NCATS - National Center for Advancing Translational Sciences Grants

NCATS - National Center for Advancing Translational Sciences

Abstract Support is requested for a Keystone Symposia conference entitled “iPSCs: Progress, Opportunities, and Challenges,” organized by Drs. Shinya Yamanaka, Yanhong Shi and Yasushi Kajii, with scientific programming input from Keystone Symposia. The meeting will take place January 26–29, 2026 at the International Conference Center (ICC) Kyoto in Kyoto, Japan. This conference is being held to mark the 20th anniversary of breakthrough discoveries in induced pluripotent stem cell (iPSC) technologies, which have matured into viable platforms for embryology and disease modeling, drug discovery, and cell-based therapy development for a variety of human diseases. Moreover, the combination of iPSC technology with three-dimensional organoids, organ-on-chip and the emerging technologies of AI and machine learning ensures that iPSC-based platforms will yield new applications in biomedical and translational science. These innovative technologies and their applications are the primary focus of this meeting. Therefore, this conference has been designed to gather the leading scientists from academia and industry to push forward basic knowledge and medical application of iPSCs, especially those being tested in clinical trials worldwide, toward new insights and potential drugs. Additionally, this Keystone Symposia conference will provide a unique opportunity for researchers, clinicians, industrial experts and investors to interact, creating unusual collaborative prospects. Moreover, this conference will provide a rare opportunity for attendees to hear from renowned stem cell researcher and Nobel Laureate, Dr. Shinya Yamanaka, who will be giving the Keynote Address and is one of the meeting organizers. The sharing of knowledge at this meeting is expected to be transformative for the field and lead to the development of new cellular platforms and therapeutic products, which will ultimately impact clinical practice favorably.

NCATS - National Center for Advancing Translational Sciences

Anti-sense oligonucleotide (ASO) drugs have shown great potential in the treatment of human diseases. However, the lack of therapeutic efficacy and intolerance of adverse drug reactions (ADRs) and toxicity have led to the termination of numerous ASO candidates in the development pipelines, as well as the discontinuation from the market after approval by the FDA. Hepatotoxicity and nephrotoxicity are two major concerns for the FDA-approved ASO drugs in the market as well as development of new ASO-based therapies. Significant knowledge gaps still exist which prevent us from better understanding the lack of effectiveness and existence of ADRs and toxicity. One of these gaps lies in how an ASO drug goes through absorption, distribution, metabolism, and excretion (ADME) in its target organs and cells. Both in vitro cellular models and in vivo animal models have been used to assess ADME and toxicity of ASO drugs; however, they have numerous limitations. While cellular models lack human complexity, animal models lack human-specificity. Microphysiological systems (MPSs) may provide a solution to overcome this unmet need by providing human specificity and complexity as an in vitro platform. Here, we propose using the Javelin Liver Tissue Chip Plus (LTC+) platform, which integrates human liver and kidney MPSs, to study distribution, metabolism, excretion, and toxicity profiles (hepato- and nephrotoxicity) of four FDA-approved ASO drugs.

NCATS - National Center for Advancing Translational Sciences

PROJECT SUMMARY/ABSTRACT The objective of this proposal is to define the molecular mechanisms and identify new therapeutic strategies for of an understudied class of myopathies, specifically X-linked myopathy with excessive autophagy (XMEA). XMEA is characterized by elevated levels of autophagy due to disruptions in the autolysosome function. One MEA of interest is X-linked myopathy with excessive autophagy (XMEA), a rare autophagic vacuolar myopathy that characterized by progressive proximal muscle weakness, high levels of serum creatine kinase and accumulation of autophagic vacuoles. XMEA is caused by pathogenic mutations in the VMA21 gene in which N- terminal loss-of-function variants result in early death by 10 years and milder pathogenic VMA21 splicing variants result in a slower disease progression. Patients with VMA21 pathogenic mutations have a defective autophagy and an impaired ability to form the autophagosomes. VMA21 is a subunit of the V-ATPase protein pump and its disruption results in a failure to properly acidify the autolysosome resulting in the formation of vacuolar inclusions in XMEA. No extensive biomarker studies have been performed in the XMEA population resulting in a dearth of knowledge and the lack of suitable XMEA models is a significant barrier towards any effective treatment. We have generated a Vma21 knock-in (Vma21 KI) mouse model based on an RNA-splice mutation identified in a set of XMEA patients observed at our Children’s of Alabama muscular dystrophy clinic. Vma21 KI mice have a progressive muscle weakness, impaired muscle function, and have vacuolar inclusions that form as they age, which phenocopies the XMEA patient symptoms. In parallel, we generated vma21 mutant zebrafish that have a severe loss-of-function (LoF) pathology resulting in muscle paralysis, vacuolar inclusion bodies, and early lethality by 10 days post fertilization (dpf). An autophagy drug library screen of our vma21 mutant zebrafish identified edaravone, an FDA-approved autophagy and oxidative stress inhibitor for ALS, as the most corrective compound out of 29 leads for XMEA zebrafish pathologies. This proposal seeks to establish molecular and therapeutic biomarkers for XMEA based on our analysis of XMEA patient cells, and VMA21-defective zebrafish and mouse models, with an emphasis on the Vma21 KI mice. Proteomic evaluation of the muscles from Vma21 KI mice will allow us to identify VMA21-dependent factors that progress with XMEA disease status. We also seek to evaluate the therapeutic mechanism of action for edaravone in a 6 month treatment of our Vma21 KI mice. These studies seek to establish the XMEA/VMA21 disease processes while advancing a promising autophagy inhibitor compound to eventually treat these XMEA patients suffering from this devastating neuromuscular disorder.

