Characterization of intravitreally delivered capsid mutant AAV2-Cre vector to induce tissue-specific mutations in murine retinal ganglion cells. (October 2016)

• Record Type: Journal Article
• Authors: Langouet-Astrie, Christophe J.; Yang, Zhiyong; Polisetti, Sraavya M.; Welsbie, Derek S.; Hauswirth, William W.; Zack, Donald J.; Merbs, Shannath L.; Enke, Raymond A.
• Is Part Of: Experimental eye research. Volume 151(2016:Oct.)
• Page Start: 61
• Page End: 67
• Publication Date: 2016-10
• Subjects: Retina -- Cre-mediated mutation -- Cre toxicity -- Retinal ganglion cell -- AAV2 -- Intravitreal injection -- Optic neuropathy -- Glaucoma; RGCs retinal ganglion cells -- AAV2 Adeno-associated virus 2 -- R26 Rosa 26 -- Vg vector genomes -- GFP green fluorescent protein -- Tuj1 βIII-tubulin -- ANOVA analysis of variance -- Y Tyrosine -- F phenylalanine -- INL inner nuclear layer -- ONL outer nuclear layer -- DLK Dual Leucine Zipper Kinase -- GCL ganglion cell layer -- sc sclera -- PR photoreceptors; Ophthalmology -- Periodicals; Eye -- Periodicals; Œil -- Périodiques; Ophthalmology; Periodicals; Electronic journals; 612.8405
• DOI: 10.1016/j.exer.2016.07.019
• Languages: English
• ISSNs: 0014-4835

Abstract

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. Graphical abstract: Highlights: An effective timing and dosage regiment for in vivo targeting of capsid mutant AAV2-Cre viral particles to murine ganglion cell layer retinal neurons is demonstrated. Unintended loxP-independent toxicity is observed in RGCs with long-term Cre expression. Short-term Cre expression strategies targeting adult murine RGCs have no measurable impact on RGC survival within a 4 week time frame. Short-term Cre expression studies can be conducted in tandem with axonal injury rodent models for genetic analysis of optic neuropathies.