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WO2000004189A1 - Oligonucleotides modifies - Google Patents

Oligonucleotides modifies Download PDF

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Publication number
WO2000004189A1
WO2000004189A1 PCT/US1999/015886 US9915886W WO0004189A1 WO 2000004189 A1 WO2000004189 A1 WO 2000004189A1 US 9915886 W US9915886 W US 9915886W WO 0004189 A1 WO0004189 A1 WO 0004189A1
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Prior art keywords
oligonucleotide
oligonucleotides
alkyl
linkages
substituent
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PCT/US1999/015886
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English (en)
Inventor
Muthiah Manoharan
Phillip Dan Cook
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Isis Pharmaceuticals, Inc.
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Priority to AU53149/99A priority Critical patent/AU5314999A/en
Publication of WO2000004189A1 publication Critical patent/WO2000004189A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids

Definitions

  • This invention is directed to modified oligonucleotides that include one or more 2 '-5' internucleotide linkages and a modified nucleotide at one of the two nucleotides that are linked by the 2 ',5' linkage. That nucleotide is modified, for example, by incorporating a substituent at its 3 '-position.
  • the modified oligonucleotides of the present invention exhibit improved properties of nuclease resistance and binding affinity, and are of use as antisense oligonucleotides.
  • Oligonucleotides are now accepted as therapeutic agents with great promise. Oligonucleotides are known to hybridize to single-stranded DNA or RNA molecules. Hybridization is the sequence-specific base pair hydrogen bonding of nucleobases of the oligonucleotide to the nucleobases of the target DNA or RNA molecule. Such nucleobase pairs are said to be complementary to one another.
  • the concept of inhibiting gene expression through the use of sequence-specific binding of oligonucleotides to target RNA sequences also known as antisense inhibition, has been demonstrated in a variety of systems, including living cells (for example see: Wagner et al .
  • hybridization arrest denotes the terminating event in which the oligonucleotide inhibitor binds to the target nucleic acid and thus prevents, by simple steric hindrance, the binding of essential proteins, most often ribosomes, to the nucleic acid.
  • Methyl phosphonate oligonucleotides Miller, P.S. and Ts'O, P.O. P. (1987) Anti - Cancer Drug Design, 2:117-128, and ⁇ -anomer oligonucleotides are the two most extensively studied antisense agents which are thought to disrupt nucleic acid function by hybridization arrest .
  • the second type of terminating event for antisense oligonucleotides involves the enzymatic cleavage of the targeted RNA by intracellular RNase H.
  • a 2 ' -deoxyribof ranosyl oligonucleotide or oligonucleotide analog hybridizes with the targeted RNA and this duplex activates the RNase H enzyme to cleave the RNA strand, thus destroying the normal function of the RNA.
  • Phosphorothioate oligonucleotides are the most prominent example of an antisense agent that operates by this type of antisense terminating event.
  • Oligonucleotides may also bind to duplex nucleic acids to form triplex complexes in a sequence specific manner via Hoogsteen base pairing (Beal et al . , Science, (1991) 251:1360-1363; Young et al . , Proc . Na tl . Acad. Sci . (1991) 88:10023-10026). Both antisense and triple helix therapeutic strategies are directed towards nucleic acid sequences that are involved in or responsible for establishing or maintaining disease conditions. Such target nucleic acid sequences may be found in the genomes of pathogenic organisms including bacteria, yeasts, fungi, protozoa, parasites, viruses, or may be endogenous in nature. By hybridizing to and modifying the expression of a gene important for the establishment, maintenance or elimination of a disease condition, the corresponding condition may be cured, prevented or ameliorated.
  • the relative ability of an oligonucleotide to bind to the complementary nucleic acid may be compared by determining the melting temperature of a particular hybridization complex.
  • T m is measured by using the UV spectrum to determine the formation and breakdown (melting) of the hybridization complex.
  • Base stacking which occurs during hybridization, is accompanied by a reduction in UV absorption (hypochromicity) .
  • Oligonucleotides may also be of therapeutic value when they bind to non-nucleic acid biomolecules such as intracellular or extracellular polypeptides, proteins, or enzymes. Such oligonucleotides are often referred to as aptamers' and they typically bind to and interfere with the function of protein targets (Griffin, et al . , Blood, (1993), 81:3271-3276; Bock, et al . , Na ture, (1992) 355: 564-566).
  • Oligonucleotides and their analogs have been developed and used for diagnostic purposes, therapeutic applications and as research reagents.
  • oligonucleotides For use as therapeutics, oligonucleotides must be transported across cell membranes or be taken up by cells, and appropriately hybridize to target DNA or RNA. These critical functions depend on the initial stability of the oligonucleotides toward nuclease degradation.
  • a serious deficiency of unmodified oligonucleotides which affects their hybridization potential with target DNA or RNA for therapeutic purposes is the enzymatic degradation of administered oligonucleotides by a variety of intracellular and extracellular ubiquitous nucleolytic enzymes referred to as nucleases.
  • the oligonucleotides should demonstrate enhanced binding affinity to complementary target nucleic acids, and preferably be reasonably stable to nucleases and resist degradation.
  • oligonucleotides need not necessarily possess nuclease stability.
  • oligonucleotides A number of chemical modifications have been introduced into oligonucleotides to increase their binding affinity to target DNA or RNA and resist nuclease degradation. Modifications have been made to the ribose phosphate backbone to increase the resistance to nucleases. These modifications include use of linkages such as methyl phosphonates, phosphorothioates and phosphorodithioates, and the use of modified sugar moieties such as 2 ' -O-alkyl ribose. Other oligonucleotide modifications include those made to modulate uptake and cellular distribution. A number of modifications that dramatically alter the nature of the internucleotide linkage have also been reported in the literature.
  • oligonucleotides usually for diagnostic and research applications, is labeling with non-isotopic labels, e.g., fluorescein, biotin, digoxigenin, alkaline phosphatase, or other reporter molecules.
  • non-isotopic labels e.g., fluorescein, biotin, digoxigenin, alkaline phosphatase, or other reporter molecules.
  • RNA hybrid duplexes frequently adopt a C3 ' endo conformation.
  • modifications that shift the conformational equilibrium of the sugar moieties in the single strand toward this conformation should preorganize the antisense strand for binding to RNA.
  • electronegative substituents such as 2 ' -fluoro or 2'-alkoxy shift the sugar conformation towards the northern pucker conformation. This pucker conformation further assisted in increasing the Tm of the oligonucleotide with its target. Large substituents at the 2'- position are, however, not well tolerated.
