Efficient Nonviral Sleeping Beauty Transposon-Based TCR Gene
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Gene Therapy (2009) 16, 1042–1049 & 2009 Macmillan Publishers Limited All rights reserved 0969-7128/09 $32.00 www.nature.com/gt SHORT COMMUNICATION Efficient nonviral Sleeping Beauty transposon-based TCR gene transfer to peripheral blood lymphocytes confers antigen-specific antitumor reactivity PD Peng, CJ Cohen, S Yang, C Hsu, S Jones, Y Zhao, Z Zheng, SA Rosenberg and RA Morgan Surgery Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA Genetically engineered lymphocytes hold promise for the genes targeting p53 and MART-1, we demonstrated sustained treatment of genetic disease, viral infections and cancer. expression and functional reactivity of transposon-engineered However, current methods for genetic transduction of peri- lymphocytes on encountering target antigen presented on pheral blood lymphocytes rely on viral vectors, which are tumor cells. We found that transposon- and retroviral-modified hindered by production and safety-related problems. In this lymphocytes had comparable transgene expression and study, we demonstrated an efficient novel nonviral platform for phenotypic function. These results demonstrate the promise gene transfer to lymphocytes. The Sleeping Beauty transpo- of nonviral ex vivo genetic modification of autologous lympho- son-mediated approach allowed for long-term stable expres- cytes for the treatment of cancer and immunologic disease. sion of transgenes at B50% efficiency. Utilizing transposon Gene Therapy (2009) 16, 1042–1049; doi:10.1038/gt.2009.54; constructs expressing tumor antigen-specific T-cell receptor published online 4 June 2009 Keywords: Sleeping Beauty transposon; TCR gene therapy; adoptive immunotherapy; nonviral vector; T-cell gene transfer Advances in tumor immunology have led to the antigen-specific TCR genes can confer tumor antigen development of effective immunotherapies for cancer. specificity to normal PBL. Using traditional murine- A number of strategies are currently employed: active leukemia g-retroviral transduction of MART-1 TCR vaccination, nonspecific cytokine infusion, passive anti- genes, adoptive transfer of gene modified PBL-mediated body therapy and adoptive cell transfer.1 Significant objective tumor regression in 13% of melanoma patients.9 clinical responses have been reported for a number of This trial demonstrated successful regression of meta- solid tumor histologies using adoptive cell-transfer static melanoma after adoptive cell treatment with gene- therapies. Most notably for metastatic melanoma, adop- modified lymphocytes, however, further investigation is tive immunotherapy with lymphocytes expanded ex vivo necessary to improve response rates. Overcoming certain from excised tumor specimens (TIL; tumor infiltrating limitations of retroviral transduction may result in lymphocytes) mediated objective tumor regression in improved clinical outcomes and facilitate mainstream 51% of patients.2,3 There are, however, several limitations adoption of autologous gene-modified adoptive transfer to this treatment approach for patients with solid tumors. therapies. Tumor-specific lymphocytes can only be consistently The development of a scalable, high-throughput, expanded from melanoma lesions, thereby severely nonviral gene transfer system for clinical use may restricting the treatable solid tumor histologies, and a circumvent a number of limitations of the current significant population of patients will not have reactive g-retroviral-based gene transduction systems. Biological lymphocytes appropriate for therapeutic transfer. Recent properties of g-retroviral-based systems allow for effi- studies have overcome these limitations through genetic cient transduction of dividing cells but are incapable modification of normal peripheral blood lymphocytes of mediating transduction of quiescent cells. Murine (PBL).4 models of adoptive immunotherapy demonstrated that Tumor antigen recognition by lymphocytes is depen- minimally stimulated or quiescent cells are more dent on cell surface T-cell receptors (TCR), composed of effective in mediating antitumor responses.10 Another a- and b-chains. TCR with tumor antigen specificity have limitation of g-retroviral vector transduction centers on been cloned for a number of tumor antigens, including transcriptional gene silencing, which compromises trans- MART-1, gp100, NY-ESO-1 and p53.5–8 Transfer of gene expression through poorly defined cellular mechan- isms dependent on viral sequences, may be absent in nonviral transposons.11,12 Additionally, g-retroviruses Correspondence: Dr RA Morgan, National Cancer Institute, have a pronounced preference for integration into 10 Center Drive, Building 10, CRC 3W-3864, Bethesda, MD, 20892, 50 promoter-containing