WO2014094645A1 - 治疗乙型病毒性肝炎的rna干扰制剂 - Google Patents

治疗乙型病毒性肝炎的rna干扰制剂 Download PDF

Info

Publication number
WO2014094645A1
WO2014094645A1 PCT/CN2013/090055 CN2013090055W WO2014094645A1 WO 2014094645 A1 WO2014094645 A1 WO 2014094645A1 CN 2013090055 W CN2013090055 W CN 2013090055W WO 2014094645 A1 WO2014094645 A1 WO 2014094645A1
Authority
WO
WIPO (PCT)
Prior art keywords
ina
nucleotides
rna
stranded
hbv
Prior art date
Application number
PCT/CN2013/090055
Other languages
English (en)
French (fr)
Inventor
崔坤元
梁东
Original Assignee
厦门成坤生物技术有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 厦门成坤生物技术有限公司 filed Critical 厦门成坤生物技术有限公司
Publication of WO2014094645A1 publication Critical patent/WO2014094645A1/zh

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • 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
    • 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
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention utilizes RNA interference (RNAi) to inhibit the replication of hepatitis B virus.
  • RNAi RNA interference
  • Double-stranded ⁇ inhibits protein expression, silencing genes, and has a wide range of potential therapeutic uses for human diseases.
  • dsRNA Double-stranded ⁇
  • siRNA small interfering RNA
  • mRNA messenger RNA Degradation of
  • Attenuation of RNA-mediated transcriptional function it is generally believed that dsRNA-induced gene interference (inhibition) refers to RNA degradation in animal cells.
  • Chemically synthesized small molecule RNAs, such as siRNA are capable of degrading intracellular mRNA by more than 95% in a submicromolar concentration.
  • RNAi can last for a long time, even for generations of cell division, while gene interference is sequence-specific inhibition. Therefore, RNAi can specifically inhibit gene expression without affecting its isoforms or other mRNAs. This specific inhibition is particularly important in the study of gene function and drug target signatures.
  • siRNA can be used to develop drug therapy: 1. Genes that are overexpressed by genes or not normally expressed; 2. Diseases caused by mutated genes.
  • RNAi can be applied to the development of new classes of drugs that differ from small molecules and proteins. Although long dsRNAs cause cellular interferon responses that cannot be directly transmitted to cells, the use of siRNA has been relatively successful. It has been widely used in research and has been clinically tested.
  • RNAi can also treat cancer by inhibiting the over-expressed genes from suppressing the division of tumor cells.
  • a very important area is the development of a safe siRNA delivery technology to ensure the clinical application of RNAi.
  • RNAi is a very promising drug development platform, whether the off-target effects of siRNA can cause some side effects also attract attention, because off-target effects can inhibit genes similar to the target gene. According to calculations, the off-target effect can reach 10%.
  • siRNA or iNA interfering nucleic acid
  • siRNA or iNA without interferon
  • design a therapeutic iNA or siRNA that can act on one or more target genes, or a different part of a target gene, in a single orientation. Or different directions, ring-shaped, ring-like, or linear. These types of iNA or siRNA differ in structure from commonly used siRNAs.
  • Hepatitis B V irus is estimated to have a population of approximately 2 billion people - one-third of the world's population, and more than 3.5 million people will become chronically infected. It is reported that 15-40% of HBV-infected patients develop cirrhosis, liver failure or liver cancer (HCC), and between 500,000 and 1.2 million people die each year from HBV infection. The prevalence of HBV in the United States is estimated to be about 0.4%. However, voluntary sampling data indicate that the prevalence of certain foreign-born ethnic minority groups exceeds 15%. In the 1990s, the number of hepatitis B virus-related diagnostic outpatients and hospitalizations increased several times. Similarly, the total cost of hospitalization has been estimated to have increased from $357 million in 1990 to $1.5 billion in 2003 and then remained at $1.3 billion.
  • HBV hepatitis B virus
  • Peginterferon alfa-2a (PEG-IFN_a) and interferon a-2a (IFN-a), nucleosides (lamivudine, entecavir and telbivudine) and nucleotide analogues (Adefovir and tenofovir) are FDA approved A drug commonly used in the anti-HBV drug market.
  • the main disadvantage of interferon therapy is its significant side effects that limit its long-term use. It is often ineffective for patients with decompensated cirrhosis and normal transaminase. In addition, only one-third of patients were anti-viral against PEG-IFN- ⁇ .
  • nucleoside (acid) analogues inhibit HBV replication and reduce hepatic necroinflammation, the virus cannot be completely eradicated. In addition, after stopping the drug, most patients observed a rebound in viremia. In addition, long-term treatment produces resistant HBV strains that result in treatment failure.
  • RNAi is the active process of intracellular regulatory genes. It was once called another name, including post-transcriptional gene silencing (PTGS). In 2006, Andrew Fire and Craig C. Mel Lo won the Nobel Prize in Physiology or Medicine in recognition of their 1998 article, which described them. RNAi phenomenon found in nematode research.
  • RNA small RNA
  • miRNAs microRNAs
  • siRNAs small interfering RNAs
  • RNAi phenomenon was found to be present in many eukaryotic cells, including animal cells, and the Dicer enzyme degrades the long dsRNA molecule into a 20-nucleotide short fragment called siRNA.
  • siRNA is depressurized into two single-stranded (ss) RNAs, the passenger strand and the guide strand, the passenger strand is hydrolyzed, and the guide strand is combined with the RISC complex.
  • the base sequence of the guide strand is complementary to the mRNA sequence molecule, and induces and activates the Argonaute protein, a catalyst component of the RISC complex, which degrades the mRNA. In some organisms, this process is spread throughout the body.
  • RNAi has been shown to be specific and active in animal experiments, with the ability to silence disease genes and treat diseases.
  • RNAi can inhibit HBV in cultured cells and infected mice. However, it has also been reported that due to the high mutation rate of HBV, not only can HBV be inhibited more effectively by inhibiting multiple sites of HBV, but also HBV gene mutation may be prevented. Summary of the invention
  • this application provides a method for treating hepatitis B virus infection by inhibiting one or more sites of the HBV genome by one or more types of siRNA or iNA.
  • the invention provides a method comprising a double-stranded iNA or siRNA, such as iNA (siRNA) ID number 1-272 (Table 1), which can be selected for one or more HBV RNA sites.
  • a double-stranded iNA or siRNA consists of two complementary strands, one or more complementary strands of which may be complementary to one or more regions of HBV RNA.
  • the double-stranded iNAs may be linear, circular, ring-like, hairpin-type, stem-loop, unidirectional, or bidirectional structures (Fig.
  • the double-stranded iNAs may have two consecutive polynucleotide strands with one or more gaps (nicks) forming one or more segmented strands between the gaps, as shown 1.
  • the double-stranded iNA or siRNA is 10-200 nucleotides in length, preferably 15 to 50 nucleotides, more preferably 19-29 nucleotides.
  • the complementary region of the double strand has a polynucleotide chain of not less than 10 nucleotides, more preferably 19-29 nucleotides.
  • any double - stranded region of a nucleotide in one strand is complementary to the other strand.
  • the double stranded iNA or siRNA can have a sticky end or a blunt end.
  • the 3' end of the iNA or siRNA comprises a length of 1 to 5 nucleosides, which can be located at one or 3' end of Table 1 HBV virus iNA (siRNA) target site .
  • the invention provides a linear segmented iNAs structure having two short chains and complementary to a continuous long chain with gaps (cuts) between the short chains, such as iNA ID N0. -241, 249-252, and 269-272.
  • the contiguous long chain comprises two segments, the sequence of each segment being complementary or identical to the HBV RNA, and the two short chains of the iNA may be identical or complementary to different sites of the HBV RNA.
  • a continuous long chain for each HBV RNA site The two sections in the middle are not in a specific order, and can be either at the front end (5' end) or at the back end (3' end), or vice versa.
  • the contiguous strand is at least 15 to 80 nucleotides in length, preferably 19 50 nucleotides in length.
  • the two short strands are at least 10 to 40 nucleotides in length, preferably 19 to 27 nucleotides.
  • Any double-stranded region of a nucleotide in the complement is complementary to the other strand.
  • the double stranded iNA or siRNA can have a sticky end or a blunt end.
  • the 3' end of the iNA or siRNA comprises a length of 1 to 5 nucleotides, which may be located in one or both.
  • the iNA numbers 242-248 of the present invention have a bidirectional circular or cyclic structure-like duplex, such as the iNA of Example 2, which can act on different regions of HBV RNA.
  • Two complementary strand RNA or RNA-like oligonucleotides form a double-stranded RNA or RNA-like oligonucleotide.
  • At least one of the chains is bidirectional, at least one of the segments has a 5' to 3' direction and the other segment has a 3' to 5' direction, and the complementation of the two different direction segments
  • Nucleotides form a cyclic or similar cyclic structure.
  • another single strand is double-stranded with a nucleotide of a single-stranded segment in the circular or similar circular bidirectional chain.
  • the circular bidirectional strand can be as short as 10 nucleotides in length, as long as one thousand nucleotides or the length of the entire RNA.
  • the length of the segment in one direction (5' to 3' or 3' to 5') should be between 5 and 80 bases, preferably 5 to 10 or 15 to 29 nucleosides. acid.
  • the circular bidirectional strand complementary to the other strand can be of any length, from 10 to 200 nucleotides, but the double stranded region formed should be from 10 to 200 nucleotides in length, preferably 15 Up to 29 nucleotides, preferably 19 to 25 nucleotides.
  • the gap between the double-stranded segments can be as short as 0 nucleotides (called a nick) and can be as long as 100 or a few thousand nucleotides (notch).
  • the short and long chains in the double-stranded region are complementary.
  • the two chains of any double-stranded region may complement each other completely or partially. If only partially complementary, one or more nucleotides in the strand may not be paired. Unpaired nucleotides can be anywhere between the 5'-end or the 3'-end, or both ends, or between the 5' end and the 3'-end.
  • the iNA in Embodiment 2 is an example.
  • the invention provides an iNAs structure having a stem-loop structure, iNA NO. 257-266 of the invention, as shown in Example 4, can be directed to different regions of RNA of HBV.
  • Two complementary strand RNA or RNA-like oligonucleotides form a double-stranded RNA.
  • At least one of the strands forms a stem-loop structure; a segment forms a loop, and a portion of the 5'-end and a portion of the nucleotide at the 3'-end of the same strand form a short double-stranded region.
  • the other strand will be complementary to the loop region of the non-double stranded region.
  • the length of the stem-like loop can be as short as 10 nucleotides, as long as several thousand nucleotides or as long as one or several mRNAs, preferably 15 to 200 nucleotides, and most preferably 19 45 The length of the nucleotide.
  • the stem-like loop may be of any length, but the double-stranded region formed should be from 10 to 200 nucleotides in length, preferably from 15 to 29 nucleotides.
  • the gap between each double-stranded region can be as short as 0 nucleotides (nick) or 100 or thousands of nucleotides (notch). Any double-stranded region of the two chains may complement each other completely or partially.
  • nucleotides in the chain may not be paired.
  • Unpaired nucleotides can be anywhere between the 5'-end or the 3'-end, or both ends, or between the 5' end and the 3'-end.
  • the iNA shown in Embodiment 4 is an example.
  • some embodiments provide an iNA molecule comprising a ribose thymidine nucleoside
  • the iNA further comprises one or more non-standard nucleosides, such as deoxyuridine, a locked nucleic acid (LNA) molecule or a universal nucleotide, or a G-clamp ( Clamp).
  • LNA locked nucleic acid
  • Clamp G-clamp
  • nucleotides include C-phenyl (C-phenyl), C-naphthy, inosine, azole carboxamide, 1_ ⁇ _D_ribofuranosyl-4-nitro 1-( 1- ⁇ -D-ribofuranosyl_4-nitroindole), 1- ⁇ -D-ribofuranosyl-1- ⁇ -D-ribofuranosyl 5-nitroguanidine
  • the RNA molecule comprises a 2'-sugar substitution component, such as 2'-0-A (2'-0-methyl), 2'-0_methoxyethyl (2'-0-methoxyethyl), 2'-0-2-methoxyethyl (2'-0-methoxyethyl) 2'- 0-allyl (2'-0-ylyl), or halogen (2,-fluoro, 2'-fluoro).
  • 2'-sugar substitution component such as 2'-0-A (2'-0-methyl), 2'-0_methoxyethyl (2'-0-methoxyethyl), 2'-0-2-methoxyethyl (2'-0-methoxyethyl) 2'- 0-allyl (2'-0-ylyl), or halogen (2,-fluoro, 2'-fluoro).
  • the INA molecule further comprises one or more first On the end cap of the chain, the second chain or the third chain a substituent group, which may independently be an alkyl group, an abasic group, a deoxy abasic group, a glyceryl group, a dinucleotide acyclic nucleotide (acycl) Ic nucleot ide ) or deoxynucleotide in the direction.
