WO2012045894A1 - Compounds and compositions for the treatment of illnesses caused by the hepatitis b virus - Google Patents

Compounds and compositions for the treatment of illnesses caused by the hepatitis b virus Download PDF

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WO2012045894A1
WO2012045894A1 PCT/ES2010/070643 ES2010070643W WO2012045894A1 WO 2012045894 A1 WO2012045894 A1 WO 2012045894A1 ES 2010070643 W ES2010070643 W ES 2010070643W WO 2012045894 A1 WO2012045894 A1 WO 2012045894A1
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seq
binds
snrna
target sequence
modified
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PCT/ES2010/070643
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French (fr)
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Lorea BLÁZQUEZ GARCÍA
María Purificación FORTES ALONSO
Sandra Jovanna GONZÁLEZ ROJAS
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Proyecto De Biomedicina Cima, S.L.
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Priority to PCT/ES2010/070643 priority Critical patent/WO2012045894A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • 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
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • 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
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/31—Combination therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2330/00—Production
    • C12N2330/50—Biochemical production, i.e. in a transformed host cell
    • C12N2330/51—Specially adapted vectors

Definitions

  • the invention relates to therapeutic methods for inhibiting the gene expression of the hepatitis B virus and, therefore, with methods for the treatment of diseases associated with infection by said virus.
  • the invention relates to compounds and compositions that act by inhibiting maturation of hepatitis B virus mRNAs and by RNA-mediated interference of the expression of said mRNAs.
  • HBV Hepatitis B virus
  • CHC hepatocellular carcinoma
  • Interferon-alpha treatment is the therapy commonly used to treat HBV infection.
  • interferon-alpha is accompanied by a series of undesirable side effects such as flu-like symptoms (fatigue, fever, myalgia, etc.), neurosychiatric effects (irritability, apathy, etc.), reduction of myeloid cells such as granulocytes and platelets, increases in triglyceride levels, thyroid abnormalities, etc.
  • Lamivudine (3TC (R)) treatment has recently been approved, which is a nucleoside analog that inhibits the synthesis of HBV DNA.
  • discontinuation of lamivudine treatment results in a reactivation of HBV replication in most patients
  • some cases of resistance to this drug have been identified in some patients.
  • Hepatitis B virus is a double-stranded circular DNA virus that encodes polymerase, protein X, core antigen and surface antigens (S and preS).
  • the HBV genome is very compact and contains open reading frames that overlap each other, so the use of expression silencing agents directed against a particular region of the HBV genome may be useful for inhibiting the expression of multiple HBV mRNAs.
  • the use of interfering RNAs directed against HBV mRNAs to inhibit HBV replication is known (Arbuthnot et al; Liver International. 2005; 25: 9-15; Chen et al; Pharm Res. 2008; 25: 72- 86 and Stein and Loomba; I infected Disord Drug Targets. 2009; 9: 105-106).
  • interfering RNAs to inhibit HBV replication has the disadvantage that it is an expensive treatment, that interfering RNAs have a relatively short serum half-life and that resistance to repeated administration of a single interfering RNA (Wu H. et al. Gastroenterology, 2005; 128: 708-16).
  • the invention relates to a compound capable of silencing the gene expression of hepatitis B virus selected from the group consisting of:
  • an Ul snRNA wherein the consensus consensus binding sequence GU of the 5 'end of the intron of said Ul snRNA is modified such that it specifically binds to a target sequence of at least one HBV mRNA so which is capable of inhibiting the maturation of said target mRNA and wherein said target sequence is selected from the group consisting of the sequences SEQ ID NO: 4-24;
  • an expression vector comprising a polynucleotide according to (ii), (iv) an interfering RNA specifically directed to a target sequence of at least one HBV mRNA that is capable of causing the silencing of said HBV mRNA wherein said target sequence is selected from the group consisting of the sequences SEQ ID NO: 29 a 35;
  • the invention relates to a composition or a kit comprising in one or several containers:
  • an expression vector comprising a polynucleotide according to (v).
  • the invention relates to a polynucleotide capable of silencing the gene expression of the hepatitis B virus comprising
  • the invention relates to a compound, a composition or kit, a polynucleotide or a vector of the invention for use in medicine or for use in the prevention or treatment of diseases caused by a hepatitis virus infection.
  • pharmaceutical compositions comprising a compound, a composition or kit, a polynucleotide or a vector of the invention and a pharmaceutically acceptable carrier.
  • Figure 1 Schematic representation of the HBV genome, of mRNAs generated from the HBV genome and of the situation in the molecule of the target regions for Ul RNAs and inter-different RNAs of the invention.
  • FIG. 1 Inhibitory activity of HBV expression produced by shRNA inhibitors.
  • Inhibition Factor represents the number of times the expression is inhibited compared to the control (Mock).
  • Figure 3 Inhibitory activity of HBV expression produced by Ulin inhibitors.
  • FI Inhibition Factor; SD: Standard deviation. (1 ⁇ g of plasmid per 1.5 x 10 5 cells).
  • FIG. 4A-4C Inhibitory activity of HBV expression produced by different combinations of shRNA and Ulin inhibitors.
  • F.I. Inhibition Factor
  • S.D. Standard deviation. Note that half of each inhibitor is used and therefore the results cannot be compared with those obtained in Figures 3 and 4.
  • FIG. 5A-5B Inhibitory activity of HBV expression produced by different shRNA and Ulin inhibitors in the transfection model with pHBV-Luc in HuH7 cells.
  • FI Inhibition Factor
  • SD Standard deviation. Doses of inhibitor plasmid were the highest dose tested (1 mg), or half this dose (1 ⁇ 2D 0.5 ⁇ ⁇ ).
  • FI Inhibition Factor; SD: Standard deviation; SI: synergy index.
  • the doses of each inhibitor plasmid used were 0.5 ⁇ g (in the combinations 0.5 ⁇ g / plasmid).
  • FIG. 7A - 13 HBV expression inhibitory activity produced by different shRNA and Ulin inhibitors in the in vivo model of transfection with pHBV-Luc.
  • [Luc] Luciferase activity expressed as photons / s / cm 2 / sr (xlO 5 ). SD: Standard deviation. The doses of the inhibitor plasmid tested were 10 ⁇ g (in the combinations also 10 ⁇ g / plasmid).
  • the administration of said modified Ul snRNAs to an animal that has been administered a plasmid comprising the HBV genome modified by replacing the S antigen with the sequence of luciferase allows to reduce statistically significant activity luciferase in liver compared to animals to which the control plasmid has been administered.
  • the invention relates to a compound capable of silencing the hepatitis B virus gene expression wherein said compound is an Ul snRNA, wherein the binding sequence to the consensus consensus sequence GU of the 5 'end of the intron of said snRNA Ul is modified so that it specifically binds to a target sequence of at least one HBV mRNA so that it is capable of inhibiting the maturation of said target mRNA and wherein said target sequence is selected from the group consisting of in the sequences SEQ ID NO: 4-24;
  • snRNA Ul is understood as a small nuclear RNA (snRNA) which is capable of being incorporated into the small nuclear ribonucleoprotein (snRNP) Ul and that, Together with other snRNPs, it forms an organelle called spliceosome that is responsible for processing or helping pre-mRNAs to give rise to mature mRNAs.
  • snRNA small nuclear RNA
  • snRNP small nuclear ribonucleoprotein
  • Ul snRNA suitable for use in the present invention include Ul snRNAs that occur naturally in different organisms such as Uls of different mammals, including, without limitation, Ul snRNA 4 of human origin (accession number NR 004421.1 in NCBI), Ul snRNA 8 of human origin (access number NR 004402.2 in NCBI), Ul snRNA 7 of human origin (access number NR 004424.2 in NCBI), Ul snRNA 1 of human origin (access number NR 004430.2 in NCBI) ; Ul snRNA 2 of human origin (access number NR 004427.2 in NCBI), Ul snRNA 3 of human origin (access number NR 004408.1 in NCBI), Ul snRNA 5 of human origin (access number NR 004400.1 in NCBI) ; Ul snRNA 9 of human origin (access number NR 004426.2 in NCBI), U l snRNA 6 of human origin (access number NR_030723.1 in NCBI), sn
  • snRNA Ul includes, according to the present invention, Ul snRNAs whose sequence differs from the sequence of native snRNA Ul and which are generically named, variant snRNA Ul genes.
  • Non-limiting examples of such genes include those collected in the GenBank database with numbers of access L78810, AC025268, AC025264 and AL592207 and the variants described in Kyriakopoulou et al. (RA (2006) 12: 1603-11).
  • the Ul corresponds to the human Ul snR A 4 (Entrez Gene ID: 6060) which corresponds to the sequence SEQ.ID.NO.2.
  • snRNA Ul includes, according to the present invention, functionally equivalent variants of the snRNA Ul defined above and whose sequence derives from that of these by insertion, substitution or elimination of one or more nucleotides and which substantially retain the ability to incorporate into snRNP Ul and promote mRNA processing.
  • Suitable methods for determining the ability of an Ul snRNA to incorporate into the Ul snRNP and to promote mRNA processing include, for example, the method described by Hamm et al.
  • the functionally equivalent variants of the snRNA Ul that appear natively show a sequence identity of at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% in the entire sequence with respect to the native Ul snRNAs mentioned above.
  • Suitable algorithms for determining sequence identity are known in the state of the art, such as BLAST, described in Altschul et al. (1990) (J Mol Biol. Oct 5; 215 (3): 403-10).
  • functionally equivalent variants of Ul snRNAs include those that hybridize with native snRNA sequences under severe hybridization conditions.
  • very severe hybridization conditions or “sequence specific hybridization conditions”.
  • sequence specific hybridization conditions The person skilled in the art can empirically determine the stability of a duplex taking into account various variables, such as the length and concentration of the base pairs of the probes, the ionic strength and the incidence of the missing base pairs, following the guidelines of the prior art (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989); and Wetmur, Critical Reviews in Biochem. And Mol. Biol. 26 (3/4): 227-259 ( 1991)).
  • severe hybridization conditions comprise, for example, a concentration between 0.15 M and 0.9 M NaCl and a temperature between 20 ° C and 65 ° C.
  • very severe hybridization conditions comprise, for example, a concentration between 0.02 M and 0.15 M NaCl and a temperature between 50 ° C and 70 ° C.
  • binding sequence to the consensus consensus GU of the 5 'end of the intron is understood the region formed by the 5' end of the snRNA Ul that interacts by base pairing with the so-called Ul site or site 5 'of the processing (5 'splice sotti) that appears in the pre-mRNA in the 5' region of the introns, characterized by the consensus sequence AG / GURAGU (where R is a purine and / indicates the exon / intron limit) and that allows to the pre-mRNA processing machinery the location of the exon-intron boundary
  • This region corresponds to nucleotides at positions 1 to 1 1 at the 5 'end of the snRNA Ul sequence (Zhuang, Y and Weiner, AM: 1986 , Cell, 46: 827-835).
  • the consensus sequence of binding to the Ul site in the pre-mRNA contains a number of mutations that result in a substantial loss in the ability of snRNA Ul to bind to the Ul site in the pre- MRNA while said snRNA Ul acquires a new complementarity that allows it to form a duplex by base pairing with a preselected region in a pre-mRNA target.
  • the preselected sequences correspond to the sequences identified as SEQ ID NO: 4 to 24 and which correspond to regions of the HBV genome. These modified Ul snRNAs are called Ulin.
  • the degree of complementarity between the Ul site binding sequence in the modified snRNA according to the present invention and the region preselected in the target pre-mRNA can vary between 3 and 16 nucleotides, preferably between 8 and 16 nucleotides and, more preferably, between 10 and 1 1 nucleotides.
  • maximum efficacy in inhibiting the expression of the target gene is observed when the 10-1 1 nucleotides that form the Ul site have been modified to make them complementary to the pre-selected site in the target pre-mRNA. Therefore, preferably, the Ulin is modified in all those nucleotides at positions 1 to 11 or 2 to 11 so that said region is completely complementary with 1 or 10 consecutive nucleotides at the preselected site of the target pre-mRNA.
  • the person skilled in the art will appreciate that, in order to generate the Ulin, it will be necessary only to modify those nucleotides in the native Ul that are different from those that appear in the target sequence.
  • Modification in the snRNA Ul sequence makes it possible to inhibit the processing of pre-mRNA, understanding as processing of the 3 ' end any of the steps necessary for the formation of the polyadenylated mRNA from a pre-mRNA without polyadenilar (consensus site processing of polyadenylation and addition of the polyA + tail).
  • These modified Ul snRNAs are incorporated into the corresponding snRNP to give rise to modified snRNPs that, instead of binding to the pre-mRNA at the intron processing site, bind to the pre-mRNA in a given region thereof depending on the sequence specificity of the modified region in snRNA Ul.
  • This interaction causes a stop of the polyadenylation machinery that results in the sequestration of the pre-mRNA in the nucleus, a decrease in the maturation of the pre mRNA and, in turn, a decrease in the production of the protein encoded by said pre -mRNA.
  • these modified Ul are known like Ulin. The effect of the Ulin is therefore to cause a decrease or an inhibition of gene expression.
  • the binding sites for a sequence of that length will appear randomly once every 10 6 nucleotides, implying that they will appear about 3000 times in the human genome.
  • exons constitute only 2% of the human genome and that snRNP-mediated inhibition with modified Ul of gene expression that comprises exons requires mating with unstructured regions in the terminal exons of the transcripts containing the signals Polyadenylation (AAUAAA), the specificity of inhibition is much higher.
  • those target sites in the HBV genome are chosen that appear a lesser number of times in the 3 ' terminal exons of the organism from which the cells in which the silencing is to be carried out.
  • the frequency of occurrence of possible targets can be determined by sequence comparison.
  • a sequence acts as the reference sequence, with which the test sequences are compared.
  • sequence comparison algorithm When a sequence comparison algorithm is used, the test sequences and the reference sequence are entered into a computer, sub-sequence coordinates are designated, if necessary, and the program sequence algorithm parameters are designated. Preferably, default program parameters may be used, or alternative program parameters may be designated.
  • the sequence comparison algorithm calculates the percentages of the sequence identities of the test sequences with respect to the reference sequence, based on the program parameters.
  • a “comparison window”, in the context of the present invention, refers to a segment of any of the contiguous positions selected from the group consisting of 19 to 600, preferably about 50 to about 200, more preferably about 100 to about 150, in which a sequence can be compared with a reference sequence with the same number of positions contiguous, once the two sequences have been optimally aligned.
  • Sequence alignment methods for comparison are widely known in the art. Optimal sequence alignments can be performed for comparison, eg, with the local homology algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2: 482), with the homology alignment algorithm of Needleman and Wunsch , (J. Mol.
  • BLAST and BLAST 2.0 algorithms are described in Altschul et al, (Nuc ⁇ . Acids Res., 1977, 25, 3389-3402) and Altschul et al, (J. Mol. Biol, 1990, 215: 403-410), respectively.
  • the modified Ul snRNAs used in the composition of the invention are selected such that their binding sequence to the consensus consensus sequence of the 5 'end of the intron, which has been modified according to the sequence of the target pre-mRNA, It is identical or substantially identical to the target sites.
  • nucleic acids or polypeptide sequences refer to two or more sequences or sub-sequences that are the same (identical) or have a percentage of residues of amino acids or nucleotides which is the same (for example, in at least 70% similarity, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, 99% and 100%.
  • polynucleotide refers to a polymer formed by a variable number of monomers wherein the monomers are nucleotides, including both ribonucleotides and deoxyribonucleotides.
  • the poly nucleotides include monomers modified by methylation or as well as unmodified forms.
  • polynucleotide and “nucleic acid” are used interchangeably in the present invention and include mRNA, cDNA and recombinant polynucleotides.
  • polynucleotides are not limited to polynucleotides as they appear in nature, but include polynucleotides in which nucleotide analogs and unnatural internucleotide bonds appear.
  • Non-limiting examples of this type of unnatural structures include polynucleotides in which sugar is different from ribose, polynucleotides in which 3 '-5' and 2 '-5' phosphodiester bonds appear, polynucleotides in which inverted bonds appear ( 3 '-3' and 5 '-5') and branched structures.
  • polynucleotides of the invention include unnatural internucleotide bonds such as nucleic acid peptides (PNA or peptide nucleic acids), closed nucleic acids (LNA or locked nucleic acids), C1-C4 alkyl phosphonate bonds of the methylphosphonate type, phosphoramidate, C1- C6 alkyl phosphotrioester, phosphorothioate and phosphorodithioate.
  • PNA nucleic acid peptides
  • LNA or locked nucleic acids closed nucleic acids
  • C1-C4 alkyl phosphonate bonds of the methylphosphonate type phosphoramidate
  • C1- C6 alkyl phosphotrioester phosphorothioate
  • phosphorodithioate phosphorodithioate
  • the polynucleotide encoding the snRNA Ul according to the invention may be operatively coupled to a promoter that allows transcription of said polynucleotide in the cell in which Ulin expression is desired.
  • Promoters suitable for carrying out the present invention include, but are not necessarily limited to, constitutive promoters such as the Ul promoter or derivatives of eukaryotic virus genomes such as polyoma virus, adenovirus, SV40, CMV, avian sarcoma virus, hepatitis B virus, the metallothionein gene promoter, the herpes simplex virus thymidine kinase gene promoter, retrovirus LTR regions, the immunoglobulin gene promoter, the actin gene promoter, the EF-lalpha gene promoter as well as inducible promoters in which gene expression depends on the addition of an exogenous molecule or signal, such as the tetracycline system, the NFkappaB / UV light system, the Cre
  • the Ulin according to the invention are to be expressed in liver cells
  • a preferred embodiment of the present invention contemplates the possibility that the polynucleotide is under operational control of a specific liver promoter.
  • Non-limiting examples of such promoters are the main urinary protein promoter or the albumin promoter.
  • liver promoter described in the database of TiProD tissue specific promoters as described by Chen, X. et al., (Nucleic Acids Res., 2006, 34) can be used in the context of the present invention. , Datábase Issue).
  • the polynucleotide encoding the Ulin according to the invention is under the control of the promoter of the gene encoding the snRNA Ul from which the Ulin is derived.
  • the invention relates to an expression vector comprising a polynucleotide according to the invention.
  • Suitable vectors for the insertion of said polynucleotides are vectors derived from prokaryotic expression vectors such as pUC18, pUC19, Bluescript and their derivatives, mpl 8, mpl9, pBR322, pMB9, CoIEl, pCRl, RP4, phage and shuttle vectors such such as pSA3 and pAT28, yeast expression vectors such as 2 micron plasmid type vectors, integration plasmids, YEP vectors, centromeric plasmids and the like, insect cell expression vectors such as pAC series vectors and the pVL series, plant expression vectors such as pIBI, pEarleyGate, pAVA, pCAMBIA, pGSA, pGWB, pMDC, pMY, pORE and the like series and Expression vectors in higher
  • Eukaryotic cell vectors preferably include viral vectors (adenovirus, adenovirus-associated viruses as well as retroviruses and, in particular, lentiviruses) as well as non-viral vectors such as pSilencer 4.1-CMV (Ambion), pcDNA3, pcDNA3.1 / hyg pHCMV / Zeo, pCR3.1, pEFl / His, pIND / GS, pRc / HCMV2, pSV40 / Zeo2, pTRACER-HCMV, pUB6 / V5-His, pVAXl, pZeoSV2, pCI, pSVL and pKSV-10, pBPV-1, pML2d and pTDTl.
  • viral vectors adenovirus, adenovirus-associated viruses as well as retroviruses and, in particular, lentiviruses
  • non-viral vectors such as pS
  • the vectors comprise a reporter or marker gene that makes it possible to identify those cells that have incorporated the vector after having been contacted with it.
  • Reporter genes useful in the context of the present invention include lacZ, luciferase, thymidine kinase, GFP and the like.
  • Marker genes useful in the context of the present invention include, for example, the neomycin resistance gene, which confers resistance to aminoglycoside G418, the hygromycin phosphotransferase gene that confers hygromycin resistance, the ODC gene, which confers resistance to the inhibitor of Ornithine decarboxylase (2- (difluoromethyl) -DL-ornithine (DFMO), the dihydrofolate reductase gene that confers methotrexate resistance, the puromycin-N-acetyl transferase gene, which confers puromycin resistance, the ble gene confers resistance to zeocin, the adenosine deaminase gene that confers resistance to 9-beta-D-xylofuranosyl adenine, the cytosine deaminase gene, which allows cells to grow in the presence of N- (phosphonacetyl) -L-aspartate, Thymidine
  • the selection gene is incorporated into a plasmid which may additionally include a promoter suitable for the expression of said gene in eukaryotic cells (for example, CMV or SV40 promoters), an optimized translation initiation site (for example a site that follows the so-called Kozak rules or an IRES), a polyadenylation site such as, for example, the polyadenylation site of SV40 or phosphoglycerate kinase, introns as, for example, the intron of the beta-globulin gene.
  • the vectors are viral vectors.
  • the vectors used for the expression of the first or second component of the invention is a viral vector selected from the group of adenovirus, associated adeno virus, retrovirus , lentivirus, herpesvirus, SV40 and alphavirus.
  • interfering RNAs capable of binding to one or more of the HBV mRNAs can inhibit the gene expression of said virus.
  • expression of said interfering RNAs in a cell previously transfected with a plasmid that allows to express the HBV mRNA inhibits the expression of the E antigen.
  • the expression of different interfering RNAs in cells expressing a modified HBV genome by replacing the S antigen gene with the sequence encoding luciferase results in a decrease in luciferase activity, indicative of an inhibition of the expression of said gene .
  • the invention relates to an interfering RNA specifically directed to a target sequence of at least one HBV mRNA that is capable of causing the silencing of said HBV mRNA wherein said target sequence is selected from the group consisting of the sequences SEQ ID NO: 29 to 35;
  • an "interfering RNA capable of causing the silencing of the expression of an mRNA” is understood, in the context of the present invention, an RNA molecule which, when inside a cell, is capable of itself or after its modification by the cellular machinery, to join the target mRNA that contains the preselected sequence and cause its degradation, thus inhibiting its translation and the production of the protein encoded by it.
  • Silencers that induce the RNAi response against a target mRNA and that could be incorporated into the second component of the invention include, without limitation, siRNA, shRNA and miRNA
  • SiRNAs are agents capable of inhibiting the expression of a target gene by RNA interference.
  • a siRNA can be chemically synthesized or can be obtained by in vitro transcription.
  • siRNAs consist of a double strand of RNA between 15 and 40 nucleotides in length and may contain a 3 'and / or 5' protruding region of 1 to 6 nucleotides. The length of the protuberant region is independent of the total length of the siRNA molecule.
  • the siRNAs act by degradation or post-transcriptional silencing of the target messenger.
  • the siRNAs of the invention are substantially homologous to a preselected region of the target mRNA.
  • substantially homologous is meant that they have a sequence that is sufficiently complementary or similar to the target mRNA so that the siRNA is capable of causing degradation of the latter by RNA interference. "Substantially homologous” can be understood as “substantially identical” as explained above. Suitable siRNAs to cause such interference include siRNAs formed by RNA, as well as siRNAs that contain different chemical modifications such as:
  • Nucleotides with modified bases such as halogenated bases (for example 5-bromouracil and 5-iodouracil), alkylated bases (for example 7- methylguanosine).
  • halogenated bases for example 5-bromouracil and 5-iodouracil
  • alkylated bases for example 7- methylguanosine
  • siRNAs of the invention can be obtained using a series of techniques known to the person skilled in the art.
  • the siRNA can be chemically synthesized from ribonucleosides protected with forphoramidite groups in a conventional DNA / RNA synthesizer.
  • the interfering RNA of the invention is shRNA (short hairpin RNA).
  • ShRNA means, in the context of the present invention, an RNA molecule formed by two antiparallel chains connected by a hairpin region and wherein the sequence of one of the antiparallel chains is complementary to a pre-selected region in the target mRNA.
  • the shRNAs are composed of a short antisense sequence (from 19 to 25 nucleotides), followed by a loop of between 5 and 9 nucleotides followed by the sense chain.
  • the shRNAs can be chemically synthesized from ribonucleosides protected with forsphoramidite groups in a conventional DNA / RNA synthesizer or they can be obtained from a polynucleotide by in vitro transcription.
  • the shRNAs are processed inside the cell by the RNase Dicer that eliminates the region of the hairpin giving rise to siRNAs as described above.
  • the shRNAs may also contain different chemical modifications as described above in the case of siRNAs. (iii) miRNA
  • the miRNA or microRNA are small RNA molecules that appear naturally in the cell and are responsible for controlling the specificity of gene expression by regulating the degradation and translation of mRNA.
  • miRNAs are endogenous molecules that act by regulating the half-life of cellular mRNA
  • the structure of an endogenous miRNA for example miR-30
  • siRNA sequences incorporated in the mir-26a stem region function as effectors of RNAi causing homologous mRNA degradation (McManus, MT et al, 2002, RNA, 8: 842-850). In both cases, the nucleotide sequence introduced into the stem of the miRNA showed 100% identity with the target gene.
  • MiRNAs suitable for use in the compositions of the invention consist of 19-24 nucleotides, preferably 21 or 22 nucleus tidos.
  • the miRNAs can be designed so that they hybridize to an RNA transcript with a high degree of specificity.
  • the miRNA is designed so that it shows 100% identity or shows a high degree of identity (for example, accepting at least 1, at least 2, at least 3 or more of bad pairings) with the given target mRNA. that a single non-complementary nucleotide may, depending on its location in the miRNA chain, decrease inhibition levels.
  • the miRNAs can be designed to address the 5 'untranslated region, the coding region or the 3' region of a target mRNA.
  • miRNA suitable for carrying out the present invention correspond to those based on the endogenous miRNA miR-30, as described in WO03093441 or based on the endogenous miRNA as described in WO03029459.
  • the invention relates to a polynucleotide whose transcription results in an interfering RNA according to the invention.
  • This polynucleotide can give rise to the siRNA, shRNA and / or miRNA described above.
  • these comprise a single promoter region that regulates the transcription of a sequence comprising the sense and antisense chains of the shRNA and miRNA connected by a hairpin or by a stem-loop region.
  • any promoter can be used for expression of shRNA and miRNA provided that said promoters are compatible with the cells in which it is desired to express the siRNAs.
  • suitable promoters for carrying out the present invention include those mentioned above for Ulin expression.
  • the promoters are RNA polymerase III promoters that act constitutively. RNA polymerase III promoters appear in a limited number of genes such as 5S RNA, tRNA, 7SL RNA and U6 RNAs.
  • the polynucleotides encoding siRNAs comprise two transcriptional units each formed by a promoter that regulates the transcription of one of the chains that forms in siRNA (sense and antisense).
  • Polynucleotides encoding siRNAs may contain convergent or divergent transcriptional units. In divergent transcription polynucleotides, the transcriptional units encoding each of the DNA chains that form the siRNA are located in tandem in the polynucleotide so that the transcription of each DNA chain depends on its own promoter, which can be same or different (Wang, J.
  • the sense and antisense chains of the siRNA are regulated by different promoters.
  • both transcriptional units are convergently oriented.
  • type III RNA polymerase promoters are promoters of the Hl and U6 genes of human or murine origin.
  • the promoters are 2 U6 promoters of human or murine origin, a mouse U6 promoter and a human Hl promoter or a human U6 promoter and a mouse Hl promoter.
  • the invention in another aspect, relates to an expression vector comprising a polynucleotide according to the invention.
  • Suitable vectors for the expression of interfering RNAs according to the invention include, without limitation, any of the vectors mentioned above in the context of the vectors for expression of the modified Ul snRNAs according to the invention.
  • compositions of the invention are provided.
  • the invention relates to a composition or a kit comprising in one or several containers:
  • an Ul snRNA wherein said Ul snRNA is modified in its binding sequence to the consensus consensus sequence GU of the 5 'end of the intron so that it specifically binds to a target sequence of at least one HBV mRNA so which is capable of inhibiting maturation of said target mRNA,
  • the first component of the compositions of the invention is a specific Ul snRNA against HBV as defined above when describing the Ul snRNA of the invention, a polynucleotide encoding an Ul RNA or an expression vector comprising a polynucleotide that encodes an RNA Ul.
  • the target sequence in the HBV genome against which the modified Ul snRNA binds is selected from the group consisting of the sequences SEQ ID NO: 4 to 28.
  • the second component of the composition of the invention is a specific interfering RNA against at least one HBV mRNA, a polynucleotide encoding said interfering RNA or an expression vector comprising said polynucleotide.
  • Particular forms of the various embodiments of said second component of the invention have been described in detail above in the context of the interfering RNAs of the invention, of the polynucleotides encoding said interfering RNAs and of the vectors containing said polynucleotides and are equally applicable. to the second component of the invention.
  • the target sequence in the HBV genome against which the interfering RNA binds is selected from the group consisting of the sequences SEQ ID NO: 29 to 35.
  • compositions of the invention comprise components characterized by their ability to bind to specific target sequences in the HBV mRNA and which are listed in Table 1.
