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Miranda L White, Frank M Dyka, Charles N de Leeuw, Seok-Hong Min, Qing Ruan, Sanford L Boye, Neal S Peachey, Elizabeth M Simpson, William W Hauswirth, Shannon Boye; Optimizing rAAV vectors to target ON bipolar cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3336.
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© ARVO (1962-2015); The Authors (2016-present)
Inaccessible by conventional vectors following subretinal or intravitreal delivery, bipolar cells in the middle retina remain a largely unexploited target for gene therapy. Mutations in bipolar specific genes are associated with Congenital Stationary Night Blindness (CSNB). X-linked CSNB (XCSNB1) is caused by mutations in NYX, which encodes nyctalopin, a protein involved in glutamate signaling in ON bipolar cells (BCs). In addition to night blindness, XCSNB1 patients have reduced acuity, high myopia, nystagmus, and strabismus. Despite having preserved retinal structure, ERG analysis reveals a lack of a b-wave. The Nyxnob mouse carries a frameshift mutation in Nyx. Like patients, it lacks a b-wave but its retina does not degenerate. The purpose of this study is to develop a novel Adeno-associated virus (AAV) vector that will drive expression of therapeutic Nyx exclusively in BCs of Nyxnob mice thereby restoring normal ERG phenotype.
eYFP- tagged Nyx was delivered by a rationally designed, AAV2-based capsid mutant- AAV2(quadY-F+T-V). “Ple155” (based on purkinje cell promoter PCP2) was chosen to drive transgene expression. AAV2(quadY-F+T-V)-Ple155-eYFP-NYX or control vector containing GFP (~2.0E13 vg/ml) were injected intravitreally or subretinally into Nyxnob or C57BL/6J mice. Four weeks post-injection, Nyxnob mice were analyzed by ERG and retinas from all mice were stained with antibodies raised against GFP and either PCP2 or PKCα to label BCs.
AAV2(quadY-F+T-V)-Ple155- mediated GFP and eYFP-NYX expression was restricted to ON BCs. No improvements in retinal function were detected in vector treated Nyxnob mice evaluated by full field ERG.
We identified an AAV serotype/cellular promoter combination that is capable of robust and highly selective transduction of murine ON BCs. This is achievable with either subretinal or intravitreal injection. Functional rescue may not have been observed because 1) an insufficient number of BCs expressed Nyx or 2) our ERG protocol lacked sufficient sensitivity. Work is underway to modify our vectors to increase transgene expression and to increase ERG sensitivity. We conclude that specific targeting of ON BCs is possible, a result which has implications for the treatment of CSNB and also for optogenetic strategies aimed to impart light sensitivity specifically to this cell type.
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