title: Transcriptional targeting of retroviral vectors to the erythroblastic progeny of transduced hematopoietic stem cells.
abstract: Targeted expression to specific tissues or cell lineages is a necessary feature of a gene therapy vector for many clinical applications, such as correction of hemoglobinopathies or thalassemias by transplantation of genetically modified hematopoietic stem cells. We developed retroviral vectors in which the constitutive viral enhancer in the U3 region of the 3' LTR is replaced by an autoregulatory enhancer of the erythroid-specific GATA-1 transcription factor gene. The replaced enhancer is propagated to the 5' LTR upon integration into the target cell genome. The modified vectors were used to transduce human hematopoietic cell lines, cord blood-derived CD34(+) stem/progenitor cells, and murine bone marrow repopulating stem cells. The expression of appropriate reporter genes (triangle upLNGFR, EGFP) was analyzed in the differentiated progeny of transduced stem cells in vitro, in liquid culture as well as in clonogenic assay, and in vivo, after bone marrow transplantation in lethally irradiated mice. The GATA-1 autoregulatory enhancer effectively restricts the expression of the LTR-driven proviral transcription unit to the erythroblastic progeny of both human progenitors and mouse-repopulating stem cells. Packaging of viral particles, integration into the target genome, and stability of the integrated provirus are not affected by the LTR modification. Enhancer replacement is therefore an effective strategy to target expression of a retroviral transgene to a specific progeny of transduced hematopoietic stem cells.
authors.affiliation: TIGET, Istituto Scientifico H.S. Raffaele, Milano, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Grande
authors.forename: B
authors.initials: B
authors.lastname: Piovani
authors.forename: A
authors.initials: A
authors.lastname: Aiuti
authors.forename: S
authors.initials: S
authors.lastname: Ottolenghi
authors.forename: F
authors.initials: F
authors.lastname: Mavilio
authors.forename: G
authors.initials: G
authors.lastname: Ferrari
mesh_terms: D000818:Animals; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics*; D004742:Enhancer Elements, Genetic; D004900:Erythroblasts / Q000166:cytology* / Q000502:physiology; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; D017930:Genes, Reporter; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000502:physiology*; D006801:Humans; D020014:K562 Cells; D004915:Leukemia, Erythroblastic, Acute; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000235:genetics; D020800:Receptor, Nerve Growth Factor; D017475:Receptors, Nerve Growth Factor / Q000096:biosynthesis / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D012190:Retroviridae; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D020298:U937 Cells
chemical_list: D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; C494688:GATA1 protein, human; C494689:Gata1 protein, mouse; D008164:Luminescent Proteins; D009687:Nuclear Proteins; D020800:Receptor, Nerve Growth Factor; D017475:Receptors, Nerve Growth Factor; D014157:Transcription Factors; D049452:Green Fluorescent Proteins