Research Awards

We are pleased to showcase our exceptional award recipients, both past and present! These individuals have demonstrated talent, dedication, and hard work. We couldn't be prouder and are honored to celebrate their success.

 

Copenhagen Award

 
The Copenhagen award was established to recognize a UCSF graduate student, postdoctoral fellow or clinical fellow mentored by a faculty member in the Department of Ophthalmology who has published outstanding work in the area of laboratory visual sciences.

2022 Nicole Tsai (Duan lab)
Tsai NY, Wang F, Toma K, Yin C, Takatoh J, Pai EL, Wu K, Matcham AC, Yin L, Dang EJ, Marciano DK, Rubenstein JL, Wang F, Ullian EM, Duan X. Trans-Seq maps a selective mammalian retinotectal synapse instructed by Nephronectin. Nat Neurosci. 2022 May;25(5):659-674. doi: 10.1038/s41593-022-01068-8. Epub 2022 May 6. PMID: 35524141; PMCID: PMC9172271.
 
2021 Nilsa La Cunza (Lakkaraju lab)
La Cunza N, Tan LX, Thamban T, Germer CJ, Rathnasamy G, Toops KA, Lakkaraju A. Mitochondria-dependent phase separation of disease-relevant proteins drives pathological features of age-related macular degeneration. JCI Insight. 2021 May 10;6(9):e142254. doi: 10.1172/jci.insight.142254. PMID: 33822768; PMCID: PMC8262309.
 
2020 Swapnil Shinde (Nachury lab)
Shinde SR, Nager AR, Nachury MV. Ubiquitin chains earmark GPCRs for BBSome-mediated removal from cilia. J Cell Biol. 2020 Dec 7;219(12):e202003020. doi: 10.1083/jcb.202003020. PMID: 33185668; PMCID: PMC7716378.
 
2019 Rachel Care (Dunn lab)
Care RA, Kastner DB, De la Huerta I, Pan S, Khoche A, Della Santina L, Gamlin C, Santo Tomas C, Ngo J, Chen A, Kuo YM, Ou Y, Dunn FA. Partial Cone Loss Triggers Synapse-Specific Remodeling and Spatial Receptive Field Rearrangements in a Mature Retinal Circuit. Cell Rep. 2019 May 14;27(7):2171-2183.e5. doi: 10.1016/j.celrep.2019.04.065. PMID: 31091454; PMCID: PMC6624172.
 
2018 Li Xuan Tan (Lakkaraju lab)
Kaur G, Tan LX, Rathnasamy G, La Cunza N, Germer CJ, Toops KA, Fernandes M, Blenkinsop TA, Lakkaraju A. Aberrant early endosome biogenesis mediates complement activation in the retinal pigment epithelium in models of macular degeneration. Proc Natl Acad Sci U S A. 2018 Sep 4;115(36):9014-9019. doi: 10.1073/pnas.1805039115. Epub 2018 Aug 20. PMID: 30126999; PMCID: PMC6130344.
 
2017 Ivan Anastassov (Dunn lab)
Anastassov IA, Wang W, Dunn FA. Synaptogenesis and synaptic protein localization in the postnatal development of rod bipolar cell dendrites in mouse retina. J Comp Neurol. 2019 Jan 1;527(1):52-66. doi: 10.1002/cne.24251. Epub 2017 Jun 12. PMID: 28547795; PMCID: PMC5745277.
 
2016 Anton Delwig (Copenhagen lab)
Delwig A, Larsen DD, Yasumura D, Yang CF, Shah NM, Copenhagen DR. Retinofugal Projections from Melanopsin-Expressing Retinal Ganglion Cells Revealed by Intraocular Injections of Cre-Dependent Virus. PLoS One. 2016 Feb 19;11(2):e0149501. doi: 10.1371/journal.pone.0149501. PMID: 26895233; PMCID: PMC4764510.
 
2015 Mao Mao (Gould lab)
Mao M, Smith RS, Alavi MV, Marchant JK, Cosma M, Libby RT, John SW, Gould DB. Strain-Dependent Anterior Segment Dysgenesis and Progression to Glaucoma in Col4a1 Mutant Mice. Invest Ophthalmol Vis Sci. 2015 Oct;56(11):6823-31. doi: 10.1167/iovs.15-17527. PMID: 26567795; PMCID: PMC4627250.
 

AMSF Fellowship Winners

 
All May See Foundation is a 501(c)(3) nonprofit that provides fundraising support for UCSF Ophthalmology and Francis I. Proctor Foundation for Research in Ophthalmology.

2023 An Cheng (Lakkaraju lab)
PROJECT: "Mechanisms of FOXO3 dysfunction and its relevance to retinal pigment epithelium injury in Stargardt disease"
 
2023 Vinod Reddy Lekkala (Lamba lab)
PROJECT: "Driving maturation of photoreceptors in early-stage retinal organoids via CRISPR activation"
 
2023 Victoria Zhao (Duan lab)
PROJECT: "Mechanisms Underlying Retinal Ganglion Cell Subtype-specific Resiliency in Glaucomatous Optic Neuropathy"
 
2022 Sangeetha Kandoi (Lamba lab)
PROJECT: "Retinal organoid-based modeling and gene editing for GUCY2D-associated Cone Rod dystrophy"
 
2021 Andrea Cozzitorto (Gould lab)
PROJECT: "Characterization of the molecular and cellular processes involved in ocular anterior segment dysgenesis"
 
2020 Yao Lu (Kim lab)
PROJECT: "Enabling direct correlation of choroidal blood flow and retinal degeneration
at the single vessel level and over time using transscleral multiphoton microscopy"

 
2020 Li Xuan Tan (Lakkaraju lab)
PROJECT: "Mechanisms of retinal degeneration in Alzheimer’s disease-related dementias"
 
2019 Joo Yeun Lee (Dunn Lab)
PROJECT: "Changes in inhibitory retinal circuits following partial cone loss"
 
2018 Alfred Yu (Ou lab)
 

NIH Funding

 

PI / Lead: ACHARYA, NISHA
Project Number: 2R01EY028739-06
Project Title: The Impact of the Herpes Zoster Vaccine on Herpes Zoster Ophthalmicus


Herpes zoster (HZ, or shingles) is a major cause of morbidity, affecting one in three Americans during their lifetime. HZ can be particularly devastating when it involves the eye (herpes zoster ophthalmicus, HZO) and can result in permanent vision loss. The FDA approved a highly effective HZ vaccine (Shingrix, recombinant zoster vaccine [RZV]) in late 2017 for ages 50 and above. Our group published the first post-licensure observational studies of real-world effectiveness of RZV using two years of data. Long-term monitoring of RZV uptake, effectiveness and safety, as well as its impact on HZ/HZO incidence is critical to guide vaccination efforts. During our current R01 grant period, we found that incidence rates of HZ/HZO increased significantly from 2007 to 2018 in the middle-aged groups. In this current proposal, we propose to assess the incidence in the era of RZV vaccination to determine if the incidence is increasing in age groups which are not currently eligible for vaccination (Aim 1). This could have ramifications for policies regarding age recommendations for RZV. Despite the high efficacy of RZV, there is concern that vaccine uptake may be low among specific demographic groups due to early shortages and healthcare disruptions during the COVID-19 pandemic. There are no studies on RZV vaccination coverage of the eligible US population, but an understanding of factors associated with lower likelihood of vaccination, including social determinants of health, could help better target vaccination efforts (Aim 2). Our previous studies were the first to demonstrate high short-term effectiveness of RZV in general practice, and we now propose to assess how long the protection against HZ and HZO will last (Aim 3). Additionally, we will provide estimates of vaccine effectiveness and waning in the immunocompromised subgroup, a group at high risk for HZ which was excluded from clinical trials. Understanding the long-term vaccine effectiveness is critical for informing vaccine policy recommendations for different groups, including the need for and timing of re- vaccination. Lastly, patients with a history of HZO are a subgroup for which there are concerns regarding RZV vaccination, as reflected in a survey conducted by our group showing hesitancy among cornea specialists to recommend RZV. This uncertainty may be due to a lack of evidence on the vaccine’s safety and efficacy to inform clinical recommendations for this patient population. We will determine whether there is an increased risk of post-RZV HZO exacerbations, and we will estimate the effectiveness of RZV in preventing future recurrences and exacerbations of HZO in patients with a history of HZO (Aim 4). Rigorous information on the relative risks and benefits of RZV will facilitate making evidence-based recommendations regarding vaccination.


PI / Lead: ACHARYA, NISHA
Project Number: 5UG1EY029658-05
Project Title: Adalimumab in Juvenile Idiopathic Arthritis-associated Uveitis Stopping Trial


Juvenile idiopathic arthritis (JIA) is the most common rheumatologic condition in children and 12-38% of patients with JIA develop chronic asymptomatic anterior uveitis typically within 4 to 7years of arthritis onset. JIA-associated uveitis can cause significant morbidity with as many as1/3 of all patients developing substantial visual impairment and up to 15% becoming legally blind.The anti-TNF- human monoclonal antibody adalimumab has shown efficacy in treating JIA-associated uveitis but is associated with a risk of serious adverse events including opportunisticinfections and malignancy. Furthermore long-term treatment with adalimumab is expensive andcauses significant financial burden for the patient and healthcare system. However stoppingadalimumab may come with risks of its own; it has been shown that stopping and restarting anti-TNF- therapy in patients with other autoimmune diseases is associated with reducedresponsiveness to the drug. Collectively these reasons contribute to a growing interest indeveloping evidence-based guidelines for stopping adalimumab treatment once control ofinflammation has been achieved.We propose a multicenter double-masked randomized controlled trial to address clinicallyrelevant questions about stopping adalimumab in patients with controlled JIA-associated uveitis.In patients with controlled JIA-associated uveitis we will compare rate of recurrence and time torecurrence of ocular inflammation in patients randomized to discontinue adalimumab comparedto those who continue treatment (Aim 1). We will also evaluate key predictors of JIA-associateduveitis recurrence by assessing clinical characteristics and potential biomarkers associated withrecurrence of uveitis (Aim 2). Finally we will determine if stopping adalimumab leads to overallless control of inflammation at the 6 and 12-month visits even if patients restart adalimumab aftera uveitis recurrence (Aim 3). By following patients from randomization to potential relapse and re-treatment we can better understand the consequences of stopping and restarting adalimumab.With the increasing use of TNF- inhibitors understanding the risks and benefits of stoppingadalimumab in patients with controlled JIA-associated uveitis is important to inform clinicalpractice for management of these patients. This study could also identify predictors of relapseand drug response that would be useful in making evidence-based treatment decisions.!


PI / Lead: ARNOLD, BENJAMIN F
Project Number: 5R01AI158884-04
Project Title: Seroepidemiology of trachoma for the elimination endgame


Trachoma caused by ocular Chlamydia trachomatis infection is the leading infectious cause of blindnessworldwide and been targeted for global elimination as a public health problem by 2030. As we approach theendgame there is broad interest in the use of serologic surveys to support control programs. IgG antibodyresponses to C. trachomatis in children enable accurate population-level assessments of trachoma endemicitybecause they integrate exposure over time and reflect recent transmission. After years of assay developmenta key gap in the field is to formalize the epidemiologic methods used for trachoma serology surveys.Our overall objective is to advance the methods used for the design and analysis of trachoma serologysurveys. We will assemble a large contemporary global dataset for trachoma serology across a gradient ofendemicity paired with clinical signs and molecular measures of infection (>100000 blood specimens tested in19 studies from 2010-2024). Aim 1 will develop robust methods to translate antibody response into population-level measures of transmission from endemic settings to post-elimination. We hypothesize that as populationsapproach elimination age-seroprevalence curves will flatten and seroconversion rates a measure of force ofinfection will approach zero. We will estimate age-seroprevalence curves semi-parametrically and derivesummary measures from the curves (e.g. seroprevalence force of infection). We will compare serologicmeasures between populations of different endemicity. We further hypothesize that different serologicsummary measures (mean IgG levels seroprevalence force of infection) will provide similar information aboutheterogeneity in transmission. We will compare serologic measures with one-another and with separatemeasures of trachoma (PCR infection clinical signs) across geographic scales from villages to districts. Aim 2will determine if model-based geostatistics improve the efficiency of serological survey design and enable finerscale targeting of control programs as populations approach elimination. We hypothesize that as trachomaapproaches elimination it will become more focal with hotspots of elevated seroprevalence among childrenthat shrink in scale from districts down to individual villages. We hypothesize that if surveys account for thisspatial structure in their design they will more efficiently monitor trachoma than random samples alone andcontrol programs that use spatial predictions to make treatment decisions at smaller spatial scales could morenarrowly target antibiotic distribution. In analyses of 11 georeferenced studies that span a range of endemicitywe will apply recent advances in geospatial design to trachoma serology and compare prevalence estimatesusing the new approach with the current standard population-based random samples. We seek to identify themost efficient sampling strategies to inform decision making as populations approach elimination and to studythe impact of using spatial predictions to target azithromycin at finer spatial scales. Completion of these aimswill lead to significant advances in the seroepidemiologic methods used to support the trachoma endgame.


PI / Lead: ARNOLD, BENJAMIN F
Project Number: 5R01AI162867-03
Project Title: Enteric Pathogen Force of Infection among Children using Serology


Enteric pathogen infections are a leading cause of the global disease burden with the largest burden amongchildren in low-resource settings. Stool-based PCR methods have dramatically improved our ability to measureenteric infections but the challenge of collecting stool and need for near-continuous monitoring to detect manyglobally important pathogens has thwarted broader use in large-scale surveillance. Large-scale population-based surveys now regularly collect blood to monitor transmission and burden of diseases such as HIV malariaand dengue. Broader testing of blood collected in such surveys with multiplex assays represents a newopportunity to measure enteric pathogen transmission and burden. Antibody-based measures could complementstool-based PCR testing because antibody responses remain elevated for many months after infection thusproviding more information in studies with infrequent measurements. Our team has developed multiplex beadassays that measure immunoglobulin G (IgG) response to diverse enteric pathogens. In preliminary studies wehave shown that IgG levels can be used to measure heterogeneity in enteric pathogen transmission betweenpopulations. We have also shown the results generalize to pathogens that span taxa. In this application wepropose to complete a series of studies that address key next steps to advance the seroepidemiology of entericpathogens in low-resource settings. We will conduct a longitudinal birth cohort in Ecuador that pairs highresolution multiplex stool-based PCR infection with longitudinal multiplex IgG and IgA measurements. Our 17-year research history at the site has documented substantial variation in enteric pathogen infection across arural-urban gradient making it an ideal setting for the research. The cohort will enroll 600 children from threesites across a rural-urban gradient and measure them frequently from birth to 24 months. On the Luminexplatform we will pair multiplex PCR assessment for 15 enteric pathogens with IgG and IgA assessment in amultiplex bead assay that includes antigens to 7 enteric pathogens: Campylobacter jejuni enterotoxigenicEscherichia coli Salmonella enterica Giardia duodenalis Cryptosporidium parvum Entamoeba histolytica andnorovirus. In Aim 1 we will use molecular and antibody-based measures to study force of infection of the entericpathogens across a rural-urban gradient. This will represent the first broad-based comparison ofseroepidemiologic measures against patent infection for enterics. In Aim 2 we will estimate enteric pathogenforce of infection by applying current-status models to population-based cross-sectional serology surveys in theregion and will benchmark the cross-sectional estimates against those obtained in the concurrent longitudinalstudy. In Aim 3 we will study IgG and IgA kinetics following infection for each of the 7 pathogens and developmodels to accurately predict recent infections and incidence from antibody levels measured in cross-sectionalserology surveys. Completion of these aims will result in generalizable seroepidemiologic methods that have thepotential to transform measurement of enteric pathogen transmission and burden in low-resource settings.


