Grants

NIGMS - National Institute of General Medical Sciences

Project Summary/Abstract The discovery of new bioactive compounds used for human medicine is driven by our ability to rapidly and efficiently generate new chemical structures. One major challenge in this aim is the development of synthetic strategies that allow us to prepare molecules with the three-dimensional structural complexity that is needed for selective recognition of cellular targets. Along these lines, it has been shown that small molecule natural products, peptides, and proteins all possess the structural features needed to efficiently and successfully recognize macromolecules of interest but remain difficult to produce on scale through traditional synthetic chemistry. The long-term goals of this research program are to develop new biocatalytic tools in the form of enzymes and metabolic pathways to address this challenge. This proposal focuses on the development of methods to discover and engineer enzymes and pathways involved in the modification of amino acids and peptides. Amino acids serve as key chiral building blocks for the cell, producing target molecules ranging from functional non- canonical amino acids, alkaloid natural products, bioactive peptides, and proteins. Only certain classes of these products, such as those made by non-ribosomal peptide synthases or the ribosome, can be readily identified through established bioinformatic methods. Our lab has defined several classes of privileged protein families involved in the transformation of amino acids and propose to discover, characterize, and engineer these new enzymes as synthetic tools for the preparation of targets ranging from small-molecule nitrogen heterocycles to modified peptides and to protein conjugates. In the proposed work, we focus on non-heme Fe(II)/α-ketoglutarate dependent enzymes including halogenases, hydroxylases, and other transformations that carry out the C-H activation of inert C(sp3)-sites to introduce synthetically useful functional groups such as Cl, Br, N3, or keto groups for downstream reactions. We also explore the enzymology of the ATP-Grasp family of enzymes, which form amide linkages between amino acids or peptides and various small molecules. Amide bonds remain essential for medicinal chemistry and we seek to develop ATP- Grasp enzymes both for solution-phase approaches for peptide synthesis as well as biocatalytic site-selective protein modification.

OD - NIH Office of the Director

Abstract The natural world is filled with fascinating proteins and the arms race between bacteria and viruses is a particularly rich reservoir for their discovery. Research in this area over the past decades has uncovered restriction enzymes for genetic mapping, T7 phage polymerase used in the production of mRNA vaccines, and Cas9 for genome editing, and motivates continued exploration into this genetic conflict for useful molecular machines. Bacteria have many defense strategies against foreign elements, including CRISPR-Cas systems, which typically provide immunity through RNA-guided nuclease activity, however, my work has uncovered new RNA-guided functions including CRISPR-associated transposases that perform RNA-targeted DNA insertion, and CRISPR- associated proteases that cleave protein substrates upon target RNA detection. These systems reveal exciting new biology and how diverse enzymes can acquire, or be acquired by, RNA-guided proteins and I believe only scratch the surface of programmable functions that exist in nature. Practically, the characterization of new RNA-guided functions will enable new capabilities in biology to alter and control cells based on genetic information. The discovery of Cas9 and programmable nucleases opened the door for genome editing technology and I envision that additional programmable modalities could similarly transform biology and provide much needed molecular tools to interrogate the function and regulation of the human genome. Advances in DNA and RNA sequencing have provided insight into gene function, mutations that cause disease, and unique cell populations based on gene expression signatures, however, we lack the ability to alter and control cells based on their genomic and transcriptomic state. My goal is to discover and characterize proteins that are regulated via nucleic acid recognition and to harness these discoveries to engineer and control human cells.

