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Efficiency of different fragment lengths of the ubiquitous chromatin opening element HNRPA2B1-CBX3 in driving human CD18 gene expression within self-inactivating lentiviral vectors for gene therapy applications
Gene 710 (2019) 265–272
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Research paper
Efficiency of different fragment lengths of the ubiquitous chromatin opening element HNRPA2B1-CBX3 in driving human CD18 gene expression within self-inactivating lentiviral vectors for gene therapy applications Chitra Gopinatha, Sarvani Chodisettya,1, Arkasubhra Ghoshb, Everette Jacob Remington Nelsona, a b
T
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Gene Therapy Laboratory, Department of Integrative Biology, School of Bio Sciences and Technology, Vellore Institute of Technology, Vellore 632 014, Tamil Nadu, India Grow Research Laboratory, Narayana Nethralaya Foundation, Bangalore 560 099, Karnataka, India
A R T I C LE I N FO
A B S T R A C T
Keywords: Leukocyte adhesion deficiency type 1 (LAD1) CD18 Ubiquitous chromatin opening element (UCOE) Gene therapy Lentiviral vectors Hematopoietic stem cells (HSCs)
Patients with leukocyte adhesion deficiency type 1 (LAD1) suffer from life-threatening bacterial infections due to mutations in the common β2 integrin subunit (CD18/ITGB2 gene). We tested different fragments of the ubiquitous chromatin opening element (UCOE) from the human HNRPA2B1-CBX3 locus for their efficiency in driving the human CD18 gene expression and compared it with that of an elongation factor 1 alpha promoter (EF1αL, 1169 bp; EF1αS 248 bp) and a murine stem cell virus (MSCV) promoter within the context of the same lentiviral vector backbone. These vectors were tested in vitro for the human CD18 gene expression on the surface of CD34+ hematopoietic stem cells (HSCs) isolated from both moderate and severe LAD1 patients. Among the promoters tested in the patients' CD34+ HSCs, only U631 bp, U652 bp, U1262 bp, 5′ 2.2 kb A2UCOE and EF1αS resulted in higher percentage of CD18+CD34+ cells comparable to that of the MSCV promoter. The U655 bp, U723 bp, U1296 bp, U2598 bp and EF1αL promoters resulted in comparatively lower numbers of CD18+CD34+ cells. This study would be useful in investigating the human CD18 gene expression in an ex vivo experiment to demonstrate the phenotypic correction of LAD1 in a pre-clinical model.
1. Introduction Leukocyte adhesion deficiency type 1 (LAD1) in humans is an autosomal recessive primary immunodeficiency that results from the lack of functional phagocytes. LAD1 is caused due to a defect in the cell surface glycoprotein complexes, such as LFA-1, Mac-1(CR3), p150,95 and CR4 resulting from mutations in the ITGB2 gene encoding the common integrin β2 or CD18 subunit (Anderson and Springer, 1987; Gahmberg et al., 1997). The disease is characterized by life-threatening bacterial infections, impaired wound healing and lack of pus formation due to a defective leukocyte adhesion cascade (Anderson et al., 1985; Fischer et al., 1983, 1988). The overall frequency of LAD1 is approximately 1 in 100,000. Previous studies carried out in a canine model of
LAD1 referred to as canine leukocyte adhesion deficiency (CLAD) showed reversal of the disease phenotype following gene therapy using gammaretroviral vectors incorporating a normal copy of the CD18 gene under the control of viral promoters (Bauer Jr et al., 2006). Viral promoters have a strong tendency to cause insertional mutagenesis which had led to the development of leukemia/myelodysplastic syndrome in a few patients in earlier gene therapy clinical trials (Hacein-Bey-Abina et al., 2003). However, a foamy virus vector incorporating the long terminal repeat (LTR) of a murine stem cell virus (MSCV) as internal promoter had been used in the treatment of CLAD dogs leading to reversal of the disease phenotype (Bauer et al., 2008). A long-term follow up by the same group further revealed that foamy virus-treated dogs continued to stay healthy for up to 7 years without any severe adverse
Abbreviations: LAD, leukocyte adhesion deficiency; CLAD, canine leukocyte adhesion deficiency; LV, lentivirus; SIN, self-inactivating; EF1α, elongation factor 1 alpha; PGK, phosphoglycerate kinase; MSCV, murine stem cell virus; ITGB, integrin beta chain; HNRP, heterogeneous nuclear ribonucleoprotein; CBX, chromobox homolog; UCOE, ubiquitous chromatin opening element; HEK, human embryonic kidney; EBV, Epstein-Barr virus; HSC, hematopoietic stem cell; BES, N,N-bis(2hydroxyethyl)-2-amino-ethanesulfonic acid; BBS, BES-buffered saline; MTA, material transfer agreement; RPMI, Roosevelt Park Memorial Institute; SCF, stem cell factor; TPO, thrombopoietin; Flt3, fms-like tyrosine kinase 3; FITC, fluorescein isothiocyanate; 7-AAD, 7-aminoactinomycin D; MOI, multiplicity of infection ⁎ Corresponding author at: Gene Therapy Laboratory, SMV124A, Department of Integrative Biology, School of Bio Sciences and Technology, Vellore Institute of Technology, Vellore 632 014, TN, India. E-mail address: [email protected] (E.J.R. Nelson). 1 Current affiliation: Research Institute, SRM Institute of Science and Technology, Chennai – 603 203, TN, India. https://doi.org/10.1016/j.gene.2019.06.016 Received 16 February 2019; Received in revised form 6 June 2019; Accepted 10 June 2019 Available online 11 June 2019 0378-1119/ © 2019 Elsevier B.V. All rights reserved.
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human CD18 gene. A pRRL.U2598.cCD18 plasmid which was originally derived from the pHR.SIN.cPPT.UCOE plasmid containing the 2.6 kb full-length UCOE served as the source for both the full-length and the 2.2 kb A2UCOE fragments. UCOE fragments were generated by PCR cloning of the full-length UCOE incorporated into the pHR.SIN.cPPT.UCOE plasmid using different sets of primers (unpublished data). The pCD18r4 plasmid containing the human CD18 gene served as the source for human CD18 cDNA. In all, six different cloning strategies were designed involving successful two-fragment and three-fragment ligations.
events related to gene therapy (Bauer et al., 2013). Meanwhile, preclinical studies conducted in CLAD dogs involving the use of promoters of housekeeping genes like human elongation factor 1 alpha (hEF1α) and human phosphoglycerate kinase (hPGK) demonstrated poor expression of the therapeutic gene in vivo (Nelson et al., 2010; Hunter et al., 2011a; Bauer et al., 2011). On the other hand, tissue-specific human CD11b and human CD18 proximal promoters were successfully tested in vivo in CLAD dogs reversing the disease phenotype (Hunter et al., 2011b). There is a great need for suitable alternative promoters to drive the expression of any therapeutic gene regardless of the cell type that could assure safety as well as efficacy for human gene therapy applications in the future. A novel human regulatory element from the heterogeneous nuclear ribonucleoprotein A2/B1–heterochromatin protein 1Hs-γ-chromobox homolog 3 (HNRPA2B1-CBX3) locus (Antoniou et al., 2003; Allen and Antoniou, 2007) known as the ubiquitous chromatin opening element (UCOE) had been previously shown to display reproducible and stable transgene expression within the context of a self-inactivating (SIN) lentiviral vector in the absence of classical enhancer activity (Zhang et al., 2007). It had also been shown to confer resistance to DNA methylation-mediated transgene silencing even upon integration into the heterochromatin regions of the host chromosome (Zhang et al., 2010; Knight et al., 2012; Pfaff et al., 2013). The UCOE could be used in combination with any specific promoter as an anti-silencing element or solely as an exogenous promoter to drive high, stable and long-term transgene expression. Elongation factor 1 alpha (EF1α) promoter incorporated into a SIN lentiviral vector has been demonstrated to drive high levels of therapeutic gene expression in hematopoietic progenitor cells (Salmon et al., 2000). The EF1α long fragment (EF1αL, 1169 bp) resulted in lower levels of CD18 expression in an EBV-transformed B cell line derived from an LAD1 patient (ZJ cells) as well as in canine CD34+ hematopoietic stem cells (HSCs) when compared to the EF1α short fragment (EF1αS, 248 bp) but its efficacy in vivo is still unknown (Nelson et al., 2010). In this study, a total of 13 novel SIN lentiviral vectors were constructed incorporating different promoters driving the human CD18 cDNA. Ten of these constructs were cloned each containing different promoter fragments of the UCOE, two containing the EF1αS and EF1αL fragments and another containing the MSCV promoter, all within the context of the same pCL20c lentiviral vector backbone. Since viral promoters had been shown to induce high levels of transgene expression in several studies conducted earlier (Hacein-Bey-Abina et al., 2003). The 2.2 kb A2UCOE and all the UCOE fragments containing only the HNRPA2B1 region resulted in higher percentage of CD18+CD34+ cells compared to the 2.6 kb full-length UCOE and the fragments containing only the CBX3 region or CBX3 along with a small portion of the HNRPA2B1. Among vector-transduced cells, only the percentage of cells staining positive with an anti-CD18 antibody were measured and not the density of CD18 molecules present on the surface of each cell. Human CD18 expression driven by EF1αS promoter was higher than that of EF1αL in vitro, similar to earlier observations with respect to canine CD18 expression (Nelson et al., 2010). In this paper, we have identified specific fragments of the UCOE that could be potentially tested and further applied to ex vivo gene therapy experiments towards the treatment of LAD1 in the future.