  • substituent group which may independently be an alkyl group, an abasic group, a deoxy abasic group, a glyceryl group, a dinucleotide acyclic nucleotide (acycl) Ic nucleot ide ) or deoxynucleotide in the direction.
  • a substituent group which may independently be an alkyl group, an abasic group, a deoxy abasic group, a glyceryl group, a dinucleotide acyclic nucle
  • Figure 1 Schematic diagram of siRNA or iNA structure for inhibition of HBV gene.
  • iNA was transfected with RFects from iNAs (5nM) to HepG2. 2. 15 cells, and changes in gene expression were analyzed by real-time quantitative RT-PCR.
  • FIG. 1 Transfection of iNAs (5nM) to HepG2. 2. 15 cells at two different sites of HBV RNA by RFect, and analysis of gene expression changes by real-time quantitative RT-PCR.
  • FIG. 4 Antiviral iNA (siRNA) encapsulated in a single intravenous delivery system of hepatitis B virus transgenic mice, specifically inhibiting replication of HBV viral genes in hepatocytes, whereas ApoB siRNA in the control group had no effect on HBV replication.
  • Figure 5. Examples of HBV DNA in HBV transgenic mice by intravenous administration of HBV1 (iNA number 267), HBV2 (iNA number 269), and AP0-B.
  • HBV1 iNA number 267)
  • HBV2 iNA number 269
  • AP0-B intravenous administration of iNA reduce HBeAg in HBV-transgenic mice.
  • FIG. 1 Example of intravenous administration of HBV1 (iNA number 267), HBV2 (iNA number 269), and AP0-B to reduce plasma HBsAg in HBV transgenic mice.
  • Figure 8 Examples of HBV RNA in HBV transgenic mice by intravenous administration of HBV1 (iNA number 267), HBV2 (iNA number 269), and AP0-B.
  • Figure 9 Examples of post-drug plasma chemical analysis of HBV transgenic mice by HBV1 (iNA number 267), HBV2 (iNA number 269), and AP0-B iNA intravenous.
  • interfering nucleic acid refers to a nucleic acid duplex having complementary strands to each other.
  • RISC complex Upon entry into the RISC complex, the RNAi mechanism that induces RNase degradation of RNA.
  • iNA regulates the increase in expression of the target gene through the RNAa mechanism of the promoter.
  • each strand of iNA is a nucleotide, mainly a ribonucleotide, but it can also be an analog of RNA, an analog of RNA and RNA, a modified nucleotide, RNA and DNA, RNA.
  • Analogs and DNA, non-nucleotides, or one strand are completely DNA, and the other strand is RNA, as long as it can induce homologous RNA degradation by RNAi mechanism, and can be used as a structure of iNA.
  • bidirectional double-stranded iNA or "bidirectional double-stranded siRNA” or “bidirectional iNA” as used herein is a generic term throughout the present invention, including the bidirectional structure of the same strand of interfering nucleic acids (iNAs), which may be in a cell. It is cleaved to form iNA or siRNA.
  • a bi-directional iNA double-chain at least one chain having one or more segments in the 5' to 3' direction and one or more Fragments in the 3' to 5' direction.
  • the other strand of the iNA has one or more moieties complementary thereto to form one or more double stranded segments. There may be a gap or cut between each double-stranded section.
  • the bidirectional iNA can be linear, stem-ring shaped, or circular.
  • the iNA of the terminal structure may be blunt or sticky (protruding or overhanging) ends, and its functional target is RNA silencing.
  • the end structure of the viscous (protruding) end is not limited to the 3' overhang portion, but may be a 5' overhang structure as long as it is capable of inducing an RNAi effect.
  • the number of pendant nucleotides is not limited to 2 or 3 reported, but may be any number as long as the RNAi effect can be induced. For example, it may be 1 to 8 or longer, or 2 to 4 bases or longer.
  • annular or ring-like iNA duplex is a generic term used in the present invention to include interfering nucleic acids (iNAs) having a circular or circular-like structural framework that can be cleaved in cells to form iNA or siRNA.
  • the circular or loop-like iNA duplex of the present invention also includes an expression vector (also referred to as an iNA expression vector) capable of producing an iNA duplex or iNA produced after transcription and/or transcription in a cell, and inducing RNAi in vivo.
  • the sense strand or antisense strand can have one or more slits or gaps.
  • the end of the iNA can be sticky (protruding) or blunt as long as it functions to silence the target RNA.
  • the viscous (protruding) end structure is not limited to the 3' overhang portion, but may also be a 5' overhang structure as long as it is capable of inducing the RNAi effect.
  • the number of overhanging nucleotides is not limited to the reported 2 or 3, and may be any number as long as the RNAi effect can be induced.
  • the overhang may be 1 to 8 bases or longer, or 2 to 4 bases.
  • Stem-loop structure refers to intracellular base pairing of a polynucleotide that can occur in single-stranded DNA, more commonly RNA.
  • a double-stranded cyclic structure refers to a single-stranded portion in which a single or multiple strands are complementary to a loop region, with gaps or slits between the fragments.
  • any segment on the stem may also have a complementary strand of one or more single-stranded regions.
  • the end structure may be blunt or sticky (protruding).
  • One or more strands may be complementary to the loop or non-annular segment with gaps or nicks between the segments, with or without protrusions at the 3' or 5' ends.
  • Length refers to the number of nucleotides in a bidirectional iNA, starting from the first base pair at the 5' end of the sense strand and ending at the last base at the 3' end of the sense strand.
  • micro- is a single-stranded RNA of approximately 21-23 nucleotides in length that regulates gene expression. miRNAs are transcribed from DNA but not translated into proteins (non-coding RNAs), which are pre-miRNAs generated by the short stem-loop structure of PRi-miRNAs called primary transcription, and finally miRNAs are generated from pre-miRNAs.
  • non-coding RNAs non-coding RNAs
  • pre-miRNAs generated by the short stem-loop structure of PRi-miRNAs called primary transcription
  • miRNAs are generated from pre-miRNAs.
  • a mature miRNA molecule is partially complementary to one or more messenger molecules whose primary function is to reduce gene expression.
  • the nucleotides modified by the iNA molecule can be in any strand.
  • a modified nucleotide can have a Northern conformation (e.g., Northern pseudorotation cycle, see Sanger, Principles of Nucleic Acid Structures, Springer-Verlag ed., 1984).
  • nucleotides having a Northern configuration examples include nucleotides of a locked nucleic acid (LNA) (eg, 2' _0, 4, -C-methylene-(D-ribofuranosyl) nucleotides), (2' -0) , 4'-C-methylene- (D-ribofuranosyl) nucleotides), 2'-methoxyethoxy (MOE) nucleotides, 2'-methyl-thio -ethyl, 2'-methyl-thio-ethyl, 2'-deoxy-2'-f luoro nucleotides, 2'-deoxy-2'-chloronucleotide ( 2'-deoxy-2'-chloro nucleotides), 2'-azido nucleotides, 2'-0-methyl nucleotides.
  • LNA locked nucleic acid
  • MOE 2'-methoxyethoxy
  • a conjugated molecule attached to a chemically modified iNA molecule can be a ligand for polyethylene glycol, human serum albumin or a cellular receptor that mediates cellular uptake.
  • An example of a chemically-modified iNA molecule and a specific conjugated molecule contemplated by the present invention can be seen in U.S. Patent No. 2,030,130, 186, issued toU.S. Pat. invention.
  • oligonucleotides are modified to enhance stability and/or enhance biological activity by modifying nuclease resistance, for example, 2'-amino, 2'-C-allyl, 2'-fluoro, 2'- Nucleotide base modification of 0-methyl, 2'-0-allyl, 2'-H. See Usman and Cedergren (USBS 17: 34, 1992; Usman, et al, Nucleic Acids Symp. Ser. 31: 163, 1994; Burgin, et al, Biochemistry 35: 14090, 1996.
  • the iNA duplex may comprise a phosphate backbone-modified iNA molecule, including: one or more phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidated urethane Ester, carboxymethyl, aC et a mid a te, polyamide, sulfonate, sulfonamide, sulfamate, forma Ce tal, thioformacetal, alkylsilyl.
  • oligonucleotide backbone modifications see Hunziker and Leumann, Nucleic Acid Analogues: Synthesis and Properties, in Modern Synthetic Methods, VCH, 1995, pp.
  • the antisense region of an iNA molecule can include a linkage between a phosphorothioate nucleoside at the 3'-end of the antisense region.
  • the antisense region can include - 5 5'-terminal phosphorothioate internucleoside linkages.
  • the 3'-terminal nucleotide of a circular or ring-like iNA molecule may comprise a chemically modified nucleic acid of a ribonucleotide or deoxyribonucleotide Sugar, base.
  • 3'-end nucleotides can include One or more universal base ribonucleotides.
  • the 3'-terminal nucleotide may comprise one or more acyclic nucleotides.
  • a chemically modified iNA may have 1, 2, 3, 4, 5, 6, 7, 8, or more phosphorothioate internucleoside linkages. There may be 1 to 8 or more phosphorothioate internucleoside linkages per chain. Phosphorothioate internucleoside linkages It may be a bidirectional iNA duplex present in one or two oligonucleotide strands, for example, may be in the sense strand, the antisense strand or both strands. In some embodiments, the iNA molecule comprises 1, 2, 3 , 4, 5, 6, 7, 8, 9, 10, or more in the sense strand, the antisense strand, or both strands of the thiol nucleoside.
  • Synthesis conditions by chemical modification of iNA molecules (1) synthesis of at least two or more RNA or RNA-like oligonucleotide molecules and complementary strands; (2) suitable conditions, two or more The complementary strands are annealed together to obtain iNA molecules.
  • the synthesis of a complementary portion of a bidirectional iNA molecule is synthesized by a solid phase oligonucleotide or by a solid phase tandem oligonucleotide.
  • Oligonucleotides can be used using techniques known in the art, such as Caruthers, et al, Methods in Enzymology 211: 3 - 19, 1992; Thompson, et al., International PCT Publ ication No. W0 99/54459; Wincott, et al., Nucleic Acids Res. 23: 2677-2684, 1995; Wincott, et al., Methods Mol. Bio. 74: 59, 1997; Brennan, et al., Biotechnol Bioeng. 61: 33-45, 1998; and Brennan, US Pat.
  • “Overlapping” refers to when two iNA fragments have sequence overlap, for example, where multiple nucleotides (nt) may be as few as 2-5 nucleotides up to 5-10 nucleotides or more. many.
  • One or more iNAs refer to iNAs that have different primary sequences from each other.
  • Target site or “target” or “targeted” refers to a sequence within a target nucleic acid (eg, RNA), a sequence of siRNA degradation mediated by the iNA antisense strand sequence.
  • RNA target nucleic acid
  • the two nucleotide phosphodiester bonds in a gap finger chain are not connected or have a gap or nick.
  • a mixed type of iNA molecule means that the double-stranded nucleic acid of iNA contains an RNA strand and a DNA strand.
  • the RNA strand is the antisense strand of the target RNA binding.
  • the hybrid iNA created by the hybridized DNA and RNA strands contains a complementary portion of the hybrid which can have at least one 3' sticky end.
  • RNA levels may include intracellular increase or decrease in RNA levels, or translation of RNA, or synthesis of a protein or protein subunit encoded thereby.
  • inhibitors means the expression, or level of a gene, a molecule encoding one or more protein or protein subunits, or the level or activity of an equivalent RNA molecule.
  • the protein subunit encoded by one or more proteins or target genes is reduced to a level lower than that before application of iNA.
  • gene silencing refers to partial or complete inhibition of gene expression in a cell, and may also be referred to as “gene knockout.” The extent of gene silencing can also be determined by methods known in the art, some of which are summarized in International Publication No. WO99/32619.
  • the iNA molecule comprises sense and antisense sequences or segments, characterized in that the nucleotides or non-nucleotides of the sense and antisense regions are covalently linked, or by non-covalent bonds, ionic bonds Role, hydrogen bonding, van der Waals interactions, hydrophobic interactions and/or stacking interactions.
  • iNAs can be assembled into two double strands by two separate oligonucleotides, one of which is a sense strand and the other is an antisense strand, wherein the antisense and sense strands are self-complementary (ie, each strand contains The nucleotide sequence of the base complements the sequence of the other strand, such as the antisense strand and the sense strand forming a biphasic or double stranded structure).
  • the base sequence of the antisense strand can be complementary to the nucleotide sequence of a target nucleic acid molecule or a portion thereof, and the nucleotide sequence of the sense strand can be identical to the target nucleic acid sequence or a portion thereof.
  • the iNA can be derived from a single oligonucleotide in which the sense and antisense regions of the self-complementary complement of the bidirectional iNA can be joined by bases of nucleic acids or non-nucleic acids.
