Ankyrin Promoter Research Articles

Fetal Hemoglobin Induction with Forced Chromatin Looping in Gene-Modified Erythroid Cells Differentiated from Rhesus Hematopoietic Progenitor Cells

Substantial advances in genome engineering offer new therapies for hemoglobin (Hb) disorders including sickle cell disease. Fetal hemoglobin reactivation remains a viable strategy, as recently demonstrated in human erythroid cells with an artificial looping molecule ZF-Ldb1, tethering Ldb1 to the γ-globin promoter by specific zinc finger proteins (Deng et al., 2014). Large animal preclinical testing can improve the therapeutic potential of such approaches by optimizing safety and efficacy issues before launching clinical trials in humans. A robust in vitro erythroid evaluation system, however, does not exist to determine if fetal hemoglobin reactivation can be modeled in the rhesus macaque. Therefore, in the current work, we aimed to develop an efficient in vitro erythroid differentiation model from rhesus hematopoietic progenitor cells with Hb production at detectable protein levels, and to evaluate rhesus fetal hemoglobin reactivation with ZF-Ldb1.To adapt a human erythroid differentiation method to rhesus cells, we used C3H or OP9 feeder cells to culture rhesus peripheral blood mononuclear cells (PBMCs) with HEMA differentiation media containing 10ng/ml stem cell factor (SCF), 1ng/ml interleukin-3 (IL3), 2U/ml erythropoietin (Epo), and 20% fetal bovine serum (FBS) (Migliaccio et al., 2010). Two weeks after erythroid differentiation, robust Hb production evaluated by HB electrophoresis was obtained on both C3H and OP9 feeder cells but not in feeder-free conditions. Increased concentration of SCF (20x), IL3 (10x), and Epo (1.5x) allowed for Hb production even in feeder-free conditions; however, fetal Hb was predominant (due to 20x SCF). Both conditions provided similar levels of enucleation (39±6.3% vs 29±5.6%, ns), indicating efficient differentiation. We then compared HEMA media with serum-free StemSpan-based differentiation media to evaluate transduction with a GFP-encoding lentiviral vector at MOI 2.5 two days after initiating differentiation. Transduction was almost completely inhibited (0.4±0.5%) in HEMA media, while StemSpan-based transduction media followed by HEMA differentiation media resulted in significant increase of transduction efficiency (41±8.5%, p<0.01). These data demonstrate that feeder cells are required for rhesus erythroid differentiation in HEMA media, while FBS strongly interferes lentiviral transduction.Next, we sought to establish a reverse phase HPLC method to quantitatively analyze rhesus globins among differentiated erythroid cells. Peaks on the HPLC chromatogram were eluted and run in liquid chromatography-mass spectrometry (LC/MS) and tandem mass spectrometry (MS/MS) for protein identification. We identified β-globin and γ-globin (HBG1 and HBG2) peaks using both rhesus peripheral blood (99±26% and 0.65±0.28%) and in vitro differentiated rhesus erythroid cells (63±13% and 37±16%), which is essential for Hb switching analysis. In addition, human β-globin and T87Q β-globin (with anti-sickling mutation) proteins were also separated from rhesus globins in the HPLC chromatogram and were detectable for quantitation.Finally, we evaluated whether fetal Hb expression can be induced by the ZF-Ldb1 molecule in rhesus erythroid cells. We transduced differentiating rhesus CD34+ cells with ZF-Ldb1-encoding lentiviral vectors that are driven by an erythroid specific ankyrin-1 promoter, and contain GFP along with a β-globin locus control region (LCR) enhancer or YFP without the LCR. After erythroid differentiation, we obtained both LCR/ZF-Ldb1/GFP+ (9.4%) and ZF-Ldb1/YFP+ (1.3%) rhesus erythroid cells, and higher γ-globin RNA expression was detected for GFP+ and YFP+ fractions (68.1±1.5% and 59.8±2.1%, respectively), as compared to GFP- and YFP- controls (28.1±3.1% and 31.8±1.9%, p<0.05). These data demonstrate successful γ-globin induction with ZF-Ldb1 vectors in the rhesus macaque.In conclusion, we developed an efficient rhesus erythroid cell differentiation protocol from hematopoietic progenitor cells with robust Hb production detectable at protein levels, analyzed in a newly established HPLC method. The ZF-Ldb1 vector transduction allows for induction of γ-globin expression in rhesus erythroid cells. Further studies are warranted to optimize gene modification and transplantation of rhesus hematopoietic progenitor cells. DisclosuresNo relevant conflicts of interest to declare.

Platelets: An Efficient Depot for Generation and Distribution of Lysosomal Idua in Enzyme-Deficient MPS I Mice.

