US 2005O153914A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2005/0153914A1 McSwiggen et al. (43) Pub. Date: Jul. 14, 2005 (54) RNA INTERFERENCE MEDIATED (60) Provisional application No. 60/413,714, filed on Sep. INHIBITION OF MDR P-GLYCOPROTEIN 26, 2002. Provisional application No. 60/358,580, GENE EXPRESSION USING SHORT filed on Feb. 20, 2002. Provisional application No. INTERFERING NUCLEIC ACID (SINA) 60/363,124, filed on Mar. 11, 2002. Provisional appli cation No. 60/386,782, filed on Jun. 6, 2002. Provi (75) Inventors: James McSwiggen, Boulder, CO (US); sional application No. 60/406,784, filed on Aug. 29, Leonid Beigelman, Longmont, CO 2002. Provisional application No. 60/408,378, filed (US); James Thompson, Lafayette, CO on Sep. 5, 2002. Provisional application No. 60/409, (US) 293, filed on Sep. 9, 2002. Provisional application Correspondence Address: No. 60/440,129, filed on Jan. 15, 2003. Provisional MCDONNELL BOEHNEN HULBERT & application No. 60/358,580, filed on Feb. 20, 2002. BERGHOFF LLP Provisional application No. 60/363,124, filed on Mar. 300 S. WACKER DRIVE 11, 2002. Provisional application No. 60/386,782, 32ND FLOOR filed on Jun. 6, 2002. Provisional application No. CHICAGO, IL 60606 (US) 60/406,784, filed on Aug. 29, 2002. Provisional appli cation No. 60/408,378, filed on Sep. 5, 2002. Provi (73) Assignee: Sirna Therapeutics, Inc., Boulder, CO sional application No. 60/409,293, filed on Sep. 9, 2002. Provisional application No. 60/440,129, filed (21) Appl. No.: 10/918,969 on Jan. 15, 2003. Provisional application No. 60/292, 217, filed on May 18, 2001. Provisional application (22) Filed: Aug. 16, 2004 No. 60/362,016, filed on Mar. 6, 2002. Provisional Related U.S. Application Data application No. 60/306,883, filed on Jul. 20, 2001. Provisional application No. 60/311,865, filed on Aug. (63) Continuation-in-part of application No. PCT/US03/ 13, 2001. Provisional application No. 60/543,480, 04250, filed on Feb. 13, 2003. filed on Feb. 10, 2004. Continuation-in-part of application No. PCT/US04/ 16390, filed on May 24, 2004, which is a continua Publication Classification tion-in-part of application No. 10/826,966, filed on Apr. 16, 2004, which is a continuation-in-part of (51) Int. Cl." ........................... A61K 48/00; CO7H 21/02 application No. 10/757.803, filed on Jan. 14, 2004, (52) U.S. Cl. ............................................. 514/44; 536/23.1 which is a continuation-in-part of application No. 10/720,448, filed on Nov. 24, 2003, which is a con tinuation-in-part of application No. 10/693,059, filed (57) ABSTRACT on Oct. 23, 2003, which is a continuation-in-part of application No. 10/444,853, filed on May 23, 2003, which is a continuation-in-part of application No. This invention relates to compounds, compositions, and PCT/US03/05346, filed on Feb. 20, 2003, and which methods useful for modulating MDR P-glycoprotein (MDR) is a continuation-in-part of application No. PCT/ gene expression using short interfering nucleic acid (SiNA) US03/05028, filed on Feb. 20, 2003. molecules. This invention also relates to compounds, com Continuation-in-part of application No. PCT/US04/ positions, and methods useful for modulating the expression 13456, filed on Apr. 30, 2004, which is a continua and activity of other genes involved in pathways of MDR tion-in-part of application No. 10/780,447, filed on gene expression and/or activity by RNA interference (RNAi) Feb. 13, 2004, which is a continuation-in-part of using Small nucleic acid molecules. In particular, the instant application No. 10/427,160, filed on Apr. 30, 2003, invention features Small nucleic acid molecules, Such as which is a continuation-in-part of application No. short interfering nucleic acid (siNA), short interfering RNA PCT/US02/15876, filed on May 17, 2002. (siRNA), double-stranded RNA (dsRNA), micro-RNA Continuation-in-part of application No. 10/727,780, (miRNA), and short hairpin RNA (shRNA) molecules and filed on Dec. 3, 2003. methods used to modulate the expression of MDR genes. Patent Application Publication Jul. 14, 2005 Sheet 1 of 24 US 2005/0153914 A1 Figure I (1) FIRST STRAND (2) SECOND STRAND iiii iliili II liiii iliili III-O-R DEPROTECTION III.iiii Iia III III III ill-O-R PURIFICATION (DETRITYLATION) SiRNA