WO2012109798A1 - Traitement du vhb - Google Patents

Traitement du vhb Download PDF

Info

Publication number
WO2012109798A1
WO2012109798A1 PCT/CN2011/071107 CN2011071107W WO2012109798A1 WO 2012109798 A1 WO2012109798 A1 WO 2012109798A1 CN 2011071107 W CN2011071107 W CN 2011071107W WO 2012109798 A1 WO2012109798 A1 WO 2012109798A1
Authority
WO
WIPO (PCT)
Prior art keywords
sequence
effector
ddrnai
sequences
hbv
Prior art date
Application number
PCT/CN2011/071107
Other languages
English (en)
Inventor
Michael Wayne Graham
Peter French
York Yuanyuan Zhu
Yixiang Lu
Tiejun Li
Yuncheng Sun
Xiaojun Tang
Li SHAN
Original Assignee
Benitec Biopharma Limited
Biomics Biotechnologies Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Benitec Biopharma Limited, Biomics Biotechnologies Co., Ltd. filed Critical Benitec Biopharma Limited
Priority to PCT/CN2011/071107 priority Critical patent/WO2012109798A1/fr
Priority to KR1020187027763A priority patent/KR20180110186A/ko
Priority to US13/882,124 priority patent/US9080174B2/en
Priority to PL16188642T priority patent/PL3124610T3/pl
Priority to DK16188642.9T priority patent/DK3124610T3/da
Priority to PCT/CN2011/081386 priority patent/WO2012055362A1/fr
Priority to RS20190704A priority patent/RS58982B1/sr
Priority to SI201131733T priority patent/SI3124610T1/sl
Priority to PT16188642T priority patent/PT3124610T/pt
Priority to BR112013010525A priority patent/BR112013010525A2/pt
Priority to TR2019/08127T priority patent/TR201908127T4/tr
Priority to HUE16188642 priority patent/HUE044426T2/hu
Priority to CN201180062884.8A priority patent/CN103370415B/zh
Priority to EP11835635.1A priority patent/EP2633051B1/fr
Priority to KR1020137013563A priority patent/KR101903778B1/ko
Priority to EP16188642.9A priority patent/EP3124610B1/fr
Priority to ES16188642T priority patent/ES2730393T3/es
Priority to AU2011320437A priority patent/AU2011320437B2/en
Priority to LTEP16188642.9T priority patent/LT3124610T/lt
Priority to RU2013124422A priority patent/RU2620966C2/ru
Priority to CA2853613A priority patent/CA2853613C/fr
Publication of WO2012109798A1 publication Critical patent/WO2012109798A1/fr
Priority to US14/731,824 priority patent/US9410154B2/en
Priority to HRP20190995TT priority patent/HRP20190995T1/hr
Priority to CY20191100583T priority patent/CY1121894T1/el

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • 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
    • C12N2310/51—Physical structure in polymeric form, e.g. multimers, concatemers
    • 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
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus

