach out to even younger students to acquaint them with this important interdisciplinary science, technology, engineering and mathematics (STEM) area. The difficulty in detecting and characterizing the transient PFAS degradation intermediates is due to their short lifetimes and low abundances. This research project will develop a novel in-situ high-resolution mass spectrometry (HRMS) approach to capture and detect short-lived degradation intermediates generated from either thermal or electrochemical degradation of PFAS. This project is expected to have a high impact in the fields of both analytical chemistry and environmental science. 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.

National Institutes of Health

Development of Interventions to Prevent and Treat Substance Use Disorders and Overdose (UG3/UH3 - Clinical Trial Optional)

NCI - National Cancer Institute

ABSTRACT Immunotherapy has revolutionized the way cancer is treated. However, limitations in efficacy combined with immune-related toxicities following systemic administration of immunotherapy motivate the development of safer, more effective approaches. Interest in intratumoral delivery of immunotherapeutics has increased in recent years. Intratumoral or intralesional injections have the potential to maximize the delivery of immunotherapies within the tumor while alleviating toxicities associated with traditional systemic delivery of broadly activating immunotherapies. Unfortunately, the feasibility and efficiency of intratumoral injections are grossly overestimated. Even with image-guidance, intratumoral injections often miss a targeted tumor and/or injected contents quickly leak out of a tumor due to high hydrostatic pressures. Viscoelastic hydrogels can resist shear forces that typically expel saline-based solutions from tumors after an injection. Formulating immunotherapeutics within hydrogels, therefore, may improve retention and overall exposure to immunotherapy within an injected lesion. Our preliminary and published data demonstrate that a novel injectable, imageable, biocompatible hydrogel, called XCSgel, can increase the retention of co-formulated immunotherapies leading to profound increases in antitumor activity. Specifically, interleukin-12 (IL-12) formulated in XCSgel (XCSgel-IL12) is retained within an injected tumor for up to 4 weeks and can safely eliminate the majority of injected and uninjected distal breast tumors. XCSgel is tunable through manipulation of several parameters, leading to multiple variations with different mechanical properties, degradation rates, release kinetics, etc. It is not obvious which type of XCSgel would maximize the antitumor potential of IL-12. Thus, it is important to optimize XCSgel-IL-12 prior to clinical translation. The overarching goal of this proposed Phase I STTR project is to identify and de-risk a lead XCSgel- IL12 candidate with demonstrated efficacy and safety profiles in stringent murine TNBC models. Aim 1 will focus on synthesizing, characterizing, and optimizing XCSgel-IL12 in a rigorous downselect process from twelve potential candidates to three. Aim 2 will evaluate the pharmacokinetics/pharmacodynamics, safety and toxicology of the top three XCSgel-IL12 candidates. Although localized XCSgel-IL12 immunotherapy may be effective in controlling a range of solid malignancies, this project will focus on triple-negative breast cancer (TNBC) as an initial indication. TNBC is an aggressive subtype of breast cancer that requires debilitating and toxic treatments and yet suffers from unacceptably high recurrence rates.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY With the most people ever in history currently living with HIV, finding a cure remains a global priority. Non-human primates (NHPs) are a clinically relevant model for developing strategies for HIV cure. The safety and efficacy of therapeutic curative approaches in ART-suppressed SIV-infected NHP have provided the basis for several strategies currently in human clinical trials. While the current daily ART regimen has advanced preclinical SIV cure research, it has its drawbacks. In addition to the high costs for staffing and drug administration, daily ART injections require daily manipulation of the NHP and unintended immune activation and perturbation from the carrier molecule Kleptose. Long-acting ART offers a novel and promising therapeutic approach as an alternative to both treat and prevent HIV. However, the impact of LA-ART on the latent viral reservoir is unknown and may alter approaches to cure HIV. Here, we are proposing to use LA-ART therapeutically in SIV-infected rhesus macaques to achieve full viral suppression. In specific aim 1, we will determine the ability of a long-acting antiretroviral regimen to achieve durable viral suppression in SIVmac239-infected rhesus macaques and assess the impact on the latent viral reservoir. In aim 2, we will characterize the safety, tolerability, and pharmacokinetics of repeated dosing of long-acting ART regimen and evaluate its effects on anti-SIV immunity. The results generated here will be directly compared to previously published historical controls that received conventional daily ART. This work will provide a safe and viable alternative to current daily antiretroviral therapies and lay the foundation for the next generation of ART in non-human primates.

NSF

A diode laser device is made from semiconductor, a material with its electrical conductivity dictated by the quantities of negatively and positively charged mobile particles within itself, which are referred as electrons and holes, respectively. An n-type semiconductor possesses more electrons than holes while a p-type semiconductor has more holes than electrons. Traditional diode laser devices contain a key component equivalent to a p-n junction, where electrons and holes meet and recombine to emit light. Diode lasers with emission wavelengths in the infrared and visible spectral bands are widely commercially available. In contrast, those devices emitting lasing with wavelengths less than 315 nanometers in the deep ultraviolet (UV) spectral bands (UV-B and UV-C) are severely underdeveloped. This is because the material used to develop those lasers, namely aluminum gallium nitride (AlGaN), has fundamental issues including weak p- and n-type conductivities. This project addresses this challenge by developing novel metal-semiconductor-metal random laser devices based on magnesium zinc oxide (MgZnO) semiconductors. These devices use the injection of high-energy electrons from power supply to generate amplified numbers of electron-hole pairs for lasing, thus circumventing the strong p-type requirement in conventional semiconductor p-n junction lasers. The principal investigator and his students will work to achieve MgZnO lasers with deep-UV emission wavelengths between 315 and 200 nanometers. In addition, a novel approach will be used to convert random lasers which emit light with multiple wavelengths in all directions into highly directional, single-wavelength MgZnO lasers. The success of this effort will enable the development of portable semiconductor lasers in the deep-UV spectral bands for next-generation data storage and recording, medical diagnosis and surgery, photodynamic therapy, biological agent detection and sterilization, and water purification. Thus, it will have a profound positive impact on national health, prosperity and welfare. This project will train PhD students and undergraduate students who will graduate with versatile skills to advance semiconductor photonics technology in industry, academia, or government. Educational outreach will be extended to K-12 students to foster their interest in semiconductors. Additionally, the project will help train semiconductor nanotechnology technicians, a workforce increasingly in demand by society. Technical Description: This project seeks to overcome fundamental challenges to the development of deep-UV semiconductor lasers. It will demonstrate novel MgZnO deep-UV laser devices with an emission wavelength range between 200 and 315 nm in the UV-B and UV-C bands using metal-semiconductor-metal junctions rather than conventional p-n junctions. Gallium-doped n-type MgZnO nano-column semiconductor thin films with various Mg mole fractions will be grown using molecular beam epitaxy. Deep-UV metal-semiconductor-metal random lasers with scalable output power as a result of impact ionization, and their mode-coupled highly directional and single-mode lasers will be simulated, fabricated and characterized. Despite the enormous success of semiconductor lasers in all other spectral ranges including near-UV, visible, infrared and terahertz, electrically driven coherent single-mode semiconductor lasers operating at wavelengths less than 315 nm at room temperature are rare. This research will fill the 200 to 315 nm range wavelength gap, which will impact areas such as information storage, display and imaging, biology and medical therapeutics, and water purification. 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

