WO1989005853A1 - Conjugue de chelate d'acide nucleique utilise comme agent therapeutique et de diagnostic - Google Patents

Conjugue de chelate d'acide nucleique utilise comme agent therapeutique et de diagnostic Download PDF

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Publication number
WO1989005853A1
WO1989005853A1 PCT/US1988/004430 US8804430W WO8905853A1 WO 1989005853 A1 WO1989005853 A1 WO 1989005853A1 US 8804430 W US8804430 W US 8804430W WO 8905853 A1 WO8905853 A1 WO 8905853A1
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sequence
chelating agent
composition
hybridizing
usually
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PCT/US1988/004430
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English (en)
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Richard H. Tullis
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Synthetic Genetics
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/547—Chelates, e.g. Gd-DOTA or Zinc-amino acid chelates; Chelate-forming compounds, e.g. DOTA or ethylenediamine being covalently linked or complexed to the pharmacologically- or therapeutically-active agent

Definitions

  • the field concerns the use of modified oligonucleotides as therapeutic agents, inhibiting maturation or expression of transcription products in vivo and in vitro.
  • one can introduce a transcription cassette comprising a promoter functional in the host and a DNA sequence which results in the production of a mRNA which is complementary to an endogenous mRNA. Again, one observes a reduction in the expression product to which the transcription construct is directed.
  • anti-sense sequences are to become a useful therapeutic agent, there are many problems and difficulties to be overcome.
  • a method must be found which allows for the transfer of the anti-sense sequence across the cellular membrane.
  • the anti-sense sequence must be designed so as to be relatively stable to degradation, particularly by nucleases.
  • there remain concerns about the specificity of the sequence particularly where one does not wish to kill the host cell.
  • Anti-sense nucleic acid sequences have been reported to selectively block translation of a number of mRNAs (Izant and Weintraub, Cell (1984) 36;1007- 1015; Izant and Weintraub, Science (1985) 229;345-352; Melton, Proc. Natl. Acad Sci. USA (1985) 82 ;144-148; Mizuno et al., Proc. Natl. Acad. Sci. USA (1984) 81 ;1966-1970). Short oligonucleotides have been reported to provide for high selectivity. (Wallace et al., Nucl. Acids Res. (1979) 6:3543-3557; Wallace et al., Nucl.
  • Oligonucleotide conjugates where a specific sequence of at least eight nucleotides is covalently linked to an ion chelating group and optionally to other groups to enhance transport across the cell membrane.
  • the resulting compositions are found to effectively block the function of a sequence complementary to the oligonucleotide.
  • the compositions find use as agents in vivo and in vitro for modulating intracellular transcription product maturation or expression.
  • oligonucleotide conjugates have at least two components: an oligonucleotide sequence of at least eight nucleotides; and a chelating agent.
  • other groups may be present which include a linker joining the chelating agent to the oligonucleotide sequence, a hydrophobia group for enhancing the transport across the membrane, or other moieties to enhance binding affinity, reduce toxicity, enhance solubility, or other characteristics of interest.
  • the subject compositions will generally have a hybridizing sequence, namely a polynucleotide unit from about 8 to 30, more usually from about 8 to 20, preferably from about 12 to 18 members.
  • the molecular weight will normally be under about 10 kD, usually under about 6 kD, although high molecular weight molecules may be used in special circumstances.
  • the chelating agent may be any one of a large number of chelating agents which are able to chelate metal ions capable of acting as scissile and/or free radical initiating agents, by themselves or in conjunction with other compounds which may be present in the host cell or introduced in the host cell.
  • the chelating agent will be able to chelate one of a variety of metals, such as iron, cobalt, nickel, molybdenum, vanadium, or other metal ion which may be encountered in the cytoplasm of the cell and may serve to initiate the formation of free radicals, resulting in the scission or other modification of the transcription product, preventing its normal function in the host cell.
  • metals such as iron, cobalt, nickel, molybdenum, vanadium, or other metal ion which may be encountered in the cytoplasm of the cell and may serve to initiate the formation of free radicals, resulting in the scission or other modification of the transcription product, preventing its normal function in the host cell.
