Γ-globin mRNA Research Articles

Identification of New and Diverse Inducers of Fetal Hemoglobin with High Throughput Screening (HTS)

AbstractAbstract 4277Pharmacologic augmentation of fetal hemoglobin (HbF, γ-globin) production, to replace diminished β-globin chains in the β-thalassemias and to inhibit HbS polymerization in sickle cell disease, is a definitive therapeutic modality. Despite long-term efforts, regulatory approval has been obtained for only one chemotherapeutic agent. Pharmacologic reactivation of high-level HbF expression with non-cytotoxic, tolerable therapeutics is still an unmet medical need for this global health burden. To investigate potential therapeutic libraries for unrecognized HbF inducers, we developed a high-throughput screening (HTS) program to interrogate diverse chemical libraries, including a library of FDA-approved and clinical stage drugs. This program has identified unexpected new and highly potent HbF-inducing drugs, some of which are already in clinical use for other medical indications and have established safety profiles. A human cell-based assay which was previously used in low throughput assays, utilizing a 1.4-kilobase (kb) KpnI-BglII fragment of the HS2 of the locus control region (LCR) linked to the γ-globin gene promoter and the enhanced green fluorescent protein (EGFP) reporter gene, was adapted for high throughput screening and employed as the primary screen. Cytotoxic activity was assayed in a simultaneous counter screen. A number of hits were identified as being more potent than positive controls (such as butyrate). Several hits were immediately eliminated from further development as potential hemoglobinopathy therapeutics because of cytotoxicity (e.g., Idarubicin) or undesirable off-target effects, but nonetheless validated the HTS itself and were validated in secondary confirmatory assays as highly-potent HbF-inducers. The HTS assay identified 8 FDA-approved drugs as potent inducers of γ-globin gene expression, with activity at 1–2 logs lower concentrations (1000-fold higher potency) than prior generation therapeutic candidates. The γ-globin-specificity of hits was determined in a secondary assay employing a stably-transfected dual-luciferase reporter construct containing the LCR and the β-globin promoter linked to renilla luciferase and the Aγ-globin promoter linked to firefly luciferase (μLCRβprRlucAγprFluc cassette). Clinical-stage or clinically-approved agents, including Ambroxol at 1 μM, Desloratadine at 1 μM, Resveratrol at 10 μM, Benserazide at 5 μM, the HDAC inhibitor MS-275 at 5 μM, and an established bioactive, NSC-95397, at 1 μM were all significantly more active in this assay than Butyrate at 2000 μM, with MS-275 and Resveratrol being the most active. These drugs were then assayed for their ability to induce γ-globin mRNA expression in cultured primary human erythroid progenitors, at concentrations which are pharmacologically achievable in humans. Drugs significantly more active in γ -globin mRNA induction than the positive control (2-fold induction) in this system included Ambroxol (3-fold), Desloratadine (up to 6-fold), Resveratrol (up to 3-fold), Benserazide (up to 5-fold), and MS-275 (up to 3.7-fold). Two agents were subsequently studied in anemic baboons, and demonstrated in vivo induction of γ-globin mRNA, HbF, and F-reticulocytes. Unexpectedly, rises in total hemoglobin (>1 gm/dL) also occurred with 2 agents. Thus, a panel of structurally- and functionally-unrelated compounds demonstrate greater HbF-inducing activity, with up to 1000-fold higher potency, than current HbF-inducers which have significant activity in clinical trials. Some of the drugs identified by HTS have entirely benign safety profiles. These candidates could be clinically evaluated rapidly and at significantly less cost than new chemical entities, which require extensive toxicology, manufacturing, and clinical evaluation. These findings demonstrate the utility of a high-throughput screening program based on γ-globin gene promoter induction. Disclosures:No relevant conflicts of interest to declare.

HbF-Inducing Cytokines and BCL11A shRNA Have Combined Effects Upon Globin Gene Reprogramming In Adult Human Erythroblasts

AbstractAbstract 2076Persistent expression of HbF ameliorates the clinical symptoms of β-thalassemia and sickle cell disease; therefore, reactivation of the γ-globin gene in adults is of substantial interest for the clinical management of β-hemoglobinopathies. The recently identified γ-globin repressor, BCL11A, has been shown to regulate HbF expression level in adult erythroblasts. Cytokine signal transduction also increases HbF expression in cultured erythroblasts due in part to reducing the chromatin occupancy of BCL11A within the β-globin locus. Cultured CD34+ cells from at least six healthy research subjects were utilized to investigate the potential for combined effects of genetic manipulation of BCL11A with signal transduction upon globin gene regulation. The cells were transduced with BCL11A shRNA lentiviral vectors under culture conditions with and without addition of HbF-inducing cytokines. Under all culture conditions, nuclear BCL11A protein levels were reduced to nearly undetectable levels by the shRNA lentiviral vectors. The combination effects of BCL11A knockdown and HbF-inducing cytokines were quantitated by QRT-PCR. The absolute copy numbers of the eight globin genes (β-like genes; ε-, γ-, δ-, β-globins and α-like genes; ζ-, μ-, α- and θ-globins) at the proerythroblast stage of maturation were determined using standard curve methods. As previously reported, cytokine-mediated induction of HbF significantly increased the expression of γ-, ε-, and ζ-globin mRNAs while β- and δ-globins were down-regulated (p-value <0.05). BCL11A knockdown similarly increased in γ-globin mRNA, decreased β- and δ-globin mRNAs, as well as an unexpected increase in ε- and ζ-globin mRNAs. A significant reduction in α-globin mRNA was additionally detected in association with BCL11A knockdown. Although the transcript levels of several individual globin genes were affected, the total α- to non-α-globin ratio remained stable. The combination of BCL11A knockdown with cytokine signal transduction resulted in further increased γ-globin mRNA to levels of 78.1 ± 3.5% of the total transcripts from the β-globin cluster. Strongly synergistic effects of BCL11A knockdown and cytokine signal transduction were also detected in the embryonic genes. As a result, ε-globin mRNA reached levels of 8.7 ± 4.7% of the total transcripts from the β-globin cluster compared with 0.0003% in baseline cultures, 0.07 ± 0.03% in HbF-inducing cytokines alone, and 0.04 ± 0.03% in BCL11A knockdown alone. Hemoglobin expression was also examined in matured cells from the same donors using high pressure liquid chromatography (HPLC). The additive effects of BCL11A knockdown and HbF-inducing cytokines were shown by HPLC: %HbF = 2.0 ± 0.7 at baseline, 28.2 ± 14.1 in HbF-inducing cytokines alone, 47.6 ± 5.7 in BCL11A knockdown alone, and 74.9 ± 2.5 in BCL11A knockdown combined with HbF-inducing cytokines. Cation exchange HPLC further revealed reduced HbA along with an unidentified peak downstream of HbA2 encompassing 1–5% of the total hemoglobin that is being investigated for embryonic globin content. These data demonstrate that synergistic and robust effects upon globin gene reprogramming in adult human erythroblasts may be achieved by combined mechanisms of signal transduction and genetic manipulation of BCL11A. Disclosures:No relevant conflicts of interest to declare.

