ARTICLE Multiplexed Gene Transfer to a Human T-cell Line by Combining Sleeping Beauty Transposon System With Methotrexate Selection Nataly Kacherovsky,1 Gary W. Liu,1 Michael C. Jensen,1,2 Suzie H. Pun1 1
Department of Bioengineering and Molecular Engineering and Sciences Institute, University of Washington, Seattle, Washington; telephone: þ206-685-3488; fax: þ1-206-543-6124; e-mail: [email protected] 2 Ben Towne Center for Childhood Cancer Research, Seattle Children’s Research Institute, Seattle, Washington; telephone: þ206-884-7900; fax: þ1-206-884-4100; e-mail: [email protected]
ABSTRACT: Engineered human T-cells are a promising therapeutic modality for cancer immunotherapy. T-cells expressing chimeric antigen receptors combined with additional genes to enhance Tcell proliferation, survival, or tumor targeting may further improve efficacy but require multiple stable gene transfer events. Methods are therefore needed to increase production efficiency for multiplexed engineered cells. In this work, we demonstrate multiplexed, nonviral gene transfer to a human T-cell line with efficient selection (50%) of cells expressing up to three recombinant open reading frames. The efficient introduction of multiple genes to T-cells was achieved using the Sleeping Beauty transposon system delivered in minicircles by nucleofection. We demonstrate rapid selection for engineered cells using methotrexate (MTX) and a mutant human dihydrofolate reductase resistant to methotrexate-induced metabolic inhibition. Preferential amplification of cells expressing multiple transgenes was achieved by two successive rounds of increasing MTX concentration. This non-viral gene transfer method with MTX step selection can potentially be used in the generation of clinical-grade Tcells housing multiplexed genetic modifications. Biotechnol. Bioeng. 2015;9999: 1–8. ß 2015 Wiley Periodicals, Inc. KEYWORDS: Sleeping Beauty transposon system; cell engineering; multiplexed gene delivery; immunotherapy
Introduction The premise of adoptive immunotherapy for cancer is augmenting, expanding, and re-infusing a patient’s own tumor-specific T-cells to facilitate the destruction of malignant cells. Once isolated, TCorrespondence to: M.C. Jensen and S.H. Pun Contract grant sponsor: Washington Research Foundation Contract grant sponsor: Ben Towne Foundation Contract grant sponsor: National Science Foundation Graduate Research Fellowship Contract grant number: 2013163401 Received 12 September 2014; Accepted 30 December 2014 Accepted manuscript online xx Month 2015; Article first published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bit.25538
ß 2015 Wiley Periodicals, Inc.
cells can be genetically engineered to recognize tumorspecific antigens and exert cytotoxic activity against cancer cells (Restifo et al., 2012). One method is to isolate patient T-cells and introduce tumor recognition capability by expressing chimeric antigen receptors (CARs), synthetic membrane proteins that contain an extracellular tumor-binding domain linked to an intracellular signaling domain via a transmembrane segment (Kershaw et al., 2013). Several promising clinical trials have used CAR-expressing Tcells, supporting the potential of this approach in fighting cancer (Kalos et al., 2011, Louis et al., 2011, Porter et al., 2011). There are now many reports indicating that co-integration of additional genes further increases the anti-tumor activity of CARexpressing T-cells (Kershaw et al., 2013). Comprehensive Tcell activation requires, in addition to initial tumor recognition and signal initiation by CARs, engagement of co-stimulatory and cytokine receptors which may not be present within the immunosuppressive environment of the tumor. To address this limitation, other groups have expressed co-stimulatory ligands such as CD80 and 4-1BBL in engineered, CAR-expressing T-cells, resulting in greater T-cell expansion due to autocostimulation compared to co-stimulation by ligands expressed on tumor cells (Stephan et al., 2007). Another challenge in T-cell immunotherapy is cell survival after infusion into patients. Induced expression of anti-apoptotic proteins has been shown to improve in vivo survival of T-cells (Charo et al., 2005). Tumor homing and infiltration has been increased by introduction of chemokine receptors in engineered T-cells; this approach may be especially useful for tumors that express chemokines that are not normally recognized by T-cells (Di Stasi et al., 2009; Kershaw et al., 2002) Finally, Tcells can be engineered to better resist the immunosuppressive tumor microenvironment through, for example, induced cytokine expression (Kerkar et al., 2010, Pegram et al., 2012, Spear et al., 2012). Thus, methods to efficiently generate engineered Tcells expressing multiple transgenes are important and advantageous for clinical translation of T-cell immunotherapy. Here, we report an engineered, non-viral gene delivery strategy comprising three key features: (1) Sleeping Beauty (SB) transposon
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system for stable gene integration, (2) minicircles for enhanced transfection by electroporation, and (3) a mutant methotrexate (MTX)-resistant human dihydrofolate reductase (DHFRdm) as a selection mechanism (Scheme 1). Efficient host integration of nonvirally delivered genes requires assistance from enzymes such as transposase. Of the available technologies, the SB transposon system has advanced the most in clinical testing (Aronovich et al., 2011). To this end, the integration efficiency of the SB transposase has been increased by enzyme engineering (Mates et al., 2009). In addition, the SB transposon system shows less tendency for intragenic integration compared with most viral vectors (Yant et al., 2005). For rapid selection of engineered cells, we employed a mechanism recently reported by the Jensen group (Jonnalagadda et al., 2013). The double mutant of human dihydrofolate reductase (DHFRdm, with amino acid mutations L22F and F31S) exhibits a 15,000-fold reduced affinity for methotrexate, a potent inhibitor of DHFR that results in the blockade of thymidylate and purine synthesis, resulting in apoptotic cell death of proliferating Tcells (Ercikan-Abali et al., 1996). Co-expression of DHFRdm therefore imparts MTX resistance to transfected cells so that MTX may be used as a selection mechanism to selectively amplify cells with integrated SB transposon. By taking advantage of the increased cell tolerance to MTX with increased number of DHFRdm gene integrations, we were able to use MTX selection pressure to derive engineered cell populations in which 50% of cells express three separate transcriptionally active open reading frames after a single multiplexed electroporation procedure.
