Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
  • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
  • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/04—Anorexiants; Antiobesity agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • 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

• the present invention provides 5'-modified bicyclic nucleosides and oligomeric compounds and compositions prepared therefrom. More particularly, the present invention provides nucleosides having a 2'-O-CH 2 -4' bridge with a further group located at the 5 '-position and oligomers and compositions prepared therefrom. In a preferred embodiment the 5'-group is in a particular configuration providing either the (R) or (S) isomer. In some embodiments, the oligomeric compounds and compositions of the present invention hybridize to a portion of a target RNA resulting in loss of normal function of the target RNA.
• Antisense technology is an effective means for reducing the expression of one or more specific gene products and can therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications.
• Chemically modified nucleosides are routinely used for incorporation into antisense sequences to enhance one or more properties such as for example nuclease resistance.
• One such group of chemical modifications includes bicyclcic nucleosides wherein the furanose portion of the nucleoside includes a bridge connecting two atoms on the furanose ring thereby forming a bicyclic ring system.
• Such bicyclic nucleosides have various names including BNA's and LNA's for bicyclic nucleic acids or locked nucleic acids respectively.
• nucleosides have been prepared and reported in the patent literature as well as in scientific literature, see for example: Mikhailov et al, Nucleosides and Nucleotides, 1991, 10, 393-343; Saha et al, J. Org. Chem., 1995, 60, 788-789; Beigleman et al, Nucleosides and Nucleotides, 1995, 14, 901-905; Wang, et al, Bioorganic & Medicinal Chemistry Letters, 1999, 9, 885-890; and PCT Internation Application WO94/22890 published October 13, 1994, the text of each is incorporated by reference herein, in their entirety.
• the present invention provides bicyclic nucleosides having the formula:
• Bx is a heterocyclic base moiety; one of Ti and T 2 is H or a hydroxyl protecting group and the other of T] and T 2 is H, a hydroxyl protecting group or a reactive posphorus group;
• Z is substituted C)-C 6 alkyl.
• each Ji and J 2 is, independently H or Cj-C 6 alkyl.
• Z is methyl, ethyl or methoxymethyl.
• Z is methyl.
• Z is ethylenyl.
• Z is substituted acyl.
• At least one of Ti and T 2 is a hydroxyl protecting group wherein a list of preferred hydroxyl protecting groups includes acetyl, t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, l-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl, diphenylmethyl, p- nitrobenzyl, triphenylmethyl (trityl), 4,4'-dimethoxytrityl, trimethylsilyl, triethylsilyl, t- butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl, triisopropylsilyl, be
• T 2 is a reactive phosphorus group wherein one list of preferred reactive phosphorus groups includes diisopropylcyanoethoxy phosphoramidite and H- phosphonate.
• T 2 is diisopropylcyanoethoxy phosphoramidite and Ti is 4,4'- dimethoxytrityl.
• the Z group is in the (i?)-conf ⁇ guration:
• the Z group is in the (S)-configuration:
• the present invention also provides oligomeric compounds comprising at least one monomer of the formula:
• Bx is a heterocyclic base moiety
• T 3 is H, a hydroxyl protecting group, a linked conjugate group or an internucleoside linking group attached to a nucleoside, a nucleotide, an oligonucleoside, an oligonucleotide, a monomelic subunit or an oligomeric compound;
• T 4 is H, a hydroxyl protecting group, a linked conjugate group or an internucleoside linking group attached to a nucleoside, a nucleotide, an oligonucleoside, an oligonucleotide, a monomelic subunit or an oligomeric compound;
• Z is substituted Ci-C 6 alkyl.
• each Ji and J 2 is, independently H or C]-C 6 alkyl.
• Z is methyl, ethyl or methoxymethyl.
• Z is methyl.
• Z is ethylenyl.
• Z is substituted acyl.
• T 3 is H or a hydroxyl protecting group.
• T 3 is an internucleoside linking group attached to a nucleoside, a nucleotide or a monomelic subunit.
• T 3 is an internucleoside linking group attached to an oligonucleoside or an oligonucleotide.
• T 3 is an internucleoside linking group attached to an oligomeric compound.
• T 4 is H or a hydroxyl protecting group.
• T 4 is an internucleoside linking group attached to a nucleoside, a nucleotide or a monomelic subunit.
• T 4 is an internucleoside linking group attached to an oligonucleoside or an oligonucleotide.
• T 4 is an internucleoside linking group attached to an oligomeric compound.
• oligomeric compounds having at least one monomer wherein the Z group is in the (/ ⁇ -configuration:
• oligomeric compounds having at least one monomer wherein the Z group is in the ( ⁇ -configuration:
• At least one of T 3 and T 4 comprises an internucleoside linking group selected from phosphodiester or phosphorothioate.
• each internucleoside linking group in the oligomeric compound is, independently, a phosphodiester or a phosphorothioate.
• oligomeric compounds are provided having at least one region of at least two contiguous 5 '-substituted bicyclic nucleoside monomers of the invention. In another embodiment oligomeric compounds are provided having at least two regions of at least two contiguous 5'-substituted bicyclic nucleoside monomers of the invention. In a further embodiment oligomeric compounds are provided having at least two separate regions of at least two contiguous 5'-modified bicyclic nucleoside monomers of the invention which comprise a gapped oligomeric compound. In one embodiment oligomeric compounds are provided having from about 8 to about 40 nucleosides and/or modified nucleosides or mimetics in length.
• oligomeric compound comprise from about 8 to about 20 nucleosides and/or modified nucleosides or mimetics in length. In an even further embodiment oligomeric compounds comprise from about 10 to about 16 nucleosides and/or modified nucleosides or mimetics in length. In another embodiment oligomeric compounds comprise from about 10 to about 14 nucleosides and/or modified nucleosides or mimetics in length.
• Also provided are methods of inhibiting gene expression comprising contacting one or more cells, a tissue or an animal with an oligomeric compound of the invention.
• the present invention provides 5'-modified bicyclic nucleosides and oligomeric compounds prepared therefrom. More particularly, the present invention provides nucleosides having 5'-modified bicyclic ribofuranosyl sugar moieties (also referred to herein as 5'-modified bicyclic nucleosides or 5'-modified-BNA's) and oligomers and compositions prepared therefrom.
• the group modifying the 5'-position has a particular configuration thereby providing either (R) or (S) chirality.
• uracil DMT phosphoramidite would have the name: (l/?,3i?,4i?,76)-7-[2-cyanoethoxy(diisopropylamino)- phosphinoxy] -1-[1-(S, R or none for racemicj-(4,4'-dimethoxytrityl)oxy-ethyl]-3-(uracil-l-yl)- 2,5-dioxa-bicyclo[2.2.1 Jheptane terminology (uracil DMT phosphoramidite for example) wherein the 1 carbon position of the ethyl is (R), (S) or racemic and the heterocyclic base which is shown as uracil- 1-yl can be substituted with any heterocyclic base described herein.
• the 5'- modified BNA's of the present invention are useful for enhancing desired properties of oligomeric
• the 5'-modified bicyclic nucleosides of the present invention have the structure shown below:
• Substitutent groups may also be introduced using functionally similar carbon homologation reactions known to those skilled in the art. Addition of nitromethane and homologation via an epoxide is described in Wang, G.; Middleton, P. J. Tetrahedron Lett. 1996, 37, 2739-2742 (see also: Wang et a., Bioorganic & Medicinal Chemistry Letters, 1999, 9, 885-890; and Saha et ah, J. Org. Chem., 1995, 60, 788-789). Additionally, appropriately functionalized Grignard or other reagents may be manipulated after addition to provide further functionalized analogs.
• allyl or vinyl magnesium bromide reagents would introduce a double bond, which could be functionalized to many different groups, including functionalities such as halomethyl, methoxymethyl, appropriatedly protected hydroxymethyl, aminomethyl and various other functional groups.
• the 5'-modified bicyclic nucleosides of the present invention are useful for modifying otherwise unmodified oligomeric compounds at one or more positions.
• modified oligomeric compounds can be described as having a particular motif.
• Motifs amenable to the present invention include but are not limited to a gapped motif, a hemimer motif, a blockmer motif, a fully modified motif, a positionally modified motif and an alternating motif.
• linkages can also be used including but not limited to phosphodiester and phosphorothioate linkages used uniformly or in combinations.
• the positioning of 6-modified bicyclic nucleosides and the use of linkage strategies can be easily optimized for the best activity for a particular target.
• Representative U.S. patents that teach the preparation of representative motifs include, but are not limited to, 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.
• stable compound and “stable structure” are meant to indicate a Compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent. Only stable Compounds are contemplated herein. Selected substituent groups within the compounds described herein are present to a recursive degree. In this context, “recursive substituent” means that a substituent may recite another instance of itself. Because of the recursive nature of such substituents, theoretically, a large number may be present in any given claim. One of ordinary skill in the art of medicinal chemistry and organic chemistry understands that the total number of such substituents is reasonably limited by the desired properties of the Compound intended. Such properties include, by way of example and not limitation, physical properties such as molecular weight, solubility or log P, application properties such as activity against the intended target, and practical properties such as ease of synthesis.
• Recursive substituents are an intended aspect of the invention.
• One of ordinary skill in the art of medicinal and organic chemistry understands the versatility of such substituents.
• substituted and substituteduent group are meant to include groups that are typically added to other groups or parent compounds to enhance desired properties or give desired effects. Substituent groups can be protected or unprotected and can be added to one available site or to many available sites in a parent compound. Substituent groups may also be further substituted with other substituent groups and may be attached directly or via a linking group such as an alkyl or hydrocarbyl group to a parent compound.
