Grants

Food and Drug Administration

Identification and Evaluation of Possible Approaches to Addressing Nitrosamine Impurities in Drugs (U01)

general

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Identification and Evaluation of Possible Approaches to Addressing Nitrosamine Impurities in Drugs (U01)

Food and Drug Administration

The Food and Drug Administration's (FDA), Center for Drug Evaluation and Research (CDER), Office of New Drugs (OND), is announcing this Funding Opportunity Announcement (FOA) for a Cooperative Agreement. The proposed work directly supports the U.S. FDA’s stated goal of protecting public health from unacceptable risks from nitrosamine impurities in human drugs. Although significant experimental and policy/regulatory initiatives have been undertaken in this area, there remains a need for further research into and development and refinement of translational and implementable practices that will protect the public against nitrosamine risks while ensuring continued safe access to critical therapeutic drugs. The aim is to improve the safety of human drugs with potential nitrosamine impurity liabilities. In addition to the work outlined above, the award recipient will assess how best to ensure that this research and practices development continues among industry members, non-profits, and/or academic institutions once the FDA funding for this cooperative agreement ends.

$350K

consumer_protectionAgriculturefood_and_nutrition

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Identification and Evaluation of WWII and Mexican Period Resources at Point Reyes National Seashore

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 the NPS' intention to fund the following project activities without full and open competition: Cooperative Agreement P19AC01148 with Sonoma State University, a partner under the Californian Cooperative Ecosystem Studies Unit (Cooperative and Joint Venture Agreement NPS-CALI-CESU-2018), for the project titled, "Identification and Evaluation of WWII and Mexican Period Resources at Point Reyes National Seashore."

Up to $130K

rolling

other

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Identification and inhibition of the novel downstream effectors of mutant Ras for mutant Ras-driven cancer targeting

NCI - National Cancer Institute

Summary KRas is the most commonly mutated oncogene in human cancers. A deregulated KRas GTPase activity is the consequence of each of the mutations. Currently, there are limited FDA approved drugs to counter tumors having activating Ras mutations. An active anti-KRas remedy would be a major advancement for the treatment of many cancers. To that end, the AM510 is an improved version of the KRasG12C inhibitor that is the first to enter to clinical testing and exhibited beneficial efficacy in 4 non-small cell lung cancer (NSCLC) patients recently approved by FDA for advanced lung cancer treatment. However, unlike other KRas mutants such as KRasG12V accounting for the vast majority of KRas mutations, KRasG12C only occurs in approximately 13% of lung adenocarcinoma, 3% of colorectal cancer and 2% of other solid cancers, thus greatly limiting the use of AM510 in targeting human cancers with other types of KRas mutations and/or KRas overexpression. To overcome this problem, it will be crucial to identify other strategies and targeted agents to combat KRas-driven cancers. Although KRas mutations are frequently found in human cancers, amplification of KRas, albeit less frequent, has also been reported in certain types of human cancers. In both cases, activation of downstream Raf/MEK/ERK (MAPK) and PI3K/Akt kinase cascades represent key mechanisms that drive malignant tumor phenotypes through Ras, establishing a molecular basis for targeting Ras-driven cancers by inhibiting downstream kinase cascades. However, combination of inhibitors simultaneously targeting MAPK and Akt cascades fail to exhibit clinical efficacy in KRas-driven cancers, suggesting that other effectors downstream of activated KRas contribute to treatment failure. By screening an amide compound library with >30,000 compound in the cell-based viability assay and in vivo tumor assays, we identify the compound #1a as a promising lead compound that selectively and potently targets mutant KRas driven cancer survival and in vivo tumor growth in xenografts, patients- derived xenografts (PDXs) and genetic models without affecting normal cell survival and causing mouse toxicity. Of note, the lead compound #1a binds to a novel downstream effector of mutant KRas, which is crucial for cancer cell survival in the context of mutant KRas activation, and disrupts its activity leading to cancer cell catastrophe. In this proposal, we proposed three specific Aims to explore the efficacy of the lead compound #1a and its derivatives and their underlying mechanisms in targeting mutant KRas driven cancers using diverse tumor models. Our proposal is highly original and innovative, as we have developed a lead compound for targeting mutant KRas cancer and utilized cutting technologies including medicinal chemistry, genetic mouse models, and PDX models to validate our provocative concept. We have assembled a unique research team with diverse expertise that is highly capable of conducting the study. Our study provides novel downstream effectors for mutant KRas signaling and develops an effective strategy/agent for targeting mutant KRas-driven cancer, thus revolutionizing our understanding of KRas signaling and therapies for targeting mutant KRas driven cancers.

Up to $693K

2031-05-31

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Identification and Investigation of CD4+ T cell Clonotypes Comprising a Polyclonal Repertoire Against Aeroallergen in a Murine Model of Allergic Asthma

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY/ABSTRACT Allergic asthma presents as either T2-high (Th2/eosinophilic) or T2-low (Th17/neutrophilic) inflammation. While external factors like co-stimulation and cytokines shape Th cell fate, the role of T cell receptor (TCR) affinity in directing Th2 vs. Th17 differentiation remains unclear. Our lab developed a fungal protease (Alp1)-driven allergic lung inflammation model and essential reagents, including an IL-5/IL-17A reporter mouse. We observed that Alp1 sensitization primarily induces a Th2 response (~30% IL-5+ Th cells, eosinophilia) but also elicits a smaller Th17 response (~5% IL-17A+ Th cells, neutrophilia). This suggests TCR-intrinsic factors influence effector fate. We hypothesize that TCR affinity dictates Th2 vs. Th17 differentiation in response to fungal protease allergens. A major limitation in allergy research is the lack of tools to study allergen-specific Th cells. We aim to develop peptide-MHCII tetramers to examine TCR affinity and its impact on Th2/Th17 fate within oligoclonal populations. Additionally, we will generate a TCR transgenic mouse and engineered protease allergens to directly test TCR affinity’s role in Th cell differentiation. Our lab has identified an immunodominant Alp1 epitope (aa 194-202) and developed a functional peptide- MHCII tetramer. We also created a transgenic mouse expressing tagged-MHCII in dendritic cells, enabling in vivo epitope discovery. With these tools, we will achieve the following aims: Aim 1: Define the relationship between TCR affinity and Th2/Th17 fate in a polyclonal response. Aim 2: Directly test the causal role of TCR affinity in Th2/Th17 differentiation. This study will advance allergy and immunology research by overcoming technical barriers in epitope discovery and immunodominance. Our transgenic models, cytokine reporters, and tetramer technology will provide a framework for understanding allergen-driven Th cell responses, with implications for new diagnostic and therapeutic strategies in allergic diseases.

