In vivo silencing of the human gamma-globin gene in murine erythroid cells following retroviral transduction

Increased expression of fetal hemoglobin can ameliorate the clinical severity of sickle cell disease. Whereas temporary induction of fetal hemoglobin can be achieved by pharmacologic therapy, gene transfer resulting in high-level expression of the fetal gamma-globin gene may provide a permanent cure for sickle cell disease. We had previously developed a high-titer, genetically stable retroviral vector in which the human gamma-globin gene was linked to HS-40, the major regulatory element of the human alpha-globin gene cluster. Based on experience in transgenic mice, the truncated promoter of the gamma-globin gene of this vector should be active in adult erythroid cells. Our earlier studies demonstrated that this retroviral vector can give rise to high-level expression of the human gamma-globin gene in murine erythroleukemia (MEL) cells. We have now utilized this vector to transduce murine bone marrow cells that were transplanted into W/W(v) recipient mice. Analysis of transduction of murine BFU-e's in vitro and peripheral blood cells from transplanted mice in vivo demonstrated efficient transfer of the human gamma-globin gene. However, in contrast to the high level of expression of the human gamma-globin gene of this vector in MEL cells, the gene was completely silent in vivo in all transplanted mice. These observations confirm that all the necessary regulatory elements responsible for the developmental stage-specific expression of the human gamma-globin gene reside in its proximal sequences. They also emphasize the differences between gene regulation in MEL cells, transgenic mice, and retroviral gene transfer vectors. For this form of globin gene therapy to succeed, the proximal regulatory elements of the human gamma-globin gene may have to be replaced with different regulatory elements that allow the expression of the gamma-globin coding sequences in adult red cells in vivo.     

High oxygen environment during pregnancy rescues sickle cell anemia mice from prenatal death

Several mouse models of sickle cell disease have been developed for the study of the pathophysiology of sickle cell disease and the investigation of drug and gene therapies. In previous years, we produced a sickle cell anemia mouse model in which the endogenous mouse α- and β-globin genes were knocked out and replaced by the human α- and β^s-globin transgenes. The β^s-globin gene was contained in a 240 kb YAC that preserved the entire native genomic context of the β-globin locus. These mice have hemolytic anemia, reticulocytosis and irreversible sickle cells in the peripheral blood, as well as other pathological features of sickle cell disease. However, in the embryo, the γ-globin, like the mouse embryonic globin, declined quickly, and was replaced by β^s-globin expression from 12 days of gestation. The low level of fetal hemoglobin expression in utero led to intrauterine sickling and fetal death so that very few live-born sickle cell anemia mice could be obtained. To rescue these mice from intrauterine death, we investigated the effect of placing the pregnant mothers in a high O₂ environment. From the tenth day of gestation onwards, we placed the mothers into a chamber containing 50% O₂ and kept them with the newborn pups in it for another 10 days after birth. The frequency of sickle cell anemia mice we obtained was increased from less than 2% to 35%. The survived sickle cell anemia mice develop congestion, atrophy, and infarcts in multiple organs similar to those found in patients with sickle cell disease. We conclude that a high oxygen environment can be used to obtain more sickle cell anemia mice in those models that have a high perinatal mortality. The higher yield of these mice has facilitated physiological and therapeutic studies of sickle cell anemia.     

Boundary sequences stabilize transgene expression from subtle position effects in retroviral vectors

Transgene expression shut-down, attenuation and/or variability from integrated retroviral vectors pose a major obstacle to gene therapy trials involving hematopoietic cells. We have undertaken a systematic assessment of the behavior of different configurations containing IFN-β SAR and/or 5′HS4 β-globin insulator sequences within a gammaretroviral vector optimized for high-level expression, focusing on the long-term achievement of stable, homogeneous transgene expression in the successfully transduced cells. Introduction of these cis regulatory elements did not perturb virus production and stability. Conversely, the SAR/5′HS4 insulator combination appeared to increase the homogeneity of EGFP expression in mass cultures. Furthermore, a clonal analysis of the dispersion of EGFP expression revealed that the IFN-SAR/5′HS4 insulator dyad was particularly effective in reducing the variability of transgene expression when both sequences were placed in opposite orientations within the retroviral backbone. These results may prove useful for the design of more stable retroviral expression cassettes able to counteract chromosomal position effects.     