• each R 33 , R bb and R cC is H, an optionally linked chemical functional group or a further substituent group with a preferred list including without limitation H, alkyl, alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic, heterocyclic and heteroarylalkyl.
• Linking groups or bifunctional linking moieties such as those known in the art are amenable to the present invention.
• Linking groups are useful for attachment of chemical functional groups, conjugate groups, reporter groups and other groups to selective sites in a parent compound.
• a bifunctional linking moiety comprises a hydrocarbyl moiety having two functional groups. One of the functional groups is selected to bind to a parent molecule or compound of interest and the other is selected to bind essentially any selected group such as chemical functional group or a conjugate group.
• the linker comprises a chain structure or an oligomer of repeating units such as ethylene glyol or amino acid units.
• bifunctional linking moieties include amino, hydroxyl, carboxylic acid, thiol, unsaturations (e.g., double or triple bonds), and the like.
• bifunctional linking moieties include 8- amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1- carboxylate (SMCC) and 6-aminohexanoic acid (AHEX or AHA).
• linking groups include, but are not limited to, substituted Ci-Cio alkyl, substituted or unsubstituted C 2 -Ci O alkenyl or substituted or unsubstituted C 2 -Ci O alkynyl, wherein a nonlimiting list of preferred substituent groups includes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.
• hydrocarbyl includes groups comprising C, O and H. Included are straight, branched and cyclic groups having any degree of saturation.
• Such hydrocarbyl groups can include one or more heteroatoms selected from N, O and S and can be further mono or poly substituted with one or more substituent groups.
• alkyl refers to a saturated straight or branched hydrocarbon radical containing up to twenty four carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl and the like.
• Alkyl groups typically include from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms (Ci-Ci 2 alkyl) with from 1 to about 6 carbon atoms being more preferred.
• the term "lower alkyl” as used herein includes from 1 to about 6 carbon atoms.
• Alkyl groups as used herein may optionally include one or more further substitutent groups.
• alkenyl refers to a straight or branched hydrocarbon chain radical containing up to twenty four carbon atoms and having at least one carbon-carbon double bond.
• alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, 1- methyl-2-buten-l-yl, dienes such as 1,3-butadiene and the like.
• Alkenyl groups typically include from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred.
• Alkenyl groups as used herein may optionally include one or more further substitutent groups.
• alkynyl refers to a straight or branched hydrocarbon radical containing up to twenty four carbon atoms and having at least one carbon-carbon triple bond.
• alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 1-butynyl, and the like.
• Alkynyl groups typically include from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred.
• Alkynyl groups as used herein may optionally include one or more further substitutent groups.
• aminoalkyl refers to an amino substituted alkyl radical. This term is meant to include C]-Ci 2 alkyl groups having an amino substituent at any position and wherein the alkyl group attaches the aminoalkyl group to the parent molecule. The alkyl or amino portions of the aminoalkyl group can be further substituted with substituent groups.
• aliphatic refers to a straight or branched hydrocarbon radical containing up to twenty four carbon atoms wherein the saturation between any two carbon atoms is a single, double or triple bond.
• An aliphatic group preferably contains from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms with from 1 to about 6 carbon atoms being more preferred.
• the straight or branched chain of an aliphatic group may be interupted with one or more heteroatoms that include nitrogen, oxygen, sulfur and phosphorus.
• Such aliphatic groups, interupted by heteroatoms include without limitation polyalkoxys, such as polyalkylene glycols, polyamines, and polyimines.
• Aliphatic groups as used herein may optionally include further substitutent groups.
• the term "alicyclic" or "alicyclyl” refers to a cyclic ring system wherein the ring is aliphatic.
• the ring system can comprise one or more rings wherein at least one ring is aliphatic.
• Preferred alicyclics include rings having from about 5 to about 9 carbon atoms in the ring.
• Alicyclic as used herein may optionally include further substitutent groups.
• alkoxy refers to a radical formed between an alkyl group and an oxygen atom wherein the oxygen atom is used to attach the alkoxy group to a parent molecule. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, w-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy and the like. Alkoxy groups as used herein may optionally include further substitutent groups.
• aryl and aromatic refer to a mono- or polycyclic carbocyclic ring system radicals having one or more aromatic rings.
• aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl and the like.
• Preferred aryl ring systems have from about 5 to about 20 carbon atoms in one or more rings.
• Aryl groups as used herein may optionally include further substitutent groups.
• aralkyl and arylalkyl refer to a radical formed between an alkyl group and an aryl group wherein the alkyl group is used to attach the aralkyl group to a parent molecule. Examples include, but are not limited to, benzyl, phenethyl and the like. Aralkyl groups as used herein may optionally include further substitutent groups attached to the alkyl, the aryl or both groups that form the radical group.
• heterocyclic radical refers to a radical mono-, or poly-cyclic ring system that includes at least one heteroatom and is unsaturated, partially saturated or fully saturated, thereby including heteroaryl groups. Heterocyclic is also meant to include fused ring systems wherein one or more of the fused rings contain at least one heteroatom and the other rings can contain one or more heteroatoms or optiohnally contain no heteroatoms.
• a heterocyclic group typically includes at least one atom selected from sulfur, nitrogen or oxygen.
• heterocyclic groups include, [l,3]dioxolane, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and the like.
• Heterocyclic groups as used herein may optionally include further substitutent groups.
• heteroaryl refers to a radical comprising a mono- or poly-cyclic aromatic ring, ring system or fused ring system wherein at least one of the rings is aromatic and includes one or more heteroatom. Heteroaryl is also meant to include fused ring systems including systems where one or more of the fused rings contain no heteroatoms. Heteroaryl groups typically include one ring atom selected from sulfur, nitrogen or oxygen.
• heteroaryl groups include, but are not limited to, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl, and the like.
• Heteroaryl radicals can be attached to a parent molecule directly or through a linking moiety such as an aliphatic group or hetero atom.
• Heteroaryl groups as used herein may optionally include further substitutent groups.
• heteroarylalkyl refers to a heteroaryl group as previously defined having an alky radical that can attach the heteroarylalkyl group to a parent molecule. Examples include, but are not limited to, pyridinylmethyl, pyrimidinylethyl, napthyridinylpropyl and the like. Heteroarylalkyl groups as used herein may optionally include further substitutent groups.
• mono or poly cyclic structure includes all ring systems that are single or polycyclic having rings that are fused or linked and is meant to be inclusive of single and mixed ring systems individually selected from aliphatic, alicyclic, aryl, heteroaryl, aralkyl, arylalkyl, heterocyclic, heteroaryl, heteroaromatic, heteroarylalkyl.
• Such mono and poly cyclic structures can contain rings that are uniform or have varying degrees of saturation including fully saturated, partially saturated or fully unsaturated.
• Each ring can comprise ring atoms selected from C, N, O and S to give rise to heterocyclic rings as well as rings comprising only C ring atoms which can be present in a mixed motif such as for example benzimidazole wherein one ring has only carbon ring atoms and the fused ring has two nitrogen atoms.
• mono or poly cyclic structures can be attached to a parent molecule directly through a ring atom, through a substituent group or a bifunctional linking moiety.
• the compounds (e.g., 5'-modified bicyclic nucleosides) described herein can be prepared by any of the applicable techniques of organic synthesis, as, for example, illustrated in the examples below. Many such techniques are well known in the art. However, many of the known techniques are elaborated in Compendium of Organic Synthetic Methods (John Wiley & Sons, New York) Vol. 1, Ian T. Harrison and Shuyen Harrison (1971); Vol. 2, Ian T. Harrison and Shuyen Harrison (1974); Vol. 3, Louis S. Hegedus and Leroy Wade (1977); Vol. 4, Leroy G. Wade Jr., (1980); Vol. 5, Leroy G. Wade Jr. (1984); and Vol. 6, Michael B.
• oligomeric compounds are modified by covalent attachment of one or more conjugate groups.
• conjugate groups modif ⁇ y one or more properties of the attached oligomeric compound including but not limited to pharmakodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge and clearance.
• Conjugate groups are routinely used in the chemical arts and are linked directly or via an optional linking moiety or linking group to a parent compound such as an oligomeric compound.
• conjugate groups includes without limitation, intercalators, reporter molecules, drug groups such as ibuprofen, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins and dyes.
• drug groups such as ibuprofen, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, r
• protecting group refers to a labile chemical moiety which is known in the art to protect reactive groups including without limitation, hydroxyl, amino and thiol groups, against undesired reactions during synthetic procedures.
• Protecting groups are typically used selectively and/or orthogonally to protect sites during reactions at other reactive sites and can then be removed to leave the unprotected group as is or available for further reactions.
• Protecting groups as known in the art are described generally in Greene's Protective Groups in Organic Synthesis, 4th Edition, Greene, T. W., and Wutz, P.G.M., John Wiley & Sons, New York (2007).
• Groups can be selectively incorporated into oligomeric compounds of the invention as precursors.
• an amino group can be placed into a compound of the invention as an azido group that can be chemically converted to the amino group at a desired point in the synthesis.
• groups are protected or present as precursor that will be inert to reactions that modify other areas of the parent molecule for conversion into their final groups at an appropriate time. Further representative protecting or precursor groups are discussed in Agrawal, et al., Protocols for Oligonucleotide Conjugates, Eds, Humana Press; New Jersey, 1994; Vol. 26 pp. 1-72.
