University Of Pennsylvania

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY This K08 proposal outlines a five-year research career development program focused on elucidating the biological role of mesenchymal stem cells (MSCs) and their interaction with neutrophils during mid-palatal suture expansion/distraction osteogenesis. The candidate is an instructor in the Department of Orthodontics at Penn Dental Medicine. Building on previous research and clinical experience in addressing transverse problems in orthodontic treatment, this proposal aims to equip the candidate with multidisciplinary skills essential for transitioning to an independent clinician-scientist with expertise in stem cell biology and osteoimmunology. Clinically, patients with maxillary transverse deficiency (MTD) exhibit varied responses to rapid maxillary expansion (RME), which is often more challenging in opening the mid-palatal suture in skeletally mature patients. While new techniques such as TAD-supported or surgical- assisted RME improve expansion efficacy, the biological basis underlying RME remains unclear, particularly concerning cell populations and tissue heterogeneity within the mid-palatal suture and the molecular mechanisms responding to mechanical forces during suture distraction osteogenesis. Preliminary studies have identified Gli1+ MSCs within mouse mid-palatal sutures, showing their proliferation in response to the RME mice model. The advent of large-scale single- cell RNA sequencing (scRNA-seq) technique has significantly advanced our ability to analyze cell heterogeneity in complex tissues. Osteoimmunology, which highlights the interaction between bone and immune systems, extends our research scope to immune cells, revealing their predominance in mid-palatal cell populations. Notably, neutrophils actively respond to mechanical stimuli, suggesting that MSCs and the immune microenvironment potentially influence RME efficacy. The aims of this proposal are: (1) To investigate the role of Gli1+ MSCs in response to mechanical stimuli during mid-palatal suture distraction osteogenesis, and (2) To identify the transcriptomic signatures of various cell populations in mouse mid-palatal suture, elucidating the interplay between MSCs and neutrophils during RME. This work may uncover previously unrecognized immune-MSC modulatory targets for maxillary expansion/distraction osteogenesis, paving the way for molecular modulation of suture growth and improved patient management, including identifying an immune molecular target that enhances mid-palatal suture growth with or without orthopedic approaches.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Bronchopulmonary dysplasia (BPD) is the most common complication affecting preterm neonates and is defined by supplemental oxygen dependence at 36 weeks postmenstrual age. Advances in neonatal care have improved overall survival of preterm neonates though BPD prevalence continues to increase. BPD develops after early-life lung injury caused by myriad pre- and postnatal factors such as the administration of supplemental oxygen. Though a lifesaving therapeutic intervention, high concentrations of oxygen (hyperoxia) arrest alveolarization and vascularization and induce inflammation. Neonates with BPD face challenges in lung function that can persist throughout adulthood. BPD has a striking male bias for both incidence and severity, demonstrating the need to interrogate sex as a biological variable in lung development, injury, and repair processes. Previous work from our lab showed that female chromosome complement (XX) is protective against lung injury, independent of gonadal sex (ovaries and testes). It is unknown if the protective effect is due to the presence of two X chromosomes or the absence of a Y chromosome. The goal of this proposal is to interrogate dosage sensitivity of sex chromosomes and an X-linked gene, Kdm6a, in an early-life hyperoxia mouse model of BPD. I hypothesize X chromosome mechanisms and dosage contribute to female resilience against hyperoxic lung injury. This hypothesis will be tested by the following: Aim 1) Determine the role of sex chromosome dosage in neonatal hyperoxic lung injury and Aim 2) Determine the role of endothelial Kdm6a in neonatal hyperoxic lung injury. In Aim 1, I will assess lung morphometry and vascularization in room air and hyperoxia-exposed in the XY star (XY*) mouse model that has XX, XO, XY, and XXY genotypes. My preliminary data shows modulation of alveolarization after injury as a function of chromosome dosage. I will then determine cell-type specific gene signatures after hyperoxia exposure using single-cell RNA sequencing. In Aim 2, I will determine the role of endothelial Kdm6a in female neonatal mouse pups during development and after hyperoxic lung injury. X chromosome inactivation (XCI) is a dosage compensation mechanism whereby supernumerary X chromosomes are transcriptionally silenced so genes from only one X chromosome are expressed. However, some genes escape XCI. Intriguingly, preliminary data from our lab show a female- specific induction of an XCI escapee, Kdm6a, after exposure to hyperoxia. I will test the hypothesis that endothelial knockdown of Kdm6a intensifies hyperoxic lung injury in female mice. To determine the role of Kdm6a in epigenetic regulation and alterations to the chromatin landscape, I will perform bulk RNA-seq, ATAC- seq, and CUT&RUN. Completion of this proposal will advance mechanistic knowledge of sex-specific responses to respiratory insults and potentially inform the development of precision medicine approaches.

NIH Research Projects · FY 2025 · 2025-08

ABSTRACT Vellus hairs are short and fine hairs that cover the human body and are essential for facilitating sweating, the major mechanism for human thermoregulatory cooling. Vellus hairs are formed during human gestation, in response to a shift in the growth cycle of human fetal hair follicles. During early fetal development, hair follicles first build large lanugo hairs, which are subsequently shed before birth. Starting around 20 weeks of gestation, fetal hair follicles undergo a reduction in the duration of their active growth phase (anagen), leading to the formation of smaller vellus hairs that predominate human skin post-partum. Determining the mechanisms that regulate the lanugo to vellus transition is critical in understanding the basis for this important thermoregulatory trait. A hallmark of the period that marks the onset of the lanugo to vellus transition in human fetal skin is the upregulated expression of a transcription factor, Engrailed1 (EN1). During this timeframe, EN1 expression is upregulated in multiple epidermal populations of the human fetal hair follicle, most notably in the hair matrix, the progenitor population from which the hair shaft derives. EN1 also becomes upregulated in the dermal papilla, the mesenchymal niche that regulates the hair cycle and hair production. In light of the concordance of EN1 upregulation and the onset of the lanugo to vellus transition, our lab turned to a humanized transgenic mouse model that allows for tunable En1 expression in the epidermis of the hair follicle in a manner consistent with human fetal skin. This model demonstrated that mice with ectopic En1 expression in the epidermis have shorter and thinner hairs due to a truncated growth phase (anagen) of the hair cycle, recapitulating key hair size diminution events observed during the lanugo to vellus transition. Considering these data, the hypothesis of this proposal is that En1 regulates hair size by altering the function of the hair matrix and the dermal papilla, two key populations involved in hair production. Accordingly, the aims of this proposal are to define the cellular and transcriptional mediators by which En1 functions in the two major skin compartments, epidermis (Aim 1) and dermal papilla (Aim 2), to alter hair follicle cycling. The proposed experiments will uncover the first mechanism underlying human vellus hair formation. Beyond their implications for understanding human-specific biology, the findings derived from this proposal will be critical for developing an integrative approach to comprehensive human skin regeneration.