Definitions

  • RNA interference RNA interference
  • This invention is directed to an RNA interference (RNAi) agent and the use of that RNAi agent to treat hepatitis B infection in individuals, as well as pharmaceutical compositions containing the RNAi agents of the invention.
  • RNAi RNA interference
  • Hepatitis is a general term meaning 'inflammation of the liver' and has a number of causes. Viral causes are among the most common, and may be caused by hepatitis A, B, C, D or E virus. Hepatitis B virus (HBV) in particular is a serious and common infectious disease of the liver, affecting millions of people throughout the world.
  • HBV Hepatitis B virus
  • HBV is a hepatotrophic DNA virus belonging to the Hepadnaviridae.
  • the full-length of the viral genome is about 3.2kb, and it has four open reading frames (ORFs) including surface antigen (the "S gene”), core antigen (the “C gene”), DNA polymerase (the “P gene”) and a gene of undetermined function referred to as the "X gene”.
  • ORFs open reading frames
  • 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.
  • HBV is transmitted by percutaneous or parenteral contact with infected bodily fluids or blood.
  • the most common route of infection is via vertical transmission from mother to her baby, and in adults through sexual intercourse or shared intravenous needles or ear-piercing equipment. Many cases of acute HBV infection occur however without a traceable route of infection.
  • HBV Hepatocellular carcinoma
  • carriers - worldwide about 350-400 million people
  • HBV causes 60- 80% of the world's primary liver cancers. Every year about 25% of the over 4 million acute clinical cases (i.e. 1 million people worldwide) die from chronic active hepatitis, cirrhosis or HBV-induced liver cancer. As a consequence, HBV ranks second only to tobacco as a known human carcinogen.
  • RNA interference RNA interference
  • RNAi pathway is initiated by the enzyme Dicer, which cleaves double-stranded RNA (dsRNA) molecules into short fragments (commonly referred to as siRNAs) of -20-25 nucleotides.
  • dsRNA double-stranded RNA
  • siRNAs short fragments
  • One of the two strands of each fragment known as the guide strand or active strand, is then incorporated into the RNA- induced silencing complex (RISC) through binding to a member of the argonaute protein family.
  • RISC RNA- induced silencing complex
  • the guide strand base-pairs with its target mRNA is thought to either inhibit a target by inhibiting translation (by stalling the translational machinery) and/or inducing cleavage of the mRNA, thereby preventing it from being used as a translation template.
  • This strand is usually the one whose 5' end is less tightly paired to its complement, and there also appears to be a clear bias for A, and to a lesser extent U, at the 5' position to facilitate binding to some argonaute proteins (Schwarz DS, Hutvagner G, Du T, Xu Z, Aronin N, Zamore PD (2003). "Asymmetry in the assembly of the RNAi enzyme complex". Cell 1 15 (2): Frank F, Sonenberg N, Nagar (2010) "Structural basis for 5'-nucleotide base-specific recognition of guide RNA by human AG02". Nature. 465(7299):818-22).
  • HBV Hepatitis B Virus
  • a DNA-directed RNA interference (ddRNAi) agent being an RNA molecule
  • an expression cassette or construct to express that agent in a cell for inhibiting expression of at least one Hepatitis B virus (HBV) gene
  • the agent comprises an effector sequence (described further below) of at least 17 nucleotides in length complementary to or substantially complementary to a predicted sequence transcribed from a target region, the target region being selected from the group consisting of any 10 or more contiguous nucleotides within a sequence from any one of SEQ ID NOS: 1-19.
  • the effector sequence is 'directed to' a target region by being sufficiently complementary in sequence to a transcript from the target region such that an RNAi agent, such as a ddRNAi agent, having a double-stranded portion containing the effector sequence, inhibits expression of the target gene sequence.
  • an RNAi agent such as a ddRNAi agent, having a double-stranded portion containing the effector sequence
  • the RNAi agent is capable of inhibiting expression of a target gene sequence because the sequence of the effector is substantially complementary to (at least) a region of the predicted target sequence (as 'effector' is defined below).
  • a target region is a region of an mRNA of a gene that is intended to be silenced or to have its expression reduced.
  • the agent is designed so that it also comprises an effector complement sequence, ie a sequence that is substantially complementary to the effector sequence such that it will tend to anneal so as to form a double stranded RNA segment - the degree of complementarity required is more particularly explained further below.
  • an effector complement sequence ie a sequence that is substantially complementary to the effector sequence such that it will tend to anneal so as to form a double stranded RNA segment - the degree of complementarity required is more particularly explained further below.
  • an effector complement sequence ie a sequence that is substantially complementary to the effector sequence such that it will tend to anneal so as to form a double stranded RNA segment - the degree of complementarity required is more particularly explained further below.
  • usually one end of the double stranded segment will be linked by a loop sequence so as to form a 'hairpin' shaped structure.
  • This is also know as an 'interrupted inverted repeat' structure, as the DNA encoding such
  • the agent has more than one effector sequence. Multiple effectors may target the same region of an HBV gene, different (possibly overlapping) regions of the same gene and/or different HBV genes.
  • RNAi agents such as ddRNAi agents, typically contain 2 or 3 different effector sequences.
  • the ddRNAi agent comprises an effector complement sequence for each effector sequence, thus forming effector - effector complement pairs (ie a first effector - first effector complement pair, a second effector - second effector complement pair, etc). These pairs may be, but need not be, contiguous to one another, as long as the RNAi agent can fold so as to permit each pair to anneal.
  • RNAi agent may be, but need not be, contiguous to one another, as long as the RNAi agent can fold so as to permit each pair to anneal.
  • embodiments of the invention include one or more of the following:
  • ddRNAi agent comprising, in a 5' to 3' direction, a first effector sequence ; a second effector sequence; second effector complement sequence ; and a first effector complement sequence
  • a ddR Ai agent comprising, in a 5' to 3' direction, a first effector sequence; a second effector sequence; a third effector sequence; a third effector complement sequence; a second effector complement sequence; and a first effector complement sequence
  • a ddRNAi agent comprising, in a 5' to 3' direction, a first effector; a first effector complement sequence; a second effector sequence; and a second effector complement sequence;
  • a ddRNAi agent comprising, in a 5' to 3' direction, a first effector sequence; a first effector complement sequence; a second effector sequence ; a second effector complement sequence; a third effector sequence ; and a third effector complement sequence;
  • a ddRNAi agent comprising, in a 5' to 3' direction, a first effector sequence; a second effector sequence; a sequence of 2 to 100 non-self-complementary nucleotides; a second effector complement sequence; and a first effector complement sequence;
  • a ddRNAi agent comprising, in a 5' to 3' direction, a first effector sequence; a sequence of 2 to 100 non-self-complementary nucleotides; a first effector complement sequence; a second effector sequence; a sequence of 2 to 100 non-self-complementary nucleotides; and a second effector complement sequence.
  • each effector sequence is at least 17 nucleotides in length selected from the group consisting of any 10 or more contiguous nucleotides within a sequence from any one of SEQ ID NOS: 1- 19.
  • the effector sequences may all be the same, or may all be different, or may be a combination, eg 2 effector sequences of at least 10 contiguous nucleotides of SEQ ID NO: l and 1 effector sequence of at least 10 contiguous nucleotides of SEQ ID NO: 4.
  • the effector sequence is selected from the group consisting of any contiguous 1 1, 12, 13, 14, 15 or 16 nucleotides within any one of SEQ ID NOS: 1-19, and most preferably 17 or more contiguous nucleotides within any one of SEQ ID NOS: 1-19.
  • the effector complement will be the same length, or about the same length (ie ⁇ 15% nucleotide length) as its corresponding effector sequence.
  • the dsRNA is comprised of 2 separate RNA strands that are annealed to form a duplex.
  • ddRNAi agents may be expressed from a DNA expression cassette inserted into any suitable vector or ddRNAi construct. Accordingly, in aspects of the invention there is provided a ddRNAi expression cassettes comprising:
  • DNA sequences preferably being sequences that encode for any 10 or more and preferably any 17 or more contiguous nucleotides within a sequence from any one of SEQ ID NOS: 1-19,
  • one promoter is operably linked to multiple effector-encoding regions such that it can drive expression of them, whereas in other embodiments, each effector-encoding region is operably linked to its own promoter. In constructs where there are multiple promoters, these may be all the same or different. Preferred promoters are U6 and HI .
  • ddRNAi expression constructs into which the ddRNAi expression cassettes are inserted for expression.
  • the vector backbone of the construct is compatible with a delivery system, the ddRNAi expression constructs are also delivery constructs.
  • the invention also provides for siRNA agents that comprise a sequence of at least 17 nucleotides in length selected from the group consisting of any 10 or more contiguous nucleotides within a sequence from any one of SEQ ID NOS: 1-19 and a sequence complement with which the sequence forms a duplex, and that are capable of inhibiting expression of an HBV gene.
  • the invention also provides for methods of treatment of acute or chronic HBV infection, the reduction of HBV viral load the reduction of the severity of symptoms associated with HBV infection, and the reduction of the infectivity of HBV, comprising administering a therapeutically effective amount of a ddRNAi construct, ddRNAi agent or siRNA agent of the invention wherein the ddRNAi construct, ddRNAi agent or siRNA agent inhibits expression of one or more target sequences in a Hepatitis B virus (HBV) gene, preferably at least the polymerase gene of HBV.
  • HBV Hepatitis B virus
  • composition comprising a ddRNAi agent, a ddRNAi expression cassette, a ddRNAi construct or a siRNA agent of the invention and a pharmaceutically acceptable carrier or diluent.
  • Figure 1A-F illustrates some of the ddRNAi agent structures of the invention.
  • Figure 2 shows the distribution of the 642 siRNA clones obtained along the HBV polymerase gene, wherein the lines denote regions corresponding to individual Entire siRNA Target (EsT) clones.
  • Figure 3 is a comparison of the RNAi effectiveness of siRNA expression cassettes (SECs) and their corresponding synthetic siRNAs on HBV polymerase mRNA levels in order to validate the initial screening results obtained with SEC inhibition of HBV polymerase expression3.
  • SECs siRNA expression cassettes
  • Figure 4 is the results of the HBV polymerase inhibition screen with 501 siRNA sequences derived from the EsT library.
  • Figure 5A is an illustration of the distribution of the top 100 most effective siRNA sequences (as identified in the large scale screen in Figure 4) along the HBV polymerase gene.
  • Figure 5B is the distribution of the top 3 most effective sequences.
  • Figure 6 is a schematic of 3 individual expression cassettes and RNAi agents encoded by them, together with the theoretical effector sequence after processing by Dicer.
  • the expression cassettes are based on SEQ ID NO: l ( Figure 6A), SEQ ID NO:4 ( Figure 6B), and SEQ ID NO:6 ( Figure 6C).
  • Figure 7 is a schematic of a multiple effector sequence expression cassette containing (a) effector sequences each operably linked to separate promoter and terminator sequences to express individual RNAi agents in the form of a short hairpin RNAi (shRNAi) agent, ( Figure 7A); or (b) a first effector sequence operably linked to a promoter, and a third effector sequence operably linked to a terminator such that a single multiple stem loop RNAi agent is expressed.
  • the expression cassettes are based on SEQ ID NO: 1, SEQ ID NO:4, and SEQ ID NO:6.
  • Figure 8 is a schematic of a multiple effector sequence expression cassette based on SEQ ID NOS: 1, 4 and 6, which gives rise to a single, long hairpin RNAi agent.
  • RNA interference refers generally to a RNA dependent gene silencing process that is initiated by double stranded RNA (dsRNA) molecules in a cell's cytoplasm.
  • dsRNA double stranded RNA
  • the dsRNA reduces the expression of a target nucleic acid sequence, which may be a DNA whose RNA expression products are reduced, or an RNA, with which the dsRNA molecule shares substantial or total homology.
  • double stranded RNA or “dsRNA” it is meant a double stranded RNA molecule that is capable of inhibiting expression of a target nucleic acid sequence with which it shares homology.
  • the dsRNA is a hairpin or stem loop structure, with a duplex region optionally linked by at least 1 nucleotide, and is referred to as a "hairpin RNA” or “short hairpin RNAi agent” or “shRNA".
  • the duplex is formed between an effector sequence and a sequence complementary to the effector sequence herein referred to as an "effector complement".
  • the effector complement will be the same length as its corresponding effector sequence.
  • the effector sequence is complementary to the target nucleic acid sequence.
  • effector sequence is the nucleotide sequence that, when part of the RISC complex, binds to the HBV target nucleotide sequence, thereby targeting that sequence for destruction by the cell. It is analogous to the "guide" strand discussed in the background section.
  • the effector sequence is 'directed to' a target region by being complementary or substantially complementary in sequence to the transcript from the target region such that an RNA agent having a double stranded portion containing the effector sequence inhibits expression of the target gene sequence.
  • effector complement which is analogous to the passenger strand discussed in the background is of sufficient complementary to the effector such that is anneals to the effector sequence. It is likely that the effector complement will be of a similar sequence to the target gene sequence, but does not necessarily have to be.
  • RNAi agent refers to a dsRNA sequence that elicits RNAi. This term may be used interchangeably with “small interfering RNAs” (siRNA agents) and small hairpin RNA (shRNAi or hpRNAi agents).
  • RNAi agent The double stranded or duplex region of the RNAi agent is at least 17 base pairs long, and usually in the range of 17 to 30 base pairs.
  • RNAi agents can be synthesized chemically or enzymatically outside of cells and subsequently delivered to cells or can be expressed in vivo by an appropriate vector in cells (see, e.g., U.S. Pat. No. 6,573,099, WO 2004/106517 and WO99/49029, both of which are incorporated herein by reference).
  • DNA-directed RNAi agent refers to an RNAi agent that is transcribed from a DNA expression cassette ("ddRNAi expression cassette").
  • the ddRNAi agent transcribed from the expression cassette may be transcribed as a single RNA that is capable of self-annealing into a hairpin structure with a duplex region linked by at least 2 nucleotides, or as a single RNA with multiple shRNA domains or as multiple transcripts each capable of folding as a single shRNA.
  • the ddRNAi expression cassette can be ligated into vectors referred to as ddRNAi vectors or ddRNAi constructs.
  • the vectors may provide sequences specifying transcription of the ddRNAi expression cassette in vivo or in vitro.
  • the vector may additionally serve as the delivery vehicle for the ddRNAi expression cassette.
  • Viral based vectors for example will generate a ddRNAi construct that is useful for expression of the ddRNAi expression cassette as well as being compatible with viral delivery.
  • a cell has been "transformed”, “transduced” or “transfected” by an exogenous or heterologous nucleic acid or vector when such nucleic acid has been introduced into the cell.
  • the transforming DNA may or may not be integrated (covalently linked) into the genome of the cell.
  • a stably transformed cell is one in which the transforming DNA has become integrated into a host cell chromosome or is maintained extra-chromosomally (episomally) so that the transforming DNA is inherited by daughter cells during cell replication. In non-replicating, differentiated cells the transforming DNA may persist as an episome.
  • “Inhibition of expression” refers to the absence or observable decrease in the level of protein and/or mRNA product from the target gene.
  • the inhibition does not have to be absolute, but may be partial inhibition sufficient for there to a detectable or observable change as a result of the administration of a RNAi or ddRNAi agent or siRNA agent or ddRNAi construct of the invention.
  • Inhibition may 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 ddRNAi agent or construct, and may be as little as 1%, 5% or 10%, or may be absolute ie 100% inhibition.
