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WO2019240504A1 - Oligonucléotides modifiés pour l'inhibition de l'expression d'un gène cible - Google Patents

Oligonucléotides modifiés pour l'inhibition de l'expression d'un gène cible Download PDF

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WO2019240504A1
WO2019240504A1 PCT/KR2019/007112 KR2019007112W WO2019240504A1 WO 2019240504 A1 WO2019240504 A1 WO 2019240504A1 KR 2019007112 W KR2019007112 W KR 2019007112W WO 2019240504 A1 WO2019240504 A1 WO 2019240504A1
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modified oligonucleotide
lnas
modified
pentose sugar
target gene
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Kyun Hwan Kim
Ah Ram Lee
Doo Hyun Kim
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Am Sciences Co Ltd
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Am Sciences Co Ltd
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    • 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
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/712—Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
    • 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
    • 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
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • 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
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • 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
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/318—Chemical structure of the backbone where the PO2 is completely replaced, e.g. MMI or formacetal
    • C12N2310/3181—Peptide nucleic acid, PNA
    • 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
    • C12N2310/321—2'-O-R Modification
    • 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
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a modified oligonucleotide that binds to a target gene sequence; a pharmaceutical composition comprising the oligonucleotide; and a method of inhibiting expression of a target gene using the oligonucleotide or the pharmaceutical composition.
  • Hepatitis B virus (hereinafter referred to as “HBV”) is the most common cause of viral infection in humans, and more than about 350 million people worldwide are infected with HBV.
  • liver diseases such as chronic hepatitis, cirrhosis, liver cancer, etc . can occur, resulting in death due to severe viral liver disease.
  • HBV carries DNA in its genome and is one of the viruses with the smallest genome among the viruses known to date (Ganem and Prince N Engl J Med (2004) 350, 1118-1129).
  • cccDNA covalently closed circular DNA
  • cccDNA is a minichromosome in the form of an episome and is known to be a major cause of chronic infection because it not only makes all RNAs of HPV but also the present therapeutic agents cannot eliminate the same (Urban et al. J Hepatol (2010) 52, 282-284).
  • pregenomic RNA which can be converted from cytoplasm to genomic DNA.
  • the HBV virions that have been successfully converted from pregenomic RNA to DNA are budded.
  • the budded HBV virions are then infected by peripheral hepatocytes or re-infected, and thus they steadily proliferate.
  • An object of the present invention is to provide a drug capable of recognizing a target gene sequence and inhibiting the target gene sequence; that is, an object of the present invention is to provide an oligonucleotide capable of forming a G-quadruplex, which comprises a consecutive guanine sequence while including a sense sequence to the target gene sequence.
  • modified oligonucleotides that bind to a target gene sequence represented by the formula: A x -G y -B z wherein, A is a sense sequence to the target gene sequence and x is an integer from 1-16, G is guanine and y is an integer from 4 to 6, and B is a sense sequence to the target gene sequence and z is an integer from 1-16, wherein at least one nucleotide of the modified oligonucleotide is a locked nucleic acid (LNA) and the modified oligonucleotide forms a G-quadruplex upon binding to the target gene sequence.
  • LNA locked nucleic acid
  • y is 4. In some embodiments, y is 5.
  • G is 6. In some embodiments, G is not an LNA. In some embodiments, G comprises a pentose sugar that is deoxyribose. In some embodiments, G comprises a pentose sugar that comprises a 2' O-methyl modification. In some embodiments, x is 4, y is 6, and z is 6. In some embodiments, x is 5, y is 5, and z is 6. In some embodiments, x is at least 4 but no more than 12, y is 5, and z is at least 4 but no more than 12. In some embodiments, LNA comprises a methylene linkage between a 2' oxygen and 4' carbon of a pentose sugar. In some embodiments, x is 4, and at least two As are LNAs.
  • x is 4, and two As are LNAs and the other two As comprise a pentose sugar that is deoxyribose.
  • z is 6, and at least four of the Bs are LNAs.
  • z is 6, and four of the Bs are LNAs and the other two Bs comprise a pentose sugar that is deoxyribose.
  • y is 6, and G comprises a pentose sugar that is deoxyribose.
  • x is 4, and two As are LNAs and the other two As comprise a pentose sugar that is deoxyribose;
  • y is 6, and G comprises a pentose sugar that is deoxyribose; and
  • z is 6, and four of the Bs are LNAs and the other two Bs comprise a pentose sugar that is deoxyribose.
  • the modified oligonucleotide comprises phosphorothioate linkages.
  • x is 4, and all four As are LNAs.
  • z is 6, and all six Bs are LNAs.
  • y is 6, and G is a pentose sugar that comprises a 2' O-methyl modification.
  • (a) x is 4, and all four As are LNAs
  • (b) y is 6, and G is a pentose sugar that comprises a 2' O-methyl modification
  • (c) z is 6, and all six Bs are LNAs.
  • the modified oligonucleotide comprises phosphorothioate linkages.
  • the modified oligonucleotide comprises a combination of phosphorothioate and phosphodiester linkages.
  • the Gs are connected by phosphodiester linkages.
  • the Gs are connected by phosphodiester linkages and the As are connected by phosphorothioate linkages and the Bs are connected by phosphorothioate linkages.
  • x is 4, and at least three As are LNAs.
  • x is 4, and three As are LNAs and the other A comprises a pentose sugar that is deoxyribose.
  • z is 6, and at least three of the Bs are LNAs.
  • z is 6, and three of the Bs are LNAs and the other three Bs comprise a pentose sugar that is deoxyribose.
  • (a) x is 4, and three As are LNAs and the other A comprises a pentose sugar that is deoxyribose; (b) y is 6, and G comprises a pentose sugar that is deoxyribose; and (c) z is 6, and three of the Bs are LNAs and the other three Bs comprise a pentose sugar that is deoxyribose.
  • the modified oligonucleotide comprises phosphorothioate linkages.
  • the target gene sequence may be included in the scope of the present invention without limitation of its sequence, size, origin, etc ., as long as it is recognized by the oligonucleotide of the present invention and is capable of binding to the oligonucleotide.
  • the target gene sequence is a viral gene sequence.
  • the target gene sequence is not a hepatitis B sequence.
  • the viral gene sequence is a sequence from a virus selected from the group consisting of HIV-1, HSV-1, EBV, KSHV, HHV-6, HCV, HPV, ZIKV, SARS Co-V, EBOV, and influenza virus, but it is not limited thereto.
  • influenza virus may be influenza A virus or influenza B virus.
  • influenza A virus may be various subtype viruses such as H1N1 and H3N2 strains, which include A/Puerto Rico/8/34 (H1N1, PR8) and A/Hong Kong/8/68 (H3N2, HK), as well as H3N8, H5N1, H5N8, H7N9, and H9N2 strains.
  • influenza B virus may be viruses of Yamagata lineage and Victoria lineage, which include B/Lee/40 (Lee), but is not limited thereto.
  • the modified oligonucleotide forms a G-quadruplex upon binding to the target gene sequence and results in inhibition of transcription of the target gene sequence.
  • the modified oligonucleotide forms a G-quadruplex upon binding to the target gene sequence and triggers degradation of the target gene sequence.
  • compositions comprising: (a) a modified oligonucleotide; and (b) a pharmaceutically acceptable excipient.
  • a modified oligonucleotide wherein the modified oligonucleotide comprises (a) at least four consecutive guanines (GGGG) and (b) at least one locked nucleic acid (LNA), wherein the modified oligonucleotide comprises a sense sequence to the target gene sequence and forms a G-quadruplex upon binding to the target gene sequence resulting in target gene expression inhibition.
  • the modified oligonucleotide comprises four consecutive guanines (GGGG).
  • the modified oligonucleotide comprises five consecutive guanines (GGGGG).
  • the modified oligonucleotide comprises six consecutive guanines (GGGGGG). In some embodiments, the at least four consecutive guanines are not LNAs. In some embodiments, the at least four consecutive guanines comprise a pentose sugar that is deoxyribose. In some embodiments, the at least four consecutive guanines comprise a pentose sugar that comprises a 2' O-methyl modification. In some embodiments, the modified oligonucleotide comprises 5 to 30 nucleotides. In some embodiments, the modified oligonucleotide comprises 12 to 24 nucleotides. In some embodiments, the modified oligonucleotide comprises 14 nucleotides.
  • the modified oligonucleotide comprises 15 nucleotides. In some embodiments, the modified oligonucleotide comprises 16 nucleotides. In some embodiments, the modified oligonucleotide comprises 17 nucleotides. In some embodiments, the modified oligonucleotide comprises 18 nucleotides. In some embodiments, the LNA comprises a methylene linkage between a 2' oxygen and 4' carbon of a pentose sugar.
  • the modified oligonucleotide of the target gene sequence is represented by the formula: A x -G y -B z wherein, A is a sense sequence to the target gene sequence and x is an integer from 1-16, G is guanine and y is an integer from 4 to 6, and B is a sense sequence to the target gene sequence and z is an integer from 1-16.
  • x is 4, and at least two As are LNAs.
  • x is 4, and two As are LNAs and the other two As comprise a pentose sugar that is deoxyribose.
  • z is 6, and at least four of the Bs are LNAs.
  • z is 6, and four of the Bs are LNAs and the other two Bs comprise a pentose sugar that is deoxyribose.
  • y is 6, and G comprises a pentose sugar that is deoxyribose.
  • (a) x is 4, and two As are LNAs and the other two As comprise a pentose sugar that is deoxyribose
  • (b) y is 6, and G comprises a pentose sugar that is deoxyribose
  • (c) z is 6, and four of the Bs are LNAs and the other two Bs comprise a pentose sugar that is deoxyribose.
  • the modified oligonucleotide comprises phosphorothioate linkages.
  • x is 4, and all four As are LNAs.
  • z is 6, and all six Bs are LNAs.
  • y is 6, and G is a pentose sugar that comprises a 2' O-methyl modification.
  • (a) x is 4, and all four As are LNAs
  • (b) y is 6, and G is a pentose sugar that comprises a 2' O-methyl modification
  • (c) z is 6, and all six Bs are LNAs.
  • the modified oligonucleotide comprises phosphorothioate linkages.
  • the modified oligonucleotide comprises a combination of phosphorothioate and phosphodiester linkages.
  • the Gs are connected by phosphodiester linkages.
  • the Gs are connected by phosphodiester linkages and the As are connected by phosphorothioate linkages and the Bs are connected by phosphorothioate linkages.
  • x is 4, and at least three As are LNAs.
  • x is 4, and three As are LNAs and the other A comprises a pentose sugar that is deoxyribose.
  • z is 6, and at least three of the Bs are LNAs.
  • z is 6, and three of the Bs are LNAs and the other three Bs comprise a pentose sugar that is deoxyribose.
  • (a) x is 4, and three As are LNAs and the other A comprises a pentose sugar that is deoxyribose
  • (b) y is 6, and G comprises a pentose sugar that is deoxyribose
  • (c) z is 6, and three of the Bs are LNAs and the other three Bs comprise a pentose sugar that is deoxyribose.
  • the modified oligonucleotide comprises phosphorothioate linkages.
  • the target gene sequence is a viral gene sequence.
  • the target gene sequence is not a hepatitis B sequence.
  • the viral gene sequence is a sequence from a virus selected from the group consisting of HIV-1, HSV-1, EBV, KSHV, HHV-6, HCV, HPV, ZIKV, SARS Co-V, EBOV, and influenza virus.
  • target gene expression inhibition comprises an inhibition of transcription of the target gene sequence.
  • the G-quadruplex triggers degradation of the target gene sequence.
  • composition comprising the oligonucleotide EH of the present invention can inhibit expression of a target gene and exhibit antiviral activity by recognizing a target sequence of a specific sequence.
