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United States Patent ( 10 ) Patent No.: US 10,653,712 B2 Hoge Et Al

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United States Patent ( 10 ) Patent No.: US 10,653,712 B2 Hoge Et Al US010653712B2 United States Patent ( 10 ) Patent No.: US 10,653,712 B2 Hoge et al. (45 ) Date of Patent : May 19 , 2020 (54 ) HIGH PURITY RNA COMPOSITIONS AND 6,528,262 B1 3/2003 Gilad et al. METHODS FOR PREPARATION THEREOF 8,383,340 B2 2/2013 Ketterer et al. 8,710,200 B2 4/2014 Schrum et al . 8,754,062 B2 6/2014 De Fougerolles et al. (71 ) Applicant: Moderna TX , Inc., Cambridge, MA 8,822,663 B2 9/2014 Schrum et al. (US ) 8,999,380 B2 4/2015 Bancel et al. 9,221,891 B2 12/2015 Bancel et al . (72 ) Inventors : Stephen Hoge , Brookline , MA (US ) ; 9,303,079 B2 4/2016 Bancel et al. 9,464,124 B2 10/2016 Bancel et al. William Issa , Dedham , MA (US ) ; 9,512,456 B2 12/2016 Wang et al. Edward J. Miracco , Arlington , MA 9,597,380 B2 3/2017 Chakraborty et al . (US ) ; Jennifer Nelson , Brookline, MA 9,868,691 B2 1/2018 Benenato et al. (US ) ; Amy E. Rabideau , Waltham , MA 9,872,900 B2 1/2018 Ciaramella et al. 10,064,934 B2 9/2018 Ciaramella et al . (US ) ; Gabor Butora , Martinsville , NJ 10,064,935 B2 9/2018 Ciaramella et al . (US ) 10,124,055 B2 11/2018 Ciaramella et al . 10,207,010 B2 2/2019 Besin et al. ( 73 ) Assignee : ModernaTX , Inc., Cambridge , MA 10,273,269 B2 4/2019 Ciaramella (US ) 2001/0001066 Al 5/2001 Cezayirli et al . 2001/0005506 A1 6/2001 Cezayirli et al . Subject to any disclaimer , the term of this 2006/0247195 A1 11/2006 Ray ( * ) Notice : 2007/0281336 A1 12/2007 Jendrisak et al . patent is extended or adjusted under 35 2008/0220471 A1 9/2008 Davis et al . U.S.C. 154 ( b ) by 0 days . 2010/0028943 Al 2/2010 Thomas et al . 2010/0048883 A1 2/2010 Ketterer et al. ( 21) Appl. No .: 16 /001,786 2010/0291156 Al 11/2010 Barner et al . 2012/0301955 A1 11/2012 Thomas et al. 2013/0102034 Al 4/2013 Schrum et al. ( 22 ) Filed : Jun . 6 , 2018 2013/0259923 A1 10/2013 Bancel et al . 2013/0266640 A1 10/2013 De Fougerolles et al . (65 ) Prior Publication Data 2014/0142290 A1 5/2014 Madden et al. US 2018/0363019 A1 Dec. 20 , 2018 (Continued ) Related U.S. Application Data FOREIGN PATENT DOCUMENTS (63 ) Continuation of application No. EP 2092064 B1 9/2010 PCT/ US2017 /051674 , filed on Sep. 14 , 2017 . WO WO 2005/118857 A2 12/2005 ( Continued ) (60 ) Provisional application No. 62/ 394,711 , filed on Sep. 14 , 2016 . OTHER PUBLICATIONS Edelmann , et al. (2014 ) Methods, v.65 ( 3 ) :333-41 . ( Year: 2014 ) .* (51 ) Int. CI. Gruegelsiepe, et al . Handbook of RNA Biochemistry , ed . R.K. C12P 19/34 ( 2006.01 ) Hartmann , Part I.1 , pp . 3-21 , 2005. ( Year: 2005) . * A61K 31/7105 ( 2006.01 ) Nedialkov , et al. ( 2003 ) NTP -driven Translocation by Human RNA COZK 14/505 ( 2006.01 ) Polymerase II. The Journal of Biological Chemistry , vol . 