THAI HIEN TAI AIUS009790495B2 AI AI AI UN TATA MONITOR (12 ) United States Patent ( 10 ) Patent No. : US 9 ,790 ,495 B2 Geller et al. (45 ) Date of Patent: Oct . 17 , 2017 ( 54 ) ANTISENSE ANTIBACTERIAL COMPOUNDS 8 , 299 ,206 B2 10 /2012 Fox et al. AND METHODS 8 , 314 , 072 B2 11/ 2012 Geller et al. 8 ,536 , 147 B2 * 9 / 2013 Weller . A61K 48 / 00 514 /44 A @(71 ) Applicants :Bruce L . Geller, Corvallis , OR (US ) ; 9 , 249 ,243 B2 * 2 /2016 Weller .. .. .. A61K 48 /00 David Greenberg , Coppell , TX (US ) 2004 /0029129 A1 * 2 /2004 Wang ** * * * . .. CO7K 14 / 195 435 /6 . 18 @(72 ) Inventors : Bruce L . Geller, Corvallis , OR (US ) ; 2005 / 0288246 Al 12/ 2005 Iversen et al. David Greenberg, Coppell , TX (US ) 2006 / 0241075 A1 * 10 /2006 McSwiggen . .. .. C12N 15 / 1138 514 /44 A @ 2006 /0270621 A1* 11/ 2006 Christiano .. .. A61K 9 /0014 ( 73 ) Assignees : Oregon State University , Corvallis , 514 /44 R OR (US ) ; Board of Regents , The 2007 / 0049542 A1 3 / 2007 Geller et al. University of Texas System , Austin , 2008 / 0194463 A1 8 / 2008 Weller et al . TX (US ) 2010 / 0016215 A1 1 / 2010 Moulton et al. 2010 / 0234281 A1 9 / 2010 Weller et al. ( * ) Notice: Subject to any disclaimer, the term of this 2010 /0261777 A1 * 10 /2010 Shaw . .. .. .. .. .. C12N 15 / 102 514 /44 R patent is extended or adjusted under 35 2012 /0040460 A1 * 2 /2012 Rigoutsos . .. C12N 15 / 111 U . S . C . 154 (b ) by 0 days . 435 /375 2012 / 0122769 A1 5 / 2012 Iversen (21 ) Appl. No. : 14 /713 , 857 2012 / 0213663 A1 8 /2012 Atieh et al. 2012 /0289457 A 11/ 2012 Hanson (22 ) Filed : May 15 , 2015 2012 / 0296087 AL 11/ 2012 Sinha et al. 2013 /0197220 Al 8 /2013 Ueda (65 ) Prior Publication Data 2016 /0106857 A14 / 2016 Geller et al . US 2015 /0361425 A1 Dec . 17 , 2015 FOREIGN PATENT DOCUMENTS KR 10 - 2013 - 0005208 * 1 / 2013 Related U . S . Application Data KR 2013 /0005208 A 1 / 2013 ( 60 ) Provisional application No . 61 / 994 , 750 , filed on May WO WO 93 /01286 A2 1 / 1993 WO WO 2004 /097017 A2 11/ 2004 16 , 2014 , provisional application No . 62 /099 , 046 , WO WO 2006 / 085973 A2 8 / 2006 filed on Dec . 31 , 2014 , provisional application No . WO WO 2008 /008113 AL 1 /2008 62/ 129 ,746 , filed on Mar. 6 , 2015 . (Continued ) (51 ) Int . Cl. C12N 15 / 11 ( 2006 . 01 ) OTHER PUBLICATIONS C12N 15 / 113 ( 2010 .01 ) Youngblood et al. Bioconjugate Chem 2007 , 18 , 50 -60 . * A61K 4748 ( 2006 . 01 ) KR 10 - 2013 - 0005208 pp . 1 -29 , Machine English Translation . * A61K 38 / 00 ( 2006 .01 ) GenBank 01251363 [KR 1020130005208 - A / 39 : Method and kit for ( 52 ) U . S . CI. detecting carbapenem resistant enterobacteriaceae using real - time CPC . .. C12N 15 / 113 (2013 .01 ) ; A61K 47/ 48246 PCR ] ( retrieved on Oct. 13 , 2015 from http : // www .ncbi . nlm .nih . ( 2013 .01 ) ; A61K 38 / 00 ( 2013 .01 ) ; C12N gov / nucleotide /662699238 ? report = genbank & log $ = nuclalign & blast _ rank = 1 & RID = 1V40 3DGD016 ] Jul . 8 , 2014 ( Jul. 8 , 2014 ) 2310 / 11 (2013 .01 ) ; C12N 2310 /3233 whole doc . ( 2013 .01 ) ; C12N 2310 / 3513 ( 2013 .01 ) ; C12N PCT/ US2015 /031150 , International Search Report and Written 2320 / 50 ( 2013. 01 ) Opinion dated Jan . 14 , 2016 . PCT/ US2015 /031213 , International Search Report and Written (58 ) Field of Classification Search Opinion dated Sep . 