WO1993020090A1

WO1993020090A1 - Paired-ion oligonucleotides and methods for preparing same

Info

Publication number: WO1993020090A1
Application number: PCT/US1993/003161
Authority: WO
Inventors: Stanley Stein; Ching-Hsuan Tung; Tianmin Zhu
Original assignee: University Of Medicine & Dentistry Of New Jersey
Priority date: 1992-04-06
Filing date: 1993-04-05
Publication date: 1993-10-14
Also published as: AU3945493A

Abstract

The present invention pertains to paired-ion oligonucleotides comprising a polyanionic oligonucleotide covalently bonded to a polycationic polymer via a cross-linking agent, wherein the polycationic polymer is represented by formula (I) or formula (II) or formula (III) or the formula R2-[[NH(CH2)b]c[NH(CH2)d]e]f[NH(CH2)g]h-NHR2 (IV), wherein X is selected from the group consisting of -NH-, -O-, and -S-; R1 is lower-alkyl; R2 is selected from the group consisting of hydrogen, acyl groups, cross-linking agents, and chemotherapeutic agents; R3 is selected from the group consisting of -NRR', -OR'', cross-linking agents, and chemotherapeutic agents; R4 is lower-alkyl; R5 is an amine group; R6 is a lower-alkyl chain; R7 is selected from the group consisting of hydrogen, alkyl groups having from 1 to 5 carbon atoms, -CH2C6H5, and -(CH2)zCOR3; R and R' are selected from the group consisting of hydrogen and lower-alkyl groups, and R'' is lower-alkyl group; wherein at least one of R2 or R3 is a cross-linking agent; a through h are defined integers; and the ratio of cations in the polycationic polymer to anions in the polyanionic oligonucleotide ranges from about 8:10 to about 16:8, respectively.

Description

PAIRED-ION OLIGONUCLEOTIDES

AND METHODS FOR PREPARING SAME

BACKGROUND OF THE INVENTION

Field of the Invention

This invention pertains to paired-ion oligonucleotides comprising a polyanionic oligonucleotide conjugated to a polycationic polymer. The cations in the polycationic polymer are paired with the anions in the polyanionic oligonucleotide to neutralize the polyanionic oligonucleotide. The paired-ion oligonucleotides have enhanced hybridization strength and may have improved ability to permeate into tissue and cellular compartments.

Description of the Background

Synthetic oligonucleotides are potentially useful for the treatment of infections and other diseases. In order to be therapeutically useful, a synthetic oligonucleotide must have: (1) the ability to hybridize to target nucleic acid sequences with high specificity and affinity, (2) reasonable stability under in vivo conditions (e.g. , nucleaεe resistant) , (3) the ability to permeate into tissue and cellular compartments, (4) low-toxicity, and (5) acceptable cost per dose, Zon, G. , "Oligonucleotide Analogs as Potential Chemotherapeutic Agents", Pharmaceutical Research 5, (1988) pp. 539-549.

Antisense oligonucleotides are of particular interest as therapeutic agents for gene regulation. Expression of a particular protein can be inhibited in a process known as translation arrest in which RNA transcripts produced by specific regulatory genes hybridize to corresponding mRNA molecules. These regulatory transcripts have been named antisense RNA because they are complementary to the "sense" RNA strand such as the mRNA that codes for a protein. Translation arrest can also be accomplished using synthetic fragments of DNA or of modified DNA (collectively known as antisense DNA) which are complementary to the mRNA that codes for a particular protein.

To improve the stability and cellular uptake of oligonucleotides, various forms of oligonucleotides have been prepared having modifications to the internucleoside backbone. For example, phosphorothioate and methylphosphonate derivatives of oligonucleotides have been synthesized and shown to have the same sequence specificity and strength of hybridization to that of unmodified oligonucleotides, i.e., short single strands of oligonucleotides, Ebright, Y. , Raska, Jr., K. , Gaur, S., Frenkel, L, Tsao, J. and Stein, S., "Antisense DNA- Analogs: Inhibition of Human Immunodeficiency Virus", New Jersey Medicine 87, (1990) pp. 1011-1015. Because of their uncharged character, methylphosphonate derivatives of oligonucleotides have enhanced uptake and access to intracellular components and are resistant to nucleases.

Concern has been expressed, however, with the toxicity of phosphorothioate and other backbone-modified DNA-analogs, S. Agrawal, J. Goodchild, M. P. Civeira, A. H. Thornton, P. S. Sarin, and P. C. Zamecnik, "Oligodeoxynucleoside Phosphoramidates and Phosphorothioates as Inhibitors of Human Immunodeficiency Virus", Proc. Natl. Acad. Sci. USA 85, (1988) pp. 7079- 7083; Y. Cheng, W. Gao, and F. Han, "Phosphorothioate Oligonucleotides as Potential Antiviral Compounds Against Human Immunodeficiency Virus and Herpes Viruses", Nucleosides & Nucleotides 10 (1991), pp. 155-166.

In another approach, a nuclease was attached to an antisense oligonucleotide to cleave a target nucleic acid, D. Pei, R. Corey, and P. G. Schultz, "Site-specific Cleavage of Duplex DNA by a Semisynthetic Nuclease via Triple-helix Formation", Proc. Natl. Acad. Sci. USA, 87 (1990) pp. 9858-9862.

Intercalating agents have also been conjugated to antisense oligonucleotides to increase the stability of the conjugate with the complementary sense strand, N. T. Thuong, U. Asseline, V. Roig, M. Takasugi, and C. Helene, "Oligo (alpha-deoxynucleotide)s Covalently Linked to Intercalating Agents: Differential Binding to Ribo- and Deoxyribopolynucleotides and Stability Toward Nuclease Digestion", Proc. Natl. Acad. Sci. USA, 84 (1987) pp. 5129-5133. The attachment of the intercalator to the antisense oligonucleotide also increased the nuclease resistance of the conjugate.

In another example, polylysine was attached to an antisense oligonucleotide resulting in a conjugate having enhanced antiviral activity in a cell culture assay, M. Lemaitre, B. Bayard, and B. Leblue, "Specific Antiviral Activity of a Poly(L-lysine)-conjugated Oligodeoxy-ribonucleotide Sequence Complementary to Vesicular Stomatitis Virus N Protein mRNA Initiation Site", Proc. Natl. Acad. Sci. USA, 84 (1987) pp. 648-652; Leonetti, J. P., Rayner, B. , Lemaire, M. , Gagnor, C. , Milhaud, P. G. , I bach, J.-L. and Lebleu, B. , "Antiviral Activity of Conjugates Between Poly(L-lysine) and Synthetic Oligodeoxyribonucleotides", Gene 72, - (1988) pp. 323-332.

Similar polylysine-antisense oligonucleotide conjugates have been shown to interfere with proliferation of HIV-1, M. Stevenson and P. L. Iversen, "Inhibition of Human Immunodeficiency Virus Type 1- Mediated Cytopathic Effects by Poly(L-lysine)-conjugated Synthetic Antisense Oligodeoxyribonucleotides", J. Gen. Virol. 70 (1989), pp. 2673-2682. The enhanced potency of these conjugates compared to non-conjugated antisense oligodeoxyribonucleotides may result from increased cellular uptake and delivery of the conjugates to the appropriate cell compartments, greater stability of the conjugates towards nucleases, and higher affinity of the conjugates to the target sequence.

In a variation of this approach, a ternary conjugate was prepared containing an antisense oligonucleotide, polylysine, and transferrin, E. Wagner, M. Zenke, M. Cotten, H. Beug, and M. L. Birnstiel, "Transferrin-polycation Conjugates as Carriers for DNA Uptake into Cells", Proc. Natl. Acad. Sci. USA 87 (1990), pp. 3410-3414. Transferrin was incorporated into the conjugate to enhance cellular uptake of the conjugate through the transferrin receptor on the surface of cells.

Polylysine-oligonucleotide complexes, however, have been reported to be poorly defined structurally, E. Wagner, M. Cotten, R. Foisner, and M. L. Birnstiel, "Transferrin-polycation-DNA Complexes: The Effect of Polycations on the Structure of the Complex and DNA Delivery to Cells", Proc. Natl. Acad. Sci. USA 88 (1991), pp. 4255-4259. Polylysine-oligonucleotide complexes are a mixture of conjugates because polylysine is a mixture of polymers having different degrees of polymerization and because the polylysine chain contains multiple points of attachment located at varying places on the polylysine chain. Hence, attachment of the oligonucleotide is random with respect to the position of the lysine -chain.

