Professor of Neurobiology, Dept. Molecular & Cell Biology
Assoc. Director, Helen Wills Neuroscience Institute
Vision Science 206D. Neuroanatomy and Neurophysiology of the Eye and Visual System
Structure and function of the neurosensory retina, photoreceptors, RPE including blood supply. Current concepts of etiology and management of major retinal conditions. Overview of diagnostic techniques in retinal imaging, electrophysiologic testing and new genetic approaches. Structure and function of the early visual pathway including retinal ganglion cells, optic nerves, lateral geniculate nucleus and visual cortex. Pupillary responses. Specialization in the visual cortex.
Cellular and Molecular Neuroscience
Retinal Degeneration and blindness result from the loss of rod and cone photoreceptors due to mutations in these cells or in their closely interacting and supportive retinal pigment epithelium (RPE), from environmental or poorly defined age-related factors, or the actions of other retinal neurons, glia or vascular elements. Relatively little is known about precisely why photoreceptors die in any of the many different retinal degenerations, and virtually no effective therapy exists for most of these diseases. One of the major goals of our laboratory is to develop therapeutic approaches that will slow or prevent the loss of rods, cones, RPE and other cells in retinal degenerations.
Current key questions in the laboratory are:
- Can a gene-based therapy be developed that would allow for the transduction of retinal cells by intravitreal injection rather than subretinal injection?
- Can neuroprotection occur by the targeting of a cell that spans the entire retina, the Müller cell?
- Can the light sensitivity of photoreceptor cells be regulated in a way to make them less sensitive to the exacerbating effects of light on opsin mutations?
- Can retinal ganglion cells be manipulated in a way to add a light-receptive function, and thereby serve to transduce light in retinas that have lost their photoreceptors due to various retinal degenerations?
- Can gene-based delivery of a number of neuroprotective agents lead to new clinical trials for retinitis pigmentosa and other photoreceptor degenerations?
These questions will be addressed using novel genetic targeting approaches for gene delivery, the pharmacological control of ion channels to regulate PR sensitivity, the targeting of retinal ganglion cells for gene-based delivery of externally gated ion channels, and the testing of a number of neuroprotective agents in models of inherited retinal degenerations using adeno-associated virus (AAV) vectors.
The important outcomes of these studies include:
- the simplification of gene therapy for PR degenerations using intravitreal injection in contrast to subretinal injections (which require retinal detachment with some inherent trauma from the procedure);
- the potential preservation of sight through a novel approach of reducing photoreceptor sensitivity to light;
- the possibility of engineering retinal ganglion cells to respond to light, for use in patients who have lost all PRs from retinal degenerations;
- The examination of gene-based delivery of two neurotrophins that have successfully achieved neuroprotection in clinical trials of patients with Parkinson’s and Alzheimer’s disease.
Ongoing studies include:
Viral Vectors for Gene Therapy of Inherited Retinal Degenerations
Gene therapy has vast potential for treating and potentially curing a number of retinal diseases including glaucoma, age-related macular degeneration, and inherited photoreceptor diseases. However, gene delivery technologies require significant improvements in cellular targeting, efficiency, and safety before promising findings in animal studies are translated to the clinic. In particular, for retinal gene therapy it would be highly advantageous to transduce a single cell type that spans the entire retina after an intravitreal injection of a gene delivery vehicle for the subsequent secretion of a general neuroprotective factor throughout the retina. Unfortunately, there is no vector capable of efficiently infecting the cell type that meets these needs, Müller cells. Vectors based on adeno-associated virus (AAV) have proven themselves to be highly promising in numerous retinal disease models, but they are also incapable of Müller cell infection. We have developed novel lentiviral vectors with new properties, including altered receptor binding, which are capable of efficient Müller cell transduction. In parallel, the basic mechanisms of AAV transduction of Müller cells will be explored in order to develop new AAV pseudotypes capable of Müller cell transduction. The novel approaches developed in this work will have general impact for the molecular engineering of enhanced viral gene delivery vehicles, and future work will focus on testing these vectors in an animal model of retinal disease.
Ush3A mRNA is expressed at Wild-Type Levels in Degenerating Rat Retina
This study is designed to examine Ush3A mRNA expression levels in wild-type and degenerating rat retina. Ush3a is the gene responsible for a significant portion of patients identified with Usher syndrome type 3. In this study, we are working to identify the cellular site of expression of the Ush3a gene product.