NIAID - National Institute of Allergy and Infectious Diseases

Abstract: Clostridioides difficile is the leading cause of nosocomial infections and antibiotic-associated diarrhea. The incidence and severity of C. difficile infections (CDI) have dramatically increased due to the overuse of antibiotics and the emergence of hypervirulent epidemic strains, which were responsible for several outbreaks globally. Even though the overuse of antibiotics is responsible for CDI, the management of CDI requires antibiotic administration. Consequently, the drawbacks associated with the current anti-CDI therapeutic arsenal highlights the critical need for developing novel strategies for the treatment of CDI and the prevention of CDI recurrence. An alternative strategy for developing novel therapeutics involves the exploitation of antisense oligomers (ASOs), such as peptide nucleic acids (PNAs) that suppress essential genes in bacterial pathogens. This research project focuses on developing targeted therapeutics to disarm virulence factors and suppress gene expression in C. difficile. Building on our successful use of antisense technology in C. difficile, we aim to investigate the potential and implication of inhibiting critical pathways within C. difficile. These pathways include transcription and translation machinery, protein translocation machinery, cell division, replication, and fatty acid synthesis. We are also planning on assessing the toxicity and in vivo efficacy in murine models of CDI and its recurrence. We believe the narrow spectrum and precision targeting of these antisense therapeutics provide an innovative new approach that selectively eliminate problematic pathogens, such as C. difficile without harming the healthy human gut microbiota. Altogether, these studies are of paramount importance and have the potential to make a significant impact, effectively leapfrogging the traditional drug development process.

NIAID - National Institute of Allergy and Infectious Diseases

ABSTRACT The overall aim of this application is to develop a new class of antimalarials targeting PfGARP for use as parenteral therapy for severe falciparum malaria in direct response to MMV’s TPP-1-“For severe malaria, a parenteral formulation of a single fast-acting TCP-1 would be appropriate” (asexual stage agent) 1. Plasmodium falciparum is a leading cause of morbidity and mortality in developing countries, infecting hundreds of millions of individuals and killing almost one-half of a million children in sub-Saharan Africa each year. The spread of parasites resistant to the artemisinin family of compounds threatens recent progress achieved by antimalarial campaigns and underscores the urgent need to identify new anti-malarial drugs. In recent work 5, we discovered PfGARP, a vaccine candidate found only in P. falciparum. PfGARP is located on the exofacial surface of iRBCs and antibodies to the highly invariant carboxyl terminal of PfGARP kill parasite in culture in the absence of immune effector molecules (complement) or cells- thus the remarkable anti-parasite effect of anti-PfGARP results from antibody binding alone. This is further supported by the killing effect of recombinant mAb and its rec monovalent Fab that target aa 443-459 of PfGARP. The Scientific Premise of this application that PfGARP is a high value druggable target is based on: 1) its surface expression on iRBCs, 2) its absence of amino acid homology with host proteins, 3) its absence of significant sequence variation in over 3,000 field isolates sequenced, 4) the requirement for PfGARP for in vivo survival, and 5) the ability of antibody binding to PfGARP to kill essentially all parasites within 12-24 hours. In the current proposal, we will: 1) conduct a targeted, high-throughput drug screen to discover drugs which mimic the lethal activity of antibodies recognizing PfGARP, 2) optimize and down select these candidates, and 3) validate these new drug candidates in a humanized mouse model of P. falciparum.

NIAID - National Institute of Allergy and Infectious Diseases

ABSTRACT Infection with human Cytomegalovirus (HCMV) is nearly universal; seroprevalence rates reach 90% in individuals older than 80. Although usually asymptomatic in the normal host, HCMV causes severe morbidity and mortality in transplant recipients and patients with AIDS. Congenital HCMV is the leading infectious cause of childhood deafness and mental retardation. There is no approved vaccine for the prevention of HCMV. The nucleoside analog ganciclovir (GCV) and its oral formulation val-GCV have improved the outcome of transplantation. GCV may also prevent hearing deterioration in congenitally infected children. However, prolonged courses of GCV or val-GCV result in intolerable toxicities to the bone marrow and select for resistant viral strains. The alternative drugs for GCV-resistant HCMV, foscarnet, and cidofovir, also target the viral DNA polymerase. Both are nephrotoxic agents and can only be administered intravenously. The HCMV UL97 kinase inhibitor, maribavir, was recently approved for adults and children (> 12 years of age) with resistant/refractory HCMV disease, and the terminase inhibitor, letermovir, is approved for HCMV prophylaxis after hematopoietic stem cell transplantation. Resistance has already been reported to these two new agents. The limited HCMV therapeutic armamentarium and the emergence of drug-resistant mutants create an urgent need to identify new HCMV inhibitors with a novel mechanism of action. Our goal is to develop a new class of HCMV inhibitors that act through a novel viral target(s), the helicase-primase (H/P). As a prototype, compound MLS8091 inhibits HCMV, including GCV-resistant strains, and we have identified that resistance to MLSS8091 resides in the primase and primase-associated factor of the H/P complex. The following Specific Aims are proposed in this R01: 1) Delineate the mechanism(s) of action of MLS8091. 2) Define the interactions and activities within the H/P complex. 3) Design and synthesize MLS8091 analogs to identify a lead compound for in vivo studies. Our program is innovative and highly significant because the target for HCMV inhibition is novel and critical for virus replication, we already identified resistant mutants, and we assembled a multi-disciplinary investigative team to develop inhibitors of the H/P complex. Our team has combined expertise in virology, proteomics, structural biology, biophysics, biochemistry, and chemistry. At the end of the project, we expect to: 1) Confirm the HCMV H/P as a druggable target, 2) Provide purified proteins, biophysical and enzymatic data of the H/P complex for future HCMV drug discovery, and 3) Identify the most favorable small molecule HCMV inhibitor(s) through drug design and synthesis.