PI / Lead: ARNOLD, BENJAMIN F
Project Number: 5R01AI166671-02
Project Title: Serologic measures of enteric pathogen transmission for intervention studies and population monitoring in low-resource settings


Enteric pathogen infections cause an immense disease burden among children in low-resource settings.Understanding pathogen-specific transmission in high burden populations and whether transmission is reducedthrough environmental intervention (reduced exposure) or improved nutrition (reduced susceptibility) is cruciallyimportant for informing global public health programs. In preliminary studies our team has developed multiplexbead assays that measure immunoglobulin G (IgG) response to diverse enteric pathogens along withseroepidemiologic methods to measure changes in transmission based on enteric pathogen antibody response.Antibody-based measures should complement stool-based PCR measures of infection in studies with infrequentmeasurement (rather than continuous monitoring) because antibody response integrates exposure over timethus providing additional information about infections that begin and resolve between measurements. Our overall objective is to use seroepidemiologic methods to measure intervention effects on entericpathogen transmission and to leverage large-scale trials to develop new methods that combine multiplex testingwith spatial epidemiology to locate communities with highest multi-pathogen burdens. Our team recentlycompleted cluster randomized trials in Kenya and Bangladesh that delivered water sanitation handwashing(WASH) and nutritional interventions to pregnant mothers and measured primary endpoints (growth diarrhea)among their newborn children through age 24 months. Each trial enrolled and randomized >700 communities.Blood samples were collected among longitudinal substudies of 1500 children in each country at ages 6 12and 24 months across factorial arms (Control WSH Nutrition Nutrition + WSH) and among ~4000 children percountry at age 24 months. We propose to test for IgG responses using a multiplex bead assay that includesantigens to nine enteric pathogens: Giardia sp. Cryptosporidium sp. Entamoeba histolytica Strongyloides sp.Ascaris sp. Campylobacter sp. enterotoxigenic Escherichia coli Salmonella sp. and norovirus. In Aim 1 we will use the factorial trial design to measure the effects of WASH and nutrition interventions onantibody-based measures of enteric pathogen transmission in each country including mean IgG responseseroprevalence and force of infection. We hypothesize that the interventions reduced transmission of entericpathogens through reduced exposure (WASH) and reduced susceptibility to infection (nutrition). In Aim 2 wewill combine multiplex antibody data with spatial models to map landscapes of enteric pathogen exposure anddevelop generalizable methods to identify communities with highest multi-pathogen burdens. We hypothesizethat antibody-based measures will align with other measures of infection and that there will be geographicoverlap in hotspots identifying communities with the highest multi-pathogen burdens. Richly characterized trialsin two countries ensure results will be rigorous and policy relevant. Completion of these aims will significantlyadvance the use of serological assays in enteric pathogen intervention studies and population surveillance.


PI / Lead: BLUMBERG, SETH
Project Number: 5R35GM147702-02
Project Title: Modeling the dynamics of disease elimination


Elimination of an infectious disease is often a goal of the public health community. Although that goal is rarelyachieved the tremendous expansion of epidemiological databases provides new opportunities to testhypotheses concerning elimination with mathematical modeling. Besides improving our scientificunderstanding of disease transmission hypotheses validated through mathematical modeling provide publichealth practitioners with a more structured quantitative assessment of how elimination of specific pathogenscan be achieved. This proposal aims to develop an interconnected set of modeling tools to support eliminationof communicable diseases. A variety of processes used to achieve disease elimination will be consideredincluding use of mass drug administration to eliminate neglected tropical diseases such as trachomavaccination for preventable diseases such as SARS-CoV-2 and antibiotic stewardship efforts to curtail drugresistant infections such as methicillin-resistant Staphylococcus aureus (MRSA). A key theme is therequirement of subcritical transmission for disease elimination meaning that the average number of newinfections each case causes is less than one. A major goal is to elucidate the transmission dynamics ofsubcritical diseases on the brink of elimination. Transmission heterogeneity may arise from manymechanisms including super-shedding of certain individuals pockets of susceptibility such as in a communitywith low vaccine uptake and contact structure in which some individuals have the potential to infect manyothers. Simulations of various patterns of disease transmission will be used to develop distinct measurementsof transmission heterogeneity. In addition new techniques to infer and compensate for observation error will bedeveloped that integrate data on the observation process such as the proportion of cases identifiedretrospectively via contact tracing programs. Models of transmission dynamics will be used to identifytransmission-hotspots and superspreaders that can jeopardize elimination. People areas or events thathave increased transmission potential can maintain endemic disease transmission even though the population-level average value of R may be less than one. In the first stage of this objective we will use existing models toconstruct a suite of in silico simulations to compare the performance of various scan statistics designed todetect disease burden beyond what is expected by chance. In the second stage we will apply these scanstatistics to observational data. Identification of transmission-hotspots and supersreaders permits optimizationof disease elimination strategies. To eliminate disease it is insufficient to merely identify transmission-hotspots or superspreading activity. A strategy is needed for suppressing the sites events or people thatcause higher levels of transmission. We will use mathematical and computational models for diseaseelimination to address 1) the impact of control interventions 2) the optimal distribution of a limited treatmentsupply and 3) monitoring of treatment efficacy.


PI / Lead: CHAN, MATILDA F
Project Number: 1R21EY035294-01
Project Title: Transcription Factor 4 (TCF4) Regulation of Corneal Health


Fuchs endothelial corneal dystrophy (FECD) is a bilateral heritable degeneration of the corneal endotheliumthat affects roughly 4% of the population older than 40 years in the United States. FECD is one of the mostcommon indications for corneal transplantation. Major advances in genomics have dramatically increased ourunderstanding of FECD and identified diverse genetic and epigenetic causes and associations. In particulartraditional linkage studies and GWAS studies identified an expanded CTG trinucleotide repeat (TNR)expansion (named CTG18.1) in the non-coding region of transcription factor 4 (TCF4) gene as the moststrongly associated genetic alteration associated with late-onset FECD. The intronic TNR expansion within theTCF4 gene is responsible for about 70% of FECD cases in European populations. Despite these substantialadvances made in identifying risk alleles and biological pathways that are involved in the development ofFECD definitive treatment of FECD has mainly remained surgical with corneal transplantation. Effectivemedical treatments for FECD are lacking. A major challenge in the field of FECD research is the absence of amouse model of the TCF4 repeat expansion. Mouse models have been developed for other genes associatedwith FECD (COL8A2 Slc4a11) but these genes are responsible for only a minority of cases. Thus currentmodels for studying TCF4 repeat expansion FECD include ex-vivo specimens primary cell cultures andimmortalized cell lines derived from tissue specimens obtained from patients with advanced FECD undergoingcorneal transplantation. An in vivo model of the TCF4 repeat expansion is needed in particular one that couldbe used to study early stage FECD. The main roadblock has been the challenge of creating animal modelswith large uninterrupted repeats due to the technical difficulties. In preliminary studies we have developed anovel method for generating knock-in mouse models of trinucleotide repeat expansion disorders. Our uniqueapproach takes advantage of CRISPR and the highly efficient microhomology-mediated end joining (MMEJ) toknock in trinucleotide repeats into gene loci. This approach has enabled us to knock in 338 CTG repeats intothe Tcf4 locus and successfully generate Tcf4-CTG338 knock-in heterozygous mice. Our overall objective is todetermine how the TCF4 repeat expansion contributes to FECD pathogenesis so that new therapeutics maybe developed. In our Specific Aims we take two major strategies: characterize mice with a short TCF4 repeat(338 repeats) that have already been generated and develop mice with a long TCF4 trinucleotide repeat (1346repeats) as repeat expansion length is correlated with corneal disease. We hypothesize that mice with a TCF4repeat expansion can serve as an in vivo model for CTG expansion-mediated FECD. We anticipate that thisproject will ultimately establish new insight into how a TCF4 repeat expansion contributes to FECD diseaseand uncover promising therapeutic targets. More broadly this work establishes a platform to develop novelmouse models for the study of trinucleotide repeat expansion diseases.


PI / Lead: CHAN, MATILDA F
Project Number: 5R01EY032161-03
Project Title: Analysis of chemical modulators for corneal endothelial dystrophies


Corneal endothelial dystrophies are a common cause of vision loss and are characterized by a slowlyprogressive bilateral dysfunction of the corneal endothelium. The main therapeutic option to restore vision inthese patients remains corneal transplantation as medical management is usually inadequate. Mutations inthe SLC4A11 gene are associated with several endothelial dystrophies including Fuchs endothelial cornealdystrophy (FECD) congenital hereditary endothelial corneal dystrophy (CHED) and Harboyan syndrome.SLC4A11 is a transporter protein that functions to maintain osmotic balance in corneal endothelium and manypoint mutations in SLC4A11 found in disease lead to misfolding of the full-length protein. There is recentevidence that assisting mutant SLC4A11 to regain proper folding is a promising therapeutic approach. A priorsmall-scale high throughput chemical screen identified the non-steroidal anti-inflammatory drug glafenine forits ability to correct folding defects in misfolded SLC4A11 and following correction the mutant SLC4A11protein regained functional activity. Though glafenine is not a candidate for clinical use due to anaphylaxis andrenal toxicity its efficacy supports an approach using other small molecule folding correctors to repair defectiveSLC4A11 in corneal endothelial dystrophies. Our long-term objective is to develop first-in-class therapeuticsfor patients with vision impairment due to protein misfolding in the cornea. We propose to test the hypothesisthat compounds identified through phenotypic screening assays will promote the correct folding of mutantSLC4A11 and restore corneal endothelial cell function. The goals of this proposal are to: Aim 1) Performfocused screening to identify correctors of SLC4A11 folding; Aim 2) Perform large unbiased chemical libraryand genetic screening to identify novel targets and mechanisms; and Aim 3) Assess cytotoxic effects ofNSAIDs on corneal cells. Using advanced experimental methods including high-content microscopy-basedscreening assays and novel CRISPR-based genetic screens the proposed studies will provide insight into thecorrection of protein folding defects as a therapeutic strategy for corneal endothelial dystrophies. Results fromthe study will be used to develop a new non-surgical treatment option for patients with vision loss due tocorneal endothelial dystrophies and establish a novel therapeutic approach to corneal disease.


PI / Lead: DOAN, THUY A
Project Number: 5R01EY032041-04
Project Title: Seasonal Conjunctivitis Outbreak Reporting for Prevention and Improved Outcomes (SCORPIO)


Seasonal and epidemic conjunctivitis (pink eye) infections are highly contagious and impose significanteconomic burden in the United States and worldwide. Long-term visual impairment can occur. In childrenconjunctivitis may herald more serious systemic complications leading to hospitalizations and even deaths.Current challenges to effective mechanisms to curtail outbreaks are the lack of tracking and reporting pooraccess to sample collection for analysis limited diagnostic testing capabilities and an incompleteunderstanding of the pathogens involved. The overall goal of this project is to combine internationalcollaboration efforts and advances in genomic technologies to identify pathogens global trends pathogenevolution and the immune responses involved in the epidemiology of conjunctivitis. We propose to enhancethe understanding of the pathogenesis of conjunctivitis with the following aims. The first aim will determine howadenovirus genome types vary within a region and across regions. The second aim will identify emergingcauses of seasonal and epidemic conjunctivitis. Finally we will identify the local immune response signaturesof children and adults with infectious conjunctivitis. We expect that conjunctival gene expression profiles willaccurately predict disease associated complications. This project has the potential to improve public healthsurveillance guide targeted interventions improve vaccine development and elevate existing diagnosticparadigms for conjunctivitis to reduce both the financial and ocular health burden worldwide.


PI / Lead: DOAN, THUY A
Project Number: 5R01EY032861-02
Project Title: Improving Corneal Ulcer Outcomes with Unbiased Pathogen and Antimicrobial Resistance Detection


Infectious corneal ulcers affect all ages and contribute to significant visual morbidity worldwide. Empirictreatment with broad spectrum antimicrobials without regard for antimicrobial resistance is presently standardof care. This is either because pathogen identification with routine microbiological testing is suboptimal ortesting is not done altogether. While empiric treatment is the preferred practice pattern for infectious keratitisworldwide this is contrary to the goals and ideals of antimicrobial stewardship. Here we propose to enhancethe understanding of the pathogenesis of infectious keratitis and antibiotic resistance profiles to improvediagnostics and therapeutics with the following aims. In the first aim we will perform worldwide surveillance ofall organisms responsible for infectious keratitis. We expect the spectrum of etiology will vary significantly withgeographic location. In addition seasonality will affect pathogen profile and disease outcomes. The secondaim will determine the frequency and richness of antimicrobial resistance (AMR) in corneal ulcer pathogensand correlate those findings with phenotypes. We expect AMR will differ by global location and predict clinicaloutcome. Finally we will define the local immune response in infectious keratitis. We hypothesize that immunesignatures can predict pathogen types and clinical outcome. Corneal opacity secondary to infectious keratitisremains one of the leading causes of blindness worldwide and we anticipate this research will guide clinicianson the best management for corneal infection to reduce both the financial and ocular health burden globally.


PI / Lead: DUAN, XIN
Project Number: 5R01EY030138-05
Project Title: Mechanisms Underlying Type II Cadherin Guided Assembly of Retinal Circuits


In the eye complex retinal circuits are wired together for precise neural computation. The diverse but precisewiring between interneurons and retinal ganglion cells serve as the structural basis for circuit processing ofdifferent visual features. These parallel circuits are wired up precisely as defects may lead to several eyediseases and neurological disorders. To investigate the mechanisms behind how diverse neuronal typesprecisely integrate into distinct parallel retinal circuits we developed methods that allow for targeted geneticaccess of the unique On-Off direction-selective circuit which conveys direction-selectivity signals as the idealmodel system. Our previous studies now position us to examine the role of Type II Cadherins (Cdhs) inassembling this circuit as individual proteins or in combinations. We showed that two Cdhs Cdh9 and Cdh8instruct parallel ON and OFF bipolar cell input to ON vs. OFF sublaminae of the ON-OFF direction-selectivecircuit thus allowing precise segregation of ON and OFF channels. However the molecular mechanismsunderlying this assembly remain elusive. To investigate the molecular mechanisms underlying the differentialfunctions of Cdh9 vs. Cdh8 we will perform a series of anatomical and functional analyses. We will identify thespecific portion of the cadherin molecule extracellular versus intracellular domains that are responsible fortheir distinct functions as well as the specific timing of their actions in forming synapses between bipolar cellsand ganglion cells. We also found that Cdh9 from bipolar neurons heterophilically recognizes the two closely-related Cdhs Cdh6 and Cdh10 from postsynaptic Ventral-pointing ON-OFF direction-selective ganglion cells(ooDSGCs) and starburst amacrine cells (SACs). We will use this established genetic system to reveal howcombinatorial Cdhs act together to wire up parallel direction-selective circuits. We will examine genetically andfunctionally how Cdh6-9-10 single double and triple combinations pattern the Ventral-ooDSGC interactionwith SACs. To further expand our understanding of the combinatorial cadherin code in neuronal patterning wewill test the role of Cdh11 which is identified as a Nasal-pointing ooDSGC enriched gene through molecularprofiling. Thus we will generate new molecularly and genetically targeted methods to examine the roles ofCdh11 and its closely related Cdh8 in the wiring of Nasal-pointing direction-selective circuits. Furthermore weestablished an in utero injection system to ectopically introduce individual Type II Cdhs onto Ventral-ooDSGCs or Nasal-ooDSGCs to pinpoint combinatorial Cdhs in regulating DS-circuit patterning. Collectivelyour studies seek to reveal how Cdh combinations control the formation of parallel but distinct DS circuits.Comprehensive studies on Type II Cdh function would be a major advance for a long-standing question inmammalian neural development. These studies will be a major step forward in understanding how multiplegenes interact to specify the wiring of complex neural circuits. The identified mechanisms will have significantrelevance to selective circuit wiring throughout the central nervous system.


PI / Lead: DUAN, XIN
Project Number: 5R01NS123912-03
Project Title: MAPPING RETINOTECTAL CIRCUITS FOR VISUAL-EVOKED INNATE BEHAVIORS


The precise assembly of neural circuits ensures accurate neurological function and behavior. Forexample to communicate specific aspects of the visual world to the brain retinal ganglion cells (RGCs) find andform synaptic contacts with specific postsynaptic partners out of the heterogeneous neuronal population ofretino-recipient areas in the brain. One such area is the superior colliculus (SC) which receives direct retinalinputs and sends commands for direct innate behaviors such as escape or prey capture. What are the moleculardeterminants for selective RGC to SC neuron wiring? How are parallel retinotectal circuits sorted onto differentSC laminae and neuronal relays? How are distinct retinotectal circuits linked to defined visual evoked behaviors?This proposed study aims to answer these questions in the mouse visual system. To accomplish this goal first we will map out parallel retinotectal circuits. We have established anintegrated anterograde-tracing and sequencing platform Trans-Seq that defines the outputome of a genetically-defined RGC subtype. We applied Trans-Seq to all RGC subtypes globally -RGCs and On-Off direction-selective-ganglion-cells and reconstructed their differential outputomes onto superficial superior-collicular (sSC)neuron subtypes. We propose to apply Trans-Seq to other major RGC subtypes representing different visualfeatures. The proposed studies will determine retinotectal circuit convergence and divergence at neuron subtyperesolution. Second we aim to understand cellular and molecular mechanisms regulating specific retinotectalcircuit wiring. We have analyzed -RGC specific outputomes and revealed a selective sSC neuron subtypeNephronectin-positive-wide-field neurons (NPWFs). The -RGC-to-NPWF circuit was genetically validated usingimaging electrophysiology and retrograde tracing. We propose to study how Nephronectin mediates -RGCselective axonal lamination onto the deep sSC layer and whether Nephronectin determines the subsequentsynaptic specificity from -RGCs to NPWFs. We will also investigate what molecular mechanisms mediateNephronectin binding and lead to a selective mammalian retinotectal circuit assembly. Third we will link specificretinotectal circuits to defined visual evoked behaviors. We propose to combine genetic and optogenetic toolsestablished above to determine whether the -RGC-to-NPWF circuit contributes to visual evoked innatebehaviors such as looming triggered defense responses. We will also examine whether molecular determinantsfor connectivity such as Nephronectin regulate this behavioral output via these retinotectal circuits. Our circuit mapping platform builds a precise connectivity map at neuronal subtype resolution. Furtherthis work will align the precise neuronal wiring diagram to innate visual evoked behaviors informing futurefunctional and behavioral analysis. The new knowledge gained here may include molecular principles underlyingmammalian circuit wiring relevant beyond the visual system.


