MUHTAUTUNELIUUS009950011B1 (12 ) United States Patent ( 10 ) Patent No. : US 9 ,950 , 011 B1 Jantz et al. (45 ) Date of Patent: * Apr. 24 , 2018 ( 54 ) GENETICALLY -MODIFIED CELLS CO7K 2319/ 40 ( 2013 .01 ) ; CO7K 2319 /74 COMPRISING A MODIFIED HUMAN T (2013 . 01) ; C12N 2510 / 00 ( 2013. 01) CELL RECEPTOR ALPHA CONSTANT ( 58 ) Field of Classification Search REGION GENE CPC .. .. .. .. A61K 35 / 17 ; C07K 14 /7051 ; C07K 14 / 70503 ; CO7K 16 / 30 ; CO7K 2317 /60 ; (71 ) Applicant: Precision BioSciences , Inc. , Durham , CO7K 2319 /33 ; CO7K 2319 /74 ; C12N NC (US ) 15 /09 ; C12N 5 / 0636 ; C12N 2510 / 00 @(72 ) Inventors : Derek Jantz , Durham , NC (US ) ; James See application file for complete search history. Jefferson Smith , Morrisville , NC (US ) ; Michael G . Nicholson , Chapel Hill, NC (US ) ; Daniel T . MacLeod , Durham , (56 ) References Cited NC (US ) ; Jeyaraj Antony , Chapel Hill , NC (US ) ; Victor Bartsevich , Durham , U . S . PATENT DOCUMENTS NC (US ) 4 ,873 , 192 A 10 / 1989 Kunkel 6 ,015 , 832 A 1 / 2000 Baker , Jr. et al. @( 73 ) Assignee : Precision BioSciences, Inc. , Durham , 6 , 506 , 803 B1 1 /2003 Baker, Jr. et al . 6 ,559 , 189 B2 5 /2003 Baker, Jr . et al . NC (US ) 6 , 635 , 676 B2 10 / 2003 Baker , Jr. et al. 8 , 021 , 867 B2 9 / 2011 Smith et al . @( * ) Notice: Subject to any disclaimer , the term of this 8 , 445 , 251 B2 5 /2013 Smith et al . patent is extended or adjusted under 35 8 , 956 , 828 B2 2 / 2015 Bonini et al . U . S . C . 154 (b ) by 0 days . 2002 /0045667 AL 4 /2002 Baker et al . 2004 /0043041 AL 3 /2004 Baker et al . This patent is subject to a terminal dis 2012 / 0321667 A112 / 2012 Sentman claimer. 2013 /0315884 A1 * 11 /2013 Galetto . .. .. .. .. .. .. C12N1 5 / 0636 424 /93 . 71 2014 / 0034902 A1 2 / 2014 Hwang et al. ( 21) Appl. No. : 15/ 865 , 075 2014 /0301990 A1 * 10 /2014 Gregory .. .. .. .. .. .. A61K 35 /26 424 / 93 .21 ( 22 ) Filed : Jan . 8 , 2018 2015 /0376650 A1 * 12/ 2015 Auerbach .. .. C12N 15 /64 435 / 34 2016 / 0081314 A1 * 3 /2016 Thurston . .. .. .. C07K 14 /7051 Related U . S . Application Data 800 / 6 (63 ) Continuation of application No. 15 /607 ,059 , filed on May 26 , 2017 , now Pat . No. 9 ,889 ,161 , which is a (Continued ) continuation of application No . PCT/ US2016 /055492 , filed on Oct . 5 , 2016 . FOREIGN PATENT DOCUMENTS WO WO 2002 /012514 A2 2 /2002 (60 ) Provisional application No . 62/ 297 , 426 , filed on Feb . WO WO 2007 /047859 A2 4 / 2007 19 , 2016 , provisional application No. 62 /237 ,394 , (Continued ) filed on Oct . 5 , 2015 . OTHER PUBLICATIONS (51 ) Int. CI. A61K 35 / 17 ( 2015 .01 ) International Search Report and Written Opinion for Application COZK 16 / 30 ( 2006 .01 ) No. PCT/ US2016 /055492 dated Feb . 3 , 2017. C12N 15 / 09 ( 2006 . 01 ) (Continued ) A61K 39 / 00 ( 2006 . 01 ) A61K 48 /00 ( 2006 .01 ) CO7K 14 /725 ( 2006 . 01 ) Primary Examiner — Kevin K Hill CO7K 14 / 705 ( 2006 .01 ) ( 74 ) Attorney , Agent, or Firm — Wolf, Greenfield & C12N 5 / 0783 ( 2010 .01 ) Sacks, P . C . C12N 15 / 113 ( 2010 .01 ) C12N 15 / 86 ( 2006 .01 ) ( 52 ) U . S . CI. ( 57 ) ABSTRACT CPC .. .. A61K 35 / 17 ( 2013 .01 ) ; A61K 39 / 0011 ( 2013 . 