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1
Ikeda, Ryuji, Chikara Kokubu, Kosuke Yusa, Vincent W. Keng, Kyoji Horie, and Junji Takeda. "Sleeping Beauty Transposase Has an Affinity for Heterochromatin Conformation." Molecular and Cellular Biology 27, no. 5 (December 18, 2006): 1665–76. http://dx.doi.org/10.1128/mcb.01500-06.
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ABSTRACT The Sleeping Beauty (SB) transposase reconstructed from salmonid fish has high transposition activity in mammals and has been a useful tool for insertional mutagenesis and gene delivery. However, the transposition efficiency has varied significantly among studies. Our previous study demonstrated that the introduction of methylation into the SB transposon enhanced transposition, suggesting that transposition efficiency is influenced by the epigenetic status of the transposon region. Here, we examined the influence of the chromatin status on SB transposition in mouse embryonic stem cells. Heterochromatin conformation was introduced into the SB transposon by using a tetracycline-controlled transrepressor (tTR) protein, consisting of a tetracycline repressor (TetR) fused to the Kruppel-associated box (KRAB) domain of human KOX1 through tetracycline operator (tetO) sequences. The excision frequency of the SB transposon, which is the first step of the transposition event, was enhanced by approximately 100-fold. SB transposase was found to be colocalized with intense DAPI (4′,6′-diamidino-2-phenylindole) staining and with the HP1 family by biochemical fractionation analyses. Furthermore, chromatin immunoprecipitation analysis revealed that SB transposase was recruited to tTR-induced heterochromatic regions. These data suggest that the high affinity of SB transposase for heterochromatin conformation leads to enhancement of SB transposition efficiency.
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Fili, A. E., A. P. Alessio, W. Garrels, D. O. Forcato, M. F. Olmos Nicotra, A. C. Liaudat, R. J. Bevacqua, et al. "242 HIGHLY EFFICIENT SLEEPING BEAUTY TRANSPOSON-MEDIATED TRANSGENESIS IN BOVINE FETAL FIBROBLASTS." Reproduction, Fertility and Development 28, no. 2 (2016): 253. http://dx.doi.org/10.1071/rdv28n2ab242.
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Active transposon-mediated transgenesis is an emerging tool for basic and applied research in livestock. We have demonstrated the effectiveness of a helper-independent piggyBac transposon (pGENIE-3) for gene transfer into the genome of bovine cells (Alessio et al. 2014 Reprod. Domest. Anim. 49, 8). Here, we extend our previous research by examining the suitability of a Sleeping Beauty (SB) transposon-based methodology to deliver transgenes into the genome of bovine fetal fibroblasts (BFF), and the ability of these cells to support in vitro embryo development upon somatic cell nuclear transfer (SCNT). In a first experiment, BFF were chemically cotransfected (JetPRIME®, Polyplus-transfection, Illkirch, France) with a helper plasmid (pCMV-SB100X), which carries an expression cassette for the SB transposase, and the donor vector (pT2/Venus/RMCE) harboring an expression cassette for a fluorescent protein (Venus) flanked by the SB inverted terminal repeats (ITR). Three different ratios of helper and donor plasmids were studied: 1 : 2, 1 : 1 and 2 : 1. After 15 days of culture, the number of fluorescent colonies was counted on an inverted microscope. When vectors were used at ratios of 1 : 1 and 2 : 1, a 78-fold and 88-fold increase (P ≤ 0.05) in the number of fluorescent colonies compared with that in the no-transposase control were calculated. In a second experiment, BFF were chemically cotransfected with the helper vector pCMV-SB100X, and 2 donor transposons: pT2/Venus/RMCE and pT2/SV40-Neo. The former harbors a neo resistance cassette framed by SB ITRs. Different ratios of helper:donors (1 : 1 : 1, 2 : 1 : 1 and 2 : 0.5 : 0.5) were studied, and each ratio compared with a no-transposase control. After 15 days of antibiotic selection, the number of G418-resistant colonies was determined. Every time a functional SB transposase vector was included, the number of fluorescent and G418-resistant colonies was markedly higher compared with that in the respective control without transposase (P ≤ 0.001). Interestingly, all G418-resistant colonies expressed Venus. Molecular characterisation of genomic insertions in 6 monoclonal cell lines was performed by PCR and splinkerette PCR. PCR analysis confirmed presence of the Venus transgene in all cell lines. Splinkerette PCR results revealed at least 15 transposase-catalyzed genomic insertions of the transgene. Individual cells from a polyclonal SB transgenic fibroblast culture were used as nuclear donors to produce zona-free SCNT embryos. Of the reconstructed embryos, 33% reached blastocyst stage and about half of them expressed Venus. In conclusion, SB transposase is able to actively transpose monomeric copies of transgenes into the genome of bovine cells, which can be reprogrammed upon nuclear transfer to generate morphologically normal embryos expressing the transgene of interest.
3
Yant, Stephen R., Julie Park, Yong Huang, Jacob Giehm Mikkelsen, and Mark A. Kay. "Mutational Analysis of the N-Terminal DNA-Binding Domain of Sleeping Beauty Transposase: Critical Residues for DNA Binding and Hyperactivity in Mammalian Cells." Molecular and Cellular Biology 24, no. 20 (October 15, 2004): 9239–47. http://dx.doi.org/10.1128/mcb.24.20.9239-9247.2004.
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ABSTRACT The N-terminal domain of the Sleeping Beauty (SB) transposase mediates transposon DNA binding, subunit multimerization, and nuclear translocation in vertebrate cells. For this report, we studied the relative contributions of 95 different residues within this multifunctional domain by large-scale mutational analysis. We found that each of four amino acids (leucine 25, arginine 36, isoleucine 42, and glycine 59) contributes to DNA binding in the context of the N-terminal 123 amino acids of SB transposase, as indicated by electrophoretic mobility shift analysis, and to functional activity of the full-length transposase, as determined by a quantitative HeLa cell-based transposition assay. Moreover, we show that amino acid substitutions within either the putative oligomerization domain (L11A, L18A, L25A, and L32A) or the nuclear localization signal (K104A and R105A) severely impair its ability to mediate DNA transposition in mammalian cells. In contrast, each of 10 single amino acid changes within the bipartite DNA-binding domain is shown to greatly enhance SB's transpositional activity in mammalian cells. These hyperactive mutations functioned synergistically when combined and are shown to significantly improve transposase affinity for transposon end sequences. Finally, we show that enhanced DNA-binding activity results in improved cleavage kinetics, increased SB element mobilization from host cell chromosomes, and dramatically improved gene transfer capabilities of SB in vivo in mice. These studies provide important insights into vertebrate transposon biology and indicate that Sleeping Beauty can be readily improved for enhanced genetic research applications in mammals.
4
Converse, Andrea D., Lalitha R. Belur, Jennifer L. Gori, Geyi Liu, Felipe Amaya, Estuardo Aguilar-Cordova, Perry B. Hackett, and R. Scott McIvor. "Counterselection and Co-Delivery of Transposon and Transposase Functions for Sleeping Beauty-Mediated Transposition in Cultured Mammalian Cells." Bioscience Reports 24, no. 6 (December 1, 2004): 577–94. http://dx.doi.org/10.1007/s10540-005-2793-9.
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Sleeping Beauty (SB) is a gene-insertion system reconstructed from transposon sequences found in teleost fish and is capable of mediating the transposition of DNA sequences from transfected plasmids into the chromosomes of vertebrate cell populations. The SB system consists of a transposon, made up of a gene of interest flanked by transposon inverted repeats, and a source of transposase. Here we carried out a series of studies to further characterize SB-mediated transposition as a tool for gene transfer to chromosomes and ultimately for human gene therapy. Transfection of mouse 3T3 cells, HeLa cells, and human A549 lung carcinoma cells with a transposon containing the neomycin phosphotransferase (NEO) gene resulted in a several-fold increase in drug-resistant colony formation when co-transfected with a plasmid expressing the SB transposase. A transposon containing a methotrexate-resistant dihydrofolate reductase gene was also found to confer an increased frequency of methotrexate-resistant colony formation when co-transfected with SB transposase-encoding plasmid. A plasmid containing a herpes simplex virus thymidine kinase gene as well as a transposon containing a NEO gene was used for counterselection against random recombinants (NEO+TK+) in medium containing G418 plus ganciclovir. Effective counterselection required a recovery period of 5 days after transfection before shifting into medium containing ganciclovir to allow time for transiently expressed thymidine kinase activity to subside in cells not stably transfected. Southern analysis of clonal isolates indicated a shift from random recombination events toward transposition events when clones were isolated in medium containing ganciclovir as well as G418. We found that including both transposon and transposase functions on the same plasmid substantially increased the stable gene transfer frequency in Huh7 human hepatoma cells. The results from these experiments contribute technical and conceptual insight into the process of transposition in mammalian cells, and into the optimal provision of transposon and transposase functions that may be applicable to gene therapy studies.
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Huang, Xin, Andrew C. Wilber, Lei Bao, Dong Tuong, Jakub Tolar, Paul J. Orchard, Bruce L. Levine, et al. "Stable gene transfer and expression in human primary T cells by the Sleeping Beauty transposon system." Blood 107, no. 2 (January 15, 2006): 483–91. http://dx.doi.org/10.1182/blood-2005-05-2133.
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AbstractThe Sleeping Beauty (SB) transposon system is a nonviral DNA delivery system in which a transposase directs integration of an SB transposon into TA-dinucleotide sites in the genome. To determine whether the SB transposon system can mediate stable gene expression in human T cells, primary peripheral blood lymphocytes (PBLs) were nucleofected with SB vectors carrying a DsRed reporter gene. Plasmids containing the SB transposase on the same molecule as (cis) or on a molecule separate from (trans) the SB transposon mediated long-term and stable reporter gene expression in human primary T cells. Sequencing of transposon:chromosome junctions confirmed that stable gene expression was due to SB-mediated transposition. In other studies, PBLs were successfully transfected using the SB transposon system and shown to stably express a fusion protein consisting of (1) a surface receptor useful for positive T-cell selection and (2) a “suicide” gene useful for elimination of transfected T cells after chemotherapy. This study is the first report demonstrating that the SB transposon system can mediate stable gene transfer in human primary PBLs, which may be advantageous for T-cell–based gene therapies.
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Zhou, Xianzheng, Xin Huang, Andrew C. Wilber, Lei Bao, Dong Tuong, Jakub Tolar, Paul J. Orchard, et al. "Stable Gene Transfer and Expression in Human Primary T-Cells by the Sleeping Beauty Transposon System." Blood 106, no. 11 (November 16, 2005): 5539. http://dx.doi.org/10.1182/blood.v106.11.5539.5539.
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Abstract The Sleeping Beauty (SB) transposon system is a non-viral DNA delivery system in which a transposase directs integration of an SB transposon into TA-dinucleotide sites in the genome. To determine whether the SB transposon system can mediate integration and long-term transgene expression in human primary T-cells, freshly isolated peripheral blood lymphocytes (PBLs) without prior activation were nucleofected with SB vectors carrying a DsRed reporter gene. Plasmids containing the SB transposase on the same (cis) (n=10) or separate molecule (trans) (n=8) as the SB transposon mediated long-term and stable reporter gene expression in human primary T-cells. We observed that delivery of SB transposase-encoding plasmid in trans effectively mediated stable gene expression in primary T-cells, exhibiting about a 3-fold increase (11% vs. 3% with 10 microgram plasmid on day 21) in potency in comparison with the cis vector (p<0.0001). In addition, a transposase mutant construct was incapable of mediating stable gene expression in human PBLs (n=6, p<0.0001), confirming that catalytic DDE domain is necessary for transposition in human primary T-cells. Immunophenotyping analysis in transposed T-cells showed that both CD4 and CD8 T-cells were transgene positive. SB-mediated high level of transgene expression in human T-cells was maintained in culture for at least 4 months without losing observable expression. Southern hybridization analysis showed a variety of transposon integrants among the 6 DsRed positive T-cell clones and no transposon sequences identifiable in the 2 DsRed negative clones. Sequencing of transposon:chromosome junctions in 5 out of 6 transposed T-cell clones confirmed that stable gene expression was due to SB-mediated transposition. In other studies, PBLs were successfully transfected using the SB transposon system and shown to stably and functionally express a fusion protein consisting of a surface receptor useful for positive T-cell selection and a “suicide” gene useful for elimination of transfected T-cells after chemotherapy. This study is the first report demonstrating that the SB transposon system can mediate stable gene transfer in human primary PBLs, which may be more advantageous for T-cell based gene therapies over widely used virus-based or conventional mammalian DNA vectors in terms of simplicity, stability, efficiency and safety.
7
Miskey, Csaba, Lisa Kesselring, Irma Querques, György Abrusán, Orsolya Barabas, and Zoltán Ivics. "Engineered Sleeping Beauty transposase redirects transposon integration away from genes." Nucleic Acids Research 50, no. 5 (February 21, 2022): 2807–25. http://dx.doi.org/10.1093/nar/gkac092.
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Abstract The Sleeping Beauty (SB) transposon system is a popular tool for genome engineering, but random integration into the genome carries a certain genotoxic risk in therapeutic applications. Here we investigate the role of amino acids H187, P247 and K248 in target site selection of the SB transposase. Structural modeling implicates these three amino acids located in positions analogous to amino acids with established functions in target site selection in retroviral integrases and transposases. Saturation mutagenesis of these residues in the SB transposase yielded variants with altered target site selection properties. Transposon integration profiling of several mutants reveals increased specificity of integrations into palindromic AT repeat target sequences in genomic regions characterized by high DNA bendability. The H187V and K248R mutants redirect integrations away from exons, transcriptional regulatory elements and nucleosomal DNA in the human genome, suggesting enhanced safety and thus utility of these SB variants in gene therapy applications.
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Kesselring, Lisa, Csaba Miskey, Cecilia Zuliani, Irma Querques, Vladimir Kapitonov, Andrea Laukó, Anita Fehér, et al. "A single amino acid switch converts the Sleeping Beauty transposase into an efficient unidirectional excisionase with utility in stem cell reprogramming." Nucleic Acids Research 48, no. 1 (November 28, 2019): 316–31. http://dx.doi.org/10.1093/nar/gkz1119.
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Abstract The Sleeping Beauty (SB) transposon is an advanced tool for genetic engineering and a useful model to investigate cut-and-paste DNA transposition in vertebrate cells. Here, we identify novel SB transposase mutants that display efficient and canonical excision but practically unmeasurable genomic re-integration. Based on phylogenetic analyses, we establish compensating amino acid replacements that fully rescue the integration defect of these mutants, suggesting epistasis between these amino acid residues. We further show that the transposons excised by the exc+/int− transposase mutants form extrachromosomal circles that cannot undergo a further round of transposition, thereby representing dead-end products of the excision reaction. Finally, we demonstrate the utility of the exc+/int− transposase in cassette removal for the generation of reprogramming factor-free induced pluripotent stem cells. Lack of genomic integration and formation of transposon circles following excision is reminiscent of signal sequence removal during V(D)J recombination, and implies that cut-and-paste DNA transposition can be converted to a unidirectional process by a single amino acid change.
9
Yusa, Kosuke, Junji Takeda, and Kyoji Horie. "Enhancement of Sleeping Beauty Transposition by CpG Methylation: Possible Role of Heterochromatin Formation." Molecular and Cellular Biology 24, no. 9 (May 1, 2004): 4004–18. http://dx.doi.org/10.1128/mcb.24.9.4004-4018.2004.
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ABSTRACT The Sleeping Beauty (SB) transposase is the most active transposase in vertebrate cells, and the SB transposon system has been used as a tool for insertional mutagenesis and gene delivery. Previous studies have indicated that the frequency of chromosomal transposition is considerably higher in mouse germ cells than in mouse embryonic stem cells, suggesting the existence of unknown mechanisms that regulate SB transposition. Here, we demonstrated that CpG methylation of the transposon region enhances SB transposition. The transposition efficiencies of a methylated transposon and an unmethylated transposon which had been targeted in the same genomic loci by recombination-mediated cassette exchange in mouse erythroleukemia cells were compared, and at least a 100-fold increase was observed in the methylated transposon. CpG methylation also enhanced transposition from plasmids into the genome. Chromatin immunoprecipitation assays revealed that histone H3 methylated at lysine-9, a hallmark of condensed heterochromatin, was enriched at the methylated transposon, whereas the unmethylated transposon formed a relaxed euchromatin structure, as evidenced by enrichment of acetylated histone H3 and reporter gene expression. Possible roles of heterochromatin formation in the transposition reaction are discussed. Our findings indicate a novel relationship between CpG methylation and transposon mobilization.
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Yant, Stephen R., and Mark A. Kay. "Nonhomologous-End-Joining Factors Regulate DNA Repair Fidelity during Sleeping Beauty Element Transposition in Mammalian Cells." Molecular and Cellular Biology 23, no. 23 (December 1, 2003): 8505–18. http://dx.doi.org/10.1128/mcb.23.23.8505-8518.2003.
