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. 2012 Mar 20;20(7):1400–1409. doi: 10.1038/mt.2012.50

The β-Globin Locus Control Region in Combination With the EF1α Short Promoter Allows Enhanced Lentiviral Vector-mediated Erythroid Gene Expression With Conserved Multilineage Activity

Claudia A Montiel-Equihua 1, Lin Zhang 1, Sean Knight 2, Heba Saadeh 3, Simone Scholz 4, Marlene Carmo 1, Maria E Alonso-Ferrero 1, Michael P Blundell 1, Aiste Monkeviciute 1, Reiner Schulz 3, Mary Collins 2, Yasuhiro Takeuchi 5, Manfred Schmidt 4, Lynette Fairbanks 6, Michael Antoniou 3, Adrian J Thrasher 1, H Bobby Gaspar 1,*
PMCID: PMC3381857  EMSID: UKMS43594  PMID: 22434141

Abstract

Some gene therapy strategies are compromised by the levels of gene expression required for therapeutic benefit, and also by the breadth of cell types that require correction. We designed a lentiviral vector system in which a transgene is under the transcriptional control of the short form of constitutively acting elongation factor 1α promoter (EFS) combined with essential elements of the locus control region of the β-globin gene (β-LCR). We show that the β-LCR can upregulate EFS activity specifically in erythroid cells but does not alter EFS activity in myeloid or lymphoid cells. Experiments using the green fluorescent protein (GFP) reporter or the human adenosine deaminase (ADA) gene demonstrate 3–7 times upregulation in vitro but >20 times erythroid-specific upregulation in vivo, the effects of which were sustained for 1 year. The addition of the β-LCR did not alter the mutagenic potential of the vector in in vitro mutagenesis (IM) assays although microarray analysis showed that the β-LCR upregulates ~9% of neighboring genes. This vector design therefore combines the benefits of multilineage gene expression with high-level erythroid expression, and has considerable potential for correction of multisystem diseases including certain lysosomal storage diseases through a hematopoietic stem cell (HSC) gene therapy approach.

Introduction

Correction of inherited diseases of the hematopoietic system through viral vector-mediated gene transfer into autologous hematopoietic stem cells (HSCs) is now a clinical reality and has been demonstrated in a number of successful clinical trials.1,2 At present this has predominantly been confined to treatment of diseases where specific hematopoietic lineages are defective. In other inherited disorders and especially enzyme defects, such as adenosine deaminase deficiency (ADA deficiency), where multiple organ systems both peripherally and centrally are affected, current methods of gene delivery to hematopoietic cells may be limited in their ability to treat nonhematopoietic abnormalities.

Erythrocytes are the most abundant cell lineage in the bloodstream and offer an attractive vehicle for expressing and delivering therapeutic proteins to several tissues. As has been shown previously, genetic modification of the erythroid lineage to express a therapeutic gene at high levels may be an effective strategy for systemic delivery.3 However, restriction of activity to the erythroid lineage may also limit efficiency where multilineage gene expression is also desirable. To achieve these characteristics in combination, we have designed a lentiviral construct in which the transgene is under the transcriptional control of the constitutively acting EFS (elongation factor 1α promoter short version, in which the first intron is deleted). In addition, the vector includes the essential elements of the locus control region of the β-globin gene (β-LCR) which is known to upregulate expression of the β-globin family to high levels specifically in erythroid cells.4 The β-LCR consists of 5 regions of erythroid-specific DNase I hypersensitivity (HS) and is functionally defined by its ability to confer on a gene linked in cis, physiological levels of gene expression that are directly proportional to gene copy number regardless of integration site in mice. Each β-LCR DNase I HS site possesses a functional core region of 200–300 bp, which contains a high density of erythroid-specific and ubiquitous transcription factor-binding elements.5 The β-LCR has previously been shownto drive high levels of erythroid-specific expression from heterologous nonerythroid promoters6,7 with the minimum requirements being a CAAT and CACCA or GC-rich (for example, Sp1) elements.4 but this has not been developed in the context of a vector delivery system.

In this study, we demonstrate the ability of this novel vector design to drive transgene expression in all hematopoietic lineages and also show that the β-LCR was able to upregulate EFS promoter activity specifically in erythrocytes. Since adenosine ADA deficiency has nonimmunological manifestations including skeletal, hepatic, and neurological defects, we generated vectors expressing the human ADA (hADA) gene and show systemic expression of this therapeutic transgene.

Results

DNAse HS sites 432 of the β-LCR are capable of upregulating EFS promoter activity in an erythroid-specific manner

We constructed a series of self-inactivating (SIN) lentiviral vectors (LV) with the EFS promoter (LV EFS GFP) alone, or in combination with the hypersensitive sites 4, 3, and 2 of the human β-globin locus control region (LV LCR EFS GFP) controlling the expression of the green fluorescent protein (GFP) gene or a codon-optimized version of the ADA gene (LV EFS ADA or LV LCR EFS ADA) (Figure 1). As controls, we also included a lentiviral vector in which the β-LCR is coupled with its native β-globin promoter (LV LCR BG GFP), a γ-retroviral construct with an intact long-terminal repeat from the spleen focus-forming virus (SFFV) long-terminal repeat (SF91 GFP) and a SIN LV construct with an internal SFFV promoter (HV SFFV GFP).

Figure 1.