NIAID - National Institute of Allergy and Infectious Diseases

The objective is to develop an oral treatment for Ebola virus infection in humans.

NIAID - National Institute of Allergy and Infectious Diseases

To develop broad-spectrum treatments for flaviviruses.

NIAID - National Institute of Allergy and Infectious Diseases

To develop novel small molecule inhibitors targeting influenza A and B viruses.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY Viral proteases are essential for the replication of many RNA and DNA viruses. Through sequence specific recognition of cleavage sites, these proteases process viral polyproteins into their functional components, while also cleaving host proteins to facilitate viral replication. Interestingly, organisms from every domain of life encode proteins known as SERPINs (SERine Protease INhibitors) that serve as natural inhibitors of serine and cysteine proteases, the types of proteases encoded by many viruses. SERPINs contain a conserved structural core and a disordered reactive center loop (RCL) that ‘baits’ a protease into cleaving it by mimicking the sequence specificity of the protease to be inhibited. Following protease-mediated cleavage of the RCL, the SERPIN undergoes a large conformation change that covalently traps and inactivates the protease, thus connecting sequence-specific recognition of the SERPIN RCL to protease inhibition. While SERPINs have been largely characterized as inhibitors of a wide range of cellular proteases, we hypothesize in this grant that SERPINs have also evolved, in a species-specific manner, to directly inhibit viral protease activity and thereby inhibit viral replication. Supporting this hypothesis, we have found that several mammalian SERPINs contain RCLs that are rapidly evolving under positive selection, consistent with SERPINs being engaged in evolutionary arms races with pathogen-encoded proteases. Moreover, we have identified primate SERPINs that have independently evolved sequences that mimic the cleavage site preferences of proteases from picornaviruses and coronaviruses, and find that these SERPINs can inhibit viral protease activity during infection. These data lead us to propose a model in which SERPINs, with their protease baiting RCLs, represent a modular platform for evolution of host-specific antiviral activity through viral protease inhibition. Based on this model, we now propose to use evolutionary biology, protease biochemistry, and virology to discover and characterize the natural variation, evolvability, protease specificity, and antiviral activity of viral protease inhibitors among mammalian SERPINs. In Aim 1, we will test the antiviral potency and specificity of primate SERPIN-mediated viral protease inhibition, while also computationally and functionally searching for additional mammalian SERPINs that can inhibit proteases from a wide range of RNA viruses. In Aim 2, we will probe the modularity and evolvability of SERPINs to determine the degree to which the SERPIN ‘core’ and RCL sequence impact function, and how sequence evolution of each can mediate selective viral protease inhibition. By identifying SERPINs as novel, rapidly evolving, host-encoded viral protease inhibitors, our work will reveal the impact of SERPIN and viral protease evolution on this new host-virus evolutionary conflict and on species-specific barriers to virus replication, and unveil the evolutionary potential of SERPINs to inhibit a wide range of viral proteases.

