Enabling Technologies

Gene Therapy (2012) 19, 15–24; doi:10.1038/gt.2011.70; published online 12 May 2011

CTF/NF1 transcription factors act as potent genetic insulators for integrating gene transfer vectors

A Gaussin1,2, U Modlich3, C Bauche4, N J Niederländer1, A Schambach3, C Duros4, A Artus4, C Baum3, O Cohen-Haguenauer4,5 and N Mermod1

  1. 1Institute of Biotechnology, University of Lausanne, Lausanne, Switzerland
  2. 2Doctoral program in Biotechnology and Bioengineering, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland
  3. 3Department of Experimental Hematology, Hanover Medical School, Hanover, Germany
  4. 4Laboratory of Biotechnology and Applied Pharmacogenetics, UMR 8113, Ecole Normale Supérieure de Cachan, Cachan, France
  5. 5Université Paris-Diderot & Hopital Saint-Louis, Paris Cedex 10, France

Correspondence: Professor N Mermod, Institute of Biotechnology, University of Lausanne, Station 6, EPFL-FSB-LBTM, Lausanne 1015, Switzerland. E-mail: Nicolas.mermod@unil.ch; Dr O Cohen-Haguenauer, Ecole Normale Supérieure de Cachan, 61 avenue du President Wilson, F-94235 Cachan Cedex, France. E-mail: odile.cohen@lbpa.ens-cachan.fr

Received 23 August 2010; Revised 15 February 2011; Accepted 2 March 2011; Published online 12 May 2011.

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Abstract

Gene transfer-based therapeutic approaches have greatly benefited from the ability of some viral vectors to efficiently integrate within the cell genome and ensure persistent transmission of newly acquired transgenes to the target cell progeny. However, integration of provirus has been associated with epigenetic repercussions that may influence the expression of both the transgene and cellular genes close to vector integration loci. The exploitation of genetic insulator elements may overcome both issues through their ability to act as barriers that limit transgene silencing and/or as enhancer-blockers preventing the activation of endogenous genes by the vector enhancer. We established quantitative plasmid-based assay systems to screen enhancer-blocker and barrier genetic elements. Short synthetic insulators that bind to nuclear factor-I protein family transcription factors were identified to exert both enhancer-blocker and barrier functions, and were compared to binding sites for the insulator protein CTCF (CCCTC-binding factor). Gamma-retroviral vectors enclosing these insulator elements were produced at titers similar to their non-insulated counterparts and proved to be less genotoxic in an in vitro immortalization assay, yielding lower activation of Evi1 oncogene expression and reduced clonal expansion of bone marrow cells.

Keywords:

oncogene; insulators; CTF/NF1; CTCF; genome integration; genotoxicity

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Introduction

Ex vivo retrovirus-mediated gene transfer into hematopoietic progenitor cells proved to be an efficient therapeutic strategy for a substantial number of patients suffering from severe combined immunodeficiency.1 However, the appearance of leukemia cases raised questions on the safety of this approach.2 The ability of retroviral vector genomic integration to activate the expression of endogenous oncogenes uncovered a new type of genotoxicity stemming from the nonspecific activation of cellular genes by viral regulatory elements.3, 4 In addition, it is now well established that integrating viral vectors have a tropism for integration into particular chromosomal regions, prompting their insertion in or near active transcriptional units.5, 6, 7 Integrating vectors used for gene therapy usually carry strong enhancers, which are able to provide high and persistent transgene expression through their capacity to withstand silencing. However, these regulatory elements are also more prone to promote the dysregulation of genes near the site of integration of the vector. Extensive studies have shown that the enhancer sequences are the major cause of cell transformation, making the design of viral vectors for life-long therapy approaches even more challenging.8, 9, 10

Insulators mark the boundaries of chromatin domains and limit the range of action of enhancers and silencers.11 They are characterized by at least one of the following properties: Enhancer blocking and/or boundary.12, 13 An insulator with enhancer-blocking properties is able to specifically block communication between an enhancer and a promoter when interposed. However, enhancer-blockers do not alter the ability of the enhancer to activate other promoters.14 Insulators with boundary properties set the borders of neighboring chromatin domains. Thus, boundaries prevent the propagation of condensed chromatin structures that silence expression and counteract the effects of chromosomal position on transgene expression.15 Particular insulators are able to act both as enhancer-blocker and boundary, such as the well-characterized 1.2-kb chicken β-globin hypersensitive site-4 (cHS4)-containing insulator.11, 16

Previous attempts to incorporate insulators into recombinant viral vectors intended for gene therapy yielded reduced transgene expression variegation and limited chromosomal position effect.17, 18, 19, 20, 21 Most of these studies were performed on gamma-retroviral and lentiviral vectors with the cHS4 insulator in various mouse and human cell types.22 Implementation of the full-length, 1.2-kb element in gamma-retroviral vectors was associated with reduced genotoxicity, but also with important constraints concerning vector design and titer of infectious particles, mainly owing to the substantial size of this sequence.18, 23, 24 A shorter sub-portion of the cHS4, namely the 250-bp insulator core element, is more suitable with viral vectors’ biology, but it failed to reproduce the activity of the full-length element.9, 20, 25, 26

