The Experts below are selected from a list of 147 Experts worldwide ranked by ideXlab platform
Punam Malik - One of the best experts on this subject based on the ideXlab platform.
-
the 3 end of the chicken hypersensitive site 4 Insulator has properties similar to the 5 Insulator core and is necessary in conjunction with the core for full Insulator activity
Blood, 2008Co-Authors: Paritha Arumugam, Fabrizia Urbinati, Chinavenmeni S Velu, Leighton H Grimes, Punam MalikAbstract:Genetic correction of hematologic defects is currently impeded by inefficient vector technology. We find that vectors that insulate the correcting transgene from position effects and genotoxicity compromise viral titers. Here we present an improved vector system which utilizes a modified Insulator Element, without sacrificing viral titers. Specifically, our genetic and epigenetic analysis of the 1.2kb chicken β-globin hypersensitive site-4 (cHS4) Insulator reveal heretofore unknown activities in regions of the chicken β-globin Insulator Element outside the canonical and well studied 250bp 5′ “core” Element. Previously, the core Insulator activity was mapped to CTCF and USF-1/2 binding sites, located only in the 5′ 250bp core. However, we find that the 5′ 250bp core alone is ineffective at shielding from position effects when it flanks transgenes, and gammaretrovirus/lentivirus vectors. In contrast, the entire 1.2kb cHS4 efficiently insulates, but significantly lowers titers of lentiviral vectors. To identify insulating activities which might be appended to the 5′ 250bp core to properly insulate transgene expression cassettes without sacrificing viral titers, we performed a structure-function analysis of the cHS4 Insulator placed within the 3′LTR of a lentivirus containing the regulatory and coding sequences of human b-globin (Table 1). We compared single-copy clonal progeny of mouse erythroleukemia cells (MEL) and primary transduced and transplanted hematopoietic stem cells for position effects. Additionally, we studied repressive and activating histone marks over the transgene promoter and cHS4 in the different proviruses. Our data indicate that while all vectors containing the core reduced the coefficient of variation (CV) of human b-globin (HbA) expression, several constructs suggested that cHS4 sequences in the most 3′ 400bp (furthest from the core) may be critical to full length Insulator activity. We next analyzed HbA expression in vector-corrected thalassemia mice, and generated single copy secondary CFU-S, the gold standard for studying chromatin position effects (Table 2). While all vectors containing the cHS4 core provided some ‘Insulator’ activity when compared to the uninsulated vector control (conceivably by reducing the CV/clonal variegation) the full length 1.2kb Insulator vector provided maximum shielding from position effects, with nearly 2.5-fold higher HbA expression compared to the uninsulated vector. These data were confirmed in secondary CFU-S. Epigenetic analyses of the vector b-globin promoter revealed that transcriptionally repressive histone modifications were decreased, and activating histone modifications increased when the last 400bp sequences of cHS4 were present. Notably, vectors carrying only the 3′ 400bp sequences of cHS4 reduced clonal variegation in MEL cells and secondary CFU-S, but did not increase HbA expressing cells both in vitro and in vivo (Table 2). However, full Insulator activity was restored in MEL clones when both the 5′ 250bp core was combined with the 3′ 400bp Element. The addition of the 3′ 400bp Element to the core was accompanied with a significant enrichment of active histone marks and minimal repressive histone marks in the provirus as seen with the 1.2Kb Insulator. These data consolidate the known insulating activity of the 5′ 250bp core Element with a novel 3′ 400bp Element which (together) constitutes a new insulated vector system with excellent insulating properties and viral titers. Our data have important implications in the design of gene therapy vectors, where optimal Insulator activity can be achieved with a minimal reduction in viral titers. Table 1. Single copy MEL clones showing effect of Insulator sequences on position effects in β-globin carrying lentiviruses Table 2. Effect of Insulator sequences on the differentiated progeny of transduced and transplanted thalassemia hematopoietic stem cells