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ABSTRACT Herein, we report that the DNA-dependent protein kinase (DNA-PK) regulates the DNA damage introduced during Sleeping Beauty (SB) element excision and reinsertion in mammalian cells. Using both plasmid- and chromosome-based mobility assays, we analyzed the repair of transposase-induced double-stranded DNA breaks in cells deficient in either the DNA-binding subunit of DNA-PK (Ku) or its catalytic subunit (DNA-PKcs). We found that the free 3′ overhangs left after SB element excision were efficiently and accurately processed by the major Ku-dependent nonhomologous-end-joining pathway. Rejoining of broken DNA molecules in the absence of Ku resulted in extensive end degradation at the donor site and greatly increased the frequency of recombination with ectopic templates. Therefore, the major DNA-PK-dependent DNA damage response predominates over more-error-prone repair pathways and thereby facilitates high-fidelity DNA repair during transposon mobilization in mammalian cells. Although transposable elements were not found to be efficiently circularized after transposase-mediated excision, DNA-PK deficiency supported more-frequent transposase-mediated element insertion than was found in wild-type controls. We conclude that, based on its ability to regulate excision site junctional diversity and transposon insertion frequency, DNA-PK serves an important protective role during transpositional recombination in mammals.
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Sumiyoshi, Teiko, Roger P. Hollis, Nathalia Holt, and Donald B. Kohn. "Optimization of the Sleeping Beauty Transposon System to Achieve Stable Transgene Expression in Human CD34+ Hematopoietic Progenitor Cells." Blood 112, no. 11 (November 16, 2008): 3527. http://dx.doi.org/10.1182/blood.v112.11.3527.3527.
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Abstract Sleeping Beauty (SB) transposon-mediated integration has been shown to achieve long-term transgene expression in a wide range of host cells. Transposon-mediated gene integration may have advantages over viral vectors, with a greater transgene carrying capacity and potentially safer integration site profile. Due to these characteristics of SB, there has been great interest in its potential use in hematopoietic stem cell (HSC) gene therapy. In this study, we optimized the SB transposon-mediated gene transfer system to achieve higher stable transgene expression in K562 human erythroleukemia cells, Jurkat human T-lymphoid cells, and primary human CD34+ hematopoietic progenitor cells. The SB transposon system was optimized by two approaches: to increase the transposition efficacy, a hyperactive mutant of SB, HSB16, was used (Baus et al.; Mol Ther12:1148, 2005); to optimize the expression of the SB transposase and the transgene cassette carried by the transposon, three different viral and cellular promoters were evaluated, including the modified MPSV long terminal repeat (MNDU3) enhancer-promoter, the human cytomegalovirus (hCMV) immediate-early region enhancer-promoter, and the human elongation factor 1 (hEF1a) promoter. SB components were delivered in trans into the target cells by nucleoporation. The SB transposon-mediated integration efficacy was assessed by integrated transgene (enhanced green fluorescent protein [eGFP]) expression using fluorescent-activated cell sorting (FACS) analysis over 3–4 weeks. The functional assay showed that HSB16 was a more efficient enzyme compared to the original SB. In purified human cord blood CD34+ cells, HSB16 achieved nearly 7-fold higher long-term transgene expression with 90% less plasmid DNA (from 10 mcg of SB reduced to 1 mcg of HSB16) than the original SB transposase. The highest level of stable transgene integration in all three cell types was achieved using the hEF1a promoter to express HSB16 in comparison to either the hCMV or MND promoter. Our data also suggested that optimal GFP reporter gene expression from the integrated transposon was influenced by the type of promoter and the target cell type. Significantly higher levels of eGFP expression (5-fold) were achieved with the hEF1a promoter in Jurkat human T cells, compared to that achieved with the MND promoter; in contrast the MND promoter expressed GFP at the highest level in K562 myeloid cells. In primary human CD34+ cord blood progenitors, optimal transgene integration and expression was achieved using the hEF1a promoter to express the SB transposase combined with the MND promoter to express GFP reporter, when studied under conditions directing myeloid differentiation. Stable transgene expression was achieved at levels up to 27% for over 4 weeks after optimized gene transfer to CD34+ cells (ave=17%, n=4). In vivo studies evaluating engraftment and differentiation of the SB-modified human CD34+ progenitor cells are currently in progress. In conclusion, the optimized SB transposon system in primary human CD34+ hematopoietic progenitors reported here has improved the stable gene transfer efficiency by 29-fold, compared to our prior published data (< 1% - Hollis et al.; Exp Hematol34:1333, 2006). The long-term stable gene expression achieved by our optimized SB transposon system shows promise for further advancement of non-viral based HSC gene therapy.
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Xue, Xingkui, Xin Huang, Sonja E. Nodland, Lajos Mátés, Linan Ma, Zsuzsanna Izsvák, Zoltán Ivics, et al. "Stable gene transfer and expression in cord blood–derived CD34+ hematopoietic stem and progenitor cells by a hyperactive Sleeping Beauty transposon system." Blood 114, no. 7 (August 13, 2009): 1319–30. http://dx.doi.org/10.1182/blood-2009-03-210005.
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Abstract Here we report stable gene transfer in cord blood-derived CD34+ hematopoietic stem cells using a hyperactive nonviral Sleeping Beauty (SB) transposase (SB100X). In colony-forming assays, SB100X mediated the highest efficiency (24%) of stable Discosoma sp red fluorescent protein (DsRed) reporter gene transfer in committed hematopoietic progenitors compared with both the early-generation hyperactive SB11 transposase and the piggyBac transposon system (1.23% and 3.8%, respectively). In vitro differentiation assays further demonstrated that SB100X-transfected CD34+ cells can develop into DsRed+ CD4+CD8+ T (3.17%-21.84%; median, 7.97%), CD19+ B (3.83%-18.66%; median, 7.84%), CD56+CD3− NK (3.53%-79.98%; median, 7.88%), and CD33+ myeloid (7.59%-15.63%; median, 9.48%) cells. SB100X-transfected CD34+ cells achieved approximately 46% engraftment in NOD-scid IL2γcnull (NOG) mice. Twelve weeks after transplantation, 0.57% to 28.96% (median, 2.79%) and 0.49% to 34.50% (median, 5.59%) of total human CD45+ cells in the bone marrow and spleen expressed DsRed, including CD19+ B, CD14+ monocytoid, and CD33+ myeloid cell lineages. Integration site analysis revealed SB transposon sequences in the human chromosomes of in vitro differentiated T, B, NK, and myeloid cells, as well as in human CD45+ cells isolated from bone marrow and spleen of transplanted NOG mice. Our results support the continuing development of SB-based gene transfer into human hematopoietic stem cells as a modality for gene therapy.
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Jia, Wenzhu, Emmanuel Asare, Tao Liu, Pingjing Zhang, Yali Wang, Saisai Wang, Dan Shen, et al. "Horizontal Transfer and Evolutionary Profiles of Two Tc1/DD34E Transposons (ZB and SB) in Vertebrates." Genes 13, no. 12 (November 29, 2022): 2239. http://dx.doi.org/10.3390/genes13122239.
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Both ZeBrafish (ZB), a recently identified DNA transposon in the zebrafish genome, and SB, a reconstructed transposon originally discovered in several fish species, are known to exhibit high transposition activity in vertebrate cells. Although a similar structural organization was observed for ZB and SB transposons, the evolutionary profiles of their homologs in various species remain unknown. In the present study, we compared their taxonomic ranges, structural arrangements, sequence identities, evolution dynamics, and horizontal transfer occurrences in vertebrates. In total, 629 ZB and 366 SB homologs were obtained and classified into four distinct clades, named ZB, ZB-like, SB, and SB-like. They displayed narrow taxonomic distributions in eukaryotes, and were mostly found in vertebrates, Actinopterygii in particular tended to be the major reservoir hosts of these transposons. Similar structural features and high sequence identities were observed for transposons and transposase, notably homologous to the SB and ZB elements. The genomic sequences that flank the ZB and SB transposons in the genomes revealed highly conserved integration profiles with strong preferential integration into AT repeats. Both SB and ZB transposons experienced horizontal transfer (HT) events, which were most common in Actinopterygii. Our current study helps to increase our understanding of the evolutionary properties and histories of SB and ZB transposon families in animals.
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Ochmann, Matthias T., and Zoltán Ivics. "Jumping Ahead with Sleeping Beauty: Mechanistic Insights into Cut-and-Paste Transposition." Viruses 13, no. 1 (January 8, 2021): 76. http://dx.doi.org/10.3390/v13010076.
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Sleeping Beauty (SB) is a transposon system that has been widely used as a genetic engineering tool. Central to the development of any transposon as a research tool is the ability to integrate a foreign piece of DNA into the cellular genome. Driven by the need for efficient transposon-based gene vector systems, extensive studies have largely elucidated the molecular actors and actions taking place during SB transposition. Close transposon relatives and other recombination enzymes, including retroviral integrases, have served as useful models to infer functional information relevant to SB. Recently obtained structural data on the SB transposase enable a direct insight into the workings of this enzyme. These efforts cumulatively allowed the development of novel variants of SB that offer advanced possibilities for genetic engineering due to their hyperactivity, integration deficiency, or targeting capacity. However, many aspects of the process of transposition remain poorly understood and require further investigation. We anticipate that continued investigations into the structure–function relationships of SB transposition will enable the development of new generations of transposition-based vector systems, thereby facilitating the use of SB in preclinical studies and clinical trials.
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Ochmann, Matthias T., and Zoltán Ivics. "Jumping Ahead with Sleeping Beauty: Mechanistic Insights into Cut-and-Paste Transposition." Viruses 13, no. 1 (January 8, 2021): 76. http://dx.doi.org/10.3390/v13010076.
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Sleeping Beauty (SB) is a transposon system that has been widely used as a genetic engineering tool. Central to the development of any transposon as a research tool is the ability to integrate a foreign piece of DNA into the cellular genome. Driven by the need for efficient transposon-based gene vector systems, extensive studies have largely elucidated the molecular actors and actions taking place during SB transposition. Close transposon relatives and other recombination enzymes, including retroviral integrases, have served as useful models to infer functional information relevant to SB. Recently obtained structural data on the SB transposase enable a direct insight into the workings of this enzyme. These efforts cumulatively allowed the development of novel variants of SB that offer advanced possibilities for genetic engineering due to their hyperactivity, integration deficiency, or targeting capacity. However, many aspects of the process of transposition remain poorly understood and require further investigation. We anticipate that continued investigations into the structure–function relationships of SB transposition will enable the development of new generations of transposition-based vector systems, thereby facilitating the use of SB in preclinical studies and clinical trials.
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Singh, Harjeet, Pallavi R. Manuri, Simon Olivares, Navid Dara, Margaret J. Dawson, Helen Huls, Dean A. Lee, et al. "CD19-Specific T Cells for Treatment of Pediatric Acute Lymphocytic Leukemia Using Sleeping Beauty Transposition." Blood 110, no. 11 (November 16, 2007): 2820. http://dx.doi.org/10.1182/blood.v110.11.2820.2820.
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Abstract Genetic modification of clinical-grade T cells is undertaken to augment function, including redirecting specificity for desired antigen. We and others have introduced a chimeric antigen receptor (CAR) to enable T cells to recognize lineage-specific tumor antigen, such as CD19, and early-phase human trials are currently assessing safety and feasibility. However, a significant barrier to next-generation clinical studies is developing a suitable CAR-expression vector capable of genetically modifying a broad population of T cells. Transduction of T cells is relatively efficient, but it requires specialized manufacture of expensive clinical-grade recombinant virus. Electro-transfer of naked DNA plasmid offers a cost-effective alternative approach, but the inefficiency of transgene integration mandates ex vivo selection under cytocidal concentrations of drug to enforce expression of selection genes to achieve clinically-meaningful numbers of CARneg T cells. We now report an improved approach to efficiently generating T cells from peripheral blood with redirected specificity. This was accomplished by introducing DNA plasmids from the Sleeping Beauty transposon/transposase system to directly express a CD19-specific CAR in both memory and effector T cells without co-expression of immunogenic drug-selection genes. The success of this approach was based upon the rationale design ofa next-generation codon-optimized CD19-specific CAR capable of coordinated signaling through chimeric CD28,CD19+ artificial antigen-presenting cells (aAPC) derived from K562 and expressing desired co-stimulatory molecules, andelectro-transfer of two SB DNA plasmids expressing CAR transposon and an improved transposase. We report that introduction of a two-component SB system into primary human T cells results in efficient (∼60-fold improved expression compared with electro-transfer without transposase) and stable CAR gene transfer (60 fold as compared to single plasmid control) which can be numerically expanded to clinically-meaningful numbers within weeks on CD19+ aAPC, without the need for addition of drug-selection, and with the outgrowth of CD8+ and CD4+ CAR+ T-cell sub-populations. The improved CAR expression is due to SB transposon/transposase integration into chromosomal DNA compared with rare non-homologous end-joining process that mediates integration by electroporation alone. We demonstrate that the CAR+ T cells expressed memory cell markers (Figure 1A) as well as redirected-killing function of an effector-cell phenotype (Figure 1B). Our data have implications for improved in vivo therapeutic potential as memory T cells are associated with long-term persistence after adoptive transfer. Figure 1. (A) Phenotypic and (B) funtional characterization of CD 19-specific T cells. Figure 1. (A) Phenotypic and (B) funtional characterization of CD 19-specific T cells.
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Kebriaei, Partow, Helen Huls, Harjeet Singh, Simon Olivares, Matthew Figliola, Sourindra Maiti, Su Shihuang, et al. "Adoptive Therapy Using Sleeping Beauty Gene Transfer System and Artificial Antigen Presenting Cells to Manufacture T Cells Expressing CD19-Specific Chimeric Antigen Receptor." Blood 124, no. 21 (December 6, 2014): 311. http://dx.doi.org/10.1182/blood.v124.21.311.311.
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Abstract Objectives: T cells can be genetically modified ex vivo to redirect specificity upon expression of a chimeric antigen receptor (CAR) that recognizes tumor-associated antigen (TAA) independent of human leukocyte antigen. We employ non-viral gene transfer using the Sleeping Beauty (SB) transposon/transposase system to stably express a 2nd generation CD19-specific CAR- (designated CD19RCD28 that activates via CD3z/CD28) in patient (pt)- or donor-derived T cells for patients with advanced B-cell malignancies. Methods: T cells were electroporated using a Nucleofector device to synchronously introduce two DNA plasmids coding for SB transposon (CD19RCD28) and hyperactive SB transposase (SB11). T cells stably expressing the CAR were retrieved over 28 days of co-culture by recursive additions of designer g-irradiated activating and propagating cells (AaPC) in presence of soluble recombinant interleukin (IL)-2 and IL-21. The aAPC were derived from K562 cells and genetically modified to co-express the TAA CD19 as well as the co-stimulatory molecules CD86, CD137L, and a membrane-bound protein of IL-15. The dual platforms of the SB system and aAPC are illustrated in figure below. Results: To date we have successfully manufactured product for 42 pts with multiply-relapsed ALL (n=19), NHL (n=17), or CLL (n=5) on 4 investigator-initiated trials at MD Anderson Cancer Center to administer thawed pt- and donor-derived CD19-specific T cells as planned infusions in the adjuvant setting after autologous (n=5), allogeneic (n=21) or umbilical cord (n=4) hematopoietic cell transplantation (HCT), or for the treatment of active disease (n=12). Each clinical-grade T-cell product was subjected to a battery of in-process and final release testing. Adjuvant trials: Twelve pts have been infused with donor-derived CAR+ T cells following allogeneic HCT, including 2 pts with cord blood-derived T cells (ALL, n=10; NHL, n=2), beginning at a dose of 106 and escalating to 5x107 modified T cells/m2. Three pts, all with ALL, remain alive and in remission at median 5 months following T cell infusion. Five pts with NHL have been treated with pt-derived modified T cells following autologous HCT at a dose of 5x108 T cells/m2, and 4 pts remain in remission at median 12 months following T-cell infusions. Relapse trials: Thirteen pts have been treated for active disease (ALL, n=8; NHL, n=3; CLL, n=2) with pt or donor-derived (if prior allo-HCT) modified T cells at doses 106-5x107/m2, and 3 remain alive and in remission at median 3 months following T-cell infusions. No acute or late toxicities, including excess GVHD, have been noted. Conclusion: We report the first human application of the SB and AaPC systems to genetically modify clinical-grade cells. Furthermore, infusing CD19-specific CAR+ T cells in the adjuvant HCT setting and thus targeting minimal residual disease may provide an effective and safe approach for maintaining remission in pts at high risk for relapse. Next steps: The SB system serves as a nimble and cost-effective platform for genetic engineering of T cells. We are implementing next-generation clinical T-cell trials targeting ROR1, releasing T cells for infusion within days after electro-transfer of SB DNA plasmid coding for CAR and mRNA coding for transposase, and infusing T cells modified with CAR designs with improved therapeutic potential. Figure: Manufacture of CD19-specific T cells from peripheral and umbilical cord blood mononuclear cells by electro-transfer of SB plasmids and selective propagation of CAR+ T cells on AaPC/IL-2/IL-21. Figure:. Manufacture of CD19-specific T cells from peripheral and umbilical cord blood mononuclear cells by electro-transfer of SB plasmids and selective propagation of CAR+ T cells on AaPC/IL-2/IL-21. Disclosures No relevant conflicts of interest to declare.