The enhancer-blocking function of the cHS4 has been attributed to the CCCTC-binding factor (CTCF), an 11-zinc-finger DNA-binding protein highly conserved in vertebrates. CTCF was implicated in diverse regulatory functions, including transcriptional activation/repression, insulation and imprinting.27 CTCF organizes higher-order chromatin structures and was associated with the formation of chromatin loops that may mediate its enhancer-blocking function.28 The boundary activity of the cHS4 element derives from the combined effect of the upstream transcription factors 1 and 2 (USF1 and USF2).29 In addition, the cHS4 insulator sequence was shown to be highly concentrated in the nuclear matrix fraction, suggesting that it may be involved in the topological organization of the genome.30

The CAAT box-binding transcription factor/nuclear factor-1 (NF1, also called CTF/NF1) consists of a family of widely expressed transcription factors that possess a barrier function.31, 32 This family comprises NF1-A, NF1-B, NF1-C and NF1-X subtypes that share the same DNA-binding domain.33, 34 Of note, the NF1-C regulatory domain prevents the propagation of repressive chromatin structures that stem from the telomere, and thereby prevent transgene silencing at mammalian and yeast cells telomeres.31, 32 NF1-C regulates DNA replication by promoting the recruitment of the DNA polymerase to the adenovirus and SV40 origins of replication, and is also involved in promoter regulation.35, 36, 37 Thus, CTF/NF1 proteins were described as barrier elements that act to control DNA transcription and replication, yet a potential enhancer-blocking function had not been assessed.

Previous identifications of insulator elements for gene therapy viral vectors have resulted in impaired vector production and/or poor insulating efficacy. We developed a quantitative procedure to screen insulator elements and assess their ability to block gene activation by the strong Friend-murine leukemia virus enhancer-containing long terminal repeat (Fr-MuLV LTR). We designed novel synthetic elements acting as binding sites for CTCF and CTF/NF1 proteins, and found that they mediate potent enhancer-blocking activities, resulting in a commensurate reduction of genotoxicity when implemented in viral gene therapy vectors.

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Results

Positive assessment of cHS4 variants in quantitative enhancer-blocking insulator activity assay

Using an enhancer-blocking insulator assay based on a neomycin (G418) resistance-conferring plasmid (construct-1 of Figure 1a), we assessed the capability of the full-length 1.2-kb cHS4 element to insulate a γ-globin promoter/neo reporter gene from activation by the mouse 5′HS2 locus control region (LCR) in several human cell lines. The presence of the 5′HS2 LCR significantly increased the occurrence of G418-resistant colonies in K562 and HeLa cells, whereas the cHS4 insulator was able to block the LCR-mediated upregulation of the selection gene when interposed between the enhancer and the promoter. The cHS4 insulator decreased the number of G418-resistant colonies nearly four-fold and 10-fold in K562 and HeLa, respectively, and fully prevented LCR-mediated upregulation to levels comparable to those observed from the γ-globin promoter without LCR and insulator (Figure 1b).

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Insulator/enhancer-blocker assay of cHS4 variants in cellular systems. (a.1) Constructs allowing semi-quantitative determination of enhancer-blocking activity. The reporter gene (neo) is driven by the γ-globin promoter under the control of either the β-globin LCR element or the Fr-MuLV enhancer-containing LTR (in both orientations). Expression of neo is assessed by the number of neomycin-resistant colonies obtained after stable transfections. The insulated neo gene is flanked by two copies of the 1.2-kb cHS4 insulator (interposed and external positions, referred to as 1 and 2, respectively), whereas its non-insulated counterpart is flanked by a single external cHS4 element. (a.2) Constructs generated for quantitative enhancer-blocker assay. A BFP reference gene is placed under the control of the promoter and the enhancer-containing Fr-MuLV LTR. A reporter GFP gene is expressed from the minimal CMV promoter and the LTR enhancer. The insulated GFP gene is flanked by two copies of the 1.2-kb cHS4, to prevent activation of the minimal CMV promoter by the LTR enhancer. GFP fluorescence is normalized to that of BFP, which serves as internal reference for potential variations in transfection efficiency and transgene expression in each analyzed cell. The cHS4 core, or a spacer, replaces the full-length cHS4 depending on experiments. (b, c) Percentage of neomycin-resistant colonies counted 3 weeks after transfection and G418 selection of HeLa (black bars) and K562 (dashed bars) cells. Presence of an enhancer (Enh) from either the β-globin LCR element (LCR) or the Fr-MuLV LTR in one orientation (LTR) or in the inverted orientation (LTRinv), and/or of an interposed cHS4 insulator, is as depicted in panel a.1. The number of resistant colonies obtained in the absence of the interposed copy of the cHS4 was set to 100%. (d) Cytofluorometric analysis of the activity of the cHS4 insulator using quantitative assay in transiently transfected HeLa cells. The panels show the data of the same two representative cell populations obtained 48h after transfection of constructs containing (blue) or not containing (red) an interposed copy of the cHS4 (as depicted in panel a.2). GFP expression of BFP-positive cells, or BFP expression of the total cell population, is shown at the left- and right-hand sides, respectively. The black profiles correspond to the background fluorescence of non-transfected cells as control. (e) Relationship between GFP and BFP fluorescence at individual cell levels. Cells transiently transfected with a construct containing or not containing an interposed cHS4 insulator, as analyzed in panel d, are depicted by blue and red dots, respectively. (f) Quantitative analysis of the enhancer-blocking activity of the cHS4 insulator. GFP fluorescence values were determined 48h after HeLa cell transfection and were normalized to BFP expression for each analyzed cell. Normalized fluorescence values are plotted for cell populations transfected with constructs containing the indicated insulator, whereas Δ ins refers to the construct with an external cHS4 but without an interposed insulator sequence. Fluorescence value ratios were normalized to the ratio obtained with a construct lacking both copies of the insulator. Elements interposed between the enhancer and the promoter driving GFP expression and their respective sizes are as indicated. Spacers refer to portions of coding sequences used as negative controls. P-value was determined by two-tailed t-test. cHS4, chicken β-globin hypersensitive site-4; Fr-MuLV, Friend-murine leukemia virus; GFP, green fluorescent protein; LCR, locus control region; LTR, long terminal repeat.

