CHA KUIN TAITEIT US009814777B2VAN DE LA MAN UNITA MAN UNITED (12 ) United States Patent ( 10) Patent No. : US 9 ,814 , 777 B2 Manoharan et al. (45 ) Date of Patent: Nov . 14 , 2017

(54 ) TARGETING (58 ) Field of Classification Search CPC . . A61K 31 /7088 ; A61K 31 /713 ; A61K 47 / 16 ; @( 71 ) Applicant: Arbutus Biopharma Corporation , A61K 47 / 22 ; A61K 47 / 28 ; A61K Burnaby, BC (CA ) 47/ 48092 ; A61K 47 / 48215 ; CO7H 21/ 02 USPC ...... 514 /44 , 777 , 784 ; 536 / 17. 9 , 5 ; 544 /258 ; @(72 ) Inventors : Muthiah Manoharan , Cambridge , MA 552 /502 ; 560/ 159 ( US) ; Kallanthottathil G . Rajeev , See application file for complete search history. Cambridge , MA (US ) ; Jayaprakash K . Nair , Cambridge , MA (US ) ; Muthusamy Jayaraman , Cambridge, (56 ) References Cited MA (US ) U .S . PATENT DOCUMENTS (73 ) Assignee : Arbutus Biopharma Corporation , 5 , 994 ,517 A 11 / 1999 Ts ' o et al. Burnaby, British Columbia ( CA ) 6 , 906 , 182 B2 6 / 2005 Ts ' o et al . 7 , 109 , 165 B2 9 / 2006 Matulic - Adamic et al . 7 , 491 , 805 B2 . 2 /2009 Vargeese et al. ( * ) Notice: Subject to any disclaimer, the term of this 7 , 803 , 397 B2 * 9 / 2010 Heyes et al...... 424 /450 patent is extended or adjusted under 35 2002 / 0086356 A1 7 / 2002 Tuschl et al. U .S .C . 154 (b ) by 0 days . 2003 /0148928 A1 8 / 2003 Beigelman et al. 2004 /0110296 Al 6 /2004 Vargeese et al . 2004 / 0259247 Al 12 / 2004 Tuschl et al . (21 ) Appl. No. : 14 / 060, 353 2006 /0148740 A17 / 2006 Platenburg 2009 /0325297 A1 12 / 2009 Tian et al . (22 ) Filed : Oct. 22, 2013 2012 /0101148 AI 4 / 2012 Aking et al. (65 ) Prior Publication Data FOREIGN PATENT DOCUMENTS US 2014 /0179761 A1 Jun . 26 , 2014 EP 0957107 11 / 1999 US 2016 /0375137 A9 Dec . 29 , 2016 WO WO 90 / 12096 10 / 1990 wo 1997043296 Al 11 / 1997 WO WO 99 /52932 10 / 1999 Related U .S . Application Data WO WO 99 /65925 12 / 1999 WO WO 00 / 44914 8 / 2000 (63 ) Continuation of application No . 12 /328 ,669 , filed on WO WO 02 /085908 10 / 2002 WO WO 02 /094185 11 /2002 Dec. 4 , 2008, now abandoned . WO WO 2004/ 024757 3 / 2004 WO WO 2004 /080406 9 /2004 (60 ) Provisional application No. 60 /992 ,309 , filed on Dec . WO WO 2004 /090108 10 / 2004 4 , 2007 , provisional application No . 61 /013 , 597 , filed (Continued ) on Dec . 13 , 2007 , provisional application No . 61/ 127 ,751 , filed on May 14 , 2008 , provisional OTHER PUBLICATIONS application No. 61/ 091, 093 , filed on Aug . 22 , 2008 , provisional application No . 61/ 097 , 261, filed on Sep . Nunez et al. Journal of Carbohydrate Chemistry, vol. 22 , No. 6 , pp . 16 , 2008 . 395 - 406 , 2003. * (Continued ) (51 ) Int. Cl. A61K 31/ 7088 ( 2006 . 01 ) A61K 31/ 713 ( 2006 .01 ) Primary Examiner — Janet Epps- Smith A61K 47/ 16 ( 2006 .01 ) (74 ) Attorney, Agent, or Firm — Viksnins Harris Padys A61K 47 / 22 ( 2006 .01 ) Malen LLP A61K 47 /28 ( 2006 .01 ) A61K 31 /7004 ( 2006 . 01 ) (57 ) ABSTRACT A61K 31 / 7052 ( 2006 .01 ) A61K 31 /70 ( 2006 . 01 ) The present invention provides targeting lipids of structure A61K 4748 ( 2006 .01 ) CO7H 21/ 02 ( 2006 .01 ) L 100 - linker- L 101 (CI ) , A61K 48 /00 ( 2006 .01 ) where L 100 is a , lipophile , alkyl , alkenyl or alkynyl , (52 ) U . S . CI. L101 is a ligand or CH , CH , (OCH , CH , ) , O (CH , ) , CH , ??? ...... A61K 47 /28 (2013 .01 ) ; A61K 31/ 70 ligand , p is 1 - 1000 , and q is 1 - 20 . In addition , the invention ( 2013 .01 ) ; A61K 31 / 7004 ( 2013 .01 ) ; A61K provides compositions and methods for the delivery of 31/ 7052 (2013 .01 ) ; A61K 31 /7088 (2013 .01 ) ; therapeutic agents to cells . In particular , these include novel A61K 31/ 713 ( 2013 . 01 ) ; A61K 47/ 16 lipids and nucleic - lipid particles that provide efficient (2013 . 01 ) ; A61K 47 / 22 ( 2013 .01 ) ; A61K encapsulation of nucleic and efficient delivery of the 47 / 48046 (2013 .01 ) ; A61K 47/ 48092 encapsulated nucleic acid to cells in vivo . ( 2013 .01 ) ; A61K 47 /48215 ( 2013 .01 ) ; C07H 21/ 02 (2013 .01 ) ; A61K 48/ 00 (2013 .01 ) ; YO2P 20 /55 (2015 . 11) 11 Claims, 20 Drawing Sheets US 9, 814 Page , 7772 B2

References Cited Krapcho et al. , “ Mono -Protected Diamines . N -tert - Butoxycarbonyl ( 56 ) a ,f3 - Alkanediamines from - O - Alkanediamines ” , Synthetic Commu nications, 2559 -2564 ( 1990 ). FOREIGN PATENT DOCUMENTS Li et al ., “ Folate- Mediated Targeting of Antisense WO WO 2004 /094595 11 / 2004 Oligodeoxynucleotides to Ovarian Cancer Cells ” , Pharmaceutical WO WO 2006 / 020768 2 / 2006 Research , vol . 15 ( 10 ), 1540 -1545 ( 1998 ). WO WO 2006 /078278 7 / 2006 Liu et al. , “ to Chemoresistant Cells : Folic Acid Derivatization of FDUMP [ 10 ] Enhances Cytotoxicity Toward 5 - FU -Resistant Human Colorector Tumor Cells ” , Journal of OTHER PUBLICATIONS Organic Chemistry , vol. 66 ( 17 ) , 5655 -5663 (2001 ) . Rensen et al. J . Med . Chem . 2004 , 47 , 5798 -5808 . * Mahato , R . I. et al. , “ Physicochemical and Disposition Characteris Sliedregt et al . Journal of Medicinal Chemistry , vol. 42, No. 4 , Feb . tics of Antisense Oligonucleotides Complexed with Glycosylated 25, 1999 , pp . 609 -618 . * Poly ( L - lysine ) ” , Biochemical Pharmacology , vol. 53 , Pergamon , Zimmermann et al. Nature Publishing Group , vol. 441, No . 7089 , Oxford , GB , vol. 53, 887 - 895, XP000197861, ISSN : 0006 - 2952 pp . 111 - 114 . * (Jan . 1997 ). Akinc et al. , “ Targeted Delivery of RNAi Therapeutics with Endog Mahato et al. , “ Modulation of Gene Expression by Antisense and enous and Exogenous Ligand - Based Mechanisms” , Molecular Antigene Oligodeoxynucleotides and Small Interfering RNA ” , Therapy vol. 18 ( 7 ) , 1357 - 1364 ( 2010 ) . Expert Opinion on Drug Delivery , vol. 2 (1 ) , 3 -28 (2005 ) . Biessen , et al ., “ The Cholesterol Derivative of a Triantennary Maier et al . , “ Synthesis of Antisense Oligonucleotides Conjugated Galactoside with High Affinity for Hepatic Asialoglycoprotein to a Multivalent Carbohydrate Cluster for Cellular Targeting” , Bioconjugate Chemistry , vol. 14 , 18 -29 , XP002510288 (2003 ) . Receptor: A Potent Cholesterol Lowering Agent” , Journal of Murata et al. , “ Design of Quaternary Chitosan Conjugate Having Medicinal Chemistry , vol . 38 ( 11) , 1846 - 1852 ( 1995 ) . Antennary Galactose Residues as a Gene Delivery Tool” , Carbo Biessen et al ., “ Synthesis of Cluster Galactosides with High Affinity hydrate Polymers , vol . 32 ( 2 ) , 105 - 109 ( 1997 ) . for the Hepatic Asialoglycoprotein Receptor” , Journal of Medicinal Nunez et al. , Journal of Carbohydrate Chemistry, vol. 22 , No. 6 , Chemistry, vol . 38 ( 9 ), 1538 -1546 . 395 - 406 (2003 ) . Chiu et al. , “ RNAi in Human Cells : Basic Structural and Functional Rensen et al ., “ Design and Synthesis of Novel Features of Small Interfering RNA ” , Molecular Cell , vol. 10 , N -Acetylgalactosamine - Terminated Glycolipids for Targeting of 549 - 561 ( 2002 ). Lipoproteins to the Hepatic Asialoglycoprotein Receptor” , Journal Choi et al. , “ Targeting Cancer Cells with DNA - Assembled of Medicinal Chemistry, vol . 47 (23 ), 5798 -5808 (2004 ) . Dendrimers: A Mix and Match Strategy for Cancer” , Cell Cycle , Sioud , “ On the Delivery of Small Interfering RNAs into Mamma vol. 4 ( 5 ) , 669 -671 ( 2005 ) . lian Cells ” , Expert Opinion on Drug Delivery , vol. 2 (4 ), 639 -651 Connolly et al. , “ Binding and Endocytosis of Cluster Glycosides by ( 2005 ) . Rabbit Hepatocytes ” , The Journal of Biological Chemistry , vol . 257 Six et al . , “ An Efficient and Stereoselective Synthesis of 1 , 2 - 0 ( 2 ) , 939 -645 ( 1982 ) . Dialkyl 3 0 Beta D Glycosyl- SN -Glycerols ” , Tetrahedron Letters , Crossman et al. , “ Synthesis of Some Second -Generation Substrate vol. 24 ( 12 ) , 1229 - 1232 ( 1983 ). Analogues of Early Intermediates in the Biosynthetic Pathway of Six et al ., “ Influence of Carbohydrate Moities on Prop Glycosylphosphatidylinositol Membrane Anchors ” , Carbohydrate erties of Dialkylglyceryletherglycosides , Simple Model Com Research , vol . 321 ( 1 - 2 ) , 2 -51 ( 1999 ) . pounds of the Glycolipids of Halophilic Bacteria ” , Journal of Dubber et al. , “ Solid - Phase Synthesis of Multivalent and Interface Science , vol. 93 ( 1 ) , 109 - 114 . Glycoconjugates on a DNA Synthesizer” , Bioconjugate Chemistry , Sliedregt et al. , “ Design and Synthesis of Novel Amphiphilic vol. 14 ( 1 ) , 239 - 246 ( 2003 ) . Dendritic Galactosides for Selective Targeting of to the Guo et al. , “ Construction of Folate -Conjugated PRNA of Hepatic Asialoglycoprotein Receptor” , Journal of Medicinal Chem Bateriophase phi29 DNA Packaging Motor for Delivery of Chime istry , vol. 42 , No . 4 , 609 -618 ( 1999 ) . ric siRNA to Nasopharyngeal Carcinoma Cells ” , Gene Therapy , Vaino et al. , " Synthesis of a D - Lactosyl Cluster -Nucleoside Con vol. 13 ( 10 ) , 814 - 820 ( 2006 ) . jugate” , Chemical Communications , 19 , 1871 -1872 ( 1997 ). Hamzavi et al. , " Modulation of the Pharmacokinetic Properties of Wolfrum et al. , “ Mechanisms and optimization of in vivo delivery PNA : Preparation of Galactosyl, Mannosyl, Fucosyl, of lipophilic siRNAs" , Nature Biotechnology 25 ( 10 ) , 1149 - 1157 N - Acetylgalactosaminyl, and N -Acetylglucosaminyl Derivatives of ( 2007 ) . Aminoethylglycine Peptide Nucleic Acid Monomers and Their Wong et al. , “ Lipid , Sugar and Liposaccharide Based Delivery Incorporation into PNA Oligomers ” , Bioconjugate Chemistry, ACS Systems” , Current Medicinal Chemistry , vol. 8 ( 9 ) , 1123 - 1136 vol. 14 ( 1) , Washington , DC , US, 941 - 954 , XP002270930 ; ISSN : ( 2001) . 1043 - 1802 (Jan . 2003 ) . Zatsepin et al ., " Synthesis and Applications of Oligonucleotide Ikeda et al. , “ Ligand - Targeted Delivery of Therapeutic siRNA ” , Carbohydrate Conjugates” , Chemistry & Biodiversity , vol. 1 ( 10 ) , Pharmaceutical Research , vol. 23 (8 ), 1631- 1640 ( 2006 ) . 1401 - 1417 ( 2004 ) . Karskela et al . , “ Synthesis and Cellular Uptake of Fluorescently Zheng et al. , “ Distribution and anti- HBV effects of antisense Labeled Multivalent Hyaluronan Disaccharide Conjugates of Oli oligodeoxynucleotides conjugated to galactosylated poly -L - lysine ” , gonucleotide Phosphorothioates” , Bioconjugate Chemistry , vol. World Journal of Gastroenterology , vol. 9 , No . 6 , 1251 - 1255 , 19 ( 12 ) , ( 2008 ) . XP002510287 ( 2003 ) . Katajisto et al. , “ An Aminooxy -Functionalized Non -Nucleosidic Zimmermann et al ., “ RNAI- Mediated Gene Silencing in Non Phosphoramidite for the Construction of Multiantennary Oligo Human Primates ” , Nature Publishing Group , vol. 441, No . 7089 , nucleotide Glycoconjugates on a Solid Support ” , Current Protocols 111- 114 (2006 ) . in Nucleic Acid Chemistry , pp . 4 . 26 .1 - 4 . 26 . 16 ( 2005 ) . Hashida , " Lipid carrier systems for targeted drug and gene deliv Katajisto et al ., “ Solid - Phase Synthesis of Oligonucleotide ery ” , Chemical and Pharmaceutical Bulletin 53 ( 11 ) , 871 - 880 Glycoconjugates Bearing Three Different Glycosyl Groups : ( 2005 ) . Orthoganally Protected Bis ( Hydroxymethyl ) - N , N ' -bis ( 3 Medina , “ Targeted liposomal drug delivery in cancer ” , Current Hydroxyproply )Malondiamide Phosphoramidite as Key Building Pharmaceutical Design 10 ( 24 ) , 2981 - 2989 (2004 ) . Block ” , Journal of Organic Chemistry , vol. 69 ( 22 ) , 7609 -7615 Noble , “ Development of ligand - targeted liposomes for cancer ( 202004 ). therapy ” , Expert Opinions on Therapeutic Targets 8 ( 4 ) , 335 - 353 Katajisto et al. , " Solid -Phase Synthesis of Multiantennary Oligo ( 2004 ) . nucleotide Glycoconjugates Utilizing On - Support Oximation ” , Pan , “ Tumour -selective drug delivery via folate receptor- targeted Bioconjugate Chemistry , vol. 15 (4 ), 890 - 896 (2004 ). liposomes ” , Expert Opinions on Drug Delivery 1 ( 1 ) , 7 - 17 ( 2004 ) . US 9, 814 , 777 B2 Page 3 ( 56 ) References Cited OTHER PUBLICATIONS Sapra , " Ligand - targeted liposomal anticancer drugs” , Progress in Lipid Research 42 (5 ), 439 -462 (2003 ) . Sapra , " Ligand -targeted liposomes for cancer treatment ” , Current Drug Delivery 2 ( 4 ), 369 -381 (2005 ). Endo et al. , “ Physical Properties and Barrier Functions of Synthetic Glyceroglycolipids” , J Biochem , 92 , 953 - 960 ( 1982) . * cited by examiner nt Nov . 14 , 2017 Sheet 1 of 20 US 9 ,814 ,777 B2

Figure 1 . Delivery Targeting system (FemeninasTether / Linker ligand

Delivery Targeting presenterametiassystem Tether /Linker ligand

PK Modulator Endosomolitic Agent

Delivery Targeting system Tether /Linker ligand

Endosomolitic Agent

PK Modulator

Delivery Targeting podensystem (FemeninterTether / Linker sentingligand U . S . Patent Nov . 14, 2017 Sheet 2 of 20 US 9 ,814 ,777 B2

Figure 2 . Delivery Targeting systempertany (Tether Treiberlinker/ Linker Belastingligand DrugDrug

Delivery Targeting system - (TetherTetertinter / Linker r ligandegning DrugDrug PK Modulator Endosomolitic PK Mostaron Agentngemalire

Delivery Targeting systemDelivery (Tether TetherLinker/ Linker ligandaisering 4 DrugDrug Endosomolitic HHH.Agent

PK Modulator

Delivery Targeting Deliverysystem ( TetherTetherLinker/ Linker ) energetingligand Drug atent Nov . 14 , 2017 Sheet 3 of 20 US 9 ,814 ,777 B2

Figure 3 . ( a )

R - Y Nih Linkage Y - R Y - R

Drug Delivery Scaffold Tether ? or

- X - R

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wa Linkage

D-X

Y - R Y - R '

Drug Delivery Scaffold Tether ( d )

Linkage

Y - R Y - R '

Drug Delivery Scaffold Y - R " Tether 1

Tether 2 atent Nov . 14 , 2017 Sheet 4 of 20 US 9 ,814 ,777 B2

Figure 4 . ( a ) R - V Linkage

[ Drug

Y - R Y - R Y - R Tether 3

Drug Delivery Scaffold Tether ( b )

Drug X - R

X-C

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C-X | Drug - 2 X

Y - R ' Y - R Tether 3

Drug Delivery Scaffold Tether

Linkage *Lxjo | Drug - 7 x-J

Y - R Y - R ' Tether 3 Y - R " Drug Delivery Scaffoldw ' n Tether 1

Tether 2 U . S . Patent Nov . 14, 2017 Sheet 5 of 20 US 9 ,814 ,777 B2

Figure 5 .

D U . S . Patent Nov. 14 , 2017 Sheet 6 of 20 US 9 ,814 ,777 B2

Figure 6 . m eyin ninyo

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Figure 7 . ???????? ????? ???? ???? U . S . Patent Nov . 14, 2017 Sheet 8 of 20 US 9 ,814 ,777 B2

Figure 8 ( A ) HO GO ?? ZIIZ p = 1 , 3, 8 , 13 , 19H IZ NH MANNH , ShithaneMNH2 se sententiam HOO IZ IZ p = 1, 2 , 7 , 12 , 18 ?? HN NH HNNY NNNH

H NH IZ HN p = 1, 2, 7 , 12 , 18 BartenN NH2 ErmittlungN N NH2 HOO OZIZI N . HN ?? the heartNNNH2 in frontandop = 1 , 3 , 8 , 13 , 19 CNNCNH , NH p = 1 , 2 , 7, 12, 18 IZ . HN BramkantNANNH2 BartomeuANNH2 HOGO ZI . HN p = 1, 2 , 7 , 12 , 18 HN postaraleNANNH2 romanenANNH U . S . Patent Nov . 14, 2017 Sheet 9 of 20 US 9 ,814 ,777 B2

Figure 8 ( B )

HOGO , IZ ?? p = 1 , 2 , 7 , 12 , 18 NH SittardMNH2 Brem habereN NH2

p = 1 , 3 , 8 , 13, 19 NANNH2 NH TANNH2 sortantHOGO de Barcelona

?? IZ NH BymillendeNNNH2NH2 belangenp = 1 , 2 , 7 , 12 , 18 NANNH U . S . Patent Nov . 14 , 2017 Sheet 10 of 20 US 9 ,814 ,777 B2

Figure 9 . NOON OHOH - hetom man y things 3g 3. 99 | H?N KHUCNH, H2N -PEG2000 -NH2 1. 95 g m o toryou to the 0 . 8 g Oyo o OH TINH HBTU , DIPEA , DMF galingFacto ngentot NannIZ an area o

CNH H ?NANYANH. w om en aynonate in FactoNNNground 1g Piperidine , NH3

?? 0 0

NH IZ ?? mammoga dengan meSNCNCNH , atent Nov . 14 , 2017 Sheet 11 of 20 US 9 ,814 ,777 B2

Figure 10 + dp ? up ; den per portat i on info tentang 3

R = - OH; - OCH3; - NH2; - NHCH3; - N ( CH3) 2; - SH; SCH8 ; - Ng; - COOH; - CONH2; - CONHNH2; - C( O) L; - OL; - N ( H) L; - O - C ( O) - NH( L) ; - o- C( O )- OL ; NH- C( O )- N ( H) L ; NH- C( O) - O( L) ; S - SL ; O- N =CL ; NH- N = CL ; C = N - OL; C = N - N ( H) O L yuz ,no : , new ] IN - V -L on y ' S L N = N NEN Titoloof homeL = Ligand to per x = 0 ; NH ; NCH3; S , CH2; S- S, - 0- N = C- ; - C ( O) - N( H) - N = C -; - C = N - O- ; - C = N - N ( H) - C( O) - : - C( 0) _ N ( Me) - N = N- ; - C = N - N ( Me) - C( O) - ; - O - C (O ) -0 - ; -O - C ( O ) -NH - ; -NH - C (O ) - 0 - ; -NH - C ( O ) -NH - ; - N (Me ) - C (O )- N (Me ) -; - N ( H )- C ( O ) - N (Me ) -; - N Me( ) - C ( O ) - N ( H ) - ; - C ( O ) - O - ; - C ( O ) -N ( H ) - ; - C ( O )- N (Me ) - ; - O - C ( O )- ; -NH - C ( O ) - ; - N (Me ) - C (O ) - ; -C = N -; - N = C - ; mother or to put together Glycerol backbone with R or S configuration or a racemic mixture of both . m = 5 - 29 ; n = 6 - 28 , p = 1 - 1000 and q = 1 - 20 U . S . Patent Nov . 14 , 2017 Sheet 12 of 20 US 9 ,814 ,777 B2

Figure 11 for you vos5 autoritate pentru o formation yang saya buatan yg samarbetering Mano duorta tangR R voor de touten voin toden oro , y yo Yoyo 10 Oy yoyote Minggo ng mgataong R photos11 VaR y yo 12 yoteO R , R = - OH ; - OCH3 ; - NH2; - NHCH3; - N ( CH3) 2 ; - SH; SCH8; - Ng ; - COOH; - CONH2; - CONHNH2; - C ( O ) L; - OL; - N ( H ) L ; - O - CO - NH( L ); - o- c ( O )- OL; - NH- C ( O )- N ( H ) L ; NH- CO - O ( L) ; - S - SL; - O - N = CL; - NH- N = CL; - C = N OL; - C = N - N ( H ) L nus , fri , fer lawn S - L ms N = N to titolo cheL = Ligand tutto in X = 0 ; NH; NGHg ; S , CH2; S - S , - O - N = C - ; - C ( O ) - N ( H )- N = C - ; - C = N - O - ; - C = N - N ( H ) -CO )- ; - C ( O ) _ N ( Me) - N = C ; -C = N -N (Me ) - C (O )- ; -O - C (O )- O -; -O - C (O )- NH - ; -NH - C (O )- O -; -NH -C (O )- NH -; -N (Me ) - C (O )- N (Me ) - ; -N (H ) C ( O ) - N (Me ) -; - N (Me ) - C (O )- N ( H )- ; - C ( O ) - 0 -; - C ( O ) - N ( H )- ; - C ( O ) - N (Me ) - ; - O - C ( O ) -; -NH -C (O )- ; - N (Me )- C (O )- ; - C = N - ; - N = C -;

Jan Juta on these tools toNon get y toNEN Glycerol backbone with R or S configuration or a racemic mixture of both . m = 5 -29 ; n = 6 - 28 , p = 1 - 1000 and q = 1 - 20 U . S . Patent Nov . 14 , 2017 Sheet 13 of 20 US 9 ,814 ,777 B2

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Yo zavyo yote 6xzoryo todas Yn 15 ár toota UMT 16 kutotolar mory zartoryota A ) zo yoyote n 17 ytojouter you gotta Yang 6 Zoy tortoHaR Y Ózo yoyote 19 yoyoda R 20 bp to your

R, R' = - OH; - OCH3; - NH2; -NHCH3 ; - N ( CH3) 2; - SH ; SCH3; - Ng; - COOH; - CONH2; - CONHNH2; - C ( O ) L ; - OL; - N ( H) L ; - o - C ( O )- NH( L) ; - o - c ( O ) - OL ; - NH- C( O )- N ( H ) L; NH- C( O )- O ( L ) ; - S - SL; O - N = CL; - NH- N = CL; - C = N OL; - C = N - N ( H ) L

, nr 5 , N | L in M S - L fors NON NEN er et meL = Ligand seperti x = 0 ; NH; NGHg ; S, CH2; S- S, O- N = N -; - C( o) - N ( H )- N = N -; - C = N - O- ; - C = N - N ( H )- C( O) -; C ( o) _ N ( Me) - N = C ; - C = N - N (Me ) - C ( O ) - ; - O - C ( O ) - O - ; - O - C ( O ) -NH -; - NH - C ( O ) - O - ; -NH - C ( O ) -NH - ; - N (Me ) - C ( O ) - N (Me ) -; - N ( H ) C (O ) -N (Me ) - ; -N (Me ) - C (O ) -N ( H )- : - C ( O )- 0 -; -C (O ) -N ( H )- ; - C (O )- N (Me ) - ; - O -C ( O )- ;- NH -C (O )- ; -N (Me ) -C (O ) - C = N - ; -N = C -; Nga punya Norte garn yang to y Sopa fors [ NSNIS NE? atent Nov . 14 , 2017 Sheet 14 of 20 US 9 ,814 ,777 B2

Figure 13 Antetop or toyota a Anthology you yolda RP dont x antophoeYo z orgroot you tota help ythose o 22 youby you yourthings x y yo yote " ne Youtonya A™ RototomYoyx qar yoyouar Rutatom qar yoyouar no 25 áv yoyote yaró 26 6w you youp" mutighet i e r for return theyou began ? to remontas bute toda * fÝ á yoyorlar Un 27 o 2828 R , R ', R " = -OH ; -OCH3 ; - NH2; - NHCH3: - N (CH3 ) 2 ; - SH ; -SCH3 ; - N3 ; - COOH ; - CONH2; - CONHNH2; - C ( O ) L : - OL; -NH ) L ; - o- c ( O) - NH ( L ) ; - o- c ( O) - OL; - NH- C( O) - N ( H )L ; NH- C( O) - O( L ) ; - S SL; - O- N = CL; - NH- N = CL ;- C = N - OL; - C = N - N( H ) L . Le mus , nes , - L Kan Y ' S - L tres NEN NEN titats i enL = Ligandperson tout x = 0 ; NH; NCH ; S, CHA; S - S, - O- N = C -; - C ( O )- N ( H) - N = C - - C = N - O- ; - C = N - N ( H) - C ( O) - ; - C( O ) _ N ( Me )- N = C ; -C =N - N (Me ) - C (O )- ; - O - C (O ) -O - ; -O -C (O )- NH -; - NH -C ( O )- O -; - NH -C (O )- NH -; -N (Me ) - C (O )- N (Me ) - ; - N (H ) C ( O )- N (Me ) - ; - N (Me ) - C (O )- N (H )- ; - C ( O )- 0 -; - C ( O )- NCH ) -; - C ( O ) -N (Me ) - ; - O - C ( O )- ; -NH -C ( O )- , - N (Me ) - C (O )- ; -C = N -; - N = C -;

Nude nard on the other N = N NON U . S . Patent Nov . 14 , 2017 Sheet 15 of 20 US 9 ,814 ,777 B2

Figure 14 peyoutou , totoo yoyott " se a yoyotessporta mo by you Rutot onger toutotary Y yoyote " x. 2030 ów you you 31ºy yoyott , a Zorto todas Oxzar fotolar öytoyota ar32 to you are 33 ou tyto 34

R , R ', R " = - OH ; -OCH3 ; -NH2 ; - NHCH3; -N (CH3 ) 2 ; - SH ; - SCH3; -N3 ; - COOH ; - CONH2; - CONHNH2; -C (O )L ; - OL; - N ( H ) L; - o - c ( O) - NH( L ); - o - c ( O ) - OL ; - NH- C ( O )- N ( H ) L ; NH - C ( O ) - O ( L) ; - S - SL; - O - N = CL; - NH- N = CL ; - C = N - OL; - C = N - N ( H ) L L rifus , nos , Ton ti target thatY S - L vstime toN = N . NON L = Ligand T x = 0 ; NH; NCH ; S, CH2; S - s, O- N = N- ; - C( O )- N ( H) - N = N- - C = N - O -; C = N - N ( H )- C( O) - ; - C( O )_ N ( Me) - N = C ; - C = N - N (Me ) - C ( O )- ; - O - C ( O ) - O - ; - O - C ( O )- NH - ; -NH - C ( O ) - O -; -NH - C ( O ) -eNH - ; o - Nco ( Me ) - C (O )- N (Me ) - ; - N (H ) C (O )- N (Me ) - ; -N (Me ) - C ( O )- N (H )- ; -C (O )- O -; - C (O ) -N (H )- ; -C (O )- N (Me ) - ; -O - C (O )- ; -NH -C (O )- , -N (Me ) - C ( O )- ; - C = N - ; - N = C - ; 10. CIO , NE more than the one that things are not Glycerol backbone with R or S configuration or a racemic mixture of both , m = 5 - 29 ; n = ange6 -28 , p = 1 - 1000lo and q = 1 -20 continuation era semi inte helt, m = atent Nov . 14 , 2017 Sheet 16 of 20 US 9 ,814 , 777 B2

Figure15

T ? ? ? ? ? ? ? , , ? ? ? ?° ? ? ?' ???HN ?????? ,' 9 ? ? ?? —??? ? — e ? ???? ? — ? — ? ," ?? ? ? ? ? ? ???? ???? ? ??????? ? ? ? ? ? ?? ?? ? ? ? ?

HN ????? ? ? ??? — — ( ? ? ), ?. ? ?? ? ???? < R = - OH; - OcH3 ; - NHz ; - NHCH ; - N ( CH3) z; - SH; - SCH ; - N ; - COOH; -CONHz ; - CONHNH : - C ( O } L; - OL; - N ( H) L ; - o - C( O) - NH ( L) : - O- C ( O) - OL; -NH - C( O) - N ( H) L; NH- C ( O) - O( L) ; -S SL ;- O - N = CL; -NH - N = CL; - C = N - OL; - c = NM - N( H} L , N ? = , ? M?W L = Ligand x = 0 ; NH; NCH3; S , CHA; S - S , - O - N = C -; - C / O ) - N ( H ) - N = C - - C = N - O - ; - C = N - N ( H ) - C ( O ) - - C ( O ) _ N ( Me) - N = C ; - C = N - N ( Me ) - C O ) - ; - O - C ( O ) - O - ) - O - C ( O ) - NH- - NH- C ( Oh - O - - NH- C ( O )- NH- ; - N ( Me ) - CO) - NKMe) - ; - N ( H ) c( O) - N( Me )- ; - N( Me) - c( O )- N{ H )- ; - C( O) - O- ; - C( O) - N( H) - ; - C { O }- N( Me) - ; - O- C{ O) - ; - NH- c( O) - ; - N( Me) - c( O )- ; - c = N -; - N = c

New ? v = W U . S . Patent Nov . 14 , 2017 Sheet 17 of 20 US 9 ,814 ,777 B2

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R , R ', R " = -OH ; -OCH3 : - NH2 ; -NHCH3 ; -N (CH3 ) 2 ; - SH ; -SCH3 ; -N3 ; -COOH ; -CONH2 ; - CONHNH2; - C (O )L ; - OL; - N ( H) L; - o - c( O) - NH (L ) ; - O- C( O) - OL; -NH - C ( O) - N ( H) L; NH- C( O) - O( L) ; - S- SL; O- N = CL; -NH - N = CL; - C = N - OL; - C = N - N ( H) L

spus , , non T S - L tus NEN NEN to hereL = Ligand to that you pro x = 0 ; NH; NCH8; , CH2 : S- S, - O- N = C- ; - C( O )- N( H) - N = C- ; - C = N - O- ; - C = N - N( H) - C( O) - ; - C ( o) _ N( Me) - N = C ; - C = N - N (Me ) - C ( O ) - ; - O - C ( O ) - O - ; - O - C ( O ) -NH - ; -NH - C ( O ) - 0 -; -NH - C ( O ) -NH - ; - N (Me ) - C ( O ) - N (Me ) - ; - N ( H ) C ( O ) - N (Me ) -; - N (Me ) - C ( O ) - N ( H )- ; - C ( O )- O - ; - C ( O )- N ( H )- ; - C ( O )- N (Me ) -; - O - C ( O )- ; -NH - C ( O ) -; - N (Me ) - C ( O )- ; - C = N - : - N = C - ;

pun Jauh of het to that theNON momentNON U . S . Patent Nov . 14, 2017 Sheet 18 of 20 US 9 ,814 ,777 B2

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R , R ', R " = - OH ; -OCH3 ; - NH2 ; - NHCH3; - N ( CH3) 2 ; - SH ; - SCH3; -N3 ; - COOH ; - CONH2; - CONHNH2; - C ( O ) L ; - OL; - N ( H) L ; - O- C( O) - NH( L) ; - o- C( O )- OL ; - NH - C ( O) - N ( H) L; NH- C ( O )- O( L ) ; - S - SL; - O - N = CL; -NH - N = CL; - C = N - OL; - C = N - N ( H)

que , s - L porn titoryoto home to N = N somNON L = Ligand x = 0 ; NH; NGHg ; S , CH2 ; S - S , O - N = N - ; - C( O )- N ( H )- N = - ; - C = N - O - ; - C = N - N ( H )- C ( O )- ; - C ( o ) _ N ( Me) - N = N ; - C = N -N (Me ) -C ( O )- ; - O - C (O )- O -; -O -C (O ) -NH -; -NH - C (O ) - O -; - NH - C ( O )- NH -; -N (Me )- C ( O )- N (Me ) -; - N (H ) C (O )- N (Me ) - ; - N (Me ) -C (O )- N (H )- ; - C (O )- O -; - C (O )- N (H )- ; -C (O )- N (Me ) - ; -O - C (O )- ; -NH -C (O )- ; -N (Me ) - C ( O )- ; - C = N -; - N = C -;

time se at hining tomention NA U . S . Patent Nov . 14 , 2017 Sheet 19 of 20 US 9 ,814 ,777 B2

Figure 18

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R , R ', R " = -OH ; - OCH3; -NH2 ; -NHCH3 ; -N (CH3 ) 2 ; -SH ; -SCH 3; -N3 ; - COOH ; - CONH2; -CONHNH2 ; - C (O )L ; - OL; - N ( H ) L; - o - C ( O )- NH ( L) ; - o - c( O )- OL; -NH - C( O )- N ( H ) L; NH- C ( O )- O( L ); - S - SL ; - O - N = CL; -NH - N = CL ; - C = N - OL; - C = N - N ( H ) 0 0 = , rs , un | porn N LN- to targetthe S - L to tryMN L = Ligand x = 0 ; NH; NCHz; S , CH2; S - s , - O - N = c ; co) - N ( H )- N = C - - C = N - O -; - C = N - N ( H ) -co ) ; - C ( O ) _ N ( Me) - N = c ; - C = N - N (Me ) - C ( O ) - ; - O - C ( O ) - O - ; - O - C ( O ) -NH - ; -NH - C ( O ) - O - ; -NH - C ( O ) -NH -; - N (Me ) - C ( O ) - N (Me ) - ; - N ( H ) C ( O )- N (Me ) - ; - N (Me ) - C (O )- N ( H )- ; - C ( O ) - 0 -; - C ( O ) -N (H )- ; - C ( O )- N (Me ) - ; - O - C ( O )- ; -NH - C ( O ) -; - N (Me ) - C (O )- ; - C = N -; - N = C -; montant tohear content LasU . S .Pieven Patent Nov . 14 , 2017 Sheet 20 of 20 US 9 ,814 ,777 B2

Figure 19

ITILD m berperanto- Y X

•0 - Y - JX abonementaraby yoyotes M o"" O - rthY - X7 more than oreYtoyotato see it IIII man" O - Y - X7 tomaten matanKUTIIL Ferfotonatentang Y = linker m totonavyoufortas20-V - motobom