The Role of p38 MAPK/CREB1 Signaling In γ-Globin Gene Regulation

AbstractAbstract 1012The clinical symptoms of sickle cell disease can be ameliorated by increased fetal hemoglobin (HbF) levels. Previous work from our laboratory demonstrated that Trichostatin A (TSA) and sodium butyrate (NaB) activate γ-globin expression via p38 MAPK signaling. In addition, cAMP response element binding protein 1 (CREB1) was shown to trans-activate the -1222 Gγ-globin cAMP response element (G-CRE) in a transient assay system. To study the role of p38 MAPK signaling in γ-globin regulation, loss of function siRNA studies were performed in K562 cells. siRNA-mediated knockdown of p38 MAPK resulted in 72% loss of γ-globin transcription. Furthermore, enforced stable expression of MKK3/6 increased the phosphorylated form of p38 MAPK by 70%, which in turn produced a 2- to 3-fold increase in γ-globin mRNA and HbF levels. Likewise, siCREB1 treatment reduced CREB1 levels by 62% and down regulated γ-globin expression 59%. In the same vein, stable expression of recombinant CREB1 activated HbF by 2-fold. These findings were subsequently confirmed in human primary erythroid cells grown in a two-phase liquid culture system. On day 11, we observed 50–70% γ-globin silencing after CREB1 and p38 MAPK siRNA knockdown with 60% target gene silencing. CREB1 enforced expression trans-activated γ-globin 4.5-fold which was accompanied by a 90% increase in HbF-FITC positive cells and HbF levels. Collectively, these data demonstrate that p38 MAPK and CREB1 are required for steady-state γ-globin gene transcription. To determine the role of the G-CRE in γ-globin regulation, the Gγ-globin promoter (-1500 to +36) was cloned into pGL4.17 Luc2/neo to produce pGγLuc2 (wild type) and mutant plasmids: -1225 G/A (m1), -1227 AC/TG (m2) and a scrambled G-CRE (m3s). Five K562 stable lines including KLuc2 (promoterless), KGγLuc2, KGγLuc2(m1), KGγLuc2(m2) and KGγLuc2(m3s) were established. Luciferase activity in KGγLuc2 was 1000-fold higher than in the control Kluc2 line; all mutations produced >90% loss of luciferase activity and a loss of γ-globin trans-activation by TSA and NaB. Next, siRNA studies were completed to determine if the G-CRE is required for γ-globin activation. A dose-dependent loss of promoter activity was observed after p38 MAPK and CREB1 siRNA knockdown of the KGγLuc2 cell line; however, promoter silencing was not observed in the mutant lines supporting a role for the G-CRE in p38 MAPK/CREB1 mediated γ-globin regulation. To study in vivo binding, chromatin immunoprecipitation (ChIP) assays were performed with CREB1 antibody in the KGγLuc2 stable line. We observed comparable 2- to 3-fold chromatin enrichment with CREB1 compared to the control IgG in the G-CRE regions of the pGγLuc2 plasmid and endogenous Gγ-globin promoter. To determine if an enhanceosome complex is bound to the G-CRE, we performed affinity column pull-down/mass spectrometry analysis. K562 nuclear extract was purified on a Heparin Sepharose column, following which fractions eluting at 0.6M NaCl showing peak gel shift binding activities with the G-CRE oligo were loaded into a size selecting Suprose 6 gel exclusion column. G-CRE eluting fractions were then identified by protein microsequencing (MS/MS). We identified CREB1, ATF2, c-Jun, BRG-1, hnRNPC1/C2, and the TCP-1 complex as major components. To determine protein co-localization, promoter pull-down assays were performed using biotinylated wild type and mutant (AC/TG) G-CRE probes and K562 nuclear extracts. We observed simultaneous CREB1, ATF-2 and cJun binding to the G-CRE which was abolished in the mutant probe. However, Brg1 was bound after NaB (2mM) induction. Subsequent co-IP studies showed interactions between ATF-2 and Brg1, CREB1, cJun, and hnRNPC1C2, which was further confirmed by co-elution profile of these molecules observed by sucrose gradient centrifugation, thus implying association as one complex. These data support complex protein-protein interactions in the G-CRE, which modulate γ-globin gene expression. Additional studies will be performed in primary erythroid cells using siRNA-based gene silencing and ChIP assays to determine novel mechanisms of γ-globin regulation and to define in vivo binding of proteins identified in the G-CRE enhanceosome complex. Disclosures:No relevant conflicts of interest to declare.

Exploiting the NRF2/Antioxidant Response Element Signaling Pathway to Induce γ-Globin Gene Expression and HbF Production