  • Table 1 Target sequences in the HBV genome versus which are directed by the Ulin and interfering RNAs according to the invention.
  • Target sequence to which it is bound Target sequence to which the snRNA Ul modified RNA interfering binds
  • SEQ ID NO: 6 SEQ ID NO: 31 Target sequence to which it is bound Target sequence to which the snRNA Ul modified RNA interfering binds
  • the invention relates to a polynucleotide capable of silencing the gene expression of the hepatitis B virus comprising:
  • component (ii) of the bifunctional polynucleotide comprises the sense and antisense chains of at least one siRNA, encodes at least one pre-miRNA or encodes at least one shRNA wherein said siRNA, miRNA and shRNA bind specifically the Ul RNA encoded by the sequence defined in (i) and which is capable of causing the in vivo silencing of said target mRNA.
  • the target sequence in the HBV genome to which the modified Ul snRNA encoded by the first sequence of the bifunctional polynucleotide of the invention binds is selected from the group consisting of the sequences SEQ ID NO: 4 to 28 .
  • the target sequence in the HBV genome to which the interfering RNA encoded by the second sequence of the bifunctional polynucleotide of the invention binds is selected from the group consisting of the SEQ sequences. ID NO: 29 to 35.
  • the bifunctional polynucleotide of the invention comprises sequences encoding particular combinations of modified snRNA U1 and interfering RNAs that show a synergistic effect on their ability to inhibit HBV gene expression.
  • the bifunctional polynucleotide of the invention comprises coding sequences for modified Ul snRNAs and interfering RNAs that bind to target sequences as described above in Table 1.
  • the bifunctional polymucleotides of the invention are usually coupled to regulatory regions of their expression that may be identical for the sequences encoding the Ulin and for the sequences encoding the interfering RNA or may be different. Suitable promoters for the expression of said polmucleotides are the same (constitutive, adjustable and tissue specific) mentioned above for the expression of the Ulin. Also, the bifunctional polymucleotides of the invention may be part of an expression vector. Thus, in another aspect, the invention relates to expression vectors comprising a bifunctional polynucleotide of the invention.
  • Suitable vectors for cloning and propagation of the polmucleotides of the invention are substantially the same as mentioned above for the expression of the polmucleotides encoding the Ulin or the polmucleotides encoding the interfering RNAs.
  • the expression vectors of the polynucleotides of the invention are viral vectors, even more preferably vectors selected from the group of adenovirus, adeno-associated virus, retrovirus, lentivirus, herpesvirus, SV40 and alphavirus.
  • the invention relates to a compound, a composition of the invention or a bifunctional polynucleotide according to the present invention for use in medicine or as a medicine.
  • both the first and second components can be administered in the form of inhibitory molecules as is, that is, component (i) can be administered in the form of snRNA Ul and component (ii) can be administered as a based silencing agent in RNA (siRNA, miRNA, shRNA).
  • the first component may be provided as a polynucleotide encoding the modified Ul snRNA and / or the second component may be DNA based, that is, it comprises a polynucleotide encoding the silencing agent.
  • the polynucleotides can be administered as a single polynucleotide comprising components (i) and (ii) or as separate polynucleotides, simultaneously-, separately- or sequentially.
  • the invention contemplates different combinations of forms of administration that may be determined by the expert according to the circumstances.
  • component (i) is an Ul RNA as such and component (ii) is an interfering RNA,
  • component (i) is an Ul RNA as such and component (ii) is a polynucleotide encoding an interfering RNA.
  • component (i) is a polynucleotide that encodes Ul RNAs and component (ii) is an interfering RNA, or component (i) is a polynucleotide encoding the sn sn AR and component (ii) is a polynucleotide that is an interfering RNA.
  • component (i) is an Ul RNA as such and component (ii) is an interfering RNA,
  • component (i) is an Ul RNA as such and component (ii) is a polynucleotide encoding an interfering RNA.
  • component (i) is a polynucleotide that encodes Ul RNAs and component (ii) is an interfering RNA,
  • component (i) is a polynucleotide that encodes Ul RNAs and component (ii) is a polynucleotide that an interfering RNA, in which case both components must be found as separate polynucleotides.
  • the first and second components of the composition of the invention are used in the form of polynucleotides encoding the active RNA molecules, said polynucleotides can be provided as part of a vector.
  • each polynucleotide will be part of a different vector.
  • simultaneous administration it is possible that both polynucleotides are part of different vectors or of the same vector.
  • the invention provides a compound, a composition, a bifunctional polynucleotide of the invention, or a vector comprising said bifunctional polynucleotide for use in the treatment or prevention of a disease caused by an HBV infection.
  • the invention relates to the use of a compound, a composition, a bifunctional polynucleotide of the invention, or a vector comprising said bifunctional polynucleotide for the preparation of a medicament for the treatment or prevention of a disease caused by an HBV infection.
  • the invention in another aspect, relates to a method for the treatment or prevention of a disease caused by an HBV infection in a subject comprising the administration to said subject of a compound, a composition, a bifunctional polynucleotide of the invention or a vector comprising said bifunctional polynucleotide.
  • a disease caused by an HBV infection refers to any disease caused by a progressive pathology of the liver and which includes acute hepatitis B, chronic hepatitis B, cirrhosis and liver carcinoma.
  • the invention relates to pharmaceutical preparations comprising the compounds of the invention, the compositions of the invention or the bifunctional polynucleotides of the invention together with one or more pharmaceutically acceptable carriers.
  • the invention contemplates pharmaceutical compositions specially prepared for the administration of the nucleic acids that form the compound of the invention, the first and the second component of the invention or for the administration of the bifunctional polynucleotides of the invention.
  • the pharmaceutical compositions can comprise both the first component and the second component, whether it is based on DNA or on AR in naked form, that is, in the absence of compounds that protect nucleic acids from their degradation by the nucleases of the organism, which entails the advantage that the toxicity associated with the reagents used for transfection is eliminated.
  • Suitable routes of administration for naked compounds include intravascular, intratumoral, intracranial, intraperitoneal, intrasplenic, intramuscular, subretinal, subcutaneous, mucosa, topical and oral (Templeton, 2002, DNA Cell BioL, 21: 857-867).
  • Initial fears regarding the ability of these compounds to induce an immune response when administered nude have been investigated by Heidel et al. (Nat. Biotechnol., 2004, 22: 1579-1582).
  • an immune response could not be observed while it was observed that the systemic administration of siRNA was well tolerated.
  • compositions and polynucleotides of the invention are administered by the so-called "hydrodynamic administration" in which the compounds are introduced into the body intravascularly at high speed and volume, resulting in high levels of transfection. with a more diffuse distribution.
  • hydrodynamic administration in which the compounds are introduced into the body intravascularly at high speed and volume, resulting in high levels of transfection. with a more diffuse distribution.
  • a modified version of this technique has allowed to obtain positive results for the silencing by means of naked siRNAs of exogenous genes (Lewis et al, 2002, Nat.Gen., 32: 107-108; McCaffrey et al, 2002, Nature, 418: 38- 39) and endogenous (Song et al, 2003, Science, Nat.Med., 9: 347-351) in multiple organs.
  • both the Ulin that forms the first component of the composition of the invention, and the RNA-based components that form the second component of the composition of the invention may contain modifications of their structure that contribute to increasing their stability. Suitable modifications to decrease nuclease sensitivity include 2'-0-methyl (Czauderna et al, 2003, Nucleic Acids Res., 31: 2705-2716), 2'-fluoropyrimidines (Layzer et al, 2004, RNA, 10: 766 -771).
  • compositions and polynucleotides of the invention can be administered as part of liposomes, cholesterol-conjugated or conjugated to compounds capable of promoting translocation through cell membranes such as the Tat peptide derived from the HIV-1 TAT protein, the third helix of the homeodomain of the Antennapedia protein of D.melanogaster, the VP22 protein of herpes simplex virus, arginine oligomers and peptides such as those described in WO07069090 (Lindgren, A. et al., 2000, Trends Pharmacol. Sci, 21: 99- 103, Schwarze, SR et al., 2000, Trends Pharmacol.
  • the second component of the invention when based on DNA, can be administered as part of a plasmid vector or a viral vector, preferably adenovirus-based vectors, adeno-associated viruses or retroviruses, particularly viruses based on murine leukemia virus (MLV) or lentivirus (HIV, IVF, EIAV).
  • a viral vector preferably adenovirus-based vectors, adeno-associated viruses or retroviruses, particularly viruses based on murine leukemia virus (MLV) or lentivirus (HIV, IVF, EIAV).
  • EXAMPLE 1 Expression vectors of inhibitors of hepatitis B virus (HBV) gene expression and other expression vectors used.
  • HBV hepatitis B virus
  • the genomic sequence of the hepatitis B virus subtype ayw Galibert was taken as reference, which is provided as SEQ.ID.NO. l.
  • the Ulin were constructed taking as a pattern the sequence of the RNA transcript whose accession number in NCBI is NR 004421.1, corresponding to the human Ul snRNA 4 (Entrez Gene ID: 6060) which is included as sequence SEQ.ID.NO.2:
  • the plasmid pGem3z +: Ul-Mscl (SEQ.ID.NO.3), already described by Fortes and cois, was used. (Proc Nati Acad Sci USA. 2003; 100: 8264-8269).
  • This plasmid was constructed on plasmid pGem (Promega), which contains the snRNA Ul gene at the BamHI site, including the promoter and termination sequences.
  • the Ul snRNA produced by this plasmid differs from the endogenous Ul snRNA in that the nucleotides at positions 98-99 and 109-110 (nucleotides 498-499 and 509-510 of SEQ.ID.NO.3; indicated below) with double underlining) have been replaced by their complementary nucleotides. These substitutions allow differentiating the exogenous Ul snRNA from the endogenous without affecting its inhibitory functionality.
  • RNUl-4 1 ATACTTACCTGGCAGGGGAGATACCATGATCACGAAGGTGGTTTTCCCAGGGCGAGGCTT 60
  • RNUl-4 61 ATCCATTGCACTCCGGATGTGCTGACCCCTGCGATTTCCCCAAATGTGGGAAACTCGACT 120
  • RNUl-4 121 GCATAATTTGTGGTAGTGGGGGACTGCGTTCGCGCTTTCCCCTG 164
  • Target sequence agent SEQ.ID Nucleotides of the target sequence
  • HBV mRNA a The Core, X prot, Pol and SAg annotations indicate in which HBV mRNA the inhibitor target is located: respectively the mRNA encoding core viral proteins, protein X, polymerase (pol) and surface antigen (SAg).
  • plasmid pGem3z + Ul-Mscl was used as a control.
  • the construction of the plasmids expressing the different Ulins tested was performed as described below:
  • the plasmids expressing the Ulin were obtained by introducing the following hybridized oligonucleotides into the Bcll and BglII sites of pGem3z +: Ul-Mscl:
  • Oligonucleotide D Oligonucleotide sequence
  • the sequence complementary to the target sequence has been highlighted in bold.
  • Plasmids for the expression of shRNAs were generated on a plasmid pSuper by cloning the oligonucleotide sequences comprising the target sequences of Table 3. To this end, the plasmid pSuper (Brummelkamp et al. Science. 2002; 296 (5567): 550-553). This plasmid was digested with BglII and HindI and in this sites the hybridized oligonucleotides indicated below were cloned:
  • Plasmid pSP65-ayw 1.3 containing a copy of HBV was provided by Dr. Chisari. Hereinafter we will call it pHBV.
  • Plasmid pRL-SV40 (Promega, E2231) was used as a transfection control of Renilla luciferase.
  • the plasmid pHBV-Luc (Ely et al. Mol Ther. 2008; 16 (6): 1105-1112) was provided by Dr. Arbuthnot and contains the HBV coding sequence in which the region of the E antigen has been replaced by a sequence encoding firefly luciferase. All clones were verified by sequencing in ABI Prism 310 genetic analyzer (Applied Biosystems). Plasmid DNA was purified with a Maxiprep kit (Marlingen) before transfection.
  • HuH7 cells HuH7 cells (ATCC), which were grown in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, at 37 ° C in an atmosphere with 5% C0 2 .
  • FBS fetal bovine serum
  • penicillin-streptomycin penicillin-streptomycin
  • HuH7 cells were transfected with calcium phosphate.
  • HuH7 cells (1.5 x 10 5 cells) were co-transfected with a. the plasmid pHBV (1 ⁇ g), which expresses the mR A of HBV; and b. the control plasmid pGem3z +: Ul-Mscl (pMock) expressing a natural Ul snRNA (1 ⁇ g); or alternatively, one or more of the inhibitor plasmids (1 ⁇ g in total) expressing an HBV inhibitor, either Ulin or shR A.
  • EAg E antigen supernatant
  • FI Inhibition Factor
  • All plasmids for the expression of one of the selected shRNAs provided inhibition factors greater than 3, those expressing shRNA2 and shRNA6 being particularly active (see Fig. 2).
  • Example 2 In the tests of Example 2 above, an evaluation of HBV inhibition was performed by a non-quantitative ELISA assessment of EAg expression. In this way a good estimate of the functionality of the inhibitors was obtained, not so much a reproducible quantification of the efficacy of inhibition.
  • the efficacy of the new Ulin in cells expressing luciferase from a plasmid having HBV sequences was evaluated.
  • the plasmid pHBV-Luc was used, which expresses an HBV in which the coding region of the S antigen (SAg) has been replaced by a coding sequence of the firefly luciferase.
  • SAg S antigen
  • expression from the modified HBV mRNA is necessary.
  • the pHBV-Luc plasmid cannot replicate in liver cells.
  • the HBV genome is so compact that the substitution of the sequence encoding SAg affects most of the polymerase coding region and the 3'UTR core (the 3 'untranslated region).
  • the protein X mRNA is not affected. Since a functional polymerase cannot be produced, HBV pregenomic RNA cannot generate new HBV genomes, and therefore viral replication cannot take place. Under these circumstances, the only way to reduce luciferase activity is to interfere with the expression of the luciferase gene. Luciferase expression should originate mostly from the Luciferase mRNA, but it is not ruled out that a fraction originates from the pregenomic HBV RNA.
  • HBV expression inhibitors selected have as their target sequence a sequence that is within the SAF ORF reading window: Ulin5, Ulinó, Ulin7, shRNA2, shRNA3, shRNA5 and shRNA6. Therefore, these inhibitors should not interfere with luciferase expression.
  • the activity inhibition assays were also performed on HuH7 cells, in the same manner as described in example 2, with the proviso that: - in this case the cells were co-transfected with 0.75 ⁇ g of the plasmid pHBV- Luc (replacing pHBV); and that 0.25 ⁇ g of plasmid pRL-SV40 was included as a transfection control
  • the doses of the inhibitor plasmid tested were the maximum dose (1 ⁇ g), or half of this dose (1 ⁇ 2D: 0.5 ⁇ g). - Two days after cotransfection, luciferase activity was quantified.
  • Luciferase activity was measured on a Berthold luminometer (Lumat LB9507) using the Dual Luciferase System kit (Promega), according to the manufacturer's instructions.
  • the FI Inhibition Factor was calculated for each inhibitor, such as the ratio of luciferase activity ([Luc]) measured in the cotransfected cells with the plasmid control (Mock) between the luciferase activity measured in the cotransfected cells with the inhibitor plasmid to be evaluated, or one of its combinations.
  • FIn +12 is the FI Inhibition Factor obtained for the combination of the two inhibitors; Fin and FI 12 are the Inhibition Factors obtained for each of the inhibitors when tested separately.
  • a positive SI value indicates that there is no synergy between the two agents; a value close to 0 indicates an additive effect, while a negative value indicates that there is no synergism.
  • EXAMPLE 4 In vivo analysis of the inhibition of HBV-Luc gene expression. The test was performed on male C56 / BL6 mice, with 5 mice per treatment group, in accordance with the requirements and approval of the Institution's Animal Experimentation Ethics Committee.
  • Each mouse received a hydrodynamic injection into the tail vein of 1.8 ml of saline containing 25 ⁇ g of plasmids, with the following composition:
  • luciferase activity in the liver of the mice was evaluated by means of a CCD camera (Xenogen). Luciferase activity was measured 5 minutes after intraperitoneal injection of 3 mg of luciferin and an anesthetic. As in example 2 above, the Inhibition Factor (FI) was also calculated, as the ratio of luciferase activity ([Luc]) measured in mice that received the control plasmid (Mock), between luciferase activity measured in mice who received the plasmid expressing the inhibitor (Inhibitor).
  • FI Inhibition Factor

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Abstract

The invention relates to agents able to inhibit the expression of hepatitis B virus genes by inhibiting the maturation of the various RNAm of said virus using U1 RNAs, by means of interferent RNAs or synergistic combinations that include both inhibiting agents. The invention also relates to the use of said agents and to compositions for the treatment of illnesses caused by a Hepatitis B virus infection.

Description

COMPUESTOS Y COMPOSICIONES PARA EL TRATAMIENTO DE ENFERMEDADES DEBIDAS A LA INFECCIÓN POR EL VIRUS DE LA COMPOUNDS AND COMPOSITIONS FOR THE TREATMENT OF DISEASES DUE TO INFECTION BY THE VIRUS OF THE
HEPATITIS B CAMPO TÉCNICO DE LA INVENCIÓN HEPATITIS B TECHNICAL FIELD OF THE INVENTION
La invención se relaciona con métodos terapéuticos para inhibir la expresión génica del virus de la hepatitis B y, por tanto, con métodos para el tratamiento de enfermedades asociadas con la infección por dicho virus. En particular, la invención se relaciona con compuestos y composiciones que actúan mediante la inhibición de la maduración de los ARNm del virus de la hepatitis B y mediante la interferencia mediada por ARN de la expresión de dichos ARNm. The invention relates to therapeutic methods for inhibiting the gene expression of the hepatitis B virus and, therefore, with methods for the treatment of diseases associated with infection by said virus. In particular, the invention relates to compounds and compositions that act by inhibiting maturation of hepatitis B virus mRNAs and by RNA-mediated interference of the expression of said mRNAs.
ANTECEDENTES DE LA INVENCIÓN BACKGROUND OF THE INVENTION
El virus de la hepatitis B (HBV) causa unos 400 millones de infecciones en todo el mundo. La infección crónica infección por VHB y el a carcinoma hepatocelular (CHC) asociado a HBV produce más de un millón de muertes al año. Desafortunadamente, el tratamiento actual de la infección crónica es menos eficaz debido a baja eficacia de los fármacos actuales y la aparición de resistencia a los medicamentos. Por lo tanto, se necesita urgentemente desarrollar un nuevo tratamiento para la infección por el VHB y el cáncer de hígado HBV asociados dado que el número de nuevos pacientes se encuentra en aumento constante. El tratamiento con interferón-alpha es la terapia comúnmente utilizada para el tratamiento de la infección con HBV. No obstante, el uso de interferón-alpha va acompañado de una serie de efectos secundarios indeseables tales como síntomas de tipo gripe (fatiga, fiebre, mialgia, etc.), efectos neurosiquiátricos (irritabilidad, apatía, etc.), reducción de células mieloides tales como granulocitos y plaquetas, aumentos en los niveles de triglicéridos, anormalidades del tiroides, etc. Recientemente ha sido aprobado el tratamiento con lamivudina (3TC(R)), que es un análogo de nucleósido que inhibe la síntesis del ADN de HBV. No obstante, la interrupción del tratamiento con lamivudina resulta en una reactivación de la replicación de HBV en la mayoría de pacientes. Además, se han identificado algunos casos de resistencia a este fármaco en algunos pacientes. Hepatitis B virus (HBV) causes about 400 million infections worldwide. Chronic HBV infection and hepatocellular carcinoma (CHC) associated with HBV causes more than one million deaths per year. Unfortunately, the current treatment of chronic infection is less effective due to low efficacy of current drugs and the emergence of drug resistance. Therefore, a new treatment for HBV infection and associated HBV liver cancer is urgently needed as the number of new patients is constantly increasing. Interferon-alpha treatment is the therapy commonly used to treat HBV infection. However, the use of interferon-alpha is accompanied by a series of undesirable side effects such as flu-like symptoms (fatigue, fever, myalgia, etc.), neurosychiatric effects (irritability, apathy, etc.), reduction of myeloid cells such as granulocytes and platelets, increases in triglyceride levels, thyroid abnormalities, etc. Lamivudine (3TC (R)) treatment has recently been approved, which is a nucleoside analog that inhibits the synthesis of HBV DNA. However, discontinuation of lamivudine treatment results in a reactivation of HBV replication in most patients In addition, some cases of resistance to this drug have been identified in some patients.
El virus de la hepatitis B es un virus ADN circular de doble cadena que codifica la polimerasa, la proteína X, el antígeno core y los antígenos de superficie (S y preS).Hepatitis B virus is a double-stranded circular DNA virus that encodes polymerase, protein X, core antigen and surface antigens (S and preS).
El genoma de HBV es muy compacto y contiene marcos abiertos de lectura que solapan entre sí, por lo que el uso de agentes silenciadores de la expresión dirigidos contra una región determinada del genoma de HBV pueden ser útiles para inhibir la expresión de múltiples ARNm de HBV. Así, es conocido el uso de ARNs interferentes dirigidos contra los ARNm de HBV para inhibir la replicación de HBV (Arbuthnot et al; Liver International. 2005; 25: 9-15; Chen et al; Pharm Res. 2008; 25: 72-86 y Stein y Loomba; Infecí Disord Drug Targets. 2009; 9: 105-106). No obstante, el uso de ARNs interferentes para inhibir la replicación de HBV tiene la desventaja de que se trata de un tratamiento costoso, de que los ARNs interferentes tienen una vida media en suero relativamente corta y que se pueden generar resistencias a la administración repetida de un único ARN interferente (Wu H. et al. Gastroenterology, 2005; 128:708-16). The HBV genome is very compact and contains open reading frames that overlap each other, so the use of expression silencing agents directed against a particular region of the HBV genome may be useful for inhibiting the expression of multiple HBV mRNAs. . Thus, the use of interfering RNAs directed against HBV mRNAs to inhibit HBV replication is known (Arbuthnot et al; Liver International. 2005; 25: 9-15; Chen et al; Pharm Res. 2008; 25: 72- 86 and Stein and Loomba; I infected Disord Drug Targets. 2009; 9: 105-106). However, the use of interfering RNAs to inhibit HBV replication has the disadvantage that it is an expensive treatment, that interfering RNAs have a relatively short serum half-life and that resistance to repeated administration of a single interfering RNA (Wu H. et al. Gastroenterology, 2005; 128: 708-16).
Por tanto, existe una necesidad en la técnica de tratamientos alternativos a la infección con HBV que solventen parcial- o totalmente los problemas anteriormente mencionados. Therefore, there is a need in the art of alternative treatments to HBV infection that partially or totally solve the aforementioned problems.
COMPENDIO DE LA INVENCIÓN SUMMARY OF THE INVENTION
En un primer aspecto, la invención se relaciona con un compuesto capaz de silenciar la expresión génica del virus de la hepatitis B seleccionado del grupo consistente en: In a first aspect, the invention relates to a compound capable of silencing the gene expression of hepatitis B virus selected from the group consisting of:
(i) un snRNA Ul, en donde la secuencia de unión a la secuencia consenso GU del extremo 5 ' del intrón de dicho snRNA Ul se encuentra modificada de forma que se una específicamente a una secuencia diana de al menos un ARNm de HBV de forma que es capaz de inhibir la maduración de dicho ARNm diana y en donde dicha secuencia diana se selecciona del grupo consistente en las secuencias SEQ ID NO:4-24;  (i) an Ul snRNA, wherein the consensus consensus binding sequence GU of the 5 'end of the intron of said Ul snRNA is modified such that it specifically binds to a target sequence of at least one HBV mRNA so which is capable of inhibiting the maturation of said target mRNA and wherein said target sequence is selected from the group consisting of the sequences SEQ ID NO: 4-24;
un polinucleótido que codifica un ARN Ul según (i);  a polynucleotide encoding an Ul RNA according to (i);
un vector de expresión que comprende un polinucleótido según (ii), (iv) un ARN interferente dirigido específicamente a una secuencia diana de al menos un ARNm de HBV que es capaz de provocar el silenciamiento de dicho ARNm de HBV en donde dicha secuencia diana se selecciona del grupo consistente en las secuencias SEQ ID NO:29 a 35; an expression vector comprising a polynucleotide according to (ii), (iv) an interfering RNA specifically directed to a target sequence of at least one HBV mRNA that is capable of causing the silencing of said HBV mRNA wherein said target sequence is selected from the group consisting of the sequences SEQ ID NO: 29 a 35;
(v) un polinucleótido que codifica un ARN de interferencia según (iv) y  (v) a polynucleotide encoding an interference RNA according to (iv) and
(vi) un vector de expresión que comprende un polinucleótido según (v).  (vi) an expression vector comprising a polynucleotide according to (v).
En un segundo aspecto, la invención se relaciona con una composición o un kit que comprende en uno o varios contenedores: In a second aspect, the invention relates to a composition or a kit comprising in one or several containers:
(a) un primer componente seleccionado del grupo de:  (a) a first component selected from the group of:
(i) un snRNA Ul, en donde dicho snRNA Ul se encuentra modificado en su secuencia de unión a la secuencia consenso GU del extremo 5 ' del intrón de forma que se una específicamente a una secuencia diana de al menos un ARNm de HBV de forma que es capaz de inhibir la maduración de dicho ARNm diana,  (i) an Ul snRNA, wherein said Ul snRNA is modified in its binding sequence to the consensus consensus sequence GU of the 5 'end of the intron so that it specifically binds to a target sequence of at least one HBV mRNA so which is able to inhibit the maturation of said target mRNA,
(ii) Un polinucleótido que codifica un ARN Ul según (i),  (ii) A polynucleotide encoding an Ul RNA according to (i),
(iii) Un vector de expresión que comprende un polinucleótido según (i) y (b) un segundo componente seleccionado del grupo de  (iii) An expression vector comprising a polynucleotide according to (i) and (b) a second component selected from the group of
(iv) Un ARN interferente dirigido específicamente a una secuencia diana del pre-ARNm de HBV que es capaz de provocar el silenciamiento de dicho pre-ARNm de HBV,  (iv) An interfering RNA specifically directed to a target sequence of the HBV pre-mRNA that is capable of causing the silencing of said HBV pre-mRNA,
(v) Un polinucleótido que codifica un ARN de interferencia según (iv) y (v) A polynucleotide encoding an interference RNA according to (iv) and
(vi) Un vector de expresión que comprende un polinucleótido según (v). En un tercer aspecto, la invención se relaciona con un polinucleótido capaz de silenciar la expresión génica del virus de la hepatitis B que comprende (vi) An expression vector comprising a polynucleotide according to (v). In a third aspect, the invention relates to a polynucleotide capable of silencing the gene expression of the hepatitis B virus comprising
(i) una secuencia que codifica al menos un snRNA Ul modificado en su secuencia de unión a la secuencia consenso GU del extremo 5 ' del sitio de corte del intrón de forma que se une específicamente a una secuencia diana de al menos un ARNm de HBV y es capaz de inhibir el procesamiento de dicho ARNm diana y  (i) a sequence encoding at least one modified Ul snRNA in its binding sequence to the consensus consensus sequence GU of the 5 'end of the intron shear site so that it specifically binds to a target sequence of at least one HBV mRNA and is able to inhibit the processing of said target mRNA and
(ii) una secuencia que codifica un ARN interferente dirigido de forma específica a una secuencia diana de al menos un ARNm de HBV y que es capaz de provocar el silenciamiento de dicho AR m de HBV. (ii) a sequence encoding an interfering RNA specifically directed to a target sequence of at least one HBV mRNA and that is capable of causing the silencing of said AR m of HBV.
En aspectos adicionales, la invención se relaciona con compuesto, una composición o kit, un polinucleótido o un vector de la invención para su uso en medicina o para su uso en la prevención o tratamiento de enfermedades causadas por una infección por el virus de la hepatitis B así como con composiciones farmacéuticas que comprenden un compuesto, una composición o kit, un polinucleótido o un vector de la invención y vehículo farmacéuticamente aceptable. In additional aspects, the invention relates to a compound, a composition or kit, a polynucleotide or a vector of the invention for use in medicine or for use in the prevention or treatment of diseases caused by a hepatitis virus infection. B as well as with pharmaceutical compositions comprising a compound, a composition or kit, a polynucleotide or a vector of the invention and a pharmaceutically acceptable carrier.
BREVE DESCRIPCIÓN DE LAS FIGURAS BRIEF DESCRIPTION OF THE FIGURES
Figura 1. Representación esquemática del genoma de HBV, de los ARNm generados a partir del genoma de HBV y de la situación en la molécula de las regiones diana para los ARN Ul y los ARN ínter ferentes de la invención. Figure 1. Schematic representation of the HBV genome, of mRNAs generated from the HBV genome and of the situation in the molecule of the target regions for Ul RNAs and inter-different RNAs of the invention.
