WO2017190197A1 - Reagents for treatment of hepatitis b virus (hbv) infection and use thereof - Google Patents

Reagents for treatment of hepatitis b virus (hbv) infection and use thereof Download PDF

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Publication number
WO2017190197A1
WO2017190197A1 PCT/AU2017/050413 AU2017050413W WO2017190197A1 WO 2017190197 A1 WO2017190197 A1 WO 2017190197A1 AU 2017050413 W AU2017050413 W AU 2017050413W WO 2017190197 A1 WO2017190197 A1 WO 2017190197A1
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set forth
sequence
sequence set
effector
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English (en)
French (fr)
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Tin Mao
David Suhy
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Benitec Biopharma Pty Ltd
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Benitec Biopharma Pty Ltd
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Priority to RU2018142797A priority Critical patent/RU2748806C2/ru
Priority to CA3023185A priority patent/CA3023185A1/en
Priority to JP2018557875A priority patent/JP2019514401A/ja
Priority to CN201780040430.8A priority patent/CN109415733A/zh
Priority to SG11201809753YA priority patent/SG11201809753YA/en
Priority to US16/098,759 priority patent/US11535851B2/en
Priority to EP17792296.0A priority patent/EP3452595A4/en
Priority to KR1020187035341A priority patent/KR102358280B1/ko
Application filed by Benitec Biopharma Pty Ltd filed Critical Benitec Biopharma Pty Ltd
Priority to AU2017260580A priority patent/AU2017260580B2/en
Publication of WO2017190197A1 publication Critical patent/WO2017190197A1/en
Priority to PH12018502320A priority patent/PH12018502320A1/en
Anticipated expiration legal-status Critical
Priority to AU2022241489A priority patent/AU2022241489A1/en
Priority to US18/056,929 priority patent/US20230093951A1/en
Ceased legal-status Critical Current

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    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
    • 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
    • A61K31/66—Phosphorus compounds
    • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
    • 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
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
    • 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
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C12N2310/141—MicroRNAs, miRNAs
    • 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
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/531—Stem-loop; Hairpin
    • 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
    • C12N2330/00—Production
    • C12N2330/50—Biochemical production, i.e. in a transformed host cell
    • C12N2330/51—Specially adapted vectors

Definitions

  • HBV hepatitis B virus
  • RNA interference RNA interference
  • HBV hepatitis B virus
  • HBV Hepatitis B virus
  • ORFs open reading frames
  • S gene surface antigen
  • C gene core antigen
  • P gene DNA polymerase
  • X gene a gene of undetermined function
  • HBV infection can cause acute and chronic type B hepatitis, and may eventually lead to the development of chronic hepatic insufficiency, cirrhosis, and hepatocellular carcinoma.
  • HBV carriers can transmit the disease for many years. Persons with chronic HBV infection i.e., carriers, have at least 12 times higher risk of developing hepatocellular carcinoma than non-carriers, and HBV causes 60- 80% of the world's primary liver cancers. As a consequence, HBV ranks second only to tobacco as a known human carcinogen.
  • the present disclosure is based, in part, on the recognition that existing vaccines and therapeutic agents for treatment and/or prevention of HBV infection are limited in their efficacy, such as where long term treatment is necessary e.g., due to the development of viral resistance to therapy and/or variation in responsiveness to therapy between genotypes of HBV.
  • the present disclosure provides DNA-directed RNA interference (ddRNAi) constructs for expressing one or more short hairpin micro-RNAs (shmiRs) targeting conserved regions of RNA transcripts produced by the HBV genome i.e., regions conserved among different genotypes of HBV.
  • shmiRs of the disclosure comprise effector sequences capable of inhibiting or reducing expression of HBV gene transcripts in
  • Exemplary shmiRs of the disclosure comprise effector sequences capable of inhibiting or reducing expression of HBV gene transcripts, reducing intracellular and extracellular HBV DNA, and reducing HBV covalently-closed circular DNA (cccDNA) in a PXB chimeric mouse infected with HBV.
  • cccDNA HBV covalently-closed circular DNA
  • the inventors provide new compounds that inhibit or reduce expression of a nucleic acid and/or protein expressed by HBV and uses of such compounds e.g., to treat a HBV infection in a subject.
  • the present disclosure provides a nucleic acid comprising a DNA sequence which encodes a short hairpin micro-RNA (shmiR), said shmiR comprising: an effector sequence of at least 17 nucleotides in length;
  • pri-miRNA primary micro RNA
  • the effector sequence is substantially complementary to a RNA transcript set forth in any one of SEQ ID NOs: 1-10, 38, 40, 42, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131 and 133.
  • the effector sequence will be less than 30 nucleotides in length.
  • a suitable effector sequence may be in the range of 17-29 nucleotides in length.
  • the effector sequence will be 20 nucleotides in length. More preferably, the effector sequence will be 21 nucleotides in length and the effector complement sequence will be 20 nucleotides in length.
  • the effector sequence may comprise 6 base pair mismatches relative to the sequence set forth in any one of SEQ ID NOs: 1-10, 38, 40, 42, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131 and 133 to which the effector sequence is substantially complementary.
  • the effector sequence comprises 5 base pair mismatches relative to the sequence set forth in any one of SEQ ID NOs: 1-10, 38, 40, 42, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131 and 133 to which the effector sequence is substantially complementary.
  • the effector sequence comprises 4 base pair mismatches relative to the sequence set forth in any one of SEQ ID NOs: 1-10, 38, 40, 42, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131 and 133 to which the effector sequence is substantially complementary.
  • the effector sequence comprises 3 base pair mismatches relative to the sequence set forth in any one of SEQ ID NOs: 1-10, 38, 40, 42, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131 and 133 to which the effector sequence is substantially complementary.
  • the effector sequence comprises 2 base pair mismatches relative to the sequence set forth in any one of SEQ ID NOs: 1-10, 38, 40, 42, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131 and 133 to which the effector sequence is substantially complementary.
  • the effector sequence comprises 1 base pair mismatch relative to the sequence set forth in any one of SEQ ID NOs: 1-10, 38, 40, 42, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131 and 133 to which the effector sequence is substantially complementary.
  • the effector sequence is 100% complementary to a region of equivalent length within a sequence set forth in any one of SEQ ID NOs: 1-10, 38, 40, 42, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131 and 133. Where mismatches are present, it is preferred that they are not located within the region corresponding to the seed region of the shmiR i.e., nucleotides 2-8 of the effector sequence.
  • the nucleic acid described herein may comprise a DNA sequence encoding a shmiR selected from the group consisting of:
  • a shmiR comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 12 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO: 12; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shmiR comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 14 with the exception of 1, 2, 3, 4, 5 or 64 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO: 14; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shmiR comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 16 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO: 16; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shmiR comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 18 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO: 18; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shmiR comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:20 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:20; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shmiR comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:22 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:22; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shmiR comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:24 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:24; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shmiR comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:26 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:26; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shmiR comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:28 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:28; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shmiR comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:30 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:30; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shmiR comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:32 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:32; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shmiR comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:34 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:34; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shmiR comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:36 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:36; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shmiR comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:38 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:38; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shmiR comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:40 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:40; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shmiR comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:42 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:42; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shmiR comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 111 with the exception of 1, 2, 3, 4, 5, or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO: 111; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shmiR comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 113 with the exception of 1, 2, 3, 4, 5, or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO: 113; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shmiR comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 115 with the exception of 1, 2, 3, 4, 5, or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO: 115; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shmiR comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 117 with the exception of 1, 2, 3, 4, 5, or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO: 117; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shmiR comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 119 with the exception of 1, 2, 3, 4, 5, or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO: 119; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shmiR comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 121 with the exception of 1, 2, 3, 4, 5, or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO: 121; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shmiR comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 123 with the exception of 1, 2, 3, 4, 5, or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO: 123; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shmiR comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 125 with the exception of 1, 2, 3, 4, 5, or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO: 125; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shmiR comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 127 with the exception of 1, 2, 3, 4, 5, or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO: 127; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shmiR comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 129 with the exception of 1, 2, 3, 4, 5, or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO: 129; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shmiR comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 131 with the exception of 1, 2, 3, 4, 5, or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO: 131; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence; and
  • a shmiR comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 133 with the exception of 1, 2, 3, 4, 5, or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO: 133; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence.
  • nucleic acid described herein may comprise a DNA sequence encoding a shmiR selected from the group consisting of:
  • a shmiR comprising an effector sequence set forth in SEQ ID NO: 11 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 11 and capable of forming a duplex therewith;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO: 13 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 13 and capable of forming a duplex therewith;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO: 15 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 15 and capable of forming a duplex therewith;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO: 17 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 17 and capable of forming a duplex therewith;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO: 19 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 19 and capable of forming a duplex therewith;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO:21 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:21 and capable of forming a duplex therewith;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO:23 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:23 and capable of forming a duplex therewith;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO:25 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:25 and capable of forming a duplex therewith;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO:27 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:27 and capable of forming a duplex therewith;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO:29 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:29 and capable of forming a duplex therewith;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO:31 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:31 and capable of forming a duplex therewith;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO:33 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:33 and capable of forming a duplex therewith;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO:35 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:35 and capable of forming a duplex therewith;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO:37 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:37 and capable of forming a duplex therewith;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO:39 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:39 and capable of forming a duplex therewith;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO:41 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:41 and capable of forming a duplex therewith;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO: 110 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 110 and capable of forming a duplex therewith;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO: 112 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 112 and capable of forming a duplex therewith;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO: 114 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 114 and capable of forming a duplex therewith;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO: 116 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 116 and capable of forming a duplex therewith;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO: 118 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 118 and capable of forming a duplex therewith;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO: 120 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 120 and capable of forming a duplex therewith;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO: 122 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 122 and capable of forming a duplex therewith;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO: 124 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 124 and capable of forming a duplex therewith;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO: 126 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 126 and capable of forming a duplex therewith;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO: 128 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 128 and capable of forming a duplex therewith;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO: 130 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 130 and capable of forming a duplex therewith;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO: 132 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 132 and capable of forming a duplex therewith.
  • the shmiR encoded by the nucleic acid described herein may comprise an effector complement sequence comprising 1, 2, 3 or 4 mismatches relative to the corresponding effector sequence, provided that the cognate effector and effector
  • complement sequences are capable of forming a duplex region.
  • nucleic acid described herein may comprise a DNA sequence encoding a shmiR selected from the group consisting of:
  • a shmiR comprising an effector sequence set forth in SEQ ID NO: 11 and an effector complement sequence set forth in SEQ ID NO: 12;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO: 13 and an effector complement sequence set forth in SEQ ID NO: 14;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO: 15 and an effector complement sequence set forth in SEQ ID NO: 16;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO: 17 and an effector complement sequence set forth in SEQ ID NO: 18;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO: 19 and an effector complement sequence set forth in SEQ ID NO: 20;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO:21 and an effector complement sequence set forth in SEQ ID NO:22
  • a shmiR comprising an effector sequence set forth in SEQ ID NO:23 and an effector complement sequence set forth in SEQ ID NO:24;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO:25 and an effector complement sequence set forth in SEQ ID NO:26;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO:27 and an effector complement sequence set forth in SEQ ID NO:28;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO:29 and an effector complement sequence set forth in SEQ ID NO:30;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO:31 and an effector complement sequence set forth in SEQ ID NO:32;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO:33 and an effector complement sequence set forth in SEQ ID NO:34;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO:35 and an effector complement sequence set forth in SEQ ID NO:36;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO:37 and an effector complement sequence set forth in SEQ ID NO:38;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO:39 and an effector complement sequence set forth in SEQ ID NO:40;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO:41 and an effector complement sequence set forth in SEQ ID NO:42;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO: 110 and an effector complement sequence set forth in SEQ ID NO: 111;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO: 112 and an effector complement sequence set forth in SEQ ID NO: 113;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO: 114 and an effector complement sequence set forth in SEQ ID NO: 115;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO: 116 and an effector complement sequence set forth in SEQ ID NO: 117;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO: 118 and an effector complement sequence set forth in SEQ ID NO: 119; a shmiR comprising an effector sequence set forth in SEQ ID NO: 120 and an effector complement sequence set forth in SEQ ID NO: 121;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO: 122 and an effector complement sequence set forth in SEQ ID NO: 123;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO: 124 and an effector complement sequence set forth in SEQ ID NO: 125;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO: 126 and an effector complement sequence set forth in SEQ ID NO: 127;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO: 128 and an effector complement sequence set forth in SEQ ID NO: 129;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO: 130 and an effector complement sequence set forth in SEQ ID NO: 131;
  • a shmiR comprising an effector sequence set forth in SEQ ID NO: 132 and an effector complement sequence set forth in SEQ ID NO: 133.
  • the shmiR encoded by the nucleic acid of the disclosure may comprise, in a 5' direction:
  • Suitable loop sequences may be selected from those known in the art. However, an exemplary stemloop sequence is set forth in SEQ ID NO: 75.
  • Suitable primary micro RNA (pri-miRNA or pri-R) backbones for use in a nucleic acid of the disclosure may be selected from those known in the art.
  • the pri- miRNA backbone may be selected from a pri-miR-30a backbone, a pri-miR-155 backbone, a pri-miR-21 backbone and a pri-miR-136 backbone.
  • the pri-miRNA backbone is a pri-miR-30a backbone.
  • the 5' flanking sequence of the pri-miRNA backbone is set forth in SEQ ID NO: 76 and the 3' flanking sequence of the pri-miRNA backbone is set forth in SEQ ID NO: 77.
  • the nucleic acid described herein comprises a DNA sequence selected from the sequence set forth in any one of SEQ ID NOs: 59-74 and 146-157.
  • a shmiR encoded by the nucleic acid of the disclosure may comprise a sequence set forth in any one of SEQ ID NOs: 43-58 and 134-145.
  • nucleic acid in accordance with the present disclosure may be combined or used in conjunction with other therapeutic agents for treating HBV. Accordingly, the present disclosure provides a nucleic acid comprising a DNA sequence encoding a shmiR as described herein in combination with one or more other agents for treating HBV.
  • a plurality of nucleic acids are provided comprising:
  • nucleic acid comprising a DNA sequence encoding a short hairpin RNA
  • RNA comprising an effector sequence of at least 17 nucleotides in length and a effector complement sequence, wherein the effector sequence is substantially complementary to a RNA sequence set forth in any one of SEQ ID NOs: 1-10, 38, 40, 42, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131 and 133;
  • shmiR encoded by the nucleic acid at (a) and the shmiR or shRNA encoded by the nucleic acid at (b) comprise different effector sequences.
  • the effector sequence of the shRNA at (b)(ii) which is substantially complementary to a RNA sequence set forth in any one of SEQ ID NOs: 1-10, 38, 40, 42, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131 and 133 will be less than 30 nucleotides in length.
  • a suitable effector sequence of the shRNA may be in the range of 17-29 nucleotides in length.
  • the shRNA may be selected from the group consisting of:
  • a shRNA comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 12 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO: 12; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shRNA comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 14 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO: 14; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shRNA comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 16 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO: 16; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shRNA comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 18 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO: 18; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shRNA comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:20 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:20; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shRNA comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:22 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:22; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shRNA comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:24 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:24; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shRNA comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:26 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:26; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shRNA comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:28 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:28; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shRNA comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:30 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:30; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shRNA comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:32 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:32; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shRNA comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:34 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:34; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shRNA comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:36 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:36; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shRNA comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:38 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:38; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shRNA comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:40 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:40; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shRNA comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:42 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:42; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shRNA comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 111 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO: 111; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shRNA comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 113 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO: 113; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shRNA comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 115 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO: 115; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shRNA comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 117 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO: 117; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shRNA comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 119 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO: 119; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shRNA comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:121 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO: 121; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shRNA comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 123 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO: 123; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shRNA comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 125 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO: 125; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shRNA comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 127 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO: 127; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shRNA comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 129 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO: 129; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • a shRNA comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 131 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO: 131; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence; and
  • a shRNA comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 133 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO: 133; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence.
  • the shRNA may be selected from the group consisting of:
  • a shRNA comprising an effector sequence set forth in SEQ ID NO: 11 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 11 and capable of forming a duplex therewith;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO: 13 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 13 and capable of forming a duplex therewith;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO: 15 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 15 and capable of forming a duplex therewith;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO: 17 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 17 and capable of forming a duplex therewith;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO: 19 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 19 and capable of forming a duplex therewith;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO:21 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:21 and capable of forming a duplex therewith;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO:23 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:23 and capable of forming a duplex therewith;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO:25 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:25 and capable of forming a duplex therewith;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO:27 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:27 and capable of forming a duplex therewith;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO:29 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:29 and capable of forming a duplex therewith;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO:31 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:31 and capable of forming a duplex therewith;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO:33 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:33 and capable of forming a duplex therewith;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO:35 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:35 and capable of forming a duplex therewith;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO:37 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:37 and capable of forming a duplex therewith;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO:39 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:39 and capable of forming a duplex therewith;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO:41 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:41 and capable of forming a duplex therewith;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO: 110 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 110 and capable of forming a duplex therewith;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO: 112 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 112 and capable of forming a duplex therewith;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO: 114 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 114 and capable of forming a duplex therewith;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO: 116 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 116 and capable of forming a duplex therewith
  • a shRNA comprising an effector sequence set forth in SEQ ID NO: 118 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 118 and capable of forming a duplex therewith
  • a shRNA comprising an effector sequence set forth in SEQ ID NO: 120 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 120 and capable of forming a duplex therewith;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO: 122 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 122 and capable of forming a duplex therewith;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO: 124 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 124 and capable of forming a duplex therewith;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO: 126 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 126 and capable of forming a duplex therewith;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO: 128 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 128 and capable of forming a duplex therewith;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO: 130 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 130 and capable of forming a duplex therewith;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO: 132 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 132 and capable of forming a duplex therewith.
