Stanford University

NIH Research Projects · FY 2025 · 2022-09

ABSTRACT Radiation therapy is a potent element of standard cancer care, used in the treatment of over half of all cancer patients. While the clinical benefits of radiotherapy are well documented, the dose to adjacent and intermeshed normal organs and subsequent toxicity remains the biggest obstacle to continued escalation of radiation doses to tumors in order to obtain cancer cures with RT. Significant number of patients will develop locally persistent/recurrent tumors after radiotherapy. Therefore, radiosensitizing compounds, radiosensitizers, have been developed to enhance tumor killing effects without escalation of radiation doses. However, their clinical applications are limited due to invasive or insufficient tumor delivery and lack of specificity or systemic toxicity. The goal of this project is to develop a prodrug-based therapeutic strategy empowered by a nanotechnology pioneered by us —in cellulo nanoassembly—to amplify and enhance radiotherapeutic efficacy for treating prostate cancer. Unlike common nanoparticle-based delivery approach, this delivery strategy does not rely on the tumor enhanced permeability and retention effect. We propose to take advantage of the intrinsic heterogeneous response to radiation therapy by targeting this initial population of apoptotic cells for depositing radiosensitizers and enhancing radiotherapeutic effects. The project will develop and characterize the new prodrug radiosensitizers for targeting apoptosis (Aim 1); investigate the pharmacokinetics, toxicity and validate the in vivo treatment mechanism (Aim 2); and develop an image-guided treatment strategy, followed by a comprehensive evaluation of the therapeutic benefit in orthotopic prostate cancer mouse models (Aim 3).

NIH Research Projects · FY 2025 · 2022-09

ABSTRACT Endogenous retroviruses (ERVs) constitute 10% of all genomes, yet they remain the wild west of biology, even though they function within the host cell in a myriad of critical ways, including as regulatory elements, as extracellular messengers, as sources of viral-like particles (VLPs), and, importantly, as somatic mutagens. Our overreaching goal is to understand how the ERV family, IAP (Intracisternal type A Protein), functions at the mouse maternal-fetal interface. Thousands of genomic IAP instances exist that consist of gag, pro, pol sequences flanked by LTRs. These instances have lost the ancestral envelope (env) sequence and form naked VLPs that remain inside the cell and can transpose. Today, only a single copy, IAPE-D1, remains with a conserved envelope (env) sequence that mediates extracellular release of its VLPs. This strong evolutionary conservation of the env in IAPE-D1 suggests that it serves an important extracellular function. Thus, IAPs function in two distinct cellular domains: one as an extracellular messenger, and the other as an intracellular source of regulation and mutation. The rigor of prior research strongly supports this hypothesis. More than 50 years ago VLPs were described in placentas of all species, including mouse and human. The source of the mouse VLPs is likely IAP as we find that it is the only ERV family expressed during placentation. Our data suggests that these VLPs are functional as, when we knock down IAP using shRNA in trophoblast stem cells (TSCs) or in mouse embryos, we observe defects in differentiation and placentation. Further, we find that IAP binds specific RNAs in TSCs, including those that mediate differentiation and imprinting, suggesting that IAP VLPs contain cargo. Importantly, we find that IAP protein is expressed in maternal tissues in the absence of transcript, suggesting that it was transported from the neighboring placental cells. Based on our own preliminary results and more than 50 years of prior research on IAP, we hypothesize that 1) the only extracellular IAP, IAPE-D1, is forming VLPs in the mouse placenta that transport specific RNAs into the maternal decidua and 2) the intracellular IAPs serve multiple functions including the generation of naked VLPs that are mutagenic and as sequences that serve as critical regulatory elements within the placenta genome. While human placentas do not have IAP, they do have HERVK, which forms VLPs that are likely to serve similar functions especially given that the placenta is a hotspot for convergent evolution. Overall, this proposal will deliver the function and mechanism of a major fraction of the unexplored genome during pregnancy.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY DNA replication is a highly conserved process essential for all life. Since eukaryotic genomes are very large, they are replicated in parallel from thousands of replication origins. Proper regulation of replication initiation is essential for maintaining genome stability. Oncogene activation dysregulates replication initiation and enables rapid cell proliferation in cancer. Key regulators of replication initiation are over-expressed in cancer, and mutations in these proteins cause rare genetic disorders. Yet, the molecular mechanisms that underlie replication initiation remain poorly understood, especially in multicellular organisms, for two main reasons: (1) replication initiation involves transient protein-protein and protein-DNA interactions that are very challenging to study using existing biochemical, genetic, and structural methods; and (2) the most comprehensive studies of replication initiation have been done in yeast, however key regulators of replication initiation are poorly conserved between yeast and metazoans. Notably, some human replication factors are 2-3x larger than their yeast counterparts and containing novel domains with unknown functions. My laboratory recently developed a single-molecule imaging platform to visualize replication initiation in real time. We will use this powerful approach to (i) provide a quantitative description of replication initiation; (ii) understand how this process changed from yeast to humans; (iii) delineate the role of key replication proteins and their domains that are unique to metazoans; and (iv) understand why mutations in some replication initiation factors cause disease. Our work will also provide valuable mechanistic insights into how cancer cells sustain rapid proliferation by over-activating replication initiation and inform how replication initiation factors may be used as biomarkers or targets for novel therapies.

NIH Research Projects · FY 2025 · 2022-09

Abstract (Overall) The central hypothesis of this program project grant is that our knowledge of tumor genomics and the microenvironment, combined with our understanding of normal tissue biology, can be exploited to protect normal tissues from radiation (RT) damage while selectively killing tumor cells, leading to an improved therapeutic index. The projects and cores that comprise this grant represent a highly integrated effort with a single focus of widening the therapeutic index of radiotherapy. Project 1 (Giaccia) will modulate the radiosensitivity of tumors and radioprotection of normal tissues via the complement pathway and will aim to understand the mechanistic basis of how inhibition of C5aR1 serves to sensitize gastrointestinal (GI) tumors and protect abdominal tissues from RT. They will also explore the role of C5aR1 inhibition in other normal tissues in collaboration with the other projects and cores. Project 2 (Le) will focus on activating Aldehyde Dehydrogenase- 3A1 (ALDH3A1) to mitigate RT-induced severe dry mouth in head and neck cancer (HNC) patients by testing d- limonene, a novel ALDH3A1 activator identified by their group, in a phase I clinical trial. While focusing on HNC, they will evaluate the effect of d-limonene in radioprotecting other normal tissues in collaboration with the other projects and cores. Project 3 (Diehn) will develop a personalized radiosensitization strategy for patients with KEAP1/NFE2L2 mutant non-small cell lung cancer (NSCLC) based on their prior work that identified mutations in this pathway as key determinants of radioresistance in NSCLC patients. They will test the hypothesis that glutaminase inhibition preferentially radiosensitizes KEAP1 mutant NSCLC without enhancing normal lung tissue toxicity. While concentrating on NSCLC, they will also evaluate the effects of glutaminase inhibition in other KEAP1/NFE2L2 mutant tumors and its effect on normal tissues in collaboration with other projects and cores. Project 4 (Rankin) tests the hypothesis that inhibition of FTO (Fat mass and obesity-associated protein), an RNA demethylases, would enhance the efficacy of RT in multiple solid tumors. This is based on their preliminary data showing that FTO is overexpressed in many cancers including cervical, lung and HN cancers, that FTO inhibition reduces cancer cell growth and enhances RT sensitivity through the inhibition of glutamine metabolism. They will determine the therapeutic effects and mechanism of action of FTO inhibition in combination with RT in multiple cancer models in collaboration with the other projects and cores. They will also study the effect of FTO inhibition on normal tissue response to RT. If successful, D-limonene, a nutraceutical, can be rapidly tested in larger phase II and III clinical trials for future clinical use. Similarly, PMX 205 (a C5aR1 inhibitor) and CB-839 (a glutaminase inhibitor) are currently being evaluated in clinical trials for other clinical indications while drugs targeting FTO are in active development. Thus the proposed projects could rapidly lead to clinical studies that could impact the management of cancer patients.

