Grants

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Interplay of heterosexual male-female partner microbiomes, Chlamydia trachomatis and transmission

NIAID - National Institute of Allergy and Infectious Diseases

SUMMARY Chlamydia trachomatis (Ct) is the leading cause of bacterial sexually transmitted infections (STI) worldwide. About 128M Ct cases are estimated to occur annually according to the WHO and 1.6M in the US. The Western Pacific Region (WPR) has over 61M of these global infections with a prevalence of ~35-40% among adolescents and young adults under the age of 25 years. About 50% of men and 80% of women are asymptomatic. Syndromic management—the reliance on signs and symptoms of STIs to determine the need for antibiotics—is often practiced in this region but is imprecise, leading to unchecked transmission to partners. Complications from these infections include infertility, pre-term birth and proctitis in addition to an increased risk of HIV and other STIs. Studies of Ct STIs focus primarily on female urogenital infections and rectal infections among men who have sex with men (MSM) with few studies of urethral infections among men who have sex only with women (MSW). While the US Centers for Disease Control (CDC) recommends annual Ct screening for women under 25 years and those at high risk (e.g., multiple partners), screening for MSW is not recommended, This leaves a gap in our knowledge about MSW urethral Ct infections and transmission dynamics to and from female partners. Our unifying hypothesis is that Ct infection, transmission and disease pathogenesis are driven/perpetuated by the interaction of male urethral and female partner endocervical, vaginal and rectal microbiomes and host immune responses that are influenced by Ct infections. For the proposed research, we will study a large cohort of male- female partners with a high prevalence of Ct in the male urethra and female endocervix, vagina and rectum. The microbiomes of these sites will be characterized by metagenome shotgun sequencing (MSS), the host immune responses by quantitative protein assays, and the Ct genomic characteristics by whole genome sequencing (WGS) and the respective analyses. Resistomes (i.e., antibiotic resistance genes (ARGs) or mutations conferring resistance to antibiotics) that are part of the microbiome will also be interrogated. Given our unique samples and metadata on all partners in this cohort, we aim to: 1) Evaluate the association between male urethral microbiomes and presence or absence of Ct; 2) Correlate male urethral microbiomes and Ct strains with similar data from female partners; 3) Model how microbiomes, including the resistome, host immune responses and Ct strains predict transmission, infection risk and disease pathogenesis. This study will create the most extensive dataset on male urethral microbiomes, resistomes, and host immune responses to date, providing a valuable resource for the medical and scientific community. Further, given the growing number of Ct STIs in the US and worldwide, the proposed research provides a unique opportunity to study male-female partners and within-host transmission to identify microbiome/resistome//inflammatory profiles that may promote Ct transmission and infection and how these data can be translated into Ct prevention and control strategies. These strategies may include identifying sentinel sites for targeted Ct screening and developing beneficial microbial therapeutics for topical therapy.

Up to $816K

2031-03-31

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Interpretable Machine Intelligence for Biomarker Discovery in Concussion

NIH

Significance to the VA: Mild traumatic brain injuries (mTBIs) are a leading cause of chronic disability among service members and Veterans. The significance of this research lies in its potential to transform the molecular-level understanding of mTBI through innovative statistical and proteomic methodologies. This project directly supports the VA mission by aiming to improve the prognostication and understanding of the molecular underpinnings of mTBI for Veterans seeking care in the VA health system. Additionally, the creation of a large proteomics dataset for Veteran TBI patients represents a valuable resource for the VA and the broader scientific community, fostering further innovation and research in TBI and related fields. Innovation and Impact: This research introduces an advanced approach to mTBI biomarker analysis, utilizing cutting-edge multivariate statistical methodologies. By moving beyond traditional univariate analyses, the project employs tools such as non-linear principal component analysis (NLPCA), interpretable supervised classifiers, and topological data analysis to uncover intricate biomarker patterns. These innovative methods promise to enhance the understanding of molecular dynamics in mTBI, identify distinct patient subgroups, and enable the development of precision medicine strategies, potentially revolutionizing care for Veterans. Specific Aims: 1) To derive multidimensional protein signatures from existing biomarker data associated with specific clinical outcomes in mTBI, leveraging a novel machine learning workflow integrating NLPCA and interpretable supervised classifiers to analyze LIMBIC-CENC data. 2) To perform comprehensive proteomic analyses of blood samples using SomaSCAN technology, supplemented by topological data analysis, to identify patient subgroups with distinct symptomatic profiles and their unique protein signatures. 3) To externally validate biomarker panels and discover new panels related to long-term outcomes using the TRACTS dataset. Methodology: This study employs advanced statistical and proteomic approaches to analyze mTBI biomarkers. The first aim uses a novel machine learning workflow integrating non-linear principal component analysis (NLPCA) with interpretable supervised classifiers to analyze data from the LIMBIC-CENC cohort. The second aim involves comprehensive proteomic profiling using SomaSCAN technology and topological data analysis to identify symptomatic subgroups and their unique protein signatures. Finally, the third aim tests and validates biomarker panels using data from the TRACTS dataset, ensuring robustness and generalizability of findings. Path to Translation/Implementation: The insights gained from this study will provide actionable knowledge for developing targeted treatment strategies and precision medicine approaches for mTBI patients. By identifying distinct biomarker patterns and symptomatic subgroups, this research can inform clinical decision-making and personalized care plans within the VA healthcare system. The creation of a comprehensive proteomics dataset also supports future translational research and its potential clinical applications, facilitating the implementation of innovative diagnostic and therapeutic tools for mTBI in Veterans.

