Human Gamma-globin Expression Research Articles

In Vivo HSC Transduction and Selection Results in Long-Term, High-Level Expression of Human Gamma Globin in Peripheral Blood Erythrocytes of Mice

Current protocols for HSC gene therapy, involving the transplantation of ex vivo lentivirus vector-transduced HSCs into myeloablated recipients, are complex and not without risk for the patient. We have developed a new approach for in vivo gene delivery into HSCs that does not require myeloablation and HSC transplantation. It involves injections of G-CSF/AMD3100 to mobilize HSCs from the bone marrow into the peripheral blood stream and the intravenous injection of an integrating, helper-dependent adenovirus (HDAd5/35++) vector system. Transgene integration is achieved (in a random pattern) using a hyperactive Sleeping Beauty transposase (SB100x). We demonstrated in adequate mouse models, using GFP as a transgene, that primitive HSCs transduced in the periphery home back to the bone marrow where they persist and stably express GFP long-term (Blood 128:2206 (2016). To achieve high level transgene marking in differentiated peripheral blood cells, we combined our in vivo HSC transduction approach with in vivo selection of transduced HSCs using the mgtmP140K mutant and low dose O6BG/BCNU treatment. Here were tested the potential of this approach for the therapy of hemoglobinopathies. Our HDAd5/35++ vector contained the human-gamma globin gene under the control of a 6kb version of the beta-globin LCR (containing HS1 to HS4 and the beta globin promoter) and the mgmtP140K gene under the control of the PGK promoter. Mobilized immunocompetent hCD46 transgenic mice were injected IV with the integrating vector system and subjected to three rounds of treatment with low-dose O6BG/BCNU followed by immunosuppression to avoid immune responses against the human transgene product. The procedure was safe and resulted in stable human gamma globin expression in >95% of erythrocytes in the peripheral blood of in vivo transduced cells mice. Human gamma globin levels were 100% of endogenous mouse beta globin levels in erythrocytes measured by HPLC. Analysis of bone marrow at week 16 after in vivo transduction showed that >60% of bone marrow LSK cells contained the integrated transgene cassette and that the beta globin LCR efficiently restricted gamma globin expression to erythroid cells. After transplantation of unsorted, lineage-depleted bone marrow cells into lethally irradiated recipients, long-term gamma-globin expression at levels seen in the primary in vivo treated mice was demonstrated. The efficacy of our approach achieved in this study indicates that it would be curative in thalassemia models or patients. Our new approach avoids the collection of HSCs, their transduction ex vivo, and subsequent transplantation into myeloablated recipients. It therefore could greatly simplify HSC gene therapy of common diseases. DisclosuresKiem:Rocket Pharmaceuticals: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

Correction of Hemoglobin Levels in a Heterozygous Humanized Mouse Model of Thalassemia after Fetal Gene Therapy

BackgroundBeta thalassaemia is a genetic blood disease that causes life-threatening anemia. Hematopoietic stem cell (HSC) transplantation successfully cures the disease but in only 30% of patients. We hypothesized that in utero gene therapy (IUGT) to the fetal HSC compartment with the corrected beta globin gene might cure the disease before birth.MethodsA humanized mouse model of thalassemia (Cooley’s anemia; CA) was used in which heterozygous animals are affected by anemia, splenomegaly and extra-medullary hematopoiesis. At E13.5 a “GLOBE” vector (HIV-2 based lentiviral vector that incorporates a mini hemoglobin beta gene, the beta-globin promoter and HS2/3 β-LCR element) was injected into the liver of each fetus (n=12). At 12 weeks of age, recipient blood, liver, spleen and bone marrow were collected for complete blood count, blood film, as well as RNA and DNA isolation. Extra-medullary hematopoiesis was examined in the spleen and liver using flow cytometry (CD71+/Ter119+ cells) and histo-pathological analysis. Gene expression of human and mouse alpha and beta globins, as well as human gamma globin was assessed by quantitative polymerase chain reaction (qPCR). High performance liquid chromatography (HPLC) was used to quantify the presence of human beta globin in the peripheral blood of treated animals. Results are expressed as mean±SEM, and statistical analysis was performed using 1-way ANOVA with Bonferroni post-hoc tests. Experimental protocols were approved by the ethical committee on animal experimentation at University College London.ResultsCompared to non-injected heterozygous pups (control), IUGT increased hemoglobin levels [11.3±0.4g/dl (n=6) vs. 7.6±0.6g/dl (n=8); p<0.01], red blood cell count [9.3±0.3*1012/L vs. 6.2±0.5*1012/L; p<0.01), and hematocrit [41.2±2.2% vs. 27.2±2.0; p<0.01]. Moreover, treated CA animals had reduced spleen weight [130±5mg vs. 310±21mg; p <0.01], as well as reduced extra-medullary hematopoiesis in the liver [0.7±0.1% (n=4) vs. 6.0±0.9% (n=5); p<0.01] and spleen [6.6±1.8 (n=4) vs. 23.1±1.4 (n=3); p<0.05]. qPCR analysis demonstrated increased gene expression of human beta globin (figure A) and reduced expression of human gamma globin in blood and bone marrow of IUGT offspring. HPLC analysis confirmed these findings at protein level (figure B). The average vector copy number in the liver was 0.1.ConclusionsIUGT resulted in phenotypic normalization in a heterozygous humanized mouse model of CA. Increased levels of beta globin and associated down-regulation of gamma globin is consistent with a switch from fetal to adult human hemoglobin, confirming successful prenatal correction of the genetic defect. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Developmental and species-divergent globin switching are driven by BCL11A

