NZ751385B2 - Complement component c5 irna compositions and methods of use thereof - Google Patents

Abstract

The invention relates to iRNA, e.g., double-stranded ribonucleic acid (dsRNA), compositions targeting the complement component C5 gene, and methods of using such iRNA, e.g., dsRNA, compositions to inhibit expression of C5 and to treat subjects having a complement component C5-associated disease, e.g. paroxysmal, nocturnal hemoglobinuria. . paroxysmal, nocturnal hemoglobinuria.

Description

[Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS COMPLEMENT COMPONENT C5 iRNA COMPOSITIONS AND METHODS OF USE THEREOF Related Applications This application claims the benefit of US Provisional Patent Application No.:61/782,531, filed on March 14, 2013, US Provisional Patent Application No.:61/837,399, filed on June 20, 2013, and US Provisional Patent Application No.:61/904,579, filed on November 15, 2013, US Provisional Patent Application No.:61/912,777, filed on December 6, 2013, and US Provisional Patent Application No.:61/942367, filed February 20, 2014. The entire contents of each of the foregoing provisional patent applications are hereby incorporated herein by reference.

Sequence Listing The t application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on March 10, 2014, is named 121301—00520_SL.txt and is 734,486 bytes in size.

Background of the Invention ment was first discovered in the 1890s when it was found to aid or “complement” the killing of bacteria by heat—stable antibodies t in normal serum (Walport, M.J. (2001) N Engl J Med. 344: 1058). The complement system consists of more than 30 ns that are either present as soluble proteins in the blood or are present as membrane—associated proteins. tion of complement leads to a sequential cascade of enzymatic reactions, known as complement activation ys, resulting in the formation of the potent anaphylatoxins C3a and C5a that elicit a plethora of logical responses that range from chemoattraction to apoptosis. Initially, ment was thought to play a major role in innate immunity where a robust and rapid response is mounted against invading ens. However, recently it is becoming singly evident that complement also plays an important role in adaptive immunity involving T and B cells that help in elimination of pathogens (Dunkelberger JR and Song WC. (2010) Cell Res. 20:34; Molina H, et al. (1996) Proc Natl Acad Sci U S A. 93:3357), in maintaining immunologic memory preventing pathogenic re—invasion, and is involved in us human pathological states (Qu, H, et al. (2009) Mol Immunol. 47: 185; Wagner, E. and Frank MM. (2010) Nat Rev Drug Discov. 9:43).

Complement activation is known to occur through three different pathways: alternate, classifiand lectin (Figure 1), involving proteins that mostly exist as inactive zymogens that [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS are then sequentially d and activated. All pathways of complement tion lead to cleavage of the C5 molecule generating the anaphylatoxin C5a and, C5b that subsequently forms the terminal complement complex (C5b—9). C5a exerts a predominant pro— inflammatory activity through interactions with the classical G—protein coupled receptor C5aR (CD88) as well as with the non—G protein coupled receptor C5L2 (GPR77), sed on various immune and non—immune cells. C5b—9 causes cytolysis through the formation of the membrane attack complex (MAC), and sub—lytic MAC and soluble C5b—9 also s a multitude of non—cytolytic immune functions. These two complement effectors, C5a and C5b—9, generated from C5 ge, are key components of the complement system responsible for propagating and/or initiating pathology in different diseases, including smal nocturnal hemoglobinuria, toid arthritis, ia—reperfusion injuries and neurodegenerative diseases.

To date, only one therapeutic that targets the C5—C5a axis is available for the treatment of complement component C5—associated diseases, the 5 antibody, eculizumab (Soliris®). gh eculizumab has been shown to be ive for the treatment of paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS) and is currently being evaluated in clinical trials for additional complement component C5—associated diseases, eculizumab therapy requires weekly high dose infusions followed by biweekly maintenance infusions at a yearly cost of about $400,000. Accordingly, there is a need in the art for alternative therapies and combination therapies for subjects having a complement component C5—associated disease.

Summary of the Invention The present invention provides iRNA compositions which effect the RNA—induced silencing complex (RISC)—mediated cleavage of RNA transcripts of a C5 gene. The C5 gene may be within a cell, e.g., a cell within a subject, such as a human. The present invention also provides methods and combination ies for treating a subject having a er that would benefit from inhibiting or ng the expression of a C5 gene, e.g., a complement component C5—associated e, such as paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS) using iRNA compositions which effect the RNA—induced silencing complex (RISC)—mediated cleavage of RNA transcripts of a C5 gene for inhibiting the expression of a C5 gene.

Accordingly, in one , the present invention provides a double—stranded ribonucleic acid (dsRNA) agent for inhibiting expression of complement component C5, wherein the dsRNA comprises a sense strand and an antisense , wherein the sense strand comprises at least 15 uous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:1 and the antisense strand comprises at least 15 [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS contiguous tides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:5.

In another aspect, the present invention provides a double—stranded ribonucleic acid (dsRNA) agent for inhibiting expression of ment component C5, wherein the dsRNA comprises a sense strand and an antisense strand, the antisense strand comprising a region of mentarity which ses at least 15 contiguous tides differing by no more than 3 nucleotides from any one of the antisense sequences listed in any one of Tables 3, 4, 5, 6, 18, 19, 20, 21, and 23.

In one embodiment, the sense and antisense strands comprise sequences selected from the group consisting of A-118320, A-118321, A-118316, A-118317, A-118332, A—118333, A-118396, A-118397, A-118386, A-118387, A-118312, A-118313, A-118324, A-118325, A- 119324, A-119325, A-119332, A-119333, A-119328, A-119329, A-119322, A-119323, A- 119324, A-119325, A-119334, A-119335, A-119330, A-119331, A-119326, A-119327, A- 125167, A-125173, A-125647, A-125157, A-125173, and A-125127. In another embodiment, the sense and nse strands comprise sequences selected from the group consisting of any of the sequences in any one of Tables 3, 4, 5, 6, 18, 19, 20, 21, and 23. In one embodiment, the dsRNA agent comprises at least one ed nucleotide.

In one aspect, the present invention provides a double—stranded ribonucleic acid (dsRNA) agent for inhibiting expression of complement component C5, wherein the dsRNA agent comprises a sense strand and an nse strand, wherein the sense strand comprises the nucleotide sequence AAGCAAGAUAUUUUUAUAAUA (SEQ ID NO:62) and wherein the nse strand comprises the nucleotide sequence UAUUAUAAAAAUAUCUUGCUUUU (SEQ ID NO:113). In one embodiment, the dsRNA agent comprises at least one modified tide, as described below.

In one , the present invention provides a double stranded RNAi agent for inhibiting sion of complement component C5 wherein the double stranded RNAi agent comprises a sense strand and an antisense strand forming a double—stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:1 and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the tide sequence of SEQ ID NO:5, wherein substantially all of the nucleotides of the sense strand and substantially all of the tides of the antisense strand are modified nucleotides, and wherein the sense strand is conjugated to a ligand attached at the 3’— terminus.

In one embodiment, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a modification.

In one embodiment, substantially all of the nucleotides of the sense strand are moditfinucleotides selected from the group consisting of a 2’—O—methyl modification, a 2’— [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS fluoro modification and a 3’—terminal deoxy—thymine (dT) nucleotide. In another embodiment, substantially all of the nucleotides of the antisense strand are modified nucleotides selected from the group consisting of a 2’—O—methyl modification, a 2’—fluoro cation and a 3’—terminal deoxy—thymine (dT) nucleotide. In another embodiment, the modified nucleotides are a short sequence of thymine (dT) tides. In another embodiment, the sense strand comprises two phosphorothioate intemucleotide linkages at the ’—terminus. In one embodiment, the antisense strand comprises two phosphorothioate intemucleotide linkages at the minus and two phosphorothioate internucleotide linkages at the 3’—terminus. In yet another embodiment, the sense strand is conjugated to one or more GalNAc derivatives attached h a branched bivalent or trivalent linker at the 3’— terminus.

In one ment, at least one of the modified nucleotides is selected from the group consisting of a 3’—terminal deoxy—thymine (dT) nucleotide, a 2'—O—methyl modified nucleotide, a 2'—fluoro modified nucleotide, a 2'—deoxy—modified tide, a locked nucleotide, an abasic nucleotide, a 2’—amino—modified tide, a 2’—alkyl—modified nucleotide, a morpholino tide, a phosphoramidate, a non—natural base comprising nucleotide, a tide comprising a sphorothioate group, and a terminal nucleotide linked to a teryl derivative or a dodecanoic acid bisdecylamide group.

In another embodiment, the modified tides comprise a short sequence of 3’— terrninal deoxy—thymine (dT) nucleotides.

In one embodiment, the region of complementarity is at least 17 nucleotides in length.

In another embodiment, the region of complementarity is between 19 and 21 nucleotides in length.

In one embodiment, the region of complementarity is 19 nucleotides in length.

In one ment, each strand is no more than 30 nucleotides in length.

In one embodiment, at least one strand comprises a 3’ overhang of at least 1 nucleotide. In another embodiment,at least one strand comprises a 3’ overhang of at least 2 nucleotides.

In one embodiment, the dsRNA agent further comprises a ligand.

In one embodiment, the ligand is conjugated to the 3’ end of the sense strand of the dsRNA agent.

In one embodiment, the ligand is an N—acetylgalactosamine (GalNAc) derivative.

In one embodiment, the ligand is [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS AcHN o o 0 o I HO n/N/\/\N o AcHN H H In one embodiment, the dsRNA agent is conjugated to the ligand as shown in the ing schematic and, wherein X is O or S.

In one ment, the X is O.

In one embodiment, the region of complementarity consists of one of the antisense sequences of any one of Tables 3, 4, 5, 6, 18, 19, 20, 21, and 23.

In one embodiment, the dsRNA agent is selected from the group consisting of AD— 58123, 11, AD-58121, AD-58116, AD—58133, AD—58099, AD—58088, AD—58642, AD-58644, AD-58641, AD-58647, AD-58645, AD-58643, AD-58646, AD-62510, AD- 62643, AD-62645, AD-62646, AD-62650, and AD-62651.

In another aspect, the present invention provides a double—stranded ribonucleic acid (dsRNA) agent for inhibiting sion of complement component C5, wherein the dsRNA agent comprises a sense strand and an antisense strand, wherein the sense strand comprises the nucleotide sequence AAGCAAGAUAUUUUUAUAAUA (SEQ ID NO:62) and wherein the antisense strand comprises the nucleotide sequence AAAAAUAUCUUGCUUUUdeT (SEQ ID NO:2899).

In another , the present invention provides a double—stranded ribonucleic acid ) agent for inhibiting expression of complement component C5, wherein the dsRNA agent comprises a sense strand and an antisense strand, wherein the sense strand comprises the nLDJtide sequence asastcAfaGfanAfouUfuuAqufauaL96 (SEQ ID NO:2876) and [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS wherein the antisense strand comprises the nucleotide sequence usAfsUqufuaAfaAfauanchqucuususudeT (SEQ ID NO:2889).

In one aspect, the present invention provides a double stranded RNAi agent capable of inhibiting the expression of complement component C5 in a cell, wherein the double stranded RNAi agent comprises a sense strand complementary to an antisense , wherein the nse strand comprises a region complementary to part of an mRNA encoding C5, wherein each strand is about 14 to about 30 tides in length, wherein the double stranded RNAi agent is represented by formula (III): sense: 5' np —Na —(X X X) i—Nb —Y Y Y —Nb —(Z Z Z)J~ —Na — nq 3' antisense: 3' '—(X'X'X')k—Nb’—Y’Y’Y'—Nb’—(Z'Z’Z')1—Na'— nq' 5' (111) wherein: i, j, k, and l are each independently 0 or 1; p, p’, q, and q’ are each independently 0—6; each Na and Na' independently represents an oligonucleotide sequence comprising 0— 25 nucleotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucleotides; each Nb and Nb’ independently represents an oligonucleotide sequence comprising 0— nucleotides which are either modified or unmodified or combinations thereof; each np, np', nq, and nq', each of which may or may not be present, independently represents an overhang tide; XXX, YYY, ZZZ, X’X'X', Y'Y'Y’, and Z’Z'Z' each independently ent one motif of three identical modifications on three utive nucleotides; modifications on N, differ from the modification on Y and modifications on Nb’ differ from the modification on Y'; and wherein the sense strand is conjugated to at least one ligand.

In one embodiment, iis 0;j is 0; iis l;j is 1; both i andj are 0; or both i andj are 1.

In one embodiment, k is 0; l is 0; k is l; l is 1; both k and l are 0; or both k and l are 1.

In one embodiment, XXX is complementary to X’X’X’, YYY is complementary to Y’Y’Y’, and ZZZ is complementary to Z’Z’Z’.

In one embodiment, the YYY motif occurs at or near the cleavage site of the sense strand.

In one embodiment, the Y’Y’Y’ motif occurs at the ll, 12 and 13 positions of the antisense strand from the .

In one embodiment, the Y’ is 2’—O—methyl.

In one embodiment, a (III) is represented by formula (Illa): sense: 5' np —Na —Y Y Y —Na - nq 3' antisense: 3' np/—Na/— Y’Y’Y’— Na/— nq/ 5' .

Danother embodiment, formula (III) is represented by formula (IIIb): [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS sense: 5' np —Na —Y Y Y —Nb —Z Z Z —Na — nq 3' nse: 3' np/—Na/— Y’Y’Y’—Nb/—Z’Z’Z’— Na/— nq/ 5' (IIIb) wherein each Nb and Nb’ independently represents an oligonucleotide sequence comprising 1- modified nucleotides.

In yet another ment, formula (III) is represented by formula (IIIc): sense: 5' np —Na —X X X —Nb —Y Y Y —Na — nq 3' antisense: 3' np/—Na/— X’X’X’—Nb/— Y’Y’Y’— Na/— nq/ 5' (IIIc) Wherein each Nb and Nb’ independently represents an oligonucleotide sequence comprising 1- modified nucleotides.

In another embodiment, formula (III) is represented by formula (IIId): sense: 5' np —Na —X X X— N, —Y Y Y —Nb —Z Z Z —Na — nq 3' antisense: 3' np/—Na/— X’X’X’— Nb/—Y’Y’Y’—Nb/—Z’Z’Z’— Na/— nq/ 5' (IIId) wherein each Nb and Nb’ independently represents an oligonucleotide sequence comprising 1—5 modified nucleotides and each Na and Na’ independently represents an oligonucleotide sequence comprising 2—10 modified nucleotides.

In one embodiment, the double—stranded region is 15—30 nucleotide pairs in length.

In one embodiment, the double—stranded region is 17—23 nucleotide pairs in length. In another embodiment, the double—stranded region is 17—25 nucleotide pairs in length. In another embodiment, the double—stranded region is 23—27 tide pairs in length. In yet another embodiment, the double—stranded region is 19—21 nucleotide pairs in length. In another embodiment, the double—stranded region is 21—23 nucleotide pairs in length.

In one ment, each strand has 15—30 nucleotides.

In one ment, the modifications on the nucleotides are ed from the group consisting of LNA, HNA, CeNA, hoxyethyl, 2’—O—alkyl, 2’—O—allyl, 2’—C— allyl, 2’— fluoro, 2’—deoxy, 2’—hydroxyl, and combinations thereof.

In one embodiment, the modifications on the tides are 2’—O—methyl or ro cations.

In one embodiment, the ligand is one or more GalNAc derivatives attached through a bivalent or ent branched linker.

In one embodiment, the ligand is [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS fifi“""‘ffi‘*\”SVAN¢"“‘TEJN \,«""\\ I N G A12???N 0 {\x an on :7 ' ~ :1 jawe‘fkbb/{3\ ,M t :t N I i ‘ a \i so , ”\F” Xx” \v"N wa {Z} was ~ t V: , {:5 ‘ i C Q iii} /OE‘i xi )Lek.» 1 7' H0i K‘xX/‘G‘th‘ \Vf\]'§"‘" {\3 w N A.‘ Q’“ a!" x . \t 33K. i‘lNV : G R H In one en'ihodiment, the ligand is attached to the 3' end ot‘the sense strand.

In one embodiment, the RNAi agent is eoningated to the ligand as shown in the following schematic ' if”. ‘,’"' -'Q an e» y \‘x/CI.

I“; H Q:E; __ 8““ “them-0w» at «rda“ ‘ l on in: :t . g {3 [mf- lit} Qi’l \1 \“pl\_; t) (1 ‘3‘ £3 H it h HQ ? \(Avmex‘fl’e‘xvri'XiWrfix‘fG‘v’ ’l E\ \fN/fflkaf’A‘xafi‘gr} {{KnHI‘é {f3 \. {3 {34) .- if: <Gi‘iO ‘ f r} satiety-karaoke“?eve £9 ASHN C: H 5.1 In one embodiment, the agent further comprises at least one phosphorothioate or methylphosphonate internneIeotide linkage.

In one embodiment, the phosphorothioate or methylphosphonate intemneleotide linkage is at. the 3 ”—termintts of one strand It} In one embodiment the strand is the antisense . In another embodiment? the strand is the sense strand.

In one embodiment, the phospliorothioate or methylphosphonate nt’ieleotide linkage is at the 5’—terminns of one strand.

In one embodiment, the strand is the antisense strand. In another embodiment, the strand is the sense strand.

In one embodiment, the orothioate or methylphosphonate intemneleotide linkage is at. the both the 5"~ and 3: ”~ten'ninns of one strand.

In one embodiment the strand is the antisense strand.

[Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS In one embodiment, the base pair at the I position of the S'nend of the antisense strand of the duplex is an All base pair.

In one embodiment, the Y nucleotides contain a '2’—tluoro modification.

In one embodiment, the Y’ nucleotides contain a 2’—O—inethyl modification.

(I: In one embodiment, p590 In one embodiment, p’===2.

In one embodiment, q’tdl, piiil), deli, and p” ng nucleotides are complementary to the target mRNA.

In one embodiment, q’=0., p=0, (3:0, and p’ overhang nucleotides are nonncomplementary ll) to the target mIiNA.

In one embodiment, the sense strand has a total off/ll nucleotides and the antisense strand has a total ol23 nucleotides.

In one embodiment, at least one np’ is linked to a neighboring nucleotide Via a phosphorothioate linkage.

In one embodiment, all np’ are linked to neighboring nucleotides Via pliosphorothioate linkages.

In one embodiment, the RNAi agent is selected from the group ofRNAi agents listed in Table 4-, 'I'able l8, 'I‘able l9, or Table 23. In another embodiment, the RNAi agent is ed from the group consisting ofAD—SS l23 AID-581 I I AD—Stl l2 l AD—Stl l l6, AD—58133, AD— , , , 2t) 58099, ADSSOSS, AISLS 8642, Alliw58644, A9686“ A358647, AD~58645, A96 8643, AD 58646, AD—éZS l 0, AID—62643, Aft—62645, AID—62646, AID—62650“, and Alli—6265 l.

In one , the present invention provides a double stranded RNAi agent e of inhibiting the expression of complement component CS in a cell, wherein said double ed RNAi agent comprises a sense strand complementary to an antisense strand, wherein said antisense strand ses a region complementary to part of an mRNA encoding complement component C5, n each strand is about l4 to about 30 nucleotides in length, wherein said double stranded RNAi agent is represented by formula (III): sense: 5' np —Na “(X X X) 3N5 ~Y Y Y ~Nb —(Z Z 2)} “Na ~ nq 3' antisense: 3‘ np’—Na’—(X'X’X’lk—Nb’~‘i"¥"‘i"—Nb'~(Z'Z’Z’)1—Na'~ nq' 5‘ (III) wherein: i, j, k, and l are each ndently 0 or 1; p, p’, q, and q' are each independently (lab; each Na and Na” independently represents an oligonucleotide sequence comprising 0—25 nucleotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucleotides; [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS each Nb and Nb' independently represents an oligonucleotide sequence sing Cult} nucleotides which are either modified or unmodified or combinations thereof; each np, np’, nq, and nq’, each h may or may not be present independently represents an overl’iang nucleotide; (I: XXX YYY, ZZZ, X’X’X', , and Z'Z’Z' each independently represent one motif ofthree identical ations on three consecutive nucleotides, and wherein the modifications are thyl or 2’stluoro modifications; modifications on Nb differ from the modification on Y and modifications on Nb' differ from the modification on Y"; and ll) wherein the sense strand is coniugated to at least one ligand. in another aspect, the present invention provides a double stranded RN Ai agent e of inhibiting the expression of ment component C5 in a cell, wherein said double stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein said antisense strand comprises a region complementary to part of an mRNA encoding complement l5 component CS, n each strand is about l4 to about 30 nucleotides in length, wherein said double stranded RNAi agent is represented by formula (lll): sense: 5' n1" "N3 {X X X) be “Y Y Y ~Nb "(Z Z le ~Na - n6 3' antisense: 3' iip'—Na’~(X’X'X")k—NbCY'Y’YZNbC(Z'Z’Z'h—Na'n nq' 5’ {lll} wherein: 2t) i, i, k, and l are each independently G or l ; each np, nq, and nq't each ofwhich may or may not he present, independently represents an overhang nuc' eotide; p, q, and d” are each independently (L6; np’ >0 and at least one np' is linlced to a neighboring nucleotide Via a phosphorothioa‘te linkage; each N1 and Na’ independently represents an oligonucleotide sequence comprising {L25 nucleotides which are either modified or unmodified or comhinations thereof, each sequence comprising at least two dit‘terently modified tides; each Nb and Nb" independently represents an oligonueleotide sequence comprising (hill (A) c. tides which are either modified or unmodified or combinations thereof; XXX, YYY, ZZZ, X'X'X', Y'Y’Y', and Z’Z'Z’ each independently represent one motif ofthree identical modifications on three consecutive nucleotides, and wherein the cations are 2'—O—methyl or 2'—tluoro tlcations; modifications on Nb differ from the modification on Y and modifications on Nb' differ from the modification on Y'; and wherein the sense stand is conjugated to at least one ligand.

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ln another aspect, the present ion provides a double stranded RNAi agent capable of inhibiting the expression of complement component CS in a cell, wherein said double stranded RNAi agent ses a sense strand complementary to an nse strand, wherein said an tisense strand comprises a region complementary to part of an mRNA encoding complement (I: component C5, wherein each strand is about l4 to about 36 nucleotides in , wherein said double stranded RNAi agent is represented by formula (Ill): sense: 5' up an {X X X) i-Nb 3r Y Y —Nb lrz z Z); N — all 3 antisense: 3' nJ—NaitXX'X’h—NJ—Y'Y’Y'~Nb’—(Z'Z’Z’)1—N;— nq' 5’ (lll) wherein: ll) i, j k, and l are each independently O or l; each np, nq, and nq’, each ofwhich may or may not he present, independently represents an o 'erhang nucleotide; p, q, and q” are each independently 0nd; np’ >0 and at least one np' is d to a neighboring nucleotide via a phosphorothioate linlcage; each Na and Na” independently represents an oligonucleotide sequence comprising 0—25 nucleotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucleotides; each N1, and N1: independently represents an oligonucleotide sequence comprising (lull) 2t) nucleotides which are either modified or unmodified or combinations thereof; XXX, YYY, ZZZ, X'X'X', Y’Y’Y’, and Z'Z’Z’ each independently represent one motif of three identical modifications on three utive nucleotides, and wherein the modifications are ECO-methyl or 2’—tluoro modifications; cations on Nb differ from the n'iodilication on Y and modifications on RE; differ from the modification on Y’; and n the sense strand is coniugated to at least one ligand, wherein the ligand is one or more GalNAc tives attached through a bivalent or trivalent branched linker. in yet another aspect, the present invention provides a double st “anded RNAi agent capable of inhibiting the expression ofcornplement component C5 in a. cell, wherein said do able (A) c. stranded RNAi agent comprises a sense strand mentary to an antisense strand, wherein said antisense strand comprises a region complementary to part of an mRNA encoding complement component (35, n each strand is about l4 to about 30 nucleotides in length, wherein said double stranded RNAi agent is represented by tormala (Ill); sense: 5' n1" "N3 ~(X X X) l—Nb “Y Y Y ~Nb "(Z Z le ~Na - nq 3' nse: 3‘ np'éNj-(X'X’X'lk—NbCY'Y’Y"-N1,'—(Z'Z’Z')t-N 3'" nq' 5‘ (ill) wherein: [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS i, j, k, and l are each independently G or l; each np, nq, and nq', each ofwhich may or may not be present, ndently represents an o Ierhang nucleotide; p, q, and q” are each independently 0—6; (I: np' >0 and at least one np' is linleed to a neighboring nucleotide Via a phosphorothioate linkage; each N, and N,” independently represents an oligonucleotide sequence comprising 0~25 nucleotides which are either ed or unmodified or ations the ieo’f, each sequence comprising at least two differently modified nucleotides; ll) each N1, and Nb' independently represents an oligonucleotide sequence comprising (lull) nucleotides which are either modified or unmodified or combinations thereof; XXX, YYY, ZZZ, X'X'X', Y’Y’Y’, and Z'Z’Z’ each independently ent one motif of three identical modifications on three consecutive nucleotides, and wherein the modifications are ECO-methyl or 2’—tluoro modifications; modifications on Nb differ from the modification on Y and modifications on Nb' differ from the rnodili cation on ‘1‘"; n the sense strand ses at least one orothioate gc; and wherein the sense strand is conjugated to at least one ligand, wherein the ligand is one or more GalNAc derivatives attached through a bivalent or ent hranched linker. 2t) ln another aspect, the present invention provides a double stranded RN Ai agent capable of inhibiting the expression of con’iplernent component C5 in a cell, wherein said double ed RNAi agent comprises a sense , complementary to an antisense strand, n said antisense strand comprises a region complementary to part of an mRNA encoding complement ent CS, wherein each strand is about lél to about 30 nucleotides in length, wherein said double stranded RNAi agent is represented by formula (lll): sense: 5' n1" "N3 —Y Y Y _ Na n nq 3‘ antisense: 3‘ rig—N,”- Y'Y'Y'- N,”- nq' 5' (lila) wherein: each np, nq, and nq', each ofwl’iich may or may not he present, independently represents (A) c. an overhang nucleotide; p, q, and q” are each independently one; np’ >0 and at least one rip' is linleed to a neighboring nucleotide Via a phosphorothioate linkage; each N1 and N,’ independently represents an oligonucleotide sequence comprising {L25 nucleotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two difterently modified nucleotides; [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS YYY and Y’Y'Y’ each independently represent one motif of three identical modifications on three consecutive nucleotides, and wherein the modifications are 2’—0-methyl or 2’~tli:toro inodili cations; wherein the sense strand con’iprises at least one phospl’iorotliioate linkage; and (I: wherein the sense strand is ated to at least one ligand, wherein the ligand is one or more GalNAc derivatives attached through a bivalent or trivalent hranched linker. in one aspect, the present invention provides a double ed RN Ai agent for inhibiting expression of complen’ient component C5, wherein the double stranded RNAi agent con’iprises a sense strand and an antisense strand g a double stranded region, wherein the sense strand it) comprises at least l5 contiguous nucleotides differing by no more than 3 tides from the tide semtence ot‘SEQ ll) Niki and the antisense strand comprises at least l5 contiguous nucleotides differing by no more than '3 nucleotides from, the nucleotide sequence of SEQ ID N025, wherein substantially all of the nucleotides ofthe sense strand comprise a modification selected from the group consisting of a 2’—O-methyl modification and a Zl—lluoro modification, l5 wherein the sense strand comprises two phosphorothioate inter‘n'ucleotide linkages at the 5’— terrninus, wl’ierein substantially all ofthe nucleotides ot‘the antisense strand comprise a modification se' ected from the group consisting of a 2"~0—inethyl modification and a Zlfluoro modification, wherein the antisense strand comprises two phosphorothioate internucleotide linkages at the 5’—terniinus and two phosphorothioate internucleotide linkages at the 3 inus, 2t) and wherein the sense st “and is conjugated to one or more NAe derivatives ed through a branched hivalent or trivalent linker at the 3 ’—tern'iinus. in one ment, all ofthe nucleotides ofthe sense strand and all ofthe nucleotides of the nse strand are modified nucleotides. in another enihodiinent, each strand has l9-3O nucleotides. ln one aspect, the present invention provides a cell containing a dsRNA agent of the invention ln one , the present invention provides a vector encoding at least one strand of a dsRNA agent, wherein the dsRNA agent ses a region of mentarity to at least a part of an mRNA encoding con’iplernent component C5, wl‘ierein the dsRNA is 30 base pairs or less (A) c. in length, and n the dsRNA agent targets the mRNA for cleavage. in one embodiment, the region of complementarity is at least 15 nucleotides in length. ln another embodiment, the region ol‘cornpleinentarity is l9 to 2l nucleotides in length. in another embodiment, each strand has l9~3 ll nucleotides. ln one aspect, the present ion provides a cell comprising a vector ofthe invention. ln one aspect, the present invention provides a pharmaceutical composition for inhibiting expression of a complement component CS gene comprising a dsRNA agent ot‘the invention.

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS In one embodiment, the RNAi agent is administered in an unbuffered solution, In one embodiment, the unbuftered solution is saline or water.

In one embodiment, the RNAi agent is administered with a butler solution.

In one embodin'ient, the butter on comprises e, citrate, prolamine, carbonate, (I: or phosphate or any combination thereof in another embodiment, the buffer solution is phosphate buffered saline (PBS), in another aspect, the present invention provides a pharmaceutical composition comprising a double stranded RNAi agent ot‘the invention and a. lipid formulation, In one embodiment, the lipid formulation comprises a LNP. in another embodiment,the ll) lipid formulation comprises a MC3.

In one aspect, the present invention provides a composition comprising an antisense cleotide agent selected iron'i the group consisting ot‘the sequences listed in any one of 'I‘a’hles 3, 4-, 5, 6, 19, IS, 2G, 21, and 2,3. in another aspect, the present invention provides a ition comprising a sense polynucleotide agent selected from the group consisting of the sequences listed in any one of Tables '3, 4, S, 6, l9, Ill, 20, El, and 23.

In yet another aspect, the present invention provides a modified antisense polynucleotide agent se ected from the group ting of the antisense sequences listed in any one of Tables 4-, 6, 18, l9, El and 23. 2t) In a further aspect, the present invention es a modified sense cleotide agent ed from the group consisting of the sense sequences listed in any one oflables 4, 6, l8, l9, 2i and 23, in one aspect the present invention provides methods ting a subject having a disease or disorder that would benefit from reduction in ment component {35 expression.

The methods include adrninistering to the subject a. therapeutically elitective amount of a dsRNA agent comprising a sense strand and an antisense strand, n the sense strand ses at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence ot‘SlEQ ID Nill and the nse strand comprises at least 15 contiguous nucleotides ring by no more than 3 nucleotides from the nucleotide sequence ofSEQ ll) N05, thereby (A) c. treating the subject, in another aspect, the present invention provides methods of preventing at least one symptom in a subject having a disease or disorder that would benefit from reduction in complement component C5 sion. The methods include stering to the t a therapeutically ive amount of a dsRNA agent comprising a sense strand and an antisense strand, wherein the sense strand comprises at least l5 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence ot‘SEQ ID Nikl and the antisense strand ation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS ses at least l5 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence ofSEQ ll) N05, thereby ting at least one symptom in the subject having a disorder that would benefit from reduction in {35 expression. ln another aspect, the present invention provides methods of treating a subject having a (I: disease or disorder that would benefit from reduction in complement component C5 expression.

The methods include administering to the subject a therapeutically effective amount of a dsRNA agent comprising a sense strand and an antisense strand, the antisense strand comprising a region ofcomplementarity which comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one ofthe antisense sequences listed in any one of Tables 3, 4., 5., 6, ill 18, l9, 20, El the subject. , 23, thereby treating ln yet another , the present invention provides methods ofpreventin g at least one symptom in a subject having a disease or disorder that would benefit from reduction in complement component C5 expression. The methods include administering to the subject a prophylactically effective amount of a dsRNA agent sing a sense strand and an antisense , the antisense strand comprising a region of complementarity which comprises at least l5 contiguous nucleotides ring by no more than 3 nucleotides from any one ofthe antisense sequences listed in any one of Tables 3., r—‘l, 5, 6, 18, 19, 20, 2t and 23, thereby preventing at least one symptom in the subject having a disorder that would benefit from ion in C5 expression. 2t) ln one aspect, the present invention provides methods oftreatin g a t having a disease or disorder that would benefit from reduction in complement component C5 expression which include administering to the subject a eutically effective amount of a double stranded RNAi agent, wherein the double stranded RNAi agent comprises a sense strand and an antisense strand forming a double stranded region, n the sense strand comprises at least l5 contiguous nucleotides (littering by no more than 3 nucleotides from the nucleotide. sequence of SEQ 1D N'Ozl and the antisense strand comprises at least l5 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ll) N025, wherein substantially all ofthe nucleotides of the antisense strand and substantially all of the nucleotides ofthe sense strand are modified nucleotides and, wl’ierein the sense strand is conjugated to one or more (A) c. ligands at the 3 ’—terminus in one ment, all ofthe nucleotides ofthe sense strand and all ofthe nucleotides of the nse strand are modified nucleotides. in one dirnent, the administration is subcutaneous administration. in one embodiment, substantially all of the nucleotides ofthe sense strand are ed nucleotides selected from the group consisting of a 2”-Oumethyl modification, a oro modification and a 3 ’~terrninal dT nucleotide. ln another embodiment, substantially all of the [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS nucleotides ofthe antisense strand are modified tides se ected from the group consisting of a 2’—O—methyl modification, a 2’—fiuoro modification and a 3 l-terminal drl‘ nucleotide. in another embodiment, the modified nucleotides are a short sequence ot‘deox3,1~tliyrniiie (dT) nucleotides. ln another embodiment, the sense strand comprises two ph osphorothioate internucleotide (I: linkages at the 5"~terminus. in one embodiment, the antisense strand comprises two phosphorothioate internucleotide linkages at the 5’—terrninus and two phosphorothioate ucleotide linkages at the 3 ’~terrninus, In yet another embodiment, the sense strand is coniugated to one or more GalNAc derivatives ed through a branched bivalent or trivalent linlrer at the 3 ’ntemiinus, ll) in another aspect, the present invention provides methods of preventing at least one symptom in a subject having a disease or disorder that would benefit from reduction in complement con'iponent CS expression which e administering to the subject a prophylactieally effective amount of a double stranded RNAi agent, n the double stranded RNAi agent comprises a sense strand and an antisense strand forming a double stranded , wherein the sense strand comprises at least l5 uous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence ol" SEQ ll) Nflzl and the antisense strand comprises at least l5 contiguous nucleotides differing by no more than 3 tides from the nucleotide sequence of SEQ ID N025, wherein substantially all ofthe nucleotides of the antisense strand and substantially all of the nucleotides ofthe sense strand are modified nucleotides and, wherein 2t) the sense strand is conjugated to a ligand at the 3 a“termirnis. in one embodiment, all of the l’luClCOlldCS ot‘the sense strand and all of the tides of the antisense strand are d nucleotides in one embodiment, the administration is subcutaneous administration. ln one embodiment, ntially all of the nucleotides ot‘the sense strand are modified nucleotides selected from the group consisting of a ’2 thyl i'nodification, a 29~tluoro modification and a 3 ”~terminal d’l" nucleotide. in another ment, substantially all of the tides of the antisense strand are modified nucleotides selected from the group consisting of a 2’~0—rnethyl modification, a 2’-tluoro modification and a 3 ’~terrninal dT nucleotide. In another embodiment, the modified nucleotides are a short sequence ot‘deoxy—thymine (dT) nucleotides.

(A) c. in r embodiment, the sense strand comprises two phospliorothioate intemucleotide linkages at the minus. in one embodiment, the antisense strand comprises two phosphorothioate intei‘n'ucleotide linkages at the 5’sterininus and two phosphorothioate internucleotide linkages at the '3 ’—terminus, In yet another embodiment, the sense strand is conjugated to one or more GalNAc tives attached through a branched bivalent or trivalent r at the 3 ’—terminus.

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS in one aspect, the present invention provides methods of treating a subject having a disease or disorder that would t from ion in complement component CS expression.

The methods include administering to the subject a therapeutically efthctive amount ofa dsRNA agent con’iprising a sense strand con’iplementary to an antisense strand“, wherein the antisen se (I: strand comprises a region complementary to part of an mRNA encoding C5, wherein each strand is about 14 to about 30 nucleotides in length, wherein the double stranded RNAi agent is represented by formula (Ill): sense: 5' np —Na ~(X X X) i—Nb ~Y Y Y ~Nb —(Z Z 2% ~Na ~ n, 3‘ antisense: 3' iip'—Na’~(X’X'X")k—N1,’—Y"‘i”Y"~N1,C(Z'Z’Z'h—NE'“ nq' 5’ (Ill) ll) wherein: i, j, k, and l are each independently G or i ; p, p”, q, and u" are each independently O~6; each N3 and N3” independently ents an oligonucleotide sequence comprising 0'25 nucleotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two ently modified nucleotides; each Nb and Nb' independently represents an oligonucleotide sequence comprising O—l t) nucleotides which are either modified or fied or combinations f; each np, np', nqi and nq', each ofwhich may or may not be present independently represents an overhang nucleotide; 2t) XXX, YYY, ZZZ, X’X'X’, Y'Y'Y', and Z'Z’Z' each independently ent one motif of three identical modifications on three utive i’iucleotides; modifications on N, differ from the modification on Y and modifications on Nb' differ from the modification on ‘r”; and n the sense strand is conjugated to at least one ligand, y treating the subject, thereby treating a suhject having a disease or disorder that would benefit from reduction in complement component C5 expression in another aspect, the present invention provides methods of preventing at least one symptom in a subject having a disease or disorder that would benefit from ion in complement con'iponent CS sion. The methods include administering to the subject a (A) c. prophylactically effective amount of a dsRNA agent comprising a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to part of an rnRNA encoding CS, wherein each strand is about M to about 30 micleotides in length, wherein the double stranded RNAi agent is represented by formula (ill): sense: 5' n1" "N3 ~(X X X) l—Nb “Y Y Y ~Nb "(Z Z le ~Na - nq 3' antisense: 3‘ np'éNj-(X'X’X'lyNfi—Y'Y’Y"-N1,'—(Z'Z’Z')i-N 3'" nq' 5‘ all) wherein: [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS i, j, k, and l are each independently G or l; p, p’, Ll: and q' are each independently 0—6; each Na and N3" independently represents an oligonucleotide sequence comprising 9—25 nucleotides which are either modified or unmodified or combinations thereof, each sequence (I: comprising at least two differently ed nucleotides; each Nb and Nb' independently represents an oligonucleotide sequence comprising O-l 0 nucleotides which are either ed or unmodilied or combinations thereof; each np, np', nq, and nq’, each of which may or may not be present, independently ents an overhang nucleotide; it) XXX, YYY, ZZZ, X’X'X’, Y'Y'Y', and Z'Z’Z' each independently represent one motif of three identical modifications on three consecutive nucleotides; from the modification on Y’; and wherein the sense strand is conjugated to at least one ligand, y preventing at least one m in the subject having a disorder that would benetit from reduction in (35 exp Tession, thereby preventing at least one symptom in a subject having a disease or disorder that would benefit from ion in complement ent C5 expression in one embodiment, the administration of the dsRNA to the subject causes a decrease in intravascular hernolysis, a stabilization of hemoglobin levels and/or a decrease in CS protein 2t) accumulation. ln one embodin'ient, the disorder is a con’iplement component {IS—associate disease, ln one ment, the complement component CSnassociated disease is se' ccted from the group consisting ofparoxysmal nocturnal hemoglobinuria (PNEl), atypical liemolytic uremic syndrome {allUS}, asthma, rheun'iatoid tis (RA); antiphospholipid antibody syndrome; lupus nepl’iritis; iscbenria~reperfusion inj ury; typical or infectious hen’iolytic uremic syndron’ie (tllUS); dense deposit disease (EDD); neuromyelitis optica ); inultifocal motor neuropatliy (MM’N); multiple sis (MS); r ration (egg, age—related macular degeneration (Alt/ll)»; hemolysis, elevated liver enzymes, and low platelets (llli’lslf) syndrome; thrombotic thrombocytopenic purpura (TTP); spontaneous fetal loss; Pauci—irnmune vasculitis; (A) c. epiderinolysis bullosa; recurrent fetal loss ; pre~eclampsia, traumatic brain injury, rnyasthenia grayis, cold inin disease, dermatomyositis bullous pernphigoid, Shiga toxin E. coil—related hemolytic uremic syndrome, (33 nephropathy, eutrophil cytoplasmic antibody-associated vasculitis, humoral and vascular transplant rejection, graft nction, myocardial infarction, an allogenic transplant, sepsis, Coronary artery disease, dermatomyositis, Graves' disease, atherosclerosis, Alzheimer’s e, systemic inflammatory response sepsis, septic shock, spinal cord irritry, rlomeiulone ihritis, llashimoto‘s thvroiditis, tv re ldiahetes isoriasis, em hious a , E) I J J 9 G 9 [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS autoimmune hemolytic anemia (AlHA), l’l‘l), Goodoasture syndrome, Degos disease, ospholinid syndrome (APSE), catastrophic APS (SAPS), a cardiovascular disorder, myocarditis, a cerebrovascular disorder, a peripheral vascular disorder, a renovascular disorder, a mesen teri e/enteri c vascular disorder, vasculitis, l’lenochSchonlein nurpura nephritis, systen‘ric (I: lupus erythematosus—associated vasculitis, vasculitis associated with rheumatoid arthritis, immune complex vasculitis, ’l'akayasu’s disease, dilated cardioniyopathy, diabetic angiopathy, Kawasaki's e {arteritis}, venous gas embolus (VGE), and restenosis following stent placement, rotational, ath erectorny, membraneous nephropathy, Guillain—Baire syndrome, and percutaneous transluminal coronary angioplasty (PICA). in another embodiment, the ill complement component CS-assoeiated disease is paroxysmal nocturnal hemoglobinuria (PNH). in yet another embodiment, the complement component sociated disease is atypical hernolytic uremic syndrome (allUS), ln one embodiment, the subject is human, lri another ment, the methods ofthe invention further include administering an omplement component (35 antibody, or antigen~binding fragment thereof, to the subj ect, in one en'ibodiment, the antibody, or antigen—binding fragn’rent thereofi inhibits clea age of complement component C5 into fragments CSa and CSb, in r ment, the antiw complement component C5 antibody is umab. in another embodiment, the methods of the ion further include administering a 2tl ococcal vaccine to the subject. ln one embodiment, eculizurnab is nistered to the subieet weekly at a dose less than about 600 mg for 4 weeks ed by a fifth dose at about one week later of less than about 900 mg, followed by a dose less than about 909 mg about every two weeks thereafter. hi another embodiment, eculizurnab is stered to the subject weekly at a dose less than about 9th mg for 4 weeks followed by a fifth dose at about one w 363k later of less than about l200 mg, followed by a dose less than about lZGQ mg about every two weeks thereafter. in one embodiment, the subject is less than l8 years of age and eculizumab is administered to the t weekly at a dose less than about 9th mg for 4 weeks followed by a fittl’r dose at about one week later ot‘less than about lZOO mg, followed by a. dose less than about (A) c. lZQO mg about every two weeks thereafter, lri another embodiment, the subject is less than l8 years of age and eculizumab is administered to the subject weekly at a dose less than about 600 mg for 2 weeks followed by a third dose at. about one week later of less than about 900 mg, followed by a dose less than about 980 mg about every two weeks thereafter. in another embodiment, the subj eet is less than l8 years of age and eculizumab is stered to the subject weekly at a dose less than about 600 mg for 2 weeks followed by a ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS third dose at about one week later of less than about 600 mg, followed by a dose less than about 600 mg about every two weeks thereafter.

In yet another embodiment, the subject is less than l8 gears of age and eculizumab is administered to the subject weekly at a dose less than about 600 mg for l week ed by a (I: second dose at about one week later of less than about 300 mg followed by a dose less than about 300 mg about every two weeks fter. in one embodiment, the subject is less than l8 years of age and ecuIizumab is administered to the t weekly at a dose less than about 300 mg for l week tbllowed by a second dose at about one week later of less than about 300 mg, ed by a dose less than ll) about 300 mg about every two weeks thereafter.

In another embodiment, the methods of the invention further include plasmapheresis or plasma exchange in the subject. In one such en‘ibodiment, eculizumab is administered to the subject at a dose less than about 609 mg or at a dose less than about 300 mg. in a further ment, the methods of the invention further include plasma infusion in the subject. In one such embodiment, eeulizumab is administered to the subject at a dose less than about 300 mg.

In one embodiment, eculizumab is administered to the subject at a dose of about OtGl nig/ltg to about l0 g or about (3.5 mg/kg to about l5 mg/hg. In r embodiment, eculizumab is administered to the subject at a dose of about 5 mg/kg to about l5 mg/kg. 2t) In one embodiment, eculizumab is administered to the subj eet at a dose selected from the group ting of 05 mg/kg, 1 rug/kg, I5 n'ig/kg, 3 mg/kg, 5 n’ig/kg, ’7 mg/Rg, I0 111ng and l5 nig/kg. in one embodiment, eculizumab is administered to the subject via an intravenous infusion .

In another embodiment“, eeulizuinab is administered to the subject subcutaneously.

In one embodiment, the dsRNA agent is administered at a dose of about (1.01 mg/kg to about ll) mg/kg or about 0.5 mg/kg to about 50 mg/kg In another embodiment, dsRNA agent is administered at a dose of about it} mg/kg to about 30 nag/kg.

(A) c. In one ment, the dsRNA agent is administered at a dose selected from the group consisting {5150.5 tug/kg l mtg/kg, 1.5 mg/kg, 3 mg/kg, 5 mg/kg, it) rug/kg, and 30 mg/kg. in one embodiment, the dsRNA agent is administered to the subject once a week. In another embodiment, the dsRNA agent is administe fed to the subject twice a week. in another embodiment, the dsRNA agent is administered to the subject twice a month.

In one embodiment, the dsRNA agent is administered to the subject subcutaneously.

[Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS In one embodiment, the dsRNA agent and the uniab are administered to the t subcutaneously. In another embodiment, the dsRNA agent and the eculizumab are administered to the subject aneously.

In one embodin'1ent, the dsRNA, agent is administered to the subject first for a period of (I: time sufficient to reduce the levels of complement ent C5 in the subject, and eeulizumab is administered subsequently at a dose less than about 691) mg.

In one embodiment, the levels oI‘coi’anement ent C5 in the t are redu ‘ed by at least about 30/0, 35 “/6, 40%, 45%, 50%, 55%, 60%, 65%, ’7 ‘3/075"m, 80%, 85%, or 90% In one embodiment, eculizuniab1s administered at a dose of about I00500 mg ll) In one embodiment, the methods otthe invention r e measuring hemoglobin and/or L'DII levels in the subject.

In one embodin'1ent, the dsRNA, is coniugated to a ligand.

In one embodiment, the ligand is ated to the 3 ’n end ofthe sense strand ofthe dsRNA.

In one embodiment the ligand1s an Nacetvloalattosainme (GaINIAc) derivative In one aspect, the present invention prov1des methods of ting complement component C5 expression in a cell Ihe methods include contacting the cell with a dsRNA agent comprising a sense strand and an antisense strand, wherein the sense strand comprises at least I5 uous nucleotides differingby no more than3 nucleotides from the nucleotide sequence of 21) SEQ ID NOzI and the antisense strand comprises at least I5 contiguous nucleotides dittering by no more than 3 nucleotides t1om the n11cleot1de seqtuence ot SEQ ID NO:5; and mainta1ning the cell ed in step (a) for a time sufficient to obtain degradation ofthe niRNA transcript of a CS gene, thereby ting expression of the C5 gene in the cell In another aspect, the present invention provides methods ot‘inhibiting complement component C5 expression in a cellt The methods include contacting the cell with a. dsRNA agent comprising a sense strand and an antisense strand, the antisense strand comprising a region of complementarity which comprises at least l5 contiguous nucleotides dit‘tering by no more than 3 nucleotides Irom any one of the antisense sequences listed in anyone ofTables 3, 4, 5, 6, Ill, 19, , El and 23; and 1'na1ntain1ng the celII produced in step (a) for a time sutticient to obtain (A) c. degradation ofthe mRNA transcript of a C 5 gene, thereby inhibiting expression of the C5 gene in the cell.

In another aspect, the present invention provides methods ot‘inhibiting complement component C5 expression in a cell, which includes contacting the cell with a dsRNA agent comprising a sense strand and an antisense strand comprising a region of complementarity, the sense strand ses at least l5 contiguous nucleotides dit‘tering by no more than 3 nucleotides trom the nucleotide sequence of SI:313 ID NII I and the antisense strand comprises at least I5 [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID N025, wherein substantially all of the nucleotides of the antisense strand and substantially all ofthe nucleotides ot‘the sense strand are modified nucleotides and, wherein the sense strand is conjugated to one or more ligands at the 3 ’—tern'iinus; and maintaining the cell (I: produ ted in the first step for a time sufficient to obtain degradation ofthe niRNA transcript of a CS gene, thereby inhibiting expression of the {.75 gene in the cell In one embodiment, all ot‘the nucleotides e sense strand and all ofthe nucleotides of the antisense strand are n'iodilied nucleotides.

In one embodiment, substantially all of the nucleotides ofthe sense strand are modified ll) nucleotides selected from the group consisting of a methyl modification, a 2’-fluoro modification and a 3 ’~ternrinal dT nucleotide. ln another embodiment, substantially all of the nucleotides of the antisense strand are modified nucleotides selected from the group consisting of a ethyl modification, a 2’—tluoro modification and a 3 ’nterniinal d'l" nucleotidei In another embodiment, the modified nucleotides are a short sequence of deoxy—thymine (d’l‘) nucleotides. l5 ln another embodiment, the sense strand comprises two phosphorothioate iiiternueleotide linkages at the 5’—terniinus In one embodiment? the antisense strand ses two phosphorothioate internucleotide linkages at the fig-terminus and two phosphorothioate internucleotide linkages at the 3 ’nterminusi In yet another embodiment, the sense strand is coniugated to one or more GalNAe derivatives attached through a branched bivalent or trivalent 2t) iinlter at the 3 inus. ln yet another aspect, the present ion es s of inhibiting con'iplen'ient component C5 expression in a cell. The methods include contacting the cell with a dsRNA agent sing a sense strand complementary to an antisense , wherein the antisense strand comprises a region complementary to part of an mRNA encoding CS, wherein eaeh strand is about l4 to about 30 nucleotides in , n the double stranded RNAi agent is represented by formula (lll); sense: 5' np "N, —(X X X) i—Nb ~Y Y Y —Nb —(Z Z le Na - nq 3' antisense: 3‘ (X'X ’X’ksNblY'Y ’Y”—Nb’-(Z 'Z ’Z ')1¥Na'~ tn“ 5‘ (ill) wherein: (A) c. i, j, l<;, and l are each independently (l or 1; p, p"', q, and LI; are each independently 0-6; each Na and 'Na' independently ents an oligomtcleotide seduen ‘e comprising 0-25 nucleotides which are either modified or unmodified or combinations the reef, each sequence comprising at least two differently modified nucleotides; each N1, and Nb," independently ents an oligonucleotide sequence comprising tl—ltl nucleotides which are either modified or unmodified or combinations thereof; [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS each 11p, np’, nq, and nq’, each of which may or may not be present, independently represents an overhang nucleotide; XXX, YYY, ZZZ, , Y'Y'Y', and Z'Z’Z' each independently represent one motif of three identical modifications on three consecutive nucleotides; (I: modifications on N, differ from the modification on Y and modifications on Nb' differ from the modification on ‘i”; and wherein the sense strand is conjugated to at least one ligand; and maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript. ofa C5 gene, thereby inhibiting expression of the C5 gene in the cell. ll) ln one embodiment, the cell is Within a subject. ln one embodiment, the t is a human. ln one embodiment, the human subject Sliders from a complement onent C5— assoeiated disease. ln one embodiment, the complement component {TSuassociated disease is ed from the group consisting ofparoxysrnal nocturnal hernoglobin'aria (PNH), atypical hernolytic uremic syndrome (allUS), asthma, rheumatoid arthritis (RA); antiphospholipid antibody syndron'ie; lupus nephritis; ischemia—reperfusion iniury; typical or infectious hemolytic uremic syndrome (tHUS’); dense deposit disease (EDD); neuromyelitis optica {NMO}; multifocal motor neuropathy (MMN); multiple sclerosis (MS); rnacular ration (cg, age—related macular 2t) ration (Ah/EBB; hernolysis, elevated liver enzymes, and low platelets (lrllilslsl’) syndrome; thrombotic thrombocytopenic purpura (TT?); spontaneous fetal loss; Pauci~irnmon e yasculitis; epidermolysis bullosa; recurrent fetal loss; pre~eclampsia, traumatic brain injury, enia graVis, cold agglutinin disease, dermatomyositis bullous pemphigoid, Shiga toxin E. coil—related henrolytic uremic syndrome, (33 nephropathy, antisneutrophil cytoplasmic antihody-associated vasculitis, humoral and vascular transplant rejection, graft ction, myocardial infarction, an allogenic transplant, sepsis, ry artery disease, dermatomyositis, Graves' disease, atherosclerosis, mer’s disease, systemic inflammatory response , septic shock, spinal cord , glomemlonephritis, llashinroto‘s thyroiditis, type I diabetes, psoriasis, pernphigus, autoimmune hemolytic anemia (,AlllA), lTP, sture syndrome, Degos disease, (A) c. antiphospholipid syndrome (APSE), catastrophic APB (CAPS), a cardiovascular disorder, myocarditis, a cerehrovascular disorder, a peripheral vascular disorder, a renovaseular disorder, a mesenteric/enteric vascular disorder, y'asculitis, llenoch~Schonlein p'urp'ara tis, ic lupus erytheniatosus—associated yascnlitis, vasculitis associated with rheumatoid arthritis, immune complex itis, 'l‘alcay’asrr’s disease, dilated eardiomyopatlry, diabetic athy, ki's disease (ar‘teritis), venous gas emholus (VGE), and osis lollowing stent placement, rotational atherectonry, neous nephropatby, Gail lainslfiarre syndrome, and [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS percutaneous translnniinal coronary angioplasty (PICA). ln another embodiment, the complement component oeiated e is paroxysmal nal obinuria (PNH). in another embodiment, the con'rplement component CEassociated disease is atypical hemolytic uremic syndrome (allUS).

(I: ln one embodiment, the methods further include contacting the cell with an anti" complement component CS antibody, or antigen—binding fragment thereof. in one embodiment, the antibody, or antigensbinding fragment thereof, inhibits cleavage oi complement component C3 into tragnrents C"a and CBl in one embodiment, the antincomnlement component C5 antibody, or antigenwbinding ll) fraagiment thereot, is eculizumab in one embodiment, the n'retliods further include contacting the cell with a meningococcal yaceine. ln one embodiment, the cell is contacted with eculizumab weekly at a dose less than about (Silt) mg for 4 weeks followed by a fifth dose at about one week later of less than about 900 mg, lollowed by a dose less than about 9th) mg about every two weeks thereatter. be another embodiment, the eellis eon'taeted with eculizurnab weekly at a dose less than about 900 mg for 4- weeks followed by a fifth dose at about one week later of less than about l20t) mg, followed by a dose less than about thltl mg about every two weeks thereafter. ln another embodiment, the cell is ted with eculizuniab weekly at a dose less than 2t) about 900 mg for 4 weeks tollowed by a fifth dose at about one week later of less than about 1200 mg, tollowed by a dose less than about lZGO mg about every two weeks fter. ln yet another embodiment, the cell is contacted with eculizunrab weekly at a dose less than about (300 mg for 2 weeks followed by a third dose at about one week later of less than about 906 mg, followed by a dose less than about 900 mgY about every two weeks thereafter. he one en'rbodiment, the cellis contacted with eculizumab weekly at a dose less than about 600 mg for 2 weeks followed by a third dose at about one week later of less than about 600 mg, ed by a dose less than about 600 mg about every two weeks thereafter. in another embodiment, the cell is contacted with umah weekly at a dose less than about 600 mg for l week edby a seeoird dose at about one week later ofless than about (A) c. 300 mg, follox 'edby a dosse less than about 300 mg about every two weeks thereafter, in one embodiment, the cell is contacted with eeulizuniab weekly at a dose less than about soc mg for 1 week tollowed by a second dose at about one week later of less than about 300 mg, followed by a dose less than about 300 mg about every two weeks thereafter. in one embodiment, the cell is within a subject ln one embodiment, the methods ot‘the ion r include plasniapheresis or plasma exchange in the subject. in one embodiment, eculizumab is administered to the subject at [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS a dose less than about 600 mg, in another embodiment, e mab is administered to the subject at a dose less than about 300 mg in one embodiment, the n'ietliods oi‘the invention turther include plasma infusion in the subject. ln one en'ibodiment, eculizumab is administered to the subject at a dose less than about (I: 300 mg, in one embodiment, the cell is contacted with eculizumab at a dose of about (Mil mg/lrg to about it) mg/kg or about 0.5 trig/leg to about l5 mg/lcg. ln another embodiment, the cell is contacted with umab at a dose of about 5 rug/kg to about l5 g. ll) in one embodiment, the cell is contacted with eculizumab at a dose ed from the group consisting ot‘Gfi ring/kg, l trig/kg, l .5 trig/kg, 3 t, Sing/leg, 7 trig/leg, l0 mg/kg, and l5 org/leg in one embodiment, eculizumab is administered to the subject Via an intravenous infusion. in another embodiment, umab is administered to the subject subcutaneously, ln one ment, the cell is contacted with the dsRNA agent at a dose of about {Bill org/leg to about it) ntg/ltg or about 0.5 n'ig,"hg to about 50 mg/kg. in another ment, the cell is contacted with the dsRNA agent at a dose of about ll) mtg/leg to about 30 rug/leg. in one embodiment, the cell is contacted with the dsRNA agent at a dose selected from 2t) the group consisting MOS mg/kg l trig/leg, l.5 mgr/kg, 3 mg/kg, 5 mg/lcg, l (l rug/leg, and 30 in one embodiment, the cell is ted with the dsRNA agent once a weelr. in another embodiment, the dsRNA agent is administered to the subject twice a weelr. in another embodiment, the cell is contacted with the dsRNA agent twice a month. in one en'ibodiment, the dsRNA agent is administered to the subject subcutaneously. in one embodiment, the dsRNA agent and the eculizumab are administered to the subject subcutaneously. in another embodiment, the dsRNA agent and the eculizumab are administered to the subject simultaneously. in one ment, the cell is contacted with the dsRNA, agent and the eculimmab (A) c. simultaneously. in one embodiment, the dsRNA agent is administered to the subject first for a period of time sutlicient to reduce the levels of complen'ient component (35 in the subject, and eculizurnab is administered subsequently at a dose less than about 690 mg. in one embodiment, the levels of complement component C5 in the subject are reduced by at least about 30%, 35%, 46%, 45%, 50%, 55%, 60%, 65%, 79%, 759/3, 80%, 85%, or 909/3, in one embodiment, eculizumab is adn'iinistei'ed at a dose of about l00~500 mg.

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ln one embodiment, the cell is contacted with the dsRNA agent first for a period oftime sufficient to reduce the levels of complement ent C5 in the cell, and the cell is subsequrntly contacted with ec’ulizumah at a dose less than about 600 mg. ln one embodin'ient, the levels of complement component C5 in the cell are reduced by at (I: least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%. in one embodiment, the cell is contacted with eeulizuniab at a dose of about lOO—SOO mg. ln one aspect, the present invention provides methods of inhibiting the expression of {35 in a subject. The methods include administering to the subject a therapeutically ellective amount of a dsRNA agent comprising a sense strand and an antisense strand, n the sense strand ill comprises at least l5 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide setptence ot‘SEQ lD NOS and the antisense strand comprises at least l5 contiguous nucleotides differing by no l’ttOl‘C than '3 nucleotides from the nucleotide sequence of SEQ ll) N025, thereby inhibiting the expression of C5 in the subject. in another aspect, the present invention provides methods of inhibiting the expression of (35 in a t. The methods include administering to the subject a therapeutically etlective amount of a dsRNA agent comprising a sense strand and an antisense strand, the antisense strand comprising a region of complementarity which comprises at least l5 contiguous nucleotides differing by no more than 3 nucleotides from any one of the antisense sequences listed in any one of’l‘ables 3, 4, 5, 6, 18, l9, 20, El and 23, thereby inhibiting the expression of {.75 in the 2t) subj ect. ln another aspect, the present invention provides s of biting complement ent C5 expression in a subject which e administering to the t a therapeutically effective amount of a dsRNA agent comprising a sense strand and an antisense strand lorming a double stranded region, wherein the sense strand comprises at least l5 contiguous nucleotides ditlering by no more than 3 nucleotides from the nucleotide sequence of SEQ lD N'Ozl and the antisense strand comprises at least l5 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ll) N025, wherein substantially all oi‘the nucleotides of the antisense strand and substantially all of the nucleotides ofthe sense strand are modified nucleotides and, wl’ierein the sense strand is conjugated to one or more (A) c. s at the 3 ’—terminus, thereby inhibiting expression ofthe C5 gene in the subject, in one embodiment, all ofthe nucleotides ofthe sense strand and all ofthe nucleotides of the antisense strand are modified tides. ln one en'ibodiment, the administration is subcutaneous stration. ln one embodiment, substantially all of the tides ofthe sense strand are modified nucleotides ed from the group consisting of a Z’uO-methyl modification, a 2’-tluoro modification and a 3 ’~terminal dT nucleotide. ln another ernbtxliment, substantially all of the [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS nucleotides ofthe antisense strand are modified tides se ected from the group consisting of a 2’—O—methyl modification, a 2’—fluoro modification and a 3 "'-terminal dri‘ nucleotide. in another embodiment, the modified nucleotides are a short sequence ot‘deoxy~tlnyrnine (dT) nucleotides. in another embodiment, the sense strand comprises two ph osphorothioate ucleotide (I: linkages at the filterininus. in one embodiment, the antisense strand comprises two phosphorothioate internucleotide linkages at the 5’—terrninus and two phosphorothioate intermitcleotide es at the 3 ’~terminns In yet another embodiment, the sense strand is coniugated to one or more GalNAc derivatives attacliied through a hranched bivalent or trivalent linker at the 3 ’ntemiinust ll) in another aspect, the present invention provides methods of inhibiting the expression of CS in a subject. The methods include administering to the subject a therapeutically etfective amount of a dsRNA, agent comprising a sense strand mentary to an antisense strand, wherein the antisense strand ses a region complementary to part of an mRNA encoding CS, wherein each strand is about l4 to about 30 nucleotides in length, wherein the double stranded RNAi agent is represented by formula (l l l }: sense: 5' np —Na {X X X) i—Nb ~Y Y Y ~Nb —(Z Z 2% ~Na ~ n, 3‘ antisense: 3' iip'—Na’~(X’X'X'lyNfi—Y'Y’Yfll‘lbC(Z'Z’Z'h—Na'n nq' 5’ (ill) wherein: i, j k, and l are each independently O or l; 2t) p, p”, q, and q' are each independently (3-6; each N3 and )1; independently represents an oligonucleotide sequence comprising {#125 nucleotides which are either modified or unmodified or ations thereof, each sequence comprising at least two differently modified nucleotides; each Nb and Nb' independently ents an oligonucleotide sequence comprising 0~l 0 nucleotides which are either n'iodified or unmodified or con'ihinations the feof; each np, np', nq, and nq', each ofwhich may or may not be present, independently represents an overhang nucleotide; XXX, YYY, ZZZ, X’X'X’, Y'Y'Y', and Z'Z’Z' each independently represent one motif of three cal modifications on three consecutive nucleotides; (A) c. modifications on N, differ from the modification on Y and modifications on Nb' differ from the cation on ‘r”; and wherein the sense strand is conjugated to at least one ligand, thereby inhibiting the expression of CS in the subject. in one ment, the methods further include administration of an antincomplement component (:5 dy, or n—binding fragment f, to the subject.

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS in one embodiment, the antineomnieinent component C5 antibody, or antigenwbinding tragment f, is eeuiizumab. in one embodiment, the antibody, or antigen-binding fragment thereof, inhibits eieavage of complement component C5 into hiagments CSa and CSb.

(I: in one embodiment, the s ofthe invention further ineiude administering a meningoeoceai vaccine to the subject. hi one embodiment, eeuiizumab is administered to the subject weekiy at a dose Eess than about 600 mg for 4 weeks tot} owed by a fifth dose at about one week later of less than about 900 mg, foiiowed by a dose iess than about 900 mg about every two weeks thereafter. it) in another ment, eeuiizumab is stered to the subject weekiy at a dose less than about 900 mg for 4 weeks tbiiowed by a titth dose at about one week iater ot‘iess than about 1200 mg, folio“ 'ed by a dose iess than about tZGO mg about every two weeks therfafter. in one embodiment, the subject is iess than 18 years of age and ecuiizumab is administered to the t weekiy at a dose iess than about 900 mg for 4 weeks followed by a fifth dose at about one week Eater of less than about EZGG mg, tbiiowed by a dose iess than about i200 ing about every two w feks thereafter. in another embodiment, the subj eet is iess than 18 years of age and ecuiizumab is administered to the subject weekiy at a dose iess than about 600 mg for 2 ‘t reeks foiioy 'ed by a third dose at about one week iater ot‘iess than about 900 mg, foiiowed by a dose less than about 2() 900 mg about every two weeks thereafter, in one ment, the subject is iess than i8 years ot‘age and eeuiizumab is administered to the subject weekiy at a dose iess than about 600 mg for 2, weeks foiiowed by a third dose at about one week Eater of iess than about 600 mg, ed by a dose less than about 600 ing about every two weeks thereatter. in another embodiment, the subject is iess than i8 years ot‘age and eeuiizumab is administered to the subject weekiy at a dose iess than about 600 mg for 1 week foiiowed by a second dose at about one week later of iess than about 300 mg, foiiowed by a dose iess than about 300 mg about every two weeks thereafter. in yet another embodiment, the subject is less than 18 years of age and eeulizumab is (A) c. administered to the subject weekiy at a dose iess than about 300 mg for 1 week foiiowed by a second dose at about one week later of iess than about 399 mg, followed by a dose Eess than about 360 ing about every two weeks thereatter. in one diment, the methods further ineiudepiasmapheresis or plasma exeh ange in the subject. in one embodiment, uinab is administered to the subjeet at a dose iess than about 600 mg. In another embodiment, ecuiizumab is stered to the subject at a dose less than about 300 mg.

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS In one embodiment, the methods r incIude plasma infusion in the subject In one embodiment, eculizumab is administered to the subject at a dose Iess than about 300 mg.

In one embodiment, eculizumab is administered to the subject at a dose of about Gill rug/kg to about it) rug/kg or about 0.5 m g/kg to about IS mg/Rg. In another embodiment, (I: eeulizuniab is administered to the subject at a dose of about 5 mg/ix'g to about l5 mg/hg in another embodiment, eculizumab is. stered to the t at a dose seiected from the group ting ol‘iié mg/kg, l rng/itg, 1.5 rug/kg, 3 mg/kg, 5 mg/itg, 7 mg/itg, It) mg/kg, and 30 rug/kg.

In one embodiment, eculizumab is administered to the subject via an intravenous it) infusion. In another embodiment, eeuiizumab is administered to the t subcutaneousiy.

In one embodiment, the dsRNA agent is administered at a dose of about 0.01 rug/leg to about it) rug/kg or about 0.5 mg/hg to about i5 mg/kg. in one embodiment, the dsRNA agent is administered at a dose of about i0 nig/lrg to about 30 mg/leg. in another embodiment,tbe dsRNA agent is administered at a dose seiected from the group consisting ol‘iié mg/kg I rug/kg, I5 mg/kg, 3 mg/kg, 5 mg/kg, 10 rug/kg, and 3t) rug/kg.

In one embodiment, the dsRNA agent is administered to the subject once a week. In another embodiment, the dsRNA agent is administered to the subject twice a week, in another embodiment, the dsRNA agent is administered to the subject twice a month. 2t) In one embodiment, the dsRNA agent is administered to the subject subcutaneousiy.

In one embodiment, the dsRNA, agent and the ecuiizumab are adn’iinistered to the subject aneousiy, In another embodiment, the dsRNA agent and the eeuiizumab are administered to the subject simuitaneousiy, In one embodiment, the dsRNA agent is administered to the t first for a period of time sufficient to reduce the ievels of complement component C5 in tl’itj subject, and eculizumab is administered subsequently at a dose less than about 600 mg.

In one embodiment, the levels of complement ent CS in the subject are d by at Ieast about 30%, 35%, 46%, 45%, 500/” 6t %, 65%, 7()%, 75%, 80%, 85%, or 90%. , 55%, In one embodiment, eculizumab is administered at a dose of about 100—500 mg.

(A) c. in one embodiment, the dsRNA agent is conjugated to a ligand, in one embodiment, the ligand is conjugated to the 3 ’_ end ofthe sense strand of the dsRNA agent.

In one en'ibodiment the li trind is an N—acet 'i talaetosamine (GaiNAc' derivative.

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS Brief Beseriptiun of the Drawings Figure l is a schematic of the three complement pathways: alternattive, classical and lectin.

Figure 2 is a graph showing the percentage ofeomplernent component C5 ren'iaining in (I: CS7BL/6 mice following a single l0 mg/lrg dose of the indicated iRNAso Figure 3 is a graph showing the percentage of complement component (:5 remaining in CS7BL/6 mice following a single it} mg/lrg dose ot‘the indicated iRNAs.

Figure 4 is a graph showing the percentage of complement component CS remaining in C57BL/6 mice 4-8 hours after a single l0 g dose ofthe indicated iRNAs. ll) Figure 5A is a graph showing the percentage of hemolysis remaining at days 4 and 7 in rats after a single 2.5 mg/l-rg, it) mg/kg, or 25 mg/lrg utaneous dose ofofAD~5 8642.

Figure 53 is a W’estern blot showing the amount nplement eon‘iponent CS remaining at day 7 in rats after a single 25 mg/hg, l0 mg/lig, or 25 mg/leg subcutaneous dose of ALE—58642. l5 Figure 6A and (SB are graphs showing the percentage ol‘compiement component CS remaining in C57BL/6 mice 5 days alter a single £425 mg/kg, ’25 mg/kg, 5 mg/kg, l0 mg/kg or rug/leg close ofADn586420 Figures 7A and 7B are graphs showing the tage ofhemolysis remaining at day 5 in 357BL/6 mice after a single l.25 mg/kg, 2.5 mgr/kg, 5 mg/ls'g, l0 mg/kg or 25 nig/kg close of 2i) 42.

Figure 8 is a W’estern blot showing the amount of ment component C5 remaining at day 5 in CS7BL/6 mice after a single l°25 mg/lig, 2.5 mg/leg, 5 mg/leg, l0 mg/lig or 25 mg/lig close MAD-58642.

Figure 9 is a graph showing the amount ot‘compiement component CS protein remaining at days 5 and 9 in mouse serum after a single {30625 mg/kg, LES nig/lrg, 2.5 trig/leg, 5.0 mg/kg, or l0 rug/leg close ofAD—SSé-tl. The lower limit of quantitation (LLOQ) of the assay is shown as a dashed line.

Figure ll) is a is a graph showing the amount of complement component {35 protein remaining at clay 8 in mouse serum after a 0.625, trig/leg, lJZS rng/Rg, or 2.5 mg/kg dose oi‘AD— (A) c. 586% at days 0,, l? 29 and 3. The lower limit of quantitation (LLOQ) of the assay is shown as a dashed line.

Figures l lA and ill? depict the efficacy and cumulative effect of repeat administration pounrl AID—5 864i in rats. Figure llA is graph depicting the hemolytie activity remaining in the serum of rats on days 0, 4,, 79 ll., l4, i8, 25, and 32 after repeat administration at 2.5 mg/kg/dose/ or 5.0 mg/hg/dose, qu X3 (twice a week for 3 weeks). Figure l EB is a Westem blot [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS showing the amount of complement component C5 protein remaining in the serum of the Figure 12 is a graph showing the amount ol‘coniplenient coi’nponent CS protein in cynomolgus macaque 56111111 at various time points before, during and after two rounds of (I: subcutaneous dosing at 2.5 mg/kg or 5 mg/kg of AB~58641 every third day for eight doses. C5 protein levels were normalized to the average ofthe three preudose samples.

Figure l3 is a graph g the percentage olysis remaining in cynomolgus macaque serum at various time points “, during and after two rounds ot‘suhcutaneous dosing at 2.5 mtg/leg or 5 mg/ltg of Al3m5 8641 every third day for eight doses. Percent hemolysis ll) was calculated relative to maximal hemolysis and to background heinolysis in l samples.

Figure 14 is a graph showing the percentage ofcon'iplement component CS protein remaining at day 5 in the serum ol‘C5’7Bl_,/6 mice following a single l nig/kg dose of the ted iRNAs.

Figure 15 is a graph showing the percentage of complement component CS protein remaining at day 5 in the serum of CSTBL/ti mice following a single (3.25 nig/hg, 0.5 trig/leg, l.0 mg/hg, or 2.0 mg/kg dose otthe indicated iRNAs.

Figure in is a graph showing the percentage of complement component C5 protein remaining in the serum of C57BL/6 mice at days 6, l3, 20, 27,, and Fir-l following a single l mg/kg dose ofthe ted iRNAs. 2t) Figure 17' is a graph showing the percentage ofheniolysis remaining in rat serum at s time points ing administration of a 5 rug/leg dose of the indicated compounds at days 0, I—‘l, and 7‘ Figure 18A shows the nucleotide sequence of Homo sopi’cns Complement Component 5 {(35) (SEQ ED N():l ); l? i gure lSB shows the nucleotide sequence of Miran/3a malaria Con’iplement Component 5 (C5) {SEQ ll) N02); Figure. lSC shows the nucleotide ce. of Mars muscular Complement Component 5 {C5} (SEQ ll) N013); Figure lSD shows the nucleotide sequence f’lls norvegicus {Complement Component 5 (CS) (SEQ lD N024); Figure l 8E. shows the reverse complement of SEQ lD NOzl (SEQ IE3 NOZS); Figure till: shows the e complement ol’SEQ ll) N02 (SEQ ll) non); Figure lSG shows the reverse (A) c. complement of SEQ lfl‘,l N023 (SEQ ll) N017); and Figure l8H shows the reverse complement of SEQ lD NQz4 (SEQ 1D NQIS).

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS Betaiied fiescription ofthe invention The present invention provides iRNA agents which effect the RNA—induced silencing comp1ex {R18C1—mediated cleavage ot‘RNA tanscripts ofa inent component C5 gene.

The iRNAs ofthe invention include an RNA strand (the antisense strand) having a region (I: which is about 30 nuc1eotides or 1ess in length, cg), 15—311, 15—29, 15—28, 15—27, 15—26, 15—25, —24, 15—23, 15—22, 15—21, 15—2G,15—19,15—18,15—17,18-30,18—29 18—28,18—27, 18—26,18— , l8 2—4 18—2.2,18—22, 1821 18 211, 19—211, 1929,1928,19-27,l9—26,19—25,19—24,19—2 19—22, 19—21, 19—211, 20—30, 20—29, 211—28, 211—27, 211—26, 211—25, 20—242 —23, 20—22, 20—21, 21— , 21—29, 21—28, 21——27 , 21 26 21—25, 21—24, 21—23, or 21—22 nncieotides1111e11gth,which 111 region is snbstantiaily complementary to at 1east part of an mR’NA ript of a C5 gene. The use ofthese iRNAs enables the targeted degradation of 1nRNAs ot‘a C5 gene in rnamma1s. Very 1ow dosages ofCS iRNAs, in particu1ar, can specitica11y and etiicient1y mediate RNA interference (RNAi), resuiting in significant inhibition of expression of a C5 gene. The present ors have demonstrated that iRNAs targeting CS can mediate RNAi in vitro and in viva, resulting in significant inhibition of expression of a 1:35 gene. Thus, methods and compositions 1111111g1711686 iRNAs are osei‘hi for treating a subj ect who wou1d benefit by a reduction in the 1evcls and/or activity of a C5 protein, such as a subject having a comp1eincnt component C5— associated disease, such as paroxysmai nocturnai hr.mogiobinnria (PNH).

The present ion also provides methods and combination therapies for treating a 211 s’uhj ect having a disorder that would benefit horn inhibiting or redncing the expression of a C5 gene, rag a comp1enient component (35—associated disease, such as paroxysnia1 nocturna1 hemogiohinuria {1312111) and atypica1hcmo1ytic uremic syndrome (aHUS) using iRNA compositions which effect the RNA—induced si1encing compiex (1118C)—rne diated c1eavage of RNA transcripts ot‘a complement component C. gene The present invention a1so provides s for preventing at 1east one symptom, rag, henioiysis, in a subject having a disorder that would benefit from inhibiting or reducing the expression of a C5 gene, eg. a cornpiement component sociated disease, such as paroxysmal nocturnal heinog1obinuria (PNH) and atypicai herno1ytic uremic syndrome (2111118).

The present invention farther es iRNA compositions which effect the RNA—induced (A) c. si1encing compiex —mediated cleavage ofRDA ripts of a compicmr.>nt component C5 gene The CS gene maybe Within a cell, cg a ce11within a t, such as a human.

The combination therapies of the present invention inciude administering to a subject having a comp1en1ent ent {IS—associate disease, an RNAi agent of the invention and an additional therapeutic, such as anti—compiement ent C5 antibody, or n—binding nt t,e ecuiiznrnab hTe combination therapies of the invention reduce C5 1eve1s in the subject (cg, by about 311%, 35%, 411%, 45%, 501343,:533‘34) 611%, 65% 711%,75‘V8, 801343, [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS 85%, 90%, 95%, or about 99%) by targeting C5 mRNA with an iRNA agent ofthe invention and, accordingly, allow the therapeutically (or prophylactically) effective amount of eculizumah ed to treat the suhj ect to he reduced, thereby decreasing the costs of treatment and permitting easier and more convenient ways of administering eculizuinah, such as subcutaneous (I: administration.

The following detailed description discloses how to malre and use compositions ning iRNAs to inhibit the expression ol‘a CS gene, as well as compositions, uses, and methods for treating suhjects having diseases and disorders that would bfil’ltjfil trorn inhibition and/or reduction of the expression ofthis gene l. Definitions in order that the present invention may he more y understood, certain terms are first defined in on, it should he noted that er a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also ed to he part ot‘this invention.

The articles “a” and “an” are used herein to refer to one or to more than one (ten, to at least one) of the grammatical object ofthe e. Byway of example, “an element” means one element or more than one element, tag, a plurality of elements.

The term ”including" is used herein to mean, and is used interchangeably with, the phrase 2t) ”including but not limited to".

The term ”or” is used herein to mean, and is used l’iangeahly with“, the term "and/or,” unless context clearly indicates otherwisei As used herein, “complement component CS,” used interchangeably With the term “C5” refers to the vv’ell-ltnown gene and polypeptide, also known in the art as CPAl‘leél, C3 and P2P“ like alph a—Z «macroglohulin domain~containing protein, anaphtlatoxin C5 a analog, l’ltjl’flOlyllC complement (He), and complement (35‘ The sequence of a human C5 rnRNA transcript can he found at, for e, CienBank ion No. Glz380l6946 (Nh/l_lllll735.2; SEQ if) NOzl).

The sequence ofrhesus (:5 mRNA can he found at, for example, GenBanl-r Accession No. (312297270262 (Xh’l___00l0957502; SEQ TD N02). The sequence ofrnouse C35, rnRNA can he (A) c. found at, for example, k Accession No. Gl229 l 575 l7l (valgggdl 0406.2; SEQ TD N023) The sequence of rat (:5 mRNA can he found at, for example, k Accession No. (3163 92346248 (XM_345342.4; SEQ ID N614). Additional examples ot‘CS rnRNA sequences are readily available using publicly available databases, age, GenBank.

The tenn“C5,” as used herein, also refers to naturally occurring DNA sequence variations ofthe (:5 gene, such as a single nucleotide polymorphism in the C5 genei Numerous SNl’s within the CS gene have been identitied and may he found at, for example, NCBT thNP (see, ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS raga, ncbi.nlm.nih.gov/snp). Non—limiting examples of SNPs Within the CS gene may be found at, NQBT dbSN’l’ Accession Nos. rs121909588 and rsl219tl9587.

As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an D’IRNA n’iolecule fornie during the transcription ofa C5 gene, including mRNA (I: that is a product ofRNA processing of a primary transcription product, In one embodiment, the target portion ofthe sequence Will be at least long enough to serve as a substrate for iRNA— directed ge at or near that n ot‘the nucleotide sequence of an inRNA n'iolecule formed during the transcription of a C5 gene, The target sequence may be from about 9—36 nucleotides in length, eg, about 15—30 11.1 tides in length. For example, the target sequence can be from about 15—31) nucleotides, 15— 29,15—28,15—27,15—26, 15—25,15—24,15-23,15—22, l5—21, 15—21),15—19,lS—18,15—l7, l8—3ll, 18—29,18—28,18—27, 18—26,18—25,18—24,18—23, 18—22, 18—21,18—20,19—30,19—29, 1928,19— 27, 19—26, 19—25, 19—2/1, 19—23, 19—22, 19—21, 19—21), 20—30, 20—29, 20—28, 20—22, 20—26, 20—25, 2G—24,20—23, 2 —22, 211—21, 21—31), 21—29, '21—’18, 21—27, 21—26, 21—25, '21—’14, 21—23, or 21—22. nucleotides in length. Ranges and s intermediate to the above recited ranges and lengths are also contemplated to be part ofthe invention.

As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain ofnucleotides that is described by the sequence referred to using the standard nucleotide nomenclature. 211 “G,” “C,” “A,” “T” and “U” each generally stand for a nucleotide that contains guanine, ne, adenine, thyrnidine and uracil as a base, tively, Howey/er, it will be understood that the term “ribonucleotide” or “nucleotide” can also refer to a modified nuc , as thither detailed below, or a surrogate replacement moiety (see, eg Table 2). The skilled person is well at ’are that guanine, ne, adenine, and uracil can be replaced by other es without ntially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For e, without limitation, a nuc eotide comprising inosine as its base can base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine can be replaced in the nucleotide sequences of dsRNA leatured in the invention by a nucleotide containing, for example, inosine ln another (A) c. example, adenine and ne re in the oligonucleotide can be replaced with guanine and uracil, respectively to form 13-1} \I‘v’obble base pairing With the target mRNA. Sequences containing such ement es are suitable for the compositions and s ed in the invention.

The terms “iRN’A”, “RNAi agent,” “iRNA agent,”, “RNA interference agent” as used interchangeably herein, refer to an agent that contains RNA as that term is defined herein, and which mediates the targeted cleavage of an RNA transcript Via an RN A—induced silencing [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS complex (RISC) pathway, iRNA directs the sequencensnecific degradation ofmRNA through a process known as RNA erence (RNAi). ’l'he iRNA modulates, cg inhibits, the expression of CS in a cell, eg a cell Within a subject, such as a mammalian subject. in one emhodin'ient, an RNAi agent oi’the invention inchides a single stranded RNA that (I: interacts with a target RNA sequence, e.g., a C5 target niRNA sequence, to direct the ge of the target RNA. Without g to be bound by theory it is believed that long double stranded RNA introduced into cells is broken down into siRNA by a Type lll endonuclease known as Dicer (Sharp er of. (ZOOl) Genes Dev, l5:485). Dicer, a ribonuclease~lll~lihe enzyme, processes the dsRNA into 1923 base pair short ering RNAs with characteristic two base 3' overhangs ltl tein, at all, {2209i} Nature 499:3 63’). The siRNAs are then orated into an RNA" induced silencing complex (RiSC) where one or more heiicases unwind the siRNA duplex, enabling the ementarfyr nse strand to guide target recognition (Nykanen, et all, (200D Cell l072309). Upon binding to the appropriate target mRNA, one or more endonucleases Within the RlSC cleave the target to induce silencing (Blhashir, er of), (200D Genes Dev. 15388) Thus, l5 in one aspect the invention relates to a single stranded RNA {siRNA) generated within a cell and which promotes the formation oi’a RlSC complex to effect silencing of the target gene, ie. a C5 gene. Accordingly, the term “siRNA” is also used herein to refer to an RNAi as described above. in another embodiment, the RNAi agent may he a singleustranded siRNA that is 2() introdu ‘ed into a cell or organism to inhibit a target rnRNA. Single—stranded RNAi agents bind to the RlSC clease, Argonaute ’2, which then cleaves the target mRNA. The sin gle— stranded siRNAs are generally l5n30 nucleotides and are chemically modified. The design and testing of single-stranded siRNAs are described in US. Patent No. 8,l01,348 and in Lima er (2]., {2612) Cell lSt'): 883-894, the entire contents oi‘each ofwhich are hereby incorporated herein by reference, Any ot‘the nse nucleotide sequences described herein may he used as a single stranded siRNA as descrihed herein or as chemically modified by the methods described in Lima 92‘ 925., (ZOlZ)(L’eii'l15il;:883—894. in another embodiment, an “iRNA” for use in the compositions, uses, and methods ot‘the invention is a double—stranded RNA and is referred to herein as a “double stranded RNAi agent,” (A) c. “douhlenstranded RNA ) molecule,” “dsRNA agent,” or “dsRNA”. The term ”, refers to a complex of cleic acid molecules, having a duplex structure sing two anti- el and substantially complementary nucleic acid strands, referred to as having “sense” and “antisense” orientations with respect to a target RNA, ten, a CS gene in some embodiments of the invention, a douhlenstranded RNA (dsRNA) triggers the degradation of a target RNA, C5;, an mRNA, through a post-transcriptional gene—silencing niechanisni referred to herein as RNA interferon ‘e or RNAi.