  • a clear correlation between substituent size at the 2 ' -position and duplex stability has been observed and reported in the literature.
  • 2'-0-alkyl substituents provide a strong positive influence on the binding affinity of oligonucleotides (Freier and Altmann, Nucleic Acids Research , (1997) 25:4429-4443). Small alkoxy groups were very favorable, and larger alkoxy groups at the 2 ' -position were found to be unfavorable. However, if the 2 ' -substituent contained an ethylene glycol motif, then a strong improvement in binding affinity to the target RNA was observed. This is suggested to arise from gauche interactions between the oxygen g to the 2 ' -oxygen atom results in a configuration of the side chain that is favorable for duplex formation.
  • 2 ' , 5/-01igoadenylates are naturally occurring RNA isomers that are implicated in the regulation of cell growth and in the antiviral mechanism of interferon. Because of the poor uptake of such oligonucleotides and the relatively nonspecific endonucleolytic action of its target RNase L, chimeric oligonucleotides that incorporate 2-5A motifs together with an antisense construct for a specific target have also been studied (Lesiak et al, Bioconj uga te Chem . , 1993, 4, 467-472).
  • 2 ' , 5/-01igonucleotides have also been the focus of research aimed at understanding the evolutionary bias towards 3 ',5' instead of 2 ',5' linked double helices to encode genetic information (Prakash et al , Angew. Chemie, 1997, 36, 1522-23) ; 2 ',5' linkages were found to be more susceptible to hydrolysis that their 3 ',5' analogs.
  • 2 ',5' phosphodiester linked oligoribonucleotides have also been studied for their binding to RNA and DNA (Giannaris and Damha, Nucl . Acids Res . , 1993, 21 , 4742-4749) .
  • duplex formed by 2 ',5' -DNA with RNA has a stability (Tm) similar to that for the 3 ' , 5 ' -DNA-RNA duplex but that this binding is of a hybrid nature (Prakash et al . , Chem . Commun . , 1996, 1793-94).
  • the present invention provides modified oligonucleotides that are easy to synthesize and exhibit good properties of nuclease resistance and hybridization to target nucleic acids. This and other objects of the invention will be apparent from a consideration of the specification as a whole .
  • the present invention provides oligonucleotides comprising a plurality of nucleotides linked together by internucleotide linkages.
  • Each nucleotide includes a sugar portion and a base portion, and at least one of the internucleotide linkages is a 2 ', 5 ' -linkage wherein at least one of the linked nucleotides bears a 3 ' -substituent of the formula : where : Z is 0, S, NH, or N-R 22 -(R 23 ) V
  • R 22 is C 1 -C 20 alkyl, C 2 -C 20 alkenyl, or C 2 -C 20 alkynyl;
  • R 23 is R 24 when Z is 0;
  • R 23 is hydrogen or R 24 when Z is S, NH, or N-R 22 - (R 23 ) v ;
  • R 24 is amino, halogen, hydroxyl, thiol, keto, carboxyl, nitro, nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl, N-dialkyl, 0- aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl, amino, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino, hydroxyalkyamino, hydroxydialkylamino, disulfide, silyl, aryl, heterocycle, carbocycle, intercalator, reporter molecule, conjugate, polyamine, polyamide, polyalkylene glycol, poly- ether, a group that enhances the pharmacodynamic properties
  • the present invention further provides oligonucleotides bearing at least one 2 ',5' internucleotide linkage wherein at least one of the linked nucleotides includes an alkoxyalkoxy , dialkoxyalkoxy , hydroxyalkoxy , dihydroxyal koxy , aminoalkoxy, al kylaminoal koxy , dialkylaminoalkoxy, dialkylaminooxyalkoxy, haloalkoxy, dihaloalkoxy or trihaloalkoxy 3 ' -substituent and protected versions of the same.
  • the present invention provides oligonucleotides bearing at the 3 '-position a substituent selected from the group consisting of, but not limited to, me t hox y e t h o x y , h y d r o x ye t h oxy , dimethylaminooxyethoxy, trifluoromethylethoxy, aminopropoxy, and protected versions of the same.
  • the present invention also provides oligonucleotides bearing methoxyethoxy substituents at one or more 2 ' -positions on the sugar portion of the nucleotides.
  • the present invention further provided oligonucleotide having at least one 2 ',5' internucleotide linkage wherein at least one of the linked nucleotides includes a 3 ' -substituent having one of the formulas:
  • the present invention further provides oligonucleotides comprising a plurality of nucleotides linked together by internucleotide linkages, wherein the linkage is selected from a group consisting of, but not limited to, phosphorus-containing and non-phosphorus-containing linkages.
  • Phosphorus-containing linkages include, but are not limited to, phosphodiester, phosphorothioate , phosphoramidate , alkylphosphonate, N3'->P5' phosphoramidate, phosphinate, phosphate, thiophosphate and phosphorodithioate linkages.
  • Non- phosphorus-containing linkages include, but are not limited to, glycol, ether, all carbon atom, urea, carbamate, amide, cyclic, amine, hydroxylamine, hydrazino, -substituted amide 3, and methylene (methylimino) linkages.
  • the internucleotide linkages present in the oligonucleotides of the present invention are either all phosphodiester or all phosphorothioate. In a further preferred embodiment, the internucleotide linkages present in the oligonucleotides of the present invention are any combination of at least one phosphodiester and at least one phosphorothioate linkage.
  • Figure 1 shows the stability of modified oligonucleotides in mouse plasma 1 h. after i . v. bolus administration.
  • Figure 2 shows the stability of modified oligonucleotides in mouse tissue 24 h. after i . v. bolus administration.
  • Figure 3 shows increased in vivo stability of modified oligonucleotides of the invention as compared to a phosphorothioate oligonucleotide in tissue samples isolated from mouse liver and mouse kidney.
  • Figure 4 shows the effects of modified oligonucleotides on c-raf mRNA expression in bEND cells.
  • the present invention provides oligonucleotides comprising a plurality of nucleotides linked together by internucleotide linkages wherein at least one of the internucleotide linkages is a 2 ' , 5 ' -linkage .
  • the nucleotide subunits may be "natural” or “synthetic" moieties.
  • Each nucleotide unit is formed from a naturally occurring or synthetic base portion and a naturally occurring or synthetic pentofuranosyl sugar portion.
  • oligonucleotide refers to a polynucleotide formed from a plurality of linked nucleotide units.
