Scripps Research Institute, The

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY/ABSTRACT Modern biomedical science enjoys an unprecedented ability to identify and describe viral pathogenic mechanisms, as well as characterize the biomolecular components that constitute them. However, we have not yet achieved a fully quantitative biophysical understanding in which modeling of component molecules is accurately predictive of viral functions in cells. Characterization of biomolecular structural dynamics, as opposed to their static structures alone, is creating to new inroads to quantitative modeling of cellular function as well as novel drug-targeting strategies. My recent work examining an RNA-protein interaction critical to HIV genome transcription suggests that highly quantitative measurements and systematic mutant design, which specifically perturbs dynamic properties, is a viable strategy for predicting viral RNA function in cells. In this proposal I plan to use a multi-modal integrative approach to quantitatively measuring RNA dynamics for the purposes of building a predictive model of RNA function and developing a novel RNA-targeting strategy. I will use the interaction of the HIV 5’-leader RNA with the Gag polyprotein as the model system for these studies as it is a well-studied yet complex interaction that involves critical interaction with the lipid bilayer and is also essential for the process of viral genome packaging, making it a relevant drug target. In Aim 1 I will develop integrative high-throughput (HTP) technologies that combine three-dimensional structural ensembles of component biomolecules with quantitative measurements of in vitro and cellular functions to build predictive models of viral activity that can be applied broadly. Specifically, I will construct a library of thousands of 5’-leader RNA mutants designed to systematically perturb its structural dynamics. I will create a plasmid library of these sequences to develop an RNA-Seq-based methodology to quantitatively measure the cellular activity of RNA mutants in HTP. I will then use existing HTP methods such as RNA-MaP to evaluate the in vitro binding affinity of the same library of sequences. Lastly, I will interpret this experimental data using structural dynamic ensembles of each RNA mutant, determined from nuclear magnetic resonance (NMR)-informed structure prediction programs, to build a predictive model of the 5’-leader:Gag interaction. By evaluating RNA mutants in HTP, I simultaneously screen for non-functional, low abundance RNA conformations in cells that could represent both attractive drug targets and tools for applications in synthetic biology. In Aim 2 I will identify and obtain ensemble descriptions of these non-functional conformations using NMR and computational modeling and target them for antiviral drug development using ensemble-based virtual screening (EBVS). I will also develop a fluorescence-based in vitro screening assay involving the lipid bilayer with which to test hits from virtual screening as well as in-house small molecule libraries. Lastly, I will use orthogonal biophysical methods to further validate hits, as well as use them to test the model constructed in Aim 1.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY Secreted proteins coordinate organ functions during homeostasis. The secreted proteome represents a poorly- characterized interorgan communication network, which is central in the etiology of various metabolic disor- ders. For example, obesity affects 40% of the US population, and the metabolic complications of obesity may result from dysfunctional interorgan communication. Despite this, the composition and activity of interorgan communication networks and their impact on obesity remain poorly defined. White adipose tissue (WAT) plays a central role in energy sensing and storage, and coordination of activities across organs to regulate systemic metabolism. In turn, energy storage in WAT is affected by the intestine in response to dietary and internal met- abolic inputs. Obesity disrupts this normal interorgan network and impacts metabolism of visceral and subcu- taneous WAT depots. We therefore anticipate that defining proteins secreted from intestine to the WAT depots will discover novel factors that regulate obesity. However, it has historically been difficult to identify such se- creted proteins due to their low abundance and lack of information on their organ(s) of origin. To address this, we established in mice a conditional BirA*G3 engineered biotin ligase system that generally labels secreted proteins within the endoplasmic reticulum of one organ, allowing identification of proteins that traffic to distal organs using affinity enrichment and quantitative mass spectrometry. This transgenic system can be ex- pressed in specific organs (e.g., intestine) to identify key secreted factors that communicate to distal organs, and provide clues to the labeled secreted factors’ underlying biology based on the organ-of-origin, the destination, and their levels in homeostatic or disease states. Using this approach in mice, we identified hun- dreds of proteins trafficking from intestinal epithelium to subcutaneous WAT, visceral WAT, brown adipose tis- sue, pancreas, and skeletal muscles in response to food intake, including the known low-abundance hormones GLP1/2, SST, FGF15, BMP8b, REG3, and CCK. We identified PLEXIN-B2 as a novel intestine-secreted pro- tein specifically targeting subcutaneous WAT. PLEXIN-B2 is produced during fasting and in diet-induced obesi- ty, and acts through SEMA4A receptor and SCRIB signaling to reduce adipocyte lipolysis and to promote adi- pogenesis. Our primary goal is to define the obesity-associated imbalances in intestine-secreted proteins, such as PLEXIN-B2, that directly impact the depot-specific differences in adipogenesis, lipolysis, lipogenesis and mitochondrial biogenesis, and contribute to systemic glucose and lipid metabolism, fibrosis, and inflammation. We will first use the BirA*G3 approach to establish the interorgan circuitry of intestine-to-visceral and subcuta- neous WAT communication during the development of obesity (Aim 1). Next, we will determine the impact of intestine-secreted proteins on WAT depots and metabolic maladaptation in obesity (Aim 2). Finally, we will de- fine the regulation of WAT depot function, metabolic homeostasis, and development of obesity by PLEXIN-B2 (Aim 3). In sum, we will discover mechanisms by which the intestine modulates WAT depot metabolic activities

