Regulated high level expression of a human gamma-globin gene introduced into erythroid cells by an adeno-associated virus vector.

The human G gamma-globin and beta-globin genes are expressed in erythroid cells at different stages of human development, and previous studies have shown that the two cloned genes are also expressed in a differential stage-specific manner in transgenic mice. The G gamma-globin gene is expressed only in murine embryonic erythroid cells, while the beta-globin gene is active only at the fetal and adult stages. In this study, we analyzed transgenic mice carrying a series of hybrid genes in which different upstream, intragenic, or downstream sequences were contributed by the beta-globin or G gamma-globin gene. We found that hybrid 5'G gamma/3'beta globin genes containing G gamma-globin sequences upstream from the initiation codon were expressed in embryonic erythroid cells at levels similar to those of an intact G gamma-globin transgene. In contrast, beta-globin upstream sequences were insufficient for expression of 5'beta/3'G gamma hybrid globin genes or a beta-globin-metallothionein fusion gene in adult erythroid cells. However, beta-globin downstream sequences, including 212 base pairs of exon III and 1,900 base pairs of 3'-flanking DNA, were able to activate a 5'G gamma/3'beta hybrid globin gene in fetal and adult erythroid cells. These experiments suggest that positive regulatory elements upstream from the G gamma-globin and downstream from the beta-globin gene are involved in the differential expression of the two genes during development.

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Long-Term Transfer and Expression of the Human β-Globin Gene in a Mouse Transplant Model

Blood ◽

10.1182/blood.v90.9.3414 ◽

1997 ◽

Vol 90 (9) ◽

pp. 3414-3422 ◽

Cited By ~ 45

Author(s):

Harry Raftopoulos ◽

Maureen Ward ◽

Philippe Leboulch ◽

Arthur Bank

Keyword(s):

Gene Therapy ◽

Gene Transfer ◽

Bone Marrow Cells ◽

High Titer ◽

Globin Gene ◽

High Level Expression ◽

Hematopoietic Stem ◽

High Level ◽

Transplant Model

Abstract Somatic gene therapy of hemoglobinopathies depends initially on the demonstration of safe, efficient gene transfer and long-term, high-level expression of the transferred human β-globin gene in animal models. We have used a β-globin gene/β-locus control region retroviral vector containing several modifications to optimize gene transfer and expression in a mouse transplant model. In this report we show that transplantation of β-globin–transduced hematopoietic cells into lethally irradiated mice leads to the continued presence of the gene up to 8 months posttransplantation. The transferred human β-globin gene is detected in 3 of 5 mice surviving long term (>4 months) transplanted with bone marrow cells transduced with high-titer virus. Southern blotting confirms the presence of the unrearranged 5.1-kb human β-globin gene-containing provirus in 2 of these mice. In addition, long-term expression of the transferred gene is seen in 2 mice at levels of 5% and 20% that of endogenous murine β-globin at 6 and 8 months posttransplantation. We further document stem cell transduction by the successful transfer and high-level expression of the human β-globin gene from mice transduced 9 months earlier into irradiated secondary recipient mice. These results demonstrate high-level, long-term somatic human β-globin gene transfer into the hematopoietic stem cells of an animal for the first time, and suggest the potential feasibility of a retroviral gene therapy approach to sickle cell disease and the β thalassemias.

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LCR/MEL: A versatile system for high-level expression of heterologous proteins in erythroid cells

Nucleic Acids Research ◽

10.1093/nar/20.5.997 ◽

1992 ◽

Vol 20 (5) ◽

pp. 997-1003 ◽

Cited By ~ 44

Author(s):

M. Needham ◽

C. Gooding ◽

K. Hudson ◽

M. Antoniou ◽

F. Grosveld ◽

...

Keyword(s):

Heterologous Proteins ◽

Erythroid Cells ◽

High Level Expression ◽

High Level ◽

In Erythroid Cells

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High-level erythroid-specific gene expression in primary human and murine hematopoietic cells with self-inactivating lentiviral vectors

Blood ◽

10.1182/blood.v98.9.2664 ◽

2001 ◽

Vol 98 (9) ◽

pp. 2664-2672 ◽

Cited By ~ 87

Author(s):

Francois Moreau-Gaudry ◽

Ping Xia ◽

Gang Jiang ◽

Natalya P. Perelman ◽

Gerhard Bauer ◽

...