This projects seeks to develop advanced materials that are able to pre-concentrate oxidized mercury compounds (HgII) to enable improved measurement of their concentrations and characterization of their chemical behavior. Better understanding the abundance and chemistry of oxidized mercury is critical for assessing mercury exposure risks in both environmental and industrial settings and for informing effective management and policy decisions. The objectives of the project are to: (1) model chemical and physical interactions between simple HgII compounds and crystalline oxide substrates, and (2) correlate HgII capture with oxide surface properties under laboratory and field conditions. This is a novel interdisciplinary project that combines surface chemistry, glass science, and atmospheric mercury research, and will support the training of students and early-career researchers in interdisciplinary environmental science. 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.

NINDS - National Institute of Neurological Disorders and Stroke

ABSTACT Migraine is a severely disabling neurologic condition and the second most disabling disorder worldwide, affecting over a billion people. The prevalence of migraine in the U.S. is increasing, with 15% of the population suffering from either episodic or chronic migraine. The global economic burden of migraine is staggering, at approximately $20 billion in the U.S. and $56 billion worldwide per year. Migraine is one of the most common reasons for emergency room visits. Despite first-line treatment recommendations, many migraine patients are still prescribed opioid pain medications, particularly in emergency departments (EDs). There is an enormous unmet need for an effective and affordable non-drug abortive therapy for acute migraine to significantly improve quality of life and substantially reduce the cost burden to our healthcare system and society. CoolTech has developed the Mi-Helper device - a novel approach to migraine treatment that is simple, safe, and drug free, using nothing but clean, dry air. Mi-Helper induces thermodynamic neuromodulation by a low flow of dry, ambient air across the nasal turbinates. This approach targets the sphenopalatine ganglion (SPG), a nerve bundle behind the upper airway that is associated with migraine symptoms. Evidence has shown that SPG inhibition, including the use of intranasal lidocaine, can reduce the pain and duration of migraines. However, the location of the SPG makes it hard to access to apply anesthetics and requires a skilled physician to administer. Neuromodulation by way of transnasal cooling is an approach that provides fast, convenient, drug-free symptom relief. We have demonstrated proof of concept of this technique’s capacity for pain and symptom reduction in multiple in-clinic, feasibility studies and in a decentralized, double-blinded, randomized controlled study in which the sham was confirmed, blinding was effective and a range of doses were tested to determine the most effective dose that was statistically significant compared to the control. Upon commercialization, Mi-Helper will offer a fast, low-cost, non-invasive treatment designed for on-demand use in an at-home setting at the earliest sign of onset. In this Direct to Phase II SBIR, CoolTech will conduct a double-blind, decentralized, randomized, sham- controlled clinical trial to demonstrate the safety and confirm the optimal dose of Mi-Helper as an acute treatment for episodic migraine, as well as optimize the usability of Mi-Helper for the target population. Additionally, CoolTech will implement design enhancements to improve the Mi-Helper device, focusing on reusability of key components, which will enhance usability, affordability, and functionality. These improvements aim to increase the benefits to migraine patients and strengthen the Mi-Helper's competitive position in the market.

NEI - National Eye Institute

Project Summary Usher syndrome type 2 (USH2) is the most common inherited condition for combined loss of hearing and vision worldwide. Several pathogenic variants in the USH2A gene have been reported to cause USH2 and non- syndromic retinitis pigmentosa (RP). The development of genetic therapies for USH2A has been mainly hampered due to the large size of the gene (15.6kb). The payload capacity of AAV restricts the USH2A gene from being included in a single vector. The USH2A protein contains several repetitive domains, and we hypothesize that some of these repetitive domains in the USH2A protein could be dispensable for its function, which is evidenced by recent exon skipping studies. This proposal exploits an innovative minigene approach by systematically deleting repetitive regions in USH2A and screening the functional efficacy of USH2A minigenes (mini-USH2A) both in vitro and in vivo. In addition, the proposed work also aims to deliver mini-USH2A to the retina and cochlea to rescue the USH2A phenotype. Our preliminary data has identified a mini-USH2A (mini- USH2A-4) and validated its functional efficacy in vitro using the Ush2a knock-out cell line. In this proposal, we aim to further characterize and functionally evaluate mini-USH2A-4 and generate novel mini-USH2A by sequential deletion of EGF-Laminin domains in mini-USH2A-4. This work will evaluate the functional efficacy of mini-USH2A through a detailed investigation of biochemical interactions using cell and animal models. The proposed work will also aim to develop an AAV-based approach to deliver mini-USH2A to the retina and cochlea to evaluate the therapeutic efficacy in the Ush2a knock-out mouse model. Successful completion of this study will set the stage for future clinical applications for treating USH2. The proposed research is clinically significant and will contribute to the mission to develop therapies targeting IRDs.

