Classifications

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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
  • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/11—Antisense
  • C12N2310/111—Antisense spanning the whole gene, or a large part of it
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/32—Chemical structure of the sugar
  • C12N2310/322—2'-R Modification

Definitions

• the invention relates to oligonucleotide compositions, pharmaceutical compositions containing such oligonucleotides, and their use for the prevention and treatment of hepatitis B infection.
• Viruses are potent infectious pathogenic agents because virions produced in one cell can invade other cells and thus cause a spreading infection. Viruses cause important functional alterations of the invaded cells, often resulting in death of the cells.
• Hepatitis viruses constitute a major medical problem throughout the world. Like the other hepatitis viruses, the hepatitis B virus (HBV) produces a whole spectrum of illnesses, ranging from acute to chronic and from subclinical or asymptomatic to fatal and fulminant. Approximately 5% of the world's population, probably at least 400 million people, are presently infected with the hepatitis B virus (HBV) . HBV presents a high risk of acute fulminant hepatitis, as well as chronic liver disease, including cirrhosis, chronic active hepatitis, and the eventual development of primary hepatoceilular carcinoma in individuals who remain chronic carriers of the virus.
• HBV hepatitis B virus
• the invention relates to antisense oligonucleotides, preferably antisense oligodeoxynucleotides, as antiviral agents against HBV; pharmaceutical compositions providing such antiviral oligonucleotides; and methods for their use in inhibiting HBV.
• Antisense oligonucleotide compositions complementary to the HBV DR2 region completely block viral transcription, antigen production, and replication.
• Such antiviral oligonucleotides can be provided to the target cell either exogenously as an antisense DNA or RNA, or by insertion of a sense DNA sequence into an expression vector capable of producing multiple copies of the antisense oligonucleotides endogenously within the target cell.
• the invention includes an oligonucleotide having antiviral activity against hepatitis B virus (HBV) , consisting essentially of a sequence substantially complementary to a portion of plus (+) strand of the HBV genome, which portion consists of the DR2 11-mer (SEQ ID NO: 44) plus 0-6 nucleotides of 5' flanking sequence and 0-30 nucleotides of 3' flanking sequence.
• HBV hepatitis B virus
• oligonucleotide preferably includes the sequence 5'-ACGTGCAGAGGTGAAGCG-3' (SEQ ID NO: 21) .
• oligonucleotides of the invention include the following:
• oligonucleotides of the invention include those having a sequence consisting essentially of one of the following:
• the oligonucleotides of the invention can be used in a method of preventing replication of HBV in a mammalian cell, which method includes the step of introducing into the cell an inhibitory amount of (a) the oligonucleotide, or (b) an expression vector containing a sequence that is transcribed within the cell to generate an oligoribonucleotide of the invention.
• an expression vector would preferably include transcription control sequences that permit it to be expressed within a hepatocyte.
• the oligonucleotide may be administered to an animal in the form of a pharmaceutical composition consisting essentially of an amount of the oligonucleotide effective to inhibit replication of HBV in the liver cells of an animal, and a pharmaceutically acceptable carrier.
• Fig. 1 is a map of the HBV genome, showing the coding organization of the four major open reading frames, as well as the 3.5 kb pregenomic RNA and the 2.1 kb subgeno ic RNA species. DR1 and DR2 are shown.
• Fig. 2 is a representation of the computer- generated putative secondary structure of the RNA sequence of HBV (HPBADW1) (n.t.s. 1500 to n.t.s. 1700), showing DR2 as part of a stem-loop structure.
• HBV Human hepatitis B virus
• hepadnaviruses hepatotropic DNA viruses
• Human HBV is classified as a hepadnavirus type 1.
• Similar viruses infect other animal species, including woodchucks, ground and tree squirrels, Pekin duck, and heron, producing acute and chronic hepatitis as well as hepatocellular carcinoma.
• Full-length molecular clones of these hepadnaviruses have been obtained and their nucleotide sequences determined.
• the coding organization of the mammalian viruses is now known to be virtually identical to that of human HBV, while the avian viruses are more divergent in terms of nucleotide sequence, biological properties, and coding organization.
• the duck HBV (DHBV) genome appears to lack an X region, and its core antigen coding region is substantially larger that that of the mammalian viruses.
• Ganem & Varmus The Molecular Biology of the Hepatitis B Viruses; Ann . Rev. Biochem . 56:651-93 (1987) .
• Replication strategy of the hepatitis B virus is discussed by Seeger et al . , Science 232:477-484 (1986); Khudyakov et al .
• Infectious human HBV virions are 42 or 43 nm double-shelled particles which include the outer coat of HBV surface antigen (HBsAg) and the HBV core antigen (HBcAg) , a basic phosphoprotein of 21 kd.
• HBV nucleocapsid core Within the HBV nucleocapsid core is a predominantly double-stranded, but partially single stranded, DNA genome measuring 3200 base pairs, as well as an endogenous DNA polymerase which directs replication and repair of HBV DNA.
• the full-length strand of HBV DNA is complementary to the viral RNAs, including the mRNAs, and by convention is designated to be of minus polarity.