-
improved human β globin expression from self inactivating lentiviral vectors carrying the chicken hypersensitive site 4 chs4 Insulator Element
Molecular Therapy, 2007Co-Authors: Paritha I Arumugam, Jessica Scholes, Natalya Perelman, Ping Xia, Jiingkuan Yee, Punam MalikAbstract:Effective gene therapy for β-thalassemia major (β-TM) requires consistent, high expression of human β-globin (hβ-globin) in red blood cells (RBCs). Several groups have now shown that lentiviral (LV) vectors stably transmit the hβ/hγ-globin genes and large Elements of the locus control region, resulting in correction of the murine thalassemia intermedia (TI) phenotype and survival of mice with the TM phenotype. However, current LVs show variable hβ/hγ-globin expression and require a high number of vector copies/cell for a therapeutic effect. To address this, we designed LVs flanked by the chicken hypersensitive site-4 (cHS4) chromatin Insulator Element and compared them with their “un-insulated” counterparts. We observed a consistent twofold-higher hβ expression from insulated vectors in single-copy mouse erythroleukemia cell clones, an increase that resulted from reduced position effect variegation (PEV) and increased probability of expression from individual integrants. This effect was confirmed in vivo: an approximately twofold increase in hβ expression was seen in the RBC progeny of murine hematopoietic stem cells, with significantly higher numbers of hβ-expressing cells in individual secondary spleen colony-forming units. In summary, cHS4-insulated hβ-globin LVs showed distinct chromatin barrier activity, resulting in higher, consistent hβ expression. These studies have important implications for vector design for clinical trials for gene therapy for hemoglobinopathies.
-
67 the chicken hypersensitive site chs4 Insulator Element reduces position effects thereby increasing expression from globin lentiviral vectors
Molecular Therapy, 2006Co-Authors: Paritha I Arumugam, Jessica Scholes, Natalya Perelman, Ping Xia, Jiingkuan Yee, Alexander Zarzuela, Punam MalikAbstract:Lentiviral vectors (LV) carrying the human |[beta]|-globin gene (h|[beta]|) and locus control region (LCR) have changed the field of gene therapy for hemoglobinopathies. However, their random integration into host cells results in variable h|[beta]| expression from chromatin position effects. We analyzed the role of a 1.2 kb chicken |[beta]|-globin locus hypersensitive site 4 Insulator Element (cHS4) in a self-inactivating (SIN) LV. The BGM vector (carrying h|[beta]|/LCR) was compared to an analogous vector BGMI, with cHS4 such that it flanks the provirus upon integration. Both vectors additionally carried the mutant P140K methyl guanine methyl transferase (MGMTP140K) cDNA to enrich for genetically modified cells. First, murine erythroleukemia (MEL) cells were transduced at <5% transduction efficiency to generate single-copy clones and transduced clones identified by PCR ('Unselected' clones) and analyzed for h|[beta]| expression by FACS and RNase Protection Assay (RPA). 'Unselected' BGMI+-MEL clones had a higher proportion of h|[beta]|+ cells (68|[plusmn]|3%) compared to BGM+ clones (36|[plusmn]|9%, n=24; P<0.001) and a reduced coefficient of variation (CV) of h|[beta]| expression: 168|[plusmn]|20 vs. 327|[plusmn]|64 in BGM (P<0.02). RPA showed a 2-fold increase in h|[beta]|/total m|[alpha]|-globin mRNA: BGMI 45|[plusmn]|6% vs. BGM 21|[plusmn]|7%, n=24, P<0.01. Next, MEL cells were selected with BG/BCNU and single-copy clones isolated ('Selected' clones). 