The 3′ Region of the Chicken Hypersensitive Site-4 Insulator Has Properties Similar to Its Core and Is Required for Full Insulator Activity

Chromatin insulators separate active transcriptional domains and block the spread of heterochromatin in the genome. Studies on the chicken hypersensitive site-4 (cHS4) element, a prototypic insulator, have identified CTCF and USF-1/2 motifs in the proximal 250 bp of cHS4, termed the “core”, which provide enhancer blocking activity and reduce position effects. However, the core alone does not insulate viral vectors effectively. The full-length cHS4 has excellent insulating properties, but its large size severely compromises vector titers. We performed a structure-function analysis of cHS4 flanking lentivirus-vectors and analyzed transgene expression in the clonal progeny of hematopoietic stem cells and epigenetic changes in cHS4 and the transgene promoter. We found that the core only reduced the clonal variegation in expression. Unique insulator activity resided in the distal 400 bp cHS4 sequences, which when combined with the core, restored full insulator activity and open chromatin marks over the transgene promoter and the insulator. These data consolidate the known insulating activity of the canonical 5′ core with a novel 3′ 400 bp element with properties similar to the core. Together, they have excellent insulating properties and viral titers. Our data have important implications in understanding the molecular basis of insulator function and design of gene therapy vectors

Genetic Therapy for Beta-Thalassemia: From the Bench to the Bedside

AbstractBeta-thalassemia is a genetic disorder with mutations in the β-globin gene that reduce or abolish β-globin protein production. Patients with β-thalassemia major (Cooley's anemia) become severely anemic by 6 to 18 months of age, and are transfusion dependent for life, while those with thalassemia intermedia, a less-severe form of thalassemia, are intermittently or rarely transfused. An allogeneically matched bone marrow transplant is curative, although it is restricted to those with matched donors. Gene therapy holds the promise of “fixing” one's own bone marrow cells by transferring the normal β-globin or γ-globin gene into hematopoietic stem cells (HSCs) to permanently produce normal red blood cells. Requirements for effective gene transfer for the treatment of β-thalassemia are regulated, erythroid-specific, consistent, and high-level β-globin or γ-globin expression. Gamma retroviral vectors have had great success with immune-deficiency disorders, but due to vector-associated limitations, they have limited utility in hemoglobinopathies. Lentivirus vectors, on the other hand, have now been shown in several studies to correct mouse and animal models of thalassemia. The immediate challenges of the field as it moves toward clinical trials are to optimize gene transfer and engraftment of a high proportion of genetically modified HSCs and to minimize the adverse consequences that can result from random integration of vectors into the genome by improving current vector design or developing novel vectors. This article discusses the current state of the art in gene therapy for β-thalassemia and some of the challenges it faces in human trials.</jats:p

Mechanism of Reduction in Titers with Vectors Flanked by Chromatin Insulator Elements