01 ) ; A61K 48 / 0008 ( 2013 .01 ) ; COOK Disclosed herein is a genetically -modified cell comprising in 14 /7051 (2013 .01 ) ; CO7K 14 / 70503 ( 2013 .01 ) ; its genome a modified human T cell receptor alpha constant CO7K 16 / 30 ( 2013 . 01 ) ; CO7K 16 / 3061 region gene , wherein the cell has reduced cell - surface (2013 . 01) ; C12N 5 /0636 ( 2013 .01 ) ; C12N expression of the endogenous T cell receptor. The present 15 /09 (2013 .01 ) ; C12N 15 / 113 ( 2013 .01 ) ; disclosure further relates to methods for producing such a C12N 15 /86 (2013 .01 ) ; A61K 2039 / 505 genetically -modified cell , and to methods of using such a (2013 .01 ) ; A61K 2039/ 5156 (2013 . 01 ) ; A61K cell for treating a disease in a subject. 2039 / 5158 ( 2013 .01 ) ; CO7K 2317/ 53 ( 2013 .01 ) ; CO7K 2319 / 02 (2013 .01 ) ; CO7K 2319 /03 (2013 .01 ) ; CO7K 2319 /33 (2013 . 01 ) ; 8 Claims, 62 Drawing Sheets US 9 , 950 ,011 B1 Page 2 (56 ) References Cited Deshayes et al ., “ Primary amphipathic cell -penetrating peptides : structural requirements and interactions with model membranes, ” U . S . PATENT DOCUMENTS Biochemistry 43 , pp . 7698 -7706 ( 2004 ) . Dinda et al . , “ Nanobiotechnology -based drug delivery in brain 2016 /0120906 A1 * 5 / 2016 Galetto .. .. .. C12N 5 / 0636 424 / 93 . 21 targeting, ” Curr . Pharm . Biotechnol . 14 , pp . 1264 - 1274 (2013 ) . 2017 /0016027 A11 / 2017 Lee et al . Dingermann et al. , “ Establishment of a system for conditional gene 2017 / 0333481 AL 11/ 2017 Jantz et al . expression using an inducible tRNA suppressor gene, " Mol. Cell 2017 /0335010 A111/ 2017 Jantz et al . Biol. 12 ( 9 ), pp . 4038 - 4045 ( 1992 ) . Durai et al ., “ Zinc finger nucleases: custom -designed molecular FOREIGN PATENT DOCUMENTS scissors for genome engineering of plant and mammalian cells ,” Nucleic Acids Res. 33 , pp . 5978 -5990 (2005 ) . WO WO 2009 /059195 A2 5 /2009 Ellebrecht et al. , “ Reengineering chimeric antigen receptor T cells WO WO 2012 / 167192 A2 12 / 2012 for targeted therapy of autoimmune disease , ” Science . Jul. 8 , WO WO 2013 /074916 A1 5 / 2013 2016 ; 353 (6295 ) : 179 - 84 . doi: 10 . 1126 / science . aaf6756 . Epub Jun . WO WO 2013 / 153391 A1 10 / 2013 WO WO 14 / 191527 * 12 /2014 30 , 2016 . Author manuscript. WO WO 2014 / 191525 Al 12 / 2014 Fu et al ., “ High - frequency off - target mutagenesis induced by WO WO 2014 / 191527 AL 12 / 2014 CRISPR - Cas nucleases in human cells ,” Nat. Biotechnol. 31, pp . WO WO 2015 /057980 A1 4 / 2015 822 - 826 ( 2013 ) . WO WO 2015 / 121454 Al 8 / 2015 Gao et al. , “ Efficient gene delivery into mammalian cells mediated WO WO 2015 / 167766 AL 11/ 2015 by a recombinant baculovirus containing a whispovirus iel pro WO WO 2017 /011519 AL 1 / 2017 moter , a novel shuttle promoter between insect cells and mammalian cells, ” J . Biotechnol . 131 ( 2 ), pp . 138 - 143 (2007 ) . OTHER PUBLICATIONS Gish et al ., “ Identification of protein coding regions by database similarity search , ” Nature Genet . 3 , pp . 266 - 272 ( 1993 ) . Airenne et al ., “ Baculovirus: an insect - derived vector for diverse Grizot et al. , “ Efficient targeting of a SCID gene by an engineered gene transfer applications, ” Mol. Ther. 21 ( 4 ) , 739 - 749 ( 2013 ) . single - chain homing endonuclease, " Nucleic Acids Res. 37 , pp . 