NIH Research Projects · FY 2025 · 2025-08

ABSTRACT: As specialized cells with limited cytoplasm, sperm have long been viewed as contributing only the paternal genome to progeny. However, in the past decade experiments in mice have revealed that sperm microRNAs (miRNAs) influence early embryogenesis and are sufficient for the paternal transmission of non-genetically inherited phenotypes. miRNAs, along with their protein cofactor Argonaute (Ago), bind messenger RNA (mRNA) targets and subsequently downregulate their expression through mRNA decay and translational repression. However, the mechanism underlying how sperm miRNAs act as inherited information remains undetermined. We hypothesize that sperm miRNAs modulate early development through the direct regulation of key mRNA targets like chromatin modifiers or signal transduction pathways, thus altering downstream gene expression programs in a manner that persists through development. Due to the difficulty of identifying targets of miRNAs in the embryo, the targets of sperm miRNAs are still currently unknown. The Fx-miRs are a cluster of miRNAs that are highly expressed in the sperm of every mammalian species analyzed. When wildtype eggs are fertilized with Fx-miR deficient sperm, resulting embryos have decreased developmental potential and demonstrate significantly altered gene expression in preimplantation embryogenesis. Despite these phenotypes, the mechanistic functions of the Fx-miRs in the early embryo, especially what mRNAs these miRNAs target and how these binding events impact overall gene expression during early development, remain unknown. I hypothesize that sperm miRNAs directly target and regulate a subset of maternally provided or zygotically expressed mRNAs postfertilization which then leads to downstream gene expression effects that modulate embryonic development. In Aim 1a, I will use ribosome profiling to evaluate the impact of individual Fx-miRs and the whole cluster on global translation to discover additional regulation by these miRNAs not captured by mRNA-seq. Here, I will use mESCs as a model for early embryonic development, as my preliminary data has demonstrated that mESCs are an efficient model to profile the molecular functions of sperm miRNAs. In Aim 1b, I will use Ago enhanced Crosslinking and Immunoprecipitation (Ago eCLIP) to identify the direct targets of the Fx-miRs on the level of individual miRNAs, also using mESCs as a model. Finally, in Aim 2, to dissect direct versus secondary effects, I will knockdown specific Fx-miR targets identified in my preliminary data via siRNA in embryos fertilized with Fx-miR deficient sperm and perform single embryo RNA-seq. Downregulation of specific Fx-miR targets and the analysis of the resulting differential gene expression will allow for the separation of direct versus downstream effects of the Fx-miRs in vivo. This project will be the first to identify the postfertilization gene regulatory functions and direct targets of an important cluster of sperm miRNAs and thus reveal mechanistically how sperm miRNAs can program embryonic development to initiate the non-genetic inheritance of offspring phenotypes.

NIH Research Projects · FY 2025 · 2025-08

Summary The goal of this proposal is to test the hypothesis that the transcriptional program mediating the process by which endothelial cells in the embryo differentiate into hematopoietic stem and progenitor cells (HSPCs) is coordinately regulated by cis-acting regulatory sequences derived from retroelements. Retroelements are repetitive DNA sequences comprising approximately 50% of mouse and human genomes. The long terminal repeats (LTRs) of the retrotransposon family of retroelements function as enhancers, promoters, and polyadenylation signals for endogenous gene expression and are highly enriched for transcription factor binding sites (TFBS). We have identified families and specific subfamilies of retrotransposons that are more highly expressed in the small population of endothelial cells in the embryo that differentiate into HSPCs (hemogenic endothelial cells) than in non-hemogenic endothelial cells, and in particular give rise to progenitors with potent lymphoid potential. Some of these subfamilies of retrotransposons contain RUNX1 motifs in their LTRs, and we hypothesize that RUNX1 regulates HSPC differentiation from hemogenic endothelial cells through these LTRs. We will use long read sequencing technology and bioinformatics approaches adapted for the purpose of analyzing retroelements to map transcriptionally active retroelements based on their expression and chromatin configuration to specific genomic coordinates and determine if they activate genes involved in HSPC formation from hemogenic endothelial cells. We will also determine if the RUNX1 transcription factor regulates the activity of a subset of these LTRs.

NSF Awards · FY 2025 · 2025-08

PART 1: NON-TECHNICAL SUMMARY The fundamental understanding of ion transport mechanisms in polymers is incomplete, while the demands for improved membrane properties increase particularly regarding separation technologies and electrochemical device applications. This project endeavors to develop this fundamental understanding through the comprehensive characterization of newly designed and synthesized polymers containing sulfonate groups. The research approach includes conductivity measurements, spectroscopy measurements, and extensive nanoscale structural characterization to establish the location and motion of ions under various conditions of temperature and solvent content. To accomplish this experimental plan, the research team will design and build environmental chambers to maintain a saturated solvent environment during in situ testing. Mirroring the working environment in the materials industry, the PI and graduate students actively collaborate with synthetic chemists and computational scientists, to holistically develop and evaluate the understanding of ion transport mechanisms. By establishing how ions move through polymers, scientists and engineers will be able to design new polymers and processing routes to optimize ion conductivity and ion selectivity that meet a variety of specific needs and drive innovation. PART 2: TECHNICAL SUMMARY This research effort aims to understand to what extent nanoscale morphologies and selective solvents promote cation transport and the underlying mechanisms of cation transport. The proposed research combines structural characterization, conductivity measurements, and spectroscopy measurements on unique polymers to further develop the understanding and demonstration of decoupled ion motion in polymer electrolytes. Aim 1 endeavors to achieve the double gyroid morphology in strictly alternating multiblock copolymers near room temperature. We will investigate a variety of multiblock copolymers and processing strategies to determine the criteria for producing the double gyroid morphology in multiblock copolymers with strongly segregated and short blocks. Aim 2 will identify solvent attributes that promote Li+ and Na+ transport in polymers with aligned nanoscale layered morphologies. To eliminate the effect of morphological orientation and grain boundaries on transport in periodic nanoscale morphologies, we will fabricate thin films with aligned layers in strictly alternating multiblock copolymers and measure the in-plane conductivity as a function of solvent swelling and solvent type. Finally, Aim 3 seeks to improve ion transport in polymers with network morphologies by the addition of solvent. For this portion of the proposed project, we will study three types of partially sulfonated polymers that self-assemble into nanoscale ionic channels without long-range order. This broader range of polymers will test the applicability of our findings about solvent-enhanced ion transport in a variety of nanostructured polymers. In addition to providing exceptional opportunities for student education, this proposal will develop an environmental chamber for grazing incident X-ray scattering and provide professional development workshops for doctoral students. 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 2025 · 2025-08

This research combines ideas and techniques from several of the most exciting directions of contemporary mathematical thought, developing powerful connections between seemingly distant fields. These include homotopy theory (the study of shapes up to continuous deformation), algebraic K-theory (a broadly applicable framework that captures how mathematical objects can be decomposed into pieces and reassembled), smooth manifolds (shapes with no edge or boundary, of fundamental importance to geometry and physics), polyhedra (the higher-dimensional version of polygons), and knots (frictionless loops of string in three-dimensional space). The PIs will pursue several new directions, building on the longstanding and successful program of combining these theories to classify important geometric objects, along with some striking new results relating K-theory to polyhedra and knots. The project also includes the training of junior mathematicians, the development of a much-needed textbook in homotopy theory, and the expansion of college-level mathematics in prison education, which was shown to curb recidivism and save taxpayer money. In this project the PIs will develop algebraic K-theory and its applications to geometric objects such as smooth manifolds, polyhedra, and knots. They plan to bring the connection between equivariant algebraic K-theory and h-cobordisms to fruition, in order to further our understanding of the homotopy type of the moduli space of G-manifolds. They will study the higher versions of Hilbert’s 3rd problem for polyhedra through scissors congruence K-theory. They will integrate recent insights from the setting of polytopes into the scissors congruence of manifolds, in order to perform computations, construct new higher invariants of smooth manifolds up to cut-and-paste relations, and investigate the tantalizing relation to cobordism categories. Lastly, they will collaborate with low dimensional topologists in order to apply stable homotopy theory and algebraic K-theory to prove new properties of Khovanov homology. 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 · 2025-08