  • compositions of the subject invention will have the following formula:
  • K represents the chelating agent capable of chelating a metal ion, which ion is capable of catalyzing a chemical reaction in the physiological medium of the cytoplasm of a cell, which results in a chemical transformation of mRNA inhibiting expression, particularly degradative modification;
  • L is a bond or linking unit derived from a polyvalent functional group having at least one atom, which functional group may be of from about 1 to 20 atoms other than hydrogen, comprising carbon, nitrogen, oxygen, sulfur, and phosphorous, where the linking group may be aliphatic, aromatic, alicyclic, heterocyclic, or combinations thereof; substituted or unsubstituted; generally having from 0 to 10 heteroatoms, usually from 0 to 6 heteroatoms, where the cyclic compounds will usually have from 1 to 2 rings, usually 1 ring, and the aliphatic groups may be branched, straight chained, heterosubstituted or unsubstituted; desirably the linking unit will have a chain of 2 to 20, usually 4 to 16 atoms normally free of linkages capable of enzymatic degradation; L may be joined through Y to the terminal phosphorous or may be joined at any convenient site of the oligonucleotide chain, being linked to P, C, N, O or S of the base (N), sac
  • Z is a monosaccharide, particularly of 5 to 6 carbon atoms, more particularly of 5 carbon atoms, which may have from 0 to 1 hydroxyl groups replaced by hydrogen, and will usually be substituted by phosphorous at the 2, 3, 5, or 6 positions, particularly at the 3 and 5 positions, and substituted at the one position, by the purine or pyrimidine, where the sugars may include such sugars as ribose, arabinose, xylylose glucose, galactose, deoxy, particularly 2-deoxy, derivatives thereof, etc.;
  • L' is a linker group which is derived from a polyvalent functional group having at least one atom, and not more than about 60 atoms other than hydrogen, usually not more than about 30 atoms other than hydrogen, having up to about 30 carbon atoms, usually not more than about 20 carbon atoms, and up to about 10 heteroatoms, more usually up to about 6 heteroatoms, particularly chalcogen, nitrogen, phosphorous, etc.;
  • M is a moiety, particularly imparting amphiphilic properties to the compound, a hydrophobic or amphiphilic moiety which will have a ratio of carbon to heteroatom of at least about 2:1, usually at least about 3:1, frequently up to greater than about 20:1, may include hydrocarbons of at least 6 carbon atoms and not more than about 30 carbon atoms, polyoxy compounds (alkyleneoxy), where the oxygen atoms are joined by from about 2 to 10 carbon atoms, usually 2 to 6 carbon atoms, preferably 2 to 3 carbon atoms, and there will be at least about 6 units and usually
  • N is any natural or unnatural base (purine or pyrimidine), capable of binding to and hybridizing with a natural purine or pyrimidine, where N may be adenine, cytidine, thymidine, guanidine, uracil, orotidine, inosine, etc.; a is at least 4, usually at least 5, and not more than about 50, usually not more than about 35; b and c are each 0 or 1.
  • the functional groups which find use with the linking groups, L and L' include functionalities such as oxy, non-oxo-carbonyl (carboxy carbonyl), oxo-carbonyl (a-ldehyde or ketone), the nitrogen or sulfur analogs thereof, e.g. imino, thiono, thio, amidino, etc., disulfide, amino, diazo, hydrazino, oximino, phosphate, phosphono, etc.
  • the linking group to the hybridizing sequence may be linked through an oxygen or sulfur present on a pyrimidine, purine, sugar or phosphorous group, to a carbon atom of a pyrimidine or purine, or to a phosphorous atom.
  • the links may be ethers to oxygen and sulfur, esters, both organic and inorganic, to oxygen and sulfur, amides, both organic and inorganic, to amines and phosphorous, and alkylamino to amino groups.
  • Esters include carboxylates, e.g. carboxy esters, carbamates, carbonates, etc., and phosphates, phosphonates, etc.
  • Of particular interest is linking at the terminal unit of the hybridizing sequence through a sugar hydroxyl, particularly at the 5' -position.
  • the phosphorous moiety may include phosphates, phosphoramidates, phosphordiamidate, phosphorothioate, phosphorothionate, phosphorothiolate, phosphoramidothiolate, phosphonates, phosphorimidate, and the like.
  • the sugar will be modified by having an amino or thio functionality at the site of binding, so that both amino and thio sugars may be employed to provide for novel linkages between the phosphorous and the sugar.
  • K is a chelating agent, having at least 3 heteroatoms, which are oxygen, nitrogen, or sulfur, usually combinations thereof, more usually having about 6 heteroatoms or more, which may serve to chelate a metal ion capable of acting to inactivate, particularly to enhance cleavage, of a nucleic acid.
  • the functionalities may include carbonyl, oxy, thiono, amino, amido, mercapto, thioether, imino, where carbonyl oxygens will normally be separated by at least 2 carbon atoms, usually up to 6 carbon atoms, more usually up to 4 carbon atoms; except for amido, heteroatoms will normally be se-parated by at least 2 carbon atoms.
  • non-oxo-carbonyl groups there will be at least 2 non-oxo-carbonyl groups frequently at least 3 non-oxo-carbonyl groups and not more than about 6, usually not more than about 5 non-oxo-carbonyl groups.
  • alkylene diamines and polyalkylene diamines having from 3 to 8, usually 4 to 6 carboxyl groups, usually as carboxymethylene groups, e.g.,
  • R 2 N(CH 2 ) m N(T)((CH) n N(J)) x (CH 2 ) p NR 2 , wherein R is a carboxyalkylene group of from 2 to 3 carbon atoms or H, at least one R on each N being carboxyalkylene, m, n and p are the same or different and are 2 to 4, usually 2 to 3, and x is 0 to 2.
  • Illustrative chelating groups include ethylenediaminetetraacetic acid, dipropyleneaminepentaacetic acid, diethylenetriaminepentaacetic acid, 2,3-bis-(2'-acetamidoethyl)succinic acid, porphyrins, phthalocyanins tetraacetic acid, and crown ethers.