  • the nucleotide units each include a nucleoside unit.
  • nucleotide units in oligonucleotides may be linked together via linkages that may be phosphorus-containing or non-phosphorus-containing.
  • oligonucleotide also includes naturally occurring species and synthetic species formed from naturally occurring or synthetic subunits. Oligonucleotides according to the present invention also can include modified subunits. Such modified subunits include modified portions, be they modified sugar moieties or modified base moieties, that function similarly to natural bases and natural sugars.
  • modified bases include deaza or aza purines and pyrimidines used in place of natural purine and pyrimidine bases; pyrimidines having substituent groups at the 5 or 6 position; and purines having altered or replacement substituent groups at the 2, 6, or 8 positions.
  • nucleobases include adenine, guanine, cytosine, uridine, and thymine, as well as other non- naturally occurring and natural nucleobases such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halo uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo uracil) , 4-thiouracil, 8-halo, oxa, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoro- methyl and other 5-substituted uracils and cytosines, 7-methyl- guanine.
  • nucleobases include
  • nucleobases include those disclosed in U.S. Patent No. 3,687,808 (Merigan, et al . ) , in chapter 15 by Sanghvi, in Antisense Research and Applica tion, Ed. S. T. Crooke and B. Lebleu, CRC Press, 1993, in Englisch et al . , Angewandte Chemie, International Edition, 1991, 30, 613-722 (see especially pages 622 and 623, and in the Concise Encyclopedia of Polymer Science and Engineering, J.I.
  • nucleosidic base is further intended to include heterocyclic compounds that can serve as like nucleosidic bases including certain "universal bases” that are not nucleosidic bases in the most classical sense but serve as nucleosidic bases. Especially mentioned as a universal base is 3-nitropyrrole.
  • modified sugars include carbocyclic or acyclic sugars, sugars having substituent groups at their 2' position, sugars having substituent groups at their 3' position, and sugars having substituents in place of one or more hydrogen atoms of the sugar.
  • Other altered base moieties and altered sugar moieties are disclosed in United States Patent 3,687,808 and PCT application PCT/US89/02323.
  • Altered base moieties or altered sugar moieties also include other modifications consistent with the spirit of this invention.
  • Such oligonucleotides are best described as being structurally distinguishable from, yet functionally interchangeable with, naturally occurring or synthetic wild type oligonucleotides. All such oligonucleotides are comprehended by this invention so long as they function effectively to mimic the structure of a desired RNA or DNA strand.
  • the oligonucleotides of the present invention preferably comprise from about 10 to about 30 subunits. It is more preferred that such oligonucleotides comprise from about 15 to about 25 subunits.
  • a subunit is a base and a sugar combination suitably bound to adjacent subunits through a linkage such as, for example, a phosphorus-containing (e.g., phosphodiester and phosphorothioate) linkage or some other non- phosphorus-containing linking moiety.
  • the nucleoside subunits need not be linked in any particular manner, so long as they are covalently bound.
  • Exemplary linkages are those between the 3' and 5' positions of adjacent nucleosides, as is observed in natural nucleic acids; such linkages are referred to as "3", 5'- linkages.” Linkages between the 2' and 5' positions of adjacent nucleosides are refereed to as “2 ', 5 ' -linkages” .
  • RNA or DNA portion which is to be modulated using oligonucleotides of the present invention be preselected to comprise that portion of DNA or RNA which codes for the protein whose formation or activity is to be modulated.
  • the targeting portion of the composition to be employed is thus selected to be complementary to the preselected portion of the targeted DNA or RNA, that is, to be an antisense oligonucleotide for that portion.
  • the present invention provides oligonucleotides comprising a plurality of linked nucleotides wherein at least one of the internucleotide linkages is a 2 ' , 5 ' -linkage .
  • a 2 ', 5 ' -linkage is one that covalently connects the 2 '-position of the sugar portion of one nucleotide subunit with the 5'- position of the sugar portion of an adjacent nucleotide subunit.
  • the actual linking moiety that accomplishes the 2 ', 5 ' -linkage may be one of any of the many linking moieties known in the art and described in the articles and patents listed above.
  • the present invention further provides oligonucleotides comprising a plurality of nucleotides linked together by internucleotide linkages, wherein the linkage is selected from a group consisting of, but not limited to, phosphorus-containing and non-phosphorus-containing linkages.
  • Phosphorus-containing linkages include, but are not limited to, phosphodiester, phosphorothioate, phosphoramidate, alkylphosphonate, N3 ' ->P5 ' phosphoramidate, phosphinate, phosphate, thiophosphate and phosphorodithioate linkages .
  • Non- phosphorus-containing linkages include, but are not limited to, glycol, ether, all carbon atom, urea, carbamate, amide, cyclic, amine, hydroxylamine, hydrazino, -substituted amide 3, and methylene (methylimino) linkages.
  • the internucleotide linkages present in the oligonucleotides of the present invention are either all phosphodiester (PO) or all phosphorothioate (PS) .
  • the internucleotide linkages present in the oligonucleotides of the present invention are any combination of at least one phosphodiester and at least one phosphorothioate linkage.
  • Oligonucleotides that bear only PO linkages are found to not only bind well to target nucleic acids but to also exhibit nuclease resistance imparted by the presence of the one or more 2 ', 5 ' -linkages .
  • Oligonucleotides that bear only PS linkages have enhanced nuclease resistance but poorer binding (due to decreased T m values for PS linkages) to their target nucleic acids.
  • Mixed phosphorothioate/phosphodiester linked oligonucleotides are advantageous because the offer improved binding because of the presence of PO linkages, while retaining nuclease resistance imparted by the PS and 2 ', 5 ' -linkages .
  • the present invention further provides oligonucleotides having at least one 2 ',5' internucleotide linkage wherein at least one of the nucleotides comprising such a linkage bears a 3 ' -substituent of the formula
  • Z is 0, S, NH, or N-R 22 -(R 23 ) V
  • R 22 is C -C ⁇ alkyl , C 2 -C 20 alkenyl , or C 2 -C 20 alkynyl ;
  • R 23 is R 24 when Z is 0 ; otherwise R 23 is hydrogen or R 24 when Z is S, NH, or N-R 22 -(R 23 ) V ;
  • R 24 is amino, halogen, hydroxyl, thiol, keto, carboxyl, nitro, nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl, N-dialkyl, 0- aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl, amino,
  • N-phthalimido imidazole, azido, hydrazino, hydroxylamino, hydroxyalkyamino, hydroxydialkylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl, heterocycle, carbocycle, intercalator, reporter molecule, conjugate, poly- amine, polyamide, polyalkylene glycol, polyether, a group that enhances the pharmacodynamic properties of oligonucleotides, or a group that enhances the pharmacokinetic properties of oligonucleotides; v is from 0 to about 10.
