ALBERT

Description: Tactile-foraging ducks are specialist birds known for their touch-dependent feeding behavior. They use dabbling, straining, and filtering to find edible matter in murky water, relying on the sense of touch in their bill. Here, we present the molecular characterization of embryonic duck bill, which we show contains a high density of mechanosensory corpuscles innervated by functional rapidly adapting trigeminal afferents. In contrast to chicken, a visually foraging bird, the majority of duck trigeminal neurons are mechanoreceptors that express the Piezo2 ion channel and produce slowly inactivating mechano-current before hatching. Furthermore, duck neurons have a significantly reduced mechano-activation threshold and elevated mechano-current amplitude. Cloning and electrophysiological characterization of duck Piezo2 in a heterologous expression system shows that duck Piezo2 is functionally similar to the mouse ortholog but with prolonged inactivation kinetics, particularly at positive potentials. Knockdown of Piezo2 in duck trigeminal neurons attenuates mechano current with intermediate and slow inactivation kinetics. This suggests that Piezo2 is capable of contributing to a larger range of mechano-activated currents in duck trigeminal ganglia than in mouse trigeminal ganglia. Our results provide insights into the molecular basis of mechanotransduction in a tactile-specialist vertebrate.

Description: Membrane-spanning proteins may interact with a variety of other integral and peripheral membrane proteins via a diversity of protein-protein interactions. Not surprisingly, defects or mutations in any one of these interacting components can impact the physical and biological properties on the entire complex. Here we use quantum dots to image the diffusion of individual band 3 molecules in the plasma membranes of intact human erythrocytes from healthy volunteers and patients with defects in one of their membrane components, leading to well-known red cell pathologies (hereditary spherocytosis, hereditary elliptocytosis, hereditary hydrocytosis, Southeast Asian ovalocytosis, and hereditary pyropoikilocytosis). After characterizing the motile properties of the major subpopulations of band 3 in intact normal erythrocytes, we demonstrate that the properties of these subpopulations of band 3 change significantly in diseased cells, as evidenced by changes in the microscopic and macroscopic diffusion coefficients of band 3 and in the compartment sizes in which the different band 3 populations can diffuse. Because the above membrane abnormalities largely arise from defects in other membrane components (eg, spectrin, ankyrin), these data suggest that single particle tracking of band 3 might constitute a useful tool for characterizing the general structural integrity of the human erythrocyte membrane.

Description: The KCl Cotransporter (KCC) is a key component of the volume regulation system of human reticulocytes, and its excessive activity in sickle cells contributes to cellular dehydration and therefore to sickling pathology. Three of the four KCC genes, are expressed in erythroid cells (Crable et al. Exp. Hematol.2005;33:624). Although the relative contribution of the three KCC isoforms to KCC fluxes and volume regulation in red cells remains unknown, KCC3 appears to be the dominant transcript in late erythroid cells. Heterogenity of the 5′ ends of the KCC3 mRNA transcripts has been described by Mount and colleagues (Mercado et al. Am J Physiol289:F1246, 2005), including two untranslated exons (ex1D and 1C) 1.2 kb upstream from ex1A of KCC3a, which contains the originally described translation initiation site. We used RT-PCR, 5′ RACE (rapid amplification of cDNA ends), and primer extension analyses to study the 5′ ends of KCC3 cDNA transcripts in erythroid cells. The large exon 1 of KCC3 was identified in fetal liver and bone marrow RNA as a 1718bp region (containing exons 1A, 1C, and 1D described by Mercado et al) that undergoes complex patterns of alternate splicing to generate 10 different transcripts. Six major splicing isoforms are expressed in hematopoietic cell RNA. One isoform incorporates the first 167bp of the exon (exon 1D) as 5′ untranslated sequence which splices to the last 208bp of exon 1, including additional 5′ untranslated sequence and an alternative in-frame initiator methionine. The translated protein exhibits a 59 amino acid N-terminal truncation of KCC3a lacking several potential phosphorylation sites (called KCC3a-Short by Mercado et al). This transcript was the most abundant isoform in hematopoietic cell RNA. A second major isoform contains 629bp of the exon immediately 3′ of exon 1D as 5′ untranslated sequence (exon 1C), then splices out the next 735bp to join the last 243bp of the exon, including additional 5′ untranslated sequence and the alternate initiator methionine of KCC3a-Short. A third novel transcript includes the entire exon, utilizing the first initiator methionine of full-length KCC3a. Two other novel transcripts were found, both of which code for KCC3a-Short. These transcripts were also identified in EST databases. We examined the genomic region around exon 1 for promoter activity using luciferase promoter constructs expressed in erythroid K562 cells. Promoter activity was minimal with constructs which spanned from exon 1D to the beginning of exon 1A, but increased substantially in constructs that included this region plus 900 bp 5′ sequence. Further deletion analysis shows that a minimal promoter containing less than 125 base pairs yields full promoter activity. A mutation of a GC box within this region reduces activity over 15 fold, implicating SP-1 as a trans-regulatory factor. Thus, KCC3 exhibits a complex pattern of alternative splicing in erythroid cells, producing several novel transcripts, some of which encode an N-terminal truncation of KCC3a. Identifying the factors modulating transcriptional and translational control of KCC3 expression and the functional behavior of this truncated protein in erythroid cells is important to understanding volume regulation in reticulocytes and its abnormalities in sickle cells.

