Boston Children'S Hospital

NIH Research Projects · FY 2026 · 2017-08

Project Summary The formation of functional neural circuits is critical for the proper functioning of the brain. To establish the most efficient synaptic circuits, synaptic connections must be refined by neural activity during development. However, the manner and molecules by which synapse refinement is regulated remain to be elucidated. We have established mouse in vivo systems, in which neural activity can be conditionally controlled, and showed that inactive synaptic connections are eliminated during development, but they are eliminated only when there are other active connections with which to compete. This suggests that active connections send a "punishment" signal to inactive ones and instruct them to leave by triggering "elimination" signals within the inactive synapses. Active connections are kept by the presence of "stabilization" signals. By performing various screens, we have identified that the tyrosine kinases Pyk2 and JAK2 serve as "elimination" signals of inactive synaptic connections during development. Pyk2 and JAK2 are turned on at inactive synapses in response to "punishment" signals sent from active synapses to drive inactive synapse elimination. Furthermore, we found that a cell adhesion molecule SIRPa from postsynaptic neurons serves as a "stabilization" signal for active synapses. Together, we propose that neurons calculate the balance between the "elimination" signals (Pyk2/JAK2) and the "stabilization" signals (SIRPa) and determine whether to eliminate or stabilize their synaptic connections. Interestingly, our recent preliminary data suggest that the elimination signals Pyk2 and JAK2 have different roles, with Pyk2 in synapse elimination and JAK2 in axon elimination. Additionally, we found that astrocytic ensheathing of synapses also plays important roles in synapse elimination. To further uncover the molecular mechanisms underlying synapse refinement in vivo, we propose to: Aim 1. Investigate the differential roles of Pyk2 and JAK2 for synapse and axon refinement and how SIRPa regulates their activity. Aim 2: Visualize the elimination signals and their regulation of activity-dependent synapse/axon refinement in vivo. Aim 3: Examine the role(s) of astrocytic sheaths in regulating the elimination signals and synapse refinement. Our project will molecularly delineate how neurons decide to establish functional synaptic connections in the mammalian brain. Many forms of mental illness including autism and schizophrenia are associated with abnormal alterations in synapse refinement. Thus, our studies should also yield novel insights into the etiology and treatment of such disorders.

NIH Research Projects · FY 2026 · 2017-08

The capacity of the immune system to govern repair after tissue damage is fundamental to protect the host against infections. The gastrointestinal tract represents an ideal tissue to explore the mechanisms underlying the exquisite balance between tissue damage and repair. Inflammatory bowel diseases (IBDs) are a group of heterogenous disorders, associated with contributing genetic and environmental factors, that are characterized by inflammatory phases kept in balance by tissue repair. Although the etiology of IBDs is not clear, a hallmark of these inflammatory diseases is a chronic and dysregulated immune response. Despite significant progress in elucidating the mechanisms that govern tissue damage, the mechanistic underpinnings controlling how the immune system affects the restitution phase following IBD flares have not been thoroughly explored. Interferons (IFNs) are key players during an immune response and are increased in IBD. IFNs belong to three families: type I, type II, and type III (also known as IFN-λ) IFNs. Type I IFNs and IFN-λ play potent anti-viral roles, both inducing a similar set of IFN stimulated genes (ISGs) that were believed to play only redundant roles. We recently challenged this paradigm and showed that IFNλ, but not type I IFNs, limits inflammation during murine colitis by dampening the tissue damaging functions of neutrophils. In keeping with a key role of IFN-λ in tuning inflammation in the gut and in maintaining a healthy intestinal environment, we have recently identified two unrelated IBD patients presenting in infancy that have rare-damaging mutations in IFN-λs that lead to defective IFN-λ receptor signaling. This potential novel monogenic disorder links directly for the first-time defects in anti- viral regulation with IBD pathogenesis. Although IFN-λ has anti-viral properties and limits inflammation and tissue damage, the involvement of this group of IFNs during tissue restitution of the gut is more controversial. In keeping with a detrimental role of IFN-λ at mucosal surfaces, we have recently demonstrated that IFNλ delays proliferation and favors apoptosis of lung epithelial cells in mouse models of persistent viral infections and in critically ill COVID-19 patients. By investigating the activity of IFNλ in multiple mouse models of intestinal damage, we now revealed a new molecular cascade initiated by IFNλ in intestinal epithelial cells that culminates in a form of cell death called pyroptosis. Our data demonstrate that IFNλ delays gut restitution. We hypothesize that along the intestinal tract, IFNλ can play opposing roles during IBD. Although IFNλ dampens tissue damage, it can delay tissue restitution because of its compartmentalized action on either neutrophils or IECs. Employing murine and human model systems and human tissue, our goal is to decipher the mechanistic basis for both the pathogenic and protective roles of IFNλ and to identify new therapeutic targets. To reach our goals we will address: i) What drives the production of IFNλ, and which cells produce, or respond to, IFNλ; ii) What are the components of the signaling cascade that controls pyroptosis induction in IECs; iii) How the production of IFNλ is regulated during early childhood and how it impacts IBD development in patients.

NIH Research Projects · FY 2025 · 2017-07

Project Summary/Abstract This application, “Translational Postdoctoral Training in Neurodevelopment (TPND)” is a competing renewal to request five additional years of funding in support of our Institutional Training Grant (T32). The TPND Program at Boston Children's Hospital is designed to provide promising postdoctoral investigators (MD, PhD or MD/PhD) with advanced training in essential translational topics ranging from preclinical considerations through implementation of clinical trials for individuals with a range of neurodevelopmental disorders. Large numbers of children, adolescents, and adults are affected by neurodevelopmental disorders that begin early in life, and are rooted in aberrant brain circuitry. Most currently available treatments have had limited impact on the course of neurodevelopmental disorders. Recent advances in genetics, neuroscience, and neuroimaging have led to dramatic gains in our understanding of the pathogenesis of neurodevelopmental disorders. We now have the possibility of developing mechanism-based treatments for many disorders in this field, and thus there is an urgent need for training young investigators who will become the leaders in initiating and executing research at the interface of these advances and ultimately improving the lives of individuals affected with these disorders. TPND leverages the significant strengths of the Translational Neuroscience Center (TNC), the Fuss Center for Neuropsychiatric Disease Research, and the Laboratories of Cognitive Neuroscience at Boston Children's Hospital and Harvard Medical School to provide trainees with research experiences ranging from pre-clinical and cognitive neuroscience labs through clinical trial involvement in neurodevelopmental disorders. A key premise of this program is that effective research training in the field of translational neuroscience requires both mentors with expertise in areas across the continuum of translational research with neurodevelopmental disorders and ongoing programs to support the trainees to conduct innovative, high impact translational research. The 17 faculty mentors will involve trainees over a 2-year period (3 new entrants per year) with a range of state of the art methods in translational neurodevelopmental science that reflect core areas of the TNC program including basic science and translational methods as well as application to clinical populations in therapeutic trials for neurodevelopmental disorders. This research experience will be supplemented with both didactic and clinical immersion experiences designed to provide trainees with the skills needed to be successful independent investigators in this critical and emerging field. Ultimately this research experience will have the potential to yield new treatments and insight that in turn will impact the field by reducing the burdens and costs of care and costs to society that is now associated with neurodevelopmental disorders. In addition, the TPND will develop models of interdisciplinary research training for promising young scientists that will have a transformative impact on the field.