  • the effects of inhibition may be determined by examination of the outward properties ie 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 agent or construct of the invention.
  • a quantitative phenotypic trait refers to a trait associated with the molecular expression of a nucleic acid in a host cell and may thus include the quantity of RNA molecules transcribed or replicated, the quantity of post-transcriptionally modified RNA molecules, the quantity of translated peptides or proteins, or the activity of such peptides or proteins.
  • a reduction of phenotypic expression of a nucleic acid where the phenotype is a qualitative trait means that in the presence of the RNAi agent of the invention, the phenotypic trait switches to a different state when compared to a situation in which the RNAi agent is absent.
  • a reduction of phenotypic expression of a nucleic acid may thus be measured as a reduction in steady state levels of (part of) that nucleic acid, a reduction in translation of (part of) that nucleic acid or a reduction in the effect the presence of the transcribed RNA(s) or translated polypeptide(s) have on the eukaryotic cell or the organism, and will ultimately lead to altered phenotypic traits.
  • the reduction in phenotypic expression of a nucleic acid of interest may be accompanied by or correlated to an observable change in phenotype.
  • the assessment may be by way of biochemical techniques such as Northern hybridisation, quantitative realtime PCR assays, gene expression assays, antibody binding, ELISA, RIA, western blotting and other assays and techniques known in the art.
  • Target nucleic acids may be either RNA or DNA, whose transcription products are targeted, coding or non-coding sequence, endogenous or exogenous.
  • the polymerase (P) gene of the DNA virus hepatitis B virus is targeted for inhibition.
  • the target nucleic acid is at least the RNA transcript of the polymerase gene.
  • An effector sequence for a target is complementary to or substantially complementary to the predicted transcript of a region of the target gene.
  • substantially complementary it is meant that the sequences are hybridisable or annealable.
  • substantially complementary is preferably about 85% complementary to a portion of the target gene. More preferably, it is at least 85-90% complementary, and most preferably at least 95, 96, 97, 98 99 or 100% complementary.
  • Substantial complementarity therefore includes 100% complementarity, but 100% complementarity may also be referred to throughout the specification as “complementary", or "being complementary”.
  • a sequence complementary to or substantially complementary to a region of a target gene has the degree of sequence complementarity across a contiguous target sequence.
  • a double stranded RNA region of the invention may be subjected to mutagenesis to produce single or several nucleotide substitutions, deletions or additions.
  • a “therapeutic composition” or “pharmaceutical composition” or “composition for treating HBV infection” refers to a composition including a ddRNAi agent, ddRNAi expression cassette, ddRNAi construct or siRNA agent.
  • beneficial or desired clinical results include, but are not limited to, alleviation of symptoms of HBV infection, reduced infectivity of HBV, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment. Treatment may not necessarily result in the complete clearance of HBV infection but may reduce or minimise complications and side effects of infection and the progression of infection.
  • terapéuticaally effective amount means an amount of a compound of the present invention that (i) treats the particular disease, condition, or disorder, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein.
  • the current invention provides a new RNAi agent, and use of the RNAi agent for targeting HBV in infected individuals. Treatment of HBV is aimed at:
  • RNAi agents expressed from DNA based ddRNAi expression cassettes are referred to as DNA-directed RNAi agents, or ddRNAi agents. They can directly target the activity of genes with minimum off-target events. By “off target events” it is meant that expression of nucleic acids other than the target are not inhibited by the RNAi or ddRNAi agents. In the case of HBV infection, this offers a unique opportunity to address the unmet clinical treatment needs for HBV. Accordingly, in one aspect of the invention, there is provided a DNA-directed RNA interference (ddRNAi) agent for inhibiting expression of one or more target sequences in a Hepatitis B virus (HBV) gene, the ddRNAi agent comprising at least:
  • the first effector sequence is substantially complementary to the predicted transcript of a region of the target gene.
  • the first effector sequence forms a double stranded region with the first effector complement sequence.
  • sequences of the ddRNAi agents of the invention have sufficient complementarity to a region of the HBV gene in order to mediate target specific RNAi.
  • substantially complementary it is meant that the sequences are hybridisable or annealable, and either:
  • the sequence of the first effector sequence is at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 or 90% complementary to at least 17 or more contiguous nucleotides of the target sequence, more preferably at least about 90, 91, 92, 92, 94 or 95% complementary and even more preferably at least about 95, 96, 97, 98 or 99% complementary or absolutely complementary (ie 100%) to 17 or more contiguous nucleotides of the target sequence; or
  • the effector sequence has at least 10 or more contiguous nucleotides that are 100% complementary with the target and preferably less than 6 nucleotides that cannot base pair with the target sequence.
  • the first effector sequence can therefore have 1, 2, 3, 4 or 5 nucleotides that will not G-C/A-U base pair with the target sequence. It is believed that this level of difference will not negatively impact on the ability of the ddRNAi agent to be able to inhibit expression of the target sequence.
  • the first effector sequence does have 1, 2, 3, 4 or 5 nucleotides that will not G-C/A-U base pair with the target sequence, it is preferred that the differences are in the first or last 5 nucleotides of the first effector sequence, with only 1 or 2 nucleotide changes in the centre portion of the effector sequence.
  • the ddRNAi agent may also comprise a first effector sequence consisting of 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length, wherein the effector sequence is substantially complementary to the predicted transcript of a region of the target gene.
  • a ddRNAi agent according to this embodiment of the invention therefore has a maximum length determined by the length and number of effector sequence/s ie each effector sequence is not comprised within a longer sequence.
  • substantially complementarity is intended to mean that the sequences are hybridisable or annealable.
  • substantially complementary sequences are able to hybridise under conditions of medium or high stringency:
  • medium stringency conditions 0.2xSSPE (or 1.OxSSC), 0.1%SDS, 50°C
  • substantially complementary would also be understood by the person skilled in the art to involve non- Watson-Crick base-pairing, especially in the context of RNA sequences, such as a so-called “wobble pair” which can form between guanosine and uracil residues in RNA.
  • “Complementary” is used herein in its usual way to indicate Watson-Crick base pairing, and “non-complementary” is used to mean non- Watson-Crick base pairing, even though such non-complementary sequences may form wobble pairs or other interactions.
  • reference to "non-pairing" sequences relates specifically to sequences between which Watson-Crick base pairs do not form.
  • the first effector sequence is at least 17 nucleotides long, preferably 17 to 50 nucleotides and most preferably 17 to 30 nucleotides. It may be 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. When the first effector sequence is longer than 17 nucleotides, it is preferred that at least 17 contiguous nucleotides of the first effector sequence forms the double stranded region with the complementary strand.
  • the ddRNAi agents of the invention inhibit expression of HBV nucleic acid sequences.
  • the HBV target gene is the nucleic acid sequence that is expressed as the polymerase (P) gene.
  • the ddRNAi agent of the invention inhibits expression of one or more target sequences in a Hepatitis B virus (HBV) polymerase gene.
  • HBV Hepatitis B virus
  • the HBV genome has overlapping open reading frames.
  • targeting particular sequences of the polymerase gene will also target the same sequences in the overlapping gene.
  • the agents of the invention therefore are capable of targeting multiple genes with a single effector sequence. In each preferred embodiment however, at least the polymerase gene is targeted.
  • the first effector sequence is selected from any 10 or more and preferably any 17 or more contiguous nucleotides within any one of the ddRNAi HBV polymerase effector sequences SEQ ID NOS: 1- 19 listed below:
  • pol corresponds to polymerase
  • HBsAg corresponds to the HBV surface antigen
  • X corresponds to the X protein
  • both strands of a dsRNA have the potential to be the effector sequence.
  • particular features of a sequence can favour one strand to enter the RISC and the other strand to be destroyed.
  • the protein Argonaut 2 (AG02) of the RISC complex has a preference for sequences with a 5' A, and to a lesser extent a 5' U.
  • a 5' mismatch is also thought to bias one strand to be incorporated into the RISC complex over the other strand. This sequence preference is reflected in preferred embodiments, but is not essential.
  • ddRNAi DNA-directed RNA interference agent for inhibiting expression of one or more target sequences in a Hepatitis B virus (HBV) gene
  • HBV Hepatitis B virus
  • the first effector sequence is substantially complementary to the predicted transcript of a region of the target gene.
  • the first effector sequence is at least 17 or more contiguous nucleotides within GAUUGACGAUAAGGGAGA (SEQ ID NO: 1).
  • the differences are preferably present in the first and/or last 5 nucleotides, and at least the centre 10 nucleotides are 100% complementary to the predicted transcript of a region of the target gene.
  • the ddRNAi agent comprises a first effector sequence of any 10 or more, preferably any 17 or more, contiguous nucleotides within SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: l l, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID: 19.
  • the first effector sequence may comprise a sequence selected from any 10 or more and preferably any 17 or more contiguous nucleotides within a sequence from the group consisting of SEQ ID NOS: 1-19, or alternatively, each effector sequence may be a variant of SEQ ID NOS: l- 19, having 1, 2, 3, 4 or 5 nucleotide variations. In yet a further embodiment, each effector sequence may consist of 22 nucleotides, of which 17, 18, 19, 20, 21 or all 22 nucleotides are contiguous nucleotides from a sequence selected from the group consisting of SEQ ID NOS: 1-19.
  • the ddRNAi agent comprises two or more effector sequences to enable targeting of more than one target sequence of the HBV genome.
  • the multiple target sequences may be in the same region of the HBV gene. For example, a 17 to 30 nucleotide region that has natural variation in the sequence between strains, or single nucleotide polymorphisms that have arisen to confer drug resistance.
  • the target sequences may be in different regions of the one target gene.
  • ddRNAi agent comprises the following (in no particular order):
  • the first and second effector sequences of a multiple targeting ddRNAi agent form a double stranded region with their respective effector complements.
  • the first and second effector sequences are 17 to 30 nucleotides in length. More preferably, the first and second effector sequence are both selected from any 10 or more and preferably any 17 or more contiguous nucleotides within any one of the sequences listed in Table 1 above, or are sequences having 1, 2, 3, 4 or 5 nucleotides difference from those sequences listed in Table 1.
  • the first effector sequence is selected from any 10 or more and preferably any 17 or more contiguous nucleotides within a sequence from any one of the group consisting of SEQ ID NOS: l- 19, and the second effector sequence is selected from any 10 or more and preferably any 17 or more contiguous nucleotides within a sequence from any one of the group consisting of SEQ ID NOS: 1-19.
  • the first and second effector sequence may both be the same sequence or may alternatively be different sequences.
  • the first and second effector sequence may each comprise a sequence selected from any 10 or more contiguous nucleotides within a sequence from the group consisting of SEQ ID NOS: 1-19, or alternatively, each effector sequence may also be a variant of SEQ ID NOS: 1-19, having 1, 2, 3, 4 or 5 nucleotide variations. In yet a further embodiment, each effector sequence may consist of 22 nucleotides, of which 17, 18, 19, 20, 21 or all 22 nucleotides are contiguous nucleotides from a sequence selected from the group consisting of SEQ ID NOS: 1-19. When there are two or more effector sequences, they may represent a combination of the 3 types described above.
  • ddRNAi agents with multiple effector sequences has the advantage of limiting the emergence of and targeting escape mutants, which is a problem of many current anti-viral therapies.
  • One aspect of the present invention neutralizes emergent escape mutants with a ddRNAi agent that contains RNAi sequences based upon the genetic sequence of the target gene and additionally sequences of the point mutations that arise to resist RNAi treatment.
  • ddRNAi agents with multiple effector sequences have the advantage of being able to target a range of sequences found in different viral genotypes or quasi-species, as well as the advantage of the additive or synergistic effects achieved with multiple effector sequences as opposed to single effector sequences.
  • HBV contains a number of overlapping reading frames, such that targeting a sequence in the overlapping regions will inhibit expression of both of the genes that contain that sequence.
  • ddRNAi agent contains more than one effector sequence
  • the ddRNAi agent is expressed as a single strand of RNA, it will fold to form different structures depending on the order of the effector sequences and the sequences complementary to the effector sequences.
  • ddRNAi DNA-directed RNA interference
  • HBV Hepatitis B virus
  • each effector sequence is substantially complementary to the predicted transcript of a region of the target gene. This will result in a ddRNAi agent with a structure as shown in Figure 1A. See also WO2004/106517, incorporated herein by reference.
  • At least one effector, and preferably both effector sequences are 100% complementary to the predicted transcript of a region of the target gene.
  • the first and second effector sequences are both selected from the group consisting of any 10 or more and preferably any 17 or more contiguous nucleotides within any one of SEQ ID NOS: 1-19.
  • ddRNAi DNA-directed RNA interference
  • HBV Hepatitis B virus
  • Each effector sequence is substantially complementary to the predicted transcript of a region of the target gene.
  • At least one effector, and preferably both effector sequences are 100% complementary to the predicted transcript of a region of the target gene.
  • ddRNAi agent for inhibiting expression of one or more target sequences in a Hepatitis B virus (HBV) gene
  • the ddRNAi agent comprising, in a 5' to 3' direction, at least: a first effector sequence of any 10 or more contiguous nucleotides within GAUUGACGAUAAGGGAGA (SEQ ID NO: 1);
  • Each effector sequence is substantially complementary to the predicted transcript of a region of the target gene.
  • At least one effector, and optionally 2 out of the 3 or all 3 of the effectors, are 100% complementary to the predicted transcript of a region of the target gene.
  • effector and effector complements can be altered, provided that a single, long hairpin structure is formed by annealing of the effector sequence with its effector complement to form dsRNA.
  • sequences may be arranged in the following exemplary 5' to 3' orders:
  • sequences may be arranged in the following exemplary 5' to 3' orders:
  • the first effector sequence may be selected from any 10 or more and preferably any 17 or more contiguous nucleotides within a sequence from the group consisting of SEQ ID NOS: 1-19;
  • the second effector sequence may be selected from any 10 or more and preferably any 17 or more contiguous nucleotides within a sequence from the group consisting of SEQ ID NOS: 1-19;
  • the third effector sequence may be selected from any 10 or more and preferably any 17 or more contiguous nucleotides within a sequence from the group consisting of SEQ ID NOS: 1- 19; and any further effector sequences may be selected from any 10 or more and preferably any 17 or more contiguous nucleotides within a sequence from the group consisting of SEQ ID NOS: 1-19.
  • each effector sequence may also be a variant of SEQ ID NOS: l- 19, having 1, 2, 3, 4 or 5 nucleotide variations.
  • the differences are present in the first and/or last 5 nucleotides, and at least the centre 1 1-12 nucleotides are 100% complementary to the predicted transcript of a region of the target gene.
  • Such embodiments are particularly useful for targeting HBV escape mutants, as well as different viral genotypes or quasi-species.
