Boston Children'S Hospital

NIH Research Projects · FY 2026 · 2025-01

Tuberous sclerosis complex (TSC) is a genetic disorder caused by heterozygous inactivating variants in TSC1 or TSC2. TSC is characterized by the growth of benign tumors in multiple organs, as well as neurological manifestations such as refractory epilepsy, autism spectrum disorder, and intellectual disability. TSC1 and TSC2 form a protein complex with TBC1D7 that acts as a key upstream negative regulator of the mechanistic target of rapamycin complex 1 (mTORC1), and mTORC1 hyperactivation is thought to be a key pathogenic driver of TSC. Treatments that inhibit mTORC1 cause arrest or regression of tumors in TSC but have shown limited efficacy in treating epilepsy and no efficacy in treating the neurocognitive symptoms of TSC. Consequently, there is a substantial unmet need for the development of new therapeutics, which will require further understanding of the molecular mechanisms of neurological symptoms in TSC. Among the several downstream consequences of mTORC1 dysregulation, disruption of primary cilia has emerged as one potential candidate for mediating neuronal dysfunction in TSC. The primary cilium is an immotile organelle that extends from the plasma membrane and contains a distinct composition of transmembrane receptors, rendering it a signaling hub. Our work has shown that in TSC, neurons are less often ciliated, and the remaining cilia are significantly lengthened. Accordingly, in the proposed research the applicant aims to investigate the molecular mechanisms underlying these alterations. Preliminary data suggests that upregulation of HSP27 in astrocytes may be a non-cell autonomous modulator of neuronal cilia in TSC. Therefore, in Aim 1 we propose to determine whether astrocytic expression of Hsp27 is necessary and/or sufficient to modulate cilia in Tsc1- deficient neurons. To enhance the translational relevance of this work, in Aim 2, we will evaluate neuronal primary cilia and astrocytic HSP27 expression in human cells. Our finding of altered neuronal cilia morphology in TSC also raises the question of whether cilia are functionally impaired. Our preliminary data suggests that ciliary calcium channels are differentially expressed in neurons in TSC, so in Aim 3 we will characterize ciliary calcium signaling in Tsc1-deficient neurons. mTOR dysregulation and cilia dysfunction are common observations across several disorders that display high rates of epilepsy and/or autism, making the proposal an excellent project to introduce the applicant to neurodevelopmental disorders and to broaden the impact of this research. The proposal contains a comprehensive training plan for the applicant to become a productive independent investigator, including plans to obtain key technical skills in stem cell culture and calcium imaging, and to increase presentation and publication opportunities. The research outlined in this proposal will take place at Boston Children’s Hospital (BCH) in the lab of Dr. Mustafa Sahin, an expert in the neurobiology of TSC. The institutional environment at BCH, including talented scientists and abundant resources and equipment, together with the mentorship of Dr. Sahin, will support the evolving needs of this project.

NIH Research Projects · FY 2026 · 2025-01

Project Summary Tetanus has been a major deadly infectious disease throughout human history. It is caused by the spore-forming anaerobic bacterial pathogen Clostridium tetani, which can germinate within the deep wound and produce a potent bacterial toxin known as tetanus neurotoxin (TeNT), which is the sole cause of tetanus’s characteristic spastic paralysis symptoms. The mysterious connection between deep puncture wounds and tetanus disease was already recognized by ancient civilizations. Modern studies have now established the structure and function of TeNT: it acts by first targeting motor nerve terminals and then mysteriously undergoes retrograde transport into the spinal cord and then re-enters inhibitory neurons and blocks inhibitory neurons, leading to hyperactivity of motoneurons and muscle spasms. However, how the toxin traffics into the spinal cord has remained a major mystery for over a century. Here our preliminary studies have identified potential host factors that may mediate trafficking of TeNT into the spinal cord and our proposal seeks to carry out a comprehensive set of studies to examine TeNT binding to host factors at the biochemical level, resolve the toxin-host factor complex structure using an X-ray crystal structure approach, examine co-trafficking of TeNT and host factors within compartmentalized neuronal cultures, and evaluate the role of host factors for TeNT in vivo dynamics and pathogenesis using genetically modified mouse models. Our proposed studies will establish the long-sought-after host factor that mediates trafficking and pathogenesis of TeNT in animal models, thus solving a century-old major puzzle in our understanding of tetanus etiology. Our studies may also reveal retrograde trafficking pathways important for neuronal health and for delivery of therapeutics into the central nervous system.

NIH Research Projects · FY 2026 · 2025-01

Project Summary/Abstract – Capillary malformations (CM) are slow-flow vascular abnormalities present at birth and predominantly manifest as cutaneous lesions. In the context of the rare neurocutaneous disorder Sturge Weber Syndrome, individuals exhibit CM not only on the skin but also within the cerebral and ocular domains. 90% of CM are caused by a somatic activating mutation in GNAQ, the gene encoding the a-subunit of the heterotrimeric G-protein - Gaq. Specifically, the somatic GNAQ mutation involves the substitution of arginine (R) at amino acid position 183 with glutamine (Q) and is notably enriched in endothelial cells (EC) isolated from CM- affected regions in both the skin and the brain. Expansion of isolated EC from patient specimens reveals up to 21% mutant allelic frequency, reflecting the mosaic nature of the mutation. To date, little is known about how the p.R183Q mutation in the Gaq activation domain leads to abnormal capillaries. CM comprises enlarged vessels lined with sprouting EC surrounded by disorganized or a lack of mural cell (pericytes and smooth muscle cells) coverage. Immunohistologic examinations indicate that blood vessels in cutaneous and cerebral lesions of SWS patients exhibit extravascular fibrin and a deficiency of the tight junction protein zona occludens-1, signifying a compromised endothelial barrier in CM. Moreover, CM tissue sections also revealed MRC1pos, LYVE1pos, CD163pos and CD68pos macrophage cells surrounding the CM vessels of the brain and skin suggesting a pro- inflammatory environment. These findings provide a premise for the central hypothesize that the mutant EC recruit macrophages and that this leads to altered and dysfunctional EC-mural cell interaction. Alternative hypothesis is that interaction between mutant EC and neighboring mural cells leads to pro-inflammatory state driving macrophage recruitment and vessel dysfunction. The proposed study will test this hypothesis through the following aims: (1) determine macrophage interaction in mosaic endothelium (2) characterize the interaction among endothelial cells, mural cells, and macrophages in a multicellular spheroid model. Development and characterization of multicellular spheroid model will serve beyond the scope of the proposed work as a predictive platform for drug screening in contrast to traditional 2D cell cultures, effectively mimicking tissue responses to drugs and allowing for the evaluation of drug efficacy and toxicity. Hence, this also meets NIH’s continuing support for non-animal model alternative methods. These studies will be conducted under the supervision of co- mentors, Dr. Joyce Bischoff (vascular biologist) and Dr. Christopher Chen (biomedical engineer). My research advisory committee members and collaborators are leading experts in macrophage biology (Dr. Ruth Franklin), organoids (Dr. Jennifer Lewis and Dr. Alessandro Fiorenzano), neuropathologist (Dr. Sanda Alexandrescu) and bioinformatician (Dr. Vitor Rezende da Costa Aguiar). With additional support from the MOSAIC UE5 awardee sponsored professional development opportunities, continued training in the K99 phase will prepare Dr. Nasim for successful transition to independence.