• the shorter complementary strand is designated the plus strand. While the 5' end of the plus strand is fixed, the position of the 3' end is variable, even within molecules of the same viral stock.
• the single- stranded gap is repaired by the addition of nucleotides to the 3' end of the plus strand DNA.
• a second asymmetry occurs at the 5' termini of the viral genome's two strands: protein is covalently linked to the 5' end of the minus strand, whereas an oligonucleotide is attached to the 5' end of the plus strand.
• Replication of HBV proceeds via reverse transcription of an RNA intermediate using protein and RNA primers for the generation of the first and second
• DNA strands Large sections of the genome are translated in more than one reading frame. Within a given reading frame, multiple proteins are expressed from overlapping transcripts, using different in-phase initiator codons. The resulting closely related gene products are posttranslationally processed and assembled into a variety of structures of differing function or subcellular distribution.
• ORFs Four major open reading frames (ORFs) encoded by the HBV minus strand have been identified and characterized: 1) the pre-S and S gene, which code for the HBsAg and several other less well characterized gene products; 2) the C gene, which codes for HBcAg and HBeAg; 3) the P gene, which codes for the viral DNA polymerase; and 4) the X gene, which codes for the transactivating X protein, HBx, often observed in patients with hepatocellular carcinoma. (HBeAg results from proteolytic cleavage of the p22 precore intermediate, and is secreted from the cell. It is found in serum as a 17 kd protein.)
• Amplification of the viral genome is believed to occur during synthesis of pregenomic RNA from cccDNA.
• HBV viral RNA serves as both the template for synthesis of genomic DNA via reverse transcription and the messenger RNA for synthesis of certain viral proteins. This is achieved by the synthesis of two classes of viral RNA, genomic (3.5 kb in length, containing the complete viral genetic information) and subgenomic (2.1 and 2.4 kb in length).
• RNAs are of plus strand polarity, unspliced, and polyadenylated at a common 3' terminus.
• HBV genome Within the HBV genome are conserved cis-acting elements that play important roles in the life cycle of the virus. Chief among these are 11-nucleotide "direct repeat" sequence designated DR1 and DR2.
• Drl and DR2 are distinguished from each other by their positions in the genome, their flanking sequences, and their biological functions.
• DR1 and DR2 are located near the 5' and the 3' ends of the HBV plus strand, respectively, and play critical roles in the initiation of viral DNA synthesis.
• DR1 and DR2 are located near the 5' and the 3' ends of the HBV plus strand, respectively, and play critical roles in the initiation of viral DNA synthesis.
• DR1 and DR2 are located near the 5' and the 3' ends of the HBV plus strand, respectively, and play critical roles in the initiation of viral DNA synthesis.
• the other major conserved sequence is the element TATAAA (SEQ ID NO: 1) found within the 5' end of the core antigen coding sequence, which forms part of the cleavage/polyadenylation signal specifying the common 3' termini of viral mRNAs.
• TATAAA SEQ ID NO: 1
• Figure 1 which sets forth a map of the HBV viral genome, showing the organizational structure of the four major ORFs and the pregenomic and subgenomic RNA species.
• Synthesis of the minus strand of viral DNA is believed to begin with the DR1 sequence that resides within the terminal repeat region, R, found at both the 5' and 3' ends of the pregenomic RNA. Initiation could occur near either the 5' or the 3' end of the RNA template, since DR1 resides within the R sequence that is terminally repeated in pregenomic RNA.
• initiation at the 3' end would permit elongation without interruption across the entire genomic sequence, ending with a second copy of the 9 nucleotide sequence found between the DRl initiation site and the 5' boundary of R, whereas initiation at the 5' end would require transfer of the growing minus strand DNA molecule to the 3' end of the same or another pregenomic RNA molecule.
• the initiation site for synthesis of plus strand DNA occurs on a minus strand DNA template at the sequence which is complementary to DR2 (DR2').
• DR2 DR2'
• the attached RNA was sequenced (Lien et al. J. Virol . 57:229-37 (1986)), the expected DR sequence was found to be flanked by 6 nucleotides from the DRl region, rather than the expected DR2 region.
• oligomer containing DRl basepairs with the DR2' site in the DNA minus strand, and serves as a primer for initiation of plus strand synthesis from that position.
• the fact that the oligomer has a 5' cap structure is evidence that the plus strand primer originates from the 5' end of the pregenomic RNA, although the reason for such a complicated priming mechanism is not immediately clear. Synthesis of the plus strand reaches a roadblock at the protein-linked 5' end of the minus strand template, requiring transfer of the partial plus strand to the 3' end of a minus strand template.
• the plus strand is usually not extended to the full length of the minus strand; instead, plus strands are incomplete and heterogeneous in length, with open circles the dominant form of virion DNA.
• the nucleotide sequence of DR2 and flanking sequences both 5' and 3' to DR2 are highly conserved among various HBV HBsAg subtypes and strains.
• the nucleotide sequence of the DR2 region of HBV HBsAg subtype ADW was compared with corresponding sequences from a number of other strains or subtypes of HBV by using sequence data available in GenBank. Sequences and designations are as listed in GenBank®. See Table I below, where the symbol ":" indicates a nucleotide identical to the corresponding position of HPBADW.
