METHOD FOR INTRODUCING ANTISENSE OLIGONUCLEOTIDES INTO EUCARYOTIC CELLS

Abstract: The present invention relates to a method for introducing one or more antisense oligonucleotides into one or more eucaryotic cells using one or more lipid formulations comprising one or more cationic lipids of Formula <i>I</i> and optionally at least one neutral lipid. In particular, the present invention relates to a method for introducing one or more antisense oligonucleotides into one or more eucaryotic cells using a lipid formulation comprising dimethyldioctadecylammonium bromide (DDAB) and at least one neutral lipid, especially dioleylphosphatidylethanolamine (DOPE). The invention also relates to kits for carrying out the invention, compositions for carrying out the invention, and compositions formed while carrying out the invention. Further, the present invention relates to a method for inhibiting or preventing cell growth or proliferation, and a method for inhibiting or preventing expression of one or more proteins.

Description: Method For Introducing Antisense Oligonucleotides Into Eucaryotic Cells Background of the Invention Field of the Invention The present invention relates to a method for introducing one or more antisense oligonucleotides into one or more eucaryotic cells using one or more lipid formulations comprising one or more cationic lipids of Formula / and optionally at least one neutral lipid. In particular, the present invention relates to a method for introducing one or more antisense oligonucleotides into one or more eucaryotic cells using a lipid formulation comprising dimethyldioctadecylammonium bromide (DDAB) and at least one neutral lipid, especially dioleylphosphatidv lethanolamine (DOPE). The invention also relates to kits for carrying out the invention, compositions for carrying out the invention, and compositions formed while carrying out the invention. Further, the present invention relates to a method for inhibiting or preventing cell growth or proliferation, and a method for inhibiting or preventing expression of one or more proteins. Related Art Antisense oligonucleotides have been described in the art as naturally occurring biological inhibitors of gene expression in both prokaryotes ( izuno el a/.. Proc. Xull. Avail. S i. i 'SA Λ7: 1966- 1970 ( 1 84)) and eukan otes (He wood, Xucleic Acids Res. 14:6771-6772 ( 1986)). and these sequences presumably function b\ ln bridi/ing to complementary mRNA sequences, resulting in hybridization arrest of translation (Paterson. l at . Proc. Xull. . laid. Sci. I 'SA, " -7:4370-4374 ( 1987)). Antisense oligonucleotides are short synthetic DNA or RNA nucleotide molecules formulated to be complementary to a specific gene or RNA message. 1 hrough the binding of these oligomers to a target DNA or mRNA sequence. transcription or translation oϊ the gene can be selectively blocked and the disease process generated b\ that gene can be halted (ΛIV. for example. Jack Cohen. Oligodeoxynucleotides, Antisense Inhibitors of Gene Expression. CRC Press ( 1989)). The cytoplasmic location of mRNA provides a target considered to be readih accessible to antisense oligodeoxynucleotides entering the cell; hence much of the work in the field has focused on RNA as a target. Currently, the use of antisense oligodeoxynucleotides provides a useful tool for exploring regulation of gene expression in vitro and in tissue culture (Rothenberg ct ul.. ./. Null. Cancer lust Λ7: 1539- 1544 ( 1989)). Antisense therapy is the administration of exogenous oligonucleotides which bind to a target polynucleotide located within the cells. For example, antisense oligonucleotides may be administered systemically for anticancer therapy (WO 90/09180). Antisense oligonucleotides are administered to a patient in order to inhibit the expression of the corresponding protein. U.S. Patent No. 5.279.833 describes a reagent for introducing nucleic acids into an animal cell. The reagent comprises a neutral lipid. such as dioleyl phosphatidylethanolamine (DOPE), and a cationic lipid. such as an ammonium salt of formula wherein R\ is a straight hv drocarbon chain of Cι to C| 8 that is saturated or unsaturated. R 2 , R 3 and R 4 are. independently of each other, hydrogen, a straight hydrocarbon chain of Cι-Cιχ that is saturated or unsaturated or an aryl, e.g.. benzv l or phenyk an A is an anion. The patent describes cetyldimethylethylammonium bromide and dimethyldioctadecylammonium bromide (DDAB) as preferred ammonium salts. Liu el ul.. J. Biol. hem. " 2: 1 1690- 1 1693 ( 1997) describe an antisense oligonucleotide treatment of drug-resistant human breast carcinoma (MCT-7'ADR) cells, wherein the antisense mixture was made b\ combining solution A containing 20 g nl I.lPOFECTACh'" in 0.25 ml of McCov ' s 5A medium without serum and solution B containing 400 nM of the antisense oligonucleotide in 0.25 ml of McCoy ' s 5A medium without serum. I.IPOFHCTACI:"' contains DDAB and DOPE in the ratio 1 :2.5, Howex er. the disclosed concentration of LIPOFKCT ACF;'" reagent (20 mg/ml) is impossible to achieve because of solubility problems. Further, Liu et al. state that the translections were performed according to the manufacturer ' s instructions. Contrary to this, LIPOFECTACE does not include instructions for antisense oligonucleotide transfection. L S. Patent No. 5,753,613 describes compositions for introducing a polyanionic material into a cell comprising a cationic compound of formula I H 3 C— (CH 2 ) n — Y— (CH 2 ) m - N + -R2 H 3 C— (CH 2 ) q — Z— (CH 2 ) p wherein R and R" are independently CV 3 alkyl and Y and Z are independently members selected from the group consisting of-CI CrTCFLCI FCTL-. ~CH=CHCH CH CH -, -CFLCH=C1 JCH 2 CH 2 -. -CH 2 CT-hCH=CHCFL-, -CHiCH 2 CH 2 CH=CH-, -CH=CHCH=CHCH 2 -, -CH=CH 2 CH 2 CH=CH-. and -CH 2 CH=CHCM=CH-, with the proviso that Y and Z are not both -CH 2 CH 2 CH 2 CH 2 CH 2 -; n and q are independently integers of from 3 to 7: and m and p are independently integers of from 4 to 9, with the proviso that the sums n+m and q+p are each integers of from 10 to 14 and X is an anion. U.S. Patent No. 5,753,613 describes that these compositions can be used, e.g., for introducing antisense oligonucleotides in the cells. It is further described that DDAB has a poor transfection efficiency. There is great potential for the use of antisense oligonucleotides to regulate gene expression. However, factors that often limit the efficacy of antisense oligonucleotides include inefficient cellular uptake, toxicity of the delivery agent, and non-specific effects seen with control oligonucleotides (Neckers. L.M.. Anli.scn.se Research and Applications. CRC Press ( 1 93) 451 and Giles. R.V.. Current Opinions in Molecular Therapeutics 2:238-252 (2000)). Thus, a need exists in the art for an efficient and non-toxic method for introducing antisense oligonucleotides into eucan tic cells. Summary of the Invention Applicants have discovered that lipid formulations comprising one or more cationic lipids of Formula / (below) are ideal for introducing one or more antisense oligonucleotides into eucaryotic cells. Applicants have found that when a lipid formulation comprising one or more cationic lipids of Formula / and optionally at least one neutral lipid is contacted with an antisense oligonucleotide, a stable complex is formed with the antisense oligonucleotide which permits efficient delivery of the antisense oligonucleotide into an eucaryotic cell. Further, introducing antisense oligonucleotides into eucaryotic cells using the above formulations can be accomplished without inducing cytotoxicity which is a serious problem in the field of antisense technology. Accordingly, the invention provides a method for introducing one or more antisense oligonucleotides into one or more eucaryotic cells, comprising (a) contacting said one or more antisense oligonucleotides with one or more lipid formulations comprising an effective amount of one or more cationic lipids of Formula / wherein R) is a straight or a branched hydrocarbon chain of Cm-mo that is saturated or unsaturated: R 2 is selected from the group consisting of a pair of electrons, hydrogen. alkyL alkenyl, alkynyl. heteroalkyl. heteroalkeny 1. heteroalkynyl, R 5 -NI-1C(0)-R ( ,. R -C(0)-0-R ( „ R . s-NH-C(OV-NH-R„. R 5 -NI I-C(SV-NI I-R„. R,-NH-C(NH)-NH-R„, alkyla inoalkyl. arylalkyl. arylalkeny 1. aryialk nyl. and ary 1. all of which can be optionally substituted; R and R 4 . independent!) of one another, are selected from the group consisting of hy drogen, alky 1. alkenyl. alkynyl, heteroalky l. heteroalkcnyl. heteroalkynyl, Rs-NHC(0)-R f) . R 5 -C(0)-0~R ( „ R -NH-QO NI l-R,,. R,-NH~C(S)-NH-R f) , R 5 -NH~C(NI 1)~NH~R,„ alky laminoalk l. arylalkyl. arylalkenyl, aryialkynyl, and ary I, all of which can be optionally substituted; wherein R 5 and R , are independently alkylene, alkenylene or alkyny ene; and A is a pharmaceutically acceptable anion when R 2 is not a pair of electrons; and optionally at least one neutral lipid to form one or more antisense oligonucleotide-lipid aggregate complexes, and (b) contacting said one or more cells with said one or more complexes. In a preferred aspect, Rj is a straight or a branched hydrocarbon chain of C i o- 3ϋ that is saturated or unsaturated. In another preferred aspect, when R 3 and R 4 in Formula / are C 1 - 3 alkyl, and one of R[ or R 2 is an unsaturated Cκ,. 