  • the iNA may contain nucleotides, non-nucleotides, or mixed nucleotide/non-nucleotide linking groups that link the sense and antisense segments of the iNA.
  • the linking portion of the nucleotide can be 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides in length.
  • the nucleotide linkage can be a nucleic acid aptamer.
  • aptamer or "nucleic acid aptamer” as used herein, includes a nucleic acid molecule that specifically binds to a target molecule, characterized in that said nucleic acid molecule comprises a sequence which is a target in its natural environment. molecule.
  • An aptamer can be a molecule that binds to a target molecule, wherein the target molecule is not a natural nucleic acid-binding molecule.
  • an aptamer can be used to bind to the active center of a protein, thereby preventing interaction with the naturally occurring ligand of the protein. See Gold, et al., Annu. Rev. Biochem. 64:763, 1995; Brody and Gold, J. Biotechnol. 74:5, 2000; Sun, Curr. Op in. Mol. Ther. 2:100, 2000; Kusser, J. Biotechnol. 74:27, 2000; Hermann and Patel, Science 287:820, 2000; and Jayasena, Clinical Chemistry 45:1628, 1999.
  • a non-nucleotide linkage can be an abasic nucleotide, polyether, polyamine, polyamide, polypeptide, carbohydrate, lipid, polyhydrocarboru or other polymer (eg, polyethylene glycol, such as Those with a unit number between 2 and 100 ethylene glycol).
  • polyethylene glycol such as those with a unit number between 2 and 100 ethylene glycol.
  • Specific examples include those by Byla and Kaiser, Nucleic Acids Res. 18:6353, 1990, and Nucleic Acids Res. 15:3113, 1987; Cload and Schepartz, J. Am. Chem. Soc. 113:6324 , 1991; Richardson and Schepartz, J. Am. Chem. Soc. 113:5109, 1991; Ma, et al., Nucleic Acids Res.
  • non-nucleotide linkage refers to a unit of one or more nucleotides in a nucleic acid strand that can be included, including any sugar and/or phosphate substituted group or compound, and allowing the remaining bases to display them. Enzymatic activity.
  • the group or compound may be abasic because it does not contain a commonly recognized nucleotide base, Such as adenosine, guanine, cytosine, uracil or thymine, as also in the C1 position of the sugar.
  • biodegradable linkage refers to a nucleic acid or non-nucleic acid joining molecule designed to biodegrade and link to another molecule. Biodegradable linkages are designed with stability in mind for specific uses, such as delivery to a particular tissue or cell type.
  • the stability of the biodegradable linking molecule of the nucleic acid can be different or tunable, for example, by combining ribonucleotides, deoxyribonucleotides, and chemically modified nucleotides, such as 2'-0-methyl, 2'-Fluoro, 2'-amino, 2'-0-amino, 2'-C-allyl, 2'-0-allyl and other 2'-modified nucleotides.
  • a dimer, a trimer, a tetramer or a longer nucleic acid molecule of a biodegradable nucleic acid linker is about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides, or may comprise a phosphorus-containing base linkage. For example, a phosphate or phosphodiester bond.
  • Biodegradable nucleic acid linker molecules can also include modifications of the nucleic acid backbone, nucleic acid sugars or nucleic acid bases.
  • Antisense nucleic acid refers to a non-enzymatic nucleic acid molecule that binds to a target RNA, RNA, or PNA (protein nucleic acid, see Egholm et al., 1993 Nature 365, 566) and alters the activity of the target RNA. (See discussion: Stein and Cheng, 1993 Science 261, 1004 and Woo If et al., US Pat. No. 5, 849-902).
  • the antisense sequence is a single sequence that is contiguous with the target sequence.
  • an antisense molecule can bind to a substrate such that the target nucleic acid molecule forms a loop and/or the bound antisense molecule itself forms a loop.
  • an antisense molecule can be complementary to a segment sequence of two (or even more) non-contiguous substrates (RNAs), a portion of a sequence of non-contiguous segments of an antisense molecule, which can be complementary to two segments of a target sequence .
  • antisense DNA can be used for target RNA, which activates RNase H and degrades target RNA through DNA-RNA interactions.
  • the antisense oligonucleotide may comprise one or more activation regions of RNase H capable of activating RNase H and cleavage of the target RNA.
  • the antisense DNA can be chemically synthesized, or can also be expressed by an expression vector or the like of the single-stranded DNA to be used.
  • Antisense RNA has a complementary target gene RNA sequence that induces RNA interference by binding to the target gene RNA.
  • Antisense RNA has a complementary sequence that binds to the target gene RNA, and induces RNA interference by RNA that binds to the target gene.
  • Justice "ribonucleic acid” has a sequence of complementary antisense RNA that anneals to its complementary antisense RNA to form an iNA. These antisense and sense RNAs can be chemically synthesized.
  • Nucleic acid refers to deoxyribonucleotides or ribonucleotides, and their polymers may be in single or double stranded form.
  • the term includes nucleic acids containing known nucleotide analogs or modified backbone residues or linkers, which can be chemically synthesized, naturally occurring, non-naturally occurring, have similar nucleic acid binding properties as a reference, and are metabolized in a similar manner. . Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonic acids, chiral methylphosphonates, 2'-0-methylribonucleotides, peptide nucleic acids (peptide nucleic acids) , PNAs).
  • RNA is meant a molecule comprising at least one ribonucleotide residue.
  • “Ribonucleotide” means that the 2' position of the BD-ribose furanosyl moiety of a nucleotide is a hydroxyl group. This term includes double-stranded RNA, single-stranded RNA, isolated RNA (e.g., partially purified RNA). Substantially pure RNA, synthetic RNA, recombinantly produced RNA, and RNA from naturally occurring RNA that are altered by addition, deletion, substitution, and/or alteration of one or more nucleotides. Such changes may include the addition of a non-nucleotide material to the end or interior of the iNA.
  • nucleotides in RNA molecules of the invention may also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs of naturally occurring RNA.
  • ribonucleic acid and "RNA” refer to a molecule containing at least one ribonucleotide residue.
  • a ribonucleotide is a hydroxyl group at the 2' position of the ⁇ -D-nuclear furanosyl moiety.
  • RNA double-stranded RNA
  • single-stranded RNA isolated RNA (eg, partially purified RNA), substantially pure RNA, synthetic RNA, recombinantly produced RNA, and different naturally occurring RNA by addition, deletion of RNA modifications and Alter, substitute, modify, and/or alter one or more nucleotides.
  • ⁇ Changes may include the addition of non-nucleotides to the end or inside of the iNA.
  • a nucleotide in an RNA molecule includes one or more non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs.
  • non-nucleotide refers to any group or compound that can be incorporated into the unit of one or more nucleotides in the nucleic acid strand, including any sugar and/or phosphate substitution, substitution, and permissibility. The remaining sequences show their enzymatic activity.
  • the group or compound is abasic because it does not contain a recognized nucleotide base such as adenosine, guanine, cytosine, uracil or thymine, and therefore lacks a base at the - position.
  • nucleotides are recognized in the art, including natural base (standard) groups, and are in the art. A well-known modified base. Such bases are typically located at the location of the nucleotide sugar. Nucleotides generally include base, sugar and phosphate groups. Nucleotides may be modified in sugar, phosphate and/or base moieties (also referred to as interchangeable nucleotide analogs, modified nucleotides, non-natural nucleotides, other unmodified or modified) Non-standard nucleotides. See, Usman and McSwiggen, supra; Eckstein, et al., International PCT Publ ication No.
  • modified base in this respect refers to a position other than or equivalent to adenine, guanine, cytosine, and uracil nucleic acid molecule.
  • a nucleotide base complementary to a base refers to a base complementary to which a hydrogen bond is formed.
  • the complementary segments or strands of the nucleic acid (added by hydrogen bonding) complement each other.
  • “Complementary” means that a nucleic acid can form a hydrogen bond with another nucleic acid sequence, either by conventional Watson-Crick or by other non-traditional bonds.
  • the sense strand of a double-stranded iNA molecule can have an end cap, such as an inverted deoxyabasic group, which can be at the 3'-end of the sense strand > 5' _end > or both ends
  • cap structure is meant a chemical modification at either end of the oligonucleotide (see Adamic, et al, US Pat. No. 5, 998, 203, incorporated herein by reference). These terminal modifications protect the nucleic acid molecule from degradation by exonucleas and can help enter the cell. It may be 5'-end (5'-cap) or 3'-end (3'-cap) or it may be present at both ends.
  • 5'-caps in non-limiting examples, include, but are not limited to, glycerides, inverted deoxy abasic residues, 4', 5'-methylene nucleotides, 1-(beta-D-erythr Furofuran) nucleotides, 4 '-thionucleotides, carbocyclic nucleotides, 1, 5-anhydrohexitol nucleotides, L-nucleotides, ⁇ -nucleotides, base-modified nucleotides , phosphorothioate linkage, threo-pentofuranosyl nucleotide (threo-pentofuranosyl nucleotide ⁇ acyclic ic 3', 4'-seco nucleotide ⁇ Acyclic ic 3, 4-dihydroxybutyl nucleotide ⁇ acyclic ic 3, 5-dihydroxypentyl nucleotide ⁇ 3'_3'
  • 3'-caps include, but are not limited to, glycerides, inverted deoxy abasic residues (parts), 4', 5'-methylene nucleotides (4', 5'-methylene nucleotides), 1_ ( ⁇ _D_ erythrofuranose) nucleotide 4' , 5' -methylene nucleotide;
  • Asymmetric hairpin is a linear iNA molecule having an antisense strand containing a circular segment, which may be composed of nucleotides or non-nucleotides, and a sense strand, wherein the sense strand contains fewer than the antisense strand. Nucleotides, but sufficient to complement the antisense strand segment, form a double strand containing a loop structure.
  • asymmetric duplex refers to an iNA molecule having two separate chains comprising a sense strand and an antisense strand.
  • the sense strand contains fewer nucleotides than the antisense strand, but is sufficient to complement the antisense strand segment to form a double strand.
  • iNA can be produced by chemical synthesis or biological production. Methods of chemical synthesis can be found in Nucleic Acids Research, 624-627, 1999 and Nucleic Acids Research, 3547-3553, 1955, respectively. Molecular cloning technology can be used for biological production.
  • siRNA small interfering RNA
  • silencing RNA is used to mean that a double-stranded RNA molecule has a length of 16-29 nucleotides and can perform a variety of biological functions. Most notably, siRNA interferes with a specific gene expression via the RNA interference pathway. In addition to the role of siRNA in RNAi-related functions, for example, antiviral mechanisms or chromatin structure formation, these complex pathways are now elucidated.
  • RNAa refers to the phenomenon of double-stranded RNA activating gene expression. This phenomenon is known as "small RNA-mediated gene activation" or RNAa.
  • small RNA-mediated gene activation or RNAa.
  • the promoter of a double-stranded RNA targeting gene induces strong gene transcription.
  • RNAa has been demonstrated in several other mammals, including non-human primates, mice, and rats.
  • RNA interference refers to the process of RNA-dependent gene silencing, which is a silencing complex induced by double-stranded RNA in a cell, where they bind to the catalytic unit of RISC ARG0NAUTE.
  • double-stranded RNA or RNA-like iNA is exogenous (from the infected RNA genome or from transfected iNA or siRNA), RNA or iNA is directly introduced into the cytoplasm and cleaved into short fragments (siRNA) by Dicer.
  • siRNA short fragments
  • Dicer short fragments
  • dsRNA can be endogenous
  • the pre-microRNA is expressed from the genome of the RNA-encoding gene.
  • the primary transcript of the gene is first processed to form a characteristic stem-loop structure of the pre-miRNA in the nucleus, which is then exported to the cytoplasm. In the case of Dicer cleavage. Therefore, there are two pathways of dsRNA, exogenous and endogenous.
  • RISC RNA-induced silencing complex
  • Argonaute protein untied and They bind to double-stranded siRNAs that degrade RNA complementary to the siRNA antisense strand.
  • Double-stranded RNA after being cleaved by Dicer, only the guide strand (antisense strand) binds to the Argonaute protein and causes gene silencing.
  • the sense strand is degraded after RISC activation.
  • the segmented structure of the iNA includes an antisense strand for two different HBV RNA sites.
