AU2022203475A1 - Lipid containing formulations - Google Patents

Abstract

Compositions and methods useful in administering nucleic acid based therapies, for example association complexes s such as liposomes and lipoplexes are described.

Description

Lipid containing fonnulations

TECHNICAL FIELD This invention relates to compositions and methods useful in administering aucleic acid based therapies, for example association complexes such as liposomes and lipoplexes. The present application is a divisional of Australian Patent Application No. 2020202816, the entirety of which is incorporated herein by reference. BACKGROUND

The opportunity to use nucleic acid based therapies holds significant promise, providing solutions to medical problems that could not be addressed with current, traditional medicines. The location and sequences of an increasing number of disease related genes are being identified, and clinical testing of nucleic acid-based therapeutic i for a variety of diseases is now underway. One method of introducingnucleicacidsinto a cell is mechanically, using direct microinjection. However this method is not generally effective forSystetic administration to asubject. Systemic delivery of a nucleic acid therapeutic requires distributing nucleic ais 5 to target cells and then transfrringthe nucleic acid across a target cell membrane intac and in form that can function in a therapeutic manner. Vialvectors have,in some instances, been used clinically successfully to administer nucleic acid based therapies. However, while viral vectors have theinheer ability to transport nucleic acids across cell membranes, they can pose risks. One such risk involves the random integration of viral genetic sequences into patient chromsomes, potentially damaging the genome and possiblyinducing a malignant transformation, Another risk is thatthe viral vector may revert to a pathogenicgenotyp either through mutation or genetic exchange with a wild type virus. Lipidbased vectorshave also been used innucleic acid therapies and have bee formulated in one of two waysInone method, thenucleic acid is introduced into preformed hiposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids,.The complexes thus formed have undefined and complicated structures and the transfectonefcfiiency is severely reduced by the presence of serum, The second meti involves the fonnation of DNA complexes with mono- orpoly-catoniclipidswithout the presence of a neutral lipid.These complexes are prepared in thepresence ofetano and are notstable in water. Additionally these complexes are adversely affected by serum (se, Ber, Acc. Chem, Res 26:274-8 (1993)).

SUMMARY The invention features novel preparations that include a polyamine compound or a lipid moiety described herein, in some embodiments, the invention features a preparation comprisng one or mOe compounds, each individually having astructure defined by fomula (f) or a pharmaceutically acceptable salt thereof

X"X R2 NIXN 'NR R n

formula (1) wherein each X' and X, for each occurrence, is independently Cj alkylene; n is 0, 1, 2, 3, 4, or 5 each R is independently H, o ,R R RI of R R-y oof b R1 Y' Y tVXY Y'

R, Ri R Rd R, wherein at least n + 2 of the R moieties in at least about 50% ofthe molecules of the compound of formula (I) in the preparation (eg, at least about 55%,at least about 60%, at least about 65%, at least. about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or substantially all) aren't; m is 1, 2 3 or 4; Y is 0, NRtor S; RIis alkyl alkenyl oralkynyl; each of which is optionallysubstituted 2$ with one or more substituents; and RisH,aikyl alkenyl or alkynyl; each ofwhich is optionally substituted each of which is optionally substituted with one or more substituents; provided that, if n =0, then at least n + 3 ofthe R moieties are not H,

In some embodiments, when R is not H, R is 1,, for example, when R is not H, R is R, foreach occurrence. In some embodiments, when R is not H, R is Rb for example, when R is not H, R is Rb, foreach occurrence. SIsome embodiments, when R is not H, R is R,, fr example, when R isnotH, R is R, for each occurrence. Insome embodiments,whenis not H, is , for example, when R is not H

R is 1 , for each occurrence. In some embodiments, when R is not H, R is R, for example, when R is not H, R is R, for each occurrence, In some embodiments, n + 2 of the R moieties offormula (I) arenot H. In some embodiments,A 3 of the R moieties offomula (I) are not H. in some embodiments, n + 4 ofthe Rmoieties offornula (I) are not H. In some embodiments, n+ 1 of the R moieties of formula (I) are not H 'In some embodiments,n>0, and at least one R ofNR of frnla (I) is H. In some embodiments, at least one R of NR- of formula (I) isH In someembodiments, at least 80% of the molecules are a single structural isomer. For exampe.n +2 of the.R moieties of fomula (I) are not H,or n + 3 of the R moieties of fbrn'ua (1) are not H, or n + 4 of the Rmoieties of formula (1) are notH. in some embodiments, n is 2 or 0. In some embodiments, X and X' are C2 alkylenc. In some embodiments, n.is 0and isethylene orpropylene. In some embodiments, n>1 and X' varies with at least one occurrence, Income embodiments, whennot H, R is

k R~ ' " . For example, Y can be 0 or N Insomeembodimentsmis 2. In some mbodinents, Yis Q or N.2 and mis 2. In some embodiments, m is L In someeiimbodiements, mis I and Y is 0 orNR In some embodiments, R for at least one occurrence is alkyl, for example, R for each occurrence is alkyl insomeembodiments, Ris alkyl and is H, for at least one occurrence, eg, for each occurrence.

In some embodiments, RI and R2 are alkyl for at leastoneoccurrence,egfor each occurrence. Insoe iembodiments, R for at least one occurrence is alkenYt In someerbodiments, R' for at least one occurrence is alkeny. a In some embodiments, when R is not H,R isRa, for at least oneoccurrence, eg., for each occurrenceandYisorNH.In some embodiments, Y is 0, In some embodiments, Y is NHi In some embodients, R is alkyl, eg., Cm-alkyl or C2 alkyl In some embodiments, n is 2 In some embodiments, Xfor each occurrence is C 2 alkylene and Xis C-, akylene, In some embodiments, m is 1 In sone embodiments, n is 2 and R, when R is not H, is R1, for at least one occurrence, e.g, for each occurrence. In some embodiments,P isky e,g, Ces alkvl orC ,askyl.insomeembodiments, YisOorNYisNiinsomeembodiments X', for eachoccurrence is C2alkyleneand Xb is 2 alkylene. insome embodiments, I is 2. a In some embodiments, at least I R of NR is H and Rxhennot H is R for at least one occurrnce, e.g. for each occurrence, and Y is 0 or NH. In some embodiments, Y is 0 or Y is NHIn some embodiments, R' is alkyl, egC salkyl or Ca alkyl. In

some embodimentsnis2.Insonicntsodiments,X for ah occurrence is C2 alkyleneand X'isC2aik'yieneIn sonic embodiments m is 2 tnsome embodiments, n is 2 and at least I R of NR is H and when R is not H, R isPRfor at least one occurrence, e.g. for each occurrence, and Y is . or NH. In some embodiments, is alkyl, e g, CO alkyl or C, alkyL In soembodiments, is 0

or Y is NH. Insone embodiments, XV, for each occurrence is C, akylneandXis C alkylne In someeimbodiments, m is 2. In some embodiments, at least I R of NR is H and R is Ra, for at least one

occurrence, e.g. for each occurrence and wherein Y is 0 or N. Insome embodiments. Y is 0 or Yis NH. In some embodiments, R is alkyl, eg,(C o i alkyl, akyl or C ikyl.In some embodiments, nis 2.insome embodiments.X foreach occurrence is C alkylene and X is C2 alkylene. Insome embodiments, m is2, 3o In some embodiments,n is 2 and at least I R ofNR2 is H and R is R for at least one occurrence,eg. for eachoccurrence,and whereinY is 0 orNH, insome embodiments,R is alkyl, e.g,0C0.1alkyl orC,2alkyl in sonicembodiments, Yis0O or Y is NH. in some embodiments, X" for each occurrence is C2 alkylene and X is C2 alkylene, insome enibodiments, m is 2. In some embodiments, the preparation comprises one or a mixture of the formula below, whereinRis riot H unless specified in thelbrmula below. R R H H R N,,-- N IN' and R ^R 5 RRK R

, in some embodiments, thei preparation consists sscndially of one or a mixture of the fonnula below R R H H R N N ,R and R'N.,--N'xN '-N'NR R R RR ,n soni embodiments, each R is

R1 0

R2 . some embodiments, each R is hisome enibodiments, R is CqCr alkyl (eg, C alky), or Co-C3o alkenyt In some embodiments, R is

SN' R2 , Insonie embodiments, R' is Q-Cj alkyl, e.g, C.,a kyl, in someembOdiments, is C2 alkyl and R2 is H. 1 In some embodiments, n is 0 and X.is propylene, in some embodiments, I R is H. In some embodiments, when R isnot H, R is Rj, forat least one occurrence, e,g. br each occurrence, In some embodiments, R is alkyl, e.g, .0.3 alkyl or C alkyl. in sonic mbodiments, Y is O or Y is NH. In some embodiments, m is 2 In some embodinents, formula (1) is 0 R ,' '' RN' or R R In some embodiments, R is R, In some embodientsRI is Cw.- 0 alkx, or ~alkenyIn socembodients.Ris

NR winsome embodiments, Ri isCw-Csalkyl, or CjC alkenyl and R is

In some cmbodiments, a is 2 X", for each occurrence is C2 alkylene and X is C2 alkylene; and wherein each R is H or 0 R-1 Y'

R, forat least one occurrence, e g. fbr each occurrence, mis 2; Y is NH or 0; R1 is C,2aikyl in some embodiments, at least 80% of the moleculesof the compound of formula (I) are a single structural isomer, In some embodiments, Y is N-, e.gwherein at least 80% of the molecues of the compound of fonnula (1) are a single structuralisomer. In some embodiments, R is RI for 5 occurrences. In some embodiments, in at least 80% of the molecules of the compound of formula (I), R isKR. for 5 occurrences, In some embodiments, Y is NH. In some embodiments, the compound of formula (I) is aninorganic ororganic salt thereof, e.ga hydrohalide salt thereof, such as a hydrochloride salt thereof. In some embodimentsthehydrochloride saltranges from a single equivalent of HCL, to n+2 equivalents of HCL. In some embodiments, the compound of formla()is sah of an organic acide.g, an acetate, for examplethe acetatesalt ranges from single equivalent of acetate, to ni-2 equivalents of acetate or aformate,orexample, the fornate saltranges from a single equivalent of acetate, ton+2 equivalents of format. insomeenbodiments, th compound of formula (I) is in the form of a hydrate. In some embodiments, R1, for atleast oneoccurrence, e.g., or each occurrence, comprises an alkenyi moiety, for example, R' comprises acis double bond In one aspect, the invention features a preparation including a compound of formula (I) and a nucleic acid (e.g, an KNA such as an siRNA or dsRNA or a DNA). In some embodiment, the preparation also includes an additional lipidsuch as a fusogenic lipid, ora PEG-lipid. In some embodiments, the preparation comprises less than 11%, by weight, of

Xa X H2N; 'N 'NH,

formula (iII)

wherein X and n are defined as in formula (I) above. In some embodiments, the preparation comprises less than 90% by weight of

` y R1

ofo rmnula (IV) wherein Y and R' are defined as in fbrmula (1) above, in some emnbodinients, the preparation comprises aplurality of compounds of

In some embodiments, the preparation comprises a mixture of compounds of the fonnulas blow: R R R3N"-x-''--ZN'Ra Nii,- ' FR M H R ' N N and R fonnula (F) formula (1") wherein in formula (I"), five of theR moieties are R In some embodiments, formula (I) andi (") are present in a ratio of from about 1:2 to about 21, in one aspect, the invention features a method of making a compound of fornuia (11'),

R$N14 N NR2 R n

fmiula (II) wherein

each X and X, for each occurrence, is independently C;.alkylene; n is 0, 1, 2, 3, 4, or 5; and wherein each R is independently H or 0 AR'

Ra; m is 2; Y is 0, NR, or S; RI is alkyl or alkenyl; Ifis or Calkloralkenyl; the method comprising reacting a compound of formula (Uf) r

, H2N 'Ni NH 2 LJ

with a compound of formula (IV), O

tbnnula (IV)

in the presence of a promoter, In one aspect, the invention features a method of making a compound of foirnla

r ,

RN 'N XNR 2 Rin

formula (II) where. each X' and X, for each occurrence, is independently Cj, akylene; n is 0, I, 2, 3, 4, or 5; and a5 wherein each R is independently H or

R"

m is 2; Y is O, NR2 , or S; R; is alky, or alkenyl; R2 is H or C alkyl orakenyl; the method comprising reacting a compoundof formula (III)

H 2N 'N 'NH2 -n forinula (Ill) with a compound of formula(IV), 0

formula (IV)

in the presence of a quencher, 1s In one aspect, the invention features a method of making a compound offormula

(II),

R2 N -aN1 NRp LNn fom'ula (II) v/herein each X" and Xt foreach occurrence, is independently C alkylene; n is 0, 1, 2, 3, 4, or 5; and wherein each R is indeperdently H or 0 y-R m is 2; Y is 0, NR or S; RIlis alkyl or alkenyl; 5WR2 is H oralkyl or alkenyl; tie method comprising reacng a compound of formula (III)

H2N N NH2z H

-formula (III1)

with a compound of formula (IV), 10

wherein the reaction mixture comprises -from about 0,8 aebouit 1.2 mnol'ar equi-valentls of a comnpound of formula (III), with from about 3M to about 6,5.fmolar equivalents of a compound of formula (V), In some embodiients, the reaction mixture comprises from abo about m8 1,2

mo~r equivalents of a compound of formula ((I ), with from about 5 to about 6.5 olar equivalents of a compound of formula (IV). In some embodimentscu, thureaction mixture cmprises about I molar equivalents of a compound of fwrnnua (1I), with from about 6 molar equivalents of a compound of formula (IV). In some embodiments, the reaction mixture comprises about I molar equivalents of a compound offnnula (iIl), with from about 5 molar equivalents of a compound of formula(TV), In one aspect, the invention features a method of making a compound of formula (Ti),

RAN 'N' 'NR2 Rax

formula (i) wherein each X and Xt for each occurrence, is independently C alkylene; n is 0, 1, 23, 4 or5; and wherein each Ris independently H or

R,,; m is 2; Y is 0, NP or S; Riis alkyl or alkenyl; R2 is H or alkyl or alkeny); the method comprising a two step process of reacting a compound of formula (HI) i X x X H2-N 'N 'NH2

is fonmula (111)

with a compound of fonnula (IV), 0

formula (IV) in the presence of boric acid and water wherein, the first step process involving the reaction mixture comprises from about 0.8 about i2 molar equivalents of a compound offormula (il1),withfrom about .8 to aboit 4,2 molar equivalents of acompound of fnnula (IV)and the second step process involvinagaddition of about 0.8 to 1.2 molar equivalent of compound of formula (IV). in one aspect, the invention features a method of makig a compound of fonula (II),

XI X R ,N NJ 'NR2 R nXl

formula (II) wherein eachX' and Xxfoir each occurrence, is independently Caiakylene; n is 0, 1,2, 3, 4, or 5; and wherein each R is hidependentlylH or 0

n is 2; Y is 0, NR2, or S; I, isalkyl orakenyl; R is R or alkyl or alkenyl; the method comprisingreacting a compound of formula (III)

r H2.7N 'N' 'NH2 !HI

formula (IIl) with a compound of formula (IV), 0

formda (IV)

and separating atleastonestructural isomer ofIbomula (1) from the reaction mixture to providea substantially purified preparation.comprisingastutural isomerofformula(1) in some embodiments, thestructural isomer of formula (H)is separated from the reaction mixture using chromatographic separation, In some embodiments, the chromatographic separationis using flash silica gel for separation of isomers. Insome

)2 embodiments, the chromatographic separation is gravityseparationofisomersusing silicagel, In someembodiments, the chromatographic separation is using moving bed chromatagraphy for separation of isomers. In some embodiments, the chromatographic separation uses liquid chromatagraphy (LC) for separation of isomers, In some embodiments, the chromatographic separation isnormal phase HPLC forseparation of isomers. In someembodiments, the chromatographic separation is reverse phase HPiLC for separation of isomers, hI some embodiments, the substantially purified preparationcomprises at least about 80% of thestmtral isomer of bmula (11), e.g, at least about 90% of the 1o structura isomer of formula (II), atleast about95% ofthe structural isomerofifrumia (II). in another aspect, the invention fares a method of making a compound of formula (V) or a pharmaceutically acceptable salt thereof,

RsN ' 3,N NR2

frmia (V) wherein each X" and X, for each ocurrence, isindependently C( akylene; n is 0, 1, 2, 3, 4, or 5; and wherein each R is independently H or 0 .Y't R4

m is 1;

Y is 0, NP2 or S; R is alkyl or alkenyl; R 2 is F or alkyl or aikenyl; the method comprising reacting a compound of formula (111)

HN t'NH2

fonnula (lI) with a compound of formula (VI),

0 0N-1 X11Y.R1 Clor Br orI

formula (VI)

to provide a compound of formula (V) or apnnaceutically acceptable salt thereof, in some embodiments, the pharmaceutically acceptable salt thereof is a hydrochloride sat of the ompound offornnula (V), in one aspect, the invention features a compound of formula (X),

4 4 R NN L2R

RQ2 LN.RS

formula (X) wherein R andR 2 are each independently H, C-C6 alkyl, optionallysubstituted with 1-4 R, (LC6alkenyl, optionallysubstituted with 1-4 R , or C(NR)(NR1)2; R and R4 are each independently alkyl, alkenyl, alkynly, each.ofwhich is optionally substituted with fluoro, chloro, bromo, oriodo; L and Lare each independently -NRC(O)-, -C(OI)NR'-,-OC(0)-,.-C(0)O-, S-S~, -N(R )C(O))N(R 6)~, -OC(O)N(R')- N(R6)C(O)O~, ~O-N=,C-, OR, OC(0)N N= - r NHC(0")NH-N=C-, L-R' and LR 4can be taken together to form an acetal, aketal, or miorthestear wherein R. and 4 are defined as above and can also be H or phenyl;

9 RI is fluoro, chloro bromo, iodo,-OR 7 >-N(R)(R ),-NSS(O)Rt

Ra is R, CC6alkyl, R is H or C-C6alkyl; each R' and Rare independently H or C-C, alkyl; R3is H orCrCa alkyl; i ¾1, 2, 3, 4, 5, or 6; n, i s 0 ,2, 3, 4, 5 or 6, and pharmaceutically acceptable salts thereof In some eibodiments, the compound is an inorganic salt thereof, for example a hvdrohalide salt thereof such as a hydrochloride salt thereof In someeibodhnents, the compound is an organic salt thereof In some embodiments, R and R2 are each independently Cr-C i alkyL In so embodiments,R is methyl. In some embodiments, Ris methyl. In some embodiments, R t and R2 are both methyl. In some embodiments, Rl is H, methyl thyl, isopropyl, or 2-hydroxyethyl. la some embodiments, R 2 .is H, In some embodiments, R 2 is methyl, ethyl, propyl, or isopropyl In sone embodiments,R is H, methyl, ethyli, isopropyl, or 2-hydroxyethyl and Ris H, ethy,ethyl, propyl, or isopropyL o In some embodiments, in is 1. In some embodiments, n is L Insome embodiments, both m and n are L In some embodiments, . is--NR 6C(O) or -C(O)NR> In some enbodinents, L is -OC(O)- or -C(Of)O In some embodiments,L' is S-S In some nbodiments, Li is -N(R)C(O)N(R)-. In some embodiments, I is -OC(O)N(Rt)- or -N(R)C(O)0-. In some embodiments, L is-O-N=C. In some embodiments, L -OC(O)NH-N=C- or ~NHC(O)NllN-=C In some embodiments,U is -NRC(O)-, or -C(OYNRt In some embodiments, U is -OC(O} or -C(0)0 In some embodiments, Lis- S

In some mbodments,Uis -N(R6 )C(O)N(R), II some embodiments, is OC(OYN(R')- or -N(R)C(O)O. In some embod ments, , i is - s-N=C~ In someembodiments, 3-C(O)NHNC or -N4C(O)NH-N='C-. Ininsoe embodiments, both L and 1 are -NR 6 C(Q)~, or -C(O)NRt In so-me embodiments, bothi iand 1 are OC(O)- or -C(O. In some embodiments, both L and 1 are S-S, Income embodiments, both L and are-N(R')C()N(Rt)t Income embodiments, both L and 1 are -OC(O)N(Re)- or-N(R)C(O)QO insome embodiments, iis -NRsC(Oand£2is-S-S In some embodiments, L is-OC(O)-and £ is -S-s. In someembodiments, L is -OC(O)N(R)or -N(R)C()O- and iis -- 4.

Insome embodiments, L'is -N(f)C(O)N(R)~ and L2 is-S Insome embodiments, L'-R3 andlI-R 4 are taken together to form an acetala ketal, or an orthoester. Insome embodiments, each Rand R 4are independentlyalky. In some embodiments, both R-nd R4 areC 5 alkyl insome embodiments, each 1 and I are independently -S-,-OC(O)N(R%)~

or-~N(R)C(O)Ds In some embodiments, Re is alkyL In some embodiments, is alkyl. In some embodinents, R is alkenyl. InsonmeembodimentsR4lis alkenyL Insome embodiments, each Rand R4 are independently alkeny, for example, 3 4 each R 3 and R4 areindependentlyC6C 3 , alkenyl oreach R and R are the same alkenyl moiety In some embodiments, each R and R4 includes two double bond moieties. In someembodiments,atleastoneofthedoublebondshaveaZconfiguration,In some embodiments, both of the double bonds have a Z configuration. In some embodiments, so atleast one of R3 and R4 is provided in formula (II) below

fomula (II) wherein x is an integer from Ito 8 and yisan integer from ~10, Insome embodiments, both oftRe and iare ofthe formula (i). insome embodiments, at'least one of the double bonds have an E v configuration, e,g both of the double bonds have an E configuration. in some embodiments, at least oneof t and Rt is provided in formula (1) below fommla (1) wherein x is an integer from I to 8; and ysan integer fIroI-i0, In some embodiments, each RI and R2 includes three double bond moieties. In some embodiments, at least one of the double bonds have a Z configuration. hi some embodiments, at least two of the double bonds have a Zconfiguration. 1 some i5 embodiments, all three of the double bonds have a Z configuration, in some embodiments, at least one of t andRW is provided in formula () below formula (IV) vherein xis aninteger from 1 to 8; and y is an integer from 1-10. In some embodiments, both of R 1 and R are as provided in fbrmula (W) In some embodiments, at least one of e double bonds have an E configuration, In some embodiments, at least two of the double bonds have an E configuration In some embodiments, all three of the double bonds have an E configurationMsome embodiments, atleast one of RI andR is provided in fbnnula (IV) below formula (V) wherein Sx is an integer from I to 8; and y is an integerfrom 1-10. insomembod entsbothof R andR2 are as provided in formula (V). In some enbodiments, R3 and R2 are each CICi alkyl (e.g.methyl), Li and LI are each ~0C(O)~, and R' and R4 are each alkenyl.In some embodiments, R3 and R4 are the same; in someembodiments, R3 and R4 both include two double bonds (e.g, 4 having cis linkages). In some embodiments R3 and are provided in formula (II) below fornmia. (11) wherein x is an integer from I to 8 eg, 5; and y is an integer from['10esg,4, In one aspect, the invention featuresa preparation including a compound of fornmia (X) in one aspect, the invention features a preparation including a compound of formula (X) and a nucleic acid (e.gan RNA such as an siRNA ordsRNA or a DNA). bn some embodiment, the preparation also includes an additional lipid such as a fusogeniclipid, ora IT-lipid, none aspect, the invention features a method of makinga compound offormula (X), fanrnua (X)i wherein R and are each independently CC alkyl, optionally substituted with 1-4

R3 is alkyl, alkenyl, alkynyl Li' is -OC(O) 0 IR is -OR -N(R)(R), -CN, SRO S(O )R S(0)2R R6 is H, C> alkyl; R' isHor C '6 alkyl; eachRta and R are independently H or CeCr alkyl; R f is H or CCe, alkyl; m iand n are each independently 1, 2, 3, 4, 5, or 6, the method comprising reacting a compound of formula (VI),

2 H ftnula (V*) with a compound of fbnnula (VII)

HO' R0 formula (VI1) in. the presence of a coupling agent, thereby providing a compound offImula (X). In some embodiments, the coupling agent is a carbodimide such as EDCL In one aspect, the invention features a compound of formula (XV) below

R 0 (1

Rs2

Sformula (XV) wherein; each L and L are independently a bond or C(O); each R and R2 are independently akyl alkenyl or alkynyl; each of which is optionally substituted with one or more substituents; X is -C(O)NH-, C(S)NH, -C(O)C.alkyC(O)NH-; or -C(O)CjalkyC()O-; m is an integer from 0-11 and n is an integer from 1-500. In some embodients, L and f are both a bond. 2 are both C(). In some enbodiments, L and insome embodiments, each R and Rareindependently alkyl, for example C C02 aikyl, e.g.,C}e&ralkyl, eg, Cr3alkyC 1 alkylCis akyl, or(>i akyL in some embodinmts, both R' and R are alkyl, e.g, straight chain aikyl having the samelength e4g, C-z, alyikyleg,CuCi alkyl, e.g,, C alkyl, C14 alkyl, C alkyl, or Caalk In some preferred embodiments, both R' and R2 are C4 alkyl. Incomeembodiments, the formula XV represents a racemic mixture In some embodiments, the compound of formula XV has an enandomeric excess ofthe R isomer, e.g., atleast about 65%,70%, 75%,,80%,85%,90%, 95%,97%,98%, or99%. in some embodiments the formula XVrepresents enantiomerically pure' isomer, insome embodiments, the compound of foinula XV has anenantiomerice xcess of the S isomer, e.g, at least about 65%, 70%,75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%. insomeembodiments the formula. XV represents enantiomerically pure 'S' isomer. insome embodiments, each R andR are independently alkenyl, fr example, each R and are independently C6-C alkenyl or each R and R are thesame alknyl moiety, In some embodiments, each R' and R 2 includes a single double bond, for example a single double bond in the E or Z configuration, insome embodiments, each t' and Re includes two double bondmoieties.In some embodiments, atleast one of the double bonds has a Z configuration, In some embodiments, both ofthe double bonds have a Z configuration, In some embodiments, at least one of R and R is provided in fonnula (II)below fornmla () wherein x is aninteger from I to 8; and y is an integer from 1-10. In sonic embodiments, both of R and Re are ofthe formula(II). In some embodiments, at leastone ofthe double bonds has an E configuration, e g, both of the double bonds have an E configuration, in some embodimentsatleast one of R and R is provided in formula (111)below formula (III) wherein xis an integer from I to 8; and y is an integer fro I-10, In some embodiments. each R' iand R2 includes threedouble bond moieties, fi some embodiments, at least one of the double bonds has a Z configuration, income enbodiments, at least two of the double bonds havea Z configuration. In some embodiments, all three of the double bonds have Z configuration. Winsome embodiments; at least one of RWand R'is provided in formula (IV) below fonnula (IV) wherein x is an integer from I to 8; and y is an integer from 1-10. In some embodiments, both of R and R2are as provided in formula (IV), In some embodiments, at least one of the double bonds has an E configuration. Insomeembodiments, at least two of the double bondshave an E configuration. In some embodiments, all three of the double bondshave an E configuration. In someembodiments, at least one of R and R2 is provided in formula (IV) below innula (V) wherein x is an integer om I to 8; and y is an integer from 1-10hI some embodiments, both of R" and R' are as provided in formula(V). in some embodiments, X is-C(O)NITIproviding a compound offbomia (XV') below:

R 1' -' 0 W

foirtla (XV') In some embodiments, each R and 2 areindependently alkyl, fr example CC 2 alkyl, e g,C(\Cj aiyl, eg., C 1 ilayl,alkyl, C alkyl, orCg alkyl, i some efbodiments, both R and R2 are alkyl, e.g, straight chain alkyl having

21.

the samelengtheg C C alkyl, wgCw alkyl,eg,, C Balkyl, CIA lkyl, C alkyl, or Ca alkyl. In somepreferred embodiments, both Rl and R are C1 alkyL In some embodiments, X is -C(O)Cj-3 alkylC(O)-. In some embodiments, m is an integerfrom 1-10fobrexanple an integerfrom 2 5 4 or an integer 2. In some eibodiments, n is an integer from 1-500, for example an integer from 40-400, from 100-350, from 40-50 or from 424, In some embodiments, the compound is a compound of ftomula (XV), 0

OL2

fomula (XV), wherein both L' and L2 are a bond, In some embodiments, each R" and R are independently alkyl,for example C6(2 alkyl, e.g,C-Cjalkyl,e .C1 alkyl

. 2 alkyl, or Ca alkyl In some embodiments, both RI andR are alkyl, e.g, straight chain alkyl having thesame length, e.g., C-C; alkyl, eg.Cwo0alkyl, e.g, Qa lkyl, Ci 1 alkyl, or Co alkyl In some preferred embodiments, both R and R2 are Ca alkyl. In some embodiments, m is an integer from1-10, for example an integer from 2-4 or an

integer 2 In some embodiments, n is an integer from 1-500, for example an integer from 40-400, orfrom 40-50, In somic embodiments, the compound is a compound ofkwmula (XV'), wherein :o LI and2 areboth bonds, RIand R2 are both alk (e.gC-C2 alkyl, e.g.,Cw-C alkyl, preferrably C)4alkyl), and n is an integer from about 40-400 in some embodiments, the comound has a formula (XVI) below 0

formula (XVI),'wherein therepeating PEG moiety has an. average molecular weight of 2000 with n value between 42 and 47. In some embodiments, the compound of formula XV has an enantiomerio excess of the R isomer, e.g, at least about 65%, 70%, 75%, 80%,85%, 90%, 95%, 97%, 98%, or 99%. In someembodiments the compound of formula XVIis astereo isomerwith preferred absolute configuration WR In oneaspect, the invention features a PEG lipid conjugated toa cholesterol moiety, For example, the compound of formula (XX) below:

Cholesterol X

ormula (XX). X is4-(G)NUP(C(S)NH,&-(O)C.:alkylC(O)NH-; or&-(O)Cuaky(G)Q~; m is an integrfkom 0-11 and n is aI integer from 1-500, in some embodiments the 0 attached to the cholesterol in formula (XX) is part of the cholesterol moiety. In some preferred embodiments, X is -C(O)NH, or -C(0)C alkyC(0)Q-, in some embodiments, the compound of formula (XX) isas provided below in formula (XX')

0

formtla(XX'), in oneaspect the invention features a PEG lipid bound to targeting moiety, for example sugar residue, For example, the compounds of formtla (XV) or (XX) are modified at the OMe terminal end with a targeting moiety In some embodiments, the targeting moiety is bound to the PEG moiety via a linker. Examplary targeted PEG lipids are provided in formulas (XXI) and (XX11) below In one embodiment, the lipid is a compound of ornAa (XXI)

R 'O x k N'T

'LZ

fo rmiuIa(X) wheorein; each L' and I are independently a bond or C(O); each'R andR 2 are independently alkyl alkenyl orakiynyl; each of which is optionally substituted with one or more substituents; each X and X' is independently -C(0JNH-.-NHC(O) - C(S)NH, C(S)NH, C(0).CalkylC(O)NH-; NHC(0)C;.alkylC(O) -;-C(O)C-aikylC(0)0- NHC(0)C alkyv; or C 3 alkylC(0)NH-; m is an integer from 0-I and n is aninteger ftom 1-500 p is an integerfrom 1-6, eg3; T isa targeting moiety such as a giycosyt moiety (ega sugar residue). OH

HO O Examplary targeting moieties include ACHN, In some embodiments, L and If are both a bond. in some embodiments, L and I are both C(O). In some embodimentseach R and R2 are independently alkyl, for example Ce S C k e C a lkyl, Ci a lkyl, e~g alkyl,Cakyl, or Ct alkyl,, In some embodiments,both. R'and R2 are alkyl, eg., straight chain alkyllhaving thesame length, e.g, C5728alkyl, e.g,Cur-Ca lkyl, e.gO alky, C alkyl, or Cakyl In some 2 are C1 alkyL preferred embodiments, both R. and In some embodiments, the fomula (XXI) reperesents a racemic mixture In someembodiments, the compound of formula (XXI) has an enantiomeric excess of the R isomergg, at least about 65%,70%, 7S%, 80%,85%,90%, 95%,97% 98%, or 99%. In some embodiments the formula (XXI) represents enaniomerically pure R' isomer. in some embodiments, the compound of formula (XXI) has an enantiomeric excess of the S isomer, e.gat least about 65%, 70%, 75%, 80%,85%, 90%, 95%,97% 98%, or 99%. Incomeembodiments the fonmua (XXI)representsnantiomericaliy pure 'S' isomer, Income embodiments, each R and R? are independently alkenyl, for example, each R, and R are independently C6C3 alkenyl or each R and Rl are the same alkeny mieity. I some embodiments, each Rl and R2includesasingledoublebondfor example a single double bond in the E or Zconfiguratio In some embodiments, eachRI andR2 includes two double bond moieties In some embodiments, at least one of the double bonds has a Z configuration, In some embodiments,both of the double bonds have a Zconfiguration. In some embodiments, at least one ofR and R2 isprovidedinforula(l)below fonnula (II) wherein i's an integer from I to 8; and v is an integer from 1-10. in some embodiments, both of R. and R2 are of the formula() In some embodiments, at least one of the double bonds has anE configuration,e.g.,both of the double bonds have an E configuration, Insome embodiments, at least one of R and R2 is provided in formula (III) below fonnul'a(l) wherein xais an integer fom I to 8; and yis an integer from I-1 In someembodimentseach R and R2 includes three double bond moieties. In some embodiments, at least one of the double bonds has a Zconfiguration in some enbodinents, at least two of the double bonds have a Z configuration, Insome embodiments, all three of the double bonds have a Z configuration. In sone embodiments, at least one of R andR2 is provided in formula (IV) below formula (IV) wherein x is an integer frm I to 8; and y is an integer from I-10. In some embodiments, both of RI andR are as provided in fomala (IV) In some embodinnts, at least one of the double bonds has an

E configuration. In some embodiments, at least two of the double bonds have an E configuration. In some embodinests, all three of the double bonds have an F configuration, .n some embodiments, atleast one of Ri and R2 is provided i, fomula (TV) below

fornula(V) wherein x is an integer from I to 8; and y is anintege ffrom 1-10. Jn someembodimentsboth. ofR and R4 are as provided in formula (V). In some enibodientts, p is 3, insome embodiments,Lis NHC(Q)Ca alkyl (e.g., NC(O)Csaikyl), In somee'mbodients, the compound of formula (XXI)is the compound of (XX) below: OH OH 0 0 4ONO N 'O AcMN H H

formula(XXP), In one embodimentthe lipid is a compound of format (XXII)

k -- 0 xO.X

formula(XXII) wherein; each X and X' is independently-(NH NHC(O)-, C(S)NH,009NH, C(O)C0 alkylC(O)NH-; NIC(O)C 3 alkylC(O)~;-C()CalkyiC(O)O-;NHC(0)C ,alky; orCalkyC(0)NH-; m is an integer from 0-11 and n is an integer from O 1-00 pisan integer from 1-6, e.g, 3;

T is a targeting moiety such as a glycosyl moiety (e.g., a sugar residue) OH O

Exampary targeting moieties include AcHN In some prefer deimbodiments, the compound of formula (XXl) is the compound of (XXIP) as provided below:

OH

HON' O N :O AcHN H H formula (XXIP) In one aspect, the invention features an associationcomplex comprising a compound preparation comprising compound described herein (e.g., a compound of formula ()or' compoundof formula (X)) and a nucleicacid suchas an RNA a single to stranded or double stranded RNA (e.g.,siRNA or dsRNA or a DNA). In some embodiments, the associationcomplex is a lipoplex or a liposome. In some embodiments the association complex includes one or moreadditiona componentssuch as a targeting moiety, a fusogenic lipid, a PEGylated lipid, such as aPEG-lipid described herein such as a PEG-lipid having the fonnula (XV),(Xv) or (XVI) or a 1s structural component.i n some embodiments, the PECipid is targeted PEG-ipid as described herein, e.g., a compound of formula (XXI), (XX'), (XXII),or (XXIP). Inone aspect, the invention features a method offorming a liposome comprising contacting a lipid preparation comprising compound described herein (e.g. a lipid described herein such as a compound offormula (1) or formula (X)) with atherapeutic agent in the presence of a buffer, wherein said buffer: is of sufficient strength that substantially all amines ofle molecules formula I ar protonated; ispresent a between 00 and 300mM; is presentat a concentration that provides significantly more protonation of than does the same buffer at 20 mM, Inone aspect, the invention features a liposone made by the method described herein,

In one aspect, the invention features a method of forming a liposome comprising contacting a lipid preparation described herein (e.ga lipid preparation comprising a compound of fonula () or a compound of fbnnula (X)) with a therapeutic agent in a mixture comprising at least about 90% ethanol and rapidly mixing thelipid preparation ,vithethetherapeutic agent to provide a particle having a diameter ofless than about200 iM. In some embodiennts, theparticle has a diameter of less than about 50 uM. In one aspect, the invention features amethod of forming a liposone comprising contacting a lipid preparation described herein (e,g, a lipid preparation composing a compound of formula (1) or a compound of formula (X)) with a therapeutic agent in the presenceof buffer, wherein said bufler has aconcentrationrabout 100 to about 300mM. In one aspect, the invention features liposome comprising a preparation described herein (e.g, a lipid preparation comprising a compound of fomula (T) or a compound of formula (X)) and a nucleic acid In some embodimentsthe preparation also includes a PEGylated lipid, for example a PEG-ipid described herein, such as a PEG-ipid having the formula (XV),,(XV') or (XVI), In some embodiments, the PEG- lipid is a targeted PEG-lipid as described herein, e.ga compound of formnla(XXI), (XXI'), (XXII) or (XXWl).In some embodiments, the preparation also includes a structural moiety such as cholesterol. Insome embodiments the preparation of association complex includes compounds of formaulac (I), (XV) andcholesterol In some embodiments, said nuclic acid is an siRNA, for example saidnucleic acid is an siRNA which has been modified to resist degradation, said nucleic acid is an siRNA which has been modiled. by modification of the polysaccharide backbone, orsaid siRNA targets the ApoB gene. in some embodiments, the liposome further comprisiesastretural moiety and a PEGylated lipid, such as a PEG-lipid described herein, wherein the ratio, by weight, of preparation (e.g, a lipid preparation comprising a compound of fomula (I) or a compound of formula (X)), a structural moiety such as cholesteroPEGylated lipid, and a nucleic acid, isS8-22:4-1I0:4-12:0.4-2.2in some embodiments, the structural moiety is cholesterol In some embodiments, the ratio isI 0-20:05-8,0:5-100.5-2,0, e g, 15:0.8:7:1. in someembodiments, the average liposom ar is between d i10 nm and 750mno,e.g.,the average liposome diameter is between 30 and 200unor the average liposome diameter is between 50 and 100 nm. In some embodiments, thepreparation is less than 15%, by weight, ofunreacted lipid. In some embodiments, the ratio of the preparation (eg, a lipid preparation comprising a compound of fornula (I) or a compound of fommula (X)),the structural moiety such as cholesterol, and the PEG lipid S is about 42/48/10 (mola ratio). In some embodiments, the total lipid to nucleie acid (eg., siRNA) is about 75% by weight, In some embodiments an association complex described herein has a weight ratio of total excipients to nucleic acid of less than about 15:1, forexample, about 10:1, 7.5: Ior about 5:1. in one aspect, the invention features a method of forming an association complex comprisinga plurality of Hpidmoieties and a therapeutic agent, the method comprising: mixing a plurality of lipid moieties in ethanol and aqueous NaOAc buffer to providea particle; and adding the therapeuticagent to the particle, thereby forming the association complex, In some embodiments, the lipid moieties are provided ina solution of 100% ethanol la some embodiments, the plurality oflipid moieties comprise a cationic lipid, in some embodiments, the cationic lipid is a lipid described herein, fbr example, the cationic lipid is a lipid of one of the following or a mixture thereof: HH

N

2901

29)

HH NN N N

H H In same -preerredembodiments, the cationic lipid is H H

^. ,N N'N N N) N-,- ~ H H o

H

In some embodiments, the plurality of lipidmoieties comprise a PG-lipidfor example, the PEG-lipid has the following structure:

0>0 0O'L2

wherein; each L and If are independently a bond or C(O); each R and R are independentlyalkyl alenyl or alkynyl; each of which is optionally substituted with one ormore substituents; X is-C(O)NH-, C(S)NH, -C0)CadkyC(O)NH-; or-C()CakyC(Q)0 m isfannteger irm 0-11 and n is an integer from 500 In some preferred embodiments, the PEG-lipid is a PEG lipd of rmula (XV0) wherein the repeating PEG moiety has an average molecular weight of 2000, for example, with an n value between 42 and 47 or the lipid provided below:

N

In some embodiments, the plurality of lipid moieties comprises a strucmral lipid, For example, the structurallipid is cholesterol, In some embodiments, the PEG-lipid is targeted PEG-lipid as described herein, e,.g, a compound of formula (XXI), XX),(XXII),. or (XXIP).