2.2. Production of lentiviruses in human embryonic kidney (HEK) 293T cells Lentivirus supernatants for each construct were collected from 4 × 15 cm dishes following transient co-transfections of HEK293T cells with each of the newly constructed transfer plasmids along with three helper plasmids such as, pCAG-KGP-1 (gag/pol), pCAG4-RTR (rev/tat), pHDM-G (env) at plasmid concentrations of 22.5, 14.6, 5.6 and 7.9 μg respectively per dish. Calcium phosphate method was used for the purpose of transfection. On Day - 1, HEK293T cells in DMEM (Thermo Fisher Scientific, Waltham, MA, USA) were seeded at an appropriate cell density in order to reach about 70% confluency on the day of transfection (Day 0). Plasmid DNAs at predetermined quantities were mixed with 2.5 M CaCl2 solution (Sigma Aldrich, St. Louis, MO, USA), which was further combined with a solution of 2× BBS, [N,N-bis(2hydroxyethyl)-2-amino-ethanesulfonic acid (BES)-buffered saline] in a drop-wise manner while being gently agitated on a vortex mixer. The mixture was then added on to the HEK293T cells and transferred to an incubator (37 °C; 3% CO2) overnight (about 16 h). On Day 1, the overnight medium was aspirated and discarded. The cells were carefully overlayed with fresh medium and transferred back to the incubator (37 °C; 10% CO2). 48 h and 72 h post-transfection the culture supernatants were harvested, filtered through a 0.45 μm filter to remove cellular debris, and further concentrated using XL-90 ultracentrifuge - SW28 swinging bucket rotor type (Beckman Coulter, Brea, CA, USA) at RCF 50,339 ×g (at rav, where r = 118.2 mm) for 2 h at 18 °C. The concentrated virus supernatants were stored in cryovials at −80 °C. 2.3. Cells Peripheral blood mobilized CD34+ HSCs obtained from moderate and severe LAD1 patients and the EBV-transformed B cell line derived from an LAD1 patient (ZJ cells) were kindly provided by Dennis Hickstein, MD, National Institutes of Health (NIH), USA as per the NIH Ethical Guidelines and Regulations following which a Materials Transfer Agreement (MTA) between NIH, VIT, and Salk Institute was signed. Normal CD34+ cells were obtained commercially (All cells, CA, USA; Cat# ABM015F). 2.4. Determination of viral titers Functional viral titers were determined in ZJ cells that lack endogenous CD18 expression. Individual wells were seeded with 100,000 ZJ cells suspended in RPMI-1640 medium (Thermo Fisher Scientific). The ZJ cells were transduced with concentrated virus supernatants added in serial dilutions to individual wells and the plates were transferred to an incubator (37 °C; 5% CO2) for 24 h. The following day (Day 1), culture media was carefully aspirated and discarded. The wells were supplemented with fresh medium, and the plates were transferred back to the incubator (37 °C; 5% CO2) for another 48–72 h. Functional viral titers were determined based on the levels of CD18 expression on the surface of ZJ cells. The transducing units per ml (TU/ml) for each 200× virus stock was calculated using the formula (% positive cells / 100) × (no. of cells) × (dilution factor) × 1000 (for ml) = TU/ml.
2. Materials and methods 2.1. Construction of SIN lentiviral vectors In silico design of cloning strategies was performed using Vector NTI Advance 9.1 software (Invitrogen Corporation, Carlsbad, CA, USA). Various pCL20c constructs with the canine CD18 gene each under the control of either the UCOE (different fragment lengths in bp), namely U655, U723, U1296, U652, U631 and U1262 or MSCV or EF1αL served as the backbone wherein the canine CD18 gene was swapped with the 266
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2.5. In vitro transduction of human LAD1 CD34+ HSCs
used as a reference standard to compare the percentage of CD18+CD34+ cells obtained from the newly made constructs. The MSCV promoter construct when tested at MOIs of 10 and 100 resulted in 20% and 19% of CD18+ cells, respectively. A 5–10% increase in surface CD18 expression above the background is reflective of successful virus-mediated gene transfer into CD34+ HSCs. About 2% of circulating CD18+ neutrophils following cell or gene therapy is considered therapeutic for patients with severe disease phenotype (< 1% endogenous CD18 expression).
All the newly cloned lentiviral constructs were tested for their efficiency of human CD18 expression by transduction into HSCs obtained from LAD1 patients. The CD34+ HSCs were pre-stimulated in X-VIVO 10 medium (Lonza, Basel, Switzerland) containing 1% HSA and a cytokine cocktail consisting of human stem cell factor (hSCF), human thrombopoietin (hTPO) and human fms-like tyrosine kinase 3 (hFlt3) ligand (PeproTech, Rocky Hill, NJ, USA) each at a final concentration of 100 ng/ml for 24 h. RetroNectin (Clontech/Takara Bio, Mountain View, CA, USA) at a concentration of 5 μg/cm2 (diluted from 100 μg/ml) was used to coat the 24 non-TC treated plates at least 24 h prior to seeding of cells. The virus supernatants generated from each of the constructs were tested at two different multiplicities of infection (MOI), 10 and 100.
3.2.1. Human CD18 expression driven by 2.6 kb full-length UCOE and 2.2 kb A2UOCE When the 2.6 kb UCOE cloned in both orientations i.e., 5′ (CBX3A2B1) and 3′ (A2B1-CBX3) were tested, no significant shift or increase in the number of CD18+ cells was observed. The 5′ 2.6 kb UCOE fragment resulted in 6.9% and 9.5% of CD18+ cells at MOIs of 10 and 100, respectively as compared to 9.2% and 7.5% by the 3′ 2.6 kb UCOE. In contrast, the 5′ A2UCOE fragment resulted in 14.8% and 18.1% of CD18+ cells at MOIs of 10 and 100 thereby achieving an actual increase of 6–10%. This was higher when compared to the levels obtained from both the 3′ A2UCOE (10.1% and 12.8%, respectively) and the EF1αS (13.8% and 14.3%, respectively) promoter fragments.
2.6. Flow cytometry analysis of human CD18 expression Percentage of surface CD18+ ZJ cells and CD34+ HSCs were assessed on Day 5 post-transduction by flow cytometry using a fluorescein isothiocyanate (FITC)-labeled mouse anti-human CD18 antibody (BD Pharmingen, clone 6.7, #555923). Due to insufficient LAD1 patient samples parallel experiments extending for 15 days post-transduction and vector copy number analysis could not be performed. FITC mouse IgG1κ served as the isotype control (clone MOPC-21, #555748) and 7aminoactinomycin D (7-AAD) was used to exclude the dead cell population (BD Pharmingen, Franklin Lakes, NJ, USA). Normal CD34+ cells obtained commercially used in the experiment served as a positive control for the percentage of normal CD18+CD34+ cell population.
3.2.2. Human CD18 expression driven by HNRPA2B1 UCOE fragments The U631 bp, U652 bp and U1262 bp fragments yielded higher numbers of CD18+ cells at MOIs of 10 and 100 respectively, such as 15.1% and 15.7% (U631 bp), 12.7% and 16.5% (U652 bp) and 11.4% and 16% (U1262 bp). The shift in the number of CD18+ cells of 3–8% observed with these fragments is comparable to those observed with the 5′ 2.2 kb A2UCOE and EF1αS fragments indicating that any of these fragments could be potentially tested further in ex vivo gene therapy experiments.
3. Results 3.1. Construction and testing of lentiviral vectors expressing hCD18
3.2.3. Human CD18 expression driven by CBX3 and CBX3-A2B1 UCOE fragments The U1296 bp fragment was not tested on CD34+ HSCs in vitro due to its poor functional viral titers as determined in the ZJ cells. The U655 bp and U723 bp promoters resulted in 11.1% and 10.4%, respectively at MOI 10 and 10.9% and 5.7% at MOI 100. These expression levels are comparatively lower when compared to those observed from the HNRPA2B1 UCOE, 2.2 kb A2UCOE (both orientations) and the EF1αS promoter fragments.