NIH Research Projects · FY 2025 · 2022-09

The application responds to PAR-21-130: Clinical Trials to Test the Effectiveness of Treatment, Prevention, and Services Interventions. With an incidence rate of about 1%, Anorexia Nervosa (AN) is a serious mental disorder associated with high mortality, morbidity and cost but becomes highly resistant once it has taken an enduring course. The first-line treatment for adolescents with AN is Family Based Treatment (FBT), but is not available to many patients. An effective strategy used with other eating disorders to address impediments to scaling effective treatments is guided self-help (GSH) versions of efficacious treatments. We developed, refined, and tested GSH-FBT in case series and pilot studies. Results of a multisite randomized feasibility study comparing GSH-FBT to FBT-V found that both treatments were similarly acceptable to families with good recruitment rates, low attrition rates, high completion rates of assessments, and were safe to deliver while achieving similar improvements in eating related cognitions and weight. However, GSH-FBT achieved these outcomes with greater efficiency (e.g., larger ratio of improvement in weight and cognitions to therapist treatment time) by utilizing approximately 75% less therapist time than FBT-V. The main aim of this proposed comparative effectiveness study is to confirm that clinical improvements in GSH-FBT are achieved with greater efficiency than FBT-V in generalizable clinical settings. If this outcome is confirmed, it will lead to an increased opportunity to utilize and scale an effective treatment for adolescent AN, promote increased access and improve outcomes for these patients. We will also explore therapist attitudes toward GSH-FBT. To conduct this study 200 adolescents with DSM-5 AN and their families will be randomized to either GSH-FBT or FBT-V in two treatment sites (Stanford Children’s Hospital Eating Disorder Clinic and the Ontario Provincial Network of Ministry-funded Specialized Treatment Services for Eating Disorders). The main outcome will be clinical efficiency (the ratio of change in weight and eating related distorted cognitions to therapist time). Parental self- efficacy will be assessed as a potential mediator of treatment effect. Family structure and severity of eating related obsessions will be examined as moderators. Weights will be collected from session 1-4 to assess early weight gain as a predictor of weight remission at the end of treatment (EOT). Data on therapists’ views of GSH-FBT implementation will be collected by quantitative measures at BL and EOT, as well as by individual semi-structured qualitative interviews. Both primary and secondary outcomes will be analyzed in line with the intention to treat principle.

NIH Research Projects · FY 2025 · 2022-09

Military spouses in relationships with a heavy drinking service member partner report high levels of depression symptoms, drinking, and social impairment, and a heightened risk for domestic violence compared to spouses who are not in a relationship with a heavy drinking partner, and these consequences are compounded when both partners drink heavily. Yet, spouses often do not seek care for their own or their partner’s problems due to multiple barriers preventing pursuit of care. Military spouses and partners —termed “concerned partners” (CPs)—may be an important gateway for motivating service members to seek care. However, CPs may first need to improve their well-being and communication to effectively support and encourage changes for their service member partner. The proposed study builds off our pilot work with Partners Connect, a 4-session web- based intervention (WBI) for military CPs. In Aim 1, we propose a Sequential Multiple Assignment Randomized Trial (SMART) design to evaluate the efficacy of an adaptive CP intervention on CP well-being and SM help- seeking. In Aim 2, we will examine how and for whom the adaptive intervention is most efficacious by looking at moderators and mediators of service-member help-seeking and supplementing these quantitative analyses with qualitative CP and service member interviews on the reasons and drivers of service member help- seeking. We will conduct a two-stage SMART design. In stage one, we will randomize CPs to either Partners Connect or communication resources from the Gottman Institute website. CPs will be considered a ‘”responder” at stage one if their service member completes their personalized normative feedback (PNF) session. CPs who are non-responders to stage one (service member has not completed PNF) will be re- randomized to receive additional communication interventions in stage two. The purpose of the stage two randomization is to test what helps stage one non-responders. We expect that by further treating those that do not respond initially to Partners Connect, we can improve CP communication, and ultimately help service members access a brief, effective PNF intervention for their drinking. Our goal is to first intervene with the service member’s spouse and improve their outcomes to better equip them to engage their service member in services. In doing so, we develop a model that increases treatment accessibility and appeal among a group that may not otherwise seek care. The optimal package for CPs is the one that improves the CP’s well-being, mental health, and communication and that ultimately engages the service member in PNF. Thus, Partners Connect is a novel “two-in-one” intervention that fills service gaps for CPs and service members.

NIH Research Projects · FY 2026 · 2022-09

PROJECT SUMMARY Almost 70% of Latino people with mental health disorders fail to receive the treatment they need due to various barriers to accessing culturally specific mental health care. We propose that particularly trusted voices might be able to decrease mental health stigma in their communities and strengthen social networks to improve mental health services connections. In Spanish-speaking communities, lay health workers called promotoras fulfill this role: promotoras act as educators, cultural brokers, and bridges between the community and health services. We will engage with a promotora learning collaborative to develop and assess a community-wide mental health education and empowerment campaign to increase culturally specific knowledge of mental health, trauma, and support services and to improve mental health services utilization in the Latino population of East San José, CA. By collaborating with deep-rooted community partners, we seek to achieve the following specific aims: 1) Map the landscape of and pathways to mental health services within the community; 2) Strengthen promotoras’ capacity to engage their community around mental health services use; and 3) Determine if engaging promotoras in a mental health education and empowerment campaign increases mental health services utilization community-wide. Under the first aim, we will work with community partners and promotoras to create a rich community asset map of culturally specific mental health services, layered with findings from a community-wide assessment of mental health services use, using geographic information system mapping (GIS). Promotoras will then be trained to facilitate Mental Health Education, (self-)Efficacy, and (outcomes) Expectancy (MHE3) gatherings – a co-created culturally specific intervention to improve mental health services use. Finally, in the third aim, we will implement MHE3 as a cluster randomized trial and determine its impact at the individual, interpersonal, and community levels. Beyond self-reported outcomes, we will investigate the impact on social networks used to identify and choose mental health services. We will combine findings from existing facility-based and claims data with the results of a newly collected annual cross-sectional survey of the community to determine broader communitylevel impact on mental health services use. Understanding how promotoras help overcome barriers to mental health and services use will deliver insights that inform collaboration with and integration of promotoras into care pathways and delivery strategies to decrease mental health gaps among Latino communities more widely. Furthermore, we believe this work can also serve as a template for how to conduct outreach to other hard-to-reach populations. Finally, the proposed research will further NIMH’s mission to strengthen the public health impact of research by testing innovating approaches to reduce differences in care access.

