University Of Washington

NIH Research Projects · FY 2026 · 2023-03

ABSTRACT Kinase signaling exquisitely orchestrates each step of neuronal development, beginning at neurogenesis to neuronal integration into functional synaptic networks. Through their highly specific substrate phosphorylation, protein kinases regulate neuronal growth, activity and their plasticity. Despite the increasing evidence for a critical and causative role of kinase dysfunction in neurodevelopmental disorders (NDD), the mechanisms through which the human kinome controls neuronal development and how its dysfunction manifests in disease remain major gaps in the field of neurodevelopmental biology. In this proposal, we will investigate the role of protein kinase TAOK1, genetic mutations in which have been strongly associated with autism spectrum disorder, macrocephaly and neurodevelopmental delay. Based on our preliminary findings, the central hypothesis of this research proposal is that TAOK1 is a pleiotropic kinase that regulates neuronal development through its ability to directly bind phospholipids and remodel the neuronal membrane, and that dysfunction in TAOK1 signaling lead to neuropathogenesis. Our data show that (a) both de novo and inherited TAOK1 mutations in NDD induce aberrant neuronal membrane extensions that disrupt neuronal morphology and function and (b) TAOK1 can directly bind phosphoinositides enriched in the plasma membrane. Through integration of innovative approaches in proteomics, chemical-genetics, structural biology, stem cell technology and human disease relevant model systems, we seek to (Aim1) understand the mechanisms through which TAOK1 signaling mediates neuronal development, (Aim2) determine the biological principles that govern TAOK1 membrane binding and remodeling, and (Aim3) generate human stem cell derived neuronal models of TAOK1 associated disease in order to determine developmental perturbations as well as phosphoproteomic changes due to deficits in TAOK1 signaling. These studies will provide a comprehensive understanding of the role of a high confidence gene in the etiology of neurodevelopmental disorders.

NIH Research Projects · FY 2025 · 2023-03

The long-term objective of this Pathway to Independence Award is two-fold: (1) support candidate Dr. Blayney in building an independent research program and (2) facilitate her transition from postdoctoral fellow to independent faculty researcher. To date, Dr. Blayney's research has focused on the risks for and consequences of sexual victimization in young adults, including the social and sexual contexts associated with sexual victimization risk. As part of postdoctoral training, Dr. Blayney's work has begun to examine the proximal influence of alcohol in sexual risk patterns for young women with and without sexual victimization histories. Dr. Blayney seeks to expand her training from basic alcohol research to developing and testing brief, technology-based interventions to reduce alcohol and sexual risk. This long-term objective will be achieved through a five-year training plan involving a carefully selected mentor team as well as targeted coursework (e.g., classes, seminars, workshops) and hands-on training experiences. The goal of the proposed research is to develop and test a specialized web-based intervention to reduce alcohol-related sexual risk behaviors among young women with sexual victimization histories, a high-risk and underserved group. During the mentored phase (K99), the intervention will be developed with user centered design (Aim 1), an innovative approach from the technology sector that incorporates the target population into all stages of development. In Aim 1a, intervention content will be drafted and presented to the target population (i.e., young women with sexual victimization histories) for user feedback. In Aim 1b, intervention design will be evaluated using rapid prototyping for user feedback before the intervention is programed. Once programed, Aim 1c will involve usability testing of intervention delivery with the target population. Following intervention development, Aim 2 will assess intervention feasibility and acceptability with a web-based open trial. During the independent phase (R00), a web-based RCT will be conducted to test intervention efficacy (Aim 3a), mechanisms of change (Aim 3b), and potential moderators (Aim 3c). Findings will serve as pilot data for an NIAAA R01 submission during the R00 phase. The training plan for this application will focus on intervention development and testing, innovative methods to enhance technology-based intervention development, and advanced statistics. Mentors (Drs. George, Bedard-Gilligan, Cue Davis, Rhew) and consultants (Drs. Billings, Widman) are committed to the candidate's training and each will provide unique expertise to the research and training plan. Support from this award will be essential to the candidate's development as an independent scientist who can contribute to alcohol research by developing and testing technology-based interventions to reduce alcohol and sexual risk. The University of Washington is well suited to provide a stellar training experience and will promote NIH's mission to develop early investigators who can be competitive for long-term research funding.

NIH Research Projects · FY 2026 · 2023-03

PROJECT SUMMARY Venezuelan equine encephalitis virus (VEEV) is a single-stranded positive-sense RNA virus transmitted by mosquitoes and is responsible for periodic epizootic/epidemic outbreaks of encephalitis in both horses and humans. The innate and interferon (IFN) responses are critical barriers for preventing the replication and spread of many viral pathogens including alphaviruses such as VEEV. As a result, viruses have evolved diverse mechanisms to inhibit or escape the innate immune response as well as antiviral effectors such as IFN-stimulated genes (ISGs). RNA structures are known to regulate basic viral processes (e.g. viral RNA transcription and translation), however, the role that viral RNA structure plays in shaping innate immune responses to viruses is understudied. We have previously shown that alphaviruses encode stable structures within their 5’-untranslated region (5’-UTR) that are critical for antagonizing IFIT1, an ISG important in restriction of non-self RNA. We have also shown that RNA structures in the 3’-UTR are important in modulating recognition of viral RNA by IFIT2. Recently we have shown that RNA structures in the 3’-UTR and E1 modulate replication of virulent and avirulent VEEV in macrophages, which are important targets of VEEV infection in vivo. The broad objectives of this proposal are to: 1) delineate E1 RNA structural determinants in virulent and avirulent VEEV that regulate macrophage replication, 2) define how E1 structural determinants recruit RBPs to the viral genome and the impact of this on host innate immune responses, 3) Define how macrophage replication fitness contributes to the differential pathogenesis in vivo of VEEV encoding virulent and avirulent RNA structures, and 4) Define how macrophage replication fitness shapes innate immune responses in vivo. Findings from these studies will provide key insight into novel mechanisms of VEEV pathogenesis and emergence of pathogenic variants. This will have broad implications for our understanding of viral emergence and development of RNA-based therapeutics.

