Title:  Antisense ogligonucleotides against Hepatitis B viral replication

United States Patent:  6,503,533

Issued:  January 7, 2003

Inventors:  Korba; Brent E. (Laurel, MD); Gerin; John L. (Bethesda, MD)

Assignee:  Georgetown University (Washington, DC)

Appl. No.:  199269

Filed:  November 25, 1998

Antisense oligonucleotides that hybridize to segments of the pres1, S, C, and .epsilon. regions of the hepatitis B virus (HBV) RNA pregenome inhibit replication of the virus. Pharmaceutical compositions which contain these oligonucleotides as the active ingredients are effective against HBV infection.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention provides a means of inhibiting the replication of hepatitis B virus (HBV), thus providing a therapy for treating chronic HBV infection. The invention is based on the use of antisense oligonucleotides which anneal to HBV-specific single-stranded RNA, and which thereby inhibit production of proteins essential to HBV replication. Inhibition of viral replication leads to alleviation of disease caused by the virus.

In accordance with the present invention oligonucleotides are provided that are designed to be complementary to portions of the mRNA coding for essential HBV proteins, or to regions of viral RNA which act as signal sequences, and thereby to disrupt the functions of these RNA's.

The present invention also includes pharmaceutical compositions comprising an effective amount of at least one of the antisense oligonucleotides of the invention in combination with a pharmaceutically acceptable sterile vehicle, as described in REMINGTON'S PHARMACEUTICAL SCIENCES: DRUG RECEPTORS AND RECEPTOR THEORY, (18th ed.), Mack Publishing Co., Easton Pa. (1990).

The HBV virion consists of a viral envelope together with a nucleocapsid made up of the virus genome, a multifunctional polymerase, and a core protein. The viral envelope contains three viral surface antigens (HBsAg, preS1, preS2) surrounding a nucleocapsid comprised of core antigen (HBcAg). An additional protein, the soluble e antigen (HBeAg) is observed in the serum of patients with active HBV infection, though its function is as yet unknown.

The virus genome consists of a relaxed circular, partially double-stranded DNA molecule held together by hydrogen bonding of the 5' cohesive termini. The minus strand has an invariable length of approximately 3.2 kb, whereas the plus strand is of variable length, ranging from 50 to 100% of the minus strand. The genome is organized into four partially overlapping open reading frames: the preC/C gene encoding the core and e proteins, the POL gene encoding the multifunctional polymerase (reverse transcriptase, DNA polymerase, Rnase H, terminal DNA binding protein), the preS/S gene encoding the envelope proteins, and the X gene encoding the transcriptional transactivator protein.

After infection, virion DNA is converted to a closed circular form, which then acts as a template for the synthesis of an RNA transcript known as the pregenome. The RNA transcript serves as a template for synthesis of minus strand DNA, which in turn is a template for synthesis of plus strand DNA. This double stranded DNA is then circularized, and the plus strand DNA is further extended to form the open circular form found in mature virus particles. It is essential for viral replication that the reverse transcription, RNase H, and DNA polymerase steps occur inside the nucleocapsid. It is known that a short, approximately 85bp sequence upstream of the preC gene, known as the .epsilon. sequence acts as a signal sequence for encapsidation when the viral core and polymerase proteins are present. See, for example, Pollack et al., J. Virol. 67: 3254 (1993). Disruption of .epsilon. by site-specific mutational analysis is known to inhibit packaging of HBV pregenomic RNA, an essential step in HBV replication. The replication pathway of hepadnaviruses produces intracellular viral DNA that, when examined by gel electrophoresis and blot hybridization techniques, presents as a heterogeneous smear (approximately 0.5 to 3.2 kb) comprised of single-stranded and partially double-stranded linear and circular viral DNA molecules. These are collectively referred to as viral DNA replication intermediates [RI]. See, for example, Fowler et al., J. Med. Virol. 13: 83 (1991). The levels of extracellular hepadnaviral virion DNA and intracellular RI are the most reliable parameters used to measure the current level of hepadnaviral replication. Accordingly, these parameters are those most commonly used to determine the effectiveness of antiviral therapy in man, experimental animal models and cell culture. See, for example, Hoofnagle, supra, Korba et al., Woodchuck Hepatitis Virus Infection As A Model For The Development Of Antiviral Therapies Against HBV In: VIRAL HEPATITIS AND LIVER DISEASE, Lemmon et al., Eds. p. 663 (1991), Korba et al., Antiviral Res. 15: 217 (1991).