• DR2 of HPBADW Hepatitis B Virus Subtype ADW
• DR2 of HPBADW consists of nucleotides 1592 through 1602, having the sequence TTCACCTCTGC (SEQ ID NO: 2) .
• the numbering listed in Table 1 is that of Hepatitis B Virus Subtype ADW from GenBank.
• the nucleotide sequences of various hepatitis virus strains can be found in Okamoto et al . , J. Gen . Virol . 69:2575-2583 (1988), and through GenBank. The teachings of these references are hereby incorporated by reference.
• HVHEPB Hepatitis B Virus (HBV 991)
• HBVXCPS Hepatiti ⁇ B Virus X, C, P and S
• HBVAYWCI Hepatitis B virus (ayw, patient CI)
• HBV Hepatitis B Virus
• antiviral antisense oligomers of the invention which are complementary to a region of HBV plus strand DNA comprising DR2; at least 3 and preferably at least 4 conserved 5' flanking nucleotides; and 0-30, preferably 0-20, more preferably 0-10, and most preferably 4-6 3" flanking nucleotides, can totally block HBV replication.
• Antisense is a term that means complementary to the sense (or plus) strand. An antisense oligonucleotide interacts in a sequence-specific manner with a cellular nucleic acid target containing a sequence complementary to the antisense molecule.
• the oligonucleotides of the invention are complementary to the DR2 region of HBV plus strand DNA and to the corresponding region of HBV RNA (which is also of plus strand polarity) , and thus interact with these regions, thereby inhibiting HBV viral replication.
• the interaction of such oligonucleotides with their complementary or "receptor" sequences may result from hybridization interactions, or through other mechanisms which are not yet fully understood.
• the therapeutic applications of antisense oligonucleotides are described, e.g., in the following review articles: Le Doan et al . , Antisense Oligonucleotides as Potential Antiviral and Anticancer Agents, Bull .
• the invention relates to a composition of matter consisting essentially of an antiviral oligonucleotide, preferably an oligodeoxynucleotide, having a nucleotide sequence substantially complementary to a portion of the plus strand of an HBV genome comprising DR2 plus certain 5" and, optionally, 3" flanking sequences, preferably both 5" and 3" flanking sequences.
• an antiviral oligonucleotide preferably an oligodeoxynucleotide, having a nucleotide sequence substantially complementary to a portion of the plus strand of an HBV genome comprising DR2 plus certain 5" and, optionally, 3" flanking sequences, preferably both 5" and 3" flanking sequences.
• Such oligonucleotides will also be complementary to the corresponding RNAs (e.g, messenger RNA or genomic RNA) , which are of plus strand polarity. Because the antisense oligonucleotides are substantially complementary to
• Antisense oligonucleotides of the invention have been shown to be capable of completely inhibiting HBV replication. Accordingly, the invention also relates to methods of inhibiting HBV replication in cells containing HBV, including methods of preventing HBV infection in an animal exposed to HBV, and methods of treating an animal infected with HBV; such animals include, for example, humans and other primates, such as chimpanzees.
• the invention also relates to pharmaceutical compositions for use in preventing HBV infection in an animal exposed to HBV, or treating an animal infected with HBV. Preferred are such pharmaceutical compositions formulated for parenteral administration. Such pharmaceutical compositions will contain an effective antiviral amount of an oligonucleotide of the invention and a pharmaceutically acceptable carrier.
• the invention also relates to a composition of matter consisting essentially of at least one antiviral antisense oligonucleotide substantially complementary to a portion of the plus strand of an HBV genome comprising DR2 plus 5" and/or 3' flanking sequences, preferably including residues complementary to all or substantially all of nucleotides 1588 through 1606, preferably all or substantially all of nucleotides 1588 through 1608, of HBV subtype ADW (HPBVADW) , or corresponding nucleotides of other HBV strains.
• a composition of matter consisting essentially of at least one antiviral antisense oligonucleotide substantially complementary to a portion of the plus strand of an HBV genome comprising DR2 plus 5" and/or 3' flanking sequences, preferably including residues complementary to all or substantially all of nucleotides 1588 through 1606, preferably all or substantially all of nucleotides 1588 through 1608, of HBV subtype ADW (HPBVADW) , or
• Corresponding sequences for strains of HBV other than HBV subtype ADW can be determined by those of ordinary skill in the art by aligning sequences for homology, e.g., by using an available database such as GenBank.
• Antiviral oligonucleotides of the invention can be supplied to a target cell either exogenously as DNA or RNA, or endogenously, by supplying a DNA sequence from which the desired oligonucleotide may be transcribed by the target cell.
• the DNA to be expressed may be supplied to the target cell, preferably a hepatocyte, as a reco binant nucleic acid (e.g., a DNA molecule) comprising a DR2 sequence and flanking oligonucleotides, wherein expression of said DNA is capable of inhibiting viral replication.