Project Abstract 80% of patients afflicted with autoimmune diseases are female, and most autoimmune diseases have no cure. Patients with female-biased autoimmune diseases such as systemic lupus erythematosus (SLE) aberrantly express X-linked genes, suggesting that impairments in X chromosome inactivation (XCI) may impact disease. XCI transcriptionally silences one X chromosome in XX females via enrichment of repressive epigenetic marks including Xist RNA, heterochromatic histone marks, and DNA methylation (DNAm) to equalize X-linked gene expression to XY males. While in vitro stimulated B cells exhibit association of all these repressive epigenetic marks to the Xi, our laboratory found that Xist RNA and heterochromatic histone modifications are delocalized from the Xi in naïve B cells. Preliminary allele-specific RNA-seq data indicate that majority of the inactive X remains ‘continuously silent’ in naïve and stimulated cells, indicating that the inactive X in B cells remains silent even in the absence of Xist RNA and heterochromatic marks. This suggests the existence of Xist RNA independent mechanisms regulating Xi-linked gene expression in female B cells. Allele-specific RNA-seq data reveal that of the 42 genes which are expressed from the inactive X in naïve cells, some are ‘continuously expressed’ in stimulated cells. In addition, some genes become ‘newly silent’ or ‘newly expressed’ in stimulated cells. Allele-specific whole genome bisulfite sequencing (WGBS) data indicate that the Xi remains highly enriched for DNAm compared to the active X and autosomes in both naïve and stimulated cells, indicating that it may be responsible for regulating gene expression from the inactive X independently of Xist RNA. As the conversion of repressive 5’-methylcytosine (5mC) to 5’-hydroxymethylcytosine (5hmC) associates with gene expression and WGBS alone does not differentiate them, I distinguished between these two forms of DNAm using the novel technique bisulfite-assisted APOEBEC-coupled epigenetic sequencing (bACE-seq) alongside WGBS. My preliminary data indicate that 5hmC is present at CpG dinucleotides within promoters of some expressed X-linked genes, and that B cell stimulation alters its enrichment levels. DNA methyltransferases (DNMT) deposit 5mC, whereas Ten Eleven Translocation (TET) enzymes remove it by generating 5hmC. I will test my central hypothesis that DNMT and TET enzymes regulate gene expression from the inactive X in stimulated B cells in an Xist RNA-independent manner. In Aim 1, I will perturb DNMT1 (Aim 1a), the DNMT responsible for 5mC maintenance during cell replication, and DNMT3a/3b (Aim 1b), the DNMTs responsible for novel deposition of 5mC, and assess the impact on 5mC enrichment and gene expression from the Xi. In Aim 2a, I will perturb TET2/3, the most highly expressed TETs in B cells, and assess the impact of perturbed 5hmC enrichment on Xi-linked gene expression. In Aim 2b, I will perturb Xist RNA expression and assess whether DNAm modulates Xi-linked gene expression independently of Xist RNA. Elucidating this mechanism will reveal how X-linked gene expression can become perturbed in female-biased autoimmune diseases.

NSF Awards · FY 2025 · 2025-08

Machine learning models have demonstrated remarkable capabilities across various domains. However, a fundamental challenge, known as "distribution shift," arises when models trained on one set of data encounter new, different data in deployment, often leading to a significant drop in performance and reliability. This issue is critical in applications like healthcare or autonomous systems, where erroneous predictions can have severe consequences. The goal of this project is to develop novel algorithms that provide provable guarantees on a model's performance even under such distribution shifts, enabling models to either make reliable predictions or to recognize when data is too different and abstain from making a prediction. This project will also contribute to training the next generation of researchers in this area and disseminate findings through workshops and publicly available educational resources. This project will develop and analyze efficient learning algorithms designed to operate robustly under distribution shift, without making any assumptions on the unseen test distribution. The investigators will extend classical learning frameworks to new paradigms, such as Testable Learning with Distribution Shift (TDS learning), where a learner can reject an entire problematic test set, and PQ learning, which allows for instance-level abstention if a significant shift is detected for a particular example. A core focus will be on circumventing the computationally intractable problem of measuring distances between training and test distributions. Instead, the algorithms will output a classifier accompanied by a proof that either the classifier performs optimally on the test set or a significant distribution shift has occurred. Research will also address sequential prediction settings where decisions are made on a sequential (potentially adversarial) stream of data. The theoretical approaches will draw from and establish new connections between machine theory, pseudorandomness, sum-of-squares proofs, robust statistics, and information theory, with empirical validation on standard benchmarks in biology and natural language. 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 · 2025-08

Project Summary Amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD) represent two extremes of an ALS- FTD spectrum, frequently characterized by TDP-43 pathological inclusions and shared genetic risk factors. We have defined normative criteria for characterizing cognitive impairment in ALS and demonstrated that 22% of ALS are cognitively impaired and, in autopsy-confirmed TDP-43 cases, there is an overall >4 hazard ratio for individuals with ALS or FTD onset to develop features of both ALS-FTD. However, prognosis is highly variable across individuals in the ALS-FTD spectrum and risk factors for developing co-occurring motor neuron dysfunction and cognitive impairment are largely unknown, even within C9orf72 repeat expansion families. The overall hypothesis of this proposal is that genetic modifiers of the FTD-ALS spectrum will identify factors predictive of ALS or FTD onset and prognosis for developing ALS-FTD. The discovery of genetic modifiers that contribute to ALS-FTD phenotypic heterogeneity will improve prognostication and clinical trial stratification while enhancing our mechanistic understanding of these highly variable and devastating disorders. This proposal will focus on two strategies for identifying genetic modifiers of binary (ALS vs FTD) and quantitative (neuropsychological and motor) traits. We will leverage the highly integrated Penn Comprehensive ALS Clinic and Penn Frontotemporal Degeneration Center, which have common procedures for neuropsychological and motor phenotyping as well as DNA and biofluid banking. First, we aim to identify polygenic modifiers of cognitive and motor features of ALS-FTD spectrum. We will generate traditional and novel module-based polygenic risk scores (PRS) of common variants associated with “disease-specific” ALS or FTD risk or associated with “domain- specific” heritable traits including cognitive and motor function. We will relate each PRS to cross-sectional and longitudinal neuropsychological and motor/functional scales. We will additionally perform Mendelian Randomization to identify causal associations between heritable traits and disease-specific outcomes. We hypothesize common genetic variation reflected by PRS will modify onset type and quantitative trait measures of cognition and motor impairment. Second, we aim to identify structural variants through long-read sequencing (LRS) of individuals and families that modify cognitive and motor features of the ALS-FTD spectrum. We will use LRS to identify structural variants (SVs) and use rare-variant aggregated and burden tests to identify SVs and genes associated with onset type and quantitative trait measures of cognition and motor. We will additionally use variants component linkage analysis in our well-characterized three-generation pedigrees to identify phenotypic modifiers. We hypothesize both strategies will uncover novel sources of common and rare genetic variation that contribute to heterogeneity in the ALS-FTD spectrum.

