WO1996031600A1

WO1996031600A1 - Method of modulating gene expression without depleting complement

Info

Publication number: WO1996031600A1
Application number: PCT/US1996/004327
Authority: WO
Inventors: Sudhir Agrawal; Denise R. Shaw
Original assignee: Hybridon, Inc.
Priority date: 1995-04-03
Filing date: 1996-03-29
Publication date: 1996-10-10
Also published as: AU5325696A

Abstract

Disclosed is a method of modulating gene expression in an animal without depleting complement. The method comprises the step of administering a therapeutic formulation comprising an oligonucleotide complementary to the gene in a pharmaceutically acceptable carrier, the oligonucleotide being hybrid and/or chimeric.

Description

METHOD OF MODULATING GENE EXPRESSION WITHOUT DEPLETING COMPLEMENT

BACKGROUND OF THE INVENTION

The present invention relates to the control of gene expression. More particularly, this invention relates to the use of synthetic oligonucleotides to modulate the expression of a gene in an animal .

The potential for the development of an antisense oligonucleotide therapeutic approach was first suggested in three articles published in 1977 and 1978. Paterson et al . (Proc. Natl. Acad. Sci. (USA) (1977) 74:4370-4374) discloses that cell-free translation of mRNA can be inhibited by the binding of an oligonucleotide complementary to the mRNA. Stephenson et al. (Proc. Natl. Acad. Sci. (USA)

(1978) 75:285-288) discloses that a 13mer synthetic oligonucleotide that is complementary to a part of the Rous sarcoma virus (RSV) genome inhibits RSV replication in infected chicken fibroblasts and inhibits RSV-mediated transformation of primary chick fibroblasts into malignant sarcoma cells.

These early indications that synthetic oligonucleotides can be used to inhibit virus propagation and neoplasia have been followed by the use of synthetic oligonucleotides to inhibit a wide variety of viruses, such as HIV (see, e.g., U.S. Patent No. 4,806,463) ; influenza (see, e.g., Leiter et al. (1990) (Proc. Natl. Acad. Sci. (USA)

87:3430-3434); vesicular stomatitis virus (see, e.g., Agris et al. (1986) Biochem. 25:6268-6275); herpes simplex (see, e.g., Gao et al. (1990) Antimicrob. Agents Chem. 34:808-812) ,^• SV40 (see, e.g., Birg et al. (1990) (Nucleic Acids Res. 18:2901-2908); and human papilloma virus (see, e.g., Storey et al. (1991) (Nucleic Acids Res. 19:4109-4114). The use of synthetic oligonucleotides and their analogs as antiviral agents has recently been extensively reviewed by Agrawal (Trends in Biotech. (1992) 10:152- 158) .

In addition, synthetic oligonucleotides have been used to inhibit a variety of non-viral pathogens, as well as to selectively inhibit the expression of certain cellular genes. Thus, the utility of synthetic oligonucleotides as agents to inhibit virus propagation, propagation of non- viral, pathogens and selective expression of cellular genes has been well established.

Improved oligonucleotides have more recently been developed that have greater efficacy in inhibiting such viruses, pathogens and selective gene expression. Some of these oligonucleotides having modifications in their internucleotide linkages have been shown to be more effective than their unmodified counterparts. For example, Agrawal et al. (Proc. Natl. Acad. Sci. (USA) (1988) 85:7079-7083) teaches that oligonucleotide phosphorothioates and certain oligonucleotide phosphoramidates are more effective at inhibiting HIV-l than conventional phosphodiester-linked oligodeoxynucleotides . Agrawal et al . (Proc. Natl. Acad. Sci. (USA) (1989) 86:7790-7794) discloses the advantage of oligonucleotide phosphorothioates in inhibiting HIV-l in early and chronically infected cells .

In addition, chimeric oligonucleotides having more than one type of internucleotide linkage within the oligonucleotide have been developed. Pederson et al . (U.S. Patent Nos. 5,149,797 and 5,220,007 discloses chimeric oligonucleotides having an oligonucleotide phosphodiester or oligonucleotide phosphorothioate core sequence flanked by nucleotide methylphosphonates or phosphoramidates. Furdon et al . (Nucleic Acids Res.

(1989) 17:9193-9204) discloses chimeric oligonucleotides having regions of oligonucleotide phosphodiesters in addition to either oligonucleotide phosphorothioate or methylphosphonate regions. Quartin et al . (Nucleic Acids Res. (1989) 17:7523-7562) discloses chimeric oligonucleotides having regions of oligonucleotide phosphodiesters and oligonucleotide methylphosphonates. Inoue et al . (FEBS Lett. (1987)

215:327-330) discloses chimeric oligonucleotides having regions of deoxyribonucleotides and 2'-0- ethyl-ribonucleotides .

Hybrid oligonucleotides having both deoxyribonucleotides and ribonucleotides have been developed. For example, Metelev et al . (International Publication No. WO 94/02498) disclose hybrid oligonucleotides having both deoxyribonucleotides and ribonucleotides or 21- substituted ribonucleotides which have superior properties of duplex formation with RNA, nuclease resistance, and the ability to activate RNase H.

Many of these modified oligonucleotides have contributed to improving the potential efficacy of the antisense oligonucleotide therapeutic approach. However, certain deficiencies remain in the known oligonucleotides, and these deficiencies can limit the effectiveness of such oligonucleotides as therapeutic agents. For example, Wickstrom [J. Biochem. Biophys. Meth. (1986) 13:97-102) teaches that oligonucleotide phosphodiesters are susceptible to nuclease- mediated degradation, thereby limiting their bioavailability in vivo . Agrawal et al. (Proc. Natl. Acad. Sci. (USA) (1990) 87:1401-1405) teaches that oligonucleotide phosphoramidates or methylphosphonates when hybridized to RNA do not activate RNase H, the activation of which can be important to the function of antisense oligonucleotides. Thus, a need for methods of controlling gene expression exists which uses oligonucleotides with improved therapeutic characteristics.

