LONG-TERM PROTECTION BY COMPLEMENT REGULATORY PROTEIN CRRY IN A MOUSE MODEL OF LIPOFUSCIN-MEDIATED RETINAL DEGENERATION

Abstract

Purpose: Mutations in the human ABCA4 gene cause recessive Stargardt disease (STGD1) and predispose to the development of age-related macular degeneration (AMD). Mice with a knockout mutation in the abca4 gene (abca4-/- mice) accumulate A2E-lipofuscin in the retinal pigment epithelium (RPE) and exhibit slow photoreceptor degeneration. Recently, we described complement activation and increased oxidative stress in abca4-/- mice due to A2E accumulation. In this study we tested the hypothesis that increasing expression of complement regulatory proteins (CRP’s) in the RPE would protect these cells from complement attack and hence protect photoreceptors from degenerating.

Methods: We generated a recombinant adeno-associated virus containing mouse CRRY (rAAV8-CRRY), which we injected subretinally into one-month-old albino abca4-/- and BALB/c mice. Negative control mice received AAV8-null or rAAV8-GFP viruses. Fundus imaging was performed at different times following the injections. Expression of CRRY was determined by qRT-PCR, immunoblotting and immunocytochemistry. Spectral-domain optical-coherence tomography (sdOCT) and electroretinography (ERG) were obtained as previously described. Visual retinoids and lipofuscin pigments were quantified by high-performance liquid chromatography.

Results: At 12-months post-injection, mice that received rAAV-CRRY showed 2 to 7-fold increased expression of CRRY by qRT-PCR and quantitative immunoblotting. Fundus images of these mice were normal, suggesting no detrimental effects of the injections. The mean scotopic a-wave and b-wave ERG’s show no significant difference between the CRRY and Null-treated eyes. We observed two-fold decreased A500-lipofuscin and a three-fold decreased A2E levels in CRRY-treated abca4-/- mice at 11-months post-injection. sdOCT showed a limited maintenance in overall retinal thickness in CRRY-treated compared to Null virus-treated mice, which correlated with enhanced preservation of photoreceptors and increase in 11-cis-retinal chromophore levels.

Conclusions: Aberrant complement activation is strongly linked to the development of AMD. For the first time, we show that inhibiting complement-convertase activity by gene therapy with a virus that increased CRRY expression may be useful in treating age-related retinal degenerations.

Keywords: 538 gene transfer/gene therapy • 555 immunomodulation/immunoregulation • 412 age-related macular degeneration