+

WO1993024507A1 - Oligomeres a conformation restreinte contenant des liaisons amide ou carbamate pour effectuer une liaison specifique d'une sequence - Google Patents

Oligomeres a conformation restreinte contenant des liaisons amide ou carbamate pour effectuer une liaison specifique d'une sequence Download PDF

Info

Publication number
WO1993024507A1
WO1993024507A1 PCT/US1993/005110 US9305110W WO9324507A1 WO 1993024507 A1 WO1993024507 A1 WO 1993024507A1 US 9305110 W US9305110 W US 9305110W WO 9324507 A1 WO9324507 A1 WO 9324507A1
Authority
WO
WIPO (PCT)
Prior art keywords
oligomer
oligomers
membered ring
dna
rna
Prior art date
Application number
PCT/US1993/005110
Other languages
English (en)
Inventor
Sundaramoorthi Swaminathan
John Munger
Robert J. Jones
Mark Matteucci
Jeff Pudlo
Xiaodong Cao
Original Assignee
Gilead Sciences, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Gilead Sciences, Inc. filed Critical Gilead Sciences, Inc.
Publication of WO1993024507A1 publication Critical patent/WO1993024507A1/fr

Links

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
    • 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
    • C07H19/06—Pyrimidine radicals
    • 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 invention relates to novel modified oligonucleotides, the synthesis thereof, their use in oligomer-based therapies and their use as diagnostic reagents. More specifically, the invention is to oligomers having modified nucleosides which are resistant to nuclease, having enhanced ability to penetrate cells, and which are and which are capable of binding target base sequences .in vitro and in vivo.
  • the modified oligonucleotides of the invention are particularly useful in oligonucleotide-based therapies utilizing the modified oligonucleotides to interrupt protein synthesis or transcription or to otherwise inactivate messenger RNA or double-stranded DNA.
  • oligonucleotides and oligonucleotide analogs for therapeutic uses represents a relatively new development in drug design and discovery.
  • oligomers oligonucleotide analogs
  • RNA sequence is identical to the antisense strand of the gene that gave rise to the RNA (Uhlmann, E. , et al., Chem Reviews (1990) 9):543-584; and Stein, CA. , et al., Cancer Res (1988) 4_8:2659-2668) .
  • Another approach, referred to herein as "triple helix” therapy utilizes oligomers that bind to duplex D ⁇ A as detailed below. Binding to a target D ⁇ A is sequence specific but involves different base pairing binding.
  • Both antisense and triple helix therapies exert therapeutic effects via binding to nucleic acid sequences that are responsible for disease conditions.
  • nucleic acid sequences are found in the genome of pathogenic organisms including bacteria, protozoa, yeasts, parasites, fungi or viruses or may be endogenous sequences (oncogenes, cytokines, etc) .
  • endogenous sequences oncogenes, cytokines, etc.
  • oligomers Another therapeutic approach that is based on the use of oligomers includes generation of "aptamer ⁇ " and is disclosed and claimed in commonly owned application nos. 745,215, 659,980 and 658,849. This approach utilizes oligomers that specifically bind to proteins thereby interfering with their function.
  • the use of oligomers that mimic the structure of certain R ⁇ A molecules that are bound by intracellular proteins has also been adduced as a therapeutic approach as described in international application no. PCT/US91/01822.
  • Antisense oligonucleotides are synthetic oligonucleotides which bind complementary nucleic acids (i.e. sense strand sequences) via hydrogen bonding, thereby inhibiting translation of these sequences.
  • Therapeutic intervention at the nucleic acid level using antisense oligonucleotides offers a number of advantages. For example, gene expression can be inhibited using antisense or triple helix oligomers. Inhibition of gene expression is more efficient than inhibition of the protein encoded by the gene since transcription of a single DNA sequence gives rise to multiple copies of mRNA which, in turn, are translated into many protein molecules.
  • Antisense and triple helix therapies for diseases whose etiology is characterized by, or associated with, specific DNA or RNA sequences are particularly useful.
  • the oligomer employed as the therapeutic agent can be directly administered and is one that is complementary to a DNA or RNA needed for the progress of the disease.
  • the oligomer specifically binds to this target nucleic acid sequence, thus disturbing its ordinary function.
  • An oligomer having a base sequence complementary to that of an mRNA which encodes a protein necessary for the initiation, maintenance or progress of the disease is useful for interfering with synthesis of the protein. By hybridizing specifically to this mRNA, the synthesis of the protein will be interrupted. However, it is also possible to bind double-stranded DNA using an appropriate oligomer capable of effecting the formation of a specific triple helix by inserting the administered oligomer into the major groove of the double-helical DNA. The resulting triple helix structure can then interfere with transcription of the target gene (Young, S.L. et al., Proc Natl Acad Sci (1991) 88;10023- 10026).
  • the backbone of the administered oligomer should contain internucleotide linkages or structures that are stable in vivo and should be structured such that the oligomer is resistant to endogenous nucleases, such as nucleases that attack the phosphodiester linkage.
  • the oligomer must also retain its ability to hybridize to the target DNA or RNA.
  • ethylphosphonates wherein one of the phosphorous-linked oxygens has been replaced by methyl
  • phosphorothioates wherein sulphur replaces one of these oxygens
  • various amidates wherein NH_ or an organic amine derivative, such as morpholidates or piperazidates, replace an oxygen
  • WO 91/15500 published October 17, 1991, teaches various oligonucleotide analogs in which one or more of the internucleotide linkages are replaced by a sulfur based linkage typically sulfamate diesters which are isosteric and isoelectric with the phosphodiester.
  • WO 89/12060 published December 14, 1989, similarly discloses linkages containing sulfides, sulfoxides, and sulfones.
  • U.S. Patent No. 5,079,151 to Lampson et al. discloses a sDNA molecule of branched RNA linked to a single strand DNA via a 2',5' phosphodiester linkage. 2',5' linkages that may be incorporated into oligomers are described in commonly owned, pending U.S. patent application attorney docket number 24610-20042, S. Swaminathan et al., inventors, filed June 1, 1992, the entire disclosure of which is incorporated herein by reference. Commonly owned, pending U.S. Patent Application
  • modified internucleotide linkages described in the 07/806,710 and 07/990,848 disclosures were identified on the basis of computer modeling studies that defined sterically the size, bond lengths, angles and torsions that were compatible with the new linkages. Synthesis of modified linkages that were compatible with linkages predicted by the computer modeling studies was accomplished. When these linkages were introduced or incorporated into oligonucleotide analogs they were found to be binding competent.
  • a binding competent conformation refers to the spatial orientation of heterocyclic bases in an oligomer required for binding to duplex or single stranded DNA or RNA in a sequence-specific manner. Conformationally restricted oligomers containing binding competent riboacetal linkages with high affinity for binding to single strand as well as double strand target nucleic acid sequences were described in 07/806,710 and 07/990,848.
  • the invention linkages were arrived at using modeling studies and were compared with the riboacetal linkage as shown in Figure 1.
  • the riboacetal linkage studies verified computer model predictions that the conformational restriction could improve affinity in some cases.
  • Nielsen et al., (Science (1991) 254:1497-1500) showed that an oligomer conjugate composed of amide linked nucleomonomers and a lysine residue had high affinity binding to single stranded oligonucleotides.
  • Figure 2c shows a composite of both structures. As shown in Figure 2c, there is a nearly identical spatial overlap between the linkages when the structures are superimposed.
  • the structural correspondence between the riboacetal linkage and the polyamide-based linkage is the most persuasive evidence supporting the underlying assumption regarding the structure of polyamide-based oligomers when bound to target nucleic acid sequences.
  • the oligomers disclosed herein fall within the same spatial group of binding competent species as the amide and riboacetal structures.