Full figure and legend (187K)

The ability of the cHS4 insulator to block activation from the potent enhancer present on the Friend-murine leukemia virus LTR was then similarly assessed in HeLa cells. Substitution of the β-globin LCR by the Fr-MuLV LTR in either orientation strongly increased the occurrence of resistant colonies (Figure 1c). The Fr-MuLV LTR proved to promote much stronger reporter gene activation than the β-globin LCR in this cell type. Nevertheless, the cHS4 was able to decrease the growth of resistant colonies nearly eight-fold when interposed between the enhancer and the promoter of the reporter construct, yielding levels similar to those obtained in the absence of an enhancer.

Previous studies have indicated that inclusion of the HS4 element and/or its core may repress the expression of nearby reporter genes, which may complicate the interpretation of insulator function assays.20, 38 We therefore designed a two-reporter gene assay implementing the accuracy of the former assay, but in which polar insulating activities can be distinguished from enhancer inhibition, global gene silencing effects or gene copy number variations (Figure 1a, construct-2). Changes in the expression of the reporter and reference genes were assessed by cytofluorometry from the green fluorescent protein (GFP) and blue fluorescent protein (BFP) fluorescence profiles of single cells within populations of transiently transfected HeLa cells. Interposition of a copy of the cHS4 insulator between the cytomegalovirus (CMV) minimal promoter driving the GFP gene and the LTR enhancer, for example, at position 1 of Figure 1a, led to a significant decrease in the expression of GFP but not BFP, as illustrated in Figure 1d. Single-cell imaging of the relative GFP and BFP levels showed a homogeneous decrease of GFP expression relative to BFP (Figure 1e). When interposed, cHS4 induced a significant decrease in GFP expression down to 20% of the enhancer-activated level (Figure 1f). Only a small proportion of this effect (approximately 20%) may be attributed to the increased distance between the enhancer and the promoter driving the GFP, as interposition of a 1.2-kb-long neutral fragment had little effect on the GFP/BFP fluorescence ratio (Supplementary Figure 1). Insertion of a single copy of the 250-bp cHS4 core did not result in a significant insulator effect in this assay, in agreement with previous studies showing that one copy of the cHS4 core does not mediate a high insulating activity.9, 20, 25, 26

Optimized CTCF and CTF/NF1 binding sites show potent enhancer-blocking activities

We designed a composite element, based on Bell et al.,22 containing multiple copies of CTCF binding sites from both the cHS4 insulator and the BEAD-1 element from the human T-cell receptor α/δ locus (CTCF nat 6x; Figure 2a and Supplementary Table 1). While much shorter in size (270bp), this element showed at least half the insulator activity of the 1.2-kb cHS4. A high insulating activity was also shown when the CTCF nat 6x was embedded within an inactivated LTR, thus mimicking the context in which the insulator would be in a retroviral or lentiviral vector (Figure 2b). Another CTCF-binding element, comprising six repeats of the consensus binding site,39 was also evaluated (CTCF cons; Figure 2a and Supplementary Table 1). Linkers were added between each binding site to make up for the size of a CTCF footprint. This consensus element showed comparable activity with native binding sites. Of relevance, doubling the number of consensus binding sites, to reach 12 consecutive elements, fully reproduced the insulation effect of the entire 1.2-kb cHS4 element (Figure 2b).

Figure 2.
Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Quantitative analysis of the enhancer-blocking activity of synthetic CTCF and CTF/NF1 binding sites. (a) Sequence and pairwise alignment of the different types of CTCF and CTF/NF1 binding sites were synthesized and assessed. The conserved nucleotides between the sequences are highlighted in red and a star indicates their position. BEAD-A and FII indicates CTCF binding sites in the chicken cHS4 insulator and in the human T-cell receptor locus, respectively, whereas adeno indicates the adenovirus type-II origin CTF/NF1-binding sequence. (b, c) Quantitative analysis of the enhancer-blocking activity of multimerized CTCF and CTF/NF1 binding sites. Transfection of HeLa cells, determination of GFP-to-BFP fluorescence ratio and normalization to the values obtained without any insulator are explained as described in Figure 1F. The number and type of adenovirus CTF/NF1 binding sites (adeno), or consensus binding sites (cons), and the spacing between adjacent binding sites are as specified. (d) Quantitative analysis of the enhancer-blocking activity of CTCF and CTF/NF1 binding sites in stable transfections. The mean GFP expression normalized to BFP expression per cell is plotted for each population 2–3 weeks after selection of transfected cells with the constructs depicted in Figure 1a.2. Fluorescence ratios were normalized to the values obtained with construct lacking both insulator copies. The elements interposed between the enhancer and the promoter of GFP are as indicated. P-values of two-tailed t-tests are shown. cHS4, chicken β-globin hypersensitive site-4; CTCF, CCCTC-binding factor; CTF/NF1, CAAT box-binding transcription factor/nuclear factor-1; GFP, green fluorescent protein.