A. Measurement of the Antiviral Effects of Antisense Oligonucleotides

In accordance with the present invention, an in vitro assay system is used which allows the accurate measurement of the antiviral effects of antisense oligonucleotides. In a preferred embodiment of the invention the assay uses a cell line of human origin which allows the replication of HBV in a manner similar to that observed in human tissue. In a particularly preferred embodiment the cell line used is the 2.2.15 cell line, available from Dr. George Acs, Mount Sinai School of Medicine, New York, N.Y. 10029. This cell line is derived from the well-known human hepatoblastoma line HepG2, and is stably transfected with a plasmid containing the entire HBV genome. HBV DNA found in these cells is both episomal, in the form of relaxed circular, covalently closed copies of the HBV genome, and chromosomally integrated. HBV virions from 2.2.15 cells produce the full spectrum of clinical disease when injected into chimpanzees. Acs et al., Proc. Natl. Acad. Sci. USA 84: 4641 (1987). 2.2.15 cells are the standard cell line known to the art to measure the effects of antiviral compositions on HBV replication and have been shown to be an accurate model for all measured aspects of cellular HBV replication and for the response of HBV to antiviral agents which have been used in vivo. Korba et al., Antiviral Res. 19: 55-70 (1992).

An in vitro assay using an HBV-producing cell line preferably allows quantitative measurement of the effects of antiviral compositions on HBV replication. Suitable quantitative methods are well known in the art. In a preferred embodiment of the present invention, effects on HBV replication are determined by measuring levels of intracellular HBV DNA, RNA, and proteins, and extracellular HBV DNA and proteins. Isolation of DNA, RNA,and-protein samples from treated cells may be carried out by standard methods. For example, Triazol.TM., a commercial reagent available from Life Technologies, Inc. (Gaithersburg, Md.) allows preparation of DNA, RNA and protein in a single step. Standard methods for measuring the levels of nucleic acids will be readily apparent to the skilled practitioner, such as the use of Southern and Northern blotting techniques. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, (1989). In a particularly preferred embodiment, HBV DNA and RNA are prepared and measured by quantitative Southern and Northern blot hybridization techniques according to Korba et al., Antiviral Res. 15: 217 (1991), and Korba et al., (1992), supra. Levels of HBV proteins found in the extracellular medium of the 2.2.15 cells can be measured by techniques well known in the art. See, for example, CURRENT PROTOCOLS IN IMMUNOLOGY, Coligan et al., Eds. at 2.1.3. In a preferred embodiment, levels of HBV proteins in the extracellular medium are measured by an enzyme-linked immunoassay as described by Muller et al., J. Infect. Dis. 165: 929 (1992).

To ensure that any observed antiviral activity is due to specific effects of the antisense oligonucleotide under test, it is appropriate to use control experiments. Suitable control experiments will be apparent to the skilled practitioner, but in a preferred embodiment, 2',3'-dideoxycytidine (2',3'-ddC) is used as a positive assay standard. 2'3'-ddC is a selective, effective antiviral agent in 2.2.15 cells; it is known to inhibit production of both HBV virions and replicative intermediates.

For an antisense oligonucleotide to be therapeutically useful it is desirable that it exhibit not only antiviral activity but also low cellular toxicity. It is envisioned therefore that each compound will also be tested for its toxic effects on 2.2.15 cells. Suitable toxicity measurements are well known in the art, but in a preferred embodiment a neutral red dye uptake assay is used, as described in Korba et al. (1992), supra.

Antisense oligonucleotides can be tested for in vivo efficacy and safety in an animal model system. A preferred animal model is one in which the animal is infected with a virus as closely related as possible to the strains of HBV that are responsible for human disease. The virus should undergo an analogous viral replication cycle, and should produce clinical symptoms analogous to those observed in human chronic hepatitis. Several animal viruses are known in the Hepadnaviridae family, for example the ground squirrel hepatitis virus of California ground squirrels, the duck hepatitis B virus of Peking ducks, and the heron hepatitis B virus of German grey herons. In a preferred embodiment of the invention, the animal model is the woodchuck Marmota Monax infected with woodchuck hepatitis virus (WHV). See Tennant et al., "The Woodchuck Model of Hepatitis B. Virus Infection" In: THE LIVER, BIOLOGY AND PATHOBIOLOGY, 3rd Ed., Arias, J. M. et al., (Eds), Chapter 76. Korba et al., "Woodchuck Hepatitis Virus Infection as a Model for the Development of Antiviral Therapies Against HBV" In: VIRAL HEPATITIS AND LIVER DISEASE, Hollinger F. B. et al. (Eds.), 632. The morphology and genetic organization of WHV is very similar to HBV, and the replication cycle appears to be identical. Woodchucks infected with WHV suffer from chronic hepatitis, and also develop hepatocellular carcinoma (HCC) at a much higher rate than uninfected animals. These similarities of WHV and HBV have led to the proposal that WHV infection in woodchucks be adopted as the standard animal model for the development of new and improved strategies for the treatment of chronic viral hepatitis and HCC induced by HBV. See Tennant et al., supra.

