University Of Pennsylvania

NIH Research Projects · FY 2026 · 2024-07

PROJECT SUMMARY Sensory environments are cluttered and dynamic, with salient features 1) often masked or degraded by other stimuli and 2) capable of holding multiple meanings depending on the surrounding context. To account for this variability, sensory systems must process information in an adaptive manner by using contextual cues and prior information to bias incoming sensory information. Though this flexibility is critical for accurate sensory processing, the mechanisms underlying adaptive processing remain poorly understood. Descending projections from hierarchically higher brain regions to lower regions are a hypothesized anatomical substrate for top-down modulation of incoming sensory information. In the central auditory system, descending connections from the auditory cortex target numerous subcortical structures and these cortico-fugal pathways have been implicated in top-down processes such as predictive coding and attentional modulation of speech in noise. Specifically, projections from the auditory cortex have been found to carry contextual information about sound statistics to the auditory midbrain, or inferior colliculus, and to enhance responses to degraded sounds in the auditory thalamus, or medial geniculate body. Though these cortico-fugal pathways have been implicated in contextual processing and perceptual adaptation to degraded sounds, the broader circuit and physiological mechanisms underlying these phenomena remain unknown. Therefore, the goal of this proposal is to 1) determine the mechanisms by which cortico-fugal neurons enable contextual processing, with the hypothesis that they induce receptive field plasticity in IC neurons, 2) determine if top-down inputs alter context-dependent network reorganization, and 3) test how top-down circuits mediate adaptation to challenging listening conditions at the physiological and network level. This proposal uses a combination of behavior, large-scale electrophysiology, two-photon calcium imaging, optogenetics, and network analysis methods to address these aims. The results of these studies will reveal the circuit and physiological mechanisms underlying auditory contextual processing and perceptual adaptation. The impact of these experiments extends beyond the auditory system, as it may reveal generalizable principles about adaptive processing and behavior.

NIH Research Projects · FY 2025 · 2024-07

Abstract The urgency of the opioid crisis (over 80,000 deaths in the past year) has encouraged research on understudied agents, such as the 5HT2A agonist psilocybin (PSI), for potential clinical benefit. Recently, PSI has been designated as a "breakthrough therapy" for depression and has demonstrated its potential benefits in substance-use disorders, including alcohol and nicotine addiction. Observational data suggest a link between PSI use and reduced odds of developing opioid use disorder (OUD). Emerging preclinical data suggest PSI may have neuroplasticity and greater neurocognitive flexibility as potential mechanisms of action in clinical disorders, but clinical trials have generally proceeded without mechanistic information in either the neurocognitive flexibility (FLEX), or the “classic” motivational (“GO”!) and regulatory (STOP!) domains. The proposed R61/R33 will address this need, examining PSI's impact on these 3 brain-behavioral domains in OUD patients. 72 individuals (R61 = 24; R33 = 48) will be prospectively assigned to receive either 25mg) or 1mg (control) of PSI, with selected pre- and post-PSI assessments that probe brain, cognition, and behavior. Aim 1 ("GO!" motivational domain), will examine brain and behavioral responses to cues and to drug-related videos. The hypothesis is PSI will reduce the brain (motivational circuitry) and behavioral (reduced positive affective bias) response to drug cues. Aim 2 ("STOP!" inhibitory domain) will assess brain responses during a valenced Go-NoGo task and during attempted inhibition of craving to drug videos and during behavioral performance of a standard (motor pre-protency) Go-NoGo task. The hypothesis is PSI will increase recruitment of STOP circuitry in the brain tasks and will reduce errors of commission in the behavioral probe. Aim 3 will investigate the "FLEX" (neurocognitive flexibility) domain using the Wisconsin Card Sort Task (brain) and Penn Conditional Exclusion Task (behavior). The hypothesis is PSI will enhance recruitment in FLEX substrates and reduce perseverative errors. Exploratory measures will include craving, withdrawal, psychedelic effects, depression, and prior adversity. Brain-behavioral probes showing large effect sizes will proceed to the R33 phase, and examinations will expand to include network-level changes in the brain. This proposal is of high importance for understanding PSI's effects on brain, cognition, and behavior in those with OUD – critical for the rational advance of PSI for OUD – and for the many other disorders with unmet need.

NIH Research Projects · FY 2025 · 2024-07

An estimated 698,000 children in the U.S. live with serious illness and significant care needs. A complex system of hospital and community providers and agencies supports these children and their families living at home, but families are still challenged by significant gaps in this system. Compounding these challenges are the socioeconomic and environmental (SEE) factors that excerbate the negative impact of serious illness on children and families. Families, particularly those from underserved communities, may also experience increased financial difficulties, greater household material hardships, and lower social support. Taken together, these health system and SEE challenges have deleterious impacts on children’s and families’ wellbeing. In response to these challenges, timely and tailored clinical actions are urgently needed to support children and families living at home. Pediatric palliative care (PPC) programs are uniquely poised to support children and families, but these programs are typically based in pediatric hospitals and few offer home services. Care is thus often provided from afar, without information about families’ day-to-day care experiences and if care is aligned with families’ needs over time. Routinely collecting and rapidly feeding back digitally-captured data about families’ home-based PPC experiences to PPC teams may a) enable tailored clinical actions to increase concordance between families’ needs and home-based PPC, b) support teams to address the health system and SEE challenges faced by children and families, and c) improve outcomes for children and families. The proposed Home-based PPC Experiences for Children with Serious Illness and Families Feedback Study (EXPERIENCE-Feedback), informed by the Chronic Care Model, the clinical decision support framework, NINR’s mission, and community-engaged research principles, will evaluate a rapid digital information feedback loop between families at home and PPC providers over time, explore longitudinal associations between home-based PPC experiences and child/family outcomes, and build a foundation for future community-engaged interventions by evaluating the feasibility and actionability of rapidly (i.e., simultaneous with family reporting) feeding back digitally-captured, family-reported experiences with home-based PPC to PPC teams over time (Aim 1), and exploring the longitudinal associations between home-based PPC experiences and child (Aim 2) and family (Aim 3) physical, mental, and social health outcomes. This award, with mentorship by an interdisciplinary research team at the University of Pennsylvania and the Children’s Hospital of Philadelphia, will support advanced training in longitudinal, community-engaged, and intervention research methods. The proposed project builds a foundation for future R01 intervention studies and supports the applicant’s career goal to advance access to high-quality care and care outcomes for all children living with serious illness and their families in homes and communities.

