Vittorio Porciatti - US grants

DESCRIPTION (provided by applicant): Glaucoma is characterized by progressive death of retinal ganglion cells (RGCs) resulting in blindness. The long-term implication of this research is prevention of RGC dysfunction and loss in patients with glaucoma. Our objective is to define the natural history of RGC dysfunction and death in an inbred strain mouse model of glaucoma (DBA/2J) with spontaneously elevated intraocular pressure (lOP). We will use non-invasive and improved methodologies such as Pattern Electroretinogram (PERG), Optical Coherence Tomography (OCT) and non-contact tonometry, as well as an unique non-glaucomatous mouse with DBA genetic background. The central hypothesis is that there is a substantial population of dysfunctional RGCs that can be detected by state-of-the-art structural-functional comparison in a longitudinal evaluation.The rationale is that the characterization of RGC dysfunction and death in a readily available mouse model of glaucoma by means of methods adapted from human clinical examination will provide a powerful experimental system for treating and even reversing the condition in humans. We will focus on two specific aims: 1) To improve methodologies for non-invasive quantification of RGC function/number and lOP, and 2) To monitor the onset and progression of retinopathy in individual animals. The proposed research is innovative, because it is based on non-invasive and sequential monitoring of key variables in glaucoma and unique strains of mice. We are particularly prepared to undertake this study because our research team combines specific expertise in visual electrophysiology, retinal imaging, mouse glaucoma models, and biophysics. Our expectation is that we will be able to determine whether RGC dysfunction precedes RGC death, determine functional and anatomical endpoints for onset and progression, and determine the visual capabilities of surviving RGCs. Such outcomes are significant, since exploiting an animal model for testing neuroprotective strategies that preserve visual function is an important component of future research on treatments for glaucoma.

[unreadable] DESCRIPTION (provided by applicant): Loss of sight in glaucomatous optic neuropathy is due to the death of retinal ganglion cells (RGC). Our long-term goal is to test the hypothesis that dysfunction of RGC precedes their death in early stages of glaucomatous optic neuropathy, and that vision may be spared if this dysfunction is first detected and then treated. Our immediate goal is to understand the relationships between potentially reversible functional changes and irreversible structural changes of RGC in the progression of glaucoma. Our collaborative research team combines expertise in glaucoma, visual electrophysiology, biophysics, and retinal imaging, working in a unique clinical-research setting that has a large number of patients with suspicion of glaucoma including high rates of individuals of African American and Hispanic descent. We will use non-invasive techniques that we developed to evaluate RGC function and structure - including the pattern electroretinogram (PERG) to measure potentially reversible functional changes of RGC, and Optical Coherence Tomography (OCT) to measure irreversible structural changes of RGC and their axons - in selected groups of these patients. We will also use non-invasive paradigms - suction cup to increase the intraocular pressure (IOP) and topical treatment to lower IOP - to establish whether PERG abnormalities depend on IOP. The specific aims are: 1) to identify and characterize PERG abnormalities in glaucoma suspects, 2) to understand the relationship between PERG losses and OCT losses, and 3) to understand the relationship between PERG abnormalities and induced changes in IOP. This combined, innovative approach will yield a better understanding of the pathophysiological mechanisms in the progression of glaucomatous neuropathy, may provide a rationale for treatment or prevention of glaucoma, and will develop a method to monitor the efficacy of treatment based on RGC function. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This P30 proposal is a request for partial funding to cover costs for core resource modules supporting research within the Miami Center for Vision Research (MCVR), the umbrella component of the Evelyn F. and William L. McKnight Vision Research Center, University of Miami School of Medicine. The modules are: Biostatistics, and Equipment. Most of the module space is located within the eight-story McKnight building. This building houses laboratory research activities of the Bascom Palmer Eye Institute, which serves as the Department of Ophthalmology for the University. Additional space is located in the adjacent Rosenstiel building, which is connected to McKnight by a sky bridge. The MCVR currently comprises the research activities of 67 faculty level scientists, physicians, and optometrists who direct or collaborate in laboratory or clinical programs in eye and vision research at the University of Miami School of Medicine. This includes 20 NEI-funded Principal Investigators who hold a total of 29 NEI grants. In addition, 10 new NEI grant applications are currently under review and 10 more are in the preparation stage. Most of the MCVR investigators hold primary appointments in the Department of Ophthalmology, but there are representatives from several other departments, as well as one investigator from a neighboring institution. Major scientific disciplines represented in this group include biochemistry, bioengineering, biophysics, biostatistics, biotechnology, cell biology, developmental biology, epidemiology, genetics, immunology, molecular biology, neurobiology, and pharmacology. All of the major categories of eye biology and disease recognized by the National Eye Institute in its current five year plan are addressed by the research of this group. Research concentrations exist in the following eight areas: 1. developmental genetics & reparative processes, 2. regenerative biology and molecular/cellular therapeutics, 3. vascular biology, 4. visual processes, 5. engineering in the eye, 6. imaging technologies, 7. surgery & treatment modalities; clinical trials & clinical outcomes, and 8. epidemiology & public health.