NIH Research Projects · FY 2025 · 2025-01

ABSTRACT/SUMMARY Rare diseases, many of which are genetic and present early in life, disproportionately contribute to pediatric morbidity, mortality, and healthcare spending. Early identification of a precise genetic diagnosis may lead to improved genetically-informed care across the lifespan. One mechanism by which early genetic diagnosis may lead to improved healthcare and health outcomes is through improved surveillance for and management of known complications of the condition. However, families face many barriers to recommended plans of care, including lack of care coordination and limited access to subspecialty services due to geographic or resource constraints. At the same time, community providers who facilitate the follow-through for these infants may have difficulty interpreting genetic testing results and determining appropriate management of these conditions. The end result is a failure to fully realize the benefits of early genetic diagnosis. We plan to address this problem via GenePAL (Portable Approach to Longitudinal Genomic Healthcare for Infants): a pilot study of a novel, web- based application (“Nest”) optimized for mobile devices to empower families to understand and obtain appropriate follow-up care via personalized healthcare plans. In Aim 1, will plan a mixed-methods approach that is informed by the Consolidated Framework for Implementation Research to refine, implement, and preliminarily evaluate Nest: we will conduct surveys and semi-structured interviews to identify priorities and vales of parents and healthcare providers, the results of which will be used to refine Nest. In Aim 2, we will implement Nest in a prospective cohort of infants diagnosed with rare genetic conditions, developing a genomic care plan for each infant. Finally, in Aim 3 we will evaluate Nest in terms of the key implementation outcomes of acceptability and appropriateness through semi-structured interviews with parents and providers who interacted with the care plans generated in Aim 2. The findings from this pilot study will inform broader implementation of Nest, toward the goal of improved health outcomes for infants, optimized management, and a fuller realization of the promise of early genetic diagnosis. The data generated from this study will also provide critical insight into the role of patient-facing clinical informatics tools for management of genomic diagnostic information in pediatric rare disease that will drive other future studies to improve implementation of genomic-empowered healthcare.

NIH Research Projects · FY 2026 · 2025-01

Social determinants of health are widely recognized to impact child health across the life course. Evidence demonstrates that social risk screening and referral in ambulatory settings benefits child health and increases family connections to community resources. Professional guidelines from the American Academy of Pediatrics therefore recommend integration of social risk screening into routine pediatric care, and multiple regulatory bodies, including the Centers for Medicare and Medicaid Services, recently launched standard-setting initiatives requiring social risk screening and referral for hospitalized patients. However, we understand very little about screening and referral in pediatric inpatient settings, leaving critical knowledge gaps regarding how to best implement social risk screening and referral in pediatric hospital environments. As patients facing social stressors are known to have less access to primary care and increased hospital use, pediatric hospitalization represents a uniquely opportunistic time to screen for social risks and intervene prior to consequent health outcomes. My long term goal is to utilize implementation science methods to promote integration of social risk screening and referral interventions into pediatric inpatient care to improve health outcomes and hospital utilization for medically and socially vulnerable children. Leveraging a natural experiment, our objective in this proposal is to gain understanding on how to best implement social risk screening and referral across all types of hospitals caring for children in the US. We plan to accomplish this goal through three specific aims: (1) survey and interview clinical leaders from a national network of hospitals, including freestanding children’s hospitals, nested pediatric hospitals, and community hospitals, assessing facilitators and barriers experienced during implementation of social risk screening and referral across hospital types; (2) create an implementation toolkit for pediatric inpatient social risk screening and referral programs, by identifying and prioritizing implementation strategies to address barriers utilizing a modified-Delphi approach with a national panel of multidisciplinary hospital stakeholders; and (3) pilot the toolkit, assessing implementation and preliminary effectiveness at one tertiary and one community hospital. Completion of this research may promote and hasten successful integration of social risk screening and referral into routine pediatric inpatient care, aligning with professional guidelines and national standard-setting initiatives, and has potential to improve child health and hospital utilization. My complimentary training in implementation science frameworks, advanced mixed methods, multistakeholder-engaged research methods, and pragmatic intervention trials will propel these aims, facilitate my leading future hybrid effectiveness-implementation trials, and will bring me closer to my career goal of becoming an independent implementation scientist, focused on developing, implementing, and evaluating interventions to improve child health and hospital utilization for vulnerable pediatric populations.

NIH Research Projects · FY 2026 · 2025-01

PROJECT SUMMARY/ABSTRACT Epilepsies with onset in the first year of life are common, affecting 1 in 1000 infants, and are associated with significant morbidity, notably drug-resistant seizures and impaired developmental outcomes, and increased mortality. Although most infantile epilepsies have presumed molecular genetic etiologies, most cases remain genetically “unsolved” and the full molecular genetic landscape is unknown, which limits our ability to develop effective precision therapies. Further, the impact of genetic diagnosis in infantile epilepsies is largely unknown, which limits our ability to broadly implement genetic testing for this population. This proposal will address these knowledge gaps by identifying novel molecular genetic etiologies (genetic diagnoses) and evaluating their impact in a prospectively enrolled, longitudinally followed cohort of infants with unexplained epilepsy. Aim 1 will apply cutting-edge, comprehensive genomic analyses to identify novel genetic etiologies of infantile epilepsies, using short-read and long-read genome sequencing to identify pathogenic germline variants (Aim 1a) and deep sequencing to identify pathogenic mosaic variants (Aim 1b). Aim 2 will evaluate the multidimensional impact of genetic diagnosis (or lack thereof), using a validated measure of clinical utility and follow up of developmental progress and seizure frequency to assess impact on infants (Aim 2a) and a validated measure of parent-perceived utility and interviews with parents to assess impact on families (Aim 2b). The insights from this proposal will advance scientific knowledge and clinical care, with immediate diagnostic and therapeutic implications for infants with epilepsy, with the ultimate goal of advancing precision medicine and improving outcomes for this vulnerable population. Dr. D’Gama is a neonatologist with prior experience in basic neurogenetics research whose career goal is to become an independent physician-scientist at an academic medical center with a translational research program focused on precision medicine for infantile epilepsies and related neurogenetic disorders. Through the proposed K23 training program, Dr. D’Gama will gain new knowledge and skills in patient-oriented clinical and translational research, including advanced genomic sequencing and analyses, neonatal and infantile epilepsy phenotyping, epidemiology and biostatistics, qualitative analyses, and human subjects and clinical trials research, which will position her to successfully transition to independence. Dr. D’Gama’s mentor, Dr. Annapurna Poduri, is a leader in pediatric epilepsy and epilepsy genetics research, and her co-mentor, Dr. Margaret Parker, is a leader in neonatology and health services research. Both Drs. Poduri and Parker have extensive mentorship track records and Dr. D’Gama has also assembled a Scientific Advisory Committee and collaborators with complementary expertise. The institutional resources and intellectual community available through Boston Children’s Hospital and Harvard Medical School are world-class and will provide an ideal environment for Dr. D’Gama’s career development as a physician-scientist.