NIGMS - National Institute of General Medical Sciences

Project Summary Currently, many genome-wide association studies (GWAS) show that single nucleotide polymorphisms (SNPs) associated with drug response and drug metabolism are in non-coding regions of the genome, requiring multi- omic data to better ascertain function. Multi-omic data exist for populations of European ancestry and have been used extensively to elucidate the target genes of associated SNPs through studies of expression quantitative trait loci (eQTLs). However, population diversity within publicly available multi-omic databases is sorely lacking, limiting the ability to utilize the greater diversity in African Ancestry populations to enable discovery for all populations. Our work will fill this gap. We plan to 1) Identify regulatory variants affecting drug-metabolizing enzymes using multi-omics data gathered in hepatocytes, 2) Determine the function of eQTLs via a massively parallel reporter assay in HepG2 and 3) Investigate the effect of eQTLs on the enzyme activity of drug- metabolizing enzymes via prime editing. The need for more predictive biomarkers is clear, and our work will fill this gap.

NIGMS - National Institute of General Medical Sciences

The translocation of polar lipids from one side of a membrane bilayer to the other is critically important in physiology. This process, termed lipid flip-flop, is variously required for biogenesis of mitochondria, growth of the endoplasmic reticulum (ER), all forms of protein glycosylation, and synthesis of glycolipid blood group antigens. It is also needed to expose the signaling lipid phosphatidylserine (PS) at the cell surface in response to physiological triggers – PS promotes blood clotting by activated platelets, marks apoptotic cells for clearance by macrophages and induces cell-cell fusion needed to produce myotubes and osteclasts. Specific proteins – termed scramblases – catalyze lipid flip-flop by acting as lipid channels. These proteins have attracted considerable recent interest, resulting in new information and new concepts concerning intracellular lipid transport and homeostasis, yet many fundamental mechanistic and biological questions remain. The overall goals of our research are to investigate these questions broadly, by discovering the molecular identity of scramblases in key processes where their activity is implicated but not yet been assigned to a specific protein(s), understanding the molecular mechanisms by which these novel lipid channels work, evaluating their specific contributions to physiological processes, and understanding how their activity is regulated. To achieve these goals, we focus here on two understudied research areas which exemplify key knowledge gaps. We recently discovered phospholipid scramblases in the outer mitochondrial membrane. These proteins likely provide the means by which polar lipids enter mitochondria to support the growth and function of the organelle. We will use a multidisciplinary suite of methods to understand how these proteins work and to define their specific contributions to mitochondrial biogenesis. Second, we are interested in the molecular identity of glycolipid scramblases that are implicated in all forms of protein glycosylation in the ER. These scramblases have eluded discovery, and their identification is a major goal for the field. We propose a multi-pronged experimental approach to identify these proteins and validate their function using biochemistry and genetics. Mitochondrial dysfunction is a common source of inborn errors of metabolism, and is associated with cancer, cardiovascular and neurodegenerative diseases, as well as with lipid syndromes such as Barth. Protein glycosylation is essential for life, and specific defects in glycosylation pathways can lead to a number of diseases including muscular dystrophy. In addition to advancing the fields of mitochondrial cell biology and glycobiology and providing unifying biophysical insights into a fundamental membrane transport process, the research proposed here and the overall program that we have conceived will contribute to an understanding of the etiology and presentation of many diseases.