  • FIG. 1 illustrates a sequence screening diagram obtained by analysis of the hepatitis B virus (HBV) genome, which illustrates the structure of the HBV genome and the positions of D1 to D9 oligonucleotides in the HBV genome
  • HBV hepatitis B virus
  • FIGS. 2A-2D illustrate oligonucleotides (D1, D2, and D6) have inhibitory effects on HBeAg levels ( FIG. 2A ), HBsAg levels ( FIG. 2B ), on replication ( FIGS. 2C-2D ) in HBV.
  • FIGS. 3A-3F illustrate oligonucleotide D2 inhibition of transcription of viral mRNAs.
  • pg/preC RNA indicates pregenomic and precore RNA
  • pre-S/S RNA indicates surface RNAs
  • HBx RNA indicates RNA encoding the HBx protein.
  • FIG. 4 illustrates oligonucleotide D2 inhibition of expression of viral surface proteins.
  • ⁇ -actin is a loading control
  • L, M, and S indicate three types of surface proteins.
  • L, M, and S represent large, medium, and small surface proteins, respectively.
  • FIGS. 5A-5C illustrate the results of luciferase reporter assays showing that oligonucleotide D2 inhibits the activity of the HBV enhancer.
  • FIGS. 6A-6F illustrate oligonucleotide D2 inhibits activity of the HBV enhancer.
  • FIGS. 7A-7B illustrate an electrophoretic mobility shift assay (EMSA) ( FIG. 7B ) showing that oligonucleotide D2 binds to HBV enhancer I and II sequences and forms a G-quadruplex.
  • EMSA electrophoretic mobility shift assay
  • FIG. 8 illustrates an EMSA result showing that oligonucleotide D2 partially forms a G-quadruplex with the region of the HBV enhancer II.
  • FIG. 9 illustrates an EMSA result showing that oligonucleotide D2 forms a G-quadruplex by recognizing its own base sequence.
  • FIGS. 10A-10C illustrate an EMSA result ( FIG. 10B ) showing oligonucleotide D3, having a point mutation, could not form a G-quadruplex. D2 was able to form a G-quadruplex.
  • FIGS. 11A-11J illustrate schematics and results of optimization experiments for length of oligonucleotides.
  • FIG. 12A illustrates a bar graph of activity of each modified D2 oligonucleotide in inhibiting HBV, which is measured by a luciferase activity assay: PS indicates phosphorothioate-modified D2, Ome indicates O-methyl-modified D2, PNA indicates PNA-modified D2, PS-Ome indicates phosphorothioate/O-methyl-modified D2, and PS-LNA indicates phosphorothioate/ locked nucleic acid (LNA)-modified D2.
  • PS indicates phosphorothioate-modified D2
  • Ome indicates O-methyl-modified D2
  • PNA indicates PNA-modified D2
  • PS-Ome indicates phosphorothioate/O-methyl-modified D2
  • PS-LNA indicates phosphorothioate/ locked nucleic acid (LNA)-modified D2.
  • FIGS. 12B-12C illustrate schematics of modified D2 oligonucleotides and experimental testing.
  • FIGS. 12D-12E illustrate graphs of HBeAg levels ( FIG. 12D ) and HBsAg levels ( FIG. 12E ) following treatment with modified oligonucleotides.
  • FIG. 13A illustrates an experimental process for HBV infection in HepG2-NT CP cells.
  • FIG. 13B illustrates the effect of PS-LNA D2 on HBV replication in the HepG2-NTCP model.
  • FIGS. 13C-13D illustrate graphs of HBeAg levels ( FIG. 13C ) and HBsAg levels ( FIG. 13D ) following treatment with PS-LNA D2.
  • FIG. 14A illustrates an experimental process for HBV infection and viral protein analysis in HepG2-NTCP cells.
  • FIGS. 14B-14F illustrate results showing the expression levels of HBV proteins. depending upon treatment of modified oligonucleotides: PS indicates phosphorothioate-modified D2, PS-Ome indicates phosphorothioate/O-methyl-modified D2, and PS-LNA indicates phosphorothioate/LNA-modified D2.
  • D2 D2, T.F
  • D2, Tr unmodified D2
  • LMV indicates lamivudine.
  • "D 3" and "D 7" indicate Day 3 and Day 7, respectively.
  • FIG. 15A illustrates an experimental process for HBV infection or IFN-alpha (IFN- ⁇ ) infection.
  • FIGS. 15B-15C illustrate graphs of HBeAg levels ( FIG. 15B ) and HBsAg levels ( FIG. 15C ) following treatment with modified D2s (PS-LNA (3, 3), PS-LNA (4, 4), PS-LNA all), IFN-alpha, or D 2 TR (unmodified D2).
  • modified D2s PS-LNA (3, 3), PS-LNA (4, 4), PS-LNA all
  • IFN-alpha IFN-alpha
  • D 2 TR unmodified D2 TR
  • FIG. 15D illustrates percentage of RC DNA and cccDNA (y-axis) following mock, PS-LNA (3, 3), PS-LNA (4, 4), PS-LNA all, IFN-alpha, and D 2 TR (unmodified D2) treatment.
  • FIG. 16A illustrates a schematic of HBV infection in PHH or HepaRG cells.
  • FIGS. 16B-16D illustrates results from HBV infection in PHH or HepaRG cells measuring HBeAg levels ( FIG. 16B ), HBsAg levels (FIG. 16C ), and cccDNA levels ( FIG. 16D ).
  • FIG. 17A illustrates an experimental process for HBV infection with PS-LNA D2, Novira, IFN-alpha, TDF, and TDF+PS-LNA.
  • FIGS. 17B-17C illustrate results from HBV infection with PS-LNA D2, Novira, IFN-alpha, TDF, and TDF+PS-LNA measuring HBeAg levels ( FIG. 17B ) and HBsAg levels ( FIG. 17C ) at Day 5 (D 5), Day 11 (D 11), and Day 16 (D 16).
  • FIGS. 18A-18D illustrates result from HBV infection with PS-LNA D2, Novira, IFN-alpha, TDF, and TDF+PS-LNA measuring HBeAg levels ( FIGS. 18A-18B ) and HBsAg levels ( FIGS. 18C-18D ) at Day 5 (D 5), Day 11 (D 11), and Day 16 (D 16).
  • FIG. 18E illustrates an experimental process for HBV infection and treatment of PS-LNA D2.
  • FIGS. 18F-18J illustrates results from HBV infection and treatment of PS-LNA D2. "D 5" indicates Day 5 and “D 11" indicates Day 11.
  • FIGS. 19A-19B illustrate experimental processes for HBV infection in HepaRG cells.
  • FIGS. 19C-19E illustrate results from HBV infection in HepaRG cells with mock, PS-LNA D2, NVR3-778, and IFN-alpha.
  • FIGS. 20A-20F illustrate data showing unmodified D2 is a pan-genotype HBV inhibitor.
  • FIGS. 21-21C illustrate schematics of modified oligonucleotides.
  • FIG. 22A illustrates an experimental process for HBV infection and viral protein analysis in primary human hepatocytes (PHHs).
  • FIGS. 22B-22C illustrate results showing the expression levels of HBV proteins depending upon treatment of modified oligonucleotides: PS indicates phosphorothioate-modified D2, PS-Ome indicates phosphorothioate/O-methyl-modified D2, and PS-LNA indicates phosphorothioate/LNA-modified D2.
  • D2 D2
  • D2 T.F
  • D2 unmodified D2
  • D2 unmodified D2
  • FIG. 23A illustrates results of a luciferase assay showing the anti-HBV activity of modified D2 species: PS indicates a modified D2 species, wherein a backbone was modified with phosphorothioate (PS), PS-Ome (4,4) indicates a modified D2 species, wherein a backbone was modified with PS and 4 nucleotides at each end of 5' and 3' ends were modified with O-methyl, PS-Ome (5,5) indicates a modified D2 species, wherein a backbone was modified with PS and 5 nucleotides at each end of 5' and 3' ends were modified with O-methyl, PS-Ome (all) indicates a modified D2 species, wherein a backbone was modified with PS and all nucleotides were modified with O-methyl, PS-LNA (2,2) indicates a modified D2 species, wherein a backbone was modified with PS and 2 nucleotides at each end of 5' and 3' ends were modified with LNA, PS-LNA (3,3) indicates a
  • FIG. 23B illustrates a HepG2 transfection model
  • FIG. 23C illustrates a graph of HBsAg from a HepG2 transfection model.
  • FIG. 23D illustrates a graph of HBeAg from a HepG2 transfection model.
  • FIG. 23E illustrates a HepG2-NTCP infection model.
  • FIG. 23F illustrates a graph of HBsAg from a HepG2-NTCP transfection model.
  • FIG. 23G illustrates a graph of HBeAg from a HepG2-NTCP transfection model.
  • FIG. 23H illustrates a PHH infection model
  • FIG. 23I illustrates a graph of HBeAg from a PHH transfection model.
  • FIG. 23J illustrates a graph of HBsAg from a PHH transfection model.
  • FIG. 24A illustrates an experimental process of HBV infection and treatment with modified D2.
  • FIGS. 24B-24D illustrate graphs of data from infection of PHH cells.
  • FIGS. 24E-24F illustrate schematic of optimized modified oligonucleotides.
  • FIGS. 25A-25E illustrate graphs of IC 50 of selected optimized modified oligonucleotides.
  • FIGS. 26A-26F illustrate data of IC 50 of selected optimized modified oligonucleotides.
  • FIGS. 27A-27F illustrate data of IC 50 of selected optimized modified oligonucleotides.
  • FIGS. 28A-28E illustrate graphs of cytokine induction after treatment with modified oligonucleotides.
  • FIG. 29A illustrates RFU (y-axis) versus number of cycles (x-axis) of RT-qPCR of PS-LNA D2 (3,3).
  • FIG. 29B illustrates a graph of Cq (y-axis) versus uM of PS-LNA D2 (x-axis).
  • FIG. 30A illustrates a schematic of PS-LNA D2 oligonucleotides transfected in HepG2 cells.
  • FIG. 30B illustrates a graph of Cq (y-axis) versus sample dilution of PS-LNA D2.
  • FIG. 31A illustrates a schedule for an in vivo experiment.
  • FIGS. 31B-31C illustrate results of measuring the expression levels of HBeAg and HBsAg viral proteins, respectively.
  • the first bar in each of ( FIG. 31B ) and ( FIG. 31C ) graphs represents mice administered with HBV DNA and an empty vector and the second bar represents mice, i.e., an experimental group, administered with HBV DNA and D2.
  • FIG. 31D illustrates Southern blot results.
  • FIG. 32A illustrates an experimental schedule for an in vivo intravenous (IV) injection.
  • FIGS. 32B-32C illustrate results showing the expression levels of HBeAg and HBsAg viral proteins, respectively.
  • FIG. 32D illustrates result of confirming with Southern blotting.
  • FIG. 33A illustrates the procedure of infecting PHHs with HBV.
  • FIGS. 33B-33C illustrate results the expression levels of HBeAg and HBsAg viral proteins, respectively.
  • FIG. 33D illustrates Southern blot results.
  • FIGS. 34A-34H illustrate graphs of a pAAV-HBV mouse model.
  • FIG. 35A illustrates a schematic of determination of injection route in chronic HBV model.
  • FIGS. 35B-35H illustrate graphs of in vivo efficacy studies to determine injection routes.
  • FIG. 35I illustrates a southern blot of in vivo efficacy studies to determine injection routes.
  • FIGS. 36A-36B illustrate graphs of tissue distribution of two experiments of modified oligonucleotide #42.
  • FIGS. 36C-36D illustrate graphs of tissue distribution of two experiments of modified oligonucleotide 3,3.
  • FIGS. 37A-37B illustrate graphs of tissue distribution of two experiments of modified oligonucleotide PS-LNA D2 (3, 3).