NIH Research Projects · FY 2025 · 2025-01

Abstract Rheumatoid arthritis (RA) is an autoimmune disease characterized by severe joint pain and debilitating inflammatory flares. There are currently no safe and effective treatments that achieve long-term remission, and therefore, RA patients are twice as likely to become chronic opioid users than non-RA pain patients. Maladaptive immune cell function is the underlying cause of RA which leads to joint inflammation and activation of nociceptor sensory neurons that trigger pain. Nociceptors, in turn, can regulate immune responses in tissues via peripheral vesicle release. Joints without sensory innervation are protected from arthritis, underscoring the key role of sensory neurons in controlling both pain and inflammation. Therefore, the neuroimmune axis is an excellent potential avenue, to treat RA. However, our understanding of the diverse sensory neurons and immune cells in the joints, how they interact with each other, and how these interactions change over the course of RA is limited. This proposal is a five-year plan of research, training, and career development focused on studying the role of neuroimmune interactions in RA pain and inflammation. In the two-year mentored phase, I will map the receptor-ligand interactions between sensory neurons and immune cells at a single-cell resolution in healthy and inflamed joints to identify neuroimmune pathways linked to arthritis, and also determine which neurons drive pain in response to immune ligands in arthritis. I will accomplish this by utilizing innovative approaches to construct receptor-ligand cell-cell interactomes, assess pain behavior in mice using machine learning and inhibit nociceptor activity in a spatially and temporally controlled manner. This scientific training will complement the career development activities selected to enhance my skills in scientific communication, leadership, mentorship, and ethics of scientific conduct. The insights and skills gained during this training will guide my research in the independent phase, elucidating how nociceptor-immune interactions contribute to the chronicity of RA. This research will uncover the biological mechanisms of joint inflammation and guide the development of novel neuroimmune-based therapies. I have assembled a diverse group of highly accomplished mentors who will ensure that I receive extensive training in pain neurobiology and in the assessment of sensory neuronal function in mice. My training will be further enhanced by the unique scientific environment of the Harvard Medical School and Boston Children’s Hospital research community, which is geared towards unifying my expertise in immunology and sensory neuroscience and enabling my successful transition into an independent academic position as a pain researcher.

NIH Research Projects · FY 2026 · 2025-01

RESEARCH SUMMARY B cells are among the most abundant tumor-infiltrating immune cells, and their presence in several solid tumors is associated with a more favorable prognosis. Yet, investigations on the role of B cells in cancer have so far yielded contradicting results, with some studies indicating a tumor-promoting role and others suggesting anticancer activity. In addition to their role in the production of antibodies, naturally occurring cancer-specific B cells can directly participate in tumor suppression by i) direct killing of tumor cells via granzyme B, TRAIL, and FasL expression and ii) by triggering robust T cell immunity, through activation of inflammatory responses and/or through their professional antigen-presenting cell (APC) function. Indeed, tumor-infiltrating B cells often form immune compartments called tertiary lymphoid structures (TLS), the presence of which in human tumors is associated with a favorable response to immunotherapy. Preclinical studies have shown that adoptive transplant of effector B cells collected from the tumor-draining lymph nodes (TDLN) of BC-bearing mice mediate the reduction of lung metastases and trigger the establishment of tumor-specific T cell immunity. However, tumor- specific B cells within TDLN are rare and difficult to specifically select and expand from the extensive polyclonal repertoire of patient B cells. Here, we hypothesized that homology-driven gene editing could be exploited to rapidly generate tumor-specific B cells that can be used to study the role of antigen-specific B cells in tumor progression, with the ultimate goal of developing innovative immunotherapeutic strategies. We will exploit an optimized B cell gene editing protocol we have recently developed to redirect the specificity of the endogenous B cell receptor (BCR) towards the HER2 tumor-associated antigen. The efficacy and tolerability of this adoptive immunotherapy strategy will be evaluated in vivo by exploiting both mouse models of breast tumors expressing human HER2 and xenogeneic murine models of human cell transplantations. The bidirectional interaction of transplanted B cells with immune cells of the tumor microenvironment will be investigated by single-cell transcriptome analyses. Establishment of epitope spreading and T cell immunity will be assessed by measuring tumor cytotoxicity and cytokine production of T cells harvested from the transplanted mice. If successful, this novel precision medicine strategy will redirect both the humoral and cellular immunity against cancer, thus introducing a new and powerful weapon to the oncologic therapeutic armamentarium and opening new avenues for empowered cancer-adoptive immunotherapies.

NIH Research Projects · FY 2026 · 2024-12

Abstract Balance and equilibrioception depend on vestibular hair cells that encode motion through mechanoelectrical transduction channels (MET) during movements of the head. Mutations in mechanoelectrical transduction channel genes (Tmc, Cib, Tmie, Lhfpl5) result in issues with hearing and balance in both human patients and mouse models. The long-term goal of this project is to better understand how heterogenous regional expression of MET components contribute to vestibular responses, and whether early expression of MET components define a critical period for gene replacement therapy. TMC1 and TMC2 are components of the MET channel and provide ionic influx of potassium and calcium during mechanical deflection whereas CIB2 and CIB3 localize to the intracellular domain of the MET channel acting as putative calcium sensors. Tmc and Cib genes are heterogeneously expressed in different regions of the mouse utricle and saccule with peak expression levels during neonatal development (P0-P7). However, it is unclear what the functional consequences of heterogenous MET subunit expression are on the development of mature vestibular responses. In Aim 1 I plan to characterize how this heterogenous expression contributes to regional differences in vestibular hair cell activity using calcium (Ca2+) imaging of utricles and saccules. Qualitative comparison of regional hair cell differences in Ca2+ influx will be evaluated with fluorescent Ca2+ indicators, and FM dyes which enter hair cells through functional MET channels. Qualitative differences between striolar versus extrastriolar hair cells will be evaluated in developing (P7) and mature hair cells (P30). Additionally, regional changes in Ca2+ influx will be assessed in Tmc2 KO and Cib2 KO mice where preliminary data shows failure of striolar hair cells to take up FM1-43 dye. In Aim 2 I will investigate whether an early period of regionally heterogenous MET expression precludes gene replacement at later stages. This will be examined by injecting adult (P60) Tmc1 KO, Cib2 KO, and Tmie KO mice with AAVs replacing a functional copy of the gene of interest (GOI). Subsequent testing of Vestiular Evoked Potential (VsEP), vestibulo ocular reflexes (VOR), and FM1-43 dye uptake will determine whether gene replacement is effective in mature hair cells. Preliminary results show that we can restore vestibular function in Tmc1, Cib2, and Tmie mice at neonatal stages, but it is unclear whether the therapeutic window extends to include mature adult vestibular hair cells. RELEVANCE: In the US alone 14.8% of patients seek clinical assistance due to balance issues, and the predominant cause of inner ear dysfunction typically includes a genetic component. Many patients receive diagnoses as adults, but precision treatment may have a limited therapeutic window of efficacy as seen in cochlear hair cells. This proposal will advance our basic understanding of vestibular hair cell function and will assist in the development of gene therapies for MET related balance dysfunction to advance precision treatment.