Several reports have been published on the development of phosphorothioate-linked oligonucleotides as potential anti-AIDS therapeutic agents. Although extensive studies on chemical and molecular mechanisms of oligonucleotides have demonstrated the potential value of this novel therapeutic strategy, little is known about the pharmacokinetics and metabolism of these compounds in vivo .

Recently, several preliminary studies on this topic have been published. Agrawal et al. (Proc. Natl. Acad. Sci. (USA) (1991) 88:7595-7599) describes the intravenously and intraperitoneally administration to mice of a 20mer phosphorothioate linked-oligonucleotide. In this study, approximately 30% of the administered dose was excreted in the urine over the first 24 hours with accumulation preferentially in the liver and kidney. Plasma half-lives ranged from about 1 hour t_12a) and 40 hours (t_{1 2β}) , respectively.

Similar results have been reported in subsequent studies (Iversen (1991) Anti-Cancer Drug Design 6:531-

538; Iversen (1994) Antisense Res. Devel. 4:43-52; and

Sands (1994) Mol. Pharm. 45:932-943) .

However, there remains a need to develop more effective therapeutic methods of modulating the expression of genes which can be easily manipulated to fit the animal and condition to be treated, and the gene to be targeted. Preferably, these methods should be simple and precise in effecting the target gene.

SUMMARY OF THE INVENTION

The present inventors have discovered that oligonucleotides that are chimeric and/or hybrid can modulate the expression of a gene to which the nucleic acid sequence oligonucleotide is complementary without depleting complement, and therefore without causing any complications which lack of complement causes in an animal.

For purposes of the invention, the term "oligonucleotide sequence that is complementary to a nucleic acid sequence" is intended to mean an oligonucleotide sequence (6 to about 50 nucleotides) that binds to the nucleic acid sequence under physiological conditions, e.g., by Watson-Crick base pairing (interaction between oligonucleotide and single-stranded nucleic acid) or by Hoogsteen base pairing (interaction between oligonucleotide and double-stranded nucleic acid) or by any other means including in the case of a oligonucleotide binding to RNA, pseudoknot formation. Such binding (by Watson-Crick base pairing) under physiological conditions is measured as a practical matter by observing interference with the function of the nucleic acid sequence.

In a first aspect of the invention, a method of modulating gene expression in an animal without depleting complement is provided. In this method, a therapeutic formulation is administered which comprises an oligonucleotide complementary to the gene in a pharmaceutically acceptable carrier, the oligonucleotide being hybrid and/or chimeric.

For purposes of the invention, the term "animal" is meant to encompass humans as well as - 7 - other mammals, as well as reptiles, amphibians, and insects.

In preferred embodiments, the oligonucleotide is a hybrid oligonucleotide, while in other preferred embodiments the oligonucleotide is chimeric. In some embodiments, the oligonucleotide is chimeric and hybrid.

In some embodiments, the oligonucleotide has at least one 2' -substituted ribonucleotide such as a 2' -O-alkyl-substituted ribonucleotide. In one particular embodiment, the oligonucleotide comprises at least one 2' -O-methyl-substituted ribonucleotide.

For purposes of the invention, the term "2'- substituted oligonucleotide" refers to an oligonucleotide having a sugar attached to a chemical group other that a hydroxyl group at its 2' position. The 2' -OH of the ribose molecule can be substituted with -O-lower alkyl containing 1-6 carbon atoms, aryl or substituted aryl or allyl having 2-6 carbon atoms, e.g., 2'-0-allyl, 2'-0- aryl, 2'-0-alkyl (such as a 2' -O-methyl) , 2'- halo, or 2' -amino, but not with 2'-H, wherein allyl, aryl, or alkyl groups may be unsubstituted or substituted, e.g., with halo, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl, carbalkoxyl or amino groups.

In some embodiments, the hybrid and/or chimeric oligonucleotide is modified, and in some particular embodiments, the oligonucleotide comprises at least one phosphorothioate internucleotide linkage.

In another aspect of the invention, a method of modulating gene expression in an animal without inhibiting normal blood clotting in the animal is provided. In this method, a therapeutic formulation is administered which comprises an oligonucleotide complementary to the gene in a pharmaceutically acceptable carrier, the oligonucleotide being hybrid and/or chimeric.

In some embodiments, the hybrid and/or chimeric oligonucleotide is modified, and in some particular embodiments, the oligonucleotide comprises at least one phosphorothioate internucleotide linkage. BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects of the present invention, the various features thereof, as well as the invention itself may be more fully understood from the following description, when read together with the accompanying drawings in which:

FIG. 1A is a graphic representation showing hemolysis in serum in the presence of different concentrations of oligonucleotide 1 (SEQ ID N0:1) in the presence or absence of protamine;

FIG. IB is a graphic representation showing hemolysis in serum in the presence of different concentrations of oligonucleotide 1 (SEQ ID N0:1) in the presence or absence of C4d fragment generation protamine;

FIG. 1C is a graphic representation showing hemolysis in serum in the presence of different concentrations of oligonucleotide 1 (SEQ ID N0:1) in the presence or absence of Bb fragment generation protamine;

FIG. 2 is a graphic representation of the anticoagulation activity of various oligonucleotides used in the method of the invention; FIG. 3 is a graphic representation of the CH50 percent of baseline at varying concentrations of oligonucleotides used in the method of the invention;

FIG. 4 is a graphic representation of the effect of different concentrations of oligonucleotides used in the method of the invention on the proliferation of murine splenic lymphocytes in vitro .

FIG. 5 is a graphic representation showing the mutual neutralization of anti-coagulation activities by different concentrations of oligonucleotide 1 and different concentrations of protamine.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. The issued U.S. patent and allowed applications cited herein are hereby incorporated by reference.

The present invention provides methods of modulating or regulating gene expression in an animal without depleting complement or without inhibiting normal blood clotting in the animal.

This method is also a means of examining the function of various genes in an animal, including those essential to animal development. Presently, gene function can only be examined by the arduous task of making a "knock out" animal such as a mouse. This task is difficult, time-consuming and cannot be accomplished for genes essential to animal development since the "knock out" would produce a lethal phenotype. The present invention overcomes the shortcomings of this model.

