US20210147837A1 - Oligomeric Compounds Comprising Bicyclic Nucleotides and Uses Thereof - Google Patents

Abstract

The present invention provides oligomeric compounds. Certain such oligomeric compounds are useful for hybridizing to a complementary nucleic acid, including but not limited, to nucleic acids in a cell. In certain embodiments, hybridization results in modulation of the amount activity or expression of the target nucleic acid in a cell.

Description

SEQUENCE LISTING
• The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled CORE0094USC1SEQ_ST25.txt, created Jun. 5, 2018, which is 12 Kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION
• Antisense compounds have been used to modulate target nucleic acids. Antisense compounds comprising a variety of chemical modifications and motifs have been reported. In certain instances, such compounds are useful as research tools, diagnostic reagents, and as therapeutic agents. In certain instances antisense compounds have been shown to modulate protein expression by binding to a target messenger RNA (mRNA) encoding the protein. In certain instances, such binding of an antisense compound to its target mRNA results in cleavage of the mRNA. Antisense compounds that modulate processing of a pre-mRNA have also been reported. Such antisense compounds alter splicing, interfere with polyadenlyation or prevent formation of the 5′-cap of a pre-mRNA.
• Certain antisense compounds have been described previously. See for example U.S. Pat. No. 7,399,845 and published International Patent Application No. WO 2008/049085, which are hereby incorporated by reference herein in their entirety.
SUMMARY OF THE INVENTION
• In certain embodiments, the present invention provides compounds comprising oligonucleotides. In certain embodiments, such oligonucleotides comprise a gapmer region. In certain embodiments, such oligonucleotides consist of a gapmer region.
• The present disclosure provides the following non-limiting numbered embodiments:
• Embodiment 1: A compound comprising:
• a modified oligonucleotide consisting of 10 to 20 linked nucleosides, wherein the modified oligonucleotide comprises:
• a 5′-wing consisting of 2 to 5 linked nucleosides;
• a 3′-wing consisting of 2 to 5 linked nucleosides; and
• a gap between the 5′-wing and the 3′-wing consisting of 6 to 14 linked 2′-deoxynucleosides; and
• wherein at least one of the 5′-wing and the 3′-wing comprises at least one bicyclic nucleoside; at least one of the 5′-wing and the 3′-wing comprises at least one 2′-substituted nucleoside; and
• wherein the nucleobase sequence of the modified oligonucleotide is complementary to the nucleobase sequence of a target nucleic acid.
• Embodiment 2: The compound of embodiment 1, wherein one of the 5′-wing or the 3′-wing comprises at least one 2′-deoxynucleoside.
• Embodiment 3: The compound of embodiments 1-2, wherein each of the 5′-wing and the 3′-wing comprises at least one 2′-deoxynucleoside.
• Embodiment 4: The compound of embodiments 1-3, wherein the 3′-wing comprises at least one 2′-deoxynucleoside.
• Embodiment 5: The compound of embodiments 1-4, wherein the 5′-wing comprises at least one 2′-deoxynucleoside.
• Embodiment 6: The compound of any of embodiments 1-5, wherein the 5′-wing comprises at least one bicyclic nucleoside.
• Embodiment 7: The compound of any of embodiments 1-6, wherein the 3′-wing comprises at least one bicyclic nucleoside.
• Embodiment 8: The compound of any of embodiments 1-7, wherein the 5′-wing comprises at least one 2′-substituted nucleoside.
• Embodiment 9: The compound of any of embodiments 1-8, wherein the 3′-wing comprises at least one 2′-substituted nucleoside.
• Embodiment 10: A compound comprising:
• a modified oligonucleotide consisting of 10 to 20 linked nucleosides, wherein the modified oligonucleotide comprises:
• a 5′-wing consisting of 2 to 5 linked nucleosides;
• a 3′-wing of 2 to 5 linked nucleosides; and
• a gap between the 5′ wing and the 3′ wing consisting of 6 to 14 linked 2′-deoxynucleosides; and
• wherein at least one of the 5′-wing and the 3′-wing comprises at least one constrained ethyl nucleoside; and
• at least one of the 5′-wing and the 3′-wing comprises at least one 2′-substituted nucleoside; and
• wherein the nucleobase sequence of the modified oligonucleotide is complementary to the nucleobase sequence of a target nucleic acid.
• Embodiment 11: The compound of embodiments 1-10, wherein and at least one of the 5′-wing and the 3′-wing comprises at least one 2′-deoxynucleoside.
• Embodiment 12: The compound of embodiments 1-11, wherein at least one of the 5′-wing and the 3′-wing comprises both at least one constrained ethyl nucleoside and at least one 2′-substituted nucleoside.
• Embodiment 13: The compound of embodiments 1-12, wherein the 5′-wing comprises at least one constrained ethyl nucleoside.
• Embodiment 14: The compound of any of embodiments 10-13, wherein the 3′-wing comprises at least one constrained ethyl nucleoside.
• Embodiment 15: The compound of any of embodiments 10-14, wherein the 5′-wing comprises at least one 2′-substituted nucleoside.
• Embodiment 16: The compound of any of embodiments 10-15, wherein the 3′-wing comprises at least one 2′-substituted nucleoside.
• Embodiment 17: The compound of any of embodiments 1-17, wherein the modified oligonucleotide has a sugar motif described by Formula I as follows:
• 
  (A)_m-(B)_n-(J)_p-(B)_r-(J)_t-(D)_g-(J)_v-(B)_w-(J)_x-(B)_y-(A)_z
• wherein:
• • each A is independently a 2′-substituted nucleoside;
  • each B is independently a bicyclic nucleoside;
  • each J is independently either a 2′-substituted nucleoside or a 2′-deoxynucleoside;
  • each D is a 2′-deoxynucleoside;
  • m is 0-4; n is 0-2; p is 0-2; r is 0-2; t is 0-2; v is 0-2; w is 0-4; x is 0-2; y is 0-2; z is 0-4; and g is 6-14;
• provided that:
  • at least one of m, n, and r is other than 0;
  • at least one of w and y is other than 0;
  • the sum of m, n, p, r, and t is from 2 to 5; and
  • the sum of v, w, x, y, and z is from 2 to 5.
• Embodiment 18: A compound comprising:
• a modified oligonucleotide consisting of 10 to 20 linked nucleosides, wherein the modified oligonucleotide has a sugar motif described by Formula I as follows:
• 
  (A)_m-(B)_n-(J)_p-(B)_r-(J)_t-(D)_g-(J)_v-(B)_w-(J)_x-(B)_y-(A)_z
• wherein:
• • each A is independently a 2′-substituted nucleoside;
  • each B is independently a bicyclic nucleoside;
  • each J is independently either a 2′-substituted nucleoside or a 2′-deoxynucleoside;
  • each D is a 2′-deoxynucleoside;
  • m is 0-4; n is 0-2; p is 0-2; r is 0-2; t is 0-2; v is 0-2; w is 0-4; x is 0-2; y is 0-2; z is 0-4; and g is 6-14;
• provided that:
  • at least one of m, n, and r is other than 0;
  • at least one of w and y is other than 0;
  • the sum of m, n, p, r, and t is from 2 to 5; and
  • the sum of v, w, x, y, and z is from 2 to 5.
• Embodiment 19: The compound of embodiment 17 or 18, wherein at least one bicyclic nucleoside is a constrained ethyl nucleoside.
• Embodiment 20: The compound of embodiment 17 or 18, wherein each bicyclic nucleoside is a constrained ethyl nucleoside.
• Embodiment 21: The compound of any of embodiments 17-19, wherein at least one bicyclic nucleoside is an LNA nucleoside.
• Embodiment 22: The compound of embodiment 17 or 18, wherein each bicyclic nucleoside is an LNA nucleoside.
• Embodiment 23: The compound of any of embodiments 1-22, wherein the 2′-substituent of the at least one 2′-substituted nucleoside is selected from among: OCH₃, F, OCH₂F, OCHF₂, OCF₃, OCH₂CH₃, O(CH₂)₂F, OCH₂CHF₂, OCH₂CF₃, OCH₂—CH—CH₂, O(CH₂)₂—OCH₃, O(CH₂)₂—SCH₃, O(CH₂)₂—OCF₃, O(CH₂)₃—N(R₄)(R₅), O(CH₂)₂—ON(R₄)(R₅), O(CH₂)₂—O(CH₂)₂—N(R₄)(R₅), OCH₂C(═O)—N(R₄)(R₅), OCH₂C(═O)—N(R₆)—(CH₂)₂—N(R₄)(R₅) and O(CH₂)₂—N(R₆)—C(═NR₇)[N(R₄)(R₅)] wherein R₄, R₅, R₆and R₇are each, independently, H or C₁-C₆alkyl.
• Embodiment 24: The compound of embodiment 23, wherein the 2′-substituent of the at least one 2′-substituted nucleoside of is selected from among: OCH₃, F, and O(CH₂)₂—OCH₃.
• Embodiment 25: The compound of embodiment 24, wherein the 2′-substituent of the at least one 2′-substituted nucleoside is O(CH₂)₂—OCH₃.
• Embodiment 26: The compound of any of embodiments 1-22, wherein the 2′-substituent of each 2′-substituted nucleoside is selected from among: OCH₃, F, OCH₂F, OCHF₂, OCF₃, OCH₂CH₃, O(CH₂)₂F, OCH₂CHF₂, OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃, O(CH₂)₂—SCH₃, O(CH₂)₂—OCF₃, O(CH₂)₃—N(R₄)(R₅), O(CH₂)₂—ON(R₄)(R₅), O(CH₂)₂—O(CH₂)₂—N(R₄)(R₅), OCH₂C(═O)—N(R₄)(R₅), OCH₂C(═O)—N(R₆)—(CH₂)₂—N(R₄)(R₅) and O(CH₂)₂—N(R₆)—C(═NR₇)[N(R₄)(R₅)] wherein R₄, R₅, R₆and R₇are each, independently, H or C₁-C₆alkyl.
• Embodiment 27: The compound of embodiment 26, wherein the 2′-substituent of each 2′-substituted nucleoside of is selected from among: OCH₃, F, and O(CH₂)₂—OCH₃.
• Embodiment 28: The compound of embodiment 27, wherein the 2′-substituent of each 2′-substituted nucleoside is O(CH₂)₂—OCH₃.
• Embodiment 29: The compound of any of embodiments 1-28, wherein the 5′-wing does not comprise a bicyclic nucleotide.
• Embodiment 30: The compound of any of embodiments 1-29, wherein the 3′-wing does not comprise a bicyclic nucleotide.
• Embodiment 31: The compound of any of embodiments 1-30, wherein the target nucleic acid is not a Huntingtin gene transcript.
• Embodiment 32: The compound of any of embodiments 1-31, wherein the modified oligonucleotide has a base sequence other than:

• 	GTGCTACCCAACCTTTCTG;	(SEQ ID NO: 1)
  	CACAGTGCTACCCAACCTT;	(SEQ ID NO: 2)
  	CAGTGCTACCCAACC;	(SEQ ID NO: 3)
  	ATATCACAGTGCTACCCAA;	(SEQ ID NO: 4)
  	GATGCTGACTTGGGCCATT;	(SEQ ID NO: 5)
  	GGGATGCTGACTTGG;	(SEQ ID NO: 6)
  	TGCCAAGGGATGCTGACTT;	(SEQ ID NO: 7)
  	AATTGTCATCACCAGAAAA;	(SEQ ID NO: 8)
  	TAAATTGTCATCACC;	(SEQ ID NO: 9)
  	ACAGTAGATGAGGGAGCAG;	(SEQ ID NO: 10)
  	ACACAGTAGATGAGG;	(SEQ ID NO: 11)
  	AAGTGCACACAGTAGATGA;	(SEQ ID NO: 12)
  	AGCTGCAACCTGGCAACAA;	(SEQ ID NO: 13)
  	GCAGCTGCAACCTGG;	(SEQ ID NO: 14)
  	or
  	GCAAGAGCAGCTGCAACCT.	(SEQ ID NO: 15)

• Embodiment 33: The compound of any of embodiments 1-31, wherein the oligonucleotide has a sugar motif other than:

• 	E-K-K-(D)9-K-K-E;
  	E-E-E-E-K-(D)9-E-E-E-E-E;
  	E-K-K-K-(D)9-K-K-K-E;
  	K-E-E-K-(D)9-K-E-E-K;
  	K-D-D-K-(D)9-K-D-D-K;
  	K-E-K-E-K-(D)9-K-E-K-E-K;
  	K-D-K-D-K-(D)9-K-D-K-D-K;
  	E-K-E-K-(D)9-K-E-K-E;
  	E-E-E-E-E-K-(D)8-E-E-E-E-E;
  	or
  	E-K-E-K-E-(D)9-E-K-E-K-E;

  
  wherein
• K is a constrained ethyl nucleoside, E is a 2′-MOE substituted nucleoside, and D is a 2′-deoxynucleoside.
• Embodiment 34: The compound of any of embodiments 1-30, wherein the 5′-wing consists of 2 linked nucleosides.
• Embodiment 35: The compound of any of embodiments 1-30, wherein the 5′-wing consists of 3 linked nucleosides.
• Embodiment 36: The compound of any of embodiments 1-30, wherein the 5′-wing consists of 4 linked nucleosides.
• Embodiment 37: The compound of any of embodiments 1-30, wherein the 5′-wing consists of 5 linked nucleosides.
• Embodiment 38: The compound of any of embodiments 1-34, wherein the 3′-wing consists of 2 linked nucleosides.
• Embodiment 39: The compound of any of embodiments 1-34, wherein the 3′-wing consists of 3 linked nucleosides.
• Embodiment 40: The compound of any of embodiments 1-34, wherein the 3′-wing consists of 4 linked nucleosides.
• Embodiment 41: The compound of any of embodiments 1-34, wherein the 3′-wing consists of 5 linked nucleosides.
• Embodiment 42: The compound of any of embodiments 1-38, wherein the gap consists of 6 linked 2′-deoxynucleosides.
• Embodiment 43: The compound of any of embodiments 1-38, wherein the gap consists of 7 linked 2′-deoxynucleosides.
• Embodiment 44: The compound of any of embodiments 1-38, wherein the gap consists of 8 linked 2′-deoxynucleosides.
• Embodiment 45: The compound of any of embodiments 1-38, wherein the gap consists of 9 linked 2′-deoxynucleosides.
• Embodiment 46: The compound of any of embodiments 1-38, wherein the gap consists of 10 linked 2′-deoxynucleosides.
• Embodiment 47: The compound of any of embodiments 1-38, wherein the gap consists of 11 linked 2′-deoxynucleosides.
• Embodiment 48: The compound of any of embodiments 1-38, wherein the gap consists of 12 linked 2′-deoxynucleosides.
• Embodiment 49: The compound of any of embodiments 1-38, wherein the gap consists of 13 linked 2′-deoxynucleosides.
• Embodiment 50: The compound of any of embodiments 1-38, wherein the gap consists of 14 linked 2′-deoxynucleosides.
• Embodiment 51: The compound of any of embodiments 1-50, wherein the oligonucleotide consists of 10 linked nucleosides.
• Embodiment 52: The compound of any of embodiments 1-50, wherein the oligonucleotide consists of 11 linked nucleosides.
• Embodiment 53: The compound of any of embodiments 1-50, wherein the oligonucleotide consists of 12 linked nucleosides.
• Embodiment 54: The compound of any of embodiments 1-50, wherein the oligonucleotide consists of 13 linked nucleosides.
• Embodiment 55: The compound of any of embodiments 1-50, wherein the oligonucleotide consists of 14 linked nucleosides.
• Embodiment 56: The compound of any of embodiments 1-50, wherein the oligonucleotide consists of 15 linked nucleosides.
• Embodiment 57: The compound of any of embodiments 1-50, wherein the oligonucleotide consists of 16 linked nucleosides.
• Embodiment 58: The compound of any of embodiments 1-50, wherein the oligonucleotide consists of 17 linked nucleosides.
• Embodiment 59: The compound of any of embodiments 1-50, wherein the oligonucleotide consists of 18 linked nucleosides.
• Embodiment 60: The compound of any of embodiments 1-50, wherein the oligonucleotide consists of 19 linked nucleosides.
• Embodiment 61: The compound of any of embodiments 1-50, wherein the oligonucleotide consists of 20 linked nucleosides.
• Embodiment 62: The compound of any of embodiments 1-50, wherein the oligonucleotide consists of 21 linked nucleosides.
• Embodiment 63: The compound of any of embodiments 1-50, wherein the oligonucleotide consists of 22 linked nucleosides.
• Embodiment 64: The compound of any of embodiments 1-30, wherein the gapmer motif is selected from among: 2-10-2, 2-10-3, 2-10-4, 2-10-5, 3-10-2, 3-10-3, 3-10-4, 3-10-5, 4-10-2, 4-10-3, 4-10- 4, 4-10-5, 5-10-2, 5-10-3, 5-10-4, 5-10-5, 2-9-2, 2-9-3, 2-9-4, 2-9-5, 3-9-2, 3-9-3, 3-9-4, 3-9-5, 4-9-2, 4-9-3, 4-9-4, 4-9-5, 5-9-2, 5-9-3, 5-9-4, 5-9-5, 2-8-2, 2-8-3, 2-8-4, 2-8-5, 3-8-2, 3-8-3, 3-8-4, 3-8-5, 4-8-2, 4-8-3, 4-8-4, 4-8-5, 5-8-2, 5-8-3, 5-8-4, and 5-8-5.
• Embodiment 65: A compound comprising a modified oligonucleotide having a sugar motif selected from among sugar motifs 1-278 as shown in Table 4.
• Embodiment 66: The compound of any of embodiments 1-65, wherein the 5′-wing has a motif selected from among the 5′-wing motifs as shown in Tables 1-3.
• Embodiment 67: The compound of any of embodiments 1-66, wherein the 3′-wing has a motif selected from among the 3′-wing motifs as shown in Tables 4-6.
• Embodiment 68: The compound of any of embodiments 66-67, wherein each A, each B, and each C are independently selected from among: HNA and F-HNA.
• Embodiment 69: The compound of any of embodiments 1-68, wherein the 5′-wing comprises at least one F-HNA.
• Embodiment 70: The compound of any of embodiments 1-69, wherein the 3′-wing comprises at least one F-HNA.
• Embodiment 71: The compound of any of embodiments 1-68, wherein the 5′-wing comprises at least one modified nucleobase.
• Embodiment 72: The compound of any of embodiments 1-69, wherein the 3′-wing comprises at least one modified nucleobase.
• Embodiment 73: The compound of embodiment 72, wherein the modified nucleobase is 2-thio-thymidine.
• Embodiment 74: The compound of any of embodiments 1-73, wherein the 5′-wing has a motif selected from among the 5′-wing motifs as shown in Tables 1-3 and the 3′-wing has a motif selected from among the 3′-wing motifs as shown in Tables 4-6.
• Embodiment 75: The compound of any of embodiments 1-74, wherein the 5′-wing has an ABABA motif, wherein each A is a modified nucleoside and each B comprises a 2′-deoxynucleoside.
• Embodiment 76: The compound of embodiment 75, wherein the modified nucleoside is a bicyclic nucleoside.
• Embodiment 77: The compound of embodiment 76, wherein the bicyclic nucleoside is cEt.
• Embodiment 78: The compound of embodiment 76, wherein the bicyclic nucleoside is LNA.
• Embodiment 79: The compound of any of embodiments 75-78 wherein the 3′-wing has a motif selected from among: AA, AB, AC, BA, BB, BC, CA, CB, and CC.
• Embodiment 80: The compound of embodiment 79, wherein the 3′-wing has an AA motif.
• Embodiment 81: The compound of embodiment 80, wherein A is a 2′-substituted nucleoside.
• Embodiment 82: The compound of embodiment 80, wherein the 2′-substituted nucleoside is selected from among: OCH₃, F, OCH₂F, OCHF₂, OCF₃, OCH₂CH₃, O(CH₂)₂F, OCH₂CHF₂, OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃, O(CH₂)₂—SCH₃, O(CH₂)₂—OCF₃, O(CH₂)₃—N(R₄)(R₅), O(CH₂)₂—ON(R₄)(R₅), O(CH₂)₂—O(CH₂)₂—N(R₄)(R₅), OCH₂C(═O)—N(R₄)(R₅), OCH₂C(═O)—N(R₆)—(CH₂)₂—N(R₄)(R₅) and O(CH₂)₂—N(R₆)—C(═NR₇)[N(R₄)(R₅)] wherein R₄, R₅, R₆and R₇are each, independently, H or C₁-C₆alkyl.
• Embodiment 83: The compound of embodiment 82, wherein the 2′-substituent of each 2′-substituted nucleoside of is selected from among: OCH₃, F, and O(CH₂)₂—OCH₃.
• Embodiment 84: The compound of embodiment 83, wherein the 2′-substituent of each 2′-substituted nucleoside is O(CH₂)₂—OCH₃.
• Embodiment 85: The compound of any of embodiments 76-84 wherein the gap between the 5′-wing and the 3′-wing consists of 6 to 11 linked 2′-deoxynucleosides.
• Embodiment 86: The compound of any of embodiments 76-84 wherein the gap between the 5′-wing and the 3′-wing consists of 7 to 10 linked 2′-deoxynucleosides.
• Embodiment 87: The compound of any of embodiments 76-84 wherein the gap between the 5′-wing and the 3′-wing consists of 10 linked 2′-deoxynucleosides.
• Embodiment 88: The compound of any of embodiments 75-87 having the sugar motif: K-D-K-D-K-(D)₆-E-E.
• Embodiment 89: The compound of any of embodiments 75-87 having the sugar motif: K-D-K-D-K-(D)₇-E-E.
• Embodiment 90: The compound of any of embodiments 75-87 having the sugar motif: K-D-K-D-K-(D)₈-E-E.
• Embodiment 91: The compound of any of embodiments 75-87 having the sugar motif: K-D-K-D-K-(D)₉-E-E.
• Embodiment 92: The compound of any of embodiments 75-87 having the sugar motif: K-D-K-D-K-(D)₁₀-E-E.
• Embodiment 93: The compound of any of embodiments 75-87 having the sugar motif: K-D-K-D-K-(D)₁₁-E-E.
• Embodiment 94: The compound of any of embodiments 75-87 having the sugar motif: K-D-K-D-K-(D)₁₂-E-E.
• Embodiment 95: The compound of any of embodiments 75-87 having the sugar motif: K-D-K-D-K-(D)₁₃-E-E.
• Embodiment 96: The compound of any of embodiments 75-87 having the sugar motif: K-D-K-D-K-(D)₁₄-E-E.
• Embodiment 97: The compound of any of embodiments 75-87 having the sugar motif: K-D-K-D-K-(D)₁₅-E-E.
• Embodiment 98: The compound of any of embodiments 1-97, wherein the 5′-wing has a BDBDB motif, wherein each B is a bicyclic nucleoside and each D comprises a 2′-deoxynucleoside.
• Embodiment 99: The compound of any of embodiments 1-97, wherein the 5′-wing has a BDBDB-(D)_6-15-AA motif, wherein each B is a bicyclic nucleoside and each D comprises a 2′-deoxynucleoside.
• Embodiment 100: The compound of any of embodiments 98-99, wherein B is selected from among: BNA, LNA, α-L-LNA, ENA and 2′-thio LNA.
• Embodiment 101: The compound of embodiment 100, wherein B comprises BNA.
• Embodiment 102: The compound of embodiment 100, wherein B comprises LNA.
• Embodiment 103: The compound of embodiment 100, wherein B comprises α-L-LNA.
• Embodiment 104: The compound of embodiment 100, wherein B comprises ENA.
• Embodiment 105: The compound of embodiment 100, wherein B comprises 2′-thio LNA.
• Embodiment 106: The compound of any of embodiments 100 to 105, wherein A comprises a 2′ substituted nucleoside.
• Embodiment 107: The compound of claim106, wherein the 2′ substituted nucleoside comprises MOE.
• Embodiment 108: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: A-B-B-(D)₈-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 109: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: A-B-B-(D)₉-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 110: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: A-B-B-(D)₁₀-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 111: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: A-A-B-(D)₈-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 112: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: A-A-B-(D)₉-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 113: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: A-A-B-(D)₁₀-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 114: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: A-D-B-(D)₈-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 115: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: A-D-B-(D)₉-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 116: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: A-D-B-(D)₁₀-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 117: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: B-D-A-(D)₈-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 118: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: B-D-A-(D)₉-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 119: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: B-D-A-(D)₁₀-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 120: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: B-A-A-(D)₈-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 121: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: B-A-A-(D)₉-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 122: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: B-A-A-(D)₁₀-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 123: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: B-B-B-(D)₈-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 124: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: B-B-B-(D)₉-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 125: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: B-B-B-(D)₁₀-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 126: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: A-A-A-(D)₈-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 127: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: A-A-A-(D)₉-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 128: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: A-A-A-(D)₁₀-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 129: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: B-B-B-(D)₈-B-B-B, wherein each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 130: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: B-B-B-(D)₉-B-B-B, wherein each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 131: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: B-B-B-(D)₁₀-B-B-B, wherein each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 132: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: A-A-A-(D)₈-B-B-B, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 133: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: A-A-A-(D)₉-B-B-B, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 134: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: A-A-A-(D)₁₀-B-B-B, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 135: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: A-D-D-B-(D)₈-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 136: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: A-D-D-B-(D)₉-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 137: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: A-D-D-B-(D)₁₀-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 138: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: B-D-D-A-(D)₈-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 139: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: B-D-D-A-(D)₉-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 140: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: B-D-D-A-(D)₁₀-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 141: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: A-A-A-B-(D)₈-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 142: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: A-A-A-B-(D)₉-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 143: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: A-A-A-B-(D)₁₀-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 144: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: B-A-A-A-(D)₈-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 145: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: B-A-A-A-(D)₉-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 146: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: B-A-A-A-(D)₁₀-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 147: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: A-A-A-A-(D)₈-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 148: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: A-A-A-A-(D)₉-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 149: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: A-A-A-A-(D)₁₀-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 150: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: B-A-A-A-(D)₈-B-B-B, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 151: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: B-A-A-A-(D)₉-B-B-B, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 152: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: B-A-A-A-(D)₁₀-B-B-B, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 153: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: B-D-D-B-(D)₈-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 154: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: B-D-D-B-(D)₉-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 155: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: B-D-D-B-(D)₁₀-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 156: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: A-A-A-A-(D)₈-B-B-B, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 157: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: A-A-A-A-(D)₉-B-B-B, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 158: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: A-A-A-A-(D)₁₀-B-B-B, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 159: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: B-B-B-B-(D)₈-B-B-B, wherein each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 160: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: B-B-B-B-(D)₉-B-B-B, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 161: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: B-B-B-B-(D)₁₀-B-B-B, wherein each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 162: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: A-A-A-A-A-(D)₈-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 163: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: A-A-A-A-A-(D)₉-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 164: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: A-A-A-A-A-(D)₁₀-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 165: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: A-A-A-A-A-(D)₈-B-B-B, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 166: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: A-A-A-A-A-(D)₉-B-B-B, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 167: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: A-A-A-A-A-(D)₁₀-B-B-B, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 168: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: A-D-A-D-B-(D)₈-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 169: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: A-D-A-D-B-(D)₉-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 170: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: A-D-A-D-B-(D)₁₀-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 171: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: A-D-B-D-A-(D)₈-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 172: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: A-D-B-D-A-(D)₉-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 173: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: A-D-B-D-A-(D)₁₀-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 174: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: B-D-A-D-A-(D)₈-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 175: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: B-D-A-D-A-(D)₉-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 176: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: B-D-A-D-A-(D)₁₀-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 177: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: A-A-A-A-B-(D)₈-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 178: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: A-A-A-A-B-(D)₉-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 179: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: A-A-A-A-B-(D)₁₀-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 180: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: A-A-B-A-A-(D)₈-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 181: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: A-A-B-A-A-(D)₉-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 182: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: A-A-B-A-A-(D)₁₀-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 183: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: B-A-A-A-A-(D)₈-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 184: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: B-A-A-A-A-(D)₉-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 185: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: B-A-A-A-A-(D)₁₀-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside
• Embodiment 186: The compound of any of embodiments 89-185, wherein at least one bicyclic nucleoside is a constrained ethyl nucleoside.
• Embodiment 187: The compound of any of embodiments 89-185, wherein each bicyclic nucleoside is a constrained ethyl nucleoside.
• Embodiment 188: The compound of any of embodiments, 89-185, wherein at least one bicyclic nucleoside is selected from among: BNA, LNA, α-L-LNA, ENA and 2′-thio LNA.
• Embodiment 189: The compound of any of embodiments, 89-185, wherein at least one bicyclic nucleoside is an LNA nucleoside.
• Embodiment 190: The compound of any of embodiments 89-185, wherein each bicyclic nucleoside is an LNA nucleoside.
• Embodiment 191: The compound of any of embodiments 89-185, wherein the 2′-substituent of the at least one 2′-substituted nucleoside is selected from among: OCH₃, F, OCH₂F, OCHF₂, OCF₃, OCH₂CH₃, O(CH₂)₂F, OCH₂CHF₂, OCH₂CF₃, OCH₂—CH—CH₂, O(CH₂)₂—OCH₃, O(CH₂)₂—SCH₃, O(CH₂)₂—OCF₃, O(CH₂)₃—N(R₄)(R₅), O(CH₂)₂—ON(R₄)(R₅), O(CH₂)₂—O(CH₂)₂—N(R₄)(R₅), OCH₂C(═O)—N(R₄)(R₅), OCH₂C(═O)—N(R₆)—(CH₂)₂—N(R₄)(R₅) and O(CH₂)₂—N(R₆)—C(═NR₇)[N(R₄)(R₅)] wherein R₄, R₅, R₆and R₇are each, independently, H or C₁-C₆alkyl.
• Embodiment 192: The compound of embodiment 191, wherein the 2′-substituent of the at least one 2′-substituted nucleoside of is selected from among: OCH₃, F, and O(CH₂)₂—OCH₃.
• Embodiment 193: The compound of embodiment 192, wherein the 2′-substituent of the at least one 2′-substituted nucleoside is O(CH₂)₂—OCH₃.
• Embodiment 194: The compound of any of embodiments 89-185, wherein the 2′-substituent of each 2′-substituted nucleoside is selected from among: OCH₃, F, OCH₂F, OCHF₂, OCF₃, OCH₂CH₃, O(CH₂)₂F, OCH₂CHF₂, OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃, O(CH₂)₂—SCH₃, O(CH₂)₂—OCF₃, O(CH₂)₃—N(R₄)(R₅), O(CH₂)₂—ON(R₄)(R₅), O(CH₂)₂—O(CH₂)₂—N(R₄)(R₅), OCH₂C(═O)—N(R₄)(R₅), OCH₂C(═O)—N(R₆)—(CH₂)₂—N(R₄)(R₅) and O(CH₂)₂—N(R₆)—C(═NR₇)[N(R₄)(R₅)] wherein R₄, R₅, R₆and R₇are each, independently, H or C₁-C₆alkyl.
• Embodiment 195: The compound of embodiment 194, wherein the 2′-substituent of each 2′-substituted nucleoside of is selected from among: OCH₃, F, and O(CH₂)₂—OCH₃.
• Embodiment 196: The compound of embodiment 195, wherein the 2′-substituent of each 2′-substituted nucleoside is O(CH₂)₂—OCH₃.
• Embodiment 197: The compound of any of embodiments 1-196, wherein the oligonucleotide comprises at least one modified internucleoside linkage.
• Embodiment 198: The compound of embodiment 197, wherein each internucleoside linkage is a modified internucleoside linkage.
• Embodiment 199: The compound of embodiment 197 or 198, wherein the modified internucleoside linkage is a phosphorothioate linkage.
• Embodiment 200: The compound of embodiment 197 or 198, wherein the modified internucleoside linkage is a methylphosphonate.
• Embodiment 201: The compound of any of embodiments 1-200 comprising a conjugate.
• Embodiment 202: The compound of any of embodiments 1-201 comprising at least one 5-methyl cytosine nucleobase.
• Embodiment 203: The compound of any of embodiments 1-202 comprising at least one modified nucleobase.
• Embodiment 204: The compound of any of embodiments 1-203, wherein the compound is an antisense compound.
• Embodiment 205: The compound of embodiment 204, wherein the compound is capable of inhibiting expression of the target nucleic acid in a cell.
• Embodiment 206: The compound of embodiment 205, wherein the compound is capable of inhibiting expression of the target nucleic acid in a cell by at least 50%.
• Embodiment 207: The compound of embodiment 205, wherein the compound is capable of inhibiting expression of the target nucleic acid in a cell by at least 80%.
• Embodiment 208: The compound of any of embodiments 205-207, wherein the cell is in an animal.
• Embodiment 209: The compound of embodiment 208, wherein the animal is a human.
• Embodiment 210: The compound of any of embodiments 1 to 209, wherein bicyclic nucleoside is selected from among: BNA, LNA, α-L-LNA, ENA and 2′-thio LNA.
• Embodiment 211: A compound of any of embodiments 1-210, comprising not more than 6 bicyclic nucleosides.
• Embodiment 212: A compound of any of embodiments 1-210, comprising not more than 5 bicyclic nucleosides.
• Embodiment 213: A compound of any of embodiments 1-210, comprising not more than 4 bicyclic nucleosides.
• Embodiment 214: A compound of any of embodiments 1-210, comprising not more than 3 bicyclic nucleosides.
• Embodiment 215: A compound of any of embodiments 1-210, comprising not more than 2 bicyclic nucleosides.
• Embodiment 216: A compound of any of embodiments 1-210, comprising not more than 1 bicyclic nucleoside.
• Embodiment 217: The compound of any of embodiments 211-216, wherein the bicyclic nucleoside comprises cEt.
• Embodiment 218: The compound of any of embodiments 211-216, wherein the bicyclic nucleoside comprises LNA.
• Embodiment 219: A pharmaceutical composition comprising the compound according to any of embodiments 1-218 and a pharmaceutically acceptable diluent.
• Embodiment 220: A method of modulating expression of a target nucleic acid in a cell comprising contacting the cell with a compound according to any of embodiments 1-218.
• Embodiment 221: A method of modulating expression of a target nucleic acid in an animal comprising administering to the animal the pharmaceutical composition according to embodiment 220.
• Embodiment 222: A method of manufacturing a compound according to any of embodiments 1-219 comprising forming chemical bonds.
• Embodiment 223: The method of embodiment 222, wherein said chemical bonds are internucleoside linkages.
• Embodiment 224: The method embodiment 222 or 223, wherein the method is performed under conditions suitable for the preparation of products for administration to humans.
• Embodiment 225: A method of manufacturing the pharmaceutical composition according to embodiment 224 comprising combining the compound according to any of embodiments 1-219 and the pharmaceutically acceptable diluent.
• Embodiment 226: The method embodiment 225, wherein the method is performed under conditions suitable for the preparation of products for administration to humans.
• Embodiment 227: A compound comprising a modified oligonucleotide having a sugar motif selected from among sugar motifs 279-615 as shown in Table 4.
• Embodiment 228: A compound comprising:
• a modified oligonucleotide consisting of 10 to 20 linked nucleosides, wherein the modified oligonucleotide comprises:
• a 5′-wing consisting of 2 to 5 linked nucleosides;
• a 3′-wing consisting of 2 to 5 linked nucleosides; and
• a gap between the 5′-wing and the 3′-wing consisting of 6 to 14 linked 2′-deoxynucleosides; and
• wherein the 5′-wing has a sugar modification motif selected from among the motifs in Table 1.
• Embodiment 229: A compound comprising:
• a modified oligonucleotide consisting of 10 to 20 linked nucleosides, wherein the modified oligonucleotide comprises:
• a 5′-wing consisting of 2 to 5 linked nucleosides;
• a 3′-wing consisting of 2 to 5 linked nucleosides; and
• a gap between the 5′-wing and the 3′-wing consisting of 6 to 14 linked 2′-deoxynucleosides; and
• wherein the 3′-wing has a sugar modification motif selected from among the motifs in Table 2.
• Embodiment 230: A compound comprising:
• a modified oligonucleotide consisting of 10 to 20 linked nucleosides, wherein the modified oligonucleotide comprises:
• a 5′-wing consisting of 2 to 5 linked nucleosides;
• a 3′-wing consisting of 2 to 5 linked nucleosides; and
• a gap between the 5′-wing and the 3′-wing consisting of 6 to 14 linked 2′-deoxynucleosides; and
• wherein the 5′-wing has a sugar modification motif selected from among the motifs in Table 1 and the 3′-wing has a sugar modification motif selected from among the motifs in Table 2.
• Embodiment 231: A compound of any of embodiments 1-16, wherein the modified oligonucleotide has a sugar motif described by Formula II as follows:
• 
  (J)_m-(B)_n-(J)_p-(B)_r-(A)_t-(D)_g-(A)_v-(B)_w-(J)_x-(B)_y-(J)_z
• wherein:
• each A is independently a 2′-substituted nucleoside;
• each B is independently a bicyclic nucleoside;
• each J is independently either a 2′-substituted nucleoside or a 2′-deoxynucleoside;
• each D is a 2′-deoxynucleoside;
• m is 0-4; n is 0-2; p is 0-2; r is 0-2; t is 0-2; v is 0-2; w is 0-4; x is 0-2; y is 0-2; z is 0-4; g is 6-14;
• provided that:
• at least one of m, n, and r is other than 0;
• at least one of w and y is other than 0;
• the sum of m, n, p, r, and t is from 1 to 5; and
• the sum of v, w, x, y, and z is from 1 to 5.
• Embodiment 232: A compound comprising:
  • a modified oligonucleotide consisting of 10 to 20 linked nucleosides, wherein the modified oligonucleotide has a sugar motif described by Formula II as follows:
• 
  (J)_m-(B)_n-(J)_p-(B)_r-(A)_t-(D)_g-(A)_v-(B)_w-(J)_x-(B)_y-(J)_z
• • wherein:
  • each A is independently a 2′-substituted nucleoside;
  • each B is independently a bicyclic nucleoside;
  • each J is independently either a 2′-substituted nucleoside or a 2′-deoxynucleoside;
  • each D is a 2′-deoxynucleoside;
  • m is 0-4; n is 0-2; p is 0-2; r is 0-2; t is 0-2; v is 0-2; w is 0-4; x is 0-2; y is 0-2; z is 0-4; g is 6-14;
• provided that:
• • at least one of m, n, and r is other than 0;
  • at least one of w and y is other than 0;
  • the sum of m, n, p, r, and t is from 1 to 5; and
  • the sum of v, w, x, y, and z is from 1 to 5.
• Embodiment 233: The compound of embodiment 231 or 232, wherein at least one bicyclic nucleoside is a constrained ethyl nucleoside.
• Embodiment 234: The compound of embodiment 233, wherein each bicyclic nucleoside is a constrained ethyl nucleoside.
• Embodiment 235: The compound of any of embodiments 231-232, wherein at least one bicyclic nucleoside is an LNA nucleoside.
• Embodiment 236: The compound of embodiments 228-232, wherein each bicyclic nucleoside is an LNA nucleoside.
• Embodiment 237: A method of treating a disease or condition.
• Embodiment 238: Use of a compound of any of embodiments 1 to 237 for the preparation of a medicament for the treatment of a disease or condition.
• In certain embodiments, including but not limited to any of the above numbered embodiments, compounds including oligonucleotides described herein are capable of modulating expression of a target mRNA. In certain embodiments, the target mRNA is associated with a disease or disorder, or encodes a protein that is associated with a disease or disorder. In certain embodiments, the compounds or oligonucleotides provided herein modulate the expression of function of such mRNA to alleviate one or more symptom of the disease or disorder.
• In certain embodiments, compounds including oligonucleotides describe herein are useful in vitro. In certain embodiments such compounds are used in diagnostics and/or for target validation experiments.
DETAILED DESCRIPTION OF THE INVENTION
• Unless specific definitions are provided, the nomenclature used in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for chemical synthesis, and chemical analysis. Certain such techniques and procedures may be found for example in “Carbohydrate Modifications in Antisense Research” Edited by Sangvi and Cook, American Chemical Society, Washington D.C., 1994; “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., 21^stedition, 2005; and “Antisense Drug Technology, Principles, Strategies, and Applications” Edited by Stanley T. Crooke, CRC Press, Boca Raton, Fla.; and Sambrook et al., “Molecular Cloning, A laboratory Manual,” 2^ndEdition, Cold Spring Harbor Laboratory Press, 1989, which are hereby incorporated by reference for any purpose. Where permitted, all patents, applications, published applications and other publications and other data referred to throughout in the disclosure are incorporated by reference herein in their entirety.
• Unless otherwise indicated, the following terms have the following meanings:
• As used herein, “nucleoside” means a compound comprising a nucleobase moiety and a sugar moiety. Nucleosides include, but are not limited to, naturally occurring nucleosides (as found in DNA and RNA) and modified nucleosides. Nucleosides may be linked to a phosphate moiety.
• As used herein, “chemical modification” means a chemical difference in a compound when compared to a naturally occurring counterpart. In reference to an oligonucleotide, chemical modification does not include differences only in nucleobase sequence. Chemical modifications of oligonucleotides include nucleoside modifications (including sugar moiety modifications and nucleobase modifications) and internucleoside linkage modifications.
• As used herein, “furanosyl” means a structure comprising a 5-membered ring comprising four carbon atoms and one oxygen atom.
• As used herein, “naturally occurring sugar moiety” means a ribofuranosyl as found in naturally occurring RNA or a deoxyribofuranosyl as found in naturally occurring DNA.
• As used herein, “sugar moiety” means a naturally occurring sugar moiety or a modified sugar moiety of a nucleoside.
• As used herein, “modified sugar moiety” means a substituted sugar moiety or a sugar surrogate.
• As used herein, “substituted sugar moiety” means a furanosyl that is not a naturally occurring sugar moiety. Substituted sugar moieties include, but are not limited to furanosyls comprising substituents at the 2′-position, the 3′-position, the 5′-position and/or the 4′-position. Certain substituted sugar moieties are bicyclic sugar moieties.
• As used herein, “2′-substituted sugar moiety” means a furanosyl comprising a substituent at the 2′-position other than H or OH. Unless otherwise indicated, a 2′-substituted sugar moiety is not a bicyclic sugar moiety (i.e., the 2′-substituent of a 2′-substituted sugar moiety does not form a bridge to another atom of the furanosyl ring.
• As used herein, “MOE” means —OCH₂CH₂OCH₃.
• As used herein the term “sugar surrogate” means a structure that does not comprise a furanosyl and that is capable of replacing the naturally occurring sugar moiety of a nucleoside, such that the resulting nucleoside is capable of (1) incorporation into an oligonucleotide and (2) hybridization to a complementary nucleoside. Such structures include rings comprising a different number of atoms than furanosyl (e.g., 4, 6, or 7-membered rings); replacement of the oxygen of a furanosyl with a non-oxygen atom (e.g., carbon, sulfur, or nitrogen); or both a change in the number of atoms and a replacement of the oxygen. Such structures may also comprise substitutions corresponding to those described for substituted sugar moieties (e.g., 6-membered carbocyclic bicyclic sugar surrogates optionally comprising additional substituents). Sugar surrogates also include more complex sugar replacements (e.g., the non-ring systems of peptide nucleic acid). Sugar surrogates include without limitation morpholinos, cyclohexenyls and cyclohexitols.
• As used herein, “bicyclic sugar moiety” means a modified sugar moiety comprising a 4 to 7 membered ring (including but not limited to a furanosyl) comprising a bridge connecting two atoms of the 4 to 7 membered ring to form a second ring, resulting in a bicyclic structure. In certain embodiments, the 4 to 7 membered ring is a sugar ring. In certain embodiments the 4 to 7 membered ring is a furanosyl. In certain such embodiments, the bridge connects the 2′-carbon and the 4′-carbon of the furanosyl.
• As used herein, “nucleotide” means a nucleoside further comprising a phosphate linking group. As used herein, “linked nucleosides” may or may not be linked by phosphate linkages and thus includes, but is not limited to “linked nucleotides.” As used herein, “linked nucleosides” are nucleosides that are connected in a continuous sequence (i.e. no additional nucleosides are present between those that are linked).
• As used herein, “nucleobase” means a group of atoms that can be linked to a sugar moiety to create a nucleoside that is capable of incorporation into an oligonucleotide, and wherein the group of atoms is capable of bonding with a complementary naturally occurring nucleobase of another oligonucleotide or nucleic acid.
• Nucleobases may be naturally occurring or may be modified.
• As used herein, “heterocyclic base” or “heterocyclic nucleobase” means a nucleobase comprising a heterocyclic structure.
• As used herein the terms, “unmodified nucleobase” or “naturally occurring nucleobase” means the naturally occurring heterocyclic nucleobases of RNA or DNA: the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) (including 5-methyl C), and uracil (U).
• As used herein, “modified nucleobase” means any nucleobase that is not a naturally occurring nucleobase.
• As used herein, “modified nucleoside” means a nucleoside comprising at least one chemical modification compared to naturally occurring RNA or DNA nucleosides. Modified nucleosides comprise a modified sugar moiety and/or a modified nucleobase.
• As used herein, “bicyclic nucleoside” or “BNA” means a nucleoside comprising a bicyclic sugar moiety.
• As used herein, “constrained ethyl nucleoside” or “cEt” means a nucleoside comprising a bicyclic sugar moiety comprising a 4′-CH(CH₃)—O-2′bridge.
• As used herein, “locked nucleic acid nucleoside” or “LNA” means a nucleoside comprising a bicyclic sugar moiety comprising a 4′-CH₂-O-2′bridge.
• As used herein, “2′-substituted nucleoside” means a nucleoside comprising a substituent at the 2′-position other than H or OH. Unless otherwise indicated, a 2′-substituted nucleoside is not a bicyclic nucleoside.
• As used herein, “2′-deoxynucleoside” means a nucleoside comprising 2′-H furanosyl sugar moiety, as found in naturally occurring deoxyribonucleosides (DNA). In certain embodiments, a 2′-deoxynucleoside may comprise a modified nucleobase or may comprise an RNA nucleobase (e.g., uracil).
• As used herein, “oligonucleotide” means a compound comprising a plurality of linked nucleosides. In certain embodiments, an oligonucleotide comprises one or more unmodified ribonucleosides (RNA) and/or unmodified deoxyribonucleosides (DNA) and/or one or more modified nucleosides.
• As used herein “oligonucleoside” means an oligonucleotide in which none of the internucleoside linkages contains a phosphorus atom. As used herein, oligonucleotides include oligonucleosides.
• As used herein, “modified oligonucleotide” means an oligonucleotide comprising at least one modified nucleoside and/or at least one modified internucleoside linkage.
• As used herein “internucleoside linkage” means a covalent linkage between adjacent nucleosides in an oligonucleotide.
• As used herein “naturally occurring internucleoside linkage” means a 3′ to 5′ phosphodiester linkage.
• As used herein, “modified internucleoside linkage” means any internucleoside linkage other than a naturally occurring internucleoside linkage.
• As used herein, “oligomeric compound” means a polymeric structure comprising two or more sub-structures. In certain embodiments, an oligomeric compound comprises an oligonucleotide. In certain embodiments, an oligomeric compound comprises one or more conjugate groups and/or terminal groups. In certain embodiments, an oligomeric compound consists of an oligonucleotide.
• As used herein, “terminal group” means one or more atom attached to either, or both, the 3′ end or the 5′ end of an oligonucleotide. In certain embodiments a terminal group is a conjugate group. In certain embodiments, a terminal group comprises one or more terminal group nucleosides.
• As used herein, “conjugate” means an atom or group of atoms bound to an oligonucleotide or oligomeric compound. In general, conjugate groups modify one or more properties of the compound to which they are attached, including, but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge and/or clearance properties.
• As used herein, “conjugate linking group” means any atom or group of atoms used to attach a conjugate to an oligonucleotide or oligomeric compound.
• As used herein, “antisense compound” means a compound comprising or consisting of an oligonucleotide at least a portion of which is complementary to a target nucleic acid to which it is capable of hybridizing, resulting in at least one antisense activity.
• As used herein, “antisense activity” means any detectable and/or measurable change attributable to the hybridization of an antisense compound to its target nucleic acid.
• As used herein, “detecting” or “measuring” means that a test or assay for detecting or measuring is performed. Such detection and/or measuring may result in a value of zero. Thus, if a test for detection or measuring results in a finding of no activity (activity of zero), the step of detecting or measuring the activity has nevertheless been performed.
• As used herein, “detectable and/or measureable activity” means a measurable activity that is not zero.
• As used herein, “essentially unchanged” means little or no change in a particular parameter, particularly relative to another parameter which changes much more. In certain embodiments, a parameter is essentially unchanged when it changes less than 5%. In certain embodiments, a parameter is essentially unchanged if it changes less than two-fold while another parameter changes at least ten-fold. For example, in certain embodiments, an antisense activity is a change in the amount of a target nucleic acid. In certain such embodiments, the amount of a non-target nucleic acid is essentially unchanged if it changes much less than the target nucleic acid does, but the change need not be zero.
• As used herein, “expression” means the process by which a gene ultimately results in a protein. Expression includes, but is not limited to, transcription, post-transcriptional modification (e.g., splicing, polyadenlyation, addition of 5′-cap), and translation.
• As used herein, “target nucleic acid” means a nucleic acid molecule to which an antisense compound hybridizes.
• As used herein, “single nucleotide polymorphism” or “SNP” means a single nucleotide variation between the genomes of individuals of the same species. In some cases, a SNP may be a single nucleotide deletion or insertion.
• As used herein, “mRNA” means an RNA molecule that encodes a protein.
• As used herein, “pre-mRNA” means an RNA transcript that has not been fully processed into mRNA. Pre-RNA includes one or more intron.
• As used herein, “object RNA” means an RNA molecule other than a target RNA, the amount, activity, splicing, and/or function of which is modulated, either directly or indirectly, by a target nucleic acid. In certain embodiments, a target nucleic acid modulates splicing of an object RNA. In certain such embodiments, an antisense compound modulates the amount or activity of the target nucleic acid, resulting in a change in the splicing of an object RNA and ultimately resulting in a change in the activity or function of the object RNA.
• As used herein, “microRNA” means a naturally occurring, small, non-coding RNA that represses gene expression of at least one mRNA. In certain embodiments, a microRNA represses gene expression by binding to a target site within a 3′ untranslated region of an mRNA. In certain embodiments, a microRNA has a nucleobase sequence as set forth in miRBase, a database of published microRNA sequences found at microrna.sanger.ac.uk/sequences/. In certain embodiments, a microRNA has a nucleobase sequence as set forth in miRBase version 12.0 released September 2008, which is herein incorporated by reference in its entirety.
• As used herein, “microRNA mimic” means an oligomeric compound having a sequence that is at least partially identical to that of a microRNA. In certain embodiments, a microRNA mimic comprises the microRNA seed region of a microRNA. In certain embodiments, a microRNA mimic modulates translation of more than one target nucleic acids. In certain embodiments, a microRNA mimic is double-stranded.
• As used herein, “targeting” or “targeted to” means the association of an antisense compound to a particular target nucleic acid molecule or a particular region of a target nucleic acid molecule. An antisense compound targets a target nucleic acid if it is sufficiently complementary to the target nucleic acid to allow hybridization under physiological conditions.
• As used herein, “nucleobase complementarity” or “complementarity” when in reference to nucleobases means a nucleobase that is capable of base pairing with another nucleobase. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). In certain embodiments, complementary nucleobase means a nucleobase of an antisense compound that is capable of base pairing with a nucleobase of its target nucleic acid. For example, if a nucleobase at a certain position of an antisense compound is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be complementary at that nucleobase pair. Nucleobases comprising certain modifications may maintain the ability to pair with a counterpart nucleobase and thus, are still capable of nucleobase complementarity.
• As used herein, “non-complementary” in reference to nucleobases means a pair of nucleobases that do not form hydrogen bonds with one another.
• As used herein, “complementary” in reference to oligomeric compounds (e.g., linked nucleosides, oligonucleotides, or nucleic acids) means the capacity of such oligomeric compounds or regions thereof to hybridize to another oligomeric compound or region thereof through nucleobase complementarity under stringent conditions. Complementary oligomeric compounds need not have nucleobase complementarity at each nucleoside. Rather, some mismatches are tolerated. In certain embodiments, complementary oligomeric compounds or regions are complementary at 70% of the nucleobases (70% complementary). In certain embodiments, complementary oligomeric compounds or regions are 80% complementary. In certain embodiments, complementary oligomeric compounds or regions are 90% complementary. In certain embodiments, complementary oligomeric compounds or regions are 95% complementary. In certain embodiments, complementary oligomeric compounds or regions are 100% complementary.
• As used herein, “hybridization” means the pairing of complementary oligomeric compounds (e.g., an antisense compound and its target nucleic acid). While not limited to a particular mechanism, the most common mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases.
• As used herein, “specifically hybridizes” means the ability of an oligomeric compound to hybridize to one nucleic acid site with greater affinity than it hybridizes to another nucleic acid site. In certain embodiments, an antisense oligonucleotide specifically hybridizes to more than one target site.
• As used herein, “fully complementary” in reference to an oligonucleotide or portion thereof means that each nucleobase of the oligonucleotide or portion thereof is capable of pairing with a nucleobase of a complementary nucleic acid or contiguous portion thereof. Thus, a fully complementary region comprises no mismatches or unhybridized nucleobases in either strand.
• As used herein, “percent complementarity” means the percentage of nucleobases of an oligomeric compound that are complementary to an equal-length portion of a target nucleic acid. Percent complementarity is calculated by dividing the number of nucleobases of the oligomeric compound that are complementary to nucleobases at corresponding positions in the target nucleic acid by the total length of the oligomeric compound.
• As used herein, “percent identity” means the number of nucleobases in a first nucleic acid that are the same type (independent of chemical modification) as nucleobases at corresponding positions in a second nucleic acid, divided by the total number of nucleobases in the first nucleic acid.
• As used herein, “modulation” means a change of amount or quality of a molecule, function, or activity when compared to the amount or quality of a molecule, function, or activity prior to modulation. For example, modulation includes the change, either an increase (stimulation or induction) or a decrease (inhibition or reduction) in gene expression. As a further example, modulation of expression can include a change in splice site selection of pre-mRNA processing, resulting in a change in the absolute or relative amount of a particular splice-variant compared to the amount in the absence of modulation.
• As used herein, “motif” means a pattern of chemical modifications in an oligomeric compound or a region thereof. Motifs may be defined by modifications at certain nucleosides and/or at certain linking groups of an oligomeric compound.
• As used herein, “nucleoside motif” means a pattern of nucleoside modifications in an oligomeric compound or a region thereof. The linkages of such an oligomeric compound may be modified or unmodified. Unless otherwise indicated, motifs herein describing only nucleosides are intended to be nucleoside motifs. Thus, in such instances, the linkages are not limited.
• As used herein, “sugar motif” means a pattern of sugar modifications in an oligomeric compound or a region thereof.
• As used herein, “linkage motif” means a pattern of linkage modifications in an oligomeric compound or region thereof. The nucleosides of such an oligomeric compound may be modified or unmodified. Unless otherwise indicated, motifs herein describing only linkages are intended to be linkage motifs. Thus, in such instances, the nucleosides are not limited.
• As used herein, “nucleobase modification motif” means a pattern of modifications to nucleobases along an oligonucleotide. Unless otherwise indicated, a nucleobase modification motif is independent of the nucleobase sequence.
• As used herein, “sequence motif” means a pattern of nucleobases arranged along an oligonucleotide or portion thereof. Unless otherwise indicated, a sequence motif is independent of chemical modifications and thus may have any combination of chemical modifications, including no chemical modifications.
• As used herein, “type of modification” in reference to a nucleoside or a nucleoside of a “type” means the chemical modification of a nucleoside and includes modified and unmodified nucleosides. Accordingly, unless otherwise indicated, a “nucleoside having a modification of a first type” may be an unmodified nucleoside.
• As used herein, “differently modified” mean chemical modifications or chemical substituents that are different from one another, including absence of modifications. Thus, for example, a MOE nucleoside and an unmodified DNA nucleoside are “differently modified,” even though the DNA nucleoside is unmodified. Likewise, DNA and RNA are “differently modified,” even though both are naturally-occurring unmodified nucleosides. Nucleosides that are the same but for comprising different nucleobases are not differently modified. For example, a nucleoside comprising a 2′-OMe modified sugar and an unmodified adenine nucleobase and a nucleoside comprising a 2′-OMe modified sugar and an unmodified thymine nucleobase are not differently modified.
• As used herein, “the same type of modifications” refers to modifications that are the same as one another, including absence of modifications. Thus, for example, two unmodified DNA nucleoside have “the same type of modification,” even though the DNA nucleoside is unmodified. Such nucleosides having the same type modification may comprise different nucleobases.
• As used herein, “separate regions” means portions of an oligonucleotide wherein the chemical modifications or the motif of chemical modifications of any neighboring portions include at least one difference to allow the separate regions to be distinguished from one another.
• As used herein, “pharmaceutically acceptable carrier or diluent” means any substance suitable for use in administering to an animal. In certain embodiments, a pharmaceutically acceptable carrier or diluent is sterile saline. In certain embodiments, such sterile saline is pharmaceutical grade saline.
• As used herein, “substituent” and “substituent group,” means an atom or group that replaces the atom or group of a named parent compound. For example a substituent of a modified nucleoside is any atom or group that differs from the atom or group found in a naturally occurring nucleoside (e.g., a modified 2′-substuent is any atom or group at the 2′-position of a nucleoside other than H or OH). Substituent groups can be protected or unprotected. In certain embodiments, compounds of the present invention have substituents at one or at more than one position of the parent compound. Substituents may also be further substituted with other substituent groups and may be attached directly or via a linking group such as an alkyl or hydrocarbyl group to a parent compound.
• Likewise, as used herein, “substituent” in reference to a chemical functional group means an atom or group of atoms differs from the atom or a group of atoms normally present in the named functional group. In certain embodiments, a substituent replaces a hydrogen atom of the functional group (e.g., in certain embodiments, the substituent of a substituted methyl group is an atom or group other than hydrogen which replaces one of the hydrogen atoms of an unsubstituted methyl group). Unless otherwise indicated, groups amenable for use as substituents include without limitation, halogen, hydroxyl, alkyl, alkenyl, alkynyl, acyl (˜C(O)R_aa), carboxyl (—C(O)O-R_aa), aliphatic groups, alicyclic groups, alkoxy, substituted oxy (—O—R_aa), aryl, aralkyl, heterocyclic radical, heteroaryl, heteroarylalkyl, amino (—N(R_bb)(R_cc)), imino(=NR_bb), amido (˜C(O)N(R_bb)(R_cc) or —N(R_bb)C(O)R_aa), azido (˜N₃), nitro (˜NO₂), cyano (—CN), carbamido (˜OC(O)N(R_bb)(R_cc) or —N(R_bb)C(O)OR_aa), ureido (—N(R_bb)C(O)N(R_bb)(R_cc)), thioureido (—N(R_bb)C(S)N(R_bb)—(R_cc)), guanidinyl (—N(R_bb)C(═NR_bb)N(R_bb)(R_cc)), amidinyl (˜C(═NR_bb)N(R_bb)(R_cc) or —N(R_bb)C(═NR_bb)(R_aa)), thiol (˜SR_bb), sulfinyl (—S(O)R_bb), sulfonyl (—S(O)₂R_bb) and sulfonamidyl (—S(O)₂N(R_bb)(R_cc) or —N(R_bb)S-(O)₂R_bb). Wherein each R_aa, R_bband R_ccis, independently, H, an optionally linked chemical functional group or a further substituent group with a preferred list including without limitation, alkyl, alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic, heterocyclic and heteroarylalkyl. Selected substituents within the compounds described herein are present to a recursive degree.
• As used herein, “alkyl,” as used herein, means a saturated straight or branched hydrocarbon radical containing up to twenty four carbon atoms. Examples of alkyl groups include without limitation, methyl, ethyl, propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl and the like. Alkyl groups typically include from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms (C₁-C₁₂alkyl) with from 1 to about 6 carbon atoms being more preferred.
• As used herein, “alkenyl,” means a straight or branched hydrocarbon chain radical containing up to twenty four carbon atoms and having at least one carbon-carbon double bond. Examples of alkenyl groups include without limitation, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, dienes such as 1,3-butadiene and the like. Alkenyl groups typically include from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred. Alkenyl groups as used herein may optionally include one or more further substituent groups.
• As used herein, “alkynyl,” means a straight or branched hydrocarbon radical containing up to twenty four carbon atoms and having at least one carbon-carbon triple bond. Examples of alkynyl groups include, without limitation, ethynyl, 1-propynyl, 1-butynyl, and the like. Alkynyl groups typically include from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred. Alkynyl groups as used herein may optionally include one or more further substituent groups.
• As used herein, “acyl,” means a radical formed by removal of a hydroxyl group from an organic acid and has the general Formula —C(O)—X where X is typically aliphatic, alicyclic or aromatic. Examples include aliphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls, aromatic sulfinyls, aliphatic sulfinyls, aromatic phosphates, aliphatic phosphates and the like. Acyl groups as used herein may optionally include further substituent groups.
• As used herein, “alicyclic” means a cyclic ring system wherein the ring is aliphatic. The ring system can comprise one or more rings wherein at least one ring is aliphatic. Preferred alicyclics include rings having from about 5 to about 9 carbon atoms in the ring. Alicyclic as used herein may optionally include further substituent groups.
• As used herein, “aliphatic” means a straight or branched hydrocarbon radical containing up to twenty four carbon atoms wherein the saturation between any two carbon atoms is a single, double or triple bond. An aliphatic group preferably contains from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms with from 1 to about 6 carbon atoms being more preferred. The straight or branched chain of an aliphatic group may be interrupted with one or more heteroatoms that include nitrogen, oxygen, sulfur and phosphorus. Such aliphatic groups interrupted by heteroatoms include without limitation, polyalkoxys, such as polyalkylene glycols, polyamines, and polyimines. Aliphatic groups as used herein may optionally include further substituent groups.
• As used herein, “alkoxy” means a radical formed between an alkyl group and an oxygen atom wherein the oxygen atom is used to attach the alkoxy group to a parent molecule. Examples of alkoxy groups include without limitation, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy and the like. Alkoxy groups as used herein may optionally include further substituent groups.
• As used herein, “aminoalkyl” means an amino substituted C₁-C₁₂alkyl radical. The alkyl portion of the radical forms a covalent bond with a parent molecule. The amino group can be located at any position and the aminoalkyl group can be substituted with a further substituent group at the alkyl and/or amino portions.
• As used herein, “aralkyl” and “arylalkyl” mean an aromatic group that is covalently linked to a C₁-C₁₂alkyl radical. The alkyl radical portion of the resulting aralkyl (or arylalkyl) group forms a covalent bond with a parent molecule. Examples include without limitation, benzyl, phenethyl and the like. Aralkyl groups as used herein may optionally include further substituent groups attached to the alkyl, the aryl or both groups that form the radical group.
• As used herein, “aryl” and “aromatic” mean a mono- or polycyclic carbocyclic ring system radicals having one or more aromatic rings. Examples of aryl groups include without limitation, phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl and the like. Preferred aryl ring systems have from about 5 to about 20 carbon atoms in one or more rings. Aryl groups as used herein may optionally include further substituent groups.
• As used herein, “halo” and “halogen,” mean an atom selected from fluorine, chlorine, bromine and iodine.
• As used herein, “heteroaryl,” and “heteroaromatic,” mean a radical comprising a mono- or poly-cyclic aromatic ring, ring system or fused ring system wherein at least one of the rings is aromatic and includes one or more heteroatoms. Heteroaryl is also meant to include fused ring systems including systems where one or more of the fused rings contain no heteroatoms. Heteroaryl groups typically include one ring atom selected from sulfur, nitrogen or oxygen. Examples of heteroaryl groups include without limitation, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl and the like. Heteroaryl radicals can be attached to a parent molecule directly or through a linking moiety such as an aliphatic group or hetero atom. Heteroaryl groups as used herein may optionally include further substituent groups.
Oligomeric Compounds
• In certain embodiments, the present invention provides oligomeric compounds. In certain embodiments, such oligomeric compounds comprise oligonucleotides optionally comprising one or more conjugate and/or terminal groups. In certain embodiments, an oligomeric compound consists of an oligonucleotide. In certain embodiments, oligonucleotides comprise one or more chemical modifications. Such chemical modifications include modifications one or more nucleoside (including modifications to the sugar moiety and/or the nucleobase) and/or modifications to one or more internucleoside linkage.
Certain Sugar Moieties
• In certain embodiments, oligomeric compounds of the invention comprise one or more modifed nucleosides comprising a modifed sugar moiety. Such oligomeric compounds comprising one or more sugar-modified nucleosides may have desirable properties, such as enhanced nuclease stability or increased binding affinity with a target nucleic acid relative to oligomeric compounds comprising only nucleosides comprising naturally occurring sugar moieties. In certain embodiments, modified sugar moieties are substituted sugar moieties. In certain embodiments, modified sugar moieties are sugar surrogates. Such sugar surrogates may comprise one or more substitutions corresponding to those of substituted sugar moieties.
• In certain embodiments, modified sugar moieties are substituted sugar moieties comprising one or more non-bridging sugar substituent, including but not limited to substituents at the 2′ and/or 5′ positions. Examples of sugar substituents suitable for the 2′-position, include, but are not limited to: 2′-F, 2′-OCH₃(“OMe” or “O-methyl”), and 2′-O(CH₂)₂OCH₃(“MOE”). In certain embodiments, sugar substituents at the 2′ position is selected from allyl, amino, azido, thio, O-allyl, O—C₁-C₁₀alkyl, O—C₁-C₁₀substituted alkyl; OCF₃, O(CH₂)₂SCH₃, O(CH₂)₂—O—N(Rm)(Rn), and O—CH₂-C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted C₁-C₁₀alkyl. Examples of sugar substituents at the 5′-position, include, but are not limited to: 5′-methyl (R or S); 5′-vinyl, and 5′-methoxy. In certain embodiments, substituted sugars comprise more than one non-bridging sugar substituent, for example, 2′-F-5′-methyl sugar moieties (see, e.g., PCT International Application WO 2008/101157, for additional 5′, 2′-bis substituted sugar moieties and nucleosides).
• Nucleosides comprising 2′-substituted sugar moieties are referred to as 2′-substituted nucleosides. In certain embodiments, a 2′-substituted nucleoside comprises a 2′-substituent group selected from halo, allyl, amino, azido, SH, CN, OCN, CF₃, OCF₃, O, S, or N(R_m)-alkyl; O, S, or N(R_m)-alkenyl; 0, S or N(R_m)-alkynyl; O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(R_m)(R_n) or O—CH₂-C(═O)—N(R_m)(R_n), where each R_mand R_nis, independently, H, an amino protecting group or substituted or unsubstituted C₁-C₁₀alkyl. These 2′-substituent groups can be further substituted with one or more substituent groups independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO₂), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.
• In certain embodiments, a 2′-substituted nucleoside comprises a 2′-substituent group selected from F, NH₂, N₃, OCF₃, O—CH₃, O(CH₂)₃NH₂, CH₂—CH═CH₂, O—CH₂—CH═CH₂, OCH₂CH₂OCH₃, O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(R_m)(R_n), O(CH₂)₂O(CH₂)₂N(CH₃)₂, and N-substituted acetamide (O—CH₂-C(═O)—N(R_m)(R_n) where each R_mand R_nis, independently, H, an amino protecting group or substituted or unsubstituted C₁-C₁₀alkyl.
• In certain embodiments, a 2′-substituted nucleoside comprises a sugar moiety comprising a 2′-substituent group selected from F, OCF₃, O—CH₃, OCH₂CH₂OCH₃, O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(CH₃)₂, —O(CH₂)₂O(CH₂)₂N(CH₃)₂, and O—CH₂-C(═O)—N(H)CH₃.
• In certain embodiments, a 2′-substituted nucleoside comprises a sugar moiety comprising a 2′-substituent group selected from F, O—CH₃, and OCH₂CH₂OCH₃.
• Certain modifed sugar moieties comprise a bridging sugar substituent that forms a second ring resulting in a bicyclic sugar moiety. In certain such embodiments, the bicyclic sugar moiety comprises a bridge between the 4′ and the 2′ furanose ring atoms. Examples of such 4′ to 2′ sugar substituents, include, but are not limited to: —[C(R_a)(R_b)_n]—, —[C(R_a)(R_b)]_n—O—, —C(R_aR_b)—N(R)—O— or, —C(R_aR_b)—O—N(R)-; 4′- CH₂-2′, 4′-(CH₂)₂-2′, 4′-(CH₂)₃-2′; 4′-(CH₂)—O-2′ (LNA); 4′-(CH₂)—S-2; 4′-(CH₂)₂—O-2′ (ENA); 4′-CH(CH₃)—O-2′ (cEt) and 4′-CH(CH₂OCH₃)—O-2′, and analogs thereof (see, e.g., U.S. Pat. No. 7,399,845, issued on Jul. 15, 2008); 4′-C(CH₃)(CH₃)—O-2′ and analogs thereof, (see, e.g., WO2009/006478, published Jan. 8, 2009); 4′-CH₂-N(OCH₃)-2′ and analogs thereof (see, e.g., WO2008/150729, published Dec. 11, 2008); 4′-CH₂-O—N(CH₃)-2′ (see, e.g., US2004/0171570, published Sep. 2, 2004); 4′-CH₂—O—N(R)-2′, and 4′-CH₂—N(R)-O-2′-, wherein each R is, independently, H, a protecting group, or C₁-C₁₂alkyl; 4′-CH₂-N(R)-O-2′, wherein R is H, C₁-C₁₂alkyl, or a protecting group (see, U.S. Pat. No. 7,427,672, issued on Sep. 23, 2008); 4′-CH₂-C(H)(CH₃)-2′ (see, e.g., Chattopadhyaya, et al.,J. Org. Chem.,2009, 74, 118-134); and 4′-CH₂—C(═CH₂)-2′ and analogs thereof (see, published PCT International Application WO 2008/154401, published on Dec. 8, 2008).
• In certain embodiments, such 4′ to 2′ bridges independently comprise from 1 to 4 linked groups independently selected from —[C(R_a)(R_b)]_n—, —C(R_a)═C(R_b)—, —C(R_a)═N—, —C(═NR_a)—, —C(═O)—, —C(═S)—, —O—, —Si(R_a)₂—, —S(═O)_x—, and —N(R_a)—;
• wherein:
• x is 0, 1, or 2;
• n is 1, 2, 3, or 4;
• each R_aand R_b, is, independently, H, a protecting group, hydroxyl, C₁-C₁₂alkyl, substituted C₁-C₁₂alkyl, C₂-C₁₂alkenyl, substituted C₂-C₁₂alkenyl, C₂-C₁₂alkynyl, substituted C₂-C₁₂alkynyl, C₅-C₂₀aryl, substituted C₅-C₂₀aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C₅-C₇alicyclic radical, substituted C₅-C₇alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃, COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)₂-J₁), or sulfoxyl (S(═O)-J₁); and each J₁and J₂is, independently, H, C₁-C₁₂alkyl, substituted C₁-C₁₂alkyl, C₂-C₁₂alkenyl, substituted C₂-C₁₂alkenyl, C₂-C₁₂alkynyl, substituted C₂-C₁₂alkynyl, C₅-C₂₀aryl, substituted C₅-C₂₀aryl, acyl (C(═O)—H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C₁-C₁₂aminoalkyl, substituted C₁-C₁₂aminoalkyl, or a protecting group.
• Nucleosides comprising bicyclic sugar moieties are referred to as bicyclic nucleosides or BNAs. Bicyclic nucleosides include, but are not limited to, (A) α-L-Methyleneoxy (4′-CH₂-O-2′) BNA, (B) β-D-Methyleneoxy (4′-CH₂-O-2′) BNA (also referred to as locked nucleic acid or LNA), (C) Ethyleneoxy (4′-(CH₂)₂-O-2′) BNA, (D) Aminooxy (4′-CH₂—O—N(R)-2′) BNA, (E) Oxyamino (4′-CH₂—N(R)-O-2′) BNA, (F) Methyl(methyleneoxy) (4′-CH(CH₃)—O-2′) BNA (also referred to as constrained ethyl or cEt), (G) methylene-thio (4′-CH₂-S-2′) BNA, (H) methylene-amino (4′-CH₂-N(R)-2′) BNA, (I) methyl carbocyclic (4′-CH₂—CH(CH₃)-2′) BNA, and (J) propylene carbocyclic (4′-(CH₂)₃-2′) BNA as depicted below.
• 

  
• wherein Bx is a nucleobase moiety and R is, independently, H, a protecting group, or C₁-C₁₂alkyl.
• Additional bicyclic sugar moieties are known in the art, for example: Singh et al.,Chem. Commun.,1998, 4, 455-456; Koshkin et al.,Tetrahedron,1998, 54, 3607-3630; Wahlestedt et al.,Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638; Kumar et al.,Bioorg. Med. Chem. Lett.,1998, 8, 2219-2222; Singh et al.,J. Org. Chem.,1998, 63, 10035-10039; Srivastava et al.,J. Am. Chem. Soc.,129(26) 8362-8379 (Jul. 4, 2007); Elayadi et al.,Curr. Opinion Invens. Drugs,2001, 2, 558-561; Braasch et al.,Chem. Biol.,2001, 8, 1-7; Orum et al.,Curr. Opinion Mol. Ther.,2001, 3, 239-243; U.S. Pat. Nos. 7,053,207, 6,268,490, 6,770,748, 6,794,499, 7,034,133, 6,525,191, 6,670,461, and 7,399,845; WO 2004/106356, WO 1994/14226, WO 2005/021570, and WO 2007/134181; U.S. Patent Publication Nos. US2004/0171570, US2007/0287831, and US2008/0039618; U.S. patent Ser. Nos. 12/129,154, 60/989,574, 61/026,995, 61/026,998, 61/056,564, 61/086,231, 61/097,787, and 61/099,844; and PCT International Applications Nos. PCT/US2008/064591, PCT/US2008/066154, and PCT/US2008/068922.
• In certain embodiments, bicyclic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by isomeric configuration. For example, a nucleoside comprising a 4′-2′ methylene-oxy bridge, may be in the α-L configuration or in the β-D configuration. Previously, α-L-methyleneoxy (4′-CH₂-O-2′) bicyclic nucleosides have been incorporated into antisense oligonucleotides that showed antisense activity (Frieden et al.,Nucleic Acids Research,2003, 21, 6365-6372).
• In certain embodiments, substituted sugar moieties comprise one or more non-bridging sugar substituent and one or more bridging sugar substituent (e.g., 5′-substituted and 4′-2′ bridged sugars). (see, PCT International Application WO 2007/134181, published on Nov. 22, 2007, wherein LNA is substituted with, for example, a 5′-methyl or a 5′-vinyl group).
• In certain embodiments, modified sugar moieties are sugar surrogates. In certain such embodiments, the oxygen atom of the naturally occurring sugar is substituted, e.g., with a sulfer, carbon or nitrogen atom. In certain such embodiments, such modified sugar moiety also comprises bridging and/or non-bridging substituents as described above. For example, certain sugar surogates comprise a 4′-sulfer atom and a substitution at the 2′-position (see, e.g., published U.S. Patent Application US2005/0130923, published on Jun. 16, 2005) and/or the 5′ position. By way of additional example, carbocyclic bicyclic nucleosides having a 4′-2′ bridge have been described (see, e.g., Freier et al.,Nucleic Acids Research,1997, 25(22), 4429-4443 and Albaek et al.,J Org. Chem.,2006, 71, 7731-7740).
• In certain embodiments, sugar surrogates comprise rings having other than 5-atoms. For example, in certain embodiments, a sugar surrogate comprises a six-membered tetrahydropyran. Such tetrahydropyrans may be further modified or substituted. Nucleosides comprising such modified tetrahydropyrans include, but are not limited to, hexitol nucleic acid (HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (see Leumann, C J.Bioorg. &Med. Chem. (2002) 10:841-854), fluoro HNA (F-HNA), and those compounds having Formula VII:
• 
• wherein independently for each of said at least one tetrahydropyran nucleoside analog of Formula VII:
• Bx is a nucleobase moiety;
• T₃and T₄are each, independently, an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound or one of T₃and T₄is an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound and the other of T₃and T₄is H, a hydroxyl protecting group, a linked conjugate group, or a 5′ or 3′-terminal group; q₁, q₂, q₃, q₄, q₅, q₆and q₇are each, independently, H, C₁-C₆alkyl, substituted C₁-C₆alkyl, C₂-C₆alkenyl, substituted C₂-C₆alkenyl, C₂-C₆alkynyl, or substituted C₂-C₆alkynyl; and
• one of R₁and R₂is hydrogen and the other is selected from halogen, substituted or unsubstituted alkoxy, NJ₁J₂, SJ₁, N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂, and CN, wherein X is O, S or NJ₁, and each J₁, J₂, and J₃is, independently, H or C₁-C₆alkyl.
• In certain embodiments, the modified THP nucleosides of Formula VII are provided wherein q₁, q₂, q₃, q₄, q₅, q₆and q₇are each H. In certain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆and q₇is other than H. In certain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆and q₇is methyl. In certain embodiments, THP nucleosides of Formula VII are provided wherein one of R₁and R₂is F. In certain embodiments, R₁is fluoro and R₂is H, R₁is methoxy and R₂is H, and R₁is methoxyethoxy and R₂is H.
• Many other bicyclo and tricyclo sugar surrogate ring systems are also known in the art that can be used to modify nucleosides for incorporation into antisense compounds (see, e.g., review article: Leumann, J. C,Bioorganic&Medicinal Chemistry,2002, 10, 841-854).
• Combinations of modifications are also provided without limitation, such as 2′-F-5′-methyl substituted nucleosides (see PCT International Application WO 2008/101157 Published on Aug. 21, 2008 for other disclosed 5′, 2′-bis substituted nucleosides) and replacement of the ribosyl ring oxygen atom with S and further substitution at the 2′-position (see published U.S. Patent Application US2005-0130923, published on Jun. 16, 2005) or alternatively 5′-substitution of a bicyclic nucleic acid (see PCT International Application WO 2007/134181, published on Nov. 22, 2007 wherein a 4′-CH₂-O-2′ bicyclic nucleoside is further substituted at the 5′ position with a 5′-methyl or a 5′-vinyl group). The synthesis and preparation of carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described (see, e.g., Srivastava et al.,J. Am. Chem. Soc.2007, 129(26), 8362-8379).
• Certain Nucleobases
• In certain embodiments, nucleosides of the present invention comprise one or more unmodified nucleobases. In certain embodiments, nucleosides of the present invention comprise one or more modifed nucleobases.
• In certain embodiments, modified nucleobases are selected from: universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases as defined herein. 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil; 5-propynylcytosine; 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (˜C≡C-CH₃) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, 3-deazaguanine and 3-deazaadenine, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases as defined herein. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, J. I., Ed., John Wiley & Sons, 1990, 858-859; those disclosed by Englisch et al.,Angewandte Chemie, International Edition, 1991, 30, 613; and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, Crooke, S. T. and Lebleu, B., Eds., CRC Press, 1993, 273-288.
• Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include without limitation, U.S. Pat. Nos. 3,687,808; 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,645,985; 5,681,941; 5,750,692; 5,763,588; 5,830,653 and 6,005,096, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.
• Certain Internucleoside Linkages
• In certain embodiments, the present invention provides oligomeric compounds comprising linked nucleosides. In such embodiments, nucleosides may be linked together using any internucleoside linkage. The two main classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus containing internucleoside linkages include, but are not limited to, phosphodiesters (P═O), phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates (P═S). Representative non-phosphorus containing internucleoside linking groups include, but are not limited to, methylenemethylimino (˜CH₂-N(CH₃)—O—CH₂-), thiodiester (˜O—C(O)—S—), thionocarbamate (—O—C(O)(NH)—S-); siloxane (—O—Si(H)₂-O-); and N,N′-dimethylhydrazine (˜CH₂-N(CH₃)—N(CH₃)-). Modified linkages, compared to natural phosphodiester linkages, can be used to alter, typically increase, nuclease resistance of the oligomeric compound. In certain embodiments, internucleoside linkages having a chiral atom can be prepared as a racemic mixture, or as separate enantiomers. Representative chiral linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing internucleoside linkages are well known to those skilled in the art.
• The oligonucleotides described herein contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations that may be defined, in terms of absolute stereochemistry, as (R) or (S), a or 13 such as for sugar anomers, or as (D) or (L) such as for amino acids etc. Included in the antisense compounds provided herein are all such possible isomers, as well as their racemic and optically pure forms.
• Neutral internucleoside linkages include without limitation, phosphotriesters, methylphosphonates, MMI (3′-CH₂-N(CH₃)—O-5′), amide-3 (3′-CH₂-C(═O)—N(H)-5′), amide-4 (3′-CH₂-N(H)—C(═O)-5′), formacetal (3′-O—CH₂-O-5′), and thioformacetal (3′-S-CH₂-O-5′). Further neutral internucleoside linkages include nonionic linkages comprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester and amides (See for example:Carbohydrate Modifications in Antisense Research; Y. S. Sanghvi and P. D. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Further neutral internucleoside linkages include nonionic linkages comprising mixed N, O, S and CH₂component parts.
• Certain Motifs
• In certain embodiments, the present invention provides oligomeric compounds comprising oligonucleotides. In certain embodiments, such oligonucleotides comprise one or more chemical modification. In certain embodiments, chemically modified oligonucleotides comprise one or more modified sugars. In certain embodiments, chemically modified oligonucleotides comprise one or more modified nucleobases. In certain embodiments, chemically modified oligonucleotides comprise one or more modified internucleoside linkages. In certain embodiments, the chemically modifications (sugar modifications, nucleobase modifications, and/or linkage modifications) define a pattern or motif. In certain embodiments, the patterns of chemical modifications of sugar moieties, internucleoside linkages, and nucleobases are each independent of one another. Thus, an oligonucleotide may be described by its sugar modification motif, internucleoside linkage motif and/or nucleobase modification motif (as used herein, nucleobase modification motif describes the chemical modifications to the nucleobases independent of the sequence of nucleobases).
• Certain Sugar Motifs
• In certain embodiments, oligonucleotides comprise one or more type of modified sugar moieties and/or naturally occurring sugar moieties arranged along an oligonucleotide or region thereof in a defined pattern or sugar modification motif. Such motifs may include any of the sugar modifications discussed herein and/or other known sugar modifications.
• In certain embodiments, the oligonucleotides comprise or consist of a region having a gapmer sugar modification motif, which comprises two external regions or “wings” and an internal region or “gap.” The three regions of a gapmer motif (the 5′-wing, the gap, and the 3′-wing) form a contiguous sequence of nucleosides wherein at least some of the sugar moieties of the nucleosides of each of the wings differ from at least some of the sugar moieties of the nucleosides of the gap. Specifically, at least the sugar moieties of the nucleosides of each wing that are closest to the gap (the 3′-most nucleoside of the 5′-wing and the 5′-most nucleoside of the 3′-wing) differ from the sugar moiety of the neighboring gap nucleosides, thus defining the boundary between the wings and the gap. In certain embodiments, the sugar moieties within the gap are the same as one another. In certain embodiments, the gap includes one or more nucleoside having a sugar moiety that differs from the sugar moiety of one or more other nucleosides of the gap. In certain embodiments, the sugar modification motifs of the two wings are the same as one another (symmetric gapmer). In certain embodiments, the sugar modification motifs of the 5′-wing differs from the sugar modification motif of the 3′-wing (asymmetric gapmer).
• Certain 5′-Wings
• In certain embodiments, the 5′-wing of a gapmer consists of 1 to 5 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 2 to 5 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 3 to 5 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 4 or 5 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 1 to 4 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 1 to 3 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 1 or 2 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 2 to 4 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 2 or 3 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 3 or 4 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 1 nucleoside. In certain embodiments, the 5′-wing of a gapmer consists of 2 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 3 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 4 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 5 linked nucleosides.
• In certain embodiments, the 5′-wing of a gapmer comprises at least one bicyclic nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least two bicyclic nucleosides. In certain embodiments, the 5′-wing of a gapmer comprises at least three bicyclic nucleosides. In certain embodiments, the 5′-wing of a gapmer comprises at least four bicyclic nucleosides. In certain embodiments, the 5′-wing of a gapmer comprises at least one constrained ethyl nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one LNA nucleoside. In certain embodiments, each nucleoside of the 5′-wing of a gapmer is a bicyclic nucleoside. In certain embodiments, each nucleoside of the 5′-wing of a gapmer is a constrained ethyl nucleoside. In certain embodiments, each nucleoside of the 5′-wing of a gapmer is a LNA nucleoside.
• In certain embodiments, the 5′-wing of a gapmer comprises at least one non-bicyclic modified nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one 2′-substituted nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one 2′-MOE nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one 2′-OMe nucleoside. In certain embodiments, each nucleoside of the 5′-wing of a gapmer is a non-bicyclic modified nucleoside. In certain embodiments, each nucleoside of the 5′-wing of a gapmer is a 2′-substituted nucleoside. In certain embodiments, each nucleoside of the 5′-wing of a gapmer is a 2′-MOE nucleoside. In certain embodiments, each nucleoside of the 5′-wing of a gapmer is a 2′-OMe nucleoside.
• In certain embodiments, the 5′-wing of a gapmer comprises at least one 2′-deoxynucleoside. In certain embodiments, each nucleoside of the 5′-wing of a gapmer is a 2′-deoxynucleoside. In a certain embodiments, the 5′-wing of a gapmer comprises at least one ribonucleoside. In certain embodiments, each nucleoside of the 5′-wing of a gapmer is a ribonucleoside.
• In certain embodiments, the 5′-wing of a gapmer comprises at least one bicyclic nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2′-substituted nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2′-MOE nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2′-OMe nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2′-deoxynucleoside.
• In certain embodiments, the 5′-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2′-substituted nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2′-MOE nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2′-OMe nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2′-deoxynucleoside.
• In certain embodiments, the 5′-wing of a gapmer comprises at least one LNA nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one LNA nucleoside and at least one 2′-substituted nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one LNA nucleoside and at least one 2′-MOE nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one LNA nucleoside and at least one 2′-OMe nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one LNA nucleoside and at least one 2′-deoxynucleoside.
• In certain embodiments, the 5′-wing of a gapmer comprises at least one bicyclic nucleoside, at least one non-bicyclic modified nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one constrained ethyl nucleoside, at least one non-bicyclic modified nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one LNA nucleoside, at least one non-bicyclic modified nucleoside, and at least one 2′-deoxynucleoside.
• In certain embodiments, the 5′-wing of a gapmer comprises at least one bicyclic nucleoside, at least one 2′-substituted nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one constrained ethyl nucleoside, at least one 2′-substituted nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one LNA nucleoside, at least one 2′-substituted nucleoside, and at least one 2′-deoxynucleoside.
• In certain embodiments, the 5′-wing of a gapmer comprises at least one bicyclic nucleoside, at least one 2′-MOE nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one constrained ethyl nucleoside, at least one 2′-MOE nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one LNA nucleoside, at least one 2′-MOE nucleoside, and at least one 2′-deoxynucleoside.
• In certain embodiments, the 5′-wing of a gapmer comprises at least one bicyclic nucleoside, at least one 2′-OMe nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one constrained ethyl nucleoside, at least one 2′-OMe nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one LNA nucleoside, at least one 2′-OMe nucleoside, and at least one 2′-deoxynucleoside.
• In certain embodiments, the 5′-wing of a gapmer has a sugar motif selected from among those listed in the following non-limiting table:

• TABLE 1
  Certain 5′-Wing Sugar Motifs

  	5′-wing
  	sugar
  	motif #	motif
  	1a	A-B-B
  	2a	A-A-B
  	3a	A-D-B
  	4a	B-D-A
  	5a	B-A-A
  	6a	B-B-B
  	7a	A-A-A
  	8a	A-D-D-B
  	9a	B-D-D-A
  	10a	A-A-A-B
  	11a	B-A-A-A
  	12a	A-A-A-A
  	13a	B-D-D-B
  	14a	A-A-A-A
  	15a	B-B-B-B
  	16a	A-A-A-A-A
  	17a	A-D-A-D-B
  	18a	A-D-B-D-A
  	19a	B-D-A-D-A
  	20a	A-A-A-A-B
  	21a	A-A-B-A-A
  	22a	B-A-A-A-A
  	1b	E-B-B
  	2b	E-E-B
  	3b	E-D-B
  	4b	B-D-E
  	5b	B-E-E
  	6b	B-B-B
  	7b	E-E-E
  	8b	E-D-D-B
  	9b	B-D-D-E
  	10b	E-E-E-B
  	11b	B-E-E-E
  	12b	E-E-E-E
  	13b	B-D-D-B
  	14b	E-E-E-E
  	15b	B-B-B-B
  	16b	E-E-E-E-E
  	17b	E-D-E-D-B
  	18b	E-D-B-D-E
  	19b	B-D-E-D-E
  	20b	E-E-E-E-B
  	21b	E-E-B-E-E
  	22b	B-E-E-E-E
  	1c	M-B-B
  	2c	M-M-B
  	3c	M-D-B
  	4c	B-D-M
  	5c	B-M-M
  	6c	B-B-B
  	7c	M-M-M
  	8c	M-D-D-B
  	9c	B-D-D-M
  	10c	M-M-M-B
  	11c	B-M-M-M
  	12c	M-M-M-M
  	13c	B-D-D-B
  	14c	M-M-M-M
  	15c	B-B-B-B
  	16c	M-M-M-M-M
  	17c	M-D-M-D-B
  	18c	M-D-B-D-M
  	19c	B-D-M-D-M
  	20c	M-M-M-M-B
  	21c	M-M-B-M-M
  	22c	B-M-M-M-M
  	1d	A-L-L
  	2d	A-A-L
  	3d	A-D-L
  	4d	L-D-A
  	5d	L-A-A
  	6d	L-L-L
  	7d	A-A-A
  	8d	A-D-D-L
  	9d	L-D-D-A
  	10d	A-A-A-L
  	11d	L-A-A-A
  	12d	A-A-A-A
  	13d	L-D-D-L
  	14d	A-A-A-A
  	15d	L-L-L-L
  	16d	A-A-A-A-A
  	17d	A-D-A-D-L
  	18d	A-D-L-D-A
  	19d	L-D-A-D-A
  	20d	A-A-A-A-L
  	21d	A-A-L-A-A
  	22d	L-A-A-A-A
  	1e	E-L-L
  	2e	E-E-L
  	3e	E-D-L
  	4e	L-D-E
  	5e	L-E-E
  	6e	L-L-L
  	7e	E-E-E
  	8e	E-D-D-L
  	9e	L-D-D-E
  	10e	E-E-E-L
  	11e	L-E-E-E
  	12e	E-E-E-E
  	13e	L-D-D-L
  	14e	E-E-E-E
  	15e	L-L-L-L
  	16e	E-E-E-E-E
  	17e	E-D-E-D-L
  	18e	E-D-L-D-E
  	19e	L-D-E-D-E
  	20e	E-E-E-E-L
  	21e	E-E-L-E-E
  	22e	L-E-E-E-E
  	1f	M-L-L
  	2f	M-M-L
  	3f	M-D-L
  	4f	L-D-M
  	5f	L-M-M
  	6f	L-L-L
  	7f	M-M-M
  	8f	M-D-D-L
  	9f	L-D-D-M
  	10f	M-M-M-L
  	11f	L-M-M-M
  	12f	M-M-M-M
  	13f	L-D-D-L
  	14f	M-M-M-M
  	15f	L-L-L-L
  	16f	M-M-M-M-M
  	17f	M-D-M-D-L
  	18f	M-D-L-D-M
  	19f	L-D-M-D-M
  	20f	M-M-M-M-L
  	21f	M-M-L-M-M
  	22f	L-M-M-M-M
  	1g	A-K-K
  	2g	A-A-K
  	3g	A-D-K
  	4g	K-D-A
  	5g	K-A-A
  	6g	K-K-K
  	7g	A-A-A
  	8g	A-D-D-K
  	9g	K-D-D-A
  	10g	A-A-A-K
  	11g	K-A-A-A
  	12g	A-A-A-A
  	13g	K-D-D-K
  	14g	A-A-A-A
  	15g	K-K-K-K
  	16g	A-A-A-A-A
  	17g	A-D-A-D-K
  	18g	A-D-K-D-A
  	19g	K-D-A-D-A
  	20g	A-A-A-A-K
  	21g	A-A-K-A-A
  	22g	K-A-A-A-A
  	1h	E-K-K
  	2h	E-E-K
  	3h	E-D-K
  	4h	K-D-E
  	5h	K-E-E
  	6h	K-K-K
  	7h	E-E-E
  	8h	E-D-D-K
  	9h	K-D-D-E
  	10h	E-E-E-K
  	11h	K-E-E-E
  	12h	E-E-E-E
  	13h	K-D-D-K
  	14h	E-E-E-E
  	15h	K-K-K-K
  	16h	E-E-E-E-E
  	17h	E-D-E-D-K
  	18h	E-D-K-D-E
  	19h	K-D-E-D-E
  	20h	E-E-E-E-K
  	21h	E-E-K-E-E
  	22h	K-E-E-E-E
  	1i	M-K-K
  	2i	M-M-K
  	3i	M-D-K
  	4i	K-D-M
  	5i	K-M-M
  	6i	K-K-K
  	7i	M-M-M
  	8i	M-D-D-K
  	9i	K-D-D-M
  	10i	M-M-M-K
  	11i	K-M-M-M
  	12i	M-M-M-M
  	13i	K-D-D-K
  	14i	M-M-M-M
  	15i	K-K-K-K
  	16i	M-M-M-M-M
  	17i	M-D-M-D-K
  	18i	M-D-K-D-M
  	19i	K-D-M-D-M
  	20i	M-M-M-M-K
  	21i	M-M-K-M-M
  	22i	K-M-M-M-M
  	1j	A-L-K
  	2j	M-E-K
  	3j	L-D-K
  	4j	K-D-A
  	5j	B-M-E
  	6j	K-L-L
  	7j	E-M-E
  	8j	E-D-D-M
  	9j	M-D-D-E
  	10j	E-M-E-B
  	11j	B-E-E-M
  	12j	E-E-E-M
  	13j	K-L-D-K
  	14j	E-M-E-M
  	15j	K-L-L-K
  	16j	E-E-M-E-E
  	17j	E-D-M-D-K
  	18j	E-D-K-D-M
  	19j	B-D-A-D-A
  	20j	E-M-E-E-L
  	21j	E-E-K-M-M
  	22j	B-E-M-E-A
  	1k	K-D-K-D-K
  	1k	A-K-L
  	2k	M-E-L
  	3k	K-D-L
  	4k	L-D-K
  	5k	L-M-E
  	6k	L-K-L
  	7k	M-E-M
  	8k	K-D-D-L
  	9k	L-D-K-E
  	10k	E-M-E-L
  	11k	L-E-E-M
  	12k	M-E-E-E
  	13k	L-K-D-L
  	14k	M-EM-E
  	15k	L-K-L-K
  	16k	M-E-E-E-M
  	17k	E-D-M-D-L
  	18k	E-D-L-D-M
  	19k	L-D-A-D-A
  	20k	E-M-M-E-L
  	21k	E-E-L-M-M
  	22k	L-E-A-M-A
  	1l	E-L-K
  	2l	E-M-K
  	3l	B-D-K
  	4l	K-B-L
  	5l	K-M-E
  	6l	L-K-K
  	7l	M-E-E
  	8l	L-D-D-K
  	9l	K-D-L-E
  	10l	E-M-E-K
  	11l	K-E-E-M
  	12l	E-M-E-E
  	13l	K-D-L-K
  	14l	E-E-M-E
  	15l	K-L-K-K
  	16l	E-E-M-M-E
  	17l	M-D-E-D-K
  	18l	M-D-K-D-E
  	19l	K-D-A-D-A
  	20l	M-E-E-E-K
  	21l	E-M-K-E-E
  	22l	K-E-A-A-A

• In the above table, “A” represents a nucleoside comprising a 2′-substituted sugar moiety; “B” represents a bicyclic nucleoside; “D” represents a 2′-deoxynucleoside; “K” represents a constrained ethyl nucleoside; “L” represents an LNA nucleoside; “E” represents a 2′-MOE nucleoside; and “M” represents a 2′-OMe nucleoside.
• In certain embodiments, an oligonucleotide comprises any 5′-wing motif provided herein. In certain such embodiments, the oligonucleotide is a 5′-hemimer (does not comprise a 3′-wing). In certain embodiments, such an oligonucleotide is a gapmer. In certain such embodiments, the 3′-wing of the gapmer may comprise any sugar modification motif.
• In certain embodiments, the 5′-wing of a gapmer has a sugar motif selected from among those listed in the following non-limiting tables:

• TABLE 2
  Certain 5′-Wing Sugar Motifs
  Certain 5′-Wing Sugar Motifs

  AAAAA	ABCBB	BABCC	BCBBA	CBACC
  AAAAB	ABCBC	BACAA	BCBBB	CBBAA
  AAAAC	ABCCA	BACAB	BCBBC	CBBAB
  AAABA	ABCCB	BACAC	BCBCA	CBBAC
  AAABB	ABCCC	BACBA	BCBCB	CBBBA
  AAABC	ACAAA	BACBB	BCBCC	CBBBB
  AAACA	ACAAB	BACBC	BCCAA	CBBBC
  AAACB	ACAAC	BACCA	BCCAB	CBBCA
  AAACC	ACABA	BACCB	BCCAC	CBBCB
  AABAA	ACABB	BACCC	BCCBA	CBBCC
  AABAB	ACABC	BBAAA	BCCBB	CBCAA
  AABAC	ACACA	BBAAB	BCCBC	CBCAB
  AABBA	ACACB	BBAAC	BCCCA	CBCAC
  AABBB	ACACC	BBABA	BCCCB	CBCBA
  AABBC	ACBAA	BBABB	BCCCC	CBCBB
  AABCA	ACBAB	BBABC	CAAAA	CBCBC
  AABCB	ACBAC	BBACA	CAAAB	CBCCA
  AABCC	ACBBA	BBACB	CAAAC	CBCCB
  AACAA	ACBBB	BBACC	CAABA	CBCCC
  AACAB	ACBBC	BBBAA	CAABB	CCAAA
  AACAC	ACBCA	BBBAB	CAABC	CCAAB
  AACBA	ACBCB	BBBAC	CAACA	CCAAC
  AACBB	ACBCC	BBBBA	CAACB	CCABA
  AACBC	ACCAA	BBBBB	CAACC	CCABB
  AACCA	ACCAB	BBBBC	CABAA	CCABC
  AACCB	ACCAC	BBBCA	CABAB	CCACA
  AACCC	ACCBA	BBBCB	CABAC	CCACB
  ABAAA	ACCBB	BBBCC	CABBA	CCACC
  ABAAB	ACCBC	BBCAA	CABBB	CCBAA
  ABAAC	ACCCA	BBCAB	CABBC	CCBAB
  ABABA	ACCCB	BBCAC	CABCA	CCBAC
  ABABB	ACCCC	BBCBA	CABCB	CCBBA
  ABABC	BAAAA	BBCBB	CABCC	CCBBB
  ABACA	BAAAB	BBCBC	CACAA	CCBBC
  ABACB	BAAAC	BBCCA	CACAB	CCBCA
  ABACC	BAABA	BBCCB	CACAC	CCBCB
  ABBAA	BAABB	BBCCC	CACBA	CCBCC
  ABBAB	BAABC	BCAAA	CACBB	CCCAA
  ABBAC	BAACA	BCAAB	CACBC	CCCAB
  ABBBA	BAACB	BCAAC	CACCA	CCCAC
  ABBBB	BAACC	BCABA	CACCB	CCCBA
  ABBBC	BABAA	BCABB	CACCC	CCCBB
  ABBCA	BABAB	BCABC	CBAAA	CCCBC
  ABBCB	BABAC	BCACA	CBAAB	CCCCA
  ABBCC	BABBA	BCACB	CBAAC	CCCCB
  ABCAA	BABBB	BCACC	CBABA	CCCCC
  ABCAB	BABBC	BCBAA	CBABB
  ABCAC	BABCA	BCBAB	CBABC
  ABCBA	BABCB	BCBAC	CBACA

• TABLE 3
  Certain 5′-Wing Sugar Motifs
  Certain 5′-Wing Sugar Motifs

  	AAAAA	BABC	CBAB	ABBB	BAA
  	AAAAB	BACA	CBAC	BAAA	BAB
  	AAABA	BACB	CBBA	BAAB	BBA
  	AAABB	BACC	CBBB	BABA	BBB
  	AABAA	BBAA	CBBC	BABB	AA
  	AABAB	BBAB	CBCA	BBAA	AB
  	AABBA	BBAC	CBCB	BBAB	AC
  	AABBB	BBBA	CBCC	BBBA	BA
  	ABAAA	BBBB	CCAA	BBBB	BB
  	ABAAB	BBBC	CCAB	AAA	BC
  	ABABA	BBCA	CCAC	AAB	CA
  	ABABB	BBCB	CCBA	AAC	CB
  	ABBAA	BBCC	CCBB	ABA	CC
  	ABBAB	BCAA	CCBC	ABB	AA
  	ABBBA	BCAB	CCCA	ABC	AB
  	ABBBB	BCAC	CCCB	ACA	BA
  	BAAAA	ABCB	BCBA	ACB
  	BAAAB	ABCC	BCBB	ACC
  	BAABA	ACAA	BCBC	BAA
  	BAABB	ACAB	BCCA	BAB
  	BABAA	ACAC	BCCB	BAC
  	BABAB	ACBA	BCCC	BBA
  	BABBA	ACBB	CAAA	BBB
  	BABBB	ACBC	CAAB	BBC
  	BBAAA	ACCA	CAAC	BCA
  	BBAAB	ACCB	CABA	BCB
  	BBABA	ACCC	CABB	BCC
  	BBABB	BAAA	CABC	CAA
  	BBBAA	BAAB	CACA	CAB
  	BBBAB	BAAC	CACB	CAC
  	BBBBA	BABA	CACC	CBA
  	BBBBB	BABB	CBAA	CBB
  	AAAA	AACC	CCCC	CBC
  	AAAB	ABAA	AAAA	CCA
  	AAAC	ABAB	AAAB	CCB
  	AABA	ABAC	AABA	CCC
  	AABB	ABBA	AABB	AAA
  	AABC	ABBB	ABAA	AAB
  	AACA	ABBC	ABAB	ABA
  	AACB	ABCA	ABBA	ABB

• In certain embodiments, each A, each B, and each C located at the 3′-most 5′-wing nucleoside is a modified nucleoside. For example, in certain embodiments the 5′-wing motif is selected from among ABB BBB, and CBB, wherein the underlined nucleoside represents the 3′-most 5′-wing nucleoside and wherein the underlined nucleoside is a modified nucleoside.
• In certain embodiments, each A comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, each A comprises a modified sugar moiety. In certain embodiments, each A comprises a 2′-substituted sugar moiety. In certain embodiments, each A comprises a 2′-substituted sugar moiety selected from among F, ara-F, OCH₃and O(CH₂)₂—OCH₃. In certain embodiments, each A comprises a bicyclic sugar moiety. In certain embodiments, each A comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, α-L-LNA, ENA and 2′-thio LNA. In certain embodiments, each A comprises a modified nucleobase. In certain embodiments, each A comprises a modified nucleobase selected from among 2-thio-thymidine nucleoside and 5-propyne uridine nucleoside. In certain embodiments, each A comprises an HNA. In certain embodiments, each A comprises an F-HNA.
• In certain embodiments, each B comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, each B comprises a modified sugar moiety. In certain embodiments, each B comprises a 2′-substituted sugar moiety. In certain embodiments, each B comprises a 2′-substituted sugar moiety selected from among F, (ara)-F, OCH₃and O(CH₂)₂—OCH₃. In certain embodiments, each B comprises a bicyclic sugar moiety. In certain embodiments, each B comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, α-L-LNA, ENA and 2′-thio LNA. In certain embodiments, each B comprises a modified nucleobase. In certain embodiments, each B comprises a modified nucleobase selected from among 2-thio-thymidine nucleoside and 5-propyne urindine nucleoside. In certain embodiments, each B comprises an HNA. In certain embodiments, each B comprises an F-HNA.
• In certain embodiments, each C comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, each C comprises a modified sugar moiety. In certain embodiments, each C comprises a 2′-substituted sugar moiety. In certain embodiments, each C comprises a 2′-substituted sugar moiety selected from among F, (ara)-F, OCH₃and O(CH₂)₂—OCH₃. In certain embodiments, each C comprises a 5′-substituted sugar moiety. In certain embodiments, each C comprises a 5′-substituted sugar moiety selected from among 5′-Me, and 5′-(R)-Me. In certain embodiments, each C comprises a bicyclic sugar moiety. In certain embodiments, each C comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, α-L-LNA, ENA and 2′-thio LNA. In certain embodiments, each C comprises a modified nucleobase. In certain embodiments, each C comprises a modified nucleobase selected from among 2-thio-thymidine and 5-propyne uridine. In certain embodiments, each C comprises a 2-thio-thymidine nucleoside. In certain embodiments, each C comprises an HNA. In certain embodiments, each C comprises an F-HNA.
• In certain embodiments, at least one of A or B comprises an unmodified 2′-deoxyfuranose sugar moiety, and the other comprises a 2′-substituted sugar moiety. In certain embodiments, at least one of A or B comprises an unmodified 2′-deoxyfuranose sugar moiety, and the other comprises a bicyclic sugar moiety.
• In certain embodiments, at least one of A or B comprises a bicyclic sugar moiety, and the other comprises a 2′-substituted sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises a 2′-substituted sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises a 2′-substituted sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside and the other of A or B comprises a 2′-substituted sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises a 2′-MOE sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises a 2′-MOE sugar moiety. In certain embodiments, one of A or B is an a -L-LNA nucleoside and the other of A or B comprises a 2′-MOE sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises a 2′-F sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises a 2′-F sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside and the other of A or B comprises a 2′-F sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises a 2′-(ara)-F sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises a 2′-(ara)-F sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside and the other of A or B comprises a 2′-(ara)-F sugar moiety.
• In certain embodiments, at least one of A or B comprises an unmodified 2′-deoxyfuranose sugar moiety, and the other comprises a 2′-substituted sugar moiety. In certain embodiments, one of A or B is an unmodified 2′-deoxyfuranose sugar moiety and the other of A or B comprises a 2′-substituted sugar moiety. In certain embodiments, one of A or B is an unmodified 2′-deoxyfuranose sugar moiety and the other of A or B comprises a 2′-MOE sugar moiety. In certain embodiments, one of A or B is an unmodified 2′-deoxyfuranose sugar moiety and the other of A or B comprises a 2′-F sugar moiety. In certain embodiments, one of A or B is an unmodified 2′-deoxyfuranose sugar moiety and the other of A or B comprises a 2′-(ara)-F sugar moiety. In certain embodiments, at least one of A or B comprises a bicyclic sugar moiety, and the other comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside and the other of A or B comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, one of A or B is an a -L-LNA nucleoside and the other of A or B comprises an unmodified 2′-deoxyfuranose sugar moiety.
• In certain embodiments, A comprises a bicyclic sugar moiety, and B comprises a 2′-substituted sugar moiety. In certain embodiments, A is an LNA nucleoside and B comprises a 2′-substituted sugar moiety. In certain embodiments, A is a cEt nucleoside and B comprises a 2′-substituted sugar moiety. In certain embodiments, A is an α-L-LNA nucleoside and B comprises a 2′-substituted sugar moiety.
• In certain embodiments, A comprises a bicyclic sugar moiety, and B comprises a 2′-MOE sugar moiety. In certain embodiments, A is an LNA nucleoside and B comprises a 2′-MOE sugar moiety. In certain embodiments, A is a cEt nucleoside and B comprises a 2′-MOE sugar moiety. In certain embodiments, A is an α-L-LNA nucleoside and B comprises a 2′-MOE sugar moiety.
• In certain embodiments, A comprises a bicyclic sugar moiety, and B comprises a 2′-F sugar moiety. In certain embodiments, A is an LNA nucleoside and B comprises a 2′-F sugar moiety. In certain embodiments, A is a cEt nucleoside and B comprises a 2′-F sugar moiety. In certain embodiments, A is an α-L-LNA nucleoside and B comprises a 2′-F sugar moiety.
• In certain embodiments, A comprises a bicyclic sugar moiety, and B comprises a 2′-(ara)-F sugar moiety. In certain embodiments, A is an LNA nucleoside and B comprises a 2′-(ara)-F sugar moiety. In certain embodiments, A is a cEt nucleoside and B comprises a 2′-(ara)-F sugar moiety. In certain embodiments, A is an α-L-LNA nucleoside and B comprises a 2′-(ara)-F sugar moiety.
• In certain embodiments, B comprises a bicyclic sugar moiety, and A comprises a 2′-MOE sugar moiety. In certain embodiments, B is an LNA nucleoside and A comprises a 2′-MOE sugar moiety. In certain embodiments, B is a cEt nucleoside and A comprises a 2′-MOE sugar moiety. In certain embodiments, B is an α-L-LNA nucleoside and A comprises a 2′-MOE sugar moiety.
• In certain embodiments, B comprises a bicyclic sugar moiety, A comprises a 2′-MOE sugar moiety, and C comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, B is an LNA nucleoside, A comprises a 2′-MOE sugar moiety, and C comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, B is a cEt nucleoside, A comprises a 2′-MOE sugar moiety, and C comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, B is an α-L-LNA nucleoside and A comprises a 2′-MOE sugar moiety.
• In certain embodiments, B comprises a bicyclic sugar moiety, and A comprises a 2′-F sugar moiety. In certain embodiments, B is an LNA nucleoside and A comprises a 2′-F sugar moiety. In certain embodiments, B is a cEt nucleoside and A comprises a 2′-F sugar moiety. In certain embodiments, B is an α-L-LNA nucleoside and A comprises a 2′-F sugar moiety.
• In certain embodiments, B comprises a bicyclic sugar moiety, and A comprises a 2′-(ara)-F sugar moiety. In certain embodiments, B is an LNA nucleoside and A comprises a 2′-(ara)-F sugar moiety. In certain embodiments, B is a cEt nucleoside and A comprises a 2′-(ara)-F sugar moiety. In certain embodiments, B is an α-L-LNA nucleoside and A comprises a 2′-(ara)-F sugar moiety.
• In certain embodiments, at least one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-substituted sugar moiety and C comprises a modified nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and C comprises a modified nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and C comprises a modified nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and comprises a modified nucleobase.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a modified nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a modified nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a modified nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and comprises a modified nucleobase.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and C comprises a modified nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and comprises a modified nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a modified nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a modified nucleobase.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a modified nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a modified nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a modified nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a modified nucleobase.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-substituted sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and C comprises a 2-thio-thymidine nucleobase.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 2-thio-thymidine nucleobase.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 2-thio-thymidine nucleobase.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises 2-thio-thymidine nucleobase.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5-propyne uridine nucleobase.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and C comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5-propyne uridine nucleobase.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5-propyne uridine nucleobase.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5-propyne uridine nucleobase.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a sugar surrogate.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and C comprises a sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a sugar surrogate.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises sugar surrogate.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a HNA sugar surrogate.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and C comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a sugar HNA surrogate.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a HNA sugar surrogate.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a F-HNA sugar surrogate.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and C comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a F-HNA sugar surrogate.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a F-HNA sugar surrogate.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5′-Me DNA sugar moiety.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5′-Me DNA sugar moiety.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5′-Me DNA sugar moiety.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety.
• In certain embodiments, at least two of A, B or C comprises a 2′-substituted sugar moiety, and the other comprises a bicyclic sugar moiety. In certain embodiments, at least two of A, B or C comprises a bicyclic sugar moiety, and the other comprises a 2′-substituted sugar moiety.
• In certain embodiments, at least two of A, B or C comprises a 2′-substituted sugar moiety, and the other comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, at least two of A, B or C comprises a bicyclic sugar moiety, and the other comprises an unmodified 2′-deoxyfuranose sugar moiety.
• Certain 3′-Wings
• In certain embodiments, the 3′-wing of a gapmer consists of 1 to 5 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 2 to 5 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 3 to 5 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 4 or 5 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 1 to 4 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 1 to 3 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 1 or 2 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 2 to 4 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 2 or 3 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 3 or 4 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 1 nucleoside. In certain embodiments, the 3′-wing of a gapmer consists of 2 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 31 inked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 4 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 5 linked nucleosides.
• In certain embodiments, the 3′-wing of a gapmer comprises at least one bicyclic nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one constrained ethyl nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one LNA nucleoside. In certain embodiments, each nucleoside of the 3′-wing of a gapmer is a bicyclic nucleoside. In certain embodiments, each nucleoside of the 3′-wing of a gapmer is a constrained ethyl nucleoside. In certain embodiments, each nucleoside of the 3′-wing of a gapmer is a LNA nucleoside.
• In certain embodiments, the 3′-wing of a gapmer comprises at least one non-bicyclic modified nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least two non-bicyclic modified nucleosides. In certain embodiments, the 3′-wing of a gapmer comprises at least three non-bicyclic modified nucleosides. In certain embodiments, the 3′-wing of a gapmer comprises at least four non-bicyclic modified nucleosides. In certain embodiments, the 3′-wing of a gapmer comprises at least one 2′-substituted nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one 2′-MOE nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one 2′-OMe nucleoside. In certain embodiments, each nucleoside of the 3′-wing of a gapmer is a non-bicyclic modified nucleoside. In certain embodiments, each nucleoside of the 3′-wing of a gapmer is a 2′-substituted nucleoside. In certain embodiments, each nucleoside of the 3′-wing of a gapmer is a 2′-MOE nucleoside. In certain embodiments, each nucleoside of the 3′-wing of a gapmer is a 2′-OMe nucleoside.
• In certain embodiments, the 3′-wing of a gapmer comprises at least one 2′-deoxynucleoside. In certain embodiments, each nucleoside of the 3′-wing of a gapmer is a 2′-deoxynucleoside. In a certain embodiments, the 3′-wing of a gapmer comprises at least one ribonucleoside. In certain embodiments, each nucleoside of the 3′-wing of a gapmer is a ribonucleoside.
• In certain embodiments, the 3′-wing of a gapmer comprises at least one bicyclic nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2′-substituted nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2′-MOE nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2′-OMe nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2′-deoxynucleoside.
• In certain embodiments, the 3′-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2′-substituted nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2′-MOE nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2′-OMe nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2′-deoxynucleoside.
• In certain embodiments, the 3′-wing of a gapmer comprises at least one LNA nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one LNA nucleoside and at least one 2′-substituted nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one LNA nucleoside and at least one 2′-MOE nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one LNA nucleoside and at least one 2′-OMe nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one LNA nucleoside and at least one 2′-deoxynucleoside.
• In certain embodiments, the 3′-wing of a gapmer comprises at least one bicyclic nucleoside, at least one non-bicyclic modified nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one constrained ethyl nucleoside, at least one non-bicyclic modified nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one LNA nucleoside, at least one non-bicyclic modified nucleoside, and at least one 2′-deoxynucleoside.
• In certain embodiments, the 3′-wing of a gapmer comprises at least one bicyclic nucleoside, at least one 2′-substituted nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one constrained ethyl nucleoside, at least one 2′-substituted nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one LNA nucleoside, at least one 2′-substituted nucleoside, and at least one 2′-deoxynucleoside.
• In certain embodiments, the 3′-wing of a gapmer comprises at least one bicyclic nucleoside, at least one 2′-MOE nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one constrained ethyl nucleoside, at least one 2′-MOE nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one LNA nucleoside, at least one 2′-MOE nucleoside, and at least one 2′-deoxynucleoside.
• In certain embodiments, the 3′-wing of a gapmer comprises at least one bicyclic nucleoside, at least one 2′-OMe nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one constrained ethyl nucleoside, at least one 2′-OMe nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one LNA nucleoside, at least one 2′-OMe nucleoside, and at least one 2′-deoxynucleoside.
• In certain embodiments, the 3′-wing of a gapmer has a sugar motif selected from among those listed in the following non-limiting table:

• TABLE 4
  Certain 3′-Wing Sugar Motifs

  3′-wing		3′-wing		3′-wing
  sugar		sugar		sugar
  motif #	motif	motif #	motif	motif #	motif
  1a	B-B-A	1d	L-L-A	1g	K-K-A
  2a	B-B-B	2d	L-L-L	2g	K-K-K
  3a	A-A-B	3d	A-A-L	3g	A-A-K
  4a	B-A-B	4d	L-A-L	4g	K-A-K
  5a	B-A-B-A	5d	L-A-L-A	5g	K-A-K-A
  6a	B-B-B-A	6d	L-L-L-A	6g	K-K-K-A
  7a	B-D-B-A	7d	L-D-L-A	7g	K-D-K-A
  8a	B-B-B-B	8d	L-L-L-L	8g	K-K-K-K
  9a	B-D-D-B	9d	L-D-D-L	9g	K-D-D-K
  10a	A-B-B-A	10d	A-L-L-A	10g	A-K-K-A
  1b	B-B-E	1e	L-L-E	1h	K-K-E
  2b	B-B-B	2e	L-L-L	2h	K-K-K
  3b	E-E-B	3e	E-E-L	3h	E-E-K
  4b	B-E-B	4e	L-E-L	4h	K-E-K
  5b	B-E-B-E	5e	L-E-L-E	5h	K-E-K-E
  6b	B-B-B-E	6e	L-L-L-E	6h	K-K-K-E
  7b	B-D-B-E	7e	L-D-L-E	7h	K-D-K-E
  8b	B-B-B-B	8e	L-L-L-L	8h	K-K-K-K
  9b	B-D-D-B	9e	L-D-D-L	9h	K-D-D-K
  10b	E-B-B-E	10e	E-L-L-E	10h	E-K-K-E
  1c	B-B-M	1f	L-L-M	1i	K-K-M
  2c	B-B-B	2f	L-L-L	2i	K-K-K
  3c	M-M-B	3f	M-M-L	3i	M-M-K
  4c	B-M-B	4f	L-M-L	4i	K-M-K
  5c	B-M-B-M	5f	L-M-L-M	5i	K-M-K-M
  6c	B-B-B-M	6f	L-L-L-M	6i	K-K-K-M
  7c	B-D-B-M	7f	L-D-L-M	7i	K-D-K-M
  8c	B-B-B-B	8f	L-L-L-L	8i	K-K-K-K
  9c	B-D-D-B	9f	L-D-D-L	9i	K-D-D-K
  10c	M-B-B-M	10f	M-L-L-M	10i	M-K-K-M
  1j	K-K-A	1k	L-K-A	1l	K-L-E
  2j	K-L-L	2k	K-K-L	2l	K-L-K
  3j	E-M-B	3k	E-M-L	3l	E-K-K
  4j	K-A-L	4k	L-A-K	4l	L-E-K
  5j	K-A-L-A	5k	L-A-K-A	5l	K-E-L-E
  6j	K-L-K-A	6k	K-K-L-A	6l	K-L-K-A
  7j	L-D-K-A	7k	K-D-L-A	7l	K-D-L-E
  8j	B-K-L-B	8k	K-L-L-L	8l	K-K-L-K
  9j	K-D-D-B	9k	K-D-D-L	9l	L-D-D-K
  10j	A-K-B-A	10k	A-K-L-A	10l	A-B-K-A
  1m	E-E

• In the above table, “A” represents a nucleoside comprising a 2′-substituted sugar moiety; “B” represents a bicyclic nucleoside; “D” represents a 2′-deoxynucleoside; “K” represents a constrained ethyl nucleoside; “L” represents an LNA nucleoside; “E” represents a 2′-MOE nucleoside; and “M” represents a 2′-OMe nucleoside.
• In certain embodiments, an oligonucleotide comprises any 3′-wing motif provided herein. In certain such embodiments, the oligonucleotide is a 3′-hemimer (does not comprise a 5′-wing). In certain embodiments, such an oligonucleotide is a gapmer. In certain such embodiments, the 5′-wing of the gapmer may comprise any sugar modification motif.
• In certain embodiments, the 5′-wing of a gapmer has a sugar motif selected from among those listed in the following non-limiting tables:

• TABLE 5
  Certain 3′-Wing Sugar Motifs
  Certain 3′-Wing Sugar Motifs

  AAAAA	ABCBB	BABCC	BCBBA	CBACC
  AAAAB	ABCBC	BACAA	BCBBB	CBBAA
  AAAAC	ABCCA	BACAB	BCBBC	CBBAB
  AAABA	ABCCB	BACAC	BCBCA	CBBAC
  AAABB	ABCCC	BACBA	BCBCB	CBBBA
  AAABC	ACAAA	BACBB	BCBCC	CBBBB
  AAACA	ACAAB	BACBC	BCCAA	CBBBC
  AAACB	ACAAC	BACCA	BCCAB	CBBCA
  AAACC	ACABA	BACCB	BCCAC	CBBCB
  AABAA	ACABB	BACCC	BCCBA	CBBCC
  AABAB	ACABC	BBAAA	BCCBB	CBCAA
  AABAC	ACACA	BBAAB	BCCBC	CBCAB
  AABBA	ACACB	BBAAC	BCCCA	CBCAC
  AABBB	ACACC	BBABA	BCCCB	CBCBA
  AABBC	ACBAA	BBABB	BCCCC	CBCBB
  AABCA	ACBAB	BBABC	CAAAA	CBCBC
  AABCB	ACBAC	BBACA	CAAAB	CBCCA
  AABCC	ACBBA	BBACB	CAAAC	CBCCB
  AACAA	ACBBB	BBACC	CAABA	CBCCC
  AACAB	ACBBC	BBBAA	CAABB	CCAAA
  AACAC	ACBCA	BBBAB	CAABC	CCAAB
  AACBA	ACBCB	BBBAC	CAACA	CCAAC
  AACBB	ACBCC	BBBBA	CAACB	CCABA
  AACBC	ACCAA	BBBBB	CAACC	CCABB
  AACCA	ACCAB	BBBBC	CABAA	CCABC
  AACCB	ACCAC	BBBCA	CABAB	CCACA
  AACCC	ACCBA	BBBCB	CABAC	CCACB
  ABAAA	ACCBB	BBBCC	CABBA	CCACC
  ABAAB	ACCBC	BBCAA	CABBB	CCBAA
  ABAAC	ACCCA	BBCAB	CABBC	CCBAB
  ABABA	ACCCB	BBCAC	CABCA	CCBAC
  ABABB	ACCCC	BBCBA	CABCB	CCBBA
  ABABC	BAAAA	BBCBB	CABCC	CCBBB
  ABACA	BAAAB	BBCBC	CACAA	CCBBC
  ABACB	BAAAC	BBCCA	CACAB	CCBCA
  ABACC	BAABA	BBCCB	CACAC	CCBCB
  ABBAA	BAABB	BBCCC	CACBA	CCBCC
  ABBAB	BAABC	BCAAA	CACBB	CCCAA
  ABBAC	BAACA	BCAAB	CACBC	CCCAB
  ABBBA	BAACB	BCAAC	CACCA	CCCAC
  ABBBB	BAACC	BCABA	CACCB	CCCBA
  ABBBC	BABAA	BCABB	CACCC	CCCBB
  ABBCA	BABAB	BCABC	CBAAA	CCCBC
  ABBCB	BABAC	BCACA	CBAAB	CCCCA
  ABBCC	BABBA	BCACB	CBAAC	CCCCB
  ABCAA	BABBB	BCACC	CBABA	CCCCC
  ABCAB	BABBC	BCBAA	CBABB
  ABCAC	BABCA	BCBAB	CBABC
  ABCBA	BABCB	BCBAC	CBACA

• TABLE 6
  Certain 3′-Wing Sugar Motifs
  Certain 3′-Wing Sugar Motifs

  	AAAAA	BABC	CBAB	ABBB	BAA
  	AAAAB	BACA	CBAC	BAAA	BAB
  	AAABA	BACB	CBBA	BAAB	BBA
  	AAABB	BACC	CBBB	BABA	BBB
  	AABAA	BBAA	CBBC	BABB	AA
  	AABAB	BBAB	CBCA	BBAA	AB
  	AABBA	BBAC	CBCB	BBAB	AC
  	AABBB	BBBA	CBCC	BBBA	BA
  	ABAAA	BBBB	CCAA	BBBB	BB
  	ABAAB	BBBC	CCAB	AAA	BC
  	ABABA	BBCA	CCAC	AAB	CA
  	ABABB	BBCB	CCBA	AAC	CB
  	ABBAA	BBCC	CCBB	ABA	CC
  	ABBAB	BCAA	CCBC	ABB	AA
  	ABBBA	BCAB	CCCA	ABC	AB
  	ABBBB	BCAC	CCCB	ACA	BA
  	BAAAA	ABCB	BCBA	ACB
  	BAAAB	ABCC	BCBB	ACC
  	BAABA	ACAA	BCBC	BAA
  	BAABB	ACAB	BCCA	BAB
  	BABAA	ACAC	BCCB	BAC
  	BABAB	ACBA	BCCC	BBA
  	BABBA	ACBB	CAAA	BBB
  	BABBB	ACBC	CAAB	BBC
  	BBAAA	ACCA	CAAC	BCA
  	BBAAB	ACCB	CABA	BCB
  	BBABA	ACCC	CABB	BCC
  	BBABB	BAAA	CABC	CAA
  	BBBAA	BAAB	CACA	CAB
  	BBBAB	BAAC	CACB	CAC
  	BBBBA	BABA	CACC	CBA
  	BBBBB	BABB	CBAA	CBB
  	AAAA	AACC	CCCC	CBC
  	AAAB	ABAA	AAAA	CCA
  	AAAC	ABAB	AAAB	CCB
  	AABA	ABAC	AABA	CCC
  	AABB	ABBA	AABB	AAA
  	AABC	ABBB	ABAA	AAB
  	AACA	ABBC	ABAB	ABA
  	AACB	ABCA	ABBA	ABB

• In certain embodiments, each A, each B, and each C located at the 5′-most 3′-wing region nucleoside is a modified nucleoside. For example, in certain embodiments the 3′-wing motif is selected from among ABB, BBB, and CBB, wherein the underlined nucleoside represents the the 5′-most 3′-wing region nucleoside and wherein the underlined nucleoside is a modified nucleoside.
• In certain embodiments, each A comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, each A comprises a modified sugar moiety. In certain embodiments, each A comprises a 2′-substituted sugar moiety. In certain embodiments, each A comprises a 2′-substituted sugar moiety selected from among F, ara-F, OCH₃and O(CH₂)₂—OCH₃. In certain embodiments, each A comprises a bicyclic sugar moiety. In certain embodiments, each A comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, α-L-LNA, ENA and 2′-thio LNA. In certain embodiments, each A comprises a modified nucleobase. In certain embodiments, each A comprises a modified nucleobase selected from among 2-thio-thymidine nucleoside and 5-propyne uridine nucleoside.
• In certain embodiments, each B comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, each B comprises a modified sugar moiety. In certain embodiments, each B comprises a 2′-substituted sugar moiety. In certain embodiments, each B comprises a 2′-substituted sugar moiety selected from among F, (ara)-F, OCH₃and O(CH₂)₂—OCH₃. In certain embodiments, each B comprises a bicyclic sugar moiety. In certain embodiments, each B comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, α-L-LNA, ENA and 2′-thio LNA. In certain embodiments, each B comprises a modified nucleobase. In certain embodiments, each B comprises a modified nucleobase selected from among 2-thio-thymidine nucleoside and 5-propyne uridine nucleoside.
• In certain embodiments, each C comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, each C comprises a modified sugar moiety. In certain embodiments, each C comprises a 2′-substituted sugar moiety. In certain embodiments, each C comprises a 2′-substituted sugar moiety selected from among F, (ara)-F, OCH₃and O(CH₂)₂—OCH₃. In certain embodiments, each C comprises a 5′-substituted sugar moiety. In certain embodiments, each C comprises a 5′-substituted sugar moiety selected from among 5′-Me, and 5′-(R)-Me. In certain embodiments, each C comprises a bicyclic sugar moiety. In certain embodiments, each C comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, α-L-LNA, ENA and 2′-thio LNA. In certain embodiments, each C comprises a modified nucleobase. In certain embodiments, each C comprises a modified nucleobase selected from among 2-thio-thymidine and 5-propyne uridine. In certain embodiments, each C comprises a 2-thio-thymidine nucleoside.
• In certain embodiments, at least one of A or B comprises an unmodified 2′-deoxyfuranose sugar moiety, and the other comprises a 2′-substituted sugar moiety. In certain embodiments, at least one of A or B comprises an unmodified 2′-deoxyfuranose sugar moiety, and the other comprises a bicyclic sugar moiety. In certain embodiments, at least one of A or B comprises a bicyclic sugar moiety, and the other comprises a 2′-substituted sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises a 2′-substituted sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises a 2′-substituted sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside and the other of A or B comprises a 2′-substituted sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises a 2′-MOE sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises a 2′-MOE sugar moiety. In certain embodiments, one of A or B is an a -L-LNA nucleoside and the other of A or B comprises a 2′-MOE sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises a 2′-F sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises a 2′-F sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside and the other of A or B comprises a 2′-F sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises a 2′-(ara)-F sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises a 2′-(ara)-F sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside and the other of A or B comprises a 2′-(ara)-F sugar moiety.
• In certain embodiments, at least one of A or B comprises an unmodified 2′-deoxyfuranose sugar moiety, and the other comprises a 2′-substituted sugar moiety. In certain embodiments, one of A or B is an unmodified 2′-deoxyfuranose sugar moiety and the other of A or B comprises a 2′-substituted sugar moiety. In certain embodiments, one of A or B is an unmodified 2′-deoxyfuranose sugar moiety and the other of A or B comprises a 2′-MOE sugar moiety. In certain embodiments, one of A or B is an unmodified 2′-deoxyfuranose sugar moiety and the other of A or B comprises a 2′-F sugar moiety. In certain embodiments, one of A or B is an unmodified 2′-deoxyfuranose sugar moiety and the other of A or B comprises a 2′-(ara)-F sugar moiety. In certain embodiments, at least one of A or B comprises a bicyclic sugar moiety, and the other comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside and the other of A or B comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, one of A or B is an a -L-LNA nucleoside and the other of A or B comprises an unmodified 2′-deoxyfuranose sugar moiety.
• In certain embodiments, A comprises a bicyclic sugar moiety, and B comprises a 2′-substituted sugar moiety. In certain embodiments, A is an LNA nucleoside and B comprises a 2′-substituted sugar moiety. In certain embodiments, A is a cEt nucleoside and B comprises a 2′-substituted sugar moiety. In certain embodiments, A is an α-L-LNA nucleoside and B comprises a 2′-substituted sugar moiety.
• In certain embodiments, A comprises a bicyclic sugar moiety, and B comprises a 2′-MOE sugar moiety. In certain embodiments, A is an LNA nucleoside and B comprises a 2′-MOE sugar moiety. In certain embodiments, A is a cEt nucleoside and B comprises a 2′-MOE sugar moiety. In certain embodiments, A is an α-L-LNA nucleoside and B comprises a 2′-MOE sugar moiety.
• In certain embodiments, A comprises a bicyclic sugar moiety, and B comprises a 2′-F sugar moiety. In certain embodiments, A is an LNA nucleoside and B comprises a 2′-F sugar moiety. In certain embodiments, A is a cEt nucleoside and B comprises a 2′-F sugar moiety. In certain embodiments, A is an α-L-LNA nucleoside and B comprises a 2′-F sugar moiety.
• In certain embodiments, A comprises a bicyclic sugar moiety, and B comprises a 2′-(ara)-F sugar moiety. In certain embodiments, A is an LNA nucleoside and B comprises a 2′-(ara)-F sugar moiety. In certain embodiments, A is a cEt nucleoside and B comprises a 2′-(ara)-F sugar moiety. In certain embodiments, A is an α-L-LNA nucleoside and B comprises a 2′-(ara)-F sugar moiety.
• In certain embodiments, B comprises a bicyclic sugar moiety, and A comprises a 2′-MOE sugar moiety. In certain embodiments, B is an LNA nucleoside and A comprises a 2′-MOE sugar moiety. In certain embodiments, B is a cEt nucleoside and A comprises a 2′-MOE sugar moiety. In certain embodiments, B is an α-L-LNA nucleoside and A comprises a 2′-MOE sugar moiety.
• In certain embodiments, B comprises a bicyclic sugar moiety, A comprises a 2′-MOE sugar moiety, and C comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, B is an LNA nucleoside, A comprises a 2′-MOE sugar moiety, and C comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, B is a cEt nucleoside, A comprises a 2′-MOE sugar moiety, and C comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, B is an α-L-LNA nucleoside and A comprises a 2′-MOE sugar moiety.
• In certain embodiments, B comprises a bicyclic sugar moiety, and A comprises a 2′-F sugar moiety. In certain embodiments, B is an LNA nucleoside and A comprises a 2′-F sugar moiety. In certain embodiments, B is a cEt nucleoside and A comprises a 2′-F sugar moiety. In certain embodiments, B is an α-L-LNA nucleoside and A comprises a 2′-F sugar moiety.
• In certain embodiments, B comprises a bicyclic sugar moiety, and A comprises a 2′-(ara)-F sugar moiety. In certain embodiments, B is an LNA nucleoside and A comprises a 2′-(ara)-F sugar moiety. In certain embodiments, B is a cEt nucleoside and A comprises a 2′-(ara)-F sugar moiety. In certain embodiments, B is an α-L-LNA nucleoside and A comprises a 2′-(ara)-F sugar moiety.
• In certain embodiments, at least one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-substituted sugar moiety and C comprises a modified nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and C comprises a modified nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and C comprises a modified nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and
Comprises a Modified Nucleobase.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a modified nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a modified nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a modified nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and comprises a modified nucleobase.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and C comprises a modified nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and comprises a modified nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a modified nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a modified nucleobase.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a modified nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a modified nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a modified nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a modified nucleobase.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-substituted sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and C comprises a 2-thio-thymidine nucleobase.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 2-thio-thymidine nucleobase.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 2-thio-thymidine nucleobase.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises 2-thio-thymidine nucleobase.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5-propyne uridine nucleobase.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and C comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5-propyne uridine nucleobase.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5-propyne uridine nucleobase.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5-propyne uridine nucleobase.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a sugar surrogate.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and C comprises a sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a sugar surrogate.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises sugar surrogate. In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a HNA sugar surrogate.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and C comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a sugar HNA surrogate.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a HNA sugar surrogate.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a F-HNA sugar surrogate.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and C comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a F-HNA sugar surrogate.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a F-HNA sugar surrogate.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5′-Me DNA sugar moiety.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5′-Me DNA sugar moiety.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5′-Me DNA sugar moiety.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety.
• In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety.
• In certain embodiments, at least two of A, B or C comprises a 2′-substituted sugar moiety, and the other comprises a bicyclic sugar moiety. In certain embodiments, at least two of A, B or C comprises a bicyclic sugar moiety, and the other comprises a 2′-substituted sugar moiety.
• In certain embodiments, at least two of A, B or C comprises a 2′-substituted sugar moiety, and the other comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, at least two of A, B or C comprises a bicyclic sugar moiety, and the other comprises an unmodified 2′-deoxyfuranose sugar moiety.
• Certain Gaps
• In certain embodiments, the gap of a gapmer consists of 6 to 20 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 to 15 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 to 12 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 to 10 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 to 9 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 to 8 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 or 7 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 7 to 10 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 7 to 9 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 7 or 8 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 8 to 10 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 8 or 9 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 7 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 8 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 9 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 10 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 11 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 12 linked nucleosides.
• In certain embodiments, each nucleotide of the gap of a gapmer is a 2′-deoxynucleoside. In certain embodiments, the gap comprises one or more modified nucleosides. In certain embodiments, each nucleotide of the gap of a gapmer is a 2′-deoxynucleoside or is a modified nucleoside that is “DNA-like.” In such embodiments, “DNA-like” means that the nucleoside has similar characteristics to DNA, such that a duplex comprising the gapmer and an RNA molecule is capable of activating RNase H. For example, under certain conditions, 2′-fluoro (arabino) nucleosides (also referred to as FANA) have been shown to support RNase H activation, and thus is DNA-like. In certain embodiments, one or more nucleosides of the gap of a gapmer is not a 2′-deoxynucleoside and is not DNA-like. In certain such embodiments, the gapmer nonetheless supports RNase H activation (e.g., by virtue of the number or placement of the non-DNA nucleosides).
• Certain Gapmer Motifs
• In certain embodiments, a gapmer comprises a 5′-wing, a gap, and a 3′ wing, wherein the 5′-wing, gap, and 3′ wing are independently selected from among those discussed above. For example, in certain embodiments, a gapmer has a 5′-wing selected from any of the 5′-wing motifs in Tables 1, 2, and 3 above and a 3′-wing selected from any of the 3′-wing motifs in Tables, 4, 5, and 6. For example, in certain embodiments, a gapmer has a 5′-wing, a gap, and a 3′-wing having features selected from among those listed in the following non-limiting table:

• TABLE 7
  Certain Gapmer Sugar Motifs

  Gapmer
  motif #	5-wing	Gap	3′-wing

  1	At least one non-bicyclic	All 2′-deoxynucleosides	At least one bicyclic
  	modified nucleoside		nucleoside
  2	At least one non-bicyclic	All 2′-deoxynucleosides	At least one LNA nucleoside
  	modified nucleoside
  3	At least one non-bicyclic	All 2′-deoxynucleosides	At least one cEt nucleoside
  	modified nucleoside
  4	At least one 2′-substituted	All 2′-deoxynucleosides	At least one bicyclic
  	nucleoside		nucleoside
  5	At least one 2′-substituted	All 2′-deoxynucleosides	At least one LNA nucleoside
  	nucleoside
  6	At least one 2′-substituted	All 2′-deoxynucleosides	At least one cEt nucleoside
  	nucleoside
  7	At least one 2′-MOE nucleoside	All 2′-deoxynucleosides	At least one bicyclic
  			nucleoside
  8	At least one 2′-MOE nucleoside	All 2′-deoxynucleosides	At least one LNA nucleoside
  9	At least one 2′-MOE nucleoside	All 2′-deoxynucleosides	At least one cEt nucleoside
  10	At least one 2′-OMe nucleoside	All 2′-deoxynucleosides	At least one bicyclic
  			nucleoside
  11	At least one 2′-OMe nucleoside	All 2′-deoxynucleosides	At least one LNA nucleoside
  12	At least one 2′-OMe nucleoside	All 2′-deoxynucleosides	At least one cEt nucleoside
  13	At least one 2′-deoxynucleoside	All 2′-deoxynucleosides	At least one bicyclic
  			nucleoside
  14	At least one 2′-deoxynucleoside	All 2′-deoxynucleosides	At least one LNA nucleoside
  15	At least one 2′-deoxynucleoside	All 2′-deoxynucleosides	At least one cEt nucleoside
  16	At least one bicyclic nucleoside	All 2′-deoxynucleosides	At least one non-bicyclic
  			modified nucleoside
  17	At least one LNA nucleoside	All 2′-deoxynucleosides	At least one non-bicyclic
  			modified nucleoside
  18	At least one cEt nucleoside	All 2′-deoxynucleosides	At least one non-bicyclic
  			modified nucleoside
  19	At least one bicyclic nucleoside	All 2′-deoxynucleosides	At least one 2′-substituted
  			nucleoside
  20	At least one LNA nucleoside	All 2′-deoxynucleosides	At least one 2′-substituted
  			nucleoside
  21	At least one cEt nucleoside	All 2′-deoxynucleosides	At least one 2′-substituted
  			nucleoside
  22	At least one bicyclic nucleoside	All 2′-deoxynucleosides	At least one 2′-MOE
  			nucleoside
  23	At least one LNA nucleoside	All 2′-deoxynucleosides	At least one 2′-MOE
  			nucleoside
  24	At least one cEt nucleoside	All 2′-deoxynucleosides	At least one 2′-MOE
  			nucleoside
  25	At least one bicyclic nucleoside	All 2′-deoxynucleosides	At least one 2′-OMe
  			nucleoside
  26	At least one LNA nucleoside	All 2′-deoxynucleosides	At least one 2′-OMe
  			nucleoside
  27	At least one cEt nucleoside	All 2′-deoxynucleosides	At least one 2′-OMe
  			nucleoside
  28	At least one bicyclic nucleoside	All 2′-deoxynucleosides	At least one 2′-
  			deoxynucleoside
  29	At least one LNA nucleoside	All 2′-deoxynucleosides	At least one 2′-
  			deoxynucleoside
  30	At least one cEt nucleoside	All 2′-deoxynucleosides	At least one 2′-
  			deoxynucleoside
  31	At least one bicyclic nucleoside	All 2′-deoxynucleosides	At least one bicyclic
  	and at least one 2′-substituted		nucleoside and at least one 2′-
  	nucleoside		substituted nucleoside
  32	At least one bicyclic nucleoside	All 2′-deoxynucleosides	At least two bicyclic
  	and at least one 2′-substituted		nucleosides
  	nucleoside
  33	At least one cEt nucleoside and	All 2′-deoxynucleosides	At least one bicyclic
  	at least one 2′-substituted		nucleoside and at least one 2′-
  	nucleoside		substituted nucleoside
  34	At least one cEt nucleoside and	All 2′-deoxynucleosides	At least two bicyclic
  	at least one 2′-substituted		nucleosides
  	nucleoside
  35	At least one LNA nucleoside and	All 2′-deoxynucleosides	At least one bicyclic
  	at least one 2′-substituted		nucleoside and at least one 2′-
  	nucleoside		substituted nucleoside
  36	At least one LNA nucleoside and	All 2′-deoxynucleosides	At least two bicyclic
  	at least one 2′-substituted		nucleosides
  	nucleoside
  37	At least one bicyclic nucleoside	All 2′-deoxynucleosides	At least one LNA nucleoside
  	and at least one 2′-substituted		and at least one 2′-substituted
  	nucleoside		nucleoside
  38	At least one bicyclic nucleoside	All 2′-deoxynucleosides	At least two LNA nucleosides
  	and at least one 2′-substituted
  	nucleoside
  39	At least one cEt nucleoside and	All 2′-deoxynucleosides	At least one LNA nucleoside
  	at least one 2′-substituted		and at least one 2′-substituted
  	nucleoside		nucleoside
  40	At least one cEt nucleoside and	All 2′-deoxynucleosides	At least two LNA nucleosides
  	at least one 2′-substituted
  	nucleoside
  41	At least one LNA nucleoside and	All 2′-deoxynucleosides	At least one LNA nucleoside
  	at least one 2′-substituted		and at least one 2′-substituted
  	nucleoside		nucleoside
  42	At least one LNA nucleoside and	All 2′-deoxynucleosides	At least two LNA nucleosides
  	at least one 2′-substituted
  	nucleoside
  43	At least one bicyclic nucleoside	All 2′-deoxynucleosides	At least one bicyclic
  	and at least one 2′-		nucleoside and at least one 2′-
  	deoxynucleoside		substituted nucleoside
  44	At least one bicyclic nucleoside	All 2′-deoxynucleosides	At least two bicyclic
  	and at least one 2′-		nucleosides
  	deoxynucleoside
  45	At least one cEt nucleoside and	All 2′-deoxynucleosides	At least one bicyclic
  	at least one 2′-deoxynucleoside		nucleoside and at least one 2′-
  			substituted nucleoside
  46	At least one cEt nucleoside and	All 2′-deoxynucleosides	At least two bicyclic
  	at least one 2′-deoxynucleoside		nucleosides
  47	At least one LNA nucleoside and	All 2′-deoxynucleosides	At least one bicyclic
  	at least one 2′-deoxynucleoside		nucleoside and at least one 2′-
  			substituted nucleoside
  48	At least one LNA nucleoside and	All 2′-deoxynucleosides	At least two bicyclic
  	at least one 2′-deoxynucleoside		nucleosides
  49	At least one bicyclic nucleoside	All 2′-deoxynucleosides	At least one LNA nucleoside
  	and at least one 2′-		and at least one 2′-substituted
  	deoxynucleoside		nucleoside
  50	At least one bicyclic nucleoside	All 2′-deoxynucleosides	At least two LNA nucleosides
  	and at least one 2′-
  	deoxynucleoside
  51	At least one cEt nucleoside and	All 2′-deoxynucleosides	At least one LNA nucleoside
  	at least one 2′-deoxynucleoside		and at least one 2′-substituted
  			nucleoside
  52	At least one cEt nucleoside and	All 2′-deoxynucleosides	At least two LNA nucleosides
  	at least one 2′-deoxynucleoside
  53	At least one LNA nucleoside and	All 2′-deoxynucleosides	At least one LNA nucleoside
  	at least one 2′-deoxynucleoside		and at least one 2′-substituted
  			nucleoside
  54	At least one LNA nucleoside and	All 2′-deoxynucleosides	At least two LNA nucleosides
  	at least one 2′-deoxynucleoside
  55	At least two 2′-substituted	All 2′-deoxynucleosides	At least one bicyclic
  	nucleosides		nucleoside and at least one 2′-
  			substituted nucleoside
  56	At least two 2′-substituted	All 2′-deoxynucleosides	At least two bicyclic
  	nucleosides		nucleosides
  57	At least two 2′-substituted	All 2′-deoxynucleosides	At least one LNA nucleoside
  	nucleosides		and at least one 2′-substituted
  			nucleoside
  58	At least two 2′-substituted	All 2′-deoxynucleosides	At least two LNA nucleosides
  	nucleosides

• In certain embodiments, a gapmer comprises a 5′-wing, a gap, and a 3′ wing, wherein the 5′-wing, gap, and 3′ wing are independently selected from among those discussed above. For example, in certain embodiments, a gapmer has a 5′-wing, a gap, and a 3′-wing wherein the 5′-wing and the 3′-wing have features selected from among those listed in the tables above. In certain embodiments, any 5′-wing may be paired with any 3′-wing. In certain embodiments the 5′-wing may comprise ABBBB and the 3′-wing may comprise BBA. In certain embodiments the 5′-wing may comprise ACACA and the 3′-wing may comprise BB. For example, in certain embodiments, a gapmer has a 5′-wing, a gap, and a 3′-wing having features selected from among those listed in the following non-limiting table, wherein each motif is represented as (5′-wing)-(gap)-(3′-wing), wherein each number represents the number of linked nucleosides in each portion of the motif, for example, a 5-10-5 motif would have a 5′-wing comprising 5 nucleosides, a gap comprising 10 nucleosides, and a 3′-wing comprising 5 nucleosides:

• TABLE 8
  Certain Gapmer Sugar Motifs
  Certain Gapmer Sugar Motifs

  	2-10-2	3-10-2	4-10-2	5-10-2
  	2-10-3	3-10-3	4-10-3	5-10-3
  	2-10-4	3-10-4	4-10-4	5-10-4
  	2-10-5	3-10-5	4-10-5	5-10-5
  	2-9-2	3-9-2	4-9-2	5-9-2
  	2-9-3	3-9-3	4-9-3	5-9-3
  	2-9-4	3-9-4	4-9-4	5-9-4
  	2-9-5	3-9-5	4-9-5	5-9-5
  	2-11-2	3-11-2	4-11-2	5-11-2
  	2-11-3	3-11-3	4-11-3	5-11-3
  	2-11-4	3-11-4	4-11-4	5-11-4
  	2-11-5	3-11-5	4-11-5	5-11-5
  	2-8-2	3-8-2	4-8-2	5-8-2
  	2-8-3	3-8-3	4-8-3	5-8-3
  	2-8-4	3-8-4	4-8-4	5-8-4
  	2-8-5	3-8-5	4-8-5	5-8-5

  
  In certain embodiments, gapmers have a motif described by Formula I as follows:
• 
  (A)_m-(B)_n-(J)_p-(B)_r-(J)_t-(D)_g-h-(J)_v-(B)_w-(J)_x-(B)_y-(A)_z
• • wherein:
  • each A is independently a 2′-substituted nucleoside;
  • each B is independently a bicyclic nucleoside;
  • each J is independently either a 2′-substituted nucleoside or a 2′-deoxynucleoside;
  • each D is a 2′-deoxynucleoside;
  • m is 0-4; n is 0-2; p is 0-2; r is 0-2; t is 0-2; v is 0-2; w is 0-4; x is 0-2; y is 0-2; z is 0-4; g is 6; and h is 14;
  • provided that:
  • at least one of m, n, and r is other than 0;
  • at least one of w and y is other than 0;
  • the sum of m, n, p, r, and t is from 2 to 5; and
  • the sum of v, w, x, y, and z is from 2 to 5.
• In certain embodiments, one or more 2′-substituted nucleoside is a 2′-MOE nucleoside. In certain embodiments, one or more 2′-substituted nucleoside is a 2′-OMe nucleoside. In certain In certain embodiments, one or more bicyclic nucleoside is a cEt nucleoside. In certain embodiments, one or more bicyclic nucleoside is an LNA nucleoside.
• In certain embodiments, a gapmer of Formula I has a motif selected from among gapmer motifs 1-58. In certain embodiments, gapmers have a motif described by Formula II as follows:
• 
  (J)_m-(B)_n-(J)_p-(B)_r-(A)_t-(D)_g-(A)_v-(B)_w-(J)_x-(B)_y-(J)_z
• wherein:
• each A is independently a 2′-substituted nucleoside;
• each B is independently a bicyclic nucleoside;
• each J is independently either a 2′-substituted nucleoside or a 2′-deoxynucleoside;
• each D is a 2′-deoxynucleoside;
• m is 0-4; n is 0-2; p is 0-2; r is 0-2; t is 0-2; v is 0-2; w is 0-4; x is 0-2; y is 0-2; z is 0-4; g is 6-14;
• provided that:
• at least one of m, n, and r is other than 0;
• at least one of w and y is other than 0;
• the sum of m, n, p, r, and t is from 1 to 5; and
• the sum of v, w, x, y, and z is from 1 to 5.
• In certain embodiments, one or more 2′-substituted nucleoside is a 2′-MOE nucleoside. In certain embodiments, one or more 2′-substituted nucleoside is a 2′-OMe nucleoside. In certain embodiments, one or more bicyclic nucleoside is a cEt nucleoside. In certain embodiments, one or more bicyclic nucleoside is an LNA nucleoside.
• In certain embodiments, each 2′-substituted nucleoside is a 2′-MOE nucleoside. In certain embodiments, each 2′-substituted nucleoside is a 2′-OMe nucleoside. In certain embodiments, each bicyclic nucleoside is a cEt nucleoside. In certain embodiments, each bicyclic nucleoside is an LNA nucleoside.
• In certain embodiments, each A is the same 2′-substituted nucleoside. In certain embodiments, each B is the same bicyclic nucleoside. In certain embodiments each A is the same 2′-modified nucleoside and each B is the same bicyclic nucleoside. In certain embodiments, each J is a 2′-modified nucleoside. In certain embodiments each J is the same 2′-modified nucleoside. In certain embodiments, each J and each A is the same 2′-modified nucleoside.
• In certain embodiments, a gapmer of Formula II has a motif selected from among gapmer motifs 1-58.
• In certain embodiments, a gapmer comprises a 5′-wing, a gap, and a 3′ wing, independently selected from among those proved in the above tables, for example as provided in the following table:

• TABLE 9
  Certain Gapmer Sugar Motifs

  	5-wing		3′-wing
  Gapmer	sugar motif		sugar motif
  motif #	(from table 1)	Gap	(from table 2)