PI / Lead: DUNCAN, JACQUE LYNNE
Project Number: 5U24EY033699-02
Project Title: Expert curation of clinically significant variants in genes for early onset retinal degeneration


The goal of this proposal is to curate clinically relevant variants in genes associated with the inheritedmonogenic diseases autosomal recessive Leber congenital amaurosis (LCA)/early-onset RetinalDegeneration (eoRD) that cause lifelong blindness beginning in infancy or childhood. More than 30 genesassociated with these phenotypes have been identified and the first gene replacement therapy was approvedfor LCA/eoRD associated with RPE65 variants while clinical trials are currently underway to treat diseasecaused by 3 other genes (AIPL1 GUCY2D and CEP290). Despite these advances it is still challenging tomake accurate clinical diagnoses and decisions based on current knowledge of variants in LCA/eoRD-associated genes. A major limitation is the lack of uniform classification criteria optimized for gene-diseasespecific features that enable accurate and consistent interpretation of the clinical relevance of variants. Toaddress this we have assembled a variant curation expert panel (VCEP) comprised of an international groupof experts with in-depth knowledge in LCA/eoRD genetics and clinical care. Further we establishedcollaborative relationships with the clinical domain working group (CDWG) oversight committee and theOcular CDWG of the NIH-sponsored Clinical Genome Resource (ClinGen) for advice and guidance. With thisleadership team we propose to curate variants in genes associated with LCA/eoRD phenotypes for whichgene therapies are available or clinical or advanced pre-clinical studies are underway. The proposed projectinvolves 2 Specific Aims: 1. Complete the approval process for the LCA/eoRD VCEP through 4 steps(assembling a group of experts for LCA/eoRD variant curation rule specification for the classification ofvariants in LCA/eoRD genes using disease-gene specific characteristic features pilot testing rules specifiedfor the curation of variants in LCA/eoRD associated genes and submitting the rules and pilot results to theClinGen Sequence Variant Interpretation Working Group for approval) and 2. Curation of variants in selectedLCA/eoRD genes by implementing the specified rules and submission to ClinVar. All steps will be carried outwith the approval of the ClinGen CDWG oversight committee utilizing a suite of variant curation tools andprotocols developed by ClinGen. The proposed project will lead to the development of variant interpretationcriteria that are in harmony with rules established for other diseases and optimized for LCA/eoRD genes andgenerate a comprehensive resource of LCA/eoRD gene variants with FDA-designated expert level variantclassifications in the ClinVar public database. This information will advance research on LCA/eoRD andenable accurate consistent high quality interpretation of genetic test results and improve patient care.Further the rules specified by the LCA/eoRD VCEP will advance development of rules for other IRDs andother hereditary diseases.


PI / Lead: DUNCAN, JACQUE LYNNE
Project Number: 5UG1EY033292-03
Project Title: NAC Attack AOSLO Reading Center


Retinitis pigmentosa (RP) is a genetically heterogeneous group of diseases affecting about 100000 people inthe United States. RP causes progressive death of rod then cone photoreceptors resulting in relentless visionloss and ultimate blindness. There are no cures and effective treatments are extremely limited. Complicatingtreatment efforts is the fact that mutations in over 65 genes cause RP. As such therapies that target commonmechanisms of photoreceptor death and promote photoreceptor survival are attractive alternatives to gene-specific methods. Although rods are lost earliest in RP it is the subsequent cone degeneration that isparticularly devastating for patients. This cone degeneration may be due in part to oxidative stress andtreatments that reduce oxidative damage such as N-acetylcysteine (NAC) have been shown to improve conefunction and survival in animal models. Recently a single-center study in patients with RP (FIGHT-RP)demonstrated improvement in visual acuity at all NAC doses studied and improvement in macular sensitivity inpatients who received the highest dose. These promising results have motivated the development of amulticenter randomized placebo-controlled Phase 3 trial (NAC Attack) to test the efficacy of NAC to slowprogression of RP. The NAC Attack trial may provide clinical evidence of the role of oxidative stress and leadto improved understanding of cone degeneration mechanisms in RP. Although the NAC Attack trial aims toimpact clinical management of RP by demonstrating improved photoreceptor survival standard outcomemeasures have limited sensitivity to detect effects of treatments on individual cones the target of NAC.Adaptive optics scanning light ophthalmoscopy (AOSLO) allows non-invasive imaging of the cone mosaic withsingle-cell resolution. Preliminary studies using AOSLO showed reduced cone loss in eyes with RP treatedwith ciliary neurotrophic factor (CNTF) compared to contralateral sham-treated eyes; however CNTF-treatedeyes showed no improvement in vision (illustrating the low sensitivity of clinical measures of visual function).These data demonstrate a critical role for AOSLO in clinical trials. The objective of this AOSLO ResourceCenter is to support the evaluation of the safety and efficacy of NAC in patients with RP. Forty patientsenrolled at 6 sites with AOSLO systems will be imaged at baseline 9 27 and 45 months after randomization toreceive oral NAC 1800 mg or a placebo twice a day. AOSLO images will be used to measure changes in conedensity cone spacing cone mosaic regularity and cone reflectivity in NAC-treated compared to placebo-treated eyes between baseline and 45 months. Successful completion of the proposed studies described herewould support FDA approval of NAC for treatment of patients with RP. The results will also provide quantitativedata to support validation of AOSLO images of cone structure as objective sensitive outcome measures ofdisease progression which could significantly reduce the time and numbers of patients required to demonstratewhether other experimental therapies are safe and effective for patients with rare IRDs.


PI / Lead: DUNN, FELICE A
Project Number: 1R01EY035028-01A1
Project Title: Retinal mechanisms underlying the optokinetic reflex


There is a fundamental gap in the mechanistic understanding of the optokinetic reflex (OKR) used in the clinicto diagnose a variety of visual and neurological disorders. The OKR provides a unique system in which the visualsystem input and eye movement output are well-controlled and quantifiable and the information bottleneck atdedicated ganglion cell types is accessible. The long-term goals are to reveal the mechanistic basis of the OKRfrom the molecular programming of circuit wiring to computations to stimulus (in)dependence and to translatethis knowledge into diagnostics. The overall objective of this proposal is to determine how the individual andpopulation properties of two specific ganglion cell types influence eye movements. This proposals focus is onthe vertical OKR which is subserved by up/Superior and down/Inferior preferring ON direction selective ganglioncells (Superior and Inferior oDSGCs). Preliminary work leads to a central hypothesis: The vertical OKR isinfluenced by properties that can be traced to retinal direction selective circuits and specifically motion encodingin Superior and Inferior oDSGCs individually and as a population. Previous studies show that the OKR is contrastsensitive and asymmetric with higher gain in the up vs. down directions. Differences in intrinsic properties e.g.dendritic morphology and synaptic inputs can explain these phenomena with contrast sensitive spike tuningcurves and greater responses in Superior vs. Inferior oDSGCs. Recent single-cell sequencing supports theasymmetry with identification of differentially expressed genes in Superior vs. Inferior oDSGCs includingmolecular guidance cues that could be integral to the development of direction selective circuits. We propose toexamine the fundamental molecular and computational mechanisms that support and preserve the vertical OKRacross diverse stimulus statistics. We will achieve this by (1) using mouse genetics to elucidate the molecularprocesses that construct and maintain direction selective circuitry and (2) measuring oDSGC populationresponses and OKR in the context of stimulus perturbations including noise. (Aim 1) Identify essential signalingpathways for the development of direction selective circuits and the OKR and (Aim 2) Elucidate the mechanismsof noise correlations among populations of oDSGCs and their impact on the OKR. The aims will be accomplishedby using stimulus manipulations genetic perturbations cellular physiology circuit mapping and computationalmodeling to identify characteristics of the reflex their potential mechanistic basis and their stability or adaptabilityunder different visual environments. The expected outcomes for Aim 1 will be a concise link between moleculesretinal circuits and behavior and for Aim 2 will be insight into how stimulus statistics including noise correlationsinfluence behavior. The proposed work is significant because it will revealfrom molecules to behaviorhowcells circuits computations and their behavioral output operate in health and change in disease in anevolutionarily conserved system with the potential for clinical application of knowledge gained in this proposal.


PI / Lead: DUNN, FELICE A
Project Number: 5R01EY029772-05
Project Title: Synaptic and circuit mechanisms of compensation following loss of cone inputs in themature mouse retina


There is a gap in knowledge of how loss of 50-80% of cone photoreceptors produces almost no change in visualacuity or sensitivity. While contributions from cortex have been examined those from retina have beenunderappreciated. The long-term goal to understand how the retina functions robustly in the face ofphotoreceptor death will generate transformative insights into how neural plasticity compensates for celldeath. Understanding this compensation is likely to lead to earlier diagnostics and more effective treatments.The overall objective of this proposal is to elucidate the fundamental synaptic and circuit-level mechanismsthat allow the retina to function while compensating for photoreceptor death. This proposal focuses on thewell-characterized circuit of the ON sustained alpha ganglion cell in mouse retina a strong model circuit withidentified cell types maps of specific connections accessibility to genetic manipulation and quantifiablestructure and function. Following genetic ablation of 50-75% of cones in adult retina with the diphtheria toxinreceptor these ganglion cells adjust receptive field structures and spike responses. The observations arecongruent with adaptation which adjusts integration and gain for stimulus statistics e.g. greater integrationand gain at lower light levels or homeostatic plasticity which involves remodeling circuitry or channelexpression. The central hypothesis is that the retina can compensate for cone loss via mechanisms ofadaptation and/or homeostatic plasticity that we will determine in two specific aims: (Aim 1) identify theextent and sites of compensation within the retinal circuit following partial cone loss in the adult and (Aim 2)determine the contributions of partial stimulation mean adaptation and homeostatic plasticity to the retina’sreaction to cone loss. The results of the first aim will identify the structural and functional consequences ofcone loss on the direct excitatory pathway from cones to type 6 cone bipolar cells to ON alpha ganglion cells.The results of the second aim will determine how adaptation changes in excitatory and inhibitory circuits andintrinsic excitability contribute to changes in ganglion cell spatial and intensity encoding following partial coneloss. The approach is innovative for the genetic control over cone ablation in mature retina the stage at whichmost human retinal diseases occur; functional and structural examination with cell-type specific resolution;and focus on synaptic and circuit mechanisms underlying a well known discrepancy between photoreceptorloss and visual function. The research is significant for (1) uncovering mechanisms that may mask visualdeficits in early stages of photoreceptor loss; (2) suggesting diagnostics that could detect earlier onset ofdiseases causing cone loss; (3) establishing knowledge about the flexibility of a sensory circuit and how thisflexibility pertains to a surviving circuit; (4) providing direct measures of how retinal function after partialcone loss is distinct from or similar to that in control retinathus potentially influencing the design oftreatments to restore retinal function following photoreceptor loss.


PI / Lead: DUNN, FELICE A
Project Number: 5R01EY030136-05
Project Title: Thresholds sites and contributions of circuit compensation following rod photoreceptorloss in mature retina


There is critical need to understand the causes extent and mechanisms of reactions to cell death so that effective treatments most appropriate for the state of the remaining circuit can be employed and so that constructive compensation can be harnessed as a potential treatment in conditions where a portion of the circuit endures. Our long-term goal is to salvage neuronal circuits. Here we will define the effects of controlled cell death on specific circuits cell types synapses and proteins for the purpose of understanding the conditions that result in constructive (e.g. compensation through increasing synaptic gain) vs. destructive (e.g. aberrant spontaneous activity that corrupts signal) response. The mouse retina is an exceptional platform for this study because the primary sensory neurons photoreceptors can be manipulated under genetic control; cell types within specific circuits are identifiable and accessible; and the functional readout can be interpreted as visual sensitivity. We propose to ablate variable populations of rods in mature retina and determine the structural and functional effects on the primary rod bipolar cell pathway the most sensitive retinal pathway: rodsrod bipolar cellsAII amacrine cellsON cone bipolar cellsON sustained alpha ganglion cells (abbr. ON alpha). ON alpha ganglion cells receive the greatest number of rod inputs thus would be the most sensitive to rod loss. Our central hypothesis is that the retina has constructive reactions to input loss with the capacity to recover normal function up to an undefined threshold; beyond this threshold destructive reactions begin. Unknown is this tipping point. Our preliminary data show that despite loss of half the rods rod-mediated light responses in ON alpha ganglion cell spikes are comparable to control suggesting compensationwithin the primary rod bipolar cell pathway. Thus the premise is strong for constructive compensation within the retina following rod loss and we will determine the induction parameters sites and contributions of this compensation to maintaining function in the following aims: (Aim 1) to determine the degree of input loss that induces constructive vs. destructive structural and functional changes and (Aim 2) to locate the site(s) and mechanism(s) of compensation within a well-defined neural circuit. The approach is innovative for genetic control over the timing and degree of rod death; synaptic- and cell-type specific structural and functional investigation of a well-defined retinal circuit; and molecular tools to distinguish between cell ablation and synapse disassembly in triggering compensatory mechanisms. The results will be significant for (1) determining the degree of rod death that triggers the remaining circuit to undergo destructive or constructive responses (2) identifying the sites and contributions of structural and functional compensation to maintaining retinal function and (3) providing knowledge essential to the optimization and deployment of therapies to treat dysfunctional photoreceptors involving stem cells genes and prostheses all of which rely on a stable retinal circuit and/or extensive knowledge of the state of the surviving retinal circuit.


PI / Lead: GOULD, DOUGLAS
Project Number: 1R21NS133610-01
Project Title: Characterization of Tagged Type IV Collagen


Mutations in type IV collagen alpha 1 (COL4A1) and alpha 2 (COL4A2) are an important cause of monogeniccerebral small vessel disease and are also significantly associated with cerebral small vessel disease hallmarksin large-scale genome wide association studies underscoring their importance for general cerebrovascular healthin humans. The broad goal of our research is to understand the biological functions COL4A1 and COL4A2 in theextracellular matrix and determine the mechanisms by which they cause genetic and acquired cerebrovasculardisease and disfunction. The specific purpose of this proposal is to validate a potentially powerful tool that willenable unveiling of tissue specific and temporal changes in collagen deposition and degradation.The inability to distinguish cell type specific or temporal changes to the extracellular matrix constitutes asignificant obstacle for understanding a fundamental aspect of tissue biology. To address this barrier wedeveloped a mouse line in which COL4A1 is fused with switchable fluorescent proteins for isoform-specificvisualization and biochemical tags for differential affinity purification. This simple but powerful tool will enableunprecedented characterization of spatial temporal biochemical and biophysical parameters of ECM that arecurrently impossible to achieve. If the detailed validations outlined in this proposal are successful we will havedeveloped a transformative tool that will provide significant insight into a fundamental aspect of tissue biologyand that can be applied to every organ of the body in normal development or in pathological settings.


PI / Lead: GOULD, DOUGLAS
Project Number: 1RF1NS128217-01A1
Project Title: Gene therapy for disorders of the extracellular matrix


COL4A1 and COL4A2 mutations cause Gould syndrome (GS) a multisystem disorder for which clinicallyheterogeneous cerebrovascular disease is the major consequence. Cerebrovascular disease in individuals withGS can range from porencephaly caused by germinal matrix hemorrhages in utero to infantile seizures to age-related cerebral small vessel disease (cSVD) and vascular cognitive impairment and dementia (VCID). Hallmarksof cSVD observed in individuals with GS include subcortical microbleeds enlarged perivascular spaces andlacunar infarcts. Importantly Col4a1 mutant mice faithfully model patient phenotypes. Moreover Col4a1 mutantmice have age-related cerebrovascular dysfunction including loss of myogenic tone and impaired hyperemicresponses that are thought to be critical to VCID progression.The extracellular insults resulting from COL4A1 and COL4A2 are heterogeneous and complex which representsa significant barrier to mechanism-based interventions. However because GS is a devastating monogenicdisease with a defined genetic cause it is an ideal candidate for correction of the root cause of the disease viagenome editing technologies. In this proposal we will leverage vastly improved CRISPR nucleases base editorsand prime editors along with novel viral vectors to test therapeutic approaches using primary GS patient cellsand mouse models of GS. This project will provide important pre-clinical data to develop the first genome editing-based therapy for this severe monogenic disorder. The successful completion of this work could eventuallyprovide a one-time lifelong treatment that prevents both childhood stroke and age-related VCID for GS patientsand create a roadmap for correction of similar diseases.