  • a wide variety of linking groups may be employed, depending upon the nature of the terminal nucleotide, the functionality selected for, whether the linking group is present during the synthesis of the oligonucleotide, the functionality present on the hydrophobic moiety and the like. A number of linking groups are commercially available and have found extensive use for linking polyfunctional compounds.
  • the linking groups include: -OCH 2 CH 2 NHCO(CH 2 ) n CONH-;-OCH 2 CH 2 NH-X-(CH 2 ) n NH-;-O-P(O)(OH)NHCO(CH 2 ) n COHN-;- OCH 2 CH 2 NHCO ⁇ S-;-NH(CH 2 ) n NH;-O(CH 2 ) n O-;-O(CH 2 CH 2 NH) m -;-NH(CH 2 ) n SYN; -CO(CH 2 ) n CO; -SCH 2 CH 2 CO-; -CO ⁇ NYS-; -(NCH 2 CH 2 ) m CH 2 N-; charged and uncharged homo- and copolymers of amino acids, such as polyglycine, polylysine, polymethionine, etc.
  • n is usually in the range of 2 to 20, more usually 2 to 12, and m is 1 to 10, usually 1 to 6.
  • the amphiphilic character imparting or solubility modifying group (M) may be a wide variety of groups, being aliphatic, aromatic, alicyclic, heterocyrop, or combinations thereof, substituted or unsubstituted, usually of at least 6, more usually at least 12 and not more than about 1000, usually not more than about 500, more usually not more than about 200 carbon atoms, having not more than about 1 heteroatom per 2 carbon atoms, being charged or uncharged, including alkyl of at least 6 carbon atoms and up to about 30 carbon atoms, usually not more than about 24 carbon atoms, fatty acids of at least about 6 carbon atoms, usually at least about 12 carbon atoms and up to about 24 carbon atoms, glycerides, where the fatty acids will generally range from about 12 to 24 carbon atoms, there being from 1 to 2 fatty acids, usually the 2 or 3 positions or both, aromatic compounds having from 1 to 4 rings, either mono- or polycyclic, fused or unfused, polyalkyleneg
  • the "M” group may be charged or uncharged, preferably being uncharged.
  • Illustrative groups include polyethylene glycol having from about 40 to 50 units, copolymers of ethylene and propylene glycol, laurate esters of polyethylene glycols, triphenylmethyl, naphthylphenylmethyl, palmitate, distearylglyceride didodecylphosphatidyl, cholesteryl, arachidonyl, octadecanyloxy, tetradecylthio, etc.
  • Functionalities which may be present include oxy, thio, carbonyl, (oxo or non-oxo), cyano, halo, nitro, aliphatic unsaturation, etc.
  • sequences will preferably be selected having greater than 40% GC content, more preferably greater than 50% and may have 60% or more GC content.
  • the melting temperature of the hybrid to be formed should be 5 to 10°C above the ambient temperature at which the hybrid forms, usually the ambient temperature being 37°C in a mammalian host. For mammalian hosts, the melting temperature will generally be chosen to be about 42-50°C.
  • the target sequence should be selected to be relatively free of secondary and tertiary structure. In many mRNA's, an open region will be present in the vicinity of the start codon (AUG).
  • a modified nucleoside is employed during the synthesis of the oligonucleotide.
  • Thymidine may be modified at the methyl group by providing for a carboxy alkyl group.
  • the carboxy group may then be further functionalized with an alkylene diamine, and the amino group emplo-yed for amide formation with a carboxy containing chelating agent.
  • the modified thymidine may then be employed as a nucleotide reagent in the automated synthesis of the oligonucleotide.
  • the final nucleotide adduct in the synthesis of the oligonucleotide may be functionalized in a variety of ways which may serve to act as a linking unit to the chelating agent. For example, after removal of the trityl protective group an aminoethanolphosphoramidite is added, as described by the supplier (Applied Biosystems, Foster City, CA) to provide for an available amino group. After deblocking and removing the oligonucleotide chain from the support, the amino group is then available for linking to the chelating agent.
  • the oligonucleotide is phosphorylated employing a polynucleotide kinase, followed by formation of a phosphoramidate using an activating agent, such as 1-methylimidazole or a water soluble carbodiimide, in the presence of an alkylene diamine, providing for an amino functionality (Chu and Orgel, DNA (1985) 4:327-331).
  • an activating agent such as 1-methylimidazole or a water soluble carbodiimide
  • alkylene diamine providing for an amino functionality
  • a further alternative is to deblock the oligonucleotide while retaining the oligonucleotide on the support, followed by treatment with carbonyldiimidazole. After removal of excess of the carbonyldiimidazole, a diamine may be added to provide an aminoalkylcarbamate (Wachter et al., Nucl. Acids Res. (1986) 14:7985-7994).
  • a mercaptan group may be provided as part of the functionalizing agent or separate from the functionalizing agent.
  • the mercaptan group may be part of the linker to the support or may be part of the functionalizing agent of the oligonucleotide, where both the chelating agent and "M” may be bound to the same linking group.
  • maleimido groups may be employed, where "M” or the chelating agent may have a mercaptan group to form a thioether.
  • Various active functionalities can be employed to produce a covalent linkage, such as isocyanates, isothiocyanates, diazo groups, imino chlorides, imino esters, anhydrides, acylhalides, sulfinylhalides, sulfonyl chlorides, etc.