2 o alky , the other one of Ri and R 2 is not an unsaturated or saturated C|(,. alkyl. In a further preferred aspect, the one or more eucaryotic cells are not drug- resistant human breast carcinoma cells. Also, the invention provides a method for introducing one or more antisense oligonucleotides into one or more eucaryotic cells, comprising (a) contacting said one or more antisense oligonucleotides with one or more lipid formulations comprising an effective amount of one or more cationic lipids of Formula // wherein Ri is a straight or a branched hy drocarbon chain of Cm-mn that is saturated or unsaturated: R is selected from the group consisting of a pair of electrons, lndrogen. alky l. alkenyl. alk\ l. heteroalkem l. heteroalkv in l. R<-NHC(0)-R,„ R 5 -C(0)-Q~R f „ R 5 _NH-C(O NH-R„. R 5 -NI I-C(S)-NH-R„. R 5 -NI-1-C(NH)-NH-R„, alky aminoalkyl. ary lalkyl. ary alkenyl, ary alky nyl, and aryl, all of which can be optionally substituted. w r herein R 5 and R,, are independently alkylene, alkenyiene or alkynylene; and A is a pharmaceutically acceptable anion when R is not a pair of electrons; and optionally at least one neutral lipid to form one or more antisense oligonucleotide-lipid aggregate complexes, and (b) contacting said one or more cells with said one or more complexes. In a preferred aspect, R| is a straight or a branched hydrocarbon chain of C 10 . 30 that is saturated or unsaturated. In another preferred aspect, when one of R] or R in Formula // is an unsaturated C)(,. 2 o alkyl, the other one is not an unsaturated or saturated C " i6-:o alkyl. In particular, the invention provides a method for introducing one or more antisense oligonucleotides into one or more eucaryotic cells, comprising (a) contacting said one or more antisense oligonucleotides with a lipid formulation comprising an effective amount of dimethyldioctadecylammonium bromide (DDAB) and at least one neutral lipid to form one or more antisense oligonucleotide-lipid aggregate complexes , and (b) contacting said one or more cells with said one or more complexes. The invention also concerns a kit, wherein the kit is preferably used for introducing one or more oligonucleotides into one or more eucaryotic cells, such kit preferably comprising at least one component selected from the group consisting of one or more cells, one or more antisense oligonucleotides. one or more lipid formulations of the invention, one or more buffering salts, one more culture media, and one or more transfection enhancers. The invention also relates to a composition for carrying out the method of the present invention, and the composition formed while carry ing out the invention. Such compositions may comprise at least one component selected from the group consisting of one or more cells, one or more antisense oligonucleotides. one or more lipid formulations of the invention, one or more buffering salts, one more culture media, and one or more transfection enhancers. Further, the invention provides a method for inhibiting or preventing cell growth or proliferation, comprising (a) contacting one or more eucaryotic cells with one or more antisense oligonucleotides and an effective amount of one or more lipid formulations comprising an effective amount of one or more cationic lipids of Formula / and optionally at least one neutral lipid to provide a composition; and (b) incubating said composition under conditions sufficient to inhibit or prevent cell growth or proliferation. Furthermore, the invention provides a method for inhibiting or preventing expression of one or more proteins, comprising (a) contacting one or more eucaryotic cells with one or more antisense oligonucleotides and an effective amount of one or more lipid formulations comprising an effective amount of one or more cationic lipids of Formula / and optionally at least one neutral lipid to provide a composition; and (b) incubating said composition under conditions sufficient to inhibit or prevent said expression of one or more proteins. Additional embodiments and advantages of the invention will be set forth in part in the description as follows, and in part will be obvious from the description, or may be learned by practice of the invention. The embodiments and advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and not restrictive of the invention, as claimed. Brief Description of the Figures FIG. 1 is a graph showing the inhibition of proliferation TRO/anϋ-c-mvc complexes in different cell lines. The black column represents the untreated sample. The white column represents cells that received only lipid and no oligonucleotide. The hatched column represents cells that received the scrambled control. The speckled column represents cells that received antisense oligonucleotide. FIG. 2 compares the ability of various transfection reagents to mediate functional oligonucleotide transfection. The black column represents untreated sample. fhe white column represents cells that received only lipid and no oligonucleotide. The hatched column represents cells that received the scrambled control. The speckled column represents cells that received antisense oligonucleotide. FIG. 3 depicts an i munoblot analysis of c-Raf protein expression in PleLa cells treated with antisense (AS) or mismatched (MM) oligonucleotides in comparison to untreated controls. Lane 1 is a cell extract from untreated HeLa cells. Lane 2 is a cell extract that received TRO but no ODN. Lane 3 is a cell extract that received the TRO/antisense ODN to c-raf complex and Lane 4 is the TRO/mismatch control ODN complex. Detailed Description of the Preferred Embodiments Applicants have surprisingly diseoλ ered an efficient and non-toxic method for introducing antisense oligonucleotides into eucaryotic cells. Accordingly, the invention relates to a method for introducing one or more antisense oligonucleotides into one or more eucaryotic cells, comprising (a) contacting aid one or more antisense oligonucleotides with one or more lipid formulations comprising one or more cationic lipids of Formula / wherein Ri is a straight or a branched hydrocarbon chain of Cm- that is saturated or unsaturated; R 2 is selected from the group consisting of a pair of electrons, hydrogen, alkyl. alkenyl. alkynyl, heteroalkyi, heteroalkenyi, heteroalkynyl, aryialkyl, R -NHC(0)-R () , R 5 -C(0)-0-R 6 , R 5 -NH-C(0)-NH-R 6 . R 5 -NH-C(S)-NH~R 6 . R 5 -NH-C(NH)-NFI-R ( „ alkylaminoalkyl, arylalkenyi, aryialkynyl. and aryi, all of which can be optionally substituted; R 3 and R 4 , independently of one another, are selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl. heteroalkyi, heteroalkenyi. heteroalkynyl, R 5 -NHC(Q)-R 6 , R 5 -C(0)-0-R ( „ R 5 -NH-C(0)-NH-R,„ R 5 -NH-C(S)-NH-R„, R 5 -NH-C(NH)-NI I-R„, alkylaminoalkyl. aryialkyl, arylalkenyi, aryialkynyl, and aryi, all of which may be optionally substituted, wherein R 5 and R ( , are independently alkylene, alkenyiene or alkynylene; and A is a pharmaceutically acceptable anion when R 2 is not a pair of electrons: and optionally at least one neutral lipid to form one or more antisense oligonucleotide-lipid aggregate complexes, and (b) contacting said one or more cells with said one or more complexes. Preferably, when R 3 and R 4 in Formula I are C|. 3 alky l. and one of R] or R is an unsaturated C| ( ,. 2 o alkyl. the other one of Ri and R 2 is not an unsaturated or saturated C|f,. 2 ø alkyl. Preferably, the one or more cells are not drug-resistant human breast carcinoma cells. Preferably 1-5 antisense oligonucleotides. more preferably 1 - 3 antisense oligonucleotides. especially one antisense oligonucleotide, are contacted w ith one or more lipid formulations. Preferably. Ri is a straight or a branched hydrocarbon chain of Cm. . ™ that is saturated or unsaturated. Preferably . R| is a straight hydrocarbon chain of C| 2 . 24 that is saturated or unsaturated: and R . R 3 and R4 are independently selected from the group consisting of hydrogen. C|. 2 o alkyl. C - 2 alkeny l, C 2 _ 2 n alkynyl. C.). o heteroalkyi, C 4 . n heteroalkenyi, C 4 . 2 o heteroalkynyl. C 12 aryi(C). o) alky l and C 12 aryi. all of which can be optionally substituted. More preferably . R ( is a straight hydrocarbon chain of C ' 14 . 