  • the shRNA encoded by the nucleic acid in the plurality of nucleic acids described herein may comprise an effector complement sequence comprising 1, 2, 3, 4, 5 or 6 base mismatches relative to the corresponding effector sequence, provided that the cognate effector and effector complement sequences are capable of forming a duplex region.
  • the shRNA may be selected from the group consisting of:
  • a shRNA comprising an effector sequence set forth in SEQ ID NO: 11 and an effector complement sequence set forth in SEQ ID NO: 12;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO: 13 and an effector complement sequence set forth in SEQ ID NO: 14;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO: 15 and an effector complement sequence set forth in SEQ ID NO: 16;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO: 17 and an effector complement sequence set forth in SEQ ID NO: 18;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO: 19 and an effector complement sequence set forth in SEQ ID NO: 20;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO:21 and an effector complement sequence set forth in SEQ ID NO:22; a shRNA comprising an effector sequence set forth in SEQ ID NO:23 and an effector complement sequence set forth in SEQ ID NO:24;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO:25 and an effector complement sequence set forth in SEQ ID NO:26;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO:27 and an effector complement sequence set forth in SEQ ID NO:28;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO:29 and an effector complement sequence set forth in SEQ ID NO:30;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO:31 and an effector complement sequence set forth in SEQ ID NO:32;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO:33 and an effector complement sequence set forth in SEQ ID NO:34;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO:35 and an effector complement sequence set forth in SEQ ID NO:36;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO:37 and an effector complement sequence set forth in SEQ ID NO:38;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO:39 and an effector complement sequence set forth in SEQ ID NO:40;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO:41 and an effector complement sequence set forth in SEQ ID NO:42;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO: 110 and an effector complement sequence set forth in SEQ ID NO: 111;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO: 112 and an effector complement sequence set forth in SEQ ID NO: 113;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO: 114 and an effector complement sequence set forth in SEQ ID NO: 115;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO: 116 and an effector complement sequence set forth in SEQ ID NO: 117;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO: 118 and an effector complement sequence set forth in SEQ ID NO: 119; a shRNA comprising an effector sequence set forth in SEQ ID NO: 120 and an effector complement sequence set forth in SEQ ID NO: 121;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO: 122 and an effector complement sequence set forth in SEQ ID NO: 123;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO: 124 and an effector complement sequence set forth in SEQ ID NO: 125;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO: 126 and an effector complement sequence set forth in SEQ ID NO: 127;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO: 128 and an effector complement sequence set forth in SEQ ID NO: 129;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO: 130 and an effector complement sequence set forth in SEQ ID NO: 131;
  • a shRNA comprising an effector sequence set forth in SEQ ID NO: 132 and an effector complement sequence set forth in SEQ ID NO: 133.
  • the shRNA may comprise a stem loop sequence positioned between the effector sequence and the effector complement sequence.
  • Suitable loop sequences may be selected from those known in the art.
  • suitable stem loops may be developed de novo.
  • a nucleic acid of the plurality described herein encoding a shRNA may comprise a DNA sequence encoding a stem loop positioned between the DNA sequences encoding the effector sequence and the effector complement sequence.
  • a shRNA encoded by a nucleic acid of the disclosure may comprise a sequence set forth in any one of SEQ ID NOs:78-93.
  • a nucleic acid in the plurality of nucleic acids described herein may comprise or consist of a DNA sequence set forth in in any one of SEQ ID NOs:94-109.
  • a plurality of nucleic acids in accordance with the present disclosure may comprise up to 10 nucleic acids, each encoding a shmiR as described herein, such as two nucleic acids or three nucleic acids or four nucleic acids or five nucleic acids or six nucleic acids or seven nucleic acids or eight nucleic acids or nine nucleic acids or ten nucleic acids.
  • the plurality of nucleic acids comprises two nucleic acids of the disclosure, each encoding a shmiR as described herein.
  • the plurality of nucleic acids comprises three nucleic acids of the disclosure, each encoding a shmiR as described herein.
  • the plurality of nucleic acids comprises four nucleic acids of the disclosure, each encoding a shmiR as described herein. In one example, the plurality of nucleic acids comprises five nucleic acids of the disclosure, each encoding a shmiR as described herein. In one example, the plurality of nucleic acids comprises six nucleic acids of the disclosure, each encoding a shmiR as described herein. In one example, the plurality of nucleic acids comprises seven nucleic acids of the disclosure, each encoding a shmiR as described herein. In one example, the plurality of nucleic acids comprises eight nucleic acids of the disclosure, each encoding a shmiR as described herein.
  • the plurality of nucleic acids comprises nine nucleic acids of the disclosure, each encoding a shmiR as described herein.
  • the plurality of RNAs comprises ten nucleic acids of the disclosure, each encoding a shmiR as described herein.
  • one or more of the nucleic acids in the plurality may encode a shRNA as described herein.
  • two or more of the nucleic acids may form separate parts of the same polynucleotide.
  • two or more of the nucleic acids in the plurality form parts of different
  • the or each nucleic acid in accordance with the present disclosure may comprise, or be in operable linkage with, one or more transcriptional terminator sequences.
  • the or each nucleic acid may comprise a transcriptional terminator sequence at the 3' terminus of the sequence encoding the shmiR or shRNA.
  • Such sequences will depend on the choice of promoter and will be known to a person of skill in the art.
  • a transcriptional terminator sequence may include ' ⁇ ' or ' XXXXX' .
  • the or each nucleic acid in accordance with the present disclosure may comprise, or be in operable linkage with, a transcription initiator sequence.
  • the or each nucleic acid may comprise a transcription initiator sequence at the 5' terminus of the sequence encoding the shmiR or shRNA. Such sequences will be known to a person of skill in the art, but may include 'G' .
  • the or each nucleic acid in accordance with the present disclosure may comprise one or more restriction sites e.g., to facilitate cloning of the nucleic acid(s) into cloning or expression vectors.
  • the nucleic acids described herein may include a restriction site upstream and/or downstream of the sequence encoding a shmiR or shRNA of the disclosure. Suitable restriction enzyme recognition sequences will be known to a person of skill in the art.
  • the nucleic acid(s) of the disclosure may include a BamHl restriction site (GGATCC) at the 5' terminus i.e., upstream of the sequence encoding the shmiR or shRNA, and a EcoRl restriction site (GAATTC) at the 3' terminus i.e., downstream of the sequence encoding the shmiR or shRNA.
  • GGATCC BamHl restriction site
  • GATTC EcoRl restriction site
  • a nucleic acid in accordance with the present disclosure may also be provided in the form of, or be comprised in, a DNA-directed RNA interference (ddRNAi) construct which is capable of expressing one or more shmiRs which is/are encoded by the nucleic acid(s) of the present disclosure.
  • ddRNAi DNA-directed RNA interference
  • one or more ddRNAi constructs comprising a nucleic acid of the disclosure is also provided.
  • ddRNAi constructs each comprising a nucleic acid encoding a shmiR as described herein is provided, wherein:
  • At least one of the plurality of ddRNAi constructs comprises a first nucleic acid of the plurality of nucleic acids as described herein;
  • At least one of the plurality of ddRNAi constructs comprises a second nucleic acid of the plurality of nucleic acids described herein;
  • first and second nucleic acids encode shmiRs that are different to one another.
  • the plurality of ddRNAi constructs described herein may comprise up to 10 ddRNAi constructs, each comprising one or more nucleic acids encoding a shmiR as described herein, such as two ddRNAi constructs or three ddRNAi constructs or four ddRNAi constructs or five ddRNAi constructs or six ddRNAi constructs or seven ddRNAi constructs or eight ddRNAi constructs or nine ddRNAi constructs or ten ddRNAi constructs of the disclosure.
  • a ddRNAi construct of the disclosure comprises a plurality of nucleic acids as described herein, such that the ddRNAi construct encodes a plurality of shmiRs targeting HBV, wherein each of the shmiRs are different to one another.
  • the ddRNAi construct comprises at least two nucleic acids of the disclosure, such that the ddRNAi construct encodes at least two shmiRs targeting HBV, each of which is different to one another.
  • An exemplary ddRNAi construct comprising at least two nucleic acids of the disclosure may comprise:
  • the ddRNAi construct of the disclosure comprises at least three nucleic acids described herein, such that the ddRNAi construct encodes at least three shmiRs targeting HBV, each of which is different to one another.
  • ddRNAi construct of the disclosure comprises:
  • nucleic acid at (b) encodes a shmiR or shRNA having an effector sequence which is different to that of the shmiRs encoded by the nucleic acid at (a) and (c).
  • a ddRNAi construct of the disclosure may comprise, preferably in a 5' to 3 'direction:
  • a ddRNAi construct of the disclosure may comprise, preferably in a 5' to
  • a ddRNAi construct of the disclosure may comprise, preferably in a 5' to 3 'direction:
  • a ddRNAi construct of the disclosure may comprise, preferably in a 5' to 3 'direction:
  • the ddRNAi construct comprises:
  • (c) a nucleic acid encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 141.
  • (a) to (c) are provided in a 5' to 3 'direction in the ddRNAi construct.
  • a ddRNAi construct of the disclosure may comprise, preferably in a 5' to 3 'direction:
  • a ddRNAi construct of the disclosure may comprise at least one nucleic acid encoding a shmiR as described herein and at least one nucleic acid encoding a shRNA targeting HBV as described herein, wherein the shmiR and shRNA encoded by the ddRNAi construct comprise different effector sequences.
  • a ddRNAi construct of the disclosure may comprise, preferably in a 5' to 3 'direction:
  • a ddRNAi construct of the disclosure may comprise, preferably in a 5' to 3 'direction:
  • a ddRNAi construct of the disclosure may comprise, preferably in a 5' to 3 'direction:
  • a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO:70.
  • a ddRNAi construct as described herein comprises a single promoter which is operably-linked to the or each nucleic acid encoding a shmiR or shRNA of the disclosure.
  • each nucleic acid encoding a shmiR or shRNA of the disclosure is operably-linked to a separate promoter.
  • the promoter(s) is(are) positioned upstream of the respective nucleic acid(s) encoding the shmiR(s) or shRNA(s).
  • the promoters may be the same or different.
  • Exemplary promoters are RNA pol III promoters, such as for example, the U6 and HI promoters.
  • Exemplary U6 promoters are U6-1. U6-8 and U6-9 promoters.
  • a ddRNAi construct of the disclosure comprises, in a 5' to 3' direction:
  • a ddRNAi construct of the disclosure comprises, in a 5' to 3' direction:
  • a ddRNAi construct of the disclosure comprises, in a 5' to 3' direction:
  • the present disclosure also provides an expression vector, comprising a ddRNAi construct of the disclosure.
  • the present disclosure also provides plurality of expression vectors each of which comprises a ddRNAi construct of the disclosure.
  • one or more of the plurality of expression vectors comprises a plurality of ddRNAi constructs as disclosed herein.
  • each of the plurality of expression vectors comprises a plurality of ddRNAi constructs as disclosed herein.
  • each of the plurality of expression vectors comprises a single ddRNAi construct as described herein.
  • the plurality of expression vectors may collectively express a plurality of shmiRs in accordance with the present disclosure.
  • the or each expression vector is a plasmid or a minicircle.
  • the plasmid or minicircle or expression vector or ddRNAi construct is complexed with a cationic DNA binding polymer.
  • the or each expression vector is a viral vector.
  • the viral vector is selected from the group consisting of an adeno-associated viral (AAV) vector, a retroviral vector, an adenoviral vector (AdV) and a lentiviral (LV) vector.
  • AAV adeno-associated viral
  • AdV adenoviral vector
  • LV lentiviral
  • the present disclosure also provides a composition comprising a ddRNAi construct and/or a plurality of ddRNAi constructs and/or expression vector and/or a plurality of expression vectors as described herein.
  • the composition may also comprise one or more pharmaceutically acceptable carriers and/or diluents.
  • the composition may further comprise another therapeutic agent known for treating HBV infection i.e., as an adjunctive therapy.
  • the other therapeutic agent known for treating HBV infection may be selected from entecavir, tenofovir, lamivudine, adefovir and/or pegylated interferon.
  • the present disclosure also provides a method of treating HBV infection in a subject, the method comprising administering a therapeutically effective amount of a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or composition described herein to the subject.
  • the present disclosure also provides a method of reducing HBV viral load in a subject infected with HBV, the method comprising administering a therapeutically effective amount of a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or composition described herein to the subject.
  • the present disclosure also provides a method of reducing severity of one or more symptoms associated with HBV infection in a subject suffering therefrom, the method comprising administering to the subject a therapeutically effective amount of a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or composition described herein to the subject.
  • the present disclosure also provides a method of reducing the infectivity of HBV in a subject infected therewith, the method comprising administering to the subject a
  • nucleic acid a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or composition described herein to the subject.
  • the present disclosure also provides a method for reducing the risk of a subject suffering from a HBV infection developing chronic hepatic insufficiency, cirrhosis, and/or hepatocellular carcinoma, the method comprising administering to the subject a
  • nucleic acid a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or composition described herein to the subject.
  • the subject is suffering from acute HBV infection.
  • the subject is suffering from chronic HBV infection.
  • the methods described herein comprise inhibiting or reducing expression of one or more transcripts encoded by the HBV genome in the subject.
  • the subject to which the nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or composition of the disclosure is/are administered has already received treatment with another therapeutic agent for treating HBV infection.
  • the subject and/or the HBV is refractory or resistant to treatment with the other agent known for treating HBV infection.
  • the nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or composition of the disclosure is administered in combination with another therapeutic agent known for treating HBV infection i.e., as an adjunctive therapy.
  • the other therapeutic agent known for treating HBV infection may be selected from entecavir, tenofovir, lamivudine, adefovir and/or pegylated interferon.
  • the other therapeutic agent known for treating HBV infection and the nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or composition of the disclosure may be administered separately or together.
  • the other therapeutic agent known for treating HBV infection may be adminiatered simultaneously with the administration of the nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or composition of the disclosure.
  • the other therapeutic agent known for treating HBV infection may be adminiatered consecutively with the administration of the nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or composition of the disclosure.
  • a composition of the present disclosure is provided in a kit.
  • a composition of the present disclosure is packaged together with one or more other therapeutic agents known for treating HBV infections.
  • Such other therapeutic agents will be known to a person of skill in the art.
  • the other therapeutic agent known for treating HBV infection may be selected from entecavir, tenofovir, lamivudine, adefovir and/or pegylated interferon.
  • the composition is packaged with instruction for use in a method of the disclosure.
  • the present disclosure also provides use of a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or composition described herein in the preparation of a medicament, e.g., for treating HBV infection in a subject and/or in a method disclosed herein.
  • the subject is suffering from acute HBV infection.
  • the subject is suffering from chronic HBV infection.
  • the present disclosure also provides a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or composition described herein for use in therapy.
  • the nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or composition may be for use in treating HBV infection in a subject and/or in a method disclosed herein.
  • the subject may be suffering from acute HBV infection. In an alternative example, the subject may be suffering from chronic HBV infection.
  • Treatment of HBV in accordance with any example described herein may comprise one or more of reducing HBV viral load in the subject, reducing severity of symptoms associated with HBV infection and/or reducing the infectivity of HBV in a subject.
  • the medicament will reduce HBV gene transcription products in the subject to which the medicament is administered.
  • Figure 1 illustrates the inhibitory activity of shmiRs designated shmiR-6, shmiR-15 and shmiR-12 relative to their shRNA counterparts (shRNA-6, shRNA-15 and shRNA-12 respectively) in Luciferase reporter assays: (A) illustrates sense and antisense strand preference inhibitory activity for shmiRs designated shmiR-6, shmiR-15 and shmiR-12 relative to their shRNA counterparts (shRNA-6, shRNA-15 and shRNA-12 respectively) in the Luciferase reporter assay; (B)-(D) illustrate and compare the abilities of shmiR-6, shmiR-15 and shmiR-12 respectively to inhibit luciferase protein expression in a Luciferase reporter assay system in a dose-dependent manner relative to their shRNA counterparts (shRNA-6, shRNA-15 and shRNA-12 respectively) at equivalent doses.
  • Figure 2 illustrates sense and antisense strand preference inhibitory activity for variants of shmiR-12 and shmiR-15 relative to the respective parental shmiRs (shmiR-12 and shmiR-15 respectively) in the Lucif erase reporter assay: (A) shows that all six variants of shmiR-12 have better strand preference to the parental shmiR; and (B) shows that four of the six variants of shmiR-15 have better strand preference to the parental shmiR.
  • Figure 3 illustrates the level of expression of RNAi effector molecules, expressed as copies per cell, in HepG2.2.15 cells transduced with ddRNAi agents: (A) shows the relative levels of expression of effector sequences for (i) shmiR- 12 expressed as a single construct, (ii) shRNA-12 expressed as a single construct, and (iii) shRNA-12 expressed as part of HBV- shRNAx3-vl, at day 3, 4, 5 and 6 post transduction; and (B) shows the relative levels of expression of effector sequences for (i) shmiR- 15 expressed as a single construct, (ii) shRNA-15 expressed as a single construct, and (iii) shRNA-15 expressed as part of HBV- shRNAx3-vl, at day 3, 4, 5 and 6 post transduction.
  • A shows the relative levels of expression of effector sequences for (i) shmiR- 12 expressed as a single construct, (ii) shRNA-12 expressed as a single construct, and (iii) sh
  • MOI HBV AdV Vectors
  • Figure 5 provides construct diagrams for ssAAV8-HBV-shRNAx3-vl, scAAV8-HBV- shRNAx3-vl and ssAAV8-HBV-shmiRx3-vl.