SIN lentiviral vectors expressing hCD18 from UCOE, EF1α or MSCV promoter was constructed using the pCL20c backbone (Fig. 1a). The 2.6 kb UCOE fragment is cloned from the region containing the EcoRI site in intron 2 of CBX3 and the TthIII I site in exon 1 of HNRPA2B1. The UCOE fragments namely, U631 bp, U652 bp and U1262 bp are exclusively from the HNRPA2B1 region whereas the U655 bp fragment is entirely from the CBX3 region. The U1296 bp, U723 bp, 2.2 kb A2UCOE and 2.6 kb UCOE fragments contain both the HNRPA2B1 and CBX3 regions. All newly constructed clones were verified by restriction digestion analysis followed by Sanger sequencing. The binding sites for key transcription factors such as c-MYB, C/EBP α and β that are responsible for myeloid differentiation, Sp1 and PU.1 in particular are pertinent to transcriptional activation of the CD18 gene as depicted in Fig. 1b (source: alggen.lsi.upc.edu). The concentrated viruses were titrated by infecting ZJ cells with a range of dilutions from an original 200× stock in conditioned media. Functional viral titers obtained from the various lentiviral constructs were in the range from 1 × 107 to 1 × 1010 TU/ml. The U1296 bp fragment resulted in very low functional viral titers and hence was not tested further in LAD1 patient CD34+ HSCs.
3.3. Gene transfer efficiency in severe LAD1 CD34+ HSCs The HNRPA2B1 UCOE fragments (U631 bp, U652 bp and U1262 bp), the 5′ 2.2 kb A2UCOE and the 5′ 2.6 kb UCOE fragments alone were further tested in CD34+ HSCs obtained from a severe LAD1 patient whose cells are almost completely devoid of endogenous CD18 expression (< 1%). The HNRPA2B1 UCOE fragments yielded CD18+ levels of 3.3% and 2.9% (U631 bp), 5% and 3.4% (U652 bp), 1.6% and 1.7% (U1262 bp) at MOIs of 10 and 100, respectively. In contrast, the 5′ 2.2 kb A2UCOE and the 5′ 2.6 kb UCOE fragments resulted in 1.5% and 2%, and 1.7% and 0.7% of CD18+ cells (Fig. 3a, b).
3.2. Gene transfer efficiency in moderate LAD1 CD34+ HSCs
4. Discussion
The percentage of cells positive for CD18 assessed on Day 5 posttransduction in CD34+ HSCs from a moderate LAD1 patient are represented in Fig. 2a, b along with normal CD34+ cells used as positive control. The untransduced CD34+ HSCs had a background fluorescence of 8.6% attributed to endogenous CD18 expression seen in moderate LAD1 patients (1–10%). The EF1αS promoter resulted in higher percentages of CD18+ cells at MOIs of both 10 and 100 (13.8% and 14.3%, respectively) when compared to the EF1αL promoter which yielded very poor levels in vitro (6.8% and 5.9%). MSCV promoter was merely
LAD1 children with < 1% circulating levels of CD18+ neutrophils are severely affected who could succumb to life-threatening bacterial infections within one year of birth unless treated with cell or gene therapy since repeated antibiotic or granulocyte infusions could only alleviate symptoms and not cure the disease. In contrast, patients with 1–10% of circulating CD18+ neutrophils have only a moderate disease phenotype with better chances of survival following management therapies for several years (Anderson et al., 1985). Even an increase of about 2% in the levels of circulating CD18+ neutrophils following cell 267
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Fig. 1. a) Schematic of the various SIN lentiviral vector constructs. The U655 bp, U723 bp, U1296 bp, U631 bp, U652 bp, U1262 bp, U2.6 kb and 2.2 kb A2UCOE (in both 5′ and 3′ orientations) fragments were cloned into the same pCL20c vector backbone. The EF1α short and long fragments (EF1αS and EF1αL), and the MSCV promoter were also cloned into the same backbone and tested simultaneously. b) Schematic of the various transcription factor binding sites across the UCOE. Binding sites for transcription factors such as c-MYB, C/EBP α and β, Sp1 and PU.1 are shown. The two bold arrows at the top indicate direction of CBX3 and HNRPA2B1 promoter activity. CBX3 promoter region (+381 to +1431) and the HNRPA2B1 region (−1383 TO +165) are relative to TSS. The thin arrows represent orientation of the promoter cloned.
standard. The U631 bp, U652 bp, U1262 bp, 5′ A2UCOE fragments all resulted in a 3–10% increase in CD18+CD34+ cells while the U655 bp, U723 bp, U1296 bp, 3′ A2UCOE and both the 5′ and 3′ 2.6 kb UCOE fragments all yielded < 4% of CD18+CD34+ HSCs. Clearly, an extended in vitro study performed in replicates could further confirm the efficiency of HNRPA2B1-CBX3 UCOE fragments as potential therapeutic gene-driving promoters since the results seem in vitro initial studies seem considerable. Further testing of these HNRPA2B1-CBX3 UCOE fragments as promoters in biological repeats an ex vivo model would help determine their efficacy in driving long-term and stable CD18 gene expression. UCOE has a unique advantage when compared to other promoters such as hEF1αS and hPGK that have been demonstrated to undergo post-transcriptional gene silencing in vivo over a period of time as previously reported (Nelson et al., 2010; Salmon et al., 2000; Chang et al., 2006). The hPGK promoter also has performed poorly as reported in a CLAD gene therapy study (Hunter et al., 2011a). The presence of histone acetylation marks in regions of DNA methylation across the HNRPA2B1-CBX3 locus indicates that the UCOE is capable of active transcription even when integrated in regions that are heavily methylated such as the heterochromatin and also regions that are prone to transcriptional gene silencing such as in the context of stem or progenitor cell types (Antoniou et al., 2003). This nature is beneficial for gene therapy studies involving stem cells in general to drive any gene that is prone to epigenetic modifications, and in particular,
or gene therapy is considered therapeutic for patients with severe disease phenotype. The UCOE tested in this study is derived from the central region of the HNRPA2B1-CBX3 locus which is an unmethylated CpG-rich island with active histone modification marks and operates as an enhancerless element. This region extends across the promoters and transcription initiation sites of dual divergently expressing genes and is about 2.6 kb in length (Zhang et al., 2007). The CBX3 promoter region extends from +381 to +1431 and the HNRPA2B1 region extends from −1383 TO +165 relative to their respective gene transcription start sites (TSS). These fragments were designed based on the distribution of transcription factor binding sites that are important for differentiation into the hematopoietic lineage, specifically driving CD18 gene expression and also due to the need for a minimal efficient promoter. The U631 bp, U652 bp, and U1262 bp promoters were derived from the HNRPA2B1 region of the element, whereas the U655 bp, U723 bp and U1296 bp were derived either exclusively from the CBX3 region or containing both the regions. The 2.6 kb UCOE and the 2.2 kb A2UCOE fragments were tested in both 5′ and 3′ orientations. All these promoter fragments were tested in vitro and compared with the human EF1α promoter and the MSCV promoter for their efficiency in driving the expression of the human CD18 gene. When tested in CD34+ HSCs obtained from a patient with moderate LAD1, the MSCV promoter resulted in 11–12% higher numbers of CD18+ cells above the background which served as a reference 268
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Fig. 2. Gene transfer efficiency in moderate LAD1 CD34+ HSCs. a) CD34+ HSCs obtained from a moderate LAD1 patient were transduced with SIN lentiviral vectors expressing human CD18 following 24 h pre-stimulation with a cytokine cocktail consisting of hSCF, hTPO and hFlt3 ligand each at a concentration of 100 ng/ml. Each vector was tested at MOIs of 10 and 100. Five days post-transduction, the cells were analyzed on a flow cytometer using a FITC-labeled mouse anti-human CD18 antibody. b) Percentages of human CD18 surface expression obtained from each of the constructs are shown along with endogenous background CD18 expression seen in untransduced cells from the same patient.