Figure 1

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Lentiviral vector architecture. ADA, codon-optimized optimized adenosine deaminase gene; cPPT, central polypurine tract; EFS, elongation factor 1α short promoter; eGFP, enhanced green fluorescence protein gene; LCR, β-globin locus control region including HS4, HS3, HS2 fragments; pA, polyadenine tail; R, repeat region; Rev-responsive element; SFFV, spleen focus-forming U3 region; U3, unique region 3; U5, unique region 5; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element; βG, β-globin mini-gene; βGp, β-globin promoter; θ, primer binding site; ψ, packaging signal.

Murine erythroleukemia cells (MEL) were transduced at an multiplicity of infection (MOI) of 3 with LV EFS GFP, LV LCR EFS GFP, or with the LV LCR BG GFP vector as a positive control. MEL cells are transformed erythroid progenitors arrested at the proerythroblast stage of development, but can be induced to undergo terminal differentiation when cultured in the presence of dimethylsulphoxide (DMSO).8 GFP expression in transduced cells was detected by flow cytometry before and after 4 days of differentiation (Figure 2a,b). As expected, the β-LCR element enhanced the activity of the β-globin promoter (BG) by threefold and had a similar effect upon the activity of the EFS promoter. In contrast, the LV EFS GFP vector, which contains no β-LCR element, showed no evidence of GFP upregulation in differentiated MEL cells.

Figure 2.

Figure 2

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The β-globin gene (β-LCR) upregulates the activity of the elongation factor 1α promoter (EFS) promoter in erythroid in vitro model systems. Cell lines were transduced at an multiplicity of infection (MOI) of 5 with lentiviral vectors; half of murine erythroleukemia cell (MEL)-transduced cultures were exposed to 2% dimethylsulphoxide (DMSO) for 4 days to induce erythroid differentiation. Enhanced green fluorescence protein (eGFP) expression was determined by flow cytometry. Graphs are representative of three experiments. (a) Cytometry of MEL cells before (no fill) and after (gray fill) differentiation. (b) Ratio of GFP expression in MEL-transduced cells before and after differentiation, mean ± SD (triplicate); MFI, mean fluorescence intensity. (c) Ratio of GFP expression in human cells transduced with LV LCR EFS GFP or LV EFS GFP, mean ± SD (triplicate).

Next, the effect of the β-LCR on the EFS promoter was evaluated in cell lines of human origin. Jurkat, U937, and K562 cells were transduced at an MOI of 5 with the GFP vectors, and the GFP expression measured by flow cytometry (Figure 2c). While the β-LCR augmented the activity of the EFS promoter by threefold in K562, a cell line that possesses erythroid properties,9 it did not modify the performance of this promoter in a T cell line (Jurkat) and showed a decrease (20%) in a myelomonocyte line (U937).

The β-LCR enhances the expression of ADA in in vitro erythroid model systems

Jurkat, U937, and MEL cells were transduced at an MOI of 5 with the ADA-expressing SIN LV, and the ADA activity determined and normalized to the average vector copy number per cell (Figure 3a and Supplementary Figure S1). In a similar manner to that observed for GFP expression, ADA activity was sevenfold higher in differentiated MEL cells transduced with the LV LCR EFS ADA vector in comparison to the LV EFS ADA construct; whereas it remained the same in Jurkat cells and MEL cells before differentiation. A reduction of ADA activity (38%) was observed in the monocyte cell line U937.

Figure 3.

Figure 3

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The β-globin gene (β-LCR) increases adenosine deaminase (ADA) activity in cell lines and human primary cells. (a) ADA activity (nmol/hours/µg protein) ratio in cells transduced with LV LCR EFS ADA or LV EFS ADA. Cell lines were infected at an multiplicity of infection (MOI) of 5 with lentiviral vectors, half of murine erythroleukemia cell (MEL) transduced cultures were exposed to 2% dimethylsulphoxide (DMSO) (+D) for 4 days to induce erythroid differentiation. ADA expression was determined by enzymatic activity assay. Graphs are average of two experiments in duplicate; mean ± SEM (n = 4). (b) ADA activity (nmol/hours/µg protein or Hb) in NK or erythroid cultures from two ADA-deficient individuals and a normal control. CD34+ cells were obtained from the bone marrow of two ADA –ve donors and the cord blood of a normal delivery, transduced with LV LCR EFS ADA or LV EFS ADA at an MOI of 25 and differentiated in vitro to NK or erythrocytes; mean ± SEM (n = 2). EFS, elongation factor 1α promoter.

To investigate the ability of the β-LCR to boost the performance of the EFS promoter in human primary cells, CD34+ cells were isolated from the bone marrow of two ADA-deficient patients, transduced with the ADA-expressing LV at an MOI of 25 and differentiated in vitro into erythroid or NK cell lineages using optimized protocols,10,11 A sample of CD34+ cells from a normal cord blood sample was differentiated in parallel as a control (Figure 3b). The ADA activity per vector copy was similar between CD56+ NK cells transduced with LVs with or without the β-LCR and the wild-type control. However, in erythroid populations (GPA+), the activity in cells transduced with LCR-EFS vector was more than tenfold higher than in cells transduced with EFS vector and these activities were 100- and 10-fold higher, respectively, over the activity in the wild-type control (of note, the ADA activity levels in wild-type and untransduced erythroid cells were too low to be shown on the graph).