In another approach, a structurally defined conjugate was prepared containing a cationic peptide attached to an antisense oligonucleotide, R. Eritja, A. Pons, M. Escareller, E. Giralt, and F. Albericio, "Synthesis of Defined Peptide-Oligonucleotide Hybrids Containing a Nuclear Transport Signal Sequence", Tetrahedron 47 (1991), pp. 4113-4120. The peptide sequence was employed to target the oligonucleotide to the nucleus of the cell but was not selected or positioned for ion pairing with the oligonucleotide.

While a number of cationic polymers have been conjugated to oligonucleotides to improve the stability and cellular uptake of the oligonucleotides, none of these conjugated oligonucleotides have been entirely satisfactory. Conjugated cationic polymers have not been designed to substantially neutralize the oligonucleotide. Accordingly, oligonucleotide conjugates having the uncharged properties of phosphoramidate , methylphosphonate, or other backbone modified oligonucleotides and the low-toxicity properties of polycationic polymeric-oligonucleotide complexes would be highly desirable. The present invention provides such oligonucleotide conjugates having improved therapeutic properties. The oligonucleotide conjugates can be synthetically modified to vary the reactivity, the specifiςity, and the general utility of the oligonucleotide to greatly facilitate gene regulation. The paired-ion oligonucleotides of the present invention may be employed with pharmaceutically acceptable carriers to provide a wide variety of pharmaceutical products. SUMMARY OF THE INVENTION

The present invention pertains to paired-ion oligonucleotides comprising a polyanionic oligonucleotide covalently bonded to a polycationic polymer via a cross- linking agent, wherein the polycationic polymer is represented by the formula:

R₂-[XRιCHCO]_a-R₃ (I)

NH₂ or the formula:

or the formula:

_R2-_[XCHCO_ro-_a-_R3 R₅-R₄ R₇ or the formula:

R₂-[[NH(CH₂)_b]_c[NH(CH₂)_d]e]f[NH(CH2)g]h-NHR2 (IV)

wherein X is selected from the group consisting of -NH-, -0-, and -S-; R- is a substituted or unsubstituted branched or unbranched lower-alkyl chain having from 1 to 4 carbon atoms in the chain; R₂ is independently selected from the group consisting of hydrogen, acyl groups, cross-linking agents covalently linking the polycationic polymer to the oligonucleotide, and chemotherapeutic agents; R₃ is selected from the group consisting of -NRR', -OR' ^r , cross-linking agents covalently linking the polycationic polymer to the oligonucleotide, and chemotherapeutic agents; R₄ is a substituted or unsubstituted branched or unbranched lower-alkyl chain having from 1 to 5 carbon atoms in the chain; R5 is selected from the group consisting of primary amines, secondary amines, tertiary amines, quaternary ammonium salts, and guanidine groups; R₆ is an alkyl chain having from 1 to 2 carbon atoms; R₇ is selected from the group consisting of hydrogen, branched and unbranched lower- alkyl groups having from 1 to 5 carbon atoms, -CH C₆H5, and -(CH )_zCOR₃, wherein z is an integer ranging from 0 to 3 and R₃ is as defined above; R and R' may be the same or different and are independently selected from the group consisting of hydrogen and branched and unbranched lower-alkyl groups, and R" is selected from the group consisting of branched and unbranched lower-alkyl groups, each lower-alkyl group having from 1 to 6 carbon atoms; wherein at least one of R₂ or R₃ is a cross-linking agent; a is an integer ranging from about 6 to about 18; b is an integer ranging from about 2 to about 5; c is an integer ranging from about 0 to about 3; d is an integer ranging from about 2 to about 5; e is an integer ranging from about 0 to about 5; f is an integer ranging from about 3 to about 9; g is an integer ranging from about 2 to about 5; h is an integer ranging from about 0 to about 5; and the ratio of cations in the polycationic polymer to anions in the polyanionic oligonucleotide ranges from about 8:10 to about 16:8, respectively.

BRIEF DESCRIPTION OF THE FIGURES

Figure l is a photograph of a ball-and-stick model of a paired-ion oligonucleotide having (delta- ornithine)n-glycine as the polycationic polymer and a random sequence 12-mer oligodeoxyribonucleotide.

Figure 2 is an expanded view of a portion of the model from Figure 1.

Figure 3 is a graph showing the elution times of a reaction mixture containing the conjugate, Fmoc-Cys- (delta-Orn)ii-Gly/MB-hexanola ine link-5'-CAT TTC TTT ATT-3' . Figure 4 is a photograph of -a gel electrophoresis plate showing the migration of a reaction mixture containing the conjugate, Fmoc-Cys- (delta-Orn)ι - Gly/MB-hexanolamine link-5'-CAT TTC TTT ATT-3'.

Figure 5 is a photograph of a gel electrophoresis plate showing the migration of a reaction mixture containing the conjugate, Fmoc-Cys- (del ta-Orn)n~ Gly/MB-hexanolamine link-5^/-CAT TTC TTT ATT-3'.

Figure 6 is a photograph of a polyacrylamide electrophoresis gel plate comparing the ability of the paired-ion antisense conjugate, Cys- (delta-Orn)n-Gly/MB- hexanolamine link-5'-CAT TTC TTT ATT-3', and the unconjugated oligonucleotide, 5'-CAT TTC TTT ATT-3', to inhibit in vitro translation of their complementary targets.

DETAILED DESCRIPTION OF THE INVENTION

The present invention pertains to paired-ion oligonucleotides comprising a polyanionic oligonucleotide conjugated to a polycationic polymer via a cross-linking agent. The cations in the polycationic polymer are attached to the backbone of the polymer in a specific and repetitive manner mimicking the pattern of attachment of the anions in the oligonucleotide. Each positively charged group in the polycationic polymer is positioned to be close to a negatively charged phosphate group in the oligonucleotide to neutralize the negative charges in the oligonucleotide through electrostatic interactions.

The paired-ion oligonucleotides of the present invention are a new class of oligonucleotides having unique properties. For example, the paired-ion oligonucleotides have an enhanced strength of hybridization of the antisense oligonucleotides to the target nucleic acid because the ionic repulsion inherent between the two polyanionic oligonucleotide strands is reduced. The paired-ion oligonucleotides also have enhanced stability in vivo because degradation is minimized when the 3'- and 5'-termini of the oligonucleotides, individually or simultaneously, are blocked by linkage to the polycationic polymer. The ability of the paired-ion oligonucleotides to enter cells is also enhanced because uncharged oligonucleotides can more readily penetrate through cellular membranes. Chemotherapeutic agents such as intercalators, cell targeting agents, transmembrane delivery agents, and nucleases may also be coupled to the polycationic polymer to further enhance the activity of the paired-ion oligonucleotides.

As set out above, the paired-ion oligonucleotides of the present invention comprise a polyanionic oligonucleotide covalently bonded to a polycationic polymer via a cross-linking agent. The polycationic polymers are represented by the formula:

R₂-[XR₁CHCO]_a-R₃ (I)

NH₂ or the formula:

R₂-[XR6COXCHCO]_a-R₃ (II)

R₇ R4-R5 or the formula:

R₂-[XCHCOXR₆CO]_a-R₃ (III)

R5-R4 R₇ or the formula:

R₂-[[NH(CH₂)_b]_c[NH(CH₂)_d]_e]_f[NH(CH₂)_g]_h-NHR₂ (IV) The polyanionic oligonucleotides in the present invention may be any natural or synthetic oligonucleotide known in the art. The polyanionic oligonucleotides may be oligodeoxyribonucleic acids (normal DNA) , oligoribonucleic acids (normal RNA) , backbone-modified oligonucleotides such as methylated RNA and phosphorothioate oligodeoxyribonucleic acids (-OP(S) (O)O-) , and combinations of normal and backbone- modified oligonucleotides. The oligonucleotide sequence may be a sense strand or an antisense strand (complementary to either a DNA or RNA sequence, i.e., the sense strand, of the gene to be inhibited) . Preferably, the oligonucleotide sequence is an antisense strand. Antisense carriers and backbone-modified oligonucleotides are more fully described in Stein et al. , New Jersey Medicine_f 87. pp. 1011-1015 (1990) , Stein et al. _f Journal of Liquid Chromatography, 11, pp. 2005-2017 (1988) , and Miller, Bio/Technology. 9., pp. 358-362 (1991) , which disclosures are incorporated herein by reference.

The oligonucleotide may be in either the 5'—>3' or the 3'—>5' orientation with respect to the polycationic polymer. While the polyanionic oligonucleotides of the present invention are not limited to any specific number of nucleotide monomers, the polyanionic oligonucleotides preferably comprise from about 6 to about 24 nucleotides, more preferably from about 8 to about 16 nucleotides, and most preferably from about 10 to about 14 nucleotides.