Up to $421K

2028-03-31

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Identification of Ancient Antiviral Defenses Using Viral Ubiquitination Modulators

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary Arthropod-borne viruses or “arboviruses” are insect-transmitted viral pathogens that pose a significant threat to human and animal health worldwide. However, we still lack effective therapeutics to combat most arbovirus infections. A deeper understanding of the conserved innate immune mechanisms that restrict arbovirus replication in insect and mammalian hosts may reveal strategies for preventing or treating arboviral disease. Thus, we sought to develop an innovative, yet simplistic, approach to identify conserved antiviral host factors affecting arbovirus replication that, at the same time, inherently provides insight into virally-encoded strategies that evade these host defenses. Here, we propose to use a novel arbovirus "rescue" assay wherein candidate immune evasion proteins (IEPs) encoded by unrelated mammalian viruses can be transiently expressed in insect cells and assayed for their ability to sensitize insect cells to arbovirus infection. The rationale behind this approach is that mammalian virus-encoded IEPs that enhance arbovirus replication in insect cells likely do so because they happen to inhibit key antiviral factors/pathways that are conserved between insect and mammalian hosts. One can then use these IEPs as “tools” to identify and characterize the conserved immunity factors these IEPs target. Thus, this screening methodology provides a mechanism to both identify novel IEPs encoded by mammalian pathogens and the functionally-relevant components of the eukaryotic innate immune response these IEPs inhibit. To discover IEPs that promote arbovirus replication in insect cells, we will screen an expression library encoding 93 “viral ubiquitination modulators (VUMs)” derived from 33 different mammalian viruses. VUMs are virally-encoded proteins that inhibit, usurp, and/or re-direct the host ubiquitination system to promote viral replication. Given that ubiquitination is a key post-translational modification that alters the stability and/or function of cellular proteins, VUMs often hijack the ubiquitination system to inhibit or degrade host proteins that are critical for antiviral defense. We hypothesize that some VUMs may target cellular antiviral factors that are conserved between insect and mammalian hosts and that are critical for restricting arboviruses. Indeed, our initial screens identified VUMs from disparate mammalian viruses that enhance arbovirus replication in insect cells. These results, along with our prior successes in using this system to identify novel IEPs and conserved antiviral responses (Rex et al., 2024, Nat. Microbiol.; Embry et al., 2024, PLoS Pathog.), suggest that we can use VUMs as tools to both inhibit, and discover, conserved antiviral immunity factors. Our study will: 1) Identify VUMs encoded by mammalian viruses that promote arbovirus replication in insect cells; 2) Identify conserved insect and mammalian factors interacting with VUM “hits” from our arbovirus rescue assays; and 3) Identify the conserved host factors interacting with VUMs that normally block arbovirus replication. Our long-term goal is to use this system to define the critical eukaryotic innate immune mechanisms that restrict arbovirus replication.

Up to $249K

2028-03-31

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Identification of attenuating mutations in the envelope protein for the rational design of a candidate West Nile live-attenuated vaccine.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary. The Orthoflavivirus genus in the family Flaviviridae contains several pathogenic arthropod- borne viruses, including West Nile virus (WNV) a mosquito-borne orthoflavivirus in the Japanese encephalitis (JE) sero-/genetic complex. The United States has reported >27,000 cases of WN neuroinvasive disease and >2,900 deaths since the introduction of WNV in 1999, making WN vaccine development a public health priority. Live-attenuated vaccines (LAVs) have successfully controlled mosquito-borne orthoflaviviruses, yellow fever and JE virus. Thus, development of a WN LAV is realistic. The development of an attenuation strategy built on multigenic mutations will support the rational design of a candidate WN LAV. To date, attenuating mutations have been identified in 3 virally encoded nonstructural (NS) proteins, but few attenuating mutations have been identified in the structural proteins, including the envelope (E) protein. Because the E protein not only mediates the binding and membrane fusion with the cell but is the primary target of neutralizing antibodies, a correlate of protection for WNV, mutations in the E protein of a candidate WN LAV must not compromise immunogenicity. This project aims to identify attenuating mutations in the E protein to be incorporated into the rational design of a candidate WN LAV alongside those in the NS proteins. The E protein is a class II fusion protein that forms a fusogenic trimer to mediate the membrane fusion process, which is the fundamental viral entry mechanism and is crucial for WNV neurotropism in vertebrate hosts and transmission by mosquitoes. The formation of the E protein trimer requires the rearrangement of its 3 domains (EDI, EDII, and EDIII). The 4 motifs between the EDI and EDII exert a hinge effect for the EDI-EDII interdomain movement to initiate the formation of the E protein trimer, hence the name EDI-EDII hinge region. Site-directed mutagenesis of the EDI-EDII hinge region in WNV strain NY99 rescued from an infectious clone (WNV-NY99ic) demonstrated that the E-A54 and E-Y201 residues each control the formation of the E protein trimer. WNV-NY99ic retained 13 alternative amino acid (aa) substitutions of the respective residues. The E-A54I and E-Y201P mutations each fully attenuated the neuroinvasive phenotype of WNV-NY99ic in outbred Swiss mice, while retaining induction of serum neutralizing antibodies. Built on the attenuated phenotype and immunogenicity of the E-A54I and E-Y201P mutants, the central hypothesis is that impairment of the membrane fusion process by mutations of either the E-A54 residue or the E-Y201 residue will attenuate murine neurotropism and mosquito transmission of WNV- NY99ic without compromising antibody-mediated neutralization. This project will select the lead mutation of the respective residues based on 1) attenuation of the mouse neuroinvasive phenotype and neurovirulence, 2) impairment of transmission by Culex species mosquitoes that are WN vectors, and 3) immunogenicity that elicits serum neutralizing antibody responses conferring passive protection to identify the E protein mutation(s) suited for the rational design of a candidate WN LAV.