NCI - National Cancer Institute

ABSTRACT Primary high-grade gliomas (pHGGs) constitute the leading cause of cancer-related mortality in children and adolescents aged. Initial treatment strategies typically consist of gross total resection, when feasible, followed by focal radiotherapy and chemotherapy. However, the choice/use of chemotherapy remains uncertain. Although numerous therapeutic approaches have been tested, outcomes remain dismal. Since there is no standard chemotherapy for pHGGs, new treatments are urgently needed. To overcome the clinical challenges related to developing new treatments for pHGGs, our primary objective for this project is to develop a new tumor-targeting therapeutic agent that is specifically designed for pHGGs on a molecular basis. Recent studies revealed significant differences in the miRNA expression profiles between pHGGs and normal brain tissues, indicating that several sets of miRNA are oncogenic and highly overexpressed in pHGGs. Specifically, our preliminary data showed that miR20a is aberrantly expressed and has an oncogenic function in pHGGs including glioblastomas (pGBM) and diffuse intrinsic pontine glioma (DIPG, diffuse midline glioma). Although pGBM and DIPG are pathologically different types of highly lethal pHGGs, they share oncogenic molecular profiles, suggesting that we can attack the same molecular targets for both pGBM and DIPG. Given that aiming to diminish oncogenic miRNA expression by using antisense-miRNA is a promising strategy, delivery of antisense-miRNA is currently the major challenge [e.g., lack of tumor targeting, poor transport, and cellular penetration]. To overcome these challenges, we developed a cell-penetrating peptide (p28)-conjugated with antisense-miR20a (namely AmiR20-p28). Our preliminary results showed that systemically injected AmiR20-p28 preferentially accumulates at the tumor lesion in the brain, and induces apoptotic cell death by significantly silencing endogenous miR20a in tumors and blood (circulating miR20a as a potential biomarker), thereby extensively prolonging the survival of mice bearing pGBM. Based on our data, we hypothesize that the development of a pHGG tumor-targeting AmiR20-p28 will provide a novel therapeutic tool. To test our hypothesis, we set realistic aims; Aim1) Determine the pharmacological activities of AmiR20- p28 in pHGGs. Aim2) Establish biomarkers, pharmacokinetics, and toxicity of AmiR20-p28. Our experimental plans were designed with multiple validations and blinded conditions, and our multi-disciplinary team integrates expertise in basic, translational, and clinical brain cancer biology, molecular biology, pathology, and biostatistics. We seek to overcome current limitations by using AmiR20-p28 for pHGGs for which there is no cure. Upon completing our aims, our unique approach can provide a broad impact on developing CPP/miRNA- based agents for pHGGs. It can also potentially provide better treatment strategies for pHGGs patients, which is a major milestone and relevant to the NIH focus area.

NHLBI - National Heart Lung and Blood Institute

Project Summary This program focuses on the development of a new class of antiarrhythmic agents that normalize dysfunctional RyR2 channels, the calcium release channel in the sarcoplasmic reticulum (intracellular) membrane. Human RyR2 mutations cause two distinct genetic arrhythmia syndromes: (i) Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) due to RyR2 gain of function mutations, and (ii), Calcium Release Deficiency Syndrome (CRDS) due to RyR2 loss of function mutations. RyR2 gain of function due to post-translational modifications has been implicated mechanistically in heart failure and atrial fibrillation. RyR2-selective modulators are lacking. The overarching goal of this transdisciplinary program is the selection of antiarrhythmic clinical candidates based on ent-verticilide, a potent inhibitor of RyR2-mediated calcium release in cardiomyocytes. Preliminary data for this first potent and selective inhibitor include efficacy in several models of disease, and PKPD in a chronic dosing study. Our collective results support the rationale that selective therapeutics can improve our understanding of RyR2 biology and lead to preclinical candidates. Since ent-verticilide is not a natural product, all compounds must be obtained by de novo chemical synthesis. Chemical synthesis has provided renewable access to drug, and served as a platform for rapid analog development to support pharmacology studies. A comprehensive study of verticilides for therapeutic development is outlined in three Aims. Aim 1 describes the use of cryo-EM and photocrosslinking studies to further elucidate mechanism of action and create a platform for structure-guided design. The goal of Aim 2 is to increase the bioavailability of ent-verticilide and test efficacy of oral dosing in atrial fibrillation and heart failure models. Aim 3 describes lead backup development using ent-verticilide B1, an 18-membered ring oligomer. A strength of this approach is its ability to adapt to changes in the state of the art in RyR2 structural biology, an effort to which we will contribute (Aim 1). Highly potent and selective compounds will be advanced to in vivo safety and efficacy studies (Aim 2) with backup lead identification to de-risk the overall program. This program is highly collaborative and transdisciplinary in nature, and the studies will advance a novel class of compounds never-before- used in therapeutic development. Preliminary results have already advanced our understanding of RyR2 molecular pharmacology and promise new antiarrhythmic agents to improve human health.