278( 20 ) : 18303 A61P 33/00 (2006.01 ) 18312. ( Year : 2003 ). * A61P 31/12 ( 2006.01) U.S. Appl . No. 15 / 089,050 , filed Apr. 1 , 2016 , Ciaramella et al . A61P 31/04 U.S. Appl . No. 15 /674,569 , filed Aug. 11 , 2017 , Ciaramella et al . ( 2006.01 ) U.S. Appl . No. 15 /674,585 , filed Aug. 11, 2017, Ciaramella et al . A61P 35/00 ( 2006.01 ) U.S. Appl. No. 15/ 674,591 , filed Aug. 11 , 2017, Ciaramella et al . C12N 15/10 ( 2006.01 ) U.S. Appl . No. 15 / 266,791 , filed Sep. 15 , 2016 , Bancel et al . GOIN 33/68 (2006.01 ) U.S. Appl. No. 15 / 905,576 , filed Feb. 26 , 2018 , Bancel et al. ( 52 ) U.S. Cl. U.S. Appl. No. 15 /674,872 , filed Aug. 11 , 2017 , Besin et al. ??? A61K 31/7105 ( 2013.01 ) ; A61P 31/04 (Continued ) (2018.01 ) ; A61P 31/12 ( 2018.01) ; A61P 33/00 ( 2018.01) ; A61P 35/00 (2018.01 ) ; CO7K Primary Examiner Jennifer Pitrak McDonald 14/505 ( 2013.01 ) ; C12N 15/10 ( 2013.01 ) ; ( 74 ) Attorney , Agent, or Firm Wolf , Greenfield & C12P 19/34 ( 2013.01 ) ; GOIN 3376851 Sacks, P.C. (2013.01 ) (57 ) ABSTRACT The invention relates to improved RNA compositions for ( 58 ) Field of Classification Search use in therapeutic applications . The RNA compositions are None particularly suited for use in human therapeutic application See application file for complete search history. ( e.g., in RNA therapeutics ) . The RNA compositions are made by improved processes, in particular , improved in (56 ) References Cited vitro - transcription ( IVT) processes. The invention also relates to methods for producing and purifying RNA ( e.g , U.S. PATENT DOCUM TS therapeutic RNAs ), as well as methods for using the RNA 5,756,264 A 5/1998 Schwartz et al . compositions and therapeutic applications thereof. 6,022,715 A 2/2000 Merenkova et al . 6,096,503 A 8/2000 Sutcliffe et al . 18 Claims, 72 Drawing Sheets 6,100,024 A 8/2000 Hudson et al . Specification includes a Sequence Listing. US 10,653,712 B2 Page 2 ( 56 ) References Cited WO WO 2018/170256 Al 9/2018 WO WO 2018/170260 A1 9/2018 U.S. PATENT DOCUMENTS WO WO 2018/170270 A1 9/2018 WO WO 2018/170347 A1 9/2018 2014/0147432 A1 5/2014 Bancel et al . WO WO 2018/175783 A1 9/2018 2014/0148502 A1 5/2014 Bancel et al . WO WO 2018/187590 A2 10/2018 2014/0193482 A1 7/2014 Bancel et al . WO WO 2018/200737 A1 11/2018 2014/0206752 Al 7/2014 Afeyan et al . WO WO 2018/232355 Al 12/2018 2014/0378538 Al 12/2014 Bancel WO WO 2018/232357 A1 12/2018 2015/0141499 A1 5/2015 Bancel et al . WO WO 2019/036670 A1 2/2019 2015/0307542 Al 10/2015 Roy et al . WO WO 2019/036682 A1 2/2019 2015/0315541 A1 11/2015 Bancel et al . WO WO 2019/036683 A1 2/2019 2016/0024141 A1 1/2016 Issa et al. WO WO 2019/036685 Al 2/2019 2016/0032273 A1 2/2016 Shahrokh et al. WO WO 2019/103993 Al 5/2019 2016/0038612 A1 2/2016 Hoge et al. 2016/0317647 A1 11/2016 Ciaramella et al. 2017/0202979 Al 7/2017 Chakraborty et al . OTHER PUBLICATIONS 2017/0340724 A1 11/2017 Ciaramella et al . 2017/0340725 A1 11/2017 Ciaramella et al . U.S. Appl. No. 15 /674,599 , filed Aug. 11 , 2017, Ciaramella et al . 