Keyword(s):

Gene Therapy ◽

Hepatitis Virus ◽

Globin Gene ◽

Hematopoietic Cells ◽

Lentiviral Vectors ◽

Specific Gene ◽

High Level Expression ◽

Specific Expression ◽

Cd34 Cells ◽

High Level

AbstractUse of oncoretroviral vectors in gene therapy for hemoglobinopathies has been impeded by low titer vectors, genetic instability, and poor expression. Fifteen self- inactivating (SIN) lentiviral vectors using 4 erythroid promoters in combination with 4 erythroid enhancers with or without the woodchuck hepatitis virus postregulatory element (WPRE) were generated using the enhanced green fluorescent protein as a reporter gene. Vectors with high erythroid-specific expression in cell lines were tested in primary human CD34+ cells and in vivo in the murine bone marrow (BM) transplantation model. Vectors containing the ankyrin-1 promoter showed high-level expression and stable proviral transmission. Two vectors containing the ankyrin-1 promoter and 2 erythroid enhancers (HS-40 plus GATA-1 or HS-40 plus 5-aminolevulinate synthase intron 8 [I8] enhancers) and WPRE expressed at levels higher than the HS2/β-promoter vector in bulk unilineage erythroid cultures and individual erythroid blast-forming units derived from human BM CD34+ cells. Sca1+/lineage− Ly5.1 mouse hematopoietic cells, transduced with these 2 ankyrin-1 promoter vectors, were injected into lethally irradiated Ly5.2 recipients. Eleven weeks after transplantation, high-level expression was seen from both vectors in blood (63%-89% of red blood cells) and erythroid cells in BM (70%-86% engraftment), compared with negligible expression in myeloid and lymphoid lineages in blood, BM, spleen, and thymus (0%-4%). The I8/HS-40–containing vector encoding a hybrid human β/γ-globin gene led to 43% to 113% human γ-globin expression/copy of the mouse α-globin gene. Thus, modular use of erythroid-specific enhancers/promoters and WPRE in SIN-lentiviral vectors led to identification of high-titer, stably transmitted vectors with high-level erythroid-specific expression for gene therapy of red cell diseases.

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A Single Vector Platform for High-Level Gene Transduction of Central Neurons: Adeno-Associated Virus Vector Equipped with the Tet-Off System

PLoS ONE ◽

10.1371/journal.pone.0169611 ◽

2017 ◽

Vol 12 (1) ◽

pp. e0169611 ◽

Cited By ~ 12

Author(s):

Jaerin Sohn ◽

Megumu Takahashi ◽

Shinichiro Okamoto ◽

Yoko Ishida ◽

Takahiro Furuta ◽

...

Keyword(s):

Virus Vector ◽

Gene Transduction ◽

Adeno Associated Virus ◽

Central Neurons ◽

High Level

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Analysis of mice containing a targeted deletion of beta-globin locus control region 5' hypersensitive site 3.

Molecular and Cellular Biology ◽

10.1128/mcb.16.6.2906 ◽

1996 ◽

Vol 16 (6) ◽

pp. 2906-2912 ◽

Cited By ~ 133

Author(s):

B A Hug ◽

R L Wesselschmidt ◽

S Fiering ◽

M A Bender ◽

E Epner ◽

...

Keyword(s):

Developmental Stages ◽

Germ Line ◽

Globin Gene ◽

Globin Genes ◽

Beta Globin ◽

Erythroid Cells ◽

Hypersensitive Site ◽

Targeted Deletion ◽

Gamma Globin ◽

Gene Switching

To examine the function of murine beta-globin locus region (LCR) 5' hypersensitive site 3 (HS3) in its native chromosomal context, we deleted this site from the mouse germ line by using homologous recombination techniques. Previous experiments with human 5' HS3 in transgenic models suggested that this site independently contains at least 50% of total LCR activity and that it interacts preferentially with the human gamma-globin genes in embryonic erythroid cells. However, in this study, we demonstrate that deletion of murine 5' HS3 reduces expression of the linked embryonic epsilon y- and beta H 1-globin genes only minimally in yolk sac-derived erythroid cells and reduces output of the linked adult beta (beta major plus beta minor) globin genes by approximately 30% in adult erythrocytes. When the selectable marker PGK-neo cassette was left within the HS3 region of the LCR, a much more severe phenotype was observed at all developmental stages, suggesting that PGK-neo interferes with LCR activity when it is retained within the LCR. Collectively, these results suggest that murine 5' HS3 is not required for globin gene switching; importantly, however, it is required for approximately 30% of the total LCR activity associated with adult beta-globin gene expression in adult erythrocytes.