PI / Lead: GOULD, DOUGLAS
Project Number: 3R33NS115132-05S1
Project Title: Development and validation of novel models for cerebral small vessel disease and vascular cognitive impairment


Vascular cognitive impairment and dementia (VCID) is any level of cognitive alteration that is attributable tocerebrovascular pathologies. VCID is second only to Alzheimer’s disease as a cause of dementia andaccounts for ~15-30% of all dementia cases. Cerebral small vessel diseases (cSVDs) are group of pathologiesafflicting the microcirculation of the brain that collectively account for up to 20% of all strokes and is the mostcommon pathology underlying VCID. The impact of cSVD and VCID is expected to increase rapidly as thepopulation of the US and other countries ages. Importantly the pathogeneses of cSVDs are incompletelyunderstood which represents a major barrier in developing strategies for prevention and treatment. Researchdescribed in this proposal will develop and validate five novel mouse models of cSVD based on genes andmutations that are demonstrated to contribute to human disease. We have assembled an interdisciplinary teamof experts that will integrate unique genetic resources vascular pressure myography patch-clampelectrophysiology calcium imaging specialized magnetic resonance imaging modalities and learning andmemory behavior assays to develop and characterize multiple novel genetic models of cSVD using genes thatcontribute to disease in humans. Our long-term objective is to develop and employ genetic models thatfaithfully recapitulate important hallmarks of human cSVD and VCID.


PI / Lead: GOULD, DOUGLAS
Project Number: 5R01EY019887-10
Project Title: Genetically testing mechanisms of ocular development and disease


Patients with ocular anterior segment dysgenesis (ASD) have a greatly elevated risk of developing severeearly onset glaucoma that is refractory to treatment. Between 50-75% of patients with ASD develop glaucomawhich occurs at significantly younger ages than in the general population leading to disproportionatelydiminished quality of life for patients and their families. We propose that intrinsic vascular defects are a primaryinsult contributing to ASD that are distinct from defects in periocular mesenchyme differentiation. We will testthis hypothesis using highly advanced ultramicroscopy innovative cell-labeling approaches state-of-the-artnanofluidics technologies and unique and complementary genetic resources. This etiological distinctionbetween migration and differentiation may have significant implications for patient stratification with respect toprognosis management of modifiable risk factors and awareness of potentially life-threatening co-morbidities all of which are important for genetic counseling family planning and patients’ psychological well being.


PI / Lead: GOULD, DOUGLAS
Project Number: 5R33NS115132-05
Project Title: Development and validation of novel models for cerebral small vessel disease and vascular cognitive impairment


Vascular cognitive impairment and dementia (VCID) is any level of cognitive alteration that is attributable tocerebrovascular pathologies. VCID is second only to Alzheimer’s disease as a cause of dementia andaccounts for ~15-30% of all dementia cases. Cerebral small vessel diseases (cSVDs) are group of pathologiesafflicting the microcirculation of the brain that collectively account for up to 20% of all strokes and is the mostcommon pathology underlying VCID. The impact of cSVD and VCID is expected to increase rapidly as thepopulation of the US and other countries ages. Importantly the pathogeneses of cSVDs are incompletelyunderstood which represents a major barrier in developing strategies for prevention and treatment. Researchdescribed in this proposal will develop and validate five novel mouse models of cSVD based on genes andmutations that are demonstrated to contribute to human disease. We have assembled an interdisciplinary teamof experts that will integrate unique genetic resources vascular pressure myography patch-clampelectrophysiology calcium imaging specialized magnetic resonance imaging modalities and learning andmemory behavior assays to develop and characterize multiple novel genetic models of cSVD using genes thatcontribute to disease in humans. Our long-term objective is to develop and employ genetic models thatfaithfully recapitulate important hallmarks of human cSVD and VCID.


PI / Lead: HAN, YING
Project Number: 1UG1EY033703-01A1
Project Title: Glaucoma Drainage Device and Endothelial Cell Density Loss Compare (DECLARE) Trial


Glaucoma drainage device (GDD) surgery has gained popularity in managing patients with medicallyuncontrolled glaucoma. However one of the major long-term complications of GDD is progressive cornealendothelial cell loss (ECL) leading to corneal decompensation which requires complex care. Although surgicaltechniques have been modified to insert the tube into the ciliary sulcus to address this issue anterior chamber(AC) tube placement remains the preferred location as there is a lack of convincing data to validate theadvantages of sulcus placement. For example (1) Direct comparison of endothelial cell loss (ECL) after sulcusversus AC tube placement is limited to three retrospective studies; (2) Studies are inconsistent regardingwhether intraocular pressure (IOP) and other surgical outcomes are similar after sulcus tube or AC tubeplacement; (3) Question remains whether a sulcus tube cause more AC microenvironment change due tohigher chance of having chronic tube-iris touch than an AC tube jeopardizing endothelial cell health IOPcontrol or other surgical outcomes of the sulcus tube. To answer these questions we propose a multi-center outcome-masked clinical trial randomizing 240subjects to sulcus tube versus AC tube placement. In this trial we will compare ECL (specific aim 1) IOPcontrol (specific aim 2) and AC microenvironment (specific aim 3) after GDD implantation with tube placementin the ciliary sulcus versus the anterior chamber. In the setting of increased use of GDDs our proposed trial toidentify better approaches to decrease its corneal complications is of substantial interest to both corneal andglaucoma specialists. Department of Ophthalmology at University of California San Francisco (UCSF) Francis I. ProctorFoundation at UCSF University of Pennsylvania (UPenn) and 4 high-volume centers for GDD implantation willjointly execute this trial. The Department of Ophthalmology at UCSF will serve as Clinical Coordinating Center.UPenn will be the Data Coordinating Center. Francis I. Proctor Foundation will be the imaging reading centerand metagenomic RNA deep sequencing analysis (MDS) center. This trial is innovative for a number of reasons including the randomization of surgery (sulcus tubeversus AC tube) and application of novel MDS analysis (examining postoperative AC microenvironment) noneof which has been prospectively studied before. It is aligned with the priorities of the NEI studying high-resolution imaging techniques such as endothelial cell imaging and anterior-segment optical coherence toguide post-operative treatment and as potential surrogate trial endpoints in future trials. This world class teamof collaborators have a proven track record for executing large NEI-funded trials in ophthalmology and are wellpositioned to answer the important questions presented in this proposal.


PI / Lead: HARRIS, SCOTT C
Project Number: 5F31EY033225-03
Project Title: Central mechanisms for integrating distinct retinal inputs during the optokinetic reflex in mice


The optokinetic reflex (OKR) is an innate visually-driven behavior that is critical to normal vision. During OKRcompensatory eye movements stabilize vision in response to slow global image motion that often results fromchanges in head position. While OKR is robust across diverse species its underlying mechanisms are unknown.This project aims to understand how distinct retinal output streams that collectively encode OKR-inducing stimuliare centrally integrated to drive eye movements. To achieve this goal a combination of physiology andbehavioral techniques will be used to measure functional readouts from three stages along the OKR pathway ofthe mouse in response to a common stimulus set: 1) The population activity of retinal ganglion cells that areresponsible for detecting and encoding OKR-inducing stimuli 2) Downstream neurons in the medial terminalnucleus a primary central hub for OKR processing and 3) The eye movements of awake animals as theyperform OKR. This novel approach to describing signal transformation along the entirety of a visual pathway willprovide a mapping of retinal activity onto behavior and offer insight into how and where information from distinctretinal output streams is centrally integrated. Broadly such findings are important for revealing general strategiesby which elements of the central nervous system integrate competing inputs from distinct sources. Furtherrevealing fundamental features of eye movement pathways will make progress towards understanding themechanisms behind a variety of common eye movement disorders including nystagmus strabismus andamblyopia. This work will be complemented by a rigorous training plan that involves tailored mentorshipcoursework presentation opportunities and career development. Along with the institutional intellectual andtraining resources available in the highly collaborative scientific environment at the University of California SanFrancisco these research and training plans will jointly facilitate the applicants development into an independentsensory neuroscientist.


PI / Lead: HORTON, JONATHAN C
Project Number: 3R01EY029703-05S1
Project Title: Horton_Admin_Supplement_EY029703


Project Summary Approximately 2% of children in the United States have strabismus a condition in which the eyes are notaligned properly. As a consequence stereovision may be lost and the deviated eye is at risk for reduced visionfrom amblyopia. There are many other potential consequences including reduced eye-hand coordinationdiminished quality of life employment discrimination social prejudice and psychological distress. The goal ofthis project is to investigate the most common form of strabismus intermittent exotropia. A translationalapproach is used that combines a prospective longitudinal observational study of patients with intermittentexotropia to define its natural history and clinical features along with analysis of data from a parallel set oflaboratory studies in nonhuman primates. Aim 1 will focus on the problem of defining the severity of intermittentexotropia. Patients wear tracking glasses that record the fixation point of each eye within the visual scene whilethey go about their normal daily activities. This technology makes it possible to measure the occurrence rateof exotropia defined as the percentage of time that an ocular deviation is present. This data will enable eyedoctors to identify which patients have a stable versus a progressive form of intermittent exotropia. The lattergroup may need surgery to re-align their eyes but there is currently no consensus regarding the criteria forrecommending surgery in part because it has not previously been possible to quantify the occurrence rate. Theproject will also stratify patients into the 3 recognized subtypes of intermittent exotropia (basic convergenceinsufficiency divergence excess) to probe the relationship between fusion loss and vergence demand. Insightsfrom that correlation could also influence surgical planning. Successful introduction of ambulatory eye trackinginto pediatric eye care would thus have a major impact on the management of intermittent exotropia. Aim 2 willexplore the contingent of retinal ganglion cells that projects to the superior colliculus a brainstem centercontrolling eye movements. Experiments have been performed in monkeys injecting a retrograde tracer intothe superior colliculus to label these cells. A different retrograde tracer has been injected in the lateral geniculatenucleus in the same monkeys. The data from these animals will be analyzed to determine the percentage ofganglion cells which is double-labeled signifying that the cells send a branching axon to both targets.


PI / Lead: HORTON, JONATHAN C
Project Number: 5R01EY029703-05
Project Title: BINOCULAR FUNCTION IN STRABISMUS


Project Summary Look me straight in the eye a parent exhorts but for some children it is impossible because one eye isdeviated. In this condition known as strabismus stereovision may be lost and the deviated eye may developamblyopia. Long term consequences include reduced eye-hand coordination diminished quality of lifeemployment discrimination social prejudice and psychological distress. The overarching goal of this project isto discover why normal binocular vision fails in some children. When children lose fusion they avoid diplopiaby suppressing portions of the visual field seen with each eye. The development of these regions calledsuppression scotomas blocks the error signal that would normally induce an adjustment in extraocular eyemuscle tone to bring the eyes back together. Ophthalmologists perform surgery to align eyes but successrates are far from satisfactory largely because the persistence of suppression robs children of the drive torecover fusion. To find better treatments it is imperative to understand the neural basis of suppression. Thisproject uses a translational approach: patients with strabismus are studied to characterize their deficits andthen these deficits are probed in nonhuman primates raised with an experimental form of strabismus thatclosely resembles the real disease. In Aim #1 studies are focused on children with exotropia an outwardsdeviation of one eye. It is usually intermittent but ophthalmologists tend to recommend surgery for fear thatan intermittent exotropia may progress to become constant resulting in permanent loss of binocular function.In a longitudinal observational cohort study patients will be outfitted with a wearable eyetracker to documentthe frequency of episodes of exotropia during the course of daily activities. The feasibility of using this deviceto track disease severity in individual patients will be investigated. Data will be collected over 5 years toelucidate the natural history of this condition. In Aim #2 a new chemogenetic technique will be used to silenceretinal ganglion cells that project to the superior colliculus. The role these cells play in generating eyemovements is unknown because until now no method has existed to selectively and reversibly block them.Exotropic monkeys will be examined to determine the impact on receptive field properties and the ability tomake alternating saccades. In Aim #3 suppression scotomas will be mapped dichoptically in monkeys withexotropia. Once their layout is established recordings will be made in the primary visual cortex to compareresponses of single cells to monocular vs. binocular stimulation. For binocular testing stimuli will be deliveredto the receptive field in one eye and to a location in the other eye that is displaced by the magnitude of theocular deviation. The hypothesis is that cells with their receptive field located in regions where perception issuppressed under binocular viewing conditions will respond more weakly during binocular stimulation thanduring monocular stimulation. These cortical recordings the first conducted in alert behaving monkeys withstrabismus may reveal the neural basis of suppression by correlating single cell firing with visual perception.


PI / Lead: KEENAN, JEREMY DAVID
Project Number: 1UG1EY033284-01A1
Project Title: Parasitic Ulcer Treatment Trial


Introduction. Acanthamoeba keratitis is a blinding corneal infection with few treatment options. Corticosteroidsare currently used by many cornea specialists for acanthamoeba keratitis but their use is controversial giventheir ability to promote acanthamoeba growth.Trial Design. This proposed study is a randomized trial assessing whether topical corticosteroids improveclinical and visual outcomes. Inclusion. 200 patients with microbiologic evidence of acanthamoeba keratitis (i.e. on culture smear or polymerase chain reaction) from 11 centers in the United States United Kingdom Brazil and India will be eligible if there is evidence of ocular inflammation after 4 weeks of anti-amoebic therapy. Pre-trial intervention. All participants will be treated with a standard of care anti-amoebic treatment with the preferred treatment being polyhexamethylene biguanide (PHMB) 0.02% eyedrops. Trial intervention. After the fourth week of anti-amoebic treatment if there is ocular inflammation (i.e. corneal episcleral or anterior chamber inflammation) then participants will be offered enrollment and randomized to either topical prednisolone sodium phosphate 1% or placebo eyedrops. Participants will continue PHMB therapy while on the study medications. Trial outcomes. The primary outcome is best corrected visual acuity with a hard contact lens assessed at months 6 and 7. Secondary outcomes include (i) time to clinical resolution (ii) time to perforation or therapeutic corneal transplantation (iii) corneal thinning (iv) eye pain and (v) quality oflife.Secondary objectives. Corneal swabs will be collected at the time of the initial diagnosis and processed withmetagenomic deep sequencing to determine if endosymbiotic bacteria living within the amoeba or theorganisms gene expression profile is predictive of severe inflammation. Conjunctival swabs will be collectedprior to randomization to determine if the host gene expression profile is predictive of severe inflammation.Impact. The trial will provide much-needed evidence using the highest-quality study design: a randomizedcontrolled trial. Moreover this will be the largest prospective cohort of acanthamoeba keratitis to date withextensive microbiological clinical and imaging data from enrollment centers on 4 continents which will allownumerous secondary analyses. The study results will be directly useful for providers who treat acanthamoebakeratitis as well as the patients currently affected by this blinding infection.


PI / Lead: KEENAN, JEREMY DAVID
Project Number: 3UG1EY028097-05S1
Project Title: Admin Supplement: Village-Integrated Eye Worker Trial II (VIEW II)


The World Health Organization estimates that 80% of blindness worldwide is avoidable. However inresource-limited settings progressive eye diseases such as glaucoma diabetic retinopathy (DR) and age-related macular degeneration (AMD) often go undiagnosed until it is too late. To address the global burden ofavoidable blindness eye care systems must determine optimal strategies for identifying people with or at riskfor visual impairment beyond opportunistic screening. Outreach programs can prevent blindness both byscreening for asymptomatic disease like age-related macular degeneration (AMD) diabetic retinopathy (DR)and glaucoma and case detection of symptomatic disease like cataract and refractive error. Eye care systemshave developed numerous community-based approaches to these identification methods including screeningusing telemedicine and case detection via cataract camps or community health worker models but few studieshave been conducted on the comparative effectiveness or cost effectiveness of these various approaches. The Village Integrated Eye Workers Trial II (VIEW II) is an ongoing cluster-randomized trial in whichcommunities in Nepal receive visual acuity screening and are subsequently randomized to receive either acommunity-based eye disease screening intervention consisting of optical coherence tomography (OCT) andintraocular pressure (IOP) assessment or to no intervention. The goal of the screening intervention is to detectcases of glaucoma diabetic retinopathy and age-related macular degenerationall of which are progressiveand cause irreversible vision loss if left untreatedand refer these cases to the local eye hospital for aconfirmatory diagnosis and follow-up management. A door-to-door census is performed four years afterstarting the screening intervention to determine the effectiveness of screening for reducing vision impairmentrelative to communities not receiving the screening intervention. Following the four-year census we willconduct ophthalmologic examinations in any eyes with incident visual impairment. This research is significant because it will provide the strongest type of evidence to guide national eyehealth programs results from a randomized controlled trial. The approach is also innovative in its use ofrecently developed portable diagnostic technology that enables mobile telemedicine-based screening on thislarge scale. Ultimately this trial will benefit blindness prevention programs worldwide in deciding whether andhow to implement eye disease screening. We maximize our chances of finding an effect by conducting thestudy in Nepal where the burden of undiagnosed eye diseases is high. If successful in Nepal future studiescould assess the generalizability of such a program to other settings such as rural communities in theindustrialized world. If successful in Nepal future studies could assess the generalizability of such a programto other settings such as rural communities in the industrialized world.