NIH Research Projects · FY 2025 · 2025-08

Project Summary/Abstract: An estimated 400 million people worldwide have experienced persisting and sometimes debilitating symptoms for months and years after SARS-CoV-2 infection, known as Long COVID (LC). No proven therapies exist to treat LC, and the underlying mechanisms driving disease remain poorly understood. Reactivation of herpesviruses such as Epstein-Barr virus (EBV) and varicella zoster virus (VZV) has been linked to LC, but it is unclear how SARS-CoV-2 infection alters immune responses to these common viruses. Recent studies have also provided emerging evidence for persistence of SARS-CoV-2 in some individuals with LC, but key questions remain unanswered, including how viral reservoirs persist, why the immune system fails to clear virus, whether persisting virus drives ongoing immune stimulation, and how therapies to treat LC will affect immune responses to SARS-CoV-2. Efforts to identify effective treatments for LC depend on answering these questions. Our preliminary data demonstrate elevated activation of SARS-CoV-2-, EBV-, or VZV-specific T cells in 40% of LC patients, providing one of the most sensitive measures of immunopathology in LC to date. Thus, there is an urgent need to investigate virus-specific T cell activation in LC to define underlying mechanisms of disease and identify promising therapeutic targets. The proposed research will respond to this need by testing three working hypotheses: first, that SARS-CoV-2-specific CD8 T cells survey tissue reservoirs of SARS-CoV-2 and sense viral antigens, but fail to clear the virus due to impaired functionality in LC; second, that therapies suppressing SARS-CoV-2 replication will reduce T cell stimulation in LC but may not achieve a durable cure; and third, that immune stimulation by common herpesviruses is associated with disease pathology in LC. These hypotheses will be tested by leveraging custom HLA-I/peptide tetramers to identify and sort rare virus-specific CD8 T cells from hundreds of longitudinal blood and tissue samples from people with LC and people who fully recovered after SARS-CoV-2 infection. Cells will be analyzed using spectral flow cytometry and single-cell sequencing approaches to elucidate the mechanisms driving LC pathology, identify promising therapeutic targets for LC, and investigate how immune stimulation by herpesviruses shapes human disease. This research will be integrated with a comprehensive training plan to develop skills in tissue immunology, single-cell sequencing, and bioinformatics, which will be reinforced with targeted coursework and professional development activities in scientific writing, responsible conduct of research, laboratory management, and mentorship of junior personnel. The research and training plan will take full advantage of the outstanding environment at the University of Pennsylvania and the wealth of expertise in Dr. John Wherry’s lab. Completing the proposed research and training will substantially advance the human immunology field and form the foundation of my research program as I open my independent laboratory, creating a pathway to my goal of being an impactful independent researcher in human infectious disease immunology.

NIH Research Projects · FY 2026 · 2025-07

PROJECT SUMMARY Pregnancy complications such as early pregnancy loss, preeclampsia, preterm birth, and fetal growth restriction or low birthweight are common, distressing, and costly to individuals and society. While their etiologies are multifactorial, aberrations in implantation and placentation are a common pathway. Vitamin D has been proposed as a possible low-cost, widely accessible intervention to improve these outcomes, but randomized controlled trials have found no benefit to supplementation among vitamin D deficient patients. The study proposed in this application will address many of the limitations that have previously made definitive conclusions elusive. First, as fertility care provides access to patients very early in pregnancy, we will be able to measure periconception vitamin D in contrast to many studies which measure vitamin D in the second or third trimesters – long after implantation and placentation are complete and too late to intervene for meaningful benefits. Second, we will measure more than the storage form of vitamin D (25-hydroxyvitamin D, or 25(OH)D, which is most commonly used in prior studies), as emerging evidence from other fields indicates that more nuanced assessments such as the vitamin D metabolite ratio (VMR) and D binding protein (DBP) isoforms are more associated with clinical outcomes. Third, we will utilize different in vitro fertilization protocols as an experimental model for dynamic hormonal environments in which vitamin D metabolites and DBP are expected to vary, allowing for broader investigation of alterations in vitamin D homeostasis. Our central hypothesis is that due to its role in implantation and placentation, periconception VMR modulates the likelihood of establishing and sustaining pregnancy as well as developing placentally-mediated obstetric complications, with varying effects depending on DBP isoform. Supported by a team of mentors/collaborators and an advisory committee with diverse expertise as well as the well-established research infrastructure at Penn, I will conduct a prospective cohort study of patients seeking fertility care at our institution in order to test this hypothesis. Aim 1 will assess the effect of different periconception hormonal environments (unassisted conceptions, natural frozen embryo transfers, and programmed frozen embryo transfers) on VMR, with stratified analysis by DBP isoform. Aim 2 will assess the association between VMR and live birth, early pregnancy loss, and obstetric complications including hypertensive disorders of pregnancy, preterm birth, and low birthweight among patients undergoing frozen embryo transfer, again with stratified analysis by DBP isoform. In addition to answering critical questions on the role of vitamin D homeostasis in reproductive outcomes, this study will provide me with valuable experience in prospective reproductive outcomes data collection. Along with mentorship from renowned epidemiologists and reproductive endocrinologists, it will also prepare me for a career as a reproductive epidemiologist as well as my longer term goal of conducting clinical trials to improve reproductive outcomes in fertility and non-fertility patients alike.

NIH Research Projects · FY 2025 · 2025-07

Intervertebral disc herniations, caused by extrusion of nucleus pulposus tissue through a defect in the annulus fibrosus (AF), affect 2 to 3% of the world population and can be a significant contributor to back pain and disability. The gold standard clinical treatment for patients with persistent pain from disc herniation that is unresolved after conservative treatment is microdiscectomy surgery, during which the herniated tissue is removed to relieve pressure on the nerve roots. However, the AF is not surgically repaired during this procedure. Given the limited endogenous healing capacity of the AF, 10 to 30% of patients will experience a symptomatic recurrent herniation. Due to the clinical burden of disc herniations and the absence of alternatives to discectomy, there is a substantial need to develop and translate novel AF repair devices which can facilitate annular healing, restore disc mechanics, and prevent re-herniation. Endogenous repair following injury is hindered by AF cell apoptosis, increased local inflammation and ultimately the formation of disorganized scar tissue. Repair strategies that address this complex biological milieu, while restoring AF mechanical function and preventing re- herniation have yet to be established and proven efficacious in large animal studies. Here, we will optimize and translate a novel tension-activated annular repair scaffold (TARS) to address both the structural and biological requirements for annular repair. The TARS implants are composed of two layers of aligned nanofibrous polymer scaffolds, containing depots of microcapsules between the scaffold layers which release their contents under mechanical loading (MAMCs). When the TARS is loaded in tension, the MAMCs are compressed, leading to release of bioactive molecules. In Aim 1, we will define the release profiles of the TARS in vitro under dynamic uniaxial tensile loading in a physiologic environment, and in situ when affixed to the AF in cadaveric goat cervical spine motion segments. From this Aim, we will validate a TARS design that can deliver bioactive molecules to the repair site over acute and chronic timescales. In Aim 2, we will target the biological sequalae of annular injury and evaluate the ability of an anti-inflammatory and pro-anabolic TARS to promote local AF repair and global spine functional restoration in vivo in a goat cervical disc injury model. AF injury and repair will be thoroughly evaluated across length scales, with a focus on the restoration of healthy AF structure, biology and mechanical function. This novel and translationally relevant AF repair technology could change clinical practice for the treatment of disc herniations, reduce the incidence of reherniation, and improve the long-term spine health of patients.