NCATS - National Center for Advancing Translational Sciences

ABSTRACT This proposal addresses the need to investigate understudied proteins associated with rare diseases, such as Brugada Syndrome (PAR-25-122). One class of proteins highlighted in this RFA—Scn2b, Scn3b, and Scn4b— belongs to a family of β subunits that associate with the large pore-forming α subunits of voltage-gated sodium channels (NaV), which regulate electrical excitability throughout the body. In total, there are four distinct β subunits that can mix and match with nine different α subunits. Beta subunits are widely recognized for their ability to regulate the gating properties, trafficking, and pharmacology of Nav channel complexes. Dysfunction of these subunits has been linked to several human diseases, including epilepsy and cardiac arrhythmias such as long QT syndrome, atrial fibrillation, and Brugada syndrome. Additionally, mutations in NaV α subunits have been implicated in rare diseases, including SCN8A encephalopathy (SCN8A/ NaV 1.6), hereditary sensory and autonomic neuropathy type 7 (SCN11A/ NaV1.9), and dilated cardiomyopathy-1E (SCN5A/ NaV1.5). A critical step in understanding how beta subunits contribute to disease is elucidating their precise modulatory effects on NaV function. Electrophysiological studies in heterologous cells have demonstrated the ability of beta subunits to influence channel gating, pharmacology, and trafficking. However, results across multiple studies have been inconsistent, often due to variability in the cell lines used. A major confounding factor is that many cell lines endogenously express beta subunits, which can interfere with exogenously introduced β subunits under investigation. To overcome this limitation, we developed a specialized cell line lacking all β subunits, including Scn2b, Scn3b, and Scn4b, as well as Scn1b, MPZ, MPZL1, MPZL2, and MPZL3. These cells, termed beHAPe cells (beta- eliminated haploid cells for expression), provide a controlled system to study NaV channel regulation. Our initial electrophysiological studies using beHAPe cells reveal novel properties of beta subunits in modulating NaV1.5, the primary α subunit in cardiac tissue. Building on these findings, we propose to produce stably-expressing human (HEK) cell lines to systematically define the roles of Scn2b, Scn3b, and Scn4b in modulating additional α subunits, including NaV1.6 (a key subunit in the central nervous system) and NaV1.7, NaV 1.8, and NaV 1.9 (which are predominant in the peripheral nervous system). This work will provide deeper insights into their function in these tissues and their associated diseases. Additionally, our new data suggest that Nav1.8 plays a previously unrecognized role in cardiac function alongside NaV1.5. Understanding how β subunits modulate pore-forming subunits could provide new insights into their involvement in cardiac arrhythmias, expanding their known roles beyond the nervous system. Taken together, in addition to providing new information on the understudied Scn2b, Scn3b, and Scn4b proteins, our newly generated stable cell lines will enable studies for the development of novel therapeutics for isoform specific modulation of specific α- and β-subunit pairs.

NCATS - National Center for Advancing Translational Sciences

The Yale Center for Clinical Investigation (YCCI) was created in 2005 to advance Yale's clinical research mission. One year later, YCCI became the home of the Yale CTSA. At YCCIs inception, Yale was a national leader in T0-T2 translational research, basic/translational science training, and it supported distinctive translational science T3-T4 fellowship programs such as the Robert Wood Johnson Clinical Scholars Program. Since then, the CTSA has had a transformative impact linking all components of the Yale community in T1-T4 research, providing the central infrastructure for the effective conduct of ethical, innovative, rigorous, and reproducible research, and in training the next generation of research leaders. By any metric of scale, breadth, quality, and impact, both the CTSA's research enterprise and its educational mission have been enormously successful for Yale. This renewal application does not simply seek to maintain excellence, but to enable YCCI to drive the continued transformation of the Yale T1-T4 translational research mission and its predoctoral and postdoctoral training mission and to promote collaboration across CTSA hubs. First, it will support informatics and computational advances that drive the emergence of a learning health system. In so doing, it will draw on the Yale New Haven Health System, a six-hospital 2,681-bed consortium that provides more than 2.4 million outpatient visits annually from patients from upper Westchester county, throughout Connecticut, and southern Rhode Island. It will also prepare young scientists to draw on this infrastructure to conduct research that influences the future of healthcare. Second, it will support technological and scientific advances in areas that will support the emergence of personalized healthcare, including multi-omics and imaging. YCCI will provide pilot grant support and training to foster the development of research careers and research teams that can deepen our insights into pathophysiology and build toward personalized treatments. Third, it will engage a broader and multidisciplinary group of faculty, trainees, and community representatives to collaborate to improve health outcomes that constitute a major burden on patients, their families, and on public health. To support this mission, YCCI will also foster the development of careers in community-based research from a multidisciplinary group of young investigators and enhance the overall clinical research workforce.

NCATS - National Center for Advancing Translational Sciences

PROJECT SUMMARY The 2026 FASEB Summer Research Conference (SRC) on The Ubiquitin System: Mechanisms, Functions, and Therapeutics (UB) will be held June 1-4, 2026, at the Galt House Hotel in Louisville, KY. The UB SRC has been held biennially for nearly four decades, since 1989, and is the premier forum for sharing and discussing the latest developments in understanding the basic biology of processes regulated by ubiquitin and ubiquitin- like (UBL) proteins, and therapeutic approaches that target or harness these systems to treat disease. This application requests support for 12 new and early career invited speakers and 2 trainee organizers of a pre- meeting career forum to attend the 2026 SRC, which will bring together approximately 150 scientists at all career stages, from graduate students and postdoctoral fellows to principal investigators, professors, and company leaders working in academia, government, and industry. Regulated protein degradation through the ubiquitin-proteasome system is a critical aspect of protein homeostasis, the cell cycle, gene expression, metabolism, and development. Ubiquitin and UBLs also regulate a myriad of other cellular functions, including vesicular trafficking, autophagy, signal transduction, and membrane protein biogenesis, via non-degradative mechanisms. Hence, ubiquitin and UBL pathways impact essentially all aspects of eukaryotic cell biology and thus human health and disease, including developmental, neurologic, and inflammatory disorders, as well as cancers. The 2026 UB SRC will feature 8 themed sessions covering a broad range of topics focused on ubiquitin and UBL systems. These sessions will include 28 invited speakers and 24 short or “lightning” poster talks that will be selected from the submitted abstracts. In addition, for the first time, the 2026 UB SRC will co- locate and run concurrently with the FASEB SRC on Protein Folding in the Cell (PF), highlighting the close connection between protein biogenesis and degradation while also maximizing cost savings. The organizers of the two SRCs have joined forces to coordinate the programs of each SRC to maximize interactions. This includes 3 joint sessions, including the opening keynote lectures, running the poster sessions concurrently, and scheduling all coffee breaks, on-site meals, and panel and roundtable discussions to be held together. In addition, there will be a joint half-day career forum before the main SRCs, aimed at introducing trainees to each other, the SRC organizers, and invited speakers. This pre-meeting forum will be organized by a peer group of 4 trainees and provides opportunities for 16 additional trainees (8 each from UB and PF) to present their work. Finally, a new outreach component will be introduced to the SRC through the invitation of local high school students to attend the poster sessions. Thus, in addition to upholding the long-standing tradition of the UB SRC in featuring the latest high-quality work from basic science to translation to the clinic, these innovations will distinctly enhance and broaden the UB community to foster a safe, welcoming, and stimulating environment for sharing nascent ideas and unpublished results and forming new collaborations.