  • active functionalities such as isocyanates, isothiocyanates, diazo groups, imino chlorides, imino esters, anhydrides, acylhalides, sulfinylhalides, sulfonyl chlorides, etc.
  • the linking arms, "M” , and the chelating moiety may be added at various times, depending upon the particular reaction scheme.
  • the chelating agent may be part of a nucleoside and be included in the synthesis of the oligonucleotide or may be added after oligonucleotide formation. "M” will normally be added after oligonucleotide formation.
  • reaction conditions will be mild and will employ polar solvents or combinations of polar and nonpolar solvents.
  • Solvents will vary and include water, acetonitrile, dimethylformamide, diethyl ether, methylene chloride, dimethylsulfoxide, etc.
  • Reaction conditions will be for the most part in the range of about -100-60°C.
  • the resulting product will be subjected to purification.
  • the manner of purification may vary, depending upon whether the oligonucleotide is bound to a support. For example, where the oligonucleotide is bound to a support, after addition of the linking arm to the oligonucleotide, unreacted chains may be degraded, so as to prevent their contaminating the resulting product. Where the oligonucleotide is no longer bound to the support, whether only reacted with the linking arm or as the conjugate to the chelating agent or as the final product, each of the intermediates or final product may be purified by conventional techniques, such as electrophoresis, solvent extraction, HPLC , chromatography, or the like. The purified product is then ready for use.
  • the subject products will be selected to have an oligonucleotide sequence complementary to a sequence of interest.
  • the sequence of interest may be present in a prokaryotic or eukaryotic cell, a virus, a normal or neoplastic cell.
  • the sequences may be bacterial sequences, plasmid sequences, viral sequences, chromosomal sequences, mitochondrial sequences, plastid sequences, etc.
  • the sequences may involve open reading frames for coding proteins, ribosomal RNA, snRNA, hnRNA, introns, untranslated 5'- and 3' -sequences flanking open reading frames, etc.
  • the subject sequences may therefore be involved in inhibiting the availability of an RNA transcript, inhibiting expression of a particular protein, enhancing the expression of a particular protein by inhibiting the expression of a repressor, reducing proliferation of viruses or neoplastic cells, etc.
  • the subject conjugates may be used in culture or in vivo for modifying the phenotype of cells, limiting the proliferation of pathogens such as viruses, bacteria, protista, mycoplasma, or the like, or inducing morbidity in neoplastic cells or specific classes of normal cells.
  • pathogens such as viruses, bacteria, protista, mycoplasma, or the like
  • one or more of the subject compositions to inhibit the transcription and/or expression of the native genes of a cell.
  • the subject compositions may be used for protection of a mammalian host from a variety of pathogens, e.g., enterotoxigenie bacteria, Pneumococcus, Neisseria, etc.; protists, such as
  • the subject compositions will be selected so as to be capable of inactivating sequences of interest, particularly mRNA, or in some circumstances the subject composition can be used with other nucleic acid moieties, e.g., tRNA, snRNA, DNA, e.g., plasmids, viruses, etc.
  • the subject compositions may bind to mRNA and provide for cleavage of the mRNA, so as to prevent the expression of a product.
  • the lifetime of the chelate conjugate may be substantially extended in the host cell, so as to have a relatively high kill ratio per sequence.
  • the subject sequences may be complementary to such sequences as sequences expressing growth factors, lymphokines, immunoglobulins, T-cell receptor sites, MHC antigens, DNA or RNA polymerases, antibiotic resistance, multiple drug resistance (mdr), genes involved with metabolic processes, in the formation of amino acids, nucleic acids, or the like, DHFR, etc. as well as introns or flanking sequences associated with the open reading frames.
  • compositions may be administered to a host in a wide variety of ways, depending upon whether the compositions are used in vitro or in vivo.
  • the compositions may be introduced into the nutrient medium, so as to modulate expression of a particular gene by transferring across the membrane into the cell interior such as the cytoplasm and nucleus.
  • the subject compositions may find particular use in protecting mammalian cells in culture from mycoplasma, for modifying phenotype for research purposes, for evaluating the effect of variation of expression on various metabolic processes, e.g., production of particular products, variation in product distribution, or the like.
  • the subject compositions may be modified by being encapsulated in liposomes or other vesicle, may be used in conjunction with permeabilizing agents, e.g., non-ionic detergents, Sendai virus, etc.
  • permeabilizing agents e.g., non-ionic detergents, Sendai virus, etc.
  • the subject compositions may be administered in a variety of ways, such as injection, infusion, tablet, etc., so that the compositions may be taken orally, parenterally, intravascularly, intraperitoneally, subcutaneously, intralesionally, or the like.
  • compositions may be formulated in a variety of ways, being dispersed in various physiologically acceptable media, such as deionized water, water, phosphate buffered saline, ethanol, aqueous ethanol, formulated in the lumen of vesicles, microencapsulated, etc.
  • compositions may be tested in conventional ways and the appropriate concentrations determined empirically.