Up to $421K

2031-05-31

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Identification of axon length-dependent gene expression programs

NINDS - National Institute of Neurological Disorders and Stroke

PROJECT SUMMARY Many neurodegenerative disorders (NDDs) preferentially affect neurons with long or complex axonal arbors. However, our understanding of this specific vulnerability is limited. We hypothesize that axon length represents a molecularly definable source of vulnerability common to many NDDs, and indeed we found that Drosophila neurons with long axons are intrinsically more vulnerable to degeneration than same-type neurons with short axons. In preliminary studies we have defined molecular markers whose expression covaries with axon length and have developed a robust platform for single cell transcriptomics, in vivo analysis of axon length-dependent effects on synaptic connectivity, and behavioral diagnostics of length-dependent axon degeneration. We will leverage the significant advantages of this system to systematically identify genes whose levels covary with axon length and assay functions for these genes in length-dependent axon degeneration. Altogether, our studies will provide fundamental insight into mechanisms of axon length-dependency in degeneration, addressing a fundamental gap in our understanding of NDDs.

Up to $428K

2028-04-30

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Identification of bacterial isolates and communities that influence male life history traits and mating success among genetically diverse Ae. aegypti mosquitoes

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary/Abstract Mosquito population control is a primary means to prevent mosquito-borne disease worldwide, and many novel approaches to population control involve traditional or modified versions of Sterile Insect Technique (SIT). One thing these methods all have in common is the need to rear healthy adult male mosquitoes en masse. These males are then released to mate with wild females, which they sterilize and prevent from producing offspring, thus causing a decline in mosquito population size. Males used for SIT must be able to compete for mates in a field setting for the method to be successful. Moreover, modifications to rearing that improve mass production of males, increase their competitive ability, or increase the duration of their field efficacy, will improve the success and reduce the cost of these programs. The microbes found in larval development water and within the mosquito body have been shown to impact multiple traits that are relevant to SIT and mass rearing, including pupation time and body size. Here, we propose to comprehensively determine the impact of the microbiota on adult male life history traits relevant to the success of SIT. We will experimentally colonize mosquitoes with multiple candidate bacterial communities and determine the impact of different communities on male longevity, flight capacity, mating success, sperm quantity, and ability to induce refractoriness to re-mating in female mosquitoes. Additionally, we will assess the impacts of these colonies on male traits in semi-field conditions We will also assess the impact of the microbiota on male life history traits across a genetically differentiated group of mosquito strains. Finally, we will assess the role of B vitamins as a mechanistic determinant of male mosquito-microbe interactions. This work will reveal, for the first time, the comprehensive impact of the microbiota on traits relevant to the success of SIT. Additionally, it has the potential to reveal specific bacterial treatments that could be used to improve male quality in mass rearing operations and the extent to which effects of the microbiota may differ depending on mosquito genetic background. Each component of the proposal is critical to understanding mosquito-microbe interactions in male mosquitoes and will provide key foundational knowledge as well as potentially actionable findings to improve the success of SIT.

Up to $526K

2031-03-31

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Identification of candidate genomic regulatory elements involved in oral clefts

NIDCR - National Institute of Dental and Craniofacial Research

Project Summary/Abstract Disturbances in the molecular pathways that control lip and palate development during embryogenesis can lead to oral clefts, but knowledge of the regulatory interactions involved in modulating the expression of genes in these pathways remains limited. Because enhancers are critical to the spatiotemporal regulation of gene expression during embryogenesis, those that are active in craniofacial tissues during lip and palate development are likely to control the expression of genes relevant to oral clefts. The overall goal of the proposed research is to identify interactions between enhancers, transcription factors that bind the enhancers, and target genes of the enhancers in lip and palate development and oral clefts. Aim 1 is to identify the subset of enhancers that drive gene expression overall in human embryonic palatal mesenchyme cells. The transcription factors with binding site motifs that are enriched in the enhancers will be identified, and binding of the transcription factors within the enhancers in human embryonic palatal mesenchyme cells will be confirmed using assays of chromatin immunoprecipitation followed by sequencing. Aim 2 is to identify enhancers that regulate transcription specifically within human embryonic palatal mesenchyme cells, because such enhancers are more likely to be involved in craniofacial-specific processes during development. Enhancers that can distinguish between human embryonic palatal mesenchyme cells and other types of tissues and cells will be considered palate-specific enhancers. The palate specificity of a subset of the enhancers will be confirmed using in vitro assays.

Up to $385K

2027-08-13

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Identification of Causative Variants in Autoimmune Risk Loci

NIH

Genome-wide Association Studies (GWAS) have emerged as a transformative approach in deciphering the complex genetic underpinnings of autoimmune diseases. Autoimmunity arises from intricate interactions between genetic susceptibility and environmental triggers. A remarkable aspect of GWAS findings is the identification of genetic variants that are associated with multiple autoimmune diseases. These shared variants suggest overlapping pathways and mechanisms underlying these diseases. For example, the HLA gene region is consistently implicated in multiple diseases such as type 1 diabetes, rheumatoid arthritis, lupus, and celiac disease. Using the Million Veterans Project (MVP) data, we created a cohort of Veterans with at least two reports of any one of 71 different autoimmune phenotypes (n= 83156) and a cohort of controls (n=335647) that did not have any autoimmune phenotypes and performed a GWAS to identify common autoimmune risk loci. Twenty-six regions of the genome were identified with variants surpassing genome wide significance (5x10-8). The largest region identified spanned the HLA region on chr 6 from 29.5 to 32.7 Mb. In the non-HLA loci, linkage disequilibrium (LD) pruning identified 44 independent associations. Thirty-one of these associations have been identified in previous autoimmune GWAS, showing the robustness of our results. Interestingly, the 13 remaining associations have not been previously identified in the GWAS catalog for an autoimmune disorder. These results suggest that these 13 associations are novel associations and provide new insight into the genetic mechanisms of autoimmune risk. GWASs only identify associations between variants and disease, they do not identify which of the associated variants cause increased risk of disease or identify which gene(s) are involved in increased risk. To fully understand the genetic mechanisms involved, we must identify the variants that impact cellular biology in an allele specific manner and which genes are impacted. The goal of this project will focus on these two specific issues. Most associations detected in autoimmunity, and in our results, are in non-protein coding regions of the genome, which suggests that disruption of gene regulation is the major mechanism increasing risk of disease. To identify all potential “causative” variants, we have used LD expansion to identify 2108 variants with an r2>0.4 with each of the non-HLA independent associations. We propose using a Massively Parallel Reporter Assay (MPRA) to screen these variants in multiple cell types relevant to autoimmunity with and without stimulation. From these experiments we will be able to determine not only which of the variants have the capacity to alter gene expression, but also the type of cells showing allele specific expression and the relevant environmental conditions that alter gene expression in an allele specific manner. While these variants would be strong candidates to be causative, the MPRA does not identify which genes the variants target. To address this issue, we propose applying a combination of bioinformatic tools and Perturb-seq techniques using either dCAS9-KRAB (suppressor) or dCAS9-PVR (activator), variant targeted gRNA’s and RNAseq to identify the genes that are regulated by our top candidate autoimmune risk variants. Four variants were identified that alter the amino acid sequence of proteins. Functional assays will be performed to determine the effect of the risk allele on protein function. Combined, these data sets will provide a deeper understanding of the genetic mechanisms that promote increased risk of autoimmunity and could lead to the design of potential therapeutics. In addition, by examining the genetic associations common to multiple autoimmune diseases, we will be able to identify Veterans that have the greatest risk of developing autoimmune disorders and potentially identify which disease will develop. This will allow earlier clinical identification of disease as well as earlier intervention which can result in better outcomes.