Abstract 3157Proviral integration into hematopoietic stem cells (HSC) by lentivirus vector (LV)-mediated gene transfer can provide the benefit of life-long therapeutic effect, yet it can also bring the risk of insertional oncogenesis. Platelets are terminally differentiated, enucleated cells full of secretory granules. Restricting transgene expression to platelets may reduce the risk for activating oncogenes in HSCs and most of their progeny, and take advantage of their rapid turnover and professional secretion function. In this study, we evaluated the feasibility of employing megakaryocyte-platelet as a depot for the over-expression and release of alpha-L-iduronidase (IDUA), the lysosomal enzyme deficient in patients with Mucopolysaccharidosis type I (MPS I, or Hurler Syndrome). Utilizing a human megakaryocytic DAMI cell line, we found that a hybrid human ankyrin-1 promoter (K) containing ALAS2 intron 8 enhancer and HS40 core element from human alpha LCR could introduce robust intracellular IDUA expression (over 500-fold of background) and significant enzyme release (120-fold). Upon introduction to megakaryocytic differentiation as verified by FACS analysis with PI staining and morphologic evaluation, the elevated intracellular IDUA levels remained unchanged, while released IDUA increased steadily by 30-fold. We then evaluated in vivo IDUA production/release from platelets in an enzyme-deficient MPS I mouse model. Lineage-negative bone marrow (BM) cells were isolated from MPS I mice using immunomagnetic cell sorting, and transduced twice with LV-K-IDUA-ires-GFP (KIiG) for a total multiplicity of infection at 30. Five-months after 1° transplantation, GFP+ platelets were detected by FACS analysis with CD41-PE staining and observed by Image X analysis. To generate animals with various transgene-dosages, 2° transplantation was performed into MPS I mice using a serial dilution of BM from 1° MPS/KIiG (51–65% transduction efficiency) with BM of MPS I. FACS analysis revealed that the percentage of GFP+ platelets was correlated directly to transduction efficiencies in 2° recipients as determined by real-time qPCR of BM 5 months post transplantation. Moreover, the intra-platelet IDUA enzyme activities were associated linearly with the changes of GFP+% platelets (r2=0.95) among MPS I mice with different transgene-dosages, suggesting that the lysosomal IDUA from transgene overexpression could be sorted and packaged into platelets with proper catalytic function. Noticeably 1% gene transfer efficiency as shown by 1% GFP+ platelets was sufficient to introduce the IDUA levels comparable to those found in wild-type platelets. To determine if platelet-derived IDUA could be released, we conducted Ca-induced platelet activation as verified by FACS analysis with Annexin V-APC and CD41-PE staining, resulting in linear correlation (r2=0.98) of released IDUA with intra-platelet IDUA. Interestingly, the plasma IDUA levels were also linearly correlated with GFP+% platelets (r2=0.96) among MPS I with different transgene-dosages. Competitive uptake assay using lymphoblastoid cells derived from a MPS I patient (LCLmps) demonstrated steady increase of intracellular IDUA with increasing amounts of extracellular enzyme levels, which was blocked by the presence of mannose-6-phosphate (M6P). This suggested that platelet-released IDUA retained its intercellular trafficking capability via M6P receptor. Finally normalization of aberrant lysosomal morphology was observed in LCLmps exposed to platelet-released IDUA as determined by in situ immunostaining with LysoTracker, indicating functional proficiency of platelet-derived IDUA to cross-correct deficit in patient’s cells. These results demonstrate for the first time that megakaryocytes/platelets are capable of over-producing, packaging and storing a lysosomal enzyme which retains proper catalytic activity, lysosomal enzyme trafficking and endogenous M6PR-mediated uptake, as well as the ability of cross-correction in patient’s cells. This data warrants further evaluation for the potential application of megakaryocytes/platelets in treating lysosomal storage diseases, especially for those, such as Gaucher disease, when the desirable enzyme is sensitive to neutral pH in serum and protection of enzyme in platelets may provide continuous, real-time enzyme release by low physiological levels of platelet activation. Disclosures:No relevant conflicts of interest to declare.

Association between promoter polymorphisms in a key cytoskeletal gene (Ankyrin 1) and intramuscular fat and water-holding capacity in porcine muscle

Ankyrin 1 (ANK1) is a positional and functional candidate gene for both bovine and porcine meat quality. The objectives of this study were: (1) to determine if the gene expression levels of ANK1 are associated with pig meat quality traits; (2) to examine polymorphisms in the promoter region of the porcine ANK1 gene for association with meat quality in diverse breeds; and (3) to search for putative transcription factor binding sites predicted to be altered by such polymorphisms. ANK1 gene expression was positively correlated with drip loss (%) in the Large White breed. Twelve novel SNPs were discovered in a 761bp region of the ANK1 promoter in three phenotypically diverse F1 cross populations. Five were subsequently selected for association analysis with meat quality traits and genotyped in three pure pig breeds (Pietrain n=98, Duroc n=99 and Large White n=98). Two of these five SNPs were associated with meat quality traits at the Bonferroni significance threshold: g.-606G>A with drip loss % in the Pietrain breed and g.-272G>A with intramuscular fat (IMF) in the LTL and SM. Following haplotype construction from SNP genotypes, Haplotype 3 was found to be associated with drip loss % at the Bonferroni level of significance in the Pietrain breed and Haplotype 5 was associated at the Bonferroni level with IMF in two muscles in the Large White breed. Further associations were observed at the nominal significance threshold. Our conclusion from this study is that SNPs in the ANK1 gene promoter could potentially contribute to genome-assisted selection SNP panels to improve IMF and water holding capacity on a breed basis.

Genome-wide detection of a TFIID localization element from an initial human disease mutation

Eukaryotic core promoters are often characterized by the presence of consensus motifs such as the TATA box or initiator elements, which attract and direct the transcriptional machinery to the transcription start site. However, many human promoters have none of the known core promoter motifs, suggesting that undiscovered promoter motifs exist in the genome. We previously identified a mutation in the human Ankyrin-1 (ANK-1) promoter that causes the disease ankyrin-deficient Hereditary Spherocytosis (HS). Although the ANK-1 promoter is CpG rich, no discernable basal promoter elements had been identified. We showed that the HS mutation disrupted the binding of the transcription factor TFIID, the major component of the pre-initiation complex. We hypothesized that the mutation identified a candidate promoter element with a more widespread role in gene regulation. We examined 17 181 human promoters for the experimentally validated binding site, called the TFIID localization sequence (DLS) and found three times as many promoters containing DLS than TATA motifs. Mutational analyses of DLS sequences confirmed their functional significance, as did the addition of a DLS site to a minimal Sp1 promoter. Our results demonstrate that novel promoter elements can be identified on a genome-wide scale through observations of regulatory disruptions that cause human disease.

A Zinc-Finger Transcriptional Activator Designed to Interact with the Gamma-Globin Gene Promoters Enhances Fetal Hemoglobin Production in Erythroid Cells Derived From Normal and Beta-Thalassemic CD34+ Cells.