• a reco binant nucleic acid e.g., a DNA molecule
• This nucleic acid molecule is characterized in that it (a) is capable of being replicated in a hepatocyte under conditions that normally prevail in the hepatocyte, and (b) is transcribed in a hepatocyte to produce an oligonucleotide substantially complementary to a portion of the plus strand of a hepatitis B viral genome consisting of DR2 and 5' and 3' flanking sequences.
• the sequence transcribed into the antiviral oligonucleotide is preferably operably linked to a cell-specific promoter to direct expression in the hepatocyte.
• the invention also includes a method for inhibiting hepatitis B virus replication in a cell by introducing the oligonucleotide molecule itself directly into the cell, or by introducing into the cell a nucleic acid which is transcribed within the cell to produce multiple copies of the antiviral oligonucleotide as an oligoribonucleotide.
• oligonucleotide compositions are provided for the inhibition of viral replication.
• the antisense oligonucleotide itself may be provided exogenously to a host cell infected with the virus or susceptible to viral infection.
• Another approach is to provide for expression of the antiviral oligonucleotide in the host cell.
• a DNA transcribable into the antisense oligonucleotide of the invention is incorporated into an expression vector downstream from, and operatively linked to, a suitable promoter which provides for tissue specific or general expression.
• the DNA of the invention may be placed downstream from a liver specific promoter, in order to induce expression by hepatocytes in the liver; however, it may not be necessary to use a liver-specific promoter, since expression of the antisense oligonucleotides in non-liver cells should be harmless to the cells.
• the DNA with the appropriate regulatory regions is provided in proper orientation to allow for expression. Methods for constructing such expression vectors are known in the art. See in particular. Molecular Cloning, A Laboratory Manual , Sambrook et al . , eds.. Cold Spring Harbor Laboratory, 2nd Edition, Cold Spring Harbor, NY (1989) .
• transcriptional regulatory sequences may be employed.
• the signals may be derived from viral sources, such as adenovirus, bovine papilloma virus, simian virus, or the like, where the regulatory signals are associated with a particular gene which has a high level of expression.
• promoters from mammalian expression products such as actin, collagen, myosin, etc. , may be employed.
• HBV DNA in eukaryotic hosts requires the use of eukaryotic regulatory regions. Such regions will, in general, include a promoter region sufficient to direct the initiation of antisense RNA synthesis.
• Typical promoters include the promoter of the mouse metallothionine I gene (Hammer, D. et al . , J. Mol . Appl . Gen . 1:273-288 (1982)); the Tk promoter of herpes virus (McKnight, S., Cell 31:355-365 (1982)); the SV40 early promoter (Benoist et al . , Nature 290:304-310 (1981)); and the like.
• liver specific promoters such as albumin, alpha- fetoprotein, alpha-1-antitrypsin, retinol-binding protein, asialoglycoprotein receptor, and viral promoters and enhancers such as those of cytomegalovirus; herpes simplex I and II viruses; hepatitis A, B, and C viruses, and Rous sarcoma virus (RSV) (Fang, X.J. et al . , Hepatology 10:781-787 (1989)).
• RSV Rous sarcoma virus
• the gene is placed downstream from a suitable promoter which provides for tissue specific or general expression.
• the DNA will be transcribed to produce RNA which will hybridize with its target RNA, thereby inhibiting viral replication.
• One particular vector useful for this task would be one based on an adenoviral system, such as described by Morsey et al . , Abstract SZ 109, "Efficient Adenoviral Gene Transduction in Human and Mouse Hepatocytes In Vitro and in Mouse Liver In Vivo" , in J. of Cellular Biochemistry, Suplement 17E, Keystone Symposia on
• a retroviral vector such as LNL6, a derivative of the Moloney murine leukemia virus, as described in Rosenberg et al . , N. Eng. J. Med . 323, No. 9:570-578 (1990).
• LNL6 a derivative of the Moloney murine leukemia virus
• Gene transfer into hepatocytes using a defective Herpes Simplex viral vector is described by Lu et al . , Abstract, page 66, Abstracts of the Meeting on Gene Therapy, Sept. 22-26, 1992, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. This latter approach is especially valuable for introducing genes into non-dividing hepatocytes, since HSV-l does not require genomic integration for expression.
• the DNA and the requisite regulatory elements may also be introduced into hepatocytes using an asialoglycoprotein carrier system as described in Wu et al. , Biotherapy 3:87-95 (1991) .
• the desired viral DNA and operably linked promoter may be introduced into a recipient cell either as a non- replicating DNA or RNA molecule, which may be a linear molecule or, more preferably, a closed covalent circular molecule. Since such molecules are incapable of autonomous replication, the expression of the desired receptor molecule occurs through the transient expression of the introduced sequence. Where more long-term expression is desired, the sequence may be integrated into the host chromosome. Alternatively, the introduced sequence may be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host; such vectors include the cDNA expression vectors described by Okayama, H. , Mol . Cell . Bio . 3:280 (1983), and others. Viral vectors include retrovirus vectors as taught in W089/07136 (specifically for expression in hepatocytes) and the references cited therein.
• either the antisense oligonucleotide itself, or a DNA which is transcribable into the antisense RNA of the invention is introduced into the cells of an animal suffering from the viral disease.
