Locus Control Region Elements Research Articles

Globin vector regulatory elements are active in early hematopoietic progenitor cells

The globin genes are archetypal tissue-specific genes that are silent in most tissues but for late-stage erythroblasts upon terminal erythroid differentiation. The transcriptional activation of the β-globin gene is under the control of proximal and distal regulatory elements located on chromosome 11p15.4, including the β-globin locus control region (LCR). The incorporation of selected LCR elements in lentiviral vectors encoding β and β-like globin genes has enabled successful genetic treatment of the β-thalassemias and sickle cell disease. However, recent occurrences of benign clonal expansions in thalassemic patients and myelodysplastic syndrome in patients with sickle cell disease call attention to the non-erythroid functions of these powerful vectors. Here we demonstrate that lentivirally encoded LCR elements, in particular HS1 and HS2, can be activated in early hematopoietic cells including hematopoietic stem cells and myeloid progenitors. This activity is position-dependent and results in the transcriptional activation of a nearby reporter gene in these progenitor cell populations. We further show that flanking a globin vector with an insulator can effectively restrain this non-erythroid activity without impairing therapeutic globin expression. Globin lentiviral vectors harboring powerful LCR HS elements may thus expose to the risk of trans-activating cancer-related genes, which can be mitigated by a suitable insulator.

Erythroid Specificity and Safety of Globin-Encoding Lentiviral Vectors

The beta-thalassemias and sickle cell disease are the most common monogenic inherited blood disorders, both arising from mutations affecting the beta-globin locus. Recent clinical trials utilizing lentiviral-mediated beta-globin gene transfer in autologous CD34+ cells have shown encouraging results in patients with non-beta-zero thalassemias and sickle cell anemia. A case report of insertional mutagenesis in a beta-thalassemia patient resulted in a prolonged clonal expansion that eventually regressed without progressing to leukemic transformation. Nonetheless, this occurrence and the need to insert multiple vector copies per cell when using globin vectors that provide insufficient globin expression to achieve curative responses from a single integrated copy per cell, raise some questions about the erythroid specificity and safety of globin vectors. We thus found it imperative to investigate globin vector expression in hematopoietic progenitors and in non-erythroid cells. To this end, we investigated what regulatory elements, including locus control region (LCR) hypersensitive sites (HS) and others, achieve the highest beta-globin expression per vector copy while at the same time minimizing non-erythroid transcriptional activity. We developed an invivo assay to track the enhancer/promoter activity of different HS elements in hematopoietic progenitor and differentiated cell subsets. We designed lentiviral vectors expressing hrGFP under the control of a short β-globin promoter (137bp) controlled by a set of LCR HS elements and/or a novel biliverdin (BLV) enhancer. Analysis of hrGFP expression in bone marrow cells derived from C57BL6 mice transplanted with transduced syngeneic bone marrow cells revealed that many of the vectors encompassing elements thought to encode erythroid-specific elements were in fact expressed in long term-HSC (LT-HSC), short term-HSC (ST-HSC), multipotent progenitors (MPP), pre-GM, Granulocyte/macrophage progenitors (GMP) and megakaryocyte progenitors (MKP). These findings confirmed that ectopically integrated erythroid regulatory elements can serve as transcriptional enhancers in non-erythroid cells. This transcriptional leakiness was confirmed in therapeutic globin vectors harboring a combination of LCR elements. To avoid these effects, we flanked the globin transcription unit with a small human enhancer-blocking element called A1, kindly provided by the late George Stamatoyannopoulos (Liu, M. et al. 2015). The insulated vector showed markedly reduced non-erythroid expression in non-erythroid cells including ST-HSC, MPP, Pre-GM and GMP. Expression in mature erythroid cells was unchanged. Our findings underscore the benefits of selecting lineage-specific regulatory elements with the least lineage promiscuity for therapeutic vectors, with the eventual addition of enhancer-blocking elements. Based on these results, we are now investigating new globin expression vectors with optimal combination of HS elements. Such novel globin vectors should minimize the risk of oncogene trans-activation in hematopoietic progenitor cells and therefore improve the safety of globin gene therapy while providing lineage-specific high-level expression. Disclosures Sadelain: Mnemo: Patents & Royalties; Fate Therapeutics: Patents & Royalties, Research Funding; Atara: Patents & Royalties, Research Funding; Takeda: Patents & Royalties, Research Funding; Minerva: Other: Biotechnologies , Patents & Royalties.

Improved Titer and Gene Transfer by Lentiviral Vectors Using Novel, Small β-Globin Locus Control Region Elements.

β-globin lentiviral vectors (β-LV) have faced challenges in clinical translation for gene therapy of sickle cell disease (SCD) due to low titer and sub-optimal gene transfer to hematopoietic stem and progenitor cells (HSPCs). To overcome the challenge of preserving efficacious expression while increasing vector performance, we used published genomic and epigenomic data available through ENCODE to redefine enhancer element boundaries of the β-globin locus control region (LCR) to construct novel ENCODE core sequences. These novel LCR elements were used to design a β-LV of reduced proviral length, termed CoreGA-AS3-FB, produced at higher titers and possessing superior gene transfer to HSPCs when compared to the full-length parental β-LV at equal MOI. At low vector copy number, vectors containing the ENCODE core sequences were capable of reversing the sickle phenotype in a mouse model of SCD. These studies provide a β-LV that will be beneficial for gene therapy of SCD by significantly reducing the cost of vector production and extending the vector supply.