  • the oligomers of the present invention are generally characterized as being comprised of a series of constrained linkers or nucleomononers which correspond in space to ⁇ even-membered rings or to seven-membered ring equivalents so as to attain a spatial conformation that is appropriate for binding of the heterocyclic base to a target nucleic acid in a sequence specific manner.
  • a seven-membered ring equivalent includes seven-sided structures or the equivalent of a seven-sided structure which may have one or more sides comprised of, for example, a hydrogen bond or a covalent structural equivalent that conforms to a seven-sided ring.
  • a seven-membered ring equivalent is any constrained arrangement of atoms which has a spatial conformation substantially identical to the formula:
  • the seven-membered ring and its equivalent is a common structural motif in the oligomers of the present invention that can be arrived at through several different structural "modes".
  • Such structural modes include structures, which when incorporated into oligomers, form a covalently linked structure that is a seven-membered ring or its - covalently linked equivalent.
  • Such structures are exemplified by the oligomers shown in Figure 10 which represents a covalently closed seven-membered ring or Figure 11 which represents a covalently closed seven- membered ring equivalent.
  • a conformation favoring the seven-membered ring or its equivalent can be attained in oligomers by structural modes that include (i) torsional constraints imposed by a covalent structure, such as a covalent ring, within the monomers that comprise an oligomer, as exemplified by the oligomers shown in Figures 9 and 13, and (ii) torsional constraints imposed by noncovalent forces such as hydrogen bonds, hydrophobic interactions, ionic interactions or steric constraints.
  • the hydrogen bond will favor a conformation in the oligomer that comprises a seven-membered ring equivalent that itself has as one of its sides, a hydrogen bond.
  • amide linkages can be characterized as having a single hydrogen bond that favors formation of the seven-membered ring or its equivalent that is required for a bonding competent conformation.
  • the constrained linkers described herein, when incorporated into oligomers, can be characterized as having a force greater than a single hydrogen bond that favors formation of the binding competent conformation.
  • the forces favoring binding competence are a hydrogen bond and the cyclic connection between C3 and N4 which locks in the cis 022-C3-N4-C5 conformation in preference over the trans 022-C3-N4-C5 conformation about the C3 and N4 bond as defined in space in Figure 1.
  • Hydroxymethyl-linked. oligomers have two hydrogen bond ⁇ that favor formation of a seven membered ring, wherein one of the hydrogen bonds is one ⁇ ide of a complimentary seven-membered ring that drives formation of the seven-membered ring to which the heterocycle is attached.
  • a covalent bond fixes 100% of the heterocycle ⁇ in an oligomer into a binding competent conformation due to the pre ⁇ ence of the covalent seven- membered ring (azepine) or its covalent equivalent (norbornyl) .
  • the nucleomonomer ⁇ of the present invention are generally characterized as moieties or residues that replace both the furanose ring and the phosphorus atom that is normally found in nucleotides.
  • the discovery of these nucleomonomers and their characteristic ⁇ is based on modeling studies that both (1) predicted such analogs are compatible with a binding competent oligomer and (2) defined the range of molecular characteristics that such nucleomonomers could assume without the loss of binding competence, when incorporated into oligomers.
  • Binding competence refers either to Wat ⁇ on-Crick ba ⁇ e pairing with single-stranded DNA or single-stranded RNA or to Hoogsteen pairing (Beal, P.A.
  • duplex nucleic acids including duplex DNA or duplex RNA.
  • Exemplary monomers and oligomers (and methods of their synthesis) of this invention are shown in Figures 3 through 13 and are conformationally more restricted relative to the phosphodiester linkages found in unmodified DNA or RNA. Thi ⁇ conformational re ⁇ triction is believed to underline, at least in part, their capacity for enhanced binding to complementary nucleic acid target sequences.
  • oligomers described herein permits synthesis of improved compounds with respect to properties such as (i) increased lipophilicity which results from eliminating the charge as ⁇ ociated with phosphodie ⁇ ter linkage ⁇ (Dalge, J.M. et al., Nucleic Acids Res (1991) .19.:1805-1810) and (ii) resistance to degradation by enzymes such as nucleases. Consequently, oligomers containing the ⁇ e nucleoside analogs are quite suitable for hybridization to target sequences or molecules, and in some aspect ⁇ are superior to unmodified phosphodiester-linked nucleoside residue ⁇ when incorporated into oligonucleotide ⁇ .
  • oligomers as described in the invention exhibit sequence-specific binding to complementary single stranded and duplex target sequences.
  • the present invention provides a multitude ⁇ of nucleomonomers that can be incorporated into binding competent oligomers.
  • the invention oligomers are resistant to nuclease digestion, are stable under physiological conditions and are neutral so as to enhance cell permeation.
  • nuclease stability and enhanced cellular permeation are important considerations for the development of oligomers that are intended to be used as therapeutic agents that function by binding to specific DNA or RNA (mRNA, hnRNA, etc.) sequences.
  • specific target sequence binding underlies their therapeutic efficacy by interfering with the normal biological function of nucleic acid sequences as ⁇ ociated with pathological condition ⁇ .
  • the present invention is ba ⁇ ed on the identification of the rationally de ⁇ igned oligomer ⁇ disclosed herein and of novel nucleomonomers and methods for their incorporation into oligomers containing the nucleomonomers.
  • the present invention i ⁇ directed to an oligomer which compri ⁇ e ⁇ the presence of one or more invention nucleomonomers which are disclo ⁇ ed herein and are exemplified by formula ⁇ I - VIII:
  • B is a purine or pyrimidine base or an analogous form thereof
  • X 1 is S, O, SO, S0 2 , CH 2 , CHF, CF 2 NR or CH- lower alkyl including CH-methyl, CH-ethyl, CH-propyl and CH-butyl, provided that adjacent X 1 are not both 0; and each R is independently H, F, OH, OMe, CH 3 or lower alkyl including ethyl, propyl and butyl provided that both R attached to the same carbon atom are not both OH or are not OH and OMe together.
  • all of the nucleomonomers of formulas I -. VIII in a given oligomer or domain of an oligomer are chirally pure at po ⁇ ition ⁇ where an X 1 or R group i ⁇ located.
  • the oligomer ⁇ of the invention contain at lea ⁇ t one domain compri ⁇ ing invention nucleomonomers that contain a seven-membered ring or a seven-membered ring equivalent that conforms in space to structures that are compatible with the binding competent oligomers described herein.
  • a domain of an oligomer, as used herein, is defined to mean a part or region of an oligomer that contains at least three linked heterocyclic base ⁇ or three linked monomer or linker residues (one or more of which residues may in some cases be abasic, i.e. not containing a heterocyclic base) .
  • amide linked domain refer ⁇ to amide linked nucleomonomer ⁇ while a "rigid domain”, as used herein, refer ⁇ to a domain that contain ⁇ other types of linked nucleomonomers or may have a mixture of nucleomonomer types and may include one or more amide linked nucleomonomer ⁇ .
  • An oligomer may contain one or more amide linked domain ⁇ that are comprised of a single type of nucleomonomer unit or may contain a mixture of nucleomonomer ⁇ .
  • the invention oligomers will be either (1) comprised solely of invention nucleomonomers or (2) comprised of a domain comprised solely of invention nucleomonomers coupled to a domain comprised of nucleomonomers coupled via linkages or via noninvention substitute linkages (e.g. thioate, ethylphosphonate, riboacetal, 3',5' for acetal, 2 ' , 5 ' formacetal and the like) .
  • the invention nucleomonomer ⁇ do not contain a furano ⁇ e moiety and can be coupled to each other via method ⁇ similar to tho ⁇ e u ⁇ ed in peptide synthesis and which are disclosed herein.