Abstract 3567Poster Board III-504Fetal hemoglobin (α2γ2; HbF) is a potent genetic modifier of the severity of beta-thalassemia and sickle cell anemia. Clinical studies indicate that moderate elevation in production of HbF achieved through heritable persistence of HbF or administration of hydroxyurea, effectively reduce the severity of beta-chain defects. Accordingly, we are exploring strategies to maintain expression of the endogenous gamma-globin genes following lentiviral vector-mediated gene transfer. The artificial zinc-finger transcription factor (GG1-VP64) was designed to interact with sequences in the proximal gamma-globin gene promoters and has been shown to enhance gamma-globin expression in human erythroleukemia cells and mouse marrow cells which are transgenic for the human beta-globin locus. Here, we describe studies designed to evaluate the impact of expression of GG1-VP64 on gamma-globin expression by maturing adult erythroblasts derived from CD34+ cells of normal and thalassemic donors. We utilized an in vitro culture model of human erythropoiesis in which late stage erythroblasts are derived from human CD34+ hematopoietic cells. In this system, cytokine-mobilized peripheral blood or steady state bone marrow CD34+ cells from adults yielded erythroblasts containing 2% or less HbF. The lentiviral vector encodes for bicistronic expression of the GG1-VP64 transactivator and GFP under transcriptional control of the beta-spectrin or ankyrin-1 promoter which give low but progressive increase in expression during erythroid development. Three normal donor CD34+ cells were transduced 48 hours after initiation of culture by overnight exposure to the GG1-VP64 vector or GFP control vector. Approximately 50-60% of the cells were successfully transduced with the control and GG1-VP64 vectors as monitored by flow cytometry analysis for GFP expression. Control vector transduction had no effect on cell proliferation or differentiation monitored by consistent increases in cell numbers and the appearance of CD71 (transferrin receptor) and CD235 (glycophorin A) on most cells (>98% and >80%, respectively) whereas GG1-VP64 gene transfer reduced cell proliferation slightly without affecting erythroid differentiation. Erythroblasts derived from GFP transduced cells expressed low levels of HbF (1.7+/−0.6%) whereas those derived from cells transduced with GG1-VP64 demonstrated induction of HbF ranging from 12-21% with an average vector copy number of 0.8 to 1.0. When cells from a normal donor were sorted into GFP- and GFP+ populations, significant levels of HbF were present only in the GFP+ fraction. We next tested the GG1-VP64 transactivator in three independent studies using bone marrow CD34+ cells from two patients with beta-thalassemia major. Gene transfer was effective as reflected by 74+/−6% (control) and 47+/− 2% (GG1-VP64) GFP marking in bulk cultures. Again, GG1-VP64 gene transfer in beta-thalassemia CD34+ cells reduced cell growth somewhat but did not perturb erythroid differentiation as monitored by the appearance of transferrin receptor (>98%) and Glycophorin A (>80%) as well as cell morphology. Erythroblasts derived from GFP transduced cells expressed levels of HbF in the range of 26+/−5% whereas those derived from cells transduced with GG1-VP64 demonstrated a 2-fold induction of HbF to 52+/−9% with an average vector copy number of 0.5-0.9. Our data show that lentiviral-mediated, enforced expression of GG1-VP64 under the control of erythroid-specific promoters induced significant amounts of HbF in normal and thalassemic erythroblasts derived from adult CD34+ cells without altering their capacity for erythroid maturation following transduction. These observations demonstrate the potential for sequence specific enhancement of HbF in patients with beta-thalassemia or sickle cell anemia. Disclosures:No relevant conflicts of interest to declare.

High-Expression Porcine FVIII Driven by Erythroid-Specific Promoters for Lentiviral Vector-Mediated Gene Therapy

Hemophilia A is an X-linked gene disorder that results in a deficiency of circulating coagulation factor VIII (fVIII) and may be ameliorated by only modest amounts of circulating protein, which makes it a logical candidate for gene therapy. Due to the potential risk of insertional mutagenesis from oncoretroviral-mediated gene therapy, cell-specific expression of transgenes using self-inactivating viral vectors may provide a safer gene therapy approach for use in humans. Therefore, we constructed simian immunodeficiency virus (SIV)-based lentiviral vectors containing a 5′ long-terminal repeat (LTR) and 3′ LTR with self-inactivating U3 deletion, the bovine growth hormone polyA signal, a packaging signal (ψ), and a single internal ankyrin-1 or β-globin promoter, designated SIV-Ank and SIV-Bg, respectively. The minimal 314-bp ankyrin-1 promoter and 180-bp β-globin promoter flanked upstream by enhancing sequences, HS2, HS3, and HS4 (Hanawa et al., Hum Gene Ther, 2002) from the locus control region were cloned into the SIV vector backbone upstream from either enhanced green fluorescent protein (eGFP) or B-domain deleted porcine factor VIII (BDDpfVIII). The erythroid-specificity of each promoter was evaluated in vitro by measurement of either eGFP or fVIII expression following transduction of SIV-Ank and SIV-Bg constructs into both K562 myelogenous leukemic cells and 293T human embryonic kidney cells. GFP expression, as measured by flow cytometry, in transduced cells revealed that the ankyrin-1 and β-globin promoters are more active in K562 cells as compared to 293T cells. The β-globin promoter yielded higher mean fluorescent intensity values for GFP compared to the ankyrin-1 promoter at similar MOIs in K562 cells, suggesting stronger β-globin promoter activity in these cells. Transduction of cells with the SIV vector encoding BDDpfVIII driven by the β-globin promoter resulted in a 14-fold higher number of transcripts per DNA copy number in K562 cells compared to 293T cells, while cells transduced with the ankyrin-l promoter had only a 1.4-fold greater number of transcripts per DNA copy number. In addition, SIV-Bg-fVIII-modified K562 cells produced a 5.2-fold greater number of transcripts per DNA copy number than SIV-Ank- fVIII-modified cells. To evaluate the usefulness of these vectors for in vivo expression of BDDpfVIII, hemophilia A mice (exon 16 knockout) were conditioned with 11 Gy total body irradiation and transplanted with gene-modified Sca-1+ cells transduced with either SIV-Ank-fVIII, SIV-Ank-eGFP, SIV-Bg-fVIII, or SIV-Bg-eGFP. The expression of eGFP from donor red blood cells in recipient mice was approximately 8–12% using both the ankyrin-1 and β-globin promoter constructs. Mice that received cells transduced with SIVAnk- fVIII demonstrated therapeutic levels of plasma fVIII up to 0.5 units/mL (i.e. 50% normal human levels). However, fVIII expression decreased over time and real-time PCR analysis of peripheral blood cells confirmed the loss of detectable fVIII transgene by 6 weeks after transplantation, suggesting there was predominantly gene transfer into short-term repopulating hematopoietic cells. Mice transplanted with SIV-Bg-fVIII-modified hematopoietic stem cells demonstrated a similar rise and fall of fVIII expression within the first 4 weeks after transplantation, and showed an increase in fVIII expression by 6 weeks. At 8 weeks post transplantation, fVIII levels greater than 300% normal human levels were observed. Red blood cell count, hemoglobin, and red blood cell morphology were normal despite the high level of expression of fVIII. Overall these data demonstrate the potential for therapeutic expression of factor VIII using a self-inactivating lentiviral vector containing an erythroid-specific internal promoter.