Material and Methods Plasmids The pMC_T3/GFP-T2A-DHFRdm minicircle (MC) plasmid that carries the T3 SB transposon cassette containing an EF1a promoter, maxGFP gene, Thosea asigna virus 2A peptide (T2A), and a double mutant of DHFRdm insensitive to MTX was constructed using the previously reported pMC_T3/eGFP_IRES_FGFR construct as a backbone (Kacherovsky et al., 2012), implementing the cloning strategy described previously to create the GFP-T2A-DHFRdm cassette (Szymczak-Workman et al., 2012). MaxGFP Lonza (Walkersville, MD. USA) and pEGFRt-T2A-IMPDHdm-T2A-DHFRdm plasmids,
reported in our previous publication, were used as templates for PCR (Jonnalagadda et al., 2013a). BmtI and BamHI sites were introduced for swapping genes for fluorescent proteins. Plasmid MC_SB100X was described previously (Kacherovsky et al., 2012). Minicircles were produced and purified according to the System Biosciences user manual for minicircle DNA vector technology. All plasmids were amplified under endotoxin-free conditions using an Endofree Plasmid Kit QIAGEN (Valencia, CA, USA). H9 Culture and Transfection H9 (derivative of HuT 78) human CD4þ T-cell line was cultured in DMEM with 10% FBS. The optimized nucleofection protocol for H9 cells (Lonza) was followed (Program X-001, Nucleofector Kit V). Per nucleofection, 1 106 cells were used with varying amounts of MC DNA. Cells were grown for a week after nucleofection to achieve stable transfection. For MTX selection, cells were cultured in DMEM with 10% FBS supplemented with different concentrations of MTX. Flow Cytometry Analysis Live cells were selected based on propidium iodide exclusion by adding propidium iodide in the flow cytometry buffer to 2 mg/mL. Flow cytometry analysis was carried out on a MACSQuant Analyzer Miltenyi Biotec (San Diego, CA, USA) and LSRII BD Bioscience (San Jose, CA, USA). Collected data were analyzed with FlowJo software. Appropriate negative controls (untransfected H9 cells with and without propidium iodide staining, as well as cells transfected with single genes for GFP, BFP, and mCherry) were used for compensation and gating. A Becton Dickinson FACSAria II was used for cell sorting. Part of the flow cytometry work was conducted at the UW Immunology Flow Cytometry Facility. Determination of Transposon Copy Number Genomic DNA was extracted with Puregene Kit A according to the manufacturer’s instructions (Qiagen), and qPCR was performed using a 7300 Real-Time PCR System Applied Biosystems (South San Francisco, CA, USA) using Universal SYBR Green Supermix Biorad (Hercules, CA, USA). Primers for qPCR were designed using Primer3 software (http://simgene.com/Primer3): maxGFP forward primer: 50 -ACAAGATCATCCGCAGCAAC-30 , maxGFP reverse primer: 50 -TTGAAGTGCATGTGGCTGTC-30 , GAPDH forward primer: 50 - ACAACTTTGGTATCGTGGAAGG-30 , GAPDH reverse primer: 50 GCCATCACGCCACAGTTTC-30 . MaxGFP primers are specific for the maxGFP gene in the transposon. Standard curves were generated using genomic DNA of a H9 clone with a single insertion of transposon (“gold standard”) obtained by limiting dilution method. Copy number was calculated using the DDCT method (Schmittgen and Livak, 2008)
Scheme 1.
Overall schematic of the gene delivery minicircle producer plasmid, MC_T3/FP-DHFRdm. Minicircle (MC) with T3 generation of SB transposon that consists of an EF1a promoter, fusion of fluorescent protein (FP; maxGFP, mCherry, or BFP), Thosea asigna virus 2A peptide (T2A), and double mutant of dihydrofolate reductase (DHFRdm) insensitive to methotrexate (MTX), positioned between inverted terminal repeats (ITRs, arrows). Recombination at attB/attP sites generates minicircle while remaining bacterial backbone is enzymatically degraded.
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Characterization of SBTS Integration Distribution A population of T3/GFP-T2A-DHFRdm transfected-H9 selected with 200 nM MTX was plated in 96 well plates concentration of 0.5 cells/well in DMEM þ10% FBS along irradiated (5000 R) H9 feeder cells at 5,000 cells/well. Plates
cells at a with were
incubated for 2–3 weeks, after which clonal populations were moved to larger plates and expanded. GFP expression was confirmed by flow cytometry. Relative RT-qPCR analysis was performed using DNA of 60 individual clones in order to determine transposon copy number.
Results Optimization of Stable Gene Transfer to H9 T-Cells Minicircles were generated as described previously by Kay and colleagues (Chen et al., 2003; Kay et al., 2010). Three reporter minicircles containing transposons expressing different fluorescent proteins (maxGFP, mCherry, or BFP) under the EF1a promoter were constructed. The selection gene, a double mutant of DHFRdm that confers metabolic resistance to MTX, was cloned in frame after the T2A sequence. The SB100X transposase gene was also prepared in a separate minicircle construct for co-delivery with transposon minicircles. We evaluated the efficiency of initial transfection (at 24 h postnucleofection) and stable transposition (at 7 days post-nucleofection) at various transposon/transposase ratios using the reporter minicircle expressing maxGFP by flow cytometry (Fig. 1). The H9 T-cell line was used as the transfection test-bed. The initial transfection efficiency (t ¼ 24 h) ranged from 47.5 2.2 to 66.9% 4.5%. In the absence of transposase, minimal stable transfection (94% GFPþ cells were obtained by 7 days post-selection under all conditions. The mean GFP fluorescence in GFPþ cells increased with selection pressure (Fig. 2B); the mean fluorescence in cells selected with 200 nM MTX was 6.4-fold higher than unselected cells and 3.3-fold higher than cells selected with 50 nM MTX. The positive correlation between mean GFP expression in GFPþ cells and MTX concentration suggests that increasing MTX concentration selects for cells with increased DHFRdm expression and therefore, multiple integration events. The expanded cell populations selected with 2 weeks of MTX treatment maintained transgene expression even upon MTX withdrawal up to 4 weeks (Fig. 3). Four weeks postMTX withdrawal, the GFPþ population remained >90% in all populations (Fig. 3A), although cells selected with 200 nM MTX had the highest GFPþ population (97%), likely due to selection of cells with multiple integration events. The mean GFP expression in all populations decreased by 21, 27, and 28% for 200, 100, and 50 nM MTX selection, respectively, by 4 weeks post-MTX withdrawal (Fig. 3B). The decrease in mean GFP expression might be due to promoter silencing or preferential expansion of cells with lower GFP expression in the absence of selective pressure.
Analysis of Distribution of Integration
Figure 1.
Optimization of transposon/transposase ratio. H9 cells were nucleofected with 2 mg of MC_T3/eGFP-T2A-DHFRdm DNA (transposon) and increasing amount of MC_SB100X (transposase) (0.5, 1, 2, 4, and 8 mg). Flow cytometry was performed 24 h (striped bars) and 7 days (black bars) after nucleofection to assess transient and stable transfection efficiency. Numbers above the bars indicate integration efficiency, which is calculated as percent of stable over transient GFP expression.
To test our hypothesis that increased MTX selection pressure would select for cells with multiple integration events, we next determined the average number of transposon copy numbers in MTXselected cell populations using RT-qPCR with GFP primers. First, a “gold standard” clone with a single copy of integrated transposon was generated by limiting dilution method. The average number of integrations in the original population before MTX selection was determined by RT-qPCR analysis of the GFPþ cells obtained by cell sorting. A trend of increasing average transposon copy number with increasing selection pressure was observed (Fig. 4A). The average integration events in cells selected with 200 nM MTX was 2.1 0.45 compared to an average of 1.1 0.02 integration events in GFPþ cells before MTX selection. RT-qPCR was performed in triplicates and data represents a single biological replica for the sorted population and three biological replicas for MTX selection. Statistical analysis performed by one-way ANOVA followed by a post-hoc Tukey’s test, *P < 0.05. We next analyzed the distribution of integration events in cells selected with 200 nM MTX. Sixty clones were generated by limiting dilution method, GFP expression confirmed by flow cytometry, genomic DNA isolated, and the number of GFP genes per haploid genome analyzed by RT-PCR. The distribution of
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Figure 2.