2031-04-30

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Interpretation and Education Best Practices and Training Needs Assessment

National Park Service

Piedmont-South Atlantic Cooperative Ecosystem Studies Unit (CESU) network. The project objective is to conduct a review and synthesis of current research regarding desired visitor and societal outcomes for Interpretation and Education and their associated best practices; and assess training needs of professionals within the career field of Interpretation and Education.

$128K

sciencetechnology

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Interpretation and Education Interns

National Park Service

This project will support education and training for four young adult interns to work with the Fort Pulaski interpretation and education program to provide programming to the public and curriculum-based programs to thousands of students who visit Fort Pulaski each year, and many more who are not able to visit the park via outreach. The participants will gain professional and technical skills that expose them to careers in the National Park Service.

$1 – $56K

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Interpretation and Information Services Interns at Fort Sumter/Moultrie and Liberty Square

National Park Service

The project objective is to support and stimulate work and/or education and training opportunities for young adults through collaborative participation at Fort Sumter National Monument (FOSU) through its partnerships, interpretation, and education programs, specifically those focusing on the efforts of youths and volunteers.

Up to $62K

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Interpretation and Resource Education Interns

National Park Service

United States Department of the Interior, National Park Service (NPS) Notice of Intent to Award. This is not a request for applications. This posting is to provide public notice of NPS' intention to fund the following project activities without full and open competition: Task Agreement P16AC00608 under master Cooperative Agreement P15AC00031 with the Student Conservation Association, for the project titled, "Interpretation and Resource Education Interns".

Up to $37K

natural resources

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Interpretation and Resource Intern at Jimmy Carter Historic Site

National Park Service

This project supports the interpretation and visitor services program at Jimmy Carter National Historic Site. This project will provide rewarding opportunities to serve park visitors and test the appeal of a national park career for the selected intern.

$1 – $46K

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Interpretation and Resource Intern at Jimmy Carter National Historic Site

National Park Service

This project will provide rewarding opportunities to serve park visitors and test the appeal of a national park career for the selected intern. Intern will assist with interpretive operations with focus on exhibit farm (working with animals and crops, providing formal and informal interpretation, assisting with curriculum-based programs).

$1 – $45K

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Interpretation and Visitor Experience Support

National Park Service

United States Department of the Interior, National Park Service (NPS) Notice of Intent to Award. This is not a request for applications. This posting is to provide public notice of NPS' intention to fund the following project activities without full and open competition: Task Agreement P16AC00074 under master Cooperative Agreement P15AC00031 with the Student Conservation Association, for the project titled, "Interpretation and Visitor Experience Support".