In some embodiments, the method includes further comprising extruding the lipid containing particlesfor example, prior to addition of the therapeutic agent. In some embodiments, the theraputic agent is a nucleic acid, forexample, an siRNA, such as ansiRNA which-hasbeen modified to resiStdegradation, an siRNA which has been modified by modification of the polysacharide backbone, or an siRNA conjugated to alipophilic moiety In some embodiments, the siRNA targets the ApoB gene. In some embodiments, the association complex comprises a cationic lipid, a structural lipid, a PEG-lipid and anucleic acid. In some embodiments, the molar ratio of the cationic lipid, structural lipid, PEG-lipid and nucleicacid is 36~48:42-54:6-14.for example, 38-46:44-52:8-12 or about 42:48:10, Insome embodiments, the weight ratio of total exipient to nucleic acid is less than about 15:1, for example, about 10:1 about 1.5:1erabout5:1. in some preferred embodiments, the cationiclipid has the fbllowing H H N-o O .N -Ns/N 0 N0 H

HH H H

structure; the PEG-lipid is a PEG lipd offormula (XVI), wherein the repeating PEG moiety has an average molecularweight of 2000, for example, with ann value between 42 and 47 or has the followingstructure: D ^o -,-o N H \ and

the structural lipid is cholesterol, for example wherein the molar ratio of the Cationic lipid, structural lipid, is PEG-ipid is 384644-52:8-12, e.g.,about 42:48:10.In some preferred embodiments, the weight ratio of total exipient to nucleic acid is less than about 15:1, eg., about 10:1, about 7.5:1, or about 5:1. in another aspect, the invention features an association complex made from a method described herein. In another aspect, the invention featuresassociationcomplexcomprisinga cationic lipid, structural lipid,a PEG-lipid and a nucleic acid, wherein the cationic lipid is is a lipid of one of the following or amixture thereof:

H

N o Hor r

H C' r'

H H

the PEG-lipidisarPEG ldofforla(XVI) wherein therepeating PEGmoietyhas an average molecularweight of 2000 for example, with ann value between 42 and47 or 0 ua has the following structure:

H\ n 'and the structurallipidis cholesterol Insome preferredembodimentsthenucleicacidisan siRNAin some preferredembodimentsthee ationic lipid hastefollowing formula:

H H

H , In some

preferred embodiments, the molarratio of theeationic lipid preparation, structuraillipid (e.g.,holesterl), PEG0-lipid and nucleic acid is 36-48:42-54:6-14, for example, 38 46:44-52:8-.2or about 42:48:10.in some preferred embodiments, the weight ratio of totale-xipient to nuclecacidbisless than about156I, for example, about 10:1, about 7.5:1, or about 5:1 In some embodiments, an association complex described herein has amean diameter or particle sizeless than about 25000rn, eag.,from about 20 to200rnm, aboutd60,or about 50 am. In some embodiments, anucleic acid as administered in anassociation complex described herein, demonstrates aserum half lie(e.g, in vitro) for atileastgabout 4hours, e.&, a! least about 6 hours, at least about 8hours, at least about 12 hours, at least about 24 hours, at least about 2 days, at least about 3 days, at least about 4 days, atleast about week, at least about 2 weeks, or at least about 3 weeks, in one aspect, the invention features a pharmaceutically acceptable omposition comprising thepreparation described herein. In one aspect, the invention features a phannacutically acceptable composition comprising a liposone described herein. SIn one aspect, the invention featuresa method of treating a mammal comprising administering to said mammal a therapeutic amount ofapharmceuticalyacceptable composition, rexample,an association complex such as a liposome described herein. Definitions The term "halo" or "haogen" refers to any radical offluorine, shrine, bromine or iodine. The term "alkyl" refers to a hydrocarbon chain that may be a straight chain or branched chain, containing the indicated number of carbon atoms, Forexample, CI-C 6 alkyl indicates that the group may have from I to 136 (inclusive) carbon atoms in it. The term "haloalkyl" refers to an alkyl in which one or more hydrogen atoms are replaced by halo, and indludesalkyl moieties in which all hydrogens have been replaced by halo (e~g,perfluoroalkyl). The terms "arylalkyl" or "araikyl'refer toan alkyl moiety in which an alkyl hydrogen atom is replaced by an ary group Aralkyl includes groups in which more than one hydrogen atom has been replaced by an aryl group, Examples of "arylalkyl" or "aralkyl" include benzyl2-phenyethyl 3-phenylpropyl,9 fluorenyl, benzhydryl, and trityl groups. The term "alkvine" refers to a divalent alkyl, e.g.,-CI9CHCHr C :ZCH'2 CH-, CH2CHC-2CH2jCHCH 2CH2 CH2CH-, and CH 2 CH 2CH2CHl 2 CiHbChr. 'he term "alkenyl" refers to a straight or branched hydrocarbon chain containing 2-36 carbon atomsand having one or more double bonds. Examples of alkenyl groups include, but arenot limited to, allyl, propenyl 2-buteny, 3-hexenyland 3-octenyl groups. One of the double bond carbons may optionally be the point of attachment of the alkenyl substituent. The term "alkynyl" refers to astraight or branched hydrocarbon chaincontaining 2-36 carbon atomsand characterized in having one ormore triple bonds, Examples ofalkynyl groups include, but arenot limited to, ethynyl,propargyl, and 3-hexynyl. One of the triple bond carbons may optionally be the point of attachment of the alkyni substituent The term "substituents"refers to a group "substituted" on an alkyl, cycloalkyl, ailkenyt alkynyl, heterocyclyl, heterocycloalkenyl, cycloalkenyl, aryl, or heteroaryl group at any atom ofthat group. Any atom can be substitutedSuitable substitutes includewithoutlimitation,alkyl (eg, C, C2, C , C4, C5, C6 C7, CS, C9; C10. ClI, C12 straight or branched chain alkyl), cycloalkyl, haloalkyl (eg., perfluoroalkyl such as CF),ary, hetcroaryl, aralkyl, heteroaralkyl, heterocyclyl, alkenyl, alkynyl, cycloalkenV, heterocycloalkenyl, alkoxy haloalkoxy (eg..perfluoroalkoxv suchas OCF3 ), halohydroxy, carboxy, carboxylate, cyano, nitro, amino, aikyl amino, S011, sulfate, phosphate, methylenedioxy (-O-CHrO- wherein oxygens are attached to sate carbon (geminal substitution) atoms), ethylenedioxy, oxo, thioxo (eg,.C=S), imino (alkyl, aryl, aralkyl), S(0),alkyl (wheren is 0-2), S(0),, aryl (where n is 0-2), S(O), heteroaryl (where n is 0-2), S(O), heterocycIy) (where n is 0-2), amine (mono-, di-, alki, cycloalkyl, aralky, heteroaralkyl, aryl, heteroary, and combinations thereof), ester (alky, araikyl, heteroaralkyl, aryl, heteroaryl), amide (mono- di-, alkyl, arakyl, heteroaralky, aryl heteroary, and combinations thereof), sufonamide (mono-, di-, alkyl, aralky heteroaralkyl, and combinations thereof), In one aspectthe substituents on a group are independently any one single, or any subset ofthe aforementioned substituents, In another aspect, a substituent may itself he substituted withany one of the above substituents, The tenn "stmetural isomer" asused herein refers to any of two or more chemical compounds, such as propyl alcohol and isopropyl alcohol, having thesame molecular fommla but different structural foaulas. The term "geometric isomer" or "stercoisomer" as used herein refers to two or more compounds which contain the same number and types ofatoms, and bonds (ie., the connectivity between atoms is the sane), but which have different spatial arrangements of the atoms, for example cis and trans isomers of a double bond, enantiomers, and diasteriomers For convenience, the meaning of certain tens and phrases used in the specification, examples, and appended claims,are provided below. If there is an apparent discrepancy between the usage of a term in other parts of this specification and its definition provided in this section, the definition in this section. shallprevail.

"s," "C," "A" and "U" each generally stand for anucleotide thatcontains guanine, cytosine, adenine, and uracil as a base, respectively, However, it will be 5 understood that the term "ribonucleotide" or"nucleotide" can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety. Theskilled person is well aware that guanine, cytosine, adenine, and uracilmay be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearingsuch replacement moiety.F or example, without limitation, a nucleotide comprising inosine as its base may base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine may bereplaced in the nucleoide sequences of the invention by a nucleotide containing, for example, inosine. Sequences comprising such replacement moietiesare embodiments of theinvention As used herein, "target sequence"refers to a contiguous portion ofthenucleotide sequence of an mRNA molecule formed during the transcription of the corresponding gene, including mRNA that is a product of RNA processing of a primarytranscription product. A target region is a segment in a target gene that is complemientaryto a portion of the RNAi agent. As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising achain of nucleotides that is described by thesequence referred to using the standard nucleotide nomenclature. As used herein, and unless otherwise indicated,the tenn complementaryy, when used to describe a first nucleotide sequence in relation to second nucleoide sequence; refers to the ability of an oligonucleodde or polynucleotide comprising the first nucleotide sequence to hybridize and fonn a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotidesequence, as will beunderstoodbyheskilledperson.Suchconditions can, forexample, be stringent conditions, where stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH6,4, 1 mM EDTA, 50T or 70"C for 12-16 hours followedby washingQOher

conditions, such as physiologically relevantconditionsasmay be encountered insidean

organism, can apply. The skilled person will be able to determine theset ofconditions nost appropriate for a test ofcomplementarity oftwo sequences in accordance with tlhe ultimate application of the hybridized nucleotides, This includes base-pairing of the oligonucleotide or polynucleotide comprising the first nucleotide sequence to the oligonucleotide or polynuleotide comprising the second nuceotide sequence over the entire length of the first and second nucleotide sequence. Such sequences can be referred to as "fullycomplementary" with respect to each other herein. However, where a first sequence is referred to as"substantially complementary" with respect to a second sequence herein, the two sequences can be fully complementary,or they may formone or more, but generally not more than 4, 3 or -2 mismatched base pairs upon hybridization, while retaining the ability to hybridize under the conditions most relevant to their ultimate application. However, where two oligonucleotidesare designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, an oligonucleotide agent comprising one oligonucleotide 21 nucleotidesin length and another oligonucleotide 23 nucleotids in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that isfully complementary to the shorter oligonucleotide, may yet bereferred to as "fully complementary" for the purposes of the invention. "Complementary"sequences, as used herein, may also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified eleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled. The terms "complementary", "fully complementaryt and substantiallyy complementary" herein may be used with respect to the base matching between the sense strand and the antisense strand of an oligonucleotide agent, or between the antisense strand ofan oligonucleotideagent and atargetsequence, as will be understood from the context of their use. As used herein, a polynucleotide which is "substantially complementary to at least part of' amessenger RNA (mRNA) refers to a polynucleotide which is 2o substantially complementary to a contiguous portion of the mRNA. of interest. For example, a polynucleotide is complementary to at least a pattof an ApoB nRNA if the sequence is substantially complementary to a non-interrupted portion ofamRNA encoding ApoB& As used herein an "oligonacleotide agent" refers to a single stranded oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or both or s modifications thereof, whichis antisensevwith respect to its target. This term includes oligonucieotides composed of naturally-occurring nucleobases, sugars andcovalent internucieoside(backbone)linkages as well as oligonucleotides having non-naturally occurring portions which function similarly. Such modified or substituted oligonudeoides are oftenpreferred over native forms because of desirable properties such as, for example, enhancedcellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence ofnucleases. Oigonucleotide agents include both nucleic acid targeting (NAT) oligonicleotide agents and protein-targeting (PT) oligonucleotide agents. NAT and PT oligonucletide agents refer to single stranded oligomers or polymers ofribonucicic acid (RNA) or deoxyribonucleic acid (DNA) or both or modifications thereof. This rtem inacldes oligonucleotides composed of naturally occurring nucleobases, sugars, and covalent intemuceoside (backbone) linkages as well asoligonucleotideshavingnon naturally-occurringportions that functionsimilarly. Such modified or substituted oligonucleotides are often preferred over native forns because of desirable properties 2 such as, for example, enhanced cellar uptake, enhanced affinity fornucleic acid target, and/or increased stability in thepresence of nucleases. NATs designed to bind to pacific RNA or DNA targets have substantial complementarity, e.g., at least 70, 80, 90, or 100% complementary, withat least 10, 20, or 30 or more bases of a target nucleic acid, and include antisense RNAs, microRNAsantagomirsand other non-duplex structures which can modulate expression. Other NAT oligonucleotideagents include external guide sequence (EGS) oligonucleotides (oligozymes), DNAzymes, and ribozymes.'The NAT oligonucleotide agents can target any nucleic acid, eg,a miRNA, a pre-miRNA, a pre-mRNA, an mRNA. or a DNA. These NAToigonucleotideagents may or may not bind via Watson-Crick complementarity to their targets PT so oligonucliotide agentsbind to protein targets, preferably by virtue of thre-dimensioni interactions, and modulate protein activity. They include decoy RNAs, aptarners, and the like.

While not wishing to be bound by theory, an oligonucleotide agentmay act by one or more of anumber of mechanisms, includinga cleavage-dependent or cleavage independent nechanism, A cleavage-based mechanism can be RNAse H dependent and/orcan include RISC complex function.Cleavageindependentmechanismsinclude S occupancybased translational arrestsuchascanbemediatedbymiRNAs,orbindingof the oligonucleotide agent to a protein, as do aptamers Oligonucleotide agents may also be used to a]ter the expression of genes by changing the choice of splice site in a pre niMRNA. Inhibition of splicing can also result in degradation of the improperly processed message, thus down-regulating gene expression,

7w Theterm "double-stranded RNA" or "dRNA". as used herein, refers to a complex of ribonucleicacid molecules, having a duplex structure comprising two anti parallel and substantially complementary, as defined above, nucleic acid strands. The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they maybeseparate RNA molecules.WhereseparateRNAmolecules, such dsRNA are often referred to in the literature as siRNA ("short interfering RNA") Where the two strands are part of one larger molecule, and thereforeare connected by an uninterrupted chain of nucleotides betweenthe 3end of one strand and the Tend of therespective otherstrand forming the duplexstructure, the connecting RNA chain is referred to asa"hairpin loop" "shorthairpin RNA" or "shRNA". Where he two strands reconnected covalently by means other than an uninterrupted chain of nucleodides between the 3'-end of one strand and the 5'end of the respective otherstrand Inning the duplex structure, the connecting structure is referred to as a "linkerThe RNA strands may have the same or a different number ofnucleodes.Themaximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minusanyoverhangsthatarepresentintheduplex.Inadditiontotheduplexstructure, a dsRNA may comprise one or more nucleotide overhangs. In additionas used in this specification, "dsRNA" may include chemical modifications to ribonucleotides, includingsubstantialmodificationsat ntiple nucleotides and including all types of modifications disclosed herein or knownin the art Any such modifications, as used in an siRNA typemolecule, are encompassed by "dsRNA" for the purposes ofthis specification and claims.

As used herein, a "nucleotide overhang" refers to the unpaired nucleotide or nuceotides that protrudefrom the duplex structure of a dsRNAwhen a 3'end of one strand of the dsRNA extends beyond the 5'-end of the other strand, or vice versa, "Blunt" or "blunt end"means that there are no unpaired nucleotides at that end of the 15 dsRNA, i,e, no nucleotide overhang. A "blunt ended" dsRNA is a dsRNA that is double-stranded over its entire length, i.e,no nucleotide overhang at either end of the molecule. For clarity, cheni'4capsornon-nucleotide chemicamoietiesconugtedto the 3' end or 5' end of an siRNA are not considered in determining whether a siRNA has an overhang or is blunt ended.

The term"antisense strand" refers to the stand of a dsRNA which includes a region that is substantially complementary to a target sequence, As used herein, the term "region of complmentarity" refers to the region on the antisense strand that is substantially complementary to a sequencefor example a target sequence, as defined herein, Where the region of complementarity is not fully complementary to the target sequence, the mismatches are most tolerated in the terminal regions and, if present, are

generally in a terminal region or regions, e.g., within 6, 5,4, 3, or 2 nuleotides of the 5' and/or 3' terminus. The term "sense strand," as used herein, refers to the strand of a dsRNA that includes a region that is substantially complementary to a region of theantisense strand.

The terms"silence" and "inhibit the expression of', in as far as they refer to a target gene, hereinlrefer to the at least partial suppression of the expression of the gene, as manifested by a reduction of the amount of mRNA transcribed from the gie which may be isolated froma first cell or group of cells in which the gene is transcribed and which has or have been treated such that the expression of the gene is ihibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells), The degree of inhibition is usually expressed in terms of

(niRNAin control cells) (mRNA in treated cells) *100% S.................... .....................

(mRNA in control cells)

Alternatively, the degree of inhibition may begiven in terms of a reduction ofa parameter that is finctionally linked to gene transcription,e.gtheamountofprotein encoded by the gene which is secreted by a cell; or the number of cells displaying a certain phenotype, e.g apoptosis. Inprinciple, gene silencing may be determined in any cell expressing the target, either constitutively or by genomic engineering, and by any appropateassay, however, when a reference is needed in order todeternewhether S a givendsRNAinhibitsthe expression of the gene by a certain degree and therefore is encompassed by the instant invention, the assay provided in the Examples below shall serve as such reference.

For example, in certain instances, expression of the gene is suppressed by at least about 20%, 25%.35%, or 50% by administration of the double-sranded oligonclotide cfthe invention. In some embodiment, the gene is suppressed by at least about 60%, 70%, or 80% by administration of hee double-stranded oligonu leotide of the invention, in some embodiments, the gene is suppressed byat least about 85%, 90%, or 95% by administration of the double-stranded oligonucleotide of the invention As used herein, the terms "treat", "treatment", and the like,refertorelieffromor 1 alleviation of pathologicalprocesses which can be mediated by down regulating a particular gene In the context of the present invention insofar as it relates to any of the other conditions recited herein below (other than pathological processes which can be mediated by down regulating the gene), the terms "treat", "treatment", and the like mean torelieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression ofsuch condition. As used herein, the phrases "therapeutically effective amount" and "prophylactically effective amount" referto an amount that provides a therapeutic benefit in the treatment prevention, or management of pathological processeswid can be mediated by down regulating the gene on or an overt symptom ofpathological 2 processes which can be mediated by down regulating the gene; The specific amount that is therapcutically effective can be readily determined by ordinary medical practitioner, and may vary depending on factors known in the art, such as, e.g the type of pathological processes which can be mediated by down regulating the gene, the patient's historyand age, the stage ofpathological processes which can be mediated by down 3o regulating gene expression, and the administration of other anti-pathologicalprocesses whichcan be mediated by down regulating gene expression. An efctiveamount,in the context of treating a subject, is sufficient to produce a therapeutic benefit. The term therapeutice benefit" as used herein refers to anything that promotes or enhances the well-being of thesubject with respect to themedical treatment of the subject's cel proliferative disease. A list of nonexhaustive examples of this includesextensionofthe patients life by any period of time; decrease or delay in the neoplastic development of 5 the disease; decrease in hyperproliferation; redaction in tumor growth; delay of metastase3s;reduction in the proliferation rate of a cancer cell, tumor cell, or any other hyperproliferative cell; induction of apoptosis in any treated cell or in anycellatffectd by a treated cell; and/or a decrease in pain to the subject that can be attributed to the patient's condition. As used hereina "pharmaceutical composition" comprises pharmacologically effective amount of an oligonucleotide agent and a pharmaceutically acceptable carrier. As used herein, "pharmacologically effective amount," "therapeutically effective amount" or simply "effective amount" refers to that amount of an RNA effective to produce the intended pharmacologicaL therapeutic or preventive result, For example,if a given clinical treatment is considered effective when there is at least a 25% reduction in a measurable parameter associated withadisease or disorder a therapeuieally effectiveamountof a drug for thetreatment of that disease or disorder is the amount necessary to effect at least a 25% reduction in that parameter, The tern "pharmaceutically acceptable carrier"refers to a carrier for 2t) administration of atherapeutic agent. Such carriers include, but arernot limited to, saline, bufferedsaline, dextrose, water, glycerol ethanol, and combinations thereof and are described in more detail below, The term specifically excludes cell culture medium. The details of one or more embodiments of the invention are set frth in the accompanying drawings and the description below. Other features, objects, and advantagesof the invention will be apparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OFDRAWINGS Fig I depicts a bar graph comparing the efficacy of various ND98 compositions. Fig. 2 depicts a bar graph comparing the efficacy of various ND98 comositions. Fig. 3 depicts aar graph demonsrating the efficacy of a 6-tailed isomer of ND98.

Fig. 4 depicts a bar graphcomparing the efficacy of association complexes prepared using two different procedures. Fig. S depicts various PEG lipid moieties, including those having various chain lengths. SFig. 6 depicts a bar graph comparing the efficacy of association complexes, Fig,7 depicts a bar graph comparing the tolerability of various complexes as the ratio of lipid to siRNA is reduced. Fig.8 is a flow chart of a process for makingan association complex loaded with nucleic acid. to Fig, 9 arebar graphs depicting the efficacy of siRNAswith two targets, FYII and ApoB. Fig. 10 is a flow chart of a process formaking an association complex loaded with nucleic acid Fig. 11 is a bargraph depicting the effect of particle size ofassociation complexes on the efficacy of a nucleic acid in asilencing assay Figs. 12aand 12b are bar graphs comparing the serum half life of nucleic acid therapeutics in unfortnlatedand formulatedfons. Fig, 13 is a bar graph comparing the efficacy of association complexes having PEG lipids withvaried chain lengths.

2A) DETAILED DESCRIPTION Lipid preparations and delivery systems useful to administer nucleic acid based therapiessuch assiRNA are described herein, Cationie LUpid compounds and lipid preparations Polyvamne lipidpreparaom Applicants have discovered that certain polyamine lipid moieties provide desiraNe properties for administration of nucleic acids, such as siRNA, For example, in some embodiments, a lipid moiety is complexed with a Factor VII-targeting siRNA and administered to ananiminal such as a mouse. The level of secreted serum Factor VII is then quantifed(24 h post administration), where the degree of Factor VII silencing so indicates the degree of in vsiRNA delivery. Accordingly, lipids providing enhanced in vio delivery of a nucleic acid such assiRNA are preferred.inparticlar,Applicants have discovered polyamines having substitutions described herein can have desirable properties for delivering siRNA, such as bioavailability, biodegradability, and tolerability In one embodiment, a lipid preparation includes a polyanine moiety having a plurality of substituents, such as acrylamide or acrylate substituents attached thereto. For example, a lipid moiety can include a polyamnemoietyasprovidedbelow, ex,-~ ~ ~ yb nwy spovd eo. H2N 'N' 'NH2 H F,,n where one or more of te hydrogen atoms are substituted,'forexample with a substiuent including a long chain alkyl, alkenyl, or alkynyl moiety, which in some embodiments is further substituted. Xa and Xbare alk-ylen moieties. In some embodiments, X and X' havethesame chain length, for exampleaX'and X areboth ethylenemoietiesiother embodiments X and X are of differing chain lengths, in someembodiments, where the polyamine includes a plurality of X* moieties, X'can vary with one or more occurrences. For example, where the polyamine is sperrnine, X in one occurrence is 1 propylene,Xain another occurrence is butylenes, and X is propylene. Applicants have discovered that in some instances it is desirable to have a relatively high degree of substitution on the polyamine. For example, in some emboditnents, Applicants have discovered that polyamine preparations where at least 80% (eg, at least about 85%, at least about 90%, at least about 95%, at least about 97%,at least about 98%, at least about 99%, or substantially all) of the polvamines in the preparation haveat least n+2 of the hydrogens substituted with. a substituent provide desirable properties, for example for use in administering a nucleic acid such as siRNA. In some instances it is desirable (preferably) to have one or more ofhetero atoms present on the substituent on the nitrogen of polyamine insomeenbodmenrits, a preparation comprises a compound of formula (1) or a pharmaceutically acceptable salt thereot

R'N N fonnula (I) each X" and X', for eachoccurrence, is independently C 6alkylene; n is 0, 1, 2 3, 4, or 5; each R is independently H,

S R 00

Rt Rb Re Rj Re Wherein at least a + 2 of the Rmoieties in at least about 80% of themolecules of the compound of formula I) in the preparation are not H; m is 1, 2, 3 or 4; Y is0,NR or o S; R'isalkylalkenyl or alkyyl; each of which is optionally substitued; and R2 is H, alkyl alkenyl or alkynyl; each of which is optionally substituted; provided that, if n=0, than at least n + 3 of the R moieties are not H. As noted above, the preparationincludes molecules containing symmetrical as well asasymmetrical polyaminc derivatives Accordingly Xa is independent for each occurrence and X"is independent of X". For example, heren is 2, XcCan either be the samefor each occurrence or can be different for each occurrence or can be the same for some occurrencesand different for one or more other occurrences. X is independentof .X"regardlcs of the number of occurrences of X' in each polyanine derivative, Xa for each occurrence and independent of X, can bemethylene, ethylenepropylene, butlene,pentylene,orhexylene.Exemplarypolyaminederivatives include those polyarnines derived ftom NcN(ethane12-diy)diethane1,2-diamine,ethane-1,> diamine, propane-I,3-diamine, spermine, spennidine,putrecine, and N<(2 Aminoethy)-propane-1,3-diamine. Preferred polyamine derivatives include propane 1,3-diamine and N'N(ethanel,2-diyl)diethane-l,2diamine 'The polyamine ofriula (1) is substituted withatleastn2.moieties that are not H In general, each non-hydrogen R moiety includes an alkyl, alkenyl, or alkynyl moiety, which is optionally substituted with one or more substituents, attached to a nitrogen of the polyamine derivative via a linker. Suitable linkers include aides, esters, thioesters. sulfones, sulfoxides, ethers, amines, and thioethers, in many instances, the linker moiety isbound to the nitrogen of the polyamine via an alkylene moiety (eg.,methyleneethylene, propylene, or butylene)For example, anainide or ester linker is attached to the nitrogen of the polyamine through a methylene or ethylene moiety, Examples of preferred amine substituents are provided below:

0 9 0 1 t-J R2

In instances where the amine is botd to the linker-R portion via an ethylene group 1,4 conjugated precursor acrylate or acrylamide can be reacted with the polyamine to provide the substituted polyamine. In instances where the amine is bound to the linker R portion viaa methylene group, an amide or ester including an alpha-halo substituent, S such as an alpha-chloro moiety, can be reacted with the polyamine to provide the substituted polyamine, In preferred embodiments, R2 is H R moieties that are not H, all requirean R moiety as provided above. In general the R moiety is a long chainmoiety, such as C-C3 a alkyl,0Cr2akenyl,or CCn alkynytL SIn some preferred embodiments, R' is an alkyl moiety, For example R is CW C1 alkyl, such as C. alkyl. Examples of especially preferred Rmoieties are provided below. 00 HN2)nCHs and (CH2),ICH 3

The preparations including a compound of formula (I) can be mixtures ofa 2 plurality ofcompoundsofformula(1),Forexamplethepreparation can inchidea mixtureofcompoundsofforla(I)having varying degrees ofsubstitution on the polyamine moiety. However, the preparations described herein are selected such thatat least n + 2 of the R moieties ina least about 80% (e.g, at least about 85%, at least about 90%, at least about 95%, at leastabout 97%, at leastabout 98%, at least about 99%, or substantiallyall) of the molecules of the compound of formula (1) in the preparation are not H, 1a some embodiments, a preparation inciades a polyamine moiety having two amino groups wherein in at least 80% (e.g, at least about 85%,at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or substantially all) of the molecules of formula (1) in the mixture are substituted with thee R moieties that are not H. Exemplary compounds of formula (I)are provided below.

R ,R and N R R

In some preferred embodiments R is 0 0 R or R

some preferred embodiments, RisCo-C~aalkyl, or Cw-C:alkenyl. in some embodiments, a preparation includes a polyamine moiety having three orfour (eg, four) amino groups wherein at leastn+2 of the R moieties in at least about 80% (e.g,.atleastabout 85%, at leastabout 90% at least about 95%. at least about 97%, at least about 98%, at least about 99%,or substantially all) of the molecules of funnula (1) are not H.Exemplarycompoundsoffommla(I)having4aminomoieties are provided below. Examples of polyamine moietywrall (ie,n+4) R moieties are not H are below: R R

R R

In some preferred embodiments R is 0 0 N' or R,'

in sonic preferred embodiments, R isC,w-Ca alkyl (e.g, C alkyl), or ClC alkenyl. Examples of polyamine moieties where five (i.e, n+3) R moieties are not H are provided below: R R H H R..Nt-xN and R' R R R RP

nsome preferred embodiments R is

NRt or R' R2

In some preferred embodiments, R iSCwoCi alkyl (e.g,, C2 alkyl), orCTC

Examples of polyamine moieties where four (i.e, n+2) R moieties are not H are provided below: R H R

R R H R R H H H R~'N N' 'N N' R R ' N Ns-NR H RR R

R andHN NNR R R3 n some preferred embodiments R is

N' or R

unmsome preferred embodiments, R isClC s alkyl (e.g.,C alkyl),or 0 Cyo aikenyl. Insome preferred embodiments, the polyamine is a compound of isomer (1) or (2) below, preferably a compound of isomer (1) H

0

N OA

isomer (1) H

'0 H

H H isomer (2),

In some embodiments, the preparation including a compound of formula (I) includes a Mixture of molecules having formula (I). For example, the mixture can include moleculeshaving the same polyamine core but differing R substituentssuch as differing degrees of R substituents that are not H. Insome embodiments, a preparation described hereinincludes acompound of fonnula (I) having a single polyamine core wherein each R of the polyamine core is Cither or a singlemoiety such as 0D 0 - -~or A'

The preparation, therefore includes a mixture of molecules having fomula (i), wherein 1) theimixture is comprised ofeither polyamnine compounds of formula (1) having a varied number of R moieties that are H and/or a polyamine compounds of finnula (T) having a single detennined numberof R moieties that are not H where the compounds of fonnula (I) are structural isomers of the polyamine, such as the structural isomers provided above, in some preferred embodiments the preparation includes molecules of fonnla (I) such that at least 80(esg., at leastabout 85%, at least about 90%,at least about 95%, at least about 97%, at least about 98%, at least about 99%, or substantially all) of the molecules are a single structural isomer. Insomeembodimentsthe preparation includes a mixture of two or more compounds of formula (I). In someembodiments, the preparation is a mixture of structural isomers of the same chemical formula, In some embodiments, the preparation is a mixture of compounds of fonnula (I) where the compounds vary in thechemical nature of the R substituents, For example, the preparation can Jncludea mixture of the following compounds:

RgN 'NE 'NR4 Rj

fonnula (I)

wherein n is 0 and each R is independently H or rY and

R 2N 'N 'NR 2

fomnula(l) 0

wherein r is 2 and each R is independently H or 'l In some embodiments, the compound of fom la (I) is in the frmn of a salt, such as apharmaceuticallyacceptable salt, A salt, for example, can be formed between an anion and a positively charged substituent (ewgamino) on a compound described herein. Suitable onions include fluoride, chloride, bromide, iodide, sulfate, bisulfate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, acetate, fumarateoleate, valerate, maleate, oxalate, isonicotinate, lactate, salicylate, tartrate, tannate, to pantothenate, bitartrate ascorbate, succinate, gentisinate, gluconate, glucaronate, sacchtrateo, fimate, benzoate, glutamate, ethanesulfonatebenzenesuifonate,p toluensdfonate, andpamoate. [n some preferred embodiments, the compound of formula (1) is a hydrohalide salt, such as a hydrochloride salt, Compounds of formula (I)can also be present in the form of hydrates (e.g., (H20)) and solvates, which are included herewith in the disclosure, BioclIeavablecationiciipii Applicants have discovered that certain cationic lipkis that include one or more biocleavable moieties can be used as a component in an association complex, such as a liposome, for the delivery of nucleic acid therapies (eg,dsRNA). For example, disclosed herein are eatioic lipidsthat are subject to ceavage in vivo, forexample, via an enzyme such as an esterase, an amidase, or a disulfide cleaving enzyme. In some instances, the lipid is cleaved chemicallyfor example by hydrolysis ofan acid labile moiety such as anacetal or ketal. In some embodiments, the lipid includes amoiety that is hydrolyzed in vitro and then subject to enzymatic cleavage by one or more of an esterase, amidase, or a disulfide cleaving enzyme This can happen in vesicular compartments of the cell such as endosomes. Another acid sensitive cleavable linkage is -thiopropionat linkage which is cleaved in the acidiccavironment ofendosomes (Jeong et at. Bioconjugate chem.2003, 4, 1426).

In some embodiments, the invention features a compound of forula (X) or a pharmaeuti acceptable salt thereof; wherein R R4 Rh LiR

forrnulad() wherein R' and R 2 are each independently H, CrC6 alky optionally substituted with 1-4 R, CZC alkenyl, optionallysubstituted with 1-4R, or CNR )(NR )2; R andR are each independently alkyl, alkenyl, alkynly, each of winch is optionally substituted with fluoro, chloro, bromo, or iodo; L and L2 areeachindependently NRC()-, -C(O)NQ-, -OC(O)-, -C(O)o-, S-S-, N(Rb)C()N(RE),-OC(Q)N(R)-,-N(R)C(OO-, -O-N=-O, OR -OC(O)NH; or L.R'andL-Rt can be taken together to form an acetal or a ketal; R5 is fluoro, chloro bromo,. iodo,-OR',-N(R )(R),-CN, SRI" S(O)R

' S(D1)2R' 1 RIis H, CC6 alkyl, R 7 is H or C-C aikyl; each R",ad R'are independently H or CrC? alkyl; R'" is H ior CrC alkyl; m is 1,2,3,4, 5, or 6; n is 0, 1, 2,3, 4, 5, or 6; and pharmaceutically acceptable salts thereof in some embodiments, R is 1-1, a lower alkylsuch as methyl, ethyl propyl, or isopropyl, or a substituted alkyl,such as 2hydroxyetbyl. In some embodiments, RIis H or a lower alkyl, such as methyl, ethyl, propyl, or isopropyL. fn some embodiments, RI or R iform a quanadinemoiety withthenitrogenof formula (X). R;-. 3 and L-R4 or the combination thereof provide at least one moiety that is cleavedinvivo. Insomeembodiments,bothULRIandL-Rarebioleavable.For example, both L'R and L-R4 are independently subject toenzymatic cleavage(e.g., by an esterase, amidase, or a disullide cleaving enzyme)n, i some embodiments, both

L and 2 are the same chemicalmoietysuch asanester, amide or disulfide. In other instances, and19 are differentfr exampeone ofl'or sanester an the other of L or 1 is a disufide. In someembodiments,LtI&and LR4 together form an acetal or ketal moiety, which is hydrolyzed in vivo. In some embodiments,one of -R3 or LR issjct toenzymatic cleavage. For example, one ofL-R, or L-R 4 is cleaved in vivo, providing a free hydroxyl moiety or free amine on thelipid, which becomes available to chemically react with the remaining L-R ortt-R moiety. Exemplaryembodiments are provided below:

X'H R X R X R8 N2 ",,A<.N'R4 N NA 4 --.......