NIH Research Projects · FY 2025 · 2024-12

Project Summary Usher Syndrome (USH) is a devastating incurable syndrome resulting in deafness, vestibular dysfunction, and retinal degeneration leading to blindness (retinitis pigmentosa). Usher Syndrome represents the most common genetic cause of deaf blindness; it affects 20% of infants with bilateral moderate to profound congenital sensorineural hearing loss, 15-30% of patients with retinitis pigmentosa and 50% of deaf-blind children. The Usher gene family includes 12 genes encoding for proteins expressed in both the photoreceptor cells of the retina and the sensory hair cells of the inner ear. Belonging to different families, the Usher proteins interact in a network that appears to be essential for the proper development, maturation and survival of the photoreceptor and inner ear hair cells. This proposal is written to support the next International Symposium on Usher Syndrome. This conference will be the fifth of its kind and is meant to convene scientists whose research and interest is closely related to Usher Syndrome, and families and patients who suffer from the disease. It will take place in Nijmegen, Netherland, June 19-21, 2025 and will include two scientific days and one family day. The goal of the symposium is to bring together scientists, clinicians and Usher patients and their families in view of 1) promoting research in emerging areas of Usher Syndrome with the goals of advancing diagnosis, prevention, treatment, and cure; 2) To present new research findings and develop future research strategies among scientists involved in Usher Syndrome research; (3) To promote and enhance collaboration among researchers and clinicians from different institutions and different research focus areas who study Usher Syndrome. (4) To educate families affected by Usher Syndrome about research advances and to promote collaboration between these families and Usher syndrome researchers. The Symposium will support the mission of the NIDCD and NEI by focusing on sharing new knowledge of a syndrome that affects hearing, balance and vision and results in significant communication impairments. The symposium will support the public health and educational mission of the NIH by including scientists, clinicians and patients/families with USH to encourage collaboration on both a formal and informal basis.

NIH Research Projects · FY 2026 · 2024-12

Motivational disruptions are at the center of mental illness, triggering depression, addiction, bipolar, and a variety of other disorders. Shifts in dopamine signaling often underlie these conditions, but the molecular mechanisms that trigger and sustain lasting dopaminergic changes are poorly understood. Elevations in dopamine that occur during drug use have long been known to trigger an enduring decrease in natural dopamine signaling. This homeostatic downregulation is a central factor in addiction, both because addicts require more and more drug and because non-drug related behaviors become devalued. To my knowledge, dopamine-triggered dopamine desensitization has never been linked to the motivational control of natural behavior. I present a new, genetically tractable system that links the devaluation that occurs after goal attainment to dopamine desensitization. This system brings the power of Drosophila genetics to this important and widespread phenomenon, making use of innate behavior in a well-defined system with genetic access to dopamine-releasing and dopamine-receiving neurons. I show that devaluation occurs over the course of repeated bouts of Drosophila mating behavior. We have localized this effect to the desensitization of the D2 dopamine receptor on a defined set of decision-making neurons. I propose to assemble a detailed molecular pathway that initiates and sustains desensitization to dopamine. We will integrate the results from new genes identified in a genetic screen with the knowledge obtained over decades in the GPCR inactivation and addiction fields to understand how the rewarding dopamine released during prior matings triggers a long-lasting desensitization of the D2 receptor. Our data implicate β- arrestin signaling, which inactivates the D2 receptor in mammals, another early indication of the broad relevance of our findings. Our ability to precisely label and manipulate the neurons that receive the dopamine signal gives us a unique opportunity to observe the mechanisms of dopamine desensitization and the molecular and physiological consequences. We have pioneered the use of 2-photon Fluorescence Lifetime Imaging (FLIM) in Drosophila neurobiology and have developed a preparation to monitor the devaluation of mating at the level of D2 receptor desensitization. We will use the FLIM-compatible biosensors for calcium and CaMKII that we have adapted for the fly, and will use them, together with our molecular genetic manipulations to visualize desensitization and its underlying mechanisms. These experiments will establish a new platform for rapidly identifying and understanding the genes involved in triggering and sustaining behaviorally-relevant shifts in dopamine reception. They will identify new pathways for control over changes in motivational states, generating a molecular-level understanding of motivational control and developing new hypotheses for treating the consequences of its disruption.

NIH Research Projects · FY 2026 · 2024-12

PROJECT SUMMARY/ABSTRACT The innate immune effector TRIM56 restricts a broad range of viruses, yet its molecular mechanisms remain elusive. I propose to uncover the underlying mechanism of TRIM56’s antiviral activity, with a focus on HIV-1, where its role is better understood. TRIM56 is an E3 ubiquitin ligase, which typically mediates the transfer of ubiquitin to a target substrate with potential impacts on stability, binding, or trafficking of the target protein. Unlike most E3 ligases, which utilize protein-interaction domains for substrate recognition, TRIM56 utilizes an NHL domain known for its RNA binding activity. This unique feature suggests that TRIM56 may employ an unconventional mode of target recognition, possibly involving direct interaction with viral RNA. My preliminary data reveal that TRIM56 interacts with the HIV-1 genome similarly to the viral packaging protein Gag, and that the ubiquitin ligase function of TRIM56 is important for restricting HIV-1. While these data do not directly demonstrate whether TRIM56 NHL binds RNA directly or indirectly, they support the two following hypotheses: (a) TRIM56 may directly bind the HIV-1 RNA, thereby blocking Gag binding and subsequent viral packaging, while ubiquitinating nearby proteins co-occupying the RNA to limit viral RNA stability or translation; (b) TRIM56 may bind HIV-1 RNA indirectly through Gag, and subsequently ubiquitinate Gag to promote its degradation. To distinguish between these hypotheses, I will (I) investigate the mode of viral genomic RNA recognition (whether direct or indirect) using biochemistry and structural biology; (II) determine the ubiquitination target of TRIM56 using proteomic analyses and viral translation and packaging assays. The proposed research promises to identify a novel mechanism for viral RNA detection and restriction. It will also lay the groundwork for further exploration of TRIM56’s actions against other viruses, with the ultimate goal to identify a unifying mechanism for broad viral sensing. I will carry out this research in the laboratory of Dr. Sun Hur at Boston Children’s Hospital (BCH)/Harvard Medical School (HMS). Dr. Hur is an expert on innate immune response to pathogen nucleic acids, and her lab additionally hosts expertise in protein biochemistry and structural biology. The BCH/HMS environment supports postdoctoral research fellows with numerous core facilities, technique trainings, and career development workshops, in addition to supporting a rich intellectual environment in which many labs collaborate. I will leverage the surrounding expertise, research seminars, and professional development courses during the fellowship period to strengthen my research and to prepare to independently head a lab.