NCI - National Cancer Institute

Project Summary Acute lymphoblastic leukemia (ALL) accounts for ~80% of all pediatric leukemia and ~20% of all adult leukemia. Since lymphocytes cannot synthesize their own asparagine, the frontline therapy for ALL includes PEG-asparaginase (PEG-ASNase; Oncaspar® and PEG), and sustained suppression of asparagine in the circulation is critical for achieving long-term remission. Unfortunately, up to half of the treated patients develop anti-PEG antibodies (APA) that specifically bind the polyethylene glycol (PEG) polymer on PEG- ASNase. The resulting immune complexes are quickly eliminated from the circulation leading to loss of efficacy, and also directly results in high rates of hypersensitivity reactions, including anaphylaxis. Currently, these immune responses force PEG-allergic patients to switch to non-PEGylated versions of ASNase that require dosing every 2 days (in contrast to every 2-3 weeks for ASNase), substantial rates of allergic reaction (since the underlying ASNase is also immunogenic), and overall nearly 100% increase in medical care costs. To overcome APA-related challenges, Mucommune has been advancing MM010, an adjunctive therapy consisting of high-molecular-weight free PEG that targets APA specifically. MM010 takes advantage of the fact that PEG alone is largely immunogenic and unable to efficiently crosslink B cell receptors on APA+ B cells, in contrast to large PEGylated proteins or PEG-coated drug carriers that potently induce APA. Free PEG can saturate and competitively inhibit pre-existing APA from binding PEGylated drugs, thereby restoring prolonged circulation. In animal models ranging from rodents to pigs, MM010 has reliably suppressed APA induction, restore drug circulation, and eliminate severe allergic reactions, making it a promising solution to enhance the safety and efficacy of PEG-ASNase. Building off these promising findings, we are seeking to extend the use of MM010 to improve the clinical management of ALL. Specifically, we will investigate whether MM010 can effectively block APA induction and restore prolonged circulation (pharmacokinetics) and suppress asparagine levels (pharmacodynamics) for PEG-ASNase in rodents repeatedly infused with Oncaspar (Aim 1A), as well as prevent Oncaspar-induced anaphylaxis in the highly sensitive swine model (Aim 1B). We will in parallel file a pre-IND package with the FDA to gain regulatory clarity on the clinical development pathway (Aim 2A), and initiate stability studies of vialed, clinical grade MM-010 (Aim 2B). Successful completion of this project will provide the foundation for rapidly advancing MM010 as an adjuvant to improve the efficacy and safety of PEG- ASNase based treatment for all ALL patients.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY Radiation induced lymphopenia (RIL) occurs in approximately one-half of cancer patients receiving external beam radiotherapy and this post-therapy condition has been shown to greatly reduce five-year survival rates. It is theorized that RIL is induced by direct lymphocyte cell kill, suggesting that assessment of the radiation dose to lymphocytes during radiotherapy is a necessary requirement for their consideration as a dose avoidance structure. In order to facilitate the computation of radiation dose to circulating lymphocytes, their supporting structures must be modeled in patient anatomic models used in tissue dosimetry. Currently, there exists no intra- organ blood vasculature nor lymphatic vasculature nor specialized lymphoid tissue structures within the International Commission on Radiological Protection’s (ICRP) reference computational phantoms – commonly used as a digital model of medical patients. Additionally, there exists major inaccuracies in the inter-organ whole- body blood vessels of these reference phantoms, and no modeling of microscale vasculature of blood or lymph has been yet undertaken to account for their large portion blood and lymph within the human body. Additionally, in applications of radiopharmaceutical therapy, blood self-dose, which is substantial for short-range radiations such as alpha particles and lower-energy electrons, emitted by many common radiopharmaceuticals, cannot be entirely quantified due to the lack of intra-organ vasculature in the ICRP phantoms. Dose to lymphatic vessels, additionally can be computed for the first time in RPT applications with these models in place. Therefore, this project hypothesizes that the development of a comprehensive model of blood and lymphatic vessels and corresponding lymphoid tissues will facilitate the computation of blood and lymphocyte radiation dose for the first time and will subsequently allow for consideration of circulating and non-circulating lymphocytes as organs-at- risk in radiotherapy treatment planning. The proposed project will be achieved by the completion of four Specific Aims: Aim 1: develop whole-body adult mesh models with intra-organ and inter-organ vasculature. Aim 2: develop corresponding lymphatic tissue and vessel models. Aim 3: develop organ specific microscale blood and lymph vasculature models. Aim 4: compute organ radionuclide S values for radiopharmaceutical therapy and lymphocyte dosimetry for external beam radiotherapy. Completion of these aims will greatly improve the computational models used as tools in reference medical dosimetry for a new assessment of dose to blood, as well as set the stage for adaptation of these models via deformable registration to expand to patient-specific modeling and personalized dosimetry.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY Sindbis virus (SINV) is a mosquito-borne alphavirus with a single-stranded positive-sense RNA genome. SINV is transmitted between mosquitoes and birds which serve as reservoir hosts for the virus but can cause spillover infections in other vertebrate hosts including humans. SINV can be classified into distinct genotypes (G1-G6) which aligns with their zoogeographical distribution. SINV causes frequent epidemic outbreaks of polyarthritis disease in Northern Europe and has also been associated with outbreaks in Africa. Epidemics in both these regions are caused by G1 viruses. SINV is widely distributed geographically but is not associated with epidemics elsewhere in the world. Despite being prevalent in mosquito populations in Australia, the SINV strains in this region (G2/3 and G6) have never been associated with epidemics of polyarthritis disease here, though isolated human cases have been reported, and serological data shows the occurrence of human infections. The Australian genotypes of SINV (G2/3, G6) are significantly divergent from the G1 viruses which cause frequent epidemics, suggesting that genetic differences between these genotypes may explain why G1 viruses have a propensity to cause epidemics but other genotypes do not. Most of the genetic tools available to study SINV are derived from G1 viruses, and there is only a single non-G1 cDNA clone (for the G4 virus XJ-160). There currently exists no molecular clones for studying other genotypes of SINV, including the divergent Australian G2 and G6 viruses. In this R03 we propose to construct cDNA clones for G2 and G6 SINV and characterize the in vitro replication properties of these clones and compare pathogenesis profiles of virus derived from these clones versus characterized G1 strains in a small animal model. We have established a subcutaneous model of SINV infection and shown that replicating virus can be detected in the skin and joints of adult mice, which reflects the pathogenesis observed in humans. Thus, this serves as a good model for examining the genetic determinants that underlie the epidemic potential of neglected SINV genotypes. These tools will be available to the research community and thus aid in further investigations of other key aspects of arbovirus transmission and pathogenesis including virus-mosquito interactions, species-specific determinants of transmission, and viral immunity.