AbstractAbstract 2074Induction of fetal hemoglobin (HbF) has proven therapeutic potential to treat sickle cell disease and β-thalassemia. However, agents known to be effective in humans, including hydroxyurea, DNMT inhibitors and butyrate derivatives are not ideal due to suppression of hematopoiesis and the possibility of long-term side effects. Two natural compounds, angelicin (Lampronti, et al, Eur J Hematol 2003) and resveratrol (Rodrigue, et al, Hematology 2001) have been found to induce γ-globin gene expression in K562 cells. These agents may be important lead compounds as they are generally non-cytotoxic and are being evaluated in ongoing human trials as cancer chemopreventative agents where these and several other agents are thought to work by activating antioxidant response pathway genes. The products of these genes are enzymes involved in antioxidant and detoxification activities and include NADPH-quinone oxireductase 1 (NQO1), glutamate-cysteine ligase (GCL) and glutathione S-transferase (GST). The activation of these genes is mediated by the transcription factor, NF-E2 related factor 2 (NRF2), which binds to a specific antioxidant response element (ARE) sequence (TGACnnnGCA) in target gene promoters. The proximal γ-globin promoter contains an ARE sequence between the two CAAT boxes, suggesting that it too may be activated by NRF2. This led us to hypothesize that drugs that activate the ARE/NRF2 pathway may provide a less toxic approach to HbF induction. To test this hypothesis, we treated K562 cells with various NRF2 pathway activators. We initially tested six compounds that are known to induce antioxidant response genes at doses that did not inhibit proliferation and found the most pronounced γ-globin induction with tert-butylhydroquinone (tBHQ) (2.8 fold). We next tested tBHQ in two different primary cell culture models: erythroid precursors isolated from normal human bone marrow and in vitro erythroid differentiation of primary human CD34+ cells. In both of these models, tBHQ treatment increased γ-globin steady state mRNA levels and induced expression of NRF2 target genes. Treatment of differentiating erythroid cells caused a dose dependent increase in γ/(γ+β) mRNA and % HbF. HPLC analysis revealed the highest non-toxic concentration of tBHQ, 5μM, produced 10% HbF while the positive control, 0.5μM of 5-Azacytidine, resulted in 12% HbF compared to the untreated control at 3%. Since similar mRNA induction was seen in K562 cells, we used these cells to characterize the mechanism of tBHQ induced γ-globin expression. First, we used siRNA to decrease NRF2 mRNA levels. This resulted in a greater than 75% knockdown of NRF2 mRNA and protein and reduced tBHQ induction of γ-globin and NQO1 gene expression by 90% and 75%, respectively, compared to samples transfected with scrambled siRNA (p < 0.01). Subsequent experiments showed that tBHQ treatment resulted in NRF2 translocation to the nucleus and binding to the NQO1 and γ-globin promoters but not at negative control sites. In addition, inducing NRF2 translocation by transiently suppressing levels of its inhibitor, KEAP1, did not result in full induction of γ-globin mRNA expression, suggesting that NRF2 translocation alone is not sufficient for γ-globin induction. However, when combined with tBHQ, suppression of KEAP1 did enhance γ-globin induction. Taken together, these results suggest that NRF2/ARE pathway activation induces γ-globin mRNA expression and HbF production in primary human erythroid cells and that this is a promising strategy for further pre-clinical development. Disclosures:No relevant conflicts of interest to declare.

Cucurbitacin D induces fetal hemoglobin synthesis in K562 cells and human hematopoietic progenitors through activation of p38 pathway and stabilization of the γ-globin mRNA

The search for novel therapeutic candidates targeting fetal hemoglobin (HbF) activation to reduce the imbalance of globin genes is regarded as a promising approach for the clinical management of sickle cell disease and β-thalassemia. For the first time, we identified cucurbitacin D (CuD), an oxygenated tetracyclic triterpenoid, as a molecular entity inducing γ-globin gene expression and HbF synthesis in K562 cells and human hematopoietic progenitors from a β-thalassemia patient. CuD demonstrated a higher potency in HbF induction when compared with hydroxyurea, which was revealed by the evidence that CuD results in a higher fetal cell percentage and greater HbF content in K562 cells, in addition, to being less cytotoxic. Moreover, CuD also promotes higher HbF expression in primary erythroid cells. In the study to elucidate the molecular mechanisms of CuD's action, our data indicated that CuD-stimulated HbF synthesis was mediated by p38 pathway activation. At the post-transcriptional level, CuD treatment led to a significant elongation of the γ-globin mRNA half-life in K562 cells. Taken together, the results suggest that CuD may be a potential therapeutic agent for β-hemoglobinopathies, including sickle cell anemia and β-thalassemia.

Identification of the Linkage of a 1.357 KB β-Globin Gene Deletion and A γ-Globin Gene Triplication in a Chinese Family

The 1.357 kb β-globin gene deletion was identified in a Chinese family by multiplex ligation-dependent probe amplification (MLPA) followed by gap-polymerase chain reaction (gap-PCR) and sequencing. Interestingly, this form of the deletion was linked to a –Gγ–AGγ–Aγ triplication. The proband, a compound heterozygote for this linked mutant gene and a β-globin gene [−28 (A>G)] mutation, had a phenotype of β-thalassemia intermedia (β-TI). She was not transfusion dependent and had the following parameters: a Hb level of 5.3 g/dL, 72.8% Hb F and 55.1% Gγ chain in Hb F. Four members of the family, who were carriers of this linked mutant gene, had a hematological phenotype of β0-thalassemia (β0-thal) with high Hb F and low Gγ chain values. RNA analyses showed decreased levels of β-globin mRNA and increased levels of γ-globin mRNA in heterozygotes. Haplotype analyses indicated that the unusual form of the β-globin gene deletion and γ-globin gene triplication in cis were linked to halotype [+ – – – – – –].

X-Ray and UV Irradiation and Heat Shock Induce γ-Globin Gene Expression in Erythroid Cells Via P38 MAPK Signaling.

Abstract 459We have recently presented a cell stress signaling model to explain the ability of a wide variety of mechanistically distinct drugs to induce fetal hemoglobin (HbF) production (Mabaera et al, Exp Hematol 2008). This model proposes that HbF inducing agents, including DNMT and HDAC inhibitors, short-chain fatty acid derivatives and cytotoxic agents induce HbF production by activating intracellular cell stress signaling pathways and that the p38 MAPK (mitogen-activated protein kinase) pathway plays a central role in the induction process. The major alternative to this model is that each class of drugs works through a distinct mechanism, such as global DNA hypomethylation or histone hyperacetylation. If our model is correct, then non-pharmacologic inducers of p38 MAPK signaling, such as X-ray and UV irradiation, heat shock, and osmotic shock should also increase γ-globin gene expression. If these physical stresses do not up-regulate γ-globin gene expression, then our model is likely to be incorrect. To mimic our previous experiments in which we treated differentiating human primary erythroid cells with inducing agents over several days, we exposed K562 cells to physical stresses daily for 5 days at doses that did not cause cell death. To test whether X-irradiation induces the transcription of γ-globin mRNA, cells were irradiated at doses ranging from 0 to 1.0 cGy/day, in the absence and presence of SB203580 (a p38 MAPK inhibitor). Experiments were first performed on K562 cells, and then during in vitro erythroid differentiation of primary human CD34+ cells. These treatments stimulated a strong, dose-dependent increase of γ-globin mRNA in both K562 cells (up to 6-fold over untreated cells) and in primary erythroid cells (up to 5-fold) as measured by quantitative RT-PCR. SB203580 abolished this effect in both cell types. The inhibitor also caused a decrease in γ-globin mRNA expression in untreated control cells, suggesting that p38 MAPK signaling plays a role in basal γ-globin gene expression. The same X-ray doses also induced phosphorylation of the down-stream p38 MAPK target, HSP-27 and up-regulated expression of stress-induced transcription factor genes including GADD34, CHOP (GADD153), and ATF3. Expression of GADD34 and CHOP genes and HSP-27 phosphorylation were inhibited by SB203580. We next tested the ability of UV light (254 nm) administered daily for 5 days to induce γ-globin mRNA in K562 cells. A dose-dependent increase in γ-globin mRNA was observed following exposure to doses as low as 10 J/m2. Cells treated with 35 J/m2 had 2.7-fold higher levels of steady state γ-globin mRNA compared to untreated control cells. This induction was also inhibited by SB203580. Preliminary experiments with heat shock have yielded similar results. Following a single 180-minute exposure to 42°C, K562 cells showed a 2.2-fold increase in γ-globin mRNA compared to untreated cells. Shorter exposures to higher temperatures (e.g., 50°C for 15 minutes) caused an approximately 3-fold increase in γ-globin steady-state mRNA after 24 hours. These 2- to 3-fold increases in expression are similar to those we have previously observed in primary human erythroid cells with 5-azacytidine and butyrate. In none of our experiments did the K562 cells become benzidine positive, indicating that increased γ-globin expression was not the result of activating an erythroid differentiation program. Together with other published studies, these data support the hypothesis that p38 MAPK pathway signaling, whether caused by drugs or physical stress, is a key component of γ-globin gene induction. This in turn suggests that the components of the p38 MAPK pathway could serve as novel targets for the development of new HbF inducing agents. Disclosures:No relevant conflicts of interest to declare.