NIH Research Projects · FY 2025 · 2024-07

Project Summary The goal of this proposal is to dissect the distinct molecular roles of Polycomb Repressive Complexes 1 and 2 (PRC1&PRC2) and upstream factors in establishing silencing at developmentally regulated genes. The PRCs are conserved chromatin modifiers that represses transcription via chemical and structural alterations of chromatin architecture. PRC1 mutations cause neurodevelopmental disorders characterized by microcephaly, intellectual disabilities, and dysmorphic body features, while mutations in PRC2 are the main cause of Weaver syndrome making it crucial to gain understanding about polycomb repression during early development. Despite ubiquitous expression, PRC1 and PRC2 can target distinct sets of genes for silencing in different cell lineages, resulting in cell-type specific expression. Though there are decades of research on Polycomb protein function, a key unanswered question is how polycomb repressive complexes select different target genes as cells differentiate into different cell types. While we understand the interplay of PRC1 and PRC2 during the maintenance of polycomb silencing, we do not understand the order of events in the establishment of new polycomb domains at de novo silenced genes. Others in the field have shown that correct PRC2 localization can be re-established de novo in mouse embryonic stem cells (mESCs) after ablation, indicating that there is information upstream of PRC2 dictating localization. Additionally, my preliminary data from a genome wide knock out screen implicates several factors of interest in regulating polycomb establishment including RNA binding protein ERH. I will investigate the regulatory role of PRC1 and these additional factors identified by the screen in PRC2 localization during the establishment of polycomb domains in mESCs with the following specific aims. In Aim 1, I will utilize acute protein degradation to investigate PRC1’s ability to re-establish correct localization on chromatin after depletion. Using a similar strategy with an orthogonal dual-degron line, I will also determine if PRC1 is necessary for correct PRC2 localization during establishment. In Aim 2, I will investigate the functional roles of candidates I identified in my genome wide KO screen during PRC2’s re-establishment on chromatin in mESCs, leveraging my lab’s expertise in designing acute protein degradation lines. The experiments in this proposal will elucidate the order of events and factors involved in the establishment of silent polycomb domains. To achieve these aims, I have developed with my sponsor a rigorous and comprehensive training program with three primary goals: 1) become an expert in epigenomics methods, 2) sharpen my scientific communication skills to broad audiences, 3) increase proficiency in bioinformatics and data analysis. I am confident that my choice of sponsor combined with my diverse training background and the collaborative nature of my training environment will enable me to achieve my goals and the proposed research plan simultaneously.

NSF Awards · FY 2024 · 2024-07

Addressing the questions of how the two-meter-long human DNA fits into the space of a cell's nucleus (~20 um) and how it is organized within this space has been among the major mysteries of cell biology. DNA is packaged into the nucleus in the form of chromatin, consisting of a complex between DNA and histone proteins. DNA wrapped in compacted histones is thought of as “repressed” and “inaccessible”, and thus chromatin compaction plays a critical role in regulating gene activity. Current chromatin modeling is based on polymer simulations at different levels of resolution. However, given the slow time scales of these processes of the order of minutes to hours, the size scales of the order of 5--10 um (typical size of nucleus) are not accessible using methods such as molecular or dissipative dynamics approaches. The objective of this project is to decode the quantitative relationship between the physical microenvironment, multiscale 3D genome organization, and transcriptional output. The project will employ a convergent research strategy that integrates super-resolution microscopy, genomics, biophysical modeling and simulation, and machine learning. The new tools developed in this project will impact many areas in biology, including normal and abnormal tissue development, tissue degeneration in disease, as well as tissue regeneration. The research team will educate future scientists and a diverse workforce with a collaborative expertise in interdisciplinary training. Additional outreach activities will include research experiences for undergraduate students and high school students. Tissue-resident cells continuously sense changes in their chemo-physical environment and use this information to maintain their phenotype and tissue homeostasis. The project will develop a predictive framework of emergent epigenetic and transcriptional features of cells in response to changes in their physical environment. The project will develop new quantitative models for the distribution of heterochromatin domains in the interior of the nucleus as well as along the nuclear periphery. Specifically, a mathematical model will be developed to study the effect of rates of histone tail acetylation, methylation, and transcription on determining the distribution of heterochromatin domains in the interior of the nucleus. The project will further extend this model to include the formation of lamina-associated domains (LADs) by incorporating the energetic interactions between chromatin and the nuclear lamina via chromatin anchoring proteins. To verify the theoretical model, cells of fixed fate and fluid fate will be grown under varying micro-environments, and their whole genome organization at the nano- and micro-scale will be visualized and quantified through super-resolution microscopy. In addition, a machine learning framework leveraging novel deep neural operators will be developed for nonlinear inverse problems to extract the high-dimensional parameter fields implicitly and explicitly from noisy experimental images. To enhance the robustness, accuracy, and efficiency of neural operators with small data, the project will endow neural operators with prior knowledge, physics, multifidelity data, and active learning. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2026 · 2024-07

The delivery of therapeutic nucleotides to the myocardium is notoriously challenging and remains an important unmet clinical need. Based on the global success of mRNA vaccines, solid lipid nanoparticles are the most widely used vehicle for RNA delivery. We recently identified species of lipid nanoparticles with unprecedented cardiotropism (cLNPs) that are efficient at delivering inhibitory RNA cargos to the heart. This supports development of cLNPs for the therapeutic inhibition of select cardiomyocyte targets. Our prior work suggests that increases in stable, post-translationally detyrosinated microtubules, as mediated by vasohibins (VASH1/2) in complex with their chaperone (SVBP), contribute to contractile dysfunction in human heart failure and clinically relevant animal models. Accordingly, the proposed research tests the hypothesis that cLNPs with inhibitory cargos that limit disease-associated microtubule network detyrosination can improve contractile dysfunction in disease models where increased VASH1 or VASH2 expression has been linked to systolic and/or diastolic dysfunction. To test this hypothesis, Aim 1 experiments will prioritize therapeutic reagents by characterizing the extent and duration of on- and off-target effects of cLNPs with alternative inhibitory cargos (siRNA, shRNA, and antisense oligonucleotides) against both constitutively expressed transcripts or against Vash1, Vash2, or Svbp in healthy rats. Studies demonstrating functional inhibition will be extended to human myocardium using ex vivo delivery of cLNPs to perfused cardiac wedge preparations derived from heart transplant recipients. Aim 2 experiments will determine whether short-term inhibition of Vash2 via delivery of cLNPs is sufficient to blunt contractile dysfunction in viable myocardium following acute myocardial infarction. Aim 3 experiments test whether cLNPs achieving sustained delivery of Vash1 can delay the progression of diastolic dysfunction in an animal model of heart failure with preserved ejection fraction. Our overall study design uses novel and complementary experimental approaches that seek to rigorously characterize inhibitory nucleic acid delivery via cLNPs, and then test them in clinically relevant models of microtubule-dependent cardiac dysfunction. Use of both acute and chronic models of cardiac dysfunction, and in turn acute and chronic therapeutic inhibition, respectively, exploits a range of therapeutic options of cLNPs. Inclusion of delivery to human myocardium furthers ultimate clinical translation. Together this work will establish whether inhibition of microtubule network remodeling is therapeutically beneficial in heart failure, defne which molecular target is best suited for each of two therapeutic scenarios, and establish the versatility of cLNP mediated delivery of inhibitory nucleic acids for the treatment of cardiac dysfunction.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY/ABSTRACT Colorectal cancer (CRC) is a leading cause of cancer mortality and is significantly affected by multifactorial influences including host genetic and epigenetic factors, diet, and microbial composition. One of the most intriguing interventions for mitigating cancer risk and progression is modifying dietary behavior, a powerful approach that has been increasingly investigated. While many studies focus on individual dietary components, it is unclear how distinct metabolic inputs from the dietary milieu integrate to influence cancer progression. Two major nutrients that have been extensively linked to CRC are fructose and fiber. Fructose is highly enriched in Western diets and promotes intestinal tumorigenesis by accelerating de novo lipogenesis (DNL) and glycolysis. Fiber is metabolized by the gut microbiota to produce short-chain fatty acids (SCFAs), which exert anticarcinogenic activity. Fructose and acetate, the most abundant SCFA, converge at a common downstream metabolite, acetyl-CoA, which can be used for DNL or histone modification, making it a central metabolite critical to metabolism and epigenetic regulation. This makes fructose and acetate prime candidates for evaluating crosstalk between multiple dietary inputs in CRC. ACSS2 is the enzyme responsible for converting acetate to acetyl-CoA and is a gene target of several transcription factors which are activated in response to fructose consumption. Thus, ACSS2 is important due to its position at the nexus of catabolic and anabolic metabolism. A key focus of this proposal is on the metabolic and epigenetic effects of dietary fiber and fructose and the role of ACSS2 in mediating these effects. My preliminary data suggest that loss of ACSS2 expression is associated with greater CRC tumor grade and progression. This is potentially due to the downregulation of cell differentiation genes and upregulation of genes relevant to CRC tumor metastasis, such as epithelial-mesenchymal transition. Using a mouse model, we found that manipulating dietary fiber and fructose led to changes to host metabolism in opposing directions, highlighting the need for understanding the integrated effects of these particular nutrients in the cancer context. I hypothesize that fructose manipulates acetyl-CoA pool utilization to prioritize biosynthetic pathways that are advantageous for tumor growth and that acetate exerts epigenetic effects on colonic differentiation gene targets, which are mediated by ACSS2-directed histone acetylation. Aim 1 will determine how acetate and fructose interact to affect CRC growth and acetyl-CoA metabolism through in vitro organoid and cell culture models and in vivo genetic mouse models. Aim 2 will identify the mechanism by which acetate, fructose, and ACSS2 regulate CRC epigenetic modifications and differentiation status through histone proteomics, RNA sequencing, and chromatin immunoprecipitation sequencing. This project will provide novel insights into the combinatorial effects of fiber and fructose and the influence of host gene-diet interactions on susceptibility to dietary impacts on CRC. Our work has significant implications for dietary interventions that can profoundly impact cancer patient care.