  • One particularly preferred 3 ' -substituents of the invention includes 3 ' -methoxyethoxy [3 ' -0-CH 2 CH 2 OCH 3 , also known as 3 ' -0- (2-methoxyethyl) or 3'-M0E], an alkoxyalkoxy group.
  • a further preferred modification includes 3'- dimethylaminooxyethoxy, i.e., a 0 (CH 2 ) 2 ON (CH 3 ) 2 group, also known as 3'-DMA0E.
  • the corresponding 2 ' -DMAOE group is described in co-owned United States patent application Serial Number 09/016,520, filed on January 30, 1998, the contents of which are herein incorporated by reference.
  • Other preferred modifications include 3 ' -methoxy (2'-0-CH 3 ) and 3 ' -aminopropoxy (3'-0CH 2 CH 2 CH 2 NH 2 ) .
  • a preferred group of compounds of the invention include oligonucleotides having at least one 2 ',5' internucleotide linkage where the nucleotide of the 2 ' side of the 2 ',5' linkage includes a 3' substituent that is an alkoxyalkoxy, dialkoxyalkoxy, hydroxyalkoxy, dihydroxyalkoxy, aminoalkoxy, alkylaminoalkoxy , dialkylaminoalkoxy, dialkylaminooxyalkoxy, haloalkoxy, dihaloalkoxy or trihaloalkoxy substituent and protected versions of the same.
  • Particularly preferred 3 ' -substituents include methoxyethoxy, hydroxyethoxy, dimethylaminooxyethoxy, trifluoromethylethoxy, aminopropoxy, and protected versions of the same.
  • This additional modification, at the 3 '-position, to the structure of oligonucleotides provides enhanced properties to the oligonucleotides of the present invention. It has been observed that incorporating these unique substitutions onto the 3 '-position of 2 ',5 '-linked oligonucleotides results in enhanced binding of the oligonucleotides to their target nucleic acids.
  • Oligonucleotides of the invention can also include sugar substitutions, particularly O-substitutions, on the ribosyl ring.
  • substitutions for ring 0 include S, CH 2 , CHF, and CF 2 , see, e.g., Secrist, et al., Abstract 21, Program & Abstracts, Tenth International Roundtable, Nucleosides , Nucleotides and their Biological Applications, Park City, Utah, Sept. 16-20, 1992, hereby incorporated by reference in its entirety.
  • oligonucleotide may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide and the 5' position of 5' terminal nucleotide.
  • one additional modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide.
  • moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553), cholic acid (Manoharan et al., Bioorg.
  • a thioether e.g., hexyl-S- tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J.
  • a phospholipid e.g., di-hexadecyl- rac-glycerol or triethylammonium 1, 2-di-0-hexadecyl-rac- glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990, 18, 3777), a polyamine or a polyethylene glycol chain (Manoharan et al .
  • alkyl includes but is not limited to straight chain, branch chain, and cyclic unsaturated hydrocarbon groups including but not limited to methyl, ethyl, and isopropyl groups.
  • Alkyl groups of the present invention may be substituted. Representative alkyl substituents are disclosed in United States Patent No. 5,212,295, at column 12, lines 41-50, hereby incorporated by reference in its entirety.
  • Alkenyl groups according to the invention are to straight chain, branch chain, and cyclic hydrocarbon groups containing at least one carbon-carbon double bond, and alkynyl groups are to straight chain, branch chain, and cyclic hydrocarbon groups containing at least one carbon-carbon triply bond. Alkenyl and alkynyl groups of the present invention can be substituted.
  • Aryl groups are substituted and unsubstituted aromatic cyclic moieties including but not limited to phenyl, naphthyl, anthracyl, phenanthryl, pyrenyl, and xylyl groups.
  • Alkaryl groups are those in which an aryl moiety links an alkyl moiety to a core structure, and aralkyl groups are those in which an alkyl moiety links an aryl moiety to a core structure .
  • hetero denotes an atom other than carbon, preferably but not exclusively N, 0, or S.
  • heterocycloalkyl denotes an alkyl ring system having one or more heteroatoms (i.e., non-carbon atoms) .
  • Preferred heterocycloalkyl groups include, for example, morpholino groups.
  • heterocycloalkenyl denotes a ring system having one or more double bonds, and one or more heteroatoms.
  • Preferred heterocycloalkenyl groups include, for example, pyrrolidino groups .
  • Oligonucleotides according to the present invention that are hybridizable to a target nucleic acid preferably comprise from about 5 to about 50 nucleosides. It is more preferred that such compounds comprise from about 8 to about 30 nucleosides, with 15 to 25 nucleosides being particularly preferred.
  • a target nucleic acid is any nucleic acid that can hybridize with a complementary nucleic acid-like compound.
  • hybridization shall mean hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding between complementary nucleobases.
  • adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds.
  • “Complementary” and “specifically hybridizable, " as used herein, refer to precise pairing or sequence complementarity between a first and a second nucleic acid-like oligomers containing nucleoside subunits. For example, if a nucleobase at a certain position of the first nucleic acid is capable of hydrogen bonding with a nucleobase at the same position of the second nucleic acid, then the first nucleic acid and the second nucleic acid are considered to be complementary to each other at that position.
  • the first and second nucleic acids are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleobases which can hydrogen bond with each other.
  • “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity such that stable and specific binding occurs between a compound of the invention and a target RNA molecule. It is understood that an oligomeric compound of the invention need not be 100% complementary to its target RNA sequence to be specifically hybridizable.
  • An oligomeric compound is specifically hybridizable when binding of the oligomeric compound to the target RNA molecule interferes with the normal function of the target RNA to cause a loss of utility, and there is a sufficient degree of complementarity to avoid nonspecific binding of the oligomeric compound to non-target sequences under conditions in which specific binding is desired, i.e. under physiological conditions in the case of in vivo assays or therapeutic treatment, or in the case of in vitro assays, under conditions in which the assays are performed.