OD - NIH Office of the Director

Summary The ability of a cell to sense, integrate, and convert mechanical stimuli into biochemical signals that result in intracellular changes is called mechanotransduction. Defects in mechanotransduction are implicated in the pathogenesis of various diseases, including arteriosclerosis, cardiomyopathies, asthma, and cancer. Thus, therapeutic means of manipulating mechanotransduction to restore mechanical homeostasis in diseased tissue represent a novel approach to treating various diseases. The goal of this R21 application is to develop a series of novel mechanotransduction-defective mouse strains that the research community can apply to multiple diseases. Vinculin, a cytoskeletal adaptor protein, plays a central role in sensing mechanical stress and mediating structural remodeling and functional responses within the cell. Site-specific tyrosine phosphorylation of vinculin has been proposed to regulate its mechanical role at both cadherin and integrin adhesions. We hypothesize that vinculin phosphorylation regulates the mechanical response of tissues to increase forces by adjusting the balance between cell-cell and cell-extracellular matrix (ECM) adhesions. This R21 proposal seeks to develop and validate a series of point mutations that alter posttranslational modification of vinculin in mice, thus allowing investigation of vinculin mechanosensing in physiology and disease. The following interrelated aims are proposed: (1) To test the hypothesis that tyrosine phosphorylation (Y822) of vinculin coordinates the mechanical response of cell adhesion structures. (2) To test the hypothesis that vinculin phosphorylation (Y100/Y1065) is required to facilitate cell-ECM interactions. Due to the vast effects of mechanobiology on disease pathogenesis, our innovative mouse models are anticipated to be widely used by scientists in an array of fields and, therefore, of relevance to multiple NIH Institutes.

NEI - National Eye Institute

Project Summary Addressing the complexities of vision at the cellular and molecular level necessitates an advanced spatial omics technology, capable of mapping the 3D distribution of specific molecular markers within tissue from the cellular level down to the ultrastructural scale. While spatial proteomics technologies have revolutionized in situ molecular profiling of tissues, limitations in resolution, epitope accessibility, sample degradation, and 3D imaging capability have hindered their widespread impact. This is the focus of our project, which proposes to combine two groundbreaking microscopy technologies, pan-Expansion Microscopy (pan-ExM) and FLASH- PAINT, into a revolutionary new method named pan-OptiX. Pan-ExM enhances spatial resolution ~20x by expanding samples physically, while retaining the proteome for subsequent labeling. FLASH-PAINT, on the other hand, allows rapid and efficient multiplexed protein labeling with minimal sample damage. By engineering these technologies for compatibility, pan-OptiX promises multi-scale and multi-color imaging, in 3D, across whole tissue sections, with no label limitations due to degradation or steric hindrance. Panluminate, Inc. is a leading spatial proteomics company which holds an exclusive license to both technologies from Yale. Our proposed project aims to develop pan-OptiX and apply it to map the circuitry of the mammalian retina. We specifically propose to (1) validate a second-generation pan-ExM protocol compatible with post-expansion DNA-conjugated antibody labeling and multiplexing, (2) develop novel FLASH-PAINT probes compatible with expansion imaging, (3) build an automated light sheet microscope designed specifically to image pan-OptiX samples, and (4) characterize the molecular and ultrastructural heterogeneity of the mouse retina. Ultimately, the developments proposed here will enable Panluminate to disseminate pan-OptiX sample preparation and imaging to every research and biopharma lab around the world.

NIAMS - National Institute of Arthritis and Musculoskeletal and Skin Diseases

Project Summary Duchenne muscular dystrophy is a severe X-linked genetic disorder characterized by progressive muscle degeneration and weakness caused by the loss of dystrophin protein. There are no curative therapies for DMD, and recently approved micro-dystrophin gene therapies have shown modest clinical benefit. Adeno-associated virus (AAV) delivered CRISPR gene editing strategies have achieved high rates of corrective gene editing in mouse and canine models of DMD but rely on dangerously high doses of AAV and result in constitutive expression of immunogenic gene editors. Lipid nanoparticles (LNPs) have emerged as a promising non-viral delivery strategy for gene editing but have lower delivery efficiency in skeletal muscle than AAV. Significant effort has been put towards improving delivery efficiency of LNPs, however, the factors that limit gene editing in skeletal muscle after delivery have not been addressed. Gene editing efficiency is not only determined by the entry of the delivery vehicle into the cell but also by the ability of the gene editor protein to populate all the myonuclei of the myofiber. Myofibers have hundreds or thousands of myonuclei that must be edited to achieve therapeutic benefit, but our preliminary data show that gene editor proteins cannot propagate to myonuclei distant from the site of transfection/transduction. In addition to the issue of low myonuclear propagation, low mRNA stability prevents the accumulation of sufficient levels of gene editor protein to edit most myonuclei. The objective of this project is to develop a muscle-optimized gene editor that can increase gene editing efficiency after non-viral delivery. This objective will be achieved by developing an in-vitro myotube based screening platform to identify modifications to the ABE8e adenine base editor that improve myonuclear propagation. An in-vivo pooled LNP screen will be performed to identify UTRs and codon optimization strategies that improve base editor mRNA stability in skeletal muscle. Modifications identified in these screens will be combined into a muscle-optimized base editor that will be used in LNP-delivered editing of a humanized mouse model of DMD. Completion of these studies will contribute to the development of a non-viral curative therapy for DMD. The insight gained from these studies will provide a basis for developing other muscle-optimized gene therapy cargos. These studies will be carried out in the laboratory of Dr. Eric Olson, PhD at University of Texas Southwestern Medical Center. All studies and career development will be performed under the mentorship of Dr. Olson and Dr. Ning Liu, PhD, pioneers in the field of skeletal muscle gene editing.