The contribution of changes in cis-regulatory elements or trans-acting factors to interspecies differences in gene expression is not well understood. The mammalian beta-globin loci have served as a model for gene regulation during development. Transgenic mice containing the human beta-globin locus, consisting of the linked embryonic (epsilon), fetal (gamma) and adult (beta) genes, have been used as a system to investigate the temporal switch from fetal to adult haemoglobin, as occurs in humans. Here we show that the human gamma-globin (HBG) genes in these mice behave as murine embryonic globin genes, revealing a limitation of the model and demonstrating that critical differences in the trans-acting milieu have arisen during mammalian evolution. We show that the expression of BCL11A, a repressor of human gamma-globin expression identified by genome-wide association studies, differs between mouse and human. Developmental silencing of the mouse embryonic globin and human gamma-globin genes fails to occur in mice in the absence of BCL11A. Thus, BCL11A is a critical mediator of species-divergent globin switching. By comparing the ontogeny of beta-globin gene regulation in mice and humans, we have shown that alterations in the expression of a trans-acting factor constitute a critical driver of gene expression changes during evolution.

The Cyclic GMP-Dependent Pathway Is Involved in the Mechanisms of Action of Hydroxyurea.

Hydroxyurea (HU) is the standard therapeutic agent for patients with sickle cell disease (SCD). Administration of HU reduces the frequency of pain crisis, increases total hemoglobin levels, and decreases the numbers of reticulocytes, leukocytes, and platelets. However, the mechanism underlying these ameliorating effects of HU on the clinical expression of SCD patients still remains to be clarified. Several lines of evidence that were obtained from our laboratory and others suggest that HU exerts its molecular effects through the cyclic GMP (cGMP)-dependent pathway. To examine whether the cGMP-dependent pathway is involved in the molecular actions of HU, we generated mice that overexpress soluble guanylate cyclase (sGC), a key enzymes of the cGMP-dependent pathway and have an activated cGMP-dependent pathway in peripheral blood cells. Adult mice expressed the transgenes encoding sGC subunits in bone marrow cells, spleen cells and peripheral leukocytes. In these cells of transgenic sGC mice, intracellular cGMP levels were elevated and the phosphorylation levels of vasodilator-stimulated phosphoprotein, which is a substrate of protein kinase G of the cGMP-dependent pathway, were increased. These results demonstrated activation of the cGMP-dependent pathway in the sGC mice. Interestingly, high-level expression of sGC subunit transgenes resulted in down-regulation of endogenous mouse sGC mRNA expression, suggesting that mRNAs encoding sGC subunits are unstable in cells which have high cGMP levels. More interestingly, adult sGC mice had elevated levels of total hemoglobin, and decreased numbers of white blood cell counts, which is consistent with a clinical picture of SCD patients who are administered with HU. In vitro colony assays demonstrated that cGMP has positive and negative effects on the differentiation of erythroid- and myeloid-lineage cells, respectively. Furthermore, in sickle cell patients treated with HU, intracellular cGMP levels of red blood cells positively correlated with total hemoglobin levels, while leukocyte counts were inversely correlated with the cGMP levels of leukocytes. These results indicate the roles of the cGMP-dependent pathway at least in part in regulating blood cell counts in sickle cell patients. We previously reported that expression of the human gamma-globin gene is induced by activating the cGMP-dependent pathway (PNAS 98: 1847, 2001). Next, we examined whether expression of human gamma-globin, which is dormant in adult erythroid cells, is induced in sGC mice. We bred sGC mice with a YAC mouse carrying the entire human beta-globin gene locus. Although human gamma-globin was not detectable by HPLC in sGC/YAC mice, low but appreciable levels of human gamma-globin expression was induced following the activation of the cGMP-dependent pathway with protoporphyrin IX, which is an activator of sGC. This demonstrated a role of the pathway in inducing gamma-globin expression in adult erythroid cells. Collectively, these results shown in this study demonstrate that sGC mice exhibit hematologic characteristics that are similar to those of SCD patients on HU therapy, suggesting that the cGMP-dependent pathway is involved at least in part in the mechanisms of action of HU.