NIH Research Projects · FY 2025 · 2024-07

Abstract Severe respiratory viral infections pose a significant risk and can result in debilitating lung diseases such as acute respiratory distress syndrome and subsequent pulmonary fibrosis, which are prominent contributors to global mortality. During severe respiratory viral injury, the lung initiates a distinct form of regeneration that is independent of the functional regeneration facilitated by distal epithelial progenitors. This secondary "dysplastic" mode of regeneration involves the activation of p63+ basal progenitor cells in the upper airways, followed by the upregulation of Krt5, expansion, and migration into the alveoli, ultimately resulting in ectopic bronchiolization of the alveolar epithelium. This dysplastic response is postulated to provide short-term benefits by restoring the epithelial barrier; however, it ultimately results in a reduction of the total functional gas-exchanging surface area of the lung. Ectopic Krt5+ cells are consistently observed in cases of both severe viral lung injury and pulmonary fibrosis but have specifically been found to be strongly associated with fibrotic regions of the lung in cases of pulmonary fibrosis. However, their specific involvement in fibrosis following viral injury and the underlying mechanisms facilitating their ectopic migration from the proximal to distal airways remains poorly understood. In my preliminary data, I observed a significant upregulation of canonical NF-κB transcription factors RelA, Nfkb1, and c-Rel in ectopic Krt5+ cells. The NF-κB pathway is a well-known pro-inflammatory signaling pathway, and its activation has been shown to promote epithelial-mesenchymal transition, migration, and proliferation. Aim 1 of this proposal will assess whether canonical NF-κB signaling is necessary for ectopic Krt5+ cell expansion and migration by utilizing a conditional deletion of IKKβ in p63+ progenitor cells. In vivo experiments will determine the impact of IKKβ deletion on the total quantity of dysplastic Krt5+ epithelium following influenza injury through immunofluorescent imaging and qPCR. Accompanying in vitro experiments will assess the specific migratory and proliferative phenotypes associated with the loss of NF-κB signaling through in vitro gap closure and EdU incorporation assays. Additional preliminary data revealed that ectopic Krt5+ cells highly express CTGF, a known secreted pro-fibrotic growth factor, and are surrounded by more Collagen 1 compared to less-injured regions following H1N1-induced lung injury. Aim 2 of this study will utilize a conditional deletion of CTGF and various metrics, including immunofluorescence and hydroxyproline content, to determine whether CTGF is necessary for the pro-fibrotic function of ectopic Krt5+ cells in vivo. Subsequently, we will elucidate the direct paracrine effect of ectopic Krt5+ cells on fibroblast extracellular matrix deposition in vitro by culturing fibroblasts in conditioned media from Krt5+ cells +/- CTGF and quantifying changes in matrix deposition. These experiments will investigate the role of NF-κB signaling in dysplastic remodeling and the pro-fibrotic function of ectopic Krt5+ cells, thereby providing insight into the underlying mechanisms governing their migration and fibrotic pathology.