treatment of Wiskott-Aldrich syndrome and was shown to drive efficient, stable and long-term expression of the therapeutic gene in HSCs both in vitro and in vivo using a foamy virus vector (Uchiyama et al., 2012). It is noteworthy to mention that this 0.6 kb A2UCOE fragment is part of the U652 bp fragment from the HNRPA2B1 region that was used in our study. Recently, a 0.7 kb fragment containing the alternate first exons and promoter of CBX3 that is known to have anti-gene silencing effects similar to that of the 1.2 kb A2UCOE studied (Zhang et al., 2010) resulted in stable but very low transgene expression levels when tested in combination with viral or cellular promoters (Muller-Kuller et al., 2015). Zhang and colleagues have also reported that the HNRPA2B1 UCOE promoters are capable of driving significant levels of transgene expression with least variations in transcription in contrast to those
hematopoietic stem cells such as in our study for the treatment of LAD1 with SIN lentiviral vectors. The 1.6 kb into HNRPA2B1 and 1 kb into CBX3 (making a total of 2.6 kb full-length UCOE that is tested here) is completely unmethylated. Despite this feature, our data demonstrates differential levels of CD18 expression driven by the various fragments of the UCOE promoter. The HNRPA2B1 UCOE and 5′ 2.2 kb A2UCOE fragments were more efficient in driving CD18 gene expression when compared to all the other fragments. This could be attributed to the presence of transcription factor binding sites such as PU.1 and Sp1 that are essential for early myeloid differentiation, in particular CD18 gene expression on the surface of neutrophils. The efficiency of a 0.6 kb A2UCOE fragment comprising only the HNRPA2B1 region, without the CBX3 had been tested in the 269
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Fig. 2. (continued)
from the CBX3 region which is known to be highly variable among cell types. Hence, it is critical to choose an appropriate fragment from the dual divergent CBX3-HNRPA2B1 promoters based on the nature of the cell type to render high or low levels of gene expression at the same time offering protection against gene silencing either by HNRPA2B1or CBX3 orientation (Zhang et al., 2010). In conclusion, from among the various fragments of the UCOE tested as internal promoters within the context of a SIN lentiviral vector, the 5′ 2.2 kb A2UCOE and the fragments derived from the HNRPA2B1 region of the UCOE were more efficient in driving the expression of the human CD18 gene when transduced into CD34+ HSCs in vitro. These potential UCOE fragments warrant further investigation in vivo to closely analyse anti-gene silencing properties, efficacy and also monitor their side effects in altering neighbouring genes of the target cell that could be potentially cytotoxic (Muller-Kuller et al., 2015; Zhang et al., 2010) before considering them for applications in gene therapy clinical trials. Fig. 2. (continued) 270
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Fig. 3. Gene transfer efficiency in severe LAD1 CD34+ HSCs. a) CD34+ HSCs obtained from a severe LAD1 patient were transduced with SIN lentiviral vectors expressing human CD18 following 24 h pre-stimulation with a cytokine cocktail consisting of hSCF, hTPO and hFlt3 ligand each at a concentration of 100 ng/ml. Each vector was tested at MOIs of 10 and 100. Five days post-transduction, the cells were analyzed on a flow cytometer using a FITC-labeled mouse anti-human CD18 antibody. b) Percentages of human CD18 surface expression obtained from each of the constructs are shown.
Declaration of Competing Interest
Chang, A.H., Stephan, M.T., Sadelain, M., 2006. Stem cell-derived erythroid cells mediate long-term systemic protein delivery. Nat. Biotechnol. 24, 1017–1021. Fischer, A., Trung, P.H., Descamps-Latscha, B., Lisowska-Grospierre, B., Gerota, I., Perez, N., Scheinmetzler, C., Durandy, A., Virelizier, J.L., Griscelli, C., 1983. Bone-marrow transplantation for inborn error of phagocytic cells associated with defective adherence, chemotaxis, and oxidative response during opsonised particle phagocytosis. Lancet 2, 473–476. Fischer, A., Lisowska-Grospierre, B., Anderson, D.C., Springer, T.A., 1988. Leukocyte adhesion deficiency: molecular basis and functional consequences. Immunodefic. Rev. 1, 39–54. Gahmberg, C.G., Tolvanen, M., Kotovuori, P., 1997. Leukocyte adhesion: structure and function of human leukocyte P-integrins and their cellular ligands. Eur. J. Biochem. 245, 215–232. Hacein-Bey-Abina, S., Von Kalle, C., Schmidt, M., Mccormack, M.P., Wulffraat, N., Leboulch, P., Lim, A., Osborne, C.S., Pawliuk, R., Morillon, E., Sorensen, R., Forster, A., Fraser, P., Cohen, J.I., De Saint Basile, G., Alexander, I., Wintergerst, U., Frebourg, T., Aurias, A., Stoppa-Lyonnet, D., Romana, S., Radford-Weiss, I., Gross, F., Valensi, F., Delabesse, E., Macintyre, E., Sigaux, F., Soulier, J., Leiva, L.E., Wissler, M., Prinz, C., Rabbitts, T.H., Le Deist, F., Fischer, A., Cavazzana-Calvo, M., 2003. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 302, 415–419. Hunter, M.J., Zhao, H., Tuschong, L.M., Bauer Jr., T.R., Burkholder, T.H., Persons, D.A., Hickstein, D.D., 2011a. Gene therapy for canine leukocyte adhesion deficiency with lentiviral vectors using the murine stem cell virus and human phosphoglycerate kinase promoters. Hum. Gene Ther. 22, 689–696. Hunter, M.J., Tuschong, L.M., Fowler, C.J., Bauer Jr., T.R., Burkholder, T.H., Hickstein, D.D., 2011b. Gene therapy of canine leukocyte adhesion deficiency using lentiviral vectors with human CD11b and CD18 promoters driving canine CD18 expression. Mol. Ther. 19, 113–121. Knight, S., Zhang, F., Mueller-Kuller, U., Bokhoven, M., Gupta, A., Broughton, T., Sha, S., Antoniou, M.N., Brendel, C., Grez, M., Thrasher, A.J., Collins, M., Takeuchi, Y., 2012. Safer, silencing-resistant lentiviral vectors: optimization of the ubiquitous chromatinopening element through elimination of aberrant splicing. J. Virol. 86, 9088–9095. Muller-Kuller, U., Ackermann, M., Kolodziej, S., Brendel, C., Fritsch, J., Lachmann, N., Kunkel, H., Lausen, J., Schambach, A., Moritz, T., Grez, M., 2015. A minimal ubiquitous chromatin opening element (UCOE) effectively prevents silencing of juxtaposed heterologous promoters by epigenetic remodeling in multipotent and pluripotent stem cells. Nucleic Acids Res. 43, 1577–1592. Nelson, E.J., Tuschong, L.M., Hunter, M.J., Bauer Jr., T.R., Burkholder, T.H., Hickstein, D.D., 2010. Lentiviral vectors incorporating a human elongation factor 1alpha promoter for the treatment of canine leukocyte adhesion deficiency. Gene Ther. 17, 672–677. Pfaff, N., Lachmann, N., Ackermann, M., Kohlscheen, S., Brendel, C., Maetzig, T., Niemann, H., Antoniou, M.N., Grez, M., Schambach, A., Cantz, T., Moritz, T., 2013. A ubiquitous chromatin opening element prevents transgene silencing in pluripotent stem cells and their differentiated progeny. Stem Cells 31, 488–499. Salmon, P., Kindler, V., Ducrey, O., Chapuis, B., Zubler, R.H., Trono, D., 2000. High-level transgene expression in human hematopoietic progenitors and differentiated blood lineages after transduction with improved lentiviral vectors. Blood 96, 3392–3398. Uchiyama, T., Adriani, M., Jagadeesh, G.J., Paine, A., Candotti, F., 2012. Foamy virus vector-mediated gene correction of a mouse model of Wiskott-Aldrich syndrome. Mol. Ther. 20, 1270–1279. Zhang, F., Thornhill, S.I., Howe, S.J., Ulaganathan, M., Schambach, A., Sinclair, J., Kinnon, C., Gaspar, H.B., Antoniou, M., Thrasher, A.J., 2007. Lentiviral vectors containing an enhancer-less ubiquitously acting chromatin opening element (UCOE) provide highly reproducible and stable transgene expression in hematopoietic cells. Blood 110, 1448–1457. Zhang, F., Frost, A.R., Blundell, M.P., Bales, O., Antoniou, M.N., Thrasher, A.J., 2010. A ubiquitous chromatin opening element (UCOE) confers resistance to DNA methylation-mediated silencing of lentiviral vectors. Mol. Ther. 18, 1640–1649.
