Differential Proteomics and Functional Research following Gene Therapy in a Mouse Model of Leber Congenital Amaurosis

Abstract

Leber congenital amaurosis (LCA) is one of the most severe forms of inherited retinal degeneration and can be caused by mutations in at least 15 different genes. To clarify the proteomic differences in LCA eyes, a cohort of retinal degeneration 12 (rd12) mice, an LCA2 model caused by a mutation in the RPE65 gene, were injected subretinally with an AAV vector (scAAV5-smCBA-hRPE65) in one eye, while the contralateral eye served as a control. Proteomics were compared between untreated rd12 and normal control retinas on P14 and P21, and among treated and untreated rd12 retinas and control retinas on P42. Gene therapy in rd12 mice restored retinal function in treated eyes, which was demonstrated by electroretinography (ERG). Proteomic analysis successfully identified 39 proteins expressed differently among the 3 groups. The expression of 3 proteins involved in regulation of apoptosis and neuroptotection (alpha A crystallin, heat shock protein 70 and peroxiredoxin 6) were investigated further. Immunofluorescence, Western blot and real-time PCR confirmed the quantitative changes in their expression. Furthermore, cell culture studies suggested that peroxiredoxin 6 could act in an antioxidant role in rd12 mice. Our findings support the feasibility of gene therapy in LCA2 patients and support a role for alpha A crystallin, heat shock protein 70 and peroxiredoxin 6 in the pathogenetic mechanisms involved in LCA2 disease process.

PRDX6 Protects ARPE-19 Cells from Oxidative Damage via PI3K/AKT Signaling

“…The mRNA and protein expression and enzymatic activity of PRDX6 markedly increase in the mouse when exposed to 100% oxygen [33]. Additionally, two-dimensional electrophoresis and mass spectrometry analyses of retinas of mice with HRD indicated higher PRDX6 expression [34]. In the present study, we demonstrated for the first time that PRDX6 could protect ARPE-19 cells against oxidative damage by H 2 O 2 .…”

Human retinal gene therapy for Leber congenital amaurosis shows advancing retinal degeneration despite enduring visual improvement

“…Similar experiments in mutant mice treated early with gene therapy have shown survival of cone (12,13,15,16) and rod photoreceptors (49). Investigation of the mechanism(s) causing photoreceptor cell loss (18,(53)(54)(55)(56) has been mostly performed in untreated eyes to date. Extension of such studies to eyes undergoing gene therapy (18) would be important to clarify molecular underpinnings of the observed modification of natural history.…”

“…Investigation of the mechanism(s) causing photoreceptor cell loss (18,(53)(54)(55)(56) has been mostly performed in untreated eyes to date. Extension of such studies to eyes undergoing gene therapy (18) would be important to clarify molecular underpinnings of the observed modification of natural history. Furthermore, the newly discovered vitamin A isomerization pathway originating in Muller cells and subserving cones (57) may be found to contribute to the differences in (cone or rod) photoreceptor survival.…”

“…To explain both findings, we postulate that there exists a critical limit in terms of accumulating molecular changes that are detrimental to photoreceptor survival under this condition of severe RPE65 dysfunction. There is evidence for molecular changes occurring in rod and cone photoreceptors in the predegenerative phase of RPE65 disease (17,18,32,54,58), and a specific translocation of one of the proapoptotic Bax isoforms from the cytosol to mitochondria has been observed to correlate with the initiation of photoreceptor cell loss (54,55). To date, there has been a paucity of murine studies of gene therapy in older animals after the onset of retina-wide rod degeneration (6,59).…”

“…It is hypothesized that correction of the underlying cellular dysfunction in a human recessive retinal degeneration will promote greater neuronal survival by slowing the natural rate of photoreceptor loss. Gene therapy experiments that have evaluated photoreceptor degeneration have been performed mainly in mouse and dog models treated at young ages (12)(13)(14)(15)(16)(17)(18) and have implied that at least a subset of photoreceptors could be rescued from degeneration. However, accumulating evidence has suggested that human RPE65-LCA, which can show degeneration prenatally or within the first years of life (19,20), is better modeled at the more advanced disease stages in older animals (3,21).…”