The β-LCR boosts erythroid-specific transgene expression in vivo

A cohort of 12 C57 BL/6 female mice were transplanted with lineage-negative (Lin-ve) cells, obtained from 12 C57 BL/6 male donors and transduced at an MOI of 50 with LV EFS GFP or LV LCR EFS GFP. Blood samples were obtained at 4, 12, and 52 weeks post-transplant and the expression of GFP measured by flow cytometry in the different cell lineages. Although a progressive decrease in the percentage of GFP-expressing cells was observed over time, particularly in the erythrocyte population (Supplementary Figure S2), the kinetics of the decrease was similar for both test groups. In erythrocytes, the decay rate for LV EFS GFP was k = 0.2381 and for LV LCR EFS GFP was k = 0.2955. A similar decrease was observed for the vector copy number (Figure 4d) and most probably relates to the preferential transduction of short-lived hematopoietic progenitors whose activity diminishes during the time period of this experiment.

Figure 4.

Figure 4

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β-Globin gene (β-LCR) enhances expression in vivo. C57BL/6 mice were transplanted with lineage-negative bone marrow cells transduced with LV LCR EFS GFP or LV EFS GFP at an multiplicity of infection (MOI) of 50. Blood samples were taken at 4 weeks, 12 weeks, and 52 weeks post-transplant. Mean ± SEM (n = 12 at 4 and 12 weeks, n = 10 at 52 weeks). (a) GFP expression in peripheral blood mononuclear cells (PBMC) at 4, 12, and 52 weeks post-transplant. (b) GFP expression in erythrocytes (TER119+) at 4, 12, and 52 weeks post-transplant. (c) GFP expression in leukocyte populations at 52 weeks post-transplant. (d) Vector copy number at 52 weeks post-transplant. EFS, elongation factor 1α promoter.

In terms of the level of transgene expression (mean fluorescence intensity), a decrease was observed over time in the peripheral blood mononuclear cell bulk population in both groups (Figure 4a). However, in erythrocytes, while there was a progressive reduction of transgene expression in the LV EFS GFP vector group, there was no reduction in the LV LCR EFS GFP cohort (Figure 4b). Notably, the MFI was significantly higher in the LV LCR EFS GFP group at all time-points and in particular, at 12 and 52 weeks post-transplant, when the MFI in LV LCR EFS GFP erythrocytes was >20-fold above the MFI in LV EFS GFP-transduced erythrocytes (Figure 4b).

Transgene expression in different leukocyte lineages at 52 week post-transplant (Figure 4c) showed no significant difference between test groups except in the NK1.1+ population where a slight reduction in MFI was observed in the cells that carried the LV LCR EFS GFP vector (P = 0.43). Of note, the GFP expression remained the same in the CD3, B220 and myeloid populations, regardless of the presence of the β-LCR.

A similar transplant experiment was performed in C57 BL/6 mice with the ADA-expressing LV (Figure 5). At 6 weeks post-transplant, the ADA activity in bulk peripheral blood mononuclear cells transduced with LV LCR EFS ADAwas not significantly different from the activity in LV EFS ADA corrected bulk peripheral blood mononuclear cells. However, strikingly the ADA activity in LV LCR EFS ADA-modified erythrocytes was again >20-fold higher than the ADA activity in LV EFS ADA-modified erythrocytes.

Figure 5.

Figure 5

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Adenosine deaminase (ADA) activity is higher in β-globin gene (β-LCR)-transduced erythroid lineage cells in vivo. C57BL/6 mice were transplanted with lineage-negative bone marrow cells transduced with LV LCR EFS ADA or LV EFS ADA at an multiplicity of infection (MOI) of 50. Blood samples were taken at 6 weeks post-transplant. Mean ± SEM (n = 4 for LV LCR EFS ADA or n = 5 for LV EFS ADA).The average vector copy number per cell (VCN) at this time point was 0.346 ± 0.102 for LV LCR EFS ADA and 1.108 ± 0.61 for LV EFS ADA. (a) ADA activity (nmol/hours/µg protein) in peripheral blood mononuclear cells. (b) ADA activity (nmol/hours/µg Hb) in erythrocytes. EFS, elongation factor 1α promoter.

Addition of the β-LCR does not alter the safety profile of the lentiviral vector

In order to analyze whether the addition of the β-LCR changes the potential of the vector to induce genotoxicity, two different in vitro mutagenesis (IM) assays were performed. The first IM assay identifies splice donor sites within the vector that can cause upregulation of cellular genes through splicing. Interleukin-3 (IL-3) independent mutant Bcl-15 cells, where the vector inserted in the growth hormone receptor gene (Ghr), activated Ghr by a vector splicing donor site being spliced to Ghr and caused cytokine-independent cell growth, were selected and analyzed.12,13 Whilst a vector with intact long-terminal repeat and internal SFFV promoter (HV SFFV eGFP) produced significant numbers of mutants by Ghr activation, neither the LV EFS GFP nor the LV LCR EFS GFP vectors gave rise to mutants that expressed vector-Ghr fusion mRNA transcripts (Table 1).

Table 1. IL-3 independence IM assay.

graphic file with name mt201250t1.jpg

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The second IM assay evaluates the capacity of the vector to transform primary hematopoietic cells by activation of proto-oncogenes in Lin-ve murine cells following high-level transduction. The self-renewal capacity of the clones was evaluated quantitatively with the WST1 cell proliferation assay (Figure 6b). The replating index was calculated by dividing the number of clones with a higher proliferation activity than the mock-treated cells by the average vector copy number per cell (Figure 6a). A retrovirus with intact SFFV based long-terminal repeat, included as a positive control, was able to produce numerous clones in this assay. In contrast, neither the LV EFS GFP nor the LV LCR EFS GFP vectors were able to generate transformed clones above background levels.

