John G. Flannery

Professor of Vision Science and Optometry; Professor of Neurobiology, Dept. Molecular & Cell Biology; Assoc. Director, Helen Wills Neuroscience Institute

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:

1. Can a gene-based therapy be developed that would allow for the transduction of retinal cells by intravitreal injection rather than subretinal injection?
2. Can neuroprotection occur by the targeting of a cell that spans the entire retina, the Müller cell?
3. 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?
4. 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?
5. 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:

1. 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);
2. the potential preservation of sight through a novel approach of reducing photoreceptor sensitivity to light;
3. the possibility of engineering retinal ganglion cells to respond to light, for use in patients who have lost all PRs from retinal degenerations;
4. 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.

Current Projects

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.

Significance
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.

Recent Publicatinos

1. Lau, D. & Flannery, J. Viral-mediated FGF-2 treatment of the constant light damage model of photoreceptor degeneration. Doc Ophthalmol 106, 89-98 (2003).
2. Adato, A. et al. USH3A transcripts encode clarin-1, a four-transmembrane-domain protein with a possible role in sensory synapses. Eur J Hum Genet 10, 339-50 (2002).
3. McGee Sanftner, L.H. et al. Recombinant AAV-mediated delivery of a tet-inducible reporter gene to the rat retina. Mol Ther 3, 688-96 (2001).
4. McGee Sanftner, L.H., Abel, H., Hauswirth, W.W. & Flannery, J.G. Glial cell line derived neurotrophic factor delays photoreceptor degeneration in a transgenic rat model of retinitis pigmentosa. Mol Ther 4, 622-9 (2001).
5. Green, E.S. et al. Two animal models of retinal degeneration are rescued by recombinant adeno-associated virus-mediated production of FGF-5 and FGF-18. Mol Ther 3, 507-15 (2001).
6. Ogueta, S.B., Di Polo, A., Flannery, J.G., Yamashita, C.K. & Farber, D.B. The human cGMP-PDE beta-subunit promoter region directs expression of the gene to mouse photoreceptors. Invest Ophthalmol Vis Sci 41, 4059-63 (2000).
7. LaVail, M.M. et al. Ribozyme rescue of photoreceptor cells in P23H transgenic rats: long-term survival and late-stage therapy. Proc Natl Acad Sci U S A 97, 11488-93 (2000).
8. Lau, D. et al. Retinal degeneration is slowed in transgenic rats by AAV-mediated delivery of FGF-2. Invest Ophthalmol Vis Sci 41, 3622-33 (2000).
9. Hauswirth, W.W., LaVail, M.M., Flannery, J.G. & Lewin, A.S. Ribozyme gene therapy for autosomal dominant retinal disease. Clin Chem Lab Med 38, 147-53 (2000).
10. Green, E.S., Menz, M.D., LaVail, M.M. & Flannery, J.G. Characterization of rhodopsin mis-sorting and constitutive activation in a transgenic rat model of retinitis pigmentosa. Invest Ophthalmol Vis Sci 41, 1546-53 (2000).
11. Flannery, JG, Geller, SF, Chen, J. Structure and Function of Rod Photoreceptors In: Retina. Ryan, S.J. and Wilkinson, C.P. Eds., Volume 1, Basic Science; St. Louis, MO, Mosby (2005).
12. Lee, ES., Burnside, B., Flannery, JG. Characterization of peripherin / rds and Rom-1 in photoreceptors of Transgenic and Knockout animals. (in press Invest Ophthalmol Vis Sci. 2006)
13. Greenberg, KP, Geller, SF., Schaffer, DV and Flannery, JG. Targeted transgene expression in Müller glia of normal and diseased retinas using lentiviral vectors. (in press Invest Ophthalmol Vis Sci. 2006).
14. Zolfaghar, I, Walsh, N, Lee, J., Hauswirth, WW., Gong, X., Xia, C, Flannery, JG AAV2-Mediated Expression of Anti-angiogenic Factors Inhibits Sub-Retinal Neovascularisation in the Vldlr -/- Mouse. (submitted, 2006).

Links

Flannery Lab website

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