NIH Research Projects · FY 2026 · 2023-03

Project Summary/Abstract My application for a Pathway to Independence Award represents the ideal next step toward reaching my goal of becoming a leading independent investigator focused on the development and evaluation of systems-based approaches for effectively delivering CIH interventions for common chronic conditions. The current application proposes the development and pragmatic pilot testing of a novel CIH-based stepped care approach for co- occurring chronic pain and PTSD. The aims of the proposal strategically align with my specific career development goals of gaining expertise in 1) systems-based care models for complex co-occurring physical and mental health conditions, 2) pragmatic clinical trial design for CIH interventions, and 3) user-centered design strategies for learning health systems. Also included is a professional development goal to 4) ensure my readiness to transition into an independent researcher and faculty member at the rank of Assistant Professor. In addition, the training plan includes 2 goals in the R00 phase of the award: 5) successfully transition into an Assistant Professor position and maintain a successful independent research program; and, 6) conduct a pilot randomized effectiveness trial and use pilot data to write and submit a competitive R01-level grant to NCCIH. The training plan includes an excellent research mentorship team with an established history of consistent independent federal funding and mentorship to ensure that these 6 goals are met. Dr. Steve Zeliadt (Primary Mentor) is an expert in pragmatic trial design, dissemination and implementation science, and quantitative data analysis; Dr. Kurt Kroenke (Co-Mentor) is an expert in systems-based and stepped care models within primary care settings for patients with chronic physical and mental health conditions; and Dr. Rhonda Williams (Co- Mentor) is an expert in pragmatic clinical trial design for CIH interventions and the development, treatment, and theoretical underpinnings of CIH interventions for individuals with complex symptom presentations. In Aim 1, we will use patient semi-structured interviews and clinic focus groups to identify provider strategies and patient perspectives relevant to treating co-occurring chronic pain and PTSD in a primary care clinic. Using proven stepped care approaches developed by mentor Dr. Kroenke for pain and by consultant Dr. Zatzick for PTSD as the foundation, our team will develop a CIH-based stepped care approach for co-occurring chronic pain and PTSD in primary care guided by current evidence of CIH interventions while including patient, provider, and clinic input. In Aim 2, we will iteratively refine the protoype using user-centered design strategies. In Aim 3, we will conduct a pragmatic pilot trial of our CIH-based stepped care approach v. treatment as usual in two primary care settings (one rural, one urban). The experiential components of each aim, combined with my mentorship plan, will ensure my readiness for independence as an expert in the development and evaluation of systems-based approaches for effectively delivering CIH interventions. The training goals and research aims align with the important NCCIH priorities of CIH interventions for co-occurring chronic pain and PTSD and well-being.

NIH Research Projects · FY 2025 · 2023-02

Suicide is the second leading cause of death among adolescents and young adults in the United States, responsible for more deaths than any single medical illness. Suicidal thoughts and behaviors are a major public health problem: they are recurrent, lead to extensive health care, and increase risk for death by suicide. The past two decades have seen notable increases in youth suicide deaths, attempts, and suicidal thinking. Approximately 1 in 5 U.S. high school students report serious suicidal thoughts in the past year and 1 in 10 have made a suicide attempt. In the absence of integrated outpatient solutions, there has been a dramatic increase in the use of emergency rooms (61% increase from 2007-15) while access to inpatient psychiatry units that care for suicidal young people has fallen (60% decrease from 1990-2008). The Suicide Care Research Center at the University of Washington will respond to this critical need by partnering with clinicians, youth who have experienced suicidality, and their families to develop outpatient solutions that are optimized for effectiveness, affordability, scalability, and efficiency and can be accessed by all Americans. Our aim is to assure our solutions are what patients and clinicians want and are feasible in primary care, pediatrics, and other medical clinics where suicidal adolescents and young adults are most likely to seek help. Our Signature project – Swift Outpatient Alternative for Rapid Stabilization (SOARS) – is an optimization trial of a just-in-time mental health intervention that offers medical providers an outpatient alternative to referring families to the emergency room. The Center also includes three Exploratory projects using human centered design to co-design just-in-time interventions with clinicians, patients, and their families. These three interventions will help medical providers, and their Integrated Behavioral Health teams to (1) better monitor and manage suicide risk via ecological momentary assessment, (2) improve risk assessment, shared decision making and safety plans using technology, and (3) use clinical narrative approaches to better treat suicidal thinking. Eight pilot projects will be selected to address other needs of medical clinics overwhelmed with management of suicidal young people. All studies will be coordinated through an Administrative Core that provides mentoring, training and experience for Collaborating Scholars from other disciplines alongside suicide researchers. A Policy Core will create and support partnerships with policy makers, primary care networks, and people with lived experience of suicidality to inform intervention development and evaluation. Center research will be centralized by the Methods Core to assure all studies are harmonized in both design and data collection including with the electronic medical record. The Center will conclude with a National Policy Briefing on Center findings to maximize dissemination.

NIH Research Projects · FY 2026 · 2023-02

PROJECT SUMMARY This application aims to build on our recent discoveries of olfactory “gating” of visual attraction in Aedes aegypti mosquitoes and determine the role of color vision in host and nectar selection. Without vision, mosquitoes cannot track odors, locate hosts, or find mates. Vision is a critical sensory modality involved in long-range olfactory search behaviors and near-host behaviors involved in landing and biting. Despite this potential importance, little is known about vision in anthropophilic mosquitoes and the neural bases of these behaviors. Mosquitoes can see a host from 2-15 m, and our recent findings show that odor “turns on” their visual attraction to hosts, thereby playing a critical role by linking long-distance odor tracking with short-range behaviors near the host. Our work also demonstrated that mosquitoes are sensitive to wavelengths reflected from human skin. However, we lack an understanding of how odor sensitizes the visual system, and the visual preferences of diverse anthropophilic mosquitoes. We have developed new tools to examine olfactory-visual integration, including new Aedes GCaMP6s mosquito lines, the generation of opsin knockout lines, and biogenic amine receptor mutants. This proposal builds on our preliminary findings that demonstrate the importance of color vision and neuromodulators in olfactory-visual integration in mosquitoes. Using semi-field and behavioral assays, calcium imaging in tethered flying mosquitoes, and molecular-genetic approaches, we propose to study the color preferences of mosquitoes and how odor modulates visual neurons. Aim 1 will allow us to characterize the colors (wavelengths) that attract different anthropophilic mosquito species and identify the odors that turn on visual search behaviors. In Aim 2, using Aedes aegypti, we will determine the neural mechanisms by which odors turn on visual search behaviors, and identify the rhodopsins that detect important wavelengths. Our preliminary results indicate that octopamine is critical for olfactory-visual integration. We will generate cell- specific knockout of the octopamine receptor to determine how olfactory-visual behaviors are compromised. In parallel, we will mutate specific long- and short-wavelength rhodopsins to suppress attraction to colors indicating hosts or nectar sources. We will also use new GCaMP6s lines to record from visual neurons in the mosquito brain and characterize how odor modulates those neurons. Aim 3 will test the wavelengths and visual features (motion, object size) that mosquitoes find attractive, and test them in new trap designs in semi-field trials. While there has been extensive work on olfaction in mosquitoes, our work emphasizes that color vision also plays a key role. Olfactory-visual integration is vital in diverse insect vectors, including tsetse flies and kissing bugs. We suggest that our proposed experiments provide a basic framework for understanding how these cues influence haematophagous insects. Furthermore, results from this work will provide information on attractive visual lures, and motivate the identification of molecular targets to cripple visual-olfactory behaviors.

