Proteomic Analysis Following AAV-Mediated Gene Therapy in rd12 Mice, a Model of Leber Congenital Amaurosis With Rpe65 Mutation

Abstract

Purpose: : To investigate the proteomic differences in retinas of treated and untreated rd12 mice, a naturally occurring mouse model of Leber congenital amaurosis with RPE65 mutation.

Methods: : 1 µl of scAAV5-smCBA-hRPE65 (2x10¹² genome containing viral particles per ml) was subretinally injected into the right eye of two-week old rd12 mice; the left eye remained uninjected as a negative control. Dark-adapted electroretinograms (ERGs) were recorded four weeks after the subretinal injection to assess the therapeutic efficacy, followed by enucleation of both rd12 eyes and age-matched normal C57BL/6J eyes for protein analysis. Retinal protein expression profiling was carried out by two-dimensional gel electrophoresis (2-D PAGE). Proteins were identified by matrix-assisted laser desorption ionization time-of-fight (MALDI-TOF) mass spectrometry using mascot software searching against NCBInr database. Immunohistochemistry was performed to locate the proteins expression in retina.

Results: : 4 weeks after treatment, dark-adapted ERG responses in treated rd12 eyes had significantly restored amplitudes, while there was no detectable rod ERG response in untreated contralateral rd12 eyes. Proteomic profiling showed no significant difference between treated rd12 eyes and normal C57BL/6J eyes, while obvious differences were observed between untreated rd12 and treated rd12 or wild-type eyes. Expression changes were detected in forty-two proteins, among which alpha A crystallin (CRYAA, a molecular chaperone), heat shock protein 8 (HSP8) and peroxiredoxin 6 were up-regulated in untreated-rd12 eyes, but near wild-type levels in treated-rd12 eyes. Immunohistochemistry showed stronger alpha A crystallin signal in the inner nuclear layer and ganglion cell layer in untreated-rd12 eyes compared to treated rd12 or normal C57BL/6J eyes.

Conclusions: : Gene therapy restores the function and normal protein expression in the rd12 retina. The up-regulation of alpha A crystalline in untreated rd12 may have potential role to prevent cell death in the process of retinal degeneration (Rao et al., 2008), while increasing expression of peroxiredoxin 6 may relate to oxidative damage (Gosbell et al., 2006).

Keywords: proteomics • gene transfer/gene therapy • retinal degenerations: hereditary