  • Other additives may be included, such as stabilizers, buffers, additional drugs, detergents, etc. These additives are conventional, and would generally be present in less than about 5 wt%, usually less than 1 wt%, being present in an effective dosage, as appropriate. For fillers or excipients, these may be as high as 99.9% of the composition, depending upon the amount of active material necessary.
  • the following examples are presented by way of illustration not by way of limitation.
  • Nucleoside addition is followed by capping of unreacted 5' hydroxyls with acetic anhydride, iodine oxidation, and 5' detritylation in trichloroacetic acid-methylene chloride.
  • the resin bound oligomer is then dried by extensive washing in anhydrous acetonitrile and the process repeated. Normal cycle times using this procedure are 12 minutes with condensation efficiencies of >98% (as judged by trityl release).
  • the product DNA containing a free 5'-hydroxyl is phosphorylated with the forward reaction of ⁇ 4 polynucleotide kinase according to Chu and Orgel, supra (1986). Phosphorylated oligomers are separated from unreacted ATP using a C-18 reverse phase column (Waters SEP-PAK) according to the direction of the manufacturer.
  • the phosphorylated oligomer is then treated with 0.1M 1 -methyl imidazole, 0.1M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, 0.1M alkyl diamine (e.g., hexane diamine), pH 7, under conventional conditions in an aqueous medium to form the desired phosphoramidate containing a free amine with the following structure
  • the diamine forms a stable carbamate linkage after a brief incubation.
  • the oligomer can then be deblocked and released from the column under conditions appropriate to the type of phosphate linkage present.
  • Attachment of the Cleavage Unit DTPA Once the amine terminated oligomer is deblocked and characterized, the cleavage unit was added using the following method. Ten units of the oligomer were dissolved in 100 ⁇ 1 dimethylformamide containing 0.1 M DTPA (bisanhydride). The mixture was incubated for 1-2 hours at room temperature, and the excess DTPA removed by gel filtration and concentration on Centricon C10 membranes.
  • the final product was dried in vacuo and dissolved to a final concentration of 1 mM in water (based on its optical density at 26 ⁇ nM) and stored frozen. This solution was stable for at least 1 month.
  • the compound was homogeneous as judged by polyacrylamide gel electrophoresis.
  • the material on the column was washed free of unreacted decane-diamine with acetonitrile and water, and then eluted from the column in concentrated ammonium hydroxide. After removal from the column, the ammonium hydroxide solution containing the oligomer conjugate was placed in a sealed vial and incubated 5 hours at 55°C. The product is then lyophilized several times from 50% aqueous ethanol and purified via reversed phase HPLC C-8 silica columns eluting 5 to 50% acetonitrile/25 mM ammonium acetate, pH 6.8 in a linear gradient.
  • the material was further purified by ion-exchange HPLC on Nucleogen DEAE 60-7 eluting 20% acetonitrile/25 mM ammonium acetate, pH 6.5.
  • the recovered product is then characterized by gel electrophoresis in 15% polyacrylamide gels carried out as described by Maxam and Gilbert (Meth. Enzymol. (1980) 68[:499-560). Oligonucleotides in finished gels are visualized using Stains-all.
  • the presence of a primary amine can be determined by two methods.
  • reaction with fluorescamine produced a fluorescent product characteristic of the presence of a primary amine while no fluorescence is observed with similarly treated control oligomers of the same type, but lacking the amine linker.
  • the decane conjugate was dissolved in 100 ⁇ l 0.1 M sodium bicarbonate to which was added 1 mg of fluorescein isothiocyanate. After 1 hour of incubation, the unreacted FITC was removed by gel filtration chromatography on Sephadex G-25 spun columns. The product was then analyzed by polyacrylamide gel electrophoresis as described above, and the fluorescent band product visualized under UV illumination.
  • a single fluorescent band is observed which corresponds to the oligomer visualized by subsequent staining with Stains-all.
  • the product of this reaction is an aminoalkyl carbamate coupled to the 5' end of the oligonucleotide.
  • the alkylcarbamate is stable to moderate exposure to concentrated base.
  • the free amino group distal to the carbamate linkage is available for subsequent derivation which can be accomplished according to the method given in EXAMPLE 1.
  • the structure of the final conjugate synthesized by this method is illustrated as:
  • EXAMPLE 3 Synthesis of DTPA Derivatives of DNAs Using Imidazole Activated Carboxylic Acid Esters and a Poly-D-lysine Linker
  • a 25 nucleotide DNA complementary to the initiation region of rabbit beta globin mRNA was synthesized according to the method given in EXAMPLE 1.
  • the CPG support containing the oligomer was treated with 80% acetic acid for 30 minutes to remove trityl from the 5' end of the molecule.
  • the solid material was then thoroughly washed with anhydrous acetonitrile and blown dry under a stream of dry argon and treated with 0.3M CDI as in
  • EXAMPLE 4 The 5' CDI activated oligomer on the column was then washed free of excess reagent with 15 ml of acetonitrile and then treated with 0.2 M poly-D-lysine (MW+1,000) dissolved in 50% acetonitrile containing 0.1 M sodium phosphate, pH 8 for 16 hours at room temperature.