NSF Awards · FY 2024 · 2024-07

Recent years have witnessed numerous exciting opportunities brought by the abundance of data and powerful machine learning algorithms. Along with the opportunities, it has been recognized that the complex nature of modern data and models makes them hard to analyze: the data can be high-dimensional and correlated in complicated ways, while the models are often of unprecedented sizes and black-box to the users. There is, therefore, a need for new statistical methodologies for understanding such data and models. This research project aims to develop new statistical tools for modern data-driven applications that provide rigorous theoretical guarantees under minimal assumptions. The results of this project will have a broad impact on applications, including genetics, personalized health, and online marketing. Open-source software for the newly developed methodologies will be released. This project will also contribute to the academic training of undergraduate and graduate students through their involvement in the research tasks. This research program has three specific aims. The first aim is to develop powerful multiple hypothesis testing procedures for dependent and high-dimensional data. The second aim is to provide valid statistical inference tools for adaptively collected data, where classical statistical inference tools often fail to deliver the promised guarantee. The third aim is devoted to distribution-free predictive inference in the face of distribution shift, with the focus on characterizing the distribution shift in different problems and developing distribution-free predictive inference tools that are robust to the corresponding distribution shift. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY While chimeric antigen receptor (CAR) T cell therapy has dramatically altered the cancer therapy landscape, the susceptibility of CAR T cells to succumb to exhaustion in response to chronic stimulation by tumor antigen remains a major challenge. Strategies to overcome CAR T cell exhaustion are critically needed and represent a key research priority. Epigenetic gene activating programs have emerged as important facilitators of CAR T exhaustion, as demonstrated by the observation that CAR T cells lacking the DNA-modifying enzyme TET2 persist in the face of chronic antigen stimulation and the previous knowledge that TET2 reverses the gene silencing program induced by DNA methylation. Taken together, these insights suggest that activation of specific genes may be sufficient and necessary to promote exhaustion. This proposal aims to identify such genes, which represent attractive targets for inhibition to generate more durable CAR T cells. Aim 1 focuses on creating the first split-engineered CRISPR/dCas9-guided epigenome editors. These TET-based technologies (seEE) inducibly activate under the control of a small molecule, enabling controllable activation of target genes in a manner that recapitulates the dynamic, inducible nature of epigenetic reprogramming. Aim 2 seeks to identify TET-regulated genes implicated in CAR T cell exhaustion, first by employing a novel inducible TET overexpression construct (seTET) to decipher the dynamic epigenetic landscape of chronically stimulated CAR T cells, and then by leveraging the unique properties of seEE to screen for genes involved in inducing CAR T cell exhaustion. Candidate genes will be targeted for reactivation and the resulting impact on CAR T cell phenotype will be evaluated, revealing actionable targets for inhibition as a strategy to improve the therapeutic efficacy of CAR T cells. The proposed research will result in the introduction of novel epigenome editing technologies and provide key insights into the epigenetics of CAR T cell exhaustion with high translational potential. Importantly, this research will also train an MD/PhD student to become a well-rounded, independent physician-scientist. In conjunction with clinical immersion activities within the Perelman School of Medicine’s Medical Scientist Training Program, opportunities for collaboration and scientific communication both inside and outside of Penn, and specialty-specific career development activities, the research performed under this funding opportunity will prepare the student for a successful career as a physician-scientist focused on the extension of cell-based immunotherapies and epigenetic prognostication strategies for various diseases of the gastrointestinal tract.

NIH Research Projects · FY 2025 · 2024-07

Project Summary The majority of the genetic risk for Alzheimer’s disease (AD) has yet to be identified. Even less is known about if and how genetics influence the age of onset and rate of progression of AD. Complex forms of genetic variation such as the ~1 million short tandem repeats (STRs) in the genome cause over 30 monogenic neurologic diseases, but the role of STRs in AD has not been explored. Inherited expansions in STRs not only cause these diseases, but somatic instability of these pathogenic expansions within brain tissues also contributes to earlier age of onset and faster rates of neurodegeneration. My preliminary studies have found that STR expansions associate with risk of AD and that they are somatically unstable in temporal lobes of patients with AD. However, it is not known how STR expansions promote neurodegeneration and whether they associate with faster progression of AD. This proposal will address these knowledge gaps to test the overarching hypothesis that inherited and somatic STR expansions promote neurodegeneration in AD. In Aim 1, I will test whether STR expansions correlate with age of onset and rate of progression in AD. I will use existing whole genome sequencing data in over 11,000 AD cases and controls and associate STR genotypes with longitudinal AD phenotypes. In Aim 2, I will test whether STRs become somatically unstable in neurons from patients with AD. I will sort neurons from multiple brain regions in the same individual and perform long-read sequencing to test for somatic STR instability. In Aim 3, I will test whether AD-associated STR expansions promote neurodegeneration in induced pluripotent stem cell models of AD. I will use genome engineering to cut- back an AD-associated STR expansion in a stem cell line from an individual with AD and test for alterations in gene expression, cell viability, and synapse formation in differentiated neurons. Together, this work will generate unprecedented insights into how an unexplored form of genetic variation underlies neurodegeneration in AD. I am uniquely suited to address these questions because of my deep expertise in human genetics and genomics in addition to my clinical expertise as a cognitive neurologist treating AD and related disorders. During my mentored training, I will develop new skills in analyzing the genetic basis of longitudinal AD phenotypes (Aim 1), performing genomics in post-mortem AD samples (Aim 2), and genome engineering in AD induced pluripotent stem cell models (Aim 3). My mentorship team of Dr. Jennifer Phillips-Cremins (expert in STRs, somatic instability, and genome engineering) and Dr. Edward Lee (expert in molecular mechanisms of neurodegeneration) are unique aligned toward these training goals. This powerful complement of skills I will develop and the insights I will derive from this work will form the foundation for a career as a physician-scientist uncovering the genetic and molecular drivers of neurodegeneration.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY Mental illness is increasingly understood through the lens of brain development, as these disorders are highly heritable and often emerge during the transition from childhood to adulthood. Thus, it is essential to investigate functional brain development and its genetic underpinnings. The adult human cortex is organized along a principal hierarchy indexed by functional MRI connectivity, anchored on one end by regions involved in perception and action and on the other by regions responsible for abstract cognition. The extent to which the cortex functionally aligns with this hierarchy has been associated with both development and diverse psychopathology, suggesting that abnormal hierarchical development may be mechanistically related to broad psychiatric vulnerability. Using a normative modeling framework, this proposal will test the overarching hypothesis that deviations in the development of the principal hierarchy are associated with transdiagnostic psychopathology burden and genetic risk. To do so, I will use four large-scale developmental datasets that include fMRI and clinical data: the Philadelphia Neurodevelopmental Cohort (PNC, N=1,559), the Lifespan Human Connectome Project in Development (HCP-D, N=1,350), the Nathan Kline Institute-Rockland Sample (NKI-RS, N=426) and the Adolescent Brain Cognitive Development study (ABCD, N=11,878). This proposal aims to 1) investigate how deviations in principal hierarchy development relate to transdiagnostic clinical psychopathology., and 2) investigate the role of the principal hierarchy as an intermediate phenotype of genetic risk for transdiagnostic psychopathology. This work will advance our understanding of functional cortical development, its genetic basis, and its relationship to the emergence of psychiatric risk. Findings from this study will lay a foundation for future endeavors to identify diagnostic markers, therapeutic targets, and interventional windows of opportunity for neuropsychiatric disorders. Study feasibility will be ensured by expert guidance from an integrated mentorship team comprised of Drs. Aaron Alexander-Bloch, Theodore Satterthwaite, Dylan Tisdall, Monica Calkins, and Laura Almasy. Mentorship from these experts, along with a coordinated program of coursework, seminars, workshops, conferences, and clinical training, will provide the applicant with rigorous training in functional MRI, developmental psychopathology, and psychiatric genetics, enabling him to pursue a future career as an independently-funded physician-scientist.