B. Preparation of Antisense Oligonucleotides

As used in this disclosure the term "oligonucleotide" encompasses both oligomeric nucleic acid moieties of the type found in nature, such as the deoxyribonucleotide and ribonucleotide structures of DNA and RNA, and man-made analogues which are capable of binding to nucleic acids found in nature. The oligonucleotides of the -present invention can be based upon ribonucleotide or deoxyribonucleotide monomers linked by phosphodiester bonds, or by analogues linked by methyl phosphonate, phosphorothioate, or other bonds. They may also comprise monomer moieties which have altered base structures or other modifications, but which still retain the ability to bind to naturally occurring DNA and RNA structures. Such oligonucleotides may be prepared by methods well-known in the art, for instance using commercially available machines and reagents available from Perkin-Elmer/Applied Biosystems (Foster City, Calif.).

Phosphodiester-linked oligonucleotides are particularly susceptible to the action of nucleases in serum or inside cells, and therefore in a preferred embodiment the oligonucleotides of the present invention are phosphorothioate or methyl phosphonate-linked analogues, which have been shown to be nuclease-resistant. See Stein et al., (1993), supra. Persons knowledgeable of this field will be able to select other linkages for use in the present invention.

C. Antiviral Effects of Antisense Oligonuclectides

The relative activity of antisense oligonucleotides directed against a specific gene is generally inversely proportional to its location relative to the AUG start codon of the target gene. In the prior art it is known that antisense oligonucleotides targeting sequences more than 60 bases downstream from the AUG start codon of chromosomally integrated HBV surface antigen [HBsAg] gene [S gene] sequences in HBsAg-producing PLC/PRF/5 cells are ineffective in inhibiting HBsAg production, while oligonucleotides placed within 20 bases of the AUG inhibit HBsAg production by 50% to 90%. In the present invention it is found that similar limitations hold for confluent cultures of 2.2.15 cells, a cell line in which over 95% of intracellular HBV DNA is episomal. As will be described in detail in Example 5, oligonucleotides targeting sequences more than 20 nucleotides upstream or downstream of the AUG have essentially no effect on HBV virion DNA and HBsAg production. Accordingly, it is preferred that an antisense oligonucleotide targeted at a specific HBV gene sequence be chosen such that the oligonucleotide hybridizes within approximately 25 bases of the AUG start codon of the gene.

To select the preferred length for an antisense oligonucleotide, a balance must be struck to gain the most favorable characteristics. Shorter oligonucleotides 10-15 bases in length readily enter cells, but have lower gene specificity. In contrast, longer oligonucleotides of 20-30 bases offer superior gene specificity, but show decreased kinetics of uptake into cells. See Stein et al., PHOSPHOROTHIOATE OLIGODEOXYNUCLEOTIDE ANALOGUES in "Oligodeoxynucleotides--Antisense Inhibitors of Gene Expression" Cohen, Ed. McMillan Press, London (1988). In a preferred embodiment this invention contemplates using oligonucleotides approximately 14 to 25 nucleotides long.

Oligonucleotides can be targeted around the AUG start codons of each of the different HBV coding regions, and their relative antiviral efficacies compared. For example, certain of the inventive oligonucleotides targeting the preS1 coding region are approximately as effective at inhibiting HBV virion production as are oligonucleotides targeting analogous regions of the S gene. For another example, oligonucleotides that target the HBeAg coding region or the HBV Pol gene do not inhibit HBV virion production in 2.2.15 cells. Oligonucleotides directed against HBeAg are, however, effective in inhibiting HBeAg production by 2.2.15 cells. The effect on HBV replication of candidate oligonucleotides directed against the HBV C gene can also be examined according to the invention. Oligonucleotides directed against regions close to, or overlapping, the AUG start codon of the C gene and that also encompass all, or part of, the single polyA signal in the HBV genome are the most effective at inhibiting HBV virion DNA and HBcAg production. Oligonucleotides directed primarily at the polyA signal are inactive or less active than those molecules directed at the beginning of the C gene.