  • the material on the column was washed free of salts and unreacted polylysine with water and acetonitrile and then eluted from the column in concentrated ammonium hydroxide. After removal from the column, the ammonium hydroxide solution containing the oligomer conjugate was incubated for 5 hours at 55°C in a sealed glass vial. The product was then lyophilized several times from 50% aqueous ethanol and purified via gel filtration chromatography on TSK G4000SW in 100 mM Tris buffer, pH 7.5. The presence of a primary amine was determined by reaction with fluorescamine. No fluorescence was observed with control oligomers lacking the polyamine linker.
  • the polyamine conjungate cannot easily be characterized by gel electrophoresis since it is electrostatically and molecularly polydisperse.
  • the complex was reacted with FITC to label the molecule and to neutralize the positive charges on the amines. This was accomplished by dissolving a portion of the material in 100 ⁇ l 0.1 M sodium bicarbonate to which was added 1 mg of fluoresceinisothiocyanate. After 1 hour of incubation, the unreacted FITC was removed by gel filtration chromatography on Sephadex G-25 spun columns (Maniatis et al., Molecular Cloning - A
  • DNA Methylphosphonates The chemical synthesis of DNA methylphophonates may be carried out using a modification of the phosphochloridite method of Letsinger (Letsinger et al., J. Am. Chem. Soc. (1975) 9 :3278; Letsinger and
  • the ultimate base may be added as the cyanoethyl phosphotriester which yields, upon cleavage in base, a 5' terminal phosphodiester. This step makes it possible to radiolabel the oligonucleotide, purify and sequence the product using gel electrophoresis at intermediate stages of preparation (Narang et al, Can. J. Biochem. (1975) 53:342-394; Miller et al., Nucl. Acids Res. (1983) 11:6225-6242).
  • the amine terminated linker arm methylphosphonate oligomer is base deblocked as follows.
  • the resin containing the DNA is removed from the column and placed in a water jacketed column and incubated in 1-2 ml phenol: ethylene diamine (4:1) for 10 hours at 40°C.
  • the resin is washed free of the phenol reagent and released base protecting groups using methanol, water, methanol and methylene chloride in succession.
  • amine terminated DNA methylphosphonate Purification of the amine terminated DNA methylphosphonate is then performed as follows. The material is first lyophilized several times from 50% aqueous ethanol and purified via reversed phase HPLC C-8 silica columns eluting 5 to 50% acetonitrile/25 mM ammonium acetate, pH 6.8 in a linear gradient. Amine containing fractions as determined by fluorescamine reactivity are pooled and the product recovered by drying in vacuo and further purified by ion-exchange
  • the purified product is then converted to the DTPA derivative as in EXAMPLE 1. Purification of the complex is then effected as previously described.
  • unbound oligonucleotide is removed by gel filtration on Sephadex G-100 or HPGFC on TSK G4000SW eluting 10 mM Tris, pH 7.5.
  • the structure of the final product of this procedure is illustrated as:
  • dimethoxytrityl nucleosides are derived by repeated lyophilization from benzene, dissolved in anhydrous acetonitrile/2,6-lutidine (8:2) and added dropwise to a stirred solution of chloro diisopropylamino ethoxyphosphine in the same solvent at -70°C.
  • the product is recovered by aqueous extraction, in vacuo drying and silica gel chromatography.
  • DNA can be carried out using slight modifications of the conventional phosphoramidite methods.
  • nucleoside phosphoramidites dissolved in anhydrous acetonitrile are mixed with tetrazole and sequentially coupled to the 5' hydroxy terminal nucleoside bound to CPG.
  • Nucleoside addition is followed by capping of unreacted 5' hydroxyls with acetic anhydride, iodine oxidation, and 5' detritylation in trichloroacetic acid-methylene chloride.
  • the resin bound oligomer is then dried by extensive washing in anhydrous acetonitrile and the process repeated.
  • the fully blocked product is base-deblocked as follows.
  • the resin containing the fully protected DNA is removed from the column and placed in a water jacketed chromatography column.
  • the resin is then incubated in 1-2 ml phenol: ethylene diamine (4:1) for 10 hours at 40°C.
  • the resin is washed free of the phenol reagent and released base protecting groups using methanol, water, methanol and methylene chloride.
  • EDA ethanol (1:1) or brief treatment with room temperature ammonium hydroxide .
  • the material is first lyophilized several times from 50% aqueous ethanol and purified via reversed phase HPLC C-8 silica columns eluting 5 to 50% acetonitrile/25 mM ammonium acetate, pH 6.8 in a linear gradient. Amine containing fractions as determined by fluorescamine reactivity are pooled and the product recovered by drying in vacuo and further purified by ion-exchange HPLC on Nucleogen DEAE 60-7 eluting 20% acetonitrile/25 mM ammonium acetate, pH 6.5. The product oligonucleotide is then suitable for coupling to DTPA and purification by the techniques previously described.
  • a preferred method for the production of the oligonucleotide triesters of variable alkane chain length is via conventional phosphate triester chemistry to synthesize the desired sequences as the p- chlorophenyl phosphate triesters.
  • the fully protected oligonucleotide chlorophenyltriesters bound to the synthesis support are subjected to ester exchange in the presence of tetrabutylammonium fluoride and the desired alcohol.