  59	1(a-i)	All 2′-deoxynucleosides	1(a-i)
  60	2(a-i)	All 2′-deoxynucleosides	1(a-i)
  61	3(a-i)	All 2′-deoxynucleosides	1(a-i)
  62	4(a-i)	All 2′-deoxynucleosides	1(a-i)
  63	5(a-i)	All 2′-deoxynucleosides	1(a-i)
  64	6(a-i)	All 2′-deoxynucleosides	1(a-i)
  65	7(a-i)	All 2′-deoxynucleosides	1(a-i)
  66	8(a-i)	All 2′-deoxynucleosides	1(a-i)
  67	9(a-i)	All 2′-deoxynucleosides	1(a-i)
  68	10(a-i)	All 2′-deoxynucleosides	1(a-i)
  69	11(a-i)	All 2′-deoxynucleosides	1(a-i)
  70	12(a-i)	All 2′-deoxynucleosides	1(a-i)
  71	13(a-i)	All 2′-deoxynucleosides	1(a-i)
  72	14(a-i)	All 2′-deoxynucleosides	1(a-i)
  73	15(a-i)	All 2′-deoxynucleosides	1(a-i)
  74	16(a-i)	All 2′-deoxynucleosides	1(a-i)
  75	17(a-i)	All 2′-deoxynucleosides	1(a-i)
  76	18(a-i)	All 2′-deoxynucleosides	1(a-i)
  77	19(a-i)	All 2′-deoxynucleosides	1(a-i)
  78	20(a-i)	All 2′-deoxynucleosides	1(a-i)
  79	21(a-i)	All 2′-deoxynucleosides	1(a-i)
  80	22(a-i)	All 2′-deoxynucleosides	1(a-i)
  81	1(a-i)	All 2′-deoxynucleosides	2(a-i)
  82	2(a-i)	All 2′-deoxynucleosides	2(a-i)
  83	3(a-i)	All 2′-deoxynucleosides	2(a-i)
  84	4(a-i)	All 2′-deoxynucleosides	2(a-i)
  85	5(a-i)	All 2′-deoxynucleosides	2(a-i)
  86	6(a-i)	All 2′-deoxynucleosides	2(a-i)
  87	7(a-i)	All 2′-deoxynucleosides	2(a-i)
  88	8(a-i)	All 2′-deoxynucleosides	2(a-i)
  89	9(a-i)	All 2′-deoxynucleosides	2(a-i)
  90	10(a-i)	All 2′-deoxynucleosides	2(a-i)
  91	11(a-i)	All 2′-deoxynucleosides	2(a-i)
  92	12(a-i)	All 2′-deoxynucleosides	2(a-i)
  93	13(a-i)	All 2′-deoxynucleosides	2(a-i)
  94	14(a-i)	All 2′-deoxynucleosides	2(a-i)
  94	15(a-i)	All 2′-deoxynucleosides	2(a-i)
  96	16(a-i)	All 2′-deoxynucleosides	2(a-i)
  97	17(a-i)	All 2′-deoxynucleosides	2(a-i)
  98	18(a-i)	All 2′-deoxynucleosides	2(a-i)
  99	19(a-i)	All 2′-deoxynucleosides	2(a-i)
  100	20(a-i)	All 2′-deoxynucleosides	2(a-i)
  101	21(a-i)	All 2′-deoxynucleosides	2(a-i)
  102	22(a-i)	All 2′-deoxynucleosides	2(a-i)
  103	1(a-i)	All 2′-deoxynucleosides	3(a-i)
  104	2(a-i)	All 2′-deoxynucleosides	3(a-i)
  105	3(a-i)	All 2′-deoxynucleosides	3(a-i)
  106	4(a-i)	All 2′-deoxynucleosides	3(a-i)
  107	5(a-i)	All 2′-deoxynucleosides	3(a-i)
  108	6(a-i)	All 2′-deoxynucleosides	3(a-i)
  109	7(a-i)	All 2′-deoxynucleosides	3(a-i)
  110	8(a-i)	All 2′-deoxynucleosides	3(a-i)
  111	9(a-i)	All 2′-deoxynucleosides	3(a-i)
  112	10(a-i)	All 2′-deoxynucleosides	3(a-i)
  113	11(a-i)	All 2′-deoxynucleosides	3(a-i)
  114	12(a-i)	All 2′-deoxynucleosides	3(a-i)
  115	13(a-i)	All 2′-deoxynucleosides	3(a-i)
  116	14(a-i)	All 2′-deoxynucleosides	3(a-i)
  117	15(a-i)	All 2′-deoxynucleosides	3(a-i)
  118	16(a-i)	All 2′-deoxynucleosides	3(a-i)
  119	17(a-i)	All 2′-deoxynucleosides	3(a-i)
  120	18(a-i)	All 2′-deoxynucleosides	3(a-i)
  121	19(a-i)	All 2′-deoxynucleosides	3(a-i)
  122	20(a-i)	All 2′-deoxynucleosides	3(a-i)
  123	21(a-i)	All 2′-deoxynucleosides	3(a-i)
  124	22(a-i)	All 2′-deoxynucleosides	3(a-i)
  125	1(a-i)	All 2′-deoxynucleosides	4(a-i)
  126	2(a-i)	All 2′-deoxynucleosides	4(a-i)
  127	3(a-i)	All 2′-deoxynucleosides	4(a-i)
  128	4(a-i)	All 2′-deoxynucleosides	4(a-i)
  129	5(a-i)	All 2′-deoxynucleosides	4(a-i)
  130	6(a-i)	All 2′-deoxynucleosides	4(a-i)
  131	7(a-i)	All 2′-deoxynucleosides	4(a-i)
  132	8(a-i)	All 2′-deoxynucleosides	4(a-i)
  133	9(a-i)	All 2′-deoxynucleosides	4(a-i)
  134	10(a-i)	All 2′-deoxynucleosides	4(a-i)
  135	11(a-i)	All 2′-deoxynucleosides	4(a-i)
  136	12(a-i)	All 2′-deoxynucleosides	4(a-i)
  137	13(a-i)	All 2′-deoxynucleosides	4(a-i)
  138	14(a-i)	All 2′-deoxynucleosides	4(a-i)
  139	15(a-i)	All 2′-deoxynucleosides	4(a-i)
  140	16(a-i)	All 2′-deoxynucleosides	4(a-i)
  141	17(a-i)	All 2′-deoxynucleosides	4(a-i)
  142	18(a-i)	All 2′-deoxynucleosides	4(a-i)
  143	19(a-i)	All 2′-deoxynucleosides	4(a-i)
  144	20(a-i)	All 2′-deoxynucleosides	4(a-i)
  145	21(a-i)	All 2′-deoxynucleosides	4(a-i)
  146	22(a-i)	All 2′-deoxynucleosides	4(a-i)
  147	1(a-i)	All 2′-deoxynucleosides	5(a-i)
  148	2(a-i)	All 2′-deoxynucleosides	5(a-i)
  149	3(a-i)	All 2′-deoxynucleosides	5(a-i)
  150	4(a-i)	All 2′-deoxynucleosides	5(a-i)
  151	5(a-i)	All 2′-deoxynucleosides	5(a-i)
  152	6(a-i)	All 2′-deoxynucleosides	5(a-i)
  153	7(a-i)	All 2′-deoxynucleosides	5(a-i)
  154	8(a-i)	All 2′-deoxynucleosides	5(a-i)
  155	9(a-i)	All 2′-deoxynucleosides	5(a-i)
  156	10(a-i)	All 2′-deoxynucleosides	5(a-i)
  157	11(a-i)	All 2′-deoxynucleosides	5(a-i)
  158	12(a-i)	All 2′-deoxynucleosides	5(a-i)
  159	13(a-i)	All 2′-deoxynucleosides	5(a-i)
  160	14(a-i)	All 2′-deoxynucleosides	5(a-i)
  161	15(a-i)	All 2′-deoxynucleosides	5(a-i)
  162	16(a-i)	All 2′-deoxynucleosides	5(a-i)
  163	17(a-i)	All 2′-deoxynucleosides	5(a-i)
  164	18(a-i)	All 2′-deoxynucleosides	5(a-i)
  165	19(a-i)	All 2′-deoxynucleosides	5(a-i)
  166	20(a-i)	All 2′-deoxynucleosides	5(a-i)
  167	21(a-i)	All 2′-deoxynucleosides	5(a-i)
  168	22(a-i)	All 2′-deoxynucleosides	5(a-i)
  169	1(a-i)	All 2′-deoxynucleosides	6(a-i)
  170	2(a-i)	All 2′-deoxynucleosides	6(a-i)
  171	3(a-i)	All 2′-deoxynucleosides	6(a-i)
  172	4(a-i)	All 2′-deoxynucleosides	6(a-i)
  173	5(a-i)	All 2′-deoxynucleosides	6(a-i)
  174	6(a-i)	All 2′-deoxynucleosides	6(a-i)
  175	7(a-i)	All 2′-deoxynucleosides	6(a-i)
  176	8(a-i)	All 2′-deoxynucleosides	6(a-i)
  177	9(a-i)	All 2′-deoxynucleosides	6(a-i)
  178	10(a-i)	All 2′-deoxynucleosides	6(a-i)
  179	11(a-i)	All 2′-deoxynucleosides	6(a-i)
  180	12(a-i)	All 2′-deoxynucleosides	6(a-i)
  181	13(a-i)	All 2′-deoxynucleosides	6(a-i)
  182	14(a-i)	All 2′-deoxynucleosides	6(a-i)
  183	15(a-i)	All 2′-deoxynucleosides	6(a-i)
  184	16(a-i)	All 2′-deoxynucleosides	6(a-i)
  184	17(a-i)	All 2′-deoxynucleosides	6(a-i)
  186	18(a-i)	All 2′-deoxynucleosides	6(a-i)
  187	19(a-i)	All 2′-deoxynucleosides	6(a-i)
  188	20(a-i)	All 2′-deoxynucleosides	6(a-i)
  189	21(a-i)	All 2′-deoxynucleosides	6(a-i)
  190	22(a-i)	All 2′-deoxynucleosides	6(a-i)
  191	1(a-i)	All 2′-deoxynucleosides	7(a-i)
  192	2(a-i)	All 2′-deoxynucleosides	7(a-i)
  193	3(a-i)	All 2′-deoxynucleosides	7(a-i)
  194	4(a-i)	All 2′-deoxynucleosides	7(a-i)
  195	5(a-i)	All 2′-deoxynucleosides	7(a-i)
  196	6(a-i)	All 2′-deoxynucleosides	7(a-i)
  197	7(a-i)	All 2′-deoxynucleosides	7(a-i)
  198	8(a-i)	All 2′-deoxynucleosides	7(a-i)
  199	9(a-i)	All 2′-deoxynucleosides	7(a-i)
  200	10(a-i)	All 2′-deoxynucleosides	7(a-i)
  201	11(a-i)	All 2′-deoxynucleosides	7(a-i)
  202	12(a-i)	All 2′-deoxynucleosides	7(a-i)
  203	13(a-i)	All 2′-deoxynucleosides	7(a-i)
  204	14(a-i)	All 2′-deoxynucleosides	7(a-i)
  205	15(a-i)	All 2′-deoxynucleosides	7(a-i)
  206	16(a-i)	All 2′-deoxynucleosides	7(a-i)
  207	17(a-i)	All 2′-deoxynucleosides	7(a-i)
  208	18(a-i)	All 2′-deoxynucleosides	7(a-i)
  209	19(a-i)	All 2′-deoxynucleosides	7(a-i)
  210	20(a-i)	All 2′-deoxynucleosides	7(a-i)
  211	21(a-i)	All 2′-deoxynucleosides	7(a-i)
  212	22(a-i)	All 2′-deoxynucleosides	7(a-i)
  213	1(a-i)	All 2′-deoxynucleosides	8(a-i)
  214	2(a-i)	All 2′-deoxynucleosides	8(a-i)
  215	3(a-i)	All 2′-deoxynucleosides	8(a-i)
  216	4(a-i)	All 2′-deoxynucleosides	8(a-i)
  217	5(a-i)	All 2′-deoxynucleosides	8(a-i)
  218	6(a-i)	All 2′-deoxynucleosides	8(a-i)
  219	7(a-i)	All 2′-deoxynucleosides	8(a-i)
  220	8(a-i)	All 2′-deoxynucleosides	8(a-i)
  221	9(a-i)	All 2′-deoxynucleosides	8(a-i)
  222	10(a-i)	All 2′-deoxynucleosides	8(a-i)
  223	11(a-i)	All 2′-deoxynucleosides	8(a-i)
  224	12(a-i)	All 2′-deoxynucleosides	8(a-i)
  225	13(a-i)	All 2′-deoxynucleosides	8(a-i)
  226	14(a-i)	All 2′-deoxynucleosides	8(a-i)
  227	15(a-i)	All 2′-deoxynucleosides	8(a-i)
  228	16(a-i)	All 2′-deoxynucleosides	8(a-i)
  229	17(a-i)	All 2′-deoxynucleosides	8(a-i)
  230	18(a-i)	All 2′-deoxynucleosides	8(a-i)
  231	19(a-i)	All 2′-deoxynucleosides	8(a-i)
  232	20(a-i)	All 2′-deoxynucleosides	8(a-i)
  233	21(a-i)	All 2′-deoxynucleosides	8(a-i)
  234	22(a-i)	All 2′-deoxynucleosides	8(a-i)
  235	1(a-i)	All 2′-deoxynucleosides	9(a-i)
  236	2(a-i)	All 2′-deoxynucleosides	9(a-i)
  237	3(a-i)	All 2′-deoxynucleosides	9(a-i)
  238	4(a-i)	All 2′-deoxynucleosides	9(a-i)
  239	5(a-i)	All 2′-deoxynucleosides	9(a-i)
  240	6(a-i)	All 2′-deoxynucleosides	9(a-i)
  241	7(a-i)	All 2′-deoxynucleosides	9(a-i)
  242	8(a-i)	All 2′-deoxynucleosides	9(a-i)
  243	9(a-i)	All 2′-deoxynucleosides	9(a-i)
  244	10(a-i)	All 2′-deoxynucleosides	9(a-i)
  245	11(a-i)	All 2′-deoxynucleosides	9(a-i)
  246	12(a-i)	All 2′-deoxynucleosides	9(a-i)
  247	13(a-i)	All 2′-deoxynucleosides	9(a-i)
  248	14(a-i)	All 2′-deoxynucleosides	9(a-i)
  249	15(a-i)	All 2′-deoxynucleosides	9(a-i)
  250	16(a-i)	All 2′-deoxynucleosides	9(a-i)
  251	17(a-i)	All 2′-deoxynucleosides	9(a-i)
  252	18(a-i)	All 2′-deoxynucleosides	9(a-i)
  253	19(a-i)	All 2′-deoxynucleosides	9(a-i)
  254	20(a-i)	All 2′-deoxynucleosides	9(a-i)
  255	21(a-i)	All 2′-deoxynucleosides	9(a-i)
  256	22(a-i)	All 2′-deoxynucleosides	9(a-i)
  257	1(a-i)	All 2′-deoxynucleosides	10(a-i)
  258	2(a-i)	All 2′-deoxynucleosides	10(a-i)
  259	3(a-i)	All 2′-deoxynucleosides	10(a-i)
  260	4(a-i)	All 2′-deoxynucleosides	10(a-i)
  261	5(a-i)	All 2′-deoxynucleosides	10(a-i)
  262	6(a-i)	All 2′-deoxynucleosides	10(a-i)
  263	7(a-i)	All 2′-deoxynucleosides	10(a-i)
  264	8(a-i)	All 2′-deoxynucleosides	10(a-i)
  265	9(a-i)	All 2′-deoxynucleosides	10(a-i)
  266	10(a-i)	All 2′-deoxynucleosides	10(a-i)
  267	11(a-i)	All 2′-deoxynucleosides	10(a-i)
  268	12(a-i)	All 2′-deoxynucleosides	10(a-i)
  269	13(a-i)	All 2′-deoxynucleosides	10(a-i)
  270	14(a-i)	All 2′-deoxynucleosides	10(a-i)
  271	15(a-i)	All 2′-deoxynucleosides	10(a-i)
  272	16(a-i)	All 2′-deoxynucleosides	10(a-i)
  273	17(a-i)	All 2′-deoxynucleosides	10(a-i)
  274	18(a-i)	All 2′-deoxynucleosides	10(a-i)
  275	19(a-i)	All 2′-deoxynucleosides	10(a-i)
  276	20(a-i)	All 2′-deoxynucleosides	10(a-i)
  277	21(a-i)	All 2′-deoxynucleosides	10(a-i)
  278	22(a-i)	All 2′-deoxynucleosides	10(a-i)
  279	1(a)-22(a)	All 2′-deoxynucleosides	1(a)-10(a)
  280	1(b)-22(b)	All 2′-deoxynucleosides	1(a)-10(a)
  281	1(c)-22(c)	All 2′-deoxynucleosides	1(a)-10(a)
  282	1(d)-22(d)	All 2′-deoxynucleosides	1(a)-10(a)
  283	1(e)-22(e)	All 2′-deoxynucleosides	1(a)-10(a)
  284	1(f)-22(f)	All 2′-deoxynucleosides	1(a)-10(a)
  285	1(g)-22(g)	All 2′-deoxynucleosides	1(a)-10(a)
  286	1(h)-22(h)	All 2′-deoxynucleosides	1(a)-10(a)
  287	1(i)-22(i)	All 2′-deoxynucleosides	1(a)-10(a)
  288	1(a)-22(a)	All 2′-deoxynucleosides	1(b)-10(b)
  289	1(b)-22(b)	All 2′-deoxynucleosides	1(b)-10(b)
  290	1(c)-22(c)	All 2′-deoxynucleosides	1(b)-10(b)
  291	1(d)-22(d)	All 2′-deoxynucleosides	1(b)-10(b)
  292	1(e)-22(e)	All 2′-deoxynucleosides	1(b)-10(b)
  293	1(f)-22(f)	All 2′-deoxynucleosides	1(b)-10(b)
  294	1(g)-22(g)	All 2′-deoxynucleosides	1(b)-10(b)
  295	1(h)-22(h)	All 2′-deoxynucleosides	1(b)-10(b)
  296	1(i)-22(i)	All 2′-deoxynucleosides	1(b)-10(b)
  297	1(a)-22(a)	All 2′-deoxynucleosides	1(c)-10(c)
  298	1(b)-22(b)	All 2′-deoxynucleosides	1(c)-10(c)
  299	1(c)-22(c)	All 2′-deoxynucleosides	1(c)-10(c)
  300	1(d)-22(d)	All 2′-deoxynucleosides	1(c)-10(c)
  301	1(e)-22(e)	All 2′-deoxynucleosides	1(c)-10(c)
  302	1(f)-22(f)	All 2′-deoxynucleosides	1(c)-10(c)
  303	1(g)-22(g)	All 2′-deoxynucleosides	1(c)-10(c)
  304	1(h)-22(h)	All 2′-deoxynucleosides	1(c)-10(c)
  305	1(i)-22(i)	All 2′-deoxynucleosides	1(c)-10(c)
  306	1(a)-22(a)	All 2′-deoxynucleosides	1(d)-10(d)
  307	1(b)-22(b)	All 2′-deoxynucleosides	1(d)-10(d)
  308	1(c)-22(c)	All 2′-deoxynucleosides	1(d)-10(d)
  309	1(d)-22(d)	All 2′-deoxynucleosides	1(d)-10(d)
  310	1(e)-22(e)	All 2′-deoxynucleosides	1(d)-10(d)
  311	1(f)-22(f)	All 2′-deoxynucleosides	1(d)-10(d)
  312	1(g)-22(g)	All 2′-deoxynucleosides	1(d)-10(d)
  313	1(h)-22(h)	All 2′-deoxynucleosides	1(d)-10(d)
  314	1(i)-22(i)	All 2′-deoxynucleosides	1(d)-10(d)
  315	1(a)-22(a)	All 2′-deoxynucleosides	1(e)-10(e)
  316	1(b)-22(b)	All 2′-deoxynucleosides	1(e)-10(e)
  317	1(c)-22(c)	All 2′-deoxynucleosides	1(e)-10(e)
  318	1(d)-22(d)	All 2′-deoxynucleosides	1(e)-10(e)
  319	1(e)-22(e)	All 2′-deoxynucleosides	1(e)-10(e)
  320	1(f)-22(f)	All 2′-deoxynucleosides	1(e)-10(e)
  321	1(g)-22(g)	All 2′-deoxynucleosides	1(e)-10(e)
  322	1(h)-22(h)	All 2′-deoxynucleosides	1(e)-10(e)
  323	1(i)-22(i)	All 2′-deoxynucleosides	1(e)-10(e)
  324	1(a)-22(a)	All 2′-deoxynucleosides	1(f)-10(f)
  325	1(b)-22(b)	All 2′-deoxynucleosides	1(f)-10(f)
  326	1(c)-22(c)	All 2′-deoxynucleosides	1(f)-10(f)
  327	1(d)-22(d)	All 2′-deoxynucleosides	1(f)-10(f)
  328	1(e)-22(e)	All 2′-deoxynucleosides	1(f)-10(f)
  329	1(f)-22(f)	All 2′-deoxynucleosides	1(f)-10(f)
  330	1(g)-22(g)	All 2′-deoxynucleosides	1(f)-10(f)
  331	1(h)-22(h)	All 2′-deoxynucleosides	1(f)-10(f)
  332	1(i)-22(i)	All 2′-deoxynucleosides	1(f)-10(f)
  333	1(a)-22(a)	All 2′-deoxynucleosides	1(g)-10(g)
  334	1(b)-22(b)	All 2′-deoxynucleosides	1(g)-10(g)
  335	1(c)-22(c)	All 2′-deoxynucleosides	1(g)-10(g)
  336	1(d)-22(d)	All 2′-deoxynucleosides	1(g)-10(g)
  337	1(e)-22(e)	All 2′-deoxynucleosides	1(g)-10(g)
  338	1(f)-22(f)	All 2′-deoxynucleosides	1(g)-10(g)
  339	1(g)-22(g)	All 2′-deoxynucleosides	1(g)-10(g)
  340	1(h)-22(h)	All 2′-deoxynucleosides	1(g)-10(g)
  341	1(i)-22(i)	All 2′-deoxynucleosides	1(g)-10(g)
  342	1(a)-22(a)	All 2′-deoxynucleosides	1(h)-10(h)
  343	1(b)-22(b)	All 2′-deoxynucleosides	1(h)-10(h)
  344	1(c)-22(c)	All 2′-deoxynucleosides	1(h)-10(h)
  345	1(d)-22(d)	All 2′-deoxynucleosides	1(h)-10(h)
  346	1(e)-22(e)	All 2′-deoxynucleosides	1(h)-10(h)
  347	1(f)-22(f)	All 2′-deoxynucleosides	1(h)-10(h)
  348	1(g)-22(g)	All 2′-deoxynucleosides	1(h)-10(h)
  349	1(h)-22(h)	All 2′-deoxynucleosides	1(h)-10(h)
  350	1(i)-22(i)	All 2′-deoxynucleosides	1(h)-10(h)
  351	1(a)-22(a)	All 2′-deoxynucleosides	1(i)-10(i)
  352	1(b)-22(b)	All 2′-deoxynucleosides	1(i)-10(i)
  353	1(c)-22(c)	All 2′-deoxynucleosides	1(i)-10(i)
  354	1(d)-22(d)	All 2′-deoxynucleosides	1(i)-10(i)
  355	1(e)-22(e)	All 2′-deoxynucleosides	1(i)-10(i)
  356	1(f)-22(f)	All 2′-deoxynucleosides	1(i)-10(i)
  357	1(g)-22(g)	All 2′-deoxynucleosides	1(i)-10(i)
  358	1(h)-22(h)	All 2′-deoxynucleosides	1(i)-10(i)
  359	1(i)-22(i)	All 2′-deoxynucleosides	1(i)-10(i)
  360	1(a-l)	All 2′-deoxynucleosides	1(a-l)
  361	2(a-l)	All 2′-deoxynucleosides	1(a-l)
  362	3(a-l)	All 2′-deoxynucleosides	1(a-l)
  363	4(a-l)	All 2′-deoxynucleosides	1(a-l)
  364	5(a-l)	All 2′-deoxynucleosides	1(a-l)
  365	6(a-l)	All 2′-deoxynucleosides	1(a-l)
  366	7(a-l)	All 2′-deoxynucleosides	1(a-l)
  367	8(a-l)	All 2′-deoxynucleosides	1(a-l)
  368	9(a-l)	All 2′-deoxynucleosides	1(a-l)
  369	10(a-l)	All 2′-deoxynucleosides	1(a-l)
  370	11(a-l)	All 2′-deoxynucleosides	1(a-l)
  371	12(a-l)	All 2′-deoxynucleosides	1(a-l)
  372	13(a-l)	All 2′-deoxynucleosides	1(a-l)
  373	14(a-l)	All 2′-deoxynucleosides	1(a-l)
  374	15(a-l)	All 2′-deoxynucleosides	1(a-l)
  375	16(a-l)	All 2′-deoxynucleosides	1(a-l)
  376	17(a-l)	All 2′-deoxynucleosides	1(a-l)
  377	18(a-l)	All 2′-deoxynucleosides	1(a-l)
  378	19(a-l)	All 2′-deoxynucleosides	1(a-l)
  379	20(a-l)	All 2′-deoxynucleosides	1(a-l)
  380	21(a-l)	All 2′-deoxynucleosides	1(a-l)
  381	22(a-l)	All 2′-deoxynucleosides	1(a-l)
  382	1(a-l)	All 2′-deoxynucleosides	2(a-l)
  383	2(a-l)	All 2′-deoxynucleosides	2(a-l)
  384	3(a-l)	All 2′-deoxynucleosides	2(a-l)
  385	4(a-l)	All 2′-deoxynucleosides	2(a-l)
  386	5(a-l)	All 2′-deoxynucleosides	2(a-l)
  387	6(a-l)	All 2′-deoxynucleosides	2(a-l)
  388	7(a-l)	All 2′-deoxynucleosides	2(a-l)
  389	8(a-l)	All 2′-deoxynucleosides	2(a-l)
  390	9(a-l)	All 2′-deoxynucleosides	2(a-l)
  391	10(a-l)	All 2′-deoxynucleosides	2(a-l)
  392	11(a-l)	All 2′-deoxynucleosides	2(a-l)
  393	12(a-l)	All 2′-deoxynucleosides	2(a-l)
  394	13(a-l)	All 2′-deoxynucleosides	2(a-l)
  395	14(a-l)	All 2′-deoxynucleosides	2(a-l)
  396	15(a-l)	All 2′-deoxynucleosides	2(a-l)
  397	16(a-l)	All 2′-deoxynucleosides	2(a-l)
  398	17(a-l)	All 2′-deoxynucleosides	2(a-l)
  399	18(a-l)	All 2′-deoxynucleosides	2(a-l)
  400	19(a-l)	All 2′-deoxynucleosides	2(a-l)
  401	20(a-l)	All 2′-deoxynucleosides	2(a-l)
  402	21(a-l)	All 2′-deoxynucleosides	2(a-l)
  403	22(a-l)	All 2′-deoxynucleosides	2(a-l)
  404	1(a-l)	All 2′-deoxynucleosides	3(a-l)
  405	2(a-l)	All 2′-deoxynucleosides	3(a-l)
  406	3(a-l)	All 2′-deoxynucleosides	3(a-l)
  407	4(a-l)	All 2′-deoxynucleosides	3(a-l)
  408	5(a-l)	All 2′-deoxynucleosides	3(a-l)
  409	6(a-l)	All 2′-deoxynucleosides	3(a-l)
  410	7(a-l)	All 2′-deoxynucleosides	3(a-l)
  411	8(a-l)	All 2′-deoxynucleosides	3(a-l)
  412	9(a-l)	All 2′-deoxynucleosides	3(a-l)
  413	10(a-l)	All 2′-deoxynucleosides	3(a-l)
  414	11(a-l)	All 2′-deoxynucleosides	3(a-l)
  415	12(a-l)	All 2′-deoxynucleosides	3(a-l)
  416	13(a-l)	All 2′-deoxynucleosides	3(a-l)
  417	14(a-l)	All 2′-deoxynucleosides	3(a-l)
  418	15(a-l)	All 2′-deoxynucleosides	3(a-l)
  419	16(a-l)	All 2′-deoxynucleosides	3(a-l)
  420	17(a-l)	All 2′-deoxynucleosides	3(a-l)
  421	18(a-l)	All 2′-deoxynucleosides	3(a-l)
  422	19(a-l)	All 2′-deoxynucleosides	3(a-l)
  423	20(a-l)	All 2′-deoxynucleosides	3(a-l)
  424	21(a-l)	All 2′-deoxynucleosides	3(a-l)
  425	22(a-l)	All 2′-deoxynucleosides	3(a-l)
  426	1(a-l)	All 2′-deoxynucleosides	4(a-l)
  427	2(a-l)	All 2′-deoxynucleosides	4(a-l)
  428	3(a-l)	All 2′-deoxynucleosides	4(a-l)
  429	4(a-l)	All 2′-deoxynucleosides	4(a-l)
  430	5(a-l)	All 2′-deoxynucleosides	4(a-l)
  431	6(a-l)	All 2′-deoxynucleosides	4(a-l)
  432	7(a-l)	All 2′-deoxynucleosides	4(a-l)
  433	8(a-l)	All 2′-deoxynucleosides	4(a-l)
  434	9(a-l)	All 2′-deoxynucleosides	4(a-l)
  435	10(a-l)	All 2′-deoxynucleosides	4(a-l)
  436	11(a-l)	All 2′-deoxynucleosides	4(a-l)
  437	12(a-l)	All 2′-deoxynucleosides	4(a-l)
  438	13(a-l)	All 2′-deoxynucleosides	4(a-l)
  439	14(a-l)	All 2′-deoxynucleosides	4(a-l)
  440	15(a-l)	All 2′-deoxynucleosides	4(a-l)
  441	16(a-l)	All 2′-deoxynucleosides	4(a-l)
  442	17(a-l)	All 2′-deoxynucleosides	4(a-l)
  443	18(a-l)	All 2′-deoxynucleosides	4(a-l)
  444	19(a-l)	All 2′-deoxynucleosides	4(a-l)
  445	20(a-l)	All 2′-deoxynucleosides	4(a-l)
  446	21(a-l)	All 2′-deoxynucleosides	4(a-l)
  447	22(a-l)	All 2′-deoxynucleosides	4(a-l)
  448	1(a-l)	All 2′-deoxynucleosides	5(a-l)
  449	2(a-l)	All 2′-deoxynucleosides	5(a-l)
  450	3(a-l)	All 2′-deoxynucleosides	5(a-l)
  451	4(a-l)	All 2′-deoxynucleosides	5(a-l)
  452	5(a-l)	All 2′-deoxynucleosides	5(a-l)
  453	6(a-l)	All 2′-deoxynucleosides	5(a-l)
  454	7(a-l)	All 2′-deoxynucleosides	5(a-l)
  455	8(a-l)	All 2′-deoxynucleosides	5(a-l)
  456	9(a-l)	All 2′-deoxynucleosides	5(a-l)
  457	10(a-l)	All 2′-deoxynucleosides	5(a-l)
  458	11(a-l)	All 2′-deoxynucleosides	5(a-l)
  459	12(a-l)	All 2′-deoxynucleosides	5(a-l)
  460	13(a-l)	All 2′-deoxynucleosides	5(a-l)
  461	14(a-l)	All 2′-deoxynucleosides	5(a-l)
  462	15(a-l)	All 2′-deoxynucleosides	5(a-l)
  463	16(a-l)	All 2′-deoxynucleosides	5(a-l)
  464	17(a-l)	All 2′-deoxynucleosides	5(a-l)
  465	18(a-l)	All 2′-deoxynucleosides	5(a-l)
  466	19(a-l)	All 2′-deoxynucleosides	5(a-l)
  467	20(a-l)	All 2′-deoxynucleosides	5(a-l)
  468	21(a-l)	All 2′-deoxynucleosides	5(a-l)
  469	22(a-l)	All 2′-deoxynucleosides	5(a-l)
  470	1(a-l)	All 2′-deoxynucleosides	6(a-l)
  471	2(a-l)	All 2′-deoxynucleosides	6(a-l)
  472	3(a-l)	All 2′-deoxynucleosides	6(a-l)
  473	4(a-l)	All 2′-deoxynucleosides	6(a-l)
  474	5(a-l)	All 2′-deoxynucleosides	6(a-l)
  475	6(a-l)	All 2′-deoxynucleosides	6(a-l)
  476	7(a-l)	All 2′-deoxynucleosides	6(a-l)
  477	8(a-l)	All 2′-deoxynucleosides	6(a-l)
  478	9(a-l)	All 2′-deoxynucleosides	6(a-l)
  479	10(a-l)	All 2′-deoxynucleosides	6(a-l)
  480	11(a-l)	All 2′-deoxynucleosides	6(a-l)
  481	12(a-l)	All 2′-deoxynucleosides	6(a-l)
  482	13(a-l)	All 2′-deoxynucleosides	6(a-l)
  483	14(a-l)	All 2′-deoxynucleosides	6(a-l)
  484	15(a-l)	All 2′-deoxynucleosides	6(a-l)
  485	16(a-l)	All 2′-deoxynucleosides	6(a-l)
  486	17(a-l)	All 2′-deoxynucleosides	6(a-l)
  487	18(a-l)	All 2′-deoxynucleosides	6(a-l)
  488	19(a-l)	All 2′-deoxynucleosides	6(a-l)
  489	20(a-l)	All 2′-deoxynucleosides	6(a-l)
  490	21(a-l)	All 2′-deoxynucleosides	6(a-l)
  491	22(a-l)	All 2′-deoxynucleosides	6(a-l)
  492	1(a-l)	All 2′-deoxynucleosides	7(a-l)
  493	2(a-l)	All 2′-deoxynucleosides	7(a-l)
  494	3(a-l)	All 2′-deoxynucleosides	7(a-l)
  495	4(a-l)	All 2′-deoxynucleosides	7(a-l)
  496	5(a-l)	All 2′-deoxynucleosides	7(a-l)
  497	6(a-l)	All 2′-deoxynucleosides	7(a-l)
  498	7(a-l)	All 2′-deoxynucleosides	7(a-l)
  499	8(a-l)	All 2′-deoxynucleosides	7(a-l)
  500	9(a-l)	All 2′-deoxynucleosides	7(a-l)
  501	10(a-l)	All 2′-deoxynucleosides	7(a-l)
  502	11(a-l)	All 2′-deoxynucleosides	7(a-l)
  503	12(a-l)	All 2′-deoxynucleosides	7(a-l)
  504	13(a-l)	All 2′-deoxynucleosides	7(a-l)
  505	14(a-l)	All 2′-deoxynucleosides	7(a-l)
  506	15(a-l)	All 2′-deoxynucleosides	7(a-l)
  507	16(a-l)	All 2′-deoxynucleosides	7(a-l)
  508	17(a-l)	All 2′-deoxynucleosides	7(a-l)
  509	18(a-l)	All 2′-deoxynucleosides	7(a-l)
  510	19(a-l)	All 2′-deoxynucleosides	7(a-l)
  511	20(a-l)	All 2′-deoxynucleosides	7(a-l)
  512	21(a-l)	All 2′-deoxynucleosides	7(a-l)
  513	22(a-l)	All 2′-deoxynucleosides	7(a-l)
  514	1(a-l)	All 2′-deoxynucleosides	8(a-l)
  515	2(a-l)	All 2′-deoxynucleosides	8(a-l)
  516	3(a-l)	All 2′-deoxynucleosides	8(a-l)
  517	4(a-l)	All 2′-deoxynucleosides	8(a-l)
  518	5(a-l)	All 2′-deoxynucleosides	8(a-l)
  519	6(a-l)	All 2′-deoxynucleosides	8(a-l)
  520	7(a-l)	All 2′-deoxynucleosides	8(a-l)
  521	8(a-l)	All 2′-deoxynucleosides	8(a-l)
  522	9(a-l)	All 2′-deoxynucleosides	8(a-l)
  523	10(a-l)	All 2′-deoxynucleosides	8(a-l)
  524	11(a-l)	All 2′-deoxynucleosides	8(a-l)
  525	12(a-l)	All 2′-deoxynucleosides	8(a-l)
  526	13(a-l)	All 2′-deoxynucleosides	8(a-l)
  527	14(a-l)	All 2′-deoxynucleosides	8(a-l)
  528	15(a-l)	All 2′-deoxynucleosides	8(a-l)
  529	16(a-l)	All 2′-deoxynucleosides	8(a-l)
  530	17(a-l)	All 2′-deoxynucleosides	8(a-l)
  531	18(a-l)	All 2′-deoxynucleosides	8(a-l)
  532	19(a-l)	All 2′-deoxynucleosides	8(a-l)
  533	20(a-l)	All 2′-deoxynucleosides	8(a-l)
  534	21(a-l)	All 2′-deoxynucleosides	8(a-l)
  535	22(a-l)	All 2′-deoxynucleosides	8(a-l)
  536	1(a-l)	All 2′-deoxynucleosides	9(a-l)
  537	2(a-l)	All 2′-deoxynucleosides	9(a-l)
  538	3(a-l)	All 2′-deoxynucleosides	9(a-l)
  539	4(a-l)	All 2′-deoxynucleosides	9(a-l)
  540	5(a-l)	All 2′-deoxynucleosides	9(a-l)
  541	6(a-l)	All 2′-deoxynucleosides	9(a-l)
  542	7(a-l)	All 2′-deoxynucleosides	9(a-l)
  543	8(a-l)	All 2′-deoxynucleosides	9(a-l)
  544	9(a-l)	All 2′-deoxynucleosides	9(a-l)
  545	10(a-l)	All 2′-deoxynucleosides	9(a-l)
  546	11(a-l)	All 2′-deoxynucleosides	9(a-l)
  547	12(a-l)	All 2′-deoxynucleosides	9(a-l)
  548	13(a-l)	All 2′-deoxynucleosides	9(a-l)
  549	14(a-l)	All 2′-deoxynucleosides	9(a-l)
  550	15(a-l)	All 2′-deoxynucleosides	9(a-l)
  551	16(a-l)	All 2′-deoxynucleosides	9(a-l)
  552	17(a-l)	All 2′-deoxynucleosides	9(a-l)
  553	18(a-l)	All 2′-deoxynucleosides	9(a-l)
  554	19(a-l)	All 2′-deoxynucleosides	9(a-l)
  555	20(a-l)	All 2′-deoxynucleosides	9(a-l)
  556	21(a-l)	All 2′-deoxynucleosides	9(a-l)
  557	22(a-l)	All 2′-deoxynucleosides	9(a-l)
  558	1(a-l)	All 2′-deoxynucleosides	10(a-l)
  559	2(a-l)	All 2′-deoxynucleosides	10(a-l)
  560	3(a-l)	All 2′-deoxynucleosides	10(a-l)
  561	4(a-l)	All 2′-deoxynucleosides	10(a-l)
  562	5(a-l)	All 2′-deoxynucleosides	10(a-l)
  563	6(a-l)	All 2′-deoxynucleosides	10(a-l)
  564	7(a-l)	All 2′-deoxynucleosides	10(a-l)
  565	8(a-l)	All 2′-deoxynucleosides	10(a-l)
  566	9(a-l)	All 2′-deoxynucleosides	10(a-l)
  567	10(a-l)	All 2′-deoxynucleosides	10(a-l)
  568	11(a-l)	All 2′-deoxynucleosides	10(a-l)
  569	12(a-l)	All 2′-deoxynucleosides	10(a-l)
  570	13(a-l)	All 2′-deoxynucleosides	10(a-l)
  571	14(a-l)	All 2′-deoxynucleosides	10(a-l)
  572	15(a-l)	All 2′-deoxynucleosides	10(a-l)
  573	16(a-l)	All 2′-deoxynucleosides	10(a-l)
  574	17(a-l)	All 2′-deoxynucleosides	10(a-l)
  575	18(a-l)	All 2′-deoxynucleosides	10(a-l)
  576	19(a-l)	All 2′-deoxynucleosides	10(a-l)
  577	20(a-l)	All 2′-deoxynucleosides	10(a-l)
  578	21(a-l)	All 2′-deoxynucleosides	10(a-l)
  579	22(a-l)	All 2′-deoxynucleosides	10(a-l)
  580	1(j)-22(j)	All 2′-deoxynucleosides	1(a)-10(a)
  581	1(k)-22(k)	All 2′-deoxynucleosides	1(a)-10(a)
  582	1(l)-22(l)	All 2′-deoxynucleosides	1(a)-10(a)
  583	1(j)-22(j)	All 2′-deoxynucleosides	1(b)-10(b)
  584	1(k)-22(k)	All 2′-deoxynucleosides	1(b)-10(b)
  585	1(l)-22(l)	All 2′-deoxynucleosides	1(b)-10(b)
  586	1(j)-22(j)	All 2′-deoxynucleosides	1(c)-10(c)
  587	1(k)-22(k)	All 2′-deoxynucleosides	1(c)-10(c)
  588	1(l)-22(l)	All 2′-deoxynucleosides	1(c)-10(c)
  589	1(j)-22(j)	All 2′-deoxynucleosides	1(d)-10(d)
  590	1(k)-22(k)	All 2′-deoxynucleosides	1(d)-10(d)
  591	1(l)-22(l)	All 2′-deoxynucleosides	1(d)-10(d)
  592	1(j)-22(j)	All 2′-deoxynucleosides	1(e)-10(e)
  593	1(k)-22(k)	All 2′-deoxynucleosides	1(e)-10(e)
  594	1(l)-22(l)	All 2′-deoxynucleosides	1(e)-10(e)
  595	1(j)-22(j)	All 2′-deoxynucleosides	1(f)-10(f)
  596	1(k)-22(k)	All 2′-deoxynucleosides	1(f)-10(f)
  597	1(l)-22(l)	All 2′-deoxynucleosides	1(f)-10(f)
  598	1(j)-22(j)	All 2′-deoxynucleosides	1(g)-10(g)
  599	1(k)-22(k)	All 2′-deoxynucleosides	1(g)-10(g)
  600	1(l)-22(l)	All 2′-deoxynucleosides	1(g)-10(g)
  601	1(j)-22(j)	All 2′-deoxynucleosides	1(h)-10(h)
  602	1(k)-22(k)	All 2′-deoxynucleosides	1(h)-10(h)
  603	1(l)-22(l)	All 2′-deoxynucleosides	1(h)-10(h)
  604	1(j)-22(j)	All 2′-deoxynucleosides	1(i)-10(i)
  605	1(k)-22(k)	All 2′-deoxynucleosides	1(i)-10(i)
  606	1(l)-22(l)	All 2′-deoxynucleosides	1(i)-10(i)
  607	1(j)-22(j)	All 2′-deoxynucleosides	1(j)-10(j)
  608	1(k)-22(k)	All 2′-deoxynucleosides	1(j)-10(j)
  609	1(l)-22(l)	All 2′-deoxynucleosides	1(j)-10(j)
  610	1(j)-22(j)	All 2′-deoxynucleosides	1(k)-10(k)
  611	1(k)-22(k)	All 2′-deoxynucleosides	1(k)-10(k)
  612	1(l)-22(l)	All 2′-deoxynucleosides	1(k)-10(k)
  612	1(j)-22(j)	All 2′-deoxynucleosides	1(l)-10(l)
  614	1(k)-22(k)	All 2′-deoxynucleosides	1(l)-10(l)
  615	1(l)-22(l)	All 2′-deoxynucleosides	1(l)-10(l)
  616	1k	All 2′-deoxynucleosides	1m

• In certain embodiments, a gapmer comprises a 5′-wing selected from among the 5′-wings provided herein and any 3′-wing. In certain embodiments, a gapmer comprises a 5′-wing selected from among 1(α-i) to 22(α-i). In certain embodiments, a gapmer comprises a 5′-wing selected from among 1(α-1) to 22(α-1). In certain embodiments, a gapmer comprises a 3′-wing selected from among the 3′-wings provided herein and any 5′-wing. In certain embodiments, a gapmer comprises a 3′-wing selected from among 1(α-i) to 10(α-i). In certain embodiments, a gapmer comprises a 3′-wing selected from among 1(α-1) to 10(α-1).
• In certain embodiments, a gapmer has a sugar motif other than: E-K-K-(D)₉-K-K-E; E-E-E-E-K-(D)₉-E-E-E-E-E; E-K-K-K-(D)₉-K-K-K-E; K-E-E-K-(D)₉-K-E-E-K; K-D-D-K-(D)₉-K-D-D-K; K-E-K-E-K-(D)₉-K-E-K-E-K; K-D-K-D-K-(D)₉-K-D-K-D-K; E-K-E-K-(D)₉-K-E-K-E; E-E-E-E-E-K-(D)₈-E-E-E-E-E; or E-K-E-K-E-(D)₉-E-K-E-K-E. In certain embodiments, a gapmer not having one of the above motifs has a sugar motif of Formula I. In certain embodiments, a gapmer not having one of the above motifs has a sugar motif selected from motifs 1-58. In certain embodiments, a gapmer not having one of the above motifs has a sugar motif of Formula I and selected from sugar motifs 1-58. In certain embodiments, a gapmer not having one of the above motifs has a sugar motif of Formula II. In certain embodiments, a gapmer not having one of the above motifs has a sugar motif selected from motifs 1-615. In certain embodiments, a gapmer not having one of the above motifs has a sugar motif of Formula II and selected from sugar motifs 1-615.
• In certain embodiments a gapmer comprises a A-(D)₄-A-(D)₄-A-(D)₄-AA motif. In certain embodiments a gapmer comprises a B-(D)₄-A-(D)₄-A-(D)₄-AA motif. In certain embodiments a gapmer comprises a A-(D)₄-B-(D)₄-A-(D)₄-AA motif. In certain embodiments a gapmer comprises a A-(D)₄-A-(D)₄-B-(D)₄-AA motif. In certain embodiments a gapmer comprises a A-(D)₄-A-(D)₄-A-(D)₄-BA motif. In certain embodiments a gapmer comprises a A-(D)₄-A-(D)₄-A-(D)₄-BB motif. In certain embodiments a gapmer comprises a K-(D)₄-K-(D)₄-K-(D)₄-K-E motif.
• Certain Internucleoside Linkage Motifs
• In certain embodiments, oligonucleotides comprise modified internucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or modified internucleoside linkage motif. In certain embodiments, internucleoside linkages are arranged in a gapped motif, as described above for sugar modification motif. In such embodiments, the internucleoside linkages in each of two wing regions are different from the internucleoside linkages in the gap region. In certain embodiments the internucleoside linkages in the wings are phosphodiester and the internucleoside linkages in the gap are phosphorothioate. The sugar modification motif is independently selected, so such oligonucleotides having a gapped internucleoside linkage motif may or may not have a gapped sugar modification motif and if it does have a gapped sugar motif, the wing and gap lengths may or may not be the same.
• In certain embodiments, oligonucleotides comprise a region having an alternating internucleoside linkage motif. In certain embodiments, oligonucleotides of the present invention comprise a region of uniformly modified internucleoside linkages. In certain such embodiments, the oligonucleotide comprises a region that is uniformly linked by phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide is uniformly linked by phosphorothioate. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate and at least one internucleoside linkage is phosphorothioate.
• In certain embodiments, the oligonucleotide comprises at least 6 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 8 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 10 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 6 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 8 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 10 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least block of at least one 12 consecutive phosphorothioate internucleoside linkages. In certain such embodiments, at least one such block is located at the 3′ end of the oligonucleotide. In certain such embodiments, at least one such block is located within 3 nucleosides of the 3′ end of the oligonucleotide.
• Certain Nucleobase Modification Motifs
• In certain embodiments, oligonucleotides comprise chemical modifications to nucleobases arranged along the oligonucleotide or region thereof in a defined pattern or nucleobases modification motif. In certain such embodiments, nucleobase modifications are arranged in a gapped motif. In certain embodiments, nucleobase modifications are arranged in an alternating motif. In certain embodiments, each nucleobase is modified. In certain embodiments, none of the nucleobases is chemically modified.
• In certain embodiments, oligonucleotides comprise a block of modified nucleobases. In certain such embodiments, the block is at the 3′-end of the oligonucleotide. In certain embodiments the block is within 3 nucleotides of the 3′-end of the oligonucleotide. In certain such embodiments, the block is at the 5′-end of the oligonucleotide. In certain embodiments the block is within 3 nucleotides of the 5′-end of the oligonucleotide.
• In certain embodiments, nucleobase modifications are a function of the natural base at a particular position of an oligonucleotide. For example, in certain embodiments each purine or each pyrimidine in an oligonucleotide is modified. In certain embodiments, each adenine is modified. In certain embodiments, each guanine is modified. In certain embodiments, each thymine is modified. In certain embodiments, each cytosine is modified. In certain embodiments, each uracil is modified.
• In certain embodiments, some, all, or none of the cytosine moieties in an oligonucleotide are 5-methyl cytosine moieties. Herein, 5-methyl cytosine is not a “modified nucleobase.” Accordingly, unless otherwise indicated, unmodified nucleobases include both cytosine residues having a 5-methyl and those lacking a 5 methyl. In certain embodiments, the methylation state of all or some cytosine nucleobases is specified.
• Certain Overall Lengths
• In certain embodiments, the present invention provides oligomeric compounds including oligonucleotides of any of a variety of ranges of lengths. In certain embodiments, the invention provides oligomeric compounds or oligonucleotides consisting of X to Y linked nucleosides, where X represents the fewest number of nucleosides in the range and Y represents the largest number of nucleosides in the range. In certain such embodiments, X and Y are each independently selected from 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50; provided that X≤Y. For example, in certain embodiments, the invention provides oligomeric compounds which comprise oligonucleotides consisting of 8 to 9, 8 to 10, 8 to 11, 8 to 12, 8 to 13, 8 to 14, 8 to 15, 8 to 16, 8 to 17, 8 to 18, 8 to 19, 8 to 20, 8 to 21, 8 to 22, 8 to 23, 8 to 24, 8 to 25, 8 to 26, 8 to 27, 8 to 28, 8 to 29, 8 to 30, 9 to 10, 9 to 11, 9 to 12, 9 to 13, 9 to 14, 9 to 15, 9 to 16, 9 to 17, 9 to 18, 9 to 19, 9 to 20, 9 to 21, 9 to 22, 9 to 23, 9 to 24, 9 to 25, 9 to 26, 9 to 27, 9 to 28, 9 to 29, 9 to 30, 10 to 11, 10 to 12, 10 to 13, 10 to 14, 10 to 15, 10 to 16, 10 to 17, 10 to 18, 10 to 19, 10 to 20, 10 to 21, 10 to 22, 10 to 23, 10 to 24, 10 to 25, 10 to 26, 10 to 27, 10 to 28, 10 to 29, 10 to 30, 11 to 12, 11 to 13, 11 to 14, 11 to 15, 11 to 16, 11 to 17, 11 to 18, 11 to 19, 11 to 20, 11 to 21, 11 to 22, 11 to 23, 11 to 24, 11 to 25, 11 to 26, 11 to 27, 11 to 28, 11 to 29, 11 to 30, 12 to 13, 12 to 14, 12 to 15, 12 to 16, 12 to 17, 12 to 18, 12 to 19, 12 to 20, 12 to 21, 12 to 22, 12 to 23, 12 to 24, 12 to 25, 12 to 26, 12 to 27, 12 to 28, 12 to 29, 12 to 30, 13 to 14, 13 to 15, 13 to 16, 13 to 17, 13 to 18, 13 to 19, 13 to 20, 13 to 21, 13 to 22, 13 to 23, 13 to 24, 13 to 25, 13 to 26, 13 to 27, 13 to 28, 13 to 29, 13 to 30, 14 to 15, 14 to 16, 14 to 17, 14 to 18, 14 to 19, 14 to 20, 14 to 21, 14 to 22, 14 to 23, 14 to 24, 14 to 25, 14 to 26, 14 to 27, 14 to 28, 14 to 29, 14 to 30, 15 to 16, 15 to 17, 15 to 18, 15 to 19, 15 to 20, 15 to 21, 15 to 22, 15 to 23, 15 to 24, 15 to 25, 15 to 26, 15 to 27, 15 to 28, 15 to 29, 15 to 30, 16 to 17, 16 to 18, 16 to 19, 16 to 20, 16 to 21, 16 to 22, 16 to 23, 16 to 24, 16 to 25, 16 to 26, 16 to 27, 16 to 28, 16 to 29, 16 to 30, 17 to 18, 17 to 19, 17 to 20, 17 to 21, 17 to 22, 17 to 23, 17 to 24, 17 to 25, 17 to 26, 17 to 27, 17 to 28, 17 to 29, 17 to 30, 18 to 19, 18 to 20, 18 to 21, 18 to 22, 18 to 23, 18 to 24, 18 to 25, 18 to 26, 18 to 27, 18 to 28, 18 to 29, 18 to 30, 19 to 20, 19 to 21, 19 to 22, 19 to 23, 19 to 24, 19 to 25, 19 to 26, 19 to 29, 19 to 28, 19 to 29, 19 to 30, 20 to 21, 20 to 22, 20 to 23, 20 to 24, 20 to 25, 20 to 26, 20 to 27, 20 to 28, 20 to 29, 20 to 30, 21 to 22, 21 to 23, 21 to 24, 21 to 25, 21 to 26, 21 to 27, 21 to 28, 21 to 29, 21 to 30, 22 to 23, 22 to 24, 22 to 25, 22 to 26, 22 to 27, 22 to 28, 22 to 29, 22 to 30, 23 to 24, 23 to 25, 23 to 26, 23 to 27, 23 to 28, 23 to 29, 23 to 30, 24 to 25, 24 to 26, 24 to 27, 24 to 28, 24 to 29, 24 to 30, 25 to 26, 25 to 27, 25 to 28, 25 to 29, 25 to 30, 26 to 27, 26 to 28, 26 to 29, 26 to 30, 27 to 28, 27 to 29, 27 to 30, 28 to 29, 28 to 30, or 29 to 30 linked nucleosides. In embodiments where the number of nucleosides of an oligomeric compound or oligonucleotide is limited, whether to a range or to a specific number, the oligomeric compound or oligonucleotide may, nonetheless further comprise additional other substituents. For example, an oligonucleotide comprising 8-30 nucleosides excludes oligonucleotides having 31 nucleosides, but, unless otherwise indicated, such an oligonucleotide may further comprise, for example one or more conjugates, terminal groups, or other substituents. In certain embodiments, a gapmer oligonucleotide has any of the above lengths.
• In certain embodiments, any of the gapmer motifs provided above, including but not limited to gapmer motifs 1-278 provided in Tables 3 and 4, may have any of the above lengths. One of skill in the art will appreciate that certain lengths may not be possible for certain motifs. For example: a gapmer having a 5′-wing region consisting of four nucleotides, a gap consisting of at least six nucleotides, and a 3′-wing region consisting of three nucleotides cannot have an overall length less than 13 nucleotides. Thus, one would understand that the lower length limit is 13 and that the limit of 10 in “10-20” has no effect in that embodiment.
• Further, where an oligonucleotide is described by an overall length range and by regions having specified lengths, and where the sum of specified lengths of the regions is less than the upper limit of the overall length range, the oligonucleotide may have additional nucleosides, beyond those of the specified regions, provided that the total number of nucleosides does not exceed the upper limit of the overall length range. For example, an oligonucleotide consisting of 20-25 linked nucleosides comprising a 5′-wing consisting of 5 linked nucleosides; a 3′-wing consisting of 5 linked nucleosides and a central gap consisting of 10 linked nucleosides (5+5+10=20) may have up to 5 nucleosides that are not part of the 5′-wing, the 3′-wing, or the gap (before reaching the overall length limitation of 25). Such additional nucleosides may be 5′ of the 5′-wing and/or 3′ of the 3′ wing.
• Certain Oligonucleotides
• In certain embodiments, oligonucleotides of the present invention are characterized by their sugar motif, internucleoside linkage motif, nucleobase modification motif and overall length. In certain embodiments, such parameters are each independent of one another. Thus, each internucleoside linkage of an oligonucleotide having a gapmer sugar motif may be modified or unmodified and may or may not follow the gapmer modification pattern of the sugar modifications. Thus, the internucleoside linkages within the wing regions of a sugar-gapmer may be the same or different from one another and may be the same or different from the internucleoside linkages of the gap region. Likewise, such sugar-gapmer oligonucleotides may comprise one or more modified nucleobase independent of the gapmer pattern of the sugar modifications. One of skill in the art will appreciate that such motifs may be combined to create a variety of oligonucleotides, such as those provided in the non-limiting Table 5 below.

• TABLE 10
  Certain Oligonucleotides

  Overall		Internucleoside	Nucleobase Mod.
  Length	Sugar motif	Linkage Motif	Motif
  12	Gapmer motif selected from 1-	uniform PS	uniform unmodified
  	278
  14	Gapmer motif selected from 1-	2-14-2 gapmer: PO in	uniform unmodified
  	278	wings and PS in gap
  14	Gapmer motif selected from 1-	uniform PS	uniform unmodified;
  	278		all C's are 5-meC
  16	Gapmer of Formula I	uniform PS	uniform unmodified;
  			no Cs are 5-meC)
  16	Gapmer of Formula I	uniform PS	uniform unmodified;
  			at least one nucleobase
  			is a 5-meC
  16	Gapmer of Formula I and having	uniform PS	uniform unmodified
  	motif selected from 1-58
  17	Gapmer of Formula I and having	uniform PO	uniform unmodified
  	motif selected from 1-58
  17	Gapmer motif selected from 1-	uniform PS	uniform unmodified
  	278
  17	Gapmer of Formula I	uniform PS	uniform unmodified
  18	Gapmer of Formula I and having	uniform PS	uniform unmodified
  	motif selected from 1-58
  18	Gapmer motif selected from 1-	uniform PS	uniform unmodified
  	278
  20	Gapmer of Formula I	uniform PS	uniform unmodified
  12	Gapmer motif selected from 1-	uniform PS	uniform unmodified
  	359
  14	Gapmer motif selected from 1-	2-14-2 gapmer: PO in	uniform unmodified
  	359	wings and PS in gap
  14	Gapmer motif selected from 1-	uniform PS	uniform unmodified;
  	359		all C's are 5-meC
  16	Gapmer of Formula II	unifonn PS	uniform unmodified;
  			no Cs are 5-meC)
  16	Gapmer of Formula II	unifonn PS	uniform unmodified;
  			at least one nucleobase
  			is a 5-meC
  16	Gapmer of Formula II and having	unifonn PS	uniform unmodified
  	motif selected from 1-359
  17	Gapmer of Formula II and having	uniform PO	uniform unmodified
  	motif selected from 1-359
  17	Gapmer motif selected from 1-	uniform PS	uniform unmodified
  	359
  17	Gapmer of Formula II	uniform PS	uniform unmodified
  18	Gapmer of Formula I and having	uniform PS	uniform unmodified
  	motif selected from 1-359
  18	Gapmer motif selected from 1-	uniform PS	uniform unmodified
  	359
  20	Gapmer of Formula II	uniform PS	uniform unmodified
  12	Gapmer motif selected from 1-	uniform PS	uniform unmodified
  	615
  14	Gapmer motif selected from 1-	2-14-2 gapmer: PO in	uniform unmodified
  	615	wings and PS in gap
  14	Gapmer motif selected from 1-	uniform PS	uniform unmodified;
  	615		all C's are 5-meC
  16	Gapmer of Formula I and having	uniform PS	uniform unmodified
  	motif selected from 1-615
  17	Gapmer of Formula I and having	uniform PO	uniform unmodified
  	motif selected from 1-615
  17	Gapmer motif selected from 1-	uniform PS	uniform unmodified
  	615
  18	Gapmer of Formula I and having	uniform PS	uniform unmodified
  	motif selected from 1-615
  18	Gapmer motif selected from 1-	uniform PS	uniform unmodified
  	615

• The above table is intended only to illustrate and not to limit the various combinations of the parameters of oligonucleotides of the present invention. Herein if a description of an oligonucleotide or oligomeric compound is silent with respect to one or more parameter, such parameter is not limited. Thus, an oligomeric compound described only as having a gapmer sugar motif without further description may have any length, internucleoside linkage motif, and nucleobase modification motif. Unless otherwise indicated, all chemical modifications are independent of nucleobase sequence.
• Certain Conjugate Groups
• In certain embodiments, oligomeric compounds are modified by attachment of one or more conjugate groups. In general, conjugate groups modify one or more properties of the attached oligomeric compound including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, cellular distribution, cellular uptake, charge and clearance. Conjugate groups are routinely used in the chemical arts and are linked directly or via an optional conjugate linking moiety or conjugate linking group to a parent compound such as an oligomeric compound, such as an oligonucleotide. Conjugate groups includes without limitation, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins and dyes. Certain conjugate groups have been described previously, for example: cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., do-decan-diol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).
• In certain embodiments, a conjugate group comprises an active drug substance, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fen-bufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indo-methicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.
• In certain embodiments, conjugate groups are directly attached to oligonucleotides in oligomeric compounds. In certain embodiments, conjugate groups are attached to oligonucleotides by a conjugate linking group. In certain such embodiments, conjugate linking groups, including, but not limited to, bifunctional linking moieties such as those known in the art are amenable to the compounds provided herein. Conjugate linking groups are useful for attachment of conjugate groups, such as chemical stabilizing groups, functional groups, reporter groups and other groups to selective sites in a parent compound such as for example an oligomeric compound. In general a bifunctional linking moiety comprises a hydrocarbyl moiety having two functional groups. One of the functional groups is selected to bind to a parent molecule or compound of interest and the other is selected to bind essentially any selected group such as chemical functional group or a conjugate group. In some embodiments, the conjugate linker comprises a chain structure or an oligomer of repeating units such as ethylene glycol or amino acid units. Examples of functional groups that are routinely used in a bifunctional linking moiety include, but are not limited to, electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. In some embodiments, bifunctional linking moieties include amino, hydroxyl, carboxylic acid, thiol, unsaturations (e.g., double or triple bonds), and the like.
• Some nonlimiting examples of conjugate linking moieties include pyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) and 6-aminohexanoic acid (AHEX or AHA). Other linking groups include, but are not limited to, substituted C₁-C₁₀alkyl, substituted or unsubstituted C₂-C₁₀alkenyl or substituted or unsubstituted C₂-C₁₀alkynyl, wherein a nonlimiting list of preferred substituent groups includes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.
• Conjugate groups may be attached to either or both ends of an oligonucleotide (terminal conjugate groups) and/or at any internal position.
• In certain embodiments, conjugate groups are at the 3′-end of an oligonucleotide of an oligomeric compound. In certain embodiments, conjugate groups are near the 3′-end. In certain embodiments, conjugates are attached at the 3′end of an oligomeric compound, but before one or more terminal group nucleosides. In certain embodiments, conjugate groups are placed within a terminal group. In certain embodiments, the present invention provides oligomeric compounds. In certain embodiments, oligomeric compounds comprise an oligonucleotide. In certain embodiments, an oligomeric compound comprises an oligonucleotide and one or more conjugate and/or terminal groups. Such conjugate and/or terminal groups may be added to oligonucleotides having any of the chemical motifs discussed above. Thus, for example, an oligomeric compound comprising an oligonucleotide having region of alternating nucleosides may comprise a terminal group.
Antisense Compounds
• In certain embodiments, oligomeric compounds of the present invention are antisense compounds. Such antisense compounds are capable of hybridizing to a target nucleic acid, resulting in at least one antisense activity. In certain embodiments, antisense compounds specifically hybridize to one or more target nucleic acid. In certain embodiments, a specifically hybridizing antisense compound has a nucleobase sequence comprising a region having sufficient complementarity to a target nucleic acid to allow hybridization and result in antisense activity and insufficient complementarity to any non-target so as to avoid non-specific hybridization to any non-target nucleic acid sequences under conditions in which specific hybridization is desired (e.g., under physiological conditions for in vivo or therapeutic uses, and under conditions in which assays are performed in the case of in vitro assays).
• In certain embodiments, the present invention provides antisense compounds comprising oligonucleotides that are fully complementary to the target nucleic acid over the entire length of the oligonucleotide. In certain embodiments, oligonucleotides are 99% complementary to the target nucleic acid. In certain embodiments, oligonucleotides are 95% complementary to the target nucleic acid. In certain embodiments, such oligonucleotides are 90% complementary to the target nucleic acid.
• In certain embodiments, such oligonucleotides are 85% complementary to the target nucleic acid. In certain embodiments, such oligonucleotides are 80% complementary to the target nucleic acid. In certain embodiments, an antisense compound comprises a region that is fully complementary to a target nucleic acid and is at least 80% complementary to the target nucleic acid over the entire length of the oligonucleotide. In certain such embodiments, the region of full complementarity is from 6 to 14 nucleobases in length.
• Certain Antisense Activities and Mechanisms
• In certain antisense activities, hybridization of an antisense compound results in recruitment of a protein that cleaves of the target nucleic acid. For example, certain antisense compounds result in RNase H mediated cleavage of target nucleic acid. RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. The “DNA” in such an RNA:DNA duplex, need not be unmodified DNA. In certain embodiments, the invention provides antisense compounds that are sufficiently “DNA-like” to elicit RNase H activity. Such DNA-like antisense compounds include, but are not limited to gapmers having unmodified deoxyfuronose sugar moieties in the nucleosides of the gap and modified sugar moieties in the nucleosides of the wings.
• Antisense activities may be observed directly or indirectly. In certain embodiments, observation or detection of an antisense activity involves observation or detection of a change in an amount of a target nucleic acid or protein encoded by such target nucleic acid; a change in the ratio of splice variants of a nucleic acid or protein; and/or a phenotypic change in a cell or animal.
• In certain embodiments, compounds comprising oligonucleotides having a gapmer motif described herein have desirable properties compared to non-gapmer oligonucleotides or to gapmers having other motifs. In certain circumstances, it is desirable to identify motifs resulting in a favorable combination of potent antisense activity and relatively low toxicity. In certain embodiments, compounds of the present invention have a favorable therapeutic index (measure of potency divided by measure of toxicity).
• Certain Target Nucleic Acids
• In certain embodiments, antisense compounds comprise or consist of an oligonucleotide comprising a region that is complementary to a target nucleic acid. In certain embodiments, the target nucleic acid is an endogenous RNA molecule. In certain embodiments, the target nucleic acid is a non-coding RNA. In certain such embodiments, the target non-coding RNA is selected from: a long-non-coding RNA, a short non-coding RNA, an intronic RNA molecule, a snoRNA, a scaRNA, a microRNA (including pre-microRNA and mature microRNA), a ribosomal RNA, and promoter directed RNA. In certain embodiments, the target nucleic acid encodes a protein. In certain such embodiments, the target nucleic acid is selected from: an mRNA and a pre-mRNA, including intronic, exonic and untranslated regions. In certain embodiments, oligomeric compounds are at least partially complementary to more than one target nucleic acid. For example, antisense compounds of the present invention may mimic microRNAs, which typically bind to multiple targets.
• In certain embodiments, the target nucleic acid is a nucleic acid other than a mature mRNA. In certain embodiments, the target nucleic acid is a nucleic acid other than a mature mRNA or a microRNA. In certain embodiments, the target nucleic acid is a non-coding RNA other than a microRNA. In certain embodiments, the target nucleic acid is a non-coding RNA other than a microRNA or an intronic region of a pre-mRNA. In certain embodiments, the target nucleic acid is a long non-coding RNA. In certain embodiments, the target RNA is an mRNA. In certain embodiments, the target nucleic acid is a pre-mRNA. In certain such embodiments, the target region is entirely within an intron. In certain embodiments, the target region spans an intron/exon junction. In certain embodiments, the target region is at least 50% within an intron. In certain embodiments, the target nucleic acid is selected from among non-coding RNA, including exonic regions of pre-mRNA. In certain embodiments, the target nucleic acid is a ribosomal RNA (rRNA). In certain embodiments, the target nucleic acid is a non-coding RNA associated with splicing of other pre-mRNAs. In certain embodiments, the target nucleic acid is a nuclear-retained non-coding RNA.
• In certain embodiments, antisense compounds described herein are complementary to a target nucleic acid comprising a single-nucleotide polymorphism. In certain such embodiments, the antisense compound is capable of modulating expression of one allele of the single-nucleotide polymorphism-containing-target nucleic acid to a greater or lesser extent than it modulates another allele. In certain embodiments an antisense compound hybridizes to a single-nucleotide polymorphism-containing-target nucleic acid at the single-nucleotide polymorphism site. In certain embodiments an antisense compound hybridizes to a single-nucleotide polymorphism-containing-target nucleic acid near the single-nucleotide polymorphism site. In certain embodiments, the target nucleic acid is a Huntingtin gene transcript. In certain embodiments, the target nucleic acid is a single-nucleotide polymorphism-containing-target nucleic acid other than a Huntingtin gene transcript. In certain embodiments, the target nucleic acid is any nucleic acid other than a Huntingtin gene transcript.
• Certain Pharmaceutical Compositions
• In certain embodiments, the present invention provides pharmaceutical compositions comprising one or more antisense compound. In certain embodiments, such pharmaceutical composition comprises a suitable pharmaceutically acceptable diluent or carrier. In certain embodiments, a pharmaceutical composition comprises a sterile saline solution and one or more antisense compound. In certain embodiments, such pharmaceutical composition consists of a sterile saline solution and one or more antisense compound. In certain embodiments, the sterile saline is pharmaceutical grade saline. In certain embodiments, a pharmaceutical composition comprises one or more antisense compound and sterile water. In certain embodiments, a pharmaceutical composition consists of one or more antisense compound and sterile water. In certain embodiments, the sterile saline is pharmaceutical grade water. In certain embodiments, a pharmaceutical composition comprises one or more antisense compound and phosphate-buffered saline (PBS). In certain embodiments, a pharmaceutical composition consists of one or more antisense compound and sterile phosphate-buffered saline (PBS). In certain embodiments, the sterile saline is pharmaceutical grade PBS.
• In certain embodiments, antisense compounds may be admixed with pharmaceutically acceptable active and/or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions depend on a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.
• Pharmaceutical compositions comprising antisense compounds encompass any pharmaceutically acceptable salts, esters, or salts of such esters. In certain embodiments, pharmaceutical compositions comprising antisense compounds comprise one or more oligonucleotide which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.
• A prodrug can include the incorporation of additional nucleosides at one or both ends of an oligomeric compound which are cleaved by endogenous nucleases within the body, to form the active antisense oligomeric compound.
• Lipid moieties have been used in nucleic acid therapies in a variety of methods. In certain such methods, the nucleic acid is introduced into preformed liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids. In certain methods, DNA complexes with mono- or poly-cationic lipids are formed without the presence of a neutral lipid. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to a particular cell or tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to fat tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to muscle tissue.
• In certain embodiments, pharmaceutical compositions provided herein comprise one or more modified oligonucleotides and one or more excipients. In certain such embodiments, excipients are selected from water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose and polyvinylpyrrolidone.
• In certain embodiments, a pharmaceutical composition provided herein comprises a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing certain pharmaceutical compositions including those comprising hydrophobic compounds. In certain embodiments, certain organic solvents such as dimethylsulfoxide are used.
• In certain embodiments, a pharmaceutical composition provided herein comprises one or more tissue-specific delivery molecules designed to deliver the one or more pharmaceutical agents of the present invention to specific tissues or cell types. For example, in certain embodiments, pharmaceutical compositions include liposomes coated with a tissue-specific antibody.
• In certain embodiments, a pharmaceutical composition provided herein comprises a co-solvent system. Certain of such co-solvent systems comprise, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. In certain embodiments, such co-solvent systems are used for hydrophobic compounds. A non-limiting example of such a co-solvent system is the VPD co-solvent system, which is a solution of absolute ethanol comprising 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80™ and 65% w/v polyethylene glycol 300. The proportions of such co-solvent systems may be varied considerably without significantly altering their solubility and toxicity characteristics. Furthermore, the identity of co-solvent components may be varied: for example, other surfactants may be used instead of Polysorbate 80™; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.
• In certain embodiments, a pharmaceutical composition provided herein is prepared for oral administration. In certain embodiments, pharmaceutical compositions are prepared for buccal administration.
• In certain embodiments, a pharmaceutical composition is prepared for administration by injection (e.g., intravenous, subcutaneous, intramuscular, etc.). In certain of such embodiments, a pharmaceutical composition comprises a carrier and is formulated in aqueous solution, such as water or physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. In certain embodiments, other ingredients are included (e.g., ingredients that aid in solubility or serve as preservatives). In certain embodiments, injectable suspensions are prepared using appropriate liquid carriers, suspending agents and the like. Certain pharmaceutical compositions for injection are presented in unit dosage form, e.g., in ampoules or in multi-dose containers. Certain pharmaceutical compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Certain solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, such suspensions may also contain suitable stabilizers or agents that increase the solubility of the pharmaceutical agents to allow for the preparation of highly concentrated solutions.
• In certain embodiments, a pharmaceutical composition is prepared for transmucosal administration. In certain of such embodiments penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
• In certain embodiments, a pharmaceutical composition provided herein comprises an oligonucleotide in a therapeutically effective amount. In certain embodiments, the therapeutically effective amount is sufficient to prevent, alleviate or ameliorate symptoms of a disease or to prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art.
• In certain embodiments, one or more modified oligonucleotide provided herein is formulated as a prodrug. In certain embodiments, upon in vivo administration, a prodrug is chemically converted to the biologically, pharmaceutically or therapeutically more active form of an oligonucleotide. In certain embodiments, prodrugs are useful because they are easier to administer than the corresponding active form. For example, in certain instances, a prodrug may be more bioavailable (e.g., through oral administration) than is the corresponding active form. In certain instances, a prodrug may have improved solubility compared to the corresponding active form. In certain embodiments, prodrugs are less water soluble than the corresponding active form. In certain instances, such prodrugs possess superior transmittal across cell membranes, where water solubility is detrimental to mobility. In certain embodiments, a prodrug is an ester. In certain such embodiments, the ester is metabolically hydrolyzed to carboxylic acid upon administration. In certain instances the carboxylic acid containing compound is the corresponding active form. In certain embodiments, a prodrug comprises a short peptide (polyaminoacid) bound to an acid group. In certain of such embodiments, the peptide is cleaved upon administration to form the corresponding active form.
• In certain embodiments, the present invention provides compositions and methods for reducing the amount or activity of a target nucleic acid in a cell. In certain embodiments, the cell is in an animal. In certain embodiments, the animal is a mammal. In certain embodiments, the animal is a rodent. In certain embodiments, the animal is a primate. In certain embodiments, the animal is a non-human primate. In certain embodiments, the animal is a human.
• In certain embodiments, the present invention provides methods of administering a pharmaceutical composition comprising an oligomeric compound of the present invention to an animal. Suitable administration routes include, but are not limited to, oral, rectal, transmucosal, intestinal, enteral, topical, suppository, through inhalation, intrathecal, intracerebroventricular, intraperitoneal, intranasal, intraocular, intratumoral, and parenteral (e.g., intravenous, intramuscular, intramedullary, and subcutaneous). In certain embodiments, pharmaceutical intrathecals are administered to achieve local rather than systemic exposures.
Nonlimiting Disclosure and Incorporation by Reference
• While certain compounds, compositions and methods described herein have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds described herein and are not intended to limit the same. Each of the references, GenBank accession numbers, and the like recited in the present application is incorporated herein by reference in its entirety.
• Although the sequence listing accompanying this filing identifies each sequence as either “RNA” or “DNA” as required, in reality, those sequences may be modified with any combination of chemical modifications. One of skill in the art will readily appreciate that such designation as “RNA” or “DNA” to describe modified oligonucleotides is, in certain instances, arbitrary. For example, an oligonucleotide comprising a nucleoside comprising a 2′-OH sugar moiety and a thymine base could be described as a DNA having a modified sugar (2′-OH for the natural 2′-H of DNA) or as an RNA having a modified base (thymine (methylated uracil) for natural uracil of RNA).
• Accordingly, nucleic acid sequences provided herein, including, but not limited to those in the sequence listing, are intended to encompass nucleic acids containing any combination of natural or modified RNA and/or DNA, including, but not limited to such nucleic acids having modified nucleobases. By way of further example and without limitation, an oligomeric compound having the nucleobase sequence “ATCGATCG” encompasses any oligomeric compounds having such nucleobase sequence, whether modified or unmodified, including, but not limited to, such compounds comprising RNA bases, such as those having sequence “AUCGAUCG” and those having some DNA bases and some RNA bases such as “AUCGATCG” and oligomeric compounds having other modified or naturally occurring bases, such as “AT^meCGAUCG,” wherein^meC indicates a cytosine base comprising a methyl group at the 5-position.
EXAMPLES
• The following examples illustrate certain embodiments of the present invention and are not limiting. Moreover, where specific embodiments are provided, the inventors have contemplated generic application of those specific embodiments. For example, disclosure of an oligonucleotide having a particular motif provides reasonable support for additional oligonucleotides having the same or similar motif. And, for example, where a particular high-affinity modification appears at a particular position, other high-affinity modifications at the same position are considered suitable, unless otherwise indicated.
• Where nucleobase sequences are not provided, to allow assessment of the relative effects of nucleobase sequence and chemical modification, throughout the examples, oligomeric compounds are assigned a “Sequence Code.” Oligomeric compounds having the same Sequence Code have the same nucleobase sequence. Oligomeric compounds having different Sequence Codes have different nucleobase sequences.
Example 1: Modified Antisense Oligonucleotides Targeting Human Target-X
• Antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. ISIS 407939, which was described in an earlier publication (WO 2009/061851) was also tested.
• The newly designed chimeric antisense oligonucleotides and their motifs are described in Table 11. The internucleoside linkages throughout each gapmer are phosphorothioate linkages (P=5). Nucleosides followed by “d” indicate 2′-deoxyribonucleosides. Nucleosides followed by “k” indicate constrained ethyl (cEt) nucleosides. Nucleosides followed by “e” indicate 2′-O-methythoxylethyl (2′-MOE) nucleosides. “N” indicates modified or naturally occurring nucleobases (A, T, C, G, U, or 5-methyl C).
• Each gapmer listed in Table 11 is targeted to the human Target-X genomic sequence.
• Activity of the newly designed gapmers was compared to a 5-10-5 2′-MOE gapmer, ISIS 407939 targeting human Target-X and is further described in U.S. Pat. No. ______, incorporated herein by reference. Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS2927 was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells, and indicate that several of the newly designed antisense oligonucleotides are more potent than ISIS 407939. A total of 771 oligonucleotides were tested. Only those oligonucleotides which were selected for further studies are shown in Table 11. Each of the newly designed antisense oligonucleotides provided in Table 10 achieved greater than 80% inhibition and, therefore, are more active than ISIS 407939.

• TABLE 11
  Inhibition of human Target-X mRNA levels by chimeric antisense
  oligonucleotides targeted to Target-X

  					Wing		SEQ
  Sequence	ISIS	%		Gap	Chemistry	SEQ	ID

  (5′ to 3′)	NO	inhibition	Motif	Chemistry	5′	3′	CODE	NO
  NkNkNkNdNdNdNdNkNd	473359	92	3-10-3	Deoxy/	kkk	eee	21	19
  NdNdNdNdNeNeNe				cEt
  NkNkNkNdNdNdNdNkNd	473360	96	3-10-3	Deoxy/	kkk	eee	22	19
  NdNdNdNdNeNeNe				cEt
  NkNkNkNdNdNdNdNdNd	473168	94	3-10-3	Full	kkk	kkk	23	19
  NdNdNdNdNkNkNk				deoxy
  NkNkNkNdNdNdNdNdNd	473317	95	3-10-3	Full	kkk	eee	23	19
  NdNdNdNdNeNeNe				deoxy
  NkNkNkNdNdNdNdNkNd	473471	90	3-10-3	Deoxy/	kkk	eee	23	19
  NdNdNdNdNeNeNe				cEt
  NkNdNkNdNkNdNdNdNd	473620	94	5-9-2	Full	kdkdk	ee	23	19
  NdNdNdNdNdNeNe				deoxy
  NkNkNdNdNdNdNdNdNd	473019	88	2-10-2	Full	kk	kk	24	20
  NdNdNdNkNk				deoxy
  NkNkNdNdNdNdNdNdNd	473020	93	2-10-2	Full	kk	kk	25	20
  NdNdNdNkNk				deoxy
  NkNkNkNdNdNdNdNdNd	473321	93	3-10-3	Full	kkk	eee	26	19
  NdNdNdNdNeNeNe				deoxy
  NkNkNkNdNdNdNdNdNd	473322	94	3-10-3	Full	kkk	eee	27	19
  NdNdNdNdNeNeNe				deoxy
  NkNkNkNdNdNdNdNdNd	473323	96	3-10-3	Full	kkk	eee	28	19
  NdNdNdNdNeNeNe				deoxy
  NkNkNkNdNdNdNdNdNd	473326	94	3-10-3	Full	kkk	eee	29	19
  NdNdNdNdNeNeNe				deoxy
  NkNkNkNdNdNdNdNkNd	473480	92	3-10-3	Deoxy/	kkk	eee	29	19
  NdNdNdNdNeNeNe				cEt
  NkNkNkNdNdNdNdNdNd	473178	96	3-10-3	Full	kkk	kkk	30	19
  NdNdNdNdNkNkNk				deoxy
  NkNkNkNdNdNdNdNdNd	473327	96	3-10-3	Full	kkk	eee	30	19
  NdNdNdNdNeNeNe				deoxy
  NkNkNkNdNdNdNdNkNd	473481	93	3-10-3	Deoxy/	kkk	eee	30	19
  NdNdNdNdNeNeNe				cEt
  NkNdNkNdNkNdNdNdNd	473630	89	5-9-2	Full	kdkdk	ee	30	19
  NdNdNdNdNdNeNe				deoxy
  NkNkNdNdNdNdNdNdNd	473029	96	2-10-2	Full	kk	kk	31	20
  NdNdNdNkNk				deoxy
  NkNkNdNdNdNdNdNdNd	472925	93	2-10-2	Full	kk	kk	32	20
  NdNdNdNkNk				deoxy
  NkNkNdNdNdNdNdNdNd	472926	85	2-10-2	Full	kk	kk	33	20
  NdNdNdNkNk				deoxy
  NkNkNkNdNdNdNdNdNd	473195	97	3-10-3	Full	kkk	kkk	34	19
  NdNdNdNdNkNkNk				deoxy
  NkNkNdNdNdNdNdNdNd	473046	90	2-10-2	Full	kk	kk	35	20
  NdNdNdNkNk				deoxy
  NkNkNdNdNdNdNdNdNd	472935	92	2-10-2	Full	kk	kk	36	20
  NdNdNdNkNk				deoxy
  NkNkNkNdNdNdNdNdNd	473089	95	3-10-3	Full	kkk	kkk	37	19
  NdNdNdNdNkNkNk				deoxy
  NkNkNkNdNdNdNdNdNd	473350	93	3-10-3	Full	kkk	eee	38	19
  NdNdNdNdNeNeNe				deoxy
  NkNkNkNdNdNdNdNdNd	473353	93	3-10-3	Full	kkk	eee	39	19
  NdNdNdNdNeNeNe				deoxy
  NkNkNdNdNdNdNdNdNd	473055	91	2-10-2	Full	kk	kk	40	20
  NdNdNdNkNk				deoxy
  NkNkNkNdNdNdNdNkNd	473392	95	3-10-3	Deoxy/	kkk	eee	41	19
  NdNdNdNdNeNeNe				cEt
  NkNkNkNdNdNdNdNdNd	473095	100	3-10-3	Full	kkk	kkk	42	19
  NdNdNdNdNkNkNk				deoxy
  NkNkNkNdNdNdNdNdNd	473244	99	3-10-3	Full	kkk	eee	42	19
  NdNdNdNdNeNeNe				deoxy
  NkNkNkNdNdNdNdNkNd	473393	99	3-10-3	Deoxy/	kkk	eee	42	19
  NdNdNdNdNeNeNe				cEt
  NkNdNkNdNkNdNdNdNd	473547	98	5-9-2	Full	kdkdk	ee	42	19
  NdNdNdNdNdNeNe				deoxy
  NkNkNdNdNdNdNdNdNd	472942	87	2-10-2	Full	kk	kk	43	20
  NdNdNdNkNk				deoxy
  NkNkNkNdNdNdNdNdNd	473098	97	3-10-3	Full	kkk	kkk	44	19
  NdNdNdNdNkNkNk				deoxy
  NkNkNkNdNdNdNdNkNd	473408	92	3-10-3	Deoxy/	kkk	eee	45	19
  NdNdNdNdNeNeNe				cEt
  NkNkNdNdNdNdNdNdNd	472958	89	2-10-2	Full	kk	kk	46	20
  NdNdNdNkNk				deoxy
  NkNkNdNdNdNdNdNdNd	472959	90	2-10-2	Full	kk	kk	47	20
  NdNdNdNkNk				deoxy
  NkNdNkNdNkNdNdNdNd	473566	94	5-9-2	Full	kdkdk	ee	48	19
  NdNdNdNdNdNeNe				deoxy
  NkNdNkNdNkNdNdNdNd	473567	95	5-9-2	Full	kdkdk	ee	49	19
  NdNdNdNdNdNeNe				deoxy
  NkNdNkNdNkNdNdNdNd	473569	92	5-9-2	Full	kdkdk	ee	50	19
  NdNdNdNdNdNeNe				deoxy
  NkNkNdNdNdNdNdNdNd	457851	90	2-10-2	Full	kk	kk	51	20
  NdNdNdNkNk				deoxy
  NkNkNdNdNdNdNdNdNd	472970	91	2-10-2	Full	kk	kk	32	20
  NdNdNdNkNk				deoxy
  NkNkNkNdNdNdNdNdNd	473125	90	3-10-3	Full	kkk	kkk	53	19
  NdNdNdNdNkNkNk				deoxy
  NkNkNkNdNdNdNdNdNd	473274	98	3-10-3	Full	kkk	eee	53	19
  NdNdNdNdNeNeNe				deoxy
  NkNkNkNdNdNdNdNkNd	473428	90	3-10-3	Deoxy/	kkk	eee	53	19
  NdNdNdNdNeNeNe				cEt
  NkNdNkNdNkNdNdNdNd	473577	93	5-9-2	Full	kdkdk	ee	53	19
  NdNdNdNdNdNeNe				deoxy
  NkNkNdNdNdNdNdNdNd	472976	97	2-10-2	Full	kk	kk	54	20
  NdNdNdNkNk				deoxy
  NkNkNdNdNdNdNdNd	472983	94	2-10-2	Full	kk	kk	55	20
  NdNdNdNdNkNk				deoxy
  NkNkNdNdNdNdNdNd	472984	90	2-10-2	Full	kk	kk	56	20
  NdNdNdNdNkNk				deoxy
  NkNkNkNdNdNdNdNd	473135	97	3-10-3	Full	kkk	kkk	57	19
  NdNdNdNdNdNkNkNk				deoxy
  NkNkNdNdNdNdNdNd	472986	95	2-10-2	Full	kk	kk	58	20
  NdNdNdNdNkNk				deoxy
  NkNkNkNdNdNdNdNd	473137	95	3-10-3	Full	kkk	kkk	59	19
  NdNdNdNdNdNkNkNk				deoxy
  NkNkNkNdNdNdNdNd	473286	95	3-10-3	Full	kkk	eee	59	19
  NdNdNdNdNdNeNeNe				deoxy
  NkNkNkNdNdNdNdNkNd	473440	88	3-10-3	Deoxy/	kkk	eee	59	19
  NdNdNdNdNeNeNe				cEt
  NkNdNkNdNkNdNdNd	473589	97	5-9-2	Full	kdkdk	ee	59	19
  NdNdNdNdNdNdNeNe				deoxy
  NkNkNdNdNdNdNdNd	472988	85	2-10-2	Full	kk	kk	60	20
  NdNdNdNdNkNk				deoxy
  NkNkNkNdNdNdNdNd	473140	96	3-10-3	Full	kkk	kkk	61	19
  NdNdNdNdNdNkNkNk				deoxy
  NkNkNdNdNdNdNdNd	472991	90	2-10-2	Full	kk	kk	62	20
  NdNdNdNdNkNk				deoxy
  NkNkNkNdNdNdNdNkNd	473444	94	3-10-3	Deoxy/	kkk	eee	63	19
  NdNdNdNdNeNeNe				cEt
  NkNkNkNdNdNdNdNd	473142	96	3-10-3	Full	kkk	kkk	64	19
  NdNdNdNdNdNkNkNk				deoxy
  NkNkNkNdNdNdNdNd	473291	95	3-10-3	Full	kkk	eee	64	19
  NdNdNdNdNdNeNeNe				deoxy
  NkNdNkNdNkNdNdNd	473594	95	5-9-2	Full	kdkdk	ee	64	19
  NdNdNdNdNdNdNeNe				deoxy
  NkNkNkNdNdNdNdNdNd	473143	97	3-10-3	Full	kkk	kkk	65	19
  NdNdNdNdNkNkNk				deoxy
  NkNkNkNdNdNdNdNd	473292	96	3-10-3	Full	kkk	eee	65	19
  NdNdNdNdNdNeNeNe				deoxy
  NkNkNkNdNdNdNdNkNd	473446	96	3-10-3	Deoxy/	kkk	eee	65	19
  NdNdNdNdNeNeNe				cEt
  NkNdNkNdNkNdNdNdNd	473595	84	5-9-2	Full	kdkdk	ee	65	19
  NdNdNdNdNdNeNe				deoxy
  NkNkNdNdNdNdNdNdNd	472994	96	2-10-2	Full	kk	kk	66	20
  NdNdNdNkNk				deoxy
  NkNkNkNdNdNdNdNdNd	473144	98	3-10-3	Full	kkk	kkk	67	19
  NdNdNdNdNkNkNk				deoxy
  NkNkNkNdNdNdNdNdNd	473293	96	3-10-3	Full	kkk	eee	67	19
  NdNdNdNdNeNeNe				deoxy
  NkNkNdNdNdNdNdNdNd	472995	96	2-10-2	Full	kk	kk	68	20
  NdNdNdNkNk				deoxy
  NkNkNkNdNdNdNdNd	473294	91	3-10-3	Full	kkk	eee	69	19
  NdNdNdNdNdNeNeNe				deoxy
  NkNdNkNdNkNdNdNdNd	473597	94	5-9-2	Full	kdkdk	ee	69	19
  NdNdNdNdNdNeNe				deoxy
  NkNkNdNdNdNdNdNdNd	472996	94	2-10-2	Full	kk	kk	70	20
  NdNdNdNkNk				deoxy
  NkNkNkNdNdNdNdNd	473295	92	3-10-3	Full	kkk	eee	71	19
  NdNdNdNdNdNeNeNe				deoxy
  NeNeNeNeNeNdNdNdNdNd	407939	80	5-10-5	Full	eeeee	eeee	72	21
  NdNdNdNdNdNeNeNeNeNe				deoxy		e
  NkNkNkNdNdNdNdNdNd	473296	98	3-10-3	Full	kkk	eee	73	19
  NdNdNdNdNeNeNe				deoxy
  NkNkNkNdNdNdNdNkNd	473450	95	3-10-3	Deoxy/	kkk	eee	73	19
  NdNdNdNdNeNeNe				cEt
  NkNkNdNdNdNdNdNdNd	472998	97	2-10-2	Full	kk	kk	74	20
  NdNdNdNkNk				deoxy
  e = 2′-MOE, k = cEt, d = 2′-deoxyribonucleoside

Example 2: Modified Antisense Oligonucleotides Comprising Constrained Ethyl (cEt) and F-HNAmodifications Targeting Human Target-X
• Additional antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. ISIS 407939 was also tested.
• The newly designed chimeric antisense oligonucleotides and their motifs are described in Table 12. The internucleoside linkages throughout each gapmer are phosphorothioate linkages (P═S). Nucleosides followed by “d” indicate 2′-deoxyribonucleosides. Nucleosides followed by “k” indicate constrained ethyl (cEt) nucleosides. Nucleosides followed by “e” indicate 2′-O-methythoxylethyl (2′-MOE) modified nucleosides. Nucleosides followed by ‘g’ indicate F-HNA modified nucleosides. “N” indicates modified or naturally occurring nucleobases (A, T, C, G, U, or 5-methyl C).
• Each gapmer listed in Table 12 is targeted to the human Target-X genomic sequence.
• Activity of the newly designed gapmers was compared to a 5-10-5 2′-MOE gapmer, ISIS 407939 targeting human Target-X. Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS2927 was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells, and demonstrate that several of the newly designed gapmers are more potent than ISIS 407939. A total of 765 oligonucleotides were tested. Only those oligonucleotides which were selected for further studies are shown in Table 12. All but one of the newly designed antisense oligonucleotides provided in Table 12 achieved greater than 30% inhibition and, therefore, are more active than ISIS 407939.