Role of HDAC9 in γ-Globin Gene Regulation.

Abstract 4074Poster Board III-1009Strategies to induce fetal hemoglobin (HbF) synthesis for the treatment of β-hemoglobinopathies will likely involve chromatin modification in the presence of histone deacetylase (HDAC)/protein complexes to promote γ-globin gene activation. The role of various HDACs in globin transcription is not very well understood therefore, the objective of our study was to identify HDACs involved in γ-gene regulation. Screening studies were performed in K562 erythroleukemia cells to determine transcription levels for HDAC genes in the absence or presence of HbF induction. Treatment with butyrate (2mM), trichostatin A (0.5μM) and the non-HDAC inhibitor control hemin (50μM) significantly reduced mRNA levels of HDAC9 and its splice variant HDRP (histone deacetylase related protein) lending indirect evidence for their involvement in drug-mediated γ-globin regulation. Subsequent studies were performed to delineate whether HDAC9 can directly modulate γ-globin gene transcription since a role for HDAC9 in hematopoiesis has been previously demonstrated. Furthermore, consensus binding sites for GATA-1 are present in the HDAC9 gene proximal promoter. Initially, we performed siRNA knockdown using Oligofectamine (Invitrogen) in K562 cells and measured γ-globin levels by real time quantitative PCR analysis. Treatment with siHDAC9 (Dharmacon) produced dose-dependent γ-globin gene silencing over an 80-320nM range; control siRNA molecules had no effect. When HDAC9 was over-expressed in K562 cells using pTarT-HDAC9 at 10-50μg concentrations, a dose dependent 2.5-fold increase in γ-globin mRNA (p<0.05) was produced. These data support a positive regulatory role for HDAC9 in γ-gene regulation. To confirm the physiological relevance of HDAC9, similar studies were performed in human primary erythroid progenitors using a two-phase liquid culture system. The 320nM siHDAC9 concentration produced 48% and 60% decrease in γ-globin mRNA at day 11 (early progenitors) and day 28 (late progenitors) respectively. Enforced HDAC9 expression increased γ-globin by 2.5-fold (p<0.05) at both days. ELISA was performed to quantify HbF protein and cytospin preps were made to visualized hemoglobin by fluorescent staining with anti-γ-FITC antibody. HDAC9 enforced expression for 72 hrs produced a 7-fold increase in HbF and γ-FITC positive cells increased >50%. Collectively these data support a positive role for HDAC9 in γ-globin regulation. Chromatin immunoprecipitation assays will be completed to elucidate the contribution of HDAC9 in maintaining an active chromatin domain in the γ-globin promoter. We will also define interactions of GATA-1 in the HDAC9 gene to coordinate expression during erythroid maturation. Disclosures:No relevant conflicts of interest to declare.

Induction of γ-Globin Gene Expression Via the Nrf2/Antioxidant Response Element Signaling Pathway.

Abstract 975Induction of fetal hemoglobin (HbF) has proven therapeutic potential to treat sickle cell disease and β-thalassemia. However, agents known to be effective in humans, including HU, DNMT inhibitors and butyrate derivatives are not ideal due to suppression of hematopoeisis and the possibility of long-term side effects including those related to DNA mutation and epigenetic changes. Recently, two natural compounds, angelicin (Lampronti, et al, Eur J Hematol 2003) and resveratrol (Rodrigue, et al, Hematology 2001) have been found to induce γ-globin gene expression in K562 cells. These agents may be important lead compounds as they are generally non-cytotoxic and are being evaluated in ongoing human trials as cancer chemoprevention agents. These and several other agents are thought to work by activating antioxidant response pathway genes. The products of these genes are enzymes involved in antioxidant and detoxification activities and include NADPH-quinone oxireductase 1 (NQO1), glutamate-cysteine ligase (GCL) and glutathione S-transferase (GST). The activation of these genes is mediated by the transcription factor NF-E2 related factor 2 (Nrf2) which binds to a specific antioxidant response element (ARE) sequence (TGACnnnGCA) in target gene promoters. The proximal γ-globin promoter contains an ARE sequence between two CAAT boxes, suggesting that it too may be activated by Nrf2. This led us to hypothesize that drugs which activate the ARE/Nrf2 pathway may provide a less toxic approach to HbF induction. Several compounds that activate this pathway are already approved for human use or are under investigation in human trials. To test this hypothesis, we treated K562 cells with various Nrf2 pathway activators. First, we determined the maximum concentration of each compound that did not inhibit proliferation of K562 cells. Using these doses, we performed time course experiments by treating K562 cells and then measuring steady-state mRNA levels of Nrf2 target genes and γ-globin using quantitative real-time PCR. We initially tested six compounds that are known to induce antioxidant response genes. We found the most pronounced γ-globin induction followed treatment with tert-butylhydroquinone (tBHQ) (2.8 fold), 3H-1,2 dithiole-3-thione (D3T) (2.4 fold) and curcumin (2.1 fold). We next tested tBHQ in erythroid precursors isolated from normal human bone marrow. In these primary cells it also significantly increased expression of γ-globin mRNA and of Nrf2 target genes NQO1, GCLM and GSTP1. To determine if Nrf2 was necessary for tBHQ induction of γ-globin mRNA we used siRNA to knockdown Nrf2 expression in K562 cells. Our results show that transiently silencing Nrf2 transcription prevented γ-globin and NQO1 gene induction by tBHQ compared to samples transfected with scrambled siRNA (p < 0.01). To determine if this requirement for Nrf2 is due to Nrf2 binding at the ARE consensus sequence in the γ-globin promoter, ChIP analysis was performed. This demonstrated that Nrf2 binding at both the γ-globin and NQO1 promoters was progressively increased at 4 and 6 hours after tBHQ treatment compared to the negative control necdin promoter where there was no change. Taken together, these results suggest that the antioxidant tBHQ induces γ-globin mRNA expression through the Nrf2/ARE pathway and that this may be a less toxic strategy for γ-globin gene induction in people with hemoglobinopathies. Disclosures:No relevant conflicts of interest to declare.

