111111111111111111111111111 11111 IR 11211JillER1111111111111111111111110111111
`
`(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2011/0112168 Al
`May 12, 2011
`Feinstein et al. (cid:9)
`(43) Pub. Date: (cid:9)
`
`(54) NOVEL SIRNA STRUCTURES
`
`Publication Classification
`
`(76) Inventors:
`
`Elena Feinstein, Rehovot (IL);
`Huseyin Aygun, Frankfurt am Main
`(DE); Rami Skaliter, Nes Ziona
`(IL); Hagar Kalinski,
`Rishon-Ie-Zion (IL); Igor Mett,
`Rehoyot (IL); James McSwiggen,
`Boulder, CO (US); Leonid
`Beigelman, San Mateo, CA (US)
`
`(21) Appl. No.:
`
`12/733,998
`
`(22) PCT Filed:
`
`Sep. 4, 2008
`
`(86) PCT No.:
`
`PCT/IL2008/001197
`
`§ 371 (c)(1),
`(2), (4) Date:
`
`Jan. 18, 2011
`
`Related U.S. Application Data
`
`(60) Provisional application No. 60/997,576, filed on Oct.
`3, 2007, provisional application No. 61/000,664, filed
`on Oct. 25, 2007, provisional application No. 61/003,
`535, filed on Nov. 15, 2007, provisional application
`No. 61/010,040, filed on Jan. 4, 2008, provisional
`application No. 61/128,519, filed on May 22, 2008,
`provisional application No. 61/134,638, filed on Jul.
`10, 2008, provisional application No. 61/189,035,
`filed on Aug. 15, 2008.
`
`(51)
`
`Int. Cl.
`A 61K 31/713
`CO7H 21/02
`A61P 27/16
`A61P 13/12
`A61P 27/06
`A61P 11/00
`A61P 25/00
`A61P 17/02
`A61P 9/00
`A61P 1/16
`
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`
`(52) U.S. Cl. (cid:9)
`
`
`
`514/44 A; 536/24.5
`
`(57)
`
`ABSTRACT
`
`The invention relates to siRNA compounds possessing novel
`sequences and structural motifs which down-regulate the
`expression of specific human genes. The invention also
`relates to pharmaceutical compositions comprising such
`compounds and a pharmaceutically acceptable carrier. The
`present invention also provides a method of treating and/or
`preventing the incidence or severity of various diseases or
`conditions associated with the genes and/or symptoms asso-
`ciated with such diseases or conditions comprising adminis-
`tering to a subject in need of treatment for such disease or
`condition and/or symptom the compound or the pharmaceu-
`tical composition in a therapeutically effective dose so as to
`thereby treat the subject.
`
`i
`
`Alnylam Exh. 1017
`
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`

`

`Patent Application Publication (cid:9) May 12, 2011 Sheet 1 of 74 (cid:9)
`
`US 2011/0112168 Al
`
`#1
`
`#2
`
`#3
`
`#4
`
`#5
`
`#6
`
`51. (cid:9)
`As1.
`
`51. (cid:9)
`As2.
`
`51. (cid:9)
`As3.
`
`S2. (cid:9)
`As1.
`
`S2. (cid:9)
`As2.
`
`S2. (cid:9)
`As3.
`
`#7
`
`#8
`
`#9
`
`#1 C
`
`S3. (cid:9)
`As1. (cid:9)
`
`S3. (cid:9)
`As2. (cid:9)
`
`S3. (cid:9)
`As3. (cid:9)
`
`S4. (cid:9)
`As1. (cid:9)
`
`S4. (cid:9)
`#1'
`As2. (cid:9)
`
`#12 S4. (cid:9)
`As3. (cid:9)
`
`Figure lA
`
`5'-gaagaaaa555ccgcaaaa-3'
`5'-u5uugcggaaauuuucuuc-3'
`
`5'-gaagaaaa555ccgcaaaa-3'
`5'-uuuugcggaaauuu57557-3'
`
`51-gaagaaaa555ccgcaaaa-3'
`5'-55uugcggaaauuuucuuc-3'
`
`5'-86686aaauuuccgcaaaa-3'
`5'-u5uugcggaaauuuucuuc-3'
`
`5'-86686aaauuuccgcaaaa-3'
`5'-uuuugcggaaauuu57557-3'
`
`5'-86686aaauuuccgcaaaa-3'
`5'-55uugcggaaauuuucuuc-3'
`
`5'-8668666655uccgcaaaa-3'
`5'-u5uugcggaaauuuucuuc-3'
`
`5'-8668666655uccgcaaaa-3'
`5'-uuuugcggaaauuu57557-3'
`
`5'-8668666655uccgcaaaa-3'
`5'-55uugcggaaauuuucuuc-3'
`
`5'-8668666655577gcaaaa-3'
`5'-u5uugcggaaauuuucuuc-3'
`
`5'-8668666655577gcaaaa-3'
`5'-uuuugcggaaauuu57557-3'
`
`5'-8668666655577gcaaaa-3'
`5'-55uugcggaaauuuucuuc-3'
`
`ii
`
`

`

`Patent Application Publication (cid:9) May 12, 2011 Sheet 2 of 74 (cid:9)
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`US 2011/0112168 Al
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`Figure 1B
`
`100% mouse serum (hr)
`
`100% rat serum (hr) (cid:9)
`
`100% human serum (hr)
`
`ds ss 0 0.5 1 3 6 16 24 (cid:9)
`
`ds ss 0 0.5 1 3 6 16 24 (cid:9)
`
`ds ss 0 0.5 1 3 6 16 24
`
`#4 •
`
`#5
`
`#6
`
`#13
`
`C
`
`Un
`
`1411101091
`
`iii
`
`(cid:9)
`

`

`Patent Application Publication (cid:9) May 12, 2011 Sheet 3 of 74 (cid:9)
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`US 2011/0112168 Al
`
`Figure 2
`
`Duplex Strand Sequence (cid:9) (5' (cid:9)
`
`-> (cid:9) 3')
`
`4
`
`5
`
`6
`
`8
`
`11
`
`2
`
`SEN
`AS
`
`SEN
`
`AS
`
`SEN
`AS
`
`SEN
`
`AS
`
`SEN
`
`AS
`
`SEN
`
`AS
`
`5' GAAGAAGAAAAUUUCCGCAAAAA 3'
`5' UUUUUGCGGAAAUUUUCUUCUUC 3'
`5' GAAGAAGAAAAUUUCCGCAAAAA 3'
`5' UUUUUGCGGAAAUUUUCUUCUUC 3'
`5' GAAGAAGAAAAUUUCCGCAAAAA 3'
`5' UUUUUGCGGAAAUUUUCUUCUUC 3'
`5' GAAGAAGAAAAUUUCCGCAAAAA 3'
`5' UUUUUGCGGAAAUUUUCUUCUUC 3'
`5' GAAGAAGAAAAUUUCCGCAAAAA 3'
`5' UUUUUGCGGAAAUUUUCUUCUUC 3'
`5' GAAGAAGAAAAUUUCCGCAAAAA 3'
`5' UUUUUGCGGAAAUUUUCUUCUUC 3'
`
`iv
`
`

`

`Patent Application Publication (cid:9) May 12, 2011 Sheet 4 of 74 (cid:9)
`
`US 2011/0112168 Al
`
`Figure 3A
`
`4+19
`
`3+19+1
`
`2+19+2
`
`1+19+3
`
`19+4
`
`Sense
`AS
`Sense
`
`AS
`
`Sense
`
`AS.
`
`Sense
`
`AS
`
`Sense
`
`AS,
`
`5 ' - CCUUGCACUCCUGAAUUUUAUCA - 3 '
`5 1 - UGAUAAAAUUCAGGAGUGCAAGG - 3 '
`5 ' - CUUGCACUCCUGAAUUUUAUCAA - 3 '
`5 ' - UUGAUAAAAUUCAGGAGUGCAAG - 3 '
`7UUGCACUCCUGAAUUUUAUCAAA - 3 '
`' ..:UUTIGAUAAAAUUCAGGAGUGCAA - 3 '
`5 ' - UGCACUCCUGAAUUUUAUCAAAC - 3 '
`5'-GUUUGAUAAAAUUCAGGAGUGCA-3'
`5HGCACUCQUGAAUUUUAUCAAACA-3
`5 - UGUUUGAUAAAAUUCAGGAGUGC -.2 !
`
`Figure 3B
`
`Ctrl GFPs' CASP2_1 4+9 3+19+1 (cid:9)
`
`1+19+3
`
`v
`
`