  • the present invention also provides oligonucleotides bearing methoxyethoxy substituents at one or more 2 ' -positions on the sugar portion of the nucleotides. When placed at the 2 ' -position of antisense oligonucleotides, these substituents are capable of improving the critical properties of nuclease resistance and binding. Therefore, the present invention also provides oligonucleotides that not only are 2 ' , 5 ' -linked-3 ' - substituted-oligonucleotides but are also PO, PS or mixed PO/PS in nature and may incorporate at one or more available 2'- positions appropriate substitutions. It is preferred that the 2 ' -substituent be a methoxyethoxy group.
  • the oligonucleotides of the present invention can be used in diagnostics, therapeutics and as research reagents. They can be used in pharmaceutical compositions by including a suitable pharmaceutically acceptable diluent or carrier. They further can be used for treating organisms having a disease characterized by the undesired production of a protein. The organism should be contacted with an oligonucleotide having a sequence that is capable of specifically hybridizing with a strand of nucleic acid coding for the undesirable protein. Treatments of this type can be practiced on a variety of organisms ranging from unicellular prokaryotic and eukaryotic organisms to multicellular eukaryotic organisms.
  • RNA-DNA transcription or RNA-protein translation as a fundamental part of its hereditary, metabolic or cellular control is susceptible to therapeutic and/or prophylactic treatment in accordance with this invention. Seemingly diverse organisms such as bacteria, yeast, protozoa, algae, all plants and all higher animal forms including warm-blooded animals, ca be treated. Further each cell of multicellular eukaryotes can be treated since they include both DNA-RNA transcription and RNA-protein translation as integral parts of their cellular activity. Many of the organelles (e.g., mitochondria and chloroplasts) of eukaryotic cells also include transcription and translation mechanisms.
  • organelles e.g., mitochondria and chloroplasts
  • single cells, cellular populations or organelles can also be included within the definition of organisms that can be treated with therapeutic or diagnostic oligonucleotides.
  • therapeutics is meant to include the eradication of a disease state, by killing an organism or by control of erratic or harmful cellular growth or expression.
  • Oligonucleotides according to the invention can be assembled in solution or through solid-phase reactions, for example, on a suitable DNA synthesizer utilizing nucleosides, phosphoramidites and derivatized controlled pore glass (CPG) according to the invention and/or standard nucleotide precursors.
  • CPG controlled pore glass
  • the nucleoside and nucleotide precursors used in the present invention may carry substituents either the 2 ' or 3' positions.
  • Such precursors may be synthesized according to the present invention by reacting appropriately protected nucleosides bearing at least one free 2' or 3 ' hydroxyl group with an appropriate alkylating agent such as , but not limited to, alkoxyalkyl halides, alkoxylalkylsulfonates, hydroxyalkyl halides, hydroxyalkyl sulfonates, aminoalkyl halides, aminoalkyl sulfonates, phthalimidoalkyl halides, phthalimidoalkyl sulfonates, alkylaminoalkyl halides, alkylaminoalkyl sulfonates, dialkylaminoalkyl halides, dialkylaminoalkylsulfonates, dialkylaminooxyalkyl halides, dialkylaminooxyalkyl sulfonates and suitably protected versions of the same.
  • an appropriate alkylating agent such as , but not limited to, al
  • Preferred halides used for alkylating reactions include chloride, bromide, fluoride and iodide.
  • Preferred sulfonate leaving groups used for alkylating reactions include, but are not limited to, benzenesulfonate, methylsulfonate, tosylate, p -b r omobe n z ene s u 1 f ona t e , triflate, tri fluoroethyl sulfonate , and ( 2 , 4 - dinitroanilino) benzenesulfonate .
  • nucleosides can be assembled into oligonucleotides according to known techniques. See, for example, Beaucage et al . , Tetrahedron, 1992, 48, 2223.
  • the ability of oligonucleotides to bind to their complementary target strands is compared by determining the melting temperature (T m ) of the hybridization complex of the oligonucleotide and its complementary strand.
  • T m melting temperature
  • the melting temperature (T m ) a characteristic physical property of double helices, denotes the temperature (in degrees centigrade) at which 50% helical (hybridized) versus coil (unhybridized) forms are present.
  • T m is measured by using the UV spectrum to determine the formation and breakdown (melting) of the hybridization complex.
  • Base stacking, which occurs during hybridization, is accompanied by a reduction in UV absorption (hypochromicity) .
  • oligonucleotides of the present invention were determined using protocols as described in the literature (Freier and Altmann, Nucl . Acids Research, 1997, 25, 4429-443) . Typically absorbance versus temperature curves were determined using samples containing 4uM oligonucleotide in 100 mM Na+, 10 mM phosphate, 0.1 mM EDTA, and 4uM complementary, length matched RNA.
  • the in vivo stability of oligonucleotides is an important factor to consider in the development of oligonucleotide therapeutics. Resistance of oligonucleotides to degradation by nucleases, phosphodiesterases and other enzymes is therefore determined.
  • Typical in vivo assessment of stability of the oligonucleotides of the present invention is performed by administering a single dose of 5 mg/kg of oligonucleotide in phosphate buffered saline to BALB/c mice. Blood collected at specific time intervals post-administration is analyzed by HPLC or capillary gel electrophoresis (CGE) to determine the amount of oligonucleotide remaining intact in circulation and the nature the of the degradation products. Increased in vivo nuclease resistance has been observed.
  • CGE capillary gel electrophoresis
  • the CGE analysis of blood plasma samples from mice dosed with the oligonucleotides of the present invention reveals the relative nuclease resistance of 2 ',5 '-linked oligomers compared to a uniform 2 ' -deoxy-phosphorothioate oligonucleotide targeted to mouse c-raf) . Because of the nuclease resistance of the 2 ', 5 ' -linkage, coupled with the fact that 3 ' -methoxyethoxy substituents are present and afford further nuclease protection the oligonucleotides of the invention were found to be more stable in plasma than the uniform 2 ' -deoxy-phosphorthioate oligonucleotide.
  • oligonucleotides of the present invention hold promise as antisense agents with longer durations of action.
  • Some oligonucleotides of the present invention have also been assessed for their activity in controlling the c-raf message in bEND cells. Comparable or better activity at controlling c-raf mRNA expression was observed with 2 ',5'- linked-3 ' -substituted oligonucleotides of the present invention.