2018/0000953 A1 1/2018 Almarsson et al. PCT/ US2017 /051674 , dated Dec. 14 , 2017 , International Search 2018/0002393 A1 1/2018 Bancel et al. Report and Written Opinion . 2018/0028645 Al 2/2018 Ciaramella et al. [No Author Listed ], MEGAscript Kit Product Manual, Oct. 27 , 2018/0028664 Al 2/2018 Besin et al. 2009. Ambion/ Invitrogen website : http://lools.invitrogen.com/contenl/ 2018/0237849 Al 8/2018 Thompson 2018/0243225 A1 8/2018 Ciaramella sfs/ manuals / cms_072987.pdf, ( last accessed Mar. 17, 2013 ) . 2018/0243230 A1 8/2018 Smith Andrews - Pfannkoch, C. et al. , Hydroxyapatite -mediated separation 2018/0271970 A1 9/2018 Ciaramella et al . of double - stranded DNA , single - stranded DNA , and RNA genomes 2018/0273977 Al 9/2018 Mousavi et al . from natural viral assemblages . Appl Environ Microbiol . Aug. 2018/0274009 A1 9/2018 Marquardt et al. 2010 ; 76 ( 15 ) :5039-45 . Epub Jun . 11, 2010 . 2018/0280496 Al 10/2018 Ciaramella et al . Devoldere et al. , Evading innate immunity in nonviral mRNA 2018/0289792 Al 10/2018 Ciaramella et al . 2018/0303929 Al 10/2018 Ciaramella et al . delivery : don't shoot the messenger. Drug Discov Today . Jan. 2018/0311336 A1 11/2018 Ciaramella et al. 2016 ;21 ( 1 ) :11-25 . doi: 10.1016 /j.drudis.2015.07.009 . Epub Jul. 23 , 2018/0312549 A1 11/2018 Ciaramella 2015 . 2018/0318409 A1 11/2018 Valiante et al. Easton et al. , Rapid , nondenaturing RNA purification using weak 2018/0363019 Al 12/2018 Hoge anion - exchange fast performance liquid chromatography . RNA . 2019/0008938 A1 1/2019 Ciaramella et al. Mar. 2010 ; 16 ( 3 ): 647-53 . Epub Jan. 25, 2010 . 2019/0099481 A1 4/2019 Ciaramella et al . Georgopoulos et al. , Use of high - performance liquid chromato 2019/0192646 Al 6/2019 Cohen et al . graphic fractionation of large RNA molecules in the assay of group I intron ribozyme activity . J Chromatogr A. Jan. 28, 2000 ;868 ( 1 ) : 109 FOREIGN PATENT DOCUMENTS 14 . Kariko et al. , Generating the optimal mRNA for therapy : HPLC WO WO 2008/077592 Al 7/2008 purification eliminates immune activation and improves translation WO WO 2011/071931 A2 6/2011 of nucleoside- modified , protein - encoding mRNA . Nucleic Acids WO WO 2013/102203 Al 7/2013 Res. Nov. 2011 ;39 ( 21 ) :e142 . WO WO 2014/140211 Al 9/2014 Kauffman et al ., Optimization of Lipid Nanoparticle Formulations WO WO 2014/152966 Al 9/2014 WO WO 2014/160243 A1 10/2014 for mRNA Delivery in Vivo with Fractional Factorial and Definitive WO WO - 2015188933 A1 * 12/2015 C12Q 1/6844 Screening Designs. Nano Lett . Nov. 11 , 2015 ; 15 ( 11 ): 7300-6 . doi: WO WO 2016/164762 A1 10/2016 10.1021/ acs.nanolett.5b02497 . Epub Oct. 20 , 2015. WO WO 2016/201377 A1 12/2016 Koch , G., et al ., Quantitative Studies on the Infectivity of ribo WO WO 2017/015457 Al 1/2017 nucleic acid from partially purified and highly purified poliovirus WO WO 2017/015463 A1 1/2017 preparations. Virology . Mar. 1960 ; 10 ( 3 ) : 329-343 . WO WO 2017/019935 Al 2/2017 Lewandowski, L.J.
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