  • FIGS. 37C-37H illustrate graphs of drug delivery ability of modified oligonucleotides in HepG2 cells or PHH cells.
  • FIG. 38A illustrates a schematic of HBV infection with modified D2 and IFN-alpha.
  • FIGS. 38B-38E illustrate graphs of HBeAg levels ( FIG. 38B ), HBsAg levels ( FIG. 38C ), relative HBV RC DNA level ( FIG. 38D ), and relative HBV cccDNA level ( FIG. 38E )
  • FIG. 39A illustrates a schematic of HBV infection with modified D2, IFN-alpha, or LMV (lamivudine).
  • FIGS. 39B-39C illustrate graphs of HBeAg levels ( FIG. 39B ) and HBsAg levels (FIG. 39C ).
  • FIG. 39D illustrates relative HBV RC DNA level and relative HBV cccDNA level.
  • FIGS. 40A-40C illustrate results of NTCP cells were infected with HBV and treated with the modified D2.
  • FIGS. 41A-41D illustrate levels of inhibition of HBeAg and HBsAg when nucleotide length was shortened on both ends (L-1 to L-4, R-1 and R-2) around the (G)6 sequence of D2 oligonucleotide.
  • FIGS. 42A-42B illustrate levels of inhibition of HBeAg and HBsAg when the nucleotide length was increased to 18, 20, 22, 24mer, and 24mer L, 24mer R around the (G)6 sequence of the D2 oligonucleotide.
  • FIGS. 43A-43B illustrates level of inhibition of HBeAg and HBsAg when one of the (G)6 sequences of the D2 oligonucleotide was substituted with C (D2-1, D2-2) or when nucleotides sequences other than (G)6 are substituted with a sequence derived from hepatitis B virus of other genotypes (D2-3, D2-4).
  • FIGS. 44A-44B illustrate level of inhibition of HBeAg and HBsAg when the length of the nucleic acid sequence was decreased (D6-1) or increased (D6-2 to D6-8) from the 5 'or the 3' end around the (G)5 sequence of the D6 oligonucleotide; (D6-9, D6-10) or when nucleotides sequences other than the (G)5 sequence are substituted with s sequence derived from the hepatitis B virus of other genotypes (D6-9, D6-10).
  • FIG. 45 illustrates antiviral activity of the D2 oligonuleotide against influenza virus PR8.
  • nucleic acid encompasses double- or triple-stranded nucleic acids, as well as single-stranded molecules.
  • nucleic acid strands need not be coextensive (i.e., a double-stranded nucleic acid need not be double-stranded along the entire length of both strands).
  • Nucleic acid sequences, when provided, are listed in the 5' to 3' direction, unless stated otherwise.
  • a "nucleic acid” as referred to herein can comprise at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, or more bases in length.
  • oligonucleotides coding or non-coding regions of a gene or gene fragment, intergenic DNA, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), small nucleolar RNA, ribozymes, complementary DNA (cDNA), which is a DNA representation of mRNA, usually obtained by reverse transcription of messenger RNA (mRNA) or by amplification; DNA molecules produced synthetically or by amplification, genomic DNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • cDNA encoding for a gene or gene fragment referred to herein may comprise at least one region encoding for exon
  • HIV human immunodeficiency virus
  • HSV herpes simplex virus
  • influenza influenza
  • the modified oligonucleotides inhibit transcription by binding to a target gene sequence and forming a G-quadruplex.
  • the modified oligonucleotides comprise a sense sequence.
  • the modified oligonucleotides comprise one or more modifications.
  • Modified oligonucleotides as described herein, in some embodiments, comprise the following formula: A x -G y -B z .
  • A is a sense sequence to the target gene sequence.
  • x is an integer from 1-16.
  • x is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.
  • x is an integer of no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.
  • G is a guanine.
  • y is an integer from 4 to 6.
  • y is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • B is a sense sequence to the target gene sequence.
  • z is an integer from 1-16. In some embodiments, z is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, z is an integer of no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.
  • modified oligonucleotides described herein comprise a sense sequence. In some embodiments, the modified oligonucleotides comprise a sense sequence to a target gene sequence.
  • the modified oligonucleotide comprises about 15 to about 30, from about 18 to about 25, from about 18 to about 24, from about 19 to about 23, or from about 20 to about 22 nucleotides in length. In some embodiments, the modified oligonucleotide comprises at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the modified oligonucleotide comprises about 10 nucleotides in length. In some embodiments, the modified oligonucleotide comprises about 12 nucleotides in length. In some embodiments, the modified oligonucleotide comprises about 14 nucleotides in length.
  • the modified oligonucleotide comprises about 15 nucleotides in length. In some embodiments, the modified oligonucleotide comprises about 16 nucleotides in length. In some embodiments, the modified oligonucleotide comprises about 17 nucleotides in length. In some embodiments, the modified oligonucleotide comprises about 18 nucleotides in length. In some embodiments, the modified oligonucleotide comprises about 20 nucleotides in length. In some embodiments, the modified oligonucleotide comprises about 25 nucleotides in length. In some embodiments, the modified oligonucleotide comprises about 30 nucleotides in length.
  • modified oligonucleotides comprising one or more modifications.
  • the modified oligonucleotide comprises natural or synthetic or artificial nucleotide analogues or bases.
  • the modified oligonucleotide comprises combinations of DNA, RNA and/or nucleotide analogues.
  • the synthetic or artificial nucleotide analogues or bases comprise modifications at one or more of ribose moiety, phosphate moiety, nucleoside moiety, or a combination thereof.
  • nucleotide analogues or artificial nucleotide base comprise a nucleic acid with a modification at a 2' hydroxyl group of the ribose moiety.
  • the modification includes an H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, wherein R is an alkyl moiety.
  • alkyl moiety includes, but is not limited to, halogens, sulfurs, thiols, thioethers, thioesters, amines (primary, secondary, or tertiary), amides, ethers, esters, alcohols and oxygen.
  • the alkyl moiety further comprises a modification.
  • the modification comprises an azo group, a keto group, an aldehyde group, a carboxyl group, a nitro group, a nitroso, group, a nitrile group, a heterocycle (e.g., imidazole, hydrazino or hydroxylamino) group, an isocyanate or cyanate group, or a sulfur containing group (e.g., sulfoxide, sulfone, sulfide, and disulfide).
  • the alkyl moiety further comprises a hetero substitution.
  • the carbon of the heterocyclic group is substituted by a nitrogen, oxygen or sulfur.
  • the heterocyclic substitution includes but is not limited to, morpholino, imidazole, and pyrrolidino.
  • the modification at the 2' hydroxyl group is a 2'-O-methyl modification or a 2'-O-methoxyethyl (2'-O-MOE) modification.
  • the 2'-O-methyl modification adds a methyl group to the 2' hydroxyl group of the ribose moiety whereas the 2'O-methoxyethyl modification adds a methoxyethyl group to the 2' hydroxyl group of the ribose moiety.
  • Exemplary chemical structures of a 2'-O-methyl modification of an adenosine molecule and 2'O-methoxyethyl modification of an uridine are illustrated below.
  • the modification at the 2' hydroxyl group is a 2'-O-aminopropyl modification in which an extended amine group comprising a propyl linker binds the amine group to the 2' oxygen.
  • this modification neutralizes the phosphate derived overall negative charge of the oligonucleotide molecule by introducing one positive charge from the amine group per sugar and thereby improves cellular uptake properties due to its zwitterionic properties.
  • An exemplary chemical structure of a 2'-O-aminopropyl nucleoside phosphoramidite is illustrated below.
  • the modification is a locked or bridged ribose modification (e.g., locked nucleic acid or LNA).
  • the oxygen molecule bound at the 2' carbon is linked to the 4' carbon by a methylene group, thus forming a 2′'- C ,4′'- C -oxy-methylene-linked bicyclic ribonucleotide monomer.
  • Exemplary representations of the chemical structure of LNA are illustrated below. The representation shown to the left highlights the chemical connectivities of an LNA monomer. The representation shown to the right highlights the locked 3′-endo ( 3 E) conformation of the furanose ring of an LNA monomer.
  • the modification comprises ethylene nucleic acids (ENA) such as for example 2'-4'-ethylene-bridged nucleic acid, which locks the sugar conformation into a C 3 '-endo sugar puckering conformation.
  • ENA ethylene nucleic acids
  • the bridged nucleic acids class of modified nucleic acids that also comprises LNA. Exemplary chemical structures of the ENA and bridged nucleic acids are illustrated below.
  • additional modifications include 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), T-O- dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamido (2'-O-NMA).
  • nucleotide analogues comprise modified bases such as, but not limited to, 5-propynyluridine, 5-propynylcytidine, 6- methyladenine, 6-methylguanine, N, N, -dimethyladenine, 2-propyladenine, 2propylguanine, 2-aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine and other nucleotides having a modification at the 5 position, 5- (2- amino) propyl uridine, 5-halocytidine, 5-halouridine, 4-acetylcytidine, 1- methyladenosine, 2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2- methylguanosine, 7-methylguanosine, 2, 2-dimethylguanosine, 5- methylaminoethyluridine, 5-methyloxyuridine, deazanucleotides such as 7-deaza-
  • Modified nucleotides also include those nucleotides that are modified with respect to the sugar moiety, as well as nucleotides having sugars or analogs thereof that are not ribosyl.
  • the sugar moieties In some embodiments are or be based on, mannoses, arabinoses, glucopyranoses, galactopyranoses, 4'-thioribose, and other sugars, heterocycles, or carbocycles.
  • the term nucleotide also includes what are known in the art as universal bases.
  • universal bases include but are not limited to 3-nitropyrrole, 5-nitroindole, or nebularine.
  • a chemically-modified sugar moiety may be generated by substituting a hydrogen atom at the 2' position of a pentose sugar within a nucleotide with a substitution selected from the list including, but not limited to: methoxyethoxy ([2'-O-CH2CH2OCH3, 2'-O-(2-methoxyethyl);MOE], Formula 1), dimethylaminooxyethoxy ([2'-O(CH2)2ON(CH3)2;DMAOE], Formula 2), dimethylaminoethyloxyethyl ([2'-OCH2CH2-O-CH2CH2-N(CH3)2;DMAEOE], Formula 3), methoxy ([2'-OCH3;Ome], Formula 4), aminopropoxy ([2'-OCH2CH2CH2NH2;AP], Formula 5) or fluorine (2'-F, Formula 6).
  • methoxyethoxy [2'-O-CH2CH2OCH3, 2'-O-(2-
  • a chemically-modified sugar moiety may be generated by replacing a sugar moiety with fluoroarabinonucleic acid (F-ANA) (2'-F- ⁇ -D-arabinofuranosyl, Formula 7).
  • F-ANA fluoroarabinonucleic acid
  • B represents a base.
  • nucleotide analogues further comprise morpholinos, peptide nucleic acids (PNAs), methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoro N3-P5'-phosphoramidites, 1', 5'- anhydrohexitol nucleic acids (HNAs), or a combination thereof.
  • Morpholino or phosphorodiamidate morpholino oligo (PMO) comprises synthetic molecules whose structure mimics natural nucleic acid structure by deviates from the normal sugar and phosphate structures.
  • the five member ribose ring is substituted with a six member morpholino ring containing four carbons, one nitrogen and one oxygen.
  • the ribose monomers are linked by a phosphordiamidate group instead of a phosphate group.
  • the backbone alterations remove all positive and negative charges making morpholinos neutral molecules capable of crossing cellular membranes without the aid of cellular delivery agents such as those used by charged oligonucleotides.
  • peptide nucleic acid does not contain sugar ring or phosphate linkage and the bases are attached and appropriately spaced by oligoglycine-like molecules, therefore, eliminating a backbone charge.