  • Oligomers are conveniently produced from dimers or tri ers as synthon ⁇ for solid phase or solution phase synthe ⁇ is using standard methods known in the art. However, oligomers of any length may be prepared including 10-mer ⁇ (10 nucleomonomer ⁇ ) , 20-mer ⁇ , 50-mer ⁇ , 100-mers, 200-mers, 500-mers or oligomer ⁇ of greater length. Oligomer ⁇ containing 2 to 30 nucleomonomer ⁇ are preferred. In general, the invention oligomer ⁇ will be ⁇ ynthe ⁇ ized by solid phase methods which sequentially add nucleomonomers to an oligomer bound to a support.
  • Additional preferred embodiments include oligomers as exemplified in Figure ⁇ 9 through 13 wherein Y and Y 1 are independently an oligomer, a blocking group such as FMOC or tBOC, hydrogen, an activated ester coupling group ⁇ uitable for ⁇ olid phase peptide synthesis a label (radioisotope, enzyme or chro ophore) or a solid support (polystyrene and the like) .
  • a blocking group such as FMOC or tBOC
  • hydrogen an activated ester coupling group ⁇ uitable for ⁇ olid phase peptide synthesis a label (radioisotope, enzyme or chro ophore) or a solid support (polystyrene and the like) .
  • an activated ester coupling group ⁇ uitable for ⁇ olid phase peptide synthesis a label (radioisotope, enzyme or chro ophore) or a solid support (polysty
  • Preferred ⁇ even-membered ring or seven-membered ring equivalent linkers or nucleomonomers have the base attached to the linker or nucleomonomer through a moiety which consists of two covalent bonds.
  • Preferred moieties contain at least one carbon atom linked to at least one of the two covalent bonds.
  • Y, Y 1 and B have the same definitions as above; and X s is a seven-membered ring or a seven- membered ring equivalent with B covalently connected to the backbone through a moiety which consi ⁇ ts of two covalent bonds.
  • the seven- membered rings or their equivalents are coupled to each other through three bonds internal to the seven-membered ring or equivalent and three covalent bonds external to the seven-membered ring or equivalent.
  • the invention is directed to methods for treating disea ⁇ e ⁇ mediated by the pre ⁇ ence of a nucleotide sequence which comprise admini ⁇ tering to a subject in need of such treatment an amount of an invention oligomer capable of specifically binding the nucleotide sequence effective so to inactivate (or modulate) the nucleotide sequence.
  • the analogs may be utilized in oligomers that contain additional modifications of other nucleomonomers that comprise the oligomer.
  • An exemplary li ⁇ t of such modifications include oligomers where (i) one or more furanose-containing nucleomonomers i ⁇ modified at the 2' po ⁇ ition, (ii) one or more cro ⁇ slinking moieties have been incorporated, (iii) switchback linkers have been incorporated as described in copending U.S. application serial no.
  • Figure 1 shows a comparison of the riboacetal and amide linkages.
  • Figures 2a, 2b and 2c show a to-scale representation generated by computer of the spatial correspondence between riboacetal and amide linkages.
  • Figure 3 depicts the synthesis of a norborynl monomer that can be incorporated into oligomer ⁇ .
  • Figure 4 depicts the synthesis of an azepine monomer that can be incorporated into oligomer ⁇ ; (scheme 2) depicts an alternative synthetic pathway.
  • Figure 5 shows the synthesis of a hydroxymethyl monomer.
  • Figure 6 depicts the synthesi ⁇ of an oligomer u ⁇ ing hydroxymethyl monomers.
  • Figure 7 shows the synthe ⁇ i ⁇ of a pyrolidone monomer; (scheme 2) shows an alternative synthetic pathway.
  • Figure 8 show ⁇ an oligomer containing an amide linkage with a ci ⁇ -fused cyclopentyl monomer.
  • Figure 9 shows a spatial comparison of an amide linked oligomer with an amide linked oligomer composed of linked pyrollidone monomers or linkers.
  • Figure 10 shows a spatial comparison of an amide linked oligomer with an amide linked oligomer compo ⁇ ed of linked azepine monomers or linkers.
  • Figure 11 shows a spatial comparison of an amide linked oligomer with an amide linked oligomer composed of linked norbornyl monomers or linkers.
  • Figure 12 show ⁇ a ⁇ patial comparison of an amide linked oligomer with an amide linked oligomer compo ⁇ ed of linked hydroxymethyl monomer ⁇ .
  • Figure 13 shows a spatial comparison of an amide linked oligomer with an amide linked oligomer composed of linked cyclopentyl monomers.
  • Structural formulas described herein are designated a ⁇ roman numeral ⁇ (I, II, etc) and chemical compound ⁇ are designated by an underlined numeral (1 , 2_, etc) .
  • Nucleomonomer means a moiety comprising (1) a base covalently linked to (2) a ⁇ econd moiety.
  • Nucleomonomer ⁇ of the invention comprise a base linked to a "second moiety" that is an amino acid, amino acid analog or related compound having a free carboxyl and a free amine group or protected forms thereof.
  • Invention nucleomonomers are exemplified by compounds of formulas I - VIII as disclosed herein.
  • the invention nucleomonomers lack a sugar or furansoe moiety ⁇ uch a ⁇ ribose or deoxyribose.
  • Nucleomonomers also include nucleosides and nucleotides.
  • Nucleomonomers can be linked to form oligomer ⁇ that bind to target or complementary base sequence ⁇ in nucleic acids in a sequence specific manner.
  • a "second moiety" a ⁇ used herein al ⁇ o include ⁇ a sugar moiety, usually a pento ⁇ e, and those species which contain modifications of the sugar moiety, for example, wherein one or more of the hydroxyl group ⁇ are replaced with a halogen, a heteroatom, an aliphatic group ⁇ , or are functionalized a ⁇ ethers, amines, thiols, and the like.
  • the pentose moiety can be replaced by a hexo ⁇ e or an alternate ⁇ tructure such a ⁇ a cyclopentane ring, a 6-member morpholino ring and the like.
  • Base "Base" a ⁇ u ⁇ ed herein include ⁇ tho ⁇ e moietie ⁇ which contain not only the known purine and pyrimidine heterocycles, but also heterocycle analogs and tautomers thereof.
  • Purine ⁇ include adenine, guanine and xanthine and exemplary purine analogs include 8-oxo-N 6 - methyladenine, 7-methyl-7-deazaguanine, 7-methyl-7- deazaadenine and 7-deazaxanthine.
  • Pyrimidines include uracil and cytosine and their analogs such as 5- methylcytosine, 5-(l-propynyluracil) , 5-(l- propynylcyto ⁇ ine) , 5-methyluracil and 4,4-ethanocyto ⁇ ine.
  • nucleo ⁇ ide include ⁇ ribonucleosides, deoxyribonucleoside ⁇ , or any other nucleoside which is an N-glycoside or C-glycoside of a ba ⁇ e.
  • Nucleo ⁇ ides include tho ⁇ e ⁇ pecie ⁇ which contain modification ⁇ of the ⁇ ugar moiety, for example, wherein one or more of the hydroxyl group ⁇ are replaced with a halogen (F, Cl, Br,or I), a heteroatom (including nitrogen and ⁇ ulfur) , an aliphatic group (1-9C alkyl, 1- 9C alkenyl and the like) , or are functionalized as ethers, amines, thiols, and the like.
  • a halogen F, Cl, Br,or I
  • a heteroatom including nitrogen and ⁇ ulfur
  • an aliphatic group (1-9C alkyl, 1- 9C alkenyl and the like
  • the pentose moiety can be replaced by a hexose or an alternate structure such as a cyclopentane ring, a 6-member morpholino ring and the like.
  • the stereochemistry of the sugar carbons can be other than that of D-ribose in one or more re ⁇ idue ⁇ .
  • the pento ⁇ e moiety can be replaced by a hexose and incorporated into oligomers as de ⁇ cribed (Augustyns, K. , et al Nucl Acid ⁇ Res (1992) 18:4711-4716).
  • Nucleoside ⁇ as defined herein also includes a purine or pyrimidine ba ⁇ e linked to an amino acid or amino acid analog having a free carboxyl group and a free amino group or protected form ⁇ thereof.
  • nucleotide means a nucleoside having a phosphate group or phosphate analog such as a thioate, methylpho ⁇ phonate, pho ⁇ phoramidate and the like.
  • sugar modification means any pentose or hexose moiety other than 2'-deoxyribose.
  • Modified sugar ⁇ include D-ribose, 2'- and 3 , -0-alkyl, 2 ' - and 3'-amino, 2 ' - and 3'-halo functionalized pento ⁇ e ⁇ , hexo ⁇ es and the like.
  • Sugars having a stereochemistry other than that of a D-ribose are also included.
  • Linkage mean ⁇ a pho ⁇ phodiester moiety (-O-P(O) (O)-0-) that covalently couples adjacent nucleomonomers.
  • substitute linkage means any analog of the native pho ⁇ phodie ⁇ ter group or any suitable moiety that covalently couples adjacent nucleomonomers and generates binding competent oligomers. Substitute linkages include 21
  • Noninvention substitute linkages include phosphodiester analogs, e.g. such as phosphorothioate and methylpho ⁇ phonate, and nonpho ⁇ phoru ⁇ containing linkages, e.g. such as acetal ⁇ and ⁇ ulfides.
  • Switchback means an oligomer having at least one region of inverted polarity. Switchback oligomers are able to bind to opposite strands of a duplex to form a triplex on both strands of the duplex.
  • the linker (“switchback linker”) joining the regions of inverted polarity is a substitute linkage.