  • iNA Sequence 231 (iNA ID #2 and #8):
  • Antisense Chain 1 UUGUCAACAAGAAAAACCCCG
  • Antisense strand 2 UGAAGCGAAGUGCACACGGUC
  • iNA ID #231A Segmented iNA designed according to iNA ID #2 and #8 iNA:
  • Anti-sense strand 1 UUGUCAACAAGAAAAACCCCG
  • Anti-sense strand 2 UGAAGCGAAGUGCACACGGUC iNA ID #232 (segmented iNA designed according to iNA ID #2 and #5iNA)
  • Ant i-sense strand 1 UUGUCAACAAGAAAAACCCCG
  • Ant i-sense strand 2 AAACGCCGCAGACACAUCCAG
  • iNA ID #233 (segmented by iNA ID #2 and #6iNA)
  • Antisense Chain 1 UUGUCAACAAGAAAAACCCCG
  • Antisense strand 2 UCCAGAAGAACCAACAAGAAG
  • iNA ID #234 Segmented iNA designed according to iNA ID #2 and #7 iNA
  • Antisense Chain 1 UUGUCAACAAGAAAAACCCCG
  • Antisense strand 2 UAAAGAGAGGUGCGCCCCGUG
  • iNA ID #235 Segmented iNA designed according to iNA ID #2 and #9 iNA
  • Antisense Chain 1 UUGUCAACAAGAAAAACCCCG
  • Antisense strand 2 UUCUUCUAGGGGACCUGCCUC
  • iNA ID #236 Segmented iNA designed according to iNA ID #2 and #11 iNA
  • Antisense Chain 1 UUGUCAACAAGAAAAACCCCG
  • Antisense strand 2 UUCACGGUGGUCUCCAUGCGA
  • iNA ID #237 Segmented iNA designed according to iNA ID #5 and #8 iNA
  • Antisense strand 1 AAACGCCGCAGACACAUCCAG
  • Antisense strand 2 UGAAGCGAAGUGCACACGGUC
  • iNA ID #238 Segmented iNA designed according to iNA ID #6 and #8 iNA
  • Antisense strand 1 UCCAGAAGAACCAACAAGAAG
  • Antisense strand 2 UGAAGCGAAGUGCACACGGUC
  • iNA ID #239 (Segmented iNA designed according to iNA ID #7 and #8 iNA)
  • Antisense strand 1 UAAAGAGAGGUGCGCCCCGUG
  • Antisense strand 2 UGAAGCGAAGUGCACACGGUC
  • iNA ID #240 (segmented iNA designed according to iNA ID #9 and #8 iNA):
  • Antisense strand 1 UUCUUCUAGGGGACCUGCCUC
  • Antisense strand 2 UGAAGCGAAGUGCACACGGUC
  • iNA ID #241 (segmented iNA designed according to iNA ID #11 and #8 iNA):
  • Antisense strand 1 UUCACGGUGGUCUCCAUGCGA
  • Antisense strand 2 UGAAGCGAAGUGCACACGGUC
  • each iNA is as follows.
  • One strand consists of two polynucleotides, opposite in direction (a 5' to 3' and another 3' to 5'), wherein the 5'-3' direction is in the same order as the sequence of the target RNA.
  • the two terminal polynucleotides are hybridized together to form a ring structure.
  • the other strand will complement the circular single-stranded region and will also be complementary to the target RNA.
  • An example of such a sequence is as follows:
  • iNA ID #242 (Class-like HBV iNA designed by iNA ID #2): Justice chain: GGGCCCGGGUUUUUCUUGUUGACAAUUCCCGGG (the part of the line is 3' ⁇ 5' direction) Antisense chain: UUGUCAACAAGAAAAACCCCG
  • iNA ID #243 (circular HBV iNA designed by iNA ID #5):
  • iNA ID #244 circular HBV ⁇ 6 designed by iNA ID #6:
  • iNA ID #245 (Class-like HBV designed by iNA ID #7):
  • Antisense chain UAAAGAGAGGUGCGCCCCGUG
  • iNA ID #246 circular HBV iNA designed by iNA ID #8
  • Antisense chain UGAAGCGAAGUGCACACGGUC
  • iNA ID #247 circular HBV iNA designed by iNA ID #9:
  • iNA ID #248 circular HBV iNA designed by iNA ID #11:
  • iNA ID #249 (Ring-like HBV iNA designed by iNA ID #2 and #8) Antisense Chain 1 : UUGUCAACAAGAAAAACCCCG
  • Antisense strand 2 UGAAGCGAAGUGCACACGGUC
  • iNA ID #250 (segmented by iNA ID #2 and #8 iNA): Antisense Chain 1: UUGUmCAAmCAAGAAAAACCCCG
  • Antisense strand 2 UGAAGCGAAGUGCACACGGUC
  • iNA ID #250A (segmented by iNA ID #2 and #8 iNA): Antisense Chain 1: UUGUmCAAmCAAGAAAAACCCCG
  • Antisense strand 2 UGAAGCGAAGUGCACACGGUC
  • iNA ID #251 (segmented by iNA ID #2 and #8 iNA): Antisense Chain 1: UUGUmCAAmCAAGAAAAACCCCG
  • Antisense strand 2 UGAAGCGAAGUGmCAmCACGGUC
  • iNA ID #252 (segmented by iNA ID #2 and #8 iNA): Antisense Chain 1: UUGUCAACAAGAAAAACCCCG
  • Antisense strand 2 UGAAGCGAAGUGmCAmCACGGUC
  • iNA ID #253 (segmented by iNA ID #2 iNA):
  • Antisense chain UUGUmCAAmCAAGAAAAACCCCG
  • iNA ID #254 (segmented by iNA ID#2 iNA):
  • Antisense chain UUGUCAACAAGAAAAACCCCG
  • iNA ID #255 (segmented by iNA ID #8 iNA):
  • Antisense chain UGAAGCGAAGUGmCAmCACGGUC
  • iNA ID #256 (segmented by iNA ID #8 iNA):
  • Antisense chain UGAAGCGAAGUGCACACGGUC
  • each iNA is a strand of polynucleotides that can form a loop, bounded by the 5'-terminal nucleotide strand and the 3'-terminal nucleotide by Watson-Crick's principle.
  • Double stranded structure Another strand will be complementary to the single stranded segment of the loop to form a double stranded iNA.
  • iNA ID #257 (Stem-ring type iNA designed according to iNA ID #2 iNA):
  • Antisense chain UUGUCAACAAGAAAAACCCCG
  • iNA ID #258 (Stem-ring iNA designed according to iNA ID #5 iNA):
  • Antisense chain AAACGCCGCAGACACAUCCAG
  • iNA ID #259 (Stem-ring type iNA designed according to iNA ID #6 iNA
  • Antisense chain UCCAGAAGAACCAACAAGAAG
  • iNA ID #260 (Stem-ring iNA designed according to iNA ID #7 iNA):
  • Antisense chain UAAAGAGAGGUGCGCCCCGUG
  • iNA ID #261 (Stem-ring iNA designed according to iNA ID #8 iNA):
  • Antisense chain UGAAGCGAAGUGCACACGGUC
  • iNA ID #262 (Stem-ring iNA designed according to iNA ID #9 iNA): Justice Chain: GGGCCCGGCAGGUCCCCUAGAAGAAUUGGGCCC
  • Antisense chain UUCUUCUAGGGGACCUGCCUC
  • iNA ID #263 Stem-ring type iNA designed according to iNA ID #11 iNA:
  • Antisense chain UUCACGGUGGUCUCCAUGCGA
  • iNA ID #264 Stem-ring type iNA designed according to iNA ID #2 iNA
  • Antisense chain UUGUCAACAAGAAAAACCCCG
  • the base of the "m” is 2, the methoxy (2 ' -0-Methyl ) RNA base.
  • the base before “d” refers to the DNA base).
  • iNA ID #265 Stem-ring type iNA designed according to iNA ID #8 iNA:
  • Antisense chain UGAAGCGAAGUGCACACGGUC
  • the base before “m” refers to the 2' methoxy (2 ' -0-Methyl ) RNA base.
  • the base before “d” refers to the DNA base).
  • iNA ID #266 Stem-ring type iNA designed according to iNA ID #8 iNA:
  • Antisense chain UGAAGCGAAGUGmCAmCACGGUC
  • the base before “m” refers to the 2' methoxy (2 ' -0-Methyl ) RNA base.
  • the base before “d” refers to the DNA base).
  • iNA ID #267 (according to iNA ID #8 HBV iNA):
  • Antisense chain UGAAGCGAAGUG [mC] A [mC] ACGGUC
  • the base before “m” refers to the 2' methoxy (2 ' -0-Methyl ) RNA base.
  • the base before “d” refers to the DNA base).
  • iNA ID #268 (according to iNA ID #2 HBV iNA):
  • Antisense chain UUGUCAACAAGAAAAACCCCG
  • the base before “m” refers to the 2' methoxy (2 ' -0-Methyl ) RNA base.
  • the base before “d” refers to the DNA base).
  • iNA ID #269 (according to iNA ID#2 and #8 HBV iNA ):
  • Antisense strand 2 UGAAGCGAAGUG [mC] A [mC] ACGGUC
  • the base before “m” refers to the 2' methoxy (2 ' -0-Methyl ) RNA base.
  • the base before “d” refers to the DNA base).
  • iNA ID #270 (according to iNA ID#2 and #8 HBV iNA):
  • Antisense strand 2 UGAAGCGAAGUG [mC] A [mC] ACGGUC
  • iNA ID #271 (according to iNA ID#2 and #8 HBV iNA):
  • Antisense strand 2 UGAAGCGAAGUG [mC] A [mC] ACGGUC
  • RNA base (The base before “m” refers to 2, methoxy (2'-O-Methyl) RNA base. " d” The former base refers to the DNA base. The number of nts in the sense region can vary from 1 to 1000, but also chemically modified RNA or DNA bases or different linkages.
  • iNA ID #272 (according to iNA ID#2 and #8 HBV iNA):
  • Antisense strand 2 UGAAGCGAAGUG [mC] A [mC] ACGGUC
  • the base before “m” refers to the 2' methoxy (2'-O-Methyl) RNA base.
  • the base before “d” refers to the DNA base.
  • the number of nts in the sense region can vary from 1 to 1000, but also chemically modified RNA or DNA bases, or different linkages.
  • D0TMA is dissolved in ethanol and mixed with siRNA to produce a water-insoluble precipitate.
  • the precipitate is dissolved in chloroform or a similar solvent and further mixed with other lipids in chloroform, such as (WO/2010/ 135207) The process. After removing the organic solvent, the dried preparation is hydrated with 9% sucrose water, and the animal can be administered.
  • iNA was transfected with RFects with iNAs (5 nM) to HepG2. 2. 15 cells. Two days later, the cells were collected and disrupted, and the changes in gene expression were analyzed by real-time quantitative RT-PCR, as shown in Fig. 2.
  • FIG. 4 Examples of iNA inhibition of HBV replication in HBV transgenic animals.
  • Hepatitis B virus transgenic mice were injected intravenously with antiviral iNA (siRNA), which specifically inhibited the replication of HBV virus genes in hepatocytes, whereas ApoB siRNA in the control group had no effect on HBV replication.
  • siRNA antiviral iNA
  • HBV transgenic mice Intravenous administration of HBV1 (iNA number 267), HBV2 (iNA number 269), and AP0-B reduces the intrahepatic HBV DNA of HBV transgenic mice.
  • HBV transgenic mice were injected with iNA three times a week via the tail vein.
  • HBV transgenic mice in the adefovir dipivoxil (ADV) group were orally administered once daily for 14 consecutive days (10 mg/kg/day). Tissue and blood were taken three days after the last administration.
  • P ⁇ 0.001 multiple comparison test using one-way analysis of variance and Bonferroni method
  • HBV transgenic mice were injected with iNA three times a week via the tail vein.
  • HBV transgenic mice in the adefovir dipivoxil (ADV) group were orally administered adefovir dipivoxi 1 once daily for 14 days (10 m g / k g / day). The material was taken three days after the last administration.
  • P ⁇ 0.001 multiple comparison test using one-way analysis of variance and Bonferroni method
  • HBV transgenic mice Intravenous administration of HBV1 (iNA number 267), HBV2 (iNA number 269), and AP0-B intravenous administration reduced HBsAg in plasma of HBV transgenic mice.
  • HBV transgenic mice were injected with the iNA wrapped in the nanocapsule delivery system three times a week via the tail vein.
  • HBV transgenic mice in the ADV group were orally administered adefovir dipivoxil once daily for 14 days (10 m g / k g / day). The material was taken three days after the last administration.
  • P ⁇ 0.001 multiple comparison test using one-way analysis of variance and Bonferroni method). The result is shown in Figure 7.
  • HBV transgenic mice Intravenous administration of HBV1 (iNA number 267), HBV2 (iNA number 269), and AP0-B reduces the intrahepatic HBV RNA of HBV transgenic mice.
  • HBV transgenic mice were injected with iNA three times a week via the tail vein.
  • HBV transgenic mice in the ADV group were orally administered adefovir dipivoxil once daily for 14 days (10 m g / k g / day). The material was taken three days after the last administration.
  • P ⁇ 0.001 multiple comparison test using one-way analysis of variance and Bonferroni method). The result is shown in Figure 8.
  • HBV transgenic mice Examples of post-pharmaceutical plasma chemical analysis of HBV transgenic mice by HBV1 (iNA number 267), HBV2 (iNA number 269), and AP0-B iNA intravenous.