NIH Research Projects · FY 2026 · 2023-02

Summary In this proposal, we will explore the role of prenylation in antiviral immunity. Prenylation is a post-translational modification that renders proteins hydrophobic, thus targeting them to the endomembrane. While the role of prenylation in immunity is unclear, our preliminary data shows compelling evidence that prenylation is a modification widely used in innate antiviral immunity. As both the host and virus utilize prenylation and the cholesterol biosynthetic pathway, we propose a dynamic host-pathogen interaction at this interface. We hypothesize that prenylation could be a potent host innovation to counter viral pathogenesis. This is based on in silica analysis that revealed an enrichment of immunity-related proteins with a prenylation motif. We followed in-silica analyses with a biochemical screen and identified several prenylated antiviral proteins that are involved in interferon responses and virus restriction, with potential to affect virus entry, replication, and egress. In this grant, we propose to identify the functional consequences of prenylated proteins on subcellular localization, interferon signaling, and antiviral function. By perturbing specific enzymes in the prenylation pathway, we will study its effect on interferon response, antiviral effectors, and virus control. This work will provide novel insights into cell-intrinsic antiviral countermeasures and the development of novel antiviral drug targets.

NIH Research Projects · FY 2025 · 2023-02

Project Summary / Abstract Decision-making requires populations of neurons in the brain to collectively process sensory evidence and select appropriate behavioral responses. Neural population dynamics (NPDs), which describe how the responses of a population of individual neurons unfold over time, can provide an insightful view into these decision processes. Much is known about how individual neurons respond in select decision making tasks. However, little is known about how populations of neurons dynamically perform decision computations, and how resulting NPDs within a brain area are structured across many decision-making tasks. Shared features in NPDs across many tasks could indicate unifying neural mechanisms of computation that underlie the multi-functionality of a given neural circuit. This proposal aims to uncover the details and structure of these decision-related NPDs in human and nonhuman primate dorsal premotor cortex, an area tightly linked to both the function and dysfunction of decision making. Particular attention will be devoted to examining how NPDs are organized across multiple decision-making tasks and how those NPDs emerge during learning of new tasks. During the K99 phase, novel analytical tools will be developed for extracting NPDs from simultaneously recorded neural population activity and, importantly, for providing interpretable links between NPDs and their role in decision-related neural computation. With the goal of identifying unifying principles of decision-related computation, large-scale analyses will then integrate existing and newly collected neurophysiological datasets involving multiple decision-making tasks performed by humans and nonhuman primates. During the R00 phase, the proposed research will pivot toward understanding how NPDs emerge during learning to make new types of decisions. The proposal postulates that the ease, speed, and efficacy of learning all hinge on the extent by which critical neural circuits can leverage pre-existing neural mechanisms of computation. These concepts will be tested in collaborative human and nonhuman primate neu- rophysiological experiments, with guidance provided by interrogations of artificial neural networks posed with similar decision-related learning tasks. Upon completion, the proposed research will provide new fundamental knowledge concerning i) the multi-functional and adaptive role of premotor cortex across many decision-making tasks and ii) unifying principles of neural computation that support this flexibility. Better understanding decision- related circuits and their neural mechanisms could ultimately elucidate the basis for the numerous psychiatric disorders that impair decision making, which could eventually lead to improved diagnosis and treatment of these debilitating conditions.

NIH Research Projects · FY 2026 · 2023-02

Project Summary/Abstract The Georgetown and South Park communities of the Duwamish Valley (DV) face higher levels of environmental pollution and poorer health outcomes compared to the rest of Seattle. For many years the communities have organized to address these environmental and health disparities, prioritizing air pollution and asthma. Our proposal is (1) directly responsive to community requests, (2) strengthens previous community-academic-government partnerships in the DV, (3) advances the environmental health interests of the community, (4) investigates low-cost tools for the measurement and control of air pollution, and (5) evaluates individual and community empowerment throughout all phases of research and action. This multilevel community engaged research (CEnR) project proposes elements that will engage and empower members of the DV community. The household-level intervention - providing participants with indoor air filters - has the goal of improving indoor air quality and reducing asthma symptoms in children. In contrast with previous studies investigating high-efficiency particulate air (HEPA) filtration, our intervention will evaluate the effectiveness of low-cost box fans equipped with lower-efficiency filters with greater airflow. The low-cost filters will be coupled with established home-based multicomponent, multi-trigger indoor air quality assessment of homes. We will conduct a randomized control trial to rigorously evaluate differences between intervention and control households in asthma outcomes such as symptoms and forced expiratory volume and in concentrations of indoor particulate matter (PM) and black carbon (BC). Promotores and healthy home consultants recruited from the community will be instrumental in carrying out the intervention. At the community level, we will expand community-driven ambient air monitoring campaigns, prioritizing the characterization of traffic-related air pollution, identification of pollutant sources, and emissions reductions. The use of a low-cost monitoring network will improve the spatial resolution of air quality data in the DV, enabling us to identify PM, BC and nitrogen dioxide hotspots. Youth from the well-established Duwamish Valley Youth Corp program will be trained in various aspects of air monitoring to help carry out the campaign. Armed with data on air quality, community members (who will also be trained in policy advocacy) can advocate for structural change including policy, infrastructure improvements such as urban green space, and targeted regulation of industrial and/or transportation emissions. Throughout the project, independent evaluation of individual and community engagement and empowerment will be tracked. From defining scientific goals, to project implementation and evaluation, our CEnR approach is designed to engage and empower a resilient community in its goal to advance health equity.

NIH Research Projects · FY 2026 · 2023-02

Project Summary The major goal of the proposed research is to develop tools to enable the study of dissolved gases on biological samples. Both blood gases (O2, and CO2) as well as trace signaling gases (NO, CO and H2S) play critical roles in regulation of wide array of tissue functions as diverse as regulation of heart rate, blood flow, immune responses, hormone and neurotransmitter secretion, as well as cytoprotective and anti-inflammatory properties. Indeed, most if not all tissues produce and are regulated by NO, CO and H2S on top of the central role of O2 in regulating bioenergetics and metabolic pathways. Research by academic institutions as well as pharmaceutical companies are endeavoring to harness the beneficial effects of gas signals in order to treat a range of conditions including diabetes, transplant rejection, sepsis, atherosclerosis and cancer. Despite the scientific and clinical importance of dissolved gases, quantitative methods to measure real time effects of dissolved gases on tissue/cells are not available. Investigators who have studied the beneficial signaling initiated by trace gases and/or who are developing drugs to trigger the same benefits almost exclusively use water soluble surrogates/donors of each signaling gas. A critical point for this proposal is that such chemical donors of gases may not allow for accurate control of gas levels within tissue, and due to their low aqueous solubility, rapidly deplete from culture media. Preliminary data we have generated and published revealed opposite effects of dissolved H2S vs. that obtained with an aqueous provider of H2S, indicating a need to re- evaluation the effects of what is established regarding NO, H2S and CO from studies using donor molecules. As most life science researchers do not have the ability to study the direct effects of trace gases at physiologically relevant concentrations, we will develop a turnkey, automated instrumentation to control the concentration and exposure time of tissue to media containing user-specified levels of 6 gases including NO, CO, H2S, O2, CO2 and N2. This automated and user-friendly system will be constructed to supply gas mixtures to a variety of widely used tissue assessment modalities including fluidics systems, static culture in plates, and cuvette systems for studying gas binding interactions to purified proteins. We have assembled a team consisting of Bio- and Mechanical/Combustion Engineers with many years of experience designing and constructing gas fluidics system applied to biological analysis, as well as an Applied Mathematician, an Analytical Chemists and Cellular Physiologists who will be involved with the validation of the instrumentation. This technology will have broad impact on fundamental research by facilitating the study of kinetic and concentration-dependent effects of signaling gases on tissue function, signaling and bioenergetics, and evaluation of therapeutics being developed to mimic the cytoprotective properties of the gases.