NIH Research Projects · FY 2025 · 2022-09

PROGRAM SUMMARY/ABSTRACT As a molecular detection platform, antibodies have growing importance in modern medical technology, ranging from diagnostic tests, to imaging, to therapeutics. The current market size for antibodies and their related products is estimated to be around $200 billion USD. The growing need for antibodies with customized specificity provides a rich environment for engineering efforts. Computational protein design has seen rapid progress in recent years. Many methods have been developed to address antibody engineering needs. Researchers have hoped that, through modeling and design, the cost for antibody development and improvements can be reduced and the pace for creating new targeting molecules can be expedited. In recent years, the experimental pipeline has been streamlined, but even so, extensive libraries and screen campaigns are usually required to get an initial binding signal. A major advancement would be to directly design a binder from scratch, providing a signal for potential optimization by artificial evolution. Current computational methods, however, have not taken a leading role due to a number of shortcomings with the current modeling approach. We have extensive expertise in protein design and have pioneered the use of generative neural network models for protein structures in recent years. We have observed several key advantages in neural network approaches over existing methods: namely, their ability to make inferences, interpolate, incorporate topological information, and accelerate sampling. These advantages can be developed independently or used in conjunction with existing methods, and they can significantly boost the performance of protein design. This project aims at leveraging several new advances we have developed to date to inspire new strategies in response to the challenges in antibody engineering, or AI-based protein design in general. We will develop new tools and design pipelines for expanding the specificities for multi- specific antibodies and customizing epitope-specific antibodies (using snake venoms and CXCR4 as targets). This project will deliver both computational methods and constructs that can be deployed in clinical settings. The results from this research will be highly impactful.

NIH Research Projects · FY 2024 · 2022-09

PROJECT ABSTRACT Viral infection is a common cause of sensorineural hearing loss (SNHL). However, the underlying mechanism is poorly understood. For most viruses, it is unclear whether SNHL is caused by direct viral infection of inner ear tissue or by a secondary effect through an immune response, or a combination of both. Antiviral drugs and steroids only improve the outcome for a small group of patients. It is therefore imperative to understand the molecular etiology of virus-induced SNHL for the development of effective preventions and treatments. The research proposes a systemic evaluation of viral effects on the inner ear using mumps virus (MuV), a human virus that causes SNHL after birth (acquired SNHL), and murine cytomegalovirus (mCMV) to understand human CMV disease that causes SNHL during embryonic development (congenital SNHL). There is no small animal model to assess SNHL caused by MuV. In Aim1, it is proposed to establish novel in vivo mouse models to assess how mCMV and MuV infections affect inner ear cells. Infection will be done locally and controlled via the posterior semicircular canal. The onset and extent of hearing loss will be measured by auditory brainstem response and distortion product otoacoustic emissions. Histochemistry will quantify apoptotic cells, virus-infected cells, and immune cells in defined anatomical locations of the cochlea from apex to base. Because the model is expected to be highly controllable, it will be optimized so that the detrimental mechanisms of different viruses can be compared to advance the mechanistic understanding of virus-induced SNHL. Aim2 focuses on understanding the etiology of the tissue damage -direct viral infection or immune response- in mCMV- and MuV- administrated cochleae, and to analyze the mechanisms at the cellular and molecular level. Susceptible cell subtypes will be quantified in P2 and P21 mice injected with mCMV and MuV before and after the onset of hearing loss by using known and recently identified markers. These include new pericochlear cell subtypes in the neonatal cochlea that were recently reported by the principal investigator (PI), as well as the new subtypes of type I spiral ganglion cells. The expression of a ganglioside called GM2, a MuV receptor which the PI identified in a previous study, and various viral host factors will be also quantified in each cochlear cell subtype to understand their correlation with cellular damage. Further, the types and numbers of infiltrating immune cells post PSC injection of mCMV and MuV in P2 and P21 mice will be assessed with flow cytometric analysis with cell type-specific markers. Together, completion of these aims will provide essential details of the mechanism of SNHL caused by mCMV and MuV, and will establish a protocol that can be applied to other hearing loss-causing viruses such as Lassa virus, which is not suitable for animal model development because of its high biosafety level. The PI envisions this project as the first step of her future research journey as a clinician-scientist with a focus on prevention and treatment of viral-induced SNHL.

NIH Research Projects · FY 2025 · 2022-09

Abstract: Hearing and balance disorders disable nearly half a billion people worldwide, yet there are virtually no pharmacological or biological therapies for these disorders. This alarming state of medicine coexist with the brighter state of science where numerous therapeutic approaches have shown efficacy in animal models. This conundrum reflects the fact that there are important differences between animal models and humans, that we have an incomplete understanding of the molecular signatures of the auditory and vestibular organs in the human inner ear, and that adult human inner ear tissues are not readily available to test promising therapeutics. We propose to solve this conundrum by defining the molecular makeup of normal, live human inner ear tissues (Aim 1), describing the three-dimensional (3D) cellular architecture of unprocessed human inner ears (Aim 2), training new and established investigators (Aim 3), and enhancing awareness of human inner ear research (Aim 4). In support of this approach, we have designed a surgical method to procure live inner ear tissues from deceased organ donors who typically have normal auditory and vestibular function. We have begun assembling a registry consisting of medical records, single-cell transcriptomes, and histologic sections of vestibular tissues (utricles). In parallel, we have augmented the registry with utricles from vestibular schwannoma patients undergoing surgical resection. Here, we propose to increase the recruitment of organ donors and vestibular schwannoma patients and expand our registry to include all inner ear sensory organs and generate a molecular cell atlas of the adult human inner ear (Aim 1). Additional tissues and perilymph will be collected, analyzed and shared with the broader scientific community for gene and protein validation. Furthermore, we will use a miniature, flexible imaging probe we developed to perform micro-optical coherence tomography (µOCT)-based endomicroscopy on rapid autopsy cadavers to generate an optical cell atlas of the 3D-intact, unprocessed human inner ear (Aim 2). A second registry will be assembled, consisting of digitized µOCT-histology images analyzed with the aid of both linear regression and artificial intelligence tools. Thirdly, we will train clinicians, clinician- scientists, and researchers on the techniques of procuring and imaging human inner ear tissues through hands- on training, simulated surgery, and didactic workshops (Aim 3). Lastly, we will raise awareness of studying human inner ear tissues through outreach activities, publicizing our resources, data sharing, and collaborations (Aim 4). Upon completion of this 5-year program, we will have assembled and shared a molecular and optical cell atlas of the human inner ear and increased awareness and utilization of this resource by the scientific community.