Insulator Elements in Erythrocyte Membrane Genes

Insulators are DNA sequences and associated binding proteins that establish and/or maintain the boundaries between euchromatin and heterochromatin. One type of insulator establishes chromatin domains to separate enhancers and promoters and prevent their interaction (enhancer-blocking insulators) whereas another type creates a barrier to protect against heterochromatin-mediated gene silencing (barrier insulators). In the well-characterized chicken beta-globin LCR 5′HS4 insulator, binding of CTCF mediates enhancer-blocking functions and USF proteins mediate barrier activity. Because varying transcripts of erythrocyte membrane protein genes are expressed in both erythroid and nonerythroid cells, we hypothesized that that insulator elements that bind USF and CTCF participate in their regulation. Advances in technology have permitted rapid identification of DNA sequences bound by transcription factors and other DNA-associated proteins on a genome-wide scale. Coupling chromatin immunoprecipitation to microarrays that contain genomic regions (ChIP-chip) is a high resolution technique available for mapping protein-DNA interactions in vivo. We used ChIP-chip to identify CTCF and USF factor binding sites with potential insulator function that regulate membrane protein gene expression in erythroid cells. ChIP was performed with K562 and HeLa cells using antibodies against CTCF, USF1, and USF2. DNA obtained from these IPs was hybridized to a custom NimbleGen high-density human genomic DNA microarray. Chip probes were ~50bp in length, Tm ≥76°C, tiled ~65bp apart. Regions of repetitive DNA excluded. The chip included 15 erythrocyte membrane protein genes, most encoding complex loci with multiple tissue-, cell-, and developmental stage-specific transcripts, including alpha spectrin, beta spectrin, ankyrin, spectrin, band 3, ƒnalpha adducin, beta adducin, gamma adducin, ICAM4, erythroid associated membrane protein, protein 4.1R, protein 4.2, dematin, beta actin, tropomodulin, and tropomyosin. Each gene plus 50 to 100kb of flanking DNA were included on the chip. Binding sites on the custom DNA array were identified using the Tamalpais peak calling algorithm using L1–L3 level of stringency (Genom Res 16:595, 2006) or Tilescope using a signal cutoff of 0.9 (Genome Biol 8:R81, 2007). In K562 chromatin, 117 sites of CTCF occupancy were identified. There were 49 sites in 5′ flanking DNA, 3 in promoters, 9 in coding sequence, 20 in introns, 1 in 3′ untranslated region, and 35 in 3′ flanking DNA. Sites of USF1 and USF2 occupancy were identified in 7/15 and 9/15 genes, respectively. 15 USF1 sites were identified; 10 in 5′ flanking DNA, 1 in a promoter (<500bp from transcription start site), and 4 in 3′ flanking DNA. 22 sites of USF2 occupancy were identified; 9 in 5′ flanking DNA, 1 in a promoter, 4 in introns, and 8 in 3′ flanking DNA. USF1 and USF2 frequently heterodimerize and 10 sites bound both USF1 and USF2. To validate ChIP-chip results, a site binding USF2 near the ankyrin gene erythroid promoter was further analyzed. This site was chosen because the USF2-associated sequence directs uniform, copy-number dependent expression of a linked reporter gene in transgenic mice, the erythroid ankyrin promoter is positioned between 2 alternate, nonerythroid ankyrin promoters, and DNaseI mapping revealed that this region is contained within an erythroid-specific chromatin domain. Quantitative ChIP confirmed USF2 occupancy, as well as USF1 occupancy, in this region. For functional analysis, we used a gene silencing/position-effect variegation (PEV) assay to determine whether this region possess barrier element function. Using a HS2-beta globin promoter-GFP cassette as negative control and a cHS4-HS2-beta globin promoter-GFP-cHS4 cassette as positive control, the ankyrin promoter region fragment (p<0.001) functioned as barrier elements in this PEV assay. These data demonstrate that there are numerous binding sites for insulator-associated DNA binding proteins in erythrocyte membrane protein genes and indicate that these elements likely play a significant role in the regulation of tissue-specific expression of many genes expressed in erythroid cells.

High Level Production of a Lysosomal Enzyme from HSC-Derived Erythroid Cells for Therapeutic Correction of Hurler Syndrome in Mice.