Transgenic knockout mice exclusively expressing human hemoglobin S after transfer of a 240-kb βs-globin yeast artificial chromosome: A mouse model of sickle cell anemia

Sickle cell anemia (SCA) and thalassemia are among the most common genetic diseases worldwide. Current approaches to the development of murine models of SCA involve the elimination of functional murine α- and β-globin genes and substitution with human α and β^s transgenes. Recently, two groups have produced mice that exclusively express human HbS. The transgenic lines used in these studies were produced by coinjection of human α-, γ-, and β-globin constructs. Thus, all of the transgenes are integrated at a single chromosomal site. Studies in transgenic mice have demonstrated that the normal gene order and spatial organization of the members of the human β-globin gene family are required for appropriate developmental and stage-restricted expression of the genes. As the cis-acting sequences that participate in activation and silencing of the γ- and β-globin genes are not fully defined, murine models that preserve the normal structure of the locus are likely to have significant advantages for validating future therapies for SCA. To produce a model of SCA that recapitulates not only the phenotype, but also the genotype of patients with SCA, we have generated mice that exclusively express HbS after transfer of a 240-kb β^s yeast artificial chromosome. These mice have hemolytic anemia, 10% irreversibly sickled cells in their peripheral blood, reticulocytosis, and other phenotypic features of SCA.     

Partial repression of human γ-globin genes by LCR element HS3 when linked to β-globin genes and LCR element HS2 in MEL cells

Clues for overcoming fetal (γ-) globin gene repression in adult human erythroid cells may come from understanding why repression of isolated γ-globin genes has not previously been achieved in the adult erythroid environment of mouse erythroleukemia cells (MEL). Repression of human γ-globin genes has been demonstrated in MEL cells when transferred as part of the entire β-globin gene cluster packaged in chromatin. Major differences in these approaches are prior packaging into chromatin and the presence of additional sequences, notably from the locus control region (LCR). In this report we focus on the contribution to γ-globin gene repression that multiple elements of the LCR may have. We first show preferential activation of β-globin genes over γ-globin genes in MEL cells when linked to each other and to LCR sequences containing the core elements of DNase I hypersensitive sites 4, 3, and 2. Removal of the HS4 element had no effect, however, removal of the 225 bp HS3 core element resulted in a five-fold increase in γ-globin gene expression. The enhancer 3′ to the Aγ-globin gene also had no apparent effect on γ-globin gene expression. These results provide first evidence of γ-globin gene repression involving the core region of HS3 in the presence of the core region of HS2 and a β-globin gene. A mechanism for repression involving sequestration of the γ-promoter away from the strong enhancer activity of HS2 is proposed. © 1996 Wiley-Liss, Inc.     

Recombinant AAV2-mediated β-globin expression in human fetal hematopoietic cells from the aborted fetuses with β-thalassemia major

Genetic correction of autologous hematopoietic stem cells has been proposed as an attractive treatment method for β-thalassemia. Our previous study has shown that recombinant adeno-associated virus 2 (rAAV2) efficiently transduces human fetal liver hematopoietic cells, and mediates the expression of the human β-globin gene in vivo. In this study, we investigated whether rAAV2 could also mediate the expression of normal β-globin gene in human hematopoietic cells from β-thalassemia patients. Human hematopoietic cells were isolated from aborted β-thalassemia major fetuses, transduced with rAAV2-β-globin, and then transplanted into nude mice. We found that rAAV2-β-globin transduced human fetal hematopoietic cells, as determined by allele-specific PCR analysis. Furthermore, β-globin transgene expression was detected in human hematopoietic cells up to 70 days post-transplantation in the recipient mice. High-pressure liquid chromatography analysis showed that human β-globin expression levels increased significantly compared with control, as indicated by a 1.2–2.8-fold increase in the ratio of β/α-globin chain. These novel data demonstrate that rAAV2 can transduce and mediate the normal β-globin gene expression in fetal hematopoietic cells from β-thalassemia patients. Our findings further support the potential use of rAAV-based gene therapy in the treatment of human β-thalassemia.     

In vivo selection of genetically modified erythroblastic progenitors leads to long-term correction of beta-thalassemia

Gene therapy for β-thalassemia requires stable transfer of a β-globin gene into hematopoietic stem cells (HSCs) and high and regulated hemoglobin expression in the erythroblastic progeny. We developed an erythroid-specific lentiviral vector driving the expression of the human β-globin gene from a minimal promoter/enhancer element containing two hypersensitive sites from the β-globin locus control region. Transplantation of transduced HSCs into thalassemic mice leads to stable and long-term correction of anemia with all red blood cells expressing the transgene. A frequency of 30–50% of transduced HSCs, harboring an average vector copy number per cell of 1, was sufficient to fully correct the thalassemic phenotype. In the mouse model of Cooley's anemia transplantation of transduced cells rescues lethality, leading to either a normal or a thalassemia intermedia phenotype. We show that genetically corrected erythroblasts undergo in vivo selection with preferential survival of progenitors harboring proviral integrations in genome sites more favorable to high levels of vector-derived expression. These data provide a rationale for a gene therapy approach to β-thalassemia based on partially myeloablative transplantation protocols.     