• TABLE 12
  Inhibition of human Target-X mRNA levels by chimeric antisense oligonucleotides targeted to Target-X

  %

  Gap

  Wing Chemistry

  SEQ

  SEQ ID

  Sequence (5′ to 3′)	ISIS No	inhibition	Motif	Chemistry	5′	3′	CODE	NO
  NgNgNdNdNdNdNdNdNd	482838	81	2-10-2	Full deoxy	gg	gg	25	20
  NdNdNdNgNg
  NgNgNgNdNdNdNdNdNd	482992	93	3-10-3	Full deoxy	ggg	ggg	28	19
  NdNdNdNdNgNgNg
  NgNgNgNdNdNdNdNdNd	482996	97	3-10-3	Full deoxy	ggg	ggg	30	19
  NdNdNdNdNgNgNg
  NgNdNgNdNgNdNdNdNd	483284	82	5-9-2	Full deoxy	gdgdg	ee	23	19
  NdNdNdNdNdNeNe
  NgNdNgNdNgNdNdNdNd	483289	70	5-9-2	Full deoxy	gdgdg	ee	27	19
  NdNdNdNdNdNeNe
  NgNdNgNdNgNdNdNdNd	483290	80	5-9-2	Full deoxy	gdgdg	ee	28	19
  NdNdNdNdNdNeNe
  NgNdNgNdNgNdNdNdNd	483294	69	5-9-2	Full deoxy	gdgdg	ee	30	19
  NdNdNdNdNdNeNe
  NgNgNdNdNdNdNdNdNd	483438	81	2-10-4	Full deoxy	gg	eeee	23	19
  NdNdNdNeNeNeNe
  NgNgNdNdNdNdNdNdNd	483444	84	2-10-4	Full deoxy	gg	eeee	28	19
  NdNdNdNeNeNeNe
  NgNgNdNdNdNdNdNdNd	483448	77	2-10-4	Full deoxy	gg	eeee	30	19
  NdNdNdNeNeNeNe
  NgNgNdNdNdNdNdNdNd	482847	79	2-10-2	Full deoxy	gg	gg	31	20
  NdNdNdNgNg
  NgNgNdNdNdNdNdNdNd	482747	85	2-10-2	Full deoxy	gg	gg	32	20
  NdNdNdNgNg
  NgNgNdNdNdNdNdNdNd	482873	81	2-10-2	Full deoxy	gg	gg	40	20
  NdNdNdNgNg
  NgNgNdNdNdNdNdNdNdNd	482874	82	2-10-2	Full deoxy	gg	gg	75	20
  NdNdNgNg
  NgNgNdNdNdNdNdNd	482875	82	2-10-2	Full deoxy	gg	gg	76	20
  NdNdNdNdNgNg
  NgNgNgNdNdNdNdNd	482896	95	3-10-3	Full deoxy	ggg	ggg	77	19
  NdNdNdNdNdNgNgNg
  NgNgNgNdNdNdNdNdNd	483019	89	3-10-3	Full deoxy	ggg	ggg	38	19
  NdNdNdNdNgNgNg
  NgNdNgNdNdNdNdNdNd	483045	92	3-10-3	Full deoxy	gdg	gdg	77	19
  NdNdNdNdNgNdNg
  NgNdNgNdNgNdNdNdNd	483194	64	3-10-3	Full deoxy	gdg	gdg	77	19
  NdNdNdNdNdNeNe
  NgNdNgNdNgNdNdNdNd	483317	79	5-9-2	Full deoxy	gdgdg	ee	38	19
  NdNdNdNdNdNeNe
  NgNgNdNdNdNdNdNdNd	483343	75	2-10-4	Full deoxy	gg	eeee	57	19
  NdNdNdNeNeNeNe
  NgNgNdNdNdNdNdNdNdNdN	483471	76	2-10-4	Full deoxy	gg	eeee	38	19
  dNdNeNeNeNe
  NgNgNdNdNdNdNdNdNd	483478	20	2-10-4	Full deoxy	gg	eeee	78	19
  NdNdNdNeNeNeNe
  NeNeNeNeNeNdNdNdNdNd	407939	30	5-10-5	Full deoxy	eeeee	eeeee	72	21
  NdNdNdNdNdNeNeNeNeNe
  NgNgNdNdNdNdNdNd	482784	83	2-10-2	Full deoxy	gg	gg	79	20
  NdNdNdNdNgNg
  NgNgNdNdNdNdNdNd	482794	91	2-10-2	Full deoxy	gg	gg	54	20
  NdNdNdNdNgNg
  NgNgNdNdNdNdNdNd	482804	80	2-10-2	Full deoxy	gg	gg	58	20
  NdNdNdNdNgNg
  NgNgNdNdNdNdNdNd	482812	81	2-10-2	Full deoxy	gg	gg	66	20
  NdNdNdNdNgNg
  NgNgNdNdNdNdNdNd	482813	92	2-10-2	Full deoxy	gg	gg	68	20
  NdNdNdNdNgNg
  NgNgNdNdNdNdNdNd	482814	94	2-10-2	Full deoxy	gg	gg	70	20
  NdNdNdNdNgNg
  NgNgNdNdNdNdNdNd	482815	81	2-10-2	Full deoxy	gg	gg	80	20
  NdNdNdNdNgNg
  NgNgNdNdNdNdNdNd	482816	71	2-10-2	Full deoxy	gg	gg	74	20
  NdNdNdNdNgNg
  NgNgNgNdNdNdNdNd	482916	90	3-10-3	Full deoxy	ggg	ggg	44	19
  NdNdNdNdNdNgNgNg
  NgNgNgNdNdNdNdNd	482932	89	3-10-3	Full deoxy	ggg	ggg	48	19
  NdNdNdNdNdNgNgNg
  NgNgNgNdNdNdNdNd	482953	93	3-10-3	Full deoxy	ggg	ggg	57	19
  NdNdNdNdNdNgNgNg
  NgNgNgNdNdNdNdNd	482962	97	3-10-3	Full deoxy	ggg	ggg	67	19
  NdNdNdNdNdNgNgNg
  NgNgNgNdNdNdNdNd	482963	96	3-10-3	Full deoxy	ggg	ggg	69	19
  NdNdNdNdNdNgNgNg
  NgNgNgNdNdNdNdNd	482965	89	3-10-3	Full deoxy	ggg	ggg	73	19
  NdNdNdNdNdNgNgNg
  NgNdNgNdNdNdNdNd	483065	69	3-10-3	Full deoxy	ggg	ggg	44	19
  NdNdNdNdNdNgNdNg
  NgNdNgNdNdNdNdNd	483092	89	3-10-3	Full deoxy	gdg	gdg	53	19
  NdNdNdNdNdNgNdNg
  NgNdNgNdNgNdNdNd	483241	79	5-9-2	Full deoxy	gdgdg	ee	53	19
  NdNdNdNdNdNdNeNe
  NgNdNgNdNgNdNdNd	483253	76	5-9-2	Full deoxy	gdgdg	ee	59	19
  NdNdNdNdNdNdNeNe
  NgNdNgNdNgNdNdNd	483258	70	5-9-2	Full deoxy	gdgdg	ee	64	19
  NdNdNdNdNdNdNeNe
  NgNdNgNdNgNdNdNd	483260	62	5-9-2	Full deoxy	gdgdg	ee	67	19
  NdNdNdNdNdNdNeNe
  NgNdNgNdNgNdNdNd	483261	76	5-9-2	Full deoxy	gdgdg	ee	69	19
  NdNdNdNdNdNdNeNe
  NgNdNgNdNgNdNdNd	483262	75	5-9-2	Full deoxy	gdgdg	ee	71	19
  NdNdNdNdNdNdNeNe
  NgNdNgNdNgNdNdNd	483263	73	5-9-2	Full deoxy	gdgdg	ee	73	19
  NdNdNdNdNdNdNeNe
  NgNgNdNdNdNdNdNd	483364	78	2-10-4	Full deoxy	gg	eeee	81	19
  NdNdNdNdNeNeNeNe
  NgNgNdNdNdNdNdNd	483395	86	2-10-4	Full deoxy	gg	eeee	53	19
  NdNdNdNdNeNeNeNe
  NgNgNdNdNdNdNdNd	483413	83	2-10-4	Full deoxy	gg	eeee	65	19
  NdNdNdNdNeNeNeNe
  NgNgNdNdNdNdNdNd	483414	76	2-10-4	Full deoxy	gg	eeee	67	19
  NdNdNdNdNeNeNeNe
  NgNgNdNdNdNdNdNd	483415	85	2-10-4	Full deoxy	gg	eeee	69	19
  NdNdNdNdNeNeNeNe
  NgNgNdNdNdNdNdNd	483416	77	2-10-4	Full deoxy	gg	eeee	71	19
  NdNdNdNdNeNeNeNe
  NgNgNdNdNdNdNdNd	483417	83	2-10-4	Full deoxy	gg	eeee	73	19
  NdNdNdNdNeNeNeNe
  e = 2′-MOE, d = 2′-deoxyribonucleoside, g = F-HNA

Example 3: Modified Antisense Oligonucleotides Comprising 2′-MOE and Constrained Ethyl (cEt) Modifications Targeting Human Target-X
• Additional antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. ISIS 403052, ISIS 407594, ISIS 407606, ISIS 407939, and ISIS 416438, which were described in an earlier publication (WO 2009/061851) were also tested.
• The newly designed chimeric antisense oligonucleotides are 16 nucleotides in length and their motifs are described in Table 13. The chemistry column of Table 12 presents the sugar motif of each oligonucleotide, wherein “e” indicates a 2′-O-methythoxylethyl (2′-MOE) nucleoside, “k” indicates a constrained ethyl (cEt) and “d” indicates a 2′-deoxyribonucleoside. The internucleoside linkages throughout each gapmer are hosphorothioate (P═S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines.
• Each gapmer listed in Table 13 is targeted to the human Target-X genomic sequence.
• Activity of the newly designed gapmers was compared to ISIS 403052, ISIS 407594, ISIS 407606, ISIS 407939, and ISIS 416438. Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS2927 (described hereinabove in Example 1) was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of Target-X, relative to untreated control cells. A total of 380 oligonucleotides were tested. Only those oligonucleotides which were selected for further studies are shown in Table 13. Each of the newly designed antisense oligonucleotides provided in Table 13 achieved greater than 64% inhibition and, therefore, are more potent than each of ISIS 403052, ISIS 407594, ISIS 407606, ISIS 407939, and ISIS 416438.

• TABLE 13
  Inhibition of human Target-X mRNA levels by chimeric
  antisense oligonucleotides targeted to Target-X

  ISIS No	Chemistry	Motif	% inhibition	SEQ CODE

  403052	eeeee-(d10)-eeeee	5-10-5	64	82
  407594	eeeee-(d10)-eeeee	5-10-5	40	83
  407606	eeeee-(d10)-eeeee	5-10-5	39	84
  407939	eeeee-(d10)-eeeee	5-10-5	57	72
  416438	eeeee-(d10)-eeeee	5-10-5	62	85
  484487	kdk-(d10)-dkdk	3-10-3	91	77
  484539	kdk-d(10)-kdk	3-10-3	92	53
  484546	kdk-d(10)-kdk	3-10-3	92	86
  484547	kdk-d(10)-kdk	3-10-3	89	87
  484549	kdk-d(10)-kdk	3-10-3	91	57
  484557	kdk-d(10)-kdk	3-10-3	92	65
  484558	kdk-d(10)-kdk	3-10-3	94	67
  484559	kdk-d(10)-kdk	3-10-3	90	69
  484582	kdk-d(10)-kdk	3-10-3	88	23
  484632	kk-d(10)-eeee	2-10-4	90	88
  484641	kk-d(10)-eeee	2-10-4	91	77
  484679	kk-d(10)-eeee	2-10-4	90	49
  484693	kk-d(10)-eeee	2-10-4	93	53
  484711	kk-d(10)-eeee	2-10-4	92	65
  484712	kk-d(10)-eeee	2-10-4	92	67
  484713	kk-d(10)-eeee	2-10-4	85	69
  484714	kk-d(10)-eeee	2-10-4	83	71
  484715	kk-d(10)-eeee	2-10-4	93	73
  484736	kk-d(10)-eeee	2-10-4	89	23
  484742	kk-d(10)-eeee	2-10-4	93	28
  484746	kk-d(10)-eeee	2-10-4	88	30
  484771	kk-d(10)-eeee	2-10-4	89	89
  e = 2′-MOE,
  k = cEt,
  d = 2′-deoxyribonucleoside

Example 4: Antisense Inhibition of Human Target-X with 5-10-5 2′-MOE Gapmers
• Additional antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. Also tested were ISIS 403094, ISIS 407641, ISIS 407643, ISIS 407662, ISIS 407900, ISIS 407910, ISIS 407935, ISIS 407936, ISIS 407939, ISIS 416446, ISIS 416449, ISIS 416455, ISIS 416472, ISIS 416477, ISIS 416507, ISIS 416508, ISIS 422086, ISIS 422087, ISIS 422140, and ISIS 422142, 5-10-5 2′-MOE gapmers targeting human Target-X, which were described in an earlier publication (WO 2009/061851), incorporated herein by reference.
• The newly designed modified antisense oligonucleotides are 20 nucleotides in length and their motifs are described in Tables 14 and 15. The chemistry column of Tables 14 and 15 present the sugar motif of each oligonucleotide, wherein “e” indicates a 2′-O-methythoxylethyl (2′-MOE) nucleoside and “d” indicates a 2′-deoxyribonucleoside. The internucleoside linkages throughout each gapmer are hosphorothioate (P═S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines.
• Each gapmer listed in Table 14 is targeted to the human Target-X genomic sequence.
• Activity of the newly designed gapmers was compared to ISIS 403094, ISIS 407641, ISIS 407643, ISIS 407662, ISIS 407900, ISIS 407910, ISIS 407935, ISIS 407936, ISIS 407939, ISIS 416446, ISIS 416449, ISIS 416455, ISIS 416472, ISIS 416477, ISIS 416507, ISIS 416508, ISIS 422086, ISIS 422087, ISIS 422140, and ISIS 422142. Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS2927 (described hereinabove in Example 1) was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of Target-X, relative to untreated control cells. A total of 916 oligonucleotides were tested. Only those oligonucleotides which were selected for further studies are shown in Tables 14 and 15.

• TABLE 14
  Inhibition of human Target-X mRNA levels by chimeric
  antisense oligonucleotides targeted to Target-X

  ISIS No	Chemistry	% inhibition	SEQ CODE

  490275	e5-d(10)-e5	35	90
  490277	e5-d(10)-e5	73	91
  490278	e5-d(10)-e5	78	92
  490279	e5-d(10)-e5	66	93
  490323	e5-d(10)-e5	65	94
  490368	e5-d(10)-e5	78	95
  490396	e5-d(10)-e5	76	96
  416507	e5-d(10)-e5	73	97
  422140	e5-d(10)-e5	59	98
  422142	e5-d(10)-e5	73	99
  416508	e5-d(10)-e5	75	100
  490424	e5-d(10)-e5	57	101
  490803	e5-d(10)-e5	70	102
  416446	e5-d(10)-e5	73	103
  416449	e5-d(10)-e5	33	104
  407900	e5-d(10)-e5	66	105
  490103	e5-d(10)-e5	87	106
  416455	e5-d(10)-e5	42	107
  407910	e5-d(10)-e5	25	108
  490149	e5-d(10)-e5	82	109
  403094	e5-d(10)-e5	60	110
  416472	e5-d(10)-e5	78	111
  407641	e5-d(10)-e5	64	112
  416477	e5-d(10)-e5	25	113
  407643	e5-d(10)-e5	78	114
  490196	e5-d(10)-e5	81	115
  490197	e5-d(10)-e5	85	116
  490208	e5-d(10)-e5	89	117
  490209	e5-d(10)-e5	81	118
  422086	e5-d(10)-e5	90	119
  407935	e5-d(10)-e5	91	120
  422087	e5-d(10)-e5	89	121
  407936	e5-d(10)-e5	80	122
  407939	e5-d(10)-e5	67	72
  e = 2′-MOE,
  d = 2′-deoxynucleoside

• TABLE 15
  Inhibition of human Target-X mRNA levels by chimeric
  antisense oligonucleotides targeted to Target-X

  ISIS No	Motif	% inhibition	SEQ CODE

  407662	e5-d(10)-e5	76	123
  416446	e5-d(10)-e5	73	103
  e = 2′-MOE,
  d = 2′-deoxynucleoside

Example 5: Modified Chimeric Antisense Oligonucleotides Comprising Constrained Ethyl (cEt) Modifications at 5′ and 3′ Wing Regions Targeting Human Target-X
• Additional antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. ISIS 407939, which was described in an earlier publication (WO 2009/061851) were also tested. ISIS 457851, ISIS 472925, ISIS 472926, ISIS 472935, ISIS 472942, ISIS 472958, ISIS 472959, ISIS 472970, ISIS 472976, ISIS 472983, ISIS 472984, ISIS 472988, ISIS 472991, ISIS 472994, ISIS 472995, ISIS 472996, ISIS 472998, and ISIS 473020, described in the Examples above were also included in the screen.
• The newly designed chimeric antisense oligonucleotides in Table 16 were designed as 2-10-2 cEt gapmers. The newly designed gapmers are 14 nucleosides in length, wherein the central gap segment comprises often 2′-deoxyribonucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment comprises constrained ethyl (cEt) modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines.
• Each gapmer listed in Table 16 is targeted to the human Target-X genomic sequence.
• Activity of the newly designed oligonucleotides was compared to ISIS 407939. Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS2927 (described hereinabove in Example 1) was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of Target-X, relative to untreated control cells. A total of 614 oligonucleotides were tested. Only those oligonucleotides which were selected for further studies are shown in Table 16. Many of the newly designed antisense oligonucleotides provided in Table 16 achieved greater than 72% inhibition and, therefore, are more potent than ISIS 407939.

• TABLE 16
  Inhibition of human Target-X mRNA levels by chimeric
  antisense oligonucleotides targeted to Target-X

  ISIS No	% inhibition	Motif	Wing Chemistry	SEQ CODE

  407939	72	5-10-5	cEt	72
  473020	90	2-10-2	cEt	25
  492465	83	2-10-2	cEt	124
  492467	74	2-10-2	cEt	125
  492492	84	2-10-2	cEt	126
  492494	91	2-10-2	cEt	127
  492503	89	2-10-2	cEt	128
  492530	91	2-10-2	cEt	129
  492534	91	2-10-2	cEt	130
  492536	90	2-10-2	cEt	131
  492541	84	2-10-2	cEt	132
  492545	89	2-10-2	cEt	133
  492566	90	2-10-2	cEt	134
  492571	82	2-10-2	cEt	135
  492572	89	2-10-2	cEt	136
  492573	90	2-10-2	cEt	137
  492574	92	2-10-2	cEt	138
  492575	88	2-10-2	cEt	139
  492593	83	2-10-2	cEt	140
  492617	91	2-10-2	cEt	141
  492618	92	2-10-2	cEt	142
  492619	90	2-10-2	cEt	143
  492621	75	2-10-2	cEt	144
  492104	89	2-10-2	cEt	145
  492105	86	2-10-2	cEt	146
  492189	88	2-10-2	cEt	147
  492194	92	2-10-2	cEt	148
  492195	90	2-10-2	cEt	149
  472925	87	2-10-2	cEt	32
  492196	91	2-10-2	cEt	150
  472926	88	2-10-2	cEt	33
  492205	92	2-10-2	cEt	151
  492215	77	2-10-2	cEt	152
  492221	79	2-10-2	cEt	153
  472935	82	2-10-2	cEt	36
  492234	86	2-10-2	cEt	154
  472942	85	2-10-2	cEt	43
  492276	75	2-10-2	cEt	155
  492277	75	2-10-2	cEt	156
  492306	85	2-10-2	cEt	157
  492317	93	2-10-2	cEt	158
  472958	92	2-10-2	cEt	46
  472959	88	2-10-2	cEt	47
  492329	88	2-10-2	cEt	159
  492331	95	2-10-2	cEt	160
  492333	85	2-10-2	cEt	161
  492334	88	2-10-2	cEt	162
  457851	89	2-10-2	cEt	51
  472970	92	2-10-2	cEt	52
  492365	69	2-10-2	cEt	163
  472976	94	2-10-2	cEt	54
  472983	76	2-10-2	cEt	55
  472984	72	2-10-2	cEt	56
  492377	70	2-10-2	cEt	164
  492380	80	2-10-2	cEt	165
  492384	61	2-10-2	cEt	166
  472988	59	2-10-2	cEt	60
  492388	70	2-10-2	cEt	167
  492389	70	2-10-2	cEt	168
  492390	89	2-10-2	cEt	169
  492391	80	2-10-2	cEt	170
  472991	84	2-10-2	cEt	62
  492398	88	2-10-2	cEt	171
  492399	94	2-10-2	cEt	172
  492401	91	2-10-2	cEt	173
  492403	78	2-10-2	cEt	174
  472994	95	2-10-2	cEt	66
  472995	91	2-10-2	cEt	68
  492404	84	2-10-2	cEt	175
  492405	87	2-10-2	cEt	176
  472996	85	2-10-2	cEt	70
  492406	43	2-10-2	cEt	177
  472998	92	2-10-2	cEt	74
  492440	89	2-10-2	cEt	178

Example 6: Modified Chimeric Antisense Oligonucleotides Comprising Constrained Ethyl (cEt) Modifications at 5′ and 3′ Wing Regions Targeting Human Target-X
• Additional antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. Also tested was ISIS 407939, a 5-10-5 MOE gapmer targeting human Target-X, which was described in an earlier publication (WO 2009/061851). ISIS 472998 and ISIS 473046, described in the Examples above were also included in the screen.
• The newly designed chimeric antisense oligonucleotides in Table 17 were designed as 2-10-2 cEt gapmers. The newly designed gapmers are 14 nucleosides in length, wherein the central gap segment comprises often 2′-deoxyribonucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment comprise constrained ethyl (cEt) modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines.
• Each gapmer listed in Table 17 is targeted to the human Target-X genomic sequence.
• Activity of the newly designed gapmers was compared to ISIS 407939. Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS2927 (described hereinabove in Example 1) was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of Target-X, relative to untreated control cells. A total of 757 oligonucleotides were tested. Only those oligonucleotides which were selected for further studies are shown in Table 17. Each of the newly designed antisense oligonucleotides provided in Table 17 achieved greater than 67% inhibition and, therefore, are more potent than 407939.

• TABLE 17
  Inhibition of human Target-X mRNA levels by chimeric
  antisense oligonucleotides targeted to Target-X

  ISIS No	% inhibition	Motif	Wing chemistry	SEQ CODE

  407939	67	5-10-5	cEt	72
  492651	77	2-10-2	cEt	179
  492652	84	2-10-2	cEt	180
  492658	87	2-10-2	cEt	181
  492725	74	2-10-2	cEt	182
  492730	78	2-10-2	cEt	183
  492731	72	2-10-2	cEt	184
  492784	72	2-10-2	cEt	185
  492816	70	2-10-2	cEt	186
  492818	73	2-10-2	cEt	187
  492877	83	2-10-2	cEt	188
  492878	79	2-10-2	cEt	189
  492913	73	2-10-2	cEt	190
  492914	82	2-10-2	cEt	191
  492928	76	5-10-5	cEt	192
  492938	80	2-10-2	cEt	193
  492991	91	2-10-2	cEt	194
  492992	73	2-10-2	cEt	195
  493087	81	2-10-2	cEt	196
  493114	80	2-10-2	cEt	197
  493178	86	2-10-2	cEt	198
  493179	69	2-10-2	cEt	199
  493182	79	2-10-2	cEt	200
  493195	71	2-10-2	cEt	201
  473046	79	2-10-2	cEt	35
  493201	86	2-10-2	cEt	202
  493202	76	2-10-2	cEt	203
  493255	80	2-10-2	cEt	204
  493291	84	2-10-2	cEt	205
  493292	90	2-10-2	cEt	206
  493296	82	2-10-2	cEt	207
  493298	77	2-10-2	cEt	208
  493299	76	5-10-5	cEt	209
  493304	77	2-10-2	cEt	210
  493312	75	2-10-2	cEt	211
  493333	76	2-10-2	cEt	212
  472998	85	2-10-2	cEt	74

Example 7: Dose-Dependent Antisense Inhibition of Human Target-X in Hep3B Cells
• Antisense oligonucleotides from the studies above, exhibiting in vitro inhibition of Target-X mRNA, were selected and tested at various doses in Hep3B cells. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.67 μM, 2.00 μM, 1.11 μM, and 6.00 μM concentrations of antisense oligonucleotide, as specified in Table 18. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human Target-X primer probe set RTS2927 was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells.
• The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented in Table 18. As illustrated in Table 18, Target-X mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells. The data also confirms that several of the newly designed gapmers are more potent than ISIS 407939 of the previous publication.

• TABLE 18
  Dose-dependent antisense inhibition of human
  Target-X in Hep3B cells using electroporation

  	666.6667	2000.0	6000.0	IC50
  ISIS No	nM	nM	nM	(μM)

  407939	47	68	85	0.7
  457851	60	80	93	<0.6
  472916	53	80	87	<0.6
  472925	62	86	95	<0.6
  472926	66	77	85	<0.6
  472935	54	84	94	<0.6
  472958	66	82	88	<0.6
  472959	64	81	93	<0.6
  472970	72	87	86	<0.6
  472976	78	92	97	<0.6
  472994	79	92	96	<0.6
  472995	61	82	93	<0.6
  472996	73	91	95	<0.6
  472998	63	90	95	<0.6
  473019	55	80	86	<0.6
  473020	61	76	85	<0.6
  473046	61	80	94	<0.6
  473055	55	84	94	<0.6
  492104	53	76	88	<0.6
  492105	62	80	90	<0.6
  492189	57	80	92	<0.6
  492194	57	83	91	<0.6
  492195	58	81	95	<0.6
  492196	62	86	95	<0.6
  492205	62	87	95	<0.6
  492215	60	78	89	<0.6
  492221	63	76	92	<0.6
  492234	51	74	91	0.5
  492276	50	56	95	0.8
  492277	58	73	81	<0.6
  492306	61	75	84	<0.6
  492317	59	80	93	<0.6
  492329	59	70	89	<0.6
  492331	69	87	95	<0.6
  492333	47	70	85	0.7
  492334	57	77	90	<0.6
  492390	72	88	95	<0.6
  492399	68	91	96	<0.6
  492401	68	89	95	<0.6
  492404	65	87	94	<0.6
  492405	44	81	90	0.7
  492406	65	82	92	<0.6
  492440	50	70	89	0.6
  492465	16	80	79	1.4
  492467	58	77	92	<0.6
  492492	45	80	94	0.7
  492494	63	82	93	<0.6
  492503	55	81	93	<0.6
  492530	70	86	90	<0.6
  492534	67	85	91	<0.6
  492536	54	81	89	<0.6
  492541	54	71	85	<0.6
  492545	59	78	89	<0.6
  492566	59	84	85	<0.6
  492571	52	81	89	<0.6
  492572	67	83	90	<0.6
  492573	69	83	92	<0.6
  492574	65	82	91	<0.6
  492575	72	83	91	<0.6
  492593	61	78	90	<0.6
  492617	62	80	93	<0.6
  492618	47	79	94	0.6
  492619	54	82	95	<0.6
  492621	44	85	92	0.6
  492651	53	66	91	0.6
  492652	61	78	88	<0.6
  492658	59	79	88	<0.6
  492725	43	84	89	0.6
  492730	51	87	93	0.4
  492731	46	82	90	0.6
  492784	56	88	96	<0.6
  492816	68	89	97	<0.6
  492818	64	84	96	<0.6
  492877	67	91	93	<0.6
  492878	80	89	93	<0.6
  492913	53	87	92	<0.6
  492914	75	89	96	<0.6
  492928	60	83	94	<0.6
  492938	70	90	92	<0.6
  492991	67	93	99	<0.6
  492992	0	82	95	2.1
  493087	54	81	90	<0.6
  493114	50	73	90	0.6
  493178	71	88	96	<0.6
  493179	47	82	95	0.6
  493182	79	87	91	<0.6
  493195	55	78	90	<0.6
  493201	87	93	96	<0.6
  493202	68	89	94	<0.6
  493255	57	79	93	<0.6
  493291	57	87	93	<0.6
  493292	70	89	93	<0.6
  493296	35	84	91	0.9
  493298	57	84	92	<0.6
  493299	65	84	93	<0.6
  493304	68	86	94	<0.6
  493312	53	82	91	<0.6
  493333	66	84	87	<0.6

Example 8: Dose-Dependent Antisense Inhibition of Human Target-X in Hep3B Cells
• Additional antisense oligonucleotides from the studies described above, exhibiting in vitro inhibition of Target-X mRNA, were selected and tested at various doses in Hep3B cells. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.67 μM, 2.00 μM, 1.11 μM, and 6.00 μM concentrations of antisense oligonucleotide, as specified in Table 19. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human Target-X primer probe set RTS2927 was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells. As illustrated in Table 19, Target-X mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells. The data also confirms that several of the newly designed gapmers are more potent than ISIS 407939.

• TABLE 19
  Dose-dependent antisense inhibition of human
  Target-X in Hep3B cells using electroporation

  	0.67	2.00	6.00	IC50
  ISIS No	μM	μM	μM	(μM)

  407939	52	71	86	0.6
  472983	49	83	97	0.5
  472984	51	82	95	0.5
  472991	49	82	95	0.5
  472998	59	88	96	<0.6
  492365	74	91	96	<0.6
  492377	56	76	91	<0.6
  492380	63	79	95	<0.6
  492384	67	84	94	<0.6
  492388	69	87	97	<0.6
  492389	62	90	96	<0.6
  492391	56	84	94	<0.6
  492398	63	80	95	<0.6
  492403	58	81	91	<0.6

Example 9: Modified Chimeric Antisense Oligonucleotides Comprising 2′-Methoxyethyl (2′-MOE) Modifications at 5′ and 3′ Wing Regions Targeting Human Target-X
• Additional antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. Also tested were ISIS 403052, ISIS 407939, ISIS 416446, ISIS 416472, ISIS 416507, ISIS 416508, ISIS 422087, ISIS 422096, ISIS 422130, and ISIS 422142 which were described in an earlier publication (WO 2009/061851), incorporated herein by reference. ISIS 490149, ISIS 490197, ISIS 490209, ISIS 490275, ISIS 490277, and ISIS 490424, described in the Examples above, were also included in the screen.
• The newly designed chimeric antisense oligonucleotides in Table 20 were designed as 3-10-4 2′-MOE gapmers. These gapmers are 17 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxyribonucleosides and is flanked by wing segments on the 5′ direction with three nucleosides and the 3′ direction with four nucleosides. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has 2′-MOE modifications. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines.
• Each gapmer listed in Table 20 is targeted to the human Target-X genomic sequence.
• Activity of the newly designed oligonucleotides was compared to ISIS 403052, ISIS 407939, ISIS 416446, ISIS 416472, ISIS 416507, ISIS 416508, ISIS 422087, ISIS 422096, ISIS 422130, and ISIS 422142. Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS2927 (described hereinabove in Example 1) was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells. A total of 272 oligonucleotides were tested. Only those oligonucleotides which were selected for further studies are shown in Table 20. Several of the newly designed antisense oligonucleotides provided in Table 19 are more potent than antisense oligonucleotides from the previous publication.

• TABLE 20
  Inhibition of human Target-X mRNA levels by chimeric
  antisense oligonucleotides targeted to Target-X

  ISIS No	% inhibition	Motif	Wing Chemistry	SEQ CODE

  403052	51	5-10-5	2′-MOE	82
  407939	78	5-10-5	2′-MOE	72
  416446	70	5-10-5	2′-MOE	103
  416472	79	5-10-5	2′-MOE	111
  416507	84	5-10-5	2′-MOE	97
  416508	80	5-10-5	2′-MOE	100
  422087	89	5-10-5	2′-MOE	121
  422096	78	5-10-5	2′-MOE	219
  422130	81	5-10-5	2′-MOE	225
  422142	84	5-10-5	2′-MOE	99
  490275	77	5-10-5	2′-MOE	90
  513462	79	3-10-4	2′-MOE	213
  513463	81	3-10-4	2′-MOE	214
  490277	74	5-10-5	2′-MOE	91
  513487	83	3-10-4	2′-MOE	215
  513504	81	3-10-4	2′-MOE	216
  513507	86	3-10-4	2′-MOE	217
  513508	85	3-10-4	2′-MOE	218
  490424	69	5-10-5	2′-MOE	101
  491122	87	5-10-5	2′-MOE	220
  513642	79	3-10-4	2′-MOE	221
  490149	71	5-10-5	2′-MOE	109
  513419	90	3-10-4	2′-MOE	222
  513420	89	3-10-4	2′-MOE	223
  513421	88	3-10-4	2′-MOE	224
  490197	77	5-10-5	2′-MOE	116
  513446	89	3-10-4	2′-MOE	226
  513447	83	3-10-4	2′-MOE	227
  490209	79	5-10-5	2′-MOE	118
  513454	84	3-10-4	2′-MOE	228
  513455	92	3-10-4	2′-MOE	229
  513456	89	3-10-4	2′-MOE	230
  513457	83	3-10-4	2′-MOE	231

Example 10: Dose-Dependent Antisense Inhibition of Human Target-X in Hep3B Cells
• Antisense oligonucleotides from the studies above, exhibiting in vitro inhibition of Target-X mRNA, were selected and tested at various doses in Hep3B cells. ISIS 403052, ISIS 407643, ISIS 407935, ISIS 407936, ISIS 407939, ISIS 416446, ISIS 416459, ISIS 416472, ISIS 416507, ISIS 416508, ISIS 416549, ISIS 422086, ISIS 422087, ISIS 422130, ISIS and 422142, 5-10-5 MOE gapmers targeting human Target-X, which were described in an earlier publication (WO 2009/061851).
• Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.625 μM, 1.25 μM, 2.50 μM, 5.00 μM and 10.00 μM concentrations of antisense oligonucleotide, as specified in Table 21. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human Target-X primer probe set RTS2927 was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells.
• The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented in Table 21. As illustrated in Table 21, Target-X mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells. The data also confirms that the newly designed gapmers are potent than gapmers from the previous publication.

• TABLE 21
  Dose-dependent antisense inhibition of human
  Target-X in Hep3B cells using electroporation

  	ISIS	0.625	1.25	2.50	5.00	10.00	IC50
  	No	μM	μM	μM	μM	μM	(μM)

  	403052	21	35	63	82	89	1.9
  	407643	29	46	67	83	90	1.4
  	407935	52	68	80	89	91	<0.6
  	407936	31	51	62	78	84	1.4
  	407939	30	61	74	83	88	1.0
  	416446	37	53	64	76	83	1.2
  	416459	51	76	83	90	92	<0.6
  	416472	37	52	66	78	85	1.2
  	416507	45	68	82	87	90	0.7
  	416508	33	56	74	84	89	1.1
  	416549	57	71	78	82	85	<0.6
  	422086	46	67	77	89	92	0.7
  	422087	50	69	74	86	91	0.6
  	422130	32	65	78	92	93	0.9
  	422142	59	73	84	86	88	<0.6
  	490103	52	57	66	83	88	0.9
  	490149	34	58	71	85	91	1.0
  	490196	26	59	66	79	84	1.3
  	490197	39	63	74	81	90	0.8
  	490208	44	70	76	83	88	0.6
  	490275	36	58	76	85	89	1.0
  	490277	37	63	73	87	87	0.8
  	490279	40	54	72	83	89	1.0
  	490323	49	68	79	86	90	<0.6
  	490368	39	62	76	86	91	0.8
  	490396	36	53	69	80	87	1.1
  	490424	45	65	69	76	82	0.6
  	490803	57	74	85	89	92	<0.6
  	513419	60	71	85	95	96	<0.6
  	513420	37	69	79	94	96	0.7
  	513421	46	64	84	95	97	0.6
  	513446	47	81	88	95	96	<0.6
  	513447	56	74	81	92	96	<0.6
  	513454	50	77	82	93	95	<0.6
  	513455	74	82	91	96	96	<0.6
  	513456	66	80	88	94	95	<0.6
  	513457	54	67	80	87	89	<0.6
  	513462	49	72	84	87	89	<0.6
  	513463	36	62	76	85	89	0.9
  	513487	42	56	73	87	93	0.9
  	513504	47	65	81	90	91	0.6
  	513505	39	50	78	85	92	1.0
  	513507	52	73	83	89	93	<0.6
  	513508	56	78	85	91	94	<0.6

Example 11: Dose-Dependent Antisense Inhibition of Human Target-X in Hep3B Cells
• Additional antisense oligonucleotides from the studies above, exhibiting in vitro inhibition of Target-X mRNA, were tested at various doses in Hep3B cells. ISIS 407935, ISIS 407939, ISIS 416446, ISIS 416472, ISIS 416507, ISIS 416549, ISIS 422086, ISIS 422087, ISIS 422096, and ISIS 422142 5-10-5 MOE gapmers targeting human Target-X, which were described in an earlier publication (WO 2009/061851).
• Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.3125 μM, 0.625 μM, 1.25 μM, 2.50 μM, 5.00 μM and 10.00 μM concentrations of antisense oligonucleotide, as specified in Table 22. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human Target-X primer probe set RTS2927 was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by^RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells. As illustrated in Table 22, Target-X mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells. The data also confirms that the newly designed gapmers are more potent than gapmers from the previous publication.

• TABLE 22
  Dose-dependent antisense inhibition of human
  Target-X in Hep3B cells using electroporation

  ISIS	0.3125	0.625	1.250	2.500	5.000	10.000	IC50
  No	μM	μM	μM	μM	μM	μM	(μM)

  407935	30	49	75	86	91	94	0.6
  407939	30	48	61	78	85	90	0.8
  416446	27	52	63	75	85	90	0.7
  416472	38	51	72	83	88	94	0.5
  416507	58	81	76	84	89	92	<0.3
  416549	52	67	75	81	88	89	0.3
  422086	48	49	68	78	86	91	0.5
  422087	30	56	66	83	72	92	0.6
  422096	47	63	70	77	83	85	<0.3
  422142	69	85	87	85	89	91	<0.3
  490103	52	57	68	78	87	93	0.4
  490149	33	64	62	77	86	93	0.5
  490197	38	46	60	75	87	93	0.7
  490208	46	62	73	83	88	91	0.4
  490209	40	54	72	79	85	94	0.5
  490275	52	61	67	78	85	91	0.3
  490277	33	59	77	79	91	94	0.5
  490323	43	61	72	69	84	87	0.4
  490368	50	64	78	83	90	92	<0.3
  490396	46	64	68	84	84	90	0.3
  490424	24	47	58	72	76	82	1.0
  490803	45	60	70	84	88	89	0.3
  513419	32	53	76	88	93	95	0.5
  513420	35	59	72	82	94	97	0.5
  513421	46	67	78	86	94	96	<0.3
  513446	26	61	77	89	91	97	0.5
  513447	22	48	60	82	91	95	0.8
  513454	25	59	76	86	94	96	0.5
  513455	60	73	85	89	95	96	<0.3
  513456	49	60	81	88	94	95	<0.3
  513457	43	50	72	77	87	92	0.5
  513462	25	48	58	76	83	88	0.8
  513463	22	45	66	73	85	88	0.9
  513487	41	56	65	79	86	90	0.4
  513504	19	48	63	76	87	92	0.9
  513505	11	21	54	73	85	90	1.4
  513507	47	55	72	82	90	91	0.3
  513508	31	59	74	85	92	93	0.5
  513642	43	55	67	80	88	92	0.4

Example 12: Tolerability of 2′-MOE Gapmers Targeting Human Target-X in BALB/c Mice
• BALB/c mice are a multipurpose mice model, frequently utilized for safety and efficacy testing. The mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.
Treatment
• Groups of male BALB/c mice were injected subcutaneously twice a week for 3 weeks with 50 mg/kg of ISIS 407935, ISIS 416472, ISIS 416549, ISIS 422086, ISIS 422087, ISIS 422096, ISIS 422142, ISIS 490103, ISIS 490149, ISIS 490196, ISIS 490208, ISIS 490209, ISIS 513419, ISIS 513420, ISIS 513421, ISIS 513454, ISIS 513455, ISIS 513456, ISIS 513457, ISIS 513462, ISIS 513463, ISIS 513487, ISIS 513504, ISIS 513508, and ISIS 513642. One group of male BALB/c mice was injected subcutaneously twice a week for 3 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.
Plasma Chemistry Markers
• To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, albumin, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.).
• ISIS oligonucleotides that did not cause any increase in the levels of transaminases, or which caused an increase within three times the upper limit of normal (ULN) were deemed very tolerable. ISIS oligonucleotides that caused an increase in the levels of transaminases between three times and seven times the ULN were deemed tolerable. Based on these criteria, ISIS 407935, ISIS 416472, ISIS 416549, ISIS 422087, ISIS 422096, ISIS 490103, ISIS 490196, ISIS 490208, ISIS 513454, ISIS 513455, ISIS 513456, ISIS 513457, ISIS 513487, ISIS 513504, and ISIS 513508 were considered very tolerable in terms of liver function. Based on these criteria, ISIS 422086, ISIS 490209, ISIS 513419, ISIS 513420, and ISIS 513463 were considered tolerable in terms of liver function.
Example 13: Dose-Dependent Antisense Inhibition of Human Target-X in Hep3B Cells
• Additional antisense oligonucleotides from the studies above, exhibiting in vitro inhibition of Target-X mRNA were selected and tested at various doses in Hep3B cells. Also tested was ISIS 407939, a 5-10-5 MOE gapmer, which was described in an earlier publication (WO 2009/061851).
• Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.074 μM, 0.222 μM, 0.667 μM, 2.000 μM, and 6.000 μM concentrations of antisense oligonucleotide, as specified in Table 23. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human Target-X primer probe set RTS2927 (described hereinabove in Example 1) was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by^RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells.
• The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented in Table 23. As illustrated in Table 23, Target-X mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells. Many of the newly designed antisense oligonucleotides provided in Table 23 achieved an IC₅₀of less than 0.9 μM and, therefore, are more potent than ISIS 407939.

• TABLE 23
  Dose-dependent antisense inhibition of human
  Target-X in Hep3B cells using electroporation

  	ISIS	0.074	0.222	0.667	2.000	6.000	IC50
  	No	μM	μM	μM	μM	μM	(μM)

  	407939	2	17	53	70	87	0.9
  	472970	17	47	75	92	95	0.3
  	472988	0	8	21	54	92	1.4
  	472996	18	59	74	93	95	0.2
  	473244	91	95	97	99	99	<0.07
  	473286	6	53	85	92	98	0.3
  	473359	2	3	20	47	67	2.6
  	473392	71	85	88	92	96	<0.07
  	473393	91	96	97	98	99	<0.07
  	473547	85	88	93	97	98	<0.07
  	473567	0	25	66	88	95	0.7
  	473589	8	47	79	94	99	0.3
  	482814	23	68	86	93	96	0.1
  	482815	6	48	65	90	96	0.4
  	482963	3	68	85	94	96	0.2
  	483241	14	33	44	76	93	0.6
  	483261	14	21	41	72	88	0.7
  	483290	0	1	41	69	92	1.0
  	483414	8	1	36	76	91	0.9
  	483415	0	40	52	84	94	0.6
  	484559	26	51	78	87	97	0.2
  	484713	6	5	53	64	88	0.9

Example 14: Modified Antisense Oligonucleotides Comprising 2′-O-Methoxyethyl (2′-MOE) and Constrained Ethyl (cEt) Modifications Targeting Human Target-X
• Additional antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. Also tested was ISIS 407939, a 5-10-5 MOE gapmer targeting human Target-X, which was described in an earlier publication (WO 2009/061851). ISIS 472998, ISIS 492878, and ISIS 493201 and 493182, 2-10-2 cEt gapmers, described in the Examples above were also included in the screen.
• The newly designed modified antisense oligonucleotides are 16 nucleotides in length and their motifs are described in Table 24. The chemistry column of Table 24 presents the sugar motif of each oligonucleotide, wherein “e” indicates a 2′-O-methythoxylethyl (2′-MOE) nucleoside, “k” indicates a constrained ethyl (cEt) nucleoside and “d” indicates a 2′-deoxyribonucleoside. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines.
• Each gapmer listed in Table 24 is targeted to the human Target-X genomic sequence.
• Activity of newly designed gapmers was compared to ISIS 407939. Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS2927 was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells and demonstrate that several of the newly designed gapmers are more potent than ISIS 407939. A total of 685 oligonucleotides were tested. Only those oligonucleotides which were selected for further studies are shown in Table 24.

• TABLE 24
  Inhibition of human Target-X mRNA levels by chimeric
  antisense oligonucleotides targeted to Target-X

  	ISIS No	% inhibition	Chemistry	SEQ CODE

  	407939	68	eeeee-d(10)-eeeee	72
  	492878	73	kk-d(10)-kk
  	493182	80	kk-d(10)-kk
  	493201	84	kk-d(10)-kk
  	472998	91	kk-d(10)-kk
  	515640	75	eee-d(10)-kkk	23
  	515637	77	eee-d(10)-kkk	232
  	515554	72	eee-d(10)-kkk	233
  	515406	80	kkk-d(10)-eee	234
  	515558	81	eee-d(10)-kkk	234
  	515407	88	kkk-d(10)-eee	235
  	515408	85	kkk-d(10)-eee	236
  	515422	86	kkk-d(10)-eee	237
  	515423	90	kkk-d(10)-eee	238
  	515575	84	eee-d(10)-kkk	238
  	515424	87	kkk-d(10)-eee	239
  	515432	78	kkk-d(10)-eee	240
  	515433	71	kkk-d(10)-eee	241
  	515434	76	kkk-d(10)-eee	242
  	515334	85	kkk-d(10)-eee	243
  	515649	61	eee-d(10)-kkk	88
  	515338	86	kkk-d(10)-eee	244
  	515438	76	kkk-d(10)-eee	245
  	515439	75	kkk-d(10)-eee	246
  	516003	87	eee-d(10)-kkk	247
  	515647	60	eee-d(10)-kkk	77
  	515639	78	eee-d(10)-kkk	34
  	493201	84	eee-d(10)-kkk	202
  	515648	36	kkk-d(10)-eee	248
  	515641	69	kk-d(10)-eeee	39
  	515650	76	kkk-d(10)-eee	44
  	515354	87	eee-d(10)-kkk	249
  	515926	87	eee-d(10)-kkk	250
  	515366	87	kk-d(10)-eeee	251
  	515642	58	kkk-d(10)-eee	252
  	515643	81	eee-d(10)-kkk	53
  	515944	84	kk-d(10)-eeee	253
  	515380	90	kkk-d(10)-eee	254
  	515532	83	kkk-d(10)-eee	254
  	515945	85	kk-d(10)-eeee	254
  	515381	82	kk-d(10)-eeee	255
  	515382	95	kkk-d(10)-eee	256
  	515948	94	eee-d(10)-kkk	256
  	515949	87	eee-d(10)-kkk	257
  	515384	89	kkk-d(10)-eee	258
  	515635	82	kk-d(10)-eeee	65
  	515638	90	kkk-d(10)-eee	67
  	515386	92	kk-d(10)-eeee	259
  	515951	84	eee-d(10)-kkk	259
  	515387	78	kkk-d(10)-eee	260
  	515952	89	kkk-d(10)-eee	260
  	515636	90	kkk-d(10)-eee	69
  	515388	84	eee-d(10)-kkk	261
  	e = 2′-MOE,
  	k = cEt,
  	d = 2′-deoxyribonucleoside

Example 15: Tolerability of Modified Oligonucleotides Comprising 2′-O-Methoxyethyl (2′-MOE) and Constrained Ethyl (cEt) Modifications Targeting Human Target-X in BALB/c Mice
• BALB/c mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.
• Additionally, the newly designed modified antisense oligonucleotides were also added to this screen. The newly designed chimeric antisense oligonucleotides are 16 nucleotides in length and their motifs are described in Table 25. The chemistry column of Table 25 presents the sugar motif of each oligonucleotide, wherein “e” indicates a 2′-O-methythoxylethyl (2′-MOE) nucleoside, “k” indicates a constrained ethyl (cEt) nucleoside and “d” indicates a 2′-deoxynucleoside. The internucleoside linkages throughout each gapmer are hosphorothioate (P═S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines.
• Each gapmer listed in Table 25 is targeted to either the human Target-X genomic sequence.

• TABLE 25
  Modified chimeric antisense oligonucleotides targeted to Target-X

  ISIS No	Chemistry	SEQ CODE

  516044	eee-d(10)-kkk	21
  516045	eee-d(10)-kkk	22
  516058	eee-d(10)-kkk	26
  516059	eee-d(10)-kkk	27
  516060	eee-d(10)-kkk	28
  516061	eee-d(10)-kkk	29
  516062	eee-d(10)-kkk	30
  516046	eee-d(10)-kkk	37
  516063	eee-d(10)-kkk	38
  516064	eee-d(10)-kkk	89
  516065	eee-d(10)-kkk	262
  516066	eee-d(10)-kkk	263
  516047	eee-d(10)-kkk	41
  516048	eee-d(10)-kkk	42
  516049	eee-d(10)-kkk	81
  516050	eee-d(10)-kkk	45
  516051	eee-d(10)-kkk	48
  516052	eee-d(10)-kkk	49
  515652	eee-d(10)-kkk	50
  508039	eee-d(10)-kkk	264
  516053	eee-d(10)-kkk	265
  515654	eee-d(10)-kkk	76
  515656	eee-d(10)-kkk	77
  516054	eee-d(10)-kkk	57
  516055	eee-d(10)-kkk	59
  515655	eee-d(10)-kkk	61
  516056	eee-d(10)-kkk	63
  516057	eee-d(10)-kkk	64
  515653	eee-d(10)-kkk	71
  515657	eee-d(10)-kkk	73
  e = 2′-MOE,
  k = cEt,
  d = 2′-deoxynucleoside

• Treatment
• Groups of 4-6-week old male BALB/c mice were injected subcutaneously twice a week for 3 weeks with 50 mg/kg/week of ISIS 457851, ISIS 515635, ISIS 515636, ISIS 515637, ISIS 515638, ISIS 515639, ISIS 515640, ISIS 515641, ISIS 515642, ISIS 515643, ISIS 515647, ISIS 515648, ISIS 515649, ISSI 515650, ISIS 515652, ISIS 515653, ISIS 515654, ISIS 515655, ISIS 515656, ISIS 515657, ISIS 516044, ISIS 516045, ISIS 516046, ISIS 516047, ISIS 516048, ISIS 516049, ISIS 516050, ISIS 516051, ISIS 516052, ISIS 516053, ISIS 516054, ISIS 516055, ISIS 516056, ISIS 516057, ISIS 516058, ISIS 516059, ISIS 516060, ISIS 516061, ISIS 516062, ISIS 516063, ISIS 516064, ISIS 516065, and ISIS 516066. One group of 4-6-week old male BALB/c mice was injected subcutaneously twice a week for 3 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.
Plasma Chemistry Markers
• To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, albumin, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.).
• ISIS oligonucleotides that did not cause any increase in the levels of transaminases, or which caused an increase within three times the upper limit of normal (ULN) were deemed very tolerable. ISIS oligonucleotides that caused an increase in the levels of transaminases between three times and seven times the ULN were deemed tolerable. Based on these criteria, ISIS 515636, ISIS 515639, ISIS 515641, ISIS 515642, ISIS 515648, ISIS 515650, ISIS 515652, ISIS 515653, ISIS 515655, ISIS 515657, ISIS 516044, ISIS 516045, ISIS 516047, ISIS 516048, ISIS 516051, ISIS 516052, ISIS 516053, ISIS 516055, ISIS 516056, ISIS 516058, ISIS 516059, ISIS 516060, ISIS 516061, ISIS 516062, ISIS 516063, ISIS 516064, ISIS 516065, and ISIS 516066 were considered very tolerable in terms of liver function. Based on these criteria, ISIS 457851, ISIS 515635, ISIS 515637, ISIS 515638, ISIS 515643, ISIS 515647, ISIS 515649, ISIS 515650, ISIS 515652, ISIS 515654, ISIS 515656, ISIS 516056, and ISIS 516057 were considered tolerable in terms of liver function.
Example 16: Efficacy of Modified Oligonucleotides Comprising 2′-O-Methoxyethyl (2′-MOE) and Constrained Ethyl (cEt) Modifications Targeting Human Target-X in Transgenic Mice
• Transgenic mice were developed at Taconic farms harboring a Target-X genomic DNA fragment. The mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for efficacy.
• Treatment
• Groups of 3-4 male and female transgenic mice were injected subcutaneously twice a week for 3 weeks with 20 mg/kg/week of ISIS 457851, ISIS 515636, ISIS 515639, ISIS 515653, ISIS 516053, ISIS 516065, and ISIS 516066. One group of mice was injected subcutaneously twice a week for 3 weeks with control oligonucleotide, ISIS 141923 (CCTTCCCTGAAGGTTCCTCC, 5-10-5 MOE gapmer with no known murine target, SEQ ID NO: 22). One group of mice was injected subcutaneously twice a week for 3 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.
• RNA Analysis
• RNA was extracted from plasma for real-time PCR analysis of Target-X, using primer probe set RTS2927. The mRNA levels were normalized using RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to control. As shown in Table 26, each of the antisense oligonucleotides achieved reduction of human Target-X mRNA expression over the PBS control. Treatment with the control oligonucleotide did not achieve reduction in Target-X levels, as expected.

• TABLE 26
  Percent inhibition of Target-X mRNA in transgenic mice

  	ISIS No	% inhibition

  	141923	0
  	457851	76
  	515636	66
  	515639	49
  	515653	78
  	516053	72
  	516065	59
  	516066	39

• Protein Analysis
• Plasma protein levels of Target-X were estimated using a Target-X ELISA kit (purchased from Hyphen Bio-Med). Results are presented as percent inhibition of Target-X, relative to control. As shown in Table 27, several antisense oligonucleotides achieved reduction of human Target-X protein expression over the PBS control. ‘n.d.’ indicates that the value for that particular oligonucleotide was not measured.

• TABLE 27
  Percent inhibition of Target-X protein levels in transgenic mice

  	ISIS No	% inhibition

  	141923	0
  	457851	64
  	515636	68
  	515639	46
  	515653	0
  	516053	19
  	516065	0
  	516066	7

Example 17: Efficacy of Modified Oligonucleotides Comprising 2′-O-Methoxyethyl (2′-MOE) and Constrained Ethyl (cEt) Modifications Targeting Human Target-X in Transgenic Mice
• Transgenic mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for efficacy.
• Treatment
• Groups of 2-4 male and female transgenic mice were injected subcutaneously twice a week for 3 weeks with 10 mg/kg/week of ISIS 407935, ISIS 416472, ISIS 416549, ISIS 422087, ISIS 422096, ISIS 473137, ISIS 473244, ISIS 473326, ISIS 473327, ISIS 473359, ISIS 473392, ISIS 473393, ISIS 473547, ISIS 473567, ISIS 473589, ISIS 473630, ISIS 484559, ISIS 484713, ISIS 490103, ISIS 490196, ISIS 490208, ISIS 513419, ISIS 513454, ISIS 513455, ISIS 513456, ISIS 513457, ISIS 513487, ISIS 513508, ISIS 515640, ISIS 515641, ISIS 515642, ISIS 515648, ISIS 515655, ISIS 515657, ISIS 516045, ISIS 516046, ISIS 516047, ISIS 516048, ISIS 516051, ISIS 516052, ISIS 516055, ISIS 516056, ISIS 516059, ISIS 516061, ISIS 516062, and ISIS 516063. One group of mice was injected subcutaneously twice a week for 3 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.
• Protein Analysis
• Plasma protein levels of Target-X were estimated using a Target-X ELISA kit (purchased from Hyphen Bio-Med). Results are presented as percent inhibition of Target-X, relative to control. As shown in Table 28, several antisense oligonucleotides achieved reduction of human Target-X over the PBS control.

• TABLE 28
  Percent inhibition of Target-X plasma
  protein levels in transgenic mice

  	ISIS No	% inhibition

  	407935	80
  	416472	49
  	416549	29
  	422087	12
  	422096	21
  	473137	57
  	473244	67
  	473326	42
  	473327	100
  	473359	0
  	473392	22
  	473393	32
  	473547	73
  	473567	77
  	473589	96
  	473630	75
  	484559	75
  	484713	56
  	490103	0
  	490196	74
  	490208	90
  	513419	90
  	513454	83
  	513455	91
  	513456	81
  	513457	12
  	513487	74
  	513508	77
  	515640	83
  	515641	87
  	515642	23
  	515648	32
  	515655	79
  	515657	81
  	516045	52
  	516046	79
  	516047	65
  	516048	79
  	516051	84
  	516052	72
  	516055	70
  	516056	0
  	516059	39
  	516061	64
  	516062	96
  	516063	24

Example 18: Dose-Dependent Antisense Inhibition of Human Target-X in Hep3B Cells
• Antisense oligonucleotides exhibiting in vitro inhibition of Target-X mRNA were selected and tested at various doses in Hep3B cells. Also tested was ISIS 407939, a 5-10-5 MOE gapmer targeting human Target-X, which was described in an earlier publication (WO 2009/061851).
• Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.074 μM, 0.222 μM, 0.667 μM, 2.000 μM, and 6.000 μM concentrations of antisense oligonucleotide, as specified in Table 29. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human Target-X primer probe set RTS2927 (described hereinabove in Example 1) was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells.
• The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented in Table 29. As illustrated in Table 29, Target-X mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells. Many of the newly designed antisense oligonucleotides provided in Table 29 achieved an IC₅₀of less than 2.0 μM and, therefore, are more potent than ISIS 407939.

• TABLE 29
  Dose-dependent antisense inhibition of human
  Target-X in Hep3B cells using electroporation

  		0.074	0.222	0.667	2.000	6.000	IC50
  	ISIS No	μM	μM	μM	μM	μM	(μM)

  	407939	0	9	21	58	76	2.0
  	515636	14	32	50	62	81	0.7
  	515639	10	24	41	61	67	1.3
  	515640	4	16	35	52	63	2.0
  	515641	0	21	27	55	66	1.9
  	515642	3	13	36	44	66	2.2
  	515648	8	10	10	5	16	>6.0
  	515653	9	35	26	55	71	1.5
  	515655	0	0	6	13	42	>6.0
  	515657	0	13	17	38	51	6.0
  	516045	0	6	15	19	40	>6.0
  	516046	0	7	32	48	69	2.1
  	516047	12	27	41	50	63	1.8
  	516051	9	8	34	52	66	2.0
  	516052	17	42	27	53	75	1.2
  	516053	9	7	28	63	77	1.3
  	516055	0	3	27	54	75	2.0
  	516056	0	4	14	52	66	2.6
  	516057	0	34	33	51	70	1.6
  	516058	13	12	25	47	74	2.0
  	516059	4	15	36	47	68	1.9
  	516060	0	1	39	29	63	3.2
  	516061	0	0	24	0	3	<6.0
  	516062	0	20	43	65	78	1.0
  	516063	0	8	10	37	61	3.8
  	516064	0	3	13	45	69	2.7
  	516065	0	14	38	63	76	1.3
  	516066	0	3	30	55	75	1.7

Example 19: Modified Oligonucleotides Comprising 2′-O-Methoxyethyl (2′-MOE) and Constrained Ethyl (cEt) Modifications Targeting Human Target-X
• Additional antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. ISIS 472998, ISIS 515652, ISIS 515653, ISIS 515654, ISIS 515655, ISIS 515656, and ISIS 515657, described in the Examples above were also included in the screen.
• The newly designed chimeric antisense oligonucleotides are 16 or 17 nucleotides in length and their motifs are described in Table 30. The chemistry column of Table 30 presents the sugar motif of each oligonucleotide, wherein “e” indicates a 2′-O-methythoxylethyl (2′-MOE) nucleoside, “k” indicates a constrained ethyl (cEt) nucleoside and “d” indicates a 2′-deoxynucleoside. The internucleoside linkages throughout each gapmer are hosphorothioate (P═S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines.
• Each gapmer listed in Table 30 is targeted to the human Target-X genomic sequence.
• Activity of newly designed gapmers was compared to ISIS 407939. Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS2927 (described hereinabove in Example 1) was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells.

• TABLE 30
  Inhibition of human Target-X mRNA levels by chimeric
  antisense oligonucleotides targeted to Target-X

  ISIS No	% inhibition	Chemistry	SEQ CODE

  472998	85	kk-d(10)-kk	74
  515652	63	eee-d(10)-kkk	50
  515653	67	eee-d(10)-kkk	71
  515654	78	eee-d(10)-kkk	86
  515655	41	eee-d(10)-kkk	61
  515656	74	eee-d(10)-kkk	87
  515657	49	eee-d(10)-kkk	73
  529265	52	eek-d(10)-keke	267
  529332	82	eek-d(10)-keke	268
  529334	78	eek-d(10)-keke	269
  529186	85	eek-d(10)-keke	213
  529223	81	eek-d(10)-kkke	213
  529129	75	eee-d(10)-kkk	270
  529149	82	kkk-d(10)-eee	270
  529177	77	eek-d(10)-keke	214
  529214	78	eek-d(10)-kkke	214
  529178	79	eek-d(10)-keke	271
  529215	82	eek-d(10)-kkke	271
  529179	71	eek-d(10)-keke	272
  529216	77	eek-d(10)-kkke	272
  529193	69	eek-d(10)-keke	273
  529230	70	eek-d(10)-kkke	273
  529136	48	eee-d(10)-kkk	274
  529156	68	kkk-d(10)-eee	274
  529194	44	eek-d(10)-keke	275
  529231	56	eek-d(10)-kkke	275
  529137	34	eee-d(10)-kkk	276
  529157	79	kkk-d(10)-eee	276
  529336	57	eek-d(10)-keke	277
  529338	73	eek-d(10)-keke	278
  529195	55	eek-d(10)-keke	279
  529232	68	eek-d(10)-kkke	279
  529340	65	eek-d(10)-keke	280
  529342	69	eek-d(10)-keke	281
  529812	69	k-d(10)-kekee	282
  529831	62	k-d(10)-kdkee	282
  529733	64	ke-d(10)-keke	283
  529753	52	ek-d(10)-keke	283
  529773	57	ke-d(10)-kdke	283
  529793	36	ek-d(10)-kdke	283
  529862	48	kde-d(10)-kdke	284
  529882	35	edk-d(10)-kdke	284
  529902	44	k-(d4)-k-(d4)-k-(d4)-ke	284
  529559	71	eek-d(10)-kke	26
  529584	57	kee-d(10)-kke	26
  529609	58	edk-d(10)-kke	26
  529634	49	kde-d(10)-kke	26
  529659	52	kddk-d(9)-kke	26
  529684	48	kdde-d(9)-kke	26
  529709	61	eddk-d(9)-kke	26
  529922	52	eeee-d(9)-kke	26
  529344	50	eek-d(10)-keke	285
  529138	32	eee-d(10)-kkk	286
  529158	75	kkk-d(10)-eee	286
  529184	75	eek-d(10)-keke	215
  529221	78	eek-d(10)-kkke	215
  529127	67	eee-d(10)-kkk	287
  529147	79	kkk-d(10)-eee	287
  529346	58	eek-d(10)-keke	288
  529348	65	eek-d(10)-keke	289
  529350	77	eek-d(10)-keke	290
  529813	20	k-d(10)-kekee	291
  529832	47	k-d(10)-kdkee	291
  529734	63	ke-d(10)-keke	292
  529754	58	ek-d(10)-keke	292
  529774	49	ke-d(10)-kdke	292
  529794	51	ek-d(10)-kdke	292
  529863	64	kde-d(10)-kdke	293
  529883	78	edk-d(10)-kdke	293
  529903	36	k-d(4)-k-d(4)-k-d(4)-ke	293
  529560	71	eek-d(10)-kke	27
  529585	70	kee-d(10)-kke	27
  529610	66	edk-d(10)-kke	27
  529635	45	kde-d(10)-kke	27
  529660	53	kddk-d(9)-kke	27
  529685	42	kdde-d(9)-kke	27
  529710	60	eddk-d(9)-kke	27
  529923	63	eeee-d(9)-kke	27
  529196	74	eek-d(10)-keke	294
  529233	80	eek-d(10)-kkke	294
  529139	75	eee-d(10)-kkk	295
  529159	62	kkk-d(10)-eee	295
  529352	74	eek-d(10)-keke	296
  529354	67	eek-d(10)-keke	297
  529197	43	eek-d(10)-keke	298
  529234	58	eek-d(10)-kkke	298
  529140	29	eee-d(10)-kkk	299
  529160	59	kkk-d(10)-eee	299
  529180	80	eek-d(10)-keke	216
  529217	79	eek-d(10)-kkke	216
  529814	51	k-d(10)-kekee	300
  529833	52	k-d(10)-kdkee	300
  529735	43	ke-d(10)-keke	301
  529755	60	ek-d(10)-keke	301
  529775	38	ke-d(10)-kdke	301
  529795	58	ek-d(10)-kdke	301
  529864	41	kde-d(10)-kdke	302
  529884	48	edk-d(10)-kdke	302
  529904	44	k-d(4)-k-(d4)-k-d(4)-ke	302
  529934	61	eek-d(10)-keke	302
  529356	71	eek-d(10)-keke	303
  529561	75	eek-d(10)-kke	28
  529586	65	kee-d(10)-kke	28
  529611	54	edk-d(10)-kke	28
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  529308	68	eek-d(10)-keke	467
  529310	59	eek-d(10)-keke	468
  529169	79	eek-d(10)-keke	469
  529206	82	eek-d(10)-kkke	469
  529312	68	eek-d(10)-keke	470
  529314	61	eek-d(10)-keke	471
  529316	62	eek-d(10)-keke	472
  529555	78	eek-d(10)-kke	59
  529580	73	kee-d(10)-kke	59
  529605	71	edk-d(10)-kke	59
  529630	64	kde-d(10)-kke	59
  529655	63	kddk-d(9)-kke	59
  529680	43	kdde-d(9)-kke	59
  529705	63	eddk-d(9)-kke	59
  529932	60	eeee-d(9)-kke	59
  529318	82	eek-d(10)-keke	473
  529170	85	eek-d(10)-keke	474
  529207	88	eek-d(10)-kkke	474
  529171	81	eek-d(10)-keke	475
  529208	84	eek-d(10)-kkke	475
  529805	40	k-d(10)-kekee	476
  529824	32	k-d(10)-kdkee	476
  529320	74	eek-d(10)-keke	477
  529726	80	ke-d(10)-keke	478
  529746	82	ek-d(10)-keke	478
  529766	63	ke-d(10)-kdke	478
  529786	69	ek-d(10)-kdke	478
  529855	39	kde-d(10)-kdke	477
  529875	40	edk-d(10)-kdke	477
  529895	27	k-(d4)-k-(d4)-k-(d4)-ke	477
  529556	72	eek-d(10)-kke	61
  529581	68	kee-d(10)-kke	61
  529606	54	edk-d(10)-kke	61
  529631	29	kde-d(10)-kke	61
  529656	74	kddk-d(9)-kke	61
  529681	32	kdde-d(9)-kke	61
  529706	41	eddk-d(9)-kke	61
  529915	51	eeee-d(9)-kke	61
  529172	88	eek-d(10)-keke	226
  529209	87	eek-d(10)-kkke	226
  529173	92	eek-d(10)-keke	227
  529210	89	eek-d(10)-kkke	227
  529183	85	eek-d(10)-keke	479
  529220	92	eek-d(10)-kkke	479
  529126	83	eee-d(10)-kkk	257
  529146	84	kkk-d(10)-eee	257
  529174	85	eek-d(10)-keke	480
  529211	86	eek-d(10)-kkke	480
  529322	71	eek-d(10)-keke	481
  529324	79	eek-d(10)-keke	482
  529326	85	eek-d(10)-keke	483
  529175	92	eek-d(10)-keke	228
  529212	92	eek-d(10)-kkke	228
  529176	89	eek-d(10)-keke	229
  529213	90	eek-d(10)-kkke	229
  529804	89	k-d(10)-kekee	259
  529823	89	k-d(10)-kdkee	259
  529166	83	eek-d(10)-keke	230
  529203	86	eek-d(10)-kkke	230
  529725	92	ke-d(10)-keke	260
  529745	91	ek-d(10)-keke	260
  529765	88	ke-d(10)-kdke	260
  529785	91	ek-d(10)-kdke	260
  529799	89	k-d(10)-kekee	260
  529818	88	k-d(10)-kdkee	260
  529854	90	kde-d(10)-kdke	230
  529874	81	edk-d(10)-kdke	230
  529894	60	k-(d4)-k-(d4)-k-(d4)-ke	230
  529167	71	eek-d(10)-keke	231
  529204	70	eek-d(10)-kkke	231
  529557	86	eek-d(10)-kke	69
  529582	86	kee-d(10)-kke	69
  529607	84	edk-d(10)-kke	69
  529632	81	kde-d(10)-kke	69
  529657	85	kddk-d(9)-kke	69
  529682	78	kdde-d(9)-kke	69
  529707	79	eddk-d(9)-kke	69
  529720	75	ke-d(10)-keke	69
  529740	70	ek-d(10)-keke	69
  529760	78	ke-d(10)-kdke	69
  529780	83	ek-d(10)-kdke	69
  529849	80	kde-d(10)-kdke	231
  529869	72	edk-d(10)-kdke	231
  529889	49	k-(d4)-k-(d4)-k-(d4)-ke	231
  529914	69	eeee-d(9)-kke	69
  529328	68	eek-d(10)-keke	484
  529558	71	eek-d(10)-kke	71
  529583	81	kee-d(10)-kke	71
  529608	68	edk-d(10)-kke	71
  529633	73	kde-d(10)-kke	71
  529658	63	kddk-d(9)-kke	71
  529683	74	kdde-d(9)-kke	71
  529708	70	eddk-d(9)-kke	71
  529909	59	eeee-d(9)-kke	71
  529192	51	eek-d(10)-keke	485
  529229	69	eek-d(10)-kkke	485
  529135	54	eee-d(10)-kkk	486
  529155	56	kkk-d(10)-eee	486
  529330	37	eek-d(10)-keke	487
  e = 2′-MOE,
  k = cEt,
  d = 2′-deoxynucleoside

Example 20: Design of Modified Oligonucleotides Comprising 2′-O-Methoxyethyl (2′-MOE) or Constrained Ethyl (cEt) Modifications
• Based on the activity of the antisense oligonucleotides listed above, additional antisense oligonucleotides were designed targeting a Target-X nucleic acid targeting start positions 1147, 1154 or 12842 of Target-X
• The newly designed chimeric antisense oligonucleotides are 16 or 17 nucleotides in length and their motifs are described in Table 31. The chemistry column of Table 31 presents the sugar motif of each oligonucleotide, wherein “e” indicates a 2′-O-methythoxylethyl (2′-MOE) nucleoside, “k” indicates a constrained ethyl (cEt) nucleoside and “d” indicates a 2′-deoxyribonucleoside. The internucleoside linkages throughout each gapmer are hosphorothioate (P═S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosine.
• Each gapmer listed in Table 31 is targeted to the human Target-X genomic sequence.

• TABLE 31
  Chimeric antisense oligonucleotides targeted to Target-X

  ISIS No	Chemistry	SEQ CODE

  529544	eek-d(10)-kke	21
  529569	kee-d(10)-kke	21
  529594	edk-d(10)-kke	21
  529619	kde-d(10)-kke	21
  529644	kddk-d(9)-kke	21
  529669	kdde-d(9)-kke	21
  529694	eddk-d(9)-kke	21
  529929	eeee-d(9)-kke	21
  529809	k-d(10)-kekee	488
  529828	k-d(10)-kdkee	488
  529730	ke-d(10)-keke	489
  529750	ek-d(10)-keke	489
  529770	ke-d(10)-kdke	489
  529790	ek-d(10)-kdke	489
  529859	kde-d(10)-kdke	490
  529879	edk-d(10)-kdke	490
  529899	k-d(4)-k-d(4)-k-d(4)-ke	490
  529545	eek-d(10)-kke	22
  529570	kee-d(10)-kke	22
  529595	edk-d(10)-kke	22
  529620	kde-d(10)-kke	22
  529645	kddk-d(9)-kke	22
  529670	kdde-d(9)-kke	22
  529695	eddk-d(9)-kke	22
  529919	eeee-d(9)-kke	22
  529548	eek-d(10)-kke	41
  529573	kee-d(10)-kke	41
  529598	edk-d(10)-kke	41
  529623	kde-d(10)-kke	41
  529648	kddk-d(9)-kke	41
  529673	kdde-d(9)-kke	41
  529698	eddk-d(9)-kke	41
  529930	eeee-d(9)-kke	41
  e = 2′-MOE,
  k = cEt,
  d = 2′-deoxynucleoside

Example 21: Modified Oligonucleotides Comprising 2′-O-Methoxyethyl (2′-MOE) and Constrained Ethyl (cEt) Modifications Targeting Human Target-X
• Additional antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. ISIS 472998 and ISIS 515554, described in the Examples above were also included in the screen.
• The newly designed chimeric antisense oligonucleotides are 16 nucleotides in length and their motifs are described in Table 32. The chemistry column of Table 32 presents the sugar motif of each oligonucleotide, wherein “e” indicates a 2′-O-methythoxylethyl (2′-MOE) nucleoside, “k” indicates a constrained ethyl (cEt) nucleoside and “d” indicates a 2′-deoxyribonucleoside. The internucleoside linkages throughout each gapmer are hosphorothioate (P═S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines.
• Each gapmer listed in Table 32 is targeted to the human Target-X genomic sequence.
• Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS2927 was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells.

