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Abstract
Gene therapy of genetic diseases requires persistent and position-independent expression of a therapeutic transgene. Transcriptional enhancers binding chromatin-remodeling and modifying complexes may play a role in shielding transgenes from repressive chromatin effects. We tested the activity of the HS2 enhancer of the GATA1 gene in protecting the expression of a β-globin minigene delivered by a lentiviral vector in hematopoietic stem/progenitor cells. Gene expression from proviruses carrying GATA1-HS2 in both LTRs was persistent and resistant to silencing at most integration sites in the in vivo progeny of human hematopoietic progenitors and murine long-term repopulating stem cells. The GATA1-HS2-modified vector allowed correction of murine β-thalassemia at low copy number without inducing clonal selection of erythroblastic progenitors. Chromatin immunoprecipitation studies showed that GATA1 and the CBP acetyltransferase bind to GATA1-HS2, significantly increasing CBP-specific histone acetylations at the LTRs and β-globin promoter. Recruitment of CBP by the LTRs thus establishes an open chromatin domain encompassing the entire provirus, and increases the therapeutic efficacy of β-globin gene transfer by reducing expression variegation and epigenetic silencing.
Highlights
Gene therapy of genetic diseases requires persistent and position-independent expression of a therapeutic transgene
We previously reported that the GATA1-HS2 element cloned in transcriptionally active viral long terminal repeats (LTRs) drives erythroid-restricted transgene expression in both MLV- and HIV-derived vectors [33,34]
GFP+ cells were detected at a significantly higher proportion in the Ter119+ compartment of mice transplanted with G-CMV-GFP-transduced hematopoietic stem cells (HSCs), compared to mice transplanted with CMV-GFPtransduced HSCs (5664% vs. 2868%, P,0.05, Figure 2A and Figure S1A)
Summary
Gene therapy of genetic diseases requires persistent and position-independent expression of a therapeutic transgene. We tested the activity of the HS2 enhancer of the GATA1 gene in protecting the expression of a b-globin minigene delivered by a lentiviral vector in hematopoietic stem/progenitor cells. Gene expression from proviruses carrying GATA1-HS2 in both LTRs was persistent and resistant to silencing at most integration sites in the in vivo progeny of human hematopoietic progenitors and murine long-term repopulating stem cells. Chromatin immunoprecipitation studies showed that GATA1 and the CBP acetyltransferase bind to GATA1-HS2, significantly increasing CBP-specific histone acetylations at the LTRs and b-globin promoter. Recruitment of CBP by the LTRs establishes an open chromatin domain encompassing the entire provirus, and increases the therapeutic efficacy of bglobin gene transfer by reducing expression variegation and epigenetic silencing
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