Figure 6.

Figure 6

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The addition of the β-globin gene (β-LCR) does not alter the safety profile of the lentiviral vector. Lineage-negative bone marrow cells were isolated from C57BL/6 mice, transduced at an multiplicity of infection (MOI) of 20, expanded in myeloid differentiation conditions, and then replated in limiting dilution so as to detect transformed clones due to proto-oncogene activation.38 (a) The replating index expresses the number of clones with a proliferation rate above the baseline established with mock-transduced cells, normalized to the vector copy number. Mean of four experiments. (b) The WST1 assay shows that in three separate experiments, clones derived from the SF91GFP vector transduction showed activity above that seen in mock-transduced cells. Any clones derived from LV LCR EFS GFP transductions were not above mock-transduced background levels in all three experiments. EFS, elongation factor 1α promoter.

Ability of the β-LCR to upregulate neighboring genes

Further experiments were performed to determine whether the LCR-EFS combination has any effect on the expression profile of genes surrounding LV-insertion sites. These experiments were performed in undifferentiated and differentiated MEL cell clones where the LCR element is known to be active (Figure 7). Eight independent MEL clones, containing a total of 36 different known insertions (ranging from clones with 1 insertion to clones with 11 insertions) were differentiated. RNA extracted from undifferentiated and differentiated cells was subject to microarray expression analysis. Levels of expression were compared to undifferentiated and differentiated MEL cells, which did not contain any vector insertions. We identified the probe sets detecting upregulation within ±200 kbp of an insertion. Only one unannotated probe set was upregulated beyond ±100 kb. We therefore made a more detailed analysis of the ±100 kb window. Of 166 probe sets within the ±100 kbp vicinity of the 36 insertions from all the clones, there were 98 upregulated probe sets in differentiated cells (Figure 7a) and 102 in undifferentiated cells (Figure 7b). Of the 98 probe sets upregulated in differentiated cells, only 15 in the ±100 kb vicinity of 36 different insertions showed upregulation of >50%. In undifferentiated MEL clones, only 7 of the probe sets surrounding the insertion sites were upregulated by >50%. A list of the genes corresponding to the probe sets upregulated >50% is provided (Supplementary Table S1). To evaluate the significance of these observations, we performed a permutation test. In each of 1,000 trials, we randomly picked an equivalent number of probe sets from all probe sets detecting upregulation, irrespective of their locations relative to insertion sites, and then counted the number of probe sets among them detecting a >50% fold-change. Comparing the observed numbers with the numbers generated by the permutation trials revealed that 15 probe sets detecting a >50% fold-change in differentiated cells was highly significant (P < 0.001). In undifferentiated cells, the observed number (seven) was not significant (P < 0.1).

Figure 7.

Figure 7

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β-Globin gene (β-LCR) elongation factor 1α promoter (EFS) upregulation of genes neighboring vector insertions sites. Log2 expression ratios (y-axis) for probe sets located within ±200 kbp of an insertion site in (a) differentiated and (b) undifferentiated murine erythroleukemia cell (MEL) cells. The x-axis represents the distance to the nearest insertion site. The three horizontal lines mark the log2 ratios (0.585, 1, and 2) that correspond to upregulation by 50, 100, and 300%, respectively. Probe sets detecting upregulation in excess of 50% have been labeled with the gene symbols from the Affymetrix annotation. (c) Significance of the gene expression enhancing cis effect of the insert in differentiated cells as a function of the distance between gene (probe set) and nearest insertion. The effect in terms of the log2 expression ratio was averaged over all probe sets within a given 100-kbp distance range from the nearest insertion (x-axis). The y-axis represents significance in terms of 1 – P value. (d) Similar to (c) for undifferentiated cells.

To determine the approximate range of the cis effect of the insert, we repeated the analysis for additional 100 kbp intervals of the distance between probe set and nearest insertion, up to a maximum interval of 450–550 kbp and using a step size of 50 kbp. We observed a sharp drop in significance between the 50–150 and 100–200 kbp intervals (Figure 7c (differentiated cells) and 7d (undifferentiated cells)), indicating that the cis effect of the insert is limited to within a distance of 150 kbp.

Discussion

This study was designed to generate a lentiviral vector design that would allow correction of inherited diseases in which multilineage yet high-level gene expression is desirable. We therefore adopted a vector design whereby the internal EFS promoter drives transgene expression in hematopoietic cells but the incorporation of essential elements of the β-globin LCR upregulates EFS activity specifically in erythrocytes. This would allow high-level transgene expression in circulating erythrocytes for systemic delivery in combination with mutilineage expression, for example allowing macrophages to permeate widespread tissues for local delivery.

The human β-LCR locus contains a cluster of related genes whose expression is limited to erythroid cells and is controlled by a number of cis-acting elements, principally the locus control region (β-LCR). The β-LCR is composed of five erythroid-specific DNAseI HS sites located upstream of entire cluster of human β-LCR-like genes and its task is to maintain an active chromatin structure, enhance expression in erythroid cells and protect the locus from silencing.14,15 Much of the transcriptional activating function of the β-LCR resides in HS sites 2 and 3, but site 4 is important in adult globin expression.16 The combination of the 432 LCR and the β-globin promoter is already used in LV to drive β-globin transcription, with the intention of limiting the expression of the transgene to the erythroid lineage.17 Previous studies both in vitro4,6,7 and in vivo15 have shown that the β-LCR can enhance erythroid-specific expression from heterologous nonerythroid promoters, although it has not previously been shown whether the inclusion of the LCR would restrict the promoter activity to the erythroid lineage and adversely affect the performance of the nonerythroid promoter in other lineages. Furthermore, this strategy and design in the context of a viral delivery system and its functioning in vivo and further clinical applicability has not been demonstrated.