Gene therapy of apolipoprotein E-deficient mice using a novel macrophage-specific retroviral vector

The use of retroviral gene transfer into hematopoietic stem cells for human gene therapy has been hampered by the absence of retroviral vectors that can generate long-lasting, lineage-specific gene expression. We developed self-inactivating retroviral vectors that incorporate gene-regulatory elements from the macrophage-restricted human CD68 gene. Through the transplantation of transduced murine hematopoietic stem cells (HSCs), we show that a vector incorporating a 342-base pair (bp) fragment of 5' flanking sequence from the CD68 gene, in addition to the CD68 first intron, was able to direct macrophage-specific expression of an enhanced green fluorescent protein (EGFP) reporter gene in inflammatory cell exudates and lymphoid organs in vivo. Levels of EGFP expression generated by this vector were greater than those generated by a standard Moloney murine leukemia retroviral vector, and they were stable for at least a year after transplantation of transduced HSCs. To evaluate the ability of this vector to generate therapeutically useful levels of gene expression, we transplanted apolipoprotein E (ApoE)-deficient HSCs transduced with a virus encoding ApoE into ApoE-deficient mice. Macrophages from these mice expressed levels of ApoE that were comparable to those from wild-type mice, and vector-driven expression of ApoE in macrophages was sufficient to reverse both hypercholesterolemia and atherosclerotic lesion development. The future application of this retroviral vector should provide a powerful tool to further elucidate macrophage function and for human gene therapy.     

Hydroxyurea therapy requires HbF induction for clinical benefit in a sickle cell mouse model

Hydroxyurea has proven clinical efficacy in patients with sickle cell disease. Potential mechanisms for the beneficial effects include fetal hemoglobin induction and the reduction of cell adhesive properties, inflammation and hypercoagulability. Using a murine model of sickle cell disease in which fetal hemoglobin induction does not occur, we evaluated whether hydroxyurea administration would still yield improvements in hematologic parameters and reduce end-organ damage. Animals given a maximally tolerated dose of hydroxyurea that resulted in significant reductions in the neutrophil and platelet counts showed no improvement in hemolytic anemia and end-organ damage compared to control mice. In contrast, animals having high levels of fetal hemoglobin due to gene transfer with a γ-globin lentiviral vector showed correction of anemia and organ damage. These data suggest that induction of fetal hemoglobin by hydroxyurea is an essential mechanism for its clinical benefits.     

Production of genetically stable high-titer retroviral vectors that carry a human gamma-globin gene under the control of the alpha-globin locus control region

The ability to generate stable high-titer vectors that give rise to high levels of expression of transduced globin genes in erythroid cells is a prerequisite for effective retroviral-mediated globin gene therapy. The human beta-globin gene with its immediate flanking sequences does not contain all the regulatory elements necessary for regulated high-level and position-independent expression in erythroid cells. The regulatory element known as the beta-globin locus control region (BetaLCR) can provide a linked Beta-globin gene with these properties. However, addition of BetaLCR sequences to a retrovirus carrying a beta-globin gene increases its genetic instability. We have developed a new generation of retroviral vectors in which a human gamma- globin gene is placed under the control of the alphaLCR, the major regulatory element of the alpha-globin gene cluster. We demonstrate that these retroviruses are genetically stable in producer cell lines and can be produced at high titers that exceed 5 x 10(6) colony-forming units (CFU)/mL. In addition, we show that the transduced gamma-globin gene can be expressed in the adult erythroid environment of mouse erythroleukemia (MEL) cells at a level comparable to that of a single endogenous Betamaj-globin gene. These retroviruses can also transduce primary murine bone marrow progenitor cells as efficiently as retroviruses that carry the neomycin resistance (neor) gene. This new generation of globin retroviral vectors may prove useful for gene therapy of human beta-globin gene disorders such as sickle cell disease and beta-thalassemia.     

Adventitious changes in long-range gene expression caused by polymorphic structural variation and promoter competition

It is well established that all of the cis-acting sequences required for fully regulated human α-globin expression are contained within a region of ≈120 kb of conserved synteny. Here, we show that activation of this cluster in erythroid cells dramatically affects expression of apparently unrelated and noncontiguous genes in the 500 kb surrounding this domain, including a gene (NME4) located 300 kb from the α-globin cluster. Changes in NME4 expression are mediated by physical cis-interactions between this gene and the α-globin regulatory elements. Polymorphic structural variation within the globin cluster, altering the number of α-globin genes, affects the pattern of NME4 expression by altering the competition for the shared α-globin regulatory elements. These findings challenge the concept that the genome is organized into discrete, insulated regulatory domains. In addition, this work has important implications for our understanding of genome evolution, the interpretation of genome-wide expression, expression-quantitative trait loci, and copy number variant analyses.