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To characterize the enhancer-blocking activity of CTF/NF1, we evaluated a panel of binding sites for CTF/NF1 proteins in the double reporter-assay system. The binding sites’ derivatives were generated to alter the nature of the last nucleotide of the binding site, to be either a thymidine, mimicking the native CTF/NF1 binding site from the adenovirus type-II origin of replication (referred to as adeno), or an adenine, to fit the consensus CTF/NF1 binding site (referred to as cons). The length of the spacing between two adjacent binding sites was also altered, with either 5 or 10 base pairs, orienting sites on similar or opposite sides of the DNA double helix (Figure 2a and Supplementary Table 2). Spacers of 5bp appeared to provide the most favorable configuration, as binding sites interspaced with 5bp are the most potent enhancer-blockers, even embedded within an LTR. Decreasing the number of repeats did not lead to a significant loss of insulating activity. A single 20-bp binding site still mediated half of the insulating effect of the full-length cHS4, like 7, 4 or 3 binding sites (Figure 2c, and data not shown). Even though the 10-bp spacing should provide sufficient length to accommodate all directly contacted nucleotides within the binding sites,40 the spacing of 5bp provided the best insulating activity for all of the tested CTF/NF1-binding sequences.

To ascertain that CTCF as well as CTF/NF1 binding sites may also show an enhancer-blocking activity in the context of a native chromatin structure, stable cell transfections were analyzed. The insulating window of the full-length cHS4 was reduced 2.5-fold of the reporter gene expression, whereas the cHS4 core did not show any significant activity (Figure 2d). The elements composed of seven CTF sites (adeno) and six CTCF native sites showed insulating effects that were comparable to those observed in transient transfections. The binding sites for CTCF (nat) and CTF/NF1 (adeno, 5bp) were combined and assessed using the double-reporter assay system. This composite element conserved significant insulator activity, but no synergistic effect could be observed as compared with the insulating activity of each element tested separately (data not shown).

In order to validate CTF/NF1 as responsible for the CTF/NF1 binding site-mediated enhancer-blocking activity, cells were co-transfected with a short interfering RNA (siRNA) targeting all CTF/NF1 isoforms. The insulator assay was performed with constructs containing either a neutral spacer of 250bp or the most active combination of the CTF/NF1 binding sites. The enhancer-blocking activity of CTF/NF1 was observed with mock-transfected cells or with cells transfected with a scrambled siRNA sequence (Figure 3a). However, the insulator activity was entirely lost upon an 80% knock-down of CTF/NF1 protein levels with the specific siRNA, demonstrating the role of the CTF/NF1 transcription factor as enhancer-blocker insulators in mammalian cells (Figures 3a and b).

Figure 3.
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CTF/NF1 proteins mediate the enhancer-blocking activity of their cognate DNA binding sites. (a) The enhancer-blocking properties of the CTF/NF1 binding sites were quantitated as described in the legend of Figure 1f in comparison with that of a 250-bp DNA control spacer. HeLa cells were transfected with siRNA targeting CTF/NF1 (controls: mock transfection or scrambled siRNA) and subsequently transfected with the insulator-containing constructs harboring either a neutral spacer of 250bp or seven binding sites for CTF/NF1 (adeno, 5-bp spacing). FACS analyses were performed 48h after transfection. The average GFP-to-BFP fluorescence ratio was determined and plotted as described in the legend of Figure 1f. Fluorescence ratios were normalized to those obtained from the mock transfection of the siRNA and the transfection of the DNA construct containing the 250-bp spacer. The P-value of two-tailed t-test is indicated. (b) Western blot analysis of cell extracts from populations analyzed in panel a. The immunoblot was performed using antibodies specific for CTF/NFI and GAPDH as loading control. CTF/NF1, CAAT box-binding transcription factor/nuclear factor-1; FACS, fluorescence-activated cell sorting; Fr-MuLV, Friend-murine leukemia virus; GFP, green fluorescent protein; siRNA, short interfering RNA.

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CTF/NF1 binding sites act as effective intra-chromosomal boundary elements

CTF/NF1 binding sites have been shown to function as barrier elements that can prevent the propagation of repressive histone modifications that stem from the telomere, and thereby prevent the silencing of telomeric genes.16, 32 Nevertheless, whether these binding sites may also function as barrier elements upon transgene integration at internal chromosomal loci has not been assessed. The CTF/NF1 adeno (5-bp spacing) or CTCF nat binding sites were sub-cloned on each side of an SV40 promoter/GFP gene cassette (Figure 4a), to address the potential barrier properties of these sequences at random chromosomal locations. A multiple cloning site (MCS) spacer element replaced insulators as negative control, whereas a matrix attachment region (MAR) element that potently abrogates silencing effects was used as positive control.41 GFP fluorescence profiles were assessed for each construct on stable polyclonal populations pooling hundreds of independent cell clones and thus distinct integration loci.41 Three GFP expression profiles delineated sub-populations termed M1, M2 and M3, which designate, respectively, low, medium, and high GFP expression ranges (Figure 4b).