  • Figure 6 provides a flow diagram for the in vivo efficacy study performed in the PhoenixBio (PXB) Chimeric mouse model using ssAAV8-HBV-shRNAx3-vl, scAAV8-HBV- shRNAx3-vl and ssAAV8-HBV-shmiRx3-vl.
  • Figure 7 illustrates that serum HBV DNA levels were reduced in mice treated with a single dose of scAAV8-HBV-shRNAx3-vl or ssAAV8-HBV-shmiRx3-vl as a monotherapy or in combination with Entecavir, whereas virus titers in saline treated control animals remained relatively constant over the course of the 91 day study.
  • Figure 8 illustrates that serum HBsAg antigen levels were reduced in mice treated with a single dose of scAAV8-HBV-shRNAx3-vl or ssAAV8-HBV-shmiRx3-vl as a
  • Figure 9 illustrates that serum HBeAg antigen levels were reduced in mice treated with a single dose of scAAV8-HBV-shRNAx3-vl or ssAAV8-HBV-shmiRx3-vl as a
  • Figure 10 illustrates that serum HBV DNA levels dropped significantly in mice treated with a single dose of scAAV8-HBV-shRNAx3-vl or ssAAV8-HBV-shmiRx3-vl as a monotherapy or in combination with pegylated interferon (where the pegylated interferon is administered twice daily), whereas serum HBV DNA levels in saline treated control animals remained relatively constant over the course of the 91 day study.
  • Figure 11 illustrates that HBV RNA levels were reduced in mice treated with pegylated interferon as a monotherapy, or a single dose of ssAAV8-HBV-shmiRx3-vl as a
  • HBV RNA levels were only modestly reduced when Entecavir was administered alone.
  • (A)-(C) show HBV RNA levels for transcript corresponding to shmiR-6, shmiR-15 and shmiR-12, respectively.
  • Figure 12 illustrates that both intracellular HBV DNA and cccDNA levels were reduced in livers of mice treated with a single dose of ssAAV8-HBV-shmiRx3-vl as a monotherapy or in combination with Entecavir or pegylated interferon.
  • Figure 13 illustrates the level of expression of RNAi effector molecules, expressed as copies per cell, in liver tissue obtained from mice treated with ssAAV8-HBV-shRNAx3-vl, scAAV8-HBV-shRNAx3-vl or ssAAV8-HBV-shmiRx3-vl as a monotherapy or in combination with Entecavir or pegylated interferon.
  • Figure 14 illustrates the predominant shRNA-6/shmiR-6 effector sequence species and numbers of same produced from ssAAV8-HBV-shRNAx3-vl, scAAV8-HBV-shRNAx3-vl and ssAAV8-HBV-shmiRx3-vl in liver tissue obtained from PXB mice, as determined by next generation sequencing (NGS).
  • NGS next generation sequencing
  • Figure 15 illustrates the predominant shRNA-15/shmiR-15 effector sequence species and numbers of same produced from ssAAV8-HBV-shRNAx3-vl, scAAV8-HBV-shRNAx3-vl and ssAAV8-HBV-shmiRx3-vl in liver tissue obtained from PXB mice, as determined by next generation sequencing (NGS).
  • NGS next generation sequencing
  • Figure 16 illustrates the predominant shRNA-12/shmiR-12 effector sequence species and numbers of same produced from ssAAV8-HBV-shRNAx3-vl, scAAV8-HBV-shRNAx3-vl and ssAAV8-HBV-shmiRx3-vl in liver tissue obtained from PXB mice, as determined by next generation sequencing (NGS).
  • NGS next generation sequencing
  • SEQ ID NO: 1 RNA transcript for target Region 1 within HBV genome.
  • SEQ ID NO: 2 RNA transcript for target Region 2 within HBV genome.
  • SEQ ID NO: 3 RNA transcript for target Region 3 within HBV genome.
  • SEQ ID NO: 4 RNA transcript for target Region 4 within HBV genome.
  • SEQ ID NO: 5 RNA transcript for target Region 5 within HBV genome.
  • SEQ ID NO: 6 RNA transcript for target Region 6 within HBV genome.
  • SEQ ID NO: 7 RNA transcript for target Region 7 within HBV genome.
  • SEQ ID NO: 8 RNA transcript for target Region 8 within HBV genome.
  • SEQ ID NO: 9 RNA transcript for target Region 9 within HBV genome.
  • SEQ ID NO: 10 RNA transcript for target Region 10 within HBV genome
  • SEQ ID NO: 11 RNA effector sequence for shmiR- 1.
  • SEQ ID NO: 12 RNA effector complement sequence for shmiR- 1.
  • SEQ ID NO: 13 RNA effector sequence for shmiR-2.
  • SEQ ID NO: 14 RNA effector complement sequence for shmiR-2.
  • SEQ ID NO: 15 RNA effector sequence for shmiR-3.
  • SEQ ID NO: 16 RNA effector complement sequence for shmiR-3.
  • SEQ ID NO: 17 RNA effector sequence for shmiR-4.
  • SEQ ID NO: 18 RNA effector complement sequence for shmiR-4.
  • SEQ ID NO: 19 RNA effector sequence for shmiR-5.
  • SEQ ID NO: 20 RNA effector complement sequence for shmiR- 5.
  • SEQ ID NO: 21 RNA effector sequence for shmiR-6.
  • SEQ ID NO: 22 RNA effector complement sequence for shmiR-6.
  • SEQ ID NO: 23 RNA effector sequence for shmiR-7.
  • SEQ ID NO: 24 RNA effector complement sequence for shmiR-7.
  • SEQ ID NO: 25 RNA effector sequence for shmiR- 8.
  • SEQ ID NO: 26 RNA effector complement sequence for shmiR- 8.
  • SEQ ID NO: 27 RNA effector sequence for shmiR-9.
  • SEQ ID NO: 28 RNA effector complement sequence for shmiR-9.
  • SEQ ID NO: 29 RNA effector sequence for shmiR- 10.
  • SEQ ID NO: 30 RNA effector complement sequence for shmiR- 10.
  • SEQ ID NO: 31 RNA effector sequence for shmiR- 11.
  • SEQ ID NO: 32 RNA effector complement sequence for shmiR- 11.
  • SEQ ID NO: 33 RNA effector sequence for shmiR- 12
  • SEQ ID NO: 34 RNA effector complement sequence for shmiR- 12.
  • SEQ ID NO: 35 RNA effector sequence for shmiR- 13.
  • SEQ ID NO: 36 RNA effector complement sequence for shmiR- 13.
  • SEQ ID NO: 37 RNA effector sequence for shmiR- 14.
  • SEQ ID NO: 38 RNA effector complement sequence for shmiR- 14.
  • SEQ ID NO: 39 RNA effector sequence for shmiR- 15.
  • SEQ ID NO: 40 RNA effector complement sequence for shmiR- 15.
  • SEQ ID NO: 41 RNA effector sequence for s shmiR- 16.
  • SEQ ID NO: 42 RNA effector complement sequence for shmiR-16
  • SEQ ID NO: 43 RNA sequence for shmiR- 1.
  • SEQ ID NO: 44 RNA sequence for shmiR-2.
  • SEQ ID NO: 45 RNA sequence for shmiR-3.
  • SEQ ID NO: 46 RNA sequence for shmiR-4.
  • SEQ ID NO: 47 RNA sequence for shmiR-5.
  • SEQ ID NO: 48 RNA sequence for shmiR-6.
  • SEQ ID NO: 49 RNA sequence for shmiR-7.
  • SEQ ID NO: 50 RNA sequence for shmiR-8.
  • SEQ ID NO: 51 RNA sequence for shmiR-9.
  • SEQ ID NO: 52 RNA sequence for shmiR-10.
  • SEQ ID NO: 53 RNA sequence for shmiR- 11.
  • SEQ ID NO: 54 RNA sequence for shmiR- 12.
  • SEQ ID NO: 55 RNA sequence for shmiR- 13.
  • SEQ ID NO: 56 RNA sequence for shmiR- 14.
  • SEQ ID NO: 57 RNA sequence for shmiR- 15.
  • SEQ ID NO: 58 RNA sequence for shmiR- 16.
  • SEQ ID NO: 59 DNA sequence coding for shmiR- 1.
  • SEQ ID NO: 60 DNA sequence coding for shmiR-2.
  • SEQ ID NO: 61 DNA sequence coding for shmiR-3.
  • SEQ ID NO: 62 DNA sequence coding for shmiR-4.
  • SEQ ID NO: 63 DNA sequence coding for shmiR- 5.
  • SEQ ID NO: 64 DNA sequence coding for shmiR-6.
  • SEQ ID NO: 65 DNA sequence coding for shmiR-7.
  • SEQ ID NO: 66 DNA sequence coding for shmiR- 8.
  • SEQ ID NO: 67 DNA sequence coding for shmiR-9.
  • SEQ ID NO: 68 DNA sequence coding for shmiR- 10.
  • SEQ ID NO- 69 DNA sequence coding for shmiR- 11.
  • SEQ ID NO 70 DNA sequence coding for shmiR- 12.
  • SEQ ID NO 71 DNA sequence coding for shmiR- 13.
  • SEQ ID NO 72 DNA sequence coding for shmiR- 14.
  • SEQ ID NO: 73 DNA sequence coding for shmiR-15.
  • SEQ ID NO: 74 DNA sequence coding for shmiR-16.
  • SEQ ID NO: 75 stemloop RNA sequence for shmiRs
  • SEQ ID NO: 76 5' flanking sequence of pri-miR-30a backbone.
  • SEQ ID NO: 77 3' flanking sequence of pri-miR-30a backbone.
  • SEQ ID NO: 78 RNA sequence for shRNA designated shRNA- 1.
  • SEQ ID NO: 79 RNA sequence for shRNA designated shRNA-2.
  • SEQ ID NO: 80 RNA sequence for shRNA designated shRNA-3.
  • SEQ ID NO: 81 RNA sequence for shRNA designated shRNA-4.
  • SEQ ID NO: 82 RNA sequence for shRNA designated shRNA-5.
  • SEQ ID NO: 83 RNA sequence for shRNA designated shRNA-6.
  • SEQ ID NO: 84 RNA sequence for shRNA designated shRNA-7.
  • SEQ ID NO: 85 RNA sequence for shRNA designated shRNA- 8.
  • SEQ ID NO: 86 RNA sequence for shRNA designated shRNA-9.
  • SEQ ID NO: 87 RNA sequence for shRNA designated shRNA- 10.
  • SEQ ID NO: 88 RNA sequence for shRNA designated shRNA-11.
  • SEQ ID NO: 89 RNA sequence for shRNA designated shRNA- 12.
  • SEQ ID NO: 90 RNA sequence for shRNA designated shRNA- 13.
  • SEQ ID NO: 91 RNA sequence for shRNA designated shRNA- 14.
  • SEQ ID NO: 92 RNA sequence for shRNA designated shRNA- 15.
  • SEQ ID NO: 93 RNA sequence for shRNA designated shRNA- 16.
  • SEQ ID NO: 94 DNA sequence coding for shRNA designated shRNA- 1.
  • SEQ ID NO: 95 DNA sequence coding for shRNA designated shRNA-2.
  • SEQ ID NO: 96 DNA sequence coding for shRNA designated shRNA-3.
  • SEQ ID NO: 97 DNA sequence coding for shRNA designated shRNA-4.
  • SEQ ID NO: 98 DNA sequence coding for shRNA designated shRNA-5.
  • SEQ ID NO: 99 DNA sequence coding for shRNA designated shRNA-6.
  • SEQ ID NO: 100 DNA sequence coding for shRNA designated shRNA-7.
  • SEQ ID NO: 101 DNA sequence coding for shRNA designated shRNA- 8.
  • SEQ ID NO: 102 DNA sequence coding for shRNA designated shRNA-9.
  • SEQ ID NO: 103 DNA sequence coding for shRNA designated shRNA- 10.
  • SEQ ID NO: 104 DNA sequence coding for shRNA designated shRNA-
  • SEQ ID NO: 105 DNA sequence coding for shRNA designated shRNA-
  • SEQ ID NO: 106 DNA sequence coding for shRNA designated shRNA-
  • SEQ ID NO: 107 DNA sequence coding for shRNA designated shRNA-
  • SEQ ID NO: 108 DNA sequence coding for shRNA designated shRNA-
  • SEQ ID NO: 109 DNA sequence coding for shRNA designated shRNA
  • SEQ ID NO: 110 RNA effector sequence for shmiR-17.
  • SEQ ID NO: 111 RNA effector complement sequence for shmiR-17.
  • SEQ ID NO: 112 RNA effector sequence for shmiR-18.
  • SEQ ID NO: 113 RNA effector complement sequence for shmiR-18.
  • SEQ ID NO: 114 RNA effector sequence for shmiR-19.
  • SEQ ID NO: 115 RNA effector complement sequence for shmiR- 19.
  • SEQ ID NO: 116 RNA effector sequence for shmiR-20.
  • SEQ ID NO: 117 RNA effector complement sequence for shmiR-20.
  • SEQ ID NO: 118 RNA effector sequence for shmiR-21.
  • SEQ ID NO: 119 RNA effector complement sequence for shmiR-21.
  • SEQ ID NO: 120 RNA effector sequence for shmiR-22.
  • SEQ ID NO: 121 RNA effector complement sequence for shmiR-22.
  • SEQ ID NO: 122 RNA effector sequence for shmiR-23.
  • SEQ ID NO: 123 RNA effector complement sequence for shmiR-23.
  • SEQ ID NO: 124 RNA effector sequence for shmiR-24.
  • SEQ ID NO: 125 RNA effector complement sequence for shmiR-24.
  • SEQ ID NO: 126 RNA effector sequence for shmiR-25.
  • SEQ ID NO: 127 RNA effector complement sequence for shmiR-25.
  • SEQ ID NO: 128 RNA effector sequence for shmiR-26.
  • SEQ ID NO: 129 RNA effector complement sequence for shmiR-26.
  • SEQ ID NO: 130 RNA effector sequence for shmiR-27.
  • SEQ ID NO: 131 RNA effector complement sequence for shmiR-27.
  • SEQ ID NO: 132 RNA effector sequence for shmiR-28.
  • SEQ ID NO: 133 RNA effector complement sequence for shmiR-28.
  • SEQ ID NO: 134 RNA sequence for shmiR-17.
  • SEQ ID NO: 135 RNA sequence for shmiR-17.
  • SEQ ID NO: 165 SEQ ID NO: 166: DNA sequence corresponding to shRNA-6 effector species 2.
  • SEQ ID NO: 167 DNA sequence corresponding to shRNA-6 effector species 3.
  • SEQ ID NO: 168 DNA sequence corresponding to shRNA-6 effector species 4.
  • SEQ ID NO: 169 DNA sequence corresponding to shRNA-6 effector species 5.
  • SEQ ID NO: 170 DNA sequence corresponding to shRNA-6 effector species 6.
  • SEQ ID NO: 171 DNA sequence corresponding to shRNA-6 effector species 7.
  • SEQ ID NO: 172 DNA sequence corresponding to shRNA-6 effector species 8.
  • SEQ ID NO: 173 DNA sequence corresponding to shRNA-6 effector species 9.
  • SEQ ID NO: 174 DNA sequence corresponding to shRNA-6 effector species 10.
  • SEQ ID NO: 175 DNA sequence corresponding to shRNA-6 effector species 11.
  • SEQ ID NO: 176 DNA sequence coding for shmiR-6, including flanking sequence of miRNA backbone.
  • SEQ ID NO: 177 DNA sequence corresponding to shmiR-6 effector species 1.
  • SEQ ID NO: 178 DNA sequence corresponding to shmiR-6 effector species 2.
  • SEQ ID NO: 179 DNA sequence corresponding to shmiR-6 effector species 3.
  • SEQ ID NO: 180 DNA sequence corresponding to shmiR-6 effector species 4.
  • SEQ ID NO: 181 DNA sequence corresponding to shmiR-6 effector species 5.
  • SEQ ID NO: 182 DNA sequence coding for shRNA-15, including flanking sequence
  • SEQ ID NO: 183 DNA sequence corresponding to shRNA-15 effector species 1.
  • SEQ ID NO: 184 DNA sequence corresponding to shRNA-15 effector species 2.
  • SEQ ID NO: 185 DNA sequence corresponding to shRNA-15 effector species 3.
  • SEQ ID NO: 186 DNA sequence corresponding to shRNA-15 effector species 4.
  • SEQ ID NO: 187 DNA sequence corresponding to shRNA-15 effector species 5.
  • SEQ ID NO: 188 DNA sequence corresponding to shRNA-15 effector species 6.
  • SEQ ID NO: 189 DNA sequence corresponding to shRNA-15 effector species 7.
  • SEQ ID NO: 190 DNA sequence corresponding to shRNA-15 effector species 8.
  • SEQ ID NO: 191 DNA sequence corresponding to shRNA-15 effector species 9.
  • SEQ ID NO: 192 DNA sequence corresponding to shRNA-15 effector species 10.
  • SEQ ID NO: 193 DNA sequence corresponding to shRNA-15 effector species 11.
  • SEQ ID NO: 194 DNA sequence corresponding to shRNA-15 effector species 12.
  • SEQ ID NO: 195 DNA sequence corresponding to shRNA-15 effector species 13.
  • SEQ ID NO: 196 DNA sequence corresponding to shRNA- 15 effector species 14.
  • SEQ ID NO: 197 DNA sequence corresponding to shRNA-15 effector species 15.
  • SEQ ID NO: 198 DNA sequence corresponding to shRNA-15 effector species 16.
  • SEQ ID NO: 199 DNA sequence corresponding to shRNA-15 effector species 17.
  • SEQ ID NO: 200 DNA sequence corresponding to shRNA- 15 effector species 18.
  • SEQ ID NO: 201 DNA sequence corresponding to shRNA-15 effector species 19.
