title: Characterization of intravitreally delivered capsid mutant AAV2-Cre vector to induce tissue-specific mutations in murine retinal ganglion cells.
abstract: Targeted expression of Cre recombinase in murine retinal ganglion cells (RGCs) by viral vector is an effective strategy for creating tissue-specific gene knockouts for investigation of genetic contribution to RGC degeneration associated with optic neuropathies. Here we characterize dosage, efficacy and toxicity for sufficient intravitreal delivery of a capsid mutant Adeno-associated virus 2 (AAV2) vector encoding Cre recombinase. Wild type and Rosa26 (R26) LacZ mice were intravitreally injected with capsid mutant AAV2 viral vectors. Murine eyes were harvested at intervals ranging from 2 weeks to 15 weeks post-injection and were assayed for viral transduction, transgene expression and RGC survival. 10(9) vector genomes (vg) were sufficient for effective in vivo targeting of murine ganglion cell layer (GCL) retinal neurons. Transgene expression was observed as early as 2 weeks post-injection of viral vectors and persisted to 11 weeks. Early expression of Cre had no significant effect on RGC survival, while significant RGC loss was detected beginning 5 weeks post-injection. Early expression of viral Cre recombinase was robust, well-tolerated and predominantly found in GCL neurons suggesting this strategy can be effective in short-term RGC-specific mutation studies in experimental glaucoma models such as optic nerve crush and transection experiments. RGC degeneration with Cre expression for more than 4 weeks suggests that Cre toxicity is a limiting factor for targeted mutation strategies in RGCs.
authors.affiliation: Department of Biology, 951 Carrier Drive, MSC 7801, James Madison University, Harrisonburg, VA, 22807, USA.
authors.forename: Christophe J
authors.initials: CJ
authors.lastname: Langouet-Astrie
authors.affiliation: Department of Surgery, University of California San Diego, 4150 Regents Park Row, La Jolla, CA, 92037, USA.
authors.forename: Zhiyong
authors.initials: Z
authors.lastname: Yang
authors.affiliation: Department of Biology, 951 Carrier Drive, MSC 7801, James Madison University, Harrisonburg, VA, 22807, USA.
authors.forename: Sraavya M
authors.initials: SM
authors.lastname: Polisetti
authors.affiliation: Department of Ophthalmology, Johns Hopkins University School of Medicine, 400 N. Broadway, Baltimore, MD, 21287, USA.
authors.forename: Derek S
authors.initials: DS
authors.lastname: Welsbie
authors.affiliation: Department of Ophthalmology, University of Florida, 1600 SW Archer Road, Gainesville, FL, 32610, USA.
authors.forename: William W
authors.initials: WW
authors.lastname: Hauswirth
authors.affiliation: Department of Ophthalmology, Johns Hopkins University School of Medicine, 400 N. Broadway, Baltimore, MD, 21287, USA; Department of Neuroscience, Johns Hopkins University School of Medicine, 400 N. Broadway, Baltimore, MD, 21287, USA; Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, 400 N. Broadway, Baltimore, MD, 21287, USA; Institute of Genetic Medicine, Johns Hopkins University School of Medicine, 400 N. Broadway, Baltimore, MD, 21287, USA; Institute de la Vision, Université Pierre et Marie Curie, 17 Rue Moreau, Paris, 75012, France.
authors.forename: Donald J
authors.initials: DJ
authors.lastname: Zack
authors.affiliation: Department of Ophthalmology, Johns Hopkins University School of Medicine, 400 N. Broadway, Baltimore, MD, 21287, USA.
authors.forename: Shannath L
authors.initials: SL
authors.lastname: Merbs
authors.affiliation: Department of Biology, 951 Carrier Drive, MSC 7801, James Madison University, Harrisonburg, VA, 22807, USA; Center for Genome & Metagenome Studies, 951 Carrier Drive, MSC 7801, James Madison University, Harrisonburg, VA, 22807, USA. Electronic address: enkera@jmu.edu.
authors.forename: Raymond A
authors.initials: RA
authors.lastname: Enke
mesh_terms: D000818:Animals; D002213:Capsid; D002478:Cells, Cultured; D000229:Dependovirus / Q000235:genetics*; D004195:Disease Models, Animal; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D005901:Glaucoma / Q000150:complications / Q000235:genetics / Q000628:therapy*; D058449:Intravitreal Injections; D051379:Mice; D009154:Mutation; D009901:Optic Nerve Diseases / Q000209:etiology / Q000235:genetics / Q000628:therapy; D011994:Recombinant Proteins / Q000008:administration & dosage*; D012165:Retinal Ganglion Cells / Q000378:metabolism* / Q000473:pathology; D014161:Transduction, Genetic
chemical_list: D011994:Recombinant Proteins
keywords: AAV2; Cre toxicity; Cre-mediated mutation; Glaucoma; Intravitreal injection; Optic neuropathy; Retina; Retinal ganglion cell