NIH Research Projects · FY 2026 · 2024-12

Project Summary Attention deficit hyperactivity disorder (ADHD) and disruptive behaviors (DBs) affect up to 9% of children. Under-diagnosis and under-treatment of ADHD and DBs disproportionately affect children with socioeconomic stress and disadvantage, and lead to early childhood morbidities (e.g., preschool expulsion, learning difficulties) and later adult adversity (e.g., mental health disorders, unemployment, suicide). Identification of infants and toddlers at risk for early ADHD and DBs, especially in families with socioeconomic disadvantage, would allow for provision of preventative interventions to minimize the negative impacts of these symptoms. Additionally, improved understanding of the neurodevelopmental pathways to ADHD and DBs would maximize the impact of developmentally and individually targeted preventative interventions. Accordingly, this study will leverage a large, currently enrolling NINDS funded sample of low-resourced, racially and ethnically diverse infants seen in an urban primary pediatric care clinic, for whom EEG, developmental and environmental data are already being collected longitudinally from 4- to 24-months old. The current investigation will collect EEG and ADHD/DB-focused measures at an additional time point (3- or 4-year well- child visit) in order to measure ADHD and DB outcomes in preschoolers. Our first aim is to characterize neurodevelopmental trajectories leading to early ADHD and DB symptoms using repeated EEG in very young children. Second, we will utilize parent survey data and medical record abstraction collected repeatedly across infancy and early childhood to integrate effects of social determinants of health (e.g., socioeconomic status, caregiver well-being, access to basic needs) into longitudinal models of brain development and behavioral regulation. Results from this study will inform future development of scalable and objective tools to identify children with increased likelihood of ADHD and DBs across resource settings, and facilitate development of interventions that use resources efficiently. Importantly, longitudinal characterization of this large cohort will provide greater understanding of what neurophysiological and environmental factors influence brain and behavioral development, and when the brain may be most sensitive to intervention.

NIH Research Projects · FY 2026 · 2024-11

Abstract The goal of this proposal is to examine the role of Candida albicans Tor1 kinase and specifically, its least- conserved domain, N-terminal HEAT repeats, in enabling this fungal pathogen to withstand oxidative stress imposed by the host immune system. We showed that C. albicans cells lacking Tor1 N-terminal HEAT repeats, which are protein-protein interaction domains, are exquisitely hypersensitive to oxidative stress. They also dysregulate dedicated oxidative stress management systems of the fungus, and their metabolic output of intermediates required in redox homeostasis is defective. Tor1 point mutants that we recently engineered, show that these severe defects are not attributable to simple hypo- or hyperactivity of the kinase. Instead, these cells’ oxidative stress management defects appear to relate to functions residing in the N-terminal HEAT repeats. Notably, we found a core enzyme of C. albicans redox homeostasis, that is structurally and functionally completely different than its human counterpart, to be dysregulated in cells lacking Tor1 N-terminal HEAT repeats. We also found that depletion of this enzyme leads to starkly decreased TORC1 signaling even in conditions of ample nutrients and absent stress. We propose to define the interactions between Tor1 and its N-terminal HEAT repeats with dedicated C. albicans oxidative stress management systems.

NIH Research Projects · FY 2025 · 2024-11

Abstract The biennial Hemoglobin Switching Conferences have been ongoing for 47 years, and there are multiple reasons for its resounding, continued success. First and foremost, the current organizers Sankaran, Zon, and Brand, following in the footsteps of the prior organizers Stamatoyannopoulos, Nienhuis, Higgs, and Engel, strive to identify and highlight new discoveries by always including early stage and diverse investigators and studies that impinge on the process of globin biosynthesis. Second, this is the only venue (other than the annual ASH meeting, with more than 30,000 participants) that brings together basic scientists and clinicians to discuss both the molecular and developmental origins of, and treatments for, the hemoglobinopathies, the most common inherited diseases in man. Third, the meetings have historically evolved with intense focus on wherever the science led, thus remaining extremely topical, and has not only been the forum for presenting the first cDNA clones, the first cloned human genomic locus (and the first mutations in same), the structure of erythropoietin and the discovery of the GATA and KLF transcription factor families, but it has also launched the careers of many of the current leaders in this field (indeed, numerous former postdoctoral fellows and current faculty first presented their work in plenary sessions at this conference). Fourth, this is the only meeting on this topic that routinely has approximately equal attendance by investigators from both inside and outside the U.S., and this fact is reflected by the biennial alternation in conference site between the U.S. and Europe. Fifth, this is the key venue where important basic science discoveries have led to key clinical advances, For example, the work by Sankaran, Orkin, and others reporting BCL11A as a key regulator of fetal hemoglobin, which was initially reported at this meeting, has now led to the recent approval in the UK of the first CRISPR curative therapy for sickle cell disease and thalassemia. In 2024 the Conference will be held in San Juan, Puerto Rico.