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ln l, the majority of nucleotides of each strand of a dsRNA molecule are cleotides, but as described in detail herein9 each or' both strands can also include one or more non~ribonucleotides9 6.55., a deoxyrihonucleotide and/or a modified nucleotide. in addition, as used in this Specification, an “RNAi agent” may include ribonucleotides with chemical (I: modifications; an RNAi agent may include substantial modifications at multiple nucleotides.

Such modifications may include all types ofrnodifications disclosed herein or known in the art.

Any such modifications, as used in a siRNA type molecule, are encompassed by “RNAi agent” for the purposes of this specification and claims.

The duplex region may he of any length that permits c degradation of a desired ll) target RNA h a RlSC pathway, and may range from about 9 to 36 base pairs in length, cg, about l5*3() base pairs in length9 for example9 about 99 ll), ll, l2, l39 l4, l59 l6, l7, l8, l9, 209 2l9 229 239 249 259 269 279 289 299 5%, 3i, 32 33 349 359 or 36 hase pairs in length, such as about15809l5—29915~289l5w279 l5269 l5n259l5—249l5239 l5—229 l5~2O915~l99l5w 18,15ul79l8—30912—9, l8-28, 18—279 l8 2—69 l88—259 l82,4 l8—23, 1822 ill—219184209 l9-309 l5 l9—299 l9s289 l9s279 l9~226, l9—259 19249 l92239 l922, l9~2l, l9 ~26, 203t), 2t)29 2t)28, 20- 279 2326, 20259 2024920239 20229 20—2l9 2l 309 2l 299 2l 289 2l—279 2l ~926 2l 259 2l 249 2i ~239 or 2l—22 base pairsin length. Ranges and lengths intermediate to the ahoye recited ranges and lengths are also contemplated to be part ofthe invention.

The two strands g the duplex structure may he different portions ofone larger 2t) RNA molecule9 or they may be separate RNA molecules. Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain ot‘nucleotides between the 3'end of one strand and the 5end of the respecetiye other strand forming the duplex structure9 the connecting RNA chainis referred to as a “hairpin loop.” A hairpin loop can comprise at least one unpaired nucleotide. in some embodiments9 the n loop can comprise at least229 atttleast 39 at least 49 at least 59 at least 69 at least 79 at least 89 at least 99 at least ltl, at least 269 at least 23 or more unpaired tides.

Where the two substantially complementary s of a dsRNA are comprised by separate RNA molecules, those molecules need not, hut can he covalently connected. Where the two strands are connected ntly hy means other than an uninterrupted chain ofnucleotides between the 3 lend of one strand and the 5 ”end of the respective other strand forming the duplex structure9 the connecting structure is ed to as a “linker.” The RNA strands may have the same or a different “ ofnucleotides. The maximum number ofbase pairs is the number of nucleotides in the shortest strand ofthe dsRNA rninus any overhangs that are present in the duplex. In on to the duplex structure, an RNAi may comprise one or more nucleotide overhangs.

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS in one embodiment, an RNAi agent ofthe invention is a dsRNA of2460 nucleotides that interacts with a target RNA sequence, eg, a (:5 target niRNA sequence, to direct the cleavage of the target RN A. Without wishing to he bound by , long double stranded RNA introduced into cells is hrolien down into siRNA by a Ty e lll endonuclease known as Dicer (Sharp at (1!.

(I: (200i) Genes Dev. 5:485). Dicer, a ribonucleasemllhlilre enzyme, processes the dsRNA into lg" '23 base pair short interfering RNAs with characteristic two base 3' overhangs (Bernstein, er a[., {260m Nature 469363) The siRNAs are then incorporated into an RNA-induced ing complex (REC) where one or more helicases unwind the siRNA , enabling the complenientaijvr antisense strand to guide target recognition (Nylianen, et all, {200i} Cell ll) M71309). Upon hinding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (lilhashir, at at, (20%) Genes Dev. l5zl 88) As used herein, the term “nucleotide ng” refers to at least one unpaired nucleotide that protrudes from the duplex structure of an iRNA, cg, a dsRNA. For example, when a 3‘nend of one strand of a dsRNA extends beyond the 5'—end of the other strand, or vice term, there is a nucleotide overhang. A dsRNA can comprise an overhang of at least one nucleotide; alternatively the overl’iang can comprise at least two tides, at least three nucleotides, at least four nucleotides, at least live nucleotides or more. A nucleotide overhang can se or consist of a nucleotide/nucleoside analog, including a dcoxynucleotide/nucleoside. ’l‘he overhangts) can he on the sense strand, the antisense strand or any combination thereof. 2t) Furthermore, the nucleotide(s) or“ an overhang can he present on the 5’—end, 3'-end or both ends of either an antisense or sense strand of a dsRNA, ln one embodiment, the antisense strand of a dsRNA has a lnltl nucleotide, eg. a l, 2, 3, 4, 5, 6, 7, 8, 9, or ll) nucleotide, overhang at the 3 ’—end and/or the 5’—end. in one enihodiment, the sense strand ot‘a dsRNA has a lell) nucleotide, eg, a l, 2, 3, 4, 5, 6, '7, 8, 9, or it) nucleotide, overhang at the 3 '—end and/or the S’~end.- in another embodiment, one or more ofthe nucleotides in the overhang is replaced with a nucleoside thiophosphate.

“Blunt” or “blunt end” means that there are no ed nucleotides at that end of the double standed RNAi agent, i.e., no nucleotide overhang. A “blunt ended” RNAi agent is a dsRNA that is double~stranded over its entire length, is, no tide overhang at either end of (A) c. the molecule. The RNAi agents ofthe invention include RNAi agents with nucleotide overhangs at one end (is, agents with one overhang and one blunt end) or with tide overhangs at both ends.

The term “antisense strand” or "guide strand" refers to the strand of an iRNA, tag, a dsRNA, which includes a region that is substantially complementary to a target sequence, eg, a :5 mRNA. As used herein, the term “region of mentarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS sequence, cg, a C5 tide sequence, as defined herein Where the region of complementarity is not fully complementary to the target sequence, the mismatches can be in the internal or terminal regions ofthe molecule. Generally, the most ted mismatches are in the al regions, 62g, within 5, 4, 3, or 2 nucleotides of the 5"~ and/or 3 9—terminus of the iRNA.

(I: The term “sense strand,” or nger“ strant " as used herein, refers to the strand of an iRNA that includes a region that is substantially complementary to a region ofthe antisense strand as that term is defined .

As used herein, the term “cleavage region” reteis to a region that is located immediately adjacent to the cleavage site. The cleavage site is the site on the target at which cleavage occurs, ll) ln some embodiments, the ge region comprises three bases on either end of, and immediately adjacent to, the cleavage site. ln some embodiments, the ge region comprises two bases on either end of, and immediately adjacent to, the cleavage site. in some embodiments, the cleavage site specifically occurs at the site bound by tides l0 and ll of the antisense strand, and the cleavage region ses nucleotides ll, l2 and 13.

As used herein, and unless otherwise indicated, the term “cornplementar‘y,” when used to describe a first nucleotide sequen e in relation to a second nucleotide Sthlfil’lCC, reters to the ability of an oligonucleotide or polynucleotide comprising the first tide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or~ polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled 2t) person. Such conditions can, for example, be stringent conditions, Where stringent conditions can include: 400 mlvl NaCl, 40 lel PIPES pill 6.4, l inM EDTA, 500C or 70°C for l2~l6 hours followed by washing (see, rag “Molecular Cloning: A Laboratory Manual, Samhroolc, er a}, if l989) Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically relevant conditions as can be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test ofcomplementarity oftwo sequences in accordance with the ultimate application of the hybridized nucleotides.

Complementary ces Within an iRNA, 9.53., Within a dsRNA as described herein, e base~pairing ofthe oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over (A) c. the entire length of one or“ both nucleotide sequences Such sequences can be referred to as “fully complementary” With respect to each other herein. However, Where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they can form one or more, but generally not more than 5, 4, 3 or 2 mismatched base pairs upon hybridization for a duplex up to 30 base pairs, While ing the ability to hybridize under the conditions most relevant to their ultimate application, cg, inhibition of gene expression via a RlSC pathway. However, Where two oligonucleotides [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches With regard to the determination of complementarity. For example, a dsRNA comprising one oiigonucleotide 2l nucleotides in length and another oligonucleotide '23 nucleotides in length, n the longer oligonucleotide ses a (I: sequence off/ll nucleotides that is fully complementary to the shorter oligonucleotide, can yet be referred to as “fully complementary” for the purposes described herein.

“Con'iplenientary” sequences, as used herein, can also include, or he iormed entirely from, [1&deatson—Cri clc base pairs and/or base pairs formed from tural, and modified nucleotides, in so far as the above requirements with t to their ability to hybridize are ll) ed. Such non—Watson—{Trick base pairs include, but are not limited to, Gzll Wobble or lloogstein base pairing.

The terms “complernentary,” “fully complementary” and “substantially complementary'” herein can be used with respect to the base ng between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of an iRNA agent and a target sequence, as l5 will be understood born the context of their use.

As used herein, a polynucleotide that is “substantially complementary to at least part ol” a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous n ofthe mRNA of interest (sign, an mRNA encoding C5), For example, a polynucleotide is complementary to at least a part ol‘a C5 mRNA if the ce is substantially 2t) mentary to a IlOYlellllSlTllplt—Ed portion of an rnRNA encoding CS. hi general, the majority of nucleotides ot‘each strand are ribonucleotides, but as described in detail herein, each or both strands can also include one or more non~ribonucleotidcs, eg, a deoxyribonucleotide and/or a modified nucleotide. ln addition, an “iRNA” may include ribonucleotides with chemical modiii rations. Such modifications may include all types of modifications disclosed herein or known in the art, Any sucl’i modifications, as used in an iRNA molecule, are encompassed by “iRNA” for the purposes ofthis specification and claims. in one aspect ofthe invention, an agent for use in the methods and compositions of the invention is a single—stranded nse RNA molecule that inhibits a target mRNA via an antisense inhibition mechanism, The single~stranded antisense RNA le is complen'ientaiy (A) c. to a sequence Within the target mRNA. The singlenstranded antisense ucleotides can inhibit translation in a stoichiometric manner by base pairing to the mRNA and physically obstructing the translation machinery, see Dias, N. at all, (2002) Mr)! Cancer Ther l:347—355.

The sin gle—stranded nse RNA, molecule maybe about l5 to about 30 nucleotides in length and have a sequence that is complementary to a target sequence, For example, the single" stranded antisense RNA molecule may comprise a sequence that is at least about l5, l6, l7, l8, [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS l9, 20, or more contiguous nucleotides from any one ofthe antisense sequences described herein, The term “lipid nanoparticle” or “LN P” is a vesicle comprising a lipid layer encapsulating a pl’iarmaceutically active molecule, such as a nucleic acid molecule, eg, an iRNA (I: or a plasmid from which an iRNA is transcribed, LNl’s are described in, for example, US.

Patent Nos. 6,858,225, €i,8l5,432, 8,l 58,601 and 8,058,969, the entire contents ofwhich are hereby o “ated herein by re terence.

As used herein, a “subject” is an animal, such as a mammal, including a prii'nate (SllCl’l as a human, a non~human e, rag, a monkey, and a chimpanzee), a non—primate (such as a ll) cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, a horse, and a whale), or a bird (8.53., a duck or a goose). in an embodiment, the subject is a human, such as a human being treated or assessed for a disease, disorder or condition that would benefit from reduction in C5 expression; a human at risk for a e, disorder or condition that would benefit from reduction in CS expression; a human having a disease, disorder or condition that would benefit from reduction in C5 expression; and/or human being treated for a disease, disorder or condition that would benetit from ion in C5 expression as described herein.

As used , the terms ing” or “treatment” re fer to a beneficial or desired result including, but not d to, alleviation or ration of one or more symptoms associated 2t) with unwanted complement y activation tag heinolysis and/or chronic inllanunation); shing the extent of unwanted con’iplement pathway activation; stabilization (la, not worsening) ofthe state of chronic inflammation and/or hemolysis; amelioration or tion of unwanted complement pathway activation (9.53., c inflammation and/or hemolysis) whether able or undetectable. ”Treatment” can also mean prolonging al as compared to expected survival in the absence ot‘treatinent.

The term “lower” in the context ofthe level of a complement component C5 in a subject or a disease marker or symptom refers to a statistically significant decrease in such level. The decrease ‘an be, for example, at least 10%, at least l5%, at least 20%, at least 25%, at least 300/” at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at (A) c. least 70%, at least 75%, at least 8 %, at least 859/ at least 90%, at least 95%, or more and is preferably down to a level accepted as within the range ofnormal for an individual without such disorder.

As used herein, “pre 'ention” or “preventing, 9'" when used in nce to a disease, disorder or condition thereof, that would benefit from a reduction in expression of a C5 gene, refers to a reduction in the likelihood that a subject will develop a symptom associated with such a disease, disorder, or condition, eg a symptom ofunwanted ment activation, such as a [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS chronic inflammation, hemolysis and/or thrombosis, The likelihood of developing a thrombosis is reduced, for example, When an individual having one or more risk factors for a thrombosis either fails to develop a thrombosis or develops a thrombosis with less severity relative to a population having the same risk, factors and not receiving ent as described herein. The (I: failure to develop a e, disorder or condition, or the reduction in the pment of a symptom associated With such a disease, disorder or condition (egg, by at least about 10% on a ally accepted scale for that disease or disorder}, or the exhibition yed symptoms delayed (cg, by days, weeks, months or years) is considered effective prevention.

As used herein, the term "complement ent CSnassociated disease” is a disease or it) er that is caused by, or associated With complement activation. Such diseases are typically associated with inflammation and/or immune system activation, cg, ne attach comples~ mediated lysis, anaphylaxis, and/or hernolysis. Non—limiting examples of con’iplement component C5~associated diseases include paroxysmal nocturnal hemoglobinuria (P‘NH), atypical tic uremic syndrome (allUS), asthma, rheumatoid arthritis (RA); antiphospholipid antibody syndrome; lupus nephritis; iia—reper'liision injury; typical or infectious hemolytic uremic syndrome (tlrlUS); dense t disease (DEB); neuromyelitis optica (Nit/l0); multifocal motor neuropathy (MR/EN); multiple sclerosis (MS); macular degeneration (eg, age~related macular degeneration {Ah/111)); hemolysis, elevated liver s, and low platelets (HELL?) syndrome; otic thrombocytopenic a (’l7’l‘l’); 2t) spontaneous fetal loss; Pauci~imniune vasculitis; epiderrnolysis bullosa; recurrent fetal loss; pre- eclampsia, traumatic brain injury, myasthenia gravis, cold agglutinin disease, dermatomyositis bullous pemphigoid, Shiga toxin E. coli—related hemolytic uremic syndrome, C3 pathy, anti—neutrophil cytoplasmic antibody-associated vasculitis (cg, granulomatosis with polyangiitis {previtnisly lenown as ‘Negener granulornatosis), ChurgStrauss syndrome, and microscopic giitis), ioral and vascular transplant rejection, graft dysfunction, myocardial infarction (gig, tissue damage and ischemia in myocardial tion), an allogenic transplant, sepsis (33:, poor outcome in sepsis), Coronary artery disease, dermatomyositis, Graves disease, atherosclerosis, Alzheimer’s disease, systemic inflammatory response sepsis, septic shock, spinal cord injury, glomeiulonephritis, llashinioto‘s thyroiditis, ty e l diabetes, psoriasis, pemphigus, (A) c. autoimmune hemolytic anemia (AlHA), l'l‘l’, Goodpasture syndrome, Degos disease, antiphospholipid syndrome (APSE), catastrophic Al’S (CAPS), a cardiovascular disorder, myocarditis, a cerebrovascular disorder, a peripheral (egg, n'iusculoslreletal} vascular disorder, a renovascular er, a eric/enteric vascular disorder, vasculitis, Henoch—Schonlein purpura nephritis, systemic lupus matosus—associated vasculitis, vasculitis associated with rheumatoid arthritis, immune complex vasculitis, ’l7alrayasu's disease, d cardiomyopathy, diabetic angiopathy, Kavvasalri’s e (arteritis), venous gas embolus (VGEE), and restenosis [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS following stent ent, rotational atherectomy, mernbraneous nephr‘opathy, Guillain~l3arre syndrome, and percutaneous transluminal ry angioplasty A) ifsee, eg, llolers (2008) immunological Reviews 2'23 :3006 l 6; llolers and Thurman (2004) Molecular immunology 41347—152; US. Patent Publication No. 2007M 72483).

(I: ln one embodiment, a complement component Cfsnassociated disease is paroxysmal nocturnal obinuria (E’Nll). The Pl‘ll’l may be classical l or' PNltl in the setting of another bone marrow e syndrome and/or myelodysplastic syndromes (MDS), cg, cytopenias, In r embodiment, a complement con'iponent CS «associated disease is atypical hemolytic uremic syndrome (allUS). ll. iRNAs of the invention The p resent invention provides iRNAs which inhibit the expression of a con’iplement ent C5 gene. in one embodiment, the iRNA agent includes double~stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of a CS gene in a cell, such as a cell within a subject, cg, a mammal, such as a human having a complement ent (ZS-associated disease, rag” PNll. The dsRNA includes an antisense strand having a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of a C5 gene, The region of complementarity is about 30 nucleotides or less in length (cg, about 30, 29, 28, 27, 26, 25, 24, 23, 22, El or l8 nucleotides or' less in length). Upon contact with a cell , 20, 19, 2t) expressing the CS gene, the iRNA inhibits the expression ofthe CS gene (eg, a human, a primate, a non—primate, or a bird C5 gene) by at least about lilo/6 as d by, for exan'iple, a PCR or~ branched DNA hased method, or by a proteinwbased method, such as by irnmunotluor‘escence analysis, using, for example, Western Blotting or tlowcytometric techniques.

A dsRNA includes two RNA strands that are complen‘ientary and idize to form a duplex structure under conditions in which the dsRNA will be used, One strand of a dsRNA (the antisense strand) includes a region of complementarity that is substantially mentary, and generally fully complementary, to a target sequence. The target sequence can be derived from the sequence of an D’IRNA torme during the expression ot‘a CS gene, The other strand (the (A) c. sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. As described ere herein and as known in the art, the complementary sequences ofa dsRNA can also be contained as seltlcomplementary regions ot‘a single nucleic acid i'nolecule, as opposed to being on separate oligonucleotides.

Generally, the duplex structure is between 15 and 30 base pairs in length, eg, between, ~29,l5-28,l5-27,l5—26,15~25,l5~24,l5-23,15—22,l5w2l,15~20,l5-l9,l5—18, l5—l7’,l8* [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS 29,18—28,18—27, 18—26.,18—25, 18—24, 18—23, 18—22, 18—21,18—20,19—30,19—29, 19—28, 19—27, 19—26, 19—25, 19—24, 19— 3, 19—22., 19—21, 19—20, 211—311, 20—29, 20—28, 20—27, 20—26, 211— , 21)—24,211—23, 20—22, 20—21 2 , 21—30, 21—29, 21—28, —2'7, 21 —26, 21—25, 21—24, 21 —23, or 21—22 base pairs in . Ranges and 1en gths intermediate to the above recited ranges and 1engths are (I: a1so ipiated to be part ofthe invention.

Simi1ariy, the region of complementarity to the target sequence is between 15 and 30 nuc1eotides in 1ength, sag, between 15—29, 15—28, 15—27, 15—26, 15—25, 15—24, 15—23, 15—22, 15— 21, 15—20,15—19,15—18, 15—17,18—30,18—29,18—28, 18—27, 18—26, 18—25,18—24,18—23, 18—22, 18—21, 1, 19—30, 19—29, 19—28., 19—27, 19—26, 19—25, 19—24, 19—23, 19—22, 19—21, 19—20, 20— 311, 20—29, 20—28, 20—27, 211—26, 211—25, 20—24,2(1—23, 20—22., 20—21, 21—30, 21—29, 2.1—2.8, 2.1—2.7, 21 —26, 21 —25, 21 —24, 21—23, or 21—22 nueieotides in 1ength. Ranges and 1engths intermediate to the above recited ranges and 1engths are a1so oonternpia‘ted to be part of the invention. 1n some embodiments, the dsRNA is between about 15 and about 20 nucieotides in 1ength, or between about 25 and about 31) nue1eotides in 1ength. 1n genera1, the dsRNA is 1ong enough to serve as a substrate for the Dicer . 11or exampEe, it is we11—1<nown in the art that dsRNAs 1onger than about 21—23 nuc1eotides in 1engt1i may serve as substrates for Dieer. As the riiy shiiied person wi11 a1so recognize, the region of an RNA targeted for eietvage wi11 most often be part of a Larger RNA mo1eeu1e, ofien an niRNA nio1ecuie. Where nt, a “part” of an niRNA target is a contiguous sequence of an rnRNA target of sufficient 1ength to a11ow it to he a substrate for RN Ai—directed ge (1.95., c1eavage through a 1118C pathway).

One of ski11 in the art wii1 aiso recognize that the dup1ex region is a y function a1 portion ofa dsRNA, eg, a duniex region of about 9 to 36 base pairs, eg, about 19—36., 11—36, 12—36,13—36,14—36, 15—36, 9—35,10—35,11—35,12—35,13—35,14—35,15—35, 9—34,10—34,11—34, 12—34,13—34,14—34, 15—34, 9—33,10—33,11—33,12—33,13—33,14—33,15—33, 9—32,10—32,11—32, 12—32,13—32,14—32, 15—32, 9—31,,10—31,11—31,12—31,13—32,14—31,15—31,15—30,15—29,15—28, —27,15—26,15—25,15—24,15—23.,15—22,15—21,15—20,15—19,15—18,15—17,18—30,18—29,18— 28,18—27,18—26,18—25, 18—24,18—23, 18—22, 18—21, 18—211, 19—311,19—29,19—28,19—27, 19—26, 19—25, 19—24, 19—23, 19—22, 19—21, 19—20, 20—30, 20—29, 211—28, 20—27, 20—26, 20—25, 20—2420— 23, 20—22, 2 .—21 21 —27, 21 ~26, 21—25, 21—24, 21 —23, or 21—22 base pairs. ,, 21—30, 21—29, 21—28, Thus, in one embodiment, to the extent that it becomes processed to a ninetionai duniex, of 9.5.3:, —30 base pairs, that s a desired RNA for e1eavage, an RNA mo1ecu1e or compiex ofRNA mo1ecu1es having a dupiex region greater than 311 base pairs is a dsRNA. Thus, an ordinariiy ski11ed artisan wi11 recognize that in one embodiment, a miRNA is a dsRNA,, 1n another embodiment, a dsRNA is not a natura11y occurring miRNA. 1n r embodiment, an iRNA agent usefui to target (:5 sion is not generated in the target ce11by c1eavage of a 1arger dsRNA. ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS A dsRNA as described herein can further include one or more singlenstranded nucleotide overhangs (is; l, '2, 3, or 4 nucleotides. dsRNAs having at least one nucleotide overhang can have unexpectedly superior inhibitory properties relative to their bluritsended counterparts. A nucleotide overhang can se. or t of a nucleotide/nucleoside analog, including a (I: deoxynucleotide/nucleoside. The overhangt‘s) can be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucleotidets) of an overhang can be present on the 5'- end, 3'-end or both ends ofeither an antisense or sense strand of a dsRNA.

A dsRNA can be synthesized by standard methods known in the art as further discussed below, rag, by use of an automated DNA synthesizer, such as are commercially ble from, ll) for example, Biosearch, Applied Biosystems, inc. iRNA compounds ofthe invention may be prepared using, a twosstep procedure. First, the individual strands of the double—stranded RNA molecule are pre ared se arately, Then, the component strands are annealed. The dual s of the siRNA compound can be prepared using solutionuphase or solid—phase organic synthesis or both. Organic synthesis offers the advantage that the oligonucleotide strands comprising unnatural or modified tides can be easily prepared, —stranded oligonucleotides of the ll’thfil’lliOH can be ed using solutionnphase or solid—phase organic synthesis or both, in one aspect, a dsRNA of the invention includes at least two tide sequences, a sense sequence and an anti-sense sequence. The sense strand is selected from the group of 2t) ces provided in any one ol‘Tables 3, 4, 5, 6, l8, l9, 2t), 2l and 23, and the corresponding antisense strand ol the sense strand is selected front the group ofsequences of any one ofTables 3, 4, 5, 6, l8, l9, 20, El, and 23, ln this aspect, one ofthe two sequences is complementary to the other oftlie two sequences, With one ofthe sequences being substantially complementary to a sequence ofan rnRNA generated in the sion of a (35 gene. As such, in this aspect, a dsRNA will include two oligonucleotides, where one oligonucleotide is described as the sense strand in any one of Tables 3, 4, 5, 6, l8, l9, 2t), 2t and 23, and the second oligonuc eotide is described as the corresponding antisense strand of the sense strand in any one of Tables 3, 4, 5, 6, l8, ll), '20, 2i and 23. in one embodiment, the substantially complementary sequences oi‘tlie dsRNA are contained on separate oligonucleotides. in another embodiment, the substantially (A) c. complementary sequences ofthe dsRNA are contained on a single oligonucleotide. it will be understood that, although some ofthe sequences in Tables 3, 4, 5, 6, l8, l9, 20, 2l and s3 are described as modified and/or conjugated sequences, the RNA ot‘tlie iRNA of the invention tag, a dsRNA ot‘the invention, may comprise any one ofthe sequences set forth in Tables 3, 4, 5, 6, l8, i9, 20, El and 23 that is unnniodified, un—coniugated, and/or modified and/or coniugated ently than described therein.

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS The skilled person is well aware that dsRNAs having a duplex structure ofbetween about and 23 base pairs, eg, 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbasliir er a!., Erlei’BO ZGGl , 205877-6888). However, others have tound that shorter or longer RNA duplex structures can also be ive (Chu and Rana (2007) RPM (I: l4-zl H44 719; Kim at al. (2905) Not Biotech 231222—226), ln the embodiments described above, by virtue ofthe nature ofthe ucleotide sequences provided in any one of Tables 3, 4, 5, 6, l8, 19, 2t), 2l and '23, dsRNAs described herein can include at least one strand ol‘a length of n'iinirnally Zl nucleotides. lt can be reasonably expected that shorter duplexes having one ofthe sequences of any one ofTables 3, 4, 5, 6, l8, l9, 20, El, and 23 minus only a few nucleotides on ll) one or both ends can be similarly ef‘ective as compared to the s described above. llence, dsRNAs having a sequence of at least l5, lo, l7, l8, l9, 20, or more contigutnis nucleotides derived from one ofthe sequences ofany one ofTables 3 4, 5, 6, lit, 19, ’20, 2l and 23, and differing in their y to inhibit the expression of a C5 gene by not more than about 5, l0, l5, , 25, or 30 % inhibition front a dsRNA sing the full sequence, are contemplated to be within the scope ofthe present ion. hi addition, the RNAs provided in any one oflables 3, 4, 5, 6, l8, l9, 20, El and 23 identify a site(s) in a CS ript that is susceptible to RlSC~niediated cleavage. As such, the present invention further features iRNAs that target within one ofthese sites. As used herein, an iliNA is said to target within a particular site of an RNA transcript ifthe iRNA promotes 2t) cleavage ofthe transcript anywhere within that particular site. Such an iRN A will generally include at least about l5 contiguous nucleotides from one ofthe ces provided in any one of ’l‘ables 3, 4, 5, 6, l8, l9, 20, El, and 23 coupled to onal nucleotide sequences talten from the region contiguous to the selected sequence in a C5 gene.

While a target sequen ‘e is lly about l5-3 {3 nucleotides in length, there is wide variation in the suitability of particular sequences in this range for directing cleavage of any given target RNA. s soft\ 'are packages and the ines set out herein provide guidance for the identification of optimal target sequences for any given gene target, but an empirical approach can also be taken in which a “window” or “mask” ofa given size (as a non—limiting example, 2i nucleotides) is literally or figuratively (including, cg, in silico) placed on the target (A) c. RNA sequence to identify sequences in the size range that can serve as target sequences. By moving the sequence “window” progressively one nucleotide upstream or downstream of an initial target sequence location, the next potential target sequence can be identified, until the complete set ot‘possible sequences is identified for any given target size selected. This process, coupled with systematic synthesis and testing ofthe identified sequences (using assays as described herein or as known in the art) to identify those sequences that m optimally can ty those RNA sequences that, when targeted with an iRNA agent, mediate the best ation] DPS None set by DPS [Annotation] DPS ionNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS inhibition oftarget gene expression. Thus, while the sequences identified, for example, in any one of’l‘ables 3, 4, 5, 6, l8, l9, 20, El and 23 represent effective target sequences, it is contemplated that titrther optimization of tion elliciehcy can he ed by progressively “walking the window” one nucleotide upstream or downstream of the given sequences to (I: identify ces with equal or better inhibition characteristics Further, it is contemplated that for any sequence identified, eg, in any one of’l'ables 3, 4, 5, 6, l8, l9, 2t), 2l and 23, further optimization could be achieved by systematically either adding or removing nucleotides to generate longer or shorter seqirtences and testing those sequences generated by walking a window of the longer or shorter size up or down the targe ll) RNA from that point. Again, coupling this approach to generating new candidate targets with testing for eftiectivehess ot‘iRNAs based on those target sequences in an inhibition assay as known in the art an d/or as described herein can lead to thither ements in the efficiency of inhibition. Further still, such optimized sequences can be adjusted by, tag, the introduction of modified nucleotides as described herein or as known in the art, addition or changes in overhang, l5 or other modifications as known in the art and/or discussed herein to titrther optimize the molecule (rag, increasing serum stability or circulating llife, increasing thermal, stability, enhancing transmembrane delivery, targeting to a particular location or cell type, increasing interaction with silencing pathway enzymes, increasing release from endosonies) as an expression inhibitor. 2t) An iRNA as bed herein can contain one or more ches to the target sequence. ln one embodiment, an iRNA as described herein contains no more than 3 misn’iatches, lfthe antisense strand of the iRNA contains mismatches to a target sequence, it is preferable that the area of mismatch is not located in the center of the region of complementarity. if the antisense strand ofthe iRNA contains mismatches to the target sequence, it is preterable that the mismatch be restricted to be within the last 5 nucleotides from either the 5’— or 3 '—end ol’the region of- complementarity, For example, for a 23 nucleotide iRNA agent the strand which is complementary to a region of a (35 e, generally does not contain any mismatch within the central l3 nucleotides. The methods described herein or methods known in the art can be used to determine whether an iRNA, containing a iniSIl’lfllTCl’l to a target sequence is et‘tective in ting (A) c. the expression of a C5 gene, Consideration ofthe efficacy of iRNAs with mismatches in inhibiting expression of a CS gene is important, especially if the ular region of complementarity in a CS gene is known to have polymorphic sequence variation within the population, [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS ill. Modified iRNAs attire invention in one embodiment, the RNA ofthe iRNA ofthe invention eg, a dsRNA, is un— modified, and does not comprise, ag al modifications and/or conjugations known in the art and bed herein. in another ment, the RNA of an iRNA of the invention, cg, a (I: dsRNA, is chemically modified to e stability or other beneficial characteristics. ln certain embodiments ofthe invention, substantially all of the nucleotides of an iRNA of the invention are modified. in other embodiments ot‘the invention, all of the nucleotides of an iRNA ofthe ion are n'iodi’lied. iRNAs oftlie ion in which “substantially all of the nucleotides are modified” are largely but not wholly modified and can include not more than 5, ll) 4, 3, 2, or l unmodified nucleotides.

The nucleic acids teatured in the invention can be synthesized and/or modified by methods well established in the art, such as those described in nt protocols in nucleic acid chemistry,” Beaucage, SL. et at. ), John Wiley & Sons, lnc., New York, NY, USA, which is hereby incorporated herein by reference Modifications include, for example, end modifications, eg, 5 ’—end modifications (phosphorylation, conjugation, inverted linkages) or 3 tend modifications (conjugation, DNA nucleotides, inverted linkages, are); base modifications, erg. , replacement with stabilizing bases, destabilizing bases, or bases that base pair with an ed repertoire ofpartners, removal ofbases c nucleotides), or coniugated bases; sugar modifications (eg, at the 2’—position or 4’uposition) or replacement of 2t) the sugar; and/or backbone modifications, including ation or replacement of the phosphodiester es. Specific examples of iRNA compounds useiiil in the en’ihodiinents described herein include, but are not limited to RNAs containing modified backbones or no natural internucleoside linkages. RNAS having ed ones e, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes reterenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonuc cosides. to some embodiments, a modified iRNA Will have a phosphorus atom in its internucleoside backbone.

Modified RNA backbones include, for example, orothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aniinoalltylphosphotriesters, methyl (A) c. and other alkyl phosphonates including 3‘nallx'ylene phosphonates and chiral phosphonates, phosphinates, phosphorainidates including 3'—arnino phosphorainidate and arninoalkylphosphorarni dates, thionophosphorarnidates, thionoalkylphosphonates, alltylpl’iosphotriesters, and boranophosph ates having norinal 3’~5’ linkages, 2’—5’—linl<ed analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked ill—5’ to 513' or 2'—5' to 512’. Various salts, mixed salts and free acid forms are also included.

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS Representative 11.8. patents that teach the preparation of the above phosphorus" containing iiiikages incinde, but are not 1imited to, US. Patent Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5286,717; ,321,151 5,399,676; 5,405,939; 5,453,496; 5 4,552;33 5 ,466, 77,5 ,476,925; 5,519,126; (I: 5536821; 5,541,316; 5,550,111; 5 5,63,253; 5,571,799, 5 587,361; 050; 6,028,188; 6,124,445; 6,160,109; 170; 6,172,209; 6, 239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 816; 7,273,933; 7,321,029; and US Pat 81339464, the entire contents ofeach of which are hereby incorporated herein by reference.

Modified RNA backbones that do not inc1ude a orus atom therein have backbones that are tortured by short chain a1ky1 orcycioaiky1 intermicEeoside s, rni’xed heteroatoins and a1ky1 or cyc1oa1ky1 internuc1eoside 1inkages, or one or more short chain 1’ieteroatornic or heterocyciic internucleoside es. These inc1ude those having morphohno es (formed in part from the sugar portion of a nuc1eoside’); si1oxane backbones; e, ide and 511116111: backbones; torrnacety1 and thiotorinacety1 backbones; methylene foimacetyi and t1’iiofonnacety1 nes; a1kene containing backbones; su11arnate backbones; met1iiy1eneiinino and methv1enehvdrazinc backbones; su1fonate and su1fonamide backbones; amide nes; and others having mixed N, 0,8and C117 component parts Representative US, patents that teach the preparation of the above o1igonuc1eosides inc1ude, but are not 1imited to, U8 Patent Nos 5,034,506; 5 ,166,315;5.. ,185444; 5,214, 134; ,216,141; 5,235,1133 ; 5,64,562; 5264,564; 5 4,115,938; 5,454,257; 5,466,677; 5,470,967; ,489,677, 5,541 ,307;5,,651 ,225;5,596,086; 5,602,240; 046; 5,610,289; ,5,618,704; ,623,070; 5,663,312; 5,633,360; 5,177,437; and, 5,677,439, the entire contents of each h are hereby incorporated herein by re thrence. 1n other embodiments, suitabie RNA rnirnetics are contemp1ated for use in iRNAs, in which both the sugar and the internuc1eoside e, 1.8., the backbone, of the nuc1eotide units are repiaced with novc1 groups, The base units are maintained for hybridization with an appropriate nuc1eic acid target compound. One such o1igorneric nd, an RNA mimetic that has been shown to have excehent hybridization properties, is reterred to as a e c acid (1115114,), 1n PNA compounds, the sugar backbone of an RNA is repiaced with an amide containing backbone, in particti1ai an aminoethy1g13cine backbone 11ie nuc1eo1-5ascs are retained and are bound direct1y or indirect1y to aza nitrogen atoms of the attitude portion of the backbone, Representative 11,8, patents that teach the preparation of 1314A con'ipounds inc1ude, but are not 1irnited to, US. Patent Nos. 082; 5,714,331; and 5,719,262, the entire ts of each of Which are hereby incorporated herein by reference, Additiona1 PNA compounds suitab1c for use [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS in the iRNAs ofthe invention are bed in, for example, in Nielsen 92‘ mi, e, l99l, 254, l497—l 500.

Some embodiments ed in the invention include RNAS with phosphorothioate backbones and oligonucleosides with l’ieteroatom backbones, and in particular g~~Nltl—~Cll2—, (I: ——CH3~~N(CH3)“Ow—CH3“[known as a methylene (methylirnino) or MMl backbone], MCHZMOM N(Cllg)--Cll2—u, --CHg--N (GHQ-N (Cllg)——Cll2—— and ——'N(Cl*l3)u{Elly-CH2“{wherein the native odiester backbone is represented as ~~O——'P~~O--Cll2»] ot‘the abovesret‘erenced US Patent, No. 5,489,677, and the amide backbones of the ahtwe—reterenced US. Patent No, ,602,240. ln some embodiments, the RNAs featured herein have morpholino backbone ll) structures of the referenced US. Patent No. 5,034,506, Modified RNAs can also contain one or more substituted sugar moieties. The iR’NAs, rig, dsRNAs, teatured herein can include one of the following at the 2‘—position: OH; F; O~, 5—, or N—alliyl; O~, 8—, or Nnalkenyl; O~, Sn or N~all<ynylg or Onalkyl—Owalliyl, wherein the alkyl, alkenyl and allrynyl can be substituted or unsubstituted (I; to Cm alkyl or C3 to C10 alken‘yl and l5 alliyllyl. Exemplary suitable modifications e ()[tCllflnO] m :H}, flflOC'llg, O(Cltl3)anlg, Ottffillg) nCl’lg, Ofljl’lflnONl’lg, and 2)n{)N[(Cl’ighClilz/Bh, Where n and m are from l to about l0. in other embodiments, dsRNAs e one ofthe following at the 2‘ position: C1 to C 10 lower alkyl, substituted lower alkyl, alkaryl, aralleyl, O—allraryl or Onaralkyl, Slit, SCllg, QCN, Cl, Br, CTN, (:Fg, OCR, SUCllg, S();{Cl’l3, ()NC’z, N02, N5, Nltlz, 2t) heterocycloalkyl, heteroeycloalkawl, arninoalkylarnino, polyalltylamino, substituted silyl, an RNA, cleaving group, a er group, an intercala‘tor, a group for improving the pharmacokinetic properties of an iRNA, or a group for improving the pharrnaeodynainie properties of an iRNA, and other substituents having similar properties. in some embodiments, the modification includes a ZKrnethoxyethoxy (:2'-(:>”CH2Cl’l2(}€l’l3, also known as 2'~()-(2— n'iethox,yeth§,/l,) or 2‘—l\/l0E) (Martin (32‘ air, Help Chim. Atria, W95, 78:486~594) tier, an alkoxy~ alleoxy group. Another exemplary modification is 2’~dimethylaininooxyetlioxy, 17.6., a ()(Cl’lztlzle-l3N(‘Ql'hh group, also known as 2'—Dl\/lAOE, as described in examples herein below, and '2'-diinethylaniinoetlioxyethoxy (also known in the art as 210-dimetliylaminoethoxyethyl or Z‘—DMAEOE), £112., 2’~O——Cll2~~0——Cll2~~N(Cll2)2, Other modifications include 2'—rnetlroxy (2’—OCHg), Z‘naminopropoxy (21 QCllzCllgflllelg) and 2‘—tluoro {2'—F). Similar modifications can also be made at other positions on the RNA of an iRNA, particularly the 3' position ot‘the sugar on the 3' terminal nucleotide or in 2'—S' linked dsRNAs and the 5‘ position of 5' tern'iinal nucleotide, iRNAs can also have sugar rnimetics such as utyl moieties in place of the uranosyl sugar.

Representative US patents that teach the preparation of such modified sugar ures e, but are not limited to, US. Pat. Nos. 4,98l,95’7; 5,l l8,§illll; 5,3 l9,08{}; 5,359,044; 5,393,878; [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS ,446,l37; 5,466,786; 5,514,785; 5,5l9,l34; 5,567,8ll; 5,576,427; 722; 5,597,909; ,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application,. The entire contents ot‘each of the foregoing are hereby incorporated herein by reterence.

(I: An iRNA can also include nucleohase (often referred to in the art simply as “base"? modifications or substitutions. As used herein, “unmodified” or “natural” nucleohases include the purine bases adenine (A) and guanine (l3), and the pyrimidine hases e (T), cytosine (C) and uracil (U). Modified nucleohases include other synthetic and natural nucleobases such as deoxynthymine {d'l‘}, 5nmetliylcytosine (5 hie—C), 5~hydroxymethyl cytosine, ne, hypoxanthine, 2-aniinoadenine, 6-inethyl and other alkyl derivatives of adenine and guanine, 2— propyl and other alky1 derivatives of adenine and gtl'imlllfi, 2~thiouracil, 2-thiothyrnine and 2“ thiocytosine, uraci1 and cytosine, 5—propyny1 uracil and ne, 6~azo uracil, cytosine and thymine, 5—uraci1 (pseudouracil), 4-thiouraci1, 8nhalo, 8—an1ino, Suthiol, 8~tliioalhy'l, 8— hydroxyl anal other Susuhstituted adenines and guanines, 5—halo, particularly 5—hronio, 5— tritluorornethyl and other 5—suhstituted uracils and cytosines, 7~rnethylguanine and '7~ n'iethyladenine, uanine and 8~azaadenine, 7~deazaguanine and 7~daazaadenine and 3— deazaguanine and 3—deazaadenine; Further bases include those disclosed in US. Pat. No; 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, llerdewiin, l). ed. Wiley—VCH, 2008; those disclosed in The Concise Encyclopedia 0f Polymer Science And ering, pages 858—859, Hiroschwitz, ,l. L, ed. John Wiley 8: Sons, 1990, these disclosed by Englisch rt all, Angewandte ie, lnternation al Edition, l99l, 30, 613, and those disclosed by Sanghvi, Y 8., Chapter 15, dsRNA Research and Applications, pages 289—302, Crooke, S '1”. and Lehleu, 8,, Ed, CRC Press, l993. Certain of these nucleohases are particularly useliil for increasing the binding, allinity of the oligomeric nds featured in the invention. These include 5—substituted pyrimidines, 6—azapyrirnidines and N—Z, N~6 and 06 tuted purines, including 2—an1inopropyladenine, 5—propynyluracil and ynylcytosine —ntethylcytosine substitutions have been shown to increase nucleic acid duplex stahility 'by 0.6— 12%: (Sanghvi, Y. S Crooke, S T. and Leble’u, 8., lids, dsRNA Research and Applications, CRC Press, Boca Raton, l993, pp, 276278) and are exemplary base substitutions, even more particularly when combined with 2‘n0~niethoxyetliyl sugar modifications.

Representative US. s that teach the ation of certain of the ahoVe noted modified nucleohases as well as other modified nucleohases include, but are not limited to, the above noted US. Patent Nos, 3,687,808, 4,845,205; 30; 5,134,066; 5,l75,273; 5,367,066; ,432,272; 5,457,l87; 5,459,255; 908; 5,502,l77; 5,525,711; 5,552,540; 5,587,469; ,594,l2l , 091; 5,614,617; 5,681,94l; 5,750,692; 6,015,886; 6,l47,200; 6,l66,l97; [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS 6,222,025; 6,235,887; 6,380,368; 640; 6,639,062; 6,6l 7,438; 7,045,610; 7,427,672; and 7,495,088, the entire contents of each ot‘which are hereby incorporated herein by reference.

The RNA of an iRNA can also he modified to include one or more ioclted nucleic acids (LNA). A locked eic acid is a tide having a modified rihose moiety in which the (I: rihose moiety comprises an extra bridge connecting the 2‘ and 4‘ carbons. This structure effectively "locks" the rihose in the 3'—endo structural contorniation. The addition of locket nucleic acids to siliNAs has been shown to increase siRNA stability in sennn, and to reduce oftL target elitects (Elinen, 3. cf (15., (2005) it"ucicic Acids Research 33(l )z439—44’7; hiloolr, OR. at (1/7., (20031113017 Caiic 1’7ch 6(3):?533—843; Grunweller, A, cf oi. , (2003) Nucleic Acids Research 3i(l2):3l85—3l93).

Representative US Patents that teach the preparation of ioclted nucleic acid nucleotides include, but are not limited to, the following: US. Patent Nos. 6,268,490; 6,670,46l; 6,794,499; 6,998,484; 207; l25; and 7,399,845, the entire contents ofeach ofwhieh are hereby incorporated herein by reference.

Potentially stabilizing modifications to the ends of RNA molecules can include N~ (acetylaniinocaproyll—4~hydroxyprolinol (Hyp—Co—NHAc), royl—tl—liydroxyprolinol (lilyp— C6), N—{acer—fl~hydroxyprolinol (HypnNHAc), thymidine—2’—0—deoxythyrnidine (ether), Nu (arninocaprovl)—4l-hydroxvprolinol {Hypfidamino}, 2—docosanoyl~uridine-3"— phosphate, inverted base driliid’l') and others. Qisclosure of this modification can he found in PCT Publication No. W0 '20l l/00586l .

A. ili’bdtfied iRi’VAs Comprising Mom‘s offlic Invention in certain aspects of the invention, the —stranded RNAi agents of the invention include agents with chemical modifications as disclosed, for example, in US Provisional Application No. 6i/56l ,7l0, tiled on November l8, 20l l in PCT/’USZOl 2,1”06569l , or tiled on November l6, 20l2, the entire contents of each h are incorporated herein by nce.

As shown herein and in Provisional ation No, til/56 l ,710 or PCT Application No.

PCT/US20l2/06569l a superior result may he obtained by introducing one or more inotil‘s of three identical rnoditications on three consecutive nucleotides into a sense strand and/or antisense strand of an RNAi agent, ularly at or near the ge site. in some embodiments, the sense strand and antisense strand of the RNAi agent may otherwise he completely modified. The introduction ofthese niotits interrupts the modification pattern, if present, of the sense and/or antisense strand; The RNAi agent may he optionally ated with a GalNAe derivative ligand, for ce on the sense . The resulting RNAi agents present superior gene silencing activity.

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS More specifically, it has been surprisingly discovered that when the sense strand and antisense strand of the double-stranded RNAi agent are completely modified to have one or more rnotils of three identical modifications on three consecutive nucleotides at or near the cleavage site of at least one strand of an RNAi agent, the gene silencing acitiVity ofthe RNAi agent was (I: superiorly ed.

Accordingly, the invention provides doubleustranded RNAi agents capable of inhibiting the expression of a target gene (£13., a complement component CS ((35) gene) in viva. The RN Ai agent comprises a sense strand and an antisense strand. Each strand ot‘the RNAi agent may range from lit—30 nucleotides in length, For example, each strand may he between M390 ll) nucleotides in length, l7—3ll nucleotides in length, 25-30 nucleotides in length, 27—30 nucleotides in length, l7-23 nucleotides in , l7s2l nucleotides in length, l7-l 9 nucleotides in length, lg—ZS nucleotides in length, l9~23 nucleotides in length, lQ—Zl tides in length, Zl—ZS tides in length, or 2l—23 nucleotides in .

The sense strand and antisense strand typically form a duplex double stranded RNA {“dsRNA”), also relerred to herein as an “RN/Ki agent.” The duplex region of an RNAi agent may be l2~30 nucleotide pairs in length. For example, the duplex region can be hen een l4~30 nucleotide pairs in , l7n30 nucleotide pairs in length, 2760 nucleotide pairs in length, l? — 23 nucleotide pairs in length, l7-2l nucleotide pairs in length, l7nl9 nucleotide pairs in length, l9—25 nucleotide pairs in , l9—“3 tide pairs in length, l9— '21 nucleotide pairs in 2t) length, '2l ~25 nucleotide pairs in length, or '2l ~23 nucleotide pairs in length. in another example, the duplex region is selected from l5, re, l7, l8, l9, 20, El, 22, '23, ’24, 25, 26, and 27 nucleotides in length. in one embodiment, the RNAi agent may contain one or more overhang regions and/or tappin g groups at the 3 lend, , or both ends of one or both strands. The overhang can he l~6 nucleotides in length, for instance 2—6 nucleotides in length, l—S nucleotides in length, 2~5 nucleotides in length, l—4 nucleotides in length, 24 nucleotides in length, 13 nucleotides in length, 2-3 nucleotides in length, or l-Z’. nucleotides in length. The overhangs can he the result of one strand being longer than the other, or the result ot‘two s ot‘the same length heing staggered. The overhang can form a mismatch with the target n'iRNA or it can be (A) c. complementary to the gene sequences being targeted or can be another sequence. The first and second s can also he joined, eg, by additional hases to form a hairpin, or by other non— base s. ln one en'ihodirnent, the nucleotides in the overhang region ot‘the RNAi agent can each independently he a modified or uninoditied tide including, but no limited to 2’—sugar d, such as, 24?, 2"’-Ornethyl, thyinidine (T), 2'—0—niethoxyethyl—Sunethyluridine (Ted), 2‘*0~rnethoxyethyladenosine (Aeo), 2‘“O-rnethoxyethth“methylcytidine (rnSCeo), and any [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS combinations thereof. For example, TT can he an overhang sequence for either end on either strand. The ng can form a mismatch with the target mRNA or it can he complementary to the gene sequences being targeted or can be another sequence.

The S’— or '3 ’— overhangs at the sense strand, antisense strand or both strands of the RNAi (I: agent may he phosphorylated In some embodiments, the overhang regiori(s) contains two nucleotides having a phosphorothioate hetween the two nucleotides, Where the two nucleotides ‘an he the same or diftierent. ln one embodiment, the overhang is present at the 3 ’—end of the sense strand, antisense strand, or both strands. In one ment, this '3 ’—overhang is present in the antisense . To one embodiment, this 3 l—overhang is present in the sense strand ll) The RNAi agent may contain only a single overhang, which can strengthen the interference activity of the RNAi, Without ing its overall ity. l3 or example, the single— stranded overhang may he located at the 3’~terminal end ot‘the sense strand or, alternatively, at the 3'nterniinal end ofthe antisense strand The RNAi may also have a blunt end, located at the ’—end ofthe antisense strand (or the 3 ”—end ot‘the sense strand) or vice vernal Generally, the antisense strand ot‘the RNAi has a nucleotide overhang at the 3 lend, and the 5 lend is hlunt~ Wl’iile not wishing to he bound by theory, the asymmetric hlunt end at the 5 9—end ofthe antisense strand and 3 ’nend overhang of the antisense strand favor the guide strand loading into RISC process. ln one emhodirnent, the RNAi agent is a double ended hluntrner of 19 nucleotides in 2t) length, wherein the sense strand contains at least one rnotif ol‘three 2’le modifications on three consecutive nucleotides at positions 7', 8, 9 front the S’end. The antisense strand ns at least one motif of three 2’n0~niethyl modifications on three consecutive nucleotides at positions ll, l2, 13 from the S’end. in another embodiment, the RNAi agent is a double ended hluntnier ot‘Zt) nucleotides in length, wherein the sense strand contains at least one ‘ol“ three 23F ations on three consecutive nucleotides at positions 8,, 9, l0 from the S’end. The antisense strand contains at least one motif ot‘three 2’—0-methyl modifications on three consecutive nucleotides at positions 11, l2, l3 from the S’end. ln yet another embodiment, the RNAi agent is a double ended hluntiner of 2 l nucleotides (A) c. in length, wherein the sense strand contains at least one motif of three Z’nl: modifications on three utive nucleotides at ons 9, 10, l l from the 5 "end. The antisense strand contains at least one inotifot‘three 2 ethyl modifications on three consecutive nucleotides at positions ll, 1'2, l3 from the Send. in one embodiment, the RNAi agent comprises a Zl nucleotide sense strand and a 23 nucleotide antisense strand, wherein the sense strand ns at least one ‘of three 21F modifications on three consecutive nucleotides at positions 9, l0, l l from the 5 ’end; the [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS antisense strand contains at least one motif of three 2 hyl modifications on three consecutive nucleotides at positions ll, 12, l3 front the send, n one end ot‘the RNAi agent is blunt, while the other end comprises a 2 tide overhang. ably, the 2 nucleotide overhang is at the 3 9—end ofthe antisense strand W’hen the 2 tide overl’iang is (I: at the 3 l—cnd of the antisense strand, there may be two orothioate internucleotide linlrages between the terminal three nucleotides, n two of the three nucleotides are the overhang nucleotides, and the third nucleotide is a paired tide next to the overhang nucleotide. ln one en’ihodiinent, the RNAi agent additionally has two phosnl’iorothioate ucleotide linkages n the terminal three nucleotides at both the 5’nend ofthe sense strand and at the S’nend of ll) the antisense strand. ln one embodiment, every nucleotide in the sense strand and the antisense strand ot‘the RNAi agent, including the nucleotides that are part ot‘the motil‘s are moditied nucleotides. ln one emhodin'ient each residue is independently modified with a ’2 ’—O~n’iethyl or 3 ’—fluoro, eg. in an alternating motif. Optionally, the RNAi agent further comprises a ligand ifpreferably GalNAcg). in one embodiment, the RNAi agent comprises a sense and an antisense strand, wherein the sense strand is 25—30 nucleotide residues in length, wherein starting item the 5’ terminal nucleotide (position l) positions l to 23 ofthe first strand comprise at least 8 ribonucleotides; the nse strand is 36~66 nucleotide residues in length and, starting from the 3' terminal nucleotide, comprises at least 8 ribonucleotides in the positions paired with positions l— 23 of 2t) sense strand to lonn a duplex; wherein at least the 3 ’terrninal nucleotide of antisense strand is unpaired with sense strand, and up to 6 coi’isecutive 3’ terminal nucleotides are unpaired with sense strand, therehy forming a 3’ single stranded overhang of ind nucleotides; wherein the 5’ terminus of antisense strand ses from 1030 consecutive nucleotides which are unpaired with sense strand, y forming a l (it—3t) nucleotide single stranded 5' overhang; n at least the sense strand 5' terminal and 3‘ terminal nucleotides are hase paired with nucleotides of antisense strand when sense and nse strands are aligned for maximum complementarity, thereby forming a substantially duplexed region between sense and antisense strands; and antisense strand is sulliciently complementary to a target RNA along at least l9 ribonucleotides of antisense strand length to reduce target gene expression when the double stranded nucleic acid (A) c. is introduced into a mammalian cell; and wherein the sense strand contains at least one nrotifof three 2’-F modifications on three consecutive nucleotides, where at least one ofthe motifs occurs at or near the cleavage site. The antisense strand contains at least one moti l‘ol’ three 2’—t}rnethyl n'iodifications on three consecutive nucleotides at or near the cleavage site. in one embodiment, the RNAi agent ses sense and antisense strands, wherein the RNAi agent comprises a first strand having a length which is at least 25 and at most '29 nucleotides and a second stand having a length which is at most 30 nucleotides with at least one [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS motif of three 2 ’—Owniethyl modifications on three consecutive nucleotides at position ll, l2, l3 from the 5 "’ end; wherein the 3 ’ end ofthe first strand and the 5” end of the second strand form a blunt end and the second strand is lsél nucleotides longer at its 3 ’ end than the tirst , wherein the duplex region region which is at least 25 nucleotides in length, and the second strand (I: is sufficiently complenienataiy to a target mRNA along at least l9 nucleotide ofthe second strand length to reduce target gene expression when the RNAi agent is introduced into a inamn'ialian cell, and n dicer cleavage ot‘the RNAi agent preferentially results in an siRNA comprising the 3 " end ot‘the second strand, thereby reducing expression ot‘the target gene in the mammal. ally, the RNAi agent r comprises a ligand. ill in one enihodiinent, the sense strand ofthe RNAi agent contains at least one motif of three identical rnoditications on three consecutive nucleotides, Where one ofthe inotits occurs at the cleavage site in the sense strand, ln one embodiment, the antisense strand ofthe RNAi agent can also contain at least one motif of three cal modifications on three consecutive nucleotides, Where one ol‘the rnotifs occurs at or near the cleavage site in the antisense strand For an RNAi agent having a duplex region of l7~23 nucleotide in , the cleavage site ofthe antisense strand is typically around the l0, ll and l2 positions from the 5’—end. Thus the motifs of three identical modifications may occur at the 9, l0, ll positions; l0, ll, l2 positions; ll, l2, 13 positions; l2, l3, l4 positions; or l3, l4, l5 positions ofthe antisense 2t) strand, the count starting from the lSt nucleotide from the 5 ”-end ofthe antisense strand, or, the count starting from the lSt paired tide within the duplex region from the S’~ end of the antisense strand. The cleavage site in the antisense strand may also change according to the length of the duplex region ofthe RNAi from the .

The sense strand ofthe RNAi agent may contain at least one rnotit‘ol’ three identical n'iodifications on three consecutive nucleotides at the cleavage site ofthe ; and the antisense strand may have at least one motif of three identical modifications on three consecutive nucleotides at or near the cleavage site of the strand. When the sense strand and the antisense strand form a dsRNA duplex, the sense strand and the nse strand can he so aligned that one rnotif o‘l’the three nucleotides on the sense strand and one n'iotil’of the three nucleotides on the (A) c. antisense strand have at least one nucleotide overlap, 119, at least one of the three nucleotides of the inotil‘in the sense strand forms a base pair with at least one of the three nucleotides of the inotit‘in the antisense strand. Alternatively, at least two nucleotides inay overlap, or all three nucleotides may overlap. in one ment, the sense strand ofthe RNAi agent may contain more than one motif ofthree identical modifications on three consecutive nucleotides. The first motif may occur at or near the cleavage site ot‘the strand and the other motifs may he a Wing rnoditication. The term [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS “wing modification” herein refers to a motif occurring at another portion ofthe strand that is separated from the motif at or near the cleavage site of the same strand. The wing cation is either adajacent to the first rnotit or is separated by at least one or more nucleotides. When the motifs are immediately adjacent to each other then the cl’iemistry of the motifs are ct li'om (I: each other and when the motifs are separated by one or more nucleotide than the chemistries can he the same or different, Two or more wing modifications may he present. For instance, when two wing modifications are present, each wing modification may occur at one end relative to the first motifwhich is at or near c eavage site or on either side ol’the lead motif.

Like the sense , the antisense strand ofthe RNAi agent may n more than one ll) motifs ofthree cal modifications on three consecutive tides, with at least one of the motil‘s occurring at or near the cleavage site of the . This antisense strand may also contain one or more wing modifications in an alignment similar to the wing modifications that inay he present on the sense strand, in one embodiment, the wing modification on the sense strand or antisense strand ofthe RNAi agent typically does not include the first one or two tern'iinal nucleotides at the 3 ’“end, 5 ‘1 end or both ends of the strand. in another embodiment, the wing modification on the sense strand or antisense strand of the RNAi agent typically does not include the first one or two paired nucleotides within the duplex region at the 3 ’—end, 5’—end or both ends of the strand. 2t) When the sense strand and the antisense strand ofthe RN Al agent each contain at least one wing modification, the wing modifications may tall on the same end of the duplex region, and, have an overlap of one, two or three tides When the sense strand and the antisense strand of the RNAi agent each contain at least two wing modifications, the sense strand and the antisense strand can he so aligned that two n'iodiiications each from one strand fall on one end of the duplex region, having an overlap of one, two or three nucleotides; two modifications each from one strand fall on the other end ofthe duplex region, haying an overlap of one, two or three nucleotides; two modifications one strand fall on each side of the lead motif, having an overlap of one, two or three nucleotides in the duplex region, (A) c. in one embodiment, every tide in the sense strand and antisense strand, ofthe RNAi agent, including the nucleotides that are part of the motifs, may be modified. Each tide may be d with the same or different modification which ‘an include one or more alteration of one or both ofthe non~linhing phosphate oxygens and/or of one or more ot‘the linl<;ing phosphate oxygens; alteration of a constituent of the rihose sugar, cg, of the 2’ hydroxyl on the ribose sugar; wholesale replacement ofthe phosphate moiety with “dephospho” linkers; [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS ed set by DPS modification or ement of a naturally occurring base; and replacement or ation of the ribose—phosphate backbone.

As nucleic acids are polymers ot‘subunits, many of the modifications occur at a position which is repeated within a nucleic acid, rag, a n'iodilication of a base, or a phosphate moiety, or a (I: nonnlinlring O of a phosphate moiety, in some cases the modification will occur at all of the subject positions in the nucleic acid but in many cases it will not, By way of example, a modification may only occur at a 3’ ’ terminal position, or 5 may only occur in a terminal region, eg, at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or l0 nucleotides ot‘a strand.

A cation may occur in a double strand region, a single strand region, or in both, A ll) modification may occur only in the double strand region of a RNA or may only occur in a single strand region ot‘a RNA. l:or example, a phospliorothioate modification at a non—linking 0 position may only occur at one or both termini, may only occur in a terminal region, tag, at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or l0 nucleotides of a strand, or may occur in double strand and single strand regions, particularly at termini. The 5’ end or ends can be phosphorylated. it may be le, 8.3;, to enhance stability, to include particular bases in overhangs, or to include modified nucleotides or nucleotide surrogates, in single strand overhangs, eg, in a 5’ ’ overhang, or 3 or in both. For e, it can be ble to include purine nue eotides in overhangs. in some embodiments all or some of the bases in a 3 ’ "’ overhang maybe or 5 2t) modified, eg, with a rnoditi ration described herein. h/loditications can include, :.g., the use of modifications at the 2’ position ot‘the ribose sugar with modifications that are known in the art, eg, the use of deoxyribonucleotides, or 2’~meetliyl modified , 2’—deoxyn2’—tluoro (a 1F) instead of the gar of the hase and modifications in the phosphate group, eg, pliosphorothioate modifications. Overhangs need not be homologous with the target sequence. ln one en'ibodiment, each residue of the sense strand and antisense strand is ndently modified with LNA, HNA, CeNA, 2 lniethoxyethyl, 2’" Onmethyl, 2’n0~allyl, 2’— C— allyl, 2 l-deoxy, Zl-liydroxyl, or 2’—tluoro. The strands can contain more than one modification. in one embodiment, each residue ofthe sense strand and antisense strand is independently moditied with 21 O~niethyl or 2’~t,luoro.

(A) c. At least two different modifications are typically present on the sense strand and antisense strand, Those two modifications may be the 2’— Qumethyl or oro modifications, or others. ln one en'ibodiment, the N; and/or Nb con’iprise n’iodilications of an alternating pattern.

The term “alternating motif’ as used herein refers to a motif haying one or more modifications, each modification occurring on alternating nucleotides of one strand, The alternating nucleotide may refer to one per every other nucleotide or one per every three nucleotides, or a similar [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS patternt For exampler if A, B and C each represent one type ofmoditieation to the tide, the ating motifcan be “ABABABABABr- J. . .,’"’ “AABBAABBAABB. . .,’"’ “AABAABAABAAB. . .,” “AA/KB AAABAA AB. . .,” “AAABBBAAABBB. . .,"‘ or “ABCABCABCABC. . .,” etc, (I: The type of modifications ned in the alternating motifmay he the same or different.

For example, if A, B, C, [3 each represent one type of modification on the tide, the alternating pattern, 518., modifications on every other nucleotide, may he the same, but each of the sense strand or antisense strand can he selected from several possihilities ofmodiiieations Within the alternating motif such as “ABABAB. , 7’, “ACACAC. . .” “BDBDBD. . .” or ll) “CDCDCQ . .,” etc. in one embodiment, the RNAi agent of the ion comprises the modification pattern for the alternating motif on the sense strand ve to the n'iodifieation pattern for the alternating motif on the antisense strand is shifted The shift may be such that the modified group of tides ofthe sense strand corresponds to a differently modifier group ofnucleotides of the nse strand and We 3 verse. l:or example, the sense strand when paired with the antisense strand in the dsRNA duplex, the ating motif in the sense strand may start with “ABABAB” from 5 ‘33 l of the strand and the alternating motif in the nse strand may start with “BABABA” from 5’_3 ’ofthe strand within the duplex region. As another example, the alternating motif in the sense strand may start With “AABBAABB” from 553 ’ ofthe strand and 2t) the alternating motifin the antisenese strand may start with “BBAABBAA” from 5 ”-3 ’ of the strand within the duplex region, so that there is a complete or partial shift ofthe modification patterns between the sense strand and the antisense strand in one embodiment, the RNAi agent comprises the pattern of the ating motif of '2'— Oi—rnethyl modifi ration ant 21F modification on the sense strand initially has a shift relative to the pattern of the alternating motif ot‘2‘—O~n’iethyl rnoditieation and 2’45 modification on the antisense strand initially, 17C. the 2‘n0~niethyl modified nucleotide on the sense strand base pairs With a 2'—F modified nucleotide on the antisense strand and Vice versa. ’l‘he 1 position ofthe sense strand may start with the 2K}? modification, and the l position ofthe antisense strand may start with the 2'— O—rnethyl modification.

(A) c. The introduction of one or more motifs of three identical modifications on three consecutive nucleotides to the sense strand and/or antisense strand interrupts the initial modification pattern present in the sense strand and/or antisense strand. This interruption ofthe n'iodification n ofthe sense. and/or antisense strand by introducing one or more motifs of three identical modifications on three consecutive nucleotides to the sense and/or antisense strand surprisingly es the gene silencing aeitivty to the target gene.

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS in one ment, when the motif ofthree identical modifications on three utive nucleotides is introduced to any of the strands, the modification of the nucleotide next to the motifis a ent modification than the n'iodi'lication of the motif. l3or example, the portion of the sequence containing the motif is “. . .NaYYYNb. . .f’ where “Y” represents the n'ioditication of (I: the motif of three identical modifications on three consecutive nucleotide, and “Na” and “Nb” represent a modification to the nucleotide next to the motif “YYY” that is different than the modification of‘i’, and where N, and Nb can he the same or dit‘thrent modifications.

Altnernatively, Na and/or Nb may he present or absent when there is a wing modification present, The RNAi agent may fiirther comprise at least one phosphorothioate or ll) methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage modification may occur on any nucleotide ofthe sense st “and or antisense strand or hoth stands in any position ot‘the st‘and. For instance, the internucleotide linkage modification may occur on every nucleotide on the sense strand and/or antisense strand; each internucleotide linkage modification may occur in an alternating pattern on the sense strand and/or antisense ; or the sense strand or antisense strand may contain both ucleotide linkage modifications in an alternating pattern. The alternating pattern of the internucleotide linkage modification on the sense strand may he the same or ent from the antisense strand, and the alternating pattern ofthe internuc eotide e modification on the sense strand may have a shift relative to the alternating pattern ofthe uclcotidc linkage modification on the 2t) antisense strand. in one embodiment, a dtnthle-standed RNAi agent comprises 6- Sphosphorothioate internucleotide es. in one embodiment, the antisense strand comprises two phosphorothioate internticleotide linkages at the niinus and two phosphorothioate ucleotide linkages at the 3 l-terminus, and the sense strand comprises at least two phosphorothioate internucleotide linkages at either the S’~terminus or the 3 ”—terrninus. ln one en'ihodiment, the RNAi comprises a phosphorothioate or rnethylphosphonate internucleotide e modification in the overhang region, For example, the overhang region may contain two nucleotides having a orothioate or methylphosphonate internuclcotide linkage between the two nucleotides. lnternucleotide linkage cations also may he made to link the overhang nucleotides with the terminal paired nucleotides within the duplex region. For (A) c. example, at least 2, 3 , 4, or all the overhang nucleotides may he linked h phosphorothioate or methylphosphonate internucleotide linkage, and optionally, there may he additional phosphorothioate or methylplriosphonate internucleotide linkages g the overhang nucleotide with a paired nucleotide that is next to the overhang nucleotide, For ce, there may he at least two phosphorothioate internucleotide linkages between the terminal thr 3e nucleotides, in which two ofthe three nucleotides are overhang nucleotides, and the third is a paired nucleotide next to the overhang nucleotide. These terminal three nucleotides may he at the ,2 ’end of the [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS antisense strand, the 3 ’end of the sense , the ill—end of the antisense strand, and/or the send ofthe antisense strand. in one embodiment, the 2 nucleotide overhang is at the 3 ”-end ofthe antisense strand, and there ae two pl’iosphorothioate internucleotide linkages hetw ten the terminal three nucleotides, (I: wherein two ofthe three nucleotides are the overhang nucleotides, and the third tide is a paired nucleotide next to the overhang nucleotide. 0ptionally, the RNAi agent may additionally have two phosphorothioate internucleotide linkages between the terminal three nucleotides at both the 5 ’~end of the sense strand and at the 5’—end of the nse strand in one embodiment, the RNAi agent comprises mismatch(es) with the , within the it) duplex, or combinations thereof. The mistrnateh may occur in the overhang region or the duplex region. The base pair may be ranked on the basis oftheir propensity to promote iation or melting (rag, on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or r analysis can also be used) in terms ofpromoting iation: Art) is red over GIC; GIU is preterred over (MT; and LC is preferred over GzC (1::inosine). Nlisrnatches, rag, norh canonical or other than canonical pairings (as described elsewhere herein) are preferred over canonical (Ail, AzU, GzC) pairings; and pairings which include a universal base are preferred over canonical pairings in one embodiment, the RNAi agent comprises at least one ofthe first l, 2, 3, 4, or 5 base 2t) pairs within the duplex regions from the 5 ’~ end of the antisense strand independently selected from the group of: All, G:U, LC, and n'iisrnatched pairs, rag, non—canonical or other than canonical pairings or pairings which include a universal base, to promote the dissociation of the antisense strand at the 5’—end of the duplex. in one embodiment, the nucleotide at the l on within the duplex region from the 5’— end in the antisense strand is selected from the group consisting of A, dA, dU, U, and ill, atively, at least one of the first l, 2 or 3 base pair within the duplex region from the 5’" end ofthe antisense strand is an All base pair. For example, the first base pair within the duplex region from the 5 ’~ end of the antisense strand is an All base pair. in r ment, the nucleotide at the 3 ”end of the sense strand is thymine (A) c. {d’l‘}. ln another embodiment, the nucleotide at the 3 lend of the antisense strand is deoxy~ thymine (d’l‘). in one embodiment, there is a short sequence of deoxy—tliyniine nucleotides, for example, two dT nucleotides on the 3 3“end of the sense and/or antisense strand. in one en'ibodiment, the sense strand sequence may he represented by tonnula (l): ’ 11p~N3~(X x x )j—NVY Y Y ~Nt—t‘z z Z )jnNannq 3' (n wherein: i and j are each independently {3 or l; [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS p and q are each independently 0~6g each N, independently represents an oligonucleotide sequence comprising 0—25 d nucleotides, each sequence comprising at least two differently modified nucleotides; each Ni, independently ents an oligonucleotide sequence coinprisin g (l—l 0 modified (I: nucleotides; each np and nq independently represent an overhang nucleotide; wherein Nb and Y do not have the same modification; and XXX, YYY and ZZZ each independently represent one n'iotifof tlii fee identical modifications on three consecutive nucleotides. Preferably Y’Y‘r’ is all 21F niodifred ll) nucleotides.

In one embodiment, the Na and/or Nb coinprise modifications of alternating pattern. in one embodiment, the YYY inotifoccurs at or near the cleavage site ofthe sense strand.

For example, when the RNAi agent has a duplex region of l7~23 nucleotides in length, the YYY inotif can occur at or the ty of the cleavage site (9.3: can occur at positions 6, 7, S, 7, 8, 9, 8, 9, ll), 9, ll}, ll, ll), l l ,l2 or ll, l2, l3) of — the sense strand, the count starting irorn the lSt nucleotide, from the 5’—end; or ally, the count starting at the lbl paired nucleotide within the duplex region, from the 5’— end. in one embodiment, i is l and j is O, or i is (l and j is l, or both i and j are l, The sense strand can therefore he ented hy the following formulas: 2t) 5' Y ‘r’~Nb—ZZZ¥N,~riq 3’ (1h); ' np—Na—XXX—NVYYY—N&—nq 3’ (1c); or ' np—Na—XXXanw‘r'YY—Nb—ZZZ~Namnq 3' (hi) When the sense strand is represented by formula (lb), Nb represents an oligonucleotide sequence comprising 0~l G, 0-7, (3-5, (L4, (L2 or 0 modified tides. Each N, independently can represent an oligonucleotide sequence comprising 220, 2~l5, or 2~l {3 modified nucleotides. “then the sense strand is ented as forniula (its), Ni, represents an oligonucleotide sequence comprising (lull), 0—7, (l—ll), 0-7, 0—5, 0—4, 0—2 or 0 modified nucleotides. Each Na can independently represent an oligonucleotide sequence comprising 290, 2~l S, or 2~l 0 modified nucleotides.

(A) c. When the sense strand is represented as formula (ld), each Nb independently represents an oligonucleotide sequence comprising ll—ltl, 0—7, 0—5, 0-4, 0—2 or 0 modified nucleotides.

Prefe “ably, Nb is O, l, '2, 3, 4, 5 or 6 lilach N, can independently represent an ucleotide ce comprising 2—20, 2~l 5 or Z—l t) modified nucleotides.

Each of X, Y and Z may be the same or different from each other.

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS in other embodiments, i is G and j is 0., and the sense strand maybe represented by the formula: ' YlpeNgYY Y~ Naan 3’ (la).

When the sense strand is represented by formula (la), each Na ndently can represent an oligonucieotide sequence comprising 2410, 2—l5, or Enid modified tides. ln one embodiment, the antisense strand sequence of the RNAi may he represented by formula (ll): ’ nq~~Na’—(Z’Z’Z’k—Nfi—Y’YY'—Nb’—(XX’X’)1—N’a~np’ 3’ (ll) wherein; ill k and l are each independently (l or i; p’ and q’ are each ndently 0-6; each Nd' independently represents an oligonucleotide sequence coinprising 6—25 modified tides, each sequence comprising at least two differently modified nucleotides; each Nb’ independently represents an oligonucleotide sequence comprising O—ll) modified tides; each np” and nq’ ndently represent an overhang nucleotide; wherein Nb’ and Y" do not have the same modification; X'X'X’, Y’Y’Y’ and Z’Z’Z’ each independently represent one motif ofthree identical 2t) modifications on three utive nucleotides. in one emhodin'ient, the Na” and/or Nb” comprise cations of alternating pattern The Y’Y’Y' motif occurs at or near the cleavage site of the antisense strand For example, when the RNAi agent has a duplex region of l 7—23nucleotidein length, the Y’Y’Y’ motif can occur at positions 9, it), ll;l(), ll, l2; ll, i2, 13; 12, l3, l4 ; or i3, l4, l5 ofthe antisense strand, with the count starting from the lSt nucleotide, iifon'i the 5 ”~end; or optionally, the count starting at the is" paired nucleotide Within the duplex region, from the 5’" end‘ Preferably, the *"Y’Y’ motif occurs at positions l l l3. , l2, ln one ment, Y'Y’Y' motifis ail 2’~Ol\/le i’noditied nucleotides. in one n'ient, it is l and l is (l, or k is 0 and l is l, or both lr and l are l.

(A) c. The antisense strand can therefore he represented by the following formulas: ’ riq=—Na’—Z’Z’Z’-Nb’—Y"i”Y"-N3"unp= 3’ (lilo); ’ iiq>—Na'~'r"‘r"‘a"-Nb'~X'X'X'-np> 3' (llc); or ’ nq~~Na’— Z’Z’Z’~Nb’~Y’Y“r”~Nh’~ X’X'XCNa’mnpv 3’ (lld). n the nse strand is represented by formula (lib), Ni; represents an oligonucleotide sequence comprising O—ll), OJ, 0—10, (3—7, 0—5, 0—4, 0—2 or 0 modified ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS nucleotides, Each Na’ ndently represents an ucleotide sequence sing 2'20, 2," , or Z—l 0 modified nucleotides.

When the antisense stand is represented as formula (tic), Nb’ represents an oligonucleotide sequence sing lit—ll), 0—7, (H {3, (VI, 05, 0—4, 0—2 or 0 modified (I: nucleotides. Each N1’ independently represents an oligonucleotide sequence comprising 25210, 2~ , or Z-l 0 modified nucleotides.

When the antisense strand is represented as formula (lid), each Nb” independently represents an oligonucleotide sequence comprising 0—10, 0—7, (H (l, (Vi, (HS, 0'4, 0—2 or 0 modified nucleotides. Each N1 independently represents an ucleotide sequence ill comprising 2—20, 2—15, or 2—10 modified nucleotides. Preferably, N11 is O, l, 2, 3, 4, 5 or 6. ln other embodiments, l; is (l and l is G and the antisense strand maybe represented by the formula: ' n112—N'3w‘r”Y"‘l”— Naa—nqr 3‘ (la).

When the antisense strand is represented as formula (lla), each N; independently represents an oiigonucleotide ce comprising 2320, 2~l S, or Eel t) niodili ed tides.

Each ofX’, Y' and Z" may he the same or difterent from each other, Each nucleotide of the sense strand and antisense strand may he independently modified with LNA, HNA, CeNA, 2 ’nmethoxyethyl, 210nmethyl, 2 ’—O—ally1, 2 ’—C~ allyl, 2, roxyl, or 2’—tluoro. For example, each nucleotide ofthe sense strand and antisense strand is independently 2t) modified with 2’—O~rnethyl or '2’~l‘luoro. Each X, Y, Z, .r ', Y' and Z', in particular, may represent a 2’—O—rnetliyl modification or a ’2 oro modification. ln one embodiment, the sense strand ofthe RNAi agent may contain YYY motif occurring at 9, l0 and l l positions of the strand when the duplex region is 21 nt, the count starting liorn the lSi nucleotide from the 5 lend, or optionally, the count starting at the l3"T paired nucleotide within the duplex region, from, the S’— end; and Y represents 2’—P modification. The sense strand may onally n XXX motif or ZZZ motifs as wing modifications at the opposite end ofthe duplex region; and XXX and ZZZ. each independently represents a 2’—{)Me modification or 2 ll“ modification. ln one n'ient the nse strand may contain YrY’Y’ inotifoceurring at positions (A) c. 11, 12, l3 oftlie strand, the count starting from the lSt nucleotide from the Slend, or optionally, the count starting at the l“ paired nucleotide Within the duplex region, from the 5 ’— end; and Y' represents 2’—(:}*methyl modification. The antisense strand may additionally contain X'X'X' motif or Z’Z’Z’ motifs as wing ations at the opposite end of the duplex region; and XX’X" and Z’" ’2’ each independently represents a 2’n0lvle modification or 2 ’nl: modification, [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS The sense strand represented by any one of the above formulas (la), (lb), (lo), and (Id) forms a duplex with a antisense strand being represented by any one of formulas (lla), (llb), (He), and (lld), respectively.

Accordingly“, the RNAi agents for use in the i’nethods of the invention may comprise a (I: sense strand and an nse strand? each strand having 14 to 39 nucleotides? the RNAi duplex represented by formula (Ill): sense: 5' n10 —Na~(X X X} -wa Y Y Y ~Nb —(Z Z Z)j*N11~nq 3‘ antisense: 3' n11LN11lor’X’X')11~Nbli"Y’YtNg—rzaiming—n; 5' (Ill) ll) wherein: l, j, k, and l are each ndently (l or l; 131 pl, q, and q’ are each ndently 0—6; each N1 and N1 independently ents an oligonueleotide ce comprising (L25 modified nucleotides, each sequence comprising at least two differently modified nucleotides; each Nb and Nbi independently represents an ollgonueleotide sequence comprising (l-l 0 modified nucleotides; wherein each np’? npe nq’i and 1111, each ofwhich may or may not be present, ndently represents an overhang nucleotide; and 2d XXX, YYY, ZZZ, XX'X’, Y’Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecuti e nucleotides. ln one embodiment i is (l and i is G; or i is l and j is (l; or i is G and} is l; or both i and j are 0; or both i and} are l1 in another embodiment, l< is l) and l is ll; or it is. l and l is O; k is. (l and l is l; or both it and l are (l; or both l; and l are l.

Exemplary ations of the sense strand and antisense strand forming a RNAi duplex include the formulas below: ' npu Na—Y Y Y -i\l11—nq 3' 3'nplN11i—Y’Y' alga; 5‘ (llla) ' np “N11 "Y Y Y UN}, "Z Z Z "Nannq 3' 3* living-Y'i'y’-Nt,’-Z’zasin ‘Ji f (lllh) ’ np—N3~ X X X ~Nh —Y Y Y — Na—nq 3' 3’ nplna’nX’X’Xszlrwry—Nam; 5' (lllc) [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ’ n1, "Na ~X X X "waY Y Y ~an Z Z Z nNa~nq 3‘ 3' n,,’_N,lX'X’xantler‘r'—Nblz'z'z'—'N,_nq’ 5* i ll ld) When the RNAi agent is represented by formula (Illa), each N; independently represents (I: an oligonucleotide sequence comprising 2—29, 245,, or 2—l0 modified tides, When the RNAi agent is represented by formula (lllh), each Nb independently represents an oligonucleotide sequence comprising lslll, 13,7, l~5 or l-4 modified nucleotides. liach Na independently represents an oligonucleotide sequence sing 2—20, 2—l S, or Z—ld modified nucleotides, ll) When the RNAi agent is represented as formula {lllc}, each Nb, Nb’ independently represents an oligonucleotide sequence comprising (H t}, (It—7’, tlwltl, 03,7, 0:5, 0-4, (3-2 or Ornodified nucleotides. Each N3 independently represents an oligonucleotide sequence comprising 23,0, 245, or Edit modified nucleotides.

When the RNAi agent is represented as formula (llld), each Nb, Nb’ independently represents an oligonucleotide ce comprising (Ht), 047, G-l G, (3-7, (It—5, (L4, 042 or Oniodified nucleotides. Each Na, N; independently represents an oligonucleotide sequence comprising 2—20, 245, or 2—ltl d nucleotides Each ofNa, Na’, Nb and Np ndently comprises modifications of alternating pattern.

Each ofX, Y and Z in as (lll), (llla’), ('lllh), (Hits), and (llld) may he the same or 2t) (lifterent from each other.

When the RNAi agent is ented by formula (Ill), (llla), (lllb), , and (llld), at least one of the Y nucleotides may form a base pair with one ofthe Y" nucleotides.

Alternatively, at least two of the Y nucleotides form base pairs With the corresponding Y" nucleotides; or all three of the Y nucleotides all form base pairs with the corresponding Y” nucleotides.

When the RNAi agent is represented by formula {lllh} or {llld}, at least one ofthe Z tides may form a hase pair With one of the 2' nucleotides. Alternatively, at least two of the 2 nucleotides form base pairs with the corresponding 2' nucleotides; or all three of the Z nucleotides all form base pairs with the corresponding 23' nucleotides, nucleotides may form a base pair with one of the X’ nucleotides. Alternatively, at least two of the X nucleotides form base pairs with the corresponding X” nucleotides; or all three of the X nucleotides all form base pairs with the corresponding X’ nucleotides. in one embodiment, the modification on the Y nucleotide is different than the modification on the Y’ nucleotide, the ation on the Z nucleotide is different than the [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS modification on the Z’ nucleotide, and/or the modification on the X nucleotide is different than the cation on the X” nucleotide. in one embodiment, when the RNAi agent is represented by formula (iiid), the Na modifications areZ’«Omethyl or77lluoio modifications in another embodiment, when the (I: RNAi agent is represented by formula {llld}, the N3 cations are ethyl or oro modifications and np' >0 and at least one np’ is linked to a neighboring nucleotide a via phosphorothioate linlcage. in yet another ment, when the RNAi agent is ented by formula (llld), the N3 modifications are 2’~0—methyl or 2’~‘lluoro modifications >0 and at , np' least one np’ is linlred toaaneighboring nucleotide via phosphorothioate linkage, and the sense it) strand is coniugated to one or more c derivatives attached through a bivalent or trivalent branched linker {described below). ln another embodiment, when the RN Ai agent is represented by forn’iula (llld), the N3 i'nodifications are 2’~O—m ethyl or 2’~lluoro modifications >0 and at , np' least one n1,” is d to a neighboring nucleotide via phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more {ialNAc derivatives attached through a bivalent or trivalent branched linlcer. in one en'ibodiment, when the RNAi agent is represented by fonnula (llla), the Na modifications are riethyl or 2'—fluoro modifications >0 and at least one np" is linlied to a , np' neighboring nuc eotide via phosphorothioate linkage, the sense strand comprises at least one orothioate linkage, and the sense strandis conjugated to one or more GalNAc derivatives 2t) attached through a bivalent or trivalent branched linker. in one embodiment, the RNAi agent is a multimer containing at least two duplexes represented by formula (iii), {llla}, (lllb), , and (iild), wherein the duplexes are connected bv a linker. The linker can be cleavable or non-cleavable. thionallv, the niultiiner further comprises a ligand. Each of the duplexes can target the same gene or two differentgenes; or each of the es can target. same gene at two ent target sites. ln one embodiment, the RNAi agent is a multimer containing three, four, five, six or more duplexes represented by formula (ill), (llla), (lllb), (lllc), and (llid), wherein the duplexes are connected by a linker. The linker can be cleavable or non—cleavable. ally, the multimer further comprises a ligand. Each ofthe duplexes can target the same gene or two (A) c. different genes; or each of the duplexes can target same gene at two different target sites. in one embodiment, two RNAi agents represented by formula (ll ),(llia), (illl ),(iiic), and (l lld) are linked to each other at the 5’ end, and one or both ofthe 3 ends and are optionally ated to to a ligand, Each ofthe agents can target the same gene or two diffi: 'ent genes; or each ofthe agents can target same gene at two different target sites, Various publications describe multimeric RNAi agents that can he used in the methods of the invention. Such publications include WGZtliti7/tl9l269, US Patent No. 7858769, [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS W020l {Ml-4 l El l WOZOGWl l 7686, 20G9/014887 and W020i l/GS 1520 the entire contents of each ofwhich are hereby incorporated herein by reference.