DUPLEX O = SOLID SUPPORT R = TERMINAL PROTECTING GROUP FOR EXAMPLE: DIMETHOXYTRITYL (DMT) wim1 = CLEAVABLE LINKER (FOR EXAMPLE: NUCLEOTIDE SUCCINATE OR (2) INVERTED DEOXYABASIC SUCCINATE) VVVVVV = CLEAVABLE LINKER (FOR EXAMPLE: NUCLEOTIDE SUCCINATE OR INVERTED DEOXYABASIC SUCCINATE) d O N-no-no 3' OAC O INVERTED DEOXYABASIC SUCCINATE GLYCERYL SUCCINATE LINKAGE LINKAGE Patent Application Publication Jul. 14, 2005 Sheet 2 of 24 US 2005/0153914 A1 Figure 2 OD BB46.96 8) 7 6850.04 4 OO 3 O Null... BSD 452O 839D Mass (m/z) Patent Application Publication Jul. 14, 2005 Sheet 3 of 24 US 2005/0153914 A1 ||| |||||=|||||| g_IIIIIIIIT.HOVNRIspIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII ••••••••-X9|dnpVNYIISTTTTTTTT RIGHOICIÎ QAQOV?Xº[duuooVNDHIS VNYHISÎ SISQU?UKSVNYI· ?THpYIKq ?l?????????????HHHH?íTALITT),mae €9.InáH Patent Application Publication Jul. 14, 2005 Sheet 4 of 24 US 2005/0153914 A1 Figure 4 SENSE STRAND (SEQ ID NO 545) ALL POSITIONS RIBONUCLEOTIDE EXCEPT POSITIONS (NN) A 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) N N N N N N N N N N N N N N N N N N N -5" ANTISENSESTRAND (SEQID NO 546) ALL POSITIONS RIBONUCLEOTIDE EXCEPT POSITIONS (NN) SENSE STRAND (SEQ ID NO 547) ALL PYRIMIDINES = 2'-FLUORO 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 (NSN) B 3'- L-(NN) NN N N N N N N N N N N N N N N N NN al ANTISENSE STRAND (SEQ ID NO 548) ALL PYRIMIDINES = 2'-FLUORO AND ALL PURINES=2'-OME EXCEPT POSITIONS (NN) SENSE STRAND (SEQ ID NO 549) ALL PYRIMIDINES = 2'-O-ME OR 2'-FLUORO 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) N N N N N N N N N N N N N N N N N N N -5' ANTISENSE STRAND (SEQ ID NO 550) ALLPYRIMIDINES = 2'-FLUORO EXCEPT POSITIONS (NN) SENSE STRAND (SEQ ID NO 551) ALLPYRIMIDINES = 2'-FLUORO EXCEPT POSITIONS (NN) AND ALL PURINES = 2'-DEOXY D 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) N N N N N N N N N N N N N N N N N N N -5 ANTISENSE STRAND (SEQ ID NO 548) ALL PYRIMIDINES = 2'-FLUORO AND ALL PURINES = 2'-O-ME EXCEPT POSITIONS (NN) SENSESTRAND (SEQ ID NO 552) ALLPYRIMIDINES = 2'-FLUORO 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' E 3'- L-(NN) N N N N N N N N N N N N N N N N N N N -5' ANTISENSESTRAND (SEQ ID NO 548) ALLPYRIMIDINES = 2'-FLUORO AND ALL PURINES = 2'-O-ME EXCEPT POSITIONS (NN) SENSE STRAND (SEQ ID NO 55) ALL PYRIMIDINES - 2'-FLUORO EXCEPT POSITIONS (NN) AND ALL PURINES = 2'-DEOXY F 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) N N N N N N N N N N N N N N N N N N N -5' ANTISENSE STRAND (SEQID NO 553) ALLPYRIMIDINES = 2'-FLUORO EXCEPT POSITIONS (NN) AND ALL PURINES = 2'-DEOXY POSITIONS (NN) CAN COMPRISE ANY NUCLEOTIDE, SUCH AS DEOXYNUCLEOTIDES (eg. THYMIDINE) OR UNIVERSAL BASES B = ABASIC, INVERTED ABASIC, INVERTED NUCLEOTIDE OR OTHER TERMINAL CAP THAT IS OPTIONALLY PRESENT L = GLYCERYL MOIETY OR BTHAT IS OPTIONALLY PRESENT S = PHOSPHOROTHIOATE ORPHOSPHORODITHIOATE THAT IS OPTIONALLY PRESENT Patent Application Publication Jul. 14, 2005 Sheet 5 of 24 US 2005/0153914 A1 Figure 5 SENSE STRAND (SEQID NO 554) A 5'- B-GA CUGU C A G CUG CUG UC U G T T-B -3' 3'- L-TT CUGA CAGU C G A CGACA GAC -5 ANTISENSE STRAND (SEQ ID NO 555) SENSE STRAND (SEQID NO 556) 5'- gac u gu cag cu g c u gu cu GTST -3' B 3'- L-TsTc u ga cagu ciga c g a Caga C -5' ANTISENSE STRAND (SEQID NO 557) SENSE STRAND (SEQ ID NO 558) 5'- B-GAC u Gu C A G c u G Cu Gu c u GTT-B -3' C 3'- L-TsT c u G A c A Gu c G A c G A c AGA c -5' ANTISENSE STRAND (SEQ ID NO 559) SENSE STRAND (SEQID NO 560) D 5'- B-GA cu Gu C A G Cu Gc u Gu cu GTT-B -3' 3'- L-TsTc u ga ca gut c g a cg a cag a c -5 ANTISENSE STRAND (SEQ ID NO 557) SENSE STRAND (SEQ ID NO 561) 5'- B-GAcu Gu c AG cu Gc u Gu cu GTT-B -3' E 3'- L-TsTC u ga ca gu ciga ciga caga C -5' ANTISENSE STRAND (SEQID NO 557) SENSE STRAND (SEQ ID NO 560) 5'- B-GA c u Gu c A G c u Gc u Gu c u GTT-B -3' F 3'- L-TST cu GA c AG u c GA c GA cA GA c -5' ANTISENSE STRAND (SEQ ID NO 562) lower case - 2'-O-Methyl or 2'-deoxy-2'-fluoro L = GLYCERYL MOIETY OR B OPTIONALLY PRESENT italica lower case =U 2'-deoxy-2'-fluoro S = PHOSPHOROTHIOATEPHOSPHORODITHOATE OR OPTIONALLY PRESENT underline = 2'-O-methyl B = ABASIC, INVERTED ABASIC, INVERTED m NUCLEOTIDE OR OTHER TERMINAL CAP ITALIC UPPER CASE = DEOXY THAT IS OPTIONALLY PRESENT Patent Application Publication Jul.