AAV Mediated Gene Transfer Restores Retinal and Visual Function in Lebercilin-/- (LCA5) Mice

Abstract

Purpose: Lebercilin, encoded by the LCA5 gene, is a ciliary protein found in the connecting cilia of photoreceptors in vivo and in the microtubules, centrioles and primary cilia of mammalian cells cultured in vitro. It is widely expressed in human tissues throughout development. Since loss of lebercilin function is associated with Leber Congenital Amsurosis (LCA), we used a gene augmentation strategy to test the ability to correct retinal degeneration in vivo in knockout (Lca5-/-) mice.

Methods: Cohorts of genotyped neonatal (P1-2) Lca5-/- mice received intravenous injections of 15 ul of AAV2/9.CBA.lebercillin.flag via the right palpebral vein. Some animals were co-injected with a smaller dose of AAV2/9.eGFP to be able to monitor transduction efficiency non-invasively in vivo. Littermates were injected with AAV2/9.eGFP as control. Retinal/visual function testing was performed at 28-35 days post injection by pupillary light response, water maze testing, and electroretinograms. Ophthalmoscopy, fundus photography and SD-OCT imaging was used for retinal structure analysis. Animals were euthanized and eyes were sectioned for immunofluorescence analysis for gene expression and retinal structure.

Results: Mice injected with AAV2/9.CBA.Lebercilin.flag,showed improvements in speed and accuracy in the water maze test, and improved pupillary light reflexes and electroretinograms, when compared to control animals. Ophthalmoscopy and fundus photos of animals receiving injection of AAV.EGFP revealed widespread GFP-positive retinal cells. SD-OCT imaging documented an increase in outer nuclear layer (ONL) thickness in experimental versus control animals. Analysis of the experimental and control AAV-injected eyes showed expression of the transgene in the inner and outer retina. Immunofluorescence analyses in AAV2/9.CBA.lebercilin.flag-injected mice revealed flag-positive cells. The ONL was thicker in AAV2/9.CBA.Lebercilin.flag-injected mice than in untreated littermates.

Conclusions: AAV mediated gene augmentation therapy via an intravenous route can restore retinal function in Lca5-/- mice. Improvements in retinal function were demonstrated using behavioral and physiologic testing and correlated with retinal structure and transgene expression. The proof-of-concept data from these studies will expedite the process of moving forward with a human clinical trial. Acknowledgements: In collaboration with the LCA5 consortium.

Keywords: 538 gene transfer/gene therapy • 695 retinal degenerations: cell biology • 533 gene/expression