. R 4 R RaC( R 4NH2 X = 0 or NH Y= O or NH

YI R X' --o H, *L ~%~R ' X-N IR x R2 ' --' -I R SH 3 ~ R 0(0). - RRY X OorNH Y =0 or NH

'Insome preferred embodiments, a carbamate or urea moiety is included in combination with an amide, ester or disulfide moiety. For example, the lipid includes an ester moiety, which upon cleavage (e.g.,enzymatic cleavage) becomes available to chemically react withthearbamate or ureamoiety. Some preferred combinations of L and i include two aides two esters, an amide and anester, two diulfides, an amide and a disulfide, an ester and a disulfide, a carbamate and a disulfideandaureaanda disulfide. Exemplary compounds areprovided below: Amide and ester linkages with Z configuration (two double bonds)

5I

R Rn R' - R'0 i ON m

R' R

'7N'

R H, Me Et propyt isoproylor2-hydroxyet'ylandH 1 to36m 1-8,i= 1-10 R:HM, Et, propy isopropyl or-hydroxyethyl andHR" Me; I =1toO 6nmz1-5, nz-1 R H)Me, Et, propyt isopropyl or2-hydroxyethyl and R" Et; i-= Ito , m t-8, n =110 R' H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R" = propy =1toi 6 m 1 1-10 R' H, Ma Et propy isopropyl or2-hydroxyethyl and R'aisopropyl; 1:1 to6, m= 1-4, a 1-10

Amide Ester linkagewith Z configuration (three double bonds)

R'

RNR iio IM o 'm

R'HN m R' HN

R'= H Me, ,*,prpy,isopropyl or 2-ydroxyethyl and R' H-,I I to 6,; 1-6.in 1-10 R'= H Me, Et, propy! isopropyl or 2-hydroxyethyl and R" = EtI = toItt, m = 6n 1-10 R' H, Me, Etpropyt isopropyl or 2-hydroxyethyl and R°= propyl; t = 1 tom -o= - -10 R' H, Me, Et, popy isopropyl or 2-hydroxyethy! and R' = isopropyt: ito 6, m = 1-8, n 10

Amides and ester linkages with E configuration(twodoublebonds)

%'N >m H 0

0< 0NMJ

R' N R' H N R:R

R= H Me E propy sopropyl or 2-hydroxyethyk and R"= H; I 1 to 6, m n 1-10 Rf H, Me, El, propy isopropyi or 2,-hydroxyethy, and R' = Me; 1 to 6, m =18n = 1-10 R H, Me, E propy isopropyl or 2-hydroxyethyll and R" = El; I =1 to 6, m zz14A, n =z 1-10 R H, Me, Et, propy, lsopropyl or 2~hydroxyethyll and R" propy -; 10 i o , m. 1-8, n = .110 R H, Me, Et prpyisopropyl or 2-hydroxyethy and R" = isnpropyl: to 6; m = 1-, a = 10

H5

Amidesand ester linkages with E conliguration (three double bonds)

R

6,m m R' HN N H", an

R' =, HMe, Et, propyl,isopropyl or 2~hydroxyethyl and R= H: t o 6, m~ i- 18 n =110 R' = H, M,-. E, propyl,isoprpyl or 2-hydroxyethyl and R= Me; I =I to 6 m =z 148 ri = 1-10 R = ii Me , Et, propyl,isopropyl or 24 ydroxyethyl and R" =Et; I = to 6, mr = 14 8 n= 1 R' = Hi, MAs; E-t propy, isopropyl or2-hydroxyethyl and R= propyl,; iz =I to 6.. m o 1-8, n =1-10 R'= ,Me, {Et;propy isopropy or24ydrxyethyl and R" ispropyt; i = 1 to 6, m =V¾ 14 1-110

Disullfde Likages

R' m

R" S

R'= H, Me, Et propyl isoprlopr 2hydroxyethyyand R" H; 1 = 1t 6, I 6-2 8 R= H-, , E, propym isopropy o2oydro xyethyl and Re Me; I = 1 t6 to 6, m 026 '=M ,MErpyyoprlyor2ydroxya hylEandR=E S-28 R'= H MMe, et, prEpiL isopropyl or 2hydroxyethyl anrd Ra'= propyl; I =1 to 6m - 628 R'P= H> Me, Et, propyr,isoproyioo2t-ydroxyethy nd and R"= isopropy; i 1to 6,m =o- 6-28

.54

Disulfide linkages with unsaturated alkyl chains, E and Z configuration

A rn RR

m \

R S SS in y 'tY"'NR"'

R, = H, M Et, propy isopropyl or 2-hydroxyethyl and R"z H; 1 to 6, m 14 , 'I = 1-1V' R H, Me, Et, propy. isopropyl or 2-hydroxyethyl and R" Mei I to 6 m 18, n =1-0 R= H, Me Et, propyl, isopropyl or 2-hydroxyethyl and R"~ Et i to 6, m = 1-8 n 1-10 R =H, Me, E, propyi isopropyl or 2-hydrcxyethyl and R" propyl;1 I to 6, m 1-6, = 1-10 R = H, Me, El, propyt isopropyl or 2-hydroxyethyl and R" isopropyt 1to, m z. 1-8, n = 1-10

Amide and disulfide linkages with saturated andunsaturated alkyl chains

N R"' s \m

R' H, Me, Et, propyl isopropyf or 2-ydroxyethy! and R" m H;I 1 to , m -2a R'= H, Me, Et, propyt isopropyt or 2-hydroxyethyl and R"= Me; = 1 to 6, m = 6-28 R' = H, Me, Et, propyl, isopropyl or 2hydroxyethyt and R'= Et:; I to 6, mt 6-28 R= H, Me, Et. propyl, isopropyt or 2-hydroxyethyl and R" = propy: i 1 to 6, m 6-28 R: H. Me, Et propyl, sopropy or 2-hydroxyethyl and R" * isopropy; I 1to 6, in 6-28

R .M m t t R% S-'--H RR' " 'nt'' 0 RHS IN' N S m

R HN R HN i R" NR A R'"'g 1 - 6 a' n

R'H HN r

R"' SA R" N' .

nn

R' H, Me t, propyt isopropyl or 2-hydroxyethyfand R" = H; i 1 to 6 m 1-, n 1-0 R' H Me, Eit, propytisopropyl or 2-hydoxyethyl and R" = Me; = I to 6,n -18 n = 1-10 R' HMe Et, propyl isopropyl or 2hydroxyethyl and R= Et,; 1 to 6, m =6n I-I R'HKM, Etpropyisopropyl or 2-hydroxyethyl and R" = propyl; I = I to5, m = 1-5,n =: TA R=H Me, t,propy propropyl or 2-hydroxyethyl and R" = isopropy; I to 6, m = 1-8, n - 1-10

Eter anddisuilde linkages with saturated and unsaturatedalkyl chains

R= HMeEt p,pror ompy or 2-hydroxyethyi and R= H;I I to 6, m = 6-28 R = H, M Et, propylsoropyl or 2hydroyethfyl andR" = Me; I to 6, m = 6,98 R' H, Me Et, propyl sopropyl or 2-hydroxyethyi and R" Et; f to 6,m = 5-28 R= H, Me Et propysopropy or 2-hydroxyethyl and R" =propyl;1= 1 to 6, m = 6-28 R- H, Me Et propysopropyl or 2 hydroxyettyi and R" sopropyl; I 1 to 6, m 6-28

F',0 -nR' t) rn( R"N kIl S R R N J s s

RR,

R =H, Me, Et, propyl isopropy or 2-hydroyethfyl and R" =H, 1 to 6, m 1-8, n 1-10 R= H MW, EL propy, OPropyOr 2hydroxytyI ahd R"= Me, = to 6, m' = 1-8, rk 1-10 R'z H, Ma, Et, propyl, isopropyl or 2 hydroxyethyl and R" = ER I =I to 6, m =- 1-8,n ='1-10 R- H, Me, E, propyl, fsopropy or 2-hydroxyetyl and R" z propyl; I =1 to 6, m z 1-8, nit 1410 R 2H Maj Et propy;| isopropyl Or 2-hydroxyetl arO, R" = isopropyl; Ei = o 6, m = 1-8, n = 1410

Carbamate orurea and disulfide linkages with alkyl chains H H O N N N Ol R' o) m R' HN m R' HN R'N S R"' S

R' H, Me, Et, propyl isopropyl or 2-hydroxyethyl and R"= H; I = I o 6, r 6-28 R' H, Me, Et propyl isopropyl or 2-hydroxyethyl and R" = Me; I = 1 to 6 r = 6-28 R' H, Me, Et, propyl isopropyl or 2-hydroxyethy and R" = Et; i = 1 to 6, m ~ 6-25 R' HMe, Et propyl isopropy or 2-hydroxyethyl and R" = propy; = 1 to 6, m = 628 R HMe, Et, propyl isopropyl or 2-hydroxyethyl and R" = isopropyl; i= 1 to 6, m = 6-28

Carbmnate or ureaand disulfide linkages with unsaturated alkyl chains H NAN

I' 'f^l"'N R` R" r H N N

R-Nt) S- ' R'N ANSNt>g!

m 8 H H

N N

H R H r R 'HR R: HN R HJNN

m jn H

R'HN r R, N fn A O-,NA O N"

nN RI 'l0 HN HR:

N HN

R H Me, Et propyl isopropyl or 2hydroxyethyl and R =H : H to 6, m =-26 R' H, Me, E propyL isopropyl or 2-hydroxyethyl and R"= Me; I = I to 6, m = 6-28 R' H, Me, Et, propyl isopropyl or 2-hydroxyethyl andR" El, I = I to 6, m = 6-2P - H, Me, Et, propyl isopropyl or 2-hydroxyethyl and R" =propyi; = I to 6, m = 6-28 R H, Me, Et, prpyi isopropyl or 2-bydroxyethyl arnd" isopropyl; I= 1 to 6n =6-28

Carbamate or ara aid disulfide linkages with unsaturated alkyl chains

R' HN i HN R"' >'m s NAN R") S

RNN m R' HN N

O Q{AN N l"

N XZA7N N"NX FN7

R"" R"' ' USm

R' H.Me, Et, propylisopropy or 2-hydroxyethyl and R H; Ito 6, m 6-28 R' H, Me, Et, propy isopropyi or 2-hydroxyathyl andHR'= Me; = I to 6, m = 6-23 R' H, Me, Et; propyl, isopropyi or 2-hydroxyethyl and R =; I to 6, m 6-28 R' i, Me, Et, propyl, sopropy or 2-hydroxyethyl and R" propyl I 11o 6, m Z 6-28 R'= H, Me, E, propyl, isopropyl or 2-ydroxyethyl and R' isopropyi; I Ito 6,im 6-2

Carbamate andurea linkages with unsaturated alkyl chains

u R,

R H R' R"N R"

H/k 0

, O N~~~~~~. ma........ K .. ~, N ..

R' e .p'6 ½

N N\ H

Htn RHN RN N NN N O

R- Me, Et, propy, isopropyl or 2-hydroxyehyl and R" H; I to 6 m 1-10,n 10 R H MEt, propy isopropyl or 2-ydroxyethyt and R" Me: Ito 6, m = 1-10n = 1-10 R H Me, Et propyl isopropyl or2-hydroxyethyland R"z Et; I to6, rm = 1-0, n =10 R' HMe, Et, propytisopropyor2-hydroxyehyl and R"t= propyl; 1 to6, I =1-10ii 1-10 RHMe, Etpropyl isopropyl or 2-hydroxyethy! andHR" isopropyi: I =1to 6,tm 110, I =1

In sonecembodiments, the lipid includes an oxime or hydrazone, which can undergo acidic cleavage. R 3 and Rzare generally long chain hydrophobic moieties, such as alkyl, alkenyl, or alkyny ,In some embodimentsR or R' are substituted with a halo moiety, for example, to provide a perfluoroalkyl or perfluoroalkenyl moiety, Each of R- and R' are 1o independent of each other. In sonie embodiments, both of R and R are the same.i n some embodiments, R: and Riare different. Insome embodiments Rand/or Rare alkyl. For example one or both of Re and/orRare Cf to C3 alkyl, e.g., Ciw to Cm alkyl, C2 to C20 alkyl, or C2 alkyL in some embodiments, R and/or are akenyL In some preferred embodiments, R" and/or R 4 include 2 or 3 double bonds, For example Rand/or R 4 includes 2 double bonds or R" and/or RO includes 3 double bonds. The double bonds can each independently have a Z or E configuration. Exemplary alkenyl Moieties are provided below whereir x is an integer from I to ;and y is an integer from 1-10. in some preferred embodiments, R 3and/or RareC6 to Co alkenyl,e CO to C akeny, CtoC a alkenyl or C7 alkenyl, for example having two double bonds, such as two double bonds withZ RonfgurationR and/orR can be the same or different. In some preferred embodiments, R and R are the same. In some embodiments, R and/or R4 are alkynyl. For example Ce to C3 alkyny, e.g., C to C, alkyyl C to C alkynyl RI and/or Recan have from to 3 triple 1o bonds, for example, one, two, or three triplebonds, in soni embodiments, the compound of formula (X)is inthe form of a salt, such as a pharmaceutically acceptable salt A salt, for example, can be formed between anan and apositively chargedsubstituent (e.g., amino) on a compound described herein, Suitableanionsinclude fluoride, chloride, bromide, iodide, sulfate, bisulfate, is nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, acetate, fumarateoleate, valerate, maleate, oxalate, isonicotinate, lactate, salicylate, tartrate, tannate, pantothenate, bitartrate, ascorbate, succinate, gentisinate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, ethanesulfonate, benzenesulfbnate toluensulfinate, and panoate. In some preferred embodiments, the compound of formula (X) isahydrohalide salt, such as a-hydrochloride sait. Compounds of formula (X) can also be present in the forn of hydrates (e.g., (H2 0),) and solvates, which are included herewith in the disclosure,

PEG4pid compounds

Applicants have discovered that certain PEG containing lipid moieties provide desirable properties for administration ofa nucleic acid agent such as single stranded or double stranded nucleic acid, for example siRNA, For example, when a PEG containing lipid, such as a lipid described herein, is ftrmiulated into an association complex with a nucleic acid moiety, such assiRNA and administered to a subject, the lipid provides enhanced delivery of the nucleic acid moiety. This enhanced delivery can be determined, for example, by evaluationin a genesilencing assay suchas silencingof FVIL In particularApplicants have discovered the PEG-lipids of formula (XV) can have desirable properties for the delivery of siRNA, including improved biavailability, diodegradability, and tolerability. In some embodiment, the PEG is attached via, a linker moiety to a structure including two hydrophobic moieties,such as a long chain alkyl moiety, Exanplary PEG-ipids are provided above, for example, thoseencompassed by fornrula (XV) (XV'), and (XVi) n some preferred embodiments, the PEG-lipid has the structure to~ below: wherein thepreferred stereochemistry of the chiral center is 'R' and the repeating PEG moiety has a total average molecular weight of about 2000daltons In some embodiments, a PEG lipid described herein is conjugated to a targeting OH OH

HO&dr2/A4 moiety, eg., a glycosyl moiety such as a ACHN some embodiments, the targeting moiety is attached to the PEG lipid through a linker, for example a linker described herein. Exemplary targeted PEG lipid compounds are compounds of fomula (XX),(XX?), (XX),and (XXI)described herein.Methods ofmakingsuch lipis are described, for example, in Examples 42 and 43, Methods ofmaking cationic lipid compounds and eationic lipid containing preparations The compounds described herein can be obtained from commercial sources (e,g, Asinex, Moscow, Russia; Bionet Canelford, England; CheinDiv, SanDiego, CA; Congenex, Budapest, Hungary; Enamine, Kiev, Ukraine; IF Lab, Ukraine; Interbiosereen, Moscow, Russia; Maybridge, Tintagel, UK; Specs, The Netherlands; Timtec, Newark, DE; Vitas-M Lab,Moscow,Russia)or sythesized by conventional methods as shown.below using commercially available startingmaterialsand reagents,

Methods ofmakingpolyamine lipjds In sone embodiments, a compound of formna (1) canbe made by reacting a polyamine of formula (Ill) as provided below

H2N NNH2 Sa y

formula (iII) wherein X, X, and n are defined as above with a 1,4 conjugated system of formula (IV) 0

1o formula(IV) wherein Y and R are defined as above to provide a compound of formula (I). Income embodiments, the compounds of formula (ill) and (IV) are reacted together neat (i.e., free of solvent); For example, the compounds of formula (iii) and 1 (V) are reacted together neat at elevated temperature (e.g., atleast about 60 'C, at least about 65 °C, at least about 70 °C, at least about 75 °C, at least about 80 C,at least about 85 °C, or at least about 90 °), preferably at about 90 °C. in some embodiments, the compounds of fomula (III) and (IV) are reacted together with a solvent (e.g., a polar aprotic solvent such as acetonitrile or DMF), For example, the compounds of formula (I) and (IV) are reacted together in solvent at an elevated temperature fromabout 50 C to about 120 °C, n some embodiments, the compounds of fomala (Ill) and (IV) are reacted together in the presence of a radical quencher orscavenger (e,g hydroquinone). The reaction conditions including a radical quencher canbe neat or in a solvent e.g., a polar aprotic solventsuch as acetonitrile or DMF, The reaction can be at an elevated temperature (eg.neat at an elevated temperature such as 90 °C or with solvent at an elevated temperature such as from about 50 ° to about 120 °C). The term "radical quencher" or "radical scavenger" as used herein refers to a chemical moiety that can absorb free radicals in a reaction mixture. Examples of radical quenchers/scavengers include hydroquinone, ascorbic acid, crsols, thiamine, 3,5-Di-tert-butyl-4 hydroxytoluenetert-Butyi~4-hydroxyansol and thiol containing moieties, in son embodiments, the compounds of forula (Ill) and (1) are reacted togetherin the presence of a reaction promoter (eg., water or a Michael addition a promoter sueh as aceticacid, boric acid, citric acid, benzoie acid, tosic acid, pentafiuorophnolpicic acid aromatic acids, salts such as bicarbonate,bisulphate mono and di-hydrogen phophates, phenols, perhaloptenols, nitrophenols, sulphonic acds,PTS, etc.), preferably boric acid such as a saturated aqueous boric acid, The reaction conditions including a reaction promotercan be neat or in a solvent e.g., a polar aproic solvent such as acetonitrile or DMF. The reaction can be at an elevated temperature(eg, neat at an elevated temperature suchas 90 °C or withsolvent at an elevatedtemperature suchas from about 50 °C to about 120 1C). The term"reaction promoter" as used herein refers to a chemical moiety that, when used in a reaction mixture, accelerates/enhances the rate of reaction. a The ratio of compounds of formula (Il) to formula (IV) can be varied,providing variability in the substitution onthe polyamine of formula (11), hi general, polyamines having at least about 50% offthe hydrogennmoieties substituted with a non-hydrogen moiety are preferred, Accordingly, ratios of compounds of formula (l)/fonnula(IV) are selected to provide for products havinga relatively high degree ofsubstituton of the free amine (eg, at least about 50%, at least about 55%, at least about 60% at least about 65%, at leastabout '70%, at least about 75%, at least about 80%, at least about 85% at least about 90%, at least about 95%, at least about 97%; at leastfabout 99%, or substantially all). In somepreferred embodiments n is 0 in the polyamine offormula (ill), and ther atio of compounds of fomiula (il) to compounds of formula (IV) is from about 1:3 to about 1:5, preferable about 1:4. In sone preferred embodiments, I is 2 in the polyamine of formula (Il), and the ratio of compound of formula (1I) to compounds of formula (IV) is from about 1:3 to about 1:6, preferably about 1:5. in someembodiments, the compounds of femmla (1i) and formula (IV) are reacted in a two step process. For example, the firs step process includes a reaction so mixture having from about 0.8 about I2 molar equivalents of a compound of fnnula (IH),with from about 3.8 to about 4.2 molar equivalents ofacompound of fonnula (V) and the second step process includes addition of about 0.8 to 1 2 molar equivalent of compound of fannula (V) to the reaction mixture. Upon completion of the reaction, one or more products having bomla (I) anbe isolated from thereaction mixture. For example, a compound of formula (1) can be isolated as a single product (e.g., asinglestructuralisomer) or as a mixture of product (e.g., a plurality of structural isomers and/or a plurality of compoundsof formula (1)). In some embodiments, one or more reaction products can be isolated and/or purified using chromatography, suchas ash chromatography, gravity chromatography(e.g gravity separation of isomers using silica gel), column chromatography (e.g ,normal a phaseHPLC or RPHPLC), or moving bed chromatography, in some embodiments, a reaction product is purified to provide a preparation containing at least about 80% ofa single Qomnpound,suchas a single structural isomer (e.g, at least about 85%, at least about 90%, at least about 95%, at least about 97%,at least about 99%), In some embodiments, a free amine product is treated with an acid such as Hl toprove an amine salt of the product (e.g.,a hydrochloride salt).In some embodiments a salt product provides improved properties, e.g., for handling and/or storage, relative to the corresponding free nine product In some embodiments, a salt product can prevent or reduce the rate of formation of breakdown product such as N-oxide or N-carbonate formation relative to the correspondingfreeamine.In some cmbodiments, a salt product can have improved properties for use in a therapeutic tfonulation relative to the corresponding free amine In some embodiments, the reaction mixture is further treated, for example, to purify one or more products or to remove-impurities such as unreacted starting materials, In some embodiments the reaction mixture is treated with an immobilized (e.gpolymer bound) thiol moiety, which can trap unreacted acrylamide. In some embodiments, an isolated product can be treated to further remove impurities, e.g., an isolated product can be treated with an imnobilized thiol moiety, trapping unreacted aerylamide compounds. in some embodiments a reaction product can be treated with an immobilized so (e.gpolyner bound) isothiocyanate. For example, a reaction product including tertiary amiines can be treated with an immobilized isothiocyanate to remove primaryand/or secondary amiesfrom the product.

In some embodiments, a compound of formula (I) can be made by reacting a polyami of fonnula (Il) as provided below

H2 N N1 NH2 Hi 'n

fonnula (111) whereinXV X", and n are defined as above with a compound of formula (VI)),

QY> R'

forimda (VI)

wherein Q is Cl Br, or 1, and Y and RI are as defined above, In some embodiments, the compound of formula (I1) and formula (VI) are reacted together neat. In some embodiments, the compound of formula (Ii) and fonmua (VI) are reacted together in the presenceof one ormore solvents, for example 1 polar aprotic solvent such as acetonitrile or DMF, In some embodiments, the reactants (fonmda (Il) and formula (VI)) are reacted together at elevated temperature (e,gat leastabout 50 °C, at least about 60°C, at least about 70 °C, at leastabout 80 °C, at least about 90 °C, at least about 100 C). In some embodiments, the reaction mixture also includes a base, for example a carbonate such as K2CO. winsome embodimentsthe reaction mixture also includes a catalyst. In some embodiments, the compound of fornula (VI) is prepared by reacting an anine moiety with an activatedacid such as an acid anhydrate or acid halide (e.4, acid chloride) to provide a compound of formula (VI). The ratio ofcompounds offormula (111) to fonula (VI) can be varied, providing variability in the substitution on the polyamine of formula ( lIn generalpolyamnes having at least about 50% ofthe hydrogen moieties substituted with anonhydrogen moiety are preferred, Accordingly, ratios of compounds of formula (I)/fonnua (VI) areselected to provide forproducts having a relatively high degree of substitution of the free amine(eg, at leastabout 50%, at least about 55%, atleast about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 99%, or substantially all),Isomepreferredembodimentsnis0inthepolyamineoftfrmuia (iI),and the ratio of compounds offormula (II) to compounds of formula (VI) is from about 1:3 to about 1:5, preferableabout 1:4, In some preferred embodiments, n is 2 in the polyamine of formula (111), and the ratio of compound of fomula (Ill) tocompoInds offormula (VI)is from about 1:3 to about 1:6 preferably about 1:5 insomeembodimentsthe compounds of forula (III) and formula (VI) are reacted ina two step process. For example, the first step process includes a reaction mixture having from about 0.8 about 1.2 molar equivalents of a compound of formula (III), with front about 3, to about 4.2 molar equivalents of a compound offormula (VI) and tie second step process includes addition of about 0,8 to 1.2 molar equivalent of compound of formula (VI) to the reaction mixture. 1 In some embodiments, one or more aminemoieties of formula (ill) are selectively protected using a protecting group prior to reacting the polyamine of formula (Ill)with a compound of formula (IV) or (VI), thereby providing improved selectivity in thesynthesisof the final product For example, one or more primary amines of the polvamine formula (111) can be protected prior to reaction with a compound of formula (IV) or (VI), providing selectivity for the compound of fomla (IV) or (VI) to react with secondary amines, Other protecting group strategies can be employed to provide for selectivity towards primary amines, for example, use of orthogonal protecting groups that can be selectively removed. Upon completion of the reaction, one or more products having formula (1)can be isolated from the reaction mixture, For example, a compound of formula (I) can be isolated as a single product (e.g. a single structural isomer) or asa mixture of product (eg,a plurality of structural isomers and/or a phrality of compounds of fimula(I)), in some embodiments, on or more reaction products can be isolated and/or purified using cWomatography, suchas flash chromatography, gravity chromatography (e.g, gravity separation of isomers using silica gel), column chromatography (e.gnormal phase HPLC or RPIPLC), or moving bed chromatography. Insomeembodimentsa reaction product is purified to provide a preparation containing at least about 80% of a single compoundsuch asasinglestructuralisomer(e.gatleastabout85%at least about 90%, at least about 95%, at least about 97 % at least about 99%). In some embodiments, a fee amine product is treated with an acid such as HCl to prove an amine salt of the product (e.g.ahydrochloride salt). In some embodinents asalt product provides improved properties, e.g., for handling and/or storage, relative to the corresponding free mine product. I some embodiments, asalt product can prevent or reduce therate of formation of breakdown product such as Noxide or N-carbonate fonationrelative to the corresponding free amine. In someembodiments, asalt product can have improved properties for use in a therapeutic formulation relative to the correspondingfree amine. in some embodimens, apolyamine cationic lipid can be made in using a regioselective synthesisapproach, Theregioselective synthetic approach provides a convenient way to make sitespecificalkylation on nitrogen(s) ofthepolyarmine backbone that leads to synthesis of specific alkylated derivatives of interest In general a compound offormula (1) is initiallyreacted with areagent thatsdectively reacts with primary amines or terminal amines to block them from reacting or interfering with further reactions and these blockages could be selectivelyremoved at appropriate stages during the synthesis of a targetcompound, After blocking terminal anines of a compound off ula(I),one or more of the secondary anines could be selectively blocked with an orthogonal amine protecting groups by using appropriate molar ratios of the reagent and reaction conditions, Selective alkylations, followed byselective deprotection of the blocked amines and. further alkylation of regenerated amines and appropriate repetition of die sequence of reactions described provides specific compound of interest. For example, terminal amines of triethylenetetramine (1) is 2s selectively blocked with primary amine specific protecting groups (e.g., trifiuoroacetamid)under appropriate reaction conditions and subsequently reacted with excess of orthogonal amine protecting reagent (Boc)20,for e.g.)] in the presence ofra base (for e,g., diisopropylethylamine) to block all internal amines (e.g, Boc), Selective removal of the terminal protecting group and subsequent alkylation oftheterminal amines, for instance with anacryamide provides a filly tenninalamineakylated derivative of compound . Deblo&king of the intemal amine protection and subsequent alkylation with calculated amount ofan acrylamide for instance yields a partially alkylaed product 7. Another approach to make compound 7 is to react terminally protectedcompound 1 with calculated amount of an orthogonal amine protecting reagent [(Boc)20, for eg)] to obtain partially protected derivatives of compound 1 Removal of the terminal amine protecting groups of partially and selectively protected I iand subsequent alkylation of all unprotected amines with an acrylamide, for insance, yields compound 7 of interest; tmoiety Methods o/making ipidshaving biocleavabe in some embodiments, a compound of fomula (X) can be made by reacting a compound of formula

R OH R2 OH formula (XI) with a compound of finnula (XII)

HO' R, formutia (X11) wherein R 1 R 2 ,andiRare as defined above. In some embodiments, the compounds of finulas (XI) and (XII) are reacted in the presence of a coupling agent such as a carbodiimide (e g, a water soluble carbodihnide such as EDCI) Other chemical reactions and starting materials.can be employedto provide a 'o compound of formula (X) having two linking groups L' and LT For example, the hydroxyl moieties of fonnla (XI) could be replaced with amine moieties to provide a precursor to aide orurea linking groups. Upon completion of the reaction, one or more products having formula (X) can be isolatedfrom the reaction mixture. For example, a compound of tonmua (X) can be isolated as a single product (e.g. a single structural isomer) or as a mixture of product (eg., a plrality ofstructuralisomers and/or a pluraity of compounds of fonua (X)). In some embodiments, on or more reaction products can be isolated and/or purified using chromatography, such as fash chromatography, gravity chromatography (e.g.

gravity separation of isomers using silica gel),column chromatography (e.g., normal 3o phase HPLCorRPHPLC),ormoving bed chromatography.insomeembodiments a

'70 reaction product is purified to provide a preparation containing at least about 80% ofa. single compoundsuch as a single structuralisomer (e.g.,at least about 85%, atleast about 90%, at least about 95%, at least about 97%, at least about 99%), in some embodiments, a freeamine product is treated with an acidsuch as BCi to prove an amine salt of the product (eg, a hydrochloridesalt) In some embodiments a sal product provides improved properties, e.g, for handling andor storage, relative to th correspondigfree amine product. In some embodiments, a salt product can prevent or reduce the rate of formation of breakdown product such asN-oxide or Ncarbonate formation relative to the corresponding free amine, In some embodiments, a salt i product canhave improved properties for use in a therapeutic form nation relative to the corresponding free amine, Methods ofmaking PEGipids ThePEG-lipid compounds can be made, for example, by reacting a glyceride moiety (eg a dimyristy glyceride, dipalmityl glyceride, or distearyl glycride) with an 1 activating moiety underappropriatconditions, for example, to provide an activated interediatethat could be subsequently reacted with a PEG componenthaving a reactive moiety such as an amine or a hydroxyl group to obtain a PEG-lipid. For example, a dalkylglyceride (e.g,dimyristyl glyceride) is initially reacted withNN' disuccinimidyl carbonate in the presence of a base (for e.g., triethylamine) and subsequent reaction of the intermediate formed with aP EGamine (e.g,mPEG2000 N12) in the presence of base such as pyridine affords a PEG-ipid of interest, Under these conditions the PEG component is attached to the lipidmoiety via a carbamate linkage, In.another instance a PEG-ipid can be made, for example, by reacting a glyceride moiety(e~g.dimyristyl glyceride, dipalmityl glyceide, distcar'yl glyceride, 255 dimyristoyl gyceride,dipalmitoyl glyceride or distearoyl glyceride) withsucinic anhydideandsubsequentactivation of the carboxyl generated followed by reaction of the activated intermediate with a PEG component with an amine or a hydroxyl group, for instance, to obtain a PEG-lipid, In one exampledimyristyl glyceride is reacted with scenic anhydride in the presence of a basesuch as DMAP toobtain a hemi-succinate The free carboxyl moiety of the hemi-suecinate thusobtained is activated using standard carboxyl activating agents such as HBTU and diisopropylethylamineand subsequent reaction of the activated carboxyl with mPEH2000-NHforinstance, yieldsaPEG- lipid. Inthis approach the PEG component is linked to thelipid component via a succinatebridge.

Associat'on compexes SThe lipid compounds and lipid preparations described herein can be used as a componentin an association complex, for example a liposome or a lipoplex. Such association complexes can be used to administer anucleic acid based therapy such as an RNA, for example a single stranded or double stranded RNA such as dsRNA. The association complexes disclosed herein can be useful for packaging an oligoanuclectide agent capableof modifying gene expression by targeting and binding to nciacid. An oligonucleotide agent can besingle-stranded or doble-stranded, and can include, e.g., a dsRNA, aa pre-mrnRNA, anmRNA, aniroRNA (miRNA), a mi RNA precursor (pnr-miRNA), plasmid or DNA, or to a protein. An oligoncleotide agent featured in the invention can be, e.g, a dsRNA anicroRNA, antisense RNA, is antagomirdecoy RNA, DNA, plasmid and aptarner. Association complexes can include aplurality of components.in sonme embodiments, an association complex-such as a liposome can include an active ingredient such as anucleic acid therapeutic (such as anoligonucleotide agent, e.g, dsRNA), a caonic lipid such as a lipid described herein, In some embodiments, the asscia t ioncomplex can includea plurality of therapeutic agents, for example two or three1sineor double stranded nucleic acid moieties targeting more than one gene or ifferntregionsofthesamegeneOthercomponents canalsobeincluded inan association complex, including a PEG-lipid suchas a PEGlipid described herein, ora structural components such as cholesterol in some embodiments the association complex also includes a fusogenic lipid orcomponentand/oratargetingmolecule. In

somepreferred embodiments, the association complex is a liposomes includingan oligonucleotide agent such as dsRNA, alipid described herein such as acompound of formula(I)or(), aPEG-lipidsuch as a PEG-lipid describedherein (e g aPEo-lipid offbrnula (XV) and structural component such as cholesterol. so Singlpgranded ribonucleid acid Oligonucleotide agents include microRNAs (miRNAs).MicroltNAs aresmall noncoding RNA molecules that are capable of causing post-transcriptional silencing of specific genes in cells such as by the inhibition of translation or through degradation of the targeted mRNA, An miRNA can be completely complementary or can have a region of noncomplementaritywith a target nucleic acid, conseuentlytesulting in a "bulge" at the region ofnon-complementarity. The region of noncomplementarity (the S bulge) can be flanked by regions of sufficient complementarity, pretbrablycomplete complenentarity to allow duplex fonnation. Preferably, the regions of complementarity are at least 8 to 10 nucleotides long (e.g8, 9, or 10 nucleotides long), A miRNA can inhibit gene expression by repressing translation, such as when themicroRNA is not completely complementary to the target nucleic acid, or by causing targetARNA degradation,which is believed to occur only when the miRNA binds its target with perfect complenmntarity. The invention also can include double-stranded precursors of miRNAsthat may or may not fbrn a bulge when bound to their targets, in a preferredembodimentan oligonucleotide agent featured in the invention can target an endogenous miRNA or pre-miRNA. The oligonucleotide agent featured in the i5 invention can include naturally occurring nucleobases, sugars, and covalent intemuclecside (backbone) linkages aswel as oligonucleotides havingnon-naturally occurring portions that function similarly Such modified or substituted ligonudeotides are often preferred over native fonns because of desirable properties such as, for example, enhanced cellular uptake,enhanced affinity for the endogenous amiRNA target, and/or increased stability in the presence of nucleases. An oligonucleotide agent designed to bind to a specific endogenous rniRNA has substantial complementarity, e.g.at least 70, 80, 90, or 100% complementary, with at least 10, 20, or 25 or more bases of the target miRNA A miRNA or pre-miRNA can be 18-100 nucleotides in length, and more preferably from 18-80 nucleotides in length. MatureamiRNAs can have length of 19 30 nucleotides, preferably21-25nucleotides,particularly21,22,23,24,or25 nucleotides.MicroRNAprecursorscanhavealengthof70-100nucleotidesandhavea hairpin conformation. MicroRNAs can be generated in vivo from pre-miRNAs by enzymes called Dicer and Drosha that specifically process long pre-miRNA into 3o functional iiRNA. The microRNAs or precursor mi-RNAs featured in the invention can besynthesized in vivo by a cell-based system or can be chemicallysynthesized, M4icroRNAs can be synthesized to include a modification that imparts a desired characteristic.For example, the modification can improve stability, hybridization themnodynamics with a target nucleic acid, targeting to a particular tissue or celttype, or cel permeability, eg by an endocytosis-dependent or -independent mechanism. Modificatons can also increase sequence specificity, and consequently decreaseof~site targeting. Methods of synthesis and chemical modifications are described in greater detail below, Given a sense strand sequence (e.g, the sequence of a sensestrand of a cDNA molecule), an miRNA can be designed according to the rules ofWatson and Crick base pairing, The miRNAcan be complementary to a portion of an RNA, e.g., a miRNA, a pre-miRNA, a pre-mRNA or an mRNA, For example, the rniRNA can be complementary to the coding region or noncoding region of an mRNA or pre-mRNA, e.g- the region surrouniding the translation start site of a pre-mRNA or mRNA, such as the 5' UTR An miRNA oligonucleotide can be, for example, from about 12 to 30 nuclotidesin length, preferably about 15 to 28 nucleotides in length (eg, 16, 17,18, 19, 20, 21, 22, 23, 24, or25 nucleotides in length). In particular, an miRNA or a pre-miRNA featured in the invention can havea chemical modification on a nucleotide in an internal (ie.,ton-terminal) region having noncompementaritywiththetargetnucleicacid. For example,amodifiednucleotide can e incorporated into the region of a miRNA that forns a bulge., Te modification caninclude a ligandattached to the miRNA, e.g, by a linker (e.g, see diagrams OT through OT-IV below). The modification can, for example, improve phamiacokinetics or stability of a therapeutic miRNA, or improve hybridization properties (e.g. hybridization thermodynamics) ofthe miRNA to a targetnucleic acid. In some embodiments, it ispreferredthat-the orientation of amodification or ligand incorporated 2$ intoortethered to the bulge region of a miRNA is oriented to occupy the space inthe bulge region. For example, the modification can include modified base or sugar on the nucleic acid strand or a ligand that functions as an intercalator. These are preferably locatedin the bulge. The intercalator can be an aromatic, e.g, apolycycic aromatic or heterocyclic aromatic compound. A polycyclic intercalator can havestacking capabilities, and can include systemswith 2, 3, or 4 fused rings. The universalbases described below can be incorporated into the miRNAs. In some embodiments, it is preferred thathe orientation of a modification or ligand incorporated into or tethered to the bulge regionof a miRNA is oriented to occupy the space in the btige region This orientation facilitates the improved hybridization properties or an othenvise desired characteristic of the miRNA, in one embodiment, an miRNA or a pre-miRNA can include an aminoglycoside ligand which can cause thei miRNA to have improved hybridization properties or improved sequence specificity. Exemplary aminoglycosides include glycosylated polylysine; glactosylated polylysine;neomycin1;tobramycin; kanamycinA;and aridin onjugates ofaminoglycosides, such as Neo-N-acridineNeo acridine,Neo C-acridine, Tobra-N-acridine, and KanaA-N-acridine. Use of an acridine analog can DQ increase sequence specificity, For example, neonycin B has a high affinity for RNA as compared to DNA, but low sequence-specificity, An aeridineanalog, neo-S-acridine has an increased affinity for the HRV Rev-response element (RRE). Income embodiments the guanidine analog (the guanidinoglycoside) of an aminoglycoside ligand is tethered to an oligonucleotide agent. In a guanidinoglycoside, the amine group a on the amino acid is exchanged for a guanidine group. Attachment of a guanidine analog canenhance cell permeability of an oligonucleotide agent. in one embodiment, the ligand can include a cleaving group that contributes to target gene inhibition by cleavage of the target nucleic acid, Preferably, the cleaving group is tethered to the miRNA in a manner such that it is positioned in thebulge region, where it can access and cleave the target RNA, The cleaving group can be, for example, a blcomycin (e.g.,bleomycin-A,bleomycin-A or bleomycin-B1),pyrene, phenanthroline(e gO-phenanthroine,apolyamine, a tripeptide (eg, lys-ty -ys tripeptide), or metal ion cheating group The metal ion chelating groupcan include, e.g an Lu(I) orEU(III) macrocycic complex, a Zn()2,9-dimethylphenanthroline derivative, a Cu(l) terpyridine, or acridinewhich canpromote the selective cleave of taraetRNAat thesiteof the bulge by free metal ions, such as Lu(II)In some embodiments, a peptide ligand can be tethered to a miRNA or pre-miRNA to promote cleavage of the target RNA, e.g.,at the bulge region. For example, 1,8-dimethyl 1,3,6,8,13~hexaazacyclotetradecane(cyclam)canhbconjugatedtoapeptide(eg.,by so an amoacid derivative) to promote target RNA cleavage. The methods and compositions featured in the invention include miRNAs that inlhibit target gene expression by acleavage or non-cleavage dependent mechanism.

AnmiRNA or a pre-niRNA can be designed and synthesized to include a region of noncompementarity (e.garegion that is 3, 4,5, or 6nucleotides long) flanked by regions of sufientcomplentenrity to forma duplex (eg regions that are 7, 8, 9, 10, or iinucleotids long), a For increased nuclase resistance and/or binding affinity to the target, the miRNA sequences include 2-0-methyl,2'-fluorine,2-O-methoxyethy 2'-0 aminopropl?2amino,and/orphosphorothioatelinkages.Inclusionoflockednuchec acids (LNA, 2-thiopyrinmidines (e,g,2-thio-U), 2-amino-A, C-clamp modifications, and ethylene nucleic acids (ENA),e.g,2 -4-ethylene-bridged nucleicacids, can also o Increasehbiniaffnity to the target. The inclusion oftfuranose sugars in the oligonuceotidebackbone can also decrease endonucleolytic cleavage, An miRNA or a pre-miRNA canbe further modified by including a 3' cationie group, or by inverting the nucleoside at the 3-teminus with a3'3$linkage.In another alternative, the 3erminus can be blocked with anaminoalkyl.group, e.gi, a 3' CS-aminoalkyldT. Other 3' conjugates can inhibit 3'-5' exonucleolytiecleavage, White not beingbound by theory, a 3'conjugate, such as naproxen or ibuprofen, may inhibit exonucleolytic cleavage by steric&llyblockingthe exonucase frombindingto the 3' end of oligonucleotide,Even samaiakylchains, aryl groups, or heterocyclic conjugates or modified sugars (D-iose deoxyribose, glucoseetc.) canblock 3-Yexonucleases, The S-terminus can be blocked with an arinoalkyl group, eg, a 5'0 alkylamino substituent. Other 5'conjugates can inhibit 5- exonucleolytic savage.

While not being bound by theory! a5' conjugate, such as naproxen or ibuprofen, may inhibit exonucleolytic cleavage by sterically blocking the exonuclease from binding to the 5' end of oligonuceotide. Even small alkyl chains, aryl gmups, orheterocyclic conjugates or modied sugars (Dribose, deoxyribose, glucose etc) can block 3'-5' exonucleases.

In one embodiment, an mIRNA or a premiRNA includes anodiicationthat improves targeting e.ga targeting modification described herein. Examples of modifications that targetmiRNA molecules to particular cell types includecrbohydrate sugars such as galactose, N-acetylgalactosamine, mannose; vitamins such as folates other ligands such as RGDs and RGD mimics; and small molecules including naproxen ibuprofen or other known protein-binding molecules,

An iRNA or a premiRNA can be constructed using chemical synthesis and/or enzymaticligation reactions using procedures known in the art. Forexample, an nmiRNA or apre-nPRNA can be chemicallysynthesized using naturally occurring nucleodes orvariously modified nucleotides designed to increase the biological stability of the moleculesor to increase the physical stability of the duplex formed between the miRNA or a pre-miRNA and target nucleic acidseg phosphorothioate derivatives and acridine substituted nucleotides can be used. Other appropriate nucleic acid modifications are described herein. Alternatively, the miRNA or pre-miRNA nucleicacid can be produced biologically using an expression vector into which a a nucleic acid has been subcloned in an antisense orietation (i.e RNA transcribed from the insertednucleic acid will ibe of an antisense orientation to a target nucleic acid of interest).