NIH Research Projects · FY 2026 · 2023-01

PROJECT SUMMARY/ABSTRACT Sensorimotor learning plays a critical role in the acquisition and refining of all skilled movements, including speech production. From early babbling to fully mature speech articulation, auditory-motor learning reflects central nervous system processes involved in acquiring and updating neural representations of the intricate motor-to-auditory transformations from motor commands to vocal tract movements and speech sound output. Auditory-motor learning is also believed to play an important role in the etiology of developmental speech disorders such as stuttering and childhood apraxia of speech. Over the past two decades, the PI’s laboratory has made important contributions to the field’s understanding of the key mechanisms underlying such speech auditory-motor learning in children and adults. We now propose a novel series of experiments, combining behavioral learning paradigms with a highly innovative neurophysiological approach that allows us to both modulate and record subcortical neural activity. Specifically, we aim to investigate (a) how speech auditory- motor learning can be enhanced and how decay of learning can be minimized, (b) whether feedforward auditory-motor learning also drives updates in feedback control policies, (c) which theoretical insights can be gained from the subset of individuals who are “followers” and paradoxically use a control strategy that leads to increasing auditory error rather than adaptation, and (d) whether, at the neural level, cerebellar and basal ganglia circuits play a distinct role in speech auditory-motor learning versus other forms of speech motor learning. The inclusion of experiments on the neural bases of auditory-motor learning leverages state-of-the-art sensing technology available in very recent Deep Brain Stimulation (DBS) implants with electrodes in either thalamic ventral intermediate nucleus (receiving cerebellar output) or subthalamic nucleus (basal ganglia). Overall, these integrated experiments will elucidate the processes, mechanisms, and neural substrates involved in speech auditory-motor learning. They will also reveal the developmental progression of auditory- motor learning, differences in auditory-motor learning between children and adults, as well as similarities and differences in speech sensorimotor learning vs. limb sensorimotor learning. Furthermore, given that auditory- motor adaptation paradigms cause speakers to implicitly alter their speech output quickly and without effort, this line of work supports the future long-term goal of developing novel, computer-assisted clinical procedures that induce adaptive changes in speech behavior by systematically altering the speaker’s auditory feedback. Thus, the work is directly relevant to public health as the generated knowledge will deepen our understanding of typical speech development, the maintenance of speech articulation skills throughout the lifespan, and fundamental sensorimotor mechanisms underlying developmental speech disorders while simultaneously paving the way for highly innovative clinical techniques for the treatment of a variety of speech disorders.

NIH Research Projects · FY 2026 · 2023-01

Project Summary/Abstract: The field of optogenetics — utilizing light to engage biological systems — is widely used for the dissection of neural circuits, cellular signaling and manipulating neurophysiological systems in awake, behaving animals. However, while many new opsins have been developed and are actively used, challenges still remain, and the current technology lacks a full toolbox for sub-cellular, spatiotemporal control of signaling — the predominant means for neuromodulator communication in the brain. Here we propose, an innovative effort combining neuroscience with structural biology and high-throughput pharmacology for the development of a series of cutting-edge novel Opto-GPCRs that will allow spatiotemporally precise and pathway-selective control of neuromodulator signaling in vitro and in freely moving animals. In four aims across five leading laboratories, we will develop and test these novel tools in vitro and in vivo. Specifically, we will work to 1) Develop and fully optimize OptoGPCR-v3.0 (Gi coupled) receptors for enhanced spectral multiplexing and altered sensitivity using structure-function analysis together with mutant-library HTS landing pad system; 2) Develop and fully optimize OptoGPCR-v3.0-Gq receptors for selective coupling to Gq signaling pathways using structure-guidance and the HTS landing pad system; 3) Utilize databases of less-explored naturally-occurring opsin-GPCRs to test, screen and further develop new optical tools with unique profiles; and 4) Assess the spectral compatibility for simultaneous use of OptoGPCR-v3.0 constructs in vivo, together with biosensors using photometry, 2p imaging and concurrent behavioral measures in vivo. Successful completion of the proposal will provide the wide neuroscience community with the long awaited capabilities of spatiotemporal manipulation of GPCR – neuromodulator signaling within neural circuits in vitro and in vivo, in awake freely behaving animals, and could be used for a wide variety of applications. This new technology will also further widen the field for unique optical approaches that allow discrete control and optodynamic simulation of neuromodulator function in brain tissue. We therefore believe that by the fulfilment of these goals we directly address the central purpose of this RFA-NS-21-027 call.

NIH Research Projects · FY 2026 · 2023-01

Project Summary/Abstract Kidney stones are prevalent and one of the costliest urologic diseases. The available treatment options such as ureteroscopy or shockwave wave lithotripsy break the stone into small fragments that can lead to future growth and recurrence of symptoms. This proposal investigates the underlying mechanisms to use acoustic radiation force produced by an ultrasound multi-element array that can trap a stone, steer it out of the kidney collecting space, and deposit it in the renal pelvis or UPJ to facilitate its natural clearance. The project seeks to answer the fundamental scientific hurdles to target and maneuver the stone toward passage. Aim 1 develops the analytical framework to optimize pulsing mechanisms to trap and manipulate natural stones. A proposed semi-analytical approach approximates the scattering with spherical functions to calculate the forces on natural stones. Predictions will be combined with the investigation of pulsing parameters to optimize trap robustness and achieve stable trapping of natural stones. Pulsing parameters such as pulse length, repetition rate, frequency, and phase excitation that control beam shape and uniformity will be adjusted to eliminate instabilities from rotation and asymmetric forces to achieve stable trapping of natural stones. The aim success is measured by performing manipulation maneuver natural stones along predetermined paths. In Aim 2, the stone acts as a target that can reflect and scatter ultrasound waves which are received back by the multi-element array. Correction algorithms use the received signal to calculate the element excitations necessary to correct for beam aberrations from the tissue heterogeneity. Hydrophone measurements will compare the beams before and after corrections with the unaberrated beam. Finally, manipulation of stones in kidney phantoms and ex vivo are performed to mimic in vivo conditions. In Aim 3, the safety and efficacy of acoustic forceps manipulation will be evaluated. First, different acoustic intensity exposures will be investigated in ex vivo porcine kidneys for thermal and mechanical injury. Afterward, natural stones of various sizes will be implemented in the kidney collecting space of live pigs. The stone will be targeted, trapped, and steered from the kidney collecting space toward the kidney exit using the acoustic forceps. The treated group will be evaluated against an untreated control group to evaluate efficacy. Tissue injury mechanisms will be assessed through histological analysis. In addition to my research, I will also pursue other activities guided by my mentors toward my career goal of becoming an independent investigator. These activities include interacting with researchers, industry partners, and clinicians through seminars and conferences; and participating in workshops on the responsible conduct of research, and grant proposals and management so that I will be able to pursue independent R-level funding toward the end of the K25 award. The Applied Physics Laboratory offers the facilities and inter-departmental collaboration necessary for successful career development in translational research.