2029-12-31

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Identification of cell type-specific insulin responsive quantitative trait loci in human skeletal muscle

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

PROJECT SUMMARY/ABSTRACT Type 2 diabetes (T2D) is a complex polygenic disease that results from a multifactorial interaction of environ- ment and lifestyle factors. The estimated heritability of T2D ranges from 30 – 70%, and over 1,000 independ- ent genetic loci have been associated with T2D. However, most loci map to non-coding regions of the genome, leading to challenges elucidating their functional significance. Non-coding genetic variation has the most proxi- mal effect on the molecules bound to DNA (epigenome), which in turn influences the expression of target genes (transcriptome) in a cell-type and context specific manner. A powerful approach to mechanistically link these layers is through identification of quantitative trait loci (QTL) for epigenomic modalities such as chromatin accessibility QTL (caQTL) and gene expression quantitative trait loci (eQTL), followed by testing whether dis- ease associated loci underlie the molecular QTL. In the current study, we will identify cell type-specific and dy- namic molecular response QTLs (rQTLs) in humans in vivo that are relevant to skeletal muscle insulin re- sistance, a core pathophysiologic defect in T2D and related comorbidities. Skeletal muscle accounts for ap- proximately 80% of all glucose uptake in the postprandial state in humans. We and others have shown that skeletal muscle insulin resistance predates glucose intolerance in normoglycemic individuals at high risk of de- veloping T2D, and is evident decades before β-cell failure and overt hyperglycemia develops. In preliminary studies sing single nuclei gene expression (snRNA-Seq) and Assay for Transposase-Accessible Chromatin with Sequencing (ATAC-Seq), we have identified novel response eQTL (r-eQTL) and caQTL (r-caQTL) that are acutely sensitive to insulin during an hyperinsulinemic-euglycemic clamp. These loci include distinct insulin- responsive rQTLs in parenchymal and non-parenchymal cells, including in type 1 and type 2 muscle fiber cells and fibro-adipogenic progenitor cells (FAPs), which are important regulators of muscle homeostasis and regen- eration. In the current application, we build on our pilot data and test the hypothesis that targeted interventional approaches in humans, combined with modern genomic techniques, will uncover novel mechanisms linking common genetic variation to relevant pathophysiological pathways in T2D. In Specific Aim 1, we will Identify cell-type specific insulin responsive molecular QTLs in human skeletal muscle using by collecting muscle biop- sies before and after a hyperinsulinemic-euglycemic insulin clamp (80 mU/m2 ⋅ min). In Specific Aim 2, we will determine acute skeletal muscle insulin resistance molecular QTLs in humans: by using an acute lipotoxicity model of insulin resistance. These studies leverage our unique combined expertise and existing productive col- laboration to integrate whole-body physiological approaches in humans with cutting edge molecular and ge- nomics techniques to reveal dynamic response QTLs that connect disease-relevant metabolic pathways to ge- netic variation.

Up to $708K

2031-03-31

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Identification of cellular functions involved in Hepatitis B Virus infection via a novel RNA sensing and editing dependent reporter system

NIAID - National Institute of Allergy and Infectious Diseases

Abstract Hepatitis B virus (HBV) chronically infects 254 million people worldwide and accounts for 1.1 million deaths per year due to cirrhosis and liver cancer. Finding a cure for this disease is urgently needed, which requires a more thorough understanding of the key molecular events driving establishment and persistence of HBV infections. Thus far, identification of HBV-hepatocyte interactions is mostly achieved through conventional focused screens or one-gene-at-a-time approaches. Due to the overlapping feature of the HBV genome, unbiased genetic screens by reporter HBV viruses allowing phenotypic selection have not been possible. To overcome this challenge, our lab recently developed an RNA sensing and editing based HBV reporter without modifying the viral genome, but instead introduces reporter genes containing stop codon (UAG) that becomes contingently translatable upon precise RNA editing (UIG, translate as UGG) guided by the complementary HBV RNA sequences. Our preliminary data demonstrated that it functions in an HBV RNA-sensing and ADAR RNA-editing dependent manner, and an HBV puromycin reporter allows for HBV infection-dependent survival of cells following puromycin treatment. Therefore, this novel cell culture-based HBV infection reporter system permits unbiased genome- wide screens using sgRNA, cDNA and chemical libraries, etc. The purpose of this R21 grant application entitled “Identification of cellular functions involved in Hepatitis B Virus infection via a novel RNA sensing and editing dependent reporter system” is to further validate the newly developed system and to identify still elusive host factors required for early steps in HBV infection and for amplification of the viral genome. We anticipate that our novel HBV infection reporter assay will allow us to perform the most comprehensive loss-of-function and gain-of-function screens in tissue culture cells to date, and most likely reveal new knowledge on virus-host interactions critical for the HBV life cycle, which will in a short-term enhance our understanding of HBV replication and advance current HBV model systems, and in a long-term, provide potential novel targets for antiviral therapies to combat chronic HBV infections.