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VandenDriessche, Thierry, Zoltán Ivics, Zsuzsanna Izsvák, and Marinee K. L. Chuah. "Emerging potential of transposons for gene therapy and generation of induced pluripotent stem cells." Blood 114, no. 8 (August 20, 2009): 1461–68. http://dx.doi.org/10.1182/blood-2009-04-210427.
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AbstractEffective gene therapy requires robust delivery of the desired genes into the relevant target cells, long-term gene expression, and minimal risks of secondary effects. The development of efficient and safe nonviral vectors would greatly facilitate clinical gene therapy studies. However, nonviral gene transfer approaches typically result in only limited stable gene transfer efficiencies in most primary cells. The use of nonviral gene delivery approaches in conjunction with the latest generation transposon technology based on Sleeping Beauty (SB) or piggyBac transposons may potentially overcome some of these limitations. In particular, a large-scale genetic screen in mammalian cells yielded a novel hyperactive SB transposase, resulting in robust and stable gene marking in vivo after hematopoietic reconstitution with CD34+ hematopoietic stem/progenitor cells in mouse models. Moreover, the first-in-man clinical trial has recently been approved to use redirected T cells engineered with SB for gene therapy of B-cell lymphoma. Finally, induced pluripotent stem cells could be generated after genetic reprogramming with piggyBac transposons encoding reprogramming factors. These recent developments underscore the emerging potential of transposons in gene therapy applications and induced pluripotent stem generation for regenerative medicine.
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Carlson, Corey M., Adam J. Dupuy, Sabine Fritz, Kevin J. Roberg-Perez, Colin F. Fletcher, and David A. Largaespada. "Transposon Mutagenesis of the Mouse Germline." Genetics 165, no. 1 (September 1, 2003): 243–56. http://dx.doi.org/10.1093/genetics/165.1.243.
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Abstract Sleeping Beauty is a synthetic “cut-and-paste” transposon of the Tc1/mariner class. The Sleeping Beauty transposase (SB) was constructed on the basis of a consensus sequence obtained from an alignment of 12 remnant elements cloned from the genomes of eight different fish species. Transposition of Sleeping Beauty elements has been observed in cultured cells, hepatocytes of adult mice, one-cell mouse embryos, and the germline of mice. SB has potential as a random germline insertional mutagen useful for in vivo gene trapping in mice. Previous work in our lab has demonstrated transposition in the male germline of mice and transmission of novel inserted transposons in offspring. To determine sequence preferences and mutagenicity of SB-mediated transposition, we cloned and analyzed 44 gene-trap transposon insertion sites from a panel of 30 mice. The distribution and sequence content flanking these cloned insertion sites was compared to 44 mock insertion sites randomly selected from the genome. We find that germline SB transposon insertion sites are AT-rich and the sequence ANNTANNT is favored compared to other TA dinucleotides. Local transposition occurs with insertions closely linked to the donor site roughly one-third of the time. We find that ∼27% of the transposon insertions are in transcription units. Finally, we characterize an embryonic lethal mutation caused by endogenous splicing disruption in mice carrying a particular intron-inserted gene-trap transposon.
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Ohlfest, John R., Joel L. Frandsen, Sabine Fritz, Paul D. Lobitz, Scott G. Perkinson, Karl J. Clark, Gary Nelsestuen, et al. "Phenotypic correction and long-term expression of factor VIII in hemophilic mice by immunotolerization and nonviral gene transfer using the Sleeping Beauty transposon system." Blood 105, no. 7 (April 1, 2005): 2691–98. http://dx.doi.org/10.1182/blood-2004-09-3496.
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AbstractHemophilia A is a lead candidate for treatment by gene therapy because small increments in the missing secreted protein product, coagulation factor VIII (FVIII), would result in substantial clinical amelioration. Clinically relevant therapy might be achieved by stably delivering a human FVIII cDNA to correct the bleeding disorder. We used the Sleeping Beauty (SB) transposon, delivered as naked plasmid DNA by tail-vein injection, to integrate B-domain–deleted FVIII genes into the chromosomes of hemophilia A mice and correct the phenotype. Since FVIII protein is a neoantigen to these mice, sustaining therapeutic plasma FVIII levels was problematic due to inhibitory antibody production. We circumvented this problem by tolerizing 82% of neonates by a single facial-vein injection of recombinant FVIII within 24 hours of birth (the remaining 18% formed inhibitors). Achievement of high-level (10%-100% of normal) FVIII expression and phenotypic correction required co-injection of an SB transposase-expressing plasmid to facilitate transgene integration in immunotolerized animals. Linker-mediated polymerase chain reaction was used to clone FVIII transposon insertion sites from liver genomic DNA, providing molecular evidence of transposition. Thus, SB provides a nonviral means for sustained FVIII gene delivery in a mouse model of hemophilia A if the immune response is prevented.
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Kebriaei, Partow, Helen Huls, Harjeet Singh, Simon Olivares, Matthew Figliola, Pappanaicken R. Kumar, Bipulendu Jena, et al. "First Clinical Trials Employing Sleeping Beauty Gene Transfer System and Artificial Antigen Presenting Cells To Generate and Infuse T Cells Expressing CD19-Specific Chimeric Antigen Receptor." Blood 122, no. 21 (November 15, 2013): 166. http://dx.doi.org/10.1182/blood.v122.21.166.166.
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Abstract Background T cells can be genetically modified ex vivo to redirect specificity upon enforced expression of a chimeric antigen receptor (CAR) that recognizes tumor-associated antigen (TAA) independent of human leukocyte antigen. We report a new approach to non-viral gene transfer using the Sleeping Beauty (SB) transposon/transposase system to stably express a 2nd generation CD19-specific CAR- (designated CD19RCD28 that activates via CD3z/CD28) in autologous and allogeneic T cells manufactured in compliance with current good manufacturing practice (cGMP) for Phase I/II trials. Methods T cells were electroporated using a Nucleofector device to synchronously introduce DNA plasmids coding for SB transposon (CD19RCD28) and hyperactive SB transposase (SB11). T cells stably expressing the CAR were retrieved over 28 days of co-culture by recursive additions of g-irradiated artificial antigen presenting cells (aAPC) in presence of soluble recombinant interleukin (IL)-2 and IL-21. The aAPC (designated clone #4) were derived from K562 cells and genetically modified to co-express the TAA CD19 as well as the co-stimulatory molecules CD86, CD137L, and a membrane-bound protein of IL-15. The dual platforms of the SB system and aAPC are illustrated in figure below. Results To date we have enrolled and manufactured product for 25 patients with multiply-relapsed ALL (n=12) or B-cell lymphoma (n=13) on three investigator-initiated trials at MD Anderson Cancer Center to administer thawed patient- and donor-derived CD19-specific T cells as planned infusions in the adjuvant setting after autologous (n=7), allogeneic adult (n=14) or umbilical cord (n=4) hematopoietic stem-cell transplantation (HSCT). Each clinical-grade T-cell product was subjected to a battery of in-process testing to complement release testing under CLIA. Currently, five patients have been infused with the CAR+ T cells following allogeneic HSCT, including one patient with cord blood-derived T cells (ALL, n=4; NHL, n=1), beginning at a dose of 106 and escalating to 107 modified T cells/m2. Three patients treated at the first dose level of 106 T cells/m2 have progressed; the patient treated at the next dose level with 107 T cells/m2 remains in remission at 5 months following HSCT. Assessment for response too early for patient treated with UCB T cells. Four patients with non-Hodgkin’s lymphoma have been treated with patient-derived modified T cells following autologous HSCT at a dose of 5x107 T cells/m2, and all patients remain in remission at 3 months following HSCT. No acute or late toxicities have been noted to date. PCR testing for persistence of CAR-modified T cells is underway. Conclusion We report the first human application of the SB and aAPC systems to genetically modify clinical-grade cells. Importantly, infusing CD19-specific CAR+ T cells in the adjuvant HSCT setting and thus targeting minimal residual disease is feasible and safe, and may provide an effective approach for maintaining remission in patients with high risk, CD19+ lymphoid malignancies. Clinical data is accruing and will be updated at the meeting. This nimble manufacturing approach can be readily modified in a cost-effective manner to improve the availability, persistence and therapeutic potential of genetically modified T cells, as well as target tumor–associated antigens other than CD19. Disclosures: No relevant conflicts of interest to declare.
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Vercellotti, Gregory M., Ping Zhang, Chunsheng Chen, Julia Nguyen, Fuad Abdulla, Phong Nguyen, Carlos Nowotny, et al. "Hemopexin Gene Therapy Inhibits Inflammation and Vaso-Occlusion in Transgenic Sickle Cell Mice." Blood 126, no. 23 (December 3, 2015): 412. http://dx.doi.org/10.1182/blood.v126.23.412.412.
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Abstract Hemolysis, oxidative stress, inflammation, vaso-occlusion, and organ infarction are hallmarks of sickle cell disease (SCD). Hemolysis releases free hemoglobin (Hb) and Hb-containing microparticles into the vasculature that upon oxidation to methemoglobin frees heme from the globin, which in turn can promote oxidative stress and activate toll-like receptor 4 (TLR4) signaling. Hemopexin (HPX), a plasma β1-glycoprotein, binds heme with a very high affinity (Kd < 10-12 M), and transports it to the liver for catabolism via CD91 receptor-mediated uptake. SCD patients have low serum HPX levels likely due to chronic hemolysis leading to increased HPX catabolism with insufficient compensatory increase in synthesis. We and others have shown that HPX transports heme to the liver, and inhibits heme toxicity and the activation of endothelial, leukocyte and platelet TLR4 signaling. Acute studies have shown HPX infusion prior to a heme challenge protects sickle mice from vaso-occlusion and developing acute pulmonary injury while chronic HPX infusion therapy modified heme toxicity to endothelium. We hypothesize that in SCD mice, hepatic overexpression of HPX will bind the proximal mediator of vascular activation, heme, and will inhibit inflammation and microvascular stasis (vaso-occlusion). To examine the protective role of HPX in SCD, we transplanted bone marrow from NY1DD SCD mice into HPX-/- or normal C57BL/6 mice. After 12 weeks, conversion to the HbS phenotype was confirmed by isoelectric focusing. Dorsal skin fold chambers (DSFC) were implanted in week 13 and microvascular stasis (% non-flowing venules) assessed in response to heme (3.2 µmol/kg) infusion. HPX-/- sickle mice had 34% ± 3% and 24% ± 2% at 1h and 4h post heme, significantly greater than HPX+/+ C57BL/6 sickle mice which had 21% ± 5% and 13% ± 8% at 1 and 4 h, (mean ± SD, p<.05), demonstrating the protective role of HPX in SCD. To further test our hypothesis, we utilized Sleeping Beauty (SB) transposon-mediated gene therapy to overexpress rat HPX in NY1DD and Townes-SS SCD mice. Rat HPX plasmid (pT2/Caggs-HPX) was delivered with an SB transposase plasmid (pK/CMV-SB 100X) and luciferase (LUC) plasmid (pT2/Caggs-LUC, as tracer) in trans into NY1DD or Townes-SS SCD mice by hydrodynamic tail vein injections. Control SCD mice were infused with the same volume of lactated Ringer's solution (LRS) or LUC plasmid with SB transposase plasmid in trans. One week later, the mice LUC bioluminescence imaging showed the liver was the primary location of expression. Four weeks later, the HPX SCD mice had marked increases in hepatic rat HPX mRNA (300-2000 copies/5ng total RNA) comparing to LRS and SB-LUC controls (0-44 copies/5ng total RNA). Plasma and hepatic HPX were significantly increased compared to LRS and SB-LUC controls. In vitro expression of the rat HPX plasmid in Chinese Hamster Ovary cells, and protein purification confirmed heme binding activity by spectroscopic scan absorbance shifts of rat HPX-heme complexes at 414nm. DSFCs were implanted 4 weeks after plasmid infusion and microvascular stasis was assessed in response to heme (3.2 µmol/kg) infusion. NY1DD and Townes-SS mice overexpressing rat HPX (SB-HPX) had significantly less stasis than LRS or SB-LUC treated SCD mice (Figure 1A and B). HPX overexpression markedly increased nuclear Nrf2 expression in the livers of sickle mice, presumably by promoting delivery of heme to the liver and activating the Keap1-Nrf2 axis. In addition, hepatic HO-1 activity and protein and CD91 protein were increased in sickle mice overexpressing HPX and NF-ĸB activation was markedly decreased as assessed by nuclear phospho-p65-NF-ĸB expression on western blots demonstrating the anti-inflammatory properties of HPX in sickle mice. In conclusion, supplementing HPX levels in transgenic sickle mice via gene therapy activates the Nrf2 anti-oxidant axis and ameliorates inflammation and vaso-occlusion. We speculate that plasma HPX supplementation may be beneficial in SCD especially during hemolytic crises or acute chest syndrome. Figure 1. Figure 1. Disclosures Vercellotti: Cydan: Research Funding; CSL Behring: Research Funding; Seattle Genetics: Research Funding; Biogen Idec: Research Funding. Belcher:CSL Behring: Research Funding; Seattle Genetics: Research Funding; Biogen Idec: Research Funding.
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Kebriaei, Partow, Helen Huls, Harjeet Singh, Simon Olivares, Matthew Figliola, Pappanaicken R. Kumar, Bipulendu Jena, et al. "Adoptive Immunotherapy Following Umbilical Cord Blood Transplantation Using The Sleeping Beauty System and Artificial Antigen Presenting Cells To Generate Donor-Derived T Cells Expressing a CD19-Specific Chimeric Antigen Receptor." Blood 122, no. 21 (November 15, 2013): 4208. http://dx.doi.org/10.1182/blood.v122.21.4208.4208.
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Abstract Background The ability to transplant across HLA disparities makes allogeneic umbilical cord blood (UCB) an attractive graft source for hematopoietic stem-cell transplantation (HSCT). Disease relapse remains a limitation, and adoptive transfer of tumor-specific T cells post UCB HSCT has not been feasible due to the functionally naïve CB T cells, and the small size as well as anonymity of the donor. We report a new approach to non-viral gene transfer using the Sleeping Beauty (SB) transposon/transposase system to stably express a 2nd generation CD19-specific chimeric antigen receptor (CAR, designated CD19RCD28) on UCB-derived T cells manufactured in compliance with current good manufacturing practice (cGMP). Methods After thawed UCB units are washed for clinical infusion 5% to 10% of cells are used to generate CAR+ T cells. The mononuclear cells are electroporated using a Nucleofector device to synchronously introduce two DNA plasmids coding for SB transposon (CD19RCD28) and hyperactive SB transposase (SB11). T cells stably expressing the CAR are retrieved over 28 days of co-culture by recursive additions of g-irradiated artificial antigen presenting cells (aAPC) in presence of soluble recombinant interleukin (IL)-2 and IL-21. The aAPC (designated clone #4) were derived from K562 cells and genetically modified to co-express the CD19 as well as the co-stimulatory molecules CD86, CD137L, and a membrane-bound protein of IL-15. Enrolled patients on our phase I trial receive two UCB units, thus two genetically modified T-cell products are made for each patient. We infuse thawed donor-derived CD19-specific CAR+ T cells from the dominant CB unit based on peripheral blood chimerism on days 40-100 post transplant in the adjuvant setting after double UCB HSCT Results To date we have successfully manufactured 8 products for 4 patients (ALL n=3, NHL=1) enrolled on trial. The median number of T cells in the starting CB aliquot was 8.6x106 (range, 2.5x106 to 54.8x106) with final modified T cell count at median 3x109 (range,1.7x108 to 4.1x1010) at time of cryopreservation days 28-32. In the final product, the median CD19-CAR+ cell purity by flow was 88% (range, 81.9% to 95.8%). The modified T cell product consisted of median 97.3% CD3+, 2.7 CD3-/CD56+ cells. All of the products exhibited CD19-specific killing by chromium assay as illustrated (Figure). Each clinical-grade T-cell product was subjected to a battery of in-process testing to complement release testing. One patient with ALL has been infused to date with a T cell dose of 106T cells/m2 and no toxicity has been observed. The patient remains alive and in continued molecular remission at 111 days post HSCT. Conclusion We combined the SB system and aAPC-mediated propagation of T cells to successfully manufacture disease-specific T cells from small aliquots of UCB used to restore hematopoiesis. Importantly, this approach allows us to employ adoptive therapy to enhance the graft-versus-tumor effect in UCB HSCT as an approach to improve overall survival for these recipients. Accrual to the trial continues and updated results will be presented at the meeting. Disclosures: No relevant conflicts of interest to declare.