NIH Research Projects · FY 2025 · 2024-09

SUMMARY Single particle cryoEM is now a ubiquitously employed methodology for high-resolution structure determination of biomedically important macromolecular complexes. CryoEM structures have revealed the detailed mechanisms underlying a wide range of cellular functions and helped understand how environmental or genetic factors perturb biological function to give rise to disease. However, all biological specimens prepared for single particle cryoEM imaging are exposed to the hydrophobic air-water interface during the sample preparation process. This interaction with the air-water interface has a destructive effect on the structural integrity of the targeted specimen, resulting in partial or complete unfolding of proteins, compositional dissociation of complexes, and widespread aggregation of the sample. This interaction with the hydrophobic air-water interface poses the single largest challenge facing the field of single particle cryoEM, and severely limits the utility of cryoEM structure determination for a wide range of samples. Particularly stable samples that maintain structural integrity after repeated interactions with the air-water interface often adopt preferred orientations relative to the hydrophobic surface, which either diminishes or completely eliminates one’s ability to determine a high-resolution structure of the targeted specimen. While there are additives and newer sample preparation devices that aim to mitigate these issues arising from the air-water interface, none of these are broadly applicable for all samples, nor do they fully overcome the air-water interface issues. Thus, cryoEM researchers often spend as much time (or more) optimizing cryoEM sample preparation methods as in establishing expression and purification conditions for a given macromolecule. Given that all commonly used cryoEM sample preparation technologies expose particles to the air-water interface, most of the acquired particle data are damaged and must be computationally discarded during image analysis. This makes cryoEM structure determination incredibly inefficient. We propose to overhaul the grid preparation process to abolish air-water interface interactions during cryoEM grid prep to accommodate preservation of fragile, hard-to-purify samples while substantially improving the efficiency of cryoEM data collection. This new technology will minimize damage to protein samples by blocking interaction with the air-water interface, which will increase applicability and efficacy of cryo-EM data collection, while also enabling robust quantification of macromolecular abundance or conformational dynamics present in a sample. Using a combination of graphene and nanocage technologies we will develop a cheap and robust methodology for cryoEM sample preparation that abolishes air-water interface interactions. Successful execution of the proposed sample preparation platform will advance our understanding of macromolecular mechanisms by enabling the preservation of challenging samples that thus far been recalcitrant to single particle cryoEM studies. This innovative cryoEM grid system will enable researchers to probe unexplored aspects of cell biology, and have a transformative impact that will reverberate throughout the structural biology community.

NIH Research Projects · FY 2025 · 2024-09

Project Summary Alzheimer’s disease (AD) and AD-related dementias (ADRD) represent the most common forms of neurodegenerative disease in patients above 65 years of age. Growing evidence has suggested that the potential causes of AD/ADRD in the aging population are multi-faceted and are likely mediated through an interaction of lifestyle, genetic, and environmental risk factors. As an example of environmental factors, air pollution caused by car exhaust and power plant emissions, contains hazardous airborne substances, such as fine particulate matter (PM) with nitric oxide-related species (NOx), and is associated with increased risk of cognitive impairment in AD. However, the molecular mechanisms underlying the interplay of genetic risk factors (such as ApoE4) and environmental air pollution exposures (termed GxE) in AD/ADRD pathogenesis remain largely unknown. In this application, we propose to study the targets of aberrant protein S-nitrosylation after exposure to PM/NOx as found in air pollution in order to better characterize this exposome and to find new potential targets of therapy for AD. In fact, we were the first group to show that increased levels of NO/NOx contribute to synaptic damage, a major correlate to cognitive decline in sporadic AD/ADRD, via aberrant protein S-nitrosylation forming SNO-proteins. For example, our recent findings in human postmortem brains from AD patients vs. age-matched controls of both sexes and diverse backgrounds has produced evidence for aberrantly S-nitrosylated proteins (SNO-proteins) in AD human brain that cause metabolic energetic compromise and resulting synaptic loss in AD, the major neuropathological correlate to cognitive decline. For our proposed work, we will use human induced pluripotent stem cell (hiPSC)-derived 2D cultures and 3D cerebral organoids as well as in vivo xenotransplantation models. In Specific Aims 1 and 2, we will determine the effect of the exposome, represented by PM/NOx present in air pollution, by assessing the S- nitrosoproteome in hiPSC-derived 2D cultures and 3D cerebral organoids as well as in mouse AD models carrying AD-related genetic variants vs. isogenic WT controls after exposure to PM/NOx. In Aim 3, we will identify and validate aberrantly S-nitrosylated proteins formed in response to the PM/NOx exposome as potential therapeutic targets in AD brain using hiPSC-derived 2D cultures and 3D cerebral organoids as well as in vivo mouse xenotransplantation models. We can determine causality of the aberrantly S-nitrosylated proteins by testing non-nitrosylatable mutant forms of the proteins, as we have previously described. Thus, the significance of these S-nitrosoproteomic changes is that they will be linked to cognitive decline in AD, and therefore S-nitrosylated protein changes arising from the exposome may represent potential new therapeutic targets.

NIH Research Projects · FY 2024 · 2024-09

PROJECT SUMMARY There exist few preventive or therapeutic modalities for vulnerable hosts against bacterial diarrheal illness. Further, the limited effectiveness of anti-infectives is being compromised by the antimicrobial resistant crisis. In response to this situation, physicians and scientists have investigated the role of probiotics. Despite promising preliminary data showing benefit in weakened immune hosts and against drug-resistant isolates, probiotic clinical trials have demonstrated inconsistent therapeutic and safety profiles. For these reasons, identifying the molecular mechanisms by which probiotics provide benefit is needed to develop new therapeutics. Notably, the Hang laboratory has discovered specific Enterococcus peptidoglycan hydrolases can improve intestinal barrier function and confer resistance against enteric infections in mouse models. Furthermore, these peptidoglycan hydrolases genetically engineered into Lactobacillus species were able to recapitulate protective effects. Given the unclear efficacy of probiotics in humans, we aim to develop orally available recombinant peptidoglycan hydrolases therapeutics that will promote intestinal barrier function to protect against enteric pathogens. We therefore propose to evaluate the activity of recombinant Enterococcus peptidoglycan hydrolase hydrogel formulations in mouse models of enteric infection (Aim 1) as well as explore peptidoglycan remodeling enzymes from other microbiota species/strains (Aim 2). The work proposed herein should afford new preventive and treatment modalities for vulnerable hosts against enteric infections.

NIH Research Projects · FY 2025 · 2024-09

Although numerous genes and loci associated with autism spectrum disorder and neurodevelopmental delay (ASD/NDD) have been identified through genome sequencing efforts, the precise mechanisms by which most of these genetic variants lead to the condition remain largely unknown. We propose to combine high-content in vivo genetic screening with whole brain cytoarchitecture spatial information from neurons with loss-of-function mutations in ASD/NDD risk genes to bridge the gap between genetic insights and mechanistic understanding. This approach, which can be scaled to interrogate large panels of genetic variants in parallel, has the power to reveal how these diverse gene variants converge to produce ASD/NDD, including identifying the specific brain regions, cell types, neural circuits, developmental time windows, and molecular networks involved in the pathogenesis of these disorders. To achieve this, we propose to use high-resolution and multimodal phenotypic characterizations to comprehensively map the functions in the neocortex and striatum of a set of 72 high-confidence ASD/NDD risk genes, many of which encode transcriptional regulators. We will adapt in vivo Perturb-seq to allow high efficiency screening across multiple developmental time points with both single-nucleus transcriptome and chromatin accessibility readouts. These rich datasets will enable us to build gene regulatory networks (GRNs) that will reveal shared and divergent molecular signatures associated with this set of ASD/NDD risk genes. In parallel, we will explore how perturbation of 5 high-confidence ASD/NDD risk genes impacts cellular migration, morphology, and long-range connectivity. Here, we will use Perturb-CAST (cytoarchitecture see-through) to combine sparse genetic perturbations in vivo with whole mount brain clearing and light-sheet imaging to examine brain-wide changes in cytoarchitecture across developmental time points. This work will expand two major technologies, in vivo Perturb-seq and Perturb-CAST, which will be broadly impactful tools for understanding the genetic basis of ASD/NDD and other complex brain disorders. By focusing on corticostriatal pathways and integrating spatial information, the proposal seeks to uncover commonalities and shared mechanisms among ASD/NDD risk genes, ultimately contributing to a more comprehensive understanding of the disorder and potentially guiding future therapeutic approaches.