Pulptura de gra :L Chol - o - Y - x n covom autoritats CholChol = = CholesterolCholesterol oyyou toVaR ea f" R, R , R " = - OH; - OCH3; -NH2 ; -NHCH3 ; - N (CH3 ) 2 ; - SH; - CH3; - Ng; - COOH; - CONH2; - CONHNH2 ; - C( O )L : LOL: - N( H) L ; - o- c( O) - NH ( L) ; - o- c( O) - OL; -NH - C ( O) - N( H )L ; NH- C( O) - O( L ) ; S - SL ; CO- N = CL; - NH- N = CL ; - C = N - OL; - C = N N ( H ) L L = , . N af N 1 VL un S - L was Non tota polis of homeL = Ligand to theO topu x = 0; NH ; NCHz; S, CHA, SUS, - O- N = C - - C( O )- N ( H) - N = C -; - C = N - O- ; - C = N - N ( H) - CO ; -; - C ( O) _ N ( Me} - N = c ; - C = N -N (Me ) - C (O )- ; -O -C (O )- O -; -O -C (O )- NH -; -NH -C (O )- O -; -NH - C (O )- NH -; -N (Me ) - C (O )- N (Me ) -; -N (H ) C (O )- N (Me ) - ; - N (Me ) - C (O )- N (H )- ; - C (O ) -0 -; - C (O ) -NCH )- ; - C ( O )- N (Me ) - ; - O -C ( O ) -; -NH - C (O )- ; - N (Me ) - C (O ) -; - C = N - ; - N = C - ; yomda ligo Ny n NN NEN onO these are that relation US 9 , 814 , 777 B2 TARGETING LIPIDS be used to identify the function of a gene. In addition , siRNA compounds offer enormous potential as a new type of RELATED APPLICATIONS pharmaceutical agent which acts by silencing disease - caus ing genes . Research is currently underway to develop inter This application is a continuation application of U . S . 5 ference RNA therapeutic agents for the treatment of many patent application Ser . No . 12 /328 ,669 , filed Dec . 4 , 2008 , diseases including central- nervous - system diseases, inflam and claims the benefit of priority of U . S . Provisional Appli matory diseases , metabolic disorders , oncology , infectious cation No . 60 /992 ,309 , filed Dec. 4 , 2007 , U . S . Provisional diseases, and ocular disease . Application No. 61/ 013 , 597 , filed Dec . 13 , 2007 , U . S . siRNA has been shown to be extremely effective as a Provisional Application No. 61/ 127 ,751 , filed May 14 , 2008 , 10 potential anti -viral therapeutic with numerous published U . S . Provisional Application No. 61 /091 , 093 , filed Aug. 22 , examples appearing recently . siRNA molecules directed 2008 , and U . S . Provisional Application No. 61/ 097 , 261, against targets in the viral genome dramatically reduce viral filed Sep . 16 , 2008 . The entire content of each of these applications is hereby incorporated herein by reference . titers by orders of magnitude in animal models of influenza 15 (lueGe et. . alAlig. , 110vProc . Natl1 . Acd . Sci. USA , 101: 8676 - 8681 (2004 ) ; Tompkins et . al ., Proc . Natl. Acd . Sci. USA , 101 : GOVERNMENT SUPPORT 8682 - 8686 (2004 ) ; Thomas et . al. , Expert Opin . Biol . Ther. The work described herein was carried out, at least in part, 5 :495 - 505 ( 2005 ) ) , respiratory synctial virus (RSV ) (Bitko using funds from the United States government under con et . al. , Nat . Med . 11 : 50 -55 (2005 ) ) , hepatitis B virus (HBV ) tract number HHSN266200600012C from the National 20 (Morrissey et . al . , Nat. Biotechnol. 23 : 1002 - 1007 ( 2005 ) ) , Institute of Allergy and Infectious Diseases /National Insti - hepatitis C virus (Kapadia , Proc . Natl . Acad . Sci. USA , tutes of Health /Department of Health and Human Services 100 : 2014 - 2018 ( 2003 ) ; Wilson et . al. , Proc . Natl. Acad . Sci. (NIAID /NIH /DHHS ) and contract number HDTRA - 1 -07 - USA , 100 :2783 - 2788 ( 2003 ) ) and SARS coronavirus (Li et . C - 0082 , from the Department of Defense and Defense al. , Nat. Med . 11: 944 - 951 ( 2005 ) ) . Threat Reduction Agency (DOD /DTRA ) . The government 25 Antisense methodology is the complementary hybridiza may therefore have certain rights in the invention . tion of relatively short oligonucleotides to mRNA or DNA such that the normal, essential functions , such as protein TECHNICAL FIELD synthesis , of these intracellular nucleic acids are disrupted . Hybridization is the sequence - specific hydrogen bonding via The present invention relates to the field of therapeutic 30 Watson - Crick base pairs of oligonucleotides to RNA or agent delivery using lipid particles . In particular, the present single - stranded DNA . Such base pairs are said to be comple invention provides targeting lipids and lipid particles com - mentary to one another . prising these lipids , which are advantageous for the in vivo The naturally - occurring events that alter the expression delivery of nucleic acids, as well as nucleic acid - lipid level of the target sequence , discussed by Cohen (Oligo particle compositions suitable for in vivo therapeutic use . 35 nucleotides: Antisense Inhibitors of Gene Expression , CRC Additionally, the present invention provides methods of Press , Inc . , 1989 , Boca Raton , Fla . ) are thought to be of two making these compositions, as well as methods of introduc types. The first, hybridization arrest , describes the terminat ing nucleic acids into cells using these compositions , e . g ., ing event in which the oligonucleotide inhibitor binds to the for the treatment of various disease conditions. target nucleic acid and thus prevents , by simple steric 40 hindrance , the binding of essential proteins , most often BACKGROUND ribosomes, to the nucleic acid . Another means by which antisense oligonucleotides alter Oligonucleotide compounds have important therapeutic the expression level of target sequences is by hybridization applications in medicine . Oligonucleotides can be used to to a target mRNA , followed by enzymatic cleavage of the silence genes that are responsible for a particular disease . 45 targeted RNA by intracellular RNase H . A 2' - deoxyribofura Gene -silencing prevents formation of a protein by inhibiting nosyl oligonucleotide or oligonucleotide analog hybridizes translation . Importantly , gene- silencing agents are a prom - with the targeted RNA and this duplex activates the RNase ising alternative to traditional small , organic compounds that H enzyme to cleave the RNA strand , thus destroying the inhibit the function of the protein linked to the disease . normal function of the RNA . Phosphorothioate oligonucle siRNA , antisense RNA , and micro -RNA are oligonucle - 50 otides are the most prominent example of an antisense agent otides that prevent the formation of proteins by gene - that operates by this type of antisense terminating event. silencing . RNA interference or “ RNAi" is a term initially The opportunity to use these and other nucleicl acid coined by Fire and co - workers to describe the observation based therapies holds significant promise , providing solu that double - stranded RNA (dsRNA ) can block gene expres - tions to medical problems that could not be addressed with sion when it is introduced into worms (Fire et al. ( 1998 ) 55 current, traditionalmedicines . The location and sequences of Nature 391, 806 -811 ). Short dsRNA directs gene- specific , an increasing number of disease- related genes are being post- transcriptional silencing in many organisms, including identified , and clinical testing of nucleic acid -based thera vertebrates , and has provided a new tool for studying gene peutics for a variety of diseases is now underway. function . RNAi is mediated by RNA - induced silencing Despite the advances in application of oligonucleotides complex (RISC ) , a sequence -specific , multi -component 60 and oligonucleotide analogs as therapeutics , the need exists nuclease that destroys messenger RNAs homologous to the for oligonucleotides having improved pharmacologic prop silencing trigger. RISC is known to contain short RNAs erties. Efforts aimed at improving the transmembrane deliv ( approximately 22 nucleotides) derived from the double ery of nucleic acids and oligonucleotides have utilized stranded RNA trigger , but the protein components of this protein carriers , antibody carriers, liposomal delivery sys activity remained unknown . 65 tems, electroporation , direct injection , cell fusion , viral siRNA compounds are promising agents for a variety of vectors , and calcium phosphate -mediated transformation . diagnostic and therapeutic purposes. siRNA compounds can However, many of these techniques are limited by the types US 9 , 814 , 777 B2 4 of cells in which transmembrane transport is enabled and by q , q24 , 228 , 234 , 238 , 244 , 24B , 254 , q5B and q5C and the conditions needed for achieving such transport . represent independently for each occurrence 0 - 20 ; To attempt to improve efficacy, investigators have also P, P24, P2B, P34, P3B, P44, P4B, PS4, P5B, PSC , T, T24 , T2B, employed lipid -based carrier systems to deliver chemically T3a , T3 , T44 , T4B , T44 , TSB and TSC are each independently modified or unmodified therapeutic nucleic acids . In Zel- 5 for each occurrence absent. NR '. O , S , C ( O ) , OC ( O ) , C ( O ) O , phati, O and Szoka , F . C . , J . Contr. Rel. 41 :99 - 119 ( 1996 ) , the authors refer to the use of anionic ( conventional) lipo NHC ( O) , C( O ) NH , NHCH , CH?, CHÚNH or CH20, somes , pH sensitive liposomes, immunoliposomes , fuso NHCH( R ^) C ( O ), C ( 0 ) CH ( R®) NH?, CO, genic liposomes , and cationic lipid /antisense aggregates . CHN O , CH2S , urea , heterocycle, heteroaryl, Similarly siRNA has been administered systemically in cationic liposomes , and these nucleic acid - lipid particles 10 have been reported to provide improved down -regulation of target proteins in mammals including non - human primates ( Zimmermann et al. , Nature 441 : 111 - 114 ( 2006 ) ) . HO In spite of this progress, there remains a need in the art for improved lipid - therapeutic nucleic acid compositions that 15 i are suitable for general therapeutic use . Preferably , these mu NH my compositions would encapsulate nucleic acids with high S - S , efficiency , have high drug : lipid ratios , protect the encapsu m si wwwsm lated nucleic acid from degradation and clearance in serum , be suitable for systemic delivery , and provide intracellular 20 delivery of the encapsulated nucleic acid . In addition , these S - S w or lipid -nucleic acid particles should be well - tolerated and w howw or provide an adequate therapeutic index , such that patient Y treatment at an effective dose of the nucleic acid is not S - Swi, associated with significant toxicity and / or risk to the patient. 25 w w The present invention provides such compositions, methods of making the compositions, and methods of using the Q , Q24 , Q2B , Q34 , Q3B , 044 , Q4B , 254 , Q5B and QSC are compositions to introduce nucleic acids into cells , including independently for each occurrence absent , (CH ) n - , for the treatment of diseases . C ( R ') ( R " )( CH2 ) , (CH2 ) mC (R ') ( R " ) , 30 (CH2CH2O ) , CH2CH2 - or (CH2CH20 ) BRIEF SUMMARY CH , CH, NH? LP is a ligand selected from a group consisting of lipo The present invention provides targeting lipids having the phile , steroid ( e . g . , uvaol , hecigenin , diosgenin ), terpene structure shown in formula (1 ) : ( e . g ., triterpene, e . g ., sarsasapogenin , Friedelin , epifriedela LA[ - P- Q -R - ] T . -LB Formula \(1 ) 35 nol derivatized lithocholic acid ), vitamin (e .g . , folate , vita wherein : min A , biotin , pyridoxal ) , ceramide or has the structure of 14 is a ligand chosen from a carbohydrate , glucose, formula (VI ) : mannose , galactose , N -acetyl - galactosamine , fucose , glu cosamine, lactose , maltose , folate , peptide , or has the struc ture shown in formula II- V : 40 Formula ( VI) R64 — L 64 Formula ( II ) | p24 – 224 – R24ta24T24 — 124 , ARB - 16B ; 45 wa R , R², R24 , R2B , R34, R3B , R44 , R4B , RS4 , RSB , RSC , R " , Yp2B – Q2B – R2B522 T2B — L 23 R64 and RB are each independently for each occurrence Fomula ( III) 50 absent , CO, NH , NR' , 0 , S, C ( O ), OC( O ), C ( O ) O , NHCO) , C ( O )NH , NHCH , CH?, CHANH or CH2O, NHCH ( R ^ ) C Ipar- q?d =R * tox7 7 –14 ( O) , C ( 0 ) CH ( R°) NH?, CO, CH?NO, WN*Ypin - q =R * fps -18 – 2 * [p * =Q4 = R * Extr- u_ z ", or Formula (IV ) 55 Nam | p4B - 04B - R4B ban - T4B — 1 4B Formula ( V ) 60 7p54 – Q5A – R541934 – T54 – [ 54 HO mustL pSB – QSB – RSB 5, 3 T5B – 15B;

p5C – Q3c = R$ CE_ $ c - T% C – 15C 65 m mn N 4 65 w nnn ? ? N US 9 , 814 , 777 B2

-continued FIGS. 10 - 19 . Schematic representation of some PEG m S - S , lipids of the invention . w w DETAILED DESCRIPTION s S - S Or 5 w In one aspect the invention provides a targeting lipid monomer having the structure shown in formula (CI ) . L 100- linker- L101 ( CI) ve n wherein : 10 L 100 is independently for each occurrence lipid , lipophile , L24 , L2B , L 34 , L3B , L44 , L 4B , L 54 , L 5B and LSC are each alkyl, alkenyl or alkynyl, each of which is optionally sub independently for each occurrence a carbohydrate , glucose , stituted with one or more substituents ; mannose , galactose , N -acetyl - galactosamine , fucose, glu L101 is independently for each occurrence a ligand or cosamine, lactose, maltose , folate or a peptide ; 15 CH CH (OCH CH2) , O (CH2 ) , CH2- ligand ; R ' and R " are each independently H , CH2, OH , SH , NH , p is 1 - 1000 ; and NRI' R20 , alkyl, alkenyl or alkynyl; alternatively , R ' and R " q is 1 - 20 . are each independently halogen ; In one embodiment, the targeting lipid monomer has the Rais H or amino acid side chain ; structure shown in formula (CII ) Riº and R20 are each independently alkyl, alkenyl or 20 L 100- linker - A - linker -L101 ( CII) alkynyl; L64 and LB are each independently alkyl, alkenyl or wherein : alkynyl, each of which is optionally substituted with one or A is O , NH , NCH3, S , CH2, S - S , - C (CH3 ) 2 - SS - , more substituents ; - CH ( CH2) - S - S — , - 0 — N — C — , - C ( O ) - N ( H ) m represent independently for each occurrence 0 - 50 ; 25- N?C- , C = N _ 0 , C = N _ N ( H ) C ( 0 ) , n represent independently for each occurrence 1 - 20 ; and ' - C ( O ) N (Me ) - N = C — , - C = N - N (Me ) - C ( O ) - , - 0 – p represent independently for each occurrence 0 - 50 . C ( O ) Oº, 20 C ( O ) NH?, — NH C ( O ) Oº, When any of q , q24 , 2B , 234 , 23B , 244 , 4B , 254 , q5 and ~ NH C ( O ) NH?, _ N ( Me ) - C ( 0 ) N ( Me) - , q is greater than 1 , the repeating unit can be the same or - N ( H ) C (O ) - N (Me ) -, - N (Me ) - C ( O ) - N (H ) — , different from each other , for example when q is 3 the unit 30 C ( O ) O - , C ( O ) - N ( H ) , C (0 ) N (Me ) - , — [ P - Q - R ]o — is expanded to — [ P - Q - R ] — [ P - Q - R ] — [ P - Q - O C ( O ) - , - NH - C (O ) - , - N (Me ) - C ( O ) - , R ] — and all of the — [ P - Q - R ] — units can be the same, C = N - , - N = C — , completely different from each other or a mixture thereof. The present invention further includes methods of pre paring lipid particles and pharmaceutical compositions , as 35 well as kits useful in the preparation of these lipid particle and pharmaceutical compositions. The method includes pro wuN viding a composition that includes an agent, e . g . an oligo nucleotide based construct that targets a selected target gene, Sun , e . g . a gene expressed in the liver , and the targeting lipid ; and 40 administering the composition to a test subject , e . g . an animal; thereby evaluating the agent and the targeting lipid , heterocycle or heteroaryl; e . g. by evaluating the expression of the target gene . L 100 is independently for each occurrence lipid , lipophile , alkyl, alkenyl or alkynyl, each of which is optionally sub BRIEF DESCRIPTION OF DRAWINGS 45 stituted with one or more substituents ; L101 is independently for each occurrence a ligand or FIG . 1 . Schematics of targeted delivery using targeting CH , CHZ( OCH CH2) , O (CH2 ) , CH2- ligand ; ligands. p is 1 - 1000 ; and FIG . 2 . Schematics of targeted delivery using targeting qis 1- 20 . ligands with conjugated therapeutic agent. 50 In one embodiment, the targeting lipid monomer has the FIG . 3 . Schematic representation of polymer drug deliv - structure shown in formula (CIII ) ery systems with one or more targeting moiety (moieties ) R separated by a tether . X and Y indicate chemical linkages L 110- A -L111 (CIII ) between the scaffold /tether and tether /ligand . R ' and/ or R " is L110 is L112 , either targeting , fusogenic , endosomal releasing groups, 55 hydrophobic /hydrophilic balancer such as saturated or unsaturated alkyls with varying length or PEG with varying R100 - L112 R100 - L 112 length or circulation enhancer like PEGS, PK modulators. FIG . 4 . Schematics of polymer drug delivery systems with therapeutic agent conjugate conjugated to the polymer back 60 or * R100 – [ 112 ; bone via a tether and linkage Z (biocleavable or stable ) . FIG . 5 . pH sensitive lipid with targeting moiety. R100 — 1112 FIG . 6 . Cationic lipid - folate conjugates. FIG . 7 . Lipid - folate conjugates . R100 is independently for each occurrence absent, CO , FIG . 8 . Folate conjugated lipids, PEG - lipids and delivery 65 NH , O , S , S - S , C ( CH3)2S - S , CH (CH3 ) S systems for targeted delivery . S , C( O ), OC( O ), C ( O ) O , NHC ( O ), C ( O ) NH, NHCH , FIG . 9 . Synthesis of folate conjugate . CH , CHANH, CHAO , CH?N20, heterogryl , heterocycle , US 9 , 814 ,777 B2 -

R100 — [ 112 R100 — 1112 ?? Nam or ma w nem R100 — 1112 and mm R100 — 1112 In one embodiment, Llll is chosen from a group consist IZ 10 ing of

L111 is L113 , L114 , R100 — 1113

R100 — 1113 mi R100 — 1113 R100 — L 113 mu R100 — L113 , R100 — 1113 R100 — [ 113 and

R100 — 1113 or L113 - R100 -

1 113 L113 - R 100 naporX R100 R100 30 1113 -R100 nin 1 . 113 . In one embodiment, L112 is alkyl , for example C3 -C31 35 alkyl , e. g . , C1 - C18 alkyl, e. g ., C14 alkyl , Cis alkyl, C16 L112 is independently for each occurrence lipid , lipophile , alkyl, C17 alkyl , C18 alkyl. alkyl, alkenyl or alkynyl , each of which is optionally sub - In one embodiment, L112 is alkenyl, for example C5 -C21 stituted with one or more substituents ; alkenyl, e . g . , C10 - C18 alkenyl, e . g . , C14 alkenyl, Cis alkenyl , L113 is independently for each occurrence CH CH2 40 C . , alkenvl. Co- alkenvl. C . alkenvl. In one embodiment ( OCH _ CH _ ), O (CH2 ) , CH2- L114 ; L114 is independently for each occurrence a ligand , L112 comprises at least one double bond . - C ( O ) - ligand , O C ( O ) - ligand , - N ( H ) - ligand , O In one embodiment , L112 is alkynyl, for example C3- Cz? C (O ) - N (H )- ligand , O - C (O ) O - ligand , - NH - C alkynyl, e. g ., C10 -C18 alkynyl, e .g . , C14 alkynyl, C15 alkynyl, (0 ) N ( H ) - ligand , - NH - C (O ) - O - ligand , S - S - li - 45 C16 alkynyl, C17 alkynyl, C18 alkynyl. In one embodiment gand , O - N - C - ligand , NH?N = C - ligand , " L " 2 comprises at least one triple bond . In one embodiment, C = N / O - ligand . -_ C = N _ N (H ) — ligand , heterocycle - L112 comprises at least one double bond and at least one ligand , heteroaryl- ligand , triple bond . In one embodiment, L112 includes one double bond , for 50 example a double bond in E or Z configuration . In one embodiment, L112 comprises two double bonds. In one embodiment, at least one double bond has a Z configu or ration . In one embodiment, both double bonds have a Z configuration . In one embodiment, at least one double bond S - Ligand 55 has an E configuration . In one embodiment , both double bonds have an E configuration . In one embodiment, L112 comprises three double bonds. In one embodiment, at least one double bond has a Z N - Ligand . 60 configuration . In one embodiment, two double bonds have a Z configuration . In one embodiment all three double bonds have a Z configuration . In one embodiment, at least one double bond has an E configuration . In one embodiment, two double bonds have an E configuration . In one embodi p is 1 - 1000 ; and 65 ment all three double bonds have an E configuration . q is 1 - 20 In one embodiment, L11° is chosen from a group consist In one embodiment, L112 is cholesterol. In one embodi ing of ment, L112 is 9 US 9 , 814 , 777 B2

nnnw?? lii? IllIIIllll

15 In one embodiment, Ll14is a targeting ligand, e. g . folate , _In une embodiinent , Lll4is chosen from group SliOwn in carbohydrate . FIG. 8 . In one embodiment, L114 has the structure shown in In one embodiment, L114 is chosen from group consisting formula (II ) - V ). of IIo H0 ?? AcHN

UH H0

HU ? NH , AcHN

H0 COH

AcHN

H0

H0 ? HO ZE HO OII

, H } NH Z AcHN _ H0

H0 ?? z AcHN US 9 , 814 , 777 B2 ? 11 ?? - continued OH

H0 H .

HO NH ) Ht H0 H }

H0 NH ,

} H H0 ? A

} H H0 ?

A ; !! ! H0?? NHAC AcHN

HOCH

OH _ H0

H0 ? AcHN OH HOH0 ?? WH } UH - AcHN OH Ht )

H0 [ AcHN Li OH H } 10 0 HU 7 ? HO ? ._ TOH0 ?? To OM _ N0 ? LOH HO ? H0 H0 OH OH ( ? 01?? ? US 9 , 814 ,777 B2 1313 US 9, 814 , 777 B2 14 -continued OH mm HO HO 20 O , HO HN HO H2N ' N ??? ??? CO2Me N

HN NH HNHÖN Knotty N

CO Me: Šand

HN NH HN " HNthen N ob HN igénynonyyOOHOH HN NH H?N N ON

In one embodiment, when L110 is chosen from a group consisting of R100 — 1112 50 R 100 — 1112 mu and R100 - L 112 R100 — 1112 and 55 mm R100 — 112 R100 — 1112 wa R100 — L112 , R 100 — 1112 60 Lllº has an enantiomeric excess of the R isomer , e . g ., at least about65 % , 70 % , 75 % , 80 % , 85 % , 90 % , 95 % , 97 % , 98 % , or 99 % . In one embodiment the L110 is an enantiomerically L 110 is a racemic mixture . 65 pure ' R ' isomer. In one embodiment, when L110 is chosen from a group In one embodiment, when Lºis chosen from a group consisting of consisting of US 9 , 814 ,777 B2 15 16 L 113 -continued R100 — 1112 L113To - R100the LR online and w R100 — L 112 - por L113 , R 100 — 1112 L111 has an enantiomeric excess of the Risomer, e . g . , at least about 65 % , 70 % , 75 % , 80 % , 85 % , 90 % , 95 % , 97 % , 98 % , or * R100 - L112 99 % . In one embodiment the Llll is an enantiomerically pure ' R ' isomer . In one embodiment, when L ' 11 is chosen from a group consisting of L11° has an enantiomeric excess of the S isomer, e . g ., at least about65 % , 70 % , 75 % , 80 % , 85 % , 90 % , 95 % , 97 % , 98 % , or 99 % . In one embodiment L110 is an enantiomerically pure R100 — 1113 ' S ' isomer. 20 In one embodiment. when Llll is chosen from a group consisting of R100 — L113, 25 R100 — 1113 R100 — L 113

R100 — L113 and

M 30 R100 — L113 , L113 — R100 1. 113 R100 R100 — 1113 35 R100 R 100 — 1113 and mi [ 113,

T 113 40 L has an enantiomeric excess of the S isomer, e . g ., at least L113 - R 100 about 65 % , 70 % , 75 % , 80 % , 85 % , 90 % , 95 % , 97 % , 98 % , or R100 99 % . In one embodiment Llll is an enantiomerically pure ' S ' isomer. In one aspect the invention provides a lipid monomer R100 45 having the structure shown in formula ( CIV ) L 113 L210- A -L211 ( CIV ) L210 is L212 L111 is a racemic mixture . 50 In one embodiment , when Llll is chosen from a group R200 — 1212 consisting of

R100 — 1113 55 muy R200 - L212 or R200 — 1212

m mm R100 — 1113 , 8 R200 — 1212 , R100 — 1113 inun R200 is independently for each occurrence absent, CO , R 100 — 1113 and 65 NH, O , S , SS, C ( CH?) SS?, CH ( CH? ) S ? S , C( O ), OC( O ), C ( O ) O , NHC ( O ), C ( O ) NH, NHCH , CH , CHANH, CHAO , CH?N20, heterogryl , heterocycle , US 9 , 814 ,777 B2 18 R200 - L212 R200 - L212

?? w Now , Or and R200 — 1212 w titoR200 — 1212 In one embodiment, L211 is chosen from a group consist to10 inglomeing of embodimen w À N R200 - L213 R200 L213 L211 is L213 , C mu * R200 — L 213 R200 L213 , wa and R200 — 1213 1213 ç L213 — R200 R200 mi - R200 R200 — 1213 , 25 tytt L213 R200 — 1213 In one aspect the present invention provides targeting R200 — L213 or 20 lipids having the structure shown in formula ( I ): L ^[ - P - Q -R -] T _ - LB Formula (1 ) than wherein : 1 213 L213 — R200 14 is a ligand chosen from a carbohydrate , glucose , R200 35 mannose , galactose , N - acetyl- galactosamine , fucose, glu cosamine, lactose , maltose , folate , peptide , or has the struc ture shown in formula II - V : R200 mann 1 .213 . forse 40 { p24– 221– 2345 , 21 124 – 124 Formula (II ) L212 is independently for each occurrence lipid , lipophile , alkyl, alkenyl or alkynyl, each of which is optionally sub stituted with one or more substituents ; optionally sub - 4545 L213 is independently for each occurrence — CH CH 4p2 - 22 – 235 20 126- 128, (OCH ,CH2 ) ,O (CH2 ) ,CH2 - L214 ; Formula ( III ) L214 is independently for each occurrence H , OH , { pat- q = t = R* 123# 134 - LM OCH39 - NH2, N ( H )CH3 , N (CH3 ) 2 , SH , SCH3, 50 N _ N , 2COOH , C ( O) NH ), C ( O ) NHNH ), - CH = CH2, C = CH or p = 28 –2 # 16– 13 Formula ( IV ) 55 par– Q61 – po * batu Tal= 144 m *9 * 8 – 9 * 5 = R * * tydp -18 –24 , or # p54 – Q54 – R54125A - T54 – L 54 Formula ( V ) mut t p5B – Q5B - R5B 105B TSB – L 5B ; p is 1 -1000 ; and p5C - Q50 – R5CD T5C – 150 q is 1 -20 In one embodiment, L 210 is chosen from a group consist- q , q24 , q2B , 234 , 23B , 244 , 24B , 254 , q5B and q5C represent ing of independently for each occurrence 0 - 20 ; US 9 , 814 , 777 B2 19 20 P , P24 , P2B , P34 , P3B , P44 , P4B , PS4 , P5B , PSC, T, T24 , T2B , - continued T32 , T3 , T44, T4B , 144 , TSB and TSC are each independently SS S - S for each occurrence absent, NR' , O , S , C ( O ), OC (O ), C (O ) O , mm om or : NHC( O ) , C( O) NH, NHCH , CH , CHÚNH or CH2O, 5 mm w NHCH( R ) C ( O ), C( O ) CHR° ) NH?, CO , CH = N / O , CH2S , urea, heterocycle , heteroaryl, L24 , L 2B , L34 , L 3B , L 44 , L4B, L54 , L 5B and L5C are each 10 independently for each occurrence a carbohydrate , glucose , mannose , galactose , N -acetyl - galactosamine , fucose , glu

w cosamine, lactose , maltose, folate or a peptide; NH ? R ' and R " are each independently H , CH3, OH , SH , NH2, no NR1°R20 , alkyl, alkenyl or alkynyl; SS , Or Ra is H or amino acid side chain ; Riº and R20 are each independently alkyl, alkenyl or my w my own 20 alkynyl; - S , L64 and L6B are each independently alkyl, alkenyl or my m w alkynyl, each of which is optionally substituted with one or Q , Q24 , Q2B , Q34 , Q3B , Q44 , Q4B , QSA , Q5B and QSC are 25 more substituents ; independently for each occurrence absent , (CH ) n - , m represent independently for each occurrence 0 -50 ; - C (R ') ( R " )( CH2 ) , – (CH2 ) mC ( R ') ( R " ) — , n represent independently for each occurrence 1 - 20 ; and - (CH2CH2O ), CH2CH2- , or (CH2CH2O ) p represent independently for each occurrence 0 -50 . CH?CH NH =; 30 When any of q , q24 , 2B , 934 , q3B , 244 , 24B, 954 , q5B and LP is a ligand selected from a group consisting of lipo - q5C is greater than 1, the repeating unit can be the same or phile , steroid ( e .g . , uvaol, hecigenin , diosgenin ) , terpene different from each other , for example when q is 3 the unit ( e. g ., triterpene , e .g ., sarsasapogenin , Friedelin , epifriedela - [ P - Q -R ] , - is expanded to [P - Q - R ] — [ P - Q - R ] — [ P - Q nol derivatized lithocholic acid ), vitamin (e .g . , folate , vita - 35 R ] — and all of the [ P - Q -R ] — units can be the same, min A , biotin , pyridoxal ), ceramide or has the structure of completely different from each other or a mixture thereof. formula (VI ) : The lipophilic moiety can be chosen , for example, from the group consisting of a lipid , cholesterol, oleyl, linoleoyl, Formula ( VI) lauroyl, docosnyl, stearoyl, retinyl, cholesteryl residues , R64 – L 64 cholic acid , adamantane acetic acid , 1 -pyrene butyric acid , dihydrotestosterone, 1, 3 -Bis - O ( hexadecyl) glycerol, gerany loxyhexyl group , hexadecylglycerol, borneol, menthol, 1, 3 R6B – L6B ; 45 propanediol, heptadecyl group, palmitic acid , myristic acid , wo 03- ( oleoyl ) lithocholic acid , 03- ( oleoyl ) cholenic acid , dimethoxytrityl, phenoxazine or a acid . A preferred R , R², R24 , R2B , R34 , R3B , R44 , R4B , RS4 , RSB , RSC, R , lipophilic moiety is cholesterol. R $ 4 and RØB are each independently for each occurrence 50 absent, CO, NH, NR' , O , S , C( O) , OC( O ), C( O ) O, NHC( O) , In one embodiment , L4 is mannose, galactose , N -acetyl C ( O )NH , NHCH , CH , CHÚNH or CHO, NHCH( R ) C( O ), galactosamine or has the structure shown in formula V . In a C( O ) CH( R' ) NH?, CO, CH?N?, preferred embodiment, 14 is mannose . In one embodiment L 4 has the structure shown in formula V. In one embodiment , L4 is

m HO HO N , . HO Nm 60 m Wat HOL S - S

w they have a65 US 9 , 814 ,777 B2 In one embodiment, L4 is 22

OH HO

HO AcHN

HO OH . HO AcHN OH HO?? HO N AcHN NH

In one embodiment both L24 and L2B are the same. 25 In one embodiment, each of L64 and LB includes two In one embodiment both L 24 and L2B are different. double bond moieties . In one embodiment, at least one of the In one embodiment both L3A and B are the same. double bonds has a Z configuration . In one embodiment, In one embodiment both L 34 and L 3B are different. both of the double bonds have a Z configuration . In one In one embodiment both L 44 and L4B are the same. 30 embodiment, at least one of R2 and R3 is provided in formula In one embodiment both L 44 and L 4B are different . (VII ) below In one embodiment all of L 54 , L 5B and L5C are the same . In one embodiment two of L 54 , L 5B and L5C are the same. In one embodiment LSA and B are the same and LSC is formula ( VII ) different . 35 In one embodiment L 54 and L5C are the same and L5B is different. In one embodiment L 5B and L5C are the same and I 54 is different. In one embodiment L64 and L6B are the same. 40 wherein : In one embodiment LÓA and LB are different. x is an integer from 1 to 8 ; and In one embodiment, each of RÓA and RB are O , C ( O ) , NH or NR '. y is an integer from 1 -10 . In one embodiment, each of L 64 and L6B are indepen - In one embodiment, both of LºA and LP are of the dently alkyl, for example C . -C29 alkyl, e . g ., C . -C18 alkyl , 45 formula ( VII ) . In one embodiment, at least one of the double e . g . , C , 4 alkyl. In one embodiment, both R2 and R3 are alkyl, bonds has an E configuration , e . g . , both of the double bonds e . g ., straight chain alkyl having the same length , e . g ., C6 -C28 have an E configuration . In one embodiment, at least one of alkyl, e . g . , C10 -C18 alkyl, e . g ., C14 alkyl or C16 alkyl. In one LA and LB is provided in formula (VIII ) below embodiment, both R² and Rºare C14 alkyl. In one embodiment, the formula VI represents a racemic 50 mixture formula (VIII ) In one embodiment, the compound of formula VI has an enantiomeric excess of the R isomer, e . g . , at least about 65 % , 70 % , 75 % , 80 % , 85 % , 90 % , 95 % , 97 % , 98 % , or 99 % . wi In one embodiment the formula VI represents enantiomeri - 55 cally pure ' R ' isomer. In one embodiment, the compound of formula VI has an wherein : enantiomeric excess of the S isomer , e. g ., at least about65 % , 70 % , 75 % , 80 % , 85 % , 90 % , 95 % , 97 % , 98 % , or 99 % . In x is an integer from 1 to 8; and one embodiment the formula VI represents enantiomerically 60 y is an integer from 1 - 10 . pure ' S ' isomer . In one embodiment , each of L64 and L6B includes three In one embodiment, each of L 5A and LB are indepen - double bond moieties . In one embodiment, at least one of the dently alkenyl, for example , each of L64 and L6B are inde - double bonds has a Z configuration . In one embodiment, at pendently C . -C30 alkenyl or each of L64 and LB are the least two of the double bonds have a Z configuration . In one same alkenyl moiety . In one embodiment, each of L64 and 65 embodiment, all three of the double bonds have a Z con LB includes one double bond , for example a double bond in figuration . In one embodiment, at least one of LÓA and LØB the E or Z configuration . is provided in formula ( IX ) below US 9 , 814 , 777 B2 24 IX ) 23 embodiment, all three of the double bonds have an E configuration . In one embodiment, at least one of LÓA and LB is provided in formula ( X ) below formula ( IX )

w formula ( X ) wherein : 10 mu x is an integer from 1 to 8 ; and y is an integer from 1 - 10 . In one embodiment, both of LÓA and LB are as provided wherein : in formula (IX ) . In one embodiment, at least one of the x is an integer from 1 to 8 ; and double bonds has an E configuration . In one embodiment, at y is an integer from 1- 10 . least two of the double bonds have an E configuration . In one In one embodiment, LP is

min .

JI

N

35 In one embodiment, LP is chosen from a group consisting of diacyl glycerol, distearylglycerol, dipalmitoylglycerol, dimyristoyl glycerol , dioleoyl glycerol, or other diacyl /steryl hydrophobic groups . In one embodiments , LP is

In one preferred embodiment, LP is mu

In another preferred embodiment, LP is

IUC US 9 , 814 , 777 B2 25 26 In one preferred embodiment formula I has the structure

HO HO HO

NH 111111 PEG2000 111111 ITIT

Di

In another preferred embodiment formula I has the HO HONHO HO —

110lllll 1111111

NH

In one preferred embodiment formula I has the structure

HO HOM HO

NH IZ PEG2000 US 9 ,814 ,777 B2 28 - continued

In another preferred embodiment formula I has the structure

H0 H } H0 H0

illll

In one preferred embodiment formula I has the structure

UH H07 H H0 AcHN _ OH ?r - H0

H0 ?? AcHN

UH ? I H0

H0 ?? z? AcHN

IIIII? Illllll: ????? US 9 , 814 , 777 B2 29 30 In another preferred embodiment formula I has the structure FIO ,QI _

AcHN

0H |HO ?? | MoH0. ? AcHN IA 0H

ACHN

TZ IllIIIIi PEG- 20f00 11IIIllll

TZ

In one preferred embodiment formula I has the structure

H0

H0 ? AcHN ? 0H ???????? H0 ,

HO AcHN N

H020 ? rri

H } TZ AcHN www US 9 , 814 , 777 B2 31 US9 , 814 , 777 B2 32 In another preferred embodiment31 formula I has the structure

OH ??

HO HungaryAcHN OH HO

HO AcHN Wyvyyut

??

HO TZ IZ bunyongeAcHN

ZI wyniPEG - 2000

In one aspect the invention features targeting lipid monomer having the structure shown in formula (VII )

(VII ) O NH

NH NH ?? Linker R300 NHO wherein R300 is a ligand . 50 In one aspect, the invention features targeting lipids of the formula (VIII -XV ):

(VIII ) O NH

NH . NH NH Z - som R 300

NH US 9 , 814 ,777 B2 33 34 -continued ( IX )

IZ

NH

mototrisR300

N R300 IZ

( XI) R3000

HN US 9 , 814 ,777 B2 35 36 -continued

NH

III emNH

( XII ) R 3000

HistoryHN US 9 , 814 ,777 B2 37 38 -continued (XIII ) R300

NH

IIIII

NH

(XIV ) R 300

NH

(XV )

NH HO 20

O = H NH NH NH NH2

Wharee60 wherein R300 is a ligand ; n is 0 - 20 ; and peptidase sensitive peptides, phosphates, triazole linkage x is an ether linkage , a thioether linkage , a carbamate derived from azide and alkyne , and / or a combination of these . linkage , a urethane linkage , a biocleavable linker ( such as In one embodiment, R has the structure shown in formula disulfides, esters , amides ) , pH sensitive linker ( such as as (II ) - ( V ) hydrazones , oximes, acetals /ketal , orthoesters, CDM (Ref : In one embodiment, R is chosen from group shown in Proc. Natl. Acad . Sci. USA 2007 , 104 (32 ), 12982 -12987 ) ), FIG . 8 . US 9 , 814 , 777 B2 40 In one embodiment , R is chosen from group consisting of

OH HO

HO AcHN HO OH

HO NH AcHN OH HO?? NH IZ AcHN

??

HON HO

OH HO

, ?? NH HN AcHN

HonestyOH HO

HO AcHN

HO OH HOI HO

HO

OH HO HO

HO NH , OH HO

HN US 9 , 814 ,777 B2 41 US9 814 ,777 B2 42 441 continued- continued 'HO ?? 1111 HO H NH NHAC AcHN

OH HO OH

HO NH BundangAcHN

??