Expression of High Levels of Human γ-Globin in Adult Mice Carrying a Transgene of the Brazilian Type of Hereditary Persistence of Fetal Hemoglobin.

The term, hereditary persistence of fetal hemoglobin (HPFH), describes a hereditary benign disease, characterized by an increase in fetal hemoglobin (HbF) during adult life. Non-deletional forms of HPFH are characterized by single-base mutations in the promoter region (most of them between −114 and −202 from the cap site) of either the Gγor Aγ-globin gene, resulting in an increase of HbF ranging from 3 to 20% in heterozygotes. Many point mutations in this region have been described, including the Aγ-195 C →G mutation that causes the Brazilian type of HPFH (HPFH-B). A previous study showed that the -195 mutations alone was not able to increase gene expression in vitro and a modest increase was observed when a LCR element fragment (HS2) was introduced in the construction. This study showed too, that the the mechanism of HbF elevation by the -195 mutation was neither mediated by the Sp-1 transcription factor nor by the creation of a CACCC box, as described for the -198 mutation. Thus, other proteins may be involved in the over-expression of the γ-globin chain and/or may depend on DNA structure and these mechanisms remain to be clarified. To better understand this mechanism we have developed HPFH-B transgenic mice. The promoter with mutation was amplified using the genomic DNA of a HPFH-B patient and cloned in a μLCRAγψβδβ cosmid. This construct, containing the micro-LCR and other essential elements of human beta-globin gene cluster was microinjected into single cell mouse embryos. To detect the differences in developmental regulation of the human gamma-globin gene expression in the transgenic mice, we analyzed the yolk sac derived embryonic blood at embryonic day 10.5 (E10.5) and the fetal liver of mouse embryos at E13.5 of both mutated and non-mutated transgenic mice by RNAse Protection Assay (RPA) and Real time PCR (RT-PCR). Levels of expression of murine alpha-globin mRNA were used as internal controls in the RPA experiment and levels of murine-GAPDH were used in RT-PCR. mRNA levels of human gamma-globin in fetal liver of transgenic mice containing mutation were clearly higher, as compared with control transgenic mice bearing cosmid construct with wild a type sequence gamma promoter. Additionally a nearly 10-fold increase in human gamma-globin expression was detected in fetal livers of HPFH transgenic mice at E13.5 compared to the yolk sac. Thus, our data indicate that the - 195 mutation is the unique cause of elevation of HbF in Brazilian HPFH, but the exact mechanism needs to be clarified.