NIH Research Projects · FY 2026 · 2024-11

Project Summary Plasmodium falciparum is a unicellular eukaryote that causes the most severe form of human malaria. In the human blood stage of malaria infection, P. falciparum undergoes asexual replication to propagate itself exponentially, resulting in the classic symptoms of malaria. This is a critical stage of the parasite’s life cycle and a compelling process to target for new therapeutics. Plasmodium utilizes a divergent form of cell division with a unique method of cytokinesis called segmentation, wherein genetic material and organelles are simultaneously partitioned into 20-36 daughter cells. This is a high-fidelity process that is largely driven by the basal complex, the putative contractile ring of the parasite. Despite its pivotal role for parasite survival and proliferation, our mechanistic understanding of the basal complex is limited. To address this knowledge gap, our lab and others have identified a dozen proteins that comprise the basal complex. Of these, three proteins emerged as a subgroup, each of which contain transmembrane domains. Previous studies demonstrate that they localize to the basal complex and my preliminary data suggests that at least one of these is important for parasite replication. It remains unclear, however, whether these proteins are associated with a membrane and what specific function they serve during segmentation. In this study, I will integrate biochemical and super-resolution microscopy approaches to decipher the precise location of the transmembrane proteins. This will provide direct evidence of a link between the basal complex and parasite membrane. I will also use direct and inducible knockout systems together with cell viability assays and live-cell microscopy to thoroughly interrogate the function of the transmembrane proteins. This will reveal how transmembrane domains contribute to the broader cytokinetic function of the basal complex. Collectively, the findings from this study will represent the first step towards a mechanistic understanding of the basal complex in Plasmodium. Further, insights from this study will enable future comparative analyses between parasites and model eukaryotes, revealing parasite-specific adaptations that can be leveraged for novel therapeutics. The proposed research will thoroughly develop my conceptual and technical expertise in Plasmodium biology. Specifically, this proposal will expand my current skills in biochemical, microscopy, and genetic techniques, honing my technical expertise and establishing a research niche. Moreover, I aim to leverage both our understanding of eukaryotic cell division and my experience in the related parasite Toxoplasma, to achieve these goals. Training at Boston Children’s Hospital and within the larger Harvard community offers a rich and stimulating environment to support this proposal and my development as a scientist and mentor. Here, I will build strong scientific relationships with leaders in microbiology and molecular biology through local meetings and international conferences and commit to training the next generation of scientists. Overall, I am well positioned to carry out this research and propel my career towards an independent investigator position.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY Intrinsically photosensitive retinal ganglion cells (ipRGCs) are neurons of the mammalian eye that capture light with a molecule called melanopsin, triggering a phototransduction cascade to generate an electrical response. This response influences dozens of brain regions and is essential for light’s normal regulation of processes that include the circadian clock, sleep, mood, and pupil constriction. Our preliminary experiments reveal ipRGC response features that are suited to these processes and qualitatively distinct from those of the classic rod and cone photoreceptors. We propose to characterize these response features, learn how they arise from molecular mechanisms within ipRGCs, and define how they shape signals sent from ipRGCs to the brain. We will apply patch-clamp electrophysiological recording to the ex vivo mouse retina and its isolated cells, introducing methods that increase the physiological relevance of our findings. Our experiments will strengthen the basic understanding of ipRGCs, sensory transduction, and ion channels. They will also provide insight into the broad question of how cells of particular types are tailored to specific tasks.

NIH Research Projects · FY 2024 · 2024-09

Idiopathic Pulmonary Fibrosis (IPF) inflicts a significant healthcare burden and high rate of mortality in the U.S. Current treatment options can slow the rate of lung function decline, but the five-year survival remains low, therefore there is clear unmet medical needs for novel therapeutics. Here, human genetics can catalyze drug development process by identifying new biological targets and elucidating the underlying pathways. As a rare polygenic disorder, however, IPF poses challenges to existing disease gene-mapping strategies due to the extensive locus heterogeneity and difficulty of assembling massive sample sizes typical of common polygenic disorders. This project aims to develop more effective gene-mapping methods for rare polygenic disorders such as IPF. Our approach is motivated by three complementary strategies for small genetic studies: (i) pleiotropy-informed SNP association tests, which can be extended to take advantage of the pleiotropy of disease SNPs with gene expression traits and to further boost power by accounting for the network connectivity to known disease genes; (ii) Polygenic Risk Scores (PRS), which can be highly useful for rare disorders by capturing the effect of disease modifiers in the genetic background; and (iii) highly modular network structure of disease genes, which can be leveraged to reduce genetic heterogeneity among cases. In Aim 1, by extending a pleiotropy-informed association test we had previously proposed, we will develop a new network model-based association test informed by pleiotropy to gene expression traits. We will apply this method to publicly available IPF GWAS data and expression Quantitative Trait Loci (eQTL) of IPF-relevant tissues and cell populations. In Aim 2, we will develop a new rare-variant association test directly accounting for the contribution of genetic background using PRS. We will apply the new method to sequencing data of ~1,500 IPF cases and ~15,000 unaffected controls from CGS-PF and TOPMed studies. In Aim 3, we will identify novel IPF genes by leveraging the association between disease gene modules and comorbidities. Known IPF genes are clustered in multiple tightly inter-connected gene modules in biological networks, and mutations disrupting each network modules cause a distinct set of comorbidities in IPF patients. We will leverage the modularity of IPF genes and comorbidity to find novel IPF genes in exome data of UK Biobank and MGB Biobank. In reverse, we also will test if genotypes of key IPF gene modules can inform the course of comorbidity development in patients by inviting 10 CGS-PF study participants to a reverse genetics study. Ultimately, the findings from these studies will uncover novel genes and pathways underlying IPF and develop new computational strategies generally applicable to rare polygenic disorders.

NIH Research Projects · FY 2025 · 2024-09

Project Summary/Abstract: Pulmonary arterial hypertension (PAH) is a progressive vascular disorder characterized by narrowing and remodeling of the distal arterioles. Current treatment options are unable to prevent disease progression, leading to a median survival time of five years after diagnosis. Therefore, there is an urgent need to develop new disease-modifying therapies. Increased pulmonary blood flow is known to stimulate vascular remodeling in patients with congenital heart disease (CHD) and after major lung resection, although the underlying mechanism is unclear. Patients with left-to-right shunts such as ventricular septal defects and atrioventricular canal defects are at high risk for PAH. A “second hit” in the context of pulmonary overcirculation, such as hypoxia, may lead to irreversible vascular remodeling and disease progression. Current rodent models of flow-induced pulmonary hypertension (PH) such as the left pneumonectomy and SUGEN rat do not fully recapitulate patients' physiological conditions. To improve patient outcomes, animal models more representative of patients are needed, along with a better understanding of the underlying molecular mechanisms. My goal is to generate a novel animal model to study flow-induced PH. Establishing a murine model of PH that mimics patients with CHD will enable the development of therapeutics capable of reversing flow-induced vascular remodeling. The second goal of this proposal is to discover key insights into the response of smooth muscle cells (SMCs) to vascular remodeling from increased pulmonary blood flow. Specifically, the aims of this proposal are to 1. Develop a new mouse model of flow-induced PH that better represents patients' physiological conditions, 2. Describe the contribution and underlying mechanisms in which SMCs contribute to shear stress- induced vascular remodeling, and 3. Discover novel therapeutic approaches for patients with PAH-CHD.