NIH Research Projects · FY 2025 · 2022-09

Project Summary/Abstract Older women are disproportionately impacted by a range of chronic diseases and conditions, such as cognitive impairments, that can be alleviated by regular physical activity (PA), including walking; yet they are the most inactive segment of the US population. While the majority of older women use smartphones, few remotely delivered PA interventions have been developed specifically to address this age + gender PA disparity, and few such interventions leverage built and social environmental factors that can increase daily PA. Using a dynamically based socio-ecological model of health behavior change, this study’s primary aim is to enhance the efficacy of a “light-touch”, remotely delivered PA educational intervention for older women by testing the added impacts of an innovative, remotely delivered citizen science program to identify and address local environmental barriers to walking and other PA (called Our Voice TM [OV]). The “light-touch” PA educational program has been delivered since 2015 to >23,000 women >70 years that have been participating in the U.S.-wide Women’s Health Initiative Strong and Healthy (WHISH) pragmatic PA trial (U01HL122280-03). WHISH, which used a pragmatic, ‘opt-out’ consent enrollment process, includes the broadest range of PA, physical function levels, and geographic regions found in PA intervention studies to date. The original WHISH educational program has resulted in significant but generally modest PA increas- es in the overall WHISH population, and surveys indicate that many WHISH participants could benefit strongly from further intervention. An enriched version of the original remote program (called enCore) will serve as the enCore Alone arm in the proposed trial. To address local built and social environmental PA barriers that many WHISH women describe, the mobile app plus web based OV program will be added to enCore (enCore+OV arm). We will randomize a total of 300 WHISH intervention women (mean age=85 yrs.) with smartphones who are insufficiently active to the two arms. We hypothesize that women receiving enCore+ OV will show higher pedometer-measured 12-month PA levels than women receiving enCore Alone. Additional questions include changes in cognitive function and sedentary behavior; exploring a theo- retically derived multi-level set of putative mediators (e.g., social cohesion, rated neighborhood walkability, community wayfinding) and baseline moderators of program success (e.g., race/ethnicity, family support, geographic region); and exploring the relative costs of the two programs for PA change. The study will add important information on the benefits and trade-offs of combining these remotely delivered, practical, and potentially scalable behavioral health delivery approaches for this fast-expanding demographic group.

NIH Research Projects · FY 2025 · 2022-09

Abstract Post-stroke dementia is an important and understudied component of the vascular contributions to cognitive impairment and dementia. Having a stroke approximately doubles the risk of incident dementia for at least a decade afterwards, even after accounting for other vascular risk factors of dementia and the initial effects of the stroke lesion on cognition. Also, silent strokes occur in nearly half of all aging individuals and are associated with dementia. We established in wildtype mice that stroke triggers chronic neuroinflammation in the stroke scar and connected brain regions, and that this causes delayed-onset cognitive decline. In humans, there is neuroinflammation in the stroke scar in about half of all chronic stroke survivors on autopsy, even decades after stroke, suggesting it may play a role in people as well. However, there are no biomarkers that can currently be used in living humans to detect who is at risk of cognitive decline and dementia after stroke. Here we propose to test the hypothesis that inflammation-induced angiogenesis in the stroke and connected regions results in immature leaky vessels that cause blood-brain barrier leakage even very late after stroke. We will recruit 200 participants with chronic stroke and 50 controls at 3 sites (Stanford School of Medicine, Columbia University, and the University of Manchester). We will test an MRI-based imaging biomarker in Aim 1 and ask whether blood-brain barrier permeability is compromised for years after stroke. In Aim 2 we will ask whether a blood biomarker of imbalanced angiogenesis is dysregulated in chronic stroke. For both, we will also look at risk factors for their development and how they relate to stroke size, location, sex, age, and NIHSS. Finally, in Aim 3 we will use both traditional multivariable and machine learning models to ask whether each biomarker separately or together predicts cognitive decline after stroke, and to identify other MRI, blood, and clinical characteristics that are associated. If we are successful, we will establish that there is chronic blood-brain barrier dysfunction after stroke and link it to dysregulated angiogenesis as a potential mechanism. This would be a fundamental change in how post-stroke dementia is conceptualized and would open avenues for novel therapy development. Our predictive models will also be useful to identify stroke survivors at high risk of cognitive decline and/or to select patients for future clinical trials. This will thus help us better understand vascular contributions to cognitive impairment and dementia.

NIH Research Projects · FY 2025 · 2022-09

Project Summary Mitotic chromosome formation is essential for the dissemination of genetic information in eukaryotes. The conversion of dispersed interphase chromatin into the iconic, X-shaped metaphase chromosome involves three principal events: the formation of a central axis (the chromosomal “scaffold”), lengthwise contraction, and condensation. Plausible theories have been advanced to explain the latter two events, with contraction occurring through the extrusion of chromatin loops and condensation occurring through a volume phase transition of the chromosomal material. In contrast, little is known about the formation of the mitotic scaffold, and its very existence has been the source of considerable controversy. Direct evidence for the scaffold's existence has recently come from the observation of a central filament in native mitotic chromosomes subjected to controlled expansion ex vivo. This filament could be liberated, intact, from its chromosomal confines by careful nucleolysis, providing a basis for further study. This proposal aims to elucidate basic principles of the mitotic chromosome scaffold, including its molecular composition (Aim 1), its three-dimensional architecture (Aim 2), and its interactions with the chromatin enveloping it (Aims 1 and 2). The information obtained will provide a global view of the core of the mitotic chromosome, explaining how its components assemble into a structure of mesoscopic proportions and how this structure organizes the genome in a manner ensuring its faithful and efficient distribution. The acquired knowledge will enable more detailed investigations into the mechanisms governing scaffold assembly and disassembly and the nature of its interactions with other cellular components (e.g., chromatin, kinetochores). This proposal also seeks to develop chemical modulators of scaffold assembly (Aim 3). As the scaffold and its constituents are increasingly understood to play important roles in human health and disease, such tools will not only enable further research on the scaffold but will also allow for its pharmacological manipulation in clinical contexts. Dysregulation of scaffold components such as the condensins, for instance, has been implicated in a growing number of malignancies, where they contribute to a particular form of genome instability known as chromosome instability. Genome instability is a key factor in the evolution of cancer cells, facilitating their escape from immune clearance and their acquisition of therapeutic resistance. By suppressing a pathway leading to chromosome instability, modulators of condensins (and other scaffold components) may act to limit the evolutionary potential of cancer cells.