2 11 that is saturated or unsaturated: R 2 is selected from the group consisting of hydrogen. C i alkyl. C,,.ιχ alkenyl. G,-tχ alkynyl. CVix heteroalkyi, CYix heteroalkenyi, CV I H heteroalkynyl, phenyi(Cr,.ιχ)alkyl, and phenyl: and R 3 and R4 are independently selected from the group consisting of hydrogen, C1- 5 alkyl, C .(, alkenyl, C - f , alkynyl, C 2 _ heteroalkyi, C 2 - 5 heteroalkenyi. C 2 -s heteroalkynyl, phenyi(C|. 5 )alkyi, especially benzyl, and phenyl, all of which can be optionally substituted. A useful group of cationic lipids of Formula / include those wherein Rj and R 2 are both C 10 - 0 saturated alkyl groups. Useful cationic lipids in the present invention included in Formula / are cationic lipids of Formula // wherein Ri is a straight or a branched hydrocarbon chain of C 1 0- 100 th t is saturated or unsaturated: R is selected from the group consisting of a pair of electrons, hydrogen, alkyl, alkenyl. alkynyl, heteroalkyi. heteroalkenyi, heteroalkynyl. R 5 -NHC(0)-R(„ R 5 -C(0)-0-R„, R 5 ^NH-C(0)-NH-R,„ R 5 -NH-C(SVNH~R<,. R ? -NH-C(NH)-NH-R(„ alkylaminoalkyl, ary ialkyl. arylalkenyi. aryialkynyl. and aryi. all of which can be optionally substituted, wherein R and R fl are independently alkyienc. alkenyiene or alkynyiene: and A is a pharmaceutically acceptable anion when R 2 is not a pair of electrons. Preferably, when one of Ri or R 2 in Formula // is an unsaturated Cif,- 2 u alky l. the other one is not an unsaturated or saturated C - 20 alky l. Preferably. Ri in Formula // is a straight or a branched hydrocarbon chain of C ' - that is saturated or unsaturated. Preferably . R] in Formula // is a straight hy drocarbon chain of C12-M that is saturated or unsaturated; and R 2 is selected from the group consisting of hydrogen. C ' 1. 2 0 alkyl, C2-21) alkenyl, C ' 2 -20 alkynyl, C ' 4-20 heteroalkyi, C4- 20 heteroalkenyi, C4-20 heteroalkynyl, C12 aryi(Cι- 2 n) alky l and C 1,-12 aryi. all which can be optionally substituted. More preferably. Ri is a straight hydrocarbon chain of C 14 - 20 that is saturated, and R 2 is selected from the group consisting of CVix alkyl, C ( ,.ιχ heteroalkyi. C ( ,.ιχ heteroalkenyi. C,,-ιχ heteroalkynyl, and phenyi(C(,-ιχ)alkyi, all of which can be optionally substituted. A is any pharmaceutically acceptable anion. These anions can be organic or inorganic. A is preferably a halogen, that is Br " . C\ ~ , F " , , or A is a sulfate, a nitrite or a nitrite. Preferably the cationic lipid of Formula / is dimethyldioctadecylammonium bromide (DDAB). Preferably, the lipid formulation contains at least one neutral lipid. Examples of neutral lipids which can be used in the present formulations are, for example, diacyiphosphatidyicholine, diacyiphosphatidyiethanolamine, ceramide, sphingomyelin, phosphatidic acid, and cholesterol. Preferably, the present formulations contain at least one neutral lipid selected from the group consisting of diacyiphosphatidyicholine, such as dioleyphosphatidyicholine, dipalmitoylphosphatidyieholine. palmitoyioleyiphosphatidyicholine, lecithin and lysolecithin. diacyiphosphatidyiethanolamine, ceramide, sphingomyelin, and cholesterol. More preferably, the neutral lipid is a diac iphosphatidyiethanolamine having 10-24 carbon atoms in the acyl group. More preferably the acyl groups are lauroyi, myristoyi, heptadecanoyi. palmitoyi, stearoyi or oleyi. Especially, the neutral lipid is dioleyiphosphatidyiethanolamine (DOPE), palmitoyloleyiphosphatidyi- ethanolamine, diheptadecanoyiphosphatidyiethanolamine. dilauroyiphosphatidyi- ethanolamine, dimyristoyiphosphatidyiethanolamine. distearoy lphosphatidyi- ethanolamine. beta-linoleyi-gamma-palmitoyiphosphatidyiethanolamine. and beta- oleyi-gamma-palmitoyiphosphatidy lethanolamine, specifically dioley lphosphatidyi- ethanolamine (DOPE). ' The ratio of the cationic lipid of Formula / or // to a neutral lipid can be widely varied depending on the particular cationic lipid employed. For example, the ratio can be from about 1 : 10 to about 1 : 1. preferably from about 1 :5 to about 1 :2.5. The ratio of antisense oligonucleotides to cationic lipids of Formula / or // should not be so high as to saturate the positive charges on the lipid aggregates, which may result in a lack of binding of the lipid aggregates to the cell surface. The lipid formulation containing one or more cationic lipids of Formula / and optionally at least one neutral lipid can be present in an amount of about 0.1 μg/ml-5 mg/ml when the antisense oligonucleotide is contacted with the lipid formulation. Preferably , the lipid formulation is present in an amount of 0.15 μg/ml-4.5 mg/ml, more preferably 0.15 μg/ml-4.2 mg/ml, more preferably 0.15 μg/ml-4.0 mg'ml, more preferably 0.2 μg/ml-3J mg/ml, more preferably 0.2 μg/ml-3,5 mg/ml. more preferably 0.2 μg/ml-3.2 mg/ml, more preferably 0.25 μg/ml-3.0 mg/ml, more preferably 0.25 μg/ml-2.8 mg/ml, more preferably 0.25 μg/ml -2.5 mg/ml, more preferably 0.25 μg/ml-2.3 mg/ml, more preferably 0.3 μg/ml-2.0 mg/ml. more preferably 0.3 μg/ml- 1.8 mg/ml. more preferably 0.3 μg/ml- 1 .6 mg/ml, more preferably 0.3 μg/ml- 1.4 mg/ml, 0.3 μg/ml- 1.1 mg/ml, more preferably 0.35 μg/ml- 0.8 mg/ml, more preferably 0.35 μg/ml-0.5 mg/ml. 0.35 μg/ml-0.3 mg/ml, more preferably 0.35 μg/ml-0.1 mg/ml, more preferably 0.35-90 μg/ml, more preferably 0.35-75 μg/ml, more preferably 0.35-60 μg/ml. more preferably 0.35-45 μg/ml, more preferably 0.35-30 μg/ml, more preferably 0.35-20 μg/ml, more preferably 0.35-14 μg/ml, more preferably OJ-14 μg/ml, more preferably about 1 -14 μg/ml, more preferably about 2-13 μg/ml, more preferably about 3-13 μg/ml, more preferably about 4-12 μg/ml. especially about 4.5- 12 μg/ml. In a preferred embodiment, the ind ention relates to a method for introducing one or more antisense oligonucleotides into one or more eucaryotic cells, comprising (a) contacting said one or more antisense oligonucleotides with a lipid formulation comprising an effective amount of dimethyidioctadccyiammonium bromide (DDAB) and at least one neutral lipid to form one or more antisense oligonucleotide-lipid aggregate complexes, and (b) contacting said one or more cells with said one or more complexes. Preferably, the neutral lipid is diacy iphosphatidy iethanolamine hax ing 10-24 carbon atoms in the acyl group, more preferably dioley lphosphatidy lethanolamine (DOPE). Preferably, the ratio of DDAB:DOPE in the present method is from about 1 :5 to about 1 : 1 , more preferably 1 :2.5. Preferably, the final concentration of the lipid formulation comprising DDAB and DOPE in the ratio of 1 :2.5 is 5.6-1 1.2 μg/ml. The present invention also relates to a kit, wherein the kit is preferably used for introducing one or more oligonucleotides into one or more eucaryotic cells. Such kit preferably comprises at least one component selected from the group consisting of one or more cells, one or more antisense oligonucleotides, one or more lipid formulations of the invention, one or more buffering salts, one more culture media, and one or more transfection enhancers. More preferably, such kit comprises one or more lipid formulations comprising an effective amount of one or more cationic lipids of Formula / and optionally at least one neutral lipid. and at least one additional component selected from the group consisting of one or more cells, one or more antisense oligonucleotides, one or more buffering salts, one or more culture media, and one or more transfection enhancers. Such kit may further include one or more cell-targeting enhancers, uptake enhancers, internalization enhancers, nuclear targeting enhancers and expression enhancers. The invention also relates to a composition for carrying out the method of the present invention, and the composition formed while carrying out the invention. Such compositions may comprise at least one component selected from the group consisting of one or more cells, one or more antisense oligonucleotides. one or more lipid formulations of the invention, one or more buffering salts, one more culture media, and one or more transfection enhancers. Preferably, such compositions comprise one or more lipid formulations comprising an effective amount of one or more cationic lipids of Formula / and optionally at least one neutral lipid. and one or more additional components selected from the group consisting of one or more cells, one or more antisense oligonucleotides. one or more buffering salts, one or more culture media, and one or more transfection enhancers. Such compositions may further include one or more cell-targeting enhancers, uptake enhancers, internalization enhancers, nuclear targeting enhancers and expression enhancers. Further, the invention relates to a method for inhibiting or cnting cell growth or proliferation, comprising (a) contacting one or more eucaryotic cells with one or more antisense oligonucleotides and an effective amount of one or more lipid formulations comprising an effective amount of one or more cationic lipids of Formula / and optionally at least one neutral lipid to provide a composition; and (b) incubating said composition under conditions sufficient to inhibit or prevent cell growth or proliferation. Furthermore, the invention relates to a method for inhibiting or preventing expression of one or more proteins, comprising (a) contacting one or more eucaryotic cells with one or more antisense oligonucleotides and an effective amount of one or more lipid formulations comprising an effective amount of one or more cationic lipids of Formula / and optionally at least one neutral lipid to provide a composition; and (b) incubating said composition under conditions sufficient to inhibit or prevent said expression of one or more proteins. Some compounds of Formula /, such as DDAB. are commercially available. Compounds of Formula / can be prepared by methods known to those of skill in the art using standard synthetic reactions (see March. Advanced Organic Chemistry. 