• substantially complementary means that an antisense oligonucleotide of the invention is capable of hybridizing with its RNA or DNA target under physiological conditions.
• HBV nucleotide sequence numbering herein is made with reference to the numbering of Hepatitis B Virus (Subtype ADW) , according to GenBank.
• Corresponding sequences for HBV strains and subtypes. other than HBV subtype ADW, e.g., the strains listed in Table 1, infra , as well as other strains of HBV that have been and will in the future be isolated and sequenced, can be determined by those of ordinary skill in the art by aligning sequences for homology using an available database such as GenBank.
• compositions of the present invention can be utilized to prevent viral infection as well as to treat viral infections.
• compositions comprising vectors containing nucleic acids transcribable into the anti-HBV oligonucleotides of the invention may be administered to prevent a virus infection or to combat the virus once it has entered the host.
• compositions of matter of the invention may be used together, either in admixture or combined in a single molecule, with other materials that do not alter the essential nature of the invention.
• antisense oligonucleotide sequences of the invention are essential to the invention, it is contemplated that they may be used in admixture or in chemical combination with one or more other materials, including other oligonucleotides antisense to other portions of HBV RNA; materials that increase the biological stability of the oligonucleotides; or materials that increase their ability to penetrate selectively their hepatocyte target cells, and reach and hybridize their target RNA.
• oligonucleotides may be modified to achieve greater stability, including backbone modifications such as phosphorothioates , methylphosphonates, phosphorodithioates, phosphoroamidates, phosphate esters, and other modifications as described in Uhlman and Peyman, Antisense Oligonucleotides: A New Therapeutic Principle. Chemical Reviews 90(4) :544-584 (1990), at 546-560, the teaching of which is hereby incorporated by reference. All such modifications are contemplated equivalents of the antisense oligonucleotides of the invention.
• the antisense oligonucleotides may be provided in stabilized form, e.g., with phosphotriester linkages, or by blocking against exonuclease attack with methylphosphonodiester linkages, with 3' deoxythymidine, as a phenylisourea derivative, or by linking other molecules such as aminoacridine or polylysine to the 3' end of the oligonucleotide. See e.g., Anticancer Research .10:1169-1182, at 1171-2 (1990), the teaching of which is incorporated herein by reference.
• antisense oligonucleotides supplied exogenously increased selectivity for hepatocytes may be achieved by linking antisense oligonucleotides of the invention to natural ligands such as ASOR (asialoorosomucoid) or to synthetic ligands that will bind to the hepatic asialoglycoprotein (ASGP) receptor.
• ASOR asialoorosomucoid
• ASGP hepatic asialoglycoprotein
• ribozymes may be targeted by linking to an oligonucleotide of the invention, since there are a number of ribozyme target cleavage sites in the DR2 region of the viral RNA. See e.g..
• a high efficiency, cell-specific delivery system for in vivo therapeutic use may utilize a number of approaches, including the following: 1) specific delivery through hepatocyte specific receptor mediated process such as the asialoglycoprotein receptor, as discussed above; 2) delivery of antisense oligodeoxynucleotides iri liposomes with or without specific targeting with monoclonal antibodies directed against specific cell surface receptors; 3) retrovirus- ediated transfer of DNA expressing the antisense construct of interest; and 4) direct targeting to cells of antisense oligodeoxynucleotides following conjugation to monoclonal antibodies that are specific for cell surface receptors that function in a receptor-mediated endocytotic process; 5) specific delivery to hepatocytes via a replication-defective HBV vector.
• the antisense compositions of the invention may be administered as individual therapeutic agents or in combination with other therapeutic agents. They can be administered alone, but are generally administered with a pharmaceutically acceptable carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.
• the dosage administered will of course vary depending upon known pharmacokinetic/ pharmacodynamic characteristics of the particular agent and its mode and route of administration, as well as the age, weight, and health (including renal and hepatic function) of the recipient, the nature and extent of disease, kind of concurrent therapy, frequency and duration of treatment, and the effect desired.
• a daily dose of active ingredient can be about 0.1 to 100 mg per kilogram of body weight. Ordinarily 0.5 to 50, and preferably 1 to 10 mg per kg of body weight per day given in divided doses or in sustained release form (including sustained intravenous infusion) will be effective to achieve the desired effects.
• Dosage forms suitable for internal administration generally contain about 1 milligram to about 500 milligrams of active ingredient per unit.
• the active ingredient will ordinarily be present in an amount of about 0.5 to 95% by weight of the total pharmaceutical preparation.
• the antisense oligonucleotide compositions of the invention may be administered parenterally (e.g., intravenously, preferably by intravenous infusion) .
• parenteral administration the compositions will be formulated as a sterile, non-pyrogenic solution, suspension, or emulsion.
• the preparations may be supplied as a liquid formulation or lyophilized powder to be diluted with a pharmaceutically acceptable, sterile, non-pyrogenic parenteral vehicle of suitable tonicity, e.g., water for injection, normal saline, or a suitable sugar-containing vehicle, e.g., D5W, D5/0.45, D5/0.2, or a vehicle containing mannitol, dextrose, or lactose.