  • HBV transgenic mice were injected with iNA three times a week via the tail vein.
  • HBV transgenic mice in the ADV group were orally administered adefovir dipivoxil once daily for 14 days (10 m g / k g / day). The material was taken three days after the last administration.
  • P ⁇ 0.05 multiple comparison test using one-way analysis of variance and Bonferroni method). The result is shown in Figure 9.
  • mice Lysis buffer.
  • the lysed cell product (80 L) was transferred to a 96-well mRNA capture plate and incubated for 1 hour at room temperature.
  • mice collected mouse liver tissue.
  • the mixture was homogenized in a lysis buffer using Polytron (Turbocapture Kit, manufactured by Qiagen Co., Ltd.). Then transfer 80 ⁇ L to a 96-well mRNA capture plate and incubate for 1 hour at room temperature. Wash three times with 100 ⁇ L of wash buffer, then add 80 of the elution buffer to each well and incubate for 5 minutes at 65 °C.
  • the elution solution (containing mRNA) was transferred to a new 96-well clear plate.
  • Real-time RT-PCR 3 L of isolated mRNA was used for real-time RT-PCR.
  • the RT-PCR method uses the SYBR Green-step RT-PCR kit (SensiMix step SYBR Green kit, BI0LINE). Mix 11 ⁇ L master mix (with reverse transcriptase), 1 forward and reverse primer (6 ⁇ ⁇ ), 0.3 L 50X SYBR Green and 2. 7 ⁇ L water.
  • the temperature of the reverse transcription reaction was 42 ° C, 30 minutes later, and then 95 ° C, 15 minutes to activate Tag polymerase; the temperature and time of the PCR cycle were 95 ° C, 15 seconds, 60 ° C, 30 seconds, 72° C, 20 seconds.
  • the change in gene expression was analyzed by the A A CT method.
  • Liver tissue homogenate in lysate Liver tissue (about 0.1 g) was homogenized with a grinding buffer containing lysis buffer (1 mM EDTA, 10 mM Tris, 10 mM NaCl, 0.5% SDS, proteinase K). To extract DNA, the homogenate was incubated at 55 ° C for 12 hours, then an equal volume of phenol (Phenol) was added. The samples were mixed and centrifuged at 12,000 xg for 10 minutes. Then, chloroform was added to the supernatant and centrifuged again. The DNA was then precipitated with NaCl and ethanol.
  • the precipitated DNA precipitate was dissolved in TE buffer (pH 8.0) containing ribonuclease A.
  • a certain amount of DNA (usually containing 40 ⁇ g of DNA), and Hindi II digestive enzyme (New England Biolabs, MA) at 37 The digestion of °C for 3 hours has been shown to be free of Hindl ll inscribed sites.
  • the digested DNA was re-extracted and separated by 1% TAE agarose gel electrophoresis.
  • the DNA was then transferred to a BioDyne B positively charged nylon membrane. The DNA is fixed to the membrane after UV irradiation.
  • Hybridization was carried out using [ 32P ]CTP-labeled, Haelll-digested HBV genome cloned into the pBluescript plasmid as a probe. Hybridization was carried out overnight at 60 °C in a solution containing 10% PEG-8000, 0.05 M sodium phosphate, 0.33 mg/ml salmon sperm DNA, and 7% SDS. Phosphor imaging method (Optiquant) The radioactivity signal is measured and the density of the radioactive bands is determined.
  • the amount of viral DNA relative to the host mouse DNA is determined by the ratio of viral DNA bands to transgenic DNA bands. The calculation was based on the fact that the cells of each host mouse contained 1.3 copies of the HBV transgene.
  • PCR method 2 minutes at 95 ° C, then 40 cycles: 10 seconds at 95 ° C, 30 seconds at 60 ° C. Determined using the standard curve method.
  • ELISA Analysis of plasma HBeAg and HBsAg: ELISA was performed according to the manufacturer's instructions (International Immunodiagnostic, Forster City, CA).
  • This application provides a method for treating hepatitis B virus infection by inhibiting one or more sites of the HBV genome by one or more types of siRNA or iNA.
  • Vvvvvvvvvv listen to honest ⁇ . Nn nn circle
  • Vvvvv. I would like to] v] g nnnnn nnn
  • Vvvvv would like vvv. I would like to]n nnnn
  • Vvvv would like WW vvv. Nn circle nn
  • Vvv please vvvvv listen. ⁇ vv nnn nnnn
  • V ⁇ vv ⁇ vv would like vvvvv. ⁇ listen to vnnn nn
  • Vv ⁇ vv listen to vvvvvv. ⁇ listen to nnnn n
  • v face let ⁇ ivnnn - i ⁇ vvi ⁇ vvv ⁇ vv: face let ⁇ vn ⁇ n - ⁇ ⁇ ⁇ ⁇ . ⁇ vvv face vvcheng vv ⁇ n n

Landscapes

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

Abstract

一种治疗乙型病毒性肝炎的RNA干扰制剂，其是一种iNA（interfering nucleic acid，干扰核酸），包括一个或多个正义链和一个或多个反义链，其中一个或多个的多核苷酸链与HBV RNA的一个或几个区域互补，该制剂可以用于治疗乙型病毒性肝炎。所述iNA为双链结构，且该双链为直链、环形、类环形、发卡型、茎-环、单向或双向形的结构。该制剂可用于治疗乙型病毒性肝炎。

Description

说明书
治疗乙型病毒性肝炎的 RNA干扰制剂
技术领域
本发明是利用 RNA干扰 （RNA interference, RNAi) 抑制乙型肝炎病毒的复制。 背景技术
双链匪 ( dsRNA)抑制蛋白的表达， 沉默基因， 具有广泛的、 潜在的治疗人类疾病的用 途。 dsRNA诱导基因沉默有三种方式： 1、转录功能的失活，这一现象是指 RNA指导下的 DNA 或组蛋白甲基化； 2、 小干扰核酸（smal l interfering RNA, siRNA) 诱导的信使 RNA (mRNA) 的降解； 3、 RNA介导的转录功能的衰减。 但一般认为 dsRNA诱导的基因干扰 （抑制） 指的 是动物细胞内的 RNA降解。 化学合成的的小分子 RNA， 如 siRNA, 能够在亚微摩尔浓度中， 酶催化下降解细胞内的 mRNA达 95%以上。
RNAi的效果能够持续很长时间，甚至持续至几代细胞分裂， 同时基因干扰是序列特异性 的抑制。 所以， RNAi能特异性的抑制基因表达， 而不影响它异构体或其他的 mRNA, 这种特 异性的抑制在研究基因功能和药物靶点的签定有特别重要作用。 siRNA能够用于开发成药物 治疗： 1、 由于基因过度表达或正常下不表达的基因； 2、 突变的基因而引起的疾病。
医学应用
RNAi 能应用于开发不同于小分子和蛋白的新一类药物， 虽然长的 dsRNA引起细胞的干 扰素反应， 不能直接传递到细胞， 但 siRNA的应用还是比较成功的。 已经成功的广泛应用于 研究并已进行了临床试验。
应用于治疗方面， 首先是用于老年性黄斑性病变和呼吸道合胞病毒。其它报道的治疗的 疾病， 包括抗病毒 HIV, 甲、 乙、 丙型肝炎、 感冒和麻疹等； 治疗神经性退行性病变， 特 别是亨廷顿氏病， 如多聚谷氨酰胺疾病也有报道。 RNAi 也可以通过抑制过度表达的基因遏 制肿瘤细胞的分裂， 治疗癌症。然而， 一个非常重要的领域是开发一个安全的 siRNA传递技 术， 才能保证 RNAi的临床应用。
尽管细胞水平的研究表明 RNAi是一个非常有前途的药物开发平台， siRNA的脱靶效应， 是否可引起一些副作用也同时引起了注意， 因为脱靶效应可抑制与靶基因相似序列的基因。 据计算， 脱靶效应可达 10%。在哺乳细胞， 长的双链 siRNA可诱导干扰素反应。 因此， siRNA 或者 iNA ( interf erring nucleic acid, 干扰核酸） 必须保持短的序列， 以避免干扰素反 应。
能够设计一个 siRNA或 iNA没有干扰素反应最好的一个方法是设计一个治疗性的 iNA或 siRNA能作用于一个或一个以上的靶基因， 或一个靶基因的不同部位， 它的结构可是一个方 向， 或不同方向， 环形的， 类似环形的、 或直线型的。 这几种类型 iNA or siRNA与常用的 siRNA的结构不同。
据估计，乙型肝炎病毒（hepatitis B Virus,HBV)感染的人口约高达 20亿人 -世界人口的三分之一， 超过 350万人会转为慢性感染。 据报道， 15— 40%的 HBV感染的患者会发展为肝硬化， 肝衰竭或肝癌 (HCC)， 每年有 50万至 120万人死于 HBV感染。 HBV在美国的患病率估计为约 0.4%。 然而， 自愿 抽检数据表明在某些外国出生的少数族裔群体患病率超过 15 %。 在 20世纪 90年代， 乙肝病毒相关的诊 断门诊和住院治疗人数增加好几倍。 同样， 住院总费用估计已经从 1990年的 3.57亿美元增加至 2003年 的 15亿美元， 然后保持在 13亿美元。