NIH Research Projects · FY 2026 · 2023-01

Hematopoietic stem cell (HSC) transplantation can provide durable HIV elimination as exemplified in the “Berlin” patient, the “London” patient, and recently, in a third (“New York”) patient. This gives a strong rationale for HSC gene therapy of HIV/AIDS. Current clinical HSC gene therapy protocols (e.g. for hemoglobinopathies) involve high-dose chemotherapy to make space in the bone marrow, and the transplantation of HSCs after ex vivo gene transfer. Because of the risk, cost, and technical complexity, it is unlikely that ex vivo protocols will be widely applicable, specifically in developing countries where the greatest demand for HIV/AIDS therapy lies. We have developed an in vivo HSC transduction approach that requires only intravenous injections and could be provided as an outpatient treatment. In this approach, HSCs are mobilized from the bone marrow into the peripheral blood stream and transduced with intravenously injected in vivo gene transfer vectors (helper-dependent adenovirus vectors) that target receptors present on primitive HSCs. HSCs transduced in the periphery return to the bone marrow, persist there long-term, and contribute to all blood cell lineages. The central goal of this application is to further develop our in vivo approach toward HIV prophylaxis and therapy with persistent eradication of HIV in target/reservoir cells. The Specific aims are. 1. Optimize HSC mobilization regimens, HSC homing, and HDAd vectors/expression systems to achieve i) efficient bone marrow homing of mobilized HSCs, ii) efficient trafficking of transduced HSC progeny cells, specifically to the brain, a main HIV reservoir tissue that is difficult to target by therapeutics, and iii) increase the level and safety of transgene expression. 2. Prevent HIV/SIV escape mutants and eliminate virus from reservoirs by a multi-modular in vivo HSC gene therapy approach. Modules will exert anti-HIV activity based on different mechanisms (e.g. opsonization of virus in blood by eCD4-Ig, protection of target cells by co-receptor knockout through in vivo genome editing, and killing of infected cells independently of MHC-I presentation by a CD4 chimeric antigen receptor (CD4-CAR) expressed on immune effector cells. 3. Demonstrate in NHPs that the optimized in vivo HSC gene therapy approach will allow for i) complete protection against SIV challenges (absence of escape mutants) and ii) SIV elimination in infected animals (including the brain). Model systems to test the safety and antiviral efficacy of the approaches will include primary HSCs/HSC- derived cells, transgenic and humanized mouse models (with and without SIV infection), as well as NHPs (in prophylaxis and therapy setting). Our efforts will address important biological obstacles in HIV therapy in the context of a technically simple, cost-efficient, and portable approach.

NIH Research Projects · FY 2026 · 2023-01

ABSTRACT This application seeks partial support for the 2023, 2025, and 2027 Conferences on Implantable Auditory Prostheses (CIAP). The 2023 CIAP is scheduled to be held at the Granlibakken Conference Center, Lake Tahoe, CA, July 9-14, 2023. The cochlear implant (CI) is the first neural prosthesis in widespread clinical application for restoring sound sensation and speech understanding to the severely hard-of-hearing population who have difficulties understanding speech with acoustic hearing aids. Over the past 30 years, dramatic improvements in patients' performance with these devices have been achieved and hence a wider population of patients can benefit from cochlear implants. Currently the average speech understanding score for implanted postlingually deafened adults is nearly 80% correct for sentence recognition in quiet and 50% correct in low levels of background noise. Congenitally deaf children who receive a CI prior to age 2 are achieving nearly normal rates of speech and language development. CIs can even provide significant improvement in communication for adults who have substantial residual acoustic hearing. These advances have benefitted considerably from the collective efforts of researchers in a broad array of scientific disciplines, from cellular biology, physiology, materials science, and signal processing, to linguistics and cognition. This interdisciplinary collaboration and cooperation has been fostered in large part through a series of biennial conferences, originating with a 1983 Gordon Conference. These conferences are the only forum related to CIs in which scientific research issues are the sole focus of active participants in the auditory community who contribute to moving the field forward. Even the best CI users though face significant limitations in music perception as well as speech perception in challenging listening environments. The long-term goal of CIAP is to generate cutting-edge research ideas to improve the design and function of auditory prostheses. The Specific Aims are to provide (1) a global forum for the presentation and discussion of the latest and highest quality research, (2) an atmosphere that is conducive to: disseminating new findings through scientific discussion, stimulating new lines of research, and facilitating collaborations through social networking, and (3) an opportunity for young investigators and many trainees in the field to present their work and network with established investigators. CIAP conferences use the Gordon Research Conference model of organization, with ample time for discussion within the program, unstructured time to encourage spontaneous informal discussions and brainstorming, and an isolated “retreat” location where participants spend most of their waking hours with other conference participants.

NIH Research Projects · FY 2026 · 2023-01

Abstract Ion channels are exquisite molecular machines that regulate the flow of ions across cell membranes in response to stimuli such as voltage and small molecule ligands (e.g. second messengers, and neurotransmitters). They underlie all electrical excitability in the brain and heart, and defects in ion channels are responsible for many human disorders. Despite decades of experiments and many high-resolution molecular structures, we still do not know, for any channel, the mechanisms for voltage- or ligand- dependent gating. The missing ingredient seems to be conformational energetics. The energetics of the different channel conformations governs the time course, voltage- dependence, and ligand-dependence of opening of the channel pore, and ultimately electrical excitability of the cell. In this proposal we will determine the mechanisms of voltage-dependent gating and ligand-dependent gating and fill important gaps in our understanding of ion channel biology. We will focus on the cyclic nucleotide-binding domain (CNBD) family of ion channels, which are structurally related, but functionally diverse. Whereas some CNBD channels are activated by depolarization, others are activated by hyperpolarization, and some members are activated by cAMP yet others are activated by cGMP. We will leverage breakthrough FRET methods we developed for measuring intramolecular distance distributions and conformational energetics using fluorescence lifetime imaging microscopy (FLIM), simultaneous with recordings of channel function using patch-clamp fluorometry (PCF). The data from multiple donor- acceptor sites throughout the channels will be compiled into a four-dimensional map (X, Y, Z, and energy) of the conformational rearrangements associated with ligand- dependent and voltage-dependent activation of CNBD channels. Our long-term vision is to understand the general themes that underlie allosteric regulation of ion channels, and these experiments promise rapid progress toward this goal. Ultimately, the methods and principles we discover will be of broad utility for elucidating mechanisms for all allosteric proteins.