Proviral integration into hematopoietic stem cells (HSC) by lentivirus vector (LV)- mediated gene transfer can provide the benefit of life-long therapeutic effect, yet it can also bring the risk of insertional oncogenesis. Restricting transgene expression to late erythroblasts and red blood cells (RBC) may reduce the risk of activating oncogenes in HSC and its offspring in all lineages. In this study, we sought to evaluate the feasibility of redirecting a fraction of robust protein-synthesis machinery in maturing erythroid cells for production of alpha-L-iduronidase (IDUA), the lysosomal enzyme missing in Mucopolysaccharidosis type I (MPS I, or Hurler Syndrome). We first utilized a murine erythroid leukemia (MEL) cell line to compare IDUA expression and release from LVs using human elongation factor 1α promoter (EF), LTR of SFFV (SF), or a erythroid specific hybrid promoter (IHK) containing human ALAS2 intron 8 erythroid specific enhancer, HS40 core element from human alpha LCR and human ankyrin-1 promoter. We have previously shown highly erythroid-specific expression in vivo using this hybrid promoter, which is sustained in secondary transplantation recipient mice and resists proviral silencing (Moreau-Gaudry et al. Blood, 2001; Mohamedali et al, Mol Ther 2004). MEL cells were introduced to differentiation with hexamethylene bis-acetamide, and progressive erythroid differentiation was confirmed by morphologic evaluation in cytospins. Relatively low levels of expression from IHK (5% of SF, and 8% of EF) were observed in non-induced MEL cells; however, it increased steadily during induction and reached a similarly high expression level as those from the SF promoter. Expression from EF promoter reduced significantly, and the levels from SF promoter remained unchanged with erythroid differentiation. A similar pattern was found in IDUA activities released from stably transduced MEL cells during induction. We then evaluated in vivo the systemic IDUA production in an enzyme-deficient MPS I mouse model using erythroid-specific LV. Lineage-negative bone marrow cells (Lin−) were isolated with 92–97% purity by lineage depletion using immunomagnetic cell sorting. After a short pre-stimulation period, Lin− cells were transduced twice with LV-IHK-IDUA-ires-GFP for a total multiplicity of infection (MOI) at 2–20, resulting in up to 71% transduction efficiency. Four-months after transplantation of Lin- cells transduced at high MOI, sustained and higher-than normal plasma IDUA levels were achieved in all treated MPS I mice (1.5 to 8-fold of normal levels) with GFP transgene expression detected in up to 7% of Ter119+ blood cells. To evaluate transgene expression pattern during erythroid differentiation in bone marrow, we defined erythroblast subpopulations by immunostaining with CD71 and Ter119 for enrichment in proerythroblasts (I), basophilic erythroblasts (II), polychromatophilic erythroblasts (III), and orthrochromatic erythroblasts and reticulocytes (IV). GFP expression was predominantly detected in population III (9–17.3%), followed by the most differentiated population IV. To determine transduction efficiency in HSC, spleen colony-forming-cell assay was performed that showed 15–25% GFP+ spleen colonies in 2o BMT recipients. Moreover, long-term systemic metabolic correction was demonstrated by normalized urinary glycosaminoglycan accumulation in all treated MPS I mice. Complete normalization of tissue pathology was observed in liver and spleen, with relatively moderate improvement in brain. These results demonstrate for the first time that a lysosomal enzyme can be produced and secreted successfully and steadily at superphysiological levels in circulation by erythroid cells using tissue-specific LV, with phenotypic correction of Hurler Syndrome in mice. This data warrants further evaluation for the potential application of erythroid-specific vectors in treating non-RBC related diseases.

650. Increasing the Activity of the Ankyrin Promoter for Globin Gene Therapy

650. Increasing the Activity of the Ankyrin Promoter for Globin Gene Therapy

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.

Erythroid-Specific Expression of β-Globin by the Sleeping Beauty Transposon for Sickle Cell Disease

Sickle cell disease (SCD) results predominately from a single monogenic mutation that affects thousands of individuals worldwide. Gene therapy approaches have focused on using viral vectors to transfer wild-type beta- or gamma-globin transgenes into hematopoietic stem cells for long-term expression of the recombinant globins. In this study, we investigated the use of a novel nonviral vector system, the Sleeping Beauty (SB) transposon (Tn) to insert a wild-type beta-globin expression cassette into the human genome for sustained expression of beta-globin. We initially constructed a beta-globin expression vector composed of the hybrid cytomegalovirus (CMV) enhancer chicken beta-actin promoter (CAGGS) and full-length beta-globin cDNA, as well as truncated forms lacking either the 3' or 3' and 5' untranslated regions (UTRs), to optimize expression of beta-globin. Beta-globin with its 5' UTR was efficiently expressed from its cDNA in K-562 cells induced with hemin. However, expression was constitutive and not erythroid-specific. We then constructed cis SB-Tn-beta-globin plasmids using a minimal beta-globin gene driven by hybrid promoter IHK (human ALAS2 intron 8 erythroid-specific enhancer, HS40 core element from human alphaLCR, ankyrin-1 promoter), IHbetap (human ALAS2 intron 8 erythroid-specific enhancer, HS40 core element from human alphaLCR, beta-globin promoter), or HS3betap (HS3 core element from human betaLCR, beta-globin promoter) to establish erythroid-specific expression of beta-globin. Stable genomic insertion of the minimal gene and expression of the beta-globin transgene for >5 months at a level comparable to that of the endogenous gamma-globin gene were achieved using a SB-Tn beta-globin cis construct. Interestingly, erythroid-specific expression of beta-globin driven by IHK was regulated primarily at the translational level, in contrast to post-transcriptional regulation in non-erythroid cells. The SB-Tn system is a promising nonviral vector for efficient genomic insertion conferring stable, persistent erythroid-specific expression of beta-globin.

Identification of a Novel Core Promoter Element That Enhances Transcription: Application to Ankyrin Promoters for Globin Gene Therapy.

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.

A Transgenic Mouse Assay To Compare the Barrier Activities of the Chicken β-Globin 5′HS4, Human β-Globin 5′HS5 and Mouse 3′HS1.

