rAAV-mediated ASAH1 overexpression prevents retinal degeneration in Farber disease

Abstract

Purpose : Farber disease (FD) is a rare monogenic lysosomal storage disorder caused by mutation of ASAH1, leading to deficiency of acid ceramidase (ACDase) and accumulation of ceramide characterized by retinopathy, macular red spots, corneal opacities, and nystagmus. As gene therapy has been demonstrated to be a promising therapeutic avenue for the treatment of recessive diseases, we evaluated the efficacy of rAAV-mediated hASAH1 over-expression in a mouse model of FD, using in vivo ocular imaging, electroretinography (ERG), post-mortem histology, and mass spectrometry (MS) to assess efficacy.

Methods : Expression and function of ACDase from the rAAV2/2[MAX].hASAH1 vector was assessed by western blot (WB) and activity assay in HEK293T cells. 3-4 week old FD mice (Asah1^P361R/P361R) and littermate controls (Asah1^+/+ and Asah1^P361R/+) received a unilateral intravitreal injection of rAAV2/2[MAX].hASAH1 (2x10¹⁰ vg/eye); the contralateral eye received a sham buffer injection of equal volume (2μL). Animals were followed up by confocal scanning laser ophthalmoscopy (cSLO), optical coherence tomography (OCT), and ERG at 5-6 and 8-9 weeks. Eyecups were harvested post-mortem for correlative histology and MS to determine the quantity of each ceramide species.

Results : WB/ceramidase activity assay showed the rAAV2/2[MAX].hASAH1 vector expressed high levels of biologically functional protein. cSLO imaging of untreated Asah1^P361R/P361R eyes (N=8) revealed increased hyperreflectivity and autofluorescence surrounding the optic nerve associated with elevated ceramide levels, retinal thickening and decreased ERG amplitudes. rAAV2/2[MAX].hASAH1 treatment (N=8 eyes) significantly reduced central retinal thickening (P≤0.038), ceramide accumulation (P=0.0006), and limited fundus hyperreflectivity and autofluorescence, but did not lead to functional rescue. Unexpectedly, ACDase over-expression in Asah1^+/+ and Asah1^P361R/+ control eyes induced abnormal retinal thickening and ceramide accumulation, similar to FD.

Conclusions : rAAV-mediated over-expression of ACDase effectively prevented the development of anatomical lesions in FD mice, but was not sufficient to rescue function. That over-expression of ACDase in control eyes resulted in increased ceramide accumulation and the development of FD-like lesions indicates that modulating ceramide levels as a therapeutic strategy in diseases other than lysosomal storage disorders may be contraindicated.

This is a 2021 ARVO Annual Meeting abstract.