RESUMEN
Retrovirus-mediated gene transfer of the human beta-globin gene into hematopoietic stem cells is an attractive approach to the therapy of human beta-globin gene disorders. However, expression of the transduced beta-globin gene linked to its proximal cis-acting sequences (-0.8 to +0.3 kb from the cap site) is considerably below the level required for a significant therapeutic effect. The discovery of the beta-locus control region (beta-LCR), organized in four major DNase I hypersensitive sites far upstream of the human beta-like globin gene cluster, provided a potential means to achieve a high level of expression of a linked human beta-globin gene, but initial attempts to incorporate beta-LCR derivatives in retroviral vectors resulted in the production of low-titer viruses with multiple rearrangements of the transmitted proviral structures. We now describe how extensive mutagenesis of the transduced beta-globin gene, eliminating a 372 bp intronic segment and multiple reverse polyadenylation and splicing signals, increases viral titer significantly and restores stability of proviral transmission upon infection of cell lines and bone marrow-repopulating cells. These optimized vectors have enabled us to analyze the expression properties of various retrovirally transduced beta-LCR derivatives in dimethylsulfoxide-induced murine erythroleukemia cells and to achieve ratios of human beta-globin/murine beta maj-globin mRNA, on a per gene basis, as high as 80%.
Asunto(s)
Vectores Genéticos , Globinas/genética , Secuencias Reguladoras de Ácidos Nucleicos , Retroviridae/genética , Animales , Secuencia de Bases , Células de la Médula Ósea , Línea Celular , ADN Viral , Células Precursoras Eritroides/metabolismo , Técnicas de Transferencia de Gen , Terapia Genética , Humanos , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Provirus/genética , ARN Mensajero/análisisRESUMEN
We have identified an erythroid-specific enhancer element far upstream of the human "beta-like globin" genes, at 10.2-11.0 kilobases 5' of the embryonic epsilon-globin gene, and thus at 53-54 kilobases 5' of the adult beta-globin gene. It is capable of enhancing the expression of a cis-linked test gene by up to 300-fold. This enhancer element is apparently developmental-stage-independent, as it is functional at the embryonic and the adult developmental stages in erythroid cells that are expressing the respective beta-like globin genes. The enhancer and globin promoter sequences work in synergy and are capable of conferring on a cis-linked gene the high transcriptional efficiency (enhancer function), erythroid specificity (enhancer and promoter functions), and developmental-stage specificity (promoter function) that are characteristic of the in vivo transcription of the beta-like globin genes in erythroid cells.
Asunto(s)
Elementos de Facilitación Genéticos , Eritrocitos/fisiología , Eritropoyesis , Regulación de la Expresión Génica , Globinas/genética , Factores de Edad , Animales , Cricetinae , Células HeLa , Humanos , Mapeo RestrictivoAsunto(s)
Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Globinas/genética , Animales , Desarrollo Embrionario y Fetal , Células Precursoras Eritroides/metabolismo , Genes , Globinas/biosíntesis , Humanos , Ratones , Especificidad de Órganos , Proteínas Recombinantes de Fusión/biosíntesis , Transcripción Genética , Células Tumorales CultivadasAsunto(s)
Cromosomas , ADN , ARN , Genes Reguladores , Histonas/análisis , Biología Molecular , ARN Nucleotidiltransferasas , Moldes GenéticosRESUMEN
The isolated chromatin of higher organisms possesses several properties characteristic of the same chromatin in life. These include the presence of histone bound to DNA, the state of repression of the genetic material, and the ability to serve as template for the readout of the derepressed portion of the genome by RNA polymerase. The important respect in which isolated chromatin differs from the material in vivo, fragmentation of DNA into pieces shorter (5 x 10(6) to 20 x 10(6) molecular weight) than the original, does not appear to importantly alter such transcription. The study of isolated chromatin has already revealed the material basis of the restriction of template activity; it is the formation of a complex between histone and DNA. Chromatin isolated by the methods now available, together with the basis provided by our present knowledge of chromatin biochemistry and biophysics, should make possible and indeed assure rapid increase in our knowledge of chromosomal structure and of all aspects of the control of gene activity and hence of developmental processes.