The HBV encapsidation signal/sequence [.epsilon.] is known to be essential for HBV replication. See Hirsch et al., J. Virol. 65: 3309 (1991). According to the present invention certain of the oligonucleotides directed at .epsilon. are the most highly effective molecules at inhibiting HBV production by 2.2.15 cells. The structure of the .epsilon. RNA transcript is thought to adopt a stem-loop secondary structure, containing both base-paired and unpaired sequences. See, for example, Knaus et al., Nucl. Acids Res. 21: 3967 (1993). The most active oligonucleotides among all the molecules tested are directed at the upper unpaired loop and the upper stem of the encapsidation sequence. Molecules that extend further into the lower stem are less active. Oligonucleotides targeting the upper stem demonstrate the importance of the inclusion of the nucleotides associated with the upper unpaired loop found in the active sequences. An oligonucleotide directed at the lower stem-and-loop structure is also effective at inhibiting HBV virion production. The relative activities of oligonucleotides directed at the unpaired nucleotides in the lower loop/bulge of .epsilon. show that these are also effective antiviral targets. Although the anti-.epsilon. oligonucleotides are targeted immediately upstream of the C gene, these molecules are relatively ineffective at lowering the intracellular levels of HBcAg.

In order to determine if the effects on HBV protein levels are correlated with the effects on HBV production, the relative levels of HBV virion DNA and HBV protein levels can be directly compared in cultures treated with the different anti-S or anti-C oligonucleotides. Strong correlation is observed between virion DNA and either HBsAg or HBcAg levels in cultures treated with either the anti-S or the anti-C oligonucleotides. A similar analysis can be performed for the anti-.epsilon. oligonucleotides. Because of the close proximity of .epsilon. to the beginning of the C gene sequence, the levels of HBcAg in cultures treated with the anti-.epsilon. oligonucleotides are compared to the levels of HBV virion DNA. Little correlation is observed between HBcAg levels and HBV virion DNA in these cultures. High concentrations of anti-.epsilon. oligonucleotides are able to effectively inhibit HBcAg levels, but at the EC₉₀ values [defined as 10-fold depression of HBV DNA levels relative to untreated (control) cultures] for virion DNA, little or no inhibition of HBcAg is observed.

D. Sequence Specificity and Toxicity

To test whether the antisense orientation of the oligonucleotides is the active antiviral component of these molecules, oligonucleotides complementary to the antisense oligonucleotides can be examined for anti-HBV activity.

The procedures described above and detailed in the examples below provide a basis for selecting oligonucleotides that selectively disrupt specific HBV functions under conditions of preexisting chronic viral replication. It is known that several different strains of HBV exist, and antisense oligonucleotides preferably are effective in inhibiting the replication of more than one strain of the virus. In accordance with this, most of the oligonucleotides revealed herein act at key control elements located in regions of the HBV genome known to be highly conserved among different sequenced isolates of HBV. For example, with the exception of specific HBe-negative mutants, the e nucleotide sequence is essentially 100% conserved among the published sequences of 35 different HBV isolates. A consensus HBV DNA .epsilon. sequence may be used to specify the antisense oligonucleotide sequences to ensure that molecules will be effective on any unspecified HBV genome.

E. Administration of Antisense Oligonucleotides to Subjects

Administration of an antisense oligonucleotide to a subject can be effected orally or by subcutaneous, intramuscular, intraperitoneal, or intravenous injection. Pharmaceutical compositions of the present invention, however, are advantageously administered in the form of injectable compositions. A typical composition for such purpose comprises a pharmaceutically acceptable solvent or diluent and other suitable, physiologic compounds. For instance, the composition may contain oligonucleotide and about 10 mg of human serum albumin per milliliter of a phosphate buffer containing NaCl.

As much as 700 milligrams of antisense oligodeoxynucleotide has been administered intravenously to a patient over a course of 10 days (i.e., 0.05 mg/kg/hour) without signs of toxicity. Sterling, "Systemic Antisense Treatment Reported," Genetic Engineering News 12: 1, 28 (1992).