`

`Patent Application Publication (cid:9) May 12, 2011 Sheet 5 of 74 (cid:9)
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`US 2011/0112168 Al
`
`Figure 4A
`
`4+19
`
`3+19+1
`
`Sense
`
`AS
`
`Sense
`
`AS
`
`2+19+2
`
`Sense
`
`1+19+3
`
`19+4
`
`AS
`
`Sense
`
`AS
`
`Sense
`
`AS
`
`5 ' - CCUUGCACUCCUGAALTUUUAUCA - 3 '
`5 ' -UGAUAAAAUUCAGGAGUGCAAGg- 3 '
`5 ' - CUUGCACUCCUGAAUULTUAUCAA- 3 '
`5 ' -UUGAUAAAAUUCAGGAGUGCAAG - 3 '
`5 ' -UTJGCACUCCUGAAUUULTA.UCAAA- 3 '
`5 ' -UUUGAUAAAAUUCAGGAGUGCAA- 3 '
`5 ' -UGCACUCCUGAAUUUUAUCAAAC - 3 '
`5 ' - GUUUGAUAAAAUUCAGGAGUGCA- 3 '
`5 1 - GCACUCCUGAAUUUUAUCAAACA- 3 '
`5 ' -UGUUUGAUAAAAUTJCAGGAGUGC - '
`
`Figure 4B
`
`Ctrl (cid:9)
`
`GFPsi CASP2_1 (cid:9)
`
`3+19+1 2+19+2 1+19+3 19+4
`
`vi
`
`

`

`Patent Application Publication (cid:9) May 12, 2011 Sheet 6 of 74 (cid:9)
`
`US 2011/0112168 Al
`
`Figure 5A
`
`4+19
`
`Sense
`
`AS
`
`3+19+1
`
`Sense
`
`AS
`
`2+19+2
`
`Sense
`
`AS
`
`1+19+3
`
`Sense
`
`AS
`
`19+4
`
`Sense
`
`AS
`
`5 ' - CCUUGCACUCCUGAAUUUUAUCA- 3 '
`5 ' - UgAUAAAAUUCAGGAGUGCAAGG- 3 '
`5 ' - CUUGCACUCCUGAAUUUUAUCAA- 3 '
`5 ' - UUGAUAAAAUUCAGGAGUGCAAG - 3 '
`5'-UUGCACUCCUGAAUUUUAUCAAA-3'
`5 ' -UUUGAUAAAAUUCAGGAGUGCAA- 3 '
`5 1 -UGCACUCCUGAAUUUUAUCAAAC - 3 '
`5 ' - GUUUGAUAAAALTUCAGGAGUGCA- 3 '
`5' - GCACUCCUGAAUUUUAUCAAACA- 3
`5 ' - UGUUUGAUAAAAUUCAGGAGUGC - 3 '
`
`Figure 5B
`
`Ctrl GFPs' CASF2_1 4+9 3+19+1 2+19+2 1+19+3 19+4
`
`vii
`
`

`

`Patent Application Publication (cid:9) May 12, 2011 Sheet 7 of 74 (cid:9)
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`US 2011/0112168 Al
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`Figure 6A
`
`9- 9
`
`# l.
`
`#2
`
`3primelQM5-s GAAGAAAAUUUCCGCA666-dT
`
`3primelQM5-as UUUUGCGGAAAUUUUC557-dT
`
`3prime2QM5-s 866GAAAAUUUCCGCAAAA-dT
`
`3prime1QM5-as UUUUGCGGAAAUUUUC557-dT
`
`#3
`
`3prime3QM5-s 866GAAAAUUUCCGCA666-dT
`3prime1QM5-as UUUUGCGGAAAUUUUC557-dT
`
`#4
`
`#5
`
`QM5-s 5' (cid:9) GAAGAAAAUUUCGCGCAAA 3'
`3prime1QM5-as UUUUGCGGAAAUUUUC557-dT
`
`3primelQM5-s GAAGAAAAUUUCCGCA666-dT
`
`3prime2QM5-as 555UGCGGAAAUUUUCUUC-dT
`
`9
`3prime2QM5-s 866GAAAAUUUCCGCAAAA-dT
`
`3prime2QM5-as 555UGCGGAAAUUUUCUUC-dT
`
`3prime3QM5-s 866GAAAAUUUCCGCA666-dT
`
`3prime2QM5-as 555UGCGGAAAUUUUCUUC-dr
`
`#6
`
`#7
`
`QM5-s 5' (cid:9) GAAGAAAAUUUCGCGCAAA 3'
`3prime2QM5-as 555UGCGGAAAUUUUCUUC-dT
`
`#8
`
`#9
`
`#10
`
`3primelQM5-s GAAGAAAAUUUCCGCA666-dT
`
`3prime3QM5-as 555UGCGGAAAUUUUC557-dT
`
`3primelQM5-s GAAGAAAAUUUCCGCA666-dT
`
`QM5-as 5' UUUUGCGGAAAUUUUCUUC 3'
`
`viii
`
`

`

`Patent Application Publication (cid:9) May 12, 2011 Sheet 8 of 74 (cid:9)
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`US 2011/0112168 Al
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`Figure 6A cont.
`
`3prime2QM5-s 866GAAAAUUUCCGCAAAA-dT
`
`3prime3QM5-as 555UGCGGAAAUUUUC5574FT
`
`3prime2QM5-s 866GAAAAUUUCCGCAAAA-dT
`
`112
`
`QMS-as 5' UUUUGCGGAAAUUUUCUUC 3'
`
`3prime3QM5-s 866GAAAAUUUCCGCA666-dT
`3prime3QM5-as 555UGCGGAAAUUUUC557-dT
`
`11'3
`
`114
`
`115
`
`QM5-s 5' GAAGAAAAUUUCGCGCAAA 3'
`
`3prime3QM5-as 555UGCGGAAAUUUUC557-dr
`
`3prime3QM5-s 866GAAAAUUUCCGCA666-d7
`
`QM5-as 5' UUUUGCGGAAAUUUUCUUC 3'
`
`Figure 6B
`
`100% mouse serum (hr) (cid:9)
`ds ss 0 0.5 1 3 6 16 24 (cid:9)
`
`100% rat serum (hr) (cid:9)
`ds ss 0 0.5 1 3 6 16 24 (cid:9)
`
`100% human serum (hr)
`ds ss 0 0.5 1 3 6 16
`
`#1
`
`#2
`
`#3
`
`C
`
`Un
`
`ix
`
`

`

`Patent Application Publication (cid:9) May 12, 2011 Sheet 9 of 74 (cid:9)
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`US 2011/0112168 Al
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`Figure 7A
`
`S-5'- GCCAGAAUGUGGAACUC*C*U*dT-3'
`AS-5'- AGGAGUUCCACAUUCUG*G*C*dT-3'
`
`S-5'- G*C*C*AGAAUGUGGAACUCCUdT-3'
`AS-5'- AGGAGUUCCACAUUCUG*G*C*dT-3'
`
`S-5'- GCCAGAAUGUGGAACUC*C*U*-3'
`AS-5'- AGGAGUUCCACAUUCUG*G*C-3'
`
`S-5'- G*C*C*AGAAUGUGGAACUCCU-3'
`AS-5'- AGGAGUUCCACAUUCUG*G*C-3'
`
`#1
`
`#2
`
`#3
`
`#4
`
`1 CASP2_4 S/
`1CASP2_4 AS
`
`2 CASP2_4 S/
`1CASP24 AS
`
`1 CASP2_4 S/
`1CASP2_4 AS
`(BLUNT)
`2 CASP2_4 S/
`1CASP2_4 AS
`(BLUNT)
`
`Figure 7B
`
`20nM p5nM
`
`10 8
`I —
`
`17
`
`11
`
`23
`
`20
`
`22
`
`16
`
`Go*
`
`c1'sP2
`
`4
` Gp1/4s04' Gpssor2. Gp.sys" G02#4
`
`x
`
`