The oligonucleotide administered is complementary to a gene of a virus, pathogenic organism, or a cellular gene in some embodiments of the invention. In some embodiments, the oligonucleotide is complementary to a gene of a virus involved in AIDS, oral or genital herpes, papilloma warts, influenza, foot and mouth disease, yellow fever, chicken pox, shingles, adult T-cell leukemia, Burkitt's lymphoma, nasopharyngeal carcinoma, or hepatitis. In one particular embodiment, the oligonucleotide is complementary to an HIV gene and includes about 15 to 26 nucleotides linked by phosphorothioate internucleotide linkages, at least one of the nucleotides at the 3' terminus being a 2'- substituted ribonucleotide, and at least four contiguous deoxyribonucleotides. In another embodiment, the oligonucleotide is complementary to a gene encoding a protein in associated with Alzheimer's disease. In yet other embodiments, the oligonucleotide is complementary to a gene encoding a protein expressed in a parasite that causes a parasitic disease such as amebiasis, Chagas' disease, toxoplasmosis, pneumocytosis, giardiasis, cryptoporidiosis, trichomoniasis, malaria, ascariasis, filariasis, trichinosis, or schistosomiasis infections.

The term "hybrid oligonucleotides" is used herein to describe a molecule comprising at least six 5' to 3' -linked nucleotides wherein both deoxyribonucleotides and ribonucleotides are present. At least one deoxyribonucleotide or at least one ribonucleotide is present in a hybrid oligonucleotide. Hybrid oligonucleotides are described in International Publication No. WO 94/02498, herein incorporated by reference.

Oligonucleotides useful in the method of the invention may also be chimeric oligonucleotides. As used herein, the term "chimeric oligonucleotides" refers to oligonucleotides having more than one type of internucleotide linkage. For example, chimeric oligonucleotides of the invention may contain a phosphodiester internucleotide linkage and at least one nonphosphodiester linkage. Chimeric oligonucleotides are described in U.S. Patent No. 5,149,797 herein incorporated by reference.

The term "non-phosphodiester-linked oligonucleotide" as used herein is an oligonucleotide in which all of its nucleotides are covalently linked via a synthetic linkage, i.e., a linkage other than a phosphodiester between the 5' end of one nucleotide and the 3' end of another nucleotide in which the 5' nucleotide phosphate has been replaced with any number of chemical groups. Preferable synthetic linkages include alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, phosphora idates, phosphoramidites, phosphate esters, carbamates, carbonates, phosphate triesters, acetamidate, and carboxymethyl esters. In one preferred embodiment of the invention, the all of the nucleotides of the oligonucleotide comprises are linked via phosphorothioate and/or phosphorodithioate linkages.

In some embodiments of the invention, the oligonucleotides administered are modified. As used herein, the term "modified oligonucleotide" encompasses oligonucleotides with modified nucleic acid(s), base(s), and/or sugar(s) other than those found in nature. For example, a 3', 5'- substituted oligonucleotide is an oligonucleotide having a sugar which, at both its 3' and 5' -_Im¬ positions is attached to a chemical group other than a hydroxyl group (at its 3' position) and other than a phosphate group (at its 5' position) .

A modified oligonucleotide may also be one with added substituents such as diamines, cholestryl, or other lipophilic groups, or a capped species. In addition, unoxidized or partially oxidized oligonucleotides having a substitution in one nonbridging oxygen per nucleotide in the molecule are also considered to be modified oligonucleotides. Also considered as modified oligonucleotides are oligonucleotides having nuclease resistance-conferring bulky substituents at their 3' and/or 5' end(s) and/or various other structural modifications not found in vivo without human intervention are also considered herein as modified.

It is known that an oligonucleotide, called an "antisense oligonucleotide," can bind to a target single-stranded nucleic acid molecule according to the Watson-Crick or the Hoogsteen rule of base pairing, and in doing so, disrupt the function of the target by one of several mechanisms: by preventing the binding of factors required for normal transcription, splicing, or translation; by triggering the enzymatic destruction of mRNA by RNase H if a contiguous region of deoxyribonucleotides exists in the oligonucleotide, and/or by destroying the target via reactive groups attached directly to the antisense oligonucleotide. Thus, because of the properties described above, such oligonucleotides are useful therapeutically by their ability to control or down-regulate the expression of a particular gene in an animal, according to the method of the present invention.

The oligonucleotides useful in the method of the invention are at least 6 nucleotides in length, but are preferably 6 to 50 nucleotides long, with 15 to 30mers being the most common. They are composed of deoxyribonucleotides, ribonucleotides, or a combination of both, with the 5' end of one nucleotide and the 3' end of another nucleotide being covalently linked by non- phosphodiester internucleotide linkages. Such linkages include alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters. Oligonucleotides with these linkages can be prepared according to known methods such as phosphoramidate or H-phosphonate chemistry which can be carried out manually or by an automated synthesizer as described by Brown (A Brief History of Oligonucleotide Synthesis. Protocols for Oligonucleotides and Analogs, Methods in Molecular Biology (1994) 20:1-8) . (See also, e.g., Sonveaux "Protecting Groups in Oligonucleotides Synthesis" in Agrawal (1994) Methods in Molecular Biology 26:1-72; Uhlmann et al. (1990) Chem. Rev. 90:543-583) . The oligonucleotides of the composition may also be modified in a number of ways without compromising their ability to hybridize to the target nucleic acid. Such modifications include, for example, those which are internal or at the end(s) of the oligonucleotide molecule and include additions to the molecule of the internucleoside phosphate linkages, such as cholesteryl or diamine compounds with varying numbers of carbon residues between the amino groups and terminal ribose, deoxyribose and phosphate modifications which cleave, or crosslink to the opposite chains or to associated enzymes or other proteins which bind to the viral genome. Examples of such modified oligonucleotides include oligonucleotides with a modified base and/or sugar such as arabinose instead of ribose, or a 3' , 5' -substituted oligonucleotide having a sugar which, at both its 3' and 5' positions is attached to a chemical group other than a hydroxyl group (at its 3' position) and other than a phosphate group (at its 5' position) . Other modified oligonucleotides are capped with a nuclease resistance-conferring bulky substituent at their 3' and/or 5' end(s) , or have a substitution in one nonbridging oxygen per nucleotide. Such modifications can be at some or all of the internucleoside linkages, as well as at either or both ends of the oligonucleotide and/or in the interior of the molecule. For the preparation of such modified oligonucleotides, see, e.g., Agrawal (1994) Methods in Molecular Biology 26; Uhlmann et al . (1990) Chem. Rev. 90:543-583) . The preparation of these unmodified and modified oligonucleotides is well known in the art (reviewed in Agrawal et al. (1992) Trends Biotechnol. 10:152-158) (see, e.g., Uhlmann et al . (1990) Chem. Rev. 90:543-584; and (1987) Tetrahedron. Lett. 28: (31) :3539- 3542) ; Agrawal (1994) Methods in Molecular Biology 20:63- 80) .