NH HO HN AcHN OH IndenynHO

HO HuntingAcHN is HO HO LOH HO HO OH H? OH HOA OH - LOH LO HO

HO HO HO ito Omma O

HO - 04 O , OH- HN HO HO HÓ e HNHNN sono ?? , Me

IZ w

HN NH HNH N Dorothy N US 9 , 814 ,777 B2 4343 44 - continued

CO Me , and

HN NH HN

HN TN H?N wanneerOOH on novely HN NH H N ' N

wherein x ???is an ether linkage , a thioether linkage , a carbam peptidase sensitive peptides , phosphates , triazole linkage ate linkage, a urethane linkage , a biocleavable linker ( such derived from azide and alkyne , and / or a combination of as disulfides , esters , amides) , pH sensitive linker ( such as these . hydrazones, oximes , acetals /ketal , orthoesters, CDM (Ref : In some embodiments , the present invention provides com Proc . Natl . Acad . Sci . USA 2007 , 104 (32 ), 12982 -12987 ) ), pounds of Table 1 . TABLE 1 Some preferred embodiments. H2NN Folate : stable COOH hydrophobic alkyl tether HN X Lipid /PEG -Lipid olyawNH . com 2 H2N N Folate : stable COOH hydrophilic HN x Lipid /PEG - Lipid tether hoolNH yumurtament 3 HN Pteroate : stable hydrophobic HN alkyl tether MeginmasNH X Lipid /PEG -Lipid 4 HNH2NN Pteroate : hydrophilicstable HN M otx - Lipid PEG -Lipid tether OS hieman teman 5 H2N Folate : COOH disulfide | linkage HN . X - Lipid /PEG -Lipid US 9 ,814 ,777 B2 45 46 TABLE 1 - continued 46 Some preferred embodiments . 6 Folate : gem H2NNV COOH dimethyldisul Lipid /PEG - Lipid fide linkage HN . ALL ?? Kayuman 7 Folate : stable H2NN ? ???? hydrophobic HN . X — Drug Delivery Scaffold alkyl tether LigiNH yetament HN . Folate : stable COOH hydrophilic HN . Dry ivery Scaffold tether NH er O lige nyheter for storming | HN, N . Pteroate : stable hydrophobic HN alkyl tether TIL DimN ondanDEIIV Scaffoldsonra HN . Pteroate : 10 HNAN stable hydrophilic tether LigitaDrug Us DeliveryDeliver Scaffold Folate : 11 HNN COOH disulfide linkage HN TS- S X — Drug Delivery Scaffold Mag " NH -iry an Folate : gem 12 H2NN . COOH dimethyldisul X4 Drug Delivery Scaffold fide linkage HN . N NH n is 0 - 20 ; x is an ether linage, a thioether linkage, a carbamate linkage , a urethane linkage, a biocleavable linker ( such as disulfides, esters , amides) , pH sensitive linker ( such as hydrazones , oximes, acetals /ketal , orthoesters, CDM (Ref : Proc . Natl . Acad . Sci. USA 2007 , 104 (32 ) , 12982 - 12987 ) ) , peptidase sensitive peptides, phosphates, triazole linkage derived from azide and alkyne , and /or a combination of these . 45 In one aspect the present invention provides drug delivery In one embodiment, tether/ linker that links the drug systems conjugated with targeting ligands . delivery system to targeting moiety is conjugated or asso Drug delivery system (also to referred to as “ drug delivery c iated with an endosomal release agent. scaffold ” herein ) can be based on a polymeric scaffold . Polymeric delivery systems include linear or branched poly - 50 Endosomal release agents include imidazoles , poly or mers , dendrimers , water soluble , biocompatible , biodegrad oligoimidazoles , PEIs , peptides , fusogenic peptides , poly able , pH sensitive, cationic , anionic , neutral, hydrophilic , carboxylates, polycations, masked oligo or poly cations or hydrophobic with or without endosomal release agent. Poly anions , acetals , polyacetals , ketals / polyketyals and / or mers also include pH sensitive masking of polyanionic or orthoesters. polycationic polymers , peptides, polysaccharides , oligosac - 55 In one aspect, the drug delivery system is based on charides, polyglycidols . Tethers and linkages between the liposomal , , micelle , membranous formulations , polymer and targeting moiety are same or similar to that of , , nano - and micro - emulsions , intra the lipid - ligand conjugates described herein . lipid , soybean based formulations , soybean fatty oil, fatty oil In one embodiment, the drug delivery system is conju - based , fish oil ( omega - 3 ) , antibody, lipidoids and dry powder gated or associated with a moiety that can modulate the PK 60 formulations. properties of the delivery system . In preferred embodiments liposomes are cationic , anionic In one embodiment, the drug delivery system is conju - or neutral . gated or associated with an endosomal release agent. In preferred embodiments are cationic , anionic In one embodiment, the drug delivery system is conju - or neutral. gated or associated with an endosomal release agent and a 65 The kidney contains a high -affinity folate binding protein moiety that can modulate the PK properties of the delivery (FBP ) concentrated in the proximal tubular cells ( Int. Rev . system . Cytol. 180 .237 - 284, 1998 ) , Therefore a nucleic acid thera US 9 ,814 ,777 B2 47 48 peutic agent ( e . g ., siRNA or antagomir ) can be targeted to lanosterol, and squalene , campesterol, sitosterol, stigmas the kidney , by targeted delivery of nucleic acid therapeutics terol, ergosterol, brassinsterol, distearyl- lithocholamide , using the targeted delivery approach of the present inven - borneol, menthol, heptadecyl group , dialkylglycerides, dia tion . cylglyceride, and bile acids . When used herein , terms to Drug in the present invention is a nucleic acid therapeutic 5 describe the lipophilic moiety such as a free acid ( such as a or an iRNA agent such as siRNA , antagomir , microRNA , free fatty acid , e . g ., palmitic acid ) are used interchangeably antisense, aptamer, plasmids, decoy RNA , immunostimula with the radical term ( e . g . , palmitoyl) . tory oligonucleotides , antisense microRNAs, splice modu - Carbohydrate lating oligonucleotides, RNA activating oligonucleotides As used herein , " carbohydrate ” refers to a compound etc . The drug is either conjugated or formulated with the 10 which is either a carbohydrate per se made up of one or more delivery system . In some embodiments , the drug is conju - monosaccharide units having at least 6 carbon atoms (which gated with the tether /linker that links the targeting moiety to may be linear , branched or cyclic ) with an oxygen , nitrogen the delivery system . or sulfur atom bonded to each carbon atom ; or a compound In a further aspect, this invention provides a method of having as a part thereof a carbohydrate moiety made up of modulating expression of a target gene , themethod includes 15 one or more monosaccharide units each having at least six administering a drug as defined herein formulated or con - carbon atoms (which may be linear, branched or cyclic ) , jugated with the drug delivery system described herein . with an oxygen , nitrogen or sulfur atom bonded to each In one aspect, this invention features a pharmaceutical carbon atom . Representative carbohydrates include the sug composition having a nucleic acid formulated or conjugated ars (mono -, di- , tri- and oligosaccharides containing from with the drug delivery system described herein and a phar- 20 about 4 - 9 monosaccharide units ), and polysaccharides such maceutically acceptable carrier. as starches, glycogen , cellulose and polysaccharide gums. In further related embodiments , the present invention Specific monosaccharides include C , and above (preferably includes a lipid particle comprising one ormore of the above C3 -C3 ) sugars ; di- and trisaccharides include sugars having lipids of the present invention . In certain embodiments , the two or three monosaccharide units (preferably C . -C2 ) . particle further comprises a targeting lipid described in this 25 The term " monosaccharide ” embraces radicals of allose , application , a cationic lipid , a neutral lipid and a lipid altrose , arabinose , cladinose , erythrose , erythrulose , fruc capable of reducing particle aggregation . In one particular tose, D - fucitol, L - fucitol, fucosamine , fucose, fuculose , embodiment, the lipid particle consists essentially of: (i ) a galactosamine , D - galactosaminitol, N -acetyl - galac targeting lipid (ii ) an amino lipid ( iii ) a neutral lipid selected tosamine , galactose , glucosamine , N - acetyl- glucosamine , from DSPC , POPC , DOPE , and SM ; (iv ) cholesterol ; and (v ) 30 glucosaminitol, glucose , glucose -6 -phosphate , gulose glyc PEG -DMG , PEG - C -DOMG or PEG -DMA , in a molar ratio eraldehyde, L -glycero - D -mannos -heptose , glycerol, glyc of about 0 . 5 -50 % targeting lipid : 20 -60 % cationic lipid : 5 erone , gulose , idose , lyxose , mannosamine , mannose , man 25 % neutral lipid :25 - 55 % Chol: 0 . 5 - 15 % PEG -DMG or nose -6 -phosphate , psicose, quinovose , quinovosamine, PEG -DMA . rhamnosamine, rhamnose , ribose , ribulose , sedoheptulose , Folate 35 sorbose , tagatose , talose , tartaric acid , threose , xylose and As used herein , the term “ folate ” is meant to refer to folate xylulose . The monosaccharide can be in D - or L - configu and folate derivatives, including pteroic acid derivatives and ration . The monosaccharide may further be a deoxy sugar analogs . The analogs and derivatives of folic acid suitable (alcoholic hydroxy group replaced by hydrogen ) , amino for use in the present invention include, but are not limited sugar ( alcoholic hydroxy group replaced by amino group ) , a to , antifolates, dihydrofloates , tetrahydrofolates, tetrahy - 40 thio sugar ( alcoholic hydroxy group replaced by thiol, or drorpterins, folinic acid , pteropolyglutamic acid , 1 - deza , CEO replaced by C = S , or a ring oxygen of cyclic form 3 -deaza , 5 -deaza , 8 - deaza , 10 - deaza , 1, 5 - deaza , 5 , 10 replaced by sulfur ) , a seleno sugar, a telluro sugar , an aza dideaza , 8, 10 -dideaza , and 5 ,8 -dideaza folates, antifolates, sugar (ring carbon replaced by nitrogen ), an sugar ( ring and pteroic acid derivatives . Additional folate analogs are oxygen replaced by nitrogen ), a phosphano sugar ( ring described in published US publication US2004 / 0 , 242 ,582 45 oxygen replaced with phosphorus ) , a phospha sugar ( ring ( published Dec . 2 , 2004 ) . carbon replaced with phosphorus ) , a C - substituted mono Lipid /Lipophile saccharide (hydrogen at a non -terminal carbon atom The terms " lipid ” and “ lipophile ” refer to any fat- soluble replaced with carbon ), an unsaturated monosaccharide, an molecule such as fats , oils , waxes , terpenes, sterols , fat - alditol ( carbonyl group replaced with CHOH group ) , aldonic soluble vitamins ( e . g ., A , D , E and K ) , monoglycerides , 50 acid ( aldehydic group replaced by carboxy group ) , a ketoal diglycerides , triglycerides , fatty acids , hopanoids and phos - donic acid , a ironic acid , an aldaric acid , and so forth . Amino pholipids. Exemplary lipophilic molecules include , but are sugars include amino monosaccharides , preferably galac not limited to , cholesterol, progestesrone , testosterone , tosamine , glucosamine, mannosamine , fucosamine, quino estradiol, norethindfrone , cortisone , cholic acid , 03 - ( oleoyl) vosamine, neuraminic acid , muramic acid , lactosediamine , lithocholic acid , cholenic acid , 03 - ( oleoyl )cholenic acid , 55 acosamine , bacillosamine, daunosamine , desosamine , foro chenodecoxy cholic acid , glycocholic acid , taurocholic acid , samine , garosamine , kanosamine, kansosamine, myca dexoycholic acid , adamantane acetic acid , 1 - pyrene butyric minose , mycosamine, perosamine, pneumosamine, purpuro acid , dihydrotestosterone, 1 , 3 - Bis - O (hexadecylglycerol , samine , rhodosamine. It is understood that the geranyloxyhexyl group , hexadecylglycerol, borneol, men - monosaccharide and the like can be further substituted . thol, 1 , 3 - propanediol, heptadecyl group , palmitic acid , 60 The terms " disaccharide” , “ trisaccharide” and “ polysac myristic acid , dimethoxytrityl, phenoxazine , polycyclic aro - charide " embrace radicals of abequose , acrabose , amicetose , matic hydrocarbons ( e . g ., phenazine , dihydrophenazine ) , amylopectin , amylose , apiose , arcanose , ascarylose , ascor lauric acid , stearic acid , arachidic acid , palmitoleic acid , bic acid , boivinose , cellobiose, cellotriose , cellulose , cha oleic acid , linoleic acid , linolenic acid , archidonic acid , cotriose, chalcose , chitin , colitose , cyclodextrin , cymarose , leukotriene A , myrcene, geraniol, carvone , chrysanthemic 65 dextrin , 2 - deoxyribose , 2 -deoxyglucose , diginose, digi acid , nepetalactone , menthofuran , alpha - pinene, camphor, talose , digitoxose , evalose , evemitrose , fructooligosach farnesol, humulene , nagione, caryophyllene , abieticv acid , haride, gentianose , gentiobiose , glucan , glucogen , glycogen , US 9 , 814 , 777 B2 49 50 hamamelose , heparin , inulin , isolevoglucosenone , isomalt TABLE 2 - continued ose , isomaltotriose , isopanose, kojibiose , lactose . lac tosamine , lactosediamine , laminarabiose , levoglucosan , List of peptides with endosomolytic activity . levoglucosenone, B -maltose , maltriose , mannan -oligosac Name Sequence ( N to C ) Ref . charide, manninotriose , melezitose , melibiose , muramic 5 acid , mycarose, mycinose , neuraminic acid , nigerose , Inf HA - 2 GLFGAIAGFIENGWEGMIDGWYG nojirimycin , noviose , oleandrose, panose , paratose , plant diINF - 7 GLF EAI EGFI ENGW EGMI DGWYGC u eose, primeverose , raffinose , rhodinose , rutinose , sarmen GLF EAI EGFI ENGW EGMI DGWYGC tose , sedoheptulose , sedoheptulosan , solatriose , sophorose , diINF3 GLF EAI EGFI ENGW EGMI DGGC a6 stachyose , streptose , sucrose , a ,a -trehalose , trehalosamine , GLF EAI EGFI ENGW EGMI DGGC turanose , tyvelose , xylobiose , umbelliferose and the like . Further, it is understood that the “ disaccharide” , “ trisaccha GLF GLFGALAEALAEALAEHLAEALAEALEALAAGGSC a ride” and “ polysaccharide ” and the like can be further substituted . Disaccharide also includes amino sugars and GALA - INF3 GLFEAIEGFIENGWEGLAEALAEALEALAAGGSC 6 their derivatives , particularly , a mycaminose derivatized at 15 INF - 5 GLF EAI EGFI ENGW EGnI DG K the C - 4 ' position or a 4 deoxy - 3 - amino - glucose derivatized GLF EAI EGFI ENGW EGNI DG at the C -6 ' position . n , norleucine Ligands References A wide variety of entities can be used as ligands for 1 . Subbarao et al . , Biochemistry , 1987 , 26 : 2964 - 2972 . conjugation according to the present invention . Preferred 20 2 . Vogel et al . , J . Am . Chem . Soc . , 1996 , 118 : 1581 - 1586 3 . Turk , M . J . , Reddy , J . A . et al . ( 2002 ) . Characterization of moieties are ligands, which are coupled , preferably cova a novel pH - sensitive peptide that enhances drug release from lently , either directly or indirectly via an intervening tether. folate - targeted liposomes at endosomal . Biochim . Biophys . Acta 1559 , 56 - 68 . In preferred embodiments, a ligand alters the distribution , 4 . Plank , C . Oberhauser , B . Mechtler , K . Koch , C . Wagner , E . ( 1994 ) . The influence of endosome - disruptive peptides on gene targeting or lifetime of the molecule into which it is incor transfer using synthetic virus - like gene transfer systems , J . porated . In preferred embodiments a ligand provides an Biol . Chem . 269 12918 - 12924 . enhanced affinity for a selected target , e . g ., molecule , cell or 5characterization . Mastrobattista ,of E . an, Koning endosome , G . - disruptiveA . et al . ( 2002peptide ) . Functional and its application in cytosolic delivery of immunoliposome - entrapped cell type, compartment, e .g ., a cellular or organ compart proteins . J . Biol . Chem . 277 , 27135 - 43 . ment, tissue , organ or region of the body, as, e . g . , compared 6 . Oberhauser , B . , Plank , C . et al . ( 1995 ) . Enhancing endosomal to a species absent such a ligand . Ligands providing Delivexit of . Strategiesnucleic acids Antisense using pH oligonucleotide - sensitive viral Therfusion . 247 peptides - 66 . . enhanced affinity for a selected target are also termed 30 targeting ligands. Ligands can improve transport, hybridization , and speci Some ligands can have endosomolytic properties. The ficity properties and may also improve nuclease resistance of endosomolytic ligands promote the lysis of the endosome the resultant natural or modified oligoribonucleotide , or a and / or transport of the composition of the invention , or its polymeric molecule comprising any combination of mono components , from the endosome to the cytoplasm of the Wellcell. 35 mers described herein and/ or natural or modified ribonucle The endosomolytic ligand may be a polyanionic peptide or otides. peptidomimetic which shows pH -dependent membrane Ligands in general can include therapeutic modifiers, e . g . , activity and fusogenicity . In certain embodiments , the endo for enhancing uptake ; diagnostic compounds or reporter somolytic ligand assumes its active conformation at endo groups e . g . , for monitoring distribution ; cross -linking somal pH . The “ active” conformation is that conformation inin 40 agagents ; and nuclease - resistance conferring moieties . General which the endosomolytic ligand promotes lysis of the endo examples include lipids , steroids , vitamins, sugars , proteins , some and / or transport of the composition of the invention , or peptides, polyamines , and peptide mimics. its components , from the endosome to the cytoplasm of the Ligands can include a naturally occurring substance , such cell. Exemplary endosomolytic ligands include the GALA as a protein ( e . g ., human serum albumin (HSA ) , low - density peptide (Subbarao et al. , Biochemistry , 1987 , 26 : 29644 -. 4545 lipoprotein (LDL ), high - density lipoprotein (HDL ), or 2972 ) , the EALA peptide ( Vogel et al. , J . Am . Chem . Soc ., globulin ); an carbohydrate ( e . g . , a dextran , pullulan , chitin , 1996 , 118 : 1581 - 1586 ), and their derivatives ( Turk et al ., chitosan , inulin , cyclodextrin or hyaluronic acid ) ; or a lipid . Biochem . Biophys . Acta , 2002 , 1559 : 56 - 68 ) . In certain The ligand may also be a recombinant or synthetic molecule , embodiments , the endosomolytic component may contain a such as a synthetic polymer , e. g ., a synthetic polyamino acid , chemical group ( e . g . , an amino acid ) which will undergoDa a 50 an oligonucleotide ( e . g . an aptamer ). Examples of change in charge or protonation in response to a change in polyamino acids include polyamino acid is a polylysine pH . The endosomolytic component may be linear or (PLL ), poly L -aspartic acid , poly L - glutamic acid , styrene maleic acid anhydride , poly ( L - lactide- co - glyco branched . Exemplary primary sequences of peptide based lied ) copolymer, divinyl ether -maleic anhydride copolymer, endosomolytic ligands are shown in Table 2 . 55 N - (2 -hydroxypropyl ) methacrylamide copolymer (HMPA ), polyethylene glycol (PEG ), polyvinyl alcohol (PVA ), poly TABLE 2 urethane, poly ( 2 - ethylacryllic acid ) , N - isopropylacrylamide List of peptides with endosomolytic activity . polymers, or polyphosphazine. Example of polyamines include : polyethylenimine, polylysine (PLL ), spermine , Name Sequence ( N to C ) Ref . 60 spermidine, polyamine , pseudopeptide- polyamine , peptido GALA AALEALAEALEALAEALEALAEAAAAGGC mimetic polyamine , dendrimer polyamine , arginine , ami -am dine , protamine , cationic lipid , cationic porphyrin , quater EALA AALAEALAEALAEALAEALAEALAAAAGGC nary salt of a polyamine, or an alpha helical peptide . ALEALAEALEALAEA Ligands can also include targeting groups, e . g . , a cell or INF - 7 GLFEAI EGFIENGWEGMIWDYG 4 65 tissue targeting agent, e . g ., a lectin , glycoprotein , lipid or protein , e . g ., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin , US 9 , 814 , 777 B2 51 52 melanotropin , lectin , glycoprotein , surfactant protein A , lectins, carbohydrates , vitamins, cofactors , multivalent lac Mucin carbohydrate , multivalent lactose , multivalent galac tose, multivalent galactose , N -acetyl - galactosamine , tose , N -acetyl - galactosamine , N - acetyl- glucosamine multi - N - acetyl- gulucosamine multivalent mannose, multivalent valent mannose , multivalent fucose , glycosylated fucose , or aptamers. The ligand can be , for example, a polyaminoacids, multivalent galactose , transferrin , bispho - 5 lipopolysaccharide , an activator of p38 MAP kinase , or an sphonate , polyglutamate , polyaspartate , a lipid , cholesterol, activator of NF -KB . The ligand can be a substance , e . g ., a drug , which can a steroid , , folate , vitamin B12, biotin , an RGD increase the uptake of the conjugate into the cell , for peptide, an RGD peptide mimetic or an aptamer . Table 3 example , by disrupting the cell ' s cytoskeleton , e . g . , by shows some examples of targeting ligands and their associ 10 disrupting the cell' s microtubules, microfilaments, and / or ated receptors . intermediate filaments . The drug can be, for example , taxon , vincristine , vinblastine , cytochalasin , nocodazole , TABLE 3 japlakinolide, latrunculin A , phalloidin , swinholide A , Targeting Ligands and their associated receptors indanocine , or myoservin . The ligand can increase the uptake of the conjugate into Liver Cells Ligand Receptor the cell by activating an inflammatory response , for 1 ) Parenchymal Galactose ASGP- R example . Exemplary ligands that would have such an effect Cell ( PC ) ( Asiologlycoprotein include tumor necrosis factor alpha ( TNFalpha ) , interleu ( Hepatocytes ) receptor ) kin - 1 beta , or gamma interferon . Gal NAC ASPG - R (n - acetyl- galactosamine ) Gal NAc Receptor 20 In one aspect , the ligand is a lipid or lipid - based molecule . Lactose Such a lipid or lipid -based molecule preferably binds a Asialofetuin ASPG - r serum protein , e. g ., human serum albumin ( HSA ). An HSA 2 ) Sinusoidal Hyaluronan Hyaluronan receptor binding ligand allows for distribution of the conjugate to a Endothelial Procollagen Procollagen receptor Cell (SEC ) Negatively charged Scavenger receptors target tissue , e . g ., a non - kidney target tissue of the body . For molecules example , the target tissue can be the liver , including paren Mannose Mannose receptors chymal cells of the liver. Other molecules that can bind HSA N - acetylGlucosamine Scavenger receptors Immunoglobulins Fc Receptor can also be used as ligands . For example , neproxin or aspirin LPS CD14 Receptor can be used . A lipid or lipid - based ligand can (a ) increase Insulin Receptor mediated resistance to degradation of the conjugate , ( b ) increase transcytosis 30 targeting or transport into a target cell or cell membrane , Transferrin Receptor mediated transcytosis and / or (c ) can be used to adjust binding to a serum protein , Albumins Non - specific e . g ., HSA . Sugar - Albumin A lipid based ligand can be used to modulate , e . g ., control conjugates the binding of the conjugate to a target tissue. For example , Mannose -6 - phosphate Mannose -6 -phosphate receptor 35 a lipid or lipid -based ligand that binds to HSA more strongly 3 ) Kupffer Mannose Mannose receptors will be less likely to be targeted to the kidney and therefore Cell (KC ) Fucose Fucose receptors less likely to be cleared from the body . A lipid or lipid - based Albumins Non - specific ligand that binds to HSA less strongly can be used to target Mannose -albumin the conjugate to the kidney . conjugates 40 In a preferred embodiment, the lipid based ligand binds HSA . Preferably , it binds HSA with a sufficient affinity such Other examples of ligands include dyes , intercalating that the conjugate will be preferably distributed to a non agents ( e . g . acridines ) , cross - linkers ( e . g . psoralen , mitomy - kidney tissue . However, it is preferred that the affinity not be cin C ) , porphyrins ( TPPC4 , texaphyrin , Sapphyrin ) , poly - so strong that the HSA - ligand binding cannot be reversed . cyclic aromatic hydrocarbons ( e . g . , phenazine , dihydro - 45 In another preferred embodiment, the lipid based ligand phenazine ) , artificial endonucleases ( e . g . EDTA ) , lipophilic binds HSA weakly or not at all , such that the conjugate will molecules , e . g ., cholesterol, cholic acid , adamantane acetic be preferably distributed to the kidney . Other moieties that acid , 1 -pyrene butyric acid , dihydrotestosterone , 1 , 3 - Bis - O target to kidney cells can also be used in place of or in (hexadecyl ) glycerol , geranyloxyhexyl group , hexadecyl - addition to the lipid based ligand . glycerol, borneol, menthol, 1, 3 - propanediol, heptadecyl 50 In another aspect, the ligand is a moiety , e. g ., a vitamin , group , palmitic acid , myristic acid , 03 -( oleoyl) lithocholic which is taken up by a target cell , e . g . , a proliferating cell . acid , O3 - (oleoyl ) cholenic acid , dimethoxytrityl, or phenox . These are particularly useful for treating disorders charac azine ) and peptide conjugates ( e . g . , antennapedia peptide , terized by unwanted cell proliferation , e . g ., of themalignant Tat peptide ) , alkylating agents , phosphate , amino , mercapto , or non -malignant type, e .g ., cancer cells . Exemplary vita PEG ( e . g ., PEG - 40K ) , MPEG , MPEG ] , polyamino , alkyl, 55 mins include vitamin A , E , and K . Other exemplary vitamins substituted alkyl, radiolabeled markers, enzymes , haptens include are B vitamin , e . g . , folic acid , B12 , riboflavin , ( e . g . biotin ) , transport/ absorption facilitators ( e . g ., aspirin , biotin , pyridoxal or other vitamins or nutrients taken up by vitamin E , folic acid ) , synthetic ribonucleases ( e . g ., imida cancer cells . Also included are HAS , low density lipoprotein zole , bisimidazole, histamine, imidazole clusters , acridine - (LDL ) and high -density lipoprotein (HDL ) . imidazole conjugates, Eu3 + complexes of tetraazamacro - 60 In another aspect, the ligand is a cell- permeation agent, cycles) , dinitrophenyl, HRP, or AP . preferably a helical cell -permeation agent. Preferably , the Ligands can be proteins, e . g . , glycoproteins, or peptides , agent is amphipathic . An exemplary agent is a peptide such e . g ., molecules having a specific affinity for a co - ligand , or as tat or antennopedia . If the agent is a peptide , it can be antibodies e . g . , an antibody, that binds to a specified cell modified , including a peptidylmimetic , invertomers , non type such as a cancer cell, endothelial cell, or bone cell . 65 peptide or pseudo -peptide linkages , and use of D -amino Ligands may also include hormones and hormone receptors . acids . The helical agent is preferably an alpha - helical agent, They can also include non -peptidic species, such as lipids, which preferably has a lipophilic and a lipophobic phase. US 9 , 814 ,777 B2 53 54 The ligand can be a peptide or peptidomimetic . A pep delivery peptides. A peptide or peptidomimetic can be tidomimetic ( also referred to herein as an oligopeptidomi encoded by a random sequence of DNA, such as a peptide metic ) is a molecule capable of folding into a defined identified from a phage -display library , or one -bead -one three - dimensional structure similar to a natural peptide. The compound (OBOC ) combinatorial library (Lam et al. , peptide or peptidomimetic moiety can be about 5 -50 amino 5 Nature , 354 : 82 - 84 , 1991 ) . Preferably the peptide or peptido acids long, e . g ., about 5 , 10 , 15 , 20, 25 , 30 , 35 , 40 , 45 , or 50 mimetic tethered to an iRNA agent via an incorporated amino acids long (see Table 4 , for example ). monomer unit is a cell targeting peptide such as an arginine TABLE 4 Exemplary Cell Permeation Peptides .

Cell Permeation Peptide Amino acid Sequence Reference Penetratin RQIKIWFQNRRMKWKK Derossi et al . , J . Biol . Chem . 269 : 10444 , 1994 Tat fragment GRKKRRQRRRPPQC Vives et al . , J . Biol . Chem . , ( 48 - 60 ) 272 : 16010 , 1997 Signal Sequence GALFLGWLGAAGSTMGAWSQPKKKR Chaloin et al . , Biochem . based peptide KV Biophys . Res . Commun . , 243 :601 , 1998 PVEC LLIILRRRIRKOAHAHSK Elmquist et al . , Exp . Cell Res . , 269 : 237 , 2001 Transportan GWTLNSAGYLLKINLKALAALAKKIL Pooga et al . , FASEB J . , 12 : 67 , 1998 Amphiphilic KLALKLALKALKAALKLA Oehlke et al . , Mol . Ther . , model peptide 2 : 339 , 2000 Arg , RRRRRRRRR Mitchell et al . , J . Pept . Res . , 56 : 318 , 2000 Bacterial cell wall KFFKFFKFFK permeating LL - 37 LLGDFFRKSKEKIGKEFKRIVORIKDFL RNLVPRTES Cecropin P1 SWLSKTAKKLENSAKKRISEGIAIAIOG GPR a - defensin ACYCRIPACIAGERRYGTCIYQGRLWA FCC B - defensin DHYNCVSSGGQCLYSACPIFTKIQGTC YRGK????? Bactenecin RKCRIVIRVCR PR - 39 RRRPRPPYLPRPRPPPFFPPRLPPRIPPGF PPRFPPRFPGKR - NH2 Indolicidin ILPWKWPWWPWRR - NH2

