MXPA99002844A - Three component chimeric antisense oligonucleotides - Google Patents

Abstract

The present application describes a novel family of oligonucleotide compounds having a novel organization of various modified nucleotides and modified chemical linkages. The application further discloses that limiting the presence and extent of specific modified nucleotides/modified linkages in the oligomers results in enhanced activation of endogenous RNase H activity.

Description

THIRD-COMPONENT CHEMICAL ANTI-SCIENTIFIC OLIGONUCLEOTIDES DESCRIPTION OF THE INVENTION This application claims priority for applications from the United States no. 60 / 026,732 filed on September 26, 1996 and 08 / 754,580 filed on November 21, 1996. This invention relates to antisense oligonucleotides that target mRNA in cells as substrates for the enzyme RNase H and therefore cause specific degradation of the MRNA objectified. Oligonucleotides have four components: an activating region of ANase H; a complementary region; an end 5 '; and an end 3. The invention optimizes each of the components to resist intracellular nucleases, to increase hybridization to target mRNA, to specifically inactivate target mRNAs in cells, and to decrease cytotoxicity.Antisense polynucleotides are useful to specifically inhibit gene expression. unwanted in mammalian cells, they can hybridize and inhibit the function of an RNA molecule, typically a messenger RNA, by activating RNase H. The use of antisense oligonucleotides has emerged as a powerful new method for the treatment of certain diseases. Preponderance of work to date has focused on the use of antisense oligonucleotides as antiviral agents or anticancer agents (ichstrom, E., Ed .; Prospects for antisense Nucleic Acid Therapy of Cancer and AIDS, New York: iley-Liss, 1991; Crooke , ST and Lebleu, B., Eds., Antisense Research and Applications, Boca Raton: CRC Press, 1993, pages 154-182; Baserga, R. And Denhardt, D.T., 1992, Antisense Strategies, New York: The New York Academy of Sciences, Vol. 660; Murray, J.A.H., Ed., Antisense RNA and DNA, New York: Wiley-Liss, 1993). There have been numerous descriptions of the use of antisense oligonucleotides as an antiviral agent. For example, Agrawal et al. reports the phosphoramidate and phosphorothioate oligonucleotides as antisense inhibitors of HIV Agrawal et al., Proc. Natl. Acad. Sci. USA 85: 7079-7083 (1988). Zamecnik et al. describes antisense oligonucleotides as inhibitors of Rous sarcoma virus replication in chicken fibroblasts. Zamecink et al., Proc. Natl. Acad. Sci.

USA 83: 4143-4146 (1986). The main mechanism by which antisense oligonucleotides affect an objective RNA molecule is by activation of the cellular enzyme RNase H, which cleaves the RNA strand of DNA / RNA hybrids. Both DNA-linked phosphodiester and phosphorothioate activate the endogenous RNase H, whereby the targeted RNA is cleaved (Agrawal, S., et al., Proc. Natl. Acad. Sci. USA 87: 1101-5 (1990); TM, et al., Nucleic Acids Res. 18: 1763-9 (1990)). However, the DNA bound to phosphodiester is rapidly degraded by cellular nucleases and, with the exception of phosphorothioate-binding DNA, resistant to nuclease, DNA derivatives that are not found naturally do not activate RNase H when they hybridize to RNA. While phosphorothioate-linked DNA has the advantage of activating RNase, DNA bound to phosphorothioate has been associated with non-specific cytotoxic effects and reduced affinity to RNA (Stein, CA, et al., Aids Res Hum Retroviruses 5: 639 -46 (1989); Woolf, TM, et al., Nucleic Acids Res. 18: 1763-9 (1990); Kawasaki, A.M., et al., J. Med. Chem. 36: 831-41 (1993)). Chimeric antisense oligonucleotides having a short extension of the phosphorothioate DNA (3-9 bases) have been used to obtain RNase H-mediated cleavage of the target RNA (Dagle, JM, et al., Nucleic Acids Res. 18: 4751-7). (1990), Agrawal, S., et al., Proc. Natl. Acad. Sci. USA 87: 1401-5 (1990), Monia, BP et al., 1993, J. Biol. Chem. 268: 14514) A minimum of 3 DNA bases are required for activation of the bacterial RNase H (Futdon, PJ, et al., Nucleic Acids Res. 17: 9193-9204; Quartin, RS, et al., Nucleic Acids Res. 17: 7235 -7262) and a minimum of 5 bases is required for mammalian Rnasa H activation (Monia, B.P., et al., J. Biol. Chem. 268: 14514-14522 (1993)). In these chimeric oligonucleotides there is a central region that forms a substrate for RNase H that is flanked by hybridizing "extremities", comprised of modified nucleotides that do not form substrates