  • Crosslinking moietv “Cros ⁇ linking moiety” include ⁇ a group or moiety in an oligomer that form ⁇ a covalent bond with a target nucleic acid. Crosslinking moieties include covalent bonding specie ⁇ that covalently link an oligomer to target nucleic acids either spontaneously (e.g. N ,N 4 -ethanocytosine) or via photoactivation (e.g. p ⁇ oralen and the like) .
  • Oligomers are defined herein as two or more nucleomonomers covalently coupled to each other by a linkage or substitute linkage moiety. Thus, an oligomer can have as few as two convalently linked nucleomonomers (a dimer) . Oligomer ⁇ can be binding competent and, thu ⁇ , can ba ⁇ e pair with cognate ⁇ ingle- ⁇ tranded or double- ⁇ tranded nucleic acid ⁇ equence ⁇ . Short oligomers (e.g. dimers - hexamers) are al ⁇ o u ⁇ eful a ⁇ ⁇ ynthon ⁇ , in particular for certain noninvention oligomers a ⁇ de ⁇ cribed herein.
  • Oligomer ⁇ can al ⁇ o contain aba ⁇ ic sites and pseudonucleosides. Invention oligomers are exemplified by the structures shown in Figures 8 - 13. Oligomer as used herein is also intended to include compound ⁇ where adjacent nucleomonomer ⁇ are linked via amide linkage ⁇ a ⁇ previou ⁇ ly de ⁇ cribed (Nielsen, P.E., et al, Science (1991) 254:1497-1500; WO 92/20702) . Elements ordinarily found in oligomers, such as the furanose ring and/or the phosphodiester linkage can be replaced with any suitable functionally equivalent element.
  • Oligomer is thus intended to include any structure that ⁇ erves as a scaffold or support for the bases wherein the scaffold permit ⁇ binding to target nucleic acid ⁇ in a ⁇ equence-dependent manner.
  • Oligomer also includes oligonucleotides, oligonucleoside ⁇ , polydeoxyribo-nucleotide ⁇ (containing 2 , -deoxy-D-ribo ⁇ e or modified form ⁇ thereof), i.e., DNA, polyribonucleo- tides (containing D-ribo ⁇ e or modified forms thereof) , i.e., RNA, and any other type of polynucleotide which is an N-glycoside or C-glycoside of a base.
  • Oligomers that are currently known can be defined into four groups that can be characterized as having (i) phosphodiester and phosphodie ⁇ ter analog (pho ⁇ phorothioate, methylpho ⁇ phonate, etc) linkages, (ii) ⁇ ubstitute linkages that contain a non-phosphorous isostere (riboacetal, formacetal, hydrazino, etc) , (iii) morpholino residues, carbocyclic residues or other furanose sugars, such as arabinose, or a hexose in place of ribose or deoxyribose and (iv) acyclic nucleomonomers linked via amide linkage ⁇ or carbamate linkages or any other suitable sub ⁇ titute linkage.
  • blocking group refer ⁇ to a substituent other than H that is conventionally coupled to oligomers or nucleomonomers, either as a protecting group, a coupling group for synthesis, OP0 3 "2 , or other conventional conjugate such as a solid support, label, antibody, monoclonal antibody or fragment thereof and the like.
  • blocking group is not intended to be construed solely as a protecting group, according to slang terminology, but is meant also to include, for example, coupling group ⁇ such as a H-phosphonate or a phosphora idite.
  • protecting group i ⁇ any group capable of protecting the O-atom, S-atom or N-atom to which it i ⁇ attached from participating in a reaction or bonding.
  • Such protecting groups for N-atoms on a ba ⁇ e moiety in a nucleomonomer and their introduction are conventionally known in the art.
  • suitable protecting groups include diisobutylformamidine, benzoyl and the like.
  • Suitable "protecting groups” for O-atoms and S-atoms are, for example, DMT, MMT, FMOC or ester ⁇ . Protecting group.
  • Protecting group a ⁇ used herein includes any group capable of preventing the O- atom, S-atom or N-atom to which it i ⁇ attached from participating in a reaction or bonding. Such protecting group ⁇ for 0-, S- and N-atoms in nucleomonomer ⁇ are de ⁇ cribed and method ⁇ for their introduction are conventionally known in the art. Protecting groups also include any group capable of preventing reactions and bonding at carboxylic acids, thiols and the like.
  • Coupling group as used herein means any group suitable for generating a linkage or substitute linkage between nucleomonomers such as a hydrogen phosphonate, a pho ⁇ phoramidite and an alkyl ether.
  • Conjugate a ⁇ u ⁇ ed herein means any group attached to the oligomer at a terminal end or within the oligomer itself.
  • Conjugates include solid supports, such as ⁇ ilica gel, controlled pore glas ⁇ and polystyrene; labels, such a ⁇ fluore ⁇ cent, chemilumine ⁇ cent, radioactive atom ⁇ or molecule ⁇ , enzymatic moieties and reporter groups; oligomer tran ⁇ port agent ⁇ , such as polycations, serum proteins and glycoproteins and polymers and the like.
  • Synthon "Synthon" a ⁇ u ⁇ ed herein means a structural unit within a molecule that can be formed and/or as ⁇ embled by known or conceivable synthetic operations.
  • Transfection refers to any suitable method that for enhanced delivery of oligomer ⁇ into cells.
  • Subject as u ⁇ ed herein mean ⁇ a plant or an animal, including a mammal, particularly a human.
  • Sequence-specific binding is used in its commonly accepted sen ⁇ e to define the binding which occur ⁇ between, for example, an oligomer and a DNA or RNA target sequence via pairs of base ⁇ which form hydrogen bonds according to conventional rules.
  • Series when used to define a number of modified nucleosides shall mean 3 or more, and especially from 3 to 100, modified nucleo ⁇ ides appearing in sequence linked one to another.
  • phosphodiester analog is meant an analog of the conventional phosphodiester linkage
  • Invention oligomer ⁇ can include any ⁇ ubstitute linkage such as riboacetal and formacetal substitute linkages. In general, such linkages will be confined to a domain of an invention oligomer that does not contain any invention nucleomonomer ⁇ .
  • Suitable riboacetal and formacetal linkage ⁇ are disclosed in copending applications having Ser. No. 07/690,786; 07/763,130, and 07/806,710, all of which are incorporated herein by reference, and include formacetal linkages ⁇ uch a ⁇ : 3'-thioformacetal (-S-CH 2 -0-) , formacetal (-0-CH 2 -0-) ,
  • Suitable riboacetal linkage ⁇ include member ⁇ of the group:
  • each R 3 is independently H, or a suitable blocking group such as P0 3 "2 , a dimethoxytrityl (DMT) moiety, a monomethoxytrityl (MMT) moiety, H-phosphonate (OP0 2 H) , methylphosphonate (OP0 2 CH 3 ) or phosphoramidite;
  • R 4 is selected from the group consisting of H, OH, F, 1-9C 0- alkyl [including OCH 3 , OC 2 H 5 , OCH 2 CHCH 2 (O-allyl, OC J H J ) , OC 3 H 7 (O-propyl) , SCH 3 , SC 2 H 5 , SCH 2 CHCH 2 (S-allyl, SC ⁇ ) , SC ⁇ H j (S-propyl)], 1-12C alkyl, 1-12C alkenyl or 1-12C alkynyl.
  • DMT dimethoxytrityl
  • MMT monomethoxytrityl
  • Methylphosphoramidite and ⁇ - cyanoethylpho ⁇ phoramidite are preferred phosphoramidite groups.
  • Each B is independently a purine or pyrimidine base or an analogous form thereof;
  • each X 3 is independently selected from the group consisting of O, S, CH 2 , CF 2 and CFH;
  • each A is independently selected from the group consisting of O, S, SO, S0 2 , CH 2 , CO, CF 2 , CS, NH and NR 6 wherein R 6 is lower alkyl (including methyl, ethyl, propyl, isopropyl, butyl and isobutyl) with the proviso that adjacent A are not both O;
  • E is selected from the group consisting of 0, S, CH 2 , CO, CF 2 , CS, NH and NR 6 ;
  • J is selected from the group consisting of 0, S, CH 2 , CO, CF 2 and CS;
  • G is selected from the
  • Bases (B) that are preferred are adenine, thymine, guanine, cytosine, 8-oxo-N*-methyladenine, N*,N*-ethanocytosine, and 5-methylcytosine, 5-propynyluracil, 5- propynylcy osine, 7-deazaxanthine and 7-deazaguanine.
  • "Derivatives" of oligomers include those conventionally recognized in the art. For instance, oligomers may be covalently linked to various moieties such as intercalators, substances which interact specifically with the minor groove of the DNA double helix and other arbitrarily chosen conjugates, such as labels (radioactive, fluorescent, enzyme, etc.).
  • intercalators such as acridine may be linked through an -R , -CH 2 -R , ⁇ attached through any available -OH or -SH, e.g., at the terminal 5', 2 ' or 3' position of RNA or DNA, the 2', 5', or 3' positions of RNA, or an OH or SH engineered into the 5 position of pyrimidine ⁇ , e.g., in ⁇ tead of the 5 methyl of cytosine, a derivatized form which contains -CH 2 CH 2 CH 2 OH or -CH 2 CH 2 CH 2 SH in the 5 po ⁇ ition.
  • terminal oligomer moieties such as -NH 2 or -COOH can be blocked or derivatized using standard procedures of routine use.
  • any -OH moieties at the terminal 3' or 5' position in oligomers with a terminal furanose-containing nucleo ⁇ ide re ⁇ idue may be replaced by pho ⁇ phonate group ⁇ , protected by ⁇ tandard protecting group ⁇ , or activated to prepare additional linkages to other nucleomonomers, or may be bound to the conjugated sub ⁇ tituent.
