Abstract:
Described are RNAi interference agents useful in modulating gene expression in a variety of applications, including use in therapeutic, diagnostic, target validation, and genomic discovery applications. Specifically, the invention relates to double stranded modified oligonucleotide molecules having blunt ends and a least one ribonucleotide near the 5′ end of a sense strand capable of mediating RNA interference (RNAi) against target nucleic acid sequences. The RNAi agents are useful in the treatment of diseases or conditions that respond to inhibition of gene expression or activity in a cell, tissue, or organism.

Description:
BACKGROUND 
       [0001]    RNA interference (RNAi) is a process by which double-stranded RNA (dsRNA) is used to silence gene expression. The process of post-transcriptional gene silencing is thought to be an evolutionarily-conserved cellular defense mechanism used to prevent the expression of foreign genes. It is currently believed that RNAi begins endogenously with the cleavage of longer dsRNAs into small interfering RNAs (siRNAs) by an RNaseIII-like enzyme, dicer. Dicer-made siRNAs are dsRNAs that are usually about 21-23 nucleotides and often contain 2-nucleotide 3′ overhangs, and 5′ phosphate and 3′ hydroxyl termini. The RNAi response also features an endonuclease complex, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single-stranded RNA (mRNA) having sequence complementary to the antisense strand of the siRNA duplex. RISC uses this siRNA strand to identify mRNA molecules that are at least partially complementary to the incorporated siRNA strand, and then cleaves these target mRNAs or inhibits their translation. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex (Elbashir et al., 2001, Genes Dev., 15, 188). The siRNA strand that is complementary to the mRNA is known as the guide strand or the antisense strand. The other siRNA strand is known as the passenger strand or the sense strand. Elbashir et al. (Nature 2001) describes RNAi induced by introduction of duplexes of synthetic 21-nucleotide RNAs in cultured mammalian cells. Synthetic siRNA have been subsequently shown to elicit RNA interference in vivo. Examples of RNA-like molecules that can interact with RISC include RNA agents containing one or more chemically modified nucleotides and/or one or more non-phosphodiester linkages. 
       SUMMARY 
       [0002]    Described herein are RNA interference (RNAi) agents (also RNAi triggers or triggers) comprising: blunt-ended double strand oligonucleotide or RNA-like molecules having a sense strand and an antisense strand wherein the sense strand and the antisense strand are each 26 nucleotides in length (26mers), the antisense strand contains at least 18 consecutive nucleotides that are at least 85% complementary to a sequence in a target mRNA, the sense strand contains at least 18 consecutive nucleotides that are at least 85% complementary to the at least 18 consecutive nucleotides in the antisense strand, and the sense strand further contains at least one ribonucleotide at the second or third position from its 5′ end. 
         [0003]    In some embodiments, the antisense strand contains at least 19 consecutive nucleotides that are at least 85% complementary to a sequence in a target mRNA and the sense strand contains at least 19 consecutive nucleotides that are at least 85% complementary to the at least 19 consecutive nucleotides in the antisense strand. 
         [0004]    In some embodiments, the antisense strand contains at least 20 consecutive nucleotides that are at least 85% complementary to a sequence in a target mRNA and the sense strand contains at least 20 consecutive nucleotides that are at least 85% complementary to the at least 20 consecutive nucleotides in the antisense strand. 
         [0005]    In some embodiments, the antisense strand contains at least 21 consecutive nucleotides that are at least 85% complementary to a sequence in a target mRNA and the sense strand contains at least 21 consecutive nucleotides that are at least 85% complementary to the at least 21 consecutive nucleotides in the antisense strand. 
         [0006]    In some embodiments, the antisense strand contains at least 22 consecutive nucleotides that are at least 85% complementary to a sequence in a target mRNA and the sense strand contains at least 22 consecutive nucleotides that are at least 85% complementary to the at least 22 consecutive nucleotides in the antisense strand. 
         [0007]    In some embodiments, the antisense strand contains at least 23 consecutive nucleotides that are at least 85% complementary to a sequence in a target mRNA and the sense strand contains at least 23 consecutive nucleotides that are at least 85% complementary to the at least 23 consecutive nucleotides in the antisense strand. 
         [0008]    Described herein are RNA interference (RNAi) agents comprising: blunt-ended double strand oligonucleotide or RNA-like molecules having a sense strand and an antisense strand wherein the sense strand and the antisense strand are each 26 nucleotides in length (26mers) and contain a base-paired (complementary) region of at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or 26 consecutive nucleotides and the sense strand further contains at least one ribonucleotide at the second or third position from its 5′ end. 
         [0009]    The herein described blunt-ended 26mer RNAi agents interact with RISC and participate in RISC-mediated inhibition of gene expression. The herein described blunt-ended 26mer RNAi agents are able to selectively and efficiently decrease expression of a target mRNA. 
         [0010]    Described herein are RNAi agents for inhibiting expression of a target gene. The RNAi agent comprises at least two sequences that are at least partially, at least substantially, or fully complementary to each other. The two RNAi agent sequences comprise a sense strand comprising a 26 nucleotide first sequence and an antisense strand comprising a 26 nucleotide second sequence. The RNAi agent sense strands comprise at least 18 consecutive nucleotides that are share at least 85% identity with an at least 18 consecutive nucleotide sequence in a target mRNA. The RNAi agent antisense strands comprise at least 18 consecutive nucleotides that are share at least 85% complementarity with an at least 18 consecutive nucleotide sequence in a target mRNA. 
         [0011]    The described RNAi agents can be linked, directly or indirectly, to a targeting group or a delivery polymer. Targeting groups and/or delivery polymers can facilitate delivery of the RNAi agent to a cell in vivo. 
         [0012]    The described RNAi agents can be used to provide therapeutic treatments of diseases. Such uses comprise administration of RNAi agent to a human being or animal. For treatment of disease of for formation of a medicament or composition for treatment of a disease, a herein described RNAi agent can be combined with one or more pharmaceutical excipients or with a second therapeutic agent or treatment including, but not limited to: a second RNAi agent or other RNAi agent, a small molecule drug, an antibody or other biologic drug product, an antibody fragment, and/or a vaccine. 
         [0013]    The RNAi agents described herein can be delivered to target cells or tissues using any known nucleic acid delivery technology known in the art. Nucleic acid delivery methods include, but are not limited to, encapsulation in liposomes, iontophoresis, or incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres, proteinaceous vectors, or DPCs (U.S. Ser. No. 14/452,626 (WO 2015/021092), US-2008-0152661-A1 (WO 2008/0022309), US-2011-0207799-A1 (WO 2011/104169), and WO 2000/053722, each of which is incorporated herein by reference). 
         [0014]    The RNAi agents or pharmaceutical compositions containing the RNAi agents described herein can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration can be topical (e.g., by a transdermal patch), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer: intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subdermal, e.g., via an implanted device; or intracranial, e.g., by intraparenchymal, intrathecal or intraventricular, administration. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0015]      FIG. 1 . Representation of a blunt-ended RNAi agent. N represents a ribonucleotide, deoxyribonucleotide, modified nucleotide, nucleotide mimic, or abasic site. u represents a 2′-O-methyl (2′-OMe) uridine nucleotide. At least one of N 25′ , N 24′ , and N 23′  is a ribonucleic acid. The duplex is substantially complementary (at least 85% complementary between the sense and antisense strand) in the region in which denoted with “|”. “:” represents optional complementarity between the two strands. SS=sense strand. AS=antisense strand. 
           [0016]      FIG. 2 . Representation of several embodiments (A-C) of blunt-ended RNAi agents. N represents a ribonucleotide, deoxyribonucleotide, modified nucleotide, nucleotide mimic, or abasic site. P represents a ribonucleotide. u represents a 2′-OMe uridine nucleotide. Z represents a 2′-modified nucleotide, a ribonucleotide, or a 2′-deoxyribonucleotide. SS=sense strand. AS=antisense strand. The duplex is substantially complementary (at least 85% complementary between the sense and antisense strand) in the region in which denoted with “|”. “:” represents optional complementarity between the two strands. 
           [0017]      FIG. 3 . Representation of several embodiments of sense strands (A) or antisense strands (B) of blunt-ended RNAi agents wherein “s” at each location represents an optional phosphorothioate linkage. For each “s” the nucleotide linkage is independently a phosphate or a phosphorothioate linkage. N represents a ribonucleotide, deoxyribonucleotide, modified nucleotide, nucleotide mimic, or abasic site. u represents a 2′-O-methyl (2′-OMe) uridine nucleotide. At least one of N 25′ , N 24′ , and N 23′  is a ribonucleic acid. 
           [0018]      FIG. 4 . Representation of several embodiments of sense strands (A) or antisense strands (B) of blunt-ended RNAi agents wherein “s” at each location represents a phosphorothioate linkage. N represents a ribonucleotide, deoxyribonucleotide, modified nucleotide, nucleotide mimic, or abasic site. u represents a 2′-O-methyl (2′-OMe) uridine nucleotide. At least one of N 25′ , N 24′ , and N 23′  is a ribonucleic acid. 
           [0019]      FIG. 5 . Representation of a blunt-ended RNAi agent having A) a frayed end, or B) fully complementary sense and antisense strands. N represents a ribonucleotide, deoxyribonucleotide, modified nucleotide, nucleotide mimic, or abasic site. u represents a 2′-O-methyl (2′-OMe) uridine nucleotide. P represents a ribonucleotide. Z represents a 2′-modified nucleotide, a ribonucleotide, or a 2′-deoxyribonucleotide. The duplex is substantially complementary (at least 85% complementary between the sense and antisense strand) in the region in which denoted with “|”. “:” indicates optional base pairing (i.e. complementarity between the two strands). “x” indicates the nucleotides are not base paired, i.e. they are not complementary. SS=sense strand. AS=antisense strand. 
       
    
    