The polycationic polymers in the present invention may be natural or synthetic polypeptides, pseudopeptides, or polyamines. The cations in the polycationic polymer are attached to the backbone of the polymer in a specific and repetitive manner mimicking the pattern of attachment of the anions in the oligonucleotide. Preferably, the cation is selected from the group consisting of primary amines, secondary amines, tertiary amines, quaternary ammonium salts, and guanidine groups. More preferably, the cation is selected from the group consisting of primary amines and secondary amines.

Pseudopeptides contain backbone modifications such as amide bond surrogates. The term "surrogate" as used herein refers to an amide bond modification which involves replacement of a naturally occurring amide bond by an unnatural bond. Nonli iting examples of amide bond modifications include ketones (-COCH₂-) , thioesters (-COS-) , substituted amides (-CONR-) , esters (-COO-) , thioamides (-CSNH-) , amines (-CH₂NH-) , alkyl groups (-CH₂CH₂-) , sulfides (-CH₂S-) , sulfoxides (-CH₂SO-) , sulfones (-CH₂S0₂-) , alkene groups (-CH=CH-) , and unnatural amide groups (-NHCO-) . The term "pseudopeptide" as used herein refers to a peptide analog containing an amide backbone modification. The term "pseudopeptide" also refers to a peptide analog having amide bonds employing side chain amines instead of alpha- amines . Peptide backbone modifications are generally employed to confer stability to a peptide against enzymatic or proteolytic degradation such as to prepare an orally active peptide or a peptide having sustained activity. Amino acids containing amide bond modifications are termed pseudoamino acids. Peptide backbone modifications are well known in the art and are discussed in detail in "Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins", Volume 7, B. Weinstein, Ed., Ch. 5, Marcel Dekker, New York, New York (1983) , which disclosure is incorporated herein by reference.

In a first embodiment, the polycationic polymers are represented by the formula:

R₂-[XR₁CHCO]_a-R₃ (I)

NH₂ In Formula (I) , the polycationic polymer is a pseudopeptide containing the monomer -XRιCH(NH )CO-. Group X is a heteroatom selected from the group consisting of -NH-, -0-, and -S-. The terminal heteroatom group X on the side chain of the monomer forms amide, ester, or thioester bonds for peptide or polymer formation instead of the alpήa-amine group. The alpha- amine group in the monomer is used as the repeating cationic group to form ion pairs with the anionic groups in the oligonucleotide. Preferably, group X is -NH-.

Group i is a lower-alkyl spacer group which may be varied to provide the desired number of carbon atoms in the skeleton of the repeating monomer. Group R^ is a substituted or unsubstituted branched or unbranched lower-alkyl chain having from 1 to 4 carbon atoms in the chain. Preferably, group R-^ contains from 2 to 3 carbon atoms in the chain.

Group R is a terminal end capping group covalently bonded to the terminal nitrogen, oxygen, or sulfur group in the polycationic polymer of Formula (I) . R₂ may be selected from the group consisting of hydrogen, acyl groups, cross-linking agents covalently linking the polycationic polymer to the oligonucleotide, and chemotherapeutic agents. Preferably, R₂ is selected from the group consisting of cross-linking agents and chemotherapeutic agents.

When R₂ is not hydrogen, R₂ will generally contain a carbonyl group for coupling R₂ to group X via an amide, ester, or thioester bond. For example, R₂ may be an acyl group having from 1 to about 15 carbon atoms, preferably from 1 to about 10 carbon atoms, and more preferably from 1 to about 6 carbon atoms.

The cross-linking agents which may be employed in the present invention are those agents which will covalently link the polycationic polymer to the oligonucleotide without interfering with the ion-pairing of the polycationic polymer and the oligonucleotide. R₂ cross-linking agents will generally contain a carbonyl group for coupling R₂ to group X via an amide, ester, or thioester bond and a hydroxyl group to form an ester bond with the phosphate group on the oligonucleotide. Group R may be a single cross-linking agent such as glycine. Group R₂ may also be a combination of compounds to form a cross-linking agent such as aminolink agents, succinate cross-linking agents, cysteine, and maleimide. Nonlimiting examples of aminolink agents include 1,6- hexanola ine, and 2,3-dihydroxy-l-aminopropane. Aminolink agents contain a hydroxyl group at one end of a chain to form an ester bond with the phosphate group on the oligonucleotide and an amine group at the other end of the chain to form an amide bond with a carbonyl group. Nonlimiting examples of succinate cross-linking agents include those agents derived from ethylene glycoJis(sulfo succinimidylsuccinate) (Sulfo-EGS) , and maleimidobenzoyl- sulfosuccinimide ester (sulfo-MBS) . Cysteine may also be coupled to the succinate cross-linking agent and then further coupled to the polycationic polymer via the terminal amine or carbonyl group. In one embodiment, the combination of cross-linking agents is an aminolink agent coupled to the oligonucleotide and further coupled to a succinate cross-linking agent, the succinate cross- linking agent is in turn coupled to a cysteine, which is in turn coupled by its carbonyl group to group X in the polycationic polymer of Formula (I) .

The chemotherapeutic agents in the present invention are those compounds which when coupled to the polycationic polymer will further enhance the chemotherapeutic activity of the paired-ion oligonucleotide without interfering with the ion-pairing of the polycationic polymer and the oligonucleotide. The R₂ chemotherapeutic agents will generally contain a carbonyl group for direct coupling to group X to form an amide, ester, or thioester bond. The chemotherapeutic agent may also be coupled to a bridging group which contains a carbonyl group for subsequent coupling to group X. Examples of chemotherapeutic agents which may be employed in the present invention include intercalators, cell targeting agents, transmembrane delivery agents, natural and synthetic nucleases, and free radical generators.

Intercalator groups are extended cyclic aromatic and heterocyclic aromatic chromophores which can interact with oligonucleotides. An intercalator group may be coupled to the polycationic polymer as a nucleic acid binding molecule to increase the strength of binding of the paired-ion oligonucleotide to the nucleic acid. The interaction between the intercalator group and the oligonucleotide occurs by means of intercalation or insertion of the intercalator group between adjacent base-pairs of the oligonucleotide without disrupting the hydrogen bonding of the oligonucleotide.

Intercalator groups are generally attached to a candidate compound by means of a bridging group. For example, when the terminal functional group of group X is nitrogen, the intercalator group may contain an aldehyde group for attachment to the amine group by formation of a Schiff base followed by reduction. When the terminal functional group of group X is oxygen or sulfur, the intercalator group may contain a halide group for attachment to the oxygen or sulfur group by alkylation. Methods for attaching intercalator groups by means of a bridging group are well known in the art and are more fully described in, for example, Gauσain et al.. Biochemistry (1978), 17, 5071-5078 and P.B. Dervan et al.. J. Am. Chem. Soc. (1982), 104, 313- 315, which disclosures are incorporated herein by reference.

Examples of intercalator groups which may be attached to group X by means of a linker or bridging compound are 2-methyl-9-acridine (Acr) , 9-aminoacridine, acridine orange, proflavine, ethidium, ellipticine, 3,5,6,8-tetramethyl-N-methyl-phenanthrolinium, 2-hydroxy- ethane-thiolato-2,2',2''-terpyridine platinum(II) , daunomycin, actinomycin, and mixtures thereof. In a preferred embodiment, the intercalator group is 2-methylacridine. Intercalator groups are more fully described in Wolfram Saenσer. Principles of Nucleic Acid Structure. Chapter 16, Springer-Verlag, New York, New York, (1984) , which disclosure is incorporated herein by reference.

Cell targeting agents are substances that bind to specific structures on the surface of the targeted cell. Nonlimiting examples of cell targeting agents are monoclonal antibodies, folic acid, transferrin, and polypeptide growth and differentiation factors.

Transmembrane delivery agents are substances that enhance cellular uptake. Nonlimiting examples of transmembrane delivery agents are those agents set out above for cell targeting agents and peptides composed of basic and hydrophobic amino acids and various types of lipids.