• hydroxyl protecting groups include, but are not limited to, t-butyl, t- butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, l-(2-chloroethoxy)ethyl, 2- trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl, diphenyl- methyl, p-nitrobenzyl, bis(2-acetoxyethoxy)methyl (ACE), 2-trimethylsilylethyl, triisopropylsilyl, [(triisopropylsilyl)oxymethyl (TOM), monomethoxytrityl, dimethoxytrityl
• DMT trimethoxytrityl
• FPMP 9-phenylxanthine- 9-yl
• MOX 9-(p-methoxyphenyl)xanthine-9-yl
• triphenylmethyl (trityl) 4,4'- dimethoxytrityl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl, benzoylformate, acetate, chloroacetate, trichloroacetate, trifluoroacetate, pivaloate, benzoate, p-phenylbenzoate, 9-fluorenyhnethyl carbonate, mesylate and tosylate.
• hydroxyl protecting groups include, but are not limited to, benzyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, benzoyl, mesylate, tosylate, dimethoxytrityl (DMT), 9- phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl (MOX).
• benzyl 2,6-dichlorobenzyl
• t-butyldimethylsilyl t-butyldiphenylsilyl
• benzoyl mesylate, tosylate, dimethoxytrityl (DMT), 9- phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl (MOX).
• amino protecting groups include, but are not limited to, carbamate- protecting groups, such as 2-trimethylsilylethoxycarbonyl (Teoc), 1 -methyl- l-(4-biphenylyl)- ethoxycarbonyl (Bpoc), t-butoxycarbonyl (BOC), allyloxycarbonyl (Alloc), 9- fluorenylmethyloxycarbonyl (Fmoc), and benzyloxycarbonyl (Cbz); amide-protecting groups, such as formyl, acetyl, trihaloacetyl, benzoyl, and nitrophenylacetyl; sulfonamide-protecting groups, such as 2-nitrobenzenesulfonyl; and imine- and cyclic imide-protecting groups, such as phthalimido and dithiasuccinoyl.
• carbamate- protecting groups such as 2-trimethylsilylethoxycarbonyl (Teoc), 1 -methyl
• thiol protecting groups include, but are not limited to, triphenylmethyl (trityl), benzyl (Bn), and the like.
• oligomeric compounds are prepared by connecting nucleosides with optionally protected phosphorus containing internucleoside linkages.
• Representative protecting groups for phosphorus containing internucleoside linkages such as phosphodiester and phosphorothioate linkages include ⁇ -cyanoethyl, diphenylsilylethyl, ⁇ - cyanobutenyl, cyano p-xylyl (CPX), N-methyl-N-trifluoroacetyl ethyl (META), acetoxy phenoxy ethyl (APE) and butene-4-yl groups. See for example U.S. Patents Nos. 4,725,677 and Re.
• orthogonal protection refers to functional groups which are protected with different classes of protecting groups, wherein each class of protecting group can be removed in any order and in the presence of all other classes (see, Barany, G. and Merrif ⁇ eld, R.B., J. Am. Chem. Soc, 1977, 99, 7363; idem, 1980, 102, 3084.)
• Orthogonal protection is widely used in for example automated oligonucleotide synthesis.
• a functional group is deblocked in the presence of one or more other protected functional groups which is not affected by the deblocking procedure. This deblocked functional group is reacted in some manner and at some point a further orthogonal protecting group is removed under a different set of reaction conditions. This allows for selective chemistry to arrive at a desired Compound or oligomeric Compound.
• the present invention provides compounds having reactive phosphorus groups useful for forming internucleoside linkages including for example phosphodiester and phosphorothioate internucleoside linkages.
• Such reactive phosphorus groups are known in the art and contain phosphorus atoms in P i ⁇ or P v valence state including, but not limited to, phosphoramidite, H- phosphonate, phosphate triesters and phosphorus containing chiral auxiliaries.
• a preferred synthetic solid phase synthesis utilizes phosphoramidites (P ⁇ i chemistry) as reactive phosphites.
• the intermediate phosphite compounds are subsequently oxidized to the P v state using known methods to yield, in preferred embodiments, phosphodiester or phosphorothioate internucleotide linkages. Additional reactive phosphates and phosphites are disclosed in Tetrahedron Report Number 309 (Beaucage and Iyer, Tetrahedron, 1992, 48, 2223-2311).
• oligomeric compounds useful in this invention include oligonucleotides containing modified e.g. non-naturally occurring internucleoside linkages.
• Two main classes of internucleoside linkages are defined by the presense or absence of a phosphorus atom.
• Modified internucleoside linkages having a phosphorus atom include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphospho triesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage.
• Oligonucleotides having inverted polarity can comprise a single 3' to 3' linkage at the 3'-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof).
• Various salts, mixed salts and free acid forms are also included.
• Modified internucleoside linkages not having a phosphorus atom include, but are not limited to, those that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
• patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S.: 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.
• the compounds described herein contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (S)-, ⁇ or ⁇ , or as (D)- or (L)- such as for amino acids et al.
• the present invention is meant to include all such possible isomers, as well as their racemic and optically pure forms.
• Optical isomers may be prepared from their respective optically active precursors by the procedures described above, or by resolving the racemic mixtures.
• the resolution can be carried out in the presence of a resolving agent, by chromatography or by repeated crystallization or by some combination of these techniques which are known to those skilled in the art. Further details regarding resolutions can be found in Jacques, et al., Enantiomers, Racemates, and Resolutions (John Wiley & Sons, 1981). When the compounds described herein contain olefinic double bonds, other unsaturation, or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers or cis- and trans-isomers. Likewise, all tautomeric forms are also intended to be included.
• oligomeric compound refers to a polymer having at least a region that is capable of hybridizing to a nucleic acid molecule.
• oligomeric compound includes oligonucleotides, oligonucleotide analogs and oligonucleosides as well as nucleotide mimetics and/or mixed polymers comprising nucleic acid and non-nucleic acid components. Oligomeric compounds are routinely prepared linearly but can be joined or otherwise prepared to be circular and may also include branching. Oligomeric compounds can form double stranded constructs such as for example two strands hybridized to form double stranded compositions. The double stranded compositions can be linked or separate and can include overhangs on the ends.
• an oligomeric compound comprises a backbone of linked monomelic subunits where each linked monomelic subunit is directly or indirectly attached to a heterocyclic base moiety. Oligomeric compounds may also include monomelic subunits that are not linked to a heterocyclic base moiety thereby providing abasic sites.
• the linkages joining the monomelic subunits, the sugar moieties or surrogates and the heterocyclic base moieties can be independently modified.
• the linkage-sugar unit which may or may not include a heterocyclic base, may be substituted with a mimetic such as the monomers in peptide nucleic acids. The ability to modify or substitute portions or entire monomers at each position of an oligomeric compound gives rise to a large number of possible motifs.
• nucleoside is a base-sugar combination.
• the base portion of the nucleoside is normally a heterocyclic base moiety.
• the two most common classes of such heterocyclic bases are purines and pyrimidines.
• Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside.
• the phosphate group can be linked to either the 2', 3' or 5' hydroxyl moiety of the sugar.
• the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound.
• oligonucleotide refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA).
• oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside linkages.
• oligonucleotide analog refers to oligonucleotides that have one or more non-naturally occurring portions. Such non-naturally occurring oligonucleotides are often desired over naturally occurring forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases.
• oligonucleoside refers to a sequence of nucleosides that are joined by internucleoside linkages that do not have phosphorus atoms. Internucleoside linkages of this type include short chain alkyl, cycloalkyl, mixed heteroatom alkyl, mixed heteroatom cycloalkyl, one or more short chain heteroatomic and one or more short chain heterocyclic.
• internucleoside linkages include, but are not limited to, siloxane, sulfide, sulfoxide, sulfone, acetyl, formacetyl, thioformacetyl, methylene formacetyl, thioformacetyl, alkeneyl, sulfamate; methyleneimino, methylenehydrazino, sulfonate, sulfonamide, amide and others having mixed N, O, S and CH 2 component parts.
• U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S.: 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.
• nucleobase or “heterocyclic base moiety” as used herein, is intended to by synonymous with “nucleic acid base or mimetic thereof.”
• a nucleobase is any substructure that contains one or more atoms or groups of atoms capable of hydrogen bonding to a base of a nucleic acid.
• heterocyclic base moiety includes, purines, pyrimidines, heterocyclic bases, modified bases, modified nucleobases and natural and non-naturally occurring nucleobases.
• unmodified or “natural” nucleobases include the purine bases adenine
• Modified nucleobases include, but are not limited to other synthetic and natural nucleobases such as for example 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine and 2-aminoadenine. Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7- deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in United States Patent No.
• Modified nucleobases include, but are not limited to, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fiuorinated bases as defined herein. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N- 2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5- propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 0 C (Sanghvi, Y.S., Crooke, S.T.
• oligomeric compounds of the present invention may also contain one or more additional nucleosides having modified sugar moieties.
• the furanosyl sugar ring can be modified in a number of ways including substitution with a substituent group, bridging to form a BNA and substitution of the 4'-O with a heteroatom such as S or N(R).
• Some representative U.S. patents that teach the preparation of such modified sugars include, but are not limited to, U.S.: 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; 5,700,920, 6,600,032 and International Application PCT/US2005/019219, filed June 2, 2005 and published as WO 2005/121371 on December 22, 2005 certain
• a representative list of preferred modified sugars includes but is not limited to substituted sugars having a 2'-F, 2'-OCH 2 or a 2'-O(CH 2 ) 2 -OCH 3 substituent group; 4'- thio modified sugars and bicyclic modified sugars.