  • a solution of adenosine (42.74 g, 0.16 mol) in dry dimethyl formamide (800 mL) at 5°C was treated with sodium hydride (8.24 g, 60% in oil prewashed thrice with hexanes, 0.21 mol). After stirring for 30 min, 2-methoxyethyl bromide (0.16 mol) was added over 20 min. The reaction was stirred at 5°C for 8 h, then filtered through Celite. The filtrate was concentrated under reduced pressure followed by coevaporation with toluene (2x100 mL) . The residue was adsorbed on silica gel (100 g) and chromatographed (800 g, chloroform-methanol 9:1-4:1).
  • Aqueous methanol (50%, 1.2 L) was added. The resulting brown suspension was heated to reflux for 5 h. The suspension was concentrated under reduced pressure to one half volume in order to remove most of the methanol. Water (600 mL) was added and the solution was heated to reflux, treated with charcoal (5 g) and hot filtered through Celite. The solution was allowed to cool to 25°C. The resulting precipitate was collected, washed with water (200 mL) and dried at 90°C/0.2 mmHg for 5 h to give a constant weight of 87.4 g (89%) of tan, crystalline solid; mp 247°C (shrinks), 255°C (dec, lit.
  • the crude product containing a ratio of 4:1 of the 2'/3' isomers, was chromatographed on silica gel (500 g, chloroform-methanol 4:1) . The appropriate fractions were combined and concentrated under reduced pressure to a semi-solid (12 g) . This was triturated with methanol (50 mL) to give a white, hygroscopic solid. The solid was dried at 40°C/0.2 mmHg for 6 h to give a pure 2' product and the pure 3' isomer, which were confirmed by NMR.
  • 3 ' -0- (2-methoxyethyl) -2, 6- diaminopurine riboside (0.078 mol) was dissolved in monobasic sodium phosphate buffer (0.1 M, 525 mL, pH 7.3-7.4) at 25°C.
  • Adenosine deaminase (Sigma type II, 1 unit/mg, 350 mg) was added and the reaction was stirred at 25°C for 60 h.
  • the mixture was cooled to 5°C and filtered.
  • the solid was washed with water (2x25 mL) and dried at 60°C/0.2 mmHg for 5 h to give 10.7 g of first crop material.
  • a second crop was obtained by concentrating the mother liquors under reduced pressure to 125 mL, cooling to 5°C, collecting the solid, washing with cold water (2x20 mL) and drying as above to give 6.7 g of additional material for a total of 15.4 g (31 % from guanosine hydrate) of light tan solid; TLC purity 97%. 2
  • the initial loading was found to be 63 ⁇ mol/g. (3.9 mg of CPG were cleaved with trichloroacetic acid. The absorption of released trityl cation was read at 503 nm on a spectrophotometer to determine the loading.) The whole CPG sample was then washed as described above and dried under P 2 0 5 overnight in vacuum oven. The following day, the CPG was capped with 25 mL CAP A (tetrahydrofuran/acetic anhydride) and 25 mL CAP B (tetrahydrofuran/pyridine/1-methyl imidazole) for approx. 3 hours on shaker. Filtered and washed with dichloromethane and ether. CPG dried under P 2 0 5 overnight in vacuum oven. After drying, 12.25 g of CPG was isolated with a final loading of 90 ⁇ mol/g.
  • CAP A tetrahydrofuran/acetic anhydride
  • CAP B tetrahydrofur
  • reaction mixture was washed three times with cold 10% citric acid followed by three washes with water. Organic phase removed and dried under sodium sulfate. Succinylated nucleoside was dried under P 2 0 5 overnight in vacuum oven.
  • the CPG was capped with 50 mL CAP A (tetrahydrofuran/acetic anhydride) and 50 mL CAP B (tetrahydrofuran/pyridine/1-methyl imidazole) for approx. 1 hour on shaker. Filtered and washed with dichloromethane and ether. CPG dried under P 2 0 5 overnight in vacuum oven. After drying, 33.00 g of CPG was obtained with a final loading of 66 ⁇ mol/g.
  • CAP A tetrahydrofuran/acetic anhydride
  • CAP B tetrahydrofuran/pyridine/1-methyl imidazole
  • reaction mixture was transferred with CH 2 C1 2 and washed with saturated NaHC0 3 (100 mL) , followed by saturated NaCl solution.
  • the organic layer was dried over anhydrous Na 2 S0 4 and evaporated to yield 3.8 g of a crude product, which was purified in a silica column (200 g) using 1:1 hexane/EtOAc to give 1.9 g (1.95 mmol, 74% yield) of the desired phosphoramidite.
  • the 5 ' -0- (dimethoxytrityl) -3 ' -0- [hexyl- ( ⁇ -N-phthal- imidoamino] uridine-2 ' -O-succinyl-aminopropyl controlled pore glass was used to synthesize the oligomers in an ABI 380B DNA synthesizer using phosphoramidite chemistry standard conditions.
  • a four base oligomer 5'-GACU * -3' was used to confirm the structure of 3 ' -O-hexylamine tether introduced into the oligonucleotide by NMR. As expected a multiplet signal was observed between 1.0-1.8 pp in : H NMR.
  • N6-Dibenzoyl-5 ' -O-Dimethoxytrityl-3 ' -O- [3- (N- trif luoroacetamido) propyl] -Adenosine 4, 4 ' -Dimethoxytrityl chloride (3.6 g, 10.0 mmol.) Is added to a solution of N 6 - (dibenzoyl) -3 ' -0- [3- (N-trifluoroacetamido) propyl) adenosine in pyridine (100 ml) at room temperature and stirred for 16 hrs. The solution is concentrated in vacuo and chromatographed on silica gel (EtOAc/TEA 99/1) to give the title compound.
  • the title compound is prepared from 3'-0-(butyl- phthalimide) -N 6 -benzoyladenosine as per Example 22.
  • the title compound is prepared from 3 ' -O- (pentyl- phthalimide) -N 6 -benzoyladenosine as per the procedure of Example 23. Chromatography on silica gel (ethylacetate, hexane, triethylamine) , gives the title compound.
  • Oligonucleotides were synthesized on a Perseptive Biosystems Expedite 8901 Nucleic Acid Synthesis System. Multiple 1- ⁇ mol syntheses were performed for each oligonucleotide. Trityl groups were removed with trichloroacetic acid (975 ⁇ L over one minute) followed by an acetonitrile wash. All standard amidites (0.1M) were coupled twice per cycle (total coupling time was approximately 4 minutes) . All novel amidites were dissolved in dry acetonitrile (100 mg of amidite/1 mL acetonitrile) to give approximately 0.08-0.1 M solutions. Total coupling time was approximately 6 minutes (105 ⁇ L of amidite delivered).