NCATS - National Center for Advancing Translational Sciences

SUMMARY We discovered mutations in Tudor family RNA-binding protein (RBP) TDRD7 (OMIM: 611258) cause congenital birth defect cataract–loss of eye lens transparency–in humans. We linked TDRD7 to defects in sperm formation, leading to the recognition of a rare novel human syndrome that includes congenital cataract and azoopermia as symptoms. Cataract occurs in neonates as a rare condition, but causes permanent vision damage, with surgery being the only treatment. Even after surgery, patients face eye complications throughout life. Thus, new therapies are urgently needed. Yet, knowledge on TDRD7, especially on its function in the eye, is limited. Thus, we will address this critical knowledge-gap by identifying potential new druggable pathways linked to TDRD7. Lens differentiation upregulates select RNAs/proteins while they undergo dramatic cell-shape changes–involving ~1000-fold length-wise increase–and migration toward lens core. A long-standing question is, what mechanisms control these complex cellular differentiation events? Our data suggests the involvement of TDRD7. TDRD7 protein has OST-HTH/LOTUS and Tudor domains that may allow it to associate with RNA and methylated arginine/lysine, respectively. Our data shows Tdrd7 knockout mice (Tdrd7KO) exhibit cataract and reduced expression of genes linked to human/animal lens defects. Further, Tdrd7 loss causes severe cellular morphology defects in mature lens fibers. Our data shows that in addition to abundant Tdrd7 protein in the fiber cytoplasm, where it participates in protein-RNA complexes, Tdrd7 protein also enters fiber nucleus beginning at midembryonic stages. These exciting findings lead to a paradigm-shifting hypothesis: TDRD7 may participate in both (1) post-transcriptional control and (2) chromatin control, to facilitate proper gene expression regulation in the lens. This will be tested by pursuing the following goals: Characterize spatiotemporal chromatin and transcriptome changes in Tdrd7KO mouse lens at the single-nucleus level and use AI-based approaches to derive regulatory networks (Aim 1). Characterize the impact of Tdrd7-loss on lens proteome and identify its protein interactions in normal lens (Aim 2). This innovative proposal will fundamentally advance knowledge on TDRD7 by: (1) defining, on single nucleus level, spatiotemporal changes in lens transcriptome and (2) changes in lens chromatin, upon Tdrd7 loss, (3) defining proteins impacting Tdrd7 function and those altered in Tdrd7KO, and (4) making this regulatory information publicly available via a web-based, user-friendly resource iSyTE for continued eye gene discovery. We will collaborate with Dr. Shinichiro Chuma (Kyoto University, Japan) who is an expert on TDRD-proteins and has developed a Tdrd7 knockout (KO) mouse model that we will investigate. While the facilities in US and Japan are similar, the targeted Tdrd7KO mouse model is not commercially available in the US and Dr. Chuma’s 20 years expertise on TDRD-proteins (e.g. advise on Tdrd7 biochemical protocols) is necessary for success of the aims. This translational research will advance knowledge on an understudied protein, TDRD7, and identify potential new drug targets/pathways for novel therapies/treatments for cataract birth defect.

NCATS - National Center for Advancing Translational Sciences

SUMMARY Lysosomal storage diseases (LSDs) are a group of inherited disorders that arise from mutations in genes responsible for encoding lysosomal enzymes or transporters, which are critical for breaking down and exporting complex macromolecules. When these enzymes or transporters fail to function properly, harmful substances accumulate within cells, leading to widespread cellular dysfunction and severe clinical abnormalities. In particular, the sphingolipidoses—a subset of LSDs—cause dysfunction in the breakdown of sphingolipids, essential components of cell membranes and regulators of critical signaling pathways. This disruption not only impairs cell function but also triggers a cascade of damaging effects that can significantly diminish quality of life. Gaucher disease (GD) is the most common sphingolipidosis. It occurs due to a deficiency in the enzyme β- glucocerebrosidase (GCase), leading to the buildup of substrate glucosylceramide (GCs) in macrophages, eventually resulting in various complications. The clinical phenotype is variable, but three clinical forms have been identified: type 1 is the most common and typically causes no neurological damage, whereas types 2 and 3 are characterized by neurological impairment. Once diagnosed, GD typically requires lifetime treatment. Regulatory T cells (Tregs) have a unique ability to cross the blood-brain barrier and to secrete anti-inflammatory factors, making them a promising foundation for new cell-based therapies. Our innovative approach allows Tregs to be engineered to deliver various therapeutic molecules directly to affected tissues and disease sites, offering a promising treatment platform for protein replacement therapies. In this application, we propose to engineer Tregs to secrete and replace GCase levels in the brain and peripheral tissues through the following aims: Aim 1: Generate GCase expression constructs with and without a MOG CAR for expression and lab-scale production testing. Aim 2: Evaluate expression and functional activity of engineered Tregs in vitro. Aim 3: Demonstrate in vivo expression of GCase and preferential localization of GCase -Tregs to the brain and peripheral tissues, local production of GCase, and reduction of GCs in affected tissues. Phase I Milestone: Select lead GCase-Treg product based on its ability to be detected in the brain and peripheral tissues within seven days of injection, and its ability to produce sufficient GCase >5-fold over vector control Tregs) to significantly reduce the levels of GCs in affected tissues. The selected candidate will be developed and advanced through IND-enabling studies in Phase II, including required studies for translation of this therapy into the clinic.

NCATS - National Center for Advancing Translational Sciences

Adeno-associated viruses (AAVs) are the leading vectors for gene therapy, valued for their safety, low immunogenicity, and broad tissue tropism. However, large-scale clinical translation remains hindered by challenges in manufacturing quality control. A critical barrier is the inability to rapidly and reliably measure capsid load and the ratio of full to empty capsids—key attributes that directly affect therapeutic potency and safety. Current assays such as ELISA (capsid titer), ddPCR (genome titer), and AUC (full/empty ratio) are costly, labor-intensive, and serotype-dependent, leading to inconsistent results across laboratories and production runs. To overcome these limitations, we will develop NANO-PAVE (NANOpore-based analysis of Proportion of full and empty Adeno-associated Virus capsids via nanopore Evaluation), a solid-state nanopore platform that analyzes individual AAV particles without labels, dyes, or antibodies. By leveraging voltage-driven translocation and electro-deformation, NANO-PAVE directly links mechanical signatures to genome packaging state, enabling real-time, quantitative discrimination between full and empty capsids. In addition, its single-particle resolution will allow accurate measurement of capsid load across serotypes, providing a powerful complement to existing bulk assays. This approach is cost-effective, scalable, and adaptable to all serotypes, making it ideally suited for integration into manufacturing pipelines. Phase I will establish feasibility by demonstrating robust particle discrimination, validating nanopore- derived measurements against orthogonal benchmarks, exploring genome-structure effects on capsid mechanics, and testing multiple AAV serotypes to ensure broad applicability of NANO-PAVE across vector types. Phase I deliverables will include a validated serotype-independent NANO-PAVE technology—integrating optimized nanopore design, recapture protocol, and machine-learning analysis—benchmarked against established assays to provide a rigorous, quantitative QC approach. This work will lay the foundation for Phase II development of a scalable, in-process analytical tool capable of distinguishing therapeutically relevant capsids from product-related impurities (empty, partially filled capsids, aggregates) and process-related impurities such as host cell proteins and media components, enabling comprehensive quality assessment. Ultimately, NANO-PAVE has the potential to become a transformative QC platform for academic, clinical, and industrial gene therapy programs, improving safety, efficacy, and manufacturing efficiency.