Figure 4.
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CTF/NF1 binding sites dampen adverse chromosomal position effects. (a) A schematic representation of the insulated GFP transgene construct. GFP expression was driven by an SV40 promoter and the effect of elements inserted on both sides of the transgene was evaluated in stable transfections of HeLa cells. (b) Results of representative FACS analysis for GFP expression of HeLa cell populations stably transfected with constructs described in panel a (16 days after transfection). The GFP transgene was flanked by either a MCS, or seven binding sites for CTF/NF1 (adeno, 5-bp spacing), or six binding sites for CTCF (nat) or the 1–68 MAR element. The profile of non-transfected cells is depicted in gray. The population of GFP-positive cells, that is, the total cell population excluding non-expressing cells, was divided in three sub-populations as following: M1 designates cells expressing low levels GFP, whereas M2 and M3 designate cells with medium or high ranges of GFP levels, respectively. (c) Relative distribution of each sub-population of cells according to GFP expression levels. The M1, M2 and M3 sub-populations are defined as described in panel b. The results are expressed as percentage of cells in the designated sub-population relative to the population of GFP-positive cells (excluding non-expressing cells). (d) Time-course FACS analysis of GFP transgene expression when flanked with the designated insulators in stably transfected HeLa cells. Results of FACS analysis were acquired 16, 20, 27 and 30 days after transfection, under constant antibiotic selection. CTF/NF1, CAAT box-binding transcription factor/nuclear factor-1; FACS, fluorescence-activated cell sorting; GFP, green fluorescent protein; MAR, matrix attachment region; MCS, multiple cloning site.

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In the presence of the MAR element, most of the cells expressed GFP and their distribution within the population of GFP-positive cells was 65% in M2 and 35% in M3 (Figure 4c). This distribution may be explained by a barrier activity of MARs that shields the transgene from silencing at the site of integration in the host cell chromosomes. The construct containing a neutral MCS sequence showed only about 2% of high-GFP-expressing cells (M3). In comparison, the GFP expression for the CTF/NF1 construct was improved relative to the MCS control, with only one-third of the cells in M1, 60% in M2 and nearly 10% in M3. The M3 cell populations for the MAR and CTF/NFI were more prominent than the MCS control (Figure 4b). A putative enhancer function of the tested CTF/NFI-binding elements was excluded by inserting them upstream from the minimal CMV promoter, in the absence of an enhancer, which did not alter positively or negatively the expression of the reporter gene as compared with the unfused minimal promoter alone (data not shown). These data strongly suggest that CTF/NF1 binding sites possesses barrier properties at internal chromosomal positions. Populations generated from MCS, CTF/NF1 and MAR constructs contained around 50, 60 and 65% of M2 cells, respectively. However, the CTCF construct showed only 10% of cells in the M2 sub-population, whereas the majority of the cells were either expressing at low levels or did not show detectable GFP fluorescence. Thus, flanking the transgene with CTCF binding sites was deleterious for long-term gene expression, suggesting that CTCF can exert a silencing activity, in agreement with previous observations of a repressive function mediated by the cHS4 CTCF binding site.20

The potential delay of transgene silencing over time was evaluated through a 30-day time-course analysis of GFP expression in stably transfected polyclonal pools (Figure 4d). Each global expression pattern was conserved over time, although a slight shift toward lower fluorescence was generally observed after 16 days. Overall, this indicated that MAR- and CTF/NF1-mediated silencing protection effects are stable and can withstand cell division.

Gain in safety of retroviral vectors flanked with CTCF and CTF/NFI binding sites

We assessed whether CTCF and CTF/NF1 may shield off the retroviral vector enhancer from activating the expression of cellular genes and/or mediating clonal cell proliferation. To this end, we used a gamma-retroviral self-inactivating (SIN) vector (SRS.SF), which contains the spleen focus-forming virus enhancer/promoter as internal promoter.42 This vector was previously shown to trigger insertional transformation events in both in vitro immortalization assays (IVIM assay) and transplanted mice.7, 9 Insulators were inserted to replace the deleted enhancer-promoter in the U3 region of the 3′LTR, ensuring their presence in both LTRs after reverse transcription and integration (Figure 5a). Inclusion of CTCF and CTF/NF1 binding sites had little effect on the titers obtained from this gamma-retroviral vector, which remained above 107 transducing units per milliliter. In both cases, <20% titer reduction was noted when compared with the non-insulated construct. While we previously observed a weak effect of the 250-bp cHS4 core insulator on transgene expression in SC-1 fibroblasts transduced at a low multiplicity of infection,9 the present insulated vectors decreased GFP transgene expression 50% compared with the control vector. The CTCF and the CTF/NF1 insulators had a similar attenuating effect on the expression of the internal promoter-driven eGFP. The composite insulators and LTRs were reverse-transcribed and integrated in an intact form in the target cells (Supplementary Figure 2).

Figure 5.
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CTF/NF1 and CTCF binding sites curtail retroviral vector genotoxicity. (a) Vector architecture of the gamma-retroviral SIN vector SRS.SF.eGFP.pre shown as provirus. It contains a splice-competent leader region and a post-transcriptional regulatory element (PRE) of the woodchuck hepatitis virus. The U3 region is almost completely deleted, leaving only the integrase attachment sites intact. eGFP is driven by the enhancer/promoter elements derived from the potent spleen focus-forming virus SF enhancer/promoter. In the insulated vectors, the insulator sequences were inserted into the U3 region of the vector's LTRs. (b, c) Introduction of insulator sequences into the LTRs of the SRS.SF.eGFP.pre vectors reduce the transformation potential. The replating frequencies of Lin cells corrected to the mean retroviral vector copy number as measured in the DNA of mass cultures were plotted for insulated vectors and for the parental un-insulated SRS.SF gamma-retroviral vector. The insulators implemented in retroviral vectors are six copies of CTCF binding sites (nat) or seven copies of CTF/NF1 binding sites (adeno, 5-bp spacing). The median replating frequency/copy number of the SRS.SF.eGFP.pre.CTCF vector was reduced ~5-fold, and that of the SRS.SF.eGFP.pre.CTF/NF1 ~4-fold. The data points shown for the SRS.SF.eGFP.pre vector contain those generated in this study (black dots) and in previously published data (gray dots; Modlich et al., 2009). The horizontal lines indicate the respective medians of the populations. (d) Quantitative real-time PCR analysis of Evi1 mRNA expression levels in mass cultures of vector-transduced lineage-negative bone marrow cells on the day of replating. Evi1 expression was scored in four independent experiments (ad) comparing CTCF and CTF/NFI binding site insulated vectors to that of a non-insulated control (SRS.SF.eGFP.pre). Basal Evi1 expression in expanded and un-transduced cells (mock) was set to 1. CTCF, CCCTC-binding factor; CTF/NF1, CAAT box-binding transcription factor/nuclear factor-1; GFP, green fluorescent protein; LTR, long terminal repeat; SIN, self-inactivating.