  • modified internucleotide linkage include, but is not limited to, phosphorothioates, phosphorodithioates, methylphosphonates, 5'- alkylenephosphonates, 5'-methylphosphonate, 3'-alkylene phosphonates, borontrifluoridates, borano phosphate esters and selenophosphates of 3'-5'linkage or 2'-5'linkage, phosphotriesters, thionoalkylphosphotriesters, hydrogen phosphonate linkages, alkyl phosphonates, alkylphosphonothioates, arylphosphonothioates, phosphoroselenoates, phosphorodiselenoates, phosphinates, phosphoramidates, 3'- alkylphosphoramidates, aminoalkylphosphoramidates, thionophosphoramidates, phosphoropiper
  • phosphorothioate backbone indicates a sugar phosphate backbone in which oxygen in a phosphate group not participating in the internucleoside linkage is substituted with sulfur.
  • a nucleic acid molecule having a phosphorothioate backbone is more stable in some conditions.
  • a phosphate group of a nucleotide may be substituted with phosphorodithioate, phosphoramidate or boranophosphate, without being limited thereto.
  • Phosphorothioate, phosphorodithioate, phosphoramidate and boranophosphate containing backbones are represented by Formulas 8 to 11 below, respectively.
  • B represents a base.
  • the modification is a methyl or thiol modification such as methylphosphonate or thiolphosphonate modification.
  • exemplary thiolphosphonate nucleotide (left) and methylphosphonate nucleotide (right) are illustrated below.
  • a modified nucleotide includes, but is not limited to, 2'-fluoro N3-P5'-phosphoramidites illustrated as:
  • a modified nucleotide includes, but is not limited to, hexitol nucleic acid (or 1', 5'- anhydrohexitol nucleic acids (HNA)) illustrated as:
  • one or more modifications further optionally include modifications of the ribose moiety, phosphate backbone and the nucleoside, or modifications of the nucleotide analogues at the 3' or the 5' terminus.
  • the 3' terminus optionally include a 3' cationic group, or by inverting the nucleoside at the 3'-terminus with a 3'-3' linkage.
  • the 3'-terminus is optionally conjugated with an aminoalkyl group, e.g., a 3' C5-aminoalkyl dT.
  • the 3'-terminus is optionally conjugated with an abasic site, e.g.
  • the 5'-terminus is conjugated with an aminoalkyl group, e.g., a 5'-O-alkylamino substituent. In some embodiments, the 5'-terminus is conjugated with an abasic site, e.g. , with an apurinic or apyrimidinic site.
  • the modified oligonucleotide comprises one or more modifications as described herein. In some embodiments, the modified oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more modifications as described herein.
  • the modifications comprise 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), T-O- dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamido (2'-O-NMA) modified, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoro N3-P5'-phosphoramidites, or a combination thereof.
  • 2'-O-NMA 2'-O-
  • the modified oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more than 25 LNAs. In some embodiments, the modified oligonucleotide comprises about 1 to about 10, about 2 to about 8, about 3 to about 7, or about 4 to about 6 LNAs. In some embodiments, the modified oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more than 25 2'-O-methyl modifications. In some embodiments, the modified oligonucleotide comprises about 1 to about 10, about 2 to about 8, about 3 to about 7, or about 4 to about 6 2'-O-methyl modifications.
  • the Gs of the modified oligonucleotide comprise a 2'-O-methyl modification.
  • the modified oligonucleotide comprises a phosphorothioate linkage.
  • the modified oligonucleotide comprises a combination of phosphorothioate and phosphodiester linkages.
  • the modified oligonucleotide comprises a combination of modifications. In some embodiments, the modified oligonucleotide comprises two or more types of modifications. In some embodiments, the modified oligonucleotide comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 modifications. In some embodiments, the two or more types of modifications comprise a nucleotide, the phosphate group of which is chemically modified into phosphorothioate, phosphorodithioate, phosphoramidate or boranophosphate, and additionally, the sugar moiety of the nucleotide may be chemically modified into the form of a locked nucleic acid (LNA) or peptide nucleic acid (PNA).
  • LNA locked nucleic acid
  • PNA peptide nucleic acid
  • the modified oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, or 8 consecutively modified nucleotides at the 5' end. In some embodiments, the modified oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, or 8 consecutively modified nucleotides at the 3' end. In some embodiments, the modified oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, or 8 consecutively modified nucleotides at the 5' and 3' ends. In some embodiments, the modified oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, or 8 consecutively modified Gs. In some embodiments, the modified oligonucleotide comprises 4, 5, or 6 consecutively modified Gs. In some embodiments, the modified oligonucleotide comprises Gs that are not modified. In some embodiments, the modified oligonucleotide comprises phosphodiester linkages, phosphorothioate linkages, or a combination thereof.
  • the modified oligonucleotide may be PS-Ome (4,4), wherein a backbone is modified with phosphorothioate (PS) and 4 nucleotides at each end of 5' and 3' ends are modified with O-methyl, or PS-Ome (5,5), wherein a backbone is modified with PS and 5 nucleotides at each end of 5' and 3' ends are modified with O-methyl.
  • the modified oligonucleotide is PS-LNA (2,2), wherein a backbone is modified with PS and 2 nucleotides at each end of 5' and 3' ends are modified with LNA. In some embodiments, the modified oligonucleotide is PS-LNA (3,3), wherein a backbone is modified with PS and 3 nucleotides at each end of 5' and 3' ends are modified with LNA.
  • the modified oligonucleotide is PS-LNA (4,4), wherein a backbone is modified with PS and 4 nucleotides at each end of 5' and 3' ends are modified with LNA; or PS-LNA (5,5), wherein a backbone is modified with PS and 5 nucleotides at each end of 5' and 3' ends are modified with LNA.
  • the modified oligonucleotide comprises at least one of: from about 5% to about 100% modification, from about 10% to about 100% modification, from about 20% to about 100% modification, from about 30% to about 100% modification, from about 40% to about 100% modification, from about 50% to about 100% modification, from about 60% to about 100% modification, from about 70% to about 100% modification, from about 80% to about 100% modification, and from about 90% to about 100% modification.
  • the modified oligonucleotide comprises at least one of: from about 10% to about 90% modification, from about 20% to about 90% modification, from about 30% to about 90% modification, from about 40% to about 90% modification, from about 50% to about 90% modification, from about 60% to about 90% modification, from about 70% to about 90% modification, and from about 80% to about 100% modification.
  • the modified oligonucleotide comprises at least one of: from about 10% to about 80% modification, from about 20% to about 80% modification, from about 30% to about 80% modification, from about 40% to about 80% modification, from about 50% to about 80% modification, from about 60% to about 80% modification, and from about 70% to about 80% modification.
  • the modified oligonucleotide comprises at least one of: from about 10% to about 70% modification, from about 20% to about 70% modification, from about 30% to about 70% modification, from about 40% to about 70% modification, from about 50% to about 70% modification, and from about 60% to about 70% modification.
  • the modified oligonucleotide comprises at least one of: from about 10% to about 60% modification, from about 20% to about 60% modification, from about 30% to about 60% modification, from about 40% to about 60% modification, and from about 50% to about 60% modification.
  • the modified oligonucleotide comprises at least one of: from about 10% to about 50% modification, from about 20% to about 50% modification, from about 30% to about 50% modification, and from about 40% to about 50% modification.
  • the modified oligonucleotide comprises at least one of: from about 10% to about 40% modification, from about 20% to about 40% modification, and from about 30% to about 40% modification.
  • the modified oligonucleotide comprises at least one of: from about 10% to about 30% modification, and from about 20% to about 30% modification.
  • the modified oligonucleotide comprises from about 10% to about 20% modification.
  • the modified oligonucleotide comprises from about 15% to about 90%, from about 20% to about 80%, from about 30% to about 70%, or from about 40% to about 60% modifications.
  • the modified oligonucleotide comprises at least about 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% modification.
  • the modified oligonucleotide comprises at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22 or more modifications.
  • the modified oligonucleotide comprises at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22 or more modified nucleotides.
  • modified oligonucleotides that bind to a target gene sequence and forms a G-quadruplex.
  • the modified oligonucleotides result in inhibition of transcription of the target gene sequence.
  • the modified oligonucleotides result in degradation of the target gene sequence.
  • the modified oligonucleotides result in inhibition of transcription of the target gene sequence and degradation of the target gene sequence.
  • the target gene sequence comprises an enriched amount of guanine.
  • the target gene sequence is a viral gene sequence.
  • the target gene sequence is not a hepatitis B sequence.
  • the viral gene sequence is a sequence from a virus selected from the group consisting of HIV-1, HSV-1, EBV, KSHV, HHV-6, HCV, HPV, ZIKV, SARS Co-V, EBOV, and influenza virus.
  • compositions and methods relating to modified oligonucleotides for targeting a gene sequence comprises administering a composition as described herein to a subject.
  • the subject is a mammal.
  • the mammal is human.
  • the mammal is non-human.
  • the subject is a vertebrate including, but not limited to, humans and other primates (for example, chimpanzees, other apes, and monkey species), farm animals (for example, cows, sheep, pigs, goats, and horses), domestic mammals (for example, dogs and cats), laboratory animals (for example, rodents such as mice, rats, and guinea pigs) and birds (for example, domestic, wild and game birds such as chickens, turkeys, and other poultry, ducks, geese, and the like).
  • humans and other primates for example, chimpanzees, other apes, and monkey species
  • farm animals for example, cows, sheep, pigs, goats, and horses
  • domestic mammals for example, dogs and cats
  • laboratory animals for example, rodents such as mice, rats, and guinea pigs
  • birds for example, domestic, wild and game birds such as chickens, turkeys, and other poultry, ducks, geese, and the like.
  • compositions and methods relating to modified oligonucleotides for targeting a gene sequence comprises contacting target cells.
  • the target cells are prokaryotic cells or eukaryotic cells.
  • eukaryotic cells include, without limitation, animal, plant, and fungal cells.
  • animal cells include, without limitation, insect, fish and mammalian cells.
  • mammalian cells include mouse, human, and primate cells.
  • Modified oligonucleotides as described herein, in some embodiments are transferred into cells done by various methods, including, without limitation, transfection, transduction, and electroporation.
  • the pharmaceutical formulations described herein are administered to a subject by multiple administration routes, including but not limited to, parenteral (e.g., intravenous, subcutaneous, intramuscular), oral, intranasal, buccal, rectal, or transdermal administration routes.
  • parenteral e.g., intravenous, subcutaneous, intramuscular, intra-arterial, intraperitoneal, intrathecal, intracerebral, intracerebroventricular, or intracranial
  • the pharmaceutical composition describe herein is formulated for oral administration.
  • the pharmaceutical composition describe herein is formulated for intranasal administration.
  • the pharmaceutical formulations include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast melt formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations (e.g., nanoparticle formulations), and mixed immediate and controlled release formulations.
  • aqueous liquid dispersions self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast melt formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations (e.g., nanoparticle formulations), and mixed immediate and controlled release formulations.
  • the pharmaceutical formulation includes multiparticulate formulations.
  • the pharmaceutical formulation includes nanoparticle formulations.
  • nanoparticles comprise cMAP, cyclodextrin, or lipids.
  • nanoparticles comprise solid lipid nanoparticles, polymeric nanoparticles, self-emulsifying nanoparticles, liposomes, microemulsions, or micellar solutions.
  • Additional exemplary nanoparticles include, but are not limited to, paramagnetic nanoparticles, superparamagnetic nanoparticles, metal nanoparticles, fullerene-like materials, inorganic nanotubes, dendrimers (such as with covalently attached metal chelates), nanofibers, nanohorns, nano-onions, nanorods, nanoropes and quantum dots.
  • a nanoparticle is a metal nanoparticle, e.g., a nanoparticle of scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, yttrium, zirconium, niobium, molybdenum, ruthenium, rhodium, palladium, silver, cadmium, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold, gadolinium, aluminum, gallium, indium, tin, thallium, lead, bismuth, magnesium, calcium, strontium, barium, lithium, sodium, potassium, boron, silicon, phosphorus, germanium, arsenic, antimony, and combinations, alloys or oxides thereof.