for RNase H. Alternatively, extracellular tests using a HeLa cell extract have been reported. contains RNase H wherein the activation region of RNase H is placed on the 5"6 3" side of the oligomer. Specifically, these tests report that a 5"or 3 'terminal RNase activation region composed of phosphodiester 2'-deoxynucleotides attached to a complementary region bound to methylphosphonate is fully active, but that an activation region of terminal RNase H "composed of phosphorothioate 2 '-deoxinucelotides bound to a complementarity region bound to methylphosphonate is only partially active. See Col. 10, United States Patent no. 5,220,007 assigned to T. Pederson et al. The nucleotides modified with 2'-0-methyl or 2'-fluro have been used for the hybridized ends of chimeric oligonucleotides. Inoue, H., et al., 1987, Nucleic Acids res. 15: 6131-48. The 2'-0-methyl group increases the affinity of the oligomer to the target RNA and increases the activity of the oligomer in cell culture. However, 2"-0-methyl bases with phosphodiester linkages are degraded by exonucleases and are therefore not suitable for use in cells or therapeutic antisense applications. Shibahara, S., et al., 1989, Nucleic Acids Res. 17: 239-52, Phosphorothioate 2"-O-methylnucleotides are resistant to nucleases as shown in the oligonucleotides uniformly modified with phosphorothioate described by Monia BP, et al. ., 1993, J. Bioll Chem. 268: 14514-14522 and 2"-0-methylribooligonucleotides substituted with phosphorothioate, Shibahara, S., et al., 1989, Nuceic Acid Res. 17: 239-252. However, oligomers totally substituted with foforothioate can cause non-specific effects including cellular toxicity. Stein, C.A. , et al., 1989, Aids Res. Hum. Retrov. 5: 639-646; Woolf, T.M .; et al; 1990, Nucleic Acids Res. 18: 1763-69; Wagner, R.W. , nineteen ninety five; antisense Res. Dev. 5: 113-115; Krieg, A.M. , & Stein, C.A. , 1995, Antisense Res. Dev. 5: 241. The effects of 2 '-fluoro-oligonucleotides on bacterial RNase H in Crooke, s.T. et al., 1995, Bioch. J. 312: 599-608 and Iwai, S. et al., 1995, FEBS Lett (Neth) 368: 315-20. Several other chemistries have been used to make "extremities" or regions of a chimeric oligomer that are not substrates for RNase H. The first chimeric oligomers use methylphosphonate or phosphoramidate linkages in the extremities (Dagle, JM, Walder, JA &Weeks, KL , Nucleic Acids res 18: 1751-7 (1990), Agrawal, S., et al., Proc. Natl. Acad. Sci. USA 87: 1401-5 (1990) .While these compounds work well in cushioned systems and Xenopus oocytes, the extremities decrease the affinity of the hybrid This decrease in affinity dramatically reduces the antisense activity of the chimeric oligomers in mammalian cell culture A number of studies have been reported for the synthesis of ethylated and methylated phosphotriester oligonucleotides and their evaluation fisocochemistry and biochemistry It is shown that dinucleotides with methyl and ethyl triesters have higher affinity to polynucleotides that possess complementary sequences (Miller, P. S., et al., J. Am. Chem. Soc. 93: 6657, (1971)). However, a few years ago, another group reported lack of binding affinity, or relatively poor binding affinity of a heptaethyl ester of oligotimidine with complementary polynucleotides (Pless, RC, and TSO, POP, Biochemistry 16: 1239-1250 ( 1977)). The methylated phosphate (p-methoxy) oligonucleotides are synthesized and found to possess resistance to endonuclease digestion (Gallo, KL, et al (Nucle.Acid Res. 18: 7405 (1986).) A P-methoxy is shown. 18-mer oligonucleotide having high Tm value in duplex with natural DNA and nloquea the process of DNA replication at room temperature (Moody, HM, et al., Nucí Acid Res. 17: 4769-4782 (1989)). et al., conclude that ethylated (p-ethoxy) phosphate oligonucleotides may have deficient antisense properties.The P-methoxy dimers of DNA bases are synthesized using FMOC as a temporary protecting group for the exocyclic amino groups (Koole, LH, et al. ., J. Org. Chem. 54; 1657-1664 (1989).) Synthesis and physicochemical properties are also determined.The thymidine and cytidine oligomers can only be prepared with methyl phosphotriester because of the difficulties encountered in keeping methyl intact. In addition, it is The methyl group has a destabilizing effect on the hybridization properties of the modified oligomers with their complementary sequences as compared to unmodified progenitor oligodeoxyribonucleotide (Vinogradeov, S., Asseline, U., Thoung, N.T., Tet. Let. 