  • the first, second and third effector sequence may each comprise a sequence selected from any 10 or more contiguous nucleotides within a sequence from the group consisting of SEQ ID NOS: 1-19, or alternatively, each effector sequence may also be a variant of SEQ ID NOS: 1-19, having 1, 2, 3, 4 or 5 nucleotide variations. In yet a further embodiment, each effector sequence may consist of 22 nucleotides, of which 17, 18, 19, 20, 21 or all 22 nucleotides are contiguous nucleotides from a sequence selected from the group consisting of SEQ ID NOS: 1-19. When there are multiple effector sequences, they may represent a combination of the 3 types described above.
  • ddRNAi DNA-directed RNA interference agent for inhibiting expression of one or more target sequences in a Hepatitis B virus (HBV) gene
  • the ddRNAi agent comprising, in a 5' to 3' direction, at least:
  • each effector sequence is substantially complementary to the predicted transcript of a region of the target gene.
  • At least one effector, and preferably both effector sequences is 100% complementary to the predicted transcript of a region of the target gene.
  • the first and second effector sequences are both selected from any 10 or more and preferably any 17 or more contiguous nucleotides within a sequence from the group consisting of SEQ ID NOS: 1- 19.
  • ddRNAi DNA-directed RNA interference
  • HBV Hepatitis B virus
  • a second effector sequence any 10 or more contiguous nucleotides within UUGAAGUCCCAAUCUGGAU (SEQ ID NO:2) or GCCGGGCAACGGGGUAAAGGUUC (SEQ ID NO:3); and
  • Each effector sequence is substantially complementary to the predicted transcript of a region of the target gene.
  • At least one effector, and preferably both effector sequences is 100% complementary to the predicted transcript of a region of the target gene.
  • ddRNAi agent has 3 effector sequences
  • ddRNAi agent for inhibiting expression of one or more target sequences in a Hepatitis B virus (HBV) gene
  • HBV Hepatitis B virus
  • Each effector sequence is substantially complementary to the predicted transcript of a region of the target gene.
  • At least one effector, and optionally 2 out of the 3 or all 3 of the effectors, is 100% complementary to the predicted transcript of a region of the target gene.
  • the first effector sequence may be any 10 or more contiguous nucleotides within a sequence selected from the group consisting of SEQ ID NOS: 1 - 19;
  • the second effector sequence may be any 10 or more contiguous nucleotides within a sequence selected from the group consisting of SEQ ID NOS: 1 - 19;
  • the third effector sequence may be any 10 or more contiguous nucleotides within a sequence selected from the group consisting SEQ ID NOS: 1- 19; and any further effector sequences may be any 10 or more contiguous nucleotides within a sequence selected from the group consisting of SEQ ID NOS: 1 - 19.
  • each effector sequence is at least 17 contiguous nucleotides.
  • Each effector sequence may also be a variant of SEQ ID NOS: l - 19, having 1 , 2, 3, 4 or 5 nucleotide variations.
  • the differences are present in the first and/or last 5 nucleotides, and at least the centre 10- 12 nucleotides are 100% complementary to the predicted transcript of a region of the target gene.
  • Such embodiments are particularly useful for targeting HBV escape mutants, as well as different viral genotypes or quasi species.
  • the first, second and third effector sequence may each comprise a sequence selected from any 10 or more contiguous nucleotides within a sequence from the group consisting of SEQ ID NOS: 1 - 19, or alternatively, each effector sequence may also be a variant of SEQ ID NOS: 1 - 19, having 1 , 2, 3, 4 or 5 nucleotide variations.
  • each effector sequence may consist of 22 nucleotides, of which 17, 18, 19, 20, 21 or all 22 nucleotides are contiguous nucleotides from a sequence selected from the group consisting of SEQ ID NOS: 1 - 19.
  • the ddRNAi agent may include additional effector sequences and corresponding complementary sequences according to one of the following formula:
  • the number of effector sequences is equal to the number of effector complement sequences.
  • the effector sequences may be the same or different.
  • the effector sequences are any 10 or more and preferably any 17 or more contiguous nucleotides within a sequence selected from the group consisting of SEQ ID NOS: 1 -19, or variants of the sequences of SEQ ID NOS: l- 19 which have 1 , 2, 3, 4 or 5 nucleotide variations.
  • the differences are present in the first and/or last 5 nucleotides, and at least the centre 10- 12 nucleotides are 100% complementary to the predicted transcript of a region of the target gene.
  • At least one effector sequence is chosen from any 10 or more and preferably any 17 or more contiguous nucleotides within a sequence selected from the group consisting of SEQ ID NOS: 1 - 19, whereas other effector sequences are variants of that chosen sequence.
  • a first effector sequence may comprise 20 nucleotides of SEQ ID NO: 1 ; the second effector sequence should therefore be a variant of SEQ ID NO: l .
  • the effector sequence hybridises with its corresponding effector complement sequence to form a hairpin structure.
  • two or more unbound nucleotides form the 'hinge' or 'loop'.
  • the unbound nucleotides are part of the effector sequence and the effector complement sequence, such that only a portion of the at least 17 nucleotides of the effector sequence will form a duplex with its corresponding effector complement sequence.
  • the effector sequence and its complement are both 22 nucleotides long, 19 of the nucleotides may base pair to form a double stranded region, leaving a total of 6 nucleotides to form a single stranded loop between and joining the effector sequence and its effector complement sequence.
  • the ddR Ai agent further includes a sequence of 2 to 100 unpaired nucleotides capable of forming a loop, more preferably, 2 to 10 unpaired nucleotides.
  • the loop includes the nucleotide sequence AA, UU, UUA, UUAG, UUACAA or NiAAN 2 , where Ni and N 2 are any of C, G, U and A and may be the same or different.
  • ddRNAi agent there may be one or more loops depending on the ddRNAi agent structure.
  • a ddRNAi agent has a long hairpin structure based on formula [effector sequence] i_i 0 [effector complement sequence]i_i 0 additional non-self-complementary sequence to give rise to a single loop structure is contained between the last effector sequence and the effector complement sequence of that last effector sequence, as illustrated in Figure ID.
  • ddRNAi DNA-directed RNA interference
  • HBV Hepatitis B virus
  • each effector sequence is substantially complementary to the predicted transcript of a region of the target gene.
  • ddRNAi agent When the ddRNAi agent has a multiple hairpin structure based on formula [effector sequence-effector complement sequence] O additional non-self-complementary sequence is contained between each effector sequence and its complementary sequence to give rise to a loop structure, as illustrated in Figure IE and F (depending on the type of expression cassette used to express it (see later in the specification).
  • ddRNAi DNA-directed RNA interference
  • HBV Hepatitis B virus
  • each effector sequence is substantially complementary to the predicted transcript of a region of the target gene.
  • the length of additional non-self-complementary sequence that forms each loop structure does not have to be the same.
  • one loop structure may have 5 nucleotides, while another loop structure may have 9 nucleotides.
  • the entire ddRNAi agent is expressed as one sequence.
  • the first effector sequence may be generated (e.g., transcribed by one DNA sequence), and the first effector complement sequence may be generated (e.g., transcribed from a separate DNA sequence).
  • a loop sequence may be attached to either transcript or part of the loop attached to the 3' end of one transcript and the 5' end of the other transcript.
  • the two transcripts then form the ddRNAi agent by hybridising through annealing between the effector sequence/s and their complement/s.
  • ddRNAi agents While it is envisaged that effective treatment of chronic HBV infection will require ddRNAi agents to be expressed in vivo from ddRNAi constructs (as will be outlined below), there may be circumstances where it is desirable to administer ddRNAi agents that are expressed in vitro or to administer siRNAs that are chemically synthesised, thereby functioning as more of a transient therapy.
  • Acute HBV infection for example may benefit from a short term treatment with siRNAs that do not integrate and replicate in the cells.
  • siRNA agent small interfering RNAi agent for inhibiting expression of one or more target sequences in a Hepatitis B virus (HBV) gene, the siRNA comprising
  • effector sequence is substantially complementary to the predicted transcript of a region of the target gene.
  • the siRNA agent may also include more than one effector sequence for multiple targeting.
  • the effector sequences preferably target the HBV polymerase gene, and most preferably, are selected from any 10 or more and preferably any 17 or more contiguous nucleotides within a sequence from the group consisting of SEQ ID NOS: 1-19.
  • siRNAs consist of dsRNA molecules with 5'-phosphate and 3'-hydroxyl residues
  • strand legths can vary from 21-29 nucleotides and may optionally be designed to include 2 nucleotide 3 ! overhangs.
  • each strand can be syntheseised as N19-27TT (where TT can be deoxynueleotides).
  • siRNAs can be readily designed based on regions of SEQ ID NOS: 1 -19 as described above and can be used therapeutically as single sequences or in any combinations.
  • siRNA agents can consist of single RNA molecules containing effector and effector complement sequences similar or identical to those expressed from ddRNAi expression cassettes.
  • siRNAs can be chemically synthesized with appropriately protected ribonueleoside phosphoramidites and a conventional synthesizer and thus are widely available commercially and able to be designed and synthesised according to routine methods in the art.
  • the siRNAs have the sequences of any 10 or more contiguous nucleotides within a sequence from one or more of SEQ ID NOS: 1-19.
  • siRNAs A number of transfection reagents have been used for delivering siRNA into different cell lines. Lipofectamine 2000 and Oligofectamine are routinely used for siRNA delivery. Naked siRNAs have also been delivered by hydrodynamic transfection methods. Other delivery methods would be known by the skilled person.
  • ddRNAi agents are expressed from DNA expression cassettes inserted into any suitable vector or ddRNAi construct.
  • ddRNAi expression cassettes comprise:
  • ddRNAi DNA-directed RNA interference
  • HBV Hepatitis B virus
  • the DNA sequence that encodes for the first effector sequence is preferably a DNA that encodes for 10 or more, preferably 17 or more, contiguous nucleotides within a sequence from any one of SEQ ID NOS: 1 - 19.
  • the sequence that encodes for the effector sequence may encode an effector sequence that varies by 1 , 2, 3, 4 or 5 nucleotides from SEQ ID NOS: 1 - 19 without affecting the ability of the effector sequence encoded to base pair with the target sequence and inhibit expression of the target sequence.
  • RNA sequence encoding any given RNA sequence is the same sequence as the RNA but having thymine (T) bases instead of uracil (U) bases.
  • the ddRNAi expression cassettes encoding ddRNAi agents having more than one effector sequence in a long hairpin structure comprise, in a 5' to 3 ' direction:
  • the DNA sequences encode first and second effector sequence selected from any 10 or more and preferably any 17 or more contiguous nucleotides within a sequence from the group consisting of SEQ ID NOS: 1 - 19.
  • the DNA sequences encode for an effector sequence that varies from SEQ ID NOS: 1 - 19 by 1 , 2, 3, 4 or 5 nucleotides without affecting the ability of the effector sequence encoded to base pair with the target sequence and inhibit expression of the target sequence.
  • the ddRNAi agent has more than one effector sequence and a multiple hairpin structure based on formula [effector sequence-effector complement sequence ]i_io expression of each [effector sequence- effector complement sequence] pair may be controlled by a single promoter, or alternatively by a separate promoter.
  • the ddRNAi expression cassette comprises, in a 5' to 3 ' direction:
  • the ddRNAi expression cassette comprises, in a 5' to 3' direction:
  • the DNA sequences preferably encode first and second effector sequence selected from any 10 or more and preferably any 17 or more contiguous nucleotides within a sequence from the group consisting of SEQ ID NOS: l-19, or . effector sequences that vary in sequence from SEQ ID NOS: 1-19 by 1, 2, 3, 4 or 5 nucleotides.
  • the ddRNAi expression cassette may alternatively be described by reference to the total length of the ddRNAi agent expressed, which is a product of the total length of sequence between the promoter and terminator.
  • the ddRNAi expression cassette will have a length of 34 to 60 nucleotides between the promoter and terminator. This length may further include 2 to 100 nucleotides of "loop" or "hinge" sequence, giving a length of between 36 to 160 nucleotides.
  • the overall length is increased proportionally.
  • ddRNAi expression cassettes of the invention One useful way of designing ddRNAi expression cassettes of the invention is to assume Dicer cuts every 22 nucleotides (also referred to as 22nt phasing).
  • the DNA sequences that encode effector sequences can therefore be designed to encode any 10 or more, and preferably any 17 or more contiguous nucleotides within a sequence from the group consisting of SEQ ID NOS: 1-19, together with appropriate spacers and other sequence requirements for the appropriate promoter. For example, some of the sequences listed in SEQ ID NOS: 1-19 are greater than 22 nucleotides; in these instances only a portion of the sequence needs to be operably linked to a promoter.
  • the DNA sequence operably linked to the promoter starts with a guanine (G) base; when a HI promoter is used, it is preferable that the DNA sequence operably linked to the promoter starts with an adenine (A) base.
  • the effector encoding sequence can therefore be modified accordingly.
  • effector sequences are shorter than 22nts; in these instances it is preferable to include HBV sequences adjacent to the HBV target sites. This would serve to maximise homology of effector sequences to HBV mRNAs and might also permit the inclusion of A or G residues to allow efficient transcriptional initiation from the U6 or HI promoters as described above.
  • effector, effector complement or loop sequences since these can act as transcriptional terminators in expression constructs which use Pol III promoters such as U6 or HI .
  • Pol III promoters such as U6 or HI .
  • each [effector sequence-effector complement sequence] pair depends on a number of factors.
  • a single promoter may be utilised to minimise interference between promoters.
  • a ddRNAi construct with only a single promoter is also smaller in size, which can be important in some cases for the stability of the construct, both during production (eg replication in E.coli) and delivery.
  • the use of a single promoter avoids the possibility of any homologous recombination between promoters.
  • the nature of the sequence may mean one sequence is expressed to higher expression levels.
  • the more highly expressed effector sequence can be paired with a weaker promoter and vice versa.
  • more efficient expression may be achieved as the length of any one sequence to be transcribed is shorter.
  • each is preferably different.
  • at least 2 and optionally all 3 are different from one another.
  • the DNA sequence encoding the effector sequence is operably linked to the promoter sequence.
  • a sequence is "operably linked" to another nucleotide sequence when it is placed in a functional relationship with another nucleotide sequence.
  • a coding sequence is operably linked to a promoter sequence, this generally means that the promoter promotes transcription of the coding sequence.
  • Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame.
  • enhancers may function when separated from the promoter by several kilobases and intronic sequences may be of variable length, some nucleotide sequences may be operably linked but not contiguous.
  • a “promoter” or “promoter sequence” or “promoter element” is generally a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a polynucleotide or polypeptide coding sequence such as mRNA or any kind of RNA transcribed by any class of any RNA polymerase.
  • the promoter and terminator may be taken from different genes, but are typically matched to each other; that is, the promoter and terminator sequences or elements are taken from the same gene in which they occur naturally. Promoters also may or may not be modified using molecular techniques, or otherwise, e.g., through mutation of regulatory elements such as enhancers, to attain higher or lower levels of transcription.
  • 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 presence or absence of a specific stimulus (e.g., heat shock, chemicals, light, etc.). Typically, constitutive promoters are capable of directing expression of a coding sequence in substantially any cell and any tissue.
  • a specific stimulus e.g., heat shock, chemicals, light, etc.