NINDS - National Institute of Neurological Disorders and Stroke

Project Summary: Development of Nav channel targeting antisense oligonucleotides as chronic pain therapeutics using an integrated platform based on machine learning and optical electrophysiology Effective treatment of chronic pain, including severe neuropathic pain conditions, such as erythromelalgia (EM) and small fiber neuropathy (SFN), remains a significant unmet medical need. Current therapies often lack full efficacy or come with serious risk, such as addiction associated with opioids. Antisense oligonucleotides (ASOs) offer a promising solution by knocking down specific mRNA transcripts with a long duration of action (~3 months) for sustained relief. ASOs have been successfully applied to patients to treat severe neurological disease. Here, we propose to develop an ASO therapeutic targeting the voltage-gated sodium channel, Nav1.7, which is highly expressed in dorsal root ganglion (DRG) neurons. Nav1.7 is a target with strong human genetics validation in pain transmission, including neuropathic pain. Reduction of channel expression via a therapeutic ASO may overcome the challenges of small molecules by mimicking the mechanism for congenital insensitivity to pain (CIP) resulting from hNav1.7 loss-of-function mutations. Administering the ASO via intrathecal (IT) injection will enable precise knockdown of Nav1.7 expression in DRG tissues in a state-independent manner with minimal risks for autonomic side effects, paving the way for an effective and targeted chronic pain therapy. To enable the selection of a Nav1.7 specific ASO with the desired profiles, Quiver has developed breakthrough technologies: (i) high throughput readout of neuronal excitability based on all-optical electrophysiology, (ii) combination of patient genetics and state-of-the-art human iPSC sensory neuron differentiation protocol for pain therapeutics validation in patient-based models, and (iii) a machine learning-guided ASO design and discovery platform for identifying the best ASO candidates with maximum therapeutic index. As an entry point, Quiver has identified a potent and selective hNav1.7 lead ASO (QV-2421) with confirmed activity in primary DRG neurons from relevant species and a clean in vivo CNS tolerability profile through in sillico prediction and empirical validation. In the UG3 phase, we will apply our breakthrough technology in combination with well-established preclinical assays to optimize the ASO lead. Quiver will collaborate with Boston Children's Hospital (BCH) to validate the functional impact of the lead ASO in sensory neurons derived from EM/SFN patients with neuropathic pain conditions. We will also evaluate long term in vivo tolerability and investigate translational biomarker approaches. Based on these studies, we aim to select one optimized ASO lead candidate with an acceptable tolerability profile and in vivo NHP PK/PD data profile by the end of the UG3 phase. In the UH3 phase, we will conduct dose-range finding toxicology studies, establish clinical biomarkers, perform large-scale manufacturing of the ASO candidate for IND-enabling studies, and initiate a Phase I clinical trial in young adult EM patients. The UH3 phase will conclude with the identification of an ASO candidate with an acceptable toxicology and preclinical profile that is advanced through IND-submission to enable the initiation of Phase I clinical trial. Our ultimate goal is to deliver a much-needed, non-opioid therapy to patients suffering from severe, chronic pain.

NIDA - National Institute on Drug Abuse

ABSTRACT Pain is a pervasive problem throughout society. It is estimated that about 105 million American adults are affected by moderate to severe pain. Opiates, such as morphine and fentanyl, have been the preferred treatment for moderate to chronic pain. Unfortunately, diversification and usage of opioids driven by fentanyl and its congeners has led to the opioid epidemic in America which is now reaching crisis levels. In addition to overdose which is lethal about 10% of patients on opiates are at risk of developing opiate use disorders (OUD). Current estimates suggest that ~5.6 million people have OUD and close to ~80k have died each year of opioid overdoses. Current treatments for OUD include buprenorphine and methadone which lack efficacy and are not well tolerated while overdose is treated with intranasal NARCAN® and OPVEE®. Both intranasal naloxone and nalmefeme are effective against counteracting overdose mediated by fentanyl while also leading to withdrawal which can be a physician’s worst nightmare. In addition, it is less clear if orthosteric site antagonists will block overdose to higher potency opioids in the nitazene class or carfentanyl class.Through this phased UG3-UH3 grant we propose to develop negative allosteric modulators (NAM’s) of the mu opioid receptors (MOR) as adjuvants in combination with/without naloxone to treat overdose and OUD while not potentiating the withdrawal mediated by naloxone. Towards this we have identified a first in class NAM named 368 identified from a large DNA encoded library (DEL) encoded library (Nature 2024; 631 (8021): 686-693). 368 modulates naloxone activity in cell lines as well as in vivo in mice. It blocks opioid antinociception as well as respiratory depression while causing less withdrawal when used in conjunction with naloxone supporting our hypothesis that NAM’s will reduce opioid withdrawal over orthosteric site antagonists used alone. In the UG3 preclinical phase of this grant we propose to optimize NAM368 for potency, ADME/PK optimization using structure-based design and focused medicinal chemistry. In addition to NAM368 template we have identified second hit we call HG3 from the DEL screening as well. cryoEM structures of both HG3 as well as 368 have been solved and will guide next generation hit to lead design. Additional DEL screening will continue to identify back-up compounds if optimization of current hits doesn’t lead to viable candidates. Compounds will be screened in a battery of biochemical, cell-based assays for potency and selectivity and eventually in ADME/PK assays both in vitro/in vivo and finally in mouse behavioral assays. The UH3 phase of the application will include CMC scale up, formulation development, pre-clinical toxicology and a full IND-enabling development plan to be carried out by ConfometRx. We have established a strong team of medicinal chemists, pharmacologists, structural biologists with expertise in academia, small biotech as well as big pharma to aid our drug development plan for this proposal to meet our milestones in both phases of this grant application.