Cellular Localization and Processing of USH3A Protein Clarin-1 in Transiently Transfected Cell Lines
Collaborators: J. Isosomppi, H. Vastinsalo E.M. Sankila Folkhälsan Institute of Genetics, Helsinki, Finland; Helsinki University Eye Hospital and Folkhälsan Institute of Genetics, Helsinki, Finland.
We are investigating the cellular localization, stability and processing of mutant and wild-type human clarin-1 in transiently transfected neural and non-neural cell lines. A human retinal cDNA library was used to amplify the main transcript of clarin-1 (accession number NM_174878). The cDNA was cloned into a hemagglutinin (HA)-tagged expression vector.
In vivo Plasmid Tracking in Electroporated Rat Retina
Delivery of exogenous DNA to the mammalian retina by in vivo electroporation is proving to be a valuable tool for genomic, therapeutic, and physiological studies of vision. We are seeking to optimize experimental parameters to obtain the highest efficiency and specificity of delivery by using rhodamine-conjugated plasmids electroporated into the wild-type rat retina.
In vivo Targeting of Müller Cells in the Rodent Retina Using Novel Lentiviral Vectors
Efficient transgene delivery and stable expression in Müller cells will be a valuable tool for therapeutic and physiological investigations of the retina. Previous studies using lentiviral and adeno-associated viral (AAV) vectors have achieved only limited Müller cell transduction or poor specificity following intra-ocular injection. Recombinant HIV-1 vectors were pseudotyped with envelope glycoproteins derived from either the Ross River Virus (RRV) or Vesicular Stomatitis Virus (VSV). Vectors were packaged by transient transfection of 293T cells and high titer viral stocks were obtained after ultracentrifugation. A panel of pseudotyped vectors were constructed in this way that contain either Müller cell-specific (glial fibrillary acidic protein, vimentin, glutamine synthetase) or promiscuous (CMV, CMV-β-actin, ubiquitin) promoters to drive GFP reporter gene expression.
Progression of the VLDLR -/- Retinal Phenotype with Age: Correlating Fundus Features with Histological and Functional Measures
The VLDLR -/- mouse provides an important model of subretinal neovascularization through which therapies for human disease may be trialed. We have sought to characterize the retinal features of this strain over a one year period through a range of measures which include fundus imaging, fluorescein angiography, histology, electrophysiology and 3D vessel reconstruction.
AAV2-Mediated Expression of Anti-Angiogenic Factors Inhibits Sub-Retinal Neovascularization in the VLDLR -/- Mouse.
Collaboration with Bill Hauswirth, Department of Ophthalmology, University of Florida, Gainesville, FL.
We are testing whether AAV-2 viral mediated delivery of PEDF, K1K3, Endostatin or the inhibitory domains of VEGF (exons 6 and 7), can decrease the growth and permeability of abnormal vessels in the VLDLR -/- mouse.
These projects are advancing the state of the art in gene therapy for retinal disease. We have made significant advances in the development of new lentiviral vectors that can transfer large cDNA’s to Muller glia and retinal neurons. We have made the first animal model of Usher Syndrome III, by knocking out the USH3a gene in a mouse model. We will begin to characterize the animal model in the coming year, and apply gene replacement therapy using AAV clarin-1 in the next project year. We will also test neurotrophin therapies for Usher syndrome in the coming year. We have made a significant advance in the development of an alternative gene transfer methodology, trans-scleral electroporation which may be very useful for short-term expression of therapeutic agents.
Pernet, V., Joly, S., Jordi, N., Dalkara, D., Guzik-Kornacka, A., Flannery, J.G. & Schwab, M.E. Misguidance and modulation of axonal regeneration by Stat3 and Rho/ROCK signaling in the transparent optic nerve. Cell death & disease 4, e734 (2013).
Pernet, V., Joly, S., Dalkara, D., Jordi, N., Schwarz, O., Christ, F., Schaffer, D.V., Flannery, J.G. & Schwab, M.E. Long-distance axonal regeneration induced by CNTF gene transfer is impaired by axonal misguidance in the injured adult optic nerve. Neurobiology of disease 51, 202-213 (2013).
Flannery, J.G. & Visel, M. Adeno-associated viral vectors for gene therapy of inherited retinal degenerations. Methods in molecular biology 935, 351-369 (2013).