  • the promoters used in the expression cassettes to transcribe the ddRNAi agents preferably are constitutive promoters, such as the promoters for ubiquitin, CMV, ⁇ -actin, histone H4, EF- 1 alfa or pgk genes whose expression is controlled by RNA polymerase II binding to the promoter, or promoter elements which are bound by RNA polymerase I.
  • a Pol II promoter such as CMV, SV40, hAAT, Ul, ⁇ -actin or a hybrid Pol II promoter is employed.
  • promoter elements bound by RNA polymerase III are used, such as the U6 promoters (U6- 1, U6-8, U6-9, e.g.), HI promoter, 7SL promoter, the human Y promoters (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)), the human MRP-7-2 promoter, Adenovirus VA1 promoter, human tRNA promoters, the 5s ribosomal RNA promoters, as well as functional hybrids and combinations of any of these promoters.
  • U6 promoters U6- 1, U6-8, U6-9, e.g.
  • HI promoter 7SL promoter
  • human Y promoters hYl , hY3, hY4 (
  • the DNA sequence operably linked to the promoter starts with a guanine (G) base; when a HI promoter is used, it is preferable that the DNA sequence operably linked to the promoter starts with an adenine (A) base.
  • G guanine
  • A adenine
  • promoters that allow for inducible expression of the multiple ddRNAi agents expressed from the ddRNAi construct.
  • a number of systems for inducible expression using such promoters are known in the art, including but not limited to the tetracycline responsive system and the lac operator-repressor system (see WO 03/022052 Al Publication; and U.S.
  • Patent Publication 2002/0162126 Al the ecdysone regulated system, or promoters regulated by glucocorticoids, progestins, estrogen, RU-486, steroids, thyroid hormones, cyclic AMP, cytokines, the calciferol family of regulators, or the metallothionein promoter (regulated by inorganic metals).
  • Promoters useful in some embodiments of the present invention may be tissue-specific or cell-specific.
  • tissue-specific refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest to a specific type of tissue in the relative absence of expression of the same nucleotide sequence of interest in a different type of tissue (e.g., brain).
  • tissue-specific as applied to a promoter refers to a promoter which is capable of directing selective expression of a nucleotide sequence of interest in a specific type of cell in the relative absence of expression of the same nucleotide sequence of interest in a different type of cell within the same tissue.
  • cell-specific when applied to a promoter also means a promoter capable of promoting selective expression of a nucleotide sequence of interest in a region within a single tissue.
  • promoters may be constitutive or regulatable. Additionally, promoters may be modified so as to possess different specificities.
  • liver specific promoters may be utilised.
  • liver specific promoters are human Alpha Antitrypsin promoter (hAAT) apolipoprotein H (ApoH) and Lecithin
  • the transthyretin or TTR promoter may be utilised.
  • the TTR promoter is derived from the mouse prealbumin gene and is also referred to as prealbumin.
  • the full length TTR promoter normally controls expression of the serum thyroxine-binding protein, which is made by hepatocytes and by the choroid plexus epithelium in adults.
  • a preferred TTR promoter is a TTR promoter in which the region which controls choroids plexus expression is deleted, but retains the regions which drive liver- specific expression. See for example WO2007/120533 and US20060189561, incorporated herein by reference.
  • enhancer elements are optionally included in the ddRNAi constructs of the invention.
  • One preferred enhancer element is ApoE.
  • the ApoE enhancer element consists of approximately 155bp derived from apolipoprotein E (ApoE). ApoE mediates binding, internalization and catabolism of lipoprotein particles and is a ligand for the low-density lipoprotein (ApoB/E) receptor and for the ApoE receptor of hepatic tissues.
  • the genetic enhancer associated with the ApoE gene is a eukaryotic control element that can increase transcription of a nucleic acid specifically in the liver.
  • the ApoE enhancer may be located up to 2000 nucleotides upstream or downstream of a liver specific promoter, and may be present in more than one copy.
  • An ApoE/hAAT is one preferred enhancer/promoter combination.
  • SynEnh a synthetic enhancer
  • the terminator sequences or elements may be the same, or different, or there may be a combination of termination elements represented only once and termination elements represented two times or more within any cassette. Whatever terminator sequences or elements are used they should be selected to ensure that they work appropriately with the liver-specific promoter used. In instances where Pol I, Pol III or Pol III promoters are used, appropriate terminator sequences should be employed.
  • Termination elements useful in the present invention include the Ul termination sequence (Ul box), the synthetic polyA terminator, and the so called minimal PolyA terminator.
  • Transcriptional pause sites such as MAZ1 and MAZ2, (See Ashfield et al EMBO J 1994 Vol 13 No 23 5656 pp and Yonaha and Proudfoot EMBO J. 2000 Jul. 17;19(14):3770-7) may be inserted upstream of the polyA terminators to assist in coupling of transcription termination and polyadenylation.
  • the sequences TTTT, TTTTT or ⁇ are commonly used as terminators. In these instances transcripts are typically terminated by the sequence UU.
  • a challenge in developing any HBV therapeutic is that virtually all hepatocytes in the patients are infected.
  • a means of achieving efficient and uniform transduction of all liver cells with the ddRNAi agent of the invention is required to provide an effective gene therapy for chronic HBV infection.
  • the ddRNAi expression cassette of the invention has to be able to be transfected into primary cells, stem cells, and non-dividing cells.
  • the ddRNAi expression cassettes of the invention are introduced into a delivery vector, preferably derived from viruses to ensure compatibility with viral delivery, to generate ddRNAi constructs.
  • the vector backbone may serve the dual purpose of being an expression vector as well as a delivery vector.
  • Generation of the construct can be accomplished using any suitable genetic engineering techniques well known in the art, including without limitation, the standard techniques of PCR, oligonucleotide synthesis, DNA synthesis, restriction endonuclease digestion, ligation, transformation, plasmid purification, and DNA sequencing.
  • the construct preferably comprises, for example, sequences necessary to package the ddR Ai construct into viral particles and/or sequences that allow integration of the ddRNAi construct into the target cell genome.
  • the viral construct also may contain genes that allow for replication and propagation of virus, though in preferred embodiments such genes will be supplied in trans.
  • the ddRNAi construct may contain genes or genetic sequences from the genome of any known organism incorporated in native form or modified.
  • the preferred viral construct comprises sequences useful for replication of the construct in bacteria.
  • the viral vector backbone may be selected from lentiviral, adenoviral (Adv), and adeno-associated viral (AAV) vectors.
  • Non-integrating viral vectors may be used for transient expression in dividing cells of ddRNAi agents of the invention or for longer term stable expression in non-dividing cells.
  • Integrating viral vectors, such as lentiviral vectors mediate stable, long term expression in both dividing and non- dividing cells.
  • minicircles such as those described in US20040214329 may be used to deliver the ddRNAi expression cassettes.
  • Minicircles provide for persistently high levels of nucleic acid transcription, and are characterised by being devoid of expression-silencing bacterial sequences.
  • AAV vectors are non-pathogenic and less immunogenic compared with other viral vectors.
  • the ability of AAV vectors to infect both dividing and non-dividing cells, and to direct long-term gene expression in these tissues makes it a useful vehicle for gene therapy.
  • AAV has a variety of pseudotypes with different tissue tropisms.
  • a preferred AAV vector is the double stranded AAV pseudotype 8 (dsAAV8).
  • the genome of AAV contains only two genes.
  • the "rep” gene codes for at least four separate proteins utilized in DNA replication.
  • the “cap” gene product is spliced differentially to generate the three proteins that comprise the capsid of the virus.
  • ITRs Inverted Terminal Repeats
  • rep and cap can be deleted from the genome and be replaced with heterologous sequences of choice.
  • the rep and cap proteins must be provided in trans.
  • helper functions normally provided by co-infection with the helper virus can also be provided in trans in the form of one or more DNA expression plasmids. Since the genome normally encodes only two genes it is not surprising that, as a delivery vehicle, AAV is limited by a packaging capacity of 4.5 single stranded kilobases (kb). However, although this size restriction may limit the genes that can be delivered for replacement gene therapies, it does not adversely affect the packaging and expression of shorter sequences such as ddRNAi vectors.
  • a ddRNAi construct comprising a viral vector into which a ddRNAi expression cassette according to the invention is inserted.
  • the expression cassette encodes for multiple RNAi agents, as either long hairpin structures or multiple hairpin structures.
  • the viral vector is an AAV vector.
  • the construct is packaged into viral particles.
  • Any method known in the art may be used to produce infectious viral particles whose genome comprises a copy of the viral ddRNAi construct.
  • One method utilizes packaging cells that stably express in trans the viral proteins that are required for the incorporation of the viral ddRNAi construct into viral particles, as well as other sequences necessary or preferred for a particular viral delivery system (for example, sequences needed for replication, structural proteins and viral assembly) and either viral-derived or artificial ligands for tissue entry.
  • the packaging cells then replicate viral sequences, express viral proteins and package the ddRNAi expression constructs into infectious viral particles.
  • the packaging cell line may be any cell line that is capable of expressing viral proteins, including but not limited to 293, HeLa, A549, PerC6, D17, MDCK, BHK, bing cherry, phoenix, Cf2Th, or any other line known to or developed by those skilled in the art.
  • One packaging cell line is described, for example, in U.S. Pat. No. 6,218, 181.
  • a cell line that does not stably express necessary viral proteins may be co-transfected with one or more constructs to achieve efficient production of functional particles.
  • One of the constructs is the viral based ddRNAi construct; the other construct comprises nucleic acids encoding the proteins necessary to allow the cells to produce functional virus as well as other helper functions.
  • the packaging cell line or replication and packaging construct may not express envelope gene products.
  • the gene encoding the envelope gene can be provided on a separate construct that is co-transfected with the viral based ddRNAi construct.
  • the viruses may be pseudotyped.
  • a "pseudotyped" virus is a viral particle having an envelope protein that is from a virus other than the virus from which the genome is derived.
  • One with skill in the art can choose an appropriate pseudotype for the viral delivery system used and cell to be targeted. In addition to conferring a specific host range, a chosen pseudotype may permit the virus to be concentrated to a very high titer.
  • Viruses alternatively can be pseudotyped with ecotropic envelope proteins that limit infection to a specific species (e.g., ecotropic envelopes allow infection of, e.g., murine cells only, where amphotropic envelopes allow infection of, e.g., both human and murine cells).
  • genetically-modified ligands can be used for cell- specific targeting, such as the asialoglycoprotein for hepatocytes, or transferrin for receptor-mediated binding.
  • the viral particles containing the ddRNAi expression cassettes are purified and quantified (titred). Purification strategies include density gradient centrifugation, or, preferably, column chromatographic methods.
  • ddRNAi agents ddRNAi constructs or siRNA agents of the invention inhibit expression of HBV genes expressed in a cell infected with HBV.
  • a method of treating HBV infection in an individual comprising providing a therapeutically effective amount of a ddRNAi agent to a patient in need of treatment, wherein the ddRNAi agent inhibits expression of one or more target sequences in a Hepatitis B virus (HBV) gene, preferably the polymerase gene of HBV.
  • HBV Hepatitis B virus
  • the ddRNAi agent to be administered to the patient may be one or more of:
  • a ddRNAi agent comprising a first effector sequence; and a first effector complement sequence; wherein the effector sequence is substantially complementary to the predicted transcript of a region of the target gene
  • ddRNAi agent comprising, in a 5' to 3' direction, a first effector sequence; a second effector sequence; a second effector complement sequence; and a first effector complement sequence, wherein each effector sequence is substantially complementary to the predicted transcript of a region of the target gene
  • a ddRNAi agent comprising, in a 5' to 3' direction, a first effector sequence; a second effector sequence; a third effector sequence; a third effector complement sequence; a second effector complement sequence; and a first effector complement sequence wherein each effector sequence is substantially complementary to the predicted transcript of a region of the target gene
  • a ddRNAi agent comprising, in a 5' to 3' direction, a first effector sequence; a first effector complement sequence; a second effector sequence; and a second effector complement sequence wherein each effector sequence is substantially complementary to the predicted transcript of a region of the target gene
  • a ddRNAi agent comprising, in a 5' to 3' direction, a first effector sequence; a first effector complement sequence; a second effector sequence; a second effector complement sequence; a third effector sequence; and a third effector complement sequence; wherein each effector sequence is substantially complementary to the predicted transcript of a region of the target gene
  • a ddRNAi agent comprising, in a 5' to 3' direction, a first effector sequence; a second effector sequence; a sequence of 2 to 100 non-self-complementary nucleotides; a second effector complement sequence; and a first effector complement sequence wherein each effector sequence is substantially complementary to the predicted transcript of a region of the target gene
  • a ddRNAi agent comprising, in a 5' to 3' direction, a first effector sequence; a sequence of 2 to 100 non-self-complementary nucleotides; a first effector complement sequence; a second effector sequence; a sequence of 2 to 100 non-self-complementary nucleotides; and a second effector complement sequence wherein each effector sequence is substantially complementary to the predicted transcript of a region of the target gene.
  • any particular effector sequence may be swapped in position with its complement in the agent.
  • each effector sequence is at least 17 nucleotides in length selected from the group consisting of any 10 or more and preferably any 17 or more contiguous nucleotides within a sequence from any one of SEQ ID NOS: 1-19.
  • the effector sequences may all be the same, or may all be different, or may be a combination ie 2 effector sequences of at least 10 contiguous nucleotides of SEQ ID NO: 1 and 1 effector sequence of at least 10 contiguous nucleotides of SEQ ID NO: 4.
  • the effector sequence is selected from the group consisting of any contiguous 1 1, 12, 13, 14, 15 or 16 nucleotides within any one of SEQ ID NOS: 1-19, and most preferably 17 or more contiguous nucleotides within any one of SEQ ID NOS: 1-19.
  • the effector complement will be the same length, or about the same length (ie ⁇ 15% nucleotide length) as its corresponding effector sequence.
  • Each of these ddRNAi agents may be administered via a ddRNAi expression cassette in a ddRNAi construct, as described in the earlier sections of the specification. Multiple targeting may be achieved by delivering two or more ddRNAi expression cassettes or constructs each capable of expressing a single ddRNAi agent, or alternatively, by delivering one or more ddRNAi expression cassettes or constructs each capable of expressing more than one ddRNAi agent.
  • each of the effector sequences may be 100% complementary to the predicted transcript of a region of the target gene, or may only vary by 1, 2, 3, 4 or 5 nucleotides.
  • the method of treating HBV infection can optionally include a preliminary step of identifying an individual having HBV infection including identification of the serotype of the HBV isolate.
  • the ddRNAi agent is provided via a ddRNAi construct of the invention ie in vivo expression of the ddRNAi agent from a ddRNAi expression cassette inserted into a suitable vector delivered to the cell.
  • the ddRNAi expression cassette comprises:
  • these components of the ddRNAi expression cassette may have different 5' to 3' arrangements, all of which are suitable for use in the methods of the invention.
  • the HBV target gene is at least the polymerase (P) gene, and potentially the surface antigen or X gene when the target sequence is contained within overlapping open reading frames