Sickle Cell Anemia in a Child with Three β-Globin Clusters (β-S/β-S, β-A): Loss of LCR and 3' HS1 in the Duplicated Wild-Type β-Globin Cluster Does Not Fully Abrogate Its Transcript

Transcriptional regulation of β-globin cluster genes follows a complex, highly conserved system of gene expression with developmental and tissue-specific control. The DNase I hypersensitivity (HS) sites in the upstream locus control region (LCR) and 3' HS1 element are thought to interact with β-globin cluster genes involving long range DNA interactions mediated by various transcription factors to drive the regulation of β-like globin gene expression. The majority of studies have focused on the role of the LCR on active transcription and globin gene switching. Various in vitro and in vivo studies have shown that the LCR interacts with one gene at a time and that absence of the LCR results in a dramatic decrease (10 -100 fold) in globin transcripts (Kiefer et. al., Mutat. Res. 2008, 647:68, Noordermeer et. al., IUBMB Life 2008, 60: 824). However, the role of the 3'HS1 downstream and other distal cis -regulatory elements are not entirely understood, with recent studies in mouse and cell-culture models suggesting they play a role in the insulation of globin genes from silencing chromatin (Bender et. al., Blood 2008, 106: 1395). Our knowledge of the β-globin LCR and 3'HS1 function is still incomplete and much can still be learned from human mutations affecting these regulatory elements.We report a unique head-to-tail duplication of the β-globin cluster in a patient phenotypically expressing homozygous HbS (sickle-cell anemia, SCA) that provides insight into the regulatory role of the β-LCR and 3'HS1 on wild-type β-globin (β-A) expression in a background of SCA. The studies were driven by an apparent discrepancy between hemoglobin analysis of an infant with a SCA phenotype and no detectable HBA, and Sanger sequencing of the β-globin genes which showed a heterozygous genotype.Analyses of parents' blood samples and DNA revealed that each were carriers for sickle cell allele. Hemoglobin analysis showed the father expressed HbS fraction at 41.3% and the mother at 33.3%, while the proband had a majority of HbS, some HbF and no detectable HbA. The reduced HbS fraction in the mother could be explained by co-inheritance of α-thalassemia (αα/α-3.7). The proband did not inherit α-thalassemia from the mother.Multiplex ligation-dependent probe amplification analysis of the proband's DNA suggested duplication of the β-globin cluster, resulting in three copies of the HBB gene in the genome. Subsequent next-generation sequencing confirmed that the duplication occurred immediately adjacent to the first iteration of sequence, in head-to-tail orientation and resulted in an intact β-S cluster having both LCR and HS1 elements, followed by the duplicated β-A cluster (β-S, β-A) that excluded a part of HBE (epsilon globin) and the upstream β-LCR regions, extending through to LINE L1LBP1 ([hg19] chr11:4640332-5290168). Further analyses revealed that the duplicated β-A cluster, which encompassed approximately 650 kb sequence, lacked a DNA segment containing the 3'HS1 element (figure 1). The proband's β genotype is thus (β-S/β-S, β-A). DNA analysis showed that the father carried the duplicated β-globin cluster with genotype (β-A/β-S, β-A), and the mother, a heterozygous HbS genotype (β-A/β-S).Reverse transcription, quantitative polymerase chain reaction (RT-qPCR) was used to assess transcription levels of β-A and β-S mRNA for each family member. Analysis of the parents' reticulocyte RNA showed that the β-globin (β-A and β-S) transcript levels were nearly balanced. RT-qPCR of proband's reticulocyte RNA showed no convincing detection of β-A transcript, but the β-A transcript was clearly detected by digitalPCR, albeit at a very low level (0.4% of total HBB transcript) (figure 2).The β-globin cluster duplication on one chromosome in a background of a phenotypically homozygous (HbSS) SCA patient has provided a unique opportunity to assess the effect of the LCR and 3'HS1 regions on the transcription of a β-A gene in an unbiased environment. Prior studies in transformed cell-lines or mouse models have shown the down regulatory effect of LCR loss and potential protective effect of the 3'HS1 element. Within the human model, the observed transcription from the duplicated, distally displaced (~650 kb) β-A cluster demonstrates that the loss of LCR and flanking HS sites does not lead to complete silencing of β-globin transcription.VD was supported by project IGA MzCR NT13587. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Dissecting the function of the adult β-globin downstream promoter region using an artificial zinc finger DNA-binding domain