NINDS - National Institute of Neurological Disorders and Stroke

Abstract Chronic pain affects millions of individuals globally, profoundly impacting their quality of life and imposing a significant burden on healthcare systems. While opioids have been the gold standard for treating moderate to severe pain, their serious adverse effects, such as dependency and tolerance, limit their clinical utility. Consequently, there is a critical need to explore new therapeutic targets for more effective and safer pain management strategies. Neuromedin U (NMU) is a neuropeptide that modulates various physiological functions, including pain perception, through its interaction with Neuromedin U Receptor 2 (NMUR2). Emerging evidence suggests that NMUR2 plays a crucial role in the transmission and modulation of pain signals within the central nervous system, and blocking this receptor could potentially alleviate pain. This proposal aims to establish a comprehensive structure-activity relationship (SAR) for two chemical series of NMUR2 antagonists, with the goal of optimizing their potency, selectivity, and drug-like properties. Upon completion of this project, we aim to provide structure-activity relationship data for compound optimization, early-stage ADME and pharmacokinetic information, laying the foundation for developing NMUR2 targeting tool compounds and drug candidates for chronic pain treatment.

NSF

With the support of the Chemical Synthesis (SYN) program in the Division of Chemistry, Professor Sarah Reisman of Caltech is studying the development of new catalytic reactions for the chemical synthesis of natural products. Natural products are molecules isolated from natural sources, which often serve as promising lead compounds for the discovery of new medicines or agrochemicals. The ability to synthesize these molecules in the laboratory can allow chemists to precisely modify their structure, study how the molecular structure affects function (e.g. biological activity), and design new molecules with improved properties. These synthetic efforts are enabled by fundamental studies aimed at developing new chemical reactions, particularly those that form carbon–carbon bonds. The experimental research seeks to develop new catalytic reactions that enable the efficient synthesis of the natural product enterocin. Although the proposed efforts will focus on enterocin, the broader impact of these studies will be to provide new general chemical reactions and strategies that can be used to synthesize related molecules of interest for applications in medicine and beyond. The rigorous training of scientists in the theory, methods, and strategies of synthesizing organic molecules will be an essential part of the funded research studies. Undergraduate, graduate student and post-doctoral researchers trained through this research experience will be poised to pursue careers in the chemical, pharmaceutical, agrochemical, biotechnology, and materials science industries. With support from the Caltech Center for Teaching, Learning and Outreach (CTLO), the Reisman team will participate in outreach programs that help students from local community colleges prepare for transfer opportunities and competitive undergraduate research programs by obtaining mentorship from Caltech graduate students. Structurally complex natural products – which often possess sterically congested three-dimensional topology and multiple reactive functional groups – challenge the limits of current synthetic methodology. This award will support the development of a radical-polar crossover annulation (RPCA) using photoredox catalysis to prepare the bridging bicyclo[3.2.1]octane core of the polyketide enterocin. Several approaches are proposed to elaborate from the RPCA product to enterocin, including the use of C–H functionalization. This work will also investigate the application of this radical-polar crossover annulation as a scaffold transposition method to access carbocycles from lactones. It is expected that these studies will advance the state of the art in catalytic C–C bond formation and demonstrate how modern synthetic methods can improve synthetic strategies. 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.

NIAID - National Institute of Allergy and Infectious Diseases

Abstract Plasma cell (PCs) antibody production is at the core of humoral immunity and inherently critical to the success of almost all existing vaccines. Their ability to persist and secret antibodies long after pathogen exposure or immunization provides long term sometimes lifelong immune protection against future encounter of the pathogens. What are the molecular pathways dictating the lifespan of PCs during their generation and subsequent maintenance remain largely unknown. Equally unknown are the regulators that control the diversity and activity of PCs reside in various lymphoid and non-lymphoid tissues. Cell lineage specific gene targeting and tracing (GT&T) with the Cre/LoxP system has been proven vital to understand the biology and regulation of various types of cells, especially rare cell populations such as PCs. However, existing mouse models for PC selective GT&T have significant drawbacks due to leakiness in either closely related cell lineage such as germinal center B cells or functionally intertwined cell lineage such as germinal center T cells. To improve selectivity in genetic dissection of PCs, we searched for genes that display high level expression in PCs but absent in the rest of lymphoid lineages. We found a single gene that fulfills this criterion and thus a good candidate for developing new mouse models with high selectivity and efficiency to drive Cre/CreERT2 expression in PCs. We propose to develop new Cre and CreERT2 models for GT&T studies in PCs. The proposed mouse model will address the immediate need for genetic and molecular investigations aimed at understanding longevity and behaviors of PCs in various tissues in response to infection or immunization.

NCI - National Cancer Institute

Abstract Breast cancer is the second leading cause of cancer-related death of women in the United States, and triple negative breast cancer (TNBC) is the most aggressive subtype. It comes back early, spreads fast, has a poor prognosis, and there are limited treatment options. Almost 90% of patients with TNBC that have spread to other parts of the body will pass away within five years, which is a lot worse compared to other subtypes, and the lungs are the most common site for TNBC to spread. Current standard therapies including platinum (Pt) drugs, taxanes, and anthracyclines are widely used but often cause severe toxicity and rapid drug resistance. TNBC responses moderately to current immunotherapies, and the overall treatment effects are heavily affected by a group of immune cells in tumor tissues, the immune-suppressing myeloid cells. This population contributes to forming immune environment which promotes tumor growth by inhibiting immune surveillance. Therefore, novel approaches are urgently needed to help these patients live longer, particularly for TNBC that spreads to distant organs such as the lungs, since they are more frequent and deadly. Reactive oxygen species (ROS) are unstable molecules within cells. While high levels of ROS can be harmful to cancer cells, they also significantly affect the behavior and interaction of cancer cells and myeloid cells in tumors. This makes them a promising target for TNBC treatment. However, most of current drugs do not produce sufficient ROS. Mild ROS not only fail to kill tumor cells but can promote tumor growth. Moreover, the immunosuppression effect remains a critical concern. It is important to design new drugs with high potency of ROS production. Accordingly, we have developed novel Pt-based therapeutics that produce robust ROS production, effectively eradicates TNBC cells rapidly, and successfully suppressed tumor growth in animal models. The goal of this project is to investigate the activities and biological mechanisms of these new drugs on both cancer and immune cells, with a special focus on TNBCs that have spread to lung. We will study the mechanisms how the drugs kill TNBC cells and assess the effectiveness when combined with current first-line TNBC treatment strategies. Moreover, we will explore their overall impact on the immune environment in lung metastatic TNBC nodules. These findings will provide a novel framework for development and assessment of drug efficacy against TNBC and other cancer types. Specific Aims: Aim 1: Elucidate how the ROS-generating Pt drug affects TNBC cells and immune-inhibiting myeloid cells. Aim 2: Assessment the performance of ROS-generating Pt drug in treating lung-spreading TNBC, along with its impacts on immune response.