     DETAILED DESCRIPTION 
       [0020]    We describe blunt-ended RNAi agents having a sense strand and an antisense strand wherein both the sense strand and the antisense strand are each 26 nucleotides in length. The 26 blunted-ended RNAi agents have the form represented in  FIG. 1  wherein at least one or more of positions 23′, 24′, and 25′ is a ribonucleotide, nucleotides at each of positions 23, 24, and 25 is optionally and independently a ribonucleotide, nucleotides at all other positions are modified nucleotides, positions 2-19 are at least 85% complementary to a sequence in a target mRNA, and positions 2′-19′ are at least 85% complementary to corresponding positions 2-19. Unless otherwise noted, when referring to “positions” in the paragraphs that follow, reference to  FIG. 1  is envisioned. Nucleotide N 1  is the nucleotide at position 1. Likewise nucleotide N 1′  is the nucleotide at position 1′. Also with respect to positions, nucleotide N 26′  is the 5′ terminal nucleotide of the sense strand, nucleotide N 25′  is the second nucleotide from the 5′ end of the sense strand, etc. 
         [0021]    In some embodiments, nucleotides at positions 2-19, 2-20, 2-21, 2-22, 2-23, 1-18, 1-19, 1-20, 1-21, 1-22, or 1-23 are at least 85%, at least 90%, or 100% complementary to a sequence in a target mRNA. In some embodiments, nucleotides at positions 2′-19′, 2′-20′, 2′-21′, 2′-22′, 2′-23′, 1′-18′, 1′-19′, 1′-20′, 1′-21′, 1′-22′, or 1′-23′ are at least 85%, at least 90%, or 100% complementary to the corresponding sequence in the antisense strand. 
         [0022]    For the RNAi agents described herein, the following notation is used: N (capital letter without additional notation), unless otherwise indicated, represents a ribonucleotide, deoxyribonucleotide, modified nucleotide, nucleotide mimic, or abasic nucleotide. N can be, but is not limited to, any of the natural or modified nucleotides described herein. P (capital letter) is a ribonucleotide. n (lower case letter) represents a 2′-OMe nucleotide. Nf represents a 2′-fluoro (2′-deoxy-2′-fluoro) nucleotide. dN represents a 2′-deoxy nucleotide. N UNA  (or NUNA) represents a 2′,3′-seco nucleotide (unlocked nucleotide). N LNA  (or NLNA) represents a locked nucleotide. Nf ANA  (or NfANA represents a 2′-F-Arabino nucleotide. NM (or 2′-MOE) represents a 2′-methoxyethyl nucleotide. X represents an abasic ribose. R represents a ribitol. (invN) represents an inverted nucleotide (3′-3′ linked nucleotide). (invdN) represents an inverted deoxyribonucleotide, (invX) represents an inverted abasic nucleotide. (invn) represents an inverted 2′-OMe nucleotide. (invN) can be, but is not limited to: (invdN), (invX), or (invn). s represents a phosphorothioate linked nucleotide. p represents a phosphate. vpdN represents a vinyl phosphonate deoxyribonucleotide. (3′OMen) represents a 3′-OMe nucleotide. 
         [0023]    The described RNAi agents contain at least one ribonucleotide in the sense strand. In some embodiments, the ribonucleotide is a ribopurine (A or G). In some embodiments, at least one of the nucleotides at positions 24′ or 25′ is a ribonucleotide or ribopurine and nucleotides at all other positions are modified. In some embodiments, at least one of the nucleotides at positions 24′ or 25′ is a ribonucleotide, at least one of the nucleotides at positions 23, 24 or 25 is a ribonucleotide, and nucleotides at all other positions are modified. 
         [0024]    In some embodiments, the nucleotide sequence at positions u 26′ N 25′ N 24′ N 23′  (5′ end of the sense strand) is selected from the group consisting of: uPuZ, uuPP, uPPu, uAuZ, uGuZ, uuAA, uuGG, uuAG, uuGA, uAAu, uGGu, uAGu, and uGAu, wherein P is a ribonucleotide or a ribopurine and Z is a 2′-modified nucleotide, a ribonucleotide, or a deoxynucleotide. 
         [0025]    In some embodiments, as represented in  FIG. 2 , position 26′ is a 2′-OMe uridine. In some embodiments, position 25′ is a ribonucleotide, a ribopurine (2′-OH adenosine (ribo-adenosine) or 2′-OH guanosine (ribo-guanosine)), or a 2′-OMe uridine. In some embodiments, if position 25′ is a ribonucleotide or a ribopurine, position 24′ is 2′-OMe uridine, a ribonucleotide or a ribopurine (2′-OH adenosine (ribo-adenosine) or 2′-OH guanosine (ribo-guanosine)). In some embodiments, if position 25′ is a 2′-OMe uridine, position 24′ is a ribonucleotide or ribopurine (2′-OH adenosine (ribo-adenosine), or 2′-OH guanosine (ribo-guanosine)). In some embodiments, if positions 25′ and 24′ are each a ribonucleotide or ribopurine, position 23′ is 2′-OMe uridine. In some embodiments, position 25′ is a ribonucleotide or a ribopurine and 24′ is a 2′-OMe uridine, position 23′ is a 2′-modified nucleotide, a ribonucleotide, a ribopurine, or a deoxynucleotide. In some embodiments, if position 25′ is a 2′-OMe uridine and 24′ is a ribonucleotide or a ribopurine, position 23′ is a ribonucleotide or a ribopurine. 
         [0026]    In some embodiments, positions 26′-24′ are uAu or uGu wherein A and G are ribonucleotides. In some embodiments, positions 26′-24′ are uuA or uuG wherein A and G are ribonucleotides In some embodiments, positions 26′-23′ are uAuA, uAuG, uGuA, uGuG or uNuN wherein A, G, and N are ribonucleotides. In some embodiments, positions 26′-23′ are uuAu, uuGA, or uUaG wherein A, G, and U are ribonucleotides. In some embodiments, positions 26′-22′ are UAUUA wherein U and A are ribonucleotides. 
         [0027]    In some embodiments the terminal 3′ nucleotide (N 1′ ) of the sense strand is Nf. In some embodiments the terminal 3′ nucleotide of the sense strand is Af. In some embodiments the terminal 3′ nucleotide of the sense strand is n. In some embodiments the terminal 3′ nucleotide of the sense strand is a. In some embodiments the terminal 3′ nucleotide of the sense strand is c. In some embodiments the terminal 3′ nucleotide of the sense strand is u. In some embodiments the terminal 3′ nucleotide of the sense strand is g. In some embodiments the terminal 3′ nucleotide of the sense strand is u. In some embodiments the terminal 3′ nucleotide of the sense strand is (invN). In some embodiments the terminal 3′ nucleotide of the sense strand is (invdN). In some embodiments the terminal 3′ nucleotide of the sense strand is (inva). In some embodiments the terminal 3′ nucleotide of the sense strand is (3′OMen). In some embodiments the terminal 3′ nucleotide of the sense strand is (3′OMea). In some embodiments the terminal 3′ nucleotide of the sense strand is NM. In some embodiments the terminal 3′ nucleotide of the sense strand is CM. 
         [0028]    In some embodiments, the terminal 5′ nucleotide (N 1 ) of the antisense strand is dN. In some embodiments, the terminal 5′ nucleotide of the antisense strand is dT. In some embodiments, the terminal 5′ nucleotide of the antisense strand is n. In some embodiments, the terminal 5′ nucleotide of the antisense strand is u. In some embodiments, the terminal 5′ nucleotide of the antisense strand is a. In some embodiments, the terminal 5′ nucleotide of the antisense strand is (invN). In some embodiments, the terminal 5′ nucleotide of the antisense strand is (invdN). 
         [0029]    In some embodiments, the terminal 5′ nucleotide of the antisense strand is (invdA). In some embodiments, the terminal 5′ nucleotide of the antisense strand is (invAbasic or invX). In some embodiments, the terminal 5′ nucleotide of the antisense strand is (invn). In some embodiments, the terminal 5′ nucleotide of the antisense strand is (invu). In some embodiments, the terminal 5′ nucleotide of the antisense strand is Abasic. In some embodiments, the terminal 5′ nucleotide of the antisense strand is (3′OMen). In some embodiments, the terminal 5′ nucleotide of the antisense strand is NM. In some embodiments, the terminal 5′ nucleotide of the antisense strand is (3′OMeu). 
         [0030]    In some embodiments the five nucleotides (5′ N 22 -N 26  3′) at the 3′ end of the antisense strand are nnnnn. In some embodiments the five nucleotides at the 3′ end of the antisense strand are nnndNdN. In some embodiments the five nucleotides at the 3′ end of the antisense strand are nnn(invdN)n. In some embodiments the five nucleotides at the 3′ end of the antisense strand are nnnNN. In some embodiments the five nucleotides at the 3′ end of the antisense strand are nnnNn. In some embodiments the five nucleotides at the 3′ end of the antisense strand are nnnNMNM. In some embodiments the five nucleotides at the 3′ end of the antisense strand are nNNNN. In some embodiments the five nucleotides at the 3′ end of the antisense strand are nNnNfn. In some embodiments the five nucleotides at the 3′ end of the antisense strand are nnNfnn. In some embodiments the five nucleotides at the 3′ end of the antisense strand are NfnnNn. In some embodiments the five nucleotides at the 3′ end of the antisense strand are NMNMnNn. 
         [0031]    Positions 1 and 1′ are modified nucleotides. In some embodiments, the nucleotide at position 1 is a modified adenosine, modified uridine, or a deoxythimidine. In some embodiments, the nucleotide at position 1′ is a modified adenosine, modified uridine, a deoxythimidine, or an inverted deoxythimidine. 
         [0032]    In some embodiments 20% or fewer of the modified nucleotides are 2′-fluoro modified nucleotides. 
         [0033]    In some embodiments, the described RNAi agent contains at least one modified backbone. In some embodiments, the modified backbone is a phosphorothioate linkage. In some embodiments, a sense strand of the described RNAi agents contains 1-4 phosphorothioate linkages. In other embodiments, an antisense strand of the described RNAi agents contains 1-4 phosphorothioate linkages. In yet other embodiments, both the sense strand and the antisense strand contain 1-4 phosphorothioate linkages. 
         [0034]    In some embodiments, each of nucleotides 1′-2′, 2′-3′, 1-2, 2-3, 19′-20′, 20′-21′, 21′-22′, 22′-23′, 23′-24′, 21-22, 22-23, 23-24, 24-25, 25-26, is optionally and independently linked via a phosphorothioate linkage (see e.g.,  FIG. 3 ). 
         [0035]    In some embodiments, the nucleotide at position 1′ is linked to the nucleotide at position 2′ via a phosphorothioate linkage. 
         [0036]    In some embodiments, the nucleotide at position 2′ is linked to the nucleotide at position 3′ via a phosphorothioate linkage. 
         [0037]    In some embodiments, the nucleotide at position 1′ is linked to the nucleotide at position 2′ via a phosphorothioate linkage and the nucleotide at position 2′ is linked to the nucleotide at position 3′ via a phosphorothioate linkage ( FIG. 4A ) 
         [0038]    In some embodiments, the nucleotide at position 19′ is linked to the nucleotide at position 20′ via a phosphorothioate linkage. 
         [0039]    In some embodiments, the nucleotide at position 20′ is linked to the nucleotide at position 21′ via a phosphorothioate linkage. 
         [0040]    In some embodiments, the nucleotide at position 19′ is linked to the nucleotide at position 20′ via a phosphorothioate linkage and the nucleotide at position 20′ is linked to the nucleotide at position 21′ via a phosphorothioate linkage. ( FIG. 4A ) 
         [0041]    In some embodiments, the nucleotide at position 20′ is linked to the nucleotide at position 21′ via a phosphorothioate linkage. 
         [0042]    In some embodiments, the nucleotide at position 21′ is linked to the nucleotide at position 22′ via a phosphorothioate linkage. 
         [0043]    In some embodiments, the nucleotide at position 20′ is linked to the nucleotide at position 21′ via a phosphorothioate linkage and the nucleotide at position 21′ is linked to the nucleotide at position 22′ via a phosphorothioate linkage ( FIG. 4A ). 
         [0044]    In some embodiments, the nucleotide at position 1 is linked to the nucleotide at position 2 via a phosphorothioate linkage. 
         [0045]    In some embodiments, the nucleotide at position 2 is linked to the nucleotide at position 3 via a phosphorothioate linkage. 
         [0046]    In some embodiments, the nucleotide at position 1 is linked to the nucleotide at position 2 via a phosphorothioate linkage and the nucleotide at position 2 is linked to the nucleotide at position 3 via a phosphorothioate linkage ( FIG. 4B ). 
         [0047]    In some embodiments, the nucleotide at position 21 is linked to the nucleotide at position 22 via a phosphorothioate linkage. 
         [0048]    In some embodiments, the nucleotide at position 22 is linked to the nucleotide at position 23 via a phosphorothioate linkage. 
         [0049]    In some embodiments, the nucleotide at position 21 is linked to the nucleotide at position 22 via a phosphorothioate linkage and the nucleotide at position 22 is linked to the nucleotide at position 23 via a phosphorothioate linkage ( FIG. 4B ). 
         [0050]    In some embodiments, the nucleotide at position 22 is linked to the nucleotide at position 23 via a phosphorothioate linkage. 
         [0051]    In some embodiments, the nucleotide at position 23 is linked to the nucleotide at position 24 via a phosphorothioate linkage. 
         [0052]    In some embodiments, the nucleotide at position 22 is linked to the nucleotide at position 23 via a phosphorothioate linkage and the nucleotide at position 23 is linked to the nucleotide at position 24 via a phosphorothioate linkage. ( FIG. 4B ). 
         [0053]    In some embodiments, the nucleotide at position 20′ is linked to the nucleotide at position 21′ via a phosphorothioate linkage, the nucleotide at position 21′ is linked to the nucleotide at position 22′ via a phosphorothioate linkage, the nucleotide at position 22 is linked to the nucleotide at position 23 via a phosphorothioate linkage and the nucleotide at position 23 is linked to the nucleotide at position 24 via a phosphorothioate linkage. 
         [0054]    In some embodiments, the nucleotide at position 19′ is linked to the nucleotide at position 20′ via a phosphorothioate linkage, the nucleotide at position 20′ is linked to the nucleotide at position 21′ via a phosphorothioate linkage, the nucleotide at position 21 is linked to the nucleotide at position 22 via a phosphorothioate linkage and the nucleotide at position 22 is linked to the nucleotide at position 23 via a phosphorothioate linkage. 
         [0055]    In some embodiments, the nucleotide at position 22′ is linked to the nucleotide at position 23′ via a phosphorothioate linkage. 
         [0056]    In some embodiments, the nucleotide at position 23′ is linked to the nucleotide at position 24′ via a phosphorothioate linkage. 
         [0057]    In some embodiments, the nucleotide at position 23 is linked to the nucleotide at position 24 via a phosphorothioate linkage. 
         [0058]    In some embodiments, the nucleotide at position 24 is linked to the nucleotide at position 25 via a phosphorothioate linkage. 
         [0059]    In some embodiments, the nucleotide at position 25 is linked to the nucleotide at position 26 via a phosphorothioate linkage. 
         [0060]    As used herein, the term “sequence” or “nucleotide sequence” refers to a succession or order of nucleobases or nucleotides, described with a succession of letters using the standard nucleotide nomenclature and the key for modified nucleotides described herein. 
