Key points are not available for this paper at this time.
We have reported earlier that the non‐viral Sleeping Beauty (SB) transposon system can mediate genomic integration and long‐term reporter gene expression in human primary peripheral blood (PB) T cells. In order to test whether this system can be used for genetically modifying both PB T cells and umbilical cord blood (UCB) T cells as graft‐versus‐leukemia effector cells, an SB transposon was constructed to coexpress a single‐chain chimeric antigen receptor (CAR) for human CD19 and CD20. PB and UCB were nucleofected with the transposon and a transposase plasmid, activated and then expanded in culture using anti‐CD3/CD28 beads. Stable dual‐gene expression was confirmed in both T‐cell types, permitting enrichment by positive selection with Rituxan. The engineered CD4+ T cells and CD8+ T cells both exhibited specific cytotoxicity against CD19+ leukemia and lymphoma cell lines, as well as against CD19 transfectants, and produced high‐levels of antigen‐dependent Th1 (but not Th2) cytokines. The in vivo adoptive transfer of genetically engineered T cells significantly reduced tumor growth and prolonged the survival of the animal. Taken together, these data indicate that T cells from PB and UCB can be stably modified using a non‐viral DNA transfer system, and that such modified T cells may be useful in the treatment of refractory leukemia and lymphoma. We have reported earlier that the non‐viral Sleeping Beauty (SB) transposon system can mediate genomic integration and long‐term reporter gene expression in human primary peripheral blood (PB) T cells. In order to test whether this system can be used for genetically modifying both PB T cells and umbilical cord blood (UCB) T cells as graft‐versus‐leukemia effector cells, an SB transposon was constructed to coexpress a single‐chain chimeric antigen receptor (CAR) for human CD19 and CD20. PB and UCB were nucleofected with the transposon and a transposase plasmid, activated and then expanded in culture using anti‐CD3/CD28 beads. Stable dual‐gene expression was confirmed in both T‐cell types, permitting enrichment by positive selection with Rituxan. The engineered CD4+ T cells and CD8+ T cells both exhibited specific cytotoxicity against CD19+ leukemia and lymphoma cell lines, as well as against CD19 transfectants, and produced high‐levels of antigen‐dependent Th1 (but not Th2) cytokines. The in vivo adoptive transfer of genetically engineered T cells significantly reduced tumor growth and prolonged the survival of the animal. Taken together, these data indicate that T cells from PB and UCB can be stably modified using a non‐viral DNA transfer system, and that such modified T cells may be useful in the treatment of refractory leukemia and lymphoma. IntroductionThe Sleeping Beauty (SB) transposon system has emerged as an effective genetic tool to achieve high‐level, persistent transgene expression from a non‐viral plasmid vector.1Ivics Z Hackett PB Plasterk RH Izsvak Z Molecular reconstruction of Sleeping Beauty, a Tc1‐like transposon from fish, and its transposition in human cells.Cell. 1997; 91: 501-510Abstract Full Text Full Text PDF PubMed Scopus (1097) Google Scholar,2Izsvak Z Ivics Z Sleeping Beauty transposon: biology and applications for molecular therapy.Mol Ther. 2004; 9: 147-156Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar SB is a “cut‐and‐paste” DNA transposon of the Tc1/mariner superfamily, and was reconstructed from sequences of teleost fish.1Ivics Z Hackett PB Plasterk RH Izsvak Z Molecular reconstruction of Sleeping Beauty, a Tc1‐like transposon from fish, and its transposition in human cells.Cell. 1997; 91: 501-510Abstract Full Text Full Text PDF PubMed Scopus (1097) Google Scholar The SB transposase mediates transposition by recognition of short inverted/directed repeat sequences that make up the termini of a constructed SB transposon. SB transposons have been known to exhibit efficient transposition in cells from a wide range of vertebrates, including in cultured mammalian cells,1Ivics Z Hackett PB Plasterk RH Izsvak Z Molecular reconstruction of Sleeping Beauty, a Tc1‐like transposon from fish, and its transposition in human cells.Cell. 1997; 91: 501-510Abstract Full Text Full Text PDF PubMed Scopus (1097) Google Scholar,3Geurts AM Yang Y Clark KJ Liu G Cui Z Dupuy AJ et al.Gene transfer into genomes of human cells by the Sleeping Beauty transposon system.Mol Ther. 2003; 8: 108-117Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar,4Yant SR Wu X Huang Y Garrison B Burgess SM Kay MA High‐resolution genome‐wide mapping of transposon integration in mammals.Mol Cell Biol. 2005; 25: 2085-2094Crossref PubMed Scopus (261) Google Scholar mouse liver and lung tissue,5Yant SR Meuse L Chiu W Ivics Z Izsvak Z Kay MA Somatic integration and long‐term transgene expression in normal and heamophilic mice using a DNA transposon system.Nat Genet. 