Figura 2. Actividad inhibidora de la expresión de HBV producida por los shRNA inhibidores. El Factor de Inhibición (F.I.) representa el número de veces que se inhibe la expresión en comparación con el control (Mock). S.D.: Desviación estándar. Figure 2. Inhibitory activity of HBV expression produced by shRNA inhibitors. Inhibition Factor (F.I.) represents the number of times the expression is inhibited compared to the control (Mock). S.D .: Standard deviation.
Figura 3. Actividad inhibidora de la expresión de HBV producida por los inhibidores Ulin. F.I.: Factor de Inhibición; S.D.: Desviación estándar. (1 μg de plásmido por cada 1,5 x 105 células). Figure 3. Inhibitory activity of HBV expression produced by Ulin inhibitors. FI: Inhibition Factor; SD: Standard deviation. (1 μg of plasmid per 1.5 x 10 5 cells).
Figura 4A-4C. Actividad inhibidora de la expresión de HBV producida por distintas combinaciones de inhibidores shRNA y Ulin. F.I. : Factor de Inhibición; S.D. : Desviación estándar. Nótese que se usa la mitad de cada inhibidor y que por ello los resultados no pueden compararse con los obtenidos en las Figuras 3 y 4. Figure 4A-4C. Inhibitory activity of HBV expression produced by different combinations of shRNA and Ulin inhibitors. F.I. : Inhibition Factor; S.D. : Standard deviation. Note that half of each inhibitor is used and therefore the results cannot be compared with those obtained in Figures 3 and 4.
Figura 5A-5B. Actividad inhibidora de la expresión de HBV producida por distintos inhibidores shRNA y Ulin en el modelo de transfección con pHBV-Luc en células HuH7. F.I. : Factor de Inhibición; S.D.: Desviación estándar. Las dosis del plásmido inhibidor ensayadas fueron la dosis máxima (1 μg), o la mitad de esta dosis (½D: 0,5 μΒ). Figura 6. Actividad inhibidora de la expresión de HBV producida por distintos inhibidores shRNA y Ulin en el modelo de transfección con pHBV-Luc en células HuH7. F.I.: Factor de Inhibición; S.D.: Desviación estándar; S.I.: índice de sinergia. Las dosis de cada plásmido inhibidor utilizadas fueron de 0,5 μg (en las combinaciones 0,5 μg/plásmido). Figure 5A-5B. Inhibitory activity of HBV expression produced by different shRNA and Ulin inhibitors in the transfection model with pHBV-Luc in HuH7 cells. FI: Inhibition Factor; SD: Standard deviation. Doses of inhibitor plasmid were the highest dose tested (1 mg), or half this dose (½D 0.5 μ Β). Figure 6. Inhibitory activity of HBV expression produced by different shRNA and Ulin inhibitors in the transfection model with pHBV-Luc in HuH7 cells. FI: Inhibition Factor; SD: Standard deviation; SI: synergy index. The doses of each inhibitor plasmid used were 0.5 μg (in the combinations 0.5 μg / plasmid).
Figura 7A - 13. Actividad inhibidora de la expresión de HBV producida por distintos inhibidores shRNA y Ulin en el modelo in vivo de transfección con pHBV-Luc. [Luc]: actividad luciferasa expresada como fotones/s/cm2/sr (xlO 5). S.D.: Desviación estándar. Las dosis del plásmido inhibidor ensayadas fueron de 10 μg (en las combinaciones también 10 μg/plásmido). Figure 7A - 13. HBV expression inhibitory activity produced by different shRNA and Ulin inhibitors in the in vivo model of transfection with pHBV-Luc. [Luc]: Luciferase activity expressed as photons / s / cm 2 / sr (xlO 5 ). SD: Standard deviation. The doses of the inhibitor plasmid tested were 10 μg (in the combinations also 10 μg / plasmid).
DESCRIPCIÓN DETALLADA DE LA INVENCIÓN Uli de la invención Los autores de la presente invención han puesto de manifiesto que el uso de snRNAs Ul modificados con capacidad de unirse a uno o varios de los ARNm de HBV permite inhibir la expresión génica de dicho virus. Así, según se demuestra en el ejemplo 2 de la presente invención, la expresión de dicho snRNA Ul en una célula previamente transfectada con un plásmido que permite expresar el ARNm de HBV inhibe la expresión del antígeno E. Asimismo, según se observa en el ejemplo 3, la expresión de distintos snRNAs Ul en células que expresan un genoma de HBV modificado mediante la sustitución del gen del antígeno S por la secuencia que codifica luciferasa resulta en una disminución de la actividad luciferasa, indicativa de una inhibición de la expresión de dicho gen. Por otro lado, según se demuestra en el ejemplo 4 de la presente invención, la administración de dichos snRNA Ul modificados a un animal al que se le ha administrado un plásmido que comprende el genoma de HBV modificado mediante la sustitución del antígeno S por la secuencia de la luciferasa permite reducir de forma estadísticamente significativa la actividad luciferasa en hígado en comparación con los animales a los que se le ha administrado el plásmido control. Así, en un primer aspecto, la invención se relaciona con un compuesto capaz de silenciar la expresión génica del virus de la hepatitis B en donde dicho compuesto es un snRNA Ul , en donde la secuencia de unión a la secuencia consenso GU del extremo 5' del intrón de dicho snRNA Ul se encuentra modificada de forma que se una específicamente a una secuencia diana de al menos un ARNm de HBV de forma que es capaz de inhibir la maduración de dicho ARNm diana y en donde dicha secuencia diana se selecciona del grupo consistente en las secuencias SEQ ID NO:4-24; DETAILED DESCRIPTION OF THE INVENTION Uli of the invention The authors of the present invention have shown that the use of modified Ul snRNAs capable of binding to one or more of the HBV mRNAs can inhibit the gene expression of said virus. Thus, as demonstrated in example 2 of the present invention, the expression of said Ul snRNA in a cell previously transfected with a plasmid that allows to express the HBV mRNA inhibits the expression of the E antigen. Likewise, as observed in the example 3, the expression of different Ul snRNAs in cells expressing a modified HBV genome by replacing the S antigen gene with the sequence encoding luciferase results in a decrease in luciferase activity, indicative of an inhibition of the expression of said gene . On the other hand, as demonstrated in example 4 of the present invention, the administration of said modified Ul snRNAs to an animal that has been administered a plasmid comprising the HBV genome modified by replacing the S antigen with the sequence of luciferase allows to reduce statistically significant activity luciferase in liver compared to animals to which the control plasmid has been administered. Thus, in a first aspect, the invention relates to a compound capable of silencing the hepatitis B virus gene expression wherein said compound is an Ul snRNA, wherein the binding sequence to the consensus consensus sequence GU of the 5 'end of the intron of said snRNA Ul is modified so that it specifically binds to a target sequence of at least one HBV mRNA so that it is capable of inhibiting the maturation of said target mRNA and wherein said target sequence is selected from the group consisting of in the sequences SEQ ID NO: 4-24;
El término "snRNA Ul ", según se usa en el contexto de la presente invención, se entiende como un ARN nuclear pequeño (snRNA por sus iniciales en inglés) que es capaz de incorporarse en la ribonucleoproteína pequeña nuclear (snRNP) Ul y que, conjuntamente con otras snRNPs forma un orgánulo llamado spliceosoma que se encarga del procesamiento o ayuste de los pre-ARNm para dar lugar a los ARNm maduros. snRNA Ul adecuados para su uso en la presente invención incluyen los snRNA Ul que aparecen de forma natural en distintos organismos tales como Uls de distintos mamíferos, incluyendo, sin limitación, Ul snRNA 4 de origen humano (número de acceso NR 004421.1 en NCBI), el Ul snRNA 8 de origen humano (número de acceso NR 004402.2 en NCBI), el Ul snRNA 7 de origen humano (número de acceso NR 004424.2 en NCBI), el Ul snRNA 1 de origen humano (número de acceso NR 004430.2 en NCBI); el Ul snRNA 2 de origen humano (número de acceso NR 004427.2 en NCBI), el Ul snRNA 3 de origen humano (número de acceso NR 004408.1 en NCBI), el Ul snRNA 5 de origen humano (número de acceso NR 004400.1 en NCBI); el Ul snRNA 9 de origen humano (número de acceso NR 004426.2 en NCBI), el U l snRNA 6 de origen humano (número de acceso NR_030723.1 en NCBI), el snRNA Ul de origen bovino (número de acceso M38483 en NCBI), el snRNA Ul de ratón (número de acceso M14121 en NCBI), el snRNA Ul de rata (número de acceso J01883en NCBI) y similares. The term "snRNA Ul", as used in the context of the present invention, is understood as a small nuclear RNA (snRNA) which is capable of being incorporated into the small nuclear ribonucleoprotein (snRNP) Ul and that, Together with other snRNPs, it forms an organelle called spliceosome that is responsible for processing or helping pre-mRNAs to give rise to mature mRNAs. Ul snRNA suitable for use in the present invention include Ul snRNAs that occur naturally in different organisms such as Uls of different mammals, including, without limitation, Ul snRNA 4 of human origin (accession number NR 004421.1 in NCBI), Ul snRNA 8 of human origin (access number NR 004402.2 in NCBI), Ul snRNA 7 of human origin (access number NR 004424.2 in NCBI), Ul snRNA 1 of human origin (access number NR 004430.2 in NCBI) ; Ul snRNA 2 of human origin (access number NR 004427.2 in NCBI), Ul snRNA 3 of human origin (access number NR 004408.1 in NCBI), Ul snRNA 5 of human origin (access number NR 004400.1 in NCBI) ; Ul snRNA 9 of human origin (access number NR 004426.2 in NCBI), U l snRNA 6 of human origin (access number NR_030723.1 in NCBI), snRNA Ul of bovine origin (access number M38483 in NCBI) , mouse snRNA Ul (access number M14121 in NCBI), rat snRNA Ul (access number J01883 in NCBI) and the like.
Asimismo, el término snRNA Ul incluye, de acuerdo con la presente invención, snRNAs Ul cuya secuencia difiere de la secuencia del snRNA Ul nativo y que se denominan de forma genérica, genes snRNA Ul variantes. Ejemplos no limitativos de dichos genes incluyen los que se recogen en la base de datos GenBank con números de acceso L78810, AC025268, AC025264 y AL592207 and las variantes descritas en Kyriakopoulou et al. (R A (2006) 12: 1603- 11). Likewise, the term snRNA Ul includes, according to the present invention, Ul snRNAs whose sequence differs from the sequence of native snRNA Ul and which are generically named, variant snRNA Ul genes. Non-limiting examples of such genes include those collected in the GenBank database with numbers of access L78810, AC025268, AC025264 and AL592207 and the variants described in Kyriakopoulou et al. (RA (2006) 12: 1603-11).
En una forma preferida, el Ul corresponde al Ul snR A 4 humano (Entrez Gene ID: 6060) que corresponde a la secuencia SEQ.ID.NO.2. In a preferred form, the Ul corresponds to the human Ul snR A 4 (Entrez Gene ID: 6060) which corresponds to the sequence SEQ.ID.NO.2.
Asimismo, el término "snRNA Ul" incluye, de acuerdo con la presente invención, variantes funcionalmente equivalentes de los snRNA Ul definidos anteriormente y cuya secuencia deriva de la de éstos mediante inserción, sustitución o eliminación de uno o más nucleótidos y que conservan sustancialmente la capacidad de incorporarse en la snRNP Ul y de promover el procesamiento de los ARNm. Métodos adecuados para determinar la capacidad de un snRNA Ul de incorporarse en la snRNP Ul y de promover el procesamiento de los ARNm incluyen, por ejemplo, el método descrito por Hamm et al. (EMBO J., 1990, 9: 1237-1244) basado en la capacidad del snRNA Ul de promover el procesamiento de un pre-ARNm co-administrado conjuntamente con dicho snRNA Ul en extractos en los que previamente se ha eliminado el Ul endógeno mediante la administración de un oligonucleótido que comprende una secuencia complementaria con el extremo 5' del Ul endógeno promoviendo así la eliminación de dicho extremo por la actividad RNasa H. OK Likewise, the term "snRNA Ul" includes, according to the present invention, functionally equivalent variants of the snRNA Ul defined above and whose sequence derives from that of these by insertion, substitution or elimination of one or more nucleotides and which substantially retain the ability to incorporate into snRNP Ul and promote mRNA processing. Suitable methods for determining the ability of an Ul snRNA to incorporate into the Ul snRNP and to promote mRNA processing include, for example, the method described by Hamm et al. (EMBO J., 1990, 9: 1237-1244) based on the ability of Ul snRNA to promote the processing of a co-administered pre-mRNA in conjunction with said Ul snRNA in extracts in which endogenous Ul has previously been removed by the administration of an oligonucleotide comprising a sequence complementary to the 5 'end of the endogenous Ul thus promoting the elimination of said end by the RNase H activity. OK
Preferiblemente, las variantes funcionalmente equivalentes de los snRNA Ul que aparecen de forma nativa muestran una identidad de secuencia de al menos 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% o 99% en la totalidad de la secuencia con respecto a los snRNA Ul nativos mencionados anteriormente. Algoritmos adecuados para determinar la identidad de secuencias son conocidos en el estado de la técnica, como por ejemplo BLAST, descrito en Altschul et al. (1990) (J Mol Biol. Oct 5 ;215(3) :403-10). Alternativamente, las variantes funcionalmente equivalentes de los snRNA Ul incluyen aquellas que hibridan con las secuencia de snRNA nativos en condiciones severas de hibridación. Las condiciones bajo las cuales hibridan cadenas de ácidos nucleicos totalmente complementarias se denominan "condiciones muy severas de hibridación" o "condiciones de hibridación específicas de secuencia". El experto en la materia puede determinar empíricamente la estabilidad de un dúplex teniendo en cuenta diversas variables, tales como, la longitud y concentración de pares de bases de las sondas, la fuerza iónica y la incidencia de los pares de bases desapareados, siguiendo las directrices del estado de la técnica (véase, por ejemplo, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. (1989); y Wetmur, Critical Reviews in Biochem. and Mol. Biol. 26 (3/4):227-259 ( 1991 )). A modo ilustrativo, en una realización particular, las condiciones severas de hibridación comprenden, por ejemplo, una concentración comprendida entre 0, 15 M y 0,9 M de NaCl y una temperatura comprendida entre 20°C y 65°C. En otra realización particular, las condiciones muy severas de hibridación comprenden, por ejemplo, una concentración comprendida entre 0,02 M y 0,15 M de NaCl y una temperatura comprendida entre 50°C y 70°C. Preferably, the functionally equivalent variants of the snRNA Ul that appear natively show a sequence identity of at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% in the entire sequence with respect to the native Ul snRNAs mentioned above. Suitable algorithms for determining sequence identity are known in the state of the art, such as BLAST, described in Altschul et al. (1990) (J Mol Biol. Oct 5; 215 (3): 403-10). Alternatively, functionally equivalent variants of Ul snRNAs include those that hybridize with native snRNA sequences under severe hybridization conditions. The conditions under which fully complementary nucleic acid chains hybridize are called "very severe hybridization conditions" or "sequence specific hybridization conditions". The person skilled in the art can empirically determine the stability of a duplex taking into account various variables, such as the length and concentration of the base pairs of the probes, the ionic strength and the incidence of the missing base pairs, following the guidelines of the prior art (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989); and Wetmur, Critical Reviews in Biochem. And Mol. Biol. 26 (3/4): 227-259 ( 1991)). By way of illustration, in a particular embodiment, severe hybridization conditions comprise, for example, a concentration between 0.15 M and 0.9 M NaCl and a temperature between 20 ° C and 65 ° C. In another particular embodiment, the very severe hybridization conditions comprise, for example, a concentration between 0.02 M and 0.15 M NaCl and a temperature between 50 ° C and 70 ° C.
Por "secuencia de unión a la secuencia consenso GU del extremo 5 ' del intrón" del snRNA Ul modificado se entiende la región formada por el extremo 5 ' del snRNA Ul que interacciona mediante apareamiento de bases con el denominado sitio Ul o sitio 5' del procesamiento (5 'splice sité) que aparece en los pre-ARNm en la región 5 ' de los intrones, caracterizado por la secuencia consenso AG/GURAGU (en donde R es una purina y / indica el límite exón/intrón) y que permite a la maquinaria de procesamiento de los pre-ARNm la localización del límite exón-intrón Esta región corresponde a los nucleótidos en posiciones 1 a 1 1 en el extremo 5 ' de la secuencia del snRNA Ul (Zhuang, Y y Weiner, A.M: 1986, Cell, 46:827-835). By "binding sequence to the consensus consensus GU of the 5 'end of the intron" of the modified snRNA Ul is understood the region formed by the 5' end of the snRNA Ul that interacts by base pairing with the so-called Ul site or site 5 'of the processing (5 'splice sité) that appears in the pre-mRNA in the 5' region of the introns, characterized by the consensus sequence AG / GURAGU (where R is a purine and / indicates the exon / intron limit) and that allows to the pre-mRNA processing machinery the location of the exon-intron boundary This region corresponds to nucleotides at positions 1 to 1 1 at the 5 'end of the snRNA Ul sequence (Zhuang, Y and Weiner, AM: 1986 , Cell, 46: 827-835).
Por "modificación" de la secuencia consenso de unión al sitio Ul en el pre-ARNm se entiende que dicha secuencia consenso contiene un número de mutaciones que resultan en una pérdida sustancial en la capacidad de snRNA Ul de unirse al sitio Ul en el pre- ARNm a la vez que dicho snRNA Ul adquiere una nueva complementariedad que le permite formar un dúplex mediante apareamiento de bases con una región preseleccionada en un pre-ARNm diana. En la presente invención, las secuencias preseleccionadas corresponden a las secuencias identificadas como SEQ ID NO:4 a 24 y que corresponden a regiones del genoma de HBV. Estos snRNA Ul modificados se denominan Ulin. El grado de complementariedad entre la secuencia de unión al sitio Ul en el snRNA modificado de acuerdo con la presente invención y la región preseleccionada en el pre-ARNm diana puede variar entre 3 y 16 nucleótidos, preferiblemente entre 8 y 16 nucleótidos y, más preferiblemente, entre 10 y 1 1 nucleótidos. Sin embargo, la máxima eficacia en la inhibición de la expresión del gen diana se observa cuando los 10-1 1 nucleótidos que forman el sitio Ul han sido modificados para hacerlos complementarios al sitio preseleccionado en el pre-ARNm diana. Por tanto, preferiblemente, el Ulin se encuentra modificado en todos aquellos nucleótidos en las posiciones 1 a 11 o de 2 a 11 de forma que dicha región sea totalmente complementaria con 1 1 o 10 nucleótidos consecutivos en el sitio preseleccionado del pre-ARNm diana. El experto en la materia apreciará que, para generar el Ulin, será necesario únicamente modificar aquellos nucleótidos en el Ul nativo que sean distintos a los que aparecen en la secuencia diana. By "modification" of the consensus sequence of binding to the Ul site in the pre-mRNA it is understood that said consensus sequence contains a number of mutations that result in a substantial loss in the ability of snRNA Ul to bind to the Ul site in the pre- MRNA while said snRNA Ul acquires a new complementarity that allows it to form a duplex by base pairing with a preselected region in a pre-mRNA target. In the present invention, the preselected sequences correspond to the sequences identified as SEQ ID NO: 4 to 24 and which correspond to regions of the HBV genome. These modified Ul snRNAs are called Ulin. The degree of complementarity between the Ul site binding sequence in the modified snRNA according to the present invention and the region preselected in the target pre-mRNA can vary between 3 and 16 nucleotides, preferably between 8 and 16 nucleotides and, more preferably, between 10 and 1 1 nucleotides. However, maximum efficacy in inhibiting the expression of the target gene is observed when the 10-1 1 nucleotides that form the Ul site have been modified to make them complementary to the pre-selected site in the target pre-mRNA. Therefore, preferably, the Ulin is modified in all those nucleotides at positions 1 to 11 or 2 to 11 so that said region is completely complementary with 1 or 10 consecutive nucleotides at the preselected site of the target pre-mRNA. The person skilled in the art will appreciate that, in order to generate the Ulin, it will be necessary only to modify those nucleotides in the native Ul that are different from those that appear in the target sequence.
El experto en la materia apreciará que las secuencias de los snRNA Ul de distintos organismos son conocidos. Por tanto, una vez que la secuencia de un determinado snRNA Ul es conocida, es posible determinar si este corresponde a un ARN Ul nativo o ha sido modificado en uno o más nucleótidos de la zona de interacción con el extremo 5 ' del intrón. The person skilled in the art will appreciate that the sequences of the snRNA Ul of different organisms are known. Therefore, once the sequence of a given Ul snRNA is known, it is possible to determine whether it corresponds to a native Ul RNA or has been modified in one or more nucleotides of the interaction zone with the 5 'end of the intron.
La modificación en la secuencia del snRNA Ul permite inhibir el procesamiento de los pre-ARNm, entendiendo como procesamiento del extremo 3 ' cualquiera de los pasos necesarios para la formación del ARNm poliadenilado a partir de un pre-ARNm sin poliadenilar (procesamiento del sitio consenso de poliadenilación y adición de la cola de poliA+). Estos snRNA Ul modificados se incorporan en la correspondiente snRNP para dar lugar a snRNP modificadas que, en lugar de unirse al pre-ARNm en el sitio de procesamiento del intrón, se unen al pre-ARNm en una región determinada de éste en función de la especificidad de secuencia de la región modificada en el snRNA Ul . Esta interacción provoca una parada de la maquinaria de poliadenilación que resulta en el secuestro del pre-ARNm en el núcleo, una disminución de la maduración del pre- ARNm y, a su vez, una disminución en la producción de la proteína codificada por dicho pre-mRNA. A la vista de la capacidad de los snRNA Ul modificados de provocar la inhibición de le expresión del pre-ARNm diana, éstos Ul modificados se conocen como Ulin. El efecto de los Ulin es por tanto el provocar una disminución o una inhibición de la expresión génica. Modification in the snRNA Ul sequence makes it possible to inhibit the processing of pre-mRNA, understanding as processing of the 3 ' end any of the steps necessary for the formation of the polyadenylated mRNA from a pre-mRNA without polyadenilar (consensus site processing of polyadenylation and addition of the polyA + tail). These modified Ul snRNAs are incorporated into the corresponding snRNP to give rise to modified snRNPs that, instead of binding to the pre-mRNA at the intron processing site, bind to the pre-mRNA in a given region thereof depending on the sequence specificity of the modified region in snRNA Ul. This interaction causes a stop of the polyadenylation machinery that results in the sequestration of the pre-mRNA in the nucleus, a decrease in the maturation of the pre mRNA and, in turn, a decrease in the production of the protein encoded by said pre -mRNA. In view of the ability of modified Ul snRNAs to cause inhibition of expression of the target pre-mRNA, these modified Ul are known like Ulin. The effect of the Ulin is therefore to cause a decrease or an inhibition of gene expression.
Dado que la mínima longitud óptima de la secuencia diana es de 10 nucleótidos, los sitios de unión para una secuencia de dicha longitud aparecerán de forma aleatoria una vez cada 106 nucleótidos, lo que implica que aparecerán unas 3000 veces en el genoma humano. Sin embargo, dado que los exones constituyen solo un 2% del genoma humano y que la inhibición mediada por snRNP con Ul modificados de la expresión de genes que comprenden exones requiere apareamiento con regiones no estructuradas en los exones terminales de los transcritos que contienen las señales de poliadenilación (AAUAAA), la especificidad de la inhibición es mucho mayor. Preferiblemente, se eligen aquellos sitios diana en el genoma de HBV que aparezcan un menor número de veces en los exones 3 ' terminales del organismo del que proceden las células en las que se va a llevar a cabo el silenciamiento. Since the minimum optimal length of the target sequence is 10 nucleotides, the binding sites for a sequence of that length will appear randomly once every 10 6 nucleotides, implying that they will appear about 3000 times in the human genome. However, since exons constitute only 2% of the human genome and that snRNP-mediated inhibition with modified Ul of gene expression that comprises exons requires mating with unstructured regions in the terminal exons of the transcripts containing the signals Polyadenylation (AAUAAA), the specificity of inhibition is much higher. Preferably, those target sites in the HBV genome are chosen that appear a lesser number of times in the 3 ' terminal exons of the organism from which the cells in which the silencing is to be carried out.
La frecuencia de aparición de las posibles dianas se puede determinar por comparación de secuencias. Típicamente, para la comparación de secuencias, una secuencia actúa como la secuencia de referencia, con la que se comparan las secuencias de prueba. Cuando se utiliza un algoritmo de comparación de secuencias, las secuencias de prueba y l a de referencia se introducen en un ordenador, se designan coordenadas de subsecuencia, si es necesario, y se designan los parámetros del algoritmo de secuencia del programa. Preferiblemente, se pueden usar parámetros de programa por defecto, o se pueden designar parámetros de programa alternativos. El algoritmo de comparación de secuencia calcula los porcentajes de las identidades de secuencia de las secuencias de prueba con respecto a la secuencia de referencia, basándose en los parámetros del programa. The frequency of occurrence of possible targets can be determined by sequence comparison. Typically, for sequence comparison, a sequence acts as the reference sequence, with which the test sequences are compared. When a sequence comparison algorithm is used, the test sequences and the reference sequence are entered into a computer, sub-sequence coordinates are designated, if necessary, and the program sequence algorithm parameters are designated. Preferably, default program parameters may be used, or alternative program parameters may be designated. The sequence comparison algorithm calculates the percentages of the sequence identities of the test sequences with respect to the reference sequence, based on the program parameters.
Una "ventana de comparación", en el contexto de la presente invención, hace referencia a un segmento de cualquiera de las posiciones contiguas seleccionadas del grupo que consiste en 19 a 600, preferiblemente alrededor de 50 a alrededor de 200, más preferiblemente alrededor de 100 a alrededor de 150, en el que una secuencia se puede comparar con una secuencia de referencia con el mismo número de posiciones contiguas, una vez que las dos secuencias se hayan alineado óptimamente. Métodos de alineamiento de secuencias para comparación son ampliamente conocidos en la técnica. Se pueden llevar a cabo alineamientos óptimos de secuencias para comparación, e.g., con el algoritmo de homología local de Smith y Waterman (Adv. Appl. Math., 1981 , 2:482), con el algoritmo de alineamiento por homología de Needleman y Wunsch, (J. Mol. Biol., 1970, 48 :443), por búsqueda de similitud con el método de Pearson y Lipman, (Proc. Nati Acad. Sci. USA, 1988, 85:2444), por implementación computerizada de estos algoritmos (GAP, BESTFIT, FASTA, y TFASTA en el Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), o por alineamiento manual e inspección visual {véase, e.g., Current Protoco ls in Molecular Biology (Ausubel et al, eds. 1995 supplement)} . A "comparison window", in the context of the present invention, refers to a segment of any of the contiguous positions selected from the group consisting of 19 to 600, preferably about 50 to about 200, more preferably about 100 to about 150, in which a sequence can be compared with a reference sequence with the same number of positions contiguous, once the two sequences have been optimally aligned. Sequence alignment methods for comparison are widely known in the art. Optimal sequence alignments can be performed for comparison, eg, with the local homology algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2: 482), with the homology alignment algorithm of Needleman and Wunsch , (J. Mol. Biol., 1970, 48: 443), in search of similarity with the method of Pearson and Lipman, (Proc. Nati Acad. Sci. USA, 1988, 85: 2444), by computerized implementation of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection {see, eg, Current Protoco ls in Molecular Biology ( Ausubel et al, eds. 1995 supplement)}.
Ejemplos preferidos de algoritmos adecuados para determinar el porcentaje de identidad de secuencia y similitud de secuencia, son los algoritmos BLAST y BLAST 2.0, que están descritos en Altschul et al, (Nucí. Acids Res., 1977, 25 :3389-3402) y Altschul et al, (J. Mol. Biol, 1990, 215:403-410), respectivamente. Preferred examples of suitable algorithms for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al, (Nucí. Acids Res., 1977, 25, 3389-3402) and Altschul et al, (J. Mol. Biol, 1990, 215: 403-410), respectively.
Preferiblemente, los snRNAs Ul modificados usados en la composición de la invención se seleccionan de tal manera que su secuencia de unión a la secuencia consenso GU del final 5 ' del intrón, que ha sido modificada de acuerdo con la secuencia del pre-ARNm diana, es idéntica o substancialmente idéntica a los sitios diana. Los términos "idéntico" o "porcentaje de identidad", en el contexto de dos o más ácidos nucleicos o secuencias polipeptídicas, se refieren a dos o más secuencias o sub-secuencias que son la misma (idénticas) o que presentan un porcentaje de residuos de aminoácidos o de nucleótidos que es el mismo (por ejemplo, en al menos 70% de similitud, preferiblemente 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% y 100%. Preferably, the modified Ul snRNAs used in the composition of the invention are selected such that their binding sequence to the consensus consensus sequence of the 5 'end of the intron, which has been modified according to the sequence of the target pre-mRNA, It is identical or substantially identical to the target sites. The terms "identical" or "percent identity", in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or sub-sequences that are the same (identical) or have a percentage of residues of amino acids or nucleotides which is the same (for example, in at least 70% similarity, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, 99% and 100%.
