title: Sleeping beauty system to redirect T-cell specificity for human applications.
abstract: The Sleeping Beauty (SB) transposon/transposase DNA plasmid system is used to genetically modify cells for long-term transgene expression. We adapted the SB system for human application and generated T cells expressing a chimeric antigen receptor (CAR) specific for CD19. Electrotransfer of CD19-specific SB DNA plasmids in peripheral blood mononuclear cells and propagation on CD19 artificial antigen presenting cells was used to numerically expand CD3 T cells expressing CAR. By day 28 of coculture, >90% of expanded CD3 T cells expressed CAR. CAR T cells specifically killed CD19 target cells and consisted of subsets expressing biomarkers consistent with central memory, effector memory, and effector phenotypes. CAR T cells contracted numerically in the absence of the CD19 antigen, did not express SB11 transposase, and maintained a polyclonal TCR Vα and TCR Vβ repertoire. Quantitative fluorescence in situ hybridization revealed that CAR T cells preserved the telomere length. Quantitative polymerase chain reaction and fluorescence in situ hybridization showed CAR transposon integrated on average once per T-cell genome. CAR T cells in peripheral blood can be detected by quantitative polymerase chain reaction at a sensitivity of 0.01%. These findings lay the groundwork as the basis of our first-in-human clinical trials of the nonviral SB system for the investigational treatment of CD19 B-cell malignancies (currently under 3 INDs: 14193, 14577, and 14739).
authors: Division of Pediatrics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
authors: Sourindra N
authors: SN
authors: Maiti
authors: Helen
authors: H
authors: Huls
authors: Harjeet
authors: H
authors: Singh
authors: Margaret
authors: M
authors: Dawson
authors: Matthew
authors: M
authors: Figliola
authors: Simon
authors: S
authors: Olivares
authors: Pullavathi
authors: P
authors: Rao
authors: Yi Jue
authors: YJ
authors: Zhao
authors: Asha
authors: A
authors: Multani
authors: Ge
authors: G
authors: Yang
authors: Ling
authors: L
authors: Zhang
authors: Denise
authors: D
authors: Crossland
authors: Sonny
authors: S
authors: Ang
authors: Hiroki
authors: H
authors: Torikai
authors: Brian
authors: B
authors: Rabinovich
authors: Dean A
authors: DA
authors: Lee
authors: Partow
authors: P
authors: Kebriaei
authors: Perry
authors: P
authors: Hackett
authors: Richard E
authors: RE
authors: Champlin
authors: Laurence J N
authors: LJ
authors: Cooper
mesh_terms: D000818:Animals; D018941:Antigens, CD19 / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D017252:CD3 Complex / Q000378:metabolism; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D018274:Electroporation; D018014:Gene Transfer Techniques; D005909:Glioblastoma / Q000276:immunology / Q000628:therapy; D006801:Humans; D016219:Immunotherapy, Adoptive; D051379:Mice; D011948:Receptors, Antigen, T-Cell / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013601:T-Lymphocytes / Q000637:transplantation*; D019895:Transposases / Q000235:genetics*
chemical_list: D018941:Antigens, CD19; D017252:CD3 Complex; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; D019895:Transposases; C494471:sleeping beauty transposase, human
references: Cancer Res. 2010 May 15;70(10):3915-24
references: 20424114
references: Gene Ther. 2011 Sep;18(9):849-56
references: 21451576
references: J Immunother. 2007 Jan;30(1):123-9
references: 17198091
references: J Immunol. 2005 Nov 15;175(10):7046-52
references: 16272366
references: Neoplasia. 2000 Jul-Aug;2(4):339-45
references: 11005568
references: Curr Opin Immunol. 2010 Apr;22(2):251-7
references: 20171074
references: J Clin Invest. 2008 Jan;118(1):294-305
references: 18060041
references: Cancer Res. 2008 Apr 15;68(8):2961-71
references: 18413766
references: Tissue Antigens. 2009 Oct;74(4):277-89
references: 19775368
references: Blood. 2006 Apr 1;107(7):2643-52
references: 16352804
references: Methods. 2001 Dec;25(4):402-8
references: 11846609
references: Nat Genet. 2009 Jun;41(6):753-61
references: 19412179
references: Hum Gene Ther. 2010 Nov;21(11):1577-90
references: 20528476
references: Hum Gene Ther. 2012 May;23(5):444-50
references: 22107246
references: N Engl J Med. 2011 Aug 25;365(8):725-33
references: 21830940
references: Blood. 2001 Feb 1;97(3):700-7
references: 11157487
references: J Immunother. 2005 May-Jun;28(3):258-67
references: 15838383
references: Nat Med. 2008 Nov;14(11):1264-70
references: 18978797
references: Sci Transl Med. 2011 Aug 10;3(95):95ra73
references: 21832238
references: Gene Ther. 2009 Aug;16(8):1042-9
references: 19494842
references: Blood. 2011 Nov 3;118(18):4817-28
references: 21849486
references: J Leukoc Biol. 2010 May;87(5):791-803
references: 20179152
references: Mol Ther. 2008 Sep;16(9):1515-6
references: 18725873
references: Blood. 2008 Sep 15;112(6):2261-71
references: 18509084
references: Mob DNA. 2011 Mar 03;2(1):5
references: 21371313
references: Sci Rep. 2012;2:249
references: 22355761
references: Nat Rev Immunol. 2009 Oct;9(10):704-16
references: 19859065
references: J Immunother. 2008 Oct;31(8):742-51
references: 18779745
references: Trends Biotechnol. 2011 Nov;29(11):550-7
references: 21663987
references: Mol Ther. 2005 Nov;12(5):933-41
references: 15979412
references: Blood. 2007 Sep 1;110(5):1439-47
references: 17468339
references: Stem Cells. 2007 Nov;25(11):2919-27
references: 17673526
references: Cancer Res. 2011 May 15;71(10):3516-27
references: 21558388
references: Proc Natl Acad Sci U S A. 2009 Oct 13;106(41):17469-74
references: 19805141
references: Blood. 2010 Nov 18;116(20):4099-102
references: 20668228
references: Nat Protoc. 2007;2(12):3153-65
references: 18079715
references: Blood. 2010 Aug 19;116(7):1035-44
references: 20439624
references: Hum Gene Ther. 2010 Apr;21(4):427-37
references: 19905893
references: Clin Cancer Res. 2012 Sep 1;18(17):4733-42
references: 22761473
references: Cancer J. 2010 Jul-Aug;16(4):336-41
references: 20693844
references: Methods Mol Biol. 2008;434:267-76
references: 18470650