US 2008O161256A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2008/0161256A1 Morrisey et al. (43) Pub. Date: Jul. 3, 2008 (54) RNA INTERFERENCE MEDIATED tinuation-in-part of application No. 10/780.447, filed INHIBITION OF GENE EXPRESSIONUSING on Feb. 13, 2004, which is a continuation-in-part of SHORT INTERFERING NUCLECACD application No. 10/427,160, filed on Apr. 30, 2003, (SINA) which is a continuation-in-part of application No. PCT/US02/15876, filed on May 17, 2002, Continua (75) Inventors: David Morrisey, Boulder, CO tion-in-part of application No. 10/727,780, filed on (US); James McSwiggen, Boulder, Dec. 3, 2003, now abandoned, Continuation-in-part of CO (US); Leonid Beigelman, application No. PCT/US05/04270, filed on Feb. 9, Longmont, CO (US) 2005. Correspondence Address: (60) Provisional application No. 60/358,580, filed on Feb. 20, 2002, provisional application No. 60/358,580, MCDONNELL, BOEHNEN, HULBERT AND filed on Feb. 20, 2002, provisional application No. BERGHOFF, LLP 60/363,124, filed on Mar. 11, 2002, provisional appli 300 SOUTHWACKER DRIVE, SUITE 3100 cation No. 60/363,124, filed on Mar. 11, 2002, provi CHICAGO, IL 60606 sional application No. 60/386,782, filed on Jun. 6, (73) Assignee: Sirna Therapeutics, Inc., Boulder, 2002, provisional application No. 60/386,782, filed on CO (US) Jun. 6, 2002, provisional application No. (Continued) (21) Appl. No.: 11/205,646 Publication Classification (22) Filed: Aug. 17, 2005 (51) Int. Cl. Related U.S. Application Data A63L/70 (2006.01) A6IP 43/00 (2006.01) (63) Continuation-in-part of application No. 1 1/098.303, (52) U.S. Cl. .......................................................... 514/44 filed on Apr. 4, 2005, now abandoned, which is a continuation-in-part of application No. 10/923,536, (57) ABSTRACT filed on Aug. 20, 2004, which is a continuation-in-part of application No. PCT/US04/16390, filed on May 24, This invention relates to compounds, compositions, and 2004, which is a continuation-in-part of application methods useful for modulating gene expression using short No. 10/826.966, filed on Apr. 16, 2004, now aban interfering nucleic acid (siNA) molecules. In particular, the doned, which is a continuation-in-part of application instant invention features Small nucleic acid molecules. Such No. 10/757,803, filed on Jan. 14, 2004, which is a as short interfering nucleic acid (siNA), short interfering continuation-in-part of application No. 10/720,448, RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA filed on Nov. 24, 2003, which is a continuation-in-part (miRNA), and short hairpin RNA (shRNA) molecules and of application No. 10/693,059, filed on Oct. 23, 2003, methods used to modulate the expression of genes, such as which is a continuation-in-part of application No. expressed pseudogenes associated with the maintenance or 10/444,853, filed on May 23, 2003, which is a continu development of diseases, disorders, traits, and conditions in a ation-in-part of application No. PCT/US03/05346, Subject or organism. The