治疗 HBV的最终目标是抑制或消除 HBV, 缓和或停止 HBV感染引起的肝损伤， 防止肝功 能衰竭和肝癌的发展。最重要短期和中期治疗目标是最大限度地提高 HBV DNA抑制率。但是， 彻底根除 B型肝炎病毒是困难的， 因为它整合到宿主基因组中，产生继续作为潜在复发倾向 的 cccDNA。 聚乙二醇干扰素 a -2a (PEG— IFN_ a )和干扰素 a -2a ( IFN- a )， 核苷类药物 (拉米夫定， 恩替卡韦和替比夫定）和核苷酸类似物（阿德福韦和替诺福韦）是 FDA批准的 抗 HBV药物市场上常用的药物。干扰素治疗的主要缺点是其显著的副作用，限制其长期使用。 它对失代偿期肝硬化与转氨酶正常患者往往是无效的。 此外， 只有三分之一的患者对 PEG-IFN- α 抗病毒有效。 虽然核苷 （酸）类似物抑制 HBV复制并能使肝脏坏死性炎症减少， 但不能完全根除病毒。 此外， 停药后， 多数患者观察到病毒血症的反弹。 此外， 长期治疗产 生耐药 HBV病毒株， 导致治疗失败。
RNAi是细胞内调节基因的活性过程。它曾被称为其他名称，包括转录后基因沉默 (PTGS ), 2006年， Andrew Fire and Craig C. Mel lo共同获得诺贝尔生理学或医学奖， 以表彰他们 1998年发表的文章， 文章描述了他们在线虫研究中发现的 RNAi现象。
两种类型的小核糖核酸 （RNA) 分子 - 微 RNA (miRNA), 小干扰 RNA ( siRNA) 是 RNA 干扰的核心。 RNA是基因的直接产物， 而这些小分子 RNA可以与其 mRNA特异性结合增加或 减少它们的活性， 例如， 抑制蛋白质的产生。 RNAi具有重要的作用， 保护细胞免受病毒和 转座子 (transposon)的影响。
RNAi现象被发现存在于许多真核细胞，包括动物细胞， 由 Dicer酶将长 dsRNA分子降解 为 20个核苷酸的被称为 siRNA的短片段。 每个 siRNA的退烧成两个单链（ss ) RNA, 即乘 客链禾口导向链 (passenger strand and the guide strand), 乘客链被水解， 导向链与 RISC 复合体结合。导向链碱基序列与 mRNA序列分子互补，诱导和激活 RISC复合物的催化剂组分 Argonaute蛋白， 将 mRNA降解。 在某些生物体内， 这个过程被传播至全身。
RNAi对基因表达的特异性抑制，无论是在细胞培养和在动物物体内，使之成为一个有价 值的研究工具， 合成的 dsRNA导入细胞可以诱导抑制特定基因。 RNAi技术也可以用于大规 模筛选，系统性的抑制细胞的每个基因，它可以帮助确定一个特定的细胞信息通路过程中所 必需的步骤，如细胞分裂。探讨信号通路是生物技术和医学上的一个行之有效的研究基因功 能的手段。 RNAi 已经在动物实验中证实了其特异性和活性， 具有沉默疾病基因和治疗疾病 的作用。
已有报道， RNAi在培养细胞和感染的小鼠， 能抑制 HBV。 但是， 也有报道由于 HBV的 高突变率， 通过抑制 HBV多个位点不仅能更有效地抑制 HBV, 也可能会阻止 HBV基因突变。 发明内容
简要地说， 此申请提供了一种方法由一种或多种类型的 siRNA或 iNA， 通过抑制 HBV基因 组的一个或多个位点治疗乙型肝炎病毒性感染。
dsRNA和 /或含有两个或多个片段的双链 RNA,适合作为 Dicer和 RISC底物，用于抑制 HBV 基因表达。 在一个方面， 本发明提供了一种方法， 包括双链 iNA或 siRNA, 如 iNA ( siRNA) ID号 1-272 (表 1 )， 可选择针对一个或一个以上 HBV的 RNA位点。双链 iNA或 siRNA由两个 彼此互补的链组成， 其中的一个或多个互补链的多核苷酸链可与 HBV RNA的一个或几个区域 互补。在其实施例中， 双链 iNAs可以是直链、环形、类环形、发卡型、茎一环（stem— loop)、 单向、 或双向形（bidirectional)的结构等 （图 1)。 在其它实施例中， 双链 iNAs (或 siRNA) 可以具有两个连续的多核苷酸链， 有一个或多个缺口 （切口）， 在间隙之间形成一个或多个分 段的链， 如图 1。 双链 iNA或 siRNA长 10-200个核苷酸， 较好的为 15〜50个核苷酸， 更好 是 19-29个核苷酸。其中双链的互补区域的多核苷酸链不少于 10个核苷酸， 更好是 19-29个 核苷酸在更进一步的实施例中， 至少 50 %， 70 % , 75 % , 80 % , 85 % , 90 % , 95 % , 96 % , 97 % , 98 % iNA ， 99 % , 或 100 %的一个链中的核苷酸的任何双链区域互补于另一条链。 在 其它实施例中，双链 iNA或 siRNA可以具有粘端或钝端。在其它实施例中， 如果 iNA或 siRNA 具有粘端， iNA或 siRNA的 3'末端包含 1至 5个核苷的长度， 可位于一条或 3 ' 末端 表 1 HBV病毒的 iNA (siRNA) 靶位点。
在一个方面， 本发明提供了一个线性分段的 iNAs结构， 其结构具有两个短链， 并互补于 一个连续的长链上， 短链之间有缺口 （切口）， 如 iNA ID N0. 231-241, 249-252, 和 269-272。 连续长链包括两个区段， 每一区段的序列与 HBV RNA互补或相同， iNA的两个短链可以相同 或互补于 HBV RNA的不同位点。 例如， 在实施例 1中， 针对每个 HBV RNA的位点， 连续长链 中的两个区段是没有特定的前后顺序的， 可以在前端 （5'端）也可以在后端 （3'端）， 相反也 可。 如 iNA ID号 231-241, 249-252, 和 269-272。 所述的连续链的长度至少长为 15至 80个 核苷酸， 最好为 19 50个核苷酸。 两个短链至少长 10至 40个核苷酸， 较好为 19至 27个核 苷酸。 在更进一步的实施例中， 至少为 50%， 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% ， 99%, 或 100% iNA的一个链中的核苷酸的任何双链区域互补于另一条链。 在 其它实施例中，双链 iNA或 siRNA可以具有粘端或钝端。在其它实施例中， 如果 iNA或 siRNA 具有粘端， iNA或 siRNA的 3'末端包含长度 1至 5个核苷酸， 可位于一条或。
在一个方面中， 本发明中的 iNA编号 242-248具有双向环状或类似环状结构的双链， 如 实施例 2中的 iNA， 可作用于 HBV RNA的不同区域。 两个互补链 RNA或 RNA样寡核苷酸形成 双链的 RNA或 RNA样寡核苷酸。 至少其中的一条链是双向的， 这条链中至少有一个区段为 5' 至 3'方向和另一个区段是 3'至 5'的方向， 并且通过这两个不同方向区段的互补核苷酸形成 一个环状或类似环状结构。 并且， 另一条单链与这条环状或类似环状双向链中的单链区段的 核苷酸通过互补方式形成双链。环形双向链的长度可以短至 10个核苷酸， 长至一千个核苷酸 或是整个 RNA的长度。 在一个方向上区段的长度 （5 '端至 3'端或 3端'至 5'端） 应该在 5至 80个碱基之间， 较好的是 5至 10或 15至 29个核苷酸。 环形的双向链互补与另一条链可以 是任何长度， 从 10到 200个核苷酸， 但形成的双链区域应该是从 10到 200个核苷酸之间的 长度， 较好的是为 15至 29个核苷酸， 最好是 19至 25个核苷酸。 双链区段之间的空隙可以 尽可能短为 0核苷酸 （称为切口）， 可长之为 100或几千个核苷酸 （缺口）。 在双链区域的短 链与长链是互补的。 任何双链区域的两条链可以完全或部分彼此互补。 如果只有部分互补， 在链中的一个或多个核苷酸可能不配对。 未配对核苷酸可以在 5'-末端或 3'-末端， 或两端， 或 5'末端和 3' -末端之间的任何地方。 实施例 2中的 iNA即是例子。
在一个方面中， 本发明提供了具有茎一环结构的 iNAs结构， 本发明中的 iNA NO.257-266, 如在实施例 4中所示， 可以针对 HBV的 RNA不同区域。 两个互补链 RNA或 RNA样寡核苷酸形 成双链 RNA。其中至少一个链形成茎一环结构； 其一段形成环， 而 5'-末端一部分和在同一条 链的 3'-末端一部分核苷酸之间形成一短的双链区域。 另一个链将与非双链区域的环状区域 互补。 类似茎一环的长度可以短至 10个核苷酸， 长至几千个核苷酸或是一个或几 mRNA的长 度， 较好为 15至 200个核苷酸， 最好为为 19 45个核苷酸的长度。类似茎一环可以是任何 长度， 但形成的双链区域应是从 10到 200个核苷酸的长度， 较好为 15至 29核苷酸。各双链 区域之间的间隙可以尽可能短为 0核苷酸 （切口）， 也可为 100或几千个核苷酸 （缺口）。 两 条链的任何双链区域可以完全或部分彼此互补。 如果只有部分互补， 在链中的一个或多个核 苷酸可能不配对。 未配对核苷酸可以在 5'-末端或 3'-末端， 或两端， 或 5'末端和 3'-末端之 间的任何地方。 实施例 4中显示的 iNA即是例子。
在本专利公开的任何方面， 一些实施例中提供了一个 iNA 分子含有核糖胸腺嘧啶核苷
(ribothymidine) 或硫代核糖胸腺嘧啶核苷 (2 - thioribothymidine) 或 2'_0_甲基 _5_甲 基尿苷 （2' -O-methyl-5-methyluridine) 在一条或多条链上代替至少一个尿苷或代替每一 个尿苷。 在进一步的实施例中， 该 iNA进一步包括一个或多个非标准核苷， 如脱氧尿苷酸、 锁核酸 （LNA) 分子或一个通用的核苷酸 （universal nucleotide), 或一个 G钳位 （clamp)。 通用核苷酸包括 C-苯基（C-phenyl )、 C_萘基（C-naphthy)、肌苷（ inosine)、唑甲酰胺（azole carboxamide) 1_ β _D_呋喃核糖基 -4-硝基吲哚 （ 1- β -D-ribofuranosyl_4-nitroindole)、 1- β -D- 呋 喃 核 糖 基 -1- β -D- 呋 喃 核 糖 5- 硝 基 吲 哚
( 1- β -D-ribofuranosyl-5-nitroindole )、 1_ β _D_呋喃核糖基 -1- β _D_呋喃核糖 6_硝基 吲哚 (1- β- D- ribofuranosyl- 6- nitroindole)、 或 1 _ β _D_呋喃核糖基 _3_硝基吡咯 (or 1- β -D-ribofuranosyl-3-nitropyrrole 在一些实施例中， RNA分子包括 2' -糖取代成分， 如 2'- 0-甲基 (2' -0-methyl), 2'_0_甲氧基乙基 (2' - 0- methoxyethyl )、 2' -0-2-甲氧基 乙基（2' -0-2- methoxyethyl )、 2'- 0-烯丙基（2' - 0- allyl)、或卤素（2， -氟， 2' - fluoro)。 在某些实施例中， INA 分子还包括一个或多个的第一链、 第二链或第三个链上一个端帽上的 取代基团， 可独立地为烷基 （alkyl )、 脱碱基位 （abasic)、 脱氧脱碱基 （deoxy abasic ) , 甘油 ( glyceryl )^ 二核苷酸 ( dinucleotide ) 无环核苷酸 ( acycl ic nucleot ide ) 或方向 的脱氧核苷酸。 在其它实施例中， 进一步包括至少一个修饰的核苷连接键， 如独立地为硫代 憐酸酉旨 (phosphorothioate ) 手性¾代憐酸酉旨 ( chiral phosphorothioate ) 二¾代 ΐ粦酸酉旨
(phosphorodithioate ) 磷酸三酉旨 ( , phosphotriester) aminoalkylphosphotriester 甲基膦酸 ( methyl phosphonate ) , 烷基膦酸盐 ( alkyl phosphonate ) , 3' -亚烷基膦酸盐
( 3 ' -alkylene phosphonate ) 5' -亚烷基膦酸盐 （5 ' -alkylene phosphonate ) 手性膦酸 盐 ( chiral phosphonate ) 、 膦 酰 基 ( phosphonoacetate ) 、 硫 代 膦 酰 基
( thiophosphonoacetate ) 勝酸酉旨 ( phosphinate ) 氛基憐酸酉旨 ( , phosphoramidate ) aminoalkylphosphoramidate 、 thionophosphoramidate ， thionoalkylphosphonate 、 thionoalkylphosphotriester selenophosphate, boranophosphate键连接键。
附图说明
图 1、 用于抑制 HBV基因的 siRNA或 iNA结构示意图。
图 2、将 iNA用 RFect转染 iNAs ( 5nM)至 HepG2. 2. 15细胞， 用实时定量 RT-PCR方法分析基 因表达的变化。
图 3、 用 RFect转染能作用于 HBV RNA两个不同位点的 iNAs ( 5nM) 至 HepG2. 2. 15细胞， 用 实时定量 RT-PCR方法分析基因表达的变化。
图 4、 乙肝病毒转基因小鼠一次静脉注射传输系统包裹的抗病毒 iNA (siRNA) , 特异性地抑 制 HBV病毒基因在肝细胞内的复制， 而对照组的 ApoB siRNA对 HBV的复制没有影响。 图 5、 HBV1 ( iNA编号 267)、 HBV2 ( iNA编号 269)、 和 AP0-B的 iNA静脉给药降低 HBV转 基因小鼠肝内 HBV DNA的例子。
图 6、 HBV1 ( iNA编号 267)、 HBV2 ( iNA编号 269)、 AP0-B的 iNA静脉给药降低 HBV转基 因小鼠血浆 HBeAg的例子。
图 7、 HBV1 ( iNA编号 267)、 HBV2 ( iNA编号 269)、 和 AP0-B的 iNA静脉给药降低 HBV转 基因小鼠血浆 HBsAg的例子。