  • SEQ ID NO: 202 DNA sequence coding for shmiR-15, including flanking sequence of miRNA backbone.
  • SEQ ID NO: 203 DNA sequence corresponding to shmiR-15 effector species 1.
  • SEQ ID NO: 204 DNA sequence corresponding to shmiR-15 effector species 2.
  • SEQ ID NO: 205 DNA sequence corresponding to shmiR-15 effector species 3.
  • SEQ ID NO: 206 DNA sequence corresponding to shmiR-15 effector species 4.
  • SEQ ID NO: 207 DNA sequence corresponding to shmiR-15 effector species 5.
  • SEQ ID NO: 208 DNA sequence corresponding to shmiR-15 effector species 6.
  • SEQ ID NO: 209 DNA sequence coding for shRNA-12, including flanking sequence.
  • SEQ ID NO: 210 DNA sequence corresponding to shRNA-12 effector species 1.
  • SEQ ID NO: 211 DNA sequence corresponding to shRNA-12 effector species 2.
  • SEQ ID NO: 212 DNA sequence corresponding to shRNA- 12 effector species 3.
  • SEQ ID NO: 213 DNA sequence corresponding to shRNA-12 effector species 4.
  • SEQ ID NO: 214 DNA sequence corresponding to shRNA-12 effector species 5.
  • SEQ ID NO: 215 DNA sequence corresponding to shRNA-12 effector species 6.
  • SEQ ID NO: 216 DNA sequence corresponding to shRNA- 12 effector species 7.
  • SEQ ID NO: 217 DNA sequence corresponding to shRNA-12 effector species 8.
  • SEQ ID NO: 218 DNA sequence corresponding to shRNA-12 effector species 9.
  • SEQ ID NO: 219 DNA sequence corresponding to shRNA-12 effector species 10.
  • SEQ ID NO: 220 DNA sequence corresponding to shRNA- 12 effector species 11.
  • SEQ ID NO: 221 DNA sequence corresponding to shRNA-12 effector species 12.
  • SEQ ID NO: 222 DNA sequence corresponding to shRNA-12 effector species 13.
  • SEQ ID NO: 223 DNA sequence corresponding to shRNA-12 effector species 14.
  • SEQ ID NO: 224 DNA sequence coding for shmiR-12, including flanking sequence of miRNA backbone.
  • SEQ ID NO: 225 DNA sequence corresponding to shmiR-12 effector species 1.
  • SEQ ID NO: 226 DNA sequence corresponding to shmiR-12 effector species 2.
  • SEQ ID NO: 227 DNA sequence corresponding to shmiR-12 effector species 3.
  • SEQ ID NO: 228 DNA sequence corresponding to shmiR-12 effector species 4.
  • SEQ ID NO: 229 DNA sequence corresponding to shmiR-12 effector species 5.
  • the recombinant DNA, recombinant protein, cell culture, and immunological techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M.
  • RNA is meant a molecule comprising at least one ribonucleotide residue.
  • ribonucleotide is meant a nucleotide with a hydroxyl group at the 2' position of a ⁇ -D-ribo- furanose moiety.
  • the terms include double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides.
  • Such alterations can include addition of non-nucleotide material, such as to the end(s) of the siNA or internally, for example at one or more nucleotides of the RNA.
  • RNA interference refers generally to RNA-dependent silencing of gene expression initiated by double stranded RNA (dsRNA) molecules in a cell's cytoplasm.
  • dsRNA double stranded RNA
  • the dsRNA molecule reduces or inhibits transcription products of a target nucleic acid sequence, thereby silencing the gene or reducing expression of that gene.
  • double stranded RNA or “dsRNA” refers to a RNA molecule having a duplex structure and comprising an effector sequence and an effector complement sequence which are of similar length to one another.
  • the effector sequence and the effector complement sequence can be in a single RNA strand or in separate RNA strands.
  • the "effector sequence” (often referred to as a “guide strand”) is substantially complementary to a target sequence, which in the present case, is a region of a RNA transcription product of the HBV genome.
  • the "effector sequence” can also be referred to as the "antisense sequence”.
  • effector complement sequence will be of sufficient complementary to the effector sequence such that it can anneal to the effector sequence to form a duplex.
  • the effector complement sequence will be substantially homologous to a region of target sequence.
  • effector complement sequence can also be referred to as the "complement of the effector sequence” or the sense sequence.
  • duplex refers to regions in two complementary or substantially complementary nucleic acids (e.g., RNAs), or in two complementary or substantially complementary regions of a single- stranded nucleic acid (e.g., RNA), that form base pairs with one another, either by Watson-Crick base pairing or any other manner that allows for a stabilized duplex between the nucleotide sequences that are complementary or substantially complementary. It will be understood by the skilled person that within a duplex region, 100% complementarity is not required; substantial complementarity is allowable. Substantial complementarity includes may include 69% or greater
  • a single mismatch in a duplex region consisting of 19 base pairs results in 94.7% complementarity, rendering the duplex region substantially complementary.
  • two mismatches in a duplex region consisting of 19 base pairs results in 89.5% complementarity, rendering the duplex region substantially complementary.
  • three mismatches in a duplex region consisting of 19 base pairs results in 84.2% complementarity, rendering the duplex region substantially complementary, and so on.
  • the dsRNA may be provided as a hairpin or stem loop structure, with a duplex region comprised of an effector sequence and effector complement sequence linked by at least 2 nucleotide sequence which is termed a stem loop.
  • a dsRNA is provided as a hairpin or stem loop structure it can be referred to as a "hairpin RNA” or “short hairpin RNAi agent” or “shRNA”.
  • dsRNA molecules provided in, or which give rise to, a hairpin or stem loop structure include primary miRNA transcipts (pri-miRNA) and precursor microRNA (pre- miRNA).
  • Pre-miRNA shRNAs can be naturally produced from pri-miRNA by the action of the enzymes Drosha and Pasha which recognize and release regions of the primary miRNA transcript which form a stem-loop structure.
  • the pri-miRNA transcript can be engineered to replace the natural stem-loop structure with an artificial/recombinant stem- loop structure. That is, an artificial/recombinant stem-loop structure may be inserted or cloned into a pri-miRNA backbone sequence which lacks its natural stem- loop structure.
  • dsRNA molecules produced using this approach are known as "shmiRNAs", “shmiRs” or “microRNA framework shRNAs”.
  • the term "complementary" with regard to a sequence refers to a complement of the sequence by Watson-Crick base pairing, whereby guanine (G) pairs with cytosine (C), and adenine (A) pairs with either uracil (U) or thymine (T).
  • a sequence may be complementary to the entire length of another sequence, or it may be complementary to a specified portion or length of another sequence.
  • U may be present in RNA
  • T may be present in DNA. Therefore, an A within either of a RNA or DNA sequence may pair with a U in a RNA sequence or T in a DNA sequence.
  • the term "substantially complementary” is used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between nucleic acid sequences e.g., between the effector sequence and the effector complement sequence or between the effector sequence and the target sequence. It is understood that the sequence of a nucleic acid need not be 100% complementary to that of its target or complement.
  • the term encompasses a sequence complementary to another sequence with the exception of an overhang. In some cases, the sequence is complementary to the other sequence with the exception of 1-2 mismatches. In some cases, the sequences are complementary except for 1 mismatch. In some cases, the sequences are complementary except for 2 mismatches. In other cases, the sequences are complementary except for 3 mismatches. In yet other cases, the sequences are complementary except for 4 mismatches.
  • a nucleic acid that encodes a shRNA or shmiR of the disclosure will comprise a DNA sequence which serves as a template for transcription of the respective shRNA or shmiR.
  • DNA-directed RNAi construct refers to a nucleic acid comprising DNA sequence which, when transcribed produces a shRNA or shmiR molecule which elicits RNAi.
  • the ddRNAi construct may comprise a nucleic acid which is transcribed as a single RNA that is capable of self-annealing into a hairpin structure with a duplex region linked by a stem loop of at least 2 nucleotides i.e., shRNA or shmiR, or as a single RNA with multiple shRNAs or shmiRs, or as multiple RNA transcripts each capable of folding as a single shRNA or shmiR respectively.
  • the ddRNAi construct may be within an expression vector i.e., "ddRNAi expression construct", e.g., operably linked to a promoter.
  • operably-linked or “operable linkage” (or similar) means that a coding nucleic acid sequence is linked to, or in association with, a regulatory sequence, e.g., a promoter, in a manner which facilitates expression of the coding sequence.
  • a regulatory sequence e.g., a promoter
  • Regulatory sequences include promoters, enhancers, and other expression control elements that are art-recognized and are selected to direct expression of the coding sequence.
  • a “vector” will be understood to mean a vehicle for introducing a nucleic acid into a cell.
  • Vectors include, but are not limited to, plasmids, phagemids, viruses, bacteria, and vehicles derived from viral or bacterial sources.
  • a "plasmid” is a circular, double-stranded DNA molecule.
  • a useful type of vector for use in accordance with the present disclosure is a viral vector, wherein heterologous DNA sequences are inserted into a viral genome that can be modified to delete one or more viral genes or parts thereof.
  • Certain vectors are capable of autonomous replication in a host cell (e.g., vectors having an origin of replication that functions in the host cell). Other vectors can be stably integrated into the genome of a host cell, and are thereby replicated along with the host genome.
  • the term "expression vector” will be understood to mean a vector capable of expressing a RNA molecule of the disclosure.
  • treating refers to clinical intervention designed to alter the natural course of the individual or cell being treated during the course of clinical pathology. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis. It follows that treatment of HBV infection includes reducing HBV viral load in a subject infected with HBV, reducing severity of symptoms associated with HBV infection, and reducing the infectivity of HBV in a subject. An individual is successfully "treated", for example, if one or more of the above treatment outcomes is achieved.
  • a “therapeutically effective amount” is at least the minimum concentration or amount required to effect a measurable improvement of a particular disease (e.g., a HBV infection).
  • a therapeutically effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient, and the ability of the shRNA or shmiR, nucleic acid encoding same, ddRNAi or expression construct to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the shRNA or shmiR, nucleic acid encoding same, ddRNAi or expression construct are outweighed by the therapeutically beneficial effects.
  • the ''subject" or “patient” can be a human or non-human animal infected with HBV.
  • the "non-human animal” may be a primate, livestock (e.g. sheep, horses, cattle, pigs, donkeys), companion animal (e.g. pets such as dogs and cats), laboratory test animal (e.g. mice, rabbits, rats, guinea pigs), performance animal (e.g. racehorses, camels, greyhounds) or captive wild animal.
  • livestock e.g. sheep, horses, cattle, pigs, donkeys
  • companion animal e.g. pets such as dogs and cats
  • laboratory test animal e.g. mice, rabbits, rats, guinea pigs
  • performance animal e.g. racehorses, camels, greyhounds
  • captive wild animal e.g. racehorses, camels, greyhounds
  • the subject or patient is a mammal.
  • the subject or patient is a primate
  • reduced expression refers to the absence or an observable decrease in the level of protein and/or mRNA product from the target gene e.g., the HBV pol gene or other HBV gene.
  • the decrease does not have to be absolute, but may be a partial decrease sufficient for there to a detectable or observable change as a result of the RNAi effected by the shmiR encoded by the nucleic acid of the disclosure.
  • the decrease can be measured by determining a decrease in the level of mRNA and/or protein product from a target nucleic acid relative to a cell lacking the shmiR or shRNA, nucleic acid encoding same, ddRNAi construct or expression construct, and may be as little as 1 %, 5% or 10%, or may be absolute i.e., 100% inhibition.
  • the effects of the decrease may be determined by examination of the outward properties i.e., quantitative and/or qualitative phenotype of the cell or organism, and may also include an assessment of the viral load following administration of a ddRNAi construct of the disclosure.
  • the present disclosure provides a nucleic acid comprising a DNA sequence which encodes a short hairpin micro-RNA (shmiR), said shmiR comprising: an effector sequence of at least 17 nucleotides in length;
  • pri-miRNA primary micro RNA
  • the effector sequence is substantially complementary to a RNA transcript set forth in any one of SEQ ID NOs: 1-10, 38, 40, 42, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131 and 133.
  • the effector sequence will be less than 30 nucleotides in length.
  • a suitable effector sequence may be in the range of 17-29 nucleotides in length.
  • the effector sequence will be 21 nucleotides in length. More preferably, the effector sequence will be 21 nucleotides in length and the effector complement sequence will be 20 nucleotides in length.
  • the shmiR comprises an effector sequence which is substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 1.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 1 and contain 6 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 1 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 1 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 1 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 1 and contain 2 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 1 and contain 1 mismatch base relative thereto.
  • the effector sequence may be 100% complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 1.
  • the shmiR comprises an effector sequence which is substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 2.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 2 and contain 6 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 2 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 2 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 2 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 2 and contain 2 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 2 and contain 1 mismatch base relative thereto.
  • the effector sequence may be 100% complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 2.
  • the shmiR comprises an effector sequence which is substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 3.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 3 and contain 6 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 3 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 3 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 3 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 3 and contain 2 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 3 and contain 1 mismatch base relative thereto.
  • the effector sequence may be 100% complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 3.
  • the shmiR comprises an effector sequence which is substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 4.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 4 and contain 6 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 4 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 4 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 4 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 4 and contain 2 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 4 and contain 1 mismatch base relative thereto.
  • the effector sequence may be 100% complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 4.
  • the shmiR comprises an effector sequence which is substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 5.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 5 and contain 6 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 5 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 5 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 5 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 5 and contain 2 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 5 and contain 1 mismatch base relative thereto.
  • the effector sequence may be 100% complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 5.
  • the shmiR comprises an effector sequence which is substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 6.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 6 and contain 6 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 6 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 6 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 6 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 6 and contain 2 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 6 and contain 1 mismatch base relative thereto.
  • the effector sequence may be 100% complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 6.
  • the shmiR comprises an effector sequence which is substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 7.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 7 and contain 6 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 7 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 7 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 7 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 7 and contain 2 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 7 and contain 1 mismatch base relative thereto.
  • the effector sequence may be 100% complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 7.
  • the shmiR comprises an effector sequence which is substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 8.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 8 and contain 6 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 8 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 8 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 8 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 8 and contain 2 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 8 and contain 1 mismatch base relative thereto.
  • the effector sequence may be 100% complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 8.
  • the shmiR comprises an effector sequence which is substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 9.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 9 and contain 6 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 9 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 9 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 9 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 9 and contain 2 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 9 and contain 1 mismatch base relative thereto.
  • the effector sequence may be 100% complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 9.
  • the shmiR comprises an effector sequence which is substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 10.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 10 and contain 6 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 10 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 10 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 10 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 10 and contain 2 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 10 and contain 1 mismatch base relative thereto.
  • the effector sequence may be 100% complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 10.
  • the shmiR comprises an effector sequence which is substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 38.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 38 and contain 6 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 38 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 38 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 38 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 38 and contain 2 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 38 and contain 1 mismatch base relative thereto.
  • the effector sequence may be 100% complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 38.
  • the shmiR comprises an effector sequence which is substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 40.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 40 and contain 6 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 40 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 40 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 40 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 40 and contain 2 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 40 and contain 1 mismatch base relative thereto.
  • the effector sequence may be 100% complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 40.
  • the shmiR comprises an effector sequence which is substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 42.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 42 and contain 6 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 42 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 42 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 42 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 42 and contain 2 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 42 and contain 1 mismatch base relative thereto.
  • the effector sequence may be 100% complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 42.
  • the shmiR comprises an effector sequence which is substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 111.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 111 and contain 6 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 111 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 111 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 111 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 111 and contain 2 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 111 and contain 1 mismatch base relative thereto.
  • the effector sequence may be 100% complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 111.
  • the shmiR comprises an effector sequence which is substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 113.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 113 and contain 6 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 113 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 113 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 113 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 113 and contain 2 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 113 and contain 1 mismatch base relative thereto.
  • the effector sequence may be 100% complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 113.
  • the shmiR comprises an effector sequence which is substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 115.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 115 and contain 6 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 115 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 115 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 115 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 115 and contain 2 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 115 and contain 1 mismatch base relative thereto.
  • the effector sequence may be 100% complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 115.
  • the shmiR comprises an effector sequence which is substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 117.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 117 and contain 6 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 117 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 117 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 117 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 117 and contain 2 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 117 and contain 1 mismatch base relative thereto.
  • the effector sequence may be 100% complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 117.
  • the shmiR comprises an effector sequence which is substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 119.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 119 and contain 6 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 119 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 119 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 119 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 119 and contain 2 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 119 and contain 1 mismatch base relative thereto.
  • the effector sequence may be 100% complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 119.
  • the shmiR comprises an effector sequence which is substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 121.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 121 and contain 6 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 121 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 121 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 121 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 121 and contain 2 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 121 and contain 1 mismatch base relative thereto.
  • the effector sequence may be 100% complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 121.
  • the shmiR comprises an effector sequence which is substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 123.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 123 and contain 6 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 123 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 123 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 123 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 123 and contain 2 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 123 and contain 1 mismatch base relative thereto.
  • the effector sequence may be 100% complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 123.
  • the shmiR comprises an effector sequence which is substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 125.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 125 and contain 6 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 125 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 125 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 125 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 125 and contain 2 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 125 and contain 1 mismatch base relative thereto.
  • the effector sequence may be 100% complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 125.
  • the shmiR comprises an effector sequence which is substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 127.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 127 and contain 6 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 127 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 127 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 127 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 127 and contain 2 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 127 and contain 1 mismatch base relative thereto.
  • the effector sequence may be 100% complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 127.
  • the shmiR comprises an effector sequence which is substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 129.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 129 and contain 6 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 129 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 129 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 129 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 129 and contain 2 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 129 and contain 1 mismatch base relative thereto.