The authors declare that they have NO affiliations with or involvement in any organization or entity pertaining to the subject matter or materials discussed in this manuscript. Acknowledgements The plasmids used in this study were originally constructed by the senior author (EJRN) during his post-doctoral training at the National Institutes of Health (NIH), Bethesda, MD, USA. Plasmids and cells used in this study were generously gifted by EJRN's former mentor at the NIH, Dennis Hickstein, MD. The authors would also like to thank Adrian Thrasher, MD, PhD (University College London, UK) and Arthur Nienhuis, MD (St. Jude Children's Research Hospital, Memphis, TN, USA) for generously gifting the pHR plasmid (UCOE) and the pCL20c backbone, respectively. Research work carried out in this article was funded by the DBT Ramalingaswami Fellowship (EJRN) and the Indian Council of Medical Research (EJRN) grants. This work was supported in part by the Fulbright-Nehru Doctoral Research Fellowship (CG) which was carried out under the supervision of Inder Verma, PhD at the Salk Institute for Biological Studies, La Jolla, CA, USA. References Allen, M.L., Antoniou, M., 2007. Correlation of DNA methylation with histone modifications across the HNRPA2B1-CBX3 ubiquitously acting chromatin open element (UCOE). Epigenetics 2, 227–236. Anderson, D.C., Springer, T.A., 1987. Leukocyte adhesion deficiency: an inherited defect in the Mac-1, LFA-1, and p150,95 glycoproteins. Annu. Rev. Med. 38, 175–194. Anderson, D.C., Schmalsteig, F.C., Finegold, M.J., Hughes, B.J., Rothlein, R., Miller, L.J., Kohl, S., Tosi, M.F., Jacobs, R.L., Waldrop, T.C., Goldman, A.S., Shearer, W.T., Springer, T.A., 1985. The severe and moderate phenotypes of heritable mac-1, LFA-1 deficiency: their quantitative definition and relation to leukocyte dysfunction and clinical features. J. Infect. Dis. 152, 668–689. Antoniou, M., Harland, L., Mustoe, T., Williams, S., Holdstock, J., Yague, E., Mulcahy, T., Griffiths, M., Edwards, S., Ioannou, P.A., Mountain, A., Crombie, R., 2003. Transgenes encompassing dual-promoter CpG islands from the human TBP and HNRPA2B1 loci are resistant to heterochromatin-mediated silencing. Genomics 82, 269–279. Bauer Jr., T.R., Hai, M., Tuschong, L.M., Burkholder, T.H., Gu, Y.C., Sokolic, R.A., Ferguson, C., Dunbar, C.E., Hickstein, D.D., 2006. Correction of the disease phenotype in canine leukocyte adhesion deficiency using ex vivo hematopoietic stem cell gene therapy. Blood 108, 3313–3320. Bauer Jr., T.R., Allen, J.M., Hai, M., Tuschong, L.M., Khan, I.F., Olson, E.M., Adler, R.L., Burkholder, T.H., Gu, Y.C., Russell, D.W., Hickstein, D.D., 2008. Successful treatment of canine leukocyte adhesion deficiency by foamy virus vectors. Nat. Med. 14, 93–97. Bauer Jr., T.R., Olson, E.M., Huo, Y., Tuschong, L.M., Allen, J.M., Li, Y., Burkholder, T.H., Russell, D.W., 2011. Treatment of canine leukocyte adhesion deficiency by foamy virus vectors expressing CD18 from a PGK promoter. Gene Ther. 18, 553–559. Bauer Jr., T.R., Tuschong, L.M., Calvo, K.R., Shive, H.R., Burkholder, T.H., Karlsson, E.K., West, R.R., Russell, D.W., Hickstein, D.D., 2013. Long-term follow-up of foamy viral vector-mediated gene therapy for canine leukocyte adhesion deficiency. Mol. Ther. 21, 964–972.
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Research paper
Efficiency of different fragment lengths of the ubiquitous chromatin opening element HNRPA2B1-CBX3 in driving human CD18 gene expression within self-inactivating lentiviral vectors for gene therapy applications Chitra Gopinatha, Sarvani Chodisettya,1, Arkasubhra Ghoshb, Everette Jacob Remington Nelsona, a b
T
⁎
Gene Therapy Laboratory, Department of Integrative Biology, School of Bio Sciences and Technology, Vellore Institute of Technology, Vellore 632 014, Tamil Nadu, India Grow Research Laboratory, Narayana Nethralaya Foundation, Bangalore 560 099, Karnataka, India
A R T I C LE I N FO
A B S T R A C T
Keywords: Leukocyte adhesion deficiency type 1 (LAD1) CD18 Ubiquitous chromatin opening element (UCOE) Gene therapy Lentiviral vectors Hematopoietic stem cells (HSCs)
Patients with leukocyte adhesion deficiency type 1 (LAD1) suffer from life-threatening bacterial infections due to mutations in the common β2 integrin subunit (CD18/ITGB2 gene). We tested different fragments of the ubiquitous chromatin opening element (UCOE) from the human HNRPA2B1-CBX3 locus for their efficiency in driving the human CD18 gene expression and compared it with that of an elongation factor 1 alpha promoter (EF1αL, 1169 bp; EF1αS 248 bp) and a murine stem cell virus (MSCV) promoter within the context of the same lentiviral vector backbone. These vectors were tested in vitro for the human CD18 gene expression on the surface of CD34+ hematopoietic stem cells (HSCs) isolated from both moderate and severe LAD1 patients. Among the promoters tested in the patients' CD34+ HSCs, only U631 bp, U652 bp, U1262 bp, 5′ 2.2 kb A2UCOE and EF1αS resulted in higher percentage of CD18+CD34+ cells comparable to that of the MSCV promoter. The U655 bp, U723 bp, U1296 bp, U2598 bp and EF1αL promoters resulted in comparatively lower numbers of CD18+CD34+ cells. This study would be useful in investigating the human CD18 gene expression in an ex vivo experiment to demonstrate the phenotypic correction of LAD1 in a pre-clinical model.
1. Introduction Leukocyte adhesion deficiency type 1 (LAD1) in humans is an autosomal recessive primary immunodeficiency that results from the lack of functional phagocytes. LAD1 is caused due to a defect in the cell surface glycoprotein complexes, such as LFA-1, Mac-1(CR3), p150,95 and CR4 resulting from mutations in the ITGB2 gene encoding the common integrin β2 or CD18 subunit (Anderson and Springer, 1987; Gahmberg et al., 1997). The disease is characterized by life-threatening bacterial infections, impaired wound healing and lack of pus formation due to a defective leukocyte adhesion cascade (Anderson et al., 1985; Fischer et al., 1983, 1988). The overall frequency of LAD1 is approximately 1 in 100,000. Previous studies carried out in a canine model of
LAD1 referred to as canine leukocyte adhesion deficiency (CLAD) showed reversal of the disease phenotype following gene therapy using gammaretroviral vectors incorporating a normal copy of the CD18 gene under the control of viral promoters (Bauer Jr et al., 2006). Viral promoters have a strong tendency to cause insertional mutagenesis which had led to the development of leukemia/myelodysplastic syndrome in a few patients in earlier gene therapy clinical trials (Hacein-Bey-Abina et al., 2003). However, a foamy virus vector incorporating the long terminal repeat (LTR) of a murine stem cell virus (MSCV) as internal promoter had been used in the treatment of CLAD dogs leading to reversal of the disease phenotype (Bauer et al., 2008). A long-term follow up by the same group further revealed that foamy virus-treated dogs continued to stay healthy for up to 7 years without any severe adverse
Abbreviations: LAD, leukocyte adhesion deficiency; CLAD, canine leukocyte adhesion deficiency; LV, lentivirus; SIN, self-inactivating; EF1α, elongation factor 1 alpha; PGK, phosphoglycerate kinase; MSCV, murine stem cell virus; ITGB, integrin beta chain; HNRP, heterogeneous nuclear ribonucleoprotein; CBX, chromobox homolog; UCOE, ubiquitous chromatin opening element; HEK, human embryonic kidney; EBV, Epstein-Barr virus; HSC, hematopoietic stem cell; BES, N,N-bis(2hydroxyethyl)-2-amino-ethanesulfonic acid; BBS, BES-buffered saline; MTA, material transfer agreement; RPMI, Roosevelt Park Memorial Institute; SCF, stem cell factor; TPO, thrombopoietin; Flt3, fms-like tyrosine kinase 3; FITC, fluorescein isothiocyanate; 7-AAD, 7-aminoactinomycin D; MOI, multiplicity of infection ⁎ Corresponding author at: Gene Therapy Laboratory, SMV124A, Department of Integrative Biology, School of Bio Sciences and Technology, Vellore Institute of Technology, Vellore 632 014, TN, India. E-mail address: [email protected] (E.J.R. Nelson). 1 Current affiliation: Research Institute, SRM Institute of Science and Technology, Chennai – 603 203, TN, India. https://doi.org/10.1016/j.gene.2019.06.016 Received 16 February 2019; Received in revised form 6 June 2019; Accepted 10 June 2019 Available online 11 June 2019 0378-1119/ © 2019 Elsevier B.V. All rights reserved.