NSF Awards · FY 2024 · 2024-07

This project addresses several questions regarding the solvability of certain partial differential equations (PDE). Solutions of these PDE are used to provide information about the models from which they are derived. One of the main challenges is to extract information about these solutions without knowing the functions explicitly. The Principal Investigator (PI) aims to refine and build upon techniques from PDE theory to overcome this challenge. The project focuses on an array of problems at the junction of several areas of mathematics. Moreover, it addresses fundamental problems involving function spaces, the theory of adhesion dynamics, and optimization. As part of this project, the PI mentors a postdoctoral scholar, disseminates results to a broad scientific audience, and continues his involvement in creating opportunities for members of underrepresented groups in mathematics. The methods of calculus of variations have been used to solve optimization problems in mathematics, physics, and engineering for hundreds of years. These methods continue to play an important role in science, and they also help bridge the gap between PDE theory and optimization. The topics studied by this project involve applications of this interplay to modern questions of interest in mathematical analysis. Morrey's inequality is one of the most important inequalities in the theory of Sobolev spaces. The PI has characterized Morrey extremals as solutions of a nonlinear PDE reminiscent of the equation which arises in the study of the classical electric dipole. In this project, he will develop ways to extend these considerations to Hardy-type inequalities in which the admissible functions are constrained to be supported in certain regions of Euclidean space. The pressureless Euler system is one of the basic model equations in cosmology. They were introduced a generation ago to understand low temperature settings in which galaxies form. The PI recently established an existence theorem for solutions in one spatial dimension. This will be further developed by establishing the uniqueness of solutions and by investigating the large time behavior of solutions. In addition, the project considers obstacle problems in which the competitor curve or shape can permeate the obstacle up to a given threshold. The PI aims to develop this optimization theory from scratch by considering the very basic problems which capture the essence of a semipermeable obstacle problem. In particular, he will study associated Hamilton-Jacobi equations and applications to minimal surfaces. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2024 · 2024-06

Advances in organ transplantation have significantly improved allograft and patient survival. However, chronic immune rejection remains a significant basis for graft failure across organ types. For example, allograft failure due to immune-mediated rejection is the most common reason for kidney re-transplantation. In cardiac transplant, allograft vasculopathy, a hallmark of chronic rejection, is one of the most common causes of death in recipients beyond 3 years of transplantation and, similarly, chronic beyond therapies lung allograft dysfunction is the leading cause of death 1 year of lung transplantation. Therefore, there is an unmet need to develop effective to halt and reverse damage due to chronic rejection.A hallmark of chronic rejection is fibrosis which progressively replaces functional tissue ultimately leading to allograft failure. This proposal seeks to modify the natural history of progressive fibrosis in the allograft. Cancer associated fibroblasts (CAFs) promote fibrosis that shield the tumor from the immune system and therapies. One approach to deplete pathogenic fibrosis within the tumor microenvironment targets fibroblast activation protein (FAP), a cell surface proteolytic enzyme that is expressed by activated fibroblasts. Pre-clinical studies have shown anti-tumor efficacy of FAP-targeted immunotherapy and have spurred clinical trials aimed at eliminating FAP+ fibroblasts. One strategy employing FAP-specific chimeric antigen receptor (CAR) T cells has yielded impressive tumor regressions in murine models. Recently, FAP-CAR T cells have also been shown to reverse pathologic cardiac fibrosis that occurs following myocardial injury. In addition to paving the way for use of CAR T cells for restoration of organ function in common non- cancer conditions, this work also clearly implicates FAP+ fibroblasts as an important cell type in collagen deposition in the context of malignancy as well as non-malignant tissue fibrosis. Our preliminary data demonstrates FAP expression in lung, kidney, and cardiac allografts undergoing rejection with associated fibrosis. The presence of FAP across species and allograft types suggests FAP/FAP+ cells are an important participant in allograft fibrosis. Therefore, we hypothesize that chronic rejection leads to FAP+ fibroblast accumulation which promotes deposition of extracellular matrix leading to fibrosis and progressive allograft dysfunction. The proposed studies seek to define the expression of FAP in the context of allograft rejection, determine the contribution of FAP and FAP+ cells to allograft dysfunction, and test a CAR T approach to mitigating fibrosis.

NIH Research Projects · FY 2026 · 2024-06

Project Summary Human papilloma virus (HPV)-positive head and neck squamous cell carcinoma (HNSCC) is a growing public health burden and has already surpassed cervical cancer as the most common HPV-related malignancy in the United States. While HPV+ HNSCC patients have generally good survival, they suffer from life-long chemoradiotherapy-related morbidities. Current data is insufficient to inform de-intensification of standard chemoradiotherapy or the development of targeted therapies. My ultimate goal is to understand the mechanisms by which HPV disrupts DNA damage response (DDR) signaling during HNSCC development, and to thereby inform the rational design of new targeted therapies. In considering new strategies to effectively control HPV+ HNSCC, I noted that HPV's oncogenic E6 and E7 proteins abrogate tumor suppressor pathways and impair DDR signaling to cause genomic instability. The Mendez lab and others have established DDR kinase WEE1 inhibition via the specific inhibitor AZD1775 (WEE1i) as a new therapeutic strategy in HNSCC, and that HPV+ HNSCC tumors are hypersensitive. WEE1 inhibition causes S-phase replication stress (RS) and irreparable DNA damage. Combined with genotoxic chemotherapy (e.g., cisplatin), WEE1i abrogates the G2/M checkpoint and causes premature mitosis. I recently showed that HPV16 E6/E7 oncoproteins sensitize HNSCC cells to WEE1i monotherapy through activation of a FOXM1-CDK1 circuit that drives mitotic gene expression and DNA damage. I also showed that elevated basal FOXM1 activity predisposes HPV+ HNSCC to WEE1i-induced toxicity. Next, I used an RNAi genetic screen to identify RS and DDR targets that synergize with WEE1i; based on my findings to date, I hypothesize that disruption of RS and DDR pathways by E6/E7 provide exploitable vulnerabilities for a combination targeted therapy that also includes WEE1i. I plan to clarify the mechanisms by which HPV sensitizes cancer cells to WEE1i-induced replication failure (Aim 1) and compromises DNA repair pathways upon WEE1 inhibition (Aim 2). I will use murine cancer models to test novel therapeutic combinations for targeting RS/DDR defects in HPV+ HNSCC and identify the situations in which they are most effective. In parallel, I will use a targeted quantitative proteomics approach to determine the E6/E7-specific RS and DDR responses to WEE1i, and multi-panel flow cytometry to determine the WEE1iassociated changes in the immune landscape of E6/E7-driven tumors in immunocompetent mice. This award will help me develop my scientific ideas and increase my competency in working with the mouse models that faithfully recapitulate human cancer. The scientific advances that I make during this training period will be critical to my ultimate goal of establishing an independent research program that focuses on how HPV drives HNSCC development and how HPV+ HNSCC might be more safely and effectively treated.

NIH Research Projects · FY 2024 · 2024-06

TAR-DNA binding protein 43 (TDP-43) is a nuclear protein that has been implicated in several neurodegenerative diseases, including Frontotemporal Lobar Degeneration (FTLD-TDP), Amyotrophic Lateral Sclerosis (ALS), and Limbic-predominant age-related TDP-43 encephalopathy (LATE-NC), among other age-related dementias. The presence of TDP-43 aggregates in the cytoplasm of neurons and glial cells, particularly in oligodendrocytes (OLs), is the neuropathological hallmark of FTLD-TDP, ALS, and LATE-NC. Similar to other misfolded proteins (i.e., α-synuclein, β-amyloid and Tau), the transmission of pathogenic TDP-43 species between cells could contribute to disease progression in neurodegenerative diseases. Our group has recently shown that pathological TDP-43 derived from FTLD-TDP brain extracts can act as pathogenic seeds and induce the formation of de novo TDP-43 pathology when injected in the brain of TDP-43 transgenic mice, and to a lesser extent in wild-type mice (WT). Under this paradigm, TDP-43 pathology spreads throughout the brain in a regional and time-dependent manner. Interestingly, as found in diseased human brains, TDP-43 pathology is present in neurons and OLs. Despite several studies indicating that TDP-43 pathology in OLs is a common feature of various neurodegenerative diseases, including AD and FTLD-TDP, the consequences of TDP-43 pathology in OLs remains unknown. The present research project aims to develop in vitro models to study the pathophysiological consequences of TDP-43 pathology in OLs and evaluate the role of OLs in facilitating the spread of TDP-43 pathogenic species between glial and neuronal cells.

