AU2015202154A1 - Lipopeptides for delivery of nucleic acids - Google Patents

Abstract

Lipopeptide compounds comprising a peptide having 2 to 100 amino acid residues, and having a lipophilic group attached to at least one terminus of the peptide or to at least one amino acid residue of the peptide, and salts and uses thereof. The lipophilic group may be attached to the N-terminus, C-terminus or both termini of the peptide. The lipophilic group may be attached to at least one interal amino acid residue (i.e., an amino acid residue that is not the N-terminus or the C-terminus amino acid residue of the peptide). The lipophilic group may be attached to either termini or both and at least one internal amino acid residue. 2800633_1.docx

Description

LIPOPEPTIDES FOR DELIVERY OF NUCLEIC ACIDS CROSS-REFERENCE TO RELATED APPLICATIONS This application is the Australian national phase application of International 5 application No. PCT/US2008/078627 filed October 2, 2008 which claims the benefit of United States Provisional Patent application No. 60/976894 filed October 2, 2007 the ful contents of each of which is incorporated by reference herein. FIELD OF THE INVENTION This invention relates to novel drug delivery enhancing agents including lipids 10 that are useful for delivering various molecules to cells. This invention provides a range of compounds, compositions, formulations, methods and uses of such agents directed ultimately toward drug delivery, therapeutics, and the diagnosis and treatment of disease! and conditions, including those that respond to modulation of gene expression or activity in a subject. More specifically, this invention relates to compounds, liposomes, lamellar 15 vesicles, emulsions, micelles, suspensions, particles, solutions and other forms of delivery enhancing compositions and formulations, as well as therapeutic methods and uses for these delivery materials. BACKGROUND The delivery of a therapeutic compound to a subject can be impeded by limited 20 ability of the compound to reach a target cell or tissue, or by restricted entry or trafficking of the compound within cells. Delivery of a therapeutic material is in general restricted by membranes of cells. These barriers and restrictions to delivery can result in the need to use much higher concentrations of a compound than is desirable to achieve a result, which brings the risk of toxic effects and side effects. 25 One strategy for delivery is to improve transport of a compound into cells using lipid or polymeric carrier molecules. These materials can take advantage of mechanisms that exist for selective entry into a cell, while still excluding exogenous molecules such as nucleic acids and proteins. For example, a cationic lipid may interact with a drug agent and provide contact with a cell membrane. Lipid molecules can also be organized into 30 liposomes or particles as carriers for drug agents. Liposomal drug carriers can protect a drug molecule from degradation while improving its uptake by cells. Also, liposomal drug carriers can encapsulate or bind certain compounds by electrostatic and other

I

interactions, and may interact with negatively charged cell membranes to initiate transp rt across a membrane. The understanding of regulatory RNA and the development of RNA interference (RNAi), RNAi therapy, RNA based drugs, antisense therapy, and gene therapy, among 5 others, has increased the need for effective means of introducing active nucleic acid agents into cells. In general, nucleic acids are stable for only limited times in cells or plasma. However, nucleic acid-based agents can be stabilized in compositions and formulations which may then be dispersed for cellular delivery. What is needed are compositions and formulations for intracellular and in vivo 10 delivery of a nucleic acid agent for use, ultimately, as a therapeutic, which maintain cytoprotection and relatively low toxicity. Furthermore, there is a need for compositions and methods to deliver double-stranded RNA to cells to produce the response of RNA interference. Moreover, there is a need for compositions and methods for delivery of interfering RNAs to selected cells, tissues, or compartments to modulate gene expression 15 in a manner that will alter a phenotype or disease state. BRIEF SUMMARY This invention satisfies these needs and fulfills additional objects and advantages by providing a range of novel compounds, compositions, formulations and methods that employ an interfering nucleic acid or precursor thereof in combination with various D components including lipopeptides, lipids, and natural or synthetic polymers. In some embodiments, this invention includes a peptide having 2 to 100 amino acid residues and a lipophilic group attached to at least one terminus of the peptide, or to at least one amino acid residue, and salts and uses thereof. The lipophilic group may be attached to the N-terminus, C-terminus or both termini of the peptide. The lipophilic 25 group may be attached to at least one interal amino acid residue (i.e., an amino acid residue that is not the N-terminus or the C-terminus amino acid residue of the peptide). The lipophilic group may be attached to either termini or both and at least one internal amino acid residue. This summary, taken along with the detailed description of the invention, as well 30 as the appended examples and claims as a whole encompasses the disclosure. 2 DETAILED DESCRIPTION This invention relates generally to the fields of RNA interference, delivery of RNA therapeutics, and to the chemistry of lipids. More particularly, this invention relate! to compositions and formulations for ribonucleic acids, and their uses for medicaments 5 and for delivery as therapeutics. This invention relates generally to methods of using ribonucleic acids in RNA interference for gene-specific inhibition of gene expression in mammals. This invention provides a range of compositions, formulations and methods whict include an interfering nucleic acid or a precursor thereof in combination with various 0 components including lipopeptides, lipids, lipoid moieties, and natural or synthetic polymers. In some aspects, this invention provides novel compositions to facilitate the delivery of nucleic acids including RNAi-inducing agents, antisense RNA agents, or DNA to cells, tissues, organs, and in living animals, for example, mammals and humans. 5 Liponeptides This invention provides a range of lipopeptides for delivery and administration of interfering-RNA agents or antisense RNAs. The lipopeptides can be cationic, or non-cationic. As used herein, non-cationic includes neutral and anionic. Cationic lipopeptides may have one or more cationic sites. D Lipopeptides of this disclosure may be formed by substituting an nucleic acid delivery-enhancing or lipoid group at the N-terminus or the C-terminus of a peptide, or both termini of the peptide. In some aspects, the nucleic acid delivery or lipoid group may be linked to the alpha-carbon of one or more an amino acid residues of the peptide or to one or more amine groups, each amine group being adjacent to the alpha-carbon of an 25 amino acid residue of the peptide, or a combination of the N-terminus, C-terminus, alpha carbon of one or more an amino acid residues of the peptide or to one or more amine groups, each amine group being adjacent to the alpha-carbon of an amino acid residue of the peptide. In some embodiments, the peptide core may include two or more sequential amino acids, or a peptide of 2 to 100 amino acid residues (or 2, 3, 4, 5, 6, 7, 8. 9, 10, 11, 30 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acid residues). 3 In some aspects, this invention provides a range of lipopeptide structures as show in Formula I: R-J- {NRN-CRlR2-(C=O)})nZ-R 4 Formula I 5 wherein R' is independently, for each occurrence, a non-hydrogen side chain of an amino acid; {NRN-CRlR 2 .(C=O)}n is a central peptide;

R

2 and RN are independently of one another hydrogen, or an organic group 0 consisting of carbon, oxygen, nitrogen, sulfur, and hydrogen atoms, and having from 1 to 20 carbon atoms, or C(1 -22)alkyl, (6 12)cycloalkyl, (6-12)cycloalkylalkyl, C(3-18)alkenyl, C(3 I 8)alkynyl, C(1 -5)alkanoyl, C(1 -5)alkanoyloxy, C(1 -5)alkoxy, C(1 -5)alkoxy 5 C( 1- 5)alkyl, C(I -5)alkoxy-C(1 -5)alkoxy, C(1 -5)alkyl-amino-C( 1 5)alkyl-, C(I-5)dialkyl-amino-C(1-5)alkyl-, nitro-C(1-5)alkyl, cyano-C(1 5)alkyl, aryl-C(1 -5)alkyl, 4-biphenyl-C(I -5)alkyl, carboxyl, or hydroxyl; R' and R4 are independently of one another, a lipophilic tail derived from i .0 naturally-occurring or synthetic lipid, phospholipid, glycolipid, triacylglycerol, glycerophospholipid, sphingolipid, ceramide, sphingomyelin, cerebroside, or ganglioside, wherein the tail may contain a steroid, or an organic group consisting of carbon, oxygen, nitrogen, sulfur, and hydrogen atoms, and having from 1 to 20 25 carbon atoms, or a substituted or unsubstituted C(1-22)alkyl, C(6 12)cycloalkyl, C(6-12)cycloalkyl-alkyl, C(3-1 S)alkenyl, C(3 18)alkynyl, C(I -5)alkoxy-C(1 -5)alkyl, or a sphinganine, (2R,3 R)-2-amino- 1,3 -octadecanediol, icosasphinganine, 30 sphingosine, phytosphingosine, cis-4-sphingenine, or a ceramide; Z is NH, 0, or a linker comprising a maleimido, thioether, amide, 4 cysteamide, cysteine, thiol, or a disulfide group, or a polyethyleneoxide or polypropyleneoxide group comprising 1-400 atoms, or a linker comprising 1-200 atoms selected from the group of C, H, F, Cl, Br, N, 0, S, Si, and P; 5 J is (C=0), 0, or a linker comprising a maleimido, thiocther, aide, cysteamide, cysteine, thiol, or disulfide group, or a polyethyleneoxide or polypropyleneoxide group comprising 1-400 atoms, or a linker comprising 1-200 atoms selected from the group of C, H, F, Cl, Br, 10 N, 0, S, Si, and P; and n is from 2 to 100. Lipopeptides of this disclosure can be prepared where, for example, the central 15 peptide is capable of binding nucleic acids. Lipopeptides of this disclosure can be prepared where, for example, the central peptide may be an amphipathic amino acid sequence. For example, the peptide may have a plurality of non-polar or hydrophobic amino acid residues that form a hydrophobic sequence domain or motif which may be linked to a plurality of charged amino acid 20 residues that form a charged sequence domain or motif, yielding an amphipathic peptide Lipopeptides of this disclosure can be prepared where, for example, the central peptide may be Poly-Lys-Trp, 41, Mw 20,000-50,000; Poly-Orn-Trp, 4:1, Mw 20,000 50,000; fragments or variants of mellitin protein, and fragments or variants of a histone protein, e.g., histone HI, histone H2A, histone H2B, histone H3 or histone H4. 25 Lipopeptides of this disclosure can be prepared where, for example, the central peptide has multiple cationic sites or multiple posivelty-charged residues. The central peptides may comprise from about 2 to about 45 positively-charged residues; from about 3 to about 45 positively-charged residues; from about 4 to about 45 positively-charged residues; from about 5 to about 45 positively-charged residues; from 30 about 10 to about 45 positively-charged residues; from about 15 to about 45 positively charged residues; from about 20 to about 45 positively-charged residues; from about 25 to about 45 positively-charged residues; from about 30 to about 45 positively-charged residues; from about 35 to about 45 positively-charged residues; from about 40 to about 45 positively-charged residues. 5 The peptides may be thiolylated substantially polylysine peptides. They may comprise at least 3, 4, 5, 6, 7, 8 or so thiol groups or may have only two thiol groups. Lipopeptides of this disclosure can be prepared where, for example, the central peptide is arginine (Arg), (SEQ ID NO: 341), homoarginine (homoArg)" (side chain 5 -(CH 2

)

4

NH(C=NH)NH

2 ), norarginine (norArg),, (side chain -(CH 2

)

2

NH(C=NH)NH

2 ), nor-norarginine (nomorArg)n (side chain -(CH 2

)NH(C=NH)NH

2 ), ornithine (Orn)n, lysine (Lys)n, (SEQ ID NO: 342), histidine (His)n (SEQ ID NO: 343), where n is 2 to 100. Lipopeptides of this disclosure can be prepared where, for example, the central peptide is (Arg,His),, (Lys,His)n, (Arg,Lys),, His-(Arg),-His (SEQ ID NO: 344), 10 His-(Lys)n-His (SEQ ID NO: 345), (His)n-(Arg)n-(His), (SEQ ID NO: 346), or (His).-(Lys), 1 -(His)n (SEQ ID NO: 347), where n is 2 to 100. Examples of the central peptide of lipopeptides of this disclosure include those shown in Table 1. Table 1 15 Peptide Structures for Lipopeptides AMINO ACID SEQUENCE SEQ ID O: (R), where n = 1-20 1 (K), where n = 1-20 2 (R) (K) where n and m are independtly for each occurence 1 20 (RH), or (HR)n where n - 1-20 4 & 318 [(RH)r(HR)m3 where n and m. are independtly for each 5 occurence 1-20 (KH), or (HK), where n = 1-20 6 & 349 [ (KH),(HK).) where n and m are independtly for each 7 occurence 1-20 H(K)nH where n = 1-20 8 H(R)nH where n = 1-20 9 (H)q (R)b 10 (H) 4 (R) 8 11 6 (H)4(R) (K) 12 (H)g(R) 8 (H)o~(K) 13

(H)

4

(R)

5

(H)

3

(K)

4 14 HHHHHKHHHKKKHKHKKK 15 KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ 16 KGS KKAVTKAQKKEGKKRKRSRKESYSVYVYKVLKQ 17 AQKKEGKKRKRSRKSYSVYVYKVLKQ 18 KRSRKESYSVYVYKVLKO 19 ESYSVYVYKVLKQ 20 YKVL-KQ 21 RVIRWFQNKRSKDKK 22 GALFLGFLGAAGS TMGAWSQPKSKRKV 23 RQI KIWFQNRRMKWKK 24 RQIKIWFQNRRMKwKK (ALL D-AMINO ACIDS) 25 GWTLNSAGYLLKINLKALAALAKKI L 26 LLNQLAGRMI PKWSQKS KRKV 2 TLDHVLDHVQTWSQKSKRKV 28 SYFILRRRRKRFYFFTDVRVAAJ 29 RRRRRIRRRRR 30 RRRRRRRR (all D-amin-o acids) 3 7 ESYSVYVYRVLRQ 35 AS YSVYVYAVLAQ 36 QKLVKYVYVSYSE (all D-amjno acids) 40 RRRRRRESYSVYVYKVLKQ 4 ESYSVVYKVKQRRRRR42 RQIK WFQN RMK KKRR RRR44 KTKI ESLI(EHGRRRRRR 45 MDVNPTLLFLKVPAQNAI STTFPYTRRRRRR 46 GLFEALLELLESLWELLLEARRRRRR 47 LLNQLAGRMI PKRRRRRR 48 TLDHVLDHVQTRRRRRR 49 GLFGAIAGFIENGWEGMT DGRRRRRR 5 KVLKQ54 KRRQRRR 55 DATTGSARTR~AASSPRV 56 VTVLALGALAGVGVG 57 8 GALFGWLAAGSMGA58 MGLG HLL LAAA QGA59 GWTLNSAGYLLKINLKALAALAKKI L 61 TPPKKKRKVEDPKKKK 62 KLALKLALKALKAALKLA 63 GLFGAIAGF'TENGWEG 64 FFGAVIGTIALGVATA 65 FLGFLLGVGSAIASGV 66 GVFVLGFLGFLATAGS 67 GA.AIGLAWI PYFGPAA 68 ACTCPYCKDSEGRGSGDPGKKKQHICHTQGCGKVYGKTSHLRAHLRWHTGERPFMC 69 ACTCPNCKDGEKRSGEQGKKKHVCHI PDCGKTERKTSLLRAHVRLETGERPFVC 70 ACTCPNCKEGGGRGTNLGKKKQHICHI PGCGKVYGKTSHLRAHLRWHSGERPFVC 71 ACSCPNCREGEGRGSNEPCKKKQHICHIEGCGKYGKTSHLRJAHLRWHTGERPFIC 72 RCTCPNCTNEMSGLPPI VGPDERGRKQHICHIPGCERLYGKASHLKTHLRWHTGERPFLC 73 TCDCPNCQEAERLGPAGVHLRKKNILHSCHIPGCGVYGKTSHLKAH TRWHTGERPFVC 74 RCTCPNCKAI!HGDRGSQHTHLCSVPGCGZKTYKKTSNLRAHLRKHTGDRPFVC 75 PQISLKKKIFFFIFSNFRGDGKSR IHICHLCNKTYGKTSHLRAHLRGHAGNKPFAC 76 WWETWKPFQCRI CMRNFS TRQARRNHRRRHR 77 GKI NLKALAALAKKIL 78 RVI RVWFQNKRCKDKK 79 GRXKRQRRPPQRKKRQRRPPQRKKRQRRPPQ80 9 GEQIAQLIAGYIDI ILKKKKSK 81 KGS KIAVT KAQKKDGKKRKRs RKES YSVYVYKVILKQ 82 KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ 83 RKESYSVYVYKVLKQ 84 KKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ 85 VTKAQKKDGKKRKRS RKESYSVYVYKVLKQ 86 AQKKDGKKRKRSRKESYSVYVYKVLK(Q 87 KDGKKRKRSRKES YSVYVYKVLKQ 88 KKRKRSRKESYSVYVYKViLKQ 89 SYSVYVYKVLKQ 90 VYVYKVLKQ 91 KGSKKAVTKAQKKEGKKRKRS RKESYSVYVYKVLKQ 92 WWHHKXRRC 93 WWHH KKRRCCRRKKH HWW 94 GRKKRRQRRRPPQ 95 KKKRKV 96 KKKRKVKKKRKV 97 GRKKRRC 98 RRBPPQC 99 CGRKKRR 100 CRRRPPQ 101. WKKKKC 102 CWKRK(K 103 10 CRRRPPQH 104 CRRRPPQ 105 CKKRRQH 106 CRR 10 7 CRRR 108 CRRRR 109 CRRRRR 110 CKK CKKK 112 CKKKK 113 CKKKKK 114 CWHRRKK 1215 C-RRKHWW 122 CKRRW 123 CKKRRW 124 CKKRHW 125 CKKRRQW 120 112 KKRRQC 127 CGRKKRR 128 GRKKRRC 129 CGRKKRRQ 130 QGRKKRRC 131 CRRH 132 CRRRH 133 CRRRRH 134 CRRRRRH 135 CKKH 136 CKKKH 137 CKKKKH 138 CKKKKKH 139 HWKKRRC 140 CHWK(KRR 141 PPHRRRC 142 CPPHRRR 143 GRKKRRVURRRPPQ 144 GRKKRRVURRKKRG 145 RRRPPQVUPPRRR 146 RRKKRGVEJGRKKRR 147 QPPRRRVURRRPPQ 148 W'KKKKVUKKKKW 149 12 KKKKWVUWKKKK 150 HQPPRRRVURRRPPQH 151 QPPRRRVURRRPPQ 152 HQRRKKVUKKRRQH 153 RRVLJRR 154 RRRVURRR 155 RRRRVURRRR 156 RRRRRVURRRRR 157 KKVUKK 158 KKKVUKKK 159 KKKKVUKKKK 160 KKKKKVUKKKKK 161 WWRRRRVURRRRWW 162 WWRRRVURRRWW 163 TWMRRVURRWW 164 WWKKVU KKWW 165 WWKKKVUKKKWh7 166 WWKKKKVUKKKKWW 167 KKRRHHWVUWHIIRRKK 168 WWHHKKRRVURRKKI HWW 169 WRRKKVU KKRRW 170 WHRRKKVUKKRRHW 171 WHHRRKKVUKKRRHHW 172 13 QRRKKVUKKRRQ 173 KKRRQVUQRRKK 174 RRKKRGVUGRKKR. 175 GRKKRRVURRKKRG 176 QRRKKRGVUGRKKRRQ 177 QGRKKRRVURRKKRGQ 178 HRRVURRH 179 HRRRVURRRH 180 HRRRRVURRRRH 181 H RRRRRVURRRRRH 182 HKKVUKKH 183 HKKKVUKKKH 184 H KKKKVtJKKKKH18 H KKKKKVUKKKKKH 186 HWKKRRVURRKKWH 187 RRKKHVUHKKRR188 PPHRRVURRHPP189 RRRH PPVLJPPHRRR 190 wwHHKKRRGGRRKKHHWW 191 WWHHKKRR 192 YYHHKKRR19 RRKKHHYY19 YYHHKKRRCCRRKKHHYY19 14 YYHKRRWRWHY CqWRRRW CWWRW 200 CWRRRRWC 200 CWRRRRWC 202 CWWRRHRRWC 203 CWWRRHRRWWC 204 CWRRRRRWC VQAAI DYING20 CWWRRRRRWWC WWRRCCRRWW CYRRRC YYRRYC C RRYYC CYYRRHRYYC 2 CYRRRRRYC21 151 CYYRRRRRYYC 219 CYYRRRRYYC 220 YYRRCCRRYY 221 YYRRVRRYY 222 WWRRHH 223 HHRRWW 224 WWRRHHCCHHRRWW 225 WWRRHHVHHRRWW 226 YYRRHH 227 HHRRYY 228 YYRRHHCCRRR-HYY 229 YY RRHHVRR-HYY 230 WWRRR 231 RRRWW 232 WWRRRCCRRRWW 233 WWRRRVRRRWW 234 YYRRR 235 RRRYY 236 YYRRRCCRRRYY 237 YYRRRVRRRYY 238 WWRRRHH239 HHRRRWW240 WWRRRHHCCHHRRRWW 241 16 WWRRRHHVHHRRRWW 242 YYRRRI{H 243 HHRRRYY 244 YYRRRHHCCRRRHHYY 245 YYRRRHHVRRRHHYY 246 WWRRRR 247 RRRRWW 248 WWRRRRCCRRRRWW 249 WWRRRRVRRRRWW 250 YYRRRR 251 RRRRYY 252 YYRRRRCCRRRRYY 253 YYRRRRVRRRRYY 254 WWRRRRHH 255 HHRRRRWW 256 WWRRRRHHiCCHHRRRRWW 257 WWRRRRHHVHHRRRRWW 258 YYRRRRHH 259 HHRRRRYY 260 YYRRRRH HCCRRRRH HYY 261 YYRRRRHHVRRRRHHYY 262 WWHHKKRRWVWRRKKH HWW 263 WWHfHRRC26 17 WWHHRRVRRHHWW 265 WWHHHRRRC 266 CWWHHHRRRC 267 CWWWH HI-HRRR 268 CWWWKKRRR 269 O-KKKWRRW 270 CWRRRWRR 271 CWWHHKKRRC 272 WWCHHKKCRR 273 1W3HHCKKRRC 274 CWWHHKKCRR 275 WWH HCCKKRR 2"76 RRWWKKHHC 277 CWWHHKK(KC 278 CWW'HHRRRRC 279 CRRRRHHC 280 C-HEKK(KC 281 CHHRRRRC 282 CYYRRRRIHC 283 CYYKKKXHHC 284 CWKC where n is from 1-20 285 CHWK,C where ni is from 1-20 286 AAASX,YXQWLXXXGPXa, where X,, is any amino acid 287 18 CKnC where n is from 1-20 288 CHKHC where n is from 1-20 289 Examples of the central peptide of lipopeptides of this disclosure include those shown in Table 2. Table 2 5 Peptide Structures for Lipopeptides AMINO ACID SEQUENCE SEQ ID 0: GALFLAFLAAALSLMGLWSQPKKKRKV 290 GALFLAFLAAALXaLMGLWXaaQXaKKKRKV 291 WSQPKKKRKV 292 XaaWSQPKKKRKVXaa 293 WXaaQPKKKRKXa 294