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human CD18 gene. A pRRL.U2598.cCD18 plasmid which was originally derived from the pHR.SIN.cPPT.UCOE plasmid containing the 2.6 kb full-length UCOE served as the source for both the full-length and the 2.2 kb A2UCOE fragments. UCOE fragments were generated by PCR cloning of the full-length UCOE incorporated into the pHR.SIN.cPPT.UCOE plasmid using different sets of primers (unpublished data). The pCD18r4 plasmid containing the human CD18 gene served as the source for human CD18 cDNA. In all, six different cloning strategies were designed involving successful two-fragment and three-fragment ligations.
events related to gene therapy (Bauer et al., 2013). Meanwhile, preclinical studies conducted in CLAD dogs involving the use of promoters of housekeeping genes like human elongation factor 1 alpha (hEF1α) and human phosphoglycerate kinase (hPGK) demonstrated poor expression of the therapeutic gene in vivo (Nelson et al., 2010; Hunter et al., 2011a; Bauer et al., 2011). On the other hand, tissue-specific human CD11b and human CD18 proximal promoters were successfully tested in vivo in CLAD dogs reversing the disease phenotype (Hunter et al., 2011b). There is a great need for suitable alternative promoters to drive the expression of any therapeutic gene regardless of the cell type that could assure safety as well as efficacy for human gene therapy applications in the future. A novel human regulatory element from the heterogeneous nuclear ribonucleoprotein A2/B1–heterochromatin protein 1Hs-γ-chromobox homolog 3 (HNRPA2B1-CBX3) locus (Antoniou et al., 2003; Allen and Antoniou, 2007) known as the ubiquitous chromatin opening element (UCOE) had been previously shown to display reproducible and stable transgene expression within the context of a self-inactivating (SIN) lentiviral vector in the absence of classical enhancer activity (Zhang et al., 2007). It had also been shown to confer resistance to DNA methylation-mediated transgene silencing even upon integration into the heterochromatin regions of the host chromosome (Zhang et al., 2010; Knight et al., 2012; Pfaff et al., 2013). The UCOE could be used in combination with any specific promoter as an anti-silencing element or solely as an exogenous promoter to drive high, stable and long-term transgene expression. Elongation factor 1 alpha (EF1α) promoter incorporated into a SIN lentiviral vector has been demonstrated to drive high levels of therapeutic gene expression in hematopoietic progenitor cells (Salmon et al., 2000). The EF1α long fragment (EF1αL, 1169 bp) resulted in lower levels of CD18 expression in an EBV-transformed B cell line derived from an LAD1 patient (ZJ cells) as well as in canine CD34+ hematopoietic stem cells (HSCs) when compared to the EF1α short fragment (EF1αS, 248 bp) but its efficacy in vivo is still unknown (Nelson et al., 2010). In this study, a total of 13 novel SIN lentiviral vectors were constructed incorporating different promoters driving the human CD18 cDNA. Ten of these constructs were cloned each containing different promoter fragments of the UCOE, two containing the EF1αS and EF1αL fragments and another containing the MSCV promoter, all within the context of the same pCL20c lentiviral vector backbone. Since viral promoters had been shown to induce high levels of transgene expression in several studies conducted earlier (Hacein-Bey-Abina et al., 2003). The 2.2 kb A2UCOE and all the UCOE fragments containing only the HNRPA2B1 region resulted in higher percentage of CD18+CD34+ cells compared to the 2.6 kb full-length UCOE and the fragments containing only the CBX3 region or CBX3 along with a small portion of the HNRPA2B1. Among vector-transduced cells, only the percentage of cells staining positive with an anti-CD18 antibody were measured and not the density of CD18 molecules present on the surface of each cell. Human CD18 expression driven by EF1αS promoter was higher than that of EF1αL in vitro, similar to earlier observations with respect to canine CD18 expression (Nelson et al., 2010). In this paper, we have identified specific fragments of the UCOE that could be potentially tested and further applied to ex vivo gene therapy experiments towards the treatment of LAD1 in the future.
2.2. Production of lentiviruses in human embryonic kidney (HEK) 293T cells Lentivirus supernatants for each construct were collected from 4 × 15 cm dishes following transient co-transfections of HEK293T cells with each of the newly constructed transfer plasmids along with three helper plasmids such as, pCAG-KGP-1 (gag/pol), pCAG4-RTR (rev/tat), pHDM-G (env) at plasmid concentrations of 22.5, 14.6, 5.6 and 7.9 μg respectively per dish. Calcium phosphate method was used for the purpose of transfection. On Day - 1, HEK293T cells in DMEM (Thermo Fisher Scientific, Waltham, MA, USA) were seeded at an appropriate cell density in order to reach about 70% confluency on the day of transfection (Day 0). Plasmid DNAs at predetermined quantities were mixed with 2.5 M CaCl2 solution (Sigma Aldrich, St. Louis, MO, USA), which was further combined with a solution of 2× BBS, [N,N-bis(2hydroxyethyl)-2-amino-ethanesulfonic acid (BES)-buffered saline] in a drop-wise manner while being gently agitated on a vortex mixer. The mixture was then added on to the HEK293T cells and transferred to an incubator (37 °C; 3% CO2) overnight (about 16 h). On Day 1, the overnight medium was aspirated and discarded. The cells were carefully overlayed with fresh medium and transferred back to the incubator (37 °C; 10% CO2). 48 h and 72 h post-transfection the culture supernatants were harvested, filtered through a 0.45 μm filter to remove cellular debris, and further concentrated using XL-90 ultracentrifuge - SW28 swinging bucket rotor type (Beckman Coulter, Brea, CA, USA) at RCF 50,339 ×g (at rav, where r = 118.2 mm) for 2 h at 18 °C. The concentrated virus supernatants were stored in cryovials at −80 °C. 2.3. Cells Peripheral blood mobilized CD34+ HSCs obtained from moderate and severe LAD1 patients and the EBV-transformed B cell line derived from an LAD1 patient (ZJ cells) were kindly provided by Dennis Hickstein, MD, National Institutes of Health (NIH), USA as per the NIH Ethical Guidelines and Regulations following which a Materials Transfer Agreement (MTA) between NIH, VIT, and Salk Institute was signed. Normal CD34+ cells were obtained commercially (All cells, CA, USA; Cat# ABM015F). 2.4. Determination of viral titers Functional viral titers were determined in ZJ cells that lack endogenous CD18 expression. Individual wells were seeded with 100,000 ZJ cells suspended in RPMI-1640 medium (Thermo Fisher Scientific). The ZJ cells were transduced with concentrated virus supernatants added in serial dilutions to individual wells and the plates were transferred to an incubator (37 °C; 5% CO2) for 24 h. The following day (Day 1), culture media was carefully aspirated and discarded. The wells were supplemented with fresh medium, and the plates were transferred back to the incubator (37 °C; 5% CO2) for another 48–72 h. Functional viral titers were determined based on the levels of CD18 expression on the surface of ZJ cells. The transducing units per ml (TU/ml) for each 200× virus stock was calculated using the formula (% positive cells / 100) × (no. of cells) × (dilution factor) × 1000 (for ml) = TU/ml.
2. Materials and methods 2.1. Construction of SIN lentiviral vectors In silico design of cloning strategies was performed using Vector NTI Advance 9.1 software (Invitrogen Corporation, Carlsbad, CA, USA). Various pCL20c constructs with the canine CD18 gene each under the control of either the UCOE (different fragment lengths in bp), namely U655, U723, U1296, U652, U631 and U1262 or MSCV or EF1αL served as the backbone wherein the canine CD18 gene was swapped with the 266
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2.5. In vitro transduction of human LAD1 CD34+ HSCs
used as a reference standard to compare the percentage of CD18+CD34+ cells obtained from the newly made constructs. The MSCV promoter construct when tested at MOIs of 10 and 100 resulted in 20% and 19% of CD18+ cells, respectively. A 5–10% increase in surface CD18 expression above the background is reflective of successful virus-mediated gene transfer into CD34+ HSCs. About 2% of circulating CD18+ neutrophils following cell or gene therapy is considered therapeutic for patients with severe disease phenotype (< 1% endogenous CD18 expression).
All the newly cloned lentiviral constructs were tested for their efficiency of human CD18 expression by transduction into HSCs obtained from LAD1 patients. The CD34+ HSCs were pre-stimulated in X-VIVO 10 medium (Lonza, Basel, Switzerland) containing 1% HSA and a cytokine cocktail consisting of human stem cell factor (hSCF), human thrombopoietin (hTPO) and human fms-like tyrosine kinase 3 (hFlt3) ligand (PeproTech, Rocky Hill, NJ, USA) each at a final concentration of 100 ng/ml for 24 h. RetroNectin (Clontech/Takara Bio, Mountain View, CA, USA) at a concentration of 5 μg/cm2 (diluted from 100 μg/ml) was used to coat the 24 non-TC treated plates at least 24 h prior to seeding of cells. The virus supernatants generated from each of the constructs were tested at two different multiplicities of infection (MOI), 10 and 100.