Developmental stage-specific expression of the β-type globin genes is regulated by many cis- and trans-acting components. The adult β-globin gene contains an E-box located 60 bp downstream of the transcription start site that has been shown to bind transcription factor upstream stimulatory factor (USF) and to contribute to efficient in vitro transcription. We expressed an artificial zinc finger DNA-binding domain (ZF-DBD) targeting this site (+60 ZF-DBD) in murine erythroleukemia cells. Expression of the +60 ZF-DBD reduced the recruitment and elongation of RNA polymerase II (Pol II) at the adult β-globin gene and at the same time increased the binding of Pol II at locus control region (LCR) element HS2, suggesting that Pol II is transferred from the LCR to the globin gene promoters. Expression of the +60 ZF-DBD also reduced the frequency of interactions between the LCR and the adult β-globin promoter. ChIP-exonuclease-sequencing revealed that the +60ZF-DBD was targeted to the adult β-globin downstream promoter and that the binding of the ZF-DBD caused alterations in the association of USF2 containing protein complexes. The data demonstrate that targeting a ZF-DBD to the adult β-globin downstream promoter region interferes with the LCR-mediated recruitment and activity of Pol II.

Neutralizing the function of a β-globin–associated cis -regulatory DNA element using an artificial zinc finger DNA-binding domain

Gene expression is primarily regulated by cis-regulatory DNA elements and trans-interacting proteins. Transcription factors bind in a DNA sequence-specific manner and recruit activities that modulate the association and activity of transcription complexes at specific genes. Often, transcription factors belong to families of related proteins that interact with similar DNA sequences. Furthermore, genes are regulated by multiple, sometimes redundant, cis-regulatory elements. Thus, the analysis of the role of a specific DNA regulatory sequence and the interacting proteins in the context of intact cells is challenging. In this study, we designed and functionally characterized an artificial DNA-binding domain that neutralizes the function of a cis-regulatory DNA element associated with adult β-globin gene expression. The zinc finger DNA-binding domain (ZF-DBD), comprising six ZFs, interacted specifically with a CACCC site located 90 bp upstream of the transcription start site (-90 β-ZF-DBD), which is normally occupied by KLF1, a major regulator of adult β-globin gene expression. Stable expression of the -90 β-ZF-DBD in mouse erythroleukemia cells reduced the binding of KLF1 with the β-globin gene, but not with locus control region element HS2, and led to reduced transcription. Transient transgenic embryos expressing the -90 β-ZF-DBD developed normally but revealed reduced expression of the adult β-globin gene. These results demonstrate that artificial DNA-binding proteins lacking effector domains are useful tools for studying and modulating the function of cis-regulatory DNA elements.

Therapeutic Transgene Expression From Genomic Safe Harbors In Patient-Specific Induced Pluripotent Stem Cells

AbstractAbstract 564Insertional oncogenesis poses a severe hurdle to current gene therapy for blood disorders. The semi-random insertion of retroviral vectors combined with the inability for prolonged ex vivo culture of hematopoietic stem cells (HSCs) prohibits prospective integration site selection. The advent of induced pluripotent stem cells (iPSCs) offers for the first time the possibility of generating patient-specific stem cells that can be extensively manipulated in vitro, thus creating a unique platform for precise genetic engineering. Here, we present a novel strategy for the genetic correction of ß-thalassemia major based on the identification and selection of patient-specific iPSC clones harboring a normal ß-globin gene integrated in genomic “safe harbor” sites that permit therapeutic levels of expression while minimizing the possibility of oncogenic risks. We generated a total of 20 iPSC lines from bone marrow stromal cells or skin fibroblasts from 4 patients with ß-thalassemia major of various genotypes using our traceable lentiviral vector system (Papapetrou et al., PNAS, 2009; Lee et al., Nature, 2009), as well as an excisable single polycistronic vector co-expressing OCT4, SOX2, KLF4 and cMYC. Additional transgene-free thal-iPSC lines were generated following Cre recombinase-mediated excision of the reprogramming vector. Seven thal-iPSC lines were selected for further characterization and were shown to fulfill multiple criteria of pluripotency, including teratoma formation. Four thal-iPSC lines were transduced with a lentiviral vector encoding the human ß-globin gene cis-linked to its hypersensitive site (HS) 2, HS3 and HS4 locus control region elements, derived from the TNS9 vector, previously shown to confer erythroid-specific ß-globin gene expression at therapeutic levels (May et al., Nature, 2000). Thal-iPSC clones harboring single vector copies were selected and vector integration sites were mapped to the human genome. To identify safe harbor sites we adopted a set of 5 criteria: (1) distance of at least 50 kb from the 5‘ end of any gene, (2) distance of at least 300 kb from any cancer-related gene, (3) distance of at least 300 kb from any miRNA, (4) location outside a transcription unit and (5) location outside ultraconserved regions. A survey of 5840 integration sites of the globin lentiviral vector that we mapped in thal-iPSCs revealed that 17.3% meet all five “safe harbor” criteria, supporting the feasibility of recovering thal-iPSC clones harboring vector integrations in “safe harbors” by screening a relatively small set of single-copy clones. Indeed, 3 “safe harbor” integrations were retrieved amongst 36 sites mapped in thal-iPSC clones. Among 13 clones randomly selected and thoroughly confirmed to harbor a single copy of the globin vector, we found one clone, thal5.10-2, with an integration that meets all five “safe harbor” criteria. Upon erythroid differentiation, 12 of the 13 single copy thal-iPSC clones expressed detectable vector-encoded ß-globin at levels ranging from 9% to 159% (mean 53%) of a normal endogenous ß-globin allele. 9 out of 13 clones expressed the ß-globin transgene at levels higher than 30%. Remarkably, the “safe harbor” clone 5.10-2, expressed 85% of a normal ß-globin allele. Microarray analysis of undifferentiated thal-iPSC clones and their erythroid progeny revealed that 3 out of 5 integrations eliminated by our “safe harbor” criteria result in perturbed expression of neighboring genes at a distance ranging between 9 and 275 kb from the vector insertion. Of note, the “safe harbor’ integration site in clone 5.10-2 is in a genomic region with no genes within 300 kb on either side, while no significant differentially expressed genes were found elsewhere in the genome. These data demonstrate that the selection of iPSC clones, wherein therapeutic levels of transgene expression without perturbation of endogenous genes is obtained from selected chromosomal sites is feasible by screening a limited number of single-copy clones and applying five “safe harbor” criteria. This study provides a framework and a strategy combining bioinformatics and functional analyses for identifying “safe harbors” for transgene integration and expression in the human genome. This approach may be broadly applicable to introducing therapeutic, suicide or marker genes into patient-specific iPSCs towards the development of safer stem cell therapies. Disclosures:No relevant conflicts of interest to declare.