Effect of methotrexate (MTX) concentration during selection. Flow cytometry analysis of H9 cell populations stably transfected with T3/GFP-T2A-DHFRdm transposon grown in the presence of increasing concentrations of MTX at 3 (white bars), 5 (parallel stripes), 7 (vertical stripes), and 10 days(black bars). (A) Percent of stably transfected cells (GFPþ/PI). (B) Mean GFP fluorescence (RFU) of GFPþ cells.
Figure 3. Transgene persistence after methotrexate (MTX) withdrawal. Flow cytometry analysis of H9 cell populations stably transfected with T3/GFP-T2A-DHFRdm transposon grown in media supplemented with different concentrations of MTX (50, 100, and 200 nM) for 2 weeks (black bars), after which MTX selection was withdrawn and data collected at different time points: 1 (parallel stripes), 2 (vertical stripes), 3 (checked bars), and 4 weeks (white bars). (A) Percent of stably transfected cells (GFPþ/PI). (B) Mean GFP fluorescence (RFU) of GFPþ cells.
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integration events is shown in Figure 4B. Most clones (65%) contained multiple copies of GFP. The average number of integration events was 1.8 which correlates well with the average transposon copy number in the cell population selected with 200 nM MTX (Fig. 4A). Demonstration of Multiplexed Gene Integration
Figure 4. (A) Transposon copy number per human haploid genome. Genomic DNA was isolated from populations of H9 cells stably transfected with T3/GFP-T2ADHFRdm transposon before and after selection with different concentrations of MTX. The average transposon copy number was determined by quantitative PCR. A ‘‘gold standard’’ was generated by the limiting dilution method. ‘‘Sorted’’ population was created by sorting original H9 population (8% of integrated transposon) to 100% GFPþcells. Statistical analysis performed by one-way ANOVA followed by a posthoc Tukey’s test, *P < 0.05. (B) Distribution of transposon integration events. Sixty clones were isolated by limited dilution method from H9 population previously selected with 200 nM MTX to 100% cells with integrated T3/GFP-T2A-DHFRdm transposon. Genomic DNA was isolated and transposon copy number determined by relative RTqPCR method. Numbers were rounded to the nearest integer value. n ¼ 60; average ¼ 1.78 0.69 (standard deviation). Probabilities of integration events and standard error were calculated from these data (inset).
As we demonstrated that a majority of the population of transfected cells amplified under 200 nM MTX selection pressure contained multiple transposon copies, we next assessed multiplexed gene integration under these conditions. H9 cells were nucleofected with three minicircles containing three different reporter genes (maxGFP, mCherry, and BFP) in transposon cassettes with DHFRdm and the SB100X transposase minicircle. Stably-transfected cells were then selected for 7 days with 200 nM and cell population assessed by flow cytometry analysis (Fig. 5). Initial transfection efficiency assessed 24 h after nucleofection was 68%, which reduced to 37 1.4% after one week, reflecting 54% integration efficiency. Of this population, 19 0.6% expressed two or three different fluorescent proteins. Stably-transfected cells grown for 1 week in the presence of 200 nM MTX were then analyzed; 23 1.0% of this selected population expressed all three reporter proteins. This concentration was determined in preliminary studies to be the highest tolerable MTX dose after initial transfection. In order to further increase the population of cells expressing triple transgenes, cells selected by 200 nM MTX were subjected to a second selection step with increased MTX concentrations (Fig. 6). Selected cells were cultured in 500 or 1000 nM MTX for an additional week, resulting in an increased population (38.5 1.0 and 53.1 0.3%, respectively) of cells expressing triple transgenes. Cell viability rebounded to 70% during the second round of selection due to further selection for overexpression of the DHFRdm gene.
Discussion In this work, we demonstrate stable transfer of up to three separate genetic payloads into H9 T-cells using a single multiplexed
Figure 5.
Multiplexed transposon delivery. Flow cytometry analysis of H9 cell populations nucleofected with 3 MCs carrying transposons with different FPs (MC_T3/GFP-T2ADHFRdm, MC_T3/BFP-T2A-DHFRdm, MC_T3/mCherry-T2A-DHFRdm) at different time points (A) 24 h after transfection (transient expression), (B) 1 week (stable integration), and (C) 1 week of selection with 200 nM of MTX. Example of dot blot flow charts are presented in Supplemental Figure S1.
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Figure 6. Distribution of expression of single, double, and triple FPs after two step selection with MTX. An H9 cell population stably transfected with three transposons was selected with 200 nM MTX for a week and then was exposed to MTX concentrations of 200, 500, or 1000 nM. Reporter protein expression was analyzed by flow cytometry (dot blot flow charts presented in Supplemental Figure S1). electroporation of minicircles containing SB transposons followed by MTX selection. Initial transfection and integration efficiencies reached >60 and >55%, respectively, through a combination of strategies: optimization of electroporation parameters, use of hyperactive SB100X transposase, use of minicircle constructs instead of plasmid vectors, and optimization of transposon to transposase ratios (Fig. 1). Stably transfected cells could then be rapidly selected by MTX such that cell populations with >94% engineered cells could be obtained with only 7 days of selection and maintain stable gene expression for at least 4 weeks. We demonstrate that average reporter gene expression increases with increasing MTX concentration (Fig. 2). Increased reporter gene expression could be due to differences in transcriptional activity based on site of integration, multiple integration events, or a combination of both factors. To confirm that cells with higher number of gene integrations were preferentially obtained by titrating selection pressure with increasing concentrations of MTX, we performed RT-qPCR analysis on both bulk populations and clonal analysis of distribution of integration events (Fig. 4). Technologies to generate tumor-specific T-cells in a costeffective manner are key in eventual broad clinical use of CARexpressing T-cells. The non-viral gene transfer system described in this work is an attractive alternative to current methods for engineering T-cells. Genetic modification of T-cells is typically accomplished using g-retroviral or lentiviral vectors. While effective, drawbacks include cost of production, limited gene packaging capacity, and potential safety issues (Kershaw et al., 2013). Plasmids containing transposon systems such as SB or piggyBac offer a nonviral approach for stably introducing genes into T-cells. For example, Kahlig et al. reported using the piggyBac system to produce stablytransfected mammalian cells expressing multiple transgenes of interest by delivery of multiple transposons (Kahlig et al., 2010). The SB system, first exploited for mammalian cell gene transfer by Ivics and coworkers (Ivics et al., 1997), has recently been used as a gene delivery modality in FDA-authorized clinical trials of Tcell immunotherapy (Huls et al., 2013). Genomic integration by SB appears to be less biased towards locations proximal to active