invention also provides Small filed on Feb. 20, 2003, which is a continuation-in-part nucleic acid molecules with reduced or attenuated immuno of application No. PCT/US03/05028, filed on Feb. 20, stimulatory properties and methods for designing and synthe 2003, Continuation-in-part of application No. PCT/ sizing Such small nucleic acid molecules having improved US04/13456, filed on Apr. 30, 2004, which is a con toxicologic properties while retaining RNAi activity. (1) F1RST STRAND (2) SEC DEPROTECTION PURFICATION (DETRTYLATION siRNADUPLEX O =SOLSUPPORT R = TERMINAL PROTECTING GROUp FOREXAME: DMETHOXYRITYL (DMT) () www. = CLEAWABLELINKER FOREXAMPLE: NUCLEOTIDESUCCNATEOR (2) INVERTEDDEoxYABASICSUCCINATE Wwww a CEAWABELNKER (FOR EXAMPLE: NUCLEOTDESUCCENATE oR INVERTEDDEoxYABASICSUCCINATE 5 5 LYCSRY SUCNATSNKAB INVERTED DEAicsuccNATENKA US 2008/01 6 1256 A1 Page 2 Related U.S. Application Data tion No. 60/440,129, filed on Jan. 15, 2003, provi sional application No. 60/440,129, filed on Jan. 15, (60) 60/406.784, filed on Aug. 29, 2002, provisional appli 2003, provisional application No. 60/292,217, filed on cation No. 60/406.784, filed on Aug. 29, 2002, provi May 18, 2001, provisional application No. 60/362, sional application No. 60/408,378, filed on Sep. 5, 016, filed on Mar. 6, 2002, provisional application No. 2002, provisional application No. 60/408,378, filed on 60/306,883, filed on Jul. 20, 2001, provisional appli Sep. 5, 2002, provisional application No. 60/409,293, cation No. 60/311,865, filed on Aug. 13, 2001, provi filed on Sep. 9, 2002, provisional application No. sional application No. 60/543,480, filed on Feb. 10, 60/409.293, filed on Sep. 9, 2002, provisional applica 2004. Patent Application Publication Jul. 3, 2008 Sheet 1 of 57 US 2008/O161256 A1 Figure I (1) FIRST STRAND (2) SECOND STRAND DEPROTECTION PURIFICATION (DETRITYLATION) siRNA DUPLEX = SOLIDSUPPORT R = TERMINAL PROTECTING GROUP FOR EXAMPLE: DIMETHOXYTRITYL (DMT) wan - CLEAVABLE LINKER (FOR EXAMPLE: NUCLEOTIDE SUCCINATE OR (2) INVERTED DEOXYABASICSUCCINATE) VVVVVV = CLEAVABLE LINKER (FOR EXAMPLE: NUCLEOTIDE SUCCINATE OR INVERTED DEOXYABASICSUCCINATE) 5 Ö O N-no- 3' OAC O INVERTED DEOXYABASIC SUCCENATE GLYCERYL SUCCINATE LINKAGE LINKAGE Patent Application Publication Jul. 3, 2008 Sheet 2 of 57 US 2008/O161256 A1 Figure 2 O S646.36 SO 7 O S850.04 O 45 3 4520 839 vastn, Patent Application Publication Jul. 3, 2008 Sheet 3 of 57 US 2008/O161256 A1 çmm’TÉHOVNRIsp =No. ||| ||-11111||1||||1||[[: vNHsÎ Xº[dnpVNCHISTITITIIT d'HPHKq £9.InáH Patent Application Publication Jul. 3, 2008 Sheet 4 of 57 US 2008/0161256 A1 Figure 4 SENSE STRAND (SEQID NO 8) ALLPOSITIONS RIBONUCLEOTIDE EXCEPT POSITIONS (NN) 5'- B-NNNN N N N N N N N N N N N N N N N (NN)-B -3' 3'- L-(NN) NNNNNNNNNNNNNNNNNNN -5' ANTISENSESTRAND (SEQID NO 9) ALLPOSITIONS RIBONUCLEOTIDE EXCEPT POSITIONS (NN) SENSE STRAND (SEQID NO 10) ALLPYRIMIDINES = 2'-F or OCF3 AND ALL PURINES = 2'-OMe EXCEPT POSITIONS (NN) 5'- N N N N N N N N N N N N N N N N N N N (NN) -3' 