Polinucleótidos y vectores que codifican los Ulin de la invención En otro aspecto, la invención se relaciona con un polinucleótido que codifica un snRNA Ulin de acuerdo a la invención. El término "polinucleótido", según se usa en la presente invención, se refiere a un polímero formado por un número variable de monómeros en donde los monómeros son nucleótidos, incluyendo tanto ribonucleótidos como desoxirribonucleótidos. Los poli nucleótidos incluyen monómeros modificados mediante metilación o así como formas no modificadas. Los términos "polinucleótido" y "ácido nucleico" se usan indistintamente en la presente invención e incluyen mRNA, cDNA y polinucleótidos recombinantes. Según se usa en la presente invención, los polinucleótidos no están limitados a polinucleótidos tal y como aparecen en la naturaleza, sino que incluye po linucleótidos en los que ap arecen análo gos de nucleótidos y enlaces internucleotídicos no naturales. Ejemplos no limitantes de este tipo de estructuras no naturales incluyen polinucleótidos en los que el azúcar es distinto a la ribosa, polinucleótidos en los que aparecen enlaces fosfodiester 3 '-5 ' y 2 '-5', polinucleótidos en los que aparecen enlaces invertidos (3 '-3 ' y 5 '-5 ') y estructuras ramificadas. Adicionalmente, los polinucleótidos de la invención incluyen enlaces internucleotídicos no naturales tales como péptidos ácidos nucleicos (PNA o peptide nucleic acids), ácidos nucleicos cerrados (LNA or locked nucleic acids), enlaces C1-C4 alquilfosfonato del tipo de metilfosfonato, fosforamidato, C1-C6 alquilfosfotrioester, fosforotioato y fosforoditioato. En cualquier caso, los polinucleótidos de la invención mantienen la capacidad de hibridar con ácidos nucleicos diana de forma similar a polinucleótidos naturales. Polynucleotides and vectors encoding the Ulin of the invention In another aspect, the invention relates to a polynucleotide encoding a Ulin snRNA according to the invention. The term "polynucleotide", as used in the present invention, refers to a polymer formed by a variable number of monomers wherein the monomers are nucleotides, including both ribonucleotides and deoxyribonucleotides. The poly nucleotides include monomers modified by methylation or as well as unmodified forms. The terms "polynucleotide" and "nucleic acid" are used interchangeably in the present invention and include mRNA, cDNA and recombinant polynucleotides. As used in the present invention, polynucleotides are not limited to polynucleotides as they appear in nature, but include polynucleotides in which nucleotide analogs and unnatural internucleotide bonds appear. Non-limiting examples of this type of unnatural structures include polynucleotides in which sugar is different from ribose, polynucleotides in which 3 '-5' and 2 '-5' phosphodiester bonds appear, polynucleotides in which inverted bonds appear ( 3 '-3' and 5 '-5') and branched structures. Additionally, polynucleotides of the invention include unnatural internucleotide bonds such as nucleic acid peptides (PNA or peptide nucleic acids), closed nucleic acids (LNA or locked nucleic acids), C1-C4 alkyl phosphonate bonds of the methylphosphonate type, phosphoramidate, C1- C6 alkyl phosphotrioester, phosphorothioate and phosphorodithioate. In any case, the polynucleotides of the invention maintain the ability to hybridize with target nucleic acids similar to natural polynucleotides.
El polinucleótido que codifica el snRNA Ul de acuerdo a la invención puede estar operativamente acoplado a un promotor que permita la transcripción de dicho polinucleótido en la célula en la que se desea la expresión del Ulin. Promotores adecuados para la realización de la presente invención incluyen, sin estar necesariamente limitados, promotores constitutivos tales como el promotor de Ul o derivados de los genomas de virus eucariotas tales como el virus del polioma, adenovirus, SV40, CMV, virus del sarcoma aviar, virus de la hepatitis B, el promotor del gen de la metalotioneina, el promotor del gen de la timidina kinasa del virus del herpes simplex, regiones LTR de los retrovirus, el promotor del gen de la inmunoglobulina, el promotor del gen de la actina, el promotor del gen EF-lalpha así como promotores inducibles en los que la expresión del gen depende de la adición de una molécula o de una señal exógena, tales como el sistema tetraciclina, el sistema NFkappaB/luz UV, el sistema Cre/Lox y el promotor de los genes de choque térmico, los promotores regulables de la ARN polimerasa II descritos en WO/2006/135436 así como promotores específicos de tejido ((por ejemplo, el promotor de PSA descrito en WO2006012221 ). En una forma de realización preferida, los promotores son promotores de la ARN polimerasa II que actúan de forma constitutiva. The polynucleotide encoding the snRNA Ul according to the invention may be operatively coupled to a promoter that allows transcription of said polynucleotide in the cell in which Ulin expression is desired. Promoters suitable for carrying out the present invention include, but are not necessarily limited to, constitutive promoters such as the Ul promoter or derivatives of eukaryotic virus genomes such as polyoma virus, adenovirus, SV40, CMV, avian sarcoma virus, hepatitis B virus, the metallothionein gene promoter, the herpes simplex virus thymidine kinase gene promoter, retrovirus LTR regions, the immunoglobulin gene promoter, the actin gene promoter, the EF-lalpha gene promoter as well as inducible promoters in which gene expression depends on the addition of an exogenous molecule or signal, such as the tetracycline system, the NFkappaB / UV light system, the Cre / Lox system and the heat shock gene promoter, the adjustable RNA polymerase II promoters described in WO / 2006 / 135436 as well as tissue specific promoters ((for example, the PSA promoter described in WO2006012221). In a preferred embodiment, the promoters are constitutive RNA polymerase II promoters.
No obstante, dado que los Ulin de acuerdo a la invención van a ser expresados en células hepáticas, una forma preferida de realización de la presente invención contempla la posibilidad de que el polinucleótido se encuentre bajo control operativo de un promotor específico de hígado. Ejemplos no limitativos de tales promotores son el promotor de la proteína urinaria principal o el promotor de la albúmina. However, since the Ulin according to the invention are to be expressed in liver cells, a preferred embodiment of the present invention contemplates the possibility that the polynucleotide is under operational control of a specific liver promoter. Non-limiting examples of such promoters are the main urinary protein promoter or the albumin promoter.
Adicionalmente, puede usarse en el contexto de la presente invención cualquier promotor específico de hígado descrito en la base de datos de promotores específicos de tejido TiProD tal como se describe por Chen, X. et al., (Nucleic Acids Res., 2006, 34, Datábase Issue). Additionally, any specific liver promoter described in the database of TiProD tissue specific promoters as described by Chen, X. et al., (Nucleic Acids Res., 2006, 34) can be used in the context of the present invention. , Datábase Issue).
En otra forma preferida de realización, el polinucleótido que codifica el Ulin de acuerdo a la invención se encuentra bajo el control del propio promotor del gen que codifica el snRNA Ul del que deriva el Ulin. In another preferred embodiment, the polynucleotide encoding the Ulin according to the invention is under the control of the promoter of the gene encoding the snRNA Ul from which the Ulin is derived.
En otro aspecto, la invención se relaciona con un vector de expresión que comprende un polinucleótido de acuerdo a la invención. Vectores adecuados para la inserción de dichos polinucleótidos son vectores derivados de vectores de expresión en procariotas tales como pUC18, pUC19, Bluescript y sus derivados, mpl 8, mpl9, pBR322, pMB9, CoIEl , pCRl , RP4, fagos y vectores "shuttle" tales como pSA3 and pAT28, vectores de expresión en levaduras tales como vectores del tipo de plásmidos de 2 mieras, plásmidos de integración, vectores YEP, plásmidos centroméricos y similares, vectores de expresión en células de insectos tales como los vectores de la serie pAC y de la serie pVL, vectores de expresión en plantas tales como vectores de la serie pIBI, pEarleyGate, pAVA, pCAMBIA, pGSA, pGWB, pMDC, pMY, pORE y similares y vectores de expresión en células eucariotas superiores, incluyendo baculovirus adecuados para la transfección de células de insectos usando cualquier sistema de baculovirus disponible comercialmente. Los vectores de células eucariotas incluyen preferiblemente vectores virales (adenovirus, virus asociados a los adenovirus así como retrovirus y, en particular, lentivirus) así como vectores no virales tales como pSilencer 4.1-CMV (Ambion), pcDNA3, pcDNA3.1/hyg pHCMV/Zeo, pCR3.1, pEFl/His, pIND/GS, pRc/HCMV2, pSV40/Zeo2, pTRACER-HCMV, pUB6/V5-His, pVAXl, pZeoSV2, pCI, pSVL and pKSV-10, pBPV-1, pML2d y pTDTl. Preferiblemente, los vectores comprenden un gen reportero o marcador que permite identificar aquellas células que han incorporado el vector tras haber sido puestas en contacto con éste. Genes reporteros útiles en el contexto de la presente invención incluyen lacZ, luciferasa, timidina quinasa, GFP y similares. Genes marcadores útiles en el contexto de la presente invención incluyen por ejemplo el gen de resistencia a la neomicina, que confiere resistencia al aminoglucósido G418, el gen de la higromicina fosfotransferasa que confiere resistencia a higromicina, el gen ODC, que confiere resistencia al inhibidor de la ornitina descarboxilasa (2-(difluorometil)-DL-ornitina (DFMO), el gen de la dihidrofolato reductasa que confiere resistencia a metotrexato, el gen de la puromicina-N-acetil transferasa, que confiere resistencia a puromicina, el gen ble que confiere resistencia a zeocina, el gen de la adenosina deaminasa que confiere resistencia a 9-beta-D-xilofuranosil adenina, el gen de la citosina deaminasa, que permite a las células crecer en presencia de N-(fosfonacetil)-L-aspartato, timidina kinasa, que permite a las células crecer en presencia de aminopterina, el gen de Xantina- guanina fosforibosiltransferasa, que permite a las células crecer en presencia de xantina y ausencia de guanina, el gen trpB de E.coli que permite a las células crecer en presencia de indol en lugar de triptófano, el gen hisD de E.coli, que permite a las células el usar histidinol en lugar de histidina. El gen de selección se incorpora en un plásmido que puede incluir, adicionalmente, un promotor adecuado para la expresión de dicho gen en células eucariotas (por ejemplo, los promotores CMV o SV40), un sitio optimizado de iniciación de la traducción (por ejemplo un sitio que sigue las denominadas reglas de Kozak o un IRES), un sitio de poliadenilación como, por ejemplo, el sitio de poliadenilación del SV40 o de la fosfoglicerato quinasa, intrones como, por ejemplo, el intrón del gen de la beta-globulina. Alternativamente, es posible usar una combinación de gen reportero y gen marcador simultáneamente en el mismo vector. En una forma preferida de realización, los vectores son vectores virales. En una forma aún más preferida de realización, los vectores usados para la expresión del primer o del segundo componente de la invención (siempre y cuando éste esté basado en ADN) es un vector viral seleccionado del grupo de adenovirus, virus adeno aso ciado, retrovirus, lentivirus, herpesvirus, SV40 y alfavirus. In another aspect, the invention relates to an expression vector comprising a polynucleotide according to the invention. Suitable vectors for the insertion of said polynucleotides are vectors derived from prokaryotic expression vectors such as pUC18, pUC19, Bluescript and their derivatives, mpl 8, mpl9, pBR322, pMB9, CoIEl, pCRl, RP4, phage and shuttle vectors such such as pSA3 and pAT28, yeast expression vectors such as 2 micron plasmid type vectors, integration plasmids, YEP vectors, centromeric plasmids and the like, insect cell expression vectors such as pAC series vectors and the pVL series, plant expression vectors such as pIBI, pEarleyGate, pAVA, pCAMBIA, pGSA, pGWB, pMDC, pMY, pORE and the like series and Expression vectors in higher eukaryotic cells, including baculoviruses suitable for transfection of insect cells using any commercially available baculovirus system. Eukaryotic cell vectors preferably include viral vectors (adenovirus, adenovirus-associated viruses as well as retroviruses and, in particular, lentiviruses) as well as non-viral vectors such as pSilencer 4.1-CMV (Ambion), pcDNA3, pcDNA3.1 / hyg pHCMV / Zeo, pCR3.1, pEFl / His, pIND / GS, pRc / HCMV2, pSV40 / Zeo2, pTRACER-HCMV, pUB6 / V5-His, pVAXl, pZeoSV2, pCI, pSVL and pKSV-10, pBPV-1, pML2d and pTDTl. Preferably, the vectors comprise a reporter or marker gene that makes it possible to identify those cells that have incorporated the vector after having been contacted with it. Reporter genes useful in the context of the present invention include lacZ, luciferase, thymidine kinase, GFP and the like. Marker genes useful in the context of the present invention include, for example, the neomycin resistance gene, which confers resistance to aminoglycoside G418, the hygromycin phosphotransferase gene that confers hygromycin resistance, the ODC gene, which confers resistance to the inhibitor of Ornithine decarboxylase (2- (difluoromethyl) -DL-ornithine (DFMO), the dihydrofolate reductase gene that confers methotrexate resistance, the puromycin-N-acetyl transferase gene, which confers puromycin resistance, the ble gene confers resistance to zeocin, the adenosine deaminase gene that confers resistance to 9-beta-D-xylofuranosyl adenine, the cytosine deaminase gene, which allows cells to grow in the presence of N- (phosphonacetyl) -L-aspartate, Thymidine kinase, which allows cells to grow in the presence of aminopterin, the Xanthine-guanine phosphoribosyltransferase gene, which allows cells to grow in the presence of xanthine and absence of guani na, the trpB gene of E.coli that allows cells to grow in the presence of indole instead of tryptophan, the hisD gene of E.coli, which allows cells to use histidinol instead of histidine. The selection gene is incorporated into a plasmid which may additionally include a promoter suitable for the expression of said gene in eukaryotic cells (for example, CMV or SV40 promoters), an optimized translation initiation site (for example a site that follows the so-called Kozak rules or an IRES), a polyadenylation site such as, for example, the polyadenylation site of SV40 or phosphoglycerate kinase, introns as, for example, the intron of the beta-globulin gene. Alternatively, it is possible to use a combination of reporter gene and marker gene simultaneously in the same vector. In a preferred embodiment, the vectors are viral vectors. In an even more preferred embodiment, the vectors used for the expression of the first or second component of the invention (as long as it is DNA based) is a viral vector selected from the group of adenovirus, associated adeno virus, retrovirus , lentivirus, herpesvirus, SV40 and alphavirus.
ARN interferentes de la invención Interfering RNAs of the invention
Los autores de la presente invención han puesto de manifiesto que el uso de ARN interferentes con capacidad de unirse a uno o varios de los ARNm de HBV permite inhibir la expresión génica de dicho virus. Así, según se demuestra en el ejemplo 2 de la presente invención, la expresión de dichos ARN interferentes en una célula previamente transfectada con un plásmido que permite expresar el ARNm de HBV inhibe la expresión del antígeno E. Asimismo, según se observa en el ejemplo 3, la expresión de distintos ARN interferentes en células que expresan un genoma de HBV modificado mediante la sustitución del gen del antígeno S por la secuencia que codifica luciferasa resulta en una disminución de la actividad luciferasa, indicativa de una inhibición de la expresión de dicho gen. The authors of the present invention have shown that the use of interfering RNAs capable of binding to one or more of the HBV mRNAs can inhibit the gene expression of said virus. Thus, as demonstrated in example 2 of the present invention, the expression of said interfering RNAs in a cell previously transfected with a plasmid that allows to express the HBV mRNA inhibits the expression of the E antigen. Likewise, as observed in the example 3, the expression of different interfering RNAs in cells expressing a modified HBV genome by replacing the S antigen gene with the sequence encoding luciferase results in a decrease in luciferase activity, indicative of an inhibition of the expression of said gene .
Así, en otro aspecto, la invención se relaciona con un ARN interferente dirigido específicamente a una secuencia diana de al menos un ARNm de HBV que es capaz de provocar el silenciamiento de dicho ARNm de HBV en donde dicha secuencia diana se selecciona del grupo consistente en las secuencias SEQ ID NO:29 a 35; Thus, in another aspect, the invention relates to an interfering RNA specifically directed to a target sequence of at least one HBV mRNA that is capable of causing the silencing of said HBV mRNA wherein said target sequence is selected from the group consisting of the sequences SEQ ID NO: 29 to 35;
Por un "ARN interferente capaz de provocar el silenciamiento de la expresión de un ARNm" se entiende, en el contexto de la presente invención, una molécula de ARN que, cuando se encuentra en el interior de una célula, es capaz bien por sí misma o bien tras su modificación por la maquinaria celular, de unirse al ARNm diana que contiene la secuencia preseleccionada y provocar la degradación de éste, inhibiendo así su traducción y la producción de la proteína codificada por éste. By an "interfering RNA capable of causing the silencing of the expression of an mRNA" is understood, in the context of the present invention, an RNA molecule which, when inside a cell, is capable of itself or after its modification by the cellular machinery, to join the target mRNA that contains the preselected sequence and cause its degradation, thus inhibiting its translation and the production of the protein encoded by it.
Agentes silenciadores que inducen la respuesta RNAi frente a un ARNm diana y que podrían incorporarse en el segundo componente de la invención incluyen, sin limitación, siRNA, shRNA and miRNA Silencers that induce the RNAi response against a target mRNA and that could be incorporated into the second component of the invention include, without limitation, siRNA, shRNA and miRNA
(i) siRNA (i) siRNA
Los siRNA son agentes capaces de inhibir la expresión de un gen diana mediante interferencia de ARN. Un siRNA se puede sintetizar químicamente o se pueden obtener mediante transcripción in vitro. Típicamente, los siRNA consisten en una cadena doble de ARN de entre 15 y 40 nucleótidos de longitud y que puede contener una región protuberante 3 ' y/o 5 ' de 1 a 6 nucleótidos. La longitud de la región protuberante es independiente de la longitud total de la molécula de siRNA. Los siRNA actúan mediante la degradación o el silenciamiento post-transcripcional del mensajero diana. Los siRNA de la invención son sustancialmente homólogos a una región preseleccionada del ARNm diana. Por "sustancialmente homólogos" se entiende que tienen una secuencia que es suficientemente complementaria o similar al ARNm diana de forma que el siRNA sea capaz de provocar la degradación de éste por interferencia de ARN. "Sustancialmente homólogos" puede entenderse como "sustancialmente idénticos" según se ha explicado anteriormente. Los siRNA adecuados para provocar dicha interferencia incluyen siRNA formados por ARN, así como siRNA que contienen distintas modificaciones químicas tales como: SiRNAs are agents capable of inhibiting the expression of a target gene by RNA interference. A siRNA can be chemically synthesized or can be obtained by in vitro transcription. Typically, siRNAs consist of a double strand of RNA between 15 and 40 nucleotides in length and may contain a 3 'and / or 5' protruding region of 1 to 6 nucleotides. The length of the protuberant region is independent of the total length of the siRNA molecule. The siRNAs act by degradation or post-transcriptional silencing of the target messenger. The siRNAs of the invention are substantially homologous to a preselected region of the target mRNA. By "substantially homologous" is meant that they have a sequence that is sufficiently complementary or similar to the target mRNA so that the siRNA is capable of causing degradation of the latter by RNA interference. "Substantially homologous" can be understood as "substantially identical" as explained above. Suitable siRNAs to cause such interference include siRNAs formed by RNA, as well as siRNAs that contain different chemical modifications such as:
- siRNA en los que los enlaces entre los nucleótidos son distintos a los que aparecen en la naturaleza, tales como enlaces fosforotioato. - siRNA in which the bonds between nucleotides are different from those that appear in nature, such as phosphorothioate bonds.
- conjugados de la cadena de siRNA con un reactivo funcional, tal como un fluoróforo. - conjugates of the siRNA chain with a functional reagent, such as a fluorophore.
- Modificaciones de los extremos de las cadenas de siRNA, en particular el extremo 3' mediante la modificación con distintos grupos funcionales del hidroxilo en posición 2'. - Nucleótidos con azúcares modificados tales como restos O-alquilados en posición 2' tales como 2'-0-metilribosa p 2'-0-fluorosibosa. - Modifications of the ends of the siRNA chains, in particular the 3 'end by modification with different hydroxyl functional groups in the 2' position. - Nucleotides with modified sugars such as O-alkylated moieties in 2 'position such as 2'-0-methylribose p 2'-0-fluorosibose.
- Nucleótidos con bases modificadas tales como bases halogenadas (por ejemplo 5-bromouracilo y 5-iodouracilo), bases alquiladas (por ejemplo 7- metilguanosina). - Nucleotides with modified bases such as halogenated bases (for example 5-bromouracil and 5-iodouracil), alkylated bases (for example 7- methylguanosine).
Los siRNA de la invención se pueden obtener usando una serie de técnicas conocidas para el experto en la materia. Por ej emplo, el siRNA puede ser sintetizado químicamente a partir de ribonucleósidos protegidos con grupos forsforamidito en un sintetizador de ADN/ARN convencional. (ii) shRNA The siRNAs of the invention can be obtained using a series of techniques known to the person skilled in the art. For example, the siRNA can be chemically synthesized from ribonucleosides protected with forphoramidite groups in a conventional DNA / RNA synthesizer. (ii) shRNA
En otra forma de realización, el ARN interferente de la invención es shRNA (short hairpin ARN). Por shRNA se entiende, en el contexto de la presente invención, una molécula de ARN formada dos cadenas antiparalelas conectadas por una región horquilla y en donde la secuencia de una de las cadenas antiparalelas es complementaria a una región preseleccionada en el ARNm diana. Los shRNA están compuestos de una secuencia antisentido corta (de 19 a 25 nucleótidos), seguida de un bucle de entre 5 y 9 nucleótidos a la que sigue la cadena sentido . Los shRNA pueden sintetizarse químicamente a partir de ribonucleósidos protegidos con grupos forsforamidito en un sintetizador de ADN/ARN convencional o bien pueden obtenerse a partir de un polinucleótido mediante transcripción in vitro. Los shRNA son procesados en el interior de la célula por la RNasa Dicer que elimina la región de la horquilla dando lugar a siRNAs según se ha descrito anteriormente. Los shRNA también pueden contener distintas modificaciones químicas tal y como se ha descrito anteriormente para el caso de los siRNA. (iii) miRNA In another embodiment, the interfering RNA of the invention is shRNA (short hairpin RNA). ShRNA means, in the context of the present invention, an RNA molecule formed by two antiparallel chains connected by a hairpin region and wherein the sequence of one of the antiparallel chains is complementary to a pre-selected region in the target mRNA. The shRNAs are composed of a short antisense sequence (from 19 to 25 nucleotides), followed by a loop of between 5 and 9 nucleotides followed by the sense chain. The shRNAs can be chemically synthesized from ribonucleosides protected with forsphoramidite groups in a conventional DNA / RNA synthesizer or they can be obtained from a polynucleotide by in vitro transcription. The shRNAs are processed inside the cell by the RNase Dicer that eliminates the region of the hairpin giving rise to siRNAs as described above. The shRNAs may also contain different chemical modifications as described above in the case of siRNAs. (iii) miRNA
Los miRNA o microRNA son pequeñas moléculas de ARN que aparecen de forma natural en la célula y que se encargan de controlar la especificidad de la expresión génica regulando la degradación y la traducción de ARNm. Aunque los miRNA son moléculas endógenas que actúan regulando la vida media de ARNm celulares, se ha demostrado que la estructura de un miRNA endógeno (por ejemplo miR-30) puede alterarse para incluir secuencias que codifican siRNA y que dichos miRNA modificadas pueden ser utilizados para promover la degradación de genes diana endógenos (WO03093441). También se ha demostrado que secuencias siRNA incorporadas en la región del tallo de mir-26a funcionan como efectores de RNAi provocando la degradación de ARNm homólogos (McManus, M.T. et al, 2002, RNA, 8:842-850). En ambos casos, la secuencia de nucléotidos introducida en el tallo del miRNA mostraba un 100% de identidad con el gen diana. The miRNA or microRNA are small RNA molecules that appear naturally in the cell and are responsible for controlling the specificity of gene expression by regulating the degradation and translation of mRNA. Although miRNAs are endogenous molecules that act by regulating the half-life of cellular mRNA, it has been demonstrated that the structure of an endogenous miRNA (for example miR-30) can be altered to include sequences encoding siRNA and that such modified miRNA can be used to promote the degradation of endogenous target genes (WO03093441). It has also been shown that siRNA sequences incorporated in the mir-26a stem region function as effectors of RNAi causing homologous mRNA degradation (McManus, MT et al, 2002, RNA, 8: 842-850). In both cases, the nucleotide sequence introduced into the stem of the miRNA showed 100% identity with the target gene.
Los miRNA adecuados para su uso en las composiciones de la invención constan de 19- 24 nucleótidos, preferiblemente de 21 o 22 núcleo tidos. Los miRNA se pueden diseñar de forma que hibriden a un transcrito de ARN con un alto grado de especificidad. Preferiblemente, el miRNA se diseña de forma que muestra un 100% de identidad o que muestran un alto grado de identidad (por ejemplo, aceptando al menos 1, al menos 2, al menos 3 o más de malos emparejamientos) con el ARNm diana dado que un solo nucleótido no complementario puede, dependiendo de su localización en la cadena de miRNA, disminuir los niveles de inhibición. Los miRNA pueden ser diseñados de forma que se dirijan a la región 5' no traducida, a la región codificante o a la región 3' de un ARNm diana. miRNA adecuados para la realización de la presente invención corresponden a aquellos basados en el miRNA endógeno miR-30, según se ha descrito en WO03093441 o basados en los miRNA endógenos según se ha descrito en WO03029459. MiRNAs suitable for use in the compositions of the invention consist of 19-24 nucleotides, preferably 21 or 22 nucleus tidos. The miRNAs can be designed so that they hybridize to an RNA transcript with a high degree of specificity. Preferably, the miRNA is designed so that it shows 100% identity or shows a high degree of identity (for example, accepting at least 1, at least 2, at least 3 or more of bad pairings) with the given target mRNA. that a single non-complementary nucleotide may, depending on its location in the miRNA chain, decrease inhibition levels. The miRNAs can be designed to address the 5 'untranslated region, the coding region or the 3' region of a target mRNA. miRNA suitable for carrying out the present invention correspond to those based on the endogenous miRNA miR-30, as described in WO03093441 or based on the endogenous miRNA as described in WO03029459.
Polinucleótidos y vectores que codifican un ARN de interferencia de acuerdo a la invención Polynucleotides and vectors encoding an interference RNA according to the invention
En otro aspecto, la invención se relaciona con un polinucleótido cuya transcripción da lugar a un ARN Interferente de acuerdo a la invención. Este polinucleótido puede dar lugar a los siRNA, shRNA y/o miRNA descritos anteriormente. En el caso de polinucleótidos que codifican un shRNA o un miRNA, éstos comprenden una única región promotora que regula la transcripción de una secuencia que comprende las cadenas sentido y antisentido de los shRNA y miRNA conectadas por una horquilla o por una región stem-loop. En principio, cualquier promotor puede ser usado para la expresión de los shRNA y miRNA siempre que dichos promotores sean compatibles con las células en las que se desea expresar los siRNAs. Así, promotores adecuados para la realización de la presente invención incluyen los que se mencionaron anteriormente para la expresión del Ulin. En una forma de realización preferida, los promotores son promotores de la ARN polimerasa III que actúan de forma constitutiva. Los promotores de la ARN polimerasa III aparecen en un número limitado de genes tales como 5S ARN, ARNt, ARN 7SL y ARNsn U6. In another aspect, the invention relates to a polynucleotide whose transcription results in an interfering RNA according to the invention. This polynucleotide can give rise to the siRNA, shRNA and / or miRNA described above. In the case of polynucleotides encoding a shRNA or a miRNA, these comprise a single promoter region that regulates the transcription of a sequence comprising the sense and antisense chains of the shRNA and miRNA connected by a hairpin or by a stem-loop region. In principle, any promoter can be used for expression of shRNA and miRNA provided that said promoters are compatible with the cells in which it is desired to express the siRNAs. Thus, suitable promoters for carrying out the present invention include those mentioned above for Ulin expression. In a preferred embodiment, the promoters are RNA polymerase III promoters that act constitutively. RNA polymerase III promoters appear in a limited number of genes such as 5S RNA, tRNA, 7SL RNA and U6 RNAs.