Natural and synthetic chemical nucleases are molecules which possess both the biochemical molecular recognition system and the required chemical reactivity to cleave nucleic acids. These nucleases generally contain a nucleic acid binding group as the biochemical molecular recognition system and a cleaving agent to provide the required chemical reactivity. The nucleic acid binding group is generally a polycationic peptide, a protein, a polyamine, or a single-stranded oligonucleotide. The cleaving agent is generally a chemically reactive group such as an oxidation-reduction (redox) reagent (generally a transitional metal complex) , an alkylating agent, or a photoreactive group. The cleaving agent may also be a catalyst for hydrolysis. The sequence-specific cleavage of nucleic acids has many applications such as in the mapping of large genomes, site directed mutagenesis, diagnostic probes, and recombinant DNA manipulations. Ribonucleases are discussed in detail in copending United States patent application serial no. 07/737,411, filed 29 July 1991, which disclosure is incorporated herein by reference.

Group R₃ is a terminal end capping group covalently bonded to the terminal carbonyl group in the polycationic polymer of Formula (I) . R₃ may be selected from the group consisting of -NRR' , -OR'', cross-linking agents covalently linking the polycationic polymer to the oligonucleotide, and chemotherapeutic agents. Preferably, R₃ is selected from the group consisting of -NRR', cross-linking agents, and chemotherapeutic agents. More preferably, R₃ is selected from the group consisting of cross-linking agents and chemotherapeutic agents.

R and R' may be the same or different and are independently selected from the group consisting of hydrogen and branched and unbranched lower-alkyl groups, and R'' is selected from the group consisting of branched and unbranched lower-alkyl groups, each lower-alkyl group having from 1 to 6 carbon atoms, preferably from 1 to 4 carbon atoms, and more preferably from 1 to 3 carbon atoms. Examples of -NRR' and -OR" groups include -NH₂, -NHCH₃, -N(CH₃)₂, -NHCH₂CH₃, -OCH₃, -OCH₂CH₃, and -OCH(CH₃)₂.

The cross-linking agents and chemotherapeutic agents discussed above as useful for group R₂ may also be used for group R₃. Because R₃ groups couple to carbonyl groups and not to heteroatom X groups (like R groups) , R₃ cross-linking agents will generally contain an amine, hydroxyl, or sulfhydryl group for coupling R₃ to the carbonyl group of the polycationic polymer of Formula (I) via an amide, ester, or thioester bond and a hydroxyl or sulfhydryl group to form an ester or thioester bond with the phosphate group on the oligonucleotide. Similarly, R₃ chemotherapeutic agents will generally contain an amine, hydroxyl, or sulfhydryl group for coupling the chemotherapeutic agent to the carbonyl group of the polycationic polymer of Formula (I) .

In a second embodiment, the polycationic polymers are represented by the formula:

In Formula (II) , the polycationic polymer is a polypeptide or pseudopeptide containing the monomer -XRg(R₇)COXCH(R4-R5)CO- which comprises two different types of amino acids or pseudo amino acids. Groups X, R₂, and R₃ are defined as set out above. A first amino acid or pseudoamino acid (-XCH(R4~R5)CO-) contains a side chain (-R4-) having a cationic group (-R5) , such as an amine group or a guanidine group, which is used as the repeating cationic group to form ion pairs with the oligonucleotide. The alpha-amine group or group X in this first amino acid forms the amide, ester, or thioester bonds for peptide or polymer formation. A second amino acid or pseudoamino acid (-XRg(R )CO-) is coupled to the first amino acid and is used as a spacer group to provide the desired skeleton size of the repeating monomer unit. R₇ may be a functionalized side chain for attaching additional groups to the polycationic polymer such as cross-linking agents and chemotherapeutic agents.

Group R4 is a lower-alkyl side chain group to which cationic group R5 is coupled. The length of the side chain may be varied to provide the desired number of carbon atoms in the chain. Group R4 is a substituted or unsubstituted branched or unbranched lower-alkyl chain having from 1 to 5 carbon atoms in the chain. Preferably, group R4 contains from 2 to 4 carbon atoms in the chain.

Group R5 is a cationic group attached to lower- alkyl side chain group R₄. The type of cationic group employed in R5 may be varied to optimize the ion-pairing properties with the particular oligonucleotide. Group R5 is selected from the group consisting of primary amines, secondary amines, tertiary amines, quaternary ammonium salts, and guanidine groups (-NHC(=NH)NH₂) . Preferably, group 5 is selected from the group consisting of primary amines and secondary amines.

Group R is a lower-alkyl spacer group which may be varied to provide the desired number of carbon atoms in the skeleton of the repeating monomer. Group Rg is an alkyl chain having from 1 to 2 carbon atoms in the chain. Preferably, group R₆ contains 1 carbon atom in the chain.

Group R₇ may be hydrogen; a side chain such as those found in alanine, leucine, isoleucine, valine, or phenyl alanine; or a functionalized side chain. When group R5 contains two carbon atoms, group R₇ may be attached to either carbon atom. Group R₇ is selected from the group consisting of hydrogen, branched and unbranched lower-alkyl groups having from 1 to 5 carbon atoms, -CH C6H5, and -(CH₂)₂-COR-₃. Preferably, the branched and unbranched lower-alkyl groups have from 1 to 4 carbon atoms, more preferably from 1 to 3 carbon atoms. The integer z may range from 0 to 3, preferably from 0 to 2. Group R₃ is as defined above. When R₇ contains a functionalized side chain such as an acyl group (-(CH₂)zCOR₃) ₁ additional groups (R₃ groups) may be attached to the polycationic polymer such as cross- linking agents and chemotherapeutic agents. Preferably, group R₇ is selected from the group consisting of hydrogen and branched and unbranched lower-alkyl groups having from 1 to 5 carbon atoms. More preferably. group R₇ is selected from the group consisting of hydrogen and branched and unbranched lower-alkyl groups having from 1 to 3 carbon atoms. Most preferably, group R is hydrogen.

In a third embodiment, the polycationic polymers are represented by the formula:

R₂-[XCHCOXR₆CO]_a-R₃ (III)

R5-R4 R₇

In Formula (III) , the polycationic polymer is a variation of the polycationic polymer set out above in Formula (II) . The polycationic polymer in Formula (III) is a polypeptide or pseudopeptide containing the monomer -XCH(R₄-R₅)COXR₆(R₇)CO-, wherein the C-terminal and N- terminal positions of the two different types of amino acids or pseudo amino acids set out above in Formula (II) are reversed. Groups X and R₂ through R are defined as set out above.

In a fourth embodiment, the polycationic polymers are represented by the formula:

R₂-[[NH(CH₂)_b]_c[NH(CH₂)_d]_e]f[NH(CH₂)_g]_h-NHR₂ (IV)

In Formula (IV) , the polycationic polymer is a polyamine containing the monomer -NH(CH₂)_]-₎- or the monomer -NH(CH₂)_d-, or both. The monomer -NH(CH₂)g- permits the polycationic polymers of Formula (IV) to contain an odd number of monomers. Each group R₂ may be the same or different and is defined as set out above. The secondary amine groups in the polyamines are an integral part of the skeleton of the polycationic polymer and are used as the repeating cationic group to form ion pairs with the oligonucleotide. In general, these polyamines are derived by hydride reduction of the amide bonds in the corresponding polypeptides to give polyamines of the spermidine and spermine type. For example, the peptide Jeta-alanine-gaiiuπa-aminobutyric acid-Jeta-alanine-carboxy amide yields spermine upon reduction. Different sequences of these amino acids or other combinations of amino acids, including cysteine, may be used to generate any desired polyamine.

In the polycationic polymers of Formulas (I) , (II) , (III) _r and (IV) , the cross-linking agent which covalently bonds the polycationic polymer to the oligonucleotide may be attached at either end of the polycationic polymer, i .e . , at R₂ or R₃, or may be attached in the interior of the polymer, i.e., at R₇ when R₇ contains an R cross-linking agent. At least one of R₂ or R₃ must be a cross-linking agent or potential cross-linking agent. Preferably, both R₂ and R₃ are cross-linking agents or potential cross-linking agents.

The integer a may range from about 6 to about 18, preferably from about 10 to about 14, and more preferably from about 11 to about 13. The integer b may range from about 2 to about 5, preferably from about 2 to about 4, and more preferably from about 3 to about 4. The integer c may range from about 0 to about 3, preferably from about 1 to about 3, and more preferably from about 2 to about 3. The integer d may range from about 2 to about 5, preferably from about 2 to about 4, and more preferably from about 3 to about 5. The integer e may range from about 0 to about 3, preferably from about 1 to about 3, and more preferably from about 2 to about 3. The integer f may range from about 3 to about 9, preferably from about 4 to about 1 , and more preferably from about 5 to about 6. The integer g may range from about 2 to about 5, preferably from about 2 to about 4, and more preferably from about 3 to about 5. The integer h may range from about 0 to about 3, preferably from about 1 to about 3, and more preferably from about 2 to about 3. In general, the ratio of cations in the polycationic polymer to anions in the polyanionic oligonucleotide will range from about 8:10 to about 16:8, preferably from about 9:10 to about 10:9, more preferably from about 9.5:10 to about 10:9.5, and most preferably about 1:1, respectively. Paired-ion oligonucleotides containing an excess of cations may have useful properties such as enhanced uptake of the paired-ion oligonucleotide into cells or into the nucleus of cells or may have ribonuclease-like activity, Perello, M. , Barbier, B. , and Brack, A., "Hydrolysis of Oligoribonucleotides by alpha-helical Basic Peptides", Int. J. Peptide Protein Res. 38 (1991), pp. 154-160.