  • the ddRNAi agent comprises ddRNAi effector sequences of any 10 or more contiguous nucleotides within a sequence from SEQ ID NOS: 1-19 listed in Table 1.
  • the sequence that encodes for the effector sequence or the sequence complementary to the first effector sequence may vary from SEQ ID NOS: l-19by 1, 2, 3, 4 or 5 nucleotides without affecting the ability of the sequence encoded to base pair with the target sequence and inhibit expression of the HBV target sequence.
  • each effector sequence forms a double stranded region with the corresponding effector complement sequence.
  • the method of treating HBV infection in an individual comprises the administration of a therapeutically effective amount of a ddRNAi construct that encodes a ddRNAi agent having more than one effector sequence, such as those listed above.
  • the HBV infection to be treated may be a chronic HBV infection.
  • chronic HBV infection it is meant that the infection with HBV is long-lasting or persistent.
  • chronic describes the course of the disease, or its rate of onset and development.
  • the ddR Ai construct is administered and either stably maintained or integrated in to the target cell to ensure longer term expression of the ddRNAi agent. As detailed above, this can be achieved with the use of a ddRNAi construct having a viral vector backbone.
  • a method of treating chronic HBV infection in an individual comprising the administration of a therapeutically effective amount of a ddRNAi construct of the invention to a patient in need of treatment.
  • Treatment of chronic HBV infection is aimed at reducing the infectivity of the virus by interfering with viral replication. This in turn lowers the risk of transmission and spread of HBV.
  • a method of reducing the infectivity of HBV in an individual infected with HBV comprising administering to the individual a ddRNAi constructs of the invention to target a HBV gene, preferably the polymerase gene.
  • the clinical endpoints used in chronic hepatitis B therapy will differ between patients with compensated hepatitis B (where sustained viral suppression, serological response, histological improvement, normalisation of liver function tests and non-clinical progression are key endpoints) and those with decompensated hepatitis B.
  • Endpoints for compensated hepatitis B are reduction of viral load with serological and biochemical improvement, histological improvement, measure of liver failure and end stage liver disease, complications, transplantation and mortality.
  • a method of minimising progression of liver disease as a result of HBV infection comprising administering to the individual a ddRNAi construct of the invention to target a HBV gene, preferably the polymerase gene.
  • a method of minimising the symptoms associated with HBV infection comprising administering to the individual a ddRNAi construct of the invention to target a HBV gene, preferably the polymerase gene.
  • the status or severity of an individual's HBV infection may be assessed by determining their viral load.
  • viral load it is meant the amount of virus in an involved body fluid. For example, it can be given in RNA copies per millilitre of blood plasma. Individuals having a high viral load are considered to have a more severe HBV infection.
  • the viral load is an indicator of the responsiveness of an individual to a particular treatment. If a treatment is working and keeping the levels of virus low, it is indicative of a successful treatment. It is therefore an objective of treatment to lower an individual's viral load. Accordingly, there is also a provided a method of lowering a viral load of an individual with an HBV infection, comprising administering to the individual a ddRNAi construct of the invention to target a HBV gene, preferably the polymerase gene.
  • Subsequent monitoring of the viral load in the individual who has received treatment with a ddRNAi construct of the invention is therefore a phenotypic indicator of the effectiveness of the ddRNAi construct and the ddRNAi agent of the invention.
  • treatment of acute HBV infection may not require long term treatment, and it may in fact be preferred to rely on the transient presence of a ddRNAi agent or siRNA agent as opposed to long term expression of ddRNAi agents from integrated or stably maintained ddRNAi constructs.
  • acute it is meant that the HBV infection has a rapid onset, and/or a short duration.
  • a method of treating acute HBV infection in an individual comprising the administration of a therapeutically effective amount of an in vitro synthesised or chemically synthesised ddRNAi agent to a patient in need of treatment, for inhibiting expression of one or more target sequences in a Hepatitis B virus (HBV) gene, the ddRNAi agent comprising at least:
  • a first effector sequence of at least 17 nucleotides in length selected from any 10 or more contiguous nucleotides within a sequence from the group consisting of SEQ ID NOS: 1-19;
  • the effector sequence is substantially complementary to the predicted transcript of a region of the target gene.
  • the ddRNAi agent of the invention is produced in vitro or chemically synthesised and provided to the cell. Any of the siRNA agents or ddRNAi agents of the invention described throughout the specification are suitable for in vitro expression and delivery to the cell.
  • the HBV target gene is at least the polymerase (P) gene, and potentially the surface antigen, core antigen or X gene when the target sequence is contained within overlapping open reading frames.
  • the ddRNAi agent inhibits expression of one or more target sequences in a Hepatitis B virus (HBV) polymerase gene.
  • the first effector sequence is preferably selected from any 10 or more and preferably any 17 or more contiguous nucleotides within a sequence from the effector sequences SEQ ID NOS: 1- 19 listed in Table 1.
  • the effector sequence may vary from SEQ ID NOS: 1-19 by 1, 2, 3, 4 or 5 nucleotides without affecting the ability of the effector sequence to base pair with the target sequence and inhibit expression of the HBV polymerase gene.
  • an siRNA agent may be administered.
  • the siRNA agents similarly to the ddRNAi agent, targets the HBV polymerase gene, and may have a sequence selected from any 10 or more and preferably any 17 or more contiguous nucleotides within a sequence from the group consisting of SEQ ID NOS: 1-19, or may vary from SEQ ID NOS: 1-19 by 1, 2, 3, 4 or 5 nucleotides without affecting the ability of the effector sequence to base pair with the target sequence and inhibit expression of the HBV polymerase gene.
  • Administration of a ddRNAi agent or siRNA agent of the invention to an individual with an acute HBV infection can also lower a viral load, reduce the severity of symptoms associated with the acute infection, and reduce the infectivity of HBV.
  • ddRNAi constructs, ddRNAi agents or siRNA agents of the invention in the preparation of medicaments for treatment of HBV infection, preferably chronic HBV infection, the reduction of HBV viral load, the reduction of the severity of symptoms associated with HBV infection, and the reduction of the infectivity of HBV.
  • ddRNAi constructs preferably chronic HBV infection, reducing HBV viral load, reducing the severity of symptoms associated with HBV infection, and reducing the infectivity of HBV.
  • composition comprising ddRNAi constructs, ddRNAi agents or siRNA agents as an active ingredient for treating HBV infection, preferably chronic HBV infection, reducing HBV viral load, reducing the severity of symptoms associated with HBV infection, and reducing the infectivity of HBV.
  • ddRNAi agents, the siRNA agents or the vectors comprising ddRNAi expression cassettes of the invention can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents. Accordingly, there is provided a pharmaceutical composition comprising a ddRNAi agent, a ddRNAi expression cassette, a ddRNAi construct or a siRNA agent of the invention and a pharmaceutically acceptable carrier or diluent.
  • the agents or the vectors comprising the ddRNAi expression cassettes may be administered alone or in association or combination with other pharmaceutically active compounds.
  • dose levels for agents or vectors comprising the ddRNAi expression cassettes will vary as a function of the nature of the delivery vehicle, the relative ease of transduction of the target cells, the expression level of the RNAi agents in the target cells and the like.
  • the ddRNAi agents, the siRNA agents or the vectors comprising ddRNAi expression cassettes of the invention can be formulated into preparations for injection or administration by dissolving, suspending or emulsifying them in an aqueous or non aqueous solvent, such as oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilisers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
  • an aqueous or non aqueous solvent such as oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol
  • conventional additives such as solubilisers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
  • Pharmaceutically acceptable carriers or diluents contemplated by the invention include any diluents, carriers, excipients, and stabilizers that are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and tricresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as plasma albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidon
  • the pharmaceutical composition may be prepared for various routes and types of administration.
  • the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and if necessary, shaping the product.
  • Formulation may be conducted by mixing at ambient temperature at the appropriate pH, and at the desired degree of purity, with physiologically acceptable carriers, i.e., carriers that are non-toxic to recipients at the dosages and concentrations employed.
  • HBV RNA-dependent DNA polymerase gene from HBV Adr- 1 was sub-cloned and used to generate an Entire siRNA Target (EsT) library.
  • EsT Entire siRNA Target
  • 5000 clones were sequenced, of which 642 were identified as having non-repeat sequences ranging from 19-23bp.
  • the sequences, which represent potential targets for the ddRNAi agents of the invention, were distributed along the target gene ( Figure 2).
  • Example 2 Large scale screening results using SECs with > 50% knock down
  • siRNA expression cassettes amplified by PCR were transfected into HepG2 2.2.15 cells. Effects on the levels of HBV polymerase mRNA expression was evaluated to identify functional siRNA sequences.
  • siRNAs that corresponded to the first SECs screened were chemically synthesised and transfected into HepG2 2.2.15 cells to validate the original polymerase mRNA knock down levels.
  • the correlation results are shown in Fig 3.
  • Example 3 Target screening by siRNA expression cassettes (SECs).
  • Figure 4 shows the results of large scale screening in vitro for inhibition of HBV mRNA accumulation (501 non- repeated siRNA agents). Of those sequences screened, 100 siRNA sequences were effective in knocking down HBV mRNA by >50%, 14 of which resulted in >70% knock down (Table 1). The distribution of the top 100 siRNA targets on the HBV polymerase gene is shown in Figure 5A; the top 3 sequences are shown in Figure 5B.
  • ddRNAi expression cassettes of the invention One useful way of designing ddRNAi expression cassettes of the invention is to assume Dicer cuts every 22 nucleotides (also referred to as 22nt phasing).
  • the DNA sequences that encode effector sequences can therefore be designed to encode any 10 or more, and preferably any 17 or more contiguous nucleotides within a sequence from the group consisting of SEQ ID NOS: l-19, together with appropriate spacers and other sequence requirements for the promoter and/or terminator.
  • ddRNAi constructs will be generated with the following structures:
  • Figure 6 illustrates the expression cassette, expressed hairpin RNAi agent and theoretical effector sequence processed by Dicer (assuming 22 nucleotide phasing) based on SEQ ID NO: 1 ( Figure 6A), SEQ ID NO:4 ( Figure 6B) and SEQ ID NO:6 ( Figure 6C).
  • Figure 6A 17 contiguous nucleotides of SEQ ID NO: l are encoded for; in the cassette of Figure 6B, 20 contiguous nucleotides of SEQ ID NO:4 are encoded for; and in the cassette of Figure 6C, 20 contiguous nucleotides of SEQ ID NO:6 are encoded for.
  • the arrows on the hairpin RNAi agent indicate where Dicer is expected to cut to produce the effector sequence shown underneath.
  • the expression cassette of Figure 6A has a DNA sequence of
  • the expression cassette of Figure 6B has a DNA sequence of
  • the expression cassette of Figure 6C has a DNA sequence of TACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGT TTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATAT AACACCGCCCCACCGCACGGAGGCCTTCAAGAGAAAGGCCTCC
  • Figure 7 illustrates the expression cassette, expressed hairpin RNAi agent and theoretical effector sequence processed by Dicer (assuming 22 nucleotide phasing) based on SEQ ID NO: 1, SEQ ID NO:4 and SEQ ID NO: 6 when arranged within the cassette to give rise to expression of individual hairpin RNAi agents ( Figure 7A) or a single RNA containing the 3 hairpin RNAi agents prior to processing ( Figure 7B).
  • SEQ ID NOS: l, 4 and 6 are the same as described above in relation to the single hairpin expression cassette.
  • the arrows on the hairpin RNAi agent indicate where Dicer is expected to cut to produce the effector sequence shown underneath.
  • the expression cassette of Figure 7A has a DNA sequence of TACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGT TTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATAT ATCTTGTGGAAAGGACGAAACACCGTCTCCCTTATCGTCAATCTTCAAGAGAAAGATTGACG
  • each effector complement is operably linked to a promoter sequence, and the corresponding effector sequence is operably linked to termination elements or sequences.
  • the first effector complement (effector 1 comp) is operably linked to a promoter and the last effector (effector 6) is operably linked to a terminator element or sequence.
  • This cassette is designed to express a single RNA molecule, and has a DNA sequence of TACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGT TTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATAT ATCTTGTGGAAAGGACGAAACACCGTGCTGTTGACAGTGAGCGTCTCCCTTATCGTCAATCT TCAAGAGAAAGATTGACGATAAGGGAGATGCCTACTGCCTCGGATTCTGCTGTTGACAGTGA GCGGTTGTCCTCTCCCGCAAATACAAGATATTTGCGGGAGGACAACTGCCTACTGCCT GGAGGCCTTCAAGAGAAAGAAAGGCCTCC
  • Figure 8 illustrates the expression cassette, expressed hairpin R Ai agent and theoretical effector sequence processed by Dicer (assuming 22 nucleotide phasing) based on (in order) SEQ ID NO: 1, SEQ ID NO:6 and SEQ ID NO:4.
  • the cassette of Figure 8 17 contiguous nucleotides of SEQ ID NO: l are encoded for and 20 contiguous nucleotides of SEQ ID NO: 6 as per the above examples, but in contrast to the cassettes of Figures 7 and 8, only 18 contiguous nucleotides of SEQ ID NO:4 are encoded for.
  • the arrows on the hairpin RNAi agent indicate where Dicer is expected to cut to produce the effector sequence shown underneath.
  • the expression cassette of Figure 8 has a DNA sequence of
  • Example 5 Assaying the effectiveness of ddRNAi agents in vitro
  • RNA agents of the invention can be transfected with RNA agents of the invention and inhibition of HBV replication assayed using techniques such as qRT PCR to determine HBV polymerase mRNA levels.
  • the ddRNAi agents would be expected to reduce levels of HBV polymerase mRNA, when compared to control cells transfected with control RNAs (such as RNAs with scrambled sequences) or non-treated Hep2 2.2.15 cells.
  • plasmid DNAs will be transfected into cells along with a suitable selectable marker genes to obtain stably transformed HepG2 2.2.15 cells. Inhibition of HBV replication will be monitored using techniques such as qRT PCR as described above. A reduction in HBV polymerase mRNA levels compared to scrambled controls or non-transformed cells would be indicative of an inhibition of HBV replication.
  • Example 6 Assaying the effectiveness of ddRNAi agents in vivo
  • HBV infected NOD/SCID mice Yang et al. 2002; Ketzinel-Gilad et al. 2006
  • transgenic mouse lines expressing HBV either or both of which could be used for these experiments.
  • Hydrodynamic injection of ddRNAi agents into mouse tail veins will be used to deliver the agents to the liver.
  • Inhibition of HBV replication can be monitored at various time points using qRT-PCR assays to determine the levels of HBV pol mRNA in liver tissues or by quantifying levels of circulating HBsAg and HBeAg in animals treated with ddRNAi agents of the invention compared to appropriate contro animals such as mice injected with scrambled sequences or irrelevant DNAs, as described above.
  • Example 7 In vivo testing of the pre-clinical candidate in a normal mouse model
  • AAV One delivery system envisaged in vivo testing of the pre-clinical candidate in a normal mouse model is AAV.
  • the ddRNAi constructs will be packaged in vitro using strategies well known to those familiar with the art and injected intravenously into HBV infected NOD/SCID mice and/or HBV transgenic mice. Inhibition of HBV replication will be monitored as described above.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Virology (AREA)
  • Wood Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biochemistry (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne un agent d'interférence d'ARN (ARNi) et l'utilisation de cet agent d'ARNi pour le traitement d'une infection d'hépatite B chez des individus, ainsi que des compositions pharmaceutiques contenant l'agent d'ARNi de l'invention.
PCT/CN2011/071107 2010-10-28 2011-02-18 Traitement du vhb WO2012109798A1 (fr)