Por otro lado, los polinucleótidos que codifican los siRNA comprenden dos unidades transcripcionales formadas cada una de ellas por un promotor que regula la transcripción de una de las cadenas que forma en siRNA (sentido y antisentido). Los polinucleótidos que codifican los siRNA pueden contener unidades transcripcionales convergentes o divergentes. En los polinucleótidos de transcripción divergente, las unidades transcripcionales que codifican cada una de las cadenas de ADN que forman el siRNA se encuentran localizadas en tándem en el polinucleótido de forma que la transcripción de cada cadena de ADN depende de su propio promotor, que puede ser igual o distinto (Wang, J. et al, 2003, Proc.Natl.Acad.Sci.USA., 100:5103-5106 y Lee, N.S., et al, 2002, Nat.BiotechnoL, 20:500-505). En los polinucleótidos de transcripción convergente, las regiones de ADN que dan lugar al siRNA se encentran formando las cadenas sentido y antisentido de una región de ADN que se encuentra flanqueada por dos promotores invertidos. Tras la transcripción de las cadenas de ARN sentido y antisentido, éstas formarán el híbrido correspondiente al siRNA funcional. Combinaciones adecuadas de promotores para los polinucleótidos que comprenden unidades transcripcionales invertidas incluyen 2 promotores U6 (Tran, N. et al., 2003, BMC Biotechnol., 3:21), un promotor U6 de ratón y un promotor Hl humano (Zheng, L., et al., 2004, Proc.Natl.Acad.Sci.USA., 101 : 135-140 y WO2005026322) y un promotor U6 humano y un promotor Hl de ratón (Kaykas, A. y Moon, R., 2004, BMC Cell Biol., 5 : 16). En una forma de realización preferida, las cadenas sentido y antisentido del siRNA se encuentran reguladas por promotores distintos. En una forma de realización aún más preferida, ambas unidades transcripcionales se encuentran orientadas de forma convergente. En una forma de realización preferida, los promotores de ARN polimerasa de tipo III son los promotores de los genes Hl y U6 de origen humano o murino. En una forma de realización aún más preferida, los promotores son 2 promotores U6 de origen humano o murino, un promotor U6 de ratón y un promotor Hl humano o un promotor U6 humano y un promotor Hl de ratón. On the other hand, the polynucleotides encoding siRNAs comprise two transcriptional units each formed by a promoter that regulates the transcription of one of the chains that forms in siRNA (sense and antisense). Polynucleotides encoding siRNAs may contain convergent or divergent transcriptional units. In divergent transcription polynucleotides, the transcriptional units encoding each of the DNA chains that form the siRNA are located in tandem in the polynucleotide so that the transcription of each DNA chain depends on its own promoter, which can be same or different (Wang, J. et al, 2003, Proc.Natl.Acad.Sci.USA., 100: 5103-5106 and Lee, NS, et al, 2002, Nat.BiotechnoL, 20: 500-505). In convergent transcription polynucleotides, the regions of DNA that give rise to the siRNA are centered forming the sense and antisense chains of a region of DNA that is flanked by two inverted promoters. After transcription of the sense and antisense RNA chains, they will form the hybrid corresponding to the functional siRNA. Suitable combinations of promoters for polynucleotides comprising inverted transcriptional units include 2 U6 promoters (Tran, N. et al., 2003, BMC Biotechnol., 3:21), a mouse U6 promoter and a human Hl promoter (Zheng, L ., et al., 2004, Proc.Natl.Acad.Sci.USA., 101: 135-140 and WO2005026322) and a human U6 promoter and a mouse Hl promoter (Kaykas, A. and Moon, R., 2004 , BMC Cell Biol., 5:16). In a preferred embodiment, the sense and antisense chains of the siRNA are regulated by different promoters. In an even more preferred embodiment, both transcriptional units are convergently oriented. In a preferred embodiment, type III RNA polymerase promoters are promoters of the Hl and U6 genes of human or murine origin. In an even more preferred embodiment, the promoters are 2 U6 promoters of human or murine origin, a mouse U6 promoter and a human Hl promoter or a human U6 promoter and a mouse Hl promoter.
En otro aspecto, la invención se relaciona con un vector de expresión que comprende un polinucleótido de acuerdo a la invención. Vectores adecuados para la expresión de los ARN interferentes de acuerdo a la invención incluyen, sin limitación, cualquiera de los vectores mencionados anteriormente en el contexto de los vectores para la expresión de los snRNA Ul modificados de acuerdo a la invención. In another aspect, the invention relates to an expression vector comprising a polynucleotide according to the invention. Suitable vectors for the expression of interfering RNAs according to the invention include, without limitation, any of the vectors mentioned above in the context of the vectors for expression of the modified Ul snRNAs according to the invention.
Composiciones de la invención Compositions of the invention
Los autores de la presente invención han demostrado que la combinación de un snRNA Ul modificado específico frente a HBV y de un ARN interferente específico frente a un ARNm de HBV permite inhibir la expresión génica de HBV de forma sinérgica más eficazmente que cada uno de los componentes actuando por separado. Dicha inhibición sinérgica ocurre independiente de que las moléculas de Ulin y ARN interferente vayan dirigidas, respectivamente, a una región del pre-ARNm y a un ARNm procedente de la misma región del genoma o no. Así, en otro aspecto, la invención se relaciona con una composición o un kit que comprende en uno o varios contenedores: The authors of the present invention have shown that the combination of a specific modified Ul snRNA against HBV and a specific interfering RNA against an HBV mRNA allows to inhibit HBV gene expression synergistically more efficiently than each of the components acting separately. Said synergistic inhibition occurs independently of whether the Ulin and interfering RNA molecules are directed, respectively, to a region of the pre-mRNA and to an mRNA from the same region of the genome or not. Thus, in another aspect, the invention relates to a composition or a kit comprising in one or several containers:
(a) un primer componente seleccionado del grupo de:  (a) a first component selected from the group of:
(i) un snRNA Ul, en donde dicho snRNA Ul se encuentra modificado en su secuencia de unión a la secuencia consenso GU del extremo 5 ' del intrón de forma que se una específicamente a una secuencia diana de al menos un ARNm de HBV de forma que es capaz de inhib maduración de dicho ARNm diana,  (i) an Ul snRNA, wherein said Ul snRNA is modified in its binding sequence to the consensus consensus sequence GU of the 5 'end of the intron so that it specifically binds to a target sequence of at least one HBV mRNA so which is capable of inhibiting maturation of said target mRNA,
(ii) Un polinucleótido que codifica un ARN Ul según (i),  (ii) A polynucleotide encoding an Ul RNA according to (i),
(iii) Un vector de expresión que comprende un polinucleótido según (i) y (iii) An expression vector comprising a polynucleotide according to (i) and
(b) un segundo componente seleccionado del grupo de (b) a second component selected from the group of
(iv) Un ARN interferente dirigido específicamente a una secuencia diana del pre-ARNm de HBV que es capaz de provocar el silenciamiento de dicho pre-ARNm de HBV,  (iv) An interfering RNA specifically directed to a target sequence of the HBV pre-mRNA that is capable of causing the silencing of said HBV pre-mRNA,
Un polinucleótido que codifica un ARN de interferencia según (iv) y Un vector de expresión que comprende un polinucleótido según (v). El primer componente de las composiciones de la invención es un snRNA Ul específico frente a HBV tal y como se ha definido anteriormente al describir los snRNA Ul de la invención, un polinucleótido que codifica un ARN Ul o un vector de expresión que comprende un polinucleótido que codifica un ARN Ul . Formas particulares de dicho primer componente se han descrito con detalle anteriormente en el contexto de los snRNA Ul modificados de la invención, de los polinucleótidos que codifican dichos snRNA Ul modificados y de los vectores que contienen dichos polinucleótidos y son igualmente aplicables al primer componente de la invención. En una forma preferida de realización, la secuencia diana en el genoma de HBV frente a la que se une el snRNA Ul modificado se selecciona del grupo consistente en las secuencias SEQ ID NO:4 a 28. A polynucleotide encoding an interference RNA according to (iv) and An expression vector comprising a polynucleotide according to (v). The first component of the compositions of the invention is a specific Ul snRNA against HBV as defined above when describing the Ul snRNA of the invention, a polynucleotide encoding an Ul RNA or an expression vector comprising a polynucleotide that encodes an RNA Ul. Particular forms of said first component have been described in detail above in the context of the modified Ul snRNAs of the invention, of the polynucleotides encoding said modified Ul snRNAs and of the vectors containing said polynucleotides and are equally applicable to the first component of the invention. In a preferred embodiment, the target sequence in the HBV genome against which the modified Ul snRNA binds is selected from the group consisting of the sequences SEQ ID NO: 4 to 28.
El segundo componente de la composición de la invención es un ARN interferente específico frente a la menos un ARNm de HBV, un polinucleótido que codifica dicho ARN Interferente o un vector de expresión que comprende dicho polinucleótido. Formas particulares de las distintas realizaciones de dicho segundo componente de la invención se han descrito con detalle anteriormente en el contexto de los ARN interferentes de la invención, de los polinucleótidos que codifican dichos ARN interferentes y de los vectores que contienen dichos polinucleótidos y son igualmente aplicables al segundo componente de la invención. En una forma preferida de realización, la secuencia diana en el genoma de HBV frente a la que se une el ARN intereferente se selecciona del grupo consistente en las secuencias SEQ ID NO:29 a 35. The second component of the composition of the invention is a specific interfering RNA against at least one HBV mRNA, a polynucleotide encoding said interfering RNA or an expression vector comprising said polynucleotide. Particular forms of the various embodiments of said second component of the invention have been described in detail above in the context of the interfering RNAs of the invention, of the polynucleotides encoding said interfering RNAs and of the vectors containing said polynucleotides and are equally applicable. to the second component of the invention. In a preferred embodiment, the target sequence in the HBV genome against which the interfering RNA binds is selected from the group consisting of the sequences SEQ ID NO: 29 to 35.
En una forma preferida de realización, las composiciones de la invención comprenden componentes caracterizados por su capacidad de unirse a secuencias diana concretas en el ARNm de HBV y que se recogen en la Tabla 1. Tabla 1: Secuencias diana en el genoma de HBV frente a la que se dirigen los Ulin y los ARN interferentes de acuerdo a la invención. In a preferred embodiment, the compositions of the invention comprise components characterized by their ability to bind to specific target sequences in the HBV mRNA and which are listed in Table 1. Table 1: Target sequences in the HBV genome versus which are directed by the Ulin and interfering RNAs according to the invention.
Secuencia diana a la que se une Secuencia diana a la que se une el el snRNA Ul modificado ARN interferente  Target sequence to which it is bound Target sequence to which the snRNA Ul modified RNA interfering binds
SEQ ID NO:4 SEQ ID NO: 29 SEQ ID NO: 4 SEQ ID NO: 29
SEQ ID NO:6 SEQ ID NO: 31 Secuencia diana a la que se une Secuencia diana a la que se une el el snRNA Ul modificado ARN interferente SEQ ID NO: 6 SEQ ID NO: 31 Target sequence to which it is bound Target sequence to which the snRNA Ul modified RNA interfering binds
SEQ ID NO: 8 SEQ ID NO: 33 SEQ ID NO: 8 SEQ ID NO: 33
SEQ ID NO: 8 SEQ ID NO: 29SEQ ID NO: 8 SEQ ID NO: 29
SEQ ID NO: 8 SEQ ID NO: 31SEQ ID NO: 8 SEQ ID NO: 31
SEQ ID NO: 8 SEQ ID NO: 32SEQ ID NO: 8 SEQ ID NO: 32
SEQ ID NO:9 SEQ ID NO: 29SEQ ID NO: 9 SEQ ID NO: 29
SEQ ID NO: 12 SEQ ID NO: 33SEQ ID NO: 12 SEQ ID NO: 33
SEQ ID NO: 12 SEQ ID NO: 29SEQ ID NO: 12 SEQ ID NO: 29
SEQ ID NO: 13 SEQ ID NO: 29SEQ ID NO: 13 SEQ ID NO: 29
SEQ ID NO: 13 SEQ ID NO: 32SEQ ID NO: 13 SEQ ID NO: 32
SEQ ID NO: 14 SEQ ID NO: 33SEQ ID NO: 14 SEQ ID NO: 33
SEQ ID NO: 14 SEQ ID NO: 29SEQ ID NO: 14 SEQ ID NO: 29
SEQ ID NO: 14 SEQ ID NO: 32SEQ ID NO: 14 SEQ ID NO: 32
SEQ ID NO: 15 SEQ ID NO: 29SEQ ID NO: 15 SEQ ID NO: 29
SEQ ID NO: 16 SEQ ID NO: 31SEQ ID NO: 16 SEQ ID NO: 31
SEQ ID NO: 17 SEQ ID NO: 31SEQ ID NO: 17 SEQ ID NO: 31
SEQ ID NO: 18 SEQ ID NO: 31SEQ ID NO: 18 SEQ ID NO: 31
SEQ ID NO:21 SEQ ID NO: 32SEQ ID NO: 21 SEQ ID NO: 32
SEQ ID NO:21 SEQ ID NO: 29SEQ ID NO: 21 SEQ ID NO: 29
SEQ ID NO:21 SEQ ID NO: 32SEQ ID NO: 21 SEQ ID NO: 32
SEQ ID NO:24 SEQ ID NO: 33SEQ ID NO: 24 SEQ ID NO: 33
SEQ ID NO:24 SEQ ID NO: 29SEQ ID NO: 24 SEQ ID NO: 29
SEQ ID NO:24 SEQ ID NO: 32SEQ ID NO: 24 SEQ ID NO: 32
SEQ ID NO:26 SEQ ID NO: 34SEQ ID NO: 26 SEQ ID NO: 34
SEQ ID NO:26 SEQ ID NO: 31 SEQ ID NO: 26 SEQ ID NO: 31
Polinucleótidos bifuncionales de la invención y vectores que comprenden dichos polinucleótidos Bifunctional polynucleotides of the invention and vectors comprising said polynucleotides
Los autores de la presente invención han demostrado que la combinación de un snRNA Ul modificado específico frente a HBV y de un ARN interferente específico frente a HBV permite inhibir la expresión de génica de HBV de forma sinérgica más eficazmente que el cada uno de los componentes actuando por separado. Por tanto, en aquellos casos en los que snRNA Ul modificado y el ARN interferente se aportan como los correspondientes ácidos nucleicos que codifican dichos ARNs, es posible la combinación de ambas secuencias en un único polmucleótido, dando lugar así a un polinucleótido (en adelante polmucleótido bifuncional de la invención) que permite la puesta en contacto del organismo a tratar con los dos componentes necesarios para obtener el efecto sinérgico de forma simultánea mediante una única administración. Así, en otro aspecto, la invención se relaciona con un polinucleótido capaz de silenciar la expresión génica del virus de la hepatitis B que comprende: The authors of the present invention have shown that the combination of a snRNA Specific modified Ul against HBV and a specific interfering RNA against HBV allows to inhibit the expression of HBV gene more synergistically than each of the components acting separately. Therefore, in those cases where modified snRNA Ul and the interfering RNA are provided as the corresponding nucleic acids encoding said RNAs, it is possible to combine both sequences into a single polymucleotide, thus giving rise to a polynucleotide (hereinafter polymucleotide bifunctional of the invention) which allows the contact of the organism to be treated with the two components necessary to obtain the synergistic effect simultaneously by a single administration. Thus, in another aspect, the invention relates to a polynucleotide capable of silencing the gene expression of the hepatitis B virus comprising:
(i) una secuencia que codifica al menos un snRNA Ul modificado en su secuencia de unión a la secuencia consenso GU del extremo 5 ' del sitio de corte del intrón de forma que se une específicamente a una secuencia diana de al menos un ARNm de HBV y es capaz de inhibir el procesamiento de dicho ARNm diana y  (i) a sequence encoding at least one modified Ul snRNA in its binding sequence to the consensus consensus sequence GU of the 5 'end of the intron shear site so that it specifically binds to a target sequence of at least one HBV mRNA and is able to inhibit the processing of said target mRNA and
(ii) una secuencia que codifica un ARN interferente dirigido de forma específica a una secuencia diana de al menos un ARNm de HBV y que es capaz de provocar el silenciamiento de dicho ARNm de HBV. En una forma preferida de realización, el componente (ii) del polinucleótido bifuncional comprende las cadenas sentido y antisentido de al menos un siRNA, codifica al menos un pre-miRNA o codifica al menos un shRNA en donde dichos siRNA, miRNA y shRNA se unen específicamente el ARN Ul codificado por la secuencia definida en (i) y que es capaz de provocar el silenciamiento in vivo de dicho ARNm diana. En una forma preferida de realización, la secuencia diana en el genoma de HBV a la que se une el snRNA Ul modificado codificado por la primera secuencia del polinucleótido bifuncional de la invención se selecciona del grupo consistente en las secuencias SEQ ID NO:4 a 28.  (ii) a sequence that encodes an interfering RNA specifically directed to a target sequence of at least one HBV mRNA and that is capable of causing the silencing of said HBV mRNA. In a preferred embodiment, component (ii) of the bifunctional polynucleotide comprises the sense and antisense chains of at least one siRNA, encodes at least one pre-miRNA or encodes at least one shRNA wherein said siRNA, miRNA and shRNA bind specifically the Ul RNA encoded by the sequence defined in (i) and which is capable of causing the in vivo silencing of said target mRNA. In a preferred embodiment, the target sequence in the HBV genome to which the modified Ul snRNA encoded by the first sequence of the bifunctional polynucleotide of the invention binds is selected from the group consisting of the sequences SEQ ID NO: 4 to 28 .
En una forma preferida de realización, la secuencia diana en el genoma de HBV a la que se une el ARN interferente codificado por la segunda secuencia del polinucleótido bifuncional de la invención se selecciona del grupo consistente en las secuencias SEQ ID NO:29 a 35. In a preferred embodiment, the target sequence in the HBV genome to which the interfering RNA encoded by the second sequence of the bifunctional polynucleotide of the invention binds is selected from the group consisting of the SEQ sequences. ID NO: 29 to 35.
Preferiblemente, el polinucleótido bifuncional de la invención comprende secuencias que codifican combinaciones particulares de snRNA U l modificado y de ARN interferentes que muestran un efecto sinérgico en su capacidad de inhibición de la expresión génica de HBV. Así, en forma preferidas de realización, el polinucleótido bifuncional de la invención comprende secuencias codificantes para snRNA Ul modificados y ARN interferentes que se unen a secuencias diana tal y como se describe anteriormente en la Tabla 1. Preferably, the bifunctional polynucleotide of the invention comprises sequences encoding particular combinations of modified snRNA U1 and interfering RNAs that show a synergistic effect on their ability to inhibit HBV gene expression. Thus, in preferred embodiments, the bifunctional polynucleotide of the invention comprises coding sequences for modified Ul snRNAs and interfering RNAs that bind to target sequences as described above in Table 1.
Los polmucleótidos bifuncionales de la invención se encuentran habitualmente acoplados a regiones reguladoras de su expresión que pueden ser idénticas para las secuencias que codifican el Ulin y para las secuencias que codifican el ARN interferente o pueden ser distintas. Promotores adecuados para la expresión de dichos polmucleótidos son los mismos (constitutivos, regulables y específicos de tejido) que se mencionaron anteriormente para la expresión de los Ulin. Asimismo, los polmucleótidos bifuncionales de la invención pueden estar formando parte de un vector de expresión. Así, en otro aspecto, la invención se relaciona con vectores de expresión que comprenden un polinucleótido bifuncional de la invención. Vectores adecuados para el clonaje y la propagación de los polmucleótidos de la invención son sustancialmente los mismos que se mencionaron anteriormente para la expresión de los polmucleótidos que codifican los Ulin o los polmucleótidos que codifican los ARN interferentes. Preferiblemente, los vectores de expresión de los polinucleótidos de la invención son vectores virales, aún más preferiblemente vectores seleccionados del grupo de adenovirus, virus adenoasociado, retrovirus, lentivirus, herpesvirus, SV40 y alfavirus. The bifunctional polymucleotides of the invention are usually coupled to regulatory regions of their expression that may be identical for the sequences encoding the Ulin and for the sequences encoding the interfering RNA or may be different. Suitable promoters for the expression of said polmucleotides are the same (constitutive, adjustable and tissue specific) mentioned above for the expression of the Ulin. Also, the bifunctional polymucleotides of the invention may be part of an expression vector. Thus, in another aspect, the invention relates to expression vectors comprising a bifunctional polynucleotide of the invention. Suitable vectors for cloning and propagation of the polmucleotides of the invention are substantially the same as mentioned above for the expression of the polmucleotides encoding the Ulin or the polmucleotides encoding the interfering RNAs. Preferably, the expression vectors of the polynucleotides of the invention are viral vectors, even more preferably vectors selected from the group of adenovirus, adeno-associated virus, retrovirus, lentivirus, herpesvirus, SV40 and alphavirus.
Usos médicos de los compuestos, las composiciones y polinucleótidos bifuncionales de la invención Medical uses of the bifunctional compounds, compositions and polynucleotides of the invention
Los autores de la presente invención han demostrado que los compuestos de la invención son capaces de inhibir la expresión génica de HBV. Asimismo, los autores de la presente invención han puesto de manifiesto que las composiciones de la invención y los polinucleótidos bifuncionales son capaces de inhibir la expresión de los genes de HBV de forma sinérgica más eficazmente que cada uno de los componentes actuando por separado. Por ello, los compuestos, composiciones y polinucleótidos bifuncionales son de utilidad para su uso en medicina. Así, en otro aspecto, la invención se relaciona con un compuesto, una composición de la invención o un polinucleótido bifuncional de acuerdo a la presente invención para su uso en medicina o como medicamento. The authors of the present invention have demonstrated that the compounds of the invention are capable of inhibiting HBV gene expression. Also, the authors of The present invention has shown that the compositions of the invention and bifunctional polynucleotides are capable of inhibiting the expression of HBV genes synergistically more efficiently than each of the components acting separately. Therefore, bifunctional compounds, compositions and polynucleotides are useful for use in medicine. Thus, in another aspect, the invention relates to a compound, a composition of the invention or a bifunctional polynucleotide according to the present invention for use in medicine or as a medicine.
En el caso de que la terapia se lleva a cabo usando las composiciones de la invención, el experto en la materia apreciará que existen distintas posibilidades para la administración de la composición de la invención dado que los dos componentes pueden ser administrados como moléculas separadas o como una única molécula. Adicionalmente, tanto el primer como el segundo componente se pueden administrar en forma de moléculas inhibidoras tal cual, es decir, el componente (i) se puede administrar en forma de snRNA Ul y el componente (ii) se puede administrar como un agente silenciador basado en ARN (siARN, miARN, shARN). Alternativamente, el primer componente puede ser aportado como un polinucleótido que codifica el snRNA Ul modificado y/o el segundo componente puede estar basado en ADN, es decir, comprende un polinucleótido que codifica el agente silenciador. En este último caso, los polinucleótidos puede ser administrados como un único polinucleótido que comprende los componentes (i) y (ii) o como polinucleótidos separados, simultánea-, separada- o secuencialmente. In the event that the therapy is carried out using the compositions of the invention, the person skilled in the art will appreciate that there are different possibilities for the administration of the composition of the invention since the two components can be administered as separate molecules or as A single molecule. Additionally, both the first and second components can be administered in the form of inhibitory molecules as is, that is, component (i) can be administered in the form of snRNA Ul and component (ii) can be administered as a based silencing agent in RNA (siRNA, miRNA, shRNA). Alternatively, the first component may be provided as a polynucleotide encoding the modified Ul snRNA and / or the second component may be DNA based, that is, it comprises a polynucleotide encoding the silencing agent. In the latter case, the polynucleotides can be administered as a single polynucleotide comprising components (i) and (ii) or as separate polynucleotides, simultaneously-, separately- or sequentially.
Así, la invención contempla distintas combinaciones de formas de administración que podrán ser determinadas por el experto según las circunstancias. Thus, the invention contemplates different combinations of forms of administration that may be determined by the expert according to the circumstances.
Administración simultánea:  Simultaneous administration:
o componente (i) es un ARNsn Ul como tal y el componente (ii) es un ARN interferente,  or component (i) is an Ul RNA as such and component (ii) is an interfering RNA,
o componente (i) es un ARNsn Ul como tal y el componente (ii) es un polinucleótido que codifica un ARN interferente.  or component (i) is an Ul RNA as such and component (ii) is a polynucleotide encoding an interfering RNA.
o componente (i) es un polinucleótido que codifica el ARNsn Ul y el componente (ii) es un ARN interferente, o componente (i) es un polinucleótido que codifica el AR sn Ul y el componente (ii) es un polinucleótido que un ARN interferente. or component (i) is a polynucleotide that encodes Ul RNAs and component (ii) is an interfering RNA, or component (i) is a polynucleotide encoding the sn sn AR and component (ii) is a polynucleotide that is an interfering RNA.
Administración separada o secuencial  Separate or sequential administration
o componente (i) es un ARNsn Ul como tal y el componente (ii) es un ARN interferente,  or component (i) is an Ul RNA as such and component (ii) is an interfering RNA,
o componente (i) es un ARNsn Ul como tal y el componente (ii) es un polinucleótido que codifica un ARN interferente.  or component (i) is an Ul RNA as such and component (ii) is a polynucleotide encoding an interfering RNA.
o componente (i) es un polinucleótido que codifica el ARNsn Ul y el componente (ii) es un ARN interferente,  or component (i) is a polynucleotide that encodes Ul RNAs and component (ii) is an interfering RNA,
o componente (i) es un polinucleótido que codifica el ARNsn Ul y el componente (ii) es un polinucleótido que un ARN interferente, en cuyo caso ambos componentes deben encontrarse como polinucleótidos separados. Alternativamente cuando el primer y el segundo componente de la composición de la invención se usan en forma de polinucleótidos que codifican las moléculas activas de ARN, dichos polinucleótidos pueden ser aportados formando parte de un vector. En el caso de la administración separada o secuencial, cada polinucleótido formará parte de un vector distinto. En el caso de la administración simultanea, es posible que ambos polinucleótidos formen parte de vectores distintos o del mismo vector.  or component (i) is a polynucleotide that encodes Ul RNAs and component (ii) is a polynucleotide that an interfering RNA, in which case both components must be found as separate polynucleotides. Alternatively, when the first and second components of the composition of the invention are used in the form of polynucleotides encoding the active RNA molecules, said polynucleotides can be provided as part of a vector. In the case of separate or sequential administration, each polynucleotide will be part of a different vector. In the case of simultaneous administration, it is possible that both polynucleotides are part of different vectors or of the same vector.
La capacidad de los compuestos, composiciones y polinucleótidos de la presente invención de inhibir la expresión génica de HBV permite el uso de dichos compuestos, composiciones y polinucleótidos para el tratamiento de enfermedades asociadas a una infección por HBV. Por tanto, en otro aspecto, la invención proporciona un compuesto, una composición, un polinucléotido bifuncional de la invención, o un vector que comprende dicho polinucleótido bifuncional para su uso en el tratamiento o la prevención de una enfermedad causada por una infección por HBV. En otro aspecto, la invención se relaciona con el uso de un compuesto, una composición, un polinucléotido bifuncional de la invención, o un vector que comprende dicho polinucleótido bifuncional para la elaboración de un medicamento para el tratamiento o la prevención de una enfermedad causada por una infección por HBV. The ability of the compounds, compositions and polynucleotides of the present invention to inhibit HBV gene expression allows the use of said compounds, compositions and polynucleotides for the treatment of diseases associated with HBV infection. Therefore, in another aspect, the invention provides a compound, a composition, a bifunctional polynucleotide of the invention, or a vector comprising said bifunctional polynucleotide for use in the treatment or prevention of a disease caused by an HBV infection. In another aspect, the invention relates to the use of a compound, a composition, a bifunctional polynucleotide of the invention, or a vector comprising said bifunctional polynucleotide for the preparation of a medicament for the treatment or prevention of a disease caused by an HBV infection.