NCATS - National Center for Advancing Translational Sciences

Project Summary INTS12 is a chromatin interacting subunit of the mammalian Integrator complex that binds to accessible chromatin and controls various aspects of transcription such as elongation and termination. INTS12 interacts with negative elongation factors as well as RNA Polymerase II and regulates its pausing and release into productive elongation. Studies on INTS12 biology in various mammalian systems such as hematopoietic tissues is lacking despite ample evidence of its presence and likely contributions to hematopoietic development and disease. For instance, INTS12 is highly expressed in early stage erythroid progenitor cells during ex vivo erythroid differentiation of human CD34+ hematopoietic stem cells, and it is significantly reduced in the rare hemolytic anemia Congenital Dyserythropoietic Anemia Type IV (CDA IV; CDAN4), which affects young children and renders them transfusion dependent. This proposal will address our core hypothesis that INTS12 function is important for transcription regulation during normal human red cell development and reduced INTS12 levels in CDA IV red cells contributes to ineffective erythropoiesis due to disrupted RNA Polymerase II elongation control. To achieve this, a newly established erythroid progenitor cell line called BEL-A that can be expanded indefinitely, and differentiated to mature erythroid cells will be used. Aim1 will focus on INTS12 chromatin binding in normal wild type (WT) and CDA IV mutant BEL-A cells, and this will be correlated with existing unpublished chromatin accessibility and transcription factor binding data from erythroid stage-matched WT and CDA IV BEL-A cells. Further, the effect of depleting or overexpressing INTS12 on red cell development in WT and CDA IV will also be characterized. In Aim2, we will explore INTS12 functions in transcription by assessing the impact of INTS12 perturbations such as depletion and overexpression on nascent gene expression and RNA Polymerase II occupancy. These data will be correlated with INTS12 occupancy determined from Aim1, and any alterations in nascent transcription leading to changes in RNA Pol II pausing and elongation due to perturbed INTS12 levels will suggest that INTS12 contributes to altered transcription regulation in CDA IV. Transcription elongation by RNA Polymerase II is highly regulated and involves many transcription co- factors and epigenetic mechanisms, some of which are chemotherapy targets for certain hematological malignancies. Further, INTS12 interacts with chromatin using a conserved PHD domain that is being investigated as a potential chemotherapy target using derivatives of a class of compounds known as Amiodarones. The knowledge harnessed from this proposal will thus enable future investigations into INTS12 and Integrator complex biology in erythropoiesis, as well as translational studies on the potential for INTS12 as a therapeutic target in CDA IV patients.

NCATS - National Center for Advancing Translational Sciences

Abstract/Summary Cell free DNA (cfDNA) analysis is a translational method that has revolutionized both basic science and clinical medicine. It has allowed basic understanding of gene function to impact clinical decisions and allowed clinical specimens to be used for basic science. It has become part of the standard of care for perinatal screening, monitoring transplants, and choosing therapy for cancer. Clinical trials evaluating cfDNA in virtually every disease, including diseases of heart function, infections, vascular disease, neurology, psychiatry, organ transplantation, eclampsia, fetal health, inflammatory/autoimmune diseases, and cancer are showing promising results. Even just the easily measured level of cfDNA is a useful screening tool for maternal risk, many other tests use relatively simple PCR. cfDNA is the basis of highly sophisticated multiple cancer early detection (MCED) and potentially screening for other diseases, even including Alzheimer’s. Most applications use blood cell free DNA (BcfDNA). BcfDNA turns over in under 2 hours, it is subject to variation over the course of the day, it needs to be collected in special tubes to be stable, or immediately processed with biohazard care, to obtain plasma, which needs to be stored frozen or immediately processed by “moderate complexity” lab cfDNA isolation methods, which yield variable amounts of BcfDNA. Typically, patients’ visits are weeks or months apart and only 1 -20 mls of blood is obtained yielding 10s of nanograms of cfDNA. For MCED, MRD, CNS diseases, and occult infections, this small amount limits sensitivity. Urine also contains cfDNA (ucfDNA), at comparable or a bit lower concentration. Urine can be collected multiple times a day and on as many days as desired at home without the cost, inconvenience and potential exposure to infectious diseases, of the often immunosuppressed patients, inherent in a clinic visit. However, existing methods to extract ucfDNA only allow small amounts of urine to be processed; they too are complex methods that have variable, generally low, efficiency. When scaled up they are prohibitively expensive. Even with preservatives, there is immediate degradation of a fraction of the DNA before the specimens reach the lab. Numerous studies show ucfDNA contains cfDNA from the blood and that this trans-renal DNA can be used to understand system functions. Included preliminary data document a use anywhere, all resource sites (even home use), simplified and inexpensive method collects 20x to 100x more cfDNA, immediately separates the cfDNA from enzymes that cause degradation by affinity binding, and allow transport, without biofluid and cold chain concerns. Each of the underlined items are in themselves novel. The aims are to confirm and extend this data to commercialize a system, which once received in the lab can generate cfDNA suitable for PCR and next generation sequencing in 10 minutes. ucfDNA collected over time complements the snapshot BcfDNA provides. Larger total amounts of cfDNA improve sensitivity and provide excess material so this clinical resource will be more available for basic research. While BcfDNA is a limited resource, ucfDNA is virtually unlimited. Such translational research is expected to continue to rapidly change clinical practice. Other methods are for collection or lab isolation, uProcess does both, less expensively and in 1/10 the lab time. Most importantly, the variation featured herein selects for the trans-renal DNA indicative of systemic disease; all current kits yield >80% urothelial DNA.

NCATS - National Center for Advancing Translational Sciences

Summary Rare diseases collectively affect approximately 30 million people in the U.S., posing a significant health and economic burden. Despite their impact, the genetic and molecular bases of many rare diseases remain poorly understood, hindering the development of effective diagnostics and therapies. This proposal focuses on elucidating the functions of a subset of understudied proteins associated with rare diseases, identified in NIH PAR-25-122, that may play critical roles in left-right (LR) patterning during embryonic development. Disruptions in LR patterning underlie heterotaxy syndrome, a rare congenital disorder characterized by mispositioned internal organs and often severe cardiac malformations. Knowledge gained on how these proteins function in LR patterning can then be leveraged to understand how the protein may function to produce rare disease phenotypes in other tissues and will identify genes that should be evaluated as causative for Heterotaxy in humans. Building on extensive expertise in zebrafish models of LR development, the project aims to (1) assess the role of candidate proteins in LR patterning using antisense and CRISPR-based approaches, (2) generate targeted mutations in genes affecting LR development, and (3) develop transgenic zebrafish lines and antibodies to enable functional studies and lay the groundwork for future drug screening efforts. Zebrafish provide a powerful in vivo system to investigate gene function and conduct high-throughput drug screens. The proposed research will advance our understanding of rare disease gene function in LR patterning and establish essential tools to support therapeutic discovery in future proposals.