Aptisense-tvicongelieotide Agents

The single-stranded oligonucleotide agents featured in the invention include antisensenucleicacids. An "antisense" nucleic acid includes a nucleotide sequence that iscomplementary to a "sense" nucleic acid encoding a gene expression product,e.g. complementary to the coding strand of a double-stranded eDNA molecule or complementary to an RNA sequence, eg, a pre-mRNA, mRNA, miRNA, or pre ,0 miRNA. Accordingly, anantisense nucleic acid can form hydrogen bonds with a sense nucleic acidtarget. Givena coding strand sequence (e.g, the sequence of a sses trand of a cDNA molecule), antisense nucleic acids can be designed according to the rules of Watson and Crick base paring The antisensenucleic acid molecule can becompementaryto a portion of the coding or noncoding region of an RNA, eg a premRNA ormRNA, F.For example, the antisense oligonucleotide can be complementary to the region surrounding the translationstart site of a prc-mRNA or nRNA, e.g, the 5' UTR, An antisense rdigonuceotiiecan be, for example, about 10 To 25 nucleotidesinlength (e.g.,l1, 12, 13,14, 1516, 18,19, 20, 21, 22, 23, or24 nucleotides in length). Anantisense oligonucleotide can also be complementary to ainiRNA or pre-niRNA. An antisense nucleic acid can be constructed using chemical synthess and/or enzymaticugation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonuleotide) can bechemiclly synthesized using natural occurring nucleotides or variouslymodified nucleotides designed to increase the biological stability of themolecules or to increase the physical stability of the duplex formed between the antisense and target nucleic acids, e.g, phosphorothioate derivatives and acridine substituted nucleotides can be used. Other appropriate nucleic acid modifications are described herein, Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which anucleic acid has been subloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisenseorientation to a targetnucleicacid of interest), An antisese agent can include ribonucleotides only, deoxyribonucleotides only (;g,,iigodeoxynucleotides)or both deoxyribonucleotides andribonucleotides. For example an antisense agent consisting only of ribonucleotides can hybridize to a c implementary RNA, and preventaccess ofthe translation machinery to the target RNA transcriptthereby preventing protein synthesis. An antisense molecule including only deoxyribonu~cleoidsor deoxyribonucleotides and ribonucleotides, eg DNA sequence Tanked by RINA sequence atthe 5 and 3' ends of theantisense agent, can hybridize to a complementary RNA, and the RNA target can be subsequently cleaved by an enzyme, eg, RNAse H. Degradation of the target RNA prevents translation, The flanking RNA sequences can include2CO-ethylated nuleoddes, nd phosphorothioate linkagesand theintermal DNA sequence can include phosphorothioate internucleotide linkages. The inteal DNA sequence is preferablyat least five nucleotides in length when targeting by RNAseH activity is desired. For increased nuclease resistance, an antisense agent can be further modified by inverting the nucleoside at the 3'terminuswvith a 3'3linkage In another alternative the 3Utenninus can be blocked with anawinoalkylgroup. In oneemibodiment, an antisense oligonucleotide agent includes a modification that improves targeting, eg a targeting modification described herein.

Deott eOigoucti~fdeAgent~s

`30 An oligonucleotide agent featured in the invention can be a decoy nucleic acid, eg,adecoyRNA. A decoynucleie acidresembles a naturalnucleic acid, but is modified insucha way as to inhibit or interrupt the activity of the natural nucleic acid

For example, a decoy RNA can mimic the natural binding domain for a ligand. The decoy RNA therefore competes with natural binding target for the binding of a specific ligand. The natural binding target can be an endogenous nucleic acid, e.g, a pre miRNAIniRNA, prerRNA,mRNA or DNAFrexampleithasbeenashownthat over-expression of HIV trans-activation response (TAR) RNA can act asa "decoy" and efficiently bind HTV tat protein, thereby preventing it from binding to TAR sequenes encoded in the HIV RNKA. In one embodunent, a decoy.RNA includes a modification that improves targeting, e.ga targeting modification described herein, The chemical modifications described above for miRNAs and antisense RNAs, and described elsewhere herein are also appropriate for use in decoy nucleic acids.

Aptaner-vpe QgonucleotideAgerts An ollgonucleotideagent featured in the invention can be an aptamer.An aptamer bindstoa non-nucleic acid ligaid, such as small organic molecule or protein, S5 e.g, a transcription or translationfactor, and subsequently modifies(e.ginhibits) activity, An aptaner can fold into aspecific structure that directstherecognition of the targetedbinding site on the non-nulei acid ligandAnaptamercaneontainanyofthe modification described herein. In one embodiment an aptainerincludes a modification that improvestargeting, e g, a targeting modification describedherein. Theemical modifications described above for miRNAs and antisense RN,,s, and described elsewhere herein, arealso appropriate for use in decoy nuclic acids. The details of one or more embodiments of the invention are set forth in the accoranwng drawings and the description below, Other features and advantages of the invention will be apparent kom the description and drawings, and from the claims. This application incorporates all cited references, patents,and patent applications by references in their entirety for all purposes. In one aspect, the invention features antagomirs. Antagomnirs are single stranded, double standed, partially double stranded and hairpi structuredchenically modiiedoligonucleotidesthat target a niicroRNA. An antagomir consisting essentially of or comprising atleast 12 or more contiguous nucleotides substantially complementary to an endogenous niRNA and

Inure particularly agents that include 12 or morecontiguous nucleotides substantially complementayto a target sequenceofanmiRNAorpre-miRNAnucotide seqene, Preferably, an antagomir featured in the invention includes a nucleotide sequence suticiently complementary to hybridize to a miRNA targetsequence of about 12 to 25 i nucleoides, preferably about 15 to 23 nucleotides. More preferably, the target sequence differs by nomore than 1, 2 or 3 nucleotides from a sequenceshown in Table 1s and in one embodiment, the antagomir is an agent shown in Table 2a-e, In oneembodiment, the antagomir includes a non-nucleotide moiety, e,g.a cholesterol moiety. The non nucleotide moiety can beattached, e.g, to the 3' or 5' end of theigonuiclotide agent, In a preferred embodiment, a cholesterol moiety is attached to the 3' end of the oligonucleotide agent. Antagomirs are stabilized against nucleolytic degradation such as by the incorporation of a modification, e.g., a nucleotide modification. In another embodiment, the antagoiir includes a phosphorothioate at at least the first, second, or third intemuclotide linkage at the 5' or 3' end of the nucleotide sequence, In yet another embodiment, theantagomir includes a 2'-modified nucleotide, e.g., a 2'-deoxy, 2' doxy-2tluoro, 2'-O-ethyl'-O-nmetoxyethyl (2QOMOE),2T-O-aminopropy! (2'0 AP), 2¾-dimethylaminoethyl(2LO-DMAE), 2'--dimethylaminopropyl (2'-0X DMAP), 2O-dimethylamainoethyloxyethyl (2'-O-DMAEOE), or 2'-O-N methylaceamio (2C-NMA).Ina particularly preferred embodiment,theantagomir includes at least one 2'-O-niethyl-modified nucleotide, and in some embodiments, a of the nucleotides of the antagomir include a2T-0-methyl modification, An antagomir that is substantially complementary to a nucleotide sequence ofan miRNA can be delivered to a cell or a human to inhibit or reduce the activity of an endogenous miRNA, such as when aberrant or undesired miRNA activity, or insufficient activity of a target mRNA that hybridizes to theendogenous niRNA, is linked toa disease ordisorder. In oneembodiment, an antagomirfeatured in the invention has a mucleotide sequence that is substantially complementary to mit-122 (see Table 1), which hybridizes to numerous RNAs, including aldolase A mRNA, N-mye so downstran regulated gene (Ndrg3) mRNA., IQmotif containing (TPase activating protein-1 (lqgapt) mRNA, HMG-CoA-reductase (Hmgcr) mRNA, and citratesynthase nRNA and others. In a prefered embodiment, the antagomir that is substantially complementary toiRil122 is antagomir-122 (Table 2a-e*).Aidolase Adeiciencies have been. found to beassociated with a variety of disorders, including hemnolytic anemia, arthrogryposis complex congenita, pituitaryectopia rhabdomyoysis, hyperkalemia. Humans suffering from alddlase A deficiencies also experience symptoms that include growth and developmental retardation, midfacial hypoplasia, hepatomegaly, as well as myopathic symptoms. Thus a human who has or who is diagnosed as having any of these disorders or symptoms is a candidate to receive treatment with an antagomir that hybridizes tomiR-122.

Doublesanded ribonucelglagidldRNA) none enhodiment, the invention provides a double-stranded ribonucleicacid (daRNA) molecule packaged in an association complex, such as a iposome,for inhibiting theexpression of a gene in a ceil or mammal, wherein the dsRNA comprises an antisense strand comprising a region of complementarity which is complementaryto Z5 at least a part of an mRNA formed in the expression of the gene, and wherein the region of compliementarity is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and wherein sid dsRNA, upon contactwithacellexpressing said gene, inhibits the expression of said gene by at least 40%, ThedsRNA comprises two RNA stnmds that are sufficiently complementary to hybridize to form a duplex structure. Onestrand ofthe dsRNA (the antisense strand) comprises a region of complementarity that is substantially complementary,andgenerally fully complementary, to a target squene, derived from the sequence ofian mRNA formed during the expression ofwa gene, the other strand (the sense strand) comprises a region Which is complementary tothe antisensestrand, such that the two strands hybridize and form a duplex structure when ,5 combined under suitable conditions. Generally, the duplex structure is between 15 and 30, more generally between 18 and 25, yet more generally between 19 and 24, and most general between 19 md 21 base pairs in. length. Similarly, the region of complementarity to the target sequence is between 15 and 30, more general between 18 and 25,yet more generally between 19 and 24,and most generally between 1.9 and 21 nucleotides in length. The dsRNA of the invention may further comprise one or more single-stranded nucleotide overhang(s). The dsRNA can besynthesized by standard methods known in the art as further discussed below, e.g., by use of an

a'I automated DNA synthesizer, such as are commercially available from, forexample, Biosearch, AppliedlBiosystems, Inc, The dsRNAs suitable for packaging in the association complexes described herein can include a duplex structure of between 18 and 25 basepairs (e.g,21 base pairs).Insome embodiments, the ds]tNAs include at least one strand that isat least 21nt long In otherenbodiments, the dsRNAs include at least one strand that is at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides. The dsRNAs suitable for packaging in the association complexes described herein can contain one or more mismatches to the targetsequence. In a preferred embodiment, the dsRNA contains no more than 3 mismatches.f the antisensestrandof the dRNA contains mismatches to a target sequence, it is preferable that the area of mismatch not be located in the center of the region ofcomplenentarity. if the antisnse strand of the dsRNA contains mismatches to the target sequence, itis preferable that the mismatch be restricted to 5 nucleotides from either end, for example 5, 4, 3, 2, orI nucleotide from either the 5' or 3' end of the region of complementarity, ione embodiment, at least one end of the dsRNA has a single-stranded nucleotide overhangof I to 4, generally I or 2 nucleotides. Generally, the single stranded overhang is located at the 3'-erminal end of the antisense strand or, altemativel, at the 3'terminal end of the sense strand. The dsRNAmay also have a 2 blunt end, generally locatedat the 5'-end of the antisense strand. Such dsRNAs have improved stability and inhibitory activity, thus, allowingadministrationat low dosages, i.e, lessthan 5 mg&g body weight of the recipient per day, Generally, the antisense strand of thc dsRNA has a nucleotide overhang at the 3-end, and the 5'-end is blunt. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate in yetanother embodiment, a dsRNA packaged in an association complex, such aa liposomne, is cmically modified to enhancestabilit, Such nucleic acids may be synthesized and/or modified by methods well establishedin the art, such as those described in "Curret protocols in nucleic acid chemistry", Beaucage, S-L eat. (Edrs.), ao John Wiley & Sons, Inc., New YorkNY, USA, which is hereby incorporated herein by referenceChemical-modificationsayinclude, but are not limited to 2' modifications, modifications at other sites of the sugar or base of an oligonucleotide, intrxluction of non-natural bases into the oligonacleotide chain, covalent attachment to a ligand or chemical moiety, and replacement of intenucleotide phosphate linkages with aftemate linkages such. as thiophosphates. More than one such modification may be employed. Chemical linking of the two separate dsRNA strands may be achieved byany of a variety of wel-known techniques, for example by introducingcovalent, ionic or hydrogen bonds; hydrophobic interactions, van der Waals or stackinginteractions; by means ofmetal-ion coordinaton, or through use of purine analogues, Such chemically linked dsRNAs are suitablefir packaging in the association complexes described herein. Generally, the chemical groups that can be used to modifythe dsRNAinclude, without limitation, methylene blue; bifunctional groupsgenerally bis-(2 chloroethyi)anine N-acetyl-NCtp-glyoxylbenzoyl)eystamine;4-thiouracil; and psoralen, In one embodiment, the linker is a hexa-ethylene glyol linker. In this case, the dRNA are produced by solid phase synthesis and the hexa-ethylene glycol linker is incorporated according to standard methods (e.g., Williams, D., andI KB. Hall, Biochem. (1996) 35:14665-14670). In a particular embodiment, the- Send of the antisense strand and the 3-end of the sense strand are chemically linked via a 'hexaethylune glycol linker, In another embodiment, at leastrone nucleotide of the dsRNA comprises a phosphorothioate or phosphorodithioate groups. Te chemrnial bond at the ends ofthe dsRNA is. generally formed by triple-helix bonds, In yet another embodiment, the nucleotides at one or both of thetwo single strands may be modified to prevent or inhibit the degradation activities of cellular enzymes, such as, for example, without limitation, certain nucleases. Techniques for inhibitingthe degradation activity of cellular enzymes againstnucleic acids are known in the art includingbut not limited to, 2T-amino modifications, 2T-aminosugar modifications,2'-F sugar modifications, 2'-F modications,2'alkylsugar modifications, 2-O-alkoxyalkyl modifications like' 2to-mnethoxyethyl, unharged and charged backbone modifications, morpholino modifications, 2'-O-methyl modifications, and phosphoramidate (see, e.g., Wagner, a, .Mee. (1995) 1:1116-8). Thus, at least one 2'-hydroxyi group of the nucleotides on a dsRNA is replaced by a chemical group, generally by a 2'-F or a 2'-O-methyl group, Also, at least one nucleotide may be modified to form a locked nucleotide. Such locked nucleotide contains a methylene bridge that connects the 2T-oxygen of ribose with the 4'-cabon of ribose. Oligonucleotides containing the locked nucleoide are described in Koshkin, AA, et al., Tetrahedron (1998), 54: 3607-3630) and Obika, S, et al.,TirahedronLett. (1998),39:5401-5404).Introductionof alockednucleotideintoanolgonucleotide IMproves the affinity for complementarysequences and increases the melting temperature byseveral degrees (Braasch D A and DR, Corey, Chem BioL (2001),8:1 7),

Conjugatinga ligand to a dsRNA can enhance its cellar absorpion as well as targeting to a particular tissue or uptake by specifictypesof cells such as liver cells. In certain instances, a hydrophobic ligand is conjugated to the dsRNA to facilitate direct prmeation of the cellular membrane and or uptake across the liver cells. Alternativevy, the ligand conjugated to the dsRNA is substrate forreceptor-mediated endocytosis. These approaches have been used to facilitate cell pemeation of antisense olignucleotides as well as dsRNA agents. For example, cholesterol has been

conjugated to various antisense oligonucleotides uling in compounds that are s substantially moreactive compared to their nonconjugated analogs, See M Manoharan Antisense &MV cleic AcidDrug Development 2002, /2, 103. Other lipophilic compounds that have been conjugated to oligonucleotides include I-pyrene butyric acid, 1,3-bis-0Jhexadecygycerol and menthol One example of a ligand for receptor mediated endocytosis is folic acid. Folic acidenters the cell by folate-receptor-mediated endocytosis.dsRNA compounds bearing folic acid would be efficiently transported into the cell via the folate-receptor-mediated endocytosis. Liand coworkers report that attachment of folic acid to the 3'-tenninus of an oligonucleotide resulted in an 8-fold increase in cellular uptakeof the oligonucleotide. Li, S.; Deslukh, H. M.;Huang L Pharn' Res. 1998, 15, 1540, Other ligands that have been conjugated to oligonucleotides include polyethylene glycols, carbohydrate clusters, cross-linking agents, porphyrin conjugates, delivery peptides and lipids such as cholesterol.,Other chemicalmodiicationsfor siRNAs have been described inManoharan MRNA interference and chemically modified small interfering RNAs. Current Opinion in Chemical Biology (2004), 8(6), 570-579,

s0 certain instances, conjugation of a cationic ligand tooligonucleotidesresults in improved resistance to nucleases Representativeexamples ofcationic ligands are propylammonium and dimethylpropylammonium. interestingly,antisense oligonuceotidswere reported to retain theirhigh binding afinity to iRNA when the eatonie ligand was dispersed throughout the oligonucleotide. See N Manoharan

Anisense& NuicwAcidDrugDevelopment 2002, 12, 103 and references therein.

Theligand-conjugated dsRN.A of the invention may be synthesized bytheuseof a dsRNA that bears a pendant reactive ftuctionality, such as that derived from the attachment of a linking molecule onto the dsRNA. This reactive oligonucleotide may be reacted directly with commercially-available ligands, ligands that are synthesized bearing any of a variety of protecting groups, or ligands that have a linking moiety attached thereto, The methods ofthe invention facilitate the synthesis ofligand o conugated dsRNA bythe use of, in some preferred embodiments, nucleoside monomers that have been appropriately conjugated with ligands and that may further be attached to asolid-support material Such ligand-nucleoside conjugates, optionally attached to a solid-support material, are prepared according to some preferred embodiments ofthe methods of the invention via reaction of a selected serum-binding ligand with a linking s moiety located on the 5' position of a nucleosideordligonueleotide.icertain instances, a dsRNA bearing an arikyl ligand attached to the3-terminus ofthe dsRNA isprepared by first covalently attaching a monomer building block to a controlled-pore glass support via a longchain aminoalkyl group. Then, nucleotides are bondedvia standard solid-phase synthesis techniques to the monomerbiding-blockboundto the solid support, The monomer building block may be a nucleoside or other organic compound that incompatible with solid-phase synthesis.

The dsRNA used in the conjugates of the invention may beonvenientlyand routinely made through the well-known techniqueof solid-phase synthesis. Equipment for such synthesis is sold by several vendors including, for exampleApplied 2s Biosystems (Foster City, CA). Any other neans forsuchsynthesis known in the art may additionally or alternatively be employed. Iisalsoknowntousesimartechniques to

prepareotheroligonucleotidessuchasthephosphorothicatesndalkylatedderivatives. Teachings regarding the synthesis of particular modified oligonucleotides may be found in the following U.S. patents: US, Pat. Nos.5,138,045 and 5,218,105, drawn 3o to polyamine conjugated oligonicleotides; USPat No, 5,212,295, drawn tomonomers for the preparation ofoligonucleotides having chiral phosphorus linkages; U.S, Pat.

Nos. 5,378,825 and 5,541,307, drawn to oligonucleotides having modified backbones; U.S. Pat. No. 5,386,023; drawn to backbone-modified oligonucleotidesand the preparation thereofthrough reductive coupling; U.S. Pat No. 5,457,191, drawn to modified nucleobases based on the 3-deazapurine ring system and methods ofsynthesis thereof; U.S. Pat. No. 5,459,255, drawn to modified nucleobases based on N-2 substitutedpurines;U.S. Pat, No. 5,521,302, drawn to processes for preparing oligonucleotides having chiral phosphorus linkages; U.S; Pat. No, 5,539,082, drawn to peptide nucleicacids; US. Pat. No. 5,554,746, drawn to oligonucleotides having fD lactam backbones; U.S, Pat. No. 5,571,902, drawn to methods and materials for the so synthesis of oligonucleotids; U. Pat No.5,578,718, drawnto nucleosides having alkylthio groups, wherein such groups may be used as linkers to other moieties attached at any of a variety of positions of th nucleoside; U.S. Pat. Nos. 5,587361 and 5,599,797, drawn to oligonucleotides having phosphorothioate linkages of high chiral purity; U.S. Pat, No, 5,506,351, drawn to processes forthe preparation of2-O-alky uanosine and related compounds, including 2,6-diaminopurine compounds; U.S. Pat. No, 5,587,469, drawn to oligonucleotides having N-2 substituted purines; U.S. Pat.No. 5,587,470, drawnto oligonucleotides having 3-deazapurines; U.S. Pat No. 5,223,168, and U.S.Pat. No. 5,608,046, both drawn to conjugated 4'-desmethyl nucleoside analogs; U;S, Pat. Nos 5,602,240, and 5,610,289, drawn to backbone-modifiedoligonucleotide analogs;U.S.Pat.Nos.6,262,241,fand5,459,255,drawnto,interalimethodsof synthesizing2'fuoro-oligonucleotides,

in the ligand-conjugated dsRNA and ligand-molecule bearingsequence-specific linked nucleosides of the invention, the oligonucleotides and oligonucleosides may be asserbed on a suitable DNA synthesizer utilizingstandard nucleotide or nucleoside precursors(or nucleotide or nucleoside conjugate precursors that already bear the linking moiety, ligand-nucleotide or nucleoside-conjugate precursors that already bear the ligand molecule, or non-nucleoside ligand-bearing building blocks.

When wsing nucleotide-conjugate precursors that already bear a linking moiety the synthesis of the sequence-specific linked nucleosides is typically completed,and the so ligand molecueis then reacted with the linking moiety to form theligand-conjugated oligonucleotide. Oligonucleotide conjugates bearing a variety of molecules such as sterods, vitamins, lipids and reporter molecules, has previouslybeen described (see

Manoharan etal, PC' Application WO 93/07883). in apreferred embodiment, the oligonucleotides or linked nucleosides of the invention are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside conjugates in addition to the standard phosphoramidites and non-standard phosphoranidites that are commercially available androutinely used in oligonucleotide synthesis. The dsRNAs packaged in the associationcomplexes described herein can include one or more modified nucleosides, e,g, a 2-0-methyl, 2'-o-ethy2T-0-propyl, 2>0-all2],Oaminoalkyi or2T-deoxy-2-fluoro group in the nucleosides; Such modifications confer enhanced hybridization properties to the oligonucleotde. Further, o oligonucleotides containing phosphorothioate backboneshave enhanced nuclease stability. Thus, functionalized, linkednucleosides can be augmented to include either or both a phosphorothioate backbone or a 2-O-methyl, 2-0-ethyl 2O-propy, 2-0 aminoalkyl, 2T-0-allyl or 2deoxy-Tfluoro group. Asummary listing of some of the oligonucleotide modifications known in te art is fund at, for example, PCT 1 Publication WO 200370918, In some embodiments, functionalized nucleoside sequences possessing an amino group at the 5-terminus are prepared using a DNA synthesizer, and then reacted with an active ester derivative of a selected ligand. Active ester derivatives are well known to those skilled in the art. Representative active esters include N-hydrosuccininide esters, 2t0 tetrafluorophenoli eesters, pentafluorophenolic esters and pentaehlorophenolicesters. The reaction of the amino group and the active ester produces an oligonueleotide in which theselectedligand is attached to the 5-position through a linking group. The amino group at the 5tterminus can be prepared utilizing a5-Amino-Modifier C6 reagent.In one embodiment, ligand molecules may be conjugaited to oliganucleotides at the 5-position by the use of a igand-nucleoside phosphoramidite wherein the ligand is linked to the hydroxy group directly or indirectly via a linked, Such ligand-ucleoside phosphoramidites are typically used at the end of an automated synthesis procedure to t provide a ligand-coningated oligonucleotide bearing the ligand at theS termnus. Examples of modified internucleoside linkages or backbones include, for so example, phsphorothioates,chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters methyl and other alkyl phosphonates including 3-alkylene phosphonates and chiral phosphonates, phosphinates, ptosphoramidates including 3-amino phosphoramnidate and aminoalkylphosphoramidates, thionophosphoramidates,thionolkylphosphonates, thionoalkyphosphotiesters,and boranophosphates having normal 3'5'linkages, 2'5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of S nuclesidunits arelinked 3l5to 5'3'or to%52Varioussalts,mixed salts and 'free-acid forms are also included. Representative United States Patents relating to the preparation of the above phosphorusatom-containinglinkages include, but are not limited to, U.S Pat Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,17',196;5,188,897;5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5455 466 ,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; and 5,697,248, each of which is herein incorporated by reference. Examples ofmodified intemuclcoside linkages or backbones that do not include i a phosphorus atom therein (i.e.,oligonucleosides) have backbones that are formed by short chain alkyl or cycloalkyl intersugar linkages, mixed heteroaton and alkyl or cyloalkyl intersugar linkages, or one or more short chain heteroatomic oreterocyclic intersugar linkages, These include those havingmorpholino linkages (fonned in part onm the sugar portion of a nuleoside); siloxane backbones; sufide, sufoideand 2o suifne backbones; formacetyl and thioformacetyl backbones;methyene fbrmacetyl and thioformacetyl backbones; alkene containing backbones;sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sufonate and sulIfonamide backbones; aide backbones; and others having mixed'N, 0, S and CH component parts. Representative United States patents relating to the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nost 5,034506 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677;5,541,307; 5561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289;5,618,704; 5,623,070; so 5,663,312; 5,633,360;5,677,437; and 5,677,439,eachofwhichishereinincorporated by reference.

In certain instances, an oligonucleotide included in an association complexsuch as a liposone, may be modified by a non-ligand group. Anumber ofnon-ligand molecules have been conjugated to oligonucleotides in order to enhancethe activity, cellular distribution or cellular uptake of the oligonuleodeand procedures for perfomiring such conjugationsareavailableinthescientificliteratureSuchnonigand moieties have included lipid moietiessuch as cholesterol (Lctsinger et at, Proc. Nati. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al, Bioorg Med. Chem. Lett,1994, 4:1053), a thioether, e.g, hexyl-S-tritylthiol (Manoharan et at, Ann.NY Acad. Sci, 1992, 660:306;Manharaniet at, Bioorg. Med. CheniLet. 1993, 3:2765),a thioholesterol (Oberhauser et al, Nuc.lAcids Res, 1992, 20:533), anaiphati chain, eg. dodecandiol or undecyl residues (Saison-Behmoaras et at, EMBO .,1991, 10:1 I1; Kabanov et aL, FEBS Lett, 1990, 259:327; Svinarchuk et alBiochiie, 1993, 75:49), a phospholipid, e~g., di-hexadecyl-rac-glycerol or triethylammonium 1,2di-0 hexadecyl-rac-glyeero-3-H-phosphonate(Manoharan et a, Tetrahedroneltt, 1995, a 36:3651; Shea et al, uc. Acids Res, 1990, 18:3777),a polyamine or a polyethylene glycol chain (Manoharan eta, Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et atTetrahedron Lett., 1995,36:3651), a palmityl moiety (Mishra et aL, BiochimniBiophys.Ata, 1995, 1264:229), oran octadecylamtine or hexyamino-carbon.l-oxchoesteromoiety (Crooke etat, L PharmacoL Exp Ther o 1996, 277:923). Representative United States patents that teach the preparation of such oligonucIeotide conjugates have been listed above. Typical conjugation protocols involve thesthehesis of oligonucleotides bearing an aminolinker at oneornore positions ofthe sequence. The amino group is then reactedwiththe molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction may be periormnedeitherwith the oligonucleotide still bound to the solid support or following cleavage of the oligonucleotide in solution phase.Purification of the oligonucleotide conjugate by HPLC typically affords the pure conjugate. The modifications described above are appropriate for use with an oligonucleotide agent as described herein,

ao Fuscgpnidcpids

The teri "fusogenic" refers to the ability of a lipid or other drug delivery system to fusewith membranes of a cell. The menibranes can beeithertheplasma memrane or membranes surrounding organeles, e.g, endosome, nucleusetc. Examples ofsuitable fusogenic lipidsinclude, but are not limited to dioleoylphosphatidylethanolanine (DOPE), DODAC, DODMADODAP, orDLinDMA. Insomeembodiments, the association complex inclde a small molecule such as an iuidzole moiety conjugated to a lipidfor example, forendosomal release.

PEGorpPEG-liids In. addition to cationicand ftsogenic lipids, the association complexes include a bilayer stabilizing component (:B )suh asan ATTAlipidoraPEG-lipid. Examplary ipids are asfolowr PEG coupled to dialkyloxypropyis (PEG-DAA) as described in, e.g.,WO 051026372,1PEG coupled to diacylglycerol (PEG-DAG)as described in, e.g, U.S. Patent Publication Nos.20030077829 and 2005008689), PEG coupled to phosphatidylethanolamine(PE) (PEG-PE), or PEG conjugated to ceranides, ora mixture thereof (seeS. PatNo5,885,613) In.a preferred embodiment, the association includes aPEG-iipiddescribedhere,forexampleaPEG-lipidof fonnua (XV), (XV') or (XVI). In one preferred embodiment, the BS(is a conjugated lipid that inhibits aggregation of the SPLPs. Suitable conjugated lipids include, but are not limited to PEG-lipid conjugates, ATTA-lipid conjugates,cationic-polymerlipidconjugates (CPLs) or mixtures thereof. in one preferred embodiment, the SPLPs conprise either a S PEG1-lipid conjugate or an ATTA-lipid conjugate together with a CPL PEG isapolyethylene glycol, a linear, water-soluble polymer of ethylene PEG repeating units with two terminal hydroxyl. groups. PEGs are classified bytheir molecular weights; for example PEG 2000 has an average molecular weight of about 2,000 daltons, and PEG 5000 has anaverage molecularweight of about 5,000 daltons, PEGsare commercially available from Sigma Chemical Co.and other companies and ancludefr example, the following: monomethoxypoyethyleneglycol (MePEG-OH) monomiethoxypoiyethyleneglycol-succinate (MPEG-S), monomethoxypoyethyIene glycol-succinimidyl succinate (MePEG-S~NHS),mnomethoxypolyethyleneglycol~ amine (MPEG-NELsub.2), monoethoxypolyethylene glycol-tresylate(NePEG 3o TRES), and monomethoxypolyethyiene glycol-imidazolycarbonyl(MePEG-M). in addition, monomethoxypolyethyleneglycolacetic acid (MePEG-CHIsub,2C00H), is particular useful forpreparing the PEG-lipid conjugates including,e.g., PG-DAA conjugates. in a preferred embodiment, the PEG has an average molecular weight of from about 550 daltons to about 10,000 dahons, more preferably of about 7150 daltons to asout 5,000 daltons, more preferably of about 1,000 daltons to about 5;000 daltons, mere preirably ofrabout 1,500 daltons to about 3,000 daltons and, even more prefIbly, of about 2,000 daitons, or about 750 daltons. The PEG can be optionally substituted by an alkyl, alkoxy, acyl or aryl group. PEG can be conjugated directly to the lipid or may be linked to the lipid via a linker moiety. Any linker moiety suitable for coupling the PEG to a lipid can be used including, e.g, non-estercontaining linker moieties and ester-containing tinker moieties. In a preferred embodiment, the linker moiety is a non-ester containing tinker moiety, As used herein, the tenn "non-ester containing lnker moiety' refers to alinker moiety that does not contain a carboxylic ester bond (-OC(0)-). Suitable non-ester containing linkermoieties include, but are not limited to, amnido (--C(O)NH-), amino (--NR--), carbonyl (-C(0)-~),carbamate( NHC(0)O-), urea (-NHC(O)NH-), disulphide (-SS-),ether (-0--), succinyl( (0)tCCHsub.2CH~sub;2C(Q)-), succinamidyi(~~NHC(0)CHsub.2C:H sub 2C(O~)NHW )ether, disulphide, etc. as well as combinations thereof (such as a linker containing both a carbaiate linker moiety and an nido linker moiety) In apreferred embodiment a carbamate linker is used to couple the PEO to the lipid. In other embodiments, an ester containing linker moietyisused to couple the PEG to the lipid. Suitable ester containing linker moieties include, e.g, carbonate ( 00(0)0-), succinoyl, phosphate esters (-O-(0)PH-0- sulfonate estersand combinations thereof, in some embodiments, the association complex includes a targetingagent. For example, a targeting agent can be included in the surface of the association complex (e.g, liposome) to help direct the association complex to a targeted area of the body An example oftargeting agents galactose, mannose, and folate, Other examples of targeting agents include sinall molecule receptors,peptdesandantibodies.Insome embodiments, the targeting agent is conjugated to the therapeutic moiety suic as

9' oligonucleotide agent, In some embodiments, the targeting moiety is attached directly to a lipid component of an association complex. In some embodiments, the targeting moiety is attached directly to the lipidcomponent via PEO preferably with PEG of average molecular weight 2000 amu. Insome embodiments, the targeting agent is unconjugated, for example on the surface of the association complex,

Strctural components In some embodiments, the association complex includes one or more components that improves the structure of the complex (e.g, liposome). In some to embodiments, a.therapeutic agents such s dsRNA can be attached (etg, conjugated) to a lipophilic compound such as cholesterol, thereby providing a lipophilic anchor to the dsRNA. [n some embodiments conjugation of dsRNA to a lipophilic moiety such as chdlesterol can improve the encapsulation efficiency of the association complex.

pj a5gciation coaLs;,es Association complexes such as liposomes are generally particles with hydrodynamic diameter ranging from about 25 nmto 500 nm. In some preferrx enbodiments, the association complexes arelessthan 500 nm, e.g from about 25 to about 400nm, e.g, from about 25nm to about 300 nm preferably about 120 nm orles. In some embodiments, the weight ratio of total excipients within the association complexto RNA is less than about 20:1, for exampleabout 15:1, insomeprefelred embodiments, the weight ratio is less than10:1, for example about 17,5:1, In some embodiments the association complex has a pKa such that the associationcomplex is protonated under endozomalconditions (e.gfacilitating the uApture of the complex), but is not protonated under physiological conditions insome embodiments, the association complex providesuinproved in vivo delivery of an oligonucleotide such as dsRNA. In vivo delivery of an oligonucleotide can be measured, usinga gene silencing assay, for example an assay measuringthe silencing of Factor VIIL M vivo Factor VU silencing experiments C57B1L/6 mice received tail vein injections of saline or various lipid formations. ILipid-formulated siRNAs are administered at varying doces in an injection volme of 10 pilJg animal body weight. Twenty-four hours after administration, serum samples are collected by retroorbital bleed. Serum Factor VII concentrations are determined using achronogenic diagnostic kit (Coaset Factor VIT Assay Kit, DiaPhanna) according to manufacturer protocols.

Methods of making association complexes Income embodiments, an association complex ismade by contacting a therapeutic agent such as an oligonucleotide with a lipid in the presence of solvent anda buffer. In some embodiments, a plurality of lipids are included in the solvent, for example, one or more of a cationic lipid (e.g, a polyamine containing lipid or a lipid including a biocleavable moiety as described herein), a PEG-lipid, a targeting lipid or a fusogenic lipid. In some embodiments, the buffer is of a strength sufficient to protonate substantially allanines of an amine containing lipid such as lipid described herein, e.g., '15 a lipid oftformula (I) or fomuda (X). In some embodiments, the buffer is an acetate buffer, such as sodium acetate (pH of about 5) in some embodiments, the buffer is present in solution at aconcentration of from about 100 mM and about 300 mM. In some embodiments, the solvent is ethanol. For example, in some embodiments, the mixture includes at least about 90% ethanol, or 100%ethanol. In some embodiments, the method includes extruding the mixture to provide association complexes having particles of a size with hydrodynanic diameter less than about 500 nm (e.g, asize from about 25 nin to about 300 nn, for example in some preferred embodimentsthe particle sizes ranges from about 40-120 nM) Itn some 2a embodinents, the method does not include extrusion of the mixture. In one embodimentaliposome is prepared by providing a solution ofa lipid described herein mixed in a solution with cholesterol, PEG,ethanol, and a 25 mM acetate buffer toprovide a mixture of about pH 5. The mixture is gently vortexed, and to the mixture is addedsucrose. The mixture is then vortexed again until the sucrose is dissolved. To this mixture is added a solution of silNA in acetatebuffer,vortexing lightly for about 20 minutes. The mixture is then extruded (e.g, at least about 10times, e.g, 11 tines or more) through at least one filter (e.g., two 200 run filters) at 40 IC, and dialyzed against PBS at pH 7.4 fbr about 90 minutes at T, in one embodiment. a liposome is prepared without extruding the liposome mixture. A lipid described herein is combined with cholesterol, PEG, and siRNA in 100% ethanol, water,and an acetate buffer having aconcentration from about 100 mM to about 300 nM (pH of about 5), Thecombination is rapidly mixed in 90% ethanol. Upon completion,the mixture is dialyzed (or treated with ultrafiltration) against an acetatebuffer having a concentrationfrom about 100 mM to about 300 nM (pH of about 5) to remove ethanol, and then dialyzed (or treated. withultrailttration) against PBS to change buffer conditions. Association complexes canbe fried in the absence ofatherapeuticagent such as single or double stranded nucleic acid, and then upon formation be treatedwith one or more thrpauticallyactive single or double stranded nucleic acid moietiesto provide loaded association complex,i.e an association complex that is loadedwith the therpaucically active nuclic acids. The nucleic acid can be entrapped within the assocIation complex,adsorbed to the surface of the association complex orboth. For example, methods of forming association complexes such as liposomes above can be used to form association complexes ftee of a therapeutic agent, such as a nucleic acid, for example a single or double strandedRNA such as siRNA. Upon formation ofthe associationcomplex, the complex can then be treated with the therapeutic agent such as siRNA to provide a loaded association complex. In one embodiment mixture including cationic lipid such as a lipid described in formula (9) preferably a cationic lipid ofthe followingformula

NN H H

cholesterol, and a PEG-ipid, for example a PEG-lipid described hereinsuchas the PEG-lipid below,

N H

are provided in ethanol (e.g, 100% ethanol) and combined with an aqueous buffer such as aqueous NaOAc, to provide unloaded association complexes, The association complexes are then optionally extruded, providing a more uniform size distribution of the association complexes, The association complexes are then treated with the thereapeutic agent such as siRNA in ethanol (e.g, 35% ethanol) to thereby provide a loadedassociation complex. in some embodiments. the association complex is then treated with a process that removes the ethanol, such as dialysis,

to Characterization of association complexes Association complexes prepared by any of the methods above arecharacterized in asimilar manner, Association complexesare first characterized by visual inspection Ingeneral, preferred association complexes are whitish transliucent solutions free from aggregates or sediment. Particlesize and particle size distribution of lipidnmoparticles are measured by dynamic light scattering using Malvern Zetasizer Nano ZS (Malvem, USA). Preferred particles are 20-300mn, more preferrably, 40-100 nm in size. Income preferred embodiments the particle size distribution is unimodal The total siRNA concentration in the formulation, as well as the entrappedfraction, is estimated using a dye exclusion assay. A sample of the formulated siRNA is incubated with the RNA 2o binding dye Ribogreen(Molecular Probes) in the presence or absence of a formulation dismpting surfactant, 0.5% Triton-X00, The totalsiRNA in the fonulation is determinedby the signal from thesample containing the surfacumt, relative to a standard curve. The entrapped fraction is determined by subtracting the "free" siRNA content (as measured bythe signal in the absence ofsurfactant) from the total siRNA content.PercententrappedsiRNAistypicaly>85%.