These oligonucleotides are provided with additional stability by having non-phosphodiester internucleotide linkages such as alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, phosphoramidates, phosphoramidites, phosphate esters, carbamates, carbonates, phosphate triesters, acetamidate, and carboxymethyl esters. Particularly useful oligonucleotides are linked with phosphorothioate and/or phosphorodithioate internucleoside linkages.

The oligonucleotides administered to the animal may be hybrid oligonucleotides in that they contain both deoxyribonucleotides and at least one 2' substituted ribonucleotide. For purposes of the invention, the term "2' -substituted" means substitution of the 2' -OH of the ribose molecule with, e.g., 2'-0-allyl, 2'-0-alkyl, 2' -halo, or 2'-amino, but not with 2'-H, wherein allyl, aryl, or alkyl groups may be unsubstituted or substituted, e.g., with halo, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl, carbalkoxyl or amino groups. The hybrid DNA/RNA oligonucleotides useful in the method of the invention resist nucleolytic degradation, form stable duplexes with RNA or DNA, and preferably activate RNase H when hybridized with RNA. They may additionally include at least one unsubstituted ribonucleotide. For example, an oligonucleotide useful in the method of the invention may contain all deoxyribonucleotides with the exception of one 2' substituted ribonucleotide at the 3' terminus of the oligonucleotide. Alternatively, the oligonucleotide may have at least one substituted ribonucleotide at both its 3' and 5' termini.

The 2' substituted ribonucleotide (s) in the oligonucleotide may contain at the 2' position of the ribose, a -0-lower alkyl containing 1-6 carbon atoms, aryl or substituted aryl or allyl having 2- 6 carbon atoms e.g., 2'-0-allyl, 2'-0-aryl, 2'-0- alkyl, 2' -halo, or 2' -amino, but not with 2'-H, wherein allyl, aryl, or alkyl groups may be unsubstituted or substituted, e.g., with halo, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl, carbalkoxyl or amino groups. Useful substituted ribonucleotides are 2'-0-alkyls such as 2' -0-methyl.

Preferably, oligonucleotides useful in the method of the invention will range from about 2 to about 50 nucleotides in length, and most preferably from about 15 to about 25 nucleotides in length. Thus, in this preferred case, oligonucleotides according to the invention will have from 14 to 24 non-phosphodiester internucleotide linkages.

The oligonucleotides according to the invention are effective in inhibiting the expression of various genes in viruses, pathogenic organisms, or in inhibiting the expression of cellular genes. The ability to inhibit such agents is clearly important to the treatment of a variety of disease states. Thus, oligonucleotides according to the method of the invention have a nucleotide sequence which is complementary to a nucleic acid sequence that is from a virus, a pathogenic organism or a cellular gene. Preferably such oligonucleotides are from about 6 to about 50 nucleotides in length.

The nucleic acid sequence to which an oligonucleotide according to the invention is complementary will vary, depending upon the gene to be down-regulated. In some cases, the target gene or nucleic acid sequence will be a virus nucleic acid sequence. The use of antisense oligonucleotides to inhibit various viruses is well known (reviewed in Agrawal (1992) Trends in

Biotech. 10:152-158) . Viral nucleic acid sequences that are complementary to effective antisense oligonucleotides have been described for many viruses, including human immunodeficiency virus type 1 (HIV-l) (U.S. Patent No. 4,806,463) , herpes simplex virus (U.S. Patent No. 4,689,320) , influenza virus (U.S. Patent No. 5,194,428) , and human papilloma virus (Storey et al . (1991) Nucleic Acids Res. 19:4109-4114 ) . Sequences complementary to any of these nucleic acid sequences can be used for oligonucleotides according to the invention, as can be oligonucleotide sequences complementary to nucleic acid sequences from any other virus . Additional viruses that have known nucleic acid sequences against which antisense oligonucleotides can be prepared include, but are not limited to, foot and mouth disease virus (see, Robertson et al. (1985) J. Virol. 54:651; Harris et al . (1980) Virol. 36:659) , yellow fever virus (see Rice et al . (1985) Science 229:726) , varicella-zoster virus

(see, Davison and Scott (1986) J. Gen. Virol.

67:2279) , Epstein-Barr virus, cytomegalovirus, respiratory syncytial virus (RSV) , and cucumber mosaic virus (see Richards et al. (1978) Virol. 89:395) .

For example, an oligonucleotide has been designed which is complementary to a portion of the HIV-l gag gene (oligonucleotide having SEQ ID N0:1) , and as such, has significant anti-HIV effects (Agrawal (1992) Antisense Res. Development

2:261-266) . The target of this oligonucleotide has been found to be conserved among various HIV-l isolates. It is 56% G + C rich, water soluble, and relatively stable under physiological conditions. This oligonucleotide binds to a complementary RNA target under physiological conditions, with the Tm of the duplex approximately being 56°C. The antiviral activity of this oligonucleotide has been tested in several models, including acutely and chronically infected CEM cells, long-term cultures mimicking in vivo conditions, human peripheral blood lymphocytes and macrophages, and isolates from HIV-l infected patients (Lisziewicz et al . (Proc. Natl. Acad. Sci. (USA) (1992) 89:11209-11213); Lisziewicz et al. (Proc. Natl. Acad. Sci. (USA) (1993) 90:3860-3864); Lisziewicz et al. (Proc. Natl. Acad. Sci. (USA) (1994) 91:7942-7946) ; Agrawal et al. (J. Ther. Biotech) in press) .