X,,X

3 8 QPKKKRKV 295 LI RLWSHLI HIWFQNRRLKWKKK 296 LIRLWXaHLIHIWFQXaaRRLKWKKK 297 QNRRLKWKKK 298 XaQNRRLKWKKKXaa 299 QXagRRLKWKKK 300 where X,. in Table 2 is independently, for each occurrence, L, A, Q, H, E, R, or K. The amino acid residues of this disclosure may be D- or L-stereocenters. Non-cationic lipopeptides can be prepared where, for example, the central peptide 10 contains only leucine, valine, alanine, serine, or combinations thereof. Lipopeptides of this disclosure can be prepared where, for example, the central peptide may be cell or tissue specific targeting peptide. For example, the peptide may 1e a ligand or fragment thereof that interacts with a cell surfact receptor or a peptide the selectively binds to lipids, peptides, sugars and combinations thereof on the cell surface 15 of a particular cell or tissue type. The central peptide may target one or more of the following cell types: hepatocytes, endothelial, neuronal, cardiacmyocytes, skeletal mus le cells (myoblast or myotube), smooth muscle cells, fibroblasts, erythrocytes, T-cells, B cells, leukocytes, osteoblasts, chondrocytes, adult stem cells, embryonic stem cells, and tumor cells. 19 Lipopeptides of this disclosure can be prepared where, for example, the central peptide may be a fusogenic peptide or fragment thereof. Lipopeptides of this disclosure can be prepared where, for example, the central peptide may be a SNARE (solutable NSF attachment receptor) peptide or fragement 5 thereof. For example, the central peptide may be a v-SNARE or t-SNARE. The central peptide may be a NSF (N-ethylamaleimide-sensitive factor) or SNAP (soluable NSF attachment protein). In some embodiments, a range of lipopeptides corresponding to Formula I are represented by Structure I RN R2 0 RI-J +N-C-C+Z-R4 n R [0 Structure 1 where R', R 2, RN, J, Z, R 3 , R 4 , and n are defined as above. In some embodiments, R 2 , RN, 3 and R may independently, for each occurrence, be C14alkyl, C16alkyl, Cl 8alkyl, or (C18:l)alkenyl. In some embodiments, R 2 , R, and R 4 may independently, for each occurrence, [5 be lipoid groups. In some embodiments, R RN, R 3 and R 4 may independently, for each occurrence, selected lipid-like tails which impart sufficient lipophilic character or lipophilicity, such as defined by water/octanol partitioning, to provide delivery across a membrane or uptak: by a cell. In some embodiments, R 2 , RN R 3 and R 4 may independently, for each 20 occurrence, selected lipid-like tails which impart sufficient lipophilic character or lipophilicity to provide cell membrane anchoring. These tails provide, when used in an amino acid lipid structure, an amphipathic molecule. Lipid-like tails may be derived front phospholipids, glycolipids, triacylglycerols, glycerophospholipids, sphingolipids, ceramides, sphingomyelins, cerebrosides, or gangliosides, among others, and may contain 25 a steroid. In certain embodiments, R 2 , RN, R 3 and R 4 may independently, for each occurrence, be a lipid-like tail having a glycerol backbone. 20 In some embodiments, R 2 , RN, R 3 and R 4 may independently, for each occurrence be C3alkyl, C4alkyl, C5alkyl, C6alkyl, C7alkyl, C8alkyl, C9alkyl, COalkyl, Cl alkyl, Cl2alkyl, Cl3alkyl, CI4alkyl, CI5alkyl, C16alkyl, C17alkyl, CI8alkyl, CI9alkyl, C20alkyl, C21alkyl, or C22alkyl. 5 In some embodiments, R 2 RN, R 3 and R 4 may independently, for each occurrence, be lipophilic tails having one of the following structures: 18:3 18:2 is:1 18:0 16:1 x 16: 0 y 14:1 14 12 10 X8 10 In the structures above, X represents Z, J, N, or the alpha-carbon of Structure I above, a is counted as one of the atoms in the numerical designation, for example, "18:3." In son e embodiments, X may be a carbon, nitrogen, or oxygen atom. In some embodiments, R2, RN, R 3 and R 4 may independently, for each occurrence, be lipophilic tails having one of the following structures: 15 x 20:4 x 20:1 Phytanoyl 21 where X is as defined above. In some embodiments,

R

2 , RN, R' and R' may independently, for each occurrenc, be selected lipid-like tails which may contain a cholesterol, a sterol, or a steroid such as 5 gonanes, estranes, androstanes, pregnanes, cholanes, cholestanes, ergostanes, campestanes, poriferastanes, stigmastanes, gorgostanes, lanostanes, cycloartanes, as well as sterol or zoosterol derivatives of any of the foregoing, and their biological intermediates and precursors, which may include, for example, cholesterol, lanosterol, stigmastanol, dihydrolanosterol, zymosterol, zymostenol, desmosterol, 10 7-dehydrocholesterol, and mixtures and derivatives thereof. In certain embodiments, R 2 , RN, R 3 and R 4 may independently, for each occurrence, be derived from fatty acid-like tails such as tails from myristic acid (C14:0)alkenyl, palmitic acid (C16:0)alkenyl, stearic acid (CI 8:0)alkenyl, oleic acid (C18:1, double bond at carbon 9)alkenyl, linoleic acid (C18:2, double bond at carbon 9 o e 15 12)alkenyl, linonenic acid (C18:3, double bond at carbon 9, 12, or 15)alkenyl, arachidonic acid (C20:4, double bond at carbon 5, 8, 11, or 14)alkenyl, and eicosapentaenoic acid (C20:5, double bond at carbon 5, 8, 11, 14, or 17)alkenyl. Other examples of fatty acid-like tails are found at Donald Voet and Judith Voet, Biochemistry, 3rd Edition (2005), p. 383. z0 In some embodiments, R 2 , RN, R' and R 4 may independently, for each occurrence, be derived from an isoprenoid. In some embodiments, R 3 may be a lipophilic group, as described above, and R 2 RN, and R 4 may be hydrogen. In some embodiments, R 4 may be a lipophilic group, as described above, and R 2 , 25 RN, and R 3 may be hydrogen. In some embodiments, R 3 and R 4 may be a lipophilic group, as described above, and R 2 and RN may be hydrogen. In some embodiments, R 3 , R 4

R

2 may be a lipophilic group, as described above, and RN may be hydrogen. 30 In some embodiments,

R

3 R, RN may be a lipophilic group, as described above, and R 2 may be hydrogen. In some embodiments,

R

3 and RN may be a lipophilic group, as described above, and R 2 and R 4 may be hydrogen. 22 In some embodiments, RN and R 4 may be a lipophilic group, as described above, and R2 and R" may be hydrogen. In some embodiments, RN and R 2 may be a lipophilic group, as described above, and R 3 and R 4 may be hydrogen. 5 In some embodiments, R 4 and R 2 may be a lipophilic group, as described above, and R 3 and RN may be hydrogen. In some embodiments, R 3 and R 2 may be a lipophilic group, as described above, and R 4 and RN may be hydrogen. In some embodiments, a lipophilic group may be linked to the alpha-carbon of any 10 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 sequential amino acid residues. In some embodiments, a lipophilic group may be 15 linked to the alpha-carbon of every other amino acid residue of the central peptide. In some embodiments, a lipophilic group may be linked to the alpha-carbon of any two sequential amino acid residues of the central peptide where the two sequential amino aci( residues are each adjacent to an amino acid residue within the central peptide that do not have a lipophilic group linked to its alpha-carbon. !0 In some embodiments, a lipophilic group may be linked to the amine group of any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 25 or 100 sequential amino acid residues where the amine groups is adjacent to the alpha carbon of the amino acid residue of the central peptide. In some embodiments, a lipophilic group may be linked to the amine group of every other amino acid residue of the central peptide. In some embodiments, a lipophilic group may be linked to the amine group of any two sequential amino acid residues of the central peptide where the two 30 sequential amino acid residues are each adjacent to an amino acid residue within the central peptide that do not have a lipophilic group linked to its amine group where the amine group is adjacent to the alpha-carbon of the amino acid residue of the central peptide. 23 In some embodiments, a range of lipopeptides corresponding to Formula I are represented by Structure 2, where z is from 0 to 20 (or 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), X is independently, for each occurance, any amino acid, R' is defined above, and n is from 0 to 98 (or 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 5 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 8 4, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, or 98). N 10 H H 0 H 0 H -C-- (X),-- -C-C --- Z- RN I I z Structure 2 15 In some embodiments, a range of lipopeptides corresponding to Formula I are represented by Structure 3, where z is from 0 to 20, X is independently, for each occurance, any amino acid, R' is defined above, and n is from 0 to 98. 20 N H H 0 H 0 H-N-C- -(X)n-N-- -- Z 25 R1 R1 Structure 3 In some embodiments, a range of lipopeptides corresponding to Formula I are 30 represented by Structure 4, where z is from 0 to 20, X is independently, for each occurance, any amino acid, R' is defined above, and n is from 0 to 98. 24 H O H H O z -N-C--C-(X),--N-C-C--OH Structure 4 10 In some embodiments, a range of lipopeptides corresponding to Formula I are represented by Structure 5, where z is from 0 to 20, X is independently, for each occurance, any amino acid, R1 is defined above, and n is from 0 to 98. 15 N H 0 H H 0 z- ~~N--C -C-(X)--N--C-C-- OH 20 I R1 R' Structure 5 In some embodiments, a range of lipopeptides corresponding to Formula I are 25 represented by Structure 6, where z is from 0 to 20, X is independently, for each occurance, any amino acid, R' is defined above, and n is from 0 to 98. H H O H H 0 30 J--- --- -- (x)n -- I---- z R' R3 Structure 6 25 In some embodiments, a range of lipopeptides corresponding to Formula I are represented by Structure 7, where z is from 0 to 20, X is independently, for each occurance, any amino acid, R' is defined above, and n is from 0 to 98. 5 N H H 0o J------ -- -(X) -N - -- C---r 10 1 R4 R' Structure 7 In some embodiments, a range of lipopeptides corresponding to Formula I are represented by Structure 8, where z is from 0 to 20, X is independently, for each 15 occurance, any amino acid, R' is defined above, and n is from 0 to 98. N 20 H H O H O -zz J--- -- c -- -- C- - R1 R' Structure 8 25 In some embodiments, a range of lipopeptides corresponding to Formula I are represented by Structure 9, where z is from 0 to 20, X is independently, for each occurance, any amino acid, R' is defined above, and n is from 0 to 98. 30 26 H 0 H H O 5 J-N-- z R Ri Structure 9 10 In some embodiments, a range of lipopeptides corresponding to Formula I are represented by Structure 10, where z is from 0 to 20, X is independently, for each occurance, any amino acid, R1 is defined above, and n is from 0 to 98. 15 N H O H H 0 20

R

1 R1 Structure 10 In some embodiments, a range of lipopeptides corresponding to Formula I are 25 represented by Structure 11, where z is from 0 to 20, X is independently, for each occurance, any amino acid, R' is defined above, and n is from 0 to 98. 30 27 5 H H 0 H O Structure 11 10 In some embodiments, a range of lipopeptides corresponding to Formula I are represented by Structure 12, where z is from 0 to 20, X is independently, for each occurance, any amino acid, R' is defined above, and n is from 0 to 98. 15 H 0 H 0 -J-- -- C -C- -- () - -- C -Z 20 Structure 12 In some embodiments, a range of lipopeptides corresponding to Formula I are 25 represented by Structure 13, where z is from 0 to 20, X is independently, for each occurance, any amino acid, R' is defined above, and n is from 0 to 98. 30 28 H 0 H 0 5 J-N--- - -()n-- - - Z R' R Structure 13 10 In some embodiments, a range of lipopeptides corresponding to Formula I are represented by Structure 14, where z is from 0 to 20, X is independently, for each occurance, any amino acid, R' is defined above, and n is from 0 to 98. N N 15 H 0 H 0 J- -N - -- - -- ( I- -- - -- z R1 R1 20 Structure 14 In some embodiments, a range of lipopeptides corresponding to Formula I are represented by Structure 15, where z is from 0 to 20, X is independently, for each occurance, any amino acid, R' is defined above, and n is from 0 to 98. 25 N N 30 H H _ J-- - -C -- (X)n- - --- - - R1 R' Structure 15 29 As used herein, the term "amino acid" includes naturally-occurring and non naturally occurring amino acids. Examples of amino acids include Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val. Thu, a lipopeptide of this invention can be made from a genetically encoded amino acid, a 5 naturally occurring non-genetically encoded amino acid, or a synthetic amino acid. Sone examples of amino acids include 2-aminoadipic acid, 3-aminoadipic acid, 2,3 diaminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 2,3-diaminobutyric acid, 2,4-diaminobutyric acid, 2-aminoisobutyric acid, 4-aminoisobutyric acid, 2-aminopimelic acid, 2,2'-diaminopimelic acid, 6-aminohexanoic acid, 6-aminocaproic 10 acid, 2-aminoheptanoic acid, desmosine, ornithine, citrulline, N-methylisoleucine, norleucine, tert-leucine, phenylglycine, t-butylglycine, N-methylglycine, sacrosine, N-ethylglycine, cyclohexylglycine, 4-oxo-cyclohexylglycine, N-ethylasparagine, cyclohexylalanine, t-butylalanine, naphthylalanine, pyridylalanine, 3-chloroalanine, 3-benzothienylalanine, 4-halophenylalanine, 4-chlorophenylalanine, 2 15 fluorophenylalanine, 3-fluorophenylalanine, 4-fluorophenylalanine, penicillamine, 2 thienylalanine, methionine, methionine sulfoxide, homoarginine, norarginine, nor norarginine, N-acetyllysine, N-aninophenylalanine, N-methylvaline, homocysteine, homoserine, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine, 6-N-methyllysine, norvaline, 0-allyl-serine, 0-allyl 20 threonine, alpha-aminohexanoic acid, alpha-aminovaleric acid, and pyroglutamic acid. As used herein, the term "amino acid" includes alpha- and beta- amino acids. Other amino acid residues can be found in Fasman, CRC Practical Handbook o Biochemistry and Molecular Biology, CRC Press, Inc. (1989). In general, a compound may contain one or more chiral centers. Compounds 25 containing one or more chiral centers may include those described as an "isomer," a "stereoisomer," a "diastereomer," an "enantiomer," an "optical isomer," or as a "racemil mixture." Conventions for stereochemical nomenclature, for example the stereoisomer naming rules of Cahn, Ingold and Prelog, as well as methods for the determination of stereochemistry and the separation of stereoisomers are known in the art. See, for 30 example, Michael B. Smith and Jerry March "March's Advanced Organic Chemistry", 5th edition, 2001. The compounds and structures of this disclosure are meant to encompass all possible isomers, stereoisomers, diastereomers, enantiomers, and/or opti al isomers that would be understood to exist for the specified compound or structure, including any mixture, racemic or otherwise, thereof. 30 The term "lipophilic group" or "lipoid group" or "lipid-like tail" or "lipiphilic ta 1" as used herein refers to an alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, alkanoyl, alkylamino, aryl, acyl, heteroaryl, heterocycle, heterocyclyl, alkanoyloxy, alkylamino, alkylaminoalkyl, aroyl, and aralkyl group including substituted variations thereof. 5 The term "alkyl" as used herein refers to a saturated, branched or unbranched, substituted or unsubstituted aliphatic group containing from 1-22 carbon atoms, This definition applies to the alkyl portion of other groups such as, for example, alkoxy, alkanoyl, aralkyl, and other groups defined below. The term "cycloalkyl" as used herein refers to a saturated, substituted or unsubstituted cyclic alkyl ring containing from 3 to 12 10 carbon atoms. The term "alkenyl" as used herein refers to an unsaturated, branched or unbranched, substituted or unsubstituted alkyl or cycloalkyl having 2 to 22 carbon atoms and at least one carbon-carbon double bond. The term "alkynyl" as used herein refers to an unsaturated, branched or unbranched, substituted or unsubstituted alkyl or cycloalkyl having 2 to 22 carbon atoms and at least one carbon-carbon triple bond. 15 The term "alkoxy" as used herein refers to an alkyl, cycloalkyl, alkenyl, or alkyn l group covalently bonded to an oxygen atom. The term "alkanoyl" as used herein refers to -C(=O)-alkyl, which may alternatively be referred to as "acyl." The term "alkanoyloxy" a used herein refers to -O-C(=O)-alkyl groups. The term "alkylamino" as used herein refers to the group -NRR', where R and R! are each either hydrogen or alkyl, and at least one of M R and R' is alkyl. Alkylamino includes groups such as piperidino wherein R and R' form a ring. The term "alkylaminoalkyl" refers to -alkyl-NRR'. The term "aryl" as used herein refers to any stable monocyclic, bicyclic, or polycyclic carbon ring system of from 4 to 12 atoms in each ring, wherein at least one ring is aromatic. Some examples of an aryl include phenyl, naphthyl, tetrahydro 25 naphthyl, indanyl, and biphenyl. Where an aryl substituent is bicyclic and one ring is non-aromatic, it is understood that attachment is to the aromatic ring. An aryl may be substituted or unsubstituted. The term "heteroaryl" as used herein refers to any stable monocyclic, bicyclic, or polycyclic carbon ring system of from 4 to 12 atoms in each ring, wherein at least one 30 ring is aromatic and contains from I to 4 heteroatoms selected from oxygen, nitrogen and sulfur. Some examples of a heteroaryl include acridinyl, quinoxalinyl, pyrazolyl, indolyl benzotriazolyl, furanyl, thienyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, and 31 tetrahydroquinolinyl. A heteroaryl includes the N-oxide derivative of a nitrogen containing heteroaryl. The term "heterocycle" or "heterocyclyl" as used herein refers to an aromatic or nonaromatic ring system of from five to twenty-two atoms, wherein from 1 to 4 of the 5 ring atoms are heteroatoms selected from oxygen, nitrogen, and sulfur. Thus, a heterocycle may be a heteroaryl or a dihydro or tetrathydro version thereof. The term "aroyl" as used herein refers to an aryl radical derived from an aroma c carboxylic acid, such as a substituted benzoic acid. The term "aralkyl" as used herein refers to an aryl group bonded to an alkyl group, for example, a benzyl group. 10 The term "carboxyl" as used herein represents a group of the formula -C(=0)Oi or -C(=0)O. The terms "carbonyl" and "acyl" as used herein refer to a group in which an oxygen atom is double-bonded to a carbon atom >C=O. The term "hydroxyl" as used herein refers to -OH or -0~. The term "nitrile" or "cyano" as used herein refers to -CN. The term "halogen" or "halo" refers to fluoro (-F), chloro (-Cl), bromo (-Br), and iodo 15 (-I). The term "substituted" as used herein refers to an atom having one or more substitutions or substituents which can be the same or different and may include a hydrogen substituent. Thus, the terms alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, alkanoyl, alkanoyloxy, alkylamino, alkylaminoalkyl, aryl, heteroaryl, heterocycle, aroy, 20 and aralkyl as used herein refer to groups which include substituted variations. Substituted variations include linear, branched, and cyclic variations, and groups havi g a substituent or substituents replacing one or more hydrogens attached to any carbon att m of the group. Substituents that may be attached to a carbon atom of the group include alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, alkanoyl, alkanoyloxy, alkylamino, 25 alkylaminoalkyl, aryl, heteroaryl, heterocycle, aroyl, aralkyl, acyl, hydroxyl, cyano, h lo, haloalkyl, amino, aminoacyl, alkylaminoacyl, acyloxy, aryloxy, aryloxyalkyl, mercap o, nitro, carbamyl, carbamoyl, and heterocycle. For example, the term ethyl includes without limitation -CH 2

CH

3 , -CHFCH 3 , -CF 2

CH

3 , -CHFCH 2 F, -CHFCHF 2 , -CHFCF 3 , -CF 2

CE

2 F,

-CF

2

CHF

2 , -CF 2

CF

3 , and other variations as described above. 30 A pharmaceutically acceptable salt of a peptide or protein composition of this invention which is sufficiently basic may be an acid-addition salt with, for example, an inorganic or organic acid such as hydrochloric, hydrobromic, sulfuric, nitric, phosphe ric, chlorosulfonic, trifluoroacetic, citric, maleic, acetic, propionic, oxalic, malic, maleic, malonic, fumaric, or tartaric acids, and alkane- or arenesulfonic acids such as 32 methanesulfonic, ethanesulfonic, benzenesulfonic, chlorobenzenesulfonic, toluenesulfonic, naphthalenesulfonic, naphthalenedisulfonic, and camphorsulfonic acid . A pharmaceutically acceptable salt of a peptide or protein composition of this invention which is sufficiently acidic may be an alkali metal salt, for example, a sodium 5 or potassium salt, or an alkaline earth metal salt, for example, a calcium or magnesium salt, or an ammonium salt or a salt with an organic base which provides a physiological y acceptable cation, for example, a salt with methylamine, dimethylamine, trimethylamin triethylamine, ethanolamine, diethanolamine, triethanolamine, ethylenediamine, tromethamine, N-methylglucamine, piperidine, morpholine or tris-(2 10 hydroxyethyl)amine. See, for example, Berge et at., J. Pharm. Sci. 66:1, 1971. Some compounds, peptides and/or protein compositions of this invention may have one or more chiral centers and/or geometric isomeric centers (F- and Z-isomers), and it is to be understood that the invention encompasses all such optical isomers, diastereoisomers and geometric isomers. 15 This invention encompasses any and all tautomeric, solvated or unsolvated, and hydrated or unhydrated forms of the compounds, peptides and/or protein compositions disclosed herein. Uses for regulatory RNA and RNA interference 20 In some aspects, this disclosure relates generally to the fields of regulatory RNA and RNA interference, antisense therapeutics, and delivery of RNA therapeutics. More particularly, this invention relates to compositions and formulations for ribonucleic acids and their uses for medicaments and for delivery as therapeutics. This invention relates generally to methods of using ribonucleic acids in RNA interference for gene-specific 25 inhibition of gene expression in cells, or in mammals to alter a disease state or a phenotype. RNA interference refers to methods of sequence-specific post-transcriptional gen silencing which is mediated by a double-stranded RNA (dsRNA) called a short interfering RNA (siRNA). See Fire, et al., Nature 391:806, 1998, and Hamilton, et al., Science 30 286:950-951, 1999. RNAi is shared by diverse flora and phyla and is believed to be an evolutionarily-conserved cellular defense mechanism against the expression of foreign genes. See Fire, et al., Trends Genet, 15:358, 1999. RNAi is therefore a ubiquitous, endogenous mechanism that uses small noncoding RNAs to silence gene expression. See Dykxhoorn, D.M. and J. Lieberman, Annu. Rev. 33 Biomed. Eng. 8:377-402, 2006. RNAi can regulate important genes involved in cell death, differentiation, and development. RNAi may also protect the genome from invading genetic elements, encoded by transposons and viruses. When a siRNA is introduced into a cell, it binds to the endogenous RNAi machinery to disrupt the 5 expression of mRNA containing complementary sequences with high specificity. Any disease-causing gene and any cell type or tissue can potentially be targeted. This technique has been rapidly utilized for gene-function analysis and drug-target discovery and validation. Harnessing RNAi also holds great promise for therapy, although introducing siRNAs into cells in vivo remains an important obstacle. 10 The mechanism of RNAi, although not yet fully characterized, is through cleava ge of a target mRNA. The RNAi response involves an endonuclease complex known as th: RNA-induced silencing complex (RISC), which mediates cleavage of a single-stranded RNA complementary to the antisense strand of the siRNA duplex. Cleavage of the targ t RNA takes place in the middle of the region complementary to the antisense strand of the 15 siRNA duplex (Elbashir, et al., Genes Dev. 15:188, 2001). One way to carry out RNAi is to introduce or express a siRNA in cells. Another way is to make use of an endogenous ribonuclease III enzyme called dicer. One activity of dicer is to process a long dsRNA into siRNAs. See Hamilton, et al., Science 286:950 951, 1999; Berstein, et al,, Nature 409:363, 2001. A siRNA derived from dicer is 20 typically about 21-23 nucleotides in overall length with about 19 base pairs duplexed. See Hamilton, et al., supra; Elbashir, et al., Genes Dev. 15:188, 2001. In essence, a long dsRNA can be introduced in a cell as a precursor of a siRNA. This invention provides a range of compositions, formulations and methods whic1 include a regulatory RNA, an interfering nucleic acid or a precursor thereof in 25 combination with various components including lipids, amino acid lipids, and natural or synthetic polymers. The term "dsRNA" as used herein refers to any nucleic acid molecule comprising at least one ribonucleotide molecule and capable of inhibiting or down regulating gene expression, for example, by promoting RNA interference ("RNAi") or gene silencing in 30 sequence-specific manner. The dsRNAs of this disclosure may be suitable substrates for Dicer or for association with RISC to mediate gene silencing by RNAi. One or both strands of the dsRNA can further comprise a terminal phosphate group, such as a 5'-phosphate or 5', 3 '-diphosphate. As used herein, dsRNA molecules, in addition to at least one ribonucleotide, can further include substitutions, chemically-modified 34 nucleotides, and non-nucleotides. In certain embodiments, dsRNA molecules comprise ribonucleotides up to about 100% of the nucleotide positions. Examples of dsRNA molecules can be found in, for example, U.S. Patent Application No. 11/681,725, U;S. Patent Nos. 7,022,828 and 7,034,009, and PCT 5 International Application Publication No. WO/2003/070897. In addition, as used herein, the term "dsRNA" is meant to be synonymous with other terms used to describe nucleic acid molecules that are capable of mediating sequence specific RNAi, for example, meroduplex RNA (mdRNA), nicked dsRNA (ndsRNA), gapped dsRNA (gdsRNA), short interfering nucleic acid (siNA), siRNA, 10 micro-RNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering substituted oligonucleotide, short interfering modified oligonucleotide, chemically-modified dsRNA, post-transcriptional gene silencing RNA (ptgsRNA), among others. The term "large double-stranded (ds) RNA" refers to any double-stranded RNA longer than about 40 base pairs (bp) to about 100 bp or more, particularly up to t5 about 300 bp to about 500 bp. The sequence of a large dsRNA may represent a segment of an mRNA or an entire mRNA. A double-stranded structure may be formed by self-complementary nucleic acid molecule or by annealing of two or more distinct complementary nucleic acid molecule strands. In some aspects, a dsRNA comprises two separate oligonucleotides, comprising a 0 first strand (antisense) and a second strand (sense), wherein the antisense and sense strands are self-complementary (i.e., each strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in the other strand and the two separate strands form a duplex or double-stranded structure, for example, wherein the double-stranded region is about 15 to about 24 base pairs or about 26 to about 40 base pairs); the antisense 25 strand comprises a nucleotide sequence that is complementary to a nucleotide sequence i a target nucleic acid molecule or a portion thereof (e.g., a human mRNA); and the sense strand comprises a nucleotide sequence corresponding (i.e., homologous) to the target nucleic acid sequence or a portion thereof (e.g., a sense strand of about 15 to about 25 nucleotides or about 26 to about 40 nucleotides corresponds to the target nucleic acid 30 or a portion thereof). In some aspects, the dsRNA may be assembled from a single oligonucleotide in which the self-complementary sense and antisense strands of the dsRNA are linked by together by a nucleic acid based-linker or a non-nucleic acid-based linker. In some embodiments, the first (antisense) and second (sense) strands of the dsRNA molecule are 35 covalently linked by a nucleotide or non-nucleotide linker as described herein and known in the art. In some embodiments, a first dsRNA molecule is covalently linked to at least one second dsRNA molecule by a nucleotide or non-nucleotide linker known in the art, wherein the first dsRNA molecule can be linked to a plurality of other dsRNA molecules 5 that can be the same or different, or any combination thereof. In some embodiments, the linked dsRNA may include a third strand that forms a meroduplex with the linked dsRNA. In some respects, dsRNA molecules described herein form a meroduplex RNA (mdRNA) having three or more strands, for example, an 'A' (first or antisense) strand, 'S ' 10 (second) strand, and S2' (third) strand in which the 'SI' and 'S2' strands are complementary to and form base pairs (bp) with non-overlapping regions of the 'A' strand (e.g., an mdRNA can have the form of A:SlS2). The SI, S2, or more strands together essentially comprise a sense strand to the 'A' strand. The double-stranded region formed by the annealing of the 'Si' and 'A' strands is distinct from and non-overlapping with the .5 double-stranded region formed by the annealing of the 'S2' and 'A' strands. An mdRNA molecule is a "gapped" molecule, meaning a "gap" ranging from 0 nucleotides up to abo t 10 nucleotides. In some embodiments, the A:SI duplex is separated from the A:S2 duplex by a gap resulting from at least one unpaired nucleotide (up to about 10 unpaired nucleotides) in the 'A' strand that is positioned between the A:S1 duplex and the A:S2 0 duplex and that is distinct from any one or more unpaired nucleotide at the 3'-end of one or more of the 'A', 'S ', or 'S2' strands. In some embodiments, the A:SI duplex is separated from the A:B2 duplex by a gap of zero nucleotides (i.e., a nick in which only a phosphodiester bond between two nucleotides is broken or missing in the polynucleotide molecule) between the A:SI duplex and the A:S2 duplex - which can also be referred to 25 as nicked dsRNA (ndsRNA). For example, A:S IS2 may be comprised of a dsRNA having at least two double-stranded regions that combined total about 14 base pairs to about 40 base pairs and the double-stranded regions are separated by a gap of about 0 to about 10 nucleotides, optionally having blunt ends, or A:S 1S2 may comprise a dsRNA having at least two double-stranded regions separated by a gap of up to 10 nucleotides 30 wherein at least one of the double-stranded regions comprises between about 5 base pairs and 13 base pairs. As described herein, a dsRNA molecule which contains three or more strands mai be referred to as a "meroduplex" RNA (mdRNA). Examples of mdRNA molecules can be found in U.S. Provisional Patent Application Nos. 60/934,930 and 60/973,398. 36 A dsRNA or large dsRNA may include a substitution or modification in which the substitution or modification may be in a phosphate backbone bond, a sugar, a base, or a nucleoside. Such nucleoside substitutions can include natural non-standard nucleosides (e.g., 5-methyluridine or 5-methyleytidine or a 2-thioribothymidine), and such backbone, 5 sugar, or nucleoside modifications can include an alkyl or heteroatom substitution or addition, such as a methyl, alkoxyalkyl, halogen, nitrogen or sulfur, or other modifications known in the art. The term "pyrimidine" as used herein refers to conventional pyrimidine bases, including standard pyrimidine bases uracil and cytosine. In addition, the term pyrimidine 10 is contemplated to embrace natural non-standard pyrimidine bases or acids, such as 5-methyluracil, 4-thiouracil, pseudouracil, dihydrouracil, orotate, 5-methylcytosine, or the like, as well as a chemically-modified bases or "universal bases," which can be used to substitute for a standard pyrimidine within nucleic acid molecules of this disclosure. Examples of pyrmidines suitable for use within a dsRNA of this disclosure include those 15 disclosed in U.S. Patent 6,846,827, hereby incorporated by reference. The term "purine" as used herein refers to conventional purine bases, including standard purine bases adenine and guanine. In addition, the term purine is contemplated to embrace natural non-standard purine bases or acids, such as N2-methylguanine, inosine, 2