Free to search & build · $99 one-time to unlock the application pack · No subscription

Interpretation, Education, Volunteer and Youth (IEVY) Fellow Program

National Park Service

United State Department of the Interior, National Park Service (NPS) Notice of Intent to Award. This is not a request for applications. This funding opportunity is to provide public notice of NPS's intention to fund the following project activities: Task Agreement #P16AC01835 under Cooperative Agreement P15AC00024 with the Conservation Legacy, for the project titled "Interpretation, Education, Volunteer, and Youth (IEVY) Fellow Program"

Up to $40K

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Interpretation, Education, Volunteers and Outreach Interns

National Park Service

United States Department of the Interior, National Park Service (NPS) Notice of Intent to Award. This is not a request for applications. This funding opportunity is to provide public notice of NPS's intention to fund the following project activities. Task Agreement P18AC00072 under Cooperative Agreement P15AC00031 with the Student Conservation Association, for the project titled "Interpretation, Education, Volunteers and Outreach Interns."

Up to $26K

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Interpreting Services

European Commission - EU Programme

Interpreting Services. Call: N/A. Programme: EU Programme. Open call from the EU Funding & Tenders Portal.

$50K

See portal

research

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Interpretive Programing and Media Project at Glacier National Park

National Park Service

This Announcement is NOT a request for applications. This announcement is to provide public notice of the National Park Service's intention to award financial assistance.

$13K – $27K

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Interpretive Programming for 50th Commemoration of Selma to Montgomery March

National Park Service

Youth providing administrative and interpretive support to National Park Service staff before during and after the 50th anniversary events commemorating the historic Selma to Montgomery March

$1 – $196K

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Interpretive Web Media for NPS Climate Change Monitoring

National Park Service

Piedmont-South Atlantic Cooperative Ecosystem Studies Unit (CESU) network. The project objective is to couple a web-based, map interactive browser with existing and new multimedia for park resources affected by climate change that will allow the public to investigate the science and stories behind managing these rapidly changing aspects of Alaska parks. This project will take a 2-phased approach and will be subject to the availability of future funding.

sciencetechnology

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Interpretive Youth Interns

National Park Service

Support and stimulate work and/or education and training opportunities for young adults through collaborative participation in the Interpretation and Visitor Services and Resource Management divisions at New River Gorge National River.

$1 – $37K

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Interrogating and targeting co-inhibitory and co-stimulatory molecules in CAR T-cell Therapies

NCI - National Cancer Institute

ABSTRACT Adoptive cell therapies (ACT) with chimeric antigen receptors (CAR) hold promise to treat cancer, yet several challenges must be overcome to achieve this goal. Clinical evidence attributes treatment failure and resistance to the lack of functional T-cell persistence, antigen escape, and a suppressive tumor microenvironment replete with inhibitory but not co-stimulatory molecules. Investigating response and relapse mechanisms and effectively targeting tumor resistance mediators are urgently needed to improve current ACT. Our analysis of clinical CAR-T products revealed intrinsic PD-L1 (int-PD-L1) expression on the T cells and trogocytic acquisition of tumor-derived PD-L1 (extrinsic; ext-PD-L1). However, the functional role of PD-L1 on CAR-T remains unknown. We hypothesize that both int-PD-L1 and ext-PD-L1 limit CAR-T activity, and that rewiring PD-L1 and CD80 signaling with synthetic receptors will boost antitumor efficacy across ACT platforms. Aim 1 will determine how int-PD-L1 regulates CAR-T under optimal and suboptimal antigen stimulation; Aim 2 will define the impact of trogocytic ext-PD-L1 transfer on CAR-T function using in vitro assays and in vivo leukemia and melanoma models. To enhance T-cell persistence and function, we developed 80BB, a synthetic dual-costimulatory receptor fusing the CD80 ectodomain to the 4-1BB endodomain. 80BB delivers CD28 and 4- 1BB signals and functions as a CTLA-4 switch receptor, driving tumor control across diverse ACT platforms. Although CD80 interaction with CD28 and CTLA-4 underlies these benefits, CD80 also binds PD-L1, yet 80BB did not protect CAR-T from PD-L1/PD-1-mediated inhibition. We therefore engineered a novel 80BB-based platform that retains CD80 interaction with CD28 and CTLA-4 while redirecting PD-L1 engagement, to overcome PD-L1-mediated suppression. Aim 3 will determine mechanisms of action and therapeutic benefit of this novel synthetic costimulatory receptor in B-cell malignancy and solid-tumor models. Our multidisciplinary approach combines basic immunology with translational immunoengineering to overcome ACT barriers. We aim to advance next-generation engineered T-cells and expand the scope of ACT, improving the survival and quality of life of cancer patients.