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Bexte, Tobias, Lacramioara Botezatu, Csaba Miskey, Julia Campe, Lisa Marie Reindl, Veronika Gebel, Winfried S. Wels, Michael Hudecek, Zoltan Ivics, and Evelyn Ullrich. "Non-Viral Sleeping Beauty Transposon Engineered CD19-CAR-NK Cells Show a Safe Genomic Integration Profile and High Antileukemic Efficiency." Blood 138, Supplement 1 (November 5, 2021): 2797. http://dx.doi.org/10.1182/blood-2021-153999.
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Abstract Background: Natural Killer (NK) cells are known for their high intrinsic cytotoxic capacity. Recently, we and others showed that virally transduced NK cells equipped with a synthetic chimeric antigen receptor (CAR) targeting CD19 induced enhanced killing of acute lymphoblastic leukemia (ALL) cells. Here, we demonstrate for the first time that primary NK cells can be engineered using the non-viral Sleeping Beauty (SB) transposon/transposase system to stably express a CD19-CAR with a safe genomic integration profile and high anti-leukemic efficiency in vitro and in vivo. Methods: Primary NK cells were isolated from PBMCs from healthy donors. SB transposons vectorized as minicircles (MC), which encode either a Venus fluorescent protein or a CD19-CAR together with truncated EGFR (tEGFR) as a marker, were introduced in combination with the hyperactive SB100X transposase into primary NK cells via nucleofection. The genetically engineered NK cells were expanded using IL-15 cytokine stimulation under feeder-cell free conditions. Vector integration sites were mapped by analyzing the genomic region around each insertion site in genomic DNA from long-term cultivated gene-modified NK cells, engineered ether by lentiviral (LV) or SB-based technology. Stable gene delivery and biological activity were monitored by flow cytometry and cytotoxicity of CD19-CAR NK cells against CD19-positive ALL and CD19-negative cell lines. Results: Applying a protocol optimized with respect to nucleofection pulses, time points and plasmid ratios, primary NK cells showed long-lasting Venus expression (up to 50%) upon SB-mediated gene delivery with similar viability as non-treated (NT) NK cells during feeder-cell free ex-vivo expansion using IL-15. Likewise, SB transposon-engineered CD19-CAR NK cells displayed high viability, durable transgene expression (Fig 1 A), and no significant change in the NK cell phenotype profile. Next, we assessed vector integration into genomic safe harbors (GSH). GSH are defined as regions of human chromosomes that fulfill the following five criteria: not ultraconserved, >300 kb away from miRNA genes, >50 kb away from transcriptional start sites (TSS), >300 kb away from genes involved in cancer and outside transcription units. CD19-CAR NK cells generated using SB100X showed a significantly higher frequency of vector integration into GSH compared to LV-transduced CAR-NK cells and a significantly more-close to random nucleotide frequency (computer-generated random positions in the genome map to GSHs; random 43.68%; LV 14.78%, SB100X 23.99%; p<0.05) (Fig 1 B). MC.CD19-CAR NK cells generated with the SB platform demonstrated significantly higher cytotoxicity compared to NT NK cells against CD19-positive Sup-B15 ALL cells after long-term cultivation for two to three weeks and no loss of natural intrinsic cytotoxicity. After 4-hour co-culture, significantly enhanced specific tumor cell lysis was found for MC.CD19-CAR NK cells vs NT NK cells at all effector to target cell ratios (E:T) tested (E:T 20:1 83.88% vs 43.13%; E:T 10:1 75.18% vs 31.32%; E:T 5:1 67.38 vs 32.22%; E:T 1:1 42.54 vs 10.19%; p<0.05) (Fig 1 C). With regard to intrinsic natural cytotoxicity of NK cells, no significant decrease in cell killing was overserved for SB-gene-modified CD19-CAR NK cells compared to NT NK cells against CD19-negative K562 cells (E:T 5:1 83%; p<0.05) (Fig 1 D). Significantly enhanced antitumor potential of SB-generated CD19-CAR NK cells was confirmed in a systemic CD19-positive lymphoma xenograft model (NSG-Nalm-6/Luc) in vivo. After injection of 0.5x10 6 tumor cells per mouse and lymphoma engraftment, animals were treated with a single dose of 10x10 6 SB-modified CD19-CAR NK cells pooled from three different donors with a mean tEGFR/CAR expression of 34%. MC.CD19-CAR NK cell therapy resulted in rapid lymphoma eradication in all treated mice (n=4; p<0.05), whereas mice receiving similar amounts of NT NK cells showed progressive lymphoma growth comparable to untreated control mice (Fig 1 E-F). Conclusion: Taken together, the Sleeping Beauty transposon system represents an innovative gene therapy approach for non-viral engineering of safe, highly functional and relatively cost-efficient CAR-NK cells that may not only be suitable for ALL therapy but also for a broad range of other applications in cancer therapy. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.
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Giotopoulos, George, Louise Van Der Weyden, Hikari Osaki, Wai-In Chan, Alistair Rust, Eshwar Meduri, Steffen Koschmieder, et al. "Modelling Cellular and Molecular Progression Of CML In The Mouse." Blood 122, no. 21 (November 15, 2013): 2706. http://dx.doi.org/10.1182/blood.v122.21.2706.2706.
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Abstract Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm, caused by a reciprocal chromosomal translocation that generates the BCR-ABL fusion protein, a constitutively activated tyrosine kinase. Patients with CML usually present in an indolent chronic phase (CP), however, if left untreated, they irrevocably progress to an aggressive form of acute leukemia, termed blast crisis (BC) that is usually fatal. Tyrosine kinase inhibitor (TKI) (e.g. Imatinib) treatment has revolutionised the treatment of CML CP. However, ∼5-10% of CP patients will progress to BC despite TKI treatment, and an additional 10-15% of patients are beyond CP at initial presentation. Upon disease progression, treatment options are very limited and prognosis is dismal. Hence, understanding the events that drive disease progression and identifying potential therapeutic targets remains an unmet clinical need. The mechanisms of BC transformation are poorly understood, but it is generally accepted that additional somatic mutations are required. To date, a small number of recurrent mutations have been reported, but these only account for a relatively small number of cases and their exact nature is not fully understood. In order to study the mechanisms of BC progression and to identify the co-operating mutations that drive this, we have utilised a published transgenic murine model of chronic phase CML (Koschmieder et al., 2005) and performed a transposon-based forward insertional mutagenesis study. In our mouse model, expression of BCR-ABL was driven in the hematopoietic stem and progenitor cell compartment (HSPC) by an SCL enhancer in a tetracycline dependant manner. Following BCR-ABL expression we conditionally induced ongoing mutations via a transposon-transposase system (SB) within HSPC and monitored disease progression from the chronic/BCR-ABL dependant phase to the transposon-mediated BC. Utilising the design of the transposon based system, it was then possible to identify these mutations by multiplexed next generation sequencing (NGS). Our experimental cohort was comprised of BC mice (which expressed BCR-ABL and transposon/transposase mediated mutation induction), CML mice (BCR-ABL only) and SB mice (mutation induction only). BC mice demonstrated a significantly shorter survival (p<0.0001, 116 vs. 147 days) compared to CML mice. Disease progression was characterised by a significantly increased disease burden, in terms of organ infiltration and leucocytosis, with around 80% of BC mice developing an exclusively acute myeloid leukemia by the Bethesda criteria. BC mice also demonstrated quantitative and functional differences within the hematopoietic stem and progenitor cell compartment in in vitro and in vivo assays in keeping with progression from a chronic to an acute leukemia. Importantly, BC mice showed a shorter survival (p=0.007, 116 vs. 128 days) compared to the SB mice, in which both acute myeloid and lymphoid leukemias were seen. Molecularly, NGS revealed insertions in both novel genes, and in genes previously implicated in CML blast crisis, hematopoiesis and leukemogenesis, such as ASXL1, FLT3 and ERG. These insertions included highly recurrent hits and were enriched for transcriptional regulators and signalling proteins, many of potential therapeutic relevance. Additionally, there was only a very modest overlap between the mutations identified in the BC and the SB cohorts, demonstrating BCR-ABL-dependant cooperation and disease progression. Considering the above data, our mouse model shows great potential in understanding the mechanisms of transformation to blast crisis, and ultimately in identifying potential therapeutic targets. Disclosures: No relevant conflicts of interest to declare.
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Petkov, S., M. Nowak-Imialek, P. Hyttel, and H. Niemann. "307 REPROGRAMMING OF PIG SOMATIC CELLS TO PLURIPOTENCY WITH SLEEPING BEAUTY TRANSPOSON VECTORS CONTAINING THE PORCINE TRANSCRIPTION FACTOR SEQUENCES." Reproduction, Fertility and Development 25, no. 1 (2013): 300. http://dx.doi.org/10.1071/rdv25n1ab307.
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Induced pluripotent stem cells (iPSC), developed by Yamanaka and co-workers (Takahashi et al., 2006), hold significant potential for the development of regenerative therapies due to the possibilities of deriving patient-specific pluripotent cells. In this aspect, the pig is an important animal model for testing iPSC-based applications for the human medicine. However, even though significant progress has been made, the derivation of porcine iPSC lines fully equivalent to those from mouse and human has been elusive. To date, most of the reported putative pig iPSC lines have been derived with the use of lentiviral or retroviral vectors harboring the mouse or human transcription factor sequences. Here, we report the construction of Sleeping Beauty (SB) transposon vectors with porcine cDNA sequences coding for OCT4, SOX2, NANOG, C-MYC, and KLF4, in addition to the human LIN28. By using standard cloning techniques, we produced 2 polycistronic SB-CAG-pOSMK-ires-Tomato and SB-Ef1a-pNANOG-ires-hLIN28 transposon vectors and we transfected them together with the SB100X transposase into pig fetal fibroblasts (pFF) harboring a mouse OCT4-GFP reporter construct (Nowak-Imialek et al., 2010). Both the basic transposon and transposase vectors were generously provided by Dr. Zoltan Ivics from Paul Ehrlich Institute, Langen, Germany. In each experiment, 2 × 106 pFF were electroporated with 3 µg of each transposon together with 0.5 µg of SB100X. Two days after transfection, the cells were transferred to mouse embryonic fibroblast (MEF) feeders and cultured with iPSC medium [DMEM with antibiotics, nonessential amino acids, 20% Knockout serum replacement, 5 ng mL–1 human recombinant basic fibroblast growth factor (bFGF), and 1000 U mL–1 ESGRO]. Two weeks post-transfection, multiple compact colonies were apparent (mean = 2195; SEM = 166; n = 3), which were 95% alkaline phosphatase-positive and ~80% expressed the OCT4-GFP reporter. Reverse transcription-PCR showed that these colonies expressed high levels of endogenous OCT4, SOX2, NANOG, REX1, UTF1, CDH1, and TDH. The cultures were passaged by trypsin disaggregation, followed by seeding on fresh feeders at density 10 × 103 cells cm–2. The established cell lines proliferated as compact, mouse iPSC-like colonies that retained their OCT4 reporter expression as well as the expression of the endogenous pluripotency genes for at least 30 passages. The expression of the transgenes was persistent and showed that no silencing had occurred, even in long-term culture. When subjected to in vitro differentiation protocols, the putative iPSC formed mainly large trophectodermal (TE) vesicles (positive for TE markers CDX2, PAG, and HAND1), fibroblast-like, and neuronal-like cells. These cells still expressed the transgenes as well as most endogenous pluripotency markers, demonstrating limited differentiation capacity. Because the stable transgene expression and the suboptimal culture conditions are the most likely causes of this limited differentiation potential, we are currently working on generating transgene-free iPSC lines under improved cell culture conditions.
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Tolar, Jakub, Mark Osborn, Scott Bell, Lily Xia, Megan Riddle, Angela Panoskaltsis-Mortari, Scott McIvor, et al. "Transgenesis of Multipotent Adult Progenitor Cells (MAPC) with Sleeping Beauty Transposons to Determine MAPC Homing and Persistence in Real-Time In Vivo." Blood 104, no. 11 (November 16, 2004): 2099. http://dx.doi.org/10.1182/blood.v104.11.2099.2099.
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Abstract MAPC are non-hematopoietic stem cells with the capacity to form most, if not all, cell types of the body. To date, the observations of homing of the MAPC have been limited to post mortem analyses. As MAPC may be useful in cellular therapies, our goal was to map their biodistribution in live organisms. To determine the real-time organ-specific homing pattern of donor MAPC, MAPC (from BM of C57BL/6J-rosa26 mice) were co-nucleofected with cDNAs encoding the red fluorescent protein DsRed2 and luciferase, using the Sleeping Beauty (SB) transposon system. Non-viral gene transfer mediated by SB is potentially advantageous to viral gene transfer because transposons may be less immunogenic since no viral proteins are present, and they are relatively easy to produce. DsRed2 and luciferase genes were cloned into plasmid vectors containing the transposase recognition sequences flanking the reporter genes (pT/CAGGS-DsRed2; pT/CAGGS-Luciferase). MAPC (106) were co-nucleofected (Amaxa, setting T-20, buffer T) with 5mcg of each marker plasmid and the SB transposase plasmid (p/CMV-HSB2) at a 1:50 ratio. 19% of MAPC expressed DsRed2 7 days after nucleofection. The MAPC were FACS sorted (1 cell per well) for cells with the highest DsRed2 expression. All MAPC tested expressed both DsRed2 and luciferase, suggesting that co-nucleofection is an efficient means of delivery of two plasmids. Two transgenic MAPC clones selected for further analysis were confirmed to be euploid by cytogenetic analysis, and maintained differentiation potential into the three germ layers. To verify transgene integration by transposition, the genomic sites of transposon integration were determined using splinkerette PCR. In the genome of MAPC clone 1, DsRed2 transposed in two sites on chromosome 5. One integration site (5qA3) was in the 3′ untranslated region of activin receptor interacting protein 1 (Acvrinp1). In clone 2 DsRed2 transposed into a single site on chromosome 10, in an intron of a gene termed SHPRH, which encodes a putative protein with SNF2/helicase and PHD-finger domains. To investigate the real time kinetics of MAPC population after infusion, 5 x 106 DsRed2 and luciferase positive MAPC (clone 2) were infused via tail vein into 8-week-old Rag2/IL-2Rgc−/− mice (T-, B- and NK-immunodeficient mice were used as a recipient to minimize the likelihood that the host would reject donor MAPC). Using whole body imaging (Xenogen) we were able to follow the distribution of the luciferase-marked MAPC over a period of 10 weeks. In addition, using DsRed2 expression the donor MAPC-derived cells in whole lung and in lung cryosections were identified. In summary, we show for the first time stable gene expression in adult stem cells using Sleeping Beauty transposon mediated non-viral gene transfer. These results show that MAPC-based cellular therapies can be monitored in vivo and suggest that transposon-based technology may be an attractive alternative to viral based gene delivery and therapy.
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Garrels, W., T. R. Talluri, R. Bevacqua, A. Alessio, A. Fili, D. Forcato, N. Rodriguez, et al. "356 SLEEPING BEAUTY TRANSGENESIS IN CATTLE." Reproduction, Fertility and Development 27, no. 1 (2015): 266. http://dx.doi.org/10.1071/rdv27n1ab356.
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Transposon-mediated transgenesis is a well-established tool for genome modification in small animal models. However, translation of this active transgenic method to large animals warrants further investigations. Here, the Sleeping Beauty (SB) transposon system was assessed for stable gene transfer into the cattle genome. The transposon plasmids encoded a ubiquitously active CAGGS promoter-driven Venus reporter and a lens-specific α A-crystallin promoter driven tdTomato fluorophore, respectively. The helper plasmid carried the hyperactive SB100x transposase variant. In total, 50 in vitro-derived zygotes were co-injected (Garrels et al. 2011 PLoS ONE 6; Ivics et al. 2014 Nat. Protoc. 9) and cultured up to blastocyst stage (Day 8). Two blastocysts were Venus-positive and were transferred to synchronized heifers, resulting in one pregnancy. The resulting calf was normally developed and vital; however, it died shortly after cesarean section due to spontaneous bleeding from an undetected aneurism. Phenotypic analysis suggested that the calf was indeed double-transgenic, showing widespread expression of Venus and lens-specific expression of tdTomato. Genotyping and molecular analyses confirmed the integration of both reporter transposons and the faithful promoter-dependent expression patterns. Subdermal tissue of an ear biopsy was used to culture fibroblasts, which were employed in somatic cell nuclear transfer experiments. In total, 39 embryos were reconstructed, of which 34 underwent cleavage, and at the end of culture 12 morulas and 12 blastocysts were obtained. Ten of the blastocysts were Venus positive, and embryo transfer of Venus-positive blastocysts is planned. In summary, we showed that the cytoplasmic injection of SB components is a highly efficient method for transgenesis in cattle. Due to the modular composition of SB plasmids, even double transgenic cattle can be generated in a one-step procedure. Importantly, the SB-catalyzed integration seems to favour transcriptionally permissive loci in the genome, resulting in faithful and robust promoter-dependent expression of the transgenes. The transposon constructs carry heterospecific loxP sites, which will be instrumental for targeted insertion of functional transgenes by Cre recombinase-mediated cassette exchange.Financial support of DFG (Ku 1586/3-1), UNRC, CONICET and Agencia Nacional de Promoción Científica y Tecnológica de la Argentina (ANPCyT) is gratefully acknowledged.