34: 5899-5902 (1993)): Other reports have suggested that P-methoxy oligonucleotides are preferable to P-ethoxy as antisense oligonucleotides because the p-methoxy oligonucleotides show stronger hybridization than methyl phosphonate or P-ethoxy oligonucleotides (van Genderen, MHP, et al., Kon. Ned, Akad, Van Wetensch.B90: 155-159 (1987), van Genderen, MHP, et al., Trav. Chim. Pays Bas 108: 28-35 (1989). )). The p-ethoxy oligonucleotides are reported by Van Genderen et al for poorly hybridizing to DNA and are thus considered less suitable for use as antisense oligonucleotides (Moody, HM, et al., Nuci Acid Res. 17: 4769-4782 ( 1989)). The p-isopropoxyphosphoramidites of several nucleosides have been synthesized (Stec, WJ, et al., Tet., 26: 2191-2194 (1985)), and few short oligonucleotides containing p-isopropoxyphosphotriesters are synthesized, and hybridization. U.S. Patent No. 5,525,719 assigned to Srivastava, s., And Raza, SK, June 11, 1996, suggests antisense oligonucleotides consisting of 2"-P-methyl nucleotides attached by phosphodiester and / or p-ethoxy moieties. or p-methoxy Currently there are no nucleic acid chemistries or chimeras that have been developed that optimally achieve all the characteristics that are necessary to provide an effective antisense oligonucleotide ie, low toxicity, high specificity, nuclease resistance, ease of synthesis, compatibility to Rnasa H. The present invention describes a class of oligonucleotides that have been optimized to objectify a specific RNA objectified for degradation by Rnasa H while resistance to the degradation by nuclease in plasma and within eukaryotic cells, especially mammals. The oligonucleotides of the invention do not contain nucleotides linked to S "- >; 3"that are not found naturally, but the invention provides oligonucleotides having two types of nucleotides: 2" -deoxyphosfortioate, which activates the RNase H, and nucleotides 2"-modified, which do not activate it. links between the modified 2"-nucleotides can be phosphodiesters, phosphothioate or p-ethoxyphosphodiester.

In addition to the S "and 3" ends, the oligonucleotides currently described comprise an activation region of RNase H, and a region of complementarity that facilitates hybridization to the target sequence. The RNase H activation region is typically a contiguous sequence containing between three and five nucleotides 2"-deoxyphosphorothioate (to activate the bacterial RNase H), and typically between about 5 and 10 nucleotides 2'-deoxyphosphorothioate to activate eukaryotic RNase H, particularly mammal The 5"and 3" ends of the oligonucleotides currently described are protected from exonuclease degradation via the incorporation of modified 5"and 3" terminal bases which are highly resistant to nuclease, particularly exonuclease and optionally by placing a terminal block group 3" . In a preferred embodiment, the RNase H activation region is composed of highly-fused nuclease-resistant foforothioate nucleotides that are placed at the S "end of the oligonucleotide.An embodiment, one embodiment of the present invention is a chimeric oligonucleotide comprising a region of activation of RNase H of between three and twelve bases linked to contiguous 2'-deoxyphosphothioate (ie, 2"- deoxyribonucleotides linked to phosphorothioate); a region of complementarity substantially resistant to endonuclease of between about nine and about fifty 2"-modified bases; a 5" terminus substantially resistant to exonuclease; and a 3"termination substantially exonuclease resistant Oligonucleotide structure The oligonucleotides of the present invention typically comprise a nucleic acid or 5" end-resistant 5-exonuclease linker, an contiguous RNase H activation region of about 3 to about ten bases in length, a terminal 3 'linked 5"-» 3", or optionally linked 3" - 3", for example," inverted "nucleoside, and from about 9 to about 50 nucleotides linked 5" - 3", at which nucleotides can be 2'-deoxynucleotides or 2'-modified nucleotides that facilitate the hybridization of the oligonucleotide to target mRNA, such as 2"-fluor, 2" -methoxy, 2"-ethoxy, 2" -methoxyethoxy, 2"- allyloxy (-0CH2CH = CH2) (after this "nucleotides 2" -modified "). The 3"terminal nucleosides can optionally be a modified 2" nucleoside. Those skilled in the art appreciate that it may not be necessary for the OH 3"from the terminal base 3" to be esterified to a phosphate or phosphate analogue. The terminal 3"residue is referred to as a nucleoside although it can be a nucleotide