  • the effector sequence may be 100% complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 129.
  • the shmiR comprises an effector sequence which is substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 131.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 131 and contain 6 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 131 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 131 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 131 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 131 and contain 2 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 131 and contain 1 mismatch base relative thereto.
  • the effector sequence may be 100% complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 131.
  • the shmiR comprises an effector sequence which is substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 133.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 133 and contain 6 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 133 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 133 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 133 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 133 and contain 2 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 133 and contain 1 mismatch base relative thereto.
  • the effector sequence may be 100% complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 133.
  • the effector sequence of a shmiR of the disclosure is substantially complementary to a HBV RNA transcript described herein and contains 1, 2, 3, 4, 5 or 6 mismatch base(s) relative thereto, it is preferred that the mismatch(es) are not located within the region corresponding to the seed region of the shmiR i.e., nucleotides 2-8 of the effector sequence.
  • the nucleic acid described herein may comprise a DNA sequence encoding a shmiR comprising: (i) an effector sequence which is substantially
  • the shmiR encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO: 11 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 11 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 11 may be the sequence set forth in SEQ ID NO: 12.
  • a shmiR in accordance with this example is hereinafter designated "shmiR-1".
  • the nucleic acid described herein may comprise a DNA sequence encoding a shmiR comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 14 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO: 14; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence.
  • the shmiR encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO: 13 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 13 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 13 may be the sequence set forth in SEQ ID NO: 14.
  • a shmiR in accordance with this example is hereinafter designated "shmiR-2".
  • the nucleic acid described herein may comprise a DNA sequence encoding a shmiR comprising: (i) an effector sequence which is substantially
  • the shmiR encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO: 15 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 15 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 15 may be the sequence set forth in SEQ ID NO: 16.
  • a shmiR in accordance with this example is hereinafter designated "shmiR-3".
  • the nucleic acid described herein may comprise a DNA sequence encoding a shmiR comprising: (i) an effector sequence which is substantially
  • the shmiR encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO: 17 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 17 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 17 may be the sequence set forth in SEQ ID NO: 18.
  • a shmiR in accordance with this example is hereinafter designated "shmiR-4".
  • the nucleic acid described herein may comprise a DNA sequence encoding a shmiR comprising: (i) an effector sequence which is substantially
  • the shmiR encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO: 19 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 19 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 19 may be the sequence set forth in SEQ ID NO:20.
  • a shmiR in accordance with this example is hereinafter designated "shmiR-5".
  • the nucleic acid described herein may comprise a DNA sequence encoding a shmiR comprising: (i) an effector sequence which is substantially
  • the shmiR encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:21 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:21 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:21 may be the sequence set forth in SEQ ID NO:22.
  • a shmiR in accordance with this example is hereinafter designated "shmiR-6".
  • the nucleic acid described herein may comprise a DNA sequence encoding a shmiR comprising: (i) an effector sequence which is substantially
  • the shmiR encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:23 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:23 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:23 may be the sequence set forth in SEQ ID NO:24.
  • a shmiR in accordance with this example is hereinafter designated "shmiR-7".
  • the nucleic acid described herein may comprise a DNA sequence encoding a shmiR comprising: (i) an effector sequence which is substantially
  • the shmiR encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:25 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:25 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:25 may be the sequence set forth in SEQ ID NO:26.
  • a shmiR in accordance with this example is hereinafter designated "shmiR-8".
  • the nucleic acid described herein may comprise a DNA sequence encoding a shmiR comprising: (i) an effector sequence which is substantially
  • the shmiR encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:27 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:27 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:27 may be the sequence set forth in SEQ ID NO:28.
  • a shmiR in accordance with this example is hereinafter designated "shmiR-9".
  • the nucleic acid described herein may comprise a DNA sequence encoding a shmiR comprising: (i) an effector sequence which is substantially
  • the shmiR encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:29 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:29 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:29 may be the sequence set forth in SEQ ID NO:30.
  • a shmiR in accordance with this example is hereinafter designated "shmiR-10".
  • the nucleic acid described herein may comprise a DNA sequence encoding a shmiR comprising: (i) an effector sequence which is substantially
  • the shmiR encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:31 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:31 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:31 may be the sequence set forth in SEQ ID NO:32.
  • a shmiR in accordance with this example is hereinafter designated "shmiR-11".
  • the nucleic acid described herein may comprise a DNA sequence encoding a shmiR comprising: (i) an effector sequence which is substantially
  • the shmiR encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:33 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:33 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:33 may be the sequence set forth in SEQ ID NO:34.
  • a shmiR in accordance with this example is hereinafter designated "shmiR-12".
  • the nucleic acid described herein may comprise a DNA sequence encoding a shmiR comprising: (i) an effector sequence which is substantially
  • the shmiR encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:35 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:35 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:35 may be the sequence set forth in SEQ ID NO:36.
  • a shmiR in accordance with this example is hereinafter designated "shmiR-13".
  • the nucleic acid described herein may comprise a DNA sequence encoding a shmiR comprising: (i) an effector sequence which is substantially
  • the shmiR encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:37 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:37 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:37 may be the sequence set forth in SEQ ID NO:38.
  • shmiR-14 A shmiR in accordance with this example is hereinafter designated "shmiR-14".
  • the nucleic acid described herein may comprise a DNA sequence encoding a shmiR comprising: (i) an effector sequence which is substantially
  • the shmiR encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:39 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:39 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:39 may be the sequence set forth in SEQ ID NO:40.
  • a shmiR in accordance with this example is hereinafter designated "shmiR-15".
  • the nucleic acid described herein may comprise a DNA sequence encoding a shmiR comprising: (i) an effector sequence which is substantially
  • the shmiR encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:41 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:41 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:41 may be the sequence set forth in SEQ ID NO:42.
  • a shmiR in accordance with this example is hereinafter designated "shmiR-16".
  • the nucleic acid described herein may comprise a DNA sequence encoding a shmiR comprising: (i) an effector sequence which is substantially
  • the shmiR encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO: 112 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 112 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 112 may be the sequence set forth in SEQ ID NO: 111.
  • a shmiR in accordance with this example is hereinafter designated "shmiR-17".
  • the nucleic acid described herein may comprise a DNA sequence encoding a shmiR comprising: (i) an effector sequence which is substantially
  • the shmiR encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO: 114 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 114 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 114 may be the sequence set forth in SEQ ID NO: 113.
  • a shmiR in accordance with this example is hereinafter designated "shmiR-18".
  • the nucleic acid described herein may comprise a DNA sequence encoding a shmiR comprising: (i) an effector sequence which is substantially
  • the shmiR encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO: 116 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 116 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 116 may be the sequence set forth in SEQ ID NO: 115.
  • a shmiR in accordance with this example is hereinafter designated "shmiR-19".
  • the nucleic acid described herein may comprise a DNA sequence encoding a shmiR comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 117 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO: 117; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence.
  • the shmiR encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO: 118 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 118 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:l 18 may be the sequence set forth in SEQ ID NO: 117.
  • a shmiR in accordance with this example is hereinafter designated "shmiR-20".
  • the nucleic acid described herein may comprise a DNA sequence encoding a shmiR comprising: (i) an effector sequence which is substantially
  • the shmiR encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO: 120 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 120 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 120 may be the sequence set forth in SEQ ID NO: 119.
  • a shmiR in accordance with this example is hereinafter designated "shmiR-21".
  • the nucleic acid described herein may comprise a DNA sequence encoding a shmiR comprising: (i) an effector sequence which is substantially
  • the shmiR encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO: 122 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 122 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 122 may be the sequence set forth in SEQ ID NO: 121.
  • a shmiR in accordance with this example is hereinafter designated "shmiR-22".
  • the nucleic acid described herein may comprise a DNA sequence encoding a shmiR comprising: (i) an effector sequence which is substantially
  • the shmiR encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO: 124 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 124 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 124 may be the sequence set forth in SEQ ID NO: 123.
  • a shmiR in accordance with this example is hereinafter designated "shmiR-23".
  • the nucleic acid described herein may comprise a DNA sequence encoding a shmiR comprising: (i) an effector sequence which is substantially
  • the shmiR encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO: 126 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 126 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 126 may be the sequence set forth in SEQ ID NO: 125.
  • a shmiR in accordance with this example is hereinafter designated "shmiR-24".
  • the nucleic acid described herein may comprise a DNA sequence encoding a shmiR comprising: (i) an effector sequence which is substantially
  • the shmiR encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO: 128 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 128 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 128 may be the sequence set forth in SEQ ID NO: 127.
  • a shmiR in accordance with this example is hereinafter designated "shmiR-25".
  • the nucleic acid described herein may comprise a DNA sequence encoding a shmiR comprising: (i) an effector sequence which is substantially
  • the shmiR encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO: 130 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 130 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 130 may be the sequence set forth in SEQ ID NO: 129.
  • a shmiR in accordance with this example is hereinafter designated "shmiR-26".
  • the nucleic acid described herein may comprise a DNA sequence encoding a shmiR comprising: (i) an effector sequence which is substantially
  • the shmiR encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO: 132 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 132 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 132 may be the sequence set forth in SEQ ID NO: 131.
  • a shmiR in accordance with this example is hereinafter designated "shmiR-27".
  • the nucleic acid described herein may comprise a DNA sequence encoding a shmiR comprising: (i) an effector sequence which is substantially
  • the shmiR encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO: 134 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 134 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 134 may be the sequence set forth in SEQ ID NO: 133.
  • a shmiR in accordance with this example is hereinafter designated "shmiR-28".
  • the shmiR encoded by the nucleic acid of the disclosure may comprise, in a 5' to 3' direction:
  • Suitable loop sequences may be selected from those known in the art. However, an exemplary stemloop sequence is set forth in SEQ ID NO: 75.
  • Suitable primary micro RNA (pri-miRNA or pri-R) backbones for use in a nucleic acid of the disclosure may be selected from those known in the art.
  • the pri- miRNA backbone may be selected from a pri-miR-30a backbone, a pri-miR-155 backbone, a pri-miR-21 backbone and a pri-miR-136 backbone.
  • the pri-miRNA backbone is a pri-miR-30a backbone.
  • the nucleic acid encoding the shmiRs of the disclosure may comprise DNA sequence encoding the sequence set forth in SEQ ID NO: 76 and DNA sequence encoding the sequence set forth in SEQ ID NO: 77.
  • the nucleic acid described herein may comprise a DNA sequence selected from the sequence set forth in any one of SEQ ID NOs: 59-74 and 146-157.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 59 and encodes a shmiR (shmiR- 1) comprising or consisting of the sequence set forth in SEQ ID NO: 43.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 60 and encodes a shmiR (shmiR-2) comprising or consisting of the sequence set forth in SEQ ID NO: 44.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 61 and encodes a shmiR (shmiR-3) comprising or consisting of the sequence set forth in SEQ ID NO: 45.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 62 and encodes a shmiR (shmiR-4) comprising or consisting of the sequence set forth in SEQ ID NO: 46.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 63 and encodes a shmiR (shmiR-5) comprising or consisting of the sequence set forth in SEQ ID NO: 47.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 64 and encodes a shmiR (shmiR-6) comprising or consisting of the sequence set forth in SEQ ID NO: 48.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 65 and encodes a shmiR (shmiR-7) comprising or consisting of the sequence set forth in SEQ ID NO: 49.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 66 and encodes a shmiR (shmiR-8) comprising or consisting of the sequence set forth in SEQ ID NO: 50.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 67 and encodes a shmiR (shmiR-9) comprising or consisting of the sequence set forth in SEQ ID NO: 51.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 68 and encodes a shmiR (shmiR- 10) comprising or consisting of the sequence set forth in SEQ ID NO: 52.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 69 and encodes a shmiR (shmiR- 11) comprising or consisting of the sequence set forth in SEQ ID NO: 53.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 70 and encodes a shmiR (shmiR- 12) comprising or consisting of the sequence set forth in SEQ ID NO: 54.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 71 and encodes a shmiR (shmiR- 13) comprising or consisting of the sequence set forth in SEQ ID NO: 55.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 72 and encodes a shmiR (shmiR- 14) comprising or consisting of the sequence set forth in SEQ ID NO: 56.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 73 and encodes a shmiR (shmiR- 15) comprising or consisting of the sequence set forth in SEQ ID NO: 57.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 74 and encodes a shmiR (shmiR- 16) comprising or consisting of the sequence set forth in SEQ ID NO: 58.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 146 and encodes a shmiR (shmiR- 17) comprising or consisting of the sequence set forth in SEQ ID NO: 134.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 147 and encodes a shmiR (shmiR- 18) comprising or consisting of the sequence set forth in SEQ ID NO: 135.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 148 and encodes a shmiR (shmiR-19) comprising or consisting of the sequence set forth in SEQ ID NO: 136.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 149 and encodes a shmiR (shmiR-20) comprising or consisting of the sequence set forth in SEQ ID NO: 137.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 150 and encodes a shmiR (shmiR-21) comprising or consisting of the sequence set forth in SEQ ID NO: 138.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 151 and encodes a shmiR (shmiR-22) comprising or consisting of the sequence set forth in SEQ ID NO: 139.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 152 and encodes a shmiR (shmiR-23) comprising or consisting of the sequence set forth in SEQ ID NO: 140.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 153 and encodes a shmiR (shmiR-24) comprising or consisting of the sequence set forth in SEQ ID NO: 141.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 154 and encodes a shmiR (shmiR-25) comprising or consisting of the sequence set forth in SEQ ID NO: 142.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 155 and encodes a shmiR (shmiR-26) comprising or consisting of the sequence set forth in SEQ ID NO: 143.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 156 and encodes a shmiR (shmiR-27) comprising or consisting of the sequence set forth in SEQ ID NO: 144.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 157 and encodes a shmiR (shmiR-28) comprising or consisting of the sequence set forth in SEQ ID NO: 145.
  • exemplary nucleic acids of the disclosure encode a shmiR selected from shmiR-6, shmiR-7, shmiR-12 and shmiR-15 as described herein.
  • nucleic acids of the disclosure encode variants of shmiR-12 selected from shmiR-23, shmiR-24, shmiR-25, shmiR-26, shmiR-27 and shmiR-28, or encode variants of shmiR-15 selected from shmiR- 17, shmiR-18, shmiR-19, shmiR-20, shmiR-21 and shmiR-22.
  • nucleic acid in accordance with the present disclosure may be combined or used in conjunction with other therapeutic agents for treating HBV. Accordingly, the present disclosure provides a nucleic acid comprising a DNA sequence encoding a shmiR as described herein (e.g., one or shmiRs designated shmiR l-shmiR-28 described herein) in combination with one or more other agents for treating HBV.
  • a plurality of nucleic acids are provided comprising:
  • nucleic acid comprising a DNA sequence encoding a shmiR as described herein;
  • nucleic acid comprising a DNA sequence encoding a short hairpin RNA
  • RNA comprising an effector sequence of at least 17 nucleotides in length and a effector complement sequence, wherein the effector sequence is substantially complementary to a RNA sequence set forth in any one of SEQ ID NOs:
  • the shmiR encoded by the nucleic acid at (a) and the shmiR or shRNA encoded by the nucleic acid at (b) comprise different effector sequences.
  • the effector sequence of the shRNA at (b)(ii) which is substantially complementary to a RNA sequence set forth in any one of SEQ ID NOs: 1-10, 38, 40, 42, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131 and 133 will be less than 30 nucleotides in length.
  • a suitable effector sequence of the shRNA may be in the range of 17-29 nucleotides in length.
  • the plurality of nucleic acids of the disclosure may comprise two or more nucleic acids encoding shmiRs as described herein, such as two,or three, or four, or five, or six, or seven, or eight, or nine, or ten nucleic acids encoding shmiRs as described herein.
  • the plurality of nucleic acids of the disclosure comprises at least one nucleic acid encoding a shmiR as described herein and at least one nucleic acid comprising a DNA sequence encoding a shRNA comprising an effector of at least 17 nucleotides in length and a effector complement sequence, wherein the effector sequence is substantially complementary to a RNA sequence set forth in any one of SEQ ID NOs: 1-10, 38, 40, 42, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131 and 133.
  • the shRNA encoded by a nucleic acid in the plurality comprises: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 12 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO: 12; and (ii) an effector complement sequence comprising a sequence which is substantially
  • the shRNA encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO: 11 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 11 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 11 may be the sequence set forth in SEQ ID NO: 12.
  • a shRNA in accordance with this example is hereinafter designated "shRNA- 1".
  • the shRNA encoded by a nucleic acid in the plurality comprises: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 14 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO: 14; and (ii) an effector complement sequence comprising a sequence which is substantially
  • the shRNA encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO: 13 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 13 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 13 may be the sequence set forth in SEQ ID NO: 14.
  • a shRNA in accordance with this example is hereinafter designated "shRNA-2".
  • the shRNA encoded by a nucleic acid in the plurality comprises: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 16 with the exception of 1, 2, 3, 4, 5 or 6base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO: 16; and (ii) an effector complement sequence comprising a sequence which is substantially
  • the shRNA encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO: 15 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 15 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 15 may be the sequence set forth in SEQ ID NO: 16.
  • a shRNA in accordance with this example is hereinafter designated "shRNA-3".
  • the shRNA encoded by a nucleic acid in the plurality comprises: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 18 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO: 18; and (ii) an effector complement sequence comprising a sequence which is substantially
  • the shRNA encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO: 17 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 17 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 17 may be the sequence set forth in SEQ ID NO: 18.
  • a shRNA in accordance with this example is hereinafter designated "shRNA-4".
  • the shRNA encoded by a nucleic acid in the plurality comprises: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:20 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:20; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence.