3'- L-(NN) NNNNNNNNNNNNNNNNNNN -5' ANTISENSESTRAND (SEQID NO 11) ALLPYRIMIDINES = 2'-F or OCF3 AND ALL PURINES = 2'-O-Me EXCEPT POSITIONS (NN) SENSE STRAND (SEQD NO 12) ALLPYRIMIDINES = 2'-F or OCF3 EXCEPT POSITIONS (NN) C B-N N N N N N N N N N N N N N N N N N N (NN)-B -3' 3'- L-(NN) NNNNNNNNNNNNNNNNNNN -5' ANTISENSESTRAND (SEQID NO 13) ALLPYRIMIDINES = 2'-F or OCF3 EXCEPT POSItIONS (NN) SENSE STRAND (SEQ ID NO 14) ALLPYRIMIDINES = 2'-F or OCF3 EXCEPT POSITIONS (NN) AND ALL PURINES = 2'-DEOXY 5'- B-NNNNNNNNNNNNNNNNNNN (NN)-B -3' 3'- L-(NN) NNNNNNNNNNNNNNNNNNN -5' ANTISENSESTRAND (SEQID NO 1) ALL PYRIMIDINES = 2'-F or OCF3 AND ALL PURINES = 2'-O-ME EXCEPT POSITIONS (NN) SENSE STRAND (SEQID NO 12) ALLPYRIMIDINES = 2'-F or OCF3 EXCEPT POSITIONS (NN) 5'- B-N N N N N N N N N N N N N N N N N N N (NN)-B -3' 3'- L-(NN) NNNNNNNNNNNNNNN NNNN -5' ANTISENSE STRAND (SEQID NO 11) ALLPYRIMIDINES = 2'-F or OCF3 AND ALL PURINES = 2'-O-Me EXCEPT POSITIONS (NN) SENSE STRAND (SEQID NO 14) ALL PYRIMIDINES = 2'-F or OCF3 EXCEPT POSITIONS (NN) AND ALL PURINES = 2'-DEOXY 5'- B-NNNNNNNNNNNNNNNNNNN (NN)-B -3' F L-(NN) NNNNNNNNNNNNNNNNNNN -5' ANTISENSE STRAND (SEQ ID NO 15) ALLPYRIMIDINES = 2'-F or OCF3 EXCEPT POSITIONS (NN) AND ALL PURINES = 2'-DEOXY POSITIONS (NN) CAN COMPRISE ANYNUCLEOTIDE, SUCH AS DEOXYNUCLEOTIDES (eg. THYMIDINE) OR UNIVERSAL BASES B = ABASIC, INVERTED ABASIC, INVERTED NUCLEOTIDE OR OTHER TERMINAL CAP THAT IS OPTIONALLY PRESENT L = GLYCERYL or B THAT IS OPTIONALLY PRESENT S = PHOSPHOROTHIOATE ORPHOSPHORODITHIOATE that is optionally absent Patent Application Publication Jul. 3, 2008 Sheet 5 of 57 US 2008/0161256A1 Figure 5 SENSE STRAND (SEQID NO 16) 5'- B- G GAGUAUGAUU C U A U U AU A TT-B -3' A 3'- L-TsTC CUC AUA CUAA GAU AAU AU -5' ANTISENSE STRAND (SEQID NO 17) SENSE STRAND (SEQID NO 18) 5'- g gag u au ga u u cu au u a u a TsT -3' B 3'- L-TsTc cu c a u a cu aaga u aau au -5' ANTISENSE STRAND (SEQID NO 19) SENSESTRAND (SEQID NO 20) 5'- B-G G A Gu Au GA u u cu Au u Au A TT-B -3' 3'- L-TST cc u c Au A cu A A GA u A A u Au -5" ANTISENSE STRAND (SEQDD NO 21) SENSE STRAND (SEQID NO 22) D 5'- B- G GA Gu Au GA u u cu A u u Au A TT-B -3' 3'- L-TsTc cuca u a cu aa gaua a u a u -5 ANTISENSESTRAND (SEQ ID NO 19) SENSE STRAND (SEQID NO 20) 5'- B- G G A Gu Au GA u u cu Au u Au ATT-B -3' E 3'- L-TsTc cu c a u a cua agaua a u a u -5 ANTISENSE STRAND (SEQID NO 19) SENSE STRAND (SEQDD NO 22) 5'- B- G GA Gu A u GA u u cu A u u Au A TT-B -3' F 3'- L-TsT cc u cAu A cu A A GA u A A u Au -5" ANTISENSE STRAND (SEQID NO 23) italic lower case = 2'-deoxy-2'-fluoro or 2'-OCF, B. SEFEEEEEEE T underline = 2'-O-methyl L = SSES6YCERYL MOIETY PRESENT or B OPTIONALLY PRESENT ITALIC UPPER CASE = DEOXY 5566.65 PHOSPHORODITHIOATE OPTIONALLY PRESENT Patent Application Publication Jul. 3, 2008 Sheet 9 of 57 US 2008/O161256 A1 •ZCINVISOEIWIXZN? x*xD-z-II–-T SHOVAVEITO?NOIJLOTRIJLS?T?HILLAA XXDOE?IJNNNNNNNNNNNNNNNNNNNNN[TRIJ-,SVX NOISNEIJLXEI-,8? x*xE-ZIJNNNNNNNNNNNNNNNNNNNNNITT-II-c asoo{} []|:NAJ?OWNOXIAVNCHUS90>{{{LOVNORICHVNTRILIS9 §2am31.I.