The internucleotide linkages of an oligonucleotide of the invention can be phosphodiester, phosphotipate or p-ethoxyphosphodiester moieties The oligonucleotide has a 3'-end and a 5-end termination. which are substantially protected from nuclease attack The oligonucleotide has a 3"terminus and a 5" terminus which are substantially protected from nuclease attack The 3"terminus is protected by having the 3" link or bonds plus 5"-» 3"which are phosphorothioate or P-alkyloxyphosphotriester linkage and / or having a substituted 3'-terminal hydroxyl, eg, a linked nucleotide 3" - > 3", wherein the alkyloxy radical is methoxy, ethoxy, or isopropoxy and, preferably, ethoxy Preferably two or three terminal 3 internucleotide bonds are phosphorothioate or p-alkyloxyphosphotriester bonds. To reduce the degradation of the nuclease, the bond 5"plus 3" - > 5"preferably should be a phosphorothioate or p-alkyloxyphosphotriester linkage. Preferably, the two 5" plus 3"-» 5 bonds should be phosphorothioate linkages or p-ethoxyphosphotriester linkages. Optionally, the 5"terminal hydroxyl portion can be esterified with a phosphorus-containing moiety, for example, phosphate, phosphate thioate or P-ethoxyphosphate, without limitation.The bound 3 'terminal nucleoside 5" - »3" has a 3"-O which may be optionally substituted by a blocking portion which prevents 3"-exonuclease degradation of the oligonucleotide In one embodiment, 3" -hydroxyl is esterified to a nucleotide via internucleotide link 3"- >3". Optionally, nucleotide 3" - »3" linked at the 3"end may be linked by a phosphorothioate moiety. By incorporating the above chemistries, the oligonucleotides currently described are substance-J resistant to exonucleases and 5"and 3" endonucleases. For purposes of the present invention, an oligomer is substantially resistant to a given endo or exonuclease when it is at least about 3 times more resistant to attack by an endogenous cellular nuclease, and is highly resistant to the nuclease when it is at least about 6 times more resistant than a corresponding oligomer comprised of unmodified DNA or RNA. In a preferred embodiment, the oligonucleotide contains, exclusive of an optional blocking nucleotide, between 15 and 50 bases and more preferably between 20 and 30 bases and in the most preferred embodiment the oligonucleotide has 25 bases in length. The oligonucleotide of the invention contains a single activation region of contiguous RNase H of from three to ten 2"-deoxyphosphorothioate nucleotides The length of the activation region of RNase H to activate the bacterial RNase H preferably has between three and five nucleotides; to activate a eukaryotic RNase H the activation region preferably has between about five and about ten or twelve nucleotides.The preferred length of the activation region of Rnasa H for the activation of mammalian Rnase has nine nucleotides.All nucleotides linked 5"3 Oligonucleotides that are not part of the activation region of RNase H are modified "2" nucleotides, which contribute to objectively link and thus form the region of complementarity determination.The complementarity region can be an adjoining region or it can be divided by the activation region of RnNsa H. In a modal Preferred condition The complementarity region is a contiguous region, and, more preferably, it is located 3"to the activation region of Rnasa H. In a preferred embodiment all bases except for one to three nucleotides 3" -terminals and / or nucleosides , the 5"terminal nucleotide, and the nucleotides of the activation region of RNase H, are linked to phosphodiester. Large amounts of contiguous phosphorothioate linkages are harmful to the function of the oligonucleotides of the invention. Accordingly, the oligonucleotides preferably do not contain twelve contiguous phosphorothioate linkages or twelve contiguous fosfortioate linked to deoxynucleotides. Additional embodiments of the chimeric oligonucleotides currently described have the structures: 5 'A: B: C 'C: B: A: B: C. wherein A is an RNase H activation region of between about 3 to about 12 nucleotides, preferably about 3 to about 10 nucleotides or 5 to about 12 nucleotides long which is also stable to the nuclease (eg, phosphorothioate DNA); B represents a region of chemistry (for example RNA substituted 2"0-methyl) which is stable against endonuclease (about 4 to about 40 nucleotides long; C represents one to four nucleotides long for exonuclease blocking which typically does not contain phosphorothioate DNA (ie