2 , 2015 . CPC .. .. C12N 15 / 113 ; C12N 2310 / 11 ; C12N PCT/ US2015 / 000280 , International Search Report and Written 2310 /3233 Opinion dated May 2 , 2016 . See application file for complete search history . Summerton et al. 1997 , " Morpholino antisense oligomers : design , preparation , and properties .” Antisense and Nucleic Acid Drug (56 ) References Cited Development ( 1997 ) ; 7 . 3 : 187 - 195 . PCT/ US2015 /031150 , International Preliminary Report on Patent U . S . PATENT DOCUMENTS ability dated Nov . 22 , 2016 , 11 pages . PCT/ US2015 /031213 , International Preliminary Report on Patent 5 , 034 , 506 A 7 / 1991 Summerton et al. ability dated Nov. 22 , 2016 , 7 pages . 5 , 142 , 047 A 8 / 1992 Summerton et al . 5 , 166 , 315 A 11/ 1992 Summerton et al. Primary Examiner — Brian Whiteman 5 , 185 , 444 A 2 / 1993 Summerton et al. (74 ) Attorney , Agent, or Firm — Cooley LLP 5 ,217 , 866 A 6 / 1993 Summerton et al . 5 , 506 , 337 A 4 / 1996 Summerton et al. (57 ) ABSTRACT 5 , 521, 063 A 5 / 1996 Summerton et al . Provided are antisense morpholino oligomers targeted 5 ,698 ,685 A 12 / 1997 Summerton et al . against bacterial virulence factors such as genes that con 6 , 245 , 747 B1 6 / 2001 Porter et al. tribute to antibiotic resistance or biofilm formation , or genes 6 , 965, 025 B2 11/ 2005 Gaarde et al. associated with fatty acid biosynthesis , and related compo 6 , 969 , 400 B2 11/ 2005 Rhee et al. sitions and methods of using the oligomers and composi 7 ,625 , 873 B2 12 / 2009 Geller et al. 7 , 790 , 694 B2 9 / 2010 Geller et al. tions , for instance , in the treatment of an infected mamma 8 , 067 ,571 B2 11 / 2011 Weller et al. lian subject. B2 12 / 2011 Reeves et al . 38 Claims, 22 Drawing Sheets US 9 ,790 ,495 B2 Page 2 ( 56 ) References Cited FOREIGN PATENT DOCUMENTS WO WO 2009 /005793 A2 1 /2009 WO WO 2009 /064471 A1 5 / 2009 WO WO 2012 /043730 4 / 2012 WO WO 2012 / 064991 Al 5 / 2012 WO WO 2012 / 150960 AL 11 /2012 wo WO2013011072 A1 * 1 / 2013 WO WO 2015 / 175977 A2 11 / 2015 WO WO 2015 / 179249 AL 11 /2015 WO WO 2016 / 108930 A2 7 / 2016 * cited by examiner atent Oct. 17 , 2017 Sheet 1 of 22 US 9 ,790 , 495 B2 Bebe BongebotV . lv 1D FIG.1 1B Direttae baino X bambina - - - - ** 1.4 ** - . ONCH) * UOMO atent Oct. 17 , 2017 Sheet 2 of 22 US 9 ,790 , 495 B2 16 NHASXARXABXKERKYRK . WOW-3269 yhose OSTEO . 2 .O '.'., mer 0 V ** * * * Wis ASTRO O ACHNRICETTEREFRETERXB. CP050305*PPMO P00734PPMO NOVO €63.Tai FIG.1 , . ecsgo « .No Mello « ACHNAXARXARXRRXKXB. OWddS2000 atent Oct. 17 , 2017 Sheet 3 of 22 US 9 ,790 ,495 B2 PPMO#5AdeA-Aba PPMOconcentration(UM) A.baumaniiAYE FIG.2 : :: : : :: : : :: : : :: : : :: : : :: : : :: : :: : : :: : : :: : : :: : : :: : : 10240 512 )ugimi ( MIC Gentamicin atent Oct. 17 , 2017 Sheet 4 of 22 US 9 ,790 ,495 B2 _ UZA32HUEwneg WoWdd . ) mL/ lug Tobramycin MIC 333Ayy?? ???