• suitable pharmaceutical carriers, as well as pharmaceutical necessities for use in pharmaceutical formulations are described in Remington 's Pharmaceutical Sciences , a standard reference text in this field, and in the USP/NF.
• the oligonucleotides or their derivatives may also be administered in liposomes or microspheres (microparticles) .
• Methods for preparing liposomes and microspheres for administration to a patient are known to those skilled in the art.
• U.S. Pat. No. 4,789,734 describes methods for encapsulating biological materials in liposomes. The material is dissolved in an aqueous solution; the appropriate phospholipids and lipids added, along with surfactants, if required; and the material dialyzed or sonicated, as necessary.
• a review of known methods is provided by G. Gregoriadis, Chapter 14, “Liposomes", Drug Carriers in Biology and Medicine, pp. 287-341 (Academic Press, 1979) .
• Microspheres formed of polymers are well known to those skilled in the art, and can be tailored for passage through the gastrointestinal tract directly into the bloodstream.
• the oligomers or their derivatives can be incorporated into microspheres, and implanted for slow release over a period of time. See, e.g., U.S. Patents Nos. 4,925,673 and 3,625,214.
• HuH 7 hepatoma cells (Cancer Research 42:3858-3863 (1982)) were seeded into 6 well plates (35mm/well) and grown to 70-90% confluency in Dulbecco's Minimal Essential Medium supplemented with 10% fetal bovine serum. Cells were transfected according to a modification of the procedure described by Chen and Okayama, Mol .Cell . Biol . 7:2745-2752 (1987). [The modification in this procedure was to incubate the cells in an atmosphere of 5% C0 2 throughout the experiment, as opposed to incubating the cells in a lower C0 2 atmosphere during the actual transfection step.
• HBV DNA sequences [head-to-tail dimer of HBV DNA HBsAg subtype adw in pGEM-72f(+) (Promega) ] , which was obtained from J. Wands, MGH, Boston [see Blum et al., J. Virol. £5(4) :1836-1842 (1991)].
• Cell supernatants were harvested post-transfection on a daily basis for up to 6 days, and cells refed daily with 2mls of medium/well.
• HBsAg is a marker for viral replication, and the ability of oligonucleotide to block HBsAg production indicates inhibition of viral replication.
• Oligonucleotides The following oligodeoxynucleotides were synthesized in a Milligen Biosearch 8750 DNA synthesizer, using asialoethyl-phosphoramidite syntheses (Tetrahedron Lett 22 : 1859-1862 (1981)):
• Antisense and scrambled oligodeoxynucleotides were purified after NH 4 OH detachment (55°C, 6 hours) and NAP 25 column (Pharmacia) desalting with 0.1 M NaHC0 3 by reverse phase HPLC (trityl on, TEAA 0.1 M, pH 7.25/acetonitrile gradient) .
• the oligodeoxynucleotides were lyophilized, deblocked with l M acetic acid for 1 hour, neutralized with 1.0 M NaHC0 3 , passed through a NAP 10 column, and then lyophilized to dryness.
• HBV 10012 SEQ ID NO: 7
• CP 10052 SEQ ID NO: 9
• DRl appears to be involved at an earlier step in HBV replication, since synthesis of the first DNA strand is believed to be initiated within DRl. It is thought that the DR2 antisense oligonucleotide may be acting at any of several possible levels.
• oligomers of the invention may bind to and have an antisense effect on the HBV 3.5 kb pregenomic RNA, the HBV 2.1 kb mRNA and/or the HBV 2.4 kb mRNA.
• the DR2 sequence is present in both the polymerase and the X protein ORFs.
• the antiviral oligonucleotides might also bind to the oligoribonucleotide primer generated during HBV replication, thereby preventing oligoribonucleotide priming at the DR2 site on minus strand DNA and inhibiting plus strand DNA synthesis. It should be recognized, however, that oligomers of the invention are also complementary to and are capable of hybridizing with plus strand HBV DNA containing DR2.
• nucleotide sequence of this region is highly conserved amoung different HBV strains, a finding which suggests that nucleotide sequence and its function in viral replication may be important, secondary and tertiary structures may also be important with respect to interaction with protein.
• the invention as claimed, however, is not predicated on any particular mechanism or mechanisms through which the DR2 oligonucleotides act to block HBV replication.
• DRl and DR2 share 11 identical nucleotides, but have different flanking sequences. Flanking regions of both DRl and DR2 are also conserved among various species and strains of HBV, indicating the importance of these regions in HBV DNA replication. Moreover, flanking sequences of DR2 of many human and woodchuck HBVs contain a two-fold symmetry, making the majority of DR2 sequence and the 5'-flanking region of DR2 into a single-stranded loop. A putative secondary structure of the DR2 region of HBV DNA is shown in Fig. 2, using a suboptimal RNA folding program (see Jaeger, JA, Turner, DH, and Zuker, M, Proc. Natl . Acad . Sci .