As described in more detail below, the RNAi agent that contains conjugations of one or more carbohydrate rnoieties to a RNAi agent can optimize one or more properties of the RNAi (I: agent. in many cases, the carbohydrate nioiety will be attached to a d subunit of the RNAi agent. For example, the ribose sugar of one or more ribonucleotide subunits of a dsRNA agent can be ed with another n'ioiety, ag, a rbohydrate (preterably cyclic) carrier to which is attached a carbohydrate ligand. A, ribonucleotide t in which the ribose sugar of the subunit has been so replaced is ed to herein as a ribose replacement modification ill subunit {ERR/ES) A cyclic carrier may be a carbocyclic ring system, 116., all ring atoms are carbon atoms, or a heterocyclic ring system, 5.93., one or more ring atorns may be a heteroatoni, (1g. , en, oxygen, sulfur, The cyclic carrier may be a monocyclic ring system, or may contain two or more rings, cg. fused rings. The cyclic carrier may be a fully saturated ring system, or it may contain one or more double bonds.

The ligand may be attached to the polynucleotide via a carrier. The carriers include (i) at least one “backbone attachment point,” preferably two “backbone ment points” and (ii) at least one “tethering attachment point.” A “backbone attachment point” as used herein refers to a functional group, cg. a hydroxyl group, or generally, a bond available for, and that is suitable for incorporation of the carrier into the bacl’hone, cg, the phosphate, or modified phosphate, rag, 2t) sulfur containing, backbone, ot‘a ribonucleic acid. A “tethering attachment point” (TAP) in some embodiments refers to a tuent ring atom of the cyclic carrier, tag, a carbon atom or a heteroatom {_distinct from an atom which provides a backbone attachment point), that connects a selected moiety. The moiety can be, cg, a carbohydrate, 51g monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide and polysaccharide. Optionally, the selected moiety is corniected by an intervening tether to the cyclic carrier, Thus, the cyclic carrier will often include a functional group, rag, an amino group, or generally, provide a bond, that is suitable for incorporation or tethering of r cheniical entity, eg, a ligand to the constituent ring.

The RNAi agents may be conjugated to a ligand via a. carrier, n the carrier can be (A) c. cyclic group or acyclic group; preferably, the cyclic group is selected from idinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [l >:olane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, ydrofuiyl and and decalin; preterably, the acyclic group is selected horn serinol ne or diethanolaniine backbone, [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS in certain specific embodiments, the RNAi agent for use in the methods ofthe invention is an agent selected front the group of agents listed in any one of'l’ahles £14, , 4, 5, 6,18,19, 2t), 21, and 23. These agents may thither comprise a ligand 1V. iRNAs Conjugated to Ligands Another modification ofthe RNA of an iRNA of the invention involves chemically linking to the RNA one 01“ more ligands, moieties or conjugates that e the activity, ar distribution or cellular uptake of the iRNA. Such es include but are not limited to lipid es such as a cholesterol moiety (Letsinger er n.1,, Proc. Natl, Acid. Sci. USA, 1989, 86: 11.1 6553-6556), cholic acid {Manoharan at at), Biorg. Med. Chem. Let, 1994, -1060), a thioether, e.g., beiy‘l—S~tritylthiol (Manoharaii at of, Ann. N, Y. Acad. Sci, 1992, 6601396309; l‘vlanoharan at all, Biorg. Anni. Chem. Let, 1993, 770), a thiocholesterol (Oberhauser er of, Natal Acids Res, 1992, 291533—538), an aliphatic chain, raga, dodecandiol or undecyi residues (Saisonuliielunoaras at (21,, EMBO J, 1991, 1—1118; y at 05., FEES Lent, 1990, 259:327-331}; Svinarcliult cf of, Biochimie, 1993, 75:4964), a pliosphoiipid, sag, di—lriexadecyl~ rac~glycerol or triethyl~ainmoniuni l,2~di—O~hexadecyl—rac~glycero—3—phosphonate (Manoharan at all, Tetrahedron Latin, 1995, 36:3651—3654; Shea 61‘ mi, Nucl. Acids Res, 1990, 1823777— 3783), a polyaniine or a polyethylene glycol chain (Manoharan e? mi, Nucleosides cot Nucleotides, 1995, 14:969—973), or adamantane acetic acid {Manoharan at 51.7., Tetrahedron Lem, 21) 1995, 3636516654), a palmityi ty (Mishra e! a!., Biochim. Biophys. Ania, 1995, 1264:1229 237), or an octadecylamine or hexyiamino—carbonyloxycholesterol moiety e at (1/7,, ,1.

Pharmacof. Exp. 1774811, 1996, 277192393 7). in one embodiment, a ligand alters the distribution, targeting or lifetime of an iRNA agent into which it is incorporated. 1n preferred embodiments a ligand provides an enhanced y for a selected target, 6g, molecule, cell or cell type, compartment, eg, a cellular or organ compartment, tissue, organ or region ofthe body, as, agn, compared to a species absent such a ligand. Preferred ligands Will not take part in duplex g in a duplexed nucleic acid, Ligands can include a naturally occurring substance, such as a protein (cg, human serum albumin (1153A), 1ow~density lipoprotein (1,131.), or globulin); carbohydrate (tag, a dextran, an, chitin, chitosan, inulin, cyclodextrin, Nnacetylgalactosamine, or hyaluronic acid); or a lipid. The ligand can also he a recombinant or synthetic molecule, such as a synthetic polymer, 8.53., a synthetic polyamino acid. Examples ofpoiyaniino acids include polyaniino acid is a polylysine (131.1), poly 1..r—aspaitic acid, poly L—glutarnic acid, styrene~nialeic acid anhydride copolyiner, l'snlactide—conglycolied) copolymer, divinyl ethernnialeic anhydride copolynier, N-(Z’. ~11ydroxypropyl)inethacrylainide nier (lli‘Vll’A), hylene glycol (PEG), polyvinyl alcohol {13VA), polyurethane, poly(2~ethylacryliic acid), ropylacrylainide polymers, or [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS polyphosphazine. Example ofpolyaniines include: polyethyleniinine, polylysine (PLL), sperrniiie, spermidine, polyaniine, pseudopeptide—polyaniine, peptidorninietic polyaniiiie, dendrimer polyarniiie, arginiiie, antidine, protarnine, ‘ElllOtllC lipid, cationic poiphyrin, (platerriary salt ot‘a polyamirie, or an alpha helical peptide, (I: Ligands can also include targeting , Cgt, a cell or tissue targeting agent, rag, a leetin, glycoprotein, lipid or protein, eg, an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can he a thyrotropin, melanotropin, lectiii, glycoprotein, surfactant protein A, Mucin carbohydrate, inultiyalerit lactose, n'iultivalent galactose, N~acetyl~ galactosainine, N~acetylngulucoseaniine inultiyalent niannose, multiyalent hicose, glycosylated ll) polyaminoacids, multiyaleiit galaetose, errin, hisphosphonate, polyglntaniate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, fol-ate, Vitamin El 2, Vitamin A, biotin, or an RGD peptide or RGD peptide n'iirnetic.

Other examples of ligands include dyes, intercalating agents (9.5,; acridines), crossnlinkers {8g psoralene, initomycin C), porphyrins (l7 PPCZl, texapliyriii, Sapphyrin), clic aromatic l5 arbons (éi.g., phenazine, dihydrophenaziiie), artificial endonucleases (cg. ESTA), lipophilic molecules, rag, cholesterol, cholic acid, adaniantane acetic acid, ne hutyric acid, diliydrotestosterone, l,3"Bis"0(hexadecyl)glycerol, loxyhexyl group, hexadecylglycerol, horneol, l, l,3 “propanediol, heptadecyl group, palniitic acid, niyristic acid,03n (oleoyl)lithocholic acid, ()3-{oleoyl)cholenic acid, diniethoxytrityl, or phenoxazinehnd peptide 2t) conjugates leg, anteiiiiapedia peptide, Tat peptide), allrylating agents, phosphate, amino, rnercapto, PEG (Cg, PEG—40K), fi, {h{lPEGl3, polyarnino, alhyl, substituted alleyl, radiolabeled markers, enzymes, haptens (cg. biotin), transport/absorption facilitators (e.g,, aspirin, Vitamin E, folic acid), synthetic ribonucleases (9.3 ole, 'hisiniidazole, histamine, irnidazole clusters, acridiiiedrnidazole conjugates, liu3+ xes of tetraazamacrocyclesl, ophenyl, HRP, or AP. s can he proteins, Cgt, glycoproteins, or es, C.g., molecules having a specific affinity for a co—ligand, or dies eg, an antibody, that binds to a specified cell type such as a hepatic cell. l_..igands can also include hormones and hormone receptors. They can also include non~peptidic species, such as , lectins, carbohydrates, Vitamins, cofactors, (A) c. multiyalent lactose, multiyalent galactose, Nnacetyl—galactosaniine, Nnacetyl~gulucosaniine multiyaleiit niannose, or niultivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator ot‘p38 MAP , or an activator ot‘NFmB.

The ligand can he a substance, tag, a drug, which can se the uptake of the iRNA agent into the cell, for example, by disrupting the cell’s cytosl'ieleton, rag, 3y disrupting the1II cell’s inicrotu‘oules microfilaments and/or intermediate filaments. 'l'he diu t can he, for 7 fi ,_ , [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS example, taxon, vincristine, stine, cytoclialasin, nocodazole, iaplaltinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoseryin. ln some embodiments, a ligand attached to an iRNA as described herein acts as a pharrnacokinetic modulator (PK modulator). PK modulators include lipophiles, bile acids, (I: steroids, phospholipid analogues, es, n binding agents, PEG, vitamins etc, Exemplary l’li modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, lglycerides, diacylgly‘ceride, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc. Oligonucleotides that comprise a number ot‘pl’iosphorothioate linkages are also known to bind to serum protein, thus short oligonucleotides, cg. , oligonucleotides of about ll) 5 bases, l0 bases, l5 bases or '20 bases, comprising multiple ofpliosphorothioate linkages in the backbone are also amenable to the present invention as ligands (eg. as PK modulating ligands). ln addition, aptaniers that bind serum components (rag serum proteins) are also suitable for use as PK modulating s in the embodiments described herein.

Ligand—coniugated oligonueleotides ofthe invention maybe synthesized by the use of an oligonucleotide that bears a pendant reactive tunctionality, such as that derived liom the attachment ot‘a linking molecule onto the oligonucleotide (described below), This reactive oligonucleotide may be reacted directly with commerciallynavailable ligands, ligands that are synthesized bearing any of a y ofprotecting groups, or ligands that have a linking moiety ed thereto. 2t) The oligonucleotides used in the conjugates ot‘tlie present invention may be con‘v'eniently and routinely made h the well—known technique id—pliase synthesis, Equipment for such synthesis is sold by several vendors including, for example, Applied tems (Foster City, Califi). Any other means for such synthesis known in the art may additionally or alternatively be employed. lt is also known to use similar techniques to prepare other oligonucleotides, such as the phosphorotl’iioates and alkylated derivatives. ln the ligand—coniugated oligonucleotides and ligand—molecule bearing sequencewspecilic linked nucleosides ot‘the present invention, the oligonucleotides and oligonucleosides may be led on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or nucleotide or nucleoside coniugate precursors that already bear the linking n'ioiety, ligand~ (A) c. tide or side~coniugate precursors that already bear the ligand molecule, or non" side ligand-bearing building blocks.

When using nucleotidesconjugate precursors that y bear a g moiety, the synthesis of the sequence—specific linked nucleosides is typically ted, and the ligand molecule is then reacted with the linking moiety to form the ligandnconjugated oligonucleotide. in some embodiments, the oligonucleotides or linked nucleosides ot‘the t invention are synthesized by an automated synthesizer using phosphoramidites derived front ligand~nucleoside [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS conjugates in addition to the standard horamidites and non—standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis. .4. Lipid Conjugates ln one einbodin'ient, the ligand or conjugate is a lipid or based molecule. Such a (I: lipid or lipidnbased molecule preferably binds a serum protein, cg, human serum albumin (BSA). An BSA binding ligand allows for distribution of the conjugate to a target tissue, eg, a idney target tissue ofthe body. For example, the target tissue can be the liver, including hymal cells of the liver. Other molecules that can bind HSA can also be used as ligands.

For example, naproxen or aspirin can be used, A lipid or lipidnbased ligand can (a) increase ll) resistance to ation of the conjugate, (b) se targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, sag, llSA.

A lipid based ligand can be used to inhibit, ag, control the binding ot‘the ate to a target tissue, For e, a lipid or lipidnbased ligand that binds to BSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipidwbased ligand that binds to llSA less strongly can be used to target the conjugate to the kidney. ln a preferred embodiment, the lipid based ligand binds BSA. ably, it binds HSA with a sufficient affinity such that the conjugate will be preferably distributed to a nonnkidney tissue. yer, it is preferred that the affinity not be so strong that the ltlSA—ligand binding 2t) cannot be reversed. ln another preferred embodiment, the lipid based ligand binds HSA weakly or not at all, such that the conjugate will be preferably distributed to the kidney. Other moieties that target to kidney cells can also be used in place of or in addition to the lipid based ligand. in another , the ligand is a moiety, 6g, a vitamin, which is talten up by a target cell, eg, a proliteratin g cell, These are particularly useful for treating disorders eh rised by unwanted cell proliferation, 9g, of the malignant or non—malignant type, Cgt, cancer cells.

Exemplary Vitamins include Vitamin A, EL, and ii, Qtlier exemplary Vitamins include are E Vitamin, e.g., folic acid, BlZ, riboflavin, biotin, pyridoxal or other Vitamins or nutrients taken up by target cells such as liver cells. Also included are HSA, and low y lipoprotein (LDL), B. Cell Permeation Agents in another aspect, the ligand is a cell—permeation agent, preferably a helical cell— perineation agent. Preferably, the agent is arnphipathic. An exemplary agent is a peptide such as tat or antennopedia, lf the agent is a peptide, it. can be modified, including a peptidylniimetic, invertomers, nonmpeptide or pseudo—peptide linkages, and use of D~anrino acids. The helical agent is preferably an helical agent, Which preferably has a lipoplrilic and a lipophobic phase. ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS The ligand can be a peptide or peptidoniimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of g into a defined three- dimensional structure similar to a natural peptide. The attachment ide and peptidomirnetics to iRNA, agents can affect pharn'iacokinetic distribution of the iRNA, such as by (I: enhancing cellular recognition and absorption, The peptide or peptidomimetic moiety can be about 5—50 amino acids long, eg, about 5, ll}, l5, 2t), 25, 30, 35, 40, 45, or 50 amino acids long.

A peptide or omimetic can be, for example, a cell permeation peptide, cationic peptide, athic peptide, or hydrophobic peptide (cg, consisting primarily onyr, Trp or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinlied ll) peptide. in another ative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic lt/lTS—containing peptide is Rlltill: having the amino acid sequence AAVALLPAVLLALLAP (SEQ ll) NO: 9). An RFGP analogue (cg, amino acid sequence AALLPVLLAAP {SEQ ll) NO: l0) containing a hydrophobic M'I'S can also be a targeting moiety. The peptide moiety can be a “delivery” peptide, Which can carry large polar molecules including peptides, oligomiicleotides, and protein across cell membranes.

For e, sequences from the lllV Tat protein (GRKKRRQRRRPPQ (SEQ ll) NO: l l) and the Drosophila Antennapedia n {RQlKlWFQNRRh/lKWKK (SEQ ll) NO; l2) have been found to be capable of functioning as delivery peptides, A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage—display 2t) library, or ad~oneecompound (0803C) atorial library {Lani at 515., Nature, 354:82 84, l99l l Examples ot‘a peptide or omimetic tethered to a dsRNA, agent Via an incorporated monomer unit for cell targeting purposes is an argininenglycine—aspartic acid (lltfiiD)—peptide, or RGD mimic. A peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any ol’the stiuctural modifications described below can be utilized.

An RGD peptide for use in the compositions and methods ofthe invention maybe linear or , and may be modified, sag glycosylated or methylated, to facilitate targeting, to a specific tissuet’s), RGD—containin g peptides and peptidiomimemtics may include D~amino acids, as well as synthetic RGD mimics. in on to RGD, one can use other moieties that target the integrin ligand. Preferred conjugates of this ligand target PECAM—l or VEGE.

A “cell permeation peptide” is capable ofpermeating a cell, eg, a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A, microbial cell— permeating peptide can be, for example, a dnhelical linear e (cg, LL37 or Ceropin Pl), a disultide bond—containing peptide leg, 0: —defensin, B—defensin or bactenecin), or a peptide containing only one or two dominating amino acids (eg, PR~39 or indolicidin). A cell [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS permeation e can also include a nuclear zation signal (NLS), For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fitsion peptide domain ot‘ltllVd gpé’ll and the NLS ot‘S‘v’élO large T antigen oni at at, Nucl. Acids Res 3l :27l7~2724l, 2003).

(I: C. Carbohydrate Cory’ugaz’es in some embodiments ofthe compositions and methods ofthe invention, an iRNA ucleotide turtlier comprises a carbohydrate. The carbohyd “ate conjugated iRNA are advantageous for the in viva delivery of nucleic acids, as well as compositions suitable for in viva therapeutic use, as described herein, As used herein, “carbohydrate” refers to a compound ltl Which is either a carbohydrate per 59 made up of one or more nionosaccharide units having at least 6 carbon atoms {which can be linear, branched or cyclic) with an oxygen, nitrogen or sulfur atom bonded to each carbon atom; or a compound having as a part thereoi’a carbohydrate moiety made up of one or more monosaccharide units each having at least six carbon atoms (which can be linear, branched or cyclic), With an oxygen, nitrogen or sulfur atoni bonded to each carbon atom. entative carbohydrates include the sugars (inono~, dis, tri- and oligosaccharides containing from about 4, 5, iii, 7, 8, or 9 nionosaccharide units)“, and polysaccharides such as starches, en, cellulose and polysaccharide gums. Specific monosaccharides include C5 and above (eg, C5, C6, C7, or C8) sugars; dim and trisaccharides include sugars having two or three nionosaccharide units (8g 15 on C7, or C8) 2t) in one embodiment, a carbohydrate conjugate for use in the compositions and methods of theinnntion is a inonosaccharide in one ment, the nionosaccharideis an N— acetylgalaetosainine such as HOACHN!WON/N’YNWNWQ 0 Formula 11.

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS In another embodiment? a carbohydrate conjugate for use in the compositions and methods ofEhHe invention is seiected from the group consisting of: \X/ANX/QWWNWNfix0 H H ACHN ‘_{O H;:;[:\:IOWYNHMEN \V/WNV,«VO\1\LM 0 (“3 ::\k/OWE§EMEfiE/LQj)”AcHN O Formuia 11, HO» HO HO' k/O A 0 “JV/«Om»,\JVANAK! Ho Ho H HO 0‘ OwGWOw/NNW”O\V;’E~‘N HE} EE 0 .

HQ \zE—QHEM J QVAC)/\,.OVI\N , , H Formula 111, ~.. —0 HQ QNAO/NN/QL OH 1 HQ / ,NM HQ%QwQ/~VO,0 ,; (J; NHAc Formula. IV, HQ {3“ “Gm/’QV/NO NHAC L\_____0 HO r _____ Q Hi) ’Qwfl’j NHAc 'Fon’mfla V, [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS 6 Formula VI, NHAC {0 OH 0» . ”Q HO O\/’\v/,1 NHAc Fenmfla VII, ,BZOX L0OBZ 82: Y’ E A 820 "’ 082 GAG a IX, Q 9’ HO ‘ 0 r”) 3“ “I,«TA/ V’\Q/\\/OV’\EE§AQO K AcHN I Formula X [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS H Fermula X1, O O“ HO‘%:EHO J 0x/\/ L . EVE {'3 Formula X11, Ha.)“Gigs/OTQk/O‘VN/jk‘NAV/VNNEO ACHN H O H) 0H HO§1¥ET 0 N. /O O\flkkm‘m’N‘n’QV/V WH AcHN E 5?01mm la X111, 0 Formula XIV, (J! 0 meul 2: XV, [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS 0 meula XVL HO O O NH HO = HO _ 0 Emma El 12; XVII, HOH OWLNW 0 F011111113 XVEEL “figiowwfimioooMW E0rmuia XIX HO 0-O aigwww U: O Eormula XX, OOHO®AHO -O WWMW 0 Eorinula XX}, [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS Hgo 'O O WNH 0 a XXll.

Another representative carbohydrate conjugate for use in the embodiments described herein includes, but is not d to AcHN H - a/ H )H Li) (J X0", \~ ,w(\ Q E 1%o 1 x . v ”WW ’A‘NV’NV/ 0 {7‘ ACHN H NHTrmN/\\IT,.~£V/\/\/I§,O ('2 A i) f O a, 'J c;- I/ i t” a / x: / /’\~/"\v"\/Jl~le‘i‘\ (I: CAN/NR {Formula XXIII), when one ofX or Y is an oligonucleotide, the other is a hydrogen. ln some embodiments, the carbohydrate conjugate further comprises one or more ll) additional ligands as described above, such as, but not limited to, a PK modulator and/or a cell permeation e.

D. Linkers in some embodiments, the conjugate or ligand described herein can be attached to an iliNA oligonueleotide with various linkers that can be cleavable or non-cleavahle.

The term ”linker" or “linking group” means an c moiety that connects two parts of a. compound, (1g, covalently attaches two parts of a compound Linkers typically se a. direct bond or an atom such as oxygen or sulfur, a unit such as NR8, C(O), C(O)NH., $0,, $03,, SOZNH or a chain of atoms, such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alleynyl, arylalkyl, arylall-renyl, arylalkynyl, heteroarfy'lalkyl, heteroaiylalkenyh heteroarylalltynyl, heteroeyclylalkyla, heteroeyclylalkenyh heteroeyclylalkynyL aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyL [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS alkylaiylallsyl, alkylaijy’lallsenyl, arylalkynyl, allcenylaiylallsyl, alkenylarylalkenyl, alkenylaiylallrynyl, alleynylaiylallryl, alliynylarylallrenyl, alkynjy'larylalkynyl, eteroawlalkyl, alkylheteroawlalkenyl, alkylheteroarylallrynyl, alkenylhetercarylallcyl, allsenylheteroarylalken yl, alkenylheteroarylallsynyl, alkynylheteroarylalkyl, (I: allsynylheteroaiylallsenyl, allcjv'nyllieteroaiylallrjy’nyl, allsyllieterocyclylalkyl, alliyllieteroejy'clylalkenyl, alkylhererocyclylallrynyl, allrenylheterocyclylalltyl, allienylheterocyclylallrenyl, allcenylheterocyclylallcynyl, alkynylheterocyclylalkyl, alleynylheterocyclylalltenyl, allrynylheterccyclylallcynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, lheteroaiyl, allsynylliereroaryl, which one or more methylenes can be ll) interrupted or terminated by O, S, 8(0), 80;, MRS), {1(0), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or tituted heterocyclic; where R8 is hydrogen, acyl, aliphatic or substituted aliphatic. ln cne embodiment, the linker is between about l—24 atoms, 2624, 3&4, $24, 5—24, 6—24, 648, 7~l8, 848 atoms, 7—l7, 847, are, 7~l6, or 8—16 atoms.

A cleavable linlcing group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together. ln a preferred ment, the cleavable linking group is cleaved at least about ill times, 20, times, times, 49 times, 50 times, 60 times, 70 times, 80 times, 90 times or more, or at least about lGQ times taster in a target cell or under a first nce condition (which can, 9g, he selected to 2t) mimic or represent ellular conditions) than in the blood ot‘a subj ect, or under a second reference condition (which can, eg, be selected to rnimi c cr represent conditions found in the blood or serum).

Cleavahle linking groups are susceptible to cleavage agents, eg, plil, redox potential or the presence ofdegradative molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood. Examples cf such degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, cg, oxidative or reductive enzymes or reductive agents such as rnercaptans, present in cells, that can degrade a reclox cleavable linking group by ion; ses; endosornes or agents that can create an acidic environment, eg, those that result in a pH of five or lower; s that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.

A cleavable linlrage group, such as a icle bond can be susceptible to pH. The pH of human serum is 7.4, while the average ellular pll is slightly lower, ranging from about ’7.l~ 7.3. Endosomes have a more acidic pH, in the range of 5.54500, and lysosomes have an even more acidic pll at around 5.0. Some linkers will have a cleavable g group that is cleaved at [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS a preferred pH, thereby ing a cationic lipid from the ligand inside the cell., or into the desired compartment ofthe cell.

A linlter can include a cleavable ng group that is cleavable by a particular enzyme.

The type of cleavable linking group incorporated into a linlcer can depend on the cell to be (I: targe ed. For example, a liverntargeting ligand can be linked to a cationic lipid through a linker that includes an ester group. Liver cells are rich in ses, and ore the linker will be cleaved more efficiently in liver cells than in cell types that are not esterase—rich. (Either cell“ types rich in ses include cells of the lung, renal cortex“, and testis Linkers that contain peptide bonds can be used when targeting cell types rich in ll) peptidases, such as liver cells and synoviocytes. ln general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. lt will also be desirable to also test the ate cleavable g group for the ability to resist cleavage in the blood or when in t with other non—target tissue. Thus, one can determine the relative susceptibility to cleavage between a lirst and a second condition, where the first is selected to be indicative ot‘cleavage in a target cell and the second is selected to be indicative of cleavage in other tissues or biological fluids e.g.,, blood or serunr The evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture? or in whole s. lt can be useful to make initial evaluations in cell-free or culture conditions and to 2t) confirm by further evaluations in whole animals. ln preferred embodiments, useful candidate compounds are cleaved at least about ’2, Ala ltli, 20, 30, All), 50, 60, '70, 809 9th or about l0!) times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions). i. Redo/it c eat/“able g grows in one embodiment, a cleavable linking group is a redox cletvable linliing group that is cleaved upon reduction or oxidation. An example of reductively cleavable g group is a disulphide linking group (“S-'8“). To determine if a candidate cleavable g group is a suitable “reductively cleavable linking group? or for example is suitable for use with a particular (A) c. iRNA moiety and particular targeting agent one can look to methods described herein‘ For e, a candidate can be evaluated by incubation with dithiothreitol (D’lll‘), or other ng agent using reagents know in the art, which mimic the rate ot‘cleavage which would be observed in a cell, egw a target cell. The candidates can also be evaluated under conditions which are selected to mimic blood or serum conditions. in one, candidate compounds are cleaved by at most about MW; in the blood. in other embodiments, useful candidate compounds are degraded at least about 2, 4, ltl, 2t), 3t), 4t), 50, 60, 70, 8t}, 9t), or about l {it} tirnes taster in the cell {or [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS under in 133770 conditions selected to mimic ellular conditions) as compared to blood {_or under in vitro conditions ed to mimic extracellular conditions). The rate of cleavage of ate compounds can be determined using standard enzyme leinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular (I: media. ii. Phosphate~bnsed Clea 37625116 linking groups in another ment, a ble linleer comprises a atesbased cieavable g group. A pl’iosphatebased cleavable linking group is cleaved by agents that degrade or hydrolyze the phosphate group. An example of an agent that cleaves phosphate groups in cells ll) are enzymes such as phosphatases in cells. Examples ofphosphate-based linking groups are -()u P(O)(0Rk)~0—, —(I>~P(S)(0Rl<)-{)», *O-MSXSRlQ—Og ~S—P(O)((}Rl<)*O-, -(}P{())(()Rl<)~S—, —S~ ?(O)(ORk)—S~, ~O—P(S)(0Rl<)~S—, —S—P(S)(ORK}~O—, —O~P(O)(Rl{)~O—, —O~P(S)(Rl{)~O—, u. ~ l’(0)(l{k)v0n, nS~P(S)(Rl()vOn, nS~P{O)(Rl<)~Sn, )( ElgnS—e Preferred embodiments are —Ow l’(())(0ll)—O-, —0—l?(S)(Qll)—Q—, -O—l’($)(Sll)-O—, 0)(0ll)u0-, -0—l’(0)(0l9l)-Sn, nS~ l5 P(O)(Oll)sS-, ~t}wP{8)(()ll)-Ss, “3-P(S)(()ll)~0—, —(I)*P{{)}(ll)~()—, —(I>~l’(S}(H)-{)w, “S-P(:(I>)(ll)—0, ~S~P(S)(ll)—O~, )(l’l)—S~, ~O—PlS)(ll)—S~. A preferred ment is ~0—l?’(0)(0l:l)~O—. ’l‘hese candidates can be evaluated using methods analogous to those described above. iii. Acid demonic [inking groups in another embodiment, a cleavable linker comprises an acid cleavable linking group. An 2t) acid cleavable linking group is a linking group that is cleaved under acidic ions. in preferred en’ihodiments acid hle linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (cg, about 6.0, 5.75, 5.5., 5025, 5.0, or lower), or by agents such as enzymes that can act as a general acid. ln a cell, specific low pll organelles, such as endosornes and lysosomes can provide a cleaving environment for acid cleavable linking groups. lilxamples of acid cleavable g groups include but are not limited to hydrazones, esters, and esters of amino acids. Acid eleavahle groups can have the general formula —C=NN—, C(O)O, or 0&0).

A preferred embodiment is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aiyi group, substituted alkyl group, or tertiary alltyl group such as dimethyl pentyl or t~butyl These candidates can he evaluated using methods analogous to those described above. iv. Ester-based [inking groups in another embodiment, a cleavable linlrer comprises an esteru'based cleavable linking group. An ester-based cleavable linking group is cleaved by enzymes such as ses and amidases in cells. Examples of ester~based cleavable linking groups include but are not limited to esters of ene, alkenylene and allcynylene groups. Ester cleavable g groups have the general formula —C(O)0—, or —0{3(())—. These candidates can be evaluated using s analogous to those described above.

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS v. Peptide-based cleaving groups in yet r embodiment, a cleavable linker comprises a peptideubased eleavabie ng group. A peptide-based eleavable linking group is cleaved by enzymes such as peptidaiess and proteases in cells. Peptide~based eleavable linking groups are peptide bonds (I: formed between amino acids to yield eptides (ego, dipeptides, tripeptides etc.) and polypeptides. lleptide—based eieavable groups do not include the amide group (—C/{OlNl’l—H). 'l'he amide group can be formed between any alkylene, lene or alkynelene. A peptide bond is a l type of arnide bond formed between amino acids to yield peptides and ns, The peptide based cleavage group is generally limited to the peptide bond (1.9., the amide bond) ll) formed between amino acids yielding peptides and proteins and does not include the entire aniide fiinetionai group. Peptidebased Cleavable linking groups have the general lorinnla NHClrlRAC(0)NllClthBC(O'}, where RA and RB are the R groups of the two adj aeent amino acidst These candidates can be evaluated using methods analogous to those described above in one embodiment, an iRNA oftlie invention is eoningated to a carbohydrate h a linker. Non—limiting examples ofiRNA carbohydrate eonj ngates with linkers oftlie compositions and methods ofthe invention include, but are not limited to, HO g :40X ”9’/-~A/o\xN/‘x‘xNx/N" AcHii YO r' L\SL/. E K>erO O N (4\ H, H ‘A/OVV\T/NVAV/er"\/O\/ H TNx/NV/\~’A\/ N 1% ACHN l 0'“ ' Hsfi’xn I4C‘IN/TLX/Cx/VNTN,N"\/\N’j; HHHH 61' H H {Forniula XXIV), HUéiilio H H0___ /;\§/OWL\Nf\/’\V/‘\¢NYO AcHN H o x—o‘ HQ 0H (j’q/O‘Y sexileo a H i n , 0A x; NWAVNYoW/i—N *r X \{Tl/‘o (:HN it H x: O y O I HO OH Nix/o C? l —113 H C' .X1 ":3 HOA/T‘QVDVN (#‘HNVH’HWANJ‘O'. y ” 1' I3 7“ V v - AcHN H {p orninla XXX/)9 HO XOH \ewo O HG «PNX/QVVK“EM/“VNYC Act-EN H G X~O HO:’Oi'i ' o idle" o H ' H I ' H HQ /::\'§/ \xAVJK 1 , .

ACHN NWNNYO\A’j»N\Tr/\\AN \HOV/jO/HNR’NMOH o , 0 rl x o y Hjji/flWOW/UMNM/“Nko’0 H E3 f x=1--’%0 y 1'15 AcHN H [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS (Formula XXVI), HO‘§,- 0 if H W EWNAVNYO HO AC;;...\,O X'O . (3.

H:E:\\VOVWNMWN-QVN/TVNNS-----ski/YE'\{’MO Z30"fi ACHN. H IO MEG 3—030 \i 2 ' .3 I" ACHN (Fgrnmia XXVH),0H :0 O ACHN H o are Hf.) G}: ’ O i. /Qty N ,0 H.. k . i.3 HAHN WH A: . O.

HOL/mOW‘KNAN/W‘NYO‘ME:\3 5*~ \ AcHN b wa z o y Hog? o x = 0—30 , H O = 1—15 ”’L y ‘30 Or _____ wivevw'Nw/VNA z 2 1.3g._ Am-iN, H {Formuia XXX/Ii i ), H0 0H0 0 W N HOLN NAV/‘W 3"0“». X‘0 ACHN H (-3 a 3 (Sm/O"!V OH 3 l'iO‘é’i‘QVi-jvw x/ NVOW”?Nxfi/“VQQV’ELQm'5‘-5W W0CHo ’0 O \ H . , - H H N ‘ ‘ H {-3 / (-3 x 1. O riO§Q:O x :1-30 = 145 e ELH f:a v W NVW\E o HO A‘s/«<3; z = 1-20 (Formuia XXLX), and \Eze O , H oVNJLHE \/V\VNE.V\:fl.‘ HO"KCHN X"O WEE i k W‘C’N o 0 o H NH MN ACHN H I J t n x z 0 V "’ +30 0“ x 1—3:} H y— 1— 5 HO Sgt/’0 (RAJ? z: 1-20 ACHN. “~va 0’” (Formula XXX), thé‘fl one ofX or Y is an eiigeriueleetide, the other is a hydrogen.

In certain embodiments of the compositinns and, methods of the invention, a iigand is one er more “CiaENAC” {N—aeet‘yigaiaetosamine) derivatives attached. through a bivaient 0r ent branched tinker. ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS In one enihediinent, a 135RNA Ofthe invention is enniugated to a bivalent 131 trivalent branched linker selected from the gtettp of ures shown in any of fiattnula (XXXI) (XXXIV): Fonnna XXXI 3311111111311 XXX” PZA'Q2A_R2A| T2A_L2A 2A )P3A“Q3A“R3A, T3A_L3A q £3 PZB-Q2B_R2B]_TZB_L2B 3634.38 2B \fi)38_(33l3_1{38 35 q ‘93 PSA-QSA-RSA T5A_L5A P4A_Q4A_R4A]_T4A-L4A 3q5A q4A P5B_Q5B_R5B ]—T5B—L5B P4B-Q413-R4Bl—4B 4B q C-R5C]—T5C—L5Cqsc , ; Fonnula XXXIH Fonnnla XXXlV 112A, 1128, 113A, 1138, 114A, 1143, 115A, 1153 and 115C represent independently fer eaeh 111:1:1111en1te GHEO and wherein the repeating unit can he the same 01 different; 2A ABA 3A B«v 4A 43 1 . —\ 4* ,13’,13 5A 3b 1) ,1? ,1? ,1? ,1) ,1} ,1>‘,11T, MZA 3 'gLB m3A ~~3B M4153 4B,1“,1”1‘, ~58 ”SC ,1 ,1, ,1 ,1 ,1 1 ’ ’/ are each independently for each occurrence absent, (It), NH, (:3, S, (KEG), N333C{()3, CH3, (33333333 111' (331330; QM, Q23, QM, Q33, QM, Q43, QSA, Q53, Q5C are independently for each occurrence , ne, substituted alkylene Whetin one 01111011: niethylenes can be intei‘inpted or terminated 233 by one or mere 03‘0, S, 53(0), 803, N(RN3, C(R’)::=C('R”3, CEC 01“ (3(0); 312““, 31.23, R.3A, R33, RT”, Rm, RSA, 32323153 are 1111531 indepemlentl) for each 011:1111e111e absent, N11, 0, s, CH3, C1010, 111011111, Naengaaegn), “c.101men11131311nm, co, CH=N~O, [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS “0711 ‘7.....N )7 “773w SWSW 581,2 ‘8 ”X W , ,ngswm heterocyciyi; LzA, 1373,1237, L73, L49, L43, LSA, LSB and L5C represent the 1igand; 1.6.. each ndentiy for each occurrence a rnonosaccharide (such as GaiNAc), disaccharide, trisaccharide, tetrasacch aride, ohgosaccharide, or po1ysaccharide; 111111113 is 1:1 or amino acid side chain.’1‘rivaient ating GaiNAe derivatives are partieu1ar1y usefui for use with RNAi agents for inhibiting the expression of a target gene, such as those oi‘fornntia (XXXV): Forn'iu1a XXXV PSA-QSA-RSA T5A_L5A PSB-Q5B_R5B]_BT5B_L5B PSC-QSC-RSC ]_CT5C_L5C wherein L5AL and L5Crepresent a inonosaceharide, such as GaiNAe derivative iesofsuitahie hivaient and triva1ent branched 1inker Oronps conjugating Ga11\Ac derivatives inc1ude, but are not 1irnite1'1 to, the ures recited above as thrntuias 11, V11, X1, X, and X111, Representative 118. patents that teach the preparation of RNA conjugates ine1ude, but are not iiinited to, US Pat, Nos, 4 ,828,979; 4948,882; 5,218,105; 5,525,465; 5,541 ,313; 5,545,731); ,552538; 5,578,",5717 31; 5,591,584;5,11)9,124; 5, 118,8112; 5,138 ,1145; 5,414,077; ,486,6113;5,512 ,4;39:,55,78,718; 5 ,,61181146; 4587,044; 4,6115,735; 125,; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; ,830; 5 63; ,214,136; 5,082,831.11 5,112,963; 5,214,136; 5,245,022.; 5,254,469; 5 ,258 ,5116; 5,262,526 ,272,250;5 ,292 ,873; 5,317 ,1198;5,371 ,241 5,,391,723; 5,416,2113, 5 ,451,463; 5 ,510,475; 667; 5,514,785;5,5655527,5 ,5157 ,810; 5,574, 142;:57,585,481; 5,587,371; 595,726;1}! ,597 6,96; 5,599,923;5 ,599928 and 5,688,94,1 6294664 6,2G,017, 6 ,783,931; ,6,576,752; 11,91111,297; 7I,1137,646; 8, 1116 1,122, the entire contents of each ofwhieh are hereby incorporated herein15y reterence. 11 is not necessary for 1111 positions in a. given compound to he uniformiy n'iodifi ed, and in fact more than one ofthe aforementioned modifications can be incorporated in a singie [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS compound or even at a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds. ric” iRNA compounds or “chimeras,” in the context ofthis invention, are iRNA compounds, prei‘erahly dsRNAs, which contain two or more chemically distinct regions, each (I: made up of at least one niononier unit, i.e., a nucleotide in the case of a dsRNA compound. 'l'hese iRNAs typically contain at least one region wherein the RNA is modified so as to confer upon the lRNA increased ance to nuclease degradation, increased cellular uptake, and/or increased binding ty for the target c acid, An additional region ot‘the iRNA can serve as a substrate for enzymes capable of cleaving RNAzlLlNA or RNAzRNA hybrids. By way of ll) example, RNase ll is a cellular endonuclease which cleaves the RNA strand of an RNAzDNA duplex~ Activation ot‘RNase ll, ore, results in cleavage of the RNA , tlierehy greatly ing the efficiency of iRNA inhibition of gene expression. Consequently, comparable results can often he obtained with shorter iRNAs when chirneric dsRNAs are used, compared to phosphorothioate deoxy dsRNAs hybridizing to the same target region. Cleavage ot‘the RNA target can he routinely detected by gel electrophoresis and, it‘necessary, associated nucleic acid hybridization techniques known in the art, in n instances, the RNA of an iRNA can be modified by a non~ligand group. A nuni ier ofnonnligand molecules have been conjugated to iRNAs in order to enhance the activity, cellular distribution or cellular uptake ofthe iRNA, and procedures for performing such 2t) conj ugations are available in the scientilic literature. Such nonsligand moieties have included lipid moieties, such as cl’iolesterol (Kuhn, T. at at, Biochem. Biophys. Res. Comm, 2007, 365(l):54m61; Letsinger at 03,, Proc. Natl. Acad. Sci. USA, l989, 8616553), cholic acid oharan at at), Bioorg. Med. Chem. Lett, l994, 42l 053), a thioether, 51g", liexyl-S—tritylthiol {h/lanoharan er al., Ann. N. l”. Amid. Sci, l992, 6; h’lanoharan er a[., Biom‘g. Med. Chem.

Let, l993, 322765), a olesterol (Oherhauser at oi, Nucl. Acids Rae, l 992, 202533;), an aliphatic chain, egg, ndiol or undecyl residues {SaisonnBehmoaras ex (217., EMBOJ., 199l, ltlzl l l; Kahanov at all, FEES Left, l99ll, 259:327; Svinarchuh at 05., Bind/limit), 1993, 75:49), a phospholipid, sag, di~hexadecylsrac-glycerol or triethylammoniurn l,2sdi—t}hexadecyl—rac~ glycero—3~H~phosplionate (Manoharan at all, Tetrahedron Left, W95, 36:365l ; Shea. at til, Nzricf.

Acids Res, l990, l823777), a polyainine or a polyethylene glycol chain (Manoharan at 0317., eoirides &: Mtcleot’ides, l995, l41969), or adamantane acetic acid (Manoharan at all, Tetrahedron Lent, l995, 36:365l), a yl moiety (Mishra at at, Biochim. Biophys. Acid, l995, l264lz229), or an octadecylaniine or mino~carbonyl~oxycliolesterol rnoiety (Crooke 61017., J. Pharmacoi. flap. Then, l996, 2772923). entative United States patents that teach the preparation of such RNA conjugates have been listed above. 'l'ypical conjugation ols involve the synthesis of an RN As hearing an aminolinlrer at one or more positions of the [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents, The conjugation reaction can be performed either with the RNA still bound to the solid support or lollowing cleavage of the RNA, in solution phase. Purification of the RNA conjugate by l*ll2l..rC lly affords the pure conjugate.

EV. Belivery of an iRNA of the invention The delivery of an iRNA of the invention to a cell eg, a cell within a subject, such as a human subject (tag, a subject, in need thereof, such as a subject having a ment component Cit—associated disease) can be achieved in a number of different ways For example, delivery ill may be performed by contacting a cell with an iRNA of the ion either in vitro or in viva. in viva delivery may also be performed directly by administering a composition comprising an iRNA, tag, a dsRNA, to a subject Alternatively in WW2) vry maybe nied indirectly by administering one or more vectors that encode and direct preession ofthe iRNA. ’l‘hese alternatives are discussed turther below ral any method ol delivering a nucleic acid molectlie (in wI‘m or in vim) can be d for use with an iRNA ofthe invention (see ag , rS“land lulian er ( l992) Trench Cch” Biol 2(5): l 39l44 and W09402595, which are incorporated hereinby reference in their entireties). For in viva delivery, factors to considerin order to deliver an iRNA molecule include for example biological stability of the delivered molecule prevention otnon-specific 29 ettects, and accumulation ot the delivered moleculein the target tissue. The nonsspecil‘ic eftects of an iRNA can be minimized by local administration, for example, by direct injection or implantation into a tissue or topically administering the preparation. Local administration to a treatment site maximizes local concentration of the agent, limits the exposure of the agent to systemic tissues that can otherwise be harmed by the agent or that can degrade the agent, and permits a lower total dose of the iRNA le to be administered Several studies have shown successful own of gene products when an iRNA is administered locally. For example, intraocular ry of a VEGF dsRNA by intravitreal injection in cynomolgus monkeys (Tolentino, Ml, at a! (2004) Return, 242l32~l 38) and subretinal injections in mice , Si, at a! (2993) Mai. We 9:2 l 92l 6) were both shown to prevent neovascularization in an (A) c. experimental model of age-related r degeneration. in addition, direct intratumoral injection ofa dsRNAin mice reduces tumor volume {l’illeJ , e! a! (2995) Moldherl l 1267- 274) and ‘an prolong survival ofturnorshearing mice (Kim, WI., 61' a! (2006) Ariel. Ther. 1434} :0 li, S, at #12007) M0! Titer l‘3.SlS5223) RNA interferitnte has also shown success with local delivery to the CNS by direct injection (Born, Gt, er til )1’Vudel'c Acids 322e49; ’l‘an, l2lil. stall2005) Gene 2225} 66; Makiniura, ll, e! a! eBil/[C Nemosci. 33l8; Shishltina GT er a! (2004) Neuroscience l '29:52l528; Thakker,l: at a! (2004) Proc. Noll.

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS ed set by DPS Acad. Sci U.S4 lGl: l727t}l7775; Alianean., 31117 (208317. 1‘7’8111‘011771319707. 602) and to the lungs 'hy intranasal administration (liloward, RA, et a! {2066) 71/707. 771er l4z476—484; Zhang, X, at 1:17 (2904) .7. 19707. Chem. 279:l 067749684; Bitlt'o, ‘17., 81 1:17 (2005) Nat. M'ea’. l l :50“ 55). For administering an iRNA systemically for the treatment ol‘a disease, the RNA can be (I: modified or alternatively red using a drug delivery system; hoth methods act to prevent the rapid degradation ofthe dsRNA by endo— and exo-nucleases 1'11 vim Modification olthe RNA or the pharmaceutical carrier can also permit targeting of the iRNA composition to the target tissue and avoid undesirable oftltarget ellects iRNA molecules can he modified by chemical conjugation to ilie groups such as cholesterol to enhance cellular uptake and prevent ll) degradation. For e, an iRNA directed against liXpoB conjugated to a lipophilic cholesterol moiety was injected systemically into mice and resulted in l-rnoclct‘lown ol‘apoB mRNA in both the liver and jejunum cheli, 7., 131‘ 117 (2004) 7‘s(171111). 432: l73~ l78) Conjugation ot an iRNA to an aptamer has heen shown to inhibit tumor growth and mediate tumor regression in a mouse model ofprostate cancer (McNamara. Kl, er a! (2006) Nat. 7370196711107. 24zltltl5ullll5). hit an alternative embodiment the iRNA can he delivered usingdrug delivery systems such as a nanoparticle. a dendrimer, a polymer, liposomes, or a cationic delivery system. hositively charged cationic delivery systems facilitate binding of an iRNA molecule (negatively charged) and also enhance interactions at the negatively d cell membrane to permit efficient uptake of an iRNA hy the cell. Cationic lipids, dendrimers, or polymers can either he hound to an iliNA, 29 or induced to form a vesicle or micelle (see cg, Kim Sill, at 1:17 (2908) Journal 21711771267) Reiease l291’2):l 07~l l6) that encases an iRNA. The tormation of vesicles or elles fitrther prevents degradation ofthe iRNA when administered ically. Methods for making and administering cationic" iRNA xes are well Within the abilities of one skilled in the art (see eg, Sorensen, BR. 18! 117200317. Moi. 77707327176l—766; Venna, UN. ct 117(2003) Clin.

Caterer Res 9 l29l~ l300; Arnold, AS er a7 (2907) 7. 12193119 25197205 which are incorporated herein by referencein their ty). Sonic nonnlimiting examples ot drug delivery systemsl selltl for systemic delivery ofiRNAs include BOlAP (Sorensen ER 91' a7(2003), supra; ‘Verma UN. 31' a! (2003),.111pra), Oil igotectamine, 'solid nucleic acid lipid les (7tinmerniann TSeta7(2130€3)1‘1117111644l :l l l—l l4), cardiolipin , FY, (31‘ a! (2005) Cancer Gene 7718?“. l2232ln328; Pal, A., 31117 (2005) 7117.7. 0111307. 26:lGS7—l09l), polyethyleneintine (Bonnet l‘vlli.., cf 911' (2008) P71511711. Res. Aug l6 Epuh ahead ofprint; Aigner, 1‘1.(2()l)6) 7. 136719071317111107 7l659) ArgGlynAsp (79619) peptidesflin .2006) M117 P71117111. 487), and polyamidoamines (Tomaliaa DA, 51,1(2007)?)10137/16121. Soc. Trans. :6l—67; ‘r'oo, ll., at 117 G999) Pizarm. Res. 1617994804). In some embodiments. an iRNA forms a complex with cyclodextrin for systemic administration. Methods for administration and [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS pharinaceutic al compositions of iRNAs and cyclodextrins can he found in US. l’atent No, 7,427,605, which is herein incorporated by reference in its entirety.

A. Va :t‘or encoded iRN/ls oft/re [retention iRNA targeting the CS gene can he expressed from transcription units inserted into DNA or (I: RNA vectors (see, egg, Couture, A, at of, T10. (1996), l225nltl; Skillem, A, at all, International PCT Publication No. W0 00/22l l3, Conrad, International PCT Publication No. Vii/O 00/22l l4, and Conrad, US Pat. No. 6,054,299). Expression can he transient (on the order ofhours to weeks) or sustained (weeks to months or longer), depending upon the specific construct used and the target tissue or cell type, These transgenes can he introduced as a linear construct, a circular ll) plasmid, or a viral vector, which can be an integrating or non—integrating vector. The transgene can also be constructed to permit it to he inherited as an extrachromosomal plasmid (Gassrnann, 83:515., Proc. Natl. Acad. Sci. USA (l 995) 921l292).

The individual strand or strands of an iRNA can he transcribed from a promoter on an expression vector, Where two separate strands are to he expressed to generate, for example, a l5 ds RNA, two separate sion vectors can he co-introduced (eg, by transfection or infection) into a target cell. Alternatively each individual strand of a dsRNA, can he transcribed by promoters both ofwhich are located on the same expression plasmid ln one embodiment, a dsRNA is sed as inverted repeat polynuc eotides joined by a linker polynucleotide sequence such that the dsRNA has a stern and loop structure. 2t) iRNA expression vectors are generally DNA plasmids or viral vectors. Expression s compatible with eukaryotic cells, preferably those compatible with vertebrate cells, can he used to e recombinant constructs for the expression of an iRNA as described herein.

Eukaryotic cell expression vectors are well lrnown in the art and are ble from a number of commercial sources. Typically, such vectors are provided ning ient restriction sites for ion of the desired nucleic acid segment, Delivery of iRNA sing vectors can he systemic, such as by intravenous or intramuscular stration, by administration to target cells ext-planted from the patient followed by oduction into the patient, or by any other means that allows for introduction into a desired target cell. iRNA, expression plasmids can he transfected into target cells as a complex with cationic (A) c. lipid rs (rag Oligofectarnine) or non—cationic lipidnhased carriers (cg sit—rl‘KOlM).

Multiple lipid transfections for iRNA-inediated knockdowns targeting different regions of a target RNA over a period ofa week or more are also contemplated by the ion. Successful uction of vectors into host cells can he rn onitored using various lrnown methods. For exarnple, ent transfiéction can he signaled with a reporter, such as a fluorescent marlrer, such as Green Fluorescent Protein (EFF). Stahle transfection of cells a): viva can he ensured [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS using markers that provide the transfected cell with resistance to specific environmental factors (cg otics and drugs), such as hygroniycin B resistance.

Viral vector systems which can be utilized with the methods and itions described herein include, but are not lirnited to, (a) ad enovirus vectors; (h) retrovirus vectors, including but (I: not limited to lentiviral vectors, ey murine leukemia virus, etc; (c) ad eno~ associated virus vectors; (d) herpes simplex virus vectors; (e) SV 40 vectors; (t) polyorna virus vectors; (g) papiilorna virus vectors; (h) picornavirus vectors; (i) pox virus vectors such as an ox, cg, vaccinia virus vectors or avipox, tag. canary pox or fowl pox; and (j) a. helper—dependent or gutless adenoviius. Replication~defective viruses can also be advantageous. Biffiérent vectors it) Will or will not become incorporated into the cells’ genome. The constructs can include viral sequences for transfection, if desired. Alternatively, the construct can be incorporated into vectors capable of nal replication, tag EPV and EBV vectors, Constructs for the recombinant expression of an iRNA will generally require regulatoijvr elements, cg, promoters, enhancers, em, to ensure the expression ofthe illNA in target cells. Other aspects to consider for s and constructs are further described below.

Vectors useiul for the delivery of an iRNA will include regulatory elen’ients (promoter, enhancer, etc.) sufficient for sion ofthe iRNA in the desired target cell or tissue The tory elements can be chosen to provide either constitutive or regulated/inducible expression. 2t) sion ofthe iRNA can be precisely regulated, for example, by using an inducible regulatory sequence that is sensitive to certain physiological regulators, (3.3;, ating glucose levels, or horrnones (Bochertv at all, l994, {‘AS'EB J. 8:20—24). Such inducible expression s, suitable for the control ot‘dsRNA expression in cells or in s include, for example, regulation by ecdysone, by estrogen, progesterone, tetracycline, al inducers of dirnerization, and isopropyl—betadfl alactopyranoside (H3176), A, person slrilled in the art would be able to choose the appropriate regulatory/promoter sequence based on the intended use ofthe iRNA transgene.

Viral vectors that contain nucleic acid sequences encoding an iRNA can be used. l:or exan'iple, a retroviral vector can he used (see Miller at all, ,Mcth. ]. 2i 7:58l «599 t @533», (A) c. These retroviral vectors n the components l’lBCCSSdly for the correct packaging ofthe viral genome and ation into the host cell DNA. The nucleic acid sequences encoding an iRNA are cloned into one or more vectors, which facilitate delivery of the nucleic acid into a patient.

More detail about retroviral vectors can be found, for example, in Boesen ct at, Biotherqpy 6:29l—302 (_ l994’), which describes the use of a retroviral vector to deliver the nidrl gene to hematopoietic stem cells in order to make the stern cells more resistant to chemotherapy. Other references rating the use of retroviral s in gene therapy are: Clowes at all, J. Clin.

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS Invest. 93:64l-4lm65l G994); Kiem ct til, Blood 83214674473 {1994); Salmons and Gunzherg, Hlmzan Gene Therapy 4zl29—l 41 0993); and nan and ‘Wilson, Curr. 0pm. in Genetics and Devel. 3 :l lilel l4 ( l993}. Lentiviral vectors contemplated for use e, for e, the lrllV based vectors described in US. Patent Nos. 6,l43,52tl; 5,665,557; and 5,98l ,276, which are (I: herein incorporated by reference, Adenoviruses are also plated for use in delivery of iRNAs of the invention.

Adenoviruses are especially tive vehicles, cg, for delivering genes to respiratory epithelia.

Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirusvbased delivery s are liver, the l nervous system, endothelial cells, ll) and muscle. Adenoviiuses have the advantage ot‘heing capable of intecting non-dividing cells. liozarsky and Wilson, Current Upsilon in Genetics and Development 32499503 @993) t a review of adenovhushased gene therapy. Bout e! at, Hummer Gene. Thcmpv 553—10 (l994l) demonstrated the use of adenovirus vectors to er genes to the respiratory lia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can he found in Rosenfeld e! oi, Science 252:43 L434 {l 99l); Rosenl’eld oi oi. Ce]! 68d 43455 , {l 992); ngeli at a]. , J. Cfin. [iii/“est. 9l 2225—234 (l993); PCT Eublication W’OQ4/ild49; and ‘Wang, (32‘ all, Gene Filtering/122775383 ( l995). A suitable AV vector for expressing an iRNA ed in the invention, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia ll 9! of. (2002), Not. Biotech. 20: l006-l (ll 0. 2t) Arlene-associated virus (AAV) s may also be used to delivery an iRNA ot‘the invention (Walsh e: mi, Proc. Soc Exp Biol. Med. 9~300 (l 993); US. Eat. No. ,436.,l4o), in one embodiment, the iRNA can he expressed as two separate, complementary single—stranded RNA molecules from a recombinant AAV vector having, for example, either the U6 or ill RNA promoters, or the galrwirus (tillVlV) promoter. Suitable AAV vectors for sing the dsRNA teatured in the invention, methods for constructing the recornhinant AV vector, and methods for delivering the vectors into target cells are described in Sainulsld R 61' of, @987), J. lfi’rol. til: 3tl96—3l0l; Fisher K .l at oi. (1996),,1. Virol, 70: 520—532; Samulski R et a]. (1989), J. Viral. 63: 38223826; US. Pat. No. 5,252,479; US. Pat. No. 5,l39,94l ; international Eatent Application No, W'O 94/l 3788; and international Patent Application No, “/0 93,1"2464l, the entire disclosures ofwhich are herein incorporated by reference.

Another viral vector suitable for delivery of an iRNA of the inevtion is a pox virus such as a vaccinia virus, for example an ated vaccinia such as Modified Virus Ankara (NEVA) or NYVAC, an avipox such as fowl pox or canary pox, The tropism of viral vectors can he modified by pscudotyping the vectors with envelope proteins or other surface ns from other viruses, or by substituting different viral capsid proteins, as appropriate. l?or example, lentiviral vectors can he pseudotyped with surtace proteins [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS from vesicular itis virus (VSV), rabies, Ehola, , and the like. AAV s can he made to target different cells by engineering the vectors to express different capsid protein serotypes; see, cg, Rahinowitz .l' E e! of. , J Vim! 761791—801 the entire disclosure of which is herein incoiporated by re ference.

(I: The pharmaceutical preparation of a vector can e the vector in an acceptahle diluent, or can include a slow release matrix in which the gene delivery vehicle is iniliedded. atively, where the complete gene delivery vector can he produced intact from recombinant cells, cg, retroviral vectors, the pl’iannaceutical preparation can include one or more cells which produce the gene delivery system.

V. Pharmaceutical Compositions of the lnvention The p resent invention also includes pharmaceutical compositions and formulations which include the iRNAs of the invention in one embodiment, ed herein are ceutical compositions containing an iRNA, as described herein, and a pharmaceutically acceptable l5 carrier.

The phrase ”pharn'iaceutically acceptahle” is employed herein to reter to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, le for use in contact with the tissues of human subjects and animal subjects Without excessive toxicity, irritation, ic response, or other problem or 2t) complication, conunensurate with a reasonable benefit/risk ratio.

The phrase naceutically—acceptahle carrier" as used herein means a pharma ieuticallywacceptahle material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (rag, lubricant, talc magnesium, m or zinc stearate, or steric acid), or solvent encapsulating al, ed in carrying or transporting the subject compound from one organ, or portion ofthe body, to another organ, or portion of the body, Each carrier must he ”acceptable” in the sense g compatible with the other ingredients of the formulation and not injurious to the subject being treated. Some examples of materials which can serve as pharmaceutically—acceptahle carriers inchide: (l) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, (A) c. such as sodium carhoxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) rnalt; to) gelatin; (7) lubricating agents, such as magnesium state, sodium lauryl e and tale; {8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cotton seed oil, satllower oil, sesame oil, olive oil, corn oil and soybean oil; ( l 0) glycols, such as propylene glycol; (l l) polyols, such as glycerin, sorhitol, inannitol and polyethylene glycol; (l 2) esters, such as ethyl oleate and ethyl laurate; t 13) agar; (l4) liuftering agents, such as magnesium hydroxide and aluminum hydroxide; (l5) alginic acid; (l 6) pyrogen— [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS free water; (_ l7) isotonic saline; (18') Ringer‘s solution; {_ l 9) ethyl alcohol; (20) pH buffered solutions; (2i ) polyesters, polycarbonates and/or polyanhydrides; (22’) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; and (22) other non—toxic compatible substances employed in pharmaceutical formulations.

(I: The pharmaceutical compositions containing the iRNA are useful for treating a disease or disorder associated with the expression or activity ofa C5 gene, er; a complement component ociated disease. Such phannaceuti ‘al compositions are ated based on the mode of delivery. One example. is compositions that are formulated for ic administration via parenteral deliveiy, e.g., by aneous (SC) or enous (IV) delivery. Another examp1e is ll) compositions that are formulated for direet delivery into the brain parencliyma, egg, by infusion into the brain, such as by continuous pump infusion. The pharn’iaeeutical compositions ot‘tlie invention may be administered in dosages ient to inhibit expression ofa C5 gene. ln general, a suitable close of an iRNA of the invention will be in the range of about (Mull to about 206.0 milligrams per kilogram body weight of the recipient per clay, generally in the range of about 1 to 50 mg per kilogram body weight per day. For example, the tisRNA can be administered at about 0.01 mgr/kg, about 0.05 rug/leg, about 0.5 rug/kg, about i rug/leg, about l.5 mtg/leg, about 2 mg/lrg, about 3 mg/kg, about l0 mtg/kg, about 20 mg/kg, about 30 mgr/leg, about 40 mgr/kg, or about 50 rug/kg per single dose.

For example, the dsRNA may be administered at a dose of about 0.} , 0.2., 0.3, 0.4, 6.5, 28 0.6, 6.7, 08,09,l,l.l,i.2,1.3,l.4,i.5,l.6,l.7,l.8,l.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.l , 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 39, 4, 4.l, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, .2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.l, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.l, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.l 9.i , 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, , 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about l0 rug/leg. Values and ranges intermediate to the recited values are also ed to be part s invention. in r embodiment, the tlsRNA is administered at a dose of about {hi to about 50 mtg/kg, about 0.25 to about 50 rug/kg, about 0.5 to about St) mg/lig, about 0.75 to about 5 0 mg/kg, about 1 to about 50 rug/ring, about l.5 to about 58 ib, about 2 to about 50 mg/l-rg, about 2.5 to about 50 rug/kg, about 3 to about 50 rug/kg, about 3 .5 to about 50 mg/kg, about 4 to about 50 mg/lrg, about 4.5 to about 50 lug/kg, about 5 to about 50 mtg/leg, about 7.5 to about 50 rug/kg, about l0 to about 50 rug/kg, about E5 to about 50 mg/kg, about 20 to about 50 mpg/kg, about 20 to about 50 rug/leg, about 25 to about 50 rug/leg, about 25 to about 56 trig/leg, about 30 to about 50 org/kg, about 35 to about 50 mg/hg, about 40 to about 50 big/kg, about 45 to about 50 rug/kg, about ill to about 45 mg/kg, about 9.25 to about 45 mgr/leg, about 0.5 to about 45 mtg/kg, about 0.75 to about 45 , about i to about 45 rug/mg, about l.5 to about 45 mg/lrb, about 2 to about 45 mg/kg, about 2.5 to about 45 rug/kg, about 3 to about 45 rug/leg, about 3.5 to [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS about 45 tug/kg, about 4 to about 45 tug/kg, about 4.5 to about 45 rug/kg, about 5 to about 45 rug/kg, about 7.5 to about 45 rug/kg, about 10 to about 45 trig/kg, about 15 to about 45 rug/kg, about 20 to about 45 rug/kg, about 20 to about 45 rug/kg, about '25 to about 45 rug/kg, about 25 to about 45 rug/Lg, about 30 to about 45 rug/Lg, about 3:5 to about 45 trig/kg, about 40 to about (I: 45 big/kg, about 0.1 to about 40 rug/leg, about 0.25 to about 40 rug/kg, about 0.5 to about 40 rug/kg, about 0.75 to about 46 g, about 1 to about 46 trig/trig, about 1.5 to about 40 nig/kb, about 2 to about 40 rug/Lg, about 2.5 to about 40 rug/kg, about 3 to about 40 rug/kg, about 3.5 to about 40 trig/kg, about 4 to about 40 trig/kg, about 4.5 to about 40 rug/Lg, about 5 to about 40 rug/kg, about 7.5 to about 40 rug/kg, about it) to about 40 tug/kg, about 15 to about 40 trig/kg, 19 about 20 to about 40 rug/kg, about 20 to about 40 rug/kg, about 25 to about 40 n1g/kg, about 25 to about 40 rug/kg, about 30 to about 40 rug/Lg, about 35 to about 40 trig/kg, about (1.1 to about org/kg, about 0.25 to about 30 tug/kg, about 0.5 to about '30 rug/kg, about (1.75 to about 30 rug/kg, about 1 to about 30 g, about 1.5 to about 30 Rig/kl), about 2, to about 30 rug/leg, about 2.5 to about 30 , about 3 to about 30 rug/kg, about 3.5 to about 30 trig/kg, about 4 to about 30 trig/Kg, about 4.5 to about 30 rug/kg, about 5 to about 30 rug/kg, about 7.5 to about 31) nag/kg, about It) to about 30 tug/kg, about 15 to about 30 tug/kg, about 20 to about '30 , about 20 to about 30 rug/kg, about 25 to about 30 rug/kg, about 0.1 to about 20 rug/kg, about {3.25 to about 20 big/kg, about 0.5 to about 20 , about 0.75 to about 20 rug/kg, about 1 to about 20 rug/mg, about 1.5 to about '20 rug/Rb, about 2 to about '20 , about 2.5 to about 20 28 rug/kg, about 3 to about 20 rug/11g, about 3.5 to about 20 rug/kg, about 4 to about '20 rug/kg, about 4.5 to about 20 , about 5 to about 20 rug/kg, about 7.5 to about 20 tug/kg, about 10 to about 21.1 rug/kg, or about l5 to about 20 big/kg. Values and ranges intermediate to the recited values are also intended to be part oftbis invention. l2or example, the dsRNA maybe administered at a dose of about Gobi , (1.02, (1.03, 0.114, {3.05, 0.06, 0.07, {3.08, 0.09, 0.1, 0.2, 0.3, 0.4, {3.5, 0.6, {1.7, 1,1.1,1.2,l.3,l.4,1.5,1.6, 1.7, lb, 1.9, 2, 2.l, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3. (Jr , 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.l , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.l or about 10 tug/kg. Values and , 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, ranges intermediate to tbe recited values are also intended to be part oftbis invention. in r embodiment, the dsRNA is administered at a dose of about 0.5 to about 50 rug/kg, about 0.75 to about 50 trig/Kg, about t to about St} trig/mg, about 1 .5 to about 50 rug/kl), about 2 to about 50 , about 2.5 to about 50 org/kg, about 3 to about 50 mg/kg, about 3.5 to about 50 tug/kg, about 4 to about 50 tug/kg, about 4.5 to about 51.1 rug/kg, about 5 to about 50 rug/kg, about 7.5 to about 50 rug/kg, about 10 to about 50 trig/kg, about 15 to about 50 rug/kg, about 20 to about 50 rug/kg, about 20 to about 50 rug/kg, about '25 to about 50 rug/kg, about 25 [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS to about 5U tug/11g, about 30 to about 5U 011;;41g, about 35 to about 50 m1;I’11g, about 40 to about 50 , about 45 to about 50 11thkg, about 0.5 to about 45 tug/111;, about 0.75 to about 45 tug/kg, about 1 to about 45 tug/tug, about 1.5 to about 45 tug/11b, about 2 to about 45 tug/111;, about 2.5 to about 45 111ng about 3 to about 45 tug/11g, about 3 .5 to about 45 tug/kg, about 4 to (I: about 45 mtg/11g, about 4.5 to about 45 tug/11g, about 5 to about 45 tug/11g, about 7.5 to about 45 tug/kg, about 10 to about 45 , about 15 to about 45 tug/kg, about 20 to about 45 mtg/11g, about 20 to about 45 tug/kg, about 25 to about 45 tug/11g, about 25 to about 45 g, about 30 to about 45 our/11g, about 35 to about 45 out/11g, about 40 to about 45 tug/11g, about 0.5 to about 40 mgIkg, about 0.7I5 to about 40 tug/111;, about 1 to about 40 tug/111g, about 1.5 to about 40 tug/11b, about 2 to about 40 ntgx’kg, about 2.5 to about 40 , about 3 to about 40 mtg/11g, about 3.5 to about 40 tug/kg, about 4 to about 40 tug/kg, about 4.5 to about 40 rug/kg, about 5 to about 40 mg/kg, about 7.5 to about 40 tug/111;, about 10 to about 40 mtg/111;, about 15 to about 40 tug/11g, about 20 to about 40 tug/kg, about 2U to about 40 tug/11g, about 25 to about 40 mtg/11g, about 25 to about 40 , about 30 to about 40 ntgx’kg, about 35 to about 40 tug/111g, about 0.5 to about 30 tug/kg about 0.75 to about 30 tug/111,1, about 1 to about 30 tug/mg, about 1 .5 to about tug/11b. about 2 to about 30 mg11g. about 2.5 to about 30 tug/kg, about 3 to about 30 , about 3.5 to about 3U tug/11g, about 4 to about 30 tug/111;, about 45 to about 30 1114kg about 5 to about 30 tug/111;, about 7.5 to about 30 tug/111;, about 10 to about 30 tug/111;, about 15 to about 30 tug/119,, about 20 to about 30 tug/1111;, about 20 to about 30 tug/11g, about 25 to about 30 111315111, about 0.5 to about 20 tug/kg, about 0.75 to about 20 11thkg, about 1 to about 20 11thmg, about 1.5 to about 20 tug/11b, about 2 to about 20 tug/11g, about 2.5 to about 20 utg/kg, about '3 to about 011;,I’11g, about 3.5 to about 20 tug/kg, about 4- to about 20 tug/kg, about 4.5 to about 20 mtg/111;, about 5 to about 20 big/11g, about 7.5 to about 20 tug/kg, about 10 to about 20 tug/kg, or about 15 to about 20 tug/kg. 111 one embodiment, the 11811111731 is tusteted at a dose of about Wing/kg; to about 30 g. Va1ues and ranges intermediate to the recited va1ueu are atso intended to be part oftbis invention.

For examp1e, subjects can be administered, 61;. suboutaneous1y or intravenously, a Sing1e therapeutic amount ofiRNA, such as about 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275, 0.3, 0.325.0355,037’5 ,0.4, 0.425, 0.45, U.5, 0.525, 0.55, 0.575, 0.6, 0625,1165, 0.675, 0.7 0.725, 0.,I75,0.775, 0.8, U.825,0.,85 0875, U9 0.925, 0.95, U.975, 1, 1.1, 1.2,1.3, 1.4, 1.,5 1.6, 1.7,1.8,1.9 ,2, 2.1, 22,23, 2.4,2.5,2.6, 2.7.2.8, 2.9,3, 3.1, 3.2 ,.33, 3 4,3- .5, 3.6, .II 39 I. 3.8, 4, 41,42, 43, 4.4,4.5,4.6, 4.7, 1., 5,5152 5.,54,55, 56, 8, 59, 6, 61, 62, 6.3,154, 6.5, 6,6 6.7 ,6.8, 69, 7 71.,7.2,73,7.4, 7.5, 76, 77, 7.8, 7.9 8, 8.1I8.2,8.3,8.4, 8.5, 8.6, 8.,7 8.8 8.9, 9, 91, 9,2 9.3, 9.4 9.5, 9.6, 97, 9.,8 99,10,105,11,115,12,12.5,13,13.5, 14,145,15,15.5,16,16.5,17,17.5,18,18.5,19,19.5, 20, 20.5, 21, 21.5, '22., 22.5, 23, '23524, 24.5, 25, 25.5, 26, 26.5, '27, 2.7.5, 28, 28.5, 29, 29.5, 30, 31, .32, 33, .34, 34, 35, 36, 37, 38,3,9 40, [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS 41, 42, 43 , 4, 4-5, 46, 47, 48, 49, or about 50 mtg/kg. Vaiues and ranges intermediate to the recited. va ues are aiso intended to be part of this invention. 111 some embodiments, subjects are administered, 6.g., suboutaneous1y or intravenous1y, m111tipie doses ofa t1111apeutie amount ofiRNA, 1 as a dose 012611101, 0.125. U. 15, 0.1 75, (I: 0.2, 0.225, 0.25, 0.275 , U.3, U. 325, 0.35, 0.375 U4, 0.425, U45, U475, 0.5, 0.525, U55, U 575, 0.6.0.625, 5.,65 0.675, 0.75775 575 0.775, 0.5, 5.525, 555, 0.,575 1.15, 5.525, 0,55 5.Q75,1, 1.1,1.2,1.3,1.41.5.16,17,15,15,2,2.1,22,73 ,2..4,25, 26,227.25, 25, 5233 3.4,3.5,3.6,3.7,3.8,3.9,4,4.1,4.2,4.3,4.4 4.5., 413, 47, 4.5., 4.5, 5, 5,1 5,2 53, 54,:5 5.15, .7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4-, 6.5, 6.6,(3. 7’ 7.9 , 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8: , 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5,12,125,13,13.5,14,14.5,15,15.5,16,16.5,17,17.5,18,18.5,19,19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23 .5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 4U, 4-1, 42, 43, 44, 45, 46, 47, 48, 4-9, or about 5U trig/11g. A muitindose regimine 111ayinc1ude administration 61a therapeutic amount 6111115178 daiiy, such as for two days, three days, four days, five days, six days, seven days, or 1onger. 1n other embodiments, subjects are administered, 6mg, Stitbeutarieousiy or intravenous1y, a repeat dose of a therapeutic amount ofiRNA, such as a dose about 0.1, 0.125, 0.15, 0.175, U2, 0.225, 0.25, U2.75 0.3, 0.325, 0.35, U375, U.4-, 0.425, U45, U.475, 0.5, U525, U55, 0.575, 0.6 0.62 5, 0.65, U675. 0.7.5 U. 8, 0.825, 0.85, U. 875, 0.9, 0.925, 0.95, 0975, , 0.7, 0.725, 0.75, , 1, 1. 1, 1.2, 1.3,1.4,1.5, 7,1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1,32 .2, 3.3, 3.4, 3.5, 3.6, 3.7 ,3.,8 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 582 9, ,162 6.3 6.4-, 65 66, 67, 6,8 69, 7, 7 .1,7.2,7.3,7.4,7.5,7.6,7.7.7.8,7.9,8, 8.,1 82, 8.3, 8.4.8.,5 8.6, 87, 8.8, 8.9 9, 9.,1 9.2, 9.3 .5,9.6,9.7,9.8,9.9, 10, 10.5, 11, 115,12,12713,13.5,14,14.5,15,15.5,16,165,17,1'7.5,18,18.5,19,19.,5 20, 20.5, 21, 21 .5,22,22.5,27,235 24, 24.5, 25, 25.5, 26, 26.5,7 ,27.5, 28, 285,129, 295, 30, 31, 32 ,33, 34. 34, 35, 36, 3’7 41 , 38, 39, 40, , 42, 43, 44-, 4-5, 46, 47, 48, 49. or about 50 1ng/1rg.A repeatndose regiinine may ineiude administration of a therapeutic amount of iRNA on a regu1ar 'hasis, such as every other day, every third day, every fourth day, twice a week, once a week, every other week, or once 11 1110111111.

The 1111ar1naeeutiea1 composition can be administered by enous on over a period ot‘time, such as over a 5, 6, 7, 8,9,10,11,12,13,14,15,16,17,18,19, 20, and2 , 22, 23, 24, or about a 25 minute period. The administration may be repeated, for exarnpie, on a reg-5.11211 basis, sueh as 5vee1€1y, 1165711611137 (1112. , every two weeks) for one month, two months, three months, four months or ionger. After an initiai ent regimen, the treatments can be administered on a iess frequent basis. For examp1e, after administration weekiy or hiweek1y for three months, administration can be repeated once per month, for six months or a year or hanger.

[Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS The pharmaceutical composition can he administered once daily, or the iRNA can he administered as two, three, or more sub—doses at appropriate intervals throughout the day or even using contirnitnis in fusion or delivery through a controlled release formulation. in that case, the iRNA contained in each strh~dose must he correspondingly smaller in order to achieve the total (I: daily dosage, The dosage unit can also he compounded for delivery over several days, egg, using a conventional ned release tormulation which es sustained release ot‘the iliNA over a several day period. Sustained release torrnulations are well known in the art and are particularly useful for delivery of agents at a particular site, such as could be used with the agents ofthe present invention. ln this embodiment, the dosage unit ns a corresponding multiple ll) ofthe daily dose. ln other en'ihodirnents, a single dose ot‘the pharmaceutical compositions can he long g, such that subsequent doses are administered at not more than '3, 4, or 5 day intervals, or at not more than l, 2, 3, or 4 week intervals. In some embodiments of the invention, a single dose ofthe ceutical compositions ofthe invention is administered once per week, in other embodiments ot‘the invention, a single dose of the pharmaceutical compositions ot‘the invention is administered tlilv The skilled artisan will appreciate that certain factors can nce the dosage and timing required to effectively treat a subject, including but not limited to the severity of the e or disorder, previous treatments, the general health and/or age ot‘the subject, and other diseases 2t) present, Moreo fer, treatment of a subject with a the “apeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates ot‘eftective dosages and in viva halfnlives for the individual illnNAs encompassed by the invention can he made using conventional methodologies or on the basis of in viva testing using an appropriate animal model, as described elsewhere herein.

Advances in mouse cs have ted a number ofrnouse models for the study of various human diseases, such as a er that would benefit from reduction in the expression of CS. Such models can he used for in vivo testing A, as well as for determining a therapeutically ellective dose. Suitable mouse models are known in the art and include, for example, collagen—induced arthritis mouse model (Courtenay, 15., er of. (l 980) Mature 283, (A) c. 666—668), myocardial ischemia (Homeister JV! and Lucchesi BR (l 994) Annu Rev Pharmacoi '[hxicoi 34:l7---40), ovalhumin induced asthma mouse models (9g, 'l‘omkinson A, at a]. .

J. Immunol. loo, S792»~5800), {N28 >11 NEW)l? l MRL/Faslm (l‘lelJ/lpr) and BXSB mouse models (ll’ieofilopoulos, A. N. and Keno, I). ll, 1999, Murine lupus models: gerievspecitic and genome“ wide studies. ln Lahita R, 6., ed, Stirszeml'c Lupus Era/itlzematosus, 3rd edn, p. li—‘lS. Academic Press, San Diego, CA), mouse allUS model (Goicoechea de Jorge cf of, (2011) The development [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS ation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ofaifiypical heifiolytic uremic syndrome departs on complement CS, JAm Soc Napkin! 222137" l45.

The pharmaceutical compositions ofthe present invention can be administered in a number of ways depending upon Whether local or systemic treatment is desired and upon the (I: area to be treated Administration can be topical (egg, by a transdermal patch), pulmonary, Cg, by inhalation or insufflation ofpowders or aerosols, including by nebulizer; intratraclieal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral station includes intravenous, intraarterial, subcutaneous, intraperitoneal or int‘amuscular injection or in fusion; subdermal, eg, via an implanted device; or intracranial, e. 53,, by intraparenchyrnal, intratliecal or ll) intraventricular, administration.

The iRNA can be delivered in a manner to target a particular tissue, such as the liver tag, the hepatocytes of the liver).

Pharmaceutical compositions and formulations for topical stration can include transdermal patches, ointrnents, lotions, , gels, drops, itories, sprays, liquids and powders. tional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like can be necessary or desirable. Coated condoms, gloves and the like can also be usetul.

Suitable topical formulations include those in which the iRNAs featured in the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid , steroids, clielating agents and surfactants. Suitable lipids and liposomes include neutral (cg, 2t) dioleoylphospliatidyl DOPE ethanolamine, dimyristoylpliospliatidyl choline DMPC, distearolyphosphatidyl e) negative (rag, dirnyristoylphosphatidyl glycerol DMPG) and ic (Cg, dioleoyltetrainethylaminopropyl DOTAP and dioleoylpbosphatidyl etlianolamine DO'l‘MA). illNAs featured in the ion can be encapsulated Within liposornes or can form complexes thereto, in particular to cationic liposomes. Alternati Iely, iRNAs can be complexed to lipids, in particular to cationic lipids. Suitable fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid, ic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, inonoolein, dilaurin, glyceiyl l ~monocaprate, l“dodecylazacycloheptan—Z—one, an acylcarnitine, an acylclioline, or a €1.30 alkyl ester (rag, isopropyln'iyristate ll’e‘l‘vl), n'ionoglyceride, diglyceride or pharmaceutically (A) c. acceptable salt f), ’l‘opical formulations are described in detail in us. Patent No. (3,7419%, which is incorporated herein by reference.

A. iRN/l ations Comprising Marni/various Molecular ,-4,rsemb!ies An iRNA for use in the compositions and n'iethods oftlie invention can be ated for delivery in a membranous lar assembly, eg, a liposome or a micelle, As used herein, the term “liposome” refers to a e composed of amphiphilic lipids ed in at least one bilayer, eg, one bilayer or a plurality yers. lgiposomes include unilamellar and [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion ns the iRNA ition. The lipophilic material isolates the aqueous or from an aqueous exterior, which typically does not include the iRNA composition, although in some es, it may. liposonies are useful for the tifanstei“ and (I: ry of active ingredients to the site of action, Because the liposonial menihrane is structurally similar to biological membranes, When liposonies are applied to a tissue, the liposoinal hilayer fuses with bilayer of the cellular membranes. As the merging of the liposoirie and cell prog fesses, the internal aqueous contents that include the iRNA are delivered into the cell where the iRNA can specifically bind to a target RNA and can e RNAi. in some it) cases the liposomes are also specifically targeted, 9.53;, to direct the iRNA to particular cell types.

A liposorne containing a RN Ai agent can he prepared by a variety ofmethods. in one exan'iple, the lipid component of a liposonie is dissolved in a detergent so that micelles are formed with the lipid ent. For example, the lipid component can he an amphipathic cationic lipid or lipid ate. The detergent can have a high critical niicelle concentration and may he nonionic. lilxeinplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and l sarcosine, The RNAi agent preparation is then added to the micelles that include the lipid component The cationic groups on the lipid interact with the RNAi agent and condense around the RNAi agent to form a liposome After condensation, the ent is removed, eg, by dialysis, to yield a liposoinal preparation ofRNAi agent. 2t) if ary a carrier compound that assists in condensation can be added during the condensation reaction, (2.3;, by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid (eg, spermine or spermidine). pH can also adjusted to favor condensation.

Methods for producing stahle polymicleotide delivery vehicles, which incorporate a polynucleotide/cationic lipid complex as structural components of the delivery vehicle, are further bed in, eg, W0 96/3719-4, the entire contents ofwhieh are incorporated herein by reference. Liposonie formation can also include one or more aspects of exemplary methods described in r, P. L. at a[., Pipe. Natl. Acad. Sci, USA 8:74l 3-74l7’, l987; LES. Pat. No. 4,897,355; US. Pat No. 5,l7l,678; Bangham, at (if. Mi ,Moi. Biol. 23:238, l965; Olson, 61 a].

(A) c. Biochim. Biophys. Acta 557:9, l979; Szoka, el’ al. Proc. Natl. Acad. Sci. 75: 4-l94, l978; l‘vlayhew, er a]. m. Biopl/nu. Acta 7752l69, l984; Kim, et (2!. Biochim Biophys. Acta 728:339, l983; and Fulmnaga, at all. Ezridocrmol. l l52757, l984. Commonly used ques for preparing lipid aggregates of appropriate size for use as delivery vehicles include tion and freeze—thawplus extrusion (see, eg. Mayer, at of. Biochim. Biophys, Acre 8581l6l , l986) lvlicrotluidization can be used When consistently small (50 to 200 nm) and relatively uniform [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS aggregates are desired (Mayhem ez‘ ail. Biochim. Biophys. Acre 7751l69, l984). These methods are readily adapted to packaging RNAi agent preparations into mes.

Liposornes fall into two broad classes. Cationic liposornes are positively charged liposornes which ii’iteract with the negatively charged c acid molecules to form a stable (I: x. The positively charged nucleic acid/liposorne complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH Within the endosome, the liposoines are ruptured, releasing their contents into the cell cytoplasm (Wang ez‘ 01L, Biochem.

Biophys. Res. CO???MW’Z., l987, l47, 5).

Liposomes which are prsensitive or negatively—charged, entrap nucleic acids rather than ll) complex with it, Since both the nucleic acid and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some nucleic acid is entrapped Within the s interior of these liposornes. pltl—sensitive liposornes have heen used to deliver nucleic acids encoding the thyniidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou e? 525., Journal quom’roz’lea’ Release, 1992, l9, 269— 274).

One major type of liposonial composition es phospholipids other than naturally~ derived phosphatidylcholine. Neutral liposome compositions, for example, can he formed from dimyristoyl phosphatidylcholine (DMPC) or iitoyl phosphatidylcholine (DFPC). Anionic liposonie compositions generally are formed from dimyristoyl phosphatidylglycerol, While 2t) anionic fusogenic ies are formed prin'iarily from dioleoyl phospliatidyletlianolamine (DOPE). Another type ofliposornal ition is lornied from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC, r type is formed from mixtures ofphospholipid and/or phosphatidylcholine and/or cholesterol.