regions of genes, which has USA. E-mail: [email protected] resulted in insertional mutagenesis and malignant 13,14 Received 31 July 2008; revised 12 November 2008; accepted 10 transformation in a human clinical trial. Finally, December 2008; published online 4 June 2009 in addition to the theoretical limitation of viral vector- Transposon-mediated TCR gene transfer PD Peng et al 1043 mediated gene transfer, there are several practical Efficient long-term transposon-mediated limitations including the cost and time required for the production of these clinical-grade viral reagents. Taken transgene expression in peripheral blood together, the inherent limitations of viral-based gene lymphocytes transfer suggest that an efficient nonviral system would We developed a highly efficient nonviral gene transfer be a significant advancement in the genetic modification system for human PBL using a two-component Sleeping of hematopoietic cells for the treatment of human disease Beauty transposon system. Transposon plasmid DNA including cancer. pT3-GFP was coelectroporated along with in vitro Nonviral gene transfer has historically been hampered transcribed mRNA encoding the hyperactive Sleeping by low transduction efficiency in primary cells. Beauty HSB5 transposase, into peripheral blood mono- Recently, transposons have been developed as a nonviral nuclear cell (PBMC). Cell mortality after electroporation integrating system of gene delivery that is dependent was approximately 70%. Cells were stimulated with on only two nonviral components for integration: (1) the OKT3 and interleukin-2 (IL2) the following day, and donor transposon plasmid and (2) the helper transpo- lymphocyte activation resulted in a 20- to 100-fold sase.15,16 The Sleeping Beauty transposon system was increase in cell number over 6 weeks of culture (data reconstructed from salmonid phylogenetic data and not shown). Titration experiments demonstrated that was demonstrated to mediate functional integration gene transfer efficiency increased with the amount of when introduced in mammalian cells in a number of input RNA and DNA (Figure 1a). preclinical disease models.17–19 Initial efforts with a Using optimal amounts of transposon plasmid and bicistronic double-plasmid first-generation Sleeping transposase RNA, PBMC were electroporated and green Beauty system in lymphocytes resulted in poor gene fluorescent protein (GFP) reporter-gene expression was transfer efficiency.20 Mutagenesis of the enzyme monitored. Fluorescence-activated cell sorting (FACS) DNA-binding domain resulted in the development of analysis demonstrated that stable GFP transgene expres- hyperactive mutants with up to a 20-fold increase in sion was maintained at an efficiency of B50% over 6 native transposition efficiency.21,22 Moreover, the use of weeks (Figure 1b). In contrast, donor pT3-GFP plasmid mRNA encoding transposase allows temporal control of without helper HSB5 transposase RNA resulted in GFP transposase expression and eliminates the risk of expression declining to background levels within 1 week recombinant elements.23 Analysis of transposon genomic after electroporation. integration does not reveal a marked preference for Studies with the Sleeping Beauty transposon system promoter regions and may indicate a safer integration have demonstrated that there was an optimal ratio of profile.24,25 Sleeping Beauty transposase to transposon. It has been In this study, we developed a novel, efficient approach previously reported that increasing the ratio results in a to introduce the two-component Sleeping Beauty transpo- decrease in transposition efficiency through mechanisms son system into primary human PBL. We demonstrated of overexpression inhibition.26 We examined transposi- efficient long-term transgene expression and conferral of tion efficiency with a range of DNA and RNA substrates antigen-specific antitumor reactivity compatible with the encoding the HSB5 transposase at a fixed amount of needs of genetically modified cells for adoptive transfer T3GFP transposon DNA (Figure 1c). Electroporation therapy. 80 80 100 μ T3-GFP 10 g HSB5 RNA DNA 20 μg HSB5 RNA T3-GFP + HSB5 60 60 75 RNA 40 40 50 20 20 25 Percent GFP + cells Percent GFP + cells Percent GFP + cells 0 0 0 5 101520 7 1421283542 5 101520 pT3-GFP DNA (μg) Time (days) Transposase nuclei acid (μg) Figure 1 Optimization of transposon-mediated gene transfer into PBL. T3 transposon (pT3) was previously described21 and modified to contain the MSCV-U3 promoter, multiple cloning site and bovine growth hormone polyadenylation signal. The green fluorescent protein (GFP) reporter gene was inserted into the multiple cloning site. The Sleeping Beauty (SB) transposase was derived plasmid HSB5 as described,38 directionally cloned into T7-promoter expression plasmid pGEM4Z/GFP/64A, replacing