4 n Ed.. Wiley-Interscienee, New York. N.Y. ( 1992)). For example, compounds of Formula /, wherein R1-R 4 are the same or different, can be prepared treating a Cm-ioo amine. preferably a C 1 0 - 30 amine, with formaldehyde and sodium cyanoborohydride under conditions that result in the reductive alkyiation of the amine to provide a tertiary amine which further is reacted with. e.g.. an optionally substituted alkyl bromide to provide a quaternary ammonium salt. Further, compounds of Formula / can be prepared by converting a fatty acid to its corresponding acid chloride with, e.g.. oxalyi chloride, thionyi chloride, p-TsCL PCI . ; or PCI 5 . and reacting the acid chloride with an optionally substituted amine to provide a corresponding amide. Reduction oϊ the amide with, e.g., lithium aluminium hydride provides a secondary amine. ' he secondary amine is further treated with optionally substituted alkyl halides to prox ide the quaternary ammonium salt. Anion exchange can then be carried to out to provide cationic lipids ing the desired pharmaceutically acceptable anion. Certain of the cationic lipids of Formula / may be insufficiently soluble in physiological media to employ for the method of the present invention. Those of ordinary skill in the art will appreciate that there are a variety of techniques available in the art to enhance solubility of such compounds in aqueous media, such as using ethanol as a co-solvent. Such methods are readily applicable without undue experimentation to the compounds described herein. In the method of the present invention, one or more cationic lipids of Formula / are used in combination with optionally at least one neutral lipid to prepare liposomes, micelles and other lipid aggregates suitable for introducing antisense oligonucleotides into target cells, either in vitro or /; vivo. Such lipid aggregates are polycationic, and are able to form stable complexes with antisense oligonucleotides. The lipid aggregate oligonucleotide complex interacts with cells making the antisense oligonucleotide available for absorption and uptake by the cell. Liposomes and micelles containing one or more cationic lipids of Formula / and optionally at least one neutral lipid can be prepared by methods well known in the art. The selection of neutral lipids is generally guided by consideration of. e.g.. liposome size and stability of the liposomes in the bloodstream. Liposomes can be generally formed by sonicating a lipid in an aqueous medium, by resuspension of dried lipid layers in a buffer or by dialysis of lipids dissolved in an organic solvent against a buffer of choice. Another method of liposome preparation is utilizing microfluidization. In this process, one or more cationic lipids of Formula / and optionally at least one neutral lipid are mixed in an organic solvent, such as chloroform, " fhe organic solvent is removed by evaporation to leave a lipid film. The lipid film is hydrated with water and past through a microfluidizer. By selecting fhe appropriate ratio, various sizes of liposomes can be prepared. For example, liposomes can be prepared as described in Szoka ft ai. Ann. Rev. Biophy.s. Bioeng. 9X67 ( 1980), U.S. Patent Nos. 4,235.871. 4.501 .728. and 4.837.028, the text Liposomes. Marc J. Ostro, ed.. Marcel Dekker. Inc.. New York. 1983. Chapter 1. and Hope el a/.. Chem. Phy . Lip. -70:89 ( 1986). Following liposome preparation, the liposomes may be sized to achieve a desired range and relatix ely narrow distribution of liposome sizes. Several techniques are av ailable for si/.ine liposomes to a desired size. One sizing method is described. for example, in U.S. Patent No. 4,737,323. Liposomes typically range in diameter from 250 angstrom units to several micrometers (the diameter of a red blood cell is roughly 10 micrometers) and are usually suspended in solution. They have two standard forms: "Onion-skimmed" multilamellar vesicles (MLV ' s), made up of several lipid bilayers separated by fluid, and unilamellar vesicles, consisting of single bilayer surrounding an entirely fluid core. The unilamellar vesicles are typically characterized as being small (SUV's) or large (LUV's). Under appropriate circumstances liposomes can absorb to almost any cell type. Once they have been adsorbed, liposomes may be endocytosed, or swallowed up. by some cells. Adsorbed liposomes can also exchange lipids with cell membranes and may at times be able to fuse with cells. When fusion takes place, the liposomal membrane is integrated into the cell membrane and the aqueous contents of the liposome merge with the fluid in the cell. Endocytosis of liposomes occurs in a limited class of cells; those that are phagocytic, or able to ingest foreign particles. When phagoeytic cells take up liposomes, the cells move the spheres into subcellular organelles known as lysosomes, where the liposomal membranes are thought to be degraded. From the lysosome, the liposomal lipid components migrate outward to become part of the cell ' s membranes and other liposomal components that resist lysosomal degradation (such as certain medications) may enter the cytoplasm. Lipid exchange involves the transfer of individual lipid molecules from the liposome into the plasma membrane (and vice versa). With lipid exchange, the aqueous contents of the liposome do not enter the cell. For lipid exchange to take place, the liposomal lipid must have a particular chemistry in relation to the target cell. Once a liposomal lipid joins the cell membrane it can either remain in the membrane for a long time or be redistributed to a v ariety of intracellular membranes. In very dilute solutions, lipid micelles may form instead of liposomes. In the methods of the present invention, the cationic lipids of Formula / may further be conjugated to or mixed with or used in conjunction with a variety of useful molecules and substances such as proteins, peptides. growth factors and the like to enhance cell-targeting, uptake, internalization. nuclear targeting and expression. See. for example. U.S. Patent Nos. 5.521.291. 5.547.932 and 5,693.509. The method of the present invention can be applied to in vitro and in vivo transfection of eucaryotic cells or tissues including animal cells, human cells, insect cells, avian cells, fish cells, mammalian cells and the like. The method oi ' this invention is useful in any therapeutic method requiring introducing of oligonucleotides into cells or tissues. In the method of the present invention, one or more antisense oligonucleotides are first contacted with one or more lipid formulations comprising an efficient amount of one or more cationic lipids of Formula / and optionally at least one neutral lipid to provide one or more antisense oligonucleotide-lipid aggregate complexes. For example, the contact can be made prior to the aggregate formation (from the cationic and neutral lipids) or subsequent to an initial lipid aggregate formation. In a preferred embodiment, the lipid aggregates of the cationic lipids and optional neutral lipids are formed first, then brought into contact with one or more antisense oligonucleotides. The antisense oligonucleotide will typically bind to the surface of the lipid aggregate as a result of the ionic attraction between the negatively charged antisense oligonucleotide and the positively charged surface of the lipid aggregate. Typically, the contact between the antisense oligonucleotide and the lipid aggregate that results in formation of a complex will be carried out at temperatures of from about 15 °C to about 45 °C, preferably at room temperature. The length of time required to complete the formation of a complex will depend on the temperature as well as