NIH Research Projects · FY 2025 · 2022-09

Clear cell ovarian cancer (ccOC) is a rare and lethal cancer with few treatment options. Based on molecular analysis ccOC appears intrinsically immunogenic but with an immunosuppressive tumor microenvironment, similar to other ovarian cancer types. However, ccOC is very distinct from high grade serous ovarian carcinoma. Strikingly, it is similar in gene expression profiles to more frequent clear cell renal cell carcinomas (ccRCC), suggesting that clear cell cancers share intrinsic mechanistic or microenvironment properties, not just morphological appearance. Around 25% of ccRCC respond well to immune checkpoint inhibitors (ICIs), but markers for predicting response are lacking. The objective response rate for monotherapy pembrolizumab in one study was 33.3% for ccOC patients; but, in general, it is unknown which clear cell cancer patients could benefit from ICI treatment. Recent work has shown that tumor behavior is driven not just by cellular composition, but also by the spatial organization of different cell types including immune and stromal cells, as well as malignant cells themselves. Knowledge of clear cell cancer tumor microenvironments and their spatial architecture is lacking. Addressing this gap will improve our understanding of mechanisms of response to ICIs in clear cell cancers, including rare ones like ccOC, and improve selection of patients for immunotherapy. This study will use systems biology approaches to (i) elucidate and compare the cell types and their transcriptional states present in ccOC and ccRCC; (ii) characterize the spatial architecture of these cells within tumors using the CODEX (CODetection by indEXing) single cell proteomic imaging platform; and (iii) model and validate cell-cell interactions in the spatial tumor microenvironment that drive clear cell cancer response to immunotherapy through extensions of causal signaling inference algorithms to incorporate spatial context, and to optimize experimental validations in mouse models that maximize the information gain about interaction networks. Similar intrinsic and tumor microenvironmental features shared by ccOC and ccRCC, will nominate common mechanisms of immunotherapy response, and identify the subset of both who might benefit from treatment with ICIs. Successful development and application of these methods to clear cell cancers will establish a framework that can be applied to other cancer types, notably to rare ones. The expected outcome of this proposal is a comprehensive definition and dissection of the tumor microenvironment of ccRCC and ccOC. It will identify common features and mechanisms between these clear cell cancers, providing a basis to extend the approach to other classes of cancer, opening new avenues for treatment, particularly in rare cancer types.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Genome-wide association studies have discovered thousands of genetic variants associated with phenotypic traits such as disease risk. Most of the associated variation lies within non-coding regions of the genome and the causative effects of those variants remain largely unknown. The sparsity of knowledge on interactions between the coding and non-coding regulatory parts of the genome makes the prediction of variant function solely from genome sequence and location impossible. We propose to experimentally uncover the functional relevance of genetic variants at a large scale, by perturbing variants and genetic elements containing variants, and reading out the direct consequences of those perturbations on gene regulation. To this end, we propose to apply our recently developed CRISPR/Cas9 functional genomics screening technology with targeted single-cell transcriptomic readouts (targeted Perturb-seq or TAP-Seq in short) to enable systematic interrogation of non- coding regions and genetic variation therein. First, we will apply our targeted Perturb-seq to decipher the regulatory circuitry encoded on an entire human chromosome by systematically perturbing all major genetic elements (enhancers, protein-coding and lncRNA genes). This extensive data set will enable to decipher the complex regulatory networks controlling gene expression on the selected chromosome. Next, we will uncover causal regulatory variants in these regions by coupling high-throughput precision genome editing to simultaneous single-cell genomic and transcriptomic readout. Using this novel approach, we will be able to decipher the functional impact of genetic variants on gene expression and derive rules by which genetic variation perturbs gene regulatory processes. We will integrate the generated data with available functional genomics data, such as transcription factor binding (ChIP-seq), chromatin accessibility (ATAC-seq, DNAse-seq) and interactions in 3D (Hi-C), in order to train machine learning models to derive rules of the observed regulatory interactions. These models will be applied to decipher the molecular mechanisms underlying the regulatory logic, and to predict regulatory interactions and variants throughout the genome and across cell types. Selected predictions will be experimentally validated using the established perturbation technologies, to verify clinically relevant predictions and improve the performance of the predictive models. Taken together, this project will answer fundamental questions in gene regulation, uncover the mechanisms by which genetic variation impacts gene expression, and create datasets and computational models as valuable tools for interpreting results from GWAS, eQTL and clinical genomic studies.

NIH Research Projects · FY 2024 · 2022-09

PROJECT SUMMARY Despite high enrollment in medical school and graduate programs, women remain under-represented in the biomedical research workforce. NIH T32 training programs are an understudied yet important contributor to the biomedical research workforce and are a key stage at which we can intervene. While roughly half of trainees covered by T32 grants are women, women remain underrepresented amongst those earning extramural research support. Given its prevalence in both medicine and science, sexual harassment is likely a contributor to this disparity. Data from the National Academies of Sciences, Engineering, and Medicine describe the damage caused by sexual harassment, ultimately leading to a costly loss of talent as women, particularly women with intersectional identities, and sexual and gender minorities leave. The drivers of sexual harassment, including factors and conditions that allow it to thrive, have been described with a conceptual model incorporating an iceberg as a metaphor. The vast majority of sexual harassment, this model contends, is the portion of the iceberg that is invisible under water. Because of this, many do not fully perceive the prevalence of sexual harassment; however, the behaviors described in this model are damaging whether they are visible (i.e., above water) or not. Despite these data, it is not yet clear what interventions can effectively decrease the occurrence of sexual harassment. Newer interventions, such as civility and upstander interventions, have been recommended by the Equal Employment Opportunity Commission but have not been rigorously tested. Data suggest that interventions that go beyond sexual harassment alone and address other related issues, such civility and upstander interventions, may be more effective than sexual harassment training alone. We will conduct a randomized, controlled trial of NIH T32 programs to test whether a multi- modal virtual intervention incorporating video game elements can increase T32 Principal Investigators’ and mentors’ confidence in their ability to intervene when they hear about or see sexual harassment and increase their knowledge on these topics. Furthermore, we will test the impact of the intervention on their mentees’ experiences with microaggressions and sexual harassment, sense of belonging, well-being, research productivity, and persistence in a biomedical research career. Finally, we will test whether the intervention improves the culture and climate of the learning environment. Rigorous evaluation of a virtual interactive intervention to address and reduce sexual harassment for NIH T32 trainees can result in a generalizable and easily scalable educational program to improve NIH training environments nationwide and ultimately improve the diversity of the biomedical research workforce.