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transcriptional units and their regulatory sequences as compared to the g-retroviral and lentiviral vectors, thus improving the safety profile by decreasing the risk of insertional mutagenesis (Vigdal et al., 2002; Yant et al., 2005). Minicircle constructs, which have bacterial plasmid sequences removed, are particularly attractive as transfection platforms for several reasons. First, the transfection efficiency of minicircles by electroporation is superior to that of their plasmid analogues (Kacherovsky et al., 2012). Second, transposition efficiency is higher when minicircles are used due to the shorter distance between the two transposon ends, which has been shown to affect transposase efficiency (Izsvak et al., 2002). Third, minicircle vectors are devoid of bacterial backbones, offering improved safety profiles compared to plasmids. For example, plasmid bacterial sequences contain unmethylated CpG motifs, which may trigger the activation of inflammatory responses (Reyes-Sandoval et al., 2004). There is also a slight risk of horizontal transfer of antibiotic resistance gene on the plasmid to pathogenic bacteria (Martinez et al., 2002). Finally, as cell viability after nucleofection decreases with increasing construct size (Iversen et al., 2005), minicircles are more advantageous given their smaller size compared to their analogous plasmids. To further improve transposition efficiency, the optimized SB100X hyperactive transposase developed by the Izsvak group was used (Mates et al., 2009) in combination with the T3 generation of SB transposon previously identified by Yant et al. (Yant et al., 2004). Two potential limitations of the SB transposon systems are overproduction inhibition and reduced transposition efficiency with increasing transposon size (Ivics and Izsvak, 2011; Wilson et al., 2007). There are four transposase binding sites in a transposon (two per inverted terminal repeat). Bound transposases are proposed to interact with each other to promote juxtaposition of the two transposon ends. Overexpression of transposase has been hypothesized to inhibit transposition due to interaction of free transposase with bound transposase, thus preventing the juxtaposition step. Therefore, the optimal transposon/transposase ratio needs to be determined if these genes are delivered on separate constructs. However, reports of the inhibition phenomenon have been varied (Geurts et al., 2003; Izsvak et al., 2000). In our studies, overproduction inhibition was not observed (Fig. 1). The efficiency of SB transposition decreases with increasing transposon size with an upper transposon size limit of around 10 kB (Izsvak et al., 2000). For example, transposition levels are reduced by over 50% by increasing transposon size from 2.5 kB to just 4 kB (Izsvak et al., 2000). Therefore, stable transfer of multiple recombinant open reading frames may require multiplexed vector delivery. The Cooper group has used electroporation of SB plasmids to co-deliver a CAR-expressing plasmid with a second plasmid for positron emission tomography (PET) imaging of engineered Tcells (Bhatnagar et al., 2013). CAR- and thymidine kinase (TK)expressing T-cells (68% of total population) were selected 35 days after initial electroporation by using drug selection for the TKcontaining transposon and by expanding CAR-expressing T cells with artificial antigen presenting cells (Bhatnagar et al., 2013). In this work, we used the double mutant of human dihydrofolate reductase, DHFRdm, as the selection marker for engineered cells. Expression of DHFRdm in T-cells imparts MTX resistance without compromising proliferative ability, expression of T-cell markers, or
cytolytic ability (Jonnalagadda et al., 2013a). Mutant DHFR genes have been investigated as a gene therapy method to protect stably transfected hematopoietic cells against drug-induced myelosuppression side effects resulting from chemotherapy treatment (Sorrentino, 2002). Additional advantages of this selection system include availability of clinical grade MTX, the use of a nongenotoxic drug, and the small gene size of DHFRdm (561 bp) (Jonnalagadda et al., 2013a). MTX is particularly attractive for selection because it is a pharmaceutical drug that is clinically simple to administer, serum drug levels are measured routinely in clinical practice, and there is an available antidote (Leucovorin). Furthermore, Jonnalagadda et al. have demonstrated in vivo selection using MTX of transduced human T-cells (Jonnalagadda et al., 2013a). Using a two-step selection method through two successive MTX selection rounds, we were able to derive MTX resistant cell lines in which >50% of cells express three separate transcriptionally-active open reading frames derived from three individual MC constructs introduced into H9 cells in a single multiplexed electroporation procedure (Fig. 6). The multiplexed cells can be further purified by selection through flow sorting or by immunomagnetic isolation of cells with “bar code” markers. In some cases, multiplexed cells will have a functional survival and/or growth advantage so that they will be selected for over time in vitro or in vivo. In addition, 32% of the cell population expressed genes from two different transposon constructs. To increase the robustness of the selection pressure, separate genes could be tethered to selection genes such as hygromycin B phosphotransferase (Blochlinger and Diggelmann, 1984) and puromycin resistance gene (Sharma et al., 2013), and a “cocktail” of the appropriate selection agents could be subsequently applied to increase the selection for triple multiplexing events. Moreover, we envision that by varying the ratio of the components within the selection agent cocktail, different ratios of genes will be integrated, resulting in different ratios of protein expression. This could potentially lead to a high degree of control over gene integration. In summary, we demonstrate efficient multiplexed gene transfer to a human T cell line using the Sleeping Beauty transposon system. Cells with multiple integration events can be selected with increasing selection pressure. A two-step selection method can be used to further increase the population of cells expressing three desired transgenes. This method can potentially be applied for generation of engineered, multiplexed T-cells for adoptive T-cell immunotherapy applications. The authors are grateful to Mark A. Kay (Stanford University) for providing the minicircle expression system (pMC.BESPX plasmid and ZYCY10P3S2T E. coli producer strain) and Zsuzsanna Izsvak (Max Delbruck Center for Molecular Medicine, Berlin, Germany) for providing the CMV(CAT)T7SB100X plasmid. We thank Lisa Rolczynski (Seattle Children’s Research Institute) for helpful discussion and advice.
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Martinez JL, Baquero F. 2002. Interactions among strategies associated with bacterial infection: Pathogenicity, epidemicity, and antibiotic resistance. Clin Microbiol Rev 15(4):647–679. Mates L, Chuah MKL, Belay E, Jerchow B, Manoj N, Acosta-Sanchez A, Grzela DP, Schmitt A, Becker K, Matrai J, Ma L, Samara- Kuko E, Gysemans C, Pryputniewicz D, Miskey C, Fletcher B, VandenDriessche T, Ivics Z, Izsvak Z. 2009. Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates. Nat Genet 41(6):753–761. Pegram HJ, Lee JC, Hayman EG, Imperato GH, Tedder TF, Sadelain M, Brentjens RJ. 2012. Tumor-targeted T cells modified to secrete IL-12 eradicate systemic tumors without need for prior conditioning. Blood 119(18):4133– 4141. Porter DL, Levine BL, Kalos M, Bagg A, June CH. 2011. Chimeric antigen receptormodified T cells in chronic lymphoid leukemia. N Engl J Med 365(8):725– 733. Restifo NP, Dudley ME, Rosenberg SA. 2012. Adoptive immunotherapy for cancer: Harnessing the T cell response. Nat Rev Immunol 12(4):269–281. Reyes-Sandoval A, Erlt HCJ. 2004. CpG methylation of a plasmid vector results in extended transgene product expression by circumventing induction of immune responses. Mol Ther 9:249–261. Schmittgen TD, Livak KJ. 2008. Analyzing real-time PCR data by the comparative CT method. Nat Prot 3(6):1101–1108. Sharma N, Cai Y, Bak RO, Jakobsen MR, Schrøder LD, Mikkelsen JG. 2013. Efficient sleeping beauty DNA transposition from DNA minicircles. Mol Ther Nucleic Acid 2(2):e74. Sorrentino BR. 2002. Gene therapy to protect haematopoietic cells from cytotoxic cancer drugs. Nat Rev Cancer 2(6):431–441.
