September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Antisense oligonucleotide delivery is an effective therapeutic approach for CEP290-associated LCA
Author Affiliations & Notes
  • Alejandro Garanto
    Human Genetics, Radboudumc, Nijmegen, Netherlands
  • Daniel C Chung
    Scheie Eye Institute, Perellman School of Medicine, university of Pennsylvania, Philadelphia, Pennsylvania, United States
  • Lonneke Duijkers
    Human Genetics, Radboudumc, Nijmegen, Netherlands
  • Julio Cesar Corral Serrano
    Human Genetics, Radboudumc, Nijmegen, Netherlands
  • Muriël Messchaert
    Human Genetics, Radboudumc, Nijmegen, Netherlands
  • Ru Xiao
    Schepens Eye Research Institute, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, United States
    Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States
  • Jean Bennett
    F.M. Kirby Center for Molecular Ophthalmology and Center for Advanced Retinal and Ophthalmic Therapeutics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States
  • Luk H Vandenberghe
    Schepens Eye Research Institute, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, United States
    Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States
  • Rob Collin
    Human Genetics, Radboudumc, Nijmegen, Netherlands
  • Footnotes
    Commercial Relationships   Alejandro Garanto, None; Daniel Chung, None; Lonneke Duijkers, None; Julio Cesar Corral Serrano, None; Muriël Messchaert, None; Ru Xiao, None; Jean Bennett, None; Luk Vandenberghe, GenSight Biologics (I), Novartis, Eleven Bio, Jefferies, Sectoral, GenSight (C), Regeneron, Cowen, GenSight (R), UPenn, ReGenX, MEEI (P); Rob Collin, Radboudumc (P)
  • Footnotes
    Support  This work is financially supported by the Netherlands Organisation for Scientific Research (NWO) (VENI 916.10.096), the Foundation Fighting Blindness (FFB) USA (TA-GT-0912-0582-RAD), the FP7-PEOPLE-2012-ITN Programme EyeTN (agreement 317472), the JANIVO stichting, the Stichting August F. Deutman Researchfonds Oogheelkunde, the Rotterdamse Vereniging Blindenbelangen, the Algemene Nederlandse Vereniging ter Voorkoming van Blindheid, the Gelderse Blindenstichting, the Stichting Winckel-Sweep and the Stichting Nederlands Oogheelkundig Onderzoek (all to RWJC), the FFB USA (C-GT-09130627-UPA02) to JB, the National Institutes of Health (DP1-OD008267) to JB and LHV, the Curing Kids Fund, FFB USA (TA-GT-0611-0571) and the Grousbeck Family Foundation (all to LHV).
Investigative Ophthalmology & Visual Science September 2016, Vol.57, No Pagination Specified. doi:
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      Alejandro Garanto, Daniel C Chung, Lonneke Duijkers, Julio Cesar Corral Serrano, Muriël Messchaert, Ru Xiao, Jean Bennett, Luk H Vandenberghe, Rob Collin; Antisense oligonucleotide delivery is an effective therapeutic approach for CEP290-associated LCA. Invest. Ophthalmol. Vis. Sci. 201657(12):.

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      © ARVO (1962-2015); The Authors (2016-present)

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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.

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