'Selected' BGMI+-MEL clones also had a higher proportion of h|[beta]|+ cells compared to BGM+ clones (80|[plusmn]|15% vs. 72|[plusmn]|20%, n=86, P<0.03), with reduced CV (140|[plusmn]|2.8 vs. 170|[plusmn]|13, P<0.01) and higher h|[beta]|-mRNA (BGMI+ 83.6%|[plusmn]|40 vs. 49.6%|[plusmn]|25, n=24, P<0.02). Two additional vectors termed BG and BGI (with cHS4) were constructed with the h|[beta]|/LCR but no MGMTP140K cassette. RPA showed that BGI+ MEL pools expressed 85% h|[beta]|/total m|[alpha]|-globin/ vector copy as compared to 38% from BG. BG and BGI single copy MEL clones showed similar results. In in vivo studies lethally irradiated normal mice were transplanted with BGM and BGMI- transduced thalassemia hematopoietic stem cells (MOI 20). Engraftment and vector copy number in both groups were similar (66%|[plusmn]|15% vs. 68%|[plusmn]|10%, and 0.21 vs. 0.17 in BGMI and BGM groups, respectively, n=12). While, 18|[plusmn]|4% of RBC expressed h|[beta]| in the BGMI group, only 4%|[plusmn]|1.5% expressed h|[beta]| in the BGM group (P<0.001). There was a 4-fold increase in chimeric hemoglobin (m|[alpha]|- h|[beta]|) in BGMI mice (13%|[plusmn]|4% vs. 3%|[plusmn]|2% in BGM mice, P<0.001). Secondary colony forming units-spleen (CFU-S) derived from these mice showed increased numbers of h|[beta]|+ cells in BGMI (21%|[plusmn]|4% vs. 8%|[plusmn]|4% BGM, n=30, P<0.03) with reduced CV (698|[plusmn]|91 vs. 987|[plusmn]|99 in BGM, P<0.04). Taken together, 'insulated' SIN-h|[beta]|/LCR LV increased the probability of expression of integrants and reduced chromatin position effects, resulting in consistent and higher expression of h|[beta]|. The enhancer blocking effect of the cHS4, although not tested here, would further improve the bio-safety of these lineage- specific, SIN LV.
Gary Felsenfeld - One of the best experts on this subject based on the ideXlab platform.
-
vezf1 regulates genomic dna methylation through its effects on expression of dna methyltransferase dnmt3b
Genes & Development, 2008Co-Authors: Humaira Gowher, Heidi Stuhlmann, Gary FelsenfeldAbstract:The zinc finger protein vascular endothelial zinc finger 1 (Vezf1) has been implicated in the development of the blood vascular and lymphatic system in mice, and has been characterized as a transcriptional activator in some systems. The chicken homolog, BGP1, has binding sites in the beta-globin locus, including the upstream Insulator Element. We report that in a mouse embryonic stem cell line deletion of both copies of Vezf1 results in loss of DNA methylation at widespread sites in the genome, including Line1 Elements and minor satellite repeats, some imprinted genes, and several CpG islands. Loss of methylation appears to arise from a substantial decrease in the abundance of the de novo DNA methyltransferase, Dnmt3b. These results suggest that naturally occurring mutations in Vezf1/BGP1 might have widespread effects on DNA methylation patterns and therefore on epigenetic regulation of gene expression.
-
the 5 hs4 chicken β globin Insulator is a ctcf dependent nuclear matrix associated Element
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Timur M Yusufzai, Gary FelsenfeldAbstract:The protein CTCF plays an essential role in the action of a widely distributed class of vertebrate enhancer-blocking Insulators, of which the first example was found in a DNA sequence Element, HS4, at the 5′ end of the chicken β-globin locus. HS4 contains a binding site for CTCF that is necessary and sufficient for Insulator action. Purification of CTCF has revealed that it interacts with proteins involved in subnuclear architecture, notably nucleophosmin, a 38-kDa nucleolar phosphoprotein that is concentrated in nuclear matrix preparations. In this report we show that both CTCF and the HS4 Insulator Element are incorporated in the matrix; HS4 incorporation depends on the presence of an intact CTCF-binding site. However the DNA sequence in the neighborhood of HS4 is not like that of canonical matrix attachment regions, and its incorporation into the matrix fraction is not sensitive to ribonuclease, suggesting that the Insulator is a distinct matrix-associated Element.