The oligonucleotides according to the invention alternatively can have an oligonucleotide sequence complementary to a nucleic acid sequence of a pathogenic organism. The nucleic acid sequences of many pathogenic organisms have been described, including the malaria organism, Plasmodium falciparum, and many pathogenic bacteria. Oligonucleotide sequences complementary to nucleic acid sequences from any such pathogenic organism can be used in oligonucleotides according to the invention. Examples of pathogenic eucaryotes having known nucleic acid sequences against which antisense oligonucleotides can be prepared include Trypanosom abrucei gambiense and Leishmania (See Campbell et al . ,

Nature 311:350 (1984)), Fasciola hepatica (See Zurita et al., Proc. Natl. Acad. Sci. USA 84:2340 (1987) .

Antifungal oligonucleotides can be prepared using a target hybridizing region having an oligonucleotide sequence that is complementary to a nucleic acid sequence from, e.g., the chitin synthetase gene, and antibacterial oligonucleotides can be prepared using, e.g., the alanine racemase gene. Among fungal diseases that may be treatable by the method of treatment according to the invention are candidiasis, histoplasmosis, cryptococcocis, blastomycosis, aspergillosis, sporotrichosis, chromomycosis, dermatophytosis, and coccidioidomycosis. The method might also be used to treat rickettsial diseases (e.g., typhus, Rocky Mountain spotted fever) , as well as sexually transmitted diseases caused by Chlamydia trachomatis or Lymphogranuloma venereum . A variety of parasitic diseases may be treated by the method according to the invention, including amebiasis, Chagas' disease, toxoplasmosis, pneumocystosis, giardiasis, cryptosporidiosis, trichomoniasis, and Pneumocystis carini pneumonia; also worm (helminthic) diseases such as ascariasis, filariasis, trichinosis, schistosomiasis and nematode or cestode infections. Malaria may be treated by the method of treatment of the invention regardless of whether it is caused by P. falcip arum, P. vivas, P. orale, or P. malaήae .

The infectious diseases identified above may all be treated by the method of treatment according to the invention because the infectious agents for these diseases are known and thus oligonucleotides according to the invention can be prepared, having oligonucleotide sequence that is complementary to a nucleic acid sequence that is an essential nucleic acid sequence for the propagation of the infectious agent, such as an essential gene. Other disease states or conditions that may be treatable by the method according to the invention are those which result from an abnormal expression or product of a cellular gene. These conditions may be treated by administration of oligonucleotides according to the invention, and have been discussed earlier in this disclosure.

Other oligonucleotides according to the invention can have a nucleotide sequence complementary to a cellular gene or gene transcript, the abnormal expression or product of which results in a disease state. The nucleic acid sequences of several such cellular genes have been described, including prion protein (Stahl et al. (1991) FASEB J. 5:2799-2807), the amyloid-like protein associated with Alzheimer's disease (U.S. Patent No. 5,015,570), and various well-known oncogenes and proto-oncogenes, such as c-myb, c- myc, c-abl, and n-ras . In addition, oligonucleotides that inhibit the synthesis of structural proteins or enzymes involved largely or exclusively in spermatogenesis, sperm motility, the binding of the sperm to the egg or any other step affecting sperm viability may be used as contraceptives. Similarly, contraceptives for women may be oligonucleotides that inhibit proteins or enzymes involved in ovulation, fertilization, implantation or in the biosynthesis of hormones involved in those processes.

Hypertension may be controlled by oligonucleotides that down-regulate the synthesis of angiotensin converting enzyme or related enzymes in the renin/angiotensin system. Platelet aggregation may be controlled by suppression of the synthesis of enzymes necessary for the synthesis of thromboxane A2 for use in myocardial and cerebral circulatory disorders, infarcts, arteriosclerosis, embolism and thrombosis. Deposition of cholesterol in arterial wall may be inhibited by suppression of the synthesis of fatty acid co-enzyme A: cholesterol acyl transferase in arteriosclerosis. Inhibition of the synthesis of cholinephosphotransferase may be useful in hypolipidemia.

There are numerous neural disorders in which hybridization arrest may be used to reduce or eliminate adverse effects of the disorder. For example, suppression of the synthesis of monoamine oxidase may be used in Parkinson's disease. Suppression of catechol o-methyl transferase may be used to treat depression; and suppression of indole N-methyl transferase may be used in treating schizophrenia.

Suppression of selected enzymes in the arachidonic acid cascade which leads to prostaglandins and leukotrienes may be useful in the control of platelet aggregation, allergy, inflammation, pain and asthma.

Suppression of the protein expressed by the multidrug resistance (mdr- 1 ) gene, which can be responsible for development of resistance of tumors to a variety of anti-cancer drugs and is a major impediment in chemotherapy may prove to be beneficial in the treatment of cancer. Oligonucleotide sequences complementary to nucleic acid sequences from any of these genes can be used for oligonucleotides according to the invention, as can be oligonucleotide sequences complementary to any other cellular gene transcript, the abnormal expression or product of which results in a disease state.

The oligonucleotides described herein are administered orally or enterally to the animal subject in the form of therapeutic pharmaceutical formulations that are effective for treating virus infection, infections by pathogenic organisms, or disease resulting from abnormal gene expression or from the expression of an abnormal gene product. In some aspects or the method according to the invention, the oligonucleotides are administered in conjunction with other therapeutic agents, e.g., AZT in the case of AIDS.

