University Of California Berkeley

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY/ABSTRACT Despite the well-known Latina birth paradox, recent data (2020) show that Latina mothers are 1.2-1.5 times more likely to have low birth weight infants compared to White mothers. Additionally, compared to White women, annual national vital statistics data from 1989 through 2020 show consistently higher rates of worse birth outcomes among Latinas. The longstanding focus on the Latina birth paradox has left a gap in the literature, resulting in a critical need for research on birth outcome disparities faced by Latinas, and the mechanisms driving those disparities. We propose two novel mechanisms as contributors to poor birth outcomes for Latinas: anticipatory racism threat and area-level racial bias. A strong body of evidence has demonstrated links between chronic social stress and poor birth outcomes. Racial discrimination, a chronic psychosocial stressor, is a prominent explanation for racial/ethnic disparities in birth outcomes. However, evidence is limited by the predominant focus on racism events and racism experiences at the individual-level. We previously developed and validated a measure of anticipatory racism threat (aRT) for African American women and found associations with hypertension, allostatic load and telomere length. These preliminary studies suggest that anticipating racism, above and beyond actual racism events, is related to biological dysregulation, including dysregulation of systems that have previously been linked with low birthweight and preterm birth. Another neglected component of racism-related stress is area-level racial bias. Emerging evidence demonstrates significant links between negative area-level racial sentiment and birth outcomes for racial/ethnic minorities groups. The specific objectives of the K99 phase are to 1) create and psychometrically validate an aRT-Latina scale and 2) examine associations with low birth weight and preterm birth in recent Latina mothers. R00 phase objectives include developing and testing associations between a novel county-level indicator of area-level racial bias towards Latines and county-level birth outcomes among Latinas. The expected impact of the proposed research is re-focusing attention on poor birth outcomes among Latinas, a long-standing disparity but largely neglected area of investigation; helping to elucidate some of the predictors and underlying mechanisms driving those disparities; and ultimately informing the types of interventions likely to ameliorate those disparities. These data will also provide preliminary data for a subsequent R01 application. Our central hypothesis is that higher anticipatory racism threat and more negative area-level racial bias toward Latines will predict worse birth outcomes (i.e., low birth weight and pre-term birth) for Latina mothers.

NIH Research Projects · FY 2026 · 2023-08

Abstract Progress toward promoting health and well-being as we age must include the identification of novel targets that are safe, powerful, inexpensive, and deployable. Our focus is on one such target—patient memory for the contents of treatment—because: (1) patient memory for treatment is poor, (2) poor memory for treatment is associated poorer adherence and poorer outcome, (3) memory support strategies can improve memory for treatment and (4) improved memory for treatment improves outcome. In this application, we propose to test a new, streamlined, and potent approach to engaging this novel target: the Memory Support Intervention (MSI). The MSI aims to improve patient memory for treatment. It was distilled from the basic, non-clinical research in cognitive science and education and is comprised of four powerful memory promoting strategies that are proactively, strategically, and intensively integrated into treatment-as-usual. Importantly, the MSI does not add to session length, or the number of sessions needed. The aim of this proposal is to conduct a confirmatory efficacy trial to test whether the MSI improves outcomes for midlife and older adults. As a “platform” for the next step in investigating this approach, we focus on sleep and circadian problems and the Transdiagnostic Intervention for Sleep and Circadian Dysfunction (TranS-C). TranS-C is a worthy platform on which to test the MSI because (1) sleep and circadian functioning, including and beyond insomnia, is highly prevalent among midlife and older adults, (2) poor sleep and circadian functioning has a wide range of serious negative consequences, including on memory and (3) TranS-C addresses a range of the most common sleep and circadian problems experienced by midlife and older adults. Promising pilot data suggest that memory for TranS-C may be poorer among midlife and older adults, relative to younger adults, and that adding memory support has potential to improve treatment adherence and treatment outcome for this age group. Over 5 years, we will recruit adults who are 50 years and older and who are experiencing sleep and circadian problems (N = 178, including 20% for attrition). The sample will be randomly allocated to TranS-C plus the MSI (“TranS- C+MSI”) vs. TranS-C alone, and all will receive eight 50-minute, weekly, individual sessions. Assessments will be conducted at baseline, post-treatment, and at 6- and 12-month follow-up. The sample will be recruited from two large community-based organizations that serve midlife and older adults who are low-income and experiencing mobility impairments. The intervention will be delivered via live telehealth to improve accessibility. We will compare the effects of TranS-C+MSI vs. TranS-C alone to determine if the MSI improves sleep and circadian functioning, daytime functioning, and well-being (Aim 1). We will determine if patient memory for treatment (the target) mediates the relationship between treatment condition and outcome (Aim 2). We will evaluate if previously reported poor treatment response subgroups moderate target engagement (Aim 3). The MSI could be added to a range of interventions to enhance intervention outcomes for midlife and older adults.

NIH Research Projects · FY 2026 · 2023-08

PROJECT SUMMARY Maintenance of proper mitochondrial function is critical to organismal health. Mitochondrial stress contributes to ageing and the pathogenesis of numerous diseases. Intracellular insults, such as oxidative stress, mitochondrial DNA mutations, and disrupted interactions with other organelles including lysosomes and the endoplasmic reticulum are relatively well identified inducers of mitochondrial dysfunction. Yet it remains largely elusive how mitochondrial homeostasis can be altered upon changes in cellular microenvironment, the extracellular matrix (ECM). In mammals, ECM remodeling occurs during aging and in multiple diseases including infections, cancers, and neurodegeneration. The remodeled ECM may liberate bioactive fragments that can promote tissue damage- related responses, such as wound healing and inflammation. TMEM2 is a plasma membrane-bound hyaluronidase that cleaves hyaluronan, a major glycosaminoglycan constituent of the ECM in vertebrates. We discovered that TMEM2 induces mitochondrial stress responses in both human cells and nematodes, suggesting a novel conserved link between the ECM and mitochondria. In aim 1 and 2 of this proposal, we will systemically characterize TMEM2-induced global changes on mitochondria, determine what specific changes to the ECM are causative of these changes, and identify essential genetic regulators of the pathway in mammals and nematodes. Both aging and infection are associated with profound changes in the ECM. We observed that TMEM2 promotes longevity and immunity in nematodes, most likely due to changes to the ECM. We hypothesize that TMEM2- induced ECM degradation may be sensed as a signal of tissue damage mimicking infection or other environmental stress, which may elicit mitochondrial stress to potentiate mitochondrial stress responses and innate immune responses, and eventually prolong the lifespan due to these protective stress responses. In aim 3, we will test whether TMEM2-induced longevity is mediated by mitochondrial signaling, and whether the ECM- mitochondria signaling may be involved in immune responses during infection. Moreover, we will perform mass spectrometry as well as functional genomic screens to systemically assess the role of ECM remodeling in regulating mitochondrial homeostasis during aging.