Up to $196K

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Identification of chromatin regulators required for enhancer hijacking in cancer genomes

NCI - National Cancer Institute

PROJECT SUMMARY Genomic instability is a hallmark of cancer, and many human cancers display structural variants such as translocations, inversions, deletions, and duplications. These structural variants can promote cancer development through various mechanisms, including "enhancer hijacking," where non-cognate enhancers aberrantly activate oncogenes. This phenomenon has recently been identified in a broad range of cancers, suggesting that enhancer hijacking is an underappreciated mechanism of oncogene dysregulation across many cancer types. Enhancer hijacking is characterized by the regulation of genes by ectopic enhancers, suggesting that the genes that are subject to enhancer hijacking are capable of being regulated promiscuously by enhancers they have never otherwise seen before in evolution. However, whether there are mechanisms that facilitate such promiscuous gene regulation events is unclear. As enhancer hijacking is a cancer specific phenomenon, understanding the mechanisms that drive enhancer hijacking may help identify potential future therapeutic targets that would be effective in diverse cancers. Therefore, there is an urgent need to better understand the processes that contribute to enhancer hijacking in cancer genomes. In preliminary data supporting this proposal, CRISPR/Cas9 genome wide knock-out screens using engineered enhancer hijacking reporters identified two paralogs, STAG1 and STAG2, as have opposing effects in regulating enhancer hijacking. STAG1 and STAG2 are mutually exclusive components of the cohesin complex, and in the CRISPR/Cas9 screens, STAG1 is critical for promoting enhancer hijacking, while STAG2 represses it. Given that STAG2 is frequently inactivated in cancers, our overarching hypothesis is that the balance of STAG1 and STAG2 containing cohesin complexes regulates ectopic gene expression in enhancer hijacking. This proposal aims to investigate the mechanisms of STAG1 and STAG2 regulation of enhancer hijacking and to identify general and lineage-specific regulators of enhancer hijacking. Specific Aim 1 will determine how STAG1 and STAG2 influence the stability of 3D chromatin interactions between hijacked enhancers and oncogenes. Specific Aim 2 will explore the impact of STAG1 and STAG2 on enhancer hijacking of different genes across diverse lineages in cell lines and patient-derived organoids. Specific Aim 3 will develop scalable genome-wide methods to identify additional regulators of enhancer hijacking, employing Perturb-seq-based approaches. The successful completion of these aims will enhance our understanding of enhancer hijacking in cancer genomes, offering insights into potential therapeutic strategies for cancers driven by these events.

Up to $795K

2031-04-30

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Identification of Drug-related and Formulation-Related Factors that Result in Alcohol Dose Dumping of Modified Release Oral Drug Products (U01) Clinical Trial Not Allowed

Food and Drug Administration

Identification of Drug-related and Formulation-Related Factors that Result in Alcohol Dose Dumping of Modified Release Oral Drug Products (U01) Clinical Trial Not Allowed

general

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Identification of Drug-related and Formulation-Related Factors that Result in Alcohol Dose Dumping of Modified Release Oral Drug Products (U01) Clinical Trial Not Allowed

Food and Drug Administration

Modified release (MR) oral drug products are considered to have a high risk for alcohol dose dumping (ADD) because they contain large quantities of drug(s), designed to release over a prolonged period of time. Accidental exposure of these products to alcohol can result in the relatively rapid release of large quantities of drug with severe side effects, including death. To mitigate this risk, the FDA recommends conducting an in vitro alcohol dose dumping assessment in 0%, 5%, 20%, and 40% alcoholic dissolution media for all prospective generic versions of MR oral drug products. To date, ADD assessments have not been harmonized globally. For instance, the U.S. FDA recommends testing up to 40% alcoholic media while the European Medicines Agency recommends testing up to 20% alcoholic media. This type of difference can present a challenge for formulators designing products for multiple markets, as historical data has shown release from MR oral products do not always follow a linear response (either increasing or decreasing) to increasing alcohol concentrations. In addition, interpretation of an ADD assessment may be limited by the inability of the test to predict in vivo behavior.  The purpose of this research is to develop tools that 1) facilitate the development of MR generic drug products that have a low potential for ADD, 2) support regulatory decision making during the assessment of such products, and 3) provide evidence that enables FDA to develop more specific recommendations for efficiently demonstrating a low or comparative potential of alcohol dose dumping for MR oral drug products containing high risk drugs.

$250K

consumer_protectionAgriculturefood_and_nutrition+1

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

Identification of enhancers that facilitate genetic manipulation of specific B-cell subsets

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary/Abstract Protective humoral immunity is mediated by both long-lived memory B cells (MBC) and antibody secreting plasma cells (ASC). Recently it has become increasingly clear that MBC and ASC are in fact composed of functionally diverse subpopulations that can be distinguished based on unique surface marker expression, tissue localization, and the B-cell receptor (BCR) isotype expressed. During a humoral immune response, naïve B cells (nB) give rise to differentiated subsets, with the ultimate composition of the MBC and ASC pool primarily influenced by the antigen, the involvement of T cells, and the cytokine environment. Given the evidence for increased heterogeneity, it is surprising that we currently lack the genetic tools to further interrogate the complexity, molecular properties, and immunological importance of the known B cell fates. A precise phenotypic analysis of MBC and ASC subsets cannot currently be performed with current Cre-lox mouse models. Cis- regulatory elements (CEs), or enhancers, are DNA sequences that act to promote cell-type and context-specific gene expression programs. These sequences are regulated by epigenetic mechanisms, which act to control the accessibility of CEs to DNA-binding transcription factors. Over the past several years, we have characterized the epigenetic architecture that controls primary humoral immune responses to T cell dependent and independent antigens, integrated newly published datasets defining MBC subsets, and generated new preliminary data defining the CEs of ASC expressing distinct BCR isotypes. These data have revealed specific CEs that are active in defined stages of B cell differentiation, including IgA ASC and extrafollicular (EF)-MBC that arise independently of a germinal center reaction. Therefore, we hypothesize that cell-type specific CEs can be co-opted to provide precise genetic manipulation that enables functional exploration of B-cell subsets. To address this, we propose two aims designed to 1) develop new Cre recombinase tools for genetic manipulation of IgA ASC and 2) map the CEs for EF-MBC that arise during influenza infection and integrate CEs specific for EF-MBC to allow precise genetic editing of this MBC subset. These aims will utilize novel hematopoietic stem cell engineering to rapidly generate chimeric mice expressing genes of interest; therefore, bypassing the need to generate transgenic animals. Completion of these aims will provide a novel set of DNA sequences that allow for MBC and ASC specific genetic manipulation. These tools are critically important begin to derive the biology, molecular properties, and importance of the entire spectrum of B-cell differentiation to humoral immunity. If successful, it also has the potential to redefine how Cre recombinase vectors are engineered and further our understanding of CE biology in B cells and the immune system.