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The Corfu delta beta zero thalassemia: a small deletion acts at a distance to selectively abolish beta globin gene expression [published erratum appears in Blood 1988 May;71(5):1509]

Blood ◽

10.1182/blood.v71.2.457.457 ◽

1988 ◽

Vol 71 (2) ◽

pp. 457-462 ◽

Cited By ~ 25

Author(s):

AE Kulozik ◽

N Yarwood ◽

RW Jones

Keyword(s):

Globin Gene ◽

Gene Promoters ◽

Globin Genes ◽

Beta Globin ◽

Promoter Regions ◽

Beta Globin Gene ◽

Gamma Globin ◽

Exon 1 ◽

Normal Gene ◽

High Level

Abstract The Corfu delta beta zero thalassemia is characterized by the clinical picture of thalassemia intermedia. In the homozygous state there is a complete absence of hemoglobin (Hb) A and Hb A2 and a high level of Hb F. A DNA fragment containing the gamma and beta globin genes has been cosmid cloned, and the deletion breakpoint region, the beta globin gene and the promoter regions of the gamma globin genes sequenced. The deletion removes 7,201 base pairs (bp) containing part of the delta globin gene and sequences upstream. The beta globin gene contains a G--- -A mutation at IVS 1 position 5. The gamma globin gene promoters are normal. Analysis of the transcription of the mutated beta globin gene in transfected HeLa cells shows that normal message is produced at a level of approximately 20% compared with a normal gene, the remaining 80% being spliced at cryptic sites in exon 1 and intron 1. This indicates that the mutation in the beta globin gene is not the sole cause of the absence of Hb A in Corfu delta beta zero thalassemia. It is concluded that the 7.2 kilobase (kb) of deleted DNA contains sequences necessary for the normal activation of the beta globin gene. Possible mechanisms for the effect of the deletion on the expression of beta and gamma globin genes are discussed.

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Controlling Long-Range Genomic Interactions to Reprogram the β-Globin Locus

Blood ◽

10.1182/blood.v120.21.280.280 ◽

2012 ◽

Vol 120 (21) ◽

pp. 280-280

Author(s):

Wulan Deng ◽

Jeremy W Rupon ◽

Hongxin Wang ◽

Andreas Reik ◽

Philip D. Gregory ◽

...

Keyword(s):

Long Range ◽

Globin Gene ◽

Chromatin Interaction ◽

Globin Genes ◽

Erythroid Cells ◽

Distal Enhancer ◽

Adult Type ◽

High Level ◽

Target Promoters

Abstract Abstract 280 Distal enhancers physically contact target promoters to confer high level transcription. At the mammalian β-globin loci long-range chromosomal interactions between a distal enhancer, called the locus control region (LCR), and the globin genes are developmentally dynamic such that the LCR contacts the embryonic, fetal and adult globin genes in a stage-appropriate fashion. LCR-globin gene interactions require the nuclear factor Ldb1. Recently, we employed artificial zinc finger (ZF) proteins to target Ldb1 to the endogenous β-globin locus to force an LCR-promoter interaction. This led to substantial activation of β-globin transcription and suggested that forced chromatin looping could be employed as a powerful tool to manipulate gene expression in vivo (Deng et al., Cell 2012). Reactivation of the fetal globin genes in adult erythroid cells has been a long-standing goal in the treatment of patients with sickle cell anemia. Therefore, building on our findings, we investigated whether the developmentally silenced embryonic globin gene βh1 can be re-activated in adult murine erythroblasts by re-directing the LCR away from the adult type globin gene and towards its embryonic counterpart. To this end, Ldb1 was fused to artificial ZF proteins (ZF-Ldb1) designed to bind to the βh1 promoter. ZF-Ldb1 was introduced into definitive erythroid cells in which only the adult but not the embryonic β-like globin gene is expressed. In vivo binding of the ZF-Ldb1 to its intended target was verified by chromatin immunoprecipitation assay. Strikingly, expression of ZF-Ldb1 re-activated βh1 transcription up to approximately ∼15% of total cellular β-globin production. This suggests that forced tethering of a looping factor to a select promoter can be employed to override a pre-existing developmental long-range chromatin interaction to reprogram a developmentally controlled gene locus. We are now in the process of testing whether our approach might be suitable to reactivate the silent fetal globin genes in adult human erythroid cells. These studies are underway and the results will be discussed at the meeting. Disclosures: Reik: Sangamo BioSciences, Inc.: Employment. Gregory:Sangamo BioSciences, Inc.: Employment.

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