-
ctcf tethers an Insulator to subnuclear sites suggesting shared Insulator mechanisms across species
Molecular Cell, 2004Co-Authors: Timur M Yusufzai, Hideaki Tagami, Yoshihiro Nakatani, Gary FelsenfeldAbstract:Insulators can block an enhancer of one gene from activating a promoter on another nearby gene. Almost all described vertebrate Insulators require binding of the regulatory protein CTCF for their activity. We show that CTCF copurifies with the nucleolar protein nucleophosmin and both are present at Insulator sites in vivo. Furthermore, exogenous Insulator sequences are tethered to the nucleolus in a CTCF-dependent manner. These interactions, quite different from those of the gypsy Insulator Element in Drosophila, may generate similar loop structures, suggesting a common theme and model for enhancer-blocking Insulator action.
-
variegated expression from the murine band 3 ae1 promoter in transgenic mice is associated with mrna transcript initiation at upstream start sites and can be suppressed by the addition of the chicken β globin 5 hs4 Insulator Element
Molecular and Cellular Biology, 2003Co-Authors: Tiffany F Frazar, Gary Felsenfeld, Jessica L Weisbein, Stacie M Anderson, Amanda P Cline, Lisa Garrett, Patrick G Gallagher, David M BodineAbstract:The anion exchanger protein 1 (AE1; band 3) is an abundant erythrocyte transmembrane protein that regulates chloride-bicarbonate exchange and provides an attachment site for the erythrocyte membrane skeleton on the cytoplasmic domain. We analyzed the function of the erythroid AE1 gene promoter by using run-on transcription, RNase protection, transient transfection, and transgenic mouse assays. AE1 mRNA was transcribed at a higher level and maintained at a higher steady-state level than either ankyrin or beta-spectrin in mouse fetal liver cells. When linked to a human gamma-globin gene, two different AE1 promoters directed erythroid-specific expression of gamma-globin mRNA in 18 of 18 lines of transgenic mice. However, variegated expression of gamma-globin was observed in 14 of 18 lines. While there was a significant correlation between transgene copy number and the amount of gamma-globin mRNA in all 18 lines, the transgene mRNAs initiated upstream of the start site of the endogenous AE1 mRNA. Addition of the Insulator Element from 5'HS4 of the chicken beta-globin cluster to the AE1/gamma-globin transgene allowed position-independent, copy-number-dependent expression at levels similar to the AE1 transcription rate in six of six lines of transgenic mice. The mRNA from the insulated AE1/gamma-globin transgene mapped to the start site of the endogenous AE1 mRNA, and gamma-globin protein was expressed in 100% of erythrocytes in all lines. We conclude that the chicken beta-globin 5'HS4 Element is necessary for full function of the AE1 promoter and that position effect variegation is associated with RNA transcription from the upstream start sites.
-
transitions in histone acetylation reveal boundaries of three separately regulated neighboring loci
The EMBO Journal, 2001Co-Authors: Michael D Litt, Melanie A Simpson, Felix Recillastarga, Marienoelle Prioleau, Gary FelsenfeldAbstract:We have studied developmentally regulated patterns of histone acetylation at high resolution across ∼54 kb of DNA containing three independently regulated but neighboring genetic loci. These include a folate receptor gene, a 16 kb condensed chromatin region, the chicken β-globin domain and an adjacent olfactory receptor gene. Within these regions the relative levels of acetylation appear to fall into three classes. The condensed chromatin region maintains the lowest acetylation at every developmental stage. Genes that are inactive show similarly low levels, but activation results in a dramatic increase in acetylation. The highest levels of acetylation are seen at regulatory sites upstream of the genes. These patterns imply the action of more than one class of acetylation. Notably, there is a very strong constitutive focus of hyperacetylation at the 5′ Insulator Element separating the globin locus from the folate receptor region, which suggests that this Insulator Element may harbor a high concentration of histone acetylases.