DESCRIPTION (provided by applicant): Retinal ganglion cell (RGC) death is the primary cause of irreversible blindness in Open Angle Glaucoma (OAG) and most optic neuropathies. The goal of this project is to prevent blindness in these diseases by obtaining the information needed to understand the etiology of RGC vulnerability, and use this understanding to develop approaches that prevent cell death. Our objective is determining RGC susceptibility to stressors such as IOP elevation, metabolic load, and neurotrophic factor (NT) deficiency in mouse models of glaucoma and optic neuropathy during the stage of progressive RGC dysfunction preceding death (critical period). Our central hypothesis is that RGC death is the result of failure of autoregulatory/adaptive processes that can no longer sustain normal RGC homeostasis, resulting in loss of RGC electrical responsiveness. During the critical period, RGC responsiveness is modifiable (plastic) upon stressors such as IOP, metabolic demand and NT support, thus providing a rationale and a target for therapeutic intervention. Using innovative methods, we will acutely modulate the levels of these stressors and simultaneously assess modifiability of RGC electrical responsiveness over time using pattern electroretinogram (PERG) and visual evoked potential (VEP). We will also use state-of-the-art optical coherence tomography (OCT) to serially monitor thickness of inner retinal layers, as well as retinal immunohistochemistry at endpoint. We will attain our goal and objective by accomplishing the following aims: 1) Test the hypothesis that RGC plasticity occurs in a specific time window in mouse models of glaucoma and optic neuropathy. Models will be the Myoc transgenic mouse of glaucoma, the ND4 transgenic mouse of multiple sclerosis, and the MOG-specific TCR transgenic mouse of optic neuritis. Controls will be corresponding non-pathological congenic mice. We will non-invasively alter either IOP with changes of body posture, or the metabolic demand with flickering light, and will measure corresponding changes of the PERG/VEP signal that precede loss of OCT signal; 2) Test the hypothesis that RGC plasticity is inducible in mouse models of chronic NT deficiency. We will perform unilateral lesions of the superior colliculus (SC) in C57BL/6J and DBA/2J mice and will quantify changes of the PERG and OCT signal in each eye. We will also induce IOP and metabolic stress in SC-lesioned mice to quantify acquired susceptibility of the PERG signal. Successful completion of our research will establish a new conceptual model of RGC susceptibility, and will improve our technical capability of detecting diseases' onset, monitoring their progression and the effect of treatment, eventually changing clinical practice. This will represent a significant advancement in the field and will be influential on future research on glaucoma and other neurodegenerative diseases involving RGC.

The Evelyn F. and William L. McKnight Vision Research Center (MVRC) is the research arm of the Bascom Palmer Eye Institute, University of Miami Miller School of Medicine. The MCVR draws together 48 eye and vision research investigators across the University of Miami and nearby institutions. Forty-two investigators hold primary appointments in the Bascom Palmer Eye Institute. This P30 core center grant is specifically directed towards MVRC investigators to integrate expertise on cellular, molecular, functional, anatomical, biophysical, computational, and clinical aspects of the visual system and its disorders, as well as share core resources and educational programs. The MVRC Team includes 21 NEI-funded Principal Investigators holding 19 qualifying NEI grants as well as 27 Principal Investigators holding non-NEI funded grants. Most of the research space is located within the eight-story MVRC. Additional space is located in the adjacent Biomedical Research Building, Rosenstiel building, Lois Pope LlFE center, Sylvester Comprehensive Cancer Center, and Clinical Research Building. We request continuing support for a Vision Research Core Grant whose specific aims are, 1) to provide and sustain core resource facilities to enhance the pace and quality of research, 2) to foster collaborative research among investigators from various disciplines, 3) to attract investigators to research on the visual system and its diseases, 4) to help investigators gathering data for new NIH grant proposals. The core grant will support four modules: I) Shared Equipment: to provide access to instrumentation beyond the reach of single labs, II) Experimental Models: to provide expertise and services on in-vivo procedures and testing. III) Biological imaging: to support the processing of biological specimens for quantitative microscopy analysis, IV) Biostatistics: to provide expertise on research design and data analysis. The Bascom Palmer Eye Institute supports additional shared resources including, V) Ophthalmic Biophysics Center: for consultation, maintenance and construction of ad-hoc instrumentation; VI) Clinical Microbiology Laboratory: providing services for clinical and basic research heeds; VII) The Mary & Edward Norton Library: housing the world's largest collection on the subject of ophthalmology.