En otro aspecto, la invención se relaciona con un método para el tratamiento o la prevención de una enfermedad causada por una infección por HBV en un sujeto que comprende la administración a dicho sujeto de un compuesto, una composición, un polinucléotido bifuncional de la invención o un vector que comprende dicho polinucleótido bifuncional. La expresión "una enfermedad causada por una infección por HBV", según se usa en la presente invención, se refiere a cualquier enfermedad causada por una patología progresiva del hígado y que incluye hepatitis B aguda, hepatitis B crónica, cirrosis y carcinoma hepático. En otro aspecto, la invención se relaciona con preparaciones farmacéuticas que comprenden los compuestos de la invención, las composiciones de la invención o los polinucleótidos bifuncionales de la invención junto con uno o más vehículos farmacéuticamente aceptables. En particular, la invención contempla composiciones farmacéuticas especialmente preparadas para la administración de los ácidos nucleicos que forman el compuesto de la invención, el primer y el segundo componente de la invención o para la administración de los polinucleótidos bifuncionales de la invención. Las composiciones farmacéuticas pueden comprender tanto el primer componente como el segundo componente, tanto si está basado en ADN como en AR en forma desnuda, es decir, en ausencia de compuestos que protejan a los ácidos nucleicos de su degradación por las nucleasas del organismo, lo que conlleva la ventaja de que se elimina la toxicidad asociada a los reactivos usados para la transfección. Rutas de administración adecuadas para los compuestos desnudos incluyen intravascular, intratumoral, intracraneal, intraperitoneal, intraesplénica, intramuscular, subretinal, subcutánea, mucosa, tópica y oral (Templeton, 2002, DNA Cell BioL, 21 :857-867). Los temores iniciales con respecto a la capacidad de éstos compuestos de inducir una respuesta inmune cuando se administración desnudos han sido investigados por Heidel et al. (Nat. Biotechnol., 2004, 22: 1579-1582). Mediante la determinación de los niveles plasmáticos de interleuquinas e interferones tras la administración intraperitoneal e intravenosa de siRNAs, no se pudo observar una respuesta inmune a la vez que se observó que la administración sistémica de siRNA fue bien tolerada. En otra forma de realización, las composiciones y polinucleótidos de la invención se administran mediante la llamada "administración hidrodinámica" en la que los compuestos se introducen en el organismo por vía intravascular a alta velocidad y volumen, lo que resulta en unos elevados niveles de transfección con una distribución más difusa. Una versión modificada de esta técnica ha permitido obtener resultados positivos para el silenciamiento mediante siRNAs desnudos de genes exógenos (Lewis et al, 2002, Nat.Gen., 32: 107-108; McCaffrey et al, 2002, Nature, 418: 38-39) y endógenos (Song et al, 2003, Science, Nat.Med., 9:347-351) en múltiples órganos. En una forma preferida de realización, tanto el Ulin que forma el primer componente de la composición de la invención, como los componentes basados en ARN que forman el segundo componente de la composición de la invención pueden contener modificaciones de su estructura que contribuyen a aumentar su estabilidad. Modificaciones adecuadas para disminuir la sensibilidad a nucleasas incluyen 2'-0- metil (Czauderna et al, 2003, Nucleic Acids Res., 31 :2705-2716), 2'-fluoropirimidinas (Layzer et al, 2004, RNA, 10:766-771). In another aspect, the invention relates to a method for the treatment or prevention of a disease caused by an HBV infection in a subject comprising the administration to said subject of a compound, a composition, a bifunctional polynucleotide of the invention or a vector comprising said bifunctional polynucleotide. The term "a disease caused by an HBV infection", as used in the present invention, refers to any disease caused by a progressive pathology of the liver and which includes acute hepatitis B, chronic hepatitis B, cirrhosis and liver carcinoma. In another aspect, the invention relates to pharmaceutical preparations comprising the compounds of the invention, the compositions of the invention or the bifunctional polynucleotides of the invention together with one or more pharmaceutically acceptable carriers. In particular, the invention contemplates pharmaceutical compositions specially prepared for the administration of the nucleic acids that form the compound of the invention, the first and the second component of the invention or for the administration of the bifunctional polynucleotides of the invention. The pharmaceutical compositions can comprise both the first component and the second component, whether it is based on DNA or on AR in naked form, that is, in the absence of compounds that protect nucleic acids from their degradation by the nucleases of the organism, which entails the advantage that the toxicity associated with the reagents used for transfection is eliminated. Suitable routes of administration for naked compounds include intravascular, intratumoral, intracranial, intraperitoneal, intrasplenic, intramuscular, subretinal, subcutaneous, mucosa, topical and oral (Templeton, 2002, DNA Cell BioL, 21: 857-867). Initial fears regarding the ability of these compounds to induce an immune response when administered nude have been investigated by Heidel et al. (Nat. Biotechnol., 2004, 22: 1579-1582). By determining the levels Plasma of interleukins and interferons after intraperitoneal and intravenous administration of siRNAs, an immune response could not be observed while it was observed that the systemic administration of siRNA was well tolerated. In another embodiment, the compositions and polynucleotides of the invention are administered by the so-called "hydrodynamic administration" in which the compounds are introduced into the body intravascularly at high speed and volume, resulting in high levels of transfection. with a more diffuse distribution. A modified version of this technique has allowed to obtain positive results for the silencing by means of naked siRNAs of exogenous genes (Lewis et al, 2002, Nat.Gen., 32: 107-108; McCaffrey et al, 2002, Nature, 418: 38- 39) and endogenous (Song et al, 2003, Science, Nat.Med., 9: 347-351) in multiple organs. In a preferred embodiment, both the Ulin that forms the first component of the composition of the invention, and the RNA-based components that form the second component of the composition of the invention may contain modifications of their structure that contribute to increasing their stability. Suitable modifications to decrease nuclease sensitivity include 2'-0-methyl (Czauderna et al, 2003, Nucleic Acids Res., 31: 2705-2716), 2'-fluoropyrimidines (Layzer et al, 2004, RNA, 10: 766 -771).
Las composiciones y polinucleótidos de la invención pueden administrarse formando parte de liposomas, conjugadas a colesterol o conjugados a compuestos capaces de promover la translocación a través de membranas celulares tales como el péptido Tat derivado de la proteína TAT de HIV-1 , la tercera hélice del homeodominio de la proteína Antennapedia de D.melanogaster, la proteína VP22 del virus del herpes simplex, oligómeros de arginina y péptidos tales como los descritos en WO07069090 (Lindgren, A. et al., 2000, Trends Pharmacol. Sci, 21 :99-103, Schwarze, S.R. et al., 2000, Trends Pharmacol. Sci., 21 :45-48, Lundberg, M et al, 2003, Mol. Therapy 8: 143- 150 y Snyder, E.L. y Dowdy, S.F., 2004, Pharm. Res. 21 :389-393). Alternativamente, el segundo componente de la invención, cuando está basado en ADN, puede administrarse formando parte de un vector plasmídico o de un vector viral, preferiblemente vectores basados en adenovirus, en virus adenoasociados o en retrovirus, particularmente virus basados en el virus de la leucemia murina (MLV) o en lentivirus (HIV, FIV, EIAV). The compositions and polynucleotides of the invention can be administered as part of liposomes, cholesterol-conjugated or conjugated to compounds capable of promoting translocation through cell membranes such as the Tat peptide derived from the HIV-1 TAT protein, the third helix of the homeodomain of the Antennapedia protein of D.melanogaster, the VP22 protein of herpes simplex virus, arginine oligomers and peptides such as those described in WO07069090 (Lindgren, A. et al., 2000, Trends Pharmacol. Sci, 21: 99- 103, Schwarze, SR et al., 2000, Trends Pharmacol. Sci., 21: 45-48, Lundberg, M et al, 2003, Mol. Therapy 8: 143-150 and Snyder, EL and Dowdy, SF, 2004, Pharm. Res. 21: 389-393). Alternatively, the second component of the invention, when based on DNA, can be administered as part of a plasmid vector or a viral vector, preferably adenovirus-based vectors, adeno-associated viruses or retroviruses, particularly viruses based on murine leukemia virus (MLV) or lentivirus (HIV, IVF, EIAV).
La invención se describe a continuación por medio del siguiente ejemplo que tiene carácter meramente ilustrativo y no limitativo del ámbito de protección. The invention is described below by means of the following example which is merely illustrative and not limitative of the scope of protection.
EJEMPLOS EXAMPLES
EJEMPLO 1. Vectores de expresión de inhibidores de la expresión génica del virus de la hepatitis B (HBV) y otros vectores de expresión utilizados. EXAMPLE 1. Expression vectors of inhibitors of hepatitis B virus (HBV) gene expression and other expression vectors used.
1.1 Plásmidos para la expresión de Ul snRNA inhibidores (Ulin) 1.1 Plasmids for the expression of Ul snRNA inhibitors (Ulin)
Para la construcción de los plásmidos que expresan los Ulin se tomó como referencia la secuencia genómica del virus de la hepatitis B subtipo ayw Galibert que se proporciona como SEQ.ID.NO. l . Los Ulin se construyeron tomando como patrón la secuencia del transcrito de ARN cuyo número de acceso en NCBI es NR 004421.1 , correspondiente al Ul snRNA 4 humano (Entrez Gene ID: 6060) que se incluye como secuencia SEQ.ID.NO.2: For the construction of the plasmids that express the Ulin, the genomic sequence of the hepatitis B virus subtype ayw Galibert was taken as reference, which is provided as SEQ.ID.NO. l. The Ulin were constructed taking as a pattern the sequence of the RNA transcript whose accession number in NCBI is NR 004421.1, corresponding to the human Ul snRNA 4 (Entrez Gene ID: 6060) which is included as sequence SEQ.ID.NO.2:
1 ATACT TACCT GGCAGGGGAG ATACCATGAT CACGAAGGTG GT T T TCCCAG 5 1 GGCGAGGCT T ATCCAT TGCA CTCCGGATGT GCTGACCCCT GCGAT T TCCC 1 0 1 CAAATGTGGG AAACTCGACT GCATAAT T TG TGGTAGTGGG GGACTGCGT T 1 5 1 CGCGCT T TCC CCTG 1 ATACT TACCT GGCAGGGGAG ATACCATGAT CACGAAGGTG GT T T TCCCAG 5 1 GGCGAGGCT T ATCCAT TGCA CTCCGGATGT GCTGACCCCT GCGAT T TCCC 1 0 1 CAAATGTGGG AAACTCGACT GCATAAT T TG TGGTAGTGCGGGGGGGGG
Para la construcción de los plásmidos de expresión para los Ulin se partió del plásmido pGem3z+:Ul-Mscl (SEQ.ID.NO.3), ya descrito por Fortes y cois. (Proc Nati Acad Sci USA. 2003; 100:8264-8269). Este plásmido fue construido sobre el plásmido pGem (Promega), que contiene en el sitio BamHI el gen de Ul snRNA, incluidos el promotor y las secuencias de terminación. El Ul snRNA producido mediante este plásmido se diferencia del Ul snRNA endógeno en que los nucleótidos en las posiciones 98-99 y 109-110 (nucleótidos 498-499 y 509-510 de la SEQ.ID.NO.3; indicados a continuación con doble subrayado) han sido sustituidos por sus nucleótidos complementarios. Estas sustituciones permiten diferenciar los Ul snRNA exógenos del endógeno sin que esté afectada su funcionalidad inhibidora. For the construction of the expression plasmids for the Ulin, the plasmid pGem3z +: Ul-Mscl (SEQ.ID.NO.3), already described by Fortes and cois, was used. (Proc Nati Acad Sci USA. 2003; 100: 8264-8269). This plasmid was constructed on plasmid pGem (Promega), which contains the snRNA Ul gene at the BamHI site, including the promoter and termination sequences. The Ul snRNA produced by this plasmid differs from the endogenous Ul snRNA in that the nucleotides at positions 98-99 and 109-110 (nucleotides 498-499 and 509-510 of SEQ.ID.NO.3; indicated below) with double underlining) have been replaced by their complementary nucleotides. These substitutions allow differentiating the exogenous Ul snRNA from the endogenous without affecting its inhibitory functionality.
SEQ . I D . O . 3 : 1 GGATCCGGTA AGGACCAGCT TCTTTGGGAG AGAACAGACG CAGGGGCGGG I KNOW THAT . I D. O. 3: 1 GGATCCGGTA AGGACCAGCT TCTTTGGGAG AGAACAGACG CAGGGGCGGG
51 AGGGAAAAAG GGAGAGGCAG ACGTCACTTC CCCTTGGCGG CTCTGGCAGC 51 AGGGAAAAAG GGAGAGGCAG ACGTCACTTC CCCTTGGCGG CTCTGGCAGC
101 AGATTGGTCG GTTGAGTGGC AGAAAGGCAG ACGGGGACTG GGCAAGGCAC 101 AGATTGGTCG GTTGAGTGGC AGAAAGGCAG ACGGGGACTG GGCAAGGCAC
151 TGTCGGTGAC ATCACGGACA GGGCGACTTC TATGTAGATG AGGCAGCGCA151 TGTCGGTGAC ATCACGGACA GGGCGACTTC TATGTAGATG AGGCAGCGCA
201 GAGGCTGCTG CTTCGCCACT TGCTGCTTCA CCACGAAGGA GTTCCCGTGC 251 CCTGGGAGCG GGTTCAGGAC CGCTGATCGG AAGTGAGAAT CCCAGCTGTG 201 GAGGCTGCTG CTTCGCCACT TGCTGCTTCA CCACGAAGGA GTTCCCGTGC 251 CCTGGGAGCG GGTTCAGGAC CGCTGATCGG AAGTGAGAAT CCCAGCTGTG
301 TGTCAGGGCT GGAAAGGGCT CGGGAGTGCG CGGGGCAAGT GACCGTGTGT 301 TGTCAGGGCT GGAAAGGGCT CGGGAGTGCG CGGGGCAAGT GACCGTGTGT
351 GTAAAGAGTG AGGCGTATGA GGCTGTGTCG GGGCAGAGGC CCAAGATCTC 351 GTAAAGAGTG AGGCGTATGA GGCTGTGTCG GGGCAGAGGC CCAAGATCTC
401 jjjjiliiillliiil 401 jjjjiliiillliiil
4 51 iiiiiiiM  4 51 iiiiiiiM
501 ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ 501 ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
551 TGGAGTTTCA AAAGTAGACT GTACGCTAAC 601 CGGATCC 551 TGGAGTTTCA AAAGTAGACT GTACGCTAAC 601 CGGATCC
Alineamiento de las secuencias SEQ.ID.NO.2 (RNUl-4) y SEQ.ID.NO.3 (Ul-Mscl): RNUl-4 1 ATACTTACCTGGCAGGGGAGATACCATGATCACGAAGGTGGTTTTCCCAGGGCGAGGCTT 60 Alignment of the sequences SEQ.ID.NO.2 (RNUl-4) and SEQ.ID.NO.3 (Ul-Mscl): RNUl-4 1 ATACTTACCTGGCAGGGGAGATACCATGATCACGAAGGTGGTTTTCCCAGGGCGAGGCTT 60
I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I  I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I
Ul-Mscl 401 ATACTTACCTGGCAGGGGAGATACCATGATCACGAAGGTGGTTTTCCCAGGGCGAGGCTT 460Ul-Mscl 401 ATACTTACCTGGCAGGGGAGATACCATGATCACGAAGGTGGTTTTCCCAGGGCGAGGCTT 460
RNUl-4 61 ATCCATTGCACTCCGGATGTGCTGACCCCTGCGATTTCCCCAAATGTGGGAAACTCGACT 120 RNUl-4 61 ATCCATTGCACTCCGGATGTGCTGACCCCTGCGATTTCCCCAAATGTGGGAAACTCGACT 120
I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I  I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I
Ul-Mscl 461 ATCCATTGCACTCCGGATGTGCTGACCCCTGCGATTTGGCCAAATGTGCCAAACTCGACT 520 Ul-Mscl 461 ATCCATTGCACTCCGGATGTGCTGACCCCTGCGATTTGGCCAAATGTGCCAAACTCGACT 520
RNUl-4 121 GCATAATTTGTGGTAGTGGGGGACTGCGTTCGCGCTTTCCCCTG 164 RNUl-4 121 GCATAATTTGTGGTAGTGGGGGACTGCGTTCGCGCTTTCCCCTG 164
I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I  I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I
Ul-Mscl 521 GCATAATTTGTGGTAGTGGGGGACTGCGTTCGCGCTTTCCCCTG 564 Para la construcción de los plásmidos de expresión de los Ulin seleccionados se introdujeron en el plásmido las sustituciones de nucleótidos necesarias para que los Ul in expresados interaccionaran con un secuencia diana del mRNA de HBV (SEQ.ID.NO. l) elegida. Las sustituciones se realizaron siempre en nucleótidos comprendidos entre los nucleótidos 402-41 1 de pGem3z+:Ul-Mscl, salvo en los plásmidos identificados como Ulin52 a Ulin55 donde las modificaciones comprendían los nucleótidos 402-415. La secuencia modificada se ha indicado en negrita y subrayado. Como ya se ha mencionado, esta es la secuencia de unión a la secuencia consenso GU del extremo 5 ' del intrón. Como resultado de las sustituciones, los Ulin producidos son capaces de unirse con una complementariedad total con una secuencia diana de entre 10 y 11 nucleótidos (14 a 16 en el caso de Ulin52 a Ulin55). En la Tabla 2 se recogen los Ulin ensayados, con sus secuencias diana en el mRNA de HBV. Ul-Mscl 521 GCATAATTTGTGGTAGTGGGGGACTGCGTTCGCGCTTTCCCCTG 564 For the construction of the expression plasmids of the selected Ulin, the nucleotide substitutions necessary for the expressed Ul to interact with a target sequence of the HBV mRNA (SEQ.ID.NO. l) chosen were introduced into the plasmid. Substitutions were always made in nucleotides between nucleotides 402-41 1 of pGem3z +: Ul-Mscl, except in the plasmids identified as Ulin52 to Ulin55 where the modifications comprised nucleotides 402-415. The modified sequence has been indicated in bold and underlined. As already mentioned, this is the sequence of binding to the consensus consensus GU of the 5 'end of the intron. As a result of the substitutions, the produced Ulin are able to bind with total complementarity with a target sequence of between 10 and 11 nucleotides (14 to 16 in the case of Ulin52 to Ulin55). Table 2 shows the Ulin tested, with their target sequences in the HBV mRNA.
Tabla 2. Secuencias diana en el mRNA de HBV para los Ulin inhibidores construidos y/o ensayados. Table 2. Target sequences in the HBV mRNA for the Ulin inhibitors constructed and / or tested.
Agente Secuencia diana SEQ.ID Nucleótidos de la secuencia diana Target sequence agent SEQ.ID Nucleotides of the target sequence
.NO. relativos a la SEQ.ID.NO. 1  .NO. relating to SEQ.ID.NO. one
UlinlB CTCCTCCAGC 4 2285-2294; Corea UlinlB CTCCTCCAGC 4 2285-2294; Core to
UlinlC GCCCCTATCC 5 2311-2320; Core y Pol  UlinlC GCCCCTATCC 5 2311-2320; Core and Pol
Ulin2 CTGGGCT T TA 6 2487-2496; Pol  Ulin2 CTGGGCT T TA 6 2487-2496; Pol
Ulin3 GTAAACCCTG 7 85-94; Pol y SAg  Ulin3 GTAAACCCTG 7 85-94; Pol and SAg
Ulin4 TGACTACTGC 8 98-107; 530-539; 2x Pol y SAg  Ulin4 TGACTACTGC 8 98-107; 530-539; 2x Pol and SAg
Ulin5 CCCAACCTCC 9 321-330; Pol y SAg  Ulin5 CCCAACCTCC 9 321-330; Pol and SAg
Ulin6 CT TGTCCTCC 10 347-356; Pol y SAg  Ulin6 CT TGTCCTCC 10 347-356; Pol and SAg
Ulin7 T TCATCCTGC 11 409-418; Pol y SAg  Ulin7 T TCATCCTGC 11 409-418; Pol and SAg
Ulin8 CCGCTGT TAC 12 796-805; Pol y SAg  Ulin8 CCGCTGT TAC 12 796-805; Pol and SAg
Ulin9 CCCAAGGTCT 13 1643-1652; X prot  Ulin9 CCCAAGGTCT 13 1643-1652; X prot
UlinlO GGGGAGGAGA 14 1745-1754; X prot  UlinlO GGGGAGGAGA 14 1745-1754; X prot
Ulinl l TCACCTCTGC 15 1593-1602; 1827-1836; 2x Pol, X prot y  Ulinl l TCACCTCTGC 15 1593-1602; 1827-1836; 2x Pol, X prot y
SAg  SAg
Ulinl2 TCATCTCT TG 16 1841-1940; Core  Ulinl2 TCATCTCT TG 16 1841-1940; Core
Ulinl3 ACCGCCTCAG 17 1999-2008; Core  Ulinl3 ACCGCCTCAG 17 1999-2008; Core
Ulinl4 ACTCAGGCAA 18 2064-2073; Core  Ulinl4 ACTCAGGCAA 18 2064-2073; Core
Ulinl5 CTCCCTCGCCT 19 2381-2391; Core y Pol  Ulinl5 CTCCCTCGCCT 19 2381-2391; Core and Pol
Ulinl6 GCCTCTCCCT T 20 106-116; Pol y SAg  Ulinl6 GCCTCTCCCT T 20 106-116; Pol and SAg
Ulinl7 CAACCCCCACT 21 1195-2205; Pol  Ulinl7 CAACCCCCACT 21 1195-2205; Pol
Ulinl8 CCCT TCTCCGT 22 1492-1502; Pol y X prot Agente Secuencia diana SEQ.ID Nucleótidos de la secuencia diana Ulinl8 CCCT TCTCCGT 22 1492-1502; Pol and X prot Target sequence agent SEQ.ID Nucleotides of the target sequence
.NO. relativos a la SEQ.ID.NO. 1  .NO. relating to SEQ.ID.NO. one
Ulinl9 CCGACCGACC 23 1511-1520; Pol y X rot  Ulinl9 CCGACCGACC 23 1511-1520; Pol and X rot
Ulin20 CATGTCCTACT 24 1853-1863; Core  Ulin20 CATGTCCTACT 24 1853-1863; Core
Ulin52 GGAGGCTGTAGGCA 25 1778-1791; X prot y core;  Ulin52 GGAGGCTGTAGGCA 25 1778-1791; X prot and core;
Ulin53 ATGCAACT T T T TCA 26 1816-1829; X prot y core;  Ulin53 ATGCAACT T T T TCA 26 1816-1829; X prot and core;
Ulin54 CCTCTGCCTAATCATC 27 1830-1845; Core;  Ulin54 CCTCTGCCTAATCATC 27 1830-1845; Core;
Ulin55 AAGCCTCCAAGCTG 28 1868-1881; Core;  Ulin55 AAGCCTCCAAGCTG 28 1868-1881; Core;
a Las anotaciones Core, X prot, Pol y SAg indican en que mRNA de HBV se localiza la diana del inhibidor: respectivamente el mRNA que codifica para las proteínas virales core, proteína X, polimerasa (pol) y antígeno de superficie (SAg). a The Core, X prot, Pol and SAg annotations indicate in which HBV mRNA the inhibitor target is located: respectively the mRNA encoding core viral proteins, protein X, polymerase (pol) and surface antigen (SAg).
Para los distintos ensayos realizados se utilizó el plásmido pGem3z+:Ul-Mscl como control.  For the different tests performed, plasmid pGem3z +: Ul-Mscl was used as a control.
La construcción de los plásmidos que expresan los diferentes Ulin ensayados se realizó como se describe a continuación: Se partió del plásmido pGem3z+:Ul-Mscl , que se digirió con los enzimas Bcll y BglII para eliminar las secuencias correspondientes al extremo 5 ' del Ul snRNA. Los plásmidos que expresan los Ulin se obtuvieron al introducir en los sitios Bcll y BglII de pGem3z+ :Ul-Mscl los oligonucleótidos hibridados siguientes: The construction of the plasmids expressing the different Ulins tested was performed as described below: The plasmid pGem3z +: Ul-Mscl, which was digested with the enzymes Bcll and BglII was split to eliminate the sequences corresponding to the 5 ' end of the snRNA Ul . The plasmids expressing the Ulin were obtained by introducing the following hybridized oligonucleotides into the Bcll and BglII sites of pGem3z +: Ul-Mscl:
SEQ.I SEQ.I
Oligonucleótido D. Secuencia del oligonucleótido  Oligonucleotide D. Oligonucleotide sequence
Inhib. Primer NO. 5' -> 3'  Inhibit First NO. 5 '-> 3'
UlinlB UliaHBVRIBs 36 GATCTCAgctggaggagGCAGGGGAGATACCAT  UlinlB UliaHBVRIBs 36 GATCTCAgctggaggagGCAGGGGAGATACCAT
UliaHBVRIBas 37 GATCATGGTATCTCCCCTGCCTCCTCCAGCTGA UliaHBVRIBas 37 GATCATGGTATCTCCCCTGCCTCCTCCAGCTGA
UlinlC UliaHBVRICs 38 GATCTCAggataggggcGCAGGGGAGATACCAT UlinlC UliaHBVRICs 38 GATCTCAggataggggcGCAGGGGAGATACCAT
UliaHBVRICas 39 GATCATGGTATCTCCCCTGCGCCCCTATCCTGA UliaHBVRICas 39 GATCATGGTATCTCCCCTGCGCCCCTATCCTGA
Ulin2 UliaHBVR2s 40 GATCTCAtaaagcccagGCAGGGGAGATACCAT Ulin2 UliaHBVR2s 40 GATCTCAtaaagcccagGCAGGGGAGATACCAT
UliaHBVR2as 41 GATCATGGTATCTCCCCTGCCTGGGCTTTATGA UliaHBVR2as 41 GATCATGGTATCTCCCCTGCCTGGGCTTTATGA
Ulin3 UliaHBVfüs 42 GATCTCAcagggtttacGCAGGGGAGATACCAT Ulin3 UliaHBVfüs 42 GATCTCAcagggtttacGCAGGGGAGATACCAT
UliaHBVfüas 43 GATCATGGTATCTCCCCTGCGTAAACCCTGTGA UliaHBVfüas 43 GATCATGGTATCTCCCCTGCGTAAACCCTGTGA
Ulin4 UliaHBVR4s 44 GATCTCAgcagtagtcaGCAGGGGAGATACCAT Ulin4 UliaHBVR4s 44 GATCTCAgcagtagtcaGCAGGGGAGATACCAT
UliaHBVR4as 45 GATCATGGTATCTCCCCTGCTGACTACTGCTGA Ulin5 Ul iaHBVR5s 46 GATCTCAggaggttgggGCAGGGGAGATACCAT Ul iaHBVR5as 47 GATCATGGTATCTCCCCTGCCCCAACCTCCTGAUliaHBVR4as 45 GATCATGGTATCTCCCCTGCTGACTACTGCTGA Ulin5 Ul iaHBVR5s 46 GATCTCAggaggttgggGCAGGGGAGATACCAT Ul iaHBVR5as 47 GATCATGGTATCTCCCCTGCCCCAACCTCCTGA
Ulin6 Ul iaHBVR6s 48 GATCTCAggaggacaagGCAGGGGAGATACCAT Ulin6 Ul iaHBVR6s 48 GATCTCAggaggacaagGCAGGGGAGATACCAT
Ul iaHBVR6as 49 GATCATGGTATCTCCCCTGCCTTGTCCTCCTGA Ul iaHBVR6as 49 GATCATGGTATCTCCCCTGCCTTGTCCTCCTGA
Ulin7 Ul iaHBVR7s 50 GATCTCAgcaggatgaaGCAGGGGAGATACCAT Ulin7 Ul iaHBVR7s 50 GATCTCAgcaggatgaaGCAGGGGAGATACCAT
Ul iaHBVR7as 51 GATCATGGTATCTCCCCTGCTTCATCCTGCTGA Ul iaHBVR7as 51 GATCATGGTATCTCCCCTGCTTCATCCTGCTGA
Ulin8 Ul iaHBVR8s 52 GATCTCAgtaacagcggGCAGGGGAGATACCAT Ulin8 Ul iaHBVR8s 52 GATCTCAgtaacagcggGCAGGGGAGATACCAT
Ul iaHBVR8as 53 GATCATGGTATCTCCCCTGCCCGCTGTTACTGA Ul iaHBVR8as 53 GATCATGGTATCTCCCCTGCCCGCTGTTACTGA
Ulin9 Ul iaHBVR9s 54 GATCTCAagaccttgggGCAGGGGAGATACCAT Ulin9 Ul iaHBVR9s 54 GATCTCAagaccttgggGCAGGGGAGATACCAT
Ul iaHBVR9as 55 GATCATGGTATCTCCCCTGCCCCAAGGTCTTGA Ul iaHBVR9as 55 GATCATGGTATCTCCCCTGCCCCAAGGTCTTGA
UlinlO Ul iaHBVRlOs 56 GATCTCAtctcctccccGCAGGGGAGATACCAT UlinlO Ul iaHBVRlOs 56 GATCTCAtctcctccccGCAGGGGAGATACCAT
Ul iaHBVRlOas 57 GATCATGGTATCTCCCCTGCGGGGAGGAGATGA Ul iaHBVRlOas 57 GATCATGGTATCTCCCCTGCGGGGAGGAGATGA
Ulinl l Ul iaHBVRl ls 58 GATCTCAgcagaggtgaGCAGGGGAGATACCAT Ulinl l Ul iaHBVRl ls 58 GATCTCAgcagaggtgaGCAGGGGAGATACCAT
Ul iaHBVRl las 59 GATCATGGTATCTCCCCTGCTCACCTCTGCTGA Ul iaHBVRl the 59 GATCATGGTATCTCCCCTGCTCACCTCTGCTGA
Ulinl2 Ul iaHBVR12s 60 GATCTCAcaagagatgaGCAGGGGAGATACCAT Ulinl2 Ul iaHBVR12s 60 GATCTCAcaagagatgaGCAGGGGAGATACCAT
Ul iaHBVR12as 61 GATCATGGTATCTCCCCTGCTCATCTCTTGTGA Ul iaHBVR12as 61 GATCATGGTATCTCCCCTGCTCATCTCTTGTGA
Ulinl3 Ul iaHBVR13s 62 GATCTCActgaggcggtGCAGGGGAGATACCAT Ulinl3 Ul iaHBVR13s 62 GATCTCActgaggcggtGCAGGGGAGATACCAT
Ul iaHBVR13as 63 GATCATGGTATCTCCCCTGCACCGCCTCAGTGA Ul iaHBVR13as 63 GATCATGGTATCTCCCCTGCACCGCCTCAGTGA
Ulinl4 Ul iaHBVR14s 64 GATCTCAttgcctgagtGCAGGGGAGATACCAT Ulinl4 Ul iaHBVR14s 64 GATCTCAttgcctgagtGCAGGGGAGATACCAT
Ul iaHBVR14as 65 GATCATGGTATCTCCCCTGCACTCAGGCAATGA Ul iaHBVR14as 65 GATCATGGTATCTCCCCTGCACTCAGGCAATGA
Ulinl5 Ul iaHBVR15s 66 GATCTCAggcgagggagGCAGGGGAGATACCAT Ulinl5 Ul iaHBVR15s 66 GATCTCAggcgagggagGCAGGGGAGATACCAT
Ul iaHBVR15as 67 GATCATGGTATCTCCCCTGCCTCCCTCGCCTGA Ul iaHBVR15as 67 GATCATGGTATCTCCCCTGCCTCCCTCGCCTGA
Ulinl6 Ul iaHBVR16s 68 GATCTCAagggagaggcGCAGGGGAGATACCAT Ulinl6 Ul iaHBVR16s 68 GATCTCAagggagaggcGCAGGGGAGATACCAT
Ul iaHBVR16as 69 GATCATGGTATCTCCCCTGCGCCTCTCCCTTGA Ul iaHBVR16as 69 GATCATGGTATCTCCCCTGCGCCTCTCCCTTGA
Ulinl7 Ul iaHBVR17s 70 GATCTCAgtgggggttgGCAGGGGAGATACCAT Ulinl7 Ul iaHBVR17s 70 GATCTCAgtgggggttgGCAGGGGAGATACCAT
Ul iaHBVR17as 71 GATCATGGTATCTCCCCTGCCAACCCCCACTGA Ul iaHBVR17as 71 GATCATGGTATCTCCCCTGCCAACCCCCACTGA
Ulinl8 Ul iaHBVR18s 72 GATCTCAcggagaagggGCAGGGGAGATACCAT Ulinl8 Ul iaHBVR18s 72 GATCTCAcggagaagggGCAGGGGAGATACCAT
Ul iaHBVR18as 73 GATCATGGTATCTCCCCTGCCCCTTCTCCGTGA Ul iaHBVR18as 73 GATCATGGTATCTCCCCTGCCCCTTCTCCGTGA
Ulinl9 Ul iaHBVR19s 74 GATCTCAggtcggtcggGCAGGGGAGATACCAT Ulinl9 Ul iaHBVR19s 74 GATCTCAggtcggtcggGCAGGGGAGATACCAT
Ul iaHBVR19as 75 GATCATGGTATCTCCCCTGCCCGACCGACCTGA Ul iaHBVR19as 75 GATCATGGTATCTCCCCTGCCCGACCGACCTGA
Ulin20 Ul iaHBVR20s 76 GATCTCAgtaggacatgGCAGGGGAGATACCAT Ulin20 Ul iaHBVR20s 76 GATCTCAgtaggacatgGCAGGGGAGATACCAT
Ul iaHBVR20as 77 GATCATGGTATCTCCCCTGCCATGTCCTACTGA Ulin52 UliaHBVR52s 78 GATCATGGTATCTCCCGGAGGCTGTAGGCATGA UliaHBVR52as 79 GATC T C ATGCCTACAGCCTCCGGG AG AT AC CATUl iaHBVR20as 77 GATCATGGTATCTCCCCTGCCATGTCCTACTGA Ulin52 UliaHBVR52s 78 GATCATGGTATCTCCCGGAGGCTGTAGGCATGA UliaHBVR52as 79 GATC TC ATGCCTACAGCCTCCGGG AG AT AC CAT
Ulin53 UliaHBVR53s 80 GATCATGGTATCTCCCATGCAACTTTTTCATGA Ulin53 UliaHBVR53s 80 GATCATGGTATCTCCCATGCAACTTTTTCATGA
UliaHBVR53as 81 GATCTCATGAAAAAGTTGCATGGGAGATACCAT UliaHBVR53as 81 GATCTCATGAAAAAGTTGCATGGGAGATACCAT
Ulin54 UliaHBVR54s 82 GATCATGGTATCTCCCTCTGCCTAATCATCTGA Ulin54 UliaHBVR54s 82 GATCATGGTATCTCCCTCTGCCTAATCATCTGA
UliaHBVR54as 83 G AT C T C AGATGATTAGGCAGAGGG AG AT AC C AT UliaHBVR54as 83 G AT C T C AGATGATTAGGCAGAGGG AG AT AC C AT
Ulin55 UliaHBVR55s 84 GATCATGGTATCTCCCAAGCCTCCAAGCTGTGA Ulin55 UliaHBVR55s 84 GATCATGGTATCTCCCAAGCCTCCAAGCTGTGA
UliaHBVR55as 85 GATCTCACAGCTTGGAGGCTTGGGAGATACCAT UliaHBVR55as 85 GATCTCACAGCTTGGAGGCTTGGGAGATACCAT
La secuencia complementaria a la secuencia diana se ha resaltado en negrita. The sequence complementary to the target sequence has been highlighted in bold.