2000; 25: 35-41Crossref PubMed Scopus (438) Google Scholar,6Belur LB Frandsen JL Dupuy AJ Ingbar DH Largaespada DA Hackett PB et al.Gene insertion and long‐term expression in lung mediated by the Sleeping Beauty transposon system.Mol Ther. 2003; 8: 501-507Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar mouse embryonic stem cells,7Luo G Ivics Z Izsvak Z Bradley A Chromosomal transposition of a Tc1/mariner‐like element in mouse embryonic stem cells.Proc Natl Acad Sci USA. 1998; 95: 10769-10773Crossref PubMed Scopus (211) Google Scholar and mouse embryos, thereby opening up potential for applications in germ‐line transgenesis and insertional mutagenesis.8Dupuy AJ Clark K Carlson CM Fritz S Davidson AE Markley KM et al.Mammalian germ‐line transgenesis by transposition.Proc Natl Acad Sci USA. 2002; 99: 4495-4499Crossref PubMed Scopus (179) Google Scholar,9Collier LS Carlson CM Ravimohan S Dupuy AJ Largaespada DA Cancer gene discovery in solid tumours using transposon‐based somatic mutagenesis in the mouse.Nature. 2005; 436: 272-276Crossref PubMed Scopus (344) Google Scholar,10Dupuy AJ Akagi K Largaespada DA Copeland NG Jenkins NA Mammalian mutagenesis using a highly mobile somatic Sleeping Beauty transposon system.Nature. 2005; 436: 221-226Crossref PubMed Scopus (420) Google Scholar,11Keng VW Yae K Hayakawa T Mizuno S Uno Y Yusa K et al.Region‐specific saturation germline mutagenesis in mice using the Sleeping Beauty transposon system.Nat Methods. 2005; 2: 763-769Crossref PubMed Scopus (99) Google Scholar,12Kitada K Ishishita S Tosaka K Takahashi R Ueda M Keng VW et al.Transposon‐tagged mutagenesis in the rat.Nat Methods. 2007; 4: 131-133Crossref PubMed Scopus (86) Google ScholarFor T‐cell gene transfer and therapy applications, the SB transposon system offers several advantages over the widely used virus‐based mammalian DNA Z Ivics Z Sleeping Beauty transposon: biology and applications for molecular therapy.Mol Ther. 2004; 9: 147-156Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar the of the SB transposon system is and the transposons and to the of gene transfer is that of AM Yang Y Clark KJ Liu G Cui Z Dupuy AJ et al.Gene transfer into genomes of human cells by the Sleeping Beauty transposon system.Mol Ther. 2003; 8: 108-117Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar in to mediated gene is for T‐cell using the of vivo culture is and in T‐cell and have that the SB transposon system can mediate expression of reporter in of human primary and T cells X L et gene transfer and expression in human primary T cells by the Sleeping Beauty transposon PubMed Scopus Google Scholar the expression of a gene useful of cells umbilical cord blood (UCB) T were gene was to whether the SB transposon system can be used for in human T cells from peripheral blood (PB) and cells be used in leukemia in lymphoma for stem cell and as a to the blood for the treatment of 2003; PubMed Scopus Google ScholarFor this in a SB a single‐chain chimeric antigen receptor (CAR) for a antigen for leukemia and and human CD20. was as a for as to T cells and to as a for T cells, using an that of CD19 and in human primary T of and was used for CD19 and in a SB transposon is of an human and human and the of human K M et receptor with cytotoxicity against 2004; 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PubMed Scopus Google Scholar not to T‐cell mediated of CD19+ cells. be these may to the CD19 that by CD19 engineered T cells from and UCB not the cells and expression of not data that may an in by the T cells. 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PubMed Scopus Google Scholar We this to coexpress CD19 in human T cells using the SB transposon system, and that the T cells from both and UCB can be to and using Rituxan. to that the engineered T cells from and CD19 were by and in and in the of a of a of with can cells the engineered were not of to in that expression was cells, the engineered and of was used for cell data in with from a that cell by expression T‐cell is to achieve of cell and to T S A SB cell by expression and to Cancer PubMed Scopus Google Scholar for cytotoxicity in in vivo have to by such as X L et gene transfer and expression in human primary T cells by the Sleeping Beauty transposon PubMed Scopus Google Scholar G et of and PubMed Scopus Google Scholar a dual‐gene expression order in the SB transposon with the advantages of the of expression efficient in selection of T in with of that T cells from and UCB CD19+ leukemia and lymphoma K M et receptor with cytotoxicity against 2004; PubMed Scopus Google et of by genetically human T by and 2003; 9: PubMed Scopus Google S et can be specific for the of the leukemia 2003; PubMed Scopus Google T S T et of cord blood T cells into for adoptive PubMed Scopus Google Scholar T cells and survival in tumor The of of the mediated by these T cells may be the of the T cells CD4+ effector cells that may adoptive and achieve a of to cells, not to cells is for and of T cells into effector and MA AJ for of CD8+ T PubMed Scopus Google CD8+ T cells into cells treatment in 2007; PubMed Scopus Google Scholar to by with CD19 and to both and T cells using NA A et cells to a of Ther. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar as to the of the T‐cell in both and a Y R M et T cells leukemia Cancer 2007; PubMed Scopus Google that engineered T cells from both and UCB CD19+ and cells as as T is an that with genetically modified T cells T cells S et can be specific for the of the leukemia 2003; PubMed Scopus Google T S T et of cord blood T cells into for adoptive PubMed Scopus Google Scholar of T cells may be a of that T cells in M M T cells in tumor 2005; PubMed Scopus Google Scholar be to both and T cells that can tumor cells in to Th1 cytokines. may be an effective in the of for tumor in to leukemia and lymphoma as in this and and were by and were from and was from cell were in of and of human was from cells using the and was using was using DNA with specific and The was into and The was with transposons and The transposon was constructed using molecular and the inverted/directed repeat sequences Z AM Liu G Hackett PB of for the Sleeping Beauty Biol. 2002; PubMed Scopus Google Scholar was into the SB transposon in the gene is with human using a of the human and a M MA A L dual‐gene transgenesis by 2004; PubMed Scopus Google Scholar In the element was of the of the human the human SB transposase T‐cell gene T‐cell gene transfer was using a with the human T‐cell X L et gene transfer and expression in human primary T cells by the Sleeping Beauty transposon PubMed Scopus Google Scholar were from from from blood from The UCB cells were by the and the in the cells from PB UCB T cells were with of transposon and of the transposase plasmid and was in with of transposon and and of not the cells were activated using and for K S et transfer of CD4+ T cells of peripheral T cells and expression in 2002; 8: PubMed Scopus Google Scholar of the the activated were in human T‐cell of human and with and Cancer and with as K S et transfer of CD4+ T cells of peripheral T cells and expression in 2002; 8: PubMed Scopus Google SR The of and to and human T Methods. PubMed Scopus Google Scholar as by the of of G with and the of G were from with mouse A and and were from was the system using of with and selection of T cells with Rituxan. was from the and were used for with and of were using the and selection of T cells with and was in with the and A T‐cell cytotoxicity was using a X AM Huang X Huang et CD8+ T‐cell to cell in with an tumor cell 2005; Google Scholar of recognition by and DA M et of CD19 in mice with 2004; PubMed Scopus Google Scholar cells were for with of effector cells. of and were in the were using and the of insertion The used for transposon the and have been LB Frandsen JL Dupuy AJ Ingbar DH Largaespada DA Hackett PB et al.Gene insertion and long‐term expression in lung mediated by the Sleeping Beauty transposon system.Mol Ther. 2003; 8: 501-507Abstract Full Text Full Text PDF PubMed Scopus (100) Google AJ Clark K Carlson CM Fritz S Davidson AE Markley KM et al.Mammalian germ‐line transgenesis by transposition.Proc Natl Acad Sci USA. 2002; 99: 4495-4499Crossref PubMed Scopus (179) Google X L et gene transfer and expression in human primary T cells by the Sleeping Beauty transposon PubMed Scopus Google Scholar the and were into and The sequences were to human of CD19 CD19 in a was from with and and into the In order to a of CD19 a was and into were by of cells in with and using vivo to mice were from the and was the of in with the mice were into a to a to T‐cell and a to T‐cell the mice were and then with the the mice that were for tumor using was the T‐cell by were and using the A was over the tumor the of the was as for and and in of A was to for the in and In order to make the to normal a was were a The of treatment was as a The of treatment and of treatment was was A of in the was and a was A was as a selection of the from using the were using were with and Methods. with cells to mediated by engineered T cells. with transfer in and with mediated cell by Rituxan. with and sequences used in a IntroductionThe Sleeping Beauty (SB) transposon system has emerged as an effective genetic tool to achieve high‐level, persistent transgene expression from a non‐viral plasmid vector.1Ivics Z Hackett PB Plasterk RH Izsvak Z Molecular reconstruction of Sleeping Beauty, a Tc1‐like transposon from fish, and its transposition in human cells.Cell. 1997; 91: 501-510Abstract Full Text Full Text PDF PubMed Scopus (1097) Google Scholar,2Izsvak Z Ivics Z Sleeping