A peptide or peptidomimetic can be , for example , a cell glycine - aspartic acid (RGD ) -peptide , or RGD mimic A permeation peptide, cationic peptide , amphipathic peptide , peptide moiety can range in length from about 5 amino acids or hydrophobic peptide ( e . g . , consisting primarily of Tyr, to about 40 amino acids . The peptide moieties can have a Trp or Phe) . The peptide moiety can be a dendrimer peptide , structural modification , such as to increase stability or direct constrained peptide or crosslinked peptide. In another alter - 55 conformational properties . Any of the structural modifica native , the peptide moiety can include a hydrophobic mem - tions described below can be utilized . brane translocation sequence (MTS ) . An exemplary hydro - An RGD peptide moiety can be used to target a tumor cell , phobic MTS -containing peptide is RFGF having the amino such as an endothelial tumor cell or a breast cancer tumor acid sequence AAVALLPAVLLALLAP. An RFGF analogue cell ( Zitzmann et al. , Cancer Res . , 62 :5139 -43 , 2002 ) . An ( e . g . , amino acid sequence AALLPVLLAAP) containing a 60 RGD peptide can facilitate targeting of an iRNA agent to hydrophobic MTS can also be a targeting moiety . The tumors of a variety of other tissues , including the lung, peptide moiety can be a " delivery ” peptide, which can carry kidney, spleen , or liver (Aoki et al. , Cancer Gene Therapy large polar molecules including peptides, oligonucleotides , 8 :783 - 787 , 2001 ) . Preferably , the RGD peptide will facili and protein across cell membranes . For example , sequences tate targeting of an iRNA agent to the kidney. The RGD from the HIV Tat protein (GRKKRRRRRRPPQ ) and the 65 peptide can be linear or cyclic , and can be modified , e. g ., Drosophila Antennapedia protein (RQIKIWFQNRRMK - glycosylated or methylated to facilitate targeting to specific WKK ) have been found to be capable of functioning as tissues . For example , a glycosylated RGD peptide can US 9 , 814 , 777 B2 55 56 deliver an iRNA agent to a tumor cell expressing außz Endosomal release agents include imidazoles , poly or (Haubner et al. , Jour. Nucl . Med ., 42: 326 - 336 , 2001 ) . oligoimidazoles , PEIs , peptides , fusogenic peptides , poly Peptides that target markers enriched in proliferating cells carboxylates, polycations, masked oligo or poly cations or can be used . E . g ., RGD containing peptides and peptidomi- anions, acetals , polyacetals , ketals / polyketyals , orthoesters , metics can target cancer cells , in particular cells that exhibit 5 polymers with masked or unmasked cationic or anionic an Ivoz integrin . Thus, one could use RGD peptides, cyclic charges, dendrimers with masked or unmasked cationic or peptides containing RGD , RGD peptides that include anionic charges . PK modulator stands for pharmacokinetic modulator. PK D - amino acids, as well as synthetic RGD mimics. In addi modulator include lipophiles , bile acids , steroids , phospho tion to RGD , one can use other moieties that target the 1, -03 10 lipid analogues, peptides , protein binding agents , PEG , integrin ligand . Generally , such ligands can be used to vitamins etc . Exemplary PK modulator include , but are not control proliferating cells and angiogeneis . Preferred con limited to , cholesterol , fatty acids, cholic acid , lithocholic jugates of this type ligands that targets PECAM -1 , VEGF, or acid , dialkylglycerides, diacylglyceride, , other cancer gene , e . g ., a cancer gene described herein . sphingolipids, naproxen , ibuprofen , vitamin E , biotin etc . A cell permeation peptide is capable of permeating a 15 Oligonucleotides that comprise a number of phosphoroth cell, e . g . , a microbial cell, such as a bacterial or fungal cell, ioate linkages are also known to bind to serum protein , thus or a mammalian cell , such as a human cell . A microbial short oligonucleotides , e . g . oligonucleotides of about 5 cell- permeating peptide can be, for example , an a -helical bases, 10 bases, 15 bases or 20 bases, comprising multiple linear peptide ( e. g. , LL -37 or Ceropin P1) , a disulfide of phosphorothioate linkages in the backbone are also ame bond - containing peptide ( e . g . , a - defensin , B -defensin or 20 nable to the present invention as ligands ( e . g . as PK modu bactenecin ) , or a peptide containing only one or two domi lating ligands ). nating amino acids ( e . g . , PR -39 or indolicidin ). A cell In addition , aptamers that bind serum components ( e . g . permeation peptide can also include a nuclear localization serum proteins ) are also amenable to the present invention as signal (NLS ) . For example , a cell permeation peptide can be PK modulating ligands. a bipartite amphipathic peptide, such as MPG , which is 25 Other ligands amenable to the invention are described in derived from the fusion peptide domain of HIV - 1 gp41 and copending applications U .S . Ser . No . 10 /916 , 185 , filed Aug . the NLS of SV40 large T antigen (Simeoni et al. , Nucl. Acids 10 , 2004 ; U . S . Ser. No . 10 / 946 , 873 , filed Sep . 21, 2004 ; U . S . Res . 31: 2717 - 2724 , 2003 ) . Ser. No. 10 /833 , 934 , filed Aug . 3 , 2007 ; U . S . Ser. No . In one embodiment, a targeting peptide can be an amphip 11 / 115 , 989 filed Apr. 27 , 2005 and U . S . Ser. No. 11 / 944 ,227 athic a -helical peptide . Exemplary amphipathic a -helical 30 filed Nov . 21 , 2007 , which are incorporated by reference in peptides include , but are not limited to , cecropins , lycotox - their entireties for all purposes . ins , paradaxins, buforin , CPF , bombinin - like peptide (BLP ) , When two ormore ligands are present, the ligands can all cathelicidins , ceratotoxins, S . clava peptides , hagfish intes - have same properties, all have different properties or some tinal antimicrobial peptides (HFIAPs ), magainines , brevi- ligands have the same properties while others have different nins- 2 , dermaseptins , melittins, pleurocidin , H , A peptides, 35 properties . For example , a ligand can have targeting prop Xenopus peptides, esculentinis - 1 , and caerins. A number of erties, have endosomolytic activity or have PK modulating factors will preferably be considered to maintain the integ - properties . In a preferred embodiment, all the ligands have rity of helix stability . For example , a maximum number of different properties . helix stabilization residues will be utilized ( e . g . , leu , ala , or Linkers / Tethers lys) , and a minimum number helix destabilization residues 40 The terms " linker” and “ tether ” means an organic moiety will be utilized ( e . g . , proline , or cyclic monomeric units . The that connects two parts of a compound . Linkers typically capping residue will be considered ( for example Gly is an comprise a direct bond or an atom such as oxygen or sulfur, exemplary N - capping residue and / or C - terminal amidation a unit such as NR , C ( O ) , C ( O )NH , SO , SO2, SO NH or a can be used to provide an extra H - bond to stabilize the helix . chain of atoms, such as substituted or unsubstituted alkyl, Formation of salt bridges between residues with opposite 45 substituted or unsubstituted alkenyl, substituted or unsub charges , separated by i 3 , or i + 4 positions can provide stituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, het stability . For example , cationic residues such as lysine , eroarylalkyl , heteroarylalkenyl, heteroarylalkynyl, hetero arginine , homo - arginine, ornithine or histidine can form salt cyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, bridges with the anionic residues glutamate or aspartate . heteroaryl , heterocyclyl , cycloalkyl, cycloalkenyl, alkylary Peptide and peptidomimetic ligands include those having 50 lalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, naturally occurring or modified peptides , e. g. , D or L alkenylarylalkenyl , alkenylarylalkynyl, alkynylarylalkyl, peptides; a , b , or y peptides ; N -methyl peptides; azapep - alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylal tides ; peptides having one or more amide, i. e ., peptide , kyl , alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenyl linkages replaced with one or more urea , thiourea , carbam heteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroary ate , or sulfonyl urea linkages ; or cyclic peptides. 55 lalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, The targeting ligand can be any ligand that is capable of alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylhet targeting a specific receptor. Examples are : folate , GalNAc , erocyclylalkenyl, alkylhererocyclylalkynyl, alkenylhetero galactose , mannose, mannose -6P , clusters of sugars such as cyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocy GalNAc cluster, mannose cluster, galactose cluster, or an clylalkynyl, alkynylheterocyclylalkyl, apatamer. A cluster is a combination of two or more sugar 60 alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, units . The targeting ligands also include integrin receptor alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenyl ligands, Chemokine receptor ligands , transferrin , biotin , heteroaryl , alkynylhereroaryl, where one or more methyl serotonin receptor ligands, PSMA, endothelin , GCPII , enes can be interrupted or terminated by O , S , S ( O ) , SO2, somatostatin , LDL and HDL ligands . The ligands can also be N ( R ) , C (O ), cleavable linking group , substituted or unsub based on nucleic acid , e . g ., an aptamer. The aptamer can be 65 stituted aryl, substituted or unsubstituted heteroaryl, substi unmodified or have any combination of modifications dis - tuted or unsubstituted heterocyclic ; where Rl is hydrogen , closed herein . acyl , aliphatic or substituted aliphatic . US 9 , 814 , 777 B2 57 58 In one embodiment, the linker / tether ( underlined ) include Specific examples of additional lipid components that may – ( CH3) , NH … ; C (O )( CH ), NH — be present are described below . - NR" " " ( CH ) NH C ( O ) (CH ), C ( 0 ) Additional components that may be present in a lipid - C ( O ) - 0 % particle of the present invention include bilayer stabilizing - C ( O ) ((CH ) ., - NHC (O -) OC ( O ) ; C ( O ) ( CH , ) . — ; 5 components such as polyamide oligomers (see , e . g . , U . S . Pat. No . 6 , 320 , 017 ) , peptides, proteins, , lipid CO NH? C ( O ) ( CH ), C ( 0 ) < ; derivatives, such as PEG coupled to phosphatidyletha - ( CH ), C( O ) 0 — — ( CH ), ; ( CH ), NH? nolamine and PEG conjugated to ceramides (see , U . S . Pat . C ( 0 ) ; C( O ) ( CH2) , — NH C( O ) ( CH2) , CH =; No . 5 , 885 ,613 ) . _ ( R " ) NH C ( O ) ( CH ) , NH C ( O ) ; In particular embodiments , the lipid particles include one - ( CH ) , C ( R ') ( R " ) — SS (CH ), NH — ; 10 or more of an amino lipid or cationic lipid , a neutral lipid , - C ( 0 ) ( CH2 ) CH ( R " ) NH _ ; C ( O ) ( CH ) , NH = ; a sterol, and a lipid selected to reduce aggregation of lipid - C ( O ) - (CH ) ,. — SS (CH , ) . particles during formation , which may result from steric CH ( R "" ) NH C ( O ) ( CH2 ) CH ( R " ) NH =; stabilization of particles which prevents charge - induced - (CH . - NH - C ( O ) - (CH ) , C ( R ') ( R " ) SS aggregation during formation . (CH ) n ; in which each n is independently 1 - 20 ( e . g ., 1 , 2 , 15 Examples of lipids that reduce aggregation of particles 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11, 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , or during formation include polyethylene glycol ( PEG ) -modi 20 ) , R ' and R " are each independently H , CH3, OH , SH , fied lipids, monosialoganglioside Gm ] , and polyamide oli NH2 , NH ( Alkyl= Me, Et, Pr , isoPr, Bu , Bn ) or gomers (“ PAO ” ) such as (described in U . S . Pat . No. 6 , 320 , N ( diAlkyl= Mez, Etz , Bn2) ; R '' is H , COOH , CONH2 , 017 ) . Other compounds with uncharged , hydrophilic, steric CONHMe, CONMe2, CONH (CH2 ) , NH2 , CONH (CH2 ) , OH , 20 barrier moieties , which prevent aggregation during CONH( CH2) COOH, CONH( CH2 ), SH, CONH( CH2 ) formulation , like PEG , Gml or ATTA , can also be coupled CONH , CONH ( CH2 ) CONHMe, CONH ( CH2 ) CONH to lipids for use as in the methods and compositions of the ( CH, CH , O ), H , CONH( CH2) CONH( CH, CHÓ) , NH , invention . ATTA - lipids are described , e . g ., in U . S . Pat . No . CONH ( CH2) CONH( CH3CH2O) CHA, CONH( CH3) , 6 , 320 ,017 , and PEG - lipid conjugates are described , e . g . , in CONH (CH ,CH , O ), COOH , or CONH (CH , ) .CONH 25 U . S . Pat. Nos. 5 , 820 ,873 , 5 ,534 ,499 and 5 , 885 ,613 . Typi cally, the concentration of the lipid component selected to (CH2CH2O ) , SH ; and R " " is C - C , alkyl, j and k are each reduce aggregation is about 1 to 15 % (by mole percent of independently 0 - 20 . Preferably, n is 2 , 5 , 6 , or 11 . In other lipids) . embodiments , the nitrogen may form part of a terminal Specific examples of PEG -modified lipids (or lipid -poly oxyamino group , e . g ., — ONH , , or hydrazino group , oxyethylene conjugates ) that are useful in the present inven - NHNH2. The linker /tether may optionally be substituted , 30 tion can have a variety of “ anchoring ” lipid portions to e . g ., with hydroxy , alkoxy, perhaloalkyl, and / or optionally secure the PEG portion to the surface of the lipid vesicle . inserted with one or more additional heteroatoms, e. g. , N , O , Examples of suitable PEG -modified lipids include PEG or S . modified phosphatidylethanolamine and phosphatidic acid , In certain embodiments , the linker is a branched linker . PEG -ceramide conjugates (e .g . , PEG - CerC14 or PEG The branchpoint of the branched linker may be at least 35 CerC20 ) which are described in co - pending U . S . Ser. No . trivalent, but may be a tetravalent, pentavalent or hexavalent 08 / 486 ,214 , incorporated herein by reference, PEG -modi atom , or a group presenting such multiple valencies . In fied dialkylamines and PEG -modified 1 , 2 -diacyloxypropan certain embodiments , the branchpoint is , - C , CH , 3 -amines . Particularly preferred are PEG -modified diacyl - C ( CH2– ) ( CH2- CH2 — , - C ( H ) ( CH2 - CH2- - N , glycerols and dialkylglycerols . - N ( 0 ) C , O C , S C , SS — C , C ( O ) N ( 0 ) — 40 In embodiments where a sterically - large moiety such as C , _ OC (O ) N (O ) - C , — N (O )C (O ) - C , or — N ( O )C (O ) PEG or ATTA are conjugated to a lipid anchor, the selection 0 C ; wherein Q is independently for each occurrence H or of the lipid anchor depends on what type of association the optionally substituted alkyl. In other embodiment, the conjugate is to have with the lipid particle . It is well known branchpoint is glycerol or glycerol derivative . that mePEG (mw2000 )- diastearoylphosphatidyletha Lipid Particles 45 nolamine (PEG -DSPE ) will remain associated with a lipo The present invention also provides lipid particles com some until the particle is cleared from the circulation , prising one or more of the targeting lipids described above . possibly a matter of days . Other conjugates, such as PEG Lipid particles include , but are not limited to , liposomes . As CerC20 have similar staying capacity . PEG -CerC14 , how used herein , a is a structure having lipid - contain - ever, rapidly exchanges out of the formulation upon expo ing membranes enclosing an aqueous interior . Liposomes 50 sure to serum , with a Tu1 less than 60 mins . in some assays . may have one or more lipid membranes . The invention As illustrated in U . S . patent application Ser. No . 08 / 486 ,214 , contemplates both single - layered liposomes, which are at least three characteristics influence the rate of exchange : referred to as unilamellar , and multi - layered liposomes , length of acyl chain , saturation of acyl chain , and size of the which are referred to as multilamellar. When complexed steric -barrier head group . Compounds having suitable varia with nucleic acids, lipid particles may also be lipoplexes, 55 tions of these features may be useful for the invention . For which are composed of cationic lipid bilayers sandwiched some therapeutic applications it may be preferable for the between DNA layers , as described , e . g . , in Feigner, Scientific PEG -modified lipid to be rapidly lost from the nucleic American . acid - lipid particle in vivo and hence the PEG -modified lipid The lipid particles of the present invention may further will possess relatively short lipid anchors. In other thera comprise one or more additional lipids and / or other com - 60 peutic applications it may be preferable for the nucleic ponents such as cholesterol. Other lipids may be included in acid - lipid particle to exhibit a longer plasma circulation the liposome compositions of the present invention for a lifetime and hence the PEG -modified lipid will possess variety of purposes , such as to prevent lipid oxidation or to relatively longer lipid anchors. attach ligands onto the liposome surface. Any of a number Other exemplary PEG - lipids include, without limitation of lipids may be present in liposomes of the present inven - 65 PEG coupled to dialkyloxypropyls (PEG -DAA ) as tion , including amphipathic , neutral , cationic , and anionic described in , e . g ., WO 05 / 026372 , PEG coupled to diacyl lipids. Such lipids can be used alone or in combination . glycerol (PEG -DAG ) as described in , e . g . , U . S . Patent US 9 , 814 , 777 B2 59 60 Publication Nos. 20030077829 and 2005008689 , PEG Anionic lipids suitable for use in lipid particles of the coupled to phosphatidylethanolamine (PE ) (PEG -PE ) , or present invention include , but are not limited to , phospha PEG conjugated to ceramides , or a mixture thereof ( see, tidylglycerol , cardiolipin , diacylphosphatidylserine , diacyl U . S . Pat . No . 5 ,885 ,613 ) . phosphatidic acid , N - dodecanoyl phosphatidylethanoloam It should be noted that aggregation preventing compounds 5 ine , N - succinyl phosphatidylethanolamine, N - glutaryl do not necessarily require lipid conjugation to function phosphatidylethanolamine , lysylphosphatidylglycerol, and properly . Free PEG or free ATTA in solution may be other anionic modifying groups joined to neutral lipids. sufficient to prevent aggregation . If the particles are stable In numerous embodiments , amphipathic lipids are after formulation , the PEG or ATTA can be dialyzed away included in lipid particles of the present invention . before administration to a subject. 10 “ Amphipathic lipids ” refer to any suitable material, wherein Neutral lipids, when present in the lipid particle , can be the hydrophobic portion of the lipid material orients into a any of a number of lipid species which exist either in an hydrophobic phase, while the hydrophilic portion orients uncharged or neutral zwitterionic form at physiological pH . toward the aqueous phase . Such compounds include , but are Such lipids include , for example diacylphosphatidylcholine , 15 not limited to , phospholipids, aminolipids, and sphingolip diacylphosphatidylethanolamine , ceramide , sphingomyelin , ids. Representative phospholipids include sphingomyelin , dihydrosphingomyelin , cephalin , and cerebrosides . The phosphatidylcholine , phosphatidylethanolamine, phosphati selection of neutral lipids for use in the particles described dylserine , phosphatidylinositol, phosphatidic acid , palmi herein is generally guided by consideration of, e . g ., lipo - toyloleoyl phosphatdylcholine , lysophosphatidylcholine , some size and stability of the liposomes in the bloodstream . 20 lysophosphatidylethanolamine , dipalmitoylphosphatidyl Preferably , the neutral lipid component is a lipid having two choline , dioleoylphosphatidylcholine, distearoylphosphati acyl groups, ( i. e. , diacylphosphatidylcholine and diacyl dylcholine , or dilinoleoylphosphatidylcholine. Other phos phosphatidylethanolamine ). Lipids having a variety of acyl phorus - lacking compounds, such as sphingolipids , chain groups of varying chain length and degree of satura glycosphingolipid families, diacylglycerols , and ß - acyloxy tion are available or may be isolated or synthesized by 25 acids , can also be used . Additionally , such amphipathic well -known techniques . In one group of embodiments, lipids lipids can be readily mixed with other lipids, such as containing saturated fatty acids with carbon chain lengths in triglycerides and sterols . the range of C10 to C20 are preferred . In another group of Also suitable for inclusion in the lipid particles of the embodiments , lipids with mono or diunsaturated fatty acids present invention are programmable fusion lipids . Such lipid with carbon chain lengths in the range of Co to C2 are used . 30 particles have little tendency to fuse with cell membranes Additionally, lipids having mixtures of saturated and unsatu - and deliver their payload until a given signal event occurs . rated fatty acid chains can be used . Preferably , the neutral This allows the lipid particle to distribute more evenly after lipids used in the present invention are DOPE , DSPC , injection into an organism or disease site before it starts POPC , or any related phosphatidylcholine . The neutral fusing with cells. The signal event can be , for example, a lipids useful in the present invention may also be composed 35 change in pH , , ionic environment, or time. In of sphingomyelin , dihydrosphingomyeline, or phospholip the latter case , a fusion delaying or “ cloaking " component, ids with other head groups , such as serine and inositol. such as an ATTA - lipid conjugate or a PEG - lipid conjugate , The sterol component of the lipid mixture , when present, can simply exchange out of the lipid particle membrane over can be any of those sterols conventionally used in the field time. By the time the lipid particle is suitably distributed in of liposome, lipid vesicle or lipid particle preparation . A 40 the body , it has lost sufficient cloaking agent so as to be preferred sterol is cholesterol. fusogenic . With other signal events, it is desirable to choose Cationic lipids suitable for use in lipid particles of the a signal that is associated with the disease site or target cell , present invention include , but are not limited to , N , N - such as increased temperature at a site of inflammation . dioleyl- N ,N -dimethylammonium chloride (“ DODAC ” ); In one exemplary embodiment, the lipid particle com N - ( 2 , 3 - dioleyloxy ) propyl- N , N - N - triethylammonium chlo - 45 prises a mixture of a targeting lipid of the present invention , ride (“ DOTMA” ) ; N , N -distearyl - N , N -dimethylammonium a cationic lipid , neutral lipids ( other than an amino lipid ), a bromide (“ DDAB ” ) ; N - ( 2 , 3 -dioleoyloxy )propyl ) - N , N , N - sterol ( e . g . , cholesterol) and a PEG -modified lipid ( e . g ., a trimethylammonium chloride (“ DOTAP ” ) ; 1 , 2 -Dioleyloxy PEG - DMG , PEG - C - DOMG or PEG -DMA ). In certain 3 - trimethylaminopropane chloride salt (“ DOTAP .C1 ” ) ; 3B - embodiments , the lipid mixture consists of or consists essen ( N - ( N ', N - dimethylaminoethane ) -carbamoyl ) cholesterol 50 tially of a targeting lipid of the present invention , a cationic ( “ DC - Chol” ) , N - ( 1 - ( 2 , 3 -dioleyloxy ) propyl) -N2 - ( spermin - lipid , a neutral lipid , cholesterol, and a PEG -modified lipid . ecarboxamido ) ethyl) - N , N - dimethylammonium trifluorace - In further preferred embodiments , the lipid particle consists tate (“ DOSPA ” ), dioctadecylamidoglycyl carboxyspermine of or consists essentially of the above lipid mixture in molar ( “ DOGS ” ) , 1 , 2 - dileoyl- sn - 3 - phosphoethanolamine ratios of about 20 - 50 % targeting lipid :20 - 70 % cationic (“ DOPE” ) , 1 , 2 - dioleoyl -3 - dimethylammonium propane 55 lipid : 5 -45 % neutral lipid : 20 -55 % cholesterol: 0 . 5 - 15 % PEG (“ DODAP ” ) , N , N - dimethyl- 2 , 3 - dioleyloxy ) propylamine modified lipid . (“ DODMA ” ), and N -( 1, 2 - dimyristyloxyprop - 3 -yl ) - N ,N -di - In a preferred embodiment, all components of the lipid methyl- N -hydroxyethyl ammonium bromide ( “ DMRIE ” ) . particle are optically pure . Additionally , a number of commercial preparations of cat- Therapeutic Agent- Lipid Particle Compositions and Formu ionic lipids can be used , such as , e . g . , LIPOFECTIN ( includ - 60 lations ing DOTMA and DOPE , available from GIBCO /BRL ) , and The present invention includes compositions comprising LIPOFECTAMINE ( comprising DOSPA and DOPE , avail - a lipid particle of the present invention and an active agent, able from GIBCO /BRL ) . In particular embodiments, a cat- wherein the active agent is associated with the lipid particle . ionic lipid is an amino lipid . In particular embodiments , the active agent is a therapeutic Other cationic lipids amenable to the present invention are 65 agent. In particular embodiments , the active agent is encap disclosed in PCT patent application PCT/ US2007 /080331 DI sulated within an aqueous interior of the lipid particle . In filed Oct. 3 , 2007. other embodiments , the active agent is present within one or US 9 ,814 ,777 B2 62 morem lipid layers of the lipid particle . In other embodiments , megastrol , melphalan , L - PAM , mesna , methotrexate , the active agent is bound to the exterior or interior lipid methoxsalen , mithramycin , mitomycin , mitoxantrone , nitro surface of a lipid particle . gen mustard , paclitaxel , pamidronate , Pegademase , pento “ Fully encapsulated ” as used herein indicates that the statin , porfimer sodium , prednisone , rituxan , streptozocin , nucleic acid in the particles is not significantly degraded 5 STI- 571, tamoxifen , taxotere , temozolamide , teniposide , after exposure to serum or a nuclease assay that would VM - 26 , topotecan (Hycamtin ) , toremifene, tretinoin , ATRA , significantly degrade free DNA . In a fully encapsulated valrubicin , velban , vinblastine , vincristine, VP16 , and system , preferably less than 25 % of particle nucleic acid is vinorelbine . Other examples of oncology drugs thatmay be degraded in a treatment that would normally degrade 100 % used according to the invention are ellipticin and ellipticin of free nucleic acid , more preferably less than 10 % and most 10 analogs or derivatives , epothilones , intracellular kinase preferably less than 5 % of the particle nucleic acid is inhibitors and camptothecins . degraded . Alternatively, full encapsulation may be deter - Nucleic Acid - Lipid Particles mined by an Oligreen® assay. Oligreen® is an ultra -sensi - In certain embodiments , lipid particles of the present tive fluorescent nucleic acid stain for quantitating oligo - invention are associated with a nucleic acid , resulting in a nucleotides and single -stranded DNA in solution (available 15 nucleic acid - lipid particle . In particular embodiments , the from Invitrogen Corporation , Carlsbad , Calif. ) . Fully encap nucleic acid is fully encapsulated in the lipid particle . As sulated also suggests that the particles are serum stable , that used herein , the term “ nucleic acid ” is meant to include any is , that they do not rapidly decompose into their component oligonucleotide or polynucleotide . Fragments containing up parts upon in vivo administration . to 50 nucleotides are generally termed oligonucleotides , and Active agents , as used herein , include any molecule or 20 longer fragments are called polynucleotides. In particular compound capable of exerting a desired effect on a cell, embodiments , oligonucletoides of the present invention are tissue, organ , or subject . Such effects may be biological, 20 -50 nucleotides in length . physiological , or cosmetic , for example . Active agents may In the context of this invention , the terms “ polynucle be any type of molecule or compound , including e . g ., otide ” and “ oligonucleotide ” refer to a polymer or oligomer nucleic acids, peptides and polypeptides , including , e . g . , 25 of nucleotide or nucleoside monomers consisting of natu antibodies , such as , e . g . , polyclonal antibodies , monoclonal rally occurring bases , sugars and intersugar (backbone ) antibodies , antibody fragments ; humanized antibodies, linkages . The terms " polynucleotide ” and “ oligonucleotide " recombinant antibodies , recombinant human antibodies , and also includes polymers or oligomers comprising non - natu PrimatizedTM antibodies , cytokines , growth factors, apop - rally occurring monomers , or portions thereof , which func totic factors , differentiation - inducing factors , cell surface 30 tion similarly . Such modified or substituted oligonucleotides receptors and their ligands ; hormones ; and small molecules , are often preferred over native formsbecause of properties including small organic molecules or compounds . such as, for example , enhanced cellular uptake and increased In one embodiment, the active agent is a therapeutic stability in the presence of nucleases. agent, or a salt or derivative thereof. Therapeutic agent Oligonucleotides are classified as deoxyribooligonucle derivatives may be therapeutically active themselves or they 35 otides or ribooligonucleotides . A deoxyribooligonucleotide may be prodrugs, which become active upon further modi- consists of a 5 - carbon sugar called deoxyribose joined fication . Thus, in one embodiment, a therapeutic agent covalently to phosphate at the 5 ' and 3 ' carbons of this sugar derivative retains some or all of the therapeutic activity as to form an alternating , unbranched polymer. A ribooligo compared to the unmodified agent, while in another embodi- nucleotide consists of a similar repeating structure where the ment, a therapeutic agent derivative lacks therapeutic activ - 40 5 -carbon sugar is ribose . ity . The nucleic acid that is present in a lipid - nucleic acid In various embodiments , therapeutic agents include any particle according to this invention includes any form of therapeutically effective agent or drug , such as anti - inflam - nucleic acid that is known . The nucleic acids used herein can matory compounds, anti- depressants , stimulants , analgesics , be single - stranded DNA or RNA , or double - stranded DNA antibiotics , birth control medication , antipyretics , vasodila - 45 or RNA , or DNA -RNA hybrids. Examples of double tors , anti -angiogenics , cytovascular agents , signal transduc - stranded DNA include structural genes , genes including tion inhibitors , cardiovascular drugs , e . g . , anti - arrhythmic control and termination regions , and self- replicating systems agents , vasoconstrictors , hormones , and steroids . such as viral or plasmid DNA . Examples of double -stranded In certain embodiments, the therapeutic agent is an oncol - RNA include siRNA and other RNA interference reagents . ogy drug , which may also be referred to as an anti- tumor 50 Single -stranded nucleic acids include , e . g . , antisense oligo drug , an anti -cancer drug , a tumor drug, an antineoplastic nucleotides, ribozymes , microRNA , antagomirs and triplex agent, or the like . Examples of oncology drugs that may be forming oligonucleotides . used according to the invention include , but are not limited Nucleic acids of the present invention may be of various to , adriamycin , alkeran , allopurinol , altretamine , amifostine , lengths, generally dependent upon the particular form of anastrozole , araC , arsenic trioxide , azathioprine, bexarotene , 55 nucleic acid . For example , in particular embodiments , plas biCNU , bleomycin , busulfan intravenous , busulfan oral, mids or genes may be from about 1 , 000 to 100 , 000 nucleo capecitabine ( Xeloda ) , carboplatin , carmustine , CCNU , tide residues in length . In particular embodiments , oligo celecoxib , chlorambucil, cisplatin , cladribine , cyclosporin nucleotides may range from about 10 to 100 nucleotides in A , cytarabine, cytosine arabinoside , daunorubicin , cytoxan , length . In various related embodiments , oligonucleotides, daunorubicin , dexamethasone , dexrazoxane , dodetaxel, 60 both single - stranded , double - stranded , and triple - stranded , doxorubicin , doxorubicin , DTIC , epirubicin , estramustine , may range in length from about 10 to about 50 nucleotides , etoposide phosphate , etoposide and VP - 16 , exemestane, from about 20 to about 50 nucleotides, from about 15 to FK506 , fludarabine , fluorouracil , 5 - FU , gemcitabine about 30 nucleotides , from about 20 to about 30 nucleotides (Gemzar ), gemtuzumab - ozogamicin , goserelin acetate , in length . hydrea, hydroxyurea, idarubicin , ifosfamide, imatinib mesy - 65 In particular embodiments , an oligonucleotide (or a strand late , interferon , irinotecan (Camptostar , CPT- 111 ) , letrozole , thereof ) of the present invention specifically hybridizes to or leucovorin , leustatin , leuprolide , levamisole , litertinoin , is complementary to a target polynucleotide. “ Specifically US 9 ,814 ,777 B2 63 64 hybridizable” and “ complementary ” are terms which are favor the antisense siRNA agent to enter into RISC are also used to indicate a sufficient degree of complementarity such envisioned . Modifications can include C3 (or C6 , C7 , C12 ) that stable and specific binding occurs between the DNA or amino linkers , thiol linkers , carboxyl linkers, non - nucleotide RNA target and the oligonucleotide . It is understood that an spacers (C3 , C6 , C9 , C12 , abasic , triethylene glycol, hexa oligonucleotide need not be 100 % complementary to its 5 ethylene glycol) , special biotin or fluorescein reagents that target nucleic acid sequence to be specifically hybridizable . come as phosphoramidites and that have another DMT An oligonucleotide is specifically hybridizable when bind protected hydroxyl group , allowing multiple couplings dur ing of the oligonucleotide to the target interferes with the ing RNA synthesis . normal function of the target molecule to cause a loss of utility or expression therefrom , and there is a sufficient 10 iRNA agents include: molecules that are long enough to degree of complementarity to avoid non - specific binding of trigger the interferon response ( which can be cleaved by the oligonucleotide to non - target sequences under condi Dicer ( Bernstein et al. 2001 . Nature , 409 :363 - 366 ) and enter tions in which specific binding is desired , i . e . , under physi a RISC (RNAi - induced silencing complex ) ) ; and ,molecules ological conditions in the case of in vivo assays or thera which are sufficiently short that they do not trigger the peutic treatment, or, in the case of in vitro assays, under 15 interferon response (which molecules can also be cleaved by conditions in which the assays are conducted . Thus , in other Dicer and / or enter a RISC ) , e . g ., molecules which are of a embodiments , this oligonucleotide includes 1, 2 , or 3 base size which allows entry into a RISC , e . g ., molecules which substitutions as compared to the region of a gene or mRNA resemble Dicer- cleavage products . Molecules that are short sequence that it is targeting or to which it specifically enough that they do not trigger an interferon response are hybridizes . 20 termed siRNA agents or shorter iRNA agents herein . RNA Interference Nucleic Acids " siRNA agent or shorter iRNA agent” as used herein , refers In particular embodiments , nucleic acid - lipid particles of to an iRNA agent, e. g ., a double stranded RNA agent or the present invention are associated with RNA interference single strand agent, that is sufficiently short that it does not (RNAi ) molecules . RNA interference methods using RNAi induce a deleterious interferon response in a human cell , molecules may be used to disrupt the expression of a gene 25 e .g . , it has a duplexed region of less than 60, 50 , 40 , or 30 or polynucleotide of interest . These RNAi molecules are nucleotide pairs . The siRNA agent, or a cleavage product also referred to as iRNA agents and described below . thereof, can down regulate a target gene, e .g . , by inducing The iRNA agent should include a region of sufficient RNAi with respect to a target RNA , wherein the targetmay homology to the target gene , and be of sufficient length in comprise an endogenous or pathogen target RNA . terms of nucleotides , such that the iRNA agent , or a frag - 30 Each strand of a siRNA agent can be equal to or less than ment thereof, can mediate downregulation of the target gene . 30 , 25 , 24 , 23 , 22 , 21 , or 20 nucleotides in length . The strand ( For ease of exposition the term nucleotide or ribonucleotide may be at least 19 nucleotides in length . For example , each is sometimes used herein in reference to one or more strand can be between 21 and 25 nucleotides in length . monomeric subunits of an RNA agent. It will be understood siRNA agents may have a duplex region of 17 , 18 , 19 , 29 , herein that the usage of the term " ribonucleotide ” or 35 21 , 22 , 23 , 24 , or 25 nucleotide pairs , and one or more “ nucleotide” , herein can , in the case of a modified RNA or overhangs , or one or two 3 ' overhangs , of 2 - 3 nucleotides . nucleotide surrogate , also refer to a modified nucleotide , or In addition to homology to target RNA and the ability to surrogate replacement moiety at one or more positions . ) down regulate a target gene , an iRNA agent may have one Thus, the iRNA agent is or includes a region which is at least or more of the following properties : partially , and in one embodiment fully , complementary to 40 A single strand iRNA agent may be sufficiently long that the target RNA . It is not necessary that there be perfect it can enter the RISC and participate in RISC mediated complementarity between the iRNA agent and the target, but cleavage of a target mRNA . A single strand iRNA agent is the correspondence must be sufficient to enable the iRNA at least 14 , and in other embodiments at least 15 , 20 , 25 , 29 , agent , or a cleavage product thereof, to direct sequence 35 , 40 , or 50 nucleotides in length . In certain embodiments , specific silencing, e . g. , by RNAi cleavage of the target RNA , 45 it is less than 200 , 100 , or 60 nucleotides in length . e . g ., mRNA . Complementarity , or degree of homology with Hairpin iRNA agents will have a duplex region equal to the target strand , is most critical in the antisense strand . or at least 17 , 18 , 19 , 20, 21, 22 , 23, 24 , or 25 nucleotide While perfect complementarity , particularly in the antisense pairs . The duplex region will may be equal to or less than strand , is often desired one embodiment can include , par - 200 , 100 , or 50 , in length . In certain embodiments, ranges ticularly in the antisense strand , one or more , or for example , 50 for the duplex region are 15 - 30 , 17 to 23 , 19 to 23 , and 19 6 , 5 , 4 , 3 , 2 , or fewer mismatches (with respect to the target to 21 nucleotides pairs in length . The hairpin may have a RNA ) . The mismatches , particularly in the antisense strand , single strand overhang or terminal unpaired region , in one are most tolerated in the terminal regions and if present may embodiment at the 3 ', and in certain embodiments on the be in a terminal region or regions , e . g ., within 6 , 5 , 4 , or 3 antisense side of the hairpin . In one embodiment, the over nucleotides of the 5 ' and /or 3 ' termini. The sense strand need 55 hangs are 2 -3 nucleotides in length . only be sufficiently complementary with the antisense strand A “ double stranded ( ds ) iRNA agent” as used herein , is an to maintain the over all double stranded character of the iRNA agent which includes more than one , and in some molecule . cases two , strands in which interchain hybridization can As discussed elsewhere herein , and in the material incor- form a region of duplex structure . porated by reference in its entirety, an iRNA agent will often 60 The antisense strand of a double stranded iRNA agent be modified or include nucleoside surrogates . Single may be equal to or at least, 14 , 15 , 16 17 , 18 , 19 , 25 , 29 , 40 , stranded regions of an iRNA agent will often be modified or or 60 nucleotides in length . It may be equal to or less than include nucleoside surrogates, e . g. , the unpaired region or 200 , 100 , or 50 , nucleotides in length . Ranges may be 17 to regions of a hairpin structure , e . g. , a region which links two 25, 19 to 23 , and 19 to 21 nucleotides in length . complementary regions , can have modifications or nucleo - 65 The sense strand of a double stranded iRNA agent may be side surrogates . Modification to stabilize one or more 3 '- or equal to or at least 14 , 15 , 16 17 , 18 , 19 , 25 , 29 , 40 , or 60 5 '- termini of an iRNA agent, e. g ., against exonucleases , or to nucleotides in length . It may be equal to or less than 200 , US 9 , 814 ,777 B2 65 66 100, or 50 , nucleotides in length . Ranges may be 17 to 25 , silenced is an endogenous gene or a pathogen gene . In 19 to 23 , and 19 to 21 nucleotides in length . addition , RNAs other than mRNA , e . g . , tRNAs , and viral The double strand portion of a double stranded iRNA RNAs, can also be targeted . agent may be equal to or at least , 14 , 15 , 16 17 , 18, 19 , 20 , As used herein , the phrase " mediates RNAi” refers to the 21, 22 , 23 , 24 , 25 , 29, 40 , or 60 nucleotide pairs in length . 5 ability to silence , in a sequence specific manner , a target It may be equal to or less than 200 , 100 , or 50 , nucleotides RNA . While not wishing to be bound by theory , it is believed pairs in length . Ranges may be 15 - 30 , 17 to 23 , 19 to 23, and that silencing uses the RNAi machinery or process and a 19 to 21 nucleotides pairs in length . guide RNA , e . g ., an siRNA agent of 21 to 23 nucleotides. In many embodiments , the ds iRNA agent is sufficiently As used herein , “ specifically hybridizable ” and “ comple large that it can be cleaved by an endogenous molecule , e .g ., mentary ” are terms which are used to indicate a sufficient by Dicer , to produce smaller ds iRNA agents, e . g ., siRNAs degree of complementarity such that stable and specific agents binding occurs between a compound of the invention and a Itmay be desirable to modify one or both of the antisense target RNA molecule . Specific binding requires a sufficient and sense strands of a double strand iRNA agent. In some 15 degree of complementarity to avoid non -specific binding of cases they will have the samemodification or the same class the oligomeric compound to non - target sequences under of modification but in other cases the sense and antisense conditions in which specific binding is desired , i. e ., under strand will have different modifications, e .g ., in some cases physiological conditions in the case of in vivo assays or it is desirable to modify only the sense strand . It may be therapeutic treatment, or in the case of in vitro assays , under desirable to modify only the sense strand , e . g . , to inactivate 20 conditions in which the assays are performed . The non it, e . g . , the sense strand can be modified in order to inactivate target sequences typically differ by at least 5 nucleotides . the sense strand and prevent formation of an active siRNA In one embodiment, an iRNA agent is " sufficiently protein or RISC . This can be accomplished by a modifica - complementary ” to a target RNA , e . g . , a target mRNA, such tion which prevents 5 '- phosphorylation of the sense strand , that the iRNA agent silences production of protein encoded e . g ., by modification with a 5 '- O - methyl ribonucleotide ( see 25 by the target mRNA . In another embodiment, the iRNA Nykänen et al. , (2001 ) ATP requirements and small inter agent is “ exactly complementary ” to a target RNA, e . g . , the fering RNA structure in the RNA interference pathway . Cell target RNA and the iRNA agent anneal, for example to form 107 , 309- 321. ) Othermodifications which prevent phospho a hybrid made exclusively of Watson - Crick base pairs in the rylation can also be used , e. g . , simply substituting the 5 '- OH region of exact complementarity . A “ sufficiently comple by H rather than O -Me . Alternatively , a large bulky group » mentary ” target RNA can include an internal region ( e .g ., of may be added to the 5' - phosphate turning it into a phos at least 10 nucleotides ) that is exactly complementary to a phodiester linkage , though this may be less desirable as target RNA . Moreover , in one embodiment , the iRNA agent phosphodiesterases can cleave such a linkage and release a specifically discriminates a single- nucleotide difference . In functional siRNA 5 '- end . Antisense strand modifications as this case , the iRNA agent only mediates RNAi if exact include 5' phosphorylation as well as any of the other 5 complementary is found in the region (e .g . , within 7 nucleo modifications discussed herein , particularly the 5 ' modifica - tides of) the single -nucleotide difference . tions discussed above in the section on single stranded iRNA As used herein , the term “ oligonucleotide” refers to a molecules . nucleic acid molecule (RNA or DNA ) for example of length The sense and antisense strandsmay be chosen such that 40 less than 100 , 200 , 300 , or 400 nucleotides . the ds iRNA agent includes a single strand or unpaired RNA agents discussed herein include unmodified RNA as region at one or both ends of themolecule . Thus , a ds iRNA well as RNA which have been modified , e . g . , to improve agent may contain sense and antisense strands, paired to efficacy , and polymers of nucleoside surrogates . Unmodified contain an overhang , e . g ., one or two 5 ' or 3 ' overhangs, or R NA refers to a molecule in which the components of the a 3 ' overhang of 2 - 3 nucleotides . Many embodiments will 45 nucleic acid , namely sugars , bases, and phosphate moieties , have a 3 ' overhang . Certain siRNA agents will have single are the same or essentially the same as that which occur in stranded overhangs , in one embodiment 3 ' overhangs , of 1 nature , for example as occur naturally in the human body . or 2 or 3 nucleotides in length at each end . The overhangs The art has often referred to rare or unusual, but naturally can be the result of one strand being longer than the other, occurring , RNAs as modified RNAs, see , e . g ., Limbach et or the result of two strands of the same length being 50 al. , ( 1994 ) Summary : the modified nucleosides of RNA , staggered . 5' ends may be phosphorylated . Nucleic Acids Res . 22 : 2183 - 2196 . Such rare or unusual In one embodiment, the length for the duplexed region is RNAs, often termed modified RNAs ( apparently because the between 15 and 30 , or 18 , 19 , 20 , 21, 22 , and 23 nucleotides are typically the result of a post transcriptionally modifica in length , e . g. , in the siRNA agent range discussed above . tion ) are within the term unmodified RNA , as used herein . siRNA agents can resemble in length and structure the 55 Modified RNA refers to a molecule in which one or more of natural Dicer processed products from long dsiRNAs. the components of the nucleic acid , namely sugars , bases , Embodiments in which the two strands of the siRNA agent and phosphate moieties , are different from that which occur are linked , e . g . , covalently linked are also included .Hairpin , in nature , for example , different from that which occurs in or other single strand structures which provide the required the human body. While they are referred to as modified double stranded region , and a 3 ' overhang are also within the 60 “ RNAs, ” they will of course, because of the modification , invention . include molecules which are not RNAs . Nucleoside surro The isolated iRNA agents described herein , including ds gates are molecules in which the ribophosphate backbone is iRNA agents and siRNA agents can mediate silencing of a replaced with a non - ribophosphate construct that allows the target RNA , e . g . , mRNA, e . g ., a transcript of a gene that bases to the presented in the correct spatial relationship such encodes a protein . For convenience , such mRNA is also 65 that hybridization is substantially similar to what is seen referred to herein as mRNA to be silenced . Such a gene is with a ribophosphate backbone , e . g . , non -charged mimics of also referred to as a target gene . In general, the RNA to be the ribophosphate backbone . US 9 ,814 , 777 B2 67 68 MicroRNAs Methods of producing antisense oligonucleotides are Micro RNAs (miRNAs ) are a highly conserved class of known in the art and can be readily adapted to produce an small RNA molecules that are transcribed from DNA in the antisense oligonucleotide that targets any polynucleotide genomes of plants and animals , but are not translated into sequence . Selection of antisense oligonucleotide sequences protein . Processed miRNAs are single stranded - 17 - 25 5 specific for a given target sequence is based upon analysis of nucleotide (nt ) RNA molecules that become incorporated the chosen target sequence and determination of secondary into the RNA - induced silencing complex (RISC ) and have structure , T . , binding energy , and relative stability . Anti been identified as key regulators of development, cell pro - sense oligonucleotides may be selected based upon their liferation , apoptosis and differentiation . They are believed to relative inability to form dimers , hairpins , or other second play a role in regulation of gene expression by binding to the 10 ary structures that would reduce or prohibit specific binding 3 -untranslated region of specific mRNAs . RISC mediates to the target mRNA in a host cell . Highly preferred target down -regulation of gene expression through translational regions of the mRNA include those regions at or near the inhibition , transcript cleavage , or both . RISC is also impli - AUG translation initiation codon and those sequences that cated in transcriptional silencing in the nucleus of a wide are substantially complementary to 5 ' regions of the mRNA . range of eukaryotes . 15 These secondary structure analyses and target site selection The number of miRNA sequences identified to date is considerations can be performed , for example , using v .4 of large and growing , illustrative examples of which can be the OLIGO primer analysis software (Molecular Biology found , for example , in : " miRBase : microRNA sequences, Insights ) and /or the BLASTN 2 .0 .5 algorithm software targets and gene nomenclature" Griffiths- Jones S , Grocock (Altschul et al. , Nucleic Acids Res . 1997 , 25 ( 17 ) :3389 -402 ) . RJ, van Dongen S , Bateman A , Enright AJ. NAR , 2006 , 34 , 20 Ribozymes Database Issue , D140 -D144 ; “ The microRNA Registry ” . According to another embodiment of the invention , Griffiths - Jones S , NAR , 2004 , 32 , Database Issue , D109 nucleic acid -lipid particles are associated with ribozymes . D111 ; and also at http : // microrna .sanger .ac .uk /sequences / . Ribozymes are RNA - protein complexes having specific Antisense Oligonucleotides catalytic domains that possess endonuclease activity (Kim In one embodiment, a nucleic acid is an antisense oligo - 25 and Cech , Proc Natl Acad Sci USA . 1987 December ; nucleotide directed to a target polynucleotide . The term 84 (24 ) :8788 -92 ; Forster and Symons, Cell . 1987 Apr. 24 ; " antisense oligonucleotide” or simply " antisense ” is meant 49 ( 2 ) :211 - 20 ) . For example , a large number of ribozymes to include oligonucleotides that are complementary to a accelerate phosphoester transfer reactions with a high degree targeted polynucleotide sequence . Antisense oligonucle - of specificity , often cleaving only one of several phospho otides are single strands of DNA or RNA that are comple - 30 esters in an oligonucleotide substrate (Cech et al. , Cell. 1981 mentary to a chosen sequence . In the case of antisense RNA , December ; 27 ( 3 Pt 2 ) :487 - 96 ; Michel and Westhof, J Mol they prevent translation of complementary RNA strands by Biol. 1990 Dec . 