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Spear P, Barber A, Rynda-Apple A, Sentman CL. 2012. Chimeric antigen receptor T cells shape myeloid cell function within the tumor microenvironment through IFN-g and GM-CSF. J Immunol 188(12):6389–6398. Stephan MT, Ponomarev V, Brentjens RJ, Chang AH, Dobrenkov KV, Heller G, Sadelain M. 2007. T cell-encoded CD80 and 4-1BBL induce auto-and transcostimulation, resulting in potent tumor rejection. Nat Med 13(12):1440–1449. Szymczak-Workman AL, Vignali KM, Vignali DA. 2012. Design and construction of 2 A peptide-linked multicistronic vectors. Cold Spring Harb Protoc 2012(2):pdb ip067876. Vigdal TJ, Kaufman CD, Izsvak Z, Voytas DF, Ivics Z. 2002. Common physical properties of DNA affecting target site selection of Sleeping Beauty and other Tc1/mariner transposable elements. J Mol Biol 323(3):441–452. Wilson MH, Coates CJ, George AL. 2007. PiggyBac transposon-mediated gene transfer in human cells. Mol Ther 15(1):139–145. Yant SR, Park J, Huang Y, Mikkelsen JG, Kay MA. 2004. Mutational analysis of the Nterminal DNA-binding domain of Sleeping Beauty transposase: Critical residues for DNA binding and hyperactivity in mammalian cells. Mol Cell Biol 24(20):9239–9247. Yant SR, Wu XL, Huang Y, Garrison B, Burgess SM, Kay MA. 2005. High-resolution genome-wide mapping of transposon integration in mammals. Mol Cell Biol 25(6):2085–2094.
Supporting Information Additional supporting information may be found in the online version of this article at the publisher’s web-site.
Department of Bioengineering and Molecular Engineering and Sciences Institute, University of Washington, Seattle, Washington; telephone: þ206-685-3488; fax: þ1-206-543-6124; e-mail: [email protected] 2 Ben Towne Center for Childhood Cancer Research, Seattle Children’s Research Institute, Seattle, Washington; telephone: þ206-884-7900; fax: þ1-206-884-4100; e-mail: [email protected]
ABSTRACT: Engineered human T-cells are a promising therapeutic modality for cancer immunotherapy. T-cells expressing chimeric antigen receptors combined with additional genes to enhance Tcell proliferation, survival, or tumor targeting may further improve efficacy but require multiple stable gene transfer events. Methods are therefore needed to increase production efficiency for multiplexed engineered cells. In this work, we demonstrate multiplexed, nonviral gene transfer to a human T-cell line with efficient selection (50%) of cells expressing up to three recombinant open reading frames. The efficient introduction of multiple genes to T-cells was achieved using the Sleeping Beauty transposon system delivered in minicircles by nucleofection. We demonstrate rapid selection for engineered cells using methotrexate (MTX) and a mutant human dihydrofolate reductase resistant to methotrexate-induced metabolic inhibition. Preferential amplification of cells expressing multiple transgenes was achieved by two successive rounds of increasing MTX concentration. This non-viral gene transfer method with MTX step selection can potentially be used in the generation of clinical-grade Tcells housing multiplexed genetic modifications. Biotechnol. Bioeng. 2015;9999: 1–8. ß 2015 Wiley Periodicals, Inc. KEYWORDS: Sleeping Beauty transposon system; cell engineering; multiplexed gene delivery; immunotherapy
Introduction The premise of adoptive immunotherapy for cancer is augmenting, expanding, and re-infusing a patient’s own tumor-specific T-cells to facilitate the destruction of malignant cells. Once isolated, TCorrespondence to: M.C. Jensen and S.H. Pun Contract grant sponsor: Washington Research Foundation Contract grant sponsor: Ben Towne Foundation Contract grant sponsor: National Science Foundation Graduate Research Fellowship Contract grant number: 2013163401 Received 12 September 2014; Accepted 30 December 2014 Accepted manuscript online xx Month 2015; Article first published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bit.25538
ß 2015 Wiley Periodicals, Inc.
cells can be genetically engineered to recognize tumorspecific antigens and exert cytotoxic activity against cancer cells (Restifo et al., 2012). One method is to isolate patient T-cells and introduce tumor recognition capability by expressing chimeric antigen receptors (CARs), synthetic membrane proteins that contain an extracellular tumor-binding domain linked to an intracellular signaling domain via a transmembrane segment (Kershaw et al., 2013). Several promising clinical trials have used CAR-expressing Tcells, supporting the potential of this approach in fighting cancer (Kalos et al., 2011, Louis et al., 2011, Porter et al., 2011). There are now many reports indicating that co-integration of additional genes further increases the anti-tumor activity of CARexpressing T-cells (Kershaw et al., 2013). Comprehensive Tcell activation requires, in addition to initial tumor recognition and signal initiation by CARs, engagement of co-stimulatory and cytokine receptors which may not be present within the immunosuppressive environment of the tumor. To address this limitation, other groups have expressed co-stimulatory ligands such as CD80 and 4-1BBL in engineered, CAR-expressing T-cells, resulting in greater T-cell expansion due to autocostimulation compared to co-stimulation by ligands expressed on tumor cells (Stephan et al., 2007). Another challenge in T-cell immunotherapy is cell survival after infusion into patients. Induced expression of anti-apoptotic proteins has been shown to improve in vivo survival of T-cells (Charo et al., 2005). Tumor homing and infiltration has been increased by introduction of chemokine receptors in engineered T-cells; this approach may be especially useful for tumors that express chemokines that are not normally recognized by T-cells (Di Stasi et al., 2009; Kershaw et al., 2002) Finally, Tcells can be engineered to better resist the immunosuppressive tumor microenvironment through, for example, induced cytokine expression (Kerkar et al., 2010, Pegram et al., 2012, Spear et al., 2012). Thus, methods to efficiently generate engineered Tcells expressing multiple transgenes are important and advantageous for clinical translation of T-cell immunotherapy. Here, we report an engineered, non-viral gene delivery strategy comprising three key features: (1) Sleeping Beauty (SB) transposon
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system for stable gene integration, (2) minicircles for enhanced transfection by electroporation, and (3) a mutant methotrexate (MTX)-resistant human dihydrofolate reductase (DHFRdm) as a selection mechanism (Scheme 1). Efficient host integration of nonvirally delivered genes requires assistance from enzymes such as transposase. Of the available technologies, the SB transposon system has advanced the most in clinical testing (Aronovich et al., 2011). To this end, the integration efficiency of the SB transposase has been increased by enzyme engineering (Mates et al., 2009). In addition, the SB transposon system shows less tendency for intragenic integration compared with most viral vectors (Yant et al., 2005). For rapid selection of engineered cells, we employed a mechanism recently reported by the Jensen group (Jonnalagadda et al., 2013). The double mutant of human dihydrofolate reductase (DHFRdm, with amino acid mutations L22F and F31S) exhibits a 15,000-fold reduced affinity for methotrexate, a potent inhibitor of DHFR that results in the blockade of thymidylate and purine synthesis, resulting in apoptotic cell death of proliferating Tcells (Ercikan-Abali et al., 1996). Co-expression of DHFRdm therefore imparts MTX resistance to transfected cells so that MTX may be used as a selection mechanism to selectively amplify cells with integrated SB transposon. By taking advantage of the increased cell tolerance to MTX with increased number of DHFRdm gene integrations, we were able to use MTX selection pressure to derive engineered cell populations in which 50% of cells express three separate transcriptionally active open reading frames after a single multiplexed electroporation procedure.