NIAID - National Institute of Allergy and Infectious Diseases

Abstract Middle East respiratory syndrome coronavirus (MERS-CoV) infection has the highest mortality rate (36%) of any of the known human-pathogenic Betacoronaviruses. MERS-CoV continues to circulate in the Middle East, and due to global travel has spread to 27 other countries making it a global health priority. A number of viruses related to MERS-CoV have been identified in mammals worldwide, and several have been found to utilize human DPP4- or ACE2-receptors for cell entry. Therefore, the MERS-related betacoronaviruses (Merbecoviruses, MERBs) have considerable zoonotic potential. While many medical countermeasures like vaccines and antibody therapeutics were developed to fight SARS-CoV-2, these countermeasures do not prevent disease caused by MERBs. The long-term goal of this proposal is to generate such MERB broadly neutralizing antibody (bnAb) therapeutics. The significance of this project includes the identification of broadly protective antibodies (Abs) and their epitopes, enabling rational design of antibody-based therapeutics and vaccines against a deadly virus. While previous research has focused on identifying potent neutralizing antibodies against MERS-CoV, no antibodies have been shown to be cross- protective against MERS-CoV and other MERBs. In preliminary studies in vaccinated rhesus macaques (RMs), we elicited robust cross-binding and cross-neutralizing plasma Abs against multiple MERBs. Post- vaccination RM B cells bound both MERS-CoV receptor binding domain (RBD) and bat MERS-CoV-related virus NL140422 RBD. Individual Abs from these RMs bound to as many as 7 different MERS-CoV-related RBDs and MERS-CoV. The objectives of this study are to 1) determine the neutralization breadth of monoclonal nAbs from these vaccinated RMs, 2) determine the critical features of the binding interface between cross-reactive nAb and virus spike RBD, and 3) determine the cross-protective efficacy of the mAbs. The innovations of this project include human, bat, and pangolin live-virus MERB models (MERS-CoV, PDF2180, NL140422 (MERS 422), MjHKU4r, HKU-5), 13 genetically diverse MERB spike and RBD antigens, MERS-CoV and MjHKU4 mouse challenge models, and the use of a machine-learning Ab optimization to improve natural Abs. The impact of this project includes the demonstration that vaccination can elicit broad MERB nAbs against multiple conserved epitopes, the development of trispecific immunotherapies, and the definition of bnAb epitopes that inform rational design of immunogens targeting cross-protective B cells. Our Abs will be potential tools to combat MERS-CoV outbreaks and prepare for future outbreaks from pre- emergent MERBs.

NCI - National Cancer Institute

Project Summary & Abstract Microbial natural products have yielded remarkable anticancer agents, such as enediyne antibiotics, bleomycin, and mitomycin, that directly target DNA and induce double-strand breaks or DNA alkylation. The recent surge in microbial genome sequencing has uncovered the genetic fingerprints for thousands of molecules within these categories. However, only a tiny fraction of this potential has been harnessed, as the discovery process is hindered by the transcriptionally silent nature of the corresponding biosynthetic genes and the extremely low production levels, even when these genes are expressed. We have established methods to access metabolites encoded by transcriptionally silent or ‘cryptic’ biosynthetic gene clusters. Using a forward chemical genetics approach, termed high-throughput elicitor screening (HiTES), we have identified >150 novel, cryptic metabolites. Very recently, we have utilized the approach in conjunction with DNA cleavage assays to identify a new, cryptic enediyne that we have named clavulyne. We are therefore poised to apply this method broadly in the current proposal to discover novel enediyne natural products, thereby expanding the chemical space of this intriguing family of metabolites. We also plan to leverage creative synthetic strategies to repurpose nine-membered ring enediynes, such as clavulyne, which are otherwise too unstable for detailed biological and chemical evaluation. To further expand these efforts, we will also target a broader group of anticancer agents. Guided by bioinformatics, we will search for products of divergent bleomycin and mitomycin gene clusters as well as exploit emerging bioassays in an untargeted manner to discover completely new classes of DNA-targeting antiproliferative agents. Successful completion of the proposed studies will contribute new and difficult-to-find enediynes and DNA-targeting metabolites, thus expanding our repertoire of antiproliferative agents derived from microbial sources. They will additionally introduce new synthetic approaches to generate next-generation designer enediynes. The synthetic and natural products that result from this proposal could serve as lead compounds, payloads for antibody-drug conjugates, or chemical probes to better investigate their biological and chemical properties.

Wildlife Conservation Society

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Wildlife Conservation Society

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Wildlife Conservation Society

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Wildlife Conservation Society

A Greener NYC

Wildlife Conservation Society

A Greener NYC

NIAID - National Institute of Allergy and Infectious Diseases

The offeror’s proposed work plan aims to identify, characterize and optimize non-toxic, low molecular weight agents as adjuvants that a) enhance protein antigen accumulation and release in non-immune and immune cells after intramuscular administration of mRNA vaccine and b) stimulate innate immune pathway-activation in antigen presenting cells.