5 ; 216 ( 3 ): 585 -610 ; Reinhold - Hurek and binding to it. Antisense DNA can be used to target a specific , Shub , Nature . 1992 May 14 ; 357 (6374 ) : 173 - 6 ) . This speci complementary ( coding or non - coding) RNA . If binding ficity has been attributed to the requirement that the sub takes places this DNA /RNA hybrid can be degraded by the 35 strate bind via specific base -pairing interactions to the enzyme RNase H . In particular embodiment, antisense oli - internal guide sequence (“ IGS ” ) of the ribozyme prior to gonucleotides contain from about 10 to about 50 nucleo - chemical reaction . tides , more preferably about 15 to about 30 nucleotides. The At least six basic varieties of naturally -occurring enzy term also encompasses antisense oligonucleotides that may matic RNAs are known presently . Each can catalyze the not be exactly complementary to the desired target gene . 40 hydrolysis of RNA phosphodiester bonds in trans (and thus Thus , the invention can be utilized in instances where can cleave other RNA molecules ) under physiological con non - target specific -activities are found with antisense , or ditions . In general, enzymatic nucleic acids act by first where an antisense sequence containing one or more mis - binding to a target RNA . Such binding occurs through the matches with the target sequence is the most preferred for a target binding portion of an enzymatic nucleic acid which is particular use . 45 held in close proximity to an enzymatic portion of the Antisense oligonucleotides have been demonstrated to be molecule that acts to cleave the target RNA . Thus , the effective and targeted inhibitors of protein synthesis , and , enzymatic nucleic acid first recognizes and then binds a consequently , can be used to specifically inhibit protein target RNA through complementary base - pairing , and once synthesis by a targeted gene . The efficacy of antisense bound to the correct site , acts enzymatically to cut the target oligonucleotides for inhibiting protein synthesis is well 50 RNA . Strategic cleavage of such a target RNA will destroy established . For example , the synthesis of polygalactauro - its ability to direct synthesis of an encoded protein . After an nase and the muscarine type 2 acetylcholine receptor are enzymatic nucleic acid has bound and cleaved its RNA inhibited by antisense oligonucleotides directed to their target, it is released from that RNA to search for another respective mRNA sequences ( U . S . Pat . No . 5 ,739 , 119 and target and can repeatedly bind and cleave new targets . U . S . Pat . No . 5 , 759, 829 ) . Further , examples of antisense 55 The enzymatic nucleic acid molecule may be formed in a inhibition have been demonstrated with the nuclear protein hammerhead , hairpin , a hepatitis à virus , group I intron or cyclin , the multiple drug resistance gene (MDG1 ) , ICAM - 1 , RNaseP RNA in association with an RNA guide sequence ) E - selectin , STK - 1 , striatalGABA , receptor and human EGF or Neurospora VS RNA motif, for example. Specific ( Jaskulski et al. , Science . 1988 Jun . 10 ; 240 (4858 ) : 1544 - 6 ; examples of hammerhead motifs are described by Rossi et Vasanthakumar and Ahmed , Cancer Commun . 1989 ; 1 (4 ) : 60 al. Nucleic Acids Res. 1992 Sep . 11 ; 20 ( 17 ) : 4559 -65 . 225 - 32 ; Penis et al. , Brain Res Mol Brain Res. 1998 Jun . 15 ; Examples of hairpin motifs are described by Hampel et al . 57 ( 2 ) :310 - 20 ; U . S . Pat . No . 5 , 801 , 154 ; U . S . Pat. No . 5 , 789, (Eur . Pat. Appl. Publ. No . EP 0360257 ) , Hampel and Tritz , 573; U . S . Pat . No . 5 ,718 ,709 and U . S . Pat. No . 5 ,610 , 288 ) . Biochemistry 1989 Jun . 13 ; 28 ( 12 ) :4929 - 33 ; Hampel et al. , Furthermore, antisense constructs have also been described Nucleic Acids Res . 1990 Jan . 25 ; 18 ( 2 ) : 299 - 304 and U . S . that inhibit and can be used to treat a variety of abnormal 65 Pat. No . 5 ,631 , 359 . An example of the hepatitis d virus motif cellular proliferations, e . g . cancer ( U . S . Pat. No. 5 ,747 ,470 ; is described by Perrotta and Been , Biochemistry . 1992 Dec . U . S . Pat. No. 5 ,591 ,317 and U . S . Pat. No. 5 ,783 ,683 ) . 1 ; 31 ( 47 ) : 11843 - 52 ; an example of the RNaseP motif is US 9 ,814 ,777 B2 69 70 described by Guerrier- Takada et al. , Cell . 1983 December; logic properties, such as enhanced tissue and cellular uptake . 35 ( 3 Pt 2 ) :849 - 57 ; Neurospora VS RNA ribozyme motif is They differ from normal RNA by , for example , complete described by Collins (Saville and Collins, Cell . 1990 May 2 - O -methylation of sugar, phosphorothioate backbone and , 18 ; 61 ( 4 ) :685 - 96 ; Saville and Collins, Proc Natl Acad Sci for example , a cholesterol- moiety at 3 ' - end . Antagomirs may USA . 1991 Oct. 1 ; 88 ( 19 ): 8826 - 30 ; Collins and Olive , 5 be used to efficiently silence endogenous miRNAs thereby Biochemistry . 1993 Mar . 23; 32 ( 11) :2795 - 9 ) ; and an preventing miRNA - induced gene silencing . An example of example of the Group I intron is described in U . S . Pat. No . antagomir -mediated miRNA silencing is the silencing of 4 ,987 ,071 . Important characteristics of enzymatic nucleic miR - 122 , described in Krutzfeldt et al, Nature , 2005 , 438 : acid molecules used according to the invention are that they 685 -689 , which is expressly incorporated by reference have a specific substrate binding site which is complemen - 10 herein , in its entirety . tary to one or more of the target gene DNA or RNA regions, Decoy Oligonucleotides and that they have nucleotide sequences within or surround - Because transcription factors can recognize their rela ing that substrate binding site which impart an RNA cleav tively short binding sequences, even in the absence of ing activity to the molecule . Thus the ribozyme constructs surrounding genomic DNA , short oligonucleotides bearing need not be limited to specific motifs mentioned herein . 15 the consensus binding sequence of a specific transcription Methods of producing a ribozyme targeted to any poly - factor can be used as tools for manipulating gene expression nucleotide sequence are known in the art. Ribozymes may in living cells . This strategy involves the intracellular deliv be designed as described in Int. Pat. Appl. Publ. No. WO ery of such “ decoy oligonucleotides ” , which are then rec 93 /23569 and Int. Pat . Appl. Publ. No . WO 94 /02595 , each ognized and bound by the target factor . Occupation of the specifically incorporated herein by reference , and synthe - 20 transcription factor 's DNA -binding site by the decoy renders sized to be tested in vitro and in vivo , as described therein . the transcription factor incapable of subsequently binding to Ribozyme activity can be optimized by altering the length the promoter regions of target genes. Decoys can be used as of the ribozyme binding arms or chemically synthesizing therapeutic agents , either to inhibit the expression of genes ribozymes with modifications that prevent their degradation that are activated by a transcription factor, or to upregulate by serum ribonucleases ( see e . g . , Int. Pat . Appl. Publ. No. 25 genes that are suppressed by the binding of a transcription WO 92 / 07065 ; Int. Pat. Appl. Publ. No. WO 93 / 15187; Int . factor. Examples of the utilization ofdecoy oligonucleotides Pat. Appl. Publ . No . WO 91 / 03162 ; Eur. Pat. Appl. Publ. No . may be found in Mann et al. , J . Clin . Invest ., 2000 , 106 : 92110298. 4 ; U . S . Pat . No. 5 , 334 ,711 ; and Int. Pat . Appl. 1071- 1075 , which is expressly incorporated by reference Publ . No . WO 94 / 13688 , which describe various chemical herein , in its entirety . modifications that can be made to the sugar moieties of 30 Nucleic Acid Modifications enzymatic RNA molecules ) , modifications which enhance Much of the discussion below refers to single strand their efficacy in cells , and removal of stem II bases to shorten molecules. In many embodiments of the invention a double RNA synthesis times and reduce chemical requirements . stranded iRNA agent , e . g . , a partially double stranded iRNA Immunostimulatory Oligonucleotides agent , is envisioned . Thus, it is understood that that double Nucleic acids associated with lipid particles of the present 35 stranded structures ( e . g ., where two separate molecules are invention may be immunostimulatory , including immunos - contacted to form the double stranded region or where the timulatory oligonucleotides ( ISS ; single - or double - double stranded region is formed by intramolecular pairing stranded ) capable of inducing an immune response when ( e . g . , a hairpin structure ) ) made of the single stranded administered to a subject, which may be a mammal or other structures described below are within the invention . Lengths patient. ISS include , e . g ., certain palindromes leading to 40 are described elsewhere herein . hairpin secondary structures ( see Yamamoto S ., et al. ( 1992 ) As nucleic acids are polymers of subunits , many of the J . Immunol. 148 : 4072 -4076 ) , or CpG motifs , as well as modifications described below occur at a position which is other known ISS features ( such as multi - G domains, see WO repeated within a nucleic acid , e . g. , a modification of a base , 96 / 11266 ) . or a phosphate moiety , or the a non - linking O of a phosphate The immune response may be an innate or an adaptive 45 moiety . In some cases the modification will occur at all of immune response . The immune system is divided into a the subject positions in the nucleic acid but in many cases it more innate immune system , and acquired adaptive immune will not. By way of example , a modification may only occur system of vertebrates , the latter of which is further divided at a 3 ' or 5 ' terminal position , may only occur in a terminal into humoral cellular components . In particular embodi- regions, e . g . , at a position on a terminal nucleotide or in the ments, the immune response may be mucosal. 50 last 2 , 3 , 4 , 5 , or 10 nucleotides of a strand . A modification In particular embodiments, an immunostimulatory may occur in a double strand region , a single strand region , nucleic acid is only immunostimulatory when administered or in both . A modification may occur only in the double in combination with a lipid particle , and is not immunos - strand region of an RNA or may only occur in a single strand timulatory when administered in its “ free form .” According region of an RNA . E .g ., a phosphorothioate modification at to the present invention , such an oligonucleotide is consid - 55 a non - linking O position may only occur at one or both ered to be immunostimulatory . termini, may only occur in a terminal regions, e . g ., at a Immunostimulatory nucleic acids are considered to be position on a terminal nucleotide or in the last 2 , 3 , 4 , 5 , or non - sequence specific when it is not required that they 10 nucleotides of a strand , or may occur in double strand and specifically bind to and reduce the expression of a target single strand regions, particularly at termini. The 5 ' end or polynucleotide in order to provoke an immune response . 60 ends can be phosphorylated . Thus , certain immunostimulatory nucleic acids may com - In one embodiment it is possible, e . g . , to enhance stability , prise a sequence corresponding to a region of a naturally to include particular bases in overhangs , or to include occurring gene or mRNA , but they may still be considered modified nucleotides or nucleotide surrogates, in single non - sequence specific immunostimulatory nucleic acids . strand overhangs, e . g ., in a 5 ' or 3 ' overhang , or in both . E . g . , Antagomirs 65 it can be desirable to include purine nucleotides in over Antagomirs are RNA -like oligonucleotides that harbor hangs . In one embodiment all or some of the bases in a 3 ' or various modifications for RNAse protection and pharmaco - 5' overhang will be modified , e . g. , with a modification US 9 ,814 ,777 B2 71 72 described herein . Modifications can include , e. g. , the use of AMINE = NH ; alkylamino , dialkylamino , heterocyclyl, ary modifications at the 2 ' OH group of the ribose sugar , e . g ., the lamino , diaryl amino , heteroaryl amino , or diheteroaryl use of deoxyribonucleotides , e .g ., deoxythymidine, instead amino , ethylene diamine, polyamino ). It is noteworthy that of ribonucleotides , and modifications in the phosphate oligonucleotides containing only the methoxyethyl group group , e . g . , phosphothioate modifications . Overhangs need 5 (MOE ) , (OCH . CHOCH , , a PEG derivative ) , exhibit nucle not be homologous with the target sequence . Unmodified oligoribonucleotides may be less than opti ase stabilities comparable to those modified with the robust mal in some applications, e . g . , unmodified oligoribonucle phosphorothioate modification . otides can be prone to degradation by e . g . , cellular nucle “ Deoxy ” modifications include hydrogen ( i. e ., deoxyri ases . Nucleases can hydrolyze nucleic acid phosphodiester 10 bose sugars , which are of particular relevance to the over bonds. However, chemical modifications to one or more of hang portions of partially ds RNA ) ; halo ( e . g ., fluoro ) ; the above RNA components can confer improved properties , amino ( e . g . , NHz; alkylamino , dialkylamino , heterocyclyl, and , e . g . , can render oligoribonucleotides more stable to arylamino , diaryl amino , heteroaryl amino , diheteroaryl nucleases . amino , or amino acid ) ; NH (CH _ CH NH ) CH2CH2 Specific modifications are discussed in more detail belowlow . 15 AMINEAN ( AMINE = NH2; alkylamino , dialkylamino , hetero The Phosphate Group cyclyl, arylamino , diaryl amino , heteroaryl amino , or dihet The phosphate group is a negatively charged species . The eroaryl amino ) , — NHC ( O ) R ( R = alkyl , cycloalkyl, aryl, charge is distributed equally over the two non -linking oxy aralkyl, heteroaryl or sugar) , cyano ; mercapto ; alkyl- thio gen atoms. However , the phosphate group can be modified alkyl; thioalkoxy ; and alkyl, cycloalkyl, aryl, alkenyl and by replacing one of the oxygens with a different substituent. 20 alkynyl, which may be optionally substituted with e .g ., an One result of this modification to RNA phosphate backbones amino functionality . Other substitutents of certain embodi can be increased resistance of the oligoribonucleotide to ments include 2 '- methoxyethyl , 2 -OCH3 , 2 - O - allyl, 2 - C nucleolytic breakdown. Thus while not wishing to be bound allyl, and 2 ' - fluoro . by theory, it can be desirable in one embodiment to intro The sugar group can also contain one or more carbons that duce alterations which result in either an uncharged linker or 25 possess the opposite stereochemical configuration than that a charged linker with unsymmetrical charge distribution of the corresponding carbon in ribose . Thus , a modified Examples of modified phosphate groups include phos - RNA can include nucleotides containing e . g . , arabinose , as phorothioate , phosphoroselenates , borano phosphates , the sugar. borano phosphate esters , hydrogen phosphonates , phospho - Modified RNA ' s can also include “ abasic ” sugars , which roamidates , alkyl or aryl phosphonates and phosphotriesters . 30 lack a nucleobase at C - 1 '. These abasic sugars can also be Phosphorodithioates have both non - linking oxygens further contain modifications at one or more of the constitu replaced by sulfur. The phosphorus center in the phospho - ent sugar atoms. rodithioates is achiral which precludes the formation of To maximize nuclease resistance , the 2 'modifications can oligoribonucleotides diastereomers . Diastereomer formation be used in combination with one or more phosphate linker can result in a preparation in which the individual diaste - 35 modifications (e . g ., phosphorothioate ) . The so -called " chi reomers exhibit varying resistance to nucleases . Further, the meric ” oligonucleotides are those that contain two or more hybridization affinity of RNA containing chiral phosphate different modifications . groups can be lower relative to the corresponding unmodi - Replacement of the Phosphate Group fied RNA species . Thus, oxygens of the phosphodiester The phosphate group can be replaced by non - phosphorus linkage can be replaced by any one of S , Se, B , C , H , N , or 40 containing connectors . While not wishing to be bound by OR ( R is alkyl or aryl) . theory , it is believed that since the charged phosphodiester The phosphate linker can also be modified by replacement group is the reaction center in nucleolytic degradation , its of a linking oxygen with nitrogen (bridged phosphoroami replacement with neutral structural mimics should impart dates ), sulfur (bridged phosphorothioates) and carbon enhanced nuclease stability . Again , while not wishing to be ( bridged methylenephosphonates ) . 45 bound by theory , it can be desirable , in some embodiment, The Sugar Group to introduce alterations in which the charged phosphate A modified RNA can include modification of all or some group is replaced by a neutral moiety . of the sugar groups of the ribonucleic acid . E . g ., the 2 Examples of moieties which can replace the phosphate hydroxyl group (OH ) can be modified or replaced with a group include siloxane , carbonate , carboxymethyl, carbam number of different “ oxy ” or “ deoxy ” substituents . While 50 ate , amide, thioether, ethylene oxide linker, sulfonate , sul not being bound by theory , enhanced stability is expected fonamide , thioformacetal, formacetal, oxime , methyl since the hydroxyl can no longer be deprotonated to form a eneimino , methylenemethylimino , methylenehydrazo , 2 ' alkoxide ion . The 2' alkoxide can catalyze degradation by methylenedimethylhydrazo and methyleneoxymethylimino . intramolecular nucleophilic attack on the linker phosphorus In certain embodiments , replacements may include the atom . Again , while not wishing to be bound by theory , it can 55 methylenecarbonylamino and methylenemethylimino be desirable to one embodiment to introduce alterations in groups . which alkoxide formation at the 2 ' position is not possible . Replacement of Ribophosphate Backbone Examples of " oxy - 2 ' hydroxyl group modifications Oligonucleotide- mimicking scaffolds can also be con include alkoxy or aryloxy (OR , e . g ., R = H , alkyl, structed wherein the phosphate linker and ribose sugar are cycloalkyl, aryl, aralkyl, heteroaryl or sugar ); polyethyl- 60 replaced by nuclease resistant nucleoside or nucleotide eneglycols (PEG ), O ( CH2CH2O ) , CH CH OR ; “ locked ” surrogates . While not wishing to be bound by theory , it is nucleic acids (LNA ) in which the 2' hydroxyl is connected , believed that the absence of a repetitively charged backbone e .g ., by a methylene bridge , to the 4' carbon of the same diminishes binding to proteins that recognize polyanions ribose sugar ; O - AMINE ( AMINE = NH , alkylamino , ( e . g . , nucleases ) . Again , while not wishing to be bound by dialkylamino , heterocyclyl, arylamino , diarylamino , het- 65 theory, it can be desirable in some embodiment, to introduce eroaryl amino, or diheteroaryl amino , ethylene diamine , alterations in which the bases are tethered by a neutral polyamino ) and aminoalkoxy, O ( CH2) , AMINE , ( e . g ., surrogate backbone . US 9 ,814 ,777 B2 73 74 Examples include the mophilino , cyclobutyl, pyrrolidine (OP40 (HO ) ( O ) P = 0 — P (HO ) ( O ) O - 5 ' ) ; and peptide nucleic acid (PNA ) nucleoside surrogates. In 5 ' -adenosine cap (Appp ) , and any modified or unmodified certain embodiments , PNA surrogates may be used . nucleotide cap structure ( N - 0 - 5 '- ( HO ) ( O ) P — 0 (HO ) Terminal Modifications ( O ) P - O — P (HO )( O ) O - 5 ') ; 5 '- monothiophosphate (phos The 3 ' and 5 ' ends of an oligonucleotide can be modified . 5 phorothioate ; (HO ) . (SP _ 0 - 5 ') ; 5 '- monodithiophosphate Such modifications can be at the 3 ' end , 5 ' end or both ends (phosphorodithioate ; (HO )( HS )( S )P — 0 - 5 ') , 5 '- phosphoro of the molecule . They can include modification or replace ment of an entire terminal phosphate or of one ormore of the thiolate ( (HO ) ( O ) P — S - 5 ') ; any additional combination of atoms of the phosphate group . E . g ., the 3 ' and 5 ' ends of an oxygen / sulfur replaced monophosphate , diphosphate and oligonucleotide can be conjugated to other functional 10 triphosphates ( e . g . , 5 ' -alpha - thiotriphosphate , 5 ' - gamma molecular entities such as labeling moieties , e . g ., fluoro thiotriphosphate, etc .) , 5 '- phosphoramidates ( HO ) (O )P phores ( e . g ., pyrene, TAMRA, fluorescein , Cy3 or Cy5 dyes ) NH - 5 ', (HO ) (NH2 ) ( O ) P - O - 5 ') , 5 '- alkylphosphonates or protecting groups (based e . g . , on sulfur, silicon , boron or ( R = alkyl= methyl , ethyl, isopropyl , propyl , etc ., e .g ., ester ) . The functional molecular entities can be attached to RP (OH ) ( O ) - 0 - 5 ' -, (OH ) 2 ( O ) P - 5 ' - CH2- ) , 5 '- alkylether the sugar through a phosphate group and/ or a spacer . The 15 phosphonates ( R = alkylether = methoxymethyl (MeOCH2 - ) , terminal atom of the spacer can connect to or replace the ethoxymethyl, etc . , e . g . , RP (OH ) ( O ) - 0 - 9 - ) . linking atom of the phosphateate group or the C - 33 ' or C - 5 ' 0 ,. N ,. Terminalmodifications can also be useful for monitoring S or C group of the sugar . Alternatively , the spacer can distribution ,, and inin such cases the groups to bebe added may connect to or replace the terminal atom of a nucleotide include fluorophores , e . g . , fluorscein or an Alexa dye , e . g . , surrogate ( e .g ., PNAs) . These spacers or linkers can include 20 Alexa 488 . Terminal modifications can also be useful for e . g ., — ( CH2) - (CH ) N — , - (CH2 ) , 0 , (CH2 ) , enhancing uptake , useful modifications for this include S - , O (CH2CH2O ) CH2CH2OH (e . g. , n = 3 or 6 ), abasic cholesterol. Terminal modifications can also be useful for sugars , amide, carboxy , amine , oxyamine , oxyimine , thio - cross- linking an RNA agent to another moiety ; modifica ether , disulfide , thiourea , sulfonamide , or morpholino , or tions useful for this include mitomycin C . biotin and fluorescein reagents . When a spacer /phosphate - 25 The Bases functional molecular entity - spacer/ phosphate array is inter - Adenine , guanine , cytosine and uracil are the most com posed between two strands of iRNA agents , this array can mon bases found in RNA . These bases can be modified or substitute for a hairpin RNA loop in a hairpin - type RNA replaced to provide RNA ’ s having improved properties. agent. The 3' end can be an – OH group . While not wishing E . g ., nuclease resistant oligoribonucleotides can be prepared to be bound by theory , it is believed that conjugation of 30 with these bases or with synthetic and natural nucleobases certain moieties can improve transport , hybridization , and ( e . g . , inosine , thymine , xanthine , hypoxanthine , nubularine , specificity properties. Again , while not wishing to be bound isoguanisine , or tubercidine ) and any one of the above by theory , it may be desirable to introduce terminal altera modifications. Alternatively, substituted or modified analogs tions that improve nuclease resistance . Other examples of of any of the above bases and “ universal bases” can be terminal modifications include dyes , intercalating agents 35 employed . Examples include 2 - aminoadenine , 6 -methyl and ( e . g . , acridines ) , cross - linkers ( e . g ., psoralen , mitomycin C ) , other alkyl derivatives of adenine and guanine, 2 - propyl and porphyrins ( TPPC4 , texaphyrin , Sapphyrin ), polycyclic aro other alkyl derivatives of adenine and guanine , 5 -halouracil matic hydrocarbons ( e . g . , phenazine , dihydrophenazine ) , and cytosine , 5 - propynyl uracil and cytosine, 6 -azo uracil, artificial endonucleases ( e . g . , EDTA ) , lipophilic carriers cytosine and thymine , 5 - uracil (pseudouracil ) , 4 - thiouracil , ( e . g ., cholesterol, cholic acid , adamantane acetic acid , 40 5 -halouracil , 5 - ( 2 - aminopropyl) uracil , 5 - amino allyl uracil, 1 -pyrene butyric acid , dihydrotestosterone , 1 , 3 - Bis - O (hexa - 8 - halo , amino , thiol, thioalkyl, hydroxyl and other 8 -substi decyl) glycerol, geranyloxyhexyl group , hexadecylglycerol, tuted adenines and guanines, 5 - trifluoromethyl and other borneol, menthol, 1 , 3 - propanediol, heptadecyl group , palm - 5 -substituted uracils and cytosines , 7 -methylguanine , 5 - sub itic acid , myristic acid , O3 - (oleoyl ) lithocholic acid , O3 stituted pyrimidines , 6 - azapyrimidines and N2, N - 6 and 0 - 6 ( oleoyl) cholenic acid , dimethoxytrityl, or phenoxazine ) and 45 substituted purines , including 2 -aminopropyladenine , 5 -pro peptide conjugates ( e. g ., antennapedia peptide, Tat peptide ), pynyluracil and 5 -propynylcytosine , dihydrouracil , 3 -deaza alkylating agents, phosphate , amino , mercapto , PEG ( e . g ., 5 - azacytosine , 2 - aminopurine , 5 - alkyluracil , 7 - alkylgua PEG -40K ) , MPEG , [MPEG ) 2 , polyamino , alkyl, substituted nine , 5 -alkyl cytosine, 7 -deazaadenine , N6 , alkyl, radiolabeled markers , enzymes , haptens ( e . g ., biotin ), N6 - dimethyladenine , 2 ,6 - diaminopurine , 5 - amino -allyl -ura transport /absorption facilitators ( e . g ., aspirin , vitamin E , 50 cil, N3- methyluracil , substituted 1 , 2 , 4 - triazoles , 2 - pyridi folic acid ) , synthetic ribonucleases ( e . g . , imidazole , bisimi- none , 5 -nitroindole , 3 - nitropyrrole , 5 -methoxyuracil , uracil dazole, histamine , imidazole clusters, acridine- imidazole 5 -oxyacetic acid , 5 -methoxycarbonylmethyluracil , conjugates, Eu3 + complexes of tetraazamacrocycles ) . 5 -methyl - 2 - thiouracil, 5 -methoxycarbonylmethyl - 2 - thioura Terminal modifications can be added for a number of cil, 5 -methylaminomethyl - 2 - thiouracil , 3 - ( 3 - amino - 3 -car reasons, including as discussed elsewhere herein to modu - 55 boxypropyl) uracil, 3 -methylcytosine , 5 -methylcytosine , late activity or to modulate resistance to degradation . Ter - N4 -acetyl cytosine , 2 -thiocytosine , N6 -methyladenine , minal modifications useful for modulating activity include N6 -isopentyladenine , 2 -methylthio - N - 6 - isopentenylad modification of the 5 ' end with phosphate or phosphate enine , N -methylguanines , or O - alkylated bases. Further analogs . E . g ., in certain embodiments iRNA agents , espe - purines and pyrimidines include those disclosed in U . S . Pat . cially antisense strands, are 5' phosphorylated or include a 60 No . 3 ,687 ,808 , those disclosed in the Concise Encyclopedia phosphoryl analog at the 5 ' prime terminus. 5 '- phosphate Of Polymer Science And Engineering , pages 858 - 859 , Kro modifications include those which are compatible with RISC schwitz , J . I . , ed . John Wiley & Sons , 1990 , and those mediated gene silencing . Suitable modifications include : disclosed by Englisch et al. , Angewandte Chemie , Interna 5 '- monophosphate ( (HO ) 2 (OP = O - 5 ') ; 5 '- diphosphate tional Edition , 1991, 30 , 613 . ( (HO ) 2 (0 ) P40 - P (HO ) (O ) O - 5' ) ; 5 '- triphosphate (( HO ) 65 Generally , base changes are not used for promoting sta 2 ( 0 ) P _ O ( HO ) ( O ) P — 0 — P (HO ) ( O )40 - 5 ') ; 5 '- guanos - bility , but they can be useful for other reasons, e . g . , some, ine cap ( 7 -methylated or non -methylated ) ( 7m - G - 0 - 5 '- (HO ) e . g ., 2 ,6 - diaminopurine and 2 amino purine , are fluorescent. US 9 , 814 ,777 B2 75 76 Modified bases can reduce target specificity . This may be 677 and in published PCT applications PCT /US92 / 04294 taken into consideration in the design of iRNA agents . and PCT /US92 /04305 ( published as WO 92 / 20822 WO and 92/ 20823, respectively ) . Formacetal and thioformacetal REFERENCES linked oligoribonucleosides can be prepared as is described 5 in U . S . Pat. Nos. 5 , 264 ,562 and 5 , 264 ,564 . Ethylene oxide The disclosure of all publications , patents , and published linked oligoribonucleosides can be prepared as is described patent applications listed herein are hereby incorporated by in U . S . Pat. No . 5, 223 ,618 . Siloxane replacements are reference . described in Cormier, J . F . et al. Nucleic Acids Res. 1988 , 16 , General References 4583 . Carbonate replacements are described in Tittensor, J . The oligoribonucleotides and oligoribonucleosides used 10 R . J. Chem . Soc . C 1971 , 1933 . Carboxymethyl replace in accordance with this invention may be with solid phase ments are described in Edge, M . D . et al. J . Chem . Soc. synthesis , see for example " Oligonucleotide synthesis , a Perkin Trans. 1 1972 , 1991 . Carbamate replacements are practical approach ” , Ed . M . J . Gait , IRL Press , 1984 ; “ Oli described in Stirchak , E . P . Nucleic Acids Res. 1989 , 17 , gonucleotides and Analogues, A Practical Approach ” , Ed . F . 6129 . Eckstein , IRL Press , 1991 (especially Chapter 1 , Modern 15 Replacement of the Phosphate - Ribose Backbone References machine -aided methods of oligodeoxyribonucleotide syn - Cyclobutyl sugar surrogate compounds can be prepared as thesis , Chapter 2 , Oligoribonucleotide synthesis , Chapter is described in U . S . Pat. No . 5 ,359 , 044 . Pyrrolidine sugar 3 , 2 - 0 -Methyloligoribonucleotide - s : synthesis and applica surrogate can be prepared as is described in U . S . Pat . No . tions, Chapter 4 , Phosphorothioate oligonucleotides, Chap - 5 ,519 ,134 . Morpholino sugar surrogates can be prepared as ter 5 , Synthesis of oligonucleotide phosphorodithioates , 20 is described in U . S . Pat. Nos . 5 , 142 ,047 and 5 ,235 ,033 , and Chapter 6 , Synthesis of oligo - 2 '- deoxyribonucleoside meth other related patent disclosures. Peptide Nucleic Acids ylphosphonates , and . Chapter 7 , Oligodeoxynucleotides (PNAs ) are known per se and can be prepared in accordance containing modified bases . Other particularly useful syn with any of the various procedures referred to in Peptide thetic procedures, reagents , blocking groups and reaction Nucleic Acids ( PNA ) : Synthesis , Properties and Potential conditions are described in Martin , P ., Helv . Chim . Acta , 25 Applications, Bioorganic & Medicinal Chemistry, 1996 , 4 , 1995 , 78 , 486 -504 ; Beaucage , S . L . and Iyer , R . P ., Tetra - 5 - 23 . They may also be prepared in accordance with U . S . hedron , 1992 , 48 , 2223 - 2311 and Beaucage , S . L . and Iyer , Pat. No . 5 ,539 , 083 . R . P ., Tetrahedron , 1993 , 49, 6123 -6194 , or references Terminal Modification References referred to therein . Modification described in WO 00 /44895 , Terminal modifications are described in Manoharan , M . et W001 / 75164 , or WO02 /44321 can be used herein . 30 al. Antisense and Nucleic Acid Drug Development 12 , 103 Phosphate Group References 128 ( 2002 ) and references therein . The preparation of phosphinate oligoribonucleotides is Base References described in U .S . Pat. No . 5 ,508 , 270 . The preparation of N - 2 substituted purine nucleoside amidites can be pre alkyl phosphonate oligoribonucleotides is described in U . S . pared as is described in U . S . Pat. No. 5 ,459 , 255 . 3 - Deaza Pat. No. 4 ,469 , 863 . The preparation of phosphoramidite 35 purine nucleoside amidites can be prepared as is described oligoribonucleotides is described in U . S . Pat. No. 5 , 256 , 775 in U . S . Pat . No. 5 , 457 , 191 . 5 , 6 -Substituted pyrimidine or U . S . Pat. No. 5 , 366 ,878 . The preparation of phosphotri - nucleoside amidites can be prepared as is described in U . S . ester oligoribonucleotides is described in U . S . Pat. No . Pat. No. 5 ,614 ,617 . 5 - Propynyl pyrimidine nucleoside amid 5 ,023 , 243 . The preparation of borano phosphate oligoribo - ites can be prepared as is described in U . S . Pat. No . nucleotide is described in U . S . Pat. Nos. 5 , 130 , 302 and 40 5 ,484 , 908 . Additional references can be disclosed in the 5 , 177 , 198 . The preparation of 3 ' -Deoxy - 3 - amino phospho - above section on base modifications . ramidate oligoribonucleotides is described in U . S . Pat. No. The nucleic acid that is used in a lipid -nucleic acid 5 ,476 ,925 . 3' -Deoxy - 3 '- methylenephosphonate oligoribo - particle according to this invention includes any form of nucleotides is described in An , H , et al. J . Org . Chem . 2001, nucleic acid that is known . Thus, the nucleic acid may be a 66 , 2789 - 2801. Preparation of sulfur bridged nucleotides is 45 modified nucleic acid of the type used previously to enhance described in Sproat et al . Nucleosides Nucleotides 1988 , nuclease resistance and serum stability . Surprisingly , how 7 ,651 and Crosstick et al. Tetrahedron Lett. 1989 , 30 , 4693 . ever , acceptable therapeutic products can also be prepared Sugar Group References using the method of the invention to formulate lipid -nucleic Modifications to the 2 ' modifications can be found in acid particles from nucleic acids that have no modification Verma, S . et al. Annu . Rev. Biochem . 1998 , 67 , 99 - 134 and 50 to the phosphodiester linkages of natural nucleic acid poly all references therein . Specific modifications to the ribose mers , and the use of unmodified phosphodiester nucleic can be found in the following references : 21 - fluoro (Kawa - acids ( i. e ., nucleic acids in which all of the linkages are saki et . al. , J . Med. Chem . , 1993 , 36 , 831- 841 ) , 2 -MOE phosphodiester linkages) is a preferred embodiment of the (Martin , P . Helv . Chim . Acta 1996 , 79 , 1930 - 1938 ) , “ LNA " invention . (Wengel , J. Acc. Chem . Res. 1999, 32 , 301 -310 ). 55 Chimeric Oligonucleotides Replacement of the Phosphate Group References It is not necessary for all positions in a given compound Methylenemethylimino linked oligoribonucleosides, also to be uniformly modified , and in fact more than one of the identified herein as MMI linked oligoribonucleosides , meth - aforementioned modifications may be incorporated in a ylenedimethylhydrazo linked oligoribonucleosides, also single compound or even at a single nucleoside within an identified herein as MDH linked oligoribonucleosides , and 60 oligonucleotide . Certain preferred oligonucleotides of this methylenecarbonylamino linked oligonucleosides, also invention are chimeric oligonucleotides. “ Chimeric oligo identified herein as amide- 3 linked oligoribonucleosides , nucleotides ” or “ chimeras, ” in the context of this invention , and methyleneaminocarbonyl linked oligonucleosides , also are oligonucleotides that contain two or more chemically identified herein as amide - 4 linked oligoribonucleosides as distinct regions , each made up of at least one nucleotide . well asmixed backbone compounds having , as for instance , 65 These oligonucleotides typically contain at least one region alternating MMI and PO or PS linkages can be prepared as of modified nucleotides that confers one or more beneficial is described in U . S . Pat . Nos. 5 ,378 , 825 , 5 ,386 ,023 , 5 ,489 , properties ( such as, e . g ., increased nuclease resistance , US 9 , 814 , 777 B2 77 78 increased uptake into cells , increased binding affinity for the acid polymer is of significance , since the modification of RNA target ) and a region that is a substrate for RNase H nucleic acids in an effort to impart nuclease resistance adds cleavage . to the cost of therapeutics while in many cases providing In one embodiment, a chimeric oligonucleotide comprises only limited resistance. Unless stated otherwise, these cri at least one region modified to increase target binding 5 teria are calculated in this specification as follows: affinity . Affinity of an oligonucleotide for its target is rou - Nucleic acid to lipid ratio is the amount ofnucleic acid in tinely determined by measuring the Tm of an oligonucle a defined volume of preparation divided by the amount of otide / target pair , which is the temperature at which the oligonucleotide and target dissociate ; dissociation is lipid in the same volume. This may be on a mole per mole detected spectrophotometrically . The higher the Tm , the 10 basis or on a weight per weight basis , or on a weight per greater the affinity of the oligonucleotide for the target. In mole basis . For final, administration -ready formulations, the one embodiment, the region of the oligonucleotide which is nucleic acid : lipid ratio is calculated after dialysis , chroma modified to increase target mRNA binding affinity com tography and / or enzyme ( e. g. , nuclease ) digestion has been prises at least one nucleotide modified at the 2 ' position of employed to remove as much of the external nucleic acid as the sugar, most preferably a 2 - O - alkyl, 2 - 0 -alkyl - O - alkyl 15 possible ; Encapsulation efficiency refers to the drug to lipid ratio of routinelyor 21 - fluoro incorporated -modified nucleotideinto oligonucleotides . Such modifications and these oliare - the starting mixture divided by the drug to lipid ratio of the gonucleotides have been shown to have a higher Tm (i . e. , final, administration competent formulation . This is a mea higher target binding affinity ) than 2 ' - deoxyoligonucleotides sure of relative efficiency . For a measure of absolute efti against a given target . The effect of such increased affinity 20 ciency , the total amount of nucleic acid added to the starting is to greatly enhance oligonucleotide inhibition of target mixture that ends up in the administration competent for gene expression . mulation , can also be calculated . The amount of lipid lost In another embodiment, a chimeric oligonucletoide com - during the formulation process may also be calculated . prises a region that acts as a substrate for RNAse H . Of Efficiency is a measure of the wastage and expense of the course , it is understood that oligonucleotides may include 25 formulation ; and any combination of the various modifications described Size indicates the size (diameter ) of the particles formed . herein . Size distribution may be determined using quasi- elastic light Another modification of the oligonucleotides of the inven - scattering ( QELS ) on a Nicomp Model 370 sub -micron tion involves chemically linking to the oligonucleotide one particle sizer. Particles under 200 nm are preferred for or more moieties or conjugates which enhance the activity, 30 distribution to neo - vascularized ( leaky) tissues, such as cellular distribution or cellular uptake of the oligonucle - neoplasms and sites of inflammation . otide. Such conjugates and methods of preparing the same Pharmaceutical Compositions are known in the art. The lipid particles of present invention , particularly when Those skilled in the art will realize that for in vivo utility , associated with a therapeutic agent, may be formulated as a such as therapeutic efficacy , a reasonable rule of thumb is 35 pharmaceutical composition , e . g . , which further comprises a that if a thioated version of the sequence works in the free pharmaceutically acceptable diluent, excipient , or carrier , form , that encapsulated particles of the same sequence , of such as physiological saline or phosphate buffer, selected in any chemistry , will also be efficacious . Encapsulated par - accordance with the route of administration and standard ticles may also have a broader range of in vivo utilities , pharmaceutical practice . showing efficacy in conditions and models not known to be 40 In particular embodiments , pharmaceutical compositions otherwise responsive to antisense therapy . Those skilled in comprising the lipid -nucleic acid particles of the invention the art know that applying this invention they may find old are prepared according to standard techniques and further models which now respond to antisense therapy. Further , comprise a pharmaceutically acceptable carrier. Generally , they may revisit discarded antisense sequences or chemis normal saline will be employed as the pharmaceutically tries and find efficacy by employing the invention . 45 acceptable carrier. Other suitable carriers include , e . g . , The oligonucleotides used in accordance with this inven - water , buffered water, 0 . 9 % saline , 0 . 3 % glycine , and the tion may be conveniently and routinely made through the like , including glycoproteins for enhanced stability , such as well -known technique of solid phase synthesis . Equipment albumin , lipoprotein , globulin , etc . In compositions com for such synthesis is sold by several vendors including prising saline or other salt containing carriers , the carrier is Applied Biosystems. Any other means for such synthesis 50 preferably added following lipid particle formation . Thus , may also be employed ; the actual synthesis of the oligo - after the lipid - nucleic acid compositions are formed , the nucleotides is well within the talents of the routineer. It is compositions can be diluted into pharmaceutically accept also well known to use similar techniques to prepare other able carriers such as normal saline . oligonucleotides such as the phosphorothioates and alky The resulting pharmaceutical preparations may be steril lated derivatives . 55 ized by conventional, well known sterilization techniques . Characteristic of Nucleic Acid - Lipid Particles The aqueous solutions can then be packaged for use or In certain embodiments, the present invention relates to filtered under aseptic conditions and lyophilized , the methods and compositions for producing lipid - encapsulated lyophilized preparation being combined with a sterile aque nucleic acid particles in which nucleic acids are encapsu - ous solution prior to administration . The compositions may lated within a lipid layer . Such nucleic acid - lipid particles , 60 contain pharmaceutically acceptable auxiliary substances as incorporating siRNA oligonucleotides, are characterized required to approximate physiological conditions , such as using a variety of biophysical parameters including : ( 1 ) drug pH adjusting and buffering agents, tonicity adjusting agents to lipid ratio ; ( 2 ) encapsulation efficiency ; and ( 3 ) particle and the like , for example , sodium acetate , sodium lactate , size . High drug to lipid rations , high encapsulation effi - sodium chloride , potassium chloride , calcium chloride , etc . ciency , good nuclease resistance and serum stability and 65 Additionally , the lipidic suspension may include lipid - pro controllable particle size , generally less than 200 nm in tective agents which protect lipids against free -radical and diameter are desirable . In addition , the nature of the nucleic lipid - peroxidative damages on storage . Lipophilic free - radi US 9 ,814 ,777 B2 79 80 cal quenchers, such as a - tocopherol and water - soluble iron can be used in the formulations of the invention using a specific chelators , such as ferrioxamine , are suitable . two - step process. First , lipid vesicles can be formed at the The concentration of lipid particle or lipid - nucleic acid lower pH with titratable cationic lipids and other vesicle particle in the pharmaceutical formulations can vary widely , components in the presence of nucleic acids. In this manner, i . e . , from less than about 0 .01 % , usually at or at least about 5 the vesicles will encapsulate and entrap the nucleic acids . 0 .05 - 5 % to as much as 10 to 30 % by weight and will be Second , the surface charge of the newly formed vesicles can selected primarily by fluid volumes, viscosities , etc . , in be neutralized by increasing the pH of the medium to a level accordance with the particular mode of administration above the pK , of the titratable cationic lipids present, i. e . , to selected . For example , the concentration may be increased to physiological pH or higher. Particularly advantageous lower the fluid load associated with treatment . This may be 10 aspects of this process include both the facile removal of any particularly desirable in patients having atherosclerosis - surface adsorbed nucleic acid and a resultant nucleic acid associated congestive heart failure or severe hypertension . delivery vehicle which has a neutral surface . Liposomes or Alternatively, complexes composed of irritating lipids may lipid particles having a neutral surface are expected to avoid be diluted to low concentrations to lessen inflammation at rapid clearance from circulation and to avoid certain toxici the site of administration . In one group of embodiments , the 15 ties which are associated with cationic liposome prepara nucleic acid will have an attached label and will be used for tions. Additional details concerning these uses of such diagnosis (by indicating the presence of complementary titratable cationic lipids in the formulation of nucleic acid nucleic acid ) . In this instance , the amount of complexes lipid particles are provided in U . S . Pat. No . 6 , 287 ,591 and administered will depend upon the particular label used , the U . S . Pat. No. 6 ,858 , 225 , incorporated herein by reference . disease state being diagnosed and the judgement of the 20 It is further noted that the vesicles formed in this manner clinician but will generally be between about 0 .01 and about provide formulations of uniform vesicle size with high 50 mg per kilogram of body weight, preferably between content of nucleic acids . Additionally , the vesicles have a about 0 . 1 and about 5 mg/kg of body weight. size range of from about 20 to about 200 nm , preferably 30 As noted above , the lipid -therapeutic agent ( e . g . , nucleic to about 150 nm , more preferably about 30 to about 90 nm . acid ) particles of the invention may include polyethylene 25 Without intending to be bound by any particular theory , it glycol ( PEG ) -modified phospholipids , PEG - ceramide , or is believed that the very high efficiency of nucleic acid ganglioside Gm?- modified lipids or other lipids effective to encapsulation is a result of electrostatic interaction at low prevent or limit aggregation . Addition of such components pH . At acidic pH (e . g. pH 4 . 0 ) the vesicle surface is charged does not merely prevent complex aggregation . Rather , it and binds a portion of the nucleic acids through electrostatic may also provide a means for increasing circulation lifetime 30 interactions. When the external acidic buffer is exchanged and increasing the delivery of the lipid -nucleic acid com - for a more neutral buffer ( e . g . pH 7 . 5 ) the surface of the lipid position to the target tissues . particle or liposome is neutralized , allowing any external The present invention also provides lipid - therapeutic nucleic acid to be removed . More detailed information on agent compositions in kit form . The kit will typically be the formulation process is provided in various publications comprised of a container that is compartmentalized for 35 (e .g ., U .S . Pat. No . 6 , 287 ,591 and U . S . Pat . No . 6 ,858 ,225 ). holding the various elements of the kit . The kit will contain In view of the above , the present invention provides the particles or pharmaceutical compositions of the present methods of preparing lipid /nucleic acid formulations . In the invention , preferably in dehydrated or concentrated form , methods described herein , a mixture of lipids is combined with instructions for their rehydration or dilution and admin - with a buffered of nucleic acid to produce istration . In certain embodiments, the particles comprise the 40 an intermediate mixture containing nucleic acid encapsu active agent , while in other embodiments, they do not. lated in lipid particles , e . g . , wherein the encapsulated nucleic Methods of Manufacture acids are present in a nucleic acid / lipid ratio of about 10 wt The methods and compositions of the invention make use % to about 20 wt % . The intermediate mixture may option of certain targeting lipids, the synthesis , preparation and ally be sized to obtain lipid -encapsulated nucleic acid par characterization of which is described in the accompanying 45 ticles wherein the lipid portions are unilamellar vesicles , Examples . In addition , the present invention provides meth - preferably having a diameter of 30 to 150 nm , more pref ods of preparing lipid particles, including those associated erably about 40 to 90 nm . The pH is then raised to neutralize with a therapeutic agent, e . g . , a nucleic acid . In the methods at least a portion of the surface charges on the lipid -nucleic described herein , a mixture of lipids is combined with a acid particles , thus providing an at least partially surface buffered aqueous solution of nucleic acid to produce an 50 neutralized lipid - encapsulated nucleic acid composition . intermediate mixture containing nucleic acid encapsulated In preparing the nucleic acid - lipid particles of the inven in lipid particles wherein the encapsulated nucleic acids are tion , the mixture of lipids is typically a solution of lipids in present in a nucleic acid / lipid ratio of about 1 wt % to about an organic . This mixture of lipids can then be dried 30 wt % , preferably 3 to 25 wt % , even more preferably 5 to form a thin film or lyophilized to form a powder before to 15 wt % . The intermediate mixture may optionally be 55 being hydrated with an aqueous buffer to form liposomes. sized to obtain lipid - encapsulated nucleic acid particles Alternatively , in a preferred method , the lipid mixture can be wherein the lipid portions are unilamellar vesicles, prefer solubilized in a water miscible alcohol, such as ethanol, and ably having a diameter of 20 to 200 nm , more preferably 30 this ethanolic solution added to an aqueous buffer resulting to 150 nm , even more preferably about 40 to 90 nm . The pH in spontaneous liposome formation . In most embodiments , is then raised to neutralize at least a portion of the surface 60 the alcohol is used in the form in which it is commercially charges on the lipid - nucleic acid particles , thus providing an available . For example , ethanol can be used as absolute at least partially surface -neutralized lipid - encapsulated ethanol ( 100 % ) , or as 95 % ethanol, the remainder being nucleic acid composition . water . This method is described in more detail in U . S . Pat. As described above, several of the cationic lipids are No. 5 , 976 , 567) . amino lipids that are charged at a pH below the pK , of the 65 In one exemplary embodiment, the mixture of lipids is a amino group and substantially neutral at a pH above the pK , . mixture of targeting lipid , cationic lipids , neutral lipids These cationic lipids are termed titratable cationic lipids and other than a cationic lipid ), a sterol (e . g ., cholesterol) and a US 9 ,814 ,777 B2 82 PEG -modified lipid (e .g ., a PEG -DMG , PEG - C - DOMG or produces a progressive size reduction down to small unila PEG - DMA ) in an alcohol solvent. In preferred embodi- mellar vesicles (SUVs ) less than about 0 .05 microns in size . ments , the lipid mixture consists essentially of a targeting Homogenization is another method which relies on shearing lipid , acationic amino lipid , a neutral lipid , cholesterol and energy to fragment large liposomes into smaller ones. In a a PEG -modified lipid in alcohol, more preferably ethanol. In 5 typical homogenization procedure , multilamellar vesicles further preferred embodiments , the first solution consists of are recirculated through a standard homogenizer the above lipid mixture in molar ratios of about 0 . 