The term "branched lower-alkyl chain" means a lower-alkyl chain or skeleton to which is attached branching lower-alkyl groups having from about l to about 4 carbon atoms, preferably from about 1 to about 3 carbon atoms. The substituent groups attached to the lower- alkyl chain or branching lower-alkyl groups may be selected from the group consisting of hydroxyl, sulfhydryl, halogen, and lower-alkoxy groups having from 1 to 4 carbon atoms. The term halogen, as used herein, refers to the chemically related elements consisting of fluorine, chlorine, bromine, and iodine.

The paired-ion oligonucleotides of the present invention have high specificity because the sequence of each particular antisense oligonucleotide can be selected to be complementary to a specific target. The paired-ion oligonucleotides also have high affinity hybridization because the ion-pairing properties of the polycationic polymer chain lowers the ionic repulsion between the two polyanionic oligonucleotides, as demonstrated by analogy with methylphosphonate oligonucleotides, R. S. Quartin and J. G. Wetmur, "Effect of Ionic Strength on the Hybridization of Oligodeoxynucleotides With Reduced Charge Due to Methylphosphonate Linkages to Unmodified Oligodeoxynucleotides Containing the Complementary Sequence", Biochemistry 28 (1989) pp. 1040-1047, and as shown by R. Eritja, A. Ponε, M. Escareller, E..Giralt, and F. Albericio, "Synthesis of Defined Peptide- Oligonucleotide Hybrids Containing a Nuclear Transport Signal Sequence", Tetrahedron 47 (1991), pp. 4113-4120. Because hybridization strength (i.e., T_m or melting temperature) is dependent upon the length and sequence of an oligonucleotide, shorter antisense chains of the strong affinity paired-ion oligonucleotides of the present invention may be sufficient for hybridizing a particular oligonucleotide with a target nucleic acid.

The paired-ion oligonucleotides also have enhanced stability in vivo because exonuclease digestion is minimized when both the 3'- and 5'-termini are blocked such as by linking the polycationic polymers of the present invention to the oligonucleotide. Similarly, polypeptide degradation is minimized when both the amino- terminus and carboxy-terminus are blocked by linkage to the oligonucleotide, thereby limiting exopeptidase digestion. Endopeptidase digestion is also minimized when pseudopeptide bonds are employed in the polypeptide.

The paired-ion oligonucleotides have enhanced bioavailability because the ability of the oligonucleotides to penetrate through cellular membranes is enhanced when the negative charges on the antisense

DNA are ion-paired, by analogy to methylphosphonate and other non-ionic antisense oligonucleotides, P. S. Miller, K. B. McParland, K. Jayaraman, and P. O. P. Ts'o,

"Biochemical and Biological Effects of Nonionic Nucleic

Acid Methylphosphonates", Biochemistry 20 (1981), pp. 1874-1880. Additional hydrophobic groups such as valine, phenylalanine, intercalators, and other groups may be coupled to the polycation polymer to enhance permeability.

Alternatively, an excess of cations in the oligonucleotide-conjugate can be used to enhance permeability. Enhanced oligonucleotide uptake occurs through binding of the positively charged paired-ion oligonucleotide to the negatively charged outer membrane of the cell followed by endocytosis, P. L. Schell, "Uptake of Polynucleotides by Mammalian Cells XIV: Stimulation of the Uptake of Polynucleotides by Poly(L- lysine", Biochim. Biophys. Acta 340, (1974) pp. 323-333). A combination of hydrophobic and positively charged groups may be synergistic with regard to the ability of the antisense substance to reach the necessary compartments in vivo , such as the nuclei of cells, R. Eritja, A. Pons, M. Escareller, E. Giralt, and F. Albericio, "Synthesis of Defined Peptide-Oligonucleotide Hybrids Containing a Nuclear Transport Signal Sequence", Tetrahedron 47 (1991), pp. 4113-4120.

The paired-ion oligonucleotides have low toxicity because the metabolic degradation products of polypeptide conjugates are amino acids and nucleotides. Concern has been expressed about the toxicity of phosphorothioate and other backbone-modified DNA-analogs, S. Agrawal, J. Goodchild, M. P. Civeira, A. H. Thornton, P. S. Sarin, and P. C. Zamecnik, "Oligodeoxynucleoside Phosphoramidates and Phosphorothioates as Inhibitors of Human Immunodeficiency Virus", Proc. Natl. Acad. Sci. USA 85, (1988) pp. 7079-7083; Y. Cheng, W. Gao, and F. Han, "Phosphorothioate Oligonucleotides as Potential Antiviral Compounds Against Human Immunodeficiency Virus and Herpes Viruses", Nucleosides & Nucleotides 10 (1991), pp. 155- 166. Increasing the potency of the antisense oligonucleotide should result in a better therapeutic index.

The cost per dose of the paired-ion oligonucleotides should be reasonable especially for a high potency product because, in the preferred size range of 5-25 nucleotide monomers, oligodeoxy-ribonucleotides and polypeptides are routinely synthesized on automated instruments. Figure 1 is a photograph of a ball-and-stick model of a paired-ion oligonucleotide having (delta- ornithine)-Li-glycine as the polycationic pseudopeptide polymer and a random sequence 12-mer oligodeoxyribonucleotide. The polypeptide is attached to the 12-mer oligonucleotide via an amide bond through the carbonyl group of glycine and the amine group of the hexanolamine linker on the oligonucleotide. The intrapeptide amide linkages are with the delta-amine group of the ornithine residues. The chemical structure of this model is as follows:

o1igodeoxyribonucleotide^₂-_mer

This model demonstrates that the chemical structure of a conjugate of an ion-paired peptide with an oligonucleotide can adopt a helical configuration which is necessary for duplex formation with a target RNA strand. In this model, the shape of the DNA strand is fixed in a three-dimensional configuration resembling that found in a DNA/DNA or DNA/RNA duplex. Furthermore, the spacing of the positive charges on the polypeptide chain allows ion-pairing with all the negative charges on the oligonucleotide chain.

Figure 2 is an expanded view of a portion of Figure 1 and shows further detail. The nitrogen (blue) atom and the five carbon (carbonyl, alpha, beta , gamma, and delta) atoms (black) in one ornithine monomer are labeled. The carbonyl oxygens (red) of the ornithine residues are not labeled. In the DNA strand, the phosphorous atoms are represented by yellow balls and the oxygen atoms are depicted in red. The ion-pairs are represented by the transparent tubing. [Note that one of the 1,2-phosphoryl oxygens is not present in the model for the sake of clarity.] Although all distances in this model are rough approximations, there should be enough flexibility in the bond angles of the polypeptide chain to allow for the configuration represented in Figures 1 and 2. In any particular embodiment, duplex or triplex strand formation with the target nucleic acid can be optimized by the choice of polycation monomers and linkers that would allow a necessary or preferred structural configuration of the antisense strand.

The present invention extends to methods for preparing the paired-ion oligonucleotides. The paired- ion oligonucleotides may be synthesized using standard techniques and apparatus known to those skilled in the art. The ultimate paired-ion oligonucleotides are readily prepared using methods generally known in the chemical and biochemical arts.

In general, the polycationic peptides can be synthesized by standard peptide chemistry coupling reactions such as by N-Fmoc [N-(9-fluorenyl- methoxycarbonyl] chemistry on a peptide synthesizer. In one embodiment, the amino acids may be coupled to a PAL™ support by BOP [benzotriazolyl-N-oxytris(dimethylamino) phosphonium hexafluorophosphate] and HOBt (1- hydroxybenzotriazole) . The side chain protecting groups may be removed by TFA (trifluoroacetic acid) at the time when the peptide is cleaved from the solid support. The peptide product may be purified by reverse-phase HPLC and the identity of the product may be confirmed by amino acid analysis, peptide sequencing, and mass spectrometry.