phosphorothioate 2"-0-methyl bonds, inverted bases, methylphosphonate, phosphoramidite, non-nucleotide linkers, amino linkers, conjugates or any other chemical consistent with nucleotide synthesis in the art, or even to discover that it is not recognized by cellular exonucleases.) Alternatively, the configuration can be inverted as follows: 5 'C: B: A if the application does not require activation of RNase H ( steric block or triple chain inactivation), the following configuration is useful: 5 'C: B: C: Synthesis of oligonucleotides The oligonucleotides of the invention can be synthesized by nucleotide synthesis of solid or liquid phase, however, the synthesis by solid phase techniques.The oligonucleotides linked to phosphodiester or phosphorothioate can be synthesized, using reagents is standard and protocols, in an automated synthesizer using methods that are well known in the art, such as, for example, those discussed in Stec et al., J. Am. Che. Soc. 106: 6077-6089 (1984); Stec et al., J. Chromatog. 326: 263-280 (1985); LaPlanche et al., Nucí Acid. Res. 14: 9081-9093 (1986); and Fasman, G.D., Practical Handbook of Biochemistry and Molecular Biology 1989, CRC Press, Boca Raton, Florida, incorporated herein by reference. The synthesis of 2"-o-alkyl-oligoribonucleotides is analyzed, where the alkyl groups are methyl, butyl, allyl or 3, 3-dimethylallyl by Lamond, Biochem. Soc. Trans. 21: 1-8 (1993). intermediates that are useful in the synthesis of 2'-o-methyl oligoribonucleotides in U.S. Patent Nos. 5,013,830, 5,525,719 and 5,214,135, which are incorporated herein by reference. "-fluorophosphodiester and 2" -fluorophosphorothioate oligonucleotides according to the teaching of Kawasaki, AM, et al., 1993, J. Med. Chem. 36: 831-41 and WO 92/03568; synthesis of linked oligonucleotides can be performed to P-alkyloxyphosphotriester and 2"-modified according to U.S. Pat. no. ,525,719, each of which are incorporated herein by reference. The synthesis of phosphorothioate oligodeoxynucleotides is taught by U.S. Patents 5,276,019 and No. 5,264,423, which are incorporated herein by reference. The synthesis of the oligonucleotides of the invention can be carried out with greater attention to quality control. It is particularly important that the phosphorothioate linkages are not contaminated with phosphodiester bonds. It is advisable to pre-test lots of individual reagents to determine that high efficiency can be obtained coupled with it to exercise all possible precautions to maintain anhydrous conditions. The quality of the oligonucleotide synthesis can be verified by testing the oligonucleotides by capillary electrophoresis and denaturing strong anions by HPLC (SAX-HPLC). The method of Begot & Egan, 1992, J. Chrom. 599: 35-42 is adequate. SAX-HPLC is particularly suitable for verifying that the phosphorothioate nucleotides are completely thiolated, ie they are not contaminated by a small percentage of phosphodiesters. The synthesis of oligonucleotides that have phosphodiester and phosphorothioate bonds is associated with a lateral reaction, so the phosphorothioate bonds are oxidized by the standard I2 treatments that are used to oxidize cyanomethylphosphoramidite. This problem can be minimized but not eliminated by reducing the concentration of I 2 as low as 0.001 M. Therefore,, in a preferred embodiment, it is found that all the phosphorothioates of the oligonucleotides of the invention are at the 5 end, such that no phosphorothioate bond is exposed to I2. Uses of Oligonucleotides The oligonucleotides of the invention can be used as antisense oligonucleotides in a variety of experimental situations in vitro to specifically degrade an mRNA of unknown function and thereby determine its physiological function. The oligonucleotides of the invention can also be used in clinical practice for any disease and against any target RNA for which it is now known that the antisense therapy is suitable or that it is to be identified. Medical conditions are reported for which antisense therapy is suitable and include respiratory syncytial virus infection, WO 95/22553 for influenza virus, Kilkuskie, WO 94/23028, and malignancies WO 94/08003. Further examples of clinical uses of antisense oligonucleotides are reviewed, in summary form, in Glaser, V., 1996, Genetic Engineering News 16, 1. Targets of antisense oligonucleotides are subject of clinical trials include Ca, ICAM-1 kinase, c-raf kinase, p53, c-myb and the bcr / abl fusion gene found in chronic myelogenous