USF and NF-E2 Cooperate to Regulate the Recruitment and Activity of RNA Polymerase II in the β-Globin Gene Locus

The human beta-globin gene is expressed at high levels in erythroid cells and regulated by proximal and distal cis-acting DNA elements, including promoter, enhancer, and a locus control region (LCR). Transcription complexes are recruited not only to the globin gene promoters but also to the LCR. Previous studies have implicated the ubiquitously expressed transcription factor USF and the tissue-restricted activator NF-E2 in the recruitment of transcription complexes to the beta-globin gene locus. Here we demonstrate that although USF is required for the efficient association of RNA polymerase II (Pol II) with immobilized LCR templates, USF and NF-E2 together regulate the association of Pol II with the adult beta-globin gene promoter. Recruitment of Pol II to the LCR occurs in undifferentiated murine erythroleukemia cells, but phosphorylation of LCR-associated Pol II at serine 5 of the C-terminal domain is mediated by erythroid differentiation and requires the activity of NF-E2. Furthermore, we provide evidence showing that USF interacts with NF-E2 in erythroid cells. The data provide mechanistic insight into how ubiquitous and tissue-restricted transcription factors cooperate to regulate the recruitment and activity of transcription complexes in a tissue-specific chromatin domain.

A Modified ANK-1 Promoter Directs Uniform, Copy Number Dependent Gamma Globin Gene Expression at Therapeutic Levels of a in a Lentivirus Vector.

Abstract 3565Poster Board III-502Successful gene therapy for hemoglobin disorders beta-thalassemia and sickle cell disease (SCD) has been hindered by inefficient expression of beta-like globin genes from viral vectors. It is estimated that transduction of ∼25% of hematopoietic stem cells (HSC) to express a beta-like globin gene at ∼20% the level of endogenous alpha-globin would be therapeutic. The expression of the beta-like globin genes in their native locus is a precisely regulated process that relies on the coordinated activities of several cis-acting enhancer elements that make up the beta-globin locus control region (LCR). Expression of therapeutic levels of beta-like globin in mouse models and patient cells requires inclusion of LCR elements. These LCR elements are susceptible to silencing, reduce virus titer and can potentially activate oncogenes at the site of proviral insertion. To minimize these risks we have developed a novel approach using non-globin erythroid promoters to drive high levels of globin gene expression independent of enhancers. Previously, we showed that a minimal 276-bp promoter of the human ankyrin gene (ANK-1) does not contain an enhancer element, but does contain a barrier element that allows erythroid-specific, uniform, position independent and copy-number dependent expression of a linked gamma-globin gene. However, the level of gamma-globin expression from the ANK-1 promoter (4% of mouse alpha-globin per copy) was sub-therapeutic. TFIID, a component of the transcription initiation complex, binds a 9-bp sequence (TGCGGTGAG) within the wild type ANK-1 promoter. A dinucleotide deletion within this sequence that disrupts the binding of TFIID and results in ankyrin deficient Hereditary Spherocytosis (Gallagher et al. Hum Mol Genet.14: 2501-9. 2005). We hypothesized that sequence modifications of the ANK1 promoter in this region would increase the affinity for TFIID and increase expression. Mutational analysis of the wildtype TFIID site identified a number of sequence variants that increase expression. An ANK-1 promoter sequence variant that differs from the wildtype TFIID sequence in the first three nucleotides (GCGGGTGAG; GC-ANK-1), results in a 7-fold increase in transcriptional activity in K562 cells. We developed five independent transgenic mouse lines that carry this GC-ANK-1 promoter linked to a human gamma globin gene. Expression in all five lines is erythroid-specific, uniform, position independent and copy-number dependent. Moreover, the average level of gamma-globin mRNA is 34 ± 14 % of mouse alpha-globin mRNA per transgene copy, a 8.5-fold increase in gamma-globin expression over mice harboring the wildtype ANK-1 promoter. We developed a simplified lentiviral vector that contains the CG-ANK-1/gamma-globin gene. This vector was produced at high titer and transduced 57% (23/40) of mouse colony forming unit Spleen (CFU-S) cells. In CFU-S containing single copies of the provirus, the level of gamma-globin mRNA was 6.8 ± 2.9 % of mouse alpha-globin per copy. In CFU-S with two and three copies of the GC-ANK-1 gamma-globin provirus, expression of gamma-globin mRNA averaged 20% ± 1.6% and 30% of mouse alpha-globin per copy respectively, demonstrating copy-number dependent of expression. Overall, an analysis of 16 CFU-S foci demonstrated that the CG-ANK-1/gamma-globin vector expressed gamma globin mRNA at levels that average 8.5 ± 2.0% of mouse alpha-globin. Our data demonstrate that just two copies of the simplified CG-ANK-1/gamma-globin lentiviral vector could potentially produce the levels of gamma-globin necessary for effective treatment of the hemoglobin disorders. Disclosures:No relevant conflicts of interest to declare.