,

6 -aminopurine, or the like, as well as a chemically-modified bases or "universal 20 bases," which can be used to substitute for a standard purine within nucleic acid molecules of this disclosure. In yet another embodiment, the mdRNA or dsRNA comprises one or more nucleotides having the formula:

R

2 zx R, Y 25 wherein, X is 0 or CH 2 , Y is 0, and Z is CH 2 ; R, is selected from the group consisting o adenine, cytosine, guanine, hypoxanthine, uracil, thymine, 2 ,6-diaminopurine, C-phenyl, C-naphthyl, inosine, azole carboxamide, 1 -p-D-ribofuranosyl-4-nitroindole, 1 -0-D ribofuranosyl-5-nitroindole, 1 -p-D-ribofuranosyl-6-nitroindole, or I -0-D-ribofuranosyl 3 -nitropyrrole, and a heterocycle wherein the heterocycle is selected from the group 37 consisting of a substituted 1,3-diazine, unsubstituted 1,3-diazine, and an unsubstituted "H imidazo[4,5}1,3 diazine; and R 2 , R 3 are independently selected from a group consisting of H, OH, DMTO, TBDMSO, BnO, THPO, AcO, BzO, OP(NiPr 2

)O(CH

2

)

2 CN, OP0 3 H, diphosphate, and triphosphate, wherein R 2 and R 3 together may be PhCHO 2 , TIPDSO 2 >r 5 DTBSO 2 . In yet another embodiment, the mdRNA or dsRNA comprises one or more are universal-binding nucleotide. Non-limiting examples of universal-binding nucleotide include C-phenyl, C-naphthyl, inosine, azole carboxamide, 1 -P-D-ribofuranosyl-4 nitroindole, 1-0-D-ribofuranosyl-5-nitroindole, 1-1-D-ribofuranosyl-6-nitroindole, or 1-B 10 D-ribofuranosyl-3-nitropyrrole. Within certain aspects, the present disclosure provides methods of using mdRNA or dsRNA that decreases expression of a target gene by RNAi, and compositions comprising one or more mdRNA or dsRNA, wherein at least one mdRNA or dsRNA comprises one or more universal-binding nucleotide(s) in the first, second or third position in the anti-codon of the antisense strand of the mdRNA or 15 dsRNA duplex and wherein the mdRNA or dsRNA is capable of specifically binding to a target sequence, such as an RNA expressed by a cell. In cases wherein the sequence of the target RNA includes one or more single nucleotide substitutions, mdRNA or dsRNA comprising a universal-binding nucleotide retains its capacity to specifically bind a targ RNA, thereby mediating gene silencing and, as a consequence, overcoming escape of the 20 target from mdRNA or dsRNA-mediated gene silencing. Non-limiting examples for the above compositions includes modifying the anti-codons for tyrosine (AUA) or phenylalanine (AAA or GAA), cysteine (ACA or GCA), histidine (AUG or GUG), asparagine (AUU or GUU), isoleucine (UAU) and aspartate (AUC or GUC) within the anti-codon of the antisense strand of the mdRNA or 25 dsRNA molecule. For example, within certain embodiments, the isoleucine anti-codon UAU, for which AUA is the cognate codon, may be modified such that the third-position uridine (U) nucleotide is substituted with the universal-binding nucleotide inosine (I) to create the anti-codon UAL Inosine is an exemplary universal-binding nucleotide that can 30 nucleotide-pair with an adenosine (A), uridine (U), and cytidine (C) nucleotide, but not guanosine (G), This modified anti-codon UAI increases the specific-binding capacity of the mdRNA or dsRNA molecule and thus permits the mdRNA or dsRNA to pair with mRNAs having any one of AUA, UUA, and CUA in the corresponding position of the 38 coding strand thereby expanding the number of available RNA degradation targets to which the mdRNA or dsRNA may specifically bind. Alternatively, the anti-codon AUA may also or alternatively be modified by substituting a universal-binding nucleotide in the third or second position of the anti 5 codon such that the anti-codon(s) represented by UAI (third position substitution) or Ul (second position substitution) to generate mdRNA or dsRNA that are capable of specifically binding to AUA, CUA and UUA and AAA, ACA and AUA. In certain aspects, mdRNA or dsRNA disclosed herein can include between abo t I universal-binding nucleotide and about 10 universal-binding nucleotides. Within 10 certain aspects, the presently disclosed mdRNA or dsRNA may comprise a sense strand that is homologous to a sequence of a target gene and an antisense strand that is complementary to the sense strand, with the proviso that at least one nucleotide of the antisense strand of the otherwise complementary mdRNA or dsRNA duplex is replaced by one or more universal-binding nucleotide. 15 It will be understood that, regardless of the position at which the one or more universal-binding nucleotide is substituted, the mdRNA or dsRNA molecule is capable cf binding to a target gene and one or more variant(s) thereof thereby facilitating the degradation of the target gene or variant thereof via Dicer or a RISC complex. Thus, the mdRNA or dsRNA of the present disclosure are suitable for introduction into cells to 20 mediate targeted post-transcriptional gene silencing of a TNF gene or variants thereof. When a mdRNA or dsRNA is inserted into a cell, the mdRNA or dsRNA duplex is then unwound, and the antisense strand annals with mRNA to form a Dicer substrate or the antisense strand is loaded into an assembly of proteins to form the RNA-induced silencing complex (RISC). 25 In yet another embodiment, the mdRNA or dsRNA comprises one or more have a 2 '-sugar substitution. Non-limiting examples of a 2'-sugar substitution include 2 '-0-methyl, 2 '-O-methoxyethyl, 2 'O-2-methoxyethyl, wherein the 2'-sugar substitution is a halogen, or wherein the 21 -sugar substitution is a 2-fluoro, or wherein the 2 '-sugar substitution is a 2t -O-allyl. 30 In yet another embodiment, the mdRNA or dsRNA comprising at least one, two, three, four, five, or more base pairs (including 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 19, 20 or more base pairs), each base pair comprising a 5-methyluridine base paired with a 2

,

6 -diaminopurine, and wherein the 5-methyluridine of each base pair is in the guide 39 strand (antisense strand) or wherein the 5 -methyluridine of each base pair is in the passenger strand (sense strand). In any one embodiment of the disclosure, a base pair comprising a 5 methyluridine base paired with a 2

,

6 -diaminopurine in a double-stranded region of the 5 RNA may be as follows: 5 -methyluridine N N NH N N HH N N N N H 2

,

6 -diaminopurine In this schematic, the 5-methyluridne:2,6-diaminopurine base pair has three hydrogen bonds (a hashed line represents a hydrogen bond). The base pair may have 1, 2 or 3 hydrogen bonds, preferably the base pair has I hydrogen bond, more preferably two 0 hydrogen bonds and most preferably three hydrogen bonds. In anyone embodiment of the disclosure, the double-stranded region of an RNA comprises at least one non-standard base pair comprising: 40

R

8 R R NyN% R5 N N R N N R In this schematic, R 4 , R5, R 6 , R 7 , and R8 are independently any one or more organic group consisting of one to twenty (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 5 or 20) atoms selected from carbon, oxygen, nitrogen, sulfur, hydrogen, selenium, silicon halogen, chlorine, fluorine, and bromine. A dashed line indicates an optional bond that is either present or absent within the structures above. In this schematic, hydrogen bonds are not indicated in the above structures; however, such hydrogen bonds would form between the hydrogen bond donor and hydrogen bond acceptor groups of R 4 and R 7 , 10 between the hydrogen bond donor group of R 5 and the nitrogen (a hydrogen bond acceptor) in the third position of the pyrimidine structure above, and between the hydrogen bond donor and hydrogen bond acceptor group of R 6 and R 9 . In an embodiment of this disclosure, R4 has a hydrogen bond donor group and R7 has a hydrogen bond acceptor group, R5 has a hydrogen bond donor group, and R6 has a hydrogen bond dono 15 group and R9 has an hydrogen bond acceptor group. In another embodiment, R4 has an hydrogen bond acceptor group and R7 has a hydrogen bond donor group, R5 has a hydrogen bond donor group, and R6 has a hydrogen bond donor group and R9 has an hydrogen bond acceptor group, In another embodiment, R4 has an hydrogen bond acceptor group and R7 has a hydrogen bond donor group, R5 has a hydrogen bond donor 20 group, and R6 has an hydrogen bond acceptor group and R9 has a hydrogen bond donor group. In anyone embodiment of the disclosure, the double-stranded region of an RNA comprises at least one non-standard base pair comprising: 41

N

8 N RRN In this schematic, R 4 , R 5 , R 6 , R 7 , Rs, and R 9 are independently any one or more organic group consisting of one to twenty (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 5 18, 19, or 20) atoms selected from carbon, oxygen, nitrogen, sulfur, hydrogen, selenium, silicon, halogen, chlorine, fluorine, and bromine. A dashed line indicates an optional bond that is either present or absent within the structures above. A hashed line between the substituent groups of the two structures above indicates the presence of a hydrogen bond (three hydrogen bonds are shown in this schematic). In another embodiment, R4 10 has a donor group and R7 has an acceptor group. In another embodiment, R4 has an acceptor group and R7 has an acceptor group. In another embodiment, R5 has a donor group and R8 has an acceptor group, In another embodiment, R5 has an acceptor group and R8 has an acceptor group, In another embodiment, R6 has a donor group and R9 ha, an acceptor group. In another embodiment, R6 has an acceptor group and R9 has an [5 acceptor group. Another aspect of the disclosure is a mdRNA or dsRNA comprising an acyclic nucleotide monomer. In a preferred embodiment, the acyclic nucleotide monomer is a 2' 3'-seco-nucleotide monomer. Preferably, the acyclic nucleotide monomer is selected from the group consisting of monomer E, F, G, H, I or J (see below). 0 Base 0 o Base o OH OH O O O 20 Monomer D Monomer E 42 U- 0 Base VO Base 0 O-R 0 S-R -O-p=O - O Monomer F Monomer G H Base O O Base 0 o Base HO 0 NH ONH 0 N

-

-0-P-0 R ~ .K PL R k N Monomer H Monomer 0- R Monomer J Other examples of acyclic nucleotide monomers are described, for example, in PCT patent application PCT/US2008/64417, hereby incorporated by reference in their entirety. 5 In addition, as used herein, the term "RNAi" is meant to be equivalent to other terms used to describe sequence specific RNA interference, such as post transcriptional gene silencing, translational inhibition, or epigenetics. For example, dsRNA molecules c f this disclosure can be used to epigenetically silence genes at the post-transcriptional leve or the pre-transcriptional level or any combination thereof. l0 In some aspects, this invention provides compositions containing one or more RNAi-inducing agents which are targeted to one or more target transcripts, along with one or more delivery components. Examples of delivery components include lipopeptides, lipids, peptides with attached lipoid moieties or attached natural or synthetic polymers, and polymeric lipids. 15 The compositions and formulations of this invention may be used for delivery of RNAi-inducing entities such as dsRNA, siRNA, mdRNA, miRNA, shRNA, or RNAi inducing vectors to cells in intact mammalian subjects, and may also be used for delivery of these agents to cells in culture. This invention also provides methods for the delivery of one or more RNAi 20 inducing entities to organs and tissues within the body of a mammal. In some embodiments, compositions containing an RNAi-inducing entity and one or more lipopeptide components are introduced by various routes to be transported within the 43 body and taken up by cells in one or more organs or tissues, where expression of a targe transcript is modulated. This invention provides pharmaceutically-acceptable nucleic acid compositions with various lipopeptides, lipids, or peptides having attached lipoid moieties or natural o 5 synthetic polymers which are useful for therapeutic delivery of nucleic acids and gene silencing RNAs. In particular, this invention provides compositions and methods for in vitro and in vivo delivery of dsRNAs for decreasing, downregulating, or silencing the translation of a target nucleic acid sequence or expression of a gene. These composition and methods may be used for prevention and/or treatment of diseases in a mammal. 10 In exemplary methods of this invention, a ribonucleic acid molecule such as a dsRNA, siRNA, mdRNA, or shRNA is contacted with a lipopeptide to formulate a composition which can be administered to cells or subjects such as mammals. In some embodiments, this invention provides methods for delivering an interfering-RNA agent such as a dsRNA, siRNA, mdRNA, or shRNA intracellularly by contacting a nucleic 15 acid-containing composition with a cell. In exemplary embodiments, this invention includes compositions containing a nucleic acid molecule, such as a double-stranded ribonucleic acid (dsRNA), a short interfering RNA (siRNA), a meroduplex RNA (mdRNA), or a short hairpin RNA (shRNA), admixed or complexed with a lipopeptide to form a composition that enhances 20 intracellular delivery of the nucleic acid molecule. In some embodiments, a delivery composition of this invention may contain an interfering-RNA agent and one, two, or more lipopeptides, as well as one or more polymeric lipids. In certain embodiments, the N/P ratio of the lipopeptide and nucleic acid is from about 0.5 to about 8, from about 1 to about 4, or about 1.1. 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8 25 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 26, 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 o about 4. The compositions of this invention can form stable particles which may incorporate an interfering RNA agent. Compositions and formulations of this invention may include further delivery-enhancing components or excipients. 30 In some embodiments, compositions of this invention contain stable RNA-lipid particles having diameters from about 5 nm to about 400 nm. In some embodiments, the particles may have a uniform diameter of from about 10 nm to about 300 nm. In some embodiments, the particles may have a uniform diameter of from about 50 nm to about 150 nm. 44 Within exemplary compositions of this invention, an interfering-RNA agent may be admixed or complexed with lipopeptides to form a composition that enhances intracellular delivery of the dsRNA as compared to contacting target cells with naked dsRNA. 5 Lipids for RNA Delivery and Administration In some aspects of this invention, lipopeptides and additional lipids are employee for delivery and administration of RNA components. More particularly, a composition cf this invention may include one or more lipopeptides, which may be cationic, along with other cationic lipids and non-cationic lipids. 10 Cationic lipids may be monocationic or polycationic. Non-cationic lipids include neutral lipids and lipids having approximately zero net charge at a particular pH, for example, a zwitterionic lipid. Non-cationic lipids also include anionic lipids. In some embodiments, a composition is a mixture or complex of an RNA component with a lipopeptide and a (non-lipopeptide) cationic lipid. In some 15 embodiments, a composition may be a mixture or complex of one or more interfering RNA agents with one or more lipopeptides and one or more cationic lipids. Examples of cationic lipids include N-[1-(2,3-dioleoyloxy)propyl]-N,N,N trimethylammonium chloride (DOTMA); 1,2-bis(oleoyloxy)-3-3 (trimethylammonium)propane (DOTAP), 1,2-bis(dimyrstoyloxy)-3-3 20 (trimethylammonia)propane (DMTAP); 1 ,2-dimyristyloxypropyl-3 dimethylhydroxyethylammonium bromide (DMRIE); dimethyldioctadecylammonium bromide (DDAB); 3 -(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol (DC-Chol); 3p-[N',N'-diguanidinoethyl-aminoethane)carbanoyl cholesterol (BGTC); 2-(2-(3-(bis(3 aminopropyl)amino)propylamino)acetamido)-N,N-ditetradecylacetamide (RPR209120); 25 pharmaceutically acceptable salts thereof, and mixtures thereof. Examples of cationic lipids include 1,2-dialkenoyl-sn-glycero-3 ethylphosphocholines (EPCs), such as 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, 1, 2 -distearoyl-sn-glycero-3-ethylphosphocholine, 1,2-dipalmitoyl-sn-glycero-3 ethylphosphocholine, pharmaceutically acceptable salts thereof, and mixtures thereof. 30 Examples of polycationic lipids include tetramethyltetrapalmitoyl spermine (TMTPS), tetramethyltetraoleyl spermine (TMTOS), tetramethlytetralauryl spermine (TMTLS), tetramethyltetramyristyl spermine (TMTMS), tetramethyldioleyl spermine (TMDOS), pharmaceutically acceptable salts thereof, and mixtures thereof. 45 Examples of polycationic lipids include 2 ,5-bis(3-aminopropylamino)-N-(2 (dioctadecylamino)-2-oxoethyl) pentanamide (DOGS); 2 ,5-bis(3-aminopropylamino)

(

2 -(di(Z)-octadeca-9-dienylamino)-2-oxoethyl) pentanamide (DOGS-9-en); 2,5-bis(3 aminopropylamino)-N-(2-(di(9Z,12Z)-octadeca-9,12-dienylamino)-2-oxoethyl) 5 pentanamide (DLinGS); 3-0-(N 4