Description: Spectrin, the major structural component of the erythrocyte membrane skeleton, is composed of α and β chains that self-associate to form tetramers. These tetramers provide the structural integrity and flexibility critical for erythrocyte stability and shape. Mutations of α spectrin have been associated with hereditary spherocytosis (HS), hereditary elliptocytosis (HE), and hereditary pyropoikilocytosis (HPP). The large size of the spectrin molecule has complicated its study. We developed a high-throughput capillary nucleotide sequencing strategy to identify mutations of the α-spectrin gene in a group of patients with spectrin-linked HS, HE, or HPP. We identified several variants including 8 nonsense, 4 splice junction, and 4 deletion/insertion mutations.(Mutations in 〉1 patient are counted only once.) We were interested in the identification of missense mutations, as we hypothesize that defects in α-spectrin occur in regions of structural and functional importance and their identification and characterization will provide important information about spectrin and the membrane skeleton. We identified 16 missense mutations in the region encoding the spectrin self-association site; 6 were proline substitutions and 2 were glycine substitutions, both predicted to disrupt the triple helical configuration of spectrin. Outside the self-association site, excluding 3 common protein polymorphisms, we identified 13 missense mutations; 3 were proline substitutions. To begin to study the functional significance of these mutations, we prepared 15 recombinant spectrin-GST fusion peptides containing residues 1–158 of α spectrin, the self-association contact site, representing wild type (WT) or 14 different missense mutations. After expression and purification, purity was assured by SDS-PAGE, absence of aggregation was verified by analytical HPLC gel filtration, and mass confirmed by MS analyses. Analyses by circular dichroism demonstrated that none of the missense mutations significantly modified secondary structure of the recombinant peptide. WT and mutant peptides exhibited a helical content of ∼65%. Ultracentrifugation studies verified that all peptides were monomeric at 4 and 30°C. Differential scanning calorimetry showed that the WT peptide was very stable with a single reversible 2-state transition with a Tm of 54.6°C. All mutations, except R34W, showed transitions similar to WT. R34W unfolded at a much lower Tm, 49.1°C, with a broader single peak transition. Analysis of spectrin tetramerization between α-spectrin peptides and a recombinant β-spectrin peptide (repeats 16, 17 and COOH-terminus) was performed using an analytical HPLC gel filtration assay. A wide range of binding affinities was observed: WT binding Kd=0.43μM at 23°C; group I: I24S, R28C, R28H, R28L, R28S, R45S, no binding; group II: I24T, R41W, L49F, much weaker binding than WT; group III: V31A, R45T, G46V, binding weaker than WT, and R34W and K48R, binding equal to WT. Quantitative thermodynamic analyses of spectrin tetramerization site formation between α and β spectrin peptides were assessed by isothermal calorimetry. These results were essentially comparable to the gel filtration data except the R34W mutant bound β-spectrin more avidly than WT. The identification and characterization of variants associated with HS, HE and HPP continues to extend our understanding and knowledge of both normal membrane biology and human disease pathogenesis.

Description: 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.