NIH Research Projects · FY 2025 · 2017-05

The pathophysiology of schizophrenia has been unknown, resulting in a lack of innovative therapeutics with novel mechanisms of action. The Conte Center for Neuroimmune Studies was created to build on our discovery that the biology underlying the most significant schizophrenia common genetic risk variants involves neuroimmune mechanisms of synaptic pruning. We have found that risk variants of the complement component C4 genes are correlated with increased C4A expression in the brain and CSF, and that overexpression of human C4A in a mouse model results in excess synaptic pruning and social behavioral deficits. We have further shown that additional neuroimmune molecules encoded at schizophrenia risk loci, CD47 and CSMD1, influence synaptic pruning and complement activity. Lastly, schizophrenia is unusual among neurodevelopmental disorders in its late-adolescent/early-adult onset, and childhood adversity is a major non-genetic risk factor for schizophrenia; yet the biological underpinnings of adolescent psychiatric vulnerability are wholly unknown. We have identified a critical period of circuit refinement in the mouse frontal cortex that we propose can be exploited to better understand the unique vulnerability of adolescent brain development to psychiatric risk factors. The goals of the Center in our next five years are to both deepen the focus on C4-mediated synaptic pruning as a pathophysiological mechanism, and expand the scope of our investigations to create a fuller picture of neuroimmune pathways, their upstream regulators, their cellular effectors, and their downstream circuit-level and behavioral impacts. Project 1 will investigate the role of astrocytes, the main source of C4 in the brain, by manipulating schizophrenia risk genes in these cells and assaying the impacts on synapse formation and function. Project 2 will spatially map the brain’s transcriptional response to C4 overexpression, and test the therapeutic hypothesis that inhibition of C4 activity may rescue over-pruning and associated behavioral phenotypes. Project 3 will examine the circuit specificity of the adolescent critical period, the impacts of gene-by-environment risk factor interactions, and the roles of the brain borders. Project 4 will explore circuit-level interactions between the basal ganglia and frontal cortex in the context of adolescent development, psychiatric risk factors, and risk/reward decision-making. We will also lay the groundwork for expanding neuroimmune studies of psychiatric risk to a non-human primate model, the marmoset. Finally, our Administrative Core will facilitate interdisciplinary collaboration that exploits the full range of expertise across the four labs, and coordinate outward- facing activities including the annual research symposium.

NIH Research Projects · FY 2026 · 2017-05

Project Summary Single-molecule measurements of DNA and RNA, and their interactions and processing have revealed how these molecules function and how their activities are regulated. Our previous mechanistic studies of DNA unwinding enzymes and CRISPR enzymes have motivated us to develop novel biotechnological tools applicable in vitro and in living cells. We have also adopted next generation sequencing to develop high throughput biophysical assays for measuring DNA mechanics and chromatin remodeling activities in a massively parallel scale. We propose to pursue three broad areas of CRISPR-Cas9, chromatin remodeling, and DNA mismatch and its repair. A permeating theme is to explore the rich sequence space that is often poorly sampled in mechanistic studies. For effective genome editing using CRISPR-Cas9, Cas9 ribonucleoprotein complexes (RNPs) need to be assembled in high concentrations, requiring accurate co-transcriptional folding of the guide RNA and its stabilization by Cas9 binding. In addition, after target DNA cleavage, a Cas9-RNP needs to dissociate rapidly for the cell to detect the lesion and mount a DNA damage response. Understanding how these two critical steps are modulated by the target sequence will help researchers optimize target site selection and guide RNA design. Chromatin remodelers are ATPases that changes the position or composition of nucleosomes through nucleosome sliding or histone exchange, respectively. Many pioneering studies, including those using single- molecule methods, have revealed the fine details of the sliding reaction, but our knowledge of the histone exchange reaction is much more limited. We propose to develop single-molecule assays to fully define the kinetics and pathways of SWR1-catalyzed exchange of H2A-H2B dimer for H2A.Z-H2B dimer. We will also discover and characterize yeast native genomic sequences that allow nucleosome formation at a sharply defined location, which we hope will replace the artificial nucleosome positioning sequence, `601', that has been used in almost all mechanistic studies involving nucleosomes. DNA mismatch repair is carried out by conserved protein machinery but despite the decades of research, we have a very limited understanding of how different mismatches under different sequence contexts are repaired. For example, most mechanistic studies have been performed on a single type of mismatch (GT mismatch). We will develop a high throughput method to measure the in vivo mismatch repair efficiencies of thousands of different mismatches. If unrepaired until histone deposition on a nascent DNA, a mismatch will become part of a nucleosome. We will study the biophysical properties of a mismatch-containing nucleosome and its interplay with chromatin remodelers, allowing us to examine how an unrepaired mismatch can influence DNA accessibility.

NIH Research Projects · FY 2026 · 2016-11

ABSTRACT The high prevalence of food allergy (FA) places many at risk of severe reactions to foods including anaphylaxis (1). Our studies on human subjects with FA and in relevant mouse models have identified changes in regulatory T (Treg) cell populations as playing a pivotal role in disease pathogenesis. Specifically, we have identified a critical role of RORgt+ Treg cells, induced by the gut commensal bacteria, in mediating oral tolerance to food allergens (2, 3). In contrast, FA is associated with defective microbiota-dependent differentiation of RORgt+ Treg cells due to dysbiosis. We identified Resistin like molecule beta (RELMβ), a gut goblet cell cytokine previously linked to the innate immune response to parasitic infections (4-6), as pivotal to FA pathogenesis by promoting dysbiosis and disrupting RORgt+ Treg cell differentiation. Reciprocally, there emerges in FA food allergen-specific Treg cells with a T helper cell type (Th2) cell like phenotype, with high expression of the transcription factor GATA3 and the Th2 cytokines IL-4 (7, 8). These reprogrammed Treg cells play an essential role in amplifying disease pathology, and they decline in patients receiving oral immunotherapy (7, 8). Our recent analysis of circulating Treg cells of human subjects with FA identified Thymic stromal lymphopoietin receptor (TSLPR) as a marker of their Th2 cell-like reprogramming. Accordingly, the focus of this proposal is to identify immune regulatory checkpoints that govern FA and means of resetting them to promote oral tolerance. Our overall hypothesis is that the evolution of FA entails two critical checkpoints each involving a distinct Treg cell population under control of dedicated innate cell and cytokine circuits. The first involves microbiota-dependent Helios– RORgt+ Treg cells generated at the peri-weaning period and thereafter which enforced oral tolerance. This circuit is negatively controlled by RELMb, itself induced by an upstream Tuft cell (IL-25)-ILC2(IL-13) axis (9-11), which predisposes to FA by promoting dysbiosis. The second involves Helios+TSLPR+ Th2 cell-like Treg cells that are positively regulated by IgE/Mast cells and which augment the FA responses. To explore this hypothesis, we will under Aim 1 examine the evolution of the pro-tolerogenic immune response in FA Il4raF709 mice following therapy with anti-RELMb mAb or the acute deletion of the RELMb gene Retnlb. We will also elucidate the mechanisms by which RELMb antagonism resets the gut microbiota to enforce oral tolerance, including the expansion of Lactobacilli species rich in indole metabolites that act via the aryl hydrocarbon receptor (AhR) to suppress FA by inducing RORgt+ Treg cells. Under Aim 2, we will dissect the role of TSLPR on Th2 cell-like reprogrammed Treg cell in disease pathogenesis. These findings will then be extending to studies under Aim 3 on FA subjects to relate changes in serum RELMb and Treg cell populations to dysbiosis and disease severity. The proposed studies will provide fundamental new insights relevant to disease pathogenesis and novel therapeutic approaches to re-establish oral tolerance in FA.