NIH Research Projects · FY 2025 · 2023-08

Project Summary/Abstract Most cell-type specific gene expression arises from transcription factors binding to enhancers and promoters12,13. The last decade has seen explosive growth in the identification of enhancers and cataloging of transcription factors binding data14, but it is still not possible to predict gene expression from genome sequence, in large part because we still have a limited understanding of transcriptional activation domains, the regions that bind coactivator proteins. Our group studies the protein sequence features that control the function of transcriptional activation domains. We seek to understand how activation domains work, how they evolve and how we can predict them from protein sequence. To study how activation domains work, we rationally design mutations to test specific hypotheses. To study activation domain evolution, we survey the diversity of extant orthologs to find highly diverse functional sequences. To predict activation domains from protein sequence, we integrate all these data with interpretable mechanistic predictors and with convolutional neural networks. We combine three approaches to study activation domain function. First, we use high throughput assays in yeast and human cell culture, rationally designed thousands of mutations to test specific hypotheses about function, and integrate these data with machine learning. Second, we use biophysical simulations to study how mutations in activation domains change the 3D structures of these intrinsically disordered regions. Third, we use high-resolution imaging to study how activation domains modulate the movements of individual transcription factor molecules in the nuclei of living cells. These three approaches will reveal the amino acid sequence features that control how activation domains control transcription. Our long term goal is to build a family of computational models that predict activation domains from protein sequence, predict the coactivators each activation domain recruits, and predict how activation domains evolve.

NIH Research Projects · FY 2025 · 2023-07

Project Summary/Abstract The now established Affordable Care Act (ACA) afforded an opportunity to increase mental health coverage and treatment for African Americans and whites and to reduce disparities. By extending Medicaid coverage to adults with incomes at or below 138% of the Federal Poverty Line (FPL), providing purchase subsidies for adults with incomes between 100% and 400% FPL, and by increasing the supply of Federally Qualified Health Centers which provide considerable mental health care nationwide, the ACA can benefit uninsured African Americans with mental health problems especially. We know little about how much ACA policies increased coverage and treatment for mentally ill Blacks and whites. Using data from the National Survey on Drug Use and Health (NSDUH), the Health Resources and Services Administration and from various Medicaid and marketplace data sources this study asks, for the first time, the following: For persons aged 18-64 with Mild and Moderate Mental Illness (MMMI), Serious Mental illness (SMI), and Serious Psychological Distress (SPD), and after controlling for individual socio-demographic variables related to insurance uptake and/or receipt of mental health treatment and key state-level differences we ask: (1) How much did the ACA Medicaid expansion increase Medicaid coverage and reduce coverage disparities? How was disparity reduction limited by some states’ supplemental Medicaid requirements? (2) How much did increases in 1) ACA Medicaid coverage (2) greater FQHC availability increase but lack of other provider availability decrease mental health treatment for African Americans and reduce African American- white disparities? How much did ACA marketplaces for subsidized purchase increase private coverage and reduce disparities? How much did increases in 1) marketplace coverage (2) and greater FQHC availability increase, but lack of other provider availability decrease mental health treatment for African Americans and reduce African American-white disparities? The ACA is status quo, and it is important to provide evidence concerning African American-white mental health coverage and treatment disparity reduction to monitor progress and guide future disparity reduction policy and administrative actions.

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY / ABSTRACT Extracellular vesicles (EVs) comprise a heterogeneous pool of membrane-enclosed compartments secreted to the extracellular milieu of cells. Eukaryotic cells release a variety of EVs subpopulations that can be classified broadly into two categories on the basis of their membrane of origin. Microvesicles are EVs that form by direct outwards budding from the plasma membrane. Exosomes are EVs that originate from the endocytic pathway. Upon fusion of a multivesicular body (MVB) with the plasma membrane, the intraluminal vesicles (ILVs) are exported to the extracellular space as exosomes. Exosomes have elicited broad interest as their intraluminal contents (e.g. miRNAs, yRNAs, and tRNAs) are distinct from their progenitor cells. An accumulating repertoire of evidence has suggested that EV-mediated intercellular propagation of miRNAs plays important roles in various aspects of cancer biology. In particular, several studies have suggested that the intercellular propagation of miR-122 through breast cancer-derived EVs promotes breast cancer metastasis by reprogramming glucose metabolism in the pre-metastatic niche in vivo. Consistent with this line of experimental evidence, our lab demonstrated previously, both in cells and a cell-free reaction, that the Lupus La antigen (La) mediates the selective sorting and enrichment of miR-122 into exosomes derived from a metastatic breast cancer cell line. However, the molecular mechanism(s) by which the La:miR-122 ribonucleoprotein (RNP) complex itself is selectively incorporated into breast cancer-derived exosomes remains unknown. In this proposed research project, Jordan Ngo seeks to elucidate the molecular mechanism(s) by which the La:miR-122 RNP is selectively sorted into ILVs. In Specific Aim #1, Jordan will assess the efficacy by which a panel of candidate endosomal receptor proteins (identified by unbiased proximity labeling proteomics) are able to invoke the capture of cytoplasmic La into ILVs prior to exosome secretion. In Specific Aim #2, Jordan will identify the domains of La required for (i) its unconventional secretion within exosomes and (ii) its interaction with miR-122. In Specific Aim #3, Jordan will establish a cell-free reaction that recapitulates the selective sorting of La into ILVs, allowing further biochemical dissection of this high-fidelity sorting mechanism. Completion of the proposed research will provide novel insights into the molecular mechanisms by which specific cytoplasmic constituents are efficiently and selectively packaged into exosomes, and by extension, identify putative therapeutic targets for metastatic breast cancer and inform efforts to engineer exosomes into efficacious delivery vehicles for therapeutic compounds and nucleic acids. At the conclusion of Jordan’s NRSA-sponsored training, Jordan will have a rigorous intellectual foundation, scientific independence, and a uniquely broad technical toolkit that will make him an outstanding candidate for an independent investigator position in the future.