NIH Research Projects · FY 2025 · 2025-07

Project Summary Gram-negative bacterial infections cause more than 325,000 cases of severe sepsis in the US and almost 10 million cases worldwide annually, with a >30% mortality rate. Gram-negative bacterial sepsis is caused by a dysregulated systemic inflammatory response to lipopolysaccharide (LPS) that is sensed at the plasma membrane by Toll Like Receptor 4 (TLR4) and in the cytosol by caspase-11 (CASP11) in mice, and its orthologs caspases-4 and -5 (CASP4 and CASP5) in humans. CASP11, CASP4, and CASP5 oligomerize into non- canonical inflammasomes in response to LPS, leading to pyroptotic cell death and release of inflammatory mediators. While excessive CASP11 activation during systemic LPS delivery or bacteremia can lead to sepsis, CASP11 is also essential for anti-bacterial defense against gram-negative bacterial infections in mouse models. Bacterial pathogens can vary the acylation status of LPS to evade innate immune sensing by TLR4 and CASP11. Intriguingly, recent findings by us and others indicate that human CASP4/5 are activated by LPS structures that are not sensed by TLR4 and CASP11, indicating that humans have evolved to respond to a broader repertoire of LPS structures than mice. The mechanisms dictating the differential responsiveness of CASP11 and CASP4/5 to distinct alterations in LPS structure and the consequences for host defense and sepsis are poorly understood. Thus, we propose to define the molecular and cellular mechanisms of non-canonical inflammasome responses to LPS in mice and humans using a combination of in vitro and in vivo models. This fundamental information will provide essential insight for the development of improved therapeutics that effectively reduce human sepsis, as well as immune adjuvants that improve host responses to immunoevasive bacterial pathogens.

NIH Research Projects · FY 2025 · 2025-07

OVERALL: PROJECT SUMMARY Our primary goal in this IPCAVD program is to develop a germline-targeting priming immunogen for the HIV Envelope V2-apex broadly neutralizing antibody (bnAb) site. We aim to test its ability to activate V2-apex bnAb precursors in preclinical animal models and manufacture it under GMP standards for human clinical testing. To address this comprehensively, in this IPCAVD grant proposal we have assembled a highly skilled team of investigators with expertise in rational protein design, bioengineering, B and T cell immunology, vaccine evaluation models, antibody discovery, and viral immunology and pathogenesis. We hypothesize that bnAb B cell germline-targeting, immunofocusing and molecularly guided affinity maturation are the essential components of an effective vaccine strategy for eliciting protective HIV Env V2-apex targeted bnAb responses. Leveraging our strong preliminary work in targeted immunogen development, novel vaccine delivery platforms, adjuvants, and immune monitoring tools, this IPCAVD proposal integrates these elements for testing rationally designed prime-boost vaccination strategies in relevant pre-clinical animal models. The overall specific aims of this IPCAVD program are: Aim #1. Rationally design HIV Env V2-apex bnAb-site-targeting vaccines through reverse vaccine engineering. Aim #2. Develop vaccination strategies for inducing epitope-targeted HIV bnAb responses using novel preclinical animal models and vaccine delivery platforms. Aim #3. GMP-manufacture the lead germline-targeting vaccine immunogen with the goal for human clinical testing to induce bnAb B cell responses. This IPCAVD application builds on a foundation of success in eliciting V2 apex bnAbs in multiple rhesus macaques (RMs) by novel simian-human immunodeficiency virus (SHIV) infections and then deconvoluting Env- Ab coevolution pathways to identify novel candidate prime and boost immunogens. From 150 SHIV-infected RMs, we identified the Q23 Env as our lead platform. Our IPCAVD comprises three projects: Project 1, through reverse vaccine engineering approaches, will further develop a Q23 Env-based germline-targeting immunogen to most efficiently engage multiple rhesus and human V2-apex bnAb B cell precursors in vivo and by ex vivo analyses. This is the crux of successful bnAb induction. Project 1 will also design mRNA-launched trimer boost immunogens for expanding the breadth of nAb responses. Project 2 will assess the in vivo priming efficiency of the germline-targeted immunogen, and neutralization breadth expansion ability of the mRNA-launched trimer boost strategies, first in the V2-apex bnAb UCA expressing KI mice, and subsequently in the NHP model. We will down-select the Q23 germline-targeting priming immunogen and this will be GMP-manufactured in collaboration with Project 3, with the goal of human clinical evaluation. If successful, our immunogen regimen could swiftly transition into human clinical testing to induce protective bnAbs against HIV.

NIH Research Projects · FY 2025 · 2025-07

ABSTRACT Breast cancer remains the second largest cause of cancer related deaths in the USA. Heat shock protein (HSP) inhibition has emerged as a promising target to treat breast cancer. However, the drawback of these drugs is cardiotoxicity. Nanoparticle delivery of HSP inhibitors should avoid this side-effect, as well as increasing delivery of drug to the tumor. Various types of nanoparticles can also enhance the effects of photothermal therapy on cancer killing. Moreover, photothermal therapy together with delivery of HSP inhibitors is a potent combination treatment. However, many of the types of nanoparticles typically used in photothermal therapy are not well-suited for drug delivery, are very slowly excreted, are expensive, are challenging to scale up and are synthesized using toxic reagents or organic solvents. We herein propose a novel polyphosphazene nanoparticle (PPNP) to treat breast cancer via enhancing photothermal therapy and delivering a HSP inhibitor. The PPNP will be also loaded with ultrasmall silver sulfide nanoparticles that provide strong photothermal therapy enhancement, but when released from the biodegradable nanoparticle, are rapidly renally cleared. The silver sulfide nanoparticles also provide contrast for multiple imaging techniques (i.e. computed tomography, mammography, photoacoustics and fluorescence), thereby allowing delivery to be assessed and guiding laser irradiation. This is a low cost platform that is synthesized by microfluidics, which enables large scale synthesis of a homogenous product. These PPNP will be synthesized, characterized, and tested for their contrast production. Drug loading and release will be assessed, as will their enhancement of photothermal heating. Breast cancer killing will be studied in vitro and in vivo. We will also perform in vivo imaging, as well as safety and clearance studies. Overall, we expect to develop a potent new tool for the treatment of breast cancer.