The therapeutic pharmaceutical formulation containing the oligonucleotide includes a physiologically acceptable carrier, such as an inert diluent or an assimilable edible carrier with which the oligonucleotide is administered. Suitable formulations that include pharmaceutically acceptable excipients for introducing compounds to the bloodstream by other than injection routes can be found in Remington 's

Pharmaceutical Sciences (181h ed. ) (Genarro, ed. (1990)

Mack Publishing Co., Easton, PA) . The oligonucleotide and other ingredients may be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the individual's diet. The oligonucleotide may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. When the oligonucleotide is administered orally, it may be mixed with other food forms and pharmaceutically acceptable flavor enhancers. When the oligonucleotide is administered enterally, they may be introduced in a solid, semi-solid, suspension, or emulsion form and may be compounded with any number of well- known, pharmaceutically acceptable additives. Sustained release oral delivery systems and/or enteric coatings for orally administered dosage forms are also contemplated such as those described in U.S. Patent Nos. 4,704,295, 4,556,552, 4,309,404, and 4,309,406.

The amount of oligonucleotide in such therapeutically useful compositions is such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit contains between from about 50 micrograms to about 200 mg per kg body weight of the animal, with 10 mg to 100 mg per kg being most preferable.

It will be appreciated that the unit content of active ingredient or ingredients contained in an individual dose of each dosage form need not in itself constitute an effective amount since the necessary effective amount can be reached by administration of a plurality of dosage units (such as capsules or tablets or combinations thereof) .

Complement Activation by Experimental Oligonucleotides (plus effects of Oligo 1 on Coagulation)

10 Oligo 1 inhibits hemolytic complement activity when normal serum samples from either humans, Rhesus monkeys or guinea pigs are spiked in vitro with Oligo 1, in a dose-dependent manner (Tables 9 and 10) .

15

TABLE 9

Hemolytic complement after in vitro treatment of human and monkey serums

Human Serum Rhesus Monkey Serum

CH50 CH10 CH50 CH10 classical alternative classical alternative

Control 352 (1.00) 118 (1.00) 173 (1.00) 130 (1.00)

Oligo 1, 177 (0.50) 51 (0.43) 130 (0.75) 73 (0.56) 500 μg/ml

DNA, 500 314 (0.89) 109 (0.92) 175 (1.01) 127 (0.98) μg/ml - 28 - TABLE 10

Oligo 1 Treatment of Guinea Pig Serum

hemolytic complement

Oligo 1, μg/ml CH50

1,000 570 (0.47)

500 775 (0.64)

250 825 (0.68)

125 1,052 (0.87)

0 1,213 (1.00)

Incubation of the oligo 1-treated sera at 37°C prior to hemolytic assay appears to increase the degree of inhibition subsequently measured (Table 11) -

TABLE 11

Effect of Adding Oligo 1 to E Hemolysis Incubation

Oligo 1 μg/ml Lysis cone. in equivalent lysis inc. serum cone. Classical Alternative

Oligo 1 in lysis inc only:

0 0 70.0 (1.00) 9.50 (1.00)

1.25 250 36.7 (0.52) 5.47 (0.58)

2.5 500 21.6 (0.31) 4.67 (0.49)

5 1000 9.9 (0.14) 2.13 (0.22)

10 2000 4.5 (0.06) 0.92 (0.10)

Pretreatment samples

0 0 55.8 (1.00) 2.64 (1.00)

1.8 357 4.4 (0.08) 0.49 (0.19)

Hemolytic complement is also inhibited when oligo 1 is present in blood during clotting to prepare serum (Table 12) .

TABLE 12

Hemolytic complement and PFl .2 after in vitro clotting of human blood with 400 μg/ml Oligo 1

Human A-neg blood Human 0-pos blood

CH50 CH10 PFl.2 CH50 CH10 PFl .2 classical alternative (nm) classical alternative (nm)

Control 354 (1.00) 111 (1.00) >60.0 305 (1.00) 98 (1.00) >60.0 Oligo 1 184 (0.52) 81 (0.73) 14.5 128 (0.42) 69 (0.70) 10.3

Oligo 1 inhibits hemolytic complement assays with both classical and alternative pathway targets (Tables 9 and 11) .

By ELISA quantitation of complement pathway by-products in treated serum, oligo 1 appears to act primarily by activation of the classical pathway (e.g., C4d generation) as compared to the alternative pathway (e.g., Bb generation) (Table 13 and Fig. 4) . However, it is possible that oligo 1 activates both pathways of complement.

TABLE 13

ELISA Determination of C4d and Bd fragments after in vitro Treatment of Human Serum with 400 μg/ml Oligo 1

μg/ml

C4d Bb serum 3.74 3.25 serum + Oligo 1 10.65 3.32 normal serum (1.2 - 8.0) (1.0 - 7.3;

(2 SD range)

Effects on coagulation by oligo 1 treatment of normal donor blood or plasma in vitro .

By visual inspection, the presence of relatively high doses of oligo 1 in vacutainers used to collect peripheral blood for preparation of serum from normal human volunteers (e.g., final concentrations of 250 μg/ml or greater) results in gross inhibition of blood clotting after 1 - 24 hours at room temperature. An apparent total inhibition of blood clotting has been observed with oligo 1 concentrations of 400-500 μg/ml.

In blood samples collected as described above, generation of PFl.2 (plasminogen fragment produced during coagulation) as assayed by the clinical ELISA method is also reduced in the presence of oligo 1 (Table 12 above) . In normal human plasma samples, the clinical clotting time measurements PT, PTT and TCT are inhibited by added oligo 1 in a dose-dependent manner Table 14) . Of the three assays, PTT was consistently prolonged by oligo 1 to the greatest extent (and thus is the most sensitive assay) .

TABLE 14

Clotting Times for normal plasma treated with Oligo 1 and assayed either immediately or after

15 min at 37°C

Oligo 1 PT, sec PTT, sec TCT, sec μg/ml 0 15' 0 15' 0 15'

0 12.4 13.1 23.5 28.6 16.9 18.5

10 12.5 12.6 26.5 27.5 18.5 17.6

50 12.9 13.1 41.7 42.8 20.2 19.0

100 13.9 14.2 57.0 62.7 20.5 19.2

200 15.2 16.3 80.8 88.6 24.6 22.1

500 19.8 23.4 159.8 194.2 58.1 37.5

Effects on serum complement by oligo 1 treatment of guinea pigs in vivo .