  • This basic method for the construction of DNA oligonucleotides is classical DNA synthesis chemistry and presents no problems. The essential chemistry is well described (Gait, Oligonucleotide Synthesis: A Practical Approach IRL Press, Washington, D.C. (1984)) and can be used with little modification.
  • An alternative phosphite based chemistry which is much more rapid and gives equivalent yields is set forth below.
  • nucleosides dissolved in anhydrous acetonitrile, 2,6-lutidine and activated in situ with chlorophenoxydichlorophosphine are sequentially added to the 5' hydroxy terminal nucleotide of the growing DNA chain bound to controlled pore glass supports via a suc ⁇ inate spacer (Matteucci and Caruthers, Tetrahedron Lett. (1980) 21:719-722).
  • Derivatized glass supports, fully block nucleosides and other synthesis reagents are commercially available through Applied Biosystems (San Francisco, CA) or American Bionuclear (Emeryville, CA) . Nucleoside addition is followed by capping of unreacted 5' hydroxyls with acetic anhydride, iodine oxidation, and 5' detritylation in trichloroacetic acid-methylene chloride.
  • the resin bound oligomer chlorophenyltriester is then dried by extensive washing in anhydrous acetonitrile and the process repeated. Normal cycle times using this procedure are 13 minutes with condensation efficiencies of >92% (as judged by trityl release).
  • the ultimate base may be added as a methyl phosphotriester which yields, upon cleavage in base, a 5' terminal phosphodiester. This step makes it possible to radiolabel the oligonucleotide and to purify and sequence the product using gel electrophoresis (Narang et al., Can. J. Biochem. (1975) 53.:392-394; Miller et al., Nucl. Acids Res. (1983) 1:6225-6242).
  • TBAF tetrabutylammonium fluoride
  • anhydrous n-propanol is used to dissolve TBAF to a final concentration of 0.2 M.
  • the solution is then percolated slowly over the resin containing the oligomer chlorophenyl triester and allowed to react for about 1 hour at room temperature.
  • the resin is then washed with methanol and acetonitrile and dried under a stream of dry argon.
  • Amine linker arm addition, deblocking and purification are then effected as in EXAMPLE 2.
  • DTPA conjugation is then performed as in EXAMPLE 1.
  • the final yield of conjugate is about 10% of the starting equivalents of nucleoside resin used.
  • EXAMPLE 7 Effect of DTPA Conjugates on the Synthesis of Hemoglobin in Mouse MEL Cells
  • the effectiveness of oligomer DTPA conjugate mediated HART was determined in cultured cells incubated in the presence or absence of the oligomer.
  • the cells used were Friend murine erythroleukemia (MEL) cells which can be induced to synthesize hemoglobin by a variety of agents including DMSO and butyric acid (of. Gusella and Houseman, Cell (1976) 8:263-269).
  • MEL Friend murine erythroleukemia
  • Friend leukemia cells were grown in culture using well known techniques in a CO 2 incubator. Hemoglobin synthesis was induced using 1.5% DMSO.
  • Induced cells expressing hemoglobin were visualized by benzidine treatment which stains globin producing cells blue (Leder et al., Science (1975) 190 :893).
  • Cells were exposed to selected oligonucleotides and DTPA conjugate at concentrations ranging from 0.1 ⁇ M to 50 ⁇ M during a 4- to 5-day induction period.
  • Controls included mock-treated cells and cells treated with unmodified oligomers of the same sequence.
  • Treated cells were scored for globin production based on staining intensity and the results compared to controls. Cell death or damage due to treatment was scored by Trypan blue exclusion.
  • Table I indicates the specific sequences synthesized.
  • Lower case letters represent nucleosides coupled to the 3' adjacent nucleoside via a methylphosphonate linkage.
  • Upper case letters represent 3' adjacent normal phosphodiester linkage.
  • C 2 derivatives are formed from the condensation of ethanolamine with a 5' terminal phosphate via an ester linkage.
  • C 6 and C 10 derivatives are the corresponding diamines coupled via an alkyl carbamate linkage to the 5' terminal hydroxyl.
  • DTPA represents diethylenetriamine pentaacetic acid.
  • conjugates of a chelating agent and an oligonucleotide sequence may be used to preferentially inhibit the expression of a sequence in a viable cell.
  • cells can be modified in a variety of ways, so as to change the phenotype or to selectively kill cells.
  • the conjugate is stable and does not require that a metal be noncovalently bound to the chelating agent prior to use in order to achieve effectiveness.
  • the subject compositions can be used in vivo or in vitro, allowing for selection of cells, enhancing activity of particular cells, reducing activity of particular cells, or permitting selection of a particular class of cells.
  • a variety of conjugates can be produced, which will have long half lives, so as to be able to provide for destruction of a large number of RNA sequences for each molecule conjugate.

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Abstract

Nouveau conjugué comprenant notamment des séquences de nucléotides stables unies à un agent de chélation. On utilise lesdits conjugués pour inhiber l'expression d'ARNm, pour tuer des pathogènes, et pour sélectionner certaines catégories de cellules ou en tuer d'autres. On utilise des agents de chélation d'acide polyamino-polycarboxylique dans les conjugués, à titre d'exemple.