  • a metal nanoparticle e.g., a nanoparticle of scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel
  • a nanoparticle includes a core or a core and a shell, as in a core-shell nanoparticle.
  • a nanoparticle is further coated with molecules for attachment of functional elements.
  • a coating comprises chondroitin sulfate, dextran sulfate, carboxymethyl dextran, alginic acid, pectin, carragheenan, fucoidan, agaropectin, porphyran, karaya gum, gellan gum, xanthan gum, hyaluronic acids, glucosamine, galactosamine, chitin (or chitosan), polyglutamic acid, polyaspartic acid, lysozyme, cytochrome C, ribonuclease, trypsinogen, chymotrypsinogen, ⁇ -chymotrypsin, polylysine, polyarginine, histone, protamine, ovalbumin or dextrin or cyclodextrin.
  • a nanoparticle comprises a graphene-coated nanoparticle.
  • a nanoparticle has at least one dimension of less than about 500nm, 400nm, 300nm, 200nm, or 100nm.
  • the nanoparticle formulation comprises paramagnetic nanoparticles, superparamagnetic nanoparticles, metal nanoparticles, fullerene-like materials, inorganic nanotubes, dendrimers (such as with covalently attached metal chelates), nanofibers, nanohorns, nano-onions, nanorods, nanoropes or quantum dots.
  • a modified oligonucleotide described herein is conjugated either directly or indirectly to the nanoparticle. In some embodiments, at least 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more modified oligonucleotides described herein are conjugated either directly or indirectly to a nanoparticle.
  • the pharmaceutical formulation comprise a delivery vector, e.g., a recombinant vector, the delivery of the modified oligonucleotide into cells.
  • the recombinant vector is DNA plasmid.
  • the recombinant vector is a viral vector.
  • Exemplary viral vectors include vectors derived from adeno-associated virus, retrovirus, adenovirus, or alphavirus.
  • the recombinant vectors capable of expressing the modified oligonucleotides provide stable expression in target cells.
  • viral vectors are used that provide for transient expression of modified oligonucleotides.
  • the pharmaceutical formulations include a carrier or carrier materials selected on the basis of compatibility with the composition disclosed herein, and the release profile properties of the desired dosage form.
  • exemplary carrier materials include, e.g., binders, suspending agents, disintegration agents, filling agents, surfactants, solubilizers, stabilizers, lubricants, wetting agents, diluents, and the like.
  • Pharmaceutically compatible carrier materials include, but are not limited to, acacia, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerine, magnesium silicate, polyvinylpyrrollidone (PVP), cholesterol, cholesterol esters, sodium caseinate, soy lecithin, taurocholic acid, phosphotidylcholine, sodium chloride, tricalcium phosphate, dipotassium phosphate, cellulose and cellulose conjugates, sugars sodium stearoyl lactylate, carrageenan, monoglyceride, diglyceride, pregelatinized starch, and the like.
  • PVP polyvinylpyrrollidone
  • the pharmaceutical formulations further include pH adjusting agents or buffering agents which include acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate and tris-hydroxymethylaminomethane; and buffers such as citrate/dextrose, sodium bicarbonate and ammonium chloride.
  • acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids
  • bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate and tris-hydroxymethylaminomethane
  • buffers such as citrate/dextrose, sodium bicarbonate and ammonium chloride.
  • acids, bases and buffers are included in an amount required to maintain pH of the composition in an acceptable range.
  • the pharmaceutical formulation includes one or more salts in an amount required to bring osmolality of the composition into an acceptable range.
  • salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.
  • the pharmaceutical formulations further include diluent which are used to stabilize compounds because they provide a more stable environment.
  • diluents which are used to stabilize compounds because they provide a more stable environment.
  • Salts dissolved in buffered solutions are utilized as diluents in the art, including, but not limited to a phosphate buffered saline solution.
  • diluents increase bulk of the composition to facilitate compression or create sufficient bulk for homogenous blend for capsule filling.
  • Such compounds include e.g., lactose, starch, mannitol, sorbitol, dextrose, microcrystalline cellulose; dibasic calcium phosphate, dicalcium phosphate dihydrate; tricalcium phosphate, calcium phosphate; anhydrous lactose, spray-dried lactose; pregelatinized starch, compressible sugar; mannitol, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate stearate, sucrose-based diluents, confectioner's sugar; monobasic calcium sulfate monohydrate, calcium sulfate dihydrate; calcium lactate trihydrate, dextrates; hydrolyzed cereal solids, amylose; powdered cellulose, calcium carbonate; glycine, kaolin; mannitol, sodium chloride; inositol, bentonite, and the like.
  • the pharmaceutical formulations include disintegration agents or disintegrants to facilitate the breakup or disintegration of a substance.
  • disintegration agents include a starch, e.g., a natural starch such as corn starch or potato starch, a pregelatinized starch such as National 1551, or sodium starch glycolate, a cellulose such as a wood product, methylcrystalline cellulose, methylcellulose, croscarmellose, or a cross-linked cellulose, such as cross-linked sodium carboxymethylcellulose, cross-linked carboxymethylcellulose, or cross-linked croscarmellose, a cross-linked starch such as sodium starch glycolate, a cross-linked polymer such as crospovidone, a cross-linked polyvinylpyrrolidone, alginate such as alginic acid or a salt of alginic acid such as sodium alginate,
  • magnesium aluminum silicate a gum such as agar, guar, locust bean, Karaya, pectin, or tragacanth, sodium starch glycolate, bentonite, a natural sponge, a surfactant, a resin such as a cation-exchange resin, citrus pulp, sodium lauryl sulfate, sodium lauryl sulfate in combination starch, and the like.
  • the pharmaceutical formulations include filling agents such as lactose, calcium carbonate, calcium phosphate, dibasic calcium phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrates, dextran, starches, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, and the like.
  • filling agents such as lactose, calcium carbonate, calcium phosphate, dibasic calcium phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrates, dextran, starches, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, and the like.
  • Lubricants and glidants are also optionally included in the pharmaceutical formulations described herein for preventing, reducing or inhibiting adhesion or friction of materials.
  • Exemplary lubricants include, e.g., stearic acid, calcium hydroxide, talc, sodium stearyl fumerate, a hydrocarbon such as mineral oil, or hydrogenated vegetable oil such as hydrogenated soybean oil, higher fatty acids and their alkali-metal and alkaline earth metal salts, such as aluminum, calcium, magnesium, zinc, stearic acid, sodium stearates, glycerol, talc, waxes, boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, a polyethylene glycol (e.g., PEG-4000) or a methoxypolyethylene glycol, sodium oleate, sodium benzoate, glyceryl behenate, polyethylene glycol, magnesium or sodium lauryl sulfate, colloidal silica, a star
  • Plasticizers include compounds used to soften the microencapsulation material or film coatings to make them less brittle. Suitable plasticizers include, e.g., polyethylene glycols such as PEG 300, PEG 400, PEG 600, PEG 1450, PEG 3350, and PEG 800, stearic acid, propylene glycol, oleic acid, triethyl cellulose and triacetin. Plasticizers also function as dispersing agents or wetting agents.
  • Solubilizers include compounds such as triacetin, triethylcitrate, ethyl oleate, ethyl caprylate, sodium lauryl sulfate, sodium doccusate, vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropylmethyl cellulose, hydroxypropyl cyclodextrins, ethanol, n-butanol, isopropyl alcohol, cholesterol, bile salts, polyethylene glycol 200-600, glycofurol, transcutol, propylene glycol, and dimethyl isosorbide and the like.
  • Stabilizers include compounds such as any antioxidation agents, buffers, acids, preservatives and the like.
  • Suspending agents include compounds such as polyvinylpyrrolidone, e.g., polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, vinyl pyrrolidone/vinyl acetate copolymer (S630), polyethylene glycol, e.g., the polyethylene glycol has a molecular weight of about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, hydroxymethylcellulose acetate stearate, polysorbate-80, hydroxyethylcellulose, sodium alginate, gums, such as, e.g., gum tragacanth and gum acacia, guar gum, xanthans, including xanthan gum, sugars, cellulosics, such as, e.g.
  • Surfactants include compounds such as sodium lauryl sulfate, sodium docusate, Tween 60 or 80, triacetin, vitamin E TPGS, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbates, polaxomers, bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide, and the like.
  • Additional surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, e.g. , polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylene alkylethers and alkylphenyl ethers, e.g. , octoxynol 10, octoxynol 40. Sometimes, surfactants is included to enhance physical stability or for other purposes.
  • Viscosity enhancing agents include, e.g., methyl cellulose, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, hydroxypropylmethyl cellulose acetate stearate, hydroxypropylmethyl cellulose phthalate, carbomer, polyvinyl alcohol, alginates, acacia, chitosans and combinations thereof.
  • Wetting agents include compounds such as oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sodium docusate, sodium oleate, sodium lauryl sulfate, sodium doccusate, triacetin, Tween 80, vitamin E TPGS, ammonium salts and the like.
  • kits and articles of manufacture for use with one or more of the compositions and methods described herein.
  • Such kits include a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in a method described herein.
  • Suitable containers include, for example, bottles, vials, syringes, and test tubes.
  • the containers are formed from a variety of materials such as glass or plastic.
  • the articles of manufacture provided herein contain packaging materials.
  • packaging materials include, but are not limited to, blister packs, bottles, tubes, bags, containers, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment.
  • the container(s) include modified oligonucleotides as described herein.
  • kits optionally include an identifying description or label or instructions relating to its use in the methods described herein.
  • a kit typically includes labels listing contents and/or instructions for use, and package inserts with instructions for use. A set of instructions will also typically be included.
  • a label is on or associated with the container.
  • a label is on a container when letters, numbers or other characters forming the label are attached, molded or etched into the container itself; a label is associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert.
  • a label is used to indicate that the contents are to be used for a specific therapeutic application. The label also indicates directions for use of the contents, such as in the methods described herein.
  • the pharmaceutical compositions are presented in a pack or dispenser device which contains one or more unit dosage forms containing a compound provided herein.
  • the pack for example, contains metal or plastic foil, such as a blister pack.
  • the pack or dispenser device is accompanied by instructions for administration.
  • the pack or dispenser is also accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the drug for human or veterinary administration. Such notice, for example, is the labeling approved by the U.S. Food and Drug Administration for prescription drugs, or the approved product insert.
  • compositions containing a compound provided herein formulated in a compatible pharmaceutical carrier are also prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
  • HepG2 and Huh7 cells Human liver cancer cell lines (HepG2 and Huh7 cells) were obtained from American Type Culture Collection (Manassas, VA, USA). HepG2 cells were transfected with a plasmid capable of expressing homo sapiens solute carrier family 10 (sodium/bile acid cotransporter family), member 1(SLC10A1) with the NCBI number of hNTCP [NM_003049.3] using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions to establish a HepG2-hNTCP cell line. Cell lines were cultured in a DMEM medium. The DMEM medium was supplemented with 10% (v/v) FBS (Gibco BRL), 1% penicillin, and 1% streptomycin.
  • DMEM medium was supplemented with 10% (v/v) FBS (Gibco BRL), 1% penicillin, and 1% streptomycin.
  • HepG2 and Huh7 cells were cultured in a CO 2 incubator set to 37°C with 5% CO 2 .
  • primary human hepatocytes PHHs
  • the PHHs were cultured in a primary maintenance medium (Gibco BRL, Oregon, USA) supplemented with CM4000 (Thermo, Rockford, USA), 1% penicillin, and 1% streptomycin.
