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Xavier GERARD, Isabelle Perrault, Arnold Munnich, Josseline Kaplan, Jean-Michel Rozet; AON intravitreal injections allows manipulating splicing in retinal cells.. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3313.
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© ARVO (1962-2015); The Authors (2016-present)
Leber congenital amaurosis (LCA) is the most common cause of incurable hereditary blindness in children. CEP290 mutations are the leading cause of the disease. It encodes a ciliary protein important to photoreceptor connecting cilium assembly and/or function. The CEP290 intronic c.2991+1655A>G change is the most common LCA-causing mutation (10% of all cases) and therefore an important therapeutic target. It introduces a cryptic exon in the mRNA and encodes a premature termination codon. Recently, we made the proof-of-concept of antisense oligonucleotides (AON)-mediated exon skipping to correct the splicing in patient fibroblasts which recovered ability to ciliate. The purpose of the present study was to make the proof-of-concept of exon-skipping in vivo using intravitreal injections of AONs.
AON sequences were designed to skip mouse Cep290 exon 36 with preservation of the reading frame. Variable concentrations of fluorescent 2’OMePS AONs that proved efficient skipping in NIH3T3 cells were injected into the vitreous of 8 week-old wildtype C57BL/6J mice. Eyes injected with sense versions and non-injected eyes were used as control. Retinal sections and mRNA were prepared at 2, 4, 8, 12, 18 and 30 days post-injections (dpi) to follow i) the distribution of oligonucleotides across cellular layers and ii) exon skipping.
In-vitro analyses identified 2’OMePS AONs allowing skipping of Cep290 exon 36; the most efficient of them and their sense versions were injected in the vitreous of animals. The pic of fluorescence in the photoreceptor layer occurred at 12 dpi. Dose-response analyses were performed at this time and showed skipping was a function of the dose of AON. Duration of action studies using 10 nmoles of AON revealed significant amounts of mRNA lacking exon 36 at day 2 post-injection which decreased progressively but mutant mRNA was still detectable at day 30 post-injection.
Here we report that single intravitreal injection of AON allows manipulating splicing in retinal cells for at least 1 month. This strategy may be regarded as an attractive alternative to gene replacement therapy for 10 % of patients affected with LCA.
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