Up to $716K

2031-04-30

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Interrogating Erythroid Differentiation and BCL-XL Axis in TP53-mutated Acute Myeloid Leukemia

NCI - National Cancer Institute

Abstract Acute myeloid leukemia (AML) is a diverse hematologic malignancy marked by rapid proliferation of poorly differentiated myeloid cells, causing ineffective hematopoiesis and bone marrow suppression. TP53 mutations, present in ~15-20% of AML patients, are associated with poor responses to conventional therapies, including the BCL-2 inhibitor venetoclax, and a grim prognosis of less than six months median survival. Despite various strategies to target TP53-mutated AML, clinical advancements have been limited, highlighting the urgent need for novel approaches for this patient subgroup. Our preliminary data support that TP53-mutated AML from primary patients have higher dependency on BCL-XL and enriched for erythroid differentiation signatures. Erythroid differentiation was correlated with both BCL-XL and GATA1 transcription factor. Notably, we recently reported that acute erythroid leukemia (AEL), a subtype of AML with pure erythroid phenotype and highly enriched for TP53 mutations, is similarly dependent on BCL-XL. This data supports the rationale to exploit BCL- XL inhibition as a promising therapeutic strategy to overcome TP53-mutated therapeutic resistance mediated via erythroid differentiation. We recently demonstrated that the novel dual BCL-XL and BCL-2 Proteolysis targeting chimera (PROTAC) 753b can indeed induce cell death in AML cell lines and primary AML samples resistant to venetoclax. However, its efficacy in TP53-AML subset and in overcoming erythroid-mediated resistance has not been tested. Further, the underlying mechanisms governing erythroid differentiation, GATA1- related erythroid differentiation and its relation to BCL-XL in TP53-mutated AML are not explored. We hypothesize that TP53-mutated AML have a marked predisposition to acquire an erythroid phenotype associated with BCL-XL dependence, which can be leveraged to overcome therapeutic resistance in TP53-mutated AML via BCL-XL or dual BCL-2/BCL-XL degradation. Aim 1 will employ multimodal characterization of primary AML patient datasets (published and unpublished) that includes single cell RNA, single cell ATAC and bulk RNA and define differentiation states and potential dependencies. We will explore the GATA1 regulation of erythroid differentiation and BCL-XL expression via performing multiome analysis and CUT&RUN to identify GATA1 binding in isogenic TP53-mutated and wildtype K562 erythroid leukemia cell lines. Using machine learning, we will identify therapeutic targets and dependencies that mediate erythroid differentiation in TP53-mutated AML. Aim 2 will test the efficacy of the BCL-XL degraders including DT-2216, clinical BCL-XL degrader that has shown safety and preliminary efficacy in the ongoing solid tumor trials, and of dual BCL-2/BCL-XL degrader 753b, in TP53-mutated AML cell lines, primary patients’ samples and in PDXs in combination with hypomethylating agents. Findings from this work have the potential to significantly advance the therapy of this challenging AML subset, providing a foundation for clinical trials aimed at improving patient outcomes.

Up to $443K

2028-03-31

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Interrogating mechanistic underpinnings of cognitive inflexibility in cannabis-exposed offspring