NIH Research Projects · FY 2024 · 2024-09

Project summary Maintenance of homeostasis at barrier tissues is essential for mammalian health. At these sites, the concerted communication of different cell lineages is central for integrating a wide range of signals, to promote responses against noxious stimuli while preventing exacerbated responses against benign stimuli. The skin represents an organ where neuro-immune interactions may be of major biological significance, as it is one of the largest interfaces between the body and the environment, integrating signals including temperature, mechanical stimulus, tissue damage, and pathogenic and commensal microbes. Both the nervous and immune systems are involved in sensing potentially damaging perturbations and mounting appropriate responses for the avoidance and clearance of noxious stimuli. Recently, we successfully identified the existence of a cellular neuro-immune circuit formed by the interaction of sensory neurons and regulatory T cells (Treg cells). This neuro-immune circuit between Treg cells and sensory nerves is mediated at least partially through Treg cell production of enkephalins, endogenous opioids that induce analgesia. Through enkephalin production Treg cells dampen nociceptor activation to prevent exacerbated skin inflammation. We additionally find that other non-neuronal skin resident populations can produce enkephalins. The work proposed in this application seeks to uncover how endogenous opioid signaling mediates communication between sensory neurons and immune cells to regulate immune homeostasis in the skin. Understanding the roles for distinct sources of endogenous opioids may lead to the therapeutic harnessing of these for the treatment of chronic conditions, and may provide alternatives to the use of exogenous opioids. In addition, this project will, at a global scale, dissect the heterogeneity of responses in neuronal populations innervating the skin to different pathological contexts, and how these responses relate to their interactions with immune cells. This project will provide new insights on the molecular underpinnings of endogenous analgesics’ role in tissue homeostasis, as well as further our understanding on how sensory neurons respond to different types of inflammation, two key concepts that may have major implications on novel therapies for chronic conditions involving pain, itch and inflammation.

NIH Research Projects · FY 2025 · 2024-09

Principal Investigator: Wu, Xiaohua Project Summary Replication stress often occurs when DNA replication is disrupted, leading to DNA double-strand break (DSB) formation. Replication stress can also be triggered by oncogene expression and is associated with tumor development. Notably, replication stress creates vulnerabilities in cancer cells that can be exploited for cancer treatment. Nevertheless, our understanding of the detailed DNA repair mechanisms upon fork breakage due to replication stress, as well as our capacity to effectively target the associated vulnerabilities for cancer treatment, remains limited. DNA polymerase θ (POLQ) is a specialized DNA polymerase critical for DSB repair, and compromised POLQ function leads to genomic instability and radiation sensitivity. Substantial evidence suggests that POLQ plays an important role in microhomology-mediated end joining (MMEJ). Given the prevalence of microhomologies at cancer breakpoints, POLQ-mediated MMEJ is thought to be involved in promoting genome instability associated with cancer, but the precise underlying mechanism remains unclear. In this study, we discovered a unique role of POLQ in repair of DSBs linked to fork breakage, revealing a new mechanism that requires POLQ to cope with replication stress. Based on the role of POLQ in repair of damaged forks, we found that inhibiting POLQ leads to cell death under replication stress, and this effect is significantly intensified when ATR is inhibited. We propose that combined therapeutic strategy utilizing POLQ inhibitors and ATR inhibitors would synergistically eradicate cancer cells experiencing high replication stress. Given the current proposed use of POLQ inhibitors is primarily for treating BRCA-deficient tumors, this study will substantially broaden the application of POLQ inhibitors. We hypothesize that the role of POLQ in repairing broken forks forms the basis for treating replication-stressed cancer cells with POLQ inhibitors and ATR inhibitors, and propose to investigate the mechanism behind POLQ-mediated MMEJ in repair of broken forks. We will examine how POLQ is engaged to repair replication-associated DSBs and study the collaborative functions of POLQ-mediated MMEJ and break-induced replication (BIR) in repairing broken forks. We will use cell-based assay and xenograft mouse model to explore cancer therapeutic strategy of using POLQ inhibitors and ATR inhibitors. This study will not only shed light on the role of POLQ-mediated MMEJ in coping with replication stress, but will also lay the groundwork for potential cancer treatments by targeting cancer vulnerabilities associated with replication stress. Since replication stress is highly associated with cancer, this study will additionally bring insights into the DNA repair mechanisms that modulate genome integrity during tumor development.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY/ABSTRACT Post-traumatic stress and alcohol use disorder (PTSD/AUD) are frequently comorbid and are a major US health burden. Individuals with comorbid PTSD/AUD manifest complex symptoms including greater risk for alcohol drinking and relapse, sleep disturbances, hyperarousal, drinking-related aggression, and increased suicidal ideations. Identification of the molecular mechanisms underlying PTSD/AUD disorder will aid the development of novel therapeutic strategies. In this proposal, my focus will be on two key components of the extended amygdala (EA), the central amygdala (CeA) and the bed nucleus stria terminalis (BNST). The CeA and BNST are regions essential for the regulation of alcohol consumption, stress, and anxiety. In the EA, overactive brain stress systems are hallmark feature of comorbid PTSD/AUD, primarily driven by the pro-stress neuropeptide, corticotrophin releasing factor (CRF), resulting in long-lasting negative emotional states. New research has highlighted that the neuropeptide somatostatin (SST) displays anxiolytic (anti-stress) and alcohol-reducing properties and may act to oppose the effects of CRF in the brain. Both CeA and BNST are abundant in SST/CRF, and blockade of CRF or activation of SST system reduces consumption of alcohol and anxiety-like behavior in various rodent models and species. In this proposal, I will apply a multidisciplinary approach (e.g., in situ hybridization, ex vivo electrophysiology, chemogenetics, viral gene transfer of SST, and site-specific behavioral pharmacology) to understand the role of SST system in the EA in PTSD/AUD comorbidity. My overarching hypothesis is that SST signaling may exert reductions of PTSD/AUD phenotypes counteracting an “overactive” CRF in the EA. During my K99 mentoring phase, I will receive critical training in in situ hybridization, and ex vivo electrophysiology to understand neuronal expression and synaptic function of SST/CRF systems in PTSD/AUD. During my independent R00 phase, I will continue to perform electrophysiology, and apply site-specific behavioral pharmacology combined with chemogenetics and viral expression of SST to examine the behavioral modulation of PTSD/AUD by SST/CRF systems. The goal of this MOSAIC K99/R00 is to enhance my training in in situ hybridization and electrophysiological methods to complement my prior training in addiction neuroscience. This award will also provide critical professional career development to guide me in building an independent research program centered around stress disorders and AUD. Collectively, the proposed work will provide novel insight into the mechanistic role of SST and its potential therapeutic use for PTSD/AUD.