`

`Patent Application Publication (cid:9) May 12, 2011 Sheet 10 of 74 (cid:9)
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`US 2011/0112168 Al
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`Figure 8A
`
`S-5'- UACUGUAGCAUGAAACA*A*A*dT-3'
`AS-5'- UUUGUUUCAUGCUACAG*U*A*dT-3'
`
`S-5'- U*A*C*UGUAGCAUGAAACAAAdT-3'
`AS-5'- UUUGUUUCAUGCUACAG*U*A*dT-3'
`
`S-5'- UACUGUAGCAUGAAACA*A*A-3'
`AS-5'- UUUGUUUCAUGCUACAG*U*A-3'
`
`S-5'- U*A*C*UGUAGCAUGAAACAAA-3'
`AS-5'- UUUGUUUCAUGCUACAG*U*A-3'
`
`Figure 8B
`
`S-5'- GAAGAAAAUUUCCGCAA*A*A*dT-3'
`AS-5'- UULTUGCGGAAAUUUUCU*U*C*dT-3'
`
`S-5'- G*A*A*GAAAAUUUCCGCAAAAdT-3'
`AS-5'- UUUUGCGGAAAUUUUCU*U*C*dT-3'
`
`S-5'- GAAGAAAAUUUCCGCAA*A*A-3'
`AS-5'- UUUUGCGGAAAUUUUCU*U*C-3'
`
`S-5'- G*A*A*GAAAAUUUCCGCAAAA-3'
`AS-5'- UUUUGCGGAAAUUUUCU*U*C-3'
`
`#1
`
`#2
`
`#3
`
`#4
`
`#1
`
`#2
`
`#3
`
`#4
`
`1 DDIT4_2 S/
`1 DDIT4_2 AS
`
`2 DDIT4_2 S/
`1 DDIT42 AS
`
`1 DDIT4_2 S/
`1 DDIT4_2 AS
` (BLUNT)
`2 DDIT4_2 S/
`1 DDIT4_2 AS
`(BLUNT)
`
`1 QM5 S/
`1 QM5 AS
`
`2 QM5 S/
`1 QM5 AS
`
`1 QM5 S/
`1 QM5 AS
`(BLUNT)
`2 QM5 S/
`1 QM5 AS
`(BLUNT)
`
`xi
`
`(cid:9)
`

`

`Patent Application Publication (cid:9) May 12, 2011 Sheet 11 of 74 (cid:9)
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`US 2011/0112168 Al
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`Figure 9A
`
`#3-dT
`
`# 3-Blunt
`
`#4-dT
`
`# 4-Blunt
`
`#5-dT
`
`# 5-Blunt
`
`#6-dT
`
`# 6-Blunt
`
`#7-dT
`
`# 7-Blunt
`
`#8-dT
`
`# 8-Blunt
`
`S - 5 ' - GAAGAAAAUUUCCGC AAAA. dT - 3 '
`AS - 5 ' - UUUUGCGGAAAUUUUCU *U*C*dT - 3 '
`S - 5 ' - GAAGAAAAUUUCCGC AAAA - 3 '
`AS - 5 ' - UUUUGCGGAAAUUUUCU *U*C - 3 '
`S - 5 ' - GAAGAAAAU UUCCGCAA *A*A* dT - 3 '
`AS - 5 ' - UUUUGCGGAAAUUUUCU *U*C*dT - 3 '
`S - 5 ' - GAAGAAAAU UUCCGCAA *A*A- 3 '
`AS - 5 ' - UUUUGCGGAAAUUUUCU *U*C - 3 '
`S - 5 - GAAGAAAAU UUCCGCAA *A*A* dT - 3 '
`AS - 5 - UUUUGCGGAAAUUUUCU *U*C*d.T - 3 '
`S - 5 ' -GAAGAAAAU UUCCGCAA *A*A- 3 '
`AS - 5 ' -UUUUGCGGAAAUUUUCU * U * C - 3 '
`S - 5 ' - G*A*A*GAAAAUUUCCGCAA *A*A*dT - 3
`AS - 5 ' - UUUUGCGGAAA.UUUUC UUC dT - 3 '
`S - 5 ' - G*A*A*GAAAAUUUCCGCAA *A*A- 3 '
`AS - 5 ' - UUUUGCGGAAAUUUUC UUC - 3 '
`S - 5 - G*A*A*GAAAAUUUCCGCAA *A*A*dT - 3 '
`AS - 5 ' -FaUUGCGGAAAUUUUC !JUG dT - 3 '
`S - 5 ' - G*A*A*GAAAAUUUCCGCAA *A*A- 3 '
`AS - 5 ' - UUUUGCGGAAAUUUUC UUC - 3 '
`S - 5 ' - G*A*A*GAAAAU UUCCGCAA *A*A*dT - 3 '
`AS - 5 - UUUUGCGGAAAUUUUC UUC dT - 3 '
`S - 5 ' - G*A*A*GAAAAU UUCCGCAA *A*A- 3 '
`AS - 5 ' - UUUUGCGGAAAUUUUC UUC - 3 '
`
`xii
`
`

`

`Patent Application Publication (cid:9) May 12, 2011 Sheet 12 of 74 (cid:9)
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`US 2011/0112168 Al
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`Figure 9B
`
`#9-dT
`
`# 9-Blunt
`
`#10 -dT
`
`# 10-Blunt
`
`#11 -dT
`
`# 11-Blunt
`
`#12-dT
`
`# 12-Blunt
`
`#13 -dT
`
`# 13-Blunt
`
`S - 5 ' - GAAGAAAAU UUCCGCAA *A*A*dT - 3 '
`AS - 5 ' - UUUUGCGGAAAUUUUC (cid:9) UUC dT - 3 '
`S - 5 ' - GAAGAAAAU UUCCGCAA *A*A - 3 '
`AS - 5 ' - UUUUGCGGAAAUUUUC (cid:9) UUC - 3 '
`*A*A* d.T - 3 '
`' - GAAGAAAAUUUCCGCAA (cid:9)
`S - (cid:9)
`AS - 5 -1113UUGCGGAAAUUURCUUCdT - 3 '
`*A*A- 3 '
`S - 5 ' - GAAGAAAAUUUCCGCAA (cid:9)
`AS - 5 ' - UUUUGCaGAAAUUUUCUUC - 3 '
`S - 5 - G*A*A*GAAAAU UUCCGCAAAAdT - 3 '
`'
`AS - 5 - UUUUGCGGAAAUUUUCUUCdT - (cid:9)
`S - 5 ' - G*A*A*GAAAAU UUCCGCAAAA - 3 '
`AS - 5 ' - UUUUaCGGAAAUUUaCUUC - 3 '
`S - 5 - GAAGAAAAUUUCCGCA (cid:9) AAA dT'- 3 '
`AS - 5 - UUUUGCGGAAAUUUUCUUCdT - 3 '
`S - 5 ' - GAAGAAAAUUUCCGCA (cid:9) AAA - 3 '
`AS - 5 ' - UUUUGCGGAAAUUUUCUUC - 3 '
`- 3 '
`S - 5 ' - GAA GAAAAUUUCCGCAAAAdT (cid:9)
`AS - 5 - UUULTaCaGAAAUUUaCRUedT - 3 '
`- 3 '
`S - 5 ' - GAA GAAAAUUUCCGCAAAA (cid:9)
`AS - 5 ' - U.UUUaCGGAAAUTJUUCUUC - 3 '
`
`xiii
`
`

`

`Patent Application Publication (cid:9) May 12, 2011 Sheet 13 of 74 (cid:9)
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`US 2011/0112168 Al
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`Figure 9C
`
`0
`
`C
`
`CN
`
`1=1
`
`r--
`
`ix)
`co
`
`0 0
`
`o_ u_
`
`V.
`
`O
`
`
`
`O
`CD
`
`CD
`1.0
`
`xiv
`
`

`

`Patent Application Publication (cid:9) May 12, 2011 Sheet 14 of 74 (cid:9)
`
`US 2011/0112168 Al
`
`Figure 9D
`
`L.C), D
`
`CO
`
`CO
`
`-1=1
`CO
`
`CN1
`
`xv
`
`

`

`Patent Application Publication (cid:9) May 12, 2011 Sheet 15 of 74 (cid:9)
`
`US 2011/0112168 Al
`
`1,0
`
`+4-
`
`Th
`
`U)
`
`„.1
`
`OW ID:60605
`
`0 LL
`
`N
`
`0
`
`0.
`0
`
`O
`
`Figure 9E
`
`20n 1V1 q 5nIVI
`
`CO
`
`O
`N
`
`O
`O
`
`xvi
`
`