  • hydroxyl ⁇ of furanose nucleoside residues may in general be derivatized to standard blocking or protecting group ⁇ a ⁇ de ⁇ cribed in the art.
  • ⁇ pecifically included are the 2'- or 3'-derivatized forms of the nucleomonomers di ⁇ clo ⁇ ed in pending U.S. application serial no. 07/425,857, as well as the formacetal/ketal type linkages disclosed in pending U.S. Patent Application Serial Nos. 07/559,957 and 07/690,786, all of which are incorporated herein by reference in their entirety. Synthesis of DNA oligomers and nucleomonomers with 2 ' modifications has been described for 2 ' fluoro compounds (Uesugi, S.
  • the ⁇ e derivative ⁇ are al ⁇ o specifically included and the chemi ⁇ try i ⁇ applicable to both 2 ' and 3' position.
  • Specific modifications that are contemplated for oligomers described in the present invention include moieties that permit duplex strand switching as de ⁇ cribed in commonly owned, pending PCT patent application No. PCT/US90/06128, moietie ⁇ such as N ⁇ N ⁇ -ethanocytosine (aziridinylcytosine) that affect covalent cros ⁇ linking- a ⁇ de ⁇ cribed in commonly owned, pending U.S. patent application Serial Nos.
  • a “constrained linker” is an organic chemical moiety (i.e.
  • nucleomonomer having two position ⁇ for covalently chemically binding to adjacent members in a modified oligomer of this invention while having internal covalent or ionic or hydrogen bonds or the like which lock or influence the structure of the linkage into one or a limited number (2 or 3) of distinct spatial conformations.
  • a 2,3- or 4,5-linked divalent seven- membered ring is an example of a "con ⁇ trained linker.” Numerou ⁇ other example ⁇ are di ⁇ closed herein. Not all invention sub ⁇ titute linkage ⁇ in the same oligomer need be identical, the only requirement being that at least one nucleomonomer is present.
  • the invention i ⁇ thu ⁇ directed to new oligomer ⁇ and nucleomonomer ⁇ which are u ⁇ eful in oligomer-ba ⁇ ed therapie ⁇ and intermediate ⁇ in their production, as well to methods to synthesize these compounds and their intermediates.
  • the invention compounds show enhanced stability with respect to nucleases by virtue of their substitute linkages, as well as enhanced ability to permeate cells.
  • nucleomonomer ⁇ corre ⁇ ponding to Formula ⁇ I - II and IV - VIII into oligomer ⁇ the carboxylate or carbamate terminus of the nucleomonomer is derivatized to an activated carboxylic acid or an active derivative thereof.
  • incorporation of nucleomonomer ⁇ corresponding to Formula III into oligomers utilizes a chloroformate derivatized compound ( 6) as ⁇ hown in Figure 3 or it ⁇ equivalent.
  • oligomers of the invention are capable of significant single-stranded or double-stranded target nucleic acid binding activity to form duplexes, triplexes or other forms of stable association
  • these oligomer ⁇ are useful in diagnosis and therapy of diseases that are associated with expression of one or more genes such a ⁇ tho ⁇ e associated with pathological conditions.
  • Therapeutic applications can employ the oligomers to specifically inhibit the expression of genes (or inhibit tran ⁇ lation of RNA ⁇ equences encoded by those genes) that are associated with either the establishment or the maintenance of a pathological condition.
  • RNAs encoded by tho ⁇ e genes that can be targeted include those that encode enzyme ⁇ , hormone ⁇ , serum protein ⁇ , tran ⁇ membrane protein ⁇ , adhesion molecules (LFA-1, GPII b /III a , ELAM-1, VACM-1, ICAM-1, E-selectin, and the like) , receptor molecules including cytokine receptors (IL-1 receptor, IL-2 receptor and the like) , cytokines (IL-1, IL-2, IL-3, IL-4, IL-6 and the like), oncogenes, growth factors, and interleukins.
  • Target gene ⁇ or RNAs can be as ⁇ ociated with any pathological condition ⁇ uch a ⁇ tho ⁇ e a ⁇ sociated with inflammatory conditions, cardiovascular disorder ⁇ , immune reaction ⁇ , cancer, viral infection ⁇ , bacterial infection ⁇ and the like.
  • Oligomers of the present invention are suitable for u ⁇ e in both in vivo and ex vivo therapeutic application ⁇ .
  • Indication ⁇ for ex vivo u ⁇ es include treatment of cells such as bone marrow or peripheral blood in conditions such as leukemia (chronic myelogenous leukemia, acute lymphocytic leukemia) or viral infection.
  • Target genes or RNAs encoded by those genes that can serve as targets for cancer treatments include oncogenes, such as ras, k-ras, bcl-2, c-myb, bcr, c-myc, c-abl or overexpre ⁇ ed ⁇ equences such as mdm2, oncostatin M, IL-6 (Kaposi's sarcoma), HER-2 and translocation ⁇ such as bcr/abl.
  • the oligmers may be used to inhibit proliferation of DNA or RNA viruse ⁇ ⁇ uch a ⁇ herpesviruses, papillomaviruses and the like.
  • Viral gene sequences or RNAs encoded by those gene ⁇ such as polymerase or reverse transcriptase gene ⁇ of herpe ⁇ viruses such as CMV, HSV-1, HSV-2, retroviruses such as HTLV-1, HIV-1, HIV-2, or other DNA or RNA viruses such as HBV, HPV, VZV, influenza virus, rhinoviru ⁇ and the like are also suitable targets.
  • Herpe ⁇ viruses such as CMV, HSV-1, HSV-2, retroviruses such as HTLV-1, HIV-1, HIV-2, or other DNA or RNA viruses such as HBV, HPV, VZV, influenza virus, rhinoviru ⁇ and the like are also suitable targets.
  • Application of ⁇ pecifically binding oligomers can be used in conjunction with other therapeutic treatment ⁇ .
  • Other therapeutic indication ⁇ for oligomers of the invention include (1) modulation of inflammatory responses by modulating expression of genes such as IL-1 receptor, IL-1, ICAM-1 or E-Selectin that play a role in mediating inflammation and (2) modulation of cellular proliferation in conditions ⁇ uch as arterial occlusion (resteno ⁇ i ⁇ ) after angiopla ⁇ ty by modulating the expre ⁇ sion of (a) growth or mitogenic factors such as non-muscle myosin, myc, fos, PCNA, PDGF or FGF or their receptors, or (b) cell proliferation factors ⁇ uch as c-myb.
  • genes such as IL-1 receptor, IL-1, ICAM-1 or E-Selectin that play a role in mediating inflammation
  • modulation of cellular proliferation in conditions ⁇ uch as arterial occlusion (resteno ⁇ i ⁇ ) after angiopla ⁇ ty by modulating the expre ⁇ sion of (a) growth or mitogenic factors such as non-muscle myosin, myc, fo
  • TGF ⁇ TGF ⁇
  • TGF ⁇ TGF ⁇
  • IL-6 IL-6
  • ⁇ INF protein kina ⁇ e C
  • tyro ⁇ ine kina ⁇ e ⁇ such as p210, pl90
  • EGF receptor, TGFo or MHC allele ⁇ may be targeted in autoimmune disease ⁇ .
  • oligomer ⁇ of the invention into cells can be enhanced by any suitable method including calcium phosphate, DMSO, glycerol or dextran transfection, electroporation or by the u ⁇ e of cationic anionic and/or neutral lipid compo ⁇ itions or liposomes by method ⁇ de ⁇ cribed (International Publication Nos. WO 90/14074, WO 91/16024, WO 91/17424, U.S. Patent 4,897,355).
  • the oligomers can be introduced into cells by complexation with cationic lipids such as DOTMA (which may or may not form liposome ⁇ ) which complex is then contacted with the cells.
  • DOTMA cationic lipids
  • Suitable cationic lipids include but are not limited to N-(2,3-di(9-(Z)- octadecenyloxyl) )-prop-l-yl-N,N,N-trimethylammonium (DOTMA) and its salts, l-0-oleyl-2-0-oleyl-3- dimethylaminopropyl- ⁇ -hydroxyethylammonium and its salts and l,2-bis(oleyloxy)-3-(trimethylammonio) propane and its salts.
  • DOTMA N-(2,3-di(9-(Z)- octadecenyloxyl) )-prop-l-yl-N,N,N-trimethylammonium
  • DOTMA N-(2,3-di(9-(Z)- octadecenyloxyl) )-prop-l-yl-N,N,N-trimethylammonium
  • DOTMA N-(2,
  • Enhanced delivery of the invention oligomers can also be mediated by the u ⁇ e of (i) viruses such as Sendai virus (Bartzatt, R. , Biotechnol Appl Biochem (1989) 11:133-135) or adenovirus (Wagner, E. , et al, Proc Natl Acad Sci (1992) £9.:6099-6013; (ii) polyamine or polycation conjugates using compound ⁇ such as polylysine, protamine or Nl, N12-bis(ethyl)spermine (Wagner, E. , et al, Proc Natl Acad Sci (1991) £8:4255-4259; Zenke, M.
  • viruses such as Sendai virus (Bartzatt, R. , Biotechnol Appl Biochem (1989) 11:133-135) or adenovirus (Wagner, E. , et al, Proc Natl Acad Sci (1992) £9.:6099-6013
  • transfection refers to any method that is suitable for delivery of oligomer ⁇ into cells.
  • reagent such as a lipid or any agent such as a virus that can be used in transfection protocol ⁇ is collectively referred to herein as a "permeation enhancing agent”.
  • Delivery of the oligomers into cells can be via cotran ⁇ fection with other nucleic acid ⁇ such as (i) expre ⁇ ible DNA fragment ⁇ encoding a protein(s) or a protein fragment or (ii) translatable RNA ⁇ that encode a protein( ⁇ ) or a protein fragment.