NCATS - National Center for Advancing Translational Sciences

Project Summary Chimeric antigen receptors (CARs) redirect T cells to eliminate antigen-expressing cells by coupling extracellular antigen binding domains to intracellular T cell signaling domains via hinge and transmembrane domains. Each domain of a CAR plays a crucial role in shaping CAR T cell phenotype and function. Despite the fundamental relationship between CAR design and T cell phenotypes, efforts to optimize CAR design have been limited and low-throughput. Consequently, most CARs in the clinic utilize the same basic design architectures, likely contributing to suboptimal performance. The overall goal of this Phase I STTR project is to develop a modular, high-throughput screening platform to optimize CAR designs for any antigen, disease, and modality. The central hypothesis is that CAR design optimization can address key challenges such as tonic signaling, antigen sensitivity, and antigen loss. To achieve the overall goal, the research proposal is structured into three specific aims focused on (1) screening large libraries of CD30 and (2) BCMA CAR designs to overcome diverse and fundamental challenges, and (3) building computational models to predict optimal CAR designs. The first aim is motivated by the profound signatures of tonic signaling of a clinically tested CD30 CAR design. Previous data suggest that multiple domains of a CAR can influence tonic signaling. To identify optimal CAR designs that reduce tonic signaling and retain antigen-specific potency, a large (~105 member) library of hinge, transmembrane, and CD3z variants will be screened using readouts of dysfunction and antigen potency. The second aim seeks to showcase the modularity of the platform by addressing a different challenge – increasing antigen sensitivity and reducing antigen loss – of a commercial BCMA CAR. The BCMA CAR exhibits impaired cytokine production in vitro and tumor control in vivo in response to tumor cells with low levels of BMCA. To identify optimal CAR designs that increase antigen sensitivity and reduce antigen loss, a large (~105 member) library of hinge, transmembrane, and CD3z variants will be screened using readouts of antigen sensitivity and antigen loss. The third aim will leverage these screening datasets to develop computational models that predict CAR activity, expanding the search space from empirical data of ~105 CARs to all possible ~1011 CAR designs. This research proposal is strongly aligned with the mission of NCATS to turn research observations into health solutions through translational science. Developing a screening platform will expedite the scale and speed at which research observations of CAR designs can happen and translate into life changing therapies. In the short- term, this proposal aims to discover optimal CD30 and BCMA CAR designs to improve CAR T cell therapy for Hodgkin Lymphoma and Multiple Myeloma. This proposal establishes the foundation of a long-term goal to engineer more effective CAR T cell therapies for indications with high unmet need.

NCATS - National Center for Advancing Translational Sciences

Human induced pluripotent stem cells (hiPSCs) are a foundational technology for regenerative medicine, disease modeling, and cell-based therapies. However, the clinical translation of hiPSC-derived therapeutics is constrained by unresolved challenges in manufacturing scalability, product consistency, and long-term storage. Current production methods, such as static culture or stirred-tank bioreactors, either lack throughput or impose damaging shear forces that compromise cell viability and stemness. Additionally, existing cryopreservation strategies are not optimized for the large-volume required for centralized manufacturing and distributed clinical deployment. To address these bottlenecks, we propose to develop a modular, low-shear, perfusion-based biomanufacturing platform coupled with controlled-rate cryopreservation for scalable and reproducible expansion of hiPSCs. This R21 will establish the technical feasibility and foundational workflows for a generalizable manufacturing system suitable for diverse therapeutic applications. In Aim 1, we will engineer and optimize a multilayer, perfusion-based bioreactor system to support hiPSC expansion at clinical scales. We will define operational parameters, evaluate multiple hiPSC lines, and develop protocols for efficient harvesting and redeployment. In Aim 2, we will implement long-term culture strategies to preserve genomic stability and pluripotency across multiple passages and optimize cryopreservation workflows using cryobags and controlled-rate freezing. Post-thaw cells will be assessed for viability, genomic integrity, and differentiation potential. Successful completion of this project will yield the first integrated platform for low-shear, current Good Manufacturing Practice (cGMP)-compatible expansion and cryostorage of hiPSC-derived therapeutic products. This innovation will reduce production variability, enhance scalability, and enable rapid deployment of hiPSC-based therapies across a range of disease indications. The resulting system will serve as a flexible and translational foundation for future commercial partnerships and larger-scale studies, consistent with the high- risk, high-reward goals of the R21 mechanism.

NCATS - National Center for Advancing Translational Sciences

Project summary/Abstract The traditional drug development pipeline is critically inefficient, slow, costly, and has high failure rates. These diff iculties are especially pronounced for patients with rare and understudied diseases, who face a significant unmet medical need. Computational drug repurposing is a promising way to address these public health issues. Existing computational drug repurposing has two key limitations in current methodologies: (1) the restricted representation of biomedical knowledge as pairwise graphs, and (2) the lack of generalizable, interpretable predictive models. To address the first limitation, we build upon the NCATS Biomedical Data Translator’s foundational knowledge graph to construct the first large-scale biomedical knowledge hypergraph (Aim 1), that explicitly captures higher-order relationships that involve more than two entities—such as how specific drug combinations benefit particular disease subtypes or patient subgroups defined by demographics or genetics. This enriched representation is critical for enabling precision medicine and the discovery of synergistic therapies, which cannot be effectively captured using traditional pairwise-only graph structures. To extract these higher-order relationships from literature, we will develop a novel reinforcement learning-enhanced large language model (RL-LLM) to extract higher-order relationships directly from biomedical literature. A key feature of our framework is a human-in-the-loop quality control process, in which domain experts curate and validate extracted relationships to ensure accuracy and biomedical relevance. To address the second limitation, we will develop HyperGAT, a hypergraph neural network framework that incorporates heterogeneous attention mechanisms, interpretability modules, and zero-shot learning capabilities to enable accurate and explainable predictions of drug repurposing opportunities and synergistic therapies (Aim 2). Our multidisciplinary team, combining expertise in graph machine learning, reinforcement learning, natural language processing, and translational science, is uniquely positioned to execute this vision. Together, these innovations will support NIH priorities in precision medicine and translational informatics. Deliverables include open-source tools, a next-generation biomedical hypergraph resource, and an interpretable predictive framework that enables scalable, context-aware therapeutic discovery beyond the capabilities of conventional KG-based approaches.