Material and Methods Plasmids The pMC_T3/GFP-T2A-DHFRdm minicircle (MC) plasmid that carries the T3 SB transposon cassette containing an EF1a promoter, maxGFP gene, Thosea asigna virus 2A peptide (T2A), and a double mutant of DHFRdm insensitive to MTX was constructed using the previously reported pMC_T3/eGFP_IRES_FGFR construct as a backbone (Kacherovsky et al., 2012), implementing the cloning strategy described previously to create the GFP-T2A-DHFRdm cassette (Szymczak-Workman et al., 2012). MaxGFP Lonza (Walkersville, MD. USA) and pEGFRt-T2A-IMPDHdm-T2A-DHFRdm plasmids,
reported in our previous publication, were used as templates for PCR (Jonnalagadda et al., 2013a). BmtI and BamHI sites were introduced for swapping genes for fluorescent proteins. Plasmid MC_SB100X was described previously (Kacherovsky et al., 2012). Minicircles were produced and purified according to the System Biosciences user manual for minicircle DNA vector technology. All plasmids were amplified under endotoxin-free conditions using an Endofree Plasmid Kit QIAGEN (Valencia, CA, USA). H9 Culture and Transfection H9 (derivative of HuT 78) human CD4þ T-cell line was cultured in DMEM with 10% FBS. The optimized nucleofection protocol for H9 cells (Lonza) was followed (Program X-001, Nucleofector Kit V). Per nucleofection, 1 106 cells were used with varying amounts of MC DNA. Cells were grown for a week after nucleofection to achieve stable transfection. For MTX selection, cells were cultured in DMEM with 10% FBS supplemented with different concentrations of MTX. Flow Cytometry Analysis Live cells were selected based on propidium iodide exclusion by adding propidium iodide in the flow cytometry buffer to 2 mg/mL. Flow cytometry analysis was carried out on a MACSQuant Analyzer Miltenyi Biotec (San Diego, CA, USA) and LSRII BD Bioscience (San Jose, CA, USA). Collected data were analyzed with FlowJo software. Appropriate negative controls (untransfected H9 cells with and without propidium iodide staining, as well as cells transfected with single genes for GFP, BFP, and mCherry) were used for compensation and gating. A Becton Dickinson FACSAria II was used for cell sorting. Part of the flow cytometry work was conducted at the UW Immunology Flow Cytometry Facility. Determination of Transposon Copy Number Genomic DNA was extracted with Puregene Kit A according to the manufacturer’s instructions (Qiagen), and qPCR was performed using a 7300 Real-Time PCR System Applied Biosystems (South San Francisco, CA, USA) using Universal SYBR Green Supermix Biorad (Hercules, CA, USA). Primers for qPCR were designed using Primer3 software (http://simgene.com/Primer3): maxGFP forward primer: 50 -ACAAGATCATCCGCAGCAAC-30 , maxGFP reverse primer: 50 -TTGAAGTGCATGTGGCTGTC-30 , GAPDH forward primer: 50 - ACAACTTTGGTATCGTGGAAGG-30 , GAPDH reverse primer: 50 GCCATCACGCCACAGTTTC-30 . MaxGFP primers are specific for the maxGFP gene in the transposon. Standard curves were generated using genomic DNA of a H9 clone with a single insertion of transposon (“gold standard”) obtained by limiting dilution method. Copy number was calculated using the DDCT method (Schmittgen and Livak, 2008)
Scheme 1.
Overall schematic of the gene delivery minicircle producer plasmid, MC_T3/FP-DHFRdm. Minicircle (MC) with T3 generation of SB transposon that consists of an EF1a promoter, fusion of fluorescent protein (FP; maxGFP, mCherry, or BFP), Thosea asigna virus 2A peptide (T2A), and double mutant of dihydrofolate reductase (DHFRdm) insensitive to methotrexate (MTX), positioned between inverted terminal repeats (ITRs, arrows). Recombination at attB/attP sites generates minicircle while remaining bacterial backbone is enzymatically degraded.
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Characterization of SBTS Integration Distribution A population of T3/GFP-T2A-DHFRdm transfected-H9 selected with 200 nM MTX was plated in 96 well plates concentration of 0.5 cells/well in DMEM þ10% FBS along irradiated (5000 R) H9 feeder cells at 5,000 cells/well. Plates
cells at a with were
incubated for 2–3 weeks, after which clonal populations were moved to larger plates and expanded. GFP expression was confirmed by flow cytometry. Relative RT-qPCR analysis was performed using DNA of 60 individual clones in order to determine transposon copy number.
Results Optimization of Stable Gene Transfer to H9 T-Cells Minicircles were generated as described previously by Kay and colleagues (Chen et al., 2003; Kay et al., 2010). Three reporter minicircles containing transposons expressing different fluorescent proteins (maxGFP, mCherry, or BFP) under the EF1a promoter were constructed. The selection gene, a double mutant of DHFRdm that confers metabolic resistance to MTX, was cloned in frame after the T2A sequence. The SB100X transposase gene was also prepared in a separate minicircle construct for co-delivery with transposon minicircles. We evaluated the efficiency of initial transfection (at 24 h postnucleofection) and stable transposition (at 7 days post-nucleofection) at various transposon/transposase ratios using the reporter minicircle expressing maxGFP by flow cytometry (Fig. 1). The H9 T-cell line was used as the transfection test-bed. The initial transfection efficiency (t ¼ 24 h) ranged from 47.5 2.2 to 66.9% 4.5%. In the absence of transposase, minimal stable transfection (94% GFPþ cells were obtained by 7 days post-selection under all conditions. The mean GFP fluorescence in GFPþ cells increased with selection pressure (Fig. 2B); the mean fluorescence in cells selected with 200 nM MTX was 6.4-fold higher than unselected cells and 3.3-fold higher than cells selected with 50 nM MTX. The positive correlation between mean GFP expression in GFPþ cells and MTX concentration suggests that increasing MTX concentration selects for cells with increased DHFRdm expression and therefore, multiple integration events. The expanded cell populations selected with 2 weeks of MTX treatment maintained transgene expression even upon MTX withdrawal up to 4 weeks (Fig. 3). Four weeks postMTX withdrawal, the GFPþ population remained >90% in all populations (Fig. 3A), although cells selected with 200 nM MTX had the highest GFPþ population (97%), likely due to selection of cells with multiple integration events. The mean GFP expression in all populations decreased by 21, 27, and 28% for 200, 100, and 50 nM MTX selection, respectively, by 4 weeks post-MTX withdrawal (Fig. 3B). The decrease in mean GFP expression might be due to promoter silencing or preferential expansion of cells with lower GFP expression in the absence of selective pressure.