  • the oligomer ⁇ can thu ⁇ be incorporated into any ⁇ uitable formulation that enhances delivery of the oligomers into cells.
  • Suitable pharmaceutical formulation ⁇ al ⁇ o include tho ⁇ e commonly u ⁇ ed in application ⁇ where compound ⁇ are delivered into cell ⁇ or ti ⁇ ue ⁇ by topical admini ⁇ tration.
  • Compound ⁇ ⁇ uch a ⁇ polyethylene glyco. , propylene glycol, azone, nonoxonyl- 9, oleic acid, DMSO, polyamine ⁇ or lipopolyamine ⁇ can be u ⁇ ed in topical preparation ⁇ that contain the oligomers.
  • the invention oligomers can be conveniently used as reagents for research or production purpo ⁇ e ⁇ where inhibition of gene expre ⁇ ion is desired.
  • Oligomer ⁇ that have been previou ⁇ ly reported to inhibit target gene expression frequently have nonspecific effects and/or do not reduce target gene expre ⁇ ion to very low levels (less than about 40% of uninhibited levels) .
  • the oligomers as described herein constitute a reagent that can be used in method ⁇ of inhibiting expre ⁇ ion of a ⁇ elected protein or protein ⁇ in a subject or in cells wherein the protein ⁇ are encoded by DNA ⁇ equence ⁇ and the proteins are translated from RNA sequences, comprising the step ⁇ cf: introducing an oligomer of the invention into the cell ⁇ ; and permitting the oligomer to form a triplex with the DNA or RNA or a duplex with the DNA or RNA whereby expre ⁇ ion of the protein or proteins is inhibited.
  • the methods and oligomers of the present invention are suitable for modulating gene expression in both procaryotic and eucaryotic cells such as bacterial, fungal parasite, yeast and mammalian cells.
  • Oligomers containing as few as about 8 modifie nucleoside ⁇ can be u ⁇ ed to effect inhibition of target protein( ⁇ ) expression by formation of duplex or triplex structure ⁇ with target nucleic acid sequences.
  • linear oligomers u ⁇ ed to inhibit target protein expression via duplex or triplex formation will preferably have from about 10 to about 20 modified nucleoside re ⁇ idues.
  • Oligomers containing modified nucleoside ⁇ of the invention can be conveniently circularized as described (International Publication No. WO 92/19732; Kool, E.T. J Am Chem Soc (1991) 111:6265-6266; Prakash, G., et al. J Am Chem Soc (1992) 111:3523-3527).
  • Such oligomer ⁇ are ⁇ uitable for binding to single-stranded or double-stranded nucleic acid target ⁇ .
  • Circular oligomer can be of variou ⁇ sizes.
  • Such oligomers in a ⁇ ize range of about 22-50 nucleomonomer ⁇ can be conveniently prepared.
  • the circular oligomer ⁇ can have from about three to about six nucleomonomer residues in the loop region that separate binding domains of the oligomer a ⁇ de ⁇ cribed (Praka ⁇ h, G. ibid) .
  • Oligomer ⁇ can be enzymatically circularized through a terminal pho ⁇ phate by ligase or by chemical means via linkage through the 5'- and 3'- terminal sugars and/or bases.
  • the oligomers can be utilized to modulate target gene expre ⁇ ion by inhibiting the interaction of nucleic acid binding protein ⁇ re ⁇ ponsible for modulating transcription (Maher, L. J. , et al. Science (1989) 245:725-730) or translation.
  • oligomers are thus suitable as sequence-specific agents that compete with nucleic acid binding proteins (including ribosomes, RNA poly erases, DNA poly erases, translational initiation factors, transcription factors that either increase or decrease transcription, protein-hormone transcription factors and the like) .
  • Appropriately designed oligomers can thus be used to increase target protein synthesis through mechanisms such as binding to or near a regulatory site that transcription factors use to repres ⁇ expression or by inhibiting the expression of a selected repressor protein itself.
  • the invention oligomers comprising additional modifications that enhance binding affinity can be designed to contain secondary or tertiary structures, such as pseudoknots or pseudo-half-knots (Ecker, D.J., et al, Science (1992) 257:958-961.. Such structures can have a more stable secondary or tertiary structure than corresponding unmodified oligomers. The enhanced stability of such structures would rely on the increased binding affinity between regions of self complementarity in a single oligomer or regions of complementarity between two or more oligomers that form a given structure. Such structures can be used to mimic structures such as the HIV TAR structure in order to interfere with binding by the HIV Tat protein (a protein that binds to TAR) .
  • HIV Tat protein a protein that binds to TAR
  • oligomers can be used to (1) disrupt or (2) bind to such structures as a method to (1) interfere with or (2) enhance the binding of proteins to nucleic acid structures.
  • the oligomers of the invention can also be applied as therapeutic or diagnostic agents that
  • SUBSTITUTE SHEET function by direct displacement of one strand in a duplex nucleic acid. Displacement of a strand in a natural duplex such as chromosomal DNA or duplex viral DNA, RNA or hybrid DNA/RNA is possible for oligomers with a high binding affinity for their complementary target sequences. Therapeutic applications of oligomer ⁇ by thi ⁇ method of u ⁇ e, referred to herein a ⁇ D-looping or "D-loop therapy" ha ⁇ not previou ⁇ ly been possible because the affinity of natural DNA or RNA for its complementary sequence is not great enough to efficiently displace a " DNA or RNA strand in a duplex.
  • Target nucleic acids include but are not limited to (i) gene sequences including exons, intron ⁇ , exon/intron junction ⁇ , promoter/enhancer region ⁇ and 5' or 3' untran ⁇ lated region ⁇ , (ii) regions of nucleic acids that utilize secondary structure in order to function (e.g.
  • oligomers can be ⁇ ynthe ⁇ ized with discrete functional domains wherein one region of an oligomer binds to a target by D-looping while an adjacent region binds a target molecule by say, forming a triple helix or binding as an aptamer to a protein.
  • a D-looping oligomer can bind to each ⁇ trand in a duplex by switching the strand to which the oligomer binds (i.e. by having one region of the oligomer that binds to one strand and another region that binds to the complementary strand) .
  • the controlling elements that dictate the mode of binding i.e. triple helix or D-loop
  • the controlling elements that dictate the mode of binding are the sequence of the oligomer and the inherent affinity built into the oligomer.
  • Base recognition rules in Watson-Crick duplex binding differ from those in Hoogsteen controlled triplex binding. Because of this, the oligomer base ⁇ equence can be u ⁇ ed to dictate the type of binding rule ⁇ an oligomer will utilize.
  • D-loop structures are formed in nature by enzyme-mediated processes (Harris, L.D. et al., J Biol Chem (1987) 262: 9285-9292) or are associated with regions where DNA replication occurs (Jacobs, H.T. et al., Nucl Acids Res (1989) 17:8949-8966). D-loops that arise from the binding of oligomers can result from a one or two step process. Direct displacement of a target strand will give rise to a D-loop by a single binding event. However, D-looping can also occur by forming a triple helix which facilitates a strand displacement event leading to the D-loop.
  • Ribozymes containing modified nucleosides of the invention can be designed in order to design species with altered characteristics. Ribozymes that cleave single stranded RNA or DNA (Robertson, D.L., et al Nature (1990) 344:467-468) have been described. Therapeutic applications for ribozymes have been postulated (Sarver, N. et al, Science (1990) 2__7:1222-1225; International Publication Number WO 91/04319) . Secondary or tertiary structure necessary for ribozyme function can be affected by design of appropriate oligomer sequences.
  • ribozymes having nuclease stable targeting domains containing modified nucleoside ⁇ of the invention can have higher affinity, while maintaining ba ⁇ e pairing ⁇ pecificity, for target ⁇ equences. Because of the higher affinity and/or nuclease stability of the invention modified nucleoside ⁇ , shorter recognition domains in a ribozyme (an advantage in manufacturing) can be designed which can lead to more favorable substrate turnover (an advantage in ribozyme function) .
  • the oligomers are utilized in a manner appropriate for treatment of a variety of conditions by inhibiting expres ⁇ ion of appropriate target genes.
  • the oligomers can be formulated for a variety of ode ⁇ of administration, including sy ⁇ temic, topical or localized admini ⁇ tration. Technique ⁇ and formulation ⁇ generally can be found in Remington's Pharmaceutical Sciences. Mack Publishing Co., Easton, PA, late ⁇ t edition.