Biological Imaging Module. Located at the 4th and 7th floors of the MVRC provides services and consultation for preparing and analyzing the structure of biological specimens in two main areas: i) Analytical Imaging ?Managed by Gabriel Gaidosh, ii) Histology ?with the technical assistance of Magda Celdran, The Module is directed by Valery Shestopalov, PhD and co-directed by Victor Perez, M.D. Technical and administrative details are in the specific sections and also mentioned in the BPEI web site http://bascompalmer.org/research-cores/histologv

4) Biostatistics Module. Located at the 15* floor of Clinical Research Building - 7 minute walk from MVRC provides expertise in experimental design and biostatistics to BPEI and Miami Eye Team investigators. The Biostatistics Center is directed by Richard K. Parrish, II, MD, co-directed by the head biostatistician Joyce Schiffman, MS and includes 2 other biostatisticians, as well as 2 research support staff. Details are in the specific section and also mentioned in the BPEI web site http://bascompalmer.org/clinical-researchunits/biostatistics-center

DESCRIPTION (provided by applicant): Glaucoma causes progressive damage and death of retinal ganglion cells (RGCs) resulting in blindness. The prevalence of the disease will rise to a projected 3 million Americans by 2020. Our long-term goal is to prevent RGC death in the early stages of glaucoma and preserve vision. The objective of this study is to identify dysfunctional RGCs and the risk of their death, together with the time window of opportunity for their recovery. Our central hypothesis is that RGCs undergo a stage of reversible dysfunction before dying, and that RGC dysfunction is modifiable in a critical period during which RGC electrical activity is responsive to artificial elevation/lowering of the intraocular pressure (IOP). Temporary IOP elevation with be obtained by means of head-down body posture; IOP lowering will be obtained by means of topical treatment. Our study will include 600 subjects with suspicion of glaucoma but with normal vision and visual field that will be longitudinally monitored over 4 years with state-of-the-art pattern electroretinogram (PERG), Spectral-domain Optical Coherence Tomography (SD-OCT), and other clinical measures. PERG losses result from reduced activity of viable RGCs as well as from lack of activity from dead/missing RGCs; OCT losses result from loss of RGC/axon tissue. Our specific aims will determine the risk of losing substantial RGC/axon tissue over 4 years based on baseline PERG susceptibility to head-down posture (aim1), baseline PERG abnormality (aim 2), and presence/absence of IOP-lowering treatment during follow up. Successful completion of our research will establish the notion that RGC death in glaucoma is preceded by a defined stage of modifiable RGC electrical activity, and that the risk of developing glaucoma can be predicted at baseline based on PERG. The outcome of our research will provide 1) the basis for new provocative tests of RGC functional susceptibility/reversibility using a non-invasive PERG method, 2) information needed for new methods to predict the risk of glaucoma development, 3) the time window for preventing it by timely treatment of high-risk individuals.

Bascom Palmer Eye Institute is a comprehensive, disease-focused academic unit that serves as the Department Of Ophthalmology, for the University of Miami Miller School of Medicine. The Anne Bates Leach Eye Hospital is Bascom Palmer's main facility for ophthalmic care. The adjacent Evelyn F. and William L. McKnight Vision Research Center (MVRC) is Bascom Palmer's research arm, and also coordinates all aspects of Bascom Palmer's research mission. Moreover, MVRC draws together NEI-funded eye and vision research investigators across the University of Miami and nearby institutions as the Miami Eye Team. This P30 core center grant application is specifically directed towards this group of investigators to share in research activities, collaborative expertise, core resources, and educational programs. The long-term goal of this P30 proposal is to strengthen the conceptual and methodological capabilities of Miami Eye Team investigators, thus expanding and enhancing their contributions to knowledge and understanding of the eye, vision, and blinding eye diseases. We request continued funding of the P30EY014801 Core Grant for Vision Research to support the following core resource modules: (1) Shared Equipment, (2) Experimental Models, (3) Biological Imaging, (4) Biostatistics. Biological Imaging combines two previous modules ?Histology & Tissue Processing and Analytical Imaging? because of their methodological interrelatedness.