NSF Awards · FY 2024 · 2024-06

This award will support students from institutions of higher learning in the United States to participate in the Northeastern Systems and Control Workshop (NECSW), to be held at the University of Pennsylvania on May 4, 2024. The workshop aims to provide a forum for systems and control researchers in the Northeastern United States to present their work, and to interact with graduate students, post-doctoral scholars, and faculty in an informal and collaborative setting. This will be the inaugural edition of the workshop. To promote interaction and community building, NESCW will be hosted by a different institution in the Northeast region each year. The workshop will include both oral and poster sessions. Importantly, and keeping in theme of providing visibility to junior researchers, all talks and poster presentations will be by students and postdoctoral scholars. The workshop will conclude with a social event and debate among leading researchers in the field on “hot topics” in systems and control research, that should be of interest to all members of the community. The workshop will help foster new relationships and collaborations across systems and control researchers in the Northeast region. Areas of research represented in confirmed registrants include dynamics, control, optimization, machine learning, robotics, interdisciplinary work at the intersections of these areas, and applications. All of these areas are of critical interest to the nation’s research enterprise. This workshop will promote STEM education and partnerships. Inclusion of early career students is a main focus of the workshop, which is aimed at providing visibility and networking opportunities to junior researchers in the field. By providing students and postdoctoral scholars the opportunity to communicate their work through presentations (via poster sessions and talks), the workshop will contribute to strengthening their technical communication and collaboration skills. Finally, through outreach and participation with industry, the workshop will promote building new partnerships between academia, industry, and government laboratories. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NSF Awards · FY 2024 · 2024-06

Humans often make guesses about what other people are like or what other people might do when making decisions, including decisions about when and whether to support, listen to, punish, or avoid particular individuals. In contrast to following simple decision rules across all contexts, these guesses can support flexibility in people’s decision-making, allowing them to use cues in the environment to tune their behavior to interactions with different people. At the same time, when whole collections of decision-makers use similar cues to make similar guesses about others, this can lead to different treatment across groups. This project aims to generate new scientific understanding of flexible human social decision-making, with implications for understanding social behavior. The plan is to combine methods from psychology, neuroscience, and behavioral economics to generate predictions of complex social behaviors and to test hypotheses about the neural circuitry that underlies them. This project aims to take a step toward a comprehensive framework that incorporates more of the richness and complexity of the social world into quantitative models of human behavior, advancing efforts in the fields of behavioral economics, social cognition, and decision neuroscience. In turn, it has the potential to generate new understanding of the origins of behavior. Additional goals are to: 1) support the development of a STEM workforce in the mind and brain sciences, 2) increase public engagement with neuroscience research, and 3) train students in computational modeling and cognitive neuroscience, as well as broadly appliable data science techniques. The broad aims of this project are to: 1) incorporate more of the psychological richness of the human social world into formal computational models of behavior, and 2) use those models to test hypotheses about the cognitive mechanisms and neural circuitry that give rise to social decisions. More specifically, the research plan includes studies that integrate information about social perception (how people see others) into computational models from behavioral economics and cognitive psychology that make predictions about how people value others’ outcomes (social valuation), value outcomes at different times (temporal discounting), learn about rewards (reinforcement learning), and value information (information seeking). The plan is to use information from these computational models with fMRI to better understand the neural basis of decisions in social contexts, with a focus on brain regions involved in social cognition and valuation. These research efforts synergize with educational and outreach initiatives to develop equitable assessment tools, engage the local community on neuroscience topics, and provide students with science mentorship, professional development, and training in broadly applicable statistical and computational modeling techniques. The research plan aims to contribute to a more widely applicable framework capable of making better predictions of human behavior in social contexts, and the results of the research should increase our understanding of specific social decisions, how people value others, and the underlying cognitive and neural mechanisms. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2026 · 2024-06

PROJECT SUMMARY Telepsychology (“telepsych”) provides a unique opportunity to use behavioral economics (BE) strategies to structure the therapy encounter in ways that nudge clinicians to deliver high-quality services. BE strategies are best suited for changing discrete provider behaviors that are observable and measurable. This R34 aims to develop and evaluate “Tele-BE,” a novel telehealth infrastructure that nudges and incentivizes clinicians to use core structural components of cognitive behavioral therapy (CBT), a leading psychosocial treatment with demonstrated efficacy and effectiveness for a range of mental health concerns. Across protocols, CBT contains many discrete components; however, nearly all CBT protocols include the following elements: (1) patient symptom tracking, (2) collaborative agenda setting at the session start, (3) out-of-session practice (“homework”) review, (4) skill instruction, (5) skill practice, and (6) homework planning. These CBT structural components are likely to be responsive to BE strategies. Structural components also represent a core and distinct CBT competency and serve as the foundation upon which specific intervention techniques are delivered; as such, they are common to other evidence-based psychosocial practices beyond CBT as well. However, our prior work illustrates that community clinicians have consistently low fidelity to these components. Aim 1 will use participatory design to refine our Tele-BE prototype in collaboration with clinicians and supervisors (our target end-users). In Aim 2, we will work closely with our web development team to field test and iteratively refine Tele- BE using rapid cycle prototyping to optimize the user experience and refine our specific BE strategies. Aim 3 will test the preliminary effectiveness of the refined Tele-BE to engage our target mechanisms and enhance clinician CBT structural component fidelity in a 12-week open trial with 30 community mental health clinicians randomized to Tele-BE or telehealth as usual (Tele-AU). The trial will include 2 patients per clinician (60 patients in total). All sessions will be recorded and coded for CBT fidelity. Clinicians and patients will complete questionnaires at Weeks 1, 5, 9, and 12 and qualitative interviews at post. Aim 3 primary outcomes will be CBT structural component fidelity, measured through coding of recorded sessions. Secondary outcomes include target implementation mechanisms (intentions and their determinants: attitudes, norms and self-efficacy), measured via mixed methods, and overall CBT fidelity. Using data from the open trial, Aim 4 will examine acceptability and feasibility of Tele-BE from patient and clinician perspectives, as well as potential ethical issues with Tele-BE. This R34 aligns with NIMH Strategic Plan Strategy 4.2.C to develop decision-support tools and technologies that increase the implementation of mental health EBPs. Results will lead to a hybrid effectiveness- implementation R01 to test strategies for optimizing Tele-BE's effect on clinician fidelity to improve patient outcomes.