Effects of Astragalus polysaccharide on the erythroid lineage and microarray analysis in K562 cells

Ethnopharmacological relevance Astragalus polysaccharide (APS), obtained from Astragalus membranaceus, displays a range of activities in many systems, including the promotion of immune responses, anti-inflammation, and the protection of vessels. It possesses potent pharmacological activity on differentiation to the erythroid lineage. Aim of the study To investigate the effects of APS on the erythroid differentiation and the mechanism of action by microarray analysis in K562 cells. Materials and methods Benzidine staining, semi-quantitative RT-PCR, Western blot and microarray methods were used to survey the effects of APS on inducing erythroid differentiation and the changes of gene expression profile in K562 cells. Results Of the 13.2% positive cells detected by benzidine staining, the induction was the highest with 200 μg/ml APS on 72 h. Gγ-mRNA expression and fetal hemoglobin synthesis were significantly up-regulated. Microarray analysis showed that 31 genes were up-regulated and 108 genes were down-regulated. These differential expression genes generally regulate protein binding, cellular metabolic process, the cell proliferation, and transcriptional activator activity. The γ-globin gene was up-regulated, the genes related with erythroid differentiation such as LMO2, Runx1 and GTF2I were up-regulated, while Bklf, Eklf, EPHB4 and Sp1 were down-regulated. Conclusions Our studies indicate that APS indicate potent activities on the erythroid differentiation by modulating genes of LMO2, Klf1, Klf3, Runx1, EphB4 and Sp1, increasing γ-globin mRNA expression and fetal hemoglobin synthesis in K562 cells.

Increase in γ-globin mRNA content in human erythroid cells treated with angelicin analogs

The aim of the present study was to identify molecular analogs of angelicin (ANG) able to increase erythroid differentiation of K562 cells and expression of gamma-globin genes in human erythroid precursor cells, with low effects on apoptosis. ANG-like molecules are well-known photosensitizers largely used for their antiproliferative activity in the treatment of different skin diseases (i.e., psoriasis, vitiligo, eczema, and mycosis fungoides). To verify the activity of these derivatives, we employed three experimental cell systems: (1) the human leukemic K562 cell line, (2) K562 cell clones stably transfected with a pCCL construct carrying green-EGFP under the gamma-globin gene promoter, and (3) the two-phase liquid culture of human erythroid progenitors isolated from normal donors and beta-thalassemia patients. The results of our study suggest that trimethyl ANG is a powerful inducer of erythroid differentiation, compared with known inducers, such as ANG, cytosine arabinoside, mithramycin, and cisplatin. These data could have practical relevance, because pharmacologically mediated regulation of human gamma-globin gene expression, with the consequent induction of fetal hemoglobin, is considered a potential therapeutic approach in hematological disorders including beta-thalassemia and sickle cell anemia.

Differences in response to fetal hemoglobin induction therapy in β-thalassemia and sickle cell disease

Inducers of fetal hemoglobin (HbF) have shown considerable promise in the treatment of sickle cell disease (SCD). However, the same agents have shown less clinical activity in β-thalassemia (β-Thal). To understand the basis of these differences in clinical effectiveness, we compared the effects of butyrate and hemin on the expression of the different globin genes in progenitors-derived erythroid cells from patients with β-Thal intermedia and SCD. Exposure to butyrate resulted in an augmentation of γ-globin mRNA levels in both SCD and β-Thal. Interestingly, butyrate exposure increased α-globin expression in β-Thal, while α-globin mRNA levels decreased in SCD in response to butyrate. As a result, the favorable effects of the butyrate-induced increase in γ-globin expression on α:β-like globin mRNA imbalance in β-Thal were reduced as a result of the associated increase in α-globin expression. Hemin had similar but less profound effects on all three globin genes in both categories of patients. Although the majority of patients with β-Thal did not correct their globin imbalance in response to butyrate or hemin induction of HbF in a minority of patients resulted in marked reduction in globin imbalance. Thus, we believe that the poor clinical response in a majority of patients with β-Thal to inducers of γ-globin expression may be a reflection of unfavorable effects of these agents on the other globin genes.

Correction of Murine Sickle Cell Disease Using γ-Globin Lentiviral Vectors to Mediate High-level Expression of Fetal Hemoglobin

Increased levels of red cell fetal hemogloblin, whether due to hereditary persistence of expression or from induction with hydroxyurea therapy, effectively ameliorate sickle cell disease (SCD). Therefore, we developed erythroid-specific, gamma-globin lentiviral vectors for hematopoietic stem cell (HSC)-targeted gene therapy with the goal of permanently increasing fetal hemoglobin (HbF) production in sickle red cells. We evaluated two different gamma-globin lentiviral vectors for therapeutic efficacy in the BERK sickle cell mouse model. The first vector, V5, contained the gamma-globin gene driven by 3.1 kb of beta-globin regulatory sequences and a 130-bp beta-globin promoter. The second vector, V5m3, was identical except that the gamma-globin 3'-untranslated region (3'-UTR) was replaced with the beta-globin 3'-UTR. Adult erythroid cells have beta-globin mRNA 3'-UTR-binding proteins that enhance beta-globin mRNA stability and we postulated this design might enhance gamma-globin expression. Stem cell gene transfer was efficient and nearly all red cells in transplanted mice expressed human gamma-globin. Both vectors demonstrated efficacy in disease correction, with the V5m3 vector producing a higher level of gamma-globin mRNA which was associated with high-level correction of anemia and secondary organ pathology. These data support the rationale for a gene therapy approach to SCD by permanently enhancing HbF using a gamma-globin lentiviral vector.