Description: Mutations in erythrocyte ankyrin, ankyrin-1, are the most common cause of typical hereditary spherocytosis. Co-inheritance of cardiac, muscular, and neurologic diseases such as cardiomyopathy, psychomotor retardation, and spinocerebellar abnormalities with hereditary spherocytosis has been described. In the nb/nb mouse, an ankyrin-1 mutation manifests in erythroid cells with ankyrin deficiency and a spherocytosis phenotype, and in neural cells with an age-dependent psychomotor disorder due to loss of cerebellar Purkinje cells. These observations highlight the importance of understanding ankyrin-1 structure, function, and regulation in erythroid and nonerythroid cells. In erythroid cells, ankyrin expression is directed by a compact promoter controlled by a single GATA-1 site. Nonerythroid ankyrin-1 isoforms have been described with diversity arising from alternate splicing, alternate polyadenylation, and, in skeletal muscle, use of an alternate, tissue-specific promoter. Using 5′ RACE, we identified 2 additional alternate first exons of the ankyrin-1 cDNA. One encoded a first exon with an initiator methionine followed by 12 amino acids, designated exon 1A, that spliced in-frame to erythroid exon 2 sequences. The other, designated 1B, encoded a novel initiator methionine followed by 40 highly charged amino acids that also spliced in-frame to the erythroid exon 2. Both exons, found in human and mouse, link directly to the downstream exons encoding the ankyrin repeat and spectrin binding domains of ankyrin-1. Exon 1B mapped to a location 98.5 kb 5′ of erythroid exon 1 (1E) and exon 1A mapped 30.1 kb 3′ of exon 1E. Northern blot and quantitative RT-PCR analyses demonstrated that 1B was expressed in heart, skeletal muscle, and brain. Similar to what we previously reported for the promoter of the erythroid-specific exon 1, 1E, DNase I hypersensitive site (HS) mapping identified a pair of HS in genomic DNA, one immediately 5′ of exon 1B and one that mapped 6.6 kb downstream of this site (chr8:41,873,229 and 41,866,593, UCSC assembly, Mar 2006). Luciferase-reporter gene expression studies with plasmids containing either a 700bp or a 278bp fragment of the 1B flanking sequence directed high-level luciferase expression in RD cells (human rhabdomyosarcoma) but no luciferase expression in K562 (erythroid) or HeLa (fibroblastoid) cells. Consistent with its tissue-restricted pattern of expression, a polyclonal antipeptide antibody raised against novel sequence in exon 1B reacted with peptides of 220, 205, 45, and 40 kDa on immunoblots prepared from muscle and brain but not erythrocytes. In contrast to erythroid-specific exon 1E and tissue-restricted exon 1B, mRNAs containing exon 1A were detected in all 18 tissues examined. Like the 1E and 1B promoters, mapping identified an HS in the 5′ flanking genomic DNA/promoter region of exon 1A and another 6 kb downstream (chr8:41,873,229 and 41,866,593). An exon 1A anti-peptide antibody reacted with peptides of 205, 195, and 190 kDa on immunoblots prepared from numerous tissues, including erythrocytes ghosts. Regulation of ankyrin-1 expression by alternate promoters directing novel NH2-termini provides the basis for a complex pattern of tissue-specific ankyrin-1 isoform diversity. Characterization of the downstream alternate exon composition of the tissue-specific exon 1B and ubiquitous exon 1A-containing transcripts will allow a systematic evaluation of whether a specific spherocytosis-linked ankyrin-1 mutation could lead to a nonerythroid phenotype.

Description: 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.