PCT/US1988/004430 1987-12-15 1988-12-12 Conjugue de chelate d'acide nucleique utilise comme agent therapeutique et de diagnostic WO1989005853A1 (fr)

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991006629A1 (fr) * 1989-10-24 1991-05-16 Gilead Sciences, Inc. Analogues d'oligonucleotides avec nouvelles liaisons
WO1991006556A1 (fr) * 1989-10-24 1991-05-16 Gilead Sciences, Inc. Oligonucleotides modifies en position 2'
WO1991014696A1 (fr) * 1990-03-29 1991-10-03 Gilead Sciences, Inc. Conjugues a base de disulfure d'oligonucleotide et d'un agent de transport
WO1991019730A1 (fr) * 1990-06-14 1991-12-26 Monsanto Company Hydrolyse/clivage d'arn
EP0490434A1 (fr) * 1990-12-10 1992-06-17 Akzo Nobel N.V. Oligonucléotides,marqués et modifiés
US5264562A (en) * 1989-10-24 1993-11-23 Gilead Sciences, Inc. Oligonucleotide analogs with novel linkages
US5495009A (en) * 1989-10-24 1996-02-27 Gilead Sciences, Inc. Oligonucleotide analogs containing thioformacetal linkages
WO1996025956A2 (fr) * 1995-02-21 1996-08-29 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Conjugue pour le dosage individuel de medicaments
US5733523A (en) * 1990-12-10 1998-03-31 Akzo Nobel N.V. Targeted delivery of a therapeutic entity using complementary oligonucleotides
US6258941B1 (en) 1990-06-14 2001-07-10 Washington University RNA hydrolysis
DE10051628A1 (de) * 2000-10-18 2002-05-02 Fresenius Hemocare Gmbh Mittel zur Inaktivierung von pathogenen Keimen und dessen Verwendung

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4508625A (en) * 1982-10-18 1985-04-02 Graham Marshall D Magnetic separation using chelated magnetic ions
US4707352A (en) * 1984-01-30 1987-11-17 Enzo Biochem, Inc. Method of radioactively labeling diagnostic and therapeutic agents containing a chelating group

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4508625A (en) * 1982-10-18 1985-04-02 Graham Marshall D Magnetic separation using chelated magnetic ions
US4707352A (en) * 1984-01-30 1987-11-17 Enzo Biochem, Inc. Method of radioactively labeling diagnostic and therapeutic agents containing a chelating group

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SUZUKI et al., "An Introduction to Genetic Analysis", published 1986, by W.H. Freeman and Company (New York), see pages 191-193. *

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6476205B1 (en) 1989-10-24 2002-11-05 Isis Pharmaceuticals, Inc. 2′ Modified oligonucleotides
WO1991006556A1 (fr) * 1989-10-24 1991-05-16 Gilead Sciences, Inc. Oligonucleotides modifies en position 2'
US5264562A (en) * 1989-10-24 1993-11-23 Gilead Sciences, Inc. Oligonucleotide analogs with novel linkages
US5495009A (en) * 1989-10-24 1996-02-27 Gilead Sciences, Inc. Oligonucleotide analogs containing thioformacetal linkages
US6911540B2 (en) 1989-10-24 2005-06-28 Isis Pharmaceuticals, Inc. 2′ Modified oligonucleotides
WO1991006629A1 (fr) * 1989-10-24 1991-05-16 Gilead Sciences, Inc. Analogues d'oligonucleotides avec nouvelles liaisons
US5792847A (en) * 1989-10-24 1998-08-11 Gilead Sciences, Inc. 2' Modified Oligonucleotides
WO1991014696A1 (fr) * 1990-03-29 1991-10-03 Gilead Sciences, Inc. Conjugues a base de disulfure d'oligonucleotide et d'un agent de transport
WO1991019730A1 (fr) * 1990-06-14 1991-12-26 Monsanto Company Hydrolyse/clivage d'arn
US6258941B1 (en) 1990-06-14 2001-07-10 Washington University RNA hydrolysis
EP0490434A1 (fr) * 1990-12-10 1992-06-17 Akzo Nobel N.V. Oligonucléotides,marqués et modifiés
US5733523A (en) * 1990-12-10 1998-03-31 Akzo Nobel N.V. Targeted delivery of a therapeutic entity using complementary oligonucleotides
WO1996025956A3 (fr) * 1995-02-21 1996-12-27 Deutsches Krebsforsch Conjugue pour le dosage individuel de medicaments
US6410695B1 (en) 1995-02-21 2002-06-25 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Individual medicament dosing conjugate
WO1996025956A2 (fr) * 1995-02-21 1996-08-29 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Conjugue pour le dosage individuel de medicaments
DE10051628A1 (de) * 2000-10-18 2002-05-02 Fresenius Hemocare Gmbh Mittel zur Inaktivierung von pathogenen Keimen und dessen Verwendung
DE10051628B4 (de) * 2000-10-18 2007-06-06 Fresenius Hemocare Beteiligungs Gmbh Einzelsträngiges Oligonukleotid und dessen Verwendung

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