1.2 Plásmidos para la expresión de shRNA inhibidores 1.2 Plasmids for the expression of shRNA inhibitors
Igualmente, se construyeron y ensayaron una serie de plásmidos para la expresión de diversos shRNA inhibidores de HBV, con las secuencias diana en el mRNA de HBV que se indican en la Tabla 2. Likewise, a series of plasmids were constructed and tested for the expression of various shRNA inhibitors of HBV, with the target sequences in the HBV mRNA indicated in Table 2.
Los plásmidos para la expresión de los shRNA se generaron sobre un plásmido pSuper clonando las secuencias de oligonucleótidos que comprenden las secuencias diana de la Tabla 3. Para ello se partió del plásmido pSuper (Brummelkamp et al. Science. 2002; 296(5567): 550-553). Este plásmido se digirió con BglII y HindI y en esto sitios se clonaron los oligonucleótidos hibridados que se indican a continuación: Plasmids for the expression of shRNAs were generated on a plasmid pSuper by cloning the oligonucleotide sequences comprising the target sequences of Table 3. To this end, the plasmid pSuper (Brummelkamp et al. Science. 2002; 296 (5567): 550-553). This plasmid was digested with BglII and HindI and in this sites the hybridized oligonucleotides indicated below were cloned:
Inhibidor Inhibitor
SEQ.ID.NO.  SEQ.ID.NO.
Oligonucleótido - Primer (5' -> 3')  Oligonucleotide - Primer (5 '-> 3')
shRNA 1  shRNA 1
86 GATCCCCGGTCTTACATAAGAGGACTTTCAAGAGAAGTCCTCTTATGTAAGACCTTTTTGGAAA g7 AGCTTTTCCAAAAAGGTCTTACATAAGAGGACTTCTCTTGAAAGTCCTCTTATGTAAGACCGGG shRNA2  86 GATCCCCGGTCTTACATAAGAGGACTTTCAAGAGAAGTCCTCTTATGTAAGACCTTTTTGGAAA g7 AGCTTTTCCAAAAAGGTCTTACATAAGAGGACTTCTCTTGAAAGTCCTCTTATGTAAGACCGGG shRNA2
GATCCCCCTCAGTTTACTAGTGCCATTTCAAGAGAATGGCACTAGTAAACTGAGTTTTTGGAAA  GATCCCCCTCAGTTTACTAGTGCCATTTCAAGAGAATGGCACTAGTAAACTGAGTTTTTGGAAA
89 AGCTTTTCCAAAAACTCAGTTTACTAGTGCCATTCTCTTGAAATGGCACTAGTAAACTGAGGGG shR A3 89 AGCTTTTCCAAAAACTCAGTTTACTAGTGCCATTCTCTTGAAATGGCACTAGTAAACTGAGGGG shR A3
90 GATCCCCCATCACATCAGGATTCCTATTCAAGAGATAGGAATCCTGATGTGATGTTTTTGGAAA 90 GATCCCCCATCACATCAGGATTCCTATTCAAGAGATAGGAATCCTGATGTGATGTTTTTGGAAA
91 AGCTTTTCCAAAAACATCACATCAGGATTCCTATCTCTTGAATAGGAATCCTGATGTGATGGGG shR A4 91 AGCTTTTCCAAAAACATCACATCAGGATTCCTATCTCTTGAATAGGAATCCTGATGTGATGGGG shR A4
92 GATCCCCGTTCAGTGGTTCGTAGGGCTTCAAGAGAGCCCTACGAACCACTGAACTTTTTGGAAA 92 GATCCCCGTTCAGTGGTTCGTAGGGCTTCAAGAGAGCCCTACGAACCACTGAACTTTTTGGAAA
93 AGCTTTTCCAAAAAGTTCAGTGGTTCGTAGGGCTCTCTTGAAGCCCTACGAACCACTGAACGGG shR A5 93 AGCTTTTCCAAAAAGTTCAGTGGTTCGTAGGGCTCTCTTGAAGCCCTACGAACCACTGAACGGG shR A5
94 GATCCCCATCAAGGTATGTTGCCCGTTTCAAGAGAACGGGCAACATACCTTGATTTTTTGGAAA 94 GATCCCCATCAAGGTATGTTGCCCGTTTCAAGAGAACGGGCAACATACCTTGATTTTTTGGAAA
95 AGCTTTTCCAAAAAATCAAGGTATGTTGCCCGTTCTCTTGAAACGGGCAACATACCTTGATGGG shR A6 95 AGCTTTTCCAAAAAATCAAGGTATGTTGCCCGTTCTCTTGAAACGGGCAACATACCTTGATGGG shR A6
96 GATCCCCGCTGGATGTGTCTGCGGCGTTCAAGAGACGCCGCAGACACATCCAGCTTTTTGGAAA 96 GATCCCCGCTGGATGTGTCTGCGGCGTTCAAGAGACGCCGCAGACACATCCAGCTTTTTGGAAA
97 AGCTTTTCCAAAAAGCTGGATGTGTCTGCGGCGTCTCTTGAACGCCGCAGACACATCCAGCGGG shR A7 97 AGCTTTTCCAAAAAGCTGGATGTGTCTGCGGCGTCTCTTGAACGCCGCAGACACATCCAGCGGG shR A7
98 GATCCCCGCAATGTCAACGACCGACCTTCAAGAGAGGTCGGTCGTTGACATTGCTTTTTGGAAA 98 GATCCCCGCAATGTCAACGACCGACCTTCAAGAGAGGTCGGTCGTTGACATTGCTTTTTGGAAA
99 AGCTTTTCCAAAAAGCAATGTCAACGACCGACCTCTCTTGAAGGTCGGTCGTTGACATTGCGGG 99 AGCTTTTCCAAAAAGCAATGTCAACGACCGACCTCTCTTGAAGGTCGGTCGTTGACATTGCGGG
Tabla 3. Secuencias diana en el mRNA de HBV para los shRNA inhibidores construidos y/o ensayados. Table 3. Target sequences in the HBV mRNA for the shRNA inhibitors constructed and / or tested.
Nucleótidos de la secuencia SEQ.ID. diana relativos a la Nucleotides of the sequence SEQ.ID. target related to
Agente Secuencia diana NO. SEQ.ID.NO.l shR A4 GT TCAGTGGT TCGTAGGGC 29 694-712 Pol y SAg shR A5 ATCAAGGTATGT TGCCCGT 30 455-473 Pol y SAg shR A6 GCTGGATGTGTCTGCGGCGT 31 374-393 Pol y SAg shR A7 GC AAT G T CAAC G AC C G AC C 32 1679-1698 X prot shRNA 1 GGTCT TACATAAGAGGACT 33 1648-1666 X prot shRNA2 CTCAGT T TACTAGTGCCAT 34 672-691 Pol y SAg shRNA3 CATCACATCAGGAT TCCTA 35 165-183 Pol y SAg  Target Sequence Agent NO. SEQ.ID.NO.l shR A4 GT TCAGTGGT TCGTAGGGC 29 694-712 Pol and SAg shR A5 ATCAAGGTATGT TGCCCGT 30 455-473 Pol and SAg shR A6 GCTGGATGTGTCTGCGGCGT 31 374-393 Pol and SAg shR A7 GC AC CG ACAC ACG GC ACAC C 32 1679-1698 X prot shRNA 1 GGTCT TACATAAGAGGACT 33 1648-1666 X prot shRNA2 CTCAGT T TACTAGTGCCAT 34 672-691 Pol and SAg shRNA3 CATCACATCAGGAT TCCTA 35 165-183 Pol y SAg
1.2 Otros plásmidos utilizados en los ensayos El plásmido pSP65-ayw 1.3 que contiene una copia del HBV (Chisari et al. Science. 1985; 230(4730): 1157-1 160) fue facilitado por el Dr. Chisari. En adelante lo denominaremos pHBV. 1.2 Other plasmids used in the assays Plasmid pSP65-ayw 1.3 containing a copy of HBV (Chisari et al. Science. 1985; 230 (4730): 1157-1 160) was provided by Dr. Chisari. Hereinafter we will call it pHBV.
El plásmido pRL-SV40 (Promega, E2231) se utilizó como control de transfección de la luciferasa de Renilla. Plasmid pRL-SV40 (Promega, E2231) was used as a transfection control of Renilla luciferase.
El plásmido pHBV-Luc (Ely et al. Mol Ther. 2008; 16(6): 1105-1112) fue proporcionado por el Dr. Arbuthnot y contiene la secuencia que codificante de HBV en la que la región del antígeno E ha sido sustituida por una secuencia que codifica la luciferasa de luciérnaga. Todos los clones se verificaron por secuenciación en ABI Prism 310 genetic analyzer (Applied Biosystems). El DNA plasmídico se purificó con un kit Maxiprep (Marlingen) antes de la transfección. The plasmid pHBV-Luc (Ely et al. Mol Ther. 2008; 16 (6): 1105-1112) was provided by Dr. Arbuthnot and contains the HBV coding sequence in which the region of the E antigen has been replaced by a sequence encoding firefly luciferase. All clones were verified by sequencing in ABI Prism 310 genetic analyzer (Applied Biosystems). Plasmid DNA was purified with a Maxiprep kit (Marlingen) before transfection.
EJEMPLO 2. Análisis in vitro de inhibición de la expresión génica de HBV. EXAMPLE 2. In vitro analysis of inhibition of HBV gene expression.
Los ensayos se realizaron sobre células HuH7 (ATCC), que se cultivaron en medio DMEM suplementado con 10% de suero bovino fetal (FBS) y 1% de penicilina- estreptomicina, a 37 °C en una atmósfera con 5% C02. The tests were performed on HuH7 cells (ATCC), which were grown in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, at 37 ° C in an atmosphere with 5% C0 2 .
Todos los reactivos para el cultivo se obtuvieron de Gibco BRL/Life Technologies. All culture reagents were obtained from Gibco BRL / Life Technologies.
Las células HuH7 fueron transfectadas con fosfato de calcio. HuH7 cells were transfected with calcium phosphate.
Las células HuH7 (1,5 x 105 células) fueron co-transfectadas con a. el plásmido pHBV (1 μg), que expresa el mR A de HBV; y b. el plásmido control pGem3z+:Ul-Mscl (pMock) que expresa un Ul snRNA natural (1 μg); o alternativamente, uno o varios de los plásmidos inhibidores (1 μg en total) que expresan un inhibidor de HBV, bien sea Ulin o shR A. HuH7 cells (1.5 x 10 5 cells) were co-transfected with a. the plasmid pHBV (1 μg), which expresses the mR A of HBV; and b. the control plasmid pGem3z +: Ul-Mscl (pMock) expressing a natural Ul snRNA (1 μg); or alternatively, one or more of the inhibitor plasmids (1 μg in total) expressing an HBV inhibitor, either Ulin or shR A.
Tres días después de la transfección, se evaluó la expresión viral mediante la valoración en el sobrenadante del antígeno E (EAg). La valoración se realizó mediante ELISA [Diasorin. ETI-EBK PLUS (HBeAg)], en 10 μΐ, de sobrenadante y siguiendo las instrucciones del fabricante. Three days after transfection, viral expression was evaluated by titration in the E antigen supernatant (EAg). The assessment was performed using ELISA [Diasorin. ETI-EBK PLUS (HBeAg)], in 10 μΐ, of supernatant and following the manufacturer's instructions.
Finalmente se calculó el Factor de Inhibición (F.I.), que representa el número de veces que se inhibe la expresión génica, y que se obtiene mediante el cociente del EAg medido en el sobrenadante de las células transfectadas con el plásmido control (Mock), entre el EAg medido en el sobrenadante de las células transfectadas con el plásmido inhibidor a evaluar: Finally, the Inhibition Factor (FI) was calculated, which represents the number of times gene expression is inhibited, and is obtained by the ratio of EAg measured in the supernatant of cells transfected with the control plasmid (Mock), between The EAg measured in the supernatant of cells transfected with the inhibitor plasmid to be evaluated:
F.I. = [ EAg ]Mock / [ EAg JlnMbidor FI = [EAg] M ock / [EAg JlnMbidor
Todos los plásmidos para la expresión de uno de los shRNA seleccionados proporcionaron factores de inhibición superiores a 3, siendo particularmente activos los que expresaban shRNA2 y shRNA6 (ver Fig.2). All plasmids for the expression of one of the selected shRNAs provided inhibition factors greater than 3, those expressing shRNA2 and shRNA6 being particularly active (see Fig. 2).
Igualmente, todos los plásmidos que expresan uno de los Ulin seleccionados fueron capaces de disminuir la expresión de EAg (ver Fig. 3), aunque con una menor eficiencia que los shRNA. Por otra parte, en ensayos paralelos se comparó la actividad inhibidora en células transfectadas con uno de los plásmidos inhibidores o co-transfectadas con 2 plásmidos inhibidores, uno que expresaba un shRNA y el otro que expresaba un Ulin. Para ello se transfectaron células HuH7 (1,5 105 células) con: a. el plásmido pHBV (1 μg), que expresa el mRNA de HBV; y b. 1 μg del plásmido control pGem3z+:Ul-Mscl (pMock) que expresa un Ul snRNA natural; o 0,5 μg del plásmido control pGem3z+:Ul-Mscl y 0,5 μg del plásmido que expresa un inhibidor, bien sea Ulin o shRNA, o 0,5 μg del plásmido que expresa un Ulin y 0,5 μg del plásmido que expresa un shRNA. Likewise, all plasmids expressing one of the selected Ulin were able to decrease EAg expression (see Fig. 3), although with a lower efficiency than shRNA. On the other hand, in parallel trials, the inhibitory activity in cells transfected with one of the inhibitor plasmids or co-transfected with 2 inhibitor plasmids was compared, one expressing a shRNA and the other expressing a Ulin. For this, HuH7 cells (1.5 10 5 cells) were transfected with: a. plasmid pHBV (1 μg), which expresses the HBV mRNA; and b. 1 μg of the control plasmid pGem3z +: Ul-Mscl (pMock) expressing a natural Ul snRNA; or 0.5 μg of the control plasmid pGem3z +: Ul-Mscl and 0.5 μg of the plasmid expressing an inhibitor, either Ulin or shRNA, or 0.5 μg of the plasmid expressing a Ulin and 0.5 μg of the plasmid that expresses a shRNA.
Nótese que en el experimento anterior (Figuras 2 y 3) se utilizaba una dosis de 1 μg del plásmido que expresa un inhibidor mientras que en este experimento se usan 0,5 μg del plásmido que expresa un inhibidor, o el otro o de cada uno de ellos. Con esto se consigue que la suma de los dos inhibidores sea de 1 μg, lo máximo que se puede poner en la mezcla de transfección. Note that in the previous experiment (Figures 2 and 3) a dose of 1 μg of the plasmid expressing an inhibitor was used while in this experiment 0.5 μg of the plasmid expressing an inhibitor is used, or the other or each one from them. With this I know it achieves that the sum of the two inhibitors is 1 μg, the maximum that can be put in the transfection mixture.
Se pudo comprobar (ver Fig. 4) que la combinación de los inhibidores que expresa un shRNA con los que expresan un Ulin aumentaba la inhibición, incluso cuando se utilizaron Ulin que por si solos no fueron inhibidores muy eficientes. Nótese que se usa la mitad de cada inhibidor y que por ello los resultados no pueden compararse con los obtenidos en las Figuras 2 y 3. It was possible to verify (see Fig. 4) that the combination of inhibitors that express a shRNA with those expressing a Ulin increased the inhibition, even when Ulin were used which alone were not very efficient inhibitors. Note that half of each inhibitor is used and therefore the results cannot be compared with those obtained in Figures 2 and 3.
EJEMPLO 3. Análisis in vitro de la inhibición de la expresión génica de HBV-Luc. EXAMPLE 3. In vitro analysis of the inhibition of HBV-Luc gene expression.
En los ensayos del ejemplo 2 anterior se realizó una evaluación de la inhibición de HBV mediante una valoración por ELISA no cuantitativo de la expresión de EAg. De esta manera se obtuvo una buena estimación de la funcionalidad de los inhibidores, no tanto una cuantificación reproducible de la eficacia de inhibición. In the tests of Example 2 above, an evaluation of HBV inhibition was performed by a non-quantitative ELISA assessment of EAg expression. In this way a good estimate of the functionality of the inhibitors was obtained, not so much a reproducible quantification of the efficacy of inhibition.
Al objeto de poder obtener valores numéricos de inhibición reproducibles, se evaluó la eficacia de los nuevos Ulin en células que expresan luciferasa a partir de un plásmido que tiene secuencias de HBV. Para generar estas células se utilizó el plásmido pHBV- Luc, que expresa un HBV en el que la región codificante del antígeno S (SAg) ha sido sustituida por una secuencia codificante de la luciferasa de luciérnaga. De esta manera, para que se genere actividad luciferasa es necesaria la expresión desde el mRNA modificado de HBV. A diferencia del plásmido pHBV utilizado en el ejemplo 2, el plásmido pHBV-Luc no puede replicarse en células hepáticas. El genoma de HBV es tan compacto que la sustitución del la secuencia que codifica SAg afecta a la mayor parte de la región codificante de la polimerasa y del core 3'UTR (la región 3 ' no traducida). El mRNA de la proteína X no se ve afectado. Dado que se no puede producir una polimerasa funcional, el ARN pregenómico de HBV no puede generar nuevos genomas de HBV, y por tanto no puede tener lugar la replicación viral. En estas circunstancias, la única manera de que se reduzca la actividad luciferasa es que se interfiera la expresión del gen de la luciferasa. La expresión de la luciferasa debería originarse mayormente desde el mRNA de la luciferasa, pero no es descartable que una fracción se origine desde el ARN pregenómico de HBV. In order to obtain reproducible numerical inhibition values, the efficacy of the new Ulin in cells expressing luciferase from a plasmid having HBV sequences was evaluated. To generate these cells the plasmid pHBV-Luc was used, which expresses an HBV in which the coding region of the S antigen (SAg) has been replaced by a coding sequence of the firefly luciferase. Thus, for luciferase activity to be generated, expression from the modified HBV mRNA is necessary. Unlike the pHBV plasmid used in example 2, the pHBV-Luc plasmid cannot replicate in liver cells. The HBV genome is so compact that the substitution of the sequence encoding SAg affects most of the polymerase coding region and the 3'UTR core (the 3 'untranslated region). The protein X mRNA is not affected. Since a functional polymerase cannot be produced, HBV pregenomic RNA cannot generate new HBV genomes, and therefore viral replication cannot take place. Under these circumstances, the only way to reduce luciferase activity is to interfere with the expression of the luciferase gene. Luciferase expression should originate mostly from the Luciferase mRNA, but it is not ruled out that a fraction originates from the pregenomic HBV RNA.
Algunos de los inhibidores de expresión de HBV seleccionados tienen como secuencia diana una secuencia que está dentro de la ventana de lectura ORF de SAg: Ulin5, Ulinó, Ulin7, shRNA2, shRNA3, shRNA5 y shRNA6. Por tanto, estos inhibidores no deberían interferir la expresión de la luciferasa. Some of the HBV expression inhibitors selected have as their target sequence a sequence that is within the SAF ORF reading window: Ulin5, Ulinó, Ulin7, shRNA2, shRNA3, shRNA5 and shRNA6. Therefore, these inhibitors should not interfere with luciferase expression.
Los inhibidores que interfieran con la poliadenilación del mRNA de la proteína core, bien sea esté sólo o conjuntamente con el mRNA de la polimerasa, no debería interferir tampoco la expresión de la luciferasa. Entre estos inhibidores se incluyen Ulinlb, Ulinlc, Ulin2, Ulin3, Ulin4 y Ulin5. Inhibitors that interfere with the polyadenylation of the core protein mRNA, either alone or in conjunction with the polymerase mRNA, should also not interfere with luciferase expression. These inhibitors include Ulinlb, Ulinlc, Ulin2, Ulin3, Ulin4 and Ulin5.
Por tanto, en este modelo se ensayaron únicamente aquellos inhibidores de expresión de HBV cuya secuencia diana está incluida en la región no codificante del mRNA de SAg. Therefore, only those HBV expression inhibitors whose target sequence is included in the non-coding region of SAg mRNA were tested in this model.
Los ensayos de inhibición de la actividad se realizaron también sobre células HuH7, de la misma manera que se ha descrito en el ejemplo 2, con la salvedad de que: - en este caso la células se cotransfectaron con 0,75 μg del plásmido pHBV-Luc (en sustitución de pHBV); y que se incluyeron 0,25 μg del plásmido pRL-SV40, como control de transfección The activity inhibition assays were also performed on HuH7 cells, in the same manner as described in example 2, with the proviso that: - in this case the cells were co-transfected with 0.75 μg of the plasmid pHBV- Luc (replacing pHBV); and that 0.25 μg of plasmid pRL-SV40 was included as a transfection control
Las dosis del plásmido inhibidor ensayadas fueron la dosis máxima (1 μg), o la mitad de esta dosis (½D: 0,5 μg). - dos días después de la cotransfección se cuantificó la actividad luciferasa. The doses of the inhibitor plasmid tested were the maximum dose (1 μg), or half of this dose (½D: 0.5 μg). - Two days after cotransfection, luciferase activity was quantified.
La actividad luciferasa se midió en un luminómetro Berthold (Lumat LB9507) mediante el kit Dual Luciferase System (Promega), de acuerdo con las instrucciones del fabricante. Luciferase activity was measured on a Berthold luminometer (Lumat LB9507) using the Dual Luciferase System kit (Promega), according to the manufacturer's instructions.
Igualmente se calculó el Factor de Inhibición F.I. para cada inhibidor, como el cociente de la actividad luciferasa ([ Luc ]) medida en las células cotransfectadas con el plásmido control (Mock) entre la actividad luciferasa medida en las células cotransfectadas con el plásmido inhibidor a evaluar, o una de sus combinaciones. Likewise, the FI Inhibition Factor was calculated for each inhibitor, such as the ratio of luciferase activity ([Luc]) measured in the cotransfected cells with the plasmid control (Mock) between the luciferase activity measured in the cotransfected cells with the inhibitor plasmid to be evaluated, or one of its combinations.
F.I. = [ LUC ]Mock [ LUC ] Inhibidor F.I. = [LUC] Mock [LUC] Inhibitor
Como se muestra en la Figura 5, todos los Ulin ensayados fueron capaces de inhibir la actividad luciferasa, aunque esta actividad disminuyó drásticamente cuando la dosis se redujo a la mitad, es decir cuando se transfectaron 0,5 μg de plásmido en vez de 1 μg. También, todos los shRNA inhibieron la actividad luciferasa, incluso a la mitad de dosis. As shown in Figure 5, all Ulins tested were able to inhibit luciferase activity, although this activity decreased dramatically when the dose was reduced by half, that is when 0.5 μg of plasmid was transfected instead of 1 μg . Also, all shRNA inhibited luciferase activity, even at half the dose.
Igualmente se ensayaron distintas combinaciones de Ulin y shRNA inhibidores, con los resultados de inhibición que se muestran en la Figura 6. Como puede observarse, la combinación de los Ulin y shRNA seleccionados proporcionó factores de inhibición superiores, incluso cuando se utilizaron Ulin que por sí solos no mostraron una gran eficiencia inhibidora. Likewise, different combinations of inhibitor Ulin and shRNA were tested, with the inhibition results shown in Figure 6. As can be seen, the combination of the selected Ulin and shRNA provided higher inhibition factors, even when Ulin were used than by themselves. alone did not show a high inhibitory efficiency.