Jeffrey R Mann - One of the best experts on this subject based on the ideXlab platform.
-
the chicken β globin Insulator Element conveys chromatin boundary activity but not imprinting at the mouse igf2 h19 domain
Development, 2002Co-Authors: Piroska E Szabo, Shih Huey E Tang, Michael R Reed, Francisco J Silva, Walter Tsark, Jeffrey R MannAbstract:Imprinting of the mouse insulin-like growth factor 2 (Igf2) and H19 genes is regulated by an imprinting control region (ICR). The hypomethylated maternal copy functions as a chromatin Insulator through the binding of CTCF and prevents Igf2 activation in cis, while hypermethylation of the paternal copy inactivates Insulator function and leads to inactivation of H19 in cis. The specificity of the ICR sequence for mediating imprinting and chromatin insulation was investigated by substituting it for two copies of the chicken beta-globin Insulator Element, (Ch beta GI)(2), in mice. This introduced sequence resembles the ICR in size, and in containing CTCF-binding sites and CpGs, but otherwise lacks homology. On maternal inheritance, the (Ch beta GI)(2) was hypomethylated and displayed full chromatin Insulator activity. Monoallelic expression of Igf2 and H19 was retained and mice were of normal size. These results suggest that the ICR sequence, aside from CTCF-binding sites, is not uniquely specialized for chromatin insulation at the Igf2/H19 region. On paternal inheritance, the (Ch beta GI)(2) was also hypomethylated and displayed strong Insulator activity--fetuses possessed very low levels of Igf2 RNA and were greatly reduced in size, being as small as Igf2-null mutants. Furthermore, the paternal H19 allele was active. These results suggest that differential ICR methylation in the female and male germ lines is not acquired through differential binding of CTCF. Rather, it is likely to be acquired through a separate or downstream process.
-
maternal specific footprints at putative ctcf sites in the h19 imprinting control region give evidence for Insulator function
Current Biology, 2000Co-Authors: Piroska E Szabo, Shih Huey E Tang, Altan Rentsendorj, Gerd P Pfeifer, Jeffrey R MannAbstract:Abstract Parent-of-origin-specific expression of the mouse insulin-like growth factor 2 ( Igf2 ) gene and the closely linked H19 gene are regulated by an intervening 2 kb imprinting control region (ICR), which displays parent-specific differential DNA methylation [1,2]. Four 21 bp repeats are embedded within the ICR and are conserved in the putative ICR of human and rat Igf2 and H19 , suggesting that the repeats have a function [3,4]. Here, we report that prominent DNA footprints were found in vivo on the unmethylated maternal ICR at all four 21 bp repeats, demonstrating the presence of protein binding. The methylated paternal ICR displayed no footprints. Significantly, the maternal-specific footprints were localized to putative binding sites for CTCF, a highly conserved zinc-finger DNA-binding protein with multiple roles in gene regulation including that of chromatin Insulator function [5,6]. These results strongly suggest that the maternal ICR functions as an Insulator Element in regulating mutually exclusive expression of Igf2 and H19 in cis .
Arthur W. Nienhuis - One of the best experts on this subject based on the ideXlab platform.
-
generation of a lentiviral vector producer cell clone for human wiskott aldrich syndrome gene therapy
Molecular therapy. Methods & clinical development, 2015Co-Authors: Matthew Wielgosz, Gael Carney, Jun Zhan, M Reddivari, Terry Coop, Richard J Heath, Scott A. Brown, Arthur W. NienhuisAbstract:We have developed a producer cell line that generates lentiviral vector particles of high titer. The vector encodes the Wiskott-Aldrich syndrome (WAS) protein. An Insulator Element has been added to the long terminal repeats of the integrated vector to limit proto-oncogene activation. The vector provides high-level, stable expression of WAS protein in transduced murine and human hematopoietic cells. We have also developed a monoclonal antibody specific for intracellular WAS protein. This antibody has been used to monitor expression in blood and bone marrow cells after transfer into lineage negative bone marrow cells from WAS mice and in a WAS negative human B-cell line. Persistent expression of the transgene has been observed in transduced murine cells 12–20 weeks following transplantation. The producer cell line and the specific monoclonal antibody will facilitate the development of a clinical protocol for gene transfer into WAS protein deficient stem cells.