Our study has focused on testing the combination of the β-LCR with the EFS, short version (EFS); a housekeeping gene promoter that has been tested in vectors for gene therapy of the hematopoietic system.18,19,20 The EFS promoter is two- to threefold less potent than the SFFV U3 region,18 but is tenfold less genotoxic21 and less prone to silencing by methylation.22 The EFS promoter has sequences that make it susceptible to LCR upregulation, that is, Sp1 elements.23 Our aim was to test whether this combination can be used to boost the expression of the transgene in erythroid cells, without affecting the activity of the promoter in cells of nonerythroid origin.

In differentiated erythroid, MEL cells or in the erythro leukemia K562 cell (Figures 2 and 3a), the β-LCR-EFS combination was able to enhance the expression of the transgene whereas in a human T cell leukemia line (Jurkat) it did not enhance, nor decrease the activity of the EFS promoter. In the monocytic cell line U937 there was a consistent decrease inexpression of the transgene by ~30% (Figures 2c and 3a). However, no such decrease was observed in the Mac1+ population, when the vectors were tested in a murine transplant model, even after 52 weeks post-transplant (Figure 4c). Whether or not the reduction in U937 cells is cell line-specific remains to be confirmed. The in vivo HSC transplant experiments either with the GFP or the ADA transgene show the erythroid specific upregulation effect even more clearly. With both transgenes, ~20 times more expression was seen in erythrocytes transduced with the LV LCR EFS vector in comparison with LV EFS construct (Figures 4 and 5). Long-term studies show that this effect is not diminished over time and even a year after transplant the erythroid-specific β-LCR upregulation effect remains intact (Figure 4b).These data correlate with previous studies in mouse models of transplantation which show that the LCR-BG combination is able to provide sustained transgene expression in red blood cells after >40 weeks post-transplant.24,25

One practical issue related to this vector design may be its size which is 4 kb larger than the control and thus may adversely affect titer and transduction efficiency. Although we did not see any reduction in vector titer, we noted a lower transduction efficiency of murine lin-ve cells (Figure 4d) which may relate to decreased viral entry and/or after entry degradation. Improving the purity of the viral preparation, altering stem cell transduction protocols or the use of novel agents such as proteasome inhibitors26 may alleviate these problems.

With respect to the safety aspects of this vector design, the EFS promoter on its own has been shown to have low mutagenicity in vitro.21 We therefore wanted to know whether the addition of the β-LCR elements would alter the safety profile of the vector. In order to address this question, IM assays were performed. In the first of these assays, where the mutagenesis potential is assessed based on the number of IL-3 independent clones arising in a population of vector-transduced cells,12 neither the LV EFS GFP nor the LV LCR EFS GFP vector were significantly different from mock-transduced cells (Table 1). Only one clone from each vector was obtained in three assay repeats; these clones were analyzed for IL-3 and growth hormone receptor expression by quantitative-PCR and no evidence of upregulation of either product was found (data not shown).In the second mutagenesis assay (IVIM) (Figure 6), where the mutagenesis potential is assessed based on the number of proliferating mutants arising in a population of transduced lineage-negative cells, neither vector was significantly mutagenic. A similar version of the β-LCR we employ here, in combination with the BG promoter, has already been tested in an IVIM assay and found to be able to produce mutant clones, but still with 100-fold less frequency than the SF91-GFP vector.27 In our hands, both the EFS- and the LCR-EFS-vectors were not able to produce “fit” transformed mutants as objectively determined by the WST1 assay, whereas the SF91 GFP vector clearly has transformation potential. This assay measures the transformation potential in early progenitors where the β-LCR is known to be active28 and these results suggest the addition of the LCR has no detrimental effect on the safety of the vector.

One caveat to these findings is that the mutagenic potential of the LCR may not be fully realized under predominantly myeloid cell culture conditions, and therefore the capacity to upregulate genes as erythroid differentiation proceeds is not determined. We therefore evaluated upregulation of neighboring genes in clonal populations where the integration site was molecularly defined.

Microarray analysis of differentiated MEL cell clones (where the β-LCR is likely to be most active) showed that of 98/166 probe sets within a ±100 kb of vector insertion were upregulated although only 15 of 166 (9%) showed upregulation of >50%. A step-size analysis revealed the upregulation effect of the β-LCR in our vector is limited to a distance of maximum 150 kb from the insertion site, shorter than that found with intact long-terminal repeat vectors.29 This is in agreement with the distance at which the β-LCR fulfils its function at its native locus (50–70 kb).4 A separate study analyzing the effect of the full human β-LCR inserted in a gene-dense region also showed that the most distal responsive gene was found ~150 kb away from the insertion.15

A previous study examining the effect of the β-globin LCR in a retroviral vector on neighboring genes in erythroid precursor clones and again using microarray expression analysis, found that 11% (6/66 transcripts) of genes within a ±300 kb window of vector insertions showed a twofold or greater difference in expression from control levels with some genes being downregulated.30 In this study, some of the erythroid clones were derived from bone marrow cells of mice that had been transplanted 6 months earlier with β-LCR vector-transduced lin-ve cells. These mice, despite evidence of gene dysregulation, showed no evidence of clonal dominance and abnormal pathology. The use of insulator sequences such as the chicken β-globin LCR HS-site 4 (cHS4) element, has been shown to decrease the transformation potential of LCR vectors in the IVIM assay and is likely to limit although not completely abrogate the effect on neighboring genes.27

