RESUMEN
BACKGROUND: Choroideremia (CHM) is a slowly progressive X-linked retinal degeneration that results in a loss of photoreceptors, retinal pigment epithelium and choroid. CHM, the gene implicated in choroideremia, encodes Rab escort protein-1 (REP-1), which is involved in the post-translational activation via prenylation of Rab proteins. METHODS: We evaluated AAV8.CBA.hCHM, a recombinant adeno-associated virus serotype 8 (rAAV8) vector, which targets retinal cells efficiently, for both therapeutic effect and safety in vitro and in vivo in a murine model. In vitro studies included western blot analyses and prenylation assays. In vivo studies included ophthalmoscopy, pupillometry, histology and immunofluorescence analysis. RESULTS: Infection with AAV8.CBA.hCHM induced the expression of REP-1 protein in a dose-responsive fashion. Transduction with AAV8.CBA.hCHM reverses the biochemical and pathogenetic defects in CHM both in vitro and in vivo and showed no safety concerns in the in vivo investigations performed in the present study. CONCLUSIONS: AAV8 is a promising vector for human clinical gene therapy trials for choroideremia.
Asunto(s)
Coroideremia/genética , Dependovirus/genética , Terapia Genética , Vectores Genéticos/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Línea Celular , Células Cultivadas , Coroideremia/terapia , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Orden Génico , Vectores Genéticos/administración & dosificación , Vectores Genéticos/efectos adversos , Heterocigoto , Humanos , Ratones , Ratones Noqueados , Retina/metabolismo , Retina/patología , Retina/fisiopatología , Transducción Genética , TransgenesRESUMEN
Choroideremia (CHM) is an X- linked retinal degeneration that is symptomatic in the 1(st) or 2(nd) decade of life causing nyctalopia and loss of peripheral vision. The disease progresses through mid-life, when most patients become blind. CHM is a favorable target for gene augmentation therapy, as the disease is due to loss of function of a protein necessary for retinal cell health, Rab Escort Protein 1 (REP1).The CHM cDNA can be packaged in recombinant adeno-associated virus (rAAV), which has an established track record in human gene therapy studies, and, in addition, there are sensitive and quantitative assays to document REP1 activity. An animal model that accurately reflects the human condition is not available. In this study, we tested the ability to restore REP1 function in personalized in vitro models of CHM: lymphoblasts and induced pluripotent stems cells (iPSCs) from human patients. The initial step of evaluating safety of the treatment was carried out by evaluating for acute retinal histopathologic effects in normal-sighted mice and no obvious toxicity was identified. Delivery of the CHM cDNA to affected cells restores REP1 enzymatic activity and also restores proper protein trafficking. The gene transfer is efficient and the preliminary safety data are encouraging. These studies pave the way for a human clinical trial of gene therapy for CHM.