Up to $413K

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Identification of Host Factors that Mediate the Nuclear Retention of Unspliced and Partially Spliced HIV-1 Transcripts

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary Transcription of a HIV-1 provirus generates a 9-kilobase full-length transcript, that either remains unspliced or undergoes extensive alternative splicing resulting in generation of over 100 transcripts. While the completely spliced viral mRNAs can easily access the host mRNA nuclear export pathway, the unspliced genomic RNA and the partially spliced mRNAs are retained in the nucleus by unknown mechanisms. Nuclear export of intron- containing HIV-1 RNAs is mediated by the viral Rev protein (translated from fully spliced mRNAs) which binds to the Rev-response element (RRE) on these RNA subsets and tethers them to the host CRM1 export protein. How intron containing HIV-1 transcripts are retained in the nucleus is a long-standing question. The majority of cellular pre-mRNAs are alternatively spliced, and recruitment of mRNA export factors is closely coupled with splicing. Accordingly, the first widely accepted model proposes that lack of splicing leads to a block in deposition of RNA export factors on the unspliced and partially spliced HIV-1 transcripts. On the other hand, it is noteworthy that ~5% of protein-coding genes in humans do not contain introns. Hence, splicing is not a strict pre-requisite for RNA export. The second model proposes that intron-containing HIV-1 transcripts are actively retained in the nucleus due to distinct features present within these RNAs. This model is supported by the finding that codon- optimization of unspliced and partially spliced viral mRNAs overcomes nuclear retention independent of splicing modulation. On the other hand, the minimal sequence features necessary and sufficient for nuclear retention and whether trans-acting host factors are involved remain unknown. A distinguishing feature of the HIV-1 genome is its unusually biased nucleotide composition, rich in adenosines (~36%) and poor in cytosines (~18%). Preliminary studies provided herein raise the possibility that this property may underlie the nuclear retention of unspliced and partially spliced HIV-1 transcripts. The central hypothesis of this application is that unspliced and partially spliced HIV-1 mRNAs are actively retained in the nucleus by host proteins that recognize adenosine- rich sequences on these transcripts. In preliminary studies, we found that altering the codon usage of multiple reporter genes (e.g. GFP, mCherry, firefly luciferase) to resemble HIV-1 codon usage, hence making them adenosine-rich, results in their nuclear retention and dependence of reporter gene expression on Rev/RRE. In Aim 1, we propose to identify the minimal features of these RNAs that result in nuclear retention and determine whether this property is conserved in other mammalian species. In Aim 2, we propose to leverage these minimal HIV-1-like reporter RNAs in genome-wide CRISPR and siRNA screens to identify putative host factors that mediate nuclear retention of HIV-1 unspliced and partially spliced transcripts. The proposed studies have the potential to significantly advance our understanding of a fundamental aspect of HIV-1 replication. Knowledge gained herein will also more broadly impact viral and eukaryotic gene regulation fields.

Up to $428K

2028-03-31

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Identification of Immune Activation DNA inside Cells

NIAID - National Institute of Allergy and Infectious Diseases

Identification of immune activation DNA inside cells Cyclic GMP-AMP synthase (cGAS) is a key sensor of cytosolic double-stranded DNA (dsDNA) that activates the STimulator of INterferon Genes (STING) pathway, triggering immune responses against infections and cellular damage. While cGAS plays a protective role in pathogen defense and tumor suppression, its aberrant activation by self-DNA is implicated in autoimmune diseases (e.g., Aicardi- Goutières Syndrome, systemic lupus erythematosus) and chronic inflammation associated with aging. Despite extensive studies on cGAS-STING signaling, the physiological dsDNA ligands that activate cGAS under various cellular conditions remain poorly characterized due to the transient and weak nature of cGAS-DNA interactions, which conventional crosslinking methods fail to capture. To address this challenge, we propose to develop and apply a novel photochemical crosslinking approach to covalently capture cGAS-DNA complexes in living cells. Aim 1 focuses on adapting and optimizing this crosslinking technology for cGAS-DNA interactions, ensuring high efficiency and specificity. Aim 2 applies this method to establish a first-in-class cGAS BFPX ChIP-seq protocol, enabling the genome-wide identification of endogenous DNA ligands that activate cGAS in Trex1 KO MEF cells—a cellular model of Aicardi- Goutières Syndrome. This innovative approach will provide a powerful tool for studying cGAS-STING pathway activation across diverse physiological and pathological contexts. By uncovering the sources and mechanisms of immune-activating DNA, our work will pave the way for novel diagnostic and therapeutic strategies targeting cGAS in autoimmune, inflammatory, and age-related diseases.