  • the shRNA encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO: 19 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 19 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 19 may be the sequence set forth in SEQ ID NO:20.
  • a shRNA in accordance with this example is hereinafter designated "shRNA-5".
  • the shRNA encoded by a nucleic acid in the plurality comprises: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:22 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:22; and (ii) an effector complement sequence comprising a sequence which is substantially
  • the shRNA encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:21 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:21 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:21 may be the sequence set forth in SEQ ID NO:22.
  • a shRNA in accordance with this example is hereinafter designated "shRNA-6".
  • the shRNA encoded by a nucleic acid in the plurality comprises: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:24 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:24; and (ii) an effector complement sequence comprising a sequence which is substantially
  • the shRNA encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:23 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:23 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:23 may be the sequence set forth in SEQ ID NO:24.
  • a shRNA in accordance with this example is hereinafter designated "shRNA-7".
  • the shRNA encoded by a nucleic acid in the plurality comprises: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:26 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:26; and (ii) an effector complement sequence comprising a sequence which is substantially
  • the shRNA encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:25 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:25 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:25 may be the sequence set forth in SEQ ID NO:26.
  • a shRNA in accordance with this example is hereinafter designated "shRNA-8".
  • the shRNA encoded by a nucleic acid in the plurality comprises: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:28 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:28; and (ii) an effector complement sequence comprising a sequence which is substantially
  • the shRNA encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:27 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:27 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:27 may be the sequence set forth in SEQ ID NO:28.
  • a shRNA in accordance with this example is hereinafter designated "shRNA-9".
  • the shRNA encoded by a nucleic acid in the plurality comprises: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:30 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO: 30; and (ii) an effector complement sequence comprising a sequence which is substantially
  • the shRNA encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:29 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:29 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:29 may be the sequence set forth in SEQ ID NO:30.
  • a shRNA in accordance with this example is hereinafter designated "shRNA-10".
  • the shRNA encoded by a nucleic acid in the plurality comprises: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:32 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO: 32; and (ii) an effector complement sequence comprising a sequence which is substantially
  • the shRNA encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:31 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:31 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:31 may be the sequence set forth in SEQ ID NO:32.
  • a shRNA in accordance with this example is hereinafter designated "shRNA- 11".
  • the shRNA encoded by a nucleic acid in the plurality comprises: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:34 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:34; and (ii) an effector complement sequence comprising a sequence which is substantially
  • the shRNA encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:33 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:33 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:33 may be the sequence set forth in SEQ ID NO:34.
  • a shRNA in accordance with this example is hereinafter designated "shRNA- 12".
  • the shRNA encoded by a nucleic acid in the plurality comprises: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:36 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:36; and (ii) an effector complement sequence comprising a sequence which is substantially
  • the shRNA encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:35 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:35 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:35 may be the sequence set forth in SEQ ID NO:36.
  • a shRNA in accordance with this example is hereinafter designated "shRNA- 13".
  • the shRNA encoded by a nucleic acid in the plurality comprises: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:38 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:38; and (ii) an effector complement sequence comprising a sequence which is substantially
  • the shRNA encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:37 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:37 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:37 may be the sequence set forth in SEQ ID NO:38.
  • a shRNA in accordance with this example is hereinafter designated "shRNA- 14".
  • the shRNA encoded by a nucleic acid in the plurality comprises: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:40 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:40; and (ii) an effector complement sequence comprising a sequence which is substantially
  • the shRNA encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:39 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:39 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:39 may be the sequence set forth in SEQ ID NO:40.
  • a shRNA in accordance with this example is hereinafter designated "shRNA-15".
  • the shRNA encoded by a nucleic acid in the plurality comprises: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:42 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:42; and (ii) an effector complement sequence comprising a sequence which is substantially
  • the shRNA encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:41 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:41 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:41 may be the sequence set forth in SEQ ID NO:42.
  • a shRNA in accordance with this example is hereinafter designated "shRNA- 16".
  • the shRNA may comprise a stem loop sequence positioned between the effector sequence and the effector complement sequence. Suitable loop sequences may be selected from those known in the art. Alternatively, suitable stem loops may be developed de novo.
  • a nucleic acid of the plurality described herein encoding a shRNA may comprise a DNA sequence encoding a stem loop positioned between the DNA sequences encoding the effector sequence and the effector complement sequence.
  • a shRNA encoded by a nucleic acid of the disclosure may comprise a sequence set forth in any one of SEQ ID NOs:78-93.
  • a nucleic acid in the plurality of nucleic acids described herein may comprise or consist of a DNA sequence set forth in in any one of SEQ ID NOs: 94-109.
  • the plurality of nucleic acids described herein comprises a nucleic acid which comprises or consists of a DNA sequence set forth in SEQ ID NO: 59 and encodes a shmiR (shmiR-1) comprising or consisting of the sequence set forth in SEQ ID NO: 43, and at least one other nucleic acid of the disclosure which encodes a shmiR or shRNA targeting HBV.
  • a nucleic acid which comprises or consists of a DNA sequence set forth in SEQ ID NO: 59 and encodes a shmiR (shmiR-1) comprising or consisting of the sequence set forth in SEQ ID NO: 43, and at least one other nucleic acid of the disclosure which encodes a shmiR or shRNA targeting HBV.
  • the plurality of nucleic acids described herein comprises a nucleic acid which comprises or consists of a DNA sequence set forth in SEQ ID NO: 60 and encodes a shmiR (shmiR-2) comprising or consisting of the sequence set forth in SEQ ID NO: 44, and at least one other nucleic acid of the disclosure which encodes a shmiR or shRNA targeting HBV.
  • a nucleic acid which comprises or consists of a DNA sequence set forth in SEQ ID NO: 60 and encodes a shmiR (shmiR-2) comprising or consisting of the sequence set forth in SEQ ID NO: 44, and at least one other nucleic acid of the disclosure which encodes a shmiR or shRNA targeting HBV.
  • the plurality of nucleic acids described herein comprises a nucleic acid which comprises or consists of a DNA sequence set forth in SEQ ID NO: 61 and encodes a shmiR (shmiR-3) comprising or consisting of the sequence set forth in SEQ ID NO: 45, and at least one other nucleic acid of the disclosure which encodes a shmiR or shRNA targeting HBV.
  • a nucleic acid which comprises or consists of a DNA sequence set forth in SEQ ID NO: 61 and encodes a shmiR (shmiR-3) comprising or consisting of the sequence set forth in SEQ ID NO: 45, and at least one other nucleic acid of the disclosure which encodes a shmiR or shRNA targeting HBV.
  • the plurality of nucleic acids described herein comprises a nucleic acid which comprises or consists of a DNA sequence set forth in SEQ ID NO: 62 and encodes a shmiR (shmiR-4) comprising or consisting of the sequence set forth in SEQ ID NO: 46, and at least one other nucleic acid of the disclosure which encodes a shmiR or shRNA targeting HBV.
  • a nucleic acid which comprises or consists of a DNA sequence set forth in SEQ ID NO: 62 and encodes a shmiR (shmiR-4) comprising or consisting of the sequence set forth in SEQ ID NO: 46, and at least one other nucleic acid of the disclosure which encodes a shmiR or shRNA targeting HBV.
  • the plurality of nucleic acids described herein comprises a nucleic acid which comprises or consists of a DNA sequence set forth in SEQ ID NO: 63 and encodes a shmiR (shmiR-5) comprising or consisting of the sequence set forth in SEQ ID NO: 47, and at least one other nucleic acid of the disclosure which encodes a shmiR or shRNA targeting HBV.
  • a nucleic acid which comprises or consists of a DNA sequence set forth in SEQ ID NO: 63 and encodes a shmiR (shmiR-5) comprising or consisting of the sequence set forth in SEQ ID NO: 47, and at least one other nucleic acid of the disclosure which encodes a shmiR or shRNA targeting HBV.
  • the plurality of nucleic acids described herein comprises a nucleic acid which comprises or consists of a DNA sequence set forth in SEQ ID NO: 64 and encodes a shmiR (shmiR-6) comprising or consisting of the sequence set forth in SEQ ID NO: 48, and at least one other nucleic acid of the disclosure which encodes a shmiR or shRNA targeting HBV.
  • a nucleic acid which comprises or consists of a DNA sequence set forth in SEQ ID NO: 64 and encodes a shmiR (shmiR-6) comprising or consisting of the sequence set forth in SEQ ID NO: 48, and at least one other nucleic acid of the disclosure which encodes a shmiR or shRNA targeting HBV.
  • the plurality of nucleic acids described herein comprises a nucleic acid which comprises or consists of a DNA sequence set forth in SEQ ID NO: 65 and encodes a shmiR (shmiR-7) comprising or consisting of the sequence set forth in SEQ ID NO: 49, and at least one other nucleic acid of the disclosure which encodes a shmiR or shRNA targeting HBV.
  • a nucleic acid which comprises or consists of a DNA sequence set forth in SEQ ID NO: 65 and encodes a shmiR (shmiR-7) comprising or consisting of the sequence set forth in SEQ ID NO: 49, and at least one other nucleic acid of the disclosure which encodes a shmiR or shRNA targeting HBV.
  • the plurality of nucleic acids described herein comprises a nucleic acid which comprises or consists of a DNA sequence set forth in SEQ ID NO: 66 and encodes a shmiR (shmiR-8) comprising or consisting of the sequence set forth in SEQ ID NO: 50, and at least one other nucleic acid of the disclosure which encodes a shmiR or shRNA targeting HBV.
  • a nucleic acid which comprises or consists of a DNA sequence set forth in SEQ ID NO: 66 and encodes a shmiR (shmiR-8) comprising or consisting of the sequence set forth in SEQ ID NO: 50, and at least one other nucleic acid of the disclosure which encodes a shmiR or shRNA targeting HBV.
  • the plurality of nucleic acids described herein comprises a nucleic acid which comprises or consists of a DNA sequence set forth in SEQ ID NO: 67 and encodes a shmiR (shmiR-9) comprising or consisting of the sequence set forth in SEQ ID NO: 51, and at least one other nucleic acid of the disclosure which encodes a shmiR or shRNA targeting HBV.
  • a nucleic acid which comprises or consists of a DNA sequence set forth in SEQ ID NO: 67 and encodes a shmiR (shmiR-9) comprising or consisting of the sequence set forth in SEQ ID NO: 51, and at least one other nucleic acid of the disclosure which encodes a shmiR or shRNA targeting HBV.
  • the plurality of nucleic acids described herein comprises a nucleic acid which comprises or consists of a DNA sequence set forth in SEQ ID NO: 68 and encodes a shmiR (shmiR- 10) comprising or consisting of the sequence set forth in SEQ ID NO: 52, and at least one other nucleic acid of the disclosure which encodes a shmiR or shRNA targeting HBV.
  • a nucleic acid which comprises or consists of a DNA sequence set forth in SEQ ID NO: 68 and encodes a shmiR (shmiR- 10) comprising or consisting of the sequence set forth in SEQ ID NO: 52, and at least one other nucleic acid of the disclosure which encodes a shmiR or shRNA targeting HBV.
  • the plurality of nucleic acids described herein comprises a nucleic acid which comprises or consists of a DNA sequence set forth in SEQ ID NO: 69 and encodes a shmiR (shmiR- 11) comprising or consisting of the sequence set forth in SEQ ID NO: 53, and at least one other nucleic acid of the disclosure which encodes a shmiR or shRNA targeting HBV.
  • the plurality of nucleic acids described herein comprises a nucleic acid which comprises or consists of a DNA sequence set forth in SEQ ID NO: 70 and encodes a shmiR (shmiR- 12) comprising or consisting of the sequence set forth in SEQ ID NO: 54, and at least one other nucleic acid of the disclosure which encodes a shmiR or shRNA targeting HBV.
  • the plurality of nucleic acids described herein comprises a nucleic acid which comprises or consists of a DNA sequence set forth in SEQ ID NO: 71 and encodes a shmiR (shmiR- 13) comprising or consisting of the sequence set forth in SEQ ID NO: 55, and at least one other nucleic acid of the disclosure which encodes a shmiR or shRNA targeting HBV.
  • the plurality of nucleic acids described herein comprises a nucleic acid which comprises or consists of a DNA sequence set forth in SEQ ID NO: 72 and encodes a shmiR (shmiR- 14) comprising or consisting of the sequence set forth in SEQ ID NO: 56, and at least one other nucleic acid of the disclosure which encodes a shmiR or shRNA targeting HBV.
  • the plurality of nucleic acids described herein comprises a nucleic acid which comprises or consists of a DNA sequence set forth in SEQ ID NO: 73 and encodes a shmiR (shmiR- 15) comprising or consisting of the sequence set forth in SEQ ID NO: 57, and at least one other nucleic acid of the disclosure which encodes a shmiR or shRNA targeting HBV.
  • the plurality of nucleic acids described herein comprises a nucleic acid which comprises or consists of a DNA sequence set forth in SEQ ID NO: 74 and encodes a shmiR (shmiR- 16) comprising or consisting of the sequence set forth in SEQ ID NO: 58, and at least one other nucleic acid of the disclosure which encodes a shmiR or shRNA targeting HBV.
  • the plurality of nucleic acids described herein comprises a nucleic acid which comprises or consists of a DNA sequence set forth in SEQ ID NO: 146 and encodes a shmiR (shmiR- 17) comprising or consisting of the sequence set forth in SEQ ID NO: 134, and at least one other nucleic acid of the disclosure which encodes a shmiR or shRNA targeting HBV.
  • the plurality of nucleic acids described herein comprises a nucleic acid which comprises or consists of a DNA sequence set forth in SEQ ID NO: 147 and encodes a shmiR (shmiR- 18) comprising or consisting of the sequence set forth in SEQ ID NO: 135, and at least one other nucleic acid of the disclosure which encodes a shmiR or shRNA targeting HBV.
  • the plurality of nucleic acids described herein comprises a nucleic acid which comprises or consists of a DNA sequence set forth in SEQ ID NO: 148 and encodes a shmiR (shmiR- 19) comprising or consisting of the sequence set forth in SEQ ID NO: 136, and at least one other nucleic acid of the disclosure which encodes a shmiR or shRNA targeting HBV.
  • the plurality of nucleic acids described herein comprises a nucleic acid which comprises or consists of a DNA sequence set forth in SEQ ID NO: 149 and encodes a shmiR (shmiR- 20) comprising or consisting of the sequence set forth in SEQ ID NO: 137, and at least one other nucleic acid of the disclosure which encodes a shmiR or shRNA targeting HBV.
  • the plurality of nucleic acids described herein comprises a nucleic acid which comprises or consists of a DNA sequence set forth in SEQ ID NO: 150 and encodes a shmiR (shmiR-21) comprising or consisting of the sequence set forth in SEQ ID NO: 138, and at least one other nucleic acid of the disclosure which encodes a shmiR or shRNA targeting HBV.
  • a nucleic acid which comprises or consists of a DNA sequence set forth in SEQ ID NO: 150 and encodes a shmiR (shmiR-21) comprising or consisting of the sequence set forth in SEQ ID NO: 138, and at least one other nucleic acid of the disclosure which encodes a shmiR or shRNA targeting HBV.
  • the plurality of nucleic acids described herein comprises a nucleic acid which comprises or consists of a DNA sequence set forth in SEQ ID NO: 151 and encodes a shmiR (shmiR-22) comprising or consisting of the sequence set forth in SEQ ID NO: 139, and at least one other nucleic acid of the disclosure which encodes a shmiR or shRNA targeting HBV.
  • a nucleic acid which comprises or consists of a DNA sequence set forth in SEQ ID NO: 151 and encodes a shmiR (shmiR-22) comprising or consisting of the sequence set forth in SEQ ID NO: 139, and at least one other nucleic acid of the disclosure which encodes a shmiR or shRNA targeting HBV.
  • the plurality of nucleic acids described herein comprises a nucleic acid which comprises or consists of a DNA sequence set forth in SEQ ID NO: 152 and encodes a shmiR (shmiR-23) comprising or consisting of the sequence set forth in SEQ ID NO: 140, and at least one other nucleic acid of the disclosure which encodes a shmiR or shRNA targeting HBV.
  • a nucleic acid which comprises or consists of a DNA sequence set forth in SEQ ID NO: 152 and encodes a shmiR (shmiR-23) comprising or consisting of the sequence set forth in SEQ ID NO: 140, and at least one other nucleic acid of the disclosure which encodes a shmiR or shRNA targeting HBV.
  • the plurality of nucleic acids described herein comprises a nucleic acid which comprises or consists of a DNA sequence set forth in SEQ ID NO: 153 and encodes a shmiR (shmiR- 24) comprising or consisting of the sequence set forth in SEQ ID NO: 141, and at least one other nucleic acid of the disclosure which encodes a shmiR or shRNA targeting HBV.
  • the plurality of nucleic acids described herein comprises a nucleic acid which comprises or consists of a DNA sequence set forth in SEQ ID NO: 154 and encodes a shmiR (shmiR-25) comprising or consisting of the sequence set forth in SEQ ID NO: 142, and at least one other nucleic acid of the disclosure which encodes a shmiR or shRNA targeting HBV.
  • the plurality of nucleic acids described herein comprises a nucleic acid which comprises or consists of a DNA sequence set forth in SEQ ID NO: 155 and encodes a shmiR (shmiR-26) comprising or consisting of the sequence set forth in SEQ ID NO: 143, and at least one other nucleic acid of the disclosure which encodes a shmiR or shRNA targeting HBV.
  • a nucleic acid which comprises or consists of a DNA sequence set forth in SEQ ID NO: 155 and encodes a shmiR (shmiR-26) comprising or consisting of the sequence set forth in SEQ ID NO: 143, and at least one other nucleic acid of the disclosure which encodes a shmiR or shRNA targeting HBV.