NIDA - National Institute on Drug Abuse

PROJECT SUMMARY Cannabis is the most used illicit drug among pregnant mothers. As the number of states with legal cannabis continues to increase, there has been a concomitant rise in the rate of maternal cannabis use. This is particularly troubling given that prenatal cannabis exposure could interfere with neurodevelopment and increase the risk for cognitive dysfunction later in life. Preclinical animal models are advantageous in that they provide fine control over potentially confounding variables. However, current models of maternal cannabis use have been plagued by methodological concerns that limit the translatability of these data to human populations. Our laboratory has generated important new data using a novel, translationally relevant model of cannabis vapor exposure in pregnant rat dams. This new method of administration uses custom-designed equipment to deliver discrete ‘puffs’ of vaporized, plant-derived cannabis extracts in a response-contingent manner. Using this approach, we have shown that prenatal cannabis exposure produces marked deficits in cognitive flexibility in offspring by impairing acquisition and maintenance of newly updated strategies. This ability is known to depend on corticostriatal neurons, namely glutamatergic projections from the prelimbic (PL) region of the medial prefrontal cortex to the nucleus accumbens (NAc). Dynamic release of glutamate and dopamine (DA) in the NAc coordinates flexible decision making and is constrained by local endocannabinoid (eCB) signaling. Our data also show sex-dependent alterations in spontaneous glutamate release onto NAc-projecting PL neurons in cannabis- exposed offspring from dams that self-administered cannabis. However, we still do not know 1) whether this corticostriatal circuit and local DA and eCB transmission are differentially recruited during flexible decision making in cannabis-exposed vs. control offspring, 2) whether long-term functional alterations occur within this pathway, and 3) whether early intervention strategies can prevent cannabis-related impairment. To address these gaps, we will use complementary projection-specific approaches to identify long-term alterations that give rise to cognitive flexibility deficits in cannabis-exposed offspring and test novel pharmacologicala and circuit- based treatment strategies for restoring behavioral and synaptic function. In Aim 1, we will conduct in vivo fiber photometry recordings of 1) Ca2+ transients in PLNAc neurons and 2) glutamate, DA, and eCB dynamics in the NAc using novel biosensors. In Aim 2, we will employ viral labeling to examine intrinsic excitability and optically evoked transmission from PLNAc neurons, as well as endocannabinoid-dependent plasticity at PLNAc synapses. In Aim 3, we will test whether in vivo depotentiation of synaptic activity at PLNAc synapses or juvenile administration of the FDA-approved steroid precursor pregnenolone can restore cognitive flexibility and recalibrate corticostriatal synaptic transmission in cannabis-exposed offspring. Completion of this work will have a broad, sustained impact by delineating cognitive effects of maternal cannabis use in exposed offspring and their neurobiological underpinnings, which is very timely given the current wave of cannabis legalization.

Up to $640K

2030-12-31

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Interrogating microglia-driven retinal ganglion cell and vascular dysfunction in metabolic syndrome associated retinopathy

NEI - National Eye Institute

PROJECT SUMMARY Retinal vascular diseases including diabetic retinopathy (DR) are a leading cause of vision loss worldwide. Current DR treatments center on reducing retinal vascular dysfunction and neovascularization. These treatments do not improve all outcomes and can reduce visual acuity underscoring the need for novel therapeutics targeting additional aspects of DR. Early neural deficits including retinal ganglion cell loss (RGC) have been identified in both patients and in DR mouse models. Microglia have been implicated in retinal neurodegenerative conditions including DR. Recent work indicates microglial mitochondrial function and metabolic flexibility drives dysfunctional responses. Yet, while human DR is linked to metabolic and mitochondrial stress how this perturbs microglia and if this drives RGC loss and microvascular damage is not clear. The goal of this project is to identify and test mechanisms by which microglia influence RGC and retinal vascular health in a DR-relevant context. Our recent work identified NZO mice (a polygenic model of metabolic syndrome) as a novel model of DR-relevant metabolic syndrome associated retinopathy. NZO mice develop hallmarks of human DR including cotton wool spots, exudates, and capillary loss. In particular, NZO mice develop microvascular dysfunction concomitant with neural deficits (initiates by 3mo) and significant RGC dysfunction and loss by 12mo. Preliminary data from single nucleus and single cell RNA-seq experiments comparing NZO (retinopathy) and B6J mice (control) suggested microglia differentially influence RGCs in B6J compared to NZO retinas. Further, NZO microglia exhibited age- dependent changes in diabetic wound healing genes, and lower expression of metabolic genes compared to B6J microglia suggesting metabolic dysregulation may underlie abnormal microglia-RGC communication. Therefore, in Aim 1, I will identify mechanisms by which microglia influence RGCs and the vascular cells using single cell RNA-seq, microglia depletion, and data alignment strategies. Additional preliminary analyses of our mouse datasets and available patient data indicate lower expression of nicotinamide metabolic enzymes in RGCs and microglia reinforcing the possibility that metabolic dysfunction may contribute to retinopathy- progression. Thus, in Aim 2 I will examine the therapeutic potential of improving cellular metabolism in NZO mice through nicotinamide and pyruvate supplementation or by increasing expression of nicotinamide mononucleotide adenylyl transferases in RGCs and microglia via AAVs. In Aim 3, in a candidate target approach I will first determine the impact of advanced glycation end products signaling in microglia on DR progression. I will then take an unbiased candidate prioritization approach and utilize data generated in Aim 1 to identify and test additional human-relevant processes that may drive pathology. Through these Aims, I will acquire essential skills to facilitate my transition to an independent investigator studying the role of microglia in retinal disease. This project will be supported by a mentoring committee of globally recognized experts in retinal neurodegeneration, microglia, and bioinformatics and excellent career development resources.