5405 - 5419 (2009 ) . Altschul et al. , “ Basic local alignment search tool , " J . Mol. Biol. Haase , et al. , “ Generation of a tumor - and tissue -specific episomal 215 , pp . 403 -410 ( 1990 ) . non -viral vector system , ” BMC Biotechnol. 13 , pp . 49 -54 ( 2013 ) . Altschul et al. , “ Gapped BLAST and PSI- BLAST : a new generation Hale et al. , “ Homology - Directed Recombination for Enhanced of protein database search programs, " Nucleic Acids Res . 25 , pp . Engineering of Chimeric Antigen Receptor T Cells ,” Molecular 3389 - 3402 ( 1997 ) . Therapy 4 , pp . 192 - 203 (2017 ) . Arnould et al ., “ Engineering of large numbers of highly specific Hegde et al. , " Current status of chimeric antigen receptor engi homing endonucleases that induce recombination on novel DNA neered T cell -based and immune checkpoint blockade - based cancer targets ,” J . Mol. Biol. 355 , pp . 443 - 458 ( 2006 ) . immunotherapies, ” Cancer Immunol Immunother 66 , pp . 1113 - 1121 Baeckseung et al ., 323 . Efficient Generation of CART Cells by ( 2017 ) . Homology Directed Transgene Integration into the TCR - Alpha Hudecz et al. , “Medium - sized peptides as built in carriers for Locus. Mol Ther. May 1 , 2016 ; 24 ( S1) : S130 . biologically active compounds, ” Med . Res . Rev . 25 , pp . 679 - 736 Baxter et al ., “ Engineering domain fusion chimeras from 1 - Onul ( 2005 ) . family LAGLIDADG homing endonucleases ,” Nucleic Acids Ibarra et al. , “ Efficient Targeted Gene Modification in Primary Research , 40 ( 16 ) , pp . 7985 - 8000 ( 2012 ) . Human Hematopoietic Cells Using Co - Delivery of Nuclease Benoist et al ., “ In vivo sequence requirements of the SV40 early mRNA and AAV Donors , ” Mol. Ther. , 23 ( suppl. 1 ) , p . S273 ( 2015 ) . promotor region , ” Nature 290 (5804 ), pp . 304 -310 ( 1981) . Jacox et al ., “ Tissue - specific and ubiquitous expression patterns Beurdeley et al ., “ Compact designer TALENs for efficient genome from alternative promoters of human genes , ” PLoS One 5 ( 8 ) , p . engineering , ” Nat . Commun . 4 , p . 1762 ( 2013 ) . e12274 ( 2010 ) . Boissel et al ., “ megaTALs: a rare -cleaving nuclease architecture for Jearawiriyapaisam et al. , “ Sustained dystrophin expression induced therapeutic genome engineering, " Nucleic Acid Research 42 ( 4 ) , pp . by peptide - conjugated morpholino oligomers in the muscles of mdx 2591 - 2601 ( 2014 ) . mice , " Mol. Ther. 16 , pp . 1624 - 1629 ( 2008 ) . Cahill et al . , “Mechanisms of eukaryotic DNA double strand break Jiang et al ., “ Cationic core - shell liponanoparticles for ocular gene repair ,” Front . Biosci. 11, pp . 1958 - 1976 (2006 ) . delivery ,” Biomaterials . 33 ( 30 ) , pp . 7621 - 7630 ( 2012 ) . Chames et al ., “ In vivo selection of engineered homing Kang et al ., “ Harnessing the capacity of cell -penetrating peptides endonucleases using double - strand break induced homologous for drug delivery to the central nervous system , ” Curr. Pharm . recombination , ” Nucleic Acids Res. 33 , p . e178 (2005 ) . Biotechnol . 15 ( 3 ) , pp . 220 - 230 ( 2014 ) . Chang et al. , “ Inducible retroviral