• Oligomeric compounds of the present invention may also contain one or more nucleosides having modified sugar moieties.
• the furanosyl sugar ring can be modified in a number of ways including substitution with a substituent group, bridging to form a BNA and substitution of the 4'-O with a heteroatom such as S or N(R).
• a representative list of preferred modified sugars includes but is not limited to substituted sugars having a 2'-F, 2'-OCH 2 or a 2'-O(CH 2 ) 2 - OCH 3 substituent group; 4'-thio modified sugars and bicyclic modified sugars.
• nucleoside mimetic is intended to include those structures used to replace the sugar or the sugar and the base not the linkage at one or more positions of an oligomeric compound such as for example nucleoside mimetics having morpholino or bicyclo[3.1.0]hexyl sugar mimetics e.g. non furanose sugar units with a phosphodiester linkage.
• sugar surrogate overlaps with the slightly broader term “nucleoside mimetic” but is intended to indicate replacement of the sugar unit (furanose ring) only.
• the oligomeric compounds in accordance with the present invention can comprise from about 8 to about 80 nucleosides and/or modified nucleosides or mimetics in length.
• One of ordinary skill in the art will appreciate that the invention embodies oligomeric compounds of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleosides and/or modified nucleosides or mimetics in length, or any range therewithin.
• the oligomeric compounds of the invention are 8 to 40 nucleosides and/or modified nucleosides or mimetics in length.
• One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleosides and/or modified nucleosides or mimetics in length, or any range therewithin.
• the oligomeric compounds of the invention are 8 to 20 nucleosides and/or modified nucleosides or mimetics in length.
• the oligomeric compounds of the invention are 10 to 16 nucleosides and/or modified nucleosides or mimetics in length.
• this embodies oligomeric compounds of 10, 11, 12, 13, 14, 15 or 16 nucleosides and/or modified nucleosides or mimetics in length, or any range therewithin.
• the oligomeric compounds of the invention are 10 to 14 nucleosides and/or modified nucleosides or mimetics in length.
• the oligomeric compounds of the invention are 10 to 14 nucleosides and/or modified nucleosides or mimetics in length.
• One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 10, 11, 12, 13 or 14 nucleosides and/or modified nucleosides or mimetics in length, or any range therewithin.
• Oligomerization of modified and unmodified nucleosides and mimetics thereof is performed according to literature procedures for DNA (Protocols for Oligonucleotides and Analogs, Ed. Agrawal (1993), Humana Press) and/or RNA (Scaringe, Methods (2001), 23, 206-217; Gait et al., Applications of Chemically synthesized RNA in RNA:Protein Interactions, Ed. Smith (1998), 1-36; Gallo et al., Tetrahedron (2001), 57, 5707-5713) synthesis as appropriate. Additional methods for solid-phase synthesis may be found in Canithers U.S. Patents Nos. 4,415,732; 4,458,066; 4,500,707; 4,668,777; 4,973,679; and 5,132,418; and Koster U.S. Patents Nos. 4,725,677 and Re. 34,069.
• RNA and related analogs have been increasing as efforts in RNAi increase.
• the primary RNA synthesis strategies that are presently being used commercially include 5'-O-DMT-2'-O-t-butyldimethylsilyl (TBDMS), 5'-0-DMT-2'-0-[l(2-fluorophenyl)-4-methoxypiperidin-4-yl] (FPMP), 2'-O- [(triisopropylsilyl)oxy]methyl (2'-O-CH 2 -O-Si(iPr) 3 (TOM), and the 5'-O-silyl ether-2'-ACE (5'- 0-bis(trimethylsiloxy)cyclododecyloxysilyl ether (DOD)-2'-0-bis(2-acetoxyethoxy)methyl (ACE).
• TDMS 5'-O-DMT-2'-O-t-butyldimethylsilyl
• FPMP 5'-0-DMT-2'-0-[l
• RNA synthesis activator advertised to reduce coupling times especially with TOM and TBDMS chemistries. Such an activator would also be amenable to the present invention.
• the primary groups being used for commercial RNA synthesis are:
• TBDMS 5'-O-DMT-2'-O-t-butyldimethylsilyl
• TOM 2'-O-[(triisopropylsilyl)oxy]methyl
• DOD/ACE (5'-0-bis(trimethylsiloxy)cyclododecyloxysilyl ether-2'-O-bis(2- acetoxyethoxy)methyl
• FPMP 5 l -0-DMT-2'-0-[l(2-fluorophenyl)-4-methoxypiperidin-4-yl] .
• hybridization means the pairing of complementary strands of oligomeric compounds, m the present invention, one mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of oligomeric compounds.
• nucleobases complementary nucleoside or nucleotide bases
• adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds.
• Hybridization can occur under varying circumstances.
• An oligomeric compound is specifically hybridizable when binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligomeric compound to non-target nucleic acid sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and under conditions in which assays are performed in the case of in vitro assays.
• “Complementary,” as used herein, refers to the capacity for precise pairing of two nucleobases regardless of where the two are located.
• a nucleobase at a certain position of an oligomeric compound is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, the target nucleic acid being a DNA, RNA, or oligonucleotide molecule
• the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be a complementary position.
• the oligomeric compound and the further DNA, RNA, or oligonucleotide molecule are complementary to each other when a sufficient number of complementary positions in each molecule are occupied by nucleobases which can hydrogen bond with each other.
• an oligomeric compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable.
• an oligonucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure).
• the oligomeric compounds of the present invention can comprise at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% sequence complementarity to a target region within the target nucleic acid sequence to which they are targeted.
• an oligomeric compound in which 18 of 20 nucleobases of the oligomeric compound are complementary to a target region, and would therefore specifically hybridize would represent 90 percent complementarity.
• the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases.
• an oligomeric compound which is 18 nucleobases in length having 4 (four) noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention.
• Percent complementarity of an oligomeric compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. MoI. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).
• oligomeric compounds such as antisense oligomeric compounds, antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other oligomeric compounds which hybridize to at least a portion of the target nucleic acid.
• these oligomeric compounds may be introduced in the form of single-stranded, double-stranded, circular or hairpin oligomeric compounds and may contain structural elements such as internal or terminal bulges or loops.
• the oligomeric compounds of the invention may elicit the action of one or more enzymes or structural proteins to effect modification of the target nucleic acid.
• single stranded oligomers are provided that hybridize to a nucleic acid target and degrade the target by recruitment of an endonuclease enzyme.
• an endonuclease enzyme is RNAse H, a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex.
• RNAse H a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex.
• RNase H Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide- mediated inhibition of gene expression. Similar roles have been postulated for other ribonucleases such as those in the RNase III and ribonuclease L family of enzymes.
• oligomeric compound is a single-stranded antisense oligonucleotide
• double-stranded RNA (dsRNA) molecules has been shown to induce potent and specific antisense-mediated reduction of the function of a gene or its associated gene products. This phenomenon occurs in both plants and animals and is believed to have an evolutionary connection to viral defense and transposon silencing.
• "suitable target segments” may be employed in a screen for additional oligomeric compounds that modulate the expression of a selected protein.
• Modulators are those oligomeric compounds that decrease or increase the expression of a nucleic acid molecule encoding a protein and which comprise at least an 8-nucleobase portion which is complementary to a suitable target segment.
• the screening method comprises the steps of contacting a suitable target segment of a nucleic acid molecule encoding a protein with one or more candidate modulators, and selecting for one or more candidate modulators which decrease or increase the expression of a nucleic acid molecule encoding a protein. Once it is shown that the candidate modulator or modulators are capable of modulating (e.g.
• the modulator may then be employed in further investigative studies of the function of the peptide, or for use as a research, diagnostic, or therapeutic agent in accordance with the present invention.
• the suitable target segments of the present invention may also be combined with their respective complementary antisense oligomeric compounds of the present invention to form stabilized double-stranded (duplexed) oligonucleotides.
• Such double stranded oligonucleotide moieties have been shown in the art to modulate target expression and regulate translation as well as RNA processsing via an antisense mechanism.
• double-stranded moieties may be subject to chemical modifications (Fire et al., Nature, 1998, 391, 806-811; Timmons and Fire, Nature 1998, 395, 854; Timmons et al., Gene, 2001, 263, 103-112; Tabara et al., Science, 1998, 282, 430-431; Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507; Tuschl et al., Genes Dev., 1999, 13, 3191-3197; Elbashir et al., Nature, 2001, 411, 494-498; Elbashir et al., Genes Dev. 2001, 15, 188-200).
• double-stranded moieties have been shown to inhibit the target by the classical hybridization of antisense strand of the duplex to the target, thereby triggering enzymatic degradation of the target (Tijsterman et al., Science, 2002, 295, 694-697).
• the oligomeric compounds of the present invention can also be applied in the areas of drug discovery and target validation.
• the present invention comprehends the use of the oligomeric compounds and targets identified herein in drug discovery efforts to elucidate relationships that exist between proteins and a disease state, phenotype, or condition.
• These methods include detecting or modulating a target peptide comprising contacting a sample, tissue, cell, or organism with the oligomeric compounds of the present invention, measuring the nucleic acid or protein level of the target and/or a related phenotypic or chemical endpoint at some time after treatment, and optionally comparing the measured value to a non-treated sample or sample treated with a further oligomeric compound of the invention.
• RNAi activity is evaluated according to existing literature (Elbashir et al, Nature (2001), 411, 494-498; Nishikura et al., Cell (2001), 107, 415- 416; and Bass et al., Cell (2000), 101, 235-238.)