`

`Patent Application Publication (cid:9) May 12, 2011 Sheet 16 of 74 (cid:9)
`
`US 2011/0112168 Al
`
`Figure 10A
`
`Dup.
`#
`1
`
`2
`
`3
`
`Sequence (cid:9)
`
`(5' (cid:9)
`
`-> (cid:9) 3')
`
`Stra
`n d
`SEN 5' GAAGAAGAAAAUUUCCGCAA*A *A *A 3'
`5' UUUUUGCGGAAAUUUUCUUC*U *U * C 3'
`AS
`SEN 5' G*A*A*GAAGAAAAUUUCCGCAAAAA 3'
`5' UUUUUGCGGAAAUUUUCUUC*U * U * C 3'
`AS
`SEN 5' G*A*A*GAAGAAAAUUUCCGCAA *A *A *A 3'
`5' UUUUUGCGGAAAUUUUCUUC*U*U * C 3'
`AS
`lalt SEN 5' GAAGAAGAAAAUUUCCGCAA*A *A*A 3"
`5' UUUUUGCGGAAAUUUUCUUCUUC 3'
`AS
`5' G*A*A*GAAGAAAAUUUCCGCAAAAA 3'
`2alt SEN
`5' UUUUUGCGGAAAUUUUCUUCUUC 3'
`AS
`3alt SEN 5' G*A*A*GAAGAAAAUUUCCGCAA *A *A *A 3'
`5' UUUUUGCGGAAAUUUUCUUCUUC 3'
`AS
`
`Figure 10B
`
`120
`
`100
`
`100
`
`98
`
`20nM
`q 5nM
`
`18
`14
`
`1 2
`
`43
`
`21
`
`5 (cid:9)
`
`7 8
`.Wr7r1
`
`10
`
`6 10 (cid:9)
`IFT-1
`
`t...co (cid:9)
`
`Pte'
`
`co 4
`
`xvii
`
`

`

`Patent Application Publication (cid:9) May 12, 2011 Sheet 17 of 74 (cid:9)
`
`US 2011/0112168 Al
`
`Figure 10C
`
`S - 5 ' - GAAGAAAAUUUCCGCAA *A*A- 3 '
`AS - 5 ' - UUUUGCGGAAAUUUUCUUC - 3 '
`incubation time in human
`
`NR NR Si
`ds 0 0.5h lh 3h 6h 811 10h 24h
`ss (cid:9)
`cis (cid:9)
`
`Figure 10D
`
`Sense
`
`AS
`
`5 ' - G*A*A*GAAGAAAAUUUCCGCAAAAA - 3 '
`
`5 ' - UUUUUGCGgAAAUUUUaUUCUUC - 3 '
`
`Incubation time in human
`
`N N S
`d 0 0.5 1 3 6 8 10 24
`d
`
`xviii
`
`(cid:9)
`

`

`Patent Application Publication (cid:9) May 12, 2011 Sheet 18 of 74 (cid:9)
`
`US 2011/0112168 Al
`
`Figure 11A
`
`4+19
`
`3+19+1
`
`2+19+2
`
`1+19+3
`
`19+4
`
`Sense
`
`AS
`
`Sense
`
`AS
`
`Sense
`
`AS
`
`Sense
`
`AS
`
`Sense
`
`AS
`
`5 ' - C*C*U*UGCACUCCUGAAUUUUAUCA3 '
`5 ' -UGAUAAAAUUCAGGAGUGCAAGG- 3 '
`5 ' - C*U*U*GCACUCCUGAAUUTJUAUCA- 3 '
`5 ' -UUGAUAAAAUUCAGGAGUGCAAG- 3 '
`5 ' -U*U*G*CACUCCUGAAUUUUAUCAA- 3 '
`5 1 -UUUGAUAAAAUUCAGGAGUGCAA- 3 '
`5 ' - U*G* C*ACUC CUGAAUUUUAUCAAC- 3 '
`5 ' -GUUUGAUAAAAUUCAGGAGUGCA- 3 '
`5 ' - G* C *A* CUCCUGAATJUUUAUCAACA- 3 '
`-UGUUUGACJAAAAITUCAGGAGUGC- 3 '
`
`Figure 11B
`
`Ctrl GFPsi CASP2_1 4+9 3+19+1 2+19+2 1+19+3 19+4
`
`xix
`
`

`

`Patent Application Publication (cid:9) May 12, 2011 Sheet 19 of 74 (cid:9)
`
`US 2011/0112168 Al
`
`Figure 12A
`
`4+19
`
`Sense
`
`AS
`
`3+19+1
`
`Sense
`
`AS
`
`2+19+2
`
`Sense
`
`AS
`
`1+19+3
`
`Sense
`
`19+4
`
`AS
`
`Sense
`
`AS
`
`5' - C*C*U*UGCACUCCUGAAULTUUAUCA - 3 '
`5 ' -UGAUAAAAUUCAGGAGUGCAA.*G*G- 3 '
`5 ' - C*U*U*GCACUCCUGAAULTUUAUCAA- 3'
`5 ' -UUGAUAAA.AUUCAGGAGUGCA*A*G- 3 '
`- U*U* G* CACUCCUGAAUUUUAUCAAA - 3 '
`5 -ULTUGAUAAAAITUCAGGAGUGC *A*A.- 3 '
`5'-U*G*C*ACUCCUGAAUUUUAUCAAAC-3 '
`5'-GUUUGAUAAAAUUCAGGAGUG*C*A-3 '
`5 ' - G* C *A* CUCCTJGAAUULTUAUCAAACA - 3
`5 ' -UGUUUGAUAAAALTUCAGGAGU*G*C - 3 '
`
`Figure 12B
`
`Ctrl
`
`GFPsi CASP2_1 4+9 3+19+1 2+19+2 1+19+3 19+4
`
`xx
`
`(cid:9)
`

`

`Patent Application Publication (cid:9) May 12, 2011 Sheet 20 of 74 (cid:9)
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`US 2011/0112168 Al
`
`No
`
`1
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`8
`
`9
`
`10
`
`11
`
`12
`
`13
`
`14
`
`15
`
`Figure 13A
`
`siRNA-duplex name
`
`Sp4QM5-S/Sp4QM5-AS
`
`Sp5QM5-S/Sp4QM5-AS
`
`Sp6QM5-S/Sp4QM5-AS
`
`QM5-S/Sp4QM5-AS
`
`Sp4QM5-S/Sp5QM5-AS
`
`Sp5QM5-S/Sp5QM5-AS
`
`Sp6QM5-S/Sp5QM5-AS
`
`QM5-S/Sp5QM5-AS
`
`Sp4QM5-S/Sp6QM5-AS
`
`Sp4QM5-S/QM5-AS
`
`Sp5QM5-S/Sp6QM5-AS
`
`Sp5QM5-S/QM5-AS
`
`Sp6QM5-S/Sp6QM5-AS
`
`QM5-S/Sp6QM5-AS
`
`Sp6QM5-S/QM5-AS
`
`Mirror nucleotide siRNA structures
`underlined characters =mirror nucs
`S-5'-GAAGAAAAUUUCCGCAAAA-dT- 3'
`AS-3'-dT-CUUCUUUUAAAGGCGITUUU- 5'
`S-5'-GAAGAAAAUUUCCGCAAAA-dT- 3'
`AS-3'-dT-CUUCUUUUAAAGGCGUUUU- 5'
`S-5'-GAAGAAAAUUUCCGCAAAA-dT- 3'
`AS-3'-dT-CUUCULTUUAAA.GGCGUUUU- 5'
`S-5'-GAAGAAAAUUUCCGCAAAA-dT- 3'
`AS-31-dT-CUUCUULUAAAGGCGUUUU- 5'
`S-51-GAAGAAAAULTUCCGCAAAA-dT- 3'
`AS-3'-dT-CUUCUUUUAAAGGCGUUUT- 5'
`S-51-GAAGAAAA1UUUCCGCAAAA-dT- 3'
`AS-3'-dT-CUUCUUUUAAAGGCGUUUT- 5'
`S-5'-GAAGAAAAUUUCCGCAAAA-dT- 3'
`AS-3'-dT-CUUCUUUUAAAGGCGUUUT- '
`S-5'-GAAGAAAAUUUCCGCAAAA-dT- 3'
`AS-3'-dT-CUUCUUUUAAAGGCGUUUT- 5'
`S-5'-GAAGAAAAUUUCCGCAAAA-dT- 3'
`AS-3'-dT-CUUCUUUTJAAAGGCGUUUT- 5'
`S-5'-GAAGAAAAUUUCCGCAAAA-dT- 3'
`AS-3'-dT-CUUCUUUUAAAGGCGUUUU- 5'
`
`S-5'-GAAGAAAAUUUCCGCAAAA-dT- 3'
`AS-3'-dT-CUUCUUUUAAAGGCGUUUT- 5'
`S-5'-GAAGAAAAUUUCCGCAAAA-dT- 3'
`AS-31-dT-CUUClUUUUAAAGGCGUUUU- 5'
`S-5'-GAAGAAAA1UUUCCGCAAAA-dT- 3'
`AS-3'-dT-CUUCUUUUAAAGGCGUUUT- 5'
`S-5'-GAAGAAAAUUUCCGCAAAA-dT- 3'
`AS-3'-dT-CUUCUU1UUAAAGGCGUUUT- 5'
`S-F-GAAGAAAAUUUCCGCAAAA-dT- 3'
`AS-3'-dT-CUUCUUUUAAAGGCGUUUU- 5'
`
`xxi
`
`