Abstract Random integration of viral vectors can result in undesirable activation of surrounding genes by enhancers in the vectors (vector genotoxicity), or result in variable expression due to effects of surrounding chromatin on the vector transgene (position effects). Vector genotoxicity has become an area of intense study since the occurrence of gene therapy related leukemias in patients in the French X-SCID trial. Additionally, variability in expression has been shown to compromize therapeutic efficacy in gene therapy for beta-thalassemia, where a high consistent level of expression is necessary. Vectors flanked by the cHS4 insulator, one of the best characterized insulator element, can reduce vector genotoxicity (Neinhuis et al, 2007) and chromatin position effects in γ-retrovirus (Rivella et al, 2000; Yannaki et al, 2002) and lentivirus vectors (Arumugam et al, 2007). Despite these favorable features, the full-length cHS4 insulator, necessary for optimal insulator activity, is infrequently used because it lowers vector infectious titers by 10–15 fold (Ramezani et al, 2003). This reduction in titers is especially limiting with vectors carrying large inserts. We analyzed mechanisms by which this occurred. Insertion of an additional 1.2kb internal cassette to the large human β-globin-LCR (hβ-LCR) lentiviral (LV) vectors did not reduce vector infectious titers. However, insertion of the 1.2Kb cHS4 element reduced titers by more than an order of magntude; showing that reduction in titers by cHS4 was not secondary to a further lengthening of vector genome but this occurred either due to a large insert in the 3′LTR or specific cHS4 sequences that bind cellular factors and hinder viral genome transcription. We then inserted varying sized fragments of cHS4, tandem repeats of the cHS4 core element, or inert DNA spacers in the 3′LTR of the hβ-LCR LV vector, sBG, resulting in vectors with 3′LTR inserts of 0, 250, 400, 500, 650, 800 and 1200bp. After a threshold length of 650bp, infectious titers fell proportional to the size of the insert into the LTR. The same effect was seen with inert DNA spacer elements, showing that this phenomenon was not sequence-specific. We next examined the stage of the vector life-cycle affected by large LTR inserts: lengthening of the 3′LTR did not increase viral readthrough transcription, as measured by northern blot analysis and an enzyme-based assay for readthrough transcription (Higashimoto et al, 2007). Equal amounts of full-length viral genomic transcripts were produced in the packaging cells with vectors with and without the insulator. Similar degree of viral genome encapsidation occurred, as measured by p24 ELISA, virus associated reverse transcriptase and viral RNA analysis, demonstrating that similar amounts of intact viral particles were produced with insulated and uninsulated vector plasmids. However, the insulated vector was inefficiently processed following target cell entry, resulting in less integrated vector; and thus lowers infectious titers. Of note, a vector carrying tandem repeats of the cHS4 core (two 250bp repeats) resulted in increased rate of recombination, with deletion of the insulator core at a high frequency. Thus, we found that large inserts in the viral 3′LTR are packaged efficiently, but have inefficient post-entry viral mRNA processing. These studies have important implications in the design of γ-retrovirus and LV vectors with insulator and other transgene/promoter/enhancer inserts into the LTR.</jats:p

Elements of a Chromatin Insulator Necessary for Consistent, High-Level Globin Gene Expression