5 - 50 % until selected liposome sizes, typically between about 0 . 1 targeting lipid : 20 -70 % cationic lipid : 5 -45 % neutral lipid : 20 - and 0 . 5 microns , are observed . In both methods , the particle 55 % cholesterol: 0 . 5 - 15 % PEG -modified lipid . size distribution can be monitored by conventional laser In accordance with the invention , the lipid mixture is 10 beam particle size determination . For certain methods combined with a buffered aqueous solution that may contain h erein , extrusion is used to obtain a uniform vesicle size . the nucleic acids . The buffered aqueous solution of is Extrusion of liposome compositions through a small- pore typically a solution in which the buffer has a pH of less than polycarbonate membrane or an asymmetric ceramic mem the pK , of the protonatable lipid in the lipid mixture . brane results in a relatively well - defined size distribution . Examples of suitable buffers include citrate , phosphate , 15 Typically , the suspension is cycled through the membrane acetate , and MES. A particularly preferred buffer is citrate one or more times until the desired liposome complex size buffer. Preferred buffers will be in the range of 1 - 1000 mM distribution is achieved . The liposomes may be extruded of the anion , depending on the chemistry of the nucleic acid through successively smaller- pore membranes , to achieve a being encapsulated , and optimization of buffer concentration gradual reduction in liposome size . In some instances , the may be significant to achieving high loading levels ( see , e . g ., 20 lipid - nucleic acid compositions which are formed can be U .S . Pat . No . 6 ,287 ,591 and U .S . Pat. No. 6 , 858, 225 ). used without any sizing . Alternatively , pure water acidified to pH 5 - 6 with chloride, In particular embodiments, methods of the present inven sulfate or the like may be useful. In this case , it may be tion further comprise a step of neutralizing at least some of suitable to add 5 % glucose , or another non -ionic solute the surface charges on the lipid portions of the lipid - nucleic which will balance the osmotic potential across the particle 25 acid compositions . By at least partially neutralizing the membrane when the particles are dialyzed to remove etha surface charges , unencapsulated nucleic acid is freed from nol, increase the pH , or mixed with a pharmaceutically the lipid particle surface and can be removed from the acceptable carrier such as normal saline. The amount of composition using conventional techniques. Preferably , nucleic acid in buffer can vary , but will typically be from unencapsulated and surface adsorbed nucleic acids are about 0 . 01 mg/ mL to about 200 mg/ mL , more preferably 30 removed from the resulting compositions through exchange from about 0 . 5 mg/ mL to about 50 mg/ mL . of buffer solutions . For example , replacement of a citrate The mixture of lipids and the buffered aqueous solution of buffer (pH about 4 . 0 , used for forming the compositions ) therapeutic nucleic acids is combined to provide an inter - with a HEPES - buffered saline (HBS pH about 7 . 5 ) solution , mediate mixture . The intermediate mixture is typically a results in the neutralization of liposome surface and nucleic mixture of lipid particles having encapsulated nucleic acids . 35 acid release from the surface . The released nucleic acid can Additionally , the intermediate mixture may also contain then be removed via chromatography using standard meth some portion of nucleic acids which are attached to the ods , and then switched into a buffer with a pH above the pKa surface of the lipid particles ( liposomes or lipid vesicles ) due of the lipid used . to the ionic attraction of the negatively - charged nucleic Optionally the lipid vesicles ( i. e ., lipid particles ) can be acids and positively -charged lipids on the lipid particle 40 formed by hydration in an aqueous buffer and sized using surface ( the amino lipids or other lipid making up the any of the methods described above prior to addition of the protonatable first lipid component are positively charged in nucleic acid . As described above , the aqueous buffer should a buffer having a pH of less than the pK , of the protonatable be of a pH below the pKa of the amino lipid . A solution of group on the lipid ) . In one group of preferred embodiments , the nucleic acids can then be added to these sized , preformed the mixture of lipids is an alcohol solution of lipids and the 45 vesicles . To allow encapsulation of nucleic acids into such volumes of each of the solutions is adjusted so that upon " pre - formed ” vesicles the mixture should contain an alco combination , the resulting alcohol content is from about hol, such as ethanol. In the case of ethanol, it should be 20 % by volume to about 45 % by volume. The method of present at a concentration of about 20 % ( w / w ) to about 45 % combining the mixtures can include any of a variety of ( w / w ). In addition , it may be necessary to warm the mixture processes, often depending upon the scale of formulation 50 of pre - formed vesicles and nucleic acid in the aqueous produced . For example , when the total volume is about buffer -ethanol mixture to a temperature of about 25° C . to 10 -20 mL or less, the solutions can be combined in a test about 50° C . depending on the composition of the lipid tube and stirred together using a vortex mixer . Large - scale vesicles and the nature of the nucleic acid . It will be apparent processes can be carried out in suitable production scale to one of ordinary skill in the art that optimization of the glassware . 55 encapsulation process to achieve a desired level of nucleic Optionally, the lipid - encapsulated therapeutic agent ( e . g ., acid in the lipid vesicles will require manipulation of vari nucleic acid ) complexes which are produced by combining able such as ethanol concentration and temperature . the lipid mixture and the buffered aqueous solution of Examples of suitable conditions for nucleic acid encapsu therapeutic agents (nucleic acids ) can be sized to achieve a lation are provided in the Examples. Once the nucleic acids desired size range and relatively narrow distribution of lipid 60 are encapsulated within the preformed vesicles, the external particle sizes. Preferably, the compositions provided herein pH can be increased to at least partially neutralize the will be sized to a mean diameter of from about 70 to about surface charge . Unencapsulated and surface adsorbed 200 nm , more preferably about 90 to about 130 nm Several nucleic acids can then be removed as described above . techniques are available for sizing liposomes to a desired Method of Use size . One sizing method is described in U . S . Pat . No . 65 The lipid particles of the present invention may be used to 4 ,737 , 323 , incorporated herein by reference . Sonicating a deliver a therapeutic agent to a cell , in vitro or in vivo . In liposome suspension either by bath or probe sonication particular embodiments , the therapeutic agent is a nucleic US 9 , 814 , 777 B2 83 84 acid , which is delivered to a cell using a nucleic acid - lipid invention include introduction of antisense oligonucleotides particles of the present invention . While the following in cells ( see , Bennett , et al. , Mol. Pharm . 41 : 1023 - 1033 description o various methods of using the lipid particles and ( 1992 ) ). related pharmaceutical compositions of the present inven - Alternatively , the compositions of the present invention tion are exemplified by description related to nucleic acid - 5 can also be used for deliver of nucleic acids to cells in vivo , lipid particles, it is understood that these methods and using methods which are known to those of skill in the art . compositions may be readily adapted for the delivery of any With respect to application of the invention for delivery of therapeutic agent for the treatment of any disease or disorder DNA or mRNA sequences , Zhu , et al ., Science 261: 209 - 211 that would benefit from such treatment. ( 1993 ) , incorporated herein by reference , describes the intra In certain embodiments, the present invention provides venous delivery of cytomegalovirus (CMV ) - chlorampheni methods for introducing a nucleic acid into a cell . Preferred col acetyltransferase (CAT ) expression plasmid using nucleic acids for introduction into cells are siRNA , immune - DOTMA -DOPE complexes . Hyde , et al . , Nature 362: 250 stimulating oligonucleotides, plasmids , antisense and 256 ( 1993 ) , incorporated herein by reference , describes the ribozymes . These methods may be carried out by contacting delivery of the cystic fibrosis transmembrane conductance the particles or compositions of the present invention with regulator ( CFTR ) gene to epithelia of the airway and to the cells for a period of time sufficient for intracellular alveoli in the lung of mice , using liposomes . Brigham , et al ., delivery to occur. Am . J. Med . Sci. 298 : 278 - 281 ( 1989 ) , incorporated herein by The compositions of the present invention can be reference, describes the in vivo transfection of lungs of mice adsorbed to almost any cell type. Once adsorbed , the nucleic 20 with a functioning prokaryotic gene encoding the intracel acid - lipid particles can either be endocytosed by a portion of lular enzyme, chloramphenicol acetyltransferase (CAT ) . the cells , exchange lipids with cell membranes , or fuse with Thus , the compositions of the invention can be used in the the cells . Transfer or incorporation of the nucleic acid treatment of infectious diseases . portion of the complex can take place via any one of these For in vivo administration , the pharmaceutical composi pathways . Without intending to be limited with respect to the 25 tions are preferably administered parenterally , i . e . , intraar scope of the invention , it is believed that in the case of ticularly , intravenously, intraperitoneally, subcutaneously , or particles taken up into the cell by endocytosis the particles intramuscularly . In particular embodiments , the pharmaceu then interact with the endosomal membrane , resulting in tical compositions are administered intravenously or intra destabilization of the endosomal membrane , possibly by the peritoneally by a bolus injection . For one example , see formation of non - bilaver phases, resulting in introduction of 30 Stadler, et al. , U . S . Pat. No . 5 ,286 , 634 , which is incorporated herein by reference . Intracellular nucleic acid delivery has the encapsulated nucleic acid into the cell cytoplasm . Simi also been discussed in Straubringer, et al. , METHODS IN larly in the case of direct fusion of the particles with the cell ENZYMOLOGY , Academic Press , New York . 101 :512 - 527 plasma membrane, when fusion takes place, the liposome ( 1983 ) ; Mannino , et al. , Biotechniques 6 :682 -690 ( 1988 ) ; membrane is integrated into the cell membrane and the 35 Nicolau , et al ., Crit. Rev . Ther. Drug Carrier Syst. 6 :239 - 271 contents of the liposome combine with the intracellular (1989 ) , and Behr, Acc . Chem . Res. 26 : 274 - 278 ( 1993 ) . Still fluid . Contact between the cells and the lipid -nucleic acid other methods of administering lipid - based therapeutics are compositions, when carried out in vitro , will take place in a described in , for example , Rahman et al. , U . S . Pat. No. biologically compatible medium . The concentration of com - 3 , 993 , 754 ; Sears , U . S . Pat. No . 4 , 145 ,410 ; Papahadjopoulos positions can vary widely depending on the particular appli - 40 et al ., U . S . Pat. No. 4 , 235 ,871 ; Schneider , U . S . Pat . No . cation , but is generally between about 1 mmol and about 10 4 , 224 , 179 ; Lenk et al. , U . S . Pat. No . 4 , 522 ,803 ; and Foun mmol. In certain embodiments , treatment of the cells with tain et al. , U . S . Pat. No. 4 , 588 , 578 the lipid -nucleic acid compositions will generally be carried In other methods, the pharmaceutical preparations may be out at physiological (about 37° C . ) for periods contacted with the target tissue by direct application of the of time from about 1 to 24 hours , preferably from about 2 to 45 preparation to the tissue . The application may be made by 8 hours . For in vitro applications, the delivery of nucleic topical , " open " or " closed ” procedures . By “ topical ,” it is acids can be to any cell grown in culture, whether of plant meant the direct application of the pharmaceutical prepara or animal origin , vertebrate or invertebrate , and of any tissue t ion to a tissue exposed to the environment, such as the skin , or type . In preferred embodiments, the cells will be animal oropharynx , external auditory canal, and the like . " Open " cells , more preferably mammalian cells , and most preferably 50 procedures are those procedures which include incising the human cells . skin of a patient and directly visualizing the underlying In one group of embodiments , a lipid -nucleic acid particle tissue to which the pharmaceutical preparations are applied . suspension is added to 60 -80 % confluent plated cells having This is generally accomplished by a surgical procedure, such a cell density of from about 10 % to about 10 cells/ mL , more as a thoracotomy to access the lungs , abdominal laparotomy preferably about 2x104 cells /mL . The concentration of the 55 to access abdominal viscera , or other direct surgical suspension added to the cells is preferably of from about approach to the target tissue . " Closed " procedures are inva 0 .01 to 20 ug/ mL , more preferably about 1 ug/ mL . sive procedures in which the internal target tissues are not Typical applications include using well known procedures directly visualized , but accessed via inserting instruments to provide intracellular delivery of siRNA to knock down or through small wounds in the skin . For example , the prepa silence specific cellular targets . Alternatively applications 60 rations may be administered to the peritoneum by needle include delivery of DNA or mRNA sequences that code for lavage . Likewise , the pharmaceutical preparations may be therapeutically useful polypeptides . In this manner , therapy administered to the meninges or spinal cord by infusion is provided for genetic diseases by supplying deficient or during a lumbar puncture followed by appropriate position absent gene products (i . e . , for Duchenne ' s dystrophy, see ing of the patient as commonly practiced for spinal anes Kunkel, et al. , Brit. Med . Bull . 45 ( 3 ) :630 -643 ( 1989 ) , and for 65 thesia or metrazamide imaging of the spinal cord . Alterna cystic fibrosis , see Goodfellow , Nature 341 : 102 - 103 tively , the preparations may be administered through (1989 )) . Other uses for the compositions of the present endoscopic devices. US 9 , 814 ,777 B2 85 86 The lipid - nucleic acid compositions can also be admin - prises a polynucleotide that specifically binds to a poly istered in an aerosol inhaled into the lungs (see , Brigham , et nucleotide that encodes the polypeptide , or a complement al ., Am . J. Sci. 298 (4 ): 278 - 281 (1989 )) or by direct injection thereof . at the site of disease (Culver , Human Gene Therapy, Mary - In another related embodiment, the present invention Ann Liebert , Inc . , Publishers , New York . pp . 70 -71 (1994 )) . 5 includes a method of treating a disease or disorder charac terized by underexpression of a polypeptide in a subject , The methods of the present invention may be practiced in comprising providing to the subject a pharmaceutical com a variety of hosts . Preferred hosts include mammalian spe position of the present invention , wherein the therapeutic cies, such as humans, non -human primates , dogs , cats , agent is a plasmid that encodes the polypeptide or a func cattle, horses, sheep , and the like . the 10 tional variant or fragment thereof. Dosages for the lipid - therapeutic agent particles of the In further embodiments , the pharmaceutical composition present invention will depend on the ratio of therapeutic is provided to the subject in combination with a vaccine or agent to lipid and the administrating physician ' s opinion antigen . Thus , the present invention itself provides vaccines based on age , weight, and condition of the patient . comprising a lipid particle of the present invention , which In one embodiment, the present invention provides a 15 comprises an immunostimulatorv oligonucleotide , and is method of modulating the expression of a target polynucle - also associated with an antigen to which an immune otide or polypeptide . These methods generally comprise response is desired . In particular embodiments, the antigen contacting a cell with a lipid particle of the present invention is a tumor antigen or is associated with an infective agent, that is associated with a nucleic acid capable ofmodulating such as, e .g ., a virus, bacteria , or parasite . the expression of a target polynucleotide or polypeptide . As 20 A variety of tumor antigens , infections agent antigens , and used herein , the term “ modulating ” refers to altering the antigens associated with other disease are well known in the expression of a target polynucleotide or polypeptide. In art and examples of these are described in references cited different embodiments , modulating can mean increasing or herein . Examples of antigens suitable for use in the present enhancing , or it can mean decreasing or reducing . Methods invention include, but are not limited to , polypeptide anti of measuring the level of expression of a target polynucle - 25 gens and DNA antigens . Specific examples of antigens are otide or polypeptide are known and available in the arts and Hepatitis A , Hepatitis B , small pox , polio , anthrax , influ include, e . g . ,methods employing reverse transcription -poly enza , typhus, tetanus, measles, rotavirus , diphtheria , pertus sis , tuberculosis , and rubella antigens. In a preferred merase chain reaction (RT - PCR ) and immunohistochemical embodiment, the antigen is a Hepatitis B recombinant anti techniques. In particular embodiments , the level of expresdor 30 gen . In other aspects , the antigen is a Hepatitis A recombi sion of a target polynucleotide or polypeptide is increased or 30 nantse antigen . In another aspect, the antigen is a tumor reduced by at least 10 % , 20 % , 30 % , 40 % , 50 % , or greater antigen . Examples of such tumor - associated antigens are than 50 % as compared to an appropriate control value . MUC - 1 , EBV antigen and antigens associated with Burkitt ' s For example , if increased expression of a polypeptide lymphoma. In a further aspect, the antigen is a tyrosinase desired , the nucleic acid may be an expression vector thatal 35 related protein tumor antigen recombinant antigen . Those of includes a polynucleotide that encodes the desired polypep skill in the art will know of other antigens suitable for use tide. On the other hand , if reduceded expressionexpression ofof aa poly in the present invention . nucleotide or polypeptide is desired , then the nucleic acid Tumor - associated antigens suitable for use in the subject may be , e . g . , an antisense oligonucleotide , siRNA , or micro - invention include both mutated and non -mutated molecules RNA that comprises a polynucleotide sequence that specifi - 40 that may be indicative of single tumor type, shared among cally hybridizes to a polynucleotide that encodes the target several types of tumors, and /or exclusively expressed or polypeptide , thereby disrupting expression of the target overexpressed in tumor cells in comparison with normal polynucleotide or polypeptide. Alternatively , the nucleic cells . In addition to proteins and glycoproteins, tumor acid may be a plasmid that expresses such an antisense specific patterns of expression of carbohydrates , ganglio oligonucleotide , siRNA , or microRNA . 45 sides, glycolipids and mucins have also been documented . In particular embodiments , the therapeutic agent is Exemplary tumor -associated antigens for use in the subject selected from an siRNA , a microRNA , an antisense oligo cancer vaccines include protein products of oncogenes, nucleotide , an antagomir and a plasmid capable of express - tumor suppressor genes and other genes with mutations or ing an siRNA , a microRNA , or an antisense oligonucleotide , rearrangements unique to tumor cells , reactivated embryonic and wherein the siRNA , microRNA , or antisense RNA 50 gene products , oncofetal antigens, tissue - specific (but not comprises a polynucleotide that specifically binds to a tumor -specific ) differentiation antigens , growth factor recep polynucleotide that encodes the polypeptide , or a comple tors , cell surface carbohydrate residues , foreign viral pro ment thereof, such that the expression of the polypeptide is teins and a number of other self proteins . reduced . Specific embodiments of tumor- associated antigens In other embodiments , the nucleic acid is a plasmid that 55 include , e . g ., mutated antigens such as the protein products encodes the polypeptide or a functional variant or fragment of the Ras p21 protooncogenes, tumor suppressor p53 and thereof, such that expression of the polypeptide or the BCR - abl oncogenes, as well as CDK4 , MUM1, Caspase 8 , functional variant or fragment thereof is increased . and Beta catenin ; overexpressed antigens such as galectin 4 , In related embodiments , the present invention provides a galectin 9 , carbonic anhydrase , Aldolase A , PRAME, Her2 / method of treating a disease or disorder characterized by 60 neu , ErbB - 2 and KSA , oncofetal antigens such as alpha overexpression of a polypeptide in a subject, comprising fetoprotein (AFP ) , human chorionic gonadotropin ( hCG ) ; providing to the subject a pharmaceutical composition of the self antigens such as carcinoembryonic antigen (CEA ) and present invention , wherein the therapeutic agent is selected melanocyte differentiation antigens such as Mart 1 /Melan A , from an siRNA , a microRNA , an antagomir , an antisense gp100 , gp75 , Tyrosinase , TRP1 and TRP2 ; prostate associ oligonucleotide , and a plasmid capable of expressing an 65 ated antigens such as PSA , PAP, PSMA, PSM - P1 and siRNA , a microRNA , or an antisense oligonucleotide , and PSM -P2 ; reactivated embryonic gene products such as wherein the siRNA , microRNA , or antisense RNA com - MAGE 1 , MAGE 3 , MAGE 4 , GAGE 1 , GAGE 2 , BAGE , US 9 ,814 ,777 B2 87 88 RAGE , and other cancer testis antigens such as NY- ESO1, moniliformis, Treponema pallidium , Treponema pertenue, SSX2 and SCP1; mucins such as Muc - 1 and Muc - 2; gan - Leptospira , Rickettsia , and Actinomyces israelli. gliosides such as GM2 , GD2 and GD3, neutral glycolipids Additional examples of pathogens include , but are not and glycoproteins such as Lewis ( y ) and globo - H ; and limited to , infectious fungi that infect mammals , and more glycoproteins such as Tn , Thompson - Freidenreich antigen 5 particularly humans. Examples of infectious fingi include , ( TF ) and sTn . Also included as tumor -associated antigens but are not limited to : Cryptococcus neoformans , Histo herein are whole cell and tumor cell lysates as well as immunogenic portions thereof , as well as immunoglobulin plasma capsulatum , Coccidioides immitis , Blastomyces der idiotypes expressed on monoclonal proliferations of B lym matitidis , Chlamydia trachomatis, Candida albicans. phocytes for use against B cell lymphomas . Examples of infectious parasites include Plasmodium such Pathogens include , but are not limited to , infectious as Plasmodium falciparum , Plasmodium malariae, Plasmo agents , e . g . , viruses, that infect mammals , and more par dium ovale , and Plasmodium vivax . Other infectious organ ticularly humans. Examples of infectious virus include , but isms (i . e. , protists ) include Toxoplasma gondii . are not limited to : Retroviridae ( e . g ., human immunodefi In one aspect the invention provides a method of modu ciency viruses , such as HIV - 1 ( also referred to as HTLV - II . 15 lating the expression of a target gene in a cell , comprising LAV or HTLV - III/ LAV , or HIV - III ; and other isolates, such providing to said cell a composition of the present invention . as HIV - LP : Picornaviridae ( e . g . , polio viruses , hepatitis A In one embodiment, the target gene is selected from the virus ; enteroviruses, human Coxsackie viruses, rhinovi group consisting of Factor VII, Eg5 , PCSK9 , TPX2 , apoB , ruses , echoviruses ) ; Calciviridae ( e . g ., strains that cause SAA , TTR , RSV , PDGF beta gene , Erb - B gene, Src gene , gastroenteritis ); Togaviridae (e .g . , equine encephalitis 20 CRK gene ,GRB2 gene, RAS gene , MEKK gene , JNK gene, viruses, rubella viruses) ; Flaviridae (e .g . , dengue viruses, RAF gene , Erk1/ 2 gene, PCNA (p21 ) gene, MYB gene , JUN encephalitis viruses , yellow fever viruses ); Coronoviridae gene , FOS gene , BCL - 2 gene , Cyclin D gene , VEGF gene , ( e. g ., coronaviruses ); Rhabdoviradae (e . g. , vesicular stoma EGFR gene , Cyclin A gene , Cyclin E gene, WNT- 1 gene , titis viruses, rabies viruses ) ; Coronaviridae ( e . g ., coronavi- beta - catenin gene , c -MET gene , PKC gene, NFKB gene , ruses ) ; Rhabdoviridae ( e . g . , vesicular stomatitis viruses, 25 STAT3 gene , survivin gene , Her2 /Neu gene, topoisomerase rabies viruses ) ; Filoviridae ( e . g ., ebola viruses ); Paramyxo - I gene , topoisomerase II alpha gene , mutations in the p73 viridae ( e . g ., parainfluenza viruses , mumps virus, measles gene , mutations in the p21 (WAF1 / CIP1 ) gene , mutations in virus , respiratory syncytial virus ) ; Orthomyxoviridae ( e . g ., the p27 (KIP1 ) gene , mutations in the PPMID gene , muta influenza viruses ) ; Bungaviridae ( e . g . , Hantaan viruses , tions in the RAS gene , mutations in the caveolin I gene , bunga viruses , phleboviruses and Nairo viruses ) ; Arena 30 mutations in the MIB I gene , mutations in the MTAI gene, viridae (hemorrhagic fever viruses ) ; Reoviridae ( e . g ., reo mutations in the M68 gene, mutations in tumor suppressor viruses , orbiviurses and rotaviruses ); Birnaviridae ; Hepad genes, and mutations in the p53 tumor suppressor gene . naviridae (Hepatitis B virus) ; Parvovirida (parvoviruses ); Definitions Papovaviridae (papilloma viruses, polyoma viruses ) ; For convenience, the meaning of certain terms and phrases Adenoviridae (most adenoviruses ) ; Herpesviridae herpes 35 used in the specification , examples , and appended claims, simplex virus (HSV ) 1 and 2 , varicella zoster virus, cyto are provided below . If there is an apparent discrepancy megalovirus (CMV ) , herpes virus; Poxyiridae (variola between the usage of a term in other parts of this specifi viruses , vaccinia viruses, pox viruses ) ; and Iridoviridae cation and its definition provided in this section , the defi ( e . g . , African swine fever virus ) ; and unclassified viruses nition in this section shall prevail . ( e. g ., the etiological agents of Spongiform encephalopathies, 40 “ G ,” “ C ” “ A ” and “ U ” each generally stand for a nucleo the agent of delta hepatitis ( thought to be a defective satellite tide that contains guanine , cytosine , adenine , and uracil as a of hepatitis B virus ) , the agents of non - A , non - B hepatitis base , respectively . However, it will be understood that the ( class 1 = internally transmitted ; class 2 = parenterally trans - term “ ribonucleotide” or “ nucleotide ” can also refer to a mitted ( i . e ., Hepatitis C ) ; Norwalk and related viruses , and modified nucleotide, as further detailed below , or a surrogate astroviruses ) . 45 replacement moiety . The skilled person is well aware that Also , gram negative and gram positive bacteria serve as guanine , cytosine , adenine , and uracil may be replaced by antigens in vertebrate animals . Such gram positive bacteria other moieties without substantially altering the base pairing include, but are not limited to Pasteurella species, Staphy - properties of an oligonucleotide including a nucleotide bear lococci species, and Streptococcus species . Gram negative ing such replacement moiety . For example , without limita bacteria include , but are not limited to , Escherichia coli, 50 tion , a nucleotide including inosine as its base may base pair Pseudomonas species , and Salmonella species . Specific with nucleotides containing adenine , cytosine , or uracil . examples of infectious bacteria include but are not limited Hence , nucleotides containing uracil, guanine , or adenine to : Helicobacter pyloris, Borelia burgdorferi, Legionella may be replaced in the nucleotide sequences of the invention pneumophilia , Mycobacteria sps (e . g. , M . tuberculosis , M . by a nucleotide containing, for example , inosine . Sequences avium , M . intracellulare , M . kansaii, M . gordonae ) , Staphy - 55 including such replacementmoieties are embodiments of the lococcus aureus, Neisseria gonorrhoeae , Neisseria menin - invention . gitidis , Listeria monocytogenes , Streptococcus pyogenes By “ Factor VII” as used herein is meant a Factor VII (Group A Streptococcus ) , Streptococcus agalactiae (Group mRNA , protein , peptide , or polypeptide . The term “ Factor B Streptococcus ), Streptococcus (viridans group ) , Strepto - VII ” is also known in the art as A1132620 , Cf7, Coagulation coccus faecalis , Streptococcus bovis , Streptococcus (an - 60 factor VII precursor, coagulation factor VII , FVII , Serum aerobic sps. ) , Streptococcus pneumoniae, pathogenic prothrombin conversion accelerator, FVII coagulation pro Campylobacter sp. , Enterococcus sp ., Haemophilus infuen - tein , and eptacog alfa . zae, Bacillus antracis , corynebacterium diphtheriae , As used herein , “ target sequence ” refers to a contiguous corynebacterium sp ., Erysipelothrix rhusiopathiae , portion of the nucleotide sequence of an mRNA molecule Clostridium perfringers , Clostridium tetani, Enterobacter 65 formed during the transcription of the gene, including aerogenes, Klebsiella pneumoniae , Pasturella multocida , mRNA that is a product of RNA processing of a primary Bacteroides sp ., Fusobacterium nucleatum , Streptobacillus transcription product . US 9 , 814 , 777 B2 89 90 As used herein , the term “ strand including a sequence ” As used herein , a polynucleotide which is “ complemen refers to an oligonucleotide including a chain of nucleotides tary , e . g ., substantially complementary to at least part of a that is described by the sequence referred to using the messenger RNA (mRNA ) refers to a polynucleotide which standard nucleotide nomenclature . is complementary , e . g ., substantially complementary, to a As used herein , and unless otherwise indicated , the term 5 contiguous portion of the mRNA of interest ( e . g ., encoding " complementary , ” when used in the context of a nucleotide Factor VII ) . For example , a polynucleotide is complemen tary to at least a part of a Factor VII mRNA if the sequence pair, means a classic Watson - Crick pair, i .e ., GC , AT, or AU . is substantially complementary to a non - interrupted portion It also extends to classic Watson - Crick pairings where one or of an mRNA encoding Factor VII . both of the nucleotides has been modified as described The term “ double - stranded RNA ” or “ dsRNA ” , as used herein , e .g ., by a rbose modification or a phosphate back herein , refers to a ribonucleic acid molecule , or complex of pone modification . It can also include pairing with an ribonucleic acid molecules , having a duplex structure inosine or other entity that does not substantially alter the including two anti -parallel and substantially complemen base pairing properties. tary , as defined above , nucleic acid strands . The two strands As used herein , and unless otherwise indicated , the temterm 15 forming the duplex structure may be different portions of " complementary, ” when used to describe a first nucleotide one larger RNA molecule, or they may be separate RNA sequence in relation to a second nucleotide sequence , refers molecules . Where the two strands are part of one larger to the ability of an oligonucleotide or polynucleotide includ - molecule , and therefore are connected by an uninterrupted ing the first nucleotide sequence to hybridize and form a chain of nucleotides between the 3 ' - end of one strand and the duplex structure under certain conditions with an oligo - 20 5 ' end of the respective other strand forming the duplex nucleotide or polynucleotide including the second nucleo structure , the connecting RNA chain is referred to as a tide sequence , as will be understood by the skilled person . " hairpin loop ” . Where the two strands are connected cova Complementarity can include, full complementarity , sub - lently by means other than an uninterrupted chain of nucleo stantial complementarity , and sufficient complementarity to tides between the 3 ' -end of one strand and the 5 ' end of the allow hybridization under physiological conditions, e . g . , 25 respective other strand forming the duplex structure , the under physiologically relevant conditions as may be encoun - connecting structure is referred to as a “ linker " The RNA tered inside an organism . Full complementarity refers to strands may have the same or a different number of nucleo complementarity , as defined above for an individual pair , at tides. The maximum number of base pairs is the number of all of the pairs of the first and second sequence . When a nucleotides in the shortest strand of the dsRNA . In addition sequence is “ substantially complementary ” with respect to a 30 to the duplex structure , a dsRNA may comprise one or more second sequence herein , the two sequences can be fully nucleotide overhangs . A dsRNA as used herein is also complementary , or they may form one or more , but generally referred to as a " small inhibitory RNA , " " siRNA ," " siRNA not more than 4 , 3 or 2 mismatched base pairs upon agent, " " iRNA agent” or “ RNAi agent. " hybridization , while retaining the ability to hybridize under As used herein , a “ nucleotide overhang ” refers to the the conditions most relevant to their ultimate application . 35 unpaired nucleotide or nucleotides that protrude from the Substantial complementarity can also be defined as hybrid - duplex structure of a dsRNA when a 3 '- end of one strand of ization under stringent conditions, where stringent condi- the dsRNA extends beyond the 5 -end of the other strand, or tions may include: 400 mM NaCl, 40 mM PIPES PH 6 .4 , 1 vice versa . “ Blunt” or “ blunt end ” means that there are no mM EDTA , 50° C . or 70° C . for 12 - 16 hours followed by unpaired nucleotides at that end of the dsRNA , i . e ., no washing . The skilled person will be able to determine the set 40 nucleotide overhang. A “ blunt ended ” dsRNA is a dsRNA of conditions most appropriate for a test of complementarity that is double - stranded over its entire length , i. e ., no nucleo of two sequences in accordance with the ultimate application tide overhang at either end of the molecule . of the hybridized nucleotides. The term “ antisense strand ” refers to the strand of a However , where two oligonucleotides are designed to dsRNA which includes a region that is substantially comple form , upon hybridization , one or more single stranded 45 mentary to a target sequence . As used herein , the term overhangs , such overhangs shall not be regarded as mis - “ region of complementarity ” refers to the region on the matches with regard to the determination of complementar antisense strand that is substantially complementary to a ity . For example , a dsRNA including one oligonucleotide 21 sequence , for example a target sequence , as defined herein . nucleotides in length and another oligonucleotide 23 nucleo - Where the region of complementarity is not fully comple tides in length , wherein the longer oligonucleotide includes 50 mentary to the target sequence , the mismatches are most a sequence of 21 nucleotides that is fully complementary to tolerated in the terminal regions and , if present, are generally the shorter oligonucleotide , may yet be referred to as “ fully in a terminal region or regions, e . g . , within 6 , 5 , 4 , 3 , or 2 complementary ” for the purposes of the invention . nucleotides of the 5 ' and / or 3 ' terminus . " Complementary ” sequences, as used herein , may also The term " sense strand, " as used herein , refers to the include, or be formed entirely from , non - Watson - Crick base 55 strand of a dsRNA that includes a region that is substantially pairs and / or base pairs formed from non - natural and modi - complementary to a region of the antisense strand . The term fied nucleotides, in as far as the above requirements with “ identity ” is the relationship between two or more poly respect to their ability to hybridize are fulfilled . nucleotide sequences , as determined by comparing the The terms “ complementary ” , “ fully complementary ” , sequences. Identity also means the degree of sequence “ substantially complementary ” and sufficient complemen - 60 relatedness between polynucleotide sequences, as deter tarity to allow hybridization under physiological conditions , mined by the match between strings of such sequences . e . g . , under physiologically relevant conditions as may be While there exist a number of methods to measure identity encountered inside an organism , may be used hereinwith between two polynucleotide sequences, the term is well respect to the base matching between the sense strand and known to skilled artisans ( see , e . g . , Sequence Analysis in the antisense strand of a dsRNA , or between the antisense 65 Molecular Biology , von Heinje , G ., Academic Press ( 1987 ) ; strand of a dsRNA and a target sequence , as will be and Sequence Analysis Primer ,Gribskov ., M . and Devereux , understood from the context of their use . J ., eds. , M . Stockton Press , New York ( 1991 ) ) . “ Substan US 9 ,814 ,777 B2 91 tially identical ," as used herein , means there is a very high the present invention insofar as it relates to any of the other degree of homology (preferably 100 % sequence identity conditions recited herein below ( e . g . , a Factor VII -mediated between the sense strand of the dsRNA and the correspond condition other than a thrombotic disorder) , the terms ing part of the target gene . However, dsRNA having greater " treat, ” “ treatment, ” and the like mean to relieve or alleviate than 90 % , or 95 % sequence identity may be used in the 5 at least one symptom associated with such condition , or to present invention , and thus sequence variations that might slow or reverse the progression of such condition . be expected due to genetic mutation , strain polymorphism , A " therapeutically relevant” composition can alleviate a disease or disorder, or a symptom of a disease or disorder or evolutionary divergence can be tolerated . Although 100 % when administered at an appropriate dose . identity is preferred , the dsRNA may contain single or 10 As used herein , the term “ Factor VII -mediated condition multiple base -pair random mismatches between the RNA or disease and related terms and phrases refer to a condition and the target gene. or disorder characterized by inappropriate , e . g . , greater than “ Introducing into a cell ” , when referring to a dsRNA , normal, Factor VII activity . Inappropriate Factor VII func means facilitating uptake or absorption into the cell , as is tional activity might arise as the result of Factor VII expres understood by those skilled in the art. Absorption or uptake 15 sion in cells which normally do not express Factor VII , or of dsRNA can occur through unaided diffusive or active increased Factor VII expression ( leading to , e . g . , a symptom cellular processes , or by auxiliary agents or devices . The of a viral hemorrhagic fever, or a thrombus ) . A Factor meaning of this term is not limited to cells in vitro ; a dsRNA VII -mediated condition or disease may be completely or may also be introduced into a cell, " wherein the cell is part partially mediated by inappropriate Factor VII functional of a living organism . In such instance , introduction into the* 20 activity . However , a Factor VII -mediated condition or dis cell will include the delivery to the organism . For example , ease is one in which modulation of Factor VII results in for in vivo delivery , dsRNA can be injected into a tissue site some effect on the underlying condition or disorder ( e . g . , a or administered systemically . In vitro introduction into a cell Factor VII inhibitor results in some improvement in patient includes methods known in the art such as electroporation well - being in at least some patients ) . and lipofection . 25 A “ hemorrhagic fever” includes a combination of ill The terms “ silence ” and “ inhibit the expression of, ” in as nesses caused by a viral infection . Fever and gastrointestinal far as they refer to the Factor VII gene , herein refer to the at symptoms are typically followed by capillary hemorrhaging . least partial suppression of the expression of the Factor VII A “ coagulopathy ” is any defect in the blood clotting gene , as manifested by a reduction of the amount of mRNA mechanism of a subject. transcribed from the Factor VII gene which may be isolated 30 As used herein , a " thrombotic disorder ” is any disorder, from a first cell or group of cells in which the Factor VII preferably resulting from unwanted FVII expression , includ gene is transcribed and which has or have been treated such ing any disorder characterized by unwanted blood coagula that the expression of the Factor VII gene is inhibited , as tion . compared to a second cell or group of cells substantially As used herein , the phrases “ therapeutically effective identical to the first cell or group of cells but which has or 35 amount” and “ prophylactically effective amount” refer to an have not been so treated ( control cells ) . The degree of amount that provides a therapeutic benefit in the treatment, inhibition is usually expressed in terms of prevention , ormanagement of a viral hemorrhagic fever, or an overt symptom of such disorder, e . g . , hemorraging , fever, weakness , muscle pain , headache , inflammation , or circula (mRNA in control cells ) - (mRNA in treated cells )- - 100 % 40 tory shock . The specific amount that is therapeutically (mRNA in control cells ) effective can be readily determined by ordinary medical practitioner, and may vary depending on factors known in Alternatively , the degree of inhibition may be given in the art, such as , e .g . the type of thrombotic disorder , the terms of a reduction of a parameter that is functionally patient' s history and age , the stage of the disease , and the linked to Factor VII gene transcription , e . g . the amount of 45 administration of other agents . protein encoded by the Factor VII gene which is secreted by As used herein , a " pharmaceutical composition ” includes a cell , or the number of cells displaying a certain phenotype , a pharmacologically effective amount of a dsRNA and a e . g . apoptosis . In principle , Factor VII gene silencingmay be pharmaceutically acceptable carrier. As used herein , “ phar determined in any cell expressing the target , either consti - macologically effective amount, " " therapeutically effective tutively or by genomic engineering , and by any appropriate 50 amount” or simply " effective amount” refers to that amount assay . However, when a reference is needed in order to of an RNA effective to produce the intended pharmacologi determine whether a given siRNA inhibits the expression of cal, therapeutic or preventive result. For example , if a given the Factor VII gene by a certain degree and therefore is clinical treatment is considered effective when there is at encompassed by the instant invention , the assays provided in least a 25 % reduction in a measurable parameter associated the Examples below shall serve as such reference . 55 with a disease or disorder , a therapeutically effective amount For example , in certain instances, expression of the Factor of a drug for the treatment of that disease or disorder is the VII gene is suppressed by at least about 20 % , 25 % , 35 % , amount necessary to effect at least a 25 % reduction in that 40 % or 50 % by administration of the double - stranded oli- parameter. gonucleotide of the invention . In a preferred embodiment, The term “ pharmaceutically acceptable carrier” refers to the Factor VII gene is suppressed by at least about 60 % , 60 a carrier for administration of a therapeutic agent. Such 70 % , or 80 % by administration of the double - stranded carriers include , but are not limited to , saline , buffered oligonucleotide of the invention . In a more preferred saline , dextrose , water, glycerol, ethanol, and combinations embodiment, the Factor VII gene is suppressed by at least thereof. The term specifically excludes cell culture medium . about 85 % , 90 % , or 95 % by administration of the double - For drugs administered orally , pharmaceutically acceptable stranded oligonucleotide of the invention . 65 carriers include , but are not limited to pharmaceutically The terms “ treat ," " treatment, ” and the like , refer to relief acceptable excipients such as inert diluents , disintegrating from or alleviation of a disease or disorder. In the context of agents , binding agents, lubricating agents , sweetening US 9 ,814 ,777 B2 93 94 agents , flavoring agents , coloring agents and preservatives. "Heteroaryl ” means a monocyclic - or polycyclic aromatic Suitable inert diluents include sodium and calcium carbon - ring comprising carbon atoms, hydrogen atoms, and one or ate , sodium and calcium phosphate, and lactose , while corn more heteroatoms, preferably , 1 to 3 heteroatoms, indepen starch and alginic acid are suitable disintegrating agents . dently selected from nitrogen , oxygen , and sulfur . As is well Binding agents may include starch and gelatin , while the 5 known to those skilled in the art , heteroaryl rings have less lubricating agent , if present, will generally be magnesium aromatic character than their all - carbon counter parts . Thus, stearate , stearic acid or talc . If desired , the tablets may be for the purposes of the invention , a heteroaryl group need coated with a material such as glyceryl monostearate or only have some degree of aromatic character. Illustrative glyceryl distearate , to delay absorption in the gastrointesti examples of heteroaryl groups include , but are not limited nal tract . 10 As used herein , a “ transformed cell ” is a cell into which to , pyridinyl, pyridazinyl , pyrimidyl, pyrazyl, triazinyl, pyr a vector has been introduced from which a dsRNA molecule rolyl , pyrazolyl, imidazolyl, ( 1, 2 ,3 ,) - and ( 1, 2 ,4 ) - triazolyl, may be expressed . pyrazinyl, pyrimidinyl, tetrazolyl. “ Alkyl” means a straight chain or branched , noncyclic or The terms “ optionally substituted alkyl” , “ optionally sub cvclic . saturated aliphatic hydrocarbon containing from 1 to 15 stituted alkenyl" , " optionally substituted alkynyl" , " option 24 carbon atoms. Representative saturated straight chain ally substituted acyl” , and “ optionally substituted hetero alkyls include methyl, ethyl, n -propyl , n -butyl , n -pentyl , cycle ” means that , when substituted , at least one hydrogen n - hexyl, and the like; while saturated branched alkyls atom is replaced with a substituent. In the case of an oxo include isopropyl, sec -butyl , isobutyl, tert -butyl , isopentyl, substituent ( = O ) two hydrogen atoms are replaced . In this and the like . Representative saturated cyclic alkyls include 20 regard , substituents include oxo , halogen , heterocycle, cyclopropyl, cyclobutyl, cyclopentyl , cyclohexyl, and the CN , — OR , — NR * R ” , — NR * C ( = O ) R ” , — NR *SO ,RY , like; while unsaturated cyclic alkyls include cyclopentenyl C ( O )R , C ( O )ORT , C ( O ) NR *R ”, - SOR and cyclohexenyl, and the like . and — SO NR * R , wherein n is 0 , 1 or 2 , R * and Ry are the “ Alkenyl” means an alkyl, as defined above , containing at same or different and independently hydrogen , alkyl or least one double bond between adjacent carbon atoms. 25 heterocycle , and each of said alkyl and heterocycle substitu Alkenyls include both cis and trans isomers . Representative ents may be further substituted with one or more of oxo , straight chain and branched alkenyls include ethylenyl, halogen . OH , CN , alkyl. - OR " , heterocycle , propylenyl, 1 -butenyl , 2 -butenyl , isobutylenyl , 1 - pentenyl, NR ' R ” , - NRC ( O )R ” , — NR * SO , R ”, C O R" , 2 -pentenyl , 3 -methyl - 1 -butenyl , 2 -methyl - 2 -butenyl , 2 ,3 -di - C ( O )OR , C ( O )NR *RY , SO R * and SO methyl- 2 -butenyl , and the like. 30 NR * R ” . “ Alkynyl” means any alkyl or alkenyl, as defined above , “ Halogen ” means fluoro , chloro , bromo and iodo . which additionally contains at least one triple bond between In one embodiment , the methods of the invention may adjacent carbons. Representative straight chain and require the use of protecting groups. Protecting group meth branched alkynyls include acetylenyl, propynyl, 1 -butynyl , odology is well known to those skilled in the art (see , for 2 -butynyl , 1 - pentynyl, 2 -pentynyl , 3 -methyl - 1 butynyl, and 35 example , PROTECTIVE GROUPS IN ORGANIC SYNTHESIS , Green , T . the like . W . et . al. , Wiley - Interscience , New York City , 1999 ). Briefly , “ Acyl” means any alkyl, alkenyl, or alkynyl wherein the protecting groups within the context of this invention are carbon at the point of attachment is substituted with an oxo any group that reduces or eliminates unwanted reactivity of group , as defined below . For example , — CEO )alkyl , a functional group . A protecting group can be added to a - C ( O )alkenyl , and — CEO )alkynyl are acyl groups . 40 functional group to mask its reactivity during certain reac “ Heterocycle ” means a 5 - to 7 -membered monocyclic , or tions and then removed to reveal the original functional 7 - to 10 -membered bicyclic , heterocyclic ring which is group . In one embodiment an “ alcohol protecting group ” is either saturated , unsaturated , or aromatic , and which con used . An " alcohol protecting group ” is any group which tains from 1 or 2 heteroatoms independently selected from decreases or eliminates unwanted reactivity of an alcohol nitrogen , oxygen and sulfur, and wherein the nitrogen and 45 functional group . Protecting groups can be added and sulfur heteroatoms may be optionally oxidized , and the removed using techniques well known in the art . nitrogen heteroatom may be optionally quaternized , includ - The compounds of the present invention may be prepared ing bicyclic rings in which any of the above heterocycles are by known organic synthesis techniques , including the meth fused to a benzene ring . The heterocycle may be attached via ods described in more detail in the Examples . any heteroatom or carbon atom . Heterocycles include het- 50 eroaryls as defined below . Heterocycles include morpholi EXAMPLES nyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl, hydantoinyl , valerolactamyl, oxiranyl , oxetanyl, tetrahydro The following examples are offered to illustrate , but not furanyl, tetrahydropyranyl, tetrahydropyridinyl , tetrahy - to limit the claimed invention . droprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, 55 tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydroth Example 1 . Synthesis of Carbohydrate Building iopyranyl, and the like . Blocks for Conjugation US 9 , 814 , 777 B2 95 96