NIAID - National Institute of Allergy and Infectious Diseases

Project summary: Development of Antivirals for Arthritogenic Alphaviruses This NIH grant proposal aims at the optimization of a New World (NW) alphavirus-specific quinazoline scaffold for the development of an anti-Old World (OW) arthritogenic alphavirus therapeutic candidate. With a particular focus on Chikungunya virus and other alphaviruses with a pandemic potential, this project responds to an urgent public health need for effective antiviral treatments for OW alphaviruses, given the recent increase in global prevalence of OW alphavirus infections. Dr. Chung's research group has made significant advancements in developing a quinazolinone-based antiviral specifically targeting NW alphaviruses like Venezuelan Equine Encephalitis Virus, exhibiting remarkable efficacy in preclinical models. This success, coupled with the compound's unique mechanism of action targeting the viral replicase complex, positions the quinazoline scaffold as a valid starting point for developing effective antivirals for other alphaviruses. However, the compound series necessitates significant enhancement in potency against OW alphaviruses. The team has identified a key residue that contributes to the NW alphavirus-specific antiviral spectrum. Here by elucidating the molecular interactions and clashes between the quinazolinone scaffold and OW alphavirus replicase complexes, the team aims to develop next- generation inhibitors with enhanced efficacy toward OW arthritogenic alphaviruses. Recognizing the need to enhance the potency of the quinazoline series against OW alphaviruses, the project proposes three Specific Aims undertaking a comprehensive and systemic approach integrating structural insights, medicinal chemistry, and preclinical evaluations. Dr. Luo's work in Specific Aim 1 focuses on mapping molecular interactions and clashes between the current quinazolinone scaffold and the OW alphavirus replicase complex, providing critical structural insights that will inform the design of new inhibitors in Specific Aim 2 led by Dr. Bannister. Specific Aim 2 efforts are directed towards identifying chemical groups leading to resistance in OW alphaviruses, designing and synthesizing new compounds tailored for OW alphaviruses, and ultimately developing lead candidates with optimized pharmacological profiles. Dr. Chung, in Specific Aim 3, will assess these novel antivirals in vitro and in vivo, supporting the structure-activity study for Aim2. Aim 3 will also study the mechanisms of action and resistance of the series. The overarching goal of the project is to deliver novel, potent therapeutic candidates for OW arthritogenic alphaviruses. With a highly integrated team of experts and a meticulous approach spanning molecular structural biology, medicinal chemistry, and virology, this project holds the promise of advancing preclinical development, ultimately addressing a critical gap in public health preparedness against OW alphavirus infections. Upon successful completion, the project will deliver promising therapeutic candidates ready for advanced preclinical development to address the critical needs for arthritogenic alphaviruses therapeutics.

NCI - National Cancer Institute

Abstract /Summary: Malignant pleural effusion (MPE) affects 200,000 individuals in the United States annually and lung cancer is the most common cancer to metastasize to the pleura. Unfortunately, cytologic examination of the pleural fluid (PF) obtained during thoracentesis only has a diagnostic sensitivity of ~50% with marginal increase upon serial sampling and limited by the need to detect malignant cells which are sparse in the PF. More invasive procedures such as thoracoscopic pleural biopsy are often required, but these procedures are associated with potential complications, hospital stay and delayed diagnosis and treatment. There is an urgent need to develop novel approaches to complement PF cytology. To address this need, we examined the potential of tumor cell products, specifically extracellular vesicles (EVs) and non-vesicular particles, for MPE diagnosis. In previous studies, we have detected and characterized EVs in PF and have identified and validated distinct EV microRNA (miRNA) signatures discriminating cytology-positive (C+) lung adenocarcinoma induced MPE (LA-MPE) from non-malignant pleural effusion (non-MPE). Additionally, because epithelial-derived surface markers are not native in the pleural space, we analyzed the expression of epithelial cell adhesion molecule (EpCAM), in cell- free PF of LA-MPE to identify tumor-derived EVs. We showed significantly higher EpCAM expression in (C+) LA-MPE compared to non-MPE. An essential next step is the evaluation of extracellular miRNA and proteins within the cytology-negative (C-) MPE populations, where current tests fail or are highly invasive. To Accomplish this goal, we will leverage samples from a diverse PF biorepository (current n=313) including (C+) and (C-) MPE and non-MPE with 35% African/Black participants and detailed tumor-specific and clinical data. Specific Aims will: 1) identify differentially expressed extracellular miRNA among patients with (C-) LA-MPE and non-MPE and validate the identified miRNA signature in an independent validation cohort, 2) identify differentially expressed extracellular proteins (particularly epithelial-derived proteins) among patients with (C-) LA-MPE and non-MPE. In addition, we will identify protein expression in (C+) and (C-) MPE due to breast cancer and compare protein expression among groups. We will evaluate the combined discriminating role of protein and miRNA expression among (C-) LA-MPE and non-MPE. Development of a diagnostic classifier, using a cell-free biofluid enriched with tumor-cell products that requires drainage and is underutilized is paradigm shifting and a crucial missing step in our MPE management which will be addressed through our proposal. Most biomarkers to date are examined in (C+) MPEs of heterogeneous populations and lack reproducibility or confirmation in (C-) MPE groups. We aim to close this gap and focus on (C-) LA-MPE populations where our findings could be clinically relevant and practice changing. This work will lead to an R01- level grant focusing on PF tumor-derived EVs and EV cargo in diagnosis, disease monitoring and prognosis of metastatic cancers of varying tissue type to the pleura.