{ SHOWdd €913 # ??? * * IIHWUBAVkuuewnegv * NIOWdd( * * * )mL / g ( Tobramycin MIC LI atent Oct. 17 , 2017 Sheet 5 of 22 US 9 ,790 ,495 B2 . PPMO#5AdeA-Aba illiu...PPMOconcentration(UM) A.baumaniiAYE FIG.4 228328 * X *X6 0 XXXX8 * AX8381983 * * * 1-5.0 )ml / ug ( MIC Tetracycline atent Oct. 17 , 2017 Sheet 6 of 22 US 9 ,790 , 495 B2 DEBOT-13025H0m1andMeropenemco 32 GermanyMDM-1*M0X2 WNOW:PPM 026NDM-1BCTBbaumannii.A Iwn)Ondd FIG.SA 0.5 meningtrate minrdirbti en mann en sammenhiberdrolabilirl bridal willann e med interne concernance internet ) mL/ ug ( Meropenem MIC atent Oct. 17 , 2017 Sheet 7 of 22 US 9 ,790 , 495 B2 .mL1#/PPMOug 8Meropenem BUM +Meropenem e mL /Bug FIG.5B 1#PPMO 1-NDM SUM PPMOScr 24Time alone Ab 100 ml/ CFU 0Time alone Ab atent Oct. 17 , 2017 Sheet 8 of 22 US 9 ,790 , 495 B2 otobolobyOZ PPMO#2concentration(UM) E.coliBCT-B036(NDM1) FIG.6 er atent Oct. 17 , 2017 Sheet 9 of 22 US 9 ,790 , 495 B2 alonePeptide Scr IIIRIIEREIII . PPMo1/??? FIG.7A * PPMOsuhB * Owddddoe ?? ? PPMONo 570 OD U . S . Patent Oct. 17 , 2017 Sheet 10 of 22 US 9 , 790, 495 B2 alonePeptide PPMOScr PPMOcepl FIG.7B PPMOsuhB PPMOacpP PPMONo 570 OD i § atent Oct. 17 , 2017 Sheet 11 of 22 US 9 ,790 ,495 B2 : : : : : FIG.8A : : W U . S . Patent Oct. 17 ,2017 Sheet 12 of 22 US 9 , 790, 495 B2 ????3 ?????????????? ??????? ??? ??? ?????? * ??????? ??? ???? ?????????? ??????? ???? ??? ???? ?????? ?????????? ??? ?????? ??????? ??????? ???????? ?????? US. Patemt o c . 17 , 2017 Sheet 13 of 22 _ US 9, 790, 495 82 ????.????? ?????? ??????????????????????? ?????????? ?????????? 8'ei ? ? ?? ?????????????????????????????????????????????????????????????????????????????????????????????????????????????????? ??????? ???????? ??? ? ? ? ? ? ? ????? U . S . Patent Oct. 17 , 2017 Sheet 14 of 22 US 9 , 790, 495 B2 (w /or ezL )Usweq01 + dos 11111111111 )mL /pg 64( Tobramycin +cepl cepi EstablishedBiofilmBreakdown FIG.9 )mL /pg 128 (Tobramycin +Scr )uglmL 64 (Tobramycin +Ser Scr PPMONo wa within mimin F01 CFU U . S . Patent Oct. 17 , 2017 Sheet 15 of 22 US 9 , 790, 495 B2 NY 2840> 16UM SUM AUM 3KM12 29#PPM0RXB ;).RFFacpr ( 28#PPMO -acpp 2627#ifcopPPMO PPMO -acpp 17#PPMO -hacpp 16#PPMO ! acp 15#PPMO -acpp 1824#acppPPMO PPMO -acpp 25#PPMO -acpp FIG.10 23#PPMO -acpP 27*PPMO -opp # 20PPMO121#PPMO -acphacpP 19#PPMQ -acpp MARS970 (35) h why &rexcio(3840-2) Bruitivorans(SH-21 .wpacia(3753) B.parja(1820) B.cepaca(1640-2) B.cpada(2299) R.duloxa(AUD$45) 8,gladiosi(HI2:37) 8.cestocepscia(1513) 8.Qanocepacia(B3 B.cenocepacia(A-245) ,cenocepacia(12315) 5.capacia(1882 28363Eacia(3. %.cegada(K43128) B.dolosa(A00158) A.gk?mae(A06208) 8.multivorans($41) pymooniaAUX:34)(B. 8.stabstis(H12230; K.anybifasia(AU0212) B.ambaska(BA4-243 B.cedoooxacia(807 8,canocepacia(K12424) 8.exacePR«X(K56 tmbAULO$4)(cenocepacia8, 8.gadol(AU15937) .multivoransAUD862) 8.stokivorals(844-147) B.multivorans(#12229) pyrodnia)(AUL114B. ?,?ibrfa{Au2 &cæ000-nacia(A137 12728Quba)crocepacia(8. B,multivos2:06AU0267 8.Pyrrocinka(A025 8.stabilisAV26756 8,vietnamlensis(AU01087 s,vietnamiensis(A118839) 24Hvietsensis8kAB. he n : S ' u?rd 0 LIOTÁLI bayS 91 JO TT S1 °06LÝ6 S6 78 22#PPMO T24 191#PPM0 T24 1 Aguo(L?ZVIH )079 B 117 ministráncele intre ense numerose momencie wwdiscovery only)H14277 (BCC TO . mL/ CFU Log FIG.11 . PPM019.T24 A only)2 -K456 (B¢¢ T24 : :: : : : : : : : : : : :: :: : : : : : : : : :: : :: : : : : : : : : : : : : : : : : : : : : : : :: : : : : : : :: : : : : :: : : :: : :: : : : : : :: : :: : . 119 m bestehet LLLLLLL met matinada la con che L .1 2 Webmx only)2-K56 (Bcc TO . ml/ CFU log U . S . Patent Oct.