Methods of using association complexes and compositions including the same

Pharmaceutica4 compositi comprisingoionucleotieagents 3o An oligonucleotide agentassembled inan association complex can be administered, e gtoacell or to a huan, in asile-stranded or double-stranded configuration. An oligonucleotide agent that is in adouble-strandedconfigurationis bound to a substantially complementary oligonucleotide strand. Delivery of an oligonuleotide agentin a double stranded configuration may confer certain advantages on the oligonucleotide agent, such as an increased resistance to nucleases. lone embodiment, the invention providespharmaceuticalcopOSitions including an oligonucleotide agent packaged in an association complex, such as a liposome, as described herein, and a pharmaceutically acceptable carrier. The fbr pharmaceuticalcomposition comprising the packaged oligonucleotide agent is useful treating a disease or disorder associated with theexpression or activity of a target gene, 1 such as a pathological process which can be mediated by down regulating gene expression. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that e formulatedfordelivery to a specific organ/tissue, such as the liver, via parenteral delivery. The pharmaceutical compositions featured in the invention are administered in dosages sufficient to inhibit expression of a target gene. In general, a suitable dose of a packaged oligonucleotide agent will be such that the oligontleotide agent delivered is in the range of 0.01 to 50milligams per kilogram body weight of the recipient per day, generally in the range of I microgramto Irmgper kilogram body weight per day, The pharmaceutical compositionmaybe administered once dy, or theoligonucleotideagent may be administered as two,three, or more sub-doses at appropriate intervals throughout the day or even using continuous infusion or delivery through a controlled release formulation. In that case, the oligonucicodde agentcontained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage, The dosage unit can also be compounded for delivery over several days, e,g.using a conventional sustained release fornulation which providessustained release of the packaged oligonueleotide agent over several day period, Sustained release formulations are well known in the art, The skilled artisan will appreciate that certain factors may influence the dosage and tiring required to effectively treat a subjectincluding but not limited to the severity of the disease or disorder, previous treatments, the general. health and/or age of thesubject,andotherdiseasespresent Moreover, treatmentof subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual oligonucleoideagents packaged in the association complexes can be made using conventional methodologiesor on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein. Advances in mouse genetics have generated a number of mouse models for the study of various human diseases. Such models are used for in vivo testing of 0ligonucleotide agentspackaged in lipophilic compositions, aswell as for determining a therapeutically effective dose. Any method canbe used to administer an oligonucleotide agent packaged in an to associationnoplex, suck as a liposore, to a manual. For example, administration can be direct; oral; or parenteral (e.g., by subcutaneous, intraventricularintramuscular, or intraperitoneal injectionorby intravenous drip). Administration can be rapid (e.g.by injection), or can occur over a period of time (e,g, by slow infusion or administration of slow release formulations), An oligonucleotide agent packaged in an association complex can be formulated into compositions such as sterile and non-sterile aqueous solutions, non-aqueous solutions uncommon solvents such as alcohols, or solutions in liquid or solid oil bases. Such solutions also can contain buffers, diluents, and other suitable additives. For parenteral, intratheca, or intraventricular administration, an ogonucleotide agent can he formulated into compositions such as sterile aqueous solutions, which alsocan contain buffers, diluents, and other suitable additives (e.g& penetration enhancers, carrier compounds, and other pharmaceutically acceptablecarriers). The oligonucleotideagents packaged in an association complex can be formulated in a pharmaceutically acceptable carrier or diluent. A "phannaceutically acceptable carrier" (also referred to herein as an "excipient") is a pharmaceutically acceptable solvent, suspendingagent, or any other phannacologically inertvehicle. Phanmaeuticcaly acceptable carriers can be liquid orsolid, and can beselected with the planned manner of administration in mind so as to provide for the desired bulk, consistency, and other pertinent transport and chemicalproperties. Typical so pharmaceutically acceptable carriers include, by way of example and not limitation: water; saline solution; binding agents (e.g., polyvinylpyrrolidone or hydroxyprupyl methyicellulose); fillers (e.g, lactose and other sugars, gelatin, or calcium sulfate); lubricants (e.g, starch,polyethylene glycol, orsodium acetate); disintegrates (e.g, starchorsodium starch glycolate); and wetting agents (e.gsodium. lauryl sulfate).

EXAMPLES

triethyenetetamineunderMichael addition condition IethodI(SchemeI) Scheme l 0 NH

seR emR RIR

A + ,

, RR

3 4 n

"(i) 90 °C, Neat, 5 days

ina350 mL pressure bottle N dodecylacrylamide 1(84 g, 0,35 mol) [Mee, Deborah }L; Romano, Suzanne J.; Yu, Jinghua; Nguyen, True N,;John, Judy K, Rahja, Neil K.; Axe, Frank ,; Jones, Todd K, Ripka William C Joumal of edaicial Chemistry (2001), 44(13), 2094-2107) was taken and the solid was melted under argon by gently heating the vessel. To this melt was added triethylenetetramine 2 (102 g 0.07 16 mol) and the mixture was heated at 90 °C for 5 days. Michael addition of triethylenetetramine 2 to the acrylamide I yielded two five and the sole six alkylated products along withminor amounts oflow alkylated products under neatreaction condition. Thereaction mixture was analyzed by TLCusing CHCb:MeQOHNEt (90:5:5) as the eluent, The T LC showed the near complete consumption of the starting acrvlamide I The reaction mixture was dissolved in dichloromethane (40 mL), loaded on a pre-packed column of silca gel and the mixture was separated using enent CH2 CL:MeOHNt(48:1;1 to 8:1:1)In order to achieve complete separation, multiple columns using the same conditions were performed and the following pure products were obtained, The required five addition products 3 and 4 were isolated along with the six addtionproduct 5 In this reaction mixture some of the'lower addition products were also detected in the TLC and the LC-MS of the crude reaction mixture N-Dodecy[3-((2-dodecylcarbamnoylethyI)2-(2-dodecylcarba noyhethyf) 2-{(2-dodecylearbamnoy1ethy-{2-(2-dodecylearbamnoy1-ethyhaunnkethyIF-aminoh a ethyl-anlno)propionamide. One of the two 5-alkylated derivatives, compound 3 (isomer ), was isolated as light yellow foam (12 g, 13%). MS l 672 (Mi21/2), 448 (M+3H1/3I) 'NMR CDb 6 0,87 (J= 6,5Hz, 151), 1.20-1.39 (m,92H), 1.46-1L57 (m,'12H), 2.20-2-50(m, 1611),2.60-278 (in, 10), 310-3,25 (m, 121),6.98 (bs, 31), 7.41 (bs 111, 7,63 (bs, 1H), 8,85 (bs, IH) C NMR CDC165 1433, 22.90 2737, to 29.59, 29,67,' 298,29-89, 29.92, 3213, 39374, 172,77 (3i(24{24{2-Bis-(2~dodecyiearbanmoybethyl-amninloIethyl-(2 dodeeylcarbamoybethyl)-auinoi-ethylamino}-ethyl)- (2-dodecylearbanioy-ethyl) amnoV-dodecyl-propionmide). Second5-alkylated derivative, ompound 4 (isomer11) was isolated as a whitepowder (13,7 g, 14%), MS m/A 672 (M+2H/2),448 (M+3'3) HINMR CDCh 0.87 (t,1=6,5Hz, 15H), 1,20-.39 (n, 92H), 1.44~L54 (m,121),.2.30-2,45 (nm K >2.46-2,54 (m, 811), 255-2.85 (,10h), 315-3,30 (m,

12H), 6.98 (bs, 31), 7.41 (bs, I H), 7.63 (bs, iH), 885 (bs, 1). "tCNMR CDC> 6 14.33,22,89,27,28,2738, 29.59,29.69,29.88, 29.89,2992,32,13, 39.65,39.74,50.84, 172,63,172.75, 17281. Alongwit this apuremixture of compounds3 and 4 (11 6 g, 12%) in 23 (3:4) aiowas aso isolated. 34{2d{2-[Bis-(2-dodeey~carbamoy-ethylamino-ethy}-(2 dodecylearamoyethy)-amiokthyl}(2dodCYiarbamoy1ethylVkanlUf]~ethyi} (2~dodecykabamyethy)-andnFN-dOdcykpropkonamlide.Thesix alkylated product 5 was isolated as cream powder (16,3 g, 17%). MS m/ 792 (M+2H/2), 528 (M+3HF/3), 'H NMR DMSO-d 6 6 0.87 (t, J= 71-1z, 18H), 1,5-1.40 (n, 11211), 1.45 153 (m, 121), 2.20-2.35 (m, 1211), 2.37-250 (m, 12H),2.64-2,8 (m, 12f), 330~3,25 (m, 12H), 7,26 (s, 411), 764 (bs 2H) C NMR CDC1 6.14.32, 2289, 17,34,27,38 29.59, 2969, 29.90,29.92, 3213, 39.77, 50.85, 17280. 1jgmpl ?jynthepssand puriication of c mnonds 3, 4 and 4 yliipf triethylen9ettraminunderMichaeladditioncondition-mhod 2 (Scheme2)

In another experiment, in order to prevent the polymerization of thestarting acrylaide Iathigh temperature, a radical quencher benzoquinone was added to the reaction mixture. Scheme 2'

RR R R

D R= N H (i) 90 C, Catalytic amount (15 mg) ofbenzoquinone, 5 days In this method;a similar reaction to that of Method I (Example 1) was performed except that, aradical quencher benzoquinone was added to the reactionmixture, In a 150 mLpressure bottleI-dodeeylacrylamide 1 (24 g, 100 ramol) was taken and to this 1o15 ng of henzoquinone was added and the solid acrylarmide wasmelted under argon by genlyheating the vessel To this melt was added triethylenetetranine2 (29g,20 mmol)and the mixture washeated at90°Cfor5daysThereationmixturewas analyzed byT LC using CH2 C1:MeOi:NEt 3(90:5-5) as the cinet. The TLC showed the near complete consumption of thestarting acrylamide 1 The reaction mixture was dissolved in dichdoromethane (40 ml) and the desired products 3, 4 and 5 were isolated as described in Example 1I In this case a slight increase in the amount of six addition productwas observed. Compound 3: The five addition product, isomer 1, was isolatedas light yellow am (3.4g, 13%). The analytical andspectral data for this compound was identical to that of 3 obtained by Method 1. Compound 4: The five addition product, isomerIt, was isolated as white powder (19 g, 14%)t The analytical and spectral data for this compound was identical to that of 4 obtained by Method 1. A pure mixture of isoners 3 and 4 (1.9 g, 7%) was also isolated,

O

Compound 5: The six.addition product was isolated as a cream powder (6,9 g, 26%), The analytical and spectral data for this compound was identical to that of5 obtained by Method 1. EampeLSvntheses and purification of cLp 3, 4 and 4alkylation of trievienp a condt-inebd3lene mteordichaelhad it eon 33 In this method the Michael addition Was performed in the presence of apromoter like boric acid (Chaudhuri, Mihir K.; Hussain, Sahid; Kantam, M, Laksmi: Neelima, 1 Tetraedron Lettrs (21005), 46(48) 8329-8331 )in order to enhance the rate of the reaction, Scheme 3a H H~ xA½A X.AX H 2 NW N

R HRN N N R R R

(16

H

()90 C aq boric acid, 2 days In this method a similar reactionto that of Method 1 (Example 1) was perfonned except that, aMichael addition promoter, saturated aqueous boric acidwas added to the reaction mixture, 1 a 150 mL pressure bodeNdodecy-acrylamide 1 (24 g, 100 mnol) was melted under argon by gently heating the vessel and to this 3 mL of aqueous boric acid was added To this melt was added triethylenetetramine 2 (2,9 g, 20 mmol) and the mixture was heated at 90 °C for 2 days, The reaction mixture was analyzed by TLC using CH(t 2 :MeOH:NEt 3 (90:5:5) as the eluent, The TLCshowed the near complete consumption ofthe starting acrylamide 1. The reaction mixture was dissolved in dichloromethae (100 mL) and the solution was stirred with solidsodium bicarbonate and the organic layer was filtered and concentrated in a rotory evaporatorThis crude product was purified bycolumn chromatography (silica gel) using CHC:MeOH:NEt (48:1: to 8:1:1), In order to achieve complete separation, multiplecolumnsusing the 2 same conditions were performed and thefollowing pure products were obtained. Under this reaction conditionan increase in yields of compound 4 (isomer I) and six addition product 5 were achieved. Compound 3: The five addition product 3, isomer 1, was isolated as light yellow foam (3.1 g, 11%). The analytical and spectral data for this compound wasidentical to that of 3 obtained by Method 1. Compound 4;The five addition product 4, isomer II, was isolated as a white powder (5.7 g, 20%), The analytical and spectraldata for this compound was identical to that o4 obtained byMiethod 1. A pure mixture of isomers 3 and 4 (2,1 g,%)wasalso isolated. Compound 5: The six addition product 5 was isolated as a cream powder (7,6

& 28%) 'The analytical and spectral data for this compound was identical to that of 5 obtainedby Method ,

Example 4: Synthesesanipurification ofen.mpounds3and 4: alkvatio of trieylenetetramineunderMichael addition condition.-method..gge4 In anotherexperiment, in order to minimize the formation of the six addition product 5,use of solvent was attempted. Scheme 4' 0H

R R 3 4

(i) 90 °C acetonitrie or DMF, 5 days inthis method a similar reaction to that of Method I (Example)andMethod2 (Example 2) was performed except that, the reactions were performed in the presence of solventsat 90 Cwith stirring.In a 150 mL pressure bottle Nr-odecylacrylamide 1 (10 g, 41,8 mmol) was dissolved in 20 mL of either acetonitrile or DMFTo this solution was added triethylenetetraniie2 (1 g, 6.8 mmol) and the mixture was heatedat 90 'C Iusing CH2CMCeOH:N"Et for 5 days. Thereactionmixture was analyzed by TLC (90:5:5) as thel chent. The TLC showed the formation of only minor amounts of the required five additionproduct, The major production this reactionwasa mixture of four addition products along with very polar lower addition products kgint& _araion f reacteerylamide from thereacionixture and/ortheisolatedproduts34 and5 To remove unrelated acrylamide'I from the reaction mixture, the reaction mixture is diluted with ethyl acetate or DMF and stirred with polystyrene or polymer bound thiol (or mercaptan) to capture all the acrylamide. The immobilized thiol was added to the solutionand gently shaken at ambient temperature andfilter offthesolid. Michael addition of immobilized thiol to acrylamide capture allureacted acrylanide 1o Traces of acrylamide as contaminant after isolation of each desired isomer could also be completely removed under the same condition. The isolated product 3 (or 4 or 5) is dissolved in DMF or ethyl acetate and gentlyshaken with the immobilized acrylamide quencher, filterand imevaporation of the fiitrate in vacuo affords a pure compound 3 (or 4 or 5) free of acrylamide contamination. amgple 6; Spagiogo4q4f iiaaseondaryamipe contaninantfRom fpgnml~d 5 After colum ehromatographic separation of compound 5, to remover traces of primary and secondary amine contaminants, the compound is dissolved in ethyl acetate or DMF and stirred with solid bound or immobilized isothiocyanate at ambient temperatureoverightFilter off the solid and evaporation of the filtrate affords apure compound 5 iee of'any primary or secondary amine contamination.

ample7: Separation ofpryarv amine contamrinas fronund 3 and 4 After the completion of the reaction the reaction mixture is treated with tetrachlorophthalic anhydride in the presence of triethylamine indichloromethane at room temperature and the solvent is evaporated and the residue stirred with ethyl acetate and the solid is filtered and the filtrate is concentrated to get the products which lacks the primary amine contaminant

Table I Methods of synthesizing products 3 and 4 Method Temperature Promoter Solvent Radical Remarks Quencher I 909°C None Neat None Fonnation of 3 and 4 in a combined isolated yield of 39%. The six addition product 5 was isolated in 17%, Reaction took six days for completion. 2 90 °C None Neat Benzoquinone Benzoquinone was used to prevent thepolvtmerization of acrylamide L The combined yield of3 and 4 was 34% However 26% of 5 was also isolated. Reaction time same as Method1L 3 90 °C Boric acid Neat None Reaction rate enhanced. The reaction was completed in two days. The combined yield of 3 and 4 was 38%, Additional 28% of 5 was also isolated. 480420 C None DMF None Reaction veryslugsh,

Only lower addition products formed.

Example 8; Methods ofparatonofthehydrochloridesales ofthe products

In order to improve the ease of handling and increase the stability of the compounds listed above, they were converted into their corresponding hydrochloride salts6,7and8. Hydrochloride of compound 3 (6) The amine 3 (9,4 g) was dissolvedin 100 mL of hot anhydrous 1,4~dioxane and 100 mL of 4M HC1 in dioxanewas added and the mixture was stirred atroom temperature ovemight. Nitrogen was bubbled into the reaction mixture for I hto remove the excess HCI and the remaining solution was concentrated to -10nL To tisheterogeneous mixture 100 mL ofEtOAC:hexanes (1I1) was added and the precipitated product wasfiltered, washed with ethyl acetate (50 m) hexanes (100 mL) and the resulting powder was dried under vacuum to get the pure product 6 (9.99 g,.96%) as a cream powder, 'H NMR CDCI8 0.83(, I J6.5Hz, 151), 120-1.39 (in,92 ,2.64-270(m,8H), 2.90-310 (n,6), 325-3,45 (m, 12H), 3.46-3.64 m,4H), 520-6,0 (bs, 211), 8.05-85 (m, 5H), 10. (bs3H), CNMR CDCb 13.83, 220 26.4828.69,28,79,28,90, 29,04,31L26,3871, 16838, 168.53, ElementalAnalysis:Caled.C:163%s,4H C3H 2 0: C, 63.05; H, I 13; N, 8.17; Cl, 919. Found: C, 6313; H 1106; N, 821; Cl, 9.21 SchemeV5

R .R

3 R

H *6(i) 4MHCI in1,4-dioxane, rt., 12h Compounid 7 The amine 4 (13.7 g 10.2 mmol) was converted to the corresponding 11Cl salt 7 using a similar procedure used above for 3 to obtain 6. The tetrahydrochloride salt 7 was isolated as a White powder (14,6, 96%).HNMR CDG8 S2.,.= 6,5H 15$), 120-1l41 (m, 921), 2,52-2.72 (m, 8H), 2,90-3.10 (m, 16H), 3.25-3.45 (m, 12H), 3.46 364 (m, 41),5,20-6.0 (bs, 2H), 805-8.15 (m, 5H), 10. (s, 3H) "CNMR CDC 8.42, 13.84.22,04,26.48,28.69,28.79, 2900, 31.26,45,44,168,53, 16860. Elemental

Analysis:Caled:%CH163NgOs4HCL2H 2 O:C, 63,79; 11,1 130;N, 8,17; CI, 9,34 Found: C, 63,78; f, 1L04;N, 8.40; Cl 9.73, Scheme 6

RH R N~ H*" R HCi

4 7

() 4MH in1,4-dioxane, rt, 12h Compound 8 The amine 5 (13.7 g, 1.2 mmol) was converted tothe corresponding HCI 8 using a procedure similar to that described above for the salt 6The tetrahydrochloride salt 8 wasisolated as a white powder (1.3 g, 96%). H NMR DMSO-4 3 0,87 (t, J=7Hz, 18H), 113-1 30 (M, 112H), 135-1,53 (i 121J), 2,10-2,25 (, 12.1)2-30-2.40 (m, 12$1), 260-2,76 (m, 1211), 3 10-3,25 (m, 121), 726 (b, 4H), 7.64 (b 211), 103i (bs, 4H). S3-heme 7a

R -R--N'4C R R R

RH

H(i) 4M HCI in 1,4-dioxane rt, 12h.

Example 9: Selective protection of am--ino iroups on trehlnttaiefor

dirogedsndieisof.oinpowd3 and4 Step 1: Preparationt of compound M0 Triethylenetetramine, 2 (20,55 g,140-52 1mol, purchased, ftrm Sigmia-AIldrich) in aceonitrile (500 mL) was cooled over an ice 20bath under constant stirring, Ethyl trifhiroacetate 10 (35.20 mnL, 295i09 mmnol) was added to the stirring solution and stirred for 20h. Solvent and volatiles were removed under reduced pressure and dried under'high vacuum to get 9 as white solid (444 g, 94%), product thus obtained could be used for the next reaction without further purification (Wender P. A. et il. O.,gani, Letters, 2005 7, 4815), 25CrudeI compound 9 (23:70,70) mmol) was dissolved in acetonitrife (400 mnL) and stirred Over an ice bah -Bnyoyabnlx)succinate (Z-O~u, 43.73g, 175 mmol, purchased from Novabiochem) and triethylamine (2340 mL, 210mmol) were added to the reaction mixtue and stirred overnight, Solvents were removed and the residue was extracted into dichloromethane (DCM), washed successivewith water (two times) and brine, dried over anhydrous sodium sulfate, Solvent was removed in vauo and residue thus obtained was purified by silica gel column chomatography (gradient elution, 30-70% EtOAc/Hexanes) to obtain compound 10 as whitesolid (382g,89%). ' NMR (MSO-d6, 400MHz) 5= 9.60-9.50(m, 2),7.40-7,20(m, 10H), 5.02(s, 41), 3.40-3.20(m, 12H).MS:CKHFeN 40 Cal 606.19 Found. 6072(M"'). Step 2: Preparation of compound 11: Compound 10 (12.60 g, 20,78 mmol) was suspended in methanol (MeOH, 150 mL) at ambient tempeatureand 3M solution of methylamine in ethanol (40 ml) was added to the suspension underconstant stirring All the solids went into solution aitrring for I h at ambient temperature, the mixture was warmed to 50C and stirred. forSh. Reaction was monitored by TLC All the is solvents were removedunder reduced pressure and the residue was purified by silica gel cohunchromatography (gradient elution, 10%MeO/DCM to 0:1080" MeGH:TEADCM) to yield the product 11(7.80g, 91%)as pale yellow gummy liquid. 'HNMR(DMSO-d6,400MHz)6S 7,80-7.40(m, 10H), 5.02-4.94(m, 4H), 3.45-3s05(m,, 8), 2370-2.55(m 4H),2.20(bs, 4K). IS: C22 N404 Cal.414.23, Found 41520(vf') Step 3: Preparation of compound 13: Compound 12 was prepared from triethylenetetramine, 100 (10.25g, 70.09mmol) as described instep I for the synthesis of compound 9 by reacting with 1,1 molar equivalent of ethyl trifluoroacetate(880mL, 77.10mmol). Crude 12 thus obtained was dissolved in anhydrous DCM (40nI) and cooled to 0C. (Boc)20 (53.53 mmol, 245.31nmol) and triethylamine (4i ml 350mmol)wereadded and reaction mixture wasallowed to stirovenight, Progress of the reaction was monitored by TLC Solvents were removed in vacuo and the residue was extracted into DCM, washed with water, brine and dried. DCM was removed id the residue was purified by silica gel chromatography (gradient elution 50%EtOAcHexane to EtOAc) to obtain the desired product 13 (34.20g, 92%)aswhite solid. 'K NMR (DM-SOd6, 400MHz) 6 9,51-9.38(mH),6,82(bs, I H), 3,30-3.00(m, 12H), 158-130(s, 27H) NS: C 4 1EF 3N4 0 Cal. 542.9, Found 543.4(M*)

Step 4: Preparation of 14: A solution of compound 13 (25g, 47.32 mml) in MeoH (200 mL) was stirred with K 2CO (50tg) in the presence of water (IniL) at 50 °C overnight. Progress of the reaction was monitored by TLC. SolidK2COwasfiltered off washed with MeOH combined washing andsolvents were removed in vacuo. Residue obtained was purified by silica gel column chromatography to yield the desired product 14 (10,2 g 50%)as white solid. H NMR (DMSO-d6,400MH z) = 6,83(bs, 1H1), 295-3.30(m, 12H), 12.62-2.50(m2H), 1l.25-1,45(m, 27H) MS: C142N40 CaL 44631, Found 447.4(M'), Scheme 8" NA N MNg/OH Obf

p o 0* Qbzz

0s

H oacetate 1e.Ethylerf

CHC

H 2HH

S.

(oc) 2O!TEA

V HiCNiTHFAVATER

fl K2CO3 Bc F NHo - NN.NfN>NH~oC

gHHsoocc14 1

SSelective protection oftriethlenetetramine nitrogens.

Step 5: Preparation of compound 15: Compound 9 (23.Og,68.02mmol) was dissolved in a mixture facetonitrldihloromethane (1:1, 300mL) and cooled to O'C,

Z-OSu (17.00g 69mmol) was added to thesolutionandstirredfor minutes. Triethylamine (23.40 mL, 210mnol) was subsequently added to the reaction mixture and allowed to stir ovemight. Solvents and triethylamine were removed in vauc and the residue was extracted ito DCM, washed with water (two times), brineanddried, After removing solvent, theresidue was purified by silicagel column chromatography (eluted initially with 20-60 %EtOAc/Hexane, then with 5% MeoH/DCM) to obtain the desired product 15 (13,3g) as white solid along with side product 10 (85g). -HNMR(DMSO d6,400MHz) 6= 9,60$(bs1H), 9.30(bs, li), 7.40728(m, 511), 5.01(s,2H),3-40 3,i0(m,S8i),.2.70~250(m, 4),MS: C 3 82 F6 N 4 04 Cal.472,15;Found 473.1(M*). SStep 6: Preparation of compound 16; Treatment of compound 15 (13,4g, 2838 mnmol) with methylaiine (50 ml, SM solution in EtOH) as described in step 2 yielded colorless liquid compound 16 (6.1Og, 79%). The product thus obtained could beused fornextreactionwithoutfurther purification. NMR (DMSO-d6, 400MHz)6 =,45~7,20(m 6H),5.07(s, 2H), 3.45-2.90(m8H), 1260-2,30(m, 41), MS:CjIHN 402 S Cal 280,19 Found 2812(M). Sheme 9

HI F 1.05Z-Osu N~

N .N v F Nbk Methy1ine(MeOH) N 50°cF

16

blocking of single secondary nitrogen of triethylenetttamine aSelective

EgipiSynthesis of 5-alkylated single isomer 4 -- Meod I 2o Step 1: Reaction of I1 with Ndadeylarylamide: Dimine 11 ( Og, 2 41 mml)andN-dodecylacrylamidec(3,47g, 14.50 mmnol) were taken togetherin a pressure tube and heated at 90C for 5 days. The reaction was monitored by TLC. Once the reaction is over, the mixture is dissolved in dichloromethane and purifed by flash chromat.graphytoget theproducts 17,18 and19. as Step 2: Preparation of compound 20: Compound 19 (2.00g 146 mmol).is dissolved in a mixture of ethylacetate and methanoi (12, 15 ml) to that2 eq. of acetic acid is added. The mixture is hydrogenated under pressure (50 psi)using palladiumcarbon (0200g 10%wt) as a catalyst to get the desired product 20.

Step 3: Preparation of single isomer 4: Compound 20 (150g, 136 mmol) and the acrylamide 1 (0.325rmmol, 1.36 mmol) is dissolved in toliene (4nL) and heated at 90'C days to form compound 4, Progress of thereaction is monitored byTLC. After completion of reaction,the mixture is cooled. to room temperature, dissolved in DCM and purinfed by flash silica gel column chromatography to obtain the desired product 4 Scheme 10 Cbr HNNNH

N-Drndecylacrylamde Neat 90W C

- N R N .R N+ N H Ctz Cbz 9

i, Pd-c, EtoAcJ~eOH|

R rN-odeicarmde R R .eq 4N,~~y

4 20

H

aj pll: 5soSlkyly t dsingl somer 4 Method2

Step 1: Preparation of compound 21: Compound 16 (LOg, 356nmol)andN dodey6acrylamide(6.00g, 7e) are taken together in a pressure tube and heated to obtam compound 21 Progress of the reaction is monitored byTLC. After completion of the reaction themixture is dissolved in DCM andpurified by flashsilica gel chromatography to afford the desired compound 21. Step 2:Preparation of compound 4 from 21: Compound 21 (2,00g,1.35 nmnol) is dissolved ina mixture of ethyl acetate and methanol (1;2, 15 ml) to that 2 eq. of acetic acid is added. The mixture is hydrogenated under pressure (50 psi) over palladium-carbon (0,200g, 10%wt) to afford the desired single isomer 4.

SchemeN1e N-Dodec(9 1ytarfamie R H2 N *N

c and showedt fortion ctrTh.racio

N N H

Exml 2 ytei of 5-alkylated sinale isomer3- Methodi Step.1:Preparationof compound 22: Compound 14 (506g 11 30mmo) and 5 KNdodeeylaryamidec(2.94g, 12.43 mmol)were taken in toluen andbeated at 90°Cfor five days. TLCwas checked and showed thetformation ofproduct.The reaction mixture was directly loaded onapre-packed column of column silica gel and purified by flash chromatography (5% MeOH{DCM) to afford compound 22 (4,82g, 62%). T H NMR (DMSO-d6,400MHz) 6 =8.17(bs IHl),660%(bs, Ii), 3.30-295(m, 12H), 270(t, 1 580Hz ), 2.60(t, J=6,00Hz, 214) 218(t, J=6,40Hz, 21H), ,35(m, 29H), 126 1.i5(m, 18108(t, J=600Hz,3H). MS: C-3 H71NO7 Cal. 685.54, Found 686.5(MP), Step 2: Preparation of compound 23: Compound 22 (475g 6.92 mmol) was dissolvedindichloroethane(00m)and cooled to OC, 2SOSu(2.5, LSq)was added to thesolution and stirred for 10minutes. The reaction mixture wassubsequently stirred with triethylamine (2,82 mL,20,76mmol) overnight, Solvent and trithylanine were removed. in vacuo and. the residue was extracted into dichloromethane, washed successvely with water (two times) andbrine, ad dried over anhydus sodium sulfate Aferremoving solvent the residue was purified by flash silica gelcolumn chromatogaphy (5-10% MeOHDCM) to obtain the desired compound 23 (5.33g, 94%)HNMR(CDdh,400MHz) 6 =749-7.25(m,, 5.1,21s,21), 3,60-3,02(m, 141), 2.45-45(m, 41), 1.50-l35(m, 271), L24-120(m,18H), 0,87(t, J=6.00Hz, 3H). 9 Cal.819,57, Found 820,7(M"), MS; C¾NfO Step 3: Preparation of compound 24: 4M HC1 in dioxane (50mn) was added into solution of compound 23 (5.30g, 650 mmol) in dioxane (100ml).The reaction mixture was then allowed to stir overnight. Product was precipitated out during the coaurseo f the reaction, Solvent and HC1 were removedunder vacuum to yield a white solid,.Thresidue was taken in MeOH containing excesstriethymine and the suspension was stirred for 1h to obtain a homogeneous solution. Solvents wereremoved in vacu and the residue was tniturated with EtOAc, filtered off the triethvlaminIe hydrochloride salt. Combined filtrate was evaporated under vacuum to obtain a gummy 5 liquid 24 (3,30g, 98%) h NMR (CDC1, 400MHz)8 7.3-7,28(n, 5),5105(s,2H), 3;60320(n, 4H), 3.10-2,70(m, 101), 240'-220(m, 41'),10 1A 130(m, 21) 1,25 i17(m,18H),0.81(t, i=6.00Hz, 3H). MS: C29 15 3N0 Cal 519.41, Found 520.4(M), Step 4: Preparation of compound 25: Compound 24 (10g, 1925 miol) and N-dodecylaerylamide (37,8eq) are taken together in a pressure tube and heated at elevated temperature to form desired cornpound 25. Formation of the product is monitored by'TLC and is subsequently purified by flash silica gel column chromatography to afford a pure compound 25. Step 5: Preparation of compound 3: Compound 25 (2,00g, 1,35 mmol) is dissolved in a mixture of ethyl acetate and methanol (1:2, 15 ml) to that 2 eq, of acetic t acid is added. Themixture is hydrogenated under pressure (50 psi) over palladium carbon (0;200g, I0%wt) to afford the desired product 3, Scheme 12 R, oiuene; heat To H H B22 14

LOsu TEA DCM V

N 'Ni ( R He) R Cb. 24 H Z TEA Qb? 23 ac

N-dodesyacylamie(excess) heat

R dR FR~..~r"NN'H,, HPdC bH

Eamnie 13: Svthesis of5-alkyad singe isomer 3 - Meth Step I: Preparation of compound 26: Benzyl bromide (125 ml L5e) to a suspension of compound 22 (480g,7.00mmol) and K 2 C0 3 (9.67g) 1Oeq)in DMF (100 miL)and the mixture was stirred overnight, Progress of the reaction was monitored by

TLC, Solids were filtered off, washed with MeOH and ethyl acetate. Combined filtrate was concentrated under reduced pressureand the residue thus obtained was purified by silicagel colunm chronatography (50-100% EtOAc/lIexane) to afford the desired compound 26 (3-30g 61%), 'H NMR (DMSO-d6,400MHz) S=7,77(bs,2), 7.28 S 7,23(n 5), 6,85-6.70(m, 1H), 3.59(s, 2H), 3.20-2.20(n 8H), L35(s,27H, 1,30 L23(m 2H), 20-L15(i,18H)6183(tJP6.00Hz,3H),S:C4 HnNSO, Cal. 775.58, Found776,5(M t )

Step 2: Preparation of compound 27: Compound 26 (3,30g, 4,25 mmol) in dioxane (50ml) was stirred with 4M HC (50 mL)in dioxane ovemight, Formation of io white precipitate wasseen during the course of the reaction, Solventand acid'were removed under vacuum and white residue thus obtainedwas redissolved inm ethanol containing excess triefylamine. The homogeneous solution was then evaporated under reducedpressure to obtain while residue.'The residue was triturated with EtOAcand filtered offtriethylanine hydrochloride salt, Filtrate was evaporated under vacuum to 1 afford the desired compound 27 (2,36g 99%) as gummy liquid. NMR (CDCI. 400MHz)J 805(t,J.5Hz, I),740-7.20(m, SH), 3:58(s, 2, 3.10-2.30(m, 18H), L40-130(m 2H), 125-15(m, 18H), 0.82(t, J= 6,00Hz, 3H). MS: Cx'fN 0 aOL 47543. Found 498A(M+Na) Step 3: Preparation of compound 28: Neat compound 27 (00g,210 nmol) andAN-dodeyacrylade(4.0g, Seq) are mixed ina pressure tube and heated to elevated temperature to form compound 28. Formation of 28ismonitored by TLC and LCAMS. After completion of the reaction the product is isolated by chromatographic purification to afford pure compound 28. Step 4: Preparation of compound 3 from compound 28: Compound 28 (2,00g1.40mmo)is dissolved in a mixture of ethyl acetate and methanol (1:2,15 m) to that 6 eq. ofacetic acid isadded.The mixtureishydrogenatedunderpressure(50psi) over palladium-carbon (0.200g, I0%wt) to obtain compound 3

Scheme 13 130C N" NHNo0 R N N R H 22 c DMF 2$oc

RHe (4M,£Dionne)

HH

H P c EOAcMeOH

H NN R H

4: Converent synthesis ofisoner3:- t hodI

Step 1: Preparation of compounds 30,31 and 32: Ethylenediamine 29 S (0,978ml, I463mmol), N-dodecylacrylamide (7.00g, 29.26mmol) and boric acid (100omg weretaken in 5 mL of water and heated at 90Cfor four days. Complete disappearance of acrylamid was ascertained by TLC analysis. Thereaction mixture was dissolved in DCM, washed with water and bicarbonate and dried over sodium sulfate. DCM was removed and the residue was purified by silica gel column 1) chromatography (2:2:96 to 10:10:80% MeOH/TEAJDCM) to get compounds 30 (.86g) H NMR (CDC.,400MHz) 6= 7.05(bs, 2H), 3,21 (q, J-630 Hz, 4H), 2,87(t J= 6.00Hz, 4), 2.73(s, 4H), 2.34(t, J600Hz, 4H),L57(bs, 21), 1,49-L45(r, 41) l28 1.19(m 40H), 0.87(t, = 6A8Hz, 6H) MS: C3 H66N 40 2Cal. 538-52, Found 53930(M+), 31 (3.50g) 'I NMR (DMS0-d6, 400MHz)6 = 8,20(bs, 11), 3.202, 15(m, 221-1), 136 56 L30(m, 6H), .25-.15(m, 30H), 0,81(t, J=6,00Hz, 9H) MS: C41 H¾N5 Q, Cal 777.74, Found 778,7(M+)and 32 (1.75g) I NMR.(DMSO-d6,400MHz) 8 3,23-2J15(m, 2814),35-1i45(m, 811), L26-1.5(m, 40H), 082(1, J: 6.00Hz, 12H), MS:CJIN04 Cal. 1016),97, Found 1018.0(M+). Step 2: Preparation of compound 33: Compound 31 (1.55g, 2moil) and o C(6g2mmo) retaken inDMF To that chloroactadehyde dimethyl acetal (0,453 ml, 4.00mmol) is addedand stirred for 24h. Reaction is monitored by TLC, filtered offK:CO 3 washed with MeOH. Solvents are removed under reduced pressure and theresidue is subjected to chromatographic purification to afford compound 33.

Step 3: Preparation of compound 34: Compound 33 (2.00, 2.31 miol) is taken in a mixture of MeOl and DCM, to thatPTSA (2.Oeq) isaddedand reaction mixture istirredovernight, The solution isneutralized with sodium bicarbonate solution and extract with DCM and dried. Compound is purified by chrornatographic separation to afford the desired product 34. Step 4: Preparationofsingle isomer 3 from 34. Compound 34 (2.00g, 243 mmol) and 30 (1. 31g2, 43 mmdo) are taken in DCM; to that activatedmolecularsieves is added and stirred for 3h, The reaction is monitored byTLC. Once the reaction is over solvents is removed Theresidue is dissolved in TIF andsodium triacetoxyborohydride (5 eq.) and acetic acid are added and stirred overnight. Solvents are removed and extracts with DCM, chromatographic separation of the residueaffords pure isomer 3. Scheme 8 Nedcrnyi H H H04 H RNR R ...........

+ 9 wates/boic ac d 031 2

-'-' o

RR R R 33 3

Examnple. 15: Convergentsynthes,,is of isomer 3 - Method 2 ,,5 Theo des ired single is4omer 3 is also prepared from compound 30 by selective protection of one of the ni.trogen to obtain compound 35. Compound 35 is subsequently reacted w ithi aldehyde 34 under reductive conditions to obtain compound 36, Acid treatment of 36 affords desired comipound 3.