  • the oligomer active ingredient is generally combined with a carrier such as a diluent or excipient which can include fillers, extenders, binders, wetting agents, disintegrants, surface-active agents, or lubricants, depending on the nature of the mode of administration and dosage forms.
  • Typical dosage form ⁇ include tablet ⁇ , powder ⁇ , liquid preparation ⁇ including ⁇ u ⁇ pensions, emulsions and solutions, granules, capsule ⁇ and ⁇ uppo ⁇ itorie ⁇ , a ⁇ well as liquid preparations for injections, including liposome preparations.
  • the oligomer ⁇ of the invention are formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank' ⁇ solution or Ringer's solution.
  • the oligomer ⁇ can be formulated in ⁇ olid form and redi ⁇ olved or suspended immediately prior to use. Lyophilized forms are also included. Dosages that can be used for systemic administration preferably range from about 0.01 mg/Kg to 50 mg/Kg administered once or twice per day.
  • sing schedules can be utilized depending on (i) the potency of an individual oligomer at inhibiting the activity of its target DNA or RNA, (ii) the severity or extent of a pathological disease state a ⁇ ociated with a given target gene, or (iii) the pharmacokinetic behavior of a given oligomer.
  • Sy ⁇ temic administration can also be by tran ⁇ muco ⁇ al or tran ⁇ dermal means, or the compounds can be administered orally.
  • penetrants appropriate to the barrier to be permeated are u ⁇ ed in the formulation.
  • penetrants are generally known in the art, and include, for example, bile salts and fusidic acid derivatives for transmuco ⁇ al administration.
  • detergents can be used to facilitate permeation.
  • Transmuco ⁇ al admini ⁇ tration can be through u ⁇ e of na ⁇ al sprays, for example, or suppo ⁇ itorie ⁇ .
  • the oligomer ⁇ are formulated into conventional oral admini ⁇ tration form ⁇ ⁇ uch as capsules, tablets, and tonics.
  • the oligomers of the invention are formulated into ointments, salve ⁇ , gels, or creams, as is generally known in the art.
  • Formulation of the invention oligomers for ocular indications such as viral infections would be based on standard compo ⁇ ition ⁇ known in the art.
  • the oligomer ⁇ of the invention can be used as diagnostic reagents to detect the pre ⁇ ence or ab ⁇ ence of the target nucleic acid ⁇ equence ⁇ to which they ⁇ pecifically bind.
  • the enhanced binding affinity of the invention oligomers is an advantage for their use as primers and probes.
  • Diagnostic test ⁇ cab be conducted by hybridization throug v either double or triple helix formation which i ⁇ then defected by conventional means.
  • the oligomers can be labeled using radioactive, fluorescent, or chromogenic labels and the pre ⁇ ence of label bound to ⁇ olid ⁇ upport detected.
  • the pre ⁇ ence of a double or triple helix can be detected by antibodie ⁇ which specifically recognize these forms.
  • Means for conducting assays using such oligomers as probes are generally known.
  • oligomers containing the invention modified nucleo ⁇ ides as diagnostic agents by triple helix formation is advantageous since triple helices form under mild conditions and the assay ⁇ can thu ⁇ be carried out without ⁇ ubjecting test specimens to harsh conditions. Diagnostic assays based on detection of RNA for identification of bacteria, fungi or protozoa sequences often require isolation of RNA from samples or organisms grown in the laboratory, which is laborious and time consuming, as RNA is extremely sensitive to ubiquitous nucleases.
  • the oligomer probe ⁇ can also incorporate additional modifications such as modified sugars and/or substitute linkages that render the oligomer especially nuclease ⁇ table, and would thu ⁇ be u ⁇ eful for assays conducted in the presence of cell or tissue extracts which normally contain nuclease activity. Oligomers containing terminal modifications often retain their capacity to bind to complementary sequences without loss of specificity (Uhlmann et al., Chemical Reviews (1990) 9 :543-584) .
  • the invention probes can also contain linkers that permit specific binding to alternate DNA strand ⁇ by incorporating a linker that permit ⁇ such binding (Froehler, B.C., et al, Biochemistry (1992) 11:1603-1609); Home et al., J Am Chem Soc (1990) 112:2435-2437) .
  • Incorporation of base analogs of the present invention into probe ⁇ that al ⁇ o contain covalent crosslinking agents ha ⁇ the potential to increa ⁇ e ⁇ ensitivity and reduce background in diagnostic or detection as ⁇ ays.
  • cros ⁇ linking agent ⁇ will permit novel a ⁇ say modifications such as (1) the use of the crosslink to increase probe discrimination, (2) incorporation of a denaturing wa ⁇ h step to reduce background and (3) carrying out hybridization and cros ⁇ linking at or near the melting temperature of the hybrid to reduce secondary structure in the target and to increase probe specificity. Modifications of hybridization condition ⁇ have been previou ⁇ ly described (Gamper et al.. Nucleic Acids Re ⁇ (1986) H:9943) . Oligomers of the invention are suitable for use in diagno ⁇ tic assays that employ methods wherein either the oligomer or nucleic acid to be detected are covalently attached to a solid support as de ⁇ cribed (U.S.
  • oligomer ⁇ are al ⁇ o suitable for use in diagno ⁇ tic a ⁇ says that rely on polymerase chain reaction techniques to amplify target sequences according to described methods (European Patent Publication No. 0 393 744) .
  • Oligomers of the invention containing a 3' terminus that can serve as a primer are compatible with polymerases used in polymerase chain reaction methods such as the Taq or VentTM (New England Biolabs) polymerase. Oligomers of the invention can thus be utilized as primers in PCR protocols.
  • the oligomers are useful as primers that are discrete sequences or as primers with a random sequence.
  • Random ⁇ equence primer ⁇ can be generally about 6, 7, or 8 nucleomonomer ⁇ in length.
  • Such primers can be used in variou ⁇ nucleic acid amplification protocol ⁇ (PCR, liga ⁇ e chain reaction, etc) or in cloning protocol ⁇ .
  • the substitute linkages of the invention generally do not interfere with the capacity of the oligomer to function a ⁇ a primer.
  • Oligomer ⁇ of the invention having 2'- modification ⁇ at ⁇ ite ⁇ other than the 3' terminal residue, other modifications that render the oligomer RNase H incompetent or otherwise nuclease stable can be advantageou ⁇ ly u ⁇ ed a ⁇ probe ⁇ or primers for RNA or DNA sequences in cellular extracts or other solutions that contain nucleases.
  • the oligomers can be used in protocols for amplifying nucleic acid in a sample by mixing the oligomer with a sample containing target nucleic acid, followed by hybridization of the oligomer with the target nucleic acid and amplifying the target nucleic acid by PCR, LCR or other suitable methods.
  • oligomers derivatized to chelating agents such as EDTA, DTPA or analogs of 1,2-diaminocyclohexane acetic acid can be utilized in various in vitro diagnostic a ⁇ says as described (U.S. Patent Nos. 4,772,548, 4,707,440 and 4,707,352).
  • oligomers of the invention can be derivatized with cros ⁇ linking agent ⁇ ⁇ uch as 5-(3-iodoacetamidoprop-l-yl)- 2'-deoxyuridine or 5-(3-(4-bromobutyramido)prop-l-yl)-2'- deoxyuridine and used in various as ⁇ ay methods or kits a ⁇ de ⁇ cribed (International Publication No. WO 90/14353) .
  • the ability of the oligomers to inhibit gene expression can be verified in in vitro sy ⁇ tems by measuring the levels of expres ⁇ ion in ⁇ ubject cells or in recombinant sy ⁇ tems, by any suitable method (Grae ⁇ smann, M. , et al.. Nucleic Acids Res (1991) 19:53-59). All references cited herein are incorporated herein by reference in their entirety.
  • the azido compound 2) (7.0 g, 17.8 mmole) was dis ⁇ olved in 40 mL of 1,4-dioxane. Triphenylphosphine (4.67 g, 17.8 mmole) was then added and the mixture was stirred for 3 hours. 10 L of water was then added to the solution dropwise. After stirring for 30 minutes at 50°C, the reaction mixture wa ⁇ evaporated to dryne ⁇ s. The residue was partitioned between CH 2 C1 2 and water. 4.7 g of compound 2_1 (72%) was obtained after purification on a flash column using methanol (0- 7%)/CH 2 Cl 2 .
  • Thyminyl acetic acid (Nielsen et al., 1991 Science 254:1497-1500; Egholm et al., 1992 J. Am. Chem. Soc. 111:1895-1897) (2.58 g, 13.2 mmole) and hydroxybenzotriazole (HOBt) (1.80 g, 13.2 mmole) were coevaporated two times with 20 mL of dry DMF and then dis ⁇ olved in 5 mL of dry DMF. Dicyclohexyl carbodiimide was added to this solution and the mixture wa ⁇ stirred for 30 minutes at 20°C. The solution of compound 212.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Saccharide Compounds (AREA)