NIH Research Projects · FY 2024 · 2024-09

Developing a CRISPR-free mammalian recombineering system Project Summary / Abstract The ability to manipulate large segments of DNA in mammalian cells is essential for research and therapeutics. However, current programmable genome editing methods for creating kilobase-scale DNA manipulations require cytotoxic double-strand breaks (DSBs) or cannot create all required manipulations1. To address this major challenge, my laboratory at Scripps Research is developing a platform for mammalian recombineering that enables programmable large DNA insertions, deletions, and substitutions without the introduction of DSBs or DNA scars. Our preliminary data demonstrates that bacterial recombinases such as RecT from Enterococcus faecium2 are sufficient to catalyze whole gene insertion in the human genome. In this proposal, I describe the further characterization, optimization, and application of this system. First, we will fully characterize the mechanism and capabilities of efmRecT to create different size insertions, deletions, and substitutions across the genome using multiple methods of detection. Then we will engineer our recombineering system using supplemental bacterial recombineering proteins, mammalian repair protein inhibitors, and other manipulations informed by our mechanistic studies. We also aim to improve the efficiency of efmRecT through directed evolution in bacteria and/or mammalian cells. Finally, we will demonstrate the utility of our developed system by applying it to cell models of Gaucher Disease. The small size of RecT enables facile biomolecule delivery as the RecT gene, homology donor, and regulatory sequences can be encoded on a single recombinant AAV genome. As such, I am proposing an “all-in-one” AAV therapeutic where a single recombinant AAV can correct any of the hundreds of mutations associated with Gaucher Disease. If developed into a functional system for efficient genome editing, this method has the potential to impact fields spanning research to the clinic, including genomic perturbations of complex cell lines and organisms, methods for mammalian synthetic biology, and therapeutics.

NIH Research Projects · FY 2024 · 2024-08

Project Summary Hepatitis B virus (HBV) infects over 250 million people worldwide and is a leading cause of liver cirrhosis and cancer in many countries. The unique life cycle of HBV involves the generation of covalently closed circular double stranded DNA (cccDNA) in the nucleus of an infected cell for viral gene transcription and persistence. Current treatments only suppress viral replication. To cure HBV, it will likely require a combination of drugs targeting the virus as well as boosting antiviral immunity. In this project, we will apply the mRNA vaccine technology as a potent vaccine and immunotherapy against HBV. A major scientific challenge is that the HBV vaccine antigen, S, can disrupt protein homeostasis (proteostasis) leading to protein accumulation and undesirable ER stress in the mammalian cells overexpressing the protein. We will investigate the viral and cellular factors that hinder effective antigen expression, and improve the vaccine antigens for elicitation of potent antibody and T cell responses to cure HBV infection. Success in this project will also create new opportunities in the development of a multivalent mRNA vaccine against common human pathogens.

NIH Research Projects · FY 2025 · 2024-08

Project Summary/Abstract The mammarenavirus Lassa (LASV) is endemic to West Africa where it infects several hundred thousand individuals yearly resulting in a high number of Lassa fever (LF) cases associated with high morbidity and mortality. There are no US FDA-approved LASV vaccines and current anti-LASV therapy is limited to an off-label use of ribavirin that has limited efficacy. LF has been included on the revised list of priority diseases for the WHO R&D Blueprint, underscoring an urgent need for vaccines to combat LF. Epidemiological studies indicate that a live-attenuated vaccine (LAV) represents the most feasible approach to control LF. Mammarenaviruses are enveloped viruses with a bi-segmented negative-strand RNA genome. Each genome segment contains two open reading frames separated by a non-coding intergenic region (IGR). The large (L) segment encodes the RNA- directed RNA polymerase, L protein, and the Z matrix protein, whereas the small (S) segment encodes the surface glycoprotein precursor (GPC) and nucleoprotein (NP). We have documented that recombinant (r) forms of lymphocytic choriomeningitis virus (LCMV) and LASV expressing a codon deoptimized (CD) GPC or containing the IGR of the S segment in both the S and L segments are stable and fully attenuated in mouse and guinea pig models of LCMV and LASV infections, respectively, but able to provide complete protection, upon a single administration, against a subsequent lethal challenge with wild-type (WT) LCMV and LASV. Recently, we have found that a rLCMV containing a CD GPC and the S-IGR in both viral S and L segments (rLCMV/IGR-CD) is fully attenuated in mice but able to trigger protective immune responses against a lethal challenge with WT LCMV. Importantly, rLCMV/IGR-CD prevented the generation of LCMV reassortants with increased virulence in co-infected mice. Following our initial studies with LCMV, we have now rescued rLASV/IGR-CD and the central goal of this application is to test the hypothesis that rLASV/IGR-CD will have excellent safety and protective efficacy features as LAV, and unbreachable attenuation. To test our hypothesis, we will carry on a comprehensive characterization of rLASV/IGR-CD in cultured cells (aim 1), assess rLASV/IGR-CD safety, immunogenicity, and protective efficacy (aim 2), examine whether rLASV/IGR-CD prevents, in coinfected cells and animals, the generation of LASV reassortants with increased virulence (aim 3), and evaluate rLASV/IGR- CD stability during multiple rounds of infection (aim 4). Our studies will provide a comprehensive assessment of the feasibility of using rLASV/IGR-CD for the development of a LASV LAV candidate able to trigger long-term protective immunity, upon a single immunization, while exhibiting unique enhanced safety features, including an unbreachable attenuated phenotype. In addition, rLASV/IGR-CD will provide a valid LASV surrogate that could be safely used, upon completion of comprehensive experimental safety training, without requiring BSL4 containment, which will accelerate research on this important human and biodefense pathogen.