         [0061]    As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence (e.g. RNAi agent sense strand or target mRNA) in relation to a second nucleotide sequence (e.g. RNAi agent antisense strand), refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize (form base pair hydrogen bonds) and form a duplex or double helical structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence. Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs and include natural or modified nucleotides or nucleotide mimics, at least to the extent that the above requirements with respect to the ability to hybridize are fulfilled. Perfectly or fully complementary means that all (100%) of the bases in a contiguous sequence of a first polynucleotide will hybridize with the same number of bases in a contiguous sequence of a second polynucleotide. The contiguous sequence may comprise all or a part of a first or second nucleotide sequence. As used herein, partial complementary means that in a hybridized pair of nucleobase sequences, at least 70% of the bases in a contiguous sequence of a first polynucleotide will hybridize with the same number of bases in a contiguous sequence of a second polynucleotide. As used herein, substantial complementary means that in a hybridized pair of nucleobase sequences, at least 85% of the bases in a contiguous sequence of a first polynucleotide will hybridize with the same number of bases in a contiguous sequence of a second polynucleotide. The terms “complementary”, “fully complementary” and “substantially complementary” herein may be used with respect to the base matching between the sense strand and the antisense strand of an RNAi agent, or between the antisense strand of a RNAi agent and a sequence of a target mRNA. 
         [0062]    Sequence identity or complementarity is independent of modification. For example, a and Af are complementary to U (or T) and identical to A for the purposes of determining identity or complementarity. 
         [0063]    The nucleic acid sequence of positions 2-19 is at least 85% complementary to a nucleotide sequence in a target mRNA. In some embodiments, the nucleic acid sequence of positions 2-19 is at least 90% complementary to a nucleotide sequence in a target mRNA. In some embodiments, the nucleic acid sequence of positions 2-19 is 100% complementary to a nucleotide sequence in a target mRNA. 
         [0064]    The nucleic acid sequence of positions 2′-19′ is at least 85% complementary to the corresponding nucleic acid sequence of positions 2-19 or identical to a nucleotide sequence in a target mRNA. In some embodiments, the nucleic acid sequence of positions 2′-19′ is at least 90% complementary to the corresponding nucleic acid sequence of positions 2-19 or identical to a nucleotide sequence in a target mRNA. In some embodiments, the nucleic acid sequence of positions 2′-19′ is 100% complementary to the corresponding nucleic acid sequence of positions 2-19 or identical to a nucleotide sequence in a target mRNA. 
         [0065]    Nucleotides N 20 , N 21 , N 22 , and N 23  (i.e. nucleotides at positions 20, 21, 22, and 23) are independently and optionally complementary to a corresponding sequence in a target mRNA. In some embodiments, the nucleotide sequence of positions 2-20, 2-21, 2-22, or 2-23 is at least 80%, at least 85%, at least 90%, or 100% complementary to a nucleotide sequence in a target mRNA. 
         [0066]    Nucleotides N 20′  and N 21′  (i.e. nucleotides at positions 20′ and 21′) are independently and optionally identical to a corresponding sequence in a target mRNA. In some embodiments, the nucleotide sequence of positions 2′-20′ or 2′-21′ is at least 80%, at least 85%, at least 90%, or 100% identical to a nucleotide sequence in a target mRNA. 
         [0067]    The nucleotide at position 20 is optionally complementary to the nucleotide at position 20′. The nucleotide at position 21 is optionally complementary to the nucleotide at position 21′. The nucleotide at position 22 is optionally complementary to the nucleotide at position 22′. The nucleotide at position 23 is optionally complementary to the nucleotide at position 23′. The nucleotide at position 24 is optionally complementary to the nucleotide at position 24′. The nucleotide at position 25 is optionally complementary to the nucleotide at position 25′. The nucleotide at position 26 is optionally complementary to the nucleotide at position 26′. 
         [0068]    In some embodiments, the nucleotide at position 20 is complementary to the nucleotide at position 20′. In some embodiments, nucleotide at position 21 is complementary to the nucleotide at position 21′. In some embodiments, the nucleotide at position 22 is complementary to the nucleotide at position 22′. In some embodiments, the nucleotide at position 23 is complementary to the nucleotide at position 23′. In some embodiments, the nucleotide at position 24 is complementary to the nucleotide at position 24′. In some embodiments, the nucleotide at position 25 is complementary to the nucleotide at position 25′. In some embodiments, the nucleotide at position 26 is complementary to the nucleotide at position 26′. 
         [0069]    In some embodiments, the nucleotide at position 20 is not complementary to the nucleotide at position 20′. In some embodiments, the nucleotide at position 21 is not complementary to the nucleotide at position 21′. In some embodiments, the nucleotide at position 22 is not complementary to the nucleotide at position 22′. In some embodiments, the nucleotide at position 23 is not complementary to the nucleotide at position 23′. In some embodiments, the nucleotide at position 24 is not complementary to the nucleotide at position 24′. In some embodiments, the nucleotide at position 25 is not complementary to the nucleotide at position 25′. In some embodiments, the nucleotide at position 26 is not complementary to the nucleotide at position 26′. 
         [0070]    In some embodiments, the nucleotides at positions 25 and 26 are not complementary to the nucleotides at position 25′ and 26′. In some embodiments, the nucleotides at positions 25 and 26 are complementary to the nucleotides at positions 25′ and 26′. In some embodiments, the nucleotides at positions 24, 25, and 26 are not complementary to the nucleotides at position 24′, 25′, and 26′ (as represented in  FIG. 5A ). In some embodiments, the nucleotides at positions 24, 25, and 26 are complementary to the nucleotides at positions 24′, 25′, and 26′. In some embodiments, the nucleotides at positions 24 and 26 are not complementary to the nucleotides at position 24′ and 26′. In some embodiments, the nucleotides at positions 24 and 26 are complementary to the nucleotides at position 24′ and 26′. In some embodiments, the nucleotides at positions 23, 24, 25, and 26 are not complementary to the nucleotides at position 23′, 24′, 25′, and 26′. In some embodiments, the nucleotides at positions 22, 23, 24, 25, and 26 are complementary to the nucleotides at position 22′, 23′, 24′, 25′, and 26′. 
         [0071]    The nucleotide at position 1′ is optionally identical to a corresponding nucleotide in a target mRNA. In some embodiments, the nucleotide at position 1′ is identical to a corresponding nucleotide in a target mRNA. In some embodiments, the nucleotide at position 1′ is not identical to a corresponding nucleotide in a target mRNA. 
         [0072]    The nucleotide at position 1 is optionally complementary to a corresponding nucleotide in a target mRNA. In some embodiments, the nucleotide at position 1 is complementary to a corresponding nucleotide in a target mRNA. In some embodiments, the nucleotide at position 1 is not complementary to a corresponding nucleotide in a target mRNA. 
         [0073]    In some embodiments, the nucleotide at position 1′ is complementary to the nucleotide at position 1. In some embodiments, the nucleotide at position 1′ is not complementary to the nucleotide at position 1. 
         [0074]    In some embodiments, the nucleotide at position 1 is complementary to the nucleotide at position 1′ and to a corresponding nucleotide in a target mRNA. In some embodiments, the nucleotide at position 1 is complementary to the nucleotide at position 1′ and not complementary to a corresponding nucleotide in a target mRNA. In some embodiments, the nucleotide at position 1 is complementary to a corresponding nucleotide in a target mRNA and not complementary to the nucleotide at position 1′. In some embodiments, the nucleotide at position 1 is not complementary to either a corresponding nucleotide in a target mRNA or the nucleotide at position 1′. 
         [0075]    In some embodiments, the nucleotide at position 1′ is complementary to the nucleotide at position 1 and identical to a corresponding nucleotide in a target mRNA. In some embodiments, the nucleotide at position 1′ is complementary to the nucleotide at position 1 and not identical to a corresponding nucleotide in a target mRNA. In some embodiments, the nucleotide at position 1′ is identical to a corresponding nucleotide in a target mRNA and not complementary to the nucleotide at position 1. In some embodiments, the nucleotide at position 1′ is not identical to a corresponding nucleotide in a target mRNA and not complementary to the nucleotide at position 1. 
         [0076]    In some embodiments, the nucleotide sequence of positions 1-19, 1-20, 1-21, 1-22, or 1-23 is at least 80%, at least 85%, at least 90%, or 100% complementary to a nucleotide sequence in a target mRNA. 
         [0077]    In some embodiments, the nucleotide sequence of positions 1′-19′, 1′-20′ or 1′-21′ is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a nucleotide sequence in a target mRNA. 
         [0078]    The sense strand and antisense strands of the described RNAi agents are at least partially complementary to each other. In some embodiments the sense strand is at least 70% complementary to the antisense strand. In some embodiments the sense strand is at least 75% complementary to the antisense strand. In some embodiments the sense strand is at least 80% complementary to the antisense strand. In some embodiments the sense strand is at least 84% complementary to the antisense strand. In some embodiments the sense strand is at least 87% complementary to the antisense strand. In some embodiments the sense strand is at least 90% complementary to the antisense strand. In some embodiments the sense strand is at least 95% complementary to the antisense strand. In some embodiments the sense strand is at perfectly complementary to the antisense strand. 
         [0079]    An RNAi agent can contain a non-nucleotide group attached to the 3′ or 5′ end of either the sense strand or the antisense strand. In some embodiments, a targeting group, linking group, or delivery vehicle is covalently linked to the sense strand. In some embodiments, the targeting group, linking group, and/or delivery vehicle is linked to the 3′ end (position 1′) and/or the 5′ end (position 26′) of the sense strand. The targeting group, linking group, and/or delivery vehicle is linked directly or indirectly via a linker to the 3′ or 5′ end of the sense strand. In some embodiments, position 1′ is covalently attached, either directly or indirectly via a linker, to a targeting group. In some embodiments, position 26′ is covalently attached, either directly or indirectly via a linker, to a targeting group. In some embodiments, a targeting group is linked to the RNAi agent via a labile, cleavable, or reversible bond or linker/spacer. 
         [0080]    A targeting group enhances the pharmacokinetic or biodistribution properties of a molecule to which they are attached to improve cell- or tissue-specific distribution and cell-specific uptake of the conjugate. Binding of a targeting group to a cell or cell receptor may initiate endocytosis. Targeting groups may be monovalent, divalent, trivalent, tetravalent, or have higher valency. Targeting groups can be, but are not limited to, compounds with affinity to cell surface molecule, cell receptor ligands, antibodies, monoclonal antibodies, antibody fragments, and antibody mimics with affinity to cell surface molecules, hydrophobic groups, cholesterol, cholesteryl groups, or steroids. In some embodiments, a targeting group comprises a cell receptor ligand. A variety of targeting groups have been used to target drugs and genes to cells and to specific cellular receptors. Cell receptor ligands may be, but are not limited to: carbohydrates, glycans, saccharides (including, but not limited to: galactose, galactose derivatives (such as N-acetyl-galactosamine), mannose, and mannose derivatives), haptens, vitamins, folate, biotin, aptamers, and peptides (including, but not limited to: RGD-containing peptides, insulin, EGF, and transferrin). 
         [0081]    In some embodiments, an RNAi agent as described herein comprises a linking group conjugated to the RNAi agent. The linking group facilitates covalent linkage of the agent to a targeting group or delivery polymer. The linking group may be linked to the 3′ or the 5′ end of the RNAi agent sense strand or antisense strand. In some embodiments, the linking group is linked to the RNAi agent sense strand. In some embodiments, the linking group is conjugated to the 5′ or 3′ end of an RNAi agent sense strand. In some embodiments a linking group is conjugated to the 5′ end of an RNAi agent sense strand. Exemplary linking groups, include, but are not limited to: Alk-SMPT-C6, Alk-SS-C6, DBCO-TEG, Me-Alk-SS-C6, and C6-SS-Alk-Me. 
         [0082]    A linker or linking group is a connection between two atoms that links one chemical group (such as an RNAi agent) or segment of interest to another chemical group (such as a targeting group or delivery polymer) or segment of interest via one or more covalent bonds. A labile linkage contains a labile bond. A linkage may optionally include a spacer that increases the distance between the two joined atoms. A spacer may further add flexibility and/or length to the linkage. Spacers may include, but are not be limited to, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, aralkynyl groups; each of which can contain one or more heteroatoms, heterocycles, amino acids, nucleotides, and saccharides. Spacer groups are well known in the art and the preceding list is not meant to limit the scope of the invention. 
         [0083]    Targeting groups and linking groups include, but are not limited to, the compounds represented by the structures below. In some of the targeting group and linking group structures shown below, the structure includes the RNAi agent, denoted by Trigger, RNA, R, or R1 or R2 (i.e. Trigger, RNA or R1 or R2 each comprises the RNAi agent). In some embodiments, the RNAi agent is linked directly to a targeting group or linking group. In other embodiments, the RNAi agent is linked to a targeting group and linking group via a linker. For (Alk-C6-Ser), (Alk-PEGS-Ser), and (Alk-PEG13-Ser), one of R1 and R2 comprises the RNAi agent and the other is a hydrogen. For linkers (C3), (C12), (Sp9), (Sp18), (Spermine), (C6-SS-C6), one of R1 or R2 comprises the RNAi agent and the other comprises a hydrogen, reactive group, targeting group, linking group, alkyl group, or substituted alkyl group. 
         [0000]    
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
     