leukemia. EXAMPLES Experimental Conditions antisense activity of oligonucleotides of the present invention using a temporal expression test which includes an mRNA encoding a luciferase protein that has been modified to include a test sequence derived from the ras gene is shown. The specific antisense effects of an oligonucleotide can be measured by comparing the luciferase production of the test cells with the production of control cells having the same expression plasmid except for the absence of the ras-derived sequence. The oligonucleotides of the invention that are tested have the sequence: 5"-TTGCCCACACCGACGGCGCCCACCA-3" (SEQ ID NO: 1). The details of the trial are as follows: Plasmid constructions. The plasmid used for the studies contains a portion of the ras gene sequence fused to the luciferase (Monia, B.P., et al., J. Biol. Chem. 267: 19954-19962 (1992)). The control luciferase plasmids do not contain the ras objective sequence. Cell culture assay. HeLa cells are grown at 40-90% confluence in DMEM / 10% FBS, supplemented with glutamine, penicline and streptomycin in gelatin-coated 24-well plates. It is necessary that the gelatin coating remains adhered during transfections. Before transfection, cells are washed twice with PBS (containing magnesium and calcium). The LIPOFECTIN ™ is mixed gently and 6.6 μl is added per milliliter of reduced serum medium (OPTI-MEM ™, Gibco / BRL, Gaithersberg, MD). The oligomers of a concentrated 50-100 μl stock are added to make a master mix. The OptiMEM / LIPOFECTIN / oligomer solution is added to the cells and incubated for 4 hours (-0.5 ml per well of a 24-well plate). An objective transfection mixture is prepared by first diluting 5 μl of lipofetin per ml of OPTI-MEM and mixing. Then add 5 μg of lucifersa and 5 μg of CMV β-galactosidase per milliliter of OPTI-MEM / LIPOFECTIN ™ mixture. The transfection mixture is mixed gently and a complex is allowed to form for approximately 15 minutes. The master mix reduces the error by ensuring that the control and experimental cells receive the same cationic / plasmid lipid complex. The concentration of the oligonucleotide in the culture medium is between 200 nM and 400 nM in all experiments. The oligonucleotide-containing media is removed from the cells and replaced with growth medium and incubated for a further 9-18 hours. The cells are rinsed with calcium-magnesium-free medium and the medium is removed. The plates are frozen at -70 ° C by >20 minutes and 100-300 μl of the reporter lysis buffer (Promega, Madison Wl). The cells are placed through two or more frozen thawing cycles, to ensure complete lysis. Luciferase assays are performed according to the manufacturer's instructions (Promega, Madison Wl) and luminescence is detected with a 96-well luminometer (Packard, Meriden CT). The β-galactosidase assays (Galacton Plus, Tropix) are performed according to the manufacturer's instructions and detected in the Packard luminometer. Experimental results The results of the luciferase assays are presented in Table I. The assays are reported as the percent specific inhibition by the formula 100 x (1- (LUCt / LUCc) oliso / (LUCt / LUCc) no oligo); wherein LUCT and LUCC are the levels of luciferase found in cells transfected with luciferase plasmids containing and lacking the ras gene insert (SEQ ID NO: 1) and the superscripts "oligo" and "no oligo" refer to the presence or absence of antisense oligonucleotides.

TABLE I Igonucleotides Formula Specific Irihibition Controls ("C") Cl 25MO 26% C2 25 s 15% C3 9DS16MO 15% C4 9Dol6MoInvT 0% C5 9Dpl6M? InvT 18% C6 9Dpl3M? 3Ms 14% Controls with all "S" Sl 25Ds 93% S2 16Ms8DsD 100% S3 8Ms9Ds7MsM 97% S4 9Dsl5 sM 95% 9Ds at the 3 'end (3") 3' I1 InvTMsl5Mo9DsInvT 59% 3 '12 2Msl4Mo9DsInvT 57% 3 '13 4Msl2Mo9DsInvT 65% 9Ds in the middle ("MI") My l 5Ms3M? 9Ds4Mo3MsM 64% MI2 2Ms6Mo9Ds7 (MsMo) InvT 71% MI3 3Ms6Mo9Ds6MoMsInvT 87% 9Ds at the 5 'end (5 T) 5'I1 9Dsl6M? InvT 83% 5' 12 9Dsl5MoMsInvT 85% 5'13 9Dsl6MoBiotin 90% 5 '14 9Dsl6Mp 91% 5' 15 9Dsl4MoMpD 90% 5 '16 9Dsl3M? 2MpD 94% 5 '17 9D = 12M? 3MpD 94% 5' 18 9Dsl4M? MsD 93% 5 '19 9Dsl3Mo2MsD 97% 5' 110 9Dsl2Mo3MsD 95% Key: M and D refer to 2"O-methyl and 2" deoxyribonucleotides, respectively. The letters "o", "s" and n "p" refer to nucleotides linked to phosphodiester phosphorothioate diester, and p-ethoxy phosphotriester. "InvT" refers to thymidine linked at 3"-3" or 5"-> 5" located at the respective 3"or 5" end of the oligomer. Table I shows the results of C1-C6 control oligonucleotides, all