NIH Research Projects · FY 2025 · 2024-08

Occupational silica exposure is linked with systemic autoimmune diseases including rheumatoid arthritis (RA) systemic lupus erythematosus (SLE), systemic sclerosis (SSc), and anti-neutrophil cytoplasmic antibody (ANCA)-related vasculitis. The health burden is significant because systemic autoimmune diseases have been reported in 6% of those employed in mining occupations, and 21% of those with severe forms of silicosis. Although several novel animal models of systemic autoimmunity induced by exposure to crystalline silica (cSi) exist, there is no experimental model in which cSi exposure results in inflammatory arthritis. This is a significant barrier to our understanding of how occupational/environment exposures contribute to the development of arthritis. The “mucosal origins hypothesis” argues that arthritis pathogenesis originates at mucosal sites and then transitions to articular joints. Crucial to this is a pre-clinical phase of autoantibody production and chronic systemic inflammation influenced by genetic susceptibility to systemic autoimmunity. Using BXD2 recombinant inbred mice, which develop generalized systemic autoimmunity and erosive arthritis, we found that transoral instillation of cSi led to an exaggerated serum anti-ENA5 autoantibody response followed by pronounced synovial inflammation, cartilage damage, bone erosion, and pannus formation of ankle joints. The anti-ENA5 response is strongly associated with cSi induced systemic autoimmunity, suggesting that its induction contributes to the aggressive nature of the inflammatory arthritis in the context of an appropriate genetic susceptibility. To study the feasibility of the BXD2 as a model of cSi induced arthritis we propose two aims to define the relationship between pulmonary exposure, systemic autoimmunity, and inflammatory arthritis. Aim 1 will address the hypothesis that severe inflammatory arthritis in cSi exposed BXD2 mice depends on a cSi driven autoantibody response that requires the autoimmune prone genetic susceptibility of the BXD2 to mature into a chronic inflammatory response leading to severe inflammatory arthritis. The proposed studies will determine if genetic predisposition is essential to development of cSi induced inflammatory arthritis. They will also examine the relationship between cSi exposure, pulmonary inflammation, systemic autoimmunity, and inflammatory arthritis to identify how immunological events occurring at a mucosal site relate to development of joint arthritis. In Aim 2 we will test the hypothesis that cSi particles find their way to joint spaces, resulting in localized inflammatory events which are then aggravated by an already existing cSi induced chronic autoantibody response leading to severe inflammatory arthritis. We will test this by asking if cSi particles can be found in ankle and knee joints, and if intra-articular injection of cSi is sufficient for arthritis or if passive transfer of IgG from anti-ENA5 positive cSi exposed BXD2 mice is necessary to enhance arthritis severity. Successful completion of these studies will result in a novel experimental model of cSi induced inflammatory arthritis, and provide a better understanding of how adverse immunological events at mucosal surfaces can impact pathology in distant tissues.

NIH Research Projects · FY 2026 · 2024-08

ABSTRACT Protein homeostasis (proteostasis) is tightly regulated by an intricate network of finely tuned cellular pathways. Among the pathways required for responding to internal or external cellular insults is the Integrated Stress Response, which halts general protein production while specifically increasing production of a select subset of “pro-survival” proteins. As we age, our cells decline in their ability to maintain proteostasis, serving as a hallmark for a range of age-related diseases such as Alzheimer’s, Parkinson’s, Huntington’s, and amyotrophic lateral sclerosis. This pathological loss of proteostasis correlates with increases in ISR activity, establishing the importance of gaining a detailed understanding of the mechanisms involved in ISR activation. Four kinases serve as upstream triggers of the ISR. Each kinase is activated by distinct forms of cellular stress, but all convergently phosphorylate the eIF2α translation complex to activate the ISR pathway. One of these kinases, Heme- Regulated Inhibitor (HRI), was recently shown to signal mitochondrial stress to the ISR via a protein called DELE1. The HRI kinase is the principal mediator of ISR activation in neurons with perturbed proteostasis, and is thus of particular relevance to age-related brain diseases. However, the detailed mechanism through which DELE1 activates this kinase to relay mitochondrial stress to the ISR is unknown. Recent structural and cellular studies on the DELE1 protein from our group have shown that that ISR activation is dependent on oligomerization of DELE1 in the cytosol. Our high-resolution single particle cryo-EM structure of DELE1, combined with biochemical and cellular studies, indicate that the higher-order assembly likely serves as a structural scaffold for HRI binding and activation. We plan to elucidate how interactions between DELE1, HRI, and eIF2α transduce mitochondrial stress to ISR activity using single-particle cryo-EM, crystallographic, and functional studies. Atomic-level descriptions of the interacting elements of this pathway could enable us to precisely counter human mitochondrial pathologies without impacting the capacity of cells to respond to other stresses, such as ER stress or viral infections. We will use a range of complementary methodologies to probe the mechanistic underpinnings of the DELE1-HRI-eIF2α pathway to gain much-needed insights into this branch of ISR activation. We will combine biophysical, biochemical, structural, and cellular studies in three Aims that: 1) tests our hypothesis that DELE1 oligomerization leads to auto- or trans-phosphorylation of bound HRI kinases; 2) defines the role of cleavage in DELE1 oligomerization and ISR activation; and 3) examine the structural details of eIF2α recruitment to HRI and the mechanism of its activation. These studies will provide a comprehensive, mechanistic description of the key interactions that relay mitochondrial stress to the ISR, and will provide novel avenues to specifically target the DELE1-HRI-eIF2α pathway to tune both the mitochondrial signaling and the adaptive ISR signaling for therapeutic interventions.