Examples er methods to uce liposornes into cells in vim) and in viva include US, Pat, No. 5,283,l 85:, US. Pat, No, 5,l 7l,678; WO 69; WO 540; WO Ell/l 6024; Felgner,J§ Biol. Chem. 26922550, l99-4; Nahel, Proc. Natl. Acad. Sci. 902l l307, l993; Nahel, Himzan Gene The}: 3:649, l992; Gershon, Biochem. 32:7l 43, 1993; and Strauss Ell/180,1 llz4l7, 1992.

Non ~ionic liposomal systems have also been ined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non—ionic surfactant and cholesterol. N on—ionic liposomal formulations comprising NovasorneTM l (glyceryl dilaurate/cholesterol/polyoxyethylene-l G-stearyl ether) and l‘slrwasomeTM ll (glyceryl distearatJ/cl’rolesterol/polyoxyetl’rylene—l 0~stearyl ether) were used to deliver cyclosporin—A into the dermis of mouse skin. Results indicated that such non—ionic mal systems were effective in facilitating the tion of cyclosporine A into difterent layers of the skin (llu et a!) S. TRPharma. Sci, l994, 4(6) 466).

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS Liposomes also include “sterically ized” liposornes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids, Examples of sterically stabilized liposoines are those in which part ol’the vesicle~fomiing (I: lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside GM; , or (B) is tized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) inoiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG~derivatized lipids, the ed circulation halfnlife of these sterically ll) stabilized liposomes derives from a reduced uptake into cells ofthe reticuloendothelial system (RES) (Allen 62‘ mi, FEBS Letters, 1987’, 223, 42; Wu et a!., Comer Res scroll, l993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in the art. l’apahadjopoulos at of. (Ann N ll [lead Seal, l987, 507, 64) reported the ability of monosialoganglioside GM; , galactocerebroside sulfate and phosphatidylinositol to improve blood half—lives of liptmornes. These s were expounded upon by (Il'abizon 62‘ al. (Prue. Nani.

Acad. Sci. USA, l9889 85, 6949). US. l3at. No. 4,; 37,028 and W0 88/049241, both to Allen ct aii, disclose liposomes comprising {_ l) sphihgomyelih and (2) the ganglioside GM; or a galactocerehroside sulfate ester. US. Fat. No, 5,543,l52 (Webb 6? cl.) ses liposonres comprising sphingornyelin. Liposomes comprising -diniyristoylphosphatidylcholine are 2t) disclosed in WO 97/l3499 (Lim et all. in one embodiment, cationic ines are used, Cationic liposorncs s the advantage of being able to fuse to the cell membrane, tionic liposomes, although not able to tuse as efficiently with the plasma membrane, are taken up by macrophages in vivo and can be used to deliver RNAi agents to macrophages. r advantages ot‘liposoines include; liposornes obtained from natural phospliolipids are biocompatihle and biodegradable; liposonies can incorporate a wide range of v 'ater and lipid soluble drugs; liposonies can protect encapsulated RNAi agents in their al compartments from lism and degradation (Resell, in "Pharmaceutical Dosage Forms,” Lieberman, Rieger and Banker (Eds), l988, volume l, p. 245). lrnpOitant considerations in the preparation (A) c. of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes, A positively charged synthetic ic lipid, N—{l—(2,3 -dioleyloxy)propyll~l\l,N,N- trimethylaniinonium chloride (DOTMA) can be used to form small n‘ies that ct spontaneously with nucleic acid to form lipid~nucleic acid xes which are capable of fusing with the negatively charged lipids of the cell membranes oftissue culture cells, resulting ’lO’l [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS in delivery of RNAi agent (see, ego, li‘elgner, P. L. et al, Proc. Natl Acad Sci, USA 8:74l3— 74W, l987 and lJ.S Pat. No 4,897,355 for a description 'MA and its use With DNA).

A DOTMA analogue, l,Z-histoleoyloxy)“3“(trirnethylamrnonia)propane (Drill AP) can he used in combination with a phospholipid to form DNA—complexing vesicles. LipofectinTM (I: Bethesda Research tories, Gaithershurg, Md.) is an effective agent for the delivery of highly anionic nucleic acids into living tissue culture cells that comprise positively charged DOTMA liposomes which interact neously with negatively charged polynueleotides to form xes. When enough positively charged liposoines are used, the net charge on the resulting complexes is also positive. Positively charged complexes prepared in this way ll) spontaneously attach to negatively charged cell surfaces, fuse With the plasma membrane, and efficiently deliver titnctional nucleic acids into, for example, tissue critlture cells. Another commercially available cationic lipid, l ,Z—his(oleoyloxy)~3,3 :nethylai'nrnonia)propane {“DO'l‘AP”) {Boehringer Mannheim, Indianapolis, lndiana) differs from DO'l‘lVlA in that the oleoyl moieties are linlred by ester, rather than ether linkages. l5 (Ether reported cationic lipid compounds e those that have been conjugated to a y of moieties including, for example, earhoxysperniine which has been conjugated to one oftwo types of lipids and includes compounds such as 5“carhoxysperniylglyeine dioctaoleoylamide (“DOGS”) ("l‘ransfiéctam’m, l’romega, Madison, Wisconsin) and dipalmitoylphosphatidylethanolamine 5—carboxyspermyl—amide (“Dl’l’BS’l) (see, eg, US. Pat, 2t) No. 5,l7l $378).

Another ic lipid conjugate includes derivatization of the lipid with cholesterol (“DC—Chol”) which has been formulated into liposomes in combination with DOPE (See, Gao, X. and Huang, L., Biochim. Biophys. Res. Con/21mm. ”9:280, l99l). Lipopolylysine, made by conjugating sine to DOPE, has been reported to he etl‘ective for ection in the presence of serum (Zhou, X. e . al, Biochim Biophys. Ania liltifizli, l99l). For certain cell lines, these liposonies containing ated ic lipids, are said to exhibit lower toxicity and provide more efficient transfection than the l‘le-containing compositions. Qtlier cornn'iercially available cationic lipid products include [)M’Rlli. and Dl‘lelliélll-l (Vical, lJa lolla, California.) and lipofectarnirie ) (Lite Technology, lne,, Gaithers‘ourg, Maryland), Other cationic lipids suitable for the delivery of oligonucleotides are descriled in WO 98/39359 and Wt) 96/37l 94. lsiposomal loririulations are particularly suited for topical zitdrninistration, liposornes present several advantages over other formulations Such advantages include reduced side effects related to high systemic absorption of the stered drug, increased accumulation of the stered drug at the desired target, and the ability to administer RNAi agent into the skin. ln some ientalions, liposomes are used for ring RNAi agent to epidermal cells [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS and also to enhance the ation ofRNAi agent into dermal tissues, aga, into skin. For example, the liposomes can he applied topieally. 'l‘opieal delivery of drugs formulated as liposornes to the skin has been docun'iented (see, eg, Weiner at a“, Journal oi"l)mg Targeting, l992, vol. 2,4054% and du Plessis at all, Antiviral Research, l8, l992, 259~265; Mannino, R. .l, (I: and Eould~Fogerite, 8., Bioteciminnes 62682—690, l988; ltani, T. at of, Gene 56:267—276. l987; Nicolau, C. at on". Mark. Enz. l492l57-l76, l987; Strauhinger, R. M. and Papahadjopoulos, D.

Math. Enz. lOl :5l2~527, l983; Wang, C. Y. and Huang, L, Pi‘OC. Nazi. Aorta". Sci. USA l - 7855, l987). nic liposomal systems have also been ed to determine their utility in the ill delivery of drugs to the shin, in ular systems comprising non-ionie surfactant and cholesterol. Non—ionic liposomal torrnulations comprising Novasome l {glyceryl dilaurate/cholesterol/polyoxyeth l O—stearyl ether) and Novasome ll (glyceryl distearate/ cholesteroll’polyoxyethyleneml 0—stearyl ether) were used to deliver a drug into the dermis of mouse skin. Such formulations with RNAi agent are useful for treating a dermatological disorder. somes that include iRNA can he made highly deformable. Such deformahility can enable the liposomes to penetrate through pore that are smaller than the average radius of the liposonie. For example, transfersomes are a type of deformable liposomes, 'l'ransferosomes can he made by adding e edge activators, usually surfactants, to a standard liposomal 2t) composition. Ti'ansi‘ersomes that include RNAi agent can he delivered, for example, subcutaneously by infection in order to deliver RNAi agent to keratinocytes in the skin. in order to cross intaet mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the inlluence of a le transdermal gradient. In addition, due to the lipid ties, these transterosornes can he seli‘loptirnizing (adaptive to the shape of pores, e.g., in the skin), seltlrepairin g, and can frequently reach their targets without fragmenting, and often oading.

Other formulations amenable to the present invention are described in United States provisional application serial Nos. 8,6l 6, tiled January '2, 26%“; ol/lll 8,6l l tiled January 2, 2008; til/039,748, tiled March 26, 2008:, 6l/(l47,087, tiled April 22, 2008 and til/05l,528, filed May 8, 2698, l’C’l‘ application no PCT/US2OG7/GSO33 l , filed October 3, 2007 also describes formulations that are amenable to the present invention.

Transiersornes are yet another type ol‘liposornes, and are highly deformable lipid aggregates which are attractive ates for drug delivery vel‘iicles. Transfersomes can he described as lipid droplets which are so highly deformable that they are easily able to ate h pores which are smaller than the droplet. ’l7ransfersomes are adaptable to the environment in which they are used, 91g, they are seliloptimizing {adaptive to the shape ofpores [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS in the shin), self~repairing, frequently reach their s Without fragmenting, and often self— loading To make transfersoines it is possihle to add surface edge—activators, usually surfactants, to a standard liposomal ition. Transfersoines have been used to deliver serum albumin to the skin The transfersome~mediated delivery of serum albumin has been shown to he as (I: efflective as subcutaneous injection of a solution containing serum albumin.

Surfactants find Wide application in formulations such as emulsions ding microemulsions) and liposomes. The most common way ot‘classifying and ranking the the hytlrophile/lipopliile balance (HLB). The nature of the hydrophilic group (also known as the l ll ”head") provides the most useful means for categorizing the different surfactants used in tormulations (Rieger, in “Pharmaceutical Dosage Forms”, Marcel Delrlrer, lnc., New York, NY, l988, p. 285). if the surfactant molecule is not ionized, it is classified as a nonionic surfactant Nonionic surfactants find Wide application in ceutical and cosmetic products and are usable over a l 5 Wide range ofplrl values. in general their 818 values range from 2 to about 18 depending on their structure Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, yl esters, polyglyceryl esters, sorbitan esters, sucrose esters, andi. ethoxylated esters. Noniomc allcanolamides and ethers such as fatty alcohol ethoxylates, proposylated alcohols, and ethoxylated/propoxylated block polymers are also included in this 2t) class. The polyoxyethylene surfactants are the most popular members ofthe ic surtactant class. if the surfactant molecule carries a negative charge when it is dissolved or sed in water, the surfactant is classified as c. Anionic surfactants include carhoxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters ofs'ul'luric acid such as alkyl sulfates and etl’ioxylated alkyl siviltates, sulfonates such as alhyl hen zene sultonates, acyl isethionates, acvl taurates and sulfosuecinates, and phosphates Tilt: most important members of the anionic surfactant class are the alkyl sulfates and the soaps. if the surfactant i’nolecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is fied as ic. Cationic surtactants include uuatemary ammonium (A) salts and etlioxylated amines. The quaternary ammonium salts are the most used members ofthis class if the surfactant molecule has the ability to tarry either a ve or negative charge, the tant is classified as ainphoteric. Ainphoteric surfactants include c acid tives, substituted allcylainides, Nnall'iylbetaines and phosphatides.

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS The use of surfactants in drug products, formulations and in ons has been reviewed (Rieger, in “Pharmaceutical Dosage Forms”, Marcel Dekker, lnc., New York, NY), P988, p. 285).

The iRNA for use in the methods of the invention can also be provided as micellar (I: formulations, “Micelles” are defined herein as a particular type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all the hydrophobic portions of the molecules are directed inward, g the hilic portions in contact with the surrounding aqueous phase. The converse arrangement exists it‘the environment is hydrophobic. ll) A mixed micellar formulation suitable for delivery h transdermal membranes may be prepared by mixing an aqueous solution ot‘the siRNA composition, an alkali metal Cg to {:22 alkyl sulphate, and a micelle forming compounds. Exemplary micel e torniing compounds include lecithin, hyaluronic acid, ceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, ile extract, cucumber extract, oleic acid, ic acid, linolenic acid, l5 monoolein, monooleates, monolaurates, borage oil, evening ot‘primrose oil, menthol, trihydroxy oxo cholanyl glycine and phannaceutically acceptable salts thereof, glycerin, ycerin, lysine, sine, in, polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethers and analogues f, chenodeoxycholate, deoxycholate, and es thereof. The e torming compounds may he added at the same time or after addition ofthe alkali metal 2t) alleyl sulphate. Mixed micelles will form with substantially any kind ot‘mixing ofthe ients but vigorous mixing in order to provide. smaller size micelles. in one method a first micellar composition is prepared which contains the siRNA composition and at least the alkali metal allryl sulphate. The first micellar composition is then mixed with at least three micelle torming compounds to form a mixed micellar composition. ln another method, the micellar composition is prepared by mixing the siRNA composition, the alkali metal alliyl sulphate and at least one ofthe micelle forming compounds, followed by addition ofthe remaining micelle forming compounds, with vigorous mixing.

Phenol and/or ol may be added to the mixed micellar composition to stabilize the lorrmilation and protect against bacterial growth. atively, phenol and/or m ~cresol may be (A) c. added with the micelle forming ingredients An ic agent such as glycerin may also be added after formation ofthe mixed micellar composition. l2or ry of the micellar formulation as a spray, the formulation can be put into an aerosol dispenser and the dispenser is charged with a propellant. The. propellant, whicl’i is under pressure, is in liquid form in the dispenser. The ratios ofthe ingredients are adjusted so that the aqueous and propellant phases become one, i.e., there is one phase. lt‘there are two , it is necessary to shake the dispenser prior to sing a portion ofthe contents, eg through a [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS metered valve. The dispensed dose ofpharmaceutical agent is led from the rnetered valve in a line spray.

Propellants may include en-containing chlorotluorocarbons, lrydrogenscontaining fluorocarbon s, dimetliyl ether and l ether. In certain embodiments, llFA l34a (l ,l ,l ,2 (I: tetralluoroetliane) may be used.

The c concentrations of the essential ingredients can be determined by relatively straightlorward experimentation. l: or absorption through the oral cavities, it is otten desirable to increase, tag. at least double or triple, the dosage for through injection or administration through the gastrointestinal tract. ll) B, Lipiripartz'ct’as iRN As, rag dsllNAs ot‘in the invention may be fully encapsulated in a lipid formulation, eg., a LNR or other nucleic acid—lipid particle.

As used herein, the term "LN?" refers to a stable nucleic acidwlipid particle. LNPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents ation of the particle (cg, a PEG—lipid conjugate). LNPs are extremely useful for systemic applications, as they exhibit extended circulation lifetin'ies following intravenous (iv) ion and accumulate at distal sites (9gp, sites physically separated from the administration site). LNl’s include "pSPLP," which \‘ an encapsulated condensing agent—nucleic acid complex as set forth in PCT Publication No. WU Oil/(Bo 33. The les ol‘the present invention nitrically have a mean 2t) er or“ about 50 run to about l5ll nrn, more typically about (all nut to about l3l) uni, more typically about 70 not to about l ll) nni, n'iost typically about ’70 nut to about 90 nni, and are substantially nontoxic, in addition, the nucleic acids when present in the nucleic acid lipid particles ol‘the present invention are resistant in aqueous solution to degradation with a nuclease Nucleic acidslipid particles and their method ot‘preparation are disclosed in, age, US Patent Nos. $376,567; 5,98l “Sill; 6,534,484; 6,586,4l 0:, 636,432; US. Publication No. 20l0/fi324l20 and PCT Publication No. W0 96/409640 in one embodiment, the lipid to drug ratio /nrass ratio) , lipid to dsRNA ratio) will be in the range ot‘frorn about l :l to about Stlzl from about l :l to about 25d front about 31l , , to about lfizl, from about 4zl to about l0:l, from about Szl to about 92l , or about ozl to about 92L Ranges intermediate to the above recited ranges are also contemplated to be part of the invention.

The cationic lipid can be, for example, N,N-dioleyl-N,N—diniethylarnrnoniurn chloride {DODAC'}, NiN—distearyl—N,N~diniethylarnmonruin e (DEAR), N—(l ~(2,3— dioleoyloxy)propyl}N,N,N—trirnetliylaninioniurn chloride (DOTAP), N—(l 42,3“ dioleyloxylpropylluN,N,N—trirnetliylarnnioniurn de (Dfllrl‘lle), Nil tliyl—Zlfidioleyloxy )propylaniine (D03DM A), l ,2“Dillinoleyloxy—N,Nsdirnetlrylarninopropane [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS (DLinDMAL linolenyloxyul‘il,N—dinietliylaminopropane {DLenDMA}, l,2~ Dilinoleylearbamoyloxy-E’i —diinetliylaminopropane {DLin-C-DAF’), l ,Z-Dilinoleyoxy-B - (dimetlriylaininolacetoxypropane (:Dliin—DAC), l,2~Dilinoleyoxy~3 ~morpliolinopropane (Ellin— MA), l,2—Dilinoleoyl—3—dirnetlijy'laniinopropane (BliinDAP), linoleyltliio—3— (I: dimethylaminopropane {DLiannDMA’}, leoyln2"linoleyloxyv’fi“dimethylaminopropane {DLin-Z—DMAP), l,2uDilinoleyloxy-3 —trimetliylarninopropane cliloride salt (DLin-TMAED, l ,lDilinoleoyl—Ei “trimetliylaminopropane de salt (DLin—TARCE), l ,2~Dilinoleyloxy—3“(N n'ietliylpiperazino)propane (Diem—R4832), or '3—U\flN~Dilinoleylamino}l iZ—propanediol (BlainAlfiy 3—(N,NwDioleylamino)—lQ—propanedio {DOAPL l,2~Dilinoleyloxo~3~(2~N,N— ll) dimetliylainino)etlioxypropane EG—DMA), l,2—DiliiiolenyloxyuN,N— dimetlriylaminopropane (DLinDMA), 2,Z—Dilinoleyl~4~dimetnylaniinonietliybll oxo lane (DLin—K—DMA) or analogs thereof, (3 aRfis,6aS)—N,N~din'ietliyl—292—di((92,1 2Z)—oetadeca—9,l2— dieiiylfietraliydrofi allncyelopentaldlll xol—5namine (ALNl 00), {62,92,28253 12)— lieptatriaeonta—oSQS,3 lutetraen-lg—yl 4"(cliinetliylaniiiioiilbntanoate (MCB), l,l 4-(2—(tf2— l5 {bistZ—liydroxydodecyl)amino)etliyl)(2—hydroxydodecylfiimino)etlryl)piperazin~l - yl)etliylazanediyl)didodecanQ—ol (Tech Gl ), or a e tliiereo'li The cationic lipid can se from about. 20 mol % to about 50 mol 9?:- or about 4-0 niol % oftlie total lipid present in the particle. in another embodiment, the compound 2,2—Dilinoleyl-él—diinetliylaminoetliyl-l:l ,3}— 2i) dioxolane can be used to prepare lipid—siRNA nanopartieles. Synthesis of2,2d)ilinoleyl—4— dimethylaminoetl’iyl—El ,3]~dioxolane is described in United States provisional patent application number til/l07,998 tiled on October 23., 2008., which is herein incorporated by reference. in one embodiment, the lipid-siRNA le includes 40% 2, 2—Dilinoleyl—4— dimetnylaminoethyl-ll ,3]*dioxolane: l0% DSPC: 40% Cholesterol: l(}% PEG-C—BOMG (rnole percent) with a le size ($63.0 :: 20 nn'i and a 0027 siRNA/Lipid Ratio.

The ionizable/nonwcationic lipid can be an anionic lipid or a neutral lipid including, but not limited to, distearoylpliospliatidylclioline (DSPC), dioleoylpnospliatidylclioline (DOPC), dipalmitoylpliospliatidylclioline (DP'PC), dioleoylpliospliatidyl glycerol (DOPG), dipalniitoylpliospliatidylglyeerol {DPPG} dioleoyl«pliospliatidylethanolaniine (DUNE), palniitoyloleoylpnospliatidylel'ioline (POFC), toyloleoylpliosphatidyletlianolamine (POPE/ll dioleoyl— iatidyletlianolamine 4"(N—nialeiinidoinetliyl)—cyelohexane—l- oarboxylate (DOPE- inal), clipalmitoyl pliosphatidyl etlianolamine (DPPEE), dimyristoylpliosplioetlianolamine (DMPE), distearoyl«pliospliatidylethanolaniine (DS?E), l6—O~niononietliyl PE, lo—O~ilin'ietliyl PE, l8~l "trans PE, l "stearoylawoleoyl— phospliatidyetnanolarnine (SOPE), cholesterol, or a mixture thereof. The non—cationic lipid can be from about 5 mol ‘34:} to about 90 mol %, about it) mol “/6, or about 58 mol ‘34; it‘eliolesterol is ed, oftlre total lipid present in the particle.

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS The conjugated lipid that inhibits aggregation ofparticles can be, for example, a hyleneglycoi (PEG)—lipid including, Without limitation, a PEG—diacyi glycerol (DAG), a PEG-diaileyloxypropyl (BAA), a PEG—phospliolipid, a PEG~ceraniide (Cer), or a mixture thereof.

The PEG~DAA conjugate can be, for example, a PEG—diiauryloxypropyl (Gig), a PEG— (I: dimyristyloxypi‘opyl (Cir), a PEG—dipalmityloxypropyl (Chg), or a PEG~ distearyloxypropyl {Cl-g). The conjugated lipid that ts aggregation icies can be from 0 rnol ‘34; to about moi “/6 or about 2 mol % ofthe total lipid present in the particle. in some en'rbodiments, the nucleic acid~lipid particle further includes cholesterol at, e.g., about l0 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle. l0 ln one embodiment, the lipidoid ND98-4liCl (MW l487) (see US. Patent Application No. l2/056,230, filed 3/2 72008, which is incorporated herein by reference), Cholesteroi (Sigma— Aldrich), and ramide era (Avanti Polar lipids) can he used to prepare lipid—dsRNA nanoparticles {1e LNPGl les). Stock solutions of each in ethanol can be prepared as follows: NDQS, l33 rug/nil; (:holesteroi, 25 mgr/nil, PEG—Ceramide era, l00 mg/nil. l”he ND98, l5 Cholesterol, and PliG~Cerarnide {316 stock solutions can then be combined in a, 6g, 42:48:10 molar ratio. The combined lipid solution can be mixed with s dsRNA (rag, in sodium acetate pli 5) such that the final ethanol concentration is about 354594) and the final sodium acetate concentration is about 100300 mhzl. Lipid~dsRNA r‘ticles typically form spontaneously upon mixing, Eepending on the desired le size distribution, the resultant nanoparticle mixture can be extruded through a polycarbonate membrane (sag, l00 nrn c’ubol’l} using, for e, a rnobarrel extruder, such as liipex Extruder (Northern Lipids, inc). In some cases, the extrusion step can be omitted, Ethanol removal and simultaneous buffer exchange can be accomplished by, for example, dialysis or tangential ilow tion. Buffer can be exchanged with, for example, ate buttered saline (PBS) at about pll 7, 655., about pll 63, about pH 7.0, about pitl 7.i about plrl 72, about pll 7.3:, or about pli 7.4.

OK?” NwMWW’N/ H K H NNAVWA‘N’lk/ANNNV“N‘Ax/NNWNVNWVV MNWNO o” lam/\MVAVK wl H N938 isomer i Formula l LNE?0l formulations are described, rag, in international Application Publication .2“Os No. Wt} 20084342973, which is hereby incorporated by reference.

[Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS Additienfi mammary 11p1d~dsRNA memEations are described in '1‘ab1e 1. 131112 1 931101119 Eipid/nen@3110“ is 10111131319/{Tatienic Lipid chuiesmml/PEG411316, can} agate 1,2{111111<11eny10xy—N,N—dimathy1aminopropane . . . . ,,/ 1:0»SLFLJL ..

(DLinDMA) . . /7. /34. /1.4) '"' :siRNA ~ 7:1 .X'1T‘(:f,/DP P (if/€1101135131011"?EG-CIMVIA iiino1ey1--dEnemy/123mm):11w}-E 1 ,3 ] __ 57.1/7.1/34.-’1/1.4 d10X01311€ (XTC) 11p1d:siRNA ~ 7:1 XTC/DSPC/Cbo1estemL/PEG-DMG 2 ,2 —Di1in01ey1—4~(iimathy1amin0'sthy1— .E1 ,3 ] ~ 57.5,1’75/315/35 dioxoiane (XTC) 2,2131}ino1ey1~4—dimet11y1aminoefilyi—U ., ,M, ]— 610140121119 (XTC) - :t/Choaestemi/PEG-DMG 2,2--Diiino1ey1--dEnemy/123mm):11w}-E 1 ,3 ] __ /31/1.5 d10X01311€ (XTC) 11p1d:siRNA ~ 6:1 XTC/DSPC/Cbo1estemL/PEG-DMG 2 ,2 —Di1in01ey1—4~(iimathy1amin0'sthy1— .E1 ,3 ] ~ 60/75/31/15, dioxoiane (XTC) 1ipid‘siRNA ,.

XTC/DSRC/Cho1estemE/PEG—DMG 2 ,2 431111101 ey1—4~dimet11y1 21011110811131 E1 ,3 ] — 50/10/38.:3/15 0121116 (X10) LipidcsiRNA 10:1 E3 aR,5 s,6a.S)—N,N~dimet11y1~2,2—(11EE92,1 22F A1111(10/1331)C/ChoiesteFOL/PEGDMG 0ctadeca~9 ,12 ~dieny1)tetrahydm—3 2111 50/10/385/15 cyc10pentaEd] E1 ,3](110x01-amine (ALN 100) iRNA 10:1 (62,92,28231 Z)1113;?iatrézicontauéfi,28,3 1 -- MC--3 .11)S 1"13/(11110136: 1 extol/PEG -- 1:}MG 118113.611—1 9—y1 4—(C1imethy1amino)butanaa§e 50,1’10/38.5/1.5 {MC3‘1 LipidzsiI’LNA 1i) :1 1,1 ‘-(2-E4~(2—((2~(1315(2- Tech (11/113? 713110163[5101/13EG—DMG hydroxydodecyDamiuo)ethy1)(2 - LNP 1 2 385/15 hydroxydedecy1)a.mino)et11y1)piperazir- — 1 — LipidcsiRNA 10:1 y1)€111y1azanediy1)didodecan~2~01 (m XTC/DSPOC1101/PEG~1J1V1G 50/10/385/15 LipidsiRNA: 33 :1 [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS MC31DSPC/ChoL/‘PEG—DMG LNP14 40/15/4116 1.11) 111311341118: 1 MCI}f1)SKY/(21101711 EEG-D3111/1113111Ara-PEG -1) SG 50/ 1 {1/35/45/05 LipmsiRNr» : 11:1 MC3/DSPC/C1101/PEG~D1V1<3 50/111/38.5/1.5 LipidsiRNA: 7: MC3,IDSPC/ChM/PEG—DSG 50/111/38.5/1.5 1.11) 111311341118: 10:1 MC},I’DSPC/CheL/PEG—Di‘vifl 50/10/385/15 LipidsiRNA: 12:1 PC/C1101/PEG~D1V1<3 1.1111) 1 9 .5 0/1 (1/3 5/5 LipidsiRNA: 8: MC3,IDSPC/ChM/PEG—DPG LNPZG /38.5/1.5 1.11) 111311341118: 10:1 C 1 2—2110/1‘} SP C/ChoL’PEG-DSQ 11111321 385/15 LipmsiRNr» : 7:1 115}?C/Cho1/11 1:G -1)SC; 1.1NP22 50/10/385/15 LipidzsiRNA: 10:1 DSPC: dis1ear0y1ph0sphatidyleh 01111:: 11131313: d1pa11111103113110sphatidybho1ine PEG—13111113: PEG-didinryristefl g1yeer01 (C1 4-1’1313, 0r1’EG—C14) (PEG with avg r1101 W1 01‘ 201.111) PEG—138G: 1?EG—disiyry1 g1yeer01 (C18—P1EG, or PEG—C18) (PEG with avg 11/101 wt 012000) MA: 1’EG—earbamoy1n1,2,—dimyristy10xypr0py1anrine (PEG with avg 11101‘WE 012000) SNAL 1? 11,2—Di111101eny10xyu'N,N—dimethflarninopropane (DLinDMAD comprising formulations are described in Internationai Publicatinn NO. W020(19/1 27060, 1111301 April 15, 211119, which is hereby incorporated by 16111176311661 X1C comprising ations are describei Cg”, in US. 1’r0v1510na1 Ser1a1N01 (31/148,366, 111ed January 29, 2009; USS. ionai Seria1'N0. 61/156,851, 111ed March 2,, 2009; US. Provisienzfl SE1‘1211N0. 111% June 11}, 211119; 11.8. ionai Seria1 NO. 61/228,373, 111ed 11,11y 24, 2069; 1.1.8. imrm Seria1Nn. 61/2399686, filed ber 3, 20099 and 1111ema‘riona1App1ieation N0. 1’C'17US2010/{1226 1-41, filed .1anuary 29, 2010, which are hereby incorporated by reference.

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS MC3 comprising formulations are described, rig, in US. Publication No. 20l0/0324 l20, tiled lune l0, 2010, the entire contents ofwhich are hereby incorporated by reference, ALNYd 00 comprising formulations are described, cg, International patent application number PCT/US09/63933, tiled on er l0, 2009, which is hereby irrem‘porated by (I: reference.

(HZ—200 comprising ations are described in US, Provisional Serial No. 63/175,770, filed May 5, 2009 and International Application No. PCT/US} (ix/33777, liled May 5, 20K), which are hereby incorporated by reference Synthesis qfiommbie/cationie s Any of the compounds, 51g, cationic lipids and the like, used in the nucleic acid—lipid particles of the invention can be prepared by Known c synthesis techniques, including the s described in more detail in the Examples. All substituents are as defined below unless indicated otherwise “Alhyl” means a straight chain or branched, noncyclic or cyclic, saturated aliphatic hydrocarbon containing from l to 24 carbon atoms. Representative saturated ht chain alkyls inchtde rn e thyl, ethyl, n—propyl, n—butyl, n~pentyl, l, and the like; while saturated ed alkyls include isopropyl, sec~bntyl, isobutyl, tertwbutyl, isopentyl, and the like.

Representative saturated cyclic allryls include cyclopropyl, cyciobtityl, cyclopentyl, cyclohexyl, and the like; while unsaturated cyclic allryls include cyclopentenyi and cyciohexenyl, and the like.

“Alkenyl” means an alkyl, as defined above, containing at least one double bond between adjaccnt carbon atoms Allrenyls include both cis and trans isoniers. Representative straight chain and branched yls include ethylenyl, propylenyl, i—butenyl, 2—butenyl, isobutyienyl, l- pentenyl, 2-pentenyl, 3-inethyl-i -bntenyl, Zarnethyl—Z—butenyl, 2,3 —din'iethyl~2 ~butenyi, and the like.

“Alliynyl” means any alliyl or allx'enyl, as defined above, which additionally contains at least one triple bond between adjacent carbons. Representative straight chain and branchec alkynyls include acetylenyl, propynyl, i —b1:ttynyl, 2~butynyl, lapentynyl, 2~pentynyh 3—rnethyl-i butynyl, and the like.

“Acyl” means any alkyl, alkenyl, or ailiynyl wherein the carbon at the point of attachment is substituted with an one group, as defined below. For example, —t;7(===())all<yi, "{3(===O)alkenyi, and 4:1}: nyl are acyl groups.

“Heterocycle” means a 5— to ’7—inembered cyclic, or 7~ to l0—n'iernbered ic, heterocyclic ring which is either saturated, unsaturated, or aromatic, and which ns from 1 or 2 heteroatonis ndently selected from nitrogen, oxygen and sulfur, and wherein the nitrogen and sulfur aton'is can be optionally ed, and the nitrogen heteroatoin can be [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ontionaiiy nized, including hicyciic rings in which any ofthe above heterocycies are fused to a benzene ring. The heterocycie can he attached via any heteroatorn or carbon atom.

Heterocycies include heteroaryis as defined heiow. Heterocycies include rnorphoiinyi, idinonyi, pyrroii dinyi, piperidinyi, piperizynyl, hydantoinyi, vaieroiactantyi, oxiran‘yi, (I: oxetanyl, tetrahydrofiiranyl, tetrahydropyranyi, tetrahydropyridinyi, tetrahydronrimidinyi, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyriinidinyi, tetrahydrothiophenyi, tetrahydrothiopyranyl, and the like.

The tenns “optionally substituted alkyi”, “optionaiiy substituted i”, “ontionaiiy substituted aileynyi”, “optionally substituted acyi”, and “optionaily substituted cycie” it) means that, when substituted, at least one en atom is replaced with a substituent. in the case of an oxo substituent (:0) two hydrogen atoms are replaced~ In this regard, substituents include oxo, halogen, heterocycie, ~CN, —OR,X,, —NRXRy, ~NR.XC(=U)Ry, —NRXSO2Ry, —C{=O)Rx, "C(ZOXJRX, NRny, —SOnRX and “SOnNRXRy, wherein n is Or 1 or 2, Ex and By are the same or dit‘terent and independently hydrogen, alkyl or heterocycie, and each of said alkyi and heterocyeie suhstituents can he titrther substituted with one or more of oxo, haiogen, ~08, ~C‘N, alkyi, —ORX,, heterocycie, —NRny, ~NRXC(=O)Ry, —NRXSOZRy, ~(T§(=O)Rx, —C(=O)ORX, NRXRy, $01in and "SOnNRny.

“Haiogen” nieans fluoro, chioro, hrorno and index in some embodiments, the methods ofthe invention can require the use ofprotecting 2t) groups~ Protecting group methodology is weli known to those sitiiied in the art (see, for e, Protective Groups in Organic Synthesis, Green, TW. cf (15., nterscience, New York City, 1999). Briefly, protecting groups within the context ofthis invention are any group that reduces or eliminates unwanted reactivity of a functionai group. A protecting group can be added to a functional group to mask its reactivity during certain reactions and then removed to reveal the original onai group, in some embodiments an ol ting group” is used.

An “aleohoi protecting group” is any group which decreases or eliminates unwanted reactivity of an aicohoi functional group. Protecting groups can he added and removed using techniques well known in the art.

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS ation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS Synthesis quormuia A ln some embodiments, nucleic acid-lipid particles of the invention are formulated using a eationic lipid offormnla A: N—R4 where Rl and R2 are independently all<yl, alkenyl or alkynyl, each can be U: optionally substituted, and R3 and R4 are independently lower alkyl or R3 and R4 can be taken together to form an optionally substituted heterocyclic ring. In some embodiments, the cationic lipid is XTC (2,Z—L‘iilinoleyl~4~dirnethylaniinoethyl~ll ,3lstlioxolane). ln general, the lipid of formula A above can be made by the following Reaction Schemes l or 2, wl’ierein all substituents are as defined above unless indicated otherwise. l0 Sclietne l Br on o MR2 Formula A Lipid A, Where Rl and RZ are independently allsyl, allienyl or l, each can be optionally substituted, and R3 and R4 are ndently lower alkyl or R3 and R4 can be taken together to form an ally substituted l‘ieterooyclic ring, can be prepared according to Scheme l. Ketone lb 1 and bromide 2 can be sed or prepared according to methods known to those of ry skill in the art. Reaction of l and 2 yields ketal 3. ’l'reatrnent oflretal 3 With amine 4 yields lipids of torniula A. The lipids ofiorrnula A ‘an be converted to the corresponding annnoninni salt with an organic salt of formula 5, where X is anion counter ion selected from halogen? hydroxide, phosphate? sulfate? or the like [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS Scheme 2 ti “3* straws? {Mllea c R-..>~i??\é Rf} Rl (Jr Alternati‘v'ely, the ketone 1 starting material can he prepared according to Scheme '2. rd reagent 6 and cyanide '7 can he purchased or prepared according to methods known to those ofordinary skill in the art Reaction ofo and 7 yields ketone l. Conversion ofketone l to the corresponding lipids of formula A is as described in Scheme l. sis QfMCS Preparation ofDLin—M—CB ~DMA , {6292,2823 l Z)-heptatriaconta—6,9,28,3 l — tetraensl 9~yl 4-{dirnetnylarnino)batanoate) was as follows. A solution of {62,923,323 l 2} heptatriacontit—6,9928,3: aen~l 9~ol (0.53 g), dirnetl’iylaniinolautyric acid hydrochloride (OOSl g), 4 ~N,N—dimethylaniinopyridine (0.6l g) and l—etliyl~3~(3 - dirnethylarninopropylkarbodiirnide hydrochloride (0.53 g) in dicltlorornetliane (5 niL) was stirred at room temperature overnight. The on was washed with dilute hydrochloric acid followed by dilute aqueous sodiurn onate. The organic fractions were dried over anl’iydrous magnesium sulphate, filtered and the solvent removed on a rotovapt The residue was passed down a silica gel column (20 g) using a 16% rnethanol/diclilorornethane elntion gradient.

Fractions containing the purified t were combined and the solvent removed, yielding a ess oil (0.54 g). Synthesis off: LAW-.100 [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS Synthesis ofketal 5 l9 l 00] was performed using the following scheme 3: ill-Ha {EHHH :Hzgosa Hit-Hale Eat? JHFHW ‘HH HHrHHe :3 HHH :3 HHHH :3 } H069 :3: (jig-g ‘LH‘H to 51$ gHm Hire 3...: r'(\r’\\/\x‘\:;;.flxa_w"\rfl,’ V %\v"vr\ae\mefi\:y—”\_X\\/ l $337637." l f""\/’\JNAW «vxxvl ““““ VfllflvWJ-A’w7 (IL/Vle\ X AV!- KL—v AK! 3 (H:" 'V‘wwwmifl n!’- * HH U: Synthesis 0326515 To a stirred suspension ofLiAlll4 (3.74 g, 0.09852 mol) in 200 ml anhydrous THF in a two neck REF UL), was added a solution of5l4 (l 0g, 0.04926rnol) in '70 mL F slowly at 0 0C under nitrogen atmosphere. After complete addition, reaction mixture was warmed to room temperature and then heated to reflux for 4- h Progress ofthe reaction was monitored by TLC. l0 After tion of reaction (hylLt; ) the mixture was cooled to 0 0C and quenched with caretul addition of saturated Na2$04 on Reaction mixture was stirred for 4 h at room temperature and filtered ott e was washed well with TlllThe ii ltrate and washings were mixed and d with 400 mL dioxane and 26 BIL eonc. lthl and stirred for 20 minutes at room temperature. The volatilities were stripped off under vacuum to furnish the hydrochloride salt of El‘3 as a white solid Yield:7l2 g lll-Nl‘le (DMSO 400MHz): 8:: 9.34 (broad, 23), 5.68 (s. 28), 374t(in, lll) 266--260 (m 211) 2.St)245 (m 5ll) 5321217335ZS of516 To a stirred solution of compound 5l5 in lOO mL dry DCM in a 250 mL two neck REE 7 niL, O2669 mol) and cooled to was added N’Et3 (37.2 l) ()6 under nitrogen atmosphere Alter a slow addition ofN—(benzyloxywarhonyloxyl—suceininiide (20 g, 0.08007 mol)in 50 rnL dry DCM on n’iixture was allowed to warm to room temperature After completion of the reaction €2~3 h by lLC) mixture was washed successively with lN HCl solution ll X. lUG mL) and saturated NalthO3 solution (l X 50 mL). The organic layer was then dried over anhyd.

NaZStfié’l and the solvent was evaporated to give crude material which was d by silica gel N iJ! column i‘hron’iatography to get Sl6 as sticky mass Yield: 1 it? (89%). lH—NMR (CDCB, 4-GOMHZ): 5: 736’27im, 5H) 569 (s 2H)5 l2 {s .96 (lira, -4 (s3 3H),2.60(rn, 28), 2.30—2.25tm, 23). LC-MS {M-v-H} 42323 80).

Symhesis off 1 7A two" 5 I 719 [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS The cyclopentene 5 l6 {_ 5 g. 0.02l64 mol) was dissolved in a solution of 220 mL acetone and water (l Ozl) in a single neelt 500 mL RBF and to it was added N—methyl morpholine-N— oxide (7.6 g, 0.06492 n'iol) followed by 4.2 ml; ot‘7.6% solution ol’lilsifll {(3.275 g, 0.00108 melt in utanol at room ature. Alter con’ipletion of the reaction (N '3 h), the mixture was (I: quenched with addition of solid l\a2 803 and resulting mixture was stirred for l5 h at room temperature. on mixture was diluted with DCM (3 00 mL) and washed with water ('2. x lllll hill) followed by saturated NallCOB (l x 50 ml.) on, water (i x Sill hill) and finally with brine (ix 50 ml.) Organic phase was dried over an .Na2S‘O4 and solvent was removed in vacuum. Silica gel column chromatographic purification ofthe crude material was ed a ll) mixture of diastereomers, which were separated by prep llPLC. Yield: — 6 g crude .5l 7A ~ Peale-l (white solid), 5.l3 g (96%). lll-NMR (DIX/ESQ, 406MHz): 5:: 7.39“ 7.3l (m, fill), 5.04(s, 28), 4.78—4.73 (m, lllL 4.48—4.47(d, 2H). 3.94—3.93(rn, 2H), 2.7l(s, 3H), 1.72 l.67{_rn. 4H). LCmMS n lM+Hln266.3, lhl+Nll4 +l—2835 present, )7.86%.

Stereochemistry confirmed by X—ray.

Symhes is of5 I8 Using a procedure ous to that described for the synthesis of compound 505, compound 5l8 (l 2 g. dig/ii) was obtained as a colorless oil. lH~NMR (CDCB, 409MHz): 5: 7.35n7.33(m. 4H), 7.30w7.27(m, lH). 5.37—5.27tm. 8H), 5.l2{s. 2H), r-‘l.75(rn.lH), 4.58— 4.57tm,2ll), 2.78-2.74(m,7ll), 2.06-2.llll(m,8ll), l.96-l .9l (m, 2H), l.62(m, 4H), l.48(m, 2H), 2t) 1.37—1.25lbrm, 36H), m, 6H). HPl’..C—98.65%.

Genera! Procedure.for the Synthesis ofCompozmd 5 1,9 A on of nd 518 (l eq) in hexane (15 mL) was added in a dropvwise fashion to an ice—cold on ofLAltl in T HE (l M, 2 eq). After complete addition, the mixture was heated at 400C over 0.5 h then cooled again on an ice bath. The mixture was lly hydrolyzed with saturated aqueous Na2SO4 then filtered through celite and reduced to an oil.

Column chromatography provided the pure 5l9 {1.3 g, 68%) which was obtained as a colorless oil. l3€ Nl‘v’lR d l30.2, l3ll.l (x2), l27.9 (x3), l l2.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9 (x2), 29.7, 29.6 (x2), 29.5 {3(3), 29.3 (X2), 27.2 (x3), 25.6, 24.5, 23.3, 226, l4.l; Electrospray MS (ti/e): lar weight for C44l5l80N02 (M + l’l)+ Calc. 654.6, Found 654.6.

Formulations prepared by either the standard or extrusionnfree method can he terized in similar manners. For example, formulations are typically characterized by visual inspection. They should be whitish translucent solutions tree horn aggregates or nt. Particle size and particle size distribution oflipid—nanoparticles can he n'ieasured by light scattering using? for example, a Malvern Zetasizer Nano ZS (lVlalvern, USA). Particles should he about 20—3 00 nm, such as 40400 nm in size. The particle size distribution should he uniniodal. The total dsRNA concentration in the torn'iulation, as. well as the entrapped fraction, ‘l‘lS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS is estimated using a dye exclusion assay. A sample ofthe formulated dsRNA can be incubated with an RNA-binding dye, such as Ribogreen ifMolecular Probes) in the presence or absence of a tbrrnulation disrupting surtactant, 8.53., 0.5% Triton—Kl 00. The total dsRNA in the formulation can be determined by the signal from the sample containing the surfactant, relative to a standard (I: curve, The entrapped fraction is determined by subtracting the “free” dsRNA content {_as measured by the signal in the absence of surfactant) from the total dsRNA content, l’erc ent entrapped dsRNA is typically >85%. l:or SNALP lation, the particle size is at least 30 nrn, at least 40 nrn, at least 50 nm, at least 60 nm, at least '70 nm, at least 80 nm, at least 90 nm, at least 100 nrn, at least ill} run, and at least l20 nrn. "l‘he suitable range is lly about at least ll) 50 nm to about at least llll run, about at least 60 nm to about at least ltlll run, or about at least 80 nm to about at least 901ml.

Compositions and formulations for oral administration include powders or granules, inicroparticulates, nanoparticulates, suspensions or solutions in water or non—aqueous niedia, capsules, gel capsules, sachets, tablets or ininitablets. ’l'hiclreners, flavoring agents, diluents, l5 errnilsiilers, dispersing aids or binders can be desirable. ln some ments, oral tbrrnulations are those in which dsRNAs featured in the invention are administered in conjunction with one or more penetration enhancer surfactants and chelators. le surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof Suitable bile acid s/salts include eoxycholic acid (CDCA) and oxychenodeoxycholic acid , cholic acid, 2t) dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocl’iolie acid, taurodeoxycholic acid, sodium tauro—Zél,25~dil’iydro~tusidate and sodium glycodiliydrofusidate. le fatty acids include donic acid, undecanoic acid, oleic acid, lauric acid, ic acid, capric acid, tic acid, palniitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, lein, dilaurin, glyceryl l~rnonocaprate, l“ dodecylazacycloheptan—Z—one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pliarinaceutically acceptable salt thereof (rag sodium). in some embodiments, combinations ofpenetration enhancers are used, for example, fatty acids/salts in combination with bile acids/salts. One ary combination is the sodiurn salt ot‘lauric acid, capric acid and UDCA. Purther penetration enhancers include polyoxyethylene~9 ~lauryl ether, (A) c. polyoxyethylene~20~cetyl ether. DsRNAs féatured in the invention can be delivered orally, in granular form including sprayed dried particles, or cornplexed to form inicro or nanoparticles.

DSRNA cornplexing' agents e polysaniino acids; nines; polyacrylates; lkylacrylates, polyoxethanes, polyalla’ylcyanoacrylates; cationi'sed gelatins, albumin s, es, acrylates, polyethyleneglycols (PEG) and es; polyall<ylcyanoacrylates; DEAEI derivatized polyiniines, pollulans, celluloses and es. Suitable conipleXing agents include chitosan, N—trirnethylchitosan, poly‘sL—lysine, polyhis ti dine, polyornithine, polysperrnines, ’l‘l? [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS protamine, nylpyridine, polythiodiethylaminoinethylethylene Pt’l‘DAE), polyaniinostyrene (9.3;, puarnino), polytmethylcyanoacrylate), polytethyl crylate), poly£hutylcyanoacrylate), poly(isohutylcyanoacrylate), poly(isohexylcynaoacrylate), DlEAlE-n'iethaciylate, DliAli~ hexylacrylate, DEAE—acrylaniide, DEAR—albumin and DEAR—dextran, polyinethylacrylate, (I: xylacrylate, poly’(B,L—lactic acid), polytDLwlacticneo—glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for dsRNAs and their preparation are bed in detail in US. Patent 6,887,906, US Puhln. No. 20030027780, and US. Patent No. 6,747,0l4, each of which is incorporated herein by nce.

Compositions and formulations for parenteral, intraparenchynial (into the brain), ll) intrathecal, intraventricular or intrahepatic administration can include sterile aqueous ons which can also contain s, diluents and other suitable additives such as, but not limited to, penetration enhancers, r con'ipounds and other pharmaceutically acceptahle carriers or excipients.

Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposorne—containing formulations. These compositions can be generated from a y ol’cornponen ts that e, but are not limited to, pretonned liquids, selfneinulsifying solids and self~eniulsifiving semisolids. Particularly preferred are formulations that target the liver when treating c disorders such as hepatic carcinoma.

The pharmaceutical formulations of the present invention, which can conveniently be 2t) presented in unit dosage torrn, can he prepared according to tional technitpies well ltnovvn in the pl’iannaceutical industry, Such techniques include the step othringing into association the active ingredients with the ceutical carrierts) or ent(s). in general, the formulations are prepared by unitormly and intimately bringing into association the active ingredients With liquid carriers or finely divided solid rs o “ both, and then, if necessary, shaping the product.

The ositions o‘l’the present invention can he formulated into any ot‘many possihle dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, solt gels, suppositories, and enemas. The compositions ofthe present invention can also he formulated as suspensions in aqueous, norraqueous or mixed media. Atpteous suspensions can further contain substances Whittl’l increase the viscosity ofthe suspension including, for example, sodium (A) c. carhoxymethylcellulose, sorhitol and/or dextran. The sion can also contain izers.

C. Additional Formulations i, Emulsions The con'ipositions ol‘ the present invention can he prepared and formulated as emulsions, Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of ts usually exceeding (Hum in diameter (see cg, Ansel‘s Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HQ, 2604, laippineott ‘l‘l8 ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS Williams 8; Wilkins (8th ed), New York, NY; ltlson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds), l988, Marcel Dekker, Inc, New York, NY” volume 1, p. 199; Rosofi‘, in Phannaeeutical Dosage Forms, herman, Rieger and Banlrer (lids), Willi, Marcel Dekher, lnc,, New York, N.Y,, Volume l in ceutical Dosage , p, 245; Block, (I: Forms, Lieberman, Rieger and Banker (Eds), l988, Marcel Deldrer, lnc,, New York, N.Y., volume 2, p. 335; lliguclii a? all, in Remington's Pharmaceutical es, Mach Publishing Co, liaston, 9a., l985, p. 30l ). Emulsions are often biphasic s comprising two immiscible liquid phases intimately mixed and dispersed with each other. in general, emulsions can he of either the waterninnoil {w/o) or the oilmin~water (o/w) variety. When an aqueous phase is finely it) divided into and dispersed as minute droplets into a hull: oily phase, the resulting composition is called a water—in-oil {w/o) emulsion. Alternatively, when an oily phase is tinely divided into and dispersed as minute droplets into a hulk aqueous phase, the ing composition is called an oil— inwwater (o/w) emulsion. Emulsions can contain onal components in addition to the sed phases, and the active drug which can he present as a solution in either the aqueous phase, oily phase or itselt‘as a separate phase. Pharmaceutical ents such as emulsifiers, stabilizers, dyes, and anti—oxidants can also he present in emulsions as needed. Pl’iairnaceutical emulsions can also he multiple ons that are comprised ofmore than two phases such as, for example, in the case of oilwinwwaterninnoil ) and waternin—oil~inn‘v 'ater (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple hinary 2t) emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w en'iulsion. se a system ofoil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic ity. (liften, the dispersed or discontinuous phase ofthe emulsion is well dispersed into the external or contintuvus phase and maintained in this form through the means ulsiiiers or the viscosity ofthe formulation.

Either ofthe phases of the emulsion can he a lid or a solid, as is the case of emulsion~style ointment bases and creams. Other means of stabilizing ons entail the use of emulsifiers that can he incorporated into either phase ofthe emulsion. limulsi’liers can broadly be classified into four categories; synthetic surfactants, naturally occurring emulsifiers, absorption bases, and (A) c. finely dispersed solids {_see 8.33., s l’harmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG, and Ansel HQ, 2004, Lippincott Williams & \I‘v’ilkins (8th ed), New York, NY; ldson, in ceutical Dosage Forms, l_..ieherman, Rieger and Banker (Eds), l988, Marcel Dekker, inc, New York, NY, volume l, p. l99).

Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (see eg, Ansel's Pharmaceutical Dosa re Forms and Dru r Deliverv Systemsh Allen, LV. Po )OVlCll NG., and , ., 9 9 [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS Ansel HQ, 2004, Lippincott Williams (51:. Wilkins {8th ed), New York, NY; Rieger, in ceutical Dosage Forms, Lieberman, Rieger and Banker (Eds), l988, Marcel Dekker, inc, New York, N.Y., volume l in Pharmaceutical Dosage Forms, man, , p. 285; ldson, Rieger and Banker (Eds), Marcel Dekker, lnc,, New York, N,Y., l988, volume l, p. l99), (I: Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion, The ratio of the hydrophilie to the hydrophobic nature ofthe surfactant has been termed the liydrophile/lipophile balance (llLlfi) and is a valuable tool in categorizing and ing surfactants in the preparation ot‘lbnnulations, Surfactants can be classified into ditlerent classes based on the nature ofthe hilic group; nonionic, c, cationic and amphoteric (see ill 9.3:, s ceutical Dosage Forms and Drug Delivery Systems, Allen, LV., l’opoyieh Nil, and Ansel EC, 2004, Lippincott Vi’illianis & Wilkins (8th ed), New York, NY Rieger, in l?harinaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds), l 988, Marcel Dekker, lnc., New York, NY, volume l, p. 1285).

Naturally ing emulsifiers used in emulsion formulations include lanolin, x, phosphatides, lecithin and acacia, Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also een used as good emulsifiers especially in combination with surfactants and in s preparations, These include polar inorganic solids, such as heavy metal ides, nonswelling clays such as bentonite, 2t) attapulgite, hectorite, kaolin, mtmtrnorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.

A large variety ofnon-emulsifying als are also included in on formulations and contribute to the ties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, tatty esters, ectants, hydrophilic colloids, preservatives and idants , in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds), l988, Marcel Dekker, inc, New York, N.Y., volume 1, p. 335; ldson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (lids), l988, Marcel Dekker, lnc,, New York, NY, volume l, p. l99). llydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic (A) c. polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose}, and synthetic polymers (for example, carbomers, cellulose ethers, and carhoxyyinyl polyi‘ners). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the sed—phase droplets and by increasing the Viscosity of the external phase. ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that can readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preser ’atives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkoniurn chloride, (I: esters ofpmhydroxyhenzoic acid, and horic acid, Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the tormulation. Antioxidants used can be free radical scavengers such as tocopherols, alkyl gall-ates, butylated yanisole, ted hydroxytoluene, or reducing agents such as ascorbic acid and sodium nietabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin, ll) The ation of on formulations via dermatological, oral and parenteral routes and methods for their nianul‘acture have been reviewed in the literature (see eg, Ansel's kharmaceutical Dosage Forms and Drug Delivery Systems, Allen, lLV., Popovich N6, and Ansel HQ, 2004-, Lippincott Williams & Wilkins (8th ed .), New York, NY; ldson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds), l988, Marcel Dekker, hid, New York, NY, volume l, p. it???) Emulsion formulations for oral ry have been very widely used because of ease ot‘forrrmlation, as well as et‘ticacy from an absorption and bioavailability standpoint {see tag, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L‘s/L, Popovich NG, and Ansel HQ, 2004, Lippincott Williams & Wilkins (8th ed), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker 2t) (lids), Willi, Marcel Dekker, lno, New York, NY, volume l, p. 245; ldson, in ceutical Dosage Forms, l_,rieberman, Rieger and Banker (Eds), l 988, Marcel Dekker, lnc., New York, NY“ volume l, p. l99). eralnoil base laxatives, oil~soluble vitamins and high fat nutritive preparations are among the materials that have ly been administered orally as o/w ermilsions. ii. Microemuisimzs in one embodiment ofthe present invention, the compositions of iRNAs and nucleic acids are formulated as microemulsions. A microemulsion can be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (see rag, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, (A) c. LV, Popovich NG, and Ansel HQ, 2004, Lippincott Williams & lit/lilkins (8th ed), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, man, Rieger and Banker (Eds), l988, l‘vlar ‘el Dekker, lnc., New York, N.Y., volume l, p. 245). Typically microemulsions are systems that are ed by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, lly an intermediate chain~length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two ible liquids that are stabilized by interlacial [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS films of surface-active molecules (Leung and Shah, in: lled Release ofDmgs: Polymers and Aggregate Systems, Rosoff, lvl, lid, l989, VCH Publishers, New York, pages l85-2l 5).

Microentulsions commonly are prepared via a combination of three to five components that include oil, water, surlactant, cosurtactant and electrolyte, ‘Whether the i'nicroernulsion is ot‘the (I: water—innoil (V '/o) or an oilninwwater (Oi/W7) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing oftlie polar heads and hydrocarbon tails of the surfactant molecules {Scliott, in Remington's Pharmaceutical Sciences, lvlack Publishing Co, Easton, Pa, l985, p. '27l ).

The phenomenological approach utilizing phase diagrams has been extensively studied ll) and has yielded a comprehensive knowlec ge, to one skilled in the art, ot‘how to formulate rnicroernulsions (see eg, Ansel’s Pharmaceutical Bosage Forms and Drug Delivery Systems, Allen, LIV” Popovich Neil, and Ansel Hill, 2004, Lippincott Williams & Wilkins (8th ed), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds), l988, Marcel r, Inc, New York, NY, volume l, p. 245; Block, in Pharmaceutical Dosage l5 Forms, Lieberman, Rieger and Banker (Eds), l988, l‘vlarcel Deklter, lnc., New York, N.‘r’., volun'ie l, p. 335), Compared to conventional emulsions, mi lsions offer the age of solubilizing waterninsolu‘ole drugs in a formulation ofthermodynamically stable droplets that are formed spontaneously, Surfactants used in the preparation of microemulsions e, but are not limited to, 2t) ionic surfactants, non~ionic surfactants, Brij 96, polyosyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol aurate (MLS l0), lycerol monooleate (MOSlO), liexaglycerol inonooleate (l303l0), hexaglycerol pentaoleate ), decaglycerol monocaprate (l‘vlCA750'), decaglycerol monooleate (MQ'YSO), ycerol sequioleate (80750), decaglycerol decaoleate (9130750), alone or in combination with cosurfactants. The cosurfactant, usually a short~chain alcohol such as ethanol, l—propanol, and l—‘outanol, serves to increase the interlacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because ofthe void space generated among surfactant molecules. mulsions can, however, he prepared Without the use ol‘cosurtactants and alcohol~free seltPeri'i'ulsifyiing microerrnilsion systems are known in the art. The aqueous phase can typically be, but is not d to, water, an (A) c. aqueous solution ofthe drug, glycerol, P136300, PEG-400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase can include, hut is not limited to, materials such as Captex 300, Captex 355, Caprnul MCM, fatty acid esters, medium chain (CS-Cl 2) mono, di, and cerides, polyoxyetl’iylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized CSIClG glycerides, ble oils and ne oil.

Microemulsions are particularly of st from the oint of drug solubilization and the enhanced absorption of drugs. Lipid based rnicroernulsions (hoth o/vv and w/o) have been [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS proposed to enhance the oral bioavailability of drugs, including peptides {_see eg, U13, Patent Nos. 6,,l 9l,l05; 7,063,869; 7,070,802; 7,157,999; Constantinides er (2!), Pharmaceutical Research, l994, ll, 13854 390; Ritschel, M’eth. Find. Exp. Clin. Pharmacofi, l993, l3, 205).

Microen’iulsions atlbrd advantages of improved drug solubilization, protection ofdiug from (I: enzymatic hydrolysis, possible enhancement of drug absorption due to surfactantninduced alterations in membrane fluidity and permeability, ease ofpreparation, ease of oral administration over solid dosage forms, ed clinical potency, and sed toxicity (see e.g., US. Patent Nos. o',l9l ,l {35; 7,063,860; 7,070,802; 7,l 57,099; Constantinides at 0,1,, Pharmaceutical Research, l99r-l, ll, l385; Ho er of, J. Pharm. Sell, l996, 85, ), Often ll) microemulsions can form spontaneously when their components are brought together at ambient temperature. This can be particularly advantageous when ating thermolabile drugs, peptides or iRNAs. hrlicroernulsions ha 1e also been effective in the transderinal delivery of active components in both cosmetic and pharmaceutical applications. it is expec ed that the microemulsion compositions and ations of the present ion Will facilitate the sed systemic absorption of iRNAs and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake ot‘iRNAs and nucleic acids, mulsions of the present ion can also contain additional ents and additives such as sorbitan monostearate (Grill 3’), Labrasol, and penetration enhancers to improve the properties ofthe formulation and to enhance the absorption ofthe iliNAs and nucleic acids of 2t) the present invention. Penetration enhancers used in the rnicroernulsions of the present ion can be classified as belonging to one of five broad categories——surtactants, fatty acids, bile salts, ing agents, and nonwchelating nonwsurfactants (Lee of (17., al Reviews in Therapeutic Drug Carrier Sir/stems, l991, p. 92'). Each ofthese classes has been discussed above. iii. particies an RNAi agent ot‘the invention may be orated into a paiticle, cg, a n’iicroparticle.

Micropaitic es can be produced by spray—drying, but may also he produced by other methods including lyophilization, evaporation, fluid bed drying, vacuum drying, or a combination ofthese techniques. iv. Permutation Enhancers (A) c. in one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly iRNAs, to the skin of s. Most drugs are present in solution in both ionized and nonionized forms. ver, usually only lipid soluble or lipophili c drugs readily cross cell membranes. it has been discove ed that even non— lipophilic drugs can cross cell membranes ifthe membrane to be crossed is treated with a penetration enhancer. in addition to aiding the diffusion of non—lipophilic drugs across cell membranes, penetration enhancers also enhance the penneability ot‘lipophilic drugs.

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS Penetration enhancers can he classified as belonging to one of five broad categories, to, surfactants, fatty acids, bile salts, ing agents, and non—chelating non—surfactants (see 6.3:, Malrnsten, M. Surfactants and polymers in drug delivery, na Health Care, New York, NY, 2002; Lee et (15,, Critical Reviews in Therapeutic Drug Carrier Systems, l99l , p.92). Each of the (I: above ned classes ofpenetration enhancers are described below in greater detail.

Surfactants (or "surface-active agents") are chemical entities which, when dissolved in an aqueous on, reduce the surface tension of the on or the acial tension hetween the aqueous solution and another liquid, with the result that tion ofiRNAs through the mucosa is enhanced. ln addition to bile salts and fatty acids, these penetration enhancers include, for ll) example, sodium lauryl sulfate, yethylene—Q—lauryl ether and polyoxyethylene—Z(l—cetyl ether) (see eg, ten, M. Surfactants and polymers in drug delivery, Intorma Health Care, New York, NY, 2002; Lee (3! (117., al Reviews in Therapeutic Drug Carrier Systems, l99l p.92); and perfluorochernical ons, such as FCH—‘lfil. 'l'ahaliaslii of (117., J. Pharm. Pizarmacoi, l988, 49, 252).

Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (i‘i—decanoic acid), niyristic acid, palmitic acid, stear‘ic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, rnonoolein (lwmonooleoylwracn glycerol), dilaurin, ic acid, donic acid, glycerol lnnioriocaprate, lw dodecylazacyclolieptanuZuone, acylcarnitines, acylcholines, €1-20 alkyl esters thereof (eg, 2t) methyl, isopropyl and t-butyl), and mono and di—glycerides thereof(i.e., ole-ate, e, caprate, rnyristate, palinitate, stearate, linoleate, cal) (see cg, Touitou, F. at al. Enhancement in Drug Delivery, CRC Press, Danvers, MA, 2006; Lee at all, Critical s in Therapeutic Drug Carrier Systems, l99l, p.92; anishi, Critical Reviews in 'llherapeutic Qrug Carrier Systems, l99t), 7', l33; El llariri at a!., J. Pharnrz. Pharmacofl, 1992, 44, till—654).

The physiological role ofhile includes the facilitation of dispersion and absorption of lipids and fatnsoluhle vitamins {see cg, Malmsten, M. Surfactants and polymers in drug delivery, lnforma Health Care, New York, NY, 2992; Butnton, Chapter 38 in: Goodman & n's The Pharmacological Basis of Therapeutics, 9th lid, llardman at at. lids, Mciiraww Hill, New York, l996, 131). 9344935), Various natural bile salts, and their tic derivatives, (A) c. act as penetration enhancers. Thus the term "bile salts" includes any of the naturally occurring components ofbile as well as any oftheir synthetic derivatives. Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptahle sodium salt, sodium cholate), del’iydrocliolic acid (sodium dehydrocholate), deoxycliolic acid m deoxycholate), glucholic acid {sodium glucliolate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid tsodium taur‘ocholate), taurodeoxycholic acid (sodium taurodeosycholatel, chenodeosycholic acid (sodium chenodeosycholate), ursodeoxycholic acid [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS (UDCA), sodium 24l-,25~dihvdro~fusidate (STDHF), sodium glycodiliydrofusitlate and polyoxyetliylene—”9—lauryl ether (FOE) (see 8.3, Malmsten, E‘s/l Surfactants and polymers1n drug delivery, lnforrna Health Care, New Yorlc, NY, 2992; Lee 62 0.12., Critical Reviewsin Therapeutic Drug Carrier Systems, l99l 39 in: ton’s Pharmaceutical , page 92:, Swinyard, Chapter (I: es, l8th lid, Gennaro, ed, Mach hing Co, , Pa, l990, pages 3; l‘vluranishi, Critical Reviews in ’l‘lierapeutic Enig Carrier Svsteins, l99ll, 7, l—33, Yamamoto a! ,J. Pharm. exp. Them l992, 263, 25; ita 212.521,]. Rheum 1322., l990, 79,5- 79—583,15? Chelating agents, as usedin tion with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the ll) result that absorption of iRNAs through the mucosa is enhanced. With regards to their use as penet 'ation enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibitedoy clielating agents (Jarrett, J Cin0122a: l,993, 6 l 8, 3 l5~ 339’). Suitable clielating agents include but are not limited to disodium ethylenedianunetetraacetate (FDTA), citric acid, salicvlates (22g, sodium salicvlate, 5- n'iethoxysalicylate and anil ate), N—acyl derivatives of collagen, laureth—9 and N—amino acyl derivatives ofbetandilcetones (enamines)(see 83,,Katdare, A c2 (27., ent development for pharmaceutical, biotechnology, and drug delivery, CRC Press, Danvers, MA, 2006; Lee 82 02., Critical Reviews in Therapeutic Drug Carrier Systems, l99l Critical , page 92; Muranishi, 29 Reviews in Therapeutic Drug Carrier s, l990, 7, l—33; Buur e! 0.12., J. Contra! Rel, l99tl, id, 43 —5 l ‘2.

As used , non~chelating non~surfactant penetration enhancing compounds can be defined as compounds that trate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of iRNAs through the tary mucosa (see eg, Muranishi, Critical Reviews in Therapeutic Ding Carrier Systems, l990, 7, l—.’).’)). This class of penetration enhancers includes, for example, unsaturated cyclic ureas, l—allryl— and in allrenylazacvclo-alharrone derivatives (Lee 92' 2:22., Critical Reviews in 'l‘herapeutic Qrug Carrier Systems, l99l and nonsteroidal antiinflammatory agents such as diclolenac sodiii,1rn , page 92); indornethacin and phenylhutazone (Yarnashita 32 (227., J. Pharm. Pizarmacoi., l9ll7, 39, 62l 4326).

(A) c. Agents that enhance uptake of iRNAs at the ar level can also he added to the pharmaceutical and other compositions ofthe present invention. For example, cationic lipids, such as lipotectin tlunichi 62 at, U3 Pat No5.,7(}5,l88), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo at 222., PCT Application ‘WO 97/3073 l ‘1, are also known to e the cellular uptake of dsRNAs. Examples of commercially available transfection reagents include, for example ctamineTM (lnvitrogen; ad, CA), lipol‘ectamine 2000““tlnvitrogen; (,arlsh21d,LCA), '293tectinTM (invitroge;n Carlsbad, CA), [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS CellfectinTM {lnvitrogeng Carlsbad, C A), Dl‘lelEnCTM (lnvitrogen; Carlshad, CA), li‘ree-Stylel'M MAX (lnvitrogen; Carlsbad, CA), LipofectarnineTM 2000 CD (lnvitrogen; Carlsbad, CA), LipofectarnineTM (lnvitrogen; Carlsbad, CA), RNAiMAX (lnvitrogen; Carlsbad, CA), OligofectamineTM (h’ivitrogen; Carlsbad, CA), OptifizctTM (lnvitrogen; Carlsbad, CA), X— (I: tremeGENE Q2 ’l‘ransfection Reagent ; Grenzaclierstrasse, Switzerland), DO'l‘AP Liposomal rlTransfection t (Grenzacherstrasse, Switzerland), DOSPER Liposornal Transfection Reagent aclierstrasse, Switzerland), or Fugene {Grenzacherstrasse rland), ectain® Reagent (Prornega; lyladison, 3W), Tram;FastTM Transfection Reagent {l’romega; n, Wl), 'l'leM~20 Reagent nega; Madison, lily/l), ’l‘fXTi‘l—SO Reagent {l’romega; Madison, WI), Dreamli‘ectl'M (OZ Biosciences; Marseille, France), Ecol'r'anstect (OZ Biosciences; lle, France), ass” Dl Transfection Reagent (New England Biolahs; Ipswich, MA, USA), Lyo‘ifecll‘l/liipoGenTM (lnvitrogen; San Diego, CA, USA), ectin 'l‘ransfection Reagent (Genlantis; San Diego, CA, USA), Neurol’OR’l‘ER 'l'ransfection Reagent (Genlantis; San Diego, CA, USA), (lonel)()l{l‘ER 'l‘ransfection reagent {(3611lfil’lll5; San Uiego, CA, US A), GenePQRTlER 2 Transl‘ection reagent (Genlantis; San Diego, CA, USA), Cytofectin TransRction t ntis; San Diego, CA, USA), BaculoPORTER ’l‘ransfiéction Reagent (Genlantis; San Diego, CA, USA), 'l'roganPOR’l‘ERl'M transfection Reagent {Genlantisg San Diego, C A, USA ), RiboFect {Biolineg 'l‘aunton, MA, USA), PlasFect ne; 'llaunton, MA, USA), UniFECTQR {EB—Bridge international; Mountain View, CA, 2t) USA), Sureliliitill'QR (B~Bridge International; Mountain View, :A, USA), or Ell}:CClTNE {B- Bridge International, Mountain View, CA, USA), among others.

Other agents can be utilized to enhance the penetration ofthe stered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2—pyrrol, azones, and terpenes such as lirnonene and rnentlione. v. Carriers Certain compositions ofthe present invention also incorporate r compounds in the tormulation. As used herein, er compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (116., does not possess biological activity per se) but is recognized as a nucleic acid by in vii/'0 processes that reduce the ilability of a nucleic acid having (A) c. biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a c acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction ofthe amount ofnucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier nd and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate dsRNA in hepatic tissue can be reduced when it is coadniinistered with polyinosinic acid, dextran sulfate, polycytidic acid or ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS 4~acetainido~4’isothiocyano“stilhene'2,Z'ndisulfonic acid (Miyao at all, DsRNA Res. Dev, l995, , l lS-lZl ; ldira at 05., DsRNA & Nucl. Acid Drug Dev, l996, 6, l77—l '93. vi. Excip lei/115; ln contrast to a. carrier compound, a “pharmaceutical carrier” or “excipient” is a (I: pharmaceutically acceptable solvent, suspending agent or any other pharniacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient can be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc, wh en combined with a nucleic acid and the other components of a given pharmaceutical composition, Typical ceutical carriers include, but are not limited ll) to, binding agents (cg pregelatinized maize starch, polyufinylpyn‘olidone or hydroxypropyl methylcellulose, 616.); tillers (cg, lactose and other sugars, inici'ociystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyaciylates or calcium l’iydrogen phospl’iate, etc); lubricants (cg, magnesium stearate, talc, , colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, hylene glycols, sodium benzoate, sodium acetate, etc); disintegrants (cg, starch, sodium starch glycolate, etc); and wetting agents (eg, sodium lauryl te, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable for non—parenteral administration which do not riously react with nucleic acids can also he used to formulate the compositions ofthe present invention. Suitable pharmaceutically acceptable carriers include, 2t) but are not limited to, water, salt ons, alcohols, polyethylene s, gelatin, lactose, se, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids can include sterile and non— sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions ofthe nucleic acids in liquid or solid oil bases. The solutions can also contain buffers, ts and other le additives. Pharmaceutically acceptable organic or inorganic ents suitable for renteral administration which do not riously react With nucleic acids can be used.

Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt (A) c. solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, ium stearate, talc, silicic acid, s paraffin, liydroxymethylcellulose, polyvinylpyrrolidone and the like. vii. Other Components The con'ipositions of the present, invention can additionally contain other adjunct ents conventionally found in pharmaceutical compositions, at their art—established usage levels Thus, for example, the compositions can contain additional, ible, pharniaceutically—active materials such as, for example, antipruritics, astrin gents, local [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS anesthetics or anti—inflammatory agents, or can contain onal materials l in physically formulating s dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacil‘iers, thicltening agents and izers.

However, such materials, when added, should not unduly ere with the biological activities (I: ofthe components ofthe compositions ofthe t invention The formulations can be sterilized and, if desired, mixed with auxiliary agents, eg, lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, b’ullers, colorings, llavorings and/or aromatic substances and the like wl’iich do not deleteriously interact with the nucleic ) of the formulation. ll) Aqueous suspensions can contain substances which increase the viscosity of the suspension including, for example, sodiurn carboxymethylcellulose, sorbitol and/or dextran. The suspension can also contain stabilizers, in some embodiments, pharmaceutical compositions featured in the invention include (a) one or more iRNA compounds and (b) one or more agents which on by a non-RNAi mechanism and which are useful in treating a hemolytic disorder. Examples of such agents include, but are not lniited to an anti~intlanimatory agent, anti~steatosis agent, anti—viral, and/or anti~fihrosis agent, ln addition, other substances commonly used to protect the liver, such as arin, can also be used in conjunction with the iRNLZlS described herein Other agents useful for treating liver diseases include telbivudine, entecav‘ir, and protease inhibitors such as telaprevir and other disclosed, for example, in Tung e! 0]., US Application ation Nos. 2005/0l48548, 2004/0l6’7l l6, and 2003/0l 442W; and in llale at all, US. ation ation No. 2001-‘l/tl l 27488. ’l‘oxicity and eutic efficacy of such compounds can be determined by rd pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the 1.3350 (the dose lethal to 50% of the population) and the EDSG (the dose therapeutically etlective in 50% ofthe population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LDSO/EDSO nds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can he used in formulating (A) c. a range of dosage for use in humans. The dosage of compositions featured herein in the invention lies lly within a range of circulating concentrations that include the £1350 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of stration utilized. For any compound used in the methods featured in the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range ot‘the compound or, when appropriate, of the polypeptide product of a target ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS sequence (egg, achieving a decreased concentration of the polypeptide) that includes the lCSG (116., the concentration ofthe est compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such inforn'iation can be used to more accurately determine usefiil doses in humans, Levels in plasma can be measured, for example, by high (I: performance liquid chromatography. in addition to their stration, as discussed above, the iRNAs featured in the invention can be administered in combination with other lcnown agents effective in treatment of pathological processes mediated by CS expression. In any event, the administering physician can adjust the amount and timing of iRNA administration on the basis ofresults observed using ll) standard measures of cy known in the art or described herein.

'Vl, Methods For inhibiting C5 Expression The t invention provides methods of inhibiting expression of C5 in a cell, The methods include contacting a cell with an RNAi agent, e.g., a double stranded RNAi agent, in an amount effective to inhibit expression of the CS in the cell, thereby inhibiting expression ofthe CS in the cell, Contacting of a cell with a double stranded RNAi agent may be done in vizr'o or in viva, Contacting a cell in vivo with the RNAi agent includes contacting a cell or group of cells within a subject, eg, a human subject, With the RNAi agent. {Tom‘oinations of in ‘vz'fm and in viva 2t) methods of contacting are also le. Contacting may be direct or indirect, as discussed above, Furthermore, contacting a cell may be accomplished via. a targeting , including any ligand described herein or known in the art, ln red embodiments, the targeting ligand is a carbohydrate moiety, eg, a {ialNAcg ligand, or any other ligand that directs the RNAi agent to a site ofinterest, eg, the liver ofa subject.

The term “inhibiting,” as used herein, is used interchangeably with “reducing; 5 “silencing,5? 44downregulating” and other similar terms, and includes any level of inhibition, The phrase “inhibiting expression of a CS” is. ed to refer to inhibition of expression of any (35 0'ene (such as, eg, a mouse (35 0em, a rat CS gene, a monkey CS gene, or a hun'ian C5 gene) as well as variants or mutants of a CS gene. Thus, the C5 gene may he a yvild~type CS (A) c. gene, a mutant C5 gene, or a transgenic C5 gene in the context of a genetically lated cell, group of cells, or organism iting expression ofa CS gene” includes any level of inhibition of a (35 gene, tag, at least partial ssion ofthe expression of a CS gene, The expression ofthe C5 gene maybe assessed based on the level, or the change in the level, of any variable associated with C5 gene expression, 6.3:, {.75 mRNA level, CS protein level, or for example, Cl’l50 ty as. a measure of total hemolytic ment, Alisa to measure the hemolytic activity of the alternate pathway of [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ment, and/or e dehydrogenase (LDH) levels as a measure of intravascular bemolysis, and/or hemoglobin . Levels of (Ba, (35b, and soluble C5b-9 complex may also be measured to assess (:5 expression. This level may be assessed in an dual cell or in a group ot‘cells, including, for example, a sample derived from a subject.

(I: lnhibition may be assessed by a decrease in an absolute or relative level of one or more les that are associated with {‘5 expression compared with a control level, The control level may be any type of control level that is utilized in the art, eg, a presdose baseline level, or a level determined item a similar subj ect, cell, or sample that is untreated or treated with a control (such as, eg , buffer only control or inactive agent control). ll) in some embodiments ofthe methods of the invention, expression of a CS gene is inhibited by at least about 5%, at least about lllll/éi, at least about l5%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about (lilo/ti, at least about 65%, at least about 7 %, at least about 75%, at least about 809/5, at least about 85%, at least about 96%, at least l 5 about 91%, at least about , at least about 93%, at least about 94%. at least about 95%, at least about 9 5%, at least about 97%, at least about 98%, or at least about 99%. inhibition ofthe sion of a C5 gene may be manifested by a reduction ofthe amount ofmRNA expressed by a first cell or group of cells (such cells may be present, for example, in a sample derived from a subject) in which a (:5 gene is transcribed and which has or have been 2t) treated (ag. the cell or cells with an RNAi agent of the invention, or by , by contacting administering an RNAi agent of the ion to a t in which the cells are or were t) such that the expression of a C5 gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but Which has not or have not been so treated (control cell(s)). In preferred embodiments, the inhibition is assessed by expressing the level ofmRNA in treated cells as a percentage of the level of n'iRNA in control cells, using the following formula: (mRNA in control cells) — (mRNA in treated cells) a l 09% {mRNA in control cells) Alternatively, inhibition ofthe sion of a C5 gene may be assessed in terms of a reduction of a parameter that is functionally linked to C5 gene expression, 9.53., CS protein 3 ll expression, liepcidin gene or protein expression, or iron levels in tissues or serum. (.75 gene silencing may be determined in any cell expressing C5, either constitutively or by c engineering, and by any assay known in the art, The liver is the major site of C5 expression.

(Ether significant sites of expression include the s and the uterus. inhibition ofthe expression ofa CS protein may be manifested by a reduction in the level w‘) U:, ot‘the C5 protein that is expressed by a cell or group ot‘cells (egg the level ofprotein expressed ation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS in a sample derived from a subject), As explained above for the assessment omeNA suppression, the inhibiton of protein expression levels in a treated cell or group of cells may similarly be expressed as a percentage ot‘the level ot‘protein in a control cell or group ot‘eells.

A control cell or group oi’cells that maybe. used to assess the inhibition of the sion (I: of a C5 gene includes a cell or group of cells that has not yet been contacted with an RN’Ai agent ofthe invention. For example, the control cell or group of cells may be derived from an individual sub} ect tag. a human or animal subject) prior to treatment of the sub} ect with an RNAi agent.

The level of C5 mRNA that is expressed by a cell or group of cells may be determined ll) using any method known in the art for assessing mRNA expression. in one embodiment, the level of expression ot‘CS in a sample is determined by detecting a transcribed cleotide, or portion f, tag, rnRNA of the C5 gene. RNA may be extracted from cells using RNA extraction techniques including, for example, using acid /guanidine isothiocyanate extraction (RNAzol B; Biogenesis), RNeasy RNA ation lrits (Qiagen) or l’AXgene {PreAnalytim Switzerland). Typical assay formats utilizing ribonucleic acid hybridization include nuclear run~on assays, RT~PCR, RNase protection assays (Melton or al, New. ic:z’s Res. l227035), Northern blotting, in site; hybridization, and microarray analysis. in one embodiment, the level of expression of C5 is determined using a nucleic acid probe. The term "probe", as used herein, refers to any molecule that is capable of selectively binding to a speeilie CS. Probes can be synthesized by one ol‘sltill in the art, or derived from appropriate biological preparations. Probes may be specifically ed to be labeled.

Examples ofmolecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules. isolated mRNA can be used in hybridization or amplili cation assays that e, but are not limited to, rn or rn es, polymerase chain reaction (PCR‘; analyses and probe arrays One method for the determination ofmRNA levels involves ting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to CS mRNA. in one embodiment, the mRNA is immobilized on a solid surtace and contacted with a probe, lbr example by running the isolated rnRNA on an agarose gel and transterring the rnRNA from the (A) (:3 gel to a membrane, such as nitrocellulose, in an alternative embodiment, the probr3(s) are lized on a solid surface and the mRNA is contacted with the probe(s), for example, in an At‘lymetrix gene chip array. A skilled artisan can y adapt known mRNA detection methods for use. in determining the level of C5 n'iRNA.

An alternative method for detemiining the level of expression of CS in a sample involves the process ofnucleic acid amplification and/or reverse transcriptase ifto prepare cDNA) of for example mRNA in the , eg, by RTsl’CR (the experimental embodiment set forth in [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS lv’lullis, l987’, US. Pat, No. 4,683,202), ligase chain reaction {Barany (l 99l) Proc. Natl. Acad.

Sci. USA SSIl 89493 self sustained sequence replication (Guatelli et al. , if l 990‘) Proc. Natl. zlcad. Sci. Lilli-4 87d 8744878), riptional amplification system (KR/Oil et all. @989) Proc.

Natl. Acad. Sci. 1.73.4 Stizl l73—l l'77), Q~Beta Replicase (Lizardi er al. (l .988) Bio/Technology (I: 61ll97), g circle replication (Lizardi et al., U18, Pat. No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those ot‘sltill in the art. These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very low numbers, lri particular aspects ofthe ion, the level of expression of C5 is determined by quantitative ll) tluorogenic RT-PCR (118., the ’l‘aql‘vlan'm System).

The expression levels ot‘CS rnRNA maybe monitored using a membrane blot {such as used in hybridization analysis such as Northern, Southern, dot, and the like), or microwells, sample tubes, gels, beads or fibers (or any solid support comprising hound nucleic acids). See US. Pat. Nos. 5,77 ,722, 5,874,2l9, 5,744,305, l95 and 5,445,934, which are l5 incorporated herein by reference. The determination ofCS expression level may also comprise using nucleic acid probes in solution, ln preferred embodiments, the level ofmRNA expression is assessed using branched DNA {bDNA} assays or real time PCR (qPCR). The use e methods is described and exemplified in the Examples presented herein.

The level ot‘CS n expression may be determined using any method known in the art for the measurement ofprotein levels. Such methods e, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer tography (TLC), hyperdiffusion chromatography, fluid or gel precipitin reactions, absorption spectroscopy, a colorimetric assays, spectrophotometric assays, tlow cytometry, mniunoassay (RlA), enzyme~linlred imniunosorbent assays (ELl-SAS), olluorescent assays, electrochemiluminescence assays, and the like.

The term “sample” as used herein refers to a collection ofsirnilar , cells, or tissues isolated item a subject, as well as fluids, cells, or tissues present within a, subject, Examples of (A) (:3 biological fluids include blood, serum and serosal fluids, plasma, lymph, urine, cerebrospinal fluid, saliva, ocular tluids, and the like, Tissue samples may include samples from tissues, organs or localized regions. l2or example, samples maybe derived from particular organs, parts of organ s, or fluids or cells within those organs. in certain embodiments, samples maybe derived from the liver (rag whole liver or certain ts of liver or certain types of cells in the liver, such as, 9.53., hepatocytes). in red embodiments, a “sample derived from a [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS subj ect” refers to blood or plasma drawn from the t. in further ments, a “sample derived from a subject” refers to liver tissue derived from the subject. in some ments ot‘the methods of the invention, the RNAi agent is administered to a subject such that the RNAi agent is delivered to a specific site within the subject. The (I: inhibition of expression of C5 may be assessed using measurements ofthe level or change in the level of C5 mliNA or (:5 protein in a sample derived from fluid or tissue from the ic site Within the subject. in preferred ments, the site is stlie liver. The site may also be a tion or subgroup of cells from any one ot‘the aforementioned sites. The site may also e cells that express a particular type of receptor. l0 The phrase “contacting a cell with an RNAi agent,” such as a A, as used herein, includes contacting a cell by any possible means. Contacting a cell with an RNAi agent includes contacting a cell in ultra with the iRNA or contacting a cell in vivo with the iRNA. The contacting may be done directly or indirectly. Thus, for example, the RNAi agent may be put into physical contact with the cell by the individual performing the method, or alternatively, the RNAi agent may be put into a situation that will permit or cause it to subsequently come into contact with the cell.

