title: Murine retroviral but not human cellular promoters induce in vivo erythroid-specific deregulation that can be partially prevented by insulators.
abstract: We are developing lentiviral vectors for gene therapy of red blood cell disorders that co-express a transgene in an erythroid-specific manner and the O(6)-methylguanine-DNA-methyltransferase (MGMT) selective gene in a constitutive way. We report that transduction of murine hematopoietic stem cells (HSCs) with a human phosphoglycerate kinase promoter-based vector at low multiplicity of infection (MOI) does not result in a selective in vivo expansion in the presence of alkylating agents. In contrast, by replacing this cellular promoter with the powerful retroviral-derived myeloproliferative sarcoma virus enhancer, negative control region-deleted, dl587rev primer-binding site substituted promoter, the vector allowed efficient chemoprotection of transduced HSCs at low MOI. However, this promoter interacted with the erythroid HS40/ankyrin enhancer/promoter driving green fluorescent protein, leading to an unexpected loss of erythroid specificity. A partial restoration of tissue-specific expression was obtained by interposition of insulator sequences between the expression units. Alternatively, we found that the strong human cellular elongation factor1-alpha promoter allows similar chemoprotection but without any deregulation of the erythroid-specific promoter in the absence of insulators. These data demonstrate that the level of in vivo deregulation induced by a promoter is not correlated with its transcriptional activity.
authors.affiliation: INSERM E0217, IFR 66, F-33000, Bordeaux, France [2] 2Université Victor Ségalen Bordeaux 2, Bordeaux, France.
authors.forename: Elodie
authors.initials: E
authors.lastname: Robert-Richard
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Richard
authors.forename: Punam
authors.initials: P
authors.lastname: Malik
authors.forename: Cécile
authors.initials: C
authors.lastname: Ged
authors.forename: Hubert
authors.initials: H
authors.lastname: de Verneuil
authors.forename: François
authors.initials: F
authors.lastname: Moreau-Gaudry
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D041905:Erythroid Cells / Q000378:metabolism*; D018628:Gene Dosage / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D008807:Mice, Inbred BALB C; D011401:Promoter Regions, Genetic / Q000235:genetics*; D013950:Thymus Gland / Q000378:metabolism