• TABLE 32
  Inhibition of human Target-X mRNA levels by chimeric
  antisense oligonucleotides targeted to Target-X

  	ISIS No	% inhibition	Chemistry	SEQ CODE

  	472998	88	kk-d(10)-kk	74
  	515554	75	eee-d(10)-kkk	493
  	534530	92	keke-d(9)-kek	491
  	534563	92	kek-d(9)-ekek	491
  	534596	88	ekee-d(9)-kke	491
  	534629	89	eke-d(9)-ekke	491
  	534662	87	eekk-d(9)-eke	491
  	534695	92	eek-d(9)-keke	491
  	534732	90	ekek-d(8)-keke	491
  	534767	92	keek-d(8)-keek	491
  	534802	93	ekk-d(10)-kke	491
  	534832	83	edk-d(10)-kke	491
  	534862	72	kde-d(10)-kke	491
  	534892	82	eek-d(10)-kke	491
  	534922	80	kddk-d(9)-kke	491
  	534952	72	kdde-d(9)-kke	491
  	534982	77	eddk-d(9)-kke	491
  	535012	70	eeee-d(9)-kke	491
  	535045	84	eeee-d(9)-kkk	491
  	535078	87	eeek-d(9)-kke	491
  	535111	63	eeeee-d(8)-kke	491
  	535144	69	ededk-d(8)-kke	491
  	535177	68	edkde-d(8)-kke	491
  	534531	61	keke-d(9)-kek	492
  	534564	30	kek-d(9)-ekek	492
  	534597	67	ekee-d(9)-kke	492
  	534630	54	eke-d(9)-ekke	492
  	534663	94	eekk-d(9)-eke	492
  	534696	68	eek-d(9)-keke	492
  	534733	44	ekek-d(8)-keke	492
  	534768	55	keek-d(8)-keek	492
  	534803	73	ekk-d(10)-kke	492
  	534833	65	edk-d(10)-kke	492
  	534863	53	kde-d(10)-kke	492
  	534893	61	eek-d(10)-kke	492
  	534923	70	kddk-d(9)-kke	492
  	534953	54	kdde-d(9)-kke	492
  	534983	58	eddk-d(9)-kke	492
  	535013	52	eeee-d(9)-kke	492
  	535046	67	eeee-d(9)-kkk	492
  	535079	57	eeek-d(9)-kke	492
  	535112	42	eeeee-d(8)-kke	492
  	535145	41	ededk-d(8)-kke	492
  	535178	35	edkde-d(8)-kke	492
  	534565	87	kek-d(9)-ekek	493
  	534598	72	ekee-d(9)-kke	493
  	534631	70	eke-d(9)-ekke	493
  	534664	94	eekk-d(9)-eke	493
  	534697	90	eek-d(9)-keke	493
  	534734	74	ekek-d(8)-keke	493
  	534769	80	keek-d(8)-keek	493
  	534804	87	ekk-d(10)-kke	493
  	534834	76	edk-d(10)-kke	493
  	534864	56	kde-d(10)-kke	493
  	534894	67	eek-d(10)-kke	493
  	534924	71	kddk-d(9)-kke	493
  	534954	54	kdde-d(9)-kke	493
  	534984	48	eddk-d(9)-kke	493
  	535014	43	eeee-d(9)-kke	493
  	535047	60	eeee-d(9)-kkk	493
  	535080	64	eeek-d(9)-kke	493
  	535113	32	eeeee-d(8)-kke	493
  	535146	31	ededk-d(8)-kke	493
  	535179	28	edkde-d(8)-kke	493
  	534533	82	keke-d(9)-kek	494
  	534566	88	kek-d(9)-ekek	494
  	534599	65	ekee-d(9)-kke	494
  	534632	69	eke-d(9)-ekke	494
  	534665	87	eekk-d(9)-eke	494
  	534698	64	eek-d(9)-keke	494
  	534735	63	ekek-d(8)-keke	494
  	534770	66	keek-d(8)-keek	494
  	534805	87	ekk-d(10)-kke	494
  	534835	68	edk-d(10)-kke	494
  	534865	66	kde-d(10)-kke	494
  	534895	57	eek-d(10)-kke	494
  	534925	82	kddk-d(9)-kke	494
  	534955	76	kdde-d(9)-kke	494
  	534985	71	eddk-d(9)-kke	494
  	535015	59	eeee-d(9)-kke	494
  	535048	69	eeee-d(9)-kkk	494
  	535081	67	eeek-d(9)-kke	494
  	535114	37	eeeee-d(8)-kke	494
  	535147	32	ededk-d(8)-kke	494
  	535180	31	edkde-d(8)-kke	494
  	534534	94	keke-d(9)-kek	234
  	534567	92	kek-d(9)-ekek	234
  	534600	92	ekee-d(9)-kke	234
  	534633	91	eke-d(9)-ekke	234
  	534666	89	eekk-d(9)-eke	234
  	534699	91	eek-d(9)-keke	234
  	534736	83	ekek-d(8)-keke	234
  	534771	80	keek-d(8)-keek	234
  	534806	96	ekk-d(10)-kke	234
  	534836	86	edk-d(10)-kke	234
  	534866	82	kde-d(10)-kke	234
  	534896	82	eek-d(10)-kke	234
  	534926	89	kddk-d(9)-kke	234
  	534956	91	kdde-d(9)-kke	234
  	534986	87	eddk-d(9)-kke	234
  	535016	83	eeee-d(9)-kke	234
  	535049	87	eeee-d(9)-kkk	234
  	535082	87	eeek-d(9)-kke	234
  	535115	77	eeeee-d(8)-kke	234
  	535148	73	ededk-d(8)-kke	234
  	535181	68	edkde-d(8)-kke	234
  	534535	66	keke-d(9)-kek	236
  	534568	85	kek-d(9)-ekek	236
  	534601	51	ekee-d(9)-kke	236
  	534634	80	eke-d(9)-ekke	236
  	534667	90	eekk-d(9)-eke	236
  	534700	88	eek-d(9)-keke	236
  	534737	65	ekek-d(8)-keke	236
  	534772	77	keek-d(8)-keek	236
  	534807	84	ekk-d(10)-kke	236
  	534837	78	edk-d(10)-kke	236
  	534867	44	kde-d(10)-kke	236
  	534897	82	eek-d(10)-kke	236
  	534927	61	kddk-d(9)-kke	236
  	534957	58	kdde-d(9)-kke	236
  	534987	49	eddk-d(9)-kke	236
  	535017	38	eeee-d(9)-kke	236
  	535050	32	eeee-d(9)-kkk	236
  	535083	43	eeek-d(9)-kke	236
  	535116	9	eeeee-d(8)-kke	236
  	535149	23	ededk-d(8)-kke	236
  	535182	18	edkde-d(8)-kke	236
  	534536	89	keke-d(9)-kek	238
  	534569	90	kek-d(9)-ekek	238
  	534602	85	ekee-d(9)-kke	238
  	534635	87	eke-d(9)-ekke	238
  	534668	90	eekk-d(9)-eke	238
  	534701	92	eek-d(9)-keke	238
  	534738	81	ekek-d(8)-keke	238
  	534773	79	keek-d(8)-keek	238
  	534808	90	ekk-d(10)-kke	238
  	534838	88	edk-d(10)-kke	238
  	534868	67	kde-d(10)-kke	238
  	534898	89	eek-d(10)-kke	238
  	534928	81	kddk-d(9)-kke	238
  	534958	78	kdde-d(9)-kke	238
  	534988	66	eddk-d(9)-kke	238
  	535018	78	eeee-d(9)-kke	238
  	535051	76	eeee-d(9)-kkk	238
  	535084	80	eeek-d(9)-kke	238
  	535117	58	eeeee-d(8)-kke	238
  	535150	51	ededk-d(8)-kke	238
  	535183	53	edkde-d(8)-kke	238
  	534537	91	keke-d(9)-kek	239
  	534570	85	kek-d(9)-ekek	239
  	534603	79	ekee-d(9)-kke	239
  	534636	72	eke-d(9)-ekke	239
  	534669	85	eekk-d(9)-eke	239
  	534702	85	eek-d(9)-keke	239
  	534739	73	ekek-d(8)-keke	239
  	534774	77	keek-d(8)-keek	239
  	534809	91	ekk-d(10)-kke	239
  	534839	86	edk-d(10)-kke	239
  	534869	71	kde-d(10)-kke	239
  	534899	82	eek-d(10)-kke	239
  	534929	83	kddk-d(9)-kke	239
  	534959	80	kdde-d(9)-kke	239
  	534989	79	eddk-d(9)-kke	239
  	535019	76	eeee-d(9)-kke	239
  	535052	79	eeee-d(9)-kkk	239
  	535085	81	eeek-d(9)-kke	239
  	535118	58	eeeee-d(8)-kke	239
  	535151	65	ededk-d(8)-kke	239
  	535184	60	edkde-d(8)-kke	239
  	534516	77	keke-d(9)-kek	495
  	534549	80	kek-d(9)-ekek	495
  	534582	73	ekee-d(9)-kke	495
  	534615	79	eke-d(9)-ekke	495
  	534648	67	eekk-d(9)-eke	495
  	534681	87	eek-d(9)-keke	495
  	534718	46	ekek-d(8)-keke	495
  	534753	68	keek-d(8)-keek	495
  	534788	84	ekk-d(10)-kke	495
  	534818	82	edk-d(10)-kke	495
  	534848	75	kde-d(10)-kke	495
  	534878	72	eek-d(10)-kke	495
  	534908	81	kddk-d(9)-kke	495
  	534938	69	kdde-d(9)-kke	495
  	534968	77	eddk-d(9)-kke	495
  	534998	76	eeee-d(9)-kke	495
  	535031	76	eeee-d(9)-kkk	495
  	535064	70	eeek-d(9)-kke	495
  	535097	57	eeeee-d(8)-kke	495
  	535130	69	ededk-d(8)-kke	495
  	535163	58	edkde-d(8)-kke	495
  	534538	71	keke-d(9)-kek	241
  	534571	64	kek-d(9)-ekek	241
  	534604	66	ekee-d(9)-kke	241
  	534637	74	eke-d(9)-ekke	241
  	534670	87	eekk-d(9)-eke	241
  	534703	72	eek-d(9)-keke	241
  	534740	56	ekek-d(8)-keke	241
  	534775	53	keek-d(8)-keek	241
  	534810	78	ekk-d(10)-kke	241
  	534840	73	edk-d(10)-kke	241
  	534870	65	kde-d(10)-kke	241
  	534900	69	eek-d(10)-kke	241
  	534930	67	kddk-d(9)-kke	241
  	534960	62	kdde-d(9)-kke	241
  	534990	66	eddk-d(9)-kke	241
  	535020	61	eeee-d(9)-kke	241
  	535053	47	eeee-d(9)-kkk	241
  	535086	61	eeek-d(9)-kke	241
  	535119	49	eeeee-d(8)-kke	241
  	535152	48	ededk-d(8)-kke	241
  	535185	57	edkde-d(8)-kke	241
  	534539	70	keke-d(9)-kek	496
  	534572	82	kek-d(9)-ekek	496
  	534605	59	ekee-d(9)-kke	496
  	534638	69	eke-d(9)-ekke	496
  	534671	89	eekk-d(9)-eke	496
  	534704	83	eek-d(9)-keke	496
  	534741	47	ekek-d(8)-keke	496
  	534776	46	keek-d(8)-keek	496
  	534811	71	ekk-d(10)-kke	496
  	534841	61	edk-d(10)-kke	496
  	534871	53	kde-d(10)-kke	496
  	534901	55	eek-d(10)-kke	496
  	534931	73	kddk-d(9)-kke	496
  	534961	53	kdde-d(9)-kke	496
  	534991	56	eddk-d(9)-kke	496
  	535021	58	eeee-d(9)-kke	496
  	535054	59	eeee-d(9)-kkk	496
  	535087	0	eeek-d(9)-kke	496
  	535120	41	eeeee-d(8)-kke	496
  	535153	44	ededk-d(8)-kke	496
  	535186	35	edkde-d(8)-kke	496
  	534573	76	kek-d(9)-ekek	497
  	534606	55	ekee-d(9)-kke	497
  	534639	72	eke-d(9)-ekke	497
  	534672	89	eekk-d(9)-eke	497
  	534705	87	eek-d(9)-keke	497
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  	535174	65	edkde-d(8)-kke	258
  	534528	94	keke-d(9)-kek	260
  	534561	0	kek-d(9)-ekek	260
  	534594	92	ekee-d(9)-kke	260
  	534627	90	eke-d(9)-ekke	260
  	534660	92	eekk-d(9)-eke	260
  	534693	95	eek-d(9)-keke	260
  	534730	93	ekek-d(8)-keke	260
  	534765	92	keek-d(8)-keek	260
  	534800	93	ekk-d(10)-kke	260
  	534830	93	edk-d(10)-kke	260
  	534860	85	kde-d(10)-kke	260
  	534890	91	eek-d(10)-kke	260
  	534920	93	kddk-d(9)-kke	260
  	534950	90	kdde-d(9)-kke	260
  	534980	88	eddk-d(9)-kke	260
  	535010	88	eeee-d(9)-kke	260
  	535043	89	eeee-d(9)-kkk	260
  	535076	88	eeek-d(9)-kke	260
  	535109	76	eeeee-d(8)-kke	260
  	535142	86	ededk-d(8)-kke	260
  	535175	71	edkde-d(8)-kke	260
  	534529	70	keke-d(9)-kek	261
  	534562	86	kek-d(9)-ekek	261
  	534595	56	ekee-d(9)-kke	261
  	534628	73	eke-d(9)-ekke	261
  	534661	64	eekk-d(9)-eke	261
  	534694	75	eek-d(9)-keke	261
  	534731	47	ekek-d(8)-keke	261
  	534766	30	keek-d(8)-keek	261
  	534801	83	ekk-d(10)-kke	261
  	534831	84	edk-d(10)-kke	261
  	534861	71	kde-d(10)-kke	261
  	534891	73	eek-d(10)-kke	261
  	534921	55	kddk-d(9)-kke	261
  	534951	61	kdde-d(9)-kke	261
  	534981	48	eddk-d(9)-kke	261
  	535011	54	eeee-d(9)-kke	261
  	535044	46	eeee-d(9)-kkk	261
  	535077	29	eeek-d(9)-kke	261
  	535110	19	eeeee-d(8)-kke	261
  	535143	15	ededk-d(8)-kke	261
  	535176	37	edkde-d(8)-kke	261
  	e = 2′-MOE,
  	k = cEt,
  	d = 2′-deoxynucleoside

Example 22: Modified Antisense Oligonucleotides Comprising 2′-O-Methoxyethyl (2′-MOE) and Constrained Ethyl (cEt) Modifications Targeting Human Target-X Targeting Intronic Repeats
• Additional Antisense Oligonucleotides were Designed Targeting the Intronic Repeat Regions of Target-X
• The newly designed chimeric antisense oligonucleotides and their motifs are described in Table 33. The internucleoside linkages throughout each gapmer are phosphorothioate linkages (P═S) and are designated as “s”. Nucleosides followed by “d” indicate 2′-deoxyribonucleosides. Nucleosides followed by “k” indicate constrained ethyl (cEt) nucleosides. Nucleosides followed by “e” indicate 2′-O-methythoxylethyl (2′-MOE) nucleosides. “N” indicates modified or naturally occurring nucleobases (A, T, C, G, U, or 5-methyl C).
• Each gapmer listed in Table 33 is targeted to the intronic region of human Target-X genomic sequence, designated herein as Target-X.
• Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human primer probe set was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells.

• TABLE 33
  Inhibition of human Target-X mRNA levels by chimeric antisense oligonucleotides
  targeted to Target-X

  	ISIS	%	SEQ	SEQ ID
  Sequence (5′ to 3′)	No	inhibition	CODE	NO
  Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds	472998	90	508	20
  Nds Nks Nk
  Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds	473327	88	30	19
  Nds Nds Nes Nes Ne
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537024	74	509	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537025	79	510	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537026	76	511	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537028	37	512	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537029	45	513	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537030	67	514	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537031	59	515	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537032	9	516	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537033	65	517	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537034	71	518	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537035	68	519	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537036	74	520	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537038	69	521	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537039	67	522	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537040	68	523	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537041	76	524	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537042	77	525	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537043	70	526	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537044	82	527	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537045	69	528	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537047	35	529	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537049	62	530	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537051	62	531	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537055	16	532	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537056	25	533	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537057	49	534	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537058	49	535	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537059	53	536	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537060	73	537	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537061	70	538	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537062	69	539	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537063	68	540	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537064	71	541	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537065	67	542	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537066	68	543	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537067	71	544	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537068	86	545	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537069	82	546	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537070	87	547	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537792	36	548	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537793	35	549	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537794	35	550	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537795	33	551	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537796	49	552	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537797	54	553	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537798	68	554	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537799	72	555	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537800	69	556	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537801	82	557	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537802	72	558	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537803	72	559	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537804	67	560	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537805	74	561	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537806	70	562	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537809	60	563	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537810	71	564	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537811	69	565	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537812	80	566	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537813	74	567	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537814	54	568	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537837	70	569	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537838	76	570	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537839	76	571	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537840	80	572	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537841	81	573	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537842	75	574	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537843	70	575	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537844	73	576	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537845	59	577	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537846	51	578	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537847	52	579	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537848	41	580	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	537849	44	581	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	538160	69	582	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	538172	24	583	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	538173	23	584	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	538185	68	585	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	538187	69	585	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	538189	81	587	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	538191	66	588	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	538192	59	589	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	538193	16	590	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	538194	10	591	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	538195	15	592	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	538196	3	593	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	538197	36	594	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	538198	49	595	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	538199	47	596	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	538200	57	597	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	538201	71	598	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	538202	60	599	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	538203	55	600	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	538204	62	601	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	538205	68	602	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	538228	63	603	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	538229	26	604	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	538230	75	605	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	538231	75	606	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	538233	52	607	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	538235	26	608	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	538237	28	609	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	538239	54	610	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	538241	73	611	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	538242	68	612	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	538243	61	613	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	538245	75	614	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	538253	37	615	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	538254	45	616	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	538361	56	617	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	538378	70	618	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	538380	68	619	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	538381	57	620	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	540361	71	621	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	540362	73	622	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	540363	78	623	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	540364	89	624	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	540365	83	625	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	540366	84	626	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	540367	65	627	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	540368	55	628	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	540369	82	629	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	540370	86	630	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	540371	74	631	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	540372	82	632	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	540373	81	633	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	540374	87	634	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	540375	78	635	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	540376	69	636	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	540377	88	637	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	540378	85	638	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	540379	77	639	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	540380	84	640	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	540381	85	641	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	540382	69	642	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	540383	85	643	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	540384	88	644	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	540385	87	645	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	540386	86	646	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	540387	77	647	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	540388	86	648	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	540389	86	649	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	540390	85	650	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	540391	83	651	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	540392	43	652	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	540393	88	653	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	540394	68	654	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	540395	87	655	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	540396	87	656	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	540397	59	657	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	540398	36	658	19
  Nds Nds Nks Nks Nk
  Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds	540399	81	659	19
  Nds Nds Nks Nks Nk

Example 23: High Dose Tolerability of Modified Oligonucleotides Comprising 2′-O-Methoxyethyl (2′-MOE) and 6′-(S)—CH₃Bicyclic Nucleoside (e.g cEt) Modifications Targeting Human Target-X in BALB/c Mice
• BALB/c mice were treated at a high dose with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.
• Additionally, the newly designed antisense oligonucleotides were created with the same sequences as the antisense oligonucleotides from the study described above and were also added to this screen targeting intronic repeat regions of Target-X.
• The newly designed modified antisense oligonucleotides and their motifs are described in Table 34. The internucleoside linkages throughout each gapmer are phosphorothioate linkages (P═S). Nucleosides followed by “d” indicate 2′-deoxyribonucleosides. Nucleosides followed by “k” indicate 6′-(S)—CH₃bicyclic nucleoside (e.g cEt) nucleosides. Nucleosides followed by “e” indicate 2′-O-methythoxylethyl (2′-MOE) nucleosides. “N” indicates modified or naturally occurring nucleobases (A, T, C, G, U, or 5-methyl C).
• Each gapmer listed in Table 34 is targeted to the intronic region of human Target-X genomic sequence, designated herein as Target-X.

• TABLE 34
  Modified antisense oligonucleotides targeted to Target-X

  		SEQ	SEQ ID
  Sequence (5′ to 3′)	ISIS No	CODE	NO
  Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne	537721	509	19
  Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne	537738	524	19
  Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne	537759	539	19
  Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne	537761	541	19
  Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne	537763	543	19
  Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne	537850	548	19
  Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne	537858	556	19
  Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne	537864	562	19
  Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne	537869	565	19
  Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne	537872	568	19
  Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne	537897	571	19
  Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne	540118	582	19
  Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne	540138	602	19
  Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne	540139	603	19
  Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne	540148	612	19
  Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne	540153	617	19
  Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne	540155	619	19
  Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne	540162	624	19
  Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne	540164	626	19
  Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne	540168	630	19
  Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne	540172	634	19
  Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne	540175	637	19
  Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne	540176	638	19
  Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne	540178	640	19
  Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne	540179	641	19
  Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne	540181	643	19
  Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne	540182	644	19
  Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne	540183	645	19
  Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne	540184	646	19
  Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne	540186	648	19
  Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne	540187	649	19
  Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne	540188	650	19
  Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne	540191	653	19
  Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne	540193	655	19
  Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne	540194	656	19
  Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne	544811	547	19
  Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne	544812	545	19
  Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne	544813	527	19
  Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne	544814	557	19
  Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne	544815	546	19
  Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne	544816	573	19
  Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne	544817	572	19
  Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne	544818	566	19
  Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne	544819	510	19
  Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne	544820	525	19
  Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne	544821	567	19
  Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne	544826	537	19
  Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne	544827	538	19
  Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne	544828	539	19
  Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne	544829	540	19
  Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne	544830	541	19
  Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne	545471	542	19
  Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne	545472	543	19
  Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne	545473	544	19
  Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne	545474	558	19
  Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne	545475	559	19
  Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne	545476	560	19
  Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne	545477	561	19
  Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne	545478	562	19
  Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne	545479	556	19
  Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne	537727	514	19

Treatment
• Male BALB/c mice were injected subcutaneously with a single dose of 200 mg/kg of ISIS 422142, ISIS 457851, ISIS 473294, ISIS 473295, ISIS 473327, ISIS 484714, ISIS 515334, ISIS 515338, ISIS 515354, ISIS 515366, ISIS 515380, ISIS 515381, ISIS 515382, ISIS 515384, ISIS 515386, ISIS 515387, ISIS 515388, ISIS 515406, ISIS 515407, ISIS 515408, ISIS 515422, ISIS 515423, ISIS 515424, ISIS 515532, ISIS 515533, ISIS 515534, ISIS 515538, ISIS 515539, ISIS 515558, ISIS 515656, ISIS 515575, ISIS 515926, ISIS 515944, ISIS 515945, ISIS 515948, ISIS 515949, ISIS 515951, ISIS 515952, ISSI 516003, ISIS 516055, ISIS 516057, ISIS 516060, ISIS 516062, ISIS 529126, ISIS 529146, ISIS 529166, ISIS 529170, ISIS 529172, ISIS 529173, ISIS 529174, ISIS 529175, ISSI 529176, ISIS 529182, ISIS 529183, ISIS 529186, ISIS 529282, ISIS 529304, ISIS 529306, ISIS 529360, ISIS 529450, ISIS 529459, ISIS 529460, ISIS 529461, ISIS 529547, ISIS 529550, ISIS 529551, ISIS 529553, ISIS 529557, ISIS 529562, ISIS 529563, ISIS 529564, ISIS 529565, ISIS 529575, ISIS 529582, ISIS 529589, ISIS 529607, ISIS 529614, ISIS 529632, ISIS 529650, ISIS 529651, ISIS 529657, ISIS 529663, ISIS 529725, ISIS 529745, ISIS 529765, ISIS 529785, ISIS 529804, ISIS 529818, ISIS 529823, ISIS 529854, ISIS 534528, ISIS 534534, ISIS 534594, ISIS 534660, ISIS 534663, ISIS 534664, ISIS 534676, ISIS 534677, ISIS 537679, ISIS 537683, ISIS 534693, ISIS 534701, ISIS 534716, ISIS 534730, ISIS 534765, ISIS 534795, ISIS 534796, ISIS 534797, ISIS 534798, ISIS 534799, ISIS 534800, ISIS 534802, ISIS 534806, ISSI 534830, ISIS 534838, ISIS 534888, ISIS 534890, ISIS 534898, ISIS 534911, ISIS 534920, ISIS 534926, ISIS 534937, ISIS 534950, ISSI 534956, ISIS 534980, ISIS 534986, ISIS 535010, ISIS 535043, ISIS 535049, ISIS 535076, ISIS 535082, ISSI 535142, ISIS 537024, ISIS 537030, ISIS 537041, ISIS 537062, ISIS 537064, ISIS 537066, ISIS 537721, ISIS 537727, ISIS 537738, ISIS 537759, ISIS 537761, ISIS 537763, ISIS 537792, ISIS 537800, ISIS 537806, ISIS 537811, ISIS 537814, ISIS 537839, ISIS 537850, ISSI 537858, ISIS 537864, ISIS 537869, ISIS 537872, ISIS 537897, ISIS 538160, ISIS 538196, ISIS 538205, ISIS 538228, ISIS 538242, ISIS 538361, ISIS 538380, ISIS 540118, ISIS 540138, ISIS 540139, ISIS 540148, ISIS 540153, ISIS 540155, ISIS 540162, ISIS 540164, ISIS 540168, ISIS 540172, ISIS 540175, ISIS 540176, ISIS 540178, ISIS 540179, ISIS 540181, ISIS 540182, ISIS 540183, ISIS 540184, ISIS 540186, ISIS 540187, ISIS 540188, ISIS 540191, ISIS 540193, ISIS 540194, ISIS 544811, ISIS 544812, ISIS 544813, ISIS 544814, ISIS 544815, ISIS 544816, ISIS 544817, ISIS 544818, ISIS 544819, ISIS 544820, ISIS 544821, ISIS 544826, ISIS 544827, ISIS 544828, ISIS 544829, ISIS 544830, ISIS 545471, ISIS 545472, ISIS 545473, ISIS 545474, ISIS 545475, ISIS 545476, ISIS 545477, ISIS 545478, and ISIS 545479. One set of male BALB/c mice was injected with a single dose of PBS. Mice were euthanized 96 hours later, and organs and plasma were harvested for further analysis.
Plasma Chemistry Markers
• To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, albumin, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.).
• ISIS oligonucleotides that did not cause any increase in the levels of transaminases, or which caused an increase within three times the upper limit of normal (ULN) were deemed very tolerable. ISIS oligonucleotides that caused an increase in the levels of transaminases between three times and seven times the ULN were deemed tolerable. Based on these criteria, ISIS 529166, ISIS 529170, ISIS 529175, ISIS 529176, ISIS 529186, ISIS 529282, ISIS 529360, ISIS 529450, ISIS 529459, ISIS 529460, ISIS 529547, ISIS 529549, ISIS 529551, ISIS 529553, ISIS 529557, ISIS 529562, ISIS 529575, ISIS 529582, ISIS 529607, ISIS 529589, ISIS 529632, ISIS 529657, ISIS 529725, ISIS 529745, ISIS 529785, ISIS 529799, ISIS 529804, ISIS 529818, ISIS 529823, ISIS 534950, ISIS 534980, ISIS 535010, ISIS 537030, ISIS 537041, ISIS 537062, ISIS 537064, ISIS 537066, ISIS 537759, ISIS 537792, ISIS 537800, ISIS 537839, ISIS 538228, ISIS 473294, ISIS 473295, ISIS 484714, ISIS 515338, ISIS 515366, ISIS 515380, ISIS 515381, ISIS 515387, ISIS 515408, ISIS 515423, ISIS 515424, ISIS 515532, ISIS 515534, ISIS 515538, ISIS 515539, ISIS 515558, ISIS 515575, ISIS 515926, ISIS 515944, ISIS 515945, ISIS 515951, ISIS 515952, ISIS 529126, ISIS 529765, ISIS 534528, ISIS 534534, ISIS 534594, ISIS 534663, ISIS 534676, ISIS 534677, ISIS 534679, ISIS 534683, ISIS 534693, ISIS 534701, ISIS 534716, ISIS 534730, ISIS 534806, ISIS 534830, ISIS 534838, ISIS 534890, ISIS 534898, ISIS 534911, ISIS 534937, ISIS 534956, ISIS 534986, ISIS 535043, ISIS 535049, ISIS 535076, ISIS 535082, ISIS 535142, ISIS 538160, ISIS 538242, ISIS 538361, ISIS 538380, ISIS 534795, ISIS 534796, ISIS 534797, ISIS 540162, ISIS 540164, ISIS 540168, ISIS 540172, ISIS 540175, ISIS 540176, ISIS 540178, ISIS 540179, ISIS 540181, ISIS 540182, ISIS 540183, ISIS 540184, ISIS 540186, ISIS 540187, ISIS 540188, ISIS 540191, ISIS 540193, ISIS 540194, ISIS 544813, ISIS 544814, ISIS 544816, ISIS 544826, ISIS 544827, ISIS 544828, ISIS 544829, ISIS 545473, and ISIS 545474 were considered very tolerable in terms of liver function. Based on these criteria, ISIS 529173, ISIS 529854, ISIS 529614, ISIS 515386, ISIS 515388, ISIS 515949, ISIS 544817, and ISIS 545479 were considered tolerable in terms of liver function.
Example 24: Tolerability of Antisense Oligonucleotides Targeting Human Target-X in Sprague-Dawley Rats
• Sprague-Dawley rats are a multipurpose model used for safety and efficacy evaluations. The rats were treated with ISIS antisense oligonucleotides from the studies described in the Examples above and evaluated for changes in the levels of various plasma chemistry markers.
Treatment
• Six-eight week old male Sprague-Dawley rats were maintained on a 12-hour light/dark cycle and fed ad libitum with Teklad normal rat chow. Groups of four Sprague-Dawley rats each were injected subcutaneously twice a week for 6 weeks with 25 mg/kg of ISIS 473286, ISIS 473547, ISIS 473567, ISIS 473589, ISIS 473630, ISIS 484559, ISIS 515636, ISIS 515640, ISIS 515641, ISIS 515655, ISIS 515657, ISIS 516046, ISIS 516048, ISIS 516051, ISIS 516052, and ISIS 516062. A group of four Sprague-Dawley rats was injected subcutaneously twice a week for 6 weeks with PBS. Forty eight hours after the last dose, rats were euthanized and organs and plasma were harvested for further analysis.
Liver Function
• To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma levels of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma levels of ALT (alanine transaminase) and AST (aspartate transaminase) were measured. Plasma levels of Bilirubin and BUN were also measured using the same clinical chemistry analyzer. ISIS oligonucleotides that did not cause any increase in the levels of transaminases, or which caused an increase within three times the upper limit of normal (ULN) were deemed very tolerable. ISIS oligonucleotides that caused an increase in the levels of transaminases between three times and seven times the ULN were deemed tolerable. Based on these criteria, ISIS 473286, ISIS 473547, ISSI 473589, ISIS 473630, ISIS 484559, ISIS 515636, ISIS 515640, ISIS 515655, ISIS 516046, and ISIS 516051 were considered very tolerable in terms of liver function. Based on these criteria, ISIS 473567, ISIS 515641, ISIS 515657, ISIS 516048, and ISIS 516051 were considered tolerable in terms of liver function.
Example 25: Tolerability of Chimeric Antisense Oligonucleotides Comprising 2′-O-Methoxyethyl (2′-MOE) Modifications Targeting Human Target-X in Sprague-Dawley Rats
• Sprague-Dawley rats were treated with ISIS antisense oligonucleotides from the studies described in the Examples above and evaluated for changes in the levels of various plasma chemistry markers.
Treatment
• Six-eight week old male Sprague-Dawley rats were maintained on a 12-hour light/dark cycle and fed ad libitum with Purina normal rat chow. Groups of four Sprague-Dawley rats each were injected subcutaneously twice a week for 6 weeks with 50 mg/kg of ISIS 407936, ISIS 416507, ISIS 416508, ISIS 490208, ISIS 490279, ISIS 490323, ISIS 490368, ISIS 490396, ISIS 490803, ISIS 491122, ISIS 513419, ISIS 513446, ISIS 513454, ISIS 513455, ISIS 513456, ISIS 513504, ISIS 513507, and ISIS 513508. A group of four Sprague-Dawley rats was injected subcutaneously twice a week for 6 weeks with PBS. Forty eight hours after the last dose, rats were euthanized and organs and plasma were harvested for further analysis.
Liver Function
• To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma levels of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma levels of Bilirubin and BUN were also measured using the same clinical chemistry analyzer.
• ISIS oligonucleotides that did not cause any increase in the levels of transaminases, or which caused an increase within three times the upper limit of normal (ULN) were deemed very tolerable. ISIS oligonucleotides that caused an increase in the levels of transaminases between three times and seven times the ULN were deemed tolerable. Based on these criteria, ISIS 416507, ISIS 490208, ISIS 490368, ISIS 490396, ISIS 490803, ISIS 491122, ISIS 513446, ISIS 513454, ISIS 513455, ISIS 513456, ISIS 513504, and ISIS 513508 were considered very tolerable in terms of liver function. Based on these criteria, ISIS 407936, ISIS 416508, ISIS 490279, and ISIS 513507 were considered tolerable in terms of liver function.
Example 26: Tolerability of Chimeric Antisense Oligonucleotides Comprising 2′-O-Methoxyethyl (2′-MOE) Modifications Targeting Human Target-X in CD-1 Mice
• CD-1 mice are a multipurpose mice model, frequently utilized for safety and efficacy testing. The mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.
Treatment
• Groups of 3 male CD-1 mice each were injected subcutaneously twice a week for 6 weeks with 50 mg/kg of ISIS 473244, ISIS 473295, ISIS 484714, ISIS 515386, ISIS 515424, ISIS 515534, ISIS 515558, ISIS 515926, ISIS 515949, ISIS 515951, ISIS 515952, ISIS 529126, ISIS 529166, ISIS 529173, ISIS 529186, ISIS 529360, ISIS 529461, ISIS 529553, ISIS 529564, ISIS 529582, ISIS 529614, ISIS 529725, ISIS 529745, ISIS 529765, ISIS 529785, ISIS 529799, ISIS 529818, ISIS 529823, ISIS 534528, ISIS 534594, and ISIS 534664. One group of male CD-1 mice was injected subcutaneously twice a week for 6 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.
Plasma Chemistry Markers
• To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, albumin, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.).
• ISIS oligonucleotides that did not cause any increase in the levels of transaminases, or which caused an increase within three times the upper limit of normal (ULN) were deemed very tolerable. ISIS oligonucleotides that caused an increase in the levels of transaminases between three times and seven times the ULN were deemed tolerable. Based on these criteria, ISIS 473295, ISIS 473714, ISIS 515558, ISIS 515926, 515951, ISIS 515952, ISIS 529126, ISIS 529166, 529564, ISIS 529582, ISIS 529614, ISIS 529725, ISIS 529765, ISIS 529799, ISIS 529823, and ISIS 534594 were considered very tolerable in terms of liver function. Based on these criteria, ISIS 515424, ISIS 515534, ISIS 515926, ISIS 529785, and ISIS 534664 were considered tolerable in terms of liver function.
Example 27: Tolerability of Chimeric Antisense Oligonucleotides Comprising 2′-O-Methoxyethyl (2′-MOE) Modifications Targeting Human Target-X in CD-1 Mice
• CD-1 mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.
Treatment
• Groups of 3 male CD-1 mice each were injected subcutaneously twice a week for 6 weeks with 100 mg/kg of ISIS 490208, ISIS 490279, ISIS 490323, ISIS 490368, ISIS 490396, ISIS 490803, ISIS 491122, ISIS 513419, ISIS 513446, ISIS 513454, ISIS 513455, ISIS 513456, ISIS 513504, ISIS 513507, and ISIS 513508. Groups of 3 male CD-1 mice each were injected subcutaneously twice a week for 6 weeks with 100 mg/kg of ISIS 407936, ISIS 416507, and ISIS 416508, which are gapmers described in a previous publication. One group of male CD-1 mice was injected subcutaneously twice a week for 6 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.
Plasma Chemistry Markers
• To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.).
• ISIS oligonucleotides that did not cause any increase in the levels of transaminases, or which caused an increase within three times the upper limit of normal (ULN) were deemed very tolerable. ISIS oligonucleotides that caused an increase in the levels of transaminases between three times and seven times the ULN were deemed tolerable. Based on these criteria, ISIS 407936, ISIS 416507, ISIS 490279, ISIS 490368, ISIS 490396, ISIS 490803, ISIS 491122, ISIS 513446, ISIS 513454, ISIS 513456, and ISIS 513504 were considered very tolerable in terms of liver function. Based on these criteria, ISIS 490208, ISIS 513455, ISIS 513507, and ISIS 513508 were considered tolerable in terms of liver function.
Example 28: Efficacy of Modified Oligonucleotides Comprising 2′-O-Methoxyethyl (2′-MOE) and Constrained Ethyl (cEt) Modifications Targeting Human Target-X in Transgenic Mice
• Transgenic mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for efficacy.
Treatment
• Groups of 2-3 male and female transgenic mice were injected subcutaneously twice a week for 3 weeks with 5 mg/kg/week of ISIS 473244, ISIS 473295, ISIS 484714, ISIS 515926, ISIS 515951, ISIS 515952, ISIS 516062, ISIS 529126, ISIS 529553, ISIS 529745, ISIS 529799, ISIS 534664, ISIS 534826, ISIS 540168, ISIS 540175, ISIS 544826, ISIS 544827, ISIS 544828, and ISIS 544829. One group of mice was injected subcutaneously twice a week for 3 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.
Protein Analysis
• Plasma protein levels of Target-X were estimated using a Target-X ELISA kit (purchased from Hyphen Bio-Med). Results are presented as percent inhibition of Target-X, relative to control. As shown in Table 35, several antisense oligonucleotides achieved reduction of human Target-X over the PBS control. ‘n.d.’ indicates that the value for that particular oligonucleotide was not measured.

• TABLE 35
  Percent inhibition of Target-X plasma
  protein levels in transgenic mice

  	ISIS No	% inhibition

  	473244	2
  	473295	13
  	484714	19
  	515926	11
  	515951	13
  	515952	0
  	516062	62
  	529126	0
  	529553	0
  	529745	22
  	529799	26
  	534664	32
  	534826	n.d.
  	540168	94
  	540175	98
  	544813	0
  	544826	23
  	544827	60
  	544828	33
  	544829	53

Example 29: Efficacy of Modified Oligonucleotides Comprising 2′-Methoxyethyl (2′-MOE) and Constrained Ethyl (cEt) Modifications Targeting Human Target-X in Transgenic Mice
• Transgenic mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for efficacy.
Treatment
• Groups of 2-3 male and female transgenic mice were injected subcutaneously twice a week for 3 weeks with 1 mg/kg/week of ISIS 407936, ISIS 490197, ISIS 490275, ISIS 490278, ISIS 490279, ISIS 490323, ISIS 490368, ISIS 490396, ISIS 490803, ISIS 491122, ISIS 513446, ISIS 513447, ISIS 513504, ISIS 516062, ISIS 529166, ISIS 529173, ISIS 529360, ISIS 529725, ISIS 534557, ISIS 534594, ISIS 534664, ISIS 534688, ISIS 534689, ISIS 534915, ISIS 534916, ISIS 534917, and ISIS 534980. One group of mice was injected subcutaneously twice a week for 3 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.
Protein Analysis
• Plasma protein levels of Target-X were estimated using a Target-X ELISA kit (purchased from Hyphen Bio-Med). Results are presented as percent inhibition of Target-X, relative to control. As shown in Table 36, several antisense oligonucleotides achieved reduction of human Target-X over the PBS control.

• TABLE 36
  Percent inhibition of Target-X plasm
  protein levels in transgenic mice

  	ISIS No	% inhibition

  	407936	28
  	490197	50
  	490275	21
  	490278	20
  	490279	59
  	490323	54
  	490368	22
  	490396	31
  	490803	30
  	491122	51
  	513446	29
  	513447	44
  	513504	45
  	516062	75
  	529166	37
  	529173	64
  	529360	43
  	529725	53
  	534557	76
  	534594	40
  	534664	14
  	534687	12
  	534688	48
  	534689	25
  	534915	40
  	534916	45
  	534917	66
  	534980	62

Example 30: Tolerability of Antisense Oligonucleotides Targeting Human Target-X in Sprague-Dawley Rats
• Sprague-Dawley rats were treated with ISIS antisense oligonucleotides from the studies described in the Examples above and evaluated for changes in the levels of various plasma chemistry markers.
Treatment
• Six-eight week old male Sprague-Dawley rats were maintained on a 12-hour light/dark cycle and fed ad libitum with Teklad normal rat chow. Groups of four Sprague-Dawley rats each were injected subcutaneously twice a week for 4 weeks with ISIS 515380, ISIS 515381, ISIS 515387, ISIS 529175, ISIS 529176, ISIS 529575, ISIS 529804, and ISIS 537064. Doses 1, 5, 6, 7, and 8 were 25 mg/kg; dose 2 was 75 mg/kg; doses 3 and 4 were 50 mg/kg. One group of four Sprague-Dawley rats was injected subcutaneously twice a week for 4 weeks with PBS. Forty eight hours after the last dose, rats were euthanized and organs and plasma were harvested for further analysis.
Liver Function
• To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma levels of transaminases ALT (alanine transaminase) and AST (aspartate transaminase) were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma levels of Bilirubin and BUN were also measured using the same clinical chemistry analyzer.
• ISIS oligonucleotides that did not cause any increase in the levels of transaminases, or which caused increase in the levels within three times the upper limit of normal levels of transaminases were deemed very tolerable. ISIS oligonucleotides that caused increase in the levels of transaminases between three times and seven times the upper limit of normal levels were deemed tolerable. Based on these criteria, ISIS 515380, ISIS 515387, ISIS 529175, ISIS 529176, ISIS 529804, and ISIS 537064 were considered very tolerable in terms of liver function. Based on these criteria, ISIS 515381 was considered tolerable in terms of liver function.
Example 31: Efficacy of Antisense Oligonucleotides Targeting Human Target-X in Transgenic Mice
• Transgenic mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for efficacy.
Treatment
• Two groups of 3 male and female transgenic mice were injected subcutaneously twice a week for 2 weeks with 0.5 mg/kg/week or 1.5 mg/kg/week of ISIS 407935 and ISIS 513455. Another group of mice was subcutaneously twice a week for 2 weeks with 0.6 mg/kg/week or 2.0 mg/kg/week of ISIS 473286. Another 16 groups of mice were subcutaneously twice a week for 2 weeks with 0.1 mg/kg/week or 0.3 mg/kg/week of ISIS 473589, ISIS 515380, ISIS 515423, ISIS 529804, ISIS 534676, ISIS 534796, ISIS 540162, ISIS 540164, ISIS 540175, ISIS 540179, ISIS 540181, ISIS 540182, ISIS 540186, ISIS 540191, ISIS 540193, ISIS 544827, or ISIS 545474. Another 3 groups of mice were injected subcutaneously twice a week for 2 weeks with 0.3 mg/kg/week of ISIS 516062, ISIS 534528 or ISIS 534693. One group of mice was injected subcutaneously twice a week for 2 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.
Protein Analysis
• Plasma protein levels of Target-X were estimated using a Target-X ELISA kit (purchased from Hyphen Bio-Med). Results are presented as percent inhibition of Target-X, relative to control. As shown in Table 37, several antisense oligonucleotides achieved reduction of human Target-X over the PBS control.

• TABLE 37
  Percent inhibition of Target-X plasma
  protein levels in transgenic mice

  ISIS No	Dose (mg/kg/wk)	% inhibition

  407935	1.5	65
  	0.5	31
  513455	1.5	64
  	0.5	52
  473286	2	67
  	0.6	11
  473589	0.3	42
  	0.1	12
  515380	0.3	64
  	0.1	32
  515423	0.3	72
  	0.1	37
  529804	0.3	36
  	0.1	24
  534676	0.3	31
  	0.1	18
  534796	0.3	54
  	0.1	43
  540162	0.3	84
  	0.1	42
  540164	0.3	25
  	0.1	17
  540175	0.3	90
  	0.1	55
  540179	0.3	29
  	0.1	24
  540181	0.3	53
  	0.1	0
  540182	0.3	78
  	0.1	21
  540186	0.3	72
  	0.1	46
  540191	0.3	62
  	0.1	35
  540193	0.3	74
  	0.1	46
  544827	0.3	28
  	0.1	19
  545474	0.3	59
  	0.1	0
  516062	0.3	33
  534528	0.3	41
  534693	0.3	34

Example 32: Tolerability of Antisense Oligonucleotides Targeting Human Target-X in Sprague-Dawley Rats
• Sprague-Dawley rats were treated with ISIS antisense oligonucleotides from the studies described in the Examples above and evaluated for changes in the levels of various plasma chemistry markers.
Treatment
• Five-six week old male Sprague-Dawley rats were maintained on a 12-hour light/dark cycle and fed ad libitum with Teklad normal rat chow. Groups of four Sprague-Dawley rats each were injected subcutaneously twice a week for 4 weeks with 50 mg/kg of ISIS 515423, ISIS 515424, ISIS 515640, ISIS 534676, ISIS 534796, ISIS 534797, ISIS 540162, ISIS 540164, ISIS 540172, ISIS 540175, ISIS 540179, ISIS 540181, ISIS 540182, ISIS 540183, ISIS 540186, ISIS 540191, and ISIS 545474. A group of four Sprague-Dawley rats was injected subcutaneously twice a week for 4 weeks with PBS. Forty eight hours after the last dose, rats were euthanized and organs and plasma were harvested for further analysis.
Liver Function
• To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma levels of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma levels of ALT (alanine transaminase) and AST (aspartate transaminase) were measured. Plasma levels of Bilirubin and BUN were also measured using the same clinical chemistry analyzer.
• ISIS oligonucleotides that did not cause any increase in the levels of transaminases, or which caused an increase within three times the upper limit of normal (ULN) were deemed very tolerable. ISIS oligonucleotides that caused an increase in the levels of transaminases between three times and seven times the ULN were deemed tolerable. Based on these criteria, ISIS 540164, ISIS 540172, and ISIS 540175 were considered very tolerable in terms of liver function. Based on these criteria, ISIS 534676, ISIS 534796, ISIS 534797, ISIS 540162, and ISIS 540179 were considered tolerable in terms of liver function.
Example 33: Dose-Dependent Antisense Inhibition of Human Target-X in Hep3B Cells
• Antisense oligonucleotides selected from the studies described above were tested at various doses in Hep3B cells. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.05 μM, 0.15 μM, 0.44 μM, 1.33 μM, and 4.00 μM concentrations of antisense oligonucleotide, as specified in Table 38. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human Target-X primer probe set RTS2927 was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells.
• The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented in Table 38. As illustrated in Table 38, Target-X mRNA levels were reduced in a dose-dependent manner in several of the antisense oligonucleotide treated cells.

• TABLE 38
  Dose-dependent antisense inhibition of human
  Target-X in Hep3B cells using electroporation

  	ISIS	0.05	0.15	0.44	1.33	4.00	IC50
  	No	μM	μM	μM	μM	μM	(μM)

  	473286	0	1	13	12	15	>4.0
  	457851	23	32	57	80	93	0.3
  	473286	3	20	43	71	88	0.5
  	473286	15	26	24	28	36	>4.0
  	473286	6	3	10	26	29	>4.0
  	473327	14	28	35	67	90	0.5
  	473589	29	53	76	89	95	0.1
  	515380	44	72	85	93	95	<0.05
  	515423	43	64	87	95	98	<0.05
  	515424	38	55	85	92	97	0.1
  	515636	21	33	74	82	93	0.2
  	516046	29	23	29	48	78	0.9
  	516048	35	24	41	67	87	0.4
  	516052	18	6	48	63	80	0.6
  	516062	24	14	21	47	68	1.6
  	529166	16	47	75	87	94	0.2
  	529173	14	49	77	91	96	0.2
  	529175	30	69	88	93	96	0.1
  	529176	34	63	85	93	96	0.1
  	529360	35	53	74	91	93	0.1
  	529725	53	69	85	92	95	<0.05
  	529804	37	41	71	90	94	0.1
  	534528	50	68	78	93	97	<0.05
  	534557	48	78	90	94	95	<0.05
  	534594	39	47	76	87	94	0.1
  	534676	29	20	40	64	87	0.5
  	534687	41	37	56	80	93	0.2
  	534688	16	56	88	94	96	0.1
  	534689	21	59	82	94	95	0.1
  	534693	18	58	81	93	95	0.1
  	534795	19	43	68	90	94	0.2
  	534796	25	59	80	93	96	0.1
  	534890	31	55	77	90	96	0.1
  	534898	22	61	80	94	97	0.1
  	534915	19	26	51	77	94	0.3
  	534916	20	36	66	86	93	0.2
  	534917	34	53	82	89	94	0.1
  	540162	40	64	84	90	92	<0.05
  	540164	34	60	83	91	92	0.1
  	540168	51	79	90	92	94	<0.05
  	540172	40	66	80	88	92	<0.05
  	540175	30	61	80	88	91	0.1
  	540176	7	17	50	75	85	0.5
  	540179	11	22	25	16	19	>4.0
  	540181	19	46	72	86	91	0.2
  	540182	16	66	83	86	92	0.1
  	540183	39	74	87	92	93	<0.05
  	540186	31	69	85	91	94	0.1
  	540191	38	54	80	88	91	0.1
  	540193	57	67	84	94	97	<0.05
  	540194	30	45	62	77	91	0.2
  	544827	37	42	67	82	96	0.1
  	544829	26	41	42	71	93	0.3
  	545473	28	27	49	80	97	0.3
  	545474	23	27	55	84	96	0.3

Example 34: Tolerability of Antisense Oligonucleotides Targeting Human Target-X in CD-1 Mice
• CD-1 mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.
Treatment
• Two groups of 4 male 6-8 week old CD-1 mice each were injected subcutaneously twice a week for 6 weeks with 50 mg/kg of ISIS 407935 and ISIS 490279. Another seven groups of 4 male 6-8 week old CD-1 mice each were injected subcutaneously twice a week for 6 weeks with 25 mg/kg of ISIS 473589, ISIS 529804, ISIS 534796, ISIS 540162, ISIS 540175, ISIS 540182, and ISIS 540191. One group of male CD-1 mice was injected subcutaneously twice a week for 6 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.
Plasma Chemistry Markers
• To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, albumin, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in Table 39. Treatment with the newly designed antisense oligonucleotides were more tolerable compared to treatment with ISIS 407935 (disclosed in an earlier publication), which caused elevation of ALT levels greater than seven times the upper limit of normal (ULN).

• TABLE 39
  Effect of antisense oligonucleotide treatment on
  liver function in CD-1 mice

  		Dose			BUN
  		(mg/kg/	ALT	AST	(mg/	Bilirubin
  	Motif	wk)	(IU/L)	(IU/L)	dL)	(mg/dL)

  PBS	—	—	37	47	28	0.2
  407935	e5-d(10)-e5	100	373	217	24	0.2
  490279	kdkdk-d(9)-ee	100	96	82	24	0.2
  473589	e5-d(10)-e5	50	93	116	22	0.2
  529804	k-d(10)-kekee	50	54	74	27	0.2
  534796	ekk-d(10)-kke	50	60	63	27	0.2
  540162	eek-d(10)-kke	50	43	55	29	0.2
  540175	eek-d(10)-kke	50	113	78	24	0.3
  540182	eek-d(10)-kke	50	147	95	26	0.1
  540191	eek-d(10)-kke	50	79	88	28	0.2
  e = 2′-MOE, k = cEt, d = 2′-deoxynucleosideBody and organ weights

• Body weights, as well as liver, heart, lungs, spleen and kidney weights were measured at the end of the study, and are presented in Table 40. Several of the ISIS oligonucleotides did not cause any changes in organ weights outside the expected range and were therefore deemed tolerable in terms of organ weight

• TABLE 40
  Body and organ weights (grams) of CD-1 mice

  		Dose	Body
  	Motif	(mg/kg/wk)	weight	Liver	Spleen	Kidney
  PBS	—	—	42	2.2	0.12	0.64
  407935	e5-d(10)-e5	100	40	2.6	0.20	0.62
  490279	kdkdk-d(9)-ee	100	42	2.8	0.17	0.61
  473589	e5-d(10)-e5	50	41	2.5	0.16	0.67
  529804	k-d(10)-kekee	50	40	2.3	0.14	0.62
  534796	ekk-d(10)-kke	50	37	2.6	0.15	0.51
  540162	eek-d(10)-kke	50	42	2.4	0.15	0.60
  540175	eek-d(10)-kke	50	39	2.2	0.11	0.62
  540182	eek-d(10)-kke	50	41	2.6	0.16	0.61
  540191	eek-d(10)-kke	50	40	2.4	0.13	0.60
  e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside

Example 35: Tolerability of Antisense Oligonucleotides Targeting Human Target-X in Sprague-Dawley Rats
• Sprague-Dawley rats were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.
Treatment
• Two groups of 4 male 7-8 week old Sprague-Dawley rats each were injected subcutaneously twice a week for 6 weeks with 50 mg/kg of ISIS 407935 and ISIS 490279. Another seven groups of 4 male 6-8 week old Sprague-Dawley rats each were injected subcutaneously twice a week for 6 weeks with 25 mg/kg of ISIS 473589, ISIS 529804, ISIS 534796, ISIS 540162, ISIS 540175, ISIS 540182, and ISIS 540191. One group of male Sprague-Dawley rats was injected subcutaneously twice a week for 6 weeks with PBS. The rats were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.
Plasma Chemistry Markers
• To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, albumin, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in Table 41. Treatment with the all antisense oligonucleotides was tolerable in terms of plasma chemistry markers in this model.

• TABLE 41
  Effect of antisense oligonucleotide treatment on liver
  function in Sprague-Dawley rats

  		Dose	ALT	AST	BUN
  		(mg/	(IU/	(IU/	(mg/	Bilirubin
  	Motif	kg/k)	L)	L)	dL)	(mg/dL)

  PBS	—	—	71	83	19	0.2
  407935	e5-d(10)-e5	100	74	96	22	0.2
  490279	kdkdk-d(9)-ee	100	96	181	22	0.4
  473589	e5-d(10)-e5	50	57	73	21	0.2
  529804	k-d(10)-kekee	50	54	78	21	0.2
  534796	ekk-d(10)-kke	50	68	98	22	0.2
  540162	eek-d(10)-kke	50	96	82	21	0.1
  540175	eek-d(10)-kke	50	55	73	18	0.2
  540182	eek-d(10)-kke	50	45	87	21	0.2
  540191	eek-d(10)-kke	50	77	104	21	0.2
  e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside

Body and Organ Weights
• Body weights, as well as liver, heart, lungs, spleen and kidney weights were measured at the end of the study, and are presented in Table 42. Treatment with all the antisense oligonucleotides was tolerable in terms of body and organ weights in this model.

• TABLE 42
  Body and organ weights (grams) of Sprague-Dawley rats

  		Dose
  		(mg/	Body	Liv-		Kid-
  	Motif	kg/wk)	weight	er	Spleen	ney
  PBS	—	—	443	16	0.8	3.5
  ISIS 407935	e5-d(10)-e5	100	337	14	1.8	3.2
  ISIS 490279	kdkdk-d(9)-ee	100	365	18	2.2	2.9
  ISIS 473589	e5-d(10)-e5	50	432	18	1.3	3.3
  ISIS 529804	k-d(10)-kekee	50	429	18	2.2	3.4
  ISIS 534796	ekk-d(10)-kke	50	434	15	1.4	3.3
  ISIS 540162	eek-d(10)-kke	50	446	18	1.1	3.3
  ISIS 540175	eek-d(10)-kke	50	467	16	1.0	3.5
  ISIS 540182	eek-d(10)-kke	50	447	22	2.5	4.5
  ISIS 540191	eek-d(10)-kke	50	471	21	1.4	3.9
  e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside

Example 36: Dose-Dependent Antisense Inhibition of Human Target-X in Cynomolgos Monkey Primary Hepatocytes
• Antisense oligonucleotides selected from the studies described above were tested at various doses in cynomolgous monkey primary hepatocytes. Cells were plated at a density of 35,000 cells per well and transfected using electroporation with 0.009 μM, 0.03 μM, 0.08 μM, 0.25 μM, 0.74 μM, 2.22 μM, 6.67 μM, and 20.00 μM concentrations of antisense oligonucleotide, as specified in Table 43. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Target-X primer probe set RTS2927 was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells. As illustrated in Table 43, Target-X mRNA levels were reduced in a dose-dependent manner with some of the antisense oligonucleotides that are cross-reactive with the rhesus monkey genomic sequence designated herein as Target-X.

• TABLE 43
  Dose-dependent antisense inhibition of Target-X in
  cynomolgous monkey primary hepatocytes using
  electroporation

  ISIS	0.009	0.03	0.08	0.25	0.74	2.22	6.67	20.00
  No	μM	μM	μM	μM	μM	μM	μM	μM

  407935	10	18	15	29	56	73	82	88
  490279	19	12	13	0	6	18	27	22
  473589	5	10	19	42	64	76	88	92
  529804	10	3	23	25	57	80	86	91
  534796	0	28	23	49	71	81	87	90
  540162	9	14	9	6	13	13	11	31
  540175	0	4	12	9	10	16	12	22
  540182	0	7	0	6	36	12	10	0
  540191	6	7	0	0	0	0	21	42

Example 37: Dose-Dependent Antisense Inhibition of Human Target-X in Hep3B Cells
• Antisense oligonucleotides from the study described above were also tested at various doses in Hep3B cells. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.009 μM, 0.03 μM, 0.08 μM, 0.25 μM, 0.74 μM, 2.22 μM, 6.67 μM, and 20.00 μM concentrations of antisense oligonucleotide, as specified in Table 44. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Target-X primer probe set RTS2927 was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells. As illustrated in Table 44, Target-X mRNA levels were reduced in a dose-dependent manner with several of the antisense oligonucleotides.

• TABLE 44
  Dose-dependent antisense inhibition of
  Target-X in Hep3B cells using electroporation

  ISIS	0.009	0.03	0.08	0.25	0.74	2.22	6.67	20.00	IC50
  No	μM	μM	μM	μM	μM	μM	μM	μM	(μM)

  407935	3	9	11	35	64	83	87	93	4.5
  473244	20	33	50	69	77	89	7	14	0.9
  473589	0	14	23	44	74	88	90	94	2.7
  490279	0	5	7	15	25	61	76	78	11.6
  515533	0	12	21	36	63	78	88	94	3.6
  515952	0	12	27	57	76	89	93	94	2.2
  516066	6	0	12	26	52	70	81	86	6.0
  529459	0	4	24	40	61	78	88	94	3.5
  529553	9	7	17	40	58	74	87	93	4.6
  529804	0	3	34	64	83	89	93	95	2.0
  534796	8	18	43	67	82	89	95	96	1.4
  537806	6	11	5	20	37	69	79	86	7.1
  540162	18	33	63	75	87	91	91	92	0.7
  540175	10	25	55	76	86	89	89	93	1.0
  540182	13	36	61	75	84	88	90	93	0.7
  540191	3	12	28	61	79	80	88	94	2.2

Example 38: Efficacy of Antisense Oligonucleotides Targeting Human Target-X in Transgenic Mice
• Transgenic mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for efficacy.
Treatment
• Eight groups of 3 transgenic mice each were injected subcutaneously twice a week for 3 weeks with 20 mg/kg/week, 10 mg/kg/week, 5 mg/kg/week, or 2.5 mg/kg/week of ISIS 407935 or ISIS 490279. Another 24 groups of 3 transgenic mice each were subcutaneously twice a week for 3 weeks with 5 mg/kg/week, 2.5 mg/kg/week, 1.25 mg/kg/week, or 0.625 mg/kg/week of ISIS 473589, ISIS 529804, ISIS 534796, ISIS 540162, ISIS 540175, or ISIS 540191. One group of mice was injected subcutaneously twice a week for 3 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.
RNA Analysis
• RNA was extracted from plasma for real-time PCR analysis of Target-X, using primer probe set RTS2927. The mRNA levels were normalized using RIBOGREEN®. As shown in Table 45, several antisense oligonucleotides achieved reduction of human Target-X over the PBS control. Results are presented as percent inhibition of Target-X, relative to control. Treatment with newly designed 2′-MOE gapmer, ISIS 490279, caused greater reduction in human Target-X mRNA levels than treatment with ISIS 407935, the 2′-MOE gapmer from the earlier publication. Treatment with several of the newly designed oligonucleotides also caused greater reduction in human Target-X mRNA levels than treatment with ISIS 407935.

• TABLE 45
  Percent inhibition of Target-X mRNA in transgenic mice

  		Dose	%
  ISIS No	Motif	(mg/kg/wk)	inhibition

  407935	e5-d(10)-e5	20.0	85
  		10.0	57
  		5.0	45
  		2.5	28
  490279	kdkdk-d(9)-ee	20.0	88
  		10.0	70
  		5.0	51
  		2.5	33
  473589	e5-d(10)-e5	5.00	80
  		2.50	62
  		1.25	44
  		0.625	25
  529804	k-d(10)-kekee	5.00	55
  		2.50	41
  		1.25	0
  		0.625	1
  534796	ekk-d(10)-kke	5.00	56
  		2.50	41
  		1.25	5
  		0.625	0
  540162	eek-d(10)-kke	5.00	97
  		2.50	92
  		1.25	69
  		0.625	78
  540175	eek-d(10)-kke	5.00	95
  		2.50	85
  		1.25	65
  		0.625	55
  540182	eek-d(10)-kke	5.00	97
  		2.50	83
  		1.25	54
  		0.625	10
  540191	eek-d(10)-kke	5.00	91
  		2.50	74
  		1.25	58
  		0.625	34
  e = 2′-MOE,
  k = cEt,
  d = 2′-deoxynucleoside

Protein Analysis
• Plasma protein levels of Target-X were estimated using a Target-X ELISA kit (purchased from Hyphen Bio-Med). As shown in Table 46, several antisense oligonucleotides achieved reduction of human Target-X over the PBS control. Results are presented as percent inhibition of Target-X, relative to control.

• TABLE 46
  Percent inhibition of Target-X plasm
  protein levels in transgenic mice

  		Dose	%
  ISIS No	Motif	(mg/kg/wk)	inhibition

  407935	e5-d(10)-e5	20	65
  		10	47
  		5	0
  		2.5	3
  490279	kdkdk-d(9)-ee	20	91
  		10	75
  		5	31
  		2.5	23
  473589	e5-d(10)-e5	5	78
  		2.5	40
  		1.25	6
  		0.625	0
  529804	k-d(10)-kekee	5	50
  		2.5	36
  		1.25	0
  		0.625	8
  534796	ekk-d(10)-kke	5	45
  		2.5	26
  		1.25	0
  		0.625	8
  540162	eek-d(10)-kke	5	98
  		2.5	96
  		1.25	78
  		0.625	74
  540175	eek-d(10)-kke	5	93
  		2.5	83
  		1.25	49
  		0.625	24
  540182	eek-d(10)-kke	5	97
  		2.5	71
  		1.25	50
  		0.625	0
  540191	eek-d(10)-kke	5	97
  		2.5	74
  		1.25	46
  		0.625	25
  e = 2′-MOE,
  k = cEt,
  d = 2′-deoxynucleoside

Example 39: Effect of ISIS Antisense Oligonucleotides Targeting Human Target-X in Cynomolgus Monkeys
• Cynomolgus monkeys were treated with ISIS antisense oligonucleotides selected from studies described above, including ISIS 407935, ISIS 490279, ISIS 473589, ISIS 529804, ISIS 534796, ISIS 540162, ISIS 540175, ISIS 540182, and ISIS 540191. Antisense oligonucleotide efficacy was evaluated. ISIS 407935, from the earlier publication, was included in the study for comparison.
Treatment
• Prior to the study, the monkeys were kept in quarantine for at least a 30-day period, during which the animals were observed daily for general health. Standard panels of serum chemistry and hematology, examination of fecal samples for ova and parasites, and a tuberculosis test were conducted immediately after the animals' arrival to the quarantine area. The monkeys were 2-4 years old at the start of treatment and weighed between 2 and 4 kg. Ten groups of four randomly assigned male cynomolgus monkeys each were injected subcutaneously with ISIS oligonucleotide or PBS using a stainless steel dosing needle and syringe of appropriate size into one of 4 sites on the back of the monkeys; each site used in clock-wise rotation per dose administered. Nine groups of monkeys were dosed four times a week for the first week (days 1, 3, 5, and 7) as loading doses, and subsequently once a week for weeks 2-12, with 35 mg/kg of ISIS 407935, ISIS 490279, ISIS 473589, ISIS 529804, ISIS 534796, ISIS 540162, ISIS 540175, ISIS 540182, or ISIS 540191. A control group of cynomolgus monkeys was injected with PBS subcutaneously thrice four times a week for the first week (days 1, 3, 5, and 7), and subsequently once a week for weeks 2-12. The protocols described in the Example were approved by the Institutional Animal Care and Use Committee (IACUC).
Hepatic Target ReductionRNA Analysis
• On day 86, RNA was extracted from liver tissue for real-time PCR analysis of Target-X using primer probe set RTS2927. Results are presented as percent inhibition of Target-X mRNA, relative to PBS control, normalized to RIBOGREEN® or to the house keeping gene, GAPDH. As shown in Table 52, treatment with ISIS antisense oligonucleotides resulted in reduction of Target-X mRNA in comparison to the PBS control.

• TABLE 52
  Percent Inhibition of cynomolgous monkey Target-X mRNA in
  the cynomolgus monkey liver relative to the PBS control

  ISIS No	Motif	RTS2927/Ribogreen	RTS2927/GAPDH

  407935	e5-d(10)-e5	90	90
  490279	kdkdk-d(9)-ee	72	66
  473589	e5-d(10)-e5	96	96
  529804	k-d(10)-kekee	90	87
  534796	ekk-d(10)-kke	80	78
  540162	eek-d(10)-kke	66	58
  540175	eek-d(10)-kke	68	66
  540182	eek-d(10)-kke	0	0
  540191	eek-d(10)-kke	34	14
  e = 2′-MOE,
  k = cEt,
  d = 2′-deoxynucleoside

Protein Levels and Activity Analysis
• Plasma Target-X levels were measured prior to dosing, and on day 3, day 5, day 7, day 16, day 30, day 44, day 65, and day 86 of treatment. Target-X activity was measured using Target-X deficient plasma. Approximately 1.5 mL of blood was collected from all available study animals into tubes containing 3.2% sodium citrate. The samples were placed on ice immediately after collection. Collected blood samples were processed to platelet poor plasma and the tubes were centrifuged at 3,000 rpm for 10 min at 4° C. to obtain plasma.
• Protein levels of Target-X were measured by a Target-X elisa kit (purchased from Hyphen BioMed). The results are presented in Table 53.

• TABLE 53
  Plasma Target-X protein levels (% reduction compared to the baseline)
  in the cynomolgus monkey plasma

  	Day	Day	Day	Day	Day	Day	Day	Day
  ISIS No	3	5	7	16	30	44	65	86

  407935	21	62	69	82	84	85	84	90
  490279	0	29	35	30	38	45	51	58
  473589	12	67	85	97	98	98	98	98
  529804	19	65	76	87	88	89	90	90
  534796	1	46	54	64	64	67	66	70
  540162	0	24	26	37	45	49	49	50
  540175	0	28	36	38	47	52	55	55
  540182	0	17	8	0	0	0	5	0
  540191	0	12	4	0	0	4	9	10

Example 40: Inhibition of Chimeric Antisense Oligonucleotides Targeting Target-Y
• A series of modified oligonucleotides were designed based on the parent gapmer, ISIS XXXX01, wherein the central gap region contains ten 2′-deoxyribonucleosides. These modified oligonucleotides were designed by having the central gap region shortened to nine, eight or seven 2′-deoxynucleosides and by introducing 2′-O-methoxyethyl (MOE) modifications at one or both wing regions. The newly designed oligonucleotides (except for ISIS XXXX09) were evaluated for their effects in reducing Target-Y mRNA levels in vitro.
• The gapmers and their motifs are described in Table 52. The internucleoside linkages throughout each gapmer are phosphorothioate linkages (P═S). Nucleosides followed by a subscript “d” indicate 2′-deoxynucleosides. Nucleosides followed by a subscript “e” indicate 2′-O-methoxyethyl (MOE) nucleosides. Nucleosides followed by a subscript “k” indicate constrained ethyl (cEt) nucleosides. “N” indicates modified or naturally occurring nucleobases (A, T, C, G, U, or 5-methyl C).
• The newly designed gapmers were tested in vitro. Mouse primary hepatocytes were plated at a density of 20,000 cells per well and transfected using electroporation with 15 μM concentration of antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-Y mRNA levels were measured by quantitative real-time PCR. Mouse Target-Y primer probe set RTS2898 was used to measure mRNA levels. Target-Y mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. The results in Table 53 are presented as Target-Y mRNA expression relative to untreated control cells (% UTC).
• The parent gapmer, ISIS XXXX01 was included in the study as a bench mark oligonucleotide against which the activity of the newly designed gapmers targeting Target-Y could be compared.
• As illustrated, most of the newly designed gapmers showed similar activity as compared to ISIS 464917.

• TABLE 52
  Chimeric antisense oligonucleotides targeting Target-Y

  Gap

  Wing chemistry

  ISIS NO.	Sequence (5′ to 3′)	Motif	chemistry	5′	3′	SEQ ID NO
  XXXX01	NkNkNkNdNdNdNdNdNdNdNd	3-10-3	Full deoxy	kkk	kkk	19
  	NdNdNkNkNk
  XXXX02	NkNkNkNdNdNdNdNdNdNdNd	3-10-3	Full deoxy	kkk	eee	19
  	NdNdNeNeNe
  XXXX03	NeNkNkNdNdNdNdNdNdNdNd	3-10-3	Full deoxy	ekk	kke	19
  	NdNdNkNkNe
  XXXX04	NeNeNkNkNdNdNdNdNdNdNd	4-9-3	Full deoxy	eekk	kke	19
  	NdNdNkNkNe
  XXXX05	NeNeNeNkNkNdNdNdNdNdNd	5-8-3	Full deoxy	eeekk	kke	19
  	NdNdNkNkNe
  XXXX06	NeNkNkNdNdNdNdNdNdNdNd	3-9-4	Full deoxy	ekk	kkee	19
  	NdNkNkNeNe
  XXXX07	NeNkNkNdNdNdNdNdNdNdNd	3-8-5	Full deoxy	ekk	kkeee	19
  	NkNkNeNeNe
  XXXX08	NeNeNkNkNdNdNdNdNdNdNd	4-8-4	Full deoxy	eekk	kkee	19
  	NdNkNkNeNe
  XXXX09	NeNeNeNkNkNdNdNdNdNdNd	5-7-4	Full deoxy	eeekk	kkee	19
  	NdNkNkNeNe
  XXXX10	NeNeNkNkNdNdNdNdNdNdNd	4-7-5	Full deoxy	eekk	kkeee	19
  	NkNkNeNeNe
  e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside

• TABLE 53
  Inhibition of modified oligonucleotides targeting Target-Y

  % UTC

  Wing chemistry

  ISIS NO.	15 μM	Motif	Gap chemistry	5′	3′

  XXXX01	8.5	3-10-3	Full deoxy	kkk	kkk
  XXXX02	9.1	3-10-3	Full deoxy	kkk	eee
  XXXX03	8.3	3-10-3	Full deoxy	ekk	kke
  XXXX04	7.1	4-9-3	Full deoxy	eekk	kke
  XXXX05	8.6	5-8-3	Full deoxy	eeekk	kke
  XXXX06	7.4	3-9-4	Full deoxy	ekk	kkee
  XXXX07	8.5	3-8-5	Full deoxy	ekk	kkeee
  XXXX08	12.5	4-8-4	Full deoxy	eekk	kkee
  XXXX10	11.2	4-7-5	Full deoxy	eekk	kkeee
  e = 2′-MOE,
  k = cEt,
  d = 2′-deoxynucleoside

Example 41: Dose-Dependent Inhibition of Chimeric Antisense Oligonucleotides Targeting Target-Y
• Additional chimeric antisense oligonucleotides were designed based on the parent gapmer, ISIS XXXX11, wherein the central gap region contains ten 2′-deoxynucleosides. These modified oligonucleotides were designed in a similar manner as the chimeric antisense oligonucleotides described in Example 40 and were evaluated for their effect in reducing Target-Y mRNA levels in vitro.
• The gapmers and their motifs are described in Table 54. The internucleoside linkages throughout each gapmer are phosphorothioate linkages (P═S). Nucleosides followed by a subscript “d” indicate 2′-deoxynucleosides. Nucleosides followed by a subscript “e” indicate 2′-O-methoxyethyl (MOE) nucleosides. Nucleosides followed by a subscript “k” indicate constrained ethyl (cEt) nucleosides. “N” indicates modified or naturally occurring nucleobases (A, T, C, G, U, or 5-methyl C).
• The newly designed gapmers were tested in vitro. Mouse primary hepatocytes were plated at a density of 20,000 cells per well and transfected using electroporation with 0.6 μM, 3.0 μM and 15 μM concentrations of antisense oligonucleotides. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-Y mRNA levels were measured by quantitative real-time PCR. Mouse Target-Y primer probe set RTS2898 was used to measure mRNA levels. Target-Y mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. The results in Table 55 are presented as Target-Y mRNA expression relative to untreated control cells (% UTC).
• The parent gapmer, ISIS XXXX11 was included in the study as a bench mark oligonucleotide against which the activity of the newly designed gapmers targeting Target-Y could be compared.
• As illustrated in Table 55, several of the newly designed gapmers exhibited similar activity as compared to ISIS XXXX11. The data also confirms that Target-Y mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells.

• TABLE 54
  Chimeric antisense oligonucleotides targeting Target-Y

  Gap

  Wing chemistry

  ISIS NO.	Sequence (5′ to 3′)	Motif	chemistry	5'	3'	SEQ ID NO.
  XXXX11	NkNkNkNdNdNdNdNdNdNdNdNd	3-10-3	Full deoxy	kkk	kkk	19
  	NdNkNkNk
  XXXX12	NkNkNkNdNdNdNdNdNdNdNdNd	3-10-3	Full deoxy	kkk	eee	19
  	NdNeNeNe
  XXXX13	NeNkNkNdNdNdNdNdNdNdNdNd	3-10-3	Full deoxy	ekk	kke	19
  	NdNkNkNe
  XXXX14	NeNeNkNkNdNdNdNdNdNdNdNd	4-9-3	Full deoxy	eekk	kke	19
  	NdNkNkNe
  XXXX15	NeNeNeNkNkNdNdNdNdNdNdNd	5-8-3	Full deoxy	eeekk	kke	19
  	NdNkNkNe
  XXXX16	NeNkNkNdNdNdNdNdNdNdNdNd	3-9-4	Full deoxy	ekk	kkee	19
  	NkNkNeNe
  XXXX17	NeNkNkNdNdNdNdNdNdNdNdNk	3-8-5	Full deoxy	ekk	kkeee	19
  	NkNeNeNe
  XXXX18	NeNeNkNkNdNdNdNdNdNdNdNd	4-8-4	Full deoxy	eekk	kkee	19
  	NkNkNeNe
  XXXX19	NeNeNeNkNkNdNdNdNdNdNdNd	5-7-4	Full deoxy	eeekk	kkee	19
  	NkNkNeNe
  XXXX20	NeNeNkNkNdNdNdNdNdNdNdNk	4-7-5	Full deoxy	eekk	kkeee	19
  	NkNeNeNe
  e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside

• TABLE 55
  Dose-dependent inhibition of chimeric
  antisense oligonucleotides targeting Target-Y

  Wing

  % UTC

  Gap

  chemistry

  ISIS NO.	0.6 μM	3.0 μM	15 μM	Motif	chemistry	5′	3′

  XXXX11	19.4	14.1	12.5	3-10-3	Full deoxy	kkk	kkk
  XXXX12	23.4	12.5	9.9	3-10-3	Full deoxy	kkk	eee
  XXXX13	29.8	13.7	11.2	3-10-3	Full deoxy	ekk	kke
  XXXX14	28.3	15.5	11.6	4-9-3	Full deoxy	eekk	kke
  XXXX15	41.3	16.7	11.6	5-8-3	Full deoxy	eeekk	kke
  XXXX16	31.6	16.7	11.7	3-9-4	Full deoxy	ekk	kkee
  XXXX17	39.2	16.8	11.1	3-8-5	Full deoxy	ekk	kkeee
  XXXX18	40.5	18.2	13.6	4-8-4	Full deoxy	eekk	kkee
  XXXX19	118.4	123.8	13.3	5-7-4	Full deoxy	eeekk	kkee
  XXXX20	52.3	27.6	12.4	4-7-5	Full deoxy	eekk	kkeee

  Saline = 100

  e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside

Example 42: Dose-Dependent Inhibition of Chimeric Antisense Oligonucleotides Targeting Target-Y
• Additional chimeric oligonucleotides were designed based on the parent gapmer, ISIS XXXX01, wherein the central gap region contains ten 2′-deoxynucleosides. These modified oligonucleotides were designed by having the central gap region shortened to eight 2′-deoxynucleosides and by introducing one or more 2′-O-methoxyethyl (MOE) modification(s) at the 3′ wing region. The modified oligonucleotides designed by microwalk were evaluated for their effects in reducing Target-Y mRNA levels in vitro.
• The gapmers and their motifs are described in Table 56. The internucleoside linkages throughout each gapmer are phosphorothioate linkages (P═S). Nucleosides followed by a subscript “d” indicate 2′-deoxynucleoside. Nucleosides followed by a subscript “e” indicate 2′-O-methoxyethyl (MOE) nucleosides. Nucleosides followed by a subscript “k” indicate constrained ethyl (cEt) nucleosides. “N” indicates modified or naturally occurring nucleobases (A, T, C, G, U, or 5-methyl C).
• The newly designed gapmers were tested in vitro. Mouse primary hepatocytes were plated at a density of 20,000 cells per well and transfected using electroporation with 0.6 μM, 3.0 μM and 15 μM concentrations of antisense oligonucleotides. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-Y mRNA levels were measured by quantitative real-time PCR. Mouse Target-Y primer probe set RTS2898 was used to measure mRNA levels. Target-Y mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. The results in Table 57 are presented as Target-Y mRNA expression relative to untreated control cells (% UTC).
• The parent gapmer, ISIS XXXX01 was included in the study as a bench mark oligonucleotide against which the activity of the newly designed gapmers targeting Target-Y could be compared.
• As illustrated in Table 57, most of the newly designed gapmers demonstrated improvement in activity at low concentrations (0.6 μM and 3.0 μM) as compared to ISIS XXXX01. The data also confirms that Target-Y mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells.

• TABLE 56
  Chimeric antisense oligonucleotides designed by microwalk targeting Target-Y

  Gap

  Wing chemistry

  ISIS NO.	Sequence (5′ to 3′)	Motif	chemistry	5′	3′	SEQ ID NO.
  XXXX01	NkNkNkNdNdNdNdNdNdNdNdNd	3-10-3	Full deoxy	kkk	kkk	19
  	NdNkNkNk
  XXXX21	NkNkNkNdNdNdNdNdNdNdNdNd	3-10-3	Full deoxy	kkk	eee	19
  	NdNeNeNe
  XXXX22	NkNkNkNdNdNdNdNdNdNdNdNk	3-8-5	Full deoxy	kkk	keeee	19
  	NeNeNeNe
  XXXX23	NkNkNkNdNdNdNdNdNdNdNdNk	3-8-5	Full deoxy	kkk	keeee	19
  	NeNeNeNe
  XXXX24	NkNkNkNdNdNdNdNdNdNdNdNk	3-8-5	Full deoxy	kkk	keeee	19
  	NeNeNeNe
  XXXX25	NkNkNkNdNdNdNdNdNdNdNdNk	3-8-5	Full deoxy	kkk	keeee	19
  	NeNeNeNe
  XXXX26	NkNkNkNdNdNdNdNdNdNdNdNk	3-8-5	Full deoxy	kkk	keeee	19
  	NeNeNeNe
  XXXX27	NkNkNkNdNdNdNdNdNdNdNdNk	3-8-5	Full deoxy	kkk	keeee	19
  	NeNeNeNe
  XXXX28	NkNkNkNdNdNdNdNdNdNdNdNk	3-8-5	Full deoxy	kkk	keeee	19
  	NeNeNeNe
  XXXX29	NkNkNkNdNdNdNdNdNdNdNdNk	3-8-5	Full deoxy	kkk	keeee	19
  	NeNeNeNe
  XXXX30	NkNkNkNdNdNdNdNdNdNdNdNk	3-8-5	Full deoxy	kkk	keeee	19
  	NeNeNeNe
  e = 2′-MOE k = cEt, d = 2′-deoxynucleoside

• TABLE 57
  Dose-dependent inhibition of chimeric antisense
  oligonucleotides designed by microwalk targeting Target -Y

  				Wing
  	% UTC		Gap	chemistry

  ISIS NO.	0.6 μM	3.0 μM	15 μM	Motif	chemistry	5′	3′

  XXXX01	83.9	94.3	8.5	3-10-3	Full deoxy	kkk	kkk
  XXXX21	39.8	21.2	9.1	3-10-3	Full deoxy	kkk	eee
  XXXX22	52.5	35.1	13.0	3-8-5	Full deoxy	kkk	keeee
  XXXX23	60.7	40.9	13.6	3-8-5	Full deoxy	kkk	keeee
  XXXX24	52.3	23.8	7.3	3-8-5	Full deoxy	kkk	keeee
  XXXX25	58.9	32.1	9.3	3-8-5	Full deoxy	kkk	keeee
  XXXX26	41.7	21.1	8.8	3-8-5	Full deoxy	kkk	keeee
  XXXX27	45.6	25.2	8.5	3-8-5	Full deoxy	kkk	keeee
  XXXX28	39.1	20.1	9.2	3-8-5	Full deoxy	kkk	keeee
  XXXX29	61.4	28.4	9.9	3-8-5	Full deoxy	kkk	keeee
  XXXX30	81.3	52.2	16.2	3-8-5	Full deoxy	kkk	keeee

  Saline = 100

  e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside

Example 43: Dose-Dependent Inhibition of Chimeric Antisense Oligonucleotides Targeting Target-Y
• Additional chimeric oligonucleotides were designed based on the parent gapmer, ISIS XXXX11, wherein the central gap region contains ten 2′-deoxynucleosides. These modified oligonucleotides were designed by in the same manner as the oligonucleotides described in Example 42. The modified oligonucleotides designed by microwalk were evaluated for their effects in reducing Target-Y mRNA levels in vitro.
• The gapmers and their motifs are described in Table 58. The internucleoside linkages throughout each gapmer are phosphorothioate linkages (P═S). Nucleosides followed by a subscript “d” indicate 2′-deoxynucleoside. Nucleosides followed by a subscript “e” indicate 2′-O-methoxyethyl (MOE) nucleosides. Nucleosides followed by a subscript “k” indicate constrained ethyl (cEt) nucleosides. “N” indicates modified or naturally occurring nucleobases (A, T, C, G, U, or 5-methyl C).
• The newly designed gapmers were tested in vitro. Mouse primary hepatocytes were plated at a density of 20,000 cells per well and transfected using electroporation with 0.6 μM, 3.0 μM and 15 μM concentrations of antisense oligonucleotides. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-Y mRNA levels were measured by quantitative real-time PCR. Mouse Target-Y primer probe set RTS2898 was used to measure mRNA levels. Target-Y mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. The results in Table 59 are presented as Target-Y mRNA expression relative to untreated control cells (% UTC).
• The parent gapmer, ISIS XXXX11 was included in the study as a bench mark oligonucleotide against which the activity of the newly designed gapmers targeting Target-Y could be compared.