5-Azacytidine Induces Human Fetal Hemoglobin Production through Activation of the p38 MAPK and Integrated Stress Response Signaling Pathways

Pharmacologic induction of fetal hemoglobin (HbF) has the potential to improve the health and quality of life of people with β-thalassemia and sickle cell disease. 5-Azacytidine (5-Aza) is a key lead compound as it, and the related drug decitabine, are active in most β-thal and SCD patients including those resistant to butyrate and because they have been shown to produce clinical benefits for selected patients. However, these drugs must be administered by injection, they depress blood counts and they are known to alter DNA structure and cause changes in genome-wide DNA methylation. Efforts to design improved inducing agents have been limited by an incomplete understanding of the mechanisms underlying pharmacologic induction of HbF. Current theories include proposals that 5-Aza and decitabine induce increased γ-globin gene expression by altering the kinetics of erythroid differentiation or by decreasing γ-globin promoter DNA methylation. We recently provided evidence that the primary action 5-Aza is not through either of these mechanisms. These results, and data from the literature, have led us to propose a new model of HbF induction based on activation of cell stress signaling pathways. To begin to evaluate this model, we used a human in vitro CD34 erythroid differentiation system to test the hypotheses that the p38 MAPK stress signaling pathway was involved in 5-Aza induction of HbF. Quantitative RT-PCR, Western blotting and HPLC were used to assess mRNA, protein and Hb levels. Our results showed that 500 nM 5-Aza causes p38 MAPK pathway activation as evidenced by phosphorylation of p38 MAPK, the downstream kinase MK2 and the downstream target Hsp27. The p38 MAPK inhibitor SB203580 (SB) prevented pathway activation and suppressed both baseline and 5-Aza-induced γ-globin mRNA production. On day +13 of differentiation, relative γ-globin mRNA levels were 1.00 (untreated), 0.40 (SB alone), 2.18 (5-Aza alone) and 0.46 (5-Aza + SB) All values were p<0.05 vs. control. HbF levels at the end of differentiation were 2.1% (untreated), 2.2% (SB alone), 24.1% (5-Aza) and 10.7% (5-Aza + SB). These results indicate that the p38 MAPK pathway is activated by 5-Aza and that inhibition of this pathway suppresses 5-Aza-induced increases in γ-globin mRNA and HbF production. However, the fact that SB did not completely inhibit HbF production suggested other pathways might be involved. The Integrated Stress Response (ISR) pathway (also known as the Unfolded Protein Response pathway) responds to a variety of stresses with phosphorylation of translation factor eIF2α. While this initially inhibits translation of most proteins, production of the ATF4 transcription factor is increased and it mediates a secondary transcriptional response. This pathway is known to be activated in differentiating erythroid cells by inadequate heme and other stresses. 500 nM 5-Aza caused pathway activation as evidenced by phosphorylation of both heme-regulated eIF2α kinase (HRI) and eIF2α and by increased levels of ATF4. Expression of a dominant-negative ATF4 protein inhibited 5-Aza induction of γ-globin mRNA by more than 50%. Independent activation of the ISR pathway by L-azetidine-2-carboxylic acid (a proline analogue that causes protein miss-folding), induced γ-globin mRNA and HbF to levels equivalent to those seen with 5-Aza. 5-Aza also alters the polysome profiles of γ and β-globin mRNA in differentiating human cells (50% increase in polysome-associated γ-globin mRNA, p=0.02; 55% decrease in β-globin mRNA, p<0.01). Taken together, these results suggest that 5-Aza induces HbF production through activation of the p38 MAPK and ISR cell signaling pathways and that this induction involves both transcriptional and translational effects. The identification of cell signaling pathways involved in HbF induction opens the possibility of future targeted drug development.

MicroRNA‐223 reversibly regulates erythroid and megakaryocytic differentiation of K562 cells

MicroRNAs (miRNAs) are thought to modulate a variety of cellular events. Several studies have revealed the functions of miR-223 in granulopoiesis. Here we analysed miR-223 expression in various human tissues, blood and leukaemia cells, and focused on its role in K562 erythroid and megakaryocytic differentiation. MiR-223 was detected not only in granulocytes but also in erythroid cells. In K562 cells, expression of miR-223 was down-regulated during haemin-induced erythroid differentiation but up-regulated during phorbol myristate acetate (PMA)-induced megakaryocytic differentiation. The overexpression of miR-223 resulted in significant decrease of γ-globin mRNA and the fraction of benzidine-positive cells in K562 cells, suggesting a suppressive effect of miR-223 on erythroid differentiation. Peaks corresponding to 4N cells in stable transfectants overexpressing miR-223 were higher than that in control K562 cells during megakaryocytic differentiation, indicating that miR-223 increases megakaryocytic differentiation. The expression of LIM domain only 2 (LMO2) reporter was suppressed in NIH-3T3 when the expression of miR-223 was enforced by both the luciferase and fluorescence system. Furthermore, LMO2 mRNA and protein levels were significantly decreased in stable K562 transfectants overexpressing miR-223. These results indicate that LMO2 is a direct target of miR-223. Thus, our results suggest that miR-223 reversibly regulates erythroid and megakaryocytic differentiation of K562 cells via down-modulation of LMO2.

Fetal Hemoglobin Inducers from the Natural World: A Novel Approach for Identification of Drugs for the Treatment of β‐Thalassemia and Sickle‐Cell Anemia

The objective of this review is to present examples of lead compounds identified from biological material (fungi, plant extracts and agro-industry material) and of possible interest in the field of a pharmacological approach to the therapy of β-thalassemia using molecules able to stimulate production of fetal hemoglobin (HbF) in adults. Concerning the employment of HbF inducers as potential drugs for pharmacological treatment of β-thalassemia, the following conclusions can be reached: (i) this therapeutic approach is reasonable, on the basis of the clinical parameters exhibited by hereditary persistence of fetal hemoglobin patients, (ii) clinical trials (even if still limited) employing HbF inducers were effective in ameliorating the symptoms of β-thalassemia patients, (iii) good correlation of in vivo and in vitro results of HbF synthesis and γ-globin mRNA accumulation indicates that in vitro testing might be predictive of in vivo responses and (iv) combined use of different inducers might be useful to maximize HbF, both in vitro and in vivo. In this review, we present three examples of HbF inducers from the natural world: (i) angelicin and linear psoralens, contained in plant extracts from Angelica arcangelica and Aegle marmelos, (ii) resveratrol, a polyphenol found in grapes and several plant extracts and (iii) rapamycin, isolated from Streptomyces hygroscopicus.