The chemotherapeutic agents may be coupled to the polycationic polymer by various methods. For example, when the terminal functional group of the polycationic polymer is an amine, such as in lysine, an intercalator such as 2-methyl-9-acridine-carboxaldehyde may be coupled to the amine group on the side chain of lysine by formation of the Schiff base followed by hydride reduction of the Schiff base to form the corresponding amine. When the terminal functional group of the polycationic polymer is a hydroxyl group, an intercalator containing a carbonyl group may be coupled to the hydroxyl group in an esterification reaction. When the terminal functional group of the polycationic polymer is a sulfhydryl group, such as in cysteine, a halogenated intercalator may be coupled to the sulfhydryl group on the side chain of cysteine by sulfide displacement of the halide group under alkaline conditions. The polycationic peptides may be purified by conventional means such as by gradient elution reverse phase high pressure liquid chromatography (HPLC) based on an N-FMOC-on and N-FMOC-off two step purification procedure.

A sense or antisense oligonucleotide having a primary amine group coupled through its 5'- or 3'- termini, or through an internal position, may be synthesized on an automated DNA synthesizer using commercially available reagents. After HPLC purification and removal of the protecting groups, the oligonucleotide may be coupled to the polycationic polymer via a cross- linking agent such as a succinimide-mediated chemical reaction. The polycationic polymer and oligonucleotide conjugate may then be deprotected and purified by HPLC or on an anion-exchange column.

In a specific embodiment, the invention is directed at a method for preparing a paired-ion oligonucleotide comprising a polyanionic oligonucleotide covalently bonded to a polycationic polymer via a cross- linking agent, wherein the polycationic polymer is represented by the formula:

R₂-[XR₁CHCO]_a-R₃ (I)

NH₂ or the formula:

R₂-[XR₆COXCHCO]_a-R₃ (II) I I

R₇ R₄-R5 or the formula:

R₂-[XCHCOXR₆CO]_a-R₃ (III)

R5-R4 R₇ or the formula:

R₂-[[NH(CH₂)_b]_c[NH(CH₂)_d]_e]f[NH(CH₂)g]_h-NHR₂ (IV)

wherein X is selected from the group consisting of -NH-, -0-, and -S-; Ri is a substituted or unsubstituted branched or unbranched lower-alkyl chain having from 1 to 4 carbon atoms in the chain; R₂ is independently selected from the group consisting of hydrogen, acyl groups, cross-linking agents covalently linking the polycationic polymer to the oligonucleotide, and chemotherapeutic agents; R₃ is selected from the group consisting of -NRR' , -OR'' , cross-linking agents covalently linking the polycationic polymer to the oligonucleotide, and chemotherapeutic agents; R₄ is a substituted or unsubstituted branched or unbranched lower-alkyl chain having from 1 to 5 carbon atoms in the chain; R5 is selected from the group consisting of primary amines, secondary amines, tertiary amines, quaternary ammonium salts, and guanidine groups; Rg is an alkyl chain having from 1 to 2 carbon atoms; R₇ is selected from the group consisting of hydrogen, branched and unbranched lower- alkyl groups having from 1 to 5 carbon atoms, -CH2C5H5, and -(CH₂)₂COR₃, wherein z is an integer ranging from 0 to 3 and R₃ is as defined above; R and R' may be the same or different and are independently selected from the group consisting of hydrogen and branched and unbranched lower-alkyl groups, and R' ' is selected from the group consisting of branched and unbranched lower-alkyl groups, each lower-alkyl group having from l to 6 carbon atoms; wherein at least one of R₂ or R3 is a cross-linking agent; a is an integer ranging from about 6 to about 18; b is an integer ranging from about 2 to about 5; c is an integer ranging from about 0 to about 3; d is an integer ranging from about 2 to about 5; e is an integer, ranging from about 0 to about 5; f is an integer ranging from about 3 to about 9; g is an integer ranging from about 2 to about 5; h is an integer ranging from about 0 to about 5; and the ratio of cations in the polycationic polymer to anions in the polyanionic oligonucleotide ranges from about 8:10 to about 16:8, respectively; which comprises the steps of: (1) providing the polyanionic oligonucleotide;

(2) providing the polycationic polymer;

(3) coupling the polyanionic oligonucleotide to the polycationic polymer via the cross linking agent.

The paired-ion oligonucleotides may be prepared using standard techniques and equipment known to those skilled in the art. The apparatus useful in accordance with the present invention comprises apparatus well known in the chemical and biochemical arts, and therefore the selection of the specific apparatus will be apparent to the artisan.

The paired-ion oligonucleotides of the present invention may be used together with pharmaceutically acceptable carriers to provide pharmaceutical compositions which can be administered to mammals such as man in amounts effective to provide a variety of therapeutic activity. Suitable carriers include propylene glycol-alcohol-water, isotonic water, sterile water for injection (USP) , emulphor™-alcohol-water, cremophor-EL^A1M¹ or other suitable carriers known to those skilled in the art. Other suitable carriers include isotonic water, sterile water for injection (USP) , alone or in combination with other solubilizing agents such as ethanol, propylene glycol, or other conventional solubilizing agents known to those skilled in the art.

Of course, the type of carrier will vary depending upon the mode of administration desired for the pharmaceutical composition as is conventional in the art. A preferred carrier is an isotonic aqueous solution of the inventive compound.

The compounds of the present invention can be administered to mammals, e.g., animals or humans, in amounts effective to provide the desired therapeutic effect. Since the activity of the compounds and the degree of the desired therapeutic effect vary, the dosage level of the compound employed will also vary. The actual dosage administered will also be determined by such generally recognized factors as the body weight of the patient and the individual hypersensitiveness of the particular patient. Thus, the unit dosage for a particular patient (human) can be as low as about 0.001 mg/kg, or about 0.1 mg in a 100 kg person, which the practitioner may titrate to the desired effect.

The compounds of the present invention can be administered parenterally, in the form of sterile solutions or suspensions, such as intravenously, intramuscularly or subcutaneously in the carriers previously described.

For parental therapeutic administration, the compounds of the present invention may be incorporated into a sterile solution or suspension. These preparations should contain at least about 0.1% of the inventive compound, by weight, but this amount may be varied to between about 0.1% and about 50% of the inventive compound, by weight of the parental composition. The exact amount of the inventive compound present in such compositions is such that a suitable dosage level will be obtained. Preferred compositions and preparations according to the present invention are prepared so that a paranteral dosage unit contains from between about 0.1 milligrams to about 100 milligrams of the inventive compound. The sterile solutions or suspensions may also include the following adjuvants: a sterile diluent, such as water for injection, saline solution, fixed oils, polyethylene glycol, glycerine, propylene glycol, or other synthetic solvent; antibacterial agents, such as benzyl alcohol or methyl paraben; antioxidants, such as ascorbic acid or sodium metabisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA) ; buffers, such as acetates, citrates or phosphates; and agents for the adjustment of tonicity, such as sodium chloride or dextrose. The parental preparations may be enclosed in ampules, disposable syringes, or multiple dose vials made of glass or plastic.

It is especially advantageous to formulate the pharmaceutical compositions in dosage unit forms for ease of administration and uniformity of dosage. The term dosage unit forms as used herein refers to physically discrete units suitable for use as a unitary dosage, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the pharmaceutical carrier.

Throughout this application, various publications have been referenced. The disclosures in these publications are incorporated herein by reference in order to more fully describe the state of the art.

The present invention is further illustrated by the following examples which are not intended to limit the effective scope of the claims. All parts and percentages in the examples and throughout the specification and claims are by weight of the final composition unless otherwise specified. Example

Cysteine-(delta-Ornithine)^-Glycine

Maleimidobenzoyl- hexanolamine link-5'-CAT TTC TTT ATT-3'

This example demonstrates the preparation of a paired-ion oligonucleotide having cysteine- (del ta- ornithine)^-glycine as the polycationic polypeptide and 5'-CAT TTC TTT ATT-3' as the 12-mer-oligodeoxy- ribonucleotide. In the polypeptide, the carboxy-terminal amino acid is glycine, the amine-terminal amino acid is cysteine, and the terminal side chain amine groups in ornithine are used for peptide bond formation. The peptide is termed a "pseudopeptide" because the amide bonds are formed with the delta-amine groups rather than the alpha-amine groups of ornithine. The peptide is linked at the cysteine group via the linking group, maleimidobenzoyl-hexanolamine link, to the 5'-terminal position of the oligonucleotide.