3.2.1. Human CD18 expression driven by 2.6 kb full-length UCOE and 2.2 kb A2UOCE When the 2.6 kb UCOE cloned in both orientations i.e., 5′ (CBX3A2B1) and 3′ (A2B1-CBX3) were tested, no significant shift or increase in the number of CD18+ cells was observed. The 5′ 2.6 kb UCOE fragment resulted in 6.9% and 9.5% of CD18+ cells at MOIs of 10 and 100, respectively as compared to 9.2% and 7.5% by the 3′ 2.6 kb UCOE. In contrast, the 5′ A2UCOE fragment resulted in 14.8% and 18.1% of CD18+ cells at MOIs of 10 and 100 thereby achieving an actual increase of 6–10%. This was higher when compared to the levels obtained from both the 3′ A2UCOE (10.1% and 12.8%, respectively) and the EF1αS (13.8% and 14.3%, respectively) promoter fragments.
2.6. Flow cytometry analysis of human CD18 expression Percentage of surface CD18+ ZJ cells and CD34+ HSCs were assessed on Day 5 post-transduction by flow cytometry using a fluorescein isothiocyanate (FITC)-labeled mouse anti-human CD18 antibody (BD Pharmingen, clone 6.7, #555923). Due to insufficient LAD1 patient samples parallel experiments extending for 15 days post-transduction and vector copy number analysis could not be performed. FITC mouse IgG1κ served as the isotype control (clone MOPC-21, #555748) and 7aminoactinomycin D (7-AAD) was used to exclude the dead cell population (BD Pharmingen, Franklin Lakes, NJ, USA). Normal CD34+ cells obtained commercially used in the experiment served as a positive control for the percentage of normal CD18+CD34+ cell population.
3.2.2. Human CD18 expression driven by HNRPA2B1 UCOE fragments The U631 bp, U652 bp and U1262 bp fragments yielded higher numbers of CD18+ cells at MOIs of 10 and 100 respectively, such as 15.1% and 15.7% (U631 bp), 12.7% and 16.5% (U652 bp) and 11.4% and 16% (U1262 bp). The shift in the number of CD18+ cells of 3–8% observed with these fragments is comparable to those observed with the 5′ 2.2 kb A2UCOE and EF1αS fragments indicating that any of these fragments could be potentially tested further in ex vivo gene therapy experiments.
3. Results 3.1. Construction and testing of lentiviral vectors expressing hCD18
3.2.3. Human CD18 expression driven by CBX3 and CBX3-A2B1 UCOE fragments The U1296 bp fragment was not tested on CD34+ HSCs in vitro due to its poor functional viral titers as determined in the ZJ cells. The U655 bp and U723 bp promoters resulted in 11.1% and 10.4%, respectively at MOI 10 and 10.9% and 5.7% at MOI 100. These expression levels are comparatively lower when compared to those observed from the HNRPA2B1 UCOE, 2.2 kb A2UCOE (both orientations) and the EF1αS promoter fragments.
SIN lentiviral vectors expressing hCD18 from UCOE, EF1α or MSCV promoter was constructed using the pCL20c backbone (Fig. 1a). The 2.6 kb UCOE fragment is cloned from the region containing the EcoRI site in intron 2 of CBX3 and the TthIII I site in exon 1 of HNRPA2B1. The UCOE fragments namely, U631 bp, U652 bp and U1262 bp are exclusively from the HNRPA2B1 region whereas the U655 bp fragment is entirely from the CBX3 region. The U1296 bp, U723 bp, 2.2 kb A2UCOE and 2.6 kb UCOE fragments contain both the HNRPA2B1 and CBX3 regions. All newly constructed clones were verified by restriction digestion analysis followed by Sanger sequencing. The binding sites for key transcription factors such as c-MYB, C/EBP α and β that are responsible for myeloid differentiation, Sp1 and PU.1 in particular are pertinent to transcriptional activation of the CD18 gene as depicted in Fig. 1b (source: alggen.lsi.upc.edu). The concentrated viruses were titrated by infecting ZJ cells with a range of dilutions from an original 200× stock in conditioned media. Functional viral titers obtained from the various lentiviral constructs were in the range from 1 × 107 to 1 × 1010 TU/ml. The U1296 bp fragment resulted in very low functional viral titers and hence was not tested further in LAD1 patient CD34+ HSCs.
3.3. Gene transfer efficiency in severe LAD1 CD34+ HSCs The HNRPA2B1 UCOE fragments (U631 bp, U652 bp and U1262 bp), the 5′ 2.2 kb A2UCOE and the 5′ 2.6 kb UCOE fragments alone were further tested in CD34+ HSCs obtained from a severe LAD1 patient whose cells are almost completely devoid of endogenous CD18 expression (< 1%). The HNRPA2B1 UCOE fragments yielded CD18+ levels of 3.3% and 2.9% (U631 bp), 5% and 3.4% (U652 bp), 1.6% and 1.7% (U1262 bp) at MOIs of 10 and 100, respectively. In contrast, the 5′ 2.2 kb A2UCOE and the 5′ 2.6 kb UCOE fragments resulted in 1.5% and 2%, and 1.7% and 0.7% of CD18+ cells (Fig. 3a, b).
3.2. Gene transfer efficiency in moderate LAD1 CD34+ HSCs
4. Discussion
The percentage of cells positive for CD18 assessed on Day 5 posttransduction in CD34+ HSCs from a moderate LAD1 patient are represented in Fig. 2a, b along with normal CD34+ cells used as positive control. The untransduced CD34+ HSCs had a background fluorescence of 8.6% attributed to endogenous CD18 expression seen in moderate LAD1 patients (1–10%). The EF1αS promoter resulted in higher percentages of CD18+ cells at MOIs of both 10 and 100 (13.8% and 14.3%, respectively) when compared to the EF1αL promoter which yielded very poor levels in vitro (6.8% and 5.9%). MSCV promoter was merely
LAD1 children with < 1% circulating levels of CD18+ neutrophils are severely affected who could succumb to life-threatening bacterial infections within one year of birth unless treated with cell or gene therapy since repeated antibiotic or granulocyte infusions could only alleviate symptoms and not cure the disease. In contrast, patients with 1–10% of circulating CD18+ neutrophils have only a moderate disease phenotype with better chances of survival following management therapies for several years (Anderson et al., 1985). Even an increase of about 2% in the levels of circulating CD18+ neutrophils following cell 267
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Fig. 1. a) Schematic of the various SIN lentiviral vector constructs. The U655 bp, U723 bp, U1296 bp, U631 bp, U652 bp, U1262 bp, U2.6 kb and 2.2 kb A2UCOE (in both 5′ and 3′ orientations) fragments were cloned into the same pCL20c vector backbone. The EF1α short and long fragments (EF1αS and EF1αL), and the MSCV promoter were also cloned into the same backbone and tested simultaneously. b) Schematic of the various transcription factor binding sites across the UCOE. Binding sites for transcription factors such as c-MYB, C/EBP α and β, Sp1 and PU.1 are shown. The two bold arrows at the top indicate direction of CBX3 and HNRPA2B1 promoter activity. CBX3 promoter region (+381 to +1431) and the HNRPA2B1 region (−1383 TO +165) are relative to TSS. The thin arrows represent orientation of the promoter cloned.
standard. The U631 bp, U652 bp, U1262 bp, 5′ A2UCOE fragments all resulted in a 3–10% increase in CD18+CD34+ cells while the U655 bp, U723 bp, U1296 bp, 3′ A2UCOE and both the 5′ and 3′ 2.6 kb UCOE fragments all yielded < 4% of CD18+CD34+ HSCs. Clearly, an extended in vitro study performed in replicates could further confirm the efficiency of HNRPA2B1-CBX3 UCOE fragments as potential therapeutic gene-driving promoters since the results seem in vitro initial studies seem considerable. Further testing of these HNRPA2B1-CBX3 UCOE fragments as promoters in biological repeats an ex vivo model would help determine their efficacy in driving long-term and stable CD18 gene expression. UCOE has a unique advantage when compared to other promoters such as hEF1αS and hPGK that have been demonstrated to undergo post-transcriptional gene silencing in vivo over a period of time as previously reported (Nelson et al., 2010; Salmon et al., 2000; Chang et al., 2006). The hPGK promoter also has performed poorly as reported in a CLAD gene therapy study (Hunter et al., 2011a). The presence of histone acetylation marks in regions of DNA methylation across the HNRPA2B1-CBX3 locus indicates that the UCOE is capable of active transcription even when integrated in regions that are heavily methylated such as the heterochromatin and also regions that are prone to transcriptional gene silencing such as in the context of stem or progenitor cell types (Antoniou et al., 2003). This nature is beneficial for gene therapy studies involving stem cells in general to drive any gene that is prone to epigenetic modifications, and in particular,
or gene therapy is considered therapeutic for patients with severe disease phenotype. The UCOE tested in this study is derived from the central region of the HNRPA2B1-CBX3 locus which is an unmethylated CpG-rich island with active histone modification marks and operates as an enhancerless element. This region extends across the promoters and transcription initiation sites of dual divergently expressing genes and is about 2.6 kb in length (Zhang et al., 2007). The CBX3 promoter region extends from +381 to +1431 and the HNRPA2B1 region extends from −1383 TO +165 relative to their respective gene transcription start sites (TSS). These fragments were designed based on the distribution of transcription factor binding sites that are important for differentiation into the hematopoietic lineage, specifically driving CD18 gene expression and also due to the need for a minimal efficient promoter. The U631 bp, U652 bp, and U1262 bp promoters were derived from the HNRPA2B1 region of the element, whereas the U655 bp, U723 bp and U1296 bp were derived either exclusively from the CBX3 region or containing both the regions. The 2.6 kb UCOE and the 2.2 kb A2UCOE fragments were tested in both 5′ and 3′ orientations. All these promoter fragments were tested in vitro and compared with the human EF1α promoter and the MSCV promoter for their efficiency in driving the expression of the human CD18 gene. When tested in CD34+ HSCs obtained from a patient with moderate LAD1, the MSCV promoter resulted in 11–12% higher numbers of CD18+ cells above the background which served as a reference 268
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Fig. 2. Gene transfer efficiency in moderate LAD1 CD34+ HSCs. a) CD34+ HSCs obtained from a moderate LAD1 patient were transduced with SIN lentiviral vectors expressing human CD18 following 24 h pre-stimulation with a cytokine cocktail consisting of hSCF, hTPO and hFlt3 ligand each at a concentration of 100 ng/ml. Each vector was tested at MOIs of 10 and 100. Five days post-transduction, the cells were analyzed on a flow cytometer using a FITC-labeled mouse anti-human CD18 antibody. b) Percentages of human CD18 surface expression obtained from each of the constructs are shown along with endogenous background CD18 expression seen in untransduced cells from the same patient.