Contacting a cell in vino may be done, for example, by incubating the cell with the RNAi agent. Contacting a cell in viva maybe done, for example, by injecting the RNAi agent into or near the tissue Where the cell is located, or by ing ecting the RNAi agent into another area, cg, the bloodstream or the subcutaneous space, such that the agent will subsequently reach the tissue Where the cell to be contacted is located. For example, the RNAi agent may n and/or be coupled to a ligand, cg, GalNAcEl, that directs the RNAi agent to a site of interest, eg, the liver.

Combinations ot‘in‘ vitro and in viva methods ot‘contacting are also possible. For example, a cell may also be contacted in vitro with an RNAi agent and uently transplanted into a subj ect. in one embodiment, contacting a cell with an iRNA includes “introducing” or “delivering the iliNA into the cell” by facilitating or effecting uptake or absorption into the cell. Absorption or uptake of an iRNA can occur through unaided dit‘t‘usive or active cellular processes, or by auxiliary agents or devices. lntroducing an iRNA, into a cell may be in vitro and/or in viva. For e, for in viva introduction, iRNA can be injected into a tissue site or administered 3% systemically. In vivo delivery can also be done by a beta—glucan delivery , such as those described in US Patent Nos. 5,032,40l and 5,607,677, and US Publication No. letlS/OZSITI'Sl the entire contents of which are hereby incorporated herein by reterence, In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein below and/or are known in the art.

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS Vli. Methods for 'i‘reating or Preventing a Complement Component CSmAssociated disorder The present invention also provides therapeutic and prophylactic methods which e administering to a t having a complement component C5~associated disease, cg, PNH or (I: aHUS, an iRNA agent, pharmaceutical compositions comprising an iRNA agent, or vector comprising an iRNA ofthe invention, in some aspects ofthe invention, the methods further include administering to the t an additional therapeutic agent, such as an anti~complement component C5 antihody, or antigen~binding fragment thereof (cg, eculizumah), in one aspect, the t invention provides methods of ng a suhject having a ll} disorder that would henefit from reduction in C5 sion, cg, a complement component C5- ated disease, 3g, PNH or aliUS The treatment methods (and uses) ofthe invention include administering to the subject, (g, a human, a tl’ierapeutically effective amount of an iRNA agent targeting a C5 gene or a. pharmaceutical composition comprising an iRNA agent targeting a CS gene, thereby treating the t having a disorder that would benefit from reduction in CS expression. in r aspect, the present invention es methods ot‘treating a suhj ect having a disorder that would henefit from reduction in C5 expression, cg a complement component C5“ associated disease, cg, PNH or aHUS, which include administering to the subject, cg, a human, a therapeutically effective amount of an iRNA agent ing a CS gene or a ceutical composition comprising an iRNA agent targeting a CS Gene, and an additional therapeutic agent, such as an anti—complement component CS antibody, or antigen—binding nt thereof (cg, eculizumah), thereby treating the subject having a disorder that would benefit from reduction in CS expression. in one aspect, the invention provides methods ofpreventing at least one m in a subject having a disorder that would benefit from reduction in C5 expression, cg, a con'iplenient component CSnassociatcd disease, c.g FNH or aHUS. The methods include administering to the subject a prohpylactically et‘tective amount of the iRNA agent, cg, dsRNA, or vector ot‘the invention, thereby preventing at least one symptom in the subject having a er that would benefit from reduction in CS expression, For example, the invention provides metl'iods for (A) (:3 preventing hcmolysis in a subject suffering from a er that would benefit from reduction in CS expression, cg, a complement component {TS-associated e, cg PNH or ailUS. in another aspect, the invention provides methods of preventing at least one symptom in a subject having a disorder that would benefit from reduction in C5 sion, cg, a con'iplenient component CSnassociatcd disease, c.g FNH or aHUS. The methods include administering to the subject a prohpylactically et‘tective amount of the iRNA agent, cg, dsRNA, or vector ot‘the invention, and an additional therapeutic agent, such as an anti—complement component CS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS antibody, or antigennhinding fragment thereof (cg, eculizumab), y preventing at least one symptom in the subject having a disorder that would benefit from reduction in CS expression.

"Therapeutically ve amount,” as used herein, is intended to include the amount of an RNAi agent or omplement component C5 antibody, or antigen—binding fragment thereof (I: tag. eculizumab), that, when administered to a subject having a complement component C5~ associated disease, is sufficient to effect treatment of the disease (eg, by shing, rating or maintaining the existing disease or one or more symptoms of disease). The ”therapeutically ive ai‘nount“ may vary depending on the RNAi agent or antibody, or antigen—binding fragment thereof, how the agent is administered, the disease and its severity and ltl the history, age, weight, family history, genetic makeup, the types ofpreceding or concomitant ents, if any, and other individual characteristics of the subject to be treated.

“Prophylactically eftbctive amount,” as used , is intended to include the amount of an iRNA agent or anti~complement component C5 antibody, or antigennhinding fragment f (eg. eculizumab), that, when stered to a subject having a complement component C: — associate disease but not yet (or currently) experiencing or displaying symptoms ofthe disease, and/or a subject at rislt of developing a complement component CS~associated disease, 6.3:, a subject having a graft and/or transplant, Cg, a sensitized or allogenic recipient, a subject haying , and/or a subject having a dial infarction, is sufficient to prevent or ameliorate the disease or one or more symptoms ofthe disease, Ameliorating the disease includes slowing the course ofthe disease or reducing the se Ierity oflater-de Ieloping disease. The ”prophylactically etl'ective amount" may vary depending on the iRNA agent or anti—complement component C5 antibody, or antigenwbinding fragment thereof, how the agent or antincomplement component C5 antibody, or antigen-binding fragment thereof, is administered, the degree of risk of disease, and the history, age, weight, family history, genetic makeup, the types ofpreceding or concomitant treatments, if any, and other individual characteristics of the patient to be treated.

A ”therapeutically effective amount" or “prophylactically effective amount” also includes an amount of an RNAi agent or anti-complement component CS antibody, or antigen—binding fragment tteg, eculizumab), that produces some desired lo ‘al or ic effect at a reason able benefit/risk ratio applicable to any treatment. iRNA agents employed in the methods (A) (:3 ofthe present invention may be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to such ent. in another , the present invention provides uses ofa therapeutically effective amount of an iRNA agent ofthe invention for treating a subiec‘, cg, a t that would benetit from a reduction and/or inhibition of C5 expression. ln another , the present invention provides uses of a therapeutically ef ective amount of an iRNA agent of the ion and an additional therapeutic agent, such as an anti“ [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS complement ent C5 antibody, or antigen—binding fragment thereof (rag ecuiizumab), for treating a subject, eg, a subject that would benefit from a reduction and/or inhibition of CS expression. in yet r aspect, the present invention es use of an iRNA agent, tag, a (I: dsRNA, ofthe invention targeting a C5 gene or a. ceutical composition comprising an iRNA agent targeting a CS gene in the manufacture of a medicament for treating a subject, eg, a subject that wouid benefit from a reduction and/or inhibition ofCS expression, such as a subject having a disorder that wouid benefit from reduction in C5 expression, eg, a con’ipiement component CSnassociated disease, eg. PNH or aHUS. it} in r aspect, the present invention provides uses of an iRNA agent, erg, a dsRNA, ofthe invention targeting a CS gene or a aceuticai composition comprising an iRNA agent targeting a CS gene in the inanutacture of a medicament for use in combination with an additional therapeutic agent, such as an antincompieinent component C5 dy, or nw binding fragment thereof (eg, ecuiizumab‘), for treating a subject, sag, a subject that would benefit from a reduction and/or tion ot’CS expression, cg, a cornpiement component (35— associated disease, cg. PNH or aHUS. in another aspect, the invention provides uses of an iRNA, cg, a dsRNA, of the invention for preventing at ieast one symptom in a subject sufqering from a er that wouid benefit from a reduction and/or inhibition of CS expression, such as a compieinent component Cfiassociated disease, cg PNH or aHUS. in yet another aspect, the ion provides uses of an iRNA agent, 6g, a dsRNA, of the invention, and an additional therapeutic agent, such as an antiwcompiement component C5 antibody, or n-binding nt thereof {cg ecuiizumab), for preventing at ieast one symptom in a subject stuttering from a disorder that wouid benefit from a reduction and/or inhibition ot‘CS expression, such as a complement ent C5~associated disease, erg. PNH or aHUS. in a further aspect, the present invention provides uses of an iRNA agent ofthe invention in the rnanutacture of a medicament for preventing at ieast one symptom in a subject stittei'ing from a disorder that would benefit from a reduction and/or inhibition of CS expression, such as a (A) (:3 a cornpiernent ent CS—associated disease, raga, PNH or aHUS, in a further aspect, the present invention provides uses of an iRNA agent of the invention in the manutacture ot‘ a medicament for use in combination with an additional therapeutic agent, such as an anti—cornpieinent component C5 antibody, or antigen~binding fragment thereotteg, ecuiizumaib), for ting at least one symptom in a subject suffering from a disorder that would benefit from a reduction and/or inhibition of CS expression, such as a a compieinent component {IS—associated disease, eg, PNH or aii US.

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS in one embodiment, an 1RNA agent targeting C5 is administered to a suhieet having a complement component C5-assoeiated disease such that C5 ieveis, 9.9., in a ce11, tissue, hiood, urine or other tissue or Einid oEthe snhieet are reducedoy at 1east about 10%, 11%, 12%, 1.31341, 14%, 15‘36, 16%, 1%7,18%, 19%, 20%,21%, 22%2.3%, 24%, 2.5%, 26%27%28%.9%, ‘J‘: 30%, 31%, 32%, 33°/E., 3441, 35%, 36%, 37%,38%, 39%, 40%, 41%, 42%43 644%,, :45%, 46%,47/0, 48%, 49‘1/1, 50%, 51%, 52% 5%3,54%, 55%, 56%, 57%, 58%, 59% , 60%, 61%, 78%,36, 62%, 64°41, ,6661341,66 %, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%0, 76%, 77%,79%, 80%, 81%, 82%, 83%, 14%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,,.72%,93%, 94%, 95%,96%,97%, 98/0, or at 1east about 99°/'0 or more ann,d suhseqnentiy, an additiona1 therapeutic Eas described heioW) is administered to the t The additionai therapeutic may he an anti~cornp1einent component C5 dy, or antigen—binding fragment or derivative thereof. 1n one embodiment, the anti—coinpieinent component C5 antibody is ecnhzuinalh (80121131817), or antigenwbinding fragment or derivative thereof. Ecuiiznmah is a humanized 1nonoc1ona11gG2/4, kappa Eight chain antibody that specificaiiy binds oomp1ement component CS with high affinity and ts eieavage ofC5 to {15a and C511, thereby inhibiting the generation 61' the termin a1 eompiement complex (.569.

Eeuhzumahis describedin 13.8. Patent No. 6,355,245, the entire contents otwhieh are orated herein by reference.

The methods ofthe ion comprising administration of an iRNA agent oE‘the invention and ec111iznniah to a t may further se administration ofa meningocoecai vaccine to the snhj eet, 1he additionai eutic,'g, een1iznmab and/or a meningococeai vaccine, may be administered to the t at the same time as the 1132th agent targeting C5 or at a different time.

Meieover, the addition a1 therapettticm. be administered to the , rnah, may snbj eet in the same formu1ation as the iRNA agent targeting C5 or in a different forinniation as the iRNA agent targeting C5. 131311112511111ah dosage ns are described in, for examp1e, the product insert 11:11” ec n11211n1ah (8013111187311211111 in 1.1.8. Patent App1ication No. 2012/0225056, the entire contents of (A) (:1 each ofwhich are incorporated herein by reference. in ary methods ofEhe invention for ng a ment component C5—associated disease, eg. 19111 or aitiUS, an iRNA agent targeting 1:35 is administered (6.9., subcutaneous1y) to the subject first, such that the {35 1eve1s in the subject are reduced Ee.g. by at ieast about 20%, 21‘” 22%, 233,6 24%, 25%, 26%, 27%, 2870, 29°.41, 30%, 31%, 32‘34,33%,34°4.1, 35%, 36%, 37% 8%,39%, 40%, 41%, 42%, 4%, 44°,45‘3/0, 46%, 47%, 48%, 49‘4 5‘04,51‘341, 52%, 53° 6,54%, 55°41, 56%, 57%, 58%, 59%, 60°41, 61%, 62%, 62%, 64%, 65 ‘14, 66%, 67% 68%,‘3669,£170°/,71 ‘141, 72%, 7.334,) 74%0, 75%0, [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS 76%, 77%, 78%, 79%, 80/0 8l“/E;, 82“7'0, 83%, 84/, 85/o , 7%, 88%, 89%, 90%, 9l%, 92“:/t:-, 39/0, 94%, 95/-,o 96% 97%, 98%, or at least about 99% or more) and subsequently eculizumab is administered at doses lower than the ones described in the t insert for SOURISQ For example, eculizumab may be atlrninsitered to the subject weekly at a dose less (I: than about 600 mg for 4 weeks followed by a fifth dose at about one week later of less than about 900 mg followedby a dosse lesss than about 900 mg about every two weelgs thereafter liculizumab may also be administered to the subj ect weekly at a dose less than about 900 mg tbr 4 weeks ed by a fifth dose at about one week later of less than about l200 mg, followed by a dose less than about l200 mg about every two weeks thereafter, if the subject is less than l8 years of age, eculizumab maybe administered to the t weekly at a dose less than about 900 mg for 4 weeks followed by a ti fth dose at about one week later ot‘less than about l200 mg, etl by a dose less than about l"200 mg aboutevery two weelgs there-alter; or it the subiectis less than l8 years of age, 3culizumab may be administered to the subject weekly at a dose less than about (300 mg for 2 weelgs followed by a third dose at about one week later of less than about 900 mg, lbllowed by a dose less than about 900 mg about every two weelgs thereafter; or if the subject is less than l 8 years of age, eculisumab may be stered to the subject weelgly at a dose less than about 600 mg for 2 weeks followed by a third dose at about one week later of less than about 600 mg, follo‘v 'ed by a dose less than about 600 mg about every two weeks thereafter; or if the subject is less than l8 years of age, eculizurnab may be administered to the subj eet weelgly at a dose less than about 600 rng for l week followed by a second dose at about one weelg later of less than about 300 m g, tbllowed by a dose less than about 300 mg about every two weeks there; fter; or if the subject is less than l8 years of age, eculizuniab may be stered to the subject weekly at a dose less than about 300 mg for l week followed by a second dose at about one weelg later of less than about 300 mg, followed by a dose less than about 300 mg about every two weeks threatter lf the t is receiving plamapheresis or plasma exchange, umab may be administered to the subject at a dose less than about 300 mg (eg, ifthe most recent does of eculizumab was about 300 mg) or less than about 600 mg (gig, ifthe most recent does ofeculizumab was about 600 mg or more). if the subject is reeeivin g plasma inttision, un’tab may be administered to the subnet t at a tloseless than (A) (:3 about 300 mg tag. ifthe most recent does o eculizumab was about 300 mg or more), The lower dosestafeculizumab allow for either subcutaneous or intravenous administration of eculizumab. ln the combination therapies ofthe present invention eon'tprising eculizumab, eculizumab may be adminisitered to the subject, cg, subcutaneously, at a dose of about 0.0l mg/lgg to about l0 mg/kg, or aboutf»mg/lgg to about l0 rug/kg or about 0.5 mg/lgg to about l5 mgkg. For example, eculizumab may be stered to the subJect, sag, subcutaneously, at a dose of 0.5 [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS ation] DPS ionNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS mg/kg, 1 nig,’1<;g, 1.5 mg/kg,_,._ mg/kg,..5mg/1<g, 3 mg/kg, 3.5 mg/irg, 4 mg/kg, 4.5 mg/h‘g, 5 mg/kg, 5.5 111g;’1<g,6 ms,I’kg, 65 mg/1<g, 7 mg/kg, 7.5 mg/kg, 8 mg/kg, 8.5 mg/kg, 9 mg/kg, 9.5 mg/kg, Kiting/1g, 10.5 mg/kg, 1 1 rng/icg, 11.5 mg/kg, 12 rng/kg, 12.5 mg/kg, 13 mg/kg, 13.5 trig/kg, 14 ing/kg. 14.5 trig/kg, or15 rng/kg.

(I: The methods and uses ofthe ion inc111de administering a composition descriLed herein such that expression ofthe target C5 gene is decreased. such as for about 1, 2’,4. 5, 6, 8, 12, 16, 18, 24, 28, 32, 36, 4t), 44, 48, 52, 56, 60, 64, 68, '72, '76, or about 81) hours. 1n one embodiment, sion ofthe target C5 gen is decreased for an extended i1iiration.r.0. at teast0: about two, three, four, five, six, seven days or more, cg. about one week, two weeks, three 111 weeks, or about four 1. ’eeks or ionger.

Administration ot‘the dsRNA according to the methods and uses ofthe invention may result in a ion oft11e severity, signs, symptoms, and/or1:nar1<ers ofsiich diseases or disorders in a patient with a comp1ement component C5nassoeiated disease. By tion” in this context is meant a statisticai1y significant decrease in such 1eve1. The reduction can be, for e, at 1east about 50/o, 1110/ 15 *1/61,02‘3/13, 250/11,-311%, “/6, 1, 50‘3-4>,:>:>" 6.60%, 650m, 70%, 75%, 80%, 85%, 9 ‘36, 95%, or about it) 0/ Efficacy oftreatment or prevention of disease can be assessed, for exanip1e by measuring disease ssion, disease remission, symptom severity, ion in pain, qnaiity of iife, dose of a medication required to sustain a treatment effect, 1eve1 of a disease marker or any other 21) rabie parameter appropriate for a given disease being treated or targeted for prevention. 1t is vve11 wit1'iin the ahihty of one shined in the art to n'ionitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination ofparameters. For exampie, efficacy oftreatment of a 11emo1ytic disorder may be assessed, for exampie, by periodic ring (31111 and {11150 1eve1s. Comparisons of the 1ater readings with the initia1 readings provide a physician an indieation 01‘w other the treatment is effectiye itis wee11 within the y of one shined in the art to monitor efficacy of treatment or prevention by 1 easnring any one of such parameters, or any combination of ters. in connection with the administration of an iRNA targeting CS or pharrnaceutica1 composition thereofettective against"a'1 comp1ernent ent (TS—associated disease indicates that administration in a c1inica11y (A) (:1 appropriate manner results in a heneficia1 effect for at 1east a. statistica11y significant fraction of patients, such as improvement of symptoms, a cure, a reduction in e, extension ofiife, improvement in qua1ityof1ife, or other effect genera11y recognized as positive by medical doctors fan’ii1iar with treating a cornp1ement component C5~associated disease and the re1ated causes.

A treatment or preventive effect is evident when there is a statistic a11y significant improvement in one or more parameters of disease , or by a fai1’nre to worsen or to devehap [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS symptoms Where they would Otherwise be anticipated As an example, a. favorable change 0f at least 111min a 111easu1a‘ole para1nete1 of disease and preferably at least 20%, 30‘30, 40‘3/o, 511/1) 01 more can be 1ndieative oi etleetiVe treatment. 1.1111eaey lot a given iRNA drug 01 111111111lation of that drug can also be judged 11sin9 an experimental animal model for the given disease as knmwn (I: in the art. When using an experimental animal model, effieaey 0f treatment is evidenced when a statistically significant reduetinn in a marker 01' symptom is ee. tively, the efficacy can be ed by a reduction in the severity of e as detemiined by one skilled in the art 61 1111191111515; based on a elinieally aeeepted disease severity 91adin9 scale, as but one example the Rheumatoid Arthritis ‘itV' Scale (RASS). AnV' pesitive 11.1 change resultin9 in e.g , ies e11in9 1afseve11tv of disease nieasnied usingthe appropriate s1: ale, ents adequate treatment 1.1sin9 an 1RNA ot1RNA 111atien as described herein.

Subjeets can be adn'iimstered a therapeutie amount of iRNA, such as ahnut {3.01 1119719, 0.02 nag/kg, 0.03 1n9/k9, 0.04 m9/lg‘9, 0.05 1119/119, 0.1 1'9, 0.l5 1119/89, 0.2, 1119419, 0.25 1n9/l<9, (3.3 :9, 11.35 n19/l<9,‘, 0.4 I"l<9, 0.45 1n9I'l<:9, 11.5 n19/l<9,‘, (3.55 n19,‘I'l<9,‘, 0.6 1119/k9, (3.65 1119/1191, 13.7 19 11 '75 1119419, {3 8 1119/k9, 0.85 1110I119 11.9 1119419, 0.9.5 1119/k9, 1.0 11119419, 1.1 1119/89, l.21’119Il<9, l 3 1119/319, l .41n9IIl<9, 1.5 Dig/k9, 1 61119119, l.7 1119/19, 1.8 nag/kg, l.9 tug/119,27 .0 1119/.k9 2 .111119Il19,7 .21n9I’l<.:9, 23 1n9/Ik9, 24 1119419, 2.5 m9I’l<.:<.; dsRNA, 2 6 rag/k9 1lsRNIA, 2.71119119 dsRN.A,2.8 ni<.;Il<:9 dsRNA, 29 m9/ls‘9 dsRNA, 3.1) ni<.;Il<:9 dsRNA, 3 .l 1119’l§9 dsRNIA,32 1119119 dsRNA, 3.3 9,‘ dsRNA34 1n9Il<:9 dsRNA 3.5 9,‘ dsRNA3 .6 m9/l~:9 21) dsRNA,3 7.1119/1-19 dsRNA, 3 .8 1119I1<9 dsRNA39 1119/1119 dsRNA, 4.0 <9 dsRNA, 4.1 1119/1119 dsRNA, 4.2 1119419 dsRNA, 4.3 169/119 dsRNA, 441:119/l<9 dsRNA, 4.5 169/119 dsRNA, 4.6 1119Il<9 dsRNA, 4.7 ni<.;I’l<:9 dsRNA 4 8 m9/k9 dsRNA, 4.9 ni<.;Il<9 dsRNA 50 m9/k9 dsRNA,53. l nigIkg dsRNA, 5.7 n19,‘I’l<9, dsRNIA, 5.3 11/19le9 dsRNA, 5.4 n19,‘Il<9, dsRNA 5.5 1119/89 dsRNA, 56 1119,,I’k9 ds RNA, 57 reg/k9,; dsRNA, 5.8 11194-19 dsRNA, 5.9 1119Il<9 dsRNA, 6.11 11194-19 dsRNA,66.l 1119,,I’k9 dsRNA, 6.2 169/119 dsRNA, 6.3 1:119/119 dsRNA, 6.4 nag/"R9 dsRNA, 6.5 1:119/119 dsRN/A, 6.6 mg/kg tlsRNIA, 6.7 m9Ik9 dsRNA, 6.8 nig/kg dsRNA, 6.9 9 dsRNA, 7.1) 11119419 dsRNA, 7.l m9I’l§9 dsRNIA, 72 1119119 dsRNA, 7.3 l<9,‘ dsRNA, 7.4 1n9I'l<:9 dsRNA, 7.5 n19,‘/l<9,‘ dsRNA, 7.6 9 dsRNA, 7.7 1119/89 dsRNA, 7.8 tug/kg, dsRNA, 7.9 1119/119 ds RNA, 8.0 tug/kg, (lsRNA, 8.1 1119/1119 dsRNA, 8.2 1119419 dsRNA, 83 169/119 dsRNA, 8.4 1:119/l<9 dsRNA, 8.5 169/119 dsRNA, 8.6 1119/89 dsRNA, 8.7 11103:0 dsRNIA, 8 8 m9/k9 dsRNA, 8.9 ni<.;/l<9 1lsRNIA,90 m9/k9 dsRNA, 9.1 mg/kg dsRNA, 9.7 n19/k9, dsRNA 93 11/19le9 dsRNA, 9.4 n19Il<9, dsRNA 95 11/19le9 dsRNA, :96 1119,)’k9 ds RNA, 9.7 1119/k9, dsRNA, 9.8 11194-19 dsRNA, 9.9 1119Il<9 dsRNA, 9.11 11194-19 dsRNA, 11) 1119/1119 dsRNA, l 5 1119/89 dsRNA, 20 1119/89 dsRN/A, 25 1:119/l<9 dsRN/A, 3O 1:n9II'l<,9 dsRN/A, 35 1:n9II'l<,9 1lsRNA, 4O n19/l19 1lsRNA, 4 5 n19/l19 dsRNA, 61 5111611150 1n9/k9 dsRNA. Values and 1an9es intermediate to the recited values are also intended to be pait oi‘tliis invention.

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS in certain embodiments, for exampie, when a composition ofthe invention comprises a dsRNA as described herein and a 1ipid, subjects can be administered a eutic amount of iRNA, such as about 11.01 mg/itg to about 5 tug/kg, about 0.01 rug/kg to about 111 rug/kg, about 0.05 trig/kg to about 5 rug/kg, about 11.05 trig/kg to about 10 trig/kg, about 0.1 org/kg to about 5 (I: tug/kg, about 0.1 mg/kg to about 10 ring/kg, about 0.2 mg/kg to about 5 mg/kg, about 0.2 tug/kg to about 111 rug/kg, about (1.3 mg/kg to about 5 mg/kg, about (1.3 rug/kg to about 10 rug/kg, about (3.4 rug/kg to about 5 rug/kg, about 0.4 mg/ieg to about 11) rug/kg, about 11.5 tug/kg to about 5 , abmtt 0.5 tug/kg to about 111 tug/kg, about 1, tug/kg to about 5 tug/kg, about 1 tug/kg to about 10 mg/kg, about 1.5 rug/kg to about 5 mg/irg, about 1.5 nig/kg to about 111 nig/kg, about 111 2 rug/kg to about about 2.5 mg/kg, about 2 mg/kg to about 10 mg/irg, about 3 mg/kg to about 5 tug/kg, about 3 tug/kg to about it} mg/itg, about 3 .5 mg/kg to about 5 mg/kg, about 4 rug/kg to about 5 mg/kg, about 4.5 tug/kg to about 5 tug/kg, about 4 rug/kg to about it} tug/kg, about 4.5 tug/kg to about 111 tug/kg, about 5 mg/kg to about 10 mtg/kg, about 5.5 mg/iig to about 10 mg/irg, about 6 mg/kg to about 10 mg/irg, about 6.5 rug/kg to about 10 rug/kg, about 7 rug/kg to about 10 rug/kg, about 7.5 rug/kg to about 111 trig/kg, about 8 mg/ieg to about 11) tug/kg, about 8.5 tug/kg to about 10 org/kg, about 9 nag/kg to about 10 org/kg, or about 9.5 g to about 10 mg/kg.

Vaiues and ranges intermediate to tbe recited vaiues are aiso intended to be part of this invention.

For exampie, the dsRNA may be administered at a dose of about 9.1, 0.2, 0.3, 9.4, 0.5, 7, 0.8.11.9, 1,1.1,1.2, 1.3,1.4,1.5, 1.6,1.7,1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 211 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, .2. 5.3, 5.4, 5.5. 5.6, 5.7, 5.8, 5.9, 6, 6.1,, 6.2, 6.3, 6.4. 6.5, (5.6, 6.7. 6.8, 6.9, 7, 7.1. 7.2, 7.3, 7.4. 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 . Values and ranges ediate to tbe recited values are a1so intended to be part s invention. 1n other embodiments, for exai’np1e, when a composition oftbe invention eon’iprises a dsRNA as described herein and an Nnacetyigaiactosaniine, subjeets can be stered a therapeutic amount of iRNA, such as a dose ofabout 0.1 to about 50 mg/irg, about 11.25 to about 50 rug/kg, about 0.5 to about 511 rug/kg, about 11.75 to about 50 rug/kg, about 1 to about 50 trig/rug, about 1.5 to about 511 itig/kb, about 2 to about 511 tug/kg, about 2.5 to about 50 trig/kg, about 3 to about 511 nig/kg, about 3.5 to about 50 rug/kg, about 4 to about 50 rug/kg, about 4.5 to about 51) mg/kg, about 5 to about 50 mtg/kg, about 7.5 to about 50 rug/kg, about 10 to about 50 rug/kg, about 15 to about 51) mg/kg, about '20 to about 51) mg/kg, about '20 to about 50 rug/kg, about 25 to about 50 rug/’kg, about 25 to about 50 g about 30 to about 50 tug/kg, about 35 to about 50 nig/kg, about 40 to about 50 nig/kg, about 45 to about 50 mtg/kg, about 0.1 to about 45 rug/kg, about 0.25 to about 45 ing/kg, about (1.5 to about 45 , about (3.75 to about 45 tug/kg, about 1 to about 45 rug/rug, about 1.5 to about 45 rug/1d), about 2 to about 45 rug/kg, [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS about 2.5 to about 45 rug/11g, about 3 to about 45 mg’kg, about 3.5 to about 45 tug/kg, about 4 to about 45 tug/kg, about 4.5 to about 45 mtg/12g, about 5 to about 45 , about 7.5 to about 45 tug/kg, about 11) to about 45 tug/kg, about 15 to about 45 1’ug/1tg, about 211 to about 45 tug/kg, about 20 to about 45 n’igxkg, about 25 to about 45 11115415 about 25 to about 45 tug/kg, about 30 (I: to about 45 mgkg, about3 5to about 45 mgkg, about 40 to about45 mg_/kg, about 0.1 to about 40 ntg/kg, about 11.25 to about 40 meg/kg, about 0.5 to about 41.1 tug/kg, about (1.75 to about 40 tug/kg, about 1 to about 40 tug/mg, about 1.5 to about 40 rug/kb, about 2 to about 40 , about 2.5 to about 40 mg/kg, about 3 to about 40 g, about 3.5 to about 40 tngx’kg, about 4 to about 40 tug/kg, about 4.5 to about 40 g, about 5 to about 40 tug/kg, about 7.5 to about 40 11.1 mg/kg, about 11) to about 41) mg/kg, about 15 to about 41) g, about 21.1 to about 41.1 tug/kg, about 21) to about 40 tug/kg, about 25 to about 411 tug/kg, about 25 to about 41) Ing/kg, about 30 to about 46 tug/kg, about 35 to about 46 111ng about 0.1 to about 39 big/kg, about 9.25 to about ntg/kg, about {1.5 to about 31) mg/kg, about 0.75 to about 30 tug/kg, about 1 to about 31.1 ing/tng, about 1.5 to about 30 ntg/kb, about 2 to about 30 meg/kg, about 2.5 to about 311 mg/kg, about 3 to about 30 tug/kg, about 3.5 to about .31) mgkg, about 4 to about .30 mg;kg, about 4.5 to about 30 tug/kg, about 5 to about 30 , about 7.5 to about '30 tug/kg, about 10 to about 30 rug/11g, about 15 to about 30 , about 21.1 to about 30 tug/kg, about 20 to about 30 tug/kg, about 25 to about 30 tug/kg, about 0.1 to about 20 tug/kg, about 0.25 to about 20 tug/kg, about (1.5 to about 20 ntg/kg, about 1.1.75 to about 21) tug/kg, about 1 to about 21) tug/mg, about 1.5 to 211 about 21) mg/kb, about 2 to about 21) rug/kg, about 2.5 to about 211 tug/kg, about 3 to about 211 tn g/kg, about 3.5 to about 20 big/1115, about 4 to about 21) nag/165, about 4.5 to about 20 tug/kg, about 5 to about 20 rug/kg, about 7.-5 to about 20 tug/kg, about 10 to about 20 tug/kg, or about 15 to about 211 ing/kg. In one embodiment, when a composiuon ofthe invention eontorises a dsRNA as alesoribed bomb; and an lga1attosanune 511056145 can be stered a therapeutic amount ot about 10 to about 31) tug/kg 01 113112441. Values and tan ges intetn‘tediate to tbe recited Vaiues are also intended to be patt oftbis invention.

For exa1np1e, subjects can be administered a tberapeut1o amount ot‘iRNA, such as about (1.1, 1.1.2, 03,114,115,116,117,11.-8,111.9,1,1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,4.8,4.9,5,5.1,5.2,53,5 4.,5.556,5.7,5,5.8 .9, 6, 6.1 6.2,6.3, 6.4 65, 66, 67, 68, 6.9 7, 7.1, 7.2, 7.3, 7.4, 7.5,:7 .6, 77, 7.8, 7.9, 8, 8.1, 8.2, 83,6.,4 6.5, 8.66.,7 8. 8, 89 9, 91, 9.2., 9.3, 94,95,961, 979.8, 99 1(1,11).5,11,11.5,12,125,13,13..5,14,145. 15, 1551,6. 16.5, , 18,18.5,19,19.5, 20, 29.5, 21, 21.5, 22. 22.. , ’23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5.3, 28, 28. 5, 29, 295, 3t), 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41,42 43, 44, 45,16, 4, 48, 49, or about 50 tug/kg. Va1ues and ranges intermediate to the recited va1ues are a1so intended to be bait ot‘tbis invention.

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS The iRNA can be administered by enous infusion over a period oftime, such as over a 5, 6, 7, 8, 9,10,11,122,13,14,15,16,17,18,19, 20, 21, '22, 23, 24, or about a 25 minute period, The administration maybe repeated, for exainp1e, on a regular basis, such as weekly, biweeidy (i. 8, every two weeks) for one. month, two months three months, four months or ‘J‘: . After an initial treatment regimen, the treatments can be administered on a less frequent basis. For exampie, after administration week1y or biweekly for three months, administration 1an be ed once per month, for six months or a year or .

Administration of the iRNA can reduce 1e veis, 1; ,in a och, tissue, hiood, urine or other compartment ofthe patient by atieast about 5%11, 6%, ‘34), 8%9/o, 10%,11‘3/1‘1, 17%1,3‘341, 111 14‘3/1, 15%, 163/11, 1 ‘341, 18%, 19“41, 2031-,213/11, 22%11, 23% ,2,4‘3/41, 25‘3/41, 26‘34'1, '2.."3/11, 28%, 29%, .30‘341,.31‘341,32‘3411,33o, 34%11, .159 ,36‘341,.3.7311, 38/0,39% ,411‘3/41,41‘341, 42341, 43341,1 44/11,45‘3410, 46%, 47%, 48.34 , 49%, 50/41,.11‘3/11, . 2%, 5‘30,54%, 55% 56%, 57%, 58%, 59 ‘341, 61%, 1%, 62%, 63%, 64"/,41 65",66‘3/1’1,6”’,68‘341,69‘3/,41 7041,7191}, %, 73‘341,7-4‘341,75‘341,76341,7'-i 941, 78%,79%o, 811%,81‘3/1, 82‘341,83%,8 341,854,, 86%, 87%, 88% 8‘341, 911%,‘3419111,,92‘3/41,93‘341, 94%, 95%, 96%, 97‘341, 983341, or at Eeast about 99% or more 1111111111 administration of a 11111 dose ofthe iRNA, patients can be administered a sinaiier dose, such as a 5% infusion, and monitored for adverse effects, such as an aiiergie reaction. In another exampie, the t can be monitored for unwanted immunostimuiatory efbcts, such as increased cytokine (9.53., ’1‘N13-a1pha or iNE-a1pha) ieveis. 211 Owing to the inhibitory effects on C5 expression, a composition according to the inv1ntion or a pharinaceuticai composition ed therefrom can enhance the quahty .

An iRNA ofthe invention may be administeredin “naked” form, or as a “free iRNA.” A. naked iRNA is stered in the absence of a pharmaceutieai composition. The naked iRNA may be in a suitabie butter so1ution, The 11111-1111 so1ution may comprise acetate, citrate, nine, carbonate, or phosphate, or any combination thereof 1n one embodiment, the buffer soiution is phosphate buffered sa1ine (1713 S). The pH and osmo’iarity of the buffer soiution containing the 11,3.th can be adjusted such that it is 1e for administering to a t 1411ternative1y, an iRN A o 1‘ the invention maybe administered as a pharmaceutica1 composition, such as a dsRNA liposomai tbrmu1ation.

(A) (:1 Subjects that wouid benefit from a reduction and/or inhibition of C5gene expression are those having a compiement component {TS-associated disease or disorder as bed herein. in one embodiment, a subject having a comp1einent component C5—associated disease has paroxysmainocturnaihemogiobinuria(131111111). In another embodiment, a subject having a ment component C5'associated disease has . in another embodiment, a subject having a compiement component sociated disease has rheumatoid arthritis. 1n yet another ment, a sub} ect having a conipiement ent CSwassociated disease has systemic [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS lupus erythmatosis, In one embodiment, a subject having a complement component C5~ associated disease has glomeiulonephritis. In another embodiment, a subject having a complement component C5~associated e has psoriasis. In yet another embodiment, a subject having a complement component (TS—associated disease has dermatomyositis s (I: pemphigoid. In one embodiment, a subject having a complement component Cfinassociated disease has atypical hemolytic uremic syndrome in another embodiment, a subject having a ment component (IS—associated disease has Shiga toxin E. coii~related hernolytic uremic me, In e embodiment, a subject having a complement ent (TS—associated disease has myasthenia gravis. In yet another embodiment, a subject having a complement ill ent sociated disease has neuromyelistis optica. in one embodiment, a subject having a ment component {ZS-associated disease has dense deposit disease. In one embodiment, a t having a complement component (IS—associated disease has C3 neuropathy, In another embodiment, a t having a complement component CSnassociated disease has age—related macular degeneration In another embodiment, a subject having a complement ent (IS—associated disease has cold agglutinin e. In one embodiment, a subject having a complement component (IS—associated disease has anti—neutrophil cytoplasmic antibody~associated itis, In r embodiment, a subject having a complement component CSnassociated disease has humoral and vascular transplant rejection. In one embodiment, a subject having a complement component sociated disease has graft dysfunction. In one embodiment, a subject having a complement component Cfisassociated disease has had a myocardial intarction. In another ment, a subject having a complement component C5~associated disease is a sensitized ent of a transplant. In yet another embodiment, a subject having a ment component {TS—associated disease has sepsis.

Treatment ol‘a subject that would benetit from a reduction and/or inhibition ofCS gene expression includes therapeutic and prophylactic tag. the subject is to undergo sensitized (or allogenic) transplant surgery) treatment.

The invention further provides methods and uses of an iRNA agent or a pharmaceutical composition thereottincluding methods and uses of an iRNA agent or a pharmaceutical composition sing an iRNA agent and an anti—complement component C5 antibody, or (A) (:3 n—bidning fragment thereof) for ng a subject that would benefit from reduction and/or inhibition of CS expression, 51g, a subject having a complement component CS ~associated disease, in combination with other pharmaceuticals and/or other therapeutic methods, eg, with known pharmaceuticals and/or known therapeutic methods, such as, for example, those which are currently employed for treating these disorders For example, in certain embodiments, an illNA targeting CS is administered in combination with, 9.53., an agent useful in treating a complement component ociated disease as described elsewhere herein.

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS For example, additional therapeutics and therapeutic inethods suitable for treating a subject that would benefit from reducton in CS expression, 8%; a subject having a complement component {IS—associated disease, include plasrnaphoresis, hoiytic therapy (cg, streptokinase), antiplateiet agents, folic acid, corticosteroids; nosuppressiye agents; (I: estrogens, inethoti‘exate, 6—MP, azathioprine salazine, mesaiazine, olsalazine, chloroquinine/hydroxychioi‘oquine, penciilamine, aurothionialate (intramuscular and oral), azathioprine, coc’hicine, corticosteroids (oral, inhaled and local injection), heta~2 adrenoreceptor ts (saihutainol, terbutaline, eterai), xanthines (theophylhne, aminophyihne), cronioglycate, nedoeroniil, fen, iprati‘opiuni and oxiti‘opiuin, cyclosporin, FKSOo, it} rapainycin, inycophenoiate rnofetil, ieflunornicle, NSAIDS, for example, ibuprofen, corticosteroids such as prednisoione, phosphodiesterase inhibitors, adensosine agonists, antithrornbotic agents, complement inhibitors, adrenergic agents, agents which interfere with signalling by proinflaniinatory cytokines, such as lNF—u or iL-i (rag, iRAK, NIK, lKK, p38 or MAP kinase inhibitors), ,lL-lB converting enzyme inhibitors, 'fNFrtconyerting enzyme (TACE) tors, T-ceil signalling inhibitors, such as kinase inhibitors, ioproteinase inhibitors, Sitiltasalazine, azathioprine, 6—i'nercaptopurines, angiotensin converting enzyme inhibitors, soluble cytohine receptors and derivatives thereof (cg. soluble p55 or p75 TNF receptors and the derivatives p75'I'NFRigG (EnbrellM and pSS’i‘NFngG (Lonerceptfi, le~lRL siLwiRll, and le—SR), antiinfiarnrnatory ines (9.53., ,EL-él, lL-l G, iL—l l, iL—13 and TGFB), celecoxib, folic acid, hydroxychloroquine sulfate, rofecoxib, cept, irnonocional antibody, naproxen, yaldecoxib, suli‘hsaiazine, rnethylprednisoione, nteloxicarn, n’iethylprednisoione acetate, gold sodium thioinalate, aspirin, triameinoione acetonide, propoxyphene ate/apap, folate, naburnetone, diciofenac, piroxicani, etocloiac, diclofenac sodium, zin, oxycodone hci, hydrocodone hitartrate/apap, enac sodiurm’ntisoprostol, fentanyl, anakinra, human recombinant, tramadol hcl, saisaiate, sulindac, cyanocohaiamin/fa/pyridoxine, acetaminophen, aiendronate sodium, prednisolone, morphine sulfate, lidocaine hydrochloride, indoniethaein, amine sul /chondroitin, aniitriptyline hcl, suifadiazine, oxycodone cetaminophen, oiopatadine hcl, ostoi, naproxen sodium, orneprazole, cyclophospharnide, rituxiinonoclonal antibody, lL—l TRAP, MRA, CTf..A4~lG, lift 8 BF, anti—H.148, An‘ti~ll..l 5, (A) (:3 BIRBn796, SCIOn469, VX5792, Alt/£6648, VX—MO, Rofluniiiast, R1485, l , Mesopram, cyciosporine, cytokine suppressive anti-inflammatory drugtts) (CSAEBS); (LTDP-S’il/BAY—l 0- 3356 (humanized anti-TNFOL antibody; Ceiltech/Bayer); cA2/infiixiinonoclonal dy (chimeric anti~TNFOt antibody; Centocor); '75 deNFR~lgG/etanercept ('75 hf) TNF receptor~lgG fusion protein; lniinunex; see eg 099-4) Artlir. Rheum. 37: S295; (l996) J. invest. Med. 44-: 235A); 55 kd’fNF—igG (55 kl) 'fNF receptor-lgG fusion protein; Hoffniann—LaRoche‘); lDEC— CEQl .1138 2l()396 (nonadepleting primatized anti—CD4 antibody; lDEC/Sniithlsiine; see eg [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS ed set by DPS (l995) Artlir. Rheum. 38: Sl 85); DAB 4864392 and/or DAB 389—an2 (lL~2 firsion proteins; Seragen; see 3.3 (l993) Artlirit. Rheum. 36: 1223); Anti-lac (humanized anti-lL—ZRoz; Protein Design oche); li..—'~’i (anti-inllaminatory ine; DNAX/Schering); llrl 0 (SCH 52000; recon’ibinant ll..~l 0, anti—intiammatow eytokine; DNAX/Schering); ll..~4; ll..~l 0 and/or ll.—4 (I: ts (6.3., agonist antibodies); anlRA (llgnl receptor antagonist; Synergen/Amgen); anakinra (Rineret®/"1®iin3en); ’l'NFubp/s—TNF (soluble ’37 NF binding protein; see 9.3 U996) Arthr. Rheum. 39(9 (supplement)): 8284; (l 995) Amer. J. Physiol. — Heart and Circ. Physiol. 268: 37—42); l (pl'iosphodiesterase Type lV inhibitor; see (2.3., (l 996) Arthr. Rheum. 39(9 (supplement): $282); MRn966 (COXUZ inhibitor; see 8.3 (1996) Arthr. Rheum. 39(9 (supplement): Stilt); st (see 6.3, {1996) Arthr. Rheum. 39(9 (supplement): $82); methotrexa’te; thalidomide (see 5.3.. (l 996) Artlir. Rheum. 39(9 (supplement): $282) and thalidomide—related drugs (:2.3 Celgen); nomide (anti~ii’illa.nunatory and eytokine inhibitor; see 9.3 (1996) Arthr. Rheum. 39(9 (supplement): $131; (l996) lnllaniin. Res. 45: l03~l07); tranexamic acid (inhibitor ofplasrninogen activation; see 9.3, (1996) Arthr. Rheum. 39(9 (supplement): $284); T~6l 4 (eytoltine inhibitor; see 5.3 0996) Arthr. Rheum. 39(9 (supplement): SL282); prostaglandin El (see 6.3., (l 996) Artliir. Rheum. 39(9 ement): $282); 'l‘enitlap (non—steroidal anti~inflammatory (hug; see (3.3:, (l 996) Artlir. Rheum. 39(9 ement): $280); Naproxen (nonnsteroidal anti~intlammatory ding; see 5.3 (l 996) Neuro.

Report 7: l209—l2l3); Meloxicam (non-steroidal anti—inflammatory drug); ibuprofen (non— steroidal anti—inl‘lan’imator;v' drug); Piroxicam (non~steroirlal antiriiitlamniatory drug); Diclol’enac teroidal anii~intlammatory drug); lndomeihacin teroidal anti~ii‘itl,a.n’unatory drug); Sulfasalazine (see 8.3 (1996) Arthr. Rheum. 39(9 (supplement): SZSl); Azathioprine (see 83. (1996) Arthr. Rheum. 39(9 (supplement): $281); lCE inhibitor ifinhibitor of the enzyme interleukinrlfl ting enzyme); zapfli) and/or lck tor (inhibitor ot‘the tyrosine kinase zap—70 or lck); VEGF inhibitor and/or VEGRR inhibitor (inhibitors ol’vascular endothelial cell growth factor or vascular endothelial cell growth factor receptor; inhibitors of angiogenesis); corticosteroid anti—inflammatory drugs (9.3, 88203580); ’l'NF—eonvertase inhibitors; anti-lL—l 2 antibodies; anti~llr18 antibodies; interle’ulrin~l i (see 8.3 (l996) Arthr. Rheum. 39(9 (supplement): $296); interleukin~l 3 (see «2.3., G996) Artbr. Rheum. 39(9 (supplement): $308); interleukin —l7 inhibitors (see C3., (l996) Artlir. Rheum. 39(9 (supplement): Sl20); gold; penicillamine; chloroquine; ehlorambncil; ychloroquine; cyclosporine; cy‘clophospharnide; total lymphoid irradiation; antisthymoeyte globulin; l—J‘l antibodies; xins; orally—administered peptides and collagen; lobenzarit disodium; Cytolrine Regulating Agents (CRAS) HPZZS and Hill-66 (Houghten Pharmaceuticals; lire); lCAlVlwl antisense phosphorothioate oligo—deoxynneleotides (lSlS 2302; lsis Pharmaceuticals, inc); soluble eom ilernent rece tor l ‘ T Cell Sciences i 9 lnc.); retinisone; ortotein; . b [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS giyoosaminoglyean iphate; minooyciine; anti—1122?; antibodies; marine and botanical lipids (fish and plant seed fatty acids; see er; a et’ of. (l 995) Rheum; Dis. Clin, North Am. 21: 7594777); aurarioiin; pheriyihutazone; niecioienamic acid; iiuteiiamie acid; intravenous immune globulin; zileuton; ine; myooplienolio aoid (RS—61443); taoroiimus (PK—5 06):, sirohmus (I: iyoin); aniiprilose (therafectin); eiadrihine (2nolilorodeoxyadenosme); methotrexate; held), inhibitors ifsee Bruneko, M. at 01'. (2007) J. Med. Chem. 50(4): 641—662); rais and immune— modulating agents, small molecule inhibitor ofKDR, small molecule inhibitor 2; n'iethotrexate; prednisone; celecoxib; folic acid; hydroxychloroquine sultate; rofeeiixih; etanereept; infiixinionoclonal antibody; leflunomide; naproxen; vaideeoxih; sulfasalazine; it} niethylprednisoione; ibuprofen; meioxicani; methyiprednisoione acetate; gold sodium thion’iaiate; aspirin; azatliioprine; triarncinolone aeetonide; propxyphene riapsyiate/apap; folate; nahumetone; dioiofenao; piroxieani; etodolac; dielofenao sodium; zin; oxyoodone hei; odone hitartrate/apan; diclofenac sodium/inisoprostoi; fentanyl; anakinra, human recombinant; oi hci; saisaiate; sulindac; cyanoeobalainin/fa/pyridoXine; acetaminophen; aiendronate sodium; prednisoione; morphine sulfate; lidoeaine hydrochloride; indomethaein; giucosamine sultate/oliondroitin; eyolosporine; amitriptyiine hcl; iazine; oxycodone hell/acetaminophen; adine hel; misoprostol; naproxen sodium; omeprazoie; myeophenolate niofetii; oyclophosphamide; nionoolonal antibody; iL~l 'I‘RAP; Mil/Ar; C'i‘LAllan; anl 8 BF; ,EL—l'Z/ZS; anti—EL l8; anti-1L 15; 96; SCH-469; VX-T’OZ; AMCi—548; VX—740; Ri’ifhirnilast; K3485; CDChOi; mesoprarn, roi, salmeteroi/i‘luticasone, montelukast sodium, thiticasone propionate, hudesonide, prednisone, salineterol xinai‘oate, levalhuteroi hoi, roi snlfate/ipratropium, prednisolone sodium phosphate, triameinolone acetonide, hecionietiiasone dipropionate, ipratropium bromide, azithromycin, pirbuteroi e, prednisoione, theophyiline anhydrous, l’Ylifjtllyipl'fidl’liSOlUYlB sodium suecinate, eiarithrornyoiri, zatiriukast, i'hri’noteroi tiirnarate, influenza virus e, prednisolone, amoxieillin trihydrate, flunisoiide, allergy injection, cromoiyn sodium, fexotenadine hydrochloride, tiunisoiide/nienthoi, amoxiciliin/eiayulanate, ieyotloxaein, inhaler assist device, guaiienesin, dexaniethasone sodium phosphate, moxiiioxacin hei, doxyeycline hyelate, nesin/d methorplian, p—ephedrine/:od/chlorphenir, gatii‘loxaein, cetirizine hydroohioride, mometasone (A) (:3 furoate, salineterol ate, benzonatate, iexin, rocodone/ohlorphenir, oetirizine hch’pseudoephed, phenylepiirine/eod/promethazine, codeine/prometliazine, cefiirozil, dexamethasone, guaifenesin/pseudoephedrine, ehiorpheniramine/liydrooodone, nedocromil sodium, terhutaiine suifate, epinephrine, inethylprednisolone, proterenol sulfate, aspirin, nitroglycerin, metoproloi tartrate; enoxaparin sodium, heparin sodium, clopidogrei bisulfate, carvediiol, atenoiol, morphine sulfate, metoproioi ate, warfarin sodium, Eisinopril, isosorbide mononitrate, digoxin, furosernide, simyastatin, rai’riipril, tenectepiase, en aiaprii [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS nialeate, torsemide, retavase, losartan potassium, quinapril hcl/mag carb, anide, alteplase, enalaprilat, amiodarone hydrochloride, tirofiban hcl m-hydrate, zein hydrochloride, captopril, rtan, valsartan, propranolol hydrochloride, lhsinopril sodium, lidocaine hloride, eptifihatide, cel’azolin sodium, atropine sall’ate, aminocaproic acid, (I: spironolactone, interferon, l hydrochloride, ium chloride, docnsate , dohutamine hcl, alprazolain, pravastatin sodium, atorvastatin calcium, midazolam hydrochloride, ineperidine hydrochloride, isosorbide dinitrate, epinephrine, dopamine hydrochloride, bivalirodin, statin, ezetimibe/sii'nvastatin, avasii’rii‘oe, and cariporide.

The iRNA agent r an anti—complement ent C5 antibody) and an additional ill therapeutic agent and/or treatment may he administered at the same time and/or in the same combination, 8.53., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or at separate times and/or by another method known in the art or described herein.

The present ion also provides methods ofusing an iRNA agent ofthe invention and/or a composition containing an iRNA agent of the invention to reduce and/or inhibit complement component C5 expression in a cell, in other aspects, the t invention provides an iRNA ofthe invention and/or a. composition comprising an iRNA of the invention for use in reducing and/or inhibiting C5 expression in a cell. ln yet other aspects, use of an iRNA of the invention and/or a composition comprising an iRNA of the invention for the mannfactuire ot‘a medicament for reducing and/or inhibiting CS expression in a cell are provided.

The methods and uses e contacting the cell with an iRNA, rag, a dsRNA, ot‘the invention and maintaining the cell for a time sufficient to obtain degradation of the mRNA transcript of a (:5 gene, thereby inhibiting expression of the CS gene in the cell.

Reduction in gene expression can be ed by any methods known in the art. For example, a reduction in the expression o‘l’CS may he detennined by determining the iriRNA, expression level of C5 using methods routine to one of ordinary skill in the art, erg. Northern ng, qRT—PCR, by determining the protein level of C5 using methods routine to one of ordinary skill in the art, such as Western blotting, iiniminological techniques, tlovv cytoinetry methods, El_,rlSA,, and/or by determining a biological ty ofCS, such as Clilgg. or A850.

(A) (:3 hemolysis assay, and/or by determining the biological activity of one or more molecules associated with the complement system, eg, {‘5 products, such as CSa and (35b (or, in an in viva setting, eflg heinolysis}. in the methods and uses ot‘the invention the cell may be contacted in nine or in viva, tie. the cell may be within a sub} ectt ln ments ofthe ion in which the cell is within a sahj ect, the methods may include further contacting the cell with an anti—complement component CS antibody, sag, ecnlizumah.

[Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS A:3e11 suitab1e for ent using the methods ofthe invention may be any :3e11 that expresses a C5 gene. A ce11 suitab1e for use in the methods and uses of the invention may be a mammalian ce11, 21g, a primate ce11 (such as a human ce11 or a non~human primate 6e11, eg, a inon11eyce1ior a chimpanzee 61311), a non—primate ce11 (such as a cow 61:11, a pig 131311, a came1 ‘J‘: ee11, a 11ama ce11, a. horse ce11. a goat ce11. a rabbit ee11, a sheep ce11, a hamster, a guinea pig ee11, a cat ce11, a dog ce11, a rat 6e11, a mouse ce11, a 11on ce11, a tiger 6e11, a bear ce11, or a o ce11), a 111111 ce11 143-8131 duck 61311 or a goose ce11), or a whaie 131311. In one embodiment, the ce11 is a human ce11, tag. a human 1iyer ce11.

C5 expression may be inhibited in the ee11 by atte1r"1st about59411, 694, 94> 8%, 9%. 10%, 111 11“:41, 12943, 1394, 1 94, 15%, 16%, 179/1, 18%, 1.994:3, 211%,21%, 22941, 2.3943, 2494, 2594, 79/1,’8294;, 2.9943, 31.190,-221% 329/ '5 5%, 34943, 359 :3,436%o, 27%, 58%, 3.9943, 4 3,941 3%, 44%,54594,o, 469m, 47%, 48%, 49%, 5 940, 51%, 52%, 53%, 54%, 55%, 5 .. , , , 0940, 61940,62%, 63%, 6494, 6594, 660/,4 679E1.689/(;,6‘~194,7094, 71%, 72%, 73%. 749/13, 7 94o, 76%o, 77% 78%, 39943, 8094, 8 9-40, 8 %, 3%, , 8594, 8694/o, 87%, 88% ,89%, 90943, 9194/o, 92%o, 939 0,0941%, 9.5943, .9694m, 97%, 98%, 99%, or about 180% The in V1120 methods and uses of the inyention may inc1ude administering to a subject a composition containing an iRNA, where the iRNA inc1udes a nu:31eotide ce that is eonip1en1enta1y to at 1east a part of an RNA transcript of the C5 gene of the niamn1a1 to be treated. When the organism to be treated is a mammai such as a human, the composition can be 211 administered by any means known in the art inciuding, but not limited to subcutaneous, enous, ora1, intraperitoneah or par:entera1 routes, inthtding intraoraniai (rag, intraventricu1ar, 1ntraparenchyma1 and intratheea1), intramuseu1ar, transdemia1. ainyay (aerosoi), 11asa1, recta1, and topica1 (inciuding bucca1 and subiingua1) administration. in certain embodiments, the itions are stered by subcutaneous or intravenous infusion or 11:11 ecti on . in some embodiments, the administration is via a depot injection. A depot injection may e the 119N141 in a tent way over a pro1onged time period. Thus, a depot ion may reduce the frequency ot‘dosing needed to obtain a d effect, e.g., a desired inhibition ofCS, or a therapeutic or prop11y1aetic 1:111:11. A depot injection may a1so provide more consistent (A) (:1 seiuin concentrations. Depot iniect1ons may inc1ude aneous injections or intraniuscu1ar injections. in preferred embodiments, the depot ion is a subcutaneous injection. 111 some embodiments, the administration is via a pump. The pump may be an externai pump or a surgica11y imp1ante11 pump In certain embodiments, the pump is a subcutaneous1y nted osmotic pump. in other embodiments, the pump is an infusion pump. An infusion pump may be used for intravenous, subcutaneous, arteria1, o1" epidurai infusions. 1n preferred [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS ed set by DPS [Annotation] DPS None set by DPS ation] DPS ionNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS embodiments, the infusion pump is a subcutaneous infusion pump. in other embodiments, the pump is a surgically implanted pump that delivers the iRNA to the liver.

The mode of administration may be chosen based upon whether local or systemic treatment is desired and based upon the area to be treated. The route and site of administration (I: may be chosen to enhance targeting. in one aspect, the present invention also provides methods for inhibiting the sion of a (35 Gene in a , ag, a human. The present invention also provides a composition comprising an iRNA, rag, a dsRNA, that targets a C5 gene in a cell of a mammal for use in inhibiting expression ofthe C5 gene in the . in another aspect, the present invention ill provides use of an iRNA, eg. a dsRNA, that targets a C5 gene in a cell of a mammal in the manufacture ot‘a medicament for inhibiting expression oftlie CS gene in the mammal.

The methods and uses include administering to the , eg, a human, a composition comprising an iRNA, age a dsRNA, that targets a C5 gene in a cell of the mammal and maintaining the mammal for a time sufficient to obtain degradation of the mRNA transcript ofthe CS gene, thereby inhibiting expression ot‘the C5 gene in the mammal. ln some embodiment, the methods further comprise administering an anti—complement component C5 dy, raga, eculizumab, to the subject. ' ednction in gene expression can be ed by any methods known it the art and by methods, eg qR’lfil’CR, described herein. Reduction in protein production can be assessed by any methods known it the art and by methods, eg. , ELISA or Western blotting, described herein. ln one embodiment, a puncture liver biopsy sample serves as the tissue material for monitoring the reduction in C5 gene and/or protein expression. ln another embodiment, a blood sample serves as the tissue material for monitoring the reduction in CS gene and/or protein expression. in other embodiments, inhibition of the expression of a (35 0rate is monitored indirectly by, for example, determining the expression and/or activity of a gene in a CS pathway, including, for example, CSa, CSb, and soluble C5b~9 (see, eg. Figure It). For e, the activity of CD59 may be monitored to determine the inhibition of expression of a CS gene. C7850, Alisa, clot tbrmation and/or serum lactate ogenase , in a sample, eg a blood or liver sample, may also be measured. le assays are thrther described in the Examples section below.

(A) (:3 Unless otherwise detined, all technical and scientific terms used herein have the same meaning as ly understood by one of ordinary skill in the ait to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the iliNAs and methods featured in the invention, suitable methods and materials are described below. All publications, patent applications, patents, and [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS other references mentioned herein are incorporated by reference in their entirety. in case of ct, the t specification, including definitions, will control. in addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

EXAMPLES Example l. iRNA Synthesis Source ofreagentr Where the source of a t is not specifically given herein, such reagent can he obtained from any supplier of ts for molecular biology at a quality/purity standard for ill ation in molecular biology.

Transcripts siRNA design was carried out to ty siRNAs targeting human, rhesus (ll/Medea malaria"), mouse, and rat C5 transcripts annotated in the NCBl Gene database (http3,1",I’Vwrv'wnchitnlm.nihgov/gene"). Design used the following transcripts from the NCBl RelSeq collection: Human - Nit/LOW 735.2; Rhesus “ lel_0(ll 0957502; Mouse — NM___(ll @4063; Rat — XMMMSMZA SiRNA es were designed in several separate batches, including hut not limited to hatches containing duplexes matching human and rhesus transcripts only; human, rhesus, and mouse transcripts only; human, rhesus, mouse, and rat ripts only; and mouse and rat transcripts only. All siRNA duplexes were ed that shared ltlt % identity with the listed human transcript and other species transcripts considered in each design hatch (above).

SHWA Design, Specypzcifgv, and Efiicucy tion The ted specificity of all possible lilmers was predicted from each sequence.

Candidate l9rners were then selected that lacked repeats longer than '7 nucleotides. These 29'7l candidate rhesus, l42 hum an/rhesus/i:nouse, 54 hurnan/rhesus/rnouse/rat, and 807 mouse/rat siRNAs were used in comprehensive searches against the appropriate transcriptonies (defined as the set ot‘Nl‘s/L and XM_ records within the human, rhesus, dog, mouse, or rat NCBI Retheq sets) using an exhaustive “hrute—thrce” algorithm implemented in the python script ‘BruteForcetpy’. The script next parsed the transcript—oligo alignments to generate a score based (A) (:3 ' on the position and number of ches between the siRNA and any ial 'off—targe transcript. ’l'he off-target score is weighted to emphasize differences in the ’seed’ region of siRNAs, in positions 2—9 from the 5'-end ofthe molecule.

Each transcript pair from the brute—force search was given a niisn'iateh score by summing the dual ch scores; mismatches in the position 2-9 were counted as 2.8, mismatches in the cleavage site positions ltlull were counted as l2, and mismatches in region 12~l 9 counted as ll). An additional offliarget prediction was carried out by comparing the [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ncy ofheptainers and octoniers derived from 3 distinct, seed—derived hexaniers of each oligo. The hexainers fren’i ons 2:; relative tn the 5” start were used to create 2 heptarners and one er. ‘lleptanierl ’ was created by adding a 3 ’~A to the liexarner; heptarnerZ was created by adding a 5 ’—A to the hexarner; the octorner was created by adding an A to both 5 ’— and (I: 3 ’nends of the hexarner‘ The frequency of octarners and heptaniers in the human, rhesus, mouse, or rat 3 l<orne (defined as the uence ofthe transcriptorne froin NCBl’s Refseq database where the end ot‘the coding region, the ‘CDS’, is clearly defined) was presealeulated.

The octanier frequency was norn'ialized to the heptanter fieriuency using the median value from the range of er ncies A ‘niirSeedScore" was then calculated by calculating the sum it} of {(3 X normalized octarner count) ('2 X heptarnerZ count) + (l X heptanierl count)).

Both siRNAs s were ed to a category of specificity according to the calctdated scores; a score above '3 qualities as highly c, equal to 3: as specific and hen reen 2.2 and 2,8 as moderately specific. The duplexes were sorted by the specificity ofthe antisense strand and those duplexes whose antisenize oligos lacked GC at the first position, lacked G at both positions l3 and E4, and had 3 or more Us or As in the seed region were selected.

For GalNaC~conjugated duplexes sense ’2 l met and antisense 2311163? oligos were designed by ing antisense l9niers (described above) to 23 nucleotides oftargetn complementary sequence. All species transcripts included in the design hatch were checked for con‘iplenientarity. {inly s that preserved lllll% sequence complementarity in at least 2 species were used. For each duplex, the sense erner was specified as the reverse complement of the first ’Zl nucleotides of the antisense strand.

SITWA ce seiecz‘iorz A total of '23 sense and 23 antisense derived rhesus, 6 sense and 6 antisense hurnan/rhesus/rnouse, 6 sense and 6 antisense derived hurnan/rhesus/rnouse/niouse/rat, and l3 sense and l3 antisense derived mouse/rat siRNA l 9nier oligos were synthesized and tbrn’ied into duplexes The above l9iner sets were extended to 21/23nier duplexes for GalN ac conjugate design and resciassitied according to their new species matches. Twenty—seven sense and 27 antisense derived hitrnanx’rhesus 1 sense and 1 sense derived human/ritesus/niousel, 3: sense and 3 antisense (A) (:3 derived human/rhesus/rah 4 sense and 4 antisense derived hunian/rhesus/rnouse/rah and 13 sense and l3 antisense derived mouse/rat Zlnier (sense) and 23rner (antisense) oligns were synthesized and formed into es.

A detailed list ot‘CS sense and antisense strand sequences is shown in Tables 346.

[Annotation] DPS None set by DPS ation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS SiRA/Zd Synthesis l Small and Medium Scale RNA S 'nthesis ure RNA oligonucleotides were synthesized at scales n 0.2400 ttinol using commercially available 5 ’—O~(4,4 ’~dii:nethoxytrityl)—2 ’ ~O—t~hutyldimethyl silyl—3 ’—O~(2— (I: cyanoethyldV;Nndiisopropyl)phosphoramidite monomers ofuridine, 4 ~Néaeetylcytidine, 6—Nn henzoyladenosine and Z—N—isobutyrylguanosine and the corresponding 2’—O—methyl and Z’ul‘luoro pliosp’horaniidites according to standard solid phase oligonucleotide synthesis protocols. The amidite solutions were prepared at ill ~(lrl5 M tration and 5~ethylthio—lll~tetrazole (021% on M in aeetonitrile) was used as the activator. l’hospliorothioate ne modifications were l0 introduced during sis using 0.2 M plrenylacetyl disultide (FADE) in lutidine:acetonitrile (l :l) (V;V) or ill M 3~(dirnethylaniinomethylene) amino-3 llél ,2,4sditliiazole*5~thione (Dill—T) in pyridine for the oxidation step. Alter completion of synthesis, the sequences were cleaved from the solid support and deprotected using methylamine followed by triethylamineSHF to remove any '2 ’—O—t—hutyldiniethylsilyl protecting groups present. l5 l?or synthesis scales between SMSOQ uniol and fully 2’ modified SfiqlllliillCGS (2’~llt:ioro and/ or 2’—0~methyl or oon'lbinations thereof} the oligonucleotides where. deprotected using '3zl (Vi/y) ethanol and concentrated (28—32%) aqueous ammonia either at 35°C l6 h or 55°C for 5.5 l1. Prior to ammonia deprotection the oligonucleotides where treated with 05 M piperidine in aeetonitrile for 20 min on the solid support The crude oligonucleotides were analyzed by LC—--MS and anion— 2t) exchange llPLC (lEX-llPLC). Purification ot‘the oligonucleotides was carried out by IEX lthLC using: 20 mM phosphate? l .%~l5% ACN, pH = 8.5 (huffer A) and 20 mM phosphate, l0%n15% ACNl l M NaBr, pH = 805 (buffer B). Fractions were analyzed for purity by analytical lll’LC. ’l‘he product—containing fractions with suitable purity were pooled and concentrated on a rotary evaporator prior to desalting. The samples were desalted by size exclusion chron‘latography and lyophilized to s. Equal Il’lfllétt‘ amounts ot‘sense and antisense strands were annealed in lX PBS buffer to prepare the corresponding siRNA duplexes.

For small scales (0.23mi panel), sis was performed on a MerMade l92 sjnthesizer in a 96 well format. in case offully 2’-modilied sequences (2’~l‘luoro and/or 2’—{?~inethyl or combinations f) the oligonucleotides where ected using ainine at room temperature for 30439 min followed by incubation at. 600C for 39 min or using 3:l (V/V) l and concentrated (28—32%) aqueous ammonia at room temperature {0180-60 min followed by incubation at 400C for l .5 hours. The entde ucleotides were then precipitated in a solution ofacetonitrile:acetone (9d) and ed hy centritugation and decanting the supernatant. Th e elude oligonucleotide pellet was re~suspended in 20 nrl‘vl NaOAe buffer and analyzed by LrCanlS and anion exchange Hills/C. The crude oligonueleotide ces were desalted in 96 deep well plates on a 5 ml; lliTrap Sephadex (325 column (GE llealthcare). In eaeh well about l5 lTlL [Annotation] DPS None set by DPS ation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS samples corresponding to an individual sequence was collectetli ’l‘hese purified desalted oligonucleotides were ed by lS and anion exchange chromatography. Duplexes were prepared by annealing eduirnolar amounts e and antisense sequences on a Tecan robot. tration of duplexes was adiusted to ill LEM in lx PBS buffer.

(I: “atalliesis.all;sills.a._____...anausat__rl__tltlt0usual...and.es..tar.£zz...l€zra.dualMus Oligonueleotides conjugated with GalNAc ligand at their 3 —terniinus were synthesized at scales between 0.26th urnol using a solid support ded with a Y-shaped linlter bearing a 4941dirnethoxytrityl (Dh’lT)—protected primary hydroxy group for oligonucleotide synthesis and a GalNAc ligand attached through a tetheri lll For synthesis of GalNAc conjugates in the scales between 5---~500 ltmol, the above synthesis protocol for RNA was followed with the ihllowing adaptions: For polvstvrenesbased sis supports 5% dichloroacetic acid in toluene was used for l)\llcleavage during synthesis Cleavage from the support and dcprotection was performed as described above Phosphorothioate-rieh sequences (usually > 5 phorphorothioates) were synthesized without removing the final $thle group (“Dl‘v’lla’in”) and, after ge and deprotection as described above, purified by rever‘e phase llPlC using 50 told annnonium acetate in water (butler A) and 50 ml‘vl animoniurnacetatein 80% acetonitirile (buffer B). Fractions were analyzed for purity by ical HFLC and/or LC~MSi 'l‘he product—containing fractions with suitable purity were pooled and concentrated on a rotary evaporator. The ‘—gi'oup was removed using 209453596 acetic acid in water until completion. The samples were desalted by size exclusion chromatography and lyophilized to dryness Equal molar amounts ofsense and antisense strands were annealedin lx PBS buffer to prepare the corresponding siRNA duplexes For small scale synthesis of ClalNAc ates --—l umol), including ces with multiple phosphorothioate linkages, the protocols described above for synthesis ol‘RNA or fully 2°~ 1/2”~Ol\/le—containing sequences on Merhrlade platlhrrn were applied. Synthesis was performed on prewpacls‘ed columns containing GalNAe—funetionalized lled pore glass support.

Example 2. In vim: screening (A) (:1 Ceii culture and fransfictions llep3l3 cells (A'l'CC, Manassas, VA) were grown to near confluence at 370C in an atmosphere of3"0 C02in s Minimum lissential lVledium (ATCC) supplemented with lllll/e- F839 streptomycin, and glutamine I) belore being released lirom the plate by tiypsinization. Cells were washed and re~suspended at 0°25xl 06 cells/ml. During translations, cells were plated onto a 96—well plate with about 20,000 cells per well [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS Primal“)r mouse liepatocytes (PMH) were freshly isolated from a C57BL/6 female rnouse (Charles River Lahortories ational, lnc. Willinington, MA) less than l hour prior to transtections and grown in primary hepatocyte media. Cells were resuspended at ().l lxl06 cells/ml in anitroGRO CF Rat (plating) mediurn (Celsis In Vitro Technologies, catalog number (I: S0l494). During transfeetions, cells were plated onto a Bl) t 96 well collagen plate (Bl), 356407) at l0,000 cells per well and incubated at 37°C in an atmosphere of5% (:03.

Cryopreserved Primary Cynomo!gus Hepatocytes (Celsis In Vitro Technologies, M003055—P} were thawed at 37°C water bath immediately prior to usage and re—suspended at 0.26le6 cells/ml in anitroGRO Cl) ng) medium (Celsis ln Vitro Technologies, catalog l0 number 299029). During transfections, cells were plated onto a Bl) BioCoat 96 well collagen plate (BID, 356407) at 25,000 cells per well and incubated at 37°C in an atmosphere ol’5% (.702.

For llep3B, PMH, and ry oigus hepatocytes, transfection was carried out by adding l4.8 all of OptinMElVl plus 0.2 til of Lipofectainine RNAih/lax per well (lnvitrogen, ad CA. catalog nuinherl3778—l50) to 5 ul of each siRNA duplex to an individual well in a 96—well plate. The mixture was then incubated at room temperature for '20 minutes. 'liiighty ul of complete growth media without antibiotic containing the appropriate cell number were then added to the siRNA mixture. Cells were ted for 24- hours prior to RNA purification.

Single dose experiments were performed at l0nlvl and 0.lnM final duplex concentration for GalNAc modified sequences or at lnl‘vl and 0.0lnM final duplex concentration for all other sequences. Dose response experiments were done at 3, l, 0.3, 0.l 0.004l 2, and , 0.037, , 0.00l3’7 nlvl linal duplex concentration for prirnary rnouse cytes and at 3, l, 0.3, 0.l 0.037, 0.0l23, 0.004l2, 0.0087, 0.00046, 0.000l5, 0.00005, and 0.0000l7 nlvl final duplex concentration for llep3B cells.

Free uptake traiisfiéction Free uptal<e experiments were performed by adding l0ul of siRNA duplexes in l’BS per well into a 96 well plate. Ninety pl of complete growth media containing riate cell number for the cell type was then added to the siRNA. Cells were incubated for 24 hours prior to RNA purification. Single dose experiments were pertorn’ied at 500anl and thd final duplex tration and dose response experiments were done at l000, 333., lll, 37, l2.3, ill/'3, LS7, 0.46 nM tinal duplex concentration.

Total RNA isolation usiizg Dl’z‘tlélBEADS n'zRNA Isolation Kit rogen, part # 61 0—1.2) Cells were harvested and lysed in l50 ul of Lysis/Binding Buffer then mixed for 5 minutes at 850 rpm using an orf ’l‘lierinoniixer (the mixing speed was the same throughout the process). Ten rnicroliters of magnetic heads and 80 ul Lysis/Binding Butler 1535 [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS mixture were added to a round bottom plate and mixed for l . Magnetic heads were captured using a magnetic stand and the supernatant was removed without disturbing the heads.

Alter removing the supernatant, the lysed cells were added te the ing heads and mixed fer minutes. After ying the supernatant, magnetic beads were washed 2 times with lle til (I: ‘Wash Buffifl' A and mixed for l minute. The heads were capturedagain and the supernatant was d. The beads were then washed with lSO pl ‘Wash Buffer B, captured and the supernatant was removed. The beads were next washed with lSl) til Elutien Stiller, captured and the supernatant removed. Finally, the heads were allowed to dry for 2 n'iinutes. After drying, 50 ul of Elution Buffer was added and mixed for 5 s at 7OOCQ The heads were captured on ll} magnet for 5 minutes. Forty—five pl of supernatant was removed and added to another 96 well pl ate.

Cit/“till synthesis using AB] High capacity CDNA reverse transcription kit (Clippiied Biosystems, Foster Citing CA, Car #4368813) A master rnix el’IZ ul lOX Butler, 0.8 pl 25X lePs, 2 ul Random primers, l ul Reverse Transcriptase, l pl RNase inhibitor and 3.2 til ol‘lrlZO per reaction as prepared. Equal s master mix and RNA were mixed for a final volume of llul for in Vifi'O screened or ZOul for in viva screened samples cDNA was generated using a d CulOOO or S—lGGG thermal cycler les, CA) through the following steps: 25°C for l0 minutes, 37°C for lZO minutes, 850C for 2t) 5 secends, and 4°C hold.

Reai time PCR ’l‘wo ul ofCDNA were added to a master mix containing Zul of 330, (3.5 ul GAPDltl TaqMan Probe (Life Technologies g number 43263 1 7l§i i‘hr llepf’ilfi cells, catalog number %? for primary mouse hepatocytes or custom probe for cyiiomelgus primary hepatocytes), Gifilul C5 'l‘aql‘s/lan probe (Life logies c catalog number Hs00l56l97wml for Hep3B cells or 4392 75_ml for Primary Mouse llepatoctyes or custom probe for cynomolgus primary hepatocytes) and Sul Lightcy‘cler 480 probe master mix {Roche catalog number 0488730l Gill) per well in a 384 well plates (Roche catalog number 0488730 l Gill). Real time PCR was performed in an Roche £0480 Real Time l’CR system (Roche) using the AACt£RQ> assay For in viiro screening, each duplex was tested with two biological replicates unless otherwise noted and each Real Time PCR was performed in duplicate technical replicates. For in viva screening, each duplex was tested in one or more experiments ('3 mice per group) and each Real Time PCR. was run in duplicate technical replicates.

To calculate relative fold change in CS mRNA levels, real time data were analyzed using the AACt method and normalized to assays performed with cells transfected with l0 nM AD- [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS l9559 or mock transfected cells. Eggs were calculated using a 4 ter tit model using XLEit and normalized to cells transfected with AD-l 955 over the same dose range, or to its own lowest dose.

The sense and antisense sequences ofAD~l 955 are: (I: SENSE: cuuAchuGAGuAcuueGAd’l‘sdT (SEQ ll) NO: 13); AN’l'lSENSE: UCGAAGuACUCAGCGuAAGd'l‘sd’l‘ (SEQ lD NQ: l4).

Table 7 shows the s ofa single dose screen in llepfllfi cells transiected with the indicated GalNAC conjugated modified iRNAs. Data are expressed as percent ofmessage remaining relative to ted cells. ll) Table 8 shows the results of a single dose transtection screen in primary mouse hepatocytes transfected with the indicated (:i'alNAC ated ied iRNAs. Data are expressed as percent ot‘message ing relative to untreated cells.

Table 9 shows the results of a single dose free uptake screen in primary szomoigus hepatocytes with the indicated GalNAC coniugated modified iRNAs. Data are expressed as l5 percent ofmessage remaining ve to untreated cells.

Table 10 shows the results of a single dose free uptake screen in y mouse hepatocytes with the indicated GalNAC conjugated modified iRNAst Data. are expressed as percent ofmessage remaining relative to untreated cells.

Table ll shows the dose response of a free uptake screen in primary Cynomo [gas hepatocytes with the indicated (:ialNAC conjugated modili ed iRNAs. The indicated leg values represent the ngo values relative to untreated cells.

Table l2 shows the dose response of a free uptake so ~een in y mouse hepatoeytes with the ted GalNAC conjugated modified iRNAsi The indicated ltjm values represent the lC5o values relative to untreated cells.

Table 13 shows the results of a single dose screen in llepSB cells transiected with llitt: indicated d and unmodified iRNAst Data are expressed as percent of message remaining relative to untreated cells. The 0.0lnl‘vl dose was a single biological transfection and the lnM dose was a duplicate biological transt‘eetion.

Table 14 shows the results ofa single dose screen in primary mouse lie atocytes (A) (:3 transfeeted with the indicated modified and unmodified iRNAst Data are expressed as percent of message remaining ve to untreated cells.

Table is shows the dose response in llep3B cells transfected with the indicated modified and unmodified iRNAs. The indicated ngQ values represent the TC50 values relative to untreated cells.

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS Table 16 shows the dose response in primary mouse eytes transfeeted with the ted modified and unmodified iRNAs. The indicated 1030 values represent the K350 values relative to untreated cells.

(I: Table 2: Abbreviations ofnueieotide monomers used in nucleic acid sequence representatinn, It Wiii he understood that these rnondniers, when present in an eligonucieotide, are niutuaiiy iinked by s ‘43 ‘~phospnediester bends.

Aiihre'viatinn Nueieotide{s) A Adcmmcirhouthdtu A t‘ 2 ’—flunr0adeuwine—3 ’«phospha’te Afs 2 ’—t‘iu0roadenesine“3 ’“phosphorothieate As ine—E’; ’—phdspherothidate C ne-B lphosnhate mwdmwithwhatc Cis 2 ’—fluereeytidine~3 ”~ph0snhorothi0ate Is cyti dined ’~ph()spnurethioate T 1. bevanwnfivheiphatu {if 2 ’—fluer0guan0sine~3 ’~ph0sphate (its 2 ’—t1uor0guanosiue~3 ’~ph()spherethioate {is guanesine—3 phorothieate LIE._________________________________________i.15..litigants!£i§ét§.§:§i:i§ii9§iztiats_______________________________________________________________________________________________t Tt‘ 2 ’~t1uoru~5~methyiuridine~3 ’—ph0sphate Its 2 e—5—inethyiuridine-3 lphospherethioate Ts Sninethyiui‘idine—3 ’—phesphornthi0ate U Urid inen3 ’nphusphate Ht:______________________________________i.E._’_:flaaiasiitiss:éiaziissdut:_________________________________________________________________________________________________i 'Ui‘s 2 ”—tiuorouridine ~3 ’~phospherethioate Us ne —3 ’—phesphorothieate N any nucleotide (G, A, C, T or U) aZOmfiMaimmth‘m ____________________________________________________________________________________ as g 210-methyiadenesinefi ‘3 phdspherothioate C 2'—O—niethyie:v’tidine—3 ’uphdsphate cs 2'~Oniriethyieytidine~3 ‘3 phosphorothiuate E_____________________________________ gs 210411ethyiguanusine—f’i 1 phospherethioate ation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS Abbreviafien Nucleutidds) t 2 ”—O—methyi—5—methyiu1‘idineu3 ’up hesphate ts ’n0~methyl~5quethyluridinefi lphosp’homfllioate .....................................................................................................................§ 1,: g2'~O—mefliyluridme—j —phosphate us 210-methy’iuridinaéfi ’—phosphorothioate s pi]osphomthiuatc e L96 N-[tris(GalNAc—alkyiij—amidedecanoylfl —4—hydr0xyprolinel Hyp— (GalNAmaEKyEB (Cit) deoxy-thymine [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS maimmmwmm 2%? mmicvfim..w, mmlcvmm HE mama ..m.3 .mmnawa . 3,0qu . .mmnwwa .mmnficoin. . 3;, 3..., xuwmcew .. H . , 9:? 3.4.4.53 303333, an w :8 851.6% 03333.4. md<fi<<w<® game“ dduuDDGB<<<<<03<< Q<<GQGQUUU835:5? 96.3832: mfizmmw j<mQuww<<Gj djwuiud ,Ujuvflquungfimizg2, 4.0430: 5 ‘ $25ko www.ma . immmml .mmmm. . T mfi . nun 323$me . Awmmw . a . w wmmmfim .Bnasu _ _ mi “2 33%:ng 3.4335:5.333.393 <<<<330303U336<Uw 323593.. 03m<<< m: E88 mm“ 3:25 D<uw30<0mww1 Duu<<03<£ $9,.me was 63 $5336:Buggies? memmmfi ESQ. 0% .fimm.§ 35m Mmfifiww . Emma?» mafia Emmmfimaa .dmmml .mmmm. . .wmmm. 2w . 08w: cmxc mmmmmgm mfifiomfi mgummm 9.3 ‘m «new $9.83 Bmfioflcms‘ agar/w M EH 3mm 23 ED H _. m [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS .344..4..4..44.4..~3 mm..4afifi.mmofi 44....4m4m- 44-33.4344 mm4-30m4...N.@E4.oflm 44-42.4444... nmeNm4-¢om4JN.mo.4943.44.... mm mm...omN..wom:m.mm443.44.... ...m..4w4..mm4.-.N.mm4. gimmméflm 4.4.44 Nam. 3.434.424 .N.mm.4.4433: .44m..m.mM44oo...444.. .mwmw4.m4m4..m..4 .N. .44m. 42.. 4.4.40.4 . .N.mmmfloo.:§z 32.43.. 34.04944. 4.44.44.45.24 .444... .. .m4m4..m.mo4o4n..§. .44.... .27 ..442 -44.... ... . 4‘} H N m ’3' .N 00 :33 (T. 01 {h 01 {h 03 (T. OH .. . ...: 3304434wU<U343333 _. 340 . 4 <05 .93 4.0334: 44443400403533: 44444833330333.404434333 4U<UUU43 4.. ,. 344.... 04.... 33440 .UUU044443 64034333.... 83.4.4444..- 44330.4..- 09.5.3.4 .. .5330... .433 ...“..30434 3.034.345.3044 43.433 5.40.4330 . 3.. 0.40.3 . 0.4444508044344344 34U34344UU3 4.444344 03330.4“..33444004 3.4334444444333444. ”444344.4464Um3333.4.4U3...<.4.4wu...< .34443u4434u . 4QU3U34U33363U33444<U .44. .0...404.0...4.3....3. 04 UUU4M<GUUH43<3<<<U9.54.444 .3. 08444000033448230.44.44.44 00044400843434.44404.4.0.4442 .0434: _ . 53038450044444 43.03.444.44..53.0330450430044444 04403044344334 £324.40: . .. .. .3 .n .. . . . . .. . . ..

. .. . .. . : . : . .... . . . T .

. .. . : : : .. .. . . . . . . . . . . Ln 4:2 .