Description: Pomalidomide, a second-generation immunomodulatory drug, is a fetal hemoglobin (HbF) inducing agent with potential implications for the treatment of β-hemoglobinopathies such as sickle cell disease (SCD). However, its mechanism of action remains unknown. Through an in-depth characterization of human erythropoiesis and globin gene regulatory networks, we now provide evidence that pomalidomide alters transcription networks involved in erythropoiesis and globin switching, thereby leading to a partial reprogramming of adult hematopoietic progenitors toward fetal-like erythropoiesis. Adult peripheral blood CD34+ cells from normal individuals were differentiated toward the red cell lineage using an adapted 3-phase culture system. At day 14 of culture, we observed a reciprocal globin gene switch at the mRNA and protein levels. These results were confirmed by high performance liquid chromatography of hemolysates (HbF/(HbF+HbA): 31.7 ± 1.4% vs. 6.5 ± 0.7% pomalidomide and vehicle, respectively). Next, we studied erythroid differentiation using flow cytometric analyses of the cell surface markers interleukin-3R (IL-3R), glycophorin A (GPA), CD34 and CD36 for early erythroid precursors (BFU-E and CFU-E) as well as GPA, α4-integrin and band3 for terminal erythroid differentiation. While there were no changes in terminal erythroblast maturation, an accumulation of BFU-E in pomalidomide-treated cultures at days 2 and 4 of differentiation was seen, indicating a delay at the BFU-E to CFU-E transition, and also, that pomalidomide exerts its effect in the early-stages of erythropoiesis. Indeed, treatment with pomalidomide during the phase of the culture system that generates erythroid progenitors led to significantly more γ-globin expression than treatment during the phase which proerythroblasts undergo terminal erythroid differentiation. At the molecular level, pomalidomide was found to rapidly and robustly decrease Ikaros (IKZF1) expression exclusively by post-translational targeting to the proteasome. Moreover, pomalidomide selectively reduced the expression of components of key globin regulatory pathways including BCL11A, SOX6, KLF1, GATA1 and LSD1 while not affecting others (e.g. CoREST, GATA2, GFI1B, and HDAC1). Pomalidomide had a transient effect on GATA1 and KLF1 expression. While shRNA knockdown of Ikaros using two different lentiviral constructs delayed erythroid differentiation, it failed to appreciably stimulate HbF production or alter BCL11A expression. These results suggest that the loss of Ikaros alone is insufficient to recapitulate the phenotype observed in pomalidomide-treated conditions. We next compared the expression levels of proteins involved in globin gene regulation among untreated peripheral blood, pomalidomide-treated peripheral blood and untreated cord blood-derived erythroid cells. We found striking similarities between cord blood and pomalidomide-treated adult cells at day 4 of differentiation. Indeed, BCL11A, KLF1, SOX6, LSD1 and GATA1 showed decreased expression levels both in cord blood and pomalidomide-treated adult peripheral blood, while the levels of CoREST, HDAC1 and GATA2 remained unchanged indicating that pomalidomide partially reprograms adult erythroid cells to a fetal-like state. Taken together, our results show that the mechanism underlying reactivation of HbF by pomalidomide involves Ikaros-independent reprogramming of adult erythroid progenitors. Finally, we found that this mechanism is conserved in SCD-derived CD34+ cells. Our work has broad implications for globin switching, as we provide direct evidence that Ikaros does not play a major role in the repression of γ-globin during adult erythropoiesis, and further supports the previously held notion that globin chain production is determined prior to or at the level of CFU-E. Disclosures Allen: Celgene: Research Funding; Bristol Myers Squibb: Equity Ownership; Onconova: Membership on an entity's Board of Directors or advisory committees; Alexion: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees.