• TABLE 58
  Chimeric antisense oligonucleotides designed by microwalk targeting Target-Y

  Gap

  Wing chemistry

  ISIS NO.	Sequence (5′ to 3′)	Motif	chemistry	5′	3′	SEQ ID NO
  XXXX11	NkNkNkNdNdNdNdNdNdNdNdNd	3-10-3	Full deoxy	kkk	kkk	19
  	NdNkNkNk
  XXXX31	NkNkNkNdNdNdNdNdNdNdNdNk	3-8-5	Full deoxy	kkk	keeee	19
  	NeNeNeNe
  XXXX32	NkNkNkNdNdNdNdNdNdNdNdNk	3-8-5	Full deoxy	kkk	keeee	19
  	NeNeNeNe
  XXXX33	NkNkNkNdNdNdNdNdNdNdNdNk	3-8-5	Full deoxy	kkk	keeee	19
  	NeNeNeNe
  XXXX34	NkNkNkNdNdNdNdNdNdNdNdNk	3-8-5	Full deoxy	kkk	keeee	19
  	NeNeNeNe
  XXXX35	NkNkNkNdNdNdNdNdNdNdNdNk	3-8-5	Full deoxy	kkk	keeee	19
  	NeNeNeNe
  XXXX36	NkNkNkNdNdNdNdNdNdNdNdNk	3-8-5	Full deoxy	kkk	keeee	19
  	NeNeNeNe
  XXXX37	NkNkNkNdNdNdNdNdNdNdNdNk	3-8-5	Full deoxy	kkk	keeee	19
  	NeNeNeNe
  XXXX38	NkNkNkNdNdNdNdNdNdNdNdNk	3-8-5	Full deoxy	kkk	keeee	19
  	NeNeNeNe
  XXXX39	NkNkNkNdNdNdNdNdNdNdNdNk	3-8-5	Full deoxy	kkk	keeee	19
  	NeNeNeNe
  e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside

• TABLE 59
  Dose-dependent inhibition of chimeric antisense
  oligonucleotides designed by microwalk targeting Target -Y

  Wing

  % UTC

  Gap

  chemistry

  ISIS NO.	0.6 μM	3.0 μM	15 μM	Motif	chemistry	5′	3′
  XXXX11	19.4	14.1	12.5	3-10-3	Full deoxy	kkk	kkk
  XXXX31	50.5	23.0	14.2	3-8-5	Full deoxy	kkk	keeee
  XXXX32	50.2	19.4	8.7	3-8-5	Full deoxy	kkk	keeee
  XXXX33	55.2	19.3	11.9	3-8-5	Full deoxy	kkk	keeee
  XXXX34	53.3	15.3	11.9	3-8-5	Full deoxy	kkk	keeee
  XXXX35	35.5	18.7	11.1	3-8-5	Full deoxy	kkk	keeee
  XXXX36	39.7	22.3	16.8	3-8-5	Full deoxy	kkk	keeee
  XXXX37	24.1	16.7	9.5	3-8-5	Full deoxy	kkk	keeee
  XXXX38	26.3	13.8	10.9	3-8-5	Full deoxy	kkk	keeee
  XXXX39	36.9	16.4	10.4	3-8-5	Full deoxy	kkk	keeee

  Saline = 100

  e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside

Example 44: Dose-Dependent Inhibition of Chimeric Antisense Oligonucleotides Targeting PTEN
• A series of modified oligonucleotides were designed based on the parent gapmer, ISIS 482050, wherein the central gap region contains ten 2′-deoxynucleosides. These modified oligonucleotides were designed by having the central gap region shortened to nine, eight or seven 2′-deoxynucleosides and by introducing 2′-O-methoxyethyl (MOE) modifications at one or both wing regions. The newly designed oligonucleotides were evaluated for their effecst in reducing PTEN mRNA levels in vitro.
• The gapmers and their motifs are described in Table 60. The internucleoside linkages throughout each gapmer are phosphorothioate linkages (P═S). Nucleosides followed by a subscript “d” indicate 2′-deoxynucleosides. Nucleosides followed by a subscript “e” indicate 2′-O-methoxyethyl (MOE) nucleosides. Nucleosides followed by a subscript “k” indicate constrained ethyl (cEt) nucleosides. “N” indicates modified or naturally occurring nucleobases (A, T, C, G, U, or 5-methyl C).
• The newly designed gapmers were tested in vitro. Mouse primary hepatocytes were plated at a density of 20,000 cells per well and transfected using electroporation with 0.6 μM, 3.0 μM and 15 μM concentrations of antisense oligonucleotides. After a treatment period of approximately 24 hours, RNA was isolated from the cells and PTEN mRNA levels were measured by quantitative real-time PCR. Mouse PTEN primer probe set RTS186 was used to measure mRNA levels. PTEN mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. The results in Table 61 are presented as PTEN mRNA expression relative to untreated control cells (% UTC).
• The parent gapmer, ISIS 482050 was included in the study as a bench mark oligonucleotide against which the activity of the newly designed gapmers targeting PTEN could be compared.

• TABLE 60
  Chimeric antisense oligonucleotides targeting PTEN

  Gap

  Wing chemistry

  ISIS NO.	Sequence (5′ to 3′)	Motif	chemistry	5′	3′	SEQ ID NO
  482050	AkTkmCkAdTdGdGdmCdTdGdmCd	3-10-3	Full deoxy	kkk	kkk	23
  	AdGdmCkTkTk
  508033	AkTkmCkAdTdGdGdmCdTdGdmCd	3-10-3	Full deoxy	kkk	eee	23
  	AdGdmCeTeTe
  573351	AeTkmCkAdTdGdGdmCdTdGdmCd	3-10-3	Full deoxy	ekk	kke	23
  	AdGdmCkTkTe
  573352	AeTemCkAkTdGdGdmCdTdGdmCd	4-9-3	Full deoxy	eekk	kke	23
  	AdGdmCkTkTe
  573353	AeTemCeAkTkGdGdmCdTdGdmCd	5-8-3	Full deoxy	eeekk	kke	23
  	AdGdmCkTkTe
  573355	AeTkmCkAdTdGdGdmCdTdGdmCd	3-9-4	Full deoxy	ekk	kkee	23
  	AdGkmCkTeTe
  573356	AeTkmCkAdTdGdGdmCdTdGdmCd	3-8-5	Full deoxy	ekk	kkeee	23
  	AkGkmCeTeTe
  573357	AkTkmCkAdTdGdGdmCdTdGdmCk	3-7-6	Full deoxy	ekk	kkeeee	23
  	AkGemCeTeTe
  573358	AeTemCeAkTdGdGdmCdTdGdmCd	4-8-4	Full deoxy	eekk	kkee	23
  	AdGkmCkTeTe
  573359	AeTemCeAkTkGdGdmCdTdGdmCd	5-7-4	Full deoxy	eeekk	kkee	23
  	AdGkmCkTeTe
  573360	AeTemCkAkTdGdGdmCdTdGdmCd	4-7-5	Full deoxy	eekk	kkeee	23
  	AkGkmCeTeTe
  e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside

• TABLE 61
  Dose-response effect of chimeric
  antisense oligonucleotides targeting PTEN

  Wing

  ISIS

  % UTC

  Gap

  chemistry

  NO.	0.6 μM	3.0 μM	15 μM	Motif	chemistry	5′	3′
  482050	45.4	23.8	8.4	3-10-3	Full deoxy	kkk	kkk
  508033	52.2	28.8	7.6	3-10-3	Full deoxy	kkk	eee
  573351	66.0	24.0	12.4	3-10-3	Full deoxy	ekk	kke
  573352	69.0	38.1	12.5	4-9-3	Full deoxy	eekk	kke
  573353	59.8	36.5	13.8	5-8-3	Full deoxy	eeekk	kke
  573355	52.1	37.4	11.4	3-9-4	Full deoxy	ekk	kkee
  573356	52.9	46.4	15.4	3-8-5	Full deoxy	ekk	kkeee
  573357	82.4	81.8	52.5	3-7-6	Full deoxy	ekk	kkeeee
  573358	67.4	46.7	14.5	4-8-4	Full deoxy	eekk	kkee
  573359	70.5	49.8	31.6	5-7-4	Full deoxy	eeekk	kkee
  573360	62.2	50.8	17.6	4-7-5	Full deoxy	eekk	kkeee

  Saline = 100

  e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside

Example 45: Dose-Dependent Inhibition of Chimeric Antisense Oligonucleotides Targeting PTEN
• Additional chimeric oligonucleotides were designed based on the parent gapmer, ISIS 482050, wherein the central gap region contains ten 2′-deoxynucleosides. These modified oligonucleotides were designed by having the central gap region shortened to eight 2′-deoxynucleosides and by introducing one or more 2′-O-methoxyethyl (MOE) modification(s) at the 3′ wing region. The modified oligonucleotides designed by microwalk were evaluated for their effects in reducing PTEN mRNA levels in vitro.
• The gapmers and their motifs are described in Table 62. The internucleoside linkages throughout each gapmer are phosphorothioate linkages (P═S). Nucleosides followed by a subscript “d” indicate 2′-deoxynucleoside. Nucleosides followed by a subscript “e” indicate 2′-O-methoxyethyl (MOE) nucleosides. Nucleosides followed by a subscript “k” indicate constrained ethyl (cEt) nucleosides.^mC indicates a 5-methyl nucleoside.
• The newly designed gapmers were tested in vitro. Mouse primary hepatocytes were plated at a density of 20,000 cells per well and transfected using electroporation with 0.6 μM, 3.0 μM and 15 μM concentrations of antisense oligonucleotides. After a treatment period of approximately 24 hours, RNA was isolated from the cells and PTEN mRNA levels were measured by quantitative real-time PCR. Mouse PTEN primer probe set RTS186 was used to measure mRNA levels. PTEN mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. The results in Table 63 are presented as PTEN mRNA expression relative to untreated control cells (% UTC).
• The parent gapmer, ISIS 482050 was included in the study as a bench mark oligonucleotide against which the activity of the newly designed gapmers targeting PTEN could be compared.

• TABLE 62
  Chimeric antisense oligonucleotides designed by microwalk targeting PTEN

  Gap

  Wing chemistry

  ISIS NO.	Sequence (5′ to 3′)	Motif	chemistry	5′	3′	SEQ ID NO.
  482050	AkTkmCkAdTdGdGdmCdTdGdmCd	3-10-3	Full deoxy	kkk	kkk	24
  	AdGdmCkTkTk
  573797	TkGkGkmCdTdGdmCdAdGdmCdTd	3-8-5	Full deoxy	kkk	keeee	25
  	TkmCemCeGeAe
  573798	AkTkGkGdmCdTdGdmCdAdGdmCd	3-8-5	Full deoxy	kkk	keeee	26
  	TkTemCemCeGe
  573799	mCkAkTkGdGdmCdTdGdmCdAdGd	3-8-5	Full deoxy	kkk	keeee	27
  	mCkTeTemCemCe
  573800	TkmCkAkTdGdGdmCdTdGdmCdAd	3-8-5	Full deoxy	kkk	keeee	28
  	GkmCeTeTemCe
  573801	AkTkmCkAdTdGdGdmCdTdGdmCd	3-8-5	Full deoxy	kkk	keeee	24
  	AkGemCeTeTe
  573802	mCkAkTkmCdAdTdGdGdmCdTdGd	3-8-5	Full deoxy	kkk	keeee	29
  	mCkAeGemCeTe
  573803	mCkmCkAkTdmCdAdTdGdGdmCd	3-8-5	Full deoxy	kkk	keeee	30
  	TdGkmCeAeGemCe
  573804	TkmCkmCkAdTdmCdAdTdGdGd	3-8-5	Full deoxy	kkk	keeee	31
  	mCdTkGemCeAeGe
  573805	TkTkmCkmCdAdTdmCdAdTdGdGd	3-8-5	Full deoxy	kkk	keeee	32
  	mCkTeGemCeAe
  e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside

• TABLE 63
  Dose-dependent inhibition of chimeric antisense
  oligonucleotides designed by microwalk targeting PTEN

  						Wing
  	% UTC				Gap	chemistry

  ISIS NO.	0.6 μM	3.0 μM	15 μM	Motif	chemistry	5′	3′
  482050	45.4	23.8	8.4	3-10-3	Full deoxy	kkk	kkk
  573797	56.8	55.4	13.1	3-8-5	Full deoxy	kkk	keeee
  573798	50.9	33.5	9.6	3-8-5	Full deoxy	kkk	keeee
  573799	62.6	27.7	10.3	3-8-5	Full deoxy	kkk	keeee
  573800	68.6	38.9	12.0	3-8-5	Full deoxy	kkk	keeee
  573801	54.6	46.3	11.8	3-8-5	Full deoxy	kkk	keeee
  573802	60.7	40.4	13.0	3-8-5	Full deoxy	kkk	keeee
  573803	47.0	29.8	8.5	3-8-5	Full deoxy	kkk	keeee
  573804	62.5	34.1	11.3	3-8-5	Full deoxy	kkk	keeee
  573805	70.3	31.6	15.2	3-8-5	Full deoxy	kkk	keeee

  Saline = 100

  e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside

Example 46: Antisense Inhibition of Target-Z mRNA in HepG2 Cells
• Antisense oligonucleotides were designed targeting a Target-Z nucleic acid and were tested for their effects on Target-Z mRNA in vitro. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. ISIS 146786, 509934, ISIS 509959, and ISIS 510100, were also included in these studies for comparison. Cultured HepG2 cells at a density of 28,000 cells per well were transfected using LipofectAMINE2000® with 70 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-Z mRNA levels were measured by quantitative real-time PCR. Viral primer probe set RTS3370 (forward sequence CTTGGTCATGGGCCATCAG, designated herein as SEQ ID NO: 33; reverse sequence CGGCTAGGAGTTCCGCAGTA, designated herein as SEQ ID NO: 34; probe sequence TGCGTGGAACCTTTTCGGCTCC, designated herein as SEQ ID NO: 35) was used to measure mRNA levels. Levels were also measured using primer probe set RTS3371 (forward sequence CCAAACCTTCGGACGGAAA, designated herein as SEQ ID NO: 36; reverse sequence TGAGGCCCACTCCCATAGG, designated herein as SEQ ID NO: 37; probe sequence CCCATCATCCTGGGCTTTCGGAAAAT, designated herein as SEQ ID NO: 38). Target-Z mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-Z, relative to untreated control cells.
• The newly designed chimeric antisense oligonucleotides and their motifs are described in Tables 64-69. The gapmers are 16 nucleotides in length, wherein the central gap region comprises ten 2′-deoxynucleosides. Nucleosides followed by ‘k’ indicate constrained ethyl (cEt) nucleosides. Nucleosides followed by “e” indicate 2′-O-methoxyethyl (2′-MOE) nucleosides. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines.
• Each gapmer listed in Tables 64-69 is targeted to the viral genomic sequence, designated herein as Target-Z. The activity of the newly designed oligonucleotides was compared with ISIS 146786, ISIS 509934, ISIS 509959, and ISIS 510100.

• TABLE 64
  Inhibition of viral Target-Z mRNA levels by chimeric
  antisense oligonucleotidesmeasured with RTS3370

  ISIS No	Motif	% inhibition

  509934	eeeee-d(10)-eeeee	30
  552787	ekk-d(10)-kke	57
  552788	ekk-d(10)-kke	60
  552789	ekk-d(10)-kke	67
  552790	ekk-d(10)-kke	67
  552791	ekk-d(10)-kke	65
  552792	ekk-d(10)-kke	44
  552793	ekkd(10)kke	0
  552794	ekk-d(10)-kke	54
  552795	ekk-d(10)-kke	55
  552796	ekk-d(10)-kke	62
  552797	ekk-d(10)-kke	59
  552798	ekk-d(10)-kke	59
  552799	ekk-d(10)-kke	58
  552800	ekk-d(10)-kke	62
  552801	ekk-d(10)-kke	65
  552802	ekk-d(10)-kke	53
  552803	ekk-d(10)-kke	67
  552804	ekk-d(10)-kke	75
  552805	ekk-d(10)-kke	72
  552806	ekk-d(10)-kke	64
  552807	ekk-d(10)-kke	68
  552808	ekk-d(10)-kke	65
  552809	ekk-d(10)-kke	60
  552810	ekk-d(10)-kke	59
  552811	ekk-d(10)-kke	64
  552812	ekk-d(10)-kke	69
  552813	ekk-d(10)-kke	64
  552814	ekk-d(10)-kke	62
  552815	ekk-d(10)-kke	61
  552816	ekk-d(10)-kke	63
  552817	ekk-d(10)-kke	42
  552818	ekk-d(10)-kke	44
  552819	ekk-d(10)-kke	56
  552820	ekk-d(10)-kke	59
  552821	ekk-d(10)-kke	76
  552822	ekk-d(10)-kke	77
  552823	ekk-d(10)-kke	73
  552824	ekk-d(10)-kke	73
  552825	ekk-d(10)-kke	51
  552826	ekk-d(10)-kke	55
  552827	ekk-d(10)-kke	67
  552828	ekk-d(10)-kke	78
  552829	ekk-d(10)-kke	72
  552830	ekk-d(10)-kke	71
  552831	ekk-d(10)-kke	69
  552832	ekk-d(10)-kke	67
  552833	ekk-d(10)-kke	65
  552834	ekk-d(10)-kke	78
  552835	ekk-d(10)-kke	70
  552836	ekk-d(10)-kke	64
  552837	ekk-d(10)-kke	65
  552838	ekk-d(10)-kke	64
  552839	ekk-d(10)-kke	60
  552840	ekk-d(10)-kke	35
  552841	ekk-d(10)-kke	62
  552842	ekk-d(10)-kke	67
  552843	ekk-d(10)-kke	77
  552844	ekk-d(10)-kke	81
  552845	ekk-d(10)-kke	63
  552846	ekk-d(10)-kke	79
  552847	ekk-d(10)-kke	47
  552848	ekk-d(10)-kke	69
  552849	ekk-d(10)-kke	59
  552850	ekk-d(10)-kke	83
  552851	ekk-d(10)-kke	90
  552852	ekk-d(10)-kke	89
  552853	ekk-d(10)-kke	83
  552854	ekk-d(10)-kke	80
  552855	ekk-d(10)-kke	75
  552856	ekk-d(10)-kke	69
  552857	ekk-d(10)-kke	68
  552858	ekk-d(10)-kke	79
  552859	ekk-d(10)-kke	79
  552860	ekk-d(10)-kke	71
  552861	ekk-d(10)-kke	68
  552862	ekk-d(10)-kke	65
  552863	ekk-d(10)-kke	70
  552864	ekk-d(10)-kke	71
  e = 2′-MOE,
  k = cEt,
  d = 2′-deoxynucleoside

• TABLE 65
  Inhibition of viral Target-Z mRNA levels by chimeric
  antisense oligonucleotidesmeasured with RTS3371

  ISIS No	Motif	% inhibition

  552787	ekk-d(10)-kke	53
  552788	ekk-d(10)-kke	45
  552789	ekk-d(10)-kke	75
  552790	ekk-d(10)-kke	68
  552791	ekk-d(10)-kke	51
  552792	ekk-d(10)-kke	38
  552793	ekk-d(10)-kke	0
  552794	ekk-d(10)-kke	44
  552795	ekk-d(10)-kke	56
  552796	ekk-d(10)-kke	45
  552797	ekk-d(10)-kke	46
  552798	ekk-d(10)-kke	53
  552799	ekk-d(10)-kke	48
  552800	ekk-d(10)-kke	54
  552801	ekk-d(10)-kke	63
  552802	ekk-d(10)-kke	49
  552803	ekk-d(10)-kke	71
  552804	ekk-d(10)-kke	64
  552805	ekk-d(10)-kke	70
  552806	ekk-d(10)-kke	67
  552807	ekk-d(10)-kke	61
  552808	ekk-d(10)-kke	83
  552809	ekk-d(10)-kke	59
  552810	ekk-d(10)-kke	56
  552811	ekk-d(10)-kke	62
  552812	ekk-d(10)-kke	66
  552813	ekk-d(10)-kke	63
  552814	ekk-d(10)-kke	65
  552815	ekk-d(10)-kke	63
  552816	ekk-d(10)-kke	88
  552817	ekk-d(10)-kke	94
  552818	ekk-d(10)-kke	82
  552819	ekk-d(10)-kke	80
  552820	ekk-d(10)-kke	84
  552821	ekk-d(10)-kke	71
  552822	ekk-d(10)-kke	85
  552823	ekk-d(10)-kke	71
  552824	ekk-d(10)-kke	81
  552825	ekk-d(10)-kke	51
  552826	ekk-d(10)-kke	64
  552827	ekk-d(10)-kke	61
  552828	ekk-d(10)-kke	76
  552829	ekk-d(10)-kke	61
  552830	ekk-d(10)-kke	59
  552831	ekk-d(10)-kke	58
  552832	ekk-d(10)-kke	64
  552833	ekk-d(10)-kke	75
  552834	ekk-d(10)-kke	84
  552835	ekk-d(10)-kke	57
  552836	ekk-d(10)-kke	51
  552837	ekk-d(10)-kke	53
  552838	ekk-d(10)-kke	48
  552839	ekk-d(10)-kke	50
  552840	ekk-d(10)-kke	54
  552841	ekk-d(10)-kke	61
  552842	ekk-d(10)-kke	71
  552843	ekk-d(10)-kke	75
  552844	ekk-d(10)-kke	78
  552845	ekk-d(10)-kke	52
  552846	ekk-d(10)-kke	76
  552847	ekk-d(10)-kke	61
  552848	ekk-d(10)-kke	72
  552849	ekk-d(10)-kke	87
  552850	ekk-d(10)-kke	76
  552851	ekk-d(10)-kke	76
  552852	ekk-d(10)-kke	79
  552853	ekk-d(10)-kke	82
  552854	ekk-d(10)-kke	85
  552855	ekk-d(10)-kke	78
  552856	ekk-d(10)-kke	77
  552857	ekk-d(10)-kke	75
  552858	ekk-d(10)-kke	75
  552859	ekk-d(10)-kke	79
  552860	ekk-d(10)-kke	71
  552861	ekk-d(10)-kke	74
  552862	ekk-d(10)-kke	66
  552863	ekk-d(10)-kke	70
  552864	ekk-d(10)-kke	73
  e = 2′-MOE,
  k = cEt,
  d = 2′-deoxynucleoside

• TABLE 66
  Inhibition of viral Target-Z mRNA levels by chimeric
  antisense oligonucleotidesmeasured with RTS3371

  ISIS No	Motif	% inhibition

  146786	eeeee-d(10)-eeeee	60
  552889	ek-d(10)-keke	59
  552890	ek-d(10)-keke	56
  552891	ek-d(10)-keke	67
  552892	ek-d(10)-keke	65
  552893	ek-d(10)-keke	68
  552894	ek-d(10)-keke	71
  552895	ek-d(10)-keke	51
  552896	ek-d(10)-keke	51
  552897	ek-d(10)-keke	43
  552898	ek-d(10)-keke	43
  552899	ek-d(10)-keke	55
  552900	ek-d(10)-keke	34
  552901	ek-d(10)-keke	42
  552902	ek-d(10)-keke	60
  552903	ek-d(10)-keke	76
  552904	ek-d(10)-keke	74
  552905	ek-d(10)-keke	66
  552907	ek-d(10)-keke	69
  552908	ek-d(10)-keke	63
  552909	ek-d(10)-keke	70
  552910	ek-d(10)-keke	72
  552911	ek-d(10)-keke	72
  552912	ek-d(10)-keke	67
  552913	ek-d(10)-keke	74
  552914	ek-d(10)-keke	75
  552915	ek-d(10)-keke	58
  552916	ek-d(10)-keke	74
  552917	ek-d(10)-keke	76
  552918	ek-d(10)-keke	75
  552919	ek-d(10)-keke	55
  552920	ek-d(10)-keke	49
  552921	ek-d(10)-keke	45
  552922	ek-d(10)-keke	83
  552923	ek-d(10)-keke	83
  552924	ek-d(10)-keke	0
  552925	ek-d(10)-keke	85
  552926	ek-d(10)-keke	50
  552927	ek-d(10)-keke	76
  552928	ek-d(10)-keke	78
  552929	ek-d(10)-keke	75
  552930	ek-d(10)-keke	78
  552931	ek-d(10)-keke	74
  552932	ek-d(10)-keke	86
  552933	ek-d(10)-keke	82
  552934	ek-d(10)-keke	74
  552935	ek-d(10)-keke	76
  552936	ek-d(10)-keke	81
  552937	ek-d(10)-keke	80
  552938	ek-d(10)-keke	78
  552939	ek-d(10)-keke	75
  552940	ek-d(10)-keke	63
  552941	ekk-d(10)-kke	78
  552942	ek-d(10)-keke	80
  552865	ekk-d(10)-kke	67
  552866	ekk-d(10)-kke	68
  552868	ekk-d(10)-kke	55
  552869	ekk-d(10)-kke	48
  552870	ekk-d(10)-kke	55
  552871	ekk-d(10)-kke	57
  552872	ekk-d(10)-kke	70
  552873	ekk-d(10)-kke	49
  552874	ekk-d(10)-kke	42
  552875	ekk-d(10)-kke	41
  552876	ekk-d(10)-kke	50
  552877	ek-d(10)-keke	39
  552878	ekk-d(10)-kke	31
  552879	ekk-d(10)-kke	5
  552880	ekk-d(10)-kke	5
  552881	ekk-d(10)-kke	10
  552882	ekk-d(10)-kke	11
  552883	ekk-d(10)-kke	27
  552884	ekk-d(10)-kke	36
  552885	ekk-d(10)-kke	12
  552886	ekk-d(10)-kke	32
  552888	ekk-d(10)-kke	1
  e = 2′-MOE,
  k = cEt,
  d = 2′-deoxynucleoside

• TABLE 67
  Inhibition of viral Target-Z mRNA levels by chimeric
  antisense oligonucleotidesmeasured with RTS3371

  ISIS No	Motif	% inhibition

  146786	eeeee-d(10)-eeeee	59
  552955	eee-d(10)-kkk	60
  552956	eee-d(10)-kkk	60
  552957	eee-d(10)-kkk	64
  552958	eee-d(10)-kkk	56
  552959	eee-d(10)-kkk	59
  552960	eee-d(10)-kkk	42
  552961	eee-d(10)-kkk	41
  552962	eee-d(10)-kkk	35
  552963	eee-d(10)-kkk	19
  552964	eee-d(10)-kkk	34
  552965	eee-d(10)-kkk	42
  552966	eee-d(10)-kkk	60
  552967	eee-d(10)-kkk	38
  552968	eee-d(10)-kkk	35
  552969	eee-d(10)-kkk	67
  552970	eee-d(10)-kkk	56
  552971	eee-d(10)-kkk	69
  552972	eee-d(10)-kkk	75
  552973	eee-d(10)-kkk	59
  552974	eee-d(10)-kkk	71
  552975	eee-d(10)-kkk	56
  552976	eee-d(10)-kkk	50
  552977	eee-d(10)-kkk	56
  552978	eee-d(10)-kkk	43
  552979	eee-d(10)-kkk	71
  552980	eee-d(10)-kkk	80
  552981	eee-d(10)-kkk	64
  552982	ek-d(10)-keke	61
  552983	eee-d(10)-kkk	77
  552984	eee-d(10)-kkk	65
  552985	eee-d(10)-kkk	41
  552986	eee-d(10)-kkk	30
  552987	eee-d(10)-kkk	41
  552988	eee-d(10)-kkk	74
  552989	eee-d(10)-kkk	85
  552990	eee-d(10)-kkk	72
  552991	eee-d(10)-kkk	73
  552992	eee-d(10)-kkk	60
  552993	eee-d(10)-kkk	52
  552994	eee-d(10)-kkk	58
  552995	eee-d(10)-kkk	70
  552996	eee-d(10)-kkk	74
  552997	eee-d(10)-kkk	59
  552998	eee-d(10)-kkk	82
  552999	eee-d(10)-kkk	70
  553000	eee-d(10)-kkk	67
  553001	eee-d(10)-kkk	67
  553002	eee-d(10)-kkk	74
  553003	eee-d(10)-kkk	72
  553004	eee-d(10)-kkk	73
  553005	eee-d(10)-kkk	67
  553006	eee-d(10)-kkk	69
  553007	eee-d(10)-kkk	60
  553008	eee-d(10)-kkk	71
  552943	ek-d(10)-keke	77
  553009	eee-d(10)-kkk	78
  552944	ek-d(10)-keke	74
  553010	eee-d(10)-kkk	78
  552945	ek-d(10)-keke	76
  553011	eee-d(10)-kkk	72
  552946	ek-d(10)-keke	71
  553012	eee-d(10)-kkk	74
  552947	ek-d(10)-keke	54
  553013	eee-d(10)-kkk	39
  552948	ek-d(10)-keke	50
  553014	eee-d(10)-kkk	37
  552949	ek-d(10)-keke	8
  553015	eee-d(10)-kkk	45
  552950	ek-d(10)-keke	44
  553016	eee-d(10)-kkk	47
  552951	ek-d(10)-keke	60
  553017	eee-d(10)-kkk	47
  552952	ek-d(10)-keke	35
  553018	eee-d(10)-kkk	30
  552953	ek-d(10)-keke	37
  553019	eee-d(10)-kkk	37
  552954	ek-d(10)-keke	40
  553020	eee-d(10)-kkk	24
  e = 2′-MOE,
  k = cEt,
  d = 2′-deoxynucleoside

• TABLE 68
  Inhibition of viral Target-Z mRNA levels by chimeric
  antisense oligonucleotidesmeasured with RTS3370

  ISIS No	Motif	% inhibition

  552889	ek-d(10)-keke	42
  552890	ek-d(10)-keke	56
  552891	ek-d(10)-keke	55
  552892	ek-d(10)-keke	53
  552893	ek-d(10)-keke	56
  552894	ek-d(10)-keke	53
  552895	ek-d(10)-keke	38
  552896	ek-d(10)-keke	43
  552897	ek-d(10)-keke	40
  552898	ek-d(10)-keke	50
  552899	ek-d(10)-keke	37
  552900	ek-d(10)-keke	43
  552901	ek-d(10)-keke	56
  552902	ek-d(10)-keke	43
  552903	ek-d(10)-keke	78
  552904	ek-d(10)-keke	75
  552905	ek-d(10)-keke	52
  552907	ek-d(10)-keke	75
  552908	ek-d(10)-keke	57
  552909	ek-d(10)-keke	66
  552910	ek-d(10)-keke	60
  552911	ek-d(10)-keke	65
  552912	ek-d(10)-keke	37
  552913	ek-d(10)-keke	76
  552914	ek-d(10)-keke	79
  552915	ek-d(10)-keke	71
  552916	ek-d(10)-keke	82
  552917	ek-d(10)-keke	78
  552918	ek-d(10)-keke	64
  552919	ek-d(10)-keke	38
  552920	ek-d(10)-keke	43
  552921	ek-d(10)-keke	49
  552922	ek-d(10)-keke	90
  552923	ek-d(10)-keke	92
  552924	ek-d(10)-keke	30
  552925	ek-d(10)-keke	81
  552926	ek-d(10)-keke	39
  552927	ek-d(10)-keke	53
  552928	ek-d(10)-keke	48
  552929	ek-d(10)-keke	68
  552930	ek-d(10)-keke	87
  552931	ek-d(10)-keke	87
  552932	ek-d(10)-keke	88
  552933	ek-d(10)-keke	75
  552934	ek-d(10)-keke	76
  552935	ek-d(10)-keke	71
  552936	ek-d(10)-keke	80
  552937	ek-d(10)-keke	81
  552938	ek-d(10)-keke	85
  552939	ek-d(10)-keke	82
  552940	ek-d(10)-keke	76
  552941	ekk-d(10)-kke	72
  552942	ek-d(10)-keke	85
  552865	ekk-d(10)-kke	70
  552866	ekk-d(10)-kke	65
  552868	ekk-d(10)-kke	36
  552869	ekk-d(10)-kke	23
  552870	ekk-d(10)-kke	49
  552871	ekk-d(10)-kke	46
  552872	ekk-d(10)-kke	73
  552873	ekk-d(10)-kke	41
  552874	ekk-d(10)-kke	18
  552875	ekk-d(10)-kke	0
  552876	ekk-d(10)-kke	49
  552877	ek-d(10)-keke	37
  552878	ekk-d(10)-kke	28
  552879	ekk-d(10)-kke	0
  552880	ekk-d(10)-kke	12
  552881	ekk-d(10)-kke	0
  552882	ekk-d(10)-kke	0
  552883	ekk-d(10)-kke	12
  552884	ekk-d(10)-kke	39
  552885	ekk-d(10)-kke	37
  552886	ekk-d(10)-kke	15
  552888	ekk-d(10)-kke	0
  e = 2′-MOE,
  k = cEt,
  d = 2′-deoxynucleoside

• TABLE 69
  Inhibition of viral Target-Z mRNA levels by chimeric
  antisense oligonucleotidesmeasured with RTS3370

  ISIS No	Motif	% inhibition

  552955	eee-d(10)-kkk	67
  552956	eee-d(10)-kkk	60
  552957	eee-d(10)-kkk	73
  552958	eee-d(10)-kkk	63
  552959	eee-d(10)-kkk	58
  552960	eee-d(10)-kkk	67
  552961	eee-d(10)-kkk	78
  552962	eee-d(10)-kkk	29
  552963	eee-d(10)-kkk	25
  552964	eee-d(10)-kkk	33
  552965	eee-d(10)-kkk	55
  552966	eee-d(10)-kkk	71
  552967	eee-d(10)-kkk	23
  552968	eee-d(10)-kkk	41
  552969	eee-d(10)-kkk	76
  552970	eee-d(10)-kkk	44
  552971	eee-d(10)-kkk	77
  552972	eee-d(10)-kkk	74
  552973	eee-d(10)-kkk	61
  552974	eee-d(10)-kkk	73
  552975	eee-d(10)-kkk	66
  552976	eee-d(10)-kkk	70
  552977	eee-d(10)-kkk	65
  552978	eee-d(10)-kkk	40
  552979	eee-d(10)-kkk	79
  552980	eee-d(10)-kkk	81
  552981	eee-d(10)-kkk	74
  552982	ek-d(10)-keke	52
  552983	eee-d(10)-kkk	78
  552984	eee-d(10)-kkk	71
  552985	eee-d(10)-kkk	38
  552986	eee-d(10)-kkk	48
  552987	eee-d(10)-kkk	54
  552988	eee-d(10)-kkk	85
  552989	eee-d(10)-kkk	84
  552990	eee-d(10)-kkk	79
  552991	eee-d(10)-kkk	53
  552992	eee-d(10)-kkk	68
  552993	eee-d(10)-kkk	67
  552994	eee-d(10)-kkk	69
  552995	eee-d(10)-kkk	62
  552996	eee-d(10)-kkk	82
  552997	eee-d(10)-kkk	58
  552998	eee-d(10)-kkk	86
  552999	eee-d(10)-kkk	63
  553000	eee-d(10)-kkk	67
  553001	eee-d(10)-kkk	70
  553002	eee-d(10)-kkk	84
  553003	eee-d(10)-kkk	83
  553004	eee-d(10)-kkk	68
  553005	eee-d(10)-kkk	57
  553006	eee-d(10)-kkk	74
  553007	eee-d(10)-kkk	62
  553008	eee-d(10)-kkk	50
  552943	ek-d(10)-keke	86
  553009	eee-d(10)-kkk	79
  552944	ek-d(10)-keke	83
  553010	eee-d(10)-kkk	74
  552945	ek-d(10)-keke	79
  553011	eee-d(10)-kkk	60
  552946	ek-d(10)-keke	68
  553012	eee-d(10)-kkk	78
  552947	ek-d(10)-keke	51
  553013	eee-d(10)-kkk	45
  552948	ek-d(10)-keke	56
  553014	eee-d(10)-kkk	53
  552949	ek-d(10)-keke	1
  553015	eee-d(10)-kkk	55
  552950	ek-d(10)-keke	52
  553016	eee-d(10)-kkk	65
  552951	ek-d(10)-keke	59
  553017	eee-d(10)-kkk	36
  552952	ek-d(10)-keke	34
  553018	eee-d(10)-kkk	20
  552953	ek-d(10)-keke	55
  553019	eee-d(10)-kkk	34
  552954	ek-d(10)-keke	51
  553020	eee-d(10)-kkk	28
  e = 2′-MOE,
  k = cEt,
  d = 2′-deoxynucleoside

Example 47: Dose-Dependent Antisense Inhibition of Target-Z mRNA in HepG2 Cells
• Antisense oligonucleotides from the study described in Example 46 exhibiting in vitro inhibition of Target-Z mRNA were selected and tested at various doses in HepG2 cells. Cells were plated at a density of 28,000 cells per well and transfected using LipofectAMINE2000® with 9.26 nM, 27.78 nM, 83.33 nM, and 250.00 nM concentrations of antisense oligonucleotide, as specified in Table 70. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-Z mRNA levels were measured by quantitative real-time PCR. Target-Z primer probe set RTS3371 was used to measure mRNA levels. Target-Z mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-Z, relative to untreated control cells.
• As illustrated in Table 70, Target-Z mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells.

• TABLE 70
  Dose-dependent antisense inhibition
  of human Target-Z in HepG2 cells

  		9.2593	27.7778	83.3333	250.0
  ISIS No	Motif	nM	nM	nM	nM

  146786	eeeee-d(10)-eeeee	10	43	74	89
  552808	ekk-d(10)-kke	13	14	55	70
  552816	ekk-d(10)-kke	38	73	87	92
  552818	ekk-d(10)-kke	29	63	87	85
  552820	ekk-d(10)-kke	58	83	90	90
  552821	ekk-d(10)-kke	33	49	71	88
  552822	ekk-d(10)-kke	24	55	74	88
  552824	ekk-d(10)-kke	8	24	65	87
  552834	ekk-d(10)-kke	11	28	68	89
  552849	ekk-d(10)-kke	12	25	73	84
  552851	ekk-d(10)-kke	13	42	74	89
  552852	ekk-d(10)-kke	4	35	70	87
  552853	ekk-d(10)-kke	19	52	86	93
  552854	ekk-d(10)-kke	28	57	80	89
  552916	ek-d(10)-keke	5	26	64	82
  552922	ek-d(10)-keke	25	44	77	89
  552923	ek-d(10)-keke	22	49	82	91
  552925	ek-d(10)-keke	33	56	80	92
  552930	ek-d(10)-keke	12	49	79	89
  552931	ek-d(10)-keke	12	40	62	82
  552932	ek-d(10)-keke	24	62	84	91
  552933	ek-d(10)-keke	20	40	75	89
  552936	ek-d(10)-keke	18	36	75	88
  552937	ek-d(10)-keke	22	51	82	88
  552938	ek-d(10)-keke	12	36	67	80
  552939	ek-d(10)-keke	17	40	65	79
  552942	ek-d(10)-keke	21	48	74	88
  552943	ek-d(10)-keke	5	39	70	85
  552944	ek-d(10)-keke	14	33	70	77
  552980	eee-d(10)-kkk	15	40	69	86
  552988	eee-d(10)-kkk	4	36	58	84
  552989	eee-d(10)-kkk	0	50	74	81
  552996	eee-d(10)-kkk	0	25	53	72
  552998	eee-d(10)-kkk	17	49	79	90
  553002	eee-d(10)-kkk	0	32	68	86
  553003	eee-d(10)-kkk	15	42	67	88
  e = 2′-MOE,
  k = cEt,
  d = 2′-deoxynucleoside

Example 48: Efficacy of Antisense Oligonucleotides Targeting Target-Z in Transgenic Mice
• Mice harboring a Target-Z gene fragment (Guidotti, L. G. et al., J. Virol. 1995, 69, 6158-6169) were used. The mice were treated with ISIS antisense oligonucleotides selected from studies described above as illustrated in Table 71 and evaluated for their efficacy in this model.
Treatment
• Groups of 10 mice each were injected subcutaneously twice a week for the first with 50 mg/kg and, subsequently, twice a week for the next 3 weeks with 25 mg/kg of ISIS 146786 or ISIS 510100. Control groups of 10 mice each were treated in a similar manner with ISIS 141923 (5-10-5 MOE gapmer with no known murine target) or ISIS 459024 (3-10-4 MOE gapmer with no known murine target). Mice were euthanized 48 hours after the last dose, and organs and serum were harvested for further analysis.

• TABLE 71
  Antisense oligonucleotides targeting Target-Z in transgenic mice

  ISIS NO.	Sequence (5′ to 3′)	Motif	SEQ ID NO.
  146786	GesTesGesAesAesGdsCdsGdsAdsAds	e5-d(10)-e5	39
  	GdsTdsGdsCdsAdsCesAesCesGesGes
  510100	GesGesmCesAdsTdsAdsGdsmCdsAds	eee-d(10)-eeee	40
  	GdsmCdsAdsGdsGesAesTesGe
  141923	mCesmCesTesTesmCesmCdsmCdsTdsGdsAds	e5-d(10)-e5	41
  	AdsGdsGdsTdsTdsmCesmCesTesmCesmCe
  459024	mCesGesGesTdsmCdsmCdsTdsTdsGdsGds	eee-d(10)-eeee	42
  	AdsGdsGdsAesTesGesmCe
  e = 2′-MOE (e.g. e5 = eeeee), d = 2′-deoxynucleoside

DNA and RNA Analysis
• RNA was extracted from liver tissue for real-time PCR analysis of Target-Z DNA, using primer probe sets RTS3370, RTS3371, or RTS3372 (forward sequence ATCCTATCAACACTTCCGGAAACT, designated SEQ ID NO: 43; reverse sequence CGACGCGGCGATTGAG, designated SEQ ID NO: 44; probe sequence AAGAACTCCCTCGCCTCGCAGACG, designated SEQ ID NO: 45). The DNA levels were normalized to picogreen. Target-Z RNA samples were also assayed with primer probe sets RTS3370 and RTS3371 after RT-PCR analysis. The mRNA levels were normalized to RIBOGREEN®. The data is presented in Table 72. Serum DNA samples were analyzed after the study period. The data is presented in Table 73, expressed relative to the levels measured in the control group. As shown in Tables 72 and 73, the antisense oligonucleotides achieved reduction of Target-Z DNA and RNA over the PBS control. Treatment with either control oligonucleotide did not cause any changes in RNA or DNA levels, as expected.

• TABLE 72
  Percent inhibition of Target-Z RNA and DNA in the liver of transgenic mice

  		%	%	%	%	%	%
  		inhibition	inhibition	inhibition	inhibition	inhibition	inhibition
  		DNA	DNA	DNA	RNA	RNA	RNA
  ISIS No	Motif	(RTS3370)	(RTS3371)	(RTS3372)	(RTS3370)	(RTS3371)	(RTS3372)

  146786	e5-d(10)-e5	97	97	95	86	85	89
  510100	eee-d(10)-eeee	95	94	94	56	64	77
  141923	e5-d(10)-e5	2	0	13	0	7	31
  459024	eee-d(10)-eeee	19	0	8	0	0	0
  e = 2′-MOE (e.g. e5 = eeeee), d = 2′-deoxynucleoside

• TABLE 73
  Percent inhibition of Target-Z DNA
  in the serum of transgenic mice

  		% inhibition	% inhibition
  ISIS No	Motif	(RTS3370)	(RTS3371)

  146786	e5-d(10)-e5	98	98
  510100	eee-d(10)-eeee	99	98
  141923	e5-d(10)-e5	0	0
  459024	eee-d(10)-eeee	0	0
  e = 2′-MOE (e.g. e5 = eeeee),
  d = 2′-deoxynucleoside

Example 49: Efficacy of Antisense Oligonucleotides Targeting Target-Z in Transgenic Mice
• Transgenic mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for their efficacy in this model.
Treatment
• A group of 6 mice was injected subcutaneously twice a week for 4 weeks with 25 mg/kg of ISIS 146786. Groups of 6 mice each were injected subcutaneously twice a week for 4 weeks with 10 mg/kg of ISIS 552803, ISIS 552903, ISIS 552817, ISIS 552822, and ISIS 552907. One group of 10 mice was injected subcutaneously twice a week for 4 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.
DNA and RNA Analysis
• RNA was extracted from liver tissue for real-time PCR analysis of Target-Z DNA, using primer probe set RTS3371. The DNA levels were normalized to picogreen. Target-Z RNA samples were also assayed with primer probe set RTS3371 after RT-PCR analysis. The mRNA levels were normalized to RIBOGREEN®. The data is presented in Table 74. Serum DNA samples were analyzed after the study period. The data is presented in Table 75, expressed relative to the levels measured in the control group. As shown in Tables 74 and 75, the antisense oligonucleotides achieved reduction of Target-Z DNA and RNA over the PBS control.

• TABLE 74
  Percent inhibition of Target-Z RNA and DNA in transgenic mice

  		Dose	% inhibition	% inhibition
  ISIS No	Motif	(mg/kg/wk)	of RNA	of DNA

  146786	e5-d(10)-e5	50	81	91
  552803	ekk-d(10)-kke	20	71	95
  552817	ekk-d(10)-kke	20	86	51
  552822	ekk-d(10)-kke	20	90	89
  552903	ek-d(10)-keke	20	56	82
  552907	ek-d(10)-keke	20	41	45
  e = 2′-MOE (e.g. e5 = eeeee),
  d = 2′-deoxynucleoside

• TABLE 75
  Serum levels of Target-Z DNA in transgenic
  mice, relative to control levels

  		Dose	Post-dose
  ISIS No	Motif	(mg/kg/wk)	DNA levels

  146786	e5-d(10)-e5	50	0.1
  552803	ekk-d(10)-kke	20	0.2
  552817	ekk-d(10)-kke	20	1.3
  552822	ekk-d(10)-kke	20	0.0
  552903	ek-d(10)-keke	20	2.9
  552907	ek-d(10)-keke	20	1.0
  e = 2′-MOE (e.g. e5 = eeeee),
  d = 2′-deoxynucleoside

Liver Function
• To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma concentrations of ALT were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.) (Nyblom, H. et al., Alcohol & Alcoholism 39: 336-339, 2004; Tietz N W (Ed): Clinical Guide to Laboratory Tests, 3rd ed. W. B. Saunders, Philadelphia, Pa., 1995). The results are presented in Table 76 expressed in IU/L. All the ISIS oligonucleotides were considered tolerable in the mice, as demonstrated by their liver transaminase profile.

• TABLE 76
  ALT levels (IU/L) of transgenic mice

  	Motif	Dose (mg/kg/wk)	ALT

  PBS	—	—	77
  ISIS 146786	e5-d(10)-e5	50	21
  ISIS 552803	ekk-d(10)-kke	20	74
  ISIS 552817	ekk-d(10)-kke	20	38
  ISIS 552822	ekk-d(10)-kke	20	47
  ISIS 552903	ek-d(10)-keke	20	57
  ISIS 552907	ek-d(10)-keke	20	28
  e = 2′-MOE (e.g. e5 = eeeee),
  d = 2′-deoxynucleoside

Example 50: Efficacy of Antisense Oligonucleotides Targeting Target-Z in Transgenic Mice
• Transgenic mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for their efficacy in this model.
Treatment
• A group of 6 mice was injected subcutaneously twice a week for 4 weeks with 25 mg/kg of ISIS 146786. Groups of 6 mice each were injected subcutaneously twice a week for 4 weeks with 10 mg/kg of ISIS 552853, ISIS 552854, ISIS 552932, and ISIS 552938. One group of 10 mice was injected subcutaneously twice a week for 4 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.
DNA and RNA Analysis
• RNA was extracted from liver tissue for real-time PCR analysis of Target-Z DNA, using primer probe set RTS3371. The DNA levels were normalized to picogreen. Target-Z RNA samples were also assayed with primer probe set RTS3371 after RT-PCR analysis. The mRNA levels were normalized to RIBOGREEN®. As shown in Table 77, the antisense oligonucleotides achieved reduction of Target-Z DNA and RNA over the PBS control. Results are presented as percent inhibition of Target-Z mRNA or DNA, relative to control.

• TABLE 77
  Percent inhibition of Target-Z DNA and RNA in transgenic mice

  		Dose	% inhibition	% inhibition
  	Motif	(mg/kg/wk)	(DNA)	(RNA)

  PBS	—	—
  ISIS 146786	e5-d(10)-e5	50	90	60
  ISIS 552853	ekk-d(10)-kke	20	94	60
  ISIS 552854	ekk-d(10)-kke	20	61	23
  ISIS 552932	ekk-d(10)-kke	20	75	70
  ISIS 552938	ek-d(10)-keke	20	67	56
  =2′-MOE (e.g. e5 = eeeee),
  d = 2′-deoxynucleoside

Example 51: Efficacy of Antisense Oligonucleotides Targeting Target-Z in Transgenic Mice
• Transgenic mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for their efficacy in this model.
Treatment
• A group of 6 mice was injected subcutaneously twice a week for 4 weeks with 25 mg/kg of ISIS 146786. Groups of 6 mice each were injected subcutaneously twice a week for 4 weeks with 10 mg/kg of ISIS 552922, ISIS 552923, ISIS 552942, ISIS 552872, ISIS 552925, ISIS 552937, and ISIS 552939. One group of 10 mice was injected subcutaneously twice a week for 4 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.
DNA and RNA Analysis
• RNA was extracted from liver tissue for real-time PCR analysis of Target-Z DNA, using primer probe set RTS3371. The DNA levels were normalized to picogreen. Target-Z RNA samples were also assayed with primer probe set RTS3371 after RT-PCR analysis. The mRNA levels were normalized to RIBOGREEN®. As shown in Table 78, the antisense oligonucleotides achieved reduction of Target-Z DNA and RNA over the PBS control. Results are presented as percent inhibition of Target-Z mRNA or DNA, relative to control.

• TABLE 78
  Percent inhibition of Target-Z DNA and RNA in transgenic mice

  		Dose	% inhibition	% inhibition
  ISIS No	Motif	(mg/kg/wk)	(DNA)	(RNA)

  146786	e5-d(10)-e5	50	52	57
  552922	ek-d(10)-keke	20	61	50
  552923	ek-d(10)-keke	20	89	76
  552942	ek-d(10)-keke	20	58	52
  552872	ekk-d(10)-kke	20	77	46
  552925	ek-d(10)-keke	20	89	65
  552937	ek-d(10)-keke	20	59	35
  552939	ek-d(10)-keke	20	57	19
  =2′-MOE (e.g. e5 = eeeee),
  d = 2′-deoxynucleoside

Example 52: Antisense Inhibition of Target-Z mRNA in HepG2 Cells
• Antisense oligonucleotides were designed targeting a Target-Z nucleic acid and were tested for their effects on Target-Z mRNA in vitro. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables. ISIS 146786, 509934, ISIS 509959, and ISIS 510100, from the studies described above, were also included. Cultured HepG2 cells at a density of 28,000 cells per well were transfected using LipofectAMINE20000 with 70 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-Z mRNA levels were measured by quantitative real-time PCR. Primer probe set RTS3370 (forward sequence CTTGGTCATGGGCCATCAG, designated herein as SEQ ID NO: 33; reverse sequence CGGCTAGGAGTTCCGCAGTA, designated herein as SEQ ID NO: 34; probe sequence TGCGTGGAACCTTTTCGGCTCC, designated herein as SEQ ID NO: 35) was used to measure mRNA levels. Levels were also measured using primer probe set RTS3371 (forward sequence CCAAACCTTCGGACGGAAA, designated herein as SEQ ID NO: 36; reverse sequence TGAGGCCCACTCCCATAGG, designated herein as SEQ ID NO: 37; probe sequence CCCATCATCCTGGGCTTTCGGAAAAT, designated herein as SEQ ID NO: 38). Target-Z mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-Z, relative to untreated control cells.
• The newly designed chimeric antisense oligonucleotides and their motifs are described in Tables 79-96. The modified oligonucleotides are 16, 17 or 20 nucleotides in length, wherein the central gap segment comprises of nine or ten 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising 2′-O-methoxyethyl (2′-MOE) modifications. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines.
• Each gapmer listed in the Tables is targeted to the viral genomic sequence, designated herein as Target-Z. The activity of the newly designed oligonucleotides was compared with ISIS 146786, 509934, ISIS 509959, and ISIS 510100, the information of which have been placed at the top of each table.

• TABLE 79
  Inhibition of viral Target-Z mRNA levels by chimeric
  antisense oligonucleotidesmeasured with RTS3370

  ISIS No	Motif	Wing chemistry	% inhibition

  146786	5-10-5	2′-MOE	50
  510100	3-10-4	2′-MOE	62
  552276	5-9-3	2′-MOE	42
  552277	5-9-3	2′-MOE	46
  552278	5-9-3	2′-MOE	31
  552279	5-9-3	2′-MOE	41
  552280	5-9-3	2′-MOE	5
  552281	5-9-3	2′-MOE	11
  552282	5-9-3	2′-MOE	20
  552283	5-9-3	2′-MOE	28
  552230	4-9-4	2′-MOE	57
  552284	5-9-3	2′-MOE	0
  552231	4-9-4	2′-MOE	29
  552285	5-9-3	2′-MOE	61
  552232	4-9-4	2′-MOE	35
  552286	5-9-3	2′-MOE	47
  552233	4-9-4	2′-MOE	38
  552287	5-9-3	2′-MOE	45
  552234	4-9-4	2′-MOE	0
  552288	5-9-3	2′-MOE	50
  552235	4-9-4	2′-MOE	0
  552289	5-9-3	2′-MOE	46
  552236	4-9-4	2′-MOE	45
  552290	5-9-3	2′-MOE	41
  552237	4-9-4	2′-MOE	44
  552291	5-9-3	2′-MOE	26
  552239	4-9-4	2′-MOE	62
  552293	5-9-3	2′-MOE	67
  552240	4-9-4	2′-MOE	61
  552294	5-9-3	2′-MOE	71
  552241	4-9-4	2′-MOE	55
  552295	5-9-3	2′-MOE	58
  552242	4-9-4	2′-MOE	60
  552296	5-9-3	2′-MOE	59
  552243	4-9-4	2′-MOE	57
  552297	5-9-3	2′-MOE	55
  552244	4-9-4	2′-MOE	33
  552298	5-9-3	2′-MOE	48
  552245	4-9-4	2′-MOE	48
  552299	5-9-3	2′-MOE	34
  552246	4-9-4	2′-MOE	81
  552300	5-9-3	2′-MOE	56
  552247	4-9-4	2′-MOE	87
  552301	5-9-3	2′-MOE	86
  552248	4-9-4	2′-MOE	72
  552302	5-9-3	2′-MOE	77
  552249	4-9-4	2′-MOE	56
  552303	5-9-3	2′-MOE	65
  552250	4-9-4	2′-MOE	52
  552304	5-9-3	2′-MOE	57
  552251	4-9-4	2′-MOE	43
  552305	5-9-3	2′-MOE	56
  552252	4-9-4	2′-MOE	62
  552306	5-9-3	2′-MOE	75
  552253	4-9-4	2′-MOE	82
  552307	5-9-3	2′-MOE	90
  552254	4-9-4	2′-MOE	74
  552255	4-9-4	2′-MOE	78
  552256	4-9-4	2′-MOE	65
  552257	4-9-4	2′-MOE	62
  552258	4-9-4	2′-MOE	72
  552259	4-9-4	2′-MOE	63
  552260	4-9-4	2′-MOE	58
  552261	4-9-4	2′-MOE	63
  552262	4-9-4	2′-MOE	50
  552263	4-9-4	2′-MOE	60
  552264	4-9-4	2′-MOE	52
  552265	4-9-4	2′-MOE	68
  552266	4-9-4	2′-MOE	62
  552267	4-9-4	2′-MOE	58
  552268	4-9-4	2′-MOE	62
  552269	4-9-4	2′-MOE	52
  552270	4-9-4	2′-MOE	54
  552271	4-9-4	2′-MOE	58
  552272	4-9-4	2′-MOE	40
  552273	4-9-4	2′-MOE	34
  552274	4-9-4	2′-MOE	34
  552275	4-9-4	2′-MOE	39

• TABLE 80
  Inhibition of viral Target-Z mRNA levels by chimeric
  antisense oligonucleotidesmeasured with RTS3370

  ISIS No	Motif	Wing chemistry	% inhibition

  146786	5-10-5	2′-MOE	49
  509959	3-10-3	2′-MOE	43
  510100	3-10-4	2′-MOE	54
  552384	2-9-5	2′-MOE	29
  552440	3-9-4	2′-MOE	58
  552385	2-9-5	2′-MOE	57
  552441	3-9-4	2′-MOE	42
  552386	2-9-5	2′-MOE	53
  552442	3-9-4	2′-MOE	53
  552387	2-9-5	2′-MOE	48
  552443	3-9-4	2′-MOE	59
  552388	2-9-5	2′-MOE	40
  552444	3-9-4	2′-MOE	51
  552389	2-9-5	2′-MOE	39
  552445	3-9-4	2′-MOE	60
  552390	2-9-5	2′-MOE	52
  552446	3-9-4	2′-MOE	54
  552391	2-9-5	2′-MOE	57
  552447	3-9-4	2′-MOE	54
  552392	2-9-5	2′-MOE	0
  552448	3-9-4	2′-MOE	58
  552393	2-9-5	2′-MOE	59
  552449	3-9-4	2′-MOE	60
  552394	2-9-5	2′-MOE	53
  552450	3-9-4	2′-MOE	53
  552395	2-9-5	2′-MOE	57
  552451	3-9-4	2′-MOE	39
  552396	2-9-5	2′-MOE	62
  552452	3-9-4	2′-MOE	57
  552238	4-9-4	2′-MOE	38
  552292	5-9-3	2′-MOE	48
  552346	6-9-2	2′-MOE	0
  552397	2-9-5	2′-MOE	63
  552453	3-9-4	2′-MOE	56
  552398	2-9-5	2′-MOE	61
  552454	3-9-4	2′-MOE	48
  552399	2-9-5	2′-MOE	52
  552400	2-9-5	2′-MOE	57
  552401	2-9-5	2′-MOE	52
  552402	2-9-5	2′-MOE	54
  552403	2-9-5	2′-MOE	74
  552404	2-9-5	2′-MOE	43
  552405	2-9-5	2′-MOE	15
  552406	2-9-5	2′-MOE	37
  552407	2-9-5	2′-MOE	37
  552408	2-9-5	2′-MOE	76
  552409	2-9-5	2′-MOE	76
  552410	2-9-5	2′-MOE	63
  552411	2-9-5	2′-MOE	70
  552412	2-9-5	2′-MOE	62
  552413	2-9-5	2′-MOE	56
  552414	2-9-5	2′-MOE	63
  552415	2-9-5	2′-MOE	52
  552416	2-9-5	2′-MOE	67
  552417	2-9-5	2′-MOE	50
  552418	2-9-5	2′-MOE	79
  552419	2-9-5	2′-MOE	70
  552420	2-9-5	2′-MOE	71
  552421	2-9-5	2′-MOE	69
  552422	2-9-5	2′-MOE	68
  552423	2-9-5	2′-MOE	65
  552424	2-9-5	2′-MOE	70
  552425	2-9-5	2′-MOE	51
  552426	2-9-5	2′-MOE	40
  552427	2-9-5	2′-MOE	35
  552428	2-9-5	2′-MOE	58
  552429	2-9-5	2′-MOE	46
  552430	2-9-5	2′-MOE	53
  552431	2-9-5	2′-MOE	51
  552432	2-9-5	2′-MOE	57
  552433	2-9-5	2′-MOE	54
  552434	2-9-5	2′-MOE	44
  552435	2-9-5	2′-MOE	46
  552436	2-9-5	2′-MOE	36
  552437	2-9-5	2′-MOE	27
  552438	2-9-5	2′-MOE	27
  552439	2-9-5	2′-MOE	13

• TABLE 81
  Inhibition of viral Target-Z mRNA levels by chimeric
  antisense oligonucleotidesmeasured with RTS3370

  ISIS No	Motif	Wing chemistry	% inhibition

  146786	5-10-5	2′-MOE	35
  509959	3-10-3	2′-MOE	52
  552496	4-9-3	2′-MOE	47
  552497	4-9-3	2′-MOE	57
  552498	4-9-3	2′-MOE	45
  552499	4-9-3	2′-MOE	52
  552500	4-9-3	2′-MOE	46
  552501	4-9-3	2′-MOE	44
  552502	4-9-3	2′-MOE	57
  552503	4-9-3	2′-MOE	52
  552504	4-9-3	2′-MOE	45
  552505	4-9-3	2′-MOE	56
  552506	4-9-3	2′-MOE	54
  552507	4-9-3	2′-MOE	34
  552508	4-9-3	2′-MOE	34
  552509	4-9-3	2′-MOE	48
  552510	4-9-3	2′-MOE	50
  552455	3-9-4	2′-MOE	66
  552511	4-9-3	2′-MOE	66
  552456	3-9-4	2′-MOE	64
  552512	4-9-3	2′-MOE	62
  552457	3-9-4	2′-MOE	14
  552513	4-9-3	2′-MOE	56
  552458	3-9-4	2′-MOE	59
  552514	4-9-3	2′-MOE	52
  552459	3-9-4	2′-MOE	69
  552515	4-9-3	2′-MOE	57
  552460	3-9-4	2′-MOE	0
  552516	4-9-3	2′-MOE	54
  552461	3-9-4	2′-MOE	20
  552517	4-9-3	2′-MOE	52
  552462	3-9-4	2′-MOE	46
  552518	4-9-3	2′-MOE	34
  552463	3-9-4	2′-MOE	48
  552519	4-9-3	2′-MOE	44
  552464	3-9-4	2′-MOE	81
  552520	4-9-3	2′-MOE	69
  552465	3-9-4	2′-MOE	84
  552521	4-9-3	2′-MOE	80
  552466	3-9-4	2′-MOE	75
  552522	4-9-3	2′-MOE	76
  552467	3-9-4	2′-MOE	65
  552523	4-9-3	2′-MOE	71
  552468	3-9-4	2′-MOE	53
  552524	4-9-3	2′-MOE	43
  552469	3-9-4	2′-MOE	51
  552525	4-9-3	2′-MOE	57
  552470	3-9-4	2′-MOE	46
  552526	4-9-3	2′-MOE	60
  552471	3-9-4	2′-MOE	54
  552527	4-9-3	2′-MOE	72
  552472	3-9-4	2′-MOE	78
  552528	4-9-3	2′-MOE	78
  552473	3-9-4	2′-MOE	67
  552529	4-9-3	2′-MOE	77
  552474	3-9-4	2′-MOE	79
  552530	4-9-3	2′-MOE	78
  552475	3-9-4	2′-MOE	74
  552531	4-9-3	2′-MOE	68
  552476	3-9-4	2′-MOE	52
  552477	3-9-4	2′-MOE	76
  552478	3-9-4	2′-MOE	70
  552479	3-9-4	2′-MOE	67
  552480	3-9-4	2′-MOE	68
  552481	3-9-4	2′-MOE	57
  552482	3-9-4	2′-MOE	51
  552483	3-9-4	2′-MOE	48
  552484	3-9-4	2′-MOE	58
  552485	3-9-4	2′-MOE	51
  552486	3-9-4	2′-MOE	55
  552487	3-9-4	2′-MOE	62
  552488	3-9-4	2′-MOE	51
  552489	3-9-4	2′-MOE	49
  552490	3-9-4	2′-MOE	51
  552491	3-9-4	2′-MOE	51
  552492	3-9-4	2′-MOE	38
  552493	3-9-4	2′-MOE	52
  552494	3-9-4	2′-MOE	17
  552495	3-9-4	2′-MOE	49

• TABLE 82
  Inhibition of viral Target-Z mRNA levels by chimeric
  antisense oligonucleotidesmeasured with RTS3370

  ISIS No	Motif	Wing chemistry	% inhibition

  146786	5-10-5	2′-MOE	47
  509959	3-10-3	2′-MOE	38
  552552	5-9-2	2′-MOE	33
  552553	5-9-2	2′-MOE	46
  552554	5-9-2	2′-MOE	54
  552555	5-9-2	2′-MOE	50
  552556	5-9-2	2′-MOE	46
  552557	5-9-2	2′-MOE	57
  552558	5-9-2	2′-MOE	55
  552559	5-9-2	2′-MOE	66
  552560	5-9-2	2′-MOE	44
  552561	5-9-2	2′-MOE	48
  552562	5-9-2	2′-MOE	52
  552563	5-9-2	2′-MOE	45
  552564	5-9-2	2′-MOE	41
  552565	5-9-2	2′-MOE	54
  552566	5-9-2	2′-MOE	56
  552567	5-9-2	2′-MOE	71
  552568	5-9-2	2′-MOE	64
  552569	5-9-2	2′-MOE	59
  552570	5-9-2	2′-MOE	60
  552571	5-9-2	2′-MOE	55
  552572	5-9-2	2′-MOE	60
  552573	5-9-2	2′-MOE	24
  552574	5-9-2	2′-MOE	34
  552575	5-9-2	2′-MOE	36
  552576	5-9-2	2′-MOE	67
  552577	5-9-2	2′-MOE	64
  552578	5-9-2	2′-MOE	75
  552579	5-9-2	2′-MOE	75
  552580	5-9-2	2′-MOE	59
  552581	5-9-2	2′-MOE	54
  552582	5-9-2	2′-MOE	61
  552583	5-9-2	2′-MOE	69
  552584	5-9-2	2′-MOE	74
  552585	5-9-2	2′-MOE	62
  552586	5-9-2	2′-MOE	79
  552587	5-9-2	2′-MOE	71
  552532	4-9-3	2′-MOE	48
  552588	5-9-2	2′-MOE	70
  552533	4-9-3	2′-MOE	43
  552589	5-9-2	2′-MOE	59
  552534	4-9-3	2′-MOE	62
  552590	5-9-2	2′-MOE	70
  552535	4-9-3	2′-MOE	55
  552591	5-9-2	2′-MOE	51
  552536	4-9-3	2′-MOE	3
  552592	5-9-2	2′-MOE	50
  552537	4-9-3	2′-MOE	14
  552593	5-9-2	2′-MOE	46
  552538	4-9-3	2′-MOE	52
  552594	5-9-2	2′-MOE	55
  552539	4-9-3	2′-MOE	47
  552595	5-9-2	2′-MOE	60
  552540	4-9-3	2′-MOE	60
  552596	5-9-2	2′-MOE	63
  552541	4-9-3	2′-MOE	60
  552597	5-9-2	2′-MOE	61
  552542	4-9-3	2′-MOE	64
  552598	5-9-2	2′-MOE	57
  552543	4-9-3	2′-MOE	46
  552600	5-9-2	2′-MOE	59
  552544	4-9-3	2′-MOE	53
  552602	5-9-2	2′-MOE	6
  552545	4-9-3	2′-MOE	33
  552604	5-9-2	2′-MOE	47
  552546	4-9-3	2′-MOE	42
  552606	5-9-2	2′-MOE	53
  552547	4-9-3	2′-MOE	51
  552608	5-9-2	2′-MOE	53
  552548	4-9-3	2′-MOE	52
  552610	5-9-2	2′-MOE	47
  552549	4-9-3	2′-MOE	38
  552612	5-9-2	2′-MOE	39
  552550	4-9-3	2′-MOE	19
  552614	5-9-2	2′-MOE	24
  552551	4-9-3	2′-MOE	24
  552616	5-9-2	2′-MOE	15

• TABLE 83
  Inhibition of viral Target-Z mRNA levels by chimeric
  antisense oligonucleotidesmeasured with RTS3370

  ISIS No	Motif	Wing chemistry	% inhibition

  146786	5-10-5	2′-MOE	51
  509934	5-10-5	2′-MOE	76
  552007	6-10-4	2′-MOE	61
  552039	7-10-3	2′-MOE	84
  552008	6-10-4	2′-MOE	48
  552040	7-10-3	2′-MOE	48
  552009	6-10-4	2′-MOE	77
  552041	7-10-3	2′-MOE	73
  552010	6-10-4	2′-MOE	63
  552042	7-10-3	2′-MOE	66
  552011	6-10-4	2′-MOE	52
  552043	7-10-3	2′-MOE	54
  552012	6-10-4	2′-MOE	73
  552044	7-10-3	2′-MOE	86
  552013	6-10-4	2′-MOE	73
  552045	7-10-3	2′-MOE	65
  552014	6-10-4	2′-MOE	76
  552046	7-10-3	2′-MOE	93
  552015	6-10-4	2′-MOE	70
  552047	7-10-3	2′-MOE	77
  552016	6-10-4	2′-MOE	61
  552048	7-10-3	2′-MOE	66
  552017	6-10-4	2′-MOE	73
  552049	7-10-3	2′-MOE	73
  552018	6-10-4	2′-MOE	98
  552050	7-10-3	2′-MOE	98
  552019	6-10-4	2′-MOE	98
  552051	7-10-3	2′-MOE	99
  551986	4-10-6	2′-MOE	92
  552020	6-10-4	2′-MOE	97
  552052	7-10-3	2′-MOE	98
  551987	4-10-6	2′-MOE	95
  552021	6-10-4	2′-MOE	97
  552053	7-10-3	2′-MOE	98
  551988	4-10-6	2′-MOE	50
  552005	5-10-5	2′-MOE	99
  552022	6-10-4	2′-MOE	99
  552054	7-10-3	2′-MOE	99
  551989	4-10-6	2′-MOE	96
  552023	6-10-4	2′-MOE	99
  552055	7-10-3	2′-MOE	98
  551990	4-10-6	2′-MOE	86
  552024	6-10-4	2′-MOE	89
  552056	7-10-3	2′-MOE	88
  551991	4-10-6	2′-MOE	0
  552025	6-10-4	2′-MOE	90
  552057	7-10-3	2′-MOE	92
  551992	4-10-6	2′-MOE	72
  552026	6-10-4	2′-MOE	88
  552058	7-10-3	2′-MOE	86
  551993	4-10-6	2′-MOE	82
  552027	6-10-4	2′-MOE	87
  552059	7-10-3	2′-MOE	88
  551994	4-10-6	2′-MOE	85
  552028	6-10-4	2′-MOE	83
  552060	7-10-3	2′-MOE	82
  551995	4-10-6	2′-MOE	84
  552029	6-10-4	2′-MOE	88
  552061	7-10-3	2′-MOE	85
  551996	4-10-6	2′-MOE	87
  552030	6-10-4	2′-MOE	88
  552062	7-10-3	2′-MOE	85
  551997	4-10-6	2′-MOE	83
  552031	6-10-4	2′-MOE	82
  551998	4-10-6	2′-MOE	85
  552032	6-10-4	2′-MOE	87
  551999	4-10-6	2′-MOE	82
  552033	6-10-4	2′-MOE	87
  552000	4-10-6	2′-MOE	83
  552006	5-10-5	2′-MOE	88
  552034	6-10-4	2′-MOE	89
  552001	4-10-6	2′-MOE	65
  552035	6-10-4	2′-MOE	60
  552002	4-10-6	2′-MOE	63
  552036	6-10-4	2′-MOE	65
  552003	4-10-6	2′-MOE	65
  552037	6-10-4	2′-MOE	58
  552004	4-10-6	2′-MOE	58
  552038	6-10-4	2′-MOE	70

• TABLE 84
  Inhibition of viral Target-Z mRNA levels by chimeric
  antisense oligonucleotidesmeasured with RTS3370

  ISIS No	Motif	Wing chemistry	% inhibition

  146786	5-10-5	2′-MOE	64
  510100	3-10-4	2′-MOE	62
  552168	3-9-5	2′-MOE	79
  552222	4-9-4	2′-MOE	79
  552169	3-9-5	2′-MOE	67
  552223	4-9-4	2′-MOE	40
  552170	3-9-5	2′-MOE	69
  552224	4-9-4	2′-MOE	64
  552171	3-9-5	2′-MOE	65
  552225	4-9-4	2′-MOE	69
  552172	3-9-5	2′-MOE	33
  552226	4-9-4	2′-MOE	48
  552173	3-9-5	2′-MOE	41
  552227	4-9-4	2′-MOE	32
  552174	3-9-5	2′-MOE	31
  552228	4-9-4	2′-MOE	42
  552175	3-9-5	2′-MOE	59
  552176	3-9-5	2′-MOE	68
  552177	3-9-5	2′-MOE	55
  552178	3-9-5	2′-MOE	66
  552179	3-9-5	2′-MOE	70
  552180	3-9-5	2′-MOE	66
  552181	3-9-5	2′-MOE	51
  552182	3-9-5	2′-MOE	69
  552183	3-9-5	2′-MOE	69
  552184	3-9-5	2′-MOE	43
  552185	3-9-5	2′-MOE	66
  552186	3-9-5	2′-MOE	54
  552187	3-9-5	2′-MOE	74
  552188	3-9-5	2′-MOE	78
  552189	3-9-5	2′-MOE	57
  552190	3-9-5	2′-MOE	39
  552191	3-9-5	2′-MOE	60
  552192	3-9-5	2′-MOE	85
  552193	3-9-5	2′-MOE	86
  552194	3-9-5	2′-MOE	68
  552195	3-9-5	2′-MOE	73
  552196	3-9-5	2′-MOE	60
  552197	3-9-5	2′-MOE	60
  552198	3-9-5	2′-MOE	61
  552199	3-9-5	2′-MOE	89
  552200	3-9-5	2′-MOE	85
  552201	3-9-5	2′-MOE	81
  552202	3-9-5	2′-MOE	76
  552203	3-9-5	2′-MOE	74
  552204	3-9-5	2′-MOE	71
  552151	2-9-6	2′-MOE	77
  552205	3-9-5	2′-MOE	78
  552152	2-9-6	2′-MOE	72
  552206	3-9-5	2′-MOE	77
  552153	2-9-6	2′-MOE	67
  552207	3-9-5	2′-MOE	81
  552154	2-9-6	2′-MOE	56
  552208	3-9-5	2′-MOE	70
  552155	2-9-6	2′-MOE	61
  552209	3-9-5	2′-MOE	63
  552156	2-9-6	2′-MOE	20
  552210	3-9-5	2′-MOE	75
  552157	2-9-6	2′-MOE	39
  552211	3-9-5	2′-MOE	75
  552158	2-9-6	2′-MOE	70
  552212	3-9-5	2′-MOE	67
  552159	2-9-6	2′-MOE	74
  552213	3-9-5	2′-MOE	70
  552160	2-9-6	2′-MOE	78
  552214	3-9-5	2′-MOE	79
  552161	2-9-6	2′-MOE	56
  552215	3-9-5	2′-MOE	61
  552162	2-9-6	2′-MOE	64
  552216	3-9-5	2′-MOE	62
  552163	2-9-6	2′-MOE	71
  552217	3-9-5	2′-MOE	58
  552164	2-9-6	2′-MOE	52
  552218	3-9-5	2′-MOE	56
  552165	2-9-6	2′-MOE	53
  552219	3-9-5	2′-MOE	33
  552166	2-9-6	2′-MOE	41
  552220	3-9-5	2′-MOE	53
  552167	2-9-6	2′-MOE	54
  552221	3-9-5	2′-MOE	31

• TABLE 85
  Inhibition of viral Target-Z mRNA levels by chimeric
  antisense oligonucleotidesmeasured with RTS3370

  ISIS No	Motif	Wing chemistry	% inhibition

  146786	5-10-5	2′-MOE	73
  509934	5-10-5	2′-MOE	76
  510100	3-10-4	2′-MOE	73
  552071	8-10-2	2′-MOE	79
  552114	2-9-6	2′-MOE	66
  552115	2-9-6	2′-MOE	70
  552116	2-9-6	2′-MOE	68
  552117	2-9-6	2′-MOE	70
  552072	8-10-2	2′-MOE	50
  552118	2-9-6	2′-MOE	66
  552119	2-9-6	2′-MOE	62
  552120	2-9-6	2′-MOE	35
  552121	2-9-6	2′-MOE	39
  552073	8-10-2	2′-MOE	80
  552122	2-9-6	2′-MOE	55
  552074	8-10-2	2′-MOE	73
  552123	2-9-6	2′-MOE	75
  552075	8-10-2	2′-MOE	78
  552124	2-9-6	2′-MOE	64
  552076	8-10-2	2′-MOE	70
  552125	2-9-6	2′-MOE	73
  552077	8-10-2	2′-MOE	83
  552126	2-9-6	2′-MOE	64
  552078	8-10-2	2′-MOE	80
  552127	2-9-6	2′-MOE	72
  552079	8-10-2	2′-MOE	86
  552128	2-9-6	2′-MOE	76
  552080	8-10-2	2′-MOE	83
  552129	2-9-6	2′-MOE	72
  552131	2-9-6	2′-MOE	61
  552132	2-9-6	2′-MOE	73
  552133	2-9-6	2′-MOE	75
  552081	8-10-2	2′-MOE	76
  552134	2-9-6	2′-MOE	58
  552135	2-9-6	2′-MOE	67
  552136	2-9-6	2′-MOE	65
  552137	2-9-6	2′-MOE	55
  552082	8-10-2	2′-MOE	98
  552138	2-9-6	2′-MOE	82
  552083	8-10-2	2′-MOE	99
  552139	2-9-6	2′-MOE	86
  552084	8-10-2	2′-MOE	99
  552140	2-9-6	2′-MOE	74
  552085	8-10-2	2′-MOE	100
  552141	2-9-6	2′-MOE	67
  552086	8-10-2	2′-MOE	100
  552142	2-9-6	2′-MOE	45
  552087	8-10-2	2′-MOE	100
  552143	2-9-6	2′-MOE	68
  552144	2-9-6	2′-MOE	78
  552145	2-9-6	2′-MOE	88
  552146	2-9-6	2′-MOE	81
  552088	8-10-2	2′-MOE	95
  552147	2-9-6	2′-MOE	88
  552089	8-10-2	2′-MOE	93
  552148	2-9-6	2′-MOE	79
  552090	8-10-2	2′-MOE	87
  552149	2-9-6	2′-MOE	81
  552091	8-10-2	2′-MOE	88
  552092	8-10-2	2′-MOE	90
  552093	8-10-2	2′-MOE	91
  552094	8-10-2	2′-MOE	88
  552063	7-10-3	2′-MOE	81
  552095	8-10-2	2′-MOE	89
  552064	7-10-3	2′-MOE	85
  552096	8-10-2	2′-MOE	92
  552065	7-10-3	2′-MOE	86
  552097	8-10-2	2′-MOE	93
  552066	7-10-3	2′-MOE	33
  552098	8-10-2	2′-MOE	88
  552067	7-10-3	2′-MOE	50
  552099	8-10-2	2′-MOE	70
  552068	7-10-3	2′-MOE	73
  552100	8-10-2	2′-MOE	70
  552069	7-10-3	2′-MOE	73
  552101	8-10-2	2′-MOE	76
  552070	7-10-3	2′-MOE	71
  552102	8-10-2	2′-MOE	64

• TABLE 86
  Inhibition of viral Target-Z mRNA levels by chimeric
  antisense oligonucleotidesmeasured with RTS3370

  ISIS No	Motif	Wing chemistry	% inhibition

  146786	5-10-5	2′-MOE	84
  510100	3-10-4	2′-MOE	76
  552330	6-9-2	2′-MOE	54
  552331	6-9-2	2′-MOE	66
  552332	6-9-2	2′-MOE	70
  552333	6-9-2	2′-MOE	55
  552334	6-9-2	2′-MOE	42
  552335	6-9-2	2′-MOE	39
  552336	6-9-2	2′-MOE	27
  552337	6-9-2	2′-MOE	74
  552338	6-9-2	2′-MOE	68
  552339	6-9-2	2′-MOE	71
  552340	6-9-2	2′-MOE	61
  552341	6-9-2	2′-MOE	58
  552342	6-9-2	2′-MOE	55
  552343	6-9-2	2′-MOE	63
  552344	6-9-2	2′-MOE	51
  552345	6-9-2	2′-MOE	65
  552346	6-9-2	2′-MOE	0
  552347	6-9-2	2′-MOE	84
  552348	6-9-2	2′-MOE	87
  552349	6-9-2	2′-MOE	74
  552350	6-9-2	2′-MOE	59
  552351	6-9-2	2′-MOE	60
  552352	6-9-2	2′-MOE	53
  552353	6-9-2	2′-MOE	0
  552354	6-9-2	2′-MOE	83
  552355	6-9-2	2′-MOE	90
  552356	6-9-2	2′-MOE	0
  552357	6-9-2	2′-MOE	45
  552358	6-9-2	2′-MOE	74
  552359	6-9-2	2′-MOE	72
  552360	6-9-2	2′-MOE	87
  552361	6-9-2	2′-MOE	96
  552308	5-9-3	2′-MOE	81
  552362	6-9-2	2′-MOE	92
  552309	5-9-3	2′-MOE	77
  552363	6-9-2	2′-MOE	92
  552310	5-9-3	2′-MOE	80
  552364	6-9-2	2′-MOE	87
  552311	5-9-3	2′-MOE	13
  552365	6-9-2	2′-MOE	84
  552150	2-9-6	2′-MOE	73
  552312	5-9-3	2′-MOE	77
  552366	6-9-2	2′-MOE	87
  552313	5-9-3	2′-MOE	64
  552367	6-9-2	2′-MOE	85
  552314	5-9-3	2′-MOE	73
  552368	6-9-2	2′-MOE	77
  552315	5-9-3	2′-MOE	75
  552369	6-9-2	2′-MOE	75
  552316	5-9-3	2′-MOE	64
  552370	6-9-2	2′-MOE	63
  552317	5-9-3	2′-MOE	99
  552371	6-9-2	2′-MOE	81
  552318	5-9-3	2′-MOE	76
  552372	6-9-2	2′-MOE	65
  552319	5-9-3	2′-MOE	55
  552373	6-9-2	2′-MOE	74
  552320	5-9-3	2′-MOE	68
  552374	6-9-2	2′-MOE	78
  552321	5-9-3	2′-MOE	74
  552375	6-9-2	2′-MOE	81
  552322	5-9-3	2′-MOE	73
  552376	6-9-2	2′-MOE	78
  552323	5-9-3	2′-MOE	75
  552377	6-9-2	2′-MOE	70
  552324	5-9-3	2′-MOE	0
  552378	6-9-2	2′-MOE	72
  552325	5-9-3	2′-MOE	70
  552379	6-9-2	2′-MOE	74
  552326	5-9-3	2′-MOE	63
  552380	6-9-2	2′-MOE	53
  552327	5-9-3	2′-MOE	30
  552381	6-9-2	2′-MOE	26
  552328	5-9-3	2′-MOE	25
  552382	6-9-2	2′-MOE	13
  552329	5-9-3	2′-MOE	33
  552383	6-9-2	2′-MOE	5

• TABLE 87
  Inhibition of viral Target-Z mRNA levels by chimeric
  antisense oligonucleotidesmeasured with RTS3370