NIH Research Projects · FY 2024 · 2023-07

Project Summary: Human oncogenic viruses are a major cause of cancer, with recent estimates that 15% of all cancers are associated with a viral infection. One such oncovirus is Kaposi’s sarcoma-associated herpesvirus (KSHV), which primarily affects untreated AIDS patients and other immunocompromised individuals. Like most viruses, KSHV relies both on host and unique viral processes for infection, each representing potential therapeutic vulnerabilities. One of these is an unusual link between the expression of an essential class of viral “late” genes and the replication of the viral dsDNA genome (vDNA). Though long recognized, the mechanism driving this link remains unknown. Here, I will use a multi-pronged functional genomics approach to identify and test new models for this process. The dependency of viral late gene transcription on vDNA replication depends on both the trans and cis viral components supporting vDNA replication. The trans component consists of the virally encoded vDNA replication factors. In Aim 1, I will use a method I have previously developed in my postdoc for high-throughput mutagenesis and phenotyping of viral mutants to identify mutations in these viral components that prevent late gene expression but still support vDNA replication. Preliminary work has already demonstrated that such mutations exist, and by expanding on this we can identify the role these viral proteins play in enabling late gene expression. In Aim 2, I will examine the viral origin of lytic replication, which is required in cis for both vDNA replication and late gene expression. While previous studies have been limited technically, new use of dCas9 will allow me to directly perturb the functional elements of this regulatory sequence and identify the distinct molecular events required to support vDNA replication and/or late gene expression. In my final aim, I will extend my work beyond the virus to identify and characterize the host components of this process. I propose to use genetic interactions to identify the host factors hijacked to support the viral life cycle. Specifically, by leveraging the mutants and functional elements discovered in my previous aims, I can further identify how the host supports or antagonizes this process both at the cis and trans levels. Each of these proposed aims will reveal new knowledge about vDNA replication and late gene expression, allow us to test models for how these processes are linked, and finally represent powerful functional genomic approaches that can be applied to many problems in KSHV and related dsDNA viruses.

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY Tuberculosis (TB) is a deadly infectious disease that kills over 1.7 million people per year. The majority of individuals are able to control Mycobacterium tuberculosis (Mtb) infection, yet others are extremely vulnerable. Life-threatening disease symptoms, including extreme weight loss or cachexia, are also heterogenous across individuals. Immunocompromised people are particularly susceptible to TB, and less extreme variations in immune system function may also contribute to susceptibility. The mammalian gut microbiome is a complex community of microbes with extreme variation across individuals. Gut microbiome composition impacts immune function and susceptibility to a variety of pathogens. Our pilot data suggests that genetically identical mice with distinct gut microbiomes have altered ability to control Mtb infection. In this proposal we will interrogate the role of the microbiome in Mtb control and cachexia. First, we will expand our comparison of mice from different housing conditions to determine the extent of variation in Mtb burden. To confirm the role of the microbiome in Mtb infection control, we will transplant fecal microbiomes of interest into germ-free mice and repeat Mtb infection. We will also profile the immune response to infection in each case. Second, we will explore the role of the gut microbiome in mediating TB associated cachexia by measuring microbial and metabolic changes over time in genetically identical mice with diverse microbiomes. In follow up, we will isolate gut bacteria of interest from these microbiomes to investigate how microbial metabolism may impact immune function. This proposal will open up a new area of research to probe the role of the gut microbiome in the pathogenesis of TB. This research has the potential to identify new microbial and host directed therapeutic targets for TB prophylaxis and treatment.

NIH Research Projects · FY 2026 · 2023-07

Summary Autism spectrum disorder (ASD) has diverse presentation but can be characterized by at core by a) rigid and repetitive behavior and b) social communication deficits. In recent decades, there is increasing confidence that identified genetic differences contribute to ASD in humans and a number of high confidence risk genes have been identified. These risk genes can be studied in mice. There is hope that convergent phenotypes and endophenotypes will illuminate key features of ASD (Hyman, 2014). One striking current area of convergence seen in mice with ASD risk genes is a gain of function in rotarod motor learning and alteration in neurotransmission onto spiny projection neurons (SPNs) of the striatum (Hyman, 2014). This was first observed in mice with neuroligin gene mutations (Rothwell et al., 2014), but has also been observed in mice with Tsc1 and Tsc2 gene mutations (Benthall et al., 2021). Tsc2 (with some studies of Tsc1 for comparison) will be the central focus of this proposal. Here we propose that a major diagnostic criteria for ASD observed in TSC patients - restricted, repetitive patterns of behavior - is mediated by changes in the activity of basal ganglia circuits that control the learning and updating of appropriate actions. Specifically, we hypothesize that Tsc2 haploinsufficiency leads to changes in corticalstriatal synapses and SPN activity that facilities striatal dependent learning and makes updating learning more inflexible. In Aim 1 we will use electrophysiological and structural imaging methods to test if specific connections are stronger in Tsc2 Het mice than WT. We posit based on studies in the dorsolateral striatum that corticostriatal connections onto D1R expressing SPNs (dSPNs) will be enhanced in the dorsomedial striatum (DMS). Aim 2 will focus on behavior and test in two different tasks if behavioral inflexibility in Tsc2 Het mice can be ameliorated by reducing reward probability during learning. Aim 3 will use in vivo imaging in the striatum to examine how dopamine and striatal activity may differ in Tsc2 mice under conditions that produce behavioral differences. In sum, these data will inform basic neurobiology surrounding a convergent gain of function phenotype seen in many ASD models: gain of function in striatal learning (rotarod being the most common test). We will translate this learning phenotype into more translatable behavior learning paradigm--cue guided action learning and seek the neural correlates of this gain of function. Finally, we will test a highly translatable therapeutic idea, the idea that using lower reward probability during training will ameliorate neural differences and allow for greater flexibility later.