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Ahrens, H. E., B. Petersen, S. Petkov, J. Hauschild-Quintern, and H. Niemann. "325 PRODUCTION OF GAL KNOCKOUT/hA20 TRANSGENIC PIGS WITH IMPROVED XENOPROTECTIVE PROPERTIES." Reproduction, Fertility and Development 25, no. 1 (2013): 310. http://dx.doi.org/10.1071/rdv25n1ab325.
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Pig-to-human xenotransplantation is promising for overcoming the shortage of suitable human donor organs, but is hampered by immunological barriers. The next immunological hurdle is the acute vascular rejection (AVR), which is associated with activation of the endothelium and the coagulation system. Recently, we demonstrated that transgenic expression of the zinc finger protein A20 protects porcine cells against apoptotic and inflammatory stimuli (Oropeza et al. 2009 Xenotransplantation 16, 522–534). Compared with other anti-apoptotic proteins, A20 also has immune-modulatory potential as shown in an CD95(Fas)Ligand assay. However, in that study, hA20 was only expressed in skeletal muscle, heart and porcine aortic endothelial cells of transgenic pigs. For use in xenotransplantation, it is critical to produce pigs with ubiquitous expression of hA20. Here, we constructed a new vector based on the Sleeping Beauty transposon plasmid pT2/HB containing the hA20 cDNA driven by the ubiquitously and strongly expressing CAGGS-promoter and co-transfected gal–/– porcine fibroblasts (Hauschild et al. 2011 Proc. Natl. Acad. Sci. USA 108, 15 010) together with the SB transposase 100X plasmid (pT2/HB and SB transposase both kindly provided by Dr. Zoltan Ivics). Cells were selected with 400 µg of G418/mL of medium for 14 days. Subsequently, cells were screened by PCR. Transfected cell clones were pooled and used as donor cells in somatic cell nuclear transfer. Reconstructed embryos were transferred to 2 synchronized sows. Both remained pregnant on Day 25 of gestation. One recipient is expected to deliver in September 2012. The second sow, which received 104 embryos, was sacrificed on Day 26 of pregnancy and 2 fetuses could be obtained (cloning efficiency 1.92%). The fetuses had integrated the transgene into their genome as shown by PCR. Real-time PCR results from fetal fibroblasts indicated similar hA20 mRNA expression levels in both fetuses, whereas wild type controls were negative. The hA20 expression level was 2.5 and 3.1 times lower than in the original pooled of transfected cells. Fetus #1, which showed a slightly higher hA20 mRNA expression, was used for recloning. In total, 3 more recipients received an average of 104 hA20 transgenic embryos each. Currently, the hA20 protein level in fetal fibroblasts is determined by fluorescence activated cell sorting analysis. Once pigs are born, the tissue distribution of hA20 will be analysed. In parallel, the function of the transgene will be studied in the CD95(Fas)Ligand assay. Hearts and kidneys of the hA20-transgenic pigs will be further tested in ex vivo perfusion assays and in a pig-to-baboon xenotransplantation. This approach is promising for advancing pig-to-human xenotransplantation to preclinical application.
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Rotiroti, Maria Caterina, Chiara Buracchi, Silvia Arcangeli, Chiara F. Magnani, Claudia Cappuzzello, Zsuzsanna Izsvak, Stefania Galimberti, et al. "Preclinical Assessment of Non-Virally Engineered CD33.CAR Cytokine-Induced Killer (CIK) Cells in Chemoresistant AML Patient-Derived Xenografts." Blood 134, Supplement_1 (November 13, 2019): 2665. http://dx.doi.org/10.1182/blood-2019-130399.
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Background Chimeric Antigen Receptor (CAR)-T cell therapy has been successfully clinically deployed in the context of B-cell malignancies, paving the way for further development also in Acute Myeloid Leukemia (AML), a still unmet clinical need in the field of oncohematology. Among the potential AML targetable antigens, CD33 is so far one of the main validated molecule. Objectives The aim of the present study was to optimize a non-viral gene transfer method to engineer Cytokine-Induced Killer (CIK) cells with a CD33.CAR by using a novel version of the Sleeping Beauty (SB) transposon system, named "SB100X-pT4". Further, a preclinical assessment of SB-modified CD33.CAR-CIK cells was performed in chemoresistant AML Patient-Derived Xenografts (PDX), in order to address the unmet need of targeting drug-resistant AML cells. Methods Donor derived-CIK cells were stably transduced with a CD33.CAR by exploiting the novel hyperactive SB100X transposase and the pT4 transposon (SB100X-pT4). The novel SB system has been in vitro compared to the previous established SB11-pT. In vitro anti-AML activity of CD33.CAR-CIK cells was assessed by flow cytometry-based cytotoxicity (AnnV-7AAD), proliferation (CFSE) and cytokine production (intracellular IFNg and IL2 detection) assays. In vivo efficacy was evaluated in NSG mice transplanted with MA9-NRas AML cell line or PDX samples. A xenograft chemotherapy model mimicking induction therapy ("5+3" Ara-C and doxorubicin) was exploited to examine the potential benefit of CD33.CAR-CIK cells on chemoresistant/residual AML cells. Results By significantly reducing the amount of DNA transposase, the novel SB100X-pT4 combination resulted in higher CAR levels than the SB11-pT. SB100X-pT4-modified CD33.CAR CIK cells showed efficient expansion after 3 weeks (median fold increase of 38.89, n=4). Both transpositions conferred to CD33.CAR-CIK cells a specific killing (up to 70%) against CD33+ AML target cell lines and primary AML cells. The anti-AML proliferative response of SB-modified CD33.CAR-CIK cells was also considerable (up to 70% of CFSE diluted CAR-CIK cells), as well as the cytokine production (up to 35% for IFN-γ and up to 25% for IL-2). To evaluate the effect of SB100X-pT4-modified CD33.CAR-CIK cells particularly on Leukemia Initiating Cells (LICs), CD33.CAR-CIK cells were administered as an "early treatment" in mice transplanted with the MA9-NRas cell line, which retains a high frequency of LICs. At sacrifice, CD33.CAR-CIK cell-treated mice showed a significant bone marrow (BM) engraftment reduction (median 27.80 for the untreated group and 22.60 for the unmanipulated CIK cells vs 6.45 for CD33.CAR-CIK cell, n=4 NSG mice per group, p= 0.02). PDX of two different AML samples at the onset were established to be used as models mimicking different disease conditions. In an "early treatment" model using secondary transplanted PDX, a setting which presumably reflects the typical LIC properties, a clear engraftment reduction in the treated cohort was observed, nearly undetectable in 2/5 mice, as compared to the untreated mice (up to 70% in BM). A significant leukemia reduction was also measured in the peripheral blood and spleen of treated mice, showing CD33.CAR-CIK cell potential of reducing AML dissemination in the periphery. When ex vivo re-exposed to CD33.CAR-CIK cells residual AML cells were still sensitive to the treatment, indicating that no resistance mechanisms occurred. CD33.CAR-CIK cells were also effective in a second model by which the treatment started when AML engraftment was clearly manifested in the BM (> 1%). Finally, when starting CD33.CAR-CIK cell treatment after disease recurrence post induction therapy, a significant disease reduction was observed in the CD33.CAR-CIK-treated group, reaching undetectable levels in half of the mice, as compared to chemotherapy-only treated mice (up to 60% of engraftment in BM)(Figure 1). Conclusions The employment of a non-viral SB-based CD33.CAR-gene transfer approach, which is overall associated to less cumbersome protocols and reduces the cost of goods, offers a unique alternative to current viral-based strategies to be explored in the setting of resistant forms of AML. Disclosures No relevant conflicts of interest to declare.
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Mattern, Larissa, Katrin Otten, Csaba Miskey, Matthias Fuest, Zsuzsanna Izsvák, Zoltán Ivics, Peter Walter, Gabriele Thumann, and Sandra Johnen. "Molecular and Functional Characterization of BDNF-Overexpressing Human Retinal Pigment Epithelial Cells Established by Sleeping Beauty Transposon-Mediated Gene Transfer." International Journal of Molecular Sciences 23, no. 21 (October 26, 2022): 12982. http://dx.doi.org/10.3390/ijms232112982.
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More and more patients suffer from multifactorial neurodegenerative diseases, such as age-related macular degeneration (AMD). However, their pathological mechanisms are still poorly understood, which complicates the development of effective therapies. To improve treatment of multifactorial diseases, cell-based gene therapy can be used to increase the expression of therapeutic factors. To date, there is no approved therapy for dry AMD, including late-stage geographic atrophy. We present a treatment option for dry AMD that transfers the brain-derived neurotrophic factor (BDNF) gene into retinal pigment epithelial (RPE) cells by electroporation using the plasmid-based Sleeping Beauty (SB) transposon system. ARPE-19 cells and primary human RPE cells were co-transfected with two plasmids encoding the SB100X transposase and the transposon carrying a BDNF transcription cassette. We demonstrated efficient expression and secretion of BDNF in both RPE cell types, which were further increased in ARPE-19 cell cultures exposed to hydrogen peroxide. BDNF-transfected cells exhibited lower apoptosis rates and stimulated neurite outgrowth in human SH-SY5Y cells. This study is an important step in the development of a cell-based BDNF gene therapy that could be applied as an advanced therapy medicinal product to treat dry AMD or other degenerative retinal diseases.
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Zhou, Xianzheng, Xin Huang, Johnthomas Kang, Hongfeng Guo, Suet Choi, Preetinder Bassi, Tom C. Zhou, et al. "Sleeping Beauty (SB) Transposon Mediated Umbilical Cord Blood (UCB) T Cell Therapy for Refractory Acute Lymphoblastic Leukemia (ALL)." Blood 108, no. 11 (November 16, 2006): 722. http://dx.doi.org/10.1182/blood.v108.11.722.722.
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Abstract UCB is a promising alternate source of hematopoietic stem cell transplantation due to a readily available graft and low risk of GVHD. However, the incidence of ALL relapse in children is relatively high (40% for high risk ALL). To test whether UCB T cells can be genetically modified as GVL effector cells, the SB transposon system was used as a delivery vehicle since we have shown that this system can mediate genomic integration and long-term reporter gene expression in 5–20% of human primary T cells without prior activation, thus reducing duration of in vitro culture and enhancing T cell function (Huang et al., Blood. 2006, 107:483). A SB bidirectional transposon was constructed to co-express a single chain chimeric antigen receptor for CD19, commonly expressed in B-ALL, and human CD20, a marker for in vitro selection of transfected T cells and a “suicide” gene for in vivo elimination by Rituxan when necessary. In preclinical studies, UCB and peripheral blood (PB) mononuclear cells were nucleofected with the bidirectional SB transposon and a SB10 transposase-expressing plasmid, and then activated and expanded by anti-CD3/CD28 beads in culture. Flow cytometric analyses confirmed the stable dual gene expression in both transfected T cell types. After sorting for the dual gene expression, engineered T cells demonstrated specific cytoxicity against CD19+ leukemia and lymphoma cell lines but not CD19− myeloid leukemia and multiple myeloma cells. Furthermore, SB engineered T cell killing was found to be CD19-specific as evidenced by killing K562 cells stably expressing CD19 but not K562 and K562 cells stably expressing eGFP. We also demonstrated that the unsorted PB T cells killed CD19+ target cells as effectively as the sorted PB T cells, suggesting that transfected T cells can be immediately infused into patients without selection and extended in vitro culture. While the mechanisms responsible for anti-leukemia cytolysis are under investigation, it is clear that both engineered CD4 and CD8 PB T cells and CD8 UCB T cells, but not engineered CD4 UCB T cells, killed CD19+ target cells. In vivo experiments are in progress to determine efficacy of CD19+ leukemia cell reduction by the engineered human T cells and the efficiency of Rituxan-mediated elimination of adoptively transferred T cells using a bioluminescent imaging technique. We conclude that the SB transposon-engineered UCB and PB T cells can stably express the therapeutic genes and mount potent anti-leukemia and lymphoma responses in vitro. Safety and therapeutic potential of engineered UCB T cells will be tested in the treatment of high risk CD19+ ALL as a strategy for reducing risk of relapse without the risk of increased acute GVHD. In addition, our approach is novel (transposon-based), simple (naked DNA), efficient (no prior T cell activation and less immunogenic), stable (integrating), and probably safe (random integration) compared to retroviral vectors and conventional plasmids.
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Kebriaei, Partow, Stefan O. Ciurea, Mary Helen Huls, Harjeet Singh, Simon Olivares, Shihuang Su, Matthew J. Figliola, et al. "Pre-Emptive Donor Lymphocyte Infusion with CD19-Directed, CAR-Modified T Cells Infused after Allogeneic Hematopoietic Cell Transplantation for Patients with Advanced CD19+ Malignancies." Blood 126, no. 23 (December 3, 2015): 862. http://dx.doi.org/10.1182/blood.v126.23.862.862.
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Background: Allogeneic hematopoietic cell transplantation (HCT) can be curative in a subset of patients with advanced lymphoid malignancies but relapse remains a major reason for treatment failure. Donor-derived, non-specific lymphocyte infusions (DLI) can confer an immune anti-malignancy effect but can be complicated by graft-versus-host-disease (GVHD). Chimeric antigen receptor (CAR)-modified T cells directed toward CD19 have demonstrated dramatic efficacy in patients with refractory ALL and NHL. However, responses are often associated with life-threatening cytokine release syndrome. Aim: We hypothesized that infusing CAR-modified, CD19-specific T-cells after HCT as a directed DLI would be associated with a low rate of GVHD, better disease control, and a less severe cytokine release syndrome since administered in a minimal disease state. Methods: We employed a non-viral gene transfer using the Sleeping Beauty (SB) transposon/transposase system to stably express a CD19-specific CAR (designated CD19RCD28 that activates via CD3z & CD28) in donor-derived T cells for patients with advanced CD19+ lymphoid malignancies. T-cells were electroporated using a Nucleofector device to synchronously introduce two DNA plasmids coding for SB transposon (CD19RCD28) and hyperactive SB transposase (SB11). T-cells stably expressing the CAR were retrieved over 28 days of co-culture by recursive additions of g-irradiated activating and propagating cells (AaPC) in presence of soluble recombinant interleukin (IL)-2 and IL-21. The AaPC were derived from K562 cells and genetically modified to co-express CD19 as well as the co-stimulatory molecules CD86, CD137L, and a membrane-bound version of IL-15. Results: To date, we have successfully treated 21 patients with median age 36 years (range 21-62) with advanced CD19+ ALL (n=18) or NHL (n=3); 10 patients had active disease at time of HCT. Donor-derived CAR+ T cells (HLA-matched sibling n=10; 1 Ag mismatched sibling n=1; haplo family n=8; cord blood n=2) were infused at a median 64 days (range 42-91 days) following HCT to prevent disease progression. Transplant preparative regimens were myeloablative, busulfan-based (n=10) or reduced intensity, fludarabine-based (n=11). All patients were maintained on GVHD prophylaxis at time of CAR T-cell infusion with tacrolimus, plus mycophenolate mofeteil for cord, plus post-HCT cyclophosphamide for haplo donors. The starting CAR+ T-cell dose was 106 (n=7), escalated to 107 (n=6), 5x107 (n=5), and currently at 108 (n=3) modified T cells/m2 (based on recipient body surface area). Patients have not demonstrated any acute or late toxicity to CAR+ T cell infusions. Three patients developed acute grades 2-4 GVHD (liver n=1, upper GI n=1, skin=1) which was within the expected range after allogeneic HCT alone. Of note, the rate of CMV reactivation after CAR T cell infusion was 24% vs. 41 % previously reported for our patients without CAR T cell infusion (Wilhelm et al. J Oncol Parm Practice, 2014, 20:257). Nineteen patients have had at least 30 days follow-up post CAR T-cell infusion and are evaluable for disease progression. Forty-eight percent of patients (n=10) remain alive and in complete remission (CR) at median 5.2 months (range 0-21.3 months) following CAR T cell infusion. Importantly, among 8 patients who received haplo-HCT and CAR, 7 remain in remission at median 4.2 months. Conclusion: We demonstrate that infusing donor-derived CD19-specific CAR+ T cells, using the SB and AaPC platform, in the adjuvant HCT setting as pre-emptive DLI may provide an effective and safe approach for maintaining remission in patients at high risk for relapse. Graft-vs-host disease did not appear increased by administration of the donor derived CAR-T cells. Furthermore, the add-back of allogeneic T cells appears to have contributed to immune reconstitution and control of opportunistic viral infection. Disclosures Huls: Intrexon and Ziopharm: Employment, Equity Ownership. Singh:Intrexon and Ziopharm: Equity Ownership, Patents & Royalties. Olivares:Intrexon and Ziopharm: Equity Ownership, Patents & Royalties. Su:Ziopharm and Intrexon: Employment. Figliola:Intrexon and Ziopharm: Equity Ownership, Patents & Royalties. Kumar:Ziopharm and Intrexon: Equity Ownership. Jena:Ziopharm Oncology: Equity Ownership, Patents & Royalties: Potential roylaties (Patent submitted); Intrexon: Equity Ownership, Patents & Royalties: Potential royalties (Patent submitted). Ang:Intrexon and Ziopharm: Equity Ownership. Lee:Intrexon: Equity Ownership; Cyto-Sen: Equity Ownership; Ziopharm: Equity Ownership.