NIH Research Projects · FY 2024 · 2024-09

Project Summary Genomic research presents a unique opportunity for molecular diagnosis of individuals with rare disease, which may empower precision therapies to alleviate the individual and public health burden of these conditions. However, minoritized populations remain underrepresented in rare disease genomic research, and optimal strategies to address this lack of representation remain poorly understood. The consequences of these knowledge gaps extend beyond the research realm and threaten the equitable provision of genomic medicine services in clinical practice. This project takes the critical next step towards further understanding, and ultimately breaking down, barriers to rare disease genomic research participation for historically underrepresented populations, in a hybrid type 3 effectiveness-implementation study design informed by the RE-AIM (Reach, Effectiveness, Adoption, Implementation, Maintenance) framework. A diversity, equity, and inclusion toolkit for genomic researchers will be developed and implemented within the Broad Institute’s Rare Genomes Project (Aim 1), with concurrent evaluation of multiple dimensions of effectiveness employing a mixed-methods approach (Aims 2 and 3). In Aim 1, stakeholder interviews will be conducted with both rare disease genomic research participants as well as providers who refer these participants to further explore barriers and facilitators of research engagement, with specific attention to study logistics and inclusive practices. This qualitative analysis will inform development and refinement of a toolkit comprising specific strategies portable to other environments to improve diversity, equity, and inclusion. This toolkit will then be implemented within the Rare Genomes Project in order to enroll a cohort of 200 participants who are historically underrepresented in rare disease genomic research, with concurrent evaluation of the following implementation outcomes: Reach, as reflected in the demographics of study participants; Adoption, as reflected in the characteristics of referring providers; and Implementation fidelity, as reflected in the proportion of participants who complete the study process from enrollment to sequencing. In Aim 2, Effectiveness of genome sequencing (GS) for this cohort of 200 participants will be evaluated, reflected in diagnostic and clinical utility, with analysis taking into account the impact of social and structural informants of health on these outcomes. In Aim 3, patient-centered utility of GS will be assessed both in this cohort as well as a larger, diverse cohort of rare disease genomic research participants to better assess longitudinal, multidimensional impact (Maintenance). This study will therefore generate utility data from a diverse population that provides real-world guidance regarding equitable applications of GS for rare disease diagnosis. Study results will inform strategies to improve access to GS and will identify key areas of clinical impact in order to direct policies related its clinical adoption. Successful completion of the proposed study will thus directly inform equitable clinical implementation of genomic medicine.

NIH Research Projects · FY 2024 · 2024-09

Project Summary ATP1A3-related disorders are a group of disorders caused by mutations in the ATP1A3 gene that encodes the α3 catalytic subunit of Na+/K+-ATPase transmembrane ion pump. Three classic phenotypes are known, which are alternating hemiplegia of childhood (AHC), rapid-onset dystonia-parkinsonism (RDP), and cerebellar ataxia, areflexia, pes cavus, optic atrophy, and sensorineural hearing loss (CAPOS). These conditions all carry significant neurological morbidity and there is no effective treatment. They are rare disorders but the prevalence of AHC may be as high as 1/100,000. Development of novel therapies is urgently needed. In order to achieve disease-modifying treatment in this condition, we need to: 1) fully understand the genotype-phenotype correlation and natural history of the disease, 2) have a patient population that is ready for clinical trial, and 3) have a novel treatment modality. In this project, we will achieve these goals by studying ATP1A3-related disorders in Brazil. Taking advantage of a large clinical whole-exome database in Brazil, we aim to build a first large cohort of patients with ATP1A3-related disorders in Latin America. We will analyze genotype- phenotype correlation and collect biospecimens from patients for induced pluripotent stem cell (iPSC) generation and biomarker identification, and two-year natural history study will prepare the population for future clinical trials. At the same time, we will develop allele-specific oligonucleotides (ASOs) against pathogenic ATP1A3 mutations or benign single nucleotide polymorphisms that are linked to pathogenic ATP1A3 mutations to knockdown mutant ATP1A3 mRNA. iPSCs from Brazilian patients will be used as a platform for ASO development. All aspects of this project will be done in close collaboration between the Brazilian and US researchers. This proposed project holds great significance because: 1) ATP1A3-related disorders are a condition with significant morbidity and mortality without effective treatment, 2) this first systematic clinical study of the condition in Brazil will lead to new insights into natural history and genotype-phenotype correlation of the disease, 3) the study will prepare the Brazilian patients for future clinical trials, 4) novel ASO treatment will be applicable not only to patients in Brazil but also worldwide, and 5) the study will help build research capacity for precision medicine in Brazil. Further, this proposed project is highly innovative because: 1) the unique, little studied patient population in Brazil fills the existing geographical gap in the study of this condition, 2) subject identification through a clinical exome database will allow enrollment of individuals with atypical and novel phenotypes and informs the pathogenesis, 3) this study develops neurons differentiated from patient-derived iPSCs as an in vitro model and uses them as a platform for therapeutic development, which has not yet been commonly done in ATP1A3-related disorders, 4) allele-specific ASOs represent a novel therapeutic approach for this condition, and 5) we will develop ASOs targeting pathogenic mutations as well as benign polymorphisms linked to pathogenic mutations, expanding the drug’s target population.

NIH Research Projects · FY 2024 · 2024-09

Novel strategies are needed when standard approaches fail to delineate mechanisms by which mutations cause disease. This is the case for Progressive Pseudorheumatoid Arthropathy of Childhood (PPAC), a degenerative joint disease caused by genetic deficiency of Wnt-inducible signaling pathway protein 3 (WISP3, also known as CCN6). Patients appear normal at birth, but during childhood develop painful, polyarticular, degenerative joint disease. In teenage years patients require hip and knee arthroplasties, and they have joints that resemble end-stage osteoarthritis. Because it is unethical to obtain prospective cartilage biopsies from children, an animal model is needed to understand the pathobiology that underlies PPAC. Wisp3 knockout mice did not model PPAC. Therefore, we knocked out WISP3 in sheep, which are larger and longer-lived animals and have cartilage that is more similar to human cartilage than mice. Like patients with PPAC, our KO sheep appeared clinically normal at birth but developed altered gaits and joint tenderness with movement and palpation by 5 months of age. Now that we have an animal model of PPAC, we propose to expand our flock of KO sheep and prospectively evaluate them clinically, radiographically, histologically, transcriptomically, Raman spectroscopically, biomechanically and biochemically. We will couple these studies with histologic and transcriptomic data we obtain from articular-like cartilage tissues that we differentiate from PPAC patient- derived induced pluripotent stem cells (iPSCs). We previously studied 2 isogenic pairs of WISP3-deficient and WISP3-sufficient human PSCs, and observed WISP3 deficiency altered several biologically plausible pathways involved in chondrocyte differentiation and cartilage homeostasis. We now want to determine if these differences can be strengthened and refined by including 3 new PPAC patient-derived iPSC lines and their isogenic controls. Pathways suggested by iPSC studies can be tested in KO sheep, and data from KO sheep can be compared to that obtained from iPSCs. Successful completion of our sheep experiments will provide insights into the genes and pathways that are altered by WISP3-deficiency in vivo. Successful completion of our iPSC experiments will inform us about the utility of using patient iPSC- derived articular-like cartilage tissue to model in vitro that which occurs in articular cartilage in vivo. Together, these complementary approaches will provide important information about the function of WISP3 in cartilage. The KO sheep and the patient-derived iPSCs also can serve as preclinical models for testing therapies to benefit patients with PPAC and, perhaps, be used to identify new approaches for protecting cartilage from common degenerative joint disorders.