NIH Research Projects · FY 2026 · 2023-01

Abstract The goal of this research is to provide a roadmap to maintain periodontal health by understanding the mechanisms which underlie the variation in inflammatory responses within the human population. Ultimately, this information will be translated to individualized preventive and treatment regimens based on host response phenotype. Periodontitis is one of the most prevalent non-communicable diseases in adults worldwide and a major public health concern. According to the latest World Workshop in Periodontics there is immense potential in studying gingivitis, an antecedent reversible disease state, as a means of primary prevention of periodontitis. Healthy periodontal tissue exists in a homeostatic relationship with its accompanying oral microbiome. In most individuals, this relationship results in a combination of host and microbial derived immune components that produce an active inflammatory surveillance protective state. This is termed healthy homeostasis. Disruptions of this healthy homeostatic state occur during episodes of both experimental and natural gingivitis, which is defined as reversible inflammation of the gingiva. The ability to induce a reversible inflammatory state in humans has provided a unique foundation to examine microbial-host interactions that dictate periodontal health and disease via the human Experimental Gingivitis (EG) model. We will capitalize on our previous work with the highly translational human experimental gingivitis model where we have identified three distinct clinical response phenotypes, which behave differently to bacterial-driven inflammation. We will use these clinical phenotypes as a foundation to explore the variability in the human inflammatory response. Specifically, we will determine the contributions of host components and microbial ecological succession patterns to the observed variations among responder types. The approach for this proposed research is to determine the bacterial and host processes in stages of health through disease using advanced parallel multi-omic measurements of both bacteria and host components coupled with ex vivo and in vitro mechanistic studies to determine the host and associated microbial functions that determine the variation in host responses. We will employ a comprehensive combination of functional meta-omics in parallel (DNA and RNA 16S sequencing, metagenomics, metabolomics, custom multiplex Immunoassay of host mediator panels) along with complementary cultivation approaches for hypothesis testing. We anticipate there will be an immediate benefit from our proposed detailed investigations in terms of the comprehensive multi-omic datasets we will generate and make available – enabling responder types to be identified and further characterized across studies. In the longer term, this fundamental mechanistic work has direct clinical and therapeutic value by identifying potential critical targets during disease initiation and development within each of the different response types that can translate to personalized treatment and intervention strategies.

NIH Research Projects · FY 2026 · 2023-01

SUMMARY In contrast to humans, some animals are able to scarlessly heal and regrow lost appendages after major injury. Appendage regeneration requires rapid and carefully regulated cell proliferation to replace lost tissue, an inherently anabolic process. Despite the fundamental need for biosynthesis as part of regenerative healing, we lack a mechanistic understanding of how injury is coupled to metabolic changes that enable cell proliferation and growth. Re-creating the metabolic conditions that enable growth is critical to being able to foster regenerative success in organisms where it is normally limited, such as ourselves. In this proposal, we leverage two advantages to articulate the metabolic requirements for vertebrate appendage regeneration. The first is a suite of mechanistic insights from other highly proliferative cell types, which rely on aerobic glycolysis to convert glucose to glucose-6-phosphate, a versatile biosynthetic precursor for nucleotide and phospholipid production. Our preliminary data suggest that this is a shared strategy in appendage regeneration. The second is the unique context-specificity of appendage regeneration in Xenopus tadpoles, which is lost or gained on the basis of nutrient source, developmental stage, and appendage type. This context specificity gives us the opportunity to directly compare regenerative structures to their non-regenerative counterparts and to other non- regenerative structures, thereby defining the specific features of the metabolic landscape that enhance or limit regenerative outcome. In this proposal we will test the central hypothesis that regenerative success is dictated by the ability of tissues to rapidly remodel their metabolic landscape and funnel nutrients toward biosynthesis. We will test this hypothesis by first defining the metabolic paradigm that dominates in regenerative conditions: glycolysis, pentose phosphate pathway, or oxidative phosphorylation. We will then identify shared and context- specific features of metabolic reprogramming, contrasting the metabolic profile of regenerative structures and non-regenerative structures at different developmental stages of the Xenopus tadpole. We conclude by building a regulatory landscape of metabolism in regeneration, functionally testing candidate regulatory transcription factors, and defining how metabolic gene expression is partitioned or integrated between cell types.

NIH Research Projects · FY 2026 · 2023-01

PROJECT SUMMARY Generating cells with different fates, functions and behaviors is critically important for the development and maintenance of tissues, organs, and multicellular organisms. Cellular diversity can be generated through Asymmetric Cell Division (ACD). Stem cells utilize ACD to create differentiating sibling cells while maintaining the stem cell in the process. In addition to the asymmetric partitioning of proteins or RNAs, other mechanisms such as mechanical cues, sibling cell size asymmetry or organelle asymmetry could potentially also contribute to binary cell fate decisions. Here, I propose to use asymmetrically dividing Drosophila neuroblasts, the neural stem cells of the developing fly central nervous system, to investigate the cell and mechanobiology of ACD in vivo. Recently, we discovered that Non-muscle Myosin II-dependent cortical flows, induced through both polarity- and spindle-dependent cues, are implicated in the generation of sibling cell size asymmetry. I will investigate how cortical flows are induced and modulated with spatiotemporal precision to achieve reproducible sibling cell size asymmetry. Our recent discovery of Protein Kinase N (PKN), and the Rho GTPase pathway as inducers of cortical flows will provide molecular entry points. I will also investigate how cell size asymmetry contributes to cell fate decisions, using RNA sequencing, immunohistochemistry, and long-term live cell imaging in vivo. A second project encompassed in this research direction is aimed at investigating the molecular mechanisms and function of molecular centrosome asymmetry, which is manifested in biased microtubule organizing center (MTOC) activity in interphase. We identified new proteins and mechanisms, such as Kinesins, Pp4 and dynamic centriolar protein localization in mitosis, regulating centrosome asymmetry. Centrosome segregation is highly stereotypic in stem cells, but whether and how centrosome asymmetry affects cell fate decisions, remains to be resolved. We will use fly neural stem cells to investigate the mechanisms and functions of centrosome asymmetry during ACD. I am particularly interested in investigating whether centrosome asymmetry provides a mechanism for biased cell fate determinant segregation, either via asymmetric RNA or sister chromatid segregation. I will also investigate whether biased MTOC activity impacts transcriptional regulation via chromatin organization. This research program will benefit from several novel and innovative tools, consisting of live cell imaging, superresolution microscopy, RNA sequencing and acute protein mislocalization and perturbation systems (nanobody, optogenetics), which my lab implemented to probe cytoskeletal dynamics with high spatial and/or temporal precision in vivo. ACD is an evolutionary conserved mechanism and the proposed research program is medically significant because defects in ACD can cause neurodevelopmental disorders or cancer.