"MH TFA

20H ? 105

103 OAC OAc / ?? ACHN AcHN Ad0Aco A0?Ac0 AcO Ac0_ 1.PPhs/H,0 THF FN3 2.TFA H,/PM(C) MeOHETOAC 0Bn TMSOTf/DCE MS TMSOTf/DCE MS

H07 104 102

0 OAC {DAC ? 101 OAC AcHN _Ac0 AcHN ?Ac0_ Ac0_ _ ?Ac0 Ac0 Ac0_

TMSOTE 500DCE,0Ac_

100 204c AcHN Ac0 Ac0_ US 9 , 814 ,777 B2 97 98 Preparation of 101: and hydrogenated under balloon pressure overnight . Filtered Galactosamine pentaacetate 100 (52 .00 g , 133 .63 mmol) through a small pad of celite ; washed with methanol and was taken in dichloroethane (300 mL ) at ambient tempera dried under high vacuum overnight to get the product (48 . 85 ture. TMSOTf (44 .55 g , 200 .44 mmol) was added that and g , 98 % ) . THNMR , 13CNMR MS: Calculated for the mixture stirred at 50 C for 90 minutes in a water bath , 5 C H , NO , 447 . 17 . Found 469. 9 ( M + Na) . heating stopped and the mixture stirred overnight at room Preparation of 104 : temperature. It was poured in to an cold sodium bicar Compound 101 (42 . 30 g, 128 .43 mmol) and the azido bonate solution ; extracted with dichloromethane , washed ethanol ( 26 g , 192. 45 mmol) were dissolved in dichloroeth with water and dried over sodium sulfate . were ane (300 mL ) , molecular sieves (50 g ) were added to that removed the residue dried under high vacuum overnight to 10 and stirred for 30 minutes. TMSOTF ( 14 . 29 g , 64. 21 mmol) get the compound as dark gum (44 . 50 g , quantitative ) . It was was added to that and the mixture stirred for overnight at used for next reaction with out any further purification . ' H room temperature . It was poured in to an ice cold solution NMR and MALDI confirmed the product formation . MS: of sodium bicarbonate and extracted with dichloromethane , Calculated for C 4H NO , , 329 . 11 . Found 352 . 1 ( M + Na ). washed with water and dried over sodium sulfate . Solvents Preparation of 102 : 15 were removed and the residue purified by chromatography Compound 101 (43 .70 g , 133. 56 mmol) and the benzyl (gradient elution : 20 - 100 % ethyl acetate /hexanes , followed ester (41 . 71 g , 200 .34 mmol) were dissolved in dichloroeth - by 5 - 10 % Methanol/ ethylacetate ) to get the required com ane (300 mL ), molecular sieves (50 g ) was added to that and pound as light brown gummy liquid (59 . 23 g , 91. 00 % ). stirred for 30 minutes. TMSOTf ( 14 .50 g , 66 .78 mmol) was 'HNMR , 13 CNMR MS : Calculated for C20H22N4011 , added to that and the mixture stirred for overnight at room 20 504 . 21 . Found 527 . 1 ( M + Na ) . temperature . It was poured in to an ice cold solution of Preparation of 105 : sodium bicarbonate and extracted with dichloromethane , Compound 104 ( 9 . 33 g , 18 .50 mmol) was dissolved in washed with water and dried over sodium sulfate . Solvents THF ( 100 mL ) to that PPhz ( 5 . 97 g , 22 . 2 mmol) was added were removed and the residue purified by chromatography and the mixture stirred for 48 h . TLC checked to see ( gradient elution : 20 - 100 % ethylacetate /hexanes ) to get the 25 complete disappearance of starting material. Water ( 1 mL , required compound as light brown gummy liquid (60 .50 g , 55 mmol) and stirred for another 24 h . TFA ( 2 . 85 mL , 23 . 12 86 % ). 1HNMR , 13CNMR MS: Calculated for C26H35NO11 , mmol) and toluene (40 mL ) were added and the solvents 537 .22 . Found 560 . 21 ( M + Na ). were removed under reduced pressure . The residue was Preparation 103 : co - evaporated with toluene ( 2x40 mL ) two times and dried Compound 102 (60 .00 g , 111 .68 mmol) was dissolved in 30 under high vacuum . It was used for the next reaction in the a mixture of Methanol/ ethylacetate and degassed with same day .MS : Calculated for C20H34N2011 , 478 .22 . Found argon . Pd/ C ( 6 .00 g , 10 wt % Degussa , wet type ) was added 500 .8 ( M + Na) . US 9 , 814 ,777 B2 99 100

NHCbz OHHBTU,DIEA HOBt

NH aremaposice 108 3CF3COOH H2N H2N1 103 H2N

TFA DCM OAC ACHN menyangAcO NHCbz Acol

107 BocHNNBocHN BocHNNBocHN BocHN.

quesetienemenyretepNH HBTU,DIEA DMF

NHCbz 20 106 HO HOO

HO. US 9 , 814 , 777 B2 :101 102 NHCbz

? 109

OAC / AcHN AcHN Ad0 Ac0_ Ac0 Ac0_ Oac AcHN Ac0 Ac0

-continued H,Pd/ MeOH Drop of acetic acid TFA NH, ?

_TFATFA

110

AcHN OAC AcHN Ac07 ??AO Ac0 ??Ad0 OAC AcHN Aco Ac0 US 9 , 814 , 777 B2 103 104 Preparation of 107 : Preparation of 109:

Compound 106 (JOC 2002) ( 6 . 94 g , 14. 73 mmol) andand GalNAc acid 103 ( 4 .00 g , 8 . 99 mmol) was dissolved in monoboc propyl amine ( 10 . 26 g , 58. 89 mmol) were dis - 5 DMF ( 50 mL ) ; HBTU ( 3 .75 g , 9 . 88 mmol) , HOBt ( 1 . 34 g , 9 .88 mmol) and DIEA ( 5 mL , 3 . 2 eq ) was added to that and solved in DMF ( 100 mL ) , to that HBTU ( 17 . 26 g , 45. 50 stirred for 3 - 4 minutes . A solution of 108 ( 2 .47 g , 2 . 50 mmol) and DIEA ( 15 . 36 mL , 88 . 14 mmol) were added and mmol) in DMF was added to that and stirred the reaction stirred overnight . Reaction mixture was poured in to ice mixture overnight. TLC was checked , solvents were water mixture and extracted with dichloromethane , washed 10 removed under reduced pressure. The residue was dissolved with sodium bicarbonate solution , brine and dried over in dichloromethane , washed with sodium bicarbonate solu sodium sulfate . Solvents were removed and the residue was tion (50 mL ) , water ( 100 mL ) and dried over sodium sulfate . purified by chromatography (Ethyl acetate , followed by Solvents were removed and the residue was purified by 2 -10 % MeOH /DCM ) to get the product as white fluffy solid 15 chromatography ( ethyl acetate , followed by gradient elution ( 10 .49 g , 76 % ). MS: Calculated for C45H , N ,014 , 939 .55 . 5 - 15 % MeOH /DCM ) to get the product 109 as a white solid Found 940 . 53 ( M + H ) . ( 4. 20 g , 87 % ). MS: Calculated for Cg7H134N1003, 1926 .89 . Found 1949 . 5 ( M + Na ) . Preparation of 108 : Preparation of 110 : 20 GalNAc derivative 109 (7 . 50 g , 4 . 18 mmol) was taken in methanol (50 mL ) degassed with argon . Pd / C (0 . 800 g , 10 wt Compound 107 (2 .40 g, 2 .56 mmol) was dissolved in % Degussa type wet) and couple of drops of acetic acid were dichloromethane (10 mL ), to that a mixture of TFA /DCM added ; the mixture was hydrogenated under balloon pressure ( 1: 4 , 10 mL ) was added and stirred for 30 minutes. Reaction110 2525 overnightV . Reaction mixture was filtered through a small pad was monitored by mass spectra . 100 mL of toluene was of celite , washed with methanol. TFA (0 .465 mL , 5. 22 added and removed the solvent under reduced pressure . The mmol) was added and removed the solvent under reduced residue was co - evaporated two times with toluene (2x100 pressure . The residue was co -evaporated with toluene ( 2 mL) and dried under high vacuum to get the compound as 30 times ) and dried under high vacuum overnight to get the its TFA salt (white gum , 2. 47 g , 99 % ). It was used for the compound as TFA salt (pale yellow solid , 7 .30 g , 99 % ). MS: next reaction with out any further purification . MS: Calcu - Calculated for C7H128N10036 , 1792 .85 . Found 1815 . 9 lated for C30H53N203, 639. 40 . Found 640 . 45 ( M + H ) . (M + Na) .

HOO OAC Aco / AcO TBTU , HOBt, AcHN HO . JOY NHCbz DIEA NHCbz DMF Aco OAC

AcoAcO . No NHCbz HOHOO AcHN N

OAC OAC AcO AcO AcO ACON AcHN AcHN 111 TFA Pd ( C ) , MeOH NH2 Drop of acetic acid 105 US 9 ,814 ,777 B2 105 106 Aco04cAc "concontinued TFA

AcO AcHN

OAC Aco

ACO NH - TFA AcHN

OAC Aco

Aco AcHN

112

Preparation of 111: white solid (5 .80 g, 68 % ) MS: Calculated for Cg1H125N , 041 , 1851. 79 . Found 1874 . 20 ( M + Na ) . The tricarboxylic acid 106 (2 . 17 g , 4 .625 mmol) and 81Preparation 1257 413 of 112 : amine ( 18 .50 mmol, crude from previous reaction ) was GalNAc derivative 111 ( 5 .75 g , 3 .09 mmol) was taken in dissolved in DMF ( 100 mL ). To that TBTU ( 5 .34 g , 16 .63 30 methanol ( 100 mL ) degassed with argon . Pd / C (0 .600 g, 10 mmol) , HOBt ( 2 . 24 g , 16 . 59 mmol) and DIEA ( 5 .64 mL , wt % Degussa type wet ) and couple of drops of acetic acid 32 .36 mmol) was added and stirred the reaction mixture for were added ; the mixture was hydrogenated under balloon 24 h . After stirring 24 hrs an additional amount of DIEA ( 4 pressure for 36 hrs . Reaction mixture was filtered through a mL ) was added continued stirring . After 48 hrs solvents small pad of celite , washed with methanol. TFA ( 0 . 354 mL , were removed under reduced pressure , the residue was 35 1 .25 eq ) and toluene (30 mL ) was added and removed the dissolved in dichloromethane , washed with 1M phosphoric solvent under reduced pressure . The residue was co - evapo acid solution , sodium bicarbonate solution , water and dried rated with toluene ( 2 times ) and dried under high vacuum over sodium sulfate . Solvents were removed and the residue overnight to get the compound as TFA salt ( 5 . 70 g , crude ) . was purified by chromatography ( ethyl acetate , followed by MS : Calculated for Cg H125N 041, 1717 .75 . Found 1740 . 5 3 - 15 % MeOH / DCM ) to get the required compound III as a ( M + Na) .

OAC Aco

Aco AcHN N

OAC Aco NHCbz AcO NH2 + HO AcHN 0 TFA OAC Aco

AcO AcHN 112 HBTU , DIEA DMF US 9 , 814 ,777 B2 107 108 -continued OAC AcO

Aco InneAcHN OAC Aco ohs

NHCbz Aco N AcHN OAC AcoAco

AcO AcHN N umet113 1 . H /Pd ( C MOH) three drops of AcOH 2 . TFA OAC AcQ

AcHN

OAC Aco - NH2 Aco . NH TFA AcHN NH OAC AcO

Acc AcHN

114

Preparation of 113 : Preparation of 114 : Z - amino caproic acid (2 . 19 g , 8 .25 mmol ) was dissolved 50 Compound 113 ( 6 . 10 g, 3 .10 mmol) was dissolved in in DMF (50 mL ) . To that HBTU ( 3 .13 g, 8 .25 mmol ) and Methanol (50 mL ), to that 1 mL of acetic acid was added . DIEA ( 7 . 19 mL , 5 . 00 eq. ) was added and stirred the mixture Degassed the reaction mixture , Pd / C (0 . 700 g , 10 wt % for few minutes . GalNAc amine 112 ( 10 . 10 g , 5 .52 mmol) Degussa wet type ) was added to that and hydrogenated was dissolved in 50 ml of DMF was added to that and stirred ss under balloon pressure for 36 hrs . Reaction mixture was for 48 hrs. TLC and MALDI were checked for product SS filtered through a small pad of celite , washed with MeOH . formation . Solvents were removed and the residue was To that 1 . 25 eq of TFA and toluene (50 mL ) were added and dissolved in DCM , washed with NaHCO3 solution and removed solvents under reduced pressure. The residue was water . Dried over sodium sulfate and removed the solvents co - evaporated with toluene two times and dried under high under reduced pressure . Residue was purified by chroma vacuum overnight night to get the required compound as an tography (eluted with ethyl acetate, followed by gradient off white solid ( 6 . 10 g , quantitative ). This compound used as elution of 5 - 15 % MeOH /DCM ) to get the required com such for the next reaction with out any further purification . pound 113 as off white solid ( 6 . 20 g , 57 % ) . MS: Calculated MS: Calculated for C 9H130N2040 , 1830 . 84 . Found 1853. 81 for Cg7H136N2042, 1964 . 88 . Found 1987 .75 ( M +Na ) . ( M + Na ) . US 9 , 814 ,777 B2 109 110

TFA MNH2 TBTUHOBt,DMF DIEA, 117 IZ om 0 NH OO 0 me N ??z Bzo| Bzo 118

Ph3P,THFH20Bz0TL TFA HO,C HO,C

N3 HO2C OBZ Bzo Bzo Bzov Bzo OBZ Bz0i OBz BzO BzOM Bzo Bz0 116 DCM:MeOH(1) www. multeaiPd/C 50eqHCO2NH4. OBz Bzo zOBzOBzo NH2 B 0 N3 NH 'YN; OON 0 ?Z www.TMSOTA EtherEther 119 HO mum CiCl NH OBZ Bzo 7 Bzo Bz0 Bzo OBZ 115 BZON OBZ OBZ Bzo Brown BZON Bz0 Bz02 Bz0 SamenBz0 BZON US 9 , 814 ,777 B2 111 112 Preparation of 116 : purified by chromatography ( ethyl acetate , followed by Mannose trichloroacetimidate 115 ( 15 .00 g , 20 .24 mmol) 3 - 15 % MeOH /DCM ) to get the required compound 118 as and azido alcohol (4 .25 g, 1. 2 eq ) were dissolved in Toluene a white solid (41 . 95 g , 67 % ) MS: Calculated for and aziotroped two times . The residue dried under high C 4 H 461 ,044 , 2598 . 93 . Found 2621. 89 ( M + Na ). vacuum overnight. Anhy . diethyl ether (30 mL ) and Molecu - 5 PrenarPreparation of 119 : lar sieves ( 10 g ) were added to that . Reaction mixture cooled Compound 133 ( 3 . 05 g , 1 . 176 mmol) was dissolved in a in an ice - water bath . TMSOTf ( 0 . 5 mL , 0 . 1 eq ) was added mixture of DCM /MeOH . To that 50 eq . of ammoniumfor to that and stirred the mixture for 10 minutes. Reaction was mate was added followed by 5 % Pd /C ( 1. 5 g, 50 wt % ) and monitored by TLC and quenched with TEA . Filtered of the 10 stirred for 8 hrs at ambient temperature. It was filtered molecular sieves and solvents were removed under reduced through small pad of celite , washed with MeOH /DCM , pressure . Residue was purified by chromatography ( 20 - 50 % solvent was removed and residue dried under high vacuum EtOAc /Hexane ) to get compound as colorless liquid ( 8 .36 g , over night to the compound as a white solid ( 2 .65 g , 92 % ) . 55 % ) . MS : Calculated for C40H3N2012 , 753 .25 . Found MS: Calculated for C 133, 22H 401+ 4422 _ 045 , 2464 . 89 . Found 776 .23 ( ( M + Na) . 2487 . 92 (M + Na ) .

OBZ OBZ Bzo Bzo BZONIBzO BzO Bz0 C1 C1 Bz0 ?? 'OBn TMSOTA OBn Ether NHNHL 115115 116 H2, Pd /C OBZ Bzo BzO Bzo

OH

117

Preparation of 117 : Preparation of 116 ' : Compound 116 ( 8 . 30 g , 11. 01 mmol) was dissolved in Mannose trichloroacetimidate 115 (15 .23 g, 20 .55 mmol) anhy. THF ( 70 mL ) , to that PPh ( 3 .46 g , 1 . 2 eq ) was added 45 and A ( 4 . 36 g , 1 . 02 eq . ) were dissolved in Toluene and and the mixture stirred for two days at ambient temperature . aziotroped two times. The residue dried under high vacuum Water ( 1 mL ) was added to that and stirred the mixture for overnight . Anhy. diethyl ether ( 30 mL ) and Molecular sieves another 24 hrs . Reaction was monitored by TLC . Trifluoro ( 10 g ) were added to that . Reaction mixture cooled in an acetic acid ( 1 . 06 mL , 1 . 25 eq ) and toluene (50 mL ) was ice -water bath . TMSOTf ( 0 . 5 mL , 0 . 1 eq ) was added to that added to that. Solvents were removed under reduced presres - 3050 and stirred the mixture for 10 minutes . Reaction was moni sure and residue was co - evaporated toluene two times and tored by TLC and quenched with TEA . Filtered of the molecular sieves and solvents were removed under reduced dried under high vacuum . This used as such for the next pressure . Residue was purified by chromatography (hexane , reaction without further purification . MS: Calculated for 15 - 25 % EtOAc /Hexane ) to get compound as colorless liquid C40H4, NO12 , 727 . 26 . Found 750 . 23 ( ( M + Na ). 55 ( 14 .52 g , 90 % ). MS: Calculated for C46H420122 786 .27 . Preparation of 118 : Found 809. 25 ( ( M + Na ) . Tricarboxylic acid (11 .05 g , 23. 45 mmol) , and amine Preparation of 117" : (68 .19 g , 94 mmol, crude from previous reaction ) was Mannose benzyl ester (14 . 30 g, 18 . 17 mmol) was dis dissolved in DMF ( 200 mL ) . To that TBTU ( 27 . 09 g , 84 solved in Ethyl acetate ( 100 mL ) to that two drops of acetic mmol) , HOBt ( 11 .34 g , 84 mmol) and DIEA ( 28 mL , 160 60 acid was added . Degassed , Pd / C ( 1 . 50 g , 10 wt % Degussa mmol) was added and stirred the reaction mixture for 24 h . wet type ) was added and hydrogenated under balloon pres After stirring 24 hrs an additional amount of DIEA ( 28 mL ) sure for 24 hrs . Reaction was monitored by TLC and was added continued stirring . After 48 hrs solvents were MALDI. It was filtered through a small pad of celite , washed removed under reduced pressure , the residue was dissolved with ethyl acetate . Solvent was removed and the residue in dichloromethane , washed with 1M phosphoric acid solu - 65 dried under high vacuum to get the compound as color less tion , sodium bicarbonate solution , and water and dried over oil ( 11. 20 g , 90 % ) . MS: Calculated for C39H36012 , 696 .22 . sodium sulfate . Solvents were removed and the residue was found 719 . 18 (( M + Na ). US 9 , 814 ,777 B2 113 114114 0 OH

CbzHN ( S) NHCbz ZT 120 HBTU , DIEA H2/ Pd ( C ) + DMF CbzHN " ( S ) NHCbz EtoAc / MeOH 122 HN was to 121 OAC AcO

OH AcO AcHN 103 H NIIN NH2 HBTU , DIEA HOBE 123

=O N OAC AcO 95 % TFA , anisole , AcO HNU DCM | NHAC AcHN OAC

Acom OAC 124

OAC OH AcO

1111" AcO H NHAC AcHN OAC

AcO OAC 125 Preparation of 122 : balloon pressure using Pd / C (500 mg) as catalyst overnight. Compound 120 (26 .55 g, 64 .06 mmol) and 121 (10 .00 g , Filtered through a small pad of celite and removed the 53 .43 mmol) were dissolved in DMF ( 150 mL ) . To that 55 solvent, this product used for the next reaction without HBTU ( 24 . 12 g , 64 mmol) and DIEA ( 46 mL , 5 eq ) were further purification . MS. MW calc . for C16H33N203: 315 . 25 . added and stirred the reaction mixture overnight. TLC Found 316 . 26 ( M + H ) . checked and the mixture was added to ice cold water and extracted with a mixture of ether and ethyl acetate dried over Preparation of 124 : sodium sulfate . Solvents were removed and the crude prod - 60 Compound 123 (5 .65 mmol) and GalNAc acid 103 (5 .81 uct was purified by chromatography ( 20 -50 % ethylacetate / g , 12 . 99 mmol) were dissolved in DMF ( 80 mL ) . To that Hexane ) to get the required product as an off white solid HBTU ( 4 . 97 g , 13 . 10 mmol) and DIEA ( 7 . 00 mL , 3 eq ) were ( 23. 20 g , 74 % ) . MS. MW calc . for C32H45N20 , : 583 .72 . added and stirred the reaction mixture overnight. Solvents Found 584 .73 ( M + H ) . were removed and the residue dissolved in DCM and Preparation of 123 : 65 washed with water and brine, dried over sodium sulfate . Compound 122 (3 .30 g, 5. 65 mmol) was dissolved in a Solvents were removed and the crude product was purified mixture of ethyl acetate /MeOH and hydrogenated under by chromatography ( EtOAc , followed by 3 - 10 % MeOH / US 9 , 814 , 777 B2 115 116 DCM ) to get the required product as an off white solid (5 .25 checked and toluene was added to the reaction mixture , g , 79 % ) . MS . MW calc . for C2H2 N 0 23, z : 1173 . 58 . Found removed the solvents under reduced pressure . Co - evapo 1196 .60 ( M + Na) . rated with toluene two times and the residue dissolved in Preparation of 125 : DCM , washed with water, dried over anhydrous sodium Biantineary GalNAc derivative 124 (5 . 15 g , 4 .40 mmol) 5 sulfate . Crude product was purified by filtration column was dissolved in 15 mL of anhydrous DCM , to that 3 mL of (10 % MeOH /DCM ) to get the required product as pale anisole and 30 mL of TFA were added and stirred the brown solid ( 4 .40 g , 91 % ) . MS. MW calc . for Cx H N O . : reaction mixture for 2 hrs at ambient temperature . TLC 1117 .52 . Found 1140 .62 (M +Na ).

OAC Aco

Aco OH AcHN 126 AcO OA homeAc AcO

AcO AcO AcHN 127

101 OAC AcO

Y - AcO SyersNH2 AcHN 128128

OAC Aco AC Aco „ NH2 AcHN rºnoNH 129

Building blocks 126 and 127 are synthesized using a procedure similar to that for synthesis of 103 . Building blocks 128 and 129 are synthesized using a procedure Sasasimilar to that for synthesis of 105 . Scheme 2 HO . HO

DSC , TEA Z - OSu NH2,Py HO . HO . a NHZNHZ BocHNBocHN -NH2 DIEA DCM 62 % 78 %

HO HO mm130 131 US 9 , 814 ,777 B2 117 118 - continued 118

BocHN

HCI/ Dioxane NHZ BocHNm y e rartbeans,

BocHN

132

HN Aco

my AcO TOH NHZ ACHNAcHN HN lynyt HBTU , DIEA m DCM /DMF

H2N 17 .50 g man133 some

AcoAco /046 AcO AcHN NH OAC AcO / Pd / C NHZ Aco H2 AcHN m N yurtout AcO

AcO AcHN Bulanses134 US 9 , 814 ,777 B2 119 120 - continued 120 OAC AcO

AcO IZ AcHN OA AcO / - NH2 Aco AcHN NH AcO

Aco AcHN NH Bundanes135

Preparation of 135 . Building block 135 is synthesized using a procedure similar to that for synthesis of 110 .