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Insulator activity was assessed using IVIM assay based on the in vitro selection of insertional mutant clones that gain a proliferative advantage after stable transduction by retroviral vectors. Immortalized mutant clones typically contain a vector insertion within the first intron of the Evi1 gene that results in the insertional upregulation of Evi1 transcription. The IVIM assay measures the replating frequency of mutant clones within the transduced culture (‘clonal fitness’), as well as the incidence of mutation events. The SRS.SF vector was shown to be transforming in every culture tested (incidence of 2 × 10−5), with a replating frequency/copy number of ~0.0035 (mean of n=10). Both CTCF and CTF/NF1 insulators reduced the number of replating clones (Figure 5b) and were able to reduce the replating frequency/copy number 5 and 4-fold, respectively, when compared with the non-insulated vector (SRS.SF CTCF versus SRS.SF P=0.055; SRS.SF CTF/NF1 versus SRS.SF P=0.043; n=7 each; Wilcoxon two-sample test; Figure 5c). The lower replating frequency was paralleled by the lower Evi1 expression levels observed in the presence of the insulated vectors (Figure 5d).

We assessed whether the reduced replating frequency and Evi1 expression reactivation observed in the presence of the insulators might have stemmed from an altered integration frequency around the Evi1 gene locus rather than from active insulation. We performed the assessment by selecting clones that arose in the IVIM assays and by mapping vector integration sites. Among the 35 insulated retroviral vector integrations sites that were mapped, five (14%) were in proximity to the Mds1/Evi1 locus (Supplementary Table 3). These data indicated that the decrease in Evi1 expression reported in Figure 5d, thus, does not stem from a lack of integration of the insulated vectors close to Evi1. Overall, we therefore conclude that the CTCF and CTF/NFI elements acted as insulators that potently reduced the genotoxic outcome of the gamma-retroviral vector.

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Discussion

Designing new generations of gene transfer viral vectors is a promising avenue to achieve safer gene therapy. Implementation of genetic insulator elements in retroviral vectors is intended to allow the transgene cassette to behave as an autonomously regulated expression unit once integrated in the host cell genome. When flanking the transgene cassette, insulators may be beneficial in two ways: (i) Enhancer-blockers would limit the range of action of the viral vector enhancer on nearby cellular genes, thus decreasing the risk of insertional activation, and (ii) barrier elements would stop the spreading of silent chromatin, to ensure long-term transgene expression, and counteract detrimental integration site position effects.29

This study outlines a standardized screening procedure, which evaluates the enhancer-blocking activity of insulator elements. Unlike approaches based on mRNA levels or reporter protein secretion, this assay can be used to efficiently process large cell populations, providing a quantitative measurement of the insulating activity at a single-cell resolution. Such quantitative assays parallel recently described assays of the barrier function of insulators specifically integrated at mammalian cell telomeres.43

Our screening evaluated a collection of novel insulating sequences comprising optimized binding sites for CTCF and CTF/NF1 insulator proteins. A 472-bp element comprising 12 CTCF binding sites reconstituted the enhancer-blocking activity of the full-length 1.2-kb cHS4. Moreover, a single copy of the cHS4 core showed little activity under experimental conditions assessing specifically its enhancer-blocking function, consistent with recent results where the 250-bp cHS4 core did not recapitulate the insulating function of the full-length element.26

The binding sites for the CTF/NF1 transcription factor family showed, even from a single copy, significant enhancer-blocking activity. Binding sites interspaced by 5bp showed the most potent enhancer-blocking activity. This may result from lower steric hindrance effects between adjacent CTF/NF1 sites lying on opposite sides of the DNA double helix. Here, insulation could be entirely attributed to CTF/NF1 proteins upon knockdown assays, thus establishing a previously unknown enhancer-blocking activity for this family of transcriptional regulators.

The compatibility of the insulator size with retroviral vectors was considered, as insertion of large elements in the 3′LTR reduces vector titers and impairs transduction efficiency.24 Therefore, insulator elements of varying sizes were designed to fit the LTR of retroviral and/or lentiviral vectors, without affecting viral vector efficacy. Whereas insulator potency correlated well with their variation in length, observed results were clearly distinct from sole distance effects, as interposition of nonspecific spacer DNA fragments between the enhancer and the promoter presented no variation. We conclude that elements as short as 20bp remain efficient for the mediation of significant enhancer-blocking function.