`

`Patent Application Publication (cid:9) May 12, 2011 Sheet 21 of 74 (cid:9)
`
`US 2011/0112168 Al
`
`Figure 13B
`
`z z
`
`cV
`0
`
`oa
`
`o (cid:9)
`
`kr3
`
`I=1
`
`P53 expression (% of Ctrl)
`
`P53 expression (% of Ctrl)
`
`xxii
`
`

`

`Patent Application Publication (cid:9) May 12, 2011 Sheet 22 of 74 (cid:9)
`
`US 2011/0112168 Al
`
`Figure 13C
`
`L-DNA structure
`S GAAGAAAAUUUCCGCA666-dT-3'
`AS 5'- UUUUGCGGAAAUUUUC557-dT-3'
`
`5: L-thymidine
`6: L-deoxyadenosine
`7: L-deoxycytidine
`NR NR
`s
`
`S
`d
`
`Incubation time in human serum
`
`0 0.5h 1h 3h 6h 811 10h 24h
`
`IC50 = 0.23 nM
`
`IC50 = 0.09 nM
`
`Figure 13D
`
`100
`
`GO
`
`GO.
`
`00•
`
`BIOmt
`
`100
`
`00
`
`GO
`
`40
`
`GO
`
`0 01
`
`0.10
`
`1.00
`
`Conc
`
`10.00
`
`100.00
`
`Con.
`
`UP Conc. WOW woos• 1/1000
`
`0,001
`
`0.010 (cid:9)
`
`0.100 (cid:9)
`
`1.000 (cid:9)
`
`10.000 (cid:9)
`
`100.000
`
`S-5' -
`AS - 3 ' -
`
`S-5' -XXXXXXXXXXXXXXXXXLL- 3 '
`AS-3' LXXXXXXXXXXXXXXXXXX
`
`m - 2'O-Me
`L -L-RNA
`
`xxiii
`
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`

`

`Patent Application Publication (cid:9) May 12, 2011 Sheet 23 of 74 (cid:9)
`
`US 2011/0112168 Al
`
`Figure 14A
`
`No
`
` LNA siRNA-duplex name
`
`1
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`8
`
`9
`
`LNA1QM5-S/LNA1QM5-AS
`
`LNA2QM5-S/LNA1QM5-AS
`
`LNA3QM5-S/LNA1QM5-AS
`
`QM5-S/LNA1QM5-AS
`
`LNA1QM5-S/LNA2QM5-AS
`
`LNA2QM5-S/LNA2QM5-AS
`
`LNA3QM5-S/LNA2QM5-AS
`
`QM5-S/LNA2QM5-AS
`
`LNA1QM5-S/LNA3QM5-AS
`
`10 LNA1QM5-S/QM5-AS
`
`11 LNA2QM5-S/LNA3QM5-AS
`
`12 LNA2QM5-S/QM5-AS
`
`13 LNA3QM5-S/LNA3QM5-AS
`
`14 QM5-S/LNA3QM5-AS
`
`15 LNA3QM5-S/QM5-AS
`
`16 QM5-S/QM5-AS
`(positive control)
`
`LNA siRNA structures
`underlined characters =LNA nucs
`S-5'-GAAGAAAAUUUCCGCAAAA-dT- 3'
`AS-3'-dT-CUUCUUUUAAAGGCGUUUU- 5'
`S-5'-GAAGAAAAUUUCCGCAAAA-dT- 3'
`AS-3'-dT-CUUCUUUUAAAGGCGUUUU- 5'
`S-51-GAAGAAAAUUUCCGCAAAA-dT- 3'
`AS-3'-dT-CUUCUUUUAAAGGCGUUUU- 5'
`S-5'-GAAGAAAAUUUCCGCA AAA-3'
`AS-3'-dT-CUUCUUUUAAAGGCGUUUU- 5'
`S-5t-GAAGAAAAUUUCCGCAAAA-dT- 3'
`AS-3'-dT-CUUCLTUUUAAAGGCGUUUU- 5'
`S-5'-GAAGAAAAUUUCCGCAAAA-dT- 3'
`AS-3'-dT-CUUCUUUUAAAGGCGUUUU- 5'
`S-V-GAAGAAAAUUUCCGCAAAA-dT- 3'
`AS-3'-dT-CUUCUUUUAAAGGCGUUUU- 5'
`S-5'-GAAGAAAAUUUCCGCAA AA-3'
`AS-3'-dT-CUUCUUUUAAAGGCGUUUU- 5'
`S-5'-GAAGAAAAUUUCCGCAAAA-dT- 3'
`AS-3'-dT-CUUCUUUUAAAGGCGUUUU- 5'
`S-5'-GAAGAAAAUUUCCGCAAAA-dT- 3'
`AS-3'-CUUCUUUUAAAGGCGUUUU- 5'
`S-5'-GAAGAAAAUUUCCGCAAAA-dT- 3'
`AS-3'-dT-CUUCUUUUAAAGGCGUUUU- 5'
`S-5'-GAAGAAAAUUUCCGCAAAA-dT- 3'
`AS-3'-CUUCUUUUAAAGGCGUUUU- 5'
`S-5'-GAAGAAAAUUUCCGCAAAA-dT- 3'
`AS-3'-dT-CUUCUUUUAAAGGCGUUUU- 5'
`S-5'-GAAGAAAAUUUCCGCAAAA-3'
`AS-3'-dT-CUUCUUUUAAAGGCGUUUU- 5'
`S-5'-GAAGAAAAUUUCCGCAAAA-dT- 3'
`AS-3'-CIJUCUUUUAAAGGCGUIJUU- 5'
`S-5'-GAAGAAAAUUUCCGCAAAA-3'
`AS-3'-CUUCUUUUAAAGGCGUUUU- 5'
`
`xxiv
`
`

`

`Patent Application Publication (cid:9) May 12, 2011 Sheet 24 of 74 (cid:9)
`
`US 2011/0112168 Al
`
`Figure 14B
`
`LU
`
`N
`
`Ql
`
`K
`
`LL
`
`O
`O
`Et
`
`C
`
`47
`
`CI
`
`P53 expression (% of Ctrl) (cid:9)
`
`O
`
`CN
`
`47 (cid:9)
`O
`P53 expression (% of Ctrl)
`
`O
`
`xxv
`
`

`

`Patent Application Publication (cid:9) May 12, 2011 Sheet 25 of 74 (cid:9)
`
`US 2011/0112168 Al
`
`Figure 15
`
`Tandem siRNA molecule
`agagcgaaaugaucuggaadTsdTuuagagaagaucuacguguuadTsdT3'
`5-5' (cid:9)
`5'
`A-3'dTsdTucucacucuacuagaccuudTsdT ucucuucuagaugcacaau (cid:9)
`
`Incubation time in human
`
`N N
`s Tande 0 0.5 1 3 6 8 10 24
`
`Tandem
`
`ds
`
`SS
`
`
`
`xxvi
`
`(cid:9)
`