Hemolytic complement was analyzed in the serum of guinea pigs administered oligo 1 intravenously. The results are shown in Table 15 below. Within 15 minutes of oligo 1 infusion, serum hemolytic complement was significantly reduced and remained so at 60 minutes post infusion, as directly compared to control guinea pigs receiving infusions of saline alone. Thus, results in guinea pigs were similar to those obtained by others with oligo 1 in monkeys. Guinea pigs may provide a more economical preclinical model for analysis of oligo 1 effects on complement and coagulation in vivo .

TABLE 15 Hemolytic Complement after in vivo Treatment of

Guinea Pigs with Oligo 1 or Saline

CH50 animal Tx -15/20 min + 15 min + 60 min

940303 Oligo 1 688 (1.00) 240 (0.35) 188 (0.27)

940309-1 saline 524 (1.00) 723 (1.38) 824 (1.57)

940309-2 Oligo 1 1210 (1.00) 531 (0.44) 591 (0.49)

940316-1 saline 858 (1.00) 919 (1.07) 785 (0.91) 940316-2 saline 711 (1.00) 841 (1.18) 754 (1.06)

940317 Oligo 1 787 (1.00) 496 (0.63) 496 (0.63)

Reversal of Oligo 1 inhibition of complement and coagulation by protamine in vitro .

Oligo 1 shares several noteworthy properties with the pharmaceutical anti-coagulant heparin: (i) both have a high density of negatively-charged residues due to sulfuration, and (ii) both act as anti-coagulants in whole blood. This led to the hypothesis that the pharmaceutical anti-heparin compound, protamine sulfate, might also exhibit neutralizing activity for the anti-coagulant and anti-complementary effects of oligo 1.

Although heparin and oligo 1 demonstrate similar anti-coagulant activities in vitro, the anti- complement activity of oligo 1 was not mimicked by heparin (Table 16) . Additionally, genomic DNA did not demonstrate significant anti-complement activity when tested in parallel with oligo 1 (Table 9 above) .

TABLE 16

Hemolytic complement and PFl.2 after in vitro clotting with Oligo 1, heparin and protamine

additives during blood clotting

(400 μg/ml) classical C alternative C PFl.2

heparin Oligo 1 protamine CH50 CH10 (nM)

265 (1.00) 103 (1.00) >455

266 (1.00) 87 (0.85) >437 293 (1.10) 92 (0.90) 13.7 258 (0.97) 76 (0.73) >479 116 (0.44) 25 (0.24) 11.9 273 (1.03) 102 (0.99) >466

Nonetheless, protamine effectively antagonized the anti-complement activity of oligo 1 treatment of human serum in vitro (Table 16, and Fig. 4 above) . Protamine was also effective in neutralizing the anti-coagulant properties of oligo 1 in plasma treated in vitro, as measured by either clotting times or PFl.2 generation (FIG. 5 and Table 16) .

The dose ratio for complete neutralization of oligo 1 by protamine in these experiments was estimated to be 0.5 μg protamine per 1.0 μg oligo 1. However, protamine at doses of ≥ 50 μg/ml alone will prolong PTT measurements (FIG. 5) .

Effects of additional oligonucleotides with either different sequences or different backbone chemistry from Oligo 1 on complement.

Initial studies were conducted with the oligo 1 C, H and L compounds. Subsequently, other oligo 1 based and control oligonucleotides were used for testing.

Estimated order of complement inhibition for the initial analogues was Oligo 1 > L (self- hybridized) > H (hybrid) >> C (chimeric) (Tables 17 - 19) .

TABLE 17

Effects of modified Oligo 1 compounds on complement

CH50 values (ratio vs control) blood clotted with serum pre-tx with sample 500 μg/ml 100 μg/ml 500 μg/ml control 394 (1.00) 580 (1.00) 580 (1.00) oligo 1 47 (0.12) 203 (0.35) 186 (0.32) oligo 1-C 235 (0.60) 457 (0.79) 353 (0.61) oligo 1-H 112 (0.28) 357 (0.62) 215 (0.37) oligo 1-L 92 (0.23) 306 (0.53) 182 (0.31)

Protamine exhibited neutralization of the anti-complementary effects of the H and L compounds, but had little effect on compound C (Table 18) .

TABLE 18

Hemolytic complement after treatment of serum with Oligo 1 compounds and protamine

inhibition in serum pre-tx with 100 μg/ml of protamine no H oligo 1 oligo

experiment 1

none 0 60 41 29 62

12.5 μg/ml 6 68 37 28 66

25 μg/ml 8 67 39 26 61

50 μg/ml 17 37 16 6 51

100 μg/ml 13 16 5 30 36

experiment 2

none 0 61 43 12 60

12.5 μg/ml 1 68 40 21 65

25 μg/ml 1 56 35 12 60

50 μg/ml 4 42 23 19 51

100 μg/ml 9 20 0 19 85

In multiple experiments, inhibition of hemolytic complement by a wide variety of oligonucleotides showed internally consistent relative activity (Table 19) which correlates with density of backbone charge. TABLE 19

Summary of multiple complement experiments with different oligonucleotides

% inhibition of C lysis

Oligo

12/13/94 12/6/94 12/1/94 11/17/94 11/17/94 11/11/94 15' 37C 15' 37C 15' 37C 15' 37C 15' 37C 30' clot 100 μg/ml 200 μg/ml 200 μg/ml 100 μg/ml 500 μg/ml 500 μg/ml

Oligo 1-PO 0 0

HYB0067 1 0

Oligo 1-C 29 6 8 21 39 40

GEM-1063 10 9

GEM-1064 13 12

GEM-1062 16 14

M-1062 18 15

M-1064 19 17

Oligo 1-H 41 27 33 38 63 72

HYB0066 49 45

Oligo 1- 60 40 49 47 69 77

HYB0074 42 51

HYB0065 77 55

Oligo 1 62 60 67 65 68 88 (PS)

Oligo 1-PS 56 70

Effects of additional oligonucleotides on coagulation.

The initial oligo 1 derived modified oligonucleotides (C, H, L) demonstrated inhibition of coagulation with potency similar to that observed for inhibition of complement hemolysis.