oligonucleotides S1-S4 phosphorothioate, and oligonucleotides of the invention having the RNase activation region at the 3 'end (3'11-3"13), in the middle (MI1-MI3) and in the 5"end (5Íl-5" -I10). The control oligonucleotides Cl, C2, C5 and C6 show low levels of specific inhibition since these oligonucleotides lack the activation region RNase H. Oligonucleotides C3 and C4 are inactive since the 3"end is not protected and since the native ssDNA is unstable respectively. All phosphorothioate oligonucleotides (Sl-S4) show specific inhibitions that are in the range between 93% and 100%, as do oligonucleotides 5"16-5'-110, which have an activation region of R-Xfasa H located in 5"and two or three nucleotides bound to modified p-ethoxy or phosphorothioate 2" o-methyl (Mp and Ms, respectively). Low levels of specific inhibition are observed when oligonucleotides with RNase H activation regions are used in 3"and means or when suboptimal 3" protecting groups are present Although the oligonucleotides of the invention have RNase H 5"activation regions they achieve specific inhibition levels which are comparable to those achieved by the uniform phosphorothiato oligonucleotides, the oligonucleotides of The invention is superior in that its use is associated with substantially less toxicity. Table II shows specific inhibition, the average metabolic activity as percent non-oligo control, as determined by the MTS assay, and the percentage of viable cells, as determined by trypan blue exclusion for conventional oligonucleotides (" C "), all phosphorothioate ("S"), 3"I, MI and 5" I. As well as for three species. TABLE II Oligonucleotide% of inh luc% of act. % of cells Metabolic of viable control Oligonucleotides 15% 94% 76% all "O" C1-C6 Oligonucleotides 96% 25? 21y all "S" S1-S4 3'I (1-4) 60% 70% 61% MI (1-3) 74% 77% 67% 5"I (1-10) 91% 71% 60% Best oligonucleotides in the diagram have high percentage values in all the columns The results show that the oligonucleotides of the invention achieve specific inhibition levels greater than four times higher than conventional oligonucleotides while showing toxicity levels that are substantially lower than oligonucleotides of phosphorothioate, the optimal group, 5"I, shows specific inhibition that is comparable to phosphorothioate oligonucleotides Effect of the location of the activation region of Rnasa H The cause of lower specific activity observed by type 3 oligonucleotides" I and MI "is investigated. One possibility is that oxidation steps using 0.02 M of I2 cause oxidation of the phosphorothioate to phosphodiester bonds, when nucleotides linked to phosphodiester 5"are added to the phosphorothioate linkages The comparison of oligonucleotides 9DS15D0D (" 5"S") and 15D09DSD ("3" s ") having the sequence of the test oligonucleotide by analytical HPLC analysis show that approximately 85% of the 5" S oligonucleotides are completely thiolated, by contrast only 26% of the 3"S oligonucleotides are completely thiolated (36% are Sl, 24% are S-2 and 14% are S-3.) Table III shows the distribution of fully thiolated and mono, di and tri-oxidized byproducts as a function of the position of the phosphorothiolated oligonucleotide region. Four thymidyl pentadecamers are synthesized using 0.02 M I2 as the oxidant for 15 nucleotides and a thiolating agent for nine nucleotides TABLE III Ts [I2] s Sl S-2 S-3 '-9Dsl5DoD03 '0.02M 96% 4% - - 5' -lD 9Dsl4DoD-3 '0.02M 85% 15% - - 5'-8Ds9Ds7DoD-3' 0.02M 41% 46% 12.5 0.5 '-15Do9DsD-3' 0.02M 32% 43% 20% 5% '-15Do9DsD-3' 0.001M 78% 14% 8% - the results show that 96% of the 5"S oligonucleotides are completely thiolated and this percentage decreases constantly as far as the phosphorothioate region is exposed to oxidation reactions more When the oxidizer concentration is reduced to 0.001 M, 78% of the fully thiolated 25-T oligonucleotides and about 60% of the oligonucleotides having the sequence of SEQ ID NO: 1 are synthesized. the publications and patents mentioned above for reference Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention Although the invention has been described in connection with preferred embodiments specific, it should be understood that the invention as claimed should not be excessively limited to such specific modalities. In effect, several modifications of the modes described above for carrying out the invention are proposed which are obvious to those skilled in the field of molecular biology or related fields to be within the scope of the following claims.