NIH Research Projects · FY 2025 · 2023-07

Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. The Biophysics Training Program (BTP) is a new interdisciplinary predoctoral training program at the University of California at Berkeley. The BTP will be the only training program at UC Berkeley focusing on biophysics. The BTP will recruit and appoint six trainees per year to train in the field of biophysics, drawn from a pool of 640 applicants and 40 matriculants per year in the Biophysics and Molecular and Cell Biology PhD programs. Trainees will be appointed for two-year terms and will continue to engage with the BTP training activities and community-building events through to graduation. We expect that 100% of our students will graduate with a PhD within 6 years, with a target mean time to degree of 5.5 years for all students. Our goal is that every student will publish a first or co-first author paper in a peer-reviewed journal recognized as excellent in the field of biophysics, and will subsequently pursue a research-related career in academia, industry, or government. Professional development will be centered on the acquisition of ten core competences designed to enable leadership in any of these workforce sectors. Training procedures will be informed by the latest scientific literature in the field of research mentorship and training, and will emphasize the acquisition of self-efficacy. Research training will be enhanced in response to the latest advances in biophysics, including the rapidly growing importance of machine learning in structural biology and data analysis. A series of training innovations have been incorporated into the BTP, including a new hands-on computational modeling course in the physics of the cell, short boot camp courses in cryo-EM and single molecule microscopy, and advanced training in laboratory safety and record keeping. The effectiveness of the training program will be rigorously evaluated on an annual basis and advice sought from newly-constituted student and external advisory groups. All 23 of the BTP training faculty are members of the Graduate Group in Biophysics (GGB), and our trainees will be drawn from the GGB and the Molecular and Cell Biology PhD program. Our faculty are world leaders in their fields and include six members of the National Academy of Sciences, six Howard Hughes Medical Institute Investigators, and a recent recipient of both the Breakthrough Prize in Life Sciences and Nobel Prize in Chemistry. All BTP faculty undergo formal mentorship training and evaluation of mentoring quality.

NIH Research Projects · FY 2025 · 2023-07

Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. Molecular Biology Across Scales (MBAS) is a new multidisciplinary predoctoral training program that will develop the next generation of researchers discovering how molecules, cells, and tissues interact to create life and how this information can advance human health. MBAS will be the only graduate program at the University of California, Berkeley that supports broad-based training rather than specialization within a particular discipline. This perspective has driven many recent transformative biological discoveries. MBAS will draw students and faculty from the Molecular & Cell Biology (MCB) Graduate Program, uniting those who tackle fundamental research problems at length scales from individual molecules to complex biochemical systems to whole organisms. It will appoint 40 students (20 first-year trainees annually for a two-year term) encouraging each to explore a range of research fields and identify those that engage their passion. Students will publish a first-author paper and graduate with a mean time to degree of 5.5 years. The curriculum will impart broad conceptual knowledge, creative and critical thinking, mastery of experimental logic and methods, application of quantitative approaches, rigorous analysis and interpretation of results, clear communication of findings, and incorporation of responsible, safe, and ethical practices. Students will choose trained mentors from a faculty of 75 nationally recognized leaders who represent a wide choice of research areas, use state-of-the-art facilities, and work in a multidisciplinary, and collaborative environment. MBAS will provide training, oversight, and support for participating faculty, and will carefully monitor the progress of its trainees. Students will be supported with peer learning communities, rigorous and clear expectations, expert advising, and a program design that offers options for filling gaps in preparation. Innovative training elements include new courses and enhanced content in responsible conduct of research and reproducibility, quantitative biology and data science, lab safety, and scientific writing. New programs will be piloted and evaluated to benefit all MCB trainees. Professional development, experiential learning, and career counseling will prepare trainees for research-related careers in academia, industry, government, and beyond. In sum, MBAS will provide an early and persistent emphasis on multidisciplinary training, centered around the student experience, for a flexible and individually directed path to a successful career in the biomedical workforce.

NIH Research Projects · FY 2026 · 2023-06

Abstract We aim to develop tools for ground-truth phantoms for quantitative and structural MRI (qMRI). qMRI aims to acquire maps of physical or chemical variables that can be measured in physical units and compared between tissue regions and among subjects. In contrast, most clinical MRI acquisitions are only qualitative, i.e. “weighted images”, and not quantitative. While qMRI has the potential to improve precision diagnostics and medicine, it has been traditionally hampered by significant barriers such as imaging speed, computational practicalities, and reproducibility and repeatability of MR measurements. The variability between scanners and human subjects and the lack of ground truth in biological tissues fundamentally challenge the development, testing and standardization of qMRI techniques. The National Institute of Standards and Technology (NIST) hosted workshops working towards standardizing qMRI. The resulting recommendation paper highlighted a list of outstanding needs. The proposed project aims to address these unmet needs by developing materials, technology, tools and processes for manufacturing quantitative anthropomorphic MRI phantoms. Current state- of-the-art solutions for manufacturing MRI phantoms often use discrete compartments or geometrical shapes filled with chemical solutions representing a single physical parameter. In contrast, our proposed novel approach will enable fabrication of phantoms that truly mimic the contrast heterogeneity of tissue in 3D. These will include proton density, T1, T2, T2* relaxation times, magnetic susceptibility, diffusion, fat fraction, air-tissue field- inhomogeneity, relative conductivity, electric permittivity and magnetic permeability. If successful, this will be the first time that such a comprehensive set of MRI parameters is accomplished in a tissue-mimicking phantom. Based on our preliminary work on quantitative anatomy mimicking slice phantoms, we propose two approaches: (a) Quantitative 3D stack of thin slices. This approach is inexpensive, easy to reproduce by labs with moderate equipment and skills. (b) An advanced approach of boundaryless fully 3D phantoms that will be fabricated via inkjet 3D printing of hydrogels and plastics and would enable true high resolution 3D structures with heterogeneity that mimics human anatomy. In collaboration with leading industrial partners, we will validate and disseminate our technology. Our proposal is motivated by a rising need for quantitative measurements in MRI driven by precision medicine and the use of data science tools for biomarker discovery. With the rise of methods such as fingerprinting, and accelerated reconstruction, quantitative MRI (qMRI) is closer to the clinics than ever. The proposed quantitative MRI phantom will mimic the complexity of tissue structure and contrast mechanism that are necessary to ensure the accuracy of qMRI. If successful, the project will greatly facilitate the development and clinical translation of qMRI, making MRI accurate, precise, and quantitative – thus enabling precision diagnostic and discoveries that will directly improve healthcare.