         [0084]    In some embodiments, a delivery vehicle may be used. A delivery vehicle is a compound which improves delivery of the RNAi agent to the cell. A delivery vehicle can be, but is not limited to: a polymer, such as an amphipathic polymer, membrane active polymer, a peptide, such as a melittin or melittin-like peptide, a reversibly modified polymer or peptide, or a lipid. 
         [0085]    In some embodiments, the targeting group is a galactose cluster. In some embodiments, an RNAi agent as described herein is linked to a galactose cluster. As used herein, a galactose cluster comprises a molecule having two to four terminal galactose derivatives. As used herein, the term galactose derivative includes both galactose and derivatives of galactose having affinity for the asialoglycoprotein receptor equal to or greater than that of galactose. A terminal galactose derivative is attached to a molecule through its C-1 carbon. In some embodiments, a galactose cluster has three terminal galactosamines or galactosamine derivatives (such as N-acetyl-galactosamine) each having affinity for the asialoglycoprotein receptor. In some embodiments, a galactose cluster has three terminal N-acetyl-galactosamines. Other terms common in the art include tri-antennary galactose, tri-valent galactose and galactose trimer. It is known that tri-antennary galactose derivative clusters are bound to the ASGPr with greater affinity than bi-antennary or mono-antennary galactose derivative structures (Baenziger and Fiete, 1980, Cell, 22, 611-620; Connolly et al., 1982, J. Biol. Chem., 257, 939-945). 
         [0086]    In some embodiments, a galactose cluster contains three galactose derivatives each linked to a central branch point. The galactose derivatives are attached to the central branch point through the C-1 carbons of the saccharides. In some embodiments, a galactose derivative is linked to the branch point via a linker or spacer. In some embodiments, the linker or spacer is a flexible hydrophilic spacer (U.S. Pat. No. 5,885,968; Biessen et al. J. Med. Chem. 1995 Vol. 39 p. 1538-1546), such as, but not limited to: a PEG spacer. In some embodiments, the PEG spacer is a PEG3 spacer. The branch point can be any small molecule which permits attachment of three galactose derivatives and further permits attachment of the branch point to the RNAi agent. Attachment of the branch point to the RNAi agent may occur through a linker or spacer. In some embodiments, the linker or spacer comprises a flexible hydrophilic spacer, such as, but not limited to: a PEG spacer. In some embodiments, a PEG spacer is a PEG3 spacer (three ethylene units). In other embodiments, the PEG spacer has 1 to 20 ethylene units (PEG 1  to PEG 20 ). 
         [0087]    In some embodiments, a galactose derivative comprises an N-acetyl-galactosamine (GalNAc or NAG). Other saccharides having affinity for the asialoglycoprotein receptor may be selected from the list comprising: galactose, galactosamine, N-formyl-galactosamine, N-acetyl-galactosamine, N-propionyl-galactosamine, N-n-butanoylgalactosamine, and N-iso-butanoylgalactosamine. The affinities of numerous galactose derivatives for the asialoglycoprotein receptor have been studied (see for example: Iobst, S. T. and Drickamer, K.  J.B.C.  1996, 271, 6686) or are readily determined using methods well known and commonly used in the art. 
         [0088]    Nucleotides at positions 1-19 of the RNAi agents described herein are modified nucleotides. In some embodiments, nucleotides at positions 1-20 are modified nucleotides. In some embodiments, nucleotides at positions 1-21 are modified nucleotides. In some embodiments, nucleotides at positions 1-22 are modified nucleotides. In some embodiments, nucleotides at positions 1-23 are modified nucleotides. In some embodiments, nucleotides at positions 1-24 are modified nucleotides. In some embodiments, nucleotides at positions 1-25 are modified nucleotides. In some embodiments, nucleotides at positions 1-26 are modified nucleotides. In some embodiments, nucleotides at positions 1-24, and 26 are modified nucleotides. In some embodiments, nucleotides at positions 1-23, 25, and 26 are modified nucleotides. In some embodiments, nucleotides at positions 1-22, 24, and 26 are modified nucleotides. 
         [0089]    Nucleotides at positions 1′-19′ of the RNAi agents described herein are modified nucleotides. In some embodiments, nucleotides at positions 1′-20′ are modified nucleotides. In some embodiments, nucleotides at positions 1′-21′ are modified nucleotides. In some embodiments, nucleotides at positions 1′-22′ are modified nucleotides. In some embodiments, nucleotides at positions 1′-23′ are modified nucleotides. In some embodiments, nucleotides at positions 1′-24′ are modified nucleotides. In some embodiments, nucleotides at positions 1′-24′ and 26′ are modified nucleotides. In some embodiments, nucleotides at positions 1′-22′, 24′ and 26′ are modified nucleotides. In some embodiments, nucleotides at positions 1′-22′, 25′, and 26′ are modified nucleotides. In some embodiments, nucleotides at positions 1′-23′ and 26′ are modified nucleotides. 
         [0090]    In some embodiments, nucleotides at positions 1′-22′, 26′, 1-22, and 26 are modified nucleotides. In some embodiments, position 1 is an inverted deoxynucleotide, a 2′-fluoro nucleotide (2′-F), a 2′-O-methyl nucleotide (2′-OMe), or a 2′-methoxyethoxy nucleotide (2′-MOE). In some embodiments, position 1′ is a 2′-F nucleotide, an inverted deoxynucleotide, a 2′-OMe nucleotide, or a 2′-MOE nucleotide. 
         [0091]    The RNAi agents described herein contain at least one ribonucleotide. Ribonucleotides include ribopurines (A, G) and ribopyrimidines (C, U). 
         [0092]    The RNAi agents described herein are contain modified nucleotides. A nucleotide base (or nucleobase) is a heterocyclic pyrimidine or purine compound which is a constituent of all nucleic acids and includes adenine (A), guanine (G), cytosine (C), thymine (I), and uracil (U). As used herein, “G,” “g”, “C,” “c”, “A”, “a”, “U”, “u”, and “T”, each generally stand for a nucleobase, nucleoside, nucleotide or nucleotide mimic that contains guanine, cytosine, adenine, uracil and thymidine as a base, respectively. Also as used herein, the term “nucleotide” may include a modified nucleotide or nucleotide mimic, abasic site, or a surrogate replacement moiety. As used herein, a “modified nucleotide” is a nucleotide, nucleotide mimic, abasic site, or a surrogate replacement moiety other than a ribonucleotide (2′-hydroxyl nucleotide). In one embodiment a modified nucleotide comprises a 2′-modified nucleotide (i.e. a nucleotide with a group other than a hydroxyl group at the 2′ position of the five-membered sugar ring). Ribonucleotide are represented herein as “N” (capital letter without further notation). Modified nucleotides include, but are not limited to: 2′-modified nucleotides, 2′-O-methyl nucleotides (represented herein as a lower case letter ‘n’ in a nucleotide sequence), 2′-deoxy-2′-fluoro nucleotides (represented herein as Nf, also represented herein as 2′-fluoro nucleotide), 2′-deoxy nucleotides (represented herein as dN), 2′-methoxyethyl (2′-O-2-methoxylethyl) nucleotides (represented herein as NM or 2′-MOE), 2′-amino nucleotides, 2′-alkyl nucleotides, 3′ to 3′ linkages (inverted) nucleotides (represented herein as invdN, invN, invn, invX), non-natural base comprising nucleotides, bridged nucleotides, peptide nucleic acids, 2′,3′-seco nucleotide mimics (unlocked nucleobase analogues, represented herein as N UNA  or NUNA), locked nucleotides (represented herein as N LNA  or NLNA), 3′-O-Methoxy (2′ internucleotide linked) nucleotide (represented herein as 3′-OMen), 2′-F-Arabino nucleotides (represented herein as NfANA or Nf ANA ), morpholino nucleotides, vinyl phosphonate deoxyribonucleotide (represented herein as vpdN), vinyl phosphonate nucleotides, and abasic nucleotides (represented herein as X or Ab). It is not necessary for all positions in a given compound to be uniformly modified. Conversely, more than one modification may be incorporated in a single RNAi agent or even in a single nucleotide thereof. The RNAi agent sense strands and antisense strands described herein may be synthesized and/or modified by methods known in the art. Modification at each nucleotide is independent of modification of the other nucleotides. 
         [0093]    Modified nucleobases include synthetic and natural nucleobases, such as 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, 5-methylcytosine (5-me-C), 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 uracil and cytosine, 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, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. 
         [0094]    Nucleotides of an RNAi agent described herein may be linked by phosphate-containing or non-phosphate-containing covalent internucleoside linkages. Modified internucleoside linkages or backbones include, for example, phosphorothioates, 5′-phosphorothioate group (represented herein as a lower case ‘s’ before a nucleotide, as in sN, sn, sNf, or sdN), chiral phosphorothioates, thiophosphate, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkyl-phosphonates, thionoalkylphosphotriesters, and boranophosphates having normal linkages, linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked to 5′-3′ or to 5′-2′. Various salts, mixed salts and free-acid forms are also included. 
         [0095]    Modified internucleoside linkages or backbones that do not include a phosphorus atom therein (i.e., oligonucleosides) have backbones that are formed by short chain alkyl or cycloalkyl inter-sugar linkages, mixed heteroatom and alkyl or cycloalkyl inter-sugar linkages, or one or more short chain heteroatomic or heterocyclic inter-sugar linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH 2  component parts. 
         [0096]    The herein described RNAi agents have blunt ends. As used herein, the terminal nucleotides of a blunt end may be complementary or may not be complementary. As used herein a frayed end refers to an end of a blunt end in which the terminal nucleotides of the two annealed strands are not complementary (i.e. do not form a non-complementary base-pair). 
         [0097]    RNA interference (RNAi) agents (also dsRNAi triggers, RNAi triggers, or triggers) are double strand oligonucleotides capable of inducing RNA interference through interaction with the RNA interference pathway machinery (RNA-induced silencing complex or RISC) of mammalian cells. RNA interference leads to degradation or inhibits translation of messenger RNA (mRNA) transcripts of a target mRNA in a sequence specific manner. 
         [0098]    An siRNA agent comprises a sense strand and an antisense strand that are at least partially complementary (at least 70% complementary) to each other. The antisense strand contains a region having a sequence that is perfectly complementary (100% complementary) or at least substantially complementary (at least 85% complementary) to a sequence in a target mRNA. This region of perfect or substantial complementarity is typically 15-25 nucleotides in length and occurs at or near the 5′ end of the antisense strand. 
         [0099]    The sense and antisense strands of the described RNAi agents are synthesized using methods commonly used in the art. Double strand RNAi agents can be formed by annealing an antisense strand with a sense strand. 
         [0100]    The described RNAi agents and methods can be used to treat a subject having a disease or disorder that would benefit from reduction or inhibition expression of the target mRNA. The subject is administered a therapeutically effective amount of any one or more of the RNAi agents. The subject can be a human, patient, or human patient. The described RNAi agents can be used to provide a method for the therapeutic treatment of diseases. Such methods comprise administration of a described herein RNAi agent to a human being or animal. 
         [0101]    We describe compositions and methods for inhibiting expression of a target mRNA in a cell, group of cells, tissue, or subject, comprising: administering to the subject a therapeutically effective amount of a herein described RNAi agent thereby inhibiting the expression of a target mRNA in the subject. Silence, reduce, inhibit, down-regulate, or knockdown gene expression, in as far as they refer to a target RNA, means that the expression of mRNA, as measured by the level of mRNA in a cell, group of cells, tissue, or subject, or the level of polypeptide, protein or protein subunit translated from the mRNA in a cell, group of cells, or tissue, or subject in which the target mRNA gene is transcribed, is reduced when the cell, group of cells, or tissue, or subject is treated with the described RNAi agents as compared to a second cell, group of cells, or tissue, or subject substantially which has not or have not been so treated. 
         [0102]    In some embodiments, we describe pharmaceutical compositions comprising at least one of the described RNAi agents. These pharmaceutical compositions are particularly useful in the inhibition of the expression of a target mRNA in a cell, a group of cells, a tissue, or an organism. The described pharmaceutical compositions can be used to treat a subject having a disease or disorder that would benefit from reduction or inhibition in expression of the target mRNA. The described pharmaceutical compositions can be used to treat a subject at risk of developing a disease or disorder that would benefit from reduction or inhibition in expression of the target mRNA. In one embodiment, the method comprises administering a composition comprising an RNAi agent described herein to a subject to be treated. In some embodiments a pharmaceutical composition comprises one or more pharmaceutically acceptable excipients (including vehicles, carriers, diluents, and/or delivery polymers). 
         [0103]    In some embodiments, the described RNAi agents are used for treating, preventing, or managing clinical presentations associated with expression of a target mRNA. In some embodiments, a therapeutically or prophylactically effective amount of one or more RNAi agents is administered to a subject in need of such treatment, prevention or management. 
         [0104]    The described RNAi agents and methods can be used to treat or prevent at least one symptom in a subject having a disease or disorder that would benefit from reduction or inhibition in expression of a target mRNA. In some embodiments, the subject is administered a therapeutically effective amount of one or more RNAi agents thereby treating the symptom. In other embodiments, the subject is administered a prophylactically effective amount of one or more of RNAi agents thereby preventing the at least one symptom. 
         [0105]    In some embodiments, expression of a target mRNA in a subject to whom an RNAi agent is administered is reduced by at least about 5%, 10%, 15%, 20% 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% relative to the subject not receiving the RNAi agent. The gene expression level in the subject may be reduced in a cell, group of cells, and/or tissue of the subject. In some embodiments, the level of mRNA is reduced. In other embodiments, the expressed protein level is reduced. Reduction in expression, mRNA levels, or protein levels can be assessed by any methods known in the art. Reduction or decrease in mRNA level and/or protein level are collectively referred to herein as a reduction or decrease in target RNA or inhibiting or reducing the expression of target mRNA. 
         [0106]    “Introducing into a cell”, when referring to an RNAi agent, means functionally delivering the RNAi agent into a cell. By functional delivery, it is meant that the RNAi agent is delivered to the cell and has the expected biological activity, sequence-specific inhibition of gene expression. 
         [0107]    The route of administration is the path by which an RNAi agent is brought into contact with the body. In general, methods of administering drugs and nucleic acids for treatment of a mammal are well known in the art and can be applied to administration of the compositions described herein. The herein described RNAi agents can be administered via any suitable route in a preparation appropriately tailored to the particular route. Thus, herein described RNAi agents can be administered by injection, for example, intravenously, intramuscularly, intracutaneously, subcutaneously, or intraperitoneally. Accordingly, in some embodiments, pharmaceutical compositions may comprise one or more pharmaceutically acceptable excipients. 
         [0108]    In one embodiment, RNAi agents described herein can be formulated for administration to a subject. 
         [0109]    The RNAi agents or compositions described herein can be delivered to a cell, group of cells, tumor, tissue, or subject using oligonucleotide delivery technologies known in the art. In general, any suitable method recognized in the art for delivering a nucleic acid molecule (in vitro or in vivo) can be adapted for use with a herein described RNAi agents. For example, delivery can be by local administration, (e.g., direct injection, implantation, or topical administering), systemic administration, or subcutaneous, intravenous, oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal and intrathecal), intramuscular, transdermal, airway (aerosol), nasal, rectal, or topical (including buccal and sublingual) administration, In certain embodiments, the compositions are administered by subcutaneous or intravenous infusion or injection. 
         [0110]    The RNAi agents can be combined with lipids, nanoparticles, polymers, liposomes, micelles. DPCs or other delivery systems available in the art. The RNAi agents can also be chemically conjugated to targeting moieties, lipids (including, but not limited to cholesterol and cholesteryl derivative), nanoparticles, polymers, liposomes, micelles, DPCs (WO 2015/021092, WO 2000/053722, WO 2008/0022309, WO 2013/158141, and WO 2011/104169), or other delivery systems available in the art. 
         [0111]    As used herein, a “pharmaceutical composition” comprises a pharmacologically effective amount of at least one kind of RNAi agent and one or more a pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients (excipients) are substances other than the Active Pharmaceutical ingredient (API, therapeutic product, e.g., RNAi agent) that have been appropriately evaluated for safety and are intentionally included in the drug delivery system. Excipients do not exert or are not intended to exert a therapeutic effect at the intended dosage. Excipients may act to a) aid in processing of the drug delivery system during manufacture, b) protect, support or enhance stability, bioavailability or patient acceptability of the API, c) assist in product identification, and/or d) enhance any other attribute of the overall safety, effectiveness, of delivery of the API during storage or use. 
         [0112]    Excipients include, but are not limited to: absorption enhancers, anti-adherents, anti-foaming agents, anti-oxidants, binders, binders, buffering agents, carriers, coating agents, colors, delivery enhancers, dextran, dextrose, diluents, disintegrants, emulsifiers, extenders, fillers, flavors, glidants, humectants, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, suspending agents, sustained release matrices, sweeteners, thickening agents, tonicity agents, vehicles, water-repelling agents, and wetting agents. A pharmaceutically acceptable excipient may or may not be an inert substance. 
         [0113]    The pharmaceutical compositions can contain other additional components commonly found in pharmaceutical compositions. The pharmaceutically-active materials may include, but are not limited to: anti-pruritics, astringents, local anesthetics, or anti-inflammatory agents (e.g., antihistamine, diphenhydramine, etc.). It is also envisaged that cells, tissues or isolated organs that express or comprise the herein defined RNAi agents may be used as “pharmaceutical compositions”. As used herein, “pharmacologically effective amount,” “therapeutically effective amount,” or simply “effective amount” refers to that amount of an RNAi agent to produce the intended pharmacological, therapeutic or preventive result. 
         [0114]    In some embodiments, an RNAi agent is conjugated to a delivery polymer. In some embodiments, the delivery polymer is a reversibly masked/modified amphipathic membrane active polyamine. 
         [0115]    The described RNAi agents can be used to provide therapeutic treatments of diseases. Such uses comprise administration of RNAi agent to a human being or animal. For treatment of disease or for formation of a medicament or composition for treatment of a disease, a herein described RNAi agent can be combined with an excipient or with a second therapeutic or treatment including, but not limited to: a second RNAi agent or other RNAi agent, a small molecule drug, an antibody, an antibody fragment, and a vaccine. 
         [0116]    The described RNAi agents and pharmaceutical compositions comprising RNAi agents disclosed herein may be packaged separately or included in a kit, container, pack, or dispenser. The RNAi agents may be packaged in pre-filled syringes or vials. 
         [0117]    The above provided embodiments are now illustrated with the following, non-limiting examples. 
       EXAMPLES 
     Example 1 
     RNAi Agent Synthesis 
     A) Synthesis. 
       [0118]    RNAi agents were synthesized according to phosphoramidite technology on solid phase used in oligonucleotide synthesis. Depending on the scale either a MerMade96E (Bioautomation) or a MerMade12 (Bioautomation) was used. Syntheses were performed on a solid support made of controlled pore glass (CPG, 500 Å or 600 Å, obtained from Prime Synthesis, Aston, Pa., USA). All DNA, 2′-modified RNA, and UNA phosphoramidites were purchased from Thermo Fisher Scientific (Milwaukee, Wis., USA). Specifically, the following 2′-O-Methyl phosphoramidites were used: (5′-O-dimethoxytrityl-N 6 -(benzoyl)-2′-O-methyl-adenosine-3′-O-(2-cyanoethyl-N,N-diisopropy-lamino) phosphoramidite, 5′-O-dimethoxy-trityl-N 4 -(acetyl)-2′-O-methyl-cytidine-3′-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite, (5′-O-dimethoxytrityl-N 2 -(isobutyryl)-2′-O-methyl-guanosine-3′-O-(2-cyano-ethyl-N,N-diisopropylamino)phosphoramidite, and 5′-O-dimethoxy-trityl-2′-O-methyl-undine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite. The 2′-Deoxy-2′-fluoro-phosphor-amidites carried the same protecting groups as the 2′-O-methyl RNA amidites. The following UNA phosphoramidites were used: 5′-(4,4′-Dimethoxytrityl)-N-benzoyl-2′,3′-seco-adenosine, 2′-benzoyl-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5′-(4,4′-Dimethoxytrityl)-N-acetyl-2′,3′-seco-cytosine, 2′-benzoyl-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5′-(4,4′-Dimethoxytrityl)-N-isobutyryl-2′,3′-seco-guanosine, 2′-benzoyl-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, and 5′-(4,4′-Dimethoxytrityl)-2′,3′-seco-uridine, 2′-benzoyl-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite. All amidites were dissolved in anhydrous acetonitrile (50 mM) and molecular sieves (3 Å) were added. In order to introduce the TEG-Cholesterol at the 5′-end of the oligomers, the 1-Dimethoxytrityloxy-3-O—(N-cholesteryl-3-aminopropyl)-triethyleneglycol-glyceryl-2-O-(2-cyanoethyl)-(N,N,-diisopropyl)-phosphoramidite from Glen Research (Sterling, Va., USA) was employed. The 5′-modifications were introduced without any modification of the synthesis cycle. 5-Benzylthio-1H-tetrazole (BTT, 250 mM in acetonitrile) was used as activator solution. Coupling times were 10 min (RNA), 180 sec (Cholesterol), 90 sec (2′OMe and UNA), and 60 sec (2′F and DNA). In order to introduce phosphorothioate linkages, a 100 mM solution of 3-phenyl 1,2,4-dithiazoline-5-one (POS, obtained from PolyOrg, Inc., Leominster, Mass., USA) in anhydrous Acetonitrile was employed. 
       B. Cleavage and Deprotection of Support Bound Oligomer. 
       [0119]    After finalization of the solid phase synthesis, the dried solid support was treated with a 1:1 volume solution of 40 wt. % methylamine in water and 28% ammonium hydroxide solution (Aldrich) for two hours at 30° C. The solution was evaporated and the solid residue was reconstituted in water (see below). 
       C. Purification. 
       [0120]    Crude Cholesterol containing oligomers were purified by reverse phase HPLC using a Waters XBridge BEH300 C4 5u Prep column and a Shimadzu LC-8 system. Buffer A was 100 mM TEAA, pH 7.5 and contained 5% Acetonitrile and buffer B was 100 mM TEAA and contained 95% Acetonitrile. UV traces at 260 nm were recorded. Appropriate fractions were then run on size exclusion HPLC using a GE Healthcare XK 16/40 column packed with Sephadex G-25 medium with a running buffer of 100 mM ammonium bicarbonate, pH 6.7 and 20% Acetonitrile. Other crude oligomers were purified by anionic exchange HPLC using a TKSgel SuperQ-5PW 13u column and Shimadzu LC-8 system. Buffer A was 20 mM Tris, 5 mM EDTA, pH 9.0 and contained 20% Acetonitrile and buffer B was the same as buffer A with the addition of 1.5 M sodium chloride. UV traces at 260 nm were recorded. Appropriate fractions were pooled then run on size exclusion HPLC as described for Cholesterol containing oligomers. 
       D. Annealing. 
       [0121]    Complementary strands were mixed by combining equimolar RNA solutions (sense and antisense) in 0.2×PBS (Phosphate-Buffered Saline, 1×, Corning, Cellgro) to form the RNAi agents. This solution was placed into a thermomixer at 70° C., heated to 95° C., held at 95° C. for 5 min, and cooled to room temperature slowly. Some RNAi agents were lyophilized and stored at −15 to −25° C. Duplex concentration was determined by measuring the solution absorbance on a UV-Vis spectrometer in 0.2×PBS. The solution absorbance at 260 nm was then multiplied by a conversion factor and the dilution factor to determine the duplex concentration. Unless otherwise stated, all conversion factor was 0.037 mg/(mL·cm). For some experiments, a conversion factor was calculated from an experimentally determined extinction coefficient. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
             