图 8、 HBV1 ( iNA编号 267)、 HBV2 ( iNA编号 269)、 和 AP0-B的 iNA静脉给药降低 HBV转 基因小鼠肝内 HBV RNA的例子。
图 9、 HBV1 ( iNA编号 267)、 HBV2 ( iNA编号 269)、 和 AP0-B的 iNA静脉给 HBV转基因小鼠 药后血浆化学分析的例子。
定义
本发明中所用技术术语的定义应理解为包括这些术语和在本领域中的技术人员已知的那 些含义， 并且， 并不意在限制本发明的范围， 并不需要重复每一次的申明。
本文中使用的术语"一"， "一个"， "这个 "和类似的术语在描述本发明，并在权利要求中， 将被解释为包括单数和复数。 术语 "包括"； "具有"； 和 "含有"将被解释为开放式术语。 它 们的意思是， 例如 "包括， 但不限于"。
应用的或设定值的范围是指在此范围内的每个单独的值， 应等同于任何单独值的描述。这 里所采用的具体值， 将被理解为示例性， 而不是限制本发明的范围。
如本文所用的术语干扰核酸（iNA)是指具有彼此链互补的核酸双链。 当进入 RISC复合物 后， 诱导 RNA酶降解 RNA的 RNAi机制。 此外， iNA通过启动子的 RNAa机制， 调节目标基因 表达的增高。 一般来说， iNA 的每一条链皆为核苷酸， 主要是核糖核苷酸， 但也可以是 RNA 的类似物， RNA和 RNA的类似物， 经修饰的核苷酸， RNA和 DNA， RNA的类似物和 DNA， 非核 苷酸， 或一个链完全是 DNA， 另一个链是 RNA， 只要是能通过 RNAi机制诱导同源 RNA降解， 均 可作为 iNA的构造。
如本文所用术语 "双向双链 iNA"或 "双向双链 siRNA"或 "双向 iNA"是一个贯穿于本 发明通用术语， 包括干扰核酸 （iNAs ) 的同一条链的双向结构， 它可以在细胞中被切割形成 iNA或 siRNA。 双向 iNA的双链至少一条链具有一个或多个 5'至 3'方向的片段和一个或多个 3'至 5'方向的片段。 iNA的另一链上有一个或多个部分与其互补， 以形成一个或多个双链区 段。 每个双链区段之间， 可以有缺口或切口。 双向 iNA可以是线性的， 茎一环形状的， 或圆 形等配置。 终端结构的 iNA可能是钝或粘性 （突出或悬垂） 末端， 其功能目标是 RNA沉默。 粘性（突出）末端的端部结构并不只限于 3'突出部分， 也可是 5'突出的结构， 只要它是能够 诱导 RNAi效应。 此外， 悬垂的核苷酸数目不限于报告的 2或 3， 但可以是任何数目， 只要能 够诱导 RNAi效应。 例如， 可能是 1至 8或更长， 或 2至 4个碱基或更长。
如本文所用， 术语"环形或类似环形的 iNA双链"是本发明用的通用术语， 包括干扰核酸 ( iNAs ) 具有环形或类环形等的结构框架， 它可以在细胞中被切割形成 iNA或 siRNA。 本发 明的环形或类环形的 iNA双链还包括表达载体 （也简称为 iNA表达载体） 能够产生 iNA双链 或在细胞中转录形成和 /或转录以后生成的 iNA， 并在体内诱导 RNAi。正义链或反义链可有一 个或多个切口或缺口。 iNA末端可以是粘性 （突出） 或钝的， 只要其功能可以使目标 RNA沉 默。 粘性 （突出） 的端部结构并不只限于 3'突出部分， 也可为 5'突出的结构， 只要它是能够 诱导 RNAi效应即可。 此外， 悬垂的核苷酸数目不限于报告的 2或 3， 可以是任何数目， 只要 能够诱导 RNAi效应。 例如， 悬垂可能是 1至 8个碱基或更长， 或 2至 4个碱基。
茎环结构是指一个多聚核苷酸分子内碱基配对， 可以发生在单链 DNA， 更常见的是 RNA。 双链环状结构是指由单个或多个链互补于环区的单链部位， 片段间有缺口或切口。 另外， 在 茎上的任何区段也可以有一个或多个单链区域的互补链。 末端结构可能是钝或粘性 （突出）。 可以有一个或多个链互补于环或非环状区段， 片段间有缺口或切口、带或不带 3'或 5'末端的 突出。
如本文所用的长度指的是双向 iNA中的核苷酸的数量， 从正义链的 5'末端的第 1个碱基 对开始， 结束于正义链的 3'末端最后一个碱基。
在遗传学上，微■ (miRNA)是单链 RNA,长度约 21-23个核苷酸，调节基因的表达。 miRNA 是从 DNA转录的，但没有翻译成蛋白质（非编码 RNA),它们是由被称为初级转录的 PRi-miRNA 的短茎环结构而生成 pre-miRNA， 最后从 pre_miRNA生成 miRNA。成熟 miRNA分子部分互补于 一个或多个的信使■分子， 它们的主要功能是降低基因的表达。
iNA分子修饰的核苷酸可以在任何链。 例如， 经修饰的核苷酸， 可以有一个 Northern的 构象（例如， Northern pseudorotation周期，见 Sanger,核酸结构的原理， Springer- Verlag ed. , 1984)。 核苷酸具有 Northern配置的实例包括锁核酸 ( LNA) 的核苷酸（如 2' _0， 4， - C- 亚甲基- ( D-呋喃核糖基） 核苷酸）， (2' -0, 4' -C-methylene- (D-ribofuranosyl) nucleotides) , 2' -甲氧基乙氧基 (Μ0Ε) 核苷酸 （2' -methoxyethoxy (MOE) nucleotides ), 2' -甲基-硫代-乙基， 2' -脱氧 -2' -氟核苷酸 （2' -methyl-thio_ethyl, 2' -deoxy-2' -f luoro nucleotides ), 2' -脱氧 -2' -氯核苷酸 （2' -deoxy-2' -chloro nucleotides ), 2' -叠氮基的核 苷酸 ( 2' -azido nucleotides ), 2' -0-甲基核苷酸 （2' -0- methyl nucleotides )。 同时保持 诱导 RNAi的能力和可以抵抗核酸酶降解的化学修饰的核苷酸。一种共轭分子连接到化学修饰 的 iNA分子可是聚乙二醇、 人血清白蛋白或一个细胞受体的配位体， 可介导细胞摄取。 可以 连接到化学修饰 iNA分子和由本发明所设想的特定的共轭分子的实例描述， 可见 Vargeese, 等人的美国专利， 公开号为 20030130186和美国专利公开号 20040110296， 其每部分均可引 用于本发明。
在本发明中有好几个例子， 描述糖， 碱和磷酸盐的修饰， 可以引入到核酸分子， 增强其核 酸酶的稳定性和有效性。例如， 寡核苷酸被修饰以增强稳定性和 /或增强生物活性， 通过修饰 抵抗核酸酶， 例如， 2' -氨基、 2' -C_烯丙基、 2' -氟代、 2' -0_甲基、 2 ' -0_烯丙基、 2' -H的 核苷酸碱基修饰。 见乌斯曼和塞德格伦 (Usman and Cedergren的论著， TIBS 17 : 34, 1992 ; Usman, et al, Nucleic Acids Symp. Ser. 31： 163, 1994； Burgin, et al, Biochemistry 35 : 14090, 1996。 已经广泛描述的现有技术中的核酸分子的糖修饰， 见 See Eckstein et al. , International Publ ication PCT No. W0 92/07065 ; Perrault, et al. Nature 344 : 565-568, 1990； Pieken, et al. Science 253 : 314—317, 1991； Usman and Cedergren, Trends in Biochem. Sci. 17 : 334-339, 1992 ; Usman et al. International Publ ication PCT No. WO 93/15187 ; Sproat, U. S. Pat. No. 5, 334, 711 and Beigelman, et al. , J. Biol. Chem. 270 : 25702, 1995 ; Beigelman, et al. , International PCT Publ ication No. WO 97/26270； Beigelman, et al. , U. S. Pat. No. 5, 716, 824 ; Usman, et al. , U. S. Pat. No. 5, 627, 053 ; Woolf, et al. , International PCT Publ ication No. W0 98/13526； Thompson, et al. , Karpeisky, et al, Tetrahedron Lett. 39 : 1131, 1998； Earnshaw and Gait, Biopolymers (Nucleic Acid Sciences) 48 : 39-55, 1998 ; Verma and Eckstein, A画. Rev. Biochem. 67 : 99-134, 1998 ; and Burl ina, et al. , Bioorg. Med. Chem. 5 : 1999-2010， 1997。 这些一般研究描述的方法 和策略，用以修饰糖，碱基和 /或磷酸。类似的修饰可以用于本发明所述的 iNA双链核酸分子， 只要 iNA的能促进细胞内的 RNAi而不是显着抑制其 RNAi的功能。
iNA双链可能包含磷酸骨架修饰的 iNA分子， 包括： 一个或多个硫代磷酸酯、 二硫代磷酸 酯、 甲基膦酸酯、 磷酸三酯、 吗啉代基、 酰胺化氨基甲酸叔丁酯、 羧甲基、 aCetamidate、 聚 酰胺、 磺酸酯、 磺酰胺、 氨基磺酸盐、 formaCetal、 thioformacetal , 烷基甲硅烷基。 寡核 苷酸骨架修饰的论述参见， Hunziker and Leumann, Nucleic Acid Analogues： Synthesis and Properties, in Modern Synthetic Methods, VCH, 1995, pp. 331-417, and Mesmaeker, et al. , "Novel Backbone Replacements for Ol igonucleotides, in Carbohydrate Modifications in Antisense Research,〃 ACS, 1994， pp. 24-39。 化学修饰的例子， 可以 包括硫代磷酸酯 nucleotide间的联接、 2' -脱氧核糖核苷酸、 核糖核苷酸的 2' _0_甲基、 2' - 脱氧 -2' -氟核糖核苷酸、 "通用碱基"核苷酸、 "无环"核苷酸、 5-C-甲基核苷酸、 终端甘油 酯和 /或倒置脱氧脱碱基残基。 一个 iNA分子的反义区可包括在所述反义区的 3' -末端的一个 硫代磷酸酯核苷间的连接。 反义区可包括 1-5个 5' -末端硫代磷酸酯核苷间的连接。 一个环 形或类似环形的 iNA分子的 3' -末端核苷酸可以包括核糖核苷酸或脱氧核糖核苷酸的化学修 饰的核酸糖、 碱。 3' -末端的核苷酸可以包括一个或多个通用碱基核糖核苷酸。 3' -末端的核 苷酸可以包括一个或多个无环核苷酸。 例如， 化学修饰的 iNA可以有 1， 2, 3， 4， 5， 6， 7， 8，或更多的硫代磷酸酯核苷间联接。每条链中可有 1至 8个或更多的硫代磷酸酯核苷间联接。 硫代磷酸酯核苷间联系可以是存在于一个或两个寡核苷酸链的双向的 iNA双体， 例如， 可在 有义链， 反义链或两条链。 在一些实施例中， iNA分子包括 1， 2, 3， 4， 5， 6， 7， 8， 9， 10， 或更多的嘌呤硫代核苷间的有义链、 反义链中或两条链中。
可以通过化学修饰 iNA分子的合成条件： （1 ) 合成至少两个或两个以上 RNA或 RNA-类似 寡核苷酸分子及互补链的； （2 ) 适合的条件下， 两个或更多个互补链一起退火， 获得 iNA分 子。 在一些实施例中， 双向 iNA分子的互补部分的合成是由固相寡核苷酸合成， 或由固相串 联的寡核苷酸合成。
寡核苷酸（例如， 某些修饰的寡核苷酸或部分缺乏核糖核苷酸的寡核苷酸）可使用本领域 已知的技术， 例如 Caruthers, et al, Methods in Enzymology 211 : 3—19， 1992 ; Thompson, et al. , International PCT Publ ication No. W0 99/54459 ; Wincott, et al. , Nucleic Acids Res. 23 : 2677-2684, 1995 ; Wincott, et al. , Methods Mol. Bio. 74 : 59， 1997 ; Brennan, et al. , Biotechnol Bioeng. 61 : 33-45， 1998 ; and Brennan, U. S. Pat. No. 6, 001, 31 1。 按照一般所描述的程序， 化学合成 RNA, 见 Usman, et al. , J. Am. Chem. Soc. 109 : 7845, 1987； Scaringe, et al. , Nucleic Acids Res. 18： 5433, 1990； and Wincott, et al, Nucleic Acids Res. 23 : 2677—2684， 1995 ; Wincott, et al, Methods Mol. Bio. 74 : 59， 1997。 任 何两个或更多个互补链或发夹■或由不同的互补的环形或类似环形 iNA链退火， 可以形成 互补 iNA的双链结构。
"重叠"（overlapping)是指当两个 iNA片段具有序列重叠，例如，其中的多个核苷酸（nt ) 可能少至 2-5个核苷酸多至 5-10个核苷酸或更多。
"一个或多个的 iNA"指的 iNA， 彼此有不同初级序列。
"靶部位"或 "靶序列 （target ) "或 "靶序列（targeted) "是指靶核酸内的序列 （例如 RNA), 是 由 iNA反义链序列介导的 siRNA降解的序列。