Analysis of Distribution of Integration
Figure 1.
Optimization of transposon/transposase ratio. H9 cells were nucleofected with 2 mg of MC_T3/eGFP-T2A-DHFRdm DNA (transposon) and increasing amount of MC_SB100X (transposase) (0.5, 1, 2, 4, and 8 mg). Flow cytometry was performed 24 h (striped bars) and 7 days (black bars) after nucleofection to assess transient and stable transfection efficiency. Numbers above the bars indicate integration efficiency, which is calculated as percent of stable over transient GFP expression.
To test our hypothesis that increased MTX selection pressure would select for cells with multiple integration events, we next determined the average number of transposon copy numbers in MTXselected cell populations using RT-qPCR with GFP primers. First, a “gold standard” clone with a single copy of integrated transposon was generated by limiting dilution method. The average number of integrations in the original population before MTX selection was determined by RT-qPCR analysis of the GFPþ cells obtained by cell sorting. A trend of increasing average transposon copy number with increasing selection pressure was observed (Fig. 4A). The average integration events in cells selected with 200 nM MTX was 2.1 0.45 compared to an average of 1.1 0.02 integration events in GFPþ cells before MTX selection. RT-qPCR was performed in triplicates and data represents a single biological replica for the sorted population and three biological replicas for MTX selection. Statistical analysis performed by one-way ANOVA followed by a post-hoc Tukey’s test, *P < 0.05. We next analyzed the distribution of integration events in cells selected with 200 nM MTX. Sixty clones were generated by limiting dilution method, GFP expression confirmed by flow cytometry, genomic DNA isolated, and the number of GFP genes per haploid genome analyzed by RT-PCR. The distribution of
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Figure 2.
Effect of methotrexate (MTX) concentration during selection. Flow cytometry analysis of H9 cell populations stably transfected with T3/GFP-T2A-DHFRdm transposon grown in the presence of increasing concentrations of MTX at 3 (white bars), 5 (parallel stripes), 7 (vertical stripes), and 10 days(black bars). (A) Percent of stably transfected cells (GFPþ/PI). (B) Mean GFP fluorescence (RFU) of GFPþ cells.
Figure 3. Transgene persistence after methotrexate (MTX) withdrawal. Flow cytometry analysis of H9 cell populations stably transfected with T3/GFP-T2A-DHFRdm transposon grown in media supplemented with different concentrations of MTX (50, 100, and 200 nM) for 2 weeks (black bars), after which MTX selection was withdrawn and data collected at different time points: 1 (parallel stripes), 2 (vertical stripes), 3 (checked bars), and 4 weeks (white bars). (A) Percent of stably transfected cells (GFPþ/PI). (B) Mean GFP fluorescence (RFU) of GFPþ cells.
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integration events is shown in Figure 4B. Most clones (65%) contained multiple copies of GFP. The average number of integration events was 1.8 which correlates well with the average transposon copy number in the cell population selected with 200 nM MTX (Fig. 4A). Demonstration of Multiplexed Gene Integration
Figure 4. (A) Transposon copy number per human haploid genome. Genomic DNA was isolated from populations of H9 cells stably transfected with T3/GFP-T2ADHFRdm transposon before and after selection with different concentrations of MTX. The average transposon copy number was determined by quantitative PCR. A ‘‘gold standard’’ was generated by the limiting dilution method. ‘‘Sorted’’ population was created by sorting original H9 population (8% of integrated transposon) to 100% GFPþcells. Statistical analysis performed by one-way ANOVA followed by a posthoc Tukey’s test, *P < 0.05. (B) Distribution of transposon integration events. Sixty clones were isolated by limited dilution method from H9 population previously selected with 200 nM MTX to 100% cells with integrated T3/GFP-T2A-DHFRdm transposon. Genomic DNA was isolated and transposon copy number determined by relative RTqPCR method. Numbers were rounded to the nearest integer value. n ¼ 60; average ¼ 1.78 0.69 (standard deviation). Probabilities of integration events and standard error were calculated from these data (inset).
As we demonstrated that a majority of the population of transfected cells amplified under 200 nM MTX selection pressure contained multiple transposon copies, we next assessed multiplexed gene integration under these conditions. H9 cells were nucleofected with three minicircles containing three different reporter genes (maxGFP, mCherry, and BFP) in transposon cassettes with DHFRdm and the SB100X transposase minicircle. Stably-transfected cells were then selected for 7 days with 200 nM and cell population assessed by flow cytometry analysis (Fig. 5). Initial transfection efficiency assessed 24 h after nucleofection was 68%, which reduced to 37 1.4% after one week, reflecting 54% integration efficiency. Of this population, 19 0.6% expressed two or three different fluorescent proteins. Stably-transfected cells grown for 1 week in the presence of 200 nM MTX were then analyzed; 23 1.0% of this selected population expressed all three reporter proteins. This concentration was determined in preliminary studies to be the highest tolerable MTX dose after initial transfection. In order to further increase the population of cells expressing triple transgenes, cells selected by 200 nM MTX were subjected to a second selection step with increased MTX concentrations (Fig. 6). Selected cells were cultured in 500 or 1000 nM MTX for an additional week, resulting in an increased population (38.5 1.0 and 53.1 0.3%, respectively) of cells expressing triple transgenes. Cell viability rebounded to 70% during the second round of selection due to further selection for overexpression of the DHFRdm gene.
Discussion In this work, we demonstrate stable transfer of up to three separate genetic payloads into H9 T-cells using a single multiplexed
Figure 5.
Multiplexed transposon delivery. Flow cytometry analysis of H9 cell populations nucleofected with 3 MCs carrying transposons with different FPs (MC_T3/GFP-T2ADHFRdm, MC_T3/BFP-T2A-DHFRdm, MC_T3/mCherry-T2A-DHFRdm) at different time points (A) 24 h after transfection (transient expression), (B) 1 week (stable integration), and (C) 1 week of selection with 200 nM of MTX. Example of dot blot flow charts are presented in Supplemental Figure S1.