               
               
               
               
               
             
               
             
               
               
               
               
               
             
               
             
               
               
               
               
               
             
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Exemplary blunt ended 26 mer RNAi agent sequences 
               
             
          
           
               
                 Duplex ID 
                 SEQ ID 
                 Antisense Sequence 
                 SEQ ID 
                 Sense Sequence 
               
               
                 No. 
                 No. 
                 (5′→3′) 
                 No. 
                 (5′→3′) 
               
               
                   
               
             
          
           
               
                 Exemplary Factor 12 26 mer RNAi agent sequences 
               
             
          
           
               
                 AD01457 
                 1 
                 TGAGAAGCUGAGGCUCAAAGCACUAU 
                 31 
                 UAUAUGCUUUGAGCCUCAGCUUCUCA 
               
               
                 AD01459 
                 2 
                 TGAGAAGCUGAGGCUCAAAGCAUAUA 
                 32 
                 UAUAUGCUUUGAGCCUCAGCUUCUCA 
               
               
                 AD01520 
                 3 
                 TGGUCUUUCACUUUCUUGGGCUCUAU 
                 33 
                 UAUAUGCCCAAGAAAGUGAAAGACCA 
               
               
                 AD01537 
                 4 
                 TCACUUUCUUGGGCUCCAAACAGUAU 
                 34 
                 UAUAUGUUUGGAGCCCAAGAAAGUGA 
               
               
                 AD01538 
                 5 
                 TUCACUUUCUUGGGCUCCAAACAUAU 
                 35 
                 UAUAUUUUGGAGCCCAAGAAAGUGAA 
               
               
                 AD01539 
                 6 
                 TUUCACUUUCUUGGGCUCCAAACUAU 
                 36 
                 UAUAUUUGGAGCCCAAGAAAGUGAAA 
               
               
                 AD01540 
                 7 
                 TUUUCACUUUCUUGGGCUCCAAAUAU 
                 37 
                 UAUAUUGGAGCCCAAGAAAGUGAAAA 
               
               
                 AD01541 
                 8 
                 TAGCUGAGGCUCAAAGCACUUCUUAU 
                 38 
                 UAUAUAAGUGCUUUGAGCCUCAGCUA 
               
               
                 AD01542 
                 9 
                 TGAAGCUGAGGCUCAAAGCACUUUAU 
                 39 
                 UAUAUGUGCUUUGAGCCUCAGCUUCA 
               
               
                 AD01543 
                 10 
                 TUUGUUGCGGUCACCACAGCCCGUAU 
                 40 
                 UAUAUGGCUGUGGUGACCGCAACAAA 
               
               
                 AD01544 
                 11 
                 TGCUUGUUGCGGUCACCACAGCCUAU 
                 41 
                 UAUAUCUGUGGUGACCGCAACAAGCA 
               
               
                 AD01545 
                 12 
                 TGGCUUGUUGCGGUCACCACAGCUAU 
                 42 
                 UAUAUUGUGGUGACCGCAACAAGCCA 
               
               
                 AD01577 
                 13 
                 TGGUCUUUCACUUUCUUGGGCUCUAU 
                 43 
                 UAUUAGCCCAAGAAAGUGAAAGACCA 
               
               
                 AD01579 
                 14 
                 TGGUCUUUCACUUUCUUGGGCUCUAU 
                 44 
                 UAUAAGCCCAAGAAAGUGAAAGACCA 
               
               
                 AD02068 
                 15 
                 UGGUCUUUCACUUUCUUGGGCUCUAU 
                 45 
                 UAUAUGCCCAAGAAAGUGAAAGACCA 
               
               
                 AD02765 
                 16 
                 UGGUCUUUCACUUUCUUGGGCUCUAU 
                 46 
                 UUAGAGCCCAAGAAAGUGAAAGACCA 
               
               
                 AD02766 
                 17 
                 UGGUCUUUCACUUUCUUGGGCUCUAU 
                 47 
                 UUAUUGCCCAAGAAAGUGAAAGACCA 
               
               
                 AD02767 
                 18 
                 UGGUCUUUCACUUUCUUGGGCUCUAU 
                 48 
                 UUGAUGCCCAAGAAAGUGAAAGACCA 
               
               
                 AD02769 
                 19 
                 UGGUCUUUCACUUUCUUGGGCUCUAU 
                 49 
                 UAUGAGCCCAAGAAAGUGAAAGACCA 
               
               
                 AD02772 
                 20 
                 TGGUCUUUCACUUUCUUGGGCUCUAU 
                 50 
                 AUAGAGCCCAAGAAAGUGAAAGACCA 
               
               
                 AD01610 
                 21 
                 UGAAGCUGAGGCUCAAAGCACUUUAU 
                 51 
                 UAUAUGUGCUUUGAGCCUCAGCUUCA 
               
               
                 AD01775 
                 22 
                 TGAAGCUGAGGCUCAAAGCACUUUAU 
                 52 
                 UAUAUGUGCUUUGAGCCUCAGCUUCA 
               
               
                 AD01856 
                 23 
                 UGGUCUUUCACUUUCUUGGGCUCUAU 
                 53 
                 UAUAUGCCCAAGAAAGUGAAAGACCA 
               
               
                 AD01975 
                 24 
                 UGGUCUUUCACUUUCUUGGGCTCUAU 
                 54 
                 UAUAUGCCCAAGAAAGUGAAAGACCA 
               
               
                 AD01994 
                 25 
                 UGGUCUUUCACUUUCUUGGGCUCUAU 
                 55 
                 UAUAUGCCCAAGAAAGUGAAAGAUAU 
               
               
                 AD02665 
                 26 
                 UAUGGUCUUUCACUUUCUUGGGCUCU 
                 56 
                 UAUGCCCAAGAAAGUGAAAGACCUAU 
               
               
                 AD02666 
                 27 
                 UAUGGUCUUUCACUUUCUUGGGCUCU 
                 57 
                 UAUGCCCAAGAAAGUGAAAGACCAAU 
               
               
                 AD02703 
                 28 
                 UAUGGUCUUUCACUUUCUUGGGCUCU 
                 58 
                 UAUGCCCAAGAAAGUGAAAGACCAUU 
               
               
                 AD02704 
                 29 
                 UAUGGUCUUUCACUUUCUUGGGCUCU 
                 59 
                 UAUGCCCAAGAAAGUGAAAGACCAUA 
               
               
                 AD02809 
                 30 
                 UAUGGUCUUUCACUUUCUUGGGCUCU 
                 60 
                 UAUGCCCAAGAAAGUGAAAGACCUAU 
               
               
                   
               
             
          
           
               
                 Exemplary LPA 26 mer RNAi agent sequences 
               
             
          
           
               
                 AD01466 
                 61 
                 TGACACCUGAUUCUGUUUCUGAGUAU 
                 79 
                 UAUAUCAGAAACAGAAUCAGGUGUCA 
               
               
                 AD01462 
                 62 
                 TGAGAAUGAGCCUCGAUAACUCUUAU 
                 80 
                 UAUAUAGUUAUCGAGGCUCAUUCUCA 
               
               
                 AD02664 
                 63 
                 TGAGAAUGAGCCUCGAUAACUCUUAU 
                 81 
                 UAUAUAGUUAUCGAGGCUCAUUCUCA 
               
               
                 AD01530 
                 64 
                 TGCGUCUGAGCAUUGUGUCAGGUUAU 
                 82 
                 UAUAUCUGACACAAUGCUCAGACGCA 
               
               
                 AD01531 
                 65 
                 TUGCGUCUGAGCAUUGUGUCAGGUAU 
                 83 
                 UAUAUUGACACAAUGCUCAGACGCAA 
               
               
                 AD01534 
                 66 
                 TAAGGGCGAAUCUCAGCAUCUGGUAU 
                 84 
                 UAUAUAGAUGCUGAGAUUCGCCCUUA 
               