Insulator elements are found at the boundary between euchromatin and heterochromatin, and are responsible for maintaining the correct chromatin configuration for a locus. The best characterized vertebrate insulator element, 5′ Hypersensitive Site (HS) 4 from the chicken β-globin locus (ch5′HS4), has two separable activities: enhancer blocking, which requires binding of the transcription factor CTCF, and barrier, which prevents transgene silencing. We have previously reported that transgenic mice carrying a wild-type erythrocyte ankyrin promoter (ANK-1E)/γ-globin gene showed uniform (γ-globin in 100% of red cells), position-independent (32/32 lines express), copy number-dependent (p=0.0005) expression of γ-globin mRNA and protein. Mutations in the ANK-1E promoter at positions −108 and −153 cause ankyrin-deficient Hereditary Spherocytosis. Transgenic mice with the −108/−153 ANK-1E/γ-globin transgene showed variegated (γ-globin in 0–80% of red cells), position-dependent (8/14 lines express), copy number-independent (p=0.27) expression of γ-globin. Flanking the −108/−153 ANK-1E/γ-globin transgene with the ch5′HS4 insulator restored uniform, position-independent (9/9 lines), copy number-dependent expression (p=0.003) at levels identical to the wild-type ANK-1E promoter. We hypothesized that we could test sequences for barrier activity by assaying their ability to restore normal expression to the −108/−153 ANK-1E/γ-globin gene in transgenic mice. In mammalian β-globin loci, human 5′HS5 and mouse 3′HS1 have been proposed to be insulator elements, similar to chicken 5′HS4, based on their ability to block enhancer element function. To test barrier function, we generated transgenic mice containing the −108/−153 ANK-1E/γ-globin transgene flanked by human 5′HS5, mouse wild-type 3′HS1, or mouse 3′HS1 with mutations that disrupt the binding of CTCF (×CTCF). A total of 5 lines of transgenic mice were generated containing the 5′HS5/−108/−153 ANK-1E/γ-globin transgene. γ-globin mRNA and protein were undetectable in 3/5 lines, indicating that expression was position-dependent, and in the two positive lines, mRNA levels did not correlate with copy number. A total of 9 lines of 3′HS1-flanked transgenic mice were generated, 3 of which did not express γ-globin mRNA and protein, demonstrating position-dependent expression. Among the 6 expressing lines, two lines showed variegated expression and the correlation between γ-globin mRNA level and copy number was significant (p=0.0117). In contrast, 3′HS1×CTCF transgenic mice expressed γ-globin in a uniform, position-independent (7/7 lines express), copy number-dependent (p=0.0005) manner. The levels of γ-globin mRNA in both the 3′HS1 and 3′HS1×CTCF transgenic mice were 2-fold greater than the levels measured in transgenic mice with the wild-type ANK-1E promoter (p=0.019; 0.0003 respectively), suggesting that 3′HS1 may contain an enhancer element. Our results indicate that while human 5′HS5 and mouse 3′HS1 block the effects of enhancer elements, neither are barrier elements as defined by the ability to prevent gene silencing. We hypothesize that the mutation of the CTCF binding sites allows the ANK-1E promoter to take greater advantage of the 3′HS1 enhancer, leading to a more uniform, position-independent, and copy number-dependent pattern of expression, as has been described for other enhancer elements in the β-globin Locus Control Region.

790. Murine Retroviral but Not Human Cellular Promoters Induce In Vivo Erythroid-Specific Promoter Deregulation That Can Be Prevented by Insulators

Top of pageAbstract We are interested in the development of lentiviral vectors that co- express a therapeutic gene in an erythroid-specific manner and the MGMT selective gene in a constitutive way for gene therapy of red blood cell (RBC) disorders. We have previously demonstrated that long-term gene expression limited to erythroid progeny was possible in vivo with a chimeric HS40 enhancer and human ankyrin promoter in the area of the constitutive human PGK promoter (PGKp) driving MGMT without loss of tissue-specificity. However, transductions were performed at high multiplicity of infection (MOI) elevating the risk of insertional mutagenesis. In this study, we found that PGKp was not strong enough at low MOI to mediate an in vivo survival advantage in the presence of alkylating agents. To be efficient at one copy per cell, the vector needs to contain strong enhancer- promoter elements. By replacing PGKp with the MND promoter (MNDp), the vector allowed an efficient chemoprotection of transduced hematopoietic stem cells (HSCs) at low MOI (75.1 |[plusmn]| 8.6 % vs 34.8 |[plusmn]| 11 % EGFP+ RBCs for treated (n=3) vs untreated group (n=3)). However, this MNDp interacts with the erythroid one, leading to a partial loss of erythroid-specificity. To avoid this promoter deregulation, the full length cHS4 insulator sequence or its minimal core element were inserted between the two expression units. Murine HSCs were transduced by lentiviral vectors and injected into 12-Gy irradiated BALB/c. Sixteen weeks after transplantation, bone marrow (BM) cells were analyzed by FACS. The core-containing vector combines a similar high chemoprotection efficiency as allowed by MNDp (up to 20-fold in vivo expansion of GFP positive RBCs) and a dramatic decrease of promoter interferences (2, 5 and up to 6-fold decrease of non specific GFP mean fluorescence intensity (MFI) in T-lymphocytes, granulocytes and monocytes respectively, as compared with the MNDp- lentivector). This study demonstrates the interest of insulators for preventing in vivo enhancer activities between 2 promoters. Alternatively, we found that the strong human cellular EF1-|[alpha]| promoter allows similar chemoprotection (86.0 |[plusmn]| 5.0 % vs 34.1 |[plusmn]| 2.5 % EGFP+ RBCs for treated and untreated groups, respectively) but without any deregulation of the erythroid-specific promoter in the absence of insulators. These data suggest that the level of in vivo deregulation induced by a promoter is not correlated with its transcriptional activity but with the presence its strong enhancers. These findings have direct implications for the design of gene therapy vectors.

Chromatin Conformation and a Distal Regulatory Element Activate the Human Erythroid Ankyrin-1 Promoter.