  • Transfection was performed using Lipofectamine 2000 according to guidelines when cells reached 80% confluence. At 15 hours after the transfection, the cell medium was replaced with a fresh medium. Cells were harvested 2 to 3 days after transfection.
  • the culture supernatant of HepAD38 cells concentrated about 100-fold was precipitated with 6% PEG8000.
  • the HBV particles were prepared in PBS containing 25% FBS.
  • the infectious HBV particles were stored at -80°C as a viral stock. Quantification of the HBV was calculated by a dot blot method.
  • PMM hepatocyte maintenance medium
  • the medium was replaced with fresh PMM.
  • the HBV-infected cells were harvested 7 days after infection.
  • Viral DNA was detected by Southern blotting. Briefly, a cell pellet was harvested by scrapping 3 days after transfection. The harvested cells were dissolved in 100 ⁇ l of a cold HEPES buffer (10 mM HEPES pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40) and then the HBV core capsids contained in the lysate were precipitated with a 26% PEG 8000 buffer. Thereafter, the HBV core capsids were digested at 37°C for 3 hours in a 0.5% SDS buffer containing 250 mg Proteinase K. HBV DNA was extracted using phenol-chloroform, followed by precipitation with NaOAC and ethanol.
  • HEPES buffer 10 mM HEPES pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40
  • Total viral DNA was separated by electrophoresis at 90 V for 3 hours on a 0.8% agarose gel and was transferred to an XL nitrocellulose membrane (GE Healthcare). Thereafter, the HBV DNA was detected by a highly pure randomized HBV probe. The relative level of HBV DNA replication was quantified using a phosphoimager.
  • HBV mRNAs were detected by Northern blotting. Briefly, total cell RNAs were extracted using TRIzol reagent (Invitrogen) according to the manufacturer's protocol, and 20 ⁇ l of the total RNAs was separated by electrophoresis at 120 V for 3 hours on a 1 % formaldehyde agarose gel and subsequently was transferred to an XL nitrocellulose membrane (GE healthcare) for 16 to 18 hours. To detect HBV-specific mRNAs, the membrane was subjected to hybridization with a highly pure and randomly primed HBV probe. The relative level of HBV RNA replication was quantified using a phosphoimager.
  • Cells were harvested 2 days after transfection and subjected to lysis at 4°C for 30 minutes in a RIPA buffer [20 mM Tris/HCl, 1% NP-40, 0.5% protease inhibitor cocktail (Sigma, St. Louis, MO), 150 mM NaCl, 2 mM KCl, pH 7.4]. Thereafter, a protein lysate obtained by the lysis process was separated by SDS-PAGE. After performing SDS-PAGE, proteins on a polyacrylamide gel were transferred to a PVDF membrane. Antibodies were used in a 1:2000 ratio, and anti-actin (Sigma), anti-HBsAg (Abcam), and anti-HBcAg (DAKO, USA) antibodies were used as primary antibodies.
  • RC relaxed circular DNA of HBV
  • total cellular DNA was extracted from HBV-infected PHH cells using a QIAamp DNA Mini kit (Qiagen).
  • the extracted DNA was treated with T5 exonuclease (NEB) before amplifying cccDNA.
  • NEB T5 exonuclease
  • Real-time PCR was performed on a 20 ⁇ l (reaction volume) of a PCR mixture composed of 20 ng of the extracted DNA, 0.5 ⁇ mol/L of each forward and reverse primer, a probe labeled with 3'-fluorescein (FL) of 0.2 ⁇ mol/L, and a probe labeled with 5'-Red640 (R640) of 0.4 ⁇ mol/L using LightCycler (Roche).
  • forward and reverse primers having sequences of 5'-CTCCCCGTCTGTGCCTTCT-3' (SEQ ID NO. 10) and 5'-GCCCCAAAGCCACCCAAG-3' (SEQ ID NO. 11), respectively, were used, and for amplifying RC DNA in the liver tissue, forward and reverse primers having sequences of 5'-CTCGTGGTGGACTTCTCTC-3' (SEQ ID NO. 12) and 5'-CTGCAGGATGAAGAGGAA-3' (SEQ ID NO. 13) respectively were used.
  • FRET hybridization probes sequences of 5'-GTTCACGGTGGTCTCCATGCAACGT-FL-3' (SEQ ID NO.
  • Total DNA of HBV was amplified using a procedure as described below: after incubation at 95°C for 10 minutes, a PCR reaction cycle composed of a denaturation step at 95°C for 10 seconds, an annealing step at 58°C for 10 seconds, and an extension step at 72°C for 15 seconds was repeated 45 times.
  • cccDNA amplification was performed as described below: after incubation at 95°C for 10 minutes, a PCR reaction cycle composed of a denaturation step at 95°C for 10 seconds, an annealing step at 58°C for 5 seconds, and an extension step at 72°C for 20 seconds was repeated 45 times.
  • PCR for amplifying a beta-globin-encoding gene was performed using LightCycler Control Kit DNA (Roche) and the expression level of beta-globin-encoding gene was used to normalize.
  • a serial dilution for a plasmid harboring an HBV monomer (pHBVEcoRI) was used as a standard for quantification.
  • An enhancer luciferase reporter assay was performed to measure the activity of the HBV enhancer.
  • HepG2 cells were added to each well of a 12-well plate at 2 ⁇ 10 5 cells/well and transfected with 0.5 ⁇ g of each Enhancer-Luc (pEnhI.II, pEnhI. ⁇ II, pEnhIXp, pXp.EnhII, pNRE.EnhII, pEnhII/cp, pEnhI ⁇ Xp-D2, pEnhIXp-D6, pEnhI ⁇ Xp-D7, and pEnhIXp-D8: see FIGS. 5 and 6) and 50 nM D2 oligonucleotide.
  • cells were harvested and subjected to lysis using a Promega lysis buffer, and then enhancer luciferase activity was measured using Luciferase Assay Reagent (Promega, Madison, WI, USA).
  • FIG. 1 is a sequence screening diagram obtained by analysis of the HBV genome. Based on the diagram, local sequences within the HBV genome having the ability to form G-quadruplexes are predicted, and oligonucleotides capable of forming the G-quadruplexes together with the local sequences are designed and can be used as anti-viral agents.
  • oligonucleotides used herein were synthesized by Cosmogenetech (Seoul, Korea) or Bio Basic (Canada). Detailed description of each oligonucleotide with consecutive G's underlined is shown in the following Table 1A . Additional oligonucleotides with consecutive G's underlined were generated as shown in Tables 1B-1D .
  • D1 to D9 are unmodified oligonucleotides.
  • D2 was modified with PS (phosphorothioate), Ome (O-methyl), PNA (peptide nucleic acid), LNA (locked nucleic acid), PS-Ome, and PS-LNA and used.
  • the oligonucleotide modified with PS easily penetrates a cell and is resistant to degradation by exonucleases.
  • the Ome variant is similar in nature to RNA, but exhibits increased stability against nuclease-mediated hydrolysis in cells.
  • a melting temperature (Tm) in a dual structure increases by 1 to 4°C.
  • the PNA is an artificially synthesized polymer similar to DNA or RNA in the structure thereof, and has a backbone composed of repeating N-(2-aminoethyl)-glycine units linked by peptide bonds.
  • the LNA-modified oligonucleotide consists of nucleotides in which each pentose sugar of nucleotides is modified with an extra bridge connecting the 2' oxygen and 4' carbon and thus exhibits a closed structure, and the LNA-modified oligonucleotide exhibits an increased Tm when hybridized and is resistant to degradation.
  • Partially modified D2 has a sequence in which the 5' and 3' ends are partially modified.
  • PS-LNA (4,4) refers to an oligonucleotide, wherein a backbone is modified with PS and 4 nucleotides at each end of 5' and 3' ends are modified with LNA.
  • D2 synthesized by partial modification includes PS-Ome (4,4), PS-Ome (5,5), PS-LNA (2,2) PS-LNA (3,3) PS-LNA (4,4), PS-LNA (5,5), and the like.
  • Electrophoretic mobility shift assay (EMSA)
  • DNAs (D2, pEnhI ⁇ Xp, pEnhI ⁇ Xp-D2, and enhancer I.II) were mixed with a buffer solution (10 mM Tris-HCl pH 7.5, 0.1M KCl, 1 mM DTT, and 10 mM MgCl 2 ), and the mixture was heated and subsequently cooled to induce folding of DNA. After reaction, BG4 antibodies (Absolute Antibody, UK) were added to the DNA mixture and a G-quadruplex was identified through DNA-protein interaction.
  • a buffer solution 10 mM Tris-HCl pH 7.5, 0.1M KCl, 1 mM DTT, and 10 mM MgCl 2
  • a DNA-DNA complex was separated by electrophoresis on a 6% polyacrylamide gel at a cold temperature. After electrophoresis, the gel was dried at 70°C for 30 minutes. A result was analyzed using a phosphoimager.
  • Plasmid DNAs (25 ⁇ g of HBV 1.2, 25 ⁇ g of D2, and 5 ⁇ g of beta-gal) were delivered into 6-week-old mice (BALB/C) using a hydrodynamic injection method. PBS equivalent to 10% of the weight of a mouse was prepared and injected into the tail vein of the mouse. Modified D2 (50 ⁇ g) was also injected into the tail vein of a mouse. PBS containing DNA was injected into the vein at a rapid rate for 4 to 6 seconds using a syringe. All animal experiments were approved by the Animal Care Committee of Konkuk University.
  • Cells were cultured on a cover glass placed on the bottom of each well of a 6-well plate.
  • the cells were infected with HBV and transfected with 500 nM modified D2, and the cells were fixed with acetone and then washed three times with PBS.
  • a blocking process was performed with PBS containing 3% BSA.
  • the cells were treated with a buffer containing 1:300 ratio of BG4 antibody (Absolute Antibody, Ab00174-1.1) and incubated overnight in a cold room for antibody reaction.
  • the cells were incubated with a buffer containing anti-Mouse Alexa 568 for one hour.
  • the nuclei of the cells were stained with DAPI for 30 minutes.
  • the cover glass was mounted on a glass slide and dried.
  • Plasmid DNAs (25 ⁇ g of HBV 1.2 and 5 ⁇ g of beta-gal) were delivered into 6-week-old mice (BALB/C) using a hydrodynamic injection method. PBS equivalent to 10% of the weight of a mouse was prepared and injected into the tail vein of the mouse. PBS containing DNA was injected into the vein at a rapid rate for 4 to 6 seconds using a syringe. The next day, 8 ⁇ g of D2 wrapped with chitosan nanoparticles was also injected into the tail vein of the mouse.
  • the chitosan nanoparticle is an effective biocompatible molecule with low cytotoxicity and immunogenicity, and is capable of effectively delivering oligonucleotides such as siRNA (see Targeted Gene Silencing Using RGD-Labeled Chitosan Nanoparticles, Hee Dong Han, Clin Cancer Res. 2010).
  • the chitosan nanoparticles used in the above experiment were prepared based on ionic gelation between chitosan (MW 50 to190 kDa) and D2.
  • TPP 0.25% w/v
  • D2 (1 ⁇ g/ ⁇ l) were added to a 1% (w/v) chitosan solution, and the mixture was incubated at room temperature to allow a continuous reaction. After incubation, centrifugation was performed at a speed of 13000 rpm at 4°C for 40 minutes to obtain a pellet. The pellet was washed three times with distilled water (DW) and stored at 4°C until use. All animal experiments were approved by the Animal Care Committee of Konkuk University.
  • a liver cancer cell line was infected with HBV and at the same time transfected with each of D1 to D9 oligonucleotides, and then the antiviral effect of the oligonucleotides was evaluated by the degree of inhibiting production of viral proteins (i.e., HBsAg and HBeAg) and inhibiting HBV replication.