NCATS - National Center for Advancing Translational Sciences

PROJECT SUMMARY Vascular diseases driven by endothelial dysfunction represent a major therapeutic challenge due to the lack of cell-specific delivery systems. While TGFβ-driven endothelial-to-mesenchymal transition (EndMT) has emerged as a key therapeutic target, current approaches are limited by off-target effects of systemic TGFβ inhibition. We have developed a novel HDL-mediated RNA delivery platform (C15-9- 900 LNP) that achieves unprecedented endothelial cell specificity through innovative ionizable lipid design. Our preliminary data demonstrate >90% targeting efficiency across multiple vascular beds, with exceptional manufacturing reproducibility at 2g scale, minimal inflammatory response, and established quality control parameters. This R21 ASCETTS proposal will advance platform development through two complementary aims. In Aim 1, we will establish critical quality attributes and manufacturing parameters through systematic evaluation of formulation conditions. Comprehensive particle characterization will include size, polydispersity index, and zeta potential analysis, complemented by quantitative biodistribution studies using endothelial lineage-traced mice. Advanced imaging and flow cytometry validation will confirm targeting specificity across multiple vascular beds. In Aim 2, we will demonstrate therapeutic efficacy using an established hypoxia- induced pulmonary hypertension model. We will evaluate the platform's ability to modulate TGFβ pathway signaling in pulmonary vascular endothelium, assess therapeutic outcomes through comprehensive hemodynamic and histological analyses, and establish clear translational parameters for clinical development. This platform technology represents a fundamental advance in targeted RNA therapeutics by enabling selective endothelial modification while minimizing systemic effects. Our systematic approach establishes standardized manufacturing parameters and analytical methods suitable for clinical translation. While initially focused on pulmonary hypertension, the platform creates a foundation for addressing multiple cardiovascular disorders where endothelial dysfunction plays a central role. Success in this R21 phase will accelerate therapeutic innovation through validated manufacturing processes and clear regulatory parameters.

NCATS - National Center for Advancing Translational Sciences

Lung complications are difficult to diagnose in critically ill children and linked to high mortality. Current diagnosis of complications such as infection and bleeding relies upon bronchoalveolar lavage (BAL), which has low yield (~30%) and high morbidity. Bronchoalveolar lavage involves lavage of a lung segment, with the fluid suctioned for infectious disease and other testing. Because current bronchoscopes cannot access all lung segments, nor the distal sites of lung infections, a large volume of fluid is required to interrogate the affected tissue. Injured lungs retain fluid, which leads to significant morbidity and respiratory compromise. This is most significant for critically ill children who have small airways and parenchyma with minimal pulmonary reserve. Lack of diagnosis is correlated to death. Other diagnostic tools are limited by higher morbidity (lung biopsy) or decreased specificity (blinded approaches such as the miniBAL or tracheal aspirate). Hence, a new approach to improve diagnosis and hence organism recovery is urgently needed for critically ill children. The main goal of this project is to develop a Robotic BAL (RoboBAL) device that allows the clinician to steer and access more distal sites and can include inaccessible sites (the right upper lobe), obviating the amount of fluid needed in critically ill children. This new system will be able to access the fourth tracheobronchiole division, beyond the current third division of BAL. We propose two specific aims (SAs): SA 1: Design and develop a 3.25mm robotically steerable BAL device with an integrated camera and light source and a 1.2mm actively steerable working channel for suction and irrigation of fluid. SA 2: Test the ability to access distal airways in five human cadaver lungs and compare the diagnostic yield and fluid retained after lavage with RoboBAL and SOC BAL. We propose innovative methods to assess the differences in phantom human models and human cadavers. In human cadavers, imaging will demonstrate the depth of the bronchoscope and proximity to a target (RoboBAL vs. SOC). Using a Alexa Fluor 488 labeled Ecoli, we will quantitate the recovery of the fluorescent dye and evaluate microbe capture by culture, modeling the yield of microbes. Finally, safety will be assessed by the wet/dry ratio as a surrogate for retained fluid, and inspection to determine any impediment to traverse small airways. Through accomplishing these aims, we will have determined whether we can access all fourth generation airways, and achieve superior yield with less fluid retained. Our highly interdisciplinary team with expertise in medical robotics (Dr. Desai), pulmonary complications of critically ill children and adults (Drs. Williams and Lama), radiographic imaging (Dr. Alazraki), and statistics (Dr. Switchenko) is poised to develop and test this new RoboBAL device that could enhance diagnosis and diminish morbidity, thereby leading to rapid, targeted treatment with downstream effects of decreased ventilator time, decreased intensive care, decreased antibiotic resistance, and improved outcomes with lower healthcare costs. The proposed RoboBAL system will improve diagnosis and permit targeted therapy to improve outcomes in critically ill children – see support letters.

NCATS - National Center for Advancing Translational Sciences

SUMMARY Severe insulin resistance syndromes, including Donohue and Rabson-Mendenhall syndromes, are rare, life- threatening disorders caused by mutations in the insulin receptor (IR) that impair insulin binding and receptor activation. There are currently no FDA-approved therapies that directly target the receptor defect, and existing interventions provide only limited, short-term benefit. This proposal aims to address this urgent unmet need in rare disease therapeutics by advancing RF-409, a first- in-class, synthetic IR agonist. RF-409 was developed using structure-guided computational protein design to engage both insulin-binding sites (site-1 and site-2) and induce conformational changes that stabilize the IR in its active state. RF-409 exhibits high affinity and specificity for IR, activates both metabolic and mitogenic signaling pathways, and demonstrates strong thermostability and bioactivity in vivo. Preclinical studies show that RF-409 lowers blood glucose more efficiently and with longer duration than insulin in wild-type, type 1 diabetic (Streptozotocin-induced), and high-fat diet–induced obese mouse models. Notably, RF-409 is also effective in activating IR mutants that are unresponsive to insulin. To enable rigorous efficacy testing, we developed a validated knock-in mouse model (IR-D707A) carrying a patient-derived, insulin-binding–defective mutation. These mice exhibit neonatal lethality and severe insulin resistance, closely mirroring human severe insulin resistance phenotypes. RF-409, but not insulin, activates IR in this mouse model, providing a robust, disease-relevant platform for therapeutic evaluation. Aim 1 will characterize the pharmacokinetic and pharmacodynamic profile of RF-409 in wild-type, diabetic, and conditional IR-D707A mice. Time-resolved LC-MS analysis will be used to define systemic exposure and tissue distribution. Glucose-lowering efficacy and downstream IR signaling will be measured to establish PK/PD relationships and guide dose selection. Aim 2 will assess the physiological and therapeutic impact of RF-409 in the IR-D707A mouse model. We will evaluate metabolic, mitogenic, and survival outcomes following chronic administration in both adult and neonatal animals to determine therapeutic benefit in a rare disease model. RF-409 is well-characterized, highly specific, and production-ready. Its activity in both preclinical and disease- specific models support its potential as a therapeutic for rare insulin receptoropathies. Completion of this project will generate critical efficacy and mechanistic data to support IND-enabling development and may lay the foundation for a new class of targeted therapies for receptor-level metabolic diseases.

NCATS - National Center for Advancing Translational Sciences

Project Abstract Among the thousands of genetic disorders in humans, most are rare and lack effective treatments. Suppressor tRNA therapy has come a long way to become a realistic treatment option for genetic diseases. Sup-tRNAs read through premature stop codons derived from genetic mutations in translation to generate full-length proteins. Compared to gene or mRNA-based therapies that deal with one gene or one mutation at a time and require safety and efficacy assessment for each therapeutic agent, a major advantage of tRNA therapy is the potential of using the same tRNA to treat many diseases that just share a common genetic mutation in many genes. However, about 30% of human genetic disorders are from missense mutations that cannot be treated with sup- tRNAs. For those disorders, missense-correcting tRNAs are needed which are charged with one amino acid but read the codon of another amino acid in translation. PI's lab has developed a missense-correcting tRNA identification platform and applied it to identify such tRNAs charged with Arg but read Gln/His/Trp/Cys codons which correspond to most frequent missense mutations in genetic disorders. Aim 1 will develop a high-throughput engineering platform for missense-correcting tRNAs that will be more efficient, and at the same time, less toxic to cells. We will screen tens of thousands of potential missense-correcting tRNAs simultaneously in cellular models. Aim 2 will screen for missense-correcting tRNA expression with reduced toxicity to cells. Our studies will establish a foundation for using missense-correcting tRNAs as a new therapeutic modality for rare diseases.