Neither DNA Hypomethylation or Changes in the Kinetics of Erythroid Differentiation Account for 5-Azacytidine's Ability To Induce Human Fetal Hemoglobin.

5-Azacytidine (5-Aza) is among the most potent inducers of fetal hemoglobin (HbF) in patients with β-thalassemia and sickle cell disease. Two models have been proposed to explain this activity. The first model is based on the drug's ability to inhibit DNA methyltransferase enzymes causing global DNA hypomethylation, including the promoters of the fetal globin genes, resulting in their expression during adult erythropoiesis. The second model is based on the drug's other well-know property - cytotoxicity. In this model, a cohort of differentiating erythroblasts either dies or undergoes growth arrest during drug exposure. The marrow then responds with a rapid proliferation of erythroblasts which express high levels of fetal globin mRNA and HbF. To determine which model best explains HbF induction by 5-Aza, we used an in vitro differentiation system in which CD34+ cells from normal donors are cultured with SCF, IL-3 and Flt-3 ligand for 7 days and then switched to Epo for an additional 14 days. This results in >90% erythroid cells, a 2,000-fold expansion in cell number and Hb expression similar to that seen in normal red cells (∼99% HbA, ∼1% HbF). To model common 5-Aza dosing schedules, we treated cultures daily with doses ranging from 0 to 1000 nM. Near-maximal induction of γ-globin mRNA (∼2-fold increase in γ/γ+β) and HbF (from 1% to 40%) occurred at the 300 nM dose level. While this dose was associated with hypomethylation of the 7 CpGs between −256 and +50 bp of the γ promoter (59% +/− 14% methylated CpGs vs. 92% +/− 8.1% in untreated controls, p < 0.001), no changes were found in cell expansion rate, differentiation kinetics (as judged by glycophorin A expression) or cell cycle distribution. These results argue against the cytotoxicity model. We also observed that global DNA methylation was not significantly changed by the 300 nM dose of 5-Aza. Because 5-Aza is incorporated into both RNA and DNA, it affects many cellular processes beyond DNA methylation. To determine if DNA hypomethylation is sufficient for γ-globin mRNA and HbF induction, we treated differentiating cells with DNMT1 siRNA. This resulted in transiently decreased DNMT1 mRNA and protein and lowered global DNA methylation. γ-globin promoter methylation decreased to levels equivalent to those seen with 300 nM 5-Aza (61% +/− 7% of CpGs methylated vs. 88% +/− 8% in controls, p < 0.001) but did not induce γ-globin mRNA or HbF. To further evaluate this unexpected finding we used a lentiviral vector expressing DNMT1 shRNA. This suppressed DNMT1 mRNA and protein throughout differentiation and decreased total γ-globin promoter methylation from 98% +/− 4% in control cells, to 20% +/− 11% (p<0.001) in treated cells. This level of γ promoter methylation is similar to that seen in fetal erythroid cells (13% +/− 7%). Global CpG methylation was also decreased vs. controls (30% +/− 10% vs. 83% +/− 11%, p<0.001). Again, despite significant γ promoter and global hypomethylation, no induction of γ-globin mRNA or HbF was observed. These results suggest that neither model explains 5-Aza's ability to induce fetal Hb and that alternative mechanisms await discovery. If verified, these findings have important implications for the future development of clinically useful HbF inducing agents.

Fetal Hemoglobin Induction in Baboons (P. Anubis) Following Admistration of a Novel Decitabine Dinucleotide (S110) Compound.

Increased fetal hemoglobin (HbF) levels can ameliorate the symptoms of patients with sickle cell disease and increase their life span. The DNA methyltransferase (DNMT) inhibitor decitabine increases HbF in non-human primates (baboons) and sickle cell patients and offers great promise as an effective therapeutic agent for the treatment of this disease. The goal of this investigation was to develop and test new compounds that inhibit DNMT, increase HbF, and offer significant advantages compared to decitabine such as increased stablity, reduced cytotoxicity, and a lower potential for mutagenicity. S110 is decitabine-guanine dinucleotide developed by Supergen, Inc., that is cytosine deaminase resistant and more stable in plasma than decitabine. Experiments were performed to compare the effect of S110 and decitabine on γ-globin expression in human erythroid progenitor cell cultures derived from CD34+ peripheral blood cells. Cultures were treated with decitabine (1 X 10−6 M) or S110 (1 and 5 X 10−6 M) on day 8. Forty eight hours later, RNA was purified for real time PCR analysis of ε- and γ-globin gene expression by the ΔΔCT method using α-globin as a control. Effects on globin polypeptide chain expression was determined by HPLC analysis of freeze-thaw lysates prepared 72 hours following drug addition. Both S110 and decitabine increased expression of ε-globin and γ-globin mRNA and the γ/γ + β polypeptide chain ratio (see Table). Effect of S110 on ε-and γ-globin mRNA and the γ / γ + β chain ratio in Human Erythroid Progenitor Cultures (n=4)DRUGCONCENTRATIONε-globin mRNA (Fold increase)γ-globin mRNA (Fold increase)Globin chain ratio (γ/γ + β)Untreated3 +/− 3.3Decitabine1 X 10-6 M3 +/− 24.82 +/− 2.88 +/− 3.2S1101 x 10-6 M5 +/− 4.24 +/− 1.18 +/− 1.9S1105 X 10−69 +/− 34.535 +/− 1.52 +/− 2.9The effect of S110 on HbF levels in vivo was then tested in non-human primates. Two baboons (PA 7256; PA 7470, P. anubis) were phlebotomized for eight days to achieve a HCT of 20 followed by treatment with S110 (1mg/kg/d; sc) for ten days. Pretreatment HbF levels were 3.1% (PA 7256) and 3.5% (PA 7470). Following S110 treatment, peak HbF levels of 46.1% (PA 7256) and 75.5% (PA 7470) were attained that were similar to levels observed in animals treated with equivalent molar doses of decitabine. Bisulfite sequence analysis showed that the level of DNA methylation of 5 CpG sites within the γ-globin promoter in purified BM erythroblasts following S110 administration was significantly lower (41.5%, PA 7256; 16.3%, PA 7470) compared to erythroblasts from bled, untreated baboons (70.8%; n=4). Pharmacokinetics was also evaluated in parallel to assess systemic exposure. These data demonstrate that S110, a newly developed decitabine-guanine dinucleotide, effectively increased HbF and reduced DNA methylation in cultured human erythroid progenitor cells and in experimental non-human primates.