  • the crude oligonucleotides synthesized in Example 49 were filtered from CPG using Gelman 0.45 ⁇ m nylon acrodisc syringe filters. Excess NH 4 OH was evaporated away in a Savant AS160 automatic speed vac. The crude yield was measured on a Hewlett Packard 8452A Diode Array Spectrophotometer at 260 nm. Crude samples were then analyzed by mass spectrometry (MS) on a Hewlett Packard electrospray mass spectrometer and by capillary gel electrophoresis (CGE) on a Beckmann P/ACE system 5000. Trityl-on oligonucleotides were purified by reverse phase preparative high performance liquid chromatography (HPLC) .
  • HPLC reverse phase preparative high performance liquid chromatography
  • HPLC conditions were as follows: Waters 600E with 991 detector; Waters Delta Pak C4 column (7.8X300mm); Solvent A: 50 mM triethylammonium acetate (TEA-Ac) , pH 7.0; B: 100% acetonitrile; 2.5 mL/min flow rate; Gradient: 5% B for first five minutes with linear increase in B to 60% during the next 55 minutes. Larger oligo yields from the larger 20 ⁇ mol syntheses were purified on larger HPLC columns (Waters Bondapak HC18HA) and the flow rate was increased to 5.0 mL/min. Appropriate fractions were collected and solvent was dried down in speed vac.
  • Solvent A 50 mM triethylammonium acetate (TEA-Ac) , pH 7.0
  • B 100% acetonitrile
  • Gradient 5% B for first five minutes with linear increase in B to 60% during the next 55 minutes. Larger oligo yields from the larger 20 ⁇ mol
  • Oligonucleotides were detritylated in 80% acetic acid for approximately 45 minutes and lyophilized again. Free trityl and excess salt were removed by passing detritylated oligonucleotides through Sephadex G-25 (size exclusion chromatography) and collecting appropriate samples through a Pharmacia fraction collector. Solvent again evaporated away in speed vac. Purified oligonucleotides were then analyzed for purity by CGE, HPLC (flow rate: 1.5 mL/min; Waters Delta Pak C4 column, 3.9X300mm), and MS. The final yield was determined by spectrophotometer at 260 nm.
  • T m melting temperature
  • T m melting temperature
  • test oligonucleotides and the complementary nucleic acid were incubated at a standard concentration of 4 ⁇ M for each oligonucleotide in buffer (100 mM NaCl, 10 mM sodium phosphate, pH 7.0, 0.1 mM EDTA) .
  • buffer 100 mM NaCl, 10 mM sodium phosphate, pH 7.0, 0.1 mM EDTA
  • Samples were heated to 90 C and the initial absorbance taken using a Guilford Response II Spectrophotometer (Corning) . Samples were then slowly cooled to 15 C and then the change in absorbance at 260 nm was monitored with heating during the heat denaturation procedure. The temperature was increased by 1 degree C/absorbance reading and the denaturation profile analyzed by taking the 1 st derivative of the melting curve. Data was also analyzed using a two-state linear regression analysis to determine the Tm' s . The results of these tests for the some of the oligonucleot
  • 2 ' -O-aminohexyluridine showed 8 signals between 7.5 and 9.0 ppm corresponding to the 8 aromatic protons.
  • Oligonucleotide identity is as follows: Oligonucleotide A is a normal 3 '-5' linked phosphodiester oligodeoxyribonucleotide of the sequence d(GGC TGU* CTG CG) where the * indicates the attachment site of a 2'- aminolinker . Oligonucleotide B is a normal 3 '-5' linked phosphodiester oligoribonucleotide of the sequence d(GGC TGU* CTG CG) where the * indicates the attachment site of either a 2 ' -aminolinker . Each of the ribonucleotides of the oligonucleotide, except the one bearing the * substituent, are 2 ' -O-methyl ribonucleotides.
  • Oligonucleotide C is has 2 '-5' linkage at the * position in addition to a 3 ' -aminolinker at this site.
  • the remainder of the oligonucleotide is a phosphodiester oligodeoxyribonucleotide of the sequence d(GGC TGU* CTG CG) .
  • the base oligonucleotide (no 2 ' -aminolinker) was not included in the study.
  • the 2 '-5' linkages demonstrated a higher melting temperature against an RNA target compared to a DNA target.
  • nucleosides with an asterisk contain 3 ' -0- (2-methoxyethyl) . All nucleosides with a # contain 2 ' -O- (2-methoxyethyl) .
  • the oligonucleotides were purified following the procedure of Example 50 and analyzed for their structure.
  • nucleosides with an asterisk contain 3 ' -0- (2-methoxyethyl)
  • nucleosides with a # contain 2 ' -0- (2-methoxy) ethyl.
  • 3' -propylphthalimido-A and 2' -propylphthalimido-A were used as the LCA-CPG solid support.
  • the required amounts of the amidites were placed in dried vials, dissolved in acetonitrile (unmodified nucleosides were made into 1M solutions and modified nucleosides were lOOmg / mL) , and connected to the appropriate ports on a Millipore ExpediteTM Nucleic Acid Synthesis System (ISIS 4) . 60mg of solid support resin was used in each column for 2Xl ⁇ mole scale synthesis (2 columns for each oligo were used) . The synthesis was run using the IBP-PS(1 ⁇ mole) coupling protocol for phosphorothioate backbones and CSO-8 for phosphodiesters . The trityl reports indicated normal coupling results .
  • the oligonucleotides were deprotected with cone, ammonium hydroxide (aq) containingl0% of a solution of 40% methylamine (aq) at 55C for approximately 16 hrs. Then they were evaporated, using a Savant AS160 Automatic SpeedVac, (to remove ammonia) and filtered to remove the CPG-resin.
  • the crude samples were analyzed by MS, HPLC, and CE . Then they were purified on a Waters 600E HPLC system with a 991 detector using a Waters C4 Prep, scale column (Alice C4 Prep. 10-16-96) and the following solvents: A: 50 mM TEA-Ac, pH 7.0 and B: acetonitrile utilizing the "MPREP2" method. After purification the oligonucleotides were evaporated to dryness and then detritylated with 80% acetic acid at room temp, for approximately 30 min. Then they were evaporated.
  • oligonucleotides were then dissolved in cone, ammonium hydroxide and run through a column containing Sephadex G-25 using water as the solvent and a Pharmacia LKB SuperFrac fraction collector. The resulting purified oligonucleotides were evaporated and analyzed by MS, CE, and HPLC.