• the oligomeric compounds of the present invention can be utilized for diagnostics, therapeutics, prophylaxis and as research reagents and kits. Furthermore, antisense oligonucleotides, which are able to inhibit gene expression with 17, specificity, are often used by those of ordinary skill to elucidate the function of particular genes or to distinguish between functions of various members of a biological pathway.
• the oligomeric compounds of the present invention can be used as tools in differential and/or combinatorial analyses to elucidate expression patterns of a portion or the entire complement of genes expressed within cells and tissues. Oligomeric compounds can also be effectively used as primers and probes under conditions favoring gene amplification or detection, respectively.
• primers and probes are useful in methods requiring the specific detection of nucleic acid molecules encoding proteins and in the amplification of the nucleic acid molecules for detection or for use in further studies.
• Hybridization of the antisense oligonucleotides, particularly the primers and probes, of the invention with a nucleic acid can be detected by means known in the art. Such means may include conjugation of an enzyme to the oligonucleotide, radiolabelling of the oligonucleotide or any other suitable detection means. Kits using such detection means for detecting the level of selected proteins in a sample may also be prepared.
• expression patterns within cells or tissues treated with one or more oligomeric compounds are compared to control cells or tissues not treated with oligomeric compounds and the patterns produced are analyzed for differential levels of gene expression as they pertain, for example, to disease association, signaling pathway, cellular localization, expression level, size, structure or function of the genes examined. These analyses can be performed on stimulated or unstimulated cells and in the presence or absence of other compounds and or oligomeric compounds which affect expression patterns.
• Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays (Brazma and ViIo, FEBS Lett., 2000, 480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serial analysis of gene expression)(Madden, et al., Drug Discov. Today,
• H 2 SO 4 (h) Uracil, BSA, TMSOTf, CH 3 CN, reflux, 2h (i) NaOH, water, dioxane O) lsobutyric anhydride, DMAP, pyridine (k) Pd/C, H 2 balloon (I) TBSCI, imidazole, DMF (m) K 2 CO 3 , MeOH (n) DMTCI, 2,6-lutidine, pyridine, 45 0 C (o) Et 3 N.3HF, Et 3 N, THF (p) (1Pr 2 N) 2 POCH 2 CH 2 CN, tetrazole, NMI, DMF.
• Methanesulfonyl chloride (0.46 mL, 5.8 mmol) was added to a cold (O 0 C) solution of crude Compound 8a, triethylamine (1.1 mL, 7.8 mmol) and 4-dimethylaminopyridine (60 mg, 0.5 mmol) in CH 2 Cl 2 (21 mL). After stirring at room temperature for 1 hour, the reaction was poured into CHCl 3 and the organic layer was sequentially washed with 5% aqueous HCl, saturated NaHCO 3 , brine, dried (Na 2 SO 4 ) and concentrated under vacuum to give Compound 9a, which was used without purification in the next step.
• N,O-Bis(trirnethylsilyl)acetamide (3.9 mL, 15.7 mmol) was added to a suspension of Compound 10a (2.7 g, 5.2 mmol) and uracil (0.73 g, 6.5 mmol) in MeCN (16 mL). After heating at 4O 0 C for 15 minutes to get a clear solution, trimethylsilyl triflate (1.23 mL, 6.8 mmol) was added to the reaction. After refluxing for 2 hours, the reaction was cooled to room temperature and poured into EtOAc. The organic layer was washed with saturated NaHCO 3 , brine, dried (Na 2 SO 4 ) and concentrated under vacuum to give Compound 11a, which was used without purification in the next step.
• DMTCl 4,4'-Dimethoxytrityl chloride
• Methanesulfonyl chloride (0.40 mL, 5.2 mmol) was added to a cold (O 0 C) solution of crude Compound 8b, triethylamine (0.88 mL, 6.3 mmol) and 4-dimethylaminopyridine (53 mg, 0.4 mmol) in CH 2 Cl 2 (16 mL). After stirring at room temperature for 1 hour, the reaction was poured into CHCl 3 and the organic layer was sequentially washed with 5% aqueous HCl, saturated NaHCO 3 , brine, dried (Na 2 SO 4 ) and concentrated under vacuum to give Compound 9b, which was used without purification in the next step.
• N,0-Bis(trimethylsilyl)acetamide (2.73 mL, 11.0 mmol) was added to a suspension of Compound 10b (2.0 g, 3.9 mmol) and uracil (0.52 g, 4.6 mmol) in CH 3 CN (11 mL). After heating at 40 0 C for 15 minutes to get a clear solution, trimethylsilyl triflate (0.87 mL, 4.8 mmol) was added to the reaction. After refluxing for 2 hours the reaction was cooled to room temperature and poured into EtOAc. The organic layer was washed with saturated NaHCO 3 , brine, dried (Na 2 SO 4 ) and concentrated under vacuum to give crude Compound 1 Ib, which was used without purification in the next step.
• Triethylamine trihydrofluoride (1.06 mL, 6.5 mmol) was added to a solution of Compound 17b (0.89 g, 1.3 mmol) and triethylamine (0.46 mL, 3.3 mmol) in THF (6.5 mL) in a polypropylene tube. After stirring at room temperature for 48 hours, the reaction was poured into EtOAc and the organic phase was sequentially washed with H 2 O, saturated NaHCO 3 , brine, dried (Na 2 SO 4 ) and concentrated under vacuum. Purification by column chromatography (SiO 2 , eluting with 30% acetone/CHCl 3 -45% acetone/CHCl 3 ) gave Compound 18b (0.73 g, 98%) as a white foam.
• Triethylamine trihydroflouride (1.00 mL, 6.1 mmol) was added to a solution of Compound 22a (0.81 g, 1.1 mmol) and triethylamine (0.35 mL, 2.5 mmol) in THF (7 mL). After stirring at room temperature for 48 hours, the reaction was poured into EtOAc and the organic layer was washed with H 2 O, saturated NaHCO 3 , brine, dried (Na 2 SO 4 ) and concentrated. Purification by column chromatography (SiO 2 , eluting with 90% EtOAc/hexanes) gave Compound 23a (0.68 g, 99%).
• Aqueous ammonia solution (5 mL) was added to a solution of triazolide 20b (from above) in 1,4-dioxane (25 mL). After stirring for 16 hours at room temperature, the reaction was concentrated to provide Compound 21b which was dried under under high vacuum for 24 hours and used without further purification in the next step.
• Compound 25a is prepared by the Vorbruggen reaction of Compound 10a using 6-N-Bz- Adenine, BSA and TMSOTf in refluxing dichloroethane. Subsequent reaction of 25a with sodium hydroxide in dioxane/water, followed by reprotection of the 4-amino group with benzoyl chloride provides nucleoside Compound 26a.
• the phosphoramidite, Compound 33a is prepared from nucleoside Compound 26a following the same steps as illustrated for Compound 19a from Compound 11a.
• nucleoside Compound 26b Subsequent reaction of 25b with sodium hydroxide in dioxane/water, followed by reprotection of the 4-amino group with benzoyl chloride provides nucleoside Compound 26b.
• the phosphoramidite, Compound 33b is prepared from nucleoside Compound 26b following the same steps as illustrated for Compound 19b from Compound 1 Ib.
• Compound 34a is prepared by the Vorbruggen reaction of Compound 10a using 2-amino- 6-chloropurine, BSA and TMSOTf in refluxing dichloroethane. Reaction of nucleoside 34a with 3-hydroxypropionitrile and sodium hydride provides the cyclized nucleoside35a.
• the phosphoramidite, Compound 42a is prepared from nucleoside Compound 35a following the same steps as illustrated for Compound 19a from Compound 1 Ia.
• Compound 34b is prepared by the Vorbruggen reaction of Compound 10b using 2-amino-6-chloropurine, BSA and TMSOTf in refluxing dichloroethane. Reaction of nucleoside 34b with 3-hydroxypropionitrile and sodium hydride provides the cyclized nucleoside 35b.
• the phosphoramidite, Compound 42b is prepared from nucleoside Compound 35b following the same steps as illustrated for Compound 19b from Compound 1 Ib.
• Tetrabutylammonium fluoride 70 mL of a IM solution in THF was added to a cold (0 0 C) stirring solution of diol 49 (62.7 mmol, 37.5 g) in THF (250 mL) after which, the reaction was allowed to warm to room temperature gradually. After stirring for an additional 72 hours, the reaction was concentrated under vacuum and the residue was poured onto crushed ice. The flask was rinsed with some additional THF (3 times) and added to the above suspension. The supernatent was removed by decantation and the solid at the bottom was added to a stirring mixture of hexanes (200 mL) and water (200 mL). After stirring for 2 hours, the flocculent solid was collected by filtration, washed with additional water and hexanes and dried under high vacuum to provide Compound 47 (20 g, 89%) as a white solid.
• Isobutyryl chloride (6.9 mmol, 0.73 mL) was added to a cold (O 0 C) solution of Compound 58 (4.6 mmol, 2.48 g), diisopropylethylamine (6.9 mmol, 0.88 mL) and dimethylaminomethylpyridine (0.68 g, 83 mg) in dichloromethane (9 mL). After 2 hours at O 0 C, additional isobutyryl chloride (6.9 mmol, 0.73 mL) and diisopropylethylamine (6.9 mmol, 0.88 mL) were added to the reaction.
• N,(9-bis-trimethylsilylamide (18.0 mmol, 4.4 niL) was added to a suspension of Compound 60 (3.6 mmol, crude from above) and uracil (7.2 mmol, 0.81 g) in acetonitrile (18 mL) and the suspension was gently heated (using a heat gun) until a solution resulted.