Up to $457K

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Identification of metabolic regulators of hepatic phosphatidylcholine synthesis

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

Abstract Hepatic phosphatidylcholine (PC) metabolism is essential for maintaining lipid homeostasis, as PCs are required for very-low-density lipoprotein (VLDL) secretion and protection against hepatic fat accumulation. Disruptions in PC synthesis are linked to metabolic dysfunction-associated fatty liver disease (MAFLD), a condition affecting over 25% of the U.S. population. However, the genetic regulation of PC metabolism in response to metabolic stress, and its relevance to MAFLD prevention and progression, remain poorly understood. Our preliminary data reveal that the transmembrane oxidoreductase VKORC1L1 regulates hepatic PC levels through a function independent of its enzymatic activity. Furthermore, loss of hepatic Vkorc1l1 in mice decreases PC levels, reduces VLDL secretion, and promotes hepatic fat accumulation. Lastly, human genetic data indicate a potential link between VKORC1L1 variants and liver steatosis, suggesting a role in MAFLD progression. Hepatic PC levels are regulated through three primary pathways: de novo synthesis from choline via the Kennedy pathway, methylation of phosphatidylethanolamine (PE), and the salvage of exogenous lipids. We aim to define the role of VKORC1L1 in hepatic PC metabolism and MAFLD by addressing three key questions. First, we will determine whether VKORC1L1 is necessary and sufficient to regulate PC levels and mitigate MAFLD progression in mouse models under varying dietary conditions, focusing on two MAFLD-relevant nutrients: fat and choline—a precursor for PC synthesis. Using liver-specific Vkorc1l1 knockout and overexpression mice, we will assess its impact on VLDL secretion, hepatic lipid accumulation, and whole-body metabolic health. Second, we will delineate the broader impact of VKORC1L1 on hepatic PC homeostasis by assessing its influence on the three major pathways that maintain PC levels—the Kennedy pathway, PE methylation, and lipid salvage—using isotope-labeled metabolic precursors and phospholipid analytical approaches. Finally, we will investigate the molecular mechanism by which VKORC1L1 influences de novo PC synthesis, building on our preliminary data showing that VKORC1L1 binds to and activates CCTα, the rate-limiting enzyme of the Kennedy pathway. By combining biochemical and structural approaches, including cryo-electron microscopy, we will test whether this interaction is influenced by membrane PC content in cells and mouse liver. By uncovering how VKORC1L1 regulates PC metabolism and VLDL secretion, our study will reveal key molecular mechanisms linking choline and PC deficiency to MAFLD. This is particularly relevant, as low-choline and high-fat diets are prevalent in segments of the U.S. population. Our findings could inform therapeutic strategies targeting VKORC1L1 to restore PC homeostasis and mitigate MAFLD progression—an urgent priority given the widespread prevalence of MAFLD and its considerable variability in severity and treatment response.

Up to $676K

2029-11-30

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Identification of New Neuroimaging Biomarkers to Quantify the Decline in Brain Connectivity in Veterans with SCI and Measure Neuroplasticity due to Exoskeletal-assisted Walking

NIH

Every 30 minutes someone suffers a spinal cord injury (SCI) in the United States. There are about 300,000 individuals with SCI in the US, with around 17,800 new cases registered each year. The Department of Veterans Affairs (DVA) has over 42,000 Veterans with SCI registered in their database, of which about 27,000 are actively followed as part of ongoing care and rehabilitation. This makes the DVA the largest healthcare system in the world providing lifelong spinal cord care. SCI drastically diminishes a person’s mobility, with life-long consequences. Individuals who experience this catastrophic event go from regular mobility to limited or no mobility below their neurological lesion, becoming wheelchair reliant to get around their home and community. Military service members when paralyzed are unable to return to active duty. This drastic change in mobility has downward spiraling effects on a person’s physical and psychological health, including chronic pain, muscle spasticity, bone health, bowel/bladder function, obesity, community isolation, depression, and premature death. An important secondary consequence of SCI and subsequent long-term immobility is the decline in neural connectivity in the corticospinal tract. Prior physical rehabilitation studies have reported partial recovery of motor function in persons with incomplete and complete SCI, alluding to a large plastic capacity and reorganization of damaged corticospinal connections after SCI. Prior studies investigating corticospinal reorganization associated with motor recovery after SCI in humans have primarily focused on the spinal cord; evidence of neural reorganization and improvements in brain connectivity in humans at the supraspinal level after SCI is non- existent and much needed. This critical gap in knowledge is primarily due to the lack of evidence-based biomarkers to quantify the decline in brain connectivity after SCI. Recent advances in technology have enabled functional near-infrared spectroscopy (fNIRS), a relatively low-cost, portable, and high temporal resolution imaging to quantify neural activity in various brain regions through hemodynamic changes based on the principle of neurovascular coupling. fNIRS-based biomarkers have tremendous potential to detect improvements in brain connectivity early in response to physical rehabilitation, prior to any detectable recovery in motor function. One mode of physical rehabilitation that is growing in popularity is exoskeletal-assisted walking (EAW). The DVA has already committed to providing a robotic exoskeleton for home use to every eligible Veteran with SCI. A question of considerable practical relevance is can EAW mimicking physiological gait patterns increase brain connectivity in Veterans with SCI? The aims of this work are to: (1) identify new fNIRS-based biomarkers to quantify the decline in brain connectivity in Veterans with SCI, and (2) assess the feasibility of improving brain connectivity to promote neuroplasticity using EAW. Forty (40) participants with chronic SCI, including both complete and incomplete injuries, and forty (40) AB age-matched participants will be recruited. The SCI group will consist of Veterans with SCI at any level with demonstrable spared hand movement. Aim 1: Resting-state Functional Connectivity, a measure of intrinsic baseline alterations in brain connectivity, will be quantified for the SCI and AB participants. Aim 2: Task-induced fNIRS-based neurovascular biomarkers of sensorimotor function, namely Task-based Functional Connectivity, Task-based Hemodynamic Response, and Breath Hold Hemodynamic Response will be quantified for the SCI and AB participants. Aims 1 and 2 will be completed in one visit. Aim 3: A subset of 30 participants with SCI will be recruited for a pilot trial to tease out potential improvements in brain connectivity in Veterans with SCI. The participants will be randomly assigned to an EAW group (n = 10) or a control group (n = 20). The EAW group will undergo walking sessions for 9 weeks (36 sessions) in a self-balancing exoskeleton, the Atalante X (Wandercraft, Paris, France). fNIRS-based biomarkers will be measured before (at baseline), in the middle (week 5), and after week 9 of the EAW trial.