NIAID - National Institute of Allergy and Infectious Diseases

The offeror proposes to discover adjuvants with two sets of unique properties – (1) directing T cell fates and (2) improving the response to mRNA vaccines. This will be accomplished by screening novel immune-potentiators that direct the innate immune response of the original vaccine or adjuvant to induce new immune phenotypes. The goal will be to discover new adjuvant systems that (1) selectively direct T cell fates toward TFH, TH17, TH1, and TH2; and (2) modify the cytokine profile of mRNA vaccinations toward safer and more effective responses while maintaining and improving antibody levels.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary The human gut microbiota plays a critical role in health, with one of its major roles being to protect the host from microbial pathogens through a phenomenon known as colonization resistance. Effective colonization resistance is associated with increased bacterial diversity within the gut microbiota, which likely enhances the functional capabilities of the community. One microbial function thought to be important for colonization resistance is the production of antimicrobial small molecules by gut bacteria. However, our current understanding of the specific gut microbial metabolites involved in colonization resistance is limited, with identified compounds being largely restricted to previously known microbial products, including short-chain fatty acids, bile acid derivatives, and antimicrobial peptides. We hypothesize that the gut microbiota produces additional metabolites with antimicrobial activity that that are important for colonization resistance. The overall objective of this application is to discover such compounds through high-throughput screening. Specifically, we will screen a library of gut bacterial culture and supernatant extracts for growth inhibitory activity against a panel of bacterial and fungal pathogens and pathobionts relevant to the human gut microbiota (Aim 1). We will then isolate and structurally characterize antimicrobial metabolites from gut bacterial supernatants or cell pellets with promising activity (Aim 2). The proposed studies are supported by a preliminary screen demonstrating the presence of antibacterial and antifungal activity from multiple extracts in the library. To our knowledge, high-throughput screening has not been used previously to identify antimicrobial metabolites from members of the human microbiota. The proposed research therefore has high potential to identify antimicrobial compounds from these organisms, setting the stage for future efforts to understand the biological activities and roles of these metabolites within the gut microbiota, including their contributions to colonization resistance. Ultimately, our findings will provide a foundation for developing novel antimicrobial therapies and enhancing the resilience of the gut microbita against infections.

NIGMS - National Institute of General Medical Sciences

Project Summary The goal of this Phase III COBRE to continue and sustain our work to discover new chemical probes and lead molecules for biological research and therapeutic discovery. Our approach is broad-based and interdisciplinary, with a focus on using chemical biology and molecular design to investigate and understand disease while leveraging our core capabilities in synthesis, biochemistry, engineering, and biology. Building on the successes of Phase I and II, our center will continue to build center momentum with the continued recruitment and development of new investigators through faculty hiring and through pilot project funding. During Phase III, we will continue to support our successful and broadly utilized cores, and we outline a plan for sustaining the cores beyond COBRE funding with a broad base of extramurally funded user groups and an outstanding University commitment to support the cores through a significant match commitment and hard-funded Ph.D. level staff. We will sponsor an annual seminar series to be hosted by COBRE investigators and the UD Allies for a Culture of Inclusive Diversity (ACID) committee. The center will develop resources for mentoring and professional development including Grant Development Awards, UD ACID activities, MIRA workshops, NIH Diversity Supplement workshops, and Career Long Collaborative workshops. Center progress will be evaluated by an Advisory Committee that will meet twice per year. Our Center will collaborate with other centers in the Delaware IDeA network, UD centers and the NIH funded Chemical-Biology Interface (CBI) Training grant centered at UD in order to maximize our research capacity. Discovery COBRE values the importance of diverse teams working together to maximize innovation. In Phase III, our center will create infrastructure for biomedical research teams who bring diverse backgrounds and expertise to our center.