Priority Applications (24)

Application Number Priority Date Filing Date Title
PCT/CN2011/071107 WO2012109798A1 (fr) 2011-02-18 2011-02-18 Traitement du vhb
HUE16188642 HUE044426T2 (hu) 2010-10-28 2011-10-27 HBV kezelés
AU2011320437A AU2011320437B2 (en) 2010-10-28 2011-10-27 HBV treatment
PL16188642T PL3124610T3 (pl) 2010-10-28 2011-10-27 Leczenie hbv
DK16188642.9T DK3124610T3 (da) 2010-10-28 2011-10-27 Hbv-behandling
PCT/CN2011/081386 WO2012055362A1 (fr) 2010-10-28 2011-10-27 Traitement d'une infection par le vhb
RS20190704A RS58982B1 (sr) 2010-10-28 2011-10-27 Lečenje hbv
SI201131733T SI3124610T1 (sl) 2010-10-28 2011-10-27 Zdravljenje HBV
PT16188642T PT3124610T (pt) 2010-10-28 2011-10-27 Tratamento do vhb
BR112013010525A BR112013010525A2 (pt) 2010-10-28 2011-10-27 tratamento de hbv
TR2019/08127T TR201908127T4 (tr) 2010-10-28 2011-10-27 Hbv tedavisi.
KR1020187027763A KR20180110186A (ko) 2010-10-28 2011-10-27 Hbv 치료
CN201180062884.8A CN103370415B (zh) 2010-10-28 2011-10-27 Hbv治疗
EP16188642.9A EP3124610B1 (fr) 2010-10-28 2011-10-27 Traitement de l'hépatite b (hbv)
KR1020137013563A KR101903778B1 (ko) 2010-10-28 2011-10-27 Hbv 치료
EP11835635.1A EP2633051B1 (fr) 2010-10-28 2011-10-27 Traitement d'une infection par le vhb
ES16188642T ES2730393T3 (es) 2010-10-28 2011-10-27 Tratamiento contra el VHB
US13/882,124 US9080174B2 (en) 2010-10-28 2011-10-27 HBV treatment
LTEP16188642.9T LT3124610T (lt) 2010-10-28 2011-10-27 Hbv gydymas
RU2013124422A RU2620966C2 (ru) 2010-10-28 2011-10-27 Лечение hbv инфекции
CA2853613A CA2853613C (fr) 2010-10-28 2011-10-27 Traitement d'une infection par le vhb
US14/731,824 US9410154B2 (en) 2010-10-28 2015-06-05 HBV treatment
HRP20190995TT HRP20190995T1 (hr) 2010-10-28 2019-05-31 LIJEČENJE HBV-a
CY20191100583T CY1121894T1 (el) 2010-10-28 2019-06-03 Θεραπεια hbv