NIH Research Projects · FY 2026 · 2023-06

All proteins sample a diverse array of conformations (folded, unfolded, and excited states) with differing free energies and dynamics depending on the environmental conditions. We can now predict a structural model for the folded state given the amino acid sequence. The sequence of a protein, however, encodes much more than just this native structure – it encodes the entire energy landscape – an ensemble of conformations whose populations (energetics) and dynamics are finely tuned and critical for proper function and cellular health. A major hurdle in going from sequence to function is our lack of understanding of the non-native regions of the landscape. These high-energy conformations are important for directing the stability, dynamics and folding of a protein, and modulations of this ensemble play a role in misfolding, protein signaling, catalytic activity, and allostery. A compromised landscape, due to either changes in the cellular milieu, intrinsic genetic defects, or the cumulative effects of cellular stresses, has been linked to disruption of proteostasis, resulting in varying misfolding diseases and pathologies. Rare and transient conformations are, by their very nature, difficult to study. For decades, biophysical chemists (including the PI) have been probing these fluctuations with high-level technologies using purified proteins in a test tube. The test tube, however, is very different from the cell. In vivo, proteins live in a crowded cellular environment, subject to quality control machinery, cellular modifications and subject to non-equilibrium effects such as protein synthesis and degradation. In order to take full advantage of the wealth of detailed, quantitative biophysical data available from in vitro studies, we need to understand how cellular factors and the cellular environment modulate the energetics and dynamics. Such complex settings, however, are inaccessible to the standard toolbox used for quantitative biophysical studies. The PI is an expert in the area of protein folding and dynamics, having devoted most of her career to developing and utilizing sophisticated technologies to probe rare and transient conformations, both at the single molecule and ensemble level. This current proposal focuses on: 1) understanding how these states are modulated by features in the cell, such as co-translational folding, post-translational modifications, and 2) understanding how the dynamics of conformational changes are controlled at the sequence level. The long- term goal is a is a molecular, quantitative, and predictive understanding of the relationship between sequence and the energy landscape, together with a predictive understanding of how the environment modulates this landscape.

NIH Research Projects · FY 2026 · 2023-06

Project Summary/Abstract For replicated chromosomes to be segregated to two daughter cells accurately, the microtubule (MT) cytoskeleton must be completely remodeled to form a bipolar spindle. A large, dynamic protein complex called the kinetochore attaches replicated chromosomes to microtubules emanating from opposite spindle poles. Proper kinetochore-microtubule attachment is vital for preservation of genomic integrity and prevention of cancer and birth defects. Therefore, mitotic spindle assembly and kinetochore attachment must be well coordinated. Challenging understanding of these processes and their coordination is the fact that mitotic spindles and kinetochores are extremely complex molecular machines (kinetochores contain >60 proteins), that they are targets of phosphoregulation by multiple protein kinases, and that they possess a striking range of biochemical activities. Kinetochore activities include: (1) lateral MT binding, (2) translocation along the MT lattice to the plus end, (3) conversion from lateral to end-binding, (4) association with dynamic MT ends while tubulin subunits are exchanged, and (5) serving as force-coupling devices between chromosomes and MT plus ends during anaphase A. Understanding how the kinetochore performs its various functions, the structural underpinnings of these activities, and how these activities are regulated post-translationally, is far from complete. A biochemical cell-lysate assay recently developed in the Barnes laboratory combines, for the first time, two of the most powerful approaches for studies of microtubule dynamics: biochemical extract studies and genetics. Dynamics of single microtubules and single kinetochores associated with these microtubules are revealed and quantitatively analyzed by highly sensitive Total Internal Reflection Fluorescence microscopy. Cell lysates synchronized to specific cell-cycle stages are made from budding yeast mutants of specific mitotic proteins. Quantitative analysis of MT dynamics and kinetochore activities will establish how these parameters are regulated in the cell cycle, and these studies will identify the specific proteins that carry out the specific behaviors. Since mitosis is a highly conserved process, lessons learned from these studies are expected to apply broadly. Unlike many other assays, this assay exclusively uses homologous sources of tubulin and interacting proteins, avoiding artefacts that arise from species mismatch incompatibilities. Proposed studies build upon recent unique observations of microtubule dynamics regulation and kinetochore dynamic activity in this lysate system. The objectives are: (1) To investigate biochemical activities of intact kinetochores and their regulation, and to relate kinetochore structure to function; and (2) To investigate how microtubule dynamics are regulated through the cell cycle in both the nucleus and the cytoplasm, focusing on Kar3 and Kip3 kinesins.

NIH Research Projects · FY 2025 · 2023-05

PROJECT SUMMARY / ABSTRACT A central promise of genome editing is its potential to treat monogenic disease. Despite early-stage clinical progress for CRISPR-Cas based approaches, monogenic neurodegenerative conditions and nucleotide triplet expansion disorders have not been a focus of any biotechnology company in this space. Our proposal brings together a team of academic investigators to develop a synergistic suite of first-in-class CRISPR-Cas based therapeutics for Huntington's disease (HD) and C9ORF72 amyotrophic lateral sclerosis (ALS). We will engineer and deploy an editing approach that excises, with IND-grade potency and mutant allele-selectivity, the disease- causing expansion repeat from human HTT and C9ORF72 loci, respectively. Our strategy is based on identifying alleles of commonly heterozygous SNPs that reside on the same haplotype as the disease-causing repeat expansion, and then engineering CRISPR-Cas9 for high selectivity of cleavage, on one or both sides of the mutant allele repeat, to drive its excision, with two tiers of delivery innovation. Our trailblazer project (Research Project 1, RP1) will develop an HD therapeutic by packaging mutant HTT-specific CRISPR-Cas9 into a newly developed serotype of adeno-associated virus (AAV) with robust and broad biodistribution in the brain parenchyma of nonhuman primates (NHP). We will implement an innovative strategy in which the CRISPR-Cas9 cassette temporally limits its own expression. We will identify and advance the preclinical lead composition through IND-enabling studies leveraging 3 dedicated Resource Cores to (i) assess molecular outcomes at the genetic level, (ii) administer reagents to animals and observe their behavior, and (iii) assess molecular and histological outcomes from cells and animal tissues. An Administrative Core led by experienced developers of genome editing-based therapeutics, will provide project-management support and lead on preparation of regulatory submissions, aiming to file an HD IND by program end. In RP2, we will apply the AAV-based excision approach to build a cognate experimental therapeutic for C9ORF72-driven ALS. Synergies with RP1 include CMC innovation to manufacture novel AAV, re-use of the self-regulating CRISPR-Cas cassette and virus harboring it, and regulatory feedback on IND-enabling pharmacology, toxicology, and biodistribution studies. We will advance RP2 through pre-IND. For RP3, we will establish a first-in-class, transformative paradigm for in vivo genome editing therapy by reformulating the preclinical lead CRISPR-Cas9 combination used in RP1 into a highly innovative “Cas9 RNP monoparticle” in which amphiphilic peptides deliver the gene editor to neurons upon injection. We will develop approaches for monoparticle manufacture to enable ex vivo and in vivo efficacy studies in HD models. Extensive synergies with RP1 project and comprehensive support by the RCs will enable us to advance this approach to pre-IND by program end. The sum total of this effort will establish a fundamentally new paradigm for in vivo genome editing applicable to all nucleotide repeat expansion disorders, and advance preclinical lead formulations for one disease, HD, to IND, and another such disease, C9ORF ALS, to pre-IND.