Abstract Vectors flanked by the chicken b-globin locus hypersensitive site-4 (cHS4) insulator provide consistent, improved expression (chromatin barrier activity) and reduced genotoxicity (enhancer-blocking activity); features important for randomly integrating viral vectors. However, full-length 1.2Kb cHS4 reduces vector titers significantly. Initial studies mapped the enhancer blocking activity of cHS4 to the first 250bp ‘core’ in a copy-number dependent manner (Felsenfeld and colleagues). However, elements required for the barrier effect are not well defined. We studied various regions of cHS4 for chromatin barrier effect. Beta-globin-LCR lentivirus vectors were constructed with the core (sBGC), 2 copies of the core (sBG2C), 400bp (sBG400) and 800bp (sBG800) fragments (both including the core) and compared to the un-insulated vector (sBG) and the vector with the full-length cHS4 (sBG-I). Barrier activity (improved probability and consistency of expression from individual integrants) in MEL cells and the clonal progeny of hematopoietic stem cells was analyzed. Single-copy MEL clones showed that sBG-I clones had a significantly higher proportion of hβ+ cells/clone. However, proportion of hβ+ cells from all other vectors carrying various fragments of cHS4 were not significantly different from the un-insulated vector (Table 1). Interestingly, all clones carrying a fragment containing the core showed reduced clonal variegation (coefficient of variation of hβ expression). We next analyzed hβ expression from the sBG, sBGC, sBG2C, sBG400 and sBG-I vectors in primary and secondary thalassemia (Hbb th3/+) mice. In the primary mice and secondary CFU-S analyzed thus far (Table 2), it appears that all vectors with cHS4 insulator fragments containing the core provide some barrier activity, but the full-length insulator is necessary for maximum barrier effect. To corroborate whether elements outside the core contribute to barrier activity, we also tested vectors with inert DNA spacers in addition to the core (sBG400−S, sBG-800−S and sBG1200−S). Single copy MEL clones confirmed that the sBG400−S, sBG800−S and 1200−S vectors had significantly lower proportion of hβ+ cells/clone [41±4.4%, 41±7% and 28±6% respectively, n=36]. These data suggest that regions of 950 bp region of cHS4 outside the core contains elements that cooperate with the core for optimal barrier activity. These studies have important implications for vector design for gene therapy for thalassemia, where variability in expression from uninsulated vectors compromizes efficacy. Table 1. β globin expression in MEL cell clones from vectors carrying portions of the cHS4 insulator sBG sBG c sBG 2c sBG 400 sBG 800 sBG-I Values are expressed as Mean±SEM, *P&amp;lt;0.05, **P&amp;lt;0.01 by ANOVA (Dunnet: comparisons of all vectors to the control vector, sBG) % hβ+ cells 59±5 49±6 51±3 51±3 49±6 84±3** CV 53±4 37±3** 40±2** 42±1.5** 42±3** 36±2** Table 2. β globin expression in primary thalassemia mice and in the clonal progeny of hematopoietic stem cells of secondary mice Primary mice (16 weeks) Mock sBG sBG c sBG 2c sBG 400 sBG-I Values are expressed as Mean ± SEM, *P&amp;lt;0.05, **P&amp;lt;0.01 by ANOVA (Dunnett: comparison of all vectors to the control, sBG) HCT (%) 20±3 32±3 36±2 37±2 37±3 39±2 Reticulocytes (%) 28±2 16±5 11±1** 13±6** 7±2** 7±2** %HbA (mαhβ by HPLC)/vector copy NA 32±19 24±5 29±3 31±3 43±4* % hβ+RBC/ vector copy NA 36±11 57±8* 67±13* 76±17* 104±18**     Secondary CFU-S (Single Copy) % hβ+ cells/clone NA 26±3 45±3* 34±2 48±9* 75±3** CV NA 88±9 68±2* 71±2* 72±3 61±2**</jats:p

Mechanism of Reduction in Titers from Lentivirus Vectors Carrying Chromatin Insulator Elements in the 3′ LTR

Abstract Random integration of viral vectors can result in undesirable activation of surrounding genes by enhancers in the vectors (vector genotoxicity), or result in variable expression due to effects of surrounding chromatin on the vector transgene (chromatin position effects). Vector genotoxicity has become an area of intense study since the occurrence of gene therapy related leukemias in 5 patients in the French X-SCID trial. Additionally, we find consistent and therefore 2–3 fold higher expression from vectors insulated by the chicken b-globin hypersensitive site (cHS4) insulator; and these vectors achieve therapeutic correction in human b0-thalassemia major, where a very high transgene expression is necessary. However, vectors that insulate the correcting transgene from position effects and genotoxicity significantly compromise viral titers by an order of magnitude. In order to define the mechanism by which this occurs and improve the titers of insulated vector systems, we placed the 1.2Kb cHS4 insulator or different regions of cHS4 that may have insulator activity and/or inert DNA spacers in the 3′LTR of self-inactivating lentiviruses carrying a large b-globin transgene and regulatory elements. We also lengthened the β-globin lentivirus vector by an additional 1.2Kb, by insertion of an internal transgene cassette. We found that addition of 1.2Kb transgene internally to a large “globin” vector did not reduce vector infectious titers. However, when cHS4 sequences or inert DNA spacers of increasing size were placed in the 3′LTR, infectious titers decreased proportional to the length of the insert. This effect occurred regardless of the type of sequence inserted in the 3′ LTR. Vectors carrying the1.2Kb cHS4 or l-DNA spacer in the 3′ LTR had the lowest titers. We next examined the stage of the vector life-cycle affected by large LTR inserts in packaging cells, the quantity and quality of the virus particles generated, and post-entry viral steps in target cells. Equal amounts of full-length viral genomic transcripts were produced in the packaging cells with vectors with or without the 1.2Kb insulator insert. All insertion in the 3′ LTR are placed in the U3 enhancer deleted region, proximal to the viral polyadenylation signal. Also, self-inactivating vectors have a U3 enhancer deletion that also deletes enhancers of polyadenylation. However, despite the insertion of cHS4 elements in this region, no increase in viral readthrough transcription (as measured by northern blot analysis and an enzyme-based assay) occurred with vectors carrying the large 1.2Kb insert. Packaging efficiency was also identical with insulated and uninsulated vectors. Similar degree of viral genome encapsidation occurred, as measured by p24 ELISA, virus associated reverse transcriptase and viral RNA analysis, demonstrating that similar amounts of intact viral particles were produced with insulated and uninsulated vector plasmids. However, lentiviruses carrying the 1.2Kb insert in the 3′LTR were inefficiently processed following target-cell entry, with reduced reverse transcription and integration efficiency. This primarily occurred from increased homologous recombination resulting in increased 1-LTR circles of the insulated vector viral DNA; resulting in reduced vector integration and hence lower transduction/infectious titers. Thus, we found that large inserts in the viral 3′ LTR are packaged efficiently, but have inefficient post-entry viral mRNA processing. In a parallel study, we also did a structure-function analysis of cHS4 fragments and identified key elements necessary for optimal insulation. Vectors constructed with this minimized 650bp cHS4 sequences had a minimal reduction in titers, yet retained full insulator activity. These studies have important implications in the design of gamma-retrovirus or lentivirus vectors with insulator, transgenes or enhancer inserts into the 3′ LTR. [FU and PA contributed equally to this work]</jats:p