NIH Research Projects · FY 2026 · 2022-12

ABSTRACT Pericytes have been implicated in lung injury and repair in a number of organs. Our research focuses on the role of lung stromal subpopulations in tissue injury and repair, and our recent work revealed lung pericytes may have multiple functional roles during injury. Data from transcriptomic analyses of activated lung pericytes reveal upregulation of multiple genes involved in inflammation, angiogenesis, and matrix remodeling. The goal of this project is to characterize the functional diversity of pericytes in their response to a clinically relevant model of lung injury – influenza infection. To achieve this goal, we will test hypotheses on the roles of pericytes in endothelial and immune regulation. In Aim 1, we will investigate the mechanisms of pericyte-endothelial cell crosstalk that lead to activation of endothelial cells and increased lung permeability. Angiopoietin-L4 (ANGPTL4) has been implicated as a hyperpermeability factor that is also highly upregulated in activated lung pericytes in our preliminary studies. We will examine how pericyte- derived ANGPTL4 affects lung endothelial function in this aim. In Aim 2, we will evaluate the role of lung pericytes in trafficking inflammatory monocytes. In murine models of influenza infection, inflammatory monocytes have been implicated in the development of lung injury. Recruitment of inflammatory monocytes in influenza occurs through a CC-chemokine receptor 2 (CCR2) dependent mechanism. Our work shows that activated pericytes highly upregulate CCL2, a major ligand for CCR2. We will evaluate the functional role of pericyte-derived CCL2, and other CCR2 ligands, in inflammatory monocyte recruitment. Finally, in Aim 3, we will characterize functional subsets in the pericyte population using single nucleus transcriptomic approaches. This proposed aim will provide insight into the diverse functional states of lung pericytes throughout the course of influenza infection and functional subtypes that mediate lung injury.

NIH Research Projects · FY 2025 · 2022-12

PROJECT SUMMARY/ABSTRACT The overall goal of this NCI K08 career development proposal is to facilitate Dr. Erin Gillespie’s development into an independent investigator in cancer health services research focusing on strategies to enhance quality and evidence-based care in radiation oncology. This proposal focuses on shortening the course of radiation from 7-9 weeks to 3-5 weeks (called “hypofractionation”) which is associated with equivalent cancer control, improved patient-reported outcomes, and decreased costs in patients with breast and prostate cancer. Nonetheless, adoption has been slow. Dr. Gillespie’s prior work has revealed that the individual radiation oncologist is highly influential in whether patients with breast cancer receive hypofractionation or a longer course of radiation. The overall hypothesis is that radiation oncologists are the linchpin in radiation treatment decision- making, and that implementation strategies that harness behavioral economics will be most likely to impact practice change. My primary objectives are to 1) identify and characterize positive deviant radiation oncologists (high users of hypofractionation) that will 2) elucidate implementation strategies and associate them with adoption of hypofractionation across various settings, and 3) pilot test a multi-pronged strategy that promotes use of hypofractionation in preparation for a large pragmatic multi-center controlled trial. The rationale that underlies the proposed research and training plan is that, with new knowledge about the levers that drive physician decision-making (beyond knowledge gaps), healthcare leaders and policymakers can optimally design and implement systems that evoke change. Dr. Gillespie will harness the resources and expertise at Memorial Sloan Kettering Cancer Center, where she is a faculty member in the Department of Radiation Oncology and the Center for Health Policy and Outcomes. She is also engaged in research in the community- based setting through the MSK Cancer Alliance. Her training plan has a foundation of implementation science that incorporates large dataset analysis, mixed methods, and behavioral economic theory. In Aim 1 of the proposed study, the investigators will analyze a Medicare dataset that includes claims linked to prescribing radiation oncologist and organization, and is supplemented by the AMA Masterfile. They will systematically conduct the first-ever evaluation of implementation strategies in radiation oncology using mixed methods. Lastly, in Aim 3 the investigators will develop and pilot test a multi-pronged strategy informed by Aims 1-2 and including audit and feedback. This proposal, together with the new skills Dr. Gillespie will acquire, will generate new knowledge about the impact of implementation strategies on de-implementation of long-course radiation – key parameters necessary to design optimal strategies to encourage physicians to adopt high value radiation. This will specifically support the development of an implementation strategy that will be tested in a subsequent prospective randomized trial to assess real world efficacy.

NIH Research Projects · FY 2025 · 2022-09

Opioid overdoses and deaths continue to increase nationally and in the Seattle area. Despite improvements in our data infrastructure and care continuum locally, significant gaps remain. We propose to use an intensive data to action framework to guide rapid data infrastructure, medical, opioid use disorder treatment, and public health interventions. We will test the impact of these activities on the rates of opioid overdose (fatal and non-fatal) as well as accessing medications for opioid use disorder (MOUD), substance related health care, and other services. In the exploratory R61 phase of this project, year 1, we will bring together a targeted, multi-sectorial group of stakeholders to identify data infrastructure issues and opportunities in order to support near real time data-driven decision-making. We will utilize Continuous Process Improvement (CPI) tools including root cause analysis and plan-do-study-act to monitor and improve data and care delivery system processes. Existing data systems will be used by Public Health-Seattle & King County including: EMS medical incident report forms, mobile integrated health case management data, and King County Medical Examiner Office data for near real time data analyses, visualization, and action planning. Initial service planning and ongoing outcome monitoring will utilize the King County integrated data hub (e.g. jail, substance use disorder/mental health treatment, housing services, Medicaid health care utilization). Each of these initial activities will have specific, concrete measurable milestones that will be met before proceeding to the next phase. For the developmental R33 phase of this project, years 2-5, we plan to create a sub-acute stabilization center (SASC) for people at high risk for opioid overdose, including those who have recently overdosed and are referred and transported by EMS. Services will also be available to those who self-refer or are referred by community agencies. The SASC will leverage the physical infrastructure and clinical expertise of Evergreen Treatment Services, a long-time provider of MOUD and community based outreach in the Seattle area. Research indicates that MOUD supports recovery, improves outcomes, and cuts mortality rates in half. Providing naloxone and substance use related supplies in partnership with existing programs will help decrease morbidity and mortality in the short term and may increase engagement and care utilization. The ongoing stakeholder group will utilize the data infrastructure and CPI processes throughout the second phase of the project to adapt services and inform an expanded cascade of care framework. The primary outcomes are to test the impact of the stabilization center on MOUD initiation and retention, acute care utilization (EMS, ED), morbidity, mortality, incarceration, and utilization of housing supports. We will also conduct cost benefit analyses from a public agency perspective.