• DRl antisense oligonucleotides require hybridization of an oligonucleotide to its complementary sequence in the RNA target. Therefore, for an antisense oligonucleotide to be effective, the complementary target sequence must be available for hybridization. In most cases, target mRNA is not single-stranded random coil, but rather contains secondary and tertiary structures.
• Target RNA structure has been shown to affect the affinity and rate of oligonucleotide hybridization, as well as the efficacy of antisense oligonucleotides [See Yoon, K. Turner, D.H., and Tinco, I, Jr., J. Mol . Biol . 99:507 (1975); Freier, S.M. and Tinoco, I., Jr., Biochemistry 14:3310 (1975); Uhlenbeck, O.C., J. Molec. Biol 65:25 (1972); Herschlag D. and Cech, T.R., Biochemistry 29:10159 (1990); and Fedor, M. J. and Uhlenbeck. O.C., Proc . Natl . Acad . Sci .
• RNA secondary structure may provide insight into the results that have been obtained experimentally.
• the oligonucleotides were tested for nonspecific cytotoxicity by means of the MTT assay.
• the assay is dependent on the reduction of the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) by the mitochondrial hydrogenase of viable cells to form a blue formazan product (D. Gerlier et al., J.
• EXAMPLE 2 In order to elucidate further the structure- activity relationships of antisense oligonucleotides complementary to plus strand HBV DNA in the DR2 region, a number of additional olignucleotides were synthesized and tested for antiviral activity in the manner described above.
• Oligo ID# CJP 110 Description DR2 antisense
• Oligo ID# CJP 100 Description DR2 antisense Sequence: 5" CGA CGT GCA GA 3' (SEQ ID NO: 18)
• DR2 antisense oligodeoxynucleotide HBV 10011 (5-* CAA CGT GCA GAG GTG AAG CGA 3'; SEQ ID NO: 6) exhibited strong anti-HBV activity, a series of other oligodeoxynucleotides complementary to the DR2 region of plus strand HBV DNA were synthesized and tested in the manner described above.
• DR2 antisense oligonucleotides was designed based on the putative secondary structure of the DR2 loop region ( Figure 1) , with the rationale being to target antisense oligonucleotides to the putative single-stranded region of the DR2 loop, which may be favorable for hybridization in a manner similar to H-ras mRNA (Lima, 1992) .
• the antiviral effect of each of these oligonucleotides is shown in Table 2, and discussed below.
• HBV 10011 is a 21-mer consisting of nucleotides complementary to the entire eleven-nucleotide DR2 as well as four 5' flanking nucleotides and six 3' flanking nucleotides.
• the oligodeoxynucleotide designated CJP 114 is a 21-mer differing from HBV 10011 only with respect to a single nucleotide at n.t.n. 1607 (numbering relative to HPBV ADW from GenBank) . Comparing the DR2 region of various HBV strains using GenBank revealed a nucleotide change from T in HBV ADW to C in HBV ADWl and HBV ADRCG.
• CJP 114 which contained a G rather than an A at n.t.n. 1607, is complementary to the plus stand of the HBV ADWl strain. Although the assay for inhibition of HBV replication employed in these experiments used an HBV ADW2 strain, which has a T at n.t.n. 1607, CJP 114 nevertheless blocked replication of HBV ADW2.
• Oligodeoxynucleotide CJP 108 is a 24-mer, differing from CJP 114 in that it has an additional three nucleotides complementary to the DR2 5' flanking region. Surprisingly, CJP 108 does not inhibit HBV replication. Similarly, CJP 109, a 21-mer lacking (relative to CJP 108) three nucleotides from the DR2 3' flanking region, does not inhibit HBV replication. Oligonucleotide CJP 126, having the sequence of HBV 10011 with a blocking group at the 3" end, also exhibited decreased antiviral activity.
• flanking sequences or groups at the 3"-end of the HBV 10011 sequence may be detrimental to antiviral activity. It appears that nucleotides complementary to the entire DR2 region as well as several nucleotides from both the 5' and 3" flanking regions are required for antiviral activity. Oligdeoxynucleotide CJP 113, a 14-mer containing nucleotides complementary to the entire DR2 but only two nucleotides from the 5" flanking region and a single nucleotide from the 3' flanking region, did not block HBV replication.
• CJP 111 containing nucleotides complementary to eight of eleven of the DR2 nucleotides and six nucleotides from the 5' flanking region, did not inhibit HBV replication.
• CJP 101 and CJP 110 both 10- mers complementary to six of the eleven DR2 nucleotides and five 5' flanking nucleotides, did not inhibit HBV replication.
• CJP 112 a 14-mer containing nucleotides complementary to four of eleven of the DR2 nucleotides plus ten nucleotides from the 5' flanking region, did not inhibit HBV replication.
• CJP 100 an 11-mer complementary to five of the eleven DR2 nucleotides and six 3" flanking nucleotides, did not inhibit HBV replication.
• antisense oligonucleotides designed to hybridize to a putative loop region of the DR2 region did not exhibit antiviral activity.
• CJP 111 and CJP 110 did not exhibit antiviral activity.
• CJP 113, CJP 109, CJP 108, CJP 112 did not exhibit antiviral activity.