The data presented here convincingly demonstrate that the β-LCR EFS vector design allows transgene expression in all blood lineages, and significantly higher expression in erythrocytes. This expression profile could be of significant benefit not only in the treatment of ADA-SCID but also in other inherited metabolic disorders, especially certain lysosomal storage diseases, where multiple organ systems are affected. Enzyme replacement therapies for a number of different diseases are available but these are limited by the inability of the exogenous enzyme to cross the blood brain barrier and effectively deliver enzyme to the central nervous system. The β-LCR EFS vector design would allow correction of both compartments through high levels of peripheral delivery by white cells and erythrocytes together with macrophage mediated delivery to the central nervous system. Clearly, further modifications such as incorporation of insulator sequences may improve the potential safety of this vector design but that withstanding; this strategy may hold considerable promise for gene therapy for a number of multisystem inherited diseases.

Materials and Methods

Vector construction. LV EFS GFP: the EFS promoter was obtained from the vector SIN-LV-EFS-γc (S.I. Thornhill, unpublished results) as a HincII/BamHI fragment, subcloned into pBluescript SK, and then removed and inserted into the P′HR-cppt-SEW vector,31 as an EcoRI/BamHI fragment replacing the SFFV promoter. LV-LCR-EFS-GFP: the β-LCRHS4 element core (275 bp) and flanking regions of 5′ 461 bp and 3′ 352 bp were amplified by PCR with the primers HS4 PCR forward-TTTGCGGCCGCTATCTCATTGCTGTTCGT and HS4 PCR reverse-TTTGCGGCCGCACAGAAGCTCATGCATT, giving the fragment NotI sites at each end. The PCR product was confirmed by sequencing and then inserted into the NotI site of the MA954 plasmid containing the HS3 (5′ 570 bp, 223 bp core, 3′ 400 bp) and the HS2 (5′ 715 bp, 388 bp core, 3′ 310 bp) fragments as employed in the GLOBE lentiviral construct.32 The β-LCR (HS4,3,2) was excised as an EcoRI fragment from the resulting MA954 HS4 and then linked upstream the EFS promoter in LV-EFS-GFP.

LV-EFS-ADA: a codon-optimized version of an ADAcDNA was commercially synthesized (GeneArt, Regensburg, Germany) and subcloned as an AfeI/SalI fragment into the pSRS11-EFS-γc vector,19 substituting the γc cDNA sequence. The fragment EFS-ADA was excised with ClaI/SalI and cloned into the plasmid pCCLsincpptW1.6hWasp-WPRE (Genethon, Evry-Cedex, France), substituting W1.6h-Wasp. LV-LCR-EFS-ADA: to insert the HS4, 3, 2 β-LCR fragment, a multicloning site was generated by aligning the primers MCS forward -CGATCTCGAGCCTGCAGGGATATCAT and MCS reverse-CGATGATATCCCTGCAGGCTCGAGAT, and cloning them into the ClaI site upstream EFS in the LV-EFS-ADA vector. The multicloning site provided the sites for XhoI and EcoRV. The β-LCR fragment was excised from the MA954 HS4 construct via XhoI/EcoRV digestion and inserted into LV-EFS-ADA. The SF91 enhanced GFP and pHV positive controls are as previously described.33,34

Cell lines. The Jurkat (human T cell leukemia), U937 (human leukemic monocyte lymphoma), and K562 (human erythroleukemia9) cell lines were maintained in RPMI medium (Invitrogen, Paisley, UK) supplemented with 10% fetal bovine serum (Sigma-Aldrich, Poole, UK) and 10 µg/ml each of penicillin and streptomycin. MEL and human embryonic kidney (HEK293T) cells were maintained in Dulbecco's modified Eagle's medium medium (Invitrogen), serum and penicillin–streptomycin as above. MS5 (mouse bone marrow stroma) cells were maintained in α-MEM medium (Invitrogen) with 20% serum, penicillin–streptomycin (Invitrogen). Cells were transduced with virus at a MOI of 5 in their corresponding culture medium; transgene expression was typically analyzed 3 days after transduction. All cells were cultured at 37 °C, 5% CO2.

MEL cell differentiation. MEL cells were seeded at a density of 2 × 105 cells/ml in Dulbecco's modified Eagle's medium medium supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 10 µg/ml each of penicillin and streptomycin (Invitrogen). Half of the cultures were induced to undergo terminal erythroid differentiation for 4 days by addition of 2% DMSO (Sigma-Aldrich), with medium replenished on day 2 after the start of the procedure.8

Lentiviral vector production and titration. Lentiviral stocks were produced in HEK293T cells by cotransfection of the packaging plasmids pMD.G2 (VSVG envelope) and pCMVΔ8.91 (gag-pol plasmid) with the corresponding LV construct, using polyethylenimine (Sigma-Aldrich) as previously described.31 The vector titre was determined in HEK293T cells transduced with serial dilutions of the viral suspension, harvested 3 days after exposure to the virus; the titer was determined by flow cytometry in the case of the GFP vectors (LV LCR EFS GFP: 0.86 × 10 E 9 virus particles/ml; LV EFS GFP: 2 × 10 E 9 virus particles/ml) or by quantitative-PCR of the WPRE region in the case of the ADA vectors that do not express GFP (LV LCR EFS ADA: 3.4 × 10 E 8 virus particles/ml, LV EFS ADA: 3.8 × 10 E 8 virus particles/ml).