NIH Research Projects · FY 2024 · 2024-06

FLASH radiotherapy utilizing ultra-high dose rate has the potential of being a substantial evolutionary advancement for cancer treatment. Ultra-high dose rate radiation provides a benefit of decreased normal tissue toxicities while maintaining equivalent tumor killing effect. Among different modalities, particle therapy has been an active area of research to integrate its unique characteristics with the FLASH effect because proton therapy remains the only clinically assessable modality that can potentially treat deep seated tumors under FLASH conditions. Thus, developing methods to enable FLASH proton therapy with clinically available accelerators is an important step to both unravel the biological mechanism using preclinical studies, clinical trials as well as explore the physical hardware requirements for clinical implementation. In FLASH proton therapy, beam modulation devices are important accessories for enabling ultra-fast dose delivery based on the clinically available cyclotrons and synchrocyclotron systems. These work by transforming an ultra-high dose rate mono-energetic proton pencil to a multi-energy spot after passing through varying thicknesses of modulating material. In this way, a multi-energy layer pencil beam scanning proton plan can be delivered completely in less than a second without the need for beam pausing for energy switching. The most common modulator is the ridge filter, which consists of uniform density spikes of varying height. The limitation of this design includes restricted structure stability and modulation flexibility. We propose to develop a new class of heterogeneous density range modulators based on the novel PixelPrint technology, to facilitate FLASH therapy. By continuously varying the ratio of filament to air, PixelPrint technology is capable of 3D printing phantoms with voxel-wise heterogeneous density. By utilizing material density as an optimization parameter, the new devices will have robust structure and high flexibility in modulation. The hypothesis is that the 3D printed heterogeneous density modulator degrades the beam energy across the transverse plane of the particle beam, based on both local material density and the stopping power distribution of the material. With greater flexibility compared to current binary modulation only design, complex modulation is achievable for application in universal range modulator and patient-specific modulators. Our deliverables will include design methods to optimize density structures for range modulation, and experimentally validated modulators for proton FLASH therapy applications. The specific aims are: Aim 1. Development of density optimization algorithms for range modulation. Aim 2. Experimental validation and characterization of 3D printed heterogeneous range modulators. Aim 3. Development of patient-specific modulators for Proton FLASH therapy. If successful, the approach will enable a new class of range modulators to enable FLASH particle therapy with flexible assembly and patient-specific modulation.

NIH Research Projects · FY 2026 · 2024-06

Squamous cell carcinoma of the head and neck (HNSCC) is a complex disorder that contains multiple cell populations, making it is difficult to study the nature and cellular function and delineate relationship among subpopulations. The analysis of global gene expressions in single cell level has evolved at an astounding pace in the past few years, and now reaching a sophisticated level to solve heterogeneity in complex organs. To understanding the in vivo tumor microenvironment, a large scale (~10,000 cells) single cell gene expression assay can elucidate transitional states and delineate relationships among subpopulations. The goals of this proposal are to characterize a distinct population of HNSCC stromal progenitors, to explore cellular mechanisms by which these cells regulate tumor progression, and to ultimately translate the findings into clinical cancer therapies. Using genetic tools and SCC animal models, data has been generated to demonstrate that: 1) a unique postnatal Gli1+ stromal population is found in the craniofacial region, 2) Gli1+ cells behave as progenitors under homeostatic and disease conditions in vivo, 3) tumor progression is supported by stromal components that are elevated in HNSCC microenvironment and serve as a chemo- attractant for tumor invasion, and 4) 3D culture of primary tumor keratinocytes with cancer associated fibroblasts (CAFs) showed that the stratified growth, cell proliferation, and differentiation are comparable between co-cultures and their respective native tissues, while they largely differed in cultures without CAFs. We hypothesize that Gli1+ cells contain a distinct subpopulation of stromal progenitors in the craniofacial region, and that Gli1+ cells play a niche role in supporting HNSCC progression through remodeling epithelial morphogenesis. During this proposal, we will explore whether Gli1+ cells as a niche support cancer progression and metastasis through lineage tracing in Gli1CreERT2;TdTomato mice and diphtheria-mediated loss-of-function in Gli1CreERT2;DTA mice (Aim 1). Subsequently, this proposal will use transcriptomic and spatial molecular analysis in single-cell level combined with computational analysis and functional validation to identify unique in vivo stromal populations in mouse and human HNSCC and elucidate their molecular and pathway signatures. We will validate these unique cancer stromal subpopulations and investigate their features, regulatory mechanism, function, and spatial and molecular characteristics. Based on the findings, by 3D organoid modeling with novel vascularized organ-on-a chip technique, newly identified tumor specific stromal progenitor populations will be utilized to generate 3D vascularized SCC organoids and discover novel therapeutic avenues for HNSCC management (Aim 2). Successful completion of the proposal will advance our understanding of the nature of in vivo stromal effects in tumor microenvironment and will develop new treatments for HNSCC.

NIH Research Projects · FY 2026 · 2024-06

PROJECT SUMMARY Phenylketonuria (PKU) is an autosomal recessive disorder caused by mutations in the gene encoding phenylalanine hydroxylase (PAH), resulting in the accumulation of phenylalanine (Phe) to neurotoxic levels. Although there are treatment options, ranging from a strict low-Phe diet to an oral medication (sapropterin, a cofactor of PAH) to an injectable enzyme substitution therapy (pegvaliase), many PKU patients find it challenging to adhere to the dietary intervention and have limited responses to or access to the medical therapies and, as a result, have impaired cognitive development and develop a range of neuropsychiatric problems. Durable and, ideally, curative therapies are needed to address the unmet medical needs of PKU patients. More than 1,000 PAH variants have been cataloged in patients. These vary in their consequences for PAH activity, from having little or no effect to eliminating PAH activity completely. Certain variants occur much more commonly than others in PKU patients. The most frequently occurring pathogenic PAH variant worldwide is the R408W (c.1222C>T, p.Arg408Trp) variant. Patients homozygous for this variant do not respond to sapropterin, limiting their treatment options. In vivo gene editing is an emerging therapeutic approach to making DNA modifications in the body of a patient, such as in the liver. Gene-editing tools include nucleases, cytosine base editors, adenine base editors, and prime editors. CRISPR base editors and prime editors are attractive because they can function efficiently for introducing precise targeted alterations without the need for double-strand breaks, in contrast to CRISPR-Cas9 and other gene-editing nucleases. We and others have demonstrated the ability of base editors to make specific DNA edits with very high efficiency and limited off-target effects in the liver in mouse models and non-human primates. We now seek to assess whether a one-time delivery of adenine base editing or prime editing can be used to permanently correct the human PAH R408W variant in human hepatocytes in vitro and in the liver in humanized mice in vivo efficiently and safely and, if so, the optimal editing system to use for this purpose. Success in completing this translational project will provide critical information on the feasibility of an in vivo genome- editing approach that could ultimately yield a one-shot, long-term therapy that permanently corrects the most frequent PAH pathogenic variant and thus serves as a potential cure for PKU patients with this variant. The proposed work, if successful, could open the door to a new therapeutic modality for many PKU patients— not just for patients with the PAH R408W variant, but those with other editable pathogenic variants in the PAH gene—as well as patients with such variants in other liver-expressed, disease-associated genes.

