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Subretinal gene therapy of mice with Bardet-Biedl syndrome type 1
Journal article   Open access   Peer reviewed

Subretinal gene therapy of mice with Bardet-Biedl syndrome type 1

Seongjin Seo, Robert F Mullins, Alina V Dumitrescu, Sajag Bhattarai, Daniel Gratie, Kai Wang, Edwin M Stone, Val Sheffield and Arlene V Drack
Investigative ophthalmology & visual science, Vol.54(9), pp.6118-6132
09/11/2013
DOI: 10.1167/iovs.13-11673
PMCID: PMC3771708
PMID: 23900607
url
https://doi.org/10.1167/iovs.13-11673View
Published (Version of record) Open Access

Abstract

To study safety and efficacy of subretinal adeno-associated virus (AAV) vector AAV-Bbs1 injection for treatment of a mouse model of Bardet-Biedl syndrome type 1 (BBS1). Constructs containing a wild-type (WT) Bbs1 gene with and without a FLAG tag in AAV2/5 vectors were generated. Viral genomes were delivered by subretinal injection to right eyes and sham injections to left eyes at postnatal day 30 (P30) to P60. Transgene expression and BBSome reconstitution were evaluated by immunohistochemistry and Western blotting following sucrose gradient ultracentrifugation. Retinal function was analyzed by electroretinogram (ERG) and structure by optical coherence tomography (OCT). Histology and immunohistochemistry were performed on selected eyes. Expression of FLAG-tagged Bbs1 was demonstrated in photoreceptor cells using antibody directed against the FLAG tag. Coinjection of AAV-GFP demonstrated transduction of 24% to 32% of the retina. Western blotting demonstrated BBS1 protein expression and reconstitution of the BBSome. ERG dark-adapted bright flash b-wave amplitudes were higher in AAV-Bbs1-injected eyes than in sham-injected fellow eyes in more than 50% of 19 animals. Anti-rhodopsin staining demonstrated improved localization of rhodopsin in AAV-Bbs1-treated eyes. WT retinas injected with AAV-Bbs1 with or without a FLAG tag showed outer retinal degeneration on ERG, OCT, and histology. In a knock-in model of BBS1, subretinal delivery of AAV-Bbs1 rescues BBSome formation and rhodopsin localization, and shows a trend toward improved ERG. BBS is challenging to treat with gene therapy due to the stoichiometry of the BBSome protein complex and overexpression toxicity.
 Immunohistochemistry Electroretinography Dependovirus - genetics Microtubule-Associated Proteins - therapeutic use Transduction, Genetic Microtubule-Associated Proteins - genetics Microtubule-Associated Proteins - metabolism Bardet-Biedl Syndrome - therapy Retinal Degeneration - etiology Retinal Degeneration - physiopathology Retinal Degeneration - metabolism Blotting, Western Animals Retinal Degeneration - therapy Subretinal Fluid Mice Photoreceptor Cells, Vertebrate - metabolism Genetic Vectors Bardet-Biedl Syndrome - complications Injections, Intraocular Disease Models, Animal Genetic Therapy - methods

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