PI / Lead: KEENAN, JEREMY DAVID
Project Number: 4R33EY033690-03
Project Title: Integrating smartphone photography for trachoma smartphone visual acuity assessment and mobile autorefraction to enhance community-based public health monitoring


The World Health Organization (WHO) aims to eliminate trachoma the world’s leading infectious cause ofblindness. To determine if trachoma treatment is needed in an area the WHO recommends eye examinationson a population-based sample of children by personnel certified in trachoma grading. The WHO’s successfultrachoma elimination campaign has created a situation in which some areas have too many to trachoma casesto declare elimination but too few trachoma cases to certify trachoma graders. Adopting smartphone-basedtrachoma telemedicine addresses this issue by allowing workers with little clinical experience to performtrachoma photo-surveys for remote trachoma diagnoses. However the WHO’s current data collection platformfor trachoma surveys Tropical Data does not currently support ocular photography. We propose here tointegrate smartphone photography with Tropical Data and add functionality that will transform it into a moreuser-friendly open access suite of smartphone-based survey modules. This would enhance flexibility andfacilitate broader health surveys that can be used to better target limited health resources.This first part of the study aims to develop and refine the hardware and software necessary to integratesmartphone conjunctival photography into the Tropical Data platform and to assess the feasibility of theresulting product in remote community-based settings in Peru. First modules for photography visual acuityassessment autorefraction sample collection and user-defined questionnaires will be added to the existingTropical Data application. Second the impact of varying smartphone models and/or ambient lighting will beassessed for each module. Third the updated mobile application will be tested in a small number of villages todetermine the feasibility of testing multiple modules per child.The second part of the study validates the components of the smartphone-based system when used in threedifferent field locations in Peru (e.g. jungle mountains desert). We will examine the sensitivity and specificityof smartphone photography relative to certified human graders smartphone visual acuity relative to standardETDRS visual acuity and autorefraction relative to human refraction and hypothesize that these metrics willbe superior to pre-defined minimally acceptable criteria.The UCSF Proctor Foundation and Cayetano University in Peru will collaborate to achieve these goals. Wewill work in close cooperation with the WHO and Tropical Data who will be actively engaged in this researchthroughout development and testing. This research has great potential for wide dissemination and positiveimpact on health.


PI / Lead: KEENAN, JEREMY DAVID
Project Number: 5UG1EY023939-10
Project Title: Sanitation Water and Instruction in face-washing for trachoma II (SWIFT II)


BRIEF SUMMARYTrachoma caused by ocular chlamydia infection is the most common infectious cause of blindness worldwide.The World Health Organization recommends the SAFE strategy for trachoma elimination: Surgery for inturnedeyelashes mass Antibiotic distributions to reduce the community burden of chlamydial infection and Facialcleanliness and Environmental improvements to reduce transmission of ocular chlamydia. However littleevidence from randomized trials exists to support the F and E components of the SAFE strategy.The present study called WUHA II is a continuation of the WUHA I trial. WUHA I is an ongoing cluster-randomized trial in rural Ethiopia designed to determine the effectiveness of water sanitation and hygiene(WASH) for trachoma. 40 communities were randomized in a 1:1 ratio either to a comprehensive WASHpackage or to no intervention. The primary outcome is ocular chlamydia monitored annually for 3 years.In WUHA II we will treat all 40 WUHA communities with a single mass azithromycin distribution after the month36 visit and then continue the WASH intervention only in the 20 communities originally randomized to theWASH arm. We perform annual monitoring visits at months 48 60 72 and 84 for the primary outcome ofocular chlamydia among 0-5 year old children. Secondary outcomes include clinically active trachoma fromconjunctival photography chlamydial load from quantitative PCR serologic tests for chlamydia andnasopharyngeal macrolide resistance each assessed in children aged 0-5 years. A second aim of WUHA II isto perform a diagnostic test accuracy study of the tests already being conducted as well as several novel testsfor trachoma surveillance. The novel tests include inexpensive point-of-care nucleic acid amplification testsperformed on conjunctival swabs a lateral flow assay for chlamydia seropositivity tested on dried blood spotsand an automated algorithm to detect clinical signs of trachoma from conjunctival photographs. The primaryobjective of the second aim is to test the sensitivity and specificity of each of these trachoma surveillance tests.By comparing the combined azithromycin-WASH communities to communities receiving mass azithromycinalone we investigate the benefit of combining the A F and E components of the SAFE strategy asopposed to focusing on antibiotics alone. This is an important question given the expense of WASHinterventions and the limited resources of trachoma programs. WUHA II also provides information on noveltrachoma surveillance tests which will become more crucial as the world nears elimination.


PI / Lead: KEENAN, JEREMY DAVID
Project Number: 5UG1EY028097-05
Project Title: Village-Integrated Eye Worker trial II (VIEW II)


The World Health Organization estimates that 80% of blindness worldwide is avoidable yet cases arenot detected early enough to prevent vision loss. To address this global burden eye care systems mustdetermine optimal methods for identifying people with or at risk for visual impairment. Worldwide systemsutilize a variety of community-based approaches to identify such cases including screening for early diseasewith telemedicine and case detection of prevalent disease with cataract camps or community health workers.Each of these models requires varying levels of resources and ophthalmic skill but no studies have beenconducted on the comparative effectiveness or cost effectiveness of these different approaches. The overall objective of this project is to determine the effectiveness of community-based approachesto prevent blindness through a cluster-randomized trial. Communities in Nepal will be randomized to one of fivearms (1) a state-of-the-art mobile screening unit employing telemedicine for screening and case detection (2)a mobile case detection unit focused on cataract and refractive error only (3) a cataract camp employingophthalmic personnel (4) a volunteer community health worker (CHW) program and (5) no intervention.Intervention arms (arms 1-4) will target all adults aged 50 years residing in intervention communities forscreening and/or case detection. Those meeting referral criteria will be referred to Bharatpur Eye Hospital for aconfirmatory diagnosis and follow-up care as needed and will be closely monitored by study staff. Four yearsafter implementation a population-based census will be conducted in all study communities and eligible adultswill undergo a visual acuity assessment. To examine effectiveness and cost-effectiveness of theseapproaches we will pursue three specific aims: 1) to determine whether screening is effective for reducingvisual impairment relative to case detection (arm 1 vs 2); 2) to determine whether a CHW program increasesthe rate of cataract surgery compared to cataract camp (arm 3 vs 4); and 3) to compare the costs per line ofvisual impairment prevented between competing outreach programs (arms 1-5). The approach is innovative inits use of recently developed portable diagnostic technology that enables mobile telemedicine-basedscreening on this large scale. In addition the post-test only population-based assessment of visual acuity in alarge trial design allows for the study of a rare event like blindness and eliminates the problem of loss to follow-up that affected previous screening trials. This research is significant because it will provide the strongest typeof evidence to guide national eye health programs results from a randomized controlled trial. Ultimately thistrial will benefit blindness prevention programs worldwide in deciding how to allocate limited resources toscreening or case detection.


PI / Lead: KIM, TYSON NAM
Project Number: 5K08EY033030-03
Project Title: Elucidating the developmental and molecular mechanism of chorioretinal anastomoses in a model of type 3 neovascular age-related macular degeneration


Neovascular age-related macular degeneration (AMD) is a leading cause of vision loss and blindness in theelderly. Among the subtype of neovascular AMD type 3 neovascularization (type 3 NV) is associated with aguarded visual prognosis and is uniquely characterized by vascular proliferation in both the retina and choroid.A defining feature of advanced type 3 NV is the development of chorioretinal anastomoses (CRA) abnormaldirect connections between the retinal and choroidal circulations. Unfortunately CRA can act as hot spots forrecurrent exudation which are resilient to available therapies.The developmental mechanism of CRA remains unclear. We established a murine model for type 3 NVwherein mice harboring a mutation of type IV collagen (Col4a1) develop features of human disease includingserous retinal detachment hemorrhage atrophy and pronounced CRA. As in patients CRA in Col4a1 mutantmice form in adulthood and mature into large de novo fusions between retinal and choroidal circulationsreminiscent of high-flow arteriovenous (AV) shunts. Recently AV specification has emerged as a potentregulator of vascular patterning and dysregulation of key genes governing AV identity induces formation of AVshunts. Notably normalizing AV specification has been shown to reverse AV shunts despite their high-flowstate in certain animal models. Elucidating a role for AV specification in CRA may offer novel insights into bothdisease pathogenesis and therapeutic strategies. Our central hypothesis is that CRA in Col4a1 mutant miceare AV shunts with a conserved developmental origin from either the retina or choroid. We have recentlyenabled multiphoton imaging of the choroid directly through the sclera of pigmented eyes using optimizedmultiphoton microscopy. The objectives of this inter-disciplinary research proposal are therefore to:1) optimize intravital imaging of chorioretinal vascular dynamics with transscleral multiphoton microscopy; 2)determine the origin blood flow and arteriovenous identity of CRA in Col4a1 mutant mice; and 3) identifycandidate pathways driving CRA in the endothelium using single cell RNA sequencing.The long-term goal of this career development research project is to provide the investigator with a cross-disciplinary skillset in advanced intravital imaging and single-cell transcriptomics that will allow him toinvestigate fundamental mechanisms of oculovascular dysgenesis toward the discovery of new therapies invascular disease.


PI / Lead: LAKKARAJU, APARNA
Project Number: 1R01EY035514-01A1
Project Title: Triple threat therapeutics for inherited retinal degenerations


ABSTRACTInherited retinal dystrophies (IRDs) cause progressive irreversible vision loss and most are currently untreatable.Pathogenic variants in over 300 genes are implicated in IRDs and variants within a single gene can causediverse retinal phenotypes which makes therapeutic development challenging. As an alternative to geneticstrategies which are disease-specific we propose that gene agnostic approaches using small molecule drugshave the potential to treat multiple IRDs independent of their genetic etiology. Our studies on mouse models ofIRDs have identified cholesterol and ceramide as common pathogenic drivers of retinal pigment epithelium(RPE) dysfunction that culminates in retinal degeneration. Accumulation of these lipids facilitates complement-induced mitochondrial injury in the RPE infiltration of microglia into the sub-retinal space and eventuallyphotoreceptor loss. Here we will evaluate therapeutic efficacy of two small molecule drugs a clinically approvedbisphosphonate that inhibits cholesterol biosynthesis and ceramide generation (Aim 1) and a pan-adiponectinreceptor agonist that stimulates ceramidase activity and promotes mitochondrial biogenesis (Aim 2) in mousemodels of Stargardt disease and Batten disease. We will test the hypothesis that these drugs act as triplethreats by lowering cholesterol and ceramide and protecting RPE mitochondria thereby preventing microglialactivation and retinal degeneration. We will use cutting-edge techniques such as intravital imaging of drugdistribution super-resolution imaging of mitochondrial dynamics in the living mouse retina lipidomicstranscriptomics and noninvasive evaluation of retinal structure and function to establish the ability of these drugsto safeguard the RPE and retina in disease models. These drugs have documented safety profiles and reachthe retina in therapeutically effective concentrations after systemic administration circumventing the needfor invasive delivery. Therefore they hold immense promise as novel powerful gene-independenttherapeutics for IRDs.


PI / Lead: LAKKARAJU, APARNA
Project Number: 3R01EY030668-04S1
Project Title: Diversity Supplement Start 05/01/23


The complement system controls the balance between homeostatic and inflammatory processes. Althoughabnormal complement activity is strongly associated with macular degenerations inhibiting this pathway hasbeen unsuccessful in halting vision loss. To date therapeutic approaches have focused on complete inhibitionof extracellular complement activity with limited insight into how complement proteins modulate retinal healthand disease. This is particularly evident in approaches targeting the complement protein C3 the core effectormolecule of the complement system which plays context-dependent roles in the retina. Countering theconventional view of C3 acting solely in the extracellular space recent studies from our group have identifiedintracellular C3 activation as a novel mechanism that modulates health of the retinal pigment epithelium (RPE)a primary site of injury in macular degenerations. In stressed or diseased RPE increased uptake and proteolysisof C3 generates biologically active C3a (intracellular C3 activation). C3a in turn activates mTOR a masterregulator of cell health. Chronic mTOR activation is detrimental to cell health because it can reprogram cellularmetabolism and cell fate decisions. Based on these exciting studies we hypothesize that abnormal intracellularcomplement activation could drive disease pathology by compromising RPE homeostasis. We propose tomolecularly dissect th intracellular C3a activity inthe RPE and identify potential points of therapeutic intervention to halt this cascade. We will identify the cellularmachinery responsible for increased intracellular C3a generation in diseased RPE (Aim 1); investigate thedynamics of C3a signaling via its cognate receptor C3aR (Aim 2); and determine how persistent C3a-C3aRsignaling disrupts RPE homeostasis and retinal function (Aim 3). We will use our expertise in innovative high-speed and super-resolution live-cell imaging and mouse models of disease to gain unprecedented spatial andtemporal information about intracellular C3 activation and its consequences for retinal health. These studies willaid the development of a unified model that links multiple features of AMD including cholesterol accumulationcomplement activation metabolic deficits and RPE dedifferentiation. Identifying molecular mechanisms thatunderlie increased intracellular C3 activation will aid the design of precision therapeutics to safeguard RPE healthand retinal function over a lifetime.


PI / Lead: LAKKARAJU, APARNA
Project Number: 3R01EY030668-04S2
Project Title: Diversity Supplement Nguyen 2023-2025


Project SummaryThe complement system controls the balance between homeostatic and inflammatory processes. Althoughabnormal complement activity is strongly associated with macular degenerations inhibiting this pathway hasbeen unsuccessful in halting vision loss. To date therapeutic approaches have focused on completeinhibition of extracellular complement activity with limited insight into how complement proteins modulateretinal health and disease. This is particularly evident in approaches targeting the complement protein C3the core effector molecule of the complement system which plays context-dependent roles in the retina.Countering the conventional view of C3 acting solely in the extracellular space recent studies from ourgroup have identified intracellular C3 activation as a novel mechanism that modulates health of the retinalpigment epithelium (RPE) a primary site of injury in macular degenerations. In stressed or diseased RPEincreased uptake and proteolysis of C3 generates biologically active C3a (intracellular C3 activation). C3ain turn activates mTOR a master regulator of cell health. Chronic mTOR activation is detrimental to cellhealth because it can reprogram cellular metabolism and cell fate decisions. Based on these excitingstudies we hypothesize that abnormal intracellular complement activation could drive disease pathology bycompromising RPE homeostasis. We propose to molecularly dissect the machinery mechanisms andconsequences of dysregulated intracellular C3a activity in the RPE and identify potential points oftherapeutic intervention to halt this cascade. We will identify the cellular machinery responsible for increasedintracellular C3a generation in diseased RPE (Aim 1); investigate the dynamics of C3a signaling via itscognate receptor C3aR (Aim 2); and determine how persistent C3a-C3aR signaling disrupts RPEhomeostasis and retinal function (Aim 3). We will use our expertise in innovative high- speed and super-resolution live-cell imaging and mouse models of disease to gain unprecedented spatial and temporalinformation about intracellular C3 activation and its consequences for retinal health. These studies will aid thedevelopment of a unified model that links multiple features of AMD including cholesterol accumulationcomplement activation metabolic deficits and RPE dedifferentiation. Identifying molecular mechanisms thatunderlie increased intracellular C3 activation will aid the design of precision therapeutics to safeguard RPEhealth and retinal function over a lifetime.


PI / Lead: LAKKARAJU, APARNA
Project Number: 5R01EY030668-04
Project Title: Mechanisms of RPE dysfunction in macular degenerations: role of intracellular complement activation


The complement system controls the balance between homeostatic and inflammatory processes. Althoughabnormal complement activity is strongly associated with macular degenerations inhibiting this pathway hasbeen unsuccessful in halting vision loss. To date therapeutic approaches have focused on complete inhibitionof extracellular complement activity with limited insight into how complement proteins modulate retinal healthand disease. This is particularly evident in approaches targeting the complement protein C3 the core effectormolecule of the complement system which plays context-dependent roles in the retina. Countering theconventional view of C3 acting solely in the extracellular space recent studies from our group have identifiedintracellular C3 activation as a novel mechanism that modulates health of the retinal pigment epithelium (RPE)a primary site of injury in macular degenerations. In stressed or diseased RPE increased uptake and proteolysisof C3 generates biologically active C3a (intracellular C3 activation). C3a in turn activates mTOR a masterregulator of cell health. Chronic mTOR activation is detrimental to cell health because it can reprogram cellularmetabolism and cell fate decisions. Based on these exciting studies we hypothesize that abnormal intracellularcomplement activation could drive disease pathology by compromising RPE homeostasis. We propose tomolecularly dissect the machinery mechanisms and consequences of dysregulated intracellular C3a activity inthe RPE and identify potential points of therapeutic intervention to halt this cascade. We will identify the cellularmachinery responsible for increased intracellular C3a generation in diseased RPE (Aim 1); investigate thedynamics of C3a signaling via its cognate receptor C3aR (Aim 2); and determine how persistent C3a-C3aRsignaling disrupts RPE homeostasis and retinal function (Aim 3). We will use our expertise in innovative high-speed and super-resolution live-cell imaging and mouse models of disease to gain unprecedented spatial andtemporal information about intracellular C3 activation and its consequences for retinal health. These studies willaid the development of a unified model that links multiple features of AMD including cholesterol accumulationcomplement activation metabolic deficits and RPE dedifferentiation. Identifying molecular mechanisms thatunderlie increased intracellular C3 activation will aid the design of precision therapeutics to safeguard RPE healthand retinal function over a lifetime.