DNA isolation and vector copy number determination. DNA was isolated from cell pellets with the DNeasy Blood and Tissue kit (Qiagen, West Sussex, UK), following the manufacturer's instructions. Average vector copy number per cell was determined by WPRE quantitative-PCR in Platinum Quantitative-PCR SuperMix-UDG with ROX (Invitrogen). Primer sequences for the WPRE region were WP forward-CGGGCCACAACTCCTCATAA and WP reverse-TTGCTTCCCGTATGGCTTTC (Invitrogen), and FAM-TCTCCTCCTTGTATAAATCCTGGTTGCTGTCTC-TAMRA probe (Eurofins-MWG Operon, Ebersberg, Germany).

ADA activity assay. Cells lysates were prepared in distilled water, typically 250 µl for 1 million cells. Three reaction tubes were prepared per sample, each containing 100 µl of phosphate buffered saline (Invitrogen), 75 µl of 10 mmol/l adenosine (Sigma-Aldrich); the tubes were prewarmed at 37 °C. Cell lysate (25 µl) was added to two reaction tubes and incubated at 37 °C for 10–30 minutes depending on the expected level of adenosine expression. To stop the reaction, 25 µl of 40% trichloroacetic acid (Sigma-Aldrich) were added to each tube. To establish the “time zero” baseline, 25 µl of cell lysate immediately followed by 25 µl of 40% trichloroacetic acid were added to the remaining reaction tube. The trichloroacetic acid was extracted from the reaction mixtures with water-saturated diethyl ether until pH >5.0 was reached. Inosine and hypoxantine levels in the clean reaction mixture, were determined on anion-pair HPLC Waters 2795 system with PDA detection (Waters, Milford, MA), using a Hyperclone ODS 5 µm column (Phenomenex, Torrance, CA) and isocratic separation system of 40 mm ammonium acetate(VWR BDH Prolabo, Lutterworth, UK) and 5 mmol/l tetrabutyl ammonium hydrogen sulphate (Sigma-Aldrich), at pH 2.70, and flow rate of 1 ml/minute. Peaks were identified by retention time and spectrum. The ADA activity (nmol/hour) was calculated using the formula: Activity = [(AUC Hx × 1.6613) + (AUC Ino × 1.6927)] × (lysate volume/injection volume) × (60 minutes/incubation time in minutes). The ADA activity was then normalized to the protein concentration, determined with the BCA kit (Pierce, Thermoscientific, Rockford, IL), or to the hemoglobin content determined by the Drabkins method.35,36

Human HSC selection and transduction. Bone marrow samples from ADA-deficient individuals and cord blood from a normal delivery were obtained with written, informed consent. The leukocytes were separated by density gradient centrifugation over a Ficoll-Paque layer (GE Healthcare, Little Chalfont, UK), and the CD34+ population was isolated using CD34 selection microbeads (Miltenyi-Biotec, Woking, UK). The cells were cultured at 37 °C, 5%CO2, at a density of 1 million/ml in serum-free Stem Span medium (Stem Cell Technologies, Grenoble, France) supplemented with 300 ng/ml hFlt3, 300 ng/ml hSCF, 100 ng/ml hTPO and 20 ng/ml hIL-3 (all Peprotech, Rocky Hill, NJ) for 16 hours and then transduced with lentivirus at a MOI of 20–25, for 16 hours.

NK and erythroid in vitro differentiation. For NK differentiation, transduced, and control CD34+ cells were seeded onto MS5 monolayers in 24-well plates at a concentration of 10,000 cells/well if obtained from bone marrow, or 1,000 cells per well if obtained from cord blood. The culture medium consisted of α-MEM medium (Invitrogen), 10% serum, 10 mmol/l HEPES (Gibco-Invitrogen, Paisley, UK), 10 mmol/l sodium pyruvate (Gibco-Invitrogen), 0.5 mg/ml Gentamicin (Gibco-Invitrogen), 10 µg/ml each of penicillin and streptomycin, 20 mmol/l L-glutamine (Gibco-Invitrogen), 2-mercaptoethanol 0.5 mmol/l (Gibco-Invitrogen), hSCF 50 ng/ml, hFlt3 50 ng/ml, hIL-3 10 ng/ml, hIL-7 20 ng/ml, and hIL-15 20 ng/ml. The medium was changed every 2–3 days and the cells were transferred to a new MS5 monolayer every week. The cells were harvested after 3–4 weeks of culture and stained with APC-CD56+ (BD Bioscience, Oxford, UK).

For erythroid differentiation, an adapted version of the protocol published by Giarratana et al. was followed.10 The basal medium formula was modified to contain serum-free Stem Span (Stem Cell Technologies), 4 mmol/l L-glutamine (Gibco-Invitrogen), 10 µg/ml each of penicillin and streptomycin, 20 mmol/l L-glutamine (Gibco-Invitrogen), ferrous nitrate 90 ng/ml (Sigma-Aldrich) and ferrous sulphate 900 ng/ml (Sigma-Aldrich). The cells were harvested on day 18 after the start of differentiation and stained for PE-Glycophorin A (eBioscience, Hatfield UK) and APC-CD71 (BD Bioscience).