10 In the coagulation experiments, aliquots of normal human plasma were treated with oligonucleotides at concentrations from 20 to 500 μg/ml and then assayed for PT, PTT and TCT (clotting times) by routine clinical assay. From clotting time prolongation data for each concentration, extrapolation of the concentration that would produce 50% prolongation was derived (Table 20) . These preliminary results indicate that properties of oligonucleotides inhibiting hemolytic complement (e.g., Table 19) may be the same as those inhibiting coagulation.

TABLE 20

Estimated μg/ml cone, of oligos that prolong coag times by 50% (1.5 X control)

Oligo PT aPTT TCT

1 330 18 130

1 (C) >>500 43 265

1 (H) >500 35 235

1 (D >500 18 145

The following examples illustrate the preferred modes of making and practicing the present invention, but are not meant to limit the scope of the invention since alternative methods may be utilized to obtain similar results. EXAMPLES

1. Hemolytic complement assays

Normal donor blood was allowed to clot at room temperature for 30 minutes, cooled on ice for 15 minutes, and then centrifuged at 4°C to recover serum. Aliquots of serum were mixed with test compounds or saline alone for the indicated final concentrations (generally 50 - 500 μg/ml) . Serum aliquots in most experiments were pre-incubated with test compounds at 37°C for 15 minutes, then chilled on ice and diluted 1:50 in ice-cold buffer for performance of standard CH50 assay (Mayer, M.M. (1961) Complement and complement fixation. In: Experimental Immunochemistry, 2nd edition, E.A.

Kabat and M.M. Mayer, editors, p. 125. CC. Thomas, Springfield, IL) . In some experiments a single dilution of serum was used and results presented as "percent lysis" instead of CH50 units.

2. Immunoassays for complement fragments

Residual aliquots of serum treated with test compounds were stored at -70°C until used in quantitative ELISA for determination of C4d, C3a and Bb using kits from Quidel (San Diego, CA) .

3. Coagulation studies

Normal donor blood anti-coagulated with sodium citrate was used to prepare platelet- depleted plasma. Aliquots of plasma were mixed with test compounds and then quickly frozen on dry ice and stored at -70°C. The activated partial thromboplastin time (PTT) , prothrombin time (PT) and thrombin clotting time (TCT) were determined by standard clinical coagulation tests.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of this invention, and are covered by the following claims.

Claims

What is claimed is:

1. A method of modulating gene expression in an animal without depleting complement, comprising the step of administering a therapeutic formulation comprising an oligonucleotide complementary to the gene in a pharmaceutically acceptable carrier, the oligonucleotide being hybrid and/or chimeric.

2. The method of claim 1 wherein the oligonucleotide is a hybrid oligonucleotide.

3. The method of claim 2 wherein the oligonucleotide is modified.

4. The method of claim 3 wherein the oligonucleotide has at least one 2' -substituted ribonucleotide.

5. The method of claim 4 wherein the oligonucleotide comprises at least one 2'-0-alkyl- substituted ribonucleotide.

6. The method of claim 5 wherein the oligonucleotide comprises at least one 2'-0- methyl-substituted ribonucleotide.

7. The method of claim 2 wherein the hybrid oligonucleotide is a chimeric oligonucleotide.

8. The method of claim 7 wherein the hybrid oligonucleotide comprises at least one phosphorothioate internucleotide linkage.

9. The method of claim 1 wherein the oligonucleotide is a chimeric oligonucleotide.

10. The method of claim 9 wherein at least one nucleotide in the oligonucleotide has a modified internucleotide linkage.

11. The method of claim 10 wherein the chimeric oligonucleotide comprises at least one phosphorothioate internucleotide linkage.

12. The method of claim 9 wherein the oligonucleotide is a hybrid oligonucleotide.

13. The method of claim 9 wherein the oligonucleotide comprises at least one phosphorothioate internucleotide linkage.

14. The method of claim 13 wherein the oligonucleotide comprises at least one 2'-0- substituted ribonucleotide.

15. The method of claim 14 wherein the oligonucleotide comprises at least one 2'-0-alkyl- substituted ribonucleotide.

16. The method of claim 15 wherein the oligonucleotide comprises at least one 2'-0- methyl-substituted ribonucleotide.

17. A method of modulating gene expression in an animal without inhibiting normal blood clotting in the animal, comprising the step of administering a therapeutic formulation comprising an oligonucleotide complementary to the gene in a pharmaceutically acceptable carrier, the oligonucleotide being hybrid and/or chimeric.

18. The method of claim 17 wherein the oligonucleotide is a hybrid oligonucleotide.

19. The method of claim 18 wherein the oligonucleotide is modified.

20. The method of claim 19 wherein the oligonucleotide has at least one 2' -substituted ribonucleotide.

21. The method of claim 20 wherein the oligonucleotide comprises at least one 2'-0-alkyl- substituted ribonucleotide.

22. The method of claim 21 wherein the oligonucleotide comprises at least one 2'-0- methyl-substituted ribonucleotide.

23. The method of claim 18 wherein the hybrid oligonucleotide is a chimeric oligonucleotide.

24. The method of claim 23 wherein the hybrid oligonucleotide comprises at least one phosphorothioate internucleotide linkage.

25. The method of claim 17 wherein the oligonucleotide is a chimeric oligonucleotide.

26. The method of claim 25 wherein at least one nucleotide in the oligonucleotide has a modified internucleotide linkage.

27. The method of claim 26 wherein the chimeric oligonucleotide comprises at least one phosphorothioate internucleotide linkage.

28. The method of claim 25 wherein the oligonucleotide is a hybrid oligonucleotide.

29. The method of claim 25 wherein the oligonucleotide comprises at least one phosphorothioate internucleotide linkage.

30. The method of claim 29 wherein the oligonucleotide comprises at least one 2'-0- substituted ribonucleotide.

31. The method of claim 30 wherein the oligonucleotide comprises at least one 2'-0-alkyl- substituted ribonucleotide.

32. The method of claim 31 wherein the oligonucleotide comprises at least one 2'-0- methyl-substituted ribonucleotide .