NIH Research Projects · FY 2026 · 2016-09

Abstract From guiding leukocyte adhesion during the immune response to influencing cell fate and tissue formation, mechanical forces play critical roles in regulating biological function. Despite growing recognition— underscored by milestones such as the Nobel Prize for mechanosensitive channel discoveries—mechanical signals remain comparatively understudied, largely because nanoscale forces are inherently difficult to measure. Indeed, precisely because forces are difficult to measure, they remain easy to overlook. Fundamental questions remain regarding how mechanical forces alter molecular structures, modulate biochemical interactions, and initiate mechanotransduction pathways that underpin diseases ranging from bleeding disorders and thrombosis, to cancer and hearing loss. Addressing these fundamental mechanobiological questions requires versatile, accessible tools capable of probing biomolecular systems under precisely controlled mechanical conditions. Single-molecule methods uniquely enable the study of biomolecules under physiological forces, out-of-equilibrium, and free from ensemble averaging, revealing the dynamics of otherwise hidden intermediate transitions. However, widespread adoption of these methods has historically been constrained by limitations in throughput and experimental capabilities, cost and technical complexity. My laboratory directly addresses these challenges by developing innovative yet accessible single-molecule technologies, including the Centrifuge Force Microscope (CFM)—a powerful, accessible, high-throughput instrument that integrates into a benchtop centrifuge—and programmable DNA nanoswitches that report biomolecular interactions with exquisite sensitivity and precision. Applying advanced single-molecule approaches, we have uncovered key molecular mechanisms such as tension-dependent activation and self-association of von Willebrand Factor (VWF), a critical regulator of hemostasis. Through innovative stroboscopic single-molecule imaging, we resolved longstanding controversies regarding shear-induced free VWF activation, suggesting that mechanical activation requires surface tethering rather than shear alone. Our development of DNA nanoswitch calipers has further advanced single-molecule proteomics, enabling fingerprinting and three-dimensional shape determination of proteins at angstrom-level precision, significantly enhancing our ability to characterize complex biomolecular structures and post- translational modifications. We will continue to democratize single-molecule and nanoscale techniques, making these transformative capabilities broadly accessible to biomedical researchers. Furthermore, we will continue addressing fundamental biological questions and critical translational challenges, further establishing mechanical force as an essential parameter for understanding biological processes. Ultimately, our efforts aim to illuminate the mechanobiological underpinnings of disease, and pave the way for new diagnostic tools, targeted therapies, and improvements in patient care.

NIH Research Projects · FY 2026 · 2016-07

Project Summary Gene therapy using autologous CD34+ cells is a promising treatment for primary immunodeficiency, particularly for individuals without optimal allogeneic donors. SCID-X1 is caused by mutations in IL2 Receptor Gamma (IL2RG) which encodes the common gamma chain (γc) of multiple cytokine receptors. Boys with SCID-X1 lack T and NK cells and their B cells fail to produce antibodies due to the lack of functional IL-7, IL-15 and IL-21 signaling. This renewal application seeks to complete our currently funded Phase I/II clinical trial that addresses two major shortcomings of previous GT using gamma retrovirus (γRV) and self-inactivating (SIN)-γRV vectors, namely lack of B cell reconstitution and insertional mutagenesis. We hypothesize that this trial will improve immune reconstitution through the introduction of low dose busulfan conditioning (Aim 1) and improve safety through the change from a gammaretroviral (γRV) vector used in previous trials to the LV vector in this trial (Aim 2). Previous trials of gene therapy for SCID-X1 have infused cells without chemotherapy conditioning, which resulted in robust T cell recovery and gene marking, but negligible gene marking in B cells and failure of humoral immune reconstitution. Initial development and marking in NK cells was also not sustained. In Aim 1, we will examine the impact of low dose busulfan conditioning on 1) cell type specific engraftment and gene marking, 2) in vivo T cell reconstitution, T cell phenotype and TRB repertoire by deep sequencing, 3) in vivo humoral immune reconstitution, B cell number, phenotype, IL-21 dependent function and IGH repertoire by deep sequencing, 4) NK cell number, phenotype and function. Previous trials of gene therapy for SCID-X1 have used a γRV vector with intact viral promoters/enhancers, which resulted in 6/20 patients developing T cell leukemia due to insertional oncogenesis. Gene therapy using an enhancer deleted, self-inactivating yRV (SIN-γRV) vector funded by NIAID in which viral enhancers have been deleted shows encouraging evidence of reduced insertion sites near lymphoid oncogenes and safety, but the insertion site pattern characteristic of γRV may still be risky. The proposed trial in this application will seek to further improve safety by using a self- inactivating LV vector. In Aim 2 we will investigate the insertion site pattern in the patients’ engrafted cells, compare samples from this current trial to previous trials using γRV and SIN-γRV to delineate clustering of insertion sites in specific genes and the effect of vector backbones on clonal dynamics and expansions.

NIH Research Projects · FY 2025 · 2016-07

PROJECT SUMMARY/ABSTRACT The lymphatic vascular system controls tissue fluid homeostasis and intestinal lipid uptake. Proper lymphatic function positively correlates with favorable outcomes for patients with cardiovascular and metabolic disorders, which accentuates the importance of this system in maintaining systemic homeostasis. Our long-term goal is to uncover molecular mechanisms and critical regulators that govern lymphatic function in health and disease, with the hope of offering new therapeutic targets to combat cardiovascular and metabolic diseases. In the previous funding period, we discovered that the Forkhead Box C2 (Foxc2) transcription factor antagonizes vascular endothelial growth factor receptor 3 (VEGFR3) signaling by inducing the expression of epsins; endocytic adaptor proteins critical for VEGFR3 degradation and vascular endothelial growth factor C (VEGF-C) signal attenuation in lymphatic endothelial cells (LECs). We also discovered that the Forkhead Box C2 transcription factor (Foxc2) was an important regulator of obesity and that restoration of lymphatic function was a potential strategy to treat metabolic diseases. As a result, in this renewal application, we sought to identify and study additional regulatory molecules of lymphatic function. We determined that the micro-ribonucleic acid miR-22 regulates lymphatic function in normal and diseased conditions. Despite its prominence in governing lymphatic pathophysiology, little is known about the role that miR-22 plays in regulating the function of this vascular system. Consequently, we generated novel, inducible lymphatic endothelial cell (LEC)-specific miR-22 loss-of-function mice and discovered that the deficiency of this molecule dramatically increased developmental lymphangiogenesis and increased the expression of the master regulator of lymphatic differentiation and fate determination Prox1 as well as fortifying VEGF-C/VEGFR3 signaling and increasing the expression of metabolic regulatory genes. Therefore, our central hypothesis is that lymphatic miR-22 represses Prox1, constrains VEGFR3 signaling, and stymies energy production by suppressing metabolic programming. Conversely, loss of lymphatic miR-22 elevates Prox1 expression, VEGFR-3 signaling, and metabolic bioenergetics; thereby, mending impaired lymphangiogenesis and lymphatic function in cardiovascular and metabolic disorders. To test our hypothesis and determine how miR-22 inhibition exerts a pro-lymphangiogenic stimulus to ameliorate cardiovascular and metabolic disease, we propose the following related, but independent, Specific Aims: 1) to determine the role of miR-22 in governing metabolic programming and VEGFR3 signaling, 2) to determine molecular mechanisms by which miR-22 governs lymphatic function in the adult, and 3) to determine the therapeutic potential of targeting miR-22 and epsins in lymphatic systems. Our findings will identify novel molecular mechanisms underlying metabolic regulation and signaling to drive reparative and regenerative lymphangiogenesis. We anticipate that therapies targeting miR-22 or epsins may be valuable for restoring the injured lymphatic vasculature to treat cardiovascular and metabolic diseases.