               
                 AD01532 
                 67 
                 TGAGAAUGAGCCUCGAUAACUCUUAU 
                 85 
                 UAUAUAGUUAUCGAGGCUCAUUCUCA 
               
               
                 AD01981 
                 68 
                 UGAGAAUGAGCCUCGAUAACUCTUAU 
                 86 
                 UAUAUAGUUAUCGAGGCUCAUUCUCA 
               
               
                 AD01979 
                 69 
                 UGAGAAUGAGCCUCGAUAACUCUUAU 
                 87 
                 UAUAUAGUUAUCGAGGCUCAUUCUCA 
               
               
                 AD02435 
                 70 
                 UGAGAAUGAGCCUCGAUAACUCUUAU 
                 88 
                 UAUAUAGUUAUCGAGGCUCAUUCUCA 
               
               
                 AD02619 
                 71 
                 UGAGAAUGAGCCUCGAUAACUCUUAU 
                 89 
                 UAUAUAGUUAUCGAGGCUCAUUCUCA 
               
               
                 AD01533 
                 72 
                 TGACACCUGAUUCUGUUUCUGAGUAU 
                 90 
                 UAUAUCAGAAACAGAAUCAGGUGUCA 
               
               
                 AD01772 
                 73 
                 UGACACCUGAUUCUGUUUCUGAGUAU 
                 91 
                 UAUAUCAGAAACAGAAUCAGGUGUCA 
               
               
                 AD01773 
                 74 
                 UGACACCUGAUUCUGUUUCUGAGUAU 
                 92 
                 UAUAUCAGAAACAGAAUCAGGUGUCA 
               
               
                 AD01774 
                 75 
                 UGACACCUGAUUCUGUUUCUGAGUAU 
                 93 
                 UAUAUCAGAAACAGAAUCAGGUGUCA 
               
               
                 AD02714 
                 76 
                 TCGUAUAACAAUAAGGGGCUGCCUAU 
                 94 
                 UAUAUCAGCCCCUUAUUGUUAUACGA 
               
               
                 AD02552 
                 77 
                 UCGUAUAACAAUAAGGGGCUGCCUAU 
                 95 
                 UAUAUCAGCCCCUUAUUGUUAUACGA 
               
               
                 AD02752 
                 78 
                 UCGUAUAACAAUAAGGGGCUGCCUAU 
                 96 
                 UAUAUCAGCCCCUUAUUGUUAUACGA 
               
               
                   
               
             
          
           
               
                 Exemplary Hif2alpha 26 mer RNAi agent sequences 
               
             
          
           
               
                 AD01295 
                 97 
                 TUUCAUGAAAUCGUUACGUUGGCUAU 
                 102 
                 UAUAUCAACGUAACGAUUUCAUGAAA 
               
               
                 AD01293 
                 98 
                 TUUCAUGAAAUCGUUACGUUGGCUGU 
                 103 
                 UAUAUCAACGUAACGAUUUCAUGAAA 
               
               
                 AD01296 
                 99 
                 TUUCAUGAAAUCGUUACGUUGGCUTT 
                 104 
                 UAUAUCAACGUAACGAUUUCAUGAAA 
               
               
                 AD01411 
                 100 
                 TUUCAUGAAAUCGUUACGUCGGCUAU 
                 105 
                 UAUAUCGACGUAACGAUUUCAUGAAA 
               
               
                 AD01294 
                 101 
                 TUUCAUGAAAUCGUUACGUCGGCUGU 
                 106 
                 UAUAUCGACGUAACGAUUUCAUGAAA 
               
               
                   
               
             
          
           
               
                 Exemplary FVII RNAi agent sequences 
               
             
          
           
               
                 AD01149 
                 107 
                 TGAGUUGGCACGCCUUUGCTT 
                 109 
                 UGUGCAAAGGCGUGCCAACUCAT 
               
               
                 AD01259 
                 108 
                 TGAGUUGGCACGCCUUUGCTT 
                 110 
                 UGUGCAAAGGCGUGCCAACUCAT 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Exemplary modified blunt-ended 26 mer RNAi agent sequences. 
               
             
          
           
               
                   
                   
                 SEQ 
                   
               
               
                 Duplex ID 
                 strand ID 
                 ID 
                   
               
               
                 No. 
                 no. 
                 No. 
                 modified antisense strand 
               
               
                   
               
             
          
           
               
                 Exemplary Factor 12 RNAi agents and sequences 
               
             
          
           
               
                   
                 AM02395-AS 
                 111 
                 dTsGfsaGfaAfgCfuGfaGfgCfuCfaAfaGfcascsuAu 
               
               
                 AD01457 
                 AM02436-SS 
                 112 
                 (Chol-TEG)uAuAusGfscUfuUfgAfgCfcUfcAfgCfuUfcUfc(invdA) 
               
               
                   
                 AM02438-AS 
                 113 
                 dTsGfsaGfaAfgCfuGfaGfgCfuCfaAfaGfcauAfsusa 
               
               
                 AD01459 
                 AM02436-SS 
                 114 
                 (Chol-TEG)uAuAusGfscUfuUfgAfgCfcUfcAfgCfuUfcUfc(invdA) 
               
               
                   
                 AM02507-AS 
                 115 
                 dTsGfgUfcUfuUfcAfcUfuUfCfuuGfgGfcucuAu 
               
               
                 AD01520 
                 AM02500-SS 
                 116 
                 (Chol-TEG)uAuAusGfcCfcAfAfgaAfaGfuGfaAfaGfaCfc(invdA) 
               
               
                   
                 AM02464-AS 
                 117 
                 dTsCfsaCfuUfuCfuUfgGfgCfuCfcAfaAfcAfsgsuAu 
               
               
                 AD01537 
                 AM02513-SS 
                 118 
                 uAuAusGfsuUfuGfgAfgCfcCfaAfgAfaAfgUfgAf(C6-SS-Alk-Me) 
               
               
                   
                 AM02466-AS 
                 119 
                 dTsUfscAfcUfuUfcUfuGfgGfcUfcCfaAfaCfsasuAu 
               
               
                 AD01538 
                 AM02514-SS 
                 120 
                 uAuAusUfsuUfgGfaGfcCfcAfaGfaAfaGfuGfaAf(C6-SS-Alk-Me) 
               
               
                   
                 AM02468-AS 
                 121 
                 dTsUfsuCfaCfuUfuCfuUfgGfgCfuCfcAfaAfscsuAu 
               
               
                 AD01539 
                 AM02515-SS 
                 122 
                 uAuAusUfsuGfgAfgCfcCfaAfgAfaAfgUfgAfaAf(C6-SS-Alk-Me) 
               
               
                   
                 AM02470-AS 
                 123 
                 dTsUfsuUfcAfcUfuUfcUfuGfgGfcUfcCfaAfsasuAu 
               
               
                 AD01540 
                 AM02516-SS 
                 124 
                 uAuAusUfsgGfaGfcCfcAfaGfaAfaGfuGfaAfaAf(C6-SS-Alk-Me) 
               
               
                   
                 AM02472-AS 
                 125 
                 dTsAfsgCfuGfaGfgCfuCfaAfaGfcAfcUfuCfsusuAu 
               
               
                 AD01541 
                 AM02517-SS 
                 126 
                 uAuAusAfsaGfuGfcUfuUfgAfgCfcUfcAfgCfuAf(C6-SS-Alk-Me) 
               
               
                   
                 AM02474-AS 
                 127 
                 dTsGfsaAfgCfuGfaGfgCfuCfaAfaGfcAfcUfsusuAu 
               
               
                 AD01542 
                 AM02518-SS 
                 128 
                 uAuAusGfsuGfcUfuUfgAfgCfcUfcAfgCfuUfcAf(C6-SS-Alk-Me) 
               
               
                   
                 AM02476-AS 
                 129 
                 dTsUfsuGfuUfgCfgGfuCfaCfcAfcAfgCfcCfsgsuAu 
               
               
                 AD01543 
                 AM02519-SS 
                 130 
                 uAuAusGfsgCfuGfuGfgUfgAfcCfgCfaAfcAfaAf(C6-SS-Alk-Me) 
               
               
                   
                 AM02478-AS 
                 131 
                 dTsGfscUfuGfuUfgCfgGfuCfaCfcAfcAfgCfscsuAu 
               
               
                 AD01544 
                 AM02520-SS 
                 132 
                 uAuAusCfsuGfuGfgUfgAfcCfgCfaAfcAfaGfcAf(C6-SS-Alk-Me) 
               
               
                   
                 AM02480-AS 
                 133 
                 dTsGfsgCfuUfgUfuGfcGfgUfcAfcCfaCfaGfscsuAu 
               
               
                 AD01545 
                 AM02521-SS 
                 134 
                 uAuAusUfsgUfgGfuGfaCfcGfcAfaCfaAfgCfcAf(C6-SS-Alk-Me) 
               
               
                   
                 AM02631-AS 
                 135 
                 dTsGfgUfcUfuUfcAfcuUfUfcUfuGfgGfcusCUAU 
               
               
                 AD01577 
                 AM02634-SS 
                 136 
                 (Chol-TEG)UAUUAGfscCfcAfaGfaaAfGfuGfaAfaGfaCfc(invdA) 
               
               
                   
                 AM02632-AS 
                 137 
                 dTsGfgUfcUfuUfcAfcuUfUfcUfuGfgGfcusCuAfu 
               
               
                 AD01579 
                 AM02635-SS 
                 138 
                 (Chol-TEG)UfaUfaAGfscCfcAfaGfaaAfGfuGfaAfaGfaCfc(invdA) 
               
               
                   
                 AM02656-AS 
                 139 
                 usGfsgUfcUfuUfcAfcuuUfcUfuGfgGfcsuscuAu 
               
               
                 AD02068 
                 AM03183-SS 
                 140 
                 (Alk-C6-C6)(Alk-C6-Ser)(Alk-C6-Ser)(Alk-C6-Ser)uAuAuGfscsCfcAfaG 
               
               
                   
                   
                   
                 faAfAfGfuGfaAfaGfaCfc(invdA) 
               
               
                   
                 AM03157-AS 
                 141 
                 usGfsgucuuUfcAfcuuUfcuugggcsuscuAu 
               
               
                 AD02765 
                 AM03571-SS 
                 142 
                 (Alk-C6-C6)uuAgagscsccaagaAfaGfugaaagacc(invdA) 
               
               
                   
                 AM03157-AS 
                 143 
                 usGfsgucuuUfcAfcuuUfcuugggcsuscuAu 
               
               
                 AD02766 
                 AM03573-SS 
                 144 
                 (Alk-C6-C6)uuAuugscsccaagaAfaGfugaaagacc(invdA) 
               
               
                   
                 AM03157-AS 
                 145 
                 usGfsgucuuUfcAfcuuUfcuugggcsuscuAu 
               
               
                 AD02767 
                 AM03575-SS 
                 146 
                 (Alk-C6-C6)uuGAugscsccaagaAfaGfugaaagacc(invdA) 
               
               
                   
                 AM03157-AS 
                 147 
                 usGfsgucuuUfcAfcuuUfcuugggcsuscuAu 
               
               
                 AD02769 
                 AM03579-SS 
                 148 
                 (Alk-C6-C6)uAugagscsccaagaAfaGfugaaagacc(invdA) 
               
               
                   
                 AM02507-AS 
                 149 
                 dTsGfgUfcUfuUfcAfcUfuUfCfuuGfgGfcucuAu 
               
               
                 AD02772 
                 AM03586-SS 
                 150 
                 (Chol-TEG)aUaGasGfcCfcAfAfgaAfaGfuGfaAfaGfaCfc(invdA) 
               
               
                   
                 AM02657-AS 
                 151 
                 usGfsaAfgCfuGfaGfgCfuCfaAfaGfcAfcUfsusuAu 
               
               
                 AD01610 
                 AM02655-SS 
                 152 
                 uAuAusGfsuGfcUfuUfgAfgCfcUfcAfgCfuUfcAf(C11-PEG3-NAG3) 
               
               
                   
                 AM02474-AS 
                 153 
                 dTsGfsaAfgCfuGfaGfgCfuCfaAfaGfcAfcUfsusuAu 
               
               
                 AD01775 
                 AM02867-SS 
                 154 
                 (Spermine)uAuAusGfsuGfcUfuUfgAfgCfcUfcAfgCfuUfcAf(C11-PEG3-NAG3) 
               
               
                   
                 AM02967-AS 
                 155 
                 usGfsgucUfuucAfcuuUfcUfugggcsuscuAu 
               
               
                 AD01856 
                 AM02960-SS 
                 156 
                 uAuAugscsccaagaaAfGfugaaagacca(C11-PEG3-NAG3) 
               
               
                   
                 AM03109-AS 
                 157 
                 usGfsgUfcUfuUfcAfcuuUfcUfuGfgGfcsTMsCMuAu 
               
               
                 AD01975 
                 AM03112-SS 
                 158 
                 uAuAuGfscsCfcAfaGfaAfAfGfuGfaAfaGfaCfCMAM(C11-PEG3-NAG3) 
               
               
                   
                 AM02656-AS 
                 159 
                 usGfsgUfcUfuUfcAfcuuUfcUfuGfgGfcsuscuAu 
               
               
                 AD01994 
                 AM03137-SS 
                 160 
                 uAuAuGfscsCfcAfaGfaAfAfGfuGfaAfaGfauAu(C6-PEG4-NAG3) 
               
               
                   
                 AM03410-AS 
                 161 
                 uAusGfsgucuuUfcAfcuuUfcuugggcsuscu 
               
               
                 AD02665 
                 AM03428-SS 
                 162 
                 uAugscsccaagaAfaGfugaaagaccsusAu(NAG13) 
               
               
                   
                 AM03410-AS 
                 163 
                 uAusGfsgucuuUfcAfcuuUfcuugggcsuscu 
               
               
                 AD02666 
                 AM03429-SS 
                 164 
                 uAugscsccaagaAfaGfugaaagacc(invdA)Au(NAG13) 
               
               
                   
                 AM03410-AS 
                 165 
                 uAusGfsgucuuUfcAfcuuUfcuugggcsuscu 
               