NIH Research Projects · FY 2026 · 2024-06

Project Summary An adaptive immunosuppressive microenvironment is a major barrier to immune-based therapies for solid tumors, including glioblastoma (GBM). Current model systems for preclinical development either lack substantial components of the immune system or rely upon different species’ immune systems, which display significant differences when compared to human immune systems. These deficiencies lead to the disconnect between preclinical and clinical research. Here we propose to develop a humanized mouse system for the study of immune system interactions with GBM. By taking hematopoietic stem cells from a GBM patient, we will engraft a human immune system in mice. From the same patient, we will obtain tumor tissue and T cells. This will allow creation of an autologous mouse system, where the components, immune system, tumor, and cell-based therapy, all come from the same source. In doing so, we aim to avoid any complications that would arise from cells coming from multiple individuals. We will validate the autologous mouse system by generating chimeric antigen receptor (CAR) T cells from the patients’ own T cells. These redirected T cells will allow for evaluation of the model system, both in terms of how the tumors respond to immunotherapy and how the existing immune system responds to immunotherapy. Results in the animal model will be compared to patients receiving the same treatment, in clinical trials at the University of Pennsylvania. Initial work for will focus on demonstrating the fidelity of the system to the originating tumor and immune microenvironment, in terms of both tumor characteristics and immune system response. Thorough characterization of immune cell subpopulations and anti-tumor activity will help identify discrepancies in the current models as well as areas in which the new, autologous models are preferable. At the conclusion of this project, we will have demonstrated the feasibility of using autologous materials for glioblastoma modeling and elucidated the aspects of tumor-immune interaction that these models are most suited for use in.

NIH Research Projects · FY 2025 · 2024-06

Project Summary My long-term career aspiration is to become a physician-scientist committed to bringing novel brain tumor therapies from bench to bedside by leading an independent basic science research group. This proposal is a first step in achieving this goal by providing crucial support for an integrated neuroscience/cancer biology-based training plan in the context of brain tumor research at the Perelman School of Medicine, University of Pennsylvania. Glioblastoma is a universally fatal brain cancer that functionally integrates into the normal neural circuitry. It has been reported that neuronal activity drives tumor progression via glutamatergic synapses onto tumor cells and, in parallel, glioblastoma cells alter neuronal excitability, remodel neural circuits, and impair cognitive function. However, little is known regarding the cellular diversity and circuit architecture of neurons that are connected with tumor cells. Understanding the anatomic and cell type distribution of these connected neurons may reveal unique functional roles of select neuronal subsets in glioma pathogenesis. We hypothesize that glioblastoma receives functional synapses from both local and long-range neuronal projections, consisting of not only glutamatergic but also neuromodulatory inputs. In the normal brain, neuromodulatory inputs project diffusely throughout the brain and play an important role in modulating neuronal excitability, and thus may also function in regulating glioma. In this proposal, we seek to define the glioblastoma connectome using monosynaptic rabies virus (RBV) for retrograde trans-synaptic tracing and monosynaptic herpes simplex virus (HSV) for anterograde trans-synaptic tracing in a xenograft mouse model. In Aim 1, we will utilize an in vivo model to map the whole-brain inputs onto glioblastoma by transplanting patient-derived glioblastoma organoids (GBOs) to various clinically relevant brain regions in immunodeficient mice. Preliminary studies revealed labeling of diverse neuronal subtypes in various brain regions, and, in particular, basal forebrain cholinergic neurons projecting to transplanted glioblastoma cells in both cortical and subcortical regions. We will confirm cholinergic inputs with HSV-based anterograde trans-synaptic tracing as well as electrophysiological recordings. In Aim 2, we will study the functional significance of these cholinergic neuromodulatory inputs onto glioblastoma by performing calcium imaging and single-cell RNA sequencing of glioblastoma organoids treated with acetylcholine as well as assess the impact of acetylcholine on tumor cell invasion in vitro and in vivo. We believe that project outcomes will elucidate the complex brain-wide interactions between tumor cells and diverse neuronal populations and investigate the significance of cholinergic inputs in glioma disease progression, thereby providing novel therapeutic avenues for glioblastoma.

NIH Research Projects · FY 2025 · 2024-06

PROJECT SUMMARY Obstructive sleep apnea (OSA) is the most common sleep disorder and a recognized risk with an estimated worldwide prevalence of one billion people. The ensuing repeated episodes of nocturnal hypoxia and hypoxemia are at the core of the disorder’s pathogenesis, leading to upregulation of neuro inflammation and oxidative stress, which predispose OSA patients to cardiac and neurovascular disease along with impaired cognitive function and neurodegeneration. The investigators have previously examined the neurovascular-metabolic alterations in terms of the cerebral metabolic rate of oxygen (CMRO2) at rest and in response to apneic challenges during wakefulness in the form of repeated cued breath-holds mimicking the hypercapnic-hypoxic events of spontaneous apnea, by means of temporally-resolved MRI-based brain oximetry. Although this work provided new insights into chronic and acute neurometabolic consequences of the disorder, the response to coached volitional apneas likely differs from that of spontaneous apneas during sleep. Also unknown are the upper airway’s morphologic changes that occur during apneas (full airway closure) and hypopneas (partial closure) that cause the metabolic alterations. Leading up to the proposed project we have been able to monitor cerebral oxygen metabolism in healthy subjects in the scanner with concurrent electroencephalography (EEG) and designed an imaging procedure that returns the vascular-metabolic parameters and upper airway morphology during continuous scanning. We illustrate the method’s potential with model apneas induced in test subjects involving the oropharyngeal phase of swallowing, causing airway closure and the expected hypoxic-hypercapnic response and, more recently, in a patient with OSA during 90 minutes of continuous scanning at six seconds temporal resolution during sleep in the scanner. The key hypothesis underlying the proposed research is that the method can evaluate state-dependent O2 brain metabolism and airway anatomy in OSA patients during wakefulness and sleep and during spontaneous apneas and further, that the acute airway structural manifestations during apneas and hypopneas correlate with the metabolic response to apneas. The project comprises three specific aims: (1) Optimize the temporally resolved interleaved structural and metabolic MRI protocol and synchronized airway plethysmography to confirm the method’s ability to simultaneously detect the metabolic and airway structural changes during induced apneas; (2) examine the state dependence of O2 metabolism and upper airway anatomy in OSA patients differing in disease severity, with the method of aim 1 and concurrent EEG monitoring; (3) evaluate the hypothesis that the transient brain metabolic and upper airway changes during apnea can be predicted by baseline measurements during respiration in the awake state along with the subjects’ biological profile obtained from blood markers of oxidative stress and neuro inflammation. The proposed research should provide new insight into the structural and neurometabolic implications of OSA and the disorder’s biological underpinnings, and ultimately guide the development of improved treatment methods.