Identification of a TFIID Binding Sequence That Increases Expression from the Ankyrin-1 Promoter: Application to Globin Gene Therapy

Effective Gene Therapy of the hemoglobin β-chain disorders β-thalassemia or Sickle Cell Disease (SCD), requires that viral vectors deliver β-like globin gene to hematopoietic stem cells (HSC) and express β-globin at levels >20% of that of endogenous α-globin. However, the β-globin gene is poorly expressed without sequences from the Locus Control Region (LCR). The LCR contains sequences that contribute to inefficient production and low titers of the recombinant virus, and pose a significant risk of insertional activation of leukemia genes. We have taken an alternative approach using enhancer independent promoters from genes expressed in erythrocytes to express sufficient β-like globin. We have focused on the erythroid ankyrin (ANK-1E) promoter, a compact GC-rich promoter with no conserved sequences that is one of the four different tissue specific promoters used to express the ANK-1 gene. We have shown that a double copy of Moloney Leukemia Virus (MLV) vector in which the ANK-1E promoter linked to a γ-globin gene replaced the promoter and enhancer sequences in the MLV Long Terminal Repeat was produced at high titer. We have also demonstrated that in mice repopulated with HSC transduced with the ANK-1E/γ-globin double copy vector, γ-globin mRNA and protein were expressed at a uniform level of 7.5% of α-globin per vector copy. To obtain the 3–4 fold increase in γ-globin expression needed to reach therapeutic levels we have taken advantage of our recent demonstration that patients with a deletion of a TG dinucleotide in the transcribed region (+4 or +32 relative to the proximal or distal mRNA start sites) of the ANK-1E promoter are ankyrin deficient due to reduced binding of the transcription initiation complex, TFIID. We hypothesized that introducing sequences with higher TFIID binding affinity into the region of the ANK-1E promoter would result in increased level of ANK-1E/γ-globin transcription needed for an effective therapy for β-thalassemia and SCD. An ANK-1E promoter library was generated using degenerate sequence in the TFIID binding region while preserving the critical TG dinucleotide (NNNNNTGNN). This library of promoters was transcribed in nuclear extract from erythroid K562 cells. The RNA transcripts were cloned by 5′RACE and analyzed by sequencing. Four different sequences were obtained: wild type (TGCGGTGAG), GGCGGTGAG, GCCGGTGAG and GGGGGTGAG. The consensus sequence derived from these clones ((G/T)(G/C)(G/C)GGTGAG) was found at two other locations in the ANK-1E promoter (−5 and −54 relative to the distal mRNA start site) as well as in 25% of 5′UTR across the human genome (p=2.2e−16). 64% of GC rich promoters contain the consensus sequence, which is enriched at +50 and −70 relative to the mRNA start site. ANK-1E promoters containing the novel sequences were linked to a luciferase reporter gene and tested individually in transient and stable transfection assays in K562 cells. The GCCGGTGAG and GGCGGTGAG promoters expressed 7- and 2.5-fold higher levels of the luciferase than the wild type promoter (p=0.001; 0.005 respectively). Electrophoretic mobility shift assays demonstrated that the two more active promoters bind more TFIID than the wild type promoter. We are evaluating the ability of these promoters to direct higher levels of γ-globin expression in primary mouse erythroid cells.

Characterization of Regulatory Elements in the Slc4a1 (Band 3) Locus To Create Safe and Effective Vectors for Globin Gene Therapy

Effective gene therapy for hemoglobin β-chain disorders, β-thalassemia and sickle cell disease, requires efficient, safe delivery of globin genes into hematopoietic stem cells (HSCs). Engraftment of ∼25% of HCSs expressing a globin gene at ∼20% the level of endogenous α-globin would be sufficient to improve both diseases. The β-globin promoter is inefficient, requiring sequences from the locus control region (LCR) to increase expression. LCR enhancers are included in globin gene therapy vectors, but unfortunately are prone to cryptic splicing and polyadenylation, resulting in low virus titer. In addition, the LCR enhancers are, in theory, capable of activating neighboring oncogenes. To improve safety and efficiency of globin vectors we have developed a novel strategy by fusing the γ-globin gene to LCR-independent, erythroid-specific promoters. Band 3/AE1 is an erythrocyte membrane protein expressed from the Slc4a1 gene. We have previously shown in transgenic mice that a 1750-bp Slc4a1 promoter linked to γ-globin gene (pSlc4a1/γ) and flanked by the chicken β-globin insulator 5′HS4 (ch5′HS4), which contains both barrier and enhancer-blocking elements, is capable of erythroid-specific, uniform γ-globin expression at therapeutic levels (∼19.8% α-globin/transgene copy). Without ch5′HS4, the pSlc4a1/γ gene was prone to silencing. Lentiviral vectors with two copies of ch5′HS4 either internal or in the Long Terminal Repeat cannot be produced at high titer. We hypothesized that flanking the pSlc4a1/γ-globin gene with distinct barrier elements would prevent recombination and gene silencing. Using this strategy, we developed first generation lentiviral vectors in which pSlc4a1/γ is flanked by combinations of the ch5′HS4 insulator and barrier elements we have identified in the ankyrin and α-spectrin loci. To test the effectiveness of these lentivirus vectors in mouse models, we pseudotyped each one with an ecotropic envelope. All three were produced at high titer (>1x106 infectious units/ml). We transduced primitive mouse hematopoietic progenitor cells and detected γ-globin mRNA in >20% of spleen foci at levels as high as 17% of endogenous α-globin. In mice repopulated with transduced stem and progenitor cells, 11–15% of peripheral blood erythrocytes were positive for γ-globin 8 to 21 weeks post-transplantation. To establish the safety of the Slc4a1 promoter we used a high throughput real-time PCR-based assay to identify DNaseI hypersensitive sites (HS) in a 119kb region including Slc4a1. We have identified 6 HS and tested each for enhancer and enhancer-blocking activity. One HS (−355 to −112) increases reporter gene expression in a position- and orientation-independent fashion consistent with the properties of an enhancer. A second HS (−112 to 0) is active in enhancer-blocking assays, and deletion analyses indicate that this region may also contain a transcriptional silencer. A third HS in intron 1 (+910 to +1581) displays enhancer-blocking activity. Three HS have no activity. We are testing a second generation of pSlc4a1/γ lentiviruses in which the Slc4a1 enhancer and silencer are deleted. A third generation of vectors flank pSlc4a1/γ upstream with ch5′HS4, and downstream with either the ankyrin or α-spectrin barrier elements plus the Slc4a1 intron 1 enhancer-blocker to prevent activation of neighboring genes. We hypothesize that these new vectors will allow safe expression of therapeutic levels of γ-globin.