  ISIS No	Motif	Wing chemistry	% inhibition

  509934	5-10-5	2′-MOE	30
  551909	2-10-8	2′-MOE	62
  551941	3-10-7	2′-MOE	74
  551973	4-10-6	2′-MOE	64
  551910	2-10-8	2′-MOE	52
  551942	3-10-7	2′-MOE	54
  551974	4-10-6	2′-MOE	51
  551911	2-10-8	2′-MOE	58
  551943	3-10-7	2′-MOE	64
  551975	4-10-6	2′-MOE	57
  551912	2-10-8	2′-MOE	59
  551944	3-10-7	2′-MOE	66
  551976	4-10-6	2′-MOE	57
  551913	2-10-8	2′-MOE	58
  551945	3-10-7	2′-MOE	56
  551977	4-10-6	2′-MOE	56
  551914	2-10-8	2′-MOE	0
  551946	3-10-7	2′-MOE	48
  551978	4-10-6	2′-MOE	53
  551915	2-10-8	2′-MOE	44
  551947	3-10-7	2′-MOE	53
  551979	4-10-6	2′-MOE	64
  551916	2-10-8	2′-MOE	57
  551948	3-10-7	2′-MOE	68
  551980	4-10-6	2′-MOE	56
  551917	2-10-8	2′-MOE	58
  551949	3-10-7	2′-MOE	64
  551981	4-10-6	2′-MOE	63
  551918	2-10-8	2′-MOE	59
  551950	3-10-7	2′-MOE	71
  551982	4-10-6	2′-MOE	63
  551919	2-10-8	2′-MOE	76
  551951	3-10-7	2′-MOE	71
  551983	4-10-6	2′-MOE	73
  551920	2-10-8	2′-MOE	68
  551952	3-10-7	2′-MOE	76
  551984	4-10-6	2′-MOE	81
  551921	2-10-8	2′-MOE	83
  551953	3-10-7	2′-MOE	82
  551985	4-10-6	2′-MOE	76
  551922	2-10-8	2′-MOE	73
  551954	3-10-7	2′-MOE	68
  551923	2-10-8	2′-MOE	59
  551955	3-10-7	2′-MOE	71
  551924	2-10-8	2′-MOE	80
  551956	3-10-7	2′-MOE	80
  551925	2-10-8	2′-MOE	82
  551957	3-10-7	2′-MOE	88
  551926	2-10-8	2′-MOE	71
  551958	3-10-7	2′-MOE	74
  551927	2-10-8	2′-MOE	68
  551959	3-10-7	2′-MOE	69
  551928	2-10-8	2′-MOE	69
  551960	3-10-7	2′-MOE	62
  551929	2-10-8	2′-MOE	54
  551961	3-10-7	2′-MOE	20
  551930	2-10-8	2′-MOE	53
  551962	3-10-7	2′-MOE	60
  551931	2-10-8	2′-MOE	47
  551963	3-10-7	2′-MOE	63
  551932	2-10-8	2′-MOE	68
  551964	3-10-7	2′-MOE	56
  551933	2-10-8	2′-MOE	72
  551965	3-10-7	2′-MOE	67
  551934	2-10-8	2′-MOE	64
  551966	3-10-7	2′-MOE	73
  551935	2-10-8	2′-MOE	68
  551967	3-10-7	2′-MOE	60
  551936	2-10-8	2′-MOE	67
  551968	3-10-7	2′-MOE	63
  551937	2-10-8	2′-MOE	47
  551969	3-10-7	2′-MOE	36
  551938	2-10-8	2′-MOE	41
  551970	3-10-7	2′-MOE	43
  551939	2-10-8	2′-MOE	53
  551971	3-10-7	2′-MOE	55
  551940	2-10-8	2′-MOE	50
  551972	3-10-7	2′-MOE	58

• TABLE 88
  Inhibition of viral Target-Z mRNA levels by chimeric
  antisense oligonucleotidesmeasured with RTS3371

  ISIS No	Motif	Wing chemistry	% inhibition

  509934	5-10-5	2′-MOE	21
  551909	2-10-8	2′-MOE	52
  551941	3-10-7	2′-MOE	62
  551973	4-10-6	2′-MOE	58
  551910	2-10-8	2′-MOE	48
  551942	3-10-7	2′-MOE	36
  551974	4-10-6	2′-MOE	45
  551911	2-10-8	2′-MOE	61
  551943	3-10-7	2′-MOE	56
  551975	4-10-6	2′-MOE	60
  551912	2-10-8	2′-MOE	53
  551944	3-10-7	2′-MOE	48
  551976	4-10-6	2′-MOE	48
  551913	2-10-8	2′-MOE	53
  551945	3-10-7	2′-MOE	54
  551977	4-10-6	2′-MOE	48
  551914	2-10-8	2′-MOE	0
  551946	3-10-7	2′-MOE	56
  551978	4-10-6	2′-MOE	36
  551915	2-10-8	2′-MOE	47
  551947	3-10-7	2′-MOE	45
  551979	4-10-6	2′-MOE	54
  551916	2-10-8	2′-MOE	44
  551948	3-10-7	2′-MOE	59
  551980	4-10-6	2′-MOE	49
  551917	2-10-8	2′-MOE	48
  551949	3-10-7	2′-MOE	60
  551981	4-10-6	2′-MOE	57
  551918	2-10-8	2′-MOE	53
  551950	3-10-7	2′-MOE	57
  551982	4-10-6	2′-MOE	57
  551919	2-10-8	2′-MOE	65
  551951	3-10-7	2′-MOE	57
  551983	4-10-6	2′-MOE	53
  551920	2-10-8	2′-MOE	57
  551952	3-10-7	2′-MOE	67
  551984	4-10-6	2′-MOE	62
  551921	2-10-8	2′-MOE	60
  551953	3-10-7	2′-MOE	57
  551985	4-10-6	2′-MOE	58
  551922	2-10-8	2′-MOE	63
  551954	3-10-7	2′-MOE	61
  551923	2-10-8	2′-MOE	50
  551955	3-10-7	2′-MOE	44
  551924	2-10-8	2′-MOE	52
  551956	3-10-7	2′-MOE	46
  551925	2-10-8	2′-MOE	54
  551957	3-10-7	2′-MOE	51
  551926	2-10-8	2′-MOE	70
  551958	3-10-7	2′-MOE	72
  551927	2-10-8	2′-MOE	60
  551959	3-10-7	2′-MOE	61
  551928	2-10-8	2′-MOE	57
  551960	3-10-7	2′-MOE	58
  551929	2-10-8	2′-MOE	49
  551961	3-10-7	2′-MOE	26
  551930	2-10-8	2′-MOE	54
  551962	3-10-7	2′-MOE	57
  551931	2-10-8	2′-MOE	46
  551963	3-10-7	2′-MOE	56
  551932	2-10-8	2′-MOE	57
  551964	3-10-7	2′-MOE	53
  551933	2-10-8	2′-MOE	65
  551965	3-10-7	2′-MOE	54
  551934	2-10-8	2′-MOE	58
  551966	3-10-7	2′-MOE	69
  551935	2-10-8	2′-MOE	63
  551967	3-10-7	2′-MOE	53
  551936	2-10-8	2′-MOE	67
  551968	3-10-7	2′-MOE	60
  551937	2-10-8	2′-MOE	51
  551969	3-10-7	2′-MOE	42
  551938	2-10-8	2′-MOE	40
  551970	3-10-7	2′-MOE	38
  551939	2-10-8	2′-MOE	32
  551971	3-10-7	2′-MOE	46
  551940	2-10-8	2′-MOE	39
  551972	3-10-7	2′-MOE	51

• TABLE 89
  Inhibition of viral Target-Z mRNA levels by chimeric
  antisense oligonucleotidesmeasured with RTS3371

  ISIS No	Motif	Wing chemistry	% inhibition

  146786	5-10-5	2′-MOE	40
  510100	3-10-4	2′-MOE	60
  552276	5-9-3	2′-MOE	44
  552277	5-9-3	2′-MOE	39
  552278	5-9-3	2′-MOE	37
  552279	5-9-3	2′-MOE	50
  552280	5-9-3	2′-MOE	2
  552281	5-9-3	2′-MOE	0
  552282	5-9-3	2′-MOE	13
  552229	4-9-4	2′-MOE	17
  552283	5-9-3	2′-MOE	27
  552230	4-9-4	2′-MOE	53
  552284	5-9-3	2′-MOE	0
  552231	4-9-4	2′-MOE	31
  552285	5-9-3	2′-MOE	56
  552232	4-9-4	2′-MOE	35
  552286	5-9-3	2′-MOE	43
  552233	4-9-4	2′-MOE	40
  552287	5-9-3	2′-MOE	44
  552234	4-9-4	2′-MOE	0
  552288	5-9-3	2′-MOE	44
  552235	4-9-4	2′-MOE	13
  552289	5-9-3	2′-MOE	21
  552236	4-9-4	2′-MOE	40
  552290	5-9-3	2′-MOE	34
  552237	4-9-4	2′-MOE	37
  552291	5-9-3	2′-MOE	34
  552239	4-9-4	2′-MOE	58
  552293	5-9-3	2′-MOE	61
  552240	4-9-4	2′-MOE	54
  552294	5-9-3	2′-MOE	62
  552241	4-9-4	2′-MOE	47
  552295	5-9-3	2′-MOE	63
  552242	4-9-4	2′-MOE	61
  552296	5-9-3	2′-MOE	61
  552243	4-9-4	2′-MOE	55
  552297	5-9-3	2′-MOE	52
  552244	4-9-4	2′-MOE	45
  552298	5-9-3	2′-MOE	27
  552245	4-9-4	2′-MOE	41
  552299	5-9-3	2′-MOE	32
  552246	4-9-4	2′-MOE	67
  552300	5-9-3	2′-MOE	57
  552247	4-9-4	2′-MOE	74
  552301	5-9-3	2′-MOE	76
  552248	4-9-4	2′-MOE	65
  552302	5-9-3	2′-MOE	68
  552249	4-9-4	2′-MOE	38
  552303	5-9-3	2′-MOE	59
  552250	4-9-4	2′-MOE	43
  552304	5-9-3	2′-MOE	30
  552251	4-9-4	2′-MOE	52
  552305	5-9-3	2′-MOE	49
  552252	4-9-4	2′-MOE	51
  552306	5-9-3	2′-MOE	56
  552253	4-9-4	2′-MOE	47
  552307	5-9-3	2′-MOE	49
  552254	4-9-4	2′-MOE	50
  552255	4-9-4	2′-MOE	64
  552256	4-9-4	2′-MOE	57
  552257	4-9-4	2′-MOE	51
  552258	4-9-4	2′-MOE	62
  552259	4-9-4	2′-MOE	59
  552260	4-9-4	2′-MOE	56
  552261	4-9-4	2′-MOE	54
  552262	4-9-4	2′-MOE	47
  552263	4-9-4	2′-MOE	45
  552264	4-9-4	2′-MOE	52
  552265	4-9-4	2′-MOE	58
  552266	4-9-4	2′-MOE	54
  552267	4-9-4	2′-MOE	43
  552268	4-9-4	2′-MOE	57
  552269	4-9-4	2′-MOE	34
  552270	4-9-4	2′-MOE	37
  552271	4-9-4	2′-MOE	42
  552272	4-9-4	2′-MOE	36
  552273	4-9-4	2′-MOE	25
  552274	4-9-4	2′-MOE	11
  552275	4-9-4	2′-MOE	38

• TABLE 90
  Inhibition of viral Target-Z mRNA levels by chimeric
  antisense oligonucleotidesmeasured with RTS3371

  ISIS No	Motif	Wing chemistry	% inhibition

  146786	5-10-5	2′-MOE	38
  509959	3-10-3	2′-MOE	49
  510100	3-10-4	2′-MOE	55
  552384	2-9-5	2′-MOE	41
  552440	3-9-4	2′-MOE	57
  552385	2-9-5	2′-MOE	53
  552441	3-9-4	2′-MOE	38
  552386	2-9-5	2′-MOE	42
  552442	3-9-4	2′-MOE	72
  552387	2-9-5	2′-MOE	43
  552443	3-9-4	2′-MOE	56
  552388	2-9-5	2′-MOE	18
  552444	3-9-4	2′-MOE	39
  552389	2-9-5	2′-MOE	24
  552445	3-9-4	2′-MOE	53
  552390	2-9-5	2′-MOE	40
  552446	3-9-4	2′-MOE	57
  552391	2-9-5	2′-MOE	51
  552447	3-9-4	2′-MOE	53
  552392	2-9-5	2′-MOE	0
  552448	3-9-4	2′-MOE	57
  552393	2-9-5	2′-MOE	52
  552449	3-9-4	2′-MOE	49
  552394	2-9-5	2′-MOE	32
  552450	3-9-4	2′-MOE	44
  552395	2-9-5	2′-MOE	33
  552451	3-9-4	2′-MOE	38
  552396	2-9-5	2′-MOE	46
  552452	3-9-4	2′-MOE	30
  552130	2-9-6	2′-MOE	46
  552184	3-9-5	2′-MOE	34
  552238	4-9-4	2′-MOE	41
  552292	5-9-3	2′-MOE	45
  552346	6-9-2	2′-MOE	0
  552397	2-9-5	2′-MOE	37
  552453	3-9-4	2′-MOE	45
  552398	2-9-5	2′-MOE	42
  552454	3-9-4	2′-MOE	39
  552399	2-9-5	2′-MOE	34
  552400	2-9-5	2′-MOE	47
  552401	2-9-5	2′-MOE	53
  552402	2-9-5	2′-MOE	47
  552403	2-9-5	2′-MOE	70
  552404	2-9-5	2′-MOE	44
  552405	2-9-5	2′-MOE	0
  552406	2-9-5	2′-MOE	25
  552407	2-9-5	2′-MOE	23
  552408	2-9-5	2′-MOE	73
  552409	2-9-5	2′-MOE	71
  552410	2-9-5	2′-MOE	52
  552411	2-9-5	2′-MOE	62
  552412	2-9-5	2′-MOE	50
  552413	2-9-5	2′-MOE	55
  552414	2-9-5	2′-MOE	64
  552415	2-9-5	2′-MOE	45
  552416	2-9-5	2′-MOE	45
  552417	2-9-5	2′-MOE	37
  552418	2-9-5	2′-MOE	73
  552419	2-9-5	2′-MOE	68
  552420	2-9-5	2′-MOE	64
  552421	2-9-5	2′-MOE	54
  552422	2-9-5	2′-MOE	60
  552423	2-9-5	2′-MOE	62
  552424	2-9-5	2′-MOE	60
  552425	2-9-5	2′-MOE	46
  552426	2-9-5	2′-MOE	48
  552427	2-9-5	2′-MOE	36
  552428	2-9-5	2′-MOE	57
  552429	2-9-5	2′-MOE	36
  552430	2-9-5	2′-MOE	42
  552431	2-9-5	2′-MOE	60
  552432	2-9-5	2′-MOE	44
  552433	2-9-5	2′-MOE	55
  552434	2-9-5	2′-MOE	46
  552435	2-9-5	2′-MOE	47
  552436	2-9-5	2′-MOE	25
  552437	2-9-5	2′-MOE	19
  552438	2-9-5	2′-MOE	25
  552439	2-9-5	2′-MOE	22

• TABLE 91
  Inhibition of viral Target-Z mRNA levels by chimeric
  antisense oligonucleotidesmeasured with RTS3371

  ISIS No	Motif	Wing chemistry	% inhibition

  509959	3-10-3	2′-MOE	49
  552496	4-9-3	2′-MOE	35
  552497	4-9-3	2′-MOE	60
  552498	4-9-3	2′-MOE	20
  552499	4-9-3	2′-MOE	45
  552500	4-9-3	2′-MOE	53
  552501	4-9-3	2′-MOE	56
  552502	4-9-3	2′-MOE	50
  552503	4-9-3	2′-MOE	36
  552504	4-9-3	2′-MOE	50
  552505	4-9-3	2′-MOE	53
  552506	4-9-3	2′-MOE	49
  552507	4-9-3	2′-MOE	35
  552508	4-9-3	2′-MOE	62
  552509	4-9-3	2′-MOE	65
  552510	4-9-3	2′-MOE	54
  552455	3-9-4	2′-MOE	60
  552511	4-9-3	2′-MOE	65
  552456	3-9-4	2′-MOE	69
  552512	4-9-3	2′-MOE	63
  552457	3-9-4	2′-MOE	4
  552513	4-9-3	2′-MOE	50
  552458	3-9-4	2′-MOE	59
  552514	4-9-3	2′-MOE	53
  552459	3-9-4	2′-MOE	69
  552515	4-9-3	2′-MOE	68
  552460	3-9-4	2′-MOE	3
  552516	4-9-3	2′-MOE	65
  552461	3-9-4	2′-MOE	37
  552517	4-9-3	2′-MOE	54
  552462	3-9-4	2′-MOE	42
  552518	4-9-3	2′-MOE	23
  552463	3-9-4	2′-MOE	28
  552519	4-9-3	2′-MOE	32
  552464	3-9-4	2′-MOE	72
  552520	4-9-3	2′-MOE	61
  552465	3-9-4	2′-MOE	68
  552521	4-9-3	2′-MOE	68
  552466	3-9-4	2′-MOE	76
  552522	4-9-3	2′-MOE	71
  552467	3-9-4	2′-MOE	72
  552523	4-9-3	2′-MOE	73
  552468	3-9-4	2′-MOE	50
  552524	4-9-3	2′-MOE	49
  552469	3-9-4	2′-MOE	65
  552525	4-9-3	2′-MOE	45
  552470	3-9-4	2′-MOE	58
  552526	4-9-3	2′-MOE	39
  552471	3-9-4	2′-MOE	30
  552527	4-9-3	2′-MOE	39
  552472	3-9-4	2′-MOE	43
  552528	4-9-3	2′-MOE	43
  552473	3-9-4	2′-MOE	25
  552529	4-9-3	2′-MOE	50
  552474	3-9-4	2′-MOE	70
  552530	4-9-3	2′-MOE	73
  552475	3-9-4	2′-MOE	64
  552531	4-9-3	2′-MOE	62
  552476	3-9-4	2′-MOE	50
  552477	3-9-4	2′-MOE	66
  552478	3-9-4	2′-MOE	68
  552479	3-9-4	2′-MOE	60
  552480	3-9-4	2′-MOE	58
  552481	3-9-4	2′-MOE	54
  552482	3-9-4	2′-MOE	44
  552483	3-9-4	2′-MOE	17
  552484	3-9-4	2′-MOE	64
  552485	3-9-4	2′-MOE	56
  552486	3-9-4	2′-MOE	26
  552487	3-9-4	2′-MOE	42
  552488	3-9-4	2′-MOE	35
  552489	3-9-4	2′-MOE	46
  552490	3-9-4	2′-MOE	41
  552491	3-9-4	2′-MOE	38
  552492	3-9-4	2′-MOE	47
  552493	3-9-4	2′-MOE	49
  552494	3-9-4	2′-MOE	22
  552495	3-9-4	2′-MOE	0

• TABLE 92
  Inhibition of viral Target-Z mRNA levels by chimeric
  antisense oligonucleotidesmeasured with RTS3371

  ISIS No	Motif	Wing chemistry	% inhibition

  146786	5-10-5	2′-MOE	56
  509959	3-10-3	2′-MOE	54
  552552	5-9-2	2′-MOE	32
  552553	5-9-2	2′-MOE	53
  552554	5-9-2	2′-MOE	48
  552555	5-9-2	2′-MOE	39
  552556	5-9-2	2′-MOE	39
  552557	5-9-2	2′-MOE	54
  552558	5-9-2	2′-MOE	41
  552559	5-9-2	2′-MOE	56
  552560	5-9-2	2′-MOE	39
  552561	5-9-2	2′-MOE	51
  552562	5-9-2	2′-MOE	56
  552563	5-9-2	2′-MOE	31
  552564	5-9-2	2′-MOE	31
  552565	5-9-2	2′-MOE	53
  552566	5-9-2	2′-MOE	46
  552567	5-9-2	2′-MOE	63
  552568	5-9-2	2′-MOE	66
  552569	5-9-2	2′-MOE	60
  552570	5-9-2	2′-MOE	60
  552571	5-9-2	2′-MOE	44
  552572	5-9-2	2′-MOE	52
  552573	5-9-2	2′-MOE	20
  552574	5-9-2	2′-MOE	36
  552575	5-9-2	2′-MOE	19
  552576	5-9-2	2′-MOE	61
  552577	5-9-2	2′-MOE	57
  552578	5-9-2	2′-MOE	71
  552579	5-9-2	2′-MOE	59
  552580	5-9-2	2′-MOE	58
  552581	5-9-2	2′-MOE	51
  552582	5-9-2	2′-MOE	40
  552583	5-9-2	2′-MOE	35
  552584	5-9-2	2′-MOE	50
  552585	5-9-2	2′-MOE	48
  552586	5-9-2	2′-MOE	74
  552587	5-9-2	2′-MOE	68
  552532	4-9-3	2′-MOE	59
  552588	5-9-2	2′-MOE	67
  552533	4-9-3	2′-MOE	52
  552589	5-9-2	2′-MOE	47
  552534	4-9-3	2′-MOE	71
  552590	5-9-2	2′-MOE	58
  552535	4-9-3	2′-MOE	59
  552591	5-9-2	2′-MOE	46
  552536	4-9-3	2′-MOE	19
  552592	5-9-2	2′-MOE	44
  552537	4-9-3	2′-MOE	26
  552593	5-9-2	2′-MOE	39
  552538	4-9-3	2′-MOE	54
  552594	5-9-2	2′-MOE	52
  552539	4-9-3	2′-MOE	50
  552595	5-9-2	2′-MOE	57
  552540	4-9-3	2′-MOE	60
  552596	5-9-2	2′-MOE	58
  552541	4-9-3	2′-MOE	68
  552597	5-9-2	2′-MOE	52
  552542	4-9-3	2′-MOE	63
  552598	5-9-2	2′-MOE	51
  552543	4-9-3	2′-MOE	44
  552600	5-9-2	2′-MOE	51
  552544	4-9-3	2′-MOE	45
  552602	5-9-2	2′-MOE	13
  552545	4-9-3	2′-MOE	42
  552604	5-9-2	2′-MOE	42
  552546	4-9-3	2′-MOE	46
  552606	5-9-2	2′-MOE	42
  552547	4-9-3	2′-MOE	38
  552608	5-9-2	2′-MOE	37
  552548	4-9-3	2′-MOE	49
  552610	5-9-2	2′-MOE	41
  552549	4-9-3	2′-MOE	34
  552612	5-9-2	2′-MOE	23
  552550	4-9-3	2′-MOE	13
  552614	5-9-2	2′-MOE	11
  552551	4-9-3	2′-MOE	8
  552616	5-9-2	2′-MOE	6

• TABLE 93
  Inhibition of viral Target-Z mRNA levels by chimeric
  antisense oligonucleotidesmeasured with RTS3371

  ISIS No	Motif	Wing chemistry	% inhibition

  146786	5-10-5	2′-MOE	47
  509934	5-10-5	2′-MOE	67
  552007	6-10-4	2′-MOE	53
  552039	7-10-3	2′-MOE	74
  552008	6-10-4	2′-MOE	47
  552040	7-10-3	2′-MOE	57
  552009	6-10-4	2′-MOE	70
  552041	7-10-3	2′-MOE	65
  552010	6-10-4	2′-MOE	51
  552042	7-10-3	2′-MOE	59
  552011	6-10-4	2′-MOE	47
  552043	7-10-3	2′-MOE	36
  552012	6-10-4	2′-MOE	62
  552044	7-10-3	2′-MOE	82
  552013	6-10-4	2′-MOE	72
  552045	7-10-3	2′-MOE	62
  552014	6-10-4	2′-MOE	73
  552046	7-10-3	2′-MOE	74
  552015	6-10-4	2′-MOE	66
  552047	7-10-3	2′-MOE	60
  552016	6-10-4	2′-MOE	67
  552048	7-10-3	2′-MOE	60
  552017	6-10-4	2′-MOE	72
  552049	7-10-3	2′-MOE	68
  552018	6-10-4	2′-MOE	89
  552050	7-10-3	2′-MOE	86
  552019	6-10-4	2′-MOE	87
  552051	7-10-3	2′-MOE	86
  551986	4-10-6	2′-MOE	64
  552020	6-10-4	2′-MOE	86
  552052	7-10-3	2′-MOE	87
  551987	4-10-6	2′-MOE	76
  552021	6-10-4	2′-MOE	84
  552053	7-10-3	2′-MOE	75
  551988	4-10-6	2′-MOE	5
  552005	5-10-5	2′-MOE	72
  552022	6-10-4	2′-MOE	80
  552054	7-10-3	2′-MOE	83
  551989	4-10-6	2′-MOE	64
  552023	6-10-4	2′-MOE	78
  552055	7-10-3	2′-MOE	57
  551990	4-10-6	2′-MOE	83
  552024	6-10-4	2′-MOE	89
  552056	7-10-3	2′-MOE	82
  551991	4-10-6	2′-MOE	0
  552025	6-10-4	2′-MOE	89
  552057	7-10-3	2′-MOE	89
  551992	4-10-6	2′-MOE	67
  552026	6-10-4	2′-MOE	84
  552058	7-10-3	2′-MOE	82
  551993	4-10-6	2′-MOE	78
  552027	6-10-4	2′-MOE	85
  552059	7-10-3	2′-MOE	85
  551994	4-10-6	2′-MOE	82
  552028	6-10-4	2′-MOE	82
  552060	7-10-3	2′-MOE	74
  551995	4-10-6	2′-MOE	81
  552029	6-10-4	2′-MOE	81
  552061	7-10-3	2′-MOE	81
  551996	4-10-6	2′-MOE	79
  552030	6-10-4	2′-MOE	86
  552062	7-10-3	2′-MOE	85
  551997	4-10-6	2′-MOE	80
  552031	6-10-4	2′-MOE	86
  551998	4-10-6	2′-MOE	74
  552032	6-10-4	2′-MOE	78
  551999	4-10-6	2′-MOE	79
  552033	6-10-4	2′-MOE	80
  552000	4-10-6	2′-MOE	84
  552006	5-10-5	2′-MOE	86
  552034	6-10-4	2′-MOE	81
  552001	4-10-6	2′-MOE	66
  552035	6-10-4	2′-MOE	55
  552002	4-10-6	2′-MOE	54
  552036	6-10-4	2′-MOE	58
  552003	4-10-6	2′-MOE	50
  552037	6-10-4	2′-MOE	43
  552004	4-10-6	2′-MOE	56
  552038	6-10-4	2′-MOE	66

• TABLE 94
  Inhibition of viral Target-Z mRNA levels by chimeric
  antisense oligonucleotidesmeasured with RTS3371

  ISIS No	Motif	Wing chemistry	% inhibition

  146786	5-10-5	2′-MOE	61
  510100	3-10-4	2′-MOE	66
  552168	3-9-5	2′-MOE	64
  552222	4-9-4	2′-MOE	76
  552169	3-9-5	2′-MOE	65
  552223	4-9-4	2′-MOE	41
  552170	3-9-5	2′-MOE	58
  552224	4-9-4	2′-MOE	58
  552171	3-9-5	2′-MOE	51
  552225	4-9-4	2′-MOE	49
  552172	3-9-5	2′-MOE	23
  552226	4-9-4	2′-MOE	36
  552173	3-9-5	2′-MOE	44
  552227	4-9-4	2′-MOE	20
  552174	3-9-5	2′-MOE	28
  552228	4-9-4	2′-MOE	29
  552175	3-9-5	2′-MOE	56
  552176	3-9-5	2′-MOE	66
  552177	3-9-5	2′-MOE	53
  552178	3-9-5	2′-MOE	57
  552179	3-9-5	2′-MOE	56
  552180	3-9-5	2′-MOE	51
  552181	3-9-5	2′-MOE	51
  552182	3-9-5	2′-MOE	63
  552183	3-9-5	2′-MOE	60
  552185	3-9-5	2′-MOE	67
  552186	3-9-5	2′-MOE	37
  552187	3-9-5	2′-MOE	68
  552188	3-9-5	2′-MOE	71
  552189	3-9-5	2′-MOE	51
  552190	3-9-5	2′-MOE	47
  552191	3-9-5	2′-MOE	50
  552192	3-9-5	2′-MOE	80
  552193	3-9-5	2′-MOE	73
  552194	3-9-5	2′-MOE	58
  552195	3-9-5	2′-MOE	60
  552196	3-9-5	2′-MOE	54
  552197	3-9-5	2′-MOE	64
  552198	3-9-5	2′-MOE	62
  552199	3-9-5	2′-MOE	57
  552200	3-9-5	2′-MOE	52
  552201	3-9-5	2′-MOE	73
  552202	3-9-5	2′-MOE	60
  552203	3-9-5	2′-MOE	60
  552204	3-9-5	2′-MOE	63
  552151	2-9-6	2′-MOE	71
  552205	3-9-5	2′-MOE	64
  552152	2-9-6	2′-MOE	69
  552206	3-9-5	2′-MOE	71
  552153	2-9-6	2′-MOE	63
  552207	3-9-5	2′-MOE	71
  552154	2-9-6	2′-MOE	56
  552208	3-9-5	2′-MOE	52
  552155	2-9-6	2′-MOE	61
  552209	3-9-5	2′-MOE	50
  552156	2-9-6	2′-MOE	40
  552210	3-9-5	2′-MOE	66
  552157	2-9-6	2′-MOE	45
  552211	3-9-5	2′-MOE	63
  552158	2-9-6	2′-MOE	66
  552212	3-9-5	2′-MOE	62
  552159	2-9-6	2′-MOE	68
  552213	3-9-5	2′-MOE	64
  552160	2-9-6	2′-MOE	78
  552214	3-9-5	2′-MOE	72
  552161	2-9-6	2′-MOE	57
  552215	3-9-5	2′-MOE	54
  552162	2-9-6	2′-MOE	54
  552216	3-9-5	2′-MOE	49
  552163	2-9-6	2′-MOE	65
  552217	3-9-5	2′-MOE	50
  552164	2-9-6	2′-MOE	48
  552218	3-9-5	2′-MOE	39
  552165	2-9-6	2′-MOE	46
  552219	3-9-5	2′-MOE	41
  552166	2-9-6	2′-MOE	42
  552220	3-9-5	2′-MOE	32
  552167	2-9-6	2′-MOE	47
  552221	3-9-5	2′-MOE	33

• TABLE 95
  Inhibition of viral Target-Z mRNA levels by chimeric
  antisense oligonucleotidesmeasured with RTS3371

  ISIS No	Motif	Wing chemistry	% inhibition

  146786	5-10-5	2′-MOE
  509934	5-10-5	2′-MOE	56
  510100	3-10-4	2′-MOE	69
  552071	8-10-2	2′-MOE	73
  552114	2-9-6	2′-MOE	64
  552115	2-9-6	2′-MOE	61
  552116	2-9-6	2′-MOE	53
  552117	2-9-6	2′-MOE	69
  552072	8-10-2	2′-MOE	39
  552118	2-9-6	2′-MOE	49
  552119	2-9-6	2′-MOE	49
  552120	2-9-6	2′-MOE	21
  552121	2-9-6	2′-MOE	27
  552073	8-10-2	2′-MOE	73
  552122	2-9-6	2′-MOE	48
  552074	8-10-2	2′-MOE	69
  552123	2-9-6	2′-MOE	68
  552075	8-10-2	2′-MOE	78
  552124	2-9-6	2′-MOE	47
  552076	8-10-2	2′-MOE	63
  552125	2-9-6	2′-MOE	72
  552077	8-10-2	2′-MOE	62
  552126	2-9-6	2′-MOE	64
  552078	8-10-2	2′-MOE	59
  552127	2-9-6	2′-MOE	65
  552079	8-10-2	2′-MOE	80
  552128	2-9-6	2′-MOE	78
  552080	8-10-2	2′-MOE	74
  552129	2-9-6	2′-MOE	68
  552130	2-9-6	2′-MOE	46
  552131	2-9-6	2′-MOE	61
  552132	2-9-6	2′-MOE	66
  552133	2-9-6	2′-MOE	78
  552081	8-10-2	2′-MOE	69
  552134	2-9-6	2′-MOE	68
  552135	2-9-6	2′-MOE	59
  552136	2-9-6	2′-MOE	39
  552137	2-9-6	2′-MOE	36
  552082	8-10-2	2′-MOE	86
  552138	2-9-6	2′-MOE	80
  552083	8-10-2	2′-MOE	85
  552139	2-9-6	2′-MOE	80
  552084	8-10-2	2′-MOE	86
  552140	2-9-6	2′-MOE	70
  552085	8-10-2	2′-MOE	83
  552141	2-9-6	2′-MOE	72
  552086	8-10-2	2′-MOE	83
  552142	2-9-6	2′-MOE	58
  552087	8-10-2	2′-MOE	77
  552143	2-9-6	2′-MOE	70
  552144	2-9-6	2′-MOE	66
  552145	2-9-6	2′-MOE	78
  552146	2-9-6	2′-MOE	63
  552088	8-10-2	2′-MOE	90
  552147	2-9-6	2′-MOE	80
  552089	8-10-2	2′-MOE	87
  552148	2-9-6	2′-MOE	74
  552090	8-10-2	2′-MOE	85
  552149	2-9-6	2′-MOE	79
  552091	8-10-2	2′-MOE	84
  552092	8-10-2	2′-MOE	86
  552093	8-10-2	2′-MOE	82
  552094	8-10-2	2′-MOE	84
  552063	7-10-3	2′-MOE	79
  552095	8-10-2	2′-MOE	85
  552064	7-10-3	2′-MOE	83
  552096	8-10-2	2′-MOE	88
  552065	7-10-3	2′-MOE	86
  552097	8-10-2	2′-MOE	90
  552066	7-10-3	2′-MOE	35
  552098	8-10-2	2′-MOE	86
  552067	7-10-3	2′-MOE	53
  552099	8-10-2	2′-MOE	66
  552068	7-10-3	2′-MOE	70
  552100	8-10-2	2′-MOE	67
  552069	7-10-3	2′-MOE	68
  552101	8-10-2	2′-MOE	65
  552070	7-10-3	2′-MOE	64
  552102	8-10-2	2′-MOE	54

• TABLE 96
  Inhibition of viral Target-Z mRNA levels by chimeric
  antisense oligonucleotidesmeasured with RTS3371

  ISIS No	Motif	Wing chemistry	% inhibition

  146786	5-10-5	2′-MOE	63
  510100	3-10-4	2′-MOE	59
  552330	6-9-2	2′-MOE	50
  552331	6-9-2	2′-MOE	46
  552332	6-9-2	2′-MOE	50
  552333	6-9-2	2′-MOE	48
  552334	6-9-2	2′-MOE	42
  552335	6-9-2	2′-MOE	30
  552336	6-9-2	2′-MOE	23
  552337	6-9-2	2′-MOE	42
  552338	6-9-2	2′-MOE	40
  552339	6-9-2	2′-MOE	50
  552340	6-9-2	2′-MOE	45
  552341	6-9-2	2′-MOE	44
  552342	6-9-2	2′-MOE	51
  552343	6-9-2	2′-MOE	44
  552344	6-9-2	2′-MOE	24
  552345	6-9-2	2′-MOE	41
  552346	6-9-2	2′-MOE	0
  552347	6-9-2	2′-MOE	75
  552348	6-9-2	2′-MOE	72
  552349	6-9-2	2′-MOE	65
  552350	6-9-2	2′-MOE	42
  552351	6-9-2	2′-MOE	45
  552352	6-9-2	2′-MOE	43
  552353	6-9-2	2′-MOE	20
  552354	6-9-2	2′-MOE	70
  552355	6-9-2	2′-MOE	66
  552356	6-9-2	2′-MOE	62
  552357	6-9-2	2′-MOE	53
  552358	6-9-2	2′-MOE	57
  552359	6-9-2	2′-MOE	46
  552360	6-9-2	2′-MOE	45
  552361	6-9-2	2′-MOE	44
  552308	5-9-3	2′-MOE	38
  552362	6-9-2	2′-MOE	51
  552309	5-9-3	2′-MOE	76
  552363	6-9-2	2′-MOE	73
  552310	5-9-3	2′-MOE	58
  552364	6-9-2	2′-MOE	66
  552311	5-9-3	2′-MOE	38
  552365	6-9-2	2′-MOE	64
  552150	2-9-6	2′-MOE	68
  552312	5-9-3	2′-MOE	75
  552366	6-9-2	2′-MOE	55
  552313	5-9-3	2′-MOE	66
  552367	6-9-2	2′-MOE	67
  552314	5-9-3	2′-MOE	56
  552368	6-9-2	2′-MOE	41
  552315	5-9-3	2′-MOE	46
  552369	6-9-2	2′-MOE	52
  552316	5-9-3	2′-MOE	55
  552370	6-9-2	2′-MOE	35
  552317	5-9-3	2′-MOE	53
  552371	6-9-2	2′-MOE	58
  552318	5-9-3	2′-MOE	59
  552372	6-9-2	2′-MOE	68
  552319	5-9-3	2′-MOE	56
  552373	6-9-2	2′-MOE	63
  552320	5-9-3	2′-MOE	62
  552374	6-9-2	2′-MOE	70
  552321	5-9-3	2′-MOE	63
  552375	6-9-2	2′-MOE	64
  552322	5-9-3	2′-MOE	52
  552376	6-9-2	2′-MOE	58
  552323	5-9-3	2′-MOE	45
  552377	6-9-2	2′-MOE	42
  552324	5-9-3	2′-MOE	49
  552378	6-9-2	2′-MOE	37
  552325	5-9-3	2′-MOE	48
  552379	6-9-2	2′-MOE	57
  552326	5-9-3	2′-MOE	50
  552380	6-9-2	2′-MOE	48
  552327	5-9-3	2′-MOE	13
  552381	6-9-2	2′-MOE	22
  552328	5-9-3	2′-MOE	9
  552382	6-9-2	2′-MOE	20
  552329	5-9-3	2′-MOE	18
  552383	6-9-2	2′-MOE	18

Example 53: Dose-Dependent Antisense Inhibition of Target-Z mRNA in HepG2 Cells
• Antisense oligonucleotides from the study described in Example 52 exhibiting in vitro inhibition of Target-Z mRNA were selected and tested at various doses in HepG2 cells. Cells were plated at a density of 28,000 cells per well and transfected using LipofectAMINE2000® with 9.26 nM, 27.78 nM, 83.33 nM, and 250.00 nM concentrations of antisense oligonucleotide, as specified in Table 97. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-Z mRNA levels were measured by quantitative real-time PCR. Target-Z primer probe set RTS3371 was used to measure mRNA levels. Target-Z mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-Z, relative to untreated control cells.
• As illustrated in Table 97, Target-Z mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells. ‘n/a’ indicates that the data for that dosage is not available.

• TABLE 97
  Dose-dependent antisense inhibition of
  human Target-Z in HepG2 cells

  	9.2593	27.7778	83.3333	250.0
  ISIS No	nM	nM	nM	nM

  146786	10	43	74	89
  509934	12	31	52	79
  509959	4	24	49	67
  510100	11	28	60	77
  510124	3	11	13	41
  551926	1	26	51	76
  551958	15	17	56	82
  551987	4	40	65	81
  551990	7	55	78	91
  551993	15	30	70	80
  551994	0	30	39	58
  551995	6	41	73	85
  551996	13	47	71	85
  551997	16	38	68	89
  551998	4	36	69	85
  551999	10	31	67	86
  552000	0	17	61	78
  552006	6	37	74	89
  552009	1	5	39	60
  552013	0	28	3	72
  552014	0	26	32	77
  552018	6	27	63	81
  552019	15	34	65	90
  552020	2	35	65	91
  552021	4	11	53	82
  552022	6	35	57	79
  552023	11	33	59	81
  552024	15	43	69	91
  552025	17	35	69	87
  552026	14	26	66	86
  552027	3	46	62	88
  552028	9	43	58	78
  552029	8	40	72	89
  552030	18	48	77	92
  552031	0	38	66	89
  552032	42	48	80	88
  552033	2	40	64	84
  552034	6	40	70	81
  552039	2	33	56	83
  552044	19	30	63	84
  552046	4	21	47	77
  552050	15	44	70	92
  552051	8	33	69	90
  552052	17	38	71	91
  552053	0	40	59	86
  552054	7	15	58	75
  552056	19	62	86	92
  552057	11	33	69	86
  552058	30	55	79	90
  552059	11	25	69	90
  552060	9	32	61	86
  552061	6	40	69	88
  552062	22	48	75	89
  552064	23	49	69	90
  552065	10	8	69	86
  552069	11	4	28	60
  552073	9	31	62	78
  552075	21	18	33	65
  552077	0	17	40	72
  552079	1	12	44	70
  552080	3	12	34	69
  552082	13	29	66	87
  552083	24	54	69	88
  552084	10	25	48	82
  552085	28	35	64	85
  552086	0	24	65	84
  552088	33	53	77	93
  552089	0	41	69	92
  552090	17	35	70	87
  552091	13	31	69	89
  552092	6	23	66	89
  552093	0	17	61	89
  552094	12	38	65	88
  552095	20	42	73	88
  552096	n/a	39	66	91
  552097	24	43	67	88
  552098	0	24	56	85
  552101	3	13	28	61
  552147	11	27	58	80
  552160	20	25	69	89
  552163	0	21	22	53
  552176	16	11	40	66
  552192	7	38	78	89
  552222	0	24	65	79
  552247	0	38	69	86
  552255	5	27	69	81
  552301	5	38	65	86
  552309	8	26	62	85
  552312	0	4	32	62
  552347	2	15	38	75
  552348	12	40	42	65
  552354	10	35	44	76
  552361	2	25	55	74
  552363	20	36	54	76
  552374	7	4	38	76
  552379	0	12	24	46
  552403	8	27	54	76
  552408	2	25	44	77
  552409	6	31	56	80
  552418	0	30	72	84
  552420	9	34	53	81
  552442	4	23	46	56
  552466	0	23	56	79
  552474	11	34	66	87
  552477	11	22	44	64
  552530	25	37	73	87
  552559	9	13	29	51

Example 54: Efficacy of Antisense Oligonucleotides Targeting Target-Z in Transgenic Mice
• Target-Z transgenic mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for their efficacy in this model.
Treatment
• Groups of 12 mice each were injected subcutaneously twice a week for 4 weeks with 50 mg/kg of ISIS 510106, ISIS 510116, ISIS 505347, or ISIS 509934. A control group of 12 mice was injected subcutaneously twice a week for 4 weeks with PBS. Mice were euthanized 48 hours after the last dose, and livers were harvested for further analysis.
DNA and RNA Analysis
• RNA was extracted from liver tissue for real-time PCR analysis of Target-Z DNA, using primer probe sets RTS3370, RTS3371, and RTS3372. The DNA levels were normalized to picogreen. Target-Z RNA samples were also assayed with primer probe sets RTS3370 and RTS3371 after RT-PCR analysis. The mRNA levels were normalized to RIBOGREEN®. The data is presented in Table 98, expressed as percent inhibition compared to the control group. As shown in Table 98, most of the antisense oligonucleotides achieved reduction of Target-Z DNA and RNA over the PBS control. Results are presented as percent inhibition of Target-Z mRNA or DNA, relative to control.

• TABLE 98
  Percent inhibition of Target-Z RNA and DNA in the liver of transgenic mice

  	%	%	%	%	%	%
  	inhibition	inhibition	inhibition	inhibition	inhibition	inhibition
  	DNA	DNA	DNA	RNA	RNA	RNA
  ISIS No	(RTS3370)	(RTS3371)	(RTS3372)	(RTS3370)	(RTS3371)	(RTS3372)

  510106	0	0	51	0	0	12
  510116	68	79	68	49	54	66
  505347	72	79	75	54	28	30
  509934	93	95	94	72	75	92

Example 55: Efficacy of Antisense Oligonucleotides Targeting Target-Z in Transgenic Mice
• Target-Z transgenic mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for their efficacy in this model.
Treatment
• Groups of 6 mice each were injected subcutaneously twice a week for 4 weeks with 50 mg/kg of ISIS 146779, ISIS 505358, ISIS 146786, ISIS 509974, ISIS 509958, or ISIS 509959. A control group of 10 mice was injected subcutaneously twice a week for 4 weeks with PBS. Mice were euthanized 48 hours after the last dose, and livers were harvested for further analysis.
DNA and RNA Analysis
• RNA was extracted from liver tissue for real-time PCR analysis of Target-Z DNA, using primer probe sets RTS3370. The DNA levels were normalized to picogreen. Target-Z RNA samples were also assayed with primer probe sets RTS3370 after RT-PCR analysis. The mRNA levels were normalized to RIBOGREEN®. The data is presented in Table 99, expressed as percent inhibition compared to the control group. As shown in Table 99, most of the antisense oligonucleotides achieved reduction of Target-Z DNA and RNA over the PBS control. Results are presented as percent inhibition of Target-Z mRNA or DNA, relative to control.

• TABLE 99
  Percent inhibition of Target-Z RNA and
  DNA in the liver of transgenic mice

  	%	%
  	inhibition	inhibition
  ISIS No	DNA	RNA

  146779	39	5
  505358	84	77
  146786	83	73
  509974	56	28
  509958	82	29
  509959	54	30

Example 56: Efficacy of Antisense Oligonucleotides Targeting Target-Z in Transgenic Mice
• Transgenic mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for their efficacy in this model.
Treatment
• Groups of 6 mice each were injected subcutaneously twice a week for 4 weeks with 25 mg/kg of ISIS 146786, ISIS 552176, and ISIS 552073. One group of 10 mice was injected subcutaneously twice a week for 4 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.
DNA and RNA Analysis
• RNA was extracted from liver tissue for real-time PCR analysis of Target-Z DNA, using primer probe set RTS3371. The DNA levels were normalized to picogreen. Target-Z RNA samples were also assayed with primer probe set RTS3371 after RT-PCR analysis. The mRNA levels were normalized to RIBOGREEN®. The data is presented in Table 100. Serum DNA samples were analyzed after the study period. The data is presented in Table 101, expressed relative to the levels measured in the control group. As shown in Tables 100 and 101, the antisense oligonucleotides achieved reduction of Target-Z DNA and RNA over the PBS control. Results are presented as percent inhibition of Target-Z mRNA or DNA, relative to control.

• TABLE 100
  Percent inhibition of Target-Z RNA and DNA in transgenic mice

  	Dose	% inhibition	% inhibition
  ISIS No	(mg/kg/wk)	of RNA	of DNA
  146786	50	81	91
  552073	50	39	22
  552176	50	55	56

• TABLE 101
  Serum levels of Target-Z DNA in transgenic
  mice, relative to control levels

  	Dose	Post-dose
  ISIS No	(mg/kg/wk)	DNA levels
  146786	50	0.1
  552073	50	2.9
  552176	50	2.1

Liver Function
• To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma concentrations of ALT were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.) (Nyblom, H. et al., Alcohol & Alcoholism 39: 336-339, 2004; Tietz N W (Ed): Clinical Guide to Laboratory Tests, 3rd ed. W. B. Saunders, Philadelphia, Pa., 1995). The results are presented in Table 102 expressed in IU/L. Both the ISIS oligonucleotides were considered tolerable in the mice, as demonstrated by their liver transaminase profile.

• TABLE 102
  ALT levels (IU/L) of transgenic mice

  	Dose
  	(mg/kg/wk)	ALT
  PBS	—	77
  ISIS 146786	50	21
  ISIS 552073	50	19
  ISIS 552176	50	27

Example 57: Efficacy of Antisense Oligonucleotides Targeting Target-Z in Transgenic Mice
• Transgenic mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for their efficacy in this model.
Treatment
• Groups of 6 mice each were injected subcutaneously twice a week for 4 weeks with 25 mg/kg of ISIS 146786, ISIS 552056, ISIS 552088, and ISIS 552309. One group of 10 mice was injected subcutaneously twice a week for 4 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.
DNA and RNA Analysis
• RNA was extracted from liver tissue for real-time PCR analysis of Target-Z DNA, using primer probe set RTS3371. The DNA levels were normalized to picogreen. Target-Z RNA samples were also assayed with primer probe set RTS3371 after RT-PCR analysis. The mRNA levels were normalized to RIBOGREEN®. As shown in Table 103, the antisense oligonucleotides achieved reduction of Target-Z DNA and RNA over the PBS control. Results are presented as percent inhibition of Target-Z mRNA or DNA, relative to control.

• TABLE 103
  Percent inhibition of Target-Z DNA and RNA in transgenic mice

  		%	%
  	Dose	inhibition	inhibition
  	(mg/kg/wk)	(RNA)	(DNA)

  ISIS 146786	50	60	90
  ISIS 552056	50	25	58
  ISIS 552088	50	8	0
  ISIS 552309	50	35	84

Example 58: Antisense Inhibition of Human Target-1 in HuVEC Cells
• Antisense oligonucleotides were designed targeting a human Target-1 nucleic acid and were tested for their effect on human Target-1 mRNA expression in vitro. The chimeric antisense oligonucleotides presented in Tables 104 and 105 were either 2-10-2 cEt gapmers or 3-10-3 cEt gapmers. The internucleoside linkages throughout each gapmer was phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer were 5′-methylcytosines.
• Cultured HuVEC cells at a density of 20,000 cells per well were transfected using electroporation with 1,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-1 mRNA levels were measured by quantitative real-time PCR. Target-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-1, relative to untreated control cells. All cEt gapmers and MOE gapmers were tested under the same conditions.
• “Human Target start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. “Human Target stop site” indicates the 3′-most nucleoside to which the gapmer is targeted human gene sequence. Each gapmer listed in Table 104 is targeted to human Target-1 mRNA. Each gapmer listed in Table 105 is targeted to the human Target-1 genomic sequence, truncated from nucleotides 4185000 to 4264000. Throughout the Examples, oligonucleotides having the same Sequence Number have the same nucleobase sequence.

• TABLE 104
  Inhibition of human Target-1 mRNA levels by cEt and MOE
  antisense oligonucleotides targeted to Target-1 mRNA

  	Human	Human
  ISIS	Start	Stop		Wing	%
  NO	Site	Site	Motif	Chemistry	inhibition

  481353	240	255	3-10-3	cEt	78
  481354	264	279	3-10-3	cEt	98
  481579	265	278	2-10-2	cEt	91
  481355	322	337	3-10-3	cEt	95
  481580	323	336	2-10-2	cEt	76
  481356	346	361	3-10-3	cEt	83
  481357	375	390	3-10-3	cEt	97
  481582	376	389	2-10-2	cEt	87
  481358	403	418	3-10-3	cEt	85
  481359	429	444	3-10-3	cEt	90
  481361	474	489	3-10-3	cEt	90
  481586	475	488	2-10-2	cEt	81
  481363	511	526	3-10-3	cEt	84
  481368	659	674	3-10-3	cEt	81
  481371	709	724	3-10-3	cEt	83
  481372	730	745	3-10-3	cEt	85
  481597	731	744	2-10-2	cEt	80
  481373	751	766	3-10-3	cEt	87
  481374	788	803	3-10-3	cEt	92
  481599	789	802	2-10-2	cEt	51
  481376	868	883	3-10-3	cEt	82
  481601	869	882	2-10-2	cEt	70
  481377	884	899	3-10-3	cEt	85
  481378	892	907	3-10-3	cEt	89
  481379	955	970	3-10-3	cEt	91
  481604	956	969	2-10-2	cEt	70
  481380	963	978	3-10-3	cEt	73
  481605	964	977	2-10-2	cEt	55
  481382	1045	1060	3-10-3	cEt	81
  481383	1053	1068	3-10-3	cEt	84
  481384	1098	1113	3-10-3	cEt	76
  481387	1225	1240	3-10-3	cEt	92
  481612	1226	1239	2-10-2	cEt	86
  481388	1269	1284	3-10-3	cEt	74
  481390	1305	1320	3-10-3	cEt	92
  481396	1480	1495	3-10-3	cEt	92
  481621	1481	1494	2-10-2	cEt	74
  481398	1542	1557	3-10-3	cEt	73
  481399	1563	1578	3-10-3	cEt	73
  481401	1589	1604	3-10-3	cEt	74
  481405	1641	1656	3-10-3	cEt	75
  481406	1691	1706	3-10-3	cEt	72
  481409	1795	1810	3-10-3	cEt	86
  481410	1825	1840	3-10-3	cEt	91
  481412	1858	1873	3-10-3	cEt	90
  481637	1859	1872	2-10-2	cEt	79
  481413	1866	1881	3-10-3	cEt	80
  481638	1867	1880	2-10-2	cEt	64
  481414	1888	1903	3-10-3	cEt	69
  481639	1889	1902	2-10-2	cEt	16
  481415	1896	1911	3-10-3	cEt	88
  481640	1897	1910	2-10-2	cEt	57
  337332	1898	1917	5-10-5	MOE	63
  481416	1901	1916	3-10-3	cEt	87
  481641	1902	1915	2-10-2	cEt	68
  337333	1903	1922	5-10-5	MOE	49
  481417	1903	1918	3-10-3	cEt	97
  481418	1904	1919	3-10-3	cEt	92
  481642	1904	1917	2-10-2	cEt	67
  481419	1905	1920	3-10-3	cEt	83
  481643	1905	1918	2-10-2	cEt	58
  481644	1906	1919	2-10-2	cEt	45
  481420	1948	1963	3-10-3	cEt	94
  481645	1949	1962	2-10-2	cEt	50
  481421	2021	2036	3-10-3	cEt	86
  481422	2036	2051	3-10-3	cEt	80
  481425	2115	2130	3-10-3	cEt	78
  481650	2116	2129	2-10-2	cEt	79
  481426	2131	2146	3-10-3	cEt	80
  481651	2132	2145	2-10-2	cEt	64
  481427	2155	2170	3-10-3	cEt	75
  481652	2156	2169	2-10-2	cEt	82
  481428	2164	2179	3-10-3	cEt	77
  481653	2165	2178	2-10-2	cEt	79
  481429	2172	2187	3-10-3	cEt	84
  481654	2173	2186	2-10-2	cEt	70
  481430	2190	2205	3-10-3	cEt	67
  481431	2206	2221	3-10-3	cEt	91
  481433	2256	2271	3-10-3	cEt	73
  481658	2257	2270	2-10-2	cEt	62
  481434	2266	2281	3-10-3	cEt	73
  345785	2267	2286	5-10-5	MOE	50
  481659	2267	2280	2-10-2	cEt	51
  481435	2269	2284	3-10-3	cEt	49
  481660	2270	2283	2-10-2	cEt	54
  481436	2275	2290	3-10-3	cEt	82
  481661	2276	2289	2-10-2	cEt	76
  481439	2371	2386	3-10-3	cEt	82
  481664	2372	2385	2-10-2	cEt	89
  481440	2387	2402	3-10-3	cEt	79
  481445	2439	2454	3-10-3	cEt	70
  481449	2503	2518	3-10-3	cEt	77
  481452	2631	2646	3-10-3	cEt	71
  481453	2681	2696	3-10-3	cEt	92
  481678	2682	2695	2-10-2	cEt	78
  481454	2702	2717	3-10-3	cEt	85
  481679	2703	2716	2-10-2	cEt	69
  481455	2722	2737	3-10-3	cEt	74
  481457	2779	2794	3-10-3	cEt	88
  481682	2780	2793	2-10-2	cEt	77
  481459	2908	2923	3-10-3	cEt	76
  481684	2909	2922	2-10-2	cEt	89
  481460	2943	2958	3-10-3	cEt	83
  481461	2969	2984	3-10-3	cEt	75
  481686	2970	2983	2-10-2	cEt	70
  481462	2984	2999	3-10-3	cEt	89
  481687	2985	2998	2-10-2	cEt	80
  481463	3001	3016	3-10-3	cEt	88
  481688	3002	3015	2-10-2	cEt	13
  481464	3016	3031	3-10-3	cEt	97
  481689	3017	3030	2-10-2	cEt	40
  481466	3047	3062	3-10-3	cEt	74
  481691	3048	3061	2-10-2	cEt	77
  481467	3097	3112	3-10-3	cEt	74
  481692	3098	3111	2-10-2	cEt	74
  481468	3112	3127	3-10-3	cEt	71
  481695	3462	3475	2-10-2	cEt	83
  481472	3491	3506	3-10-3	cEt	76
  481697	3492	3505	2-10-2	cEt	63
  481474	3521	3536	3-10-3	cEt	80
  481475	3536	3551	3-10-3	cEt	93
  481700	3537	3550	2-10-2	cEt	89
  481476	3551	3566	3-10-3	cEt	92
  481701	3552	3565	2-10-2	cEt	60
  481477	3567	3582	3-10-3	cEt	95
  481702	3568	3581	2-10-2	cEt	89
  481478	3585	3600	3-10-3	cEt	84
  481479	3600	3615	3-10-3	cEt	80
  481485	3717	3732	3-10-3	cEt	90
  481710	3718	3731	2-10-2	cEt	88
  481486	3730	3745	3-10-3	cEt	75
  481711	3731	3744	2-10-2	cEt	74
  481490	3833	3848	3-10-3	cEt	78
  481715	3834	3847	2-10-2	cEt	79
  481491	3848	3863	3-10-3	cEt	70
  481716	3849	3862	2-10-2	cEt	68
  481495	3940	3955	3-10-3	cEt	92
  481498	3992	4007	3-10-3	cEt	90
  481723	3993	4006	2-10-2	cEt	49
  481499	4007	4022	3-10-3	cEt	43
  481724	4008	4021	2-10-2	cEt	17
  481500	4022	4037	3-10-3	cEt	92
  481501	4048	4063	3-10-3	cEt	91
  481502	4063	4078	3-10-3	cEt	85
  481727	4064	4077	2-10-2	cEt	70
  481510	4237	4252	3-10-3	cEt	95
  481735	4238	4251	2-10-2	cEt	22
  481513	4290	4305	3-10-3	cEt	85
  481738	4291	4304	2-10-2	cEt	70
  481514	4305	4320	3-10-3	cEt	85
  481739	4306	4319	2-10-2	cEt	60
  481515	4325	4340	3-10-3	cEt	88
  481740	4326	4339	2-10-2	cEt	71
  481516	4364	4379	3-10-3	cEt	78
  481741	4365	4378	2-10-2	cEt	80
  481517	4394	4409	3-10-3	cEt	87
  481742	4395	4408	2-10-2	cEt	64
  481518	4425	4440	3-10-3	cEt	67
  481743	4426	4439	2-10-2	cEt	75
  481519	4437	4452	3-10-3	cEt	29
  481744	4438	4451	2-10-2	cEt	69
  481520	4439	4454	3-10-3	cEt	73
  481745	4440	4453	2-10-2	cEt	74
  481521	4459	4474	3-10-3	cEt	86
  481746	4460	4473	2-10-2	cEt	67
  481522	4474	4489	3-10-3	cEt	92
  481747	4475	4488	2-10-2	cEt	95
  481523	4489	4504	3-10-3	cEt	95
  481524	4530	4545	3-10-3	cEt	70
  481749	4531	4544	2-10-2	cEt	70
  481525	4541	4556	3-10-3	cEt	93
  481750	4542	4555	2-10-2	cEt	94
  481526	4543	4558	3-10-3	cEt	82
  481528	4579	4594	3-10-3	cEt	77
  481753	4580	4593	2-10-2	cEt	71
  481530	4630	4645	3-10-3	cEt	87
  481755	4631	4644	2-10-2	cEt	84
  481532	4664	4679	3-10-3	cEt	65
  481757	4665	4678	2-10-2	cEt	81
  481533	4666	4681	3-10-3	cEt	80
  481758	4667	4680	2-10-2	cEt	62
  481534	4693	4708	3-10-3	cEt	79
  481759	4694	4707	2-10-2	cEt	74
  481535	4767	4782	3-10-3	cEt	78
  481760	4768	4781	2-10-2	cEt	78
  481536	4782	4797	3-10-3	cEt	91
  481761	4783	4796	2-10-2	cEt	78
  481537	4830	4845	3-10-3	cEt	84
  481538	4844	4859	3-10-3	cEt	92
  481763	4845	4858	2-10-2	cEt	96
  481541	4934	4949	3-10-3	cEt	71

• TABLE 105
  Inhibition of human Target-1 mRNA levels by cEt and MOE
  antisense oligonucleotides targeted to Target-1 Genomic Sequence

  	Human	Human
  ISIS	Start	Stop		Wing	%
  NO	Site	Site	Motif	Chemistry	inhibition

  481543	1996	2011	3-10-3	cEt	84
  481768	1997	2010	2-10-2	cEt	95
  481546	2113	2128	3-10-3	cEt	70
  481771	2114	2127	2-10-2	cEt	75
  481547	2121	2136	3-10-3	cEt	87
  481548	2705	2720	3-10-3	cEt	78
  481549	6476	6491	3-10-3	cEt	96
  481774	6477	6490	2-10-2	cEt	56
  481553	10364	10379	3-10-3	cEt	96
  481554	15469	15484	3-10-3	cEt	86
  481779	15470	15483	2-10-2	cEt	60
  481555	24588	24603	3-10-3	cEt	73
  481780	24589	24602	2-10-2	cEt	60
  481353	40968	40983	3-10-3	cEt	78
  481354	40992	41007	3-10-3	cEt	98
  481579	40993	41006	2-10-2	cEt	91
  481355	41050	41065	3-10-3	cEt	95
  481580	41051	41064	2-10-2	cEt	76
  481356	41074	41089	3-10-3	cEt	83
  481581	41075	41088	2-10-2	cEt	31
  481357	42778	42793	3-10-3	cEt	97
  481582	42779	42792	2-10-2	cEt	87
  481358	42806	42821	3-10-3	cEt	85
  481359	42832	42847	3-10-3	cEt	90
  481360	42862	42877	3-10-3	cEt	75
  481585	42863	42876	2-10-2	cEt	77
  481361	42877	42892	3-10-3	cEt	90
  481586	42878	42891	2-10-2	cEt	81
  481368	50122	50137	3-10-3	cEt	81
  481559	50668	50683	3-10-3	cEt	72
  481784	50669	50682	2-10-2	cEt	79
  481371	50673	50688	3-10-3	cEt	83
  481372	50694	50709	3-10-3	cEt	85
  481597	50695	50708	2-10-2	cEt	80
  481373	50715	50730	3-10-3	cEt	87
  481376	51705	51720	3-10-3	cEt	82
  481601	51706	51719	2-10-2	cEt	70
  481378	51905	51920	3-10-3	cEt	89
  481603	51906	51919	2-10-2	cEt	60
  481379	51968	51983	3-10-3	cEt	91
  481604	51969	51982	2-10-2	cEt	70
  481380	51976	51991	3-10-3	cEt	73
  481382	55443	55458	3-10-3	cEt	81
  481383	55451	55466	3-10-3	cEt	84
  481384	55496	55511	3-10-3	cEt	76
  481387	55748	55763	3-10-3	cEt	92
  481612	55749	55762	2-10-2	cEt	86
  481388	55792	55807	3-10-3	cEt	74
  481390	57969	57984	3-10-3	cEt	92
  481396	60034	60049	3-10-3	cEt	92
  481621	60035	60048	2-10-2	cEt	74
  481398	63306	63321	3-10-3	cEt	73
  481399	63327	63342	3-10-3	cEt	73
  481401	63353	63368	3-10-3	cEt	74
  481405	64459	64474	3-10-3	cEt	75
  481409	64729	64744	3-10-3	cEt	86
  481410	64759	64774	3-10-3	cEt	91
  481411	65859	65874	3-10-3	cEt	72
  481412	65877	65892	3-10-3	cEt	90
  481637	65878	65891	2-10-2	cEt	79
  481413	65885	65900	3-10-3	cEt	80
  481638	65886	65899	2-10-2	cEt	64
  481566	66127	66142	3-10-3	cEt	62
  481791	66128	66141	2-10-2	cEt	73
  481415	66133	66148	3-10-3	cEt	88
  481640	66134	66147	2-10-2	cEt	57
  337332	66135	66154	5-10-5	MOE	63
  481416	66138	66153	3-10-3	cEt	87
  481641	66139	66152	2-10-2	cEt	68
  337333	66140	66159	5-10-5	MOE	49
  481417	66140	66155	3-10-3	cEt	97
  481418	66141	66156	3-10-3	cEt	92
  481642	66141	66154	2-10-2	cEt	67
  481419	66142	66157	3-10-3	cEt	83
  481420	66185	66200	3-10-3	cEt	94
  481645	66186	66199	2-10-2	cEt	50
  481421	66374	66389	3-10-3	cEt	86
  481422	66389	66404	3-10-3	cEt	80
  481423	66430	66445	3-10-3	cEt	69
  481424	66446	66461	3-10-3	cEt	70
  481425	66468	66483	3-10-3	cEt	78
  481650	66469	66482	2-10-2	cEt	79
  481426	66993	67008	3-10-3	cEt	80
  481651	66994	67007	2-10-2	cEt	64
  481427	67017	67032	3-10-3	cEt	75
  481652	67018	67031	2-10-2	cEt	82
  481428	67026	67041	3-10-3	cEt	77
  481653	67027	67040	2-10-2	cEt	79
  481429	67034	67049	3-10-3	cEt	84
  481654	67035	67048	2-10-2	cEt	70
  481430	67052	67067	3-10-3	cEt	67
  481431	67068	67083	3-10-3	cEt	91
  481433	67118	67133	3-10-3	cEt	73
  481658	67119	67132	2-10-2	cEt	62
  481434	67128	67143	3-10-3	cEt	73
  345785	67129	67148	5-10-5	MOE	50
  481659	67129	67142	2-10-2	cEt	51
  481435	67131	67146	3-10-3	cEt	49
  481660	67132	67145	2-10-2	cEt	54
  481436	67137	67152	3-10-3	cEt	82
  481661	67138	67151	2-10-2	cEt	76
  481568	72290	72305	3-10-3	cEt	85
  481793	72291	72304	2-10-2	cEt	93
  481569	72430	72445	3-10-3	cEt	62
  481794	72431	72444	2-10-2	cEt	81
  481570	72438	72453	3-10-3	cEt	79
  481440	72586	72601	3-10-3	cEt	79
  481443	72622	72637	3-10-3	cEt	78
  481444	72630	72645	3-10-3	cEt	66
  481445	72638	72653	3-10-3	cEt	70
  481670	72639	72652	2-10-2	cEt	60
  481449	73690	73705	3-10-3	cEt	77
  481452	73818	73833	3-10-3	cEt	71
  481453	73868	73883	3-10-3	cEt	92
  481678	73869	73882	2-10-2	cEt	78
  481454	73889	73904	3-10-3	cEt	85
  481679	73890	73903	2-10-2	cEt	69
  481455	73909	73924	3-10-3	cEt	74
  481457	73966	73981	3-10-3	cEt	88
  481682	73967	73980	2-10-2	cEt	77
  481459	74095	74110	3-10-3	cEt	76
  481684	74096	74109	2-10-2	cEt	89
  481460	74130	74145	3-10-3	cEt	83
  481685	74131	74144	2-10-2	cEt	36
  481461	74156	74171	3-10-3	cEt	75
  481686	74157	74170	2-10-2	cEt	70
  481462	74171	74186	3-10-3	cEt	89
  481687	74172	74185	2-10-2	cEt	80
  481463	74188	74203	3-10-3	cEt	88
  481688	74189	74202	2-10-2	cEt	13
  481464	74203	74218	3-10-3	cEt	97
  481689	74204	74217	2-10-2	cEt	40
  481466	74234	74249	3-10-3	cEt	74
  481691	74235	74248	2-10-2	cEt	77
  481467	74284	74299	3-10-3	cEt	74
  481692	74285	74298	2-10-2	cEt	74
  481468	74299	74314	3-10-3	cEt	71
  481695	74649	74662	2-10-2	cEt	83
  481472	74678	74693	3-10-3	cEt	76
  481697	74679	74692	2-10-2	cEt	63
  481474	74708	74723	3-10-3	cEt	80
  481475	74723	74738	3-10-3	cEt	93
  481700	74724	74737	2-10-2	cEt	89
  481476	74738	74753	3-10-3	cEt	92
  481701	74739	74752	2-10-2	cEt	60
  481477	74754	74769	3-10-3	cEt	95
  481702	74755	74768	2-10-2	cEt	89
  481478	74772	74787	3-10-3	cEt	84
  481479	74787	74802	3-10-3	cEt	80
  481485	74904	74919	3-10-3	cEt	90
  481710	74905	74918	2-10-2	cEt	88
  481486	74917	74932	3-10-3	cEt	75
  481711	74918	74931	2-10-2	cEt	74
  481487	74933	74948	3-10-3	cEt	66
  481490	75020	75035	3-10-3	cEt	78
  481715	75021	75034	2-10-2	cEt	79
  481491	75035	75050	3-10-3	cEt	70
  481716	75036	75049	2-10-2	cEt	68
  481492	75050	75065	3-10-3	cEt	61
  481495	75127	75142	3-10-3	cEt	92
  481720	75128	75141	2-10-2	cEt	63
  481498	75179	75194	3-10-3	cEt	90
  481500	75209	75224	3-10-3	cEt	92
  481725	75210	75223	2-10-2	cEt	88
  481501	75235	75250	3-10-3	cEt	91
  481502	75250	75265	3-10-3	cEt	85
  481727	75251	75264	2-10-2	cEt	70
  481510	75424	75439	3-10-3	cEt	95
  481735	75425	75438	2-10-2	cEt	22
  481513	75477	75492	3-10-3	cEt	85
  481738	75478	75491	2-10-2	cEt	70
  481514	75492	75507	3-10-3	cEt	85
  481739	75493	75506	2-10-2	cEt	60
  481515	75512	75527	3-10-3	cEt	88
  481740	75513	75526	2-10-2	cEt	71
  481516	75551	75566	3-10-3	cEt	78
  481741	75552	75565	2-10-2	cEt	80
  481517	75581	75596	3-10-3	cEt	87
  481742	75582	75595	2-10-2	cEt	64
  481518	75612	75627	3-10-3	cEt	67
  481743	75613	75626	2-10-2	cEt	75
  481744	75625	75638	2-10-2	cEt	69
  481520	75626	75641	3-10-3	cEt	73
  481745	75627	75640	2-10-2	cEt	74
  481521	75646	75661	3-10-3	cEt	86
  481746	75647	75660	2-10-2	cEt	67
  481522	75661	75676	3-10-3	cEt	92
  481747	75662	75675	2-10-2	cEt	95
  481523	75676	75691	3-10-3	cEt	95
  481524	75717	75732	3-10-3	cEt	70
  481749	75718	75731	2-10-2	cEt	70
  481525	75728	75743	3-10-3	cEt	93
  481750	75729	75742	2-10-2	cEt	94
  481526	75730	75745	3-10-3	cEt	82
  481528	75766	75781	3-10-3	cEt	77
  481753	75767	75780	2-10-2	cEt	71
  481530	75817	75832	3-10-3	cEt	87
  481755	75818	75831	2-10-2	cEt	84
  481757	75852	75865	2-10-2	cEt	81
  481533	75853	75868	3-10-3	cEt	80
  481758	75854	75867	2-10-2	cEt	62
  481534	75880	75895	3-10-3	cEt	79
  481759	75881	75894	2-10-2	cEt	74
  481535	75954	75969	3-10-3	cEt	78
  481760	75955	75968	2-10-2	cEt	78
  481536	75969	75984	3-10-3	cEt	91
  481761	75970	75983	2-10-2	cEt	78
  481537	76017	76032	3-10-3	cEt	84
  481538	76031	76046	3-10-3	cEt	92
  481763	76032	76045	2-10-2	cEt	96
  481539	76047	76062	3-10-3	cEt	19
  481541	76121	76136	3-10-3	cEt	71

Example 59 Antisense Inhibition of Murine Target-1 in b.END Cells
• Antisense oligonucleotides tested in the study described in Example 58 were also tested for their effects on Target-1 mRNA in b.END cells. Cultured b.END cells at a density of 20,000 cells per well were transfected using electroporation with 7,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-1 mRNA levels were measured by quantitative real-time PCR. Target-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®.
• Certain sequences complementary to the Target-1 mouse gene sequence showed good inhibition in b. END cells. Results are presented in Table 106 as percent inhibition of Target-1, relative to untreated control cells. The human oligonucleotides in Table 106 were compared to the mouse Target-1genomic sequence. “Mouse Target start site” indicates the 5′-most nucleotide to which the gapmer is targeted in the murine sequence. “Mouse Target stop site” indicates the 3′-most nucleotide to which the gapmer is targeted murine sequence.

• TABLE 106
  Inhibition of human Target-1 mRNA levels by certain
  antisense oligonucleotides complementary to Murine Target-1

  	Mouse	Mouse
  ISIS	Start	Stop	%
  NO	Site	Site	inhibition

  481549	5283	5298	96
  481553	9913	9928	94
  481768	3189	3202	91
  481356	30356	30371	83
  481548	4045	4060	82
  481554	14662	14677	82
  481426	48328	48343	82
  481580	30333	30346	81
  481412	47413	47428	81
  481417	47636	47651	81
  481418	47637	47652	80
  481355	30332	30347	79
  481396	43120	43135	79
  481416	47634	47649	79
  481420	47681	47696	79
  481358	32842	32857	78
  481363	33520	33535	78
  481570	51870	51885	78
  481382	37857	37872	77
  481378	36560	36575	76
  481431	48403	48418	76
  481453	53034	53049	76
  481621	43121	43134	75
  481641	47635	47648	75
  481637	47414	47427	74
  481380	36631	36646	73
  481574	53000	53015	73
  481601	36392	36405	71
  481419	47638	47653	71
  481371	35938	35953	70
  481642	47637	47650	70
  481542	3180	3195	69
  481547	3313	3328	69
  481772	3314	3327	69
  481362	32929	32944	69
  481653	48362	48375	69
  481786	38812	38825	68
  481415	47629	47644	68
  481543	3188	3203	67
  481793	51714	51727	67
  481443	52060	52075	67
  481684	53229	53242	67
  481398	45226	45241	66
  481560	36394	36409	65
  481643	47638	47651	65
  481430	48387	48402	65
  481440	52024	52039	65

Example 60: Tolerability of Antisense Oligonucleotides Targeting Target-1 in BALB/c Mice
• Forty antisense oligonucleotides exhibiting a high level of activity, selected from among the 452 compounds evaluated in Example 58, were further tested for in vivo tolerability.
• Groups of 2-4 male BALB/c mice were injected subcutaneously twice a week for 3 weeks with 25 mg/kg of ISIS antisense oligonucleotides. One group of 4 male BALB/c mice was injected subcutaneously twice a week for 3 weeks with PBS. This group of mice was utilized as a control group to which the treatment groups were compared. One day after the last dose, body weights were taken, mice were euthanized, and organs and plasma were harvested for further analysis.
• The body weights of the mice were measured pre-dose and at the end of the treatment period. Percent increase over the initial body weight was calculated. Liver, spleen, and kidney weights were measured at the end of the study and were compared to PBS treated mice.
• To evaluate the effect of ISIS oligonucleotides on metabolic function, plasma concentrations of transaminases and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma concentrations of ALT (alanine transaminase), AST (aspartate transaminase), and BUN were measured.
• Among the forty antisense oligonucleotides tested, certain antisense oligonucleotides, including ISIS 481374, ISIS 481390, ISIS 481420, ISIS 481431, ISIS 481453, ISIS 481464, ISIS 481475, ISIS 481495, ISIS 481500, ISIS 481501, ISIS 481525, ISIS 481548, ISIS 481549, ISIS 481597, ISIS 481695, ISIS 481700, ISIS 481702, ISIS 481710, ISIS 481725, ISIS 481750, and ISIS 481763 met tolerability thresholds for body weight, organ weight, ALT, AST, and BUN parameters.
Example 61: Dose-Dependent Antisense Inhibition of Human Target-1 in HuVEC Cells
• Gapmers from Examples 58 and 59 exhibiting in vitro inhibition of Target-1 were tested at various doses in HuVEC cells. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 31.25 nM, 62.5 nM, 125 nM, 250 nM, 500 nM, and 1,0000 nM concentrations of antisense oligonucleotide, as specified in Table 107. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-1 mRNA levels were measured by quantitative real-time PCR. Target-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-1, relative to untreated control cells.
• The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented in Table 107 and was calculated by plotting the concentrations of oligonucleotides used versus the percent inhibition of Target-1 mRNA expression achieved at each concentration and noting the concentration of oligonucleotide at which 50% inhibition of Target-1 mRNA expression was achieved compared to the control.

• TABLE 107
  Dose-dependent antisense inhibition of human
  Target-1 in HuVEC cells using electroporation

  	31.25	62.5	125.0	250.0	500.0	1000.0	IC50
  ISIS No	nM	nM	nM	nM	nM	nM	(μM)

  481355	19	15	36	61	75	89	0.18
  481374	25	42	52	72	82	88	0.10
  481390	17	37	44	60	73	86	0.15
  481420	23	20	40	60	81	92	0.16
  481453	21	37	52	69	79	88	0.12
  481464	57	73	81	90	94	94	<0.03
  481475	22	46	54	78	83	92	0.10
  481500	25	37	42	75	83	90	0.12
  481501	32	57	69	82	94	94	0.05
  481523	35	60	74	85	90	93	0.04
  481525	36	53	60	79	89	92	0.06
  481549	0	16	60	81	90	96	0.15
  481554	0	15	28	49	70	86	0.25
  481597	8	18	39	48	64	83	0.24
  481695	15	27	39	50	64	80	0.22
  481700	0	17	44	58	80	88	0.20
  481710	12	39	65	79	86	90	0.11
  481715	11	26	32	44	53	69	0.36
  481725	27	40	56	77	89	93	0.09
  481750	7	24	46	63	83	89	0.16
  481755	17	28	30	54	68	80	0.20
  481768	7	21	27	44	67	85	0.26

Example 62: Dose-Dependent Antisense Inhibition of Target-1 Following Free Uptake of Antisense Oligonucleotide in SK-BR-3 Cells
• Gapmers from Example 61 were tested at various doses in SK-BR-3 cells. Cells were plated at a density of 4,000 cells per well. Cells were incubated with 0.02 μM, 0.1 μM, 0.5 μM, 1 μM. 2.5 μM, and 10 μM concentrations of antisense oligonucleotide, as specified in Table 108. After approximately 24 hours, RNA was isolated from the cells and Target-1 mRNA levels were measured by quantitative real-time PCR. Target-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-1, relative to untreated control cells. The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented in Table 108.

• TABLE 108
  Dose-dependent antisense inhibition of human Target 1 by
  free-uptake of ISIS oligonucleotide by SK-BR-3 cells

  							IC50
  ISIS No	0.02 μM	0.1 μM	0.5 μM	1 μM	2.5 μM	10 μM	(μM)

  481374	10	18	18	16	8	25	15.9
  481390	0	10	11	12	40	72	3.2
  481453	14	13	27	45	58	79	1.3
  481464	23	32	57	70	85	93	0.5
  481475	0	0	35	49	72	88	1.0
  481500	7	9	26	45	49	75	1.7
  481501	0	0	4	5	53	65	2.7
  481523	9	24	56	67	83	92	0.5
  481525	0	17	13	15	32	68	4.4
  481549	0	0	0	16	33	54	8.2
  481597	1	0	11	14	22	44	10.6
  481695	0	0	0	0	0	0	—
  481710	5	0	10	13	27	66	6.0
  481725	29	45	47	39	39	63	2.6
  481750	19	24	36	42	71	80	1.1
  481763	30	38	51	63	81	89	0.6
  481768	12	5	34	25	32	35	12.4

Example 63: Dose-Dependent Antisense Inhibition of Target-1 Following Free Uptake of Antisense Oligonucleotide in U251-MG Cells
• Gapmers from Example 62 were further tested at various doses in U251-MG cells. Cells were plated at a density of 4,000 cells per well. Cells were incubated with 0.02 μM, 0.1 μM, 0.5 μM, 1 μM. 2.5 μM, and 10 μM concentrations of antisense oligonucleotide, as specified in Table 109. After approximately 24 hours, RNA was isolated from the cells and Target-1 mRNA levels were measured by quantitative real-time PCR. Target-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-1, relative to untreated control cells. The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented in Table 109.