NIH Research Projects · FY 2025 · 2022-09

Project Summary/Abstract Social deficits occur across a wide array of neuropsychiatric (NPD) and neurodevelopmental (NDD) disorders and contribute to poor outcomes and sizable public health costs. However, the lack of adequate characterization of mechanisms underpinning social impairments by the current categorical diagnostic systems has significantly stifled the development of etiologically based, individually tailored treatments. A fully dimensional alternative to the categorical frameworks offered by the National Institute of Mental Health’s Research Domain Criteria (RDoC) holds particular promise for a better understanding of mechanisms behind social deficits. More specifically, the RDoC initiative operationalizes an initial set of basic, biologically meaningful components that underpin social functioning, and if disrupted, can impede one’s ability to navigate the complexities of the social world. These processes can therefore be used to better understand social deficits seen across NPD/NDD and inform personalization of treatments. However, we currently lack dedicated measures able to comprehensively capture components of social functioning across clinical, at risk and normative populations which has significantly impeded the translation and adoption of this potentially promising framework. Therefore, the overarching aim of this project is to further expand a recently developed RDoC-based social processes scale: The Stanford Social Dimensions Scale (SSDS). The goal is to refine, factorize, validate and establish regression-based norms of the updated SSDS (SSDS-2). We will also aim to construct a preliminary computerized adaptive testing (CAT) version of the SSDS-2 that will enable individually tailored item selection and administration. These objectives will be achieved by: 1) obtaining feedback on a preliminary item bank from experts and parents of children from normative and clinical groups in order to evaluate the content validity, developmental appropriateness and clinical relevance of the items and to guide the item refinement (Specific Aim 1); 2) utilizing advanced psychometric approaches including exploratory structural equation modeling and item response theory on a large online recruited clinically diverse and normative sample to establish the factor structure (Specific Aim 2); 3) confirming factor structure of the SSDS-2, establishing regression based and standardized change norms and constructing a preliminary CAT version (Specific Aim 3); and further validating the SSDS-2 through an in-person multi-method assessment protocol encompassing interview, observational and experimental methodology with a transdiagnostic sample of youth with a range of social abilities and typically developing youth and their parents. An additional goal is to examine the association between the SSDS-2 subdomains with the neural networks subserving corresponding social processes in a transdiagnostic subsample of youth (Specific Aim 4). This project will lay the foundation for future investigations aimed at: (i) extending the new measure to different age groups (2-5 years and adulthood); (ii) developing a companion clinician-rated structured interview, and (iii) testing its utility as a clinical outcome measure.

NIH Research Projects · FY 2024 · 2022-09

Project Summary/Abstract Social deficits occur across a wide array of neuropsychiatric (NPD) and neurodevelopmental (NDD) disorders and contribute to poor outcomes and sizable public health costs. However, the lack of adequate characterization of mechanisms underpinning social impairments by the current categorical diagnostic systems has significantly stifled the development of etiologically based, individually tailored treatments. A fully dimensional alternative to the categorical frameworks offered by the National Institute of Mental Health’s Research Domain Criteria (RDoC) holds particular promise for a better understanding of mechanisms behind social deficits. More specifically, the RDoC initiative operationalizes an initial set of basic, biologically meaningful components that underpin social functioning, and if disrupted, can impede one’s ability to navigate the complexities of the social world. These processes can therefore be used to better understand social deficits seen across NPD/NDD and inform personalization of treatments. However, we currently lack dedicated measures able to comprehensively capture components of social functioning across clinical, at risk and normative populations which has significantly impeded the translation and adoption of this potentially promising framework. Therefore, the overarching aim of this project is to further expand a recently developed RDoC-based social processes scale: The Stanford Social Dimensions Scale (SSDS). The goal is to refine, factorize, validate and establish regression-based norms of the updated SSDS (SSDS-2). We will also aim to construct a preliminary computerized adaptive testing (CAT) version of the SSDS-2 that will enable individually tailored item selection and administration. These objectives will be achieved by: 1) obtaining feedback on a preliminary item bank from experts and parents of children from normative and clinical groups in order to evaluate the content validity, developmental appropriateness and clinical relevance of the items and to guide the item refinement (Specific Aim 1); 2) utilizing advanced psychometric approaches including exploratory structural equation modeling and item response theory on a large online recruited clinically diverse and normative sample to establish the factor structure (Specific Aim 2); 3) confirming factor structure of the SSDS-2, establishing regression based and standardized change norms and constructing a preliminary CAT version (Specific Aim 3); and further validating the SSDS-2 through an in-person multi-method assessment protocol encompassing interview, observational and experimental methodology with a transdiagnostic sample of youth with a range of social abilities and typically developing youth and their parents. An additional goal is to examine the association between the SSDS-2 subdomains with the neural networks subserving corresponding social processes in a transdiagnostic subsample of youth (Specific Aim 4). This project will lay the foundation for future investigations aimed at: (i) extending the new measure to different age groups (2-5 years and adulthood); (ii) developing a companion clinician-rated structured interview, and (iii) testing its utility as a clinical outcome measure.

NIH Research Projects · FY 2025 · 2022-09

The mission of our proposed Autism Center of Excellence (ACE) is to examine if dysregulation of sleep is central to the development and exacerbation of symptoms in ASD. Animal data demonstrate that sleep is essential for the maturation of fundamental brain structures, neuronal development and synaptic plasticity. Sleep dysregulation is one of the most burdensome symptoms in individuals with ASD. Late sleep onset, frequent nighttime awakening, sleep fragmentation and abnormal sleep quantity hallmark sleep in ASD. Sleep EEG studies indicate less REM sleep and increased Non-REM Sleep. Despite its central role in brain development and function, sleep impairments are frequently considered as secondary. The main goal of our Center for Sleep in ASD is to determine if sleep disturbances reflect convergent pathways that can act as causal for, and/or co-aggravating factors of, core, behavioral and cognitive symptoms in ASD. We propose a multi-modal, human subjects and animal models program which encompasses four synergistic projects aimed at characterizing the role of sleep fragmentation and physiology on the core symptoms of ASD. We will examine Sleep EEG, daytime awake, resting EEG, and actigraphy in 150 individuals with ASD, 4 to 17 years, compared to 75 age- and sex-matched Typical Developing (TD) controls and determine the impact of these sleep parameters on core symptoms (Project 1). Using a target engagement approach, we will determine if normalization of sleep is associated with improvements in the core symptoms (Project 2). We will examine if these findings are recapitulated in genetic animal models of ASD (Mice: Project 3; & Zebrafish: Project 4). The evolutionary conservation of Sleep EEG signatures and behavior makes this a powerful translational approach as the same physiological parameters, biological endpoints and behavioral phenotypes can be compared across species, revealing if there is a convergence of the impact of sleep phenotypes across different genetic models of ASD, or alternatively differential pathways from sleep phenotypes to core symptoms. Specific Aim 1: To leverage comparative biology across humans with ASD and controls and complementary genetic animal models of ASD and wild type to examine if multisystem sleep measurements across species a) converge on a common phenotype of sleep fragmentation and architecture in ASD; or b) capture different sub- phenotypes of sleep dysregulation and sleep architecture across species. Specific Aim 2: Examine if a) the sleep phenotypes identified are differentially associated with the core, behavioral and cognitive symptoms of ASD across humans with ASD and complementary genetic animal models of ASD; b) if sleep normalization in humans with ASD and in complementary genetic animal models of ASD, alleviate the core, behavioral and cognitive symptoms of ASD; and c) if these effects are moderated by age and/or sex. Specific Aim 3: Provide research and collaborative opportunities to junior and established researchers new to the field of autism, or established in the field of autism but new to the research emphases of the ACE Center.