Ankyrin forms the bridge between the spectrin/actin network of the erythrocyte membrane skeleton and the red cell membrane by binding to both β-spectrin and band 3. The erythrocyte ankyrin promoter (Ank-1E) is active only in erythroid cells, while two other Ank-1 promoters located 20 kb downstream and 40 kb upstream of Ank-1E are active in the cerebellum and muscle cells respectively. We have been studying the mechanism by which the Ank-1E promoter becomes active in erythroid cells by studying the cis acting regulatory elements and the chromatin structure of the Ank-1 promoter region. We have previously shown that the sequences between −296 and −15 of the Ank-1E promoter are fully sufficient for erythroid specific, copy number dependent uniform expression of reporter genes in transgenic mice. We have also mapped a DNase I Hypersensitive site (5′HS) between −300 and −100 of the human and mouse Ank-1E promoters in human K562 and mouse fetal liver cells. Both the mouse and human 5′HS are capable of preventing the silencing of a β-globin/GFP reporter gene in K562 cells, establishing that they function as barrier elements. Consistent with this observation, the human and mouse 5′HS are hyperacetylated in erythroid cells. The chromatin 10 kb 5′ to the 5′HS is DNase I resistant (associated with inactive chromatin) in human and mouse erythroid and non-erythroid cells. Approximately 6 kb 3′ to 5′HS are two adjacent HS (3′HS1, 3′HS2). Beyond 3′HS2 the chromatin is also DNase I resistant in both human and mouse erythroid and non-erythroid cells. Between 5′HS and 3′HS1 the 6kb region is DNase I sensitive (active) in erythroid cells but not in other cell types. We hypothesized that this 6 kb region contains regulatory elements that activate the Ank-1E promoter. To screen for regulatory elements we isolated overlapping segments of a 10 kb region extending from 2 kb upstream of 5′HS to 2 kb downstream of 3′HS2. We inserted these fragments into a plasmid vector containing the Ank-1E promoter linked to a luciferase reporter gene and transfected these constructs into K562 cells. A single region up regulated Ank-1E/luciferase expression. This region mapped to a 211bp segment that included 3′HS1, but did not include 3′HS2. A fragment containing only 3′HS2 did not up regulate an Ank-1E/luciferase reporter gene, but 3′HS2 was capable of preventing the silencing of a β-globin/Green Fluorescent Protein reporter gene in K562 cells, demonstrating barrier activity. The region around 3′HS1 and 2 was also a site of histone hyperacetylation. The sequence of the 211 bp fragment containing 3′HS1 does not contain consensus sequences for any known erythroid-specific transcription factors, but does contain potential binding sites fro Sp1, AP-1 and E-box binding proteins. Using the Chromatin Conformation Capture assay we demonstrated that 5′HS and 3′HS1 and 2 are in close proximity in K562 chromatin, but are not closely associated in chromatin from other cell types. We propose that an erythroid-specific chromatin loop brings 3′HS1 and 2 into proximity with 5′HS, adjacent to the Ank-1E promoter. This interaction translocates the positive regulatory element in 3′HS1 to the Ank-1E promoter allowing the Ank-1E promoter to become active in erythroid cells.

A dinucleotide deletion in the ankyrin promoter alters gene expression, transcription initiation and TFIID complex formation in hereditary spherocytosis

Ankyrin defects are the most common cause of hereditary spherocytosis (HS). In some HS patients, mutations in the ankyrin promoter have been hypothesized to lead to decreased ankyrin mRNA synthesis. The ankyrin erythroid promoter is a member of the most common class of mammalian promoters which lack conserved TATA, initiator or other promoter cis elements and have high G+C content, functional Sp1 binding sites and multiple transcription initiation sites. We identified a novel ankyrin gene promoter mutation, a TG deletion adjacent to a transcription initiation site, in a patient with ankyrin-linked HS and analyzed its effects on ankyrin expression. In vitro, the mutant promoter directed decreased levels of gene expression, altered transcription initiation site utilization and exhibited defective binding of TATA-binding protein (TBP) and TFIID complex formation. In a transgenic mouse model, the mutant ankyrin promoter led to abnormalities in gene expression, including decreased expression of a reporter gene and altered transcription initiation site utilization. These data indicate that the mutation alters ankyrin gene transcription and contributes to the HS phenotype by decreasing ankyrin gene synthesis via disruption of TFIID complex interactions with the ankyrin core promoter. These studies support the model that in promoters that lack conserved cis elements, the TFIID complex directs preinitiation complex formation at specific sites in core promoter DNA and provide the first evidence that disruption of TBP binding and TFIID complex formation in this type of promoter leads to alterations in start site utilization, decreased gene expression and a disease phenotype in vivo.

Defining the Active Chromatin Domain of the Erythroid Ankyrin Promoter.

Erythrocyte ankyrin forms the bridge between the spectrin/actin cytoskeleton and the red cell membrane protein band 3. Mutations resulting in defective or deficient ankyrin are the most common cause of Hereditary Spherocytosis. The erythroid ankyrin promoter (ankR) lies between two other ankyrin promoters, one that is active in neuronal cells and the other in many cell types. Active genes are generally found in DNase I sensitive regions of chromatin while regulatory sequences (promoters, enhancers, boundary elements) are associated with DNase I Hypersensitive Sites (HS). We hypothesized that the chromatin surrounding ankR determines its activity. In human K562 cells and mouse fetal liver cells a DNase I HS (5′HS) maps between −300 and −100 of ankR. 5′HS marks the end of a DNase I resistant chromatin extends at least 10 kb upstream, indicating that this chromatin is inactive in erythroid cells. We have previously shown that a 300 bp fragment containing ankR was sufficient to direct erythroid specific, copy number dependent, position independent and uniform expression of a linked γ-globin gene in transgenic mice. Identical results were obtained with mice containing −2.7 kb and −656 bp ankyrin promoters, as expected, and 5′ HS was present in fetal liver chromatin of all 8 lines examined. Downstream of 5′HS is a 5.7 kb DNase I sensitive region that includes a 500 bp enhancer-like element near the 3′ end. At the 3′ end of the DNase I sensitive domain are 2 HS (3′HS) that mark the beginning of a DNase I resistant region that extends at least 3 kb downstream. AnkR is inactive in non-erythroid HeLa, Jurkat and Sh-SY5Y (neuronal) cells. Although both 5′HS and 3′HS are present in these cells, the intervening 5.7 kb region is DNase I resistant indicating that erythroid specific factors are required for the activation of ankR. Boundary elements (insulators) are found between DNase I resistant and DNase I sensitive chromatin and have the ability to prevent the transgene silencing at ectopic sites in the genome. To test whether the 5′HS and 3′HS function as boundary elements, a β-globin promoter/EGFP transgene was flanked by 200 bp fragments containing either 5′HS or 3′HS. While 0/8 K562 cell lines with the transgene alone expressed GFP, GFP expression was observed in 12/12 5′HS and 11/12 3′HS K562 cell lines (χ2=12.0, p<0.001; χ2=9.0, p<0.01 respectively), indicating boundary function. We have previously shown that mutations at positions −153 and −108 in 5′HS associated with Hereditary Spherocytosis cause position dependent and variegated expression of the γ-globin transgene in mice. When the −153/−108 sequence was used to flank the β-globin/EGFP transgene gene silencing was observed in 5/10 K562 cell lines (χ2=5.7,p<0.02). Because boundary elements are sites of high levels of histone acetylase (HA) activity we hypothesized that the −153/−108 mutations disrupted histone acetylation in ankR. EMSA analysis showed that the −153/−108 mutations disrupted the binding of a complex that includes both CTCF (a zinc finger protein associated with boundary elements in the β-globin cluster) and Brg-1 (the ATP dependent subunit of the HA complex). Chomatin Immune Precipitation analysis of transfected K562 cells showed that histone acetylation in sequences containing the −153/−108 mutations was 4-fold less than in wild type sequences (p<0.02). These studies have defined the active chromatin domain of the ankR and have demonstrated that the −153/−108 mutations cause Hereditary Spherocytosis by inhibiting histone acetylation in ankR.