NIH Research Projects · FY 2025 · 2025-07

Abstract While the incidence of Opioid Use Disorder (OUD) continues at epidemic levels, a necessary step in the development of improved OUD therapeutics is to understand the adapting molecular architecture and functional dynamics neural circuits responding to opioids. Despite extensive research to date, it remains unclear how opioidergic circuits in various brain regions modulate neural activities and opioid gene expression patterns across stages of the addiction cycle (e.g., drug naïve, acute exposure, chronic exposure, and abstinence) within the same animals. To better capture the molecular and activity underpinnings of OUD, our collaborative team of bioengineers and circuit physiologists will develop a new approach for long-term monitoring of opioid-induced changes in neural population activity and opioid receptor gene expression with a simple blood test, called Released Markers of Activity—RMAs—that translocate from neurons into the peripheral blood stream. Thus, the breakthrough potential of RMAs for OUD discoveries are illustrated by their device-free and within-subjects measurement features with around-the-clock, weeks-long monitoring, as well as modular use for both neural activity and genetic alterations as provided by our novel viral promoters that give genetic access to active populations and opioid receptor-expressing cell-types. In Aim 1, we will clone our 2nd generation “erasable” RMAs (eRMAs; protease cleavable variants for higher signal-to-noise detection amplification across time) under an activity-dependent viral promoter system to measure increased and/or decrease neural activity in two key brain regions for OUD—before, during and after abstinence from morphine and fentanyl exposure, within the same animal. In Aim 2, we will clone eRMAs under a new opioid receptor promoter system that would permit a new class of long-term gene expression measurement related to the up- or down-regulation of the mu-opioid receptor following chronic morphine and fentanyl exposure. Across both aims we will target this new blood-based monitoring system to key brain regions implicated in OUD: ventral tegmental area, nucleus accumbens, and central amygdala. The RMAs for OUD approach will enable us to understand how opioid exposure influences neural population functions and opioid receptor expression that are related to OUD-related behavior in mice. Beyond our lab, this innovative and team-driven project will result in the creation of an easy-to-implement and fast, device-free blood monitoring system for researchers to accelerate the pace of research in opioid addiction and future development of new OUD treatments. In principle, RMAs for OUD are not limited to mice but any genetically intractable model system, such as rats, human iPSC brain organoids, pigs, and non-human primates, which we will explore after successful completion of the CEBRA R21 goal: To develop Release Markers of Activity for detecting neural activities and opioid gene expression across the OUD cycle in individual mice.

NIH Research Projects · FY 2025 · 2025-07

Project Summary/Abstract In this proposal, we request funds from the Shared Instrumentation Grant mechanism for the procurement of an Olympus FV4000-MPE Multiphoton Laser Scanning Microscope equipped with a Spectra-Physics tunable infrared Ti:Sapphire pulsed laser. This equipment will be used to establish the first Multiphoton Intravital Microscopy (MP-IVM) imaging facility at the University of Pennsylvania Perelman School of Medicine. This resource will be accessible to all investigators at Penn and across the greater Philadelphia area, offered as a service by the Cutaneous Phenomics and Transcriptomics (CPAT) Core of the Penn Skin Biology and Disease Resource-based Center (SBDRC). Intravital microscopy, which involves the direct imaging of live mice and other small animals, enables the visualization of tissues and cells in their natural environment. Multiphoton microscopy is a powerful laser scanning imaging modality that has revolutionized the ability to visualize biological processes in living organisms with high spatial and temporal resolution. Unlike conventional microscopy, multiphoton excitation reduces light scattering, photodamage, and phototoxicity. It enables the non-invasive study of cellular dynamics, signaling pathways, and tissue architecture in real-time. Additionally, it offers unprecedented depth penetration, allowing for the visualization of tissue structures up to a millimeter deep, surpassing the capabilities of traditional confocal microscopy. These attributes make multiphoton microscopy ideal for the study of the skin and other accessible organs. This technology is rapidly becoming an invaluable tool for studying dynamic processes such as tissue regeneration, wound healing, aging, and disease progression. With the Olympus FV4000-MPE microscope, the new MP-IVM facility in the SBDRC CPAT Core will be broadly available and easily accessible to investigators studying skin biology, as well as those interested in biological processes accessible to intravital imaging, including immunology, vascular biology, neurobiology, ocular biology, musculoskeletal biology, and stem cell biology. The research projects outlined in this application are the product of a diverse array of NIH-funded investigators at the University of Pennsylvania School of Medicine, each with unique expertise and research interests. They leverage the extraordinary capability of multiphoton microscopy to visualize their cells and tissues of interest at high spatiotemporal resolution, conducting multidisciplinary experiments. The availability of a multiphoton microscope will significantly empower and expand the research directions of these investigators and open new avenues of research for new users of this resource. The requested Olympus FV4000-MPE Multiphoton Laser Scanning Microscope will be the only instrument configured specifically for mouse intravital imaging, available on a shared-user basis for the entire University of Pennsylvania Medical School.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY / ABSTRACT Essential tremor (ET) is the most common movement disorder in adults, yet its underlying pathophysiology is poorly understood. Non-invasive neural recordings have implicated oscillations at tremor frequency in the cerebello-thalamo-cortical network of ET, yet peri-operative invasive thalamic recordings have not consistently shown such oscillations. Deep brain stimulation (DBS) to the ventral intermediate nucleus (Vim) of the thalamus is a treatment option for medically refractory tremor, and new DBS technology that chronically senses neurophysiologic signals from the stimulation site offers a novel window into studying neural signatures of ET and the effects of DBS. The central hypothesis of this proposal is that ET tremor is associated with the propagation of tremor frequency oscillations from the cerebellum to motor cortex via the Vim thalamus, and that thalamic DBS interrupts this propagation. The central hypothesis will be tested by pursuing three Specific Aims. Aim 1: Identify the relationship of chronically recorded Vim thalamus oscillatory activity to tremor intensity. Aim 2: Assess the directionality of oscillatory activity within the cerebello-thalamo- cortical network. Aim 3: Assess the effect of DBS on oscillatory activity within the cerebello-thalamo-cortical network and its relationship to tremor. To pursue these aims, this study will use multimodal neurophysiology, including novel methods such as chronically recorded thalamic local field potentials and combined cerebellar and motor cortex electroencephalography, to study the relationship of Vim oscillations with manifested tremor and other oscillations in the cerebello-thalamo-cortical network. Results from the proposed research will make significant contributions to our understanding of the underlying neurophysiology of ET, thus providing a framework for using sense-enabled DBS systems to further study mechanisms of DBS in ET and to improve DBS technology. This proposed project will provide career development training to Dr. Hammer, who is establishing herself as a physician-scientist conducting patient-centered research in the field of deep brain stimulation (DBS) for movement disorders. This K23 will propel Dr. Hammer towards scientific independence as she develops expertise in electroencephalography, cerebellar network physiology, network neuroscience, and trial design and statistics. She has assembled a multidisciplinary mentoring group including Dr. Brian Litt (expert in neural engineering in the setting of epilepsy), Dr. Casey Halpern (expert in DBS for novel indications in mental health), Dr. Sheng-Han Kuo (expert in cerebellar physiology in tremor and ataxia), and Dr. Svjetlana Miocinovic (expert in DBS for movement disorders). This K23 training will provide the foundation for a future R01 application using chronically sensed thalamic neural signals from DBS implants to study mechanisms behind declining efficacy of DBS for ET over time (i.e., “habituation”).