-(N',N

8 dicarbobenzoxyspermidine)carbamoyl)cholesterol (GL-67); (9Z,9'Z)-2-(2,5-bis(3 aminopropylamino)pentanamido)propane- 1,3 -diyl-dioctadec-9-enoate (DOSPER); 2,3 dioleyloxy-N-[ 2 (sperminecarboxamido)ethyl]-N,N-dim ethyl-i-propanaminium trifluorc acetate (DOSPA); pharmaceutically acceptable salts thereof, and mixtures thereof. 10 Examples of cationic lipids include those shown in Table 3. Table 3 Examples of Cationic Lipids Compound Name FA Chains M.W. CAS registry # DS404-28 BGTC Cholesterol 642.96 182056-06-0 DOSPER C18:1 848.34 178532-92-8 GL-67 Cholesterol 615.00 179075-30-0 RPR209120 Myristoyl C14 695.16 433292-13-8 DOGS C18:0 807.37 12050-77-7 DOGS (9-en) C18:1 803.34 DLinGS C18:2 799.31 DOTMA C18:1 712.57 104162-48-3 Examples of cationic lipids are described in U.S. Patent Nos. 4,897,355; 15 5,279,833; 6,733,777; 6,376,248; 5,736,392; 5,334,761; 5,459,127; 5,208,036; 5,264,61; 5,283,185; 5,753,613; 5,785,992; and U.S. Patent Publication No. 2005/0064595. In some embodiments, the composition is a mixture or complex of an RNA component with a lipopeptide and a non-cationic lipid. In some embodiments, the composition is a mixture or complex of one or more RNA components with one or more 20 lipopeptides and one or more non-cationic lipids. Non-cationic lipids include neutral, zwitterionic, and anionic lipids. In some embodiments, a composition is a mixture or complex of an RNA component with a cationic lipopeptide and a (non-lipopeptide) non-cationic or neutral lipid. 25 In some embodiments, a composition is a mixture or complex of one or more RNA components with one or more cationic lipopeptides, one or more (non-lipopeptide) cationic lipids, and one or more (non-lipopeptide) non-cationic or neutral lipids. 46 Examples of non-cationic lipids include 1, 2 -Dilauroyl-sn-glycerol (DLG); 1,2-Dimyristoyl-sn-glycerol (DMG); 1, 2 -Dipalmitoyl-sn-glycerol (DPG); 1,2-Distearoy sn-glycerol (DSG); 1, 2 -Dilauroyl-sn-glycero-3-phosphatidic acid (sodium salt; DLPA); 1, 2 -Dimyristoyl-sn-glycero-3-phosphatidic acid (sodium salt; DMPA); 1,2-Dipalmitoyl, 5 sn-glycero-3-phosphatidic acid (sodium salt; DPPA); 1, 2 -Distearoyl-sn-glycero-3 phosphatidic acid (sodium salt; DSPA); 1, 2 -Diarachidoy-sn-glycero-3-phosphocholine (DAPC); 1, 2 -Dilauroyl-sn-glycero-3-phosphocholine (DLPC); 1,2-Dimyristoyl-sn glycero-3 -phosphocholine (DMPC); 1, 2 -Dipalmitoyl-sn-glycero-o-ethyl-3 phosphocholine (chloride or triflate; DPePC); 1, 2 -Dipalmitoyl-sn-glycero-3 10 phosphocholine (DPPC); 1,2-Distearoyl- sn-glycero-3-phosphocholine (DSPC); 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE); 1,2-Dimyristoyl-sn-glycero-I phosphoethanolamine (DMPE); 1, 2 -Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE); 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE); 1,2-Dilauroyl-sn glycero-3-phosphoglycerol (sodium salt; DLPG); 1, 2 -Dimyristoyl-sn-glycero-3 15 phosphoglycerol (sodium salt; DMPG); 1, 2 -Dimyristoyl-sn-glycero-3-phospho-sn-I 1 glycerol (ammonium salt; DMP-sn-1-G); 1, 2 -Dipalmitoyl-sn-glycero-3+phosphoglycerol (sodium salt; DPPG); 1, 2 -Distearoyl-sn-glycero-3-phosphoglycero (sodium salt; DSPG); 1, 2 -Distearoyl-sn-glycero-3-phospho-sn- 1-glycerol (sodium salt; DSP-sn-i-G); 1,2 Dipalmitoyl-sn-glycero-3-phospho-L-serine (sodium salt; DPPS); 1-Palmitoyl-2 20 1 inoleoyl-sn-glycero-3 -phosphocholine (PLinoPC); I -Palmitoyl-2-oleoyl-sn-glycero-3 phosphocholine (POPC); 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (sodium salt; POPG); I-Palmitoyl.- 2 -oleoyl-sn-glycero-3-phosphoglycerol (sodium salt; POPG); I-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (ammonium salt; POPG); 1 Palmitoyl-2-4o-sn-glycero-3-phosphocholine (P-lyso-PC); I -Stearoyl-2-lyso-sn-glycero 25 3-phosphocholine (S-lyso-PC); and mixtures thereof. Examples of non-cationic lipids include polymeric compounds and polymer-lipid conjugates or polymeric lipids, such as pegylated lipids having PEG regions of 300, 500, 1000, 1500, 2000, 3500, 5000, or 10,000 molecular weight, including polyethyleneglycols, N-(Carbonyl-methoxypolyethyleneglycol.2000)-1,2-dimyristoyl-sn 30 glycero-3-phosphoethanolamine (sodium salt; DMPE-MPEG-2000); N-(Carbonyl methoxypolyethyleneglycol-5000)-1, 2 -dimyristoyl-sn-glycero-3-phosphoethanolamine (sodium salt; DMPE-MPEG-5000); N-(Carbonyl-methoxypolyethyleneglycol 2000)-1,2 dipalmitoyl-sn-glycero-3-phosphoethanolamine (sodium salt; DPPE-MPEG-2000);

N

(Carbonyl-methoxypolyethyleneglycol 5000)-1,2-dipalmitoyl-sn-glycero-3 47 phosphoethanolamine (sodium salt; DPPE-MPEG-5000); N-(Carbonyl methoxypolyethyleneglycol 750)-1, 2 -distearoyl-sn-glycero-3-phosphoethanolamine (sodium salt; DSPE-MPEG-750); N-(Carbonyl-methoxypolyethyleneglycol 2000)-1,2 distearoyl-sn-glycero-3-phosphoethanolamine (sodium salt; DSPE-MPEG-2000);

N

5 (Carbonyl-methoxypolyethyleneglycol 5000)-1,2-distearoyl-sn-glycero-3 phosphoethanolamine (sodium salt; DSPE-MPEG-5000); sodium cholesteryl sulfate (SCS); pharmaceutically acceptable salts thereof, and mixtures thereof. Examples of non-cationic lipids include polymeric lipids such as DOPE-PEG, DLPE-PEG, DDPE-PEG DLinPE-PEG, and diacylglycerol-PEG-2000, -5000 or -10,00C. 10 Examples of non-cationic lipids include polymeric lipids such as multi-branched pegylated compounds, for example DSPE-PTE020 and DSPE-AM0530K. Examples of non-cationic lipids include polymeric lipids such as DSPE-PG8G polyglycerine lipids. Examples of non-cationic lipids include dioleoylphosphatidylethanolamine 15 (DOPE), didecanoylphosphatidylcholine (DDPC), diphytanoylphosphatidylethanolamine (DPhPE), 1, 2 -Dioleoyl-sn-Glycero-3-Phosphocholine (DOPC), and 1,2-Diphytanoyl-sn Glycero-3-Phosphocholine (DPhPC). Examples of non-cationic lipids include cholesterols, sterols, and steroids such as gonanes, estranes, androstanes, pregnanes, cholanes, cholestanes, ergostanes, 20 campestanes, poriferastanes, stigmastanes, gorgostanes, lanostanes, cycloartanes, as well as sterol or zoosterol derivatives of any of the foregoing, and their biological intermediates and precursors, which may include, for example, cholesterol, lanosterol, stigmastanol, dihydrolanosterol, zymosterol, zymostenol, desmosterol, 7-dehydrocholesterol, and mixtures and derivatives thereof. 25 Examples of non-cationic lipids include pegylated cholesterols, and cholestane 3-oxo(C 1 -22acyl) derivatives such as cholesteryl acetate, cholesteryl arachidonate, cholesteryl butyrate, cholesteryl hexanoate, cholesteryl caprylate, cholesteryl n decanoate, cholesteryl dodecanoate, cholesteryl myristate, cholesteryl palmitate, cholesteryl behenate, cholesteryl stearate, cholesteryl nervonate, cholesteryl pelargonate, 30 cholesteryl n-valerate, cholesteryl oleate, cholesteryl elaidate, cholesteryl erucate, cholesteryl heptanoate, cholesteryl linolelaidate, cholesteryl linoleate, and mixtures and derivatives thereof. 48 Examples of non-cationic lipids include compounds derived from plant sterols including phytosterols, beta-sitosterol, campesterol, ergosterol, brassicasterol, delta-7 stigmasterol, delta-7-avenasterol, and mixtures and derivatives thereof. Examples of non-cationic lipids include bile acids, cholic acid, chenodeoxycholi4 5 acid, glycocholic acid, taurocholic acid, deoxycholic acid, lithocholic acid, methyl lithocholic acid, and mixtures and derivatives thereof. Examples of non-cationic lipids include compounds derived from steroids including glucocorticoids, cortisol, hydrocortisone, corticosterone, A 5 -pregnenolone, progesterone, deoxycorticosterone, 1 7-OH-pregnenolone, 17-OH-progesterone, 11 10 dioxycortisol, dehydroepiandrosterone, dehydroepiandrosterone sulfate, androstenedione, aldosterone, 18-hydroxycorticosterone, tetrahydrocortisol, tetrahydrocortisone, cortisone prednisone, 6a-methylpredisone, 9a-fluoro-16a-hydroxyprednisolone, 9a-fluoro-16a methylprednisolone, 9a-fluorocortisol, and mixtures and derivatives thereof. Examples of non-cationic lipids include compounds derived from steroids 15 including adrogens, testosterone, dihydrotestosterone, androstenediol, androstenedione, androstenedione, 3a,5a-androstanediol, and mixtures and derivatives thereof. Examples of non-cationic lipids include compounds derived from steroids including estrogens, estriols, estrones, estradiols, and mixtures and derivatives thereof. Examples of non-cationic lipids include compounds derived from lumisterol and 20 vitamin D compounds. Examples of non-cationic lipids include lipids ranging from C10:0 to C22:6 phosphocthanolamine as shown in Table 4. Table 4 Examples of Non-cationic Lipids Name FA chains M W. CAS Registry # DDPE CIO:_ 523.64 253685-27-7 DLPE C12:0 579.76 59752-57-7 DSPE C18:0 748.08 1069-79-0 DOPE C18:1 744.05 4004-05-1 DLinPE C18:2 740.01 20707-71-5 DLenPE C18:3 735.98 34813-40-6 DARAPE C20:4 788.06 5634-86-6 DDHAPE C22:6 836.10 123284-81-1 DPhPE 16:0[(CH3)4] 804.19 201036-16-0 25 Examples of anionic lipids include phosphatidylserine, phosphatidic acid, phosphatidylcholine, platelet-activation factor (PAF), phosphatidylethanolamine, 49 phosphatidyl-DL-glycerol, phosphatidylinositol, phosphatidylinositol (pi(4)p, pi(4,5)p2) cardiolipin (sodium salt), lysophosphatides, hydrogenated phospholipids, sphingoplipids, gangliosides, phytosphingosine, sphinganines, pharmaceutically acceptable salts thereof and mixtures thereof. 5 In certain aspects, the lipopeptide is about from 10% to about 100% mole percentage of delivery components, not including the nucleic acid (e.g., RNA). In other embodiments, the lipopeptide is from about 20% to about 80%, or from about 30% to about 70%, or from about 40% to about 60% mole percentage of delivery components, cr about 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, r 10 95% mole percentage of delivery components. Additional delivery lipids In some aspects of this invention, amino acid lipids and additional non-amino acid lipids may be employed for delivery and administration of regulatory RNA components, RNA antagonists, interfering RNA, or nucleic acids. More particularly, a composition o f 15 this invention may include one or more amino acid lipids along with non-amino acid cationic lipids and non-amino acid non-cationic lipids. Non-amino acid cationic lipids may be monocationic or polycationic. Some non-amino acid cationic lipids include neutral lipids and lipids having approximately zero net charge at a particular pH, for example, a zwitterionic lipid. Non-amino acid non 20 cationic lipids also include anionic lipids. In some embodiments, a composition is a mixture or complex of an RNA component with an amino acid lipid and a non-amino acid cationic lipid. In some embodiments, a composition may be a mixture or complex of one or more regulatory or interfering RNA agents with one or more amino acid lipids and one or more non-amino 25 acid cationic lipids. The compounds and compositions of this disclosure can be admixed with, or attached to various targeting ligands or agents to deliver an active agent to a cell, tissue, organ or region of an organism. Examples of targeting agents include antibodies, ligand; for receptors, peptides, proteins, lectins, (poly)saccharides, galactose, mannose, 30 cyclodextrins, nucleic acids, DNA, RNA, aptamers, and polyamino acids. Examples of non-amino acid cationic lipids include N-[l -(2,3 dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA); 1,2-bis(oleoyloxy 3 - 3 -(trimethylammonium)propane (DOTAP), 1,2-bis(dimyrstoyloxy)-3-3 50 (trimethylammonia)propane (DMTAP); 1, 2 -dimyristyloxypropyl-3 dimethylhydroxyethylammonium bromide (DMRIE); dimethyldioctadecylammonium bromide (DDAB); 3 -(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol (DC-Chol); 30-[N',N'-diguanidinoethyl-aminoethane)carbamoyI cholesterol (BGTC); 2-(2-(3 -(bis(3 5 aminopropyl)amino)propylamino)acetamido)-NN-ditetradecylacetamide (RPR209120); pharmaceutically acceptable salts thereof, and mixtures thereof Examples of non-amino acid cationic lipids include 1, 2 -dialkenoyl-sn-glycero-3 ethylphosphocholines (EPCs), such as 1, 2 -dioleoyl-sn-glycero-3-ethylphosphocholine, 1, 2 -distearoyl-sn-glycero-3-ethylphosphocholine, 1, 2 -dipalmitoyl-sn-glycero-3 10 ethylphosphocholine, pharmaceutically acceptable salts thereof, and mixtures thereof. Examples of non-amino acid cationic lipids include 1,2-distearyloxy-N,N dimethyl-3-aminopropane (DSDMA), 1, 2 -dioleyloxy-NNdimethyl-3-aminopropane (DODMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), and 1,2 dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA). 5 Examples of non-amino acid polycationic lipids include tetramethyltetrapalmitoy spermine (TMTPS), tetramethyltetraoleyl spermine (TMTOS), tetramethlytetralauryl spermine (TMTLS), tetramethyltetramyristyl spermine (TMTMS), tetramethyldioleyl spermine (TMDOS), pharmaceutically acceptable salts thereof, and mixtures thereof. Examples of non-amino acid polycationic lipids include 2,5-bis(3 .O aminopropylamino)-N-(2-(dioctadecylamino)-2-oxoethyl) pentanamide (DOGS); 2,5 bis( 3 -aminopropylamino)-N-(2-(di(Z)-octadeca-9-dienylamino)-2-oxoethyl) pentanamide (DOGS-9-en); 2 ,5-bis(3-aminopropylamino)-N-(2-(di(9Z,1 2 Z)-octadeca-9,12 dienylamino)-2-oxoethyl) pentanamide (DLinGS); 3-beta-(N 4

-(N',N

8 dicarbobenzoxyspermidine)carbamoyl)cholesterol (GL-67); (9Z,9'Z)-2-(2,5-bis(3 25 aminopropylamino)pentanamido)propane-1, 3 -diyl-dioctadec-9-enoate (DOSPER); 2,3 dioleyloxy-N-[ 2 (sperminecarboxamido)ethyl]-N,N-dimethyl- I -propanaminium trifluoro acetate (DOSPA); pharmaceutically acceptable salts thereof, and mixtures thereof. Examples of non-amino acid cationic lipids include DS404-28 BGTC (CAS 182056-06-0), DOSPER (CAS 178532-92-8), GL-67 (179075-30-0), RPR209120 (CAS 30 433292-13-8), DOGS (12050-77-7), DOGS (9-en, C18:1), DLinGS (C18:2), and DOTMA (104162-48-3). Examples of non-amino acid cationic lipids are described in U.S. Patent Nos. 4,897,355; 5,279,833; 6,733,777; 6,376,248; 5,736,392; 5,334,761; 5,459,127; 2005/0064595; 5,208,036; 5,264,618; 5,279,833; 5,283,185; 5,753,613; and 5,785,992. 51 In some embodiments, the composition is a mixture or complex of an RNA component with an amino acid lipid and a non-amino acid non-cationic lipid. In some embodiments, the composition is a mixture or complex of one or more RNA component. with one or more amino acid lipids and one or more non-amino acid non-cationic lipids. 5 Non-amino acid non-cationic lipids include neutral, zwitterionic, and anionic lipids. Thus, a non-cationic zwitterionic lipid may contain a cationic head group. Examples of non-amino acid non-cationic lipids include 1, 2 -Dilauroyl-sn-glycerc1 (DLG); 1, 2 -Dimyristoyl-sn-glycerol (DMG); 1, 2 -Dipalmitoyl-sn-glycerol (DPG); 1, 2 -Distearoyl-sn-glycerol (DSG); 1, 2 -Dilauroyl-sn-glycero-3-phosphatidic acid sodiumn 10 salt; DLPA); 1, 2 -Dimyristoyl-sn-glycero-3-phosphatidic acid (sodium salt; DMPA); 1, 2 -Dipalmitoyl-sn-glycero-3-phosphatidic acid (sodium salt; DPPA); 1,2-Distearoyl-sn glycero-3-phosphatidic acid (sodium salt; DSPA); 1,2-Diarachidoyl-sn-glycero-3 phosphocholine (DAPC); 1, 2 -Dilauroyl-sn-glycero-3-phosphocholine (DLPC); 1, 2 -Dimyristoyl-sn-glycero-3-phosphocholine (DMPC); 1, 2 -Dipalmitoyl-sn-glycero-0 15 ethyl-3 -phosphocholine (chloride or triflate; DPePC); 1, 2 -Dipalmitoyl-sn-glycero-3 phosphocholine (DPPC); 1,2-Distearoyl- sn-glycero-3-phosphocholine (DSPC); 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE); 1, 2 -Dimyristoyl-sn-glycero-3 phosphoethanolamine (DMPE); 1, 2 -Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE); 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE); 1,2-Dilauroyl-sn !0 glycero-3-phosphoglycerol (sodium salt; DLPG); 1, 2 -Dimyristoyl-sn-glycero-3 phosphoglycerol (sodium salt; DMPG); 1, 2 -Dimyristoyl-sn-glycero-3-phospho-sn-1 glycerol (ammonium salt; DMP-sn-1-G); 1, 2 -Dipalmitoyl-sn-glycero-3-phosphoglyceroI (sodium salt; DPPG); 1, 2 -Distearoyl-sn-glycero-3-phosphoglycero (sodium salt; DSPG); 1,2-Distearoyl-sn-glycero-3-phospho-sn-I-glycerol (sodium salt; DSP-sn-I -G); 1,2 25 Dipalmitoyl-sn-glycero-3-phospho-L-serine (sodium salt; DPPS); I -Palmitoyl-2 linoleoyl-sn-glycero-3-phosphocholine (PLinoPC); 1-Palmitoyl-2-oleoyl-sn-glycero-3 phosphocholine (POPC); I-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (sodium salt; POPG); 1 -Palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (sodium salt; POPG); I-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (ammonium salt; POPG); 1 30 Palmitoyl- 2

-

4 o-sn-glycero-3-phosphocholine (P-lyso-PC); I-Stearoyl-2-lyso-sn-glycero 3-phosphocholine (S-lyso-PC); and mixtures thereof. Examples of non-amino acid non-cationic lipids include polymeric compounds and polymer-lipid conjugates or polymeric lipids, such as pegylated lipids having PEG regions of 300, 500, 1000, 1500, 2000, 3500, or 5000 molecular weight, including 52 polyethyleneglycols, N-(Carbonyl-methoxypolyethyleneglycol-2000)-1,2-dimyristoyl-su glycero-3-phosphoethanolamine (sodium salt; DMPE-MPEG-2000); N-(Carbonyl methoxypolyethyleneglycol-5000)- 1, 2 -dimyristoyl-sn-glycero-3-phosphoethanolamine (sodium salt; DMPE-MPEG-5000); N-(Carbonyl-methoxypolyethyleneglycol 2000)-1,2 5 dipalmitoyl-sn-glycero-3-phosphoethanolamine (sodium salt; DPPE-MPEG-2000);

N

(Carbonyl-methoxypolyethyleneglycol 5000)-1, 2 -dipalmitoyl-sn-glycero-3 phosphoethanolamine (sodium salt; DPPE-MPEG-5000); N-(Carbonyl methoxypolyethyleneglycol 750)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (sodium salt; DSPE-MPEG-750); N-(Carbonyl-methoxypolyethyleneglycol 2000)-1,2 10 distearoyl-sn-glycero-3-phosphoethanolamine (sodium salt; DSPE-MPEG-2000);

N

(Carbonyl-methoxypolyethyleneglycol 5000)-1,2-distearoyl-sn-glycero-3 phosphoethanolamine (sodium salt; DSPE-MPEG-5000); sodium cholesteryl sulfate (SCS); pharmaceutically acceptable salts thereof, and mixtures thereof. Examples of non-amino acid non-cationic lipids include polymeric lipids such as 15 DOPE-PEG, DLPE-PEG, DDPE-PEG DLinPE-PEG, and diacylglycerol-PEG-2000 or -5000. Examples of non-amino acid non-cationic lipids include polymeric lipids such as multi-branched pegylated compounds, for example DSPE-PTE020 and DSPE-AM0530<. Examples of non-amino acid non-cationic lipids include polymeric lipids such as 20 DSPE-PG8G polyglycerine lipids. Examples of non-amino acid non-cationic lipids include dioleoylphosphatidylethanolamine (DOPE), diphytanoylphosphatidylethanolamine (DPhPE), 1,2-Dioleoyl-sn-Glycero-3-Phosphocholine (DOPC), and 1,2-Diphytanoyl-sn Glycero-3-Phosphocholine (DPhPC). 25 Examples of non-amino acid non-cationic lipids include cholesterols, sterols, and steroids such as gonanes, estranes, androstanes, pregnanes, cholanes, cholestanes, ergostanes, campestanes, poriferastanes, stigmastanes, gorgostanes, lanostanes, cycloartanes, as well as sterol or zoosterol derivatives of any of the foregoing, and their biological intermediates and precursors, which may include, for example, cholesterol, 30 lanosterol, stigmastanol, dihydrolanosterol, zymosterol, zymostenol, desmosterol, 7 -dehydrocholesterol, and mixtures and derivatives thereof. Examples of non-amino acid non-cationic lipids include pegylated cholesterols, and cholestane 3-oxo(CI-22acyl) derivatives such as cholesteryl acetate, cholesteryl arachidonate, cholesteryl butyrate, cholesteryl hexanoate, cholesteryl caprylate, 53 cholesteryl n-decanoate. cholesteryl dodecanoate, cholesteryl myristate, cholesteryl palmitate, cholesteryl behenate, cholesteryl stearate, cholesteryl nervonate, cholesteryl pelargonate, cholesteryl n-valerate, cholesteryl oleate, cholesteryl elaidate, cholesteryl erucate, cholesteryl heptanoate, cholesteryl linolelaidate, cholesteryl linoleate, and 5 mixtures and derivatives thereof Examples of non-amino acid non-cationic lipids include compounds derived fror I plant sterols including phytosterols, beta-sitosterol, campesterol, ergosterol, brassicasterol, delta-7-stigmasterol, delta-7-avenasterol, and mixtures and derivatives thereof. 10 Examples of non-amino acid non-cationic lipids include bile acids, cholic acid, chenodeoxycholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, lithocholic acid, methyl-lithocholic acid, and mixtures and derivatives thereof. Examples of non-amino acid non-cationic lipids include compounds derived front steroids including glucocorticoids, cortisol, hydrocortisone, corticosterone,