PI / Lead: LEE, JOO YEUN
Project Number: 5K99EY033858-02
Project Title: The contribution of plasticity to the recovery of retinal function following gene therapy


Gene replacement therapy for inherited retinal degenerations has improved visual function in animal modelswhich has built momentum to curing blindness in humans. Optimal therapy is the return of normal visual functionhowever current clinical trials face challenges associated with variability and durability of recovery due to thelack of rigorous mechanistic understanding of the retinal circuit’s reaction to the therapies and any potentialhinderance to full recovery. To restore vision it is essential to understand how surviving retinal neurons modifysynaptic connections upon vision restoration treatment and how retinal plasticity can be leveraged to improvevisual function. Our long-term goal is to elucidate fundamental mechanisms that enable the retina to establishfunctional connections following gene therapy. The objectives of the proposed work are to determine underlyingmechanisms of functional recovery at cellular and circuit resolution using a mouse model of achromatopsiawhich restores selective loss of cone-mediated function after gene therapy. In Aim 1 we will determine therecovery of spatial and temporal processing in ON and OFF pathways after gene therapy. We will measurespatio-temporal receptive fields of specific ganglion cell types. In Aim 2 we will determine the contribution ofsynaptic remodeling and transmitter release homeostasis to the structure and function of cone bipolar cellsfollowing gene therapy. Achieving robust and sustained therapies require understanding of how gene therapyrestores rewiring and neurotransmitter release from first- and second-order synapses. Imaging andelectrophysiology will allow us to determine the wiring patterns of outer and inner retina and how cones andbipolar cell release rates potentially adapt to changes in inputs to reach homeostasis. The approach is innovativefor a new perspective on restoring vision in the context of the retinal plasticity investigating the effects of genetherapy on connectivity patterns and functional properties at the single-cell level of the retinal circuit. The resultswill be significant for (1) revealing whether retinal plasticity is constructive toward restoring visual function (2)determining mechanisms that allow the remaining retinal neurons to re-establish functional connections withnewly rescued cones and (3) providing knowledge essential for maximizing function after photoreceptorrecovery.


PI / Lead: LIETMAN, THOMAS M
Project Number: 2R01EY025350-05A1
Project Title: Forecasting Trachoma Control


Trachoma programs have been enormously successful but they fell short of their goal of GlobalElimination of Trachoma by 2020. Nearly one third of endemic districts still had not met the threshold for control.Why? One reason may be that the 2020 goals were set long before sufficient evidence was available. In theoriginal grant period we used program surveys to forecast the distribution of active trachoma worldwide. Wealso used NEI clinical trial data to better define the relationship between clinical activity and actual chlamydialinfection. New clinical infection and serology data will now enable more precise estimates. In carrying out theaims of this current proposal we will use these enriched information sources to forecast which districts willachieve control under current efforts. A second factor is that the trachoma intervention guidelines have essentially been one size fits all. Herewe propose to assess the enhanced interventions necessary to achieve control in hotspots where current effortsare not sufficient for trachoma control. Finally the WHO plan acknowledged that resurgence may happen. In the original grant period wedemonstrated that surveys of trachoma prevalence sometimes switched from supporting control to questioningcontrol. In this current proposal we propose to model resurgence so that surveillance of resurgence can beimproved. In summary we propose: (a) real-time open-access forecasts that set realistic interim and eliminationgoals to help keep our specific collaborating stakeholder’s programs on track; (b) models for better targeting hotspots with enhanced interventions to accelerate success; (c) models for rational surveillance for resurgence tomaintain control once success has been declared.


PI / Lead: LIETMAN, THOMAS M
Project Number: 3R01EY025350-05A1S1
Project Title: Forecasting Trachoma Control - Diversity Supplement


Annual mass azithromycin distribution dramatically reduces the prevalence strains of Chlamydia trachomatisthat lead to blindness. Current World Health Organization guidelines indicate that annual mass azithromycindistribution should be continued until district-level prevalence of the clinical sign of trachoma trachomatousinflammation-follicular (TF) drops below 5%. However TF doesnt correlate well with true prevalence and thereis no gold standard to detect this. Here we propose that a Hidden Markov Model 1) will identify the trueprevalence in hypo-endemic areas that can be used as a signal that the mass azithromycin protocol wassuccessful and an indicator to stop antibiotics and 2) identify hyperendemic areas that the protocol has not beensuccessful to change the treatment regimen. We anticipate that results will provide evidence to support alteringthe modification of current interventions for trachoma.


PI / Lead: LIETMAN, THOMAS M
Project Number: 5UG1EY028518-05
Project Title: Steroids and Cross-linking for Ulcer Treatment (SCUT II)


Investigating factors that mitigate the inflammatory response to infection may have the greatest impact onclinical outcomes in bacterial keratitis. Well-designed randomized controlled trials comparing different topicalantibiotics have been unable to identify any significant difference of treatment success. Activated immune cellssecret cytokines and collagenases which lead to destruction of corneal tissue. Simultaneous treatment ofinflammation may reduce corneal astigmatism and opacity thus improving visual acuity outcomes. TheSteroids for Corneal Ulcer Trial (SCUT) was unable to identify a benefit or harm to the use of adjuvant steroidsin the treatment of bacterial ulcers overall however hypothesis-generating subgroup analysis consistentlysuggested a benefit in severe non-Nocardia ulcers treated with early steroids.Here we propose the Steroids and Cross-linking for Ulcer Treatment II (SCUT II) an internationalrandomized double-masked clinical trial designed to evaluate the benefit of adjuvant corneal cross-linking(CXL) and early steroids in the treatment of bacterial ulcers. Patients presenting to one of the Aravind EyeHospitals in India or to UCSF with smear-positive typical (i.e. non-Nocardia or Mycobacteria) bacterial cornealulcers and moderate to severe vision loss defined as Snellen visual acuity of 20/40 or worse will be eligible forinclusion. Those who agree to participate will be randomized to one of three treatment groups:Group 1: Standard Therapy Group topical 0.5% moxifloxacin plus topical placebo plus sham CXLGroup 2: Early Steroid Group topical 0.5% moxifloxacin plus topical 0.05% difluprednate plus sham CXLGroup 3: CXL Group topical 0.5% moxifloxacin plus topical 0.05% difluprednate plus CXLThis approach is innovative for a number of reasons including its testing of novel treatments such as topicaldifluprednate 0.05% and corneal cross-linking. It is also aligned with the priorities of the NEI including the studyof infectious processes as well as studying new high-resolution imaging techniques such as confocalmicroscopy anterior-segment optical coherence and Pentacam Scheimflug imaging to guide treatment ofcorneal ulceration and as potential surrogate trial endpoints in future trials. Proctor has a proven track record ofstudying the optimal treatment of corneal ulceration with large NEI-funded trials. Corneal opacity remains oneof the leading causing of blindness worldwide and we anticipate that this research will guide clinicians on thebest management of cornea infection to reduce the morbidity associated with this condition.


PI / Lead: NACHURY, MAXENCE V
Project Number: 2R01GM089933-15
Project Title: Quality Control of the cilia proteome


Primary cilia organize signaling pathways such as vision olfaction and Hedgehog signaling and dysfunctionof cilia causes a host of symptoms in disease collectively known as ciliopathies. The movements of signalingreceptors into inside and out of cilium are critical for the correct regulation of these pathways. Past work fromthe lab identified two paths for the regulated exit of signaling receptors out of cilia. One route goes back into thecell and has now been extensively characterized. Another route packages signaling receptors into extracellularvesicles (EVs) and its mechanisms remain poorly understood. The major goal of this proposal is to determine the molecular mechanisms for signaling receptor packaginginto ciliary EVs. A major goal of this proposal is the generate genetically encoded disrupters of ciliary EVshedding. These tools will enable a rigorously test of the physiological importance of ectocytosis.


PI / Lead: NACHURY, MAXENCE V
Project Number: 5R01EY031462-04
Project Title: Structural basis of BBSome-mediated ciliary exit


Primary cilia organize signaling pathways such as vision olfaction and Hedgehog signaling. Proper functioningof these pathways is critically dependent on the movements of molecules into inside and out of cilia yet ourunderstanding of the basic mechanisms governing trafficking through cilia remains fragmentary. Past workfrom the lab identified and characterized the BBSome a protein complex that ferries signaling receptors out ofcilia and clears photoreceptor outer segments of unwanted proteins. The relevance of the BBSome to humanhealth and disease is evidence by the fact that BBSome dysfunction causes Bardet-Biedl Syndrome (BBS) ahereditary disease characterized by obesity retinal degeneration polydactyly and kidney malformations.The major goal of this proposal is to determine the structure and function of the molecular cogs and leverswithin the BBSome that enable selective removal of proteins from cilia. The proposed studies will cast new lighton ciliary trafficking and lay the basis of future therapeutic interventions.


PI / Lead: NAIR, KAYARAT SAIDAS
Project Number: 5R01EY032666-02
Project Title: Characterizing the Molecular Mechanisms of PRSS56-Dependent Ocular Growth and Refractive Error


Refractive errors are a major cause of vision loss worldwide and the rising prevalence of myopia andassociated blinding conditions is a significant public health concern. Regulation of ocular axial growth is criticalfor normal refractive development to ensure that a focused image falls directly on the retina. Our goal is todecode the molecular and genetic program that governs ocular axial growth. Ocular growth is driven by anintrinsic genetic process during prenatal and postnatal development (vision-unadjusted) and by a postnatalvision-guided process emmetropization thought to interact with intrinsic ocular growth such that the eye’saxial length matches its optical power. Enhanced intrinsic ocular growth and defective emmetropization arethought to cause a mismatch between ocular axial length and optical power leading to myopia. Ocular axialgrowth relies on signals from the retina to the sclera to promote extracellular matrix remodeling and ocularelongation. However the mechanisms by which the signals translate to ocular axial growth remain elusive. Ourstudies suggest that PRSS56 a secreted serine protease is a component of the intrinsic machinery thatsupports ocular axial growth. However it is not known whether Prss56 has a direct role in emmetropization.We propose to uncover the molecular and cellular processes underlying PRSS56-dependent refractivedevelopment and associated errors and assess the role of PRSS56 in vision-guided ocular growth.Despite evidence that altered expression of PRSS56 affects ocular axial length the factors that regulate itsexpression and mediate its effect are not known. The Wnt-mediated pathway is associated with myopiapathogenesis and we have found that Prss56 responds to Wnt signaling agonists. In Aim 1 we will elucidatethe link between Wnt and Prss56 by modulating WNT activity in genetic mouse models and studying the effecton the retinal expression of PRSS56 and ocular growth (Aim 1.1). We will also determine in conditional mousemodels whether retinal pigment epitheliumlocalized Serpine3which we identified as a candidate mediatorof PRSS56-dependent growthhelps relay PRSS56-dependent signals that support ocular growth (Aim 1.2).In Aim 2 we will characterize the function of PRSS56 to guide the identification of its substrate(s) and targetedtherapies. In Aim 3 we will test the role of PRSS56 in emmetropization and PRSS56-dependent regulation ofocular axial growth by temporarily inactivating PRSS56 in conditional mutant mice and using experimentalparadigms that induce axial elongation in response to visual blur or optical defocus.The proposed studies will provide a molecular and genetic framework to understand the mechanisms of oculargrowth and guide us to potential therapeutic targets to manage myopia.


PI / Lead: NAIR, KAYARAT SAIDAS
Project Number: 5R01EY033015-02
Project Title: Determining Molecular Mechanisms of Human Glaucoma Genes


ABSTRACTGlaucoma a major cause of blindness worldwide is a significant public health concern. In the U.S. it affectsover 2.7 million people and its prevalence will rise to 7.3 million by 2050. Targeted therapies are needed toprevent glaucoma or slow its progression. A major risk factor is high intraocular pressure (IOP) typically due toimpaired aqueous humor (AqH) outflow. However the genes and pathways involved are poorly understood.We have identified GLIS1 encoding the transcription factor GLIS1 as a susceptibility gene for primary open-angle glaucoma (POAG) and showed that Glis1/ mice have pathophysiological hallmarks of glaucoma. Wealso found that Glis1 is predominantly expressed in the trabecular meshwork (TM) a key component of theocular drainage tissue regulating AqH outflow and that Glis1/ mice exhibit progressive TM degenerationleading to high IOP and glaucomatous optic neuropathyhighlighting the relevance of this model for studiesof glaucoma. Our preliminary functional genomic analysis suggested that GLIS1 interacts with GLIS3 andFOXC1 transcription factors previously implicated in elevated IOP to regulate gene expression in TM cells.Moreover reduced or increased GLIS1 activity can impair the integrity of ocular drainage tissues. Using uniquemouse models genetic and functional genomic approaches and in vitro assays we propose to characterizethe GLIS1-dependent transcriptional regulatory network and determine its role in homeostasis and dysfunctionof ocular drainage tissue. In Aim 1 we will test the hypothesis that increased GLIS1 expression contributes toPOAG-associated ocular drainage tissue defects and determine whether the POAG-associated variants weidentified in GLIS1 enhancer regions increased its transcriptional activity in primary human TM cells. We willalso test whether GLIS1 overexpression in the mouse TM leads to high IOP and ocular drainage tissue defectssimilar to those in POAG. Finally we will assess the potential role of dexamethasone and TGF2 previouslyimplicated in IOP elevation as upstream regulators of GLIS1. In Aim 2 we will test for potential geneticinteractions between Glis1 and Foxc1 and/or Glis3 in ocular drainage tissue homeostasis. We will determinewhether mice heterozygous for null alleles of both Glis1 and Foxc1 or Glis1 and Glis3 develop TM defects andaltered IOP regulation. In parallel we will characterize the transcriptional program and molecular pathwaysimplicated in TM maintenance and function. These studies will provide important mechanistic insight into oculardrainage tissue homeostasis and dysfunction and could reveal targets for therapies to manage glaucoma.


PI / Lead: OATTS, JULIUS
Project Number: 1K23EY034893-01
Project Title: Community-based amblyopia screening using a novel device


Worldwide more than 500000 children become blind each year and the majority of blind children live in low-and middle-income countries (LMICs) where rates of preventable vision loss and blindness are significantlyhigher. Amblyopia is the leading cause of vision loss in children worldwide. Vision loss from amblyopia can beprevented through early effective amblyopia screening though current screening devices have high sensitivityand low specificity which leads to a high number of false positive referrals. This ultimately limits the success ofamblyopia screening programs due to increased cost and resource utilization which is of particular concern inLMICs and low resource settings. This proposal will evaluate the efficacy and cost-effectiveness of twoamblyopia screening devices and screening strategies: a current standard device autorefractor (AR) whichonly detects amblyopia risk factors and a novel device retinal birefringence scanner (RBS) which detectsamblyopia directly. Aim 1 of this proposal will compare the performance of the AR and RBS devices fordetecting amblyopia in a clinic-based setting in Nepal. Aim 2 will compare the devices in a community-basedsetting in Nepal. Aim 3 will compare the cost-effectiveness of these two amblyopia screening strategies anddevices. The feasibility of these Aims is anchored by collaboration with the Village Integrated Eye Worker II(VIEW II) trial a cluster-randomized trial led by the candidates primary mentor Dr. Jeremy Keenan. The VIEWII trial randomizes adults in peri-urban communities in Nepal to undergo a vision screening intervention bytrained community outreach workers. This proposal incorporates several innovations. First it uniquelyleverages a well-established adult vision screening infrastructure to create and evaluate a screening programfor children. Second it will be the first study to directly compare the performance and cost of two amblyopiascreening strategies. The candidate Dr. Julius Oatts is a pediatric ophthalmologist at the University ofCalifornia San Francisco (UCSF) whose long-term goal is to become an independent investigator withexpertise in diagnostic accuracy studies for novel technologies to diagnose and monitor preventable vision lossin children nationally and internationally. To successfully complete this research Dr. Oatts will focus on fourrelevant domains of training: diagnostic accuracy studies clinical trial design cost-effectiveness analysis andepidemiology and biostatistics. His exceptional mentorship team includes his primary mentor Dr. JeremyKeenan Director of International Programs at the Proctor Foundation and PI of the VIEW II trial and co-mentors Dr. David Hunter Chief of Ophthalmology at Boston Childrens Hospital and Dr. Ying Han Director ofthe UCSF Glaucoma service. This team combined with the environment of the Proctor Foundation and UCSFDepartment of Ophthalmology will support his development into an NIH-funded independent investigator.