NIH Research Projects · FY 2025 · 2025-07

Project Summary Type 2 diabetes (T2D) pathology is complex and multifactorial. Typically, individuals develop insulin resistance and beta cell compensation that ultimately succumbs to chronic hyperglycemia leading to beta cell failure and death. By increasing our understanding of the mechanisms and predispositions leading to beta cell demise, we can begin to develop better strategies and medicines to treat people living with this debilitating disease. It is well known that beta cells harness transcriptional and post transcriptional controls to increase both the gene and protein expression of critical proteins that support insulin secretion in response to nutrient stimulation. However, due to the technical limitations in assessing translational control as opposed to high throughput methods used to study transcriptional control such as RNA-seq, we have a limited understanding of the mechanisms that coordinate the rapid translational response of proteins after beta cell stimulation. Our lab has shown that glucolipotoxicity to mimic the metabolic environment of individuals with T2D stimulates a robust increase in the translation but not transcription of the transcription factor JUND. This is dependent upon the activity of the RNA binding protein, hnRNPK, that binds the RNA helicase DDX3X stimulating 18S and preinitiation complex recruitment and translation initiation. Therefore, the goals of this proposal are to determine the in vivo function of hnRNPK in beta cells and to decipher the mechanism by which beta cell hnRNPK stimulates translation in response to metabolic stress. In the first aim, we will utilize our beta cell hnRNPK deletion mouse line to elucidate the metabolic outcomes of hnRNPK deficiency using glucose and insulin tolerance tests to determine glucose homeostasis. We will also perform morphometric analyses to determine any effects on beta cell mass or survival following hnRNPK deletion. In the second aim, we will use co-immunoprecipitation, immunofluorescence, and gene deletion or reduction to understand how hnRNPK and its interactors regulate the translatome in response to metabolic stress. Our preliminary data show that male mice devoid of hnRNPK are glucose intolerant and appear to have reduced beta cell mass suggesting an important role for hnRNPK in beta cells. Furthermore, hnRNPK and DDX3X share 10 common interactors determined by mass spectrometry upregulated in glucolipotoxicity conditions that are closely linked to mRNA metabolism and translation in the cell. Using polysome profiling, we will determine how hnRNPK controls translation under metabolic stress and identify translationally upregulated transcripts by dissociating mRNA from polysome fractions followed by RNA sequencing. Using CRISPR-Cas9 and shRNAs in MIN6 cells and isolated islets we can determine the functional impact hnRNPK or interactor removal has on beta cell translation. Together, these proposed aims will uncover novel mechanisms underlying protein translation in beta cells mediated by the RNA binding protein hnRNPK. This will have important implications in our understanding of beta cell function that could be leveraged to aid the development of better treatments focused on improving beta cell insulin output and survival.

NIH Research Projects · FY 2025 · 2025-07

X-linked myotubular myopathy (XLMTM) is a devastating childhood muscle disease characterized clinically by severe weakness and early death and pathologically by small myofibers that contain disorganized organelles and myonuclei with aberrant appearance and localization. XLMTM is caused by mutations in the MTM1 gene. How MTM1 mutations cause these phenotypes is not well known, and this lack of knowledge presents a key barrier for disease understanding and therapy development. Myotubularin (MTM1) encodes a 3-position phosphoinositide phosphatase that in vitro localizes to the endosome and has role(s) in regulating endosomal vesicular sorting. In exciting new data, we have discovered for the first time subpopulation(s) of MTM1 at the nuclear envelope and within the nucleus. We also reveal that Mtm1 knockout mice have structurally abnormal myonuclei, display an arrest in muscle development and abnormalities in the myogenic transcriptome, and have altered genome organization and a shift in chromatin accessibility. Further, we show that expression of MTM1 exclusively in the nucleus can rescue aspects of the knockout phenotype. Based on these data, and our previous work, we hypothesize that MTM1 regulates nuclear architecture and regional chromatin modifications, and thereby participates as a key modulator of myogenesis. We additionally hypothesize that a critical consequence of MTM1 mutation is to alter genome topography and interrupt the normal progression of the myogenic expression program, resulting in failure of muscle development and disruption of myofiber growth. We will test these novel, conceptually innovative hypotheses using our mouse model of XLMTM in three specific aims. Aim 1 will characterize the localization and interactome of nuclear MTM1, and determine how these change with Mtm1 knockout. Aim 2 will define the alterations in nuclear structure, genome organization, and myogenic transcription associated with MTM1 mutation. Aim 3 will test the impact of nuclear vs non-nuclear MTM1 during muscle development using a series of AAV driven rescue constructs. These Aims utilize state- of-the field experimental approaches, including 3D block face electron microscopy, in vivo miniTurboID, spatial transcriptomics and single nucleus RNAseq, and a novel HiC approach (scHiCAR) for defining genome architecture. Findings from this project will be highly significant as they will advance understanding of the role of MTM1 in normal nuclear function and in the regulation of an understudied area of muscle development (postnatal sequential progression of the myogenic transcriptional program). In addition, this study will improve our knowledge of XLMTM pathogenesis by addressing a key fundamental unknown (i.e. the contribution of the pathognomonic nuclear abnormalities to the disease process) and by providing new knowledge that will lay the groundwork for therapy development. To ensure success of this project and enable its highest impact, we have assembled a collaborative investigator team expert in XLMTM and the key experimental approaches.

NIH Research Projects · FY 2026 · 2025-07

Despite anti-retroviral therapies (ART), HIV-1 continues to cause a considerable medical and economic burden, and there continues to be a pressing need for an HIV-1 cure. The goal of this Program is to generate a chimeric antigen receptor (CAR) approach that can control viral replication below the limit of detection and eliminate the viral reservoir. We recently completed a Phase I clinical trial that infused HIV resistant, HIV specific CAR T cells (CARTs) in people with HIV (PWH) in which 50% of the individuals showed post rebound control of HIV replication. A major goal of this consortium to develop strategies that improve the effector function, trafficking, and persistence of these T cells. The elements of our proposal are: 1) Engineering viral- specific T cells with improved function and persistence (Project 1, John Wherry). This project will use well- characterized models to search for factors or pathways that augment CAR T cell function and persistence to chronic infection. 2) In vivo targeting of T cells to enable an HIV cure (Project 2, Mike Betts and Drew Weissman). This project seeks to develop mRNA-LNP strategy to deliver HIV-specific CARs to effector T cells and supply the trafficking information to move these cells in the lymph tissue where HIV hides. 3) Engineering CAR T cells to provide a durable control of HIV replication (Project 3, Jim Riley). Project 3 seeks build upon the success of HIV CARTs in the clinic by increasing their sensitivity, function, and persistence. 4) Multiplex gene editing in CAR T cells and hematopoietic stem cells to enable an HIV cure (Project 4 Saar Gill and Hans- Peter Kiem). In this project we will implement a genetically engineered cell therapy platform to eradicate the cellular HIV reservoir using the power of chimeric antigen receptor T cells. The CART cells are redirected against a pan-leukocyte antigen, and coupled with an autologous stem cell transplant that is resistant to HIV as well as to the CART cells. Three industry partners (Gilead Sciences, Synthekine, and Acuitas Therapeutics) will provide proprietary reagents and know-how to assist investigators develop these HIV cure strategies and if appropriate help them perform Phase I testing, and eventually commercialize this strategy so it can be available to PWH. The Program is supported by 3 Cores: Core A is the administrative Core (PI, Jim Riley); Core B is the Genome Engineering Core (PIs, Rick Bushman, Junwei Shi, and Rahul Kohli); and NHP Core (PIs Mirko Paiardini, and Hans-Peter Kiem). In addition, our Program takes advantage of existing School of Medicine and CFAR Cores to promote cost sharing and avoid duplication of resources.