2030-02-28

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Identification of novel botanical derived NRF2 activating agents with potential against Alzheimer’s disease from Malagasy plants

FIC - John E. Fogarty International Center for Advanced Study in the Health Sciences

The incidence of Alzheimer’s disease (AD) in the USA is expected to increase from 7.2 million to 13.8 million by 2060, highlighting the urgent need to discover new treatments. Madagascar is known for its unique plant biota and hosts about 12,000 plant species, of which 80% are endemic. Malagasy flora has provided clinically used bioactive compounds such as the anticancer drug vincristine. Madagascar is now among the top priority conservation sites in the world since the forest is deteriorating due to urban expansion, pollution, bushfires, and poverty. The overall goals of this proposal are (1) to assist in conserving the unique plants of Madagascar by developing a model program for bioprospection and conservation, (2) to identify plants from Madagascar that contain phytochemicals that can be used against Alzheimer’s disease (AD), and (3) to promote scientific training and capacity development in the island. This project will focus on three main objectives involving medicinal plant inventory and conservation, exploration/discovery, and training and capacity building. Plants have been used in traditional medicines for central nervous system (CNS)-related conditions and many are known to produce antioxidant compounds. We aim to perform an ethnobotanical survey and develop conservation approaches for plants that are being used in traditional medicine for brain-related diseases including dementia and contain neuroprotective constituents from among the rich biodiversity of flora in Madagascar. Oxidative stress can impair the functions and activities of the CNS and has been shown to contribute to the onset and progression of many neurodegenerative conditions including AD. Our previous studies have shown that that activation of NRF2, which is an antioxidant regulatory transcription factor, elicits neuroprotection both in vitro and in vivo, can attenuate decreases in dendritic arborization and synaptic density, reduces oxidative stress, and improves mitochondrial function in hippocampal neurons isolated from AD mice as well as in the brains of AD mice treated with NRF2 activators. These data suggest that identification of NRF2-activating compounds could be a useful approach to discovering novel AD therapeutic compounds. Based on the structures of previously isolated bioactive compounds and their plant families of origin, we hypothesize that plant species with a high level of endemicity from the Malagasy flora will produce many previously unknown NRF2-activating compounds. The project will assist aging populations developing AD diseases by not only identifying and standardizing plants that have already been used in traditional medicine but also discovering new chemical scaffolds for discovering AD drugs.

Up to $3.4M

2030-05-31

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Identification of Novel Innate Immune Checkpoint Receptors

NCI - National Cancer Institute

PROJECT SUMMARY Immunotherapies targeting adaptive immune checkpoints have improved cancer outcomes, but innate immune checkpoints also play crucial roles in cancer immune evasion and are promising targets for immunotherapy. The innate immune system uses "eat-me" and "don’t-eat-me" signals to regulate phagocytosis, essential for maintaining tissue homeostasis and preventing cancer. Phagocytic cells have also recently emerged as new key actors in the success of immunologically mismatched allograft transplants through human leucocyte antigens (HLA) allorecognition. Thus, identifying the molecular patterns and receptors governing phagocytosis is vital for understanding cancer clearance and transplantation. Recently published studies of the PI revealed novel functions for Vascular Cell Adhesion Molecule-1 (VCAM1) on healthy and malignant hematopoietic stem cells (HSCs). We have found that VCAM1 is highly expressed on healthy HSCs, serving as an innate immune checkpoint for entry into the bone marrow by providing a "don't-eat-me" signal in the context of major histocompatibility complex (MHC) class-I presentation. In addition, we found that leukemia cells exploit this tolerance mechanism to avoid innate immune recognition, suggesting that the VCAM1-receptor axis is a promising target for immunotherapy. However, the specific receptor mediating this interaction remains unknown. In preliminary studies, we employed proteomics and AlphaFold modeling to identify novel VCAM1 receptor candidates on phagocytic cells. Our Specific Aim 1 focuses on identifying the VCAM1 receptor promoting immune tolerance and leukemia evasion and validating its function in vitro and in vivo using mouse and human models of leukemia. Specific Aim 2 will assess the impact of inhibiting or deleting VCAM1 receptor signaling on myeloid and lymphoid leukemia cell clearance, as well as allogeneic transplantation outcomes. Successful completion of this research will advance knowledge of innate immune recognition mechanisms, identify new leukemia immunotherapy targets, and improve outcomes in stem cell transplantation. 1

Up to $412K

2027-04-30

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Identification of Novel Targets for Enhancing Targeted RNA Degradation in Antiviral Discovery

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY / ABSTRACT The development of targeted RNA degradation (TRD) technologies, such as RNA-degrading chimeras, holds significant promise for antiviral therapies by reducing viral RNA levels to prevent viral replication. Despite their potential, current TRD mechanisms exhibit limited potency. In various literature reports, ribonuclease- targeting chimeras (RIBOTACs) exemplify this problem, achieving only ~75% maximum degradation of distinct RNA targets in cells. For example, our preliminary work optimized a synthetic RNA ligand, C34, which binds robustly to an RNA G bulge in the 5’ untranslated region of SARS-CoV-2’s RNA genome with a low nanomolar dissociation constant. However, the minimum inhibitory concentration (MIC) of the C34-based RIBOTAC remained moderately high at 20 μM in SARS-CoV-2-infected cells, indicating insufficient cellular potency. RIBOTAC is a drug-induced TRD modality utilizing an endogenous ribonuclease, RNase L. Interestingly, this RIBOTAC potency cap in cells was not observed in cell-free assays using purified recombinant RNase L, indicating the possible presence of cellular factors that inhibit the RNase L degradation complex. To address this potency limitation, the proposed project aims to discover novel cellular targets to significantly enhance RNase L activity and brand-new TRD mechanisms for future drug development through two specific aims. Aim 1 focuses on identifying cellular determinants that inhibit RNase L-dependent RIBOTAC activity using a genome-wide CRISPR knockout screen. By targeting these inhibitory genes, we aim to significantly boost the efficacy of existing RIBOTACs. Aim 2 focuses on discovering novel cellular targets for TRD by performing genome-wide screenings in three screening platforms. We will utilize a plasmid library comprising ~14,000 open reading frames from the human genome to identify candidate genes that can effectively induce TRD through drug-induced proximity. Both aims will leverage a SARS-CoV-2 cellular model and our optimized RNA ligand C34 to validate these new mechanisms in antiviral research. Ultimately, this project seeks to expand our chemical genetics toolbox by unveiling new mechanisms and targets for RNA degradation. These advancements will be pivotal in developing new chimeric molecules designed to combat a variety of infectious diseases, significantly enhancing our capacity to address global health challenges. 1

Up to $451K