A post‐transcriptional process contributes to efficient γ‐globin gene silencing in definitive erythroid cells

The expression of human gamma globin is developmentally regulated through mechanisms that affect the transcriptional activity of its encoding gene. The current manuscript investigates whether the efficiency of this process might be enhanced though an unrecognized post-transcriptional event that defines the stability of gamma-globin mRNA. Experiments were conducted in vivo in transgenic mice expressing human gamma globin in their adult erythroid cells. The expression of gamma-globin protein was manipulated by breeding the transgene into animals producing different levels of endogenous mouse beta-globin. Changes in the expression of gamma globin were then correlated to measures of gamma-globin mRNA stability in vivo. Human gamma globin was expressed at higher levels in thalassemic than in than non-thalassemic control transgenics, paralleling a highly significant increase in the stability of gamma-globin mRNA. Other molecular events-including possible transcriptional induction of the transgene, or an increase in the stability of the gamma-globin protein-did not appear to contribute to the observed increase in transgene expression. As anticipated, the stability of gamma-globin mRNA also fell in bitransgenic animals that co-expressed human beta-globin mRNA. Our results are consistent with a model for dynamic post-transcriptional control of gamma-globin gene expression, through modulation of the stability of its encoding mRNA. Moreover, the stability of gamma-globin mRNA appears to be inversely related to ambient levels of co-expressed beta-globin mRNA. This data suggests that therapeutic gene-reactivation and/or gene-replacement therapies may be particularly effective in individuals with severe forms of beta-thalassemia.

The degree of phenotypic correction of murine β-thalassemia intermedia following lentiviral-mediated transfer of a human γ-globin gene is influenced by chromosomal position effects and vector copy number

Increased fetal hemoglobin (HbF) levels diminish the clinical severity of beta-thalassemia and sickle cell anemia. A treatment strategy using autologous stem cell-targeted gene transfer of a gamma-globin gene may therefore have therapeutic potential. We evaluated oncoretroviral- and lentiviral-based gamma-globin vectors for expression in transduced erythroid cell lines. Compared with gamma-globin, oncoretroviral vectors containing either a beta-spectrin or beta-globin promoter and the alpha-globin HS40 element, a gamma-globin lentiviral vector utilizing the beta-globin promoter and elements from the beta-globin locus control region demonstrated a higher probability of expression. This lentiviral vector design was evaluated in lethally irradiated mice that received transplants of transduced bone marrow cells. Long-term, stable erythroid expression of human gamma-globin was observed with levels of vector-encoded gamma-globin mRNA ranging from 9% to 19% of total murine alpha-globin mRNA. The therapeutic efficacy of the vector was subsequently evaluated in a murine model of beta-thalassemia intermedia. The majority of mice that underwent transplantation expressed significant levels of chimeric m(alpha)(2)h(gamma)(2) molecules (termed HbF), the amount of which correlated with the degree of phenotypic improvement. A group of animals with a mean HbF level of 21% displayed a 2.5 g/dL (25 g/L) improvement in Hb concentration and normalization of erythrocyte morphology relative to control animals. gamma-Globin expression and phenotypic improvement was variably lower in other animals due to differences in vector copy number and chromosomal position effects. These data establish the potential of using a gamma-globin lentiviral vector for gene therapy of beta-thalassemia.

THE EFFECT OF ADENO-ASSOCIATED VIRUS MEDIATED BRAIN DERIVED NEUROTROPHIC FACTOR IN AN ANIMAL MODEL OF NEUROGENIC IMPOTENCE

THE EFFECT OF ADENO-ASSOCIATED VIRUS MEDIATED BRAIN DERIVED NEUROTROPHIC FACTOR IN AN ANIMAL MODEL OF NEUROGENIC IMPOTENCE

Genome scan identifies a locus affecting gamma-globin level in human beta-cluster YAC transgenic mice.

Genetic factors affecting postnatal gamma-globin expression--a major modifier of the severity of both beta-thalassemia and sickle cell anemia--have been difficult to study. This is especially so in mice, an organism lacking a globin gene with an expression pattern equivalent to that of human gamma-globin. To model the human beta-cluster in mice, with the goal of screening for loci affecting human gamma-globin expression in vivo, we introduced a human beta-globin cluster YAC transgene into the genome of FVB/N mice. The beta-cluster contained a Greek hereditary persistence of fetal hemoglobin (HPFH) gamma allele, resulting in postnatal expression of human gamma-globin in transgenic mice. The level of human gamma-globin for various F1 hybrids derived from crosses between the FVB/N transgenics and other inbred mouse strains was assessed. The gamma-globin level of the (C3HeB/FeJ x FVB/N)F1 transgenic mice was noted to be significantly elevated. To map genes affecting postnatal y-globin expression, we performed a 20-centiMorgan (cM) genome scan of a (C3HeB/FeJ x FVB/N)F1 transgenics x FVB/N backcross, followed by high-resolution marker analysis of promising loci. From this analysis we mapped a locus within an 18-cM interval of mouse Chromosome (Chr) 1 (LOD = 4.3) that contributes 10.9% of variation in gamma-globin level. Combining transgenic modeling of the human beta-globin gene cluster with quantitative trait analysis, we have identified and mapped a murine locus that impacts on human gamma-globin level in vivo.