Up to $140K

2028-04-30

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Interrogating the Role of Chromatin in Nuclear Receptor Structure and Function

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY/ABSTRACT The nuclear receptors (NRs) named retinoic acid receptor-related orphan receptor γ isoform 2 (RORγt) and glucocorticoid receptor (GR) are powerful transcription factors that mediate central functions of the adaptive and innate immune system, respectively. NRs are a major family of drug targets and mechanistic studies are needed to better understand how drugs impact NR function. NRs function by binding to their respective small molecules, localizing to specific DNA sequences in the genome called response elements, and then by recruiting coregulatory protein complexes that (1) modify histones, (2) promote chromatin remodeling, and (3) recruit transcription initiation machinery. This proposal seeks to unveil the mechanisms of RORγt- and GR-mediated histone acetylation – a hallmark of NR target gene activation. This will be done using a combination of chromatin biochemistry, structural biology, and functional genomic analyses. The underlying hypothesis is that the chromatin context provides important cues for how target genes are regulated by RORγt and GR, as well as by their associated coregulatory protein complexes. In support of the hypothesis, exciting preliminary data is disclosed showing how the precise positioning of the DNA response element on the nucleosome (the fundamental unit of chromatin) dictates if and how RORγt and GR engage with chromatin. Going further, the first structure of RORγt bound to the nucleosome is presented, which shows an unexpected and novel positioning of domains on the nucleosome. This project will extend from these studies by building up RORγt:coregulator complexes using model coregulatory proteins named SRC3 and p300. Previous biochemical studies with NRs have reduced these large multidomain proteins to small peptide fragments or have failed to achieve high- resolution. Solving structures of full length SRC3 and p300 with RORγt will define how NRs and chromatin direct these important proteins to their substrates. In addition, this project aims to complement in vitro biochemical and biophysical analyses with novel functional analyses of RORγt in a relevant primary T cell model biological system. Specifically, the candidate (Tim) will be trained to isolate and culture primary naïve T cells as well as to perform a technique called CUT&RUN. The goal of these experiments is to learn how RORγt and coregulators work together to drive the expression of pro-inflammatory IL-17 cytokine in naïve T cells. These results will uncover the specific changes in chromatin PTMs and nucleosome positioning during RORγt-mediated activation of its target genes. Finally, in the independent phase, the project aims to extend the methodologies that Tim has learned during his postdoctoral training to GR and its fundamental role in macrophage biology. This extension would form a new biological system that Tim will investigate in his future career. Overall, this innovative and interdisciplinary research strategy will answer important biological questions regarding NR structure and function, with significant impact on human health. The career development plan will take place at The Salk Institute and provide an important opportunity for training and that will prepare Tim to start independent research career.

Up to $123K

2028-01-31

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Interrogating the role of H3K4 & H3K27 methylation in hematopoiesis with novel histone tools