NCATS - National Center for Advancing Translational Sciences

PROJECT SUMMARY/ABSTRACT Vici syndrome (VS) is a rare pediatric genetic disorder characterized by profound developmental delay, seizures, immunodeficiency, cardiomyopathy, and progressive motor decline, with a median survival of just 42 months, This devastating disorder is caused by pathogenic variants in the EPG5 gene, which encodes a critical regulator in autophagy. Loss of EPG5 function results in the accumulation of toxic intracellular material and progressive cellular dysfunction. VS is a member of a broader class of diseases known as 'Congenital Disorders of Autophagy' (CDAs), in which core components of autophagy are defective. There are essentially no treatment options for children with VS or other CDAs. We hypothesize that agents that induce pharmacological enhancement of autophagy in VS and other CDAs represent a viable therapeutic strategy. In collaboration with VS families, we have developed and characterized critical preclinical models for VS for therapeutic discovery and validation, including patient-derived induced pluripotent stem (iPS) cells and novel genetically engineered mouse models. These Epg5 mutant mice recapitulate a range of neurological phenotypes seen in VS including biochemical deficits in autophagy, progressive motor dysfunction, and a strong molecular and cellular signature indicative of neuroinflammation. In parallel, we have established a novel engineered iPS cell-based platform for high throughput cell-based screens, which has identified rapamycin-related class of compounds (rapalogs) as enhancers of autophagy in the VS cells. This project will evaluate the therapeutic potential of the rapalog everolimus, an FDA-approved drug with over 14 years of safety data, including well-described use in pediatric populations, leveraging the VS mouse models for in vivo preclinical proof-of-principle studies. In Aim 1, we will define the pharmacokinetics (PK) and pharmacodynamics (PD) of everolimus in the VS mouse model, optimizing dosing regimens to sustain autophagy induction in vivo after extended use. In Aim 2, we will assess the ability of everolimus to slow or halt disease progression in vivo using the VS mouse model. Neurological function and inflammatory response in the VS animal model will be monitored during longitudinal treatment with everolimus. Phenotypic monitoring will include whole animal behavioral assays of motor function and molecular and histological biomarkers of neuroinflammation in various CNS tissues. Successful completion of this project will provide critical preclinical data supporting the repurposing of everolimus for clinical proof of concept in VS patients. Our multidisciplinary team, combining expertise in drug discovery, preclinical disease modeling and analysis in mice, clinical treatment of VS patients, and rare disease research, is well-positioned to advance this therapeutic strategy toward clinical translation in partnership with highly engaged VS families.

NCATS - National Center for Advancing Translational Sciences

PROJECT SUMMARY Pulmonary arterial hypertension (PAH) is a rare and progressive cardiopulmonary disorder characterized by the remodeling of pulmonary vascular cells, which leads to narrowing and the obstruction of pulmonary arteries. This results in increased pulmonary vascular resistance and pressure, ultimately causing right ventricular failure. Despite current therapies targeting key pathogenic pathways, the morbidity and mortality associated with PAH remain unacceptably high, highlighting the urgent need for new treatments. A hallmark pathological feature of PAH is the phenotypic shift of pulmonary artery smooth muscle cells (PASMCs) to a “cancer-like” state, which is characterized by hyperproliferation and resistance to apoptosis. Previous studies have suggested that abnormal histone methylation contributes to this pathological phenotypic switching, promoting uncontrolled vascular remodeling during the early stages of PAH. However, the precise molecular mechanisms involved are still poorly understood. Menin (Men1), a scaffold protein that interacts with the histone methyltransferase Mixed Lineage Leukemia 1 (MLL1 or KMT2A), plays a crucial role in regulating gene expression through epigenetic mechanisms, including the trimethylation of H3K4me3 histone marks, which influences oncogenic pathways and cell cycle control. While its role in cancer is well established, the involvement of the Men1/MLL1 complex in the pathogenesis of PAH has yet to be elucidated. In this proposal, we aim to elucidate the role of this complex by investigating how Men1 contributes to the phenotypic reprogramming of PASMC and vascular remodeling in PAH. Importantly, we will evaluate the therapeutic potential of Revumenib, a potent and selective Men1/MLL1 complex interaction inhibitor, in both in vitro and in vivo preclinical models of PAH. Our preliminary results show an upregulation of Men1 and MLL1 levels in both human PAH lung tissues and a rat model of PAH. Treatment with Revumenib led to a reduction in Men/MLL1 expression and interaction, accompanied by a decrease in H3K4me3 histone marks and reduction of PASMC hyperproliferation and migration. Based on these findings, we hypothesize that Men1 exacerbates vascular remodeling in PAH via MLL1-mediated H3K4me3 marks and associated transcriptional gene programs, thereby promoting the reprogramming of PASMCs. SA1 will evaluate the in vitro efficacy of Revumenib in PAH-PASMCs by examining its effects on cell proliferation, apoptosis, and gene expression signatures associated with Men1/MLL1 activity. SA2 will assess the in vivo therapeutic potential of Revumenib in healthy and in rat models of PAH. Biochemical and cellular assays will be used to evaluate drug distribution, Men1/MLL1 complex disruption, and downstream epigenetic modifications and signaling in the lungs. Given Revumenib’s promising safety profile in previous human and animal studies in acute myeloid leukemia, we anticipate minimal adverse effects in the context of PAH. The successful completion of this project will enhance our understanding of epigenetic regulation in PAH and may establish Revumenib as a foundation for the development of a novel, mechanism-based treatment strategy for this rare, deadly disease.