A

5 15 pregnenolone, progesterone, deoxycorticosterone, 1 7 -OH-pregnenolone, 17-OH progesterone, 11 -dioxycortisol, dehydroepiandrosterone, dehydroepiandrosterone sulfate androstenedione, aldosterone, 1 8 -hydroxycorticosterone, tetrahydrocortisol, tetrahydrocortisone, cortisone, prednisone, 6a-methylpredisone, 9a-fluoro-16a hydroxyprednisolone, 9a-fluoro-1 6 a-methylprednisolone, 9a-fluorocortisol, and mixtures 20 and derivatives thereof. Examples of non-amino acid non-cationic lipids include compounds derived front steroids including adrogens, testosterone, dihydrotestosterone, androstenediol, androstenedione, androstenedione, 3a,5a-androstanediol, and mixtures and derivatives thereof. 25 Examples of non-amino acid non-cationic lipids include compounds derived front steroids including estrogens, estriols, estrones, estradiols, and mixtures and derivatives thereof. Examples of non-amino acid non-cationic lipids include compounds derived front lumisterol and vitamin D compounds. 30 Examples of non-amino acid non-cationic lipids include lipids having tails ranging from C10:0 to C22:6, for example, DDPE (C10:0) (CAS 253685-27-7), DLPE (C1.2:0) (CAS 59752-57-7), DSPE (C18:0) (CAS 1069-79-0), DOPE (C18:1) (CAS 4004-05-1), DLinPE (C18:2) (CAS 20707-71-5), DLenPE (C18:3) (CAS 34813-40-6), DARAPE 54 (C20:4) (CAS 5634-86-6), DDHAPE (C22:6) (CAS 123284-81-1), DPhPE (16:0[(CH 3 ) 1) (CAS 201036-16-0). Examples of non-amino acid anionic lipids include phosphatidylserine, phosphatidic acid, phosphatidylcholine, platelet-activation factor (PAF), 5 phosphatidylethanolamine, phosphatidyl-DL-glycerol, phosphatidylinositol, phosphatidylinositol (pi(4)p, pi(4,5)p2), cardiolipin (sodium salt), lysophosphatides, hydrogenated phospholipids, sphingoplipids, gangliosides, phytosphingosine, sphinganines, pharmaceutically acceptable salts thereof, and mixtures thereof. Compositions and Formulations for Administration 10 The nucleic acid compositions and formulations of this invention may be administered by various routes, for example, to effect systemic delivery via intravenous, parenteral, or intraperitoneal routes. In some embodiments, an siRNA may be delivered intracellularly, for example, in cells of a target tissue such as lung or liver, or in inflamed tissues. Included within this disclosure are compositions and methods for delivery of an 15 siRNA agent by removing cells of a subject, delivering an siRNA agent to the removed cells, and reintroducing the cells into a subject. In some embodiments, this invention provides a method for delivery of siRNA in vivo. A nucleic acid composition may be administered intravenously, subcutaneously, or intraperitoneally to a subject. In some embodiments, the invention provides methods for in vivo delivery of interfering RNA to 20 the lung of a mammalian subject. In some embodiments, this invention provides a method of treating a disease or disorder in a mammalian subject. A therapeutically effective amount of a composition o F this invention containing an interfering RNA, a lipopeptide, and optionally a non-cationie lipid, a polymeric lipid, and one or more delivery-enhancing components or excipients 25 may be administered to a subject having a disease or disorder associated with expression or overexpression of a gene that can be reduced, decreased, downregulated, or silenced by the composition. This invention encompasses methods for treating a disease of the lung such as respiratory distress, asthma, cystic fibrosis, pulmonary fibrosis, chronic obstructive 30 pulmonary disease, bronchitis, or emphysema, by administering to the subject a therapeutically effective amount of a composition. 55 This invention encompasses methods for treating rheumatoid arthritis, liver disease, encephalitis, bone fracture, heart disease, viral disease including hepatitis and influenza, or cancer. Compositions and formulations of this disclosure may be used for delivery of dru Y 5 agents or biologically active agents to a variety of cells in vitro. Examples of cells for which in vitro delivery is encompassed include epithelial cells such as A549, immortal cell lines such as HeLa, hepatoma cells such as HepG2, rat gliosarcoma cells such as 9L/LacZ, human monocyte cells such as THP-1, Madin-Darby canine kidney cells (MDCK), various fibroblast cell lines, and primary cells in culture in the presence or 10 absence of various sera, among others. Compositions and formulations of this disclosure may be used for delivery of drug agents or biologically active agents to a variety of cells, tissues or organs in vivo. Modalities for delivering an agent in vivo include topical, enteral, and parenteral routes. Examples of modalities for delivering an agent in vivo include inhalation of particles or 15 droplets, delivery of nasal or nasal-phamgyl drops, particles, or suspensions, transderma I and transmucosal routes, as well as injection or infusion by intramuscular, subcutaneous intravenous, intraarterial, intracardiac, intrathecal, intraosseus, intraperitoneal, and epidural routes. In some embodiments, an agent can be administered ex vivo by direct exposure to 20 cells, tissues or organs originating from a mammalian subject. A drug agent or biologically active agent to be delivered using a composition or formulation of this disclosure may be found in any form including, for example, a pure form, a crystalline form, a solid form, a nanoparticle, a condensed form, a complexed form, or a conjugated form. 25 This invention also provides methods for the delivery of one or more RNAi inducing entities to organs and tissues within the body of a mammal. In some embodiments, compositions containing an RNAi-inducing entity, one or more amino acid lipids, and one or more additional lipid components are introduced by various routes to be transported within the body and taken up by cells in one or more organs or tissues, where 30 expression of a target transcript is modulated. Ribonucleic acid agents useful for this invention may be targeted to various gene;, Examples of human genes suitable as targets include TNF, PLKI, BIRC5, APOB, FLTI the VEGF family, the ERBB family, the PDGFR family, BCR-ABL, and the MAPK family, among others. Examples of human genes suitable as targets and nucleic acid 56 sequences thereto include those disclosed in PCT/US08/55333, PCT/US08/55339, PCT/US08/55340, PCT/US08/55341, PCT/US08/55350, PCT/US08/55353, PCT/US08/55356, PCT/JS08/55357, PCT/US08/55360, PCT/US08/55362, PCT/US08/55365, PCT/US08/55366, PCT/US08/55369, PCT/US08/55370, 5 PCT/US08/55371, PCT/US08/55372, PCT/US08/55373, PCT/US08/55374, PCT/US08/55375, PCT/US08/55376, PCT/US08/55377, PCT/US08/55378, PCT/US08/55380, PCT/US08/55381, PCT/US08/55382, PCT/US08/55383, PCT/US08/55385, PCT/US08/55386, PCT/US08/55505, PCT/US08/55511, PCT/US08/55515, PCT/US08/55516, PCT/US08/55519, PCT/US08/55524, 10 PCT/US08/55526, PCT/US08/55527, PCT/US08/55532, PCT/US08/55533, PCT/US08/55542, PCT/US08/55548, PCT/US08/55550, PCT/US08/55551, PCT/US08/55554, PCT/US08/55556, PCT/US08/55560, PCT/US08/55563, PCT/US08/55597, PCT/US08/55599, PCT/US08/55601, PCT/US08/55603, PCT/US08/55604, PCT/US08/55606, PCT/US08/55608, PCT/US08/55611, 15 PCT/US08/55612, PCT/US08/55615, PCT/US08/55618, PCT/US08/55622, PCT/US08/55625, PCT/US08/55627, PCT/US08/55631, PCT/US08/55635, PCT/US08/55644, PCT/US08/55649, PCT/US08/55651, PCT/US08/55662, PCT/US08/55672, PCT/US08/55676, PCT/US08/55678, PCT/US08/55695, PCT/USO8/55697, PCT/US08/55698, PCT/US08/55701, PCT/US08/55704, 20 PCT/US08/55708, PCT/US08/55709, PCT/US08/5571 1, U.S. Patent Application No. 61/086,435, and U.S. Patent Application No. 61/086,445. The compositions and methods of the invention may be administered to subjects by a variety of mucosal administration modes, including by oral, rectal, vaginal, intranasal, intrapulmonary, or transdermal delivery, or by topical delivery to the eyes, 25 ears, skin or other mucosal surfaces. In some aspects of this invention, the mucosal tissue layer includes an epithelial cell layer. The epithelial cell can be pulmonary, tracheal, bronchial, alveolar, nasal, buccal, epidermal, or gastrointestinal. Compositions of this invention can be administered using conventional actuators such as mechanical spray devices, as well as pressurized, electrically activated, or other types of actuators. 30 Compositions of this invention may be administered in an aqueous solution as a nasal or pulmonary spray and may be dispensed in spray form by a variety of methods known to those skilled in the art. Pulmonary delivery of a composition of this invention may be achieved by administering the composition in the form of drops, particles, or spray, which can be, for example, aerosolized, atomized, or nebulized. Pulmonary 57 delivery may be performed by administering the composition in the form of drops, particles, or spray, via the nasal or bronchial passages. Particles of the composition, spray, or aerosol can be in a either liquid or solid form. Preferred systems for dispensin y liquids as a nasal spray are disclosed in U.S. Patent No. 4,511,069. Such formulations 5 may be conveniently prepared by dissolving compositions according to the present invention in water to produce an aqueous solution, and rendering said solution sterile. The formulations may be presented in multi-dose containers, for example in the sealed dispensing system disclosed in U.S. Patent No. 4,511,069. Other suitable nasal spray delivery systems have been described in Transdermal Systemic Medication, Y.W. Chien 10 ed., Elsevier Publishers, New York, 1985; and in U.S. Patent No. 4,778,810. Additional aerosol delivery forms may include, for example, compressed air-, jet-, ultrasonic-, and piezoelectric nebulizers, which deliver the biologically active agent dissolved or suspended in a pharmaceutical solvent, for example, water, ethanol, or mixtures thereof. Nasal and pulmonary spray solutions of the present invention typically comprise 15 the drug or drug to be delivered, optionally formulated with a surface active agent, such as a nonionic surfactant (e.g., polysorbate-80), and one or more buffers. In some embodiments of the present invention, the nasal spray solution further comprises a propellant. The pH of the nasal spray solution may be from about pH 6.8 to 7.2. The pharmaceutical solvents employed can also be a slightly acidic aqueous buffer of pH 4-6 20 Other components may be added to enhance or maintain chemical stability, including preservatives, surfactants, dispersants, or gases. In some embodiments, this invention is a pharmaceutical product which includes a solution containing a composition of this invention and an actuator for a pulmonary, mucosal, or intranasal spray or aerosol. 25 A dosage form of the composition of this invention can be liquid, in the form of droplets or an emulsion, or in the form of an aerosol. A dosage form of the composition of this invention can be solid, which can be reconstituted in a liquid prior to administration. The solid can be administered as a powder. The solid can be in the form of a capsule, tablet or gel. 30 To formulate compositions for pulmonary delivery within the present invention, the biologically active agent can be combined with various pharmaceutically acceptable additives or delivery-enhancing components, as well as a base or carrier for dispersion of the active agent(s). Examples of additives or delivery-enhancing components include pH control agents such as arginine, sodium hydroxide, glycine, hydrochloric acid, citric acid, 58 and mixtures thereof, Other additives or delivery-enhancing components include local anesthetics (e.g., benzyl alcohol), isotonizing agents (e.g., sodium chloride, mannitol, sorbitol), adsorption inhibitors (e.g., Tween 80), solubility enhancing agents (e.g., cyclodextrins and derivatives thereof), stabilizers (e.g., serum albumin), and reducing 5 agents (e.g., glutathione). When the composition for mucosal delivery is a liquid, the tonicity of the formulation, as measured with reference to the tonicity of 0.9% (w/v) physiological saline solution taken as unity, is typically adjusted to a value at which no substantial, irreversible tissue damage will be induced in the mucosa at the site of administration. Generally, the tonicity of the solution is adjusted to a value of about 1/3 10 to 3, more typically 1/2 to 2, and most often 3/4 to 1.7. The biologically active agent may be dispersed in a base or vehicle, which may comprise a hydrophilic compound having a capacity to disperse the active agent and any desired additives. The base may be selected from a wide range of suitable carriers, including but not limited to, copolymers of polycarboxylic acids or salts thereof, 15 carboxylic anhydrides (e.g., maleic anhydride) with other monomers (e.g., methyl (meth)acrylate, acrylic acid, etc.), hydrophilic vinyl polymers such as polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone, cellulose derivatives such as hydroxymethylcellulose, hydroxypropylcellulose, etc., and natural polymers such as chitosan, collagen, sodium alginate, gelatin, hyaluronic acid, and nontoxic metal salts 20 thereof. A biodegradable polymer may be selected as a base or carrier, for example, polylactic acid, poly(lactic acid-glycolic acid) copolymer, polyhydroxybutyric acid, poly(hydroxybutyric acid-glycolic acid) copolymer and mixtures thereof. Alternatively or additionally, synthetic fatty acid esters such as polyglycerin fatty acid esters, sucrose fatty acid esters, etc., can be employed as carriers. Hydrophilic polymers and other 25 carriers can be used alone or in combination, and enhanced structural integrity can be imparted to the carrier by partial crystallization, ionic bonding, crosslinking and the like. The carrier can be provided in a variety of forms, including, fluid or viscous solutions, gels, pastes, powders, microspheres and films for direct application to the nasal mucosa. The use of a selected carrier in this context may result in promotion of absorption of the 30 biologically active agent. The biologically active agent can be combined with the base or carrier according to a variety of methods, and release of the active agent may be by diffusion, disintegration of the carrier, or associated formulation of water channels. In some circumstances, the active agent is dispersed in microcapsules (microspheres) or nanocapsules (nanospheres) 59 prepared from a suitable polymer, for example, isobutyl 2-cyanoacrylate (see, e.g., Michael, et al., J. Pharmacy Pharmacol. 43:1-5, 1991), and dispersed in a biocompatibI dispersing medium applied to the nasal mucosa, which yields sustained delivery and biological activity over a protracted time. 5 Formulations for mucosal, nasal, or pulmonary delivery may contain a hydrophil c low molecular weight compound as a base or excipient. Such hydrophilic low molecula weight compounds provide a passage medium through which a water-soluble active agent, such as a physiologically active peptide or protein, may diffuse through the base t> the body surface where the active agent is absorbed. The hydrophilic low molecular 10 weight compound optionally absorbs moisture from the mucosa or the administration atmosphere and dissolves the water-soluble active peptide. The molecular weight of the hydrophilic low molecular weight compound is generally not more than 10,000 and preferably not more than 3,000. Examples of hydrophilic low molecular weight compounds include polyol compounds, such as oligo-, di- and monosaccarides including 15 sucrose, mannitol, lactose, L-arabinose, D-erythrose, D-ribose, D-xylose, D-mannose, D galactose, lactulose, cellobiose, gentibiose, glycerin, polyethylene glycol, and mixtures thereof. Further examples of hydrophilic low molecular weight compounds include N methylpyrrolidone, alcohols (e.g., oligovinyl alcohol, ethanol, ethylene glycol, propylene glycol, etc.), and mixtures thereof. 20 The compositions of this invention may alternatively contain as pharmaceutically acceptable carriers substances as required to approximate physiological conditions, such : ;11 r fng agents, tonicity adjusting agents, and wetting agents, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and mixtures thereof. For solid 25 compositions, conventional nontoxic pharmaceutically acceptable carriers can be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. In certain embodiments of the invention, the biologically active agent may be 30 administered in a time release formulation, for example in a composition which includes a slow release polymer. The active agent can be prepared with carriers that will protect against rapid release, for example a controlled release vehicle such as a polymer, microencapsulated delivery system or bioadhesive gel. Prolonged delivery of the active agent, in various compositions of the invention can be brought about by including in the 60 composition agents that delay absorption, for example, aluminum monosterate hydrogen s and gelatin. Within certain embodiments of this invention, the siNA composition may conta n one or more natural or synthetic surfactants. Certain natural surfactants are found in 5 human lung (pulmonary surfactant), and are a complex mixture of phospholipids and proteins that form a monolayer at the alveolar air-liquid interface and reduces surface tension to near zero at expiration and prevents alveolar collapse. Over 90% (by weight of pulmonary surfactant is composed of phospholipids with approximately 40-80% bei g DPPC and the remainder being unsaturated phosphatidylcholines POPG, POPC and 10 phosphatidylglycerols. The remaining 10% (by weight) of surfactant is composed of plasma proteins and apoproteins, such as surface proteins (SP)-A, SP-B, SP-C and SP-D. Examples of natural surfactants that may be used in this invention include SURVANTAM (beractant), CUROSURFIm (poractant alfa) and INFASURFM (calfactant), and mixtures thereof. 15 Examples of synthetic surfactants include sinapultide; a combination of dipalmitoylphosphatidylcholine, palmitoyloleoyl phosphatidylglycerol and palmitic aci d; SURFAXINTM (lucinactant); and EXOSURFTM (colfosceril); components which may contain tyloxapol, DPPC, and hexadecanol; and mixtures thereof. Compositions of this invention can be prepared by methods known in the art. 20 Methods of making the lipid compositions include ethanol injection methods and extrusion methods using a Northern Lipids Lipex Extruder system with stacked polycarbonate membrane filters of defined pore size. Sonication using probe tip and bath sonicators can be employed to produce lipid particles of uniform size. Homogenous and monodisperse particle sizes can be obtained without the addition of the nucleic acid 25 component. For in vitro transfection compositions, the nucleic acid component can be added after the transfection agent is made and stabilized by additional buffer compone ts. For in vivo delivery compositions, the nucleic acid component is part of the formulati n. A mixing procedure involving the graded substitution of ethanol for buffer can be used to create a narrow homodisperse particle size distribution. The lipid components 30 and lipopeptides can be dissolved in USP absolute ethanol and the RNA component c n be dissolved in an aqueous buffer, for example, at 0.9 mg/mL. Both mixtures can be injected through an HPLC mixing tee into a 20 mM citrate buffer at pH 7.2. The initial concentration of ethanol may be 90% for the lipids and 0% for the RNA component. After mixing into a 45% ethanol mixture, the RNA-lipid particles can be immediately 61 diluted into citrate buffer with a final concentration of ethanol at 30%. The ethanol and citrate buffer can be exchanged for PBS, pH 7.2 by overnight dialysis in a Pierce dialysis cassette with a 2K MWCO membrane. Particle sizing and PAGE gel analysis of each siRNA lipid particle can be performed to confirm successful entrapment. PAGE gel 5 results can confirm that an siRNA is intact after exposure to multiple processing steps. The nucleic acid component, lipopeptides, and any additional components may be mixed together first in a suitable medium such as a cell culture medium, after which one or more additional lipids or compounds may be added to the mixture. Alternatively, the lipopeptides can be mixed together first in a suitable medium such as a cell culture 10 medium, after which the nucleic acid component can be added. Within certain embodiments of the invention, a dsRNA is admixed with one or more lipopeptides, or a combination of one or more lipopeptides and non-cationic lipids. The interfering RNA agent may also be complexed with, or conjugated to a lipopeptide or a polymeric lipid, and admixed with one or more non-cationic lipids, or a 15 combination of one or more non-cationic and cationic lipids. An interfering RNA agent and a lipopeptide may be mixed together first, followed . by the addition of one or more non-cationic lipids, or a combination of non-cationic and cationic lipids added in a suitable medium such as a cell culture medium. Alternatively, the lipopeptides and lipid components may be mixed first, followed by the addition of th 20 RNA agent in a suitable medium. RNA Therapeutics and RNA Interference This invention provides compositions and methods for modulating gene expression by RNA interference. A composition of this invention can deliver a ribonucleic acid agent to a cell which can produce the response of RNAi. Examples of 25 nucleic acid agents useful for this invention include double-stranded nucleic acids, modified or degradation-resistant nucleic acids, RNA, siRNA, siNA, mdRNA, shRNA, single-stranded nucleic acids, DNA-RNA chimeras, antisense nucleic acids, and ribozymes. As used herein, the terms siRNA, siNA, and shRNA include precursors of siRNA, siNA, and shRNA, respectively. For example, the term siRNA includes an RNA 30 or double-stranded RNA that is suitable as a dicer substrate. Meroduplex RNA (mdRNA) is described in U.S. Provisional Application No. 60/934,930, as well as International Publication No. WO/2007/056153. 62 Ribonucleic acid agents useful for this invention may be targeted to various gen s. For example, a siRNA agent of this invention may have a sequence that is complement ry to a region of a TNF-alpha gene. In some embodiments of this invention, compounds and compositions are useful to regulate expression of tumor necrosis factor-a (TNF-a), TN F 5 a can be linked, for example, to inflammatory processes which occur in pulmonary diseases, and can have anti-inflammatory effects. Blocking TNF-ca by delivery of a composition of this invention can be useful to treat or prevent the signs and/or symptom s of rheumatoid arthritis. This invention provides compositions and methods for modulating expression and activity of TNF-a by RNA interference. 10 Expression and/or activity of TNF-a can be modulated by delivering to a cell, for example, the siRNA molecule Inm-4. Inm-4 is a double stranded 21-nt siRNA molecu e with sequence homology to the mouse TNF-a gene. Inm-4 has a 3' dTdT overhang on the sense strand and a 3' dAdT overhang on the antisense strand. The primary structure of Inm-4 is: 15 (SEQ ID NO: 301) sense 5'-CCGUCAGCCGAUUUGCUAUdTdT (SEQ ID NO: 302) antisense 5'-AUAGCAAAUCGGCUGACGGdTdT Expression and/or activity of TNF-a can be modulated by delivering to a cell, ff>r 20 example, the siRNA molecule LC20. LC20 is a double stranded 21-nt siRNA molecul: with sequence homology to the human TNF-a gene. LC20 is directed against the 3'-U TR region of human TNF-a. LC 20 has 19 base pairs with a 3' dTdT overhang on the sen se strand and a 3' dAdT overhang on the antisense strand. The molecular weight of the sodium salt form is 14,298. The primary structure of LC20 is: 25 (SEQ ID NO: 303) sense (5') GGGUCGGAACCCAAGCUUAdTdT (SEQ ID NO: 304) antisense (5') UAAGCUUGGGUUCCGACCCdTdA A p-galactoside reporter cell line was used to assay the RNAi activity of various 30 formulations. The structure of Lac-Z is: Sense: CN2938. (SEQ ID NO: 305) 63 5'-CUACACAAAUCAGCGAUUUdTdT-3' Antisense: CN2939. (SEQ ID NO: 306) 5'-AAAUCGCUGAUUUGUGUAGdTdC-3' A siRNA of this invention may have a sequence that is complementary to a region 5 of a viral gene. For example, some compositions and methods of this invention are usel to regulate expression of the viral genome of an influenza. In this context, this invention provides compositions and methods for modulatin, expression and infectious activity of an influenza by RNA interference. Expression and/or activity of an influenza can be modulated by delivering to a cell, for example, a 10 short interfering RNA molecule having a sequence that is complementary to a region of a RNA polymerase subunit of an influenza. For example, in Table 5 are shown double stranded siRNA molecules with sequence homology to an RNA polymerase subunit of an influenza. 64 Table 5 Double-Stranded siRNA Molecules Targeted to Influenza siRNA Subunit SEQUENCE G3789 PB2 (SEQ ID NO 307) CGGGACUCUAGCAUACUUAdTdT (SEQ ID NO 308) UAAGUAUGCUAGAGUCCCGdTdT G3807 PB2 (SEQ ID NO 309) ACUGACAGCCAGACAGCGAdTdT (SEQ ID NO 310) UCGCUGUCUGGCUGUCAGUdTdT G3817 PB2 (SEQ ID NO 311) AGACAGCGACCAAAAGAAUdTdT (SEQ ID NO 312) AUUCUUUUGGUCGCUGUCUdTdT G6124 PBI (SEQ ID NO 313) AUGAAGAUCUGUUCCACCAdTdT (SEQ ID NO 314) UGGUGGAACAGAUCUUCAUdTdT G6129 PB1 (SEQ ID NO 315) GAUCUGUUCCACCAUUGAAdTdT (SEQ ID NO 316) UUCAAUGGUGGAACAGAUCdTdT G8282 PA (SEQ ID NO 317) GCAAUUGAGGAGUGCCUGAdTdT (SEQ ID NO 318) UCAGGCACUCCUCAAUUGCdTdT G8286 PA (SEQ ID NO 319) UUGAGGAGUGCCUGAUUAAdTdT (SEQ ID NO 320) UAAUCAGGCACUCCUCAAdTdT G1498 NP (SEQ ID NO 321) GGAUCUUAUUUCUUCGGAGdTdT (SEQ ID NO 322) CUCCGAAGAAAUAAGAUCCdTdT A siRNA of this invention may have a sequence that is complementary to a region )f a RNA polymerase subunit of an influenza. 5 This invention provides compositions and methods to administer siNAs directed against a mRNA of an influenza, which effectively down-regulates an influenza RNA and thereby reduces, prevents, or ameliorates an influenza infection. In some embodiments, this invention provides compositions and methods for inhibiting expression of a target transcript in a subject by administering to the subject a 10 composition containing an effective amount of an RNAi-inducing compound such as a shc rt interfering oligonucleotide molecule, or a precursor thereof. RNAi uses small interfering RNAs (siRNAs) to target messenger RNA (mRNAs) and attenuate translation. A siRNA ;s used in this invention may be a precursor for dicer processing such as, for example, a long dsRNA processed into a siRNA. This invention provides methods of treating or preventing 15 diseases or conditions associated with expression of a target transcript or activity of a peptide or protein encoded by the target transcript. 65 A therapeutic strategy based on RNAi can be used to treat a wide range of diseases y shutting down the growth or function of a virus or microorganism, as well as by shutting down the function of an endogenous gene product in the pathway of the disease. In some embodiments, this invention provides novel compositions and methods for 5 delivery of RNAi-inducing entities such as short interfering oligonucleotide molecules, and precursors thereof. In particular, this invention provides compositions containing an RNAi-inducing entity which is targeted to one or more transcripts of a cell, tissue, and/or organ of a subject. A siRNA can be two RNA strands having a region of complementarity about l0 19 nucleotides in length. A siRNA optionally includes one or two single-stranded overhang s or loops. A shRNA can be a single RNA strand having a region of self-complementarity. Th< single RNA strand may form a hairpin structure with a stem and loop and, optionally, one o more unpaired portions at the 5' and/or 3' portion of the RNA. 5 The active therapeutic agent can be a chemically-modified siNA with improved resistance to nuclease degradation in vivo, and/or improved cellular uptake, which retains RNAi activity. A siRNA agent of this invention may have a sequence that is complementary to a region of a target gene. A siRNA of this invention may have 29-50 base pairs, for example, a ,O dsRNA having a sequence that is complementary to a region of a target gene. Alternately, the double-stranded nucleic acid can be a dsDNA. In some embodiments, the active agent can be a short interfering nucleic acid (siNA, short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA, or short hairpin RNA (shRNA) that can modulate expression of a gene product. 25 Comparable methods and compositions are provided that target expression of one or more different genes associated with a particular disease condition in a subject, including ar y of a large number of genes whose expression is known to be aberrantly increased as a causa or contributing factor associated with the selected disease condition. The RNAi-inducing compound of this invention can be administered in conjunction 30 with other known treatments for a disease condition. 66 In some embodiments, this invention features compositions containing a small nucleic acid molecule, such as short interfering nucleic acid, a short interfering RNA, a double-stranded RNA, a micro-RNA, or a short hairpin RNA, admixed or complexed with, or conjugated to, a delivery-enhancing compound. 5 As used herein, the terms "short interfering nucleic acid," "siNA," "short interfering RNA," "siRNA," "short interfering nucleic acid molecule," "short interfering oligonucleotic e molecule," and "chemically-modified short interfering nucleic acid molecule," refer to any nucleic acid molecule capable of inhibiting or down regulating gene expression or viral replication, for example, by mediating RNA interference (RNAi) or gene silencing in a 10 sequence-specific manner. In some embodiments, the siNA is a double-stranded polynucleotide molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence in a target ribonucleic acid molecule for down regulating expression, or a portion thereof, and the sene 15 region comprises a nucleotide sequence corresponding to (i.e., which is substantially identical in sequence to) the target ribonucleic acid sequence or portion thereof "siNA" means a small interfering nucleic acid, for example a siRNA, that is a short-length double-stranded nucleic acid, or optionally a longer precursor thereof. The length of useful siNAs within this invention will in some embodiments be preferred at a 20 length of approximately 20 to 50 bp. However, there is no particular limitation to the lengt of useful siNAs, including siRNAs. For example, siNAs can initially be presented to cells n a precursor form that is substantially different than a final or processed form of the siNA th it will exist and exert gene silencing activity upon delivery, or after delivery, to the target cell. Precursor forms of siNAs may, for example, include precursor sequence elements that are 25 processed, degraded, altered, or cleaved at or after the time of delivery to yield a siNA that s active within the cell to mediate gene silencing. In some embodiments, useful siNAs will have a precursor length, for example, of approximately 100-200 base pairs, or 50-100 base pairs, or less than about 50 base pairs, which will yield an active, processed siNA within th target cell. In other embodiments, a useful siNA or siNA precursor will be approximately 30 10 to 49 bp, or 15 to 35 bp, or about 21 to 30 bp in length. 