NIH Research Projects · FY 2023 · 2025-07

Summary Over 600 human proteins have been recently prioritized as key cancer targets, with nearly half being considered ‘intractable’ by standard small-molecule inhibition approaches, due to target instability and active site accessibility constraints. By redirecting the ubiquitin-proteasomal pathway (UPS) for targeted protein degradation, the proteolysis-targeting chimera (PROTAC) technology provides a potential solution, enabling rapid and continuous target consumption as well as the stronger pharmacological effects than small molecule inhibition. Nonetheless, PROTACs suffer from similar developmental hurdles as small molecules and cannot be easily designed for motif or post-translational modification-specific targeting. To address these hurdles, research efforts have shifted toward gene therapy approaches by introducing the concept of protein-mediated protein degradation. Here, E3 ubiquitin ligases are redirected by replacing their natural substrate binding domains with “off-the-shelf” binding domains, including nanobodies, antibodies, and DARPins, to generate target-specific ubiquibodies. To augment this platform, we recently exploited natural protein-protein interaction information to develop algorithmic pipelines that prioritize target-selective peptides which can be fused to the E3 ubiquitin ligase conjugation domains to induce target protein degradation. In this project, we will augment our current methods to enable the development of these ubiquibodies (uAbs) for any protein, including those deemed ‘intractable’ by small molecule-based means. To do this, we will automate a bipartite algorithmic pipeline that leverages recent advancements in protein language modeling as well as existing co-complex databases to design peptide binders to diverse protein targets, including those with solved co-crystals as well as those with minimal structural information. Specifically, our pipeline will take user-specified target proteins as inputs, and generate prioritized lists of candidate peptide binders as outputs, enabling subsequent generation of uAbs for target degradation. Through library-on-library fluorescence-based assays in human cells and subsequent encapsulation of uAb mRNA in lipid nanoparticles (LNPs), we will develop a scalable method to test and translate our degraders for downstream in vivo validation. In total, this work will generate a robust peptide design tool that will enhance targeted protein degradation efforts and lay the foundation for programmable proteome editing.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY Hepatocellular carcinoma (HCC) is the predominant subtype of liver cancer and arises almost exclusively in the context of chronic inflammation. The healthy liver is a modulator of systemic immune tolerance, recognizing harmless dietary and environmental antigens from the gut and degrading them without inducing inflammation. Dysregulation of this tightly controlled response can lead to chronic inflammation, compensatory hepatocyte regeneration, and eventually fibrosis and hepatocarcinogenesis. While viral hepatitis is the predominant risk factor for HCC, metabolic disorders such as metabolic-associated fatty liver disease (MAFLD) are expected to increase the global incidence of HCC in the coming decades. With treatment options limited due to the mutational heterogeneity of HCC and poor immunotherapeutic responses in patients with MAFLD-associated HCC, there is a clinical need to identify the metabolic vulnerabilities of cancer and immune cells in the tumor micro- environment to exploit specific dependencies of HCC and improve cancer therapy. To this end, we have identified one of the most downregulated metabolic pathways in HCC to be catabolism of the branched chain amino acids (BCAAs). The BCAAs are leucine, isoleucine, and valine, all of which are essential amino acids that contribute to protein synthesis or undergo catabolism to support anaplerotic processes. The majority of BCAA catabolic enzymes are often downregulated in HCC, apart from two enzymes responsible for the first step in the pathway: BCAT1 and BCAT2. Both enzymes catalyze the reversible transamination of BCAAs by transferring the amino group to α-ketoglutarate to generate glutamate and the respective branched chain ketoacids. Loss of BCAA catabolism downstream of the BCATs may represent a unique vulnerability of HCC and confer dependence on BCAT1 or BCAT2 expression for tumor growth. Here, I demonstrate that high BCAT1 expression, but not BCAT2, correlates with worse overall survival in patients according to data from The Cancer Genome Atlas. Moreover, BCAT1 protein expression is high in many well-characterized human cell lines of HCC, and BCAT1 knockdown inhibits growth in a high BCAT1-expressing line, which can be rescued by BCAT1 re-expression. This suggests that tumors with high BCAT1 still utilize the BCAA pathway to support their growth, and this could impact BCAA availability within the tumor microenvironment as a result. Thus, I hypothesize that BCAT1 promotes HCC cell growth through a tumor-intrinsic mechanism, and this suppresses anti-tumor immunity by reducing the amount of BCAAs available to immune cells in the tumor microenvironment. For my first aim, I will determine how BCAT1 knockdown leads to growth inhibition with metabolite supplementation and will confirm the relevance of BCAT1 to HCC growth in vivo. In my second aim, I will investigate whether BCAT1 expression regulates BCAA uptake and how changes in BCAA levels impact immune cell populations in the liver. Collectively, these findings will provide insight into BCAA utilization in the context of BCAT1 and establish a critical crosstalk between tumor and immune cell metabolism in HCC.

NSF Awards · FY 2025 · 2025-07

In the rapidly expanding field of data science, the ability to understand group actions in data analysis is pivotal for a broad spectrum of scientific tasks. In mathematical terms, a “group” is a collection of elements combined with an operation that links any two elements to form a third, adhering to closure, associativity, identity, and invertibility principles. A “group action” involves applying elements of a group to another set’s elements, transforming them in structured ways, such as through rotations or reflections. These transformations are crucial in many data processing applications, including cryo-electron microscopy (cryo-EM), image registration, and multi-reference alignment. Each observation in these problems involves a common, unknown signal and an unknown group element, with the primary goal being to infer both the signal and the group elements accurately. This project aims to significantly advance statistical understanding and develop effective methodologies for handling data influenced by group actions. The wide existence of such data ensures that the progress we make towards our objectives will have a great impact not only on the statistics and machine learning community but also on a much broader scientific community, including fields such as structural biology, computer vision, and signal processing. This project will have educational outcomes that result in curriculum development, teaching, and outreach activities, including activities to K-12 students through the University of Pennsylvania Data Science Academy. The project will advance applications in image recognition and time series alignment, which have broad application in areas like medical imaging. This project is structured around three main aims, each designed to tackle distinct aspects of group actions. First, the PI will improve the accuracy of orbit recovery in scenarios where the prior distributions of group elements are non-uniform, developing computationally efficient procedures that are effective under realistic conditions. Second, the PI will develop theories and methods for group synchronization problems, particularly under high noise levels and in situations with incomplete data, aiming to reduce the error of group recovery and provide entrywise inference. Third, the PI will address theoretical and computational challenges in the multi-reference alignment problem, developing procedures specifically designed for the cyclic structural nature of data, thereby enabling more precise uncertainty quantification. Together, these aims will not only enhance the theoretical understanding of and the ability to analyze group actions but also lead to the development of accurate and computationally efficient algorithms designed to tackle real-world challenges in data analysis where group actions are integral. This research project will have impacts more broadly, in that it will result in software development and in the education of technical experts. These experts will use this software to advance applications in image recognition and time series alignment, which have broad application in areas like medical imaging. These activities will then advance applications in image recognition and time series alignment, which have broad application in areas like medical imaging. 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.