Dalkara, D., Byrne, L.C., Klimczak, R.R., Visel, M., Yin, L., Merigan, W.H., Flannery, J.G. & Schaffer, D.V. In vivo-directed evolution of a new adeno-associated virus for therapeutic outer retinal gene delivery from the vitreous. Science translational medicine 5, 189ra176 (2013).
Pernet, V., Joly, S., Dalkara, D., Schwarz, O., Christ, F., Schaffer, D., Flannery, J.G. & Schwab, M.E. Neuronal Nogo-A upregulation does not contribute to ER stress-associated apoptosis but participates in the regenerative response in the axotomized adult retina. Cell death and differentiation 19, 1096-1108 (2012).
Lee, S.H., Kwan, A.C., Zhang, S., Phoumthipphavong, V., Flannery, J.G., Masmanidis, S.C., Taniguchi, H., Huang, Z.J., Zhang, F., Boyden, E.S., Deisseroth, K. & Dan, Y. Activation of specific interneurons improves V1 feature selectivity and visual perception. Nature 488, 379-383 (2012).
Dalkara, D., Byrne, L.C., Lee, T., Hoffmann, N.V., Schaffer, D.V. & Flannery, J.G. Enhanced gene delivery to the neonatal retina through systemic administration of tyrosine-mutated AAV9. Gene therapy 19, 176-181 (2012)
Caporale N, Kolstad KD, Lee T, Tochitsky I, Dalkara D, Trauner D et al. LiGluR Restores Visual Responses in Rodent Models of Inherited Blindness. Mol Ther 2011; 19(7): 1212-9.
Dalkara D, Kolstad KD, Guerin KI, Hoffmann NV, Visel M, Klimczak RR et al. AAV Mediated GDNF Secretion From Retinal Glia Slows Down Retinal Degeneration in a Rat Model of Retinitis Pigmentosa. Mol Ther 2011.
Vastinsalo, H., Jalkanen, R., Dinculescu, A., Isosomppi, J., Geller, S., Flannery, J.G., Hauswirth, W.W. & Sankila, E.M. Alternative splice variants of the USH3A gene Clarin 1 (CLRN1). Eur J Hum Genet 19, 30-35 (2011).
Kolstad, K.D., Dalkara, D., Guerin, K., Visel, M., Hoffmann, N., Schaffer, D.V. & Flannery, J.G. Changes in adeno-associated virus-mediated gene delivery in retinal degeneration. Hum Gene Ther 21, 571-578 (2010).
Klimczak, R.R., Koerber, J.T., Dalkara, D., Flannery, J.G. & Schaffer, D.V. A novel adeno-associated viral variant for efficient and selective intravitreal transduction of rat Muller cells. PLoS One 4, e7467 (2009).
Tackenberg, M.A., Tucker, B.A., Swift, J.S., Jiang, C., Redenti, S., Greenberg, K.P., Flannery, J.G., Reichenbach, A. & Young, M.J. Muller cell activation, proliferation and migration following laser injury. Mol Vis 15, 1886-1896 (2009).
Isosomppi, J., Vastinsalo, H., Geller, S.F., Heon, E., Flannery, J.G. & Sankila, E.M. Disease-causing mutations in the CLRN1 gene alter normal CLRN1 protein trafficking to the plasma membrane. Mol Vis 15, 1806-1818 (2009).
Liu, H., Wang, M., Xia, C.H., Du, X., Flannery, J.G., Ridge, K.D., Beutler, B. & Gong, X. Severe retinal degeneration caused by a novel rhodopsin mutation. Invest Ophthalmol Vis Sci 51, 1059-1065 (2010).
Geller, S.F., Guerin, K.I., Visel, M., Pham, A., Lee, E.S., Dror, A.A., Avraham, K.B., Hayashi, T., Ray, C.A., Reh, T.A., Bermingham-McDonogh, O., Triffo, W.J., Bao, S., Isosomppi, J., Vastinsalo, H., Sankila, E.M. & Flannery, J.G. CLRN1 is nonessential in the mouse retina but is required for cochlear hair cell development. PLoS Genet 5, e1000607 (2009).