The synthesis of the peptide was carried out in the following manner. N-alpha-t-Boc-delta-ornithine was treated with N-(9-fluorenylmethoxycarbonyloxy)- succinimide (Fmoc-NHS) to give N-alp a-t-Boc-N-Fmoc- ornithine. This monomer was then used in 11 repetitive cycles followed by one cycle with trityl-protected Fmoc- cysteine in a Milligen/Biosearch automated peptide synthesizer on a solid support to which a carboxy- terminal glycine residue was appended. The pseudopeptide, Fmoc-Cys- (delta-Orn)n-Gly, was cleaved from the solid support using trifluoroacetic acid. The product was treated with dithiothreitol (to maintain the thiol in reduced form) and purified by reverse-phase HPLC using a typical gradient of increasing acetonitrile in about 0.1% trifluoroacetic acid.

The 12-mer-oligodeoxyribonucleotide, 5'-CAT TTC TTT ATT-3' with a 5'-hexanolamine linker, was synthesized on an Applied Biosystems automated DNA synthesizer. The 5'-hexanolamine linker reagent (Applied Biosystems) was used in the last cycle to provide a primary, aliphatic amine at the 5'-terminus of the 12-mer for subsequent covalent attachment of the pseudopeptide. The DNA oligo er was purified on an EM Lichrospher 100RP-18, 5uM column (4x125mm) . Mobile phase A was 95% 0.1M triethylammonium acetate, pH 7, and 5% acetonitrile. Mobile phase B was 5% 0.1M triethylammonium acetate, pH 7, and 95% acetonitrile. The gradient was 100% A for 5 minutes, then 100% A to 50% A/50% B over 50 minutes. The flow rate was 1 ml/minute. The peak eluting at about 20 minutes was collected as the 12-mer-oligodeoxy- ribonucleotide, hexanolamine link-5'-CAT TTC TTT ATT-3'.

The hexanolamine link-5'-CAT TTC TTT ATT-3' (20 units, A260) was then mixed with 4mg of maleimidobenzoylsulfosuccinimide ester (sulfo-MBS) in lOOul of 0.1M NaHC0₃ solution for 30 minutes. The product was purified by anion-exchange chromatography on a Nucleogen 60-7 DEAE column (4 x 125mm) . Mobile phase A was 60% 20mM sodium acetate, pH 6.5, and 40% acetonitrile. Mobile phase B was mobile phase A containing 0.7M lithium chloride. The gradient was 100% A for 10 minutes, then 100% A to 88% A over 20 minutes, then 88% A to 50% A over one minute. The flow rate was 1 ml/minute. The peak eluting at about 35 minutes was collected as the MB-hexanolamine link-5'-CAT TTC TTT AIT¬ S'.

Approximately 12 Units (&2₆0- ^{of MB}~ hexanolamine link-5'-CAT TTC TTT ATT-3' were treated with 5 g of Fmoc-Cys-(delta-Orn)_1:L-Gly for 12 hours in the eluent solution collected from the anion-exchange column. The mixture was concentrated under vacuum and purified on a Lichrospher 100RP-18, 5 urn column. Mobile phase A was 95% 0.1M triethylammonium acetate, pH 7, and 5% acetonitrile. Mobile phase B was 5% 0.1M triethylammonium acetate, pH 7, and 95% acetonitrile. The gradient was 100% A for 5 minutes, then 100% A to

50% A/50% B over 50 minutes. The flow rate was

1 ml/minute. A DNA-MB calibration marker eluted at 22 minutes.

As set out in Figure 3, the reaction mixture gave a small peak at 22 minutes corresponding to unreacted oligonucleotide, a peak at 29 minutes corresponding to the Fmoc-Cys- (delta-Orn)n-Gly/MB- hexanolamine link-5'-CAT TTC TTT ATT-3' conjugate, and a peak at 31 minutes corresponding to unreacted Fmoc- peptide. The material eluting at 29 minutes was taken to dryness, treated with piperidine/water (1:1), taken to dryness again, redissolved, and analyzed on the same reverse-phase Lichrospher 100RP-18, 5 um column system. The retention time of the hydrolyzed material shifted to 22 minutes, signifying removal of the Fmoc group and formation of the Cys-(delta-Orn)^-Gly/MB-hexanolamine link-5'-CAT TTC TTT ATT-3' conjugate. The reaction components having retention times of 22 minutes and 29 minutes contained glycine and ornithine as determined by acid hydrolysis and amino acid analysis. The presence of the DNA moiety was confirmed by spectrophotometry (lambda max 260 nm) .

The components in the reaction mixture were also analyzed by gel electrophoresis (Figures 4 and 5) . Electrophoresis was carried out using the Phast System from Pharmacia/LKB on homogeneous 20%PhastGel and native buffer strips at pH 8.8 (the positive electrode was at the bottom) . The gel was developed by silver staining.

As set out in Figure 4, Lane 1 contains the 12- mer-oligodeoxyribonucleotide control marker, 5'-CAT TTC TTT ATT-3'. Lane 2 contains the Cys-(delta-Orn)n- Gly/MB-hexanolamine link-5'-CAT TTC TTT ATT-3' conjugate after piperidine treatment of the Fmoc-Cys- (del ta-Orn)n- Gly/MB-hexanolamine link-5'-CAT TTC TTT ATT-3' conjugate. Lane 3 contains the Fmoc-Cys-(delta-Orn)n-dy B- hexanolamine link-5'-CAT TTC TTT ATT-3' conjugate after purification on HPLC. Lane 4 contains a control sample consisting of a mixture of the hexanolaminolink-12-mer and the Fmoc-peptide. Since no malei ide group is present on the hexanolaminolink-12-mer, these components cannot link. This control demonstrates that the migration of the hexanolaminolink-12-mer on the gel is not influenced by the presence of peptide, i.e., unless a covalent linkage is formed between the peptide and the DNA, the DNA will run at the position set out in Lane 1. Lane 5 contains Fmoc-Cys-( delta-Orn)n-Gly. Lane 6 contains the reaction mixture before HPLC purification.

In Figure 5, Lane 1 contains the Fmoc-Cys- ( elta-Orn)ιι-Gly. Lane 2 contains the reaction mixture before HPLC purification. Lane 3 contains a control sample consisting of a mixture of the hexanolaminolink- 12-mer and the Fmoc-peptide. Lane 4 contains the Fmoc- Cys-(delta-Orn)ιι-Gly/MB-hexanolamine link-5'-CAT TTC TTT ATT-3' conjugate after purification on HPLC. Lane 5 contains the Cys-(delta-Orn)^-Gly/MB-hexanolamine link¬ s'-CAT TTC TTT ATT-3' conjugate after piperidine treatment and HPLC purification of the Fmoc-Cys-(del a- Orn)^-Gly/MB-hexanolamine link-5'-CAT TTC TTT ATT-3' conjugate. Lane 6 contains the 12-mer- oligodeoxyribonucleotide control marker, 5'-CAT TTC TTT ATT-3'.

The ability of the paired-ion antisense conjugate, Cys- (delta-Orn)^-Gly/MB-hexanolamine link-5'- CAT TTC TTT ATT-3', and the unconjugated antisense oligonucleotide, 5'-CAT TTC TTT ATT-3', to inhibit in vitro translation of their complementary targets was then compared in a cell free translation system where information from messenger RNA "m transcript" of killer virus of yeast was translated into protein molecules. The translation reaction from each test sample, conjugated and unconjugated antisense nucleotide, was analyzed on a polyacrylamide electrophoresis gel using autoradiography to measure the radioactive amino acids incorporated into the protein. This method is discussed in detail in Leibowitz et al. , Methods in Enzymology 194. pp. 536-545, Academic Press (1991) , which disclosure is incorporated herein by reference.

Figure 6 shows a photograph of the polyacrylamide electrophoresis gel (SDS-PAGE gel analysis) after 7 days exposure at -70° C. with intensifier. As set out in Figure 6, Lane 1 contains a ten fold excess of the paired-ion peptide and antisense oligonucleotide conjugate and m transcript. Lane 2 contains a hundred fold excess of the paired-ion peptide and antisense oligonucleotide conjugate and m transcript. Lane 3 contains a thousand fold excess of the paired-ion peptide and antisense oligonucleotide conjugate and m transcript. Lane 4 contains a ten fold excess of normal (unpaired) antisense oligonucleotide and m transcript. Lane 5 contains a hundred fold excess of normal antisense oligonucleotide and m transcript. Lane 6 is blank. Lane 7 contains a thousand fold excess of normal antisense oligonucleotide and m transcript. Lane 8 contains m transcript only through a mock hybridization and a normal translation procedure (no antisense DNA) . Lane 9 contains m transcript only through a normal translation reaction (no antisense DNA) . Lane 10 contains translation without m transcript. Lane 11 contains molecular weight markers.