NIA - National Institute on Aging

PROJECT SUMMARY Dementias are projected to become the most burdening group of diseases, expected to affect over 150 million people worldwide by 2050, and Alzheimer’s disease (AD) is the most common among them. Currently, over 6 million Americans are living with AD. While there have been several recently-approved drugs for AD, these drugs target amyloid beta plaques – just one facet of the disease – and have not proven effective in all patients. Non-neuronal cell types in the brain such as microglia (the brain’s immune cells) and astrocytes (star- shaped helper cells) play a critical role in disease progression but have been traditionally understudied. Microglia are known to be activated by amyloid beta plaques and they were ascribed both a protective and a detrimental role. They were shown to induce a toxic state in astrocytes. The microglia’s behavior is likely influenced by patient genetics, which might explain why the majority of AD risk genes are expressed in microglia. To determine which of the 90 known AD-associated genetic variants exert their effect through microglia, we need a better understanding of the functional links between a variant and the disease. This requires large-scale studies of diseased, human cells from genetically diverse patients, as there is a wide variety of genetic variants that can influence AD risk. This project proposes to develop an automatable protocol for the creation of human induced pluripotent stem cell (iPSC)-derived microglia (iMG) and their co-culture with primary astrocytes. Standardized co-cultures of iMGs from AD patients and primary astrocytes will allow scientists to observe how these cells interact in a diseased environment, better understand known variants, and possibly identify new risk variants. Attaining sufficient statistical power for the identification of novel variants requires many cell lines, which can only be achieved with robotic automation. This study will serve as a proof of concept to demonstrate that such co-culture systems be automated and will later be scaled up to include additional cell lines. It will furthermore help to understand how certain genetic variants contribute to AD, which might inspire new therapeutic approaches.

NIEHS - National Institute of Environmental Health Sciences

Project Summary Our goal is to identify gene-environment interactions (GxE) with acute and chronic exposure to the ambient air pollutant ozone (O3) that contribute to common, chronic respiratory diseases like asthma and COPD. However, detecting GxE with O3 exposure in human epidemiologic studies is extremely challenging for a number of reasons, including statistical power and precision of exposure assessment. To overcome these challenges, we have exploited genetically diverse, multi-parental mouse populations, namely the Diversity Outbred (DO) and Collaborative Cross (CC, inbred), as GxE discovery platforms because in these models we can maximize information about both genetic variation and O3 exposure. Previously, we utilized the CC to identify a large-effect GxE quantitative trait loci (QTL) with acute O3 on chromosome (Chr) 15 that affected airway injury and inflammation. At this locus, two candidate genes emerged, Angpt1 and Rspo2. We also found that a common variant at the orthologous locus in humans (rs10086579, located between ANGPT1 and RSPO2) affected response to acute O3 in humans, demonstrating proof of concept for our approach. This variant also exhibited GxE with chronic air pollution in COPD, indicating it likely affects response to both acute and chronic exposure. Here, we propose to first identify the causal gene on Chr 15 in mice (Angpt1 vs. Rspo2), then identify new GxE QTL for both acute and chronic O3 responses. In all cases, we will translate our findings from mouse to human through targeted analysis of GxE with common variants in human orthologs of the genes we identify in mice using existing genotype and O3 exposure data from 3 human studies. The human studies include (1) an acute O3 challenge dataset (n=191) in which inflammation was quantified, (2) the Multi-Ethnic Study of Atherosclerosis Air Pollution Study (MESA-Air, n~6000) in which associations between chronic O3 exposure and COPD (emphysema and lung function) have been quantified, and (3) the Children’s Health Study (CHS, n~1800) in which associations between chronic O3 and childhood-onset asthma have been quantified. In Aim 1, we will evaluate Angpt1 and Rspo2 using knockdown/knockout and rescue experiments in mice. In parallel, we will conduct an expanded human genetic analysis of acute and chronic O3 response with rs10086579 and nearby variants using the 3 human datasets. In Aim 2, we will identify new GxE for acute O3 response in mice by performing a QTL mapping study involving two CC strains that are phenotypically divergent but not due to variation at the Chr 15 QTL. Gene expression QTL (eQTL) mapping in alveolar macrophages (AM) and mediation analysis will be used to identify candidate genes. We will then test for GxE in the human studies. In Aim 3, we will identify GxE for chronic O3 response using DO mice, employing a longitudinal study design in which lung function is measured prior to and after O3 exposure. After mapping QTL at high resolution and identifying candidate genes, we will test for GxE in the CHS and MESA-Air. In total, our work will reveal novel genes associated with that affect O3-induced asthma and COPD onset and/or progression.