Other pharmaceutically acceptable excipients include non-aqueous or aqueous solutions and non-toxic compositions including salts, preservatives, buffers and the like. Examples of non-aqueous solutions are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate. Aqueous solutions include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components the pharmaceutical composition are adjusted according to routine skills in the art.

Antisense oligonucleotides may be administered by injection as an oily suspension. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides. Moreover, antisense oligonucleotides may be combined with a lipophilic carrier such as any one of a number of sterols including cholesterol, cholate and deoxycholic acid. A preferred sterol is cholesterol. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the suspension also contains stabilizers.

An alternative formulation for the administration of antisense PTN oligonucleotides involves liposomes. Liposome encapsulation provides an alternative formulation for the administration of antisense PTN oligonucleotides. Liposomes are microscopic vesicles that consist of one or more lipid bilayers surrounding aqueous compartments. See, generally, Bakker-Woudenberg et al., Eur. J. Clin. Microbiol. Infect. Dis. 12 (Suppl. 1): S61 (1993), and Kim, Drugs 46: 618 (1993). Liposomes are similar in composition to cellular membranes and as a result, liposomes can be administered safely and are biodegradable. Depending on the method of preparation, liposomes may be unilamellar or multilamellar, and liposomes can vary in size with diameters ranging from 0.02 .mu.m to greater than 10 .mu.m. A variety of agents can be encapsulated in liposomes: hydrophobic agents partition in the bilayers and hydrophilic agents partition within the inner aqueous space(s). See, for example, Machy et al., LIPOSOMES IN CELL BIOLOGY AND PHARMACOLOGY (John Libbey 1987), and Ostro et al., American J. Hosp. Pharm. 46: 1576 (1989). Moreover, it is possible to control the therapeutic availability of the encapsulated agent by varying liposome size, the number of bilayers, lipid composition, as well as the charge and surface characteristics of the liposomes.

Liposomes can adsorb to virtually any type of cell and then slowly release the encapsulated agent. Alternatively, an absorbed liposome may be endocytosed by cells that are phagocytic. Endocytosis is followed by intralysosomal degradation of liposomal lipids and release of the encapsulated agents. Scherphof et al., Ann. N.Y. Acad. Sci. 446: 368 (1985).

After intravenous administration, conventional liposomes are preferentially phagocytosed into the reticuloendothelial system. However, the reticuloendothelial system can be circumvented by several methods including saturation with large doses of liposome particles, or selective macrophage inactivation by pharmacological means. Claassen et al., Biochim. Biophys. Acta 802: 428 (1984). In addition, incorporation of glycolipid- or polyethelene glycol-derivatised phospholipids into liposome membranes has been shown to result in a significantly reduced uptake by the reticuloendothelial system. Allen et al., Biochim. Biophys. Acta 1068: 133 (1991); Allen et al., Biochim. Biohys. Acta 1150: 9 (1993) These Stealth.RTM. liposomes have an increased circulation time and an improved targeting to tumors in animals. Woodle et al., Proc. Amer. Assoc. Cancer Res. 33: 2672 (1992). Human clinical trials are in progress, including Phase III clinical trials against Kaposi's sarcoma. Gregoriadis et al., Drugs 45: 15 (1993).

Antisense oligonucleotides can be encapsulated within liposomes using standard techniques. A variety of different liposome compositions and methods for synthesis are known to those of skill in the art. See, for example, U.S. Pat. No. 4,844,904, U.S. Pat. No. 5,000,959, U.S. Pat. No. 4,863,740, and U.S. Pat. No. 4,975,282, all of which are hereby incorporated by reference.

Liposomes can be prepared for targeting to particular cells or organs by varying phospholipid composition or by inserting receptors or ligands into the liposomes. For instance, antibodies specific to liver associated antigens may be incorporated into liposomes, together with antisense oligonucleotides, to target the liposome more effectively to the liver. See, for example, Zelphati et al., Antisense Research and Development 3: 323-338 (1993), describing the use "immunoliposomes" containing antisense oligonucleotides for human therapy.

In general, the dosage of administered liposome-encapsulated antisense oligonucleotides will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition and previous medical history. Dose ranges for particular formulations can be determined by using a suitable animal model.

Claim 1 of 16 Claims

What is claimed is:

1. A method for treating an HBV infection in a patient, comprising administering to said patient an effective amount of a single stranded antisense oligonucleotide sufficient to bind the E encapsidation sequence of the single stranded mRNA intermediate derived from the HBV DNA genome to inhibit HBV replication.
 

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