NIH Research Projects · FY 2025 · 2016-07

Abstract Nucleotide-binding domain (NBD)-like and leucine-rich repeat (LRR)-containing proteins (NLRs) perform diverse functions in cellular biology, mediating a broad set of fundamental biological pathways from immune signaling to embryonic development. In immunity, several NLRs form supramolecular protein signaling complexes called inflammasomes. Inflammasomes activate caspase-1, an inflammatory protease that processes the cytokine interleukin-1β (IL-1β) and the pore-forming protein gasdermin D to potentiate pyroptotic cell death. One such inflammasome- forming protein, NLRP1, is directly activated in response to intracellular pathogens and the inhibition of DPP9, an endogenous peptidase, serving as both a pattern recognition receptor and a signaling complex. While most NLRs share a common domain architecture, the multifunctionality and regulation of NLRP1 requires additional structural components. In addition to the characteristic NBD and LRR domains, human NLRP1 contains an N- terminal pyrin domain (PYD) and a rare function-to-find domain (FIIND) followed by a caspase activation and recruitment domain (CARD) on its C-terminus. The only other protein with a FIIND is CARD8, which has also been shown to form inflammasomes, leading to cytokine secretion and cell death. Mechanistically, the NLRP1 and CARD8 FIIND domains constitutively catalyze autoproteolytic cleavage, leading to the formation of two noncovalently associated peptides: an autoinhibitory N-terminal fragment and an inflammatory C-terminal fragment. Additionally, Dipeptidyl peptidase 8 or 9 (DPP8/9) binds and inhibits NLRP1 and CARD8, and small molecule inhibitors of DPP8/9 induces NLRP1 and CARD8 activation through some poorly understood pathway. In this application, we propose to elucidate the activation and regulation of NLRP1 and CARD8 using a structure-guided approach. We will determine structures of NLRP1 or CARD8 in complex with DPP8 or DPP9, both WT and mutants. We will analyze the structures and perform additional biochemical and cellular biological experiments to test our hypotheses. In one central aim, we propose that NLRP1 and CARD8 are stress sensors for endogenous cellular dysregulation and play important roles in unwanted inflammation and diseases.

NIH Research Projects · FY 2025 · 2016-06

ABSTRACT- limited to 30 lines The corneal epithelium is a rapidly self-renewing epithelial surface essential for maintaining a clear cornea and normal vision. The limbus contains a small subpopulation of limbal stem cells (LSCs) that continually repopulate the corneal epithelium and patients with limbal stem cell deficiency (LSCD) are unable to regenerate the corneal epithelium, resulting in "conjunctivalization" that ultimately leads to blindness. We previously identified that the ABCB5 gene is expressed by LSCs in both mouse and human tissues and that normal function of ABCB5+ LSCs is required for corneal development, homeostasis, and repair. Moreover, ABCB5 is a cell surface protein and specific monoclonal antibodies are capable of isolating pure ABCB5+ LSCs for study and transplantation. Research from our prior grants supported the first clinical trials using purified in vitro-expanded human allogeneic ABCB5+ LSCs for patients with bilateral LSCD (ClinicalTrials.gov: Identifier NCT03549299). Despite our success, the use of donor allogeneic ABCB5+ LSC transplants is suboptimal because it requires immunosuppressive treatment to prevent immune rejection of donor cells. Allogeneic donor ABCB5+ LSCs were used because LSCD patients lack a source of healthy autologous ABCB5+ LSCs since both eyes are involved. To solve this problem and to treat bilateral LSCD patients with autologous ABCB5+ LSCs, this grant renewal will study three novel approaches. The first approach uses transplantation of ABCB5+ iLSCs (induced LSCs derived from iPSCs generated from skin) using full reprogramming of autologous skin cells, using the Yamanaka genes OSKM (Oct4, Sox2, Klf4, c-Myc) to produce human induced pluripotent stem cells (iPSCs) that are then differentiated in vitro into 2D eye-like organoids that remarkably contain ABCB5+ LSCs (Watanabe et al., iScience, 2021). The second approach uses transplantation-independent restoration and expansion of residual dysfunctional or quantity-deficient LSCs, using in vivo partial epigenetic reprogramming, a new form of reprogramming discovered by Co-PI Dr. Bruce Ksander and collaborators (Lu et al., Nature, 2020), which uses transient expression of three Yamanaka genes, OSK, to restore and rescue injured dysfunctional cells in vivo. The third approach uses niche-produced LAMA5 ligand-based signaling activation of the BCAM molecular pathway that we just discovered is critical to ABCB5+ LSC and Transient Amplifying Cell (TAC) expansion (Sasamoto et al. Cell Reports, 2022, in press), with resultant stem cell proliferation programs that enhance corneal regeneration. Thus, our hypothesis is that it is possible to manufacture and expand autologous ABCB5+ LSCs from patients with bilateral LSCD using (i) full reprogramming in vitro, (ii) partial epigenetic reprogramming in vivo, and (iii) niche-produced LAMA5 ligand-based activation of LSCs through specific BCAM/ABCB5 signal transduction pathways. These autologous ABCB5+ LSC will fully regenerate and maintain a normal limbus and corneal epithelium. This hypothesis will be tested in three independent specific aims that are thematically related.

NIH Research Projects · FY 2025 · 2016-05

Project Summary The first critical step for enveloped viruses, such as HIV-1, to enter host cells is viral membrane fusion. Viral fusion proteins are fascinating protein folding machineries capable of adopting completely different conformations during the fusion process; they are also important vaccine and therapeutic targets. Previous studies have revealed both pre- and post-fusion conformations of the soluble fragments of many viral fusion proteins, but less is known for structures of their fusion peptide, transmembrane and membrane-proximal regions in the context of lipid bilayers. There is strong evidence for functional roles of the membrane- interacting regions in fusion, and yet mechanistic studies on how they exert their functions remain scarce. We hypothesize that membrane-interacting regions of other fusion proteins related to HIV-1 envelope protein (Env) adopt defined oligomeric structures that are critical for the stability, function and antigenicity of the full-length proteins in membrane. In the studies that we completed during the previous funding period, we have determined the structures of the TM, membrane proximal external region, and cytoplasmic tail of HIV-1 Env in bicelles that mimic lipid bilayers using the latest NMR technology. We find that these regions all form well-ordered trimeric clusters and are conformationally coupled, and that disrupting them can reduce fusion and alter the antigenic structure of the entire Env. In this application, we propose to apply our NMR/bicelle technology to investigate the membrane regions of SIV Env and the recently emerged SARS- CoV-2 spike (S), and to use cryo-electron microscopy to determine structures of the full-length proteins reconstituted in lipid nanodiscs. We will define roles in membrane fusion of critical structural elements of these regions by deep mutagenesis and functional assays. We will purse the following specific aims: 1) we will investigate the membrane-interacting components of SIV Env; 2) we will investigate the membrane-interacting components in the postfusion arrangement; 3) we will determine structures of the full-length SIV Env and SARS-CoV-2 S in the context of membrane; 4) we will elucidate roles of the membrane-interacting domains of HIV/SIV Env and SARS-CoV-2 S in their stability, function and antigenicity.