  • the plurality of nucleic acids described herein comprises a nucleic acid which comprises or consists of a DNA sequence set forth in SEQ ID NO: 156 and encodes a shmiR (shmiR- 27) comprising or consisting of the sequence set forth in SEQ ID NO: 144, and at least one other nucleic acid of the disclosure which encodes a shmiR or shRNA targeting HBV.
  • the plurality of nucleic acids described herein comprises a nucleic acid which comprises or consists of a DNA sequence set forth in SEQ ID NO: 157 and encodes a shmiR (shmiR-28) comprising or consisting of the sequence set forth in SEQ ID NO: 145, and at least one other nucleic acid of the disclosure which encodes a shmiR or shRNA targeting HBV.
  • a nucleic acid which comprises or consists of a DNA sequence set forth in SEQ ID NO: 157 and encodes a shmiR (shmiR-28) comprising or consisting of the sequence set forth in SEQ ID NO: 145, and at least one other nucleic acid of the disclosure which encodes a shmiR or shRNA targeting HBV.
  • the plurality of nucleic acids may comprise two or more nucleic acids encoding shmiRs or shRNAs as described herein, such as two,or three, or four, or five, or six, or seven, or eight, or nine, or ten nucleic acids encoding shmiRs as described herein, provided at that at least one of the nucleic acids encodes a shmiRs of the disclosure.
  • the plurality of nucleic acids comprises two nucleic acids encoding a shmiR or shRNA described herein, with the proviso that at least one of the nucleic acids encodes a shmiR as described herein. In one example, the plurality of nucleic acids comprises three nucleic acids encoding a shmiR or shRNA described herein, with the proviso that at least one of the nucleic acids encodes a shmiR as described herein. In one example, the plurality of nucleic acids comprises four nucleic acids encoding a shmiR or shRNA described herein, with the proviso that at least one of the nucleic acids encodes a shmiR as described herein.
  • the plurality of nucleic acids comprises five nucleic acids encoding a shmiR or shRNA described herein, with the proviso that at least one of the nucleic acids encodes a shmiR as described herein. In one example, the plurality of nucleic acids comprises six nucleic acids encoding a shmiR or shRNA described herein, with the proviso that at least one of the nucleic acids encodes a shmiR as described herein. In one example, the plurality of nucleic acids comprises seven nucleic acids encoding a shmiR or shRNA described herein, with the proviso that at least one of the nucleic acids encodes a shmiR as described herein.
  • the plurality of nucleic acids comprises eight nucleic acids encoding a shmiR or shRNA described herein, with the proviso that at least one of the nucleic acids encodes a shmiR as described herein. In one example, the plurality of nucleic acids comprises nine nucleic acids encoding a shmiR or shRNA described herein, with the proviso that at least one of the nucleic acids encodes a shmiR as described herein. In one example, the plurality of nucleic acids comprises ten nucleic acids encoding a shmiR or shRNA described herein, with the proviso that at least one of the nucleic acids encodes a shmiR as described herein.
  • the effector sequence of a shmiR or shRNA encoded by one of the nucleic acids in the plurality is substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 4.
  • Suitable nucleic acids encoding a shmiR or shRNA having an effector sequence which is substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 4 are described herein.
  • the effector sequence of a shmiR or shRNA encoded by one of the nucleic acids in the plurality is substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 5.
  • Suitable nucleic acids encoding a shmiR or shRNA having an effector sequence which is substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 5 are described herein.
  • the effector sequence of a shmiR or shRNA encoded by one of the nucleic acids in the plurality is substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 9.
  • Suitable nucleic acids encoding a shmiR or shRNA having an effector sequence which is substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 9 are described herein.
  • the effector sequence of a shmiR or shRNA encoded by one of the nucleic acids in the plurality is substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 40.
  • Suitable nucleic acids encoding a shmiR or shRNA having an effector sequence which is substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 40 are described herein.
  • the effector sequence of a shmiR encoded by a nucleic acid in the plurality is substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 4 and the effector sequence of a shmiR encoded by a nucleic acid in the plurality is substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 9.
  • the effector sequence of a shmiR encoded by a nucleic acid in the plurality is substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 4 and the effector sequence of a shmiR encoded by a nucleic acid in the plurality is substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 9; and the effector sequence of a shmiR encoded by a nucleic acid in the plurality is substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 5.
  • the effector sequence of a shmiR encoded by a nucleic acid in the plurality is substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 4 and the effector sequence of a shmiR encoded by a nucleic acid in the plurality is substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 9; and the effector sequence of a shmiR encoded by a nucleic acid in the plurality is substantially complementary to a RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 40.
  • Exemplary nucleic acids of the disclosure encoding shmiRs which target HBV, including shmiRs comprising effector sequences which are substantially complementary to RNA transcripts set forth in SEQ ID NO: 4, 5, 9 or 40, are described herein and shall be taken to apply mutatis mutandis to each example in which a plurality of nucleic acids of the disclosure is described.
  • the plurality of nucleic acids of the disclosure comprises: (i) a nucleic acid comprising a DNA sequence encoding a shmiR comprising an effector sequence consisting of the sequence set forth in SEQ ID NO: 21 and an effector complement sequence consisting of the sequence set forth in SEQ ID NO: 22; and
  • nucleic acid comprising a DNA sequence encoding a shmiR comprising an effector sequence consisting of the sequence set forth in SEQ ID NO: 33 and an effector complement sequence consisting of the sequence set forth in SEQ ID NO: 34.
  • the plurality of nucleic acids of the disclosure comprises:
  • nucleic acid comprising a DNA sequence encoding a shmiR comprising an effector sequence consisting of the sequence set forth in SEQ ID NO: 21 and an effector complement sequence consisting of the sequence set forth in SEQ ID NO: 22;
  • nucleic acid comprising a DNA sequence encoding a shmiR comprising an effector sequence consisting of the sequence set forth in SEQ ID NO: 33 and an effector complement sequence consisting of the sequence set forth in SEQ ID NO: 34;
  • At least one other nucleic acid comprising a DNA sequence encoding a shmiR or shRNA comprising an effector sequence of at least 17 nucleotides in length which is substantially complimentary to a RNA transcript of the HBV genome.
  • the other nucleic acid of the disclosure is a nucleic acid described herein which encodes a shmiR or shRNA having an effector sequence which is different to that of the shmiRs encoded by the nucleic acids at (i) and (ii)
  • the plurality of nucleic acids of the disclosure comprises:
  • nucleic acid comprising a DNA sequence encoding a shmiR comprising an effector sequence consisting of the sequence set forth in SEQ ID NO: 21 and an effector complement sequence consisting of the sequence set forth in SEQ ID NO: 22;
  • nucleic acid comprising a DNA sequence encoding a shmiR comprising an effector sequence consisting of the sequence set forth in SEQ ID NO: 39 and an effector complement sequence consisting of the sequence set forth in SEQ ID NO: 40;
  • nucleic acid comprising a DNA sequence encoding a shmiR comprising an effector sequence consisting of the sequence set forth in SEQ ID NO: 33 and an effector complement sequence consisting of the sequence set forth in SEQ ID NO: 34.
  • the plurality of nucleic acids of the disclosure comprises: (i) a nucleic acid comprising a DNA sequence encoding a shmiR comprising an effector sequence consisting of the sequence set forth in SEQ ID NO: 21 and an effector complement sequence consisting of the sequence set forth in SEQ ID NO: 22;
  • nucleic acid comprising a DNA sequence encoding a shmiR comprising an effector sequence consisting of the sequence set forth in SEQ ID NO: 23 and an effector complement sequence consisting of the sequence set forth in SEQ ID NO: 24;
  • nucleic acid comprising a DNA sequence encoding a shmiR comprising an effector sequence consisting of the sequence set forth in SEQ ID NO: 33 and an effector complement sequence consisting of the sequence set forth in SEQ ID NO: 34.
  • the plurality of nucleic acids of the disclosure comprises:
  • nucleic acid comprising a DNA sequence encoding a shmiR comprising an effector sequence consisting of the sequence set forth in SEQ ID NO: 21 and an effector complement sequence consisting of the sequence set forth in SEQ ID NO: 22;
  • nucleic acid comprising a DNA sequence encoding a shRNA comprising an effector sequence consisting of the sequence set forth in SEQ ID NO: 39 and an effector complement sequence consisting of the sequence set forth in SEQ ID NO: 40;
  • nucleic acid comprising a DNA sequence encoding a shmiR comprising an effector sequence consisting of the sequence set forth in SEQ ID NO: 33 and an effector complement sequence consisting of the sequence set forth in SEQ ID NO: 34.
  • the plurality of nucleic acids of the disclosure comprises:
  • nucleic acid comprising a DNA sequence encoding a shmiR comprising an effector sequence consisting of the sequence set forth in SEQ ID NO: 21 and an effector complement sequence consisting of the sequence set forth in SEQ ID NO: 22;
  • nucleic acid comprising a DNA sequence encoding a shmiR comprising an effector sequence consisting of the sequence set forth in SEQ ID NO: 116 and an effector complement sequence consisting of the sequence set forth in SEQ ID NO: 117.
  • the plurality of nucleic acids of the disclosure comprises:
  • nucleic acid comprising a DNA sequence encoding a shmiR comprising an effector sequence consisting of the sequence set forth in SEQ ID NO: 21 and an effector complement sequence consisting of the sequence set forth in SEQ ID NO: 22; and (ii) a nucleic acid comprising a DNA sequence encoding a shmiR comprising an effector sequence consisting of the sequence set forth in SEQ ID NO: 124 and an effector complement sequence consisting of the sequence set forth in SEQ ID NO: 125.
  • the plurality of nucleic acids of the disclosure comprises:
  • nucleic acid comprising a DNA sequence encoding a shmiR comprising an effector sequence consisting of the sequence set forth in SEQ ID NO: 21 and an effector complement sequence consisting of the sequence set forth in SEQ ID NO: 22;
  • nucleic acid comprising a DNA sequence encoding a shmiR comprising an effector sequence consisting of the sequence set forth in SEQ ID NO: 116 and an effector complement sequence consisting of the sequence set forth in SEQ ID NO: 117;
  • nucleic acid comprising a DNA sequence encoding a shmiR comprising an effector sequence consisting of the sequence set forth in SEQ ID NO: 124 and an effector complement sequence consisting of the sequence set forth in SEQ ID NO: 125.
  • the plurality of nucleic acids of the disclosure comprises:
  • nucleic acid comprising a DNA sequence comprising or consisting of the sequence set forth in SEQ ID NO: 64 (encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 48);
  • nucleic acid comprising a DNA sequence comprising or consisting of the sequence set forth in SEQ ID NO: 70 (encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 54).
  • the plurality of nucleic acids of the disclosure comprises:
  • nucleic acid comprising a DNA sequence comprising or consisting of the sequence set forth in SEQ ID NO: 64 (encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 48);
  • nucleic acid comprising a DNA sequence comprising or consisting of the sequence set forth in SEQ ID NO: 73 ( encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 57);
  • nucleic acid comprising a DNA sequence comprising or consisting of the sequence set forth in SEQ ID NO: 70 (encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 54).
  • the plurality of nucleic acids of the disclosure comprises: (i) a nucleic acid comprising a DNA sequence comprising or consisting of the sequence set forth in SEQ ID NO: 64 (encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 48);
  • nucleic acid comprising a DNA sequence comprising or consisting of the sequence set forth in SEQ ID NO: 73 (encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 57);
  • nucleic acid comprising a DNA sequence comprising or consisting of the sequence set forth in SEQ ID NO: 70 (encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 54).
  • the plurality of nucleic acids of the disclosure comprises:
  • nucleic acid comprising a DNA sequence comprising or consisting of the sequence set forth in SEQ ID NO: 64 (encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 48);
  • nucleic acid comprising a DNA sequence comprising or consisting of the sequence set forth in SEQ ID NO: 99 (encoding a shRNA comprising or consisting of the sequence set forth in SEQ ID NO: 83);
  • nucleic acid comprising a DNA sequence comprising or consisting of the sequence set forth in SEQ ID NO: 70 (encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 54).
  • the plurality of nucleic acids of the disclosure comprises:
  • nucleic acid comprising a DNA sequence comprising or consisting of the sequence set forth in SEQ ID NO: 64 (encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 48);
  • nucleic acid comprising a DNA sequence comprising or consisting of the sequence set forth in SEQ ID NO: 149 (encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 137).
  • the plurality of nucleic acids of the disclosure comprises:
  • nucleic acid comprising a DNA sequence comprising or consisting of the sequence set forth in SEQ ID NO: 64 (encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 48); and (ii) a nucleic acid comprising a DNA sequence comprising or consisting of the sequence set forth in SEQ ID NO: 153 (encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 141).
  • the plurality of nucleic acids of the disclosure comprises:
  • nucleic acid comprising a DNA sequence comprising or consisting of the sequence set forth in SEQ ID NO: 64 (encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 48);
  • nucleic acid comprising a DNA sequence comprising or consisting of the sequence set forth in SEQ ID NO: 149 (encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 137);
  • nucleic acid comprising a DNA sequence comprising or consisting of the sequence set forth in SEQ ID NO: 153 (encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 141).
  • two or more of the nucleic acids may form separate parts of the same polynucleotide.
  • two or more of the nucleic acids in the plurality form parts of different
  • the plurality of nucleic acids described herein are provided as multiple components e.g., multiple compositions.
  • each of the nucleic acids of the plurality may be provided separately.
  • at least one of the nucleic acids may be provided separately and two or more of the plurality provided together.
  • the or each nucleic acid in accordance with the present disclosure may comprise, or be in operable linkage with, additional elements e.g., to facilitate transcription of the RNA.
  • the or each nucleic acid may comprise a promoter operably linked to the sequence encoding a shmiR or shRNA described herein.
  • Other elements e.g., transcriptional terminators and initiators, are known in the art and/or described herein.
  • the or each nucleic acid in accordance with the present disclosure may comprise one or more restriction sites e.g., to facilitate cloning of the nucleic acid(s) into cloning or expression vectors.
  • the nucleic acids described herein may include a restriction site upstream and/or downstream of the sequence encoding a shmiR or shRNA of the disclosure. Suitable restriction enzyme recognition sequences will be known to a person of skill in the art.
  • the nucleic acid(s) of the disclosure may include a BamHl restriction site (GGATCC) at the 5' terminus i.e., upstream of the sequence encoding the shmiR or shRNA, and a EcoRl restriction site (GAATTC) at the 3' terminus i.e., downstream of the sequence encoding the shmiR or shRNA.
  • GGATCC BamHl restriction site
  • GAATTC EcoRl restriction site
  • the or each nucleic acid of the disclosure is provided in the form of, or is comprised in, a DNA-directed RNAi (ddRNAi) construct.
  • ddRNAi DNA-directed RNAi
  • the present disclosure provides a ddRNAi construct comprising a nucleic acid as described herein.
  • the present disclosure provides a ddNAi construct comprising a plurality of nucleic acids described herein. Exemplary nucleic acids encoding shmiRs or shRNAs comprising effector sequences targeting HBV transcripts are described herein and shall be taken to apply mutatis mutandis to this example of the disclosure.
  • the ddRNAi construct comprises a nucleic acid of the disclosure operably linked to a promoter.
  • each of the nucleic acids may be operably-linked to a promoter.
  • the nucleic acids in the ddRNAi construct may be operably linked to the same promoter.
  • the nucleic acids in the ddRNAi construct may be operably linked to different promoters.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 59 and encodes a shmiR (shmiR- 1) comprising or consisting of the sequence set forth in SEQ ID NO: 43.
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence selected from the sequences set forth in any one of SEQ ID NOs: 60-74 and 146-157.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 60 and encodes a shmiR (shmiR-2) comprising or consisting of the sequence set forth in SEQ ID NO: 44.
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence selected from the sequences set forth in any one of SEQ ID NOs: 59, 61-74 and 146-157.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 61 and encodes a shmiR (shmiR-3) comprising or consisting of the sequence set forth in SEQ ID NO: 45.
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence selected from the sequences set forth in any one of SEQ ID NOs: 59-60, 62-74 and 146-157.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 62 and encodes a shmiR (shmiR-4) comprising or consisting of the sequence set forth in SEQ ID NO: 46.
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence selected from the sequences set forth in any one of SEQ ID NOs: 59-61, 63-74 and 146-157.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 63 and encodes a shmiR (shmiR-5) comprising or consisting of the sequence set forth in SEQ ID NO: 47.
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence selected from the sequences set forth in any one of SEQ ID NOs: 59-62, 64-74 and 146-157.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 64 and encodes a shmiR (shmiR-6) comprising or consisting of the sequence set forth in SEQ ID NO: 48.
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence selected from the sequences set forth in any one of SEQ ID NOs: 59-63, 65-74 and 146-157.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 65 and encodes a shmiR (shmiR-7) comprising or consisting of the sequence set forth in SEQ ID NO: 49.
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence selected from the sequences set forth in any one of SEQ ID NOs: 59-64, 66-74 and 146-157.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 66 and encodes a shmiR (shmiR-8) comprising or consisting of the sequence set forth in SEQ ID NO: 50.
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence selected from the sequences set forth in any one of SEQ ID NOs: 59-65, 67-74 and 146-157.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 67 and encodes a shmiR (shmiR-9) comprising or consisting of the sequence set forth in SEQ ID NO: 51.
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence selected from the sequences set forth in any one of SEQ ID NOs: 59-66, 68-74 and 146-157.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 68 and encodes a shmiR (shmiR- 10) comprising or consisting of the sequence set forth in SEQ ID NO: 52.