• HBV 10011 and CJP 114 which contain a single nucleotide difference, did exhibit antiviral activity. This suggests that two nucleotides at the 5' end of antisense oligonucleotides HVB 10011 and CJP 114 may not be important for antiviral activity.
• oligonucleotide CJP 140 identical to HBV 10011 and CJP 114 except that the two 5' nucleotides of the latter were deleted, was synthesized and tested. CJP 140 was found to inhibit completely HVB replication. Accordingly, it appears that more than four 5' flanking nucleotides are unnecessary for full antiviral activity. While up to twelve, preferably only up to six, 5' flanking nucleotides may be included, oligomers having only up to four 5' flanking nucleotides are more preferred for ease of synthesis and pharmaceutical delivery.
• oligonucleotide HBV 1018 (SEQ ID NO: 19) , which is an 11-mer complementary to the DR2 sequence alone, without either 3' or 5' flanking sequences, was synthesized and tested. It failed to inhibit HBV replication.
• Oligonucleotide CJP 150 (SEQ ID NO: 20) , which is identical to CJP 114 except for a single nucleotide deletion at the 3' end, reduced the amount of HBsAg in transfected cell supernatants to 5% of levels found in supernatants of cells transfected with the HBV plasmid DNA alone. Since oligonucleotide CJP 150 exhibits a slightly decreased antiviral activity relative to oligonucleotides HBV 10011 and CJP 114, it appears that the A residue complementary to nucleotide 1588 of the HBV genome may be necessary for full antiviral activity. A modified oligonucleotide having the sequence 5'- ACGTGCAGAGGTGAAGCG-3' (SEQ ID NO: 21) would also be expected to have antiviral activity.
• oligonucleotides targeted to a narrow region of DR2 sequence exhibit antiviral activity.
• Table 2 There appears to be no apparent correlation between the putative secondary structure (Fig. 1) and antiviral activity observed experimentally. However, not only RNA structure but also its interaction with proteins may be important for antiviral activity. This must be determined experimentally.
• Preliminary evidence indicates that the oligonucleotides of the invention exhibit specific antiviral activity against HBV since these oligonucleotides are non-toxic in a mitochondrial enzyme assay and there is no evidence, by gross morphology, of toxicity to hepatocytes in culture.
• (+) indicates positive antiviral activity
• (+) indicates lack of antiviral activity
• HBV 10011 and CJP 114 both of which map to nucleotide positions 1588 to 1609 inclusive of the hepatitis B genome (numbering relative to HPBV ADW from GenBank) , prevent the accumulation of detectable levels of HBsAg in cell supernatant.
• HBV 10011 and CJP 114 differ from each other only at position 1607, with HBV 10011 containing an A and CJP 114 containing a G.
• Antiviral oligonucleotides CJP 101 which corresponds to positions 1588 to 1598, does not inhibit HBV surface antigen expression. This indicates that some or all of the nucleotides mapping from positions 1599 to 1609 are essential for an antiviral effect. Oligonucleotide CJP 100, which corresponds to position 1599 to 1609, has no effect on the accumulation of Hepatitis B surface antigen. This indicates that some or all of the nucleotides corresponding to positions 1588 to 1597 are also critically important for the inhibition effected by oligonucleotides HBV 10011 and CJP 114.
• Oligonucleotide CJP 113 corresponds to positions 1590 to 1603 and is not active as an inhibitor of Hepatitis B replication. This indicates that all or some of the nucleotides mapping at positions 1588, 1589, 1604, 1605, 1606, 1607, 1608, and 1609 are required for antiviral activity. Oligonucleotide CJP 108 is identical to CJP 114, except that CJP 108 contains an additional three nucleotides at the 3' end; however, CJP 108 does not exhibit antiviral activity. One or more of the extra three 3' nucleotides appear to abolish the antiviral activity.
• Oligonucleotide CJP 109 also includes the same three 3' terminal nucleotides as does CJP 108, and CJP 109 is also inactive in the assay. Similarly, oligonucleotides CJP 112 and CJP 111 exhibit no antiviral activity in the assay. Both contain a 3" nucleotide or nucleotides which may abolish activity, as well as lacking important 5" sequences present in HBV 10011 and CJP 114.
• DR2 oligonucleotides (shown 5' to 3') would also be expected to exhibit anti-HBV activity: OLIGO CJP 141 CGTGCAGAGGTGAAGCGA (SEQ ID NO: 25) ; OLIGO CJP 142 GTGCAGAGGTGAAGCGA (SEQ ID NO: 26) ; OLIGO CJP 143 TGCAGAGGTAAGCGA (SEQ ID NO: 27) ; OLIGO CJP 144 GCAGAGGTGAAGCGA (SEQ ID NO: 28) ; OLIGO CJP 145 CAGAGGTGAAGCGA (SEQ ID NO: 29) ; OLIGO CJP 146 AGAGGTGAAGCGA (SEQ ID NO: 30); OLIGO CJP 147 GAGGTGAAGCGA (SEQ ID NO: 31) ; OLIGO CJP 148 AGGTGAAGCGA (SEQ ID NO: 32); OLIGO CJP 149 CGACG

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