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Petkov, S. G., W. A. Kues, and H. Niemann. "337 PROMOTER-DEPENDENT SILENCING OF REPROGRAMMING TRANSCRIPTION FACTORS IN MOUSE INDUCED PLURIPOTENT STEM CELLS PRODUCED WITH SLEEPING BEAUTY TRANSPOSON VECTORS." Reproduction, Fertility and Development 27, no. 1 (2015): 257. http://dx.doi.org/10.1071/rdv27n1ab337.
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Epigenetic silencing of the transgenes has been considered a prerequisite for complete reprogramming of mouse somatic cells to induced pluripotent stem cells (miPSC). Here, we examined the activity status of the reprogramming transcription factors in miPSC produced with Sleeping Beauty (SB) transposon vectors carrying expression cassettes with the porcine OCT4, SOX2, c-MYC, and KLF4 (pOSMK) under the control of doxycycline (DOX)-inducible (TetO) or constitutive (CAG) promoters. Mouse embryo fibroblasts (MEF) were electroporated with SB-TetO-rTA-SV40pA-TetO-pOSMK-IRES-tdTomato-bGHpA (TetO group) or with SB-loxP-CAG-pOSMK-IRES-tdTomato-SV40pA-loxP (CAG group) together with SB100x (SB transposase). The cells were cultured on mitotically inactivated MEF feeders with DMEM supplemented with 20% knockout serum replacement, 2 mM l-glutamine, penicillin-streptomycin, nonessential amino acids, 0.1 mM 2-mercaptoethanol, 1000 U mL–1 of ESGRO, and 5 µg mL–1 of DOX. The miPSC colonies were individually picked, disaggregated to single cells, and propagated further under the same culture conditions. Three cell lines from each experimental group were examined for pluripotency characteristics, and the activity of the transgenes was monitored by the presence of tdTomato fluorescence and by RT-PCR. The miPSC produced with TetO vector silenced the transgene expression within 11 days post-transfection (in the presence of DOX) and upregulated the endogenous pluripotency genes Oct4, Sox2, Nanog, Rex1, and Utf1. These cells showed typical miPSC morphology and ability to differentiate into cells from the 3 primary germ layers in vitro and in vivo (teratomas). At the same time, the miPSC from the CAG group did not silence the transgenes even after 20 passages of continuous propagation, although they upregulated the endogenous pluripotency genes similarly to the TetO group. Moreover, these cells also showed ability to differentiate in vitro into cells from the 3 germ layers (contracting cardiac myocytes, neurons, epithelia) expressing differentiation markers Afp, Sox17, Gata4, Gata6, cardiac troponin, nestin, and PGP 9.5. Following Cre-mediated excision of the reprogramming cassette, the miPSC from the CAG group continued to self-renew and the expression of pluripotency markers Oct4, Sox2, Nanog, and Rex1 did not change significantly, as evidenced by real-time RT PCR (all P > 0.1), showing that these cells were not dependent on the transgenes for maintaining their pluripotency characteristics. Currently, we are investigating the ability of the miPSC from the CAG group to differentiate in vivo by producing teratomas and chimeras. The results from our preliminary investigations suggest that porcine transcription factors can be used for production of miPSC and that the silencing of the reprogramming transcription factors in miPSC is promoter-dependent, but may not be absolutely necessary for complete reprogramming to pluripotency.
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Wang, Saisai, Yali Wang, Dan Shen, Li Zhang, Songlei Xue, Hengmi Cui, Chengyi Song, and Bo Gao. "Efficient Gene Transfer into Chicken Gonads by Combining Transposons with Polyethylenimine." Journal of Agricultural Science 8, no. 10 (September 7, 2016): 63. http://dx.doi.org/10.5539/jas.v8n10p63.
Full textAbstract:
<p>Transposon mediated transfection is a promising, safe, and convenient way to generate transgenic chicken compared with virus-mediated technology and the in vitro modification of primordial germ cells (PGCs). To establish a simple method for in vivo transfection of chicken PGCs, we applied four different transposon systems (PB, SB, Tol2, and ZB) to investigate the gene transfer efficiency of chicken gonads via direct injection of a mixture of transposon and transposase plasmids and transfection reagent (polyethylenimine, PEI) into the subgerminal cavity of Hamburger and Hamilton stage 2-3 chick embryos. We also compared the effect of the amount of plasmids injected on the gene transfer efficiency of chicken gonads. We found that over 70% of the gonads were green fluorescent protein (GFP)-positive across all four transposon groups, and that the proportion of GFP-positive gonads was not significantly different between different transposons. Some GFP positive cells in gonads were confirmed as germ cells by co-labeling with the germ cell specific antibody. We also found that the proportions of GFP-positive gonads decreased significantly with a decrease of plasmid dose from 100 ng to 20 or 50 ng. Here we revealed that a combination of transposons with PEI is a simple and efficient method for gene transfer into chicken gonads and able to transfect PGCs in vivo that could be used for the production of transgenic chickens.</p>
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Masihi, Meher Beigi, Catherine Lee, Grace A. Furnari, Alexandra Garancher, and Robert J. Wechsler-Reya. "MBRS-12. A TRANSPOSON MUTAGENESIS SCREEN IDENTIFIES Rreb1 AS A DRIVER FOR GROUP 3 MEDULLOBLASTOMA." Neuro-Oncology 22, Supplement_3 (December 1, 2020): iii400. http://dx.doi.org/10.1093/neuonc/noaa222.529.
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Abstract Medulloblastoma (MB) is the most common malignant childhood brain tumor. MB can be divided into four major subgroups – WNT, Sonic hedgehog (SHH), Group 3 (G3), and Group 4 (G4) – that exhibit distinct genetic alterations, gene expression profiles, and clinical outcomes. Patients with G3-MB have the worst prognosis, and a deeper understanding of this disease is critical for development of new therapies. Most G3-MBs express high levels of the MYC oncogene, suggesting that MYC plays an important role in tumorigenesis. To identify genes that cooperate with MYC to promote formation of G3-MB, we performed an in vivo mutagenesis screen using mice expressing the Sleeping Beauty (SB) transposon. Cerebellar stem cells from transposon/transposase-expressing mice were infected with viruses encoding Myc, and transplanted into the cerebellum of adult hosts. The resulting tumors were sequenced to identify transposon-targeted genes, and these genes were functionally analyzed to determine whether they could cooperate with Myc to drive G3-MB. These studies identified the transcription factor Ras-responsive element binding protein 1 (Rreb1) as a potent Myc-cooperating gene. Tumors driven by Myc and Rreb1 resemble G3-MB at a histological and molecular level. Moreover, RREB1 is overexpressed in human G3-MB, and knockdown of RREB1 impairs growth of G3-MB cell lines and patient-derived xenografts. Ongoing studies are aimed at identifying the mechanisms by which Rreb1 contributes to tumor growth. Our studies demonstrate an important role for RREB1 in G3-MB, and provide a new model that can be used to identify therapeutic targets and develop more effective therapies for medulloblastoma.
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Garrels, W., S. Holler, C. Struckmann, U. Taylor, C. Ehling, D. Rath, H. Niemann, Z. Ivics, and W. A. Kues. "328 ANALYSIS OF FLUOROPHORE-EXPRESSING SPERMATOZOA FROM TRANSGENIC BOARS PRODUCED BY SLEEPING BEAUTY TRANSPOSITION." Reproduction, Fertility and Development 23, no. 1 (2011): 260. http://dx.doi.org/10.1071/rdv23n1ab328.
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The pig is an important model for biomedical research. Recently, we described a method for producing transgenic pigs using a nonautonomous Sleeping Beauty (SB) transposon1 (Garrels et al. 2010 Reprod. Domest. Anim. 45, 65 abst.). Briefly, in vivo developed porcine zygotes were co-injected with a CAGGS-Venus transposon and hyperactive SB100. A total of 141 in vivo developed zygotes were injected and transferred to synchronized foster sows. Subsequent analysis revealed specific transposase-mediated integration of 1 to 5 copies of the Venus transposon in fetuses and piglets. This method results in highly efficient SB-mediated transgene transposition into the porcine genome: 57% of the fetuses examined and 42% of piglets were transgenic, representing 6.4% of the treated zygotes. The piglets showed persistent expression of the Venus reporter. Here, we present cellular analysis of 2 founder boars. Expression of the Venus reporter was observed in skin, cultured fibroblasts, leukocytes, and spermatozoa of both animals. However, flow cytometric measurement of leukocytes and cultured ear fibroblasts revealed that these boars carried both a Venus-fluorescence-positive population and a Venus-fluorescence-negative cell population. PCR analysis revealed that the Venus-fluorescence-negative cells were genotypically negative, indicating transgene mosaicism. Interestingly, all spermatozoa tested were Venus-positive and gave a distinct fluorescence peak in repeated flow-cytometric measurements (n = 6). Fluorescence microscopy revealed localisation of the Venus fluorophore in the sperm tail, in the midpiece, and in the equatorial segment of the sperm head. Motility of the transgenic sperm as measured by computer-assisted sperm analysis (Hamilton-Thorne, Beverly, MA, USA) indicated no decrease in percentage of motile sperm and the movement patterns. Sorting Hoechst 33342–stained transgenic sperm into X- and Y-chromosome bearing populations did not reveal any differences in Venus fluorescence between these 2 groups. To test the fertility of the transgenic sperm, 6 wild-type sows were artificially inseminated. Four pregnancies were established, 2 of these sows were sacrificed on Day 29 of gestation and a total of 9 Venus-positive normally developed fetuses and 2 degenerated fetuses were recovered. The other 2 pregnancies are ongoing at the time of writing. This is the first characterisation of spermatozoa from transposon transgenic pigs. The results show that Venus transposon–bearing transgenic spermatozoa are fertile and demonstrate germline transmission to the F1 offspring. Sleeping Beauty-mediated transposition is thus a promising approach for genetic modification of the pig genome.
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Lock, Dominik, Razieh Monjezi, Caroline Brandes, Stephan Bates, Simon Lennartz, Karin Teppert, Leon Gehrke, et al. "Automated, scaled, transposon-based production of CAR T cells." Journal for ImmunoTherapy of Cancer 10, no. 9 (September 2022): e005189. http://dx.doi.org/10.1136/jitc-2022-005189.
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BackgroundThere is an increasing demand for chimeric antigen receptor (CAR) T cell products from patients and care givers. Here, we established an automated manufacturing process for CAR T cells on the CliniMACS Prodigy platform that is scaled to provide therapeutic doses and achieves gene-transfer with virus-free Sleeping Beauty (SB) transposition.MethodsWe used an advanced CliniMACS Prodigy that is connected to an electroporator unit and performed a series of small-scale development and large-scale confirmation runs with primary human T cells. Transposition was accomplished with minicircle (MC) DNA-encoded SB100X transposase and pT2 transposon encoding a CD19 CAR.ResultsWe defined a bi-pulse electroporation shock with bi-directional and unidirectional electric field, respectively, that permitted efficient MC insertion and maintained a high frequency of viable T cells. In three large scale runs, 2E8 T cells were enriched from leukapheresis product, activated, gene-engineered and expanded to yield up to 3.5E9 total T cells/1.4E9 CAR-modified T cells within 12 days (CAR-modified T cells: 28.8%±12.3%). The resulting cell product contained highly pure T cells (97.3±1.6%) with balanced CD4/CD8 ratio and a high frequency of T cells with central memory phenotype (87.5%±10.4%). The transposon copy number was 7.0, 9.4 and 6.8 in runs #1–3, respectively, and gene analyses showed a balanced expression of activation/exhaustion markers. The CD19 CAR T cell product conferred potent anti-lymphoma reactivity in pre-clinical models. Notably, the operator hands-on-time was substantially reduced compared with conventional non-automated CAR T cell manufacturing campaigns.ConclusionsWe report on the first automated transposon-based manufacturing process for CAR T cells that is ready for formal validation and use in clinical manufacturing campaigns. This process and platform have the potential to facilitate access of patients to CAR T cell therapy and to accelerate scaled, multiplexed manufacturing both in the academic and industry setting.
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Beigi Masihi, Meher, Catherine Lee, Alexandra Garancher, Grace Furnari, and Robert Wechsler-Reya. "TMOD-30. IDENTIFYING NEW DRIVERS OF GROUP 3 MEDULLOBLASTOMA." Neuro-Oncology 22, Supplement_2 (November 2020): ii234. http://dx.doi.org/10.1093/neuonc/noaa215.980.
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Abstract Medulloblastoma (MB) is the most common malignant childhood brain tumor. MB can be divided into four major subgroups – WNT, Sonic hedgehog (SHH), Group 3 (G3-MB), and Group 4 (G4-MB) – that exhibit distinct genetic alterations, gene expression profiles, and clinical outcomes. Patients with G3-MB have the worst prognosis, and a deeper understanding of this form of the disease is critical for development of new therapies. Most G3-MBs express high levels of the MYC oncogene, suggesting that MYC plays an important role in tumorigenesis. However, MYC overexpression is not sufficient to drive tumor formation. To identify genes that cooperate with MYC to promote development of G3-MB, we performed an in vivo mutagenesis screen using mice expressing the Sleeping Beauty (SB) transposon. Cerebellar stem cells isolated from transposon/transposase-expressing transgenic mice were infected with viruses encoding Myc, and these cells were transplanted into the cerebellum of adult hosts. Tumors that arose were subjected to DNA and RNA sequencing to identify candidate genes, and these genes were subjected to functional analysis to determine whether they could cooperate with Myc to drive G3-MB. These studies identified the transcription factor Ras-responsive element binding protein 1 (Rreb1) as a potent Myc-cooperating gene. Tumors driven by Myc and Rreb1 (MR tumors) resemble G3-MB at a histological and molecular level. Moreover, RREB1 is overexpressed in human G3-MB, and knockdown of RREB1 expression impairs growth of G3-MB cell lines and patient-derived xenografts. Ongoing studies are aimed at identifying the molecular mechanisms by which Rreb1 contributes to tumor growth. Our studies demonstrate an important role for RREB1 in G3-MB, and provide a new model that can be used to identify therapeutic targets and develop more effective and less toxic therapies for this devastating pediatric brain tumor.
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Gaipa, Giuseppe, Chiara Francesca Magnani, Daniela Belotti, Giada Matera, Sarah Tettamanti, Benedetta Cabiati, Stefania Cesana, et al. "Clinical-Grade Transduction of Allogeneic Cytokine Induced Killer (CIK) Cells with CD19 Chimeric Antigen Receptor (CAR) Using Sleeping Beauty (SB) Transposon: Successful GMP-Compliant Manufacturing for Clinical Applications." Blood 132, Supplement 1 (November 29, 2018): 196. http://dx.doi.org/10.1182/blood-2018-196.