.. .. . . . K? 4: . m ... .. <33344<4<<3<T n:0<333444<44344.3..03< 334344444444403 334U<<43 3ww33344w3u44<w431Uu3 3J4U3U3434u44<uw ...4.4 .30 3U 44.443324: 33339444. ...4,4..;4.- 34.34.044.444 4.3. 3U. 944403433 ...34444U3u444/4Um43u3343 .U<4<Um43 u<33m43334<44<<34m3uu 040.444.404.38... 44033344454044.3240 4443.33.50.43; 44345444044444.4404. .43_.4 D.n. UU. , U UUUU33U 4.4..80.430444044044443...43.44 U 394033344<H444u<44a4u3<w<u 43.4..30“.,430<.40<.4u<§4...334 5.4.4.444...4....44mn 444440.403. 33.443.443.404.4.443.434.4040 34444348440304.404.434.840 2.5844440334344008:4.0 4434.10000430 ..mfifiwmfi ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS .8. m 83%. mamémm 331%? mvmv $38322 mummies Nfimmflaa $3.39. Nfiowaflc 22 E2 22 E mafia 333353 3333 amvfimstummdfl sgéfiosbén 3&3 $93.9; 3:) 3335 <0<anum 3333u033U3<3<<<<fi3<33<3 duwzvguwfljamnmnjdxuqmj unDUJ 332 fi V a “£03.40.ny S : .q :aeumfiauaasmfi mOCmVJUMm ~33333<m5£<m Eameumzfiaz .1323E333gfiésm 3%, . gMde‘me 0m: éabmwszmEm £5533 mmbazumfififliamm mtg wmficdx Q Euamflé :35: 5: 33.4.5$§é¢3<fiui§ 55%;“wa mmgmymwwmmxuagfiaasmEfinm E.3£433mufinmwwflduwdnmj Dwmmj‘. Nmmfl. mm mm m5 mm mm «a Em m EU «O «in 3 333% 3 3540 3040 TE 8.333 3<<U 3‘34de 33 33 3<§3 Edda» wjzfiufmt mmcqgafiu <0 33 mm¢:s.q3<;bmbSEEififléuam “5.3335&meeflfiqfiwsbubx: mocqgmrfizegEEBNESEGGE mEEaumEx2333333333¢ 3,6533%?53E 32x33? «and jfijflm<duw<< D<D<w<<uw<< D<flu<wfl<<u fi<vflu<<du 3&433 <w< 1.235.vade 34040 ququ wUCWwSUUm mwjbzuwzuzfi 3.4.528 “Cut 32335 egg3T3.EEKEIEE3 fluegeflfifimfimw: MmCmvm 33¢ <3 qumfisémb 3 50 E ,3 Cfiéfim .Emfimégawgmfim3:33mm? ,mfizbtzmfiefihnfiq E MEMEQS32333qu m3bm33.53%?23523mewan dfiflflwhamfigqgmfi mmm: .wmmmflzd mmomWQ [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS Ln \D .'\ .x. O) «a. 0 O O O O N N N N N N J mmmmu‘ mu? :mmmmmwu:&m:m§ aim: ,. :5 we .m 33 mmwxumthUststwU mmmmusmgfi: 3383qu . ., .n émaama 1.333% i .. v may" w<meUwfi3w3 533$:nnmwiaaésfiam 3533333252 33E33$?ng:mfiamefiwanm sflnmwflwmhwfifihugfiu35333430: :mbmfimmfiafiug.383u33fim2m5a mmkomflumga3303380808“), jmhqfiu 3m; 359$nugflmfijnmiwa334%» weoflafi . «£3333 : 3 ENE: Bum iambflamsflqfiunwDEdem 5333<3..§w:§Jauflafiamfimm Dmszmwowflmvmmsmk033.333 <33§<3<3me$ 3.2 ‘ ‘ ‘ . . ‘ . . _ . . H434, .mmmmfl-< , . .

. . V V V ‘ ‘ ‘ mmtnmhwfi. 9333:nmqmfiqmmdmwaxmwfiuuwjfl_:. . . , mad: , 3: a n5. @3533 weu€303qu?qu”35% r a éfimgefif; Squmw: ‘ 33022303: : $333333? mmdnfinufifljfid‘flm wflfiqufiiwwqfluSumhjfid. . a 843$»? .5333; smjmgzmwwmbfi . 33$ :0 a ,wDfiqugufiflqzflwamhmmmwufiD mmdnmhqéDEnmvuaflwu33<£nmwwflu m”. £52 E<£<fi333mamh< 0343.33:38333(34333a 0 ‘ ‘ ‘ I . . .. .. ‘ mdmmm.

I ‘ , , Hzafimm..mq flmcfimwéd‘ fimjdmé flmfifiMméd‘ flmmammé 32mg HAmomé m m 23mg ym H‘mgmmlm, 42¢:ng figmmmln, [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS mEm: mm..mwfl.flfl:w.n2 Siammzmfl Baum I ....N,mm:8. 80% ..mmR:N.mmD81$_ :mzmsmpfiummmnmwauflam mmUmmmSuflnmmwmnmS $6QO .5; mm:mcm,vmm.Ndnwaagfii nmmhxmmyuéj wmommuhoo .

JJ :msmfiaummfinfiamamfiqflflfijflj :mmEmmEmmENflD :mzflmnflamfldm mpwwmfi mucmsuwm .q “093$ .

Em: . T I wmvsmwmunmemmvfimmmUm u nmw<mn 33m: sanflwen:meménflqflofiua mwmanmm ummmmflfim fit mafifimmfi mmcmmzcd 03w¢3433<<30<<03<<< 304030<3<33<<36<¢ 303333<333<m3u<3<33 thqqJinx. S<<<<<ufl03304303 H an“ . mg ‘ .mNmmHHV. $88 ,3 , .

H ‘ , fiamqbw ‘ may mmdDEBEUmEmuSEagzflwm amJ ,wo muumgwwm 40% ‘ 3.1 mugsgm ‘mmsfiw ww<£<uh$<m<£1 uwfloflosm wm . <wmq<<qnu :noEcn u<m43<<3¢30<u finqqaqajnmnqj gnauuqamqquqquqqqan «moquawauanqqoqun «ququw3:3<uw<<mnu< u3w3<<<<amwgw3u33mq 0%. ammmmmfig 33<U3_;3:<u< fl E wfinmuflw mmpfimmfl mmcmm 64.43 wqwnjanqm was auu<<w2<un wwmn<u J wofimoa ‘ mg «gum H was m.¢mmm:.< , dmmmqu dmmmmfizq mama ammo wwwmwafimnfl ..mq .mqmmgq 339mm .m LG: xmmmaa mefimfiaq Hdmfimfiaq . «Emu.

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS mWJGHVSwfifs, a mm..~mm$.mo3..m. mm...mflm,.mflm...m mm. 2:,Nmi:~.mm\.8e:§a m:Namfimmfi..N.mm:8:§a mWMNNéHV : 3.33mi, , mm:2E,mmfi:m.mm\.8a:§a .

.. , V .nnDae,._>.;, AMNHGGIEa :3 H N Ln \0 5 00 G .4 N m m a H N N (“4 .. N P4 N N Nu m M (‘1‘! m w Q‘ m {‘11 N1 :‘n N1 m m m m N1 {‘11 .. ,... _ _ m m m <34fl®<w3<33333344<3 . ‘ <UD<DZDDDD<<< 363333<33$< .3U<3<33303<< 3:<33G<G3;. 333<<dnudq 3&84 8803333353344 3<m<DDGDDG<u<Du<<UE 833353433. <33033<4<<UT.. ..;.>:3 V‘ 950.434.3533 wnm§<33<,.:._w<8..;.\<< 3323392 . CUDDD<<Uuwo<<UE ‘ ‘ ‘ ‘ ‘ ‘ . ‘ ‘H . . m : . . . V. ‘ .2; .2; : «“ .«“ , , , .. ‘ .. . «. . . “ ‘ .‘ _ _ _ ‘ ‘ u<uzu<<<<3<6<6wwnuu uw<463<wn<<< <333<4<<<<3<03UU3<3 33<3333Uw<uw33364<0 46033<j<u3<w33fi<<w <<<3wh 33<U<<<3<36fi swanjafiwaufiquqa anfinqumqugfi. <033G<2030< agglufiawmnafigifio Dquj<<dm3333<<w<<m D<Uu33fi< Snamqquwiqmw («5:33. 33322385853 <<:<:u< «mm m<<3fi3< <m< 33m00323<fi<flwjuauun :<<D<Du<u3u<<:<<<.

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS mmimvaéwanNImNficcifi; mfiwmma$flm.w, wwwmmmmmfiwg, mmimamémm..m.nn:8:3»a mmiwoxwxvvxvi mmgaafi&mm:mx mfiammvmsh,s, mm:a¢mfimmmfi:mdnsfiaa:Eg mmimmmfisfimfi, Mmfigamfi:wmnwma:§g madamemm:wnnsma:§g mfiNmmwgfistw mmloooaxmmmlu. mmlmmmwém 2 .2 .

, . .. ; V V V . mmNHaQegv _ ‘ . . . . ‘2 . .

. .. V_ _ ‘ ,,_‘ ,_‘ .‘ , .

.A.‘ (3.533" wqw .‘ . 336 DOQUD—Juquq <mo<o<uu2 _. . 33<U<<<<fiu<g jw<u33UwD<<fl U<O<mu:3.fi.\<uw 33<u<<<<<u<w 4 guqmon <wjunnju<m<u<unjuun 504360.4«4. . .3. ‘. «namqwamqmqmaqqanq sojnnmqmamqwamngmn «quwquu:_nq<wuww<<‘ .wqwmw<n<nn< quuwmq uw33U3<3<<<<q3<33<3 awaamnau<3ww<<< au<w<u<unauun ”3.393 ‘ ‘ <33u<03w<:<33 m032un<n<<<<<2<23<n (mmjunnju<w<uflunnuun ‘ <336403®<3fi33<fi33<< <<umow<< <3<33< GDDGEDuxwawflqa.‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ . , . , . : .N , . . . .y ,. up . , .

.:= I f‘ h“ .. . ‘; ‘z . , , , , ‘ ‘ . , .. :. . ‘ . .‘ ‘“ .h ‘. .. ‘ ‘ ‘ _ _ _ _ _ ‘ ‘ ‘ ‘ ‘awdafiqduqdafim <60<<0303030<<<0<UD <4.~wj<0333<vw<<msu< unwaqqqqamogwn : ”uquaqqmqjuqumuqqn .<:<:u<unu<qnq<<<u< :nauu<3w<<u<<u<<<3n <ww<<oaoauao<<<wqua <4.~wj<v333<ww<<msu< u3w3<<<<3393w3 g<m33u<anq )93.,3<fi3<3u<uDmdfln <<3<3u<unu<<n<<<<u< anuu¢032<<<ow. aqqnqnuqu2m<qaqqq<m <3<<3<33333<3<o<<um ,:w<<unoa V‘ gaqq :330w<3®<<u<<u<<<33 iuwflddbmujuawfiflflwflun V maiwm “‘ \. gmfimma dammwé ‘oa§%;z ‘Nwmmmxn, .mvmmmym «mmmwm, .cwmmmé ,mmmmmxn, [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS mm. mm .mmmfizfiflmfii Hmmm.mmmm magma; mm Nmmémx...” mm..83;mmm.w 3.3mv,mR¢..N.mm\.Sa:Ea 3?QO v . ammo . mama? n .. .. ..

N m Ln LO [x :x. N . H ,.. .. .

(Ti m {‘11 N1 hwmhijw< hanging Egmmazas . 3%:303353533 Du¢<m<u<w<<3333<uflw 63w<3<33<<30<<uz<<< j<3<33<<3mu<d Snag/«33$? (wwdwduuan. H quuamaqfiumwfimn <§33w33w33§9m<§ 3G<UDDijqd<ujunDD nufl<fi<u<u<<3333 mmgmsawm <33? . . mmcmmucd w<flu3<fl< qsqiéqzwqq aquamqi, 3 brawn333<<<<<u<03304363 ‘ u<0 3.2%% , , ‘ ‘ ‘ a. . #3qu . fimmw. mg 02%: HM . . , .. .. . ,.,. , .w\.mm._u._u.< < < < .. ,. . . .3 ‘ mmumgwnwm n: dam1 ‘ . : .

H m H m m . ..

‘ ‘ ‘ H u.

.. ‘ . Djuq33<<adau<usu<<m afluuww33<<<ww3. figbm 3d<<<3303w3033w< 33w<33<<3<3u<u <<3<3UflUDUq<3<<<<ud .. . <3<<j<33333 fiuqfiqig mmjfldflfijbwjm . .. ammumwfimfi JUDDOQ 35:33. 5 . .

‘ “Ea ‘ wmcmm fiqfifiafiafis :Suwwsuiéméfig $3433 $3.36::uuwmnndflxmwjuau hamha<a<<u<s:3:3<:fl0<<w® hm<u<aw<<u3wmazzswudd‘ . mmamw 33m: . ,ccmhm .. . ‘ ‘ mmmwwweg 32mm. (.mmwmfie.< H.mmqm,:..< . . :mmwmfifi , .fimNMmé .mmmmmé é umsmrw .a< .mflmmdq 5&9? .mm,flmm..mq ..mmfimm.m< [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS vaknxfius FER—.030 km NW tag v.9 Panhan— .%m%.muw<mu3ju<m3w3<fi<ujfi 3 3m“; .63 majwwjdxuquafiwj Dd<<4u3u Dudnwwmxdd 0/4 D Jquafluflnwwqfluflmjj wvmhnu.DQ<003<9§G<<3<332 4569.33.4333..53<<<: _.um.%u33Ufi<4u03u04<<<nfi< i ,. , ,. $63 ‘4 (0. , : >4 rfl ham_.u<dmfi<<:wnnuu<<mnfiwn by wwuu<usu<<d<nfiw<mafia”. ram,—U<<<3<33005<3<um5Q raflnqnnnwifiaiwzgwsuon $2 . mumuno J ,_§.§ao snuwdumnnnw‘ :«ku/u/fiw :(UD fifixxw m”. 4:333:33? ragga35433<<<<§_._<w_._8 FEEKNEoifiafiawifiao Z£3sqéiéiwqqfiénn «.6 fififiqgéna a:aquamvsqnfiaifiugwm r53253:wagéimnq ma Saba/E3iqaméfiémwa5 mQWSuq [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS ation] DPS Unmarked set by DPS U33 rum U.3...33U33.. ..3U.3U ...3U3UU: 9393334 ..3U.3<<333U. #3.“.34‘U3U333U4U4U4033UU3 U.3U.33 4.U3U4.~.3<U.33<<U3U4.< ”3333.3 ~3...U<UU...3U .U,3m...3U3U<3<:...<.4.3U<<u3<<.4... #3334... 3:33U J3. U<U . 3U3<3I4.4.I<34.33<3 U<4.4.<<UU.U33U.<3 U <33U4.U 30<Us®<3<3 3<<<U 3<U3UM.4..<UM<. .3 333 3 UU<<U3 U3 .U33U43UU<<4. UNU.4...U.w...4....U3....4.U 3<<3®<< .33:<:..U.3.UU.U_..<1.3 Memmii 5% .VIQ. $.34 U31. 33. 34.53 U.3m.U,3U3U3U3. . U.3/~3<<UU .53. ..4.U ..UMUU. 83.4.3.3: ..)...U U3<<34.3U4.U. 3_U .33 3U .—3.—3<UU<4U3U3U <<UUU3UU3U3U U4..UMUU3UU3.UUU3 .3UU 3.3 34.<3<3U<U <<3<<<<Ud 3U<<<U<U 3U 3.393.434. UUUUMU3U</.\UU.U.~..4.UUU4:4.U<U :42; 3 .ququU.

.J. 3 .3. 3U3U3<UuU<<UU3Uud UU3.UUU3U3.U< 4.33.4...U33Uuzzqqsduuq. £5.43...2.3.4:...4U<4U.4.....3.4U..U....:4U<: .Uu.4......4U H.mmfiMm.Q< WNUNMmfi .mmNMm. .wmmmmfi wmfimmym .Qmmmm-m [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS Pvmhndww<w<mu .w JmhvmnnfimQ<1JO<waw<3<33< flumqwfiunnsqfiuomfio: :<u<<q<fi<m§u<:o: 5353843343 $£3m83355332.83 :3.Emfinfiwsifinfiwé qufififlén .Ehamnmqaqni ._.u.m.%m2‘_m<mnw<$n3445 :4th2m::_5<330.<0:w<:<32 mmu Sjfiqqqqqsqsaqn 33,) 3<<uuwwd< 3qu 35:43» s<<<UD<Umus q w<03w<3<33< : ummmeHLQ ,moomflfl.< AK? ,m “.mhm‘ Hm- womfiflkx. 4h. 351% :ofl unfit Uqunnuumwaéqqamnunu kudUQJOrnqunmufinnnnmufiqd gumbo magma? Efluqfiwi .MEfinogqfiqs5532qu .Fumrgsiw .ZumHUw—Jud aauuqsmqfiqfiqién w Fugifiosqow mccammanm “546.3qu 3453534 u<§<§<8fi Qfizxufiuquwguqfifi PUWPGQH‘S<3u<unu<<3<<<<u< PUMFDGSUUGUSDQQJNUMVDUSUUS .FE.§<<:<BEB§:<<<§ $3uqunnuumwsfiudqwmnunu $§<§§§2§3m<fiw 3433.534 FEE:agiéqszwnofls mam HE Erica , Em Ur..nuvmfifi 082 , SEC .N\.NMm.a .NMNMmé .m\.NMm.a ‘mmNMmzm .mvmmmym .mmNMmé .cmmmmé .memmxm, .mvmmmym .Nmmmmxm, 33m [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS Tame 7 C5 o 59. {19 a HepSB celis ‘4 smg SS §CFQ€B m ‘Wl”:h (IalNAC eunjugated iRNAs J\J a.m eX I1) atL5 8 11 0“U 1 atL5 8 11 0 .1 1 AIL3.5 on .J 0 7 1 4 on 4 S 1. 0 1x, o.6 7 7,.i mm, 5 8 .J 2 0“ 1 7 65 1 O57 r05 8 3 56 m_5 8 1 AmI 1 8 A1 5 l 5 3 9 7“ 0. oo [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [AD—58132“: ‘r91.1 Ail-58103.1 Mock Untreated "fame S _ C5 single dame transfecfitm screen in primary mmsse hepatecytes with GaENAC wnjugated {RNAS .................fi Duplex ID 1011M .................4 ADI-5 8093. ......... -58099. i 1.65 ... ................. ........ ........ ........ ----1r AD—SSOSS. 0.84 ........

---- Aim—58094, 1134 ........ ----- ........ ---- ........ ----1r---- AD—smss. 1.74 .........+........ ---- 14!)»581164 i 1,76 ........

---- ......... ---- ........ Umrcaxed E 1 GO ......... 100 [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS Tame 9° (35 Single. {1959 §cmen in primary {yiwmolgm ytes with GalNAC canjugated ERNAS 500mm SHM SOQHM AVG 511M AVG STDEV STDEV AD—SXWB. 9“ 13.65 AD» 580999 9 21.95 AID—581059 1 1.48 ALL-38117. ,, 11,0; 97 1'3 .06 7777 [.114 97 90962 21‘ AID—580889 980949 Ali-581069 40.65 ’7:- ‘g 3 AD» 581189 AD—581309 ________.|.

AD—58089. ________ Ali—58095. _________ AD»581019 ________ AD—SSl 13_ ________.|.________ AD~581 J"). ________ Ali-581259 ________ ________ AID—581089 9413—5981149 ALE—58120.

Ali—58126. 105.90 2.39 7.96 [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS txD- 58-391. 1 1\D—58103.1 Tame m u (35 Singie {1959 free uptake screen in primary mama hepamcyiw with GaENAC umjugamd ERNAS 50019.4 A VG 31.62 AD» 58100.

ALE—58133.

AD—SX‘: if).

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS AD95809’819? (5.218 3.487 ADP—58113396) 3.540 9999999999- 99.99 999999999 AD-58108.i 0.95.3 10.080 AD~581201 36,170 8817K) AD »5 8099 3 .777 AID-58111 0.622 ALE—58133 0,549 .................AL........

Ali—58088 if)581 21 ........AL........

........ ........ AID—58644. ........

AD—5865 1. .........

AD—SS64L ........

AD—58648. ........ ........ ................. 549 ........ 645. ........

AID-58652.

ALE—58643, ........AL........ 50. ........

........ AID—58653. ........4.

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS Tame 13 C5 Single. {1039 “men in Hepififi celis with madified and unmedified ERNAS 111M 0.011134 QQEHM AVG ................. S'I‘DEV STDEV 100.58 ........+........ 3 47 2 9‘» 76.24 ........ 102.30 .. ........ ........+........ ........+................. ........ ........ ........ 9.41 56.7" E J 9} 1 [’5 ADSfim 68.91 1 m 0.34 1.94 2.03 1 71 4.77 1 05 0,00 J 63 0.59 1 88 2.04 4 72 3.52 oEIIIIIIIIIIIIIIIIIIIIIIII IIIIIIIIIIIIIIIIIIIIIIIIIIIIE..L A1108 98.86 0 70 4 I0 43.83 2 G6 2 A: AD—58170. 39.59 E a) 96 J 36 _______________________1..3}........... 0.90 0 SS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ......

........ ......... ................. ........ ........ ......... ................. ......... ......... ........ ........ ........ AD—58193. .........+........ 0.00 ALE—58199. ........ 1640 7.05. ........ 0.77 ........ 0.48 .........+........

........ ......... ........ ........ ........+........ ......... ........ ........ ......... ................. [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS 1.96 5.79 ALE—58243, 86.05 1.11 2,95 Ara—58249. 05,4.) 15‘ J 4 0-95 AD» 51255. ___________+____________gQQ___________j Ian—58279. 5.52 0.4 '3 28 4.91 24 0,45 7 19 0.00 91) 193 _____ 1 27 1.63 .90 1 12 .......................a1.119.___________ ........ E 7.44 2 3: ........ ........ 38' I") 0.65 ' A 72 , , . 5': .........+........ 14 36 AD—1955 72.30 82.77, ........ 19.54 49.99 Untreated 100,00 100.00 ........ .7} 68 ”.6 IX Table 14 — CS singie dOSQ screen in y mange yms with d am} mmwdified ERNAS Duplex ID 1111“ STDEV 0.11134 STDEV AD—58143. ’ .. 1 .. . ........AL........ 18) 7311. 3.11 ........

Ali-58155 ........ ......... ........ ........4........

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [mmm5..J J 50 J J , ..

J, 00./J0+.I J J (0“ 4 Lmu0 c 1 _ J _ oo 1 56 J A1x, 5J 8 J .b2 J r\JL1.. .3 on 1.. 0Apr .

AJ c _ J 08 , _ 002 J 3 1 7A... 3 9 00 6O 27 4;.“ 5 A1x, 5 S2 M.J . J AJa. 3 on1 >\.pr AL1. :3 00 11 9 J. 1 r . , 0J DJ9 50 .3 48 3. 5 .fi J _..... J ...u....u....muB_ 7;u J .u.u..m.uu.n9m3“0“Q;.m.IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII .mu.u.u.uuuum51m9;1.u J .u.u...m.n“«3.2mJ“..1 AIa. 5 (,8.J 7..N”H . H J .J 1 . J J A1)sJJ.QJ an.“% J m“J.) 4 80..., J 3 A“%., mlI J .0 AE,J .. J awe2 H . HJ O J J J. (0..Wu .J. \H r ) . 1.. 54 aNJJ)” A. J J“w.inS. n 2“Em J“u..... ....u....u....mum. uu ..u.u..m.uu.umnm.m.IIIIIIII.mu.u.u.uuuummm_n ......u.u.u...m.n7:.9“u. 9 G fl A1J)A. y .

J “X”. J 7..M,n IIIIIIII u“ V.1 m;a. 90J : J w». J M 2 01\J. (no;\I 1L mm J 5. J .00+ A13sJ S.m/A.% J Aia. . a on2w. um .

JwsS2% .

A13s. ., .

.Sa/ .mm J 7,H "M, . ”M ........................................ 0“ M», J J J Q5.

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS MW 1% _________________________ .22... 90.61 3.42 :33 330 4 3o 3 an ........ ........+........ .........n. 68.67 112108 ........ * ........

.P:7 '»/I CZ} I\) U] _A JA:\1 :: ,_d ,1 L2) LI) i\) :2 ....... 4n 3‘» xv \J r4 p ........ xV’.nr U CC?\ k)- a w 37 rzx ()0 50 “\1 U — 4 . so N u; i\> CO 4.\ ........ .........4......... ........ . . 1 ..+........ 1 .........n......... ....... . no. \x no \9 '17 u .43 <7 m ........

AD—SSZSZ. P“ C>\u uC‘ .a C: .a N Ll) 12.02 ........

.................. ........ ALE—1955 4.08 Untmamd 10000 10000 ....... 1525 1837 Table 15 4C5“ data in Hep3B cells with madified and unmadified iiWAS Duplex ID 143.1 AD»SS149, 1 AD~58161J AD—SSBG.‘ 151358242X: [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS Table Hi ”le4; data in primary mouse liepateeytes with modified and unmodified iRNAs l 391a}? .........

AD~5826CL 11015 E 019670 E AD—SSMQ. E 11309 1.749 t Airssista 1.991 11477 E l i , l 1.8 i + +_____________2£191.11“,____________Emelt/“.2... 01887 (3-1613 3 11+) 073m L144 19.88 3.224 5475 Example 3. in viva screening A subset of seven GalNAC coniugated iRNAs was selected for further in Vii/‘0 evaluation C57BL/6 mice (N13 per group) were injected subcutaneously with lilnig/lrg of GalNAc conjugated duplexes or an equal volume of hi Bulbecco’s Phosphate—Buffered Saline (DEBS) (Life Technologies, Catti l4040l33}. Forty-eight hours later, mice were euthanized and the livers were ted and flash frozen in liquid nitrogen. Livers were ground in a 2000 Geno/Grinder (SPEX Saniplel’rep, lvletuchenr NJ"). Approximately l0nig of liver powder per sample was used for RNA isolation. Samples t ’ere lirst homogenized in a ue Lyserll n lnc, Valencia, CA) and then RNA was extracted using a RNeasy 96 Universal Tissue Kit (Qiagen inc, , Catll7488l) ing i’nanutacturerls protocol using vacuun‘i/spin technology.

RNA concentration was measured by a. op 8000 {'l‘liernio ific, Wilmington DE) and was adjusted to l00ng/ul. eDNA and R'l‘—l’CR t ’ere performed as described above.

The results ot‘the single dose screen are ed in Figure 2. Table l7 shows the results of an in viva single dose scrfen with the indicated GalNAC conjugated modified iRNAs Data are expressed as percent ofrnRNA remaining relative to DPBS d mice. Tilt: “Experiments” column lists the number ot‘experiinents from which the average was calculated. The standard deviation is ated from all mice in a group across all experiments analyzed. ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS Table 17 min viva C5 single dose ecreen Dunlex 1D r------*-----------------«r 1- 53580332 2 [AD-5864421 i 6511 i 1 ADSSOQQQ 2 ----. . ----1r ---- ----w----- ---- ---- ---- ----. ----- ---- ----v----1r---- .

........ ----- AD—58l33 '7. ........ 51.98 13445 ---- AD»5864 6.1 ........ 28267 ---- 43.02 ----1r .........+........ 1 00.00 Two ofthe most efficacious GalNAC conjugated iRNAs were Ember modified to include additional phosphorothioate linkages (Table l8) and the efficacy of these duplexes was determined in viva as described above The results ofthe single dose screen are depicted in Figure 3 and demonstrate that the RNA agents with additional phosphorothiate linkages are we re efficacious than those iRNA agents without or with fewer phosphorothioate linkagesg [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS E5 «my? «3% % Sam mmacw m MESH “3&5 “3&5 mmm.» mDfiDflGED? mmwiflmotw‘ @285 mwmégw:5.31$;E»<5»NEEEEE wDfiDfixfi? 3 w Enamflafl {Emafiflm mmomumapfiuaasrEwaufiwcfiwafimm fiOEDfldfiHOEUm msmmggau 3mENE:3aunfiéumfi<fi<.§émafia m, 001$)» m. flfiflvfiQEmeflflflflflfiflu mflD (Edwin, 3.2me M EomDEDflxa fimmfia 353 agmfifigimém um «www.mn‘fiflflb ummem wwr $me wmfiu wmafim Mimozé M .m M6 M w m m 353m w M mm T< T< 33$ M_m$mm,m< [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS Given the impact ofthe onal phospliorothioate linkages on the silencing ability of the iRNA agents described above, the efficacy of additional GalNAC conjugated iRNA duplexes including pliosphoriotliioate linkages (Table l9} was determined in vivo as described above.

The results of this single dose screen are depicted in Figure 4.

(I: The duration of silencing of ADn5 86-42 in viva v 'as determined by administering a single 2.5 mg/kg, ll) nig/kg, or 25 mg/kg dose to rats and determining the amount of CS protein {Figure SB) t on day 7 and the activity ot‘CS protein (Fi gure 5A) present on days 4 and 7'. As demonstrated in Figure 5, there is a 509/6 reduction in the activity of {35 protein by Day 4 at a 25 nig/lrg dose and at Day 7, a greater than 70% reduction in the actiyity of C5 protein ll) The amount ofC5 protein was determined by V‘v’estern blot is e serum. The activity ol‘CS protein was determined by a hernolysis assay. Briefly, a fixed dilution ot‘lnirnan C5 ed human serum \ 'as mixed with mouse serum and incubated with antibodyvcoated sheep red blood cells for l hour. The hemoglobin ance was measured and the ‘34) liemolysis as compared to a re erence curve red using a dilution series of mouse serum) l5 was calculated.

The efficacy ol’AD—S 8642 in viva was also assayed in mice following a single subcutaneous injection of l .25 rug/kg, 2.5 , 5 mg/kg, ll) rug/kg, and 25 mg/lig ofAD— 58642. At day 5 C5 mRNA was assayed in liver samples using qPCR, C5 activity was assayed for liemolysis, and the amount of {.75 n was determined by Western blot analysis ot‘whole 2t) serurn.

As depicte in Figures 6A and 63, although there is only a minor iinproyement (126., about 5%) in efficacy of ADn5 86-42 to inhibit C5 niRNA at a dose of25 nig/lrg as compared to a. nig/lrg dose, there is an average of 85% silencing with a '25 rug/leg dose, ln addition, there is a dose response eltect with an K359 ot‘ahout 2.5 rug/leg.

Figures 7A and 7B and 8 demonstrate that AD~58642 is ious for sing the amount of C5 protein (Figure 8) and C5 protein activity (Figures 7A and 7B).

The duration of ing of AD-S 8641 in arrive was also determined by subcutaneously administering a single 0.625 g, l.25 trig/kg, 2.5 mg/kg, 5.0 mg/kg, or ll) rug/leg dose of AD~ 58641 to C57 l/6 (n=3) mice and determining the amount oi’CS protein present in these animals (A) (:3 on days 5 and 9 "cy ELlSA, Briefly, serum was collee ed on day 0, prewbleed, day 5, and day 9 and the levels of CS proteins were quantified by ELlSA. C5 protein levels were ized to the day ll pre~hleed level. As depicted in Figure 9, the results demonstrate that there is a dose dependent potent and durable knockdown ot‘CS serum protein. (The single dose Elks was 0.6 nig/lrg).

Compound AD—S 86554 was also tested for efficacy in C57Bl/o mice using a multi—dosing administration protocol. Mice were aneously administered compound All-586M at a [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS 00625 nig/lig, li25 nig/ltg, or 2.5 nig/lig dose at days 0, l, 2, and, 3. Serum was ted at days 0 and 8 as illustrated in Figure l0 and analyzed for {75 protein levels by ELlSA, CS levels were ized to the day 0 eed level. Figure 10 shows that rnultiwdosing ot‘AD—Stieéll es silencing of CS protein at all of the does tested, with a r than 93% silencing of CS (I: protein at a dose of 2.5 mg/kg.

Compound All—5 864i was flirtlier tested for efficacy and to evaluate the cumulative effect oftne compound in rats using a repeat administration protocol. Wild~type Sprague Dawley rats were subcutaneously it’ljeillffitl with compound AD~S 8644 at a 205 nig/kg/dose or 5.0 nig/ltg/dose twice a week for 3 weeks (qlw x3). Serum was collected on days 0, 4,, 73 11., 14, iii, it) 25, and 32. Seiuin neniolytic activity was quantified using a lieinolysis assay in which a lzlSO dilution ot‘rat serum was incubated with sensitized sheep rat blood cells in (Wlfieleet buffer for 1 hour and hemoglobin e was quantified by ing absorbanee at 4i 5 nm (see Figure 11A} The amount ofC5 protein present in the samples was also determined by ELlSA (Figure 118’). ’l‘ he results demonstrate a dose dependent potent and durable decrease in liernolytic activity. achievino about 90% liernolvtie activity inhibition. .1 , It) a a [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS figmufiflm E1 mm. w: mmvymmmfl. 5 ad: QEEES Augmfimm msfim mug/MET <Aa€m§i magmas: QEEES Emmzmagafimm Augmfimm EMEJZ EMEE E Esau» 1: Manda tam maflwmam £333:93:33:: ummsmmmf .3 (imam .53 EEfiDwmm $me mm 033%:me 3 qw:fisméma‘gwg.“ .1mQ34l33nwm4wan“ EEMam 00:05“me mfic 93m? gammafié a: an :2 mmflvmmwfldx 35%:vang “5&5. ,mDEmUfi m_:m4:mdq 43443 «$53?meng m; F35 Sign” ummmmflmd Egg? om wan: owxmmDsm vm‘mm wwum SEESEEEEQ Q 4me 1., .rywfiufi : £3 tm 840m hstmmxfi, m<mm 40m :m4mfivm 3,43 Vménfimfixm/Bm/ummd‘ .. 4434.: Dum<m<mm %, :2 144.? mrm 1mm Manama? mummagm imd‘.“ (am m om< ‘ “Emm. wmh s mmfihww “WES uwnnum Manama [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS EEfiEEv a: i $2535 523 I H eSMMmfixf‘, :Sémafié fiimflmur a? an; HuwDfiQa «35,5? Eaé qdwfi waifl<fi “Eggs Usfl mogamfifiDagw .E.Efisfififigm [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS Example 4: flesign, Synthesis? and in Wire Screening of Additional sillNAs SITRNA design (35 duplexesj l9 nucleotides long for both the sense and antisense strand, were designed using the human C5 inRNA sequence set forth in GenBank Accession No NM___GOl 735.2. Five~ hundred and nine duplexes were initially identified that did not contain repeats longer than 7 nucleotides, spanning substantially the entire 5480 nucleotide transcript. All 569 duplexes are then scored for predicted cy according to a linear model that evaluates the nucleotide pair at each duplex position, and the dose and cell line to be used for screening. The duplexes are also matched against all transcripts in the human RelSed collection using a custom brute force lll algorithm, and scored for lowest ers ot‘inisrnatches (per strand) to transcripts other than Ci Duplexes to be synthesized and screened are then ed ‘t the 569, according to the following SCl’lfil’nC; Beginning at the 5’ end of the transcript, a duplex is selected within a “window” of every l ll :1: 2 nucleotides that l) had the highest predicted ellicacy, l5 2’) had at least one mismatch in both s to all ripts other than SERl’lNCl 3) had not already been synthesized and screened as part of other duplex sets. l t‘no duplex is identified within a given window that ed all criteria, that window was skipped.

A detailed list ofthe 569 CS sense and antisense strand sequences is shown in Table 20.

The in vitro efficacy ot‘duplexes comprising the sense and antisense sequences listed in Table 20 is determined using the following methods.

Cell culture and zmnsfhcz‘ions ll’epGZ’. cells (Arl‘CC, Manassas, VA) are grown to near confluence at 370C, in an atmosphere ol‘5% CO2 in Eagle's Minintuin Essential Medium ) supplemented with ltl% FEES, omycin, and glutainine C) before being released from the plate by nizationi 'l‘ransfiéction is carried out by adding l4 .Stll of OptinMElVl plus 02nd of Lipofectaniine RNAiMax per well (lnvitrogen, Carlsbad CA. cat #— l37’78-l 50) to Sul of each of the l64 siRNA duplexes to an individual well in a 96—well plate‘ The mixture is then incubated at room ature for l5 minutes. hfiul ol‘cornplete growth media without antibiotic containing ~25 le4 Elepr/I cells is then added to the siRNA mixture. Cells are incubated for 24 hours [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS prior to RNA purification. Experiments are performed at ZGnM and included naive cells and cells trarisleeted with , a liieiteifase targeting siRNA as negative controls Eater! RNA inflation using DlWABEADS mRNA ‘z'mz Kit (Invitmgen, part #. 6id~12j (J! Cells are ted and lysed in 150w ofLysis/Binding Buffer then mixed for 5 minute at 700 rpm on a platform shaker (the mixing speed was the same throughout the process) Ten mi eroliters ofmagrietie heads and SOul Lysis.«’Binding Butter“ n'iixture are added to a round bottom plate and mixed for l minute. Magnetic heads are captured using magnetic stand and the supernatant is removed Without disturbing the heads. After removing atant, the lysed cells are added to the remaining heads and mixed for 5 minutes. After removing supernatant, magnetic beads are washed 2, times with 150m Wash Buffer A and mixed for 1 minute. Beads are captured again and supernatant retrieved. Beads are then washed with lStlul Wash Buffer l3, captured and supernatant is removed. Beads are next washed with £50m Elution Buffer, captured and supernatant removed. Beads are allowed to dry for 2 minutes. After drying SOul affiliation Butler is added and mixed for 5 minutes at 70°C. Beads are captured on magnet, for 5 s Forty ul of atant, containg the isolated RNA is removed and added to another 96 well plate.

GEN-4 synthesis using AB? High capacity CDNA l"€i‘/€FS€ transcription kit (Applied Biasystems, Foster City, CA, Cat #1368813} A master rnix offiZul lth limiter, (liélul 25X dNTl’s, IZul Random primers, lul Reverse Transcriptasey lul RNase inhibitor and 3,2ul ofHZO per on is added into ldul total RNA, cDNA is generated using a Bio~Rad C—ltltltl or S-l GOG l eyeler ll’lercules, CA) through the tellowing steps: 25%? l0 min, 37%;? 120 min, 85°C 5 sec, 4°C hold.

Real time PCR Two» ‘ul of CDNA is added to a master mix containing 0.5 ul human GAPBH Taql‘vl an Probe (Applied Biosystems Cat #4326317E), 0.5ul human SERPlNCl 'l'aqMan prohe (Applied Biosystems eat # lilsill)892758_ml) and Sal Lighteycler 480 prehe master mix (Reehe Cat 3t) #0488730l GGl ) per well in a ll plate (Roche cat # O48873llltltll). Real time PCR is med in an [13483 Real Time PCR machine (Rachel [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS TO caieulate relative feid change, reai time data is analyzed using the [3,53th method and normalized ’EO assays performed with cefls transfected with 2011M ADvE 955.

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS Q3 ........... ........... 00m .m.

H02 on: 3.: ...

HHQ4Q3DH33H33433--§H44am. mm: 4:3 mm: mu: an: cm: Hm: mm: E: mm: 30w: H ...

.H.H4 0044.400 000004444003.H.3334.H304H< 0033< H.3<44..3<4.4.<4.4HHD.4.H0<4.0H0 04.4.00 031R 0334.. .00HD 0004. HH< H3044..4H0HHH4.4H4:HH<33H.HH .H 0003030030093:03454. DH,.HP..30H.H<H.3HHH0405444030 0344.404 .0030H0H4.2533433. H344 30:.” “Home: 03309 34x Em .3H .Hmfim..HHs< 00<4s400<00<H>H 330040093 0003333300400 Ha H...

.H .0 00H,.HUH.33HHHH.H<0H.3H.HH>HH3434.40 03H034HHHH03H3H. 0333044....31034H30 HH..30H0:<4.<0 m .H. 3.053335... 33 UWw H02 H.330 New 40¢ m8 com new wow wow 03¢ HG NE HHHHHHHHHHHHHHHSHHHHZHHH: HHS m3 43 3.3 . . 3E3; 4400:. P38QQQ34334400333. H.304H .H4H.HH>HH.3.. .0033 0H033044<4H0H <0 .3 H0 :0 3<3 H34. 434.<<4.4.. ..................H.H3.H.H.H...H.....HHHmeHH...HH..H.H.H.wwW H3H.H34HH H3<< .044 H33 0030 33H. 0.0 HHHHFHWHHHE .5... 0:3 03.4.4.3 .H3H.3<. H4504 3H.HH0H0H 00433003309 H.H<<00033H.HH 33303..) H4 334.004.

HQ): 04000033044 H04:HH<00.HH00404.00. H403?) 3..H<. 4 3.9.3 34.34.4.<<4.H .H<4H<<H.330H> 3:2“ 3030:3343 .3H..3<4.

H3 0H macaw H_00H.33.HH .30 3000.00 H.34<H. 5H 30030.).5H .44.40 3H0<033HH< 34 03.34.0433.

H.033. 3 00 .30 .30 04.00 2.00400 H4H<4<0 34.0 00 . . 35333320:ng .H 3:03.39 Lo: 3 3273 344333444. ”am 0.8.sz $33th @5330 ii: [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS ........... ........... !\ ww: a3 4 .4..

GO m4: :1 4 .40: 444:. N44: .4. .4; 44:. 24: m4... $.44... 4.44.4.4. 3N4. N444... .4- .444 . .......... ... . .........+.........+......... ... .......... wwwflVme <QQDD©4QD<MV<41 33.44441 .44. 4.4 0.4.4.34. <44 .U _.V4\)».V<.<_). 444.443.45.245: 4.44.4.4.:“44.444444544444444 .......M.4..“.4..4.w.....m.4.”www44.4w4u..w..m..4nm.4.w44.fim.. .454444.44.44.44444434.4.4..4.44.4.44 444444.3445.4.4.4449444.444.44.424 n44.445.45.4553 544>.44.4.m....4.m...4.4..w...4...n4. 9.544.. 4.44.54 4.4.44).44.4.4Q4.4.34.4.4..4444<.4 44444<44444444444<444+ .4->:.4§>44:JP.4.44:4+ <<$334Q3< QVDPQDD H.444. .UMFKUDK.. .0334)». 44.3.4449?)4 .4434. 4:14 “.454... 4.4.4.4.... 40.444448444444944... .HVWVQQngQ 4.344%DLQDDQA. 4443.4. 44.44.4444444.44.4.444.4..4 WVD44x4J4444444034).» $34.54Q34VD4Q U433<UUQVDD<DDm _ _- . _ {M4 44.44:?) 3.4..) . 4.5.4.4 444.4 ....T DS. 44:4. :6 4.4.44.4. 44:3...“ b.4444. -44. 5444.4. 34.4.5.) ..4.QU_) 444.444..) 4.4.44.4...s4.<<3<m43 4444.34.44. 4.45.4.9. Q<<M44V4 Q4444. 4.............L4<4 43.4.4 424 V 4.439.444... .3444QUDQD4444. .0942.) <QJ<<U 4.43.5...

Rm." 4444444445.... .4.._m.4.m44:.4.a:494:m U33 4424. 4.443.444: .4544443S44444w4“44.44444 4.4444045443345443: 4.4.“4434.4244.444444444440444 444.454334.444.45.434 44.44.443.444344441445.4 ..4...4.4.4.4.4...44.444344.4....4.......4.w4.m....... 44.44.444.44444444644444444.4444 44444444444444 S 4:44. <:.G§<<< .4 44:42-55 V -N: 9.9sz 444.444 84.944 . 3..

N4... ...44.4...4....mmN 4444-44.44 9.4-44 N.44N .44....4444 44....

[Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS . .......... ......... C ...C.....+.........+......... ......... ..........

“COG: mCMCCCCC CowC £9: MCDCCCCH DEC)? DR CCvCC C. .1» CCCCCCCCCC CUQCCDCCCQC CCCCCCQCCWCCHCCQDCCCCMCDCC CCC:CCCCC CDCCCC mCCCCCHCCCCCCCD MCCCCCCCCCCCCC BCDGC CCCCCCCCCCUMC (4 :CDCVMCCCCCCCCMCD.” “CDC CC..CCDC.UC.CCCCCCC ,C CC nCCCCCCCWCCCCCCUC D Dru/CU DDCCCDHCDDQCCCCDCC 32 E :7 CtC <C .CCCDC v CA C OCCCCCCCCCCCCVWCCC U Q D CC.

C)C_r <7: C E CCfiC CCCDCCC CCUDDCCDDDCCCCCCQCC CCmCCC/CUCC.CQDCC.CC.CUUCCCC CDmCCCCCCHCCCCCCCCCCCCCCCDCCC CCCCUMCCCCCCCCCCC CCCC..CC.CCC..CCCCC.CCDDCQC.CCC CACCCCDCCC DUDUCJCCCCCC.3CCCCCCCDCCCCC.>CCC.CCC«CCU mCCCCCDDDOCUCDDCMCGCCVDCCCCCCD CDCMCQCCCCGCCCCCCCUDCCCC ,CCCCCCGCCCDDCCCCQCCCMCCDCM C.) DCCC CsCCCvC. CCCCCCCCCCCD WCCCCC UCCC..CCD.CCCC.CCWCCCU C r:‘ C C)” UUCCCCCCCCDCCCCQCCCDDCCCCC DDDCCCCMMCCDCCCCCCCCCDCCCCCCCCCCCCU CCCCH C .CCCL DCCCCCUCCm DU?) DCC C. CDC CCD CCCC CC.CUUCCCCCUCCCC CCCCC: J. C,CCCC..C.CC.C.CCC CC); .CCCCCDCCCmCr :CCCC DCCCCCC CC...C.C3C DCCWCCCCCVCCCCCCCDCGCCCCCCCDCH CCGCCCCC CCC «CCU: CCCCCG .DCC DUDCC UCCCvCC CCDCCCCCC CCUC CC .CCCC C, CCCCCC CCDCDCCCCC MCmCDCC GCCCC DCCU: DDCCCCC. CDC DCCCCDCUUCCCCCCCCCCCCCC:CCMC .CCCCCUDCCC DCCUDDD C.CCDC.UCCCCCCC UCCCCCCCCDCC :CCCCCUUCCCCCCUCCGCCC >C>CCC CCCSDJCCHCCC CC).

CU C .3 mCflCCCmCCCCCCC CCCCCCC CCCCDCCCCCCWCCCCCCCCCCC G39. CmCCC.CC> C0,... CCC.CCC Q CCCCC CCCCCDCCDU.» CC. CCCC CC) CCCCCC v CDCC DDCCCUJD .CCCUCCCCCCU CC DCCC CCCCCCCC CDCCCC CCCCC mCDCCCCCCm CCUCCCV O CCCCCCCCCCCQQCCC C C.C:UCCCC( CDC.

CCCCCCCCCC U C MCCCC CDUDGCCCLCCCCC mCCCCCCCCDCCCCCwCCCCCCCv CC HCCCMCD .CCCCCCCCCUCCCCw DDCCCCCUG MCDCCCWCCCDCCWCDQDDDDCJCMCDCMC oNC; CC @31me Cmm-mCCm, mm: wmelCCwa m ClCoC -me. mCCmACCmCC. m NxCC N. mi. mmCxC m .. wCCC.. C as ca C5 N.m.i.25 as E 2 C2. E E [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS . .......... ......... . .........+.........+......... ......... .......... 3.36.633 MUG <3<UUM G GLUL U33 334 MG MM Q3333m3<<. 333360369 Q63<33<QQ<333<3<Q 30<3U3<3<Dr 34. 34<MMUM<6 333(64 3H3 .MMM 3me 3M 33330<< U3MM36.6.M.M .M36.MM. M, . 6r M3<<M3LU ,2m 36.M)M<3M..M3UL..:MH <64. M36L<LU<MM33 ML 333365664499.3385.M L..3<M..M3<6. 1.333334% M6. 3.6.34.

QM3mM6<<<M3MJM .MvaM M <M 33... 3M336.M)M<U6.MV<<UM3M.JM3M3L3<6 <06. MUM M3333... M.MM..M3Q.3M3BUM3<D< M.M M4._r <33 .M.

QMMt3 L..:MM3L3M3M34...1M 6MM< 36.34.33 36. D<G<M33L3 3.3M r3MM 36.MM 333334. 3M.MM.M<6.M.MMr.M M.M<M M2...33M..M...1M33M...<6.6.M3 <>6 36.M..M<< 336.6. 3M.M33.>MM3M.M3 .M...M4.D<6.4<<34.3M..M4.D.L.>.3M..M..l.M 6.333 <3 .3 M M.MM..M M64< .ML.>M M3 $343 L3 << M G < M <..1.MDL..:MM..ML3M <6, 4.3L. 3333 MM..MGM3L 6.333 33 6.<3....MMwM.U6.6.336M336..ULUMMMU3 MIMD<U<HMMMU 63(3336. <6.M.M436.< M<Q M..MD<6 Mi: M33<3<Mu<3QQM3<<3Q3L >.a Mv<< 33366 U6.MVM.JM<M361<6.L3M<3< 334.<U6<r33333U6M3Q<M3 336.3<QQQU<<OMU3Q<<D M3M.M.<4UM.MM.M3M.M43<UMMU M.>MQM.MMH.M..>M6.MM<<M3<<6. 3:333M..M....M3.M3M..MDL.. 33,33M3M MM3M..MD6. M336<M3<<UM<M U M<L M6 M MMM 33 356533 3M34< 34<U :34. m36.....M....MMwM<M3M..MD<6.M..M 3333:... .3<<M..MDL..:MQ<<U <3M3<<<3<<36ML.3M..ML1M.M3< U6. 3.0. um 34. 3M6. ofifimmw MMS-..MM. $0.... x...L- M...MM.ML.MM-M.L%-N. $3-33.. w a9..- ms. mm N.mm\.MMMMM 3-2.2 8.2 L MMM/M [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ...M $qu MMo- Mu . .......... ......... .....+..........+......... ......... ..........

QDMU MD» r)MmVMMM MHDM.uMaM.MMMv<_m.M.DMv<QM.MMMH ’0 M). <7 <3< MHMM: 4M.» ,M Ma. <1 MAMMDMDM G<<DDDDMD UHE.HMD.¢MMvDM.MMMM..MDM.MMMUEDD. HQ<.M.Df DEBUGMM.MM.M«MUQMMDMDMMMQDMVMMD MugMM mmD3<< $95-5 DDDMr <1 Mx H M DMMVMMG U .MDMDM MMUQDMHQQDDMr H M3 <¢M<D< M H M“ M M..MDMDMMvDUmHH M<M M.

M M¢M<_u: «MMMHDMD.

MMMMMMMM D ,M .u_m.MDqu<DM.MMMMvD MM UOMMMMUDMMHv<m M.) MUHHQUKMMMDMSMMMMMKM vflMmMMMM QMVMMMUD HN ..MMDMMH. ..

M MD M M“ M H ODDMMHHMM D MDMDMMM MMDMDM UDD< (MMDMMMJMM MHM MMMDMGDSSEMO .....-m.mMmmam”mQfififi.Mwmmmm- QSDMMMMMJDMMMZMMMMMMC GMMMMMMDMMn«MMMMMMYDMMMKJMMD. MH.MD.Q<MMGMVM‘MMJMMMMMDMMMMDMMMMMMMMMGMMM UMMMMMDMMMD ......... ......... ..................... .......... .... .........+++ ...................... .......... ......... M..........+..........+......... ......... .......... w: mmm. mwm. Mmme mnw owe MMMww m. 0‘ M we ac Mo 0% Maw N99 mow woe mwo mac Mm 2.6 09M. E.

............ UMMMMv<GMHM< DMD): «MJQMD «MJwaMMU a: Dash. «M «MM. ,.

.. «MDMMMU QQQHMGDQQGMDMMOP. .MHQ<.M MMMxMUMMMmefiMMM. <<<<DMMUMMMMVMMMJMMHMM <M<<<<UD <Mua<<< <B<MVM DMMMUaMQMDMGjMM DMMH: U3. .

{DDHMDMMMDM MxMMMMMDMM. MMUD<MV¢M<<<MDMMHM {Maia HMDMMMNDQ‘MMMQAMMMMJMQQD‘MMMG beMexmulmMP MMMvaMMMM 23%)-:M5320? .

MMUMM .UDMMMMMM‘QMDM4. DUMMMUxMaMJMMMvMMDMHMxM .MMMMMMVMMMMJMQMHHMMMMMMUMV DU: . MIMMVMMMUDMMMMD UBG<DG<< 4.

DMMH. ODD MUM}? ,.

MxMUMeMU MVMMMMMMMMDMMM. UDMQMMvDaMMv 944% .WKMJMUMVKMMUXM 39$}:an GD<<U MDMUUMHwMgMMvG UM.MM.MD.M.MM MMMDMMGD MMDMMMMMMMQMMMMDGMMufidfiai mmfifiwfimmflmmmfififififi-...M.M..Mw.m...mmm MamMMMMiMPMM,M0:¥M:MMMMM Max . <0 mum. .M.< D CMHM.M.JMM.DMM..M<D.

..... MNm- m mew REM; wggamo .H M33? 53.98; wacfoma . m . :32.me 3N9 :0. Mm _ mam" 4..”an =3me NMMOM Nm Nm N. mm! m mMaMcMM N.mm\.McQ mMaMMMMM MM. 9313 M5 M2. M M2. MMMM Z MMMM/M .............................................................................. ........... z [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS ..................... ........... f MENU UUxNU UwNU CwNU mme meU E.

+ . ++ ........ DDUUUU4UU.U4D.UU.U.U4UC.D.m U UUUDUCCU4DUUUUU4UUUU444 USU)». 4 ..........

UUDCCCJU4U4UPUUUUU 15%: UU U. UU U.

UU4 UUCDUC. CH UDrUU 44U 43.3 UCUUUUU)» U..\UCCU..U ..C U». UUDUUUD UUU UU. OUCUUUD4 CUUU U.U4 DUUUUU.) C4UUCU4 UUDLD.

U: UCJCCUUCUUUUUCU .U..UD4UUDUUCUUUUU.

D. UUDU44UU4UUUUUW UUUU . U4UU4U4U4UUUCCU.UU U44U U,U4 .49)» UCU UUUUU UUCU UU4UUUUCUD U4UU4U4U ...CUUUUU ....................................................................... DESESCECQCD$5355....EU4U UUCCU4444UUCUD UCCU4444UU4“HUDUCUUUUHUCCUU UUD44UUUU4D4UUUD 4CUUrUU4C UUUUU4U ..HUCCUUDUUUUDUU U4U4UD ......... ..................... .......... .......... ............ fl»............H.UCUUU...mammUfimUU ......... + .................. o U. U E U: N4. UC.U.....C:.U..::UC:...:Eiélséfi v4.4. TC: 4.: 2.5.33.5..55..3 52:?33330 ............. ............. .............

DUCCW4C CUUUD4U U4 UTUUCU. 4CC4U .................................................................................................................................................................................................... .4UDUUUUUUUUDU... UU...~U..UUC U UDUCUUUUUUUCU4U44C.DUCUUU4U UUHUUC m ... C49 4 U .UU UUU4 .UC UU UU. HUfiC 4C 7244.. .UUUUDUDUUUUUDU CUU D4UU4U UU4...

U4 U UU4U U ...UCCUUU 4DUUJU UCCU4 U4 D UU U4UU UUM UC U3UU4 UCUHUUC CU ..CU UDrUCC U UUCUC UUUD- :34 U44 U4UUDU4U4 4UU U4U44U ...CCCUUC U....UDUU..UUDU UUDU (8:00.21: UUUU ..U C EUCQUUUECZC: ......4%.UCCU...U.U.UU.UUUMU.& UU4UDUUC4U444U4UUD44 UC4U\UCCC ..UUC CUC4 C UU U U U U a .....

«CNQU ,.

UTCU. UUNUU-NQU 4.3.42: ....U...UU.U... .3 Cg. wUNUéomU 4.NN.U.@CUUNU meUéMNU Q 3.. «.3 w 8283.. C C U: UN U- NU- Ti: U- U mm 9 ............................................................... 3 2 .......................................................................... UAUUUNU m:UNU UHNU 4.3. N6 NM NCm «4 mCaUUUUU ..4 Tics Nmm4..oUU 312.2 8‘2. U2. .............................. ........... 3. U2 [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS . ........... mm. .

NV mo”... .

. NVVmV 82 . 82 ...th E 33.3... . 33...... 33...... .........+.........+..........<.......... 33...... ........................ 33...... 3333. . 3333.++ mV Q C. . 33C._m.VC.mVV3_m.VC.C...V.VV3C.VMVV7.VD.V.3HV UDCMVVVV)V 33? V337 33.4.7 “VC.V3 MVmVCCdDC. VVMVDC. MVVMVCnVVVD”VC.C..V.._.33..3. C.MVC.VMVV3N .VD.C.HVVVCmVVV3.\V.V..3C53.33.13“ .............Gmfiwfiqmg..V..7.Vm._......V.V..V..3.V 37.5.... MVMVVVVV1VQMVMVVVV....VVVVV.C1VVVVVVV1V HVV4...4.._..3.VV7.VVDMVmVVJVDC.3.33.3343 3.4....3HVVVVDC33VVCJVVV ....................................................................... .<V...Vr....<.V7.VV3(V7.VC. .

. UmVVtVV VflVmVCC. MVDC.

MVmV<<mVVVVC<CmVC C. .

VVC. V3Vr.VU.<...VC.C. .

V. me .

V....V .54 .

.-.)VUDQVVNVVDQQQVVGVV V..... . 3.7V.r . 34.333V3V<3...3VGC.C 3333.0 4.V1Vu.>VVVV.VV .

D<fiVV3mVfiVmVD<.VC.C mVC.V7.V_m.VmVV.3.4.mVC.7V7VV3 . 3V3. .

. V<<V3 wVDVC. 3C. . .V.VD.VMV<.VDC. 033CHVDC>>VHVV3 V.4.V.,VC. MV3.V7.VV.VDV..VV7.VV33.13 . 33.13.3333 UmVCVMVmVV. . 389.313 .

MVmVC.C.V3 . 33.333 33.33 .

V. . 3 . MVVHVC.C3.V.3C.V\3< 3.333333353333333.mVC. VV3.4.V.3 mmm CNN m m. C m.VT.......................... .

Nu an. N... wN... NV. om... 3m wa 3t. . .3... {F VS R... mm... Q. 9mm . . : 33mm NE mi.

C3.DV.V 333333 a . mVC.C.. .

VV.333VVVw <33 ......VVmV 3335 3......“ .3331... QQDQVV C. CCC. . .77VV . . 33.3.33. 3333.003. V3333? .

DQCC. . .53 .4.>VVMVC.V . 4.. MVMVVHVC. .

LC.C. 33C.VtVV V....VHVVVVVV VDWVKEHVVHVV. V.4. .VDWVDHV3C VDC. .

C . ... 3.33.. . . 53.0.5. C.V3.4.MVHV<<DC.VVVV3 33 <C..V.r 3 VV VC. 33V.) .7 VC. mVC.7 7V...V.4.m UVDHVC. VDUDU . . .... . 3. .

G... .VVC.C33..»L. 51 3C. . .

...VC. .

V VG3333.3333.33.3333 33HVV3.4.MVHVHV3.VDC.C.V V<OQDQD V7VV3.V D.C..V...V V7V7VC. 03:33.0 37VV3< .

Vn . 333. . . . ....37. VDMVVVV VVVV<VV5 .

. MVCqVVVVD3C .

DDVC. DC V .3V7VC3V1VV3VG .4.MVMVVWHVVMVDCVVVDMVC.C..43.“VDVVVD. G .1}.0..V.m.«..wwfiww..........mfi.... V..Cum.w.<VV....VmV.u.<....V1V<<CC.UV... VVV.V..CVVVVVV....V..V.VVV 3.139 .13 .......................................................................... V: .33.33 mV cm .............J................... . . cm N . . . 3.. 3- . . 3- NMNV . ._ .

. NVCV. VVCCVNNCV N33 ....................................u.......................... .

.H . .H . _ 2 . cm . 3 .

NA . 33:25 . . .3335 32. . . 32.

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ....

NM VF w. .---1 .HM MMNM MM: MNMEM ”1 --¢ MNNM NNNM MNMM 5M4 mm ,5an NNMM MMNMM MMNM MMMMM MMNM NNNM MN: ... ......... ............... ........... .........

M5<5MM<MM5M5M5MM<M> w.

Dmowaufidr Q<DUUDQDDQD< CD<§< <MM M.MMM M.M.MM.<. M.MMVMMMMVMMMM M5MM<M> >5M5. w...<.m<m.

UDDMQ. 5553555.. .MMUMM MMMMMM5M5M55MM.MMM<MM M55555 5<MM<< MMMM<5 M<MM<<5MMM ..... MM<<5MMM5M>MMM5M>M>< MMM.55M5.M<< <DQ<UUUDQD D<< MudQDD D<< M5<<MMMMMMMMVMMMHMMMMMMM 5M55MM<5<5<<5 .........5...MM.MmmmwmwwwmMmflmflm...sm..m.. 55b55555<>5<<555 5<MM<<5MMM5MM MM<MM<<5MM5M>MMM5 ........MH.MM.5...MM.M.5.M»MMMNM.M.<MM<5.5>M M......mmmw..M..M.m.m....M.,M.mM.M..M.m._...M.M..M.m._...M...... MMMMMMMM MMMMM MM MMMMMMM5<MMMMM5 fly“....mmm.mmmmmm .......fifimfimflm.M...M.M...M.m.....M.m..m.M....M.M.r.M.aw <<5MMM5MMMMMM53.5.

«VD D<<MMM <<MMMMM5MM5 MMMMMMMMM MMMM <55 <M..M<<,MMMMMMMMMMMMM<MMMM5<MMMMM5 55<M5MM5< _. _ . M , ................................. : _.

.. . M : . ............. .

MMMM55M55¢<MM<55<<MM<M5MM5 <Q<QGQDO<DQODDDQQQ< $5M)». 5.. 5<5MM .

M.MDMMMMDDMMMMM.MMxEMUMMMUDaMU 6.59.55555<MMMM5M.MMM.<M..MMM<MM M5MMMM<MM<MM<555M5<<§MM fiwfi....... n 5M.MMMM:MM.<:<5<M.MMMM 55 ..........fimmmflmfifiwwmmwfimfiww .Mwmwummfiw <5<.5M.MMM<M.M<M>M<5555<<<< MMM<5<M5MMMM<MM<MMMMJMMMMMMVMM<< <<5<M5MMMM<MM<M>M<555M5<< ..........<fiflmfiflfl.Mfimflfimmmmfl. Mamm.M..MM......M....M.:3»me 5<<M.M<<<<M<5MMMM<MM< MM M>M5<5MM5<<5<<¢<5<555< ................................................................................................ ............................................................................................ .

MM- .........

M.MEAMQ Em. .

.. . ..... .. , E ......

... MMNMMMMMMMM 32.32 MM ................................................................................................................................................. M .3 . MMMNM.

M EMS -8 MM EMMMM 22-82 ..N.MNM. N.

MUMaMMMMM NMNMMMMMM :N: N $12.2 ....................................... MUMaMMMMM NMNMMMMMM M2. Ma MMM..M...m.MM.M....M....N..mHN.H.M.MMM...Mm./.M.. LmMooerMMoMz ...................................................................................................................................................................................................... .......... M2. SM.

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS 2.2+22<N2222<22222<22§22<<2222..+2 2 2 ........... ........... ........... ........... 22.2.2 2.232 2. 2. 2<222r<)<£<p22<<2+9-953.222... N22 2.22.2 2.2.2 m 2... $2.12 222.2 2.22.2 N222 2.....2 - 2.2+ ....... 3.2.2322222.5222..:.2..222<u223.22 <92)». Q. 222 2.2th 22.2.2 2222.252 222.2222. 22.3.2 222.222.2222 ESQD<DD©<QDr 22. 32.223.2Q<2..WDU222 22222222222222.2232:5.2.2... 22m<222u222...22:22<2.2<222.23.22.22 22222222222222...2222M).N22.2W.3. <22N2..22...2.2.<.2.2222..:..2.22.23223.22.... .2<N2..22N2..<:2..2.2..<.J222 2.2222.22.222.32;H.M.2..w2.2...m.....2.2....2...22.m...2.<..2.....2..2.-r. 53.32.222.122) 2.3.222..2<2..2..2222...2..

QDqu <N2.22<.<.2222.2.<.23.<<<a2.2222 232.322.532.3322222.w. 22+222.22.222.22.222.222.52.052.N22- 2.22.1292 @DGfiiUQGDm GD 22:22.2.3222.

U<.D<D 2.222.222.2232. <3. 3}; 222222.2222.23.3.32.2.2222222223 22.2.2.2 2... USUUUDM .212 .9.2.2. .2. .2222 ......... ..................... .......... .......... ......... .222.2222.22>_2..2<<2.2m ......... $2. can won. $9 22.22. E EN 2.2. 2E at. 222.22. ma 2.2K m . . ............ <32 2.22.22 22:. 2.. ...22...D....2..22...2< .2..<22<:2222.222..22225.23.22.22. .2 {2.2- 2..:2) {$.qu 222..2<..32.<<<<N2<222 D<DQ<UDQ<4D<<J<1<Q<GD 22< 222222222222 qu<<D<<<< 2222<N2P2<<Q<< ...........N.2.m.2...2w.m.2.22.«fi..2.2.<..2flfim 2.2252222.222.222.22.252 2.233.223.3234. 23.22.52 22.2.22 3 <22.2.2..>22222.22:2. .2223 2.<2.22. 2.22.2272: 2: 2222233.22N2<<2u2<2222<2a ..........N.2.2A...m.......2.2...mm.flmm..2...2.fim.m........2.m..2..... .2R2..2.2..2.«w.2..2.m2.m.2.<.2...H.M.2.<..2.....2..2.fl2.2.2....... .............................................................................................................................. 2.222292222222222:3.2.52.2.<22 fimflmfiflfim........ <25 UG2UQD22L<U .................................................................................................................. . .

. . «Rm . . __ mm 22-22%.2-22222..N.2N2.22.222...2.22z 222.2. 2.222- 3 .. m 2.2N2 22.2 2 ES N. $2222.22 .........2....2..2..w.2...Hw.2....N..m....u.2n.2.2m...2m...... N9221N .................................................................................................. N. N 222.2222 222.2222 2 222 2.2. ........... 2222 32 [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ............... ........... ........... 83 ES mmmL meQ womQ E Q : L: 3.? 3.2 + ++ ........ ..........

QQQQL DquJLALLQQLQQDL QGfiQLmQQJLDQL‘QQL QQQL<. QQ QQL ,”L133QQ..\Q<.LLMQLQQQQLLQQQQLLL m Q.L <0 QQQQQQ QQQQQ QQQ ,5 Q QU<<QO<<G QQQLLQQLQQLLQQ. QaLQQQLQ VQLQLQQQQL QQQQQQQQQLQQ<<<<QQLMQQQ QL QQLDQQQQLQQQ U Q LQLLQQ Q< QVQ big, {QQQLQQQQQLL :Q QL: QQ.QQ.QQ<L<<QQ<QQQQL Q< QQ<D<PQ QQLQQQLLQ.,Q:QQL<50L. QQ<<QQ Q L :Q QLQLLQ QQ MQQQQLLQQQQQQQLLLQELL 43 QQQ QQQL HALL mQ<<QQ QQ<QQQQQQQQLQQ<< ....................................................................... Lfbiééwafiéfi .QUQLUQLLLQJLQQQDQLLQQanLLLULmQDW QQQLMQQQ QQLQQQQQQQQLQQQQQDGQQQQQQLMQQQ :QQQQLLLLQLJLLQQLL LQLLQQLL.QQ<Q<QQQQLLMQQQLQ CD QQQLLQQ .QQQQQQLL‘QQ ......... ......... ..................... .......... .......... .........L. tam mg can won sow Qom 25:33:23 ............. ....... <QQ< QQ<QQQQLD<QQ<<< QLLQQQLQLLQQQLL. QQLLMQQQQQL LLOQLLQQQQQQQQLQQLQQQ .L «LmaLQQQmQQD QLLQ << my: LLZ.:::L:L:§LLL..... LEL....L..QL.ME..me....L..LQL.LL..L...............

Hr)» QQL). QLUQLLLQC? “Q 388953 Q:L<QQ<QQQ AQ<<<QQ<QQ LQQ<<LQQ<QQQQLQQ< CGLdQ<<§<Q<Q LQ< QLQLLQLLQQJLALQQL: <L QQL QQQQLLAQLQQQQQLQQ.

L< L. QQL“QL.QQQQL‘LQLLMQQQLJLQQQL.

Q<QLQQ .4143 UQQLQLL Q <4444QQQQQQAQIQQLOQQLQL QQQAQLQQQ JLQQ QQQQL QQQQ QQQ “QQQQ mQQQ QL< ,QQ4aLMQ‘qLQQQwMQ<<MQ<QQQQ<Q LEELLLEELELiasiiifiL: LLQLLSBSLLZL;LLLzLLeL.L.LL.:LsL <QDL<D<<<DQQCQQQQQQLQQQLLQQ L<<<LQLQ QL L Q QLLJQLQQAQL‘ LLLLL...L.....L.mfim§fimm Q QQ<.QQQ TQLQLm 33.33 «SE; 3:- 3.3 .................................................................................................................................................................................................... .................................................................................................. sifmmw L LLL: m m em‘QLstQQ Jaggimgesz QQEQ. 33 meLQQQLQL mLaQQLQQ .mmLa...

Q5 Nmiba Q2. ........... Q2. Q2.

[Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS 1%..MMMMM1MMMM<M1MM1M<>MSM<+ME: 11w T ........... ........... .

PM .

M. w . 11.16.31. 311M- 1me £2 11353111113219.3111: M<§MM<<1MMMCMMMMMMA as: 119M 1N1: a: . +.........+......... ......... .......... ........ 1M.MMMMMM1.M1M..M Q< .1533:magi: .MMMMMMMM1MM1M11um:a:..fiu<:§. m .MDDxflMUDQQQ 1.111.131.1131 Quagiau...muwmufiMuMMm DEE QMMMMMMDQMSM»MMMM11§18MMMMM1 .11.: 35mMC.M<QMM.1..M.MM.MMM.11M5 wmmmmfimmmmmmmM...11_.Mumnmmm..M111 GA «1 9 .1MM1MMMMMMMM1M M50213» 1.

QMflmfi .w...1.1..1.M.M.1.

M 1.15.1U1U.M1..1.>U1v11199304313$.91 UDQ<MM11DGCM<1< MM_M.M<QM1..M.11MM<MM<.11 MDMmfi hadL.) (MDM11MM1 . .1UWMGM1.:.M<U U.<M..1.C.UM1.MQD<MMM r MMMMMMM1M11MMMMM do 535M111. 188$ Db MJMVQU 191910 HTUMx UG<§DG<DD ......... ......... ..................... .......... .......... ......... MMSW .......... ..... ... ......... 1..........p..........+......... ......... .......... 91% MMM1 3:11.11MMQ1MP1M..M; Em mew Em 2M1 m; 1M; 1.; MMNMM MNMM NNMM 11.1.11 wmw mmw 1 1 3....1525mMMMvmufiMuamugM. «EQDQQDMwDMflmDDD QEQQG. ............. 1.61 1 4.11.1.3 -9114 MM.111.M.M.M.MMMM<H1MM.11 11.1%....wmmw1. 11.111113. mum: ..... DUQQDUUwa 1M1DMv1M1 ..MQDf MMMMQQMMMM. thQD G911. SM BUM h .11MacawMMMMMMMMMMMMMMMMMM < .314 $13.6 Q2111 (M. 15.11.11 ,.

MSHMMQ.

M 5 ..........M.M.m1..1m.1.m..H...1..M.M.M..w.1.m.1.fl.m......m.M.m.1.m.M.Mm wfimwfifiwfiw M1M1MBMM§1UMMM1MMM1M1M1MMQME< 1MMMMMMMMMM1M><21.1MHMMMMMM1MMMMM11MHMM1 M5111MM.M.M.11MM<.M1.M1MMM.5.MMQ8.0.11 ............M1..MMmmmfiflwmfiwfimmfi$111.. MM<M1M11MM191M>1<M1HMX ...MMM.1MM3MM23.11.11Scum ESQ:.uMMM1<uuMMMM<<MMM1M1u MMMM11mM.>.MMM1w<M1w<1M wfiwwm...wmw.1mw....M.mm.M....MWMH1.MM.M............ 1.M1.M13MQM.MGM1.M1M1MM1U 1G .................................................................................................. .............................................................................................. 1 1 1.53-311. Swfmm: _ . SM: €11.21 .3: M1M1w 1. :31 M1 21:81: M1321 11..MW111MM-MM11111M M1115. MM a- :wm mg 3th :13 MM BSAQE MwmoM .3va T313 ..EMM. .

N11 N11 1.1.3 NMMHMMMMMM mfg N.mm\1 9111.312 as M1,... M21. Emmwmm...fi .................................. 3.1 E [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS ed set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS <mKDKU DDDQK HKKKKKQUm U45 KKKKKKK K DDD<UODQD<UDr G<GMKGDDDDDDD<QD<UDD $044.94 KKKB<4KKKK<GDDDGGKKKDKHKDDDKKKK $vaDODDUDEMKUKU<QQDDD Kv.u_m.K4QODDDK)K KKKUDDDKKKK MK<U UUK‘FéKKKK HKHKMKUQDD DD<UUDDD4KDKKKDUD DD:UDKKKD<4KWK<UD DQD44DUDDDDDD<D DDMKD r UKDDUD<DUD KUKVKZD DD< KKKKKKKKDKKKHKDKKK <KL©KUDD U444 KKK: 4D< 4KKKK.KDD4D<DDDCDMK4K DKUD<<GDU<DU<UDDUDDD MK<4K ........................................................................ KKKKKKKW Kw4ma4.KKKKKKKKKKK4KK4“Know: ..........m..K.m4&3meHmfimmmflKKK).+ KKKKrKKKKKKKKaaKKKKKLKKKKZxKKKKKKi D<<4KDUUUUUKKKDU K<LKKKKK<KU1KDKKDHK DD<KUDDDUKU<4<KUD$<QED ......... ... ..................... .......... .......... .........+---------+--- ....... .......... ...................... ................. on omw Nmm mmm 4mm m emw 2w & mm N} : ..K 00 co w KKKKKK KKKKK. ~44 m MK 44.4. 34m 54w ,Kvw.

K ............... ............. 4m <4 4 K DD D4KKKK< 3: 4K4< DDU KKK? 044 KGK UK) <<©D< D<44 («KKKKJAKKKKJQDUDE <4<UGD <KU<D<$<<G<41<4KD®DUKUD <D<<<G<<U DD Q14 U<< KG K434? KKKKK444KKKK pu K <44<Q44D UGQ<< .K4KK‘KDMKKKDUMK44KKK4<<KMK< 4<D4D< GK, Wu D4: 46K)» UnKmKKUmeK U44<DUDDUU K D4 D KKKKKKKKKKKKKKKKKKKK <K 3534037?.3 .K4K4K v4fi5 KKKK<<K1 93444 Q44K4KUD<MKKUD<KKK< <©<DOD4<< KMKDHKKKK C4<G4K4Kfl J KV44 KKD K DKKDDKU<D KKK4KKK D <<4K UUDKKL 4 KG“ D<U< mK4K <4 KK D46 UK KG 3G ..........Kw.K.K.K.K..K..K.wK.K>2..mK.m.K..K.mK.w.K....K.K.4.K.KM.. KKKKKKKKKPKKKKKKKKKKKKKEKKKKKKKKKKUKK 4133mm KSKK4KKKK4 m.KKKw.m.4“memewmmmmfimmmmm: KSQKKKxKSKKKKKKaKKKKKKLK MK KQQUKUG K ..... an 3 KW ww .QK am. K ._ N4 q.

K3 ND Sew , H am 304 ...r..m...K..m.K...K..K.Kr 43$me ,KKNKKKEN QNKKKK «Knot: D N LN QNNN.NQNN ...................

KKKKKKKcKKmeKKKKKKKNQKKKKKKKKKKKZ KaoKKN. m m N, «KemN. 33m. ........Km...mm.m.4...KK..K.K..K.m «SSE cw a MN N QNNN.NQNN NM NM miss mK: :5 N.mm§¢© 3. .................................. ........... 3. E [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ........ . ....... .............................. ........ 554.. 554.54.454.5455555555 55455555555555.5455». N.5.55.<55.455454554. 54.555455.5.5.5555 55.555554.55.55. 5 55544555554313 w. m. <-5>5545N555>555<5+NN 55.45... 5 .55 :55 555555.55 5555555: 554.4455 4.5554545 5.554.. 4555....554.555555555555m 55555555555.».....4.4.N.N555 ...N.m...m.N.N..N.N..4.fiNM_Hm?444495y 5455445455.... 5 5455 2.5554 N 55555 5.5.55W www.mwflflfifiwmmwfl5N:EN 44 4554.54.<54.<555555555 ........N.55N.N.NmmNN.N.MMN.NM5N.NNm.N.5.NNM55mm.55”.5.m...m.....................N..N..H.... 55555553555.55..55.555 5.3% 55454.454.5545555554 5555554.555555.44.5555 ..................

% QNN 55535554555554-3555 NNN N55 EN EN 5N NNN N555. New $5 EN New N5 .. ............. . . 5555? .

N.555.555N.5.5.5<4.5 <5. 4.. ......m..m.fi.NN5.N.mwwwfinmmmmmfinm.. 4.5. 5.5. 5 5555 :5 55555 53545544354555 4554. <54.“ 55 55 .55 45 54555555455535 :55: 55$. 544.455 54555545555 5555555544. N55 55555 N.,..4 4555555555 N54.4.,.55...5<4<55N <<555455N555 5 5555 $545,445.45 53:..5N545545 55.5.4... .55. LN..NNmmfimwu....55.4.fiw.m .........._N.mm.¢.fi5m.¢.m5.m5.m5..N.N.N......m5.«.5...5.m5.m.¢.. 54.4.4545 .55 455.45....555.55.N_N.5.N5,.55<55 ............mm.5.NN.N...55N...5.m4uw.4..«.m4...mmmfiwflmm.. 55555555555555:5 5554.5 554.455555555555 5554.555.

....................................................................................... ............. . NNNNNNNNNN NNNNNNN . NNNN. _.. NNNN. ._ g NN N...N...N...N.NNr H . NNNN -mmmeMNm ...................... c WNNNNNNNNNNNE3.22 N N N N s.N.mm“.mmmmsmflmm.§€%fl- ENNNNNN NNNN. NNNNiNN NTN.

SNN SNN N558 T. T .55 555.55 5.55 52 N»... E. ............................................................................................ .......................................................................... 5...

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ........... .

NNVNV $3. NVVNV 3.3 .mV.N+ .33.. NVNNV NVVVVV NVVVVNV N ..me 3.3 .NNVV. $3 . .

- +.........+......... ......... .......... ........ ........ HVVVNV...VNVNNVDVV.VNVNVHVNVNVNVNVNV UUNANUUUNN UD<4A<NUQUDQDU<UU<D<D U<D.<N. NV NV. ..VNV NU VVV NM UUDD.

ENUNVNUNVJV... . 131.93?) V. .HVVNVVNNDVVVVDVV:...VNV :VVVVVVVNVVNVNN <UUJUUUDVVVV.‘ NmVDNUAN V.“ NVNVNVVNNVVNDVVNVNVVNNNVNH .DU V..V-V.V.NVVVNVVVVNNV.NVVNV.VNHVHVV.V .HVVNN .VVNVNVNVV.VVNVVVVVNV<VVNVN+ NVDVSNVNVNVNNNVVVNVVPNZ NVNNVVNVNNVNVNVVVDNNVVVwNNVVNDW HVVVNVNVVNNVNVVVNVVNV..V..N.NV.,VV..VNV<.N DVNNV..V:NVV.NHVNVNNV.NV..VV.V.VNVNVNVVVV VCNNVVVVVVVVLVNVVDVNNVVSNNV. anVVVVVVVEVVVNVVViV V.N.NV..VNVNVVVNVNVNNVNVVNVVVVV .mmNNVVVVVVNVVVVVVJVVNVNVNVN+ «VVNNNVNNV.NHVVVN<.>EV.VD.....VVVNVN.m. <NNVVNWV>NDVN3VU<G<UU<U<UUD ....... .mVDNVmV HVVVNV DU DUNJVUUUDDD ......... ......... ...............

.................. EN ... . N. w mmw mum NNN NNN ....NN EN 9a m... NVN NNN NNN NNN www 3w cam . .............

.NVVVVNVVVVVNVNVVVVVVN DUwNUVNHNVdNUPQNNxN <3<DUQDU<U<NNJVUDN U<V.V<UVNMUVUDDNUDUVUNVVNNVNNVDU .NVVVVVV? NV VVVVVVVVVVVV. «.VVVNNVNVVN VVNVNVDVVNVN PVV. ...VNVUN UEVU <<<UNUUW .NNVVVVNVVN .VVVV.VV..NV.VVVNV.VNVVV NVNVVVNVNVVVVNNNHVVVNVNNVNVNV9V VVNV.VNNVVNV<VVVN..NN,VVVNV.Vuu 3.. 5 VG.) VVVVVNNNVNVNVNN .NVNVVVNVV .VmVDNNNNVVV. U:VDUN Had .HVVVNVaxNVNNV UUN.

...VNVVVVVVN<V:..NV<VVVVN U3 . VVVPV V.....

VV.. :3 55.

V4. «Ed Eu VVVNV NVHVVNNVNVVVNVNVVVVNV...VNNVVN NNVVVNmNVfiwfiVNfiww. VNNVNVVNNVVVVVVVNVVNNVV NNNVNVNNNVV .V..V.NV§NVVVVNVVVVVVVVVVNMVN V....UDNNJVNVJNVNVVD :N ...NVNHVVVVNV..NV.VVVNVD NVVNVVNVVVVVVNV UVNVVUC3U:«NANVVUCGNNVVUGDUDU UNEJNVUG SVNNNNN : .r N.NN.NN NNVVrN .... m..NVV...N.NV.V.VN..N.NNNVNVVV...VVNz V....VVV V.. m .H N..NNV N N N VVVVVNNNNN wgmgccmm mg N NAN NNVVVVV mica N... .4 .............................. N2.

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ENE NEE E: $3 ENE ........

NEEEENEEMEEEE...M<NEE<.M.ENE EEEEE<EEE$.EEEM.MEEMNEMEEE MEEMEEEEEENEEEEMEEM. :EM..EENEEEE._ MENEEE... EMEEMENEEEEENEEEE<EEEEEEE EEEEMEMNNMENME NE.

ENEEEENEEErdEE<EEEE+NaN M.EEEEEME.EENENENEEMQNEENE...W .EE...M......,ENEMEM..EMEEEEMEMEEMEM.NEMN EENEEEE. E EM......EEE ENE E EENEEE .NME. .._M.E....EM .N EM: M. <NEEEM.NENEME ...«.wE.M.......MN«m.M.

.ENEENEMEEEEEEE E.E.E..M...ENE.EENEE ENEMEMEEEEENRMEMJA... fimfiwu .EM.M.E EZEEM....M..EEENEE EMEN ......... ENEMEM: E<M.M.EEM..E ENSEMEEMEM. .MEME.EE<NEEEENEEMENE...M..E .ME<...NE....E<M.NEME§ <EM...NENEEENEEEEEENEEMEM.NE + .l..EEEEEEiEMEMEENEENi ENNEEEEENNEW .................. ................. .................. .................

ENE NNN .N. .N.

NW NON ENE. ENE NNMN 02. ENE... mm. 3N 8N. EON. Mam .LEE+EEEM.EEM.mE.m.m.N.E.M.ME.m.E.MM.mmE..M...EM..+NMEN NR. EN. 2...

.................................................................................................................................................................. ............. ............. ............ ............ «NE2E...EEMEEEEEMEEMEEE MEMEMN EM M. MQ..E.“Em........E.M..........MwE.M.EENE.mEENE..m......

M1}. ......M.NEM.. EMEMENM...ME<NEME<. EEG M.M..NENEE. ENE :EE ENE M.E.....E....N.E...mmm....... EEM..NEP.E. E... _. EEME EfidMNEEMEN EENEMEM..<< EMEE. NEEEENEME EMENM MEEEMME...E<E<E M....M..<E.M..M....M....EME <M..MEMMEM..M.M....EE.EEM MNEENEM...M..<ME..EMEM..M..M. NLNEEENEE EMENEMEM...

ENE... EM. .MEM.EE ENS ENE... EM.

EENE >..M. E< EE EEME mm”.................MEmma...”.E....M.E.”mmmwmmwfimflfifl ENEEEENEPaEgEEENEEEEE ENE EE «JEENE «333E ..E.NEE...mm.gm.E.«NE...._.N.m.E.......N.E.wE.M..... 5E ENEME «NEEEMNE.MJ§.<EEMEMEEEENE...< wN.Em.EE.E..Nwmwmmwfimmfimfimfifl E...M...EM. .EEMENE EEEEEENE .................................................................................................. ................................................................................................... .

ENEN. EN .. . __ NNEN . _.