treatment of Wiskott-Aldrich syndrome and was shown to drive efficient, stable and long-term expression of the therapeutic gene in HSCs both in vitro and in vivo using a foamy virus vector (Uchiyama et al., 2012). It is noteworthy to mention that this 0.6 kb A2UCOE fragment is part of the U652 bp fragment from the HNRPA2B1 region that was used in our study. Recently, a 0.7 kb fragment containing the alternate first exons and promoter of CBX3 that is known to have anti-gene silencing effects similar to that of the 1.2 kb A2UCOE studied (Zhang et al., 2010) resulted in stable but very low transgene expression levels when tested in combination with viral or cellular promoters (Muller-Kuller et al., 2015). Zhang and colleagues have also reported that the HNRPA2B1 UCOE promoters are capable of driving significant levels of transgene expression with least variations in transcription in contrast to those
hematopoietic stem cells such as in our study for the treatment of LAD1 with SIN lentiviral vectors. The 1.6 kb into HNRPA2B1 and 1 kb into CBX3 (making a total of 2.6 kb full-length UCOE that is tested here) is completely unmethylated. Despite this feature, our data demonstrates differential levels of CD18 expression driven by the various fragments of the UCOE promoter. The HNRPA2B1 UCOE and 5′ 2.2 kb A2UCOE fragments were more efficient in driving CD18 gene expression when compared to all the other fragments. This could be attributed to the presence of transcription factor binding sites such as PU.1 and Sp1 that are essential for early myeloid differentiation, in particular CD18 gene expression on the surface of neutrophils. The efficiency of a 0.6 kb A2UCOE fragment comprising only the HNRPA2B1 region, without the CBX3 had been tested in the 269
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Fig. 2. (continued)
from the CBX3 region which is known to be highly variable among cell types. Hence, it is critical to choose an appropriate fragment from the dual divergent CBX3-HNRPA2B1 promoters based on the nature of the cell type to render high or low levels of gene expression at the same time offering protection against gene silencing either by HNRPA2B1or CBX3 orientation (Zhang et al., 2010). In conclusion, from among the various fragments of the UCOE tested as internal promoters within the context of a SIN lentiviral vector, the 5′ 2.2 kb A2UCOE and the fragments derived from the HNRPA2B1 region of the UCOE were more efficient in driving the expression of the human CD18 gene when transduced into CD34+ HSCs in vitro. These potential UCOE fragments warrant further investigation in vivo to closely analyse anti-gene silencing properties, efficacy and also monitor their side effects in altering neighbouring genes of the target cell that could be potentially cytotoxic (Muller-Kuller et al., 2015; Zhang et al., 2010) before considering them for applications in gene therapy clinical trials. Fig. 2. (continued) 270
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Fig. 3. Gene transfer efficiency in severe LAD1 CD34+ HSCs. a) CD34+ HSCs obtained from a severe LAD1 patient were transduced with SIN lentiviral vectors expressing human CD18 following 24 h pre-stimulation with a cytokine cocktail consisting of hSCF, hTPO and hFlt3 ligand each at a concentration of 100 ng/ml. Each vector was tested at MOIs of 10 and 100. Five days post-transduction, the cells were analyzed on a flow cytometer using a FITC-labeled mouse anti-human CD18 antibody. b) Percentages of human CD18 surface expression obtained from each of the constructs are shown.
Declaration of Competing Interest
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The authors declare that they have NO affiliations with or involvement in any organization or entity pertaining to the subject matter or materials discussed in this manuscript. Acknowledgements The plasmids used in this study were originally constructed by the senior author (EJRN) during his post-doctoral training at the National Institutes of Health (NIH), Bethesda, MD, USA. Plasmids and cells used in this study were generously gifted by EJRN's former mentor at the NIH, Dennis Hickstein, MD. The authors would also like to thank Adrian Thrasher, MD, PhD (University College London, UK) and Arthur Nienhuis, MD (St. Jude Children's Research Hospital, Memphis, TN, USA) for generously gifting the pHR plasmid (UCOE) and the pCL20c backbone, respectively. Research work carried out in this article was funded by the DBT Ramalingaswami Fellowship (EJRN) and the Indian Council of Medical Research (EJRN) grants. This work was supported in part by the Fulbright-Nehru Doctoral Research Fellowship (CG) which was carried out under the supervision of Inder Verma, PhD at the Salk Institute for Biological Studies, La Jolla, CA, USA. References Allen, M.L., Antoniou, M., 2007. Correlation of DNA methylation with histone modifications across the HNRPA2B1-CBX3 ubiquitously acting chromatin open element (UCOE). Epigenetics 2, 227–236. Anderson, D.C., Springer, T.A., 1987. Leukocyte adhesion deficiency: an inherited defect in the Mac-1, LFA-1, and p150,95 glycoproteins. Annu. Rev. Med. 38, 175–194. Anderson, D.C., Schmalsteig, F.C., Finegold, M.J., Hughes, B.J., Rothlein, R., Miller, L.J., Kohl, S., Tosi, M.F., Jacobs, R.L., Waldrop, T.C., Goldman, A.S., Shearer, W.T., Springer, T.A., 1985. The severe and moderate phenotypes of heritable mac-1, LFA-1 deficiency: their quantitative definition and relation to leukocyte dysfunction and clinical features. J. Infect. Dis. 152, 668–689. Antoniou, M., Harland, L., Mustoe, T., Williams, S., Holdstock, J., Yague, E., Mulcahy, T., Griffiths, M., Edwards, S., Ioannou, P.A., Mountain, A., Crombie, R., 2003. Transgenes encompassing dual-promoter CpG islands from the human TBP and HNRPA2B1 loci are resistant to heterochromatin-mediated silencing. Genomics 82, 269–279. Bauer Jr., T.R., Hai, M., Tuschong, L.M., Burkholder, T.H., Gu, Y.C., Sokolic, R.A., Ferguson, C., Dunbar, C.E., Hickstein, D.D., 2006. Correction of the disease phenotype in canine leukocyte adhesion deficiency using ex vivo hematopoietic stem cell gene therapy. Blood 108, 3313–3320. Bauer Jr., T.R., Allen, J.M., Hai, M., Tuschong, L.M., Khan, I.F., Olson, E.M., Adler, R.L., Burkholder, T.H., Gu, Y.C., Russell, D.W., Hickstein, D.D., 2008. Successful treatment of canine leukocyte adhesion deficiency by foamy virus vectors. Nat. Med. 14, 93–97. Bauer Jr., T.R., Olson, E.M., Huo, Y., Tuschong, L.M., Allen, J.M., Li, Y., Burkholder, T.H., Russell, D.W., 2011. Treatment of canine leukocyte adhesion deficiency by foamy virus vectors expressing CD18 from a PGK promoter. Gene Ther. 18, 553–559. Bauer Jr., T.R., Tuschong, L.M., Calvo, K.R., Shive, H.R., Burkholder, T.H., Karlsson, E.K., West, R.R., Russell, D.W., Hickstein, D.D., 2013. Long-term follow-up of foamy viral vector-mediated gene therapy for canine leukocyte adhesion deficiency. Mol. Ther. 21, 964–972.
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