A zinc-finger transcriptional activator designed to interact with the γ-globin gene promoters enhances fetal hemoglobin production in primary human adult erythroblasts

Wilber, Andrew; Tschulena, Ulrich; Hargrove, Phillip W.; Kim, Yoon-Sang; Persons, Derek A.; Barbas, Carlos F.; Nienhuis, Arthur W.

doi:10.1182/blood-2009-08-240556

Cited by 78 publications

(80 citation statements)

References 52 publications

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“…It is well recognized that the mouse has a developmentally limited fetal hematopoietic stem cell population that gives rise to unique hematopoietic lineages, 12,13 including B-1 B cells, which generate "natural" antibodies. [14][15][16] Human fetal T cells 11 and erythrocytes 17 also appear to arise from distinct fetal progenitors. Because hematopoietic stem cells have multilineage potential, we hypothesized that human fetal myeloid cells (specifically, fetal monocytes) arising from these cells would also have distinct functional characteristics compared with their adult counterparts.…”

Section: Foxp3mentioning

confidence: 99%

Distinct functional programming of human fetal and adult monocytes

Krow-Lucal¹,

Kim²,

Burt

et al. 2014

Blood

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Key Points• Human fetal and adult classical monocytes have distinct baseline transcriptional and signaling programs.• Transcriptional and signaling differences in fetal monocytes underlie stronger responses to cytokine stimulation.Preterm birth affects 1 out of 9 infants in the United States and is the leading cause of long-term neurologic handicap and infant mortality, accounting for 35% of all infant deaths in 2008. Although cytokines including interferon-g (IFN-g), interleukin-10 (IL-10), IL-6, and IL-1 are produced in response to in utero infection and are strongly associated with preterm labor, little is known about how human fetal immune cells respond to these cytokines. We demonstrate that fetal and adult CD14 1 CD16 2 classical monocytes are distinct in terms of basal transcriptional profiles and in phosphorylation of signal transducers and activators of transcription (STATs) in response to cytokines. Fetal monocytes phosphorylate canonical and noncanonical STATs and respond more strongly to IFN-g, IL-6, and IL-4 than adult monocytes. We demonstrate a higher ratio of SOCS3 to IL-6 receptor in adult monocytes than in fetal monocytes, potentially explaining differences in STAT phosphorylation. Additionally, IFN-g signaling results in upregulation of antigen presentation and costimulatory machinery in adult, but not fetal, monocytes. These findings represent the first evidence that primary human fetal and adult monocytes are functionally distinct, potentially explaining how these cells respond differentially to cytokines implicated in development, in utero infections, and the pathogenesis of preterm labor. (Blood.

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Section: Foxp3mentioning

confidence: 99%

Distinct functional programming of human fetal and adult monocytes

Krow-Lucal¹,

Kim²,

Burt

et al. 2014

Blood

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“…24 Culture conditions used for expansion and differentiation of purified CD34 ϩ cells have been previously described. 22 Cell numbers and viability were determined by trypan blue exclusion. Cell morphology was assessed by Wright-Giemsa staining of cytocentrifuge preparations and images acquired with an Olympus Bx41 Upright microscope equipped with a DP70 digital camera and DP manager software v03.02 (Olympus).…”

Section: Purification Of Human Cd34 ؉ Cells and Erythroid Culture Conmentioning

confidence: 99%

“…(3) GG1-VP64/GFP: The details regarding the construction of the ␤-spectrin regulated bicistronic expression vector encoding for the rhesus variant of the designer zinc-finger transcription factor (GG1-VP64) and GFP have been described previously. 22 (4) shBCL11A/GFP: Vectors encoding for control (pLKO.1-PGK-GFP) and U6 regulated expression of shRNA-encoding sequences specific to the ␥-globin gene repressor protein BCL11A (pLKO.1-shBcl11A-PGK-Puro) were previously described. 23 pLKO.1-shBCL11A-PGK-Puro was modified to replace the Puro marker with the GFP marker.…”

Section: Lentiviral Vector Designmentioning

confidence: 99%

“…[18][19][20][21] Using a different approach, we recently reported the successful use of an artificial transcription factor (GG1-VP64) designed to interact with proximal ␥-globin gene promoters to increase production of HbF in erythroid progeny of normal human CD34 ϩ cells. 22 The recent discovery that the transcription factor BCL11A has a significant role in silencing of ␥-globin expression in adult erythroid cells has led to yet another potential genetic approach to induce HbF. 23 These results led us to undertake studies designed to comparatively evaluate enhancement of HbF levels through lentiviral gene transfer of (1) an erythroid-specific, ␥-globin lentiviral vector (V5m3) further modified to include a 400-bp chicken HS4 insulator element (V5m3-400), (2) the GG1-VP64 zinc-finger transcription The online version of this article contains a data supplement.…”

Section: Introductionmentioning

confidence: 99%

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

Wilber

Hargrove

Kim

et al. 2011

Blood

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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 of normal adults or steady-state bone marrow from patients with β-thalassemia major. Lentiviral vectors encoding (1) a human γ-globin gene with or without an insulator, (2) a synthetic zinc-finger transcription factor designed to interact with the γ-globin gene promoters, or (3) a short-hairpin RNA targeting the γ-globin gene repressor, BCL11A, were tested. Erythroid progeny of normal CD34+ cells demonstrated levels of HbF up to 21% per vector copy. For β-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 γ-globin gene addition and genetic reactivation of endogenous γ-globin genes have potential to provide therapeutic HbF levels to patients with β-globin deficiency.

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“…Early work on the use of engineered zinc-finger transcription factors revealed that synthetic transcriptional modulators are effective tools for a broad range of applications, enabling such tasks as inhibiting viral replication (Papworth et al 2003;Reynolds et al 2003;Segal et al 2004;Eberhardy et al 2006), modulating the expression of disease-associated loci (Graslund et al 2005;Wilber et al 2010), inducing angiogenesis for accelerated wound healing (Rebar et al 2002), and genomic screening of cellular targets for cancer progression and drug resistance Blancafort et al 2005Blancafort et al , 2008. Facilitated by many of the insights gained from zinc-finger transcription factor technology, both TALEs and CRISPR-Cas9 have now further expanded the possibilities of engineered transcriptional activators and repressors.…”

Section: Applications Of Targeted Transcriptional Regulationmentioning

confidence: 99%

Genome-Editing Technologies: Principles and Applications

Gaj

Sirk

Shui

et al. 2016

Cold Spring Harb Perspect Biol

262

142

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Targeted nucleases have provided researchers with the ability to manipulate virtually any genomic sequence, enabling the facile creation of isogenic cell lines and animal models for the study of human disease, and promoting exciting new possibilities for human gene therapy. Here we review three foundational technologies-clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9), transcription activator-like effector nucleases (TALENs), and zinc-finger nucleases (ZFNs). We discuss the engineering advances that facilitated their development and highlight several achievements in genome engineering that were made possible by these tools. We also consider artificial transcription factors, illustrating how this technology can complement targeted nucleases for synthetic biology and gene therapy.

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A zinc-finger transcriptional activator designed to interact with the γ-globin gene promoters enhances fetal hemoglobin production in primary human adult erythroblasts

Cited by 78 publications

References 52 publications

Distinct functional programming of human fetal and adult monocytes

Distinct functional programming of human fetal and adult monocytes

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

Genome-Editing Technologies: Principles and Applications

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