`

`Patent Application Publication (cid:9) May 12, 2011 Sheet 26 of 74 (cid:9)
`
`US 2011/0112168 Al
`
`Figure 16A
`
`.
`rn
`H
`rd
`ta
`EA
`V
`'n 0I
`0I M
`O M I
`01 0
`O M
`01 0
`o 01
`M I 0
`I-1 0
` M
`1 ril 0 0)
`
`
`0 0I m I
`0
`M M
`01
`01
`
`I 0M
`M 0 I
`t DI
`m
`0I
`01 0
`. M 0 I
`M • Vito
`O
`isl
`vi.
`H
`05
`6
`H
`6
`c^
`H
`N
`H
`N
`al (cid:9)
`(cid:9) N
`al
`cc'
`E-1 (cid:9)
`
`eP 45 IS)
`rd (cid:9)
`V,
`. ca
`co
`6 66\ *
`Ln El (cid:9)
`V (cid:9)
`6
`I al
`(6\ (6\
`• ail
`‘`
`01 01
`o\
`u 01
`6 6
`M I 0
`e>\ 0 15\
`0) 01
`0\ e>\
`M I 0
`6 6
`O M I
`e\ 6\
`01 01
`m 0 I
`x5A\ tlk
`0 I al
`0 6
`u 01
`e\ 6\
`0 61\ 0\
`0 I M
`• 01
`0
`1 0
`u 01
`N ‘P
`0 I M
`45 1st
`O 01
`I M (cid:9)
`tA
`E-1 - (cid:9)
`0:5
`rd LI)
`ta (cid:9)
`Cr\
`H 3
`(A
`'744 N6
`tfl
`
`m
`
`xxvii
`
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`

`

`Patent Application Publication (cid:9) May 12, 2011 Sheet 27 of 74 (cid:9)
`
`US 2011/0112168 Al
`
`Figure 16B
`
`Incubation time in human
`
`N N
`
`K
`star 0 0.5 1 3 6 8 10 24
`
`ds
`
`SS
`
`xxviii
`
`(cid:9)
`

`

`Patent Application Publication (cid:9) May 12, 2011 Sheet 28 of 74 (cid:9)
`
`US 2011/0112168 Al
`
`Figure 17A
`
`duple
`x No
`
`siRNA-duplex
`name
`
`N = 2'Ome
`Bold underlined (cid:9)
`Small case italicized = 2'-
`5'bond plus 31 0me
`
`1 31 0me-1QM5-as/
`31 0me-1-QM5-s
`
`2 31 0me-1QM5-as/
`310me-2-QM5-s
`
`3 3'Ome-1QM5-as/
`QM5-s
`
`4 3'Ome-2-QM5-as/
`31 0me-1-QM5-s
`
`S-5I-GAAGAAAAUUUCCGCAaaA-3'
`AS-5' -UITUUGCGGAAAUUUUCuuC- 3'
`S-5,-gaaGAAAAUUUCCGCAAAA-3'
`AS-5'-UUUUGCGGAAAUUUUCuue- 3'
`S-5'- GAAGAAAAUUUCCGCAAAA- 3'
`AS-5'-UUUUGCGGAAAUUUUCuuC- 3'
`S-5,-GAAGAAAAUUUCCGCAaaA-3'
`AS-5,-uuuUGCGGAAAUUUUCUUC- 3'
`S-5'-gaaGAAAAUUUCCGCAAAA-31
`AS-5'-uuuUGCGGAAAUUUUCUUC- 3'
`S-5'- GAAGAAAAUUUCCGCAAAA- 3'
`AS-5,-uuuUGCGGAAAUUUUCUUC- 3'
`S-51 -GAAGAAAAUUUCCGCAaaA_-31
`
`AS-5'-UUUUGCGGAAAUUUUCUUC- 3'
`S-5'-gaaGAAAAUUUCCGCAAAA-3'
`AS-5,-UUUUGCGGAAAUUUUCUUC- 3'
`
`5 3'Ome-2-QM5Las/
`3'Ome-2-QM5-s
`
`6 3'Ome-2-QM5-as/
`QM5-s
`
`7 QM5as/
`31Qme-1-QM5-s
`8 QM5as/
`3'Ome-2-QM5-s
`
`Activity
`20nM- %
`target
`gene
`post KD
`
`35;17
`
`57
`
`100
`
`100
`
`100
`
`26
`(5nM) ;
`13
`16 (cid:9) ; (cid:9) 16
`
`xxix
`
`

`

`Patent Application Publication (cid:9) May 12, 2011 Sheet 29 of 74 (cid:9)
`
`US 2011/0112168 Al
`
`Figure 17B
`
`siRNA-
`duplex
`No
`
`siRNA-
`duplex
`name
`
`Italicized small case=
`P-Ethoxy-dN;
`N_= 2'-Ome
`
`1
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`8
`
`S-5'-GAAGAAAAUUUCCGCAaaa-dT-3'
`Y1QM5-as/
`Y1QM5-s
`AS-5'-UUUUGCGGAAAUUUUCuuc-dT-3'
`
`Activity at
`20nM-
`% target
`gene KD
`55 (5nM)
`
`S-51-gaaGAAAAUUUCCGCAAAA-dT-3,
`Y1QM5-as/
`Y2QM5-s
`AS-5'-UUUUGCGGAAAUUUUCuuc-dT-3'
`
`S-5'-GAAGAAAAUUUCCGCAAAA- 3'
`Y1QM5-as/
`QM5-s
`AS-5'-UUUUGCGGAAAUVUUCuuc-dT-3'
`
`S-5,-GAAGAAAAUUUCCGCAaaa-dT-3'
`Y2QM5-as/
`Y1QM5-s
`AS-5'-uuuUGCGGAAAUUUUCUUC-dT-3'
`
`S-51-gaaGAAAAUUUCCGCAAAA-dT-3'
`Y2QM5-as/
`Y2QM5-s
`AS-5'-uuuUGCGGAAAUUUUCUUC-dT-3,
`
`S-5,-GAAGAAAAUUUCCGCAAAA- 3'
`Y2QM5-as/
`QM5-s
`AS-5'-uuuUGCGGAAAUUUUCUUC-dT-3,
`
`100
`
`38
`
`87
`
`100
`
`100
`
`QM5-as/
`Y1QM5-s
`
`QM5-as/
`Y2QM5-s
`
`S-51-GAAGAAAAUUUCCGCAaaa-dT-3,
`
`27 (5nM)
`
`AS-3,-CUUCUUUUAAAGGCGUUUU- 5'
`
`S-51-gaaGAAAAUUUCCGCAAAA-dT-3,
`
`19
`
`AS-3'-CUUCUUUUAAAGGCGUUUU- 5'
`
`xxx
`
`

`

`Patent Application Publication (cid:9) May 12, 2011 Sheet 30 of 74 (cid:9)
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`Figure 17C
`
`siRN
`A-
`dupl
`ex
`No
`4
`
`siRNA-duplex
`name
`
`CASP2 _ 4 _ DS _1
`
`5
`
`CASP2 _ 4 _ DS _2
`
`Activity
`at 20nM-
`% target
`gene KD
`(residual)
`11 (cid:9) (8)
`
`10 (cid:9) (8)
`
`6
`
`11
`
`17
`
`3
`
`6
`
`10
`
`4
`
`8
`
`7
`
`6
`
`18
`
`17
`
`3'-
`end
`Pi
`
`ITALICS CAPITAL =
`2'-5'-nucs;
`n=LNA;
`N - 2, -Ome
`italics small - L-DNA
`y
`gccAGAAUGUGGAACUCCU
`AGGAGUUCCACAUUCUGGC y
`y
`gccAGAAUGUGGAACUCCU
`y
`AGGAGUUCCACAUUCUGGC
`GCCAGAAUGUGGAACUCCU
`y
`AGGAGUUCCACAUUCUGGC y
`
`GCCAGAAUGUGGAACUCCU y-2'
`AGGAGUUCCACAUUCUGGC
`y
`GCCAGAAUGUGGAACUCCU y-2'
`AGGAGUUCCACAUUCUGGC
`y
`y
`GCCAGAAUGUGGAACUCcu
`AGGAGUUCCACAUUCUGGC
`y
`GCCAGAAUGUGGAACUCCu
`y
`AGGAGUUCCACAUUCUGGC y
`GCCAGAAUGUGGAACUccu
`y
`AGGAGUUCCACAUUCUGGC
`y
`GCCAGAAUGUGGAACUCcu
`y
`AGGAGUUCCACAUUCUGGC y
`y
`GCCAGAAUGUGGAACUCCu
`AGGAGUUCCACAUUCUGGC
`y
`y
`GCCAGAAUGUGGAACUCcu
`AGGAGUUCCACAUUCUGGC y
`GCCAGAAUGUGGAACUCCU
`y
`AGGAGUUCCACAUUCUGGC y
`GCCAGAAUGUGGAACUccU
`y
`AGGAGUUCCACAUUCUGGC
`y
`GCCAGAAUGUGGAACUCCU
`y
`AGGAGUUCCACAUUCUGGC y
`
`xxxi
`
`4 CASP2 4 S-2-5-
`_ _
`Pi-3'/
`CASP2_4_AS-Pi
`CASP2_4_81/
`CASP2 _ 4 AS _alt
`_
`CASP2_4_S2/
`CASP2 _ 4 AS _alt
`CASP2_4_S5/
`CASP2 _ 4 _ AS _alt
`CASP2_4_S6/
`CASP2 _ 4 _ AS _alt
`CASP2_4_S7/
`CASP2 _ 4 _ AS _alt
`CASP2_4_S8/
`CASP2 _ 4 _ AS _alt
`CASP2_4_89/
`CASP2 _ 4 _ AS _alt
`10 CASP2_4_810/
`CASP2 _ 4 AS _alt
`11 CASP2_4_811/
`CASP2 _ 4 AS _alt
`12 CASP2_4_S12/
`CASP2 _ 4 _ AS _alt
`CASP2_4_S3/
`CASP2_4_AS_alt
`
`1
`
`2
`
`5
`
`6
`
`7
`
`8
`
`9
`
`3
`
`