NIH Research Projects · FY 2025 · 2024-08

Project Summary Antisynthetase syndrome (ASSD) is a chronic autoimmune disorder characterized by myositis and interstitial lung disease (ILD). A key feature of ASSD is the presence of autoantibodies against aminoacyl-tRNA synthe- tases (aaRSs), among which histidyl-tRNA synthetase (HARS or HisRS) causing anti-Jo-1 antibodies is the most common. aaRSs are the enzymes that attach amino acids to their appropriate tRNA and are thus essential components of the intracellular translation machinery. Interestingly, some aaRSs are also secreted and function extracellularly to regulate inflammation and immune responses. However, the mechanisms by which aaRSs are released from cells, become targets of autoimmunity, and cause symptoms are not well understood. Importantly, tRNA, a key partner of aaRS, has not been considered at all in the context of ASSD. There is growing evidence suggesting the involvement of endosomal Toll-like receptor TLR7 and type I interferon (IFN-I) signaling in sus- taining and spreading inflammation in the autoimmune disease. The ligands that activate TLR7 are single strand RNAs (ssRNAs). Therefore, we hypothesize that tRNAs or tRNA fragments bound to aaRSs play critical roles in the pathogenesis of ASSD. We propose a two-year project to explore the hypothesis using ASSD-relevant cells and to generate a novel and clinically relevant animal model for mechanistic study and future therapeutic devel- opment. The results obtained from this study would uncover a previously unrecognized involvement of tRNAs in the pathogenesis of ASSD and suggest new targets for therapeutic development of the disease.

NIH Research Projects · FY 2025 · 2024-08

Project Summary/Abstract Somatic mutations alter the folding and therewith function of oncogene and tumor suppressor proteins. The alterations in protein structure drive malignant transformation of cells. While structural changes of somatic mutated proteins have been extensively characterized with high spatial resolution, conformational alterations of other nonmutated effector proteins in the cancer proteome remain elusive. The heterogeneity of protein conformations and protein-protein interactions in tumors at the single cell level remains unknown, and it is unclear whether tumor cells with the same genetic background differ in protein conformation or protein-protein interactions in non-mutated proteins. Here, we propose to implement a new mass spectrometry-based protein footprinting technique to measure protein conformations in single cells. We recently developed Covalent Protein Painting which infers protein structural information with a chemical protein footprinting technique that surveys the chemical reactivity of lysine residues in proteins to determine alterations in protein conformation or protein-protein interactions in a proteome. We propose new versions of CPP that will overcome the current limitations in sample preparation and analysis of single cell proteomes (CPP-SCP). We will quantify lysine site accessibility in single cells with CPP-SCP, and we will establish bioTMT-CPP-SCP, a variation of the CPP-SCP method that can directly compare changes in the 3D proteome between several (>10) single cells with high sensitivity. We propose to analyze intact single cells that are isolated from two cancer cell lines and from murine tumor tissues. Our goal is to find out whether measurements at single cell level recapitulate structural alterations that we observed in bulk tumor samples. Specifically, it remains unclear whether aberrant protein conformations per protein are observed at equal abundance across all cells or if they are confined to a subset of cells in a cancer cell line or tumor. We hope that CPP-SCP will allow us to differentiate between intra- and intercellular variation in protein conformation and folding.

NIH Research Projects · FY 2025 · 2024-08

The growth factors and transcriptional activators that give rise to diverse populations of functionally distinct circulating monocytes and tissue-resident macrophages (MFs) have largely been identified. In contrast, the influence of metabolic signals from the local tissue microenvironment on MF differentiation and function remains essentially unexplored. One endogenous signal that been associated to MF differentiation in homeostatic conditions is the essential metabolite heme, the body’s main purveyor of iron, which directs red pulp MF (RPM) differentiation. As RPMs degrade senescent red blood cells, they accrue large amounts of hemoglobin-derived heme. This buildup of intracellular heme triggers degradation of the heme-regulated transcriptional repressor BACH1, thus inducing expression of SPIC1, a transcription factor (TF) required for RPM development. Beyond its role in regulating RPM differentiation, our preliminary data show that BACH1 plays a previously unappreciated outsized role in myeloid cells. We find that BACH1 is part of the small set of core factors that regulate MF identity and function, and that it integrates the transcriptional response to signaling heme and pro-inflammatory stimuli. Supporting this notion, mice lacking BACH1 in myeloid cells have altered numbers of tissue-resident MFs, and cells derived from them show reduced fitness in bone marrow transplant repopulation studies. Moreover, myeloid BACH 1 KO mice have alterations in the inflammatory/repair process in the setting of acute muscle injury, and, unlike WT littermates, quickly perish when given with a sublethal dose of LPS. Given that BACH1 activity is regulated by heme, these unexpected findings stress the extent to which heme signaling regulates MF biology and highlight the need to gain insight into the pathways of intracellular heme delivery that control BACH1 function. Heme is a vital metabolite for life, but due to its highly oxidative iron content, free heme is very cytotoxic. As such, heme intracellular mobilization requires a protein network to enable its transfer throughout the cell, the components of which are largely unknown. We have discovered in adipocytes an intracellular heme trafficking pathway mediated by the poorly characterized proteins Progesterone Receptor Component 1 and 2 (PGRMC1, PGRMC2), that delivers heme to proteins in the ER and the nucleus, including heme-responsive TFs such as BACH1. We recently found that the PGRMC1/2 heme trafficking pathway is active in MFs, and that PGRMC1/2 DKO MFs have increased BACH1 levels and functional defects. Our hypothesis is that in monocytes/MFs, the PGRMC1/2 pathway is central for delivery of heme to the nucleus, and that BACH1 is a principal mediator of the effects of heme signaling on MF differentiation and function under normal and pathological conditions. In this project, we leverage unique mouse models with reduced nuclear heme, chemical tools that increase signaling heme flux to the nucleus, and new mouse mutants of the key mediator BACH1 to reveal a molecular pathway for metabolite control of MF differentiation/function.

NIH Research Projects · FY 2025 · 2024-08

Scripps Research is becoming a leader in the field of translational research. This leadership establishes itself on three strengths: the quality of its faculty, its outstanding and holistic training programs, and its interdisciplinary culture. Ours is one of the few institutions in the US where a basic science idea can be taken seamlessly to a drug candidate and a clinical trial. Reciprocally, we can also bring patients' data or specimens from the clinic to the most basic aspects of science, including genomics, structural biology, and chemistry. This translational ability requires early career scientists to be trained across disciplines and in many emerging areas of data acquisition and processing. These areas include single cell data acquisition, genomic studies, big data analysis, sophisticated biological measurements, and animal models, and ultimately a deep knowledge of the disease being studied. In addition to this interactive scientific multilayered education, our program benefits from integration of the post-doctoral training program within the larger educational goals of the Scripps Research CTSA, including interns, predoctoral students, and clinical research fellows. This mentee to mentor approach is exemplified by the pairing of each T32 postdoctoral scholar with two T32 predoctoral scholars, and a K12 scholar, and the T32 trainee summer supervision of interns from the SURF program. This aspect of our training program will be enhanced by an already existing mentorship structure and an added mentor training program. Our T32 funding will support three T32 postdoctoral trainees for 2-3 years and will train them for a successful career in translational science and medicine. A particular emphasis will be put on individual grant writing and submission to establish research independence. Translational science coursework, created and directed by SRTI research team leaders, will incorporate protocol design, literature review, ethical research practice, transdisciplinary teamwork, effective communication, and advanced translational methodologies.