PI / Lead: O'BRIEN, KIERAN
Project Number: 1R01AI175250-01A1
Project Title: Rsistance Evalue contre la Vie des Enfants au Niger-Implementation et Recherche (REVENIR). Community antimicrobial resistance after azithromycin distribution: selection spillover co-selection


Azithromycin mass drug administration (MDA) to children 1-59 months old reduces child mortality and is beingconsidered for inclusion in child survival programs. However azithromycin MDA leads to emergence ofantimicrobial resistance (AMR). The World Health Organization (WHO) thus suggests azithromycin MDA belimited to children 1-11 months old to reduce the risk of AMR. Several high mortality West African countrieshave since initiated azithromycin MDA though key questions about its impact on AMR remain. In particularthe impact of long-term selection pressure is not well understood trachoma studies treating all ages foundthat AMR continues to increase with additional distributions but studies treating only children suggest thatresistance may plateau after an initial increase. Understanding AMR patterns with long-term MDA is essentialto define the duration of future programs. In addition spillover of AMR from treated to untreated groups isplausible though has yet to be demonstrated in this context. If present the risks of AMR with this interventionmay be greater than anticipated. Similarly previous studies on the impact of azithromycin MDA on co-selectionfor resistance in non-macrolide antibiotic classes have had mixed results. The emergence of co-selection withazithromycin MDA would amplify the risks of this intervention as well threatening the efficacy of other essentialantibiotics. The Bill & Melinda Gates Foundation-funded AVENIR trial is a large trial that randomizes more than3000 communities in Niger to 4 years of biannual MDA of 1) azithromycin to 1-11-month-olds with placebo to12-59-month-olds 2) azithromycin to 1-59-month-olds or 3) placebo to 1-59-month-olds. AVENIRs primaryendpoints are mortality and AMR compared across the 3 arms. The trial will also collect rectal andnasopharyngeal samples from several treated and untreated groups in 150 communities after 2 and 4 years ofdistributions. In addition AVENIR includes a subset of communities that received 5 years of azithromycin MDAin a prior study resulting in very long-term distributions between the two studies. This presents a uniqueopportunity to study key questions beyond the scope of the main trial but essential to understanding how MDAdrives community AMR. Our large sample size enables adequate power to elucidate the relationship betweenantibiotic use and population-level AMR emergence including long-term effects spillover effects to non-targetgroups and co-selection in other antibiotic classes. Moreover this project proposes metagenomic deepsequencing to characterize the respiratory and gut resistome in order to complement the proposed phenotypicAMR monitoring. We propose to leverage this trial-based infrastructure large sample bank and our labs high-throughput genomic approaches to provide evidence to directly impact mass azithromycin programs and theWHO guidelines on this intervention.


PI / Lead: OLDENBURG, CATHERINE ELIZABETH
Project Number: 1R01HD111532-01A1
Project Title: Azithromycin as adjunctive treatment for uncomplicated severe acute malnutrition: the AMOUR trial


Amoxicillin is recommended by the World Health Organization (WHO) as adjunctive therapy for the treatmentof uncomplicated severe acute malnutrition (SAM). Because children with uncomplicated SAM may haveasymptomatic infection due to immune suppression presumptive treatment with a broad-spectrum antibioticmay be beneficial by clearing any existing infection and improving outcomes. Two randomized placebo-controlled randomized trials have evaluated amoxicillin for uncomplicated SAM and have found conflictingresults. These results may indicate either that antibiotics are not helpful for the management of uncomplicatedSAM or that a better antibiotic is needed. Recently we demonstrated that biannual mass azithromycindistribution as a single oral dose reduces all-cause child mortality in sub-Saharan Africa. Children withuncomplicated SAM who have an elevated risk of mortality relative to their well-nourished peers mayparticularly benefit from presumptive azithromycin treatment. Our pilot data demonstrated feasibility in rapidenrollment of children with uncomplicated SAM in our study area and showed no significant differencebetween azithromycin and amoxicillin demonstrating equipoise for a full-scale trial. Here we propose anindividually randomized trial in which children will be randomized to a) azithromycin b) amoxicillin or c)placebo and evaluated for differences in weight gain nutritional recovery and the gut microbiome. The resultsof this study will strengthen the evidence base for policy related to the use of antibiotics as part of themanagement of uncomplicated SAM including additional evidence of amoxicillin versus placebo as well asevaluation of an antibiotic class that has not been considered for uncomplicated SAM which may lead tochanges in guidelines for treatment.


PI / Lead: OLDENBURG, CATHERINE ELIZABETH
Project Number: 3UG1EY030833-04S1
Project Title: Azithromycin Reduction to Reach Elimination of Trachoma


Annual mass azithromycin distribution dramatically reduces the prevalence of the ocular strains of Chlamydiatrachomatis that lead to blinding trachoma. Current World Health Organization guidelines indicate that annualmass azithromycin distribution should be continued until district-level prevalence of the clinical sign oftrachoma trachomatous inflammation-follicular (TF) drops below 5%. However TF does not correlate wellwith infection after multiple rounds of azithromycin treatment. Specifically any decrease in TF lags well behindthe decrease in infection. Thus the TF threshold may lead to overuse of antibiotics and depletion of scarceresources. Here we propose a community randomized controlled trial and diagnostic test study to evaluatewhether 1) azithromycin distribution can be discontinued in communities with TF prevalence up to 20% and 2)alternative indicators of trachoma transmission can better measure true infection. We anticipate that results willprovide evidence to support discontinuation of azithromycin treatment earlier and evidence of that true localelimination of infection can be achieved altering the goal of the trachoma program from control to eradication.


PI / Lead: OLDENBURG, CATHERINE ELIZABETH
Project Number: 5UG1EY028088-05
Project Title: Kebele Elimination of Trachoma for Ocular Health


7The World Health Organization (WHO) Trachoma Program aims to eliminate trachoma as a public healthconcern by 2020strictly speaking this would be a control program as infection is not being eliminated. TheWHO recommends three to five annual mass azithromycin distributions to districts with measured prevalenceof follicular trachoma (TF) above 10% before re-assessment. However despite as much as a decade ofannual mass azithromycin distribution TF prevalence in some districts of Amhara Ethiopia remains over 25%.Elimination of infection defined as reduction to zero in the incidence of infection in a specific definedgeographic area is feasible and may actually be more sustainable than control as it may be less likely forinfection to return in a community that has achieved zero new infections. Here we propose to test twoalternative antibiotic-based strategies for intensive targeting of core groups to achieve elimination at the kebele(administrative unit consisting of ~15 villages or development teams) level. We propose a three-arm clusterrandomized trial to test whether quarterly treatment of (1) all children aged 0-9 in the kebele plus annual massazithromycin distribution or (2) all children aged 0-9 in the kebele with ocular C. trachomatis infection by PCRplus annual mass azithromycin distribution results in lower prevalence of ocular chlamydia and elimination inthe kebele compared to (3) WHO-recommended annual mass azithromycin distribution alone. The results ofthis trial are expected to lead to evidence of alternative antibiotic-based strategies that may more reliably leadto complete elimination in geographic areas larger than a village.


PI / Lead: OLDENBURG, CATHERINE ELIZABETH
Project Number: 5UG1EY030833-04
Project Title: Azithromycin Reduction to Reach Elimination of Trachoma


Annual mass azithromycin distribution dramatically reduces the prevalence of the ocular strains of Chlamydiatrachomatis that lead to blinding trachoma. Current World Health Organization guidelines indicate that annualmass azithromycin distribution should be continued until district-level prevalence of the clinical sign oftrachoma trachomatous inflammation-follicular (TF) drops below 5%. However TF does not correlate wellwith infection after multiple rounds of azithromycin treatment. Specifically any decrease in TF lags well behindthe decrease in infection. Thus the TF threshold may lead to overuse of antibiotics and depletion of scarceresources. Here we propose a community randomized controlled trial and diagnostic test study to evaluatewhether 1) azithromycin distribution can be discontinued in communities with TF prevalence up to 20% and 2)alternative indicators of trachoma transmission can better measure true infection. We anticipate that results willprovide evidence to support discontinuation of azithromycin treatment earlier and evidence of that true localelimination of infection can be achieved altering the goal of the trachoma program from control to eradication.


PI / Lead: OU, YVONNE
Project Number: 1R01EY034973-01
Project Title: Retinal circuit disassembly in primate glaucoma


/ ABSTRACT During development specific synaptic partners connect in order to ensure proper neural circuit functionbut how these connections are disassembled during neurodegeneration is less well understood. In rodentexperimental glaucoma (EG) models synapse loss occurs early preceding retinal ganglion cell (RGC) dendriteretraction and cell death. Converging evidence in rodents suggests that specific RGC types are more susceptibleto elevated intraocular pressure but little is known about retinal circuit disassembly in glaucomatous primateretina. Indeed significant differences in mice which lack a lamina cribrosa and macula and have dissimilar RGCtypes limit the translation and generalizability of findings to humans. It is critically important to address thisknowledge gap in order to advance successful development of clinically relevant diagnostics and novel treatmentapproaches such as neuroprotection gene therapy and cell-based vision restoration strategies. Here weassemble a highly collaborative team of investigators with complementary expertise well-matched to our goalof systematically determining the connectivity function and transcriptomes of RGCs undergoing circuitand synapse disassembly in glaucomatous primate retina. Our approach builds on a well-established rhesusmacaque non-human primate (NHP) model of experimental glaucoma that closely recapitulates structural andfunctional changes observed in human glaucoma and permits detailed and precise staging of disease. Basedon our studies in mice and preliminary data in NHP we hypothesize that specific microcircuits in the injuredadult NHP retina may exhibit susceptibility in connectivity and function which is reflected in differentialgene expression. To test this hypothesis we apply rigorous quantitative electrophysiological anatomical andmolecular assessments focusing on the four main RGC types in NHP retina: ON and OFF midget and parasolganglion cells. Aim 1 will use high-density multielectrode arrays single cell recordings and Patch-seq to identifythe functional RGC types that are vulnerable in NHP EG and probe their transcriptomes to reveal mechanisticinsights and novel therapeutic targets. Aim 2 will determine the specificity and patterns of circuit and synapsedisassembly in NHP EG from both lamina-specific and cell type-specific perspectives using detailed circuit andsynapse mapping. The proposal is innovative because it brings together multi-modal function morphologic andmolecular analyses and is significant because it focuses on the four main RGC types in primate that account forthe majority of human vision and are affected in glaucoma. We will generate significant resources for the scientificcommunity and reveal insights into retinal circuit disassembly and the potential for circuit repair in a highlyclinically relevant model of glaucoma.


PI / Lead: OU, YVONNE
Project Number: 3R01EY034973-01S1
Project Title: Retinal circuit disassembly in primate glaucoma - Diversity Supplement


/ ABSTRACT During development specific synaptic partners connect in order to ensure proper neural circuit functionbut how these connections are disassembled during neurodegeneration is less well understood. In rodentexperimental glaucoma (EG) models synapse loss occurs early preceding retinal ganglion cell (RGC) dendriteretraction and cell death. Converging evidence in rodents suggests that specific RGC types are more susceptibleto elevated intraocular pressure but little is known about retinal circuit disassembly in glaucomatous primateretina. Indeed significant differences in mice which lack a lamina cribrosa and macula and have dissimilar RGCtypes limit the translation and generalizability of findings to humans. It is critically important to address thisknowledge gap in order to advance successful development of clinically relevant diagnostics and novel treatmentapproaches such as neuroprotection gene therapy and cell-based vision restoration strategies. Here weassemble a highly collaborative team of investigators with complementary expertise well-matched to our goalof systematically determining the connectivity function and transcriptomes of RGCs undergoing circuitand synapse disassembly in glaucomatous primate retina. Our approach builds on a well-established rhesusmacaque non-human primate (NHP) model of experimental glaucoma that closely recapitulates structural andfunctional changes observed in human glaucoma and permits detailed and precise staging of disease. Basedon our studies in mice and preliminary data in NHP we hypothesize that specific microcircuits in the injuredadult NHP retina may exhibit susceptibility in connectivity and function which is reflected in differentialgene expression. To test this hypothesis we apply rigorous quantitative electrophysiological anatomical andmolecular assessments focusing on the four main RGC types in NHP retina: ON and OFF midget and parasolganglion cells. Aim 1 will use high-density multielectrode arrays single cell recordings and Patch-seq to identifythe functional RGC types that are vulnerable in NHP EG and probe their transcriptomes to reveal mechanisticinsights and novel therapeutic targets. Aim 2 will determine the specificity and patterns of circuit and synapsedisassembly in NHP EG from both lamina-specific and cell type-specific perspectives using detailed circuit andsynapse mapping. The proposal is innovative because it brings together multi-modal function morphologic andmolecular analyses and is significant because it focuses on the four main RGC types in primate that account forthe majority of human vision and are affected in glaucoma. We will generate significant resources for the scientificcommunity and reveal insights into retinal circuit disassembly and the potential for circuit repair in a highlyclinically relevant model of glaucoma.


PI / Lead: OU, YVONNE
Project Number: 5K12EY031372-04
Project Title: UCSF-Proctor Clinician Vision Scholars K12 Program


We propose the establishment of a UCSF-Proctor Clinician Vision Scholars K12 program for the mentoring andtraining of talented and diverse faculty Scholars in high quality reproducible scientific research and thedevelopment of successful academic careers in ophthalmology and vision sciences. UCSF is one of the worldsleading health sciences institutions and this program will build on existing strengths in clinical and translationalsciences bioengineering and career development. The objectives of the program are to 1) mentor and trainexceptional and diverse junior faculty in developing methodological expertise and impactful research programs;and 2) transition the K12 Scholars to become independent investigators and successful academic careers. Twoclinician-scientists with complementary scientific expertise and leadership skills will co-direct the program. Anaccomplished group of Mentors with broad expertise and strong records of mentorship and collaboration willprovide the Scholars with structured mentorship across three Tracks. Track 1 is focused onepidemiology/randomized clinical trials/global health Track 2 on basic and translational discovery sciencespecifically around the themes of ocular genetics & therapeutics visual system injury plasticity & regenerationand neurodegeneration and Track 3 on bioengineering and innovation investigations. Key activities in thetraining plan include: 1) high-quality mentored research; 2) a customized didactic curriculum tailored to the Track;3) career development and academic success skills; 4) detailed plan for transition to independence; and 5)training in the responsible conduct of research. The career development and transition to independence activitiesemphasize oral and written communication grant writing leadership networking responsible conduct ofresearch and scientific management skills. An Advisory Committee will work with the program directors to selectK12 Scholars monitor their progress and identify opportunities for program quality improvement. An ExternalAdvisory Board will also provide input on program evaluation and strategic planning. The planned duration ofappointments is three years and the projected number of junior faculty Scholars is two training at any given time.The intended outcomes for Scholars are engagement in rigorous reproducible research the development offunded independent ophthalmology and vision science research programs with a lasting impact on the field anda focus on improving human health and alleviating blindness and the advancement of successful academiccareers as leaders and mentors in the field.


PI / Lead: OU, YVONNE
Project Number: 5R01EY028148-05
Project Title: Neuronal plasticity in glaucoma


Precise connections between synaptic partners are shaped during development to ensure proper neural circuitfunction but how these connections are disassembled and rearranged after injury is less well understood.Glaucoma provides an excellent model to explore circuit plasticity when a postsynaptic neuron is injured.Furthermore the ability to diagnose and treat glaucoma at an optimal stage before irreversible retinal ganglioncell (RGC) loss occurs requires a comprehensive understanding of inner retina circuit disassembly andplasticity. However major gaps exist in knowledge about how RGCs are disconnected from and potentiallyrewired with their excitatory presynaptic partners bipolar cells (BCs) how this remodeling affects RGCfunction and the potential mechanistic role of microglia in circuit disassembly. Such knowledge is required tosuccessfully develop optic nerve regeneration strategies that depend on functional circuit rewiring. The overallobjective of this application is to determine the connectivity function and potential mechanisms of circuitdisassembly and remodeling following intraocular pressure (IOP) elevation. The central hypothesis is thatspecific microcircuits in the injured adult retina may exhibit plasticity in terms of connectivity and function withmicroglia playing an important role in synapse pruning. The hypothesis will be tested in the following specificaims: 1) Determine the specificity and timing of anatomic circuit rewiring in diseased adult retina; 2) Determineif diseased adult retina has the capacity for functional plasticity; 3) Identify the contributions of microglia in themechanism of circuit disassembly. The approach includes biolistic transfection of individual RGCs sub-micronimaging electrophysiological measurements and novel genetic tools to study bipolar-ganglion cellconnectivity RGC function and microglia in experimental rodent glaucoma. The proposal is innovativebecause it examines the potential for cell-type specific rewiring circuit-specific synapse pruning and functionalplasticity concepts that shift the paradigm in understanding RGC degeneration in glaucoma. The proposedresearch is significant because the resulting identification of both vulnerable and resilient retinal microcircuitsto target will open new research horizons particularly in novel psychophysics testing paradigms drugdevelopment and RGC regeneration or neuroprotection strategies. Finally these experiments willfundamentally expand knowledge of how adult neural circuits react and rearrange in the face of injury.