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

SUMMARY Developmental gene expression is tightly regulated by the dynamic interplay of H3K4 methylation (H3K4me) and H3K27 methylation (H3K27me) associated with active and repressed genes, respectively. However, our understanding of the individual and combinatorial roles these histone modifications play in adult physiological contexts remains incomplete. To overcome these limitations, we have recently generated histone mutant transgenic tools to uncover a previously unappreciated role for H3K4me in adult hematopoiesis. Adult mice globally depleted for all forms of H3K4me via expression of an inducible histone H3 lysine-4-to-methionine (H3K4M) mutant allele succumbed to a severe loss of all major mature blood cell types. Unexpectedly however, H3K4M-expressing hematopoietic stem cells (HSCs) and most committed progenitors were present at normal numbers and persisted upon transplantation into recipient mice, suggesting that H3K4me is dispensable for the maintenance and early commitment of HSCs and progenitors but essential for the terminal maturation of progenitors. Mechanistically, we showed that H3K4me opposes the deposition of repressive H3K27me at differentiation-associated genes bivalently marked by H3K4me3 and H3K27me3 in HSCs or progenitors. Indeed, by concomitantly suppressing H3K27me in H3K4me-depleted mice with an H3K27M transgene, we could rescue the acute lethality, hematopoietic failure and gene dysregulation. Thus, our results reveal that H3K4me guides hematopoiesis by opposing repressive H3K27me at fate-instructive bivalent genes, providing the first evidence for the functional interaction between these crucial chromatin marks in mammalian tissue homeostasis. These preliminary data raise fundamental questions with clinical relevance that will be addressed in 3 complementary aims. In Aim 1, we will further define the consequences of H3K4me loss on the function of HSCs and progenitors using self-renewal and differentiation assays. Additionally, we will assess whether any observed defects are reversible upon restoration of H3K4me. In Aim 2, we will identify epigenetic regulators that mediate the H3K4M- dependent arrest and the H3K27M-dependent rescue by purifying proteins associated with H3K4M and H3K27M; measuring changes to all major histone modifications; and testing select candidates for their ability to phenocopy the effects of H3K4M and H3K27M. In Aim 3, we will dissect the molecular basis by which H3K4me/H3K27me safeguard hematopoiesis with a focus on fate-instructive cytokine receptors and transcription factors dysregulated in H3K4M mice but normalized in H3K4M/H3K27M mice. Moreover, we will investigate the contribution of other epigenetic marks to the H3K4M phenotype using DNA methylation inhibitors and a novel histone mutant library. Collectively, this proposal will leverage novel tools to probe the direct, physiological impact of two antagonizing chromatin marks on hematopoiesis. As arrested differentiation and disrupted H3K4me/H3K27me have been implicated in diverse hematological conditions, our results will elucidate the underlying mechanisms and may pave the way for novel therapeutic interventions.

Up to $762K

2030-01-31

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Interrogating the roles of truncated/mutated inverted terminal repeat sequences in adeno-associated virus vectors in transgene stability through episome formation and host cell genome integration

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY Gene therapies based on adeno-associated virus (AAV) vectors have been a revolutionary medical advancement in treating human genetic diseases. With a single dose, AAV-mediated gene therapies can confer long-term correction or abatement of disease for the lifetime of the patient. However, recent findings have found that vector DNA integration into the host-cell genome occurs at much higher frequencies than previously thought. Furthermore, integrated transgenes seem to substantially contribute to the long-term stable expression of the therapy. These reports have drastically shifted the safety status of AAVs. The only viral elements retained in AAV vectors used in gene therapy are the inverted terminal repeat (ITR) sequences found at both the ends of the vector genome. The ITRs are essential for the stability for the AAV vector genome after entry into the host cell. We recently revealed that vectors manufactured for gene therapy can harbor truncated or mutated (t/m)ITRs with fairly high frequencies. Interestingly, these types of imperfect ITRs match the truncated ITRs observed in integration sites. I, therefore, hypothesize that truncated/mutated ITRs in AAV viruses and in packaged vectors can influence the frequency of integration. This project proposal is divided into three main aims to define if and how truncated/mutated ITRs can alter the outcomes of AAV genomes, with paradigm-shifting implications for AAV-based gene therapy applications. • Aim 1: To examine ITR structures among wildtype AAVs. Examination of wildtype ITRs, which are proven to also integrate into the host cell infected with AAV, will help to inform on how ITR integrity in gene therapy vectors influences integration. We will perform advanced NGS-based identification of ITRs among natural proviral AAV genomes found in non-human primate tissues and test the ability of wtAAVs to spontaneously form t/mITRs. • Aim 2: To track the post-entry outcomes of vectors that bear a high percentage of t/mITRs. We will test vectors that harbor t/mITRs or wildtype (wt)ITRs for their ability to integrate into the host cell genome. We will develop novel cell culture and NGS-based workflows to interrogate t/mITR-mediated integration in cultured human hepatocytes and in humanized liver mouse models. • Aim 3: To find correlation between integration frequencies in treated non-human primate tissues with the prevalence of t/mITRs in test vectors. Examination of tissues from human patients receiving gene therapy is hard to justify. Non-human primate models are the best proxy to track the kinetics of integration. We will, therefore, examine tissues treated with AAV vectors sourced from previous studies to determine the rate of integration and the structure of ITRs in multiple treated tissues of NHPs.

Up to $1.7M

2030-04-30