`

`Patent Application Publication (cid:9) May 12, 2011 Sheet 31 of 74 (cid:9)
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`Figure 18
`
`Strand name
`CASP2 4-S-dn 1
`CASP2 4-S-dn2
`CASP2 4-S-dn3
`CASP2 4-S-dn4
`CASP2_4-S-dn5
`CASP2_4-S-dn6
`
`19-mer - Sense - (5'>3')
`GCCAGAAUGUG GAACTCCT
`GCCAGAAUGUGGAACTCCT
`GCCAGAAUGUGGAACTCCT
`GCCAGA AUGUGGAACUCCU
`GCCAGA AUGUGGAACTCCT
`GCCAG AAUGUGGAACTCCT
`
`Strand name
`CASP2 4-AS-dn 1
`CASP2 4-AS-dn2
`CASP2 4-AS-dn3
`CASP2 4-AS-dn4
`CASP2 4-AS-dn5
`CASP2_4-AS-dn6
`
`19-mer-Antisense (553')
`AGGAGTTC CACAUUCUGGC
`A GGA GTTCCACAUUCUGGC
`A GGA GT UCCACAUUCUGGC
`AGGAG UUCCACAUUCUGGC
`AGGAG UUCCACAUUCTGGC
`6g8a8u5c7a7a5u7u8g7
`
`xxxii
`
`

`

`Patent Application Publication (cid:9) May 12, 2011 Sheet 32 of 74 (cid:9)
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`Figure 19A
`
`Strand sequence 5'>3' Strand
`Sense
`GcCAGAAUGUGGAACUCcU
`AS
`AGGAGUUCCACAUUCUGGC
`
`GcCAGAAUGUGGAACUccU
`AGGAGUUCCACAUUCUGGC
`Legend
`n = L-DNA
`N - 2'-OMe ribonucleotide
`
`Sense
`AS
`
`Figure 19B
`(N'y) ' (cid:9) 19-mer (cid:9) (5'>3')
`Sense Strand (cid:9)
`GcCAGAAUGUGGAACUccU
`GccAGAAUGUGGAACUccU
`GcCAGAAUGUGGAACUCcU
`GccAGAAUGUGGAACUCcU
`Lc- GCCAGAAUGUGGAACUccU
`Lc- GCCAGAAUGUGGAACUccU
`Lc- GCCAGAAUGUGGAACUCcU
`Lc -GCCAGAAUGUGGAACUCcU
`ab-GCCAGAAUGUGGAACUccU
`ab-GCCAGAAUGUGGAACUccU
`ab-GCCAGAAUGUGGAACUCcU
`ab-GCCAGAAUGUGGAACUCcU
`(5 >3 1
`(N,) x; (cid:9) 19-mer (cid:9)
`AS Strand (cid:9)
`AGGAGUUCCACAUUCUGGC
`AGGAGUUCCACAUUCUGGC
`AGGAGUUCCACAUUCUGGC
`
`ILagend
`n = L-DNA (L-deoxyribocytidine)
`N - 2°-0Me ribonucleotide
`Lc- L-Deoxy cytidine 5'-overhang
`ab- Abasic 2'deoxyribo moiety
`xxxiii
`
`

`

`Patent Application Publication (cid:9) May 12, 2011 Sheet 33 of 74 (cid:9)
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`Figure 19C
`
`OLIGO NAME
`
`CASP2 4 S1 _ _
`CASP2 4_ S2
`_ _
`CASP2 4 S3
`CASP2 4 S4
`CASP2 _ 4 _S5
`CASP2 4 S6
`CASP2 4 S7
`CASP2 4 S8
`
`SEQUENCE (5' (cid:9) -> 3')
`5' GCCAGAAUGUGGAAdCUCcA
`5' GC CAGAAUGUGGAAdC cC cA
`5' I GCCAGAAUGUGGAAdCUC cA
`5' c GC CAGAAUGUGGAAdCUC cA
`5 ' C 6 - GC CAGAAUGUGGAAdCUC cA
`5' C 6 - GC CAGAAUGUGGAAdC cC cA
`5' GcCAGAAUGUGGAAdCUC cA
`5' G cCAGAAUGUGGAAdC cC cA
`
`CASP2 4 AS1
`CASP2 4 AS2
`
`5' AGGAGUUCCACAUUCUGGC
`5 ' AGGAGUUCCACAUUCUGGC
`
`n— = L-ENA (L-deoxyribonucleotide)
`N - 2'-OMe ribonucleotide
`dN- 2'deoxynucleotide
`I- INVERTED Abasic 2'deoxyribo moiety
`C6- C6 imino Pi
`
`Figure 19D
`
`NR NR
`ds (cid:9)
`ss ds 0 0.5 1 3 6 8 10 1 (cid:9)
`
`Human Serum hrs
`
`24
`
`S1/AS2
`
`S3/AS2
`
`S5/AS2
`
`S7/AS2
`
`1116* (cid:9)
`
`0* 08. (cid:9)
`
`41110.
`
`xxxiv
`
`(cid:9)
`

`

`Patent Application Publication (cid:9) May 12, 2011 Sheet 34 of 74 (cid:9)
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`DUPLEX motifs
`(5' (cid:9) > 3') (cid:9) SEN/AS
`
`Figure 19E
`3'end
`Pi
`
`Activity at 20nM
`% target gene KD
`(= residual target gene
`activity)
`
`GAAGGATCTTCGGAATGAT
`ATCATTCCGAAGATCCTTC
`GAAGGATCTTCGGAATGAT
`ATCATTCCGAAGATCCTTC
`GAAGGATCTTCGGAATG*A*T
`ATCATTCCGAAGATCCTTC
`GAAGGATCTTCGGAATG*A*T
`ATCATTCCGAAGATCCTTC
`GAAGGATCTTCGGAATG*A*T
`ATCATTCCGAAGATCCTTC
`GAAGGATCTTCGGAATGat
`ATCATTCCGAAGATCCTTC
`GAAGGATCTTCGGAATGat
`ATCATTCCGAAGATCCTTC
`GAAGGATCTTCGGAATGAT
`ATCATTCCGAAGATCCTTC
`GAAGGATCTTCGGAATGAT
`ATCATTCCGAAGATCCTTC
`GAAGGATCTTCGGAATG*A*T
`ATCATTCCGAAGATCCTTC
`GAAGGATCTTCGGAATG*A*T
`ATCATTCCGAAGATCCTTC
`GAAGGATCTTCGGAATG*A*T
`ATCATTCCGAAGATCCTTC
`GAAGGATCTTCGGAATGat
`ATCATTCCGAAGATCCTTC
`GAAGGATCTTCGGAATGat
`ATCATTCCGAAGATCCTTC
`
`Y
`
`Y
`N
`Y
`
`N
`Y
`
`Y
`Y
`
`Y 2'
`Y
`
`Y
`Y
`
`N
`Y
`
`Y
`N
`
`N
`N
`
`N
`N
`
`Y
`N
`
`Y 2'
`N
`
`Y
`N
`
`N
`N
`
`xxxv
`
`1 . 7
`
`2 . 1
`
`3 . 4
`
`3 . 2
`
`3 . 2
`
`2 . 2
`
`2 . 5
`
`3
`
`6
`
`8
`
`4
`
`5
`
`4
`
`4
`
`