the nature of the antisense oligonucleotide and the lipid aggregate itself. When contact temperatures of about room temperature are used, the length of time to form a complex will be about 15 minutes to about 1 hour. Alternativ ely, the antisense oligonucleotide can be incorporated into the interior of liposomes prepared from the cationic lipids and optional neutral lipids of the invention by methods known to those of skill in the art. One method may involve encapsulation and can be carried out by a variety of techniques. Following formation of antisense oligonucleotide-lipid aggregate complexes, the complexes are contacted with the cells to be transfected. Once adsorbed, the lipid aggregates, including the complexes, can either be cndocytosed by a portion of cells. exchange lipids with the cell membranes or fuse with the cells as described above. Transfer or incorporation of the oligonucleotide part of the complex can take place via one of the abov e mentioned pathways. In particular, when a liposomal fusion takes place. the liposomal membrane and the antisense oligonucleotide-lipid aggregate complex combine with the intracellular fluid. Contact between the cells and the antisense oligonucleotide-lipid aggregate complexes, when carried out in vitro, will take place in a biologically compatible medium. The concentration of lipid can vary widely. Treatment of the cells with the antisense oligonucleotide-lipid aggregate complexes will generally be carried out at physiological temperatures (about 37 °C) for periods of time of from 1 to about 6 hours, preferably from 2 to 4 hours. For / ' /; vitro applications, the delivery of antisense oligonucleotides can be to any eucaryotic cell grown in culture. The cells are preferably mammalian cells, more preferably human cells. Definitions Useful alkyl groups include straight-chained and branched CVix alkyl groups, preferably C M O alkyl groups, more preferably C1. 5 alkyl groups, i ' ypical ix alkyl groups include methyl, ethyl, propyi, isopropyi, butyl, .vff-butyi, ttrt-butyl, 3-pentyi, hexyl, octyl, decyl, dodeeyi, tetradecyl, hexadecyl and octadecyi groups. Useful alkenyl groups are C 2 -1 8 alkenyl groups, preferably CV 10 alkenyl, more preferably C 2 - 6 alkenyl groups. Typical CVix alkenyl groups include ethenyi, propenyi. isopropenyl. butenyl. Λt-r-butenyi, hexenyi, octeneyi, decenyl, dodecenyi, tetradecenyl. especially 9-tetradecenyi, hexadecenyl. especially 9-hexadecenyi, and octadeeenyi. especially 9-octadecenyl, groups. Useful alkynyl groups are CVix alkynyl groups, preferably C 2 - 10 alkynyl, more preferably CVo alkynyl groups. Typical CVix alkynyl groups include ethynyi. propy nyi, butynyi, 2-butynyi, hexynyi. octynyi, decyny l. dodecynyi. tetradecynyi. hexadecyny l. and octadecy nyi groups. Typical heteroalkyi groups include any of the above-mentioned CVix alkyl groups having one or more Ci I 2 groups replaced with O or S. ' I ' ypical heteroalkenyi groups include any oϊ the above-mentioned CVis alkenyl groups hav ing one or more GIF groups replaced with 0 or S. i ' ypical heteroalkyny l groups include any oϊ the above-mentioned CVix alkynyl groups having one or more Ci f groups replaced with 0 or S. Typically alkylaminoalkyl groups are R -NH-Rx, wherein R? and R^ are alkyiene groups as defined above. Useful aryi groups are CVπ aryi, especially CVio aryi. i ' ypical C i4 aryi groups include phenyl. naphthyi, phenanthryl, anthracyi, indenyl, azulenyi. biphenyi, biphenyienyi and lluorenyl groups. Useful aryialkyl groups include any of the above-mentioned Cι_ι 8 alkyl groups substituted by any oϊ the above-mentioned C f ,.i4 aryi groups. Useful values include benzyl, phenethyl and naphthyimethyi. Useful arylalkenyi groups include any of the above-mentioned CVix alkenyl groups substituted by any of the above-mentioned CVu aryi groups. Useful aryialkynyl groups include any of the above-mentioned C 2 -ιx alkynyl groups substituted by any of the above-mentioned C 6 -u aryi groups. Useful values include phenyiethynyi and phenyipropynyi. Useful halo or halogen groups include fluorine, chlorine, bromine and iodine. Useful haloalkyi groups include CVio alkyl groups substituted by one or more fluorine, chlorine, bromine or iodine atoms, e.g. fluoromethyi, difluoromethyi, trifluoromethyi, pentafluoroethyl, 1 , 1 -difluoroefhyl and trichloroinefhyi groups. Useful hydroxyalkyi groups include CVio alkyl groups substituted by hydroxy, e.g. hydroxymethyi, hydroxyethyl. hydroxypropyi and hydroxybutyi groups. Useful alkoxy groups include oxygen substituted by one of the CVio alkyl groups mentioned above. Useful alkylthio groups include sulfur substituted by one of the CVio alkyl groups mentioned above. Useful aeyiamino groups are any acyl group, particularly CV, alkano i or C b .ιo aryi(C 2 -ι,)alkanoyi attached to an amino nitrogen, e.g. acetamido. propionamido. butanoyiamido, pentanoyiamido. hexanoyiamido, and benzoyi. Useful acyio.xy groups are any CVι» acy l (alkanoyi) attached to an oxy (-0-) group, e.g. acetoxy. propionoy loxy. butanovloxv. pentanoyloxy. hexanoyioxy and the like. Useful alkyiamino and dialkyiamino groups are — NHRu and — RoRm. wherein Ro and Ri are CVio alkyl groups. Λminocarbonyi group is — - C ' l ONI f. Useful alkylthiol groups include any of the above-mentioned mentioned CV alkyl groups substituted by a ■ — I-I group. A carboxy group is -COOI I. An ureido group is -NH- OVNI-f- An amino group is -NH 2 . Optional substituents on R], R 2 . R and R 4 include any one of halogen, halo(CVo) alkyl. Cu, alkyl, CV<, alkenyl, C 2 . f , alkynyl, amino (Cι-(,)alkyi, carboxy(CV())alkyi, alkoxy(CV 6 )alkyl, nitro, amino, ureido, acylamino. hydroxy, thiol. acyioxy, alko.xy, carboxy, aminocarbonyi. and CV( > alkylthiol groups mentioned above. Preferred optional substituents include: hydro.xy(Ci- ( ,)alkyl, amino(Ci. () )alkyl. hydroxy, carboxy. nitro. . b alkyl, alkoxy, thiol and amino. Pharmaceutically acceptable anion. Anions of inorganic or organic acids that provide non-toxic salts in pharmaceutical preparations. Antisense Oligonucleotide. An antisense oligonucleotide is a DNA or RNA molecule or a derivative of a DNA or RNA molecule containing a nucleotide sequence which is complementary to that of a specific mRNA. An antisense oligonucleotide binds to the complementary sequence in a specific mRNA and inhibits or prevents translation of the mRNA. There are many known derivatives of such DNA and RNA molecules. See. for example, U.S. Patent Nos. 6,031.086, 5,929,226, 5,886,165, 5.693,773, 6.054.439, 5,919,772, 5,985,558, 5,595,096, 5.916,807, 5,885.970, 5,877.309. 5,681.944, 5.602,240. 5,596,091 , 5.506.212, 5.521.302. 5.541.307, 5.510,476. 5,514.787. 5,543.507, 5,512,438, 5,510,239, 5.514.577. 5,519.134, 5.554.746. 5.276.019, 5,286.717, 5,264,423. as well as WO96/35706, W096/32474. W096/29337 (thiono triester modified antisense oligodeoxynucleotide phosphorothioates). WO94/17093 (oligonucleotide alkyiphosphonates and alkylphosphothioates). WO94/08004 (oligonucleotide phosphothioates. methyl phosphates, phosphoramidates. dithioates, bridged phosphorothioates, bridge phosphoramidates. sulfones. sul fates, ketos. phosphate esters and phosphorobutyiamines (v an der fvrol ft al. Biotech. 6:958-976 ( 1988): Uhlmann ci al, Chem. Rev W;542-585 ( 1990)). W094O2499 (oligonucleotide alky lphosphonothioates and arylphosphonothioates). and WO92/20697 (3'-end capped oligonucleotides). Further, useful antisense oligonucleotides include derivatives such as S-oligonucleotides (phosphorothioate derivatives or S-oligos, see. Jack Cohen, Oligodeoxynucleotides. Antisense Inhibitors of Gene Expression, CRC Press (1989) which can be prepared, e.g. , as described by Iyer et al.