Defective Erythropoiesis in Transgenic Mice Expressing Dominant-Negative Upstream Stimulatory Factor

Transcription factor USF is a ubiquitously expressed member of the helix-loop-helix family of proteins. It binds with high affinity to E-box elements and, through interaction with coactivators, aids in the formation of transcription complexes. Previous work demonstrated that USF regulates genes during erythroid differentiation, including HoxB4 and beta-globin. Here, we show that the erythroid cell-specific expression of a dominant-negative mutant of USF, A-USF, in transgenic mice reduces the expression of all beta-type globin genes and leads to the diminished association of RNA polymerase II with locus control region element HS2 and with the beta-globin gene promoter. We further show that the expression of A-USF reduces the expression of several key erythroid cell-specific transcription factors, including EKLF and Tal-1. We provide evidence demonstrating that USF interacts with known regulatory DNA elements in the EKLF and Tal-1 gene loci in erythroid cells. Furthermore, A-USF-expressing transgenic mice exhibit a defect in the formation of CD71(+) progenitor and Ter-119(+) mature erythroid cells. In summary, the data demonstrate that USF regulates globin gene expression indirectly by enhancing the expression of erythroid transcription factors and directly by mediating the recruitment of transcription complexes to the globin gene locus.

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

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

Expression of green fluorescent protein under the regulation of human locus control region elements HS2 and HS3 in transgenic mice

Human beta-globin locus control region (LCR) is composed of five DNase I hypersensitive sites (HSs). We previously demonstrated that when HS2 and HS3 were constructed together in one vector and integrated into one position in transgenic mice, the two cis-elements showed a marked synergy in regulating the spatial and temporal expression of beta-globin transgene. This study is to investigate whether these two elements still show a synergy or superposition when they are not located in one construct. HS2/GFP and HS3/GFP transgenic mice, with the transgene encoding green fluorescent protein (GFP) driven by beta-globin promoter and under the control of HS2 (HS2/GFP mice) and HS3 (HS3/GFP mice), were mated to generate double-heterozygotes (HS2/GFP + HS3/GFP mice). The expression and integration status of GFP genes in transgenic mice were analyzed. Our data showed that there was no difference in the percentages of GFP positive cells in peripheral blood between double-heterozygotes and their corresponding founders (HS2/GFP or HS3/GFP). Interestingly, it was observed that exogenous GFP genes were highly expressed in another transgenic mouse pedigree with HS2 element integrated twice into the same chromosome at two different loci. However, when one site was lost in some offspring because of recombination, the percentages of GFP positive cells in peripheral blood as well as in some erythroid tissues were decreased obviously. These results demonstrated that HS2 and HS3 elements would not show any synergy or superposition if they were located on different chromosomes in transgenic mice, and that HS2 element containing the core and flanking sequences might not be able to overcome the position effects and control position dependent expression of linked genes in transgenic mice.