M. MM MM ENMEN.NNEN..NN. as: ENNENN SEN.

NNNN GNNNNNN N N..

NE NEE NNNNSE 32 .............................. .................................................................................................. ........... E... 3..

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS ...........MN..4N.....m........m.NN4..N.mm.N.N.HMfifimmfimflmmmm:N .ww:+ T ........... .

FE NW: NNWEW NS N3 m N; NNE NNE NN: ............NN..m.N...m.......m...“N.N.N.mNmm...“N...@Efimflmmfimm: . . 3% N :wa +.........+......... ......... ....

........ ......... N m < 4044NNNN4N<<NNNNNN4NNNN4NNNN QQD< 0.49%.," u<4N.. $9.3m UG<DQQQQ<D<<4~N U944N~414~<D<UQ<DQOD414~D NJNN 5&va NfDUCDUNumxv.La DNNNNNNNNNNNNNNNN4.4.NNNNNN muuNNNNNNfiNNNNNNNNN.NSNNN 44NNNN<44NN§©8NNS+NN NNNNNNNNNNNNSNEBNNNN. N.”upon:34....NNNENNNNNNN3444. N5:8.34;.ND..N4NN4.:NNNN fimflmmfififlflwafi @4504.NNNNNNNENNEN454w :4NN<4Q4NNNNNNNNNNNN44:43 GN.ND<N..N< 04d". O‘NQODUNWNH 4mafiaNNNNNNNNNNNNN: MNU/NQQNN/NQQGNNQQNNN NUUPQN:NUUUN.NU.< HENDUUUN 40.2% N :3 34 E 1ND QNNVMNMNDONNVNNNUWNQ ......... ......... ..................... .......... .......... ......... .......... .......... ......... 4..........+..........+......... ......... .......... ............ 3N1 0‘1 4.. m m N p-N Nu N. on C3\ Ch m 3. NN ....N NNN NNN N4NNNNNNNQNN€4NNNN NNN NNN ....NN N94). om) 0mm) .N.N .3 Nam ............. . ............. 34 4. 4 NN DANNQDDNNVDGGQOMNDUDDNNNNUQ ~QQ<DQOD4~ 4x414: NN4.NN4NN. 446244.. 4.44:4.L4u G4®< 534 NU<NN<UJ4~N. Q N4 .....

.. Dmifivmv NNN N.) Q45 N<LUU4JNNDUU DD4ND < 4QNNNNNNNNNN4NNNNNJNNNNNNNNNNN fim...wmmNflw.mmH G<<< J2 .. N4 4.NN4NN D4 DO #3450 an 54 .NN <\.N44<4:NNNNNN NNNNNNNNNNNNNNNNNN4NN3444NN4N4 NNNNNNN4NNNN444 .........mmmmmfiwfiwwNfig5i. m..N.4....N.N.N.mfi.N.NN.N. Nuwmv<fifiw<<<UUUUDUUNNUU 34mm.QUfi4fi5hm§<§4~QUEU 5.59NDNQNNNSNN. <03 UUUQ . .NN.HENNdENEMMmfimfifi.. cw - mm. cmwm. .

N mm . . mm mmwm mafimwwm SNNNNNN NNNNNNN .NNNNNNNN .............................................................................................................. r .................................................................................................. . .

N..NNNN.NNNN..N.NNN. N N m NNNN.N..NNN NNNN.

NNN NNNNNNNN 33.26 NN NNNNNNNN....N..... N NNNNNNN NHNNN NNNNNNNN N5...NN2 mites 2 N2. 2. NNNNNCNNNNNWN.N.NN...NNN ........................ 3 [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS 82 mam .

. NNmV - +++ ...................... .......... ......... ..........+.........+......... ......... ..........

Va VVVVV<HVHVDENVVVDVV VVDDG.D<<<<<VD<. N N 3:83: VVV66._r (I) N ... . VD VV N 6 VVMVD6 3.3m VVVVDN V6.. VV 6. 6< 6V“ VV 6VCN VDDVVVD. EDD/66% VVJD6V VVV<VV NVVHV6VNV-..VV VVVDV .DD66. VVHV6VVVV. 6.VD6V DV. DNV VVVVVV .flVmuuflégfiV .06.D<V<6.<6~6:V V06; VD V...VNVMVVVV..VVNV .VV6 .V<.V<VV. _.VD6V6 VmVVV6..Vr VNV NVV. VVVVV V<VVV6. 2 6<m<VEunqu<V VDVNVVVVVVDVDVVVD DVVVD6.VV<<VDVVVD66V VVVVDVD 6.V6.mVVHV6.NVmVD.VVNVDD6.r_ .V.HV<V.VNV<VVV6.< <DV<G6V<DNVDV<V<H ...).V MV6VV (V 3VV V V36. DVV.VVV.. DVD: E mV6VVDV D_VV. 6 N VmV6VVV VVVVV .V DDDVVVDVD DVD VD VNV<6V:NVVVV NV 6.V..VVDV VVVVVDD MV.6V 6V.V.VV<<D..VVV.VDV “HVVHV<6VV..VVNV.HVDVVmV6V.VJVV<MVVVV<<6 6.VVH.V6V6V6.VVNVVD.V<DVVV<<VVD ......... ......... ..................... .......... .......... .........+++ ...................... ..... ... ......... ...........p..........+......... ......... .......... ._. V... m V.. a mm. cmo. V..? mg a? own. mm. V1. ox N66 N66 6.3 mg V66 V..; w; a; 0mm» Vmw Nma Nmm VVDVVVVVVVV:<0 VDDIDV_ D6.DV ( {mVDVNVD mVVD < VD < 3V N (DD ..6.VmVV.VV 6VVNV<6V6. 6VmV6 6.6. 06 6V6.

.V N DVV6..

D V66. (EV D6. V<V.6V6.NVD. VNVMVVVVVVVN 66.

. VVVVVV V....NV6V D 1VD6VVV .V NVV.

V6.V DDVVDAVVDVDDVVV DVVV VV.V D6VV: HV<6V6VVVV .VDNVDDD ..VVV666V6VVVDV VVD...V6. .DD6AVVV6VNVVVNV.mVVV<mVVVVmV.6V .VVV/VVNVDVDD V 6. 66.6.:VVV1V6V6V6V6IVVVV {mVVVVV6V64VV6V46VANDD6.<66.6.< .VD6 <66. V<.VV.V6. .DVV VDVVV 66. <06. N ..V<V .VHVNVVVVVVVVVVH VVHV< VHVV 606.9. V..

DVr6V VD6 V DNV:DD .V6: 66:..VVVV mVDVV DVD VflVV: V:VN 66VVNVDVVNVVD66DVDJDVVN V.6VV..VV..

..V mVVVV .V VU6V6.< V.VHVDVVVVJVV rVVVVeVmV VD {ODDNVDVVNVVVANV6VDDVDV<D 06. D.V..V.VVV.. <VVVVDVVVV<NVVV<NV6V<DDDVVVVD wagon.» . . oVNc. NV . _. _- _ _. NOV _ .m _ _ N _ VVVONNVVHV.V8-2.2 w m m w m m 136:- NNVN $3.33. VONNVVMWVN mmNméNNm EVNNNV NVNNN DVNNNVVN V.Voc N N.mm\.25 N.mm\.25 N. N. mites E... E E S ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ........... . mm. wmm... R... NE. Em. ............3.mm.....m........m..mfimwwmflm...mfimw..mmmmm:m w. E3... 2... ...........m.mm.....m.............wmmmawflm$333.43....mmm...m..................m.....m...................................mm............m.3.......flw.3...3M... . .43.. .33+..3...4J....4J..3..J.J..:4+soc .Er...©4a3mguJ.33..4.3403J4+Sm 5... Em. . 4 r...4......mm.....m..m.............m...m..4..........3...... ++ Q49.344so......3..5.4.J.4DPJ..4D .UDDD<DQD4.DDD4.DQU4.DQ D4 . ..

DP.. DDUD...MUDDDQD.. Q43344w444:44u4uua4 3.3.33.69.43.D.....4D...4H5W :.......uu:3....3.3.......,.:.:uu...:... 0.434....:38.J.D.D.J.3..4c.u._ub .32....3359. 289......83.4.5.5.9433... Q44. “5.3.3....D4 . . «<4... magma...M..B.J...:bw.u. .

. ED .DQQ4.DDDQGDDDD .D. . .3... . . 3me .: .

. . D... v3.3.3.3.c3... .«:uu4.4..ww44n 4.6.3.3.DLDUDij4. . («€4.45D463.

. DD “5.3.4.4.3U.....DDUUC<4.<GQD QUG<<<OQD4wD<D40$..va ............ .

E... m 0 w . mm mm 5.00. 3:. Em. Sm. m... E... mm. k... 3.... 4.... .

: N3 m3 4...... ..........m...4......m...«.wmm.w.fl.mmmw.@.......m...m......... ...... .................... . 33333244....flfi.... . 43444 @344 4.44 3.44.35...) .5 . . < . 4.

. U.

DO Q<QG<Q4 3.... .J....u...J 4.34 44m . 3v.

. D . V . DD. v.

Q44 . 544.34 :D DD.

D4145 4.444.? .4495 .33.... . a 34:. 3.0.5.33... . wDUfT 3.4... . 5% D4 DD. 53344 www.mmwu......¢.m . 4... 4.44... 4..3.w..,.3.3.............M 44043453. ............. 5 .

. D4545 .43.... «D3444 D4404Qm 3.3.3.4 DDUUWZ . 3344.0 3.0.....3.3..4..3.3......43R5 4.4.4.D4...... . . 45:; VEU 533.354.. 535344 a43uJ344w4aafi4a Dum.w.4...35:3.3.44 33......333. 3.3.3.443. $44.53. .:.J 4«mm.m.4.4.:.mNmMmMNHH... . D14 D4. .5 .3. . (.3:.. .44 .............................................................................................. . 94......5 G ..D3 ....... . . .. u . _. .................................................................................................. ..M....mm..mmm.miaa $44.33 . . . _4 _ _ . _ ................... . “-33.“..wm.m._...m..m..u..n.mm....m.. LE8 . ...a..m...wmm..mmm...3-4.2 .. . ... . r . n.mmrm .......................... .

. Wmmica . E ..... .

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS D T ........... ........... ........... Dem. .

DDED DE D. SE DDS E2 m DD. $2 DDS; «DP: . . mom: mom . D we? : ++ ...DD...DDDDDDDDDDDfiDDflDDD 0.0.4 mDrDDQDmDDQmDDD.Q¢ MDUDDNZDD DD...§.DD<.DDD§.D DDmDDDDDmDD.<D..D.D.DDDD DDDDD. .DD...DDDD,.D<DD 9 DDDDDDDfiaDDDDDDD.DDDDDDDD. UDUDDN .

. UKJDDD. $.13“.

DDDDDDDVQQDDr «EDDDDDDDDDJQDDD. . _>. .

. DDDD<DCDD gig. .

.D .

. .DDDrDrDagD fiflD<QU<< .

.. DUthDEQ. . DDDDDDDDD€ .

D DDDUMDDDDUDD, QED? DD<<DDD DDUDDUOAD .

. DDtDD .uuDD.DD... A . DDUUM D... DDDDDDDDDDDDDDDDDDJDDD.DDDDDEDDD- DDDDDDDDSDDD: D5...aDDDDDDD:D<DDDDDDDD2DDDD+DD. 2+DDMDDDDDDDDDDDDDDDEDDD+gm«DHDDDDDDJDDD DDDDD. .DDDDDD... rmDDDDEDDDDDDDDDDaDSDD... CL(DDDDDDSDDDDD...D.D.D..D...,.D.D..D.m...,.wmmm...:D DDDDDDDDDDDJDDDDD. Di... DDDDDDDfiDgDDDDDDDDfiiDDDD: .DDJDDDDDDDDDDDDD¢D<+DD¢ UMD<<DDDD .

. DD DflDmDasa. USED : + 2.... 03 Em x? D3 9.3 ma 3WD a... 33 DD? Nam» . : mam.

. . {DDDDD.H.H......_.D...D.DD...D..D...D..D.DD. .

DDJ}...H.D...D...D.D.fiD..H.D.DDwmwmmmmw: D.DD.3%..D...D..D.meD.D.D.m..a.D.D.D.D......wDD.D.. .

DDDDDDDDDDDDDDDDDDSDDDDfiD «D DDDDDDDDDDDDDDDDDDD:DD...» . .

D5 DD DDDDDD§.D¢D¢DD:<DDDDDDD «$2.5 DDDDDDDDD..,DDDD . 9&vaODD/.21...DD<<DDDDDUD D§D€<<< DD .

DDDDD<D<DDEDDDDD$DD DDDD .

DDDDDDDDDQD . .1. .DDDDD .

DDDDUDD . 4D DDDD......DDD.DDDDDDDDDDD:D DDDDDD... . . <<<MDDD . .

«GD .

D: D.DmD<u<DEDD. DDDUDU . . .9): DDD: .

DD .

DfififiwfiDmDQ DDDDDDDDDDDDDJDD. D . DD DDDD DDDflDDDDDDDD . <.< . .

DPDDDD .

...DDDDDD . «tum. ............................................................................................................ .DIDD... . <D DDDDDDDDDE. MD<U<3r .

. DzDDDmDDDDt .............................................................................................. .3“:.. . . .. . . . . . . g _. . _ .. _ . ugméam 3D :33me D. . m DNDm.mDDDD_. . mm.~.m.D..D_.:N.mDDD.DDDDD...D.Dz . . $3.33 3. EDDDDDDDD . .......................... . .23 .

. Wmmica E DD .

. E [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS V V93+VVVVQSVVVVVVVVVVVQVVVJVVVVVZ2V3 V T ........... ........... ........... ........... . wMVmVV VVmV 9P: .

VVV+ m5 ES £2 VV. m . . a: ,0.me .

......... ......... ......... V 2m ....+......... ......... .......... ........ gm DD<Q<<ODDQD<P 034V VU<G< ........

VVVVVVVVVVVVVVVV m VMVVVVVVV.V<VV<VV.,V«‘VVVVVVVVVVVVVV VVVVVVEVVVVVVVVVVVVVEVVV+..........w.m”mHMM.mwwmwfiwmmfifififlmmm VVVV<<VV VVVVVVVVVVVVVVVVV VVVVEVV Vm<VV§VV$VV<V§5VVVVSE r:rV<<._.......m..V.mV.uV.V.V...V.mV..V.V.Vm.V..V.m.V....V39 <VVVV<UVVPVV<VVVVVVV<VVVV «VVVVVVVVVVV VVDVVVVVVVV<VVVVVVVVVV<VVVVV<< mMmHMammmmmfifififiwfiw: <VVV<<UVVVDQGMVDUV, @er mVVVVVVwamVVVVDVVVQVU @D< UV): )9: :1: V‘VV V<VVV VVVrV MODE VagVVVVerVVV: 1V VD VVUVUUGWVVQ .V HQDVUDQMVVN DDKVVUD; «@4me '1 :VVVVVV G3< VDD a: rV VVVVVVVVUVVV 4 VVVVVVVVVVVVV VVVVVVVVVV ,V< BSDUa G)(VV D DQD B VVVMVVKUUVVV V3VVV VVVVVVVVVVVVVVVVMVPVVW VV:‘VVVVVVVVVVVSVVVVVVEVVV VVVVVVVV V‘VVUDGDD DOUG VB ......... ......... ............... .......... .......... ......... .......... .......... ......... V..........+..........+......... ......... .......... m m cam mam a3 VVVVVVV 32 3V: 3.: VVVVOV VVVVVV :2 32 9: V V 3 V: V: a .............. V V <«15:0D<<Q<VU<<<Q < DmVV. VVVVVVVVVVV «mi; V «VVVVVVVVVVVVVVVVVVV<VVVVVVVVVVV,VVVVV < «V VUV Qd‘mVafiViq VVVVVVaaoerVVfiVrV VV VVVVEVVVfVVVV: VVVSVVVVVVVVHVmfiV..V..V.V.H. V, ,VVVVVVVVVVVVVV<VVVVV<VVVVVVV3<VV .VVVJVVVVVVVVVV‘ <<U< V:<UU 594 a? (VVVVVV 34% (.395 {Vflwwmxfifir VV LDfiDQDLDv VVVVVVEVVVVVV VV<VVVVVVV V<VVVVVVV VVVVVV<MV<<<MVVVVV.,V< VVVVVV VVVVV VVV mVG/VVQDDVQ‘VHVRVV. VDVVVJVVUDVVVJVV VVV V<<<VVVV<VVV u<©fi<uuUVVVVVVVMVDDVVVVD Q;QD<< @er VVVV MEGS/m V5 QDVVEVV G VDVJ VVrVVVVV 3 VVV VVV Vagflme .3 Q ........................................................................................... V V wmmméV VVVV Raw. mm .................................................................................................. VNVVVVVVVVVV. SEW $me mefiVVVV mmam [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ............wam.K...W........M.K.mm.mwmmmKflfim....mwwfimmjm. .2;..K3.uwo3.3KS.K3.K-33NK3P.K. . . .......... .......... a: 3.2 333W 3.32 333K M. N. 33K N52 82 5.: .333. 39: 3.3.3 8.3K ......... ........ 3m 33H... K.l w b05335.13.@303...va3.DU UKQG<©Q<QQ<3N DQ3463QDQDKQD<QQDG§O 09:3.m3gN3fi3KSm R535:3.3.K..K3KK>.:KlK3935W .KNK.K_.3..K KENKKKKKNE?$3.38? $533.3.KK03K33 D<3KKNK36KKNK 35: -3.” KNK3.3.3.K.K.K:K3>K>KK33.K+ 253;... 33 KKKKKNKSK .uKl P 3:353. KN... 2.5..

DPKQ KKK 3:33 33.3.9350 u ...3.3...m..K.........m...K.aw..w...3....3.K.m.m.K.z.K.m......... .KKlKéKlK K...l.K3..KKKK:umafiiouw: wwwfim.....K.l.K.mK.w..m.K..:.K.mm...m. .K..KK«KK.K3.,K..K3KK>.:K.KB a:m2...9&353K3.KH.KK..KKKK..KNKK.Km 9.333%.lKKNKKKQKSNKKKKKNK3S NS K. 2 ..................................... ..................................... $039,333.02NK.3.K.K.K3.KKNKNK3. 3.3..D<A\<DDQ©DU ...K.3...KN.K33 3.33: 3.3.KK3K3. 3.333.633.3K3Kfi. K33... N. w.3....u 3K. 33$...

K.K._K3 ...... .

... KKKKKK... 3.35:.

L3 5.33.023: KK3NKK K:3.3.: K353 3.3.u.<3.K.K.uK..K3K SK 3.3.K K3: 3.3>.KK.K:NK..L.NK .9 3.59 .KK3..KKK.NNK3.K @3033? 5:35 3... N3... .KNK3K3.,K.. 3.1.3.3.? KKK3K3 DD 3.3.9 uKKNK K..K33. 33.3.3...,KNK3K33K3K KKKKKNKDKK3K3...K<3.NK 3.mamnw.....K3N.K.K3nmfiwmmww...mmmmmwwm K.33.33%..K...H.$3...H.K..3.K...K.N&...3.N.K.m.¢....m.3..K,.K.w..K.m...K.m...........VTKKEEK3N3KKSZ KlK_lKK>.:,K..K 334533:$33.3KKKKKK3.m3 53G...“ wNKKKKQKKQKKENKKVNKKKNK....K.NN.K.U< NKNKuKlKNKuwuNK NC 3.3K 3NK.K3NK..K3333.>K3.KKK».K3K.KNK KK§N.NNK..N K K. N8... . 3...... .

Ni. $3me3 333N333 ONKK33N..>KKK maize... KKKKKJNKKKKK. 3312.33 WNKEN; ...................

KK.......3..NN.NN Leia.... -3332. N..mflw.mmqm..m.mfl.am...fi./.N. w .. 3 KK KKllKKKKKaNNKKK..- 3 .K .K 3833.03 K333. 358.323 33.35393 NKKEKNKKK. :K.

NKKKK: :3 3:13.33 NllN.mNK.KKKKKlllK.Kz N,NK.K.NKKK N. N: N. N. N3 3:3 KKKKKKKK N33KNKKKKK 2 3.. K... 3..

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS ation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ........... ........... .

E... .3. . 2... NE. .181 .181 2... 1...... :2 .1. m .

U.m...m..........w.mm.m”wwmmmwmmw......«.3.m.......m._..1w...m...............m..m..... Ec<m...m.......w..wmfi.w...31.:$9... am... 3.. mm... .1S........w1.w.1....w...mmfimmmwm...www.mm..m.1m. . . mm... a”... $.01. 1 ++ ........ ........ . 3......1. m .

. Ba . $33... . ww<<bm 3..1.<.1..._u<c<5mm... <umuum..u<<<urmuw§. <QQDDO§1<<Df 8.1.1.1:9.....NH...1.11..1..§GQ.. 8.1.11.1...14n5.3......D11GQQ... mmmmmmm 1.1.11)1u..1..11..o1.1.1.P1.+ .1..._....<<:w......:cu@.1. was...:93“....1..fl.<...._u..u $50.... $1.13... ...11.w<u.1. . wD<u§ . $391...

. H...1Hv...: $05 5.1. M...,....<w3...m.:<m.1.1uu.....<cw...m 32......5111Q.<9...m DD<UU.....1..D<.£1.<1O$1.<1 H.333... .

.......................... . . MGD...: Do. . . 31“ 1.03.0... . Hu1wmv<< 351.1. . 59W . . 3.): . .159). HQ + + JE.Q<1.1..1D»<U:U<M ............... ............... ............... . mm: mm... 911...: :16. 1%.: $12 mm... wmm..: 2...: E: £1... .8. .198. “mo. we. . : own: .32 wmc. .............. . . . 1.91«Mmmm.wflwmwmmwmflfl1 ..111.13%...«ww«EMMEMWHMw...:..m.......m.. . ..............0m.w......3%............w.1w......m.....am.1..... . 11......«u..u.).u.u<..m1$u<<< p.554u0915.1...11.uo1...m..1. 141 <n.D»U<. <15......1......<.........9.5.1.311... . 1.4.... 1.3.6.31. €2.31...) 1......<3...uwu<35.1.1.1. ...,......M.M.c.).u..<mu...2......21...... ....1n..1..1;§<§ 35.1.3539....H....1.A...:>§ ..<_m...,1.§.uw.).<13.2.9.511... . . < $.15...“.1..h...m......mm.1.1.u...mfim.w.. .

G . . <<<<..D.

R :5 . 31.0“... . r w..<<C...?><x . .41 . 1..) 9.1.13.3..1151ww 35.1%.... <05 . .x.<1<<.u.1. . .30504 . . gu<1v<< ..n.<1.1<um.u .01.... . . <Um..3$< . via...

SD»... . 1...: 1.1.11 .

. DU»..U...<<GMVDU<Dm.UU....1.U M31121... 13 .3“: . . m.

.................................................................................................. ........................................................................................... . . . Swims. ammvmmfi. 3.1%.... .....mwm..mw.w w: 3815.2. .13 . w . cm . 33.1. .113 .

, .H . z. . _ . mm. $1:me .................. . 11w 1. 1. .N . m m. . .1...” m1.o..1m...11~m:..o12, 3.15. 3.1.33... ...m..mHN.H.mm...mm,111 ...m....u....1..mm1m...m 33.1.9“: NEGQ :51QS mm...m . 3%... N... . . mmmfimmmw N m. . mt... $22.... .981 .......................... .

. Wmmicc $13.2 1. .2. 3., .

. E [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS .VmV+ V38+VV<V4VVVVVVV-V€VVVVV§+ T ........... ..... .

SS 3: Va: 2.3 VVV m V. .

VEVVVVVVVVVVVQVVJVVVaV}7.9... :1: 3.2 V..: .VV .

..V<VVVVV.€VVVVV<VV<€<V.3 . 4 +.........+......... ... .......... ........ -5 m QGEQVVNDUPH DDDQQQQVVNDUDVVUDDMVVNDAV. VVVVVVVVVVVVVVVVVVVVVVVSVVVVVVVVVVVV VVVVVVVVVVVVVVVV.VVV.VV.V.V.VV<<.V .V.m.V.V.V...VV..VV.V.w.meVmVVV<V4.3.VV+ VVVVD .VVV.V<.V.V ..VV..VV.. VV.VV.<V§.VV.VVVVV>VVVVV.V. V...V§.VVVVV<<VV.VV<.VV .VVVV.VV<V..<.VVVV. GDUVVVUVVV WVrVDQZrVQQVtV .UDH VVVVVVViaVVVVV VVVVVVVV «.«VVVV§.V<V<VVVVVV.V MfumtVrVDV. <042va3<©<.

VVVVVVVVJV ..V...VVVVV...<VVVVVSVVVVVV.W .:<VVVVV.<< VVV<VVVV VV<VVVVV VVV<VV<VVVVVVVVV QVVVVDD . V.....VVEVVV.V.4..VVVV<V.VVVVVVVV<<< QDVUGDVU ED .«DD:mVDUwaAVVUmVD 2&3wa ............... ............... 3V: 09.: SE meg $2 EVE mm W mm... Va $2 $2 VVVVVV ”EV 2.2 V.....VVV V.....VVV VS: RV: mi: EV: .................. . .

VVVVVVVVVVVVVJ4<DVVVSV<<u Sf «S. <9. VVVV @03ch VVVVAVV. 4... V....VJ.VVV «DVVVVV. <34 VDUVVTV V.4.

VVVV.

VV<_VVV<V... V..VVVVV.VVV< V....VVV.V<VV {.VVVVVVVVfDVVVV. VV V...» Gngr VDVVquVVu <<VVV<VVVVVVV V.....VV V..? .4..VVVV.EVV.VVV.V.VV>VV VVV.4.. ...........V...q..a.mV.VVV4.V....V.V.V............H..VV..V.VMV.....W.V..H.V. «.0..mem.V.V...V.V...mV..m.V..V..V.V....V.....VVw.....V..m $32 D<<WV3 <QQ<VVVVVQVVHVVQa VVVVVE G 13.. -5VVVVVVVVVVVVVVVVVVVVV. VVVVV < DO r .. VV. VVVVVQV< V.....VflVVVV 4:3 .3590. QGVVJVQ DUVVJVVUDVV. V.<<<V .VVV 5.

.V< DEV VVVVVVVVV VVVVVV V4V..VVMVMVVV.mfiV...V.mV..V.w. .VVVV..H.H”www.mm.m....mV.wV.V....V.m.....m....... .VEVVVVVVVVVV.VV.<V...VV.VV..VVVVV>VVVV. <VQVVVVL<>VVVVKV 5V... VaVV: VVV VVVV V.....VVDUVVVUVVVU Vt CG JGDGtU . . _... ._ ._ wmmv: .. .

. .................................................................................................. ........................................................................................... VVVVVJNNVV. VVmV...NmVVV. «.mVVwVVVS. ...V.H..H.V..:V.VVV. 3323 VVVVESV 5.9. 91.4%;qu v.3. m.VVV.lemV.-N.mVV. :3».V....mum.H.V.VV.V...VVV...... V V. m m V $3.8... :2. mo? bmmfigmv ZVmemmVV amemew wmmficvmw m. m. NA. mica Wmmica $12.2 3.. E [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS EmSEE EEEmEE+o¢uEEE<EE<rErEEEEEb<EEmEE ...........

NEE SEE E: EEEE 33E E $3 $2 $2 BE: E63 $3 m + ........

E ......... 111E.EEEEEEEEEEEEEEEEEEQEE:EEE Q§EEQEEEE<D<EEEEEEDD1EEQE EEEEQPDbEEEEdEErEEJrEEEJE «EDEEEEBEEEEEGEEuEEEEuuauEEEE EEEEEENENEEEEEEEEEEEE EEEE<.EEEEEEE.E<E m ,1: u :1qu EEEEEEE .EauEEEE EEEmEEEEEEEGEEEE.EEEE.1E EEEEEEEEEEEEEENEEEJEE ,EE,EEEE< .EESEQEEEEEEEE w.m.E.mME¢.mmE.E.E.Emfi.mE.mEM.E.M.E.M.EM/E ENEEEEEEEEEEEEEE) 3% EEEEEEEEEEEEEGNEEE1?EEEEEEEEEE EEEEEEEEEEEEEEEEEEEEEEEEE EEEEEEEE<EE<,EEEEEEEEEEEEEEEEEE ...... QEEEEE EEEEEEEEEEEEEEE<<EEEEEEEEEEESEEEEEEE EEEE3.34..EEEEEfiuEEEEEEEEREEafiuE Em8+1E.E.E.Em.E.m.E.mmgEEMMEEEEMQEMEEEE..EM..EWE EEEEEEEEEEEEEEE><EEEEEEEE1EEEC<EE+ 155.2% EEEEEEEEEEEEEEE E,EEEE»E..E<EEEEEEEEEEEEEEEEEE ................. ................. ............... ............... 32 EwEEE £9... 9W2 3E: 9E2 m N m N. ma WE $2 $2 EEEEEEE E?,EEEEEEEEEEEEEEEEEEEEESE03 SEEEaiEEzECiEfiEiE E9 $2 $2 mm... w: mEEEEE EEEEEEE 8: . ............. . ..............

EEnEmEEEEEEmEEE<EEE.EE_EEEnEauEEfiEEE EEEEEEEE<E1EEEEEEEEEEEEEEEEEEEEEJEEEEEEE .,EEEEEMEEEEEEVEEEE ............................... .EEEEEEEEEEEEEEEEE<>EEEEE< 3 $11.31 m.EEEwmm.E.E.EmE.mMfiwwmmwfiwmmm EEEEEEE .EEEEEEEEEEEEEEEEEEEE 13EE3111amfiwfimfifi: «330.31: ..........Emwfi.E.E.E.EmmmwmmEmmmmfimfifi.. ..........mumMm«.w.w.....E.E.mE.wnm.E.,.Efim.. ESE :EEEEEEE§ EE .1 a? 3E...... EEEEEEEEEEEEEE EEEEmEE <<EEEEE1EEEEEEEEQ1<EEEE EEG EEEEEE EEEEEEEQEE .EEEEE EEEEEEEEEEEEEE 131:: ESEEEEEEEE 32E?wfiEmEEEEEEEU: EEEEEE1mEEEEEEEEEEEEEEEEEEEEEEEEE E21 . ....... . 311$. E .E....m. $9 .....

E :EEEAENEE EVEEEEEEEE E3. $8? ENSEE? ...EHE.m........N..E. EWEEEEEE. @3meEmm_E...N.m.EEE.E3-2.2 ii: ........EE......_E”w........E...............m..mm.E..EE..m...EEE.EE..... ................. E m m :EEEEENEEE. «:3.

N,m.E..:EEEE N EMNEEnmE.mm...m 2.

EEEIEEEEE 12 E ................................................................................................... mimwmmwmfimmfl: .................... ...........

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS UU...UUUUUUUU<UUUUU35§4UUUJ+ .FUU+ T .... 4 USU 4 EU UEUUW UEUU UUUWUU UM: 9...: UUUUUU am: .UUU 4 +.........+......... ......... ..........

.. ........ DUUUDDUUUUmUaU4&4UUDUUUUUD UUUUU4U<UUD<D<U<DU <D<U<DUDIDDDU<UUUUDD U.

UUUUU.

U(U._.Ur U.U<.UUU Um? D.DUU<U<< UUU<UUUUUUUUUU UUUUUUUUUUUUUU .UDUUUU.UUUHUDUUUUUUUUUUUUUUU UUU QUUUSUUUUUU<<<UUUUfi A K.U... UUUUUUUUUU.UUU..U.UU<<U.UUUUU.U:U.UUU UUK.DUB«NU/UDUMUUUUJUUUUUUU 3.: . DDID :UUUUUUU UrdUDPUUUraUUUUUUUU5+ UUEUUUUUsUUUUUUUUUUUUUUUUW 9......UUUUUU<UUUUUU<UUUU..U..U:U.Um UUUSUUUUUUUUUUUUUUUUUflUU mUE.m.U....UU.U.U.Um.U...U...mwfimM.U.U.m.Um.U.m....m...............m.U..U..U... UUUUUUUUUUUULUUUUUU: UUUUUUUUUUUUUUUUUUUUUUU UUUUU...DUUUUUUUUUUgUU U: UUUUEUUDUUUUUUUUUUUUUEUUU UD .UUUU.U.UUDDUU.\.<UMUUUEUUUDUU< ND UUD<<UDD<DDUU<UUUUUUU<DU ............... ............... ................

Us: me. .33 UUUUU U UH. mU D UUUUU Mm... moi: 3: UUUU UUUU ..U..U..U. U 2: E: UUUU U 3.: 3N: UN: ............. . .............

UDUUDDUUUEUUUaU «flaw UUUUUU.UU<UU..U..UUUUUUUUUUUUUUU U. ..........EMU.fl.m.Ufimmem..m.UUmme.UmmfiU.U. UUUUUDUUPDHUUCJUU}. U. UUUUUUUUUE: UUUHUUUUUUU.U<UU.._U UUUUUU .........mm.....U.mum..UUUMUm..U.H.U.m.UUm.H.U.UmUU.w.. U.U. SUUUUUUU:UUUUUUUUUUUUUUUUUUU U... $323..” UUUUU...UU.U <06... U.D.UU<U.

UUUU)U .UQUUUU. UUUUUUUUUUUrgUEDUr 5D. ..

U. UDDD.

UUUUUU. 34. .UU... $4.1...UU. UUUmDUUUUUUDUUQUUDU.

UUUVDUDf UUU UNUSUUDUQM .5 UUUUPUU..UUU UUUUU $9.2... .UflUUUUUUUUUUUUUUfiU UUDDUUUUUUUUD... .DUUD :UUU. U...

USU <UUUUUU<<U<UU>$ U<UU .UUU.U<UU.UU<UUUU.UU<UUUUUU UtDU.

D EUU EUU D<UUmU _. .................................................................................................. ........................................................................................... .U.._ .. _.

U . . A...mUUUmfi.UmmmUmUm.mU..U.U.U.U..§.U.U.m.............._ U. 30+... .. 3.5..

.. U _. ........ . .5 253%. UUAUENUUUU mg cwawamwflu 03. m _. U m .U U. U m m ._ UUUEUUUU. UNUSED. UonUU.UNU.Nw.UM...NU.W..UWN.U.UUUU...UUU.U UUoUz U.;U.U.\..9MUUU..U...N9 133$ UUmUUU.NU~UUU UUUUUUANQUU mUUUUUUWUmoUU UUQUUWNUUQU UmmmenmUw 3.3%an : N .3. n. N. NM 9:03 ., UKUUUUU EMUUUUUU :8 mica Wmmicc 22 U2. UUU DU 3.2 U2. E [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS 444.4...44434344444044.4442... 4444.44 ........... 844 4 4.44444 444444 :4: m .4 4.440444444444444.444444444 44444 4.44: 8: 4.4 4.44.4: 444: - .54 4 ........ ......... ....... 4.4 4-4. 4. 4.4.4 .44.. 44444444444444.444544.4.4454 43.4444443444444444444u44m54d 44.45.434.443-8344 “44444444444. 4444444444. 5444.4 44.44................. .444. 44.44.. 444.444.44.44-44444... 44.444433484494454 «6.4444444.4.4444444440444445 4.4444.4444 443444444444. 44444444444444 4.444.444 4 44.444.44.44444444444.444.44.44. mummwamflflwmmfi...m 4444414444444 44444.4... 4.4.4.44. 4444.4. :64. .4544444444.44.444.444444 44444444444444.4444 44344444444432.3444 443:444444445944444.34-4444 444.44%}...4444.444444.444.44.44444 4.44444444444444444444444444444 4.4m4.444444.44444434444444.4444 4.4 4.44.. 44 4.4 . ..................................... 444444.444.4.44.44.54.44.454.4445 4.44.. > 444.4444 444.4 444444445444 4.4.44.4 4.444 44.4.44. .4444, 454.4444 .444:- 4.444.444.4434 444444.44444444444444444 444.444.44.444 44uw4-44m4u4 4.44m44444u4...44<4.44.4.444m4444.4.4.. 444.444.444.4444444444444.444.:44 ....4.4.........4.44..444.Mm4.44....44.4.4.4.m4m...4.fl.w.. m..-.4.4.m....mmmaw..4.4..4....4.4.4...4..4.....w.4.4. 444 444.444.494444444444 a444.44.44.44.4444.444444444445444444 444.4444.444.4444444.n44.444.4.4.>4444.444 4.4.44444444. 444.444.4444.) ...) 44.44 4.444444444444. 4144444444.. .4444 444444.444. 4444.44. .84. 4444444.... .444 .4. .444 4.4.444. 4544 4.444444444444444 4.44 444.44. 4.44.044 4444444 444.444 ..44444444444604444 4 4.4.44.4.4:44.454404454444444. . ...... 444.444.444.44 4.. .4.-4mm? 44.44-44.444 R4324. -444.44 4.44.4.»444441.44”:4444-44442 44444. 4.... 34.. 444444.484 .4.-4.4444 44.44.44.444 44.... 4.44. 4.64444.449....N...W...4M.4.4444...444 ...............................................................................

[Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS .............................. ........... my... W VN: VN: EV .31 :5 EV .........

....VVVEVVVVVVafidumflVVVVVDVVVV. D.) VV4V)44)«)4VVVVVVVV: VVVVVVEVVVV «2.4V. ..V.4VVV)V.V,V.4.V4.<.VV V):V4)VVV)V4VVVVWV<)4V)+ WV. .W .VVV. VVVVVVVVV.) E4 EPVVVV. .4 V5.44.) .4VVVVV4VVEVV EV V4<VVV)V.E<VVVV4 VVVVVEVVVVVVVVVVVVVV VVVV<VV..V.

V mmmwmflmmgefl EVVVV.V..........V.V...SmWflflwwWme...VrV... .VVVVVEVVVVV VVVVVVDVVVVVVV: 4VVVVVEVVVV44 ...........WWW.V...m........HWMWWW.V.V.Vme.Vm.V.V.V.mWm.V.w.VWm...mWVWV...m...............WW..V..V... EVVVSVVVSVVVV. VVVVVVVVVV~ VV<EVVVVVVVV<VVEVVVVVEVVVVVVVE V..........._WWW.V....m...........VV.V.V.WV.ME.W...HWWWmeWflWflVVMVVWWVmem.JW. .WV.WV«NVNVHVWVVWWWVmWWVVWmmWWVVWWVm.)W. VVVVUVVVVVEVV VVVEVVVVVVVV VVVVVVVEVVVVVVVVDVV; VVVNVVVVVVBVAV VV...VVV<.44 V)E.VVV4.:.VVVVE 995%wa V VEVVVV VVVVVVVVVVE ..V.VVVVVV.V<V Pam VVVVVVVVVVVVVV 5:5 ............... ............... ............... ............... 2.: VVVVVV VV: 3V: Va: W W W W mm... VVVV NV: in: rm: W.V..V... a: mm: V..3. WWW s: NV: 3: . . . ............. ..............

VV ..VV V.4..V V549). .VV. 34 VVVVV <<4VVVV. <VVEV A VEVVVVVV.

.VVVV.)4VV4.,VV.4VVVVV< :VVVQVV4VVQVVu4 4VV4VV45V .4.V<V4.VV.4)<V4.V V3: :VVVVVVVfiVVVVVVVVVVVVEVEE VV.) ..VVVVVVVVVVVVV4VVVVVV VVVVVVVVVVVVV .9 VSVVVVVVVVVVVVVVVEUV). .4.4 ................V.V..V....W.V..V...V.V..V.WV..WV...4.....)VWWV...V.VWNVVWVV.V. 4VVVV4VV4VVVVV4V-)V4 VVVV44VV,VVVVVV...V V4.4. .............WW4..WV.WV.Wm.VVW«VMWVMWHWVNWVWVWWV.. ...........W.HVWVWVWWVMW........“.WV.V..V.WV.V.VV...V...V....V.V.WV.VV.V.. .VVVVJVVVVVV:VV<VVV44VVVVVVVVV. V_.VV<.: .VVVVVVVVVVVVVV4VV4VVV EV..V.fl.V...WWWWV..VVWWVVV...V.V.V..W.WV...V.WW...WH ...Vnnmmfififl.H....V.V.V..V.V..V..V.V...V.V.V.WV. <VV.4)4.4)4)4.VVV4:VVV4:VV4V.V.V, : VVUVVVVVVV V.. V...V.VV,VVV V VVEVV . .

VVVNV-NVVVV ................................................................................................... ......................................................................................... .................................................................................................. 3m _. _.

. . VV VV VV VVNVAVWVV..N.VVVV.VVVVV-VVV2 3%.

VVVNV ENVNVVVNV VVVVVVVVVWVV VHWVVVVV NVVVEVVVV NVVVEVVVV V2. Va, ........V....V..H.WWV.WWWW....W..W.VHW.H.WW...VW,. 2.

..... ............................................................................................ .................................................................................................. [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS (’7 KI? r“; (‘3 r- ;\ . ..................... .......... .......... 395mafiuggflmfin UGqufiQGEQDDDQDDDb QDDDUDQHQ‘IQNDGQH r a m r ,3 Jana r: wflmfixw r" 3< i; $3.44 L.) m :2) 1:»: :9 DDDOGGxVUDUfi .35qu 7: ,Sr’ 5me Q ‘05.”va ......... ... ..................... .......... ..........

E: mo: mo no xw .0? 4 N M M I w.Baum:35muun<u€<<< <<<<u§AQdDDDGDbDUUQ «AadeflaQw < DDUgfix<QU<U<<UG<<UU $3va <VHVD‘AEEQJKXN 33359qu <D<<<<Q<44Q puumfidabmm M amfii flag. , flaw. amm;m.m.fl.§122 :“Esmmflsefiz [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS e 521:3 Viva €35 Silencing Groups ot‘three female cynornolgus macaques were treated with CS ~siRNA AD—5864l subcutaneously in the scapular and mid~dorsal areas of the back at 2.5 rug/leg or 5 rug/leg doses or a, vehicle controll Two rounds ol‘dosing were administered with eight doses in each round given every third day. Serum CS was ted and evaluated using an ELISA assay ic for C5 detection (Ahcam) at the ted time points (Figure l3). CS levels were normalized to the average ofthree presdose samples. Samples collected prior to dosing, and on day '23 (24 hours alter the last dose administered in the first round ot‘treatment) were analyzed by con'iplete serun'i chemistry, hematology and coagulation panels. ll) Analysis of serum CS protein levels relative to pre—treatnient serurn CS protein levels derritmstrated that the 5 rng/lrg AD-5864l dosing regirnen reduced serum {35 n levels up to 98% (Figure 1'2). The average serum C5 levels were reduced by 97% at the nadir“, indicating that the majority culating CS is hepatic in origin. There was potent, dosewdepehdent and durable kneels—down ot‘seruni (35 protein levels with subcutaneous administation ofADfi 864l. l5 No changes in hematology, seruin chemistry or coagulation parameters v ’ere identified 24 hours alter the first round of dosing.

Serum heniolytic ty was also analyzed using a sensitized sheep erythrocyte assay to measure classical pathway activity. The percent heinolysis was calculated relative to maximal heinolysis and to background heinolysis in control samples. Mean hernolysis values H" the SEM for three animals were calculated and analyzed {Figure l3). llernolysis was reduced up to 94% in the 5 mg/ltg dosing regimen with an average inhihiti on of 929/2:- at the nadir. The ion in sis was maintained for greater than two weeks following the last dose. e 62: In Vim Screening of onal siRNAs The CS sense and antisense strand sequences shown in ’l'ahle 20 were modified at the 3 ’— terminus with a short sequence of deoxydhyrnine nucleotides (d’l‘) ('l'ahle Ell The in vitro y of duplexes sing the sense and antisense sequences listed in Table El was determined using the following methods 2‘l9 [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS Cali (:uitztre and Veterinarians llep3B cells (ATCC, Manassas, VA) were grown to near contluence at 37°C in an atmosphere of5% CO; in EMEM (Al—CC) mented with lilo/2) F853, hethre being ed from the plate by nization, Trai’isteetion was carried out by adding Sgl of Upti—h’lEM plus Oil ul ofliipofectaniine RNAiMaX per well (lnyitrogen? Carlsbad CA. cat # l37'78—l 56) to Sui of siRNA duplexes per well into a 384~well plate and incubated at room ten'iperature for l5 minutes. 40M of te growth media containing ~55 xltl3 llepBB cells were then added to the siRNA mixture. Cells were incubated for 24 hours prior to RNA purification. Experiments were med at l0nl‘vl linal duplex concentration.

Total RN34 isolation using D YN/l BEADS mRN/l ion Ki! (In n, part # 610—12) RNA isolation was performed using a semi—automated process of a Bioteh Eli 405 washer. Briefly, cells were lysed in 75 til of Lysis/lflindin g Buffer containing 2rd onynaheads, then mixed for it) minutes on setting 7 of an electromagnetic shaker (Union Scientific).

Magnetic heads were captured using magnetic stand and the supernatant was removed. Alter removing supernatant, ic heads were washed with 90rd ‘Wash Buffer A, followed by 90rd of Wash buffer B. Beads were then washed twice with lOOul ofElution buffer which was then aspirated and CDNA generated directly on head hound RNA in the 384 well plate.

CDN/l synthesis using AB! High capacitor.» CDM/l reverse Zranseriptinn {cit {applied Biosaa’tems, Foster (Tin), CA, Cat #4368813) A master mix of Zul lOX Buffer, (3.8 til 25X der‘l’s, Zul Random primers, l ul Reverse 'l‘ranscriptase, lul RNase inhibitor and 3.2ul of H30 per on were added ly to the head hound RNA in the 384 well plates used for RNA isolation Plates were then shaken on an electromagnetic shaker for l0 minutes and then placed in a 370C incubator for 2 hours ing this incubation, plates were place on a. shake in an 80°C incubator for 7 minutes to inactivate the enzyme and elute the RNA/CDNA from the heads Real time PCR 2 ul ofCDNA were added to a master mix containing 0‘ 5 ul GAPDH 'l‘aqlvlan Probe {_Applied Biosystems Cat #43263 l 7E), 0.5nl C5 Taqlvlan probe (Applied Biosysteins eat it [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS 11300? 56} 97___Mi) and 5.141 1.,ighteyeiei 480 ptebe master mix (Reehe Cat #0488730} {30} ) pet weii iii a 384 well plates (Roche eat # 04887361661} Real time PCR was done in a Reehe LC480 Real Time PCR system ). Each dupiex was tested in in at ieast two independent transfeetiens and each traiisteetieii was assayed in duplicate.

To calculate relative fold ehan gm real time data were analyzed using the AACt method and ized to assays pertained with eelis transfeeted with MUM [RD-1955, or meek tiansteeted eelis.

Table 22 shows the tesitits efa singte dese Screen in Hep3B eetis transfeeted with the indicated dT modified . Data are expressed as t ofmessage remaining relative to it) untreated eeits.

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS .ww.ww¢§<$$ Fawn» HWHUGWEGGDDUG3&va 93%QUQGU<<<{DUMB—x .§.§QQDD< EMEQG DU ‘ 8ngme U4<<GO<UQ<DGG<GGDD . «1% D<<<GD QWGGD Q93 4 U e: $0§U<Q<<UQ @9291 93m um wax is: an Hflwwfixw<GG<UUUUDD dJQ‘qQQQQ wawQQDaVDGDwanDDQ ‘ Omvmq‘ 1:): @062me \ 3‘“,va<<U<<<<¢><xQ DEG 5% H331<Dm<<aw<um<waw U DGQDDDDWED D<<<D thmUDDQGD4<<h3<flflvabwma $<G<D<<DDD HwE3Ugafimmgfidufia Ecuwmwammwwgufiémx UU‘QVD m ‘ Exam, .m D news ‘ ‘ ‘ ‘ ‘ ‘ Ssoaza Nmmioc M . . w _ _ . _\ a- __ a . _ ._ @707 m3 TS. _ . . . ‘ ‘ ‘ ‘ ‘ ‘ ‘ ._ . .

. H . H __ . . _ . . . . . . _ . _ _ _ . _, H H H % Hme<<Um $wa Esta? hmhfivm Hwhwfl Hmth< % flaw/«DOGS H .53 ,5 \ d . wflZMfl Maw mBadgnibflwi‘ggU<3<<GU HWQUUDDMD‘AEND < Q<U<DQCGDUO<<<<GJ D< N§<D<<<<<DU<UG< DflvflvmiflwmeDUDDDGO UUCGJ m m M3333 13% \ .93 Mw<<<m DMD wwgfiamfiwéflu HEESQQDDGD UGDGUU 1» i @qu wwwxwag 15% . vidmvwfi ago“: Hggmowwfimuumufl .1; GDD<D<<<¢AVDOD<UO< u) 343‘ 595m.» DDDU UgvfimflvQDDQ Q<QGG DUDE/mu fidfi<<<<<uu<ow Q HwHwGPQDxQQAQAwQDanQ<D<QG ,Hwhwaqfiwwéowfi<GDDD<UG<<Q UD<<U<§H¢D HmhfimmwflgagdfigfldmmwDu. 535 kw ‘ ‘ AN m. N. m. m3. gm. Nflmw mam £me .Lvim: m m m on my on m. b Q4 gm Q4 m3 TVA? [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS HwHw<<©D< {EH ,mvbg Esq. Hmbgbmfifibfixfix Hvafixx. gfi PMFWWD<U< bfiwuuggm HGHHUKQFKDNE hwkwxéafim HGHEVNVQDDDUDNE {Rwrflfimvfifim .EwrwwxH33§><x<mv<mv< .wckwmv33.53333sz hmrwaxgxfifiubmz .ngwmfivmgflvmv3mg... Evin.“ rm.w.§_..w U<31R 3U: wDD< bywwmvxum 3393.33.“ <<< 3.3%GQAEUBD $3.339). wax DEBUG G<Q< .4D<QG<ODDDQDD buwafiflgwwflthVDODDOQ933)» buHwQDDQODPQDODDfiNQQGANQ ~3th3.MD<<UGU<.UUDUUDUDDN HmeUnfiMVDMQPQGQGDU<<<C<< UUEQ «JV...» m3~waD<QD kcHWWDGDDDANMJFDDGKVD<UD”.33..wa UngDxVQUDaEJV HfirgfiizxfivimaDQDQDQDDDGCAQ. 3.

.U< 33a... <<D<< DKNDDNFJKUQ 002.. < L.50<Q< mw<<flpy Q .UDDn $3“va M32". 33..." 59,“; 5352.3 wUCD<<<<<Qw Q33 .H.Mm33.._mv<<m <3: GDUEGDDBDUDUDE—gm.» U DUQD/m A w ..:.HD3..3U...WD UUGPQ/SDQ DD ND<U<U<D<< U<<U< .U.< U<<<D<m§ DUGDU< G3U<D3w5©<< 2 $33.53U3 3.5. S my)“.3.5.C<<<UD< mfiflsmw mmwémv wnvécv $34.33 (’1 “Ci" m \‘Q {\ 00 Q (‘7 “Ci" m 6' ‘ (n ~ In x , . . HT. “xi? \rJ m ‘5'; “xi? 3; L~~ g\. g\. r- [\ 00 00900000 00 ¥ ‘. p. 5‘. I“ 5‘ r~~ r~~ p. [3. r\_ i". . v—«w-«v-awd "-1 7.4 rhubufimawaqdmaWDQQDxVfi: [SHEEN .3..va . HHHEE {$3.84. HEM...» .EVHVGM.“ .m ?) HmeGDUDU<<<<UUDD<<<3.34% <3..wa Hwhc<<o kwhwu<< HwHwDUD U $.63?”wa <U<< %wh3§1§0bxfi. HGHGQDQDQDDMHN Hmrwm<<<m< afivflxfibfg HWHfiDmH§><x<U< (waxwmfiax3.3.534... .H.M§%<<QDDDE 13.93.5393?wa .Hmw.<<fimwNED<DQDQD< .

DEM.“D<UG<<<E.FA.§ D<<<<6.UD<<<<U¢EDD J<<UDQD<< .HD<QGD<< 3..? Dfigbbgq UU<G< Sb<a><x<3m HwHwUB«EMvDDCUDm (www.mhgmgzmaxaaQUQ<QH3V<<m3n U<<< .JJVQKNMéUDU DDUDaxfivmngDU wwwflfidwflmfi Oxyajowwflmu va<.Gm3.WUUGDw§\< UU< G<U< <<U< ..WDQ<< «£3. ND<<UUQ «$3.239.» Q33U3UDw5<CDD G<U<D<H UU<<<U<G< DUDUKNDaéiqbr <quv<uu< C<DDG<<<¢Y 94¢ng QM £33.“:“VENDDUDQDHE u U .UQ .UQ <<U DD<U a,” D G< GU{.m.rfl<<m&3§b G<QQ m4 N39” m N.wowG-Q< m. N.w m N. mdww N. m N. m Nd m m N. N. N. cow N3 m VNw mmm mom EwEAm< ham 3 3 me Ndmw Nwom Now N: w w mmm wm 4 mCaflxu .. wE-Q< E N.vwn$-m< 3. M my- a. 9% a. my- 0-3.x.» my- .3 .meu 0-3.x.» a. alga a. a. .0: Q< Q< Q< man. fix» wsmgéfix fix» man. N.$Nm$.d< m..Q< Q< fix» mix [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS HDVHDVuV.VV.VnVnVD4.V HwHVVrVP. HwVHVV0VD44V0D4.D4.4._rVrVDD444D MVUDVV4. HvHDVmV44VHV44.4x4.mVVr.V4.HVV..V HVVHVVDVV44. VV HGHDVVVMV44DHVVV44D4. HVVHVV4U44HVHVV. HVVHVVmV HnH HVVHVVmVDflVHVVV HcHDVVVDD V.VV.V.VVDVHVVV4 .D..».V.D.D_... HVVHVVmVDV..V4.D4.U4.VHV HDVHDVVHVVVD.. .HVVDVVVV4. H. .V.VV.V.VV .VDVVDVw ($4.? DVVV HDVVV V.VV.V.VVVDHVV...RV.VV4. .V7.. .V..VVV.VV_V.VV QDDD4.4. .meV4V.V.4. V: V44D4.V. V.VV...V44 “V44 .mV 4.5. DVV.4. V44V D3344 4.34. V.V..VV.

D. VDVD mVrVD V..4.V..VMVVD V444.HVVV444 {:44 $44.: V.

V. 444 .V ..444 DUDVV 4.4.

.VD4...VD4.D U4.mV44 V..VV.VnVD4.4. VVrVD4VVD .VV.V.V.VDV VmV444.4.44V.V .. V..,VV. D..,Vw Va4.

V.V4. VV.V.V.

JVV4:V MwVwV4. .VmeVDVV Bang VD“ V 440... 4444444404.

VrV4x4....VV..VmVD VHVVV .mVVVU4. V. V....D4 .V...V.V..HV44V.V44 4.4.4. a TV D4. V.VV V 44 004. 4.VtVV. DUD4. D404. V....DVMV4. VVHVVVVV .VD mVV. 41.4.0. 4 VVVV.

.VU44 244x D.4..V.V..DV.4.4.44VV.V.V4444HVHV4. 444 V .DHDV .mV44VVVV D44>V meVD D: VanVmV4.V..VDD4.D44V.DV..V V44444.44.VrVD VuV...VDV..V4..0 V444VDV.VV. V..DHV4..4.DDVVUDD44V44HV4.$44. D44. ..V44V..V4.VrVDV.D DHVVV .V..DHVV.VD44V.V4.DVD 4. V.

V V4 D 4.4.VVV.V 44V.VMV4.D44VV 5 HVVHDVVVJEVDQQDVN HVVHDV44D HDVHDV4U4MVV. HGH H.. ..VV H.

HDVVV V.V.V EV. HDVHVV444DDVVVVMV HDVHDVVHVVV4. HLHD4VtiVVDVtV HVVHDV.4.4.V..V 444D4D444.4.DUVVMV4N44HVV..V4. VHVV44UDV. HVVHDVVVD>V4.4. HVVHDVDV..VV.V44V.D4. .VDVVVVDVDHVV... H©H14.4. V44.4.:44V.V4.44VV.4.UmeV<VVUD4.4.V .HVVHVV .V.VV.V.VV44VVV.VDHV4.4. .HVVHVV4VVVDVVV...RV.4.4. D. ..VV ...V..V 44.4 V.V44VD4.:_V.V44V.VD444 HFHDV4.

DUUDV 44DDDVVHV<V.V..V. VD4. V 4.V..VVV4.4..1 D.VVV.V4.4..HV4.HVV.. 40C4. V4. mVVVV...H .VDDDVVUD44VVD444.mVVr.V D4.mV44DHVU5V4x44VVNVDVHVHVHV 4 4V..V V44. VDDVV4. 4.4443344 V.( VDUDV VA V44 .4.V.. 04 MV V .V4444>VV VVrVDV VrVD VmV4.V.,V.V.. G44 V. MVVVw 444.344 .V D40. ....V_.V.>. 091544044. V.VV.VV.. D4. VV.V4.MV44 NVMVUHV44. ..4. VV. V.

WV a D424. 4mV4. .4.V..

.V:VD V4.D4.mV4.DHVHV VD Va a V44V.VV V. V..D 15.4% VVDDQ4.4. 4V.V.4. mV44VVmV 4.4. .VDDDU44. V.

V VV.V.. VVVVVHV .DV.

D41V .V.4.44V.V .VVVMVDD MVDVVNV4. DVHVVV .4.V.. VHVV.V.4. V44 D4 .meV V4 VVV4.4444 V..V MVVHVVVNV<44HV V..DV.V:..DV.4..V.V44MVVVQ mVVD4d44V.4.VH.V4><44.DDV.,V444 V:VV..VV..V4.D4.4.V..V $4.944qu DVVVVVHV 3.344 mV4s4.V...RV.VVD4.44VV4. 4.4V .4mV VV mom...

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS wove HwHFD HUUHUUMUMU HMUHUUMU HMOHMVUW .M.M.MU..

»Dr.UUmUPU HMUHMUU HUUHMUMUCC .H1MUUMU11 HMuHmUMUH HMUHMUU 1U.mU1U.U_1.M..

NUCCUUUC [UHMztUUMUC .U1C1CCCMU HcHUUQCCMUMU HMUHMUUUUCUMUCC HMUHMUUUCUD» MM. U..HUUM (UMUHMUMUMUBH ..C.M.

DMU1U HUUHHUCM1MHHUM1UMU1UUU HUCHUUUUMUUCCUU. UMUMUH :11UUMUC11MCCCC1UMUC.

UC.C<CCC.1M«CUUMMUCCM1.M .D»M U »MUMUCCCCCU MUUC MUC..CfiC.M.

MD» UUUMUCi HMUHMUUMUCDUUUMUUUDMUUUMU1U UM.M UM.MUDMUUM1MH MDCC M1MDCCUCC1UUD». UCCMUM1 1‘ UCCCMUCC UMUC M1M...UCD.1U.11.

CCUCUCCCC1C1UCCMU MUUC UMUMU MC (UMUHMUMUUDUCCMUCDMUM. UCCU UMULUCCCCU UMUUCCMD UUUC1 MM UM1.M»tU H UUUUUUC HU1MCCM1MMM MUUUM1MH D»1UC.U CC UUUM1M1.D»1 M1MMHUmUMUMU<mUMUM1M .HUCCCUM1MCC «UiCCCM M1.».MUM1MUCC1MUMU.UC.C..1UMU U MUM1M MUCCCCCUU CU tUUU .UM.MM1M MUUM1MM1MMDMUU C M. M_r 1UCC1UC.

MCC UMUM1 USU/UCMUMU UUU .UMU UUUC U UMU UCCUUC.M1MMD.1U M1MDUUCUUDUUC. MC C M.M.U»MU.U MUMUMU MUCCMCUMU UUMUUM HUREUMUDCUUC.DCUMUCCC1M1MCCMUCCCCUCUC. U.MUUC.MHUMUC.1. .MMMHUMU UM1MCCCC. MUMU1UM1UD_.HU..D»MU:CCMUMHUUCCU mmoMémMMM .2 HMUHUC. Hm. HMUHMUUCUUMDMUUMUUC HwHMoCmUMU. HwHMCCCCCmUC1 HUUHUCCJCCCCH HMUHMUCCCJCCCCCUMUC1UM1MC HUUHMCCUD CJC HMU[U 1M... HU.

HMUCC1U1U 8H EC HE.

MUCCC CCCCCDCCUM1 CM. MUMU HMUHMUM. Hm: MCCCU» HMUHMUUUCUCr MUMMMDCCMUM1MMD»MMCnUCCUMMUCCC HMUHUUCCUUCm .HMUHMUUC1M1M HUUHMUC HMUHMUUCC CCCCUM1 .U.U_1..M.MU<D1..UM1. UMUMUHMMUC1M1MCCM. .U.U_1..M.MU<DC.UM1MmUCa .M.MUHMUCCMUCCDHUMUMUC Ma .UC1 M h: C.C1 U» .UC “HUCC .1 1U» MM1M1.D» MUCCC H MM C».C1C (UC : UCCUM. 1MUCC» CC UC UDCCC HUM.MC UHMUCC 1” UCUUH UUCUCCC UUMUCC 1MUDUMUC.M1MCC.C. CM UMU .C. 1 Uar 1UCCC MrUCCUM1 M1UMUM1 C U1. MUMUM) 1UCCCMUC CCCC.C1 UCCMUCCCCUMUCCCCCC MCC UUU MMHUCCUMU MUM1M UCJC UZH 1UC.M.MCC UCCMHUUMUMHUCUC1 .M1MU.M.MU.UC.UMHUUC1MUCCCCMU.1U1UD mUCCHUMU CC MUC UU.D» “HUCC C >C1UCC M1U1U M1M U CMU» UUCUD UCCUCHKUMUUU CCC.C1UUM.MC«C1U..UC .MU1UCC .1UC.C UCJCCCCC C.1UC «C UCCC. C1MUC .HUU »UC.1U UM.M M1UMMMD H MUMHUCC U<CC CCU UCCC M.MM1MnU U1UC.,1UDCCMU C..11UUMUC.MUDCCCJMC C UC CC UC.,1UDUUMUDCCC..11U M1.MC1CC.1UMU C.C1»MUUUUM1MDCCMUM1MUM1MC MHUM. CC.“ UCCC1mUCM1MMM».1.UC1UCUUUC.M1MCCU D1UMU.MC 1.MmUC.C.M MUMUCCUC. CCCC.D MUM1MCCM.

C NU .

U U .C.M1.UMHU UCCMUCCCU N1mmmU1U1hMCC N m. 1m. 1m 1. 1m. meW Cm MUMU .M M112”. w m NQUUW . Um MCHC M. MmUWMCUAUC. omeW... EHCCWM U1QCC 1U- CC 1U- CC 1NUAUNU1QCC 1wCMUNC1UC. U- CUAUC. 1U1hMCC 1.0 MNU.

QC. QCC EC QCC EC QCC MUMMMUNUAUC. MUUWAUMMU13CC M1NmMUNC1UC. 1w©¢mU1mMCC QC. mUAUmUAMCC [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS 33% vam HwHUU HDUEHWUUDC1U313DDDU1. HUUH3U<UC1UU<U< HHHDU. {US U313 HUUHDUUUC. HDUHDUCJU H3UH3U< .31DU131DUU31 DU HDUHUUUDC HUUHDUU» 131DU131UU< UH 13 .U13U.313UU<3 UU EH)3 C131 HUUHUUUU313m DC1C1U1 HDUHDUUU313U. DU< H31U1H3U1UD313. 3C131U3 1H3UH3UU31U<Um «31x UH 13.131U1H3U1UD<»U»11 ISUUU<C.

UUU<1U1ar1 ~13U< 14. 33U3. 3.1.33.3 .D3U1HUU313313U33UU .3331.

DUU<UUUD<U DU DU UC1U311UU<313U3D313D rUUC1C HwHUUUU<3DU<UU<UC<<<UU<U3D HwHDUU<U<<11~<UU UUUDCJQ. 3x1.U»U»UD HDUHUUUDUUCJUDD33 C1UU< U3..3.»U.).1U U< 31U<3UU HDUHH3113DU3..3313«11\<U3.133<.1 3.333..113U311U31.1HU3 MUD31U1UU3U«<131HU UUC1<31U11U< 313U<U31.3311UUU«<1U311U <U<UU<31U<U UU.3.< UU3.13«11D<»UUU3.13 .UDDUUCUUD U»UDU1U<1U1D UDC1C1U3UC< D:UUDU<<UN«113..3 DUU3UUH 3..3U1UD .3U«11UD313«11UU3.13UD »13.1UD UDU<<U1 3 U C131UDD»UU.)1.D UDU31 U3.. 33. UC<1U3< <<U U 1U311UD313DDU<U<D313 HQHDUU.)1.D31133131U<UC133.).311U313UU<U1U1«1 U<< 3.33 <1U<U31UU3D111U1311U< <.1H.U»U«11 3»UH 3314.111)? 3C1U< UU313DU UU<<< 1UU<<313 3<».U<U1U3U«.11U.D»U1UUU1.11UU 1D1U<<<33 134115103 DDD U 3U<U 3U U313 1UU3 UUD D313 "-1 mm: f- if} '1"l '1"l if} ’1‘: 00 (>3 (>3 DO 2 "-1 "-1 334,3 HwHw<C<U31C3‘1 HDUHDUUDU3131UUDU313 HUUHDU<<<U«11D.1.U.1.UUDUDU3UUDU3D HEUUDUUm HwHHC. HDUHUUU 13131531 HmUHMUCUUDUU<<U313< HDUHUUUJUDm. .3133UH3UUD313 HD1HUUCDU3U331. .3131UU13U1UDU1U1 HUUHUUUC HDUHDU<UCU311UD§U HMUH Hm. 3 D... 13x1. HUUHDU<C1UU<<C1313 HDUUUUC. HDUUUUC. .

U31 .HwHUuUC. DDUD1 1Hm313.m11<mUmU313 .3133U.313U3D313313U.11.. .HUUHmU31113U31U? U3) UD UUU3..3C.<1U1.U a 31.1 .UUDP 3U» U<C13.13 U311 DU313< 1 3U»): << U:1.DUU3131.).UU.1.U)D3.13«N<U UUU<< H UU3. DUUH UU< UU3. 11. 1Um 31U3U1U1 31.111 H391.D.».UDU<1U1«x<U»1U«x<U1U31 U4. U3<U31 3 U3..3 U»1UU«13 .313U.3.3U<<31331.1DU< U313U31 UUUDr C 1U .3131 33D. U»1UD» 3313«11 U3) »..U DCUU 313 UU« 31331331 UU3 D<<H 3.13. UC. U<UUU313 U3131U< U<31U< 1UU1U UU<3DC<<U< 31.1 .U»1 13313» U D3 UDUC. .UUDP 11.U<r. <U3.13 U<< (1.111 ..1 33 13» DUUDH U<UU31 3U 33DU D<U»UU31 UUUDC< U«x<1 131U 3313 U<»UU U<C13.13 UU3. UU<1 3133U<U313 U 1» DU313< U<U311UH UU DU UU 31.111 UUC. DUUU3.13D UU<313U31U33.U13111...1U.( 3UU<313 U 31x1.UU U .U C1.1H.U»U m3U<U «11U 3..3<»UD3.13<.1.111UCJ1. 1.31113. 3«1U U«11C13.13»11UDU<.<C1<U Ufi< 313U<U31UD<U313 .31U3«<1C3..3DU.1U U UU UU<UH 3 3UU 3 3.933333%- D.moDNU1D< :Umammfijw 3 1 . 811.13 "11:15 .meNUADC. 3&1 do Hwy: 253$. 1.133 U931? Qkx at UNUADC. .UQUNU1QC1 a: DC. DC. m3< [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS ation] DPS ionNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ...mvm 55mg .554. HnH H55H55D55UD<5545< 5.55H55DDD5. HHQHUDDDDDDDQ D5Dm5D 55.5% .5.. HvH 5555.555. 55.5%.. .5.. 55.5.. 55 55.. HwH......DD<:3<5L.4.D5. 5.55H55N5DD55DDD4.4.555.5m5.<......4.. DD4454. m55.54.4.r5m5D5D45r 5.55H55w54.DDD5.5D5.5D.5 5.55.55.05.54. .5 D<5D53 5.55.5:5D 555.555.5555..

D5.54.DD4..M5.D H3555. 5. ..5. ..5 5.an......5542..5.54N<D4..4..53 55:55.55555.55.55.5DD5D55D5 .5455 5.355.551... 55... 55 .55.5DD5..55 DD1D .5D4..5.L.DDDD5L53 5...D.....5.55.D<5t553 .555.5D.<5.54..4. 5.5 5.54.5 ..L.5r. D5.5D5r.5DD5. 5.55 505.5 5 5.55DD5D55D5.54..D.¢5 .35....

DDDDD<DD 5.5.54...” .5..555.55m55.54..D<5..:.< DDD¢ 54.5.5.4. 54.555...

.D5t5.. 5. 4.54). D4555 5.4 5555:. 4.54. .....D .5.. 54.4..555... .54.. .5.D.5 5.

D DW .L.

DD 5.« 54.4.9.2... 5.55m55D5DH5D55395555555554534.3454. 4.555.555 D555.

D ..L.D..5 053.559.699.555 . DDDDD D 5.54.555 ODDS. ..L.D..D .5DDD5D5555.54.54.55454.9.55553535 DDD D4554. D4554.4.555.54x<555.55.5m5< DDDD<D m5 .....5.5.54N<D5.5m5 5 5.55.55... .5D555..5<D.5 55.5D<5.5.5..5m5 Rm... i' (“I 1r. \0 {\ 00 O‘ r- [\ r\ L‘~ r\ L‘~ H- 00 (X1 DO 00 0:1 co 0:1 4X) ... “4 5.. .5 5.. .5 5. 5..; HwH3.1.5.DDDD5D4.5D5D5D4..4.4..4.DD5DD 55.55.555.555.535555555455555 b...t;.<<<... 5.5.5....46554.55.555543 H55H5..<m5U.D.15.53 Hch<D<DD<D4fiwD5D<DD H55H5545m5DD5. H©H554545<DD5D45 HnH H55H55DD5...D5... H5..H554..D.< 55.559555 H55H5. .555 H5. .55.5 45V 5.5.5.5555. 5.5.1555 ..... 5.554.554}. 55.5.9.4}. .5.55.H55<5¥ 55.55.5504. 54.4... 5.5455345 55.5 5 H55H554.D......5t55 4. .4334... (555.55....

D555 55.4% 54.45.545.55555 LD<D..5D4.D4. 4.D....553.5DL4.D5555<.....555555 5.5.54.4.D45L53 m5D5D454.D4.. D5.

D55 D554 5D5.L.4 5UD<D< H©H555....5.5DD5.5D.<5.5D4.5.54.4.D5.55D.<D DDDDD<55DD5.5554.5..55.54.D 154.34. 54. $555.54.5.54555.<DD...DD4N<454.. 4D 5.54.5551}.54.553554534f5.5..”5DD4IJ. 54.555255D<5..54.D.....UUM5.5 54755534 47......55455555555< 5.55.5554.D4.4..4.4.D.........DD5.54..D¢ :55554..4..:41.415155:54.34.3485. 5.545414...

.DD4.4.5D5D5U .5DD5..5U5.54.D5.5U4.D 4.505. 5.. 5.54. .5554.5.54..5DD..5._.5._ 5.54..

.D5..5t5.. w5n54x<5545 DD5D5.5D5.5U5..55U< 4.. 5.5.5554 D5... 5559554.. 5555.546 ._5<55<Q 94...). m55..5.L.5r .1 .55... :4. 5. 4.5.5.4. DDDD .D5.54.5.D DDD 5D 4.1.15 5 .54.. 5 4.43.5555 H5 5M5D5D454x<< 4.4.. 5.545545555555554. 5555 5.5.5.5 5 4.m555.5..54.554..3<55 ~.l 5. 5 5 5. .. 55mvx 5.55.5... mm 5.05 WNW 55 %. “my: 5.83934. 53.55.55... m55mo 5). v.55).

D5... D4. 559555-34. ofik 5.5N5N45AD555 D5... 35555-34. $555.34. 39554.. 5.553934. 53.55.55... 5.wm5mm..m54. 5.4553955... 5.55w5mm..m54. 5554.. 5.mm5m.55..m54. D5... 39554.. .555m55..m54.

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS mp. mmwm V 9. 6m E ...........

HwHw.) $ch D V36<05..V....VmSVVVVD.....VV>VV.VVm.DVV HwHVVD6V6VQDmVDD) HV..H..066.r HVVHVXV. H. H.

V.VHVVVDVVV HVV. V.VHVV.VD6.V.V HVV. HvHVV6.VVV) IVVVV {.VV .VVV E:D E6D6. HEM. HVVHVXV. V.V..VV D.. .VV.

.«DMV. 0......VVVVV0VVVVVJV .V.. 6. :6V VV1 5661.. V.) V:V6V 6VV..VV :VVVD VxVDVVx 3.36.6 .HVgHVVVVwV6V6VV pg V6DVV DDVV6V. VV..V6 D6.V.VD6 .V.V..V.VV._1.V6..1.V.DVMV6.6. 5D... V.VVVmVVVVV6s6V. VV.VV QVHMY E VVVDVVDD HVVHVVMV.6VVVV.V.HV6.VV6 D....VV. 66.VVVD .1VV. V)V.,V6.6V. V.6.V..VmVV.VV V.V .9 VD6 VDVVDDVVVV6VDVVMV.6VV.VDmV6.V.V6V VD6VVVDV1V6.VV V6VVVVVmV6VV6DV.V6V.6V VDVU6.VVV) V6VVD VmV6VVV6V. fVVVVVV<wV6VVVD6V< VmVV V6V6 DH. DV.V6. V.VmV6 .V66V1VV. DVV6V6VV. V. V.D6V6 VD6.6.6V6. V.V Vb .VV.V<VVV.VVV<VV :2 DDV.V6 DDmV6VVDmV6VD VV..V6:V6VV.VVV6V6V 6.V.V V6... V.V._)V..V.D.VV6..1.V...V VV..V .00.. VVV6VD6DD66. V.V.VVDVVtV6V1VVVVrVVV66DD66V1DVV<6V .mV6V6.VVD6V6..VV6VVV6. .VV...V..VmVV.VD .VVV66. 3.66. ..

VVD VDMVMVVV VD6V6. 6.6VV .V.: VMV6V6....VV.V6V VV..V.VV.D6VV.V VV)V6.6..VV.1V DVV6. 6VVVmV6V6VVVV €VD66.. 6.6V. V6VVV VV6.V. 1.V.mVVMVV..VD662...“ V. HVVHVVVVV:.VVMVDMVV.VDVV.1.V.6><V6VV...VDMV 6V6V D6..VV6V6VV.V V6V .VmV VVmVVV VmVVVVVV ._)V..V..VV6VD V V.V.V VV (”"1 G V. m ”1 VP if) ‘0 f- m .w. G .6.

V N (‘N C.) {.3 (3 C) C.) C) CD. {.3 6:. C) 1—1 (>3 7‘1 0‘ G\ "‘N G\ Ch 0\ 31 G\ 7‘1 0‘ 0‘ (SN "-1 “1 7-6 V.‘ "-1 “1 "-1 V. "-1 V. V. V.

HVuH..0V-V....VVVVVDUVJVp....>........§:.. VEE V3.6 V HVVHG6VDQ6 HVVHVVVVV V HVVHVV6VV.VV.V6V6VV.V6 HVVHD..V.V._V HVVHVVDV6VVD66. HLHYVV HVVHVVDVVKVM .HVVHwVVDDVV . HVVHVVVV) .1......D .V.VVHVV6VHV. Hm... H.

.V.:V6V .VQH Hm...

V.V...V 6 .V_.1V... 63666:...<.......u<9..0...... DV. V6D. D6. VV) V6.V).V. .HVVVV. VVV AVVV .VD. V6.6..V VD Va V6.V.V .V.VD6VV VV.

D6666 V6. 6 V6..V DV.: V.V«VVVVVV V.V 6.6.D666V6VQ6MVDD< DU.6V...VV.V (5066..V 6 .V.) V VDVVV6D... V.VVV VVV6.V .V.VVHVV<V.VD6V6.< 6. VD6 VDDVV V..V6V :1 V66 D6.V V...V ._1 VHVVVVV VV6VV...V6VVMV..,VVH.V6VVMV“V6. _......V.V..V.1V...m.V>V a VV.VDV.V6.VH.V._)VV .V V .V V6V V 6. V6.

V6VVD:) Vm V V6VV .VVVVV.V h HVHVVV6V V6616).V V6.6 V ...VDHV6.6V. V.V..V6V. V6.V.V.. VV VVV<V1 V. 6.6 .VV. DVD 66.....V6V V.

V. VV 6.6. V .DV.VV.V6VV..VV.VD.>V6.V.VD V.VVV). .3606.VHVVV<VVDVVD6VVVVVV1DH 6 D.>V6.UDU6..VV.D6V66. 6V.>VV.VV6V.6VV.V.V..6V mVD...V6.

D6D6. V6 .V.VD D6666.

V6.1V.VV..VHV.6...,V:DVV6.: V.V 6V V6VUDU<6V V66VV..V....V)VU6.... V.VVV6V D6. DD 0.6.0 D6. VV6.DMV6. .VV6VD6.D..V ..6.V.V6VV.V 6.56 6.6VD VV6. 6VV..V._)> V..:V)VHV6..

V. mV6V6nVVVD6VVVVV 6..1.V.VmV 1V VMVVVVV6VVMVVV V V N :36 :3 Vdm 7m“. V.V..m VdV VNVV- mexVV 66:...

D6. Simmd... D6 V.mVVmV-D6V VNVV-D6. V.VVNmeVao6V VNVV-D6. V.VmemV1D6V VNVV-D6. V1D6V VNVV-D6. mVmeV1D6V VNVV-D6. V.V.UNVNVV..Q6V V.VmVNV.D6. V.HmeV-D6V D6 Q6V V.mmVNV.D6. NVVAV6V V.omVNV.D6. V.VNVNVVAV6V V.NmVNV.D6. V.memV..mV6V [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS HwHwDfiJflmGDGDdNGGDdNGaJyDDN DVDDGG GDDDG<D< {EDGE<<GGDG<DDGDGDGDDGD GDGDDGD‘qGGwaGG<<G HUHUGGDJVGGAQANGDDVGGGGDGAQAN HDWPDWGGGQDGKJVGDQG HmeU ($5vame .DGDFDVGDGGDUPDGD‘D HDWHDVGDDGDGDDDXSGD PDVDGDWDaKGDVGGxD HDWHDWG EVDD DQGG (DDDrDDGDaxGx‘G (DDDrDDGaVGJKGGQ .DDMEWDDDGGQ (DEG .DGDW. (SUB D<G<<D<GDDDG¢< DU< G<w§~< G G .D G<G< GGDDD D «KGDGDUQGDGDDGDGD GD<< HDWHDVDDDPDDDDKGDDGDGDDGGDU hmHmDGDDGGDGGxD HDWHDWGDDQGUGDGDDKGG HmeUGDfiDxfixGxD D: {,HDDxEVDD DG G EEG?DGGGGDDD:G<D< D<< DG<G< D,DGD<GD<GDGD GDU< .DNGGGDDDJKDDGGDGDEG D< G DDDU<<GD<G< D<< G<G< A < NGG3x§~GG< DD GGDG D<<D GDGD‘DUGDE‘D G<G<G<GD {ONE D<D<<GD D<<GEGDDDDGDDD GGDGDGG<<GD GG<D3<GGDGMGD GG<G GGDGDDDGGDGDD G<{EDDQGDMGKGGGDGMD hwbfix<DD<3<GGGDDDGDDDUGDDDDQ G<G< G<< GU GDDG HwHwD<DDGD<GGD<G<GDDD HDWHDVG<§GD<DG<<<DD<DDGDDD HmhmmzGGDDGD<G<<<D<DG<<<G HDVHDV HDuHUDDGxVGG HDFDE HDVHDVG<G<UDGGD< HDQDJGG <G< HE.DEGDGDJDGKGG .5;kaDDDGGDGDGJN HHHDEGGDDKGG DFDJQK HEDGGDD HHHDEGUD‘DDGG<GGDUG<GG<< ,MDNMDWDGGDDEGDDQGD:DGG<GG 53% “EDD; (DDQDEG DD< H©H©<<< <D<<G<U< GG<D<< G FUDGE? GG<< <G<GG .DGwGwalQ‘DQGUGG .D.w.DGwGDVDGamD._\ DGDGGDDDDGDQDG GG<U< D <<<G<< GGGGxD .DGMVGHVGAQ‘GGDG< G<GG 2x64 UGGDGD DD< DD< G n5D<D<GGD<G< (D.D_..rD.D5._DGDDQGMGDDD ._\ BEE)”«JADDGDQDV .de G<DDDJAVGGDGG GG DGDGDDGD G<SD G G G<DDGD<DDDGDGG D D {Dam <U<<G<GG<D DD<<G<UG<G GDDGE DD< DIE: G G DGG GD<<<<< DDUUDGDGDGM DG<< <sz {DEGDDDVG n _ DD: G D.» D DGE‘FV DGD um DGDDUw D D 31% D GGDDDD<D< GGGD<DD<< GDH Dam «JG Dd G DUB GDG G<G 9G BUG GDDQG {G G GG< G GG ~GGGG< 3G D3 a DDGDDGMD GGG DGDDRFKGDSGDQG G G G G G G G G G G G G G G G G 2% DGmmDmmamxV DGNQ -Q< DGgDNm‘DDw D Gam 6m . . D. do G G G G ma Sm, mm do .mn .n. Gan Gwm Di» G?» D m m ma mm wx DAV Dam DNQ- Dmm- D Dmm- D D D Dmm- D Dmm- D DNQ- D DNm. D D D no: no: we- no: we: we: mm: we: Dmm- DNm. mm: DNm. mm: DGOGNNm.

D< D< Q< D< Q< D< Q< D< Q< D< Q< D< 34 QDQG Q< D< GDDanm..mw< QDQG 34 QDQG 34 QDQG Dmm.d< [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS HGHGUO‘QQMLQ<D<UUDQ<QQDDD Ha {gin UDUN hang: HwDfiDQDDxVfiQQAwQDDDfi‘GED waEVEQDDfidfigDUDQQDDD Habflfl chv‘fiabxawam FMFMGUUUD m [$155 Hair)»; {GS E‘EDDxN DD< 1%vamgmflxBUDaQUD.U< "wag Hwhwagmv:H<DD<U§DDD< .wawmaflvmamx‘: (wwrwm..mvmv¢2~m ExtraDD<<Q<CDUUGCDDUPDUD DDUa wawmammz HFEQWHEQCXNDD ENE:DDDAE‘DDQAQ .w.c,m.mxu<3v< hfiHFflaD/fi _ Dr DDDUD <5 93;: DUNE fl DUUDQO. UGPDDPDUQGUAR UUDDDUB U<UD< UUDD‘a‘Ux» DDUUKNQDDGDDKV UQGDD< Gaming 531% DDUE; Unvfiaufimaagmv: UU< fi DDUR .UUme Q fi N VHVDDQDDDKV D<D<< D<UUUC< UDDDD n2 U<U<<QDfiamvfilw 123% N 03m..." <<Q{<33ng . GDWZ 3x <DDDFuDDDM‘sa‘DJQ 1534“ ._\ a: D 159x: U<<G HEAR DQGd DD DQDUD U3: $3 15655 D D 3 K15 Q<<Q<< QQDQ mwmfcmwm \ 53,33 33%me wfizéag c3733 miémg Dmonma $3:ng maofi‘oma $3-an Ra ”50W 05R: Daom‘ T33 :3 m 5.0m DSNREN QR wmcm..cwom EwFEJfiwUUDWQQQDAQVDQDQDDQ Hch<<<DUU<CD<G<< hang:unaUD/igxfi» bwkaDxa magma HMFWWDUU Habfig Hakim .6va .Evbgax Hakim (Drag (figmg (agavfiaw Laid/Dina ,Hfitm‘wfiam Mamba m D Gaza» g 1% 3.44 Dwain» HwdeQwfia‘dewmafii DUQDDD UUfimaU REBDDQDGDKE HarwgfiafiquUUam fl «7% GGD< mv<<< <3DU<G DQDUGQD «VUDDUD << uDadDDx» .éflabbDufiaD 1% G<D<G< HE.~..<4343“}me Q UDDDQD, maDHama< (DQMEJKGD<GD<<<<m UQDUGU<< DCDDGD<<.4» UUUDQ‘QD ,...

ONE Esq DQUDQCAQ‘ $92“.me U D<< DDU< maaé‘vomawfii‘iD .Q< {ME DDDDUUUGKV HUHUdNDDQDDfiwQDDDdNDQdNQwa DD‘QJNDPDD DARE DURFKDD Harv?»UDUUDGDUGDUUd13VUGD (Kiln<<DMDD<<U<<UDUUUGDU UUDD<<U<< D D<<U DUO/Q0 .U UUDKJVDUJQUD UUD<<< <<UUMDQ m<$<<<DCDDUU D<< maag/Q‘mfixfiau GazflVJQKQWEUGU m<$<<$<B<C<<C<<< D<<<G< Q<<<D¢fi<<<3mwma3mm.» ,vaxflafliaxnim mys<wDG<B$ ..."D,VD<<< mfgg .3 £393 .mm a . .