(J. Org. Chem. 55:4693-4698 (1990) and J Am. Chem. Soc. 7/2: 1253-1254 (1990)). Complementary DNA (eDNA). A "complementary DNA," or "cDNA" gene includes recombinant genes synthesized by reverse transcription of mRNA and from which intervening sequences (introns) have been removed. Eucaryotic Cell. Eukaryotic cells can be of any type and from any source. Types of eukaryotic cells include epithelial, libroblastic, neuronal. hematopoietic cells and the like from primary cells, tumor cells or immortalized cell lines. Sources of such cells include any animal such as human, canine, mouse, hamster, cat, bovine, porcine, monkey, ape, sheep, fish, insect, fungus and any plant including crop plants, ornamentals and trees. Delivery is used to denote a process by which a desired compound is transferred to a target cell such that the desired compound is ultimately located inside the target cell or in. or on, the target cell membrane. In many uses of the compounds of the invention, the desired compound is not readily taken up by the target cell and delivery via lipid aggregates is a means for getting the desired compound into the cell. In certain uses, especially under in vivo conditions, delivery to a specific target cell type is preferable and can be facilitated by compounds of the invention. Lipid Aggregate is a generic term which includes liposomes of all types both unilamellar and multilameller as well as micelles and more amorphous aggregates of cationic lipids mixed with neutral lipids. Target Cell refers to any cell to which a desired compound is delivered, using a lipid aggregate as carrier for the desired compound. Introducing is intended to include, e.g., transfecting, transforming, and delivering. Transfection. Transfection is used herein to mean the delivery of an antisense oligonucleotide to a target cell, such that the antisense oligonucleotide is expressed or has a biological function in the cell. The term "expression " means any manifestation of the functional presence of the nucleic acid within the cell including, without limitation, both transient expression and stable expression. Functional aspects include inhibition of expression by oligonucleotides or protein delivery. Kit refers to transfection or protein expression kits. Such kits are preferably used for introducing one or more oligonucleotides into one or more eucaryotic cells. Such kits preferably comprise at least one compound selected from the group consisting of one or more cells, one or more antisense oligonucleotides. one or more lipid formulations of the invention, one or more buffering salts, one more culture media, one or more transfection enhancers, etc. Such kits may comprise a carrying means being compartmentalized to receive in close confinement one or more container means such as vials, test tubes and the like. Each of such container means comprises components or a mixture of components needed to perform transfection. The invention will be further clarified by the following examples, which are intended to be purely exemplary of the invention. All reagents and media used in the examples were from Invitrogen Corporation, Life Technologies Division (Rockville, MD) unless otherwise stated. EXAMPLES Synthesis of Oligonucleotides Synthesis and high-performance liquid chromatography (HPLC) purification of antisense phosphorothioate oligonucleotide (S-ODN) 5 * -AACGTTGAGGGGCAT- 3 ' (SEQ ID NO: l ) complementary to the initiation codon of human c-myc mRNA and a scrambled phosphorothioate oligonucleotide containing the same base composition in random order 5 ' -GAΛCGGAGACGGTTT-3 " (SEQ ID NO:2) were performed as described by Wickstrom el al. (Proc. Xatl. Acad Sci. C.S.Λ. 65: 1028- 1032 ( 1988) and Cancer Res. 52:6741 -6745 ( 1 92)). Synthesis and high-performance liquid chromatography (HPLC) purification of antisense phosphorothioate oligonucleotide 5 " -T CCCGCCTGTGACATGCAi T-3 " (SEQ ID NO:3) complementary to the initiation codon of human -raf . and a 7 bp mismatch phosphorothioate oligonucleotide 5 ' -TCCCGCGCACTTGATGCΛi " f-3 " (SEQ ID NO:4) were performed as described by Monia el al. (Proc. Nail. Acad. Sci. l.'S.A. 93: 15481 - 15484 ( 1996)). Cell Cultures All cell lines were maintained at subconfluent levels and below passage 20 in a humidified incubator with a 5° o CO 2 atmosphere at 37 °C for all experiments described. For transfection, cells were seeded onto 96-well microplates at specific plating densities (HeLa & HeLaS3: 2000 cells/well, HEK293: 3000 cells/well, CHOKl & CHO-S: 1000 cells/well. 562; 1200 cells/well) in serum-containing medium. Adherent cells were seeded 24 hours before transfection and suspension cells were seeded 4 hours before translection. Except for FleLa cells, all cells were then washed one time with serum-free growth medium and then treated for 4 hours in serum-free growth medium or with mixtures containing the tested transfection reagents and oligonucleotides. After 4 hours the appropriate grovvth medium containing 3X serum was added to the cells. HeLa cells were grown in high-glucose Dulbecco's-modified Eagle ' s medium (DMEM: 4500 mg/L glucose, 862 mg/L L-alanyi-L-glutamine. 1 10 mg/L sodium pyruvate) containing 10% (v/v) heat-inactivated, certified, fetal bovine serum (FBS). Human endothelial kidney (HE 2293) cells were plated in high-glucose Dulbecco ' s-modified Eagle ' s medium (DMEM) containing 10% (v/v) heat- inactivated, certified, fetal bovine serum (FBS), and 0.1 mM non-essential amino acids (NEAA). Chinese Hamster Ovary (CHO-K1 , adherent) and adapted for suspension growth (CHO-S) cells were grown in high-glucose DMEM. 10% FBS containing 0.1 mM NEAA, 1 % proline. and 10% (v/v) heat-inactivated, certified, fetal bovine serum (FBS). HeLaS3 (adapted for suspension grovvth) were grown in minimum essential medium with Earle ' s salts (S-MEM). 10% (vV) heat-inactivated horse serum, and 4 mM L- lutamine. K562 were grown in Iscove's modified Dulbecco's medium (IMDM: 4500 mg/L glucose, 862 mg/L L-alanyi-L-glutamine. 1 10 mg/L sodium pyruvate) containing 10% (v/v) heat-inactivated, certified, fetal bovine serum (FBS). Example I The cell lines HeLa, CHO-K1. CHO-S. 293F, K562, and HeLaS3 were transfected and assayed for a specific response to c-inyc antisense oligonucleotides to investigate the potency of TRO (a 1 :2.5 w/w liposome formulation of the cationic lipid dimethyl dioctadecyiammonium bromide (DDAB) and dioley l phosphatidylethanolamine (DOPE)) as a non-toxic and specific means of delivery for antisense oligonucleotides. TRO is sold under the trademark LIPOFECTACE'". Transfection Procedure The day before transfection. cells were plated in 96-well plates at an optimal seeding density according to each cell line described above. No antibiotics were used during these experiments. 200 nM of oligonucleotide (concentration calculated for a final volume of 100 μl) was added into 16 μl OPiT-MEM I Reduced Serum Medium. In a second tube. TRO was diluted 1 :5 in OPTT-MEM I Reduced Serum Medium and was incubated for 5- 10 minutes at room temperature. Diluted TRO was then added to diluted oligonucleotide (the final concentration of TRO added per w r ell was 8.4 μg/mL). mixed gently and incubated at room temperature for 15 minutes. 20 μl volumes of complexed TRO and oligonucleotides were added to washed cells containing 80 μl of fresh serum-free medium. Complexes were incubated in serum- free medium for 4 hours at 37 °C. 3X Serum-containing medium was then added to make a final concentration of I X serum. 48 hours post-transfection. complexes were removed, cells washed and fresh growth media added. Cells were assay ed for inhibition of proliferation at 24 hours, 48 hours, and 72 hours post-transfection. Both antisense and scrambled phosphorothioate oligonucleotides were transfected as described above. i he control samples were prepared similarly w ithout oligonucleotide or without oligonucleotide and TRO. The optimal concentration of TRO was found to be between 5.6 μg/ml and 1 1 .2 μg/ml. Measurement of Cell Proliferation Proliferation was measured with alamarBlue (Trek Diagnostics, Westlake, Ohio) which is a non-toxic redox indicator that yields a signal that can be detected with either fluorescent-based or absorbent based instrumentation in response to metabolic activity. alamarBlue 11 ^ was added to the cells at a 10% final volume of the reactions at 48 hours post-transfection. The absorbance of each well was read at two wavelengths, 570 nm and 600 nm, using a Molecular Devices Vmax® microplate reader and SOFTmaxΦPro 3.1 software (Molecular Devices, Sunnyvale, CA). Plates were then placed in the CO 2 incubator and readings were taken at 24 hours, 48 hours, and 72 hours according to Voytik-Harbin et al. (J. Cell. Biochem. 