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Abstract Background: Acute lymphoblastic leukemia (ALL) is a malignant disorder with a long-term remission of less than 50% of adult patients and of nearly 80% of children. Relapsed and refractory (r/r) adult and childhood B-ALL patients, have significant unmet medical needs. Adoptive transfer of patient-derived T cells engineered to express a chimeric antigen receptor (CAR) by viral vectors has achieved complete remission and durable response in highly refractory populations (June CH et al. Science 2018). In addition, unmodified Cytokine Induced Killer (CIK) cells (CD3+, CD56+ T cells) have clearly demonstrated a high profile of safety in ALL patients (Introna M et al. Biol Blood Marrow Transplant. 2017). Here, we demonstrate the feasibility and reproducibility of a GMP-compliant clinical-grade culture and gene-modification protocol of allogeneic CIK cells using the non-viral Sleeping Beauty (SB) transposon system (Singh H et al, Plos One 2013) to obtain CD19CAR expressing CIK cells (Magnani CF et al, Oncotarget 2016, Magnani CF et al, Hum Gene Ther. 2018, Biondi A et al. J Autoimmun. 2017) starting from a limited amount of an easily available material such as peripheral blood (PB). Methods: Fifty mL of PB were centrifuged on Ficoll gradient to obtain mononuclear cells (PBMCs). PBMCs were then simultaneously electro-transferred with the SB GMP-grade DNA transfer CD19.CAR/pTMNDU3 plasmid (human 3rd generation anti-CD19CD28OX40z CAR under the pTMNDU3 promoter), and transposase pCMV-SB11 plasmid (kindly provided by L. Cooper, MDACC, Houston, TX, USA). CIK populations (Introna M et al, Haematologica 2007) were then generated according to the method enclosed in the filed patent EP20140192371 (Magnani CF et al, Oncotarget 2016). The manufacturing process and the quality control tests were performed in a good manufacturing practices (GMP) academic cell factory authorized by Agenzia Italiana del Farmaco (AIFA) in the context of an ongoing phase I clinical trial (NCT03389035) for children and adults with relapsed/refractory B-cell precursor ALL post hematopoietic stem cell transplantation (HSCT). Results: We manufactured nine batches by seeding a mean of 102.52x106 allogeneicPBMCs derived from 50 ml of peripheral blood (range 46.1 - 193.17x106). After 21-22 days of culture the mean harvesting was 5.0x109 nucleated cells (range 1.15 - 16.00x109) with a mean viability of 97.56% (min. 95.24% - max 99.43%). These cells were mostly CD3+ lymphocytes (mean 98.54%, min. 94.85% - max 99.68%) which had a median fold increase of 87.3. Expanded CD3+ cells expressed CD56+ and surface CAR at variable levels among the batches (mean 44.79% and 43.78%, respectively) and had a median vector copy number (VCN) of 2.56 VCN/cells, according to pre-clinical data (Magnani CF et al, Hum Gene Ther. 2018). In all the nine batches, CARCIK-CD19 cells demonstrated potent and specific in vitro cytotoxicity towards the CD19+ REH target cell line (mean 82.96%, min. 61.89% - max 97.72%). Cell products appear to be highly polyclonal and no signs of genotoxicity by transposon insertions could be observed by integration site (IS) analysis performed by Sonication Linker Mediated (SLiM)-PCR and Illumina MiSeq sequencing. The GMP batches were released after about 10 days after the end of production. Quality control release specifications and results are reported in Table 1. Conclusions: Overall, these results demonstrate that clinical-grade SB transduction of allogeneic CIK cells with CD19 CAR is feasible and allows rapid and efficient expansion of highly potent CARCIK-CD19 cells starting from easily available small amounts of PB, with important implications for non-viral technology. In summary our data represent a solid ground for the future development of further SB-based platforms. A clinical trial investigating allogeneic CARCIK-CD19 in r/r pediatric and adult ALL post HSCT is currently ongoing (NCT03389035). Disclosures Gritti: Autolus: Consultancy. Rambaldi:Celgene: Consultancy; Omeros: Consultancy; Novartis: Consultancy; Italfarmaco: Consultancy; Pfizer: Consultancy; Amgen Inc.: Consultancy; Roche: Consultancy.
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Zong, Shan, Laurence J. N. Cooper, George T. McNamara, and Hiroki Torikai. "Personalization of T-Cell Therapy Using a High-Throughput Platform to Identify Tumor-Specific T-Cell Receptors." Blood 128, no. 22 (December 2, 2016): 3359. http://dx.doi.org/10.1182/blood.v128.22.3359.3359.
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Abstract Background T-cell receptors (TCRs) can be used to redirect the specificity of T-cells for human application. This has particular appeal for the targeting of neoantigens. However, efficient identification, cloning, and characterization of antigen (Ag)-specific TCRs is needed to enable the timely adoptive transfer of T-cells genetically modified to express therapeutic TCRs. We have harnessed next generation sequencing (NGS) to identify desirable TCRs. This approach enables us to simultaneously identify hundreds of Ag-specific TCRs, along with the expression of genes to characterize functional and phenotypical values of individual Ag-specific T-cells (e.g. related to cytotoxicity, exhaustion, etc...). To take advantage of NGS technology, a sister high-throughput technology was developed to evaluate harvested TCRs. A reporter system was implemented using (immortalized) Jurkat T-ALL cells genetically modified to (i) enforce expression of CD8aβ, (ii) conditionally express GFP under minimal elements of NR4A1 promoter, and (iii) prevent expression of both endogenous TCRα and β chains. Sequenced CDR3 regions were coded within DNA plasmids from Sleeping Beauty (SB) transposons as TCR Vα and Vβ libraries that were expressed on the reporter cell to identify both TCR specificity as well as TCR avidity. Thus, we implemented (i) a TCR cloning system based on CDR3 sequencing of Ag-specific T-cells identified by NGS and (ii) a novel reporter cell based on the TCR-mediated induction of GFP expression. As a proof-of-concept to evaluate the entire platform, we used HLA-A2-restricted CMV peptide (NLVPMVATV: CMV/A2) and NY-ESO-1 peptide (SLLMWITQC: NY-ESO-1/A2) as model Ags. Results The reporter cell was initially genetically modified with either high- or low-avidity TCRs against a NY-ESO-1/A2. Upon stimulation with HLA-A2+ 721.221 immortalized B cells, it was found that the expression of GFP positively correlated with the avidity of TCRs (Figure). Next, we isolated naïve Ag-specific T-cells from umbilical cord blood using CMV/A2 tetramer. Single CMV/A2-specific CD8+ T-cells were sorted and their TCRαβ CDR3 sequences were amplified by reverse transcription and PCR with bar-coded probes. The pooled PCR products were sequenced in MiSeq Sequencer (illumina) to obtain TCRαβ CDR3 regions and analyzed in silico using IMGT (International Immunogenetics Information System). The efficiency of identifying individual TCRαβ pairs was 65% to 76% (using 96-well plate or 384-well plates, respectively). Individual SB-derived DNA transposons expressing TCRαβ constructs were synthesized by Gibson assembly and electroporated, with SB transposase, into the reporter cell. These cells were co-cultured with HLA-A2+ 721.221 stimulator cells loaded with graded doses of cognate peptide. The percentage and intensity of GFP expression was evaluated by high-throughput flow-cytometer (IntelliCyt) which revealed high-avidity Ag-specific TCRαβs. Conclusion Our new high-throughput system can identify and characterize, based on specificity and avidity, Ag-specific TCRαβs within a week. This system will be used to generate neoantigen-specific TCRαβs for human application. Disclosures Zong: ZIOPHARM Oncology, Inc.: Equity Ownership, Patents & Royalties; Intrexon: Equity Ownership, Patents & Royalties; Immatics US, Inc: Equity Ownership, Patents & Royalties. Cooper:Ziopharm Oncology: Employment, Equity Ownership, Patents & Royalties; Intrexon: Equity Ownership; City of Hope: Patents & Royalties; Targazyme, Inc.,: Equity Ownership; Immatics: Equity Ownership; Sangamo BioSciences: Patents & Royalties; MD Anderson Cancer Center: Employment; Miltenyi Biotec: Honoraria. McNamara:GeoMcNamara: Consultancy, Other: Consultant in immuno-oncology field; ZIOPHARM Oncology, Inc: Equity Ownership, Patents & Royalties; Intrexon: Equity Ownership, Patents & Royalties. Torikai:intrexon: Equity Ownership, Patents & Royalties; ZIOPHARM Oncology, Inc.: Equity Ownership, Patents & Royalties; Immatics US, Inc: Equity Ownership.
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Hauschild-Quintern, J., B. Petersen, D. Herrmann, A. Lucas-Hahn, S. Petkov, R. Schwinzer, and H. Niemann. "327 PRODUCTION OF TRIPLE TRANSGENIC hHO-1/GGTA-1–/–/hCD55 TRANSGENIC PIGS USING SLEEPING BEAUTY TRANSPOSITION AND ZINC-FINGER NUCLEASES." Reproduction, Fertility and Development 25, no. 1 (2013): 311. http://dx.doi.org/10.1071/rdv25n1ab327.
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Advances in xenotransplantation (pig-to-baboon or human transplantation) require multi-transgenic pigs with a homozygous knockout (KO) of the α1,3-galactosyltransferase gene (GGTA-1, encoding for Gal-epitopes) to control the hyperacute rejection response. To achieve prolonged survival of the porcine xenograft, transgenic expression of additional immune modulatory genes on the GGTA-1–/– background is considered the solution of choice. The acute vascular rejection (AVR) is primarily caused by endothelial cell activation and leads to rejection of GGTA-1–/– pig organs. Here, we set out to produce triple transgenic pigs with high expression of the human complement regulatory gene hCD55 (human decay acceleration factor, hDAF) and the anti-apoptotic and anti-inflammatory gene human heme oxygenase-1 (hHO-1), respectively. Porcine ear fibroblasts carrying a hHO-1 transgene (Petersen et al. 2011 Xenotransplantation 18, 355–368) were transfected with zinc-finger nucleases (ZFN) targeting the GGTA-1 gene, leading to a biallelic KO of the GGTA-1 gene in ~1% of the transfected cells (Hauschild et al. 2011 Proc. Natl. Acad. Sci. USA 108, 15 010). Somatic cell nuclear transfer (SCNT) was accomplished with cells from double transgenic cell line (hHO-1/GGTA-1–/–), and one pregnancy was terminated to obtain fetal cell cultures. Fluorescence-activated cell sorting (FACS) analysis revealed that all 11 fetuses (C6F1-F11) were lacking Gal-epitopes. One pure fetal cell culture (C6F5) was used for co-transfection with the newly designed SB-CAGGS-hDAF-Puror transposon, based on the Sleeping Beauty transposon plasmid pT2/HB, together with the SB transposase 100X plasmid (both kindly provided by Dr. Zoltan Ivics). After 14 days of selection with puromycin, cell colonies were picked and expanded to obtain cells for analysis and as a backup for SCNT. The PCR amplification with hDAF-specific primers revealed that ~50% of picked colonies had integrated the transgene. Two hDAF-positive colonies were pooled and used for SCNT. One pregnancy was sacrificed and 4 hHO-1/GGTA-1–/–/hCD55 fetuses were obtained for real-time PCR analysis and subsequent use in SCNT. Real-time PCR showed elevated hCD55 expression representing a 0.9-fold (0.7- to 1.0-fold) expression of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH), while FACS analysis confirmed absence of Gal-epitopes in all 4 fetuses. Binding of human-specific anti-CD55 antibodies showed high expression of hCD55 by FACS measurement. Triple transgenic fetuses C8F2 and C8F4 showed higher expression of hCD55 by real-time PCR and by FACS analysis compared with fetuses C8F1 and C8F3. Fetuses C8F3 and C8F4 were used for re-cloning experiments to produce live offspring, which will further be characterised and used in transplantation experiments. This work underlines the importance of new genomic technologies to further improve the efficiency of the generation of transgenic pigs suitable for xenotransplantation.
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Magnani, Chiara F., Renier Myburgh, Norman F. Russkamp, Steve Pascolo, Judith A. Shizuru, Dario Neri, and Markus G. Manz. "Anti-CD117 CAR T Cells Incorporating a Safety Switch Eradicate Acute Myeloid Leukemia and Deplete Human Hematopoietic Stem Cells." Blood 138, Supplement 1 (November 5, 2021): 2808. http://dx.doi.org/10.1182/blood-2021-145195.
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Abstract Introduction Acute Myeloid Leukemia (AML) arises from the accumulation of mutations within the hematopoietic stem and progenitor cells (HSPC), leading to the emergence of a population of malignant leukemia-initiating cells (LIC). AML-LICs maintain high phenotypic similarity with their cells-of-origin and can cause post-treatment relapse. Immunotherapy with chimeric antigen receptor (CAR) T cells is an innovative approach to tackle cancer via surface-expressed cancer-associated antigens. We recently proposed the use of CAR T cells specific for the CD117 antigen to deplete LIC and replace HSPC by allogeneic hematopoietic stem cell transplantation (HSCT) (Myburgh R et al. Leukemia 2020). This concept implies early termination of CAR T-cell activity to prevent subsequent graft rejection. Here, we exploit a non-viral technology for the generation of anti-CD117 CAR T cells incorporating a safety switch. Methods We designed a Sleeping Beauty (SB) transposon vector that includes the inducible Caspase 9 (iC9) switch and the anti-CD117CAR, separated by a 2A peptide. SB allows the generation of CAR T cells with potent anti-leukemic activity (Magnani CF et al. J Clin Invest. 2020). The vector has an optimized donor vector architecture and allows for the stoichiometric expression of the two transgenes. iC9 allows for rapid termination of CAR T cells by activation of the apoptotic pathway in case of treatment with a small molecule that acts as a chemical inducer of dimerization (CID). The hyperactive SB100X transposase, supplied as plasmid DNA or mRNA, catalyzes transgene integration. As an alternative approach, we used mRNA encoding an anti-CD117 CAR in human T cells. Results With the purpose of transduction optimization, we compared total PBMC and selected T cells as starting material in the presence of different concentrations of plasmids or mRNA. The procedure of generating CAR T cells with SB did not affect T cell memory differentiation but increased the CD8/CD4 proportion compared to non-transduced (NT) cells (75.63% vs. 41.63%, p= 0.0124). Based on higher transduction efficiency and favored in vitro expansion, we defined the lead protocol (selected T cells, PT4:SB100X plasmid 3:1 ratio, or PT4:SB100X mRNA 1:2 ratio). CAR T cells had a high level of viability, retained a high proportion of naïve-like (mean 36.38%, SEM 8.80) and T stem cell memory populations (mean 39.21%, SEM 8.43), and showed low levels of the exhaustion markers PD-1 (mean 2.21%, SEM 1.04), LAG3 (mean 61.20%, SEM 9.61), and TIM3 (mean 35.72%, SEM 10.79). Anti-CD117 CAR T cells exhibited potent cytotoxicity against the AML cell line MOLM-14, transduced and sorted to express human CD117, luciferase, and GFP. The addition of 200nM of the CID to cultures of anti-CD117 CAR T cells induced apoptosis of transduced CAR T cells within 24h but had no effect on the viability of NT cells. Anti-CD117 CAR T cells mediated depletion of CD117+ MOLM-14 cells in vivo, leading to a significant survival advantage compared to mice treated with NT cells (median overall survival for NT= 22.5 days vs. SB= not reached, p= 0.0122, Mantel-Cox). Notably, SB-transduced CAR T cells were as efficient as CAR T cells transduced with lentiviral vectors. In NSG mice reconstituted with human CD34+ cord blood cells, anti-CD117 CAR T cells were able to achieve complete CD117+ HSPC depletion. Treatment with a combination of CID and anti-thymocyte globulin (ATG) eliminated anti-CD117 CAR T cells and T cells of the previous transplant donor. Finally, transient expression of anti-CD117 CAR by mRNA conferred T cells the ability to kill CD117+ targets throughout 72 hours post mRNA electroporation. The cytotoxic activity decreased over time as mRNA-electroporated CAR T cells proliferate and lose CAR expression upon 3-5 divisions. Treatment of humanized NSG mice with two subsequent doses of anti-CD117 CAR mRNA T cells resulted in HSPC depletion. Conclusions Anti-CD117 CAR T cells engineered with the SB vector showed anti-leukemic activity and completely depleted healthy HSPC in vivo. iC9 transgene induced CAR T cell apoptosis and allowed rapid CAR T cell depletion that alternatively also could be achieved with mRNA electroporation of the anti-CD117 CAR. The ability to control CAR T cell pharmacokinetic properties is attractive to enable subsequent HSCT and to terminate unexpected toxicities. Anti-CD117 CAR T cells could be used prior to HSCT in refractory or minimal residual disease AML. Disclosures Myburgh: University of Zurich: Patents & Royalties: CD117xCD3 TEA. Shizuru: Forty seven Inc: Other: Inventor on a patent licenses by Forty Seven. Forty seven was acquired by Gilead in 2020; Jasper Therapeutics, Inc.: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Other: Chair of scientific advisory board. Neri: Philogen S.p.A.: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Multiple patents on vascular targeting; ETH Zurich: Patents & Royalties: CD117xCD3 TEA. Manz: CDR-Life Inc: Consultancy, Current holder of stock options in a privately-held company; University of Zurich: Patents & Royalties: CD117xCD3 TEA.
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Tolar, Jakub, In-Hyun Park, Lily Xia, Mark Osborn, Ron T. McElmurry, Paul J. Orchard, George Q. Daley, and Bruce R. Blazar. "Patient-Specific Induced Pluripotent Stem Cells in Hurler Syndrome." Blood 112, no. 11 (November 16, 2008): 386. http://dx.doi.org/10.1182/blood.v112.11.386.386.