Murine HSC transplant. All experiments were conducted according to Home Office animal welfare legislation. C57BL/6 mice were housed in specified pathogen-free conditions in individually ventilated cages (Tecniplast, Italy) and supplied with sterile food, water, and bedding. Lin-ve cells were isolated from the hind legs of male mice 8–10 weeks of age, using the lineage depletion kit (Miltenyi-Biotec). The cells were cultured at 37 °C, 5 %CO2, at a density of 1 million/ml in serum-free Stem Span (Stem Cell Technologies) supplemented with murine stem cell factor 100 ng/ml, mFlt3 100 ng/ml, hIL-11 100 ng/ml, and mIL-3 20 ng/ml (Peprotech), and transduced at a MOI of 50 with the LV for 16 hr. Recipient female mice 10–12 weeks of age were lethally irradiated with a dose of 10 Gy (Cs 137), split between 24 and 2 hours before injection. Each recipient was injected with at least 300,000 lineage-negative cells via the tail vein. Blood samples were taken via the tail vein at 4, 12 and 52 weeks post-transplant. Whole blood samples (2 µl) were stained with TER119-PE (eBioscience). For peripheral blood mononuclear cell isolation, red cell lysis was performed with RBC lysis buffer (Biolegend, San Diego, CA), and the leukocyte populations were marked with PE-CD3 (eBioscience), APC-B220 (BD Bioscience), APC-NK1.1 (BD Bioscience), PE-Cy7-Gr1 (eBioscience), or APC-Mac1 (eBioscience).

IM assay. As described in refs. 12,13. Briefly, 3.6 × 107 Bcl-15 cells were transduced with each vector at an MOI of 10 in the presence of IL-3 (10% WEHI supernatant). After 96 hours expansion the cells were replated in supernatant containing 1 µg/ml bGH (Prospec, East Brunswick, NJ), in the absence of IL-3 to select IL-3 independent mutants.

IM-WST1 assay. A modified version of the IVIM protocol37,38 was followed. After the 2-week expansion in 96-well plates step, half of the cells in each “clone” well were transferred into a new well containing 10 µl of WST1 reagent (Roche, Penzberg, Germany), and incubated at 37 °C, 5 %CO2 for 4 hours. The absorbance was measured with a FLUOstar Optima colorimeter (BMG Labtech, Offenburg, Germany) at a wavelength of 450 nm. Clones were considered positive when absorbance was over the baseline established with the mock-transduced cells. The replating efficiency was then calculated using L-Cal software (Stem Cell Technologies), normalized with the virus copy number and expressed as replating index.

MEL cell clones. MEL cells were transduced with LV LCR EFS GFP at an MOI of 3. The GFP+ cells were sorted on a MoFlo XDP (Beckman Coulter, High Wycombe, UK) using a 70 µm nozzle, voltage of 2,000–4,000 V and sheath pressure of 60 PSI, and seeded in limiting-dilution (1 cell/well) in a 96-well plate with conditioned medium (50% culture medium from unsorted transduced cells + 50% fresh culture medium). Sixteen clones were chosen based on their GFP expression pattern (low, medium, or high) and the insertion sites were determined by nrLAM-PCR and pyrosequencing.39 Five clones with multiple insertions (4, 6, or 11), and four clones with single insertions were chosen for microarray analysis. The chosen clones were cultured in the presence or absence of 2% DMSO (basal or differentiated state) and RNA was isolated using the RNEasy kit (Qiagen).

Microarray analysis. RNA samples were processed and hybridized on Mouse 1.0 ST arrays (Affymetrix, Fremont, CA). Summarization and between-array quantile normalization were performed using RMA.

Statistical analysis. T-test or Mann–Whitney tests were run in the GraphPad Prism4 Software and the significance level is expressed as follows: (***) if P < 0.001; (**) if 0.01 > P > 0.001; (*) if 0.05 > P >0.01; (ns) if P > 0.05.

SUPPLEMENTARY MATERIAL Figure S1. Percentage of GFP+ cells at 4, 12, and 52 weeks post-transplant. Figure S2. Erythroid differentiation characterization. Table S1. List of upregulated genes in MEL clones.

Acknowledgments

This work has been funded by grants from the Medical Research Council (C.A.M.-E. and L.Z.) and the NIHR Biomedical Research Centre at Great Ormond Street Hospital (C.A.M.-E. and A.J.T). The authors acknowledge the support of GOSHCC (H.B.G), Wellcome Trust (A.J.T and M.P.B.), European Commission's 7th Framework Program Contract 222878 (PERSIST) (S.S. and M.S.), UK Health and Safety Executive, Department of Health (M.C., Y.T., and S.K.), Histiocytosis Research Trust (M.C.), Research Councils UK (R.S.) and King's College London (H.S.).

Supplementary Material

Figure S1.

Percentage of GFP+ cells at 4, 12, and 52 weeks post-transplant.

Click here for additional data file. (12.3MB, tiff)
Figure S2.

Erythroid differentiation characterization.

Click here for additional data file. (234.9MB, tiff)
Table S1.

List of upregulated genes in MEL clones.

Click here for additional data file. (40.5KB, doc)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1.

Percentage of GFP+ cells at 4, 12, and 52 weeks post-transplant.

Click here for additional data file. (12.3MB, tiff)
Figure S2.

Erythroid differentiation characterization.

Click here for additional data file. (234.9MB, tiff)
Table S1.

List of upregulated genes in MEL clones.

Click here for additional data file. (40.5KB, doc)

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