Derivatives of CTF/NF1- and CTCF-binding insulator sequences yielded reduced genotoxicity when inserted in SIN gamma-retroviral vectors, without significantly altering virus titers. These insulators led to a five-fold reduction of retroviral vector genotoxicity in an IVIM assay. Furthermore, the decreased occurrence of clonal cell proliferation correlated well with the 5- to 10-fold lower expression of Evi1 noted in presence of the CTF/NF1 insulator. This implies that the enhancer-blocking activity obtained with a plasmid-based assay was preserved for integrating a retroviral vector. Here, our results introduce novel insulator elements suitable for implementation in viral vectors, endorsing previous reports on insulator elements able to reduce insertional genotoxicity.9, 26, 44, 45

The barrier activity of the novel insulating elements was probed in the context of random transgene chromosomal integration upon stable transfection. Flanking the transgene with CTCF binding sites led to a decrease in its expression, stably propagated over cell division. Prior studies on cHS4 insulators showed that deletion of the CTCF binding sites was associated with loss of enhancer-blocking activity, but did not alter the barrier function of the element.22, 46 However, earlier work on the natural cHS4 locus could not readily assess a potential silencing effect of CTCF in addition to its enhancer-blocking activity.27 Analysis of CTCF in several genomic contexts brought forward its key function in the organization of the chromatin architecture. Although current models of CTCF action rely on a looping mechanism, CTCF might also orchestrate genome architecture through epigenetic chromatin modifications such as recruitment of chromatin-modifying proteins. It has been implicated in imprinting as well as repression and activation of transcription, suggesting a versatile mode of action upon a biological context. As such, implementing CTCF-binding synthetic sequences in viral vectors that integrate at multiple and potentially random loci in the genome could yield effects unanticipated from its activity at the cHS4 or at imprinted loci.47 Large-scale analyses of CTCF-insulated and non-insulated vectors consequences will be required to address these issues.

Transgenes flanked with binding sites for CTF/NF1 appeared to be protected from silencing effects when integrated at random chromosomal loci. This observation is consistent with previous studies where CTF/NF1 proteins act as barrier elements that block the propagation of silent chromatin structures, and thus protect transgenes from silencing effects mediated by repressive chromatin structures.31, 32, 43 Hence, CTF/NF1 binding sites act both as enhancer-blockers and as barrier insulator elements. Maintenance of a euchromatic status of the provirus should favor the production of retroviral vectors. This could also be advantageous when using tissue-specific promoters to drive transgene expression, as these are often weaker than promoters of viral origin, which may lead to the silencing of the therapeutic gene over time.

The tropism of retroviral vectors for specific genomic regions such as certain proto-oncogenes still remains a major issue for gene therapy safety,6, 7 despite reassuring reports of long-term follow-up in gene therapy-treated adenosine deaminase patients.48 Using insulators to counteract position effects and the occurrence of poor expression of some integrated vectors may favor therapeutic outcome from lower multiplicities of infection and reduced vector integration events,24 which should further reduce the risk of both activating and inactivating integration events.

Ensuring complete insulation of provirus sequences in large populations of cells may be difficult to demonstrate, as insulator function depends potentially on the chromatin state at the site of integration and/or on the expression of the insulator protein in the targeted cells or tissues. In that respect, the ubiquitous expression of CTF/NF1 family members is a favorable feature, and their use should further improve already safer SIN lentiviral vectors.

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Materials and methods

Plasmid vectors and insulator sequences

The plasmid constructs shown in Figure 1a.1 were constructed from the pJC5-4 plasmid provided by Dr Gary Felsenfeld,49 where the 5′HS2 LCR was substituted by the Friend-murine leukemia virus LTR (Fr-MuLV, FB29 strain; see reference Cohen-Haguenauer et al.50), either in its 5′–3′ native orientation or in the inverted orientation. The plasmid construct shown in Figure 1a.2 was obtained as follows: The eGFP gene expressed from a minimal CMV promoter was PCR-amplified from a pcDNA3-EGFP plasmid excluding the CMV enhancer, and the EBFP gene was PCR-amplified from the pEBFP-N1 plasmid (Clontech Laboratories, Inc., Mountain View, CA, USA). Both reporters were sub-cloned in pBS2-SKP (Stratagene, La Jolla, CA, USA). The Fr-MuLV LTR was inserted upstream from the EBFP gene such that transcription from the LTR is directed toward EBFP, and a copy of the 1.2-kb cHS4 was inserted downstream from the eGFP gene. Insulator sequences were inserted between the Fr-MuLV LTR and the minimal CMV promoter driving eGFP expression. The 250-bp cHS4 core was PCR-amplified from the full-length cHS4 (GenBank accession number: U78775.2; amplification from position 1–250). A series of neutral DNA spacers of various lengths were PCR-amplified from the mouse utrophin cDNA (GenBank accession number: BC062163.1; amplifications from position 355 to positions 605 or 1555). The plasmid construct shown in Figure 4a was based on a plasmid previously described by Girod et al.41

Binding sites for CTCF and CTF/NF1 were obtained by annealing complementary oligonucleotides. Native CTCF binding sites refer to alternate combinations of three binding sites from each of BEAD-A and FII sequences.22 Consensus CTCF binding sites correspond to direct repeats of the consensus binding motif.39 CTF/NF1 binding sites from the adenovirus type-II origin of replication were isolated from pNF7CAT.51 The consensus CTF/NF1 binding site was obtained from SELEX-SAGE experiments.40 Spacer sequences separating adjacent CTCF or CTF/NF1 binding sites were randomly chosen in order to limit the occurrence of repetitive DNA sequences. The GenBank accession number of the 1–68 MAR is EF694965.