1 15

Scheme 1.5 RN

R R R pss 34

RR

R= t N N N

Etsxmple16 Cnvenagetsynthesis of isomer 3 - Method3 The desired single isomer 3 is also prepared from monobenzyl ethylenediamine a 37. Alkylation of 37 with 1 affords a mixture compounds 38, 39 and 40 Compound 40 is reacted with aldehyde 34 underreductive conditions to obtain compound 41. H ydrogenoiysis of 41 affords the desired compound 3. Scheme 16

NHBn a¾.'Rr + nHNr ~N'n 5NN~ 37 R

3N R 34K.o i

RN R N 4

R H

ExConvergent synthesis iso r4 Mtd Step 1: Preparation of compounds 43: 'ina 150 nL pressure bottleNdodecyV acrylamde 1 (16.4 g, 688 minmol) was melted under argon by gently heating the vessel and to this 3 mL ofaqueous boric acid was added Tothismelt was added Boo protected ethylenediamine 42 (5 g, 31.2 mmol) and the mixture was heated at 90 °C ovemight. s The reactionmixture was analyzed by TLC using CH 2 CI 2 :MeOH:NEt (90:5:5) as the eluent. The TLC showed the near complete consumption of thestarting acrylamide. Thereaction mixture was dissolved in dichloromethane (100 mL) and the solution was stirred with solid sodium bicarbonate and the organic layer was filtered and concentrated in a rotory evaporator. This crude productwas purified by olunin chromatography ,o (silica gel) using CH 2ClAIMeOH:NEt(48:1:1 to 8:1:1), ThenIjor product in this reaction is the double addition product 43. Minor amounts of mono adduct was also observed, Step 2: Preparation of compound 44: Compound 43 (200g, 3.13 nmol) is taken in dioxane (50 mL) to that HC1 (20 mL,4M solution in dioxane) is added and s stirredovernight. Solvent is removed to get the compound 44. Step 3: Preparation of single isomer 4 from 34 and 44: Compound 34 (2(0g, 2.43 inmol) and 44 (31g., 2.43 mmol) are taken inDCM; to that activated molecular sieves is added and stirred for 3h The reaction is monitored by'TL.Once the reaction is over solvents are removed, The residue is dissolved in THF and sodium triacetoxy o horohydride (5 eq.) and acetic acid are added and stirred ovemight. Solvents are removed and extracts with DCM, chromatographic separation of the residue affords pure isomer 4. Scheme 17

H2 soc44NT,$ BOcHN NR.''''. HR..N.. R

42 wtrorcai

4 R

Example 18:Addition ofN dodeevlacrylamide to I,3~diannoproyane and aehction of the amide to amine Inorder to study the effect ofnumber of charges in the cationic lipid the Michael adducts of acrylamide I with 1,3-diaminopropane 45 was investigated.

II7

Scheme 18'

H ,N NH2 H45

NH + RNN N

49, SOand 5 47 4APH

R

R- R R 53 54

H

() 90 TC,aq boricacid, 16h; (ii) 4M HCIin 1,4-dioxane, rt, 1Zh and (iii)lBlt Step 1: Synthesis of 46, 47 and 48: hi a 150ml pressure bottle Adodecyl acrylamide 1 (15.4 g,64 mmol) was melted under argon by gently heating thevessel and to this 3 mL of aqueous boric acid was added. To this melt was added 1,3 diamnopropane 44 (158 g,21 mmol) and the mixture was heated at 90 °C oveight, The reaction mixture was analyzed by TLC using0H2 C1:MeOH:NEt 3 (90:5:5) as the ciuent.'The'TI showed the near complete consumption of the starting acrylamide1. The reaction mixture was dissolved in dichloromethane (100 ml) and the solution was stirred with. solidsodium bicarbonate and theorganic layer was filtered and concentrated in a rotory evaporator. This crude product was purified by column chromatography (silicagel) using CH2CU:MeOH:NEt 3(48:1:1 to 8:1:1). The major product in this reaction is the triple addition product 46 Minor amountsof tetra addut 47 andh is adduct 48 were also isolated, N-Dodecy1.-3-{{2-dodecylerbamoly-thyI)-3-(2dOdcy1carbaml ethylamino)-propylI-amino)-propionfafmide 46. The three addition product 46 was isolatedas a white powder (5.7 g, 35%). MS m/ 793 (M )'H NMR CDC, 8 0.87( J;- 6.61-z, 9H),1.20-130 (in, 601-), 1.42-.66 (m, 6H), 233 (t, J= 61z, 41),238-2,46

l'8

(m414), 2,60-270 (in, 44), 2.84 (,211),3,15-3 28 (m,611), 6.65 (bs, 111),6.99 (bs, 3H), 4-[{3-[Bis-(2-.dodecyicarbamnoyl-ethyf)-andno)-propyl)-(2 dedecykarbanwyl-cthyl)amino)-N-dodey-batyramilde47. The fouraddition 5 product 47 was also isolated in minor amounts. N-Dodecyl-+(2-dodecylearbamoyi-ethfaniino)-propyain.I propionamide 48.The diadduct 48 was isolated as a cream powder (1.6 , 10%), MS m/h553(M4).:)HiNMR CDd 8 0.89 (t, J 6,6Hz, 6H),1L.10-L:20 (mn4014),1L42 1.66 (m, 4H),2.2J0 ( ;;=6Hz, 414),2-55 (t,4H) 2.60 (t, 41),300 (i,4H), 8.00 (bs, 1g0 2H),

Step 2: Conversion of anines 4, 35 and 36 to their corresponding hydrochloride salts 49, 50 and 5. Theamine 46 (5.5 g) was converted to the corresponding HC 49 using a procedure similar to the described in Example 8 and the dihydoeloride salt 49 was isolated asawhitepowder(5.73g,92%). 'H NMRDMSO-d5 6 0.88(t i=7Hz,9H) 172-L.30t(m,6614),135-l45(, 6H), 2,10-2.25 (m, 2H), 255-2 70 (m, 6),295-35 (m, 10H), 3.20-3.35 (m, 61),816(t IH), 8.24(t, 1H-),9.15 (bs, 11),10.65 (bs, 11). iasimilar procedure to that described in Example 8 the amine 47 is treated with 4M1101 to obtain the dihydrochloride salt 5, in a similar procedure to that described in Example 8 theamine 48 is treated with 4M HC toobtain the dihydrochloridesalt 5L Step 3: Reduction ofanides 46,47 and 48 to amines 52,53 and54: Amine 46 is refluxed inTHFwith excess of diborane overnightand subsequent treatment with 4M - 1 affords hydrochloride salt of polyamine 52. Similar treatment of amines 47 and 48 affords thecorresponding reduced product 53and 54 as their respective hydrochloride salt. Egam le 19: Reduction of polyamides 3 4 and 5 to the corresponding poyvnedeadme Compound 3 is refluxed with largeexcess of diboranein THF to obtain the 3 corresponding reduced product 55. After completion. of the reaction, the reaction mixture is treated with 4M1C1prior to work-up and the product is isolated as its

I19 hydrochloride salt. Hydrohoride saltsof56 and 57 are also obtained from the corresponding precursors 4 and 5respectively, Scheme I

NN NR R H

R RRR s H 56 51

~N,

*(i) Bl.THF, reflux Exappk1 : Iogyagmino alkyllipids-egduction gmttlie Preparation of polyamines 60 from 32: Compound 32 (10X2g, Iunol)is taken inTHF (20 ml), to that BlH.THF (60 nt, I M in THF) is added and refluxed for two days, Reaction is monitored byTLC. Removal of THFgives a white residue, whih S is treated withI 1 HCI and extracts into DCM Chromatographie separation of thecmde products yields pure compound 60 Preparation of polyamines 58 and 59 from 30 and 31; Reduction of amides 30 and 31 undersimilar conditions described for the preparation 60 respectively affords 58 and 59. Scheme 20 RII-IIN RN H .

H 30

it 35 54

N RN R RRN

ESde=:ayhesis of ynkylatiorgis

usipg kalbgdo Step I: preparation of compound 62: A solution of chloroacety! chloride (10.31 mI, 129.37 mmol) in DCM (200 ml) was cooled over an ice bath and to this a solution of dodecylamine (61, 20.00g, 107.81 mmol) in dichloromethane containing TEA (36.70 ml,269.5 mmol) was added dropwise over period of 1 hrThe reaction mixture tuned brownish-black by this time, continued the stirring for another hour at O'CIThereaction mixture was filtered through a sintered funnel, washedwith EtOAc, dilutedwith chlorofom, washed successively with water, sodium bicarbonate solution, 1n I IM HC1and brine, Organic layer was dried over sodium sulfate. Solvents wereremoved and the residue was purified by silica gelcolumnchromatography (550% EtOA/Hexane) to aford compound 62 (26.00g, 92%) as brown solid. NMR (CDCh, 400MHz)8 = 6,59(bs, IH), 4.03(s, 211) 3,25(q, J=6,00Hz, 2H), 154-1.49m, 2H), 1.45-1.15(, 18H), 0.86(t, k6.00Hz 3H). 3MS: C14sCINO Cal, 261.19, Found 262.20(M). Step 2: Preparation of 63,64 and 65Triethylenetetramine2 (OOg, 6,83 mmol) and chloroacetamide 62 (10,00g, 55 eq) are taken together in a mixture of CH 3CN/DMF (1:3), to that K 2 0C0 (9,43 g, 10 eq) and K (50 mg)areadded and heated at 85 °C for three days. Thereaction mixture isfiltered to remove solids, wash with DCM, solvents are removed in vacuo and chroatographicseparationof the crude residueaflfords pure compounds 63, 64 and 65.

Scheme 21

C 0

~Cat K N N CICN, DMP 2 H

NH

tN ..*., I0 HN NH N 0C H H 0 63 o

NH HNZ N'½'

' N 0€ N N AN-N

65C

H NH HN

Step I1 Preparation of 67: Chloroacetyl chloride (4.05ml, 51 mmlwstaken in C 10m)adcoiddw o0C To this a dichIlorome thane solution of NN~ N~r.ANA~xNNZZ~f7NNXAN.At' ~~>rNN NAN22.

di dadecyhami-ne (66, 1 5.00g, 42.41 immol) and TEA (I 4A 3 mi, 2I5 eq.) were added time., dropwvis.e oYver a period of I hr. The reaction mixture tuned brownish-black by this The after thle addition the reaction mixture was stirred fo~r 214 h at ambi-net temperature. reaction mixture was filtered through a sinitered funnel, washied with EtOAC, diluted

with corfnw She uccessively wvith water, sodium bicarbonate solution, 'IM HCl and brine, Organic layer was dried over sodi um sulfate. Solvens wer removed in vacuo and the rsidue was puriied by silica ge~l column chromatography (5-50%

EOAc/Rexane to obtian the required product 67 ( 1215g69%,,) as brownish liquidL '11

NMR.(CDCh,400MHz)= 4.04(s,I2H) 3 30m4, 11.50-.45(m,2H), 1404.20(m, ISH), 0,7(t,J=6,00Hz, 3H).MS:C04 2CINO Cal 430.15, Found 431.2(M), Step 2: Preparation of 68,69 and70: Triethyenetetramine2(0.500g 683 mmol) and hloroacetamide67 (8.10g, 5.5 eq) are taken together in a mixture of CH3CNDMF (1:3),to that K 2 003 (4,72g, 10 eq) and KJ (30mg)areaddedandheated at 85°C forthree daysThe reaction mixture was filtered to remove insolublesolids, washwith DCM, solvents are removed and chromatographic separation of the residue affords t 68, 69 and 70,

Scheme 22 0 Q

66 3 Cat KJ R§ HAN"~k- " i ~.bH2NDMFN 85 c

\- "N NN N N

66

NN

0N N- 08 N

N N N

Enm ~ ddtionffN-dialkylacrlmdtjlaie SIn order to sftudy the effect of adding more hydrophobic chains to the Qationic lipids, didodecylainine was used as a precursor to the acrylmide,

Scheme 2 3 a d

6e6T

NH H 2N .H

R RR R N R N R N

HydoChtoride salts r, 76 and 77 "(i) Acryloyl chloride,-10-0 C, DIPEA, ClIC 4h, (ii) 90 °C, Neat, 5 days

and (iii) HC/Dioxane

Step I: Synthesis of NN-Didodecylaeryimide 71 Toasolution of didodecylamine 66 (25 g70.7 mmol) and diisopropyethyamine(18 g 141 mnol) inanhydrous CH2C (700 mL) at -10 "C, a solution of acryloyl cloride (7,68 g, 85 mmol) in CHC (100mL) was added 1c dropwise over a period of 20 min. After the completion of the addition the reaction mixturewasstirredor 4 h at 0 °Caerwhichthe TLC of the reaction mi turshowed the completion of the reaction.The reaction mixture was washed withsad, NaHCO3 soluion (200 mU), water (200 mL), brine (100 iL) and dried overNaSO 4 Concentration of the organic layer provided the product 71 (28,4 g, 100%) which was used assuch in the next step, 'H NMR CDC1 6(94 (t, J= 65Hz, H), 1.05-1,69 (m, 40H1 3.15-3,60 (dt, 4H),1564 (d, 1), 6.36 (d, IH), 6.63 (m, lI). Step 2: Reaction of triethyelentetramne 2 and 71 Theacrylarmide 71 is treatedwiththe amine 2 and afer usual work-up and column purification the Michael addition products 72, 73 and 74 are isolated,

Step 3: Synthesis of hydrochloride salts 75, 76 and 77: Each single compound obtained is taken in dioxaneaid 4M HCI in dioxane is added to the solution and stirred as described in example 8 to yield the corresponding hydrochloride salt Elampie24utdevlyaion of olvamines usine mono unsaturated47afkyl gqacylanthounider Michael addition codtn In order to study the effect ofdouble bond in. the alky chain oleiamine was used as a precursor to the acrylanide 79, Scheme 24'

78 78

H2N H

HH R NR

Bsyrnxamle sal 83,4 and 80

(i) Acryloyl chloride, 10-0 T, DIPEA,C '* C1 4h, f(ii) 90 'C, Neat,$days and (iii) ICI/Dioxane Step 1: Synthesis of compound 79:To a solution ofoleylamine 78 (26,75g, 100 mmol)and triethylamine (20 g, 200 nunol) in anhydrous CH2C12 (200 mL) at -10 °C; a solution of acryloyl chloride (9,9 g, 110 mmOl) in CH 2 CtZ (100 ml..) was added t5 dropwise over a period of 20 min. After the completion of the additionthe reaction mixture was stirred for 4 h at0C after which the'TLC of the reactionmixture showed the completion of the reaction The reaction mixture was washed with sad. NaHCO solution (200 mL), water (200 mL), brine (100 mL) and dried over NaSO 4 ,

Concenrationof theorganic layer provided the product 79 (32 g, 100%) which was used as such in the next step. H NMR CDCl 3 5 0,91 (t, J= 6.5Hz, 3H), 1.05-1,35 (n,

24H), 142 (t 2H), 1.96 (m, 4H), 5,31 (tI),5.33-536 (in, 1H), 554 (dd, 1H), 6.02 (dd, 1),618 (dd, 1H), 8,03 (bs,I H). Step:2: Reaction of compound 79 with triethylentetranine The acrylamide 79 is treated with triethylenetetramine 2 and after usual work-up Sand columnpurification of the Michael addition products affords pure compounds80S, 81 and 82. Step 3: Synthesis of hydrochloride salts 83, 84 and 85: Each singlecompound (80, 81 or82) obtained is taken in dioxane and 4M HC1 in dioxane is added to the solution and tired as described in example 8 to yield the corresponding hydrochloride salt -uxample25;:Akenyiationofdiamineusingmouartedzhy

aurvanmide under Michael addition condition Scheme 25 N N- +H-yN'c NH

H

R R R 8687 88 80.890 Id 91

N R

(i) 90 'C, aq. boric acid, 16h and (ii) HCIDioxane in asimilar procedure to that of Example 24 the acrylamide 79is treated with the diamine 45 andafter usual work-up and colunn purification theMichael addition products8i, 87 aid 88 are isolated .Treatment of the free amine thus obtained with 1H1 indioxane affbrds the corresponding hydrochloride salts 89, 90 and 91 respectively, xamole 26;Akenylation of polvamnines usina nol? unsaturated N-alkyi acrvamide under Michael addition condition In orderto study theeffectof polyunsaturationin thealkylchain lnoleylamine 92 was used as a precursor to the acrylamide 93.

Schfeme 26'

H

R 155

---------- _

h73 at6 9

(i) (iii) H7./Dioxan chloride, -10-0 °C, DIPEA, CH2,Cl" 4h, (ii) 90 °C, NeAt, 5 days and Acrioyl

S Step 1: Compound 93: Linolylamine 92 is treated with acryloyl chloride in a similar procedure to that of Example 24, step I and the corresponding aerylamide 93 is isolated, Step 2: Reaction of compound 93 with triethylenetetramine Theacrylunide 93 is treated with triethylenettraine2 inthepresence of boric acid as described in Example 3 and after usual work-up and column purification of the Michael addition products aflords pure compounds 94, 95 and 96. Step 3; Synthesisof hydrochloride salts 97, 98 and 99 Each single compound (94,95 or 96) obtained is taken in dioxane and 4M HC in dioxane is added to the solutionand stirredas described in example 8 to yieldthe correspondinghydrochloride salt. Exanple.27Alkeniation ofdiainesusinpolvunsaturated 4l arlamide under Michael addition condition

Scheme 27 0 AH 2N NH2 N 45 93 H

+ + HNN'RR-y r R R R 1 R -IO 101 12135 n 0 H R

`(i) 90 C, aq. boric acid 16h and (i) ICI/Dioxane In asimilar procedure to that of Example 3 the acrylamide 93 is treated with the a diamnie 4inthepresence of boric acid andafter usual work-p and column purification the Michael addition products 100, 101 and 102 are isolated. Treatment of the fee amine thus obtained with H1C in dioxane affords the corresponding hydrochloride salts 103, 104 and 105 rspectivcly. cxampleS28; Alkenvlition of polymnies siAtkylayltes nder M Ja additionconditi Scheme 2 8

2 NH

06

R R

1I? 108

i) Methanol-water, 40 °C orMethanol,waterboric acid, room temperature Method 1: iDxiecyacrylate (106) is stirred with triethyenetetramine 2 in methanol-water at 40 °C to obtain compounds 107, 108 and 109 The products are isolated by chromatographic separation. Method 2: n-Dodecylacrylate (106) is stiredwith triethylenetetramine 2 in the presence of boric acid inmethanol-water at 40 °C to obtain compounds 107, 108 and 109, The products are isolated bychromatographic separation.

Examle 29: ALkenvlation of diamines gsin kylarylates under Michael addition condition Scheme 29

R R R R

0

*(i) Methanobwatr, 40 C or Nethanol, water, boric acid, room tempernature Method 1: n-Dodecylacrylate (106) is stirredwith triethylenctetramine 2 in nethannolwater at40 ° to obtain compounds 110, 111 and 112. The products are isolated by chroatographicseparation. Method 2: n-Dodecylacrylate (106) isstirred with triethylenetetraine 2 in the presence of boric acidin methanol-water at 40 C to obtain compounds 110 111 and 112. The products arc isolated by chromatographic separation. Example 30: Synthesis of Octadeca-92-igo cid3-nyetyjamino2 octadeca-l2-dienoyioxv-propyl ester 3

~~ 2 1 N 0

To a solution of the linoleic acid (25 g, 89.1 mmol) in anhydrous DM (60 mL) diisopropyl ethdylamine (17 mL, 100 mil) was added at room temperature with stirring followed by 3dimethlamino)-1,2-propanediol(48 g, 405minol) and EDC (17,25 g 89.9 mmol) and the mixture wasstirred at mom temperature overnight. ThelTLC of the reaction mixture (eluent 20% EtOAc in hexanes) showed the completion of the reaction The reaction mixture was poured into ice water and extracted with ethyl acetate (2 x

100 mL), The combined organic layers were washed with water (100OmL) saturated NaCO3(10mL)anddredover Na 2 SOs.Concentrationoftheorganiclayerprovided the crude productwhichwaspurified by column chromatography (silica gelenrlt: 20%,EtOAc in hexanes). The fractions containing pure product was pooled and s concentratedThe pureester was isolated as a clear liquid (5.7 g, 22%), MS m/z 645 (MH41). - NMR CDCbL0.88 (t, J=6.3Hz, 611), 120-1.39m, 28H)1L61 (t, J= 4.9 Hz, 121), 2.03-2.08 (m,8), 226-2.38 (m 10), 2,44-2.56 (m,2H),2.76 (; J= 63 Hz, 4h114,09 (dd, J= 6.1 Hz & 119 Hz, 1 ), 4,36 (dd,= 3.3 & 11 9Hz, 1H), 5.29-5.34 (m,n1H 5.34-<.41 (m,8H). "C' NMR CDC1 6 14.30, 22,79, 2508, 25,10, 2,83, S 27,40, 2926, 29,30, 29,34,29,42, 29.55, 29.83, 31.73 3432,34 58,46,01, 59.37, 64.02, 128.08,128,24, 130.2L 130.42, 173,39, 17365. Examnple31; Exemnplary procedure forimaking aiposomneusing.extrusion Prepare stock solutions of ND98 (120 mg/ml), cholesterol (25 mg/ml), and CI6 PEG-Cer-2000 (100 mg/ml) in 100% ethanol. Store at -20°C. Warm in 37C water bath iS prior to preparing formulations (up to 30 minutes is helpful - it takes a while for the cholesteroltodissolve completely).

2x 2mIPrep To a I5ml Falcon tube, add: 1)125ul of lipid 2)200ul of cholesterol 3)70ul of PEG 415u of 100% ethanol 5)600u1 of 25 mM sodium acetate pH 5 6)Mixgently (setting 5) on a vortex 7)Add20Omg sucrose 8)Vortex again until sucrose has dissolved 9)Add I ml. of a freshly-prepared (in a new Falcon tube) I ig/m solution of siRNA in 25 mM sodium acetate (=100 ul of 10mgiml siRNA + 900 ul of 25 mM 3K sodium acetate) 10)Vortex tightly (setting 1, wth Falcon tube holder adapter) for 20 minutes I)Afier 15 minutes (5 minutes remaining), clean extruder l2)Extrude 11 times through two 200 n filters at 40 C 13)Dialyze against'PBS, pH 7,4 for 90 minutes at RT in 3,500 lMWCO Pierce cassettes

5 Examptr 32;_Jxmngplaoedfor makingjlipsonie wyithlut -wing 27nusion Prepare stock solutions ofND98 (120 mg/ml), cholesterol (25 mg/mh), and.C16 PEG-Cer-2000 (100 mg/ml) in 100% ethanol. Store at -20°C. Warm in 37C water bath prior to preparing tamulations (up to 30 minutes is helpful- it takes a while for the cholesterol to dissolve completely)

To a 15ml Falcon tube, add: 1)125ul of lipid 2)2001. of cholesterol 3)70u of PEG 4)495 I of 100% ethanol 5)1Oul of water 6)Prepare I ml of I mg/mI siRNA in 100-300 mM sodium acetate, pH5 7)Rapidymix lipids in 90% ethanol with siRNA in acetate buffer 8)Dialyze (or use ultrafiltration) against 100-300 mM sodium acetatepH -5 to removeethanol

9)Dialvze (or use ultrafiltration) against PBS to change butter conditions

Example 33: Exemplary protocol for quantification of RNA in aliposoe sample The procedure below can be used to quantify (1) the proportion of entrapped siRNA and (2) the total amount of siRNA in a liposome,

Materials: RiboGreen (Molecular Probes) 2 %Triton X- 100 TE buffer

Protocol (96-well plate format):

L. Dilutesamples to be testedin TE buffer such that siRNA concentration is - 2 ug/mL(0:4 -4ug/mL). Note dilution of samples. 2Array 50uL of each sample into 2 wells (e.g. samplesarrayed into 2 rows of miscroplate) 3. Add 50 uL of TE buffer to one of each of the 2 samples (eg. toprow samples). This sample will be used to determine"free" siRNA, 1o 4. Add 50uL of2%VTriton X-100 to the remaining of the 2 samples (e.g bottorn row samples). This sample will be-used to detennine "tota"siRNA. 5, Prepare standard siRNA dilutions by usingknownamountsof the siRNA to be quantified. Start with 50 uLof4 ug/rL, anddo 2-fold dilutions. Add0uLof 2% Triton X-100 to each of the standard sample dilutions. 1 6.Ihcubate for 15 min at room temperature. 7. Add 100 uhof diluted RiboGreen to all of the samples. Diluted RiboGreen to be used at 1:100 dilution. 8, Read plate in fluorimeter (Victor2) using FITC settings.

Calculations:

Final volumein wells will be 200 uL RiboGreen will beat 1:200 final dilution, TritonkX-100 will be at 0.5%. Standards willbe dilutions starting from I ug/nL

Plot Standard Curve, perform linear fit. Determine Entrapment % l00*(-"fre" signal "total" signal) Determine (siRNAJ First convert "total"signal to concentration using the standard curve, then multiply by dilution factor.

Example 34: Conparison of Lipid moieties as fbnmlated intoLiposomes The effectivenessof lipid compositions can be tested by determining the relative ability of alipidto deliveran siRNA moiety to a target. For example, the silencingofa as taretindicates that the siRNA delivered into the cell Applicants have compared liposome complexes that include each of the following.lipid moieties together with siRNA that is used tosilenceFactorVU(FV).

initially unpurified reaction mixtures were used. Different ND98 reaction mixtures were generated by synthesizing product at different ND:98 monomer ratios: ND8 :1,:1,:1, 5:1,and 6:1 ND98 is generated by reacting ND, the structure of which is provided below: 0 N H , with amine 98, thestructure of which is provided below H H 2N .N 'N.'NH 2

in the ratios provided above (i.e., ND:98 1:1,2:1, 3:1,4:1, 5:1, and 6:1). Liposomes wereformulated at ND98:cholestero:FED2000-CerCI:siRNA s 15:0,8:7:1 (wtratios). Liposomes prepared with ND98 = 1;1 and 2:1 precipitated drinig formulation and were not characterized further. Table 1, below provides the average particle size and percent entrapment of the hposomes using the various monomer ratios (i.e, the number indicating the ratio of ND relative to 98), as Table 1: Z-Avg. Particle size (nm)% Entrapment

ND983 56 95

ND98 456 95

D98 581 93

ND)8 6 2 74

Figure 1 provides the results of the FVl1 siliencing assay for the various monomer ratios using ar experimental dosing of 2 mg/kg siRNA. The results sugest that the ND98 5 tail moiety and/or ND 98 6 tail moiety are the active species as these are the most abundants species on the ND98 6:1 preparation, As described a.5 tailnoiety indicates a compowdwhere 5 of the hydrogens on the starting amine 98 have been reacted with a startingacrylamidemoiety ND. A 6 tail moiety indicates a compound where 6 of the hydrogens on the stating amine 98 have been reacted with an acrylamide moiety ND. Accordingly, the numer of "tails" indicates the number of reacted hydrogens on the starting nine.

Example: Determination of preferred lipid isomer Applicants purified ND98 lipid products. ND98 lipid moietiesarethe lipid moieties resultingin the reaction of NID, the structure of which is provided below: 0 N H , with amine 98, thestructure ofwhich is provided below H H2 Nx-'N N NH, H

Applicants tested 4-taiimixed isomers of ND98S(i.e..where four of the amine bydorgens havebeenreactedwiththeNDacrylamide above single structural isomers of5-tail ND98 (iewhere for of theamine hydrogens have been reacted with the ND acrylanide above). Examples of the two 5 tail isomers are provided below: R R H H R' N '-"N'R and R'N NN.r A '- R R R R

Liposomes of the purified ND98 products were formulated with the following components in the following ratios: ND98:choesterol:PB02000.CerCi6:siRNA= 15:5:7:1 (wt ratios). Table 2, below provides the average particle size and percent entrapment of the liposomesusing the various monomer ratios (i,e, the number idicating the ratio of ND relative to 98).

Table 2: ZAvg Particle size % Entrapment

ND98 1 88 295

ND98 2 104 86

D98 3 115 86

ND984 92 >95

For the purposes of table 2 and Figure 2: ND98 1 5-tailed (isomer 1); ND98 2 5-tailed (isomer I - ;ID ND98 3 = 5-tailed (isomer)and ND98 4 = 4-tailed. The liposomieswhere administered with siRNA at a does of22,5 ngg,and a evaluatedfor the silencing ofFVRFigure 2 provides the results of the 4 tailed isomer mixture, the single 5 tailed isomers (i., isomer I and II) and the mixture of 5 tailed isomers(i.e, isomer Iand 1).

Exmpal36: Determination of preferredND98 isomer 1 A purified isomer of6 tailed ND98 was prepared and purified. ND98 structure corresponds with those described inexamples 34 and 35 above, The 6 tail indicates that all of the hydrogens of amine 98 have been reacted with the ND starting material. With this lipid starting material, liposomeswere formulated at the following ratios ND98:cholesterol:EG2000-CerCI6:siRNA = 15:5:7:1 (t ratios), Figure 3 1s demonstrates the effectiveness of the ND98 6 tail isomer in delivery of siRNA, which effectivelysilenced FV1

Exampe37: Losome particle size using various ND98 lipid startingmaterials A plurality of lipid starting materials having the ND98 structures (as provided in examples 34 and 35 above) were fomulated into liposomes. The particle size of the liposores were evaluated, the results of which are provided in table 3 below

Formulation Particle DiameterQ(n} ND98 3 (Exp 1) 56 ND98 4 (Exp 1) 56

ND98 5 (Exp 1) 81 ND986(Exp1) 72 ND98 I (Exp 2) 88

ND982(Exp2) 104 ND98 3 (Exp 2) 115

ND98 4 (Exp 2) 92 6-tailed ND98 (Exp 3) 127

EamLe 38 Extrusion free Iposome formulation Liposome complexes were prepared using ND98 lipids. The formulations nrcluideetheolowingratios:ND98:choleterol:PEG200-CerG16:siRNA= 15:57:1 (wt.hratios). The liposomes were prepared without extrusion, as generally described in Example 32 above. Two samples were prepared, a first sample having the following: 100 mM = siRNA prepared in 100 mM sodium acetate with a first dialysisstep in 100 m acetate; and a secondsample having 300 mM = siRNA prepared in 300 mM sodium acetatewith a first dialysis step in 300 mM acetate. Figure 4 shows theresults ofan FVI silening assay,demonstrating the comparative activity ofthe formulations made using the various processes.

Examnle 39IRegioselective synthesis of cationic lipid 7- strategy I Scheme 31'

21 eq, Ethyl trifluroacetate H CH 3CN, 0 0C-RT N'N "NN F ~N N SNNONNNH2 P H H F

(Boc)20DPEA THF/CHrCl 2

MeNH 2/MeOH F -' N F NN,N NH"- H F VC F 113 Boo 0 114 B0

0 -N' H Boic acid, Water

BR c R NN 'R HCl, Dioxane R H

115 2 NaHCO H 0 N H I

H R H R.N NrN' R RN <N N 'R 4HOI R 7 '117

aRegioseletive synthesis of cationic lipid 7- Approach I

Step 1 Preparation of compound 9:.Tethyleneteiranine, 1 (4,83 g, 0,334 mol, purchased from Sigma-Aldrich) in anhydrous acetonitrile (500 mL) wascooled to over an ice bath under constant stirring Ethyl trifroactate (79,6 mL, 0.668 mol) was added to the solution and after completion of the addition the reaction mixture was allowed to warm to room temperature and stirred for 20h, Solvent and volatiles were removed under reduced pressure and the residue was dissolved in minimum amount of warm dichloromethane (100 mL) and to it cold hexanes was added with stirring. The precipitatedproduct was cooled in ice and filtered to get a whitesolid (1122 g, 99%),

Step 2. Synthesis of (2-tert-utoxyearbony[2-2,trifluorn 5acetlamnino)ethyij-amino)-2-(222-trifluoro-acetyiamine)ethy11bearbamieacidiert butyl ester113 TheW ifluroacetamide 9 (112,2 g, 0.332 mol) was dissolved in CH2C1JTHF(600 mil100 nl) and to it diisopropylethylamine (12925 g, I mol) was added and stirred over an ice bath. Di-wrtrbutyi dicarbonale (145 g, 0664 mal, purchased. from Sigma Aldrich) in C 2 C12 (100 mL) was added drop wise to the reactionmixture and stirred overnigt.Solvents were removed and the residue was stirred with asaturated solution of Na HC0(400 mL) and filtered and washed with hexanes (100 nL) and dried in vacuo at 45 °C ovemight to obtain the pure diboe compound as a white solid (167 g, 94%). 'H NMR for 113 (DMSO-d6, 400MHz) 8=960-9.40(m, 211), 3.35-3,15(m, 1211), .36(, 18H) MS: C 5 It 4 F-N404 Cal, 438.17, Found 439.20(Mt) MS: CntZF6N40sCal. 538.22, Found 539.20(M).

Step 3. Synthesis of (2-amino-ethyl)-2-{2amino-ethy1}-err batoxyearbonyl-amninu]-ethy1caramicacid tert-butyl ester The acetamide 113 (167 g, 0.31 mol) was taken in a stainless steel pressure reactor and to it a solution ofmethylamine (33% by wt) in ethanol (200m) was added, The mixture was wamed to 90°C and stirred for 24 h. Reaction was monitored by mass spectra. All thesolvents were removed under reduced pressure and the residue was subjected to high vacuum at 80 T to yield the product 114 (103 g, 96%) as gummy liquidandthis compound could be used for the next reaction with out further purification. 'H NMR (CDC3 , 400MHz) 8 :: 3,20~3.00(m, 4H),2.62438(m, 8), 1.32(s, 9H) MS: C H N402 Cal. 246,21, Found 246.20(Mft ).

Step 4. Synthesis of Michael addition product 115 cc The diamine 114 (103 g 0.297 uol),N-dodecylacrylanide (356 g, 1.487 mol) and saturated solution of boric acid in water (30 ml) were takentogether in a pressure reator and heated at 90'C for 4 days. The reaction was monitored by TLC and Mass spectra. The reaction mixture was extracted intodichoroethan (DCM) washed successively with NalCO3 solution and brine, dried overanhydrous sodium sulfate. Solvent was removed in vacuo and residue thus obtained was purified by silica gel column chromatography (gradient elution- Ethyl acetate then 340% MeOH/DCM) to obtain 115 as a pale yellow solid (228 g, 59%). MS:, CHalNsO at130316, Found 1304,20(NfO) Step 5. Preparation of diaminel16 4M HC] in dioxane (500 mL) was added to a solution of the dibo compound 115 (228 g, 0175 mol) in methanol (100 niL) and the mixture was stirred atroom o temperature for 2 days. The reaction was monitored by Mass spectra. After the complete disappearane of the starting dibo compound, the precipitated hydrochloride salt was filtered, washed with THF (100 mL) and dried to get the pure salt as white powder (178 g 93%), The above salt was treated with saturated NaHCO3 (L) andextracted withdichloromethane (3 x 600 mL). The combined organic tracts were dried and concentrated to isolate the tetramer as a white solid (164 g, 85%),MS: CUH 4N0O4 Cal 1103,05. Found 1104,10(M) Step 6, Synthesis of 117: Compound 11.6 (164 g, 149 mmol) , N dodecylacrylaide (35.6 g, 149 mrnol) and saturated solution of boric acid in water (30 nL) were taken together in a pressure reactor and heated at 90'C for 3 days. Progress of the reaction was monitored byTLC and Mass spectra. The reaction mixture extracted into dichoromethane (DCM), washed successivly with NaHCOs solutionand brine, dried over anhydrous sodium suliate, Solvent was removed in vacuo and residue thus obtained was purified by silica gel (2 Kg) column homatography (gradient eluion 0:5:95-10:10:80% TBA/MeOH/DOM) to obtain 117 as a pale yellow solid (83.8 g, 42%), MS: C 6 5 N 5 0Cal 1303.16, Found 130420(M),The material was compared with authentic sample'1LC (qualitative), HPLC and Mass spectra. MS: Cg136 N9 05 Cal. 1342.28, Found 134330(M). Step 7. Synthesis ofthe hydrochloride salt 7 The amine 117 (54 g, 40mmol) was dissolved ethanol(100 mL) and to it 200 so ml of 2M HCI in ether was added andthe mixture was stirred atroom temperature ovemight. Nitrogen was bubbled to the reaction mixture and the outlet was passed through dryrite and to a 10% solution of KOHI After 30minute, the reaction mixture was concentrated to dryness and the residue was re-dissolved in 500 ml ofanhydrous ethanol and the mixture was concentrated ni a rotary evaporator. Thisprocess was again repeated once again and the thus obtained residuewas dried in a vacuum oven at 43 C overnight. The pure product was isolated as a cream powder (59.5 g, 99%). R Bixie40,Reogoeleeyevnhesisoif niejipi d strategyI Method 1 21 e Ethyl trfiuroacetate o H nt i F 0 CH 3CN, C-RT F 'N N N

' H2 N "'AN - F HHr H 1 1.0 q(oc)2 0 DEA

MeNHZIMeOH NY0 F IN, N2A IN H'NA'NNANNH W"" N 0 H F H 90 °C2days) H 102

N 90 C H so-rc ac4d Water 0 H H

o 4 H

H. H

HCJDioxneor Ether H y ~ O N~A

N N N XA"rN'

H H

Step1: Triethylenetetramine, I (200gg,&L37 mol, purchased from Siguna Aldrich) in. acetonitrile (2 L) in a 4 neck 5L fask with overhead stirrer was cooled over an. ice bath under constant stirring. Ethyl trifluroacetate (388.5 g,274 ol) was added to the stirring solution and stirred for 20h. Solvent and volatdles were removed under reduced pressure; the residue was triturated with a mixture of DCM/Hiexane and fitered to get 101 as white solid (429 g, 93%). The productthus obtained could bused for the next reaction without further purification. MS: CIFN402 Cat 338,12, Found 339.0(M) Step2: Crude compound 101 (427g, 1.26 mol) was dissolved in amixture of solvents (3 L, THF/DCM (1:2)) and stirred over an ice-water bath. Didert-butyl dicarbonate ((Boc)20,270 g, 1.26 mot, purchased from SigmaAldrich) and DIEA (500 mL2,86 mol) were added to the reaction mixture and stirred overnight. Solvents were removed and theresidue wasextractd into dichloromethane (DCM, 1000 mL), washed successively with NaIHC03 solution (500 iL), water (500 mL x2) and brine, dried over anhydrous sodium sulfate. Solvents were removed in vacuo and residue thus obtained was triturated with DCM/flexane (2:1) and filtered, Solvents were removed and the residue was dried under high vacuum to get the compound 102 as gummy liquid (5 2 3g). Part of the compound 102 was purified by silica gel chromatography (gradient elution, Ethyl acetate, followed by 3~10% MeOH/DCM) to obtain compound 102 as gummy liquid (102.00g). 'H NMR for 102 (DMSO-d6, 400MHz) 8 = 9.60-910(m, 311), 3.35-3.25(n, 4H), 1325-3,20(2, 2H), 3.20-3.10(n, 2H-2,68-258(m, 4H), 1-35(s, 9H). MS:;CH. 4 F 6N40 Cal. 438.17, Found 439.20(M),

Step 3: Purified compound 102 (102,Og, 233.40 mmol) was dissolved in EithanoVMethyl amine (400 ml, 33 wt% methylamine solution in EtOH) at ambient temperature in apressure reactor. The mixture was warmed to 90C and stirred for two days. Reaction was monitored by mass spectra. All the solvents were removed under reduced pressure and the residue was subjected to high vacuum at 80 'C to yield the product 103 (58.00 g, 99 %) as gmmiy liquid and this compound could be used for the next reaction with out further purification. H NMR (CDCla, 400MHz) = 3.20 3.00(m, 41)2.62-2,38 (m, 81), 1,32(s, 9H). MS: C1 H2 Cal 246.21, Found .NA 247.20(Mr). Step 4: Triamine 103 (56.00 g, 227,64 mmol), Ndod(eyacryamide(327,00 g, 1365 mniol) and saturated solution of boric acid in water (50 mL) weretaken together in pressure reactor and heated at 90'C for 6 days. The reaction was monitored by TLC and Mass spectra. Thereaction mixture extracted into dichloromethane (DCM), washed sucessvwith NaHCOU solution (400mnAt) and dried over anhydrous sodiumsulfate Solvent was removed in vacuo and residue thus obtained was purified by silica gel columnchromatography (gradient eltion- Ethyl acetate then 310%MeOH/DCM) to obtain 104 as a pale yellow solid (186 g 57%). 'H NMR (CDCI 3 400Mliz) b

7 20(bsfH), 7:05(bs, 1H), 6,85(bs, 1H), 674(1s,H). 3,25-3,03(1, 1-), 2.80-2,60 (i, H), 2.55-2.21(m, 1211) -52.45(m, 10H), 142(s, 9-1), 34-120(m, 100H), 0.87(t, J= 6,5Hz, 15H). MS: CsIN907 Cal. 1442,33, Found 1443.30(MI), Step 5: 4M1 H in dioxane (400 nL) was added into a solution of compound 105 (184.00 g, .127,23 mmol) in dioxane (300 mL). The reaction mixture was then allowed to stir for overnight. The reaction was monitored by Mass spectra. Excess If(I was removed by passing nitrogen through the solution, Solvents were removed under vacuum and residue was co evaporated three ies with ethanol (500 mL X 3) to yield a material pale yellow gummy solid 7 (186.0g , 98%) as tetra hydrochloride salt. The was compared with authentic sampleTLC (qualitative), HPLC and Mass spectra. MS: QO !,sHNOs Cal, 1342,28, Found 134330(I),

Method:2 Compound 102 was prepared as described in Method 1: steps I and 2.The crde without further product obtained from step 2 of Method I was used for thenext reaction purification. Step 1: Compound 102 (103A5g, 238.90 mmol, crude compound nom step2 Method I was dissolved inEthanol/Methyl amine (400 ml, 33 wt% niethylamine solution in EtOH) at ambient temperature in a pressure reactor. The mixturewas warmed to 90'C and stirred for two days, Reaction was monitoredby mass spectra. All the solventswere removed under reduced pressure and the residue was subjected to high vacuum at 80 ° over a water bath toyield the product 103 (6350 g) aspale yellow further gumy liquid and this compound could be used for the next reaction with out purification. Step 4: Triamine 103 (63,50 g 238 mmol), N-dodecylacrylamide (32000g, in 3o 1338 niol) and saturated solution ofboric acid in water (50 nL) were taken together I The a pressure reactor and heated at 90"C for 6 days as described in step 4, Method reactionwas monitored by TLC and Mass spectra, The reaction mixture extracted into dichloromthanc (DCM), washed successively with Na1CO3 solution (400 mL) and dried over anhydrous sodium sulfate. Solvent was removed in vacuo and residue thus obtained was purified by silica gel column chromatography (gradient elution- Ethyl acetate then 3-10% MeOH/DCM) to obtain 104 asapale yellow solid (652 g, 20%), Step 5: 2M HC in ether (800 m) was added to compound 105 (65.00 g, 45 mmol). lhe reaction mixture was then allowed to stir for ovemight, The reaction was monitored by Mass spectra, Excess CI was removed by passing nitrogen through the solution. Solvents were removed under vacuumand residue was co evaporated three times with ethanol (500 mL X 3) to yield a pale yellow gummysolid 7(66g , 98%) as 1 tetra hydrochloride salt. The material was compared with authentic sample TLC (qualitative), HPLC and Mass spectra. MS: Cgjl(nNOs Cal, 1342,28, Found 1343.30(M).