-
suppression of clonal dominance in cultured human lymphoid cells by addition of the chs4 Insulator to a lentiviral vector
Molecular Therapy, 2006Co-Authors: Marguerite V Evansgalea, Hideki Hanawa, Matthew M Wielgosz, Deo Kumar Srivastava, Arthur W. NienhuisAbstract:Lentiviral vectors efficiently transduce quiescent stem cells and are being evaluated for gene therapy of blood dis-orders. The risk of genotoxicity as a result of insertional mutagenesis is an important safety consideration. The hy-persensitive site 4 Insulator from the chicken β -globin locus (cHS4) possesses chromatin bar-rier and enhancer-blocking functions. A control lentiviral vector encoding green fluorescent protein was compared with a vector in which the cHS4 Insulator Element flanked the green fluorescent protein expression cassette in single cell isolates of transduced human T cells (Jurkat) after 9 days in culture. The Insulator had minimal effect on mean fluorescent intensity and only modestly reduced the variability of green fluorescent protein expression among indi-vidual single cell isolates. Most unique integration sites were within genes, but the Insulator-containing vector had a moderate predilection to integrate near the transcriptional start site compared with the control vector. Clonal domi-nance developed in cultures of cells containing the integrated vector genomes, as reflected by the recovery of mul-tiple single cell isolates containing the same integration site. We infer that certain integrations conferred a prolifera-tive or survival advantage by affecting gene expression through insertional mutagenesis, leading to this clonal dominance. This effect was diminished by including the Insulator Element in the vector genome.
Paritha I Arumugam - One of the best experts on this subject based on the ideXlab platform.
-
improved human β globin expression from self inactivating lentiviral vectors carrying the chicken hypersensitive site 4 chs4 Insulator Element
Molecular Therapy, 2007Co-Authors: Paritha I Arumugam, Jessica Scholes, Natalya Perelman, Ping Xia, Jiingkuan Yee, Punam MalikAbstract:Effective gene therapy for β-thalassemia major (β-TM) requires consistent, high expression of human β-globin (hβ-globin) in red blood cells (RBCs). Several groups have now shown that lentiviral (LV) vectors stably transmit the hβ/hγ-globin genes and large Elements of the locus control region, resulting in correction of the murine thalassemia intermedia (TI) phenotype and survival of mice with the TM phenotype. However, current LVs show variable hβ/hγ-globin expression and require a high number of vector copies/cell for a therapeutic effect. To address this, we designed LVs flanked by the chicken hypersensitive site-4 (cHS4) chromatin Insulator Element and compared them with their “un-insulated” counterparts. We observed a consistent twofold-higher hβ expression from insulated vectors in single-copy mouse erythroleukemia cell clones, an increase that resulted from reduced position effect variegation (PEV) and increased probability of expression from individual integrants. This effect was confirmed in vivo: an approximately twofold increase in hβ expression was seen in the RBC progeny of murine hematopoietic stem cells, with significantly higher numbers of hβ-expressing cells in individual secondary spleen colony-forming units. In summary, cHS4-insulated hβ-globin LVs showed distinct chromatin barrier activity, resulting in higher, consistent hβ expression. These studies have important implications for vector design for clinical trials for gene therapy for hemoglobinopathies.