Self-Inactivating Lentiviral Vectors Resist Proviral Methylation but Do Not Confer Position-Independent Expression in Hematopoietic Stem Cells

Oncoretroviral expression is transcriptionally silenced in embryonic and hematopoietic stem cells (HSCs). This is associated with methylation of viral and internal promoters. We determined whether self-inactivating (SIN) lentiviral vectors (LV) would circumvent proviral silencing in HSCs. We studied long-term expression, methylation, and position effects (PE) from two GFP-encoding SIN-LV containing erythroid enhancers and the human ankyrin-1 promoter (h-Ank-P) using the murine secondary bone marrow (BM) transplant assay. Proviral expression was detected in RBC 6–11 months following transplant only in 28 of 49 secondary mice, with 0.9 ± 0.2 copy/cell and oligoclonally integrated provirus in BM, spleen, and thymus. Twenty-one of 49 secondary mice lacked integrated provirus. Secondary mice containing provirus also had GFP-expressing RBCs, although proviral copy number did not always correlate with expression, suggesting either proviral methylation or chromatin PE. The endogenous h-Ank-P was partially methylated in nonerythroid cell lines and unmethylated in erythroid cell lines. However, h-Ank-P in the provirus was unmethylated in erythroid and nonerythroid cells within secondary murine BM. Despite lack of methylation, GFP expression was variable in secondary BFU-e and in single-copy mouse erythroleukemia cell clones. Taken together, these data show that erythroid-specific SIN-LV express long term and resist methylation-associated proviral silencing, but may require additional elements to confer position-independent expression.

High-level erythroid lineage-directed gene expression using globin gene regulatory elements after lentiviral vector-mediated gene transfer into primitive human and murine hematopoietic cells.

Lentiviral vectors efficiently transduce primitive human hematopoietic cells and are capable of transferring complex genomes. Vectors were designed with hypersensitive sites containing regulatory elements from the beta-globin locus control region linked to the beta-globin gene promoter to drive expression of the enhanced green fluorescent protein marker to facilitate analysis of the pattern of gene expression in various hematopoietic lineages. Such vectors gave higher level, induced expression in mouse erythroleukemia cells than a previously described vector that utilized an enhancer from the alpha locus and the ankyrin-1 promoter [Moreau-Gaudry, F., Xia, P., Jiang, G., Perelman, N.P., Bauer, G., Ellis, J., Surinya, K.H., Mavilio, F., Shen, C.K., and Malik, P. (2001). Blood 98, 2664-2672]. The addition of gamma-globin intron sequences further augmented vector expression. Expression was also effectively targeted to the erythroid lineage in cultured human cells from peripheral blood and in mouse red blood cells in vivo, although lower levels of expression were also observed in other lineages. Thus, these newly described vectors provide a means to achieve high-level gene expression, predominantly in erythroid cells, an outcome that may have potential therapeutic application.

High-level erythroid-specific gene expression in primary human and murine hematopoietic cells with self-inactivating lentiviral vectors

AbstractUse of oncoretroviral vectors in gene therapy for hemoglobinopathies has been impeded by low titer vectors, genetic instability, and poor expression. Fifteen self- inactivating (SIN) lentiviral vectors using 4 erythroid promoters in combination with 4 erythroid enhancers with or without the woodchuck hepatitis virus postregulatory element (WPRE) were generated using the enhanced green fluorescent protein as a reporter gene. Vectors with high erythroid-specific expression in cell lines were tested in primary human CD34+ cells and in vivo in the murine bone marrow (BM) transplantation model. Vectors containing the ankyrin-1 promoter showed high-level expression and stable proviral transmission. Two vectors containing the ankyrin-1 promoter and 2 erythroid enhancers (HS-40 plus GATA-1 or HS-40 plus 5-aminolevulinate synthase intron 8 [I8] enhancers) and WPRE expressed at levels higher than the HS2/β-promoter vector in bulk unilineage erythroid cultures and individual erythroid blast-forming units derived from human BM CD34+ cells. Sca1+/lineage− Ly5.1 mouse hematopoietic cells, transduced with these 2 ankyrin-1 promoter vectors, were injected into lethally irradiated Ly5.2 recipients. Eleven weeks after transplantation, high-level expression was seen from both vectors in blood (63%-89% of red blood cells) and erythroid cells in BM (70%-86% engraftment), compared with negligible expression in myeloid and lymphoid lineages in blood, BM, spleen, and thymus (0%-4%). The I8/HS-40–containing vector encoding a hybrid human β/γ-globin gene led to 43% to 113% human γ-globin expression/copy of the mouse α-globin gene. Thus, modular use of erythroid-specific enhancers/promoters and WPRE in SIN-lentiviral vectors led to identification of high-titer, stably transmitted vectors with high-level erythroid-specific expression for gene therapy of red cell diseases.