67 In some embodiments of this invention, polynucleotide delivery-enhancing polypeptides are used to facilitate delivery of larger nucleic acid molecules than convention a] siNAs, including large nucleic acid precursors of siNAs. For example, the methods and compositions herein may be employed for enhancing delivery of larger nucleic acids that 5 represent "precursors" to desired siNAs, wherein the precursor amino acids may be cleaver or otherwise processed before, during or after delivery to a target cell to form an active siNA for modulating gene expression within the target cell. For example, a siNA precursor polynucleotide may be selected as a circular, single-stranded polynucleotide, having two or more loop structures and a stem comprising 10 self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof, and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siNn 15 molecule capable of mediating RNAi. siNA molecules of this invention, particularly non-precursor forms, can be less than 30 base pairs, or about 17-19 bp, or 19-21 bp, or 21-23 bp. siRNAs can mediate selective gene silencing in the mammalian system. Hairpin RNAs, with a short loop and 19 to 27 base pairs in the stem, also selectively silence 20 expression of genes that are homologous to the sequence in the double-stranded stem. Mammalian cells can convert short hairpin RNA into siRNA to mediate selective gene silencing. RISC mediates cleavage of single stranded RNA having sequence complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA takes place within the 25 region complementary to the antisense strand of the siRNA duplex. siRNA duplexes of 21 nucleotides are typically most active when containing two-nucleotide 3'-overhangs. Replacing the 3'-overhanging segments of a 21 -mer siRNA duplex having 2-nucleotide 3' overhangs with deoxyribonucleotides may not have an adverse effect on RNAi activity. Replacing up to 4 nucleotides on each end of the siRNA with 30 deoxyribonucleotides can be tolerated whereas complete substitution with deoxyribonucleotides may result in no RNAi activity. 68 Alternatively, the siNAs can be delivered as single or multiple transcription produc s expressed by a polynucleotide vector encoding the single or multiple siNAs and directing their expression within target cells. In these embodiments the double-stranded portion of a final transcription product of the siRNAs to be expressed within the target cell can be, for 5 example, 15 to 49 bp, 15 to 35 bp, or about 21 to 30 bp long. In some embodiments of this invention, the double-stranded region of siNAs in which two strands are paired may contain bulge or mismatched portions, or both. Double-strandel portions of siNAs in which two strands are paired are not limited to completely paired nucleotide segments, and may contain nonpairing portions due to, for example, mismatch 10 (the corresponding nucleotides not being complementary), bulge (lacking in the corresponding complementary nucleotide on one strand), or overhang. Nonpairing portion! can be contained to the extent that they do not interfere with siNA formation. In some embodiments, a "bulge" may be I to 2 nonpairing nucleotides, and the double-stranded region of siNAs in which two strands pair up may contain from about I to 7, or about I to 15 bulges. In addition, "mismatch" portions contained in the double-stranded region of siNAs may be present in numbers from about I to 7, or about 1 to 5. Most often in the case of mismatches, one of the nucleotides is guanine, and the other is uracil. Such mismatching may be attributable, for example, to a mutation from C to T, G to A, or mixtures thereof, in a corresponding DNA coding for sense RNA, but other causes are also contemplated. 20 The terminal structure of siNAs of this invention may be either blunt or cohesive (overhanging) as long as the siNA retains its activity to silence expression of target genes. The cohesive (overhanging) end structure is not limited to the 3' overhang, but includes the 5' overhanging structure as long as it retains activity for inducing gene silencing. In addition, the number of overhanging nucleotides is not limited to 2 or 3 nucleotides, but can be any 25 number of nucleotides as long as it retains activity for inducing gene silencing. For example, overhangs may comprise from I to about 8 nucleotides, or from 2 to 4 nucleotides. The length of siNAs having cohesive (overhanging) end structure may be expressed in terms of the paired duplex portion and any overhanging portion at each end. For exampl:, a 25/27-mer siNA duplex with a 2-bp 3' antisense overhang has a 25-mer sense strand anda 30 27-mer antisense strand, where the paired portion has a length of 25 bp. 69 Any overhang sequence may have low specificity to a target gene, and may not be complementary (antisense) or identical (sense) to the target gene sequence. As long as the siNA retains activity for gene silencing, it may contain in the overhang portion a low molecular weight structure, for example, a natural RNA molecule such as a tRNA, an rRNA 5 a viral RNA, or an artificial RNA molecule. The terminal structure of the siNAs may have a stem-loop structure in which ends o one side of the double-stranded nucleic acid are connected by a linker nucleic acid, for example, a linker RNA. The length of the double-stranded region (stem portion) can be, fol example, 15 to 49 bp, or 15 to 35 bp, or about 21 to 30 bp long. Alternatively, the length of 10 the double-stranded region that is a final transcription product of siNAs to be expressed in a target cell may be, for example, approximately 15 to 49 bp, or 15 to 35 bp, or about 21 to 30 bp long. The siNA can contain a single stranded polynucleotide having a nucleotide sequenc complementary to a nucleotide sequence in a target nucleic acid molecule, or a portion 15 thereof, wherein the single stranded polynucleotide can contain a terminal phosphate group such as a 5'-phosphate (see e.g. Martinez, et al., Cell. 110:563-574, 2002, and Schwarz, el al., Molecular Cell 10:537-568, 2002, or 5',3'-diphosphate. As used herein, the term siNA molecule is not limited to molecules containing only naturally-occurring RNA or DNA, but also encompasses chemically-modified nucleotides 20 and non-nucleotides. In some embodiments, the short interfering nucleic acid molecules of the invention lack 2-hydroxy (2'-OH) containing nucleotides. In some embodiments, short interfering nucleic acids do not require the presence of nucleotides having a 2'-hydroxy gro ap for mediating RNAi and as such, short interfering nucleic acid molecules of this invention optionally do not include any ribonucleotides (e.g., nucleotides having a 2'-OH group). si A 25 molecules that do not require the presence of ribonucleotides within the siNA molecule to support RNAi can, however, have an attached linker or linkers or other attached or associate ed groups, moieties, or chains containing one or more nucleotides with 2'-OH groups. siNA molecules can comprise ribonucleotides in at least about 5, 10, 20, 30, 40, or 50% of the nucleotide positions. 30 As used herein, the term siNA encompasses nucleic acid molecules that are capable of mediating sequence specific RNAi such as, for example, short interfering RNA (siRNA' 70 molecules, double-stranded RNA (dsRNA) molecules, micro-RNA molecules, short hairpi RNA (shRNA) molecules, short interfering oligonucleotide molecules, short interfering nucleic acid molecules, short interfering modified oligonucleotide molecules, chemically-modified siRNA molecules, and post-transcriptional gene silencing RNA 5 (ptgsRNA) molecules, among others. In some embodiments, siNA molecules comprise separate sense and antisense sequences or regions, wherein the sense and antisense regions are covalently linked by nucleotide or non-nucleotide linker molecules, or are non-covalently linked by ionic interactions, hydrogen bonding, van der waals interactions, hydrophobic interactions, and/o 10 stacking interactions. "Antisense RNA" is an RNA strand having a sequence complementary to a target gene mRNA, that can induce RNAi by binding to the target gene mRNA. "Sense RNA" is an RNA strand having a sequence complementary to an antisense RNA, and anneals to its complementary antisense RNA to form a siRNA. 15 As used herein, the term "RNAi construct" or "RNAi precursor" refers to an RNAi-inducing compound such as small interfering RNAs (siRNAs), hairpin RNAs, and other RNA species which can be cleaved in vivo to form a siRNA. RNAi precursors herein also include expression vectors (also referred to as RNAi expression vectors) capable of giving rise to transcripts which form dsRNAs or hairpin RNAs in cells, and/or transcripts 10 which can produce siRNAs in vivo. A siHybrid molecule is a double-stranded nucleic acid that has a similar function to siRNA. Instead of a double-stranded RNA molecule, a siHybrid is comprised of an RNA strand and a DNA strand. Preferably, the RNA strand is the antisense strand which binds tc a target mRNA. The siHybrid created by the hybridization of the DNA and RNA strands have 25 a hybridized complementary portion and preferably at least one 3'overhanging end. siNAs for use within the invention can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self-complementary (i.e., each strand comprises nucleotide sequence that is complementary to nucleotide sequence in the other strand; such 30 as where the antisense strand and sense strand form a duplex or double stranded structure, for example wherein the double stranded region is about 19 base pairs). The antisense strand 71 may comprise a nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof, and the sense strand may comprise a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. Alternatively, the siNA can be assembled from a single oligonucleotide, where the 5 self-complementary sense and antisense regions of the siNA are linked by means of a nucle c acid-based or non-nucleic acid-based linker(s). In some embodiments, siNAs for intracellular delivery can be a polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises a 0 nucleotide sequence that is complementary to a nucleotide sequence in a separate target nucleic acid molecule or a portion thereof, and the sense region comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. Examples of chemical modifications that can be made in an siNA include phosphorothioate internucleotide linkages, 2'-deoxyribonucleotides, 2'-O-methyl 5 ribonucleotides, 2'-deoxy-2-fluoro ribonucleotides, "universal base" nucleotides, "acyclic" nucleotides, 5-C-methyl nucleotides, and terminal glyceryl and/or inverted deoxy abasic residue incorporation. The antisense region of a siNA molecule can include a phosphorothioate internucleotide linkage at the 3'-end of said antisense region. The antisense region can M comprise about one to about five phosphorothioate internucleotide linkages at the 5-end of said antisense region. The 3'-terminal nucleotide overhangs of a siNA molecule can include ribonucleotides or deoxyribonucleotides that are chemically-modified at a nucleic acid suger, base, or backbone. The 3'-terminal nucleotide overhangs can include one or more universa base ribonucleotides. The 3-terminal nucleotide overhangs can comprise one or more 25 acyclic nucleotides. For example, a chemically-modified siNA can have 1, 2, 3, 4, 5. 6, 7, 8, or more phosphorothioate internucleotide linkages in one strand, or can have I to 8 or more phosphorothioate internucleotide linkages in each strand. The phosphorothioate internucleotide linkages can be present in one or both oligonucleotide strands of the siNA 30 duplex, for example in the sense strand, the antisense strand, or both strands. 72 siNA molecules can comprise one or more phosphorothioate internucleotide linkages at the 3-end, the 5'-end, or both of the 3'- and 5'-ends of the sense strand, the antisense strand, or in both strands. For example, an exemplary siNA molecule can include 1,2,3,4,5, or more consecutive phosphorothioate internucleotide linkages at the 5'-end of the sense 5 strand, the antisense strand, or both strands. In some embodiments, a siNA molecule includes 1,2,3,4,5,6,7,8,9,10, or more pyrimidine phosphorothioate internucleotide linkages in the sense strand, the antisense strand, or in both strands. In some embodiments, a siNA molecule includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more [0 purine phosphorothioate internucleotide linkages in the sense strand, the antisense strand, oi in both strands. A siNA molecule can include a circular nucleic acid molecule, wherein the siNA is about 38 to about 70, for example, about 38,40,45,50,55,60,65, or 70 nucleotides in length, having about 18 to about 23, for example, about 18, 19, 20, 21, 22, or 23 base pairs, wherei 1 15 the circular oligonucleotide forms a dumbbell-shaped structure having about 19 base pairs and 2 loops. A circular siNA molecule can contain two loop motifs, wherein one or both loop portions of the siNA molecule is biodegradable. For example, the loop portions of a circus r siNA molecule may be transformed in vivo to generate a double-stranded siNA molecule 20 with 3'-terminal overhangs, such as 3'-terminal nucleotide overhangs comprising about 2 nucleotides. Modified nucleotides in a siNA molecule can be in the antisense strand, the sense strand, or both. For example, modified nucleotides can have a Northern conformation (e.g. Northern pseudorotation cycle; see e.g., Saenger, Principles ofNucleic Acid Structure, 25 Springer-Verlag ed., 1984). Examples of nucleotides having a Northern configuration include locked nucleic acid (LNA) nucleotides (e.g., 2'-0, 4'-C-methylene-(D-ribofuranosyl) nucleotides), 2'-methoxyethoxy (MOE) nucleotides, 2'-methyl-thio-ethyl, 2'-deoxy-2-fluor> nucleotides, 2'-deoxy-2-chloro nucleotides, 2'-azido nucleotides, and 2'-O-methyl nucleotides. 30 Chemically modified nucleotides can be resistant to nuclease degradation while at e same time maintaining the capacity to mediate RNAi, 73 The sense strand of a double stranded siNA molecule may have a terminal cap moiety such as an inverted deoxyabasic moiety, at the 3-end, 5'-end, or both 3' and 5'-ends of the sense strand. Examples of conjugates include conjugates and ligands described in Vargeese, et al. 5 U.S. Application Serial No. 10/427,160, filed April 30, 2003, incorporated by reference herein in its entirety, including the drawings. In some embodiments of this invention, the conjugate may be covalently attached to the chemically-modified siNA molecule via a biodegradable linker. For example, the conjugate molecule may be attached at the 3-end of either the sense strand, the antisense 0 strand, or both strands of the chemically-modified siNA molecule. In some embodiments, the conjugate molecule is attached at the 5'-end of either the sense strand, the antisense strand, or both strands of the chemically-modified siNA molecule. In some embodiments, the conjugate molecule is attached both the 3'-end and 5'-end of either the sense strand, the antisense strand, or both strands of the chemically-modified siNA 5 molecule, or any combination thereof. In some embodiments, a conjugate molecule comprises a molecule that facilitates delivery of a chemically-modified siNA molecule into a biological system, such as a cell. In some embodiments, a conjugate molecule attached to the chemically-modified siNA molecule is a polyethylene glycol, human serum albumin, or a ligand for a cellular 0 receptor that can mediate cellular uptake. Examples of specific conjugate molecules contemplated by the instant invention that can be attached to chemically-modified siNA molecules are described in Vargeese, el al., U.S. Patent Publication Nos. 2003/0130186 and 2004/0110296. A siNA may be contain a nucleotide, non-nucleotide, or mixed nucleotide/non 25 nucleotide linker that joins the sense region of the siNA to the antisense region of the siNA. In some embodiments, a nucleotide linker can be 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In some embodiments, the nucleotide linker can be a nucleic acid aptamer. As use( herein, the terms "aptamer" or "nucleic acid aptamer" encompass a nucleic acid molecule ti at binds specifically to a target molecule, wherein the nucleic acid molecule contains a 30 sequence that is recognized by the target molecule in its natural setting. Alternately, an 74 aptamer can be a nucleic acid molecule that binds to a target molecule where the target molecule does not naturally bind to a nucleic acid. For example, the aptamer can be used to bind to a ligand-binding domain of a protein, thereby preventing interaction of the naturally occurring ligand with the protein. See, for 5 example, Gold, et al., Annu; Rev. Biochem. 64:763, 1995; Brody and Gold, J Biotechnol. 74:5, 2000; Sun, Curr. Opin. Mol. Ther. 2:100, 2000; Kusser, J Biotechnol. 74:27, 2000; Hermann and Patel, Science 287:820, 2000; and Jayasena, Clinical Chemistry 45:1628, 1999. A non-nucleotide linker can be an abasic nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, polyhydrocarbon, or other polymeric compounds 10 (e.g., polyethylene glycols such as those having between 2 and 100 ethylene glycol units). Specific examples include those described by Seela and Kaiser, Nucleic Acids Res. 18:6353, 1990, and Nucleic Acids Res. 15:3113, 1987; Cload and Schepartz, J Am. Chem. Soc. 113:6324, 1991; Richardson and Schepartz, J. Am. Chem. Soc. 113:5109, 1991; Ma, et al., Nucleic Acids Res. 21:2585, 1993, and Biochemistry 32:1751, 1993; Durand, et al., Nucleic 15 Acids Res. 18:6353, 1990; McCurdy, et al., Nucleosides & Nucleotides 10:287, 1991; Jaschke, et al., Tetrahedron Lett. 34:301-304, 1993; Ono, et al., Biochemistry 30:9914, 1991; Arnold, et al., International Publication No. WO/1989/02439; Usman, et al., International Publication No. WO/1995/06731; Dudycz, et al, International Publication No. WO/1995/11910, and Ferentz and Verdine, J Am. Chem. Soc. 113:4000, 1991. 20 A "non-nucleotide linker" refers to a group or compound that can be incorporated ir to a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity. The group or compound can be abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine, for 25 example at the C1 position of the sugar. In some embodiments, modified siNA molecule can have phosphate backbone modifications including one or more phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, 30 and/or alkylsilyl substitutions. Examples of oligonucleotide backbone modifications are given in Hunziker and Leumann, Nucleic Acid Analogues: Synthesis and Properties, in 75 Modern Synthetic Methods, VCH, pp. 331-417, 1995, and Mesmaeker, et al., Novel Backb ne Replacements for Oligonucleotides, in Carbohydrate Modifications in Antisense Research, ACS, pp. 24-39, 1994. siNA molecules, which can be chemically-modified, can be synthesized by: 5 (a) synthesis of two complementary strands of the siNA molecule; and (b) annealing the tvo complementary strands together under conditions suitable to obtain a double-stranded siNA molecule. In some embodiments, synthesis of the complementary portions of the siNA molecule is by solid phase oligonucleotide synthesis, or by solid phase tandem oligonucleotide synthesis. 10 Oligonucleotides (e.g., certain modified oligonucleotides or portions of oligonucleotides lacking ribonucleotides) are synthesized using protocols known in the ar, for example, as described in Caruthers, et al., Methods in Enzymology 211:3-19, 1992; Thompson, et aL, International Publication No. WO/1999/54459; Wincott, et al., Nucleic Acids Res. 23:2677-2684, 1995; Wincott, et al., Methods Mol. Bio. 74:59, 1997; Brennan et 15 al., Biotechnol Bioeng. 61:33-45, 1998; and Brennan, U.S. Patent No. 6,001,311. Synthe is of RNA, including certain siNA molecules of the invention, follows general procedures a described, for example, in Usman, et al., J. Am. Chem. Soc. 109:7845, 1987; Scaringe, et al., Nucleic Acids Res. 18:5433, 1990; and Wincott, et al., Nucleic Acids Res. 23:2677-2684, 1995; Wincott, et al., Methods Mol. Bio. 74:59, 1997. 20 An "asymmetric hairpin" as used herein is a linear siNA molecule comprising an antisense region, a loop portion that can comprise nucleotides or non-nucleotides, and a sense region that comprises fewer nucleotides than the antisense region to the extent that the sense region has enough complementary nucleotides to base pair with the antisense region and form a duplex with loop, 25 An "asymmetric duplex" as used herein is a siNA molecule having two separate strands comprising a sense region and an antisense region, wherein the sense region comprises fewer nucleotides than the antisense region to the extent that the sense region has enough complementary nucleotides to base pair with the antisense region and form a du lex. To "modulate gene expression" as used herein is to upregulate or downregulate 30 expression of a target gene, which can include upregulation or downregulation of mRN \ 76 levels present in a cell, or of mRNA translation, or of synthesis of protein or protein subuni s, encoded by the target gene. The terms "inhibit," "down-regulate," or "reduce expression" as used herein mean tf at the expression of the gene, or level of RNA molecules or equivalent RNA molecules 5 encoding one or more proteins or protein subunits, or level or activity of one or more protein s or protein subunits encoded by a target gene, is reduced below that observed in the absence of the nucleic acid molecules (e.g., siNA) of the invention. "Gene silencing" as used herein refers to partial or complete inhibition of gene expression in a cell and may also be referred to as "gene knockdown." The extent of gene 0 silencing may be determined by methods known in the art, some of which are summarized in International Publication No. WO/1999/32619. As used herein, the terms "ribonucleic acid" and "RNA" refer to a molecule containing at least one ribonucleotide residue. A ribonucleotide is a nucleotide with a hydroxyl group at the 2' position of a P-D-ribo-furanose moiety. These terms include doubl 5 stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified and altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution, modification, and/or alteration of one or more nucleotides. Alterations of an RNA can include addition of non-nucleotide material, such as to the end(s) of a siNA or 0 internally, for example at one or more nucleotides of an RNA. Nucleotides in an RNA molecule include non-standard nucleotides, such as non naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs. By "highly conserved sequence region" is meant, a nucleotide sequence of one or 25 more regions in a target gene does not vary significantly from one generation to the other or from one biological system to the other. By "sense region" is meant a nucleotide sequence of a siNA molecule having complementarity to an antisense region of the siNA molecule. In addition, the sense region of a siNA molecule can comprise a nucleic acid sequence having homology with a target 0 nucleic acid sequence. 77 By "antisense region" is meant a nucleotide sequence of a siNA molecule having complementarity to a target nucleic acid sequence. In addition, the antisense region of a siNA molecule can include a nucleic acid sequence having complementarity to a sense regi n of the siNA molecule. 5 By "target nucleic acid" is meant any nucleic acid sequence whose expression or activity is to be modulated. A target nucleic acid can be DNA or RNA. By "complementarity" is meant that a nucleic acid can form hydrogen bond(s) with another nucleic acid sequence either by traditional Watson-Crick or by other non-traditional modes of binding. 0 The term "biodegradable linker" as used herein, refers to a nucleic acid or non-nucleic acid linker molecule that is designed as a biodegradable linker to connect one molecule to another molecule, for example, a biologically active molecule to a siNA molecule or the sense and antisense strands of a siNA molecule. The biodegradable linker is designed such that its stability can be modulated for a particular purpose, such as delivery to a particular ,5 tissue or cell type. The stability of a nucleic acid-based biodegradable linker molecule can be variously modulated, for example, by combinations of ribonucleotides, deoxyribonucleotidcs, and chemically-modified nucleotides, such as 2'-0-methyl, 2'-fluoro, 2T-amino, 2'-0-amino, 2'-C-allyl, 2-O-allyl, and other 2-modified or base modified nucleotides. The biodegradable nucleic acid linker molecule can be a dimer, trimer, tetramer or longer nucleic acid molecule, !0 for example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length, or can comprise a single nucleotide with a phosphorus-based linkage, for example, a phosphoramidate or phosphodiester linkage. The biodegradable nucleic acid linker molecule can also comprise nucleic acid backbone, nuclei: acid sugar, or nucleic acid base modifications. 25 In connection with 2-modified nucleotides as described herein, by "amino" is mean 2'-NH 2 or 2'-O-NH 2 , which can be modified or unmodified. Such modified groups are described, for example, in Eckstein, et al., U.S. Patent No. 5,672,695 and Matulic-Adamic, ,t al., U.S. Patent. No. 6,248,878. Supplemental or complementary methods for delivery of nucleic acid molecules for 30 use within then invention are described, for example, in Akhtar et al., Trends Cell Bio. 2:13 1992; "Delivery Strategies for Antisense Oligonucleotide Therapeutics," ed. Akhtar, 1995, 78 Maurer et al., Mol. Membr, Biol, 16:129-140, 1999; Hofland and Huang, Handb. Exp. Pharmacol. 137:165-192, 1999; and Lee et al., A CS Symp. Ser. 752:184-192, 2000. Sulliv n, et al., International Publication No. WO/1 994/02595, further describes general methods for delivery of enzymatic nucleic acid molecules. 5 Nucleic acid molecules can be administered within formulations that include one or more additional components, such as a pharmaceutically acceptable carrier, diluent, excipient, adjuvant, emulsifier, buffer, stabilizer, or preservative. As used herein, the term "carrier" means a pharmaceutically acceptable solid or liquid filler, diluent or encapsulating material. A water-containing liquid carrier can contain 10 pharmaceutically acceptable additives such as acidifying agents, alkalizing agents, antimicrobial preservatives, antioxidants, buffering agents, chelating agents, complexing agents, solubilizing agents, humectants, solvents, suspending and/or viscosity-increasing agents, tonicity agents, wetting agents or other biocompatible materials. Examples of ingredients of the above categories can be found in the US. Pharmacopeia National 15 Formulary, 1990, pp. 1857-1859, as well as in Raymond C. Rowe, et al., Handbook of Pharmaceutical Excipients , 5th ed., 2006, and "Remington: The Science and Practice of Pharmacy," 21st ed., 2006, editor David B. Troy. Examples of preservatives include phenol, methyl paraben, paraben, m-cresol, thiomersal, benzylalkonium chloride, and mixtures thereof. io Examples of surfactants include oleic acid, sorbitan trioleate, polysorbates, lecithin, phosphotidylcholines, various long chain diglycerides and phospholipids, and mixtures thereof. Examples of phospholipids include phosphatidylcholine, lecithin, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, and 25 phosphatidylethanolamine, and mixtures thereof. Examples of dispersants include ethylenediaminetetraacetic acid. Examples of gases include nitrogen, helium, chlorofluorocarbons (CFCs), hydrofluorocarbons (HFCs), carbon dioxide, air, and mixtures thereof. In certain embodiments, the siNA and/or the polypeptide can be encapsulated in 30 liposomes, or reside either internal or external to a liposome, or exist within liposome layer , or be administered by iontophoresis, or incorporated into other vehicles, such as hydrogels, 79 cyclodextrins, biodegradable nanocapsules, bioadhesive microspheres, or proteinaceous vectors. See, for example, O'Hare and Normand, International Publication No. WO/2000/53722. Alternatively, a nucleic acid composition can be locally delivered b) direct injection or by use of an infusion pump. Direct injection of the nucleic acid molecul s 5 of the invention, whether subcutaneous, intramuscular, or intradermal, can take place using standard needle and syringe methodologies, or by needle-free technologies such as those described in Conry et al., Clin. Cancer Res. 5:2330-2337, 1999, and Barry el al., International Publication No. WO/1999/31262. The compositions of this invention can be effectively employed as pharmaceutical 10 agents. Pharmaceutical agents prevent, modulate the occurrence or severity of, or treat (alleviate one or more symptom(s) to a detectable or measurable extent) of a disease state o other adverse condition in a patient. In some embodiments, this invention provides pharmaceutical compositions and methods featuring the presence or administration of one or more polynucleic acid(s), .5 typically one or more siNAs, combined, complexed, or conjugated with a lipid, which may further be formulated with a pharmaceutically-acceptable carrier, such as a diluent, stabiliz( r, or buffer. Typically, the siNA will target a gene that is expressed at an elevated level as a causal or contributing factor associated with the subject disease state or adverse condition. In this .0 context, the siNA will effectively downregulate expression of the gene to levels that preven , alleviate, or reduce the severity or recurrence of one or more associated disease symptoms. Alternatively, for various distinct disease models where expression of the target gene is not necessarily elevated as a consequence or sequel of disease or other adverse condition, down regulation of the target gene will nonetheless result in a therapeutic result by lowering gene 25 expression (i.e., to reduce levels of a selected mRNA and/or protein product of the target gene). Alternatively, siNAs of the invention may be targeted to lower expression of one gene, which can result in upregulation of a "downstream" gene whose expression is negatively regulated by a product or activity of the target gene. This siNAs of the present invention may be administered in any form, for example 30 transdermally or by local injection (e.g., local injection at sites of psoriatic plaques to treat psoriasis, or into the joints of patients afflicted with psoriatic arthritis or RA). In more so detailed embodiments, the invention provides formulations and methods to administer therapeutically effective amounts of siNAs directed against of a mRNA of TNF-a, which effectively down-regulate the TNF-a RNA and thereby reduce or prevent one or more TNF-a-associated inflammatory condition(s). Comparable methods and compositions are 5 provided that target expression of one or more different genes associated with a selected disease condition in animal subjects, including any of a large number of genes whose expression is known to be aberrantly increased as a causal or contributing factor associated with the selected disease condition. The compositions of the present invention may also be formulated and used as table ts, 10 capsules or elixirs for oral administration, suppositories for rectal administration, sterile solutions, suspensions for injectable administration, and the other forms known in the art. A pharmacological composition or formulation refers to a composition or formulati n in a form suitable for administration, for example, systemic administration, into a cell or patient, including for example a human. Suitable forms, in part, depend upon the use or the 15 route of entry, for example oral, transdermal, transepithelial, or by injection. Such forms should not prevent the composition or formulation from reaching a target cell (i.e., a cell to which the negatively charged nucleic acid is desirable for delivery). For example, pharmacological compositions injected into the blood stream should be soluble. Other factors are known in the art, and include considerations such as toxicity. 