RESUMEN
Mutations in CEP290 are the most common cause of Leber congenital amaurosis (LCA), a severe inherited retinal degenerative disease for which there is currently no cure. Autosomal recessive CEP290-associated LCA is a good candidate for gene replacement therapy, and cells derived from affected individuals give researchers the ability to study human disease and therapeutic gene correction in vitro. Here we report the development of lentiviral vectors carrying full-length CEP290 for the purpose of correcting the CEP290 disease-specific phenotype in human cells. A lentiviral vector containing CMV-driven human full-length CEP290 was constructed. Following transduction of patient-specific, iPSC-derived, photoreceptor precursor cells, reverse transcriptase-PCR analysis and western blotting revealed vector-derived expression. As CEP290 is important in ciliogenesis, the ability of fibroblast cultures from CEP290-associated LCA patients to form cilia was investigated. In cultures derived from these patients, fewer cells formed cilia compared with unaffected controls. Cilia that were formed were shorter in patient-derived cells than in cells from unaffected individuals. Importantly, lentiviral delivery of CEP290 rescued the ciliogenesis defect. The successful construction and viral transfer of full-length CEP290 brings us closer to the goal of providing gene- and cell-based therapies for patients affected with this common form of LCA.
Asunto(s)
Antígenos de Neoplasias/genética , Células Madre Pluripotentes Inducidas/trasplante , Amaurosis Congénita de Leber/terapia , Lentivirus/genética , Proteínas de Neoplasias/genética , Células Fotorreceptoras/metabolismo , Retina/metabolismo , Animales , Antígenos de Neoplasias/metabolismo , Proteínas de Ciclo Celular , Células Cultivadas , Cilios/metabolismo , Cilios/patología , Proteínas del Citoesqueleto , Modelos Animales de Enfermedad , Fibroblastos/metabolismo , Fibroblastos/patología , Vectores Genéticos/farmacología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Amaurosis Congénita de Leber/genética , Amaurosis Congénita de Leber/patología , Ratones , Proteínas de Neoplasias/metabolismo , Retina/patología , Transducción GenéticaRESUMEN
MOTIVATION: In gene discovery projects based on EST sequencing, effective post-sequencing identification methods are important in determining tissue sources of ESTs within pooled cDNA libraries. In the past, such identification efforts have been characterized by higher than necessary failure rates due to the presence of errors within the subsequence containing the oligo tag intended to define the tissue source for each EST. RESULTS: A large-scale EST-based gene discovery program at The University of Iowa has led to the creation of a unique software method named UITagCreator usable in the creation of large sets of synthetic tissue identification tags. The identification tags provide error detection and correction capability and, in conjunction with automated annotation software, result in a substantial improvement in the accurate identification of the tissue source in the presence of sequencing and base-calling errors. These identification rates are favorable, relative to past paradigms. AVAILABILITY: The UITagCreator source code and installation instructions, along with detection software usable in concert with created tag sets, is freely available at http://genome.uiowa.edu/pubsoft/software.html CONTACT: tomc@eng.uiowa.edu
Asunto(s)
Sistemas de Administración de Bases de Datos , Etiquetas de Secuencia Expresada , Biblioteca de Genes , Almacenamiento y Recuperación de la Información/métodos , Programas Informáticos , Algoritmos , Animales , Secuencia de Bases , Modelos Genéticos , Modelos Estadísticos , Datos de Secuencia Molecular , Ratas , Reproducibilidad de los Resultados , Análisis de Secuencia de ADN , Lugares Marcados de SecuenciaRESUMEN
Autism is a severe neurodevelopmental disorder defined by social and communication deficits and ritualistic-repetitive behaviors that are detectable in early childhood. The etiology of idiopathic autism is strongly genetic, and oligogenic transmission is likely. The first stage of a two-stage genomic screen for autism was carried out by the Collaborative Linkage Study of Autism on individuals affected with autism from 75 families ascertained through an affected sib-pair. The strongest multipoint results were for regions on chromosomes 13 and 7. The highest maximum multipoint heterogeneity LOD (MMLS/het) score is 3.0 at D13S800 (approximately 55 cM from the telomere) under the recessive model, with an estimated 35% of families linked to this locus. The next highest peak is an MMLS/het score of 2.3 at 19 cM, between D13S217 and D13S1229. Our third highest MMLS/het score of 2.2 is on chromosome 7 and is consistent with the International Molecular Genetic Study of Autism Consortium report of a possible susceptibility locus somewhere within 7q31-33. These regions and others will be followed up in the second stage of our study by typing additional markers in both the original and a second set of identically ascertained autism families, which are currently being collected. By comparing results across a number of studies, we expect to be able to narrow our search for autism susceptibility genes to a small number of genomic regions. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 88:609-615, 1999.