β-Globin LCR and Intron Elements Cooperate and Direct Spatial Reorganization for Gene Therapy

The Locus Control Region (LCR) requires intronic elements within β-globin transgenes to direct high level expression at all ectopic integration sites. However, these essential intronic elements cannot be transmitted through retrovirus vectors and their deletion may compromise the therapeutic potential for gene therapy. Here, we systematically regenerate functional β-globin intron 2 elements that rescue LCR activity directed by 5′HS3. Evaluation in transgenic mice demonstrates that an Oct-1 binding site and an enhancer in the intron cooperate to increase expression levels from LCR globin transgenes. Replacement of the intronic AT-rich region with the Igμ 3′MAR rescues LCR activity in single copy transgenic mice. Importantly, a combination of the Oct-1 site, Igμ 3′MAR and intronic enhancer in the BGT158 cassette directs more consistent levels of expression in transgenic mice. By introducing intron-modified transgenes into the same genomic integration site in erythroid cells, we show that BGT158 has the greatest transcriptional induction. 3D DNA FISH establishes that induction stimulates this small 5′HS3 containing transgene and the endogenous locus to spatially reorganize towards more central locations in erythroid nuclei. Electron Spectroscopic Imaging (ESI) of chromatin fibers demonstrates that ultrastructural heterochromatin is primarily perinuclear and does not reorganize. Finally, we transmit intron-modified globin transgenes through insulated self-inactivating (SIN) lentivirus vectors into erythroid cells. We show efficient transfer and robust mRNA and protein expression by the BGT158 vector, and virus titer improvements mediated by the modified intron 2 in the presence of an LCR cassette composed of 5′HS2-4. Our results have important implications for the mechanism of LCR activity at ectopic integration sites. The modified transgenes are the first to transfer intronic elements that potentiate LCR activity and are designed to facilitate correction of hemoglobinopathies using single copy vectors.

Locus control region elements HS1 and HS4 enhance the therapeutic efficacy of globin gene transfer in β-thalassemic mice

Globin gene transfer in autologous hematopoietic stem cells is a promising therapeutic option for subjects with beta-thalassemia major. In this approach, high level, erythroid-specific globin transgene expression should correct ineffective erythropoiesis and hemolytic anemia following the delivery of only 1 to 2 vector copies per cell. The generation of vectors that provide high-level globin expression and require low vector copy (VC) integration is therefore essential for both safety and efficacy. We show here the major roles played by 2 lesser-known locus control region elements, termed HS1 and HS4. Partial deletions within HS4 markedly reduce in vivo globin expression requiring multiple VC per cell to correct the anemia. Most strikingly, addition of HS1 to HS2-3-4 increases globin expression by 52%, yielding 9 g Hb/VC in beta-thalassemic mice. Thus, while vectors encoding HS2-3-4 provide curative levels of hemoglobin at 1 to 2 copies per cell, adding HS1 is a promising alternative strategy if upcoming clinical trials prove higher levels of expression to be necessary.

Improved Human β-globin Expression from Self-inactivating Lentiviral Vectors Carrying the Chicken Hypersensitive Site-4 (cHS4) Insulator Element

Effective gene therapy for beta-thalassemia major (beta-TM) requires consistent, high expression of human beta-globin (hbeta-globin) in red blood cells (RBCs). Several groups have now shown that lentiviral (LV) vectors stably transmit the hbeta/hgamma-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 hbeta/hgamma-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 hbeta 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 hbeta expression was seen in the RBC progeny of murine hematopoietic stem cells, with significantly higher numbers of hbeta-expressing cells in individual secondary spleen colony-forming units. In summary, cHS4-insulated hbeta-globin LVs showed distinct chromatin barrier activity, resulting in higher, consistent hbeta expression. These studies have important implications for vector design for clinical trials for gene therapy for hemoglobinopathies.

Locus Control Region Transcription Plays an Active Role in Long-Range Gene Activation

Activation of eukaryotic genes often relies on remote chromatin determinants. How these determinants function remains poorly understood. The hGH gene is activated by a 5'-remote locus control region (LCR). Pituitary-specific DNase I hypersensitive site I (HSI), the dominant hGH LCR element, is separated from the hGH-N promoter by a 14.5 kb span that encompasses the B-lymphocyte-specific CD79b gene. Here, we describe a domain of noncoding Pol II transcription in pituitary somatotropes that includes the hGH LCR and adjacent CD79b locus. This entire "LCR domain of transcription" is HSI [corrected] dependent and terminates 3' to CD79b, leaving a gap in transcription between this domain and the target hGH-N promoter. Insertion of a Pol II terminator within the LCR blocks CD79b transcription and represses hGH-N expression. These data document an essential role for LCR transcription in long-range control, link "bystander"CD79b transcription to this process, and support a unique model for locus activation.

The Binding of GATA-1 to a β-Globin LCR HS Core Element in Nuclear Chromatin Requires Multiple Associated Factor Binding Sites.