HOMHO . OH HOHOA HOV HO CSA phimOH 136

OBz Bzo

Bzo BzO HO ?? O NH HO HO C13C 138 137 BF3•Et20 /DCM 75 %

OBZ Bzo

Bzo Bzo Bzo OBZ OH Ac20 Bzo 01 DMAP /Py Bzo 89 %

139 US 9 , 814 , 777 B2 121 122 121 - continued OBZ Bzo N BxeBzo 1. OsO4, NaI04, BzOBzó Lutidine/ Dioxane / Bzo OBz Water NOAC 2 . Oxone/ DMF 66 % ?oBz0Bzo N Bzo

140 OBZ BzO /

Bzo BromoBzó JOBZ Bzo OAC Aco Bzo - 1 96 % Bzo OH

141

30 Preparation of 137 : 70 . 30 , 70. 26 , 70 . 05 , 68 .31 , 68 . 23 , 67. 41 , 66 . 11, 62 .63 , Mannose (10 .00 g, 55 . 53 mmol) and Decinol (100 g , 62 .08 , 33 .96 , 29. 65 , 29 .58 , 29. 53 , 29 .58 , 29. 08 , 26 .20 . MS. solvent) and CSA (500 mg) were stirred at 110° C . in an oil Molecular weight calculated for C34H22024 , Cal. 1474 .52 . bath for overnight. The color of the decinol turned to dark Found 1497 .60 ( M + Na) . brown overnight . Bulk of the decinol was distilled out under 35 Preparation of 140 : reduced pressure . The residue was dissolved in DCM and Compound 139 ( 0 . 104 g , 0 .07 mmol ) was dissolved in a neutralized with TEA . Extracted the solution with water and mixture of DCM / Py ( 10 mL , 1 : 1 ) . Ac, O ( 0 . 5 mL, excess ) dried over sodium sulfate . Solvent was removed and the and DMAP ( 0 . 050 g ) and stirred the reaction overnight. The residue was purified by filtration through a small pad of reaction was quenched with MeOH , solvents were removed silica gel, first ethyl acetate followed by 10 - 15 % MeOH / 40 and residue was purified by chromatography ( gradient elu DCM to get the product ( 7 . 52 g , 42 % ). ' H NMR (CDC13 , tion 10 - 30 % EtoAc/ Hexane ) to the compound was white 400 MHz ) 400 MHz) ( 7 .52 g , 42 % ) . ol ) and Decinol ( 100 fluffy solid ( 0 . 108 g , 99 % ) . ' H NMR (CDC12 , 400 MHz ) : g , solvent) and CSA (500 mg) were stirred at 110° C . in an 400 MHz) excess ) and DMAP (0 .050 g ) and stirred the oil bath . reaction overnight. The reaction was quenched with MeOH , Preparation of 139 : 45 solvents were remove 07 ( m , 13H ), 3. 90 -3 .80 (m , 1H ) , Compound 137 (0 .172 g , 0 .541 mmol) was dissolved in 3 .69 - 3 .61 ( m , 1H ), 3 . 36 - 3 .28 (m , 1H ) , 2 .98 -2 .81 (m , 1H ), anhydrous DCM ( 10 mL ) under argon . MS was added to that 2 . 08 ( s , 3H ) , 2 . 10 - 2 . 01 ( m , 4H ) , 1 . 35 ( s , 3H ) , 1 .42 - 1 . 20 ( m . and cooled the reaction in an ice bath . BF3. Et20 ( 10u ( 102 12H ). 13C NMR (CDC13 , 100 MHz) 8 100 MHz ) excess ) and g , 0 . 541 mmol) was dissolved in anhydrous DCM ( 10 mL ) DMAP ( 0 .050 g ) and stirred the reaction overnight. The under argon . MS was ad138 ( 1 . 00 g . 1 . 35 mmol) in 5 mL of 50 reaction 7 .82 , 10 .43 Hz, 1H ) , 5 .65 -5 .47 ( m , 2H ) , 5 . 10 - 4 . 07 DCM was added drop wise over a period of 15 minutes . ( m , 13H ) , 3 . 90 - 3 . 80 ( m , 1H ) , 3 .69 - 3 .61 ( m , 1H ) , 3 . 36 - 3 . 28 Reaction was monitored by TLC , once the acceptor was ( m , 1H ) , 2 . 98 - 2 .81 ( m , 1H ) , 2 .08 ( s , 3H ) , 2 . 10 - 2 .01 ( m , 4H , finished the reaction was quenched with TEA and diluted 128 .47 , 128 . 40 , 114 . 35 , 102 . 32 , 99. 58 , 96 .64 , 74 .51 , 72 . 11, with DCM , filtered off MS and dried . The residue was 71. 91 , 71. 46 , 71. 21, 6978 , 6972 , 69 .51 , 69 .28 , 68 .19 , 68 .03 , purified by chromatography ( gradient elution 10 - 40 % 55 67 . 82 , 67 . 12 , 61. 97 , 61 . 83 , 33 . 94 , 29 .63 , 29 .61 , 29 .55 , EtOAc /Hexane ) to the compound as a white fluffy solid 29. 49 , 29 .27 , 29 . 20 , 29 . 05 , 26 . 11 , 21. 06 , 20 .02 . MS: ( 0 .550 g , 69 % ). ' H NMR (CDC12 , 400 MHz) 8 400 MHz) Molecular weight calculated for Cg3H36026 , Cal. 1558 .54 . 35 mmol) in 5 mL of DCM was added drop wise over a Found 1581. 8 ( M +Na ) . period of 15 minutes . Reaction was monitored by TLC , once Preparation of 141 : the acceptor was finished the reaction was quenched with 60 Compound 141 ( 1 . 36 g , 0 . 873 mmol) was dissolved in a TEA and diluted with DCM , filter 13C NMR (CDC1z , 100 mixture of Dioxane : Water (40 mL , 3 :1 ) . To the reaction MHz ) 8 100 MHz) 35 mmol) in 5 mL of DCM was added mixture lutidine ( 0 . 203 mL, 2 eq ) , followed by OsO solu drop wise over a period of 15 minutes . Reaction was tion ( 1 mL . 0 . 05M solution in 'Butanol ) were added . Sodium monitored by TLC , once the acceptor was finished the periodate ( 0 .774 g , 4 eq ) was added and stirred for 4 hr ' s at reaction was quenched with TEA and 2H ) , 1 . 30 - 0 . 92 ( m , 65 room temperature . Reaction was monitored by TLC , once 12H . 63 , 128 .61 , 128 .54 , 128 . 47 , 128 .44 , 114 .37 , 102 .74 , the starting material was consumed ; the mixture was diluted 102. 68 , 98 .81 , 85 .27 , 72 .43 , 71. 96 , 71. 37 , 71. 31 , 71 .01 , with water and extracted with DCM (3 times ) and dried over US 9 ,814 ,777 B2 123 124 sodium sulfate. All the solvents were removed and the 4 . 90 - 4 .35 ( m , 7H ) , 4 . 10 - 3 .55 ( m , 4H ) , 3 .30 - 3 .20 ( m , 1H ) , residue was directly used next reaction . Residue from the 2 . 96 - 2 .87 ( m , 1H ) , 2 . 18 - 2 . 10 ( m , 2H ) , 1. 96 ( s , 3H ) , 2 .01 above reaction was dissolved in DMF ( 20 mL ) to that Oxone 1 .95 ( m , 1H ) , 1 .51 - 1 .39 ( m , 2H ) , 1 .27 ( s , 3H ) , 1 .20 - 1 .01 ( m , ( 0 . 590 g , 1 .05 eq ) and stirred at ambient temperature for 3 12H ) . 13C NMR (CDC12 , 100 MHz) 8 100 MHz ) - d eq ) and h . Once the starting material was consumed , 2 mL of 1M 5 stirred at ambient temperature for 3 h . Once the starting HCl was added and diluted with Ethyl acetate . Washed with material was consumed , 2 mL of 1 .60 , 133 .49 , 130 . 18 , water , brine and dried over sodium sulfate . Solvents were 130 . 08 , 128 .85 , 129 .61 , 129 . 52 , 129 .44 , 129 . 20 , 129 . 13 , removed and the residue was purified by chromatography 128 .91 , 128 .89 , 128 .81 . 128 .78 , 128 .71 , 128 .51 , 128 .45 , ( gradient elution 20 -40 % EtoAc/ hexane ) to get the com - 102 . 34 , 99. 67 , 96 .65 , 74 .60 , 72 .17 , 71. 94 , 71 .49 , 71 .21 , pound as a white solid ( 1 . 08 g 79 % ) . ? H NMR (DMSO - da, 10 69. 82 , 69 . 79 , 69 . 59 , 69 . 37 , 68 . 22 , 68. 11 , 67 .81 , 67 .20 , 400 MHz) 8 400 MHz) - d eq ) and stirred at ambient tem - 64 . 55 , 61. 99 , 61. 85 , 60 .59 , 44 . 06 , 33 .96 , 30 .79 , 29 .39 , perature for 3 h . Once the starting material was consumed , 29 . 31 , 29 . 24 , 29. 20 , 29. 17 , 29 .08 , 26 . 08 , 24 . 85 , 24 . 79 , 2 mL of 1M HC1 was added and diluted with Ethyl acetate . 22 .20 , 21 . 24, 21. 11 , 20 .07 . MS: Molecular weight calculated Washed with water, brine and dried 0 . 15 (d , J = 7 . 8 Hz, 1H ) , for C3 -H34028 , Cal. 1576 .51 . Found 1599 .50 ( M + Na) .

OH OH HO ?? 1 . BFz•Et20 , Ether 2 . Ac20 , Py BzO - OBZ DMAP , DCM Bz0 BzO NH

Bzo OBZ Bz0 Bz0 Bzo BzO2 OBZ OAC B20D79Bz0

142

RuCl3 , Na104 DCM / CH3CN /water BzoBzO OBzQBZ Bz0Bzot Bz02 OBZ 2 BzO QAC - AcO BzoBzo LOH

143 OH OBZ ?? OBz HO HBr BzCI, DMAP Bzo -N o ( AcOH ) H0 ? DMAP BzoBzota DCM HO DMAP Bzo Bzo 0C . HO BzoBzó SungguhnyaOH OBz 144 145 US 9 , 814 ,777 B2 125 US 9,814 , 77 B2 126

OBZ - continuedBz OBz OBz Bzo Bzo . CC13CN , AgCO3 DBU BzoBzO . _ Acetone, , * Bzo _ 1 DCM Bzo BroBzo water Bzo Bzo BzO Bzo 7 Bzó Br Bzó OH 146 147

OBZ OBz Bzo BottBzo Bzó Bzo Y 1 - Decinol , B20Bzo 0 LNH BF•EtO OC . 148

OBZ OBZ OSO4, Bzo NaI04, OXONE Bzo BzotaBzOBzó Bzo Bzo 149 OBZ www OBZ Bzo Bzo OH BzO Bzo Bz0 150 my

Preparation of 148 . 129 .62 , 129 .55 , 129 . 50 , 129 .47 , 129. 41, 129 .40 , 129 .29 , Compound 148 was synthesized according to the reported 45 129 . 14 , 129. 11, 129 .03 , 129128. .47 96 ; , 128. .8787 , , 128 128 . 84 , 128 .83 , procedure (Martin , C . ; Karen , P .; Laurence , V . Chem . 128 . 78 , 128 .76 , 128 .63 , 128 . 56 , 128 .54 , 128 . 48 , 128 .37 , Pharm . Bull. 2004 , 52, 965 - 971. ) 128 . 26 , 114 . 33 , 114 . 26 , 100 .92 , 100 .84 , 97 .04 , 96 .52 ,128 , 75. .37 36 ;, Preparation of 149 : 75 . 17 , 74 .84 , 73 . 37 , 72 . 95 , 72 . 90 , 72 .81 , 72 .57 , 72 .507 , 1 - Decinol ( 0 . 300 g , 1 .92 mmol) and trichloroacetimidate 71. 94 , 71. 58 , 71. 05 , 70 . 37 , 70 . 27 , 70 .19 , 70 . 06 , 69 . 86 , 148 ( 2 .33 g, 1 .2 eq ) was dissolved in anhydrous DCM ( 10 50 69. 24 , 69. 19 , 69 . 02 , 63 .71 , 63 .56 , 63. 20 , 62 .93 , 62. 69 , mL ) under argon . MS was added to that and cooled the 33. 96 , 33 . 91 , 32 . 93 , 29 .60 , 29. 53 , 29 . 50 , 29. 46 , 29 .42 , reaction in an ice bath . BFz. Et20 (30u (30 g , 1 . 2 eq ) was 29. 33 , 29 .30 , 29. 22 , 29 . 14 , 29 .06 , 29. 00 . MS . Molecular dissolved in anhydrous DCM ( 10 mL ) under argon . MS was weight calculated for C7H68018 , Cal. 1208 .44 . Found added to that and cooled the reaction in an ice bath . 1231 .4 ( M + Na ) . BF130 .08 , 128 . 85 , 129 .61 , 129 . 52 , 129 .44 , 129 . 20 , 129 . 13 , 55 Preparation of 150 : 128 .91 , 128 . 89 , 128 .81 . 128 .78 , 128 .71 , 128 .51 , 128 .45 , Compound 149 ( 7 . 26 g , 6 mmol) was dissolved in a 102. 34 , 99 .60 % EtoAc/ Hexane ) to the compound as a white mixture of Dioxane : Water ( 100 mL , 3 : 1) . To the reaction fluffy solid ( 2 .01 g , 86 % ). ' H NMR (CDC13 , 400 MHz ) 8 mixture lutidine ( 0 .7 mL , 2 eq ), followed by OsO4 solution 400 MHz ) 2 eq ) was dissolved in anhydrous DCM ( 10 mL ) (5 mL . 0 .05M solution in Butanol) were added . Sodium under argon . MS was added to that and cooled the reaction 60 periodate ( 5 . 11 g , 4 eq ) was added and stirred for 4 hr ' s at in an ice bath . BF130 . 08 , 128 . 85 , 129 .61 , 129 . 10 ( m , 4H ) , room temperature . Reaction was monitored by TLC , once 1 . 00 - 1 .60 ( m , 11H ) . 13C NMR (CDC12 , 100 MHz ) / 100 the starting material was consumed ; themixture was diluted MHz ) 2 eq ) was dissolved in anhydrous DCM ( 10 mL ) with water and extracted with DCM ( 3 times ) and dried over under argon . MS was added to that and cooled the reaction sodium sulfate . All the solvents were removed and the in an ice bath . BF130 .08 , 128 .85 , 129 .61 , 129 . 10 ( m , 4H ) , 65 residue was directly used next reaction . Residue from the 1 . 00 - 1 .60 ( m , 11H ) . 8 .91 , 128 .81 , 130 . 12 , 130 . 05 , 129 . 98 , above reaction was dissolved in DMF (60 mL ) to that Oxone 129. 95 , 129. 92 , 129 . 88 , 129. 80 , 129 . 77 , 129. 73 , 129 .68 , ( 3 . 86 g , 1 . 05 eq ) and stirred at ambient temperature for 3 h . US 9 , 814 , 777 B2 127 128 Once the starting material was consumed , 10 mL of 1M HC1 -continued was added and diluted with Ethyl acetate . Washed with water, brine and dried over sodium sulfate . Solvents were removed and the residue was purified by chromatography ( gradient elution 20 -40 % EtoAc /hexane ) to get the com - 5 OH pound 150 as a white solid ( 5 .50 g 75 % ) . ' H NMR (DMSO da , 400 MHz ) 8 400 MHz ) - dolid ( 5 .50 g 75 % ). iodate ( 5 . 11 HN ' N g , 4 eq ) was added and stirred for 4 hr ' s at room temperature . Re13C NMR (DMSO - do, 100 MHz) 8 100 MHz )- dolid ( 5 .50 g 75 % ). iodate ( 5 . 11 g , 4 eq) was added and stirred for 4 hr' s IN N N 0 CF2 at room temperature . Reaction was monitored by TLC , once the starting material was consumed ; the mixture was dilut99 , 164 . 88 , 164 . 75 , 164 . 70 , 164 .60 , 164 . 54 , 164 .50 , 133 . 80 , 133 .71 , 133 . 58 , 133 . 42 , 133 . 29 , 133 . 15 , 129 .88 , 129 .42 , 152 129 . 36 , 129 . 29 , 129 .23 , 129. 20 , 129 . 12 , 129. 07 , 129 .05 , 129. 03 , 128 . 91 , 128 . 88 , 128 . 72 , 128 .59 , 128 .48 , 128 . 38 , 15 99 . 96 , 99. 29 , 99 .22 , 95 . 96 , 95 .64 , 95 .22 , 93 . 10 , 75 .61 , Synthesis of 4 - [ ( 2 -isobutyrylamino - 4 - oxo - 3 , 4 - di 74 .86 , 74 .57 , 74 . 37 , 74 . 15 , 73 . 59 , 73 . 14 , 72 .58 , 71 .46 , hydro -pteridin - 6 - ylmethyl) - ( 2 , 2 , 2 - trifluoroacetyl) 71 . 15 , 70 .48 , 70 .31 , 70 .09 , 69. 97 , 69. 00 , 68 . 87 , 68 . 22 , amino ] benzoic acid 152 67 .81 , 63 .65 , 62 . 49 , 60 . 73 , 59 .76 , 43 .01 , 33 .68 , 33 .62 ,· 20 To a suspension of pteroic acid ( 25 g , 61 . 2 mmol) and 32 .54 , 28 .84 , 28 .82 , 28 .61 , 28 . 55 , 28 . 47 , 28 . 40 , 25 . 47 , DMAP ( 11 .25 g , 92 mmol) in anhydrous pyridine (400 mL ) , 25 .21 , 24 . 52 , 24 .43 , 20 .45 . MS . Molecular weight calculated TBDPS chloride (42 g , 153 mmol) was added . The reaction for C70H66020 , Cal. 1226 .41 . Found 1249. 4 ( M + Na) . mixture was stirred at room temperature for 30 h after which isobutric anhydride (14 . 6 g , 92 mmol ) was added and the Example 2 . Synthesis of Pteroic Acid Precursors mixture was slightly warmed . An additional 60 mL of for Conjugation pyridine was also added and the reaction mixture was stirred at room temperature overnight. The reaction mixture Appropriately substituted pteroic acid precursor 110 was became homogenous after which pyridine and other vola prepared as follows. tiles were concentrated in a rotary evaporator . The residue was stirred with EtoAc ( 1 L ) and acetic acid ( 100 mL ) and 30 water (500 mL ) for 24 h . The thus obtained slurry was filtered , the residue was washed with water (500 mL ), 1 . TBDPS, EtOAC ( 1 L ) and dried to obtain the pure product as a white OH DMAP , solid ( 26 . 1 g , 89 % ) . ' H NMR (DMSO - da , 400 MHz ) d = 8 . 87 Py ( s , 1H ) , 7 .95 ( d , J = 8 .6 Hz, 2H ) , 7 .67 ( d , J = 8 . 6 Hz, 2H ), 5 . 21 HN 2 . Isobutyric ? 35 ( s , 2H ) , 2 .79 - 2 .74 ( m , 1H ) , 1 . 12 ( d , J = 6 .83 Hz , 6H ) , 13C anhydride NMR (DMSO - da ) S = 180 .72 , 166 .49 , 159 . 25 , 149 . 87 , 147 . 68 , 142 . 69 , 136 . 34 , 134 .45 , 130 . 54 , 129 . 16 , 128 . 86 . HANNN CF3 127 .49 , 34 . 96 , 33 .09 , 26 . 52 , 18 . 88 , 18. 74 . 9F NMR 151 (DMSO - d . ) 8 - 64 . 32 . MS. Molecular weight calculated for osole C20H F3N605, Cal. 478 . 12 . Found 479 . 12 (MH +) . US 9 , 814 ,777 B2 129129 130

COH CO2Me EZ

0 CF3 DIPEA DMF / 153 obtainto 154 HN HN HN HN

DSC,pyCH2Cl2 COOMe VSH HBTU,DIPEA DMF H2N OH toolstheWhen -CO2Me SH 152152 CO2Me N sthese0with CF3 HN HN O 155

N HN HNHN US 9 , 814 ,777 B2 131 132

HO

N * ?? H2N V CO Bu 156 N NH HBTU , DIPEA DMF

Loredana152

O ' Bu NH

CH3CFz o NH

malacotte157 | TFA CH2Cl2

HOOC

NH haredCEROCF3 NH 158

In order to synthesize an appropriately substituted more mixture showed completion of the reaction . The reaction versatile precursor of Folic acid amenable for RNA synthe - mixture was slowly poured in ice with vigorous stirring . The sis , the following strategy was used . In this method the sn precipitated product was filtered to get the product 157 as a protected Folic acid 152 was treated with the y - tert -butyl , MHz ) d = 12 .33 ( s , 1H ) , 11. 94 ( s , 1H ) , 8 .88 ( s , 1H ) , 8 .82 ( d , a -Me ester of glutamic acid , 156 to obtain the ester 157 J = 7 . 3 Hz, 1H ) , 7 . 90 ( d , J = 8 . 6 Hz, 2H ) , 7 .68 ( d , J = 8 . 4 Hz, which on treatment with TFA /CH2Cl2 provided the precur 2H ) , 5 .22 (s , 2H ), 4 . 46 -4 .40 (m , 1H ) , 3 .62 (s , 3H ), 2. 86 - 2. 73 sor 158 . (m , 1H ), 2 .32 ( t, J = 7 . 4 Hz, 2H ) 2 .05 - 1. 90 ( m , 2H ), 1. 35 (m , 55 9H ) . 1 . 12 ( d . J = 6 . 8 Hz, 6H ) . 13C NMR DMSO - da ) Synthesis of 2 - { 4 - [ (2 - isobutyrylamino -4 -oxo - 3 ,4 d = 180 .75 , 172 . 13 , 171 .45 , 165 .64 , 159 .10 , 154 . 80 , 149 . 97 , dihydro - pteridin - 6 -ylmethyl ) - ( 2 , 2 , 2 -trifluoroacetyl ) 149 .79 , 147 .72 , 141 .75 , 134 . 15 , 130 .53 , 128 . 70 , 128 .49 , amino } - pentanedioic acid 5 -tert -butyl ester 117 . 50 , 114 .64 , 79 .79 , 51 . 96 , 51. 91 , 34 . 96 , 31 . 22 , 27 .68 , 1 -methyl ester 157 25 . 71 , 18 . 72 . MS. Molecular weight calculated for 60 C30H34F3N 0g, Cal. 677. 63 . Found 676 . 72 ( M - H - ) . In a representative procedure, the pteroic acid precursor 152 ( 2 . 4 g , 5 mmol ) was dissolved in anhydrous DMF ( 20 Synthesis of 2 - { 4 - [ ( 2 - isobutyrylamino - 4 -oxo - 3 , 4 mL ), HBTU ( 1 .9 g , 1 eq . ) followed by DIEA ( 1 mL , 5 eq .) dihydro -pteridin - 6 -ylmethyl ) - ( 2, 2 ,2 - trifluoroacetyl) were added and stirred for 20 minutes. To this reaction amino } -pentanedioic acid 1 -methyl ester 158 mixture the amine hydrochloride 156 ( 1 . 2 g , 1 eq ) was added 65 as a solution in DMF (6 mL ) . Reaction was monitored by The ester 157 ( 2 g , 2 . 9 mmol) was dissolved in 20 mL of TLC ( 8 % MeOH /DCM , PMA stain ) . TLC of the reaction 50 % TFA in dichloromethane and the solution was stirred at US 9 ,814 ,777 B2 133 134 room temperature for 30 min . after which the TLC showed - continued the complete disappearance of the starting ester. The reac OOH tion mixture was concentrated and the residue was crystal lized from CH2C12 :Hexanes (2 : 3 ) and crystallized product 5 was filtered off and dried to obtain the pure product 158 ( 1 . 76 g , 96 % ) as off white powder. ' H NMR (DMSO - 06, 400 MHz) 8 = 12 .32 (bs , 1H ) , 11 .94 (s , 1H ), 8 .88 (s , 1H ) , 8 .84 (d , J = 7 . 4 Hz, 1H ) , 7 . 90 ( d , J = 8 . 3 Hz, 2H ) , 7 .69 ( d , J = 8 . 3 Hz , 10 2H ) , 5 .22 ( s , 2H ), 4 . 45 - 4 . 41 ( m , 1H ), 3 .62 ( s , 3H ) , 2 . 78 - 2 . 75 ( m , 1H ), 2 .35 (t , J = 7. 4 Hz , 2H ) 2 .07 -1 .92 ( m , 2H ), 1. 12 ( d , HN J = 6 .8 Hz , 6H ). 13C NMR DMSO -do ) d = 180 .77 , 173 .70 , - NH 172 .19 , 165 .70 , 159. 21 , 155 .54 , 149. 93 , 149. 84 , 147. 75 , 15 NH 141. 78 , 134 . 18, 130 .53 , 128 .71 , 128 .49 , 117 .51 , 114 .64 , 53 .98 , 52 . 06 , 51. 93 , 34 .97 , 30 . 11 , 25 .68 , 18. 73 . MS. 161 Molecular weight calculated for C26H26F3N Os, Cal . 621. 18 . Found 620 . 18 ( M - H -) . 20 Synthesis of 160 . OOH To suspension of Compound 159 (0 .2 g , 0 .000671 moles) in anhydrous pyridine (5 mL) was added DMAP (0 .13 g , 25 0. 0010 moles ) , followed by isobuytric anhydride (0 . 6 mL , 0 .0040 moles ) at room temperature . The resulting mixture was then refluxed for 4 hr. After completion of reaction ( by isobutyric anhydride TLC ), the mixture was poured onto ice -HCl / hexane and DMAP , Pyridine stirred well . The resulting solid was filtered , washed with haxane and used directly for further reactions . Yield (0 . 1 g ,

HN 34 % ). ' H NMR (DMSO , 400 MHz ): 8 = 12. 08 ( s , 1H ), 11. 48 ( s, 1H ), 7. 85 (d , 2H ), 7. 35 (d , 2H ), 7 .22 (s , 1H ) , 4 .33 (m , HN AN 1H ) , 2 . 98 ( m , 2H ) , 2 . 96 ( m , 2H ), 2 . 81 ( m , 1H ), 1 .19 ( d , 6H ) , 1 . 14 (d , 6H ) . 13C NMR (DMSO ) : d = 179. 85 , 175. 21, 167 .00 , 159 156 .64 , 147 .81 , 147 .34 , 146 .73 , 129 .08 , 128 .32 , 128 . 14 , 121. 27 , 116 .00 , 106 .07 , 35. 02 , 34 .51 , 33. 37 , 26 . 90 , 18 .72 , 40 18 .64 . MS (MH ) *: 439 .40 . Synthesis of 197 . To a stirring solution of 160 (0 . 4 g , 0 .00091 moles) in 5 45 ml MeOH , DIPEA (0 .036 mL , 0 . 00278 moles ), was added at room temperature . (Note : After addition of DIPEA reac tion mixture becomes clear ). After 10 minutes DMAP (cata OH lytic ) was added to themixture . The completion of reaction was monitored by TLC ( & LCMS) . MeOH was then con centrated and the residue was diluted with water (5 mL ) . Acidification with dilute HCl was followed by extraction with ethyl acetate . Organic layer was separated , dried over Na2SO4 and concentrated . The crude mixture was pure DIPEA / DMAP enough and was used directly for further reactions . Yield : Methanol 260 mg ( 54 % ). ' H NMR (DMSO , 400 MHz) , 8 = 12. 77 (bs , HN 1H ), 11. 68 ( s , 1H ), 11. 34 ( s , 2H ), 7 .84 ( d , 2H ) , 7 .32 ( d , 2H ) , 6 .64 ( s, 1H ) , 3 .01 (m , 2H ), 2 .94 (m , 2H ), 2. 74 ( m , 1H ), 1. 09 ( d , 6H ). MS (MH ) * : 369 . 10 . youtube In order to synthesize azido functional group containing 65 folate conjugates the following strategy was used . The azido 160 amine tether 165 was synthesized starting from the com mercially available diamine 162 as shown below . US 9 , 814 ,777 B2 135 136 (Boc ) 20 , NEt3 , CH2Cl2 HNHAN VO moNNH- NH2, -22 BOCHNoBocHN Co CNHNH2, 162 163 TfN3, CH3CN , Cu S04

2M ??? in ether H2N Nz + etner BocHNBocHNY N3 HCl 164 165

Synthesis of Amine 163 . stirred for 30 min after which the TLC and MS showed the To a solution of the diamine ( 22 g , 0 . 1 mol) in dichlo - complete disappearance of starting amine . The reaction romethane (300 mL ) , triethylamine ( 15 mL ) was added and mixture was concentrated in a rotary evaporator and the the mixture was cooled in ice bath . To this cold solution a residue was purified by chromatography on silica gel ( elu ent: dichloromethane -methanol ) to obtain the pure azide 164 solution of (Boc ) , in CH ,C1 , ( 100 mL ) was added drop ( 1 g , 96 % ) as an oil. MS: MW Calc . for CisH2N _ 05: wise and the mixture was stirred overnight. The reaction 25 346 .42 . Found 347 .41 (MH + ) . IHNMR (CDCI 400 MHz ) mixture was washed with satd . NaHCO3 (200 mL ) , water 8 = 4 .68 (bs , 1H ), 3. 40 - 3 .30 ( m , 12H ), 3 . 16 (t , J = 6 . 4 Hz , 2H ), ( 300 mL ) , brine (300 mL ) and dried (Na2SO4 ) . Concentra 3 . 00 - 2 . 95 ( m , 2H ), 1 .68 - 1. 54 ( m , 4H ) , 1 . 04 ( s , 9H ) . tion of this organic layer followed by column purification Synthesis of 165 . provided the pure mono Boc amine 202 in 55 % yield . MS : The azide 203 (1 g , 2. 88 mmol) was dissolved in ethanol MW Calc . for C15H22N205: 320 . 42 . Found 321 .41 (MH * ) . 30 ( 10 mL ) and to this a 2M solution ofHCl in ether was added Synthesis of Azide 164 . and the mixture was stirred at room temperature overnight. The triflic azide stock solution was prepared as reported The MS showed the absence of starting material . The in Tetrahedron Letters 47 ( 2006 ) 2382 - 2385 . The amine reaction mixture was concentrated and the thus obtained oil ( 0 . 96 g, 3 mmol) , sodium bicarbonate (0 .85 mg, 10 mmol) was used as such for the next reaction without further and copper ( II ) sulfate pentahydrate ( 22 mg, 0 . 1 mmol) were 35 purification . MS: MW Calc . for C10H23CIN 03: 246 . 17 . dissolved in water ( 3 mL ) . Triflic azide stock solution ( 5 Found 247 . 17 (MH + ) . " HNMR (DMSO -do 400 MHz ) mL ) was added , followed by the addition of methanol ( 20 8 = 8 . 96 (bs , 1H ), 7 . 92 (bs , 2H ), 3 .52 - 3 .40 ( m , 12H ), 3 .37 ( t , mL ) to yield a homogeneous system . The blue mixture was J = 6 . 8 Hz, 2H ) , 2 . 85 - 2 .77 ( m , 2H ) , 1 . 81 - 1 . 70 ( m , 4H ) .

COOM NH HBTU , DIPEA , DMF HnNovoHN NN 0 CF3 165

158 COOMe NH HN

HN N O CFz

166

LiOH US 9 ,814 ,777 B2 137 138 COOH- continued NH 03 HN

H2N ' N N ? 167

Coupling of the amine 165 (0 .6 g ) with the acid 158 (1 .2 acetate ( 100 mL ) . The precipitated product was filtered off g ) provided the coupled azide 166 ( 1 . 68 g , 93 % ) as a light and washed with water and ethyl acetate and dried under yellow . ' H NMR (DMSO - do, 400 MHz ) = 12 . 34 (s , 15 vacuo at 40° C . overnight to get the pure azide 167 (0 .455 g 55 % ) as an orange solid . ' H NMR (DMSO -d6 , 400 MHz) 1H ), 11. 95 (s , 1H ), 8 .89 (s , 2H ), 7 . 92 (d , J = 8 . 4 Hz , 2H ), 7 .81 = 8 . 59 ( s , 1H ) , 7 . 85 (bs , 1H ) , 7 . 72 (bs , 1H ) , 7 .56 ( d , J = 8 . 4 ( m , 1H ) , 7 .70 ( d , J = 8 . 4 Hz, 2H ), 5 . 22 ( s , 2H ), 4 .40 - 4 .34 ( m , Hz, 2H ) , 6 . 88 (bs , 1H ) , 6 .65 ( d , J = 8 . 4 Hz, 2H ), 4 .45 (s , 2H ), 1H ) , 3 .62 ( s , 3H ) , 3 . 50 - 3 . 31 ( m , 15H ) , 3 . 09 - 3 . 00 ( m , 2H ) , 4 . 00 - 4 .02 (m , 1H ), 3 .50 - 3 . 33 ( m , 14H ) , 3 . 04 - 3 . 00 (m , 2H ), 2 .80 - 2 .72 (m , 1H ) , 2 . 20 ( t, J = 7 . 4 Hz, 2H ) 2 . 10 - 1. 89 (m , 2Hn ) , 2020 202 .07 - 1. 83 ( m , 4H ) , 1. 76 - 1 .54 (m , 4H ) . MS. Molecular 1 . 76 - 1 .54 ( m , 4H ) , 1 . 12 ( d , J = 6 . 8 Hz, 6H ) . MS. Molecular weight calculated for C29H39N1108 , Cal. 669 .69 . Found weight calculated for C36H46F3N11010 , Cal. 849. 81 . Found 668 . 2 ( M - H - ) . 850 . 2 (MH + ) . In another embodiment, the alkyne containing folic acid Synthesis of 167 : is synthesized as follows. In this case the protected pteroic The azide 166 ( 1 g ) was dissolved in THF (20 mL ) and to 25 acid 158 was coupled with the protected lysine 168 to get the it an aqueous solution of lithium hydroxide (100 mg in 2 mlmL. coupled product 169 which on Cbz deprotection provided of water ) was added and the solution was stirred at room the amine 170 . Coupling of the amine 170 with the acid 171 temperature for 4 h after which the MS showed the complete provided the coupled product 172 which after purification disappearance of SM . The reaction mixture was acidified to andand deprotection provided the product 173 as described pH 5 using acetic acid and the RM was diluted with ethyl below .

OH

HN OF fons158 ce COOMte

HBTU , NHCbz DIPEA , NH COOMe DMF H2, Pd / C HN H2N NHCbz HN N OP CF2

w 168 169 COOMe

HBTU . DIPEA , HN DMF OH futhingHN N 0 oltinCF3 wa 170 171 US 9 , 814 ,777 B2 139 140140 - continued O COOMe N

HN LiOH lotenHN N 0 obtinenCF3 172

COOH

HN

HNHN ANH 173

Synthesis of 169 : 30 Synthesis of 172 : Using a similar procedure to that used for the synthesis of Coupling of the amine 170 with the acid 171 using a 166 , coupling of the acid 158 with the lysine derivative 168 procedure to that used for the synthesis of 166 provided the provided the coupling product 169 as a white solid in 95 % couple product 172 in high yields. yield . Synthesis of 173 : Synthesis of 170 : 35 The deprotection of the protecting groups is achieved The compound 169 on hydrogenation with Pd /C provided using a similar procedure as described for the synthesis of the deprotected amine 170 as a yellow solid . 167 to isolate the fully deprotected alkyne 173 .

OH NH NHS, DCC DMSO HN N H N N N Inoltre174

O OH

HN NH HONHN HorostyTNCN 175 US 9 , 814 , 777 B2 141 142 Preparation of 175 . dark for 18 h . The reaction mixture was filtered and to the Treatment of folic acid 174 with DCC followed by N -hydroxysuccinimide provided the activated ester 175 in filtrate EtOAC ( 1 L ) was added and the precipitated product 80 % yield . In a typical procedure , folic acid (5 g , 11. 33 was filtered , washed with ethyl acetate (500 mL ) , ether (200 mmol) was dissolved in anhydrous DMSO ( 100 mL ) and to 5 mL ) and dried under vacuum to isolate the product as a this solution was added , triethylamine ( 2. 5 mL ), DCC 47(4 .7 yellow powder . The purity of the product was found to be g , 22 .6 mmol) and N -hydroxysuccinimide (2 .6 g , 22 .6 83 % by HPLC . This product was used as such for the mmol ) and the solution was stirred at room temperature in coupling steps without further purification .

1 . ETFA , ACN HN - NH2 2 . Cbz - OSU F C N o Ph

176 177

THF/ H20 LiOH CooMe OH NH HN Y ' N HN N Ph HN 0 CF3 + HNNY

furtzorg158 178178 DIPEA, HBTU , DMF

COOM N Ph HN Siguenye HN 07 -CF3

praten ordent179

Pd / C

COOMe N KodeNH2

AN N N 0 CF3 front of180

OH

181 US 9 , 814 ,777 B2 143 144 -continued OMe N

HN toglangin yang N 0 CF3

182 fotos LiOH

COOH NH HN H?N ??????? 183

Preparation of 183 . LiOH provided the amine 178 in quantitative yield . Cou The ketal 176 was synthesized using a reported procedure 30 pling of this amine 178 ( 0. 5 g ) with the protected folic acid ( Paramonov , S . E .; Bachelder, E . M . ; Beaudette , T . T .; 158 ( 1 g ) provided the coupled product 179 ( 1 . 1 g ) which on Standley , S . M . ; Lee , C . C . ; Dashe, J . ; Frechet, Jean M . J . hydrogenation provided the amine 180 in quantitative yield . Fully Acid -Degradable Biocompatible Polyacetal Micropar ticles for Drug Delivery. Bioconjugate Chemistry (2008 ), 19 Coupling of amine 180 was carried out with the maleimi ( 4 ) , 911 - 919 ) . The transient protection of the ketal was 35 dopropionic acid 181 to give the coupled product 182 in carried out in two steps in one pot first by treating the good yields. The final deprotection of all the protecting diamine with one equivalent of ethyltrifluoroacetate fol group in 182 is carried out using ice - cold aqueous LiOH in lowed by one equivalent of Cbz -OSu to provide the di THF to afford the precursor 183 as an orange solid . protected derivative 177 in 80 % yield after column purifi cation . The protected amine 177 on treatment with aqueous Example 3. Synthesis of Lipid Conjugates

HO

200

DSC , TEA, 0° C .

o

201

OMe CIH HN Py, DCMOC. US 9 , 814 ,777 B2 145145 146 -continued N

202 boyfrowneLiOH MOH /DCM uso/ H , O

HO .

203

HBTU , DIEA HO Toto- NH2 DMF/ DCM

HOM NH

204

Preparation of 201 : 45 Preparation of 203 : 1 , 2 -Dioctadecyl sn glycerol ( 8 .50 g , 14 .23 mmol) and Compound 202 (4 . 50 g , 5 . 86 mmol) was dissolved in a DSC ( 5 . 47 g , 1 . 5 eq . ) were dissolved in DCM ( 100 mL ) and mixture of THF /MeOH /Water ( 2 : 2 : 1 ) and cooled in an ice cooled in an ice -water bath . Triethylamine (6 .00 mL , 44 bath . LiOH ( 1 .23 g, Seq .) was added and the mixture stirred mmol) was added and stirred the mixture overnight. The 50 overnight. TLC checked and the mixture neutralized with mixture was transferred to a separatory funnel diluted with AcOH . Solvents were removed and the residue extracted DCM , washed with bicarbonate solution and water. DCM with dichloromethane, washed with water . Solvents were layer separated and dried over sodium sulfate . Solvents were removed and the residue purified by chromatography to get removed and the residue dried under high vacuum over night. It was used for the next reaction with out further 55 msthe required compound as a white solid ( Yield , 4 .32 g , 97 % ) . purification ( Yield , 11. 50 g ) . MS: Calculated for C46H , NO6, 753 .68 . Found 752. 70 Preparation of 202 : (M - H ). Compound 201 ( 4 .00 g, 5 .42 mmol) and 6 -aminohexano Preparation of 204 : ate hydrochloride ( 1 .477 g , 1 . 5 eq .) were dissolved in DCM Compound 203 ( 0 . 832 g , 1 . 10 mmol) and HBTU ( 0 . 461 and cooled in an ice bath . Pyridine ( 5 mL ) was added to the 60 g , 1 .21 mmol) were dissolved in a mixture of DCM /DMF to mixture and stirred overnight. Solvents were removed and that DIEA ( 0 . 573 mL ) was added and stirred the mixture for the residue dried under high vacuum . The residue extracted 5 minutes . PEG amino acid ( 2 . 00 g , 0 .921 mmol ) was added with dichloromethane , washed with bicarbonate and water. and stirred the mixture overnight. Solvent was removed and Crude product was purified by chromatography (Gradient the residue purified by chromatography ( Ethyl acetate , then elution of EtoAc /Haxane ) to get the required product 202 65 5 - 10 % MeOH /DCM ) to get the required product ( 2 . 58 g , ( Yield -3 .80 g , 91 % ). MS : Calculated for C49H ,3NO , 95 % ). MS calculated Average MWt. 2700 - 2900 . Found 767 .70 . Found 768 .68 (M + H ) . 2720 - 2960 . US 9 , 814 ,777 B2 147 148

HO 1111111

II

205 myysHN HBTU , DIEA , DCM / DMF

11111|

N

206

MeOH / DCM /water LiOH

HO In

TIT. qurtainment207 HBTU , DIEA HO??????? NH2 DMF /DCM ? US 9 , 814 ,777 B2 149 150 - continued N HOW NH

208

Preparation of 206 : 15 with AcOH . Solvents were removed and the residue Compound 205 ( 2 . 11 g , 3 . 15 mmol) and methylamino extracted with dichloromethane, washed with water. Sol hexanoate ( 0 .688 g , 1 . 2 eq .) were dissolved in a mixture of vents were removed and the residue purified by chromatog DMF/ DCM (50 mL ) . To that HBTU ( 1 . 31 g 1 . 05 eg ) and raphy to get the required compound as a white solid ( Yield , 2 . 12 g , 96 % ) . MS : Calculated for C49H 6N205, 782 .65 . DIEA (2 mL, excess) were added . The mixture stirred 2020 Found 781 .70 (M - H ). overnight at ambient temperature overnight. The mixture Preparation of 208 : was poured into ice -water mixture and extracted with ether . Compound 207 (0 .865 g , 1 . 10 mmol) and HBTU (0 .461 Ether layer separated and dried over sodium sulfate . Sol g , 1. 21 mmol) were dissolved in a mixture of DCM /DMF to vents were removed and the residue purified by chromatog - that DIEA ( 0 . 573 mL ) was added and stirred the mixture for raphy to get the required product 206 (yield - 2 .27 g , 90 % ) . 25 5 minutes. PEG amino acid ( 2 .00 g , 0 .921 mmol) was added Preparation of 207 : and stirred the mixture overnight. Solvent was removed and Compound 206 (2 . 25 g, 2 .925 mmol) was dissolved in a the residue purified by chromatography (Ethyl acetate , then mixture of THF /MeOH /Water ( 50 mL , 2 : 2 : 1 ) and cooled in 5 - 12 % MeOH /DCM ) to get the required product ( 1. 60 g , an ice bath . LiOH ( 0 .614 g , Seq .) was added and the mixture 59 % ) . MS calculated Average MWt. 2750 -2950 . Found stirred overnight. TLC checked and the mixture neutralized 2730 - 2970 . US 9 , 814 , 777 B2 151 152

TFA -NH,

TZ 110

IllIlli OAC AcHN OAC AcHN Ac0 Ac0_ Ac0 Ac0_ OAC TZ ? AcHN NaoMe/MeOH Ac0 Ad0 207 ??DMF 209 HBTU,DIEA]

TZ TZ

OAC AcHN AcHN Ac0_ Ac0 Ac0_ OAC AcHN Ac0 Ac0 ???? US 9 , 814 ,777 B2 153 154

HN

III

continued- 210

HN fabushingBuyukN HO AcHN HO NHV OH HO OH OH HO AcHN Irland HO OH