Method I Comnpound 102 was prepared as described in Method I:stepsI iand 2. The crude product obtained from step 2 of Method I was used for the nextreaction without further puriication Step3: Compound 102 (105.20g, 240 mmol, crude compound from method 1) was dissolved in Ethanol/Methyl amine (400 ml, 33 wt% methylamine solution in EtOH) at ambient temperature in a pressurereactor. The mixturewas warmed to 90'C andstied for two days, Reaction was monitored by mass spectra, All the solvents were removed under reduced pressureand the residue was subjected to high vacuum at 80 C over water bath to yield the product 103 (64.70 g) as pale yellow gummy liquid and this compound could be used for the next reaction with out further purification Step 4: Trianine 103 (64,70 g, 240 mmol) N-dodecylacrylamide (370.00 g 1569 mmol) and saturated solution of boric acid in water (50 mL) weretaken togetherin a pressure reactor and heated at 90C for 6 days. The reaction was monitored by TLC and Mass spectra. The reaction mixtureextracted into dichloromethane (DCM), washed successively with NaHCO 3 solution (400 mL) and dried over anhydrous sodium sulfate, s Solvent was removed in vacuo and residue thus obtained was purified by silica gel column chromatography (gradient elution- Ethyl acetate then 3-10% MeOH/DCM) to obtain 104 as a pale yellow solid (192 g),

Step 5: The desired compound 7 was obtained as hydrochloride salt from compound 104 as described in step 5, Method I of Example 40, Compound 7: 194g (98%) as tetra hydrochloride salt The material was compared with authentic sample TLC (qualitative), HPLC and Mass spectra. MS: CsjrNsO5 Cal 134228, Found a 1343.30(M)).

__p_4_.Comparisonativitf siRNAfonulatedintovaris asociation Udin gPiflnilidmoieties: The effectiveness of lipid compositions can be tested by determining the relative abilityofalipidtodeliveransiRNAmoietytoa targetFor example, the silencingofa target indicatesthat thesiRNAis delivered into the cell, Applicants have compared association complexes that include one of 13 different PEG-4ipid moieties asprovided in Figure 5, together with siRNA that is used to silence Factor V.1 (FVU), PEG-lipids 1-13 were synthesized using the following procedures

Scheme la

taR= C14 H 2 Ib RC

DSC, TEA DyM H2NNOOY'N.sOMe GVC-RT t TOR 3R l ..... 0 0 mnPE0 2001yNH 2 Rga gN ^ ~ R - y,g ---.- --,----- .AHi Py /DCMR R 00RT 4a R =Cdhe 2) R CaH2 4b R = CAb H 2b R = CtHn 4C R= 2c R=C.ZiHa7 4 "Scheme 1: iPEG2000-,2Di-0-alkyl-sn3-caromoylglyceride Preparation of compound 5: 2-Di-Q-teradecysn-giyceride1 (30 g6180 nmol) and NN'succiniidylCarbonte(DSC, 2376 g, 1.5q) were taken in 2-0 dichloromethane (DCM, 500 mL) and stirred over an ice water mixture. Triethylamine (2530 mL, 3eq) was added to stirring solution and subsequently therection mixture was allowed tostirovemight at ambient temperature. Progress of the reaction was monitored by TLC. The reaction mixture was diluted with DCM (400 mE) and the organic layer was washed with water (2X500 mL), aqueous NaFCO3 solution (500 ml) followed by standard work-up. Residue obtained was dried at ambienttemperature under hig vacuum overnight. After drying the omde carbonate 3 thus obtained was 5 dissolved in dichloromethane (500 mL) and stirred over an ice bath; To the stirring solution mPEG 2 5 N(4, 103,00 g, 4720 mml, purchased from NCF Corporation, Japan) and anhydrous pyridine (80 mL, excess) were added under argon. The reaction mixture was then allowed stir at ambient temperature overnight. Solvents and volatiles were removed under vacuum and the residue was dissolved in DCM (200 mL) and i charged on a column of silica gel packed in ethyl acetate. The column was initially luted with ethyl acetate and subsequently with gradient of 5-10 % methanol in dichloromnethane to afford the desired PEG-Lipid 5 as a white solid (105.30g, 83%). H NMR (CDCI, 400 M1z) 3 = 5.20-5.12(m, 1H), 4.18-401(m, 2H), 3,80-370(m, 211) 3.70-3.20(m, -0-C4ZrC-1,0- PEG-CH12), 2,10-2,01(m, 2H), 1,70.1L60 (m, 2H), L.56 L45(m, 4H), 131-L15(m, 481), 0,84(t, J:: 6.5Hz, 611). MS rangefound: 26602836.

g, Preparation of 4b: 1,2-Di-O-hexadeyl-sn-glyceride 1b (100 1848mnmol)

andDSC (0.710 g,.1 Seq) were taken together in dichIloromethane (20 mL)and cooled down to VC in an ice water mixture. Triethylamine (100 ml, 3eq) was added to that and stirred ovemight. The reaction was followed by TLC, diluted with :DCM, washed with water (2 times), NaEHCOQ solution and dried over sodium sulfae, Solvents were removed under reduced pressure and the residue 2b under high vacuum oveight, This compound was directly used for the next reactionwithout further purification. MPEG NH 2 3 (1.50g 0,687 minol, purchased from NOF Corporation, Japan) and compound ftom previous step 2b (0.702g,I 5eq)were dissolved in dichloromethane (20 mL) underargon. The reaction was cooled to 0°C. Pyridine (I mL, excess) was added to that and stirred overnight. The reaction was monitored by TLC Solvents and volatiles were removed under vacuum and the residue was purified by hromatography (first Ethyl acetate then 5-10% MeO1/DCM as a gradient elution) to get the required 3' compound 4b as white solid (146 g, 76 %), HNMR (CDCI2_400 MHz) 8= 5.17(t,Y3 5.5Hz, 11), 4.13(dd, J= 4.00Hz, 11.00 lHz, 1I), 4.05(dd, J= 5,00z, 11.00 Hz, 1H), 3,82-35(m, 211), 30-3,20(m, -O-C"fA ' CHl'O-, PEG-CH '), 22H), 80

1.70 (m,2H,2161-L415(m, 6), 135~1,17(m,56), 0,85(t, J= 6,5Hz, H),MS range found: 2716-2892,

Preparation of 4e: i,2-Di-O-otadecyl-sn-glyceride le (40 g, 6,70 mmol) and DSC (2.58 g, 15eq) were taken together in dichlromethane (60 mL) and cooled down to( °C in an ice water mixture. Triethylzamine (2.75 mL, 3eq) was added to that and stirred overnight. The reaction was followed by TLC, diluted with DCM, washed with water (2 times). NaHCOy solution and. dried over sodium sulfate, Solvents were removedunder reduced pressure and the residue under high vacuum ovemight. This compound was directly used fbr thenext reaction with further pifiication, MPEG2 co NH 3 (15Og, .687nmmol, purchased from NOF Corporation, Japan) and compound from previous step 2c (0 760g, 1,5eq) were dissolved in dichloromethane (20 mU) under argon. The reaction was cooled to 0°C. Pyridine (1 mL, excess) was added to that and stirred overnight. The reaction was monitored by TLC, Solvents and volatiles were removed under vacuum and the residue was purified bychromatography (first Ethyl acetate then 5-10% MeOH/DCM as a gradient elution) to get the required compound 4 C as white solid (092 g 48 %). 'H INMR (CDCt, 400 MHz) 6 = 522-5.15(m, IH), 4,16(dd, J 4,00Hz, 1100 Hz, I ), 4.6(dd; J= 5,00z, 1100 Hz, 1H), 381-3,75(m. 2H), 3.0-3.20(m, -G-CH2-CHrO-,PEG-C2), 1.80-1.70 (m, 2H), 1601.48(m, 411), 1.3l- II5(m, 641), 0.85(t, J=6.5z, 611), S range found: 2774-2948,

Scheme 2'

R' la Rk =-CH24 1L R ICIAHr

0 00 - O1 -Me

HBTU/DiEA R.. 0 ,,Oie DMFFDCM R 0 6Sa R CjjH2, R CtiHa 6eb R= 50 RV=C3a1 6r .p C,?

5 *aScheme2:mPEG2000I,2-Di-Oalkyl-sn3-succinylglyceride Preparation of compomud 6a: 12i-Oetradecyn-glyceride la (1.00 g, 2.06 mmol), suecinic anhydride (0.416 g 2 eq) and DvAP (0,628g,2eq) were taken together in dichiormethane (20 mL.) and stirred overnight. The reaction was bfllowed S by1TLC, diluted with DCM. washed. wih cold dilute citric acid, waterand dried over sodiumsulfIte. Solvents were removed under reduced pressure and the residue under high vacuum overnight. This compound was directly used Bor the nexl. reaction with father purification MPEG2cawrNH2 3 (150g, 0.687 mmol, purchased from NOF Corporation, Japan), compound from previous step 5a (0.66g, 12 eq) and HBTU 1 (0.430g, .13 mnol) were dissolved in a mixture of dichloromethane/DMF (2:1, 20 TnL) under argon. DIEA (0.358 nL, 3 eq.) was added to that andstirred overnight. The -ea-tion mixture was transferred to a large Ilask and removed the solvents and volatile

under reduced pressure. The residue was dried under high vacuum overnight and purified by chromatography (first ethyl acetate then 5-10% MeOH/DOM as a gradient clution) to get the required compound 6a as white solid (0.822g, 43 %). 1 NMR. (CDCI, 400 MHz) 8 = 6.34-630(m, 1H), 46(dd, J-4.00Hz, 11.00 Hz, IH), 4.08(dd, J- 5,00Hz, 11.00 Hz, IH), 3.82-3.78(m, 2'H), 3,70-3,30(m, -O-CH 2 -CHr-O PEG CH2), 2.64 (t, J- 7.00Hz, 2H), 2.43(t, J=6.80Hz, 2H),.76-172(nx2H),1.56~L48(m, 41), L34-1.I6(m,4811), 0,95(t, J= 6,5z, 611), MS range found 2644-2804,

Preparation of compound 6b: 2-Di-hexadecy-sn-glyceridlb (1.00 g, 1848 munol), sucinik anhydride (0.369 g, 2 eq) and DMAP (0563 g 2,5eq) were taken together in dichloromethane (20 mL) and stirred overnight. The reaction was fNlowed by TLC, diluted wita DCM, washed with cold dilute citric acid, water and dried over sodium sulfate. Solvents were removed under reduced pressure and the residue under high vacuum overnight. This compound was directly used for the next reaction with further purification.MPEGtrNt3(L.50g,0.687immol,purchasedfrom NOF Corporation, Japan), compound from previous step 5b (0.66g, 103 mmol) and 1o HBTU (0400g, 1,05 mmol) were dissolved in a mixture of dichioromethan'DMF (2;1, 20 iL) under argon, DIEA (0.358 mL, 3 eq.) was added to that and stirred overnight The reaction mixture was transferred to a large flask and removed the solvents and volailes under reduced pressure. The residue was dried under high vacuum ovemight and purified by chromatography (first ethyl acetate then 5-10% MeOH/DCM as a gadient ehtion) to get the required conpounid 6b as white solid (0300g, 16 %). 'H NMR. (CDCI, 400MHz) 8 = 6.33~-6.28(m, MH), 4,18(dd, P= 4.00Hz, 'a1.00 Hz, Ili), 408(dd J= 5.0Hz, 11.00 Hz, 1H) 3.82-376(m, 211),70330(m,-~O-ClzCit4> PEG-CH 2) 265 (t, J= 7.08Hz, 21), 2.44(t, J= 683Hz, 211), 176-.68 (n, 21),1 57 1t48(m,41), 132-M17(n, 56H), 0.86(t, J6.6Hz, 61-1). MS rangeund2640-2822,

Preparation of compound 6c:1,2-i--octadecyl-sn-glyceridec(5.00 g, 8.37 mmol), succinic anhydride (1,70 g,2 eq) and DMAP (2.55 g, 25e) were taken together in dihloromethane(50 mL),and stirred ovemight. The reaction was followed by TLC, diluted with DCM washed with cold dilute citric acid, water and dried over sodium sulfate. Solvents were removed under reduced pressure and the residue under high vacuum overnight This compound was directly used for the next reaction with further purification. MPEGyNH 2 3 (15g,0.687mnuol, purchased from NOF Corporation, Japan), compound from previous step 5e (0.718g, 103 mmol) andHBTU (0.410g, 1.08 mmol) were dissolved in a mixture of dichloromethanwDMF (2:1,20 mL) under argon. 3O DIEA (0.350 mL, 3 eq) was added to that and stirred ovemight. The reaction mixture was transferred to a large flask and removed the solvents and volatiles underreduced pressure. The residue was dried under high vacuum overnight and purified by chromatography (first ethyl acetate then 5-10% MeODCMas a gradient elation) to get therequired compound 6eas white solid (L1 .g, 56 %). 1H NMR (CDCI, 400 MHz) 8 6,38-6.33(m, 1HI), 419(dd, J= 4,00Hz, 11.00 Hz, 11H), 407(dd, J 5,00Hz, 11.00 Hz, i-), 3.813.4(m, 21), 3.70-3.20(m, -O-CHR-CH-O- PEG-CH, 2,63 (t, J= s 7,03Hz, 21) 2,43(t, J6.87Hz, 2H), 1.76-1,68 (in, 2H), 157-148(m, 41), 132-17(m, 641), 086(t J 6,60Hz, 6H). MS rmge found: 2680-2922 Scheme 3'

OO t. ~DOC DCM RO

R OH a R:; C 4H29 RO 11b R CjrH3.

ibn R =cjjqg IC. R CjeH3;

aScheme 3 : mPEG2000-1,2~Di-0-alkyl-sn3-s-ccinylglyceride

Preparation of compound 8a: 1,2Di-0-tetradecyl-sn-glyceride la (0.300 g, 0.618 mmol) MPEG-Succinate 7 (1.00g, 0.476 nmol, purchased from NOF 1 Corporation, Japan), DCC (0127 g, 3eq) and DMAP (0,058 g0.476 mmol) were takenin dichloromethane (20 mL) under agon and stirred overnight. Reaction was monitored. yT iC, The reaction mixtureas cooled to 0C afterstirring ovemight and filtered off the precipitated solid Volatiles and solvents were removed under reduced pressure and the resulting residue was purified by chromatography (first elted with EtOAc, followedby5-10 % DCM!MeOH gradient elution) to get the compound Sa as a white solid (0.590g, 48%) H NMR (CD h, 400 MHz) 6 = 4.25-4,18(m,21), 4,08(dd, J= 5,60Hz. 11-50 liz, 11), 3.80-373(n, 211), 3,70-3,30(m, -O-CH2-CHr'~, PEG C , 1.56-L47(m, 4H), 1-30-1I5(m, 48H), 0.85(t, J=6.60Hz, 61H) MS rangefound: 2440-2708

Preparation of compound 8b:1,2-Di--hexadecyl-snglycride1 0334g, 0,618 nmmol), MPEG-Succinate 7(1.00g, 0,476 tmmol, purchased firom NOF

Corporation, Japan), DCC (0,127 g,13eq) and DMAP (0.058 g0.476 mmol) were taken in dichioromethane(20 mL) under argon and stirred ovemight. Reaction was monitored byTLC. The reaction mixture was cooled to 0°C afterstirring ovenight and filtered off the precipitated solid. Volatiles and solvents were removedunder reduced pressure and the resulting residue was purified by chromatography (first eluted with ,EtOAcfollowed by 5-10 % DCM/MeOH gradient elation) to get the compound 8b as a white solid (10930 g, 74%). 1 H NMR (CDCh, 400 MHz) t = 4.25-4.17(m 2,H), 409(dd, J= 5.50Hz, 11.50 Hz, H), 3.81-3,73(n, 2H), 3,70-3.30(m -- CHCH 2 ~O PEG CH2), L58-.47(m, 41), 130-,17(m, 56H), 0,86(t, J= 6,60Hz, 611) MS range found 2452-2760.

Preparation of compound Sc: ,2Di-Ootadecyi-sn-glyceride c(0.369g, 0.618 mnol), MPEG-Succinate 7 (1.00g, 0,476 mmol, purchased from NOP Corporation, Japan), DCC (0127 g, 1.3eq) and DMAP (0.058 g, 0,476mmol) were taken in dihioromethane(20 nL) under argon and stirred overmigt.Reactionwas monitor by TLC The reaction mixture was cooled to 0 °C afterstirring overnight and filtered off the precipitated solid. Voatiles and solvents were removed under reduced pressure and the resulting residue was purified by chromatography (first fluted with EtOAc, followed by5-10 % DCM/MeOH gradient elution) to get the compound Sc as a white solid (0.960 g 75%). 'H NMR (CDCb, 400 MHz) 8 = 4.27-4,20(m, 21), 4.10(dd, J= 5.801z, 11 50 Hz, fH),33-3.74(m, 211) 370-3.35(m., -O-CH-CHr-., PEG-CH), 154-146(m, 4H), 130-117(m, 64H), 0,86(t, J= 660Hz611). MS range fiud;: 2508-2816,

Scheme 4'

OH

DCM R.O 0

' * 10a R= R.1 1 0---10O6 R Ca R 0

90a RP CIRH27 9b R =C Haj

*Scheme 4; mPEG2000-1,2-Di--acyi-sn3-succinylglyceride Preparation ofcompound 10a: 1,2Dinyristoybisn-glyceroi9a (0317 g, 0,618 Smmol), nIMPEG-Succinate 7 (1,Og, 0,476 mmol, purchased from NOF Corporation, Japan), DCC (0127 g, 1 3 eg) and MAP (Q058 g, 0.476 mmol) were taken in dichloronethane (20 mL) under argon and stirred overnight, Reaction was monitored by TLC The reaction mixture was cooled to OC after stirring ovemight andfiltered off the precipitated solid, Volatiles and solvents were removed under reduced pressure and the resulting residue was purified by chromatography (first elied with EtOAc, followed by 5-10 % DCMMeOIgradient elution) to get the compound Iaas a white solid (0,960 g, 78%) UH NMR (CDCL, 400 MHz) 6 = 5.26-5.20(m, 1H), 4.30-4.09(m., H)! 3,81 3,73(m, 21) 3.70-3 4 0(m -O-C>rCH-O-, PEG-CH 2), 2.65-2.60(m, 4H), 2.35-2, 2 8 (nk 4H), L63-1.52(m, 4H), .30-1,15(m, 44H), 0.86(t, J= 6,60Hz, 6H), MS ange found: 1 2468-2732,

Preparation of compound 10b: 1,2-Dipamitoy~-sn-glycerol9b (0g352 g 0618 mmol) MPEG-Succinate 7 (00g 0.476 mmol, purchased from NOP Corporation, Japan). DCC (0,127 g, 33 eg) and DMAP (0.058 g, 0.476 mmol) were taken in dichloromethmne (20 mL) under argon and stirred overight. Reaction was monitored by TLC. The reaction mixture was cooled to 0°C afterstirring overnightand filtered off the precipitated solid.Volatiles and solvents were removed under reduced pressure and the resulting residue was purified by chromatography (first elated with EtOAc,followed by 5-10 %DCM/MOH gradient elution) to get the compound 10b as white solid (102 g, 81%), H NMR (CDCV, 400 MHz) 6 = 5:26-5,19(, 1H), 4.30-4.05(m, 61), 380 3,40(m-O-CHr-CHr'O-PEG-C), 2.65-2.60(m, 4H), 233-2,24(m, 4H), 163-1,50(m, 41), 30-1 .15(m, 5211), 085(t J> 6:60Hz, 6f). MS range found 2524-2792. 1,52

Preparation of compound 10c: 9,2-Distearoyksn(0.387g -ycerol 0618 mmol),MPE-Suecinate7 (1.00g, 0476 mmol, purchased fIom.NOF Corporation, Japan), DCC (0127 g I3eq) and DMAP (0,058 g, 0,476 nnol) were taken in S dichioromethane (20mL) under argon and stirred overnight. Reaction was monitored by TLC. The reaction mixture was cooled to 0 after stirring overnight and filtered off theprecipitated solidVolatiles andsolvents wereremoved underreduced pressureand the resulting residue was purified by chromatography (first eluted with.EtOAc, followed by 5-10 % DCM/MeOH gradient elution) to get the compound 10c asa white solid (1.04 g, 80%), 'H NMR (CDCi,400 MHz) S= 5.26-S.19(m, 111), 430-4,05(n, 6H), 3.80-340(m, -CH 2 -CH 2 -- , PEG-Cz>), 2.66-2.59(m, 4H), 231-2.26(m, 4H), 1,63~

1L52(nm 411) i.30~1.15(m, 52H),0,85(t, J= 6.60Hz, 6H). MS range flund: 2540-2844,

Scheme 5'

HBTU,DIEA /0 DMF/DCM 4 -~ 0 Z 0

'Scheme 5: Cholesteryl-mPEG2000 Preparation of compound 13: mPEG 2 eco-OH 11 (6,00g, 3nmol, purchased from Sigma-Aldrich), Cholesterol hemisuc ate12 (1.50 g, 3.08 mmol mmol) and. io HBTU (1.23g, 3,23 minmol) were dissolved in a mixtureuf dichloromethne/DMF (2:1, 100 mL) under argon. DIEA (L60 mL, 3 eq.) was added to that and stirred overnight. Solvents and volatiles wereremoved under reduced pressure. The residue was dried under high vacuum ovemight and purified by chronmtography (first ethyl acetate then 5 10% MeGDOCM as a gradient elution) to get therequired compound 13 as white solid 1 (5.05g, 68 %). 'H NMR (CDC1 3,400 MIH)& = 5.35-5.25(m, 1l), 4,60-4.3(m, 11), 422-4.138(n, 211), 3,80-376(m, 2H), 3.72-3,40(m, -O~C ClO~,PEG-C4), 2.64 2.56m 41)2.31-2.20(mH311), 2.01~08(m, 44H)M.S range found:23902654,

E'xanple 42,'ageedPEG-igi

14 PY 2^CM, 0 0o0ocN* 0.-RT n H2' f

rOs A HBTU,jDAz

15 Ato HNDM/DO

AO OAc o AcHN~ rOO H d H

AcH H19 nH

19

Preparaton of 19: Step 1: Conipnd 14 (2.00 g1.01 innol) and cholesterol ichlorofornmte 15 (0.453 g1.01mmol) were taken together in dichloromethane (20 mL). The mixture was s cooled in an ice-water bath Triethylamine (0.448 ml) was addd and the reaction mixture was stirred overnight. Reaction was monitored by TLC. Solvent was removed and the residue was purified by silica gel chromatography (Ethyl acetate followed by 5 10% MeOHDM)togetthe desird compound 1.6 (1.Og, 45,40 %). 'HNMR (CDC, 400 MHz) 6 = 5.35(m, 11), 5.15(m, IH),3.40-3.85(m, O&CIYCHrQ), 3.10-3,25(m, l1H), 0.80-2-38(m,441, Cholesterol). MS range found: 2220-2490.

Step 2: Compound 16 (L0g, 0.417 mmol), 17 (0.235g, 0.542 mmol) and U1 _(09, 05 mmol) were taken in a mixture of DCM/DMF (20 mL,2:1). To that DIEA was added and stirred overnight, Reaction was monitored by TVC, solvents were removed under reduced pressure and the residue was purified by chromatography (5 '10%MeOH/DCM) to get the desired compound 18 (1.02g, 87%). iH NMR (DMSO

d6, 400 MHz) $ 7.52(d, J= 8.06 Hz, 1N), 733(t, J= 7,02 Hz, I i) 7.25(t, J= 7,32 Hz, 1H), 5,27(m, 1H), 5J8(d, J- 3,2 Hz, 1), 4.92(dd,f= 3.17, 11.23 Hz, 1H) 4.43(m, 1H), 3.60-4.02(m5H), 3,203.55(m, -CHrCI-0), 2.90-3,10(m, IOH) 2.05(s, 3H),96(s,

'15

3H), 18s,3H) 177(s, 3H), 0.80-238(m, 4411, Cholestero). MS range found: 2680 2990.

Step 3: Compound 18 (1,02g, 0.362 mmol) was dissolved in a mixture of fMeOH/DCM (10 ml) tothat 0.5 M solution of NaOMe in methanol excesss) was added and stirred ovcnight. Progress ofthe reaction was monitored byTLC The mixture was neutralized with AcOH. Solvents were removed under vacuum and the residue was purified by chromatography (5-10 % MeOH /DCM) to get compound 19 (280 mg, 30%), '1 NMR (CDC%, 400 MHz) 6= 5,38(m, lH), 4.02-4,06(n, 7H), 3,30-3,80(m, 0 HC-01 2 -0), 3.20-32 9 (mi 8208(s,33H), 0.80-2.38(m, 44, Cholesterol) MS range found: 2600-2900. Example 43: Targeted PEG-lipids

14 OcM,Py Q O O-RT

ACO 9 0 121 HBTU.ODEA AcQ HDMEOCMI AcHN

OcCAC 0 0" AcHN M H 22 NaOMe DOCMIMeOH

HO, 0

AcHN H a H 23

Preparation of 23: Step 1: Compound 14 (2,00 g, 1.01 mnol) and compound 20 (0.453 g, L 1mmol) were taken together in dichloromethane (20 ml). The mixture was cooled in an ice-water bath. Pyridine (1mL, excess) was added and the reaction mixture was stirred overnight.Reaction was monitoredby TLC. Solvent wasremoved and the residue was purified by silica gel chromatography (Ethyl acetate followed by 5-10% MeiIHDCM) toget the desired cmpound 21 (400 mg, 15 %'H NMR (CDCl3 , 400 MHz) 8 5,20(m,1H),405-4.20(m,2H, 3,20-380(mnO~CMrCH5)), 130-.82(m, S 4H), 1,50-i.61(m, 21), L18-.38(m, 60H), 0.87(t, J= 6.30 Hz, 6H), MS range found; 2400-2750,

Step 2: Compound 21 (0415 g, 0.159 mmol), 17 (0.100g 1.3 eq) and MTU (090g9, 15eq ) were taken i1 a mixture ofDCMDMF (20 mL, 2:1). To that DILA (02 t ml) was added and stirred overnight Reaction was monitored byTLC, solvents were removed under reduced pressure and the residue was purified bychromatography (3 10% MeOHDCM) to get the desired compound 22 (0,450g, 94%). 'H NMR()CDb, 400 MHz) -= 6.21(J 8.70 Hz, IH), 5,33(d, J= 2.70 z, 1I), 5.15-5.20(m, 21),

4,55(d, J= 8,15 Hz, 1), 4.01-4.20(m, 41), 320-390(m, O-Cr1 2 -Cf-O), 234(s, 3), 2.03(s311), M99(s3H), 1.93(s, 31), 70-L,82(m, 4H),L50-L61(m,4H> 1A 7-1.38(m, 6M0H)0.86(0, J=6.32 Hz, 6H), MS rangefound: 2800-3200.

Step 3: Compound 22 (0.450 g, 0.359 nimol) was dissolved in a mixture of MeOl/DCM (5 mL) to that 0.5 Msolution of NaOMe in methanol (excess) was added and stirred overnight.Progressof the reaction was monitored by TLC. The mixture was neutralized with AcOH Solvents were removed under vacuum and the esidue was purified by chromatography (5-10 % MeOH/DCM) to get compound 23 (365 ng, 85 %),'HNMR (CDC1, 400 MlHz) 6 5.18(m, 1H),4.05-4.20(n. 41), 320-3.90(m, O CHrC) 2 -0, 2.05(s, 311),l.71-l80(m 4H), 1.50-L61(m, 4H), 1.17~.138(m, 60H), 0:87(t, J= 6.32 Hz, 6H). MS range fund: 2760-3000. As provided in Figure 6, the formulations, when administered to a subject, provided a varying degree of silencing of FVIL For example, ihnumlation 3 provideda rlatve high degree of silencing ofIFV, as did formultion 5, 6, and 12,

so Exampl4; Tolerabiitvof formulation LN iaspdosed in mice Emptyliposomes with composition ND98:cholesterol:PEO-C4 42:48:10 (molar ratio) were prepared as described in Example 45, Different amounts ofsiRNA were then added to the pre-nbrned, extuded empty liposomes to yield formulations with initial total excipientsiRNA ratios of 30:1, 20:1, 15:1, 10:1,and 5:1 (wtt Preparation ofa fonnulation ata total excipient:siRNA ratio of 5:1 results in an excess of siRNA in the formulation, saturating the lipid loading capacity. Excess siRNA was s then removed by tangentialflow filtration using a 100,000 MWCO membrane against volumes ofPBS. The resulting rommlations were then administered to C57BL/6 mice via tail vein injection at 10 mg/kgsiRNA dose. Tolerability of the formulations was assessed by measuring the body weight gain of theanimals 24 h and 48 hipost administrationof the formulation, the results of which are provided in Figure7,

Examppe45rmation of association comniexes byfirstbntnngunloadedl complexes andthenetreating thenloadedcomplexeswthsiRNAand administration of association complexes includingtwotherapeutic agents Association complexes having two different nucleic acid moieties were prepared s asfollows. Stock solutions of ND98, cholesterol, and PEG-C14 in ethanol were preparedat the following concentrations: 133 mg/mi , 25 mg/m.l and 100 mg/mL for ND98, cholesterol, and PEG-C4,,respectively. The lipidstockswere thenmixed to yield ND98:cholesterol:PEG-C4 molar ratios of42:48:10, This mixture was then added to aqueous buffer resulting in the spontaneous formulation oflipid nanopartiIes in35% ethanol,100 mM sodium acetate, pH 5. The unloaded lipidnanoparticles were then passed twicethrough a 008 pm membrane (Whatman, Nucleopore) using an extruder (Lipex, Northern Lipids) to yield unimodal vesiedes 20-100 nmin size.The appropriate amount of siRNA in 35% ethanol was then added to the pre-sized, unloaded vesicles ata total excipient-siRNA ratio of 7.5:1 (wtvwt). The resulting mixture was then incubated at 37 °C for 30 min to allow for loading of siRNA into the lipid nanoparticles. After incubation, ethanol removal and buffer exchange wasperformed by either dialysis or tangentialflow filtration against PBS. The final formulation was then sterile filtered through a 0.2 pm filter, Aflow chart demonstrating the order of addition of exhipients and therapeutic agents is provided in Figure 8. A 1:1 mixture ofsiRNAs targeting ApoB and Factor VIlI were fomnulated as described in Example 44, Separately, the same ApoB- and Factor VI-targetingsiRNAs wereidiidualyrmuatedasdescribedinExample31,Thethreennuationswere ihen administered at varyingdoses in an injection volume of 10 pL/g animal.body weight. Forty-eight hours after administration, serum samples were collected by retroorbital bleed, animals were sacrificed, and livers were harvested, Serum Factor VII concentrations were determined using a chromogenic diagnostic kit (Coaset Factor VII s Assay Kit, DiaPharma) according to manufacturer protocols. Liver mRNA levels of ApoB and Factor VII were detenined using a branched-DNA (bDNA) assay (Quantigene, Panomics), the results of which are provided in figure 9. No evidence of inhibition between the two therapeutic agents was observed. Rather, both ofthe therapeutic agents demonstrated effectiveness when administered.

ExamnMie46: Methods of making associationcomplexes using prefored

Lipid Stock Preparation Stock solutions of lipidoid ND98-4HCI (MW 1487), cholesterol, and PEG-C14 were prepared in ethanol at the following concentrations; 133 mg/nL, 25 mg/mL uand 200 mgmLforrND98cholesterol, andPEG-Cl4,respectively Stocksolutionswere warmed at 50'C to assist in bring lipids into solution.

Empty Vesicle Preparation The hid stocks were then mixed according to the volumes listed below to yield ND98:cholesterol:PEG-Cl4molarratios of42:48:10. Anaqueousmixture wasalso prepared according to the volumes listed in the table below,

Volume Upid Mixture (mL) ... _....... ND98 ......... - -. CholesteroI A. PEG jTotal 5.250 ~ 90.000 3.011770

Aqueous Mixture (mL) 3M Water NaOAc Ethano T-otal 378,000 27.000 40.327 445,32

The ethanolic Lipid Mixture was then added to the Aqueous Mixture while rpidly stirring on amagneticstir plate, Upon mixinglipidoid esiesforned spontaneously, The resulting vesicles were then exuded (2 passes) through a 0.08 ienbrane (Whatman,Nucleopore) to size the empty vesicles. All manipulations were performed at room temperature.

Loading of Empty Vesices with siRNA An siRNA stock solution was prepared by dissohing dsalted duplex siRNA in 50 mMsodium acetate pH 5at a concentration of 10mg/mLAnappropriatevolumeo this siRNA stock was mixed with the appropriate volume of ethanol to yield a diluted siRNA solution in 35% (vol) ethanol (see table below). 1) sIRNA Dlutioi

Stock sIRNA (mrmL) (50 nM NaOAc) Ethanol Total ______ 180.000 96.923 2.923

277 mL of diluted siRNA solution was added to 623 mL of empty veicle mixture while rapidly stirring on amagnetic stir plate The resulting combined mixture was then incubated at 37°C for 30min to allow for loading of siRNA.

Ultrafitration and Terminal 0.2 g Filtration After incubation, the 900 mL loaded nanopartice mixture was diluted into 1,8. L of PBS to yielda 2.7 L diluted mixture. This dilutedmixture was then concentrated to ~ 1UL and diaitered bytangentialfow ftratin against 10 volumes of PBS using a Sartorius TFF system utilizing two stacked 100,000 MWCO cartridges. No back pressure was applied to the eartridge and the pump speed was set to 300 rpm. After buffer exchange the resulting solution was concentrated to roughly 2 mg/mL siRNA, Terminal filtration was performed by passing thesolution through a 0,2,p filter capsule (Whatman, Polycap 36 AS). A flow chartillustrating this process is shown in Figure 10.

Association complexes werefomied using theprocedure generally described in Example 46. However, because the complexes were being evaluated based onsize, different extrusion membranes were used to produce particles having the following 6 diameters 150 nm,85m, 60 n, and 50 nm. The siRNAs loaded in the muplexes targeted factor VII The particles were evaluated ina Factor VII silencing assay, demonstrating that the 50 nm paticleswere the mot efficacious relative to the 150 nm, 85nm, and 60 nm particles. Theresults of the assayare depicted inFigure 11. SExample 48: Comparison of half life ofnucleic acid agents unfomulated versus formulated into an association complex The half life of siRNA fonnulated inassociation complexes was evaluated in vitro in human serum at 37 C. The association complexes were prepared asin Example 46 For purposes of comparison, unfonnulated siRNA was also evaluated in vitro in human seru. The percent of full length product detennined by 1iLCNwas evaluated for both the formulated and unformulated siRNA, As demonstrated in Figure 12, the fonnulated siRNA had a significanly improvedhalf life in vitro in humanserum. Exmpl 49:Comoarison of efficacy of association having PEG lipids of varied chain lenah Association complexes were prepared as in Example 46 with variation on the length of the alkyl chainof the PEG lipid. Alkyl chain lengths of 10, 11, 12, 134, 115, and 16 were evaluatedand compared for efficacy in a FactorVIlsilencing assay: As shown in Figure 13, chain lengths of 13,14, and 15 demonstrated the most silencing as measured in the assay.

Number ofembodimentsof the invention have been described. Nevertheless, it will. be understood thatvarious modifications may be made without ceparting from thespirit and scope of the invention. Accordingly, other embodiments are withinthe scope of the following claims.,

Claims (1)

1. CLAIMS:

   L A preparation comprising one or more compounds, each individually having a structuredefined by formula (I) or a pharmaceuticady acceptable salt thereof;

   xaX R2N VN NR2 FR

   formula (I) wherein each Xand X for each occurrence, is independntly Cmalkyene; n is 0, 1, 2, 3, 4, or 5; each R.is independently H,

   10 R .RI S R1 SyR' Yor eR1 Y,

   Ra Rb R, Rc R, Wherein at least n 2of the R moieties in atleast about 80% of themolecules of the conpounx of fonnula (I) in the preparation are not H; m is 1, 2, 3or 4; Y isO, NR 2, or S; RI is alkyl alkenyl or alkynyl; each of which is optionallysubstituted with one or more substituents; and R2 isHalkylakenyl oralkynyl; eachof which is optionallysubstituted 2D each of which is optionally substituted with one or more substituents; provided that, if n=0, then at least n + 3 of the R moieties are not.H.

   2, The preparation of claim 1, wherein when R is not H, R isR

   3. The preparation of claim 1, wherein when R is not H, R is Ri.

   4. The preparation of claim 1, wherein when R is not H, R is R,

   5, The preparation of claim I, wherein when R is notfH, R is R

   6. The preparation of claim i, wherein when. R is not H R is R,

   7, The preparation of claim 1, wherein n + 2 of the R moieties offonnuila (f) are not 1

   8& The preparation of claim 1, wherein n+ 3 of the R moities of formula (1) aren't H

   100 9. The preparation of claim 1, wherein+ 4 ofthe R moieties of formula (1) are notiH.