High levels of human gamma-globin gene expression in adult mice carrying a transgene of deletion-type hereditary persistence of fetal hemoglobin.

Persistent expression of the gamma-globin genes in adults with deletion types of hereditary persistence of fetal hemoglobin (HPFH) is thought to be mediated by enhancer-like effects of DNA sequences at the 3' breakpoints of the deletions. A transgenic mouse model of deletion-type HPFH was generated by using a DNA fragment containing both human gamma-globin genes and HPFH-2 breakpoint DNA sequences linked to the core sequences of the locus control region (LCR) of the human beta-globin gene cluster. Analysis of gamma-globin expression in six HPFH transgenic lines demonstrated persistence of gamma-globin mRNA and peptides in erythrocytes of adult HPFH transgenic mice. Analysis of the hemoglobin phenotype of adult HPFH transgenic animals by isoelectric focusing showed the presence of hybrid mouse alpha2-human gamma2 tetramers as well as human gamma4 homotetramers (hemoglobin Bart's). In contrast, correct developmental regulation of the gamma-globin genes with essentially absent gamma-globin gene expression in adult erythroid cells was observed in two control non-HPFH transgenic lines, consistent with autonomous silencing of normal human gamma-globin expression in adult transgenic mice. Interestingly, marked preferential overexpression of the LCR-distal (A)gamma-globin gene but not of the LCR-proximal (G)gamma-globin gene was observed at all developmental stages in erythroid cells of HPFH-2 transgenic mice. These findings were also associated with the formation of a DNase I-hypersensitive site in the HPFH-2 breakpoint DNA of transgenic murine erythroid cells, as occurs in normal human erythroid cells in vivo. These results indicate that breakpoint DNA sequences in deletion-type HPFH-2 can modify the developmentally regulated expression of the gamma-globin genes.

Single-copy transduction and expression of human gamma-globin in K562 erythroleukemia cells using recombinant adeno-associated virus vectors: the effect of mutations in NF-E2 and GATA-1 binding motifs within the hypersensitivity site 2 enhancer [published erratum appears in Blood 1995 Feb 1;85(3):862

The use of recombinant adeno-associated virus (rAAV) vectors provides a new strategy to investigate the role of specific regulatory elements and trans-acting factors in globin gene expression. We linked hypersensitivity site 2 (HS2) from the locus control region (LCR) to a A gamma-globin gene (A gamma*) mutationally marked to allow its transcript to be distinguished from endogenous gamma-globin mRNA. The vector also contains the phosphotransferase gene that confers resistance to neomycin (NeoR). HS2 region mutations within the NF-E2 motifs prevented NF-E2 binding while preserving AP-1 binding. Another set in the GATA-1 motif prevented binding of the factor. Several NeoR K562 clones containing a single unrearranged RAAV genome with the A gamma* gene linked to the native HS2 core fragment (WT), mutant NF-E2 HS2 (mut-NFE2), mutant GATA-1 HS2 (mut-GATA1), or no HS [(-)HS] were identified. In uninduced K562 cells, mut-NFE2 clones expressed A gamma* mRNA at the same level as the WT clones, compared with a lack of A gamma* signal in the (-)HS2 clones. However, hemin induction of mut- NFE2 clones did not result in an increase in the A gamma* signal above the level seen in uninduced cells. Mut-GATA1 clones expressed the A gamma* mRNA at the same level as WT clones in both uninduced and induced cells. Thus, GATA-1 binding to this site does not appear to be required for the enhancing function of HS2 in this context. This single- copy rAAV transduction model is useful for evaluating the effects of specific mutations in regulatory elements on the transcription of linked genes.