title: Antisense oligonucleotide-based therapy in human erythropoietic protoporphyria.
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.
authors.affiliation: Institut National de la Santé et de la Recherche Médicale, U1149, Centre de Recherches sur l'Inflammation, F-75018 Paris, France.
authors.forename: Vincent
authors.initials: V
authors.lastname: Oustric
authors.affiliation: Institut National de la Santé et de la Recherche Médicale, U1149, Centre de Recherches sur l'Inflammation, F-75018 Paris, France.
authors.forename: Hana
authors.initials: H
authors.lastname: Manceau
authors.affiliation: Institut National de la Santé et de la Recherche Médicale, U1149, Centre de Recherches sur l'Inflammation, F-75018 Paris, France.
authors.forename: Sarah
authors.initials: S
authors.lastname: Ducamp
authors.affiliation: Institut National de la Santé et de la Recherche Médicale, U1149, Centre de Recherches sur l'Inflammation, F-75018 Paris, France.
authors.forename: Rima
authors.initials: R
authors.lastname: Soaid
authors.affiliation: 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.
authors.forename: Zoubida
authors.initials: Z
authors.lastname: Karim
authors.affiliation: 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.
authors.forename: Caroline
authors.initials: C
authors.lastname: Schmitt
authors.affiliation: Institut National de la Santé et de la Recherche Médicale, U1149, Centre de Recherches sur l'Inflammation, F-75018 Paris, France.
authors.forename: Arienne
authors.initials: A
authors.lastname: Mirmiran
authors.affiliation: Institut National de la Santé et de la Recherche Médicale, U1149, Centre de Recherches sur l'Inflammation, F-75018 Paris, France.
authors.forename: Katell
authors.initials: K
authors.lastname: Peoc'h
authors.affiliation: 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.
authors.forename: Bernard
authors.initials: B
authors.lastname: Grandchamp
authors.affiliation: 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.
authors.forename: Carole
authors.initials: C
authors.lastname: Beaumont
authors.affiliation: 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.
authors.forename: Said
authors.initials: S
authors.lastname: Lyoumi
authors.affiliation: 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.
authors.forename: François
authors.initials: F
authors.lastname: Moreau-Gaudry
authors.affiliation: 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.
authors.forename: Véronique
authors.initials: V
authors.lastname: Guyonnet-Dupérat
authors.affiliation: 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.
authors.forename: Hubert
authors.initials: H
authors.lastname: de Verneuil
authors.affiliation: 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.
authors.forename: Joëlle
authors.initials: J
authors.lastname: Marie
authors.affiliation: 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.
authors.forename: Herve
authors.initials: H
authors.lastname: Puy
authors.affiliation: 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.
authors.forename: Jean-Charles
authors.initials: JC
authors.lastname: Deybach
authors.affiliation: 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.
authors.forename: Laurent
authors.initials: L
authors.lastname: Gouya
mesh_terms: D002460:Cell Line; D005260:Female; D005294:Ferrochelatase / Q000235:genetics*; D006801:Humans; D008297:Male; D016376:Oligonucleotides, Antisense / Q000627:therapeutic use*; D010375:Pedigree; D011110:Polymorphism, Genetic; D046351:Protoporphyria, Erythropoietic / Q000628:therapy*; D011524:Protoporphyrins / Q000378:metabolism; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics
chemical_list: D016376:Oligonucleotides, Antisense; D011524:Protoporphyrins; D012333:RNA, Messenger; C028025:protoporphyrin IX; D005294:Ferrochelatase