20 By "systemic administration" is meant in vivo systemic absorption or accumulation of drugs in the blood stream followed by distribution throughout the entire body. Administration routes which lead to systemic absorption include, without limitation: intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular. 25 Examples of agents suitable for formulation with the nucleic acid molecules of this invention include: P-glycoprotein inhibitors (such as Pluronic P85), which can enhance entry of drugs into the CNS (Jolliet-Riant and Tillement, Fundam. Clin. Pharmacol. 13:16-26, 1999); biodegradable polymers, such as poly (DL-lactide-coglycolide) microspheres for sustained release delivery after intracerebral implantation (Emerich, D.F., et al., Cell 30 Transplant 8:47-58, 1999, Alkermes, Inc., Cambridge, Mass.); and loaded nanoparticles, such as those made of polybutylcyanoacrylate, which can deliver drugs across the blood 81 brain barrier and can alter neuronal uptake mechanisms (Prog. Neuropsychopharmacol Bio. Psychiatry 23:941-949, 1999). Other examples of delivery strategies for the nucleic acid molecules of the instant invention include material described in Boado, et al., J. Pharm. Sci 87:1308-1315, 1998; Tyler, et al., FEBS Lett. 421:280-284, 1999; Pardridge, et al., PNAS 5 USA. 92:5592-5596, 1995; Boado, Adv. Drug Delivery Rev. 15:73-107, 1995; Aldrian-Herrada et al., Nucleic Acids Res. 26:4910-4916, 1998; and Tyler, et al., PNAS US L 96:7053-7058, 1999. The present invention also includes compositions prepared for storage or administration, which include a pharmaceutically effective amount of the desired compoun s 1.0 in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's PharmaceuticalSciences, Mack Publishing Co. (A.R. Gennaro ed. 1985). For example, preservatives, stabilizers, dyes and flavoring agents may be provided. These include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. In addition, 5 antioxidants and suspending agents may be used. A pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence of, treat, or alleviate a symptom to some extent of a disease state. An amount ol from 0.01 mg/kg to 50 mg/kg body weight/day of active nucleic acid should be administered L Aqueous suspensions contain the active materials in admixture with excipients 10 suitable for the manufacture of aqueous suspensions. Such excipients are suspending agent for example sodium carboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents can be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or 25 condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial ester! derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions 30 can also contain one or more preservatives, for example ethyl, or n-propyl 82 p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents. such as sucrose or saccharin. Oily suspensions can be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oi 5 such as liquid paraffin. The oily suspensions can contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents and flavoring agents can be added to provide palatable oral preparations. These compositions can be preserved by the addition of an anti-oxidant such as ascorbic acid. Dispersible powders and granules suitable for preparation of an aqueous suspension 10 by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Additional excipients, for example sweetening, flavoring and coloring agents, can also be present. Pharmaceutical compositions of the invention can also be in the form of oil-in-watel emulsions. The oily phase can be a vegetable oil or a mineral oil or mixtures of these. 5 Suitable emulsifying agents can be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and ester or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions can also contain sweetening 10 and flavoring agents. The pharmaceutical compositions can be in the form of a sterile injectable aqueous er oleaginous suspension. This suspension can be formulated according to the known art usin those suitable dispersing or wetting agents and suspending agents that have been mentioned above. The sterile injectable preparation can also be a sterile injectable solution or 25 suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono-or diglycerides. In addition, fatty 30 acids such as oleic acid find use in the preparation of injectables. 83 The siNAs can also be administered in the form of suppositories, for example, for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials 5 include cocoa butter and polyethylene glycols. The siNAs can be modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-fluoro, 2-O-methyl, 2'-H. F r a review see Usman and Cedergren, TIBS 17:34, 1992; Usman, et al., Nucleic Acids Symp. Ser. 31:163, 1994. SiNA constructs can be purified by gel electrophoresis using general 0 methods or can be purified by high pressure liquid chromatography and re-suspended in water. Chemically synthesizing nucleic acid molecules with modifications (base, sugar and/or phosphate) can prevent their degradation by serum ribonucleases, which can increase their potency. See for example, Eckstein, et aL, International Publication 5 No. WO/1992/07065; Perrault et al,, Nature 344:565, 1990; Pieken, et al., Science 253, 314 1991; Usman and Cedergren, Trends in Biochem, Sci. 17:334, 1992; Usman, et aL, International Publication No. WO/1993/15187; and Rossi et al., International Publication N WO/1991/03162; Sproat, U.S. Patent No. 5,334,711; and Gold, et al., U.S. Patent No. 6,300,074. All of the above references describe various chemical modifications that can 0 be made to the base, phosphate and/or sugar moieties of the nucleic acid molecules described herein. There are several examples in the art describing sugar, base and phosphate modifications that can be introduced into nucleic acid molecules with significant enhancement in their nuclease stability and efficacy. For example, oligonucleotides are 25 modified to enhance stability and/or enhance biological activity by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-fluoro, 2'-0-methyl, 2'-0 allyl, 2'-H, nucleotide base modifications. For a review, see Usman and Cedergren, TIBS 17:34, 1992; Usman, et aL, Nucleic Acids Symp. Ser. 31:163, 1994; Burgin, et al., Biochemistry 35:14090, 1996. Sugar modification of nucleic acid molecules have been 30 extensively described in the art. See Eckstein et al., International Publication No. WO/1 992/07065; Perrault, et al Nature 344:565-568, 1990; Pieken, et al Science 84 253:314-317, 1991; Usman and Cedergren, Trends in Biochem. Sci. 17:334-339, 1992; Usman et al. International Publication No. WO/1993/15187; Sproat, U.S. Patent No. 5,334,711 and Beigelman, et alJ Biol. Chem. 270:25702, 1995; Beigelman, et aL, International Publication No. WO/1 997/26270; Beigelman, et al., U.S. Patent No. 5,716,824; 5 Usman, et al., U.S. Patent No. 5,627,053; Woolf, et al,, International Publication No. WO/1998/13526; Thompson, et al., Karpeisky, et al., Tetrahedron Lett. 39:1131, 1998 Earnshaw and Gait, Biopolymers (Nucleic Acid Sciences) 48:39-55, 1998; Verma and Eckstein, Annu. Rev. Biochem. 67:99-134, 1998; and Burlina, et al., Bioorg. Med. Chem. 5:1999-2010, 1997. Such publications describe general methods and strategies to determine 10 the location of incorporation of sugar, base and/or phosphate modifications and the like inte nucleic acid molecules without modulating catalysis. In view of such teachings, similar modifications can be used as described herein to modify the siNA nucleic acid molecules o the instant invention so long as the ability of siNA to promote RNAi in cells is not significantly inhibited. [5 While chemical modification of oligonucleotide internucleotide linkages with phosphorothioate, phosphorodithioate, and/or 5'-methylphosphonate linkages improves stability, excessive modifications can cause some toxicity or decreased activity. Therefore, when designing nucleic acid molecules, the amount of these internucleotide linkages should be minimized. The reduction in the concentration of these linkages should lower toxicity, 10 resulting in increased efficacy and higher specificity of these molecules. In one embodiment, the invention features modified siNA molecules, with phosphate backbone modifications comprising one or more phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, 25 and/or alkylsilyl, substitutions. For a review of oligonucleotide backbone modifications, see Hunziker and Leumann, Nucleic Acid Analogues: Synthesis and Properties, in Modern Synthetic Methods, VCH 1995, pp. 331-417, and Mesmaeker, et al., "Novel Backbone Replacements for Oligonucleotides, in Carbohydrate Modifications in Antisense Research,' ACS, 1994, pp. 24-39. 30 Methods for the delivery of nucleic acid molecules are described in Akhtar, et al., Trends Cell Bio. 2:139, 1992; "Delivery Strategies for Antisense Oligonucleotide 85 Therapeutics," ed. Akhtar, 1995; Maurer, et al., Mol. Membr. Biol. 16:129-140, 1999; Hofland and Huang, Handb. Exp. Pharmacol. 137:165-192, 1999; and Lee, et al, AC Svn p. Ser. 752:184-192, 2000. Beigelman, et al., U.S. Patent No. 6,395,713, and Sullivan et al., International Publication No. WO/1994/02595 further describe the general methods for 5 delivery of nucleic acid molecules. These protocols can be utilized for the delivery of virtually any nucleic acid molecule. Nucleic acid molecules can be administered to cells by a variety of methods known to those of skill in the art, including, but not restricted to, encapsulation internally or externally by liposomes, by iontophoresis, or by incorporation into other vehicles, such as biodegradable polymers, hydrogels, cyclodextrins (see e.g. 10 Gonzalez, et al, Bioconjugate Chem. 10:1068-1074, 1999; Wang, et al., International Publication Nos. WO/2003/47518 and WO/2003/46185), poly(l actic-co-glycol ic)ac- id (PLGA) and PLCA microspheres (see e.g. U.S. Patent No. 6,447,796 and U.S. Patent Application Publication No. US 2002/130430), biodegradable nanocapsules, and bioadhesi Ve microspheres, or by proteinaceous vectors (O'Hare and Normand, International Publication 15 No. WO/2000/53722). Alternatively, the nucleic acid/vehicle combination is locally delivered by direct injection or by use of an infusion pump. Direct injection of the nucleic acid molecules of the invention, whether subcutaneous, intramuscular, or intradermal, can take place using standard needle and syringe methodologies, or by needle-free technologies such as those described in Conry, et aL, Clin. Cancer Res. 5:2330-2337, 1999, and Barry, et 20 al., International Publication No. WO/1999/31262. The molecules of the instant invention can be used as pharmaceutical agents. Pharmaceutical agents prevent, modulate the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) o'a disease state in a subject. By "RNA" is meant a molecule comprising at least one ribonucleotide residue. By 25 "ribonucleotide" is meant a nucleotide with a hydroxyl group at the 2' position of a P-D-rib furanose moiety. The terms include double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinant produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alteratio is 30 can include addition of non-nucleotide material, such as to the end(s) of the siNA or internally, for example, at one or more nucleotides of the RNA. Nucleotides in the RNA 86 molecules of the instant invention can also comprise non-standard nucleotides, such as nor naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA. By "cap structure" is meant chemical modifications, which have been incorporated at 5 either terminus of the oligonucleotide (see, e.g. Adamic, et al., U.S. Patent No. 5,998,203, incorporated by reference herein). These terminal modifications protect the nucleic acid molecule from exonuclease degradation, and may help in delivery and/or localization with n a cell. The cap may be present at the 5'-terminus (5'-cap) or at the 3'-terminal (3'-cap) or n ay be present on both termini. In non-limiting examples, the 5'-cap includes, but is not limited 10 to, glyceryl, inverted deoxy abasic residue (moiety); 4',5'-methylene nucleotide; I-( p-D erythrofuranosyl) nucleotide, 4'-thio nucleotide; carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; acyclic 3,4 dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3'-3'inverted nucleotide 15 moiety; 3'-3T-inverted abasic moiety; 32-Tinverted nucleotide moiety; 3-2-inverted abasic moiety; 1,4-butanediol phosphate; 3'-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3'-phosphate; 3-phosphorothioate; phosphorodithioate; or bridging or non bridging methylphosphonate moiety. Examples of the 3-cap include, but are not limited to, glyceryl, inverted deoxy aba ic 20 residue (moiety), 4',5'-methylene nucleotide; 1-( P-D-erythrofuranosyl) nucleotide; 4'-thio nucleotide, carbocyclic nucleotide; 5'-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate; 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate; I,5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; threo-pentofuranosyl 25 nucleotide; acyclic 3',4'-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5'-5'-inverted nucleotide moiety; 5'-5'-inverted abasic moiety; 5'-phosphoramidate; 5'-phosphorothioate; 1,4-butanediol phosphate; 5'-amino; bridging and/or non-bridging 5'-phosphoramidate, phosphorothioate and/or phosphorodithioate, bridging or non bridging methylphosphonate and 5-mercapto moieties (for more details see 30 Beaucage and Lyer, Tetrahedron 49:1925, 1993; incorporated by reference herein). 87 By the term "non-nucleotide" is meant any group or compound which can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, includin either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity. The group or compound is abasic in that it does not contain a common] v 5 recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine and therefore lacks a base at the l'-position. By "nucleotide" as used herein is as recognized in the art to include natural bases (standard), and modified bases well known in the art. Such bases are generally located at th: ' position of a nucleotide sugar moiety. Nucleotides generally comprise a base, sugar and t 10 phosphate group. The nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and other; see, e.g. Usman and McSwiggen, supra; Eckstein, et al., International Publication No. WO/1 992/07065; Usman, et al, International Publication No. WO /1993/15187; Uhlman & Peyman, supra, al [5 are hereby incorporated by reference herein). There are several examples of modified nucleic acid bases known in the art as summarized by Limbach, et al., Nucleic Acids Res. 22:2183, 1994. Some of the non-limiting examples of base modifications that can be introduced into nucleic acid molecules include, inosine, purine, pyridin-4-one, pyridin-2-on , phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, 0 aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g. 6 methyluridine), propyne, and others (Burgin, et al., Biochemistry 35:14090, 1996; Uhlman k Peyman, supra). By "modified bases" in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at 1' position or their equivalents. 25 By "target site" or "target sequence" or "targeted sequence" is meant a sequence within a target nucleic acid (e.g., RNA) that is "targeted" for cleavage mediated by a siNA construct which contains sequences within its antisense region that are complementary to t e target sequence. The siNA molecules can be complexed with cationic lipids, packaged within 30 liposomes, or otherwise delivered to target cells or tissues. The nucleic acid or nucleic acic complexes can be locally administered to through injection, infusion pump or stent, with or 88 without their incorporation in biopolymers. In another embodiment, polyethylene glycol (PEG) can be covalently attached to siNA compounds of the present invention, to the polypeptide, or both. The attached PEG can be any molecular weight, preferably from about t 2,000 to about 50,000 daltons (Da). 5 The sense region can be connected to the antisense region via a linker molecule, suc as a polynucleotide linker or a non-nucleotide linker. "Inverted repeat" refers to a nucleic acid sequence comprising a sense and an antisense element positioned so that they are able to form a double stranded siRNA when th3 repeat is transcribed. The inverted repeat may optionally include a linker or a heterologous 0 sequence such as a self-cleaving ribozyme between the two elements of the repeat. The elements of the inverted repeat have a length sufficient to form a double stranded RNA. Typically, each element of the inverted repeat is about 15 to about 100 nucleotides in length preferably about 20-30 base nucleotides, preferably about 20-25 nucleotides in length, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. 5 "Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers there f in single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference .0 nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O-methyl ribonucleotides, peptide-nucleic acids (PNAs). "Large double-stranded RNA" refers to any double-stranded RNA having a size greater than about 40 bp for example, larger than 100 bp or more particularly larger than 25 300 bp. The sequence of a large dsRNA may represent a segment of a mRNA or the entire mRNA. The maximum size of the large dsRNA is not limited herein. The double-stranded RNA may include modified bases where the modification may be to the phosphate sugar backbone or to the nucleoside. Such modifications may include a nitrogen or sulfur heteroatom or any other modification known in the art. 89 The double-stranded structure may be formed by self-complementary RNA strand such as occurs for a hairpin or a micro RNA or by annealing of two distinct complementary RNA strands. "Overlapping" refers to when two RNA fragments have sequences which overlap b) a 5 plurality of nucleotides on one strand, for example, where the plurality of nucleotides (nt) numbers as few as 2-5 nucleotides or by 5-10 nucleotides or more. "One or more dsRNAs" refers to dsRNAs that differ from each other on the basis of primary sequence. "Target gene or mRNA" refers to any gene or mRNA of interest. Indeed any of the 0 genes previously identified by genetics or by sequencing may represent a target. Target genes or mRNA may include developmental genes and regulatory genes as well as metabol e or structural genes or genes encoding enzymes. The target gene may be expressed in those cells in which a phenotype is being investigated or in an organism in a manner that directly or indirectly impacts a phenotypic characteristic. The target gene may be endogenous or t 5 exogenous. Such cells include any cell in the body of an adult or embryonic animal or plan including gamete or any isolated cell such as occurs in an immortal cell line or primary cell culture. All publications, references, patents, patent publications and patent applications cite herein are each hereby specifically incorporated by reference in entirety. !0 While this invention has been described in relation to certain embodiments, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that this invention includes additional embodiments, and that some of the details described herein may be varied considerably without departing from this invention. This invention includes such additional embodiments, modifications and equivalents. In 25 particular, this invention includes any combination of the features, terms, or elements of the various illustrative components and examples. The use herein of the terms "a," "an," "the" and similar terms in describing the invention, and in the claims, are to be construed to include both the singular and the plural. The terms "comprising," "having," "including" and "containing" are to be construed as ope 30 ended terms which mean, for example, "including, but not limited to." Recitation of a rang: of values herein refers individually to each and any separate value falling within the range as 90 if it were individually recited herein, whether or not some of the values within the range ar expressly recited. For example, the range "4 to 12" includes without limitation the values !, 5.1, 5.35 and any other whole, integer, fractional, or rational value greater than or equal to 4 and less than or equal to 12. Specific values employed herein will be understood as 5 exemplary and not to limit the scope of the invention. Definitions of technical terms provided herein should be construed to include with ut recitation those meanings associated with these terms known to those skilled in the art, anc are not intended to limit the scope of the invention. Definitions of technical terms provide J herein shall be construed to dominate over alternative definitions in the art or definitions 10 which become incorporated herein by reference to the extent that the alternative definition conflict with the definition provided herein. The examples given herein, and the exemplary language used herein are solely for the purpose of illustration, and are not intended to limit the scope of the invention. When a list of examples is given, such as a list of compounds or molecules suitable: 15 for this invention, it will be apparent to those skilled in the art that mixtures of the listed compounds or molecules are also suitable. 91 EXAMPLE 1 In Vitro Assay for LacZ Gene Expression Knockdown in 9L Cells 9L/LacZ is a rat gliosarcoma cell line stably expressing the LacZ gene that encodes bacterial galactosidase. LacZ gene knockdown measurements can be used as a primary 5 activity-based in vitro assay for interfering RNA delivery formulations. For LacZ gene knockdown measurements, 9L/LacZ cells were transfected with an RNAi formulation, and a -galactosidase assay was performed on cells harvested at day 3 post transfection. An additional assay was performed to quantify protein concentration. 9L/LacZ cells were plated at 8000 cells/well (96-well) and incubated overnight in .0 medium. Confluency was about 15-20% at the time of transfection. Transfection complex was prepared by adding an interfering RNA to OptiMEMTM medium and vortexing, separately adding a delivery formulation to OptiMEMTM medium and vortexing, and finally mixing the interfering RNA in medium with the delivery formulation in medium to make th transfection complex. The medium for incubated cells was replaced with fresh no-serum 5 media (OptiMEMTM without serum) and transfection complex was added to each well. Cells were incubated 5 hrs, then after the addition of 100 microliters complete medium (DMEM plus 10% fetal bovine serum) were incubated overnight at 37'C and 5% CO 2 . The next day 24 hours after transfection, the medium was changed to fresh complete medium and the cell were incubated another 48 hrs at 37*C and 5% CO 2 . 0 For LacZ gene knockdown, the harvested 9L/LacZ cells were washed in PBS, lysed in M-PERT m Reagent (Pierce), and incubated at room temperature for 15 minutes. Lysate wis taken from each well for protein assay with a Micro BCA kit (Pierce, Thermo Fisher Scientific) and O-gal assay with All-in-OneTM P-Galactosidase Assay Reagent (Pierce). 25 In Vitro Assay for PPIB Gene Expression Knockdown in A549 cells Cyclophilin B (PPIB) gene knockdown measurements can be used as a primary activity-based in vitro assay for interfering RNA delivery formulations. Cyclophilin B (PPIB) gene expression knockdown was measured in A549 human alveolar basal epithelial cells. For PPIB gene knockdown measurements, A549 cells were transfected with an 0 interfering RNA formulation, total RNA prepared 24 hours after transfection, and PPIB 92 mRNA assayed by RT-PCR. QRT-PCR of 36B4 (acidic ribosomal phosphoprotein PO) mRNA expression was performed for normalization. A549 cells were seeded at 7,500 cells/well (96-well) and incubated overnight in medium. Confluency was about 50% at the time of transfection. Transfection complex was 5 prepared by adding an interfering RNA to medium (OptiMEMTM) and vortexing, separately adding a delivery formulation to medium (OptiMEMTM) and vortexing, and finally mixing the interfering RNA in medium with the delivery formulation in medium and incubating 20 minutes at room temperature to make the transfection complex. The medium for incubated cells was replaced with fresh OptiMEMTM and transfection complex was added to each well 0 Cells were incubated for 5 hrs at 37*C and 5% CO 2 , then complete medium was added (to a final fetal bovine serum concentration 10%) and incubation continued until 24 hours post transfection. For PPIB gene knockdown cells were lysed and RNA prepared (Invisorb RNA Cell HTS 96-Kit/C, Invitek, Berlin, or RNeasy 96 Kit, Qiagen). Quantitative RT-PCR was 5 performed using One-Step qRT-PCR kit (Invitrogen) on a DNA Engine Opticon2 thermal cycler (BioRad). Primers used for PPIB were: (SEQ ID NO: 323) 5'-GGCTCCCAGTTCTTCATCAC-3' (forward) and 0 (SEQ ID NO: 324) 5'-CCTTCCGCACCACCTC-3' (reverse) with (SEQ ID NO: 325) 5'-FAM-CTAGATGGCAAGCATGTGGTGTTTGG-TAMRA-3' for the probe. 25 For 36B4, primers were: (SEQ ID NO: 326) 5'-TCTATCATCAACGGGTACAAACGA-3' (forward) and (SEQ ID NO: 327) 5'-CTTTTCAGCAAGTGGGAAGGTG-3' (reverse) with 30 (SEQ ID NO: 328) 5'-FAM-CCTGGCCTTGTCTGTGGAGACGGATTA-TAMRA-3' for the probe. 93 In Vivo Assay for Influenza Viral Titer Knockdown in Mouse Influenza viral titer knockdown measurements in mice can be used as an in vivo gauge of efficacy for interfering RNA lipopeptide delivery formulations. In this assay, typically 50 uL of a interfering RNA lipopeptide formulation, or PBS 5 for a control group, was administered intranasally in 7-9 week old Balb/C mice anesthetized with ketamine/xylazine. Daily dosing was performed for 3 consecutive days on days -2, -1, and 0. Infection was induced 4 hours after the last dosing, Influenza infection was induced with Influenza A/Puerto Rico/8/34 (PR8, subtype HINI). For infection, 50 p.l of 20 pfu PR8 diluted in 0.3%BSA/lXPBS/PS was administered 0 intranasally into mice anesthetized with ketamine/xylazine. 48 hours after infection, the lungs were harvested and homogenized in 600 uL 0.3%BSA/IXPBS/PS. The homogenates were frozen and thawed twice to release the virus. A TCID50 assay (Tissue-Culture Infectious Dose 50) was performed to titer virus in lung homogenates. Flat-bottom, 96-wel plates were seeded with 2x10 4 MDCK cells per well, and 24 hours later, the serum 5 containing medium was removed. 30 uL of lung homogenates, either undiluted or diluted from 10- to 10 7 -fold (in 10-fold steps), was added into quadruplicate wells. After incubation for I hr, 170 i1 of infection medium (DMEM/0.3%BSA/10 mM HEPES/PS) containing 4 j.g/ml trypsin was added to each well. After incubation for 48 hours at 37*C, the presence or absence of virus in the culture supernatants was determined by hemagglutination of chicken ,0 red blood cells. The virus titers were estimated using the Spearman and Karber formula. SYBRTM Gold Assay for siRNA Concentrations The concentration of dsRNA in a formulation can be determined by SYBRTM Gold assay as follows: 10 ul of dsRNA formulation is added to 100 ul MeOH and incubated for 5 minutes at 550C. 50 ul Heparin (200mg/ml) is added, and the solution is incubated for 25 5 minutes at 55*C. 790 ul PBS (phosphate buffered saline) is added, and the sample is spur down in microfuge to pelletize. A 90 ul sample of the pellet is incubated with 10 ul SYBR'M Gold reagent (Molecular Probes, Eugene, Oregon). Fluorescence is read at Em 535 nm with excitation at 495 nm. 94 EXAMPLE 2 The structure of some double-stranded RNAs of this disclosure are shown in Table Table 6 Double-stranded RNAs (SEQ ID NO:329) DX3030 Sense 5'-GGAUCUUAUUUCUUCGGAGACAAdTdG-3' Influenza (SEQ ID NO:330) Antisense 5'-CAUUGUCUCCGAAGAAAUAAGAUCCUU-3' DX2816 (SEQ ID NO:331) Non-target Sense 5'-UUCUCCGAACGUGUCACGUdTdT-3' Qneg (SEQ ID NO:332) Qneg _ Antisense 5'-ACGUGACACGUUCGGAGAAdTdT-3' (SEQ ID NO:333) DX2940 Sense 5'-CUACACAAAUCAGCGAUUUdTdT-3' LacZ (SEQ ID NO:334) Antisense 5-AAAUCGCUGAUUUJGUGUAGdTdC-3' DX2742 (SEQ ID NO:335) PPIB Sense 5'-GGAAAGACUGUUCCAAAAAUU-3' MoCypB (SEQ ID NO:336) Antisense 5'-UUUUUGGAACAGUCUUUCCUU-3' DX 2744 (SEQ ID NO:337) G1498 Sense 5'-GGAUCUUAUUUCUUCGGAGdTdT-3' influenza (SEQ ID NO:338) Antisense 5'-CUCCGAAGAAAUAAGAUCCdTdT-3' DX 2918 (SEQ ID NO:339) Inm4 Sense 5'-CCGTCAGCCGATTTGCTATTT-3 TNFa (SEQ ID NO:340) modified Antisense 5'-p-AUAGCAAATCGGCTGACGGTT-3 5 EXAMPLE 3 Active RNA formulations of this disclosure can be prepared by dissolving an interfering RNA in buffer or cell culture medium and vortexing, separately admixing a delivery formulation with buffer or cell culture medium and vortexing, and finally admixin 10 the interfering RNA mixture with the delivery formulation mixture to make an active RNAi transfection formulation. To prepare a delivery formulation, lipopeptides along with other lipids and/or excipients can be solubilized in CHCl 3 /MeOH, dried down under N 2 , and hydrated in 10 mA HEPES with 5 % dextrose at pH 7.4. The mixture can be sonicated, or extruded, dialyzed, 15 and/or tangential flow filtered, 95 An exemplary interfering RNA formulation of this disclosure is shown in Table 7. I this example, the lipopeptide provides its own formulation for intracellular delivery of an interfering siRNA therapeutic. The amount of lipopeptide is given as the mole percentage of delivery components, not including the active RNA agent. 5 Table 7 siRNA Formulation Component Amount siRNA 50 nM lipopeptide 100 mole % An exemplary interfering RNA formulation of this disclosure is shown in Table 8. 1 this example, the lipopeptide provides its own formulation for intracellular delivery of an 0 interfering siRNA therapeutic. The amount of lipopeptide is given as the mole percentage of delivery components, not including the active RNA agent. The length of the lipophilic group for each peptide on the proceeding tables is represented by "(CX)" where "X" represents the number of carbons in the lipophilic group. For example, C20 represents a lipophilic group with a 20 carbon chain length. 5 Table 8 dsRNA Formulation Component Amount dsRNA 50 nM (C16)-(H)8(R)g(H)sK-NH-(C16) (SEQ ID NO: 13) 100 mole % An exemplary RNAi formulation of this disclosure is shown in Table 9. In this example, the lipopeptide is combined with a non-cationic lipid in a co-delivery formulation 20 Table 9 DX3030 Formulation Component Amount siRNA DX3030 50 nM (C20)-(H)(R)s(H) 8 K-NH-(C20) (SEQ ID NO: 13) 50 mole % 1, 2 -Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) 50 mole % 96 An exemplary RNAi formulation of this disclosure is shown in Table 10. In this example, a lipopeptide is combined with a cationic lipid, a non-cationic lipid, and a pegylat ed lipid in a multicomponent delivery formulation. 5 Table 10 dsRNA Formulation Component Amount dsRNA 25 nM (C20)-(H)g(R)s(H)sK-NH-(C20) 50 mole % (SEQ ID NO: 13) DSPC 49 mole % DSPE-PEG2000 I mole % Examples of additional lipids for RNAi formulations of this disclosure are shown in Table 11. 10 Table 11 Lipids for Delivery Formulations Material MW DOTAP Avanti 890890C 698.55 DOPE Avanti 850725C 744.04 DPhyPE Avanti 850402P 804.18 EXAMPLE 4 The knockdown activities of example formulations of interfering RNA compositic ns 15 of this disclosure are shown in Table 12. Table 12 Knockdown for Delivery Formulations NT SEQ % KD (vs % D Ratio ID Lipopeptide QNeg) (vs Neg) NO: A549/PPIB 9L/ acZ 2 -- RNAiMAXTm 73.4 8 .4 2 350 (C18)-HHHHRRRRRRRR-Cysteamide 15.3 - .8 2 351 (Cl18)-PEG27-HHHHHHHHRRRRRRRR-amide 30.6 C .2 2 352 Ac-GALFLAFLAAALSLMGLWSQPKKKRKV-Cysteamide 20.5 2 .1 2 353 Ac-GALFLAFLAAALSLMGLWSQPKSKRKV-Cysteamide 15.5 2 .5 97 N: SEQ % KD (vs %KD Ratio ID Lipopeptide QNeg) (vsQNe ) NO: A549/PPIB 9L/lac 2 296 Ac-LTRLWSHLIHIWFQNRRLKWKKK-amide 8.7 -19.9 2 13 (C20)-(H) 8