   10 The preparation of claim 1, wherein n>0, at least one R of NR of is formula (1) is H.

   11, The prepanaion of claim 1, wherein at least one R of NR2 of formula (I) is H.

   12. The preparation of claim 1, wherein at least 80% of the molecules are a single structuralisomer.

   13, The preparation of claim 12, wherein n+ 2 of the It moieties of fonbua (I)are not H

   14, The preparation of claim 12, whereinn + 3 of the R moieties of fomua (I) are not H.

   1. The preparation of claim 12, wherein n +4 of the R moieties of fonnula (1)are notH.

   16. The preparation of laim 1, wherein at least n + 2 of the R moieties of ima(I) in at least about 90% of the compound. of fonnula (1) are not H.

   17, The preparation of claim 1, wherein at least n4 2 ofthe R moieties of formula (I) in at least about 95% of thecompound of formula () are notH.

   18 Thepreparation of claim , wherein at least n+2 of the It moieties of fonnula (1) in at leastabout 99% of the compound of formula (I) arenotH.

   9 The preparation of claim 1, wherein is 2

   20. The preparation of claim 1, wherein n is 0.

   21. The preparation of claim 1, wherein Xnd X0 are Calkylene,

   22, The preparation of claim 1, wherein n is 0 and X is ethylene or propylene,

   23, The preparation of claim 1, wherein n >1 and X" varies with at least one occurrence,

   24. The preparation of claim 1, wherein when R not H R is y

   25, The preparation of claim 24, wherein Y is 0 or NR

   26. Tle preparation of claim 24, wherein m is 2,

   2 27. The preparationof claim 24 wherein Y is O orN andmis2.

   28 The preparation of claim 24, whereinin is 1.

   29, The preparation of claim 1, wherein R for at least one ocenrence is alkyL.

   30 The preparation of claim 1, wherein Rfor each occurrence is alkL

   31. The preparation of claim 1, wherein R is alkyl and R is F

   32. The preparation of claim 1, wherein R' and R2iare alkyl.

   33, The preparationof claim 1, wherein R for at least one occurrence is alkenyt

   34, The preparation of claim 1, wherein Rfor at least one occurrence is alkenyl.

   35, The preparation of claim 1,wherein when R is not H, R is R,, and wherein Y is 0 or NH,

   36. The preparation of claim 35, wherein Y is 0,

   37. The preparation of claim 35, wherein Y is NI.

   38. The preparation of claim 35, wherein R is alky.

   39, The preparation of claim 38, wherein R is C.o alkyL.

   40. The preparation of claim 39, wherein R is C1 2 alky,

   so 41, The preparation ofclaim 35, wherein n is 2.

   42, The preparaion of claim 41, wherein X', for each occurrence is C2 aikylene and X" is C2 alkylene.

   43. The preparation of claim 35, wherein in is 2.

   44. Thepreparation of clain 1, whrein n is 2 and R, when R is not H, is R,

   45. 'The preparation of claim 44, wherein Ri is alkyl.

   1 46. The preparation of claim 45, wherein 1 is Cjog aalkyl,

   47, The preparation of claim 46, whrin R 1 is C' alky.

   48. The preparation of claim 44, wherein Y is 0,

   49, The preparation of claim 44, wherein Y is NIL

   50, The preparation of claim 44, wherein X", for each occurrence isC2 alkylene and X, is C2 alkylene.

   51, The preparation of claim 44, wherein m is 2.

   52. The preparation of claim 1, wherein at least i R of Ni is H and R, when not H is R, and wherein Y is 0 or NHL

   53. The preparation of claim 52, wherein Y is 0.

   54, The preparation of elaim 52, wherein Y is NH.

   5. The preparation of claim 52, wherein Ri is alkyL

   56. The preparation ofclain 55, wherein Ri is Cmjalkyt

   57. Thepreparation of claim 56,wherein I is C alkyL

   58 The preparation of claim 52, wherein is 2.

   59, Thepreparation of claim 58, wherein Xt for each occurrence is C2 alkyleneandXbisC 2 alkylen.

   60. The preparation of daim 52, wherein m is2.

   61. The preparation of claim 1, wherein n is 2 and at east 1. R ofNR is H and when R is not , R isR and wherein Y is0 or NH.

   62. The preparation of claim 61, wherein R' is akyL

   63, The preparation ofclaim 62, wherein R is CM alkyl.

   64. The preparation of claim 63, wherein R' is C alkyl.

   65, The preparation of claim 61, wherein Y is 0.

   66. The preparation of claim 61, wherein Y is NH

   67. Thepreparation of claim 61w, herein, for each occurrence is C2 alkylene and X" is C2 alkylene.

   68. The preparation of claim 61, wherein m is 2,

   69. The preparation of claim 1, wherein at least I R of NR is H and I is R, and wherein Y is 0 or N-.

   70. The preparation of claim 69, wherein Y is 0.

   71. The preparation of claim 69, wherein Y is NH.

   72. The preparation of claim 69, wherein Ris alkyl

   73. The preparation of claim 72, wherein R' isCo 1 alkyl.

   74. The preparation of claim 73,,wherein RI is C, alkyl

   75. The preparation of claim 69, wherein n is 2.

   76. The preparation of claim 69, wherein X, for each occurrence is-2 alkylene and X"is C2 alkylene,

   77. The preparation of claim 69, wherein m is 2.

   78. The preparation of claim 1, wherein n is 2 and at least 1 R of NR is H and R is R,, and wherein Y is 0 or NIH.

   79. The preparation of claim 78, wherein R is alkyl.

   80, The preparation of claim 79, wherein R is Caa alkyl.

   8 1 [he preparation of claim 80, wherein R is Cz alkyl.

   82. The preparation of claim 78, wherein Y is 0.

   83. The preparation of claim 78, wherein Y is NH.

   84. The preparation of claim. 78, wherein X, for each occurrence is C2 .

   alkylene and X, is C2 akylene.

   85. The preparation of claim 78, wherein m is 2.

   86. The preparation of claimi1, wherein n is 0 and X is propylene. 5

   87, The preparation of claian86, wherein I R is H.

   $8. The preparation of claim 86, wherein when R is not H,.R is R,

   89. The preparation of claim 86, wherein R is alkyl.

   90. The preparation of claim 89, wherein Ris Co-3 alkyl.

   91. The preparation of claim 90, wherein R is C alkyl '15

   92. The preparation of claim 86, wherein Y is 0.

   93. The preparation of claim 86, wherein Y is Nil

   94. The preparation of claim 86, wherein m is 2.

   95, iThepreparation of claim 1, wherein n is 2; X1 for each occurrence is C, alkyleneandXs2alkyene; and wherein each R is H or 0 H Y'R

   R, mis 2; SY i NH or 0; R is C 2 alkyl,

   96, The preparation of claim 95,wherein at least 80% of the molecules ofthe compound of formula (1) are a singlestructural isomer

   S97 The preparation of claim 95, whereinY is NH.

   98, The preparation of claim 97, wherein at least 80% of the molecules of the compound of formula (1) are asingle structural isomer,

   99, The preparation of claim 98, wherein Ris R, for 5 occurrences,

   1.00. The preparation of claim 95, whereiin at least 80% of the molecules of thecompound of fomula (1), R is R. for 5 occurrences.

   101, The preparation of claim 100, wherein Y is NH.

   102. The preparation of claim 95, wherein the compound of formula (1) is an inorganic or organic salt thereof

   103, The preparation of claim 102, wherein the compound of formula (1) is a hydrohalidesalt thereof

   104. Thepreparation of claim 103, the compound of formula (1) is a hydrochloride salt thereof.

   105. The preparation of claim 1, wherein the hydrochloride salt ranges from a single equivalent of HCL, to n,2 equivalents of HCL

   106, The preparation of claim 1, comprising a hydrate of the compound of so formula (1).

   107. The preparation of claim 1, wherein the compound offormula (1) is salt of an organic acid,

   108. The preparation of claim 107, wherein the salt isan acetate.

   109. The preparation of claii 108, wherein the acetate salt ranges from single equivalent of acetate, to n+2 equivdents of acetate.

   11'. The preparation of claim 107, wherein the salt is an fonnate,

   11. The preparation of claim 108,wherein thefomate salt ranges from a single equivalent ofacetate, to n+2equivalents of format

   112 151 The preparation of claim 1, wherein R.' comprises an alkenyl moiety,

   13. The preparation of claim 112, wherein RI comprises a cis double bond.

   114. The preparation of claim 1, wherein the preparation comprises less than

   H 2 N 'N NH2 H 11N, by weight, of fall),

   wherein X and a are defined as in formula (1) of claim 1

   115 The preparation ofclaim 1, wherein the preparation comprises less than 90% by weight of 0 R

   formla (IV) wherein Y and R 1 are defined as in formula (1) of claim

   17)

   116. The preparation of claimI the preparation comprisinga plurality of compounds of formula (1),

   117. The preparation of claim 116, the preparation comprising a mixture of compounds ofthefannulas below:

   R R RR t'NN R N - R Rn and R R formula (F) formula (l") Whereinfin tormula (I")at least five of the R moieties are R'

   118. mhe preparation of claim 117, wherein formula (F) and (F)arc present in a ratio offrom abot1:2 to about 2:1

   119. Amethod ofmaking a compoundof-formula(I1), '15

   R{4N A NR 2

   fonnula (II) wherein each Xaand X, for each occurrence, is independently Cs alkylene; n is , 1, 2, 3, 4, or5;. and wherein each R is independently H or

   R,; m is 2; 2n Y is 0, NR2, or S; R'is alkyl or alkenyl; R2 is H or C akyl or alkenyl; the method comprising reacting a compound of formula (111)

   H2N xz$N 'Nt H

   fonnula (Ill) with a compound of fonnula (IV), 0 ..R

   formula (IV)

   in the presence of a promoter.

   120. A method ofmaking a compound of formula (II),

   xa |Xt R2 Ni 'N 'NR 2 n

   formula (II) wherein each Xa and X, for each occurrence, is independently C6 alkylene; n is 0, 1, 2 3 4, or 5; and wherein each Ris independentlyiH or 0

   R

   mis 2; Y is 0, NR2 , or S; R is alky or alkenil; Re is H or C alkyl or alkenyl; themethod comprising reactinga compound ofibrmula(i)

   H 2N 'N 'NH[ L n

   fominua (III) with a compound of formula (IV),

   formula (IV)

   in the presence of a quencher.

   2. A method of making a compnd of formula (II),

   Rp)N' 'N ANR2 n

   formula (II)

   eacXa and X for each occTencc, is independently C alkylene; n is O 1, 2, 3, 4, or 5; and wherein each R is independently 11 or 0 Rl Ra

   >0 m is 2; YisONR or S; R is alkyl or alkenyl; R is H or alkyl or akenyl; the method comprising reacting a compound of funiula (III)

   H2N'41X 1NH2 Xa X formula(I) with acompound of formula (IV),

   5 formula (V)

   wherinthe reaction mixture comprises from about .8 about 1.2 molar equivalents of a compound of formula (119) with from about 3.8 to about 6.5 molar

   equivalents of a compound of fornula (IV).

   122. The method of claim 121,wherein the reaction mixture comprises from about 0.8 about 12 molar equivalents of a compound of formula (111), with from about55btout 6.5 molar equivalents of a compound of formula (IV)

   123. The method of claim 122, wherein the reactionmixturecomprises about Imolar equivalents of a compound of formula (Il, with from about 6 molar equivalents of acompound Of formula (IV)

   124, The method of claim 121,wherein the reaction mixture comprises about 1molar equivalents of a compound of formula (III), with from about 5 molar equivalents ofa compound of formula (IV).

   125, A method of making a compound offormula (1),

   X 1X R2N XN XNR, LRj

   formula (II) wherein each Xa and X, foreach ocurrece, is independently C. alkylene; n is 0, 1, 2, 3, 4, or 5; and wherein each R is independently H or

   Ri

   is 2; Y is O NR or S; Rl is alkyl or ulkenyl; 1o IC is H or alkyl or alkenyl; the method comprising a two step process of reactinga compound of formula(i)

   X" X H2N' 'N 'NH2

   formula (1l) with a compound of formula (IV), 0

   fornmla (IV) in the presence of boric acid and water wherein, the first step process involving the reaction mixture comprises from about 08 about 1.2 molar equivalents of a compound of formula(Ui), with from about 3,81about 4.2 molar equivalents of a compound of formula (IV) and the second step process involving addition ofabout 0.8 to 12 molar equivalent of compound of formula (IV).

   2.5 126. A method of making a compound of formula (Ii),

   RN' N 4xNR2

   formula (11) wherein each X" and.Xb foreach occurrence, is independently C alkylene; a is O, 1,2, 3, 4, or 5; and wherein each R is independently R or 0

   Rl

   1i mis 2; Y is O NR or S; Rxisalkyl oralkenyl; R" is H or alkyl or alkenyl; themethod comprising reactinga compound of formula (il)

   Ft- H ? 1HI

   formula (11I1) with- a compound of formula (IV), 0

   formula (IV)

   and separating at least one structural isomer of formula (II) from the reaction mixture to provide substantially purified preparation comprising astructural. isomer of formula (II).

   127. Themethodofclaim126,whereinthestructuralisomerofforma(I)is separated from the reactionmixture using chromatographic separation.

   128, method of claim 127, wherein thechromatographic separation is Them using fhash silica gei for separationof isomers.

   129. The method of claim 128, wherein the chromatographic separation is gravityseparation of isomers using silica gel.

   130, Themethod of claim 128, Wherein the hromatographicseparation is using moving bed chromatagraphy forseparation of isomers

   131 The method of claim 128, wherein the chromatographic separation is using liquid chromatagraphy (1C) for separation of isomers

   132, The method of claim 131, wherein the chromatographic separation using normal phase HP LC for separation of isomers.

   133. The method of claim 131,wherein the hromatographic separation is using reverse phase HPLC for separation of isomers,

   134. The method ofclaim 126, wherein thesubstantially purifiedpreparation comprises atleast about 80% of the structural isomer of fenla (11),

   135. The method of claim 134, wherein the substantially purified preparation comprises at least about 90% of the structural isomer offormula (II).

   13 6. The method of claim 135,wherein the substantially purified preparation comprises at least about 95% of thestructural isomer of formula (11).

   s 137. Amethodofmakingacompoundofformula(V)orapharmaceutically acceptable salt thereof,

   I78

   R2N XNX 'NR 2 Sn

   formula (V) wherein each X and X for each occurrence isindependently Ca alkylene; is 0, 1, 2, 3, 4, or 5; and wherein each R is independently H or

   0 R'

   Rr to m is l; Y is 0, NR, or S; R is alkyl or alkenyl; R.is H or alkyi or alkenyl; the method comprising reacting a compound of fonula (III)

   H 2N {XNZ NH, -n formula(II) with a compound of formula (VI),

   0 0N>A y.R 0 = CI rror 1

   formula (VI)

   to provide a compound of formula (V) oraphamaceuticallyacceptable sai thereof,

   138. The method of claim 137, wherein the phamaceutically acceptable salt thereof is a hydrochloride salt of the compound offormula (V).

   139. A compound of formula (X),

   R N 7-,L2 R4

   'AR3

   tfornula (X) wherein R 1 and R 2 are each independently H, CrC6alkyl, optionally substituted with 1-4 R- CrC alkenyl, optionally substituted with 1~4 R' or C(NR')(NR)2; RI and Rare each independently alkyl, alkenyl, alkyly, each of which is optionallysubstituted with fluoro, chloro, bromo, or iodo;

   [! and L2 are each independently -NR 6 C(O),C(O)NRt, OC(),-C(0)0, S-S~, -NT(R)C(ON(R)-, -OC(O)N(Ry)-, -N(Rl)C(O)O- -ON=C-, OR, -OC(O)NH N=C-, or -NHC(Q}NH-N=C-, .1-R' and L'R4 can be taken together to form an acetal, a ketal, or anorthoester is wherein Rand R4 are defmed as above and can. also be H or phenyl; R is fluoro, ehloro, bromo, iodo-OR, -N(R)(R), ~CN, SR S(O)R S(O) 2 R Re is H, C-Cs akyl, R is H or C,-C alkyl; each R' and R'are independently H or CC alkyl; Rt"' is H or Cr s alkyl; m is 1, 2,3, 4,5, or 6; n is 0, 1 ,2, 3,4, 5, or 6; andpharmaceutically acceptablesalts thereof

   140. The compound of claim 139, wherein the compoundis aninorganic salt thereof,

   141. The compound of claim 140, wherein the compound is a hydrohalide salt so thereof

   142. The compound of claim 141, wherein the compound is a hydrohoride salt thereof,

   143, The compound of claim 139, whereinthe compound is anorganicsalt a thereof

   144. The compound of claim 139, wherein R and R are each independently C-CI alkyl.

   145. The compound of claim 139, wherein R is methyl.

   146. The compound of claim 139, wherein R is methyl

   147. The compound of claim 139, wherein R and R are both methy.

   or 2-hydroxyethyL

   149, The compound of clain 148, wherein Re isti,

   150. The compound of claim 148, wherein R.is methyl, 202

   i51. Thecompound of claim 148, wherein R2 is ethyL

   152, The compound of claim 148, wherein R2 is propyl

   153. The compound of claim 148, wherein R2 is isopropyl,

   154. The compound of claim 139, wherein R 2 is H, methyl, ethyl, propyor isopropyl.

   155, The compound of claim 139, wherein R is H, methyI, ethyl, isopropyl, or 2-hydroxyethyl and R2 is H, methyl, ethyl, propyl, or isopropyl,

   156. 'hecompound of claim 139, wherein is 1.

   157. The compound of claini 139, wherein n is 1

   158, The compound of claim 139, Wherein both n and n are

   . 159, The compound of claim 139, wherein C is NRC(0), or -C(0)NPK

   160. The compound of claim 139, wherein L'is -OC(O)- or -C(O)O-.

   161 The compound of dlaim 139, wherein 1 is S-S,

   162. The compound of claim 139, wherein L' is -N(Ra)C(O)N(R),

   163, The compound of claim139, wherein L" is -OC(0)N( or N(R)C(0)O-.

   164. The compound ofclaim 139, wherein Li is -O-N=C

   165. The compound of claim 139, wherein L. -OC(O)NH-N-C, or ~ NrHC(O)NH-N=C(>

   166, The compound of claim 139, wherein L2 is -NRC(O), or C(Q)NR

   167. The compound of claim 139, wherein . is -OC(O)- or -C(0)0

   168. The compound of claim 139, wherein L' is S-8,

   169 The compotud of claim 139, wherein , is-N( )N(R)

   170. The compound of claim 139, wherein is -OC(0)N(R or N(R)C(0)O-.

   171. The compound of claim 139, wherein C is -O-N=zC.

   172, The compoundofclaim139,.whereinU-OC()NH-N=-t or NHC(O)NH-N=C

   173. The compound of claim 139, wherein both L' and 1,are-NRC(0-, or C(0)NR

   174. The compound of claim 139,wherein both L and 0 are -OC(O)- or ~

   17$. The compound of claim 139, wherein both . and L are S-S~

   176. The compound of claim 139, wherein both L and Q are N(R%)C(0)N( )

   177. The compound of claim'139, wherein both I'and U are -OC(O)N(R) or-N(Rt)C(OO

   178, The compoundof claim 139,whereinUIis--NRkC(O)- and L is -S-.

   179. The compound ofclaim 139, wherein L'is-OC(O)-and is-- S-.

   180. The compound ofclaim 139,whereinL'is-OC(0)N(RC)or N(R)C(0)O- and 0 is

   181, The compound of claim 139, wherein L' is -N(R')C(O)N(R,)- and L2 is

   182. The compound of claim 139, wherein I¾R3and IR are taken together to fom an acetal, a ketal, or an orthoester,

   183. The compound of claim 139, wherein each R' andR are independently alkyl.

   184. The compound of claim 139, wherein bothRi and R are C(as alkyl,

   185. The compound of claim-184, wherein each0and 1are indepedently 3-S,-OC(O)N(R)- or -N(Rt)C(O)O,

   186. The compond of claim 139, wherein R3 is alkyl

   187. The compound ofclaim 139, wherein Ri is alkyl,

   188L The compound of claim 139, Wherein R3 isalkenylt

   189. The compound of claim 139, wherein R4 is alkenyl.

   4 are independently 190. The compound of claim 139, wherein each R and alkenyl,

   alkenvi. 191. The compound of claim 190, wherein each R and Rare independently C6-Ca alkenyi,

   192. The compound of claim 190, wherein each R and R4 are thesame alkenyi moiety,

   so 193. The compound of claim 139, wherein each R and R includes two double bond moieties,

   194, The compound of claim 193, wherein at least one ofthe doublebonds have a Z configuration.

   19. The compound of claim 193, wherein both of the double bonds have Z configuration.

   196. The compound of claim 190, whereinat least one of R3 and R4 is provided in formula (1) below

   formula (I) wherein x is an integer from 1 to 8; and

   y is an integer from 1~10.

   i5 197. The compound of claim 196, wherein both of R and R are of the formtda ().

   198. The compound of claim 190, wherein at least one of the double bonds have an E configuration.

   199. The compound of claim 198, wherein both of the doublebonds have an E confAguration,

   200, The compound of chaim 198, wherein at least one of R and i0 is provided in formula (Il) below

   formula (HI) wherein x is anintegertfrom 1 to 8; and y ia an integer from1-10.

   201. The compound of claim 200, whereinbofh of R and R2 are as provided in fonula (111).

   202. The compound of claim 190, wherein each R' and R2 includes three double bond moieties.

   203, The compound of claim 202, wherein at least one of the double bonds have Z configuration.

   204, The compound of claim 203, wherein at least two of the double bonds have a Z configuration.

   205. The compound of claim 204, wherein all three of the double bonds have a Zconfiguration.

   206. The compound of claim 190, wherein at least oneof R1 and R is provided in formula (IV) below

   formula (IV) wherein x is an integer from to 8; and y is an integer from 1-10

   207. T'e compound of claim 206, wherein both of R and R2 are as provided informnula (IV).

   208. The compound of claim 190,wherein at least one of the double bonds have an E configuration.,

   209. The compound of clain 208, wherein at least two of the double bonds have an Econfiguration.

   210. Thecompound of claim 209,wherein all threeof thedoublebonds have an E configuration,

   211, The compound of claim 210, wherein at least one of R and R is a provided in formula (IV) below

   formula (V) wherein x is aninteger from I to 8; and y is an integer from 1-10.

   212. 'Thecompound ofclaim212, whereinboth ofR and.R2 are asprovided in fbnnula (V),

   213, A preparation comprising a compound offormula (X).

   214, A method of making a compound of formula(X),

   R2L R3 Ri Ltga

   fbnnula (X) wherein R1 and1 are each independently C-C6 alkyl, optionallysubstituted with 1-4 RW; Ra is alkyl, alkenyl, alkynyl Uis -0C(0) R 5is -OR', -N(R)(R), -CN, SR, S(O)R IS(O) 2 R R is H, Cr alkyl,\ R is H or C-C6 alkyl; eachlR and R9 are independently H or CrC- alkyl; S-isH or CC alkyl; i and n are each independently -12,3, 4, 5, or 6, the method comprising reacting a compound of formula (VI), R1

   R2 QH fonnula (VI) with a compound of fonula (VII)

   C

   fonnula (VII) in the presence of a coupling agent, therby providing a compound of formula (X)X

   215. The method of claim 214, wherein the coplingagent is a carbodiinide,

   216. Themethodofclaim215,whereinthecouplingreagentis[DCL

   i5 21.7 A method of forming an association complex comprisingacontacting a lipid preparation of claim I or claim 213 with atherapeutic agent in the presence of a buffer, wherein said buffer: is ofsufficient strength that substantially all amines of the molecules frmula.are protonated; is present at between 100 and 300mM is present at a concentration that provides significantly more protonation of than does the same buffer at 20 mM,

   218. An association complex made by the method ofclain217.

   219, A method of forming an association complex comprising contacting a lipid preparationof claim I or claim 213 witha therapeutic agent in a mixture comprising at least about 90% ethanol and rapidly mixing ie lipid preparation with the therapeutic agent to provide a particle having a diameter of less than about 200 uM

   220. Themethod ofclaim 219, wherein the particle has a diameter ofless than about 50aM.

   221. A method of farming an association complex comprising contacting a lipid preparation of claim I or claim 213 with a therapeutic agent in the presence of a Tuffer, wherein said buffer has a concentration from about 100 to about 300mM.

   222. An association complex comprisinga preparationof claim I orclaim 213 and a nucleic acid.

   223. The association. complex of claim 222, further comprising aPEGylated lipid.

   224. The association complex of claim 222, further comprising astructural moiety.

   225, The association complex of claim 224, wherein thestructural moiety is cholesterot.

   226. The association complex of claim 222, wherein said nucleic acid is an siRNA,

   227. Theassociation complex of claim 226, whereinsaid nucleic acid is an siRNA which has been modified to resist degradation.

   228, The association complex of claim 226, wherein said nucleic acidis an siRNA which has been modified by modification of the polysaccharide backbone,

   229. The association complex of claim 226, wherein the siRNA targets a gene 3o or genes of interest

   230., Theassociationcomplexofclaim229,whereinthegeneorgeneso interest is an endogeneously expressed gene in liver,

   231. The association complex of claim 230, wherein the gene of interest is apoB.

   232, The association complex of clain 230, wherein the gene of interest is

   233. The association complex of claim 230, wherein the gene ofinterest is PCSK9,

   234. The association coipiex of claim 230, wherein the gene of interest is VEGF,

   235. The association complex of claim 230, wherein the gene of interest is KSP (eg5)

   236. The association complex of claim 230,whain the gene of interest is hepeidin.

   237. The association complex of claim 230, wherein the gene of interest is HCV,

   238. The association complex of claim 222,wherein said nucleic acid is a single stranded nucleic acid or derivatives thereof

   239. The association complex of claim 238, wherein the nucleic acid is ai anitisensenucleic acid.

   240, The association complex of claim 238, wherein the nuclie acid is a. miroRNA,

   241. The association complex of claim 238,xwhereinthe nucleic acid is an antisenseeoligonucleotide ofmicroRNA (antagomir).

   242. The association complex of claim 241, wherein the nucleic acid is against microRNA-122

   243. The association complex of claim 241,wherein thenucleic acid isagainst microRNA-181.

   244, The association complex of claim 241, wherein the nucleic acid isagainst microRNA-155.

   245. The association complex of claim 241, wherein the nucleic acid is against microRNA-16,

   246. The association complex of claim 222, further comprising structural moiety and a PEGylated lipid, wherein the ratio, by weight, of preparation of claim I or claim 213, structural moiety, PEGylated lipid, and a nucleic acid, is 8-22:0.4-10:0.4 12,0.4-2..2.

   247. Theassociationcomplexofclaim246,whereinthe sircturalmoietyis cholesteroTl

   247. The association complexof claim 247,wherein the ratiois 10-20:0&5 8.0:5-100.5-2. 0.

   24& The association complexofclaim248,whereintheratio is15:0:7:.

   249. Theassociation complex of claim 246,wherein the average liposome diameter is between 10 nm and 750un.

   19i

   250. The association complex of claim 249 wherein the average association complex diameter is between 30 and 200 mn.

   251. The association complex of claim 250, wherein theaverage association complex diameter is between 50 and 100I n.

   252. Theassociationcomplexofclaim222,whereinthepreparationisLess than 15%, by weight, of unreacted lipid

   253. A pharmacuticallyaccptable composition comprising the preparation of claim I or claim 213,

   254. A pharmaceutically acceptable composition comprising the association complex of claim 222

   255. A method of treating a mammal comprising administering to said mammal atherapeutic amount of anassociation complex ofclaim 2222.

   256. A preparation of claim 1, wherein the preparation comprises one or a mixture of the fornmla below, wherein R is not H unless specified in the fominla below. R R H H R and R' N R R'N R R R R ,

   257. The preparation of claim 1, wherein the prparaton consists essentially of one or amixture of the formula below

   H iH RNl N 'N ' RN'N and RN ¾ N ,-NR RR R R

   258. Thepreparation of claim 257, wherein each R is

   R2

   259, The preparation of claim 258 wherein each R is 0

   )CN,<N 0 R'

   260, The preparationof claims 256 or 257,wherein R' isC-CB alkyl(e.g, Caalkyl), or CI(C akenyl,

   261. The preparation of claim 256w herein R is

   N'R R2,

   262. The preparation of claim 261, wherein R'isur-C alkyl,

   263. The preparation of clai261, wherein is aaky andt2isH,

   264. The preparation of claim 253, wherein formula (f) is providedbelow, wherein R. is no H unless specificaly recited: R H E R' Na N NR RR

   R-R N, Ris

   265. The prepration of claim264, wherein R 1is C1 alkyl and R is H.

   266, Thepreparation of claim 256, wherein formula (1) is provided below, wherein R is not H unless specifically recited:

   RH R'N''NN' RR R

   N' R is R2

   267, Thepreparation of claim 266, wherein R' is C2 alkyl and R2 isH,

   268, The preparation of claim 1,wherein formula(I) isprovided below, wherein R is not 1 unless specially recited H R,N N,'R

   269, The preparation of claim 268, wherein R is 0o N' or R R2 T 270. he preparation of claim 269, wherein, R isC alkyl, orCt

   alkenylt

   271. The preparation ofclaim 268,whereinRis

   N' R' R; Y

   272. The preparation of claim 271, wherein R isvCc, alkylor Cwty alkenyl and R 2 is H.

   271. A method of forming an association complex comprising a plurality of lipid moieties and a therapeutic agent, the method comprising: mixing a plurality of lipid moieties in ethanol andaqueous NaOAc buffer to provide a particle; and adding the therapeutic agent to the particle;thereby formingthe association complex,

   272. The method of claim 271, wherein the lipid moieties are provided in a solution of 100% ethanol,

   271 The method of claim 271, wherein the plurality of lipid moieties comprises a cationic lipid,

   274, The method of claim 273, wherein the cationic lipid is a lipid of claim or Claim 213.

   275. The method of claim 274, wherein the cationic lipid is a lipid of one of the following or a mixture thereof: H H

   o - H ~N -N 0

   or H

   0

   U 0

   H H

   276. The method of clain 271,wherein theplurality of lipid moieties cumpises a PEG-lipid.

   277. The method of claim 276, wherein the PEG-ipid has the fIlowing structure:

   Hm L2 4

   wherein; eachI an d 2are independently a bond or C(0); each R and R areindependently alkyl alkenyl or alkynyl; each of which is optionally substiautd with one or more substituents; X is-C()NH-, C(S)NH, -C()CalkylC(0)NI-; or -C(O)C 3 alkyC(0)O-; m is ai integer from 0-11 iand n is an integer from 1-500.

   278, The method of claim 277,whereinthe PEGlipid is 0 N H \ 'a

   279, Themethodofclaim271,whereinthepluralityoflipid moieties comprises a structural lipid.

   28(. The method of claim 279, wherein the structural lipid is cholesterol.

   28i, The method of claim 271, further comprising extrudingthe lipid containing particles.

   282. Themthod ofclaim271,wherein the lipid containing particles are extruded prior to addition of the therapeutic agent

   283. The method of claim 271, wherein the therapeutic agent is a nucleic acid

   284, The method of claim 283 wherein the nuclic acid isan siRNA,

   284. The method of claim 283, wherein said nucleic acid is an siRNA which has been modified to resist degradation.

   285. Theimethodof claim283, wherein said nucleic acid is ansiRNAwhich has ben modified by modification of the polysacchaide backbone,

   286. Themethod of claim 283, siRNAis conjgatd toaLipphilic moiety.

   287. The method of claim 284, wherein the siRNA targets a gene or genes of interest

   288. The method of claim 287, wherein the gene or genes ofis an endogeneously expressed gene in liver,

   IS 289. The methodof claim 288, wherein thegee of interst is apoB.

   290. The method of claim 288, wherein the gene of is FVIL

   29. The method of claim 288 wherein the gene of is PCSK9,

   292. The method ofchim 288, wherein the gene of is VEGF.

   293. The method of claim 288, wherein the gene of is KSP (eg5),

   294. Themethodofclaim288, wherein thegeneofis hepcidin,

   295,, The method ofclaim 288, wherein the gene ofis HCV.

   296. The method of clim 283, wherein saidnucleic acid is asingle stranded so nucleic acid or derivatives thereof

   297. The method of claim 296, wherein the nucieacid is an antisense nuceic acid,

   298, The method of claim 296, wherein the nucleic acid is amicroRNA.

   299, The method of claim 296, Wherein the nucleic acid is an antimicroRNA (antagomir).

   300. The method of claim 271, wherein the association complex comprises a cationic ipid, a structural lipid, a PEG-lipid and a nucleic acid,

   301. The method of claim 300, wherein the molar ratio of the eationic lipid, structural lipid, PEG-ipid and nucleicacid is 36-48:42-54:6-14,

   302, Themethod ofclaim 301, wherein the molarratio of the cationic lipid, structural lipid, PEG-lipid and nucleic acid is 3846:44-52:8 12.

   303. Themethod of claim 302, wherein themolarratio of the cationic lipid, structural lipid, PEG-lipid and nucleic acid is about 42:48:A0.

   304. The method of claim 271, wherein the weight ratio of total exipient to nucleic acid is Zess than about 151.

   305, The method of claim 304, whereinthe weight ratio oftotal exipient to nucleic acid is about 10:1.

   306, The method of claim 305, wherein the weight ratio of total exipient to nmoleic acid is about 7:1.

   307. The method of claim 305, wherein the weight ratio of totalexipient to nucleic acid is about 5:1.,

   308. The method of claim 300, wherein the cationic lipid has the following. H

   N N H N' )H

   structure; ; the PEGlipid has the following structure:

   and the stnxtural lipid is cholesterol

   309. The method of claim 308, wherein the molar ratio of the cationic lipid, structurallipid and PEGipidis38-46:44-52:8-12.

   310. The method of claim 309, wherein the molar ratio of the cationic lipid structural lipid, PEGipid and nucleic acid is about 42:48:10,

   310, The method of claim 308, wherein the weight ratio of total exipient to nucleic acid is less than about 15:1,

   311. The method of claim 310, wherein the weight ratio of total exipient to nucleic acid is about 10: 1.

   312. The method of claim 310, wherein the weight ratio of total exipient to nucleic acid is about 7.5:1.

   313. Themethod of claim 310; wherein the weight ratio of total exipient to nucleic acid is about 5^1,

   314. An association complex made y a method of any of claims 271-313.

   315. An association complex comprising a cationic lipid, a structural lipid,a PEG-lipid and a nucleic acid, wherein the cationic lipid is a lipid of one of the following or a mixture thereof;

   N H H

   o H

   H H 0 or

   o H' the.PEG- pid has the foi ng structure:

   thePG-lpidhsth~lloingtrucure H H

   the structuadlipid iscQholesterol.

   3116. The association complex of31,hrite iai isiRN.A,

   H ;and 317 71e msciation compl xof315 .wherein th catioi cpidihastie lb lowingfnua

   318, Themethod ofcamiwhriteoartofeaoelpd structural lipid, PEG-lipid and nucleic acid is 36-48:42-54:6-14,

   319, The method of claim 318 wherein themolar ratio of the atonic lipid, structural lipid, PEG-lpid and nucleic acid is 38-46:44-528-12.

   320, The method of claim 319, whereint he molar ratio of the cationic lipid structurallipid, PEG-lipid andnucleic acidis about 42:48:10.

   321, The method of claim 315, wherein the weight ratio of total exipient to nucleic acidis less than. about 15:1

   322 Themethod of claim 321, wherein the weight ratio of total excientto nucic acid is about 10:1L

   323. The method of claim 321, wherein the weight ratio of total excient to nucleicacid is about7.5:1.

   324. The method of claim 321, wherein the weightratio of total excient to nucie acid is about 5:1,

   325. A compound of fomnula (XV)

   (Xq

   fonnula (XV)

   each L' and 2 are independently a bond or C(0); a eachR and R2 are independentlyalkyl alkenyl or alkynyl; each of which is optionallysubstitutedwith one or more substituents; X is -C()N'l-, -C(S)NH-, -C()C 0 alkylC(O)NH-; or -C(O)C 3 akyC(O)O-; m is an integer from 0-1 and n is an integer from 1-500.

   326. The compound of claim 325;wherein Li and L are both a bond,

   327.The compound of claim 325, wherein Li and I are both C(O).

   328, The compound of claim 325, wherein each R and R2 are independently a alkyl. 329, The compmmd of caiWm 328, wherein each R1 and Raare independently C6gaG alkyl, eg,C-Cis aikyl, e.g, C1 4 alkyL

   330, Thecompound of claim 325, wherein both R and R2 are alkyl, e.g, straightchain alkyl having the same length, eg, Q-C 2 alkyleg,CwC alkyle.g., C alkyl orC 6 alkyl

   331. The compound of claim 330, wherein both RI and 2 are C4 alky

   332. The compound of claim 325, wherein formula XVreperesents a racemic Mixture

   333. The compound of claim 325,whereinformulaXV represents enantiomerically pure R isomer (e.g.a compound having an enmtiomeric excess of R isomer, e,g.,at least about 95% ee or greater than 97%ce, e.g, 98%, or 99%).

   334. The compound of claim 325, wherein formula XV represents enantiomericaly pure'S' isomer (e.g., a compound having an enantiomeric excessof R isomere,g, at least about 95% ce, or greater than 97% ee,e.g, 98%, or 99%),

   2 are independently 335. Thecompoundof claim 325,wherein eachR and alkenyl..1btexample, each R' and R2 are independently C6 -Cm alkeny or each R and R are the same alkenyl moiety,

   336 The compound of claim 335, wherein each R and R2 includes a single double bond, for example a single double bond in the E or Z configuration.

   337, The-compound of claim 335, whereineach R and R2 includes two double bondmoieties

   338 The compound of claim 325, wherein X is -C(O)NH-, providinga compound of farnula (XV) below:

   L2

   formala (XV'). 339, TIe compound of claim 325, wherein, X is -C()CjaIkyC(O)I-O

   340. The compound of claim 325, wherein m is an integer from 1-10, for example an integer from'2-4 or an integer 2,

   341, Thecompoundofcliaim325,wherein, n isan integerfrom 1-500, for example aninteger from 40-400, from 100-350, from 40-50 or from 42-47.

   342 The compound of claim 325, wherein thecompoundis compound of fomlmula (XV(), 0

   H H rn

   thbrmula (XV'), wherein both L and C are a bond.

   343. The compound of claim 342, wherein eachR andR2are independently alkyl, for example C-C 2 alkyl, e.g,Cw-CS alkyl, e.g., C1 alkyL.

   344. Thecompoundof claim 343 wherein, both W vmR are alkyl, e.g., straight chain alkyl having the same length, e.g., CCz, alkyl, e.g,Cj-C5 alkyl, e.g., C1 alkyl orC1 alkyl.

   345 Thecompound of claim 342, wherein m is an integer from 1-10, for example an integer from 2-4 or an integer 2

   346. The compound of claim 342, wherein, n is an integer from 1-500, for example an integer from 40-400, or from 40-50.

   347, The compound of claim 342, wherein, the compound is a conpund of formula (XV).whereinL'zand C are both bonds, R andi R arebAth alkyl (e.g.,CC alky, e.g,,C 1 -Cj alkyl, preferrably C 1alkyl), and n is an integer fromaout 40400.

   348. The compound of claim 325, wherein, the comound has a formula (XVI) below:

   0 01 0 H

   s formma (XVI), wherein the repeating PEG moiety has an average molecular weight of 2000 with n value between 42 and 47.

   349. The compound of claim 348, wherein the compound of formula XVI is a stereo isomerwith preferred absolute configuration R' (eg, having an enantiomeric L excess of R isomer such as 90%, 95%, 97%,98%, 99%),