Dalkara, D., Kolstad, K.D., Caporale, N., Visel, M., Klimczak, R.R., Schaffer, D.V. & Flannery, J.G. Inner limiting membrane barriers to AAV-mediated retinal transduction from the vitreous. Mol Ther 17, 2096-2102 (2009).
Koerber, J.T., Klimczak, R., Jang, J.H., Dalkara, D., Flannery, J.G. & Schaffer, D.V. Molecular evolution of adeno-associated virus for enhanced glial gene delivery. Mol Ther 17, 2088-2095 (2009).
Geng, Y., Greenberg, K.P., Wolfe, R., Gray, D.C., Hunter, J.J., Dubra, A., Flannery, J.G., Williams, D.R. & Porter, J. In vivo imaging of microscopic structures in the rat retina. Invest Ophthalmol Vis Sci 50, 5872-5879 (2009).
Tian, G., Zhou, Y., Hajkova, D., Miyagi, M., Dinculescu, A., Hauswirth, W.W., Palczewski, K., Geng, R., Alagramam, K.N., Isosomppi, J., Sankila, E.M., Flannery, J.G. & Imanishi, Y. Clarin-1, encoded by the Usher Syndrome III causative gene, forms a membranous microdomain: possible role of clarin-1 in organizing the actin cytoskeleton. J Biol Chem 284, 18980-18993 (2009).
Geng, R., Geller, S.F., Hayashi, T., Ray, C.A., Reh, T.A., Bermingham-McDonogh, O., Jones, S.M., Wright, C.G., Melki, S., Imanishi, Y., Palczewski, K., Alagramam, K.N. & Flannery, J.G. Usher
syndrome IIIA gene clarin-1 is essential for hair cell function and associated neural activation. Hum Mol Genet 18, 2748-2760 (2009).
Guerin, K., Gregory-Evans, C.Y., Hodges, M.D., Moosajee, M., Mackay, D.S., Gregory-Evans, K. & Flannery, J.G. Systemic aminoglycoside treatment in rodent models of retinitis pigmentosa. Exp Eye Res 87, 197-207 (2008).
Geller, S.F., Ge, P.S., Visel, M. & Flannery, J.G. In vitro analysis of promoter activity in Muller cells. Mol Vis 14, 691-705 (2008).
Paskowitz, D.M., Greenberg, K.P., Yasumura, D., Grimm, D., Yang, H., Duncan, J.L., Kay, M.A., Lavail, M.M., Flannery, J.G. & Vollrath, D. Rapid and stable knockdown of an endogenous gene in retinal pigment epithelium. Hum Gene Ther 18, 871-880 (2007).
Chen, Y., Hu, Y., Lu, K., Flannery, J.G. & Ma, J.X. Very low density lipoprotein receptor, a negative regulator of the wnt signaling pathway and choroidal neovascularization. J Biol Chem 282, 34420-34428 (2007).
Geller, S.F., Ge, P.S., Visel, M., Greenberg, K.P. & Flannery, J.G. Functional promoter testing using a modified lentiviral transfer vector. Mol Vis 13, 730-739 (2007).
Lee, E.S. & Flannery, J.G. Transport of truncated rhodopsin and its effects on rod function and degeneration. Invest Ophthalmol Vis Sci 48, 2868-2876 (2007).
Greenberg, K.P., Geller, S.F., Schaffer, D.V. & Flannery, J.G. Targeted transgene expression in muller glia of normal and diseased retinas using lentiviral vectors. Invest Ophthalmol Vis Sci 48, 1844-1852 (2007).
Greenberg, K.P., Lee, E.S., Schaffer, D.V. & Flannery, J.G. Gene delivery to the retina using lentiviral vectors. Adv Exp Med Biol 572, 255-266 (2006).
Lee, E.S., Burnside, B. & Flannery, J.G. Characterization of peripherin/rds and rom-1 transport in rod photoreceptors of transgenic and knockout animals. Invest Ophthalmol Vis Sci 47, 2150-2160 (2006).
Flannery, J.G. & Greenberg, K.P. Looking within for vision. Neuron 50, 1-3 (2006).
Hauswirth, W.W., Li, Q., Raisler, B., Timmers, A.M., Berns, K.I., Flannery, J.G., LaVail, M.M. & Lewin, A.S. Range of retinal diseases potentially treatable by AAV-vectored gene therapy. Novartis Foundation symposium 255, 179-188; discussion 188-194 (2004).