• the reaction was cooled in an ice bath and TMSOTf (7.2 mmol, 1.3 mL) was added to the reaction. After the addition was complete, the ice bath was removed and the reaction was refluxed for 2 hours after which it was cooled to room temperature, diluted with EtOAc and carefully quenched with saturated NaHCO 3 solution. The organic layer was further washed with brine, dried (Na 2 SO 4 ) and concentrated to provide crude Compound 61, which was used without any further purification.
• DDQ (3.8 mmol, 0.86 g) was added to a solution of Compound 64 (1.9 mmol, 1.0 g) in dichloromethane (19 mL) and water (1 mL). After stirring at room temperature for 24 hours, the reaction was concentrated under reduced pressure. The residue was dissolved in EtOAc and the organic layer was washed with water, 10% sodium bisulfite, saturated NaHCO 3 , brine, dried (Na 2 SO 4 ) and concentrated. Purification by column chromatography (SiO 2 , eluting with 75% EtOAc in hexanes) provided Compound 64 (0.74 g, quantitative).
• TBSCl (5.8 mmol, 0.87 g) was added to a solution of Compound 64 (1.9 mmol, 0.75 g) and imidazole (11.6 mmol, 0.79 g) in DMF (5 mL). After stirring at room temperature for 16 hours, the reaction was diluted with EtOAc and the organic layer was washed with water, brine, dried (Na 2 SO 4 ) and concentrated. Purification by column chromatography (SiO 2 , 50% EtOAc in hexanes) provided Compound 65 (0.89 g, 94%) as a white foam.
• Phosphoramidite compound 69 is prepared from compound 68 according to the procedure described for the preparation of phosphoramidite 19a from compound 18a in example 1.
• Vorbrugen reaction of Compound 60 with N-benzoyl-cytosine, BSA and TMSOTf in refluxing acetonitrile provides nucleoside Compound 74.
• Treatment of 74 with aqueous NaOH solution effects cyclization to Compound 75. Protection of the 5'-hydroxyl group and the exocyclic amine with BzCl in pyridine provides Compound 76.
• Further processing of Compound 76 to phosphoramidite Compound 71 is similar to the procedures described for the preparation of phosphoramidite Compound 69 from Compound 63.
• Vorbrugen reaction of Compound 60 with N-benzoyl-adenine, BSA and TMSOTf in refluxing dichloroethane provides nucleoside Compound 80.
• Treatment of 80 with aqueous NaOH solution effects cyclization to Compound 81. Protection of the 5'-hydroxyl group and the exocyclic amine with BzCl in pyridine provides Compound 82.
• Further processing of Compound 82 to phosphoramidite Compound 88 is similar to the procedures described for the preparation of phosphoramidite Compound 69 from Compound 63.
• Compound 97 is prepared using the same general procedures described for the preparation of phosphoramidite Compound 69 from Compound 60 in example 11.
• Vorbrugen reaction of Compound 60 with 2-amino-6-chloropurine, BSA and TMSOTf in refluxing dichloroethane provides nucleoside Compound 89.
• Treatment of Compound 89 with 3- hydroxypropionitrile and sodium hydride effects cyclization to Compound 90.
• Transient protection of the 5'hydroxyl group as the trimethylsilyl ether is followed by protection of the exocyclic amino group with isobutyryl chloride.
• Phosphoramidite compound 110 is prepared from compound 102 using the same general procedure described for the preparation of phosphoramidite compound 19a from compound 17a in example 1.
• Phosphoramidite Compounds 111-113 are prepared from nucleoside Compounds 79, 85 and 94. Hydrogenation of the double bond using catalytic Palladium on carbon and hydrogen provides Compounds 98-101 respectively. Protection of the 5' hydroxyl group as the dimethoxytrityl ether followed by removal of the silyl protecting group and a phosphitilation reaction (as described in example 1) provides phosphoramidite Compounds 110-113.
• Compounds 117a-d are prepared from Compounds 116a-d by treatment with a fluorinating agent such as DAST using dichloromethane as the solvent.
• Compounds 118a-d are prepared from Compounds 116a-d by first oxidizing the primary hydroxyl group with Dess- Martin periodinane or under Swern conditions followed by treatment of the resulting aldehyde with DAST.
• Compounds 119a-d are prepared from Compounds 116a-d by first oxidizing the primary hydroxyl group with Dess-Martin periodinane or under Swern conditions followed by reductive amination of the resulting aldehyde with a primary or a secondary amine in the presence of glacial acetic acid and a reducing agent such as sodium cyanoborohydride.
• Compounds 120a-d are prepared from Compounds 116a-d by converting the hydroxyl group to a thiocarbonate derivative followed by a radical deoxygenation procedure using 11Bu 3 SnH.
• Compounds 121a-d are prepared from Compounds 116a-d by converting the hydroxyl group to a leaving group (mesylate, tosylate, halide) followed by heating with excess sodium azide.
• Compounds 124a-d are prepared from Compounds 116a-d by oxidation of the primary alcohol to a carboxylic acid followed by reaction with a amine in the presence of HATU or any other peptide coupling reagent.
• Compounds 125a-b are prepared from Compounds 116a-d by activating the hydroxyl group with carbonyl diimidazole followed by reaction with a amine.
• Compounds 126a-d are prepared from Compounds 116a-d by oxidizing the primary alcohol under Swern or Dess-Martin conditions followed by reaction with a suitable organometallic reagent.
• Compounds 128a-d are prepared from Compounds 116a-d by converting the hydroxyl group to a leaving group followed by displacement with a thiol nucleophile.
• Compounds 122a-d are prepared from Compounds 121a- d by reduction of the azide group followed by reaction with an isocyanate or an isothiocyanate.
• Compounds 123a-d are prepared from Compounds 121a-d by reduction of the azido group and reaction with FmocNCS to provide an activated thiourea. Further reaction of the fmoc activated thiourea with an amine in the presence of EDC provides the substituted guanidine. Removal of the fmoc protecting group liberates Compounds 123a-d.
• Compounds 130-132 are prepared by hydrogenation of the benzyloxymethyl protecting group using catalytic palladium on carbon and hydrogen gas. Protection of the 5' hydroxyl group as the dimethoxytrityl ether followed by removal of the silyl protecting group and a phosphitilation reaction (as described in example 1) provides phosphoramidite Compounds 142- 144.
• nucleoside phosphoramidites The preparation of nucleoside phosphoramidites is performed following procedures that are illustrated herein and in the art such as but not limited to US Patent 6,426,220 and published PCT WO 02/36743.
• Oligonucleotide and oligonucleoside synthesis The oligomeric compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, CA). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives.
• Alkyl phosphonate oligonucleotides can be prepared as described in U.S. Patent 4,469,863.
• 3 '-Deoxy-3 ' -methylene phosphonate oligonucleotides can be prepared as described in
• Phosphoramidite oligonucleotides can be prepared as described in U.S. Patent, 5,256,775 or U.S. Patent 5,366,878.
• Alkylphosphonothioate oligonucleotides can be prepared as described in published PCT applications PCT/US94/00902 and PCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively).
• 3'-Deoxy-3'-amino phosphoramidate oligonucleotides can be prepared as described in U.S. Patent 5,476,925.
• Phosphotriester oligonucleotides can be prepared as described in U.S. Patent 5,023,243.
• Borano phosphate oligonucleotides can be prepared as described in U.S. Patents
• Ethylene oxide linked oligonucleosides can be prepared as described in U.S. Patent 5,223,618.
• the oligonucleotides or oligonucleosides are recovered by precipitation out of 1 M NH 4 OAc with >3 volumes of ethanol. Synthesized oligonucleotides are analyzed by electrospray mass spectroscopy (molecular weight determination) and by capillary gel electrophoresis. The relative amounts of phosphorothioate and phosphodiester linkages obtained in the synthesis is determined by the ratio of correct molecular weight relative to the -16 amu product (+/-32 +/-48).
• oligonucleotides are purified by HPLC, as described by Chiang et al., J. Biol. Chem. 1991, 266, 18162-18171. Results obtained with HPLC-purif ⁇ ed material are generally similar to those obtained with non-HPLC purified material.
• Oligonucleotides can be synthesized via solid phase P(III) phosphoramidite chemistry on an automated synthesizer capable of assembling 96 sequences simultaneously in a 96-well format.
• Phosphodiester internucleotide linkages are afforded by oxidation with aqueous iodine.
• Phosphorothioate internucleotide linkages are generated by sulfurization utilizing 3,H- 1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile.
• Standard base- protected beta-cyanoethyl-diiso-propyl phosphoramidites are purchased from commercial vendors (e.g.
• Non-standard nucleosides are synthesized as per standard or patented methods. They are utilized as base protected beta-cyanoethyldiisopropyl phosphoramidites. Oligonucleotides are cleaved from support and deprotected with concentrated NH 4 OH at elevated temperature (55-6O 0 C) for 12-16 hours and the released product then dried in vacuo. The dried product is then re-suspended in sterile water to afford a master plate from which all analytical and test plate samples are then diluted utilizing robotic pipettors.
• the concentration of oligonucleotide in each well is assessed by dilution of samples and UV absorption spectroscopy.
• the full-length integrity of the individual products is evaluated by capillary electrophoresis (CE) in either the 96-well format (Beckman P/ACETM MDQ) or, for individually prepared samples, on a commercial CE apparatus (e.g., Beckman P/ACETM 5000, ABI 270).