NIH Research Projects · FY 2024 · 2022-09

Project Summary Patient-derived tumor organoids (PDO), involving the ex vivo culture of fresh tumor fragments, have emerged as promising models for predicting patient drug response for personalized cancer therapy. PDOs recapitulate the tumor micro-environment (TME), resemble the source tumor phenotypically and genomically, and are compatible with high-throughput drug screening. However, the lack of preservation of immune cells in PDOs has been a major roadblock to modeling immunotherapy. Our team recently demonstrated a new type of PDO that cultures tumor fragments as a cohesive unit, allowing the in situ preservation of diverse immune cell types alongside tumor cells without artificial reconstitution. This approach has enabled the modeling of patient-specific responses to immune checkpoint inhibitors. One of the first steps in the generation of PDOs is the dissection of patient tumor specimen into small fragments. Mechanical dissection, instead of enzymatic digestion, is critical in preserving the in vivo association between tumor cells and endogenous immune and non-immune elements. The ability to preserve endogenous immune cells, including tumor-infiltrating lymphocytes (TIL), is particularly important for personalized immunotherapy testing. However, current mechanical dissection relies primarily on manual mincing of tumor specimen into small fragments. It results in fragments with a broad size range, and is imprecise and irreproducible. Fragments that are too large suffer from inadequate nutrient supply, suboptimal oxygenation and viability, and poor drug penetration. Fragments that are too small are unlikely to preserve sufficient stromal cells to support PDO growth, and/or endogenous immune cells which may be present at low concentrations. As such, there is an unmet need for a better way to generate tumor fragments of controllable and uniform size, and identify optimal size(s) to increase the reproducibility and yield of viable PDOs that can preserve the cellular contexture and tumor architecture. This project aims to address this need by developing a new method to mechanically dissect tumor specimen into uniform fragments. Performance measures include fragment size uniformity, PDO viability, preservation of immune cells, and tumor cytotoxicity in response to immunotherapy. Other cutting methods including manual mincing will be used as benchmarks.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY/ABSTRACT Reproductive success and survival of progeny in organisms that undergo sexual reproduction are contingent on social interactions, and in humans of course these behaviors are also critical for mental well-being and success in our professional lives. There have been significant advances in understanding how the nervous system of diverse animals, ranging from worms to mammals, enables successful display of social interactions. In mice for example, central components of neural circuits underlying reproductive behavior, territorial aggression, and parental care have been clearly identified in both males and females using molecular genetic approaches. Recent studies have also provided insights into how specific chemosensory cues act on these neural circuits to guide ongoing social interactions. How social interactions and their outcomes are internally sensed to modulate downstream long term changes in the organism is less well understood. Mammalian females show dramatic changes following reproductive behavior as they transition from seeking mates to preparing for pregnancy, childbirth, and subsequent nursing. The neural circuits that sense and mediate these major transitions are poorly characterized. Our goal is to characterize these neural circuits mechanistically in order to understand how they sense changes in peripheral, visceral reproductive organs to guide subsequent behavioral transitions. In unpublished findings, we have identified a population of neurons in the female mouse brain that specifically senses successful culmination of reproductive behavior but not the social interactions that precede it. In Aim 1 of this project, we propose to image ongoing activity of these neurons to define how and when they sense this behavioral endpoint. In Aim 2, we will functionally characterize these neurons to understand their contribution to post-reproductive behavior related transitions in females. In Aim 3, we will characterize projection targets of, and presynaptic inputs to, these neurons in order to understand mechanistically how this neural circuit senses an internal event to mediate these transitions; in addition, we will use molecular genetic approaches to determine the identity of these neurons. If successful, our project will provide insights into neural circuit mechanisms that regulate flexibility in female mouse social behaviors centered around reproduction. Women also experience major transitions in behavior and physiology centered around reproduction, and our work has the potential to shed light on this important aspect of human biology in health as well as potentially the many disease conditions that can impact women during this process.

NIH Research Projects · FY 2025 · 2022-09

Microbial communities drive important biogeochemical cycles, from the ocean to the soil to the human gut. High rates of cell turnover provide these ecosystems with an enormous potential for rapid evolutionary change: billions of new mutations are produced within each of our gut microbiota every day. We and others have recently shown that these genetic changes can sweep through resident populations of gut bacteria on timescales ranging from a few months to a few days. This rapid pace of evolution is hypothesized to have important practical consequences, from the spread of antibiotic resistance mutations to the success of fecal microbiome transplants and other personalized therapies. Despite the potential importance of these effects, we currently know very little about the evolutionary forces that operate within complex communities like the gut microbiota, or how they influence (or are influenced by) the composition of the surrounding community. A central challenge is that we lack a population genetic framework for predicting how mutations spread in communities with large numbers of ecologically interacting strains. This limits our ability to predict how microbiota will evolve in response to environmental perturbations such as drugs or diet, or to interpret functional changes that we observe in these communities over time. My lab aims to address this gap in our understanding by combining biophysical and population genetic modeling with the development of computational tools for measuring in situ evolution in both natural and synthetic gut communities. Our long-term goal is to decipher the population genetic “rules” that govern the short-term evolution of the gut microbiota, and to use these insights to guide future experimental and therapeutic efforts. In the next five years, we will pursue this goal through a multi-pronged strategy: First, we will develop new time-series methods for analyzing the trajectories of linked mutations in longitudinally sampled human gut metagenomes. These methods will allow us to address key open questions about the strength and duration of natural selection on sweeping genetic variants, and whether they are correlated with broader shifts in taxonomic or functional composition. Second, we will develop new methods for leveraging widely deployed transposon insertion libraries to measure the rates and fitness effects of spontaneous beneficial mutations in vivo in ex-germ-free mice, and we will quantify for the first time how this landscape varies across species, diets, and community contexts. Finally, to interpret these new data and to craft driving hypotheses, we will develop a mechanistic modeling framework for predicting how ecological diversity influences short-term evolutionary dynamics in highly diverse communities that compete for common metabolites. Together, this work will provide unprecedented insight into the short-term evolution of our gut microbiota, and will constitute a crucial step toward the development of truly predictive models of microbial community dynamics.