Self-Inactivating Lentiviral Vectors Flanked by a Chromatin Insulator Element Result in Increased, Consistent Expression of Human Beta Globin.

Abstract Lentiviral vectors carrying the human β-globin gene (hβ) and hypersensitive sites 2, 3 and 4 of the hβ locus control region (LCR) have revolutionized the field of gene therapy for hemoglobinopathies, resulting in correction in mouse and human models of β-thalassemia. However, their random integration into host chromosome results in highly variable hβ expression, dependent on the flanking host chromatin (chromosomal position effects). Moreover, the recent occurrence of leukemogenesis from activation of a cellular oncogene by the viral enhancer elements calls for safer vector designs, with expression cassettes that can be ‘insulated’ from flanking cellular genes. We analyzed the role of the chicken β-globin locus hypersensitive site 4 insulator element (cHS4) in a self-inactivating (SIN) lentiviral vector. The BGM vector carrying the hβ/LCR was compared to an analogous vector BGMI, where the cHS4 was inserted in the SIN deletion to flank the hβ/LCR at both ends upon integration. Both vectors additionally carried the methyl guanine methyl transferase (MGMT) cDNA to enrich for genetically modified cells. First, murine erythroleukemia (MEL) cells were transduced at &lt;5% transduction efficiency in three separate experiments to generate single copy clones, transduced clones identified by PCR (‘Unselected’ clones) and analyzed for hβ protein by FACS and mRNA by RNase Protection Assay (RPA). ‘Unselected’ BGMI+-MEL clones had a higher proportion of hβ+ cells (68±3%) compared to BGM+ clones (36±9%, n=24; p&lt;0.001). Additionally, the coefficient of variation of hβ expression (CV) in each clone was reduced in BGMI+ clones: 168±20 vs. 327±64 in BGM (P&lt;0.02). RPA showed a 2-fold increase in hβ/total mα-globin mRNA with the BGMI vector [BGMI 45±6% vs. BGM 21±7%, n=24, p&lt;0.01]. Next, the same pool of MEL cells transduced with BGM and BGMI at &lt;5% transduction efficiency were selected with BG/BCNU and 86 single-copy clones isolated (‘Selected’ clones). ‘Selected’ BGMI+-MEL clones also had a higher proportion of hβ+ cells (BGMI 80±15%, n=54 vs. BGM 72±20%, n=32; p&lt;0.03), with reduced CV (140±2.8 vs. 170±13, P&lt;0.01) and higher hβ-mRNA [BGMI+ 83.6%± 40 vs. 49.6%±25 in BGM+ clones (n=24, p&lt;0.02)]. In vivo studies confirmed MEL cell results: 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±15% vs. 68±10%, and 0.21 vs 0.17 in BGMI and BGM groups, respectively, n=12). While, 18±4% of murine RBC expressed hβ in the BGMI group, only 4±1.5% expressed hβ in the BGM group of mice by FACS (p&lt;0.001). There was a 4-fold increase in chimeric hemoglobin (mα-hβ) in the BGMI mice (13% ± 4 vs. 3±2% in BGM mice) by hemoglobin electrophoresis (p&lt;0.001). Secondary colony forming units-spleen (CFU-S) derived from these mice 12 weeks after transplant showed increased numbers of hβ+ cells in BGMI CFU-S (21±4% vs. 8±4% BGM-CFU-S, n=30, p&lt;0.03) with reduced CV in BGMI CFU-S (698±91 vs. 987±99 in BGM CFU-S, p&lt;0.04). MGMT selection and secondary transplants in BGM and BGMI mice are underway. Taken together, ‘insulated’ SIN-hβ/LCR lentiviral vectors increased the probability of expression of integrants and reduced chromatin position effects, resulting in consistent and higher hβ expression for gene therapy of β-thalassemia. The enhancer blocking effect of the cHS4, although not tested here, would further improve bio-safety of these lineage-specific, self-inactivated vectors.</jats:p