               
                 AD02703 
                 AM03479-SS 
                 166 
                 uAugscsccaagaAfaGfugaaagaccauu(NAG13) 
               
               
                   
                 AM03410-AS 
                 167 
                 uAusGfsgucuuUfcAfcuuUfcuugggcsuscu 
               
               
                 AD02704 
                 AM03480-SS 
                 168 
                 uAugscsccaagaAfaGfugaaagaccaua(NAG13) 
               
               
                   
                 AM03410-AS 
                 169 
                 uAusGfsgucuuUfcAfcuuUfcuugggcsuscu 
               
               
                 AD02809 
                 AM03634-SS 
                 170 
                 uAugscsccaagaAfaGfugaaagaccuAu(NAG18) 
               
               
                   
               
             
          
           
               
                 Exemplary Factor VII RNAi agents and sequences 
               
             
          
           
               
                   
                 AM00026-AS 
                 171 
                 dTsGfaGfuUfgGfcAfcGfcCfuUfuGfcdTsdT 
               
               
                 AD01149 
                 AM01952-SS 
                 172 
                 (Alk-C6)uGuGfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT) 
               
               
                   
                 AM00026-AS 
                 173 
                 dTsGfaGfuUfgGfcAfcGfcCfuUfuGfcdTsdT 
               
               
                 AD01259 
                 AM02094-SS 
                 174 
                 (DBCO-TEG)uGuGfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT) 
               
               
                   
               
             
          
           
               
                 Exemplary LPA RNAi agents and sequences 
               
             
          
           
               
                   
                 AM02412-AS 
                 175 
                 dTsGfsaCfaCfcUfgAfuUfcUfgUfuUfcUfgAfsgsuAu 
               
               
                 AD01466 
                 AM02445-SS 
                 176 
                 uAuAusCfsaGfaAfaCfaGfaAfuCfaGfgUfgUfcAf(C6-SS -Alk-Me) 
               
               
                   
                 AM02404-AS 
                 177 
                 dTsGfsaGfaAfuGfaGfcCfuCfgAfuAfaCfuCfsusuAu 
               
               
                 AD01462 
                 AM02441-SS 
                 178 
                 uAuAusAfsgUfuAfuCfgAfgGfcUfcAfuUfcUfcAf(C6-SS -Alk-Me) 
               
               
                   
                 AM03427-AS 
                 179 
                 dTsGfaGfaAfuGfaGfccuCfgAfuAfaCfuCfuuAu 
               
               
                 AD02664 
                 AM03426-SS 
                 180 
                 (Chol-TEG)uAuAusAfgUfuAfuCfgAfGfGfcUfcAfuUfcUfc(invdA) 
               
               
                   
                 AM02532-AS 
                 181 
                 dTsGfscGfuCfuGfaGfcauUfgUfgUfcAfgGfsusuAu 
               
               
                 AD01530 
                 AM02538-SS 
                 182 
                 uAuAusCfsuGfaCfaCfaAfUfGfcUfcAfgAfcGfcAf(C11-PEG3-NAG3) 
               
               
                   
                 AM02533-AS 
                 183 
                 dTsUfsgCfgUfcUfgAfgcaUfuGfuGfuCfaGfsgsuAu 
               
               
                 AD01531 
                 AM02539-SS 
                 184 
                 uAuAusUfsgAfcAfcAfaUfGfCfuCfaGfaCfgCfaAf(C11-PEG3-NAG3) 
               
               
                   
                 AM02536-AS 
                 185 
                 dTsAfsaGfgGfcGfaAfucuCfaGfcAfuCfuGfsgsuAu 
               
               
                 AD01534 
                 AM02542-SS 
                 186 
                 uAuAusAfsgAfuGfcUfgAfGfAfuUfcGfcCfcUfuAf(C11-PEG3-NAG3) 
               
               
                   
                 AM02534-AS 
                 187 
                 dTsGfsaGfaAfuGfaGfccuCfgAfuAfaCfuCfsusuAu 
               
               
                 AD01532 
                 AM02540-SS 
                 188 
                 uAuAusAfsgUfuAfuCfgAfGfGfcUfcAfuUfcUfcAf(C11-PEG3-NAG3) 
               
               
                   
                 AM03119-AS 
                 189 
                 usGfsaGfaAfuGfaGfccuCfgAfuAfaCfuCMsTMsuAu 
               
               
                 AD01981 
                 AM03122-SS 
                 190 
                 uAuAusAfsgUfuAfuCfgAfGfGfcUfcAfuUfcUfCMAM(C11-PEG3-NAG3) 
               
               
                   
                 AM02857-AS 
                 191 
                 usGfsaGfaAfuGfaGfccuCfgAfuAfaCfuCfsusuAu 
               
               
                 AD01979 
                 AM03122-SS 
                 192 
                 uAuAusAfsgUfuAfuCfgAfGfGfcUfcAfuUfcUfCMAM(C11-PEG3-NAG3) 
               
               
                   
                 AM03255-AS 
                 193 
                 usGfsaGfaAfugaGfccuCfgauaaCfuCfsusuAu 
               
               
                 AD02435 
                 AM03238-SS 
                 194 
                 uAuAusAfsgUfuAfuCfgAfGfGfcUfcauucuca(C11-PEG3-NAG3) 
               
               
                   
                 AM03377-AS 
                 195 
                 usGfsagaauGfaGfccuCfgauaacucsusuAu 
               
               
                 AD02619 
                 AM03243-SS 
                 196 
                 uAuAusasguuaucgAfGfGfcucauucuca(C11-PEG3-NAG3) 
               
               
                   
                 AM02535-AS 
                 197 
                 dTsGfsaCfaCfcUfgAfuucUfgUfuUfcUfgAfsgsuAu 
               
               
                 AD01533 
                 AM02541-SS 
                 198 
                 uAuAusCfsaGfaAfaCfaGfAfAfuCfaGfgUfgUfcAf(C11-PEG3-NAG3) 
               
               
                   
                 AM02863-AS 
                 199 
                 usGfsaCfaCfcUfgAfuucUfgUfuUfcUfgAfsgsuAu 
               
               
                 AD01772 
                 AM02541-SS 
                 200 
                 uAuAusCfsaGfaAfaCfaGfAfAfuCfaGfgUfgUfcAf(C11-PEG3-NAG3) 
               
               
                   
                 AM02864-AS 
                 201 
                 usgsaCfaCfcUfgAfuucUfgUfuUfcUfgAfsgsuAu 
               
               
                 AD01773 
                 AM02541-SS 
                 202 
                 uAuAusCfsaGfaAfaCfaGfAfAfuCfaGfgUfgUfcAf(C11-PEG3-NAG3) 
               
               
                   
                 AM02865-AS 
                 203 
                 usgsaCfaCfcUfgAfuucUfgUfuUfcUfgasgsuAu 
               
               
                 AD01774 
                 AM02541-SS 
                 204 
                 uAuAusCfsaGfaAfaCfaGfAfAfuCfaGfgUfgUfcAf(C11-PEG3-NAG3) 
               
               
                   
                 AM03490-AS 
                 205 
                 vpdTCfsgUfaUfaAfcAfauaAfgGfgGfcUfgCfscsuAu 
               
               
                 AD02714 
                 AM03492-SS 
                 206 
                 uAuAusCfsaGfcCfcCfuUfAfUfuGfuUfaUfaCfga(NAG13) 
               
               
                   
                 AM03107-AS 
                 207 
                 usCfsgUfaUfaAfcAfauaAfgGfgGfcUfgCfscsuAu 
               
               
                 AD02552 
                 AM03289-SS 
                 208 
                 uAuAuscsagccccuUfAfUfuguuauacga(C11-PEG3-NAG3) 
               
               
                   
                 AM03283-AS 
                 209 
                 usCfsgUfaUfaacaauaAfgGfgGfcugcscsuAu 
               
               
                 AD02752 
                 AM03546-SS 
                 210 
                 uAuAuscsagccccuUfAfUfuguuauacga(NAG13) 
               
               
                   
               
             
          
           
               
                 Exemplary Hif2alpha RNAi agents and sequences 
               
             
          
           
               
                   
                 AM02145-AS 
                 211 
                 dTsUfsuCfaUfgAfaAfucgUfuAfcGfuUfggscsuGu 
               
               
                 AD01293 
                 AM02149-SS 
                 212 
                 uAuAusCfsaAfcGfuAfaCfGfAfuUfuCfaUfgAfaAf(C6-SS-Alk-Me) 
               
               
                   
                 AM02146-AS 
                 213 
                 dTsUfsuCfaUfgAfaAfucgUfuAfcGfuCfggscsuGu 
               
               
                 AD01294 
                 AM02163-SS 
                 214 
                 uAuAusCfsgAfcGfuAfaCfGfAfuUfuCfaUfgAfaAf(C6-SS-Alk-Me) 
               
               
                   
                 AM02147-AS 
                 215 
                 dTsUfsuCfaUfgAfaAfucgUfuAfcGfuUfggscsuAu 
               
               
                 AD01295 
                 AM02149-SS 
                 216 
                 uAuAusCfsaAfcGfuAfaCfGfAfuUfuCfaUfgAfaAf(C6-SS-Alk-Me) 
               
               
                   
                 AM02150-AS 
                 217 
                 dTsUfsuCfaUfgAfaAfucgUfuAfcGfuUfggcusdTsdT 
               
               
                 AD01296 
                 AM02149-SS 
                 218 
                 uAuAusCfsaAfcGfuAfaCfGfAfuUfuCfaUfgAfaAf(C6-SS-Alk-Me) 
               
               
                   
                 AM02346-AS 
                 219 
                 dTsUfuCfaUfgAfAUNAAfuCfgUfuAfcGfuCfggcsuAu 
               
               
                 AD01411 
                 AM02365-SS 
                 220 
                 uAuAusCfgAfcGfuAfaCfgAfuUfuCfaUfgAfaAf(C6-SS -Alk-Me) 
               
               
                   
               
             
          
         
       
     
       Example 2 
     In Vivo Analysis of 26Mer Factor XII (F12) RNAi Agent Efficacy In Vivo 
     A) Administration and Sample Collection. 
       [0122]    In order to evaluate the efficacy of 26mer F12 RNAi agents in vivo, wild-type mice were used. For some experiments, cholesterol-conjugated 26mer F12 RNAi agents were administered to mice using MLP delivery polymer on day 1. Each mouse received an intravenous (IV) injection into the tail vein of 200-250 μL solution containing a dose of RNAi agent+MLP delivery polymer (1:1 w/w RNAi agent: MLP delivery polymer in most cases). For other experiments, the indicated 26mer F12 RNAi agent was administered by subcutaneous injection. Control serum (pre-treatment) samples were taken from the mice pre-injection on days −7, −5, −4, or −1. Post injection serum samples were taken from the mice days 4, 8, 15, 22, 29, 36, 43, 50, 53, 57, 64, and/or 71. 
       B) Factor 12 Serum Protein Levels. 
       [0123]    F12 protein (mF12) levels in serum were monitored by assaying serum from the mice using an ELISA for mouse F12 (Molecular Innovations) until mF12 expression levels returned to baseline. For normalization, mF12 level for each animal at a time point was divided by the pre-treatment level of expression in that animal to determine the ratio of expression “normalized to pre-treatment”. Expression at a specific time point was then normalized to the saline control group by dividing the “normalized to day pre-treatment” ratio for an individual animal by the mean “normalized to day pre-treatment” ratio of all mice in the saline control group. This resulted in expression for each time point normalized to that in the control group. Experimental error is given as standard deviation. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Serum F12 protein levels in wild-type mice following 
               
               
                 administration of 26mer F12 RNAi agents. Cholesterol- 
               
               
                 onjugated 26mer F12 RNAi agents were administered 
               
               
                 to mice using MLP delivery polymer. 
               
             
          
           
               
                   
                   
                   
                 Delivery 
                   
               
               
                   
                 Duplex ID 
                 RNAi agent 
                 Polymer 
                 Relative F12 
               
               
                   
                 no. 
                 (mg/kg) 
                 (mg/kg) 
                 levels 
               
               
                   
                   
               
             
          
           
               
                   
                 AD01457 
                 2 
                 2 
                 0.088 
               
               
                   
                 AD01459 
                 2 
                 2 
                 0.197 
               
               
                   
                 AD01520 
                 2 
                 2 
                 0.012 
               
               
                   
                 AD01537 
                 10 
                 10 
                 0.588 
               
               
                   
                 AD01538 
                 10 
                 10 
                 0.705 
               
               
                   
                 AD01539 
                 10 
                 10 
                 0.788 
               
               
                   
                 AD01540 
                 10 
                 10 
                 0.661 
               
               
                   
                 AD01541 
                 10 
                 10 
                 0.577 
               
               
                   
                 AD01542 
                 10 
                 10 
                 0.470 
               
               
                   
                 AD01543 
                 10 
                 10 
                 0.774 
               
               
                   
                 AD01544 
                 10 
                 10 
                 0.647 
               
               
                   
                 AD01545 
                 10 
                 10 
                 0.820 
               
               
                   
                 AD01577 
                 2 
                 2 
                 0.790 
               
               
                   
                 AD01579 
                 2 
                 2 
                 0.538 
               
               
                   
                 AD02068 
                 5 
                 5 
                 0.038 
               
               
                   
                 AD02765 
                 0.4 
                 4 
                 0.010 
               
               
                   
                 AD02766 
                 0.4 
                 4 
                 0.013 
               
               
                   
                 AD02767 
                 0.4 
                 4 
                 0.014 
               
               
                   
                 AD02769 
                 0.4 
                 4 
                 0.014 
               
               
                   
                 AD02772 
                 2 
                 2 
                 0.700 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Serum F12 protein levels in wild-type mice following administration 
               
               
                 of 26mer F12 RNAi agents. 26mer F12 RNAi agents were 
               
               
                 administered to mice by subcutaneous injection. 
               