-
67 the chicken hypersensitive site chs4 Insulator Element reduces position effects thereby increasing expression from globin lentiviral vectors
Molecular Therapy, 2006Co-Authors: Paritha I Arumugam, Jessica Scholes, Natalya Perelman, Ping Xia, Jiingkuan Yee, Alexander Zarzuela, Punam MalikAbstract:Lentiviral vectors (LV) carrying the human |[beta]|-globin gene (h|[beta]|) and locus control region (LCR) have changed the field of gene therapy for hemoglobinopathies. However, their random integration into host cells results in variable h|[beta]| expression from chromatin position effects. We analyzed the role of a 1.2 kb chicken |[beta]|-globin locus hypersensitive site 4 Insulator Element (cHS4) in a self-inactivating (SIN) LV. The BGM vector (carrying h|[beta]|/LCR) was compared to an analogous vector BGMI, with cHS4 such that it flanks the provirus upon integration. Both vectors additionally carried the mutant P140K methyl guanine methyl transferase (MGMTP140K) cDNA to enrich for genetically modified cells. First, murine erythroleukemia (MEL) cells were transduced at <5% transduction efficiency to generate single-copy clones and transduced clones identified by PCR ('Unselected' clones) and analyzed for h|[beta]| expression by FACS and RNase Protection Assay (RPA). 'Unselected' BGMI+-MEL clones had a higher proportion of h|[beta]|+ cells (68|[plusmn]|3%) compared to BGM+ clones (36|[plusmn]|9%, n=24; P<0.001) and a reduced coefficient of variation (CV) of h|[beta]| expression: 168|[plusmn]|20 vs. 327|[plusmn]|64 in BGM (P<0.02). RPA showed a 2-fold increase in h|[beta]|/total m|[alpha]|-globin mRNA: BGMI 45|[plusmn]|6% vs. BGM 21|[plusmn]|7%, n=24, P<0.01. Next, MEL cells were selected with BG/BCNU and single-copy clones isolated ('Selected' clones). 'Selected' BGMI+-MEL clones also had a higher proportion of h|[beta]|+ cells compared to BGM+ clones (80|[plusmn]|15% vs. 72|[plusmn]|20%, n=86, P<0.03), with reduced CV (140|[plusmn]|2.8 vs. 170|[plusmn]|13, P<0.01) and higher h|[beta]|-mRNA (BGMI+ 83.6%|[plusmn]|40 vs. 49.6%|[plusmn]|25, n=24, P<0.02). Two additional vectors termed BG and BGI (with cHS4) were constructed with the h|[beta]|/LCR but no MGMTP140K cassette. RPA showed that BGI+ MEL pools expressed 85% h|[beta]|/total m|[alpha]|-globin/ vector copy as compared to 38% from BG. BG and BGI single copy MEL clones showed similar results. In in vivo studies lethally irradiated normal mice were transplanted with BGM and BGMI- transduced thalassemia hematopoietic stem cells (MOI 20). Engraftment and vector copy number in both groups were similar (66%|[plusmn]|15% vs. 68%|[plusmn]|10%, and 0.21 vs. 0.17 in BGMI and BGM groups, respectively, n=12). While, 18|[plusmn]|4% of RBC expressed h|[beta]| in the BGMI group, only 4%|[plusmn]|1.5% expressed h|[beta]| in the BGM group (P<0.001). There was a 4-fold increase in chimeric hemoglobin (m|[alpha]|- h|[beta]|) in BGMI mice (13%|[plusmn]|4% vs. 3%|[plusmn]|2% in BGM mice, P<0.001). Secondary colony forming units-spleen (CFU-S) derived from these mice showed increased numbers of h|[beta]|+ cells in BGMI (21%|[plusmn]|4% vs. 8%|[plusmn]|4% BGM, n=30, P<0.03) with reduced CV (698|[plusmn]|91 vs. 987|[plusmn]|99 in BGM, P<0.04). Taken together, 'insulated' SIN-h|[beta]|/LCR LV increased the probability of expression of integrants and reduced chromatin position effects, resulting in consistent and higher expression of h|[beta]|. The enhancer blocking effect of the cHS4, although not tested here, would further improve the bio-safety of these lineage- specific, SIN LV.