(R)

8 (H),K-NH-(C20) 29.7 60.1 2 354 (C16)-HHHHHKHHHKKKHKHKKK-Cysteamide 14.7 12.0 2 355 (C18)-HHHHHKHHTKKKHKHKKK-cysteamide 25.5 -11.3 2 356 Ac-GALFLGFLGAAG STMGAWSQPKSKRKV-amide -8.1 -0.4 4 10 (CI8)-PEG27-HHHHHHHRRRRRRRR-amide 7.4 -16.1 4 353 Ac-GALFLAFLAAALSLMGLWSQPKSKRKV-Cysteamide 16.0 -9.3 4 296 Ac-LIRLWSHLIHIWFQNRRLKWKKK-amide -28.8 -4.5 4 13 (C20)-(H)s(R)s(H)gK-NH-(C20) 65,8 57.5 4 354 (C16)-HHHHHKHHHKKKHKHKKK.Cysteanide -19.8 12.4 4 355 (C18)-HHHHHKHHHKKKHKHKKK-cysteamide 21.6 -19.4 4 356 Ac-GALFLGFLGAAG STMGAWSQPKSKRKV-amide -3.7 -1.6 For the lipopeptides in Table 12, the results showed that at least two lipopeptides, (C20)-(H)s(R)8(H) 8 K-NH-(C20) (SEQ ID NO: 13) and (C I 8)-HHHHHKHHHKKKHKHKKK-cysteamide (SEQ ID NO: 355), exhibited significan 5 knockdown in at least the A549/PPIB assay as compared to both Qneg and vehicle (data for vehicle not shown). 98

Claims (10)

1.I Thenropound claim n Comprising the suncure shownin Formul I: R Ni-3-RONR orma I $ whereto R is independently, for each occurrence, a non-hydrogen side chain of an amino acid; (NRKCIURK(C-0)}Z is a central peptide; R2 and RN are independently of one another hydrogen, or an organic group consisting 10 of carbon, oxygen, nitrogen, sulfur, and hydrogen atoms, and having from 1 to 20 carbon atoms, or 0(1-22)alkyt, (6-12)cycloalkyL (6-12)cycioalkyialkyi, C(3-18)alkenyl, C(3-18)alkyny1, 0(1-5)alkanoyl, (1 5)alkanoyloxy, 0(1-5)aikoxy, C(1-5)alkoxy-C(1-5)alkyl, C(1-5) alkoxy-C( 1-5)alkoxy, C(I -5)aikyl-amiino-C0(1-5 )alkyi-, C(1 S5 )dialkyl-amino-C(I~5)alkyl-, nitro-C(1-$)alkl, cyano-C(I-5)alkyl, aryl-C(1-5)alkyi, 4-biphenylC(I-5)alkyl carboxyl, or hydroxyl; RI and R 4 arc independently of one another,a lipophilic tail derived front a naturally occurring or synthetic lipid, phospholipid, glycolipid, triacylglyccrol glycerophospholipid, sphingolipid, ceramide, sphingomyein, cerebroside, or 20 ganglioside, wherein the tail may contain a steroid, or an organic group consisting of carbon, oxygen, nitrogen, sulfur, and hydrogen atoms, and having from Ito 20 carbon atoms, or a substituted or unsubstituted (1 -22)alky., C(6-12)ycloalkyt (6-2)ycloalkykalkyl, C(3-18)alkenyl. C(3~18)alkynyi, 0(1 5)alkoxy-C(1 5 )alky or a sphinganine, (2R,31R)-2-amino-I,3-octadecanedio icosasphiuganine 25 Sphingosine, phytosphingosinc, cis-4-sphingenine, or a ceramide; Z is NI-I 0, or a linker comprising a mialeirmido, thiocther, amide, cysteamnide, cysteine, thiol, or a disulfide group, or a polyethyleneoxide or polypropylencoxide group comprising 1-400 atoms, or a linker comprising 1-200 atoms selected fron the group of C, Q , F, CA1 Br, N, 0, 5, Si, and P; i or a inker comprsing a nakddo thnerarnde cye de 4 cvstee, duel or iuiddagroup, or a poiyehyienexde o polypropyleneoxie group comprising 00 atoms, or a inker camping 100 os isdeyd fom the group of C. H, W PI M S Si and P an 5 where na2 t o 10-0.

2. The compound of claim 1, wherein the amino acid sequence of the central peptide is selected from the group consisting of SEQ ID NOs: 1-300 and 350-356.

3. The compound of claim , wherein the amino acid sequence of the central 10 peptide is selected from the group consisting of GALFL AFLAAALXILMGLWXaQXaKKKRKV (SEQ ID NO: 291) WSQPKKKR RK (SEQ ID NO: 292, WWHHKKRRCCRRKKHHWW (SEQ ID NO:94), XL.WSQPKKKRKVX$ (SEQ ID NO0:293), WXeQPKKKRKX, (SEQ ID NO:294) XPXN'iQPKKKRKV(SEQ ID NO:295), LRLWXAHLIHIWFQXfRRLKWKKK (SEQ ID 15 NO: 297), QNRRLKWKKK (SEQ ID NO:298), XaQNRRLKWKKXJSEQ ID NO:299), and QXMRRIKWKK.K (SEQ 1D NO3 00), where X, is indepedenly, for each occurance, L, A, Q, Q, H, , or K., 4, The compound of claim 1, wherein a side chain is linked to the central peptide, wherein the side chain contains a releasing functional group selected from 3,5 20 diiodo-tyrosine, 1-methyhistidine, 2-methylbutanoiacicid, 2d-,anisylpropa noic acid, meso tartaric acid, 4,6-dimethylpyrimidinarmine, p-phthalic acid, creairnine, butano acid. NN dimethyl-1I-naphthlaminc, pentanoic acid, 4-methy lperntanoic acid, N-methylaniline, 1,10~ phenanthrol ine, 3-pyri dinecarboxyli c acid, hexanoice acid, propanoic acid, 4-anirnohenzo ic acid, 2-mnethyiproanoie acid, heptanoic acid, netano ic acid, cyclohexaniecarboxyl ic acid, 25quinoline, 3-quinolinamnine, 2-aminobenzaoic acid, 4-pyridinecarboxylic acid, nonanic acid, mol amine, 8quinoiinol, trimethylacetic acid, 6-methoxyquinoline, 4-(methylamino)benzoic acid, pjmethylaniline, 3-(methyiamino)benzoic acid, malie acid, -ethylaniline, 2 benzylpyridine. 3,6-dinitrophenol, N,N-dimecthylaniline, 2,S-dimethylpiperazine, p pheneti dine, 5 -methykquinioline 2-phenyl benzimi dazote, pyri dine, picolini c ac id, 3,5- diiodityrosinc, p-anisidine, 2(m ethylamino)benzcic acid, 2-thiazolNmine, glutaric acid, adipic acid, isoquinoline, itaconic acid, o-phthaiic acid, benimidazole, piperazine, heptanedioic acid, acridine, phenanthridine, succinic acid, rnethylsuccinic acid, 4 moethylquinoline, 3-methylpyridine, 7-soquino inol, masonic acid, methymalonic acid, 2 n riethylquinoline, 2-tiylpyridine, 2-rnethylpyrdine, 4-methylpyridine, histamine, histidine maleic acid, is-,2~cyclohexanediamine, 3,5-dimethylpyridine, 2-ethylbenzimidazole, 2-methylbe nzimidazole, cacodylic acid, perimidine, citric acid, isocitric acid, 2,5 -dimethylpyridine, papaverine, 6-hydroxy-4-methypteri dine, L-thyroxinc, 3,4-dimethylpyridinc, methoxypyridine, trans- ,2-cylohcxanedi amine, 2,5-pyridincdiamine, 10 ( 1 methylhistidine, -methyihistidine, 2 ,3-dimethylpyridine, xanthoptern 2 propanediamine, NN-diethylaniline. ailoxanic acid, 2,6-dimethylpyridine, L-canosince, 2 pyridinamine, N-b--aianylhistidine, plocarpine, 1 -amethylir idazol, i H-imidazolo, 24~ dimothylpyridire, 4-nitrophenol, 2-nitropheno!, tyrosineanide, 5-hydoxxyquinazoline, cyclopropancdicarboxyl ic acid, 2,4,6-trimtedhypyridine, veronal, 2,3-dichioropheno 1, 1,2 5 ethanediamine. 1-isoquinoinamine, and combinations thereof. A pharmace composition omnipLng an neng RNA agentand on rme compounds of claim I orha u ticat a pa ltshheof 7 The cpoiinof clair 5, weenteitreigRAaeti ntdt 20 8' A pharmaceutical composition comprising an interfering RNA agent, one or more compounds of claim I or pbarmaceuticaily-acceptable thereof, one or more non~ cationic Uipids, and a polymeric lipid, . hueomposiin oAim 8, whereirahheinteren RNA agentiunN

10. The compositon of cNai 8. wherein teitreigRAaeii nmRA 25 11. The composition of claim 8, wherein the one or more compounds is (C20)~(H3(RtIHsKNH-(C20) (SEQ ID NO 13) I to-

12. The composition of claim 8, wherein the non-cationic lipid is selected from DDPE. DULPE SPI, DOPE, DinPE, DLeniPE, DARAPE, DDHAPE, DPhPE, DSP, DPPC, DDPC DP'S, DSPS, cholesterol, lanosterol, stigmastanol, dihydrolanosterol, zymosterol, zyrmostenol, desmosterol, 7-dehydrocholesterol, pegylated cholesterol, S cholesterol acetate, cholesteryl arachidonate, cholesterol butyrate, cholesterol hexanoate, cholesteryi caprylate, cholesteryl n-decanoate, cholesteryl dodecanoate, choiesteryl myristate, eholesteryl pal mitate, eholesteryl behenate, eholesteryl stearate, cholesterol nervonate, cholesteryl pelargonate, choiesteryi n-vaierate, eholestoryl sleate, cholesteryl el aidate, e holesteryl erucate, cholesteryl heptanoate, cholestery!I linolelaidate, cho lesteryl 10 linolcare, and mixtures and derivatives thereof,

13. The compositon of claim 8, wherein the polymeric lipid is selected from DMP.E-MPEG0-2000 DMPEMPEG-5000, DPPE-MPEG~2000, DPPW MPE-$S000, DSPE-MPEG-750, DSPE~M.PEG-2000, DSPE-MPEG-5000, sodium cholesteryl sulfate, pegylated cholesterol, pharmaceutically acceptable salts thereof, and mixtures thereof,

14. A method for delivering an interfering PNTA agent to a cell comprising preparng a composition according to claim 8 and treating a cell with the composition

15. A method for inhibiting expression of a gene in a cdl comprising preparing a composition according to claim 8 and treating a cell with the composition.

16. A method for inhibiting expression of a gene in a mammal comprise ng preparing a composition according to claim 8 and administering the composition to the . A se of a composting ofcim f he manfactu of a medicament i the atment ofa dieae disorder condin

18. The use of claim 17, wherein the disease or condition is cancer, a metabolic 25 disease or disorder, or inflammatory disease, 9 A use of a pot o m 8 for the ma"noutt oa medicamenit for the teatmem o a viral infection 2015202154 27 Apr 2015 2015202154 27 Apr 2015 2015202154 29 Apr 2015 2015202154 27 Apr 2015 2015202154 27 Apr 2015 2015202154 29 Apr 2015 2015202154 27 Apr 2015 2015202154 27 Apr 2015 2015202154 29 Apr 2015 2015202154 27 Apr 2015 2015202154 27 Apr 2015 2015202154 29 Apr 2015 2015202154 27 Apr 2015 2015202154 27 Apr 2015 2015202154 29 Apr 2015 2015202154 27 Apr 2015 2015202154 27 Apr 2015 2015202154 29 Apr 2015 2015202154 27 Apr 2015 2015202154 27 Apr 2015 2015202154 29 Apr 2015 2015202154 27 Apr 2015 2015202154 27 Apr 2015 2015202154 29 Apr 2015 2015202154 27 Apr 2015 2015202154 27 Apr 2015 2015202154 29 Apr 2015 2015202154 27 Apr 2015 2015202154 27 Apr 2015 2015202154 29 Apr 2015 2015202154 27 Apr 2015 2015202154 27 Apr 2015 2015202154 29 Apr 2015 2015202154 27 Apr 2015 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