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2011/071107 WO2012109798A1 (fr) 2011-02-18 2011-02-18 Traitement du vhb

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US13/882,124 Continuation US9080174B2 (en) 2010-10-28 2011-10-27 HBV treatment
PCT/CN2011/081386 Continuation WO2012055362A1 (fr) 2010-10-28 2011-10-27 Traitement d'une infection par le vhb

Publications (1)

Publication Number Publication Date
WO2012109798A1 true WO2012109798A1 (fr) 2012-08-23

Family

ID=46671928

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2011/071107 WO2012109798A1 (fr) 2010-10-28 2011-02-18 Traitement du vhb

Country Status (1)

Country Link
WO (1) WO2012109798A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2018515136A (ja) * 2015-05-06 2018-06-14 ベニテック バイオファーマ リミテッド Ｂ型肝炎ウイルス（ｈｂｖ）感染を治療するための試薬及びその使用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1733916A (zh) * 2005-07-01 2006-02-15 中国人民解放军第三军医大学 抑制Stat3基因表达的siRNA及其制备方法
WO2006047394A1 (fr) * 2004-10-22 2006-05-04 Benitec, Inc. Agents arni therapeutiques pour traiter le psoriasis
WO2006116756A1 (fr) * 2005-04-28 2006-11-02 Benitec, Limited. Cassettes d'expression d'arni multiples en vue de la delivrance simultanee d'agents d'arni associes a des motifs d'expression heterozygotes

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006047394A1 (fr) * 2004-10-22 2006-05-04 Benitec, Inc. Agents arni therapeutiques pour traiter le psoriasis
WO2006116756A1 (fr) * 2005-04-28 2006-11-02 Benitec, Limited. Cassettes d'expression d'arni multiples en vue de la delivrance simultanee d'agents d'arni associes a des motifs d'expression heterozygotes
CN1733916A (zh) * 2005-07-01 2006-02-15 中国人民解放军第三军医大学 抑制Stat3基因表达的siRNA及其制备方法

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2018515136A (ja) * 2015-05-06 2018-06-14 ベニテック バイオファーマ リミテッド Ｂ型肝炎ウイルス（ｈｂｖ）感染を治療するための試薬及びその使用
EP3292202A4 (fr) * 2015-05-06 2019-04-10 Benitec Biopharma Limited Réactifs pour le traitement de l'infection par le virus de l'hépatite b (vhb) et utilisation de ceux-ci
US10898505B2 (en) 2015-05-06 2021-01-26 Benitec Biopharma Limited Reagents for treatment of hepatitis B virus (HBV) infection and use thereof
AU2016257150B2 (en) * 2015-05-06 2022-03-31 Benitec IP Holdings Inc. Reagents for treatment of hepatitis B virus (HBV) infection and use thereof
JP7191690B2 (ja) 2015-05-06 2022-12-19 ベニテック バイオファーマ リミテッド Ｂ型肝炎ウイルス（ｈｂｖ）感染を治療するための試薬及びその使用

Similar Documents

Publication Publication Date Title
US9410154B2 (en) HBV treatment
US10093927B2 (en) Reduction of off-target RNA interference toxicity
US20180320180A1 (en) Age-related macular degeneration treatment
US9523093B2 (en) Huntington's disease therapeutic compounds
AU2005222084B2 (en) Multiple promoter expression cassettes for simultaneous delivery of RNAI agents
US9790502B2 (en) HBV treatment
WO2012109798A1 (fr) Traitement du vhb

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11858997

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11858997

Country of ref document: EP

Kind code of ref document: A1