Beauty transposon: biology and applications for molecular therapy.Mol Ther. 2004; 9: 147-156Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar SB is a “cut‐and‐paste” DNA transposon of the Tc1/mariner superfamily, and was reconstructed from sequences of teleost fish.1Ivics Z Hackett PB Plasterk RH Izsvak Z Molecular reconstruction of Sleeping Beauty, a Tc1‐like transposon from fish, and its transposition in human cells.Cell. 1997; 91: 501-510Abstract Full Text Full Text PDF PubMed Scopus (1097) Google Scholar The SB transposase mediates transposition by recognition of short inverted/directed repeat sequences that make up the termini of a constructed SB transposon. SB transposons have been known to exhibit efficient transposition in cells from a wide range of vertebrates, including in cultured mammalian cells,1Ivics Z Hackett PB Plasterk RH Izsvak Z Molecular reconstruction of Sleeping Beauty, a Tc1‐like transposon from fish, and its transposition in human cells.Cell. 1997; 91: 501-510Abstract Full Text Full Text PDF PubMed Scopus (1097) Google Scholar,3Geurts AM Yang Y Clark KJ Liu G Cui Z Dupuy AJ et al.Gene transfer into genomes of human cells by the Sleeping Beauty transposon system.Mol Ther. 2003; 8: 108-117Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar,4Yant SR Wu X Huang Y Garrison B Burgess SM Kay MA High‐resolution genome‐wide mapping of transposon integration in mammals.Mol Cell Biol. 2005; 25: 2085-2094Crossref PubMed Scopus (261) Google Scholar mouse liver and lung tissue,5Yant SR Meuse L Chiu W Ivics Z Izsvak Z Kay MA Somatic integration and long‐term transgene expression in normal and heamophilic mice using a DNA transposon system.Nat Genet. 2000; 25: 35-41Crossref PubMed Scopus (438) Google Scholar,6Belur LB Frandsen JL Dupuy AJ Ingbar DH Largaespada DA Hackett PB et al.Gene insertion and long‐term expression in lung mediated by the Sleeping Beauty transposon system.Mol Ther. 2003; 8: 501-507Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar mouse embryonic stem cells,7Luo G Ivics Z Izsvak Z Bradley A Chromosomal transposition of a Tc1/mariner‐like element in mouse embryonic stem cells.Proc Natl Acad Sci USA. 1998; 95: 10769-10773Crossref PubMed Scopus (211) Google Scholar and mouse embryos, thereby opening up potential for applications in germ‐line transgenesis and insertional mutagenesis.8Dupuy AJ Clark K Carlson CM Fritz S Davidson AE Markley KM et al.Mammalian germ‐line transgenesis by transposition.Proc Natl Acad Sci USA. 2002; 99: 4495-4499Crossref PubMed Scopus (179) Google Scholar,9Collier LS Carlson CM Ravimohan S Dupuy AJ Largaespada DA Cancer gene discovery in solid tumours using transposon‐based somatic mutagenesis in the mouse.Nature. 2005; 436: 272-276Crossref PubMed Scopus (344) Google Scholar,10Dupuy AJ Akagi K Largaespada DA Copeland NG Jenkins NA Mammalian mutagenesis using a highly mobile somatic Sleeping Beauty transposon system.Nature. 2005; 436: 221-226Crossref PubMed Scopus (420) Google Scholar,11Keng VW Yae K Hayakawa T Mizuno S Uno Y Yusa K et al.Region‐specific saturation germline mutagenesis in mice using the Sleeping Beauty transposon system.Nat Methods. 2005; 2: 763-769Crossref PubMed Scopus (99) Google Scholar,12Kitada K Ishishita S Tosaka K Takahashi R Ueda M Keng VW et al.Transposon‐tagged mutagenesis in the rat.Nat Methods. 2007; 4: 131-133Crossref PubMed Scopus (86) Google ScholarFor T‐cell gene transfer and therapy applications, the SB transposon system offers several advantages over the widely used virus‐based mammalian DNA Z Ivics Z Sleeping Beauty transposon: biology and applications for molecular therapy.Mol Ther. 2004; 9: 147-156Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar the of the SB transposon system is and the transposons and to the of gene transfer is that of AM Yang Y Clark KJ Liu G Cui Z Dupuy AJ et al.Gene transfer into genomes of human cells by the Sleeping Beauty transposon system.Mol Ther. 2003; 8: 108-117Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar in to mediated gene is for T‐cell using the of vivo culture is and in T‐cell and have that the SB transposon system can mediate expression of reporter in of human primary and T cells X L et gene transfer and expression in human primary T cells by the Sleeping Beauty transposon PubMed Scopus Google Scholar the expression of a gene useful of cells umbilical cord blood (UCB) T were gene was to whether the SB transposon system can be used for in human T cells from peripheral blood (PB) and cells be used in leukemia in lymphoma for stem cell and as a to the blood for the treatment of 2003; PubMed Scopus Google ScholarFor this in a SB a single‐chain chimeric antigen receptor (CAR) for a antigen for leukemia and and human CD20. was as a for as to T cells and to as a for T cells, using an that
Huang et al. (Tue,) studied this question.