As set out in Figure 6, the specific protein

"P" disappeared, i.e., was totally inhibited, when a 1000-fold excess of the paired-ion antisense conjugate, Cys-(delta-Orn)ii-Gly/MB-hexanolamine link-5'-CAT TTC TTT ATT-3' was employed as shown in Lane 3. The unconjugated antisense oligonucleotide, 5'-CAT TTC TTT ATT-3', was much less effective inhibiting the specific protein "P" at the same concentration as shown in Lane 7. While the invention has been particularly described in terms of specific embodiments, those -skilled in the art will understand in view of the present disclosure that numerous variations and modifications upon the invention are now enabled, which variations and modifications are not to be regarded as a departure from the spirit and scope of the invention. Accordingly, the invention is to be broadly construed and limited only by the scope and spirit of the following claims.

Claims

We claim:

1. A paired-ion oligonucleotide comprising a polyanionic oligonucleotide covalently bonded to a polycationic polymer via a cross-linking agent, wherein the polycationic polymer is represented by the formula:

R₂-[XR₁CHCO]_a-R₃ (I)

NH₂ or the formula:

_R2-_tXR6COXC„CO_]a-_R3

R₇ R₄-R5 or the formula:

^R2 R-5^[x-Rr4 °τR₇ ^oia-^R3 or the formula:

R₂-[[NH(CH₂)_b]_c[NH(CH₂)_d3_e]_f[NH(CH₂)_g]_h-NHR₂ (IV)

wherein X is selected from the group consisting of -NH-, -0-, and -S-; R± is a substituted or unsubstituted branched or unbranched lower-alkyl chain having from 1 to 4 carbon atoms in the chain; R₂ is independently selected from the group consisting of hydrogen, acyl groups, cross-linking agents covalently linking the polycationic polymer to the oligonucleotide, and chemotherapeutic agents; R₃ is selected from the group consisting of -NRR' , -OR'', cross-linking agents covalently linking the polycationic polymer to the oligonucleotide, and chemotherapeutic agents; R₄ is a substituted or unsubstituted branched or unbranched lower-alkyl chain having from 1 to 5 carbon atoms in the chain; R5 is selected from the group consisting of primary amines, secondary amines, tertiary amines, quaternary ammonium salts, and guanidine groups; R₆ is an alkyl chain having from 1 to 2 carbon atoms; R₇ is selected from the group consisting of hydrogen, branched and unbranched lower- alkyl groups having from 1 to 5 carbon atoms, -CH₂CgH5, and -(CH₂)_zCOR_3r wherein z is an integer ranging from 0 to 3 and R is as defined above; R and R' may be the same or different and are independently selected from the group consisting of hydrogen and branched and unbranched lower-alkyl groups, and R" is selected from the group consisting of branched and unbranched lower-alkyl groups, each lower-alkyl group having from 1 to 6 carbon atoms; wherein at least one of R2 or R3 is a cross-linking agent; a is an integer ranging from about 6 to about 18; b is an integer ranging from about 2 to about 5; c is an integer ranging from about 0 to about 3; d is an integer ranging from about 2 to about 5; e is an integer ranging from about 0 to about 5; f is an integer ranging from about 3 to about 9; g is an integer ranging from about 2 to about 5; h is an integer ranging from about 0 to about 5; and the ratio of cations in the polycationic polymer to anions in the polyanionic oligonucleotide ranges from about 8:10 to about 16:8, respectively.

2. The paired-ion oligonucleotide according to claim 1, wherein the oligonucleotide is an antisense oligonucleotide.

3. The paired-ion oligonucleotide according to claim 1, wherein X is -NH-.

4. The paired-ion oligonucleotide according to claim 1, wherein R-^ has from 2 to 3 carbon atoms in the chain.

5. The paired-ion oligonucleotide according to claim 1, wherein R₂ is selected from the group consisting of cross-linking agents and chemotherapeutic agents.

6. The paired-ion oligonucleotide according to claim 1, wherein R₃ is selected from the group consisting of -NRR', cross-linking agents, and chemotherapeutic agents.

7. The paired-ion oligonucleotide according to claim 1, wherein R₄ has from 2 to 4 carbon atoms in the chain.

8. The paired-ion oligonucleotide according to claim 1, wherein group R5 is selected from the group consisting of primary amines and secondary amines.

9. The paired-ion oligonucleotide according to claim 1, wherein group Rg has 1 carbon atom.

10. The paired-ion oligonucleotide according to claim 1, wherein group R₇ is selected from the group consisting of hydrogen and branched and unbranched lower- alkyl groups having from 1 to 5 carbon atoms.

11. The paired-ion oligonucleotide according to claim 1, wherein the cross-linking agent comprises an aminolink agent, a succinate cross-linking agent, cysteine, and maleimide.

12. The paired-ion oligonucleotide according to claim 1, wherein the chemotherapeutic agent is selected from the group consisting of intercalators, cell targeting agents, transmembrane delivery agents, natural and synthetic nucleases, and free radical generators.

13. The paired-ion oligonucleotide according to claim 1, wherein both R₂ and R₃ are cross-linking agents.

14. The paired-ion oligonucleotide according to claim 1, wherein a is an integer ranging from about 10 to about 14, b is an integer ranging from about 2 to about 4, c is an integer ranging from about 1 to about 3, d is an integer ranging from about 2 to about 4, e is an integer ranging from about l to about 3, f is an integer ranging from about 4 to about 7, g is an integer -ranging from about 2 to about 4, and h is an integer ranging from about 1 to about 3.

15. The paired-ion oligonucleotide according to claim 1, wherein the ratio of cations in the polycationic polymer to anions in the polyanionic oligonucleotide ranges from about 9:10 to about 10:9, respectively.

16. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the paired-ion oligonucleotide according to claim 1.

17. A method for preparing a paired-ion oligonucleotide comprising a polyanionic oligonucleotide covalently bonded to a polycationic polymer via a cross- linking agent, wherein the polycationic polymer is represented by the formula:

R₂-[XR₁CHCO]_a-R₃ (I)

NH₂ or the formula:

R₂-[XR₆COXCHCO]_a-R₃ (II)

R₇ R₄-R5 or the formula:

R₂-[XCHCOXR₆CO]_a-R₃ (III)

R5-R4 R₇ or the formula:

R₂-[[NH(CH₂)_b]_c[NH(CH₂)_d]_e]_f[NH(CH₂)_g]_h-NHR₂ (IV)

wherein X is selected from the group consisting of -NH-, -0-, and -S-; R_x is a substituted or unsubstituted branched or unbranched lower-alkyl chain having from 1 to 4 carbon atoms in the chain; R₂ is independently selected from the group consisting of hydrogen, acyl . groups, cross-linking agents covalently linking the polycationic polymer to the oligonucleotide, and chemotherapeutic agents; R₃ is selected from the group consisting of -NRR' , -OR' ' , cross-linking agents covalently linking the polycationic polymer to the oligonucleotide, and chemotherapeutic agents; R₄ is a substituted or unsubstituted branched or unbranched lower-alkyl chain having from 1 to 5 carbon atoms in the chain; R5 is selected from the group consisting of primary amines, secondary amines, tertiary amines, quaternary ammonium salts, and guanidine groups; Rg is an alkyl chain having from 1 to 2 carbon atoms; R₇ is selected from the group consisting of hydrogen, branched and unbranched lower- alkyl groups having from 1 to 5 carbon atoms, -CH₂C₆H5, and -(CH₂)_zCOR₃, wherein z is an integer ranging from 0 to 3 and R is as defined above; R and R' may be the same or different and are independently selected from the group consisting of hydrogen and branched and unbranched lower-alkyl groups, and R" is selected from the group consisting of branched and unbranched lower-alkyl groups, each lower-alkyl group having from 1 to 6 carbon atoms; wherein at least one of R₂ or R₃ is a cross-linking agent; a is an integer ranging from about 6 to about 18; b is an integer ranging from about 2 to about 5; c is an integer ranging from about 0 to about 3; d is an integer ranging from about 2 to about 5; e is an integer ranging from about 0 to about 5; f is an integer ranging from about 3 to about 9; g is an integer ranging from about 2 to about 5; h is an integer ranging from about 0 to about 5; and the ratio of cations in the polycationic polymer to anions in the polyanionic oligonucleotide ranges from about 8:10 to about 16:8, respectively; which comprises the steps of:

(1) providing the polyanionic oligonucleotide;

(2) providing the polycationic polymer;