title: Subretinal gene therapy of mice with Bardet-Biedl syndrome type 1.
abstract: OBJECTIVE
  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).
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Ophthalmology, University of Iowa, Iowa City, Iowa.
authors.forename: Seongjin
authors.initials: S
authors.lastname: Seo
authors.forename: Robert F
authors.initials: RF
authors.lastname: Mullins
authors.forename: Alina V
authors.initials: AV
authors.lastname: Dumitrescu
authors.forename: Sajag
authors.initials: S
authors.lastname: Bhattarai
authors.forename: Daniel
authors.initials: D
authors.lastname: Gratie
authors.forename: Kai
authors.initials: K
authors.lastname: Wang
authors.forename: Edwin M
authors.initials: EM
authors.lastname: Stone
authors.forename: Val
authors.initials: V
authors.lastname: Sheffield
authors.forename: Arlene V
authors.initials: AV
authors.lastname: Drack
mesh_terms: D000818:Animals; D020788:Bardet-Biedl Syndrome / Q000150:complications* / Q000628:therapy; D015153:Blotting, Western; D000229:Dependovirus / Q000235:genetics*; D004195:Disease Models, Animal; D004596:Electroretinography; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D007150:Immunohistochemistry; D056965:Injections, Intraocular; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism / Q000627:therapeutic use*; D020419:Photoreceptor Cells, Vertebrate / Q000378:metabolism; D012162:Retinal Degeneration / Q000209:etiology / Q000378:metabolism / Q000503:physiopathology / Q000628:therapy*; D058471:Subretinal Fluid; D014161:Transduction, Genetic
chemical_list: C506280:Bbs1 protein, mouse; D008869:Microtubule-Associated Proteins
keywords: AAV vector; Bardet Biedl syndrome; gene therapy; mouse model; retinal degeneration
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