`

`Patent Application Publication (cid:9) May 12, 2011 Sheet 35 of 74 (cid:9)
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`Figure 20
`
`N- 2'OMe ribonucleotide
`Underlined (cid:9)
`ab - abasic pseudonucleotide
`1 - inverted abasic pseudonucleotide
`Italicized 5N= 5-OMe DNA
`•• (cid:9) -
`- (cid:9)
`k (cid:9) -
`italicized small case underlined : L-DNA
`otrikcthrough (cid:9)
`mismatchco
`
`Figure 20A
`
`Seti-Sense (cid:9) strands(5'>3')
`Name
`CASP2 4 S1
`5'-GCCAGAAUGUGGAACUCcU (cid:9) (19)
`CASP2 4 S2
`5'-GCCAGAAUGUGGAACUccU (cid:9) (19)
`CASP2 4 S3
`5'-5G CCAGAAUGUGGAACUccU (19)
`CASP2 4 S4
`5'-5G CCAGAAUGUGGAACUCcU (19)
`CAS
`4 S5
`5'-I-GCCAGAAUGUGGAACUCcU (20)
`CASP2 4 S6
`5'-I-GCCAGAAUGUGGAACUccU (20)
`Name
`Seti- (cid:9) AS (cid:9) strands (cid:9) (5'-3')
`CASP2 4 AS1 5'- AGGAabUUCCACAUUCUGGC (19)
`CASP2 4 AS2 5'- AGGAGabUCCACAUUCUGGC (19)
`CASP2 4 AS3 5 , - AGGAGUUCCACAUUabUGGC (19)
`CASP2 4 AS4 5'- AGGAGUUCCACAUabCUGGC (19)
`CASP2 4 ASx 5'- AGGAGUUCCACAUUabUGGC (19)
`CASP2 4 ASy 5'- AGGAabUUCCACAUUCUGGC (19)
`
`xxxvi
`
`(cid:9)
`

`

`Patent Application Publication (cid:9) May 12, 2011 Sheet 36 of 74 (cid:9)
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`Figure 20B
`
`Sett-Sense (cid:9) strands(5'>3')
`Name
`CASP2 4 (cid:9) S7 5'-I CCAGAAUGUGGAACabababab (19)
`CASP2 4 S8 5'-5G CAGAAUGUGGAAababababab (19)
`CASP2 4 (cid:9) S9 5 , -I abCAGAAUGUGGAACUababab (19)
`CASP2 4 (cid:9) S1 5'-I ababAGAAUGUGGAACUCabab (19)
`CASP2 4 (cid:9) S1 5'-I abababGAAUGUGGAACUCCab (19)
`CASP2 4 (cid:9) S1 5'-I ababababAAUGUGGAACUCCU (19)
`CASP2 (cid:9) 4 (cid:9) S1 5'-5G CCAGMAUGUGGAAababababab(19)
`(19)
`5'-I ababababMIAUGUGGAMCUCCU
`CASP2_4_S1
`Sett- (cid:9) AS (cid:9) strands (cid:9) (5'-3')
`Name
`CASP2 4 AS 5'- AGGAGUUCCACAUUCUGGC (cid:9) (19)
`CASP2 4 AS 5'- AGGAGUUCCACAUUCUGGC (cid:9) (19)
`
`Figure 20C
`
`Set3-duplex sequences(5'>3')
`Name
`CASP2 4 S1 5'-I GCCAGAAUGUGGAAWUCCU (20)
`CASP2 4 AS 5'- AGGAPEUUCCACAUUCUGGC (19)
`
`CASP2 4 S1 5'-I GCCAGAAUGUGGAAUUccU (20)
`CASP2 4 AS 5'- AGGAMTUCCACAUUCUGGC (19)
`
`CASP2 4 S1 5'-I GCCAGAAUGUGGAGCUCcU (20)
`CASP2 4 AS 5'- AGGAGOUCCACAUUCUGGC (19)
`
`CASP2 4 S1 5'-I GCCAGAAUGUGGAGCUCCU (20)
`CASP2 4 AS 5'- AGGAGOUCCACAUUCUGGC (19)
`
`CASP2 4 S1 5'-I GCCAGGAUGUGGAACUCCU (20)
`CASP2 4 AS 5 , - AGGAGUUCCACAUUCUGGC (19)
`
`CASP2 4 S2 5'-I GCCAGGAUGUGGAACUCCU (20)
`CASP2 4 AS 5'- AGGAGUUCCACAUUCUGGC (19)
`
`xxxvii
`
`

`

`Patent Application Publication (cid:9) May 12, 2011 Sheet 37 of 74 (cid:9)
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`Figure 20D
`
`Oligo Name
`
`Set4- (cid:9) SEN strands (cid:9) (5'>3')
`
`CASP2 4 S1
`CASP2 4 S2
`CASP2 4 S3
`CASP2 4 S4
`CASP2 4 S5
`CASP2 4 S6
`
`5'-GCCAGAAUGUGGAACUCcU (19)
`5 , -GCCAGAAUGUGGAACUccU (19)
`5'-5G CCAGAAUGUGGAACUCcU (19)
`5'-5G CCAGAAUGUGGAACUccU (19)
`5'-I GCCAGAAUGUGGAACUCCU (20)
`5'-I GCCAGAAUGUGGAACUCCU (20)
`
`Oligo Name
`CASP2 4 AS10
`CASP2 _ 4 AS11
`CASP2 4 AS12
`CASP2 4 AS13
`CASP2 4 AS14
`CASP2 4 AS15
`CASP2 4 AS16
`CASP2 4 AS17
`
`Set4- (cid:9) AS (cid:9) strands (cid:9) (5'-3')
`5'- AGGAabUUCCACAUUCUGGC (cid:9) (19)
`5'- AGGAGbaUCCACAUUCUGGC (cid:9) (19)
`5'- AGGAGUUCCACAUUabUGGC (cid:9) (19)
`5'- AGGAGUUCCACAUabCUGGC (cid:9) (19)
`5'- AGGAabUUCCACAUUCUGGC (cid:9) (19)
`5'- AGGAabUUCCACAUUCUGGC (cid:9) (19)
`5'- AGGAGUUCCACAUUabUGGC (cid:9) (19)
`5'- AGGAGUUCCACAUUabUGGC (cid:9) (19)
`
`xxxviii
`
`

`

`Patent Application Publication (cid:9) May 12, 2011 Sheet 38 of 74 (cid:9)
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`Figure 20E
`
`Oligo Name
`
`Set5-Sense strands (5' >3 ' )
`Same sense strands as in SET1
`
`CASP2 4 Si 5 ' -GCCAGAAUGUGGAACUCcU (19)
`(19)
`CASP2 (cid:9) 4 (cid:9) S2 5 ' -GCCAGAAUGUGGAACUccU (cid:9)
`CASP2 4 S3 5'-5G CCAGAAUGUGGAACUCcU (19)
`CASP2 4 S4 5' -5G CCAGAAUGUGGAACUccU
`(19)
`(20)
`CASP2 4 S5 5 ' -1 GCCAGAAUGUGGAACUCcU
`CASP2 4 S6 5 ' -1 GCCAGAAUGUGGAACUccU (20)
`
`Oligo Name
`
`Set5- AS strands (cid:9) (5'-3')
`
`CASP2 4 AS18
`CASP2 4 AS19
`
`CASP2 4 AS20
`
`CASP2 4 AS21
`
`CASP2 4 AS22
`
`CASP2 4 AS23
`
`CASP2 4 AS24
`
`5 ' - AGGAGUabCCACAUUCUGGC
`5 ' - AGGAGLTUabCACAUUCUGGC (19)
`
`( 1 9 )
`
`5'- AGGAGUUCabACAUUCUGGC (19)
`5 ' - AGGAGUUCCabCAUUCUGGC (19)
`5 ' - AGGAGUUCCAabAUUCUGGC (19)
`5 ' - AGGAGUUCCACabUUCUGGC (19)
`5 ' - AGGAGUUCCACAabUCUGGC (19)
`
`xxxix
`
`

`

`Patent Application Publication (cid:9) May 12, 2011 Sheet 39 of 74 (cid:9)
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`Figure 20F
`
`Oligo Name
`CASP2 4 SO
`
`Set6-Sense (cid:9) strand(5'>3')
`5'-GCCAGAAUGUGGAACUCCU (19)
`
`Oligo Name
`Set6- (cid:9) AS (cid:9)