NIH Research Projects · FY 2024 · 2016-03

Project Summary Our ability to visually interpret the world around us depends on rapid bottom-up computations that extract relevant information from the sensory inputs, but it also depends on our accumulated core knowledge about the world providing top-down signals based on prior experience. The goal of this proposal is to study the mechanisms by which visual information is integrated spatially and temporally to combine bottom-up and top- down knowledge. Towards this goal, we combine behavioral measurements, invasive neurophysiological recordings, invasive electrical stimulation, and computational models. We focus on the ubiquitous challenge of visual search, exemplified by searching for your phone using exclusively visual cues. The behavioral data will provide critical constraints about human integrative abilities, particularly through eye movements and the dynamics of recognition and object location. The invasive neurophysiological data will provide high spatiotemporal resolution of neural activity along the inferior temporal cortex and the interactions with the pre- frontal cortex, which are hypothesized to be critical for conveying the type of top-down signals required for recognition and attention modulation during visual search. Ultimately, a central goal of our proposal is to formalize our understanding of these integrative processes via a quantitative computational model. This computational model should be able to capture the behavioral and physiological results and provide testable predictions. During the current award, we have made progress towards elucidating the mechanisms underlying pattern completion used by the visual system to infer the identity of objects from partial information, the effects of contextual information during object recognition, and computational models of visual search. We have strong preliminary evidence that suggests that state-of-the-art purely bottom-up theories of recognition instantiated by deep convolutional networks cannot explain human behavior and physiology. Therefore, the proposed work aims to establish a strong computational, behavioral and physiological framework that merges bottom-up and top-down processing. Furthermore, we will move beyond correlative measures by using electrical stimulation to stress test the models and establish causal links between key nodes in the circuitry and visual search behavior. Understanding the neural mechanisms by which core knowledge is incorporated into sensory processing is arguably one of the greatest challenges in Cognitive Science and may have important implications for many neurological and psychiatric conditions that are characterized by dysfunctional top-down signaling and remain poorly understood.

NIH Research Projects · FY 2025 · 2016-03

PROJECT SUMMARY The dialogue between innate and adaptive branches of the immune system is a central paradigm of modern immunology and is vital for protection against infections as well as for the pathogenesis of autoimmune, allergic and inflammatory diseases. According to the current model, innate immune sentinels dispersed throughout peripheral tissues sense, via their pattern recognition receptors (PRRs), the presence of microbial clues or endogenous moieties released during an infection, are activated and migrate to the draining lymph node (dLN). This process enables a transfer of “information” from peripheral tissue to the dLN, where the antigen-dependent adaptive immune response against the pathogen is initiated. The dLN also hosts an initial antigen-independent, innate immune response governed by migrating phagocytes that enables expansion of the LN and establishes a pro-inflammatory milieu. These events are required for the development and polarization of the adaptive immune response. Here, we focused our attention on the capacity of ligands derived from the cell wall of Candida (C.) albicans to dictate the LN innate response. Our working hypothesis is that the size and solubility of stimuli that activate the PRRs affect not only the LN innate response itself, but also the final outcome of the immune response. Also, that the LN innate response initiated by soluble fungal ligands can be harnessed to develop a potent and protective adaptive immune response to prevent life-threatening systemic fungal infections. Our preliminary data demonstrate that the physical form of fungal ligands dictates the location where the initial immune response takes place, and thereby determines the activation of adaptive immunity. In particular, we have found that small soluble fungal ligands that are immunosilent in the periphery and do not cause an inflammation in the tissue, become potent immunogens once they reach the dLN. Also, that the LN innate response initiated by these ligands completely bypasses the need of phagocyte migration from the periphery into the dLN and, instead, requires a unique gene signature that is characterized by the production of interferons and that is driven by the activation of the noncanonical NFkB transcription factor RelB in subcapsular sinus macrophages. Notably, Dectins are required for this process but CARD9, the key signaling adaptor downstream of Dectins, is largely dispensable. Plus, the initial innate response to the dLN instructs a potent type 1 adaptive immunity and allows the production of antibodies directed against the most external layer of the fungal cell wall. Fungal diseases are a global health problem and Candida species are the most common cause of invasive fungal infections. We propose to unravel how physical properties of the PAMPs can be harnessed as a therapeutic intervention against systemic fungal infections that are a major medical problem in the US. We anticipate that identifying new features of the immune response that is anatomically restricted to the LN will help with the design of an improved vaccine against poorly controlled pathogens.

NIH Research Projects · FY 2025 · 2016-03

Nephrotic syndrome (NS) is a rare form of chronic kidney disease resulting from glomerular filtration barrier failure and massive proteinuria. To achieve increasingly effective care for NS, a more precise understanding of its underlying molecular mechanisms is necessary. Human genetics studies in NS using family- and population- based methods have proven effective in doing so. In the US, chronic and end stage kidney disease (CKD/ESKD) disproportionally affects African Americans. Social determinants certainly contribute to these health disparities. However, it is now clear that a major contributor to the large excess risk of kidney disease for Black people in the US is a genetic factor, namely common variants in apolipoprotein L1 (APOL1). One in eight Black Americans (13%) carry two APOL1 risk variants, which constitutes a high-risk (HR) genotype. The APOL1 HR genotype is associated with a 15-20x increased odds of focal segmental glomerulosclerosis (FSGS). The combination of the HR genotype's high frequency in the Black population and its outsized effect size for FSGS results means that African Americans with a HR genotype have a 5-10% lifetime risk of FSGS and that ~70% of all Black patients with FSGS have APOL1-mediated FSGS (AMFSGS). Based on our own work and that of our colleagues globally in the fields of both APOL1 kidney diseases specifically and Precision Medicine of diverse human diseases more broadly, our overarching conceptual model is that one day we will be able to treat, cure, or even prevent the majority of AMFSGS cases by using genomics and multiomics to gain a deep understanding of the molecular anatomy of this disease. To do this we must discover the full set of (1) genetic and environmental modifiers that increase the penetrance of FSGS among people (of any race or ethnicity) with a HR genotype, and (2) mechanistic pathways that, upon onset of AMFSGS, cause initial kidney cell injury and progressive glomerular injury, failure, and tubulointerstitial inflammation and fibrosis. In Aim 1, we will discover genetic modifiers of AMFSGS via genome-wide association studies (GWAS). In Aim 2, we will integrate GWAS data, multi-tissue bulk and single-cell omics datasets, and functional assays to functionally characterize the pathobiology of genetically-driven modifiers of AMFSGS. In Aim 3, we will discover the transcriptome-wide consequences of a HR genotype and APOL1's cis-regulation at cell-specific resolution using kidney snRNA-seq and snRNA-seq + ATAC-seq datasets and functional assays. This proposal will result in the discovery of multiple genetic modifiers of AMFSGS and specific genes, pathways, and cell types underlying its pathology. By discovering multiple molecular drivers of this condition, we will help pave the way for subsequent development of targeted therapies that can be used alone or in combination to treat, or even cure, people who already have AMFSGS. Beyond this, for healthy children and adults in our community with a HR genotype, discovering genetic factors that increase the penetrance of AMFSGS would allow efforts to be undertaken to devise pharmacologic or lifestyle strategies to prevent disease from ever occurring in community members at a greater risk of developing it.