Description: Abstract 565 Erythroid Kruppel-Like Factor (EKLF; KLF1) is the founding member of the Kruppel family of C2H2 zinc finger transcription factors. First identified as an activator of the beta-globin locus, EKLF facilitates chromatin remodeling and transcriptional activation of target genes, at least in part through recognition of a 9-base consensus motif (NCNCNCCCN). By comparing the transcriptional profiles of E13.5 wild type and Eklf-/- mice, we demonstrated that the lethal failure to complete definitive erythropoiesis in the fetal liver (FL) was due in part to dysregulation of an EKLF target gene, the cell cycle control factor, E2F2 (Pilon et al. 2008). To identify further direct targets of EKLF activation that affect erythropoiesis, we are coupling chromatin immunoprecipitation with ultra high-throughput massively parallel sequencing (ChIP-seq). ChIP-seq is increasingly being used to map protein-DNA interactions in vivo, allowing simultaneous genome-wide analysis of transcription factor occupancy, defining an ‘interactome‘. Using mice whose endogenous Eklf gene was replaced with a fully functional HA-tagged form of EKLF, chromatin was isolated at E13.5 from immature erythroid progenitors and maturing erythroblasts by ChIP. Using a highly specific high-affinity anti-HA antibody, libraries of HA-EKLF-bound chromatin were subjected to fluorescent in situ sequencing on a Solexa 1G platform, providing 36-base signature tags that were mapped to the mouse genome using the Eland software package. A control library was derived from E13.5 FL chromatin that was not enriched for HA-EKLF occupancy. For both progenitors and erythroblasts, 〉1.1×107tags were obtained. 72.5% and 78.7% of progenitor and erythroblast tags mapped to unique sites within the genome, respectively. The tags were highly enriched in the ∼10% of the genome within genes (genic; 42% of tags), sites ≤10 kb from the nearest gene (adjacent; 15%), as opposed to the ∼90% of the genome that is 〉10 kb from the nearest gene (intergenic; 22%) or in repetitive DNA (21%) p=2.2 ×10-16. Using the MACS software package clustered peaks of EKLF occupancy were identified throughout the genome, defining the EKLF ‘interactome‘. The vast majority of peaks were mapped to non-repetitive regions of the genome (98% in progenitors; 95% in erythroblasts). Progenitors contained 4,383 peaks of EKLF occupancy, while erythroblasts contained 15,396 peaks. Only 100 peaks were common between populations. This 〉3.5-fold increase in genomic EKLF occupancy between progenitors and erythroblasts (p=1×10-5) reflects the shift in the expression and activity of EKLF protein in erythropoiesis described previously (Bouilloux et al. 2008; Lohmann & Bieker 2008). To identify potential EKLF target genes, we partitioned the genome into 3 categories, relative to annotated RefSeq coordinates (genic) as well as adjacent and intergenic. In progenitors, the majority of EKLF binding (54%) occurred in intergenic regions, with a minority within (38%) or adjacent (7%) to genes. By contrast, the EKLF binding profile in erythroblasts was reversed, with 62% of the peaks in genic regions, and a minority at intergenic (26%) or adjacent (12%) sites.To assess the effect of this shift in EKLF binding on gene transcription, we used publicly availabel data from the inducible G1E model of erythroid maturation (GEO: GSE628) to correlate our ChIP-seq data with mRNA expression. Informatic analyses using MetaCore demonstrated that 〉2,200 EKLF-associated genes were differentially expressed during maturation (949 increasing expression; 1,298 decreasing expression, all p

Description: Gene Therapy for the hemoglobin β-chain disorders, Sickle Cell Disease (SCD) or beta thalassemia (β-thal), requires both efficient globin gene transfer into hematopoietic stem cells (HSC) as well as expression of globin mRNA and protein at levels that are 〉20% of the level of α-globin. Expression of the β-like globin genes in either transgenic mice or from RNA virus vectors integrated into the genome of erythroid cells requires sequences from the Locus Control Region (LCR). The LCR sequences contain cryptic splicing and polyadenylation sequences that contribute to inefficient production and low titers of recombinant virus particles. In addition, the powerful LCR enhancer elements pose a significant risk of insertional activation of leukemia genes. We hypothesized that enhancer-independent promoters from other genes expressed in erythrocytes could be used to express sufficient amounts of β-like globin to treat SCD or β-thal. We have focused on the erythroid ankyrin (ANK-1E) promoter, a compact GC-rich promoter with no conserved sequences that is one of the 4 different tissue-specific promoters used to express the ANK-1 gene. We have previously demonstrated that a “double copy” 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. In mice repopulated with HSC transduced with the ANK-1E/γ-globin double copy vector, γ-globin mRNA and protein was expressed at uniform level of 7.5 % of α-globin per vector copy (Sabatino et al. PNAS 97:13294–9, 2000). 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 (−73/−72) dinucleotide in the ANK-1E promoter are ankyrin deficient due to reduced binding of the transcription initiation complex, TFIID (Gallagher et al. Hum Mol Gen 14:2501–9, 2005). We hypothesized that altering the sequence in the TFIID binding region of the ANK-1E promoter would increase TFIID binding, resulting in the increased level of ANK-1E/γ-globin transcription needed for an effective therapy for SCD and β-thal. We generated a library of ANK-1E promoters with degenerate sequence in the TFIID binding region, 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 from the sequencing results: the wild type sequence (TGCGGTGAG), GGCGGTGAG, GCCGGTGAG and GGGGGTGAG. The consensus sequence derived from these clones: (T/G)(G/C)(G/C)GG(T/A)GAG was found in similar locations relative to the transcriptional start site in the three other ANK-1 promoter regions, as well as in 22% of 〉4000 human loci with GC-rich promoters that lack TATA boxes and other consensus promoter sequences. 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 mRNA and protein than the wild type promoter (p=0.001; 0.005 respectively). We are evaluating the ability of these promoters to direct higher levels of γ-globin expression in primary mouse erythroid cells.