No abstract provided

Administrative Core Summary The Administrative Core manages all activities of the McKnight Vision Research Center (MVRC) investigators related to the use of Core Grant modules (Experimental Models; Biological Imaging; Multi- Omics). Specific Aims are: 1) To provide administrative support for the use of shared Core modules prioritizing NEI-R01 studies, 2) To review the quality, efficiency, progress and impact of Core Modules through Module Directors, Executive Committee, and the Advisory Committee of external members, 3) To manage financial operations of all core grant activities and the grant budget, 4) to provide the annual progress reports to the National Eye Institute. Administrative Director is the Center Core PI with the assistance of: 1) Administrative Manager, 2) Module Directors (Module Directors + Module Manager), 3) Executive Committee (PI + Module Directors + Administrative Manager), 3) Advisory committee (Three external advisors + PI + Module Directors) (see Figure 1 below). The Administrative Manager handles record keeping, billing, equipment maintenance/repair, and the operating funds for all Modules of the core P30 grant. Module Directors oversee the daily duties of Module Managers of three grant Modules (Experimental Models, Biological Imaging, Multi Omics) and meet with them weekly or on an as needed basis for prompt resolution of outstanding issues; module directors and managers also meet with the users of the modules regarding experimental design and monitor usage through the scheduling websites. The Executive Committee meets quarterly to assess module usage, adherence to usage priority, equipment maintenance/repair and service contracts, budget, or any other activity relevant to the function of the Modules. The Executive Committee also meets on an as needed basis for issues that require prompt interventions such as the core grant budget or conflict with module users. The Advisory Committee meets once a year to assess efficiency and impact of Core Modules on the productivity of MVRC Investigators and to formulate strategic decisions for the next year. Strategic decisions that involve technological expansion and module reconfiguration are discussed, if necessary, with the Chairs of the Departments of participating MRVC investigators, and the Dean the School of Medicine.

Research Summary Mutations in mitochondrial DNA (mtDNA) lead to a spectrum of neurodegenerative diseases for which no effective treatment exists. The most common of these is Leber hereditary optic neuropathy (LHON) caused by mutations in NADH dehydrogenase subunit genes (ND1, ND4 or ND6), which is complex I of the respiratory chain. Therapies for LHON in common with all disorders caused by mutated mtDNA are inadequate, in large part because of the barrier in delivering DNA into the organelle. We have successfully broken through this barrier by developing a pioneering adeno-associated virus (AAV) vector to which a mitochondrial targeting sequence (MTS) was appended to the viral capsid. The modified vector delivered the ND4 gene directly to the mitochondria for reversal of visual loss in mice with mutated G11778A ND4 responsible for more than half of all LHON cases, the rest caused by mutated ND1 and ND6. We will now design, modify and test the efficacy and safety of a clinically relevant vector for treatment of this mitochondrial disease by delivery of genes encoding the promoter and regulatory elements of a normal mitochondrially encoded human ND4 subunit to affected cells and tissues for rescue of cultured human LHON cells harboring the NADH dehydrogenase subunit 4 mutation causing LHON and then transgenic mice and primates with mutated ND4. The ND4 mouse we developed by injection of a MTS AAV containing mutated G11778A ND4 into the rodent blastocyst has visual loss progressing to blindness a year after birth, optic nerve head swelling followed by atrophy and degeneration of retinal ganglion cells, which are the characteristic hallmarks of LHON patients. Our goals are: (A) To facilitate translational studies for LHON by developing a clinical grade MTS AAV vector that accommodates the wild-type ND4 LHON gene and regulatory elements in a single AAV cassette. (B) To test expression to rescue respiration in cybrid cells and the visual system of mice and primates with mutated G11778A ND4. (C) To evaluate biological effects of intravitreal delivery of MTS AAV vectors in normal rodents and primates that result in mitochondrial gene transfer without adverse effects. (D) To develop an IND application for a single AAV containing normal ND4 for a future phase I/II clinical trial designed to restore the vision of patients with the commonest and most severe LHON mutation and prevent visual loss in their family members in a U10 application to follow successful completion of this R24.