NIH Research Projects · FY 2026 · 2023-05

Obesity has become a global epidemic and is associated with type 2 diabetes and other chronic diseases. While white adipose tissue is the primary energy storage organ, brown adipose tissue (BAT) dissipates energy through non-shivering thermogenesis. Discovery of the presence of BAT/BAT-like tissues in human adults has generated a considerable interest in BAT biology to design strategies against obesity and insulin resistance. Recently, we have identified a new microprotein of 76 aa in length, highly expressed in BAT compared to other tissues, and is induced upon cold exposure. Microproteins in general function by affecting protein-protein interaction between signaling molecules. Our microprotein contains consensus docking motifs for Protein Phosphatase 2B. Our preliminary studies showed that overexpression of this microprotein in differentiated BAT cells increased oxygen consumption rate (OCR), while knockdown decreased OCR in basal and forskolin stimulated conditions. Moreover, we detected higher PKA activity without changes in cAMP levels upon overexpression of this microprotein in BAT cells. To document its physiological function, we have generated conditional knockout mice and transgenic mice overexpressing the microprotein in UCP1+ cells and in adipocytes. We propose that, our microprotein interacts with PP2B to regulate classic b-adrenergic downstream signaling and potentiates PKA activity for promotion of thermogenesis. Aim 1 is to study its effect on thermogenesis in differentiated brown adipocytes in culture. Aim 2 is to dissect the biochemical basis of its function in promoting thermogenesis. Finally, Aim 3 is to evaluate its in vivo function in thermogenesis by loss- and gain-of function studies in mice. This research may allow us to devise small molecule therapeutics to increase thermogenesis for preventing obesity and improving insulin sensitivity in the future.

NIH Research Projects · FY 2026 · 2023-05

ABSTRACT The molecular mechanisms through which cells sense nutrients remain largely unknown, but their elucidation is key to our understanding of metabolic regulation both in normal and disease states. At the center of nutrient sensing and growth regulation is an ancient protein kinase known as the mechanistic Target of Rapamycin Complex 1 (mTORC1). In response to the combined action of metabolic inputs such as nutrients, growth factors, energy and oxygen, mTORC1 translocates from the cytoplasm to the surface of lysosomes, where it becomes activated. Accumulating evidence indicates that aberrant mTORC1 activation at the lysosome could be a driving force in diseases ranging from cancer to type-2 diabetes to neurodegeneration. Thus, a deep mechanistic understanding of how mTORC1 is activated and then inactivated in response to nutrients could point the way to novel therapeutic strategies in these diseases. My lab has made important contributions to the understanding of mTORC1 pathway organization, and how its function is integrated with the many activities of the lysosome. In particular, we have identified a dedicated signaling pathway via which cholesterol, an important building block for cellular membranes, promotes mTORC1 recruitment to the lysosome and activation of its downstream programs. We have uncovered membrane contact sites between lysosomes and the endoplasmic reticulum as key nodes where mTORC1 activation by cholesterol occurs, thus implicating inter-organelle communication as an important aspect of mTORC1 regulation. Furthermore, we found that excess mTORC1 signaling, caused by cholesterol accumulation in the lysosome, drives cellular dysfunction and could be a driving force in a neurodegenerative and metabolic disease, Niemann-Pick type C (NPC). These discoveries directly lead to deep questions on the organization of cellular nutrient sensing, which are at the core of the current MIRA proposal. One key challenge is to elucidate the mechanisms and physiological roles of lipid-dependent mTORC1 regulation, specifically whether dedicated cholesterol sensors exist in the lysosomal membrane, and how they couple the abundance of sterol molecules to mTORC1 activation and to overall metabolic regulation at the cell and organism level. Based on our finding that cholesterol sensing by mTORC1 involves physical communication between the lysosome and the ER, another major goal of the proposal is to delineate the machinery that mediates communication and metabolite exchange between the lysosome and the ER, and how this machinery participates in regulation of mTORC1 as well as another major metabolic kinase, protein kinase A. Finally, the pathogenic role of dysregulated mTORC1 in NPC, and the ability of mTORC1 inhibition to restore several parameters of NPC cell function, strongly support mTORC1 as a prime target in neurodegenerative disease. We will thus determine how lysosomal mTORC1 controls neuronal cell homeostasis, and how dysregulated mTORC1 signaling contributes to neuronal degeneration. Together, these studies will shed light on fundamental principles of metabolic organization in health and disease states.