Para estas combinaciones se calculó el Indice de Sinergia (S.I.) mediante la siguiente fórmula: For these combinations, the Synergy Index (S.I.) was calculated using the following formula:
S.I. = (FI„+i2 - (FI„ + FI12)) / (Fin + FI:2) YES = (FI „ + i2 - (FI„ + FI12)) / (End + FI : 2 )
FIn+12 es el Factor de Inhibición F.I. obtenido para la combinación de los dos inhibidores; Fin y FI12 son los Factores de Inhibición obtenidos para cada uno de los inhibidores cuando se ensayaron separadamente. Un valor de S.I. positivo indica que no hay sinergia entre los dos agentes; un valor próximo a 0 indica un efecto aditivo, mientras que un valor negativo indica que no hay sinergismo. FIn +12 is the FI Inhibition Factor obtained for the combination of the two inhibitors; Fin and FI 12 are the Inhibition Factors obtained for each of the inhibitors when tested separately. A positive SI value indicates that there is no synergy between the two agents; a value close to 0 indicates an additive effect, while a negative value indicates that there is no synergism.
Como se observa en la Figura 6, las combinaciones de inhibidores resaltadas en negrita mostraron una actividad inhibidora sinérgica. EJEMPLO 4. Análisis in vivo de la inhibición de la expresión génica de HBV-Luc. El ensayo se realizó sobre ratones C56/BL6 macho, con 5 ratones por grupo de tratamiento, de acuerdo con los requerimientos y aprobación del Comité de Etica en Experimentación Animal de la institución. As seen in Figure 6, the combinations of inhibitors highlighted in bold showed synergistic inhibitory activity. EXAMPLE 4. In vivo analysis of the inhibition of HBV-Luc gene expression. The test was performed on male C56 / BL6 mice, with 5 mice per treatment group, in accordance with the requirements and approval of the Institution's Animal Experimentation Ethics Committee.
Cada ratón recibió una inyección hidrodinámica en la vena de la cola de 1,8 mi de suero salino conteniendo 25 μg de plásmidos, con la siguiente composición: Each mouse received a hydrodynamic injection into the tail vein of 1.8 ml of saline containing 25 μg of plasmids, with the following composition:
- un plásmido pHBV-Luc (5 μg); y - a plasmid pHBV-Luc (5 μg); Y
- 20 μg de un plásmido control o 10 μg de un plásmido control y 10 μg de un plásmido que expresa uno de los inhibidores o 10 μg de un plásmido que expresa uno de los inhibidores basados en Ulin y 10 μg del plásmido que expresa el shRNA contra HBV a ensayar. - 20 μg of a control plasmid or 10 μg of a control plasmid and 10 μg of a plasmid expressing one of the inhibitors or 10 μg of a plasmid expressing one of the Ulin-based inhibitors and 10 μg of the shRNA-based plasmid against HBV to be tested.
A distintos tiempos después de la inyección hidrodinámica (1, 3, 5 y 7 días) se evaluó la actividad luciferasa en el hígado de los ratones mediante una cámara CCD (Xenogen). La actividad luciferasa se midió 5 minutos después de la inyección intraperitoneal de 3 mg de luciferina y un anestésico. Como en ejemplo 2 anterior, también se calculó el Factor de Inhibición (F.I.), como el cociente de la actividad luciferasa ([ Luc ]) medida en los ratones que recibieron el plásmido control (Mock), entre la actividad luciferasa medida en los ratones que recibieron el plásmido que expresa el inhibidor (Inhibidor). At different times after hydrodynamic injection (1, 3, 5 and 7 days) luciferase activity in the liver of the mice was evaluated by means of a CCD camera (Xenogen). Luciferase activity was measured 5 minutes after intraperitoneal injection of 3 mg of luciferin and an anesthetic. As in example 2 above, the Inhibition Factor (FI) was also calculated, as the ratio of luciferase activity ([Luc]) measured in mice that received the control plasmid (Mock), between luciferase activity measured in mice who received the plasmid expressing the inhibitor (Inhibitor).
Cuando se ensayó la actividad inhibidora de una combinación de dos plásmidos que expresan un Ulin y un shRNA respectivamente, se calculó el Indice de Sinergia (S.I.) mediante la fórmula descrita en el ejemplo 2. When the inhibitory activity of a combination of two plasmids expressing a Ulin and a shRNA respectively was tested, the Synergy Index (S.I.) was calculated by the formula described in Example 2.
Se realizaron 5 estudios independientes. 5 independent studies were performed.
Como se observa en la Figura 7, la actividad luciferasa en los animales tratados con el plásmido control permaneció constante. Igualmente, el primer día después de la inyección la expresión de la luciferasa no varió en los animales que recibieron plásmidos inhibidores, lo que sugiere que la luciferasa podría ser expresada más rápidamente que los inhibidores. Sin embargo, la inyección de los plásmidos que expresan shRNAl o shRNA4 produjo una reducción de la actividad luciferasa en todos los animales. Sorprendentemente, la expresión de los inhibidores Ulin4 o Ulin20 produjo una inhibición mayor a la que se observó en los ensayos in vitro, a la vez que mostraron una actividad inhibidora sinérgica cuando se administraron en combinación un shRNA inhibidor (Figura 7). La inyección de shRNA inhibidores con Ulin no funcionales produjo inhibiciones similares a las obtenidas para los mismos shRNA administrados solos. Por otra parte e inesperadamente, la administración en combinación de los inhibidores Ulin 10 y shRNAl produjo una inhibición sinérgica. As seen in Figure 7, luciferase activity in animals treated with the control plasmid remained constant. Similarly, on the first day after injection the expression of luciferase did not vary in animals that received inhibitor plasmids, suggesting that luciferase could be expressed more rapidly than inhibitors. However, the injection of plasmids that Expressing shRNAl or shRNA4 produced a reduction in luciferase activity in all animals. Surprisingly, the expression of the Ulin4 or Ulin20 inhibitors produced a greater inhibition than was observed in the in vitro assays, while showing a synergistic inhibitory activity when a shRNA inhibitor was administered in combination (Figure 7). Injection of shRNA inhibitors with non-functional Ulin produced inhibitions similar to those obtained for the same shRNA administered alone. On the other hand and unexpectedly, the combination administration of the Ulin 10 and shRNAl inhibitors produced a synergistic inhibition.

Claims

REIVINDICACIONES
1. Un compuesto capaz de silenciar la expresión génica del virus de la hepatitis B seleccionado del grupo consistente en: 1. A compound capable of silencing the gene expression of hepatitis B virus selected from the group consisting of:
(i) un snRNA Ul, en donde la secuencia de unión a la secuencia consenso GU del extremo 5 ' del intrón de dicho snRNA Ul se encuentra modificada de forma que se una específicamente a una secuencia diana de al menos un ARNm de HBV de forma que es capaz de inhibir la maduración de dicho ARNm diana y en donde dicha secuencia diana se selecciona del grupo consistente en las secuencias SEQ ID NO:4-24;  (i) an Ul snRNA, wherein the consensus consensus binding sequence GU of the 5 'end of the intron of said Ul snRNA is modified such that it specifically binds to a target sequence of at least one HBV mRNA so which is capable of inhibiting the maturation of said target mRNA and wherein said target sequence is selected from the group consisting of the sequences SEQ ID NO: 4-24;
(ii) un polinucleótido que codifica un snARN Ul según (i);  (ii) a polynucleotide encoding an Ul snRNA according to (i);
(iii) un vector de expresión que comprende un polinucleótido según (ii),  (iii) an expression vector comprising a polynucleotide according to (ii),
(iv) un ARN interferente dirigido específicamente a una secuencia diana de al menos un ARNm de HBV que es capaz de provocar el silenciamiento de dicho ARNm de HBV en donde dicha secuencia diana se selecciona del grupo consistente en las secuencias SEQ ID NO:29 a 35;  (iv) an interfering RNA specifically directed to a target sequence of at least one HBV mRNA that is capable of causing the silencing of said HBV mRNA wherein said target sequence is selected from the group consisting of the sequences SEQ ID NO: 29 a 35;
(v) un polinucleótido que codifica un ARN de interferencia según (iv) y  (v) a polynucleotide encoding an interference RNA according to (iv) and
(vi) un vector de expresión que comprende un polinucleótido según (v). 2. Una composición o un kit que comprende en uno o varios contenedores:  (vi) an expression vector comprising a polynucleotide according to (v). 2. A composition or kit comprising in one or several containers:
(a) un primer componente seleccionado del grupo de:  (a) a first component selected from the group of:
(i) un snRNA Ul, en donde dicho snRNA Ul se encuentra modificado en su secuencia de unión a la secuencia consenso GU del extremo 5 ' del intrón de forma que se una específicamente a una secuencia diana de al menos un ARNm de HBV de forma que es capaz de inhibir la maduración de dicho ARNm diana,  (i) an Ul snRNA, wherein said Ul snRNA is modified in its binding sequence to the consensus consensus sequence GU of the 5 'end of the intron so that it specifically binds to a target sequence of at least one HBV mRNA so which is able to inhibit the maturation of said target mRNA,
(ii) Un polinucleótido que codifica un ARN Ul según (i),  (ii) A polynucleotide encoding an Ul RNA according to (i),
(iii) Un vector de expresión que comprende un polinucleótido según (i) y (iii) An expression vector comprising a polynucleotide according to (i) and
(b) un segundo componente seleccionado del grupo de (b) a second component selected from the group of
(iv) Un ARN interferente dirigido específicamente a una secuencia diana del pre-ARNm de HBV que es capaz de provocar el silenciamiento de dicho pre-ARNm de HBV, (v) Un polinucleótido que codifica un ARN de interferencia según (iv) y(iv) An interfering RNA specifically directed to a target sequence of the HBV pre-mRNA that is capable of causing the silencing of said HBV pre-mRNA, (v) A polynucleotide encoding an interference RNA according to (iv) and
(vi) Un vector de expresión que comprende un polinucleótido según (v). (vi) An expression vector comprising a polynucleotide according to (v).
Una composición o un kit según la reivindicación 2 en donde la secuencia diana a la que se une el snRNA Ul modificado se selecciona del grupo consistente en las secuencias SEQ ID NO:4 a 28. A composition or kit according to claim 2 wherein the target sequence to which the modified Ul snRNA binds is selected from the group consisting of the sequences SEQ ID NO: 4 to 28.
Una composición o un kit según las reivindicaciones 3 ó 4 en donde la secuencia diana a la que se une el ARN interferente se selecciona del grupo consistente en las secuencias SEQ ID NO:29 a 35. A composition or kit according to claims 3 or 4 wherein the target sequence to which the interfering RNA binds is selected from the group consisting of the sequences SEQ ID NO: 29 to 35.
Una composición o un kit según la reivindicación 4 en donde: A composition or kit according to claim 4 wherein:
(i) la región preseleccionada a la que se une el snRNA Ul modificado es SEQ ID N 0:4 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO: 29,  (i) the preselected region to which the modified snRNA Ul binds is SEQ ID N 0: 4 and the target sequence to which the interfering RNA binds is SEQ ID NO: 29,
(ii) la región preseleccionada a la que se une el snRNA Ul modificado es SEQ ID NO: 6 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO: 31,  (ii) the preselected region to which the modified Ul snRNA binds is SEQ ID NO: 6 and the target sequence to which the interfering RNA binds is SEQ ID NO: 31,
(iii) la región preseleccionada a la que se une el snRNA Ul modificado es SEQ ID NO: 8 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO: 33,  (iii) the preselected region to which the modified Ul snRNA binds is SEQ ID NO: 8 and the target sequence to which the interfering RNA binds is SEQ ID NO: 33,
(iv) la región preseleccionada a la que se une el snRNA Ul modificado es SEQ ID NO: 8 y la secuencia diana a la que se une ARN interferente es SEQ ID NO: 29,  (iv) the preselected region to which the modified Ul snRNA binds is SEQ ID NO: 8 and the target sequence to which interfering RNA binds is SEQ ID NO: 29,
(v) la región preseleccionada a la que se une el snRNA Ul modificado es SEQ ID NO : 8 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO: 31,  (v) the preselected region to which the modified snRNA Ul binds is SEQ ID NO: 8 and the target sequence to which the interfering RNA binds is SEQ ID NO: 31,
(vi) la región preseleccionada a la que se une el snRNA Ul modificado es SEQ ID NO :8 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO: 32, (vii) la región preseleccionada a la que se une el snRNA Ul modificado es SEQ ID NO :9 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO: 29, (vi) the preselected region to which the modified Ul snRNA binds is SEQ ID NO: 8 and the target sequence to which the interfering RNA binds is SEQ ID NO: 32, (vii) the preselected region to which the modified Ul snRNA binds is SEQ ID NO: 9 and the target sequence to which the interfering RNA binds is SEQ ID NO: 29,
(viii) la región preseleccionada a la que se une el snRNA Ul modificado es SEQ ID NO: 12 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO: 33,  (viii) the preselected region to which the modified snRNA Ul binds is SEQ ID NO: 12 and the target sequence to which the interfering RNA binds is SEQ ID NO: 33,
(ix) la región preseleccionada a la que se une el snRNA Ul modificado es SEQ ID NO : 12 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO: 29,  (ix) the preselected region to which the modified Ul snRNA binds is SEQ ID NO: 12 and the target sequence to which the interfering RNA binds is SEQ ID NO: 29,
(x) la región preseleccionada a la que se une el snRNA Ul modificado es(x) the preselected region to which the modified snRNA Ul binds is
SEQ ID NO: 13 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO: 29, SEQ ID NO: 13 and the target sequence to which the interfering RNA binds is SEQ ID NO: 29,
(xi) la región preseleccionada a la que se une el snRNA Ul modificado es SEQ ID NO : 13 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO : 32,  (xi) the preselected region to which the modified Ul snRNA binds is SEQ ID NO: 13 and the target sequence to which the interfering RNA binds is SEQ ID NO: 32,
(xii) la región preseleccionada a la que se une el snRNA Ul modificado es SEQ ID NO: 14 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO: 33,  (xii) the preselected region to which the modified Ul snRNA binds is SEQ ID NO: 14 and the target sequence to which the interfering RNA binds is SEQ ID NO: 33,
(xiii) la región preseleccionada a la que se une el snRNA Ul modificado es SEQ ID NO: 14 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO: 29,  (xiii) the preselected region to which the modified Ul snRNA binds is SEQ ID NO: 14 and the target sequence to which the interfering RNA binds is SEQ ID NO: 29,
(xiv) la región preseleccionada a la que se une el snRNA Ul modificado es SEQ ID NO: 14 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO: 32,  (xiv) the preselected region to which the modified Ul snRNA binds is SEQ ID NO: 14 and the target sequence to which the interfering RNA binds is SEQ ID NO: 32,
(xv) la secuencia diana a la que se une el snRNA Ul modificado es SEQ(xv) the target sequence to which the modified snRNA Ul binds is SEQ
ID NO: 15 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO: 29, ID NO: 15 and the target sequence to which the interfering RNA binds is SEQ ID NO: 29,
(xvi) la región preseleccionada a la que se une el snRNA Ul modificado es SEQ ID NO: 16 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO: 31, (xvii) la región preseleccionada a la que se une el snRNA Ul modificado es SEQ ID NO : 17 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO: 31, (xvi) the preselected region to which the modified Ul snRNA binds is SEQ ID NO: 16 and the target sequence to which the interfering RNA binds is SEQ ID NO: 31, (xvii) the preselected region to which the modified snRNA Ul binds is SEQ ID NO: 17 and the target sequence to which the interfering RNA binds is SEQ ID NO: 31,
(xviii) la región preseleccionada a la que se une el snRNA Ul modificado es SEQ ID NO: 18 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO: 31 ,  (xviii) the preselected region to which the modified Ul snRNA binds is SEQ ID NO: 18 and the target sequence to which the interfering RNA binds is SEQ ID NO: 31,
(xix) la región preseleccionada a la que se une el snRNA Ul modificado es SEQ ID NO:21 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO: 33,  (xix) the preselected region to which the modified Ul snRNA binds is SEQ ID NO: 21 and the target sequence to which the interfering RNA binds is SEQ ID NO: 33,
(xx) la región preseleccionada a la que se une el snRNA Ul modificado es SEQ ID NO :21 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO: 29,  (xx) the preselected region to which the modified Ul snRNA binds is SEQ ID NO: 21 and the target sequence to which the interfering RNA binds is SEQ ID NO: 29,
(xxi) la región preseleccionada a la que se une el snRNA Ul modificado es SEQ ID NO:21 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO: 32,  (xxi) the preselected region to which the modified Ul snRNA binds is SEQ ID NO: 21 and the target sequence to which the interfering RNA binds is SEQ ID NO: 32,
(xxii) la región preseleccionada a la que se une el snRNA Ul modificado es SEQ ID NO :24 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO: 33,  (xxii) the preselected region to which the modified Ul snRNA binds is SEQ ID NO: 24 and the target sequence to which the interfering RNA binds is SEQ ID NO: 33,
(xxiii) la región preseleccionada a la que se une el snRNA Ul modificado es SEQ ID NO:24 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO: 29,  (xxiii) the preselected region to which the modified Ul snRNA binds is SEQ ID NO: 24 and the target sequence to which the interfering RNA binds is SEQ ID NO: 29,
(xxiv) la región preseleccionada a la que se une el snRNA Ul modificado es SEQ ID NO:24 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO: 32,  (xxiv) the preselected region to which the modified Ul snRNA binds is SEQ ID NO: 24 and the target sequence to which the interfering RNA binds is SEQ ID NO: 32,
(xxv) la región preseleccionada a la que se une el snRNA Ul modificado es SEQ ID NO :26 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO: 34 o  (xxv) the preselected region to which the modified snRNA Ul binds is SEQ ID NO: 26 and the target sequence to which the interfering RNA binds is SEQ ID NO: 34 or
(xxvi) la región preseleccionada a la que se une el snRNA Ul modificado es SEQ ID NO :26 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO: 31  (xxvi) the preselected region to which the modified snRNA Ul binds is SEQ ID NO: 26 and the target sequence to which the interfering RNA binds is SEQ ID NO: 31
Un polinucleótido capaz de silenciar la expresión génica del virus de la hepatitis B que comprende una secuencia que codifica al menos un snR A Ul modificado en su secuencia de unión a la secuencia consenso GU del extremo 5 ' del sitio de corte del intrón de forma que se une específicamente a una secuencia diana de al menos un AR m de HBV y es capaz de inhibir el procesamiento de dicho ARNm diana y A polynucleotide capable of silencing the gene expression of hepatitis B virus comprising a sequence encoding at least one snR A Ul modified in its binding sequence to the consensus consensus sequence GU of the 5 'end of the intron shear site so that it specifically binds to a target sequence of at least one mR of HBV and is able to inhibit the processing of said target mRNA and
una secuencia que codifica un ARN interferente dirigido de forma específica a una secuencia diana de al menos un ARNm de HBV y que es capaz de provocar el silenciamiento de dicho ARNm de HBV.  a sequence that encodes an interfering RNA specifically directed to a target sequence of at least one HBV mRNA and that is capable of causing the silencing of said HBV mRNA.
Un polmucleótido según la reivindicación 6 en donde la secuencia diana a la que se une el snRNA Ul modificado se selecciona del grupo consistente en la secuencias SEQ ID NO:4 a 28. A polymucleotide according to claim 6 wherein the target sequence to which the modified Ul snRNA binds is selected from the group consisting of the sequences SEQ ID NO: 4 to 28.
Un polmucleótido según las reivindicaciones 6 ó 7, en donde la secuencia diana a la que se une el ARN interferente se selecciona del grupo consistente en la secuencias SEQ ID NO:29 a 35. A polymucleotide according to claims 6 or 7, wherein the target sequence to which the interfering RNA binds is selected from the group consisting of the sequences SEQ ID NO: 29 to 35.
Un polmucleótido según la reivindicación 8 en donde: A polymucleotide according to claim 8 wherein:
(i) la región preseleccionada a la que se une el snRNA Ul modificado es SEQ ID NO:4 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO: 29,  (i) the preselected region to which the modified snRNA Ul binds is SEQ ID NO: 4 and the target sequence to which the interfering RNA binds is SEQ ID NO: 29,
(ii) la región preseleccionada a la que se une el snRNA Ul modificado es SEQ ID NO: 6 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO: 31,  (ii) the preselected region to which the modified Ul snRNA binds is SEQ ID NO: 6 and the target sequence to which the interfering RNA binds is SEQ ID NO: 31,
(iii) la región preseleccionada a la que se une el snRNA Ul modificado es SEQ ID NO : 8 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO: 33,  (iii) the preselected region to which the modified Ul snRNA binds is SEQ ID NO: 8 and the target sequence to which the interfering RNA binds is SEQ ID NO: 33,
(iv) la región preseleccionada a la que se une el snRNA Ul modificado es SEQ ID NO: 8 y la secuencia diana a la que se une ARN interferente es SEQ ID NO: 29, (v) la región preseleccionada a la que se une el snRNA Ul modificado es SEQ ID NO: 8 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO: 31, (iv) the preselected region to which the modified Ul snRNA binds is SEQ ID NO: 8 and the target sequence to which interfering RNA binds is SEQ ID NO: 29, (v) the preselected region to which the modified snRNA Ul binds is SEQ ID NO: 8 and the target sequence to which the interfering RNA binds is SEQ ID NO: 31,
(vi) la región preseleccionada a la que se une el snRNA Ul modificado es SEQ ID NO :8 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO: 32,  (vi) the preselected region to which the modified Ul snRNA binds is SEQ ID NO: 8 and the target sequence to which the interfering RNA binds is SEQ ID NO: 32,
(vii) la región preseleccionada a la que se une el snRNA Ul modificado es SEQ ID NO :9 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO: 29,  (vii) the preselected region to which the modified Ul snRNA binds is SEQ ID NO: 9 and the target sequence to which the interfering RNA binds is SEQ ID NO: 29,
(viii) la región preseleccionada a la que se une el snRNA Ul modificado es(viii) the preselected region to which the modified snRNA Ul binds is
SEQ ID NO : 12 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO: 33, SEQ ID NO: 12 and the target sequence to which the interfering RNA binds is SEQ ID NO: 33,
(ix) la región preseleccionada a la que se une el snRNA Ul modificado es SEQ ID NO: 12 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO : 29,  (ix) the preselected region to which the modified Ul snRNA binds is SEQ ID NO: 12 and the target sequence to which the interfering RNA binds is SEQ ID NO: 29,
(x) la región preseleccionada a la que se une el snRNA Ul modificado es SEQ ID NO : 13 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO: 29,  (x) the preselected region to which the modified Ul snRNA binds is SEQ ID NO: 13 and the target sequence to which the interfering RNA binds is SEQ ID NO: 29,
(xi) la región preseleccionada a la que se une el snRNA Ul modificado es SEQ ID NO : 13 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO: 32,  (xi) the preselected region to which the modified Ul snRNA binds is SEQ ID NO: 13 and the target sequence to which the interfering RNA binds is SEQ ID NO: 32,
(xii) la región preseleccionada a la que se une el snRNA Ul modificado es SEQ ID NO: 14 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO: 33,  (xii) the preselected region to which the modified Ul snRNA binds is SEQ ID NO: 14 and the target sequence to which the interfering RNA binds is SEQ ID NO: 33,
(xiii) la región preseleccionada a la que se une el snRNA Ul modificado es(xiii) the preselected region to which the modified snRNA Ul binds is
SEQ ID NO : 14 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO: 29, SEQ ID NO: 14 and the target sequence to which the interfering RNA binds is SEQ ID NO: 29,
(xiv) la región preseleccionada a la que se une el snRNA Ul modificado es SEQ ID NO : 14 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO: 32, (xv) la secuencia diana a la que se une el snRNA Ul modificado es SEQ ID NO: 15 y la secuencia diana a la que se une el ARN interf érente es SEQ ID NO: 29, (xiv) the preselected region to which the modified Ul snRNA binds is SEQ ID NO: 14 and the target sequence to which the interfering RNA binds is SEQ ID NO: 32, (xv) the target sequence to which the modified snRNA Ul binds is SEQ ID NO: 15 and the target sequence to which the interfering RNA binds is SEQ ID NO: 29,
(xvi) la región preseleccionada a la que se une el snRNA Ul modificado es SEQ ID NO: 16 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO: 31,  (xvi) the preselected region to which the modified Ul snRNA binds is SEQ ID NO: 16 and the target sequence to which the interfering RNA binds is SEQ ID NO: 31,
(xvii) la región preseleccionada a la que se une el snRNA Ul modificado es SEQ ID NO: 17 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO: 31,  (xvii) the preselected region to which the modified snRNA Ul binds is SEQ ID NO: 17 and the target sequence to which the interfering RNA binds is SEQ ID NO: 31,
(xviii) la región preseleccionada a la que se une el snRNA Ul modificado es(xviii) the preselected region to which the modified snRNA Ul binds is
SEQ ID NO : 1 8 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO: 31 , SEQ ID NO: 1 8 and the target sequence to which the interfering RNA binds is SEQ ID NO: 31,
(xix) la región preseleccionada a la que se une el snRNA Ul modificado es SEQ ID NO:21 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO: 33,  (xix) the preselected region to which the modified Ul snRNA binds is SEQ ID NO: 21 and the target sequence to which the interfering RNA binds is SEQ ID NO: 33,
(xx) la región preseleccionada a la que se une el snRNA Ul modificado es SEQ ID NO:21 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO: 29,  (xx) the preselected region to which the modified Ul snRNA binds is SEQ ID NO: 21 and the target sequence to which the interfering RNA binds is SEQ ID NO: 29,
(xxi) la región preseleccionada a la que se une el snRNA Ul modificado es SEQ ID NO:21 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO: 32,  (xxi) the preselected region to which the modified Ul snRNA binds is SEQ ID NO: 21 and the target sequence to which the interfering RNA binds is SEQ ID NO: 32,
(xxii) la región preseleccionada a la que se une el snRNA Ul modificado es SEQ ID NO:24 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO: 33,  (xxii) the preselected region to which the modified Ul snRNA binds is SEQ ID NO: 24 and the target sequence to which the interfering RNA binds is SEQ ID NO: 33,
(xxiii) la región preseleccionada a la que se une el snRNA Ul modificado es(xxiii) the preselected region to which the modified snRNA Ul binds is
SEQ ID NO :24 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO: 29, SEQ ID NO: 24 and the target sequence to which the interfering RNA binds is SEQ ID NO: 29,
(xxiv) la región preseleccionada a la que se une el snRNA Ul modificado es SEQ ID NO:24 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO: 32, (xxv) la región preseleccionada a la que se une el snRNA Ul modificado es SEQ ID NO:26 y la secuencia diana a la que se une el AR interferente es SEQ ID NO: 34 o (xxiv) the preselected region to which the modified Ul snRNA binds is SEQ ID NO: 24 and the target sequence to which the interfering RNA binds is SEQ ID NO: 32, (xxv) the preselected region to which the modified snRNA Ul binds is SEQ ID NO: 26 and the target sequence to which the interfering AR binds is SEQ ID NO: 34 or
(xxvi) la región preseleccionada a la que se une el snRNA Ul modificado es SEQ ID NO:26 y la secuencia diana a la que se une el ARN interferente es SEQ ID NO: 31  (xxvi) the preselected region to which the modified snRNA Ul binds is SEQ ID NO: 26 and the target sequence to which the interfering RNA binds is SEQ ID NO: 31
10. Un vector de expresión que comprende un polinucleótido según cualquiera de las reivindicaciones 6 a 9. 10. An expression vector comprising a polynucleotide according to any one of claims 6 to 9.
11. Un compuesto según la reivindicación 1 , una composición o kit según cualquiera de las reivindicaciones 2 a 5 , un polinucleótido según cualquiera de las reivindicaciones 6 a 9 o un vector según la reivindicación 10 para su uso en medicina. 11. A compound according to claim 1, a composition or kit according to any of claims 2 to 5, a polynucleotide according to any of claims 6 to 9 or a vector according to claim 10 for use in medicine.
12. Una composición farmacéutica que comprende un compuesto según la reivindicación 1 , una composición o kit según cualquiera de las reivindicaciones 2 a 5, un polinucleótido según cualquiera de las reivindicaciones 6 a 9 o un vector según la reivindicación 10 y un vehículo farmacéuticamente aceptable. 12. A pharmaceutical composition comprising a compound according to claim 1, a composition or kit according to any of claims 2 to 5, a polynucleotide according to any of claims 6 to 9 or a vector according to claim 10 and a pharmaceutically acceptable carrier.
13. Un compuesto según la reivindicación 1, una composición o kit según cualquiera de las reivindicaciones 2 a 5 , un polinucleótido según cualquiera de las reivindicaciones 6 a 9 o un vector según la reivindicación 10 para su uso en la prevención o tratamiento de enfermedades causadas por una infección por el virus de la hepatitis B. 13. A compound according to claim 1, a composition or kit according to any of claims 2 to 5, a polynucleotide according to any of claims 6 to 9 or a vector according to claim 10 for use in the prevention or treatment of diseases caused for a hepatitis B virus infection.
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