  • viral proteins i.e., HBsAg and HBeAg
  • HepG2 cells were transfected with an HBV 1.2 plasmid and oligonucleotides (D1 to D9 corresponding to SEQ ID NO. 1 to 9, respectively). The cells and supernatant thereof were cultured for 3 days after transfection. To determine the expression levels of the HBV proteins, the secreted amounts of HBeAg and HBsAg were measured. The amounts of HBeAg and HBsAg secreted into culture media were analyzed using an HBeAg and HBsAg ELSIA kit (Wantai Pharm Inc., Beijing, China). HBV DNA was measured by Southern blotting.
  • oligonucleotide D2 was used in further experiments examining inhibition of expression of HBV RNA. Based on the results shown in FIGS. 2A-2D , it is expected that oligonucleotides D1 and D6 would behave similarly.
  • Huh7 cells and HepG2 cells were transfected with a plasmid containing a 1.2-mer HBV replicon and the level of HBV mRNA was analyzed using Northern blotting.
  • FIGS. 3A-3B it was confirmed that D2 inhibited the expression of HBV RNA in a dose-dependent manner. Further, as shown in FIGS. 3C-3F , D2 strongly inhibits HBV transcription and antigen expression (HepG2). Accordingly, based on the result that D2 inhibited the expression of HBV RNA, indicating D2 acted on the stage of RNA transcription of the virus and inhibits transcription.
  • Huh7 cells were transfected with a plasmid containing a 1.2-mer HBV replicon and D2, and the expression levels of cell surface proteins were measured by Western blot analysis.
  • the activities of the HBV enhancer I and II were suppressed to about 80% by the presence of D2.
  • the result confirmed that D2 inhibits both enhancer I and II activities.
  • no effect was observed in a plasmid construct, such as pEnhI ⁇ Xp, in which the upstream region of the enhancer I (EnhI) remained.
  • the enhancer activity of the construct was suppressed to about 48% by the presence of D2.
  • D2 exhibits an antiviral effect by reducing the activities of enhancer I and II of HBV, thus indicating that D2 regulates the transcription of HBV RNA.
  • a luciferase reporter assay was performed after preparing the aforementioned reporter plasmid.
  • sequences corresponding to D2, D6, D7, and D8 in Table 1 which are the guanine (G)-rich sequence motifs of HBV, were introduced into the promoter region of the reporter plasmid. See also FIGS. 6C-6D .
  • FIG. 6B As a result, as shown in FIG. 6B , no effect was observed in a pEnhI ⁇ Xp luciferase clone, whereas luciferase activity was strongly suppressed by a pEnhI ⁇ Xp luciferase clone containing a D2 or D6 motif. See also FIGS. 6E-6F .
  • D2 has no effect on a pEnhI ⁇ Xp reporter, in which the upstream region of the enhancer I (EnhI) remains, D2 exerts a powerful inhibitory effect on a reporter, which is prepared by introducing the sequence of D2 into the promoter region of the pEnhI ⁇ Xp reporter.
  • D6 having a nucleotide sequence similar to D2 exhibited a similar result as in the case of D2.
  • an in vitro electrophoretic mobility shift assay was performed using D2 and a 32 P-labeled HBV enhancer sequence ( FIG. 7A ).
  • the result of the EMSA experiment showed that D2 partially forms a G-quadruplex with the enhancer I and II sequences.
  • FIG. 7B the presence of a super-shifted band generated by binding of BG4 antibodies specific to a G-quadruplex indicates that a G-quadruplex is formed.
  • the image of FIG. 7B is a gel image visualized by phosphorimaging.
  • D2 inhibits the activity of the HBV enhancer by forming a G-quadruplex, which is generated by physical binding between D2 and the HBV enhancer region.
  • the result of the EMSA experiment showed that D2 partially forms a G-quadruplex with the enhancer II sequence. Formation of a G-quadruplex was validated by the presence of a super-shifted band generated by binding of BG4 antibodies specific to a G-quadruplex. Based on the result of FIG. 8 , it was confirmed that D2 forms a G-quadruplex structure with the HBV enhancer II region.
  • the result of the EMSA experiment showed that D2 forms a perfect G-quadruplex with the region of the HBV genome, in which the region has the same sequence as D2.
  • Formation of a G-quadruplex was validated by the presence of a super-shifted band generated by binding of BG4 antibodies specific to a G-quadruplex.
  • a gel was visualized by phosphorimaging.
  • D2 did not bind to the enhancer I region (EnhI ⁇ Xp), whereas D2 formed a perfect G-quadruplex structure with a construct (EnhI ⁇ Xp-D2), which was prepared by introducing the sequence of D2. This result indicates that D2 recognizes its own sequence to form a G-quadruplex structure. G-quadruplex formation correlated with inhibition of activity of HBV.
  • D3 did not exhibit any antiviral effect. Furthermore, D3 exhibited a failure to form a G-quadruplex structure, as shown in the EMSA result of FIGS. 10 A -10C . Taken together, these results indicate that formation of a G-quadruplex structure correlates with antiviral action.
  • FIGS. 11A-11F shows the various lengths tested and results. As seen in FIGS. 11A-11F , a 16-mer was the optimal size. Having determined the length (16mer) and core G-rich region, the allowance range of the rest bases was tested ( FIG. 11G ). Further, as seen in FIG. 11H , the anti-viral effects of the various D2's were tested.
  • the length D6 was also optimized as seen in FIG. 11I and the anti-viral effects of the various D6's were tested ( FIG. 11J ).
  • Modified D2 penetrates cell and inhibits HBV enhancer activity
  • HepG2 cells were transfected with a plasmid containing HBV enhancer I and II. Before transfection, the HepG2 cells were pretreated with each of several modified D2 oligonucleotides (PS, OPMe, PNA, LNA, PS-Ome, and PS-LNA) at a final concentration of 500 nM. The next day, the culture medium of the HepG2 cells was replaced with a fresh medium (DMEM) containing each of the modified D2 oligonucleotides at a concentration of 500 nM. After transfection, the cells were cultured for 24 hours, and then luciferase activity was measured using the Steady-Glo Luciferase Assay System.
  • PS modified D2 oligonucleotides
  • the PS-modified D2 oligonucleotide exhibited cell permeability and HBV inhibitory activity.
  • modification of the backbone of an oligonucleotide with phosphorothioate (PS) or locked nucleic acids (LNA) improves the permeability of the oligonucleotide, and consequently increases an antiviral effect of the same.
  • FIGS. 12D-12E show the results of the different modification on HBeAg ( FIG. 12D ) and HBsAg ( FIG. 12E ) levels.
  • the potency of a modified D2 was tested in various HBV infection models.
  • the infection models included HepG2-NTCP, HepaRG, and PHH.
  • a schematic for the HepG2-NTCP is seen in FIG. 13A .
  • FIGS. 13B-13D show that PS-LNA D2 reduces HBV replication in the HepG2-NTCP model.
  • the IC 50 was 0.21 uM when PS-LNA D2 was treated in the media, and the IC 50 was less than 25 nM when PSA-LNA D2 was transfected.
  • HepG2-NTCP cells were infected with HBV and then treated with PS-modified D2 oligonucleotides (PS, PS-Ome, and PS-LNA).
  • PS-modified D2 oligonucleotides PS, PS-Ome, and PS-LNA.
  • FIG. 14A an experimental process for HBV infection and viral protein analysis in HepG2-NTCP cells is as follows: HepG2-NTCP cells were infected with HBV at 2000 genome equivalents (GEq)/cell, which had been cultured in a PHH maintenance medium (PMM, Gibco) containing 2% DMSO and 4% PEG8000 for 16 to 20 hours.
  • PMM PHH maintenance medium
  • HBeAg and HBsAg were measured.
  • the amounts of HBeAg and HBsAg secreted into a culture medium were analyzed using an HBeAg and HBsAg ELSIA kit (Wantai Pharm Inc., Beijing, China).
  • D2 D2, T.F
  • D2, Tr unmodified D2
  • LMV indicates lamivudine.
  • PS-modified D2 oligonucleotides PS, PS-Ome, and PS-LNA
  • PS, PS-Ome, and PS-LNA PS-modified D2 oligonucleotides also inhibit the activity of HBV in HepG2-NTCP cells, a HBV infectible cell line, indicating that modified D2 oligonucleotides have antiviral effects.
  • PS-modified D2 with IFN-alpha was also compared in HepG2-NTCP cells as seen in FIGS. 14D-14E .
  • D2 showed improved results as compared to interferon-alpha.
  • FIG. 15A shows a schematic of the experimental protocol. As seen in FIGS. 15B-15D , modified D2's decrease HBeAg levels, HBsAg levels, and percentage of RC DNA and cccDNA.
  • Modified D2 eliminates preexisting cccDNA in PHHs
  • FIG. 16A shows a schematic of the experimental protocol. As seen in FIGS. 16B-16D , modified D2 (PS-LNA D2) was found to reduce preexisting cccDNA in PHHs.
  • PS-LNA D2 modified D2
  • PS-LNA D2 A modified D2 (PS-LNA D2) inhibits the HBV rebound after cessation of drug in PHHs
  • FIGS. 18A-18D show HBeAg and HBsAg levels following treatment with a modified D2 (PS-LNA-D2). As seen in FIGS. 18A-18D , PS-LNA-D2 inhibited HBV rebound after cessation of drug in PHHs.
  • PS-LNA-D2 a modified D2
  • PS-LNA D2 Modified D2 was also found to eliminate preexisting cccDNA in PHHs ( FIGS. 18F-18J ) following an experimental protocol as seen in FIG. 18E .
  • a modified D2(PS-LNA D2) suppresses preexisting HBV in HepaRGcells
  • FIGS. 19A -19B A modified D2 (PS-LNA D2) was tested in HepaRG according to experimental protocols as seen in FIGS. 19A -19B .
  • FIGS. 19C-19E show that PS-LNA D2 suppressed preexisting HBV in HepaRGcells.
  • D2 is a pan-genotype HBV inhibitor
  • D2 is a pan-genotype HBV inhibitor.
  • PHH cells were isolated from human liver tissue of the remnant after liver surgery, and the isolated PHH cells were infected with HBV and then treated with the indicated modified D2 oligonucleotides.
  • FIG. 22A an experimental process for HBV infection and viral protein analysis in PHH cells is as follows: PHH cells were infected with HBV at 5000 genome equivalents (GEq)/cell, in which HBV had been cultured in a PHH maintenance medium (PMM, Gibco) containing 2% DMSO and 4% PEG8000 for 16 to 20 hours.
  • PMM PHH maintenance medium
  • HBeAg and HBsAg were washed three times with 500 ⁇ l of PBS, and cultured for 7 days with PMM containing 2% DMSO.
  • PMM containing 2% DMSO 2% DMSO.
  • HBeAg and HBsAg secreted into a culture medium were analyzed using an HBeAg and HBsAg ELSIA kit (Wantai Pharm Inc., Beijing, China). Cells transfected with unmodified D2 were used as a negative control group. LMV indicates lamivudine.
  • modified D2 oligonucleotides exhibit antiviral effects.
  • PS-LNA-modified D2 showed the most excellent inhibitory effect.
  • the result indicates that the modified D2 oligonucleotides also have antiviral effects in human cells.
  • D2 oligonucleotides having three (3, 3), four (4, 4), or five (5, 5) modified nucleotides at the ends were tested for inhibitory effects on HBV.
  • HepG2 cells were transfected with an HBV enhancer I.II construct for the analyses. Before transfection, HepG2 cells were pretreated with each of various modified D2 oligonucleotides (PS, PS-Ome (4,4), PS-Ome (5,5), PS-Ome (all), PS-LNA (2,2), PS-LNA (3,3), PS-LNA (4,4), PS-LNA (5,5), and PS-LNA (all)) at a final concentration of 500 nM.