• Base and backbone composition is confirmed by mass analysis of the oligomeric compounds utilizing electrospray-mass spectroscopy. All assay test plates are diluted from the master plate using single and multi-channel robotic pipettors. Plates are judged to be acceptable if at least 85% of the oligomeric compounds on the plate are at least 85% full length.
• oligomeric compounds on target nucleic acid expression can be tested in any of a variety of cell types provided that the target nucleic acid is present at measurable levels. This can be routinely determined using, for example, PCR or Northern blot analysis. Cell lines derived from multiple tissues and species can be obtained from American Type Culture Collection (ATCC, Manassas, VA).
• b.END cells The mouse brain endothelial cell line b.END was obtained from Dr. Werner Risau at the Max Plank Institute (Bad Nauheim, Germany). b.END cells were routinely cultured in DMEM, high glucose (Invitrogen Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Carlsbad, CA).
• Cells were routinely passaged by trypsinization and dilution when they reached approximately 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #353872, BD Biosciences, Bedford, MA) at a density of approximately 3000 cells/well for uses including but not limited to oligomeric compound transfection experiments.
• Oligonucleotide is mixed with LIPOFECTINTM Invitrogen Life Technologies, Carlsbad, CA) in Opti-MEMTM-1 reduced serum medium (Invitrogen Life Technologies, Carlsbad, CA) to achieve the desired concentration of oligonucleotide and a LIPOFECTINTM concentration of 2.5 or 3 ⁇ g/mL per 100 nM oligonucleotide.
• This transfection mixture is incubated at room temperature for approximately 0.5 hours. For cells grown in 96-well plates, wells are washed once with 100 ⁇ L OPTI-MEMTM-1 and then treated with 130 ⁇ L of the transfection mixture.
• Cells grown in 24-well plates or other standard tissue culture plates are treated similarly, using appropriate volumes of medium and oligonucleotide. Cells are treated and data are obtained in duplicate or triplicate. After approximately 4-7 hours of treatment at 37 0 C, the medium containing the transfection mixture is replaced with fresh culture medium. Cells are harvested 16-24 hours after oligonucleotide treatment.
• transfection reagents known in the art include, but are not limited to, CYTOFECTINTM, LIPOFECTAMINETM, OLIGOFECTAMINETM, and FUGENETM.
• Other suitable transfection methods known in the art include, but are not limited to, electroporation.
• Antisense modulation of a target expression can be assayed in a variety of ways known in the art.
• a target mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR.
• Real-time quantitative PCR is presently desired.
• RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA.
• One method of RNA analysis of the present invention is the use of total cellular RNA as described in other examples herein. Methods of RNA isolation are well known in the art.
• Northern blot analysis is also routine in the art.
• Real-time quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISMTM 7600, 7700, or 7900 Sequence Detection System, available from PE-Applied Biosystems, Foster City, CA and used according to manufacturer's instructions.
• Protein levels of a target can be quantitated in a variety of ways well known in the art, such as imrnunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA) or fluorescence-activated cell sorting (FACS).
• Antibodies directed to a target can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, MI), or can be prepared via conventional monoclonal or polyclonal antibody generation methods well known in the art. Methods for preparation of polyclonal antisera are taught in, for example, Ausubel, F.M. et al., Current
• Western blot (immunoblot) analysis is standard in the art and can be found at, for example, Ausubel, F.M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley & Sons, Inc., 1997.
• Enzyme-linked immunosorbent assays (ELISA) are standard in the art and can be found at, for example, Ausubel, F.M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley & Sons, Inc., 1991.
• the oligomeric compounds are further investigated in one or more phenotypic assays, each having measurable endpoints predictive of efficacy in the treatment of a particular disease state or condition.
• Phenotypic assays, kits and reagents for their use are well known to those skilled in the art and are herein used to investigate the role and/or association of a target in health and disease.
• Representative phenotypic assays which can be purchased from any one of several commercial vendors, include those for determining cell viability, cytotoxicity, proliferation or cell survival (Molecular Probes, Eugene, OR; PerkinElmer, Boston, MA), protein-based assays including enzymatic assays (Panvera, LLC, Madison, WI; BD Biosciences, Franklin Lakes, NJ; Oncogene Research Products, San Diego, CA), cell regulation, signal transduction, inflammation, oxidative processes and apoptosis (Assay Designs Inc., Ann Arbor, MI), triglyceride accumulation (Sigma- Aldrich, St. Louis, MO), angiogenesis assays, tube formation assays, cytokine and hormone assays and metabolic assays (Chemicon International Inc., Temecula, CA
• cells determined to be appropriate for a particular phenotypic assay i.e., MCF-7 cells selected for breast cancer studies; adipocytes for obesity studies
• a target inhibitors identified from the in vitro studies as well as control compounds at optimal concentrations which are determined by the methods described above.
• treated and untreated cells are analyzed by one or more methods specific for the assay to determine phenotypic outcomes and endpoints.
• Phenotypic endpoints include changes in cell morphology over time or treatment dose as well as changes in levels of cellular components such as proteins, lipids, nucleic acids, hormones, saccharides or metals. Measurements of cellular status which include pH, stage of the cell cycle, intake or excretion of biological indicators by the cell, are also endpoints of interest. Measurement of the expression of one or more of the genes of the cell after treatment is also used as an indicator of the efficacy or potency of the a target inhibitors. Hallmark genes, or those genes suspected to be associated with a specific disease state, condition, or phenotype, are measured in both treated and untreated cells. In vivo studies
• the individual subjects of the in vivo studies described herein are warm-blooded vertebrate animals, which includes humans.
• PoIy(A)+ mRNA isolation PoIy(A)+ mRNA is isolated according to Miura et al., (Clin. Chem., 1996, 42, 1758-
• Plates are incubated for 60 minutes at room temperature, washed 3 times with 200 ⁇ L of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plate is blotted on paper towels to remove excess wash buffer and then air-dried for 5 minutes. 60 ⁇ L of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70 0 C, is added to each well, the plate is incubated on a 90 0 C hot plate for 5 minutes, and the eluate is then transferred to a fresh 96-well plate.
• wash buffer 10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl
• 60 ⁇ L of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70 0 C, is added to each well, the plate is incubated on a 90 0 C hot plate for 5 minutes, and the eluate is then
• RNA Isolation Total RNA is isolated using an RNEASY 96TM kit and buffers purchased from Qiagen
• RNA is then eluted by pipetting 140 ⁇ L of RNAse free water into each well, incubating 1 minute, and then applying the vacuum for 3 minutes.
• the repetitive pipetting and elution steps may be automated using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia CA). Essentially, after lysing of the cells on the culture plate, the plate is transferred to the robot deck where the pipetting, DNase treatment and elution steps are carried out.
• Quantitation of a target mRNA levels was accomplished by real-time quantitative PCR using the ABI PRISMTM 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, CA) according to manufacturer's instructions.
• ABI PRISMTM 7600, 7700, or 7900 Sequence Detection System PE-Applied Biosystems, Foster City, CA
• This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in real-time.
• PCR polymerase chain reaction
• products in real-time quantitative PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes.
• a reporter dye e.g., FAM or JOE, obtained from either PE-Applied Biosystems, Foster City, CA, Operon Technologies Inc., Alameda, CA or Integrated DNA Technologies Inc., Coralville, IA
• a quencher dye e.g., TAMRA, obtained from either PE-Applied Biosystems, Foster City, CA, Operon Technologies Inc., Alameda, CA or Integrated DNA Technologies Inc., Coralville, IA
• TAMRA obtained from either PE-Applied Biosystems, Foster City, CA, Operon Technologies Inc., Alameda, CA or Integrated DNA Technologies Inc., Coralville, IA
• annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5'-exonuclease activity of Taq polymerase.
• cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated.
• additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular intervals by laser optics built into the ABI PRISMTM Sequence Detection System.
• a series of parallel reactions containing serial dilutions of mRNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples.
• primer-probe sets specific to the target gene being measured are evaluated for their ability to be "multiplexed" with a GAPDH amplification reaction.
• GAPDH amplification reaction In multiplexing, both the target gene and the internal standard gene GAPDH are amplified concurrently in a single sample.
• mRNA isolated from untreated cells is serially diluted.
• Each dilution is amplified in the presence of primer-probe sets specific for GAPDH only, target gene only ("single-plexing"), or both (multiplexing).
• primer-probe sets specific for GAPDH only target gene only
• target gene only target gene only
• multiplexing target gene only
• standard curves of GAPDH and target mRNA signal as a function of dilution are generated from both the single-plexed and multiplexed samples. If both the slope and correlation coefficient of the GAPDH and target signals generated from the multiplexed samples fall within 10% of their corresponding values generated from the single-plexed samples, the primer-probe set specific for that target is deemed multiplexable.
• Other methods of PCR are also known in the art.
• RT and PCR reagents were obtained from Invitrogen Life Technologies (Carlsbad, CA).
• RT real-time PCR was carried out by adding 20 ⁇ L PCR cocktail (2.5x PCR buffer minus MgCl 2 , 6.6 niM MgCl 2 , 375 ⁇ M each of dATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5 Units MuLV reverse transcriptase, and 2.5x ROX dye) to 96-well plates containing 30 ⁇ L total RNA solution (20-200 ng).
• PCR cocktail 2.5x PCR buffer minus MgCl 2 , 6.6 niM MgCl 2 , 375 ⁇ M each of dATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverse primer,
• the RT reaction was carried out by incubation for 30 minutes at 48°C. Following a 10 minute incubation at 95 0 C to activate the PLATINUM® Taq, 40 cycles of a two-step PCR protocol were carried out: 95°C for 15 seconds (denaturation) followed by 60 0 C for 1.5 minutes (annealing/extension).