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

Abstract 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 MEL Clones Uninsulated 5′ 250bp Core 5′ 400bp 2 Cores 5′800bp 1.2Kb 3′ 400bp HbA+ cells (%) 59±5 49±6 51±3 52±3 49±6 84±3** 59±3 CV of HbA expression 53±4 37±3* 40±2* 42±1* 42±3 37±1* 38±1* Table 2. Effect of insulator sequences on the differentiated progeny of transduced and transplanted thalassemia hematopoietic stem cells Primary transplants (24 wks) Mock Uninsulated 5′250 bp 5′400bp 2 cores 1.2Kb 3′ 400bp *P&lt;0.05; **P&lt;0.01; ***P&lt;0.001; HbA = human β and mouse α globin tetramers Statistical analysis was performed by ANOVA (Dunnell’s multiple comparison test to the control vector sBG) *Data has not been nomialized far vector copies; CFU-S represent those screened for carrying single integrants by qPCR Note: Copy number of vector with 1.2kb cHS4 insert is significantly lower compared to uninsulated vector ^RBC (M/μl) 6.6±0.4 8.6±0.5 8.5±0.3 7.9±0.2 7.5±0.2 8.9±0.2 7.9±0.2 ^Hematecrit (%) 24±2 32±3 36±1 36±1 35±2 38±1 34±1 ^MCHC (g/dL) 24±1 20±1 29±2 29±2 27±2 33±1* 28±1 ^Reticulocyte (%) 29±2 11±4 10±2 12±7 11±13 7±1 12±2 Vector Copy (VC)/Cell 0 1.2±0.18 0.90±0.12 0.97±0.13 1.2±0.26 0.63±0.10 0.76±0.16 HbA (%)/VC (HPLC) 19±6 26±4 30±3 22±3 43±3** 18±3 HbA+ RBC/VC (FACS) 40±8 56±5 62±7 51±9 100±5** 32±7 ^CV 618±171 375±36* 384±29* 294±29** 281±16** 270±44** Secondary CFU-S (single copy) ^HbA+cells/CFUS (%) 37±4 53±4 53±5 42±4 85±2** 8±1*** ^CV 98±15 70±5* 75±3* 91±5 57±2** 59±2**</jats:p