  • PS, PS-Ome (4,4), PS-Ome (5,5), PS-Ome (all) PS-LNA (2,2), PS-LNA (3,3), PS-LNA (4,4), PS-LNA (5,5), and PS-LNA (all)
  • the culture medium of the HepG2 cells was replaced with a fresh medium (DMEM) containing each of the modified D2 oligonucleotides at a concentration of 500 nM.
  • DMEM fresh medium
  • the luciferase activity of HBV enhancer was analyzed using the Luciferase Assay System (Promega, Madison, WI, USA) according to the protocol.
  • each of the modified D2 oligonucleotides exhibited anti-HBV effects.
  • PS-LNA (4,4) in which four nucleotides at both the 5' and 3' ends of D2 were modified with LNA, exhibited the strongest anti-viral effect.
  • oligonucleotides 7, 18, 19, 42, 52, and (3, 3) exhibited improved cccDNA reduction as compared to PS-LNA-D2.
  • FIG. 24E shows a schematic of oligonucleotides with good activity in HepG2 cells and
  • FIG. 24F shows a schematic of oligonucleotides with good activity in PHH cells.
  • the modified oligonucleotides with good activity were tested for IC 50 as seen in FIGS. 25A-25E . Measurements of the IC 50 were repeated as seen in FIGS. 26A-26F and FIGS. 27A-27F .
  • RT Reverse transcriptase
  • Table 3 The RT primers were combined, and the PS-LNA-D2 (3, 3) template was serially diluted according to Table 4 .
  • the PS-LNA-D2 (3, 3) template was heat denatured (95 °C for 10 min, immediately ice chilled). Tables 5A-5B show the primer and probes used.
  • the tube or plate was incubated at 85 °C for 5 min, then at 60 °C for 5 min.
  • the denatured template was placed on ice.
  • 7 uL of RT master mix was added to the denatured RT primer and mixed.
  • the tube or plate was incubated on ice for 5 minutes.
  • the tube or plate was thermal cycled according to Table 6 .
  • a PCR reaction was prepared according to Table 7 . 19 uL of the master mix was dispensed into a real-time PCR tube and 1 uL of the RT reaction was added. The tube or plate was thermal cycled according to Table 8 .
  • Results from the real-time PCR reaction are seen in FIGS. 29A-29B and Table 9 .
  • a mouse model for HBV infection was used. Experiments were performed according to the experimental schedule shown in FIG. 31A . 6-week-old male mice were used for each group. PBS-injected mice were used as a control group (Mock).
  • the hydrodynamically injected (HI) DNA is prepared as follows: 25 ⁇ g of a plasmid containing a 1.2-mer HBV replicon, 25 ⁇ g of an empty vector or D2, and 5 ⁇ g of beta-gal. Mice injected with beta-gal were used as a control group. Mice were sacrificed to obtain blood samples.
  • Mouse serum was diluted with PBS (1:50 for HBeAg and 1:2000 for HBsAg). The levels of viral proteins (HBeAg and HBsAg) were measured using an ELISA kit.
  • mice injected with D2 exhibited a significant antiviral effect.
  • FIG. 31D Southern blot analysis showed that the level of HBV DNA was reduced in mice injected with D2.
  • a mouse model for HBV infection was used. In vivo experiments were performed according to the experimental schedule shown in FIG. 32A . 6-week-old male mice were used for each group. Mice injected with only HBV were used as a control group. 25 ⁇ g of a plasmid containing a 1.2-mer HBV replicon and 5 ⁇ g of a beta-gal plasmid were hydrodynamically injected. Then 50 ⁇ g of each of the modified D2 oligonucleotides (PS, PS-Ome and PS-LNA) was intravenously injected for 3 days. At 4 days after hydrodynamic injection, mice were sacrificed to obtain blood samples.
  • PS, PS-Ome and PS-LNA modified D2 oligonucleotides
  • mice injected with beta-gal were used as a control group.
  • Mouse serum was diluted with PBS (1:50 for HBeAg and 1:2000 for HBsAg).
  • the levels of viral proteins (HBeAg and HBsAg) were measured using an ELISA kit.
  • mice injected with the modified D2 exhibited an antiviral effect.
  • FIG. 32D Southern blot analysis showed that the level of HBV DNA was reduced in mice injected with the modified D2. This result confirms that, when the modified D2 was injected, it was delivered to the liver of mouse and exhibited an antiviral effect.
  • mice injected with beta-gal were used as a control group.
  • Mouse serum was diluted with PBS (1:50 for HBeAg and 1:2000 for HBsAg).
  • the levels of viral proteins (HBeAg and HBsAg) were measured using an ELISA kit.
  • mice injected with the D2 wrapped with nanoparticles exhibited an antiviral effect.
  • the first bar represents mock
  • the second bar represents HBV
  • the third bar represents HBV and D2 wrapped with chitosan nanoparticles
  • the fourth bar represents HBV and D4 wrapped with chitosan nanoparticles.
  • Mice infected with the D4 wrapped with chitosan nanoparticles were used as a negative control that did not inhibit the activity of HBV at all.
  • FIG. 33D Southern blot analysis showed that the level of HBV DNA was reduced in mice injected with the D2 wrapped with chitosan nanoparticles. This result confirms that, when the D2 wrapped with chitosan nanoparticles was injected, it was delivered to the liver of the mouse and exhibited a strong antiviral effect.
  • FIGS. 34A-34H show the results.
  • the in vivo efficacy was measured to determine an improved injection route.
  • An experimental schematic is seen in FIG. 35A .
  • the genotype was pAAV-C
  • an amount of HBV DNA injected was 5 ⁇ g ( ⁇ -gal: 5 ⁇ g)
  • the oligonucleotide injection route was intravenous (IV) subcutaneous (SC)
  • the oligonucleotide dose was 50ug/inj/mouse
  • the number of injections/mouse was 2 times
  • the mouse strain was C57/BL6 at age 6 weeks
  • the number of mice was 2-3/group. Additional experimental details are seen in Tables 12-13 .
  • Blood, heart, brain, liver, lung, kidney, and spleen of the mice were analyzed.
  • HBV DNA was determined in the liver and serum
  • HBeAg and HBsAg was determined in the serum.
  • HB Elisa's were also performed as seen in FIGS. 35B-35I .
  • tissue distribution of the PS-LNA-D2 (3, 3) and modified oligonucleotide #42 was determined as seen in FIGS. 36A-36D and Tables 14-17 . As seen in the results, there was a greater than 10 fold liver targeting of (3,3) as compared to #42 via IV route.
  • the modified oligonucleotides were tested for drug delivery ability in HepG2 cells. Results of the various modified oligonucleotides are seen in FIGS. 37A-37H .
  • Modified D2 exhibits an anti-HBV effect from the time the modified D2 is treated after HBV infection.
  • FIG. 38A Interferon- ⁇ (IFN- ⁇ ) was used as a positive control, and unmodified D2 was used as a negative control that did not inhibit the activity of HBV at all.
  • FIGS. 38D-38E show the result of quantitative real time PCR.
  • cells treated with modified D2 oligonucleotides including PS-LNA (3,3), PS-LNA (4,4), and PS-LNA (all) exhibited reduced levels of HBeAg and HBsAg compared to cells infected with HBV alone.
  • the level of HBV rcDNA was effectively reduced by treatment of the partially modified D2 oligonucleotides including PS-LNA (3,3), PS-LNA (4,4), and PS-LNA (all).
  • cccDNA In order to ultimately treat HBV infection, cccDNA must be removed.
  • the level of HBV cccDNA was reduced by treatment of the partially modified D2 oligonucleotides including PS-LNA (3,3), PS-LNA (4,4), and PS-LNA (all).
  • Modified D2 inhibits HBV even when sufficient cccDNA is generated
  • FIGS. 39B-39C cells treated with the modified D2 oligonucleotide with different concentrations exhibited reduced levels of HBeAg and HBsAg compared to cells infected with HBV alone.
  • FIG. 39D shows the result of confirming the difference in the amount of HBV DNA by electrophoresis after performing general PCR.
  • the levels of HBV rcDNA and cccDNA were reduced by the modified D2 in a concentration-dependent manner.
  • HepG2-NTCP cells were infected with HBV, treated with D2, and were examined using a microscope.
  • the cells were treated with the modified D2 from 5 days after HBV infection and the cells were fixed on day 7. Then, a slide glass was prepared to detect a red signal corresponding to G-quadruplexes using BG4 antibodies.
  • FIG. 40A As a result, as shown in FIG. 40A , after NTCP cells were infected with HBV and treated with the modified D2, the formation of a guanine polymer between the modified D2 and cccDNA generated from the infected HBV was confirmed using BG4 antibodies recognizing a G-quadruplex. In addition, cells infected with HBV alone exhibited a normal level of HBeAg, and HBV-infected cells treated with the modified D2 exhibited a reduced level of HBeAg, indicating that the modified D2 has an antiviral effect. Based on the graphs shown in FIGS. 40B-40C and the number of foci detected by BG4 antibodies shown in the bottom of FIG.
  • FIGS. 41A-41D illustrates levels of inhibition of HBeAg and HBsAg when nucleotide length was shortened on both ends (L-1 to L-4, R-1 and R-2) around the (G)6 sequence of D2 oligonucleotide.
  • FIGS. 42A-42B illustrate levels of inhibition of HBeAg and HBsAg when the nucleotide length was increased to 18, 20, 22, 24mer, and 24mer L, 24mer R around the (G)6 sequence of the D2 oligonucleotide.
  • FIGS. 41A-41D illustrates levels of inhibition of HBeAg and HBsAg when nucleotide length was shortened on both ends (L-1 to L-4, R-1 and R-2) around the (G)6 sequence of D2 oligonucleotide.
  • FIGS. 42A-42B illustrate levels of inhibition of HBeAg and HBsAg when the nucleotide length was increased to 18, 20, 22, 24mer, and 24
  • 43A-43B illustrates level of inhibition of HBeAg and HBsAg when one of the (G)6 sequences of the D2 oligonucleotide was substituted with C (D2-1, D2-2) or when nucleotides sequences other than (G)6 are substituted with a sequence derived from hepatitis B virus of other genotypes (D2-3, D2-4).
  • 44A-44B illustrate level of inhibition of HBeAg and HBsAg when the length of the nucleic acid sequence was decreased (D6-1) or increased (D6-2 to D6-8) from the 5 'or the 3' end around the (G)5 sequence of the D6 oligonucleotide; (D6-9, D6-10) or when nucleotides sequences other than the (G)5 sequence are substituted with s sequence derived from the hepatitis B virus of other genotypes (D6-9, D6-10).
  • the entire nucleotide sequence of the PR8 influenza virus was analyzed, and it was confirmed that there is one site in the PA(polymerase acidic protein) gene for the site where 6 consecutive sequences of guanine (G) appeared as in D2.
  • D2 and D4 were transfected into A549 cells using lipofectamine. After 3 hours, the A549 cells were infected with PR8 influenza virus (A/Puerto Rico/8/34 (H1N1)) at 0.01 MOI (10 4 PFU), and the virus titration was determined by a plaque assay using culture media every 24 hours (24 hours, 48 hours, and 72 hours).
  • PR8 influenza virus A/Puerto Rico/8/34 (H1N1)
  • D2 is considered to exhibit anti-influenza activity by targeting a site having 6 consecutive sequences of guanine in the PA gene.

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Abstract

L'invention concerne des compositions et des procédés comprenant des oligonucléotides qui forment des G-quadruplexes avec des séquences cibles. De tels oligonucléotides peuvent comprendre une ou plusieurs modifications.
PCT/KR2019/007112 2018-06-12 2019-06-12 Oligonucléotides modifiés pour l'inhibition de l'expression d'un gène cible Ceased WO2019240504A1 (fr)

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WO2018089914A1 (fr) * 2016-11-11 2018-05-17 Alios Biopharma, Inc. Stratégie de ciblage d'oligonucléotide pour l'adnccc du vhb
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