Cell culture and transfection assays

DNA transfection of K562 cells was performed as previously described,49 and resistant colonies were counted after 3 weeks of selection for G418 resistance. HeLa cells were transfected using FuGENE 6 (Roche Diagnostics, Basel, Switzerland) according to the manufacturer's recommendations. Equimolar amounts of the various plasmids were transfected in each experiment (using the pBS2-SKP backbone plasmid as carrier). To obtain stable populations, the reporter constructs were co-transfected with a puromycin resistance-encoding plasmid (pPUR; Clontech) at a molar ratio of 10:1 and cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 0.5μgml−1 puromycin (all from Gibco, Carlsbad, CA, USA). Fluorescence analyses were performed using the FACS Cyan (Dakocytomation, Copenhagen, Denmark). Data analysis of the double-reporter assay consisted of normalizing GFP fluorescence to BFP fluorescence for each cell and averaging these values over the total cell population of several hundreds of independent clones. This method ensured to discriminate variations in GFP expression owing to the insulator effect from differences in expression levels resulting from variability in transfection efficiency. For the boundary assay, cells whose fluorescence profile overlapped with the profile of non-transfected cells were considered as non-expressing cells.

HeLa cells were transfected with 50nmoles of an siRNA targeting the mRNA of all CTF/NF1 isoforms (sc-43561; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or with a non-targeting control (scrambled siRNA, sc-37007; Santa Cruz Biotechnology) using Oligofectamine (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The cells were transfected with the double-reporter construct 24h later and fluorescence-activated cell sorting (FACS) analyses were performed after 48h.

Western blot analysis

Western blotting was performed following standard protocols: Protein extracts from a defined number of cells were separated by denaturing PAGE and transferred to a nitrocellulose membrane (Schleicher und Schuell, Dassel, Germany), and incubated with the primary antibodies anti-NF1 (H-300; Santa Cruz Biotechnology; dilution 1:200) applied overnight or anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (sc-32233; Santa Cruz Biotechnology) applied 2h after blocking the membrane in 5% dried-milk in phosphate-buffered saline. After incubation with a goat anti-rabbit horseradish peroxidase-coupled secondary antibody (Sigma-Aldrich, St Louis, MO, USA) or a goat anti-mouse horseradish peroxidase-coupled secondary antibody (Jackson Laboratory, Bar Harbor, ME, USA), the membrane was subjected to immunodetection by enhanced chemiluminescence (Amersham, Munich, Germany). Band intensity was quantified using the ImageJ software (http://rsb.info.nih.gov/ij/).

Retroviral vectors

The gamma-retroviral SIN vector has been described previously.52 The insulator sequences were inserted into the NheI site of the 3′ ΔU3 region, which is copied into the 5′LTR after reverse transcription, and thus results in a design flanking the introduced expression cassette. Gamma-retroviral supernatant production was performed using 293T cells as previously described, with co-expression of ecotropic envelope proteins.42, 52 Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100Uml−1 penicillin/streptomycin and 2mM glutamine. Viral titers, determined on SC-1 cells by flow cytometry, were in the range of 5 × 106 to 2 × 107IUml−1 in un-concentrated supernatants.

In vitro immortalization assay

Lineage-negative (Lin) bone marrow cells of untreated C57BL6/J mice (Charles River Laboratories, Wilmington, MA, USA) were obtained and transduced as previously described.53 DNA samples for real-time PCR analysis (copy numbers) and flow cytometry (FACSCalibur; Becton-Dickinson, Franklin Lakes, NJ, USA) were obtained 4 days after the last transduction. Quantitative PCR was performed using the Applied Biosystems 7300 Real-Time PCR System (Foster City, CA, USA), using the Quantitect SYBR Green kit (Qiagen, Hilden, Germany), as described previously.8 The IVIM assay was performed as described previously.54 After retroviral transduction, bone marrow cells were expanded for 14 days, plated onto 96-well plates at a concentration of 100 cells per well and positive wells were counted. The frequency of replating cells was calculated based on Poisson statistics and differences in replating frequency/copy number between cultures were evaluated by Wilcoxon two-sample test.

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Conflict of interest

The authors declare no conflict of interest.

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Acknowledgements

This work was performed with support from EC-DG research (FP6-NoE), CLINIGENE (LSHB-CT-2006-018933 to NM, OCH and CB) and the University of Lausanne (NM). UM and CB also received support from the Food and Drug Administration (project FDA-09-1056211-KP) and the German Ministry of Research and Education (BMBF project iGene, 01GU0813). The kind gift of plasmids by Dr G Felsenfeld is acknowledged. We thankJulien DeRoyer (LBAP, ENSC); Johanna Krause (Department of Experimental Hematology, Hannover); Dr Jérémy Catinot and Pieric Doriot for technical help.

AUTHOR CONTRIBUTION

AG designed and performed experiments on the identification and assay of insulators, analyzed data and wrote the paper with NN under the supervision of NM; C Bauche constructed insulators and insulated hybrid LTRs; CD and AA performed experiments on the implementation of insulators in viral vectors, and analyzed data under the supervision of OCH who initiated the program with NM; and UM and AS performed experiments to assay the cytotoxicity of insulated viral vectors, and analyzed data under the supervision of C Baum.

Supplementary Information accompanies the paper on Gene Therapy website