The GATA-1 transcription factor has been shown to interact in vitro with many gene regulatory elements including promoters, enhancers, silencers and locus control region elements. The GATA-1 consensus binding sequence is generally considered to be WGATAR, yet it can also bind to other related sequences that do not perfectly fit this consensus. This raises the question of whether all potential GATA-1 binding sites identified by in vitro DNA binding assays or sequence analysis are actually bound by GATA-1 in nuclear chromatin. We hypothesized that it was unlikely that all, or even most, predicted GATA-1 sites were bound in vivo. If correct, this would imply that additional mechanisms, beyond DNA sequence, are required to target GATA-1 to specific genetic regulatory elements. To address this hypothesis we first performed GATA-1 ChIP assays in MEL cells at 10 WGATAR sites which spanned the mouse β-globin locus but were not associated with known regulatory elements. Positive controls for GATA-1 binding were the core region of LCR HS2 and the -2.8 mGATA-1 gene upstream enhancer. Negative controls included two regions from the β-globin locus which do not contain WGATAR elements and a WGATAR element from the proximal mGATA-1 gene promoter previously shown by the Bresnick lab to not bind GATA-1. While enrichment of the positive control sites using anti-GATA-1 antibody was 10-20-fold more than with IgG (p<0.001), binding at none of the 10 test WGATAR sites was enriched. This result implied that conditions other than the presence of the WGATAR consensus sequence are required to specify GATA-1 binding in nuclear chromatin. To better understand this process we decided to more closely evaluate the binding of GATA-1 to β-globin LCR HS core elements. We first tested the idea that GATA-1 was able to bind these sites due to high-level affinity based on the presence of tandem-inverted WGATAR elements and the known ability of GATA-1 molecules to self-interact. However, when tested in gel shift assays, GATA-1 binding to the LCR HS core elements had a very rapid off-rate which was much faster than a known high-affinity control site from the murine α-globin promoter. This led us to next test the idea that surrounding factor binding sites were required for GATA-1 binding to the LCR HS cores. Using human LCR HS4 as a model, we systematically assembled combinations of binding sites (3 KLF sites and 1 NF-E2 site) known to flank the tandem-inverted WGATAR elements and tested these artificial HS cores for GATA-1 binding after integration into MEL cell chromatin. The wild-type HS4 core served as a positive control and a fragment of luciferase cDNA served as a negative control for GATA-1 binding. Results for each cell line were normalized to the internal positive control -2.8 mGATA-1 gene site. Our experiments showed that only when all known flanking sites were present was GATA-1 binding detected. Similarly, DNase HS formation and histone H3 acetylation only occurred when all of the surrounding sites were present. Finally, we used ChIP to show that the assembled complex, with all sites present, includes BRG1 a component of the swi/snf nucleosome remodeling complex. These results indicate that GATA-1 targeting to the LCR HS cores involves complex, multi-protein interactions including histone acetylation and nucleosome displacement. Similar mechanisms are likely to be involved in targeting this and other transcription factors to functionally important sites in the genome.

10. The Effect of Globin Locus Control Region (LCR) Elements or the MSCV-LTR on Promoter Trapping by Integrated Lentiviral Vector Genomes

Integrating vectors are desirable for gene therapy of inherited blood disorders to achieve permanent genetic modification and stable expression of transgenes in hematopoietic cells. However, three patients with severe combined immunodeficiency enrolled in a gene therapy trial of a retroviral vector developed leukemia and activation of the LMO2 proto-oncogene by vector integration has been demonstrated in two. Retroviral vectors prefer the transcriptional start sites for integration while lentiviral vector integration sites are distributed throughout the active genes. Thus, the safety profile of integrating vectors is a critical issue. As a strategy to evaluate interaction of regulatory elements in the vectors with genes into which they have integrated, a series of lentiviral promoter trap vectors were designed to compare the effect of regulatory elements within the vector on the potential for promoter trapping. The trapping cassette consists of a splice acceptor followed by the GFP coding sequences and a polyadenylation site. The cassette was inserted immediately upstream of the 3|[prime]| LTR in a reverse orientation in vectors containing either a globin LCR element or the oncoretroviral MSCV-LTR also in a reverse orientation to the lentiviral LTR. As a control, a vector containing the cassette without regulatory elements was also constructed. HeLa or erythroid K562 cells were transduced with these vectors and analyzed for GFP expression by flow cytometry. Transduction frequency was estimated by Southern hybridization and the efficiency of promoter trapping was then calculated as a percentage of GFP expressing cells in transduced cells. Our results showed 5% promoter trapping in HeLa cells transduced with the control vector. This efficiency is similar to that recently reported by others (De Palma et al, Blood 2004 Nov 12). Vectors containing either the globin LCR or the MSCV-LTR gave 15 to 20% trapping efficiency. Similarly, in K562 cells, the percentage of promoter trapping of vectors containing either the globin or MSCV regulatory elements was significantly higher (up to 6-fold) of that of the control vector. To further investigate the effect of the globin LCR or the MSCV-LTR on promoter trapping, we isolated single cell clones from GFP-positive HeLa and K562 cells that had been transduced with a trapping vector. Many clones had lost GFP expression when reanalyzed, but the proportion (43%) in which more than 60% of the cells were positive was higher with vectors containing the globin LCR compared to those containing the MSCV-LTR (27%). In addition, the MFI of globin LCR clones was, on average, slightly higher than that of MSCV-LTR clones (20 and 17, respectively). Eighteen clones having a single integrated proviral genome were identified and 15 integration sites have been mapped to date. Ten integration sites were in genes and 8 of these were in the first intron with the promoter trap in tandem with the natural transcript. Our results to date indicate that the presence and nature of regulatory elements within a lentiviral vector influences the probability of vector genome transcription either by trapping of a natural promoter or by activation of cryptic promoters.

