Intravitreal Injection of Splice-switching Oligonucleotides to Manipulate Splicing in Retinal Cells.

Xavier Gérard,Isabelle Perrault,Jean-Michel Rozet,Josseline Kaplan,Arnold Munnich

doi:10.1038/mtna.2015.24

Abstract

Leber congenital amaurosis is a severe hereditary retinal dystrophy responsible for neonatal blindness. The most common disease-causing mutation (c.2991+1655A>G; 10–15%) creates a strong splice donor site that leads to insertion of a cryptic exon encoding a premature stop codon. Recently, we reported that splice-switching oligonucleotides (SSO) allow skipping of the mutant cryptic exon and the restoration of ciliation in fibroblasts of affected patients, supporting the feasibility of a SSO-mediated exon skipping strategy to correct the aberrant splicing. Here, we present data in the wild-type mouse, which demonstrate that intravitreal administration of 2'-OMePS-SSO allows selective alteration of Cep290 splicing in retinal cells, including photoreceptors as shown by successful alteration of Abca4 splicing using the same approach. We show that both SSOs and Cep290 skipped mRNA were detectable for at least 1 month and that intravitreal administration of oligonucleotides did not provoke any serious adverse event. These data suggest that intravitreal injections of SSO should be considered to bypass protein truncation resulting from the c.2991+1655A>G mutation as well as other truncating mutations in genes which like CEP290 or ABCA4 have a mRNA size that exceed cargo capacities of US Food and Drug Administration (FDA)-approved adeno-associated virus (AAV)-vectors, thus hampering gene augmentation therapy.

Highlights

Retinal diseases are a leading cause of incurable severe visual dysfunction worldwide and a major public health problem
We present data in the wild-type mouse, which demonstrate that intravitreal administration of 2’-OMePS-splice-switching oligonucleotides (SSO) allows selective alteration of Cep[290] splicing in retinal cells, including photoreceptors as shown by successful alteration of Abca[4] splicing using the same approach. We show that both SSOs and Cep[290] skipped mRNA were detectable for at least 1 month and that intravitreal administration of oligonucleotides did not provoke any serious adverse event
For each of the two exons, we produced a set of three 2′-OMePS oligonucleotides

Summary

Introduction

Retinal diseases are a leading cause of incurable severe visual dysfunction worldwide and a major public health problem. Leber congenital amaurosis (LCA, MIM204000) is the earliest and most severe of these diseases and a leading cause of blindness in childhood. It is characterized by major clinical, genetic, and physiopathological heterogeneity with non syndromic and syndromic forms, variable visual outcomes, and more than 45 disease genes with variable inheritance, pattern of expression, and retinal function.[1] The safety and efficacy of AAV-based gene replacement therapy in LCA patients harboring RPE65 mutations have paved the way for treating retinal diseases.[2,3,4] FDA-approved AAV vector genomes are limited in size. The 7.9 kb CEP290 cDNA are currently not amenable to AAV-based gene therapy

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