NIH Research Projects · FY 2025 · 2023-05

PROJECT SUMMARY Antibiotic resistance is a significant threat to human health. Human therapeutic antibiotic use, the majority of which occurs in outpatient settings for non-severe infections, is a major, modifiable driver of resistance. Acute respiratory infections (ARIs) account for large proportions of outpatient antibiotic use and vaccines have been recognized as important mechanisms to combat antibiotic use and resistance, especially for ARIs. However, uncertainties remain in understanding the contributions of common respiratory pathogens to outpatient antibiotic use and quantifying the full potential of vaccines to reduce antibiotic use, impeding informed policymaking and priority-setting for vaccine research and development. The proposed research addresses these gaps in the context of both existing and pipeline vaccines for three common respiratory pathogens in children: Streptococcus pneumonaie, influenza virus, and respiratory syncytial virus (RSV). The objective of this proposed work is to estimate impacts of both existing and potential vaccine strategies on outpatient antibiotic prescriptions for ARIs in children. More specifically, the research focuses on the following aims: 1) estimate the attributable fractions of ARI-associated pediatric outpatient antibiotic use due to vaccine-preventable respiratory pathogens; 2) evaluate the impact of pneumococcal vaccination strategies on outpatient antibiotic use for ARIs in children; and 3) evaluate the impact of viral (influenza, RSV) vaccination strategies on outpatient antibiotic use for ARIs in children. This project focuses specifically on ARIs in children as they are both major drivers of outpatient antibiotic use and targets of existing and pipeline vaccines. The work will leverage multiple, complementary, large-scale data sources including both claims data (Optum Clinformatics, IBM MarketScan) and nationally-representative outpatient medical surveys (National Ambulatory Medical Care Survey and National Hospital Ambulatory Medical Care Survey), each with millions of pediatric ARI visits and outpatient antibiotic prescriptions per year, to address these aims. The breadth of data and statistical and epidemiologic methods used in this proposed research will enable a novel and comprehensive understanding of the potential for vaccines to reduce outpatient antibiotic use for ARIs in children. This work will inform urgently-needed antibiotic resistance mitigation strategies and priority setting and decision-making for vaccine research/development and policy. The proposed methods will further provide a novel framework for evaluating current and future vaccines as outpatient infections and antibiotic use are not commonly considered endpoints, despite their substantial burden. This research will be enhanced by and complementary to my proposed training plan, which emphasizes development and experience in epidemiologic and statistical methods, big data methods and applications, subject matter expertise, and additional skills necessary for me to become an independent investigator. UC Berkeley, particularly the School of Public Health, provides extensive resources and support to enable me to successfully complete the proposed fellowship and fulfill my long-term goal of becoming a research-oriented infectious disease epidemiologist.

NIH Research Projects · FY 2025 · 2023-04

ABSTRACT / PROJECT SUMMARY Sickle-cell disease (SCD) is an autosomal recessive disorder that causes considerable morbidity and mortality, affecting an estimated 100,000 individuals in the US, and millions more worldwide. Multiple editing-based cures for SCD are currently in clinical development, however, there are no clinical-grade laboratory tests available capable of characterizing the biophysical and rheological properties of RBCs derived from genome-edited SCD HSPCs. Assays capable of characterizing RBC quality are urgently needed to assess the potency of emerging editing-based genomic therapies for SCD, regardless of the editing modality. One of the central challenges that has impeded the development of a highly performant potency assay for evaluating the functional efficacy of editing-based genomic therapies for SCD has been the lack of laboratory technologies capable of sensitively, accurately, and precisely capturing the biophysical properties of SCD-RBCs utilizing only a small number of cells. In recent years, several innovations have emerged that now make the development and analytical validation of a potency assay for editing-based SCD genomic therapies feasible. One of these has been the advent of microfluidics-based diagnostic devices capable of functionally characterizing the health of RBCs at unprecedented levels of resolution and sensitivity. This proposal seeks to leverage (1) an existing suite of these aforementioned next-generation RBC biophysical and functional characterization devices, (2) conventional hematologic assays, and (3) a well-established machine learning approach to develop and analytically validate a first-in-kind potency assay for editing-based therapies for SCD. To achieve this, we will first construct a panel of comprehensively profiled, gold-standard reference samples of HSPCs that simulate a representative range of genome editing outcomes in SCD and prepare data for machine learning training. A machine learning model will then be trained to predict the percentage of RBCs that functionally exhibit a non-SCD phenotype. Once trained, we will validate the performance of the new potency assay, a panel of HSPCs affected by SCD will be therapeutically edited using at least three different modalities (e.g. homology-directed repair, base-editing, etc.).

NIH Research Projects · FY 2026 · 2023-04

ABSTRACT Cells tightly regulate translation initiation in order to control which proteins they synthesize and how much of each protein they produce. This regulation of protein synthesis matches translation levels with the cell's translational capacity and physiological needs. Translation initiation, in particular, is a key point for both global and transcript-specific regulation. In the canonical pathway for translation initiation, an mRNA is first activated by the formation of a closed-loop complex bridging between the 5'-methylguanosine cap and the 3'-polyadenylate tail. A small ribosomal subunit, accompanied by a variety of other initiation factors, is recruited to the mRNA and scans in order to begin translation at the first AUG. Recent evidence suggests that translation initiation does not proceed down such a uniform pathway. Individual translation factors are subject to regulation downstream of major signaling pathways, including MAP kinase cascades, mTOR kinase signaling, and the integrated stress response. Activation or inhibition of core translation initiation factors can produce transcript- specific changes in translation, leading to broad translational reprogramming. Translation of developmentally regulated genes also depends on cryptic initiation factors such as eIF2A, eIF2D, and DENR/MCTS-1. Our motivating hypothesis is that this heterogeneous landscape of translation initiation complexes underlies dynamic, mRNA-specific control of protein synthesis. Here, we propose to use proximity labeling of protein and RNA in order to survey the composition of translation initiation complexes that assemble in vivo and understanding how this changes in response to physiological and environmental signals. We will couple this with an analysis of translational across the transcriptome. Together, these results will reveal the full diversity of pathways for translation initiation in vivo and show how these different pathways mediate translational expression programs.

NIH Research Projects · FY 2026 · 2023-04

Summary Non-topographic, intermixed representations (salt-and-pepper maps) of sensory information are common in cerebral cortex, but how neural coding and plasticity are organized within them is unclear. We propose that salt-and-pepper maps contain distinct pyramidal (PYR) subnetworks with differential roles in coding stability and flexibility (including learning and attentional modulation). To test this, we study the whisker map in layer 2/3 of mouse somatosensory cortex (S1), where PYR cells tuned for the columnar whisker (CW) and for non- columnar (non-CW) whiskers are intermixed in each column. We recently discovered that non-CW tuned cells show marked tuning instability across days, while CW-tuned cells have stable tuning. This reveals that the L2/3 salt-and-pepper map has two components: a stable columnar map of CW-tuned cells, intermixed with non-CW tuned cells that are unstably tuned and have little columnar topography. We propose that CW- and non-CW tuned cells are distinct PYR subcircuits with different roles in coding and plasticity. This is a novel model of S1 circuit function. We predict that the CW network provides coding stability, while non-CW cells are the primary site for plasticity and learning. Based on preliminary data, we hypothesize that tuning instability in non-CW cells is internally driven, and acts to sample novel sensory codes which may then be stabilized by experience or reward. This is a novel hypothesis for how sensory maps balance stability and plasticity—by segregating these functions in different subcircuits. In Aim 1, we use longitudinal 2-photon calcium imaging to understand the nature and origins of tuning instability, and to test whether experience or reinforcement stabilizes whisker tuning. In Aim 2, we evaluate whether CW and non-CW networks represent distinct functional networks with different sensory coding and plasticity properties. We test our central hypothesis that non-CW cells are the primary locus of sensory plasticity and learning within the map. Aim 3 asks how attention modulates neural coding within intermixed maps. We developed a selective attention task in which mice use history-dependent cues to guide attention to a specific whisker to improve detection performance. Mice show robust spatial attention to cued whiskers. Attention lasts ~10 sec and is driven by recent pairing of whisker stimuli with reward. Preliminary data show that attention enhances whisker-evoked activity of PYR cells encoding the attended whisker in S1. This establishes S1 as a powerful site to study cortical mechanisms of attention. We will use 2-photon imaging and Neuropixels recording to study how attention modulates sensory coding in S1, including measuring the size and CW- or non-CW network specificity of the attentional spotlight. In a major effort, we use imaging and optogenetics to identify the control circuits for attention in S1, with initial focus on VIP interneurons. Together, these studies will reveal how plasticity and attentional modulation are organized within a canonical salt-and-pepper map.

