Genetic correction of sickle cell disease: Insights using transgenic mouse models

Blouin, Marie-José; Beauchemin, Hugues; Wright, Andrew; Paepe, ME De; Sorette, Martin; Bleau, Anne-Marie; Nakamoto, Betty; Ou, Ching-Nan; Stamatoyannopoulos, G; Trudel, Marie

doi:10.1038/72279

Cited by 60 publications

(32 citation statements)

References 35 publications

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“…Indeed, by using a lower viral titer stock, we observed lower integration copy number, averaging 1 per stem cell, human ␤ globin expression in only 30% of repopulated erythroid cells, and no improvement in any hematopoietic parameters. This finding is in contrast to previous estimates from transgenic mouse models that chimerism at levels of Ϸ20% of RBC with ''normal'' ␤ globin expression is sufficient for cure (26,27). In an effort to decrease further position effect variegation even at single proviral copy, we are now incorporating chromatin insulators at various locations of the [␤ globin gene͞LCR] lentiviral vector (28).…”

Section: Discussioncontrasting

confidence: 54%

Permanent and panerythroid correction of murine β thalassemia by multiple lentiviral integration in hematopoietic stem cells

Imren

Payen

Westerman

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

182

157

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The viral titers produced were high enough to achieve transduction of virtually all of the hematopoietic stem cells in the graft with an average of three integrated proviral copies per genome in all transplanted mice; the transduction was sustained for >7 months in both primary and secondary transplants, at which time Ϸ95% of the red blood cells in all mice contained human ␤ globin contributing to 32 ؎ 4% of all ␤-like globin chains. Hematological parameters approached complete phenotypic correction, as assessed by hemoglobin levels and reticulocyte and red blood cell counts. All circulating red blood cells became and remained normocytic and normochromic, and their density was normalized. Free ␣ globin chains were completely cleared from red blood cell membranes, splenomegaly abated, and iron deposit was almost eliminated in liver sections. These findings indicate that virtually complete transduction of the hematopoietic stem cell compartment can be achieved by high-titer lentiviral vectors and that position effect variegation can be mitigated by multiple events of proviral integration to yield balanced, panerythroid expression. These results provide a solid foundation for the initiation of human clinical trials in ␤ thalassemia patients.

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Section: Discussioncontrasting

confidence: 54%

Permanent and panerythroid correction of murine β thalassemia by multiple lentiviral integration in hematopoietic stem cells

Imren

Payen

Westerman

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

182

157

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“…Similarly, in a study using an SCD mouse model, c-globin mRNA expression in the majority of reticulocytes at 19 to 24% of the levels of the endogenous globin genes resulted in corresponding levels of HbF protein of 16 to 25%, significantly improving the disease phenotype and life span of the animals (Blouin et al, 2000). Using a different SCD mouse model, Perumbeti and colleagues (2009) corroborated the aforementioned findings.…”

Section: Discussionsupporting

confidence: 70%

The New Self-Inactivating Lentiviral Vector for Thalassemia Gene Therapy Combining Two HPFH Activating Elements Corrects Human Thalassemic Hematopoietic Stem Cells

et al. 2012

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To address how low titer, variable expression, and gene silencing affect gene therapy vectors for hemoglobinopathies, in a previous study we successfully used the HPFH (hereditary persistence of fetal hemoglobin)-2 enhancer in a series of oncoretroviral vectors. On the basis of these data, we generated a novel insulated selfinactivating (SIN) lentiviral vector, termed GGHI, carrying the A c-globin gene with the -117 HPFH point mutation and the HPFH-2 enhancer and exhibiting a pancellular pattern of A c-globin gene expression in MEL-585 clones. To assess the eventual clinical feasibility of this vector, GGHI was tested on CD34+ hematopoietic stem cells from nonmobilized peripheral blood or bone marrow from 20 patients with b-thalassemia. Our results show that GGHI increased the production of c-globin by 32.9% as measured by high-performance liquid chromatography ( p = 0.001), with a mean vector copy number per cell of 1.1 and a mean transduction efficiency of 40.3%. Transduced populations also exhibited a lower rate of apoptosis and resulted in improvement of erythropoiesis with a higher percentage of orthochromatic erythroblasts. This is the first report of a locus control region (LCR)-free SIN insulated lentiviral vector that can be used to efficiently produce the anticipated therapeutic levels of c-globin protein in the erythroid progeny of primary human thalassemic hematopoietic stem cells in vitro.

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“…The mechanism by which the ␤-globin gene repression reported here actually occurs will require further investigation using technology capable of identifying all proteins in a multisubunit complex, such as matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry and peptide mass fingerprinting (42). It will also be of interest to determine whether ferritin-H will repress human ␤-globin and/or activate ␥-globin sufficiently in a transgenic mouse model (43) to alleviate the sickle cell phenotype.…”

Section: Figmentioning

confidence: 99%

Specific repression of β-globin promoter activity by nuclear ferritin

Broyles

Belegu²,

DeWitt³

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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Developmental hemoglobin switching involves sequential globin gene activations and repressions that are incompletely understood. Earlier observations, described herein, led us to hypothesize that nuclear ferritin is a repressor of the adult ␤-globin gene in embryonic erythroid cells. Our data show that a ferritin-family protein in K562 cell nuclear extracts binds specifically to a highly conserved CAGTGC motif in the ␤-globin promoter at ؊153 to ؊148 bp from the cap site, and mutation of the CAGTGC motif reduces binding 20-fold in competition gel-shift assays. Purified human ferritin that is enriched in ferritin-H chains also binds the CAGTGC promoter segment. Expression clones of ferritin-H markedly repress ␤-globin promoter-driven reporter gene expression in cotransfected CV-1 cells in which the ␤-promoter has been stimulated with the transcription activator erythroid Krü ppel-like factor (EKLF). We have constructed chloramphenicol acetyltransferase reporter plasmids containing either a wildtype or mutant ␤-globin promoter for the ؊150 CAGTGC motif and have compared the constructs for susceptibility to repression by ferritin-H in cotransfection assays. We find that stimulation by cotransfected EKLF is retained with the mutant promoter, whereas repression by ferritin-H is lost. Thus, mutation of the ؊150 CAGTGC motif not only markedly reduces in vitro binding of nuclear ferritin but also abrogates the ability of expressed ferritin-H to repress this promoter in our cell transfection assay, providing a strong link between DNA binding and function, and strong support for our proposal that nuclear ferritin-H is a repressor of the human ␤-globin gene. Such a repressor could be helpful in treating sickle cell and other genetic diseases.

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Genetic correction of sickle cell disease: Insights using transgenic mouse models

Cited by 60 publications

References 35 publications

Permanent and panerythroid correction of murine β thalassemia by multiple lentiviral integration in hematopoietic stem cells

Permanent and panerythroid correction of murine β thalassemia by multiple lentiviral integration in hematopoietic stem cells

The New Self-Inactivating Lentiviral Vector for Thalassemia Gene Therapy Combining Two HPFH Activating Elements Corrects Human Thalassemic Hematopoietic Stem Cells

Specific repression of β-globin promoter activity by nuclear ferritin

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