• Gene target quantities obtained by RT, real-time PCR are normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RIBOGREENTM (Molecular Probes, Inc. Eugene, OR).
• GAPDH expression is quantified by real time RT-PCR, by being run simultaneously with the target, multiplexing, or separately.
• Total RNA is quantified using RiboGreenTM RNA quantification reagent (Molecular Probes, Inc. Eugene, OR). Methods of RNA quantification by RIBOGREENTM are taught in Jones, L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374).
• RIBOGREENTM working reagent RIBOGREENTM working reagent diluted 1 :350 in 1OmM Tris-HCl, 1 mM EDTA, pH 7.5
• RIBOGREENTM reagent diluted 1 :350 in 1OmM Tris-HCl, 1 mM EDTA, pH 7.5 is pipetted into a 96-well plate containing 30 ⁇ L purified, cellular RNA.
• the plate is read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 485nm and emission at 530nm.
• Probes and primers may be designed to hybridize to a target sequence, using published sequence information.
• the following primer-probe set was designed using published sequence information (GENBANKTM accession number U92436.1, SEQ ID NO: 1).
• Forward primer AATGGCTAAGTGAAGATGACAATCAT (SEQ ID NO: 2)
• Reverse primer TGCACATATCATTACACCAGTTCGT (SEQ ID NO: 3)
• the PCR probe FAM-TTGCAGCAATTCACTGTAAAGCTGGAAAGG-TAMRA (SEQ ID NO: 4), where FAM is the fluorescent dye and TAMRA is the quencher dye.
• Western blot analysis of target protein levels Western blot analysis (immunoblot analysis) is carried out using standard methods.
• Cells are harvested 16-20 h after oligonucleotide treatment, washed once with PBS, suspended in Laemmli buffer (100 ⁇ l/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gels are run for 1.5 hours at 150 V, and transferred to membrane for western blotting.
• Appropriate primary antibody directed to a target is used, with a radiolabeled or fluorescently labeled secondary antibody directed against the primary antibody species. Bands are visualized using a PHOSPHORIMAGERTM (Molecular Dynamics, Sunnyvale CA).
• nuclease stability of 5'-(S) and (JI)-CH 3 -BNA modified oligomers treated with SVPD The nuclease stability of 5'-CH 3 -BNA modified oligomers was determined using snake venom phosphodiesterase (SVPD). Each oligomer was prepared as a 500 ⁇ L mixture containing: 5 ⁇ L lOO ⁇ M oligomer, 50 ⁇ L phosphodiesterase I @ 0.5 Units/mL in SVPD buffer (50 mM Tris- HcL, pH 7.5, 8 mM MgCl 2 ) final concentration 0.05 Units/mL, 445 ⁇ L SVP buffer. Samples were incubated at 37°C in a water bath. Aliquats (100 ⁇ L) were taken at 0, 1, 2 and 4 days with fresh enzyme added at days 1 and 2. EDTA was added to aliquats immediately after removal to quench enzyme activity. Samples were analyzed on IP HPLC/MS.
• All internucleoside linkages are phosphodiester, subscript S or R indicates the configuration at the 5' carbon atom for 5'-CH 3 -BNA nucleosides which also have a 4'-CH 2 -O-2' bridge group.
• a subscript e indicates 2'-O-MOE nucleosides and subscript 1 indicates 4'-CH 2 -O- 2' modified nucleosides.
• the 5-methyl substituted BNA-containing compounds (392746 and 392747) had a marked improvement over the unsubstitued BNA-containing compound (392745).
• the nuclease stability of 5'-CH 3 -BNA modified oligomers was determined using snake venom phosphodiesterase (SVPD). Each oligomer was prepared as a 90 ⁇ L mixture containing 5 ⁇ L oligomer (2 ⁇ L of 5 ⁇ M oligomer and 3 ⁇ L of 5' 32 P-labled oligomer) 75 ⁇ L H 2 O, and 10 ⁇ L 1OX buffer (500 mM Tris-HCl, 700 mM NaCl, and 140 mM MgCl 2 at pH 8.6).
• SVPD snake venom phosphodiesterase
• oligomeric compounds were synthesized and tested for their ability to reduce PTEN expression over a range of doses.
• b.END cells were treated with the 5'-CH 3 -BNA modified oligomers at concentrations of 0.3125, 0.0625, 1.25, 2.5, 5, 10 or 20 nM using methods described herein.
• Expression levels of PTEN were determined using real-time PCR and normalized to RIBOGREENTM as described in other examples herein.
• the percent reduction of PTEN mRNA relative to untreated control cells (%UTC) at a drug concentration of 20 nM is tablulated below. Resulting dose-response curves were used to determine the IC50 of 392747 as shown below.
• Tm's were assessed in 100 mM phosphate buffer, 0.1 mM EDTA, pH 7, at 260 nm using 4 ⁇ M 5'-CH 3 -BNA modified oligomers and 4 ⁇ M complementary RNA.
• All internucleoside linkages are phosphorothioate and subscripts R and S indicate the configuration at the 5' carbon atom for 5'-CH 3 -BNA nucleosides which also have a 4'-CH 2 -O-2 T bridge group.
• mice Six week old Balb/c mice (Jackson Laboratory, Bar Harbor, ME) were injected twice weekly for 3 weeks with a 5'-CH 3 -BNA modified oligomers (either 5'-(S) or 5'-(R)) targeted to PTEN at a dose of 0.5 or 2 ⁇ mol/kg. The mice were sacrificed 48 hours following the final administration. Liver tissues were homogenized and mRNA levels were quantitated using real- time PCR as described herein for comparison to untreated control levels (%UTC).
• composition (5' to 3') dose %l
• All internucleoside linkages are phosphorothioate and subscripts R and S indicates the configuration at the 5' carbon atom for 5'-CH 3 -BNA nucleosides which also have a 4'-CH 2 -O-2' bridge group.
• mice Six week old Balb/c mice (Jackson Laboratory, Bar Harbor, ME) were injected once with a 5'-(S)-CH 3 -BNA modified oligomer targeted to PTEN at a dose of 1 , 2, 4 or 8 ⁇ mol/kg. The mice were sacrificed 72 hrs following administration. Liver tissues were homogenized and mRNA levels were quantitated using real-time PCR as described herein for comparison to untreated control levels (%UTC). SEQ ID NO. Composition (5' to 3') dose %UTC /ISIS NO. ( ⁇ mol/kg) saline 100
• internucleoside linkages are phosphorothioate and subscript S indicates the configuration at the 5' carbon atom for 5'-CH 3 -BNA nucleosides which also have a 4'-CH 2 -O-2' bridge group.
• mice Six week old Balb/c mice (Jackson Laboratory, Bar Harbor, ME) were injected twice weekly for three weeks with 5'-(S)-CH 3 -BNA (2-10-2, 14-mer), 4'-CH 2 -O-2'-BNA (2-10-2, 14- mer) and 2'-0-MOE (5-10-5, 20-mer) modified oligomers targeted to PTEN at a dose of 3.2, 1.0,
• mice were sacrificed 48 hrs following last administration. Liver tissues were homogenized and mRNA levels were quantitated using real-time PCR as described herein for comparison to untreated control levels (%UTC). Plasma chemistries and liver weights were determined after sacrifice.
• liver weights for 5'-(S)-CH 3 BNA (392747, 3.2 ⁇ mol/Kg dose group) containing oligomers were 121% relative to saline.
• Liver weights for 2'-O-MOE containing oligomers (116847, 1.0 ⁇ mol/Kg dose group) were 116% relative to saline.
• mice Six week old Balb/c mice (Jackson Laboratory, Bar Harbor, ME) were injected once with modified 5'-(S)-CH 3 (396569), 4'-CH 2 -O-2' BNA 2-10-2 gapped oligomers targeted to PTEN at a dose of 2.5, 5, 10 and 20 ⁇ mol/kg (only 5 and 10 ⁇ mol/Kg data shown). The mice were sacrificed 66 hrs following administration. Liver tissues were homogenized. SEQ ID NO. Composition (5' to 3') dose ALT
• PTEN in vivo study
• Six week old Balb/c mice (Jackson Laboratory, Bar Harbor, ME) were injected once with 5'-CH 3 -BNA modified oligomers targeted to PTEN at a dose of 2 or 10 ⁇ mol/kg.
• the mice were sacrificed 72 hrs following administration.
• Liver tissues were homogenized and mRNA levels were quantitated using real-time PCR as described herein for comparison to untreated control levels (% UTC).
• CiTiGCTAGCCTCTGGATT 1 Ti 4'-CH 2 -O-2' BNA 13/400521 CsUsGCTAGCCTCTGGATUsUs 5'-(S)-CH 3
• All internucleoside linkages are phosphorothioate
• subscripts R and S indicate the configuration at the 5' carbon atom for 5'-CH 3 -BNA nucleosides which also have a 4'-CH 2 -O-2' bridge group
• subscript e indicates 2'-0-MOE nucleosides
• subscript 1 indicates 4'-CH 2 -O-2' nucleosides
• Me C indicates a 5'-methyl cytosine nucleoside.
• oligonucleotide 400521 containing the 5'(S)- Me modification is essentially equally efficacious as 394425.
• the ALT elevations for 400521 are modest (152) as compared to 394425 (2453.2) clearly indicating that the 5'- substitution results in a greatly improved therapeutic index.

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