PI / Lead: PASRICHA, NEEL DAVE
Project Number: 5K08EY033859-02
Project Title: Human Ocular Surface Electrophysiology


This is a 5-year mentored K08 award to facilitate the transition of Dr. Neel Pasricha an ophthalmologist withcornea subspecialty training to an independent investigator in ocular surface eye translational research. Astrong group of experienced mentors at UCSF scientific advisors and didactic work will assist Dr. Pasrichasresearch skills transfer including training in ocular surface electrophysiology human corneal epithelial cellculture models high-throughput screening for small-molecule drug discovery experimental mouse modelsand human subject clinical studies.The research is focused on dry eye disease (DED) a major unmet need in ophthalmology characterized byimpaired tear film homeostasis accompanied by ocular symptoms. There are currently just four FDA-approvedtherapies for DED each targeting only the inflammatory pathway and having limited efficacy. The ocularsurface comprised of the cornea and conjunctiva is lined by stratified epithelial cells expressing ion transportproteins that facilitate active fluid secretion or absorption to regulate tear fluid volume and osmolarity. The goalof the proposed research is to discover and advance drug candidates to promote tear fluid secretionby epithelial cells lining the ocular surface.This research utilizes a novel ocular surface potential difference (OSPD) method introduced in animal studiesand advanced for use in humans during my residency at UCSF. OSPD measures the electrical potentialdifference generated across epithelia from apical and basal membrane ion transporters. In Aim 1 in vivoOSPD measurements in mice will investigate the role of ion transporters in ocular surface fluid transport withparticular focus on chloride and potassium channels. Aim 2 will use ex vivo high-throughput screening inprimary human corneal epithelial cell cultures and in vivo experimental mouse studies to advance potentialdrug candidates that target calcium-activated chloride channels to increase tear fluid secretion for treatment ofDED. Aim 3 will use in vivo human OSPD measurements in healthy adults to test a pro-secretory drugcandidate in phase 2 clinical trial that activates the CFTR chloride channel. The long-term careerdevelopment goal is to build a robust cross-disciplinary research program that advances thefundamental understanding of ocular surface ion transport and translates that knowledge into noveldiagnostic and therapeutic strategies for ocular surface diseases including DED.


PI / Lead: PORCO, TRAVIS CHRISTIAN
Project Number: 5R01GM130900-04
Project Title: Ebola modeling: behavior asymptomatic infection and contacts


Project Summary! The impact of unrecognized Ebola virus (EBOV) infection (asymptomatic and symptomatic) ontransmission dynamics during the 20132016 West Africa Ebola outbreak is poorly understood. Individualswho had asymptomatic EBOV infection or unrecognized symptomatic Ebola virus disease (EVD) representtwo groups who may have had different levels of exposure and rates of EBOV transmission. Increasinglyprotective behaviors to avoid contact with EVD cases may have resulted in lower levels of exposure andthese exposures may be associated with asymptomatic EBOV infection. On the other hand individuals whohad symptomatic EVD but were never diagnosed may be disproportionately important to transmissiondynamics because some of these individuals were part of transmission chains leading to Ebola outbreaks inpreviously unaffected communities. Our research question focuses on understanding the drivers of EBOV transmission leading toepidemic decline. Competing hypotheses were centered around issues of preventive behaviors health-seeking behaviors saturation of transmission among contacts and asymptomatic EBOV infection. Newlyavailable detailed serologic social network behavioral ethnographic and vaccination data from researchcollaborations in Liberia Sierra Leone and Democratic Republic of Congo will allow us to test competinghypotheses in the following aims: 1) Dynamical effects of unrecognized EBOV infection in social networkstructure 2) Unrecognized symptomatic EVD cases barriers to care and preventive behaviors and 3)Causes of asymptomatic EBOV infection. These findings have the potential to quantify what ended the Ebolapandemic and improve mathematical models. Mathematical modeling applications will improve forecastingduring new outbreaks and inform ways to deliver vaccines to contacts by ring vaccination or novel socialnetwork algorithms. As Ebola outbreaks continue to occur two in 2018 this R01 proposal will provide lessons learnedthat are immediately applicable to future outbreaks of EBOV other viral hemorrhagic fevers and emerginginfectious diseases.!


PI / Lead: SHANTHA, JESSICA
Project Number: 5K23EY030158-05
Project Title: Ophthalmic Disease and Laboratory Surveillance in a Viral Hemorrhagic Fever Zone


Project SummaryEmerging infectious diseases can lead to devastating epidemics and pose a threat to global health security. The WorldHealth Organization has identified pathogens that are likely to cause severe and imminent outbreaks which includeEbolavirus (EBOV) and Lassa fever virus all of which require urgent attention in research. The West African Ebola virusdisease (EVD) outbreak of 2013-2016 is the most devasting EBOV outbreak in history with over 28600 infected and11300 deaths. To date there continue to be isolated outbreaks with significant morbidity and mortality. There areapproximately 17000 EVD survivors who are at-risk for Post-Ebolavirus Disease Syndrome a recently described entitywhich includes systemic conditions ocular disease and viral persistence in immune privileged sites (aqueous humorsemen and central nervous system). Viral persistence is of public health importance given its initiation of EBOVtransmission events and its association with end-organ damage.Uveitis a potentially blinding inflammatory eye disease is the most common ocular complication occurring inapproximately 13-34% of EVD survivors in West Africa. We have described vision loss due to ocular complications inretrospective cohorts in both Sierra Leone and Liberia. Specifically within Liberian EVD survivors with uveitisdeveloped vision impairment was noted in 60% and blindness (vision worse than 20/400) in approximately 40% of EVDsurvivors. Currently we have insufficient knowledge about the disease course of EVD-associated uveitis long-termvisual acuity outcomes and appropriate treatment for this sight-threatening disease. Besides our recent studies related toclinical uveitis in EVD survivors our recent investigation of EBOV persistence raises questions about the potential for theeye to serve as a novel tissue site for infectious disease surveillance particularly in a viral hemorrhagic fever zoneendemic for other emerging pathogens of public health significance.The objectives of this study are to 1. Determine the prevalence of uveitis incident vision loss over time and risk factorsfor uveitis development in EVD survivors 2. Explore ophthalmic manifestation in Lassa fever virus survivors 3. Validateocular fluid sampling as method for detection for surveillance of infectious diseases in collaboration with the Child Healthand Mortality Prevention Surveillance study.The career development aims for this K-23 proposal are to 1. Obtain methodological skills in disease surveillance systems2. Gain competency in molecular diagnostics of ocular fluid in the diagnosis of infectious pathogens 3. Continue todevelop skills in clinical research in international settings 4. Further my training in statistical and epidemiology methodsnecessary for success in clinical studies. To accomplish these goals I have designed a training plan that includescoursework mentorship with international leaders attendance and presentation at local and national researchmeetings/conferences and directed hands-on training. This K23 mentored career development award will be thefoundation for the skills necessary to become a successful independent clinician scientist.


PI / Lead: SMITH, ALEXANDER JAMES
Project Number: 1R21AG078873-01
Project Title: Measuring impairment of extracellular solute transport in Alzheimer's disease


/abstractThe extracellular space (ECS) of the brain consists of a highly interconnected network of narrow tunnel andsheet-like structures with width of 10-80 nm. Perivascular spaces surrounding blood vessels connect with theECS and act as conduits for transport of solutes throughout the parenchyma and to the CSF at the surface ofthe brain. Nutrient delivery to tissues clearance of toxic protein aggregates and therapeutic delivery to targetsrequire transport through the ECS and considerable uncertainty exists regarding the rates and mechanisms oftransport in vivo. Specifically neither the rate direction or regulation of solute transport in the perivascularspaces are well understood. Recently we demonstrated that multiphoton fluorescence recovery afterphotobleaching (MP-FRAP) of exogenous fluorophores introduced into the ECS can be used to directlymeasure solute transport rates deep within the brain parenchyma in vivo. Experiments to be performed herewill adapt this technique to measure the rates and direction of perivascular solute transport in mouse brain andthereby resolve basic questions regarding the physiology of these spaces. In Alzheimers disease (AD)amyloid plaques form in the parenchyma and around blood vessels causing extensive changes in structureand function of the surrounding glia. The consequences of these changes for transport of solutes in theperivascular space remain largely unknown and we will utilize AD model mice to determine if deposition of Aaround vessels impairs perivascular solute movement. It is hoped that these experiments will lead to aquantitative and well substantiated understanding of perivascular extracellular transport in the brain andsuggest approaches for enhancing endogenous clearance mechanisms and improving therapeutic delivery.


PI / Lead: SUN, CATHERINE QING
Project Number: 5K23EY032637-03
Project Title: Real-world outcomes of proliferative diabetic retinopathy


:Real-world outcomes of proliferative diabetic retinopathyVision loss from diabetic retinopathy remains the leading cause of preventable blindness in working-agedadults in the United States (US). Advanced diabetic retinopathy is referred to as proliferative diabeticretinopathy (PDR). In many patients blindness associated with PDR can be prevented with appropriate andtimely diagnosis and treatment. Unfortunately some patients at high risk for PDR are not receiving adequateeye care. More knowledge is needed about PDR outcomes in a real-world setting and the differences betweenpublished study outcomes and real-world effectiveness. Electronic health records (EHRs) are used in nearly90% of outpatient physician offices and can be a powerful tool for studying PDR in a real-world setting. Thegoal of this proposal is to develop and validate EHR-based methods to improve outcomes in PDR. The studyaims are: (1) to classify patients with PDR in the EHR system using an automated method that incorporatesstructured (e.g. diagnosis code medications labs) and unstructured data (e.g. clinical notes) (2) to predictthe progression of non-proliferative diabetic retinopathy to PDR using a forecasting model with time-varyingcovariates and (3) to determine the real-world effectiveness of treatments for PDR in a large nationwide eyedataset. The study will utilize data from the University of California San Franciscos (UCSF) De-IdentifiedClinical Data Warehouse a de-identified EHR with over 1 million patients that has available eye examinformation and the Intelligent Research in Sight (IRIS) registry a nationwide comprehensive eye databasethat includes data from over 15000 eye providers in the US with over 1 million patients with PDR. Theinnovative methods and tools from this study can be applied to other eye conditions to facilitate future EHR-based clinical studies in ophthalmology. The candidate Dr. Catherine Sun is an ophthalmologist whose long-term goal is to study real-world clinical outcomes in ophthalmology by conducting EHR-based pragmaticclinical trials and using large-scale EHR data. While she possesses the foundational skills additional mentoredtraining and coursework in data analytics biomedical informatics biostatistics and advanced clinical trialdesign and implementation will help her reach her goals. Her outstanding mentorship team of primary mentorDr. Nisha Acharya and co-mentors Dr. Travis Porco and Dr. Joshua Stein and the exceptional environment ofthe Department of Ophthalmology and the F.I. Proctor Foundation at UCSF will support Dr. Suns developmentinto an R01-funded independent investigator.


PI / Lead: TSAI, NICOLE Y
Project Number: 5F30EY033201-03
Project Title: Investigating the Role of Nephronectin in Establishing Retinotectal Circuit Specificity


PROJECT ABSTRACTAn impressive feature of the nervous system is the specificity of its synaptic connections. Within the brain asingle neuron is able to correctly identify and synapse onto its proper signaling partner over billions of otherneurons. How this degree of specificity is achieved is the subject of intense research. While great progress hasbeen made in identifying molecules that participate in long-range axon guidance and topographic positioninglittle is known in what molecules guide axons into their proper lamina once they reach their destined target region.Through screening for extracellular matrix proteins differentially expressed by wide-field neurons in theretinorecipient stratum griseum superficiale (SGS) of the superior colliculus (SC) I identified nephronectin (Npnt)as a candidate for mediating synaptic specificity in retinotectal circuits. Npnt belongs to the EGF-like superfamilyof extracellular matrix glycoproteins and has been biochemically well-characterized to bind strongly andspecifically to integrin 81 (Itg81).My preliminary data show that 1) Npnt is exclusively expressed by wide-field neurons of the SC whose cellbodies define a lamina within the lower SGS; 2) Npnt deposits on the somata and proximal dendrites of wide-field neurons 3) loss of Npnt in the SGS results in ectopic RGC axons within the upper SGS and 4) alpharetinal ganglion cells (RGCs) express Itg81during developmentconsistent with Itg81 acting as thereceptor for Npnt on RGCs. Taken altogether I hypothesize that Npnt instructs RGC axons to laminate withinthe lower SGS through binding Itg81 on RGCs thereby conferring synaptic specificity of RGCs onto wide-field neurons. This proposal will address how RGCs form precise connections to their neuronal partners withinthe SC by studying how Npnt regulates axon laminar targeting and synapse formation. In Aim 1 I will determinehow Npnt mediates axon laminar targeting of RGCs by focusing on its interactions with Itg81. In Aim 2 I willdetermine how Npnt affects RGC synaptic partner choice within the SC. Completion of the proposedexperiments will reveal unique molecular and cellular mechanisms regarding the role of Npnt in specifyingneuronal pairing from RGCs to wide-field neurons and will shed light on the function of Npnt in the mammaliannervous system.


PI / Lead: ULLIAN, ERIK M
Project Number: 5P30EY002162-45
Project Title: P30 - Core Grant for Vision Research


The broad objective of this NEI Center Core Grant for Vision Research application is to facilitate studyof the structure development and function of the visual system in health and in blinding diseases with the aimof preventing mitigating or curing such diseases or the restoration of lost vision through the application of themost sophisticated available techniques. Four resource and service Cores will help achieve the broadobjective as follows: I. Image Analysis and Graphics Core: Morphometric analysis; computer-aided image analysis;production of graphics for data analysis presentation and publication II. Morphology Core: Histology (paraffin and plastic embedding sectioning and staining frozensectioning for immunohistochemistry) ocular imaging (slit lamp examination and photography Micron IIIfundus photography (with fluorescein angiography) and optical coherence tomography (OCT) biometricBioptigen OCT) and visual functional testing (ERG OptoMotry and IOP) and in vivo rodent eye injection withelectroporator set up. Microscopy (light microscopy – brightfield darkfield phase contrast DIC andfluorescence) and photomicrography and advanced microscopy using a combination of spinning disk andconfocal microscopes). III. Computer/IT Core: Programming for custom research needs; assistance in computer andinformation technology hardware and software selection installation instruction in use maintenance and minorrepairs. IV. Rapid-Prototyping and Design Core: Design of new unique research equipment plus educationand training of users empowering them to participate in the manufacturing construction maintenance andupgrading of their unique research equipment designs. This is an application of a NEI Center Core Grant for Vision Research competing renewal submitted bythe Principal Investigator and 19 other vision scientists who hold 23 active NEI R01 research grants. Inaddition the UCSF vision research community supported by the NEI Vision Core Grant comprises 14 NEI-supported scientists with grant mechanisms other than active R01 4 with other NIH funding 1 with FDA R01funding and 5 with private funding. There are 41 Core Investigators with active research programs overalleach using at least one Core at a moderate or extensive level. Using traditional and innovative approachesthis Core Vision Research Grant has been highly successful and instrumental in enhancing the productivityand impact of vision research attracting scientists to vision research and facilitating collaborative studies onthe visual system at UCSF.


PI / Lead: ULLIAN, ERIK M
Project Number: 5R01EY032197-04
Project Title: Mechanistic analysis and allellic genome editing of iPSC-derived dominant LCA model


Leber congenital amaurosis (LCA) is a group of devastating early-onset retinal dystrophies affectingroughly 1/50000 to 1/33000 newborns. LCA-associated variants in the CRX gene result in a severe autosomaldominant form of the disease for which no effective treatments are currently available. Importantly both mouseand human studies suggest that haploinsufficiency is not responsible for disease manifestation in dominant CRX-associated LCA and one copy of wildtype CRX is enough to allow for mostly normal photoreceptor maturationand function. Despite substantial progress being made in the field there is a critical need to uncoverpathophysiology and establish reliable treatment options for CRX-associated LCA. The overall goal of thisproposal is to bring together two major unsolved problems in vision research: (1) the ability to accuratelyrecapitulate dominant LCA in a scalable in vitro model system to study variant-specific disease mechanismsand (2) the ability to efficiently and specifically eliminate dominant disease alleles leaving healthy alleles torestore photoreceptor cell function. In Aim 1 we will develop and characterize iPSC-based disease models from two different dominantvariants of CRX. We will validate disease phenotypes using retinal organoids. In Aim 2 variant-specific diseasemechanisms responsible for the onset of LCA will be examined by generating a retinal organoid model systemfrom patient-derived induced pluripotent stem cells. In Aim 3 mutant CRX alleles will be inactivated with CRISPRtools within the human retinal organoid model to study rescue of disease phenotypes. Completion of this aim willprovide the field with a proof-of-concept study for the development of patient-specific CRISPR-based therapeuticstrategies. Taken together the proposed studies will contribute to our basic understanding of the pathophysiologicalmechanisms underlying photoreceptor dysfunction in dominant CRX-associated LCA and will enable thedevelopment of targeted gene therapies to treat affected individuals.



 
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