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Am J Hum Genet. 2014 Apr 3;94(4):611-7. doi: 10.1016/j.ajhg.2014.02.010. Epub 2014 Mar 27.

Antisense oligonucleotide-based therapy in human erythropoietic protoporphyria.

Author information

1
Institut National de la Santé et de la Recherche Médicale, U1149, Centre de Recherches sur l'Inflammation, F-75018 Paris, France.
2
Institut National de la Santé et de la Recherche Médicale, U1149, Centre de Recherches sur l'Inflammation, F-75018 Paris, France; Université Paris Diderot, F-75018 Paris, France.
3
Institut National de la Santé et de la Recherche Médicale, U1149, Centre de Recherches sur l'Inflammation, F-75018 Paris, France; Université Paris Diderot, F-75018 Paris, France; Assistance Publique-Hôpitaux de Paris, Centre Français des Porphyries, Hôpital Louis Mourier, 178 Rue des Renouillers, F-92701 Colombes, France.
4
Institut National de la Santé et de la Recherche Médicale, U1149, Centre de Recherches sur l'Inflammation, F-75018 Paris, France; Université Paris Diderot, F-75018 Paris, France; Assistance Publique-Hôpitaux de Paris, Laboratoire de Biochimie Hormonale et Génétique, Hôpital Bichat, F-75018 Paris, France.
5
Institut National de la Santé et de la Recherche Médicale, U1149, Centre de Recherches sur l'Inflammation, F-75018 Paris, France; Université de Versailles Saint Quentin en Yvelines, F-78035 Versailles, France.
6
Institut National de la Santé et de la Recherche Médicale, U1035, Biothérapies des Maladies Génétiques et Cancers, Laboratoire d'Excellence du Globule Rouge, F-33000 Bordeaux, France; Université Bordeaux Segalen, F-33000 Bordeaux, France.
7
Centre de Génétique Moléculaire, Centre National de la Recherche Scientifique, UPR 3404, Avenue de Terrasse, 91198 Gif-sur-Yvette, Université Paris-Sud, 91400 Orsay, France.
8
Institut National de la Santé et de la Recherche Médicale, U1149, Centre de Recherches sur l'Inflammation, F-75018 Paris, France; Université Paris Diderot, F-75018 Paris, France; Assistance Publique-Hôpitaux de Paris, Centre Français des Porphyries, Hôpital Louis Mourier, 178 Rue des Renouillers, F-92701 Colombes, France; Assistance Publique-Hôpitaux de Paris, Laboratoire de Biochimie Hormonale et Génétique, Hôpital Bichat, F-75018 Paris, France.
9
Institut National de la Santé et de la Recherche Médicale, U1149, Centre de Recherches sur l'Inflammation, F-75018 Paris, France; Université Paris Diderot, F-75018 Paris, France; Assistance Publique-Hôpitaux de Paris, Centre Français des Porphyries, Hôpital Louis Mourier, 178 Rue des Renouillers, F-92701 Colombes, France; Assistance Publique-Hôpitaux de Paris, Laboratoire de Biochimie Hormonale et Génétique, Hôpital Bichat, F-75018 Paris, France. Electronic address: jc.deybach@wanadoo.fr.
10
Institut National de la Santé et de la Recherche Médicale, U1149, Centre de Recherches sur l'Inflammation, F-75018 Paris, France; Assistance Publique-Hôpitaux de Paris, Centre Français des Porphyries, Hôpital Louis Mourier, 178 Rue des Renouillers, F-92701 Colombes, France; Université de Versailles Saint Quentin en Yvelines, F-78035 Versailles, France; Assistance Publique-Hôpitaux de Paris, Laboratoire de Biochimie Hormonale et Génétique, Hôpital Ambroise Paré, F-92100 Boulogne Billancourt, France; Laboratory of Excellence GR-Ex, 75000 Paris, France.

Abstract

In 90% of people with erythropoietic protoporphyria (EPP), the disease results from the inheritance of a common hypomorphic FECH allele, encoding ferrochelatase, in trans to a private deleterious FECH mutation. The activity of the resulting FECH enzyme falls below the critical threshold of 35%, leading to the accumulation of free protoporphyrin IX (PPIX) in bone marrow erythroblasts and in red cells. The mechanism of low expression involves a biallelic polymorphism (c.315-48T>C) localized in intron 3. The 315-48C allele increases usage of the 3' cryptic splice site between exons 3 and 4, resulting in the transcription of an unstable mRNA with a premature stop codon, reducing the abundance of wild-type FECH mRNA, and finally reducing FECH activity. Through a candidate-sequence approach and an antisense-oligonucleotide-tiling method, we identified a sequence that, when targeted by an antisense oligonucleotide (ASO-V1), prevented usage of the cryptic splice site. In lymphoblastoid cell lines derived from symptomatic EPP subjects, transfection of ASO-V1 reduced the usage of the cryptic splice site and efficiently redirected the splicing of intron 3 toward the physiological acceptor site, thereby increasing the amount of functional FECH mRNA. Moreover, the administration of ASO-V1 into developing human erythroblasts from an overtly EPP subject markedly increased the production of WT FECH mRNA and reduced the accumulation of PPIX to a level similar to that measured in asymptomatic EPP subjects. Thus, EPP is a paradigmatic Mendelian disease in which the in vivo correction of a common single splicing defect would improve the condition of most affected individuals.

PMID:
24680888
PMCID:
PMC3980518
DOI:
10.1016/j.ajhg.2014.02.010
[Indexed for MEDLINE]
Free PMC Article
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