  • mice 9 male BALB/c mice (Charles River, Wilmington, MA) weighing about 25 g were used. Following a one week acclimatization the mice received a single tail-vein injection of oligonucleotide (5 mg/kg) administered in phosphate buffered saline (PBS), pH 7.0.
  • PBS phosphate buffered saline
  • One retro-orbital bleed either at 0.25, 0.5, 2 or 4 h post-dose
  • a terminal bleed either 1, 3, 8, or 24 h post-dose
  • the terminal bleed (approximately 0.6-0.8 ml) was collected by cardiac puncture following ketamine/xylazine anasthesia.
  • the blood was transferred to an EDTA-coated collection tube and centrifuged to obtain plasma.
  • the liver and kidneys were collected from each mouse.
  • Plasma and tissue homogenates were used for analysis to determine intact oligonucleotide content by CGE. All samples were immediately frozen on dry ice after collection and stored at -80C until analysis.
  • the CGE analysis inidcated the relative nuclease resistance of 2 ',5 '-linked oligomers compared to ISIS 11061 (uniformly 2 ' -deoxy-phosphorothioate oligonucleotide targeted to mouse c-raf) . Because of the nuclease resistance of the 2 ', 5 ' -linkage, coupled with the fact that 3 ' -methoxyethoxy substituents are present and afford further nuclease protection the oligonucleotides ISIS 17176, ISIS 17177, ISIS 17178, ISIS 17180, ISIS 17181 and ISIS 21415 were found to be more stable in plasma, while ISIS 11061 was not.
  • FIG. 1 A plot of the percentage of full length oligonucleotide remaining intact in tissue 24 hours following administration of an i . v. bolus of 5 mg/kg oligonucleotide is shown in Figure 2.
  • CGE traces of test oligonucleotides and a standard phosphorothioate oligonucleotide in both mouse liver samples and mouse kidney samples after 24 hours are shown in Figure 3. As is evident from these traces, there is a greater amount of intact oliogonucleotide for the oligonucleotides of the invention as compared to the standard seen in panel A.
  • the oligonucleotide shown in panel B included one substituent of the invention at each of the 5' and 3' ends of a phosphorothioate oligonucleotide while the phosphorothioate oligonucleotide seen in panel C included one substituent at the 5' end and two at the 3' end.
  • the oligonucleotide of panel D include a substituent of the invention incorporated in a 2 ',5' phosphodiester linkage at both its 5' and 3' ends. While while less stable than the oligonucleotide seen in panels B and C, it is more stable than the full phosphorothioate standard oligonucleotide of panel A.
  • an in vitro cell culture assay was used that measures the cellular levels of c-raf expression in bEND cells .
  • the bEnd.3 cell line a brain endothelioma, was the kind gift of Dr. Werner Risau (Max-Planck Institute) .
  • Opti-MEM, trypsin-EDTA and DMEM with high glucose were purchased from Gibco-BRL (Grand Island, NY).
  • Dulbecco' s PBS was purchased from Irvine Scientific (Irvine, CA) .
  • Sterile, 12 well tissue culture plates and Facsflow solution were purchased from Becton Dickinson (Mansfield, MA) .
  • Ultrapure formaldehyde was purchased from Polysciences (Warrington, PA) .
  • NAP-5 columns were purchased from Pharmacia (Uppsala, Sweden) .
  • Oligonucleotide Trea tmen t Cells were grown to approximately 75 % confluency in 12 well plates with DMEM containing 4.5g/L glucose and 10 % FBS . Cells were washed 3 times with Opti-MEM pre-warmed to 37 °C. Oligonucleotide was premixed with a cationic lipid (Lipofectin reagent, (GIBCO/BRL) and, serially diluted to desired concentrations and transferred on to washed cells for a 4 hour incubation at 37 °C. Media was then removed and replaced with normal growth media for 24 hours for northern blot analysis of mRNA.
  • a cationic lipid Lipofectin reagent, (GIBCO/BRL)

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Abstract

On décrit des oligonucléotides modifiés contenant au moins une liaison 2',5'-internucléotidique. Les oligonucléotides de l'invention peuvent également comprendre des substituants supplémentaires aux positions 2'- et 3'-.
PCT/US1999/015886 1998-07-14 1999-07-13 Oligonucleotides modifies WO2000004189A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003008576A3 (fr) * 2001-07-12 2003-10-23 Aventis Pharma Gmbh Oligonucleotides synthetiques a double brin utilises pour inhiber de façon ciblee l'expression genique
WO2003008595A3 (fr) * 2001-07-12 2004-01-22 Aventis Pharma Gmbh Derives d'oligoribonucleotide permettant l'inhibition orientee de l'expression genique
CN108424432A (zh) * 2017-07-13 2018-08-21 上海兆维科技发展有限公司 一种3’-氧-甲氧乙基核苷的制备方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5188897A (en) * 1987-10-22 1993-02-23 Temple University Of The Commonwealth System Of Higher Education Encapsulated 2',5'-phosphorothioate oligoadenylates
US5434257A (en) * 1992-06-01 1995-07-18 Gilead Sciences, Inc. Binding compentent oligomers containing unsaturated 3',5' and 2',5' linkages

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5188897A (en) * 1987-10-22 1993-02-23 Temple University Of The Commonwealth System Of Higher Education Encapsulated 2',5'-phosphorothioate oligoadenylates
US5434257A (en) * 1992-06-01 1995-07-18 Gilead Sciences, Inc. Binding compentent oligomers containing unsaturated 3',5' and 2',5' linkages

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003008576A3 (fr) * 2001-07-12 2003-10-23 Aventis Pharma Gmbh Oligonucleotides synthetiques a double brin utilises pour inhiber de façon ciblee l'expression genique
WO2003008595A3 (fr) * 2001-07-12 2004-01-22 Aventis Pharma Gmbh Derives d'oligoribonucleotide permettant l'inhibition orientee de l'expression genique
AU2002325305B2 (en) * 2001-07-12 2007-05-17 Sanofi-Aventis Deutschland Gmbh Novel oligoribonucleotide derivatives for the targeted inhibition of gene expression
US8772469B2 (en) 2001-07-12 2014-07-08 Sanofi-Aventis Deutschland Gmbh Synthetic double-stranded oligonucleotides for specific inhibition of gene expression
CN108424432A (zh) * 2017-07-13 2018-08-21 上海兆维科技发展有限公司 一种3’-氧-甲氧乙基核苷的制备方法

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