M .oa .aa g,“ annual Rn .3 Ma Raina: .mw .mm .3 Ra. Ra. .acmma. RNNa.

D< Ra-fl< FV $4? Ra-fl< awtmafifiu $4? Ra-fl< D< Ra-fl< amcmmaaoxu Ra-fl< ‘waRa..Q< Katmafifiu Ra-fl< AwRa..Q< m3 m3 ‘msmma:fl< Rma..fl< m3 m3 ‘wmmma..fl< [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS 555mm Ia .555.555.5555. H55H5555< H55H55D< HwHw5 H55H55555. H55H5555r 5.555.555 5555.55.55. 5.ch 555(5. 5555.55.55 5.555.555 .1 ..

UH H55H55DD< H55H555<55H .555 5i 55555H 55.55 555556.555... H55H55555Dr5 DD? E55E55>555<< H55H5555<5555555 H55H5555<555 5.5555 55D555<55 5.55 5.5.5 .5...5.5.55.155...55_.5.15 5.55.55 .5. 51555.155. D555 . 5.55.5.555555554555555. .5.m..5.5555.1551.5.55.5 55.5«5555.5.55._ 55.5555 5.5.1.. 55 5.55 5.5.5.. 5.555% 5< .1 5.5% 55 . 5155.5 5.1535565 <5<5< 51555.5. 55.155555D5 .55 5555 5.55. 555.555 55555.5 55.5 55555555543555 .D. .5 5.55 55% 55.55. >5 5555.5. (5.555.555..55.5555555.555.55.555554555.555 5.5555515553511555.55.15.5555?5.55. .5D....5. 5.555. 5555555155... (5.5)5 555% 5.55555.5555H5555x5D5555x5 DD 555155.55... 555.5.55t5x5515 55D<<D5<< $5.5.»55D53353P H55H55555555D5..5...55.5<5555.5<55.5DD5 H55H.555D5.<55...51555.55r.5<<....5.5.5D55.55r_515D H5555:D5..5...55.55555.5.55555.5«555555555.5D 55565555555155.5555D555 5555.5.5D....5.D..<555.55 55555555555 555555555. EDD 55.5 55555.5 5.55.15 5553 5H 5... 5555.55. 5< 55.5.55. 55.5 5 .5.< 55.5 .5 5H 5 5555.5 51555.5«55.5555555555155355555395 555.55.5.5.555.555.535.55555 55.55555... 5.5. .55.555 5.55.5 5.55 55 .555NN555N CNrOF ..Nm5 5 .11....) {N o1. HmMN$5N NN omNN-555NN HmNN-o55MN H55H55555551555DD555D555<55D<55 5555555515525 H5. H55 .55.55... H55H55<. < H55H55< 5.. H55H55H H5555... .. 5555.55. H55H55555w5n5m 5.55555 55.155.55.35 .5...5.5.55<55555. .1 5.1555555. 55 U<5.<5.5 ...55 .5555551555555555 555.5 555% .H55H555..:5555.55.15 . 5.155555555555165. 5555555555555. 5<DD<55555.55D55<5..5 HwHw5§55555<DD555 H55H555<<<5D<555D. H55H5555.....5.5.5D5<55.5D55 55DU...55.5.15...<5(5555.5..555555515555 H55H55555..55n5...555.555..5D 5.555.559.5555:55.555.555.55555555D55 H55H555..5...55.5H5D55.5.5«51<... 555.55.5H5D. 55.51 5.55.55. {55.525.55.555 53.155555 5.55.5555.5.5.545555555855555554 H55H55<5w55555D<55J55 55.55 5.155 5.55D5..55.5.55.15555.5... 525 5.. 55.... ... .55 .1 .1 .55. <55 . 55.55.55 55.55555 5.55.55.53.15» 5555.55. 5155555555 < 425555555 5.5.55 .1555. 5.5455555 D5 55.55.45 5D<5D «D.D D515. D 5.5.5. 555.555.55.51 .5 555595.531 3.55.5.5 5555 55.55 5.15.5555 55555.4. 5515 5 55.55.55. 55555531555555 55555.1 .5 55 {.5525 5 5555555555 551555.551 5.3.555 55.55x5 55.5«55 55.55.5.55455555555555.1555:455.53.5555 5 mNNm55- .51. .555 55.NN¢. N35 Nmm..m5< NNm. .55NNN5..A5¢5 .OMNNm. 5.N55.NNm.

Q< at D555. .wmmmm..m5< D555. D555. D555. 5.w55Nm55..m5< [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS UUamN NU..- .U..

Hw HUUHUUU... Hm. HwHUUUUU4..444<UU. HUU .UU Hm.

HwUU<UUU44<U44U<UUD HUUUH H4644. HUUHUU44UU44 U...UU4.. HLHUUUUUDR HUUHUUU4U44 949634 U..UHUU.UUUU HMUHU UU....UUUA HcHU. 43r 54.4.9444. HimUUUUU UAU. U. .UUUUUUUUUUA ..UUUU UUUUUUUUUU...

U.UUU4U4.4..Ur. 4.4 HUUUUUUUUUU4UM RUU.U 4..UD HUUHUUUUUDUU. r(.UUAU.4. A U UUUUUU4U U... U .U 4. RDU.

.U,..UU.D U UUU4...Ur HUUU444..UU4.. UUUUUUU44U44U... UA DU UU .U.UU.HUUU.UU..U4.U<4. A UUUA U4. U UU.

U..U4. U4 .. U..U U.

... (U4.U U4. US$4.04.» UUUA UUU.

UUDRUU UUU. U4 A 4..4..4.... USU D44UU44U4.U4..44. A UU.U4. U4...UU4UU..U.UUUr. 444.UUAU..4UU..U,. .D4.U4..U.. U. .U.UU UUUU DRUU..U UU..UAU4.U..U4..U4UU..UUU4U4.U. U4..U4...4..r. .UU.U U4U.U4.. UUUUA D.U4 U4. U. UUU UU..

U .U4.U .U4 U44 UDRUU U.4.4...Ur. U44. UUU4. 4...: (UUU <4 U R U34 U444.UU.U4. ... UU .U4444UU R.UU. UU4 UUU.4 .UUD U UUUU U.444.,U.U.44.,.. .U444.4....U UU.4..A 4.4... 4.4 U4.. U4.UU.U4.. UUU4><UU UUUUUUUUUUAUUUUUUUDU4EU.DU .UAU4...U4..Ur. UU.... .UA U4...

.UU .. .UU44. 4. U ..,.U,..r U.D...UU. UUD.

.Ur..U .UU4.. .U4..U.. U4U4..U.U .. U4 U4.. U, UA U D UUU..UU U, .UU4..UUU UAU .4..4, .UU44. A .UUU..U U4... U, UU (UUU U.4U4. D443 4.4 ..DR 4.. .. ..UA 4.4.UUUUUDUUUUU UAUU4UU..UU .UU U4.3UUAU UA UA U 4.U..U 4.. -UU:U4UU..UU..UUU4U4.U.U.U..UUUU..U4. U .U444. U4. .UUD UUU U.U....

U. 4UU.,UUU U4.U..UU4. 334.63% 3.... UUUmN-mm4N mcmN .Um UUU m: N.oUU4N ENNmUmN UUN. mmom.

RUN mmmNéNcN Uch..mmom U.. 4N QUUcN UEN NNUUN mmom .UUN 4404 gnaw 4404 m- . NmUUN . W. H HwHwAUUU<UU44<DUUUP HUUHUUUU4U44 HUUHUU4UUUUUUAUUUU Hm. HUUHUU.4.4.Um HUUHUU4UUD. .U..UUHUUUDRUUD HMUHUU4UUUU4.UU4.UUUUAUU4UUUAU HUUHUUUU44.4U Hm. HUUHUU UU....UUU44 UUUHUU4.U HUU. .UUU Hm...

UUH. .U.UU.U.UUU.U4.4.UAU UUU HUU4..r. HUUUr. {UUU U .U....UAD..UU..UU.D UUUU U.DU UUUU4. U444.4...U U.4.4...U4. UUU AUUUUU 44.44.444.44 444.44.4UU UUU. U..U4..44.UUU.U444.4..UUU. .UUUU. UUU4U44 D.U UDR.UU..U UU.4..AU4.UUU444.U4... U... U44..UU.,UAU..UU 44444.UUU.UU..

UDUUUAU . UA A UAUUUUU. U.U,..4..UUUUUUUAUUUU. .UUU4. HUUHUUUUUUAUU444. .U.4.. UUU U UUUUU U44U4U4...U, 4..Ur4 .. U 4..4....UU.UU4U4.4..4.UUU. 4..U4... UUU.UUUAU .4U 4.. UUU.U4.. .U4.U.U4U4 4.4..444.U..U...U.. .U.... 44U4UUUU. .UU. ,4UU. U..U4. U<UU.U.4 4 U4..4..

U44..U.4UU.U. UUU.” 4UDUU ..U..U4.. .4.UU4.. . U4.. U..UU4..4.UUU44 .UUUHUU4UUUAUU.U4.UAU.U4.UUUU4..4..4.. UAU.4 .U..UU.U4UUU4...

UU4. 4.4.U UAUUDUU U44U..U4..AU4.4...UU U4.. .UU.UU.,U4.

U4U4. UU.. U4..U4..4.UU 4.4.U UU..U4.UUUAU4.U..UUU..UUU UAUUUUUUU 4U.UAU444.4..44..4U3 U4.UUUUAU.4UU.U.U ...UU UUU UU .U U4.UU.,UU U.UmNNU-Q44. U.....mNNm-Q4. U. U. U. U U. - waNNUAU4... QNNUUAU4. U.U4UNN4U-4U4. UUUNNNUYD4. U44.

N.. UNmNNUYD4. meNUAU4... U4U4NNUU- U.U4....~WU4UU-4U44. U.UUmmU.UU..mU4. :wa UNomU mam U433 2: UU U4. gm UmmU U U. 4-34... .UUAU4. U. UUUUUUAF. N4 U-D4. .U..

D4. 34. UwaUUAUE. D4. Q4... Q44. Q44. $.34.

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS 3.3mm USN ..Q. a 8 UN 8.3.3 NNUN ..NUN UNUN HCHDQUQDDCUCUCUU H.3U3UQUUUUQ3UDU3: U.DQU.DQ3..3U<HD HCHDQ1ODUUCC .3...3U3.3QUUD.. DH .H3U.H3Q.U UUQHDQUUCJUUC. UUQHDQD3H3D3. HDQEDQUDUCUU EU.

EUCH DUQUUQUUUQC. 133:3. U.DQ.U.DUU .HUUHUUU U..UQ.3.3Q UUQUUm 3.3Q..33.Q ...Q3..3 UH3. H.QC. Q3 HQ »U3..3. 3....

H U.3QU.3Q3Q.«<H.»H .Q.D . QC.C.H .3.D....3.3Q3HQDU3.QU..UH ... U.3U.U.m.U...QOQUUUQH 3U 3U. »333 Q Qt DU...QC.... 1QUQC H 33 Q .HUUEUQUUCAQUDUUUU UUC HUHUODEUDUC.UU«UUCA«UU<U« 3C.C...Q..QU.Q.QC 3CCxC.C.fU.. UUD3H .rDQU.DU3..3H3C.mQ.«,UUDUC.3 .33.QDC. ..3DUC.3 .QU..Q UDUU 33.. ..U D33 3 ......Q31QD..3...Q..( ..QCDCJUCDUUUU CC.“ .3....U3. .... 3DU. D..U QUQ3U ..mQCAQUC. C.,<C.U UUQUUQDUUH3UCxU...U..UmQU3.. UUU.

.UC.1UUC....Q3.. 3... H333.

UUUUDU3 .3U3.

D«C C Q 3DU Q3. 3 Q3 .QtDH ..Q Q3.. H .D3DUU3.3.«U..3..3 U.3..33 Q....UU U..QD.. UDmQxUC.UUC.C.C U:..UUC....Q 3UUUUC.H HQDUH3H U . U..}.D DUCC. 3DC 3.... .U3. DU D.QUU..H.U Q.

UH MUD UH 33.QDUUH3<DUUHQUCCH .... CC. C.

DD 3QC. .... U ..D3Q3.3..HQ..Q. 3Q. ..UC. 3DUQ UUD3 C QC Q...U3.33.3D3..3...QUU3.3D QHQCC: HQUUUQH . 3..mQ3QC.H3U »C. QDC.C.H3 ....DUUUHDUDUUQUHQHHDD 3. DC. 3UD U.....HQUUUQQHQDD3 QHQDD DHUNNHUN 33.0...NNUWUQN UUUKN. URHN. .13th mi. EmN. 3.3 w DEN USN 3.3.....

UHN..-c. .1 ..N.om3.N ?....r cnmNNch .meN HCwN$NwN DUwN.mCxN owwNwaN QUUMWNNwwN AQUQCNNMUMWN mmwm ..me UmUNéUQaN swamNNaN maNimmom ..EQUwCC1OC.UUUUUDUDmQD«C1U .HUQHDQUUUHQCAQQU UU. U..c.H UD...

UUQ3 U.DUU...U CUDDC UU..[. HUQHDQDH3H3D3< .3.3U.H3Q< D3 .HDUUUUMQH3 HE. DUUU. ..3...UHD»UQ3 3... .HDUUUU EFTDU» UCC.UC....Q3.3HD H3QC.3UC..1QUQ CC. . .HDUUUU. 1% 3.1QC. 3H .3.3QU.3Q.UC.

QC.3UDUQUU. U3UD3..33.3U..QU.. 3... .3C.U «Cch .Q .UUQH3 3.33.3..«D3C ..U..

.U.D....3.3QH3C.UU..U< ...U DC.

.U . .UUQ3UC. DUCH 3.3..QUD . 3UUC.C.C. U.3UU.3UU...U..U<D3.QUU C.

....U3.3C.

D3Q3 .QCSQC. C. C.C.C. ......UQHQU UUC.3.33U QC U... «U H3U.3.«QHQ.1QC.

DC. C.

UQC...NC.MQC. .Q.QDU. .1QC....Q HQ...

UmQ QC.

..U3. .DUD...QC.r. 3..Q<UD33U 3 UDC..U..<U..QC:U..CxC...H.U C...H.UU<D3C UUUUCCJUC UDmQH3 mQ3Q3 3..33..3..33UU..QU..H.U.U .33...UH3.QC. .UDC. UUUD3 DC.

QUC.3.QC DUCUM U<C..QC. ...Q3UDUQ3U .1Q3.

H «3.33.3 QHQU “.QCCCJU. »UC.3U<. UH 3. 33:33C. .3.3Q.3.3UC>x1.Q.Q.QUH3nQUQ.33CD...

UUUUU UD3 U..QD3.33.3 DDCH 3..33UC. U13UDUUUUDDUC.UD .HCHQDUDUUDDUUUDUDUC.UDD1U D3.3.U..<C.3H.U3U QC. 3< .H3Q<UD3.3<U.C..«.UUC.C.3.3.UC.3..3H3U3..3.U.:U.. UC.C.C< .UUQCC UDDU .1QUQ3 UDmQH3 3 H C. 3U UmQ C. UUC. UU .3H3UD3HQ UU3.<1QUQ D3.U3U .UUC.3.3HQ nQUQCC>.UQU«1QC U.....UQHQC. . ..QUUUU.

D 3C.C.C.3HQC.QC. 3 3.3.3.3 3. .

.U n. 3N m Tm 3 a» 3.3333 3.9.5 .mmw .maw 38335 3.3% 369w M33“. 3 3823. vwmmU .. 3...

UUAUC. DUHwDUADC. 3.NwwG-QC. U- Yo... UUAUC. U- UUAUC. 3.2.93... .wSUUUAUC. VG... UUAUC. 3amm3.3-h3< UUAUC. SEWHF. a. U- a. a. a. 3... UdSTUAUC. .wnfifiHfi. QC. 3.33wm3UH3C. QC. QC. .N33U-m3< 3... QC. DCUUUDWHF.

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS 2.32m 2.32m 1.2.2.2 1222M 23222.1 gem 2.22H2221222DDD¢<<<Q222<U<2 .22 .1122. 92 .2. .222 2212U.<12.<12122212- 22.222222212212124 292.62%. 522222214. 22.221222222-r £222.12.<,2.21222< 22222222122 2.12.12 2.12.222 2.2212122 224. 12121222222. 22222222122 ...T2-.

J<< 212212224 2222221212 222112.22 ...2.212... 2.225212022122132. 2222222212122}. 221221221212122212<122< 2.12211222221212.<12<<m2<22< H22H@U«.x..x2<<m22) 2.22m212.<12122-21 222.. 1222-122 2 2.222.222.1221... 2.221.121 (22.11221. 2... ..212<<.-2 212 0332.12 22212212212 122.212.222.2212.212212221212226. 2121200221 22.22. 21222121212... 1212221212 12126.4. (212122 2.2222221213224222... 2.222.112 2.2. 204.2% 12212222r 2122211( 21201212.2310.<21.2 212221.26. 212.212.122.24 21222122121221 «12.2122 .2222.1<<22<122222121212w><2212<<1 ..<<2222 22122224112222.2121222: 22 21.21.112.221212 122.126.2212.). .2122121212121222<<22<1241122221212121212. 1222222 (2.22.211.212<1222<1222222212122.12<<121222 121 1222m2x21212<<D H2<221222< 12< 212222.43. 2.0.022 22.2... 2< 2 2.121 12<< .2 22.. 4r .2 22222 2.2.21.2 .2<12<.2 32221212 22.21.2122,.K 222221 2 3.21222 212121 2.2.22.K 2... 12.<... <12< <.2.2...2<1 .)22 2.22 2.< .22 24. H2<<<<<12 21212222 2.<2212< 212 21221222122 212 22212 2212 12121212. <12<12 12<1222 21222 2222.25 H. NmSN Hch22<¢<121222m2<2222122222<0m2<<< .2... 2....22222 22.22... 22 2.221122212112222) Hwh22<222m2<< 2122.22.22.22.1212.12.2-2<222.<<12222.2.w2122122...<12 9229221212221 .12 2.22.2.

ED229522. 222.222.....2....51.212212221222321 .2.2_...2.22.2\.122.2122.. 2.3. 2.225521% 2221.22.42. 2222.25.22.22 _-2121.2.2...2222. 22222<w2 2922121222122) 2.2.2.2212t2212-2)222 2.22222122.212.<<1212 22.22124N 2122.( )22.2221 22922122221223}. 2.22.2232...12122222122 <22 << 2.22.2221...21212122212.<< 2121 .212< 2.222.224.1222122.222.21.24}.. 12122.. 3.122 212<1222121. 2. .. .222. 1.22222. F2222<<222222221.2.21222212222 2.121 .12 1222 121121 <2<122 22.2. 5212.22.21... ).>22 2.2221212222212221 12222221222121. 22212<1222 124221212121222212222.. 2. 22. 222 22(UU12.<<.)2.>2.>21<12<<212 12.<1212122-2m2122.2m2n2 .2-21212<1212<1...2> n2<<<2212221212 <2222 )222212 £21212 .1 12.<1.2m2<.<<<122212<<2.212<.>2.>2 O<<221212<<<22<2122112}. <<§2<<U1212<< <<<<<< 2.222212124122222.121222.221.2222212222121242. 121222.22 12222212. <12<1222122222212126.2212122212122 .2<2.2 .22-12: 2.222.322-2112... 2fimw2w§2< 2.mo¢2m-m1< 212212.22-qu 222-222... 2. 2. 2 .2 21.1 N12... 21221.2... 2.32.2... 21222 22- 1.22 .22.. 12< 211< 2.121.622-1112... 22:12.1 E22222... .222-2«1 2.321.222-1112... 2.1N12222..12< 2.222122229122222. 22:12.1 2.122.222-1112... 22.222.222.131 21121229122222. 2.wm@2.22..12< 2.3122m.12< 32.22.22-131 2.122122229122222. 12< [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS Nacm 3cm wwom wwco owom HwHwMDU<U HMMHMMUUM.<UU HLHM...J..M EMUFMM<Ume HnHMMMan .M.M HMMHMMU HMUMMM .M.M..[M .M.m..M.M...MU ..MM M....M .M..M.M MM HMMH MMH HimMMMMU D (f HMMHMMU.<M. HMMHMMDMMMM »<¢ ¢.M.Mm M.MMM.M<.4M. MUUMJM UMU¢.

..M .MMUHwUxuflfl.

..UmaUUDMDUUUMLUUDMD HMMHMMUMUKMDUMUMMM..M.<¢.UU<..<MMMM.MM..MD..U D HMMHMMDMMMMUK 143$ HMMHwUxMDMU¢DMMU MM.. M...K HLHM.»U<<¢.MUM U4. U¢.HM<MUM. MMMDS GUMMUMM «..D. M..1......MMU.MmU§¢ 4.4.1.» E. .4... .M.

MDUMM..U»U<DU»UDMU< »MD.U»U< ..D<¢.» O<<UUD UDMUMMMD MMUU< HMMHMMMM<D¢MUDMM<< UUD¢.»U< <¢...UM.M D»D M424. UUMU< UH M..... H M1MMU¢ MM.U»M1UM MMMHM<<DUHMM M < MM UDUMUMMUDMMUMUDM. U MD».<¢ M414. MMMMUH .MM..MDUM..UMMM<¢. MMM <¢< »U¢DD. MD.<M.MM.MD¢.M.MM..MU¢.M.MU ..rUMM M.MUMM.4M: MDDMUHMDMM DU<¢ ¢.M.M< UMM M... m M MM¢.< HMMHMMMMD<<MUDD¢.<.<U¢.M.MD¢.< UMU¢. -MMUMM.

.MM..MMMUM.MD U4. H .MMU¢ DUMM<DMM< MMDMMUUH UMUUxM. < M44. «.5 .UMMM UM.MUDM..M MM..DDMM<..<MMUM.M.M.M MM..: .4349 M4M.MMU.UM.M1UMU¢...M.M< D .M 34¢. UUMU¢ UDM..M.MM<D¢.»UDM.M.MM UUD MDMM D M.DMUMMH »MMHM¢><MMM HMMHMMDMMMMD¢.¢.D.UM..MUUU..<MMMU.U.<¢.U ...M<¢.UM.

M .D»”MMMMMMMMMM M< M<MU..4..4MDMUM.M..MMMUMUKMJN .....D».U¢.MHM.4MUD MMM MMM M. MMUMUMMmU MmMM...mMM.MM4m HMUHMM4V¢ HMMHMMDU HMMHMMMM<U<¢MMDMUMUD HMMHMMDMMMMUMUDMMDDUWZ MMNMU HMMHMMxVfiM¢¢AwMDmMM414VU¢dMUMDDU HLHMMMUMM HMMHMM<¢fiM<UMUKEU< HM.

UMMD HMMHM HMJJM .. .D».<¢.».

.M.MMHMUM< .MLMMMM M<<UMMK EU.» HHHMMKJM. HMMHMXM .M.M....M.MM< .M.MM.M.MM< U»U.1UM. HMMHMXMU¢UMMD¢.<<¢.»U.U.<¢.UM.MDMMU M.MM.M.MM<<.U. .M.M....M.MMHM..D»U. M¢. HFHMMMMMMU DMUM) .M.M<M »»UMUMU¢.¢ »U< MUDDUMVDMUML DMUUMM UDMM M< ».<¢M. MMU. MEUM. .UD<¢.M U¢..MJMMM. MMHM§¢:4. MUMMDU. MMM. UDMMMM D¢.¢. .<¢ .MtMU¢ <<UM.MDDUM.M44005324. ...D M UUUUMUDMM.

M¢ M<M¢. HnHMMMUDDUUx.DMUMUM...<U¢.M.MD¢:MM<< U<¢.MH.M<MU¢.MUUUUMU MUM mM»rM UMUxMU<M MMUUDMUKJM. .M.. MM.. D»D¢DDM MM¢.(MU¢»UDM MM..“ M .4. M. M.

MM .M MMU¢1 MUM}. (3.43M MM»UU< UU<¢M.. M1 DM. .4: U94: MM.M ...M.D» .HMUHMM<MU:DMMMU.MM..\.»MMM.M..M..”MDMMM U mUMMMUMMMDUUUUUMMM MUMUDMUDMUMJMU».U U DD¢<r M..... rM< ...M.

UMUMM .M..D»M M.1M.M< .<M.» MM..).M . .4244 MD».<M.MD Ma MM»U .

M U: G M<D MM...M UMUU .UM ..¢.M.MDMU»U UMMD ¢.M.M<. M....MUMUMMMU MM.MD¢;MMUDMM.MM< MD MDU¢.M.MU.UMUM.MM..MDM.M< M»U<DU 3.414N < MMM..M .» ». MMM..M .UMU¢ UMM<MU<MM<MUUU MM. mU U UMUMU.MM<¢.MU.4MM.MM..M<<MU “MUD M¢.HM<MM.4.M.M Mdm MMM..M..mDM M....Mmm... M9M3¢. MAKE. .wwm. 34mm... :36. MUMMMM... mmm. ..M .».mm. .mMMMMM Dem. M69“. MAME“. mm... 9fl< .M93¢. 93< 9D< 9D< 9 9D< 9 9 9 3¢. 9D< 93¢. 3¢. 9D< 93¢. 9Q< @329-qu 93¢. .M....MMMMMM..AM< 3.4. ax M.meMMMm.3¢. MMMMMMMMMVAMJK 3.4. M.me.MM.MM..mM< [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS Cam-m mama Macm 3cm MMMMMMN W W (M.MMHMMCDDCCD HMMHDCMD HMMHDVDMDMCCMMCCMC.MM.M«CCCCM.MM.MDDM.L;CC M.M... HMC PM. M.M...

CDMDMDKMMDM .MCMM-MCMMMDCCC-MCCCMDCCUMMCCCC..DM14 HUDMDMDDCCDMDM HMMMCMMD MMDMDWMCCCCCC: HMMHMMDC Chm-M.M.MMMMCCDMDDKC HMMMMDVDMCCJCCCDMMM.M HMMHDCDMDNK HMMM. EMMMLMD CDCMMCMDDM DMD-EEC. .M.M...CM.MKM CDMCEMM CDMMM DMDMtMmC .DKMCCCCDUMr DCMMDCDMLMMMCDML DDC CDMCDCMMDMMMK CM.M...CM.MMMD<CCM .DMMDMCCCMCCCCCMC .M..1MCC .D DMDMDCCC .MCMMMCMMDMK DMKMD ..1 M1 DD?) MCMM. DDMC MCD .> M.CCM.M D. MDCCDCCMM DC .MDMDMK MMCCCCUDM) .L.xC MMC.M.DM.M.r MMDM. C DMDM.M .DMwaC D MCCCACMDDCC MCCCCxCMDCCM.M. MDDDC MDMDCCDDCCMM CMMCC MCCM M DD D MMthC MDM.MKMCCCCUKCCCCCMDKMDMDMCCACCCDMC.MM.M CCMMMDMMDDMDNKMDMC.MM.MnMDCCMCCC«CC MMDMCDMDMDDCC CCCC M.MDMK M MCCDMDxCxCM DMMCCDMDMMM MDMC.MMMM.DCCMCCJCCDMDM.M CDMC DM.M.CC«CCDDMDMwMD CMDKC .MDCC DMDM.M DMCCCDCCCMDMMMDCC DMDnD 1MMM MDCCszCDDCCxC CCC. DMDCCKMDMKMM .MDCCCCUKCJCCC DDfiC HMM-M-MKMM.MMCCmMmMM.MMMMDMDMDCCCCCCDCCMDUKCCC MCCCCKCCCCCDQ MC CCCCDMMMDDMCMCCKMMCDMDMKMDMCM DDDMDMM MmMDDMDDDCC DCCKMMDMC M.MDC.

DDMDMD MMD .C. DCCMHMCCCC .MCCMDMDCfCC DD DMDD MDDM-LMDMMD .CCM.KM.DMC.M DM-DDCC .MM-.MMD K K MDDMDCCK ,MM.

MD DMMDDDMDMDD MD MDM.M CCDMDCCC mMDCCMMCC .MMDM CM MCC MCC HUHMCDDMDCCD EDD-M.MMCCCAC HMMCwMMMCCC .MCDVEMMCCMDM wawCCrDCCDCJCCCCCCDM HUHUCCDDQMMCCD .M-MMMMCMMCCDCCMKMDDDMCCC EDDEMMMCDCCCM CM.M...CM.M.MM.MM. HMMD-DCMDUUNKMDMDD:DOM. HM. (M.M.

CM.MM.CC< EMMM-MCCCMva .M-MM-MCMMMDMDMD .MCMMM-SMDMC.MM.MMMMDM.M«CCCCDM.MM.MmMDM. HMMCwMMxCxC HMM-M-MMMMDMMDMDNMDCCMCCC .DCMMCDCDMCC HUD.

CCM MMMCCCMM1 .MCMMDCMCCCDMMCC. (M.M...CMMM waCCM-DMDNM M.MMMCMMMCDCC MDDMDM C...MCC ..MM. MDMDM. CCCK ..M .M M.MMCMMMDDCCM ..M UK DDC. MDC IMMMCMMMDDMKM CDC-MD CC. MDMDM. C (MCCDM. MDM.M MACC M. CL..CC DDMC mMCCCCCMCMC. MCDMCC M-D MCCC CC M.M...M...M.mMMD.C C .DCCCJCC .MD M-DCC DM.MM.MDDMDCC C DCCCCC DC.

DDCJMCCCCCCMMDMDMM .M- MC.MM M-MMD D .KMDDMKMC LMCCC ..1 DCCM. C M.1M.CC.C.DMCCCCC.. Mm K M.L..CC mMCC CCCCDCC DC-LMDDCCDCCD DDCCMDDC MCDDMDC .CMC CCUDNMDC nMMDMDM.M CCCMC MCCDM CC CM... MDMDDMDCKMDC C CMDDDM.

C.M«CM-D DM.M.CC«CCMDDM.M DD MUM.MM>MCC.C.DMCCCDMDM.MUU.CC D MnMiCCCCxCDCCM. M.MDMM MCCCMCMM.MMD.DMDMKMDMDCCCCCMDMCCC. DMDMDCCCCCCK MC. DCCKC DCC ..L.DMDCC.. DMDDMLM DMDMK M.1 KMrM M-DMDMCMxC-MCC MC .MDCCC.

D MMMMDD DDDMDDCDDDDD CCCCCJCC DMDCC DMD .MxC MED DMDDCC MCDCCU MD DCCCCMMMDMCDKMNM mMCCMD D MMCD MCCCCCC MCDDDCCKMDMDDMMMD .DCMMDCMCMCMCCKMDDCCMDMDDDCCKMmMMDMM..DCCDCC -MDMD DD MD .CMCMC .owaMC- .wova .CCMMC- CCMCMC CCCMQ Mfimm- .MCCMC- .Cova M .95 .Nmm. ..CCmMCC Mamm- m. DRE? @- CVWMMMCMMMw- m. CwEMmm- .NMMMNC. Miam- maim- QCC MD DJC MD QCC MD MD CC CC CC QCC CC QCC AMCC DCC DJC DCC AMCC DCC AMCC DCC AMCC mMCC [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS HMLHLMQCLCJLDMQCLQDMQQDQCLQCLQL). HMLHLDMQCLCL. HLHLCLM HLHLCLMMCL HLHLDL HLHMLC. HL. ..MM M.M..MML HM.

MMmLM. HLLHLCLCLLLD HLHLM..(.. HLHLMQMQLQM .MLHLMQUw HLHLDQMMCL. HLHLMQL DM .MLLLMMLMQDM. HLMMDC LDQC .MLMLMLMLMQC.

.MCLDM QMM D» M DLD M 1. LL UM .L. MMCLTDL La L.

UDMMMMUDCMMCLDMMDCLC.M.M .L .

M QQQDM. HLHLQDQQC...r M a MCL LQDDCLLUDM. QMMC. LD. CLCLDM.

D»..CLC. DC LQLQC HLHMLMQDCLCLQMMDD .DMQL(.M.DM MCL LMQC.L(. L.-Q MD MCL. CLCUL MMC Ma M MQQ UCLCJLL. QLQDCLDM Q M QCLLLQDD DCLLQCLC. QM.M (M DC CLCL LMMQD...MQCLL LU .M. Cf QLLQL QLLQMQCL. MMC. CL QCLM..MCL M .ML QL (.LLUDQMMUUCL. QM .LLMQw UM ..QCL LLQL QLLQL.

CL MMMM .LM.MHMDC.M.MCL MM LDLDM.L.L( »QQCLQ..MLLCL ..L.D.CLCL.L.QM.M.r .LC.M.MLLQM. MCLCL Cw: UDLLQ QC... CLC. .LD” QLQCDLLQC.

LQM MMQCLMQQCL QCLCLMM (MLLQLLQC. LDM QCsCLQQMQMM QM ML .M MEDLUMM .MM D MDD HLHLDCuflDDMQMQMQDQCNMQDCfiQDDDC. HLHLCLQDCNLLQDDDCLUQMQCLDCLMQDDD D C. DLMQ MDMMLQLDLD QMMLQDDMMDCLMQMMDCQLLQLUMMC. .5 DUMMD DU MDCLQUC.CLM.MM.MDUM.MM.M MDDMMDUCLUDQMQCCLU QQDMQDCLLLQ Q .MCCL CLM..MQDQ “MU .MCL MCLQMMMQDCLLLQL H MM H MMMm-MMmem Hnomdgm 0L3 mwamémam Hma ... Mwm.r Hm©m§§m 0% mymmam wrawdm m. mam. (Com. mom m MMMML..mLLLom HNQLéLQC MMLLMMMMJMNOML. HMLOML.ONMMML meMMwMMLoML.

HLHLCCLLQDDDMQ .M... HL LMM...

.LHLDCLM HLHL HL[M .MMLM MLM La HLHL MLQ HLM. HL HLHLM. LMMLLML MML CLM.M HLHLDCVCCC HLHLCL MD .MLMLHLMMC. CJLL. MCL LL M.CLLQC. LDLC QQQCLCL. HLHLCLMQCLr» HLHLCLDLLQMQC. QL HMLHMMUCLCLQDM. M.MM.M LMMLLMLUDC .MLMLHLMQLLQCL LL ..L.L(MDL HMCL MQLQCLLUM. HMLHMMLLLCLLUMQUC. CLQCCLCL. HLHLUMMMMUDQQMMM CL..MD... QLQC MDCLC. MQCLMQCR ... DLQL C. BUD LCLDCSM CLCL LDCM. UDCLQ CLD.r ..L.L( MMCLCL CUDCLQDQCLLD .MQCLM QCLQM ..DQMM.

Mi MCLDM.MM.MLD DDQUL. DM rQCL 3CD DCCLDL .MLMLMLMLCLQCLQDCLCCLC LC. .M ML(M QC.L..L.MQMM ...LMMM.MQMMC. U .M .MC MCLLQLLLCxCLLQM. DDLQDCLMM DLUCLQ LUMQDLL LMQCLCL MNMC L.L..DL DM 0 UM. Mr.

LA .QCLLUDCLQQUC. HMLHLMMCLQQMQM.M<CL.L.QD<CL.L.QC. MDCLLQQLLD HLHLLUMQMMUUDMQCQDDCDCLCLQDQCL CLL CJLL. LM..M.L .D. CL CLLQC.DM .MQCLQC. QMQLLQDDQQMLMDMMCCLMQCLC. QMQCLMQ .MQMMM.

.QCLCMMDL ML MCL. LLQLQL(MDLQL( LCLC. LLULQCL. .QLCLL MM »M.MM. ..DLM DLCL QQCJML DC. M MG CL .QMMC.C....

C. L CLM.

C. :LCL LDDLQ ML DC.

LD QDMMCL. QDMM DM QMMCCLCL GL9»: MCLM M ..DQMM.

.LLU DLLQC. DCLM.M ANCLQDCLQD LUCLMQMMCLD MLQLLQCLLUCL MCLLLQ D DCLMQLQCL. UDCLCLM. MCLQ MUUDUCLMQMMMM MDLLQ Q.L.MH.LDMMMQMQ..QQ QMMCLQCLMQLLQ CU ULLQCLCLQ .MMMQQMQDLQ .MMC...MCL .M CL MUM QQ MMMCQ .M .wHLMMM. .33 .MMOLMMM. Lam. ..rdL .

LSMLLLLC .mmm. 89m mQL Mm? NM? LAMCL L. MM- L. ...o L. ....o C.. 0..

DC. QCL DC. QCL LECL LLVDC. 33.0..AMCL DC. DCL mMCL mMCL [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS HDVHVV6.D H mHDV6VDDVD66. HwHDVDDV63DDDD HVV HDV HUDDDDDD6.D6.DD6.6.DDDHVVDD HVVHDVD.6.V.VD66:..VV.VVDDVDHVDVVVDDD6.V.V HDVHDVVVVDDVVDDD6H HvHDV6.DV..Vr HDVHDV6V. HVVHDVD6.6.6 HLHDVVV HDVHD... HVLVDVV. .V..VV HDV». HimVVDVV ..V HVVHDVDDV..V6V. .V..V.VV) HDVHDVDVD6DVDV..rV HDHDVVHVD .HVV.HVV<.VV .VVVHVVVHVDDVDDHVD<DVVVHVD6.VV6V DVDV. HVLVDVVDV. .D.DV.D.DVVVHVVD6.V...VH.V VDD6..VVDHVDV..V6.DV6.DHVDV6.6V. HDVVVVVHV<6.VVDVDV6.V.V V6. VV.

DDVV6.VDV) DDD6.6.6 6: VDV ..V DV)_ V..V V6 D66x DV..VD....V»D< ..6.V.

DDV.VV.VD6...H.VDD6.V.V H VDVDV. VH ..V..VVHVV. 6. .DV. D.6.. VD DD. .VVV6V. VVD< DD6.HV V6.6...VV.V. DVDV.V.. ..V 6.6V. V6.0 V.) V6.6.DV..VHV V. .6.6D6.VVV) DDDVV.VD6.DDVH.V6.6 DVD DHVDDD6V. VV6..D._ .D. VD». 6. VmV.6...H.V6. .VV. VD. VVVD6DVHV6AVVVVVD6.. .66.DDV..VV.V D6. VVDVV6. D666. VDVVV. VVVHVD .DDVD 6.D»D DD6. VV. .V.,VHV<V.,V6.. VVDVD.VDHV .VV .D.6.DV.V..D..DH VVD6DVDV. D6 H .D6. D.3). VVD6...V V.VD.

V6x V6.0 ..VV. ..HVDD6.

DDD DDDID DVV6f DDHVH VVV6DV.V.VDVV).. .V». .V....D. ..HVDVVVVVD VCV..VV..VV.VD»DDDDDV.VH V6..DV....V HVDH VV V6. VHVDVVV D 6.....VHVH V 6. .D.6.VVV.V 36. VD V VDVV VD HVHVD DDVV V VDD . mV<VVHV<DVVVVVV6V..HVD6. . ..HVDVVV6. D<DDV..VHV6.VVV..VD6K DDD HDVHw6.D6.DVD6.DDDDD6.DDDD6.DD HDVHDV<DVVVD HGHDVVVVVDDDVVHVVDDV6.D»D.DV..VHVD6. HVVHDVD6.D HVVHHV6.6.D6.6.DVVD HDVHVV6~6.6.D<DPDD<DDDD6.<D<6. HDVHDV6V. HDVHDV HvH HVVHDV<666 HDV HDVHDV.6.6.VDVDV..V HDVHDVD6... HD....V.DV6.. HDVHVV .DD... HVVHDVV HDVHD... 6...H.VD6.V..VV.V DVVVV 6..

H HDV6V... 6V. 6..V HDVHDV6V6N6DVVDD<V..VDV..VV..V6.D6.D.6.V.VD6 HDVHDV.6.6.6. H VDVVVA HvHDV6.6. HDV6...HV DVD .. VDD. DmV.6.6. V .... VV V.VV.V.VVVD6.VV6V .HVV.HVVD.VV...V.

DDV.V6.V..V..H.V6 V V...6. V.DV6..H VD» VVHV.DDDVV.. MDV V06. VV..V6.D..D6x MVV..VV.VVD.6.V. V.V6.DDV. 6. V6. .DVVVD6 D6.6..».V.D V 6V.DV..V.6.6.DDV. D6.DV.V6 VV6>H H: D6.D.

D.6;.V». HVD.6....V VVV66V VHV6..VV,V6..HV.VV6 DVD6.D..DD.D V6..DVV,VHVDD .V.DV.V.DVHV6.VVV..VVHVDVVVVVVHV.6.VVV..

DDDV.VVDV..V6V.6.V..VVV6.D DDVV..V6..DVV..V VHVDDDVVV..V6.D6.V...VVHVD65 36.6..DVVVHVVHV6D ... 6 DD .6.. m 6V D 6.6.6m V VHVDV.VDV.V V6666... V6gDV..VD6.V) ....V m ..D6.DVD6 D6.V.V DH V6» DDVV. 6. ..V». 6.DV.VV.V.D6.V.VV..V .D VDV. ....VV.V<DVV6.6.V..V VD 6.VV6V 6.6V.

...V6.<V,V».V.D H VVV6DDVDVVD VVHV< .».V6.DV..VVD VDDDD -D6.DDD 6.....VD66x VHVD V6.VD6...H.V»D 6.

..VVVV. D36. VV..

.DVDD ..6.D6 .V.V.VV..VD VHVV. VHVDDD6.

D. .6}.V D .HV<DV.,V VD6.....VVD VDV..V6. VHV<.VV...6.<VV D V DD 6..V».V6.D VHVDVDVVHV6AVDVV V V 3% DDEVVNV. Dd ..V: C6. D6. VDVNV..HV6.

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS HVUHVUD. HVVHD... HVVHD... HVVHD... HDVHVVVV .VVVVVU .V..VV :VV..

U: .VDVDVVUVDV. V... HVUDVUVUDV V...).V ...VV D..V.VD.D_..U.V VVDVVV MVUVVVJV. HVVHVUUDDUUUUKV. HwHwD1UDDU<D§ HDUHVVVUVDU1.VDEVVDUMUDDDVVVVUDDD HVUHVUDVVVDDD. UUVVUDKV. HVUHDU<VU< HVUHVUUUVUVVV.

DVVVVVVVr UDMVJVVVVVHVUVVVVVQVUVVVUKV. HVUHDUUVVVV..VUUDVVVJVV HVVHDVVU.UUD<D<<<UDDVU< HDUHVVV..VU<UVV.V.<.«<VUD.VV.V U<. .VVVVVUaVVVVVUanV HVUHVUUVUV U< U UUDVUVD VVUVVVVHVU DHVVV U<< < _.VVVUHVDVVHVVKVVVVUHVUDV.:VUVVVVUJV DV.,VV .VDH UDDUV ..DVr «UH .UVVH «(D V<< UVUVVUUDVVUUUUVUVVUUVV< .DDV..VUD.<UUV..V VD.VV..V..V .UUVV <1V (VtVVVV. VUDDVVJV. VUHV<VVVU< VV.VmV V.V VUDH .V.VD UUDDVVVVUDV V<. 3.4.: V..V_.HV<VUVVV._.HV VVDVV.. UV..V .<.V..V.VU..<VUV..V VVU. UVr V. .VDUVUU. V..:VVVVVV..V< U U.<UV..V<. VUUUUD. .VUVVVV. VDUV. .VVUDUVVVV UVVVDVVVUHVVV.

DDUDVUEVDDDD V .U VVU D r VDVV. VUVVVr UVUVJVVUU V .VVV < DH UMVVUVUKJV. UwVaUDgDD .UDVVV. «...V.VUUDVVVJVV. VfiVUV .<.V..V.UVUVV;VV.<. V U<<< U<UU< VUDVVVVVDQV ..HVMVVVDVUUH ...VVUUDVU UVDVVUM VVUVUVV V.

U<<D .VUDV. ...UDUVVV V<<<w U<< V<DVVV.VVUDD UVVRVDVVVVV V1.3}. UVVVVmVDV V.. 9.2 {V..UmVVDVVVeV VVD...<<V.

U Vx. .VUV U ...V. VD .V..V<D V ..UUVVVU V..V_.HV< m3: QVVVMVVMNVVVV. DVmVVWNmeVV 03.? V-mmVV.VV QmeVMUVV .341... m. V V £1... m3.

XV Qamfimwww mVvaévaw H©H@§.<<DDU1VU41VU<<UD<<< HVUHVUUD HVVH HVVHVU?VV Hub? HVVHD... EV .V.V HVVD. [ D....V.V HLHDVVUH VVVVV EV HVVDDV ...V.V :D .V.VV.V.VV.VV DDVDVVDV V.._V HDUHVVVVJEVUH HDUHVVDVUVVV. .UHVUDVVVVUr .V.V..U.HVU.UV.DV.V HVUHVUDU.

VDVVV VD VDVUV V... .HVUHVUVVVVEHVUa UxVUUVVVVV <U< UU< UUUVVVVVV.

...V...VUUV..V:VV:.<V< .V<VUVUVV.UVVmUm U<<UVUDUDV HVVHVUDDDVVUDDVVV rV<<UVxV<UVVVVmVVUUmVVVVVVVU VDVDUVVVUV ..VfiDH VDUD VV DVVU< VU UMVVDDVV DVV mVVUVVV. 1U V<4AV1U VtUVVVUUDVVVM UDUV UVVUUDD DUKJVVU... VfiUDUVVV VUVVU- DVVV.

DVU <<D<U<UU<U VHUDVUVVV. HVUHVU<DDVDVDVVHVDVV VVVQVVVVVUVVV. .V.

V..<V.VV.VUV..V V< <{VHUDVVUVUVHVUUVVV VtDDVV V.. «...VMVV ..HVDV. VDVUH DUHUDDUU VDDVVVDUVVNVVVUVDVQ. U<VDD<VUDU1U VDUVVVVUxfigVU V<< U<<. UU< U Dim UUDDUUUVVDVVVVUUD VDV DU VVV. UtVUVV D<VVUVVDDDVKEVDUKVfUVU. ..VxVxVU 1VV.V(UUV..VUUU .VV.VDUUV.VV.VDDV..V.U DDDVUVUVU VfiUUVVVDVU VVVDVVV.V.V<U.VUM MV<<UDDVVUDU< DUVVVUHVUVHV VH V <<UU<DUVVU<UVVVU <U< 721.4. mVVUVVV.

(VDVV. VD V4. UUVVV ..VDVVVV VVU VV Davemm- V V V V V.

V. V.... um :3. VVm, H? VVnm Vdmm Dm mm .(rq: ..V.. m ...... Ummmo ...:V- mmo ..V.. Ne. NVWVV- cc. ..V..

D1 DVV. at V.VVmDVmVU-D< of Q< vumNUADVVV. at Q< Dd. Q4 Dd. Q< QWAVMVV [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS HV..3...0 HVVHVVNVC. 3333. HVVHNVNVNV3VVNVC..VNVV.V HVVHNVNVC HVVHVVNVNVV. HN.H3...N.V_D..3 HVVHVV. HN.H3...N.V HVVHVVNVNVVVC HN.H3...C.C3C.V..V HNVHNV». HNVHNVC. HVVHVV3NVVVNV HNVHNVC. 3V.. .V.

V.. N ....VV....VVNV .V..VVV.VV .V.N....V.V.N HVVHVVC. 13.33.? NVQCHV NVCa 3NVN 3NVN ...V.

.V3C.C... 3h .3 V3C. HVVHVVCVVC 3.

V.r V...... 3.

VQV3h VV. .NV NV3NVV.V »V. NV3N.VC. V.VN V.V3. ._ V<C.V.VN N V...VC>.N mV.VV3..VfVC.UC VV..VC.... D»V V VV. .VNVN D..3..VC.C3.V.» D»NVV. .VC. .fi V.VNV.NVNV< 33HVVNVNVCNV33.

VN.VC C.C.N.VC.NVNVV. UNVCC. VC.NV.» NVC.V..VUV. VNCV . 3NVN 33.... NVCV V.VC. ..HV..

..VN ..:3 V3C.C.. .VCVVVNVVV. 3.chH.33UV33CC...V..V3C.MVC.VV VC.JC:VC. VUNVNV3NVV.V_D »tV 3C.UMV<C.V..VC C.»wV3V..VUC.

NV.C.C.C.CNC.NVC.»«V3V..VNV 3..VN.VNVC.

.VUV.V :33 .3V. VC.3V.VNVNVNVV.VNV3.VV..V UNV3C. ..VNVC.NVN V_D» V .. V »NVV. HVVHVVNVCQUDUNV3NVWVC..V .VN C.3N VNVC. 3.

V3 .V. .333...NVNV.NVC.NVC.NVV.3NVNVC}.NV NV.C.V..V»wV3V..VNV3NVC. VN V 3...

.VC..V.V.VN.V N 3V.VV.V NVC.N 3V.: D..3N V..VNVC..VC. V._ D..3C..wV»wV3 3NV3.»NV VC. V VNVC...V. 3C.C. 3 VN . ..VNVC.NVN VwVC..VC 3U.VNV.. VNVC.V .VN.VC.3NVNV nVanVNV33 ..CUNVC..VV.V V3NVNV NVNVCC. C.C.NVV.V...C.N.»3V.VNV NVNVC. VNV.1 333 .VNV3V..V VC. .VC.3.. VnV3NVC.

..VNV »NVNVfi V ..D»V NV< VNVC. N NV V C. U.C.C.V..V VNV3 N VNV 3 V.

V «..CxC. NV NV3 3 NV... VNV NV\N HwHmeVCuflNUC.3NVV3mV H3. HN NVHVVNVN HNV. 33.3. 3.33.

H3. HNVHNVC. D..3V. 33.13. 33.32...

VNV.» HVVNV3V. .HVVHVVNVVVC. .HVVHVVC..VC. .N.N..N.N...NVV.V 3.233....

..VNV1 VNVV.VC.C.C.VNVWVNVV3N... » HNVHVVCC....VC.NV3..V3C.NVMV33CC.OCC.NV HVVHVVC.NVC.NVC..»VNV3H.30V3C.C..VC.MV33 HNVHNVCVCN<NVCC NVNVCQCC. .HVVHVV3V..VUVV3C.V..VC 3.N.H3...C.V.V3N.VC.CNC.... NV.C.N.VC. .VV.

.HVVHVVCCHVN NV< .V.VV.V.VVC.3NVN NV HVVHVVC.3NVNVNV<NVV..VC N.N.VC.C.V3NVNV3V. . VNVC.C... .V.N....V.V.NVC.3C.NVNVV .3.VV.HVVC.C.C.NV<333C H3..HNVCCNVNV»VN .HVVHVVCNVVVHVNVV NVC. VNVNVNVC.V.VNV.C.NVC .CsCCN VNVNV ..VC» ..V.VC. ..VCC.NV»VC.VNVV. .C.C.»wVC. U.C.C.C VC..VNVC.. VCCxC.C.NVC.V.V» 3V.

V. VV..V.VC.C.C.C... V.V3NVmVC.C.NVmVNVNVV.VwV CsC.NVC. NVVVN CC. NV3NVNVNV<C -C.a ( .... .VVVHVvC.CCNVNVNVNVNVNVC. N V CHVCC. .VND».C.»VC D. V .V.

V VJV »mVCA. NV ..VNV CHVC. 3NVV.VC..VNVC. VVLVVV. 33C. NVU3CHVC .VV.V3NVV.V<C.C.C VmVCC. N VN .3..

V VVNVNVN V.NVNVC.N VNVNVC}. .NVC.3NVN NVNVC.

CC. D. .3C. C.C. V ..C.V. VCCA ....V VC.3NV.V .NVVVNVN. C.

.VN NV3NVNVV VNVV3. NV CsC.C.N VCxC.C. NVC.N.VC NV .V3.VC. V ..VV V3.V» 35 NVNV NV.C.C.NV 3VVNVN nVC. C.NVNV C.UNV VC.V..V... NV3CU NVV.V»V3V..VV..V VNVN. 3N UNV NVC. ..C. V. VC3 .VfiN VNV C.NV 3NVC. VNVNVCCN V V V 3.33. NVNV 2... V V V V V. V.mmmmmV-QC. 3....

E. Vdm VVVNV 36m (.r.rxfifl ...mm \((TGV mm mmo- mum- NWNNV..NVC. «V...

NCQC. No. .V.. rmmxm- C. wCNWJNV- we. mNm.

QC. QC. 3C. V.CNmmVV-DC. V.wm.N..mNV..NVC. 3C. CC. 3C. QC. 3C. QC.

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS HDUHDU4KKDKD.) HDUHKK HDUHwDPD. H..QHDU H.DK HDU 1U: UK HKUHDUrUU4ymK. HLHDUUU4.4. HDUHDUU.K HDKHDUUKDUD. U HDKHDUU4K HKUHDUD. DUK HDU...DK U.KU4 U4. .D..K.K.D.D...UU..)K .U...UK .DK4KKU4.

KK4UDD UKU4KK.UKUD.UD4: 444. 4 .D.DU.D.D44.DU4K.4K 4K KUDD4. 4..DUD. U4K.4KUDDU 4.4..UD4.U ..K)K DD .4. HDUUKUUK..KDUDD .D.D....D.DUDHU4>.4.4K.4><KK HDUHDUUUDD4UU .D.DU.D.D4DDK..U4K4KDD4K. HUD..K.K4.4><>.4.4K.K U4 UK U4.

D UD.)4.4.4 UDDUK. K (DUUU4KUUKDUD4D4D UDUU4. HDUHDUU4UUUU4D4Dr.K DDUDK UDDDDUUDDKKK D.K.. U4. KU4.

U4KK. 4 U4K4K4KDDUK4D D4K4KU.K K4.D DK. KK...KK4 UUKKDU4K 44.4.4.

)KDDKD4KDD.K D UD4.. U4KU4.U 44.x HDUHDUKDUD4UKDD4.4.44KUU.K U4KKUKU.K U44 4K4KK 4. DDKDDDD4K U4. 444 UKD4. UD44U4. U4K4KUK4D U4.U.K.UKD4\4.UK4DD4KDD HDUHDUUUD44.UDDD4KDU4UUUU4D4DU )KUDDUD .U4K4KKUK. DDDUUD .. 4K K)KUKU4.. .444.DDUD.4.UD.K)D U. .....U4K4..4K. .K4KD UK..U4K. 4 DDDUDD .UDDD. 4K4KD UU4K. DDKUDK DHUKKDU4K.4><KUK..UDU4. UK. .UKKDDDU4K4KK..UU4>4. U4>KK UDDD4. 4.4.

UD DU UU4. U4KU K4.4.

DD UDD 4:). 4.4 DDDDD UIUD4KKUKU4KDKKD UDDD HDUHDU4KDDDDDD4.4.U.U4.U4DDU4.4..UDU U4..KK4 U44U4. DDDU4K 444.4. 444.4K. DD.K..KDU.K..K4><K mwwWDva mémew D.Do.K4-mmwK4 N. D3404. DWDUQm. m D .75..

D 7.54.. c.DKKUm U3. maw KU4D444N444 ... D34 DUm-DUKUom Sam. 0.4.9.0-mmom Docméwom DHom-mmDUm Rewramom @meDHDUm om HDUHUK) x HDUHDU4K. HDUHDUDD444K. HwHDK4U. HDUHGU<DUD 4K. HDUHDUDDUU4K HDKHD44KD HDUHDUU4.K4D HDUHD HDUHDUUDDKKKK Hm HDUHDU4.4.K4DUU4.K4DDDD HD... H_. .DK HDUDDD4.UU4K.4KUDDUD. HDUHDUDDDD UHDUD4.K. H4 .HDUHDU4K4nK2KUUDD4K. HDUHDU4K4K. HDUHDUU4UKDDU4K4K. .D..K4.D.D....K..KDU4.U4K HDUHDUKKD4. DE. .HDUHDU .KUKK HWHFDK: K.4.D- 4.U44x D4.U4K. DP) 4.4.U .U4KU4.4.. D4 :44 U4.K4D4x4.4.4.4 UUUUDD DD“ DK. KDD 44.9. K... 4. K4.K U4K. .UDD.K 5.) U..K4. U4K4KUUUU UD.

DK..KKKD U. DDU4K4KDUDDDU D 4.. DKU.

U4.DU4. UUUa KKDK..K.U..K>.. K UUK. UKD DD.K..K D. UDKK D4K UUDDUUUU44U4K .U44UDDD44. U4.DUU4..U KKK).K U 4.U4K. .U4.K4DDDUUU4..DUDD4...U4K. UDDDDUUDDD4K .UUDDUU4KUDDKDUU U4KDD4K4K. U4K.U4..D 4.KUDD4U4KDD U44. H DDa .U4. D D4K KKDK.4.UU..)KUK.,U4K 4U D< DDUU: 4. UUDDUU U44D4. DDDU4K DDUU UDDD4.UU RDKUUDDU U4 UKD4KUDDK4D 4.UU4K4KUDDU 4.4.K. 4U 4.34KKU4K4KU D4K4s4..K..KDKKDDDDU UDDUUDDD44K<4D4DUUD UDDUUDD.D4..4K. UKK 4.UUDDDK2KU4K4U4K54K4UKU .U4KKUKUD4KD DKU4K.4KUK.. 4.4.: 4.44K DDUK. U4KU D4..

WU UUU4. . UU UU 4.4.3 ..KU4K UU .DUDD -Q4K .aDUUKNKUAD4. 4% K .wxmmm. .mommm-D4K .wammm- .4svmm..fl4K .DUD $4 Dw mum- wmmm..Q4K .me 4N4. .334. mm .KU- man]? 34K U4. D4. U4. 4K $4. Q4K NUADKKK. Q4K [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS H54 H.HUH Hw144..43.44144.fi HHUHUUU4.»U4414.H.H441414.HUH.4.H.HUUH.HU5U14.5UH.H HHUHUUD1U44144.D,U HwHHUDU<UUUHUmU430mU HHUHUUUHU44 $5.55... HHUHHU14. H©H5UHU445UUUHU441414U44 HHUHUUUUUHU5U14. H.HUHHUHUHU44 155155.55 HHUHUUHUHU44 .H.5..H.5..H.H4.>4. H5934 ...HU...HUH.UH .U.HU.H.HU H14.UU U U).5HH 11.4..»U5UHH5U14.“U,.5.U4.. UUH U14. .UUHUHHH. H.HU.H.HUH.UH HHH UH.U44 U,»U4.1UH.U»U1414.< 4414. 54.» 44 ..... .H.H.HU4...UHU4..

H.U»U14. U0 H5H5UH.H.. 3.44.1... HHH UUHH5 H..U H».UH. .U.HU.U.HUUHU5U<14.

HbHUHUD44444UHU1afi rUU4.»U4414. D.) U.) .UH H U14.

UH .5355.HH5U.4.U4..<14.U....U44 H4.57414.HHHU5UUUUHH44 1U.U4. .U..U1U4 HH.U5 .»HHHH U,rU.4.U »4.4..44.5UH. UrUU4. U U UUUH. U14.4.1.4.5H5UHHH.H. 4UU5U14145 .5U5UH.H U14.

»...) HH HH.U.5U4.». H H441414.U4..5U 5).»5UH... UU... 5 UH.H D: U1U1.4 5 UUmU144. UU .»HHUH.H5 »»U.4. 3.4.34.2... U344. H.HH5U/4UMU4. 04.4.. )»UU./4.4.. U5 UVHU5U5U4.4414.MU....U44 H)» HH .»HHH..HUH.U1.4. U... 44 UDHH5U14.HH5H5UUU .....5H1U4.._rUU5U,»U4.1.UUH.U UUUUHUUUWUD4..UH.HH)»UUU»U H.1UUU.HmU.)UU.H4.5U1U ..44UU5U5UUU .RUHUUUHUMUUUHUMUUUUUHHDUHHH.H14.H.HH.H5U .H<HH...H5.»..UU5U.U1U. U4.»U1UHH.1.141U.UHH 5.» ...UHU4UMUUUHHHUUU4.5U1UU5U1U.<»U. .H.5..U.HU»U14.UUHU»UDHU4UMUUUUU5H34.914414. UUU5UHUUU5HD U5H..)».5UH.

H 5U UH.H 341..r...r. 1..me Ummm H.595 H .51 .H5. .5. .H5. .55.

:UHH .52.

.UU HHUHUU45U44 HUUFHU4UMUHUHHH.HUU...UHU5U14.HU1.4.44 HHUHUUU4.»U4414. HUUFHUNEH HHUHUUUU44.5HUU1..UVU1414. U.4.H.4.44 .14 ,HHUH34 $3.64. 4.1UU5UH.U4. HHUHUUUUHH: ...HHU ...HHU U... 4,4..5U .H.HU.U.HU4.U.1....4.5U1.4.44<.5UUUHUHU5UHU5H5U.5UH.H 33.05 ....U4.HH5H4..HHH.U<.1U.UH.U111.4 .....U5U45 1.31.4414 0.3.4.»4.. UH.U4..445U1.4.»U14.UUH.H44 44 34.4.. 4.»U44 34.4.. ..U4..1.1U.UHH.1. 144.5UHHUUH.H“HUU14.U.4.. U4. ...UH.H4..14.HU»U44 U UHUH.U.5UUU5H_..U5UH 4..

UU.4.HHHU,.r H 14 .1...5U14.

PB) 44 . U.,»4. U141 1411.421... .44. UU5 4.4..

U44.4.4....DUHU.1U UHUmU U4.14. UUH 4.3.. .1...HU..U.

H ..H 14. 4UH .U UUH 5UH.UUH.H5UHHU4414.H.U»U H14. :3)» 5UUU»U1414..U.U5HUU MUUUU44 H444 14 1414.44, UUUHU4.. U..5U1.4.UU<14. .1... .HU.5UH.HH.H..5U5UHHH.H5U.UUHU5U5.» .UH.UH H 34444.0 US UUU»U1U4. U.,.... U.

.H.. ..UUUU )»UHU 5U14.5U U14. 54443.3 5U.5UH.H5.> UH.H ...UHU U5UUUUU UU14.UUHU H5H..)»UU5H4.14. $444.14. 14 H5H_..U ..U ..5U4..HHH.H4..HH....U<.5UUU .U44414..... U5U H H. H. . 8.1.4 H320- H.Nwmmm-m4. .H H.411: :5. m cam Um :4 ..U 4.4.4-154. 3.4.4-3 “$39314. $0.34.? .UHUVNUAUU4. 33.0-34. 534$. S H3934. H3934. mNm. 434...? mNm. 533934.. 534$. .. 34. Q44 Q44 5H4. $4.. Q44 www-mfix [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS HwkaDMéU GD<< DE‘GUUKN I“if" C) wwhfizfigjwuufimfii‘éfidw :ficé <<<wu<u<<mw<<3 UU<<<DD<UD< FUDUKJV 33%;? E $$o< [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ’E‘abie 22. CS smgle {i099 screen (1331371) in HepEB cells with 61‘ madified iEmAs Dupiex ED Avg. “/6 message remaining AD—617792 43.2 AD-61803.2 3 9 869.2 75.1 AD—51815.2 90.7 Ari-61821.2 33.7 lap-61730.2 53.5 AD~61786.2 34.4 922 27.5 AD~61798.2 23.3 {3.0618042 23.6 IRE—61810.2 33.4 AD—617992 91 ACE—61865.2 22.1 AD—51811.2 90.9 Ari-61817.2 26.1 71061823.: 41.3 710617322 42.5 3.9617832 28.9 AD~61794.2 133.5. 00.: 27.9 i AD-emom 4.2.8 122 26.9 ""“ii'fiiéi'é'i'éfé""""306 [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS Ari-61824.2 29.3 AD-61.783.2 61.3 AD»-6178§§.2 25.5 43617352 34.2 mama-1.2 24.2 {3.0618072 42.8 AD-618132 31 4361319.). 42.2 494318252 31 A331’7842 34.1 """'2§'51'é'i'§§'53""""2m """"l4" 06179::2546 """'2§'51'é'i'§5'£3""""30 Ari-61808.2 23.5 AD-61.814.2 45.3 Ari-61820.2 56 AD»6‘J_826.2 31.5 ACE—61832.2 36.2 40-61833: 39.7 Ari-61844.2 37 4061850.: 66.3 AD-618562 172.6 4361352.). 41.3 """"+322 """"K D618272927 """" """""""""""""" mm“ 293 839.2 41.5 452 23.7 {3.0618512 37 AD—51857.2 34.3 ACE—61863.2 33.3 40-61853: 38.2 Ari-61828.2 30.3 40-61834: 27.1 [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS 1111-1318402 (-34.3 119618462 4?.

AD-618522. 25.2 AD»6'J.858.2 95.7 AD-618642 29.6 {10618702 30.5 AD»-6182§}.2 92.7 AD»6'J.83.‘3.2 24.8 1111—1318412 59.2 {10618472 30.9 fil'é'i'é'é'é'i'z'""""352 """"L1" 0618551240 """EQSZEEEEEE""""421 1111-1318712 55.3 119618302 162.9 1111-1318352 28.8 AD»6'J.842.2 18.2 ACE—61848.2 25 110618542 42.3 1111-5185112 41.7 110618562 28.9 722 64.7 AD»6'J.83'J..2 15.9 111112322""""+249 """"LE1151842275‘ ""“xli'iii'é'i'éléfé""""+2.98"""""""""""""" ACE—61855.2 20 AD—51861.2 28.6 8572 18 210520521 22 ACE—62068.1 29.9 110620741 411.2. 1111-5203111 30.4 110620361 21 [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS Ari-62092.1 20 4.9620981 38.4 wane-4:1. 42.7 496205-31 25.5 AD-ezosm 55.6 43620731 114.4 mama“. 21.2 4.9620871 33.8 49—52093; 26.3 91 23.9 ""“2351'6571'633""""30 """"L4" 95703142 """'2{'51'é'£6'7"5?1""""1337 Ari-62076.1 84.3 AD-62082.1 42.3 7x04320331 36.5 4.9620941 66 49521-331 66.4 40-62mm 33.9 7x04320531 33 711 38.4 AD-ezm'm. 27.8 831 44.7 """"+4.27 mam; """"K 3520931453 ""“KSE‘EESITI""""393"""""""""""""" 49521-371 2.9.9 AD—52066.1 33.5 404520.721 27.5 40520731 49.9 49520341 117.6 901 44 7x04320931 33.5 4.0621021 39.2 [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS 301321081 (-39.5 3.0620671 323 301320731 811 30620791 45.8 30520351 31.6 30320911 32 30320971 35.3 30621031 35.6 91 24.7 30321151 25.7 """Ei'fii'é'ii'91'3""""21 """"3" 0671271563 """'2{'51'é'£71'§'§f1""""seq""""""""""""""" 301321391 841 30621451 90.8 30521511 56.9 30621101 26 51 145.5 3.0621221 1923.7 281 178.4 3.0621331 52. 30321401 55.6 61 47.2 30521171 46.2 30521231 95.1 30521291 156.2 30621351 62 30521411 128.1 30521471 146.2 3.0621531 35.5 30521121 43 30621181 32 [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS AID-~62 124.1 {SD-62130.1 AD-fi}: 136,1 42.1 AD-fi}: 148,1 Afi~621541 AD-fi}: 113,1 AD~62119.1 IRE—62125.1 Afi~621311 AD—52149.l Ari-62155.1 IRS-62114.1 AID-~62 120.1 AD~62126.1 ACE—62132.1 {SD-62138.1 Ari-62144.1 {3.0621501 AD-fi}: 156,1 AD~62162.1 IRE—62120.1 ACE—62185.1 AD—SZlfiZJ Afi~621981 AD—52157.l ACE—62163.1 15.4 {SD-62169.1 236 Ari—62175.1 29.6 Ail-62181.1 264 [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS #3821371 28.8 #3821831 22.9 #3821991 16.4 #3821531 18.5 #3821841 191 #3821701 15 61 6.2.7 #3821821 73.8 #3821881 811 41 63.6 """"#'51'é'£§5'53""""218 """"l1" 0671591428 """"#'51'é'£71'€§3""""277 #3821711 31.9 #3821771 29.6 #3821331 25.2 #3821891 32.7 #3821951 73.1 #3822011 35.6 #3821831 56.5 #3821881 115.1 #3821721 107.4 31 71.3 #111811""""+1.72 """"7;" 3821901372 """" """"""""""""""" #382161 195 #3822021 1.9.4 #3821811 23.7 #3821871 24.4 #3821731 36 #3821791 50.5 #3821851 43.5 #3821911 39.3 #3821871 39.4 [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ACE-{32203.1 34.1 139622091 34.6 jsxj. 31 21.1 15.3 AD-ezzzm. 68.5 {3.0622331 34.3 AD-ezzam 37.2 AD»6224.‘3.1 31.2 IRE—62204.1 33 2101 29 '"""'2§Si'é'£§'i'éf'i""""38/ """"A" 95727134», """'2§Si'é'£§'2"§f'i""""m Ari-62234.1 15.2 AD-62240.1 26.2 am 40.4 AD»6220‘3.1 17.1 ACE—62211.1 20.9 Ail-62217.1 49.8 Ari-62223.1 40 Ail-62229.1 25.7 AD-ezzasxj. 21.5 AD»6224‘J..1 46.2 """"+404 AH"221%".

""""Kmmmmz‘ ""“KSEEEEETE""""+517"""""""""""""" ACE—62218.1 26 AD—52224.l 40.3 {3.0622301 32.8 AD—52236.l 52.4 ACE—62242.1 33.1 AD-62248.1l8 Ari-62207.1 19.7 Ail-62213.1 43.4 [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS 7151-1322131 39.3 719622251 34.3 7111-1322311 37.2 AD»6223?.1 25.9 7111-1322431 19.3 {3.0622491 13.8 322031 13.7 AUSZRMJ 16.6 7111—1322201 25.2 2261 27 """73351'552'3'33""""3132 """"I1" 0673815315 """73351'553221213""""13 7151-1318741. 27.1 83301 30.8 7111-1318351 30.4 AD»6‘J.8§2.1 48.9 ACE—61898.1 24.7 1.9041 125.9 7111-1319101 45.7 710-619161 25.7 AD-61875J. 33.4 AD»6‘J.88‘J..1 64 711111351""""+357 """"K {161891223 """" """""""""""""" 11.51191 84S 710—519-351 32.1 7111—5111111 23.7 4111519171 22.1 7111—5153761 47.3 ACE—61882.1 26.5 {AD-61888.1 27.7 7111-1318941. (-34.8 710-6191101 89.8 [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS 7101313061 22.4 710613121 13.8 710519131 37.1 AD»6‘J.87’?.1 145 7101313331 31.5 710618831 33.3 7101313351 37.5 AD»6‘J.99‘J..1 26.1 710613071 33 710613131 33.1 """"7{'51'é'1'§'i'§3""""3616 """"71" 3518781163 """"7{'51'é'1'§'g"£1'3""""11"""""""""""""" 7101318901 37.2 710618361 41.7 7101313021 58.6 4.0613631 28 710613141 31.4 710613201 27.1 7101318731 33.1 710618351 33.7 7101313311 41.3 AD»6‘J.8§'.7.1 33.4 710613151 122.4. 710613211 66.4 AD—51927.l 40.5 710619331 27.7 39.l 28.1 710613451 30 710613511 33.7 7101313571 32.6 710613631 17 [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS 391319221 32.9 3.9619281 28.3 AD-61934J. 24 39619401 28.2 AD»-61946.1 33.2 39619521 197.9 39619981 37 39619641 30.6 39519231 51.2 39619291 29.4 """'2{'51'é'1'§§'§i1""""61 """"L4" 9519411196 """EQSZEEQEEI""""289 391319531 23.7 3.9619391 189 391319691 17 39619241 24.1 39619391 31.9 3.9619361 369 391319421 13.8 3.9619481 40.2 AD»-61954.1 41.8 39619601 24.1 """"+189 ALH‘BGJ """"3' {1619291924 """"/3{E§i€i'§§'i'f'1'""""1.63""""""""""""""" 39619371 1.9.1 39619431 27.8 39619491 26.5 51 83.8 39619511 26 671 16.3 39619261 17.8 3.9619321 18.6 [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS AID-{31938.1 31.9 AD-61.944.1 29.5 AD-emam 57.8 AUS‘JBSGJ 42.; mama/2.1 30 [3.0619681 29.1 AD-61974.1 50.8 AUS‘JBSGJ 19.7 926.1 36.4 {3.0619921 36.3 """'2{'51'é'i'ic3§'§fi""""T""""""""""""""i"24 """"A" 9570-14. """'2{'51'é'£6'i'5f1""""5(8 Ari-61969.1 30 AD-61.975.1 51.1 7x04319314. 37.6 AUS‘JBS'ZJ 32.5 ACE—61993.1 23.4 IRS-6191991 43.8 7x04320031 23.3 Ail-62011.1 32.7 AD-61970J. 39.6 AUS‘JBYGJ 27.5 mmgafl""""+549 """"K mmsngg """" """""""""""""" 1 :05 ACE—62005 1 42.1 12.l 21 {3.0619711 27.1 AD—51977.l 23.4 ACE—61983.1 57.5 AD-61.989.1 25.8 7x04319931 18.2, Ail-62061.1 29.7 [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS 30620071 106.4 3.0620131 361 30619721. 40.5 61 49.1 30619641. 24.3 30619901 38.8 30619961. 40.5 30620021 32.5 30620081 35.3 30620141 23.6 '51'é'i'§'7"§f1""""334 """"3" 660731.03 """'2{'51'é'i'§'21'§f1""""11 30619911 34.9 3.0619371 29.2 30620031 25.9 30620091 21.1 30620561 16.3 3.0620151 133.3 30620211 36.4 3.0620271 42.4 31. 62 30620391 35.2 """7QEIEEESZEH""""+308 """"3' 6620511223 ""“xli'fiié'iiiii‘iii""""313"""""""""""""" 30620161 23.2 30620221 36.9 31 52.6 30620331 31 30620401 30.7 3.0620461 28.2 30620521 23.7 3.0620561 77.3 ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS Ari-62017.1 41 AD-62023.1 27 ZQIJ. 31.8 AD»6203.‘3.1 45.4 AD-SXOMIJ. 25.3 {3.0620471 20 AD-SXO‘BJ. 37.1 AD»62059.1 31 1113—5201531 37.8 {104320241 34.7 ------------------------------------- .............................................................................................

AD—52042.l 1101320481 28.3 AD-62054.1 55.6 1101320501 26.9 AD»62019.1 29 ACE—62025.1 78.5 Ail-62031.1 152.23 1101320371 27.3 Ail-62043.1 33.8 AD-SXOMB‘J. 46 .‘3.1 24.5 1113—5211261 24.9 ACE—62032.1 23 AD—52038.l 21.2 {3.0620441 34.1 AD—SZOSO.1 22.4 {ms—52329.1 16.6 110623261 16.6 1101323321 15.4 {AD-62338.1 41.9 [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS 40623441. 19.6 40623301 323 40623561. 20.4 40623621 27.8 40623211. 18.7 40623271 14.8 40623331. 22.2 40623391 134.5 406234.51 321 40623511 35.5 """'2{'51'é'£':£§§f1""""31 """"4" 6631262 """'2{'51'é'£':£§£f1""""46"""""""""""""" 40623281 301, 40623341 39.1 40623461 24.3 61 35.4 40623521 33.8 40623561 45.7 40623641 19.7 40623231 40.5 40623291. 57.5 51 27.5 IRE—62353.1 40623591 361 40623651 23.6 40623241 271 40623301 251 40623361 25.3 40623421 45.4 40623481 91.6 40623541 1321 [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS ation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS ation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS Ari-62360.1 31.6 AD-62366.1 14.2 AD-SBZSJ. 27.9 AD»62331.1 31.5 AD-6233'7IJ. 33.9 [3.0623431 36.1 AD-6234QIJ. 37.6 AD»623SS.1 38.8 IRE—62361.1 46.1 3671 23.6 """'2{'51'é'£':£'7"§i1""""3.2 """"A" 067731296 ""“EQSZ'é'EEéEE""""37 Ari-62391.1 . 3.7 730823371 54.1 Ari-{32403.1 34.8.

Auezmm 28.2 ACE—62368.1 29.7 730823743 29.6 Ari-{32380.1 30.6 730823363 23.4 AD~62392§L 30.5 AD»623§8.1 48.7 """"i§611§2~_'2i€3;3""""+1.48 """"K D621101213 ""“xiifiié'iéié'éf'i""""1.74"""""""""""""" ACE—62375.1 31.9 AD—52381.1 27.3 {3.0623871 77 33.1 93.3 ACE—62399.1 13-32 730824053 28.5 Ari-{32411.1 19.4 730823703 16.3 [Annotation] DPS None set by DPS [Annotation] DPS ionNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS AID-{32376.1 48.2. 49623321 28.5 49623331 49.9 4.9623941 29.9 49624001 45.2 49624061 23 496241.21 45.9 4.9623711 66.5 49623771 49.5 49623831 73.8 """'2§Si'é'£':£'zl'§f'1'""""6.24 """"4" 957991513 '"""'2§Si'é'£2£5'i'f'1'""""12 491324071 30.2. 49624131 28.1 49 32372 1 43 4962373 1 17.9 4 1 2.9.6 49623901 37.7 491323951 26 21 31.6 49624031 46.6 4.9624141 27.2 """7ILSIEEZEIEH""""+174 9424141253‘ """"/3{"1§i€2'2£'i‘§f'1'""""+363"""""""""""""" 49617792 43.2 52 22.5 49617912 27.3 49617972 39.5 49618-332 30.9 49618092 75.: 491318152 90.7 49618212 33.7 [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS 730.2 53.5 All-617922 Example 7:312 l-”ivn Screening of Additional siRNAs Based on the sequence of AME—58643“, an additional tour sense and three antisense sequences were synthesized and used to prepare twelver 2l/25 mer compounds (Table 23). In general, the antisense strands of these compounds were extended with a d’l'd’l‘ and the duplexes had fewer fluorosrnodiiied nucleotides.

C57BL/6 mice W23 per group) were injected subcutaneously with l nig/kg ofthese GalNAc conjugated duplexes, serum was collected on day t) pren‘nleede and day 5, and the levels of CS ns were quantified by ELlSA. CS protein levels were normalized to the day 0 pre- ll) bleed level.

Figure l4 shows the results of an in viva single dose screen with the indicated iRNAs.

Data are expressed as percent of CS protein ing relative to eed levels. These iRNAs having improved y as compared to the parent compound included ADn625 l0? ADn626-43t ALE-62645, ABE-62646, ABE-62650, and All—(3265i 'l‘hese iRNAs also demontsrated similar potencies (Kiss of about 23-59 plvl).

The efficacy of these iRNAs was also tested in CS'7Bl/6 mice using a —dosing administration protocol. Mice were subcutaneously administered ADwtiES l 0, All—62643 AD- 62645, AID—62646, 2650, and AD-(i'Zt’iSl at a 0. 25 mg/kg, (3.5 mg/kg, ill mg/lrg, or 2.5 mg/ltg dose. Serum was collected at days (l and S and analyzed for CS protein levels by liLlSA.

C5 levels were normalized to the day 0 eed level.

Figure l5 shows that there is a dose response with all of the tested iRNAs and that single~ dosing of all of these iRNAs achieved silencing of (:5 protein similar to or better than AD- 58641.

The duration encing ot‘ADeZSlO, Fifi—62643, AD—626459 AD—626469 AD—62650, and AD~6265l in viva was determined by administering a single ill mgx’kg dose to C57Bl/6 mice and determining the amount of {.75 protein present on days 6, 13, 2t), 27, and 34 by ELlSA.

CS levels were normalized to the day (l pre-hleed level.

As demonstrated in Figure l6“, each of the iRNAs tested has the same recovery cs as AD~62643 trending toward the ‘ est silencing, but within the error of the assay.

[Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS ed set by DPS Ali—625 it), AD~62643, AD~62645, AD~62646, AD~62650, 62651 were further tested for efficacy and to evaluate the eumuiative effect cr‘the iRNAs in rats using a repeat administration protccei. WiEd—typc Spragrie Dawiey rats were sutmutanecusiy irij ected with each 0f the iRNAs at a 5.0 mg/kg/dcse On days (in, 4a, and 7. Serum was ted 0111 days {3“, 4, 7, ii, (I: 14-, 18, 25? 289 and 32,. Serum hemoiytic activity was quantified as described above.

The resuits depicted in Figure 17 dcineristratc that aii Ofthe tested iRNAs have a potent and (hirabie decrease in hemoiytie activity and a simi iar recovery ot‘heriioiysis t0 that Observed wi th AD—S 864i 'treaimen t. ation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS [Annotation] DPS Unmarked set by DPS [Annotation] DPS None set by DPS [Annotation] DPS MigrationNone set by DPS ation] DPS Unmarked set by DPS H3335?“ H3335?“ flywwnw .m<mew EDQSSEEVSEV EDQSSEEEEQ whwgsugswdfibzwdwzfiflfie mm HwhwsmsmsmfiwfimfiDms‘ww<fi<mfi<fimmm<ma Bdfivfifififlmumfixufiafiwa‘: <E<EDE<E E<EDE<€ Ama‘ws E HuHEmax806EDEEEEEEEEDE (a My?mm3mBEHGEDQMDmsfixwmwflfifiwa:EmmaaE .w.ww§§§<dCfibfi3msmfiuéyfiafigfi3%;me HmwhmsmsmssfififiDfiDmexfigmfixxfiDmm/EE wages.sousodqlzwmmvwo . . A . . _. . . _ _ __ Ia‘mwmmT/«LEL _ _ .. \ . . .. _ I A ._ 5 , I , 3253, E33,1 um E E wwfifiw :MSEEE E .g .\ _ on I cal? >E$ a<.w..w.m....w.»...v.m...m\ .w , ‘3 m “Haggis”: EE< ‘3 S .mmnmmézsiwbfl:i 12m .m, m: Eu.m.fl.....w.w. .. . 32:?me 35m gnaw ., ,, _.. Egan? 353,4 a :Nmfimfip‘q _, GEES? . ‘ e a E W q _ : , 39% f ‘p i , .mm a: mEmH wax A, M5 $£wn< H2 E33 38-3 mwwmmA?‘ “unwavmd wwmmmAfix ESEQ wwfiNWQE omwmc.m< Semwxflfix

Claims (17)

We claim :

1. A -stranded RNAi agent for inhibiting expression of complement component wherein said double ed RNAi agent comprises a sense strand and an nse strand forming a double stranded region, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of 5’- UUAUAGUGAGUUAUUUUGUCAAU-3’ (SEQ ID NO:105), wherein substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, and wherein the sense strand is conjugated to at least one ligand which is one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.

2. The double stranded RNAi agent of claim 1, wherein all of the nucleotides of said sense strand and all of the nucleotides of said antisense strand comprise a modification.

3. The double stranded RNAi agent of claim 1, wherein at least one of said modified nucleotides is selected from the group consisting of a 3’-terminal deoxy-thymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro ed nucleotide, a 2'-deoxymodified nucleotide, a locked nucleotide, an abasic tide, a 2’-amino-modified nucleotide, a 2’-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a tide comprising a 5'-phosphorothioate group, and a al nucleotide linked to a cholesteryl derivative or a noic acid bisdecylamide group.

4. The double stranded RNAi agent of claim 1, wherein each strand is no more than 30 nucleotides in length.

5. The double stranded RNAi agent of claim 1, wherein at least one strand comprises a 3’ ng of at least 1 nucleotide; or at least one strand comprises a 3’ overhang of at least 2 nucleotides.

6. The double stranded RNAi agent of claim 1, further comprising a ligand.

7. The double stranded RNAi agent of claim 6, wherein the ligand is conjugated to the 3’ end of the sense strand of the dsRNA agent.

8. The double stranded RNAi agent of claim 6, wherein the ligand is an N- galactosamine (GalNAc) derivative.

9. The double stranded RNAi agent of claim 8, wherein the ligand is HO OH O H H HO O N N O AcHN HO OH O H H HO O N N O AcHN O O O HO OH HO O N N O AcHN H H O .

10. The double stranded RNAi agent of claim 9, wherein the double stranded RNAi agent is conjugated to the ligand as shown in the ing schematic and, wherein X is O or S.

11. The double stranded RNAi agent of claim 1, wherein the double-stranded region is 15-30 nucleotide pairs in length; 17-23 nucleotide pairs in length; 17-25 nucleotide pairs in length; 23-27 tide pairs in length; 19-21 tide pairs in length; or 21-23 nucleotide pairs in length.

12 The double stranded RNAi agent of claim 1, wherein each strand is independently 15- 30 nucleotides in length; or 19-30 nucleotides in length.

13. The double stranded RNAi agent of claim 1, wherein the agent further comprises at least one phosphorothioate or phosphonate internucleotide linkage.

14. An isolated cell containing the double stranded RNAi agent of any one of claims 1 to

15. A pharmaceutical composition for inhibiting expression of a complement component C5 gene comprising the double stranded RNAi agent of any one of claims 1 to 13.

16. A method of inhibiting complement component C5 expression in a cell in vitro or ex vivo, the method comprising: (a) contacting the cell with the double ed RNAi agent of any one of claims 1 to 13, or the pharmaceutical composition of claim 15; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain ation of the mRNA transcript of a complement component C5 gene, thereby inhibiting expression of the complement component C5 gene in the cell.

17. The method of claim 16, wherein the complement component C5 expression is inhibited by at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98% or about 100%.