NIH Research Projects · FY 2025 · 2024-08

The quest for a broadly protective vaccine against Influenza has been stymied in part to the antigenic diversity of the flu virus, and in part due to pre-existing, non-protective immunity from past exposures, so called immunological imprinting. Further complicating the elicitation of broadly protective antibodies against Influenza is that the most highly immunodominant regions of hemagglutinin tend to be in regions of high antigenic variability, which generally only result in strain specific or seasonal responses [1]. In recent years, multiple groups have isolated antibodies directed towards highly conserved regions of hemagglutinin located in the stem [2-4], including towards the newly identified anchor epitope, which is analogous to the membrane proximal external region (MPER) of the HIV-1 envelope protein. These broadly neutralizing anchor antibodies have exhibited the ability to bind and neutralize H1, H2 and H5 viruses [5, 6]. Thus, anchor bnAbs are an attractive class of antibodies for passive infusion or vaccine elicitation, either in isolation or in concert with other epitopes where they would likely enhance protection breadth. Though the stem region of hemagglutinin tends to be more conserved, the recessed and immunologically subdominant nature of the epitope restricts the frequency of these responses, however the development of epitope scaffold immunogens could overcome these barriers for antibody access. Rigorous prior research has demonstrated (i) germline targeting epitope scaffolds can elicit highly specific antibodies against HIV in both preclinical and clinical models [7-11]; (ii) influenza neutralizing antibodies are influenza correlates of protection [12-14]; and (iii) tuning Influenza humoral responses towards conserved regions can improve cross strain reactivity of elicited responses [6, 15]. This project seeks to develop immunogens that present the anchor epitope in a native-like context with the aim of eliciting anchor class antibodies.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY PIEZO1 and PIEZO2 are mechanosensitive ion channels that convert force into chemoelectric signals. PIEZOs play diverse roles in physiological processes such as touch, proprioception, breathing, and vascular development. Pathogenic mutations in PIEZOs can cause extensive sensory defects and debilitating neurological diseases such as distal arthrogryposis. Despite recent advances in elucidating the physiological roles of PIEZO ion channels, the underlying structural correlates of function are largely unknown. PIEZOs are homotrimeric ion channels with extensive blades of transmembrane domains that are thought to be the principal transducers of mechanical force, but static structural snapshots and in vitro studies are poorly equipped to describe which structural rearrangements underlie channel gating. To address these fundamental gaps in knowledge, this proposal will use fluorescence nanoscopy to determine the structural mechanics of PIEZO ion channels in a cell at single molecule resolution. These structural mechanics will then be correlated with the functional output of the channel using electrophysiology. Aim 1 will determine which unique structural mechanics underlie the functional differences between PIEZO1 and PIEZO2. This will be accomplished by examining how the cellular environment and intrinsic structural mechanics shape the functional output of each channel. Aim 2 will define how the interplay between the protein and the plasma membrane shapes the activity of PIEZOs, especially in the context of pathogenic gain-of-function mutations that cause distal arthrogryposis. This will be accomplished by manipulating the lipid composition of the plasma membrane and by assessing how diverse modulators shape the structure of the channel. This proposal will ultimately provide fundamental insight into how PIEZO ion channels function inside of a cell and provide a foundation for the study of other sensory ion channels.

NIH Research Projects · FY 2024 · 2024-07

The Participant Center (TPC) will 1) maintain capabilities that enable full All of Us Research Program (All of Us) participation across the United States (US) and 2) lead innovation in the participant experience in support of program goals. TPC is an alliance of multiple partners, led by Scripps Research. TPC makes it possible for interested individuals anywhere in the U.S. to join and remain engaged in All of Us. TPC will reach individuals where they are through approaches ranging from light touch digital outreach to high touch, in-person one-on-one support. TPC strategies and tactics aim to support the recruitment of over 200,000 new baseline participants, while upholding the program’s goal to reflect broad groups from across the US. We aim to maintain at least 75% active retention, in line with TPC’s current active retention rate of 86%. Drawing on our experience in All of Us since 2016, including the recruitment of over 97,000 participants and support of biospecimen collection from over 44,000 participants, we will improve on the processes and relationships we have built to further the program’s mission to enable individualized health care. TPC will use innovative methods that accelerate progress toward program goals. Building on this innovation we aim to combine novel data collection with technology to return personalized information and improve participant health.

NIH Research Projects · FY 2025 · 2024-06

The NLRP3 inflammasome is a key regulator of inflammatory responses. These include responses to infection by pathogens but also are a component of many chronic inflammatory diseases, including neurodegenerative diseases. The end point of the inflammasome response is the secretion of mature inflammatory cytokines, IL-1β and IL-18. To accomplish this, the NLRP3 inflammasome is assembled and activated through a complex series of events, some of which remain poorly understood. The first step in NLRP3 inflammasome activation is the de novo biosynthesis of NLRP3 itself through transcriptional upregulation, and the assembly or newly synthesized NLRP3 molecules into inactive oligomers. How this occurs is only partially understood. We have evidence that the ubiquitin ligase SCFFBXW7 is required for this particular process. The objective of the proposed research is to test a hypothesis where SCFFBXW7 places ubiquitin chains on the N-terminal end of NLRP3 which are bound and capped by a UIM (ubiquitin interacting motif) near the C-terminus. The constraints enforced by this interaction would lock NLRP3 in a conformation that would promote the assembly of inactive oligomers.