             
          
           
               
                   
                   
                 RNAi agent 
                   
               
               
                   
                 Duplex ID No. 
                 (mg/kg) 
                 F12 activity 
               
               
                   
                   
               
             
          
           
               
                   
                 AD01610 
                 10 
                 0.637 
               
               
                   
                 AD01775 
                 10 
                 0.660 
               
               
                   
                 AD01856 
                 10 
                 0.034 
               
               
                   
                 AD01975 
                 5 
                 0.444 
               
               
                   
                 AD01994 
                 5 
                 0.903 
               
               
                   
                 AD02665 
                 3 
                 0.241 
               
               
                   
                 AD02666 
                 3 
                 0.151 
               
               
                   
                 AD02703 
                 3 
                 0.308 
               
               
                   
                 AD02704 
                 3 
                 0.288 
               
               
                   
                 AD02809 
                 3 
                 0.610 
               
               
                   
                   
               
             
          
         
       
     
       Example 3 
     In Vivo Analysis of 26Mer Factor VII RNAi Agent Efficacy In Vivo 
       [0124]    A) 120 μg polyacrylate polymer (1095-126) was modified with 2×AC-NAG and 6×AC-PEG12. The modified polymer was then conjugated to 12 μg of AD-01149 26mer FVII RNAi agent and administered to ICR mice by subcutaneous injection. Samples were collected on day 5 and assayed for Factor VII. 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Relative Factor VII expression following 
               
               
                 administration of 26mer FVII RNAi agent 
               
             
          
           
               
                   
                   
                 Relative Factor VII 
               
               
                   
                 Treatment 
                 expression 
               
               
                   
                   
               
               
                   
                 isotonic glucose 
                   1 ± 0.06 
               
               
                   
                 AD-01149 
                 0.65 ± 0.18 
               
               
                   
                   
               
             
          
         
       
     
         [0125]    B) 20 μg MLP was modified with 2×CDM-NAG followed by 3×CDM-NAG. The modified MLP was combined with 30 μg of AD-01259 26mer FVII RNAi agent and administered to ICR mice by intravascular injection. Samples were collected on day 5 and assayed for Factor VII. 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Relative Factor VII expression following 
               
               
                 administration of 26mer FVII RNAi agent. 
               
             
          
           
               
                   
                 Treatment 
                 Relative expression 
               
               
                   
                   
               
               
                   
                 isotonic glucose 
                   1 ± 0.34 
               
               
                   
                 AD-01259 
                 0.12 ± 0.05 
               
               
                   
                   
               
             
          
         
       
     
       Example 4 
     In Vivo Analysis of 26Mer Hif2α RNAi Agent Efficacy In Vivo 
       [0126]    RGD Targeted HiF2α-RNAi agent delivery conjugates were formed using RGD mimic-PEG-HyNic masking. 400 μg 126 or 100 A polymer was modified with 8×PEG 12 -ACit-PABC-PNP/0.5× aldehyde-PEG 24 -FCit-PABC-PNP (with RGD mimic #1-PEG-HyNic using protocol #1) (WO 2012/092373 and WO 2015/021092) and 80 μg of the indicated Hif2α RNAi agent. Kidney RCC tumor-bearing mice were generated as described and treated with a single tail vein injection of isotonic glucose or the indicated Hif2α-ITG-DPC (Hif2α-ITG-DPC=Hif2α RNAi agent-delivery polymer conjugate. The delivery polymer was modified with RGD ligand and PEG masking agents). Mice were euthanized 72 h after injection and total RNA was prepared from kidney tumor using Trizol reagent following manufacture&#39;s recommendation. Relative HiF2α mRNA levels were determined by RT-qPCR as described below and compared to mice treated with delivery buffer (isotonic glucose) only. 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 Hif2α knockdown in mice following Hif2α RNAi agent 
               
               
                 delivery. RNAi agents were conjugated to the indicated 
               
               
                 reversibly masked delivery polymer. 
               
             
          
           
               
                   
                 Relative Expression 
               
             
          
           
               
                 RNAi agent 
                 Polymer 
                   
                 low error/ 
               
             
          
           
               
                 duplex number 
                 μg 
                 number 
                 μg 
                 day 4 
                 high error 
               
               
                   
               
             
          
           
               
                 isotonic glucose 
                 0 
                   
                 0 
                 1.00 
                 0.06/0.06 
               
               
                 AD01293 
                 80 
                 126 
                 400 
                 0.20 
                 0.01/0.01 
               
               
                 AD01294 
                 80 
                 126 
                 400 
                 0.17 
                 0.01/0.02 
               
               
                 AD01295 
                 80 
                 126 
                 400 
                 0.22 
                 0.02/0.02 
               
               
                 AD01296 
                 80 
                 126 
                 400 
                 0.21 
                 0.04/0.06 
               
               
                 AD01411 
                 150 
                   100A 
                 300 
                 0.36 
                 0.01/0.01 
               
               
                   
               
             
          
         
       
     
       Quantitative Real-Time PCR Assay. 
       [0127]    In preparation for quantitative PCR, total RNA was isolated from tissue samples homogenized in TriReagent (Molecular Research Center, Cincinnati, Ohio) following the manufacturer&#39;s protocol. Approximately 500 ng RNA was reverse-transcribed using the High Capacity cDNA Reverse Transcription Kit (Life Technologies). For human (tumor) Hif2α (EPAS1) expression, pre-manufactured TaqMan gene expression assays for human Hif2α (Catalog #4331182) and CycA (PPIA) Catalog #: 4326316E) were used in biplex reactions in triplicate using TaqMan Gene Expression Master Mix (Life Technologies) or VeriQuest Probe Master Mix (Affymetrix). For human (tumor) VegFa (VEGFA) expression, pre-manufactured TaqMan gene expression assays for human VegFa (Catalog #4331182, Assay ID: Hs00900055) and CycA (Part#: 4326316E) were used in biplex reactions in triplicate using TaqMan Gene Expression Master Mix (Life Technologies) or VeriQuest Probe Master Mix (Affymetrix). Quantitative PCR was performed by using a 7500 Fast or StepOnePlus Real-Time PCR system (Life Technologies). The ΔΔC T  method was used to calculate relative gene expression. 
         [0128]    Polymer APN 1095-126 (126): propyl acrylate/ethoxyethylamine acrylate membrane active amphipathic copolymer. 
         [0000]    
       
         
               
               
               
               
               
               
               
             
           
               
                   
               
               
                 MW 
                 Theoretical MW 
                   
                 % Amine 
                 % Alkyl 
                 % End Group 
                 Azides Per 
               
               
                 (protected) 
                 (deprotected) 
                 PDI 
                 Incorporation 
                 Incorporation 
                 Removal 
                 Polymer 
               
               
                   
               
             
             
               
                 66,670 
                 47,606 
                 1.11 
                 56 
                 44 
                 0 
                 4.1 
               
               
                   
               
             
          
         
       
     
         [0129]    Polymer APN 1170-100 A (100 A) propyl acrylate/ethoxyethylamine acrylate membrane active amphipathic copolymer. 
         [0000]    
       
         
               
               
               
               
               
               
               
               
             
           
               
                   
               
               
                   
                 MW 
                 Theoretical MW 
                   
                 % Amine 
                 % Alkyl 
                 % End Group 
                 Azides/ 
               
               
                 Polymer 
                 (protected) 
                 (deprotected) 
                 PDI 
                 Incorp. 
                 Incorp. 
                 Removal 
                 Polymer 
               
               
                   
               
             
             
               
                 APN 1170-100A 
                 64,430 
                 45,765 
                 1.22 
                 56 
                 44 
                 0 
                 1.25 
               
               
                   
               
             
          
         
       
     
       Protocol 1. 
       [0130]    The indicated polymer was reacted with SMPT at a weight ratio of 1:0.015 (polymer: SMPT) in 5 mM HEPES, pH 8.0 buffer for 1 h at RT. The SMPT-modified polymer was then reacted with aldehyde-PEG-dipeptide masking agent (aldehyde-PEG 12 -FCit or aldehyde-PEG 24 -ACit) at desired ratios for 1 h at RT. The modified polymer was then reacted with PEG 12 -dipeptide masking agent (PEG 12 -FCit, PEG 12 -ACit or PEG 24 -ACit) at a weight ratio of 1:2 (polymer:PEG) in 100 mM HEPES, pH 9.0 buffer for 1 h at RT. The modified polymer was then reacted overnight with SATA-RNAi agent at a weight ratio of 1:0.2 (polymer:SATA-RNAi agent) in 100 mM HEPES, pH 9.0 buffer at RT to attach the RNAi agent. Next, the modified polymer was reacted with protease cleavable PEG (PEG12-FCit or PEG12-ACit or PEG24-ACit) at a weight ratio of 1:6 (polymer:PEG) in 100 mM HEPES, pH 9.0 buffer for 1 h at RT. The resultant conjugate was purified using a sephadex G-50 spin column. 
         [0131]    RGD-HyNic (Example 6B) was attached to the modified polymer to form the full delivery conjugate by reaction with the modified polymer at a weight ratio of 1:0.7 (polymer:RGD-HyNic mimic) in 50 mM MES, pH 5.0 buffer for a minimum of 4 h at RT. The conjugate was purified using a sephadex G-50 spin column. RGD ligand attachment efficiency was determined as described above. 
       Example 5 
     In Vivo Analysis of 26Mer LPA RNAi Agent Efficacy In Vivo 
       [0132]    For some experiments, a plasmid containing LPA target sequences inserted into the 3′ UTR of secreted placental alkaline phosphatase (SEAP) was injected into wild-type mice by hydrodynamic tail vein injection. At four to five weeks post HTV injection, RNAi agents were administered to these transiently transgenic SEAP-LPA HTV mice. 
         [0133]    For other experiments, apo(a) and Lp(a) transgenic mice (Frazer K A et al 1995, Nature Genetics 9:424-431) were used. The apo(a) transgenic mice expresses human apo(a) from a YAC containing the full LPA gene (encoding apo(a) protein) with additional sequences both 5′ and 3′. Lp(a) mice were bred by crossing apo(a) YAC-containing mice to human apoB-100 expressing mice (Callow M J et al 1994, PNAS 91:2130-2134, Lawn R M et al. 1992 Nature 360(6405): 670-672). 
       A) Intravascular Administration of 26Mer LPA RNAi Agent: 
       [0134]    Polymer ARF1164-106A-5 was masked with AC-NAG and AC-PEG12 and conjugated to the 26mer LPA RNAi agent. Each mouse received an intravenous (IV) injection into the tail vein of 200-250 μL solution containing a dose of 26mer LAP RNAi agent attached to protease-masked polymer. Control serum (pre-treatment) samples were taken from the mice pre-injection on day −1. Post injection serum samples were taken from the mice on various days. Polymer ARF1164-106A-5 is a propyl acrylate and ethyl ethoxy amino acrylate (54%) copolymer having a PDI of 1.043. 
       B) Subcutaneous Administration of 26Mer LPA RNAi Agent: 
       [0135]    The indicated 26mer LPA RNAi agent was administered by subcutaneous injection of 100 μl to 300 μl RNAi agent in buffer into the loose skin on the back between the shoulders. 
       C) Target Gene Knockdown Evaluation. 
       [0136]    SEAP protein (SEAP) levels in serum were monitored by assaying serum from the mice using a chemiluminescent substrate (Tropix® Phospha-Light™, Applied Biosystems) until SEAP levels returned to baseline. For normalization, the SEAP level for each animal at a time point was divided by the pre-treatment level of expression in that animal to determine the ratio of expression “normalized to pre-treatment”. Expression at a specific time point was then normalized to the saline control group by dividing the “normalized to day pre-treatment” ratio for an individual animal by the mean “normalized to day pre-treatment” ratio of all mice in the saline control group. This resulted in expression for each time point normalized to that in the control group. Experimental error is given as standard deviation. For LP(a) transgenic mice, Apo(a) levels were measured by ELISA and LP(a) levels were measured by clinical chemistry analyzer (Cobas). A decrease in target gene expression was observed following administration of all the 26mer LPA RNAi agents tested. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 8 
               
             
             
               
                   
               
               
                 Target gene knockdown in mice following administration of 26mer 
               
               
                 LPA RNAi agents. Cholesterol-conjugated 26mer LPA RNAi agents 
               
               
                 were administered to mice using MLP delivery polymer. 
               
             
          
           
               
                   
                 RNAi agent 
                 Delivery Polymer 
                 Target gene 
               
               
                 Duplex ID No. 
                 (mg/kg) 
                 (mg/kg) 
                 knockdown 
               
               
                   
               
             
          
           
               
                 AD01466 
                 0.5 
                 2 
                 0.329 
               
               
                 AD01462 
                 0.5 
                 2 
                 0.319 
               
               
                 AD02664 
                 2 
                 2 
                 0.001 
               
               
                 AD01530 
                 1 
                 2 
                 0.264 
               
               
                 AD01531 
                 1 
                 2 
                 0.220 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 9 
               
             
             
               
                   
               
               
                 Target gene knockdown in mice following administration 
               
               
                 of 26mer LPA RNAi agents. 26mer LPA RNAi agents were 
               
               
                 administered to mice by subcutaneous injection. 
               
             
          
           
               
                   
                   
                 RNAi agent 
                 Target gene 
               
               
                   
                 Duplex ID No. 
                 (mg/kg) 
                 knockdown 
               
               
                   
                   
               
             
          
           
               
                   
                 AD01534 
                 10 
                 0.398 
               
               
                   
                 AD01532 
                 10 
                 0.434 
               
               
                   
                 AD01981 
                 3 
                 0.049 
               
               
                   
                 AD01979 
                 3 
                 0.057 
               
               
                   
                 AD02435 
                 10 
                 0.038 
               
               
                   
                 AD02619 
                 3 
                 0.024 
               
               
                   
                 AD01533 
                 10 
                 0.299 
               
               
                   
                 AD01772 
                 10 
                 0.247 
               
               
                   
                 AD01773 
                 10 
                 0.2759 
               
               
                   
                 AD01774 
                 10 
                 0.370 
               
               
                   
                 AD02714 
                 3 
                 0.064 
               
               
                   
                 AD02552 
                 10 
                 0.033 
               
               
                   
                 AD02752 
                 3 
                 0.081