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Figure 6. Distribution of expression of single, double, and triple FPs after two step selection with MTX. An H9 cell population stably transfected with three transposons was selected with 200 nM MTX for a week and then was exposed to MTX concentrations of 200, 500, or 1000 nM. Reporter protein expression was analyzed by flow cytometry (dot blot flow charts presented in Supplemental Figure S1). electroporation of minicircles containing SB transposons followed by MTX selection. Initial transfection and integration efficiencies reached >60 and >55%, respectively, through a combination of strategies: optimization of electroporation parameters, use of hyperactive SB100X transposase, use of minicircle constructs instead of plasmid vectors, and optimization of transposon to transposase ratios (Fig. 1). Stably transfected cells could then be rapidly selected by MTX such that cell populations with >94% engineered cells could be obtained with only 7 days of selection and maintain stable gene expression for at least 4 weeks. We demonstrate that average reporter gene expression increases with increasing MTX concentration (Fig. 2). Increased reporter gene expression could be due to differences in transcriptional activity based on site of integration, multiple integration events, or a combination of both factors. To confirm that cells with higher number of gene integrations were preferentially obtained by titrating selection pressure with increasing concentrations of MTX, we performed RT-qPCR analysis on both bulk populations and clonal analysis of distribution of integration events (Fig. 4). Technologies to generate tumor-specific T-cells in a costeffective manner are key in eventual broad clinical use of CARexpressing T-cells. The non-viral gene transfer system described in this work is an attractive alternative to current methods for engineering T-cells. Genetic modification of T-cells is typically accomplished using g-retroviral or lentiviral vectors. While effective, drawbacks include cost of production, limited gene packaging capacity, and potential safety issues (Kershaw et al., 2013). Plasmids containing transposon systems such as SB or piggyBac offer a nonviral approach for stably introducing genes into T-cells. For example, Kahlig et al. reported using the piggyBac system to produce stablytransfected mammalian cells expressing multiple transgenes of interest by delivery of multiple transposons (Kahlig et al., 2010). The SB system, first exploited for mammalian cell gene transfer by Ivics and coworkers (Ivics et al., 1997), has recently been used as a gene delivery modality in FDA-authorized clinical trials of Tcell immunotherapy (Huls et al., 2013). Genomic integration by SB appears to be less biased towards locations proximal to active
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transcriptional units and their regulatory sequences as compared to the g-retroviral and lentiviral vectors, thus improving the safety profile by decreasing the risk of insertional mutagenesis (Vigdal et al., 2002; Yant et al., 2005). Minicircle constructs, which have bacterial plasmid sequences removed, are particularly attractive as transfection platforms for several reasons. First, the transfection efficiency of minicircles by electroporation is superior to that of their plasmid analogues (Kacherovsky et al., 2012). Second, transposition efficiency is higher when minicircles are used due to the shorter distance between the two transposon ends, which has been shown to affect transposase efficiency (Izsvak et al., 2002). Third, minicircle vectors are devoid of bacterial backbones, offering improved safety profiles compared to plasmids. For example, plasmid bacterial sequences contain unmethylated CpG motifs, which may trigger the activation of inflammatory responses (Reyes-Sandoval et al., 2004). There is also a slight risk of horizontal transfer of antibiotic resistance gene on the plasmid to pathogenic bacteria (Martinez et al., 2002). Finally, as cell viability after nucleofection decreases with increasing construct size (Iversen et al., 2005), minicircles are more advantageous given their smaller size compared to their analogous plasmids. To further improve transposition efficiency, the optimized SB100X hyperactive transposase developed by the Izsvak group was used (Mates et al., 2009) in combination with the T3 generation of SB transposon previously identified by Yant et al. (Yant et al., 2004). Two potential limitations of the SB transposon systems are overproduction inhibition and reduced transposition efficiency with increasing transposon size (Ivics and Izsvak, 2011; Wilson et al., 2007). There are four transposase binding sites in a transposon (two per inverted terminal repeat). Bound transposases are proposed to interact with each other to promote juxtaposition of the two transposon ends. Overexpression of transposase has been hypothesized to inhibit transposition due to interaction of free transposase with bound transposase, thus preventing the juxtaposition step. Therefore, the optimal transposon/transposase ratio needs to be determined if these genes are delivered on separate constructs. However, reports of the inhibition phenomenon have been varied (Geurts et al., 2003; Izsvak et al., 2000). In our studies, overproduction inhibition was not observed (Fig. 1). The efficiency of SB transposition decreases with increasing transposon size with an upper transposon size limit of around 10 kB (Izsvak et al., 2000). For example, transposition levels are reduced by over 50% by increasing transposon size from 2.5 kB to just 4 kB (Izsvak et al., 2000). Therefore, stable transfer of multiple recombinant open reading frames may require multiplexed vector delivery. The Cooper group has used electroporation of SB plasmids to co-deliver a CAR-expressing plasmid with a second plasmid for positron emission tomography (PET) imaging of engineered Tcells (Bhatnagar et al., 2013). CAR- and thymidine kinase (TK)expressing T-cells (68% of total population) were selected 35 days after initial electroporation by using drug selection for the TKcontaining transposon and by expanding CAR-expressing T cells with artificial antigen presenting cells (Bhatnagar et al., 2013). In this work, we used the double mutant of human dihydrofolate reductase, DHFRdm, as the selection marker for engineered cells. Expression of DHFRdm in T-cells imparts MTX resistance without compromising proliferative ability, expression of T-cell markers, or
cytolytic ability (Jonnalagadda et al., 2013a). Mutant DHFR genes have been investigated as a gene therapy method to protect stably transfected hematopoietic cells against drug-induced myelosuppression side effects resulting from chemotherapy treatment (Sorrentino, 2002). Additional advantages of this selection system include availability of clinical grade MTX, the use of a nongenotoxic drug, and the small gene size of DHFRdm (561 bp) (Jonnalagadda et al., 2013a). MTX is particularly attractive for selection because it is a pharmaceutical drug that is clinically simple to administer, serum drug levels are measured routinely in clinical practice, and there is an available antidote (Leucovorin). Furthermore, Jonnalagadda et al. have demonstrated in vivo selection using MTX of transduced human T-cells (Jonnalagadda et al., 2013a). Using a two-step selection method through two successive MTX selection rounds, we were able to derive MTX resistant cell lines in which >50% of cells express three separate transcriptionally-active open reading frames derived from three individual MC constructs introduced into H9 cells in a single multiplexed electroporation procedure (Fig. 6). The multiplexed cells can be further purified by selection through flow sorting or by immunomagnetic isolation of cells with “bar code” markers. In some cases, multiplexed cells will have a functional survival and/or growth advantage so that they will be selected for over time in vitro or in vivo. In addition, 32% of the cell population expressed genes from two different transposon constructs. To increase the robustness of the selection pressure, separate genes could be tethered to selection genes such as hygromycin B phosphotransferase (Blochlinger and Diggelmann, 1984) and puromycin resistance gene (Sharma et al., 2013), and a “cocktail” of the appropriate selection agents could be subsequently applied to increase the selection for triple multiplexing events. Moreover, we envision that by varying the ratio of the components within the selection agent cocktail, different ratios of genes will be integrated, resulting in different ratios of protein expression. This could potentially lead to a high degree of control over gene integration. In summary, we demonstrate efficient multiplexed gene transfer to a human T cell line using the Sleeping Beauty transposon system. Cells with multiple integration events can be selected with increasing selection pressure. A two-step selection method can be used to further increase the population of cells expressing three desired transgenes. This method can potentially be applied for generation of engineered, multiplexed T-cells for adoptive T-cell immunotherapy applications. The authors are grateful to Mark A. Kay (Stanford University) for providing the minicircle expression system (pMC.BESPX plasmid and ZYCY10P3S2T E. coli producer strain) and Zsuzsanna Izsvak (Max Delbruck Center for Molecular Medicine, Berlin, Germany) for providing the CMV(CAT)T7SB100X plasmid. We thank Lisa Rolczynski (Seattle Children’s Research Institute) for helpful discussion and advice.
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