NIH Research Projects · FY 2025 · 2022-09

Project Summary Neuroscience research and training have seen tremendous growth in recent years. Of note, this progress has extended into the study of psychiatric disorders, which has increased our knowledge around the neurobiology and mechanisms underlying drug use and addiction. Nonetheless, this growth has not translated into training opportunities for historically marginalized groups, including ethnic minorities, people from economically disadvantaged groups and people with disabilities. These disparities in training, mentoring and education are particularly concerning for the study of addiction, as the harms related to drug use and addiction disproportionally affect minority and underserved communities. Thus, it remains critically important that our mentoring of the next generation of neuroscientists is reflective of all the diverse groups in our society. We recognize that research experience is not only a key factor in bolstering awareness and interest in neuroscience careers, but also in the admittance and successful completion of neuroscience PhD programs. Furthermore, we believe that the historic underrepresentation of marginalized groups in neuroscience stems, in part, from a lack of research opportunities in these groups during their undergraduate education. Thus, the primary objective of the University of Washington Substantial Opportunities in Addiction Research (UW-SOAR) Doctoral Readiness Program is to close this gap of training by providing diverse groups with a mentored research experience in a world-class neuroscience laboratory in the UW NAPE (Neurobiology of Addiction, Pain and Emotion) Center. This experience will be coupled to unique educational and professional development opportunities to facilitate their recruitment and success in neuroscience doctoral programs. To achieve this goal, our program aims to 1) increase representation across multiple dimensions of difference (race, ethnicity, gender, sex, geography, ability, etc.) among postbaccalaureate researchers to improve the quality and diversity of science, 2) advance the matriculation and retention of diverse groups of trainees in Neuroscience PhD programs, and 3) determine the effectiveness of the SOAR program through an evaluation process.

NIH Research Projects · FY 2024 · 2022-09

Hypertrophic cardiomyopathy (HCM) is the most common inherited heart disease, characterized by progressive thickening of the left ventricular walls and potential for sudden cardiac death. Twenty-five percent of HCM mutations occur in the sarcomere protein cardiac myosin binding protein-C (cMyBP-C). Currently, there is no cure for HCM, only management of symptoms and disease progression, left ventricular obstruction surgery, or heart transplantation. As such, there is great need to better understand the pathological mechanisms that underly specific HCM mutations in order to better inform development of targeted therapeutics. For this project, I will study a highly penetrant mutation in cMyBP-C, c.772G>A (p.E258K), that has an identified founder effect in the north-east Tuscany region of Italy. To better explore the direct impacts of the mutation, I have generated patient induced pluripotent stem cells (iPSCs) from six HCM patients carrying the E258K mutation and a representative isogenic cell line using CRISPR/Cas9 by correcting the mutation. Initial studies have been performed on myectomy samples from three of the above six HCM patients with the E258K mutation, however, such patient tissue is limited and provides results from late stage of disease. Utilizing our patient iPSC lines, I can more thoroughly probe mechanisms underlying HCM from an almost unlimited supply of tissue specific cells. I propose to study multiple patient-derived iPSC lines all harboring the same E258K mutation, allowing me to probe the mechanism of the E258K mutation as well as investigate how other factors such as gender and age of onset may affect said mechanisms. Within the E258K patient cohort at the Careggi University Hospital, myectomy samples demonstrate consistently lower expression of full-length cMyBP-C protein, suggesting a potential haploinsufficiency disease mechanism. At the level of the sarcomere, myectomy samples indicate accelerated cross-bridge cycling, accompanied by a greater energetic cost of tension generation. Taken together, I hypothesize that the E258K mutation 1) destabilizes cMyBP-C’s ability to recruit and regulate myosin, leading to reduced expression and/or incorporation of cMyBP-C into the sarcomere (haploinsufficiency) and 2) shifts the sarcomere to a state of excessive ATP utilization during contraction (energetic inefficiency). To test this hypothesis, I will use our patient iPSCs differentiated to cardiomyocytes, and their isogenic control lines, cultured on linear, aligned substrate surfaces to enhance maturation of cardiomyocyte structure and function. My hypothesis will be tested with multiple modalities: myofibril cross-bridge kinetics, evaluation of myosin confirmations by stopped flow (disordered relaxed state vs. super-relaxed state), cMyBP-C expression and stoichiometry in the sarcomere using mass spectrometry (MS) based proteomics, cellular metabolism via Seahorse assay, substrate utilization via MS based metabolomics and energetic cost of tension generation using engineered heart tissue (EHT) constructs. If successful, this study will help uncover the mechanism of this highly penetrant HCM mutation and inform preclinical screening of potential therapeutics.

NIH Research Projects · FY 2025 · 2022-09

ABSTRACT. This project seeks funding to complete manufacturing and IND-supporting pharmacokinetic, efficacy, and safety/toxicology preclinical studies for a Phase I clinical trial of a candidate humanized monoclonal antibody (mAb) to treat and reverse toxicity associated with overdose involving fentanyl and its potent analogs. The incidence of fatal drug overdoses has dramatically increased due to the proliferation and widespread availability of fentanyl and its analogs, often found in street drug mixtures. Fatal drug overdoses totaled more than 92,000 in 2020. Current medications are not always sufficient to prevent or reverse overdose from fentanyl and its analogs. As a complementary strategy to current medications, our team has developed humanized mAbs against fentanyl and its analogs. Lead mAbs are effective in preventing and reversing drug-induced respiratory depression and bradycardia in rodents exposed to fentanyl and carfentanil. Anti-drug mAbs selectively sequester the target drug from circulation. Due to their selectivity for the target(s), our mAbs do not interfere with endogenous ligands, FDA-approved medications for treating opioid use disorders (OUD) and overdose, and other critical medications. Anti-fentanyl mAbs can be co-administered with standard of care treatments for OUD and/or overdose, and may offer longer-lasting clinical benefits over opioid receptor antagonists. Translation of mAbs will benefit those with an OUD and other individuals at high-risk of fatal overdoses from accidental or deliberate exposure to fentanyl and fentanyl analogs. This UG3/UH3 project proposes a milestone-driven iterative developmental plan with predetermined go/no go criteria to advance a mAb against fentanyl and its analogs to the clinic. AIM1 focuses on the GMP manufacturing of a lead candidate mAb in collaboration with a partner CDMO. AIM2 focuses on IND-enabling potency and efficacy studies in key animal models. AIM3 focuses on the GLP toxicology and safety of the lead mAb. AIM4 focuses on Phase I clinical trials to first test mAb safety, pharmacokinetics, and immunogenicity, and then mAb efficacy against fentanyl in healthy human subjects. Completion of this project will provide clinical data to support mAb testing in future Phase II-III trials.