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence selected from the sequences set forth in any one of SEQ ID NOs: 59-67, 69-74 and 146-157.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 69 and encodes a shmiR (shmiR- 11) comprising or consisting of the sequence set forth in SEQ ID NO: 53.
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence selected from the sequences set forth in any one of SEQ ID NOs: 59-68, 70-74 and 146-157.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 70 and encodes a shmiR (shmiR- 12) comprising or consisting of the sequence set forth in SEQ ID NO: 54.
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence selected from the sequences set forth in any one of SEQ ID NOs: 59-69, 71-74 and 146-157.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 71 and encodes a shmiR (shmiR-13) comprising or consisting of the sequence set forth in SEQ ID NO: 55.
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence selected from the sequences set forth in any one of SEQ ID NOs: 59-70, 72-74 and 146-157.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 72 and encodes a shmiR (shmiR-14) comprising or consisting of the sequence set forth in SEQ ID NO: 56.
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence selected from the sequences set forth in any one of SEQ ID NOs: 59-71, 73-74 and 146-157.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 73 and encodes a shmiR (shmiR- 15) comprising or consisting of the sequence set forth in SEQ ID NO: 57.
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence selected from the sequences set forth in any one of SEQ ID NOs: 59-72, 74 and 146-157.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 74 and encodes a shmiR (shmiR- 16) comprising or consisting of the sequence set forth in SEQ ID NO: 58.
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence selected from the sequences set forth in any one of SEQ ID NOs: 59-73 and 146-157.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 146 and encodes a shmiR (shmiR- 17) comprising or consisting of the sequence set forth in SEQ ID NO: 134.
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence selected from the sequences set forth in any one of SEQ ID NOs: 59-74 and 147-157.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 147 and encodes a shmiR (shmiR- 18) comprising or consisting of the sequence set forth in SEQ ID NO: 135.
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence selected from the sequences set forth in any one of SEQ ID NOs: 59-74, 146 and 148-157.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 148 and encodes a shmiR (shmiR-
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence selected from the sequences set forth in any one of SEQ ID NOs: 59-74, 146, 147 and 149-157.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 149 and encodes a shmiR (shmiR-
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence selected from the sequences set forth in any one of SEQ ID NOs: 59-74, 146-148 and 150-157.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 150 and encodes a shmiR (shmiR-
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence selected from the sequences set forth in any one of SEQ ID NOs: 59-74, 146-149 and 151-157.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 151 and encodes a shmiR (shmiR-
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence selected from the sequences set forth in any one of SEQ ID NOs: 59-74, 146-150 and 152-157.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 152 and encodes a shmiR (shmiR- 23) comprising or consisting of the sequence set forth in SEQ ID NO: 140.
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence selected from the sequences set forth in any one of SEQ ID NOs: 59-74, 146-151 and 153-157.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 153 and encodes a shmiR (shmiR-
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence selected from the sequences set forth in any one of SEQ ID NOs: 59-74, 146-152 and 154-157.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 154 and encodes a shmiR (shmiR-
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence selected from the sequences set forth in any one of SEQ ID NOs: 59-74, 146-153 and 155-157.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 155 and encodes a shmiR (shmiR-
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence selected from the sequences set forth in any one of SEQ ID NOs: 59-74, 146-154 and 156-157.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 156 and encodes a shmiR (shmiR-
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence selected from the sequences set forth in any one of SEQ ID NOs: 59-74, 146-155 and 157.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 157 and encodes a shmiR (shmiR- 28) comprising or consisting of the sequence set forth in SEQ ID NO: 145.
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence selected from the sequences set forth in any one of SEQ ID NOs: 59-74 and 146-156.
  • a ddRNAi construct comprising a plurality of nucleic acids of the discloses may comprise:
  • nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO:64 (coding for shmiR-6); and (b) a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 149 (coding for shmiR-20).
  • a ddRNAi construct comprising a plurality of nucleic acids of the discloses may comprise:
  • the present disclosure also provides a ddRNAi construct comprising at least three nucleic acids described herein, such that the ddRNAi construct encodes at least three shmiRs targeting HBV, each of which is different to one another.
  • the disclosure provides a ddRNAi construct comprising:
  • nucleic acid at (b) encodes a shmiR or shRNA having an effector sequence which is different to that of the shmiRs encoded by the nucleic acid at (a) and (c).
  • the ddRNAi construct of the disclosure comprises, preferably in a 5' to 3 'direction:
  • a ddRNAi construct of the disclosure may comprise, in a 5' to
  • a ddRNAi construct of the disclosure comprises, preferably in a 5' to 3 'direction:
  • a ddRNAi construct of the disclosure may comprise, in a 5' to
  • ddRNAi construct comprising:
  • nucleic acid at (b) encodes a shmiR or shRNA having an effector sequence which is different to that of the shmiRs encoded by the nucleic acid at (a) and (c).
  • the disclosure provides a ddRNAi construct comprising:
  • nucleic acid at (b) encodes a shmiR or shRNA having an effector sequence which is different to that of the shmiRs encoded by the nucleic acid at (a) and (c).
  • the ddRNAi construct of the disclosure comprises, preferably in a 5' to 3 'direction:
  • a ddRNAi construct of the disclosure may comprise, in a 5' to
  • a ddRNAi construct of the disclosure may comprise at least one nucleic acid encoding a shmiR as described herein and at least one nucleic acid encoding a shRNA targeting HBV as described herein, wherein the shmiR and shRNA encoded by the ddRNAi construct comprise different effector sequences.
  • a ddRNAi construct of the disclosure may comprise, preferably in a 5' to 3 'direction:
  • a ddRNAi construct of the disclosure may comprise, preferably in a 5' to 3 'direction:
  • a ddRNAi construct of the disclosure may comprise, in a 5' to
  • the or each nucleic acid comprised therein may be operably linked to a promoter.
  • the ddRNAi construct as described herein may comprise a single promoter which is operably-linked to the or each nucleic acid comprised therein e.g., to drive expression of one or more shmiRs and/or shRNAs from the ddRNAi construct.
  • each nucleic acid encoding a shmiR or shRNA of the disclosure comprised in the ddRNAi construct is operably-linked to a separate promoter.
  • the promoters can be the same or different.
  • the construct may comprise multiple copies of the same promoter with each copy operably linked to a different nucleic acid of the disclosure.
  • each promoter operably linked to a RNA of the disclosure is different.
  • the three nucleic acids encoding the shmiRs are each operably linked to a different promoter.
  • a ddRNAi construct encoding three or more shmiRs two (or more) of the nucleic acids encoding the shmiRs are linked to the same promoter and one (or more) of the nucleic acids encoding the shmiR is linked to a different promoter.
  • the promoter is a constitutive promoter.
  • constitutive when made in reference to a promoter means that the promoter is capable of directing transcription of an operably linked nucleic acid sequence in the absence of a specific stimulus (e.g., heat shock, chemicals, light, etc.).
  • constitutive promoters are capable of directing expression of a coding sequence in substantially any cell and any tissue.
  • the promoters used to transcribe shmiRs or shRNAs from the nucleic acid(s) of the disclosure include promoters for ubiquitin, CMV, ⁇ -actin, histone H4, EF-la or pgk genes controlled by RNA polymerase II, or promoter elements controlled by RNA polymerase I.
  • a Pol II promoter such as CMV, SV40, Ul, ⁇ -actin or a hybrid Pol II promoter is employed.
  • a promoter controlled by RNA polymerase III is used, such as a U6 promoter (U6-1, U6-8, U6-9), HI promoter, 7SL promoter, a human Y promoter (hYl, hY3, hY4 (see Maraia, et al, Nucleic Acids Res 22(15):3045-52(1994)) and hY5 (see Maraia, et al, Nucleic Acids Res 24(18):3552-59(1994)), a human MRP-7-2 promoter, an Adenovirus VA1 promoter, a human tRNA promoter, or a 5s ribosomal RNA promoter.
  • U6 promoter U6-1, U6-8, U6-9
  • HI promoter 7SL promoter
  • a human Y promoter hYl, hY3, hY4 (see Maraia, et al, Nucleic Acids Res 22(15):3045-52(
  • Suitable promoters for use in a ddRNAi construct of the disclosure are described in US Patent No. 8,008,468 and US Patent No. 8,129,510.
  • the promoter is a RNA pol III promoter.
  • the promoter is a U6 promoter (e.g., a U6-1, U6-8 or U6-9 promoter).
  • the promoter is a HI promoter.
  • each of the nucleic acids in the ddRNAi construct is operably linked to a U6 promoter e.g., a separate U6 promoter.
  • the promoter in a construct is a U6 promoter.
  • the promoter is a U6-1 promoter.
  • the promoter is a U6-8 promoter.
  • the promoter is a U6-9 promoter.
  • promoters of variable strength are employed.
  • use of two or more strong promoters may tax the cell, by, e.g., depleting the pool of available nucleotides or other cellular components needed for transcription.
  • use of several strong promoters may cause a toxic level of expression of RNAi agents e.g., shmiRs or shRNAs, in the cell.
  • RNAi agents e.g., shmiRs or shRNAs
  • one or more of the promoters in the multiple -promoter ddRNAi construct is weaker than other promoters in the construct, or all promoters in the construct may express the shmiRs or shRNAs at less than a maximum rate.
  • Promoters may also be modified using various molecular techniques, or otherwise, e.g., through modification of
  • PSE Proximal Sequence Element
  • Promoters useful in some examples of the present disclosure can be tissue-specific or cell- specific.
  • tissue specific as it applies to a promoter refers to a promoter that is capable of directing selective transcription of a nucleic acid of interest to a specific type of tissue (e.g., liver tissue) in the relative absence of expression of the same nucleotide sequence of interest in a different type of tissue (e.g., muscle).
  • cell-specific as applied to a promoter refers to a promoter which is capable of directing selective
  • a ddRNAi construct of the disclosure may additionally comprise one or more enhancers to increase expression of the shmiRs or shRNAs encoded by the nucleic acids described herein.
  • Enhancers appropriate for use in examples of the present disclosure include the Apo E HCR enhancer, a CMV enhancer (Xia et al, Nucleic Acids Res 31- 17(2003)), and other enhancers known to those skilled in the art. Suitable enhancers for use in a ddRNAi construct of the disclosure are described in US Patent No. 8,008,468.
  • a ddRNAi construct of the disclosure may comprise a
  • the transcriptional terminator linked to a nucleic acid encoding a shmiR or shRNA of the disclosure In the case of a ddRNAi construct comprising a plurality of nucleic acids described herein i.e., encoding multiple shmiRs and/or shRNAs, the terminators linked to each nucleic acid can be the same or different. According to an example in which a RNA pol III promoter is employed, the terminator may be a contiguous stretch of 4 or more or 5 or more or 6 or more T residues.
  • the terminators can be different and are matched to the promoter from the gene from which the terminator is derived.
  • Such terminators include the SV40 poly A, the AdV VAl gene, the 5S ribosomal RNA gene, and the terminators for human t-RNAs.
  • promoters and terminators may be mixed and matched, as is commonly done with RNA pol II promoters and terminators.
  • the promoter and terminator combinations used for each nucleic acid in a ddRNAi construct comprising a plurality of nucleic acids is different to decrease the likelihood of DNA recombination events between components.
  • One exemplary ddRNAi construct of the disclosure comprises a nucleic acid encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO:48 operably linked to a U6 promoter e.g., a U6-9 promoter.
  • the ddRNAi construct of the disclosure comprises a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO:64 operably linked to a U6 promoter e.g., a U6-9 promoter.
  • Another exemplary ddRNAi construct of the disclosure comprises a nucleic acid encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO:57 operably linked to a U6 promoter e.g., a U6-1 promoter.
  • the ddRNAi construct of the disclosure comprises a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO:73 operably linked to a U6 promoter e.g., a U6-1 promoter.
  • Another exemplary ddRNAi construct of the disclosure comprises a nucleic acid encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 54 operably linked to a U6 promoter e.g., a U6-8 promoter.
  • the ddRNAi construct of the disclosure comprises a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO:70 operably linked to a U6 promoter e.g., a U6-8 promoter.
  • Another exemplary ddRNAi construct of the disclosure comprises a nucleic acid encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO:49 operably linked to a U6 promoter e.g., a U6-1 promoter.
  • the ddRNAi construct of the disclosure comprises a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO:65 operably linked to a U6 promoter e.g., a U6-1 promoter.
  • Another exemplary ddRNAi construct of the disclosure comprises a nucleic acid encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 137 operably linked to a U6 promoter e.g., a U6-1 promoter.
  • the ddRNAi construct of the disclosure comprises a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 149 operably linked to a U6 promoter e.g., a U6-1 promoter.
  • Another exemplary ddRNAi construct of the disclosure comprises a nucleic acid encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 141 operably linked to a U6 promoter e.g., a U6-1 promoter.
  • the ddRNAi construct of the disclosure comprises a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 153 operably linked to a U6 promoter e.g., a U6-8 promoter.
  • the ddRNAi construct of the disclosure comprises a plurality of nucleic acids described herein
  • the ddRNAi construct may comprise:
  • a nucleic acid encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO:48 operably linked to a U6 promoter e.g., a U6-9 promoter;
  • a nucleic acid encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO:54 operably linked to a U6 promoter e.g., a U6-8 promoter.
  • the ddRNAi construct may comprise:
  • U6 promoter e.g., a U6-9 promoter
  • nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO:70 operably linked to a U6 promoter e.g., a U6-8 promoter.
  • the ddRNAi construct of the disclosure comprises a plurality of nucleic acids described herein
  • the ddRNAi construct may comprise:
  • a nucleic acid encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO:48 (shmiR-6) operably linked to a U6 promoter e.g., a U6-9 promoter;
  • a nucleic acid encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 141 (shmiR-24) operably linked to a U6 promoter e.g., a U6-8 promoter.
  • the ddRNAi construct may comprise:
  • nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO:64 (coding for shmiR-6) operably linked to a U6 promoter e.g., a U6-9 promoter;
  • nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 153 (coding for shmiR-24) operably linked to a U6 promoter e.g., a U6-8 promoter.
  • the ddRNAi construct of the disclosure comprises a plurality of nucleic acids described herein
  • the ddRNAi construct may comprise:
  • a nucleic acid encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO:48 (shmiR-6) operably linked to a U6 promoter e.g., a U6-9 promoter;
  • a nucleic acid encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 137 (shmiR-20) operably linked to a U6 promoter e.g., a U6-1 promoter.
  • the ddRNAi construct may comprise:
  • nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 149 (coding for shmiR-20) operably linked to a U6 promoter e.g., a U6-1 promoter.
  • the ddRNAi construct of the disclosure comprises three nucleic acids described herein
  • the ddRNAi construct comprises: (a) a nucleic acid encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO:48 (shmiR-6) operably linked to a U6 promoter e.g., a U6-9 promoter;
  • a nucleic acid encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO:57 (shmiR- 15) operably linked to a U6 promoter e.g., a U6-1 promoter;
  • a nucleic acid encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO:54 (shmiR- 12) operably linked to a U6 promoter e.g., a U6-8 promoter.
  • the ddRNAi construct may comprise:
  • nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO:64 (coding for shmiR-6) operably linked to a U6 promoter e.g., a U6-9 promoter;
  • a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO:73 (coding for shmiR-15) operably linked to a U6 promoter e.g., a U6-1 promoter;
  • nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO:70 (coding for shmiR- 12) operably linked to a U6 promoter e.g., a U6-8 promoter.
  • the ddRNAi construct of the disclosure comprises three nucleic acids described herein, the ddRNAi construct comprises:
  • a nucleic acid encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO:48 (shmiR-6) operably linked to a U6 promoter e.g., a U6-9 promoter;
  • a nucleic acid encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 137 (shmiR-20) operably linked to a U6 promoter e.g., a U6-1 promoter;
  • a nucleic acid encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 141 (shmiR-24) operably linked to a U6 promoter e.g., a U6-8 promoter.
  • the ddRNAi construct may comprise:
  • nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO:64 (coding for shmiR-6) operably linked to a U6 promoter e.g., a U6-9 promoter;
  • nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 149 (coding for shmiR-20) operably linked to a U6 promoter e.g., a U6-1 promoter; and (c) a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 149 (coding for shmiR-20) operably linked to a U6 promoter e.g., a U6-1 promoter; and (c) a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 149 (coding for shmiR-20) operably linked to a U6 promoter e.g., a U6-1 promoter; and (c) a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 149 (coding for shmiR-20) operably linked to a U6 promoter e.g., a U6-1 promoter; and (c) a nucleic acid comprising or consisting of
  • the ddRNAi construct of the disclosure comprises three nucleic acids described herein, the ddRNAi construct comprises:
  • a nucleic acid encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO:48 (shmiR-6) operably linked to a U6 promoter e.g., a U6-9 promoter;

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CN201780040430.8A CN109415733A (zh) 2016-05-05 2017-05-05 用于治疗乙型肝炎病毒(hbv)感染的药剂及其使用
SG11201809753YA SG11201809753YA (en) 2016-05-05 2017-05-05 Reagents for treatment of hepatitis b virus (hbv) infection and use thereof
US16/098,759 US11535851B2 (en) 2016-05-05 2017-05-05 Reagents for treatment of hepatitis B virus (HBV) infection and use thereof
KR1020187035341A KR102358280B1 (ko) 2016-05-05 2017-05-05 B형 간염 바이러스(hbv) 감염 처리용 시약 및 그 용도
AU2017260580A AU2017260580B2 (en) 2016-05-05 2017-05-05 Reagents for treatment of hepatitis B virus (HBV) infection and use thereof
RU2018142797A RU2748806C2 (ru) 2016-05-05 2017-05-05 Средства для лечения инфекции вируса гепатита в (hbv) и их применение
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