Home Faculty and Research Faculty by Name John G. Flannery

John G. Flannery

Affiliated Professor of Neurobiology

Lab Homepage: http://mcb.berkeley.edu/labs/flannery/

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Research Interests

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

Using light-activated ion channels to confer light-sensitivity on retina lacking functional rods and cones.

We are developing a new type of retinal prosthetic by using light-activated ion channels to convert “blind” retinal ganglion cells (RGC’s) into intrinsically photoresponsive cells. Blinding diseases such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD) result from progressive degeneration of rods and cones. Downstream retinal neurons that receive and process signals from the photoreceptors are preserved for years after the onset of blindness in these diseases, giving hope that visual sensitivity might be restored by allowing the artificial input of information to these surviving cells. Previous retinal prosthetic devices used electrode arrays driven by a CCD video camera to control the firing of surviving retinal interneurons. While electrical stimulation of the retina produced light perception in patients receiving these devices, spatial and temporal resolution was poor, probably because of the relatively large size and imprecise positioning of electrodes in the arrays with relation to individual RGCs. The materials used in building these devices also present serious challenges of long-term biocompatibility. We propose using light activated ion channels as an alternative therapeutic approach to avoid these problems. In preliminary studies, we have packaged the cysteine-modified Shaker K+ (SPARK) channel fused to GFP and under control of the synapsin promoter into an AAV serotype 2 vector and transferred it into RGCs in vivo by intravitreal injection. Light-activated switching of action potentials in a wildtype rat retinal flat mount preparation by patch clamp recording. We are currently expressing these SPARK channels in RGCs of the s334ter rat model after the photoreceptors have been lost.

 

 

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.Västinsalo E.M. Sankila Folkhalsan Institute of Genetics, Helsinki, Finland; Helsinki University Eye Hospital and Folkhalsan 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.

Selected Publications

Stephanie Szobota, Pau Gorostiza,  Rika Numano, Matthew Volgraf, Kate Kolstad,  Holly Aaron, Steve Ruzin, John Flannery, Richard Kramer, Dirk Trauner and Ehud Isacoff,  Remote control of neuronal activity with a light-gated glutamate receptor Neuron 54:535-545 (2007).

Aarnisalo AA, Pietola L, Joensuu J, et al. Anti-clarin-1 AAV-delivered ribozyme induced apoptosis in the mouse cochlea. Hearing Research (2007).

Greenberg KP, Geller SF, Schaffer DV, Flannery JG. Targeted transgene expression in Müller glia of normal and diseased retinas using lentiviral vectors. Investigative ophthalmology & visual science 48:1844-1852 (2007).

 Scott Geller, Phillip Ge , Meike Visel, and John Flannery,   Functional promoter testing using a modified lentiviral transfer vector. Molecular Vision 13:730-739 (2007).

 Lee ES, Flannery JG. Transport of truncated rhodopsin and its effects on rod function and degeneration. Investigative ophthalmology & visual science 48:2868-2876 (2007).

 Greenberg KP, Lee ES, Schaffer DV, Flannery JG.   Gene delivery to the retina using lentiviral vectors. Adv Exp Med Biol. 572:255-66 (2006).

 Flannery JG, Greenberg KP.Looking within for vision. Neuron. Apr 6 50(1):1-3 (2006).

 Lee, E., Burnside, B. & Flannery, J. Characterization of Peripherin/rds and Rom-1 Transport in Photoreceptors of Transgenic and Knockout Animals. Invest Ophthalmol Vis Sci May 47(5):2150-60 (2006).

 Flannery, J., Geller, S. & Chen, J. Structure And Function Of Rod Photoreceptors. in Retina, Vol. 1 (eds. Ryan, S.J. & Wilkinson, C.P.) 45 (Mosby, St. Louis , MO , 2005).

 Hauswirth WW, Li Q, Raisler B, Timmers AM, Berns KI, Flannery JG, LaVail MM, Lewin AS.Range of retinal diseases potentially treatable by AAV-vectored gene therapy. Novartis Found Symp. 255:179-88 (2004).

Lau, D. & Flannery, J. Viral-mediated FGF-2 treatment of the constant light damage model of photoreceptor degeneration. Doc Ophthalmol 106, 89-98 (2003).

 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).

 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).

 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).

 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).

 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).

 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 USA 97, 11488-93 (2000).

 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).

 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).

 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).

Last Updated 2007-07-27