• TABLE 109
  Dose-dependent antisense inhibition of Target-I mRNA
  levels by free-uptake of ISIS oligonucleotide by U251-MG cells

  							IC50
  ISIS No	0.02 μM	0.1 μM	0.5 μM	1 μM	2.5 μM	10 μM	(μM)

  481374	0	0	10	0	12	25	15.7
  481390	0	4	10	8	16	31	13.9
  481453	4	3	15	16	20	42	11.0
  481464	13	11	41	42	54	79	1.3
  481475	3	13	26	37	41	67	2.6
  481500	2	12	14	12	25	38	11.7
  481501	0	0	2	1	14	47	10.3
  481523	22	27	39	45	63	83	1.1
  481525	1	1	17	17	35	60	6.3
  481549	0	0	0	0	9	29	14.5
  481597	3	3	12	18	18	47	10.1
  481695	0	14	12	22	25	33	12.9
  481710	0	0	0	0	6	23	16.8
  481725	0	0	5	7	20	38	11.8
  481750	4	15	18	18	17	33	13.2
  481763	15	16	25	36	36	64	3.2
  481768	22	16	18	22	21	37	12.2

Example 64: Dose-Dependent Antisense Inhibition of Target-1 Following Free Uptake of Antisense Oligonucleotide in U251-MG Cells
• ISIS 481464 and ISIS 481549, from the studies described above, were further tested at different doses in U251-MG cells. Cells were plated at a density of 4,000 cells per well. Cells were incubated with 0.1 μM, 1 μM, 5 μM, 10 μM, and 20 μM concentrations of antisense oligonucleotide, as specified in Table 110. After approximately 24 hours, RNA was isolated from the cells and Target-1 mRNA levels were measured by quantitative real-time PCR. Target-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-1, relative to untreated control cells.

• TABLE 110
  Dose-dependent antisense inhibition of Target-1 mRNA levels
  by free-uptake of ISIS oligonucleotide by U251-MG cells

  						IC50
  ISIS No	0.1 μM	1 μM	5 μM	10 μM	20 μM	(μM)

  481464	0	30	69	80	79	2.3
  481549	0	0	26	35	38	>20

Example 65: Dose-Dependent Antisense Inhibition of Target-1 Following Free Uptake of Antisense Oligonucleotide in MDA-MB-231 Cells
• ISIS 481464 and ISIS 481549 were further tested at different doses in MDA-MB-231 cells. Cells were plated at a density of 4,000 cells per well. Cells were incubated with 0.02 μM, 0.2 μM, 1.0 μM, 5.0 μM, and 10.0 μM concentrations of antisense oligonucleotide, as specified in Table 111. After approximately 24 hours, RNA was isolated from the cells and Target-1 mRNA levels were measured by quantitative real-time PCR. Target-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-1, relative to untreated control cells.

• TABLE 111
  Dose-dependent antisense inhibition of Target-1 mRNA levels
  by free-uptake of ISIS oligonucleotide by MDA-MB-231 cells

  						IC50
  ISIS No	0.02 μM	0.2 μM	1.0 μM	5.0 μM	10.0 μM	(μM)

  481464	0	25	71	85	87	0.6
  481549	0	2	33	49	66	4.4

Example 66: Dose-Dependent Antisense Inhibition of Target-1 Following Free Uptake of Antisense Oligonucleotide in A431 Cells
• ISIS 481464 and ISIS 481549 were further tested at different doses in A431 cells. Cells were plated at a density of 4,000 cells per well. Cells were incubated with 0.02 μM, 0.2 μM, 1.0 μM, 5.0 μM, and 10.0 JIM concentrations of antisense oligonucleotide, as specified in Table 112. After approximately 24 hours, RNA was isolated from the cells and Target-1 mRNA levels were measured by quantitative real-time PCR. Target-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-1, relative to untreated control cells.

• TABLE 112
  Dose-dependent antisense inhibition of Target-1 mRNA levels
  by free-uptake of ISIS oligonucleotide by A431 cells

  						IC50
  ISIS No	0.02 μM	0.2 μM	1.0 μM	5.0 μM	10.0 μM	(μM)

  481464	79	93	98	98	98	<0.02
  481549	0	38	68	82	84	0.6

Example 67: Dose-Dependent Antisense Inhibition of Target-1 Following Free Uptake of Antisense Oligonucleotide in H460 Cells
• ISIS 481464 and ISIS 481549 were further tested at different doses in H460 cells. Cells were plated at a density of 4,000 cells per well. Cells were incubated with 0.02 μM, 0.2 μM, 1.0 μM, 5.0 μM, and 10.0 JIM concentrations of antisense oligonucleotide, as specified in Table 113. After approximately 24 hours, RNA was isolated from the cells and Target-1 mRNA levels were measured by quantitative real-time PCR. Target-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-1, relative to untreated control cells.

• TABLE 113
  Dose-dependent antisense inhibition of Target-1 mRNA levels
  by free-uptake of ISIS oligonucleotide by H460 cells

  						IC50
  ISIS No	0.02 μM	0.2 μM	1.0 μM	5.0 μM	10.0 μM	(μM)

  481464	46	89	96	97	98	0.01
  481549	8	53	78	96	98	0.23

Example 68: Antisense Inhibition of Human Target-1 in HuVEC Cells
• Antisense oligonucleotides were designed targeting a human Target-1 nucleic acid and were tested for their effect on human Target-1 mRNA expression in vitro. Cultured HuVEC cells at a density of 20,000 cells per well were transfected using electroporation with 1,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-1 mRNA levels were measured by quantitative real-time PCR. Target-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-1, relative to untreated control cells.
• The chimeric antisense oligonucleotides in Table 114 were designed as 3-10-3 MOE, deoxy, and cEt gapmers as indicated in the Table. The chemistry column of Table 114 presents the sugar motif of each gapmer, wherein ‘e’ indicates a 2′-MOE nucleoside, 1′ indicates a constrained ethyl (cEt) nucleoside, and indicates a 2′-deoxynucleoside. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5′-methylcytosines.
• “Human Target start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. “Human Target stop site” indicates the 3′-most nucleoside to which the gapmer is targeted in the human gene sequence. Each gapmer listed in Table 114 is targeted to human Target-1 mRNA.

• TABLE 114
  Inhibition of human Target-1 mRNA levels by chimeric
  antisense oligonucleotides targeted to Target-1 mRNA

  Human	Human
  Start	Stop	ISIS		%
  Site	Site	No	Chemistry	inhibition

  250	265	528204	e-e-e-d(10)-k-k-k	83
  251	266	528205	e-e-e-d(10)-k-k-k	72
  252	267	528206	e-e-e-d(10)-k-k-k	44
  253	268	528207	e-e-e-d(10)-k-k-k	49
  263	278	528208	e-e-e-d(10)-k-k-k	73
  264	279	528209	e-e-e-d(10)-k-k-k	81
  265	280	528210	e-e-e-d(10)-k-k-k	78
  266	281	528211	e-e-e-d(10)-k-k-k	72
  267	282	528212	e-e-e-d(10)-k-k-k	81
  268	283	528213	e-e-e-d(10)-k-k-k	46
  270	285	528214	e-e-e-d(10)-k-k-k	80
  271	286	528215	e-e-e-d(10)-k-k-k	69
  433	448	528269	e-e-e-d(10)-k-k-k	69
  434	449	528270	e-e-e-d(10)-k-k-k	73
  435	450	528271	e-e-e-d(10)-k-k-k	71
  867	882	528378	e-e-e-d(10)-k-k-k	72
  1146	1161	528501	e-e-e-d(10)-k-k-k	67
  1147	1162	528502	e-e-e-d(10)-k-k-k	76
  1153	1168	528503	e-e-e-d(10)-k-k-k	68
  1154	1169	528504	e-e-e-d(10)-k-k-k	69
  1155	1170	528505	e-e-e-d(10)-k-k-k	68
  1206	1221	528518	e-e-e-d(10)-k-k-k	80
  1207	1222	528519	e-e-e-d(10)-k-k-k	61
  1208	1223	528520	e-e-e-d(10)-k-k-k	63
  2699	2714	528833	e-e-e-d(10)-k-k-k	77
  2980	2995	528845	e-e-e-d(10)-k-k-k	65
  2981	2996	528846	e-e-e-d(10)-k-k-k	80
  2982	2997	528847	e-e-e-d(10)-k-k-k	72
  2983	2998	528848	e-e-e-d(10)-k-k-k	46
  2984	2999	528849	e-e-e-d(10)-k-k-k	59
  3001	3016	528850	e-e-e-d(10)-k-k-k	10
  3008	3023	528851	e-e-e-d(10)-k-k-k	61
  3010	3025	528852	e-e-e-d(10)-k-k-k	88
  3012	3027	528853	e-e-e-d(10)-k-k-k	91
  3016	3031	518349	e-e-e-d(10)-k-k-k	85
  e = 2′-MOE,
  k = cEt,
  d = 2′-deoxynucleoside

Example 69: Dose-Dependent Antisense Inhibition of Human Target-1 in HuVEC Cells
• Gapmers from the study described in Example 68, above, exhibiting in vitro inhibition of Target-1 were tested at various doses in HuVEC cells. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 23.4375 nM, 93.75 nM, 375.0 nM, and 1,500.0 nM concentrations of antisense oligonucleotide, as specified in Table 115. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-1 mRNA levels were measured by quantitative real-time PCR. Target-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-1, relative to untreated control cells.

• TABLE 115
  Dose-dependent antisense inhibition of
  human Target-1 in HuVEC cells

  	23.4375	93.75	375.0	1500.0	IC50
  ISIS No	nM	nM	nM	nM	(μM)

  518340	0	8	28	63	1.0
  518349	13	30	68	90	0.2
  528189	8	13	43	71	0.5
  528204	4	24	53	79	0.3
  528205	0	9	59	80	0.4
  528208	0	19	56	84	0.3
  528209	0	28	58	90	0.3
  528210	0	16	49	87	0.3
  528211	0	10	47	86	0.4
  528212	0	16	42	83	0.4
  528214	0	25	55	88	0.3
  528215	3	16	53	82	0.3
  528237	13	19	33	73	0.6
  528245	3	16	53	78	0.4
  528263	0	3	32	76	0.6
  528264	9	0	19	50	>1.5
  528268	0	7	25	63	1.0
  528269	0	11	39	77	0.5
  528270	5	9	48	79	0.4
  528271	0	14	37	81	0.5
  528327	0	0	26	72	0.8
  528347	0	2	25	69	0.9
  528357	0	17	36	69	0.6
  528389	0	3	19	82	0.7
  528501	0	17	40	69	0.6
  528502	0	10	35	76	0.6
  528503	3	1	38	70	0.7
  528504	0	19	45	72	0.5
  528505	0	7	41	73	0.6
  528518	0	24	51	81	0.3
  528534	0	8	32	72	0.7
  528539	0	7	39	73	0.6
  528557	0	9	26	53	>1.5
  528565	4	12	31	57	1.3
  528567	8	13	25	54	>1.5
  528569	9	19	37	60	0.8
  528574	5	17	32	62	0.9
  528622	10	4	29	68	0.9
  528623	0	13	24	62	1.1
  528626	1	0	34	68	0.8
  528627	22	19	30	64	1.0
  528664	0	14	37	74	0.5
  528675	0	10	28	62	1.0
  528689	0	16	33	65	0.7
  528691	0	3	34	61	0.9
  528695	1	4	36	66	0.8
  528697	3	15	39	72	0.5
  528710	13	16	28	63	1.0
  528711	8	13	14	62	>1.5
  528726	0	8	36	72	0.6
  528757	4	10	29	76	0.6
  528758	1	5	28	62	1.1
  528772	0	2	21	63	1.2
  528773	9	8	28	70	0.8
  528791	4	9	41	69	0.6
  528822	0	0	40	46	>1.5
  528833	0	23	47	82	0.4
  528846	10	19	49	85	0.3
  528847	0	19	45	75	0.4
  528852	5	33	66	93	0.2
  528853	19	46	77	95	0.1

Example 70: Antisense Inhibition of Human Target-1 in HuVEC Cells
• Antisense oligonucleotides were designed targeting a human Target-1 nucleic acid and were tested for their effect on human Target-1 mRNA expression in vitro. The chimeric antisense oligonucleotides in Tables 116 and 117 are gapmers16 or 17 nucleotides in length having various chemical modifications, as indicated in Tables 18 and 19, below. The chemistry column of Tables 116 and 117 provides the sugar motif of each gapmer, wherein ‘e’ indicates a 2′-MOE nucleoside, ‘k’ indicates a constrained ethyl (cEt) nucleoside, and indicates a 2′-deoxynucleoside. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5′-methylcytosines.
• Cultured HuVEC cells at a density of 20,000 cells per well were transfected using electroporation with 1,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-1 mRNA levels were measured by quantitative real-time PCR. Target-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-1, relative to untreated control cells.
• “Human Target start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. “Human Target stop site” indicates the 3′-most nucleoside to which the gapmer is targeted in the human gene sequence. Each gapmer listed in Table 116 is targeted to human Target-1 mRNA. Each gapmer listed in Table 117 is targeted to human Target-1 genomic sequence, truncated from nucleotides 4185000 to 4264000).

• TABLE 116
  Inhibition of human Target-1 mRNA levels by chimeric
  antisense oligonucleotides targeted to Target-1 mRNA 1

  Human

  Human

  Start	Stop			%
  Site	Site	ISIS No	Chemistry	inhibition

  728	743	530423	k-d(10)-k-e-k-e-e	70
  729	745	530053	e-e-k-d(10)-k-e-k-e	84
  729	744	530373	e-k-d(10)-k-e-k-e	85
  730	745	530121	e-k-k-d(10)-k-k-e	77
  730	745	530168	e-e-k-d(10)-k-k-e	75
  730	745	530218	e-d-k-d(10)-k-k-e	61
  730	745	530268	e-d-d-k-d(9)-k-k-e	76
  730	745	530318	e-e-e-e-d(9)-k-k-e	27
  786	801	530424	k-d(10)-k-e-k-e-e	42
  787	803	530058	e-e-k-d(10)-k-e-k-e	73
  787	802	530374	e-k-d(10)-k-e-k-e	71
  788	803	530122	e-k-k-d(10)-k-k-e	80
  788	803	530169	e-e-k-d(10)-k-k-e	72
  788	803	530219	e-d-k-d(10)-k-k-e	55
  788	803	530269	e-d-d-k-d(9)-k-k-e	76
  788	803	530319	e-e-e-e-d(9)-k-k-e	30
  897	912	528403	e-e-e-d(10)-k-k-k	72
  962	977	528424	e-e-e-d(10)-k-k-k	42
  1023	1038	528458	e-e-e-d(10)-k-k-k	70
  1899	1914	530425	k-d(10)-k-e-k-e-e	73
  1900	1916	530054	e-e-k-d(10)-k-e-k-e	75
  1900	1915	530375	e-k-d(10)-k-e-k-e	77
  1901	1916	530123	e-k-k-d(10)-k-k-e	86
  1901	1916	530170	e-e-k-d(10)-k-k-e	87
  1901	1916	530220	e-d-k-d(10)-k-k-e	74
  1901	1916	530270	e-d-d-k-d(9)-k-k-e	87
  1901	1916	530320	e-e-e-e-d(9)-k-k-e	17
  1946	1961	530426	k-d(10)-k-e-k-e-e	55
  1947	1963	530059	e-e-k-d(10)-k-e-k-e	73
  1947	1962	530376	e-k-d(10)-k-e-k-e	77
  1948	1963	530124	e-k-k-d(10)-k-k-e	79
  1948	1963	530171	e-e-k-d(10)-k-k-e	69
  1948	1963	530221	e-d-k-d(10)-k-k-e	64
  1948	1963	530271	e-d-d-k-d(9)-k-k-e	73
  1948	1963	530321	e-e-e-e-d(9)-k-k-e	44
  2204	2219	530427	k-d(10)-k-e-k-e-e	43
  2205	2221	530060	e-e-k-d(10)-k-e-k-e	77
  2205	2220	530377	e-k-d(10)-k-e-k-e	66
  2206	2221	530125	e-k-k-d(10)-k-k-e	65
  2206	2221	530172	e-e-k-d(10)-k-k-e	59
  2206	2221	530222	e-d-k-d(10)-k-k-e	48
  2206	2221	530272	e-d-d-k-d(9)-k-k-e	63
  2206	2221	530322	e-e-e-e-d(9)-k-k-e	55
  2681	2696	530126	e-k-k-d(10)-k-k-e	70
  2681	2696	530173	e-e-k-d(10)-k-k-e	62
  2681	2696	530223	e-d-k-d(10)-k-k-e	44
  2681	2696	530273	e-d-d-k-d(9)-k-k-e	63
  2681	2696	530323	e-e-e-e-d(9)-k-k-e	63
  3012	3027	530513	k-d(10)-k-e-k-e-e	88
  3013	3028	530507	e-k-d(10)-k-e-k-e	86
  3013	3028	530514	k-d(10)-k-e-k-e-e	80
  3014	3029	530430	k-d(10)-k-e-k-e-e	87
  3014	3029	530468	e-k-k-d(10)-k-k-e	81
  3014	3029	530476	e-e-k-d(10)-k-k-e	82
  3014	3029	530484	e-d-k-d(10)-k-k-e	74
  3014	3029	530492	e-d-d-k-d(9)-k-k-e	83
  3014	3029	530500	e-e-e-e-d(9)-k-k-e	56
  3014	3029	530508	e-k-d(10)-k-e-k-e	83
  3015	3031	530062	e-e-k-d(10)-k-e-k-e	94
  3015	3030	530380	e-k-d(10)-k-e-k-e	94
  3015	3030	530469	e-k-k-d(10)-k-k-e	91
  3015	3030	530477	e-e-k-d(10)-k-k-e	87
  3015	3030	530485	e-d-k-d(10)-k-k-e	87
  3015	3030	530493	e-d-d-k-d(9)-k-k-e	81
  3015	3030	530501	e-e-e-e-d(9)-k-k-e	74
  3015	3030	530515	k-d(10)-k-e-k-e-e	87
  3016	3031	481464	k-k-k-d(10)-k-k-k	93
  3016	3031	518349	e-e-e-d(10)-k-k-k	58
  3016	3031	519637	e-k-k-d(10)-k-k-e	96
  3016	3031	530175	e-e-k-d(10)-k-k-e	93
  3016	3031	530225	e-d-k-d(10)-k-k-e	85
  3016	3031	530275	e-d-d-k-d(9)-k-k-e	91
  3016	3031	530325	e-e-e-e-d(9)-k-k-e	91
  3017	3032	530470	e-k-k-d(10)-k-k-e	91
  3017	3032	530478	e-e-k-d(10)-k-k-e	87
  3017	3032	530486	e-d-k-d(10)-k-k-e	84
  3017	3032	530494	e-d-d-k-d(9)-k-k-e	60
  3017	3032	530502	e-e-e-e-d(9)-k-k-e	64
  3017	3032	530509	e-k-d(10)-k-e-k-e	80
  3018	3033	530471	e-k-k-d(10)-k-k-e	83
  3018	3033	530479	e-e-k-d(10)-k-k-e	74
  3018	3033	530487	e-d-k-d(10)-k-k-e	71
  3018	3033	530495	e-d-d-k-d(9)-k-k-e	68
  3018	3033	530503	e-e-e-e-d(9)-k-k-e	53
  3460	3476	530055	e-e-k-d(10)-k-e-k-e	45
  3460	3475	530381	e-k-d(10)-k-e-k-e	74
  3461	3476	530128	e-k-k-d(10)-k-k-e	52
  3461	3476	530176	e-e-k-d(10)-k-k-e	66
  3461	3476	530226	e-d-k-d(10)-k-k-e	51
  3461	3476	530276	e-d-d-k-d(9)-k-k-e	70
  3461	3476	530326	e-e-e-e-d(9)-k-k-e	52
  3595	3610	530390	k-d(10)-k-e-k-e-e	83
  3596	3611	530340	e-k-d(10)-k-e-k-e	89
  3597	3612	528869	e-e-e-d(10)-k-k-k	83
  3597	3612	530088	e-k-k-d(10)-k-k-e	90
  3597	3612	530135	e-e-k-d(10)-k-k-e	91
  3597	3612	530185	e-d-k-d(10)-k-k-e	85
  3597	3612	530235	e-d-d-k-d(9)-k-k-e	28
  3597	3612	530285	e-e-e-e-d(9)-k-k-e	86
  3597	3612	530391	k-d(10)-k-e-k-e-e	79
  3598	3614	530021	e-e-k-d(10)-k-e-k-e	87
  3598	3613	530341	e-k-d(10)-k-e-k-e	88
  3599	3614	530089	e-k-k-d(10)-k-k-e	71
  3599	3614	530136	e-e-k-d(10)-k-k-e	66
  3599	3614	530186	e-d-k-d(10)-k-k-e	51
  3599	3614	530236	e-d-d-k-d(9)-k-k-e	74
  3599	3614	530286	e-e-e-e-d(9)-k-k-e	56
  3715	3731	530022	e-e-k-d(10)-k-e-k-e	80
  3715	3730	530342	e-k-d(10)-k-e-k-e	70
  3715	3730	530393	k-d(10)-k-e-k-e-e	46
  3716	3732	530023	e-e-k-d(10)-k-e-k-e	74
  3716	3731	530090	e-k-k-d(10)-k-k-e	78
  3716	3731	530137	e-e-k-d(10)-k-k-e	76
  3716	3731	530187	e-d-k-d(10)-k-k-e	68
  3716	3731	530237	e-d-d-k-d(9)-k-k-e	36
  3716	3731	530287	e-e-e-e-d(9)-k-k-e	56
  3716	3731	530343	e-k-d(10)-k-e-k-e	68
  3716	3731	530394	k-d(10)-k-e-k-e-e	49
  3717	3732	518343	e-e-e-d(10)-k-k-k	5
  3717	3733	530024	e-e-k-d(10)-k-e-k-e	79
  3717	3732	530091	e-k-k-d(10)-k-k-e	81
  3717	3732	530138	e-e-k-d(10)-k-k-e	81
  3717	3732	530188	e-d-k-d(10)-k-k-e	78
  3717	3732	530238	e-d-d-k-d(9)-k-k-e	29
  3717	3732	530288	e-e-e-e-d(9)-k-k-e	69
  3717	3732	530344	e-k-d(10)-k-e-k-e	85
  3718	3733	530092	e-k-k-d(10)-k-k-e	85
  3718	3733	530139	e-e-k-d(10)-k-k-e	79
  3718	3733	530189	e-d-k-d(10)-k-k-e	77
  3718	3733	530239	e-d-d-k-d(9)-k-k-e	61
  3718	3733	530289	e-e-e-e-d(9)-k-k-e	75
  3720	3735	528880	e-e-e-d(10)-k-k-k	65
  4022	4037	518344	e-e-e-d(10)-k-k-k	89
  4234	4249	530395	k-d(10)-k-e-k-e-e	71
  4235	4250	528936	e-e-e-d(10)-k-k-k	71
  4235	4251	530025	e-e-k-d(10)-k-e-k-e	90
  4235	4250	530345	e-k-d(10)-k-e-k-e	93
  4235	4250	530396	k-d(10)-k-e-k-e-e	71
  4236	4251	528937	e-e-e-d(10)-k-k-k	73
  4236	4252	530026	e-e-k-d(10)-k-e-k-e	87
  4236	4251	530093	e-k-k-d(10)-k-k-e	95
  4236	4251	530140	e-e-k-d(10)-k-k-e	89
  4236	4251	530190	e-d-k-d(10)-k-k-e	82
  4236	4251	530240	e-d-d-k-d(9)-k-k-e	50
  4236	4251	530290	e-e-e-e-d(9)-k-k-e	69
  4236	4251	530346	e-k-d(10)-k-e-k-e	89
  4237	4252	528938	e-e-e-d(10)-k-k-k	72
  4237	4252	530094	e-k-k-d(10)-k-k-e	88
  4237	4252	530141	e-e-k-d(10)-k-k-e	80
  4237	4252	530191	e-d-k-d(10)-k-k-e	74
  4237	4252	530241	e-d-d-k-d(9)-k-k-e	53
  4237	4252	530291	e-e-e-e-d(9)-k-k-e	68
  4242	4257	528942	e-e-e-d(10)-k-k-k	77
  4320	4335	528945	e-e-e-d(10)-k-k-k	74
  4439	4454	530096	e-k-k-d(10)-k-k-e	72
  4439	4454	530143	e-e-k-d(10)-k-k-e	74
  4439	4454	530193	e-d-k-d(10)-k-k-e	62
  4439	4454	530243	e-d-d-k-d(9)-k-k-e	34
  4439	4454	530293	e-e-e-e-d(9)-k-k-e	59
  4488	4504	530063	e-e-k-d(10)-k-e-k-e	74
  4488	4503	530382	e-k-d(10)-k-e-k-e	17
  4488	4503	530465	e-k-k-d(10)-k-k-e	63
  4488	4503	530473	e-e-k-d(10)-k-k-e	45
  4488	4503	530481	e-d-k-d(10)-k-k-e	14
  4488	4503	530489	e-d-d-k-d(9)-k-k-e	13
  4488	4503	530497	e-e-e-e-d(9)-k-k-e	7
  4488	4503	530512	k-d(10)-k-e-k-e-e	21
  4489	4504	519638	e-k-k-d(10)-k-k-e	86
  4489	4504	530177	e-e-k-d(10)-k-k-e	71
  4489	4504	530227	e-d-k-d(10)-k-k-e	51
  4489	4504	530277	e-d-d-k-d(9)-k-k-e	70
  4489	4504	530327	e-e-e-e-d(9)-k-k-e	61
  4490	4505	530466	e-k-k-d(10)-k-k-e	82
  4490	4505	530474	e-e-k-d(10)-k-k-e	62
  4490	4505	530482	e-d-k-d(10)-k-k-e	53
  4490	4505	530490	e-d-d-k-d(9)-k-k-e	42
  4490	4505	530498	e-e-e-e-d(9)-k-k-e	45
  4490	4505	530506	e-k-d(10)-k-e-k-e	70
  4539	4554	530433	k-d(10)-k-e-k-e-e	62
  4540	4555	528958	e-e-e-d(10)-k-k-k	66
  4540	4556	530056	e-e-k-d(10)-k-e-k-e	73
  4540	4555	530383	e-k-d(10)-k-e-k-e	64
  4541	4556	518345	e-e-e-d(10)-k-k-k	80
  4541	4556	519636	e-k-k-d(10)-k-k-e	90
  4541	4556	530178	e-e-k-d(10)-k-k-e	86
  4541	4556	530228	e-d-k-d(10)-k-k-e	77
  4541	4556	530278	e-d-d-k-d(9)-k-k-e	86
  4541	4556	530328	e-e-e-e-d(9)-k-k-e	80
  4542	4557	528959	e-e-e-d(10)-k-k-k	73
  4621	4636	528973	e-e-e-d(10)-k-k-k	71
  4782	4797	530329	e-e-e-e-d(9)-k-k-e	61
  4813	4829	530032	e-e-k-d(10)-k-e-k-e	74
  4813	4828	530099	e-k-k-d(10)-k-k-e	73
  4813	4828	530146	e-e-k-d(10)-k-k-e	70
  4813	4828	530196	e-d-k-d(10)-k-k-e	67
  4813	4828	530246	e-d-d-k-d(9)-k-k-e	39
  4813	4828	530296	e-e-e-e-d(9)-k-k-e	67
  4813	4828	530352	e-k-d(10)-k-e-k-e	67
  4814	4829	530100	e-k-k-d(10)-k-k-e	77
  4814	4829	530147	e-e-k-d(10)-k-k-e	84
  4814	4829	530197	e-d-k-d(10)-k-k-e	71
  4814	4829	530247	e-d-d-k-d(9)-k-k-e	53
  4814	4829	530297	e-e-e-e-d(9)-k-k-e	75
  4814	4829	530403	k-d(10)-k-e-k-e-e	77
  4815	4831	530033	e-e-k-d(10)-k-e-k-e	65
  4815	4830	530353	e-k-d(10)-k-e-k-e	83
  4816	4831	530101	e-k-k-d(10)-k-k-e	59
  4816	4831	530148	e-e-k-d(10)-k-k-e	79
  4816	4831	530198	e-d-k-d(10)-k-k-e	54
  4816	4831	530248	e-d-d-k-d(9)-k-k-e	32
  4816	4831	530298	e-e-e-e-d(9)-k-k-e	73
  4827	4842	530404	k-d(10)-k-e-k-e-e	67
  4828	4844	530034	e-e-k-d(10)-k-e-k-e	69
  4828	4843	530354	e-k-d(10)-k-e-k-e	85
  4828	4843	530405	k-d(10)-k-e-k-e-e	55
  4829	4845	530035	e-e-k-d(10)-k-e-k-e	69
  4829	4844	530102	e-k-k-d(10)-k-k-e	71
  4829	4844	530149	e-e-k-d(10)-k-k-e	70
  4829	4844	530199	e-d-k-d(10)-k-k-e	58
  4829	4844	530249	e-d-d-k-d(9)-k-k-e	47
  4829	4844	530299	e-e-e-e-d(9)-k-k-e	47
  4829	4844	530355	e-k-d(10)-k-e-k-e	72
  4830	4845	530103	e-k-k-d(10)-k-k-e	77
  4830	4845	530150	e-e-k-d(10)-k-k-e	73
  4830	4845	530200	e-d-k-d(10)-k-k-e	63
  4830	4845	530250	e-d-d-k-d(9)-k-k-e	59
  4830	4845	530300	e-e-e-e-d(9)-k-k-e	65
  4842	4857	530435	k-d(10)-k-e-k-e-e	62
  4843	4859	530057	e-e-k-d(10)-k-e-k-e	69
  4843	4858	530385	e-k-d(10)-k-e-k-e	70
  4844	4859	529005	e-e-e-d(10)-k-k-k	64
  4844	4859	530130	e-k-k-d(10)-k-k-e	85
  4844	4859	530180	e-e-k-d(10)-k-k-e	82
  4844	4859	530230	e-d-k-d(10)-k-k-e	65
  4844	4859	530280	e-d-d-k-d(9)-k-k-e	75
  4844	4859	530330	e-e-e-e-d(9)-k-k-e	52
  e = 2′-MOE,
  k = cEt,
  d = 2′-deoxynucleoside

• TABLE 117
  Inhibition of human Target-1 mRNA levels by chimeric antisense
  oligonucleotides targeted to Target-1 Genomic Sequence

  Human	Human
  Start	Stop			%
  Site	Site	ISIS No	Chemistry	inhibition
  1794	1809	529022	e-e-e-d(10)-k-k-k	69
  1796	1811	529023	e-e-e-d(10)-k-k-k	72
  1906	1921	529024	e-e-e-d(10)-k-k-k	64
  1907	1922	529025	e-e-e-d(10)-k-k-k	73
  1966	1981	529026	e-e-e-d(10)-k-k-k	78
  1968	1983	529027	e-e-e-d(10)-k-k-k	92
  2409	2425	530038	e-e-k-d(10)-k-e-k-e	71
  2409	2424	530358	e-k-d(10)-k-e-k-e	46
  2410	2425	530106	e-k-k-d(10)-k-k-e	70
  2410	2425	530153	e-e-k-d(10)-k-k-e	50
  2410	2425	530203	e-d-k-d(10)-k-k-e	43
  2410	2425	530253	e-d-d-k-d(9)-k-k-e	33
  2410	2425	530303	e-e-e-e-d(9)-k-k-e	40
  2670	2686	530039	e-e-k-d(10)-k-e-k-e	73
  2670	2685	530359	e-k-d(10)-k-e-k-e	82
  2671	2686	530107	e-k-k-d(10)-k-k-e	77
  2671	2686	530154	e-e-k-d(10)-k-k-e	57
  2671	2686	530204	e-d-k-d(10)-k-k-e	28
  2671	2686	530254	e-d-d-k-d(9)-k-k-e	3
  2671	2686	530304	e-e-e-e-d(9)-k-k-e	22
  2703	2718	530429	k-d(10)-k-e-k-e-e	60
  2704	2720	530065	e-e-k-d(10)-k-e-k-e	70
  2704	2719	530379	e-k-d(10)-k-e-k-e	54
  2705	2720	530127	e-k-k-d(10)-k-k-e	80
  2705	2720	530174	e-e-k-d(10)-k-k-e	69
  2705	2720	530224	e-d-k-d(10)-k-k-e	32
  2705	2720	530274	e-d-d-k-d(9)-k-k-e	38
  2705	2720	530324	e-e-e-e-d(9)-k-k-e	32
  5000	5015	530410	k-d(10)-k-e-k-e-e	53
  5001	5017	530040	e-e-k-d(10)-k-e-k-e	67
  5001	5016	530360	e-k-d(10)-k-e-k-e	70
  5002	5017	530108	e-k-k-d(10)-k-k-e	70
  5002	5017	530155	e-e-k-d(10)-k-k-e	53
  5002	5017	530205	e-d-k-d(10)-k-k-e	44
  5002	5017	530255	e-d-d-k-d(9)-k-k-e	33
  5002	5017	530305	e-e-e-e-d(9)-k-k-e	22
  5699	5714	530411	k-d(10)-k-e-k-e-e	91
  5700	5716	530041	e-e-k-d(10)-k-e-k-e	89
  5700	5715	530361	e-k-d(10)-k-e-k-e	88
  5701	5716	530109	e-k-k-d(10)-k-k-e	89
  5701	5716	530156	e-e-k-d(10)-k-k-e	91
  5701	5716	530206	e-d-k-d(10)-k-k-e	89
  5701	5716	530256	e-d-d-k-d(9)-k-k-e	33
  5701	5716	530306	e-e-e-e-d(9)-k-k-e	83
  6475	6491	530066	e-e-k-d(10)-k-e-k-e	82
  6475	6490	530386	e-k-d(10)-k-e-k-e	53
  6476	6491	530131	e-k-k-d(10)-k-k-e	97
  6476	6491	530181	e-e-k-d(10)-k-k-e	82
  6476	6491	530231	e-d-k-d(10)-k-k-e	75
  6476	6491	530281	e-d-d-k-d(9)-k-k-e	69
  6476	6491	530331	e-e-e-e-d(9)-k-k-e	53
  6846	6861	529039	e-e-e-d(10)-k-k-k	31
  8079	8095	530042	e-e-k-d(10)-k-e-k-e	78
  8079	8094	530362	e-k-d(10)-k-e-k-e	76
  8080	8095	530110	e-k-k-d(10)-k-k-e	84
  8080	8095	530157	e-e-k-d(10)-k-k-e	69
  8080	8095	530207	e-d-k-d(10)-k-k-e	55
  8080	8095	530257	e-d-d-k-d(9)-k-k-e	39
  8080	8095	530307	e-e-e-e-d(9)-k-k-e	77
  9123	9138	530413	k-d(10)-k-e-k-e-e	73
  9862	9877	530414	k-d(10)-k-e-k-e-e	61
  9863	9879	530044	e-e-k-d(10)-k-e-k-e	78
  9863	9878	530364	e-k-d(10)-k-e-k-e	59
  9864	9879	530112	e-k-k-d(10)-k-k-e	84
  9864	9879	530159	e-e-k-d(10)-k-k-e	69
  9864	9879	530209	e-d-k-d(10)-k-k-e	54
  9864	9879	530259	e-d-d-k-d(9)-k-k-e	57
  9864	9879	530309	e-e-e-e-d(9)-k-k-e	46
  9864	9879	530415	k-d(10)-k-e-k-e-e	51
  9865	9881	530045	e-e-k-d(10)-k-e-k-e	73
  9865	9880	530365	e-k-d(10)-k-e-k-e	78
  9866	9881	530113	e-k-k-d(10)-k-k-e	60
  9866	9881	530160	e-e-k-d(10)-k-k-e	54
  9866	9881	530210	e-d-k-d(10)-k-k-e	28
  9866	9881	530260	e-d-d-k-d(9)-k-k-e	0
  9866	9881	530310	e-e-e-e-d(9)-k-k-e	26
  9873	9888	530416	k-d(10)-k-e-k-e-e	57
  9874	9890	530046	e-e-k-d(10)-k-e-k-e	76
  9874	9889	530366	e-k-d(10)-k-e-k-e	75
  9874	9889	530417	k-d(10)-k-e-k-e-e	66
  9875	9891	530047	e-e-k-d(10)-k-e-k-e	75
  9875	9890	530114	e-k-k-d(10)-k-k-e	80
  9875	9890	530161	e-e-k-d(10)-k-k-e	81
  9875	9890	530211	e-d-k-d(10)-k-k-e	73
  9875	9890	530261	e-d-d-k-d(9)-k-k-e	78
  9875	9890	530311	e-e-e-e-d(9)-k-k-e	82
  9875	9890	530367	e-k-d(10)-k-e-k-e	80
  9876	9891	530115	e-k-k-d(10)-k-k-e	74
  9876	9891	530162	e-e-k-d(10)-k-k-e	68
  9876	9891	530212	e-d-k-d(10)-k-k-e	58
  9876	9891	530262	e-d-d-k-d(9)-k-k-e	23
  9876	9891	530312	e-e-e-e-d(9)-k-k-e	52
  9876	9891	530418	k-d(10)-k-e-k-e-e	59
  9877	9893	530048	e-e-k-d(10)-k-e-k-e	82
  9877	9892	530368	e-k-d(10)-k-e-k-e	85
  9878	9893	530116	e-k-k-d(10)-k-k-e	90
  9878	9893	530163	e-e-k-d(10)-k-k-e	79
  9878	9893	530213	e-d-k-d(10)-k-k-e	72
  9878	9893	530263	e-d-d-k-d(9)-k-k-e	73
  9878	9893	530313	e-e-e-e-d(9)-k-k-e	61
  12345	12360	530414	k-d(10)-k-e-k-e-e	61
  12346	12362	530044	e-e-k-d(10)-k-e-k-e	78
  12346	12361	530364	e-k-d(10)-k-e-k-e	59
  12347	12362	530112	e-k-k-d(10)-k-k-e	84
  12347	12362	530159	e-e-k-d(10)-k-k-e	69
  12347	12362	530209	e-d-k-d(10)-k-k-e	54
  12347	12362	530259	e-d-d-k-d(9)-k-k-e	57
  12347	12362	530309	e-e-e-e-d(9)-k-k-e	46
  12347	12362	530415	k-d(10)-k-e-k-e-e	51
  12348	12364	530045	e-e-k-d(10)-k-e-k-e	73
  12348	12363	530365	e-k-d(10)-k-e-k-e	78
  12349	12364	530113	e-k-k-d(10)-k-k-e	60
  12349	12364	530160	e-e-k-d(10)-k-k-e	54
  12349	12364	530210	e-d-k-d(10)-k-k-e	28
  12349	12364	530260	e-d-d-k-d(9)-k-k-e	0
  12349	12364	530310	e-e-e-e-d(9)-k-k-e	26
  12356	12371	530416	k-d(10)-k-e-k-e-e	57
  12357	12373	530046	e-e-k-d(10)-k-e-k-e	76
  12357	12372	530366	e-k-d(10)-k-e-k-e	75
  12357	12372	530417	k-d(10)-k-e-k-e-e	66
  12358	12374	530047	e-e-k-d(10)-k-e-k-e	75
  12358	12373	530114	e-k-k-d(10)-k-k-e	80
  12358	12373	530161	e-e-k-d(10)-k-k-e	81
  12358	12373	530211	e-d-k-d(10)-k-k-e	73
  12358	12373	530261	e-d-d-k-d(9)-k-k-e	78
  12358	12373	530311	e-e-e-e-d(9)-k-k-e	82
  12358	12373	530367	e-k-d(10)-k-e-k-e	80
  12359	12374	530115	e-k-k-d(10)-k-k-e	74
  12359	12374	530162	e-e-k-d(10)-k-k-e	68
  12359	12374	530212	e-d-k-d(10)-k-k-e	58
  12359	12374	530262	e-d-d-k-d(9)-k-k-e	23
  12359	12374	530312	e-e-e-e-d(9)-k-k-e	52
  12359	12374	530418	k-d(10)-k-e-k-e-e	59
  12360	12376	530048	e-e-k-d(10)-k-e-k-e	82
  12360	12375	530368	e-k-d(10)-k-e-k-e	85
  12361	12376	530116	e-k-k-d(10)-k-k-e	90
  12361	12376	530163	e-e-k-d(10)-k-k-e	79
  12361	12376	530213	e-d-k-d(10)-k-k-e	72
  12361	12376	530263	e-d-d-k-d(9)-k-k-e	73
  12361	12376	530313	e-e-e-e-d(9)-k-k-e	61
  15469	15484	530132	e-k-k-d(10)-k-k-e	74
  15469	15484	530182	e-e-k-d(10)-k-k-e	48
  15469	15484	530232	e-d-k-d(10)-k-k-e	21
  15469	15484	530282	e-d-d-k-d(9)-k-k-e	19
  15469	15484	530332	e-e-e-e-d(9)-k-k-e	20
  16863	16878	530419	k-d(10)-k-e-k-e-e	75
  16864	16880	530049	e-e-k-d(10)-k-e-k-e	88
  16864	16879	530369	e-k-d(10)-k-e-k-e	92
  16865	16880	530117	e-k-k-d(10)-k-k-e	73
  16865	16880	530164	e-e-k-d(10)-k-k-e	65
  16865	16880	530214	e-d-k-d(10)-k-k-e	37
  16865	16880	530264	e-d-d-k-d(9)-k-k-e	48
  16865	16880	530314	e-e-e-e-d(9)-k-k-e	42
  25105	25120	530717	e-e-e-d(10)-k-k-k	77
  50692	50707	530423	k-d(10)-k-e-k-e-e	70
  50693	50709	530053	e-e-k-d(10)-k-e-k-e	84
  50693	50708	530373	e-k-d(10)-k-e-k-e	85
  50694	50709	530121	e-k-k-d(10)-k-k-e	77
  50694	50709	530168	e-e-k-d(10)-k-k-e	75
  50694	50709	530218	e-d-k-d(10)-k-k-e	61
  50694	50709	530268	e-d-d-k-d(9)-k-k-e	76
  50694	50709	530318	e-e-e-e-d(9)-k-k-e	73
  51905	51920	528400	e-e-e-d(10)-k-k-k	57
  51910	51925	528403	e-e-e-d(10)-k-k-k	72
  64959	64974	529082	e-e-e-d(10)-k-k-k	20
  66136	66151	530425	k-d(10)-k-e-k-e-e	73
  66137	66153	530054	e-e-k-d(10)-k-e-k-e	75
  66137	66152	530375	e-k-d(10)-k-e-k-e	77
  66138	66153	530123	e-k-k-d(10)-k-k-e	86
  66138	66153	530170	e-e-k-d(10)-k-k-e	87
  66138	66153	530220	e-d-k-d(10)-k-k-e	74
  66138	66153	530270	e-d-d-k-d(9)-k-k-e	87
  66138	66153	530320	e-e-e-e-d(9)-k-k-e	83
  66184	66200	530059	e-e-k-d(10)-k-e-k-e	73
  66184	66199	530376	e-k-d(10)-k-e-k-e	77
  66185	66200	530124	e-k-k-d(10)-k-k-e	79
  66185	66200	530171	e-e-k-d(10)-k-k-e	69
  66185	66200	530221	e-d-k-d(10)-k-k-e	64
  66185	66200	530271	e-d-d-k-d(9)-k-k-e	73
  66185	66200	530321	e-e-e-e-d(9)-k-k-e	56
  67067	67083	530060	e-e-k-d(10)-k-e-k-e	77
  67067	67082	530377	e-k-d(10)-k-e-k-e	66
  67068	67083	530125	e-k-k-d(10)-k-k-e	65
  67068	67083	530172	e-e-k-d(10)-k-k-e	59
  67068	67083	530222	e-d-k-d(10)-k-k-e	48
  67068	67083	530272	e-d-d-k-d(9)-k-k-e	63
  67068	67083	530322	e-e-e-e-d(9)-k-k-e	45
  71616	71631	530120	e-k-k-d(10)-k-k-e	78
  71616	71631	530167	e-e-k-d(10)-k-k-e	69
  71616	71631	530217	e-d-k-d(10)-k-k-e	47
  71616	71631	530267	e-d-d-k-d(9)-k-k-e	64
  71616	71631	530317	e-e-e-e-d(9)-k-k-e	60
  73868	73883	530126	e-k-k-d(10)-k-k-e	70
  73868	73883	530173	e-e-k-d(10)-k-k-e	62
  73868	73883	530223	e-d-k-d(10)-k-k-e	44
  73868	73883	530273	e-d-d-k-d(9)-k-k-e	63
  73868	73883	530323	e-e-e-e-d(9)-k-k-e	37
  74199	74214	530513	k-d(10)-k-e-k-e-e	88
  74200	74215	530507	e-k-d(10)-k-e-k-e	86
  74200	74215	530514	k-d(10)-k-e-k-e-e	80
  74201	74216	530430	k-d(10)-k-e-k-e-e	87
  74201	74216	530468	e-k-k-d(10)-k-k-e	81
  74201	74216	530476	e-e-k-d(10)-k-k-e	82
  74201	74216	530484	e-d-k-d(10)-k-k-e	74
  74201	74216	530492	e-d-d-k-d(9)-k-k-e	83
  74201	74216	530500	e-e-e-e-d(9)-k-k-e	56
  74201	74216	530508	e-k-d(10)-k-e-k-e	83
  74202	74218	530062	e-e-k-d(10)-k-e-k-e	94
  74202	74217	530380	e-k-d(10)-k-e-k-e	94
  74202	74217	530469	e-k-k-d(10)-k-k-e	91
  74202	74217	530477	e-e-k-d(10)-k-k-e	87
  74202	74217	530485	e-d-k-d(10)-k-k-e	87
  74202	74217	530493	e-d-d-k-d(9)-k-k-e	81
  74202	74217	530501	e-e-e-e-d(9)-k-k-e	74
  74202	74217	530515	k-d(10)-k-e-k-e-e	87
  74203	74218	481464	k-k-k-d(10)-k-k-k	93
  74203	74218	518349	e-e-e-d(10)-k-k-k	58
  74203	74218	519637	e-k-k-d(10)-k-k-e	96
  74203	74218	530175	e-e-k-d(10)-k-k-e	93
  74203	74218	530225	e-d-k-d(10)-k-k-e	85
  74203	74218	530275	e-d-d-k-d(9)-k-k-e	91
  74203	74218	530325	e-e-e-e-d(9)-k-k-e	91
  74204	74219	530470	e-k-k-d(10)-k-k-e	91
  74204	74219	530478	e-e-k-d(10)-k-k-e	87
  74204	74219	530486	e-d-k-d(10)-k-k-e	84
  74204	74219	530494	e-d-d-k-d(9)-k-k-e	60
  74204	74219	530502	e-e-e-e-d(9)-k-k-e	64
  74204	74219	530509	e-k-d(10)-k-e-k-e	80
  74205	74220	530471	e-k-k-d(10)-k-k-e	83
  74205	74220	530479	e-e-k-d(10)-k-k-e	74
  74205	74220	530487	e-d-k-d(10)-k-k-e	71
  74205	74220	530495	e-d-d-k-d(9)-k-k-e	68
  74205	74220	530503	e-e-e-e-d(9)-k-k-e	53
  74648	74663	530128	e-k-k-d(10)-k-k-e	52
  74648	74663	530176	e-e-k-d(10)-k-k-e	66
  74648	74663	530226	e-d-k-d(10)-k-k-e	51
  74648	74663	530276	e-d-d-k-d(9)-k-k-e	70
  74648	74663	530326	e-e-e-e-d(9)-k-k-e	52
  74734	74749	528866	e-e-e-d(10)-k-k-k	60
  74735	74750	528867	e-e-e-d(10)-k-k-k	47
  74772	74787	530086	e-k-k-d(10)-k-k-e	58
  74772	74787	530133	e-e-k-d(10)-k-k-e	53
  74772	74787	530183	e-d-k-d(10)-k-k-e	52
  74772	74787	530233	e-d-d-k-d(9)-k-k-e	29
  74772	74787	530283	e-e-e-e-d(9)-k-k-e	32
  74782	74797	530390	k-d(10)-k-e-k-e-e	83
  74783	74798	530340	e-k-d(10)-k-e-k-e	89
  74784	74799	528869	e-e-e-d(10)-k-k-k	83
  74784	74799	530088	e-k-k-d(10)-k-k-e	90
  74784	74799	530135	e-e-k-d(10)-k-k-e	91
  74784	74799	530185	e-d-k-d(10)-k-k-e	85
  74784	74799	530235	e-d-d-k-d(9)-k-k-e	28
  74784	74799	530285	e-e-e-e-d(9)-k-k-e	86
  74784	74799	530391	k-d(10)-k-e-k-e-e	79
  74785	74801	530021	e-e-k-d(10)-k-e-k-e	87
  74785	74800	530341	e-k-d(10)-k-e-k-e	88
  74786	74801	530089	e-k-k-d(10)-k-k-e	71
  74786	74801	530136	e-e-k-d(10)-k-k-e	66
  74786	74801	530186	e-d-k-d(10)-k-k-e	51
  74786	74801	530236	e-d-d-k-d(9)-k-k-e	74
  74786	74801	530286	e-e-e-e-d(9)-k-k-e	56
  74902	74918	530022	e-e-k-d(10)-k-e-k-e	80
  74902	74917	530342	e-k-d(10)-k-e-k-e	70
  74902	74917	530393	k-d(10)-k-e-k-e-e	46
  74903	74919	530023	e-e-k-d(10)-k-e-k-e	74
  74903	74918	530090	e-k-k-d(10)-k-k-e	78
  74903	74918	530137	e-e-k-d(10)-k-k-e	76
  74903	74918	530187	e-d-k-d(10)-k-k-e	68
  74903	74918	530237	e-d-d-k-d(9)-k-k-e	36
  74903	74918	530287	e-e-e-e-d(9)-k-k-e	56
  74903	74918	530343	e-k-d(10)-k-e-k-e	68
  74903	74918	530394	k-d(10)-k-e-k-e-e	49
  74904	74919	518343	e-e-e-d(10)-k-k-k	5
  74904	74920	530024	e-e-k-d(10)-k-e-k-e	79
  74904	74919	530091	e-k-k-d(10)-k-k-e	81
  74904	74919	530138	e-e-k-d(10)-k-k-e	81
  74904	74919	530188	e-d-k-d(10)-k-k-e	78
  74904	74919	530238	e-d-d-k-d(9)-k-k-e	29
  74904	74919	530288	e-e-e-e-d(9)-k-k-e	69
  74904	74919	530344	e-k-d(10)-k-e-k-e	85
  74905	74920	530092	e-k-k-d(10)-k-k-e	85
  74905	74920	530139	e-e-k-d(10)-k-k-e	79
  74905	74920	530189	e-d-k-d(10)-k-k-e	77
  74905	74920	530239	e-d-d-k-d(9)-k-k-e	61
  74905	74920	530289	e-e-e-e-d(9)-k-k-e	75
  74907	74922	528880	e-e-e-d(10)-k-k-k	65
  75209	75224	518344	e-e-e-d(10)-k-k-k	89
  75421	75436	530395	k-d(10)-k-e-k-e-e	71
  75422	75437	528936	e-e-e-d(10)-k-k-k	71
  75422	75438	530025	e-e-k-d(10)-k-e-k-e	90
  75422	75437	530345	e-k-d(10)-k-e-k-e	93
  75422	75437	530396	k-d(10)-k-e-k-e-e	71
  75423	75438	528937	e-e-e-d(10)-k-k-k	73
  75423	75439	530026	e-e-k-d(10)-k-e-k-e	87
  75423	75438	530093	e-k-k-d(10)-k-k-e	95
  75423	75438	530140	e-e-k-d(10)-k-k-e	89
  75423	75438	530190	e-d-k-d(10)-k-k-e	82
  75423	75438	530240	e-d-d-k-d(9)-k-k-e	50
  75423	75438	530290	e-e-e-e-d(9)-k-k-e	69
  75423	75438	530346	e-k-d(10)-k-e-k-e	89
  75424	75439	528938	e-e-e-d(10)-k-k-k	72
  75424	75439	530094	e-k-k-d(10)-k-k-e	88
  75424	75439	530141	e-e-k-d(10)-k-k-e	80
  75424	75439	530191	e-d-k-d(10)-k-k-e	74
  75424	75439	530241	e-d-d-k-d(9)-k-k-e	53
  75424	75439	530291	e-e-e-e-d(9)-k-k-e	68
  75429	75444	528942	e-e-e-d(10)-k-k-k	77
  75492	75507	528944	e-e-e-d(10)-k-k-k	28
  75507	75522	528945	e-e-e-d(10)-k-k-k	74
  75626	75641	530096	e-k-k-d(10)-k-k-e	72
  75626	75641	530143	e-e-k-d(10)-k-k-e	74
  75626	75641	530193	e-d-k-d(10)-k-k-e	62
  75626	75641	530243	e-d-d-k-d(9)-k-k-e	34
  75626	75641	530293	e-e-e-e-d(9)-k-k-e	59
  75676	75691	519638	e-k-k-d(10)-k-k-e	86
  75676	75691	530177	e-e-k-d(10)-k-k-e	71
  75676	75691	530227	e-d-k-d(10)-k-k-e	51
  75676	75691	530277	e-d-d-k-d(9)-k-k-e	70
  75676	75691	530327	e-e-e-e-d(9)-k-k-e	61
  75677	75692	530466	e-k-k-d(10)-k-k-e	82
  75677	75692	530474	e-e-k-d(10)-k-k-e	62
  75677	75692	530482	e-d-k-d(10)-k-k-e	53
  75677	75692	530490	e-d-d-k-d(9)-k-k-e	42
  75677	75692	530498	e-e-e-e-d(9)-k-k-e	45
  75677	75692	530506	e-k-d(10)-k-e-k-e	70
  75726	75741	530433	k-d(10)-k-e-k-e-e	62
  75727	75742	528958	e-e-e-d(10)-k-k-k	66
  75727	75743	530056	e-e-k-d(10)-k-e-k-e	73
  75727	75742	530383	e-k-d(10)-k-e-k-e	64
  75728	75743	518345	e-e-e-d(10)-k-k-k	80
  75728	75743	519636	e-k-k-d(10)-k-k-e	90
  75728	75743	530178	e-e-k-d(10)-k-k-e	86
  75728	75743	530228	e-d-k-d(10)-k-k-e	77
  75728	75743	530278	e-d-d-k-d(9)-k-k-e	86
  75728	75743	530328	e-e-e-e-d(9)-k-k-e	80
  75729	75744	528959	e-e-e-d(10)-k-k-k	73
  75808	75823	528973	e-e-e-d(10)-k-k-k	71
  75969	75984	528995	e-e-e-d(10)-k-k-k	64
  75969	75984	530129	e-k-k-d(10)-k-k-e	79
  75969	75984	530179	e-e-k-d(10)-k-k-e	74
  75969	75984	530229	e-d-k-d(10)-k-k-e	64
  75969	75984	530279	e-d-d-k-d(9)-k-k-e	55
  75969	75984	530329	e-e-e-e-d(9)-k-k-e	61
  75999	76014	530402	k-d(10)-k-e-k-e-e	60
  76000	76016	530032	e-e-k-d(10)-k-e-k-e	74
  76000	76015	530099	e-k-k-d(10)-k-k-e	73
  76000	76015	530146	e-e-k-d(10)-k-k-e	70
  76000	76015	530196	e-d-k-d(10)-k-k-e	67
  76000	76015	530246	e-d-d-k-d(9)-k-k-e	39
  76000	76015	530296	e-e-e-e-d(9)-k-k-e	67
  76000	76015	530352	e-k-d(10)-k-e-k-e	67
  76001	76016	530100	e-k-k-d(10)-k-k-e	77
  76001	76016	530147	e-e-k-d(10)-k-k-e	84
  76001	76016	530197	e-d-k-d(10)-k-k-e	71
  76001	76016	530247	e-d-d-k-d(9)-k-k-e	53
  76001	76016	530297	e-e-e-e-d(9)-k-k-e	75
  76001	76016	530403	k-d(10)-k-e-k-e-e	77
  76002	76018	530033	e-e-k-d(10)-k-e-k-e	65
  76002	76017	530353	e-k-d(10)-k-e-k-e	83
  76003	76018	530101	e-k-k-d(10)-k-k-e	59
  76003	76018	530148	e-e-k-d(10)-k-k-e	79
  76003	76018	530198	e-d-k-d(10)-k-k-e	54
  76003	76018	530248	e-d-d-k-d(9)-k-k-e	32
  76003	76018	530298	e-e-e-e-d(9)-k-k-e	73
  76014	76029	530404	k-d(10)-k-e-k-e-e	67
  76015	76031	530034	e-e-k-d(10)-k-e-k-e	69
  76015	76030	530354	e-k-d(10)-k-e-k-e	85
  76015	76030	530405	k-d(10)-k-e-k-e-e	55
  76016	76032	530035	e-e-k-d(10)-k-e-k-e	69
  76016	76031	530102	e-k-k-d(10)-k-k-e	71
  76016	76031	530149	e-e-k-d(10)-k-k-e	70
  76016	76031	530199	e-d-k-d(10)-k-k-e	58
  76016	76031	530249	e-d-d-k-d(9)-k-k-e	47
  76016	76031	530299	e-e-e-e-d(9)-k-k-e	47
  76016	76031	530355	e-k-d(10)-k-e-k-e	72
  76017	76032	530103	e-k-k-d(10)-k-k-e	77
  76017	76032	530150	e-e-k-d(10)-k-k-e	73
  76017	76032	530200	e-d-k-d(10)-k-k-e	63
  76017	76032	530250	e-d-d-k-d(9)-k-k-e	59
  76017	76032	530300	e-e-e-e-d(9)-k-k-e	65
  76029	76044	530435	k-d(10)-k-e-k-e-e	62
  76030	76046	530057	e-e-k-d(10)-k-e-k-e	69
  76030	76045	530385	e-k-d(10)-k-e-k-e	70
  76031	76046	529005	e-e-e-d(10)-k-k-k	64
  76031	76046	530130	e-k-k-d(10)-k-k-e	85
  76031	76046	530180	e-e-k-d(10)-k-k-e	82
  76031	76046	530230	e-d-k-d(10)-k-k-e	65
  76031	76046	530280	e-d-d-k-d(9)-k-k-e	75
  76031	76046	530330	e-e-e-e-d(9)-k-k-e	52
  e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside

Example 71: Dose-Dependent Antisense Inhibition of Human Target-1 in HuVEC Cells
• Gapmers from the study described in Example 70 exhibiting in vitro inhibition of Target-1 were tested at various doses in HuVEC cells. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 39.1 nM, 156.3 nM, 625.0 nM, and 2,500.0 nM concentrations of antisense oligonucleotide, as specified in Table 118. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-1 mRNA levels were measured by quantitative real-time PCR. Target-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-1, relative to untreated control cells.

• TABLE 118
  Dose-dependent antisense inhibition of human
  Target-1 in HuVEC cells

  		39.1	156.3	625.0	2500.0	IC50
  	ISIS No	nM	nM	nM	nM	(μM)

  	481464	6	51	84	94	0.2
  	518345	0	9	56	84	0.6
  	518349	16	3	47	83	0.6
  	519636	16	41	75	89	0.2
  	519637	24	43	84	94	0.2
  	519638	6	34	70	92	0.3
  	528403	0	4	39	77	0.9
  	528458	0	15	46	81	0.7
  	528475	1	10	51	76	0.7
  	528476	0	11	42	80	0.7
  	528869	25	19	67	86	0.3
  	528880	0	3	45	76	0.8
  	528937	0	1	49	82	0.8
  	528938	0	9	50	82	0.7
  	528942	0	20	59	88	0.5
  	528959	0	4	55	79	0.7
  	529022	0	0	52	81	0.8
  	529023	0	0	53	90	0.6
  	529024	0	0	47	80	0.8
  	529025	0	11	50	90	0.6
  	529026	0	31	73	96	0.4
  	529027	0	7	36	80	0.9
  	530021	6	30	69	92	0.3
  	530025	10	33	73	92	0.3
  	530026	3	18	52	80	0.6
  	530041	0	28	72	91	0.4
  	530048	0	22	53	83	0.5
  	530049	2	16	69	92	0.4
  	530053	0	16	66	90	0.5
  	530062	4	56	85	94	0.2
  	530066	0	12	46	84	0.7
  	530088	2	39	77	93	0.3
  	530091	3	12	59	84	0.5
  	530092	7	27	65	85	0.4
  	530093	7	46	79	96	0.2
  	530094	0	17	63	89	0.5
  	530109	9	30	72	94	0.3
  	530110	0	23	61	83	0.5
  	530112	0	13	42	90	0.6
  	530114	0	21	62	79	0.6
  	530116	22	40	71	92	0.2
  	530123	8	19	72	93	0.3
  	530130	0	33	64	89	0.4
  	530131	4	34	81	93	0.3
  	530135	22	38	79	94	0.2
  	530138	6	23	57	86	0.4
  	530140	4	22	62	91	0.4
  	530147	0	15	51	83	0.6
  	530156	7	41	81	96	0.2
  	530161	0	20	46	78	0.7
  	530170	0	29	67	90	0.4
  	530175	37	52	84	95	0.1
  	530178	8	24	70	86	0.4
  	530180	0	0	61	82	0.6
  	530181	0	27	52	86	0.5
  	530185	0	22	54	86	0.5
  	530190	17	17	60	87	0.4
  	530206	8	29	73	93	0.3
  	530225	0	27	67	91	0.4
  	530228	11	16	64	86	0.4
  	530261	5	25	57	91	0.4
  	530270	7	11	62	91	0.4
  	530275	14	34	73	91	0.3
  	530278	1	27	60	85	0.4
  	530285	5	20	61	82	0.5
  	530306	3	14	66	85	0.5
  	530311	6	27	59	86	0.4
  	530320	3	17	56	85	0.5
  	530325	5	35	70	92	0.3
  	530328	4	34	61	87	0.4
  	530340	8	34	74	90	0.3
  	530341	2	23	77	89	0.4
  	530344	16	20	64	89	0.4
  	530345	15	35	77	94	0.2
  	530346	5	24	66	92	0.4
  	530353	7	25	57	83	0.5
  	530354	2	24	60	81	0.5
  	530359	0	4	44	89	0.7
  	530361	13	30	59	92	0.3
  	530365	0	0	45	88	0.7
  	530367	0	15	49	88	0.5
  	530368	0	27	64	89	0.4
  	530369	10	28	78	95	0.3
  	530373	13	29	64	92	0.3
  	530375	0	14	53	90	0.5
  	530380	8	40	80	94	0.2
  	530390	11	21	66	90	0.4
  	530391	20	7	49	86	0.5
  	530411	5	19	81	95	0.3
  	530430	0	8	53	91	0.6
  	530466	0	4	53	87	0.6
  	530468	4	17	65	90	0.4
  	530469	8	38	86	94	0.2
  	530470	5	39	78	91	0.3
  	530471	0	21	69	91	0.4
  	530476	7	9	32	89	0.7
  	530477	0	12	64	87	0.5
  	530478	0	14	59	90	0.5
  	530485	0	10	61	85	0.5
  	530486	0	17	64	80	0.5
  	530492	0	25	71	89	0.4
  	530493	4	23	58	88	0.4
  	530507	5	17	65	82	0.5
  	530508	0	14	56	89	0.5
  	530509	0	17	54	86	0.5
  	530513	6	24	74	91	0.3
  	530514	1	7	52	78	0.7
  	530515	0	19	73	89	0.4

Example 72: Antisense Inhibition of Human Target-1 in HuVEC Cells
• Additional antisense oligonucleotides were designed targeting a Target-1 nucleic acid and were tested for their effects on Target-1 mRNA in vitro. Cultured HuVEC cells at a density of 20,000 cells per well were transfected using electroporation with 1,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-1 mRNA levels were measured by quantitative real-time PCR. Target-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-1, relative to untreated control cells.
• The chemistry column of Table 119 presents the sugar motif of each gapmer, where ‘e’ indicates a 2′-MOE nucleoside, ‘k’ indicates a constrained ethyl (cEt) nucleoside, and indicates a 2′-deoxynucleoside. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5′-methylcytosines.
• “Human Target start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. “Human Target stop site” indicates the 3′-most nucleoside to which the gapmer is targeted in the human gene sequence. Each gapmer listed in Table 119 is targeted to human Target-1 mRNA. Each gapmer listed in Table 120 is targeted to human Target-1 genomic sequence, truncated from nucleotides 4185000 to 4264000).

• TABLE 119
  Inhibition of human Target-1 mRNA levels by chimeric antisense
  oligonucleotides targeted to Target-1 mRNA

  Human	Human
  Start	Stop			%
  Site	Site	ISIS No	Chemistry	inhibition

  730	745	530011	k-k-k-d(10)-e-e-e	73
  1901	1916	529974	e-e-e-d(10)-k-k-k	83
  1901	1916	530012	k-k-k-d(10)-e-e-e	73
  2206	2221	530015	k-k-k-d(10)-e-e-e	38
  3016	3031	481464	k-k-k-d(10)-k-k-k	94
  3461	3476	529975	e-e-e-d(10)-k-k-k	54
  3461	3476	530013	k-k-k-d(10)-e-e-e	58
  3584	3600	530018	e-e-k-d(10)-k-e-k-e	46
  3585	3600	529944	e-e-e-d(10)-k-k-k	44
  3585	3600	529977	k-k-k-d(10)-e-e-e	66
  3592	3608	530019	e-e-k-d(10)-k-e-k-e	43
  3593	3608	529945	e-e-e-d(10)-k-k-k	22
  3593	3608	529978	k-k-k-d(10)-e-e-e	49
  3596	3612	530020	e-e-k-d(10)-k-e-k-e	85
  3597	3612	529979	k-k-k-d(10)-e-e-e	86
  3599	3614	529946	e-e-e-d(10)-k-k-k	46
  3599	3614	529980	k-k-k-d(10)-e-e-e	25
  3716	3731	529947	e-e-e-d(10)-k-k-k	68
  3716	3731	529981	k-k-k-d(10)-e-e-e	83
  3718	3733	529948	e-e-e-d(10)-k-k-k	75
  3718	3733	529982	k-k-k-d(10)-e-e-e	84
  4236	4251	529983	k-k-k-d(10)-e-e-e	96
  4237	4252	529984	k-k-k-d(10)-e-e-e	91
  4437	4452	529949	e-e-e-d(10)-k-k-k	48
  4437	4452	529985	k-k-k-d(10)-e-e-e	37
  4439	4454	529950	e-e-e-d(10)-k-k-k	58
  4439	4454	529986	k-k-k-d(10)-e-e-e	72
  4646	4661	529987	k-k-k-d(10)-e-e-e	0
  4664	4679	529951	e-e-e-d(10)-k-k-k	38
  4664	4679	529988	k-k-k-d(10)-e-e-e	40
  4782	4797	530016	k-k-k-d(10)-e-e-e	60
  4813	4828	529952	e-e-e-d(10)-k-k-k	65
  4813	4828	529989	k-k-k-d(10)-e-e-e	63
  4814	4829	529953	e-e-e-d(10)-k-k-k	65
  4814	4829	529990	k-k-k-d(10)-e-e-e	75
  4816	4831	529954	e-e-e-d(10)-k-k-k	79
  4816	4831	529991	k-k-k-d(10)-e-e-e	52
  4829	4844	529955	e-e-e-d(10)-k-k-k	52
  4829	4844	529992	k-k-k-d(10)-e-e-e	23
  4830	4845	529956	e-e-e-d(10)-k-k-k	60
  4830	4845	529993	k-k-k-d(10)-e-e-e	51
  4844	4859	530014	k-k-k-d(10)-e-e-e	67
  e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside

• TABLE 120
  Inhibition of human Target-1 mRNA levels by chimeric antisense
  oligonucleotides targeted to Target-1 Genomic Sequence

  Human	Human
  Start	Stop		%
  Site	Site	ISIS No	Chemistry	inhibition

  74203	74218	481464	k-k-k-d(10)-k-k-k	94
  74772	74787	529944	e-e-e-d(10)-k-k-k	44
  74780	74795	529945	e-e-e-d(10)-k-k-k	22
  74786	74801	529946	e-e-e-d(10)-k-k-k	46
  74903	74918	529947	e-e-e-d(10)-k-k-k	68
  74905	74920	529948	e-e-e-d(10)-k-k-k	75
  75624	75639	529949	e-e-e-d(10)-k-k-k	48
  75626	75641	529950	e-e-e-d(10)-k-k-k	58
  75851	75866	529951	e-e-e-d(10)-k-k-k	38
  76000	76015	529952	e-e-e-d(10)-k-k-k	65
  76001	76016	529953	e-e-e-d(10)-k-k-k	65
  76003	76018	529954	e-e-e-d(10)-k-k-k	79
  76016	76031	529955	e-e-e-d(10)-k-k-k	52
  76017	76032	529956	e-e-e-d(10)-k-k-k	60
  2340	2355	529957	e-e-e-d(10)-k-k-k	21
  2385	2400	529958	e-e-e-d(10)-k-k-k	10
  2410	2425	529959	e-e-e-d(10)-k-k-k	51
  2671	2686	529960	e-e-e-d(10)-k-k-k	30
  5002	5017	529961	e-e-e-d(10)-k-k-k	52
  5701	5716	529962	e-e-e-d(10)-k-k-k	91
  8080	8095	529963	e-e-e-d(10)-k-k-k	55
  9125	9140	529964	e-e-e-d(10)-k-k-k	18
  11263	11278	529964	e-e-e-d(10)-k-k-k	18
  9864	9879	529965	e-e-e-d(10)-k-k-k	52
  12347	12362	529965	e-e-e-d(10)-k-k-k	52
  9866	9881	529966	e-e-e-d(10)-k-k-k	51
  12349	12364	529966	e-e-e-d(10)-k-k-k	51
  9875	9890	529967	e-e-e-d(10)-k-k-k	80
  12358	12373	529967	e-e-e-d(10)-k-k-k	80
  9876	9891	529968	e-e-e-d(10)-k-k-k	56
  12359	12374	529968	e-e-e-d(10)-k-k-k	56
  9878	9893	529969	e-e-e-d(10)-k-k-k	69
  12361	12376	529969	e-e-e-d(10)-k-k-k	69
  16865	16880	529970	e-e-e-d(10)-k-k-k	41
  26063	26078	529971	e-e-e-d(10)-k-k-k	32
  48404	48419	529972	e-e-e-d(10)-k-k-k	30
  71616	71631	529973	e-e-e-d(10)-k-k-k	49
  66138	66153	529974	e-e-e-d(10)-k-k-k	83
  74648	74663	529975	e-e-e-d(10)-k-k-k	54
  2705	2720	529976	e-e-e-d(10)-k-k-k	25
  74772	74787	529977	k-k-k-d(10)-e-e-e	66
  74780	74795	529978	k-k-k-d(10)-e-e-e	49
  74784	74799	529979	k-k-k-d(10)-e-e-e	86
  74786	74801	529980	k-k-k-d(10)-e-e-e	25
  74903	74918	529981	k-k-k-d(10)-e-e-e	83
  74905	74920	529982	k-k-k-d(10)-e-e-e	84
  75423	75438	529983	k-k-k-d(10)-e-e-e	96
  75424	75439	529984	k-k-k-d(10)-e-e-e	91
  75624	75639	529985	k-k-k-d(10)-e-e-e	37
  75626	75641	529986	k-k-k-d(10)-e-e-e	72
  75833	75848	529987	k-k-k-d(10)-e-e-e	0
  75851	75866	529988	k-k-k-d(10)-e-e-e	40
  76000	76015	529989	k-k-k-d(10)-e-e-e	63
  76001	76016	529990	k-k-k-d(10)-e-e-e	75
  76003	76018	529991	k-k-k-d(10)-e-e-e	52
  76016	76031	529992	k-k-k-d(10)-e-e-e	23
  76017	76032	529993	k-k-k-d(10)-e-e-e	51
  2340	2355	529994	k-k-k-d(10)-e-e-e	44
  2385	2400	529995	k-k-k-d(10)-e-e-e	0
  2410	2425	529996	k-k-k-d(10)-e-e-e	65
  2671	2686	529997	k-k-k-d(10)-e-e-e	44
  5002	5017	529998	k-k-k-d(10)-e-e-e	35
  5701	5716	529999	k-k-k-d(10)-e-e-e	91
  8080	8095	530000	k-k-k-d(10)-e-e-e	80
  9125	9140	530001	k-k-k-d(10)-e-e-e	21
  11263	11278	530001	k-k-k-d(10)-e-e-e	21
  9864	9879	530002	k-k-k-d(10)-e-e-e	74
  12347	12362	530002	k-k-k-d(10)-e-e-e	74
  9866	9881	530003	k-k-k-d(10)-e-e-e	67
  12349	12364	530003	k-k-k-d(10)-e-e-e	67
  9875	9890	530004	k-k-k-d(10)-e-e-e	83
  12358	12373	530004	k-k-k-d(10)-e-e-e	83
  9876	9891	530005	k-k-k-d(10)-e-e-e	77
  12359	12374	530005	k-k-k-d(10)-e-e-e	77
  9878	9893	530006	k-k-k-d(10)-e-e-e	89
  12361	12376	530006	k-k-k-d(10)-e-e-e	89
  16865	16880	530007	k-k-k-d(10)-e-e-e	21
  26063	26078	530008	k-k-k-d(10)-e-e-e	58
  48404	48419	530009	k-k-k-d(10)-e-e-e	59
  71616	71631	530010	k-k-k-d(10)-e-e-e	75
  50694	50709	530011	k-k-k-d(10)-e-e-e	73
  66138	66153	530012	k-k-k-d(10)-e-e-e	73
  74648	74663	530013	k-k-k-d(10)-e-e-e	58
  76031	76046	530014	k-k-k-d(10)-e-e-e	67
  67068	67083	530015	k-k-k-d(10)-e-e-e	38
  75969	75984	530016	k-k-k-d(10)-e-e-e	60
  2705	2720	530017	k-k-k-d(10)-e-e-e	46
  74771	74787	530018	e-e-k-d(10)-k-e-k-e	46
  74779	74795	530019	e-e-k-d(10)-k-e-k-e	43
  74783	74799	530020	e-e-k-d(10)-k-e-k-e	85
  e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside

Example 73: Dose-Dependent Antisense Inhibition of Human Target-1 in HuVEC Cells
• Gapmers from the study described in Example 72 exhibiting in vitro inhibition of Target-1 were tested at various doses in HuVEC cells. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 39.1 nM, 156.3 nM, 625.0 nM, and 2,500.0 nM concentrations of antisense oligonucleotide, as specified in Table 121. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-1 mRNA levels were measured by quantitative real-time PCR. Target-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-1, relative to untreated control cells.

• TABLE 121
  Dose-dependent antisense inhibition of human
  Target-1 in HuVEC cells

  		39.1	156.3	625.0	2500.0	IC50
  	ISIS No	nM	nM	nM	nM	(μM)

  	481464	41	78	92	91	0.04
  	529962	30	51	86	95	0.12
  	529979	0	43	81	95	0.27
  	529982	0	0	70	90	0.56
  	529983	31	67	87	94	0.08
  	529984	17	44	83	97	0.19
  	529999	29	51	83	96	0.13
  	530006	18	38	77	94	0.22
  	530020	2	39	75	92	0.28

Claims (22)

1-238. (canceled)

239. A compound comprising:

a modified oligonucleotide consisting of 10 to 20 linked nucleosides, wherein the modified oligonucleotide has a sugar motif described by Formula I as follows:

(A)_m-(B)_n-(J)_p-(B)_r-(J)_t-(D)_g-(J)_v-(B)_w-(J)_x-(B)_y-(A)_z

wherein:

each A is independently a 2′-substituted nucleoside;

each B is independently a bicyclic nucleoside;

each J is independently either a 2′-substituted nucleoside or a 2′-deoxynucleoside;

each D is a 2′-deoxynucleoside;

m is 0-4; n is 0-2; p is 0-2; r is 0-2; t is 0-2; v is 0-2; w is 0-4; x is 0-2; y is 0-2; z is 0-4; and

g is 6-14;

provided that:

at least one of m, n, and r is other than 0;

at least one of w and y is other than 0;

the sum of m, n, p, r, and t is from 2 to 5; and

the sum of v, w, x, y, and z is from 2 to 5.

240. The compound ofclaim 239, wherein the 5′-wing consists of 2-5 linked nucleosides, the 3′-wing consists of 2-5 linked nucleosides, and the gap between the 5′-wing and the 3′-wing consists of 7 to 10 linked 2′-deoxynucleosides.

241. The compound ofclaim 239, wherein each 2′-substituted nucleoside independently comprises a 2′-substituent selected from OCH₃, F, OCH₂F, OCHF₂, OCF₃, OCH₂CH₃, O(CH₂)₂F, OCH₂CHF₂, OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃, O(CH₂)₂—SCH₃, O(CH₂)₂—OCF₃, O(CH₂)₃—N(R₄)(R₅), O(CH₂)₂—ON(R₄)(R₅), O(CH₂)₂—O(CH₂)₂—N(R₄)(R₅), OCH₂C(═O)—N(R₄)(R₅), OCH₂C(═O)—N(R₆)—(CH₂)₂—N(R₄)(R₅) and O(CH₂)₂—N(R₆)—C(═NR₇)[N(R₄)(R₅)] wherein R₄, R₅, R₆and R₇are each, independently, H or C₁-C₆alkyl.

242. The compound ofclaim 239, wherein the oligonucleotide comprises at least one modified internucleoside linkage, wherein the at least one modified internucleoside linkage is a phosphorothioate or a methylphosphonate linkage.

243. The compound ofclaim 239 comprising a conjugate.

244. The compound ofclaim 239 comprising at least one 5-methyl cytosine nucleobase.

245. The compound ofclaim 239, comprising not more than 4 bicyclic nucleosides.

246. The compound ofclaim 239, wherein the oligonucleotide consists of 14-20 linked nucleosides.

247. A compound comprising:

a modified oligonucleotide consisting of 10 to 20 linked nucleosides, wherein the modified oligonucleotide has a sugar motif described by Formula II as follows:

(J)_m-(B)_n-(J)_p-(B)_r-(A)_t-(D)_g-(A)_v-(B)_w-(J)_x-(B)_y-(J)_z

wherein:

each A is independently a 2′-substituted nucleoside;

each B is independently a bicyclic nucleoside;

each J is independently either a 2′-substituted nucleoside or a 2′-deoxynucleoside;

each D is a 2′-deoxynucleoside;

m is 0-4; n is 0-2; p is 0-2; r is 0-2; t is 0-2; v is 0-2; w is 0-4; x is 0-2; y is 0-2; z is 0-4; and

g is 6-14;

provided that:

at least one of m, n, and r is other than 0;

at least one of w and y is other than 0;

the sum of m, n, p, r, and t is from 1 to 5; and

the sum of v, w, x, y, and z is from 1 to 5.

248. The compound ofclaim 247, wherein at least one bicyclic nucleoside is a constrained ethyl nucleoside or an LNA nucleoside.

249. A compound comprising a modified oligonucleotide consisting of 10-20 linked nucleosides, wherein the modified oligonucleotide comprises a 5′-wing having an ABABA motif, a 3′-wing consisting of 2-5 linked nucleosides, a gap between the 5′-wing and the 3′-wing consisting of 6 to 15 linked nucleosides, wherein each A is a modified nucleoside and each B comprises a 2′-deoxynucleoside, and wherein the nucleobase sequence of the modified oligonucleotide is complementary to the nucleobase sequence of a target nucleic acid.

250. The compound ofclaim 242, wherein the 3′-wing has a motif selected from: AA, AB, AC, BA, BB, BC, CA, CB, and CC, wherein each C is a modified nucleoside or a 2′-deoxynucleoside.

251. The compound ofclaim 249, wherein the modified oligonucleotide has a ABABA-(D)_6-15-CC motif, wherein each D is a 2′-deoxynucleoside, and each C is a modified nucleoside.

252. The compound ofclaim 251, wherein A is selected from BNA, LNA, α-L-LNA, ENA and 2′-thio LNA.

253. The compound ofclaim 249, wherein the modified oligonucleotide has a sugar motif selected from K-D-K-D-K-(D)₆-E-E, K-D-K-D-K-(D)₇-E-E, K-D-K-D-K-(D)₈-E-E, K-D-K-D-K-(D)₉-E-E, K-D-K-D-K-(D)₁₁-E-E, K-D-K-D-K-(D)₁₂-E-E, K-D-K-D-K-(D)₁₃-E-E, K-D-K-D-K-(D)₁₄-E-E, and K-D-K-D-K-(D)₁₅-E-E, wherein each K is a constrained ethyl nucleoside, each D is a 2′-deoxynucleoside, and each E is a 2′-substituted nucleoside.

254. A method of modulating expression of a target nucleic acid in a cell comprising contacting the cell with a compound according toclaim 239.

255. A method of modulating expression of a target nucleic acid in an animal comprising administering to the animal the pharmaceutical composition according toclaim 239.

256. A method of modulating expression of a target nucleic acid in a cell comprising contacting the cell with a compound according toclaim 247.

257. A method of modulating expression of a target nucleic acid in an animal comprising administering to the animal the pharmaceutical composition according toclaim 247.

258. A method of modulating expression of a target nucleic acid in a cell comprising contacting the cell with a compound according toclaim 249.

259. A method of modulating expression of a target nucleic acid in an animal comprising administering to the animal the pharmaceutical composition according toclaim 249.