NIH Research Projects · FY 2024 · 2016-01

Project Summary Our program vision is to unravel the information buried in health-related narratives by advancing text-processing methods in a unified way across all the genres of health texts and distributing them through an advanced NLP software platform under solid governance and sustainability. The crosscutting theme is the investigation of methods for health NLP made possible by big data, fused with health knowledge. The underlying theme of this renewal is the development of methods towards generalizable, efficient and knowledge-rich models in the context of modern machine learning techniques, particularly models implementing attention mechanisms and using large unlabeled datasets. There is growing penetration of deep learning approaches in the field of health natural language processing. Our proposal aims to address critical methodological gaps and understudied areas in the current unprecedented fast-paced environment. Therefore, our renewal lays out novel and much needed explorations of health NLP research which we will advance through our specific aims. Our datasets will continue to span the spectrum of health-related data – Electronic Medical Records clinical narrative, patient-authored on- line community posts, and health-related social media. The evaluation of the methods we will develop will be performed on the key clinical tasks of concept extraction, relation extraction, and phenotyping with comparisons to other traditional or deep learning algorithms as baselines. We will demonstrate impact of our methods and tools through several use cases, ranging from clinical point of care to public health, to translational and precision medicine. Finally, we will disseminate our work through community activities to advance the state of the art in health natural language processing.

NIH Research Projects · FY 2026 · 2015-12

ABSTRACT Telomerase plays a vital role in human health and longevity, yet its regulation in human cells remains poorly understood. This gap in knowledge limits the ability to develop therapies for a growing spectrum of telomere biology disorders (TBDs) linked to telomerase dysfunction. The long-term goal of this project is to enable therapeutic manipulation of telomerase in human cells, with a primary focus on the hematopoietic system. Hypomorphic mutations affecting telomerase reverse transcriptase (TERT) and the telomerase RNA component (TERC) are known to cause a range of degenerative disorders, including dyskeratosis congenita, bone marrow failure (BMF), myelodysplastic syndrome (MDS) / leukemia, cardiovascular disease, pulmonary fibrosis, and liver cirrhosis. Recent genetic studies highlight non-coding RNA turnover and nucleotide metabolism as key regulators of TERC and TERT, respectively, suggesting new therapeutic avenues. What remains lacking is a detailed understanding of how specific genetic variants associated with TBDs disrupt telomerase function and which strategies for modulating telomerase would be most effective. The overall objectives of this proposal are to: (1) define mechanisms by which genetic variants impact TERC RNA metabolism, (2) understand how thymidine nucleotide metabolism regulates telomerase in hematopoietic stem and progenitor cells (HSPCs), and (3) determine how telomerase genetic variants respond to interventions targeting RNA and nucleotide metabolism. The central hypothesis is that specific TBD-associated mutations respond differently to metabolic manipulation of TERC and TERT. This work is grounded in the rationale that understanding the molecular mechanisms behind telomerase deficiency and therapeutic response will inform precision treatment approaches for BMF and other degenerative diseases. The central hypothesis will be tested by pursuing three Specific Aims: (1) Identify the mechanisms by which TERC variants impair telomerase activity, (2) Define how nucleotide metabolism affects telomere biology in hematopoiesis, (3) Align targeted metabolic strategies with specific telomerase defects. Aim 1 will involve a comprehensive functional analysis of TERC variants in human cells and HSPCs. In Aim 2, thymidine metabolism and nucleotide salvage and turnover will be manipulated in HSPCs to understand impacts on stem cell function in vivo. Aim 3 will evalute the efficacy of RNA and nucleotide metabolism modulating interventions in the context of distinct telomerase genetic variants. The approach is innovative because it exploits robust human genetic insights, along with novel small molecules, enzyme targets, and RNA-based tools to elucidate molecular disease mechanisms and nominate therapeutic candidates. The proposed research is significant, because it is expected to yield tailored strategies for telomerase modulation in a range of disorders such as BMF, MDS / leukemia, and pulmonary and hepatic diseases, where telomerase dysfunction plays a critical role and curative treatments are lacking.

NIH Research Projects · FY 2025 · 2015-07

ABSTRACT A specific subset of T cell lymphoma (TCL) called Anaplastic Large Cell Lymphoma (ALCL) frequently harbors chromosomal translocations involving the Anaplastic Lymphoma Kinase (ALK) gene. Chemotherapy is the current standard of care for ALK+ ALCL, but fails in approximately 30% of patients. Most ALK+ ALCL that fail chemotherapy respond well to the ALK tyrosine kinase inhibitors (TKIs), such as crizotinib, with higher responses in children than in adults and FDA agency recently granted the breakthrough therapy designation for crizotinib for the treatment of patients with relapsed/refractory ALK+ ALCL. Based on these exciting results, it is not impossible to think that in the future ALK TKIs will become the first-line therapy for ALCL, thus overcoming the long-term toxicity of chemotherapy. A similar switch has happened in the case of ALK+ non- small cell lung cancer (NSCLC) that is currently treated in first-line with ALK TKIs. Despite most ALK+ ALCL patients refractory to chemotherapy achieve complete remission when treated with ALK TKIs, still a fraction of patients quickly develop resistance. In addition, responder patients are not completely cured as discontinuation of crizotinib is associated with rapid lymphoma relapse even after many years of complete remission with undetectable disease. Therefore, ALCL can develop molecular mechanisms that protect lymphoma cells from the activity of ALK TKIs. To achieve the ambitious goal of treating ALK+ ALCL with targeted therapy and replace chemotherapy as much as possible, there is need to completely understand these mechanisms that lead to ALK TKI resistance in ALCL. By genetic screenings, extensive sequencing and mouse models, we identified three main mechanisms leading to ALK TKI resistance and possibly sustaining the long-term persistence of ALCL cells: - we identified a phosphatase-mediated mechanism when we discovered that PTPN1 and PTPN2 are phosphatases of ALK and together with the SHP2 phosphatase regulate the sensitivity of ALCL cells to ALK TKIs; - we discovered that activation of PI3Kγ signaling supports survival of persister cells during ALK inhibition; - we elucidated the key role of the Rho family GTPases to mediate ALK signaling in ALCL. For this project, we hypothesize that targeting these three main pathways with specific combined therapies could cope with resistance and lead to the eradication of persister cells for a complete cure of ALK+ ALCL. In this project, we will validate this concept with in vitro and in vivo models that will be used to test different therapeutic strategies. Thus, ALK+ ALCL could become the first T cell lymphoma to be completely cured without chemotherapy, with obvious long-term benefits for children and adults affected by this disease. In addition, our results could pave the way to broaden these therapeutic concepts to other incurable TCL.

