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Therapeutic levels of fetal hemoglobin in erythroid progeny of ?-thalassemic CD34+ cells after lentiviral vector-mediated gene transfer.

ABSTRACT: ?-Thalassemia major results from severely reduced or absent expression of the ?-chain of adult hemoglobin (????;HbA). Increased levels of fetal hemoglobin (????;HbF), such as occurs with hereditary persistence of HbF, ameliorate the severity of ?-thalassemia, raising the potential for genetic therapy directed at enhancing HbF. We used an in vitro model of human erythropoiesis to assay for enhanced production of HbF after gene delivery into CD34(+) cells obtained from mobilized peripheral blood of normal adults or steady-state bone marrow from patients with ?-thalassemia major. Lentiviral vectors encoding (1) a human ?-globin gene with or without an insulator, (2) a synthetic zinc-finger transcription factor designed to interact with the ?-globin gene promoters, or (3) a short-hairpin RNA targeting the ?-globin gene repressor, BCL11A, were tested. Erythroid progeny of normal CD34(+) cells demonstrated levels of HbF up to 21% per vector copy. For ?-thalassemic CD34(+) cells, similar gene transfer efficiencies achieved HbF production ranging from 45% to 60%, resulting in up to a 3-fold increase in the total cellular Hb content. These observations suggest that both lentiviral-mediated ?-globin gene addition and genetic reactivation of endogenous ?-globin genes have potential to provide therapeutic HbF levels to patients with ?-globin deficiency.

SUBMITTER: Wilber A

PROVIDER: S-EPMC3062294 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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Therapeutic levels of fetal hemoglobin in erythroid progeny of β-thalassemic CD34+ cells after lentiviral vector-mediated gene transfer.

Wilber Andrew A Hargrove Phillip W PW Kim Yoon-Sang YS Riberdy Janice M JM Sankaran Vijay G VG Papanikolaou Eleni E Georgomanoli Maria M Anagnou Nicholas P NP Orkin Stuart H SH Nienhuis Arthur W AW Persons Derek A DA

Blood 20101214 10

β-Thalassemia major results from severely reduced or absent expression of the β-chain of adult hemoglobin (α₂β₂;HbA). Increased levels of fetal hemoglobin (α₂γ₂;HbF), such as occurs with hereditary persistence of HbF, ameliorate the severity of β-thalassemia, raising the potential for genetic therapy directed at enhancing HbF. We used an in vitro model of human erythropoiesis to assay for enhanced production of HbF after gene delivery into CD34(+) cells obtained from mobilized peripheral blood o ...[more]

PMID: 21156846

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Project description:The importance of retinal glial cells in the maintenance of retinal health and in retinal degenerations has not been fully explored. Several groups have suggested that secretion of neurotrophic proteins from the retina's primary glial cell type, the Müller cell, holds promise for treating retinal degenerations. Tight regulation of transgene expression in Müller cells is likely to be critical to the efficacy of long-term neuroprotective therapies, due to the genetic heterogeneity and progressive nature of retinal disease. To this end, we developed a modified lentiviral (LV) transfer vector (pFTMGW) to accelerate the testing and evaluation of novel transcriptional regulatory elements. This vector facilitates identification and characterization of regulatory elements in terms of size, cell specificity and ability to control transgene expression levels.A synthetic multiple cloning site (MCS) which can accept up to five directionally cloned DNA regulatory elements was inserted immediately upstream of an enhanced green fluorescent protein (eGFP) reporter. A cytomegalovirus (CMV) promoter, required for tat-independent viral packaging, is located around 2 kb upstream of the eGFP reporter and is capable of directing transgene expression. A synthetic transcription blocker (TB) was inserted to insulate the MCS/eGFP from the CMV promoter. We evaluated eGFP expression from pFTMGW and control constructs using flow cytometry and quantitative reverse transcriptase polymerase chain reaction (RT-PCR). We also tested and compared the activity and cell specificity of a computationally identified promoter fragment from the rat vimentin gene (Vim409) in transfection and lentiviral infection experiments using fluorescence microscopy.Transfection data, quantitative RT-PCR, and flow cytometry show that around 85% of expression from the CMV promoter was blocked by the TB element, allowing direct evaluation of expression from the Vim409 candidate promoter cloned into the MCS. Lentiviruses generated from this construct containing the Vim409 promoter (without the TB element) drove robust eGFP expression in Müller cells in vitro and in vivo.The TB element efficiently prevented eGFP expression by the upstream CMV promoter and the novel MCS facilitated testing of an evolutionarily conserved regulatory element. Additional sites allow for combinatorial testing of additional promoter, enhancer, and/or repressor elements in various configurations. This modified LV transfer vector is an effective tool for expediting functional analysis of gene regulatory elements in Müller glia, and should prove useful for promoter analyses in other cell types and tissues.

Dataset Information

Therapeutic levels of fetal hemoglobin in erythroid progeny of ?-thalassemic CD34+ cells after lentiviral vector-mediated gene transfer.

Publications

Therapeutic levels of fetal hemoglobin in erythroid progeny of β-thalassemic CD34+ cells after lentiviral vector-mediated gene transfer.

Similar Datasets

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