NIH Research Projects · FY 2026 · 2022-09

PROJECT SUMMARY/ABSTRACT Background. Visual impairment has been strongly associated with Alzheimer’s disease and related dementias (ADRD) in numerous cross-sectional and longitudinal studies, and we have found that worse baseline vision is tied to increasingly higher risk of subsequent dementia. Neurosensory deprivation from visual impairment may place greater demands on cognitive resources, accelerating cognitive decline and increasing the incidence of cognitive impairment. Conversely, improving vision could improve cognitive outcomes by increasing neurosensory input and reducing cognitive demand for processing visual information. Cataracts are the most common cause of visual impairment—fortunately reversible with surgery, however, we have found that ADRD patients are only half as likely to undergo cataract surgery as those without ADRD. This may reflect concerns regarding less potential benefit and greater perceived risks. Objectives. Our long-term goal is to evaluate cataract surgery as a potential intervention to “bend the curve” for risk of ADRD onset and progression, including optimizing patient selection and timing for surgery. The objective of this proposal is to investigate how cataract surgery may affect incidence and progression of mild cognitive impairment (MCI) and ADRD, develop models to predict individual patients’ ADRD/MCI outcomes following cataract surgery, and identify key confounders, mediators, and effect modifiers. We hypothesize that cataract surgery is associated with (1) reduction in incidence of new MCI and ADRD and (2) reduced cognitive decline and impairment progression among patients with baseline MCI or ADRD, and that (3) we will be able to predict individual patient outcomes. We propose to use methods our group has developed to archive and analyze electronic health record (EHR) data, to develop a curated data set and achieve three Aims: (1) Determine impact of cataract surgery on ADRD and MCI incidence; (2) Determine impact of cataract surgery on cognitive decline and impairment among patients with baseline ADRD or MCI, and (3) Develop patient-level predictive models for ADRD and MCI outcomes after cataract surgery. Impact. EHR-based machine learning analysis has not been applied to ADRD research to date, and the influence of cataract surgery on cognitive outcomes is not yet known. Finding that a widely-available cataract surgery intervention improves cognitive outcomes would be transformative. We estimate a potential unmet need for cataract surgery affecting almost 350,000 patients annually—just among the subset of patients with existing Alzheimer’s disease. Results from this work will directly inform discussion of cataract surgery risks and benefits and will also facilitate future research, including pragmatic clinical trial design. By developing and disseminating open source EHR-based algorithms to identify and classify cognitive and visual impairment, this proposal will enable investigation of other ADRD risk factors and interventions, eye disease research, and a more precise approach to managing individual patients.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY The convergence of human induced pluripotent stem cells (hiPSCs), organoids, synthetic biology and 3D bioprinting promises a future of patient-specific lab-grown organs for patients suffering from organ failure. However, to realize this organ engineering vision, biofabrication researchers sorely need thousand-liter-scale cultures of hiPSCs to generate enough material to begin high-throughput experimentation. Solving the myriad challenges in organ construction, vascularization, maintenance, maturation, and characterization will require decades of painstaking research. Yet, deriving patient-specific cells at this scale remains two orders of magnitude too expensive for academic laboratories due, in large part, to the expensive growth factors required for hiPSC maintenance and differentiation. Furthermore, existing protocols to generate organoids from stem cells are cumbersome, slow, and inefficient, limiting the number of organoids that can be derived for 3D bioprinting applications. In these proposed studies, we detail novel methods to dramatically reduce the cost of stem cell maintenance and increase the scale of organoid production. To reduce the costs of large-scale hiPSC growth by two orders of magnitude, we propose to engineer growth factor-free hiPSCs by programming them to express constitutively-active growth factor receptors which can be excised prior to differentiation. To enhance the scale and throughput in generating multicellular cardiac organoids, we propose engineering hiPSCs to undergo simultaneous multicellular differentiation without requiring growth factors. To achieve this, we propose a novel stochastic Cre-lox recombination system to upregulate one-of-three transcription factors, EOMES, Nkx3.1, or ETV2, to generate tri-cellular synthetic cardiac organoids containing cardiomyocytes, fibroblasts, and endothelial cells, respectively. By culturing millions of these synthetic cardiac organoids in suspension culture, we will derive therapeutically-relevant quantities of densely cellular myocardial bioink for 3D bioprinting. We will next use synthetic cardiac organoid bioink to derive a human-scale, thick-walled, and vascularized ventricle model. These bioprinted ventricles will be housed in a custom perfusion bioreactor for studying how mechanical and electrical stimulation can maintain vascular perfusion, enhance cardiomyocyte maturation and alignment, and affect organ- scale contractility and ejection fraction. The highly scalable stem cell and organoid culture methods presented here are applicable across many organ systems, and could revolutionize the scale and pace of organ biofabrication research.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY/ABSTRACT A central goal of modern neuroscience is to link genetically defined neural circuits with specific behaviors. Deep sequencing studies have revealed an incredible transcriptional heterogeneity of neuronal subtypes within populations previously thought to be homogeneous. Despite the discovery of such molecular heterogeneity, the functional and behavioral relevance of these recently genetically defined neuronal cell types is largely unknown. Our application addresses this gap in knowledge by focusing on the neuronal control of social behaviors. Social interactions, ranging from non-vocal communication to complex language to core behaviors such as courtship or aggression, are important for mental well-being and reproductive success. The neural circuits underpinning these diverse behaviors have been difficult to disentangle, with non-genetically targeted approaches yielding phenotypes in multiple behavioral domains. Here we propose to characterize how progesterone and estrogen receptor alpha expressing neurons (Pvl neurons) in the ventromedial hypothalamus ventrolateralis (VMHvl) regulate social behaviors. Our deep sequencing studies reveal distinct neuronal cell types within the population of Pvl neurons, with one of them restricted exclusively to females. Using newly developed Cre and Flpo mouse strains that mark this female-restricted neuronal cell type within Pvl neurons, we will determine its activity (Specific Aim 1), behavioral function (Specific Aim 2), and functional connectivity (Specific Aim 3) as these relate to female social behaviors. Together, our studies will uncover the activity dynamics, function, and connectivity of a genetically defined neuronal cell type of Pvl neurons; in parallel, we will also characterize the complementary set of remaining Pvl neurons and test their participation ins social behaviors. Thus, our research aims to understand how genetically defined cellular heterogeneity within a neuronal population relates to functional specificity in the control of social behaviors, a critical issue in neuroscience. Health Relatedness: Diverse neurodegenerative conditions and mental illnesses manifest with devastating clinical symptoms as well as a range of deficits in social and emotional behaviors. Pvl neurons within the VMHvl represent a critical hub that regulates diverse core, developmentally programmed social behaviors that differ between the sexes. Rare case reports of lesions in proximity to this region show dramatic alterations in social behaviors. Our studies therefore will provide new insights into fundamental questions in neuroscience, and they also have the potential to shed light on how dysfunction in pathways emanating from the VMHvl alters social behavior in disease states.