NIH Research Projects · FY 2025 · 2015-07

While food allergy has emerged as a major health issue, treatment options are quite limited. There is an urgent need to identify the specific environmental and endogenous factors that prime and then sustain allergic responses to foods so that new mechanism-based therapies can be developed. We have found that, IgE- activated mast cells, best known for causing immediate hypersensitivity reactions including food anaphylaxis, actually play a separate but critical role as food allergen sensors and adjuvants for Type 2 immune responses and that the activating effects of food-specific IgE on mast cells can be countered by IgG antibodies of corresponding specificity. Important questions remain unanswered, however: 1) What specific aspects of Type 2 immunity (Th2, ILC2, IgE+ B cells) are enhanced by IgE-activated mast cells?, 2) Are these effects mediated by mast cell cytokines?, 3) Is the protective effect of IgG antibodies in vivo, suppressing Th2 and enhancing Treg responses, mediated by their negative signals, delivered via FcgR2b, specifically in mast cells and can responses to food allergens be attenuated by using specific monoclonal IgG antibodies to activate this suppressive pathway? These questions will be answered in this project under the following aims: AIM 1: Establish how IgE-activated mast cells promote food allergy: 1.1 Test the impact of IgE-activated mast cells in vivo on emergence of Th2, Tfh and IgE+ B cells and evaluate the contributions of mast cell derived Th2 cytokines 1.2 Examine mast cell effects on emerging effector RORgt+ vs. pathogenic GATA-3+, IRF-4+, IL-4+ Treg 1.3 Evaluate reciprocal TGFb-mediated inhibitory effects of Treg on mast cell functions 1.4 Examine the influence of IgE-activated mast cells on ILC2 induction in vivo: Roles of mast cell- vs. gut epithelial cell-derived cytokines AIM 2: Determine how IgG:FcgR2b signals can reset the immune response in food allergy: 2.1 Evaluate the direct role of IgG inhibitory signals in mast cells in suppression of Type 2 and restoration of functional Treg responses in food allergy 2.2 Evaluate the effects of high-affinity human monoclonal peanut specific IgG antibodies (and their IgG subclass swap variants) cloned from single B cells transcriptomes on IgE-mediated basophil activation 2.3 Use our humanized mouse model of peanut allergy to test the effects of monoclonal peanut-specific IgE on immediate hypersensitivity responses (systemic anaphylaxis). 2.4 Test IgG effects on Th, Tfh and Treg responses in the humanized mice

NIH Research Projects · FY 2026 · 2015-07

PROJECT SUMMARY More than $17 billion dollars are spent per year on health care for children with neurologic impairment (NI). The main driver of these high costs is aspiration pneumonias, which cause lengthy and repeated hospitalizations, higher acuity hospitalizations, and significant mortality. The dogma has long been that these pneumonias are caused by aspiration of refluxed acidic gastric contents and thus should be treated with acid suppression and anti-reflux surgeries. However, these therapies not only have failed to reduce the development of aspiration pneumonias, but also may hasten the development of lung disease by increasing pneumonia risk by altering the aerodigestive microbiome, in the case of acid suppression, or by obstructing esophageal outflow or delaying gastric motility, in the case of anti-reflux surgery. Esophageal and gastric dysmotility are particularly concerning because they cause fluid stagnation with secondary changes in chemical and microbial composition. If these altered esophageal and gastric contents are aspirated, lung disease likely ensues. But questions remain: Which specific motility abnormalities result in fluid stasis and do these abnormalities predispose children with NI to more frequent aspiration pneumonias? Which components of static fluid portend worse clinical outcomes? Will correcting the dysmotility improve clinical outcomes? The proposed research will build on the current literature by addressing the following hypotheses: (1) distinct esophageal and gastric motility patterns result in stasis of esophageal and gastric contents; (2) this stasis alters the microbial and chemical milieu of esophageal and gastric fluids; (3) the stagnant gastric and esophageal contents, when aspirated, cause measurable microbiome and inflammatory changes that predispose patients to developing aspiration pneumonias; and (4) treatment of esophageal and gastric dysmotility improves aspiration-related symptoms and results in measurable changes in esophageal and gastric dysmotility. First, using a longitudinal cohort study design, the investigators will determine which unique esophageal and gastric motility patterns cause stasis and predict the development of aspiration pneumonias in children with NI. Second, using a randomized, blinded crossover design, investigators will test whether prucalopride, a 5HT4 agonist, improves both aspiration-related symptoms and esophageal and gastric motility. This research will: 1) attack a common and costly problem, aspiration pneumonias, in a medically vulnerable population; (2) move beyond GERD to pursue a novel, paradigm-shifting pathophysiologic mechanism, dysmotility, underlying development of aspiration pneumonias; (3) progress beyond standard testing to include not only motility parameters, but also the microbiome and chemical consequences of dysmotility; and (4) offer an innovative therapeutic intervention for aspiration symptoms that may improve health outcomes and reduce health care costs in a vulnerable population. Ultimately, this research can be used to devise additional novel therapeutic motility interventions to treat extraesophageal symptoms.

NIH Research Projects · FY 2025 · 2014-12

Project Summary Age-related macular degeneration (AMD) is a major cause of blindness in the elderly, associated with altered lipid (cholesterol) metabolism and altered immunity (complement). Chronic subretinal inflammation occurs during aging in clinical and experimental AMD, and is associated many other neurodegenerative diseases. Resolving harmful persistent inflammation is important to protect the retinas from age-related damage. Previous work identified that retinoic-acid-receptor-related orphan receptor alpha (RORα) a lipid (cholesterol)-sensing nuclear receptor, is genetically linked with the risk for wet AMD. RORα is a transcription factor that regulates lipid homeostasis and inflammation, both important for AMD. Our preliminary results from mouse models of aging and retinal degeneration indicate that: 1) RORα deficiency induces subretinal deposits, accumulation of lipid- enriched microglia/macrophages in the subretinal space in aging mice; 2) RORα deficiency worsens light- induced retinal degeneration; 3) Loss of RORα induces microglia/macrophage lipogenesis and chronic inflammation in RPE/choroid with induction of PPARγ, a key lipid metabolic regulator; 4) RORα deficiency alters complement factors and suppresses complement inhibitory factor H (CFH, one of the strongest AMD susceptibility genes) in the liver and in the eyes; and 5) RORα directly regulates both CFH and PPARγ transcription. Based on these findings, we hypothesize that during aging, RORα links lipid dysregulation with subretinal microglia/macrophage lipogenesis and complement alteration, to suppress chronic pathogenic subretinal inflammation; RORα activation may resolve chronic inflammation associated with early AMD. We will test this hypothesis with three aims. Aim I: To determine whether RORα deficiency exacerbates pathological subretinal inflammation and retinal degeneration in RORα deficient mice during aging and with a light-induced retinal degeneration model. Aim II: To assess if RORα deficiency induces chronic subretinal inflammation by accelerating microglia/macrophage recruitment, lipogenesis, and function through PPARγ, and/or by controlling systemic and/or local CFH function and complement cascade. Aim III: to determine if RORα activation resolves chronic inflammation and protects the retinas in chronic dry AMD models and in light-induced retinal degeneration model. This work will uncover the potential role of RORα as a key mediator of lipid homeostasis and altered innate immunity in chronic subretinal inflammation associated with AMD, and develop potential new treatments via activating RORα to resolve persistent inflammation during aging and protect retinas.