6 " 478-4 1 (1997)). The percentage of inhibition of cellular proliferation was defined as the relative absorbance of sample versus untreated control cells. Results The results of the readings at 72 hours post-transfection are shown in FIG. 1. The numbers are presented according to the alamarBlue I protocol. The results are expressed as a mean + SEM. Each assay represents the mean of replicates of 8 performed in a minimum of three separate experiments. The results show that TRO-complexed ODN targeted to the c-myc start codon produces a significant reduction in cell grovvth and survival. In six different cell lines, TRO consistently prov ided a specific inhibition of proliferation when compared to untreated cells. In HeLa cells, the inhibition was as great as 95% of the untreated sample, " fhe variation in the magnitude of effect seen across cell lines can be understood as a function of the sensitiv ity of the specific cell line to -myc down- regulation. Importantly , no cytotoxicity either with TRO or with TRO complexed to a scrambled ODN was observed w ith these complexes. Example 2 HeLa cell line was transfected and assayed for a specific response to c-inyc antisense oligonucleotides using the following transfection reagents: TR1 (LIPOFLCTIN IM ): LIPOFEC IN" (a 1 : 1 w/w liposome formulation of the cationic lipid N-[l -(2,3-dioleyioxy)propyi]-N.N,N- trimethyiammonium chloride (DOTMA) and dioleyi phosphatidyiethanolamine (DOPE in membrane filtered water) was diluted in OPTI-MEM I and incubated for 30 minutes at room temperature prior to complexation. Final concentration of LIPOFECTIN" added was 0.3 μl/mL. TR2 (CellFECTIN' ): The final concentration of CellFECTIlsT (a 1 : 1.5 M/M liposome formulation of a cationic lipid tetramethyipalmitylspermine (TMTPS) and DOPE) added per well was 0.2 μg/mL. TR3 (DMRIE-C' ): The final concentration of DMRIE-C" (a 1 : 1 M/M liposome formulation of a cationic lipid N-(2-hydiOxyethyi)-N,N-dimethy 1-2,3- bis(tetradeeyioxy)-l -propanaminium bromide (DMRIE) and cholesterol) added per well was 0.15 μg/mL. TR4 (LipofectAMINE'"): The final concentration of LipofectAMINE'" (a 3: 1 w/w liposome formulation of a polycationic lipid 2.3-dioleyioxy-N-[2- sperminecarboxamido)ethyi]-N,N-dimethyi- l -propanaminium (DOSPA) and DOPE) added per well was 0.3 μg/mL. TR5 (LipofectAMINE 200θ' M ): The final concentration of LipofectAMINE 2000'" added per cell was 0.2 μg/mL. The transfections and measurement of cell proliferation followed the procedures described in Example 1. The results of the readings at 72 hours post- transfection are shown in FIG. 2. The numbers are presented according to the alamarBlue protocol, fhe results are expressed as a mean + SEM. Each assay represents the mean of replicates of 8 performed in a minimum of three separate experiments, " fhe results for TRO from Example 1 are presented in FIG. 2 for comparison. FIG. 2 shows that I R0 produced the greatest reduction in cell growth and surv ival with little or no toxic effects. Of other c transfection reagents tested, only TR 1 showed a specific inhibition of proliferation. However, TR1 only inhibited proliferation 40% to that of the untreated sample (a 95% inhibition seen with ' FRO). FR2 and TR3 showed an inhibition of proliferation in both the antisense/TR complex and the scrambled/TR complex. This effectively eliminates these reagents as viable for antisense research since a non-specific effect is not desirable. Complexes formed with TR4 and TR5 showed no response to antisense targeting. Example 3 Western Blot A nalysis The ability of TR0/ODN complexes to inhibit c-Raf protein expression was examined by western blot analysis. Transfections were performed in 6-well plates using HeLa cells plated at 60,000 cells/well. Cells were treated for 6 hours with 200nM of c-raf antisense or mismatch oligonucleotide complexed to TRO (undiluted reagent was added for a final amount of 3 μl/well). The same treatment was repeated after 24 hours according to the procedure described by Lau el al. (Oncogene 76: 1899- 1 02 ( 1998)). Supernatant was transferred to a fresh micro fuge tube. For immunoblot analysis, cells were harvested at 24 hours and 48 hours and washed with I X PBS without QX or Mg + ~. Cellular extracts were prepared using 1 mL of boiling lysis buffer ( 1 % SDS. 1.0 mM sodium orthovanadate (Sigma-Aldrich. St. Louis, MO), and 10 mM " fris-HCl. pli 7.4). Typically, about 400 ng of protein were then separated and by electrophoresis on a 4-12% NuPageΦ Bis- ' fris SDS- polyaeryiamide mini-gel (Invitrogen Corporation, Carlsbad. CA). Once transferred to nitrocellulose, membranes were treated for 1 hour with a monoclonal antibody that specifically recognizes c-Raf kinase protein (BD Transduction Laboratories. Franklin Lakes, N.0 at a dilution of 1 : 1.000. Detection was performed with WesternBreeze 1 1 Kit (Invitrogen Corporation. Carlsbad. CA) and goat anti-mouse antibody (BD T ransduction Laboratories. Franklin Lakes. N.I). he control samples that received only TRO without oligonucleotide were prepared accordingly . The results at 48 hours after treatment are shown in FIG. 3. Inhibition of c- Raf was observed only in the presence of the TRO/antisense c-raf complex. No inhibition of c-Raf expression was seen with the untreated samples, samples treated with TRO alone, or with the TRO/mismatch ODN complex. Those skilled in the art will recognize that while specific embodiments have been illustrated and described, various modifications and changes may be made without departing from the spirit and scope of the invention. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims. All publications, patent applications and patents cited herein are fully incorporated by reference.

Claims: What is Claimed Is: 1 . A method for introducing one or more antisense oligonucleotides into one or more eucaryotic cells, comprising (a) contacting said one or more antisense oligonucleotides with one or more lipid formulations comprising an effective amount of one or more cationic lipids of Formula / wherein Ri is a straight or a branched hydrocarbon chain of Cιo-ιon that is saturated or unsaturated; R 2 is selected from the group consisting of a pair of electrons, hydrogen, alkyl, alkenyl, alkynyl, heteroalkyi, heteroalkenyi, heteroalkynyl, R 5 -NHC(0)-R 6 , R ? -C(0)-0-R 6 . R 5 -NH-C(0)-NH-R 6 . R 5 -NH-C(S)-NH~R , R 5 -NH-C(NH)-NH-R () . alkylaminoalkyl, aryialkyl. ar lalkenyi, aryialkynyl, and aryi, all of which can be optionally substituted; R 3 and R4. independently of one another, are selected from the group consisting of hydrogen, alkyl, alkenyl. alky nyl. heteroalkyi, heteroalkeny i, heteroalkynyl, R 5 -NHC(0)-R r ,. R 5 -C(0)-Q-R ( „ R 5 -NH-C(0)-NH-R t) . R 5 -NH-C(S)-NH~R ( „ R 5 -NH-C(NI 1)-NH-R ( ,. alkylaminoalkyl. aryialkyl. arylalkeny i. aryialkynyl. and ary i. all of which can be optionally substituted; wherein R and R fl are independently alkyiene. alkeny lene or alkynylene; and A is a pharmaceutically acceptable anion when R2 is not a pair of electrons: and optionally at least one neutral lipid to form one or more antisense oligonucleotide-lipid aggregate complexes, and (b) contacting said one or more cells with said one or more complexes. 2. The method according to claim 1 , wherein when R 3 and R 4 are Cj. 3 alky l, and one of R| or R 2 is an unsaturated Cκ,- 2 alkyl. the other one of Ri and R 2 is not an unsaturated or saturated CV- 20 alkyl. 3. The method according to claim 1. wherein said one or more cells are not drug- resistant human breast carcinoma cells. 4. The method according to claim 1. wherein Ri is a straight or branched hydrocarbon chain of C 1 0- 3 0 that is saturated or unsaturated. The method according to claim 4. wherein R| is a straight hydrocarbon chain of C 1 2 - 2 4 that is saturated or unsaturated: and R 2 . R 3 and R4 are independently selected from the group consisting of hydrogen. C M S alky l. CVis alkeny l. C 2 - 1 X alkynyl. C4-18 heteroalkyi, CVix heteroalkenyi. C 4 -ιχ heteroalkynyl, CV12 ary l(CVi 8 ) alkyl and CV1 2 aryi. all of which can be optionally substituted. 6. The method according to claim 5. wherein Ri is a straight hydrocarbon chain of C - 2 that is saturated or unsaturated: R 2 is selected from the group consisting of hydrogen, CVix alkyl. G,.ιχ alkeny l. CVix alky nyl. CVix heteroalkyi, CVix heteroalkeny i. G,_ιχ heteroalkynyl. phenyi(CVιχ)alkyi. and phenyl; and R 3 and R 4 are independently selected from the group consisting of hy drogen, CV 5 alkyl. CV <> alkeny l. C 2 -ι, alky ny l. CV* heteroalky i. CV 5 heteroalkeny i. CV5 heteroalky ny l, pheny l(Cι_ 5 )alky L and phenyl. all of which can be optionally substituted. 7. " I he method according to claim 6. wherein said cationic lipid of Formula / is dimethvidioctadec lammonium bromide (DDAB). 8. The method according to claim 1 , wherein said lipid formulation comprises a neutral lipid. 9. The method according to claim 8, wherein said neutral lipid is diacyiphosphatidyiethanolamine having 10-24 carbon atoms in the acyl group. 10. The method according to claim 9 wherein said neutral lipid is dioleyiphosphatidyiethanolamine (DOPE). 1 1. The method according to claim 1 , wherein said cationic lipid is the cationic lipid of Formula //: wherein Ri is a straight or a branched hydrocarbon chain of Cio-ioo that is saturated or unsaturated: R 2 is selected from the group consisting of a pair of electrons, hydrogen, alkyl, alkeny l, alkynyl, heteroalky i, heteroalkenyi, heteroalkynyl. R.-NI IC(0)-R 6 . R,-C(0)-0-R„. Rs-NH-C(0)-NI 1~R„. R 5 -NI VC(S)-NH-R () . R 5 -NH-C(NI I)-NH-RV alkylaminoalkyl. aryialkyl. ary lalkeny i. ary ialkynyl. and ary i, all of which can be optionally substituted: wherein R and R ( , are independently alkyiene. alkenyiene or alkyny lene; and A is a pharmaceutically acceptable anion when R 2 is not a pair of electrons. 12. The method according to claim 1 1 , wherein when one of Ri or R is an unsaturated Cu,. 