Fonds de recherche du Québec – Nature et technologies · FY 2023-2024 · 2023-04

Volet: Bourses de doctorat en recherche; Domaine: Structures abstraites; Objet: Inférence paramétrique et non paramétrique; Objet: Statistique informatique; Mots-clés: ESTIMATION, COVARIANCE MATRIX, HIGH DIMENSIONS, CROSS-VALIDATION, DIMENSION REDUCTION, SEMIPARAMETRICS

Fonds de recherche du Québec – Nature et technologies · FY 2023-2024 · 2023-04

Volet: Bourses de recherche postdoctorale; Domaine: Matériaux; Objet: Technologies biomédicales; Objet: Micro-organismes; Application: Sciences et technologies; Application: Fondements et avancement des connaissances; Mots-clés: MICROFLUIDICS, ORGAN-ON-A-CHIP, INESTINE, GUT MICROBIOME, BIOLOGICAL MODELING, PATHOGENS

NIH Research Projects · FY 2026 · 2023-04

PROJECT SUMMARY Much of our understanding of the genetic basis of adaptation derives from studies of simple traits in which a large proportion of the phenotypic variation is controlled by one or a few genes of major effect. However, much of evolution involves changes in complex traits that are controlled by many genes of small to modest effect. Complex traits also underlie most phenotypic differences among humans, including those related to human health. The proposed research will study the genetic basis of environmental adaptation in house mice, Mus musculus, the best mammalian model for humans. House mice have recently expanded into the Americas from their native range in Western Europe. By combining studies of genetic and phenotypic variation in natural populations and in the lab, this project will make explicit links between genotype and phenotype for several complex traits. This work will utilize recent large-scale surveys of 28 populations of house mice collected across the Americas from 55° S latitude to 54° N latitude. New inbred lines of mice from different environments form a critical resource for the proposed work. Mice from colder environments have evolved to become larger (Bergmann’s rule) and have shorter extremities (Allen’s rule), conforming to two of the best-documented eco- geographic patterns in mammals. In addition, mice from different environments differ in many metabolic traits, including activity levels, body mass index, and aspects of blood chemistry. We have two major goals for the next five years. First, we will identify the genetic architecture and specific loci underlying complex adaptive traits using (1) QTL mapping with wild-derived inbred lines of mice from different environments, (2) expression studies, including the identification of cis-eQTL, to identify specific genes within broad QTL intervals, (3) studies of chromatin accessibility to identify potential regulatory changes, (4) association studies of traits in large samples of mice from natural populations, and (5) studies of inbred lines reared in different laboratory environments to measure the effects of environmental perturbations on both gene expression and organismal- level traits. Second, we will expand on our previous work studying patterns of SNP variation of wild mice by using a combination of long-read PacBio sequencing of mice from natural populations and long-read PacBio sequencing and Hi-C scaffolding of genomes from wild-derived inbred strains to study structural variation across the genome, including (1) copy-number variation contributing to environmental adaptation, (2) transposable element insertion polymorphisms underlying adaptive differences, and (3) larger structural variants such as inversion polymorphisms. The impact of structural variation on gene expression will be assessed using RNAseq from the same animals. Together this combination of approaches will provide an unparalleled picture of the genomic details underlying polygenic adaptation in mice and will identify the genetic basis of traits likely to be relevant for understanding differences among humans.

NIH Research Projects · FY 2026 · 2023-03

Project Summary Microglial inflammation has been implicated the pathology of a host of neurological conditions, including neurodevelopmental disorders such as autism and Down Syndrome; neurogenerative disorders such as Alzheimer's disease (AD), Parkinson’s disease, amyotrophic lateral sclerosis (ALS), and Huntington’s disease; and neuropathic pain. Gene therapy utilizing adeno-associated viral (AAV) vectors has emerged as a highly promising strategy for treating central nervous system (CNS) disorders, and an immunosuppressive gene therapy to inhibit immune signaling pathways in microglia would thus be highly promising for treating this broad range of chronic conditions. However, this signaling pathway serves protective roles in other CNS cells including neurons, such that therapeutic delivery would need to be not only efficient but targeted to microglia. By leveraging our expertise in viral engineering, single cell analysis, machine learning, and human and non-human primate models, we propose to develop a technology platform for genetically accessing specific cell types in the adult primate brain, in particular microglia. We will integrate directed evolution of AAV with molecular barcoding, single cell next generation sequencing (NGS), machine learning, and human tissue and non-human primate (NHP) brain models to develop AAVs for selective delivery to primate microglia. Additionally, to further enhance the specificity of these technologies, we will apply analogous library selection, NGS, and machine learning approaches to engineer short, synthetic promoters and to identify endogenous enhancers for selective microglial gene expression. Finally, these capabilities will be applied to deliver potential therapeutic gene cargoes to microglia in vitro and in vivo. In sum, we propose a high-risk, innovative research program that will, if successful, advance our capacity to selectively modulate immune signaling in microglia, work that if successful will have implications for treating a broad range of neurological conditions. Furthermore, this work will establish a broadly impactful technology platform that integrates vector engineering, next generation sequencing, and machine learning to engineer tools for cell specific genetic manipulation, which can in principle be applied to in principle any cell or tissue in the central nervous system or body. We thus anticipate that our experienced, multidisciplinary team can offer strong contributions to technology development, neuroscience, and fundamental and translational biology in other systems.