Claims (20)

1. CLAIMS 1. A chimeric antisense oligonucleotide comprising: a 5"terminus;" 3"terminus; and about 11 to about 59 5"to 3" linked nucleotides independently selected from the group consisting of 2'-deoxyphosphorothioate nucleotides, 2"-modified phosphorothioate nucleotides, 2-modified" phosphodiester nucleotides, 2-modified "p-alkyloxyphosphotriester nucleotides, because: a) the oligonucleotide incorporates an RNase H activation region of between about 3 and about 12 bases linked to contiguous 2"-deoxyphosphorothioate; b) the 5 'nucleotide link plus 5"to 3" is a phosphorothioate or p-alkyloxyphosphodiester linkage; c) the 3"plus 5" to 3 'nucleotide linkage is a phosphorothioate or p-alkyloxyphosphodiester linkage or the 3' termination is blocked; and d) the oligonucleotide contains no more than 12 contiguous 2'-deoxyphosphorothioate bonds.
2. 2. The oligonucleotide according to claim 1, characterized in that the 3"terminus is not blocked by the nucleotide bound to 3" to 3"phosphorothioate 3. The oligonucleotide according to claim 1, characterized in that the 3" terminus is blocked by a portion comprising the nucleotide linked 3"to 3" phosphorothioate. The oligonucleotide according to claim 1, characterized in that the 3 'terminus is blocked by a portion comprising the nucleotide linked 3"to 3" phosphodiester. 5. The oligonucleotide according to claim 4, characterized in that the nucleotide bond 3"plus 5" to 3"is a phosphorothioate bond or a p-ethoxyphosphotriester bond 6. The oligonucleotide according to claim 4, characterized in that the link nucleotide 5 'plus 5"to 3" is a phosphorothioate bond or a p-ethoxyphosphotriester linkage 7. The oligonucleotide according to claim 1, characterized in that the phosphorothioate 2"-modified nucleotides are present at both the 3' endings and the termination. 5'. The oligonucleotide according to claim 1, characterized in that the RNase H activation region is located at the 5"terminus 9. The oligonucleotide according to claim 8, characterized in that the bond 3" plus 5"to 3" nucleotide is a phosphorothioate bond or a p-ethoxyphosphotriester bond. 10. The oligonucleotide according to claim 9, characterized in that two 3"plus 5" - "3" bonds are independently either a phosphorothioate bond or a p-ethoxyphosphotriester linkage 11. The oligonucleotide according to claim 9, characterized because the activation region Rnasa H is contiguous with the nucleotide bond 3 'plus 5"- > 3. The oligonucleotide according to claim 11, characterized in that the 2'-modified phosphodiester nucleotide is a 2'-methoxy or 2"-fluor nucleotide. The oligonucleotide according to claim 11, characterized in that it additionally comprises at least thirteen nucleotides 2"-methoxyphosphodiester 14. The oligonucleotide according to claim 11, characterized in that it has between 15 and 50 nucleotides. according to claim 14, characterized in that it additionally comprises at least eight 2"-methoxyphosphodiester nucleotides. 16. The oligonucleotide according to claim 14, characterized in that it additionally comprises at least thirteen 2"-methoxyphosphodiester nucleotides. 17. The oligonucleotide according to claim 1, characterized in that the activation region comprises the termination 3"18. The oligonucleotide according to claim 1, characterized in that the phosphodiester 2-modified nucleotides are selected from the group consisting of 2 '. -fluorine and 2'-methoxynucleotides. 19. The oligonucleotide according to claim 4, characterized in that the Rnasa H activation region is present at the 5 'terminus followed by four to approximately forty 2'-methoxynucleotides linked 5"to 3" and the 3"terminus is blocked by a deoxyribonucleotide linked 3"to 3" phosphodiester 20. A method for specifically cleaving an RNA in a cell containing RNase H which is characterized in that it comprises administering an effective amount of an oligonucleotide complementary to the RNA, the oligonucleotide comprising a "; a 3"terminus, and about 11 to about 59 5" to 3"linked nucleotides independently selected from the group consisting of 2" -deoxyphosphorothioate nucleotides, phosphorothioate nucleotides 2'-modified, 2'-modified phosphodiester nucleotides, 2'-modified p-alkyloxyphosphotriester nucleotides, where | : a) the oligonucleotide incorporates an RNase H activation region of between approximately 3 and approximately 12 2"-deoxyphosphorothioate adjoining phosphorothioate; b) the 5" plus 5"to 3 'nucleotide linkage is a phosphorothioate or p-linkage; alkyloxyphosphodiester, c) the 3 'plus 5' to 3 'nucleotide linkage is a phosphorothioate or p-alkyloxyphosphodiester linkage or the 3' termini is blocked, and d) the oligonucleotide contains no more than 12 contiguous 2'-deoxyphosphorothioate bonds.