Antisense oligonucleotide delivery is an effective therapeutic approach for CEP290-associated LCA

Abstract

Purpose : Leber congenital amaurosis (LCA) is a genetically heterogeneous disorder characterized by severe visual impairment starting in the first year of life. The most frequent genetic cause of LCA, present in up to 15% of all LCA cases in some Western European and North American populations, is an intronic mutation in CEP290 (c.2991+1655A>G) that creates a cryptic slice donor site and results in the insertion of an aberrant pseudoexon (exon X) into CEP290 mRNA. Previously, we have shown that antisense oligonucleotides (AONs) effectively restore normal CEP290 splicing in patient-derived lymphoblastoid cells. Given the safety and efficacy of adeno-associated viruses (AAVs) used in ongoing clinical trials for other genetic subtypes of retinal dystrophy, we aimed to explore the therapeutic potential of both naked AONs and AON-containing AAVs in vitro and in vivo.

Methods : We used naked chemically modified AON molecules and generated AONs cloned into a modified U7snRNA construct that were packaged into AAV particles. Naked AONs or AAV2/2-AONs were delivered to patient-derived fibroblasts. Subsequently, the effect was assessed by RT-PCR, Western blot and immunocytochemical analyses. For in vivo studies, we employed our transgenic humanized Cep290 mouse model that carries the deep-intronic CEP290 mutation. Mice of 2 months old were injected with either naked AONs (intravitreally) or AAV2/9-AONs (subretinally). By RT-PCR analysis, the amount of aberrantly spliced Cep290 was assessed. Morphological and stress analyses were performed by toluidine-blue staining and immunodetection of the gliosis marker GFAP.

Results : In patient fibroblasts, naked AON or AAV2/2-AON delivery fully restored normal CEP290 pre-mRNA splicing and significantly increased CEP290 protein levels. Moreover, a ciliary phenotype present in these fibroblasts was completely rescued upon treatment. Intravitreal (naked AONs) or subretinal (AAV2/9-AONs) delivery to our humanized mouse model resulted in a statistically significant decrease of exon-X containing Cep290 transcripts without compromising retinal structure, as shown by the absence of any abnormalities at the morphological level or of gliosis.

Conclusions : Together, our data show that naked AONs and AAV-AONs are excellent therapeutic molecules to restore splice defects in the retina, and highlight the tremendous potential of AONs for the treatment of CEP290-associated LCA.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.