Gene Therapy for β-Thalassemia

Gene transfer for beta-thalassemia requires gene transfer into hematopoietic stem cells using integrating vectors that direct regulated expression of beta globin at therapeutic levels. Among integrating vectors, oncoretroviral vectors carrying the human beta-globin gene and portions of the locus control region (LCR) have suffered from problems of vector instability, low titers and variable expression. In recent studies, human immunodeficiency virus-based lentiviral (LV) vectors were shown to stably transmit the human beta-globin gene and a large LCR element, resulting in correction of beta-thalassemia intermedia in mice. Several groups have since demonstrated correction of the mouse thalassemia intermedia phenotype, with variable levels of beta-globin expression. These levels of expression were insufficient to fully correct the anemia in thalassemia major mouse model. Insertion of a chicken hypersensitive site-4 chicken insulator element (cHS4) in self-inactivating (SIN) LV vectors resulted in higher and less variable expression of human beta-globin, similar to the observations with cHS4-containing retroviral vectors carrying the human gamma-globin gene. The levels of beta-globin expression achieved from insulated SIN-LV vectors were sufficient to phenotypically correct the thalassemia phenotype from 4 patients with human thalassemia major in vitro, and this correction persisted long term for up to 4 months, in xeno-transplanted mice in vivo. In summary, LV vectors have paved the way for clinical gene therapy trials for Cooley's anemia and other beta-globin disorders. SIN-LV vectors address several safety concerns of randomly integrating viral vectors by removing viral transcriptional elements and providing lineage-restricted expression. Flanking the proviral cassette with chromatin insulator elements, which additionally have enhancer-blocking properties, may further improve SIN-LV vector safety.

Diminished Mobilization of Self-Inactivating (SIN) Lentiviral Vectors Containing Globin Regulatory Elements Compared to Those Containing a Retroviral Long Terminal Repeat.

Stem cell transfer was successful in treating severe combined immunodeficiency due to deficiencies of the common γ-chain of the lymphoid cytokine receptor (N Engl J Med 346:1185, 2002) and adenosine deaminase (Science 296:2410, 2002) but 2 of 10 children in the common γ-chain trial developed a lymphoproliferative disease secondary in part due to activation of the LMO2 proto-oncogene by the retroviral long terminal repeat (LTR) (Science 302:415, 2003). Both oncoretroviral and lentiviral vectors integrate preferentially into transcriptionally active genes so that vector design to improve safety is important. In focusing on the interactions between vector sequences and the genome surrounding integration sites, we have used self-inactivating (SIN) lentiviral vectors with transcriptionally inactive LTRs. One assay detects mobilization of the vector genome by rescue of a GFP marker while a second detects vector encoded tat transcription (Blood 102:249a, 2003). Approximately 1 in 1,000 to 1 in 3,000 vector genomes containing the Mouse Stem Cell Virus (MSCV) LTR are transcribed with transcription arising from cryptic promoters within the vector genome or from nearby genomic sequences. The frequency of genome transcription was diminished by removal of the MSCV LTR enhancer or by addition of an insulator element from the β-globin locus control region (LCR) to the lentiviral SIN LTR. We have now evaluated the effect of globin regulatory elements on transcription of integrated SIN lentiviral vector genomes. Initially, we substituted the LTR GFP cassette in vector MSCV-U3 for one in which elements from the β-globin LCR were linked to the β-globin promoter driving GFP in the reverse transcriptional orientation (d432βGFPim). Two additional vectors were also studied; the globin LCR β-promoter GFP cassette was reversed and the globin RNA processing signals removed to derive vector Fd432βGFP and then it was modified by substituting larger LCR elements to derive vector FmLARβV5GFP. Vector preparations made by co-transfection of 293T cells with vector and packaging plasmids had transducing titers from 1.8 x 107 to 3.0 x 107 TU/ml as assayed on HeLa cells. Three separate polyclonal 293T cell populations transduced 3 times at high vector concentration had copy numbers of the SIN-proviral genomes, as measured by RealTime PCR, that averaged 41 with a range of 23–68. Vector mobilization was evaluated by transfection of these populations with packaging plasmids. Conditioned media harvested from the transfected cells were then assayed for transfer of the GFP marker on HeLa cells. The mobilized titers were normalized based on vector copy number. The mobilized titer of the MSCV-U3 was 38,000 ± 3500 and that of d432βGFPim was only 810 ± 100 (p = 0.0004). The mobilized titers of the vectors in which the globin regulatory elements were in the forward orientation were also significantly lower than MSCV-U3; Fd432βGFP was 2100 ± 250 (p = 0.005) and FmLARβV5GFP was 1600 ± 120 (p = 0.0005). Those data suggest that the globin LCR elements are less likely than the retroviral LTR to induce transcription of the integrated vector genome in nonerythroid cells. Our results combined with ongoing analysis of the influence of vector integration on expression of surrounding genes in separate studies will provide a safety profile of globin lentiviral vectors to guide the development of future clinical protocols.