2 o alkyl. the other one is not an unsaturated or saturated CV,- 2 o alkyl. 13. The method according to claim 1 1 , wherein Rj is a straight or branched hydrocarbon chain of C 1 - 30 that is saturated or unsaturated. 14. The method according to claim 13, wherein R) is a straight hydrocarbon chain of C 12 - 2 4 that is saturated or unsaturated; and R 2 is selected from the group consisting of hydrogen, CVix alkyl, CVix alkenyl, CVix alkynyl. C.j.ix heteroalkyi, CVix heteroalkenyi, C4-1 heteroalkynyl, C(,-i aryi(CVιχ) alkyl and C(,-i aryi, all of which can be optionally substituted. 15. The method of claim 14, wherein R| is a straight hydrocarbon chain of Ci 4. 20 that is saturated or unsaturated: and R 2 is selected from the group consisting of hydrogen, C ,.ιχ alkyl, C ( ,_ιχ alkenyl, C ( ,.ιs alkynyl, C 6 -ιχ heteroalkyi, C 6 -ιχ heteroalkenyi, CVix heteroalkynyl, phenyi(CVιχ)alkyi, all of which can be optionally substituted. 16. The method according to claim 15, wherein Ri is a straight hydrocarbon chain of C 14-20 that is saturated, and R is selected from the group consisting of C ( ,.ιχ alkyl. CVix heteroalkyi, ix heteroalkenyi. C f ,_ιχ heteroalkynyl. and phenyi(C(,.ιχ)alkyi. all of which can be optionally substituted. 17. he method according to claim 1 or claim 1 1. wherein A is selected from the group consisting of a halogen, a sulfate. a nitrite or a nitrite. 18. ' fhe method according to claim 17. wherein A is a bromide. 19. " fhe method according to claim 1 or claim 1 1. wherein said optional SLibstituent is selected from the group consisting of halogen, halo(CVr,) alky l. Cu, alky l. CV, alkeny l. CV f , alkynyl. hydroxy(Cu,)alkyl. amino(Cu,)alkyl. carboxy(CVf > )alkyi, alkoxy(Cu,)alkyi, nitro, amino, ureido. acyiamino, hydroxy, thiol, acyioxy, alkoxy. carboxy, aminocarbonyi, and C ' u, alkylthiol. 20. " fhe method according to claim 19, wherein said optional substituent is selected from the group consisting of hydroxy(Cu,)alky l, amino(CV<,)alkyl. hydroxy, carboxy, nitro, CV <5 alkyi, alkoxy, thiol and amino. 21. The method according to claim 1 , wherein said lipid formulation is present in an amount of about 0.1 μg/ml-5 mg/ml. 22. The method according to claim 21. wiierein said lipid formulation is present in an amount of about 0.35-14 μg/ml. 23. The method according to claim 22, wherein said lipid formulation is present in an amount of about 2- 13 μg/ml. 24. The method according to claim 23, wherein said lipid formulation is present in an amount of about 4.5- 12 μg/ml. 25. The method according to claim 24, wherein said lipid formulation is present in an amount of about 5.6-1 1.2 μg/ml. 26. A method for introducing one or more antisense oligonucleotides into one or more eucaryotic cells, comprising (a) contacting said one or more antisense oligonucleotides with a lipid formulation comprising an effective amount of dimethyldioctadecy lammonium bromide (DDAB) and at least one neutral lipid to form one or more antisense oligonucleotide-lipid aggregate complexes . and (b ) contacting said one or more cells w ith said one or more complexes. 27. The method according to claim 26, wherein the ratio of said DDAB and said neutral lipid is from about 1 :5 to about 1 : 1. 28. The method according to claim 27, wherein said ratio is 1 :2.5. 29. The method according to any one of claims 26-28. wherein said neutral lipid is diacyiphosphatidyiethanolamine having 10-24 carbon atoms in the acyl group. 30. The method according to claim 29, wherein said neutral lipid is dioleyiphosphatidyiethanolamine (DOPE). 31. The method according to claim 30, wherein said lipid formulation is present in an amount of about 2- 13 μg/ml. 32. The method according to claim 3 1 , wherein said lipid formulation is present in an amount of about 4.5-12 μg/ml. 33. The method according to claim 32. wherein said lipid formulation is present in an amount of about 5.6- 1 1.2 μg/ml. 34. A kit for introducing one or more oligonucleotides into one or more eucaryotic cells, comprising at least one component selected from the group consisting of one or more cells, one or more antisense oligonucleotides. one or more lipid formulations comprising an effective amount one or more cationic lipids of Formula / and optionally at least one neutral lipid. one or more buffering salts, one more culture media, and one or more transfection enhancers. 35. The kit according to claim 34. wherein said kit comprises one or more lipid formulations comprising an effectiv e amount of one or more cationic lipids of Formula / wherein Ri is a straight or a branched hydrocarbon chain of Cio-ioo that is saturated or unsaturated; R 2 is selected from the group consisting of a pair of electrons, hydrogen, alkyl, alkenyl, alkyny l. heteroalkyi, heteroalkenyi, heteroalkynyl, R 5 -NHC(0)-R t) , R 5 -C(O)-O~R b , R 5 -NH-C(OVNH-R 6 , R 5 -NH-C(SVNH-R b , R 5 ~NH-C(NHVNH~R<„ alkylaminoalkyl, aryialkyl, arylalkenyi. aryialky nyl, and ary i. all of which can be optionally substituted; R 3 and R 4 , independently of one another, are selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, heteroalkyi, heteroalkenyi, heteroalkynyl, R 5 ~NHC(0)-R„. R -C(0)-0-R ( „ R 5 -NH-C(OVNH-R 6 , R 5 -NH-C(S)-NH-R(„ R 5 -NH-C(NH)-NH~R ( „ alkylaminoalkyl. aryialkyl. arylalkenyi, aryialkynyl, and aryi, all of which can be optionally substituted; wherein R 5 and R ) are independently alkyiene. alkeny lene or alkynyiene; and Λ is a pharmaceutically acceptable anion when R 2 is not a pair of electrons; and optionally at least one neutral lipid. and at least one additional component selected from the group consisting of one or more cells, one or more antisense oligonucleotides. one or more buffering salts, one or more culture media, and one or more transfection enhancers. 36. A composition comprising at least one component selected from the group consisting of one or more cells, one or more antisense oligonucleotides, one or more lipid formulations comprising an effectiv e amount o\ ' one or more cationic lipids of Formula / and optionally at least one neutral lipid, one or more buffering salts, one more culture media, and one or more transfection enhancers. 37. The composition according to claim 36. wherein said composition comprises one or more lipid formulations comprising an effective amount of one or more cationic lipids of Formula / wherein Ri is a straight or a branched hydrocarbon chain of Cio- o that is saturated or unsaturated; R 2 is selected from the group consisting of a pair of electrons, hydrogen, alkyl. alkenyl. alkynyl. heteroalkyi. heteroalkenyi, heteroalkynyl, R -NHC(0)-R 6 , R 5 -C(0)-0-R () , R 5 -NFf-C(0)-NH-R () . R 5 -NH-C(S)-NH-R (1 , Rj-NH-C(NH)~NH~R„. alkylaminoalkyl. aryialkyl. arylalkenyi, aryialkynyl, and aryi. all of which can be optionally substituted; R 3 and R4, independently of one another, are selected from the group consisting of hydrogen, alkyi, alkenyl, alkynyl. heteroalkyi, heteroalkenyi, heteroalkynyl, R 5 -NHC(0)-R () . R 5 -C(0)-0-R 6 , R 5 ~NH-C(S)-NI f-R(,. R 5 -NH-C(NI 1)-NH-R 6 . alkylaminoalkyl. aryialkyl, arylalkenyi. aryialkynyl. and aryi. all of which can be optionally substituted; wherein IV- and R ( , are independently alkyiene, alkenyiene or alkynyiene; and A is a pharmaceutically acceptable anion when R 2 is not a pair of electrons; and optionally at least one neutral lipid. and one or more additional components selected from the group consisting of on or more cells, one or more antisense oligonucleotides. one or more buffering salts, one or more culture media, and one or more transfection enhancers. 38. A method for inhibiting or preventing cell growth or proliferation, comprising (a) contacting one or more eucaryotic cells with one or more antisense oligonucleotides and an effective amount of one or more lipid formulations comprising an effective amount of one or more cationic lipids of Formula / and optionally at least one neutral lipid to provide a composition; and (b) incubating said composition under conditions sufficient to inhibit or prevent cell growth or proliferation. 39. A method for inhibiting or preventing expression of one or more proteins, comprising (a) contacting one or more eucaryotic cells with one or more antisense oligonucleotides and an effective amount of one or more lipid formulations comprising an effective amount of one or more cationic lipids of Formula / and optionally at least one neutral lipid to provide a composition: and (b) incubating said composition under conditions sufficient to inhibit or prevent said expression of one or more proteins.

Inventors: FOX DONNA K, GEBEYEHU GULILAT, OGILVIE MARTHA K, FOX, DONNA, K., GEBEYEHU, GULILAT, OGILVIE, MARTHA, K.
Applicants: INVITROGEN CORP, INVITROGEN CORPORATION
Application year: 2001
Main classification: C12N
IPC codes: A61K31, A61K47, A61K48, A61P35, C12N1, C12N5, C12N15