NIH Research Projects · FY 2026 · 2024-06

We propose in this application a novel therapeutic approach for the eradication of CoV-2. We developed a new strategy, which consists of hijacking the viral replication machinery to trigger the death of CoV-2-infected cells, while preserving uninfected cells. We propose to administer intranasally human ACE2 transgenic mice a “tailored” RNA encoding the diphtheria toxin fragment A (DTA) called {CoV-2 Hijack DTA} that is only recognized and transcribed by the CoV-2 polymerase (Pol/RdRp) present in infected cells, triggering DTA expression and rapid death of infected cells. Since DTA cannot cross the cellular membrane, it cannot kill uninfected cells. Because RNA can be easily broken down in the body, it needs to be transported within a protective carrier. Noninvasive aerosol inhalation is a well-established method of drug delivery to the respiratory tract and represents an ideal route for nucleic-acid-based therapeutics as demonstrated in various clinical trials. We propose to design degradable polymer-lipid nanoparticles (LNPs) that can deliver RNAs by nebulization (inhalation) to the respiratory tract. We propose to synthesize hyperbranched poly-beta amino esters (hPBAEs) to enable nanoformulation by nebulizer of stable and concentrated polyplexes suitable for inhalation. This strategy should achieve uniform distribution of RNAs throughout lungs resulting in high levels of proteins of interest 24h post-inhalation of hPBAE polyplexes without local or systemic toxicity due to rapid degradation of hPBAE vectors. The safety and antiviral efficacy of nebulized {CoV-2 Hijack DTA} RNA LNPs stably protected by degradable hPBAEs will be analyzed in CoV-2 variant-infected mice. Our in vivo imaging IVIS Lumina S5 system permits a daily bioluminescence (NanoLuc-CoV-2) or fluorescence (mNeonGreen CoV-2) quantification of the {CoV-2 Hijack DTA} RNA LNPs-mediated killing of infected lungs in live mice. Viral and host events required for the beneficial therapeutic effect of {CoV-2 Hijack DTA} RNA LNPs will be investigated including caspase pathway activation, membrane permeability and chromosomal degradation of infected lung cells as well as the prevention of the development of interstitial pneumonia and cytokine cascade. In summary, this application proposes to demonstrate the safety and efficacy of this novel therapeutic approach that consists of hijacking the enzymatic viral replication machinery to trigger the specific killing of CoV-2-infected cells, but not of uninfected cells. Importantly, {CoV-2 Hijack DTA} RNA LNPs will be administered at different time points of the infection in order to compare the preventive and therapeutic efficacy of our novel approach.

NIH Research Projects · FY 2026 · 2024-06

Dry eye is a prevalent condition that significantly impacts the lives of millions of elderly adults worldwide, causing ocular irritation and blurred vision, thereby severely affecting their overall quality of life. The lacrimal gland (LG), responsible for producing the aqueous layer of the tear film, undergoes age-related changes characterized by immune cell infiltration and impaired regenerative capacity. While calorie restriction (CR) has been shown to slow down functional decline in various tissues, its specific effects on LG inflammation remain to be fully understood. In our preliminary studies, we successfully generated the first single-cell RNA sequencing (scRNAseq) atlas of the LG from both young and old mice fed ad libitum (AL) and those subjected to a 40% calorie-restricted (CR) diet for a duration of six months. Our preliminary findings indicate that the aging process is associated with a decline in mitochondrial and lipid metabolism, a process regulated by peroxisome proliferator-activated receptor (PPAR)-α. This metabolic decline contributes to the accumulation of lipids within the epithelial cells of the lacrimal gland. Additionally, we observed an upregulation of the inflammasome pathway, leading to inflammasome activation in the LG epithelial cells, production of inflammatory cytokines such as IL-1β and IL-18, and Gasdermin-D cleavage. Notably, our research revealed a compelling observation: CR effectively reversed the primary dry eye phenotype associated with aging. In the LG, CR led to an elevation in PPAR-α and retinoid X receptor (RXR) levels, thereby enhancing lipid and mitochondrial metabolism. Importantly, CR also resulted in a significant enhancement of the expression of the epithelial progenitor cell markers. Our data suggests that the molecular pro-resolving mechanism underlying CR involves the activation of PPAR-α signaling, that in turn, leads to a reduction in lipid accumulation within the epithelial cells, thereby mitigating cell damage, inflammasome formation and activation of immune cells. We hypothesize that the increase in PPAR-α is a key mechanism by which CR protects epithelial cells from damage and enhances the function of lacrimal gland (LG) progenitor cells. To test these hypotheses, our research will employ several approaches: 1) We will utilize Nanostring GeoMX spatial transcriptomics combined with immunostaining and flow cytometry to elucidate the specific effects of CR on epithelial cells and macrophages within the LG. 2) We will evaluate the impact of modulating PPAR-α on the function of aging LG epithelial cells and the inflammatory responses of immune cells. 3) Furthermore, we aim to determine the effect of PPAR-α modulation on LG regeneration, as well as the growth, proliferation, and differentiation of progenitor cells in LG organoids. By investigating the mechanisms underlying CR, we hold the potential to develop interventions for age-associated LG inflammation and dry eye. This research offers promising prospects for addressing these conditions and advancing the field of ocular health.

NIH Research Projects · FY 2026 · 2024-05

Development of potent and safe CRISPR tools for in vivo gene editing using directed evolution Abstract Genome editing, or the ability to precisely manipulate DNA, is an emerging technology that has the potential to be a permanent cure for deadly and debilitating genetic diseases. The development of CRISPR- Cas9 as a genome editing tool has catapulted this field from primary research to clinical trials within the past decade. CRISPR has already been successfully used to treat multiple diseases in human clinical trials, including hereditary blindness, neurodegeneration, blood disorders, and cancer. As these treatments grow in complexity and enter more patients, we increasingly need advanced CRISPR-Cas9 tools that enable potent gene correction while improving safety from aberrant toxic and immunogenic effects. Cas9 is favored for many genome editing approaches due to the ease of reprogramming; the CRISPR single guide RNA (sgRNA) dictates target site through base pairing to a user-defined sequence. However, eukaryotic cells display robust cellular and immunogenic responses towards RNAs, leading to sgRNA instability and toxicity. Chemical RNA modification overcomes this issue by protecting the oligonucleotide from cellular RNA-recognizing machinery, leading to improved stability, distribution, cellular uptake, and safety. Importantly, full chemical modification (modification at every residue) has been essential for the therapeutic success of established RNA therapeutics, like silencing RNA (siRNA). Despite the promises of full chemical modification, fully modified sgRNA remain poorly active for Cas9 genome editing, likely due to the complex interactions between the Cas9 and sgRNA being incompatible with chemical modification. This proposal describes an approach to engineer Cas9 proteins towards compatibility with fully chemically modified sgRNA. These Cas9:sgRNA pairs would enable highly efficient, easy to deliver, and immune evasive genome editing, supporting genome editing applications including multiplexed targets, transient editing, redosing, and vector inactivation.