NIH Research Projects · FY 2025 · 2024-09

Summary Opioids are commonly prescribed for a variety of acute and chronic pain states. Unfortunately, prescribed opioids can be tampered with or diverted, both of which can have severe consequences (e.g., lethal overdose). Tamper-resistant formulations have had limited success, and cannot prevent diversion of drugs. Injectable formulations that can deposit an extended course of treatment in the body would make diversion nearly impossible once administered, but most, if stolen, can still be tampered with (and the opioid extracted) by relatively simple means. Importantly, once administered to patients, they deliver drugs at a rate that does not change with the varying needs of the patient. They are therefore unusable for acute pain: if opioids were continuously released at a rate adequate for acute pain, patients would be narcotized for extended periods. Here, we propose to develop depot formulations made of polymers which are attached covalently to opioids by photolabile linkers. The depots would be injected subcutaneously by healthcare providers, for example, prior to discharge after a procedure. The covalent linkers would render the depots difficult to tamper with, as the drug cannot be easily removed from stolen devices by simple dissolution in organic solvents. Moreover, having been injected into the body the formulations could not be diverted from patients. The covalent bonding prevents the drug from being active even after injection, and the drug is only released by irradiation – not polymer degradation. However, since the bonds are photolabile, the patient would be able to release the drug with a simple handheld or wearable light source. This approach would allow patients to determine the timing, intensity, and duration of analgesia throughout the postoperative period.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY Hematopoietic stem cell (HSC) gene therapies with lentiviral or CRISPR based gene modification are demonstrating favorable safety profiles and remarkable efficacy to treat severe monogenic blood disorders with unmet clinical need, including X-linked Severe Combined Immunodeficiency (X-SCID) and Sickle Cell Disease (SCD), with feasibility of rapid progress from target discovery and preclinical validation to first-in-human trials. Despite these successes, the technologies and procedures used in these trials require complex ex vivo bespoke manufacturing of autologous cell products, are expensive and difficult to scale to treat many patients, and intrinsically associated with significant risks related to ex vivo manipulation, myeloablative chemotherapy and bone marrow transplantation. We here propose a novel technology platform based on alpha-retroviral vectors that shares many of the advantages with the currently dominating delivery platform, lentiviral vectors, but has increased flexibility to deliver diverse therapeutic payloads, including non-integrating Cas9-based genome editors, high potential for in vivo gene delivery, and compatibility with scalable production systems. The long- term goal of our proposal is to further develop this platform for the delivery of gene therapy payloads (integrating DNA and Cas9/base editor ribonucleoprotein complexes into HSCs in vivo to enable simple, more economical and widespread application of gene therapies to serve diverse patient populations. A shorter term goal is to employ the same technology for improved ex vivo delivery of gene therapy payloads to HSCs. Our central hypothesis is that a single platform based on alpha-retroviral vectors is suitable for ex vivo and in vivo delivery of genetic therapies with diverse requirements in terms of the type of therapeutic payload and targeting specificity. In preliminary studies, we have demonstrated that both integrating vectors and non-integrating genome editors can be delivered into HSC ex vivo and in vivo. Here, we will capitalize on these results and further expand the reach of these innovative genetic engineering tools with the objectives to: i) develop integrating vectors to deliver a curative transgene to HSCs in vivo as a modality to treat X-SCID; ii) engineer virus-like particles for the delivery of Cas9 into quiescent HSC with minimal toxicity both ex vivo and in vivo to treat SCD; and iii) to modify virus-like particles for the transfer of base editors into HSCs for correction of the common mutation underlying the bone marrow failure disorder Shwachman-Diamond Syndrome. The alpha- retroviral based viral particle technology will be used to develop treatments for this exemplary group of diseases and is also intended to address key general shortcomings of current GT approaches and could be readily transferable to many other monogenic diseases.

NIH Research Projects · FY 2024 · 2024-09

Project Summary/Abstract: African American (AA) individuals with prostate cancer (PCa) face significantly worse clinical outcome than their European American (EA) counterparts. There is a strong correlation between the stage of disease (localized versus metastatic) at diagnosis and long-term survival. While patients diagnosed with localized disease have a 99% 5-year survival, patients presenting with metastatic disease have significantly worse prospects, an about 32% 5-year survival. A minimally invasive, plasma based diagnostic method could significantly improve chances of detecting PCa at an early stage and thus reduce PCa related mortality. There are two, next generation sequencing (NGS)-based methods that likely have the required sensitivity and specificity of early detection of PCa from liquid, plasma biopsies. The first is based on the altered fragmentation profile of cancer derived cell free DNA (cfDNA). In this, whole genome sequencing (WGS) is applied to plasma derived cfDNA and the ratio of short to normal fragment size indicates the presence of cancer. The second method is based on a specific pattern of methylated DNA loci across the genome and combines cell-free methylated DNA immunoprecipitation with high throughput sequencing. We found that the altered cell free DNA fragmentation profile is a highly sensitive, robust indicator of the presence of metastatic prostate cancer. In this proposal we will investigate whether this method can identify cases when AA men present at diagnosis or at later time-point after post-primary treatment with metastatic PCa. We will also investigate whether a clinically useful sensitivity and specificity is retained as we analyze samples at earlier stages of disease. We will benchmark and optimize an experimental and computational protocol to detect prostate cancer at various stages based on the fragmentation profile of plasma derived cell free DNA. This will be applied to AA patients that presented with metastatic disease at diagnosis or later developed metastatic disease, to AA PCa patients with localized or locally advanced disease, and to AA PCa cases where sequential plasma was collected at various times, ranging from right before surgery to ten years before disease progression. We will determine the sensitivity and specificity of a fragmentomics profile-based method for early detection of PCa of various disease stages. Similarly, we will benchmark and optimize an experimental and computational protocol to detect prostate cancer at various stages based on cell free methylated DNA profiles. The next generation sequencing based methylation profiling of plasma derived cfDNA samples will be applied to AA PCa of various disease stages and we will determine the sensitivity and specificity of DNA methylation profile-based method for early detection of PCa of various disease stages. This will establish a non-invasive method for the early detection of PCa of AA men.