Abstract

Nouveaux nucléosides et oligomères modifiés, leur production et leur utilisation dans des thérapies à base d'oligonucléotides. L'invention se rapporte plus spécifiquement à de nouveaux oligonucléotides qui présentent des liaisons modifiées entre nucléosides résistantes aux nucléases, qui présentent une plus grande capacité de pénétration dans les cellules, et qui peuvent lier des séquences d'oligonucléotides cibles in vitro et in vivo. Les oligonucléotides modifiés décrits sont particulièrement utiles dans des thérapies à base d'oligonucléotides, utilisant les oligonucléotides modifiés pour interrompre la synthèse ou la transcription protéique ou pour inactiver d'une autre manière l'ARN messager ou l'ADN à double brin.
PCT/US1993/005110 1992-05-28 1993-05-28 Oligomeres a conformation restreinte contenant des liaisons amide ou carbamate pour effectuer une liaison specifique d'une sequence WO1993024507A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US88973692A 1992-05-28 1992-05-28
US07/889,736 1992-05-28
US89439792A 1992-06-05 1992-06-05
US07/894,397 1992-06-05

Publications (1)

Publication Number Publication Date
WO1993024507A1 true WO1993024507A1 (fr) 1993-12-09

Family

ID=27128923

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1993/005110 WO1993024507A1 (fr) 1992-05-28 1993-05-28 Oligomeres a conformation restreinte contenant des liaisons amide ou carbamate pour effectuer une liaison specifique d'une sequence

Country Status (2)

Country Link
AU (1) AU4525093A (fr)
WO (1) WO1993024507A1 (fr)

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993012135A1 (fr) 1991-12-12 1993-06-24 Gilead Sciences, Inc. Oligomeres stables a la nuclease et aptes aux liaisons et methodes d'utilisation
WO1996020212A3 (fr) * 1994-12-28 1996-09-26 Buchardt Dorte & Lf Acide nucleique peptidique a squelette chiral
US5734040A (en) * 1996-03-21 1998-03-31 University Of Iowa Research Foundation Positively charged oligonucleotides as regulators of gene expression
US5859259A (en) * 1994-04-29 1999-01-12 Perseptive Biosystems, Inc. Activated esters of 1-phenylpyrazolin-5-one for labeling amine-functionalized molecules
US5977296A (en) * 1991-05-24 1999-11-02 Nielsen; Peter Chiral peptide nucleic acid monomers and oligomers
US6007992A (en) * 1997-11-10 1999-12-28 Gilead Sciences, Inc. Pyrimidine derivatives for labeled binding partners
US6028183A (en) * 1997-11-07 2000-02-22 Gilead Sciences, Inc. Pyrimidine derivatives and oligonucleotides containing same
US6274313B1 (en) 1996-03-21 2001-08-14 Pioneer-Hybrid International, Inc. Oligonucleotides with cationic phosphoramidate internucleoside linkages and methods of use
US6331617B1 (en) 1996-03-21 2001-12-18 University Of Iowa Research Foundation Positively charged oligonucleotides as regulators of gene expression
US6670393B2 (en) 1995-06-07 2003-12-30 Promega Biosciences, Inc. Carbamate-based cationic lipids
US8071737B2 (en) 1995-05-04 2011-12-06 Glead Sciences, Inc. Nucleic acid ligand complexes

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHEMICAL REVIEWS, Volume 90, No. 4, issued June 1990, UHLMANN et al., "Antisense Oligonucleotides: A New Therapeutic Principle", pages 543-584. *
SCIENCE, Volume 254, Number 5037, issued 06 December 1991, NIELSEN et al., "Sequence-Selective Recognition of DNA by Strand Displacement with a Thymine Substituted Polyamide", pages 1497-1500. *

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6201103B1 (en) 1991-05-24 2001-03-13 Peter E. Nielsen Peptide nucleic acid incorporating a chiral backbone
US5977296A (en) * 1991-05-24 1999-11-02 Nielsen; Peter Chiral peptide nucleic acid monomers and oligomers
WO1993012135A1 (fr) 1991-12-12 1993-06-24 Gilead Sciences, Inc. Oligomeres stables a la nuclease et aptes aux liaisons et methodes d'utilisation
US5859259A (en) * 1994-04-29 1999-01-12 Perseptive Biosystems, Inc. Activated esters of 1-phenylpyrazolin-5-one for labeling amine-functionalized molecules
WO1996020212A3 (fr) * 1994-12-28 1996-09-26 Buchardt Dorte & Lf Acide nucleique peptidique a squelette chiral
US8071737B2 (en) 1995-05-04 2011-12-06 Glead Sciences, Inc. Nucleic acid ligand complexes
US6670393B2 (en) 1995-06-07 2003-12-30 Promega Biosciences, Inc. Carbamate-based cationic lipids
US6274313B1 (en) 1996-03-21 2001-08-14 Pioneer-Hybrid International, Inc. Oligonucleotides with cationic phosphoramidate internucleoside linkages and methods of use
US6331617B1 (en) 1996-03-21 2001-12-18 University Of Iowa Research Foundation Positively charged oligonucleotides as regulators of gene expression
US5734040A (en) * 1996-03-21 1998-03-31 University Of Iowa Research Foundation Positively charged oligonucleotides as regulators of gene expression
US6028183A (en) * 1997-11-07 2000-02-22 Gilead Sciences, Inc. Pyrimidine derivatives and oligonucleotides containing same
US6414127B1 (en) 1997-11-07 2002-07-02 Isis Pharmaceuticals, Inc. Pyrimidine derivatives for labeled binding partners
US6800743B2 (en) 1997-11-07 2004-10-05 Isis Pharmaceuticals, Inc. Pyrimidine derivatives for labeled binding partners
US6951931B2 (en) 1997-11-07 2005-10-04 Isis Pharmaceuticals, Inc. Pyrimidine derivatives for labeled binding partners
USRE39324E1 (en) * 1997-11-07 2006-10-03 Isis Pharmaceuticals, Inc. Pyrimidine derivatives and oligonucleotides containing same
US7511125B2 (en) 1997-11-07 2009-03-31 Carlsbad Pyrimidine derivatives for labeled binding partners
US6007992A (en) * 1997-11-10 1999-12-28 Gilead Sciences, Inc. Pyrimidine derivatives for labeled binding partners

Also Published As

Publication number Publication date
AU4525093A (en) 1993-12-30

Similar Documents

Publication Publication Date Title
EP0643720B1 (fr) Oligomeres aptes a la fixation contenant des liaisons 2', 5'
US5792608A (en) Nuclease stable and binding competent oligomers and methods for their use
AU662298B2 (en) Modified internucleoside linkages
US5434257A (en) Binding compentent oligomers containing unsaturated 3',5' and 2',5' linkages
JP3484197B2 (ja) アミン誘導化ヌクレオシドおよびオリゴヌクレオシド
US5955599A (en) Process for making oligonucleotides containing o- and s- methylphosphotriester internucleoside linkages
US6140482A (en) Primary phosphoramidate internucleoside linkages and oligonucleotides containing the same
US6066720A (en) Modified oligonucleotides, their preparation and their use
JPH07501527A (ja) 修飾されたピリミジンを含有するオリゴマーを使用する増強された三重らせんおよび二重らせんの成形
WO1994024144A2 (fr) Formation a helice triple et double a l'aide d'oligomeres contenant des purines modifiees
WO1992010590A1 (fr) Inhibition de transcription par formation de triple helice
JPH06511492A (ja) キラルリン結合を有するオリゴヌクレオチド
US5646261A (en) 3'-derivatized oligonucleotide analogs with non-nucleotidic groupings, their preparation and use
WO1993024507A1 (fr) Oligomeres a conformation restreinte contenant des liaisons amide ou carbamate pour effectuer une liaison specifique d'une sequence
US5834607A (en) Amines and methods of making and using the same
US5969135A (en) Oligonucleotide analogs with an amino acid or a modified amino alcohol residue
WO1994006811A1 (fr) Analogues d'oligonucleotides a modification siloxy
AU693622B2 (en) Amino acid nucleic acids
WO1993012135A1 (fr) Oligomeres stables a la nuclease et aptes aux liaisons et methodes d'utilisation
EP0931090B1 (fr) Vecteurs oligonucleotidiques chimeres ameliores
WO1998027425A1 (fr) Purification a grande echelle d'oligonucleotides de longueur totale par extraction par affinite solide-liquide
CN119351403A (zh) 一种反义寡核苷酸-正义寡核苷酸偶联分子及其应用
WO1994015620A1 (fr) Nouveaux oligonucleotides modifies par des groupes de pontage non nucleotidiques
Li Novel oligonucleotide mimics for antisense therapeutics: Design, syntheses, biophysical and sequence-selective DNA cleavage studies of hydroxamate linked oligomers
WO2008101515A1 (fr) Nouveaux synthons utilisés dans la synthèse d'acides nucléiques peptidiques chiraux et leurs procédés de préparation

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP KR US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载