NIH Research Projects · FY 2025 · 2014-09

Advances in genetics have illustrated that many neurodevelopmental disorders such as autism spectrum disorder (ASD) and intellectual disability (ID) include a spectrum of rare disorders, and that pathogenic variants in hundreds of genes may result in susceptibility to ASD/ID. This heterogeneity presents significant challenges, but at the same time unique opportunities for research in the field of ASD/ID. Many of the genes implicated in ASD/ID appear to converge on a few common pathways, suggesting that there may be a common dysfunction at the cellular or systems level. Deeper understanding of the shared pathophysiology of these diseases may serve as gateways for understanding mechanisms of other causes of ASD/ID and for shared treatment possibilities. The Developmental Synaptopathies Consortium (DSC) was formed and funded in 2014 to perform mechanistic analysis of three genetic disorders with high penetrance of ASD/ID and investigate molecular pathways and mechanism-based therapeutic targets relevant to ASD/ID. We propose to continue our focus on genetic syndromes with abnormal synapse structure or function and that are associated with high penetrance for ASD/ID: TSC1/2 (Tuberous Sclerosis Complex or TSC), PTEN (PTEN Hamartoma Tumor Syndrome or PHTS) and SHANK3 (Phelan McDermid Syndrome or PMS) pathogenic variants. Over the last ten years, DSC has developed potential biomarkers and clinical outcome assessments for clinical trials, initiated clinical treatment trials in TSC and PHTS, developed longitudinal natural history data used to achieve FDA-approval for a first-in-human gene therapy trial for PMS, created consensus guidelines for the assessment and treatment of these conditions and trained and mentored several junior investigators who are now taking leadership positions in the field. Here we will add another genetic condition, SYNGAP1-related intellectual disability (SYNGAP1-ID), to test the generalizability of our approach and infrastructure. In the next grant cycle, DSC proposes four specific aims: (1) comprehensively characterize the phenotypes of individuals with these rare diseases across the lifespan, including cognition, communication, sleep, sensory deficits and neuropsychiatric symptoms while developing neurophysiological biomarkers to accelerate clinical trial readiness; (2) support pilot projects using strategic priorities of each disorder in collaboration with the patient advocacy groups; (3) continue to cultivate a pipeline of highly skilled new investigators within a collaborative and translational framework, emphasizing clinical trialist training and community engagement; (4) build on our current Administrative Core by expanding the leadership structure, continuing successful oversight of the activities of the DSC, disseminate DSC information to physicians, researchers, patient families and the lay public and planning future sustainability of this Consortium beyond RDCRN funding.

NIH Research Projects · FY 2026 · 2014-09

Project Summary/ Abstract Our lab has made substantial strides in understanding how human genetic variation affects erythropoiesis and fetal hemoglobin (HbF) levels, both critical factors in conditions such as sickle cell disease and β-thalassemia, as well as other forms of anemia. Utilizing cutting-edge genetic studies, we have mapped thousands of genetic variants influencing red blood cell phenotypes and HbF levels. We have also functionalized these variants through the use of innovative techniques such as massively parallel reporter assays and base editing in primary human hematopoietic stem and progenitor cells that were developed with support we received over the past ten years from this grant. In this competitive renewal application for this foundational grant, “Systematic Genetic Dissection of Human Erythropoiesis,” we propose a deeper investigation into the genetic network regulating human erythropoiesis and HbF expression. Specifically, we aim to 1) use massively parallel base editing to pinpoint key nucleotide determinants of HbF-related regulatory elements; 2) chart high-resolution interaction maps between these regulatory elements, their target genes, and other interactive genomic elements across different stages of erythropoiesis; and 3) conduct multiplexed base editing of key transcription factors and identified regulatory nucleotides to understand their genetic interactions. Our research will provide invaluable insights into the intricate genetic mechanisms regulating erythropoiesis and HbF expression, potentially paving the way for innovative therapeutic approaches for hemoglobinopathies. This project signifies a critical foundational step in leveraging genetic knowledge for the development of clinical interventions in debilitating hematological conditions.

NIH Research Projects · FY 2025 · 2014-08

Project Summary Diarrheal disease is a leading cause of morbidity and mortality in resource-poor areas. In order to colonize the intestine and cause disease, successful bacterial pathogens must sense and respond to intestinal signals by altering both metabolism and virulence factor expression. We hypothesize that by understanding the critical intestinal signals and the bacterial regulatory networks they activate, we can devise simple dietary alterations that prevent or mitigate morbidity and mortality. We focus on Vibrio cholerae, the agent of the severe diarrheal disease cholera. Because the metabolic pathways we study are highly conserved, these findings also serve as a paradigm for other bacteria that cause diarrhea. The goal of this work is to elucidate the complex and highly conserved regulatory network that is activated when V. cholerae enters the intestinal environment. In the first 4-year funding period of this grant, we explored the role of a global regulator of metabolism and virulence known as glucose-specific Enzyme llA. We showed that this regulator is membrane-associated through an N-terminal amphipathic helix and that membrane association is critical for its interaction with integral membrane protein partners that it regulates. Based on this work, we hypothesize that the inner membrane of the bacterial cell may act as a platform for regulatory proteins that sense and respond to nutritional signals in the intestinal environment. During the previous funding period, we discovered that the subcellular location of the global transcription factor, the cAMP receptor protein (CRP), is regulated in response to environmental conditions. In the current funding period, we propose to follow up on these observations by investigating regulation of CRP subcellular localization, the mechanism by which subcellular localization alters CRP activation of gene transcription, and the functional significance of CRP subcellular localization for V. cholerae virulence. We hypothesize that if the subcellular localization of CRP can be controlled via host diet, it may be possible to reduce pathogen virulence and thereby the morbidity and mortality caused by cholera.

NIH Research Projects · FY 2024 · 2014-07

Project Summary Neutrophils protect against infection but also mediate inflammatory tissue injury. As a result, targeting neutrophils therapeutically will require a detailed understanding of their basic biology, focused on domains wherein the defensive and pathogenic functions of neutrophils may diverge. In the first cycle of the present award, we explored two related GPI-linked neutrophil surface proteins of poorly-characterized function, Ly6G in mice and CD177 in humans. We showed that both associate at a molecular level in cis with neutrophil surface β2 integrins and that their ligation thereby attenuates neutrophil migration. Taking advantage of the experimental potential of murine inflammatory models, we found that Ly6G ligation selectively reduced integrin-mediated migration typical for neutrophil diapedesis toward sterile triggers, leaving integrin-independent migration to infectious stimuli largely unperturbed. Our preliminary data now show that Ly6G differentiates subphenotypes within murine neutrophils, while in humans CD177pos and CD177neg neutrophils differ in gene expression and potentially cytokine production. Together with the productivity of the first cycle of the award, these findings support deeper investigation of the role of these Ly6-family molecules in neutrophil biology. We propose two new and independent specific aims. Aim I pursues the mechanisms by which Ly6G and CD177 alter neutrophil β2 integrin function, including a search for novel endogenous counterligands. Aim II develops preliminary RNAseq data distinguishing neutrophil subtypes based on expression of Ly6G and CD177, from healthy donors as well as from adults and children with inflammatory arthritis and the transient but intensely inflammatory vasculitis Kawasaki disease. Together, these studies will advance the understanding of neutrophils by defining how Ly6 family members regulate 2 integrins to control cell migration and by identifying neutrophil phenotypes reflected in differential expression of Ly6G and CD177.