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Abstract Hurler syndrome (HS; mucopolysaccharidosis type I) is caused by severe mutations in the iduronidase (IDUA) gene, leading to multi-organ system dysfunction due to the toxic accumulation of glycosaminoglycans. Although allogeneic hematopoietic cell transplantation (HCT) has been shown to provide the IDUA protein and to reverse many of the manifestations of HS, allogeneic HCT is associated with significant morbidity and mortality. We hypothesized that an advantageous alternative strategy may be to induce gene-corrected autologous pluripotent cells to become hematopoietic stem cells, which then provide the missing IDUA enzyme. Because patient-specific embryonic stem cell isolation is not practical, recent strategies have been developed that reprogram adult cells to acquire pluripotency. Such induced pluripotent stem (iPS) cells can be created from fibroblasts or mesenchymal stromal cells (MSCs). As a first step in testing of iPS cells for gene-corrected HS treatment, we isolated host MSCs from the bone chips of a 9-year-old boy with HS who had undergone spinal surgery 8 years after successful allogeneic HCT. HS-MSCs expressed no IDUA, confirming a lack of contamination from either donor-derived hematopoietic cells or MSCs. To create HS-iPS cells, HS-MSCs were transduced with viral vectors carrying reprogramming transcription factors (OCT4, SOX2, KLF4, and c-MYC) that are typically associated with pluripotency and expressed at high levels in embryonic but not adult stem cells. Transduced cells were cultured on supportive stroma of irradiated mouse embryo fibroblasts. Within several weeks, colonies of iPS cells emerged from the two-dimensional culture. When compared to MSCs, the HS-iPS cells showed persistent mRNA expression of OCT3/4 and SOX2 and transient mRNA expression of c-MYC and KLF4, which is expected to occur in the wild-type iPS cells. HS-iPS cells expressed protein markers characteristic of reprogrammed immature cells: OCT3/4, NANOG, stage-specific embryonic antigens (SSEA) 3 and 4, tumor rejection antigens (TRA) 1–60 and 1–81, and alkaline phosphatase. HS-iPS cells had normal male karyotype as determined by chromosomal G-banding. As a second step in creating gene-corrected HS-iPS cells, we employed the non-viral Sleeping Beauty (SB) transposon system (because of the less random pattern of genome integration when compared to viral vectors). Human HS-iPS cells were co-nucleofected with an SB transposon that harbored the human IDUA gene and an expression cassette of the green fluorescent protein (GFP) along with an SB transposase plasmid that provides the enzymatic machinery necessary for integration into TA dinucleotide sites within the genome. Two weeks after nucleofection 10%-15% of HS-iPS cells expressed GFP. Total glycosaminoglycans (a hallmark of the biochemical defect in HS) in unsorted cultures were decreased to wild-type levels. IDUA expression in unsorted cultures was approximately 10% of wild-type IDUA levels, which is within the range sufficient for phenotypic rescue in HS patients after allogeneic HCT. Experiments are ongoing, and data will be presented in regards to: a) map transposon insertions in the genome to prove stable transgenesis by transposition; b) characterization of the differentiation potential of the corrected HS-iPS cells into various mesodermal lineages relevant to rescue of the clinical phenotype associated with HS (hematopoietic, chondrogenic, and osteogenic); c) assessment of development and consequences of cellular pathology in numerous tissue types affected by IDUA deficiency. To our knowledge these are the first data to report that autologous iPS cells can be obtained from HS patients. In summary, HS-iPS cells present an opportunity to use the hematopoietic progeny of gene-corrected autologous cells clinically in a manner that may preclude the immunologic complications of allogeneic transplantation.
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Monga, Satdarshan, Sungjin Ko, Laura Molina, Junyan Tao, Aatur D. Singhi, and Aaron Bell. "Hepatocyte-derived intrahepatic cholangiocarcinoma requires Yap and Sox9: A clinical and preclinical analysis." Journal of Clinical Oncology 38, no. 4_suppl (February 1, 2020): 582. http://dx.doi.org/10.1200/jco.2020.38.4_suppl.582.
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582 Background: Intrahepatic cholangiocarcinoma (ICC) is a liver tumor of increasing incidence and devastating prognosis. WGS and WES have identified numerous molecular pathways and fusions in ICC. Recent studies have also suggested hepatocyte as a cell source in a subset of ICCs, especially those associated with chronic liver insult such as non-alcoholic steatohepatitis (NASH) or primary sclerosing cholangitis (PSC). Methods: Since co-expression of myristoylated AKT (myrAKT) & Notch intracellular domain (NICD) in hepatocytes using sleeping beauty transposon/transposase-based hydrodynamic tail vein injection (SB-HTVI) lead to ICC, we initiated a comprehensive analysis of mechanism of ICC development in patients and in this preclinical model. Results: Over 90% of CC samples exhibited high levels of nuclear SOX9 & YAP, in addition to a significant positivity for pAKT in ICCs as compared to extrahepatic CC. We also identified upregulation of p-AKT, SOX9 & YAP in hepatocytes of patients with PSC and NASH. This was also seen in many murine models of cholestatic injury and NASH. While co-expression of myrAKT+NICD led to hepatocyte-derived ICC, conditional deletion of either Yap or Sox9 significantly delayed and almost completely abrogated ICC development. While Yap deletion impaired the initial HC-to-BEC fate conversion, Sox9 elimination had no such effect on reprograming. Interestingly, following deletion of either Yap or Sox9 we observed a few AKT/NICD-driven ICC tumors expressing either Sox9 or Yap but not both. This also occurred in a small subset of human CC tumors which may be Sox9+Yap- (4%) or Sox9-Yap+ (3.7%), showing that deletion of Yap or Sox9 is not sufficient to completely abrogate ICC development. We finally demonstrated that conditional deletion of both Yap & Sox9 completely blocked development of ICC tumors in the myrAKT+NICD model. Conclusions: Thus, we show that cholestatic injury or NASH in humans and mice induces hepatocyte-to-cholangiocyte reprograming to increase the risk of ICC development. We also provide evidence for critical but distinct roles of Yap and Sox9 in ICC development and demonstrate the therapeutic potential of targeting these factors for treatment of subsets of ICC.
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Patel, Krina, Simon Olivares, Harjeet Singh, Lenka V. Hurton, Mary Helen Huls, Muzaffar H. Qazilbash, Partow Kebriaei, Richard E. Champlin, and Laurence J. N. Cooper. "Combination Immunotherapy with NY-ESO-1-Specific CAR+ T Cells with T-Cell Vaccine Improves Anti-Myeloma Effect." Blood 128, no. 22 (December 2, 2016): 3366. http://dx.doi.org/10.1182/blood.v128.22.3366.3366.
Full textAbstract:
Abstract Adoptive transfer of T cells expressing chimeric antigen receptor (CAR) has demonstrated clinical effectiveness in early phase clinical trials, with persistence of effector cells typically leading to improved outcomes. Most CARs directly dock with cell-surface antigens, but this limits the number of tumor-derived targets. Thus, we have adapted two technologies to target intracellular antigens and improve survival of infused T cells. This was accomplished by expressing a CAR on T effector cells that functions as a mimetic of T-cell receptor (TCR) to recognize NY-ESO-1 in the context of HLA A2 and adapting HLA-A2+ T cells to serve as antigen presenting cells (T-APC) by expressing NY-ESO-1 antigen. NY-ESO-1 is a desirable target for T-cell therapy of high risk multiple myeloma (MM) with efficacy in trials infusing T cells expressing TCR recognizing this antigen. We hypothesized combined immunotherapy with NY-ESO-1-specific CAR+ T cells and an NY-ESO-1+ T-APC vaccine will lead to enhanced anti-myeloma efficacy due to improved persistence of the CAR+ T effector cells. An NY-ESO-1-specific CAR and control TCR were expressed on primary T cells using the Sleeping Beauty (SB) transposon/transposase system. T-APC was generated by electro-transfer of DNA plasmids from SB system coding for NY-ESO-1 and membrane-bound IL-15 (mbIL15). The tethered cytokine functions as co-stimulatory molecule to improve the potency of the vaccine. In vitro studies confirmed the NY-ESO-1-specific CAR+ (and TCR+) T cells could be numerically expanded upon co-culture with T-APC. A mouse model of NY-ESO-1+HLA-A2+(CD19neg) multiple myeloma was used to compare tumor growth for CAR+ T effector cells with and without T-APC. The NY-ESO-1-specific CAR+ T effector cells displayed anti-tumor effect that was superior to control mice without T cells and mice receiving CD19-specific control CAR+ T cells. Mice receiving both NY-ESO-1-specific CAR+T effector cells and T-APC exhibited further improvement in anti-myeloma activity. This group demonstrated superior persistence of T effector cells with recovered cells exhibiting a memory phenotype. In summary, T cells can target intracellular NY-ESO-1 using a TCR mimetic CAR. Improved anti-tumor effect attributed to better persistence can be achieved by co-infusion of T-APC vaccine. These data provide the foundation to assess T cells targeting NY-ESO-1 in a clinical trial. Disclosures Patel: Ziopharm Oncology: Equity Ownership, Patents & Royalties; Intrexon: Equity Ownership, Patents & Royalties. Olivares:Ziopharm Oncology: Equity Ownership, Patents & Royalties; Intrexon: Equity Ownership, Patents & Royalties. Singh:Ziopharm Oncology: Equity Ownership, Patents & Royalties; Immatics: Equity Ownership, Patents & Royalties; Intrexon: Equity Ownership, Patents & Royalties. Hurton:Ziopharm Oncology: Equity Ownership, Patents & Royalties; Intrexon: Equity Ownership, Patents & Royalties. Huls:Ziopharm Oncology: Equity Ownership, Patents & Royalties; Intrexon: Employment, Equity Ownership, Patents & Royalties. Cooper:City of Hope: Patents & Royalties; Intrexon: Equity Ownership; Ziopharm Oncology: Employment, Equity Ownership, Patents & Royalties; Targazyme, Inc.,: Equity Ownership; Immatics: Equity Ownership; Sangamo BioSciences: Patents & Royalties; MD Anderson Cancer Center: Employment; Miltenyi Biotec: Honoraria.
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Tolar, Jakub, Scott Bell, Ron McElmurry, Lily Xia, R. Scott McIvor, Stephen R. Yant, Mark A. Kay, Christopher H. Contag, Catherine M. Verfaillie, and Bruce R. Blazar. "Real-Time In Vivo Biodistribution of Multipotent Adult Progenitor Cells (MAPC): Role of the Immune System in MAPC Resistance in Non-Transplanted and Bone Marrow Transplanted Mice." Blood 104, no. 11 (November 16, 2004): 507. http://dx.doi.org/10.1182/blood.v104.11.507.507.
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Abstract MAPC are non-hematopoietic stem cells derived from adult BM with the potential for a wide differentiation pattern in vitro and in vivo. MAPCs are MHC class I and thus may be a target of natural killer (NK) cell mediated elimination in the syngeneic setting. To determine whether MAPC are susceptible targets for NK mediated killing, splenocytes from poly I:C (an inducer of NK activity) treated C57BL/6 mice were mixed with Yac-1 (H2a; a NK sensitive target) or MAPC (from C57BL/6J-rosa26) in a chromium release assay. Effector:target ratios indicated that MAPC were susceptible to NK lysis albeit less so than Yac-1 cells. To assess in vivo immune responses to MAPC, we infused MAPC into mice with various degrees of T-, B-, and NK- cell immune competence. To follow biodistribution of MAPC in live animals with whole body imaging (WBI), we labeled MAPC with red fluorescent protein DsRed2 and luciferase, using Sleeping Beauty transposons. MAPC (106) were co-nucleofected (Amaxa) with 5mcg of each pT/CAGGS-DsRed2 and pT/CAGGS-Luciferase and an SB transposase-encoding plasmid (p/CMV-HSB2) at a 1:50 ratio. Selected double transgenic MAPC (MAPC DL) clones were euploid, and maintained their characteristic trilineage differentiation. MAPC DL (106) were injected IV into cohorts (n=5–6) of adult C57BL/6 (B6), Rag2−/− (T- and B-cell deficient) and B6 Rag2/IL-2Rgc (T-, B- and NK deficient mice). Additional cohorts of B6 and Rag2−/− were given anti-NK1.1 mAb 2x/wk to deplete NK cells. In B6 mice, MAPC DL were detected on d4 but not d14 or d30. In Rag2−/− mice, MAPC DL were detected throughout the 30d period. NK depletion did not substantially increase MAPC DL number in B6 mice. However, in Rag2/IL-2Rgc mice MAPC DL were persistent and in 50% of mice they increased in number from d4‡d30. Post-mortem analysis revealed MAPC DL cells in all but B6 wild type mice: Rag2/IL-2Rgc ≥ Rag2−/− with NK depletion>> Rag2−/−. These data suggest that endogenous NK cells and T cells resist MAPC DL. Interestingly, in vitro studies indicate that MAPCs suppress an allogeneic mixed lymphocyte reaction (MLR) culture. Therefore, the T cell resistance to MAPC may be due to an immune response generated to the multiple foreign reporter proteins expressed by these cells. Since MAPCs may be useful as cellular therapies for the treatment of regimen-related toxicity, studies were performed in which B10.BR mice were lethally irradiated (TBI) and given B6 BM ± MAPC DL (106). MAPC DL were seen in the chest, abdomen, face, and paws on d4, d7, d10 and d28 at high numbers suggesting that TBI conditioning overcomes both NK and T cell mediated resistance resuting in a widespread homing/migration of MAPC. These data are the first to illustrate the immune responses to MAPCs and indicate that TBI conditioning may be advantageous in the long-term survival and widespread homing of MAPCs.
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Zhou, Yiting, Guangwei Ma, Jiawen Yang, Zenghong Gao, and Yabin Guo. "The Integration Preference of Sleeping Beauty at Non-TA Site Is Related to the Transposon End Sequences." Frontiers in Genetics 12 (March 10, 2021). http://dx.doi.org/10.3389/fgene.2021.639125.
Full textAbstract:
Recently, we proved that Sleeping Beauty (SB) transposon integrates into non-TA sites at a lower frequency. Here, we performed a further study on the non-TA integration of SB and showed that (1) SB can integrate into non-TA sites in HEK293T cells as well as in mouse cell lines; (2) Both the hyperactive transposase SB100X and the traditional SB11 catalyze integrations at non-TA sites; (3) The consensus sequence of the non-TA target sites only occurs at the opposite side of the sequenced junction between the transposon end and the genomic sequences, indicating that the integrations at non-TA sites are mainly aberrant integrations; and (4) The consensus sequence of the non-TA target sites is corresponding to the transposon end sequence. The consensus sequences changed following the changes of the transposon ends. This result indicated that the interaction between the SB transposon end and genomic DNA (gDNA) may be involved in the target site selection of the SB integrations at non-TA sites.
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Ramos, Aline Lisie, Fernanda Soares Niemann, Adriana Silva Santos Duarte, Karla Priscila Ferro, Irene Santos, Carolina Louzão Bigarella, Antonio Filareto, and Sara Teresinha Olalla Saad. "Comparison of different methods to overexpress large genes." Journal of Biological Research - Bollettino della Società Italiana di Biologia Sperimentale 91, no. 2 (October 26, 2018). http://dx.doi.org/10.4081/jbr.2018.7249.
Full textAbstract:
Gain-of-function of very large transgene constructs can lead to genetic perturbations, providing researchers with the alternative of a powerful tool to identify pathway components which remain undetected when using traditional loss-of-function analysis. To promote longer-term expression, various systems for transgene integration have been developed, however large cDNA sequences are often difficult to clone into size-limited expression vectors. We attempted to overexpress ARHGAP21, a 5.874 kb gene, using different methodologies as plasmid, lentiviral and Sleeping Beauty (SB) transposon based gene transfer. Using lentiviral based transduction; an enormous amount of lentiviral supernatant was produced to obtain a satisfactory titration after double ultracentrifugation. However, U937 transduced cells showed only 50% of gene expression increase, which vanished after 5 days. SB transposon system application to overexpress ARHGAP21 was a complete success. Nucleofecting SB-based vector plus SB100x transposase vector resulted in an expressive increase of gene and protein expression. Furthermore, the overexpression was maintained even after freezing and thawing processes. In conclusion, our work shows that the SB transposon system is the best choice for those seeking a stable and high gene expression. Once the overexpression is achieved, freezing cells and using them for a long time becomes possible.
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Prommersberger, Sabrina, Michael Reiser, Julia Beckmann, Sophia Danhof, Maximilian Amberger, Patricia Quade-Lyssy, Hermann Einsele, Michael Hudecek, Halvard Bonig, and Zoltán Ivics. "CARAMBA: a first-in-human clinical trial with SLAMF7 CAR-T cells prepared by virus-free Sleeping Beauty gene transfer to treat multiple myeloma." Gene Therapy, April 13, 2021. http://dx.doi.org/10.1038/s41434-021-00254-w.
Full textAbstract:
AbstractClinical development of chimeric antigen receptor (CAR)-T-cell therapy has been enabled by advances in synthetic biology, genetic engineering, clinical-grade manufacturing, and complex logistics to distribute the drug product to treatment sites. A key ambition of the CARAMBA project is to provide clinical proof-of-concept for virus-free CAR gene transfer using advanced Sleeping Beauty (SB) transposon technology. SB transposition in CAR-T engineering is attractive due to the high rate of stable CAR gene transfer enabled by optimized hyperactive SB100X transposase and transposon combinations, encoded by mRNA and minicircle DNA, respectively, as preferred vector embodiments. This approach bears the potential to facilitate and expedite vector procurement, CAR-T manufacturing and distribution, and the promise to provide a safe, effective, and economically sustainable treatment. As an exemplary and novel target for SB-based CAR-T cells, the CARAMBA consortium has selected the SLAMF7 antigen in multiple myeloma. SLAMF7 CAR-T cells confer potent and consistent anti-myeloma activity in preclinical assays in vitro and in vivo. The CARAMBA clinical trial (Phase-I/IIA; EudraCT: 2019-001264-30) investigates the feasibility, safety, and anti-myeloma efficacy of autologous SLAMF7 CAR-T cells. CARAMBA is the first clinical trial with virus-free CAR-T cells in Europe, and the first clinical trial that uses advanced SB technology worldwide.