NCATS - National Center for Advancing Translational Sciences

PROJECT SUMMARY Alport Syndrome (AS) is a monogenic disease primarily caused by a deficiency of functional type IV collagen a chains (COL4A3/4/5), matrix proteins that play a crucial role in maintaining the structural integrity of basement membranes in the kidneys, ears, and eyes, leading to progressive chronic kidney disease (CKD), hearing loss and ocular impairment, respectively. Although all three manifestations in this triad are equally devastating and diminish the quality of life for affected individuals, kidney involvement is particularly severe and often life- threatening. Importantly, AS is the second most prevalent genetic kidney disease that leads to CKD and, ultimately, kidney failure (KF). Despite its relatively high prevalence among the rare genetic diseases, estimated to be 1 in 2,000 to 5,000 people, current clinical pharmacological therapy for AS-related CKD is limited to the treatment with angiotensin-converting enzyme inhibitors, which only partially delays the progression of CKD. Currently, there is no curative therapy for the disease. Therefore, there is an urgent, unmet need for innovative and more effective CKD therapies for AS. In recent years, significant progress has been made in the field of adeno-associated virus (AAV) vector-mediated gene therapy for monogenic diseases, leading to the approval of commercial products. Despite the immense potential of AAV vectors to treat genetic diseases and the compelling nature of genetic kidney diseases, including AS, as gene therapy targets, AAV vector-mediated gene therapy for genetic kidney diseases has remained underexplored. This is primarily due to the challenge in effectively delivering genes to the kidney even with AAV vectors. Moreover, AAV gene therapy for AS faces an additional challenge in that the size of the therapeutic gene payload encoding COL4A3, COL4A4, and COL4A5 chains exceeds the packaging capacity of AAV vectors. In this regard, notably, the Nakai (PD/PI) lab has recently achieved a breakthrough by devising a novel AAV vector approach that can mediate effective expression of the full-length COL4A5 protein in podocytes and allows its expression, secretion, and deposition in the glomerular basement membrane in the kidneys of AS mouse models. This breakthrough has warranted assessment of its efficacy using clinically relevant large animal models. While non-human primate models for AS do not exist, a well-established AS dog model is available, the X-linked AS (XLAS) dog model that has been extensively studied and maintained by the Nabity (MPI) lab. Given this background, this collaborative, exploratory multi-PI project between the Nakai and Nabity labs aims to demonstrate proof of concept of AAV vector-mediated gene therapy for AS in an XLAS dog model and investigate AAV vector biology, pharmacokinetics, biodistribution, off-target effects, and immune responses in this dog model. Success in the project will significantly spur the development of AAV gene therapy for AS and further our knowledge of AAV vector biology in the AS context, which is essential for successful clinical translation. Furthermore, the project outcomes will have substantial implications for the treatment of other CKDs, whether of genetic or non-genetic etiologies.

NCATS - National Center for Advancing Translational Sciences

PROJECT SUMMARY/ABSTRACT Cystic fibrosis (CF) is a multisystemic, autosomal recessive disorder with the majority of morbidity and mortality extending from lung disease. Given the benefits that older children and adults with CF have derived from cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapies, it is anticipated that early childhood – or even in utero – treatment with CFTR modulator therapies may significantly delay or even prevent the development of CF lung disease. The role of CFTR protein in fetal lung development, and thus the potential impact of early CFTR modulator therapies, has yet to be fully elucidated. The proposed research project aims to better understand the pathogenesis of CF lung disease and the impacts of deficient CFTR protein on fetal lung development while overcoming a general roadblock in the study of many pediatric lung diseases, the scarcity of available human material. Previous immunohistochemistry studies in the fetal CF lung described a three-week delay in the developmentally regulated pattern of CFTR protein expression as well as an early, intrinsic, pro- inflammatory state. Leveraging a three-dimensional, in vitro and in vivo, lung organoid model that undergoes branching morphogenesis and alveologenesis – thus recapitulating early fetal lung development – we will characterize CFTR gene transcript expression and CFTR protein expression and function and articulate the transcriptome and proteome of normal and CF lung organoids. We hypothesize that CFTR protein deficiency in the fetal CF lung results in pro-inflammatory transcriptomic and proteomic profiles in respiratory epithelial cell populations and that this can be reasonably demonstrated using a lung organoid model. To test our hypotheses and validate the findings in the lung organoids, we will also articulate the cellular multi-omes in fetal normal and CF lungs, characterizing any disease-related cellular, transcriptomic, epigenomic, and proteomic changes. The proposed research project will provide critical insights into the pathogenesis of CF lung disease and establish a new, well characterized, in vitro and in vivo, model system of the CF lung that can be leveraged in future research endeavors (e.g. viral disease modeling, therapeutic testing). The proposed research project has the potential to identify the earliest pathogenic changes in the CF lung, determine the pulmonary impact of the application of in utero CFTR modulator therapies, and determine the need for alternative, novel treatment modalities to more readily change the early trajectory of CF lung disease.

NCATS - National Center for Advancing Translational Sciences

Multiple hereditary exostoses (MHE) is an autosomal dominant disorder that affects one in every 50,000 children worldwide and is characterized by the formation of cartilage-capped osteochondromas, known as exostoses, adjacent to the growth plates of long bones and other skeletal elements. Due to their location and size, exostoses can cause skeletal deformities, growth retardation, chronic pain, and undergo malignant transformation in ~5% of the patients. Currently, there are no approved treatments for this disorder besides surgery and pain management; therefore, there is an urgent need to develop new therapeutic approaches to prevent and/or slow the progression of the disease. Greater than 90% of cases are caused by heterozygous loss-of-function genetic mutations in exostosin-1 (EXT1) or exostosin-2 (EXT2), genes that encode enzymes responsible for the biosynthesis of heparan sulfate (HS), which can lead to truncation of the HS chains and a consequent decrease in HS levels in various tissues. Current evidence suggests that a decrease in HS content disrupts multiple signaling pathways through which growth factors regulate the organization and function of chondrocytes in the growth plate. Regardless of the mechanism, the primary defect is in the assembly of heparan sulfate, suggesting that restoring the level of heparan sulfate would diminish the frequency of exostoses. Here, we propose that over-stimulating the expression of the normal EXT allele to compensate for the activity of the mutant allele could be a promising therapeutic approach to restore functionally normal levels of HS and homeostasis at the growth plate. Recently, we developed drug repurposing screens to search for small molecule agents that could upregulate EXT expression and HS levels in cells. From these screens, we identified the clinically approved PI3Kα inhibitor, BYL-719 (alpelisib), as a potent activator of EXT1/EXT2 expression and HS biosynthesis in human and murine chondrocytes. Subsequent studies confirmed the PI3K/AKT pathway as a promising target to enhance HS assembly and inhibit chondrogenesis. BYL-719 is a potent, selective, and orally active PI3Kα inhibitor and is clinically approved for treatment of metastatic breast cancer and PIK3CA-related overgrowth spectrum (PROS) disorders in patients as young as two years old. Additionally, it was recently shown to inhibit ectopic bone and cartilage formation in a mouse model of heterotopic ossification (HO). In this project, we aim to leverage an approved drug with known properties and clinical approval status to expedite a potential therapeutic application for MHE. The objective of this R21 proposal is to investigate the therapeutic efficacy of BYL-719 to reduce exostoses in an established mouse model of MHE. To accomplish this, we will (i) evaluate BYL-719’s ability to reduce and/or slow exostoses formation in Ext1+/-Ext2+/- mice, and (ii) assess its in vivo mechanism of action via analysis of isolated exostoses and primary chondrocytes. The successful completion of these aims will provide essential preclinical data supporting the feasibility of BYL-719 as a viable treatment for MHE, thus improving the quality of life for patients with this devastating disease.