一个空隙指链中的两个核苷酸磷酸二酯键之间不连接或有缺口或切口。
一种混合型 iNA分子是指 iNA的双链核酸含有一个 RNA链和 DNA链。 优选 RNA链是靶 RNA 结合的反义链。 通过杂交的 DNA和 RNA链创建的混合 iNA含有杂交的互补部分， 可至少有一 个 3'粘性端。
为了 "调节基因的表达"这里所用的增加或降低靶基因的表达， 其可以包括细胞内增高或 降低 RNA水平， 或 RNA的翻译， 或由其编码的蛋白或蛋白亚基的合成。
术语 "抑制"， "降低 "或 "减少的表达"这里意思是基因的表达， 或水平， 编码一种或多 种蛋白质或蛋白质亚基的■分子， 或等效的 RNA分子的水平或活性中的一种或多种蛋白或 靶基因所编码的蛋白亚基， 降低到低于应用 iNA前的水平。
本文所用的"基因沉默"是指在细胞内部分或完全抑制基因的表达，并且也可以被称为"基 因敲除"。基因沉默的程度， 也可以通过本领域中已知的方法来确定， 其中一些被总结在国际 公开号 W099 / 32619中。
在一些实施例中， iNA分子包括正义和反义序列或区段， 其特征在于正义和反义区的核苷 酸或非核苷酸以共价键连接， 或通过非共价键、 离子键相互作用、 氢键、 范德华相互作用， 疏水相互作用和 /或堆积 （stacking) 相互作用。
iNAs 可以由两个单独的寡核苷酸组装成一个双链， 其中一条链是正义链和另一个是反义 链， 其中， 所述的反义和正义链自身互补 （即每一条链所含的碱基的核苷酸序列与另一条链 的序列相补， 如其中的反义链和有义链形成了一个双相或双链结构）。反义链的碱基序列可与 一个靶核酸分子或其部分的核苷酸序列互补， 有义链的核苷酸序列可于靶核酸序列或其一部 分相同。 iNA可以从单一的寡聚核苷酸而来， 其中双向 iNA的自身互补的正义和反义区域可 由核酸的碱基或非核酸的基团连接。
iNA可能含有核苷酸， 非核苷酸， 或混合的核苷酸 /非核苷酸连接基团， 将 iNA的正义和 反义区段连接在一起。 在一些实施例中， 核苷酸的连接部分可以是 3， 4, 5， 6， 7， 8， 9或 10个核苷酸的长度。 在一些实施例中， 核苷酸链接可以是一种核酸适体。 如本文所用， 术语 "适配体"或 "核酸适体"包括特异性结合靶分子的核酸分子， 其特征在于， 所述的核酸分 子， 包含一个序列， 该序列是在其天然环境中的靶分子。 适体可以是结合到靶分子， 其中所 述靶分子不是自然的核酸结合的分子。例如， 适配体可以用于绑定到一种蛋白质的活性中心， 由此防止与蛋白质的天然存在的配位体相互作用。 参见 Gold, et al. , Annu. Rev. Biochem. 64:763, 1995; Brody and Gold, J. Biotechnol. 74:5， 2000; Sun, Curr. Op in. Mol. Ther. 2:100, 2000; Kusser, J. Biotechnol. 74:27, 2000; Hermann and Patel, Science 287:820, 2000; and Jayasena, Clinical Chemistry 45:1628, 1999。
一个非核苷酸的链接可以是一个脱碱基的核苷酸、 聚醚、 聚胺、 聚酰胺、 聚肽、 碳水化合 物、 脂质， polyhydrocarboru 或其他的聚合物 （例如， 聚乙二醇， 如那些具有单元数为 2和 100之间的乙二醇）。 具体的例子见 Specific examples include those described by Seela and Kaiser, Nucleic Acids Res. 18:6353, 1990， and Nucleic Acids Res. 15:3113, 1987; Cload and Schepartz, J. Am. Chem. Soc. 113:6324, 1991； Richardson and Schepartz, J. Am. Chem. Soc. 113:5109, 1991; Ma, et al. , Nucleic Acids Res. 21:2585, 1993, and Biochemistry 32:1751, 1993; Durand, et al. , Nucleic Acids Res. 18:6353, 1990; McCurdy, et al, Nucleosides & Nucleotides 10:287, 1991； Jschke, et al. , Tetrahedron Lett. 34:301, 1993; Ono, et al. , Biochemistry 30:9914, 1991; Arnold, et al. , International Publication No. W089/02439; Usman, et al. , International Publication No. W095/06731; Dudycz, et al. , International Publication No. W095/11910, and Ferentz and Verdine, J. Am Chem. Soc. 113:4000, 1991。 A "非核苷酸连接"是指可以被纳入的核酸链中的一个 或多个核苷酸的单位， 包括任一糖和 /或磷酸盐取代的基团或化合物， 并允许剩余碱基展示他 们的酶促活性。 基团或化合物可以是脱碱基的， 因为它不含有一种常用公认的核苷酸碱基， 如腺苷、 鸟嘌呤、 胞嘧啶、 尿嘧啶或胸腺嘧啶， 也如在糖的 C1位置。
本文所用的术语"生物降解的连接"， 是指一种核酸或非核酸的连接分子， 被设计为可生 物降解的连接另一个分子。 可生物降解连接的设计考虑其稳定性可以针对特定用途， 例如传 递到一个特定的组织或细胞类型。核酸的生物降解的连接分子的稳定性可以是不同或可调的， 例如， 通过组合的核糖核苷酸， 脱氧核糖核苷酸和化学修饰的核苷酸， 如 2' -0-甲基、 2' -氟 代、 2' -氨基、 2' -0_氨基、 2' -C_烯丙基、 2' -0-烯丙基和其他 2' -修饰核苷酸。 生物降解的 核酸连接分子的二聚体， 三聚体， 四聚体或更长的核酸分子的寡核苷酸为约 2、 3、 4、 5、 6、 7、 8、 9、 10、 11、 12、 13、 14、 15、 16、 17、 18、 19、 20 或更多个核苷酸， 或者可以包括 一个含磷的碱基连接。 例如， 磷酸酯或磷酸二酯键。 生物降解性的核酸连接分子还可以包括 核酸骨架， 核酸糖或核酸碱基的修饰。
"反义核酸"， 是指非酶的核酸分子， 结合到靶 RNA的 RNA、 DNA或 PNA (蛋白质核酸， 见 Egholm et al. , 1993 Nature 365, 566 ) 的分子， 并改变了靶 RNA的活性 (见论述: Stein and Cheng, 1993 Science 261, 1004 and Woo If et al. , U. S. Pat. No. 5, 849— 902)。 通 常情况下， 反义序列是一单一的连续与靶序列完全互补的序列。 然而， 在某些实施例中， 反 义分子可以结合到底物， 使得靶核酸分子形成了一个环和 /或结合的反义分子本身形成一个 环。 因此， 反义分子可以与两个（或甚至更多） 非连续的底物 （RNA) 的区段序列互补、 反义 分子非连续区段序列的部分， 可以互补于靶序列的两个区段。 此外， 反义 DNA可以用于靶基 因 RNA， 通过 DNA-RNA的相互作用， 激活 RNA酶 H， 降解靶 RNA。 反义寡核苷酸可以包括一个 或多个 RNA酶 H的激活区， 能够激活 RNA酶 H， 裂解靶 RNA。 反义 DNA可以化学合成， 或者也 可以通过使用的单链 DNA的表达载体或等表达。 "反义 RNA"具有互补靶基因 RNA序列， 通过 结合到靶基因 RNA可以诱导 RNA干扰。 反义 RNA具有结合靶基因 RNA的互补序列， 通过结合 到靶基因的 RNA诱导 RNA干扰。 正义 "核糖核酸"具有互补反义 RNA的序列， 与其互补的反 义 RNA退火形成 iNA。 这些反义和正义 RNA可以化学合成。
"核酸"是指脱氧核糖核苷酸或核糖核苷酸， 它们的聚合物可是单链或双链形式。 该术 语包括含由已知的核苷酸类似物或修饰骨架残基或联接子的核酸， 可以化学合成， 天然存在， 非天然存在， 具有相似的作为基准的核酸结合特性， 以及类似方式被代谢。 这种类似物的实 例包括， 但不限于硫代磷酸酯、 氨基磷酸酯、 甲基膦酸、 手性甲基膦酸盐、 2' -0-甲基核糖核 苷酸、 肽核酸 （肽核酸， PNAs )。
所谓 "RNA"是指包含至少一个核糖核苷酸残基的分子。 "核糖核苷酸"是指核苷酸的 BD- 核糖呋喃糖部分的 2'位是一个羟基基团。 这一术语包括双链 RNA、 单链 RNA、 分离的 RNA (例 如部分纯化的 RNA)。 基本上纯的 RNA、 合成 RNA、 重组产生的 RNA, 以及从天然存在的 RNA通 过添加、 缺失、 取代不同和 /或改变一个或多个核苷酸改变的 RNA。 这样的改变可包括添加非 核苷酸材料到 iNA的末端或内部。 例如一个或多个核苷酸。 发明中的 RNA分子中的核苷酸还 可以包括非标准核苷酸， 例如非天然存在的核苷酸或化学合成的核苷酸或脱氧核苷酸。 这些 改变的 RNA可称为天然存在的 RNA的类似物。 如本文所用， 术语 "核糖核酸"和 "RNA"指含 有至少一个核糖核苷酸残基的分子。 核糖核苷酸是含有 β -D-核呋喃糖基部分的 2'位的羟基 核苷酸。 这些术语包括双链 RNA、 单链 RNA、 分离的 RNA (例如部分纯化的 RNA)， 基本上纯的 RNA, 合成 RNA， 重组产生的 RNA， 以及不同于天然存在的 RNA通过添加， 缺失 RNA修饰和改 变、 替代、 修改和 /或改变一个或多个核苷酸。 ■的改变可包括添加非核苷酸到 iNA的末端 或内部。 例如 RNA分子中的核苷酸包括一个或多个非标准核苷酸， 例如非天然存在的核苷酸 或化学合成的核苷酸或脱氧核苷酸。 这些改变的 RNA可称为类似物。
所用的术语 "非核苷酸"是指任何基团或化合物， 该化合物可以被纳入核酸链中的一个 或多个核苷酸的单位， 包括任一糖和 /或磷酸盐取代、代替、 并允许剩余序列展示他们的酶活 性。 基团或化合物是脱碱基的， 因为它不包含一个公认的核苷酸碱基， 如腺苷、 鸟嘌呤、 胞 嘧啶、 尿嘧啶或胸腺嘧啶， 因此在 -位缺乏碱基。
本文所用的 "核苷酸"在本领域中所承认的， 包括天然的碱 （标准） 基， 并在本领域中 公知的经修饰的碱基。 这类碱基一般位于核苷酸糖 的位置。 核苷酸一般包括碱， 糖和磷酸 基团。 核苷酸可以在糖， 磷酸盐和 /或碱基部分修饰（也可简称为可互换的核苷酸类似物、 经 修饰的核苷酸、 非天然核苷酸、 其他未修饰或修饰的非标准核苷酸。 参见， Usman and McSwiggen, supra ; Eckstein, et al. , International PCT Publ ication No. WO 92/07065； Usman, et al, International PCT Publ ication No. WO 93/15187； Uhlman & Peyman, supra, 所有在此引入本文作为参考）。核酸研究作为总结可参考 Limbach, et al, Nucleic Acids Res. 22 : 2183, 1994。一些非限制性实施例可以被引入到核酸分子的修饰基团包括，次黄嘌呤核苷、 嘌呤、 吡啶 -4-酮、 吡啶 -2-酮、 苯基、 pseudouraci l、 2, 4, 6-三甲氧基苯、 3—甲基尿嘧啶、 dihydrouridine 萘基、氨基苯基、 5-alkylcytidines (例如， 5_甲基胞苷)、 5-alkyluridines (例如， ribothymidine )、 5-halouridine (例如， 5—漠尿昔) 或 6-azapyrimidines 或 6-alkylpyrimidines (例如 6—甲基尿昔）、 丙块禾口其它基团 (Burgin, et al. , Biochemistry 35 : 14090, 1996 ; Uhlman & Peyman, supra)。 在这方面的所谓 "经修饰的碱基"是指在 的位置腺嘌呤、 鸟嘌呤、 胞嘧啶和尿嘧啶核苷酸碱基以外或等同物。
碱基互补的核苷酸碱基是指彼形成氢键的碱基互补。腺嘌呤（A)与胸腺嘧啶（T)或 RNA 中的尿嘧啶 （U)、 鸟嘌呤 （G) 与胞嘧啶 （C)。 核酸的互补链段或链（加入通过氢键）彼此互 补。 "互补"是指核酸可以与另一核酸序列形成氢键， 可以通过传统的 Watson-Crick或由 其他非传统键结合。
双链 iNA分子的正义链可以有末端帽，如一个倒置 deoxyabasic基团，可在正义链的 3' - 末端 > 5' _末端 > 或两个末端
所谓 "帽结构"指的是在寡核苷酸任一末端的化学修饰 （参见 Adamic, et al, U. S. Pat. No. 5, 998, 203, 在此引入作为参考）。 这些终端修饰可保护核酸分子不被外切酶降解， 可以 帮助进入到细胞内。可以是 5' -末端（5' -帽）或 3' -末端（3' -帽）也可能是存在于两个末端。 5' -帽在非限制性实施例中， 包括， 但不限于甘油酯、 倒置的脱氧脱碱基残基、 4'， 5' -亚甲 基核苷酸、 1- ( β -D-赤呋喃糖）核苷酸， 4 ' -硫代核苷酸、碳环核苷酸、 1， 5-anhydrohexitol 核苷酸、 L-核苷酸、 α -核苷酸、 碱基修饰的核苷酸、 硫代磷酸酯键、 苏式-呋喃戊糖基核苷 酸 （threo-pentofuranosyl nucleotide ^ 无环的 3'、 4' -塞科核苷酸 （acycl ic 3' , 4' -seco nucleotide )^ 无环的 3, 4_二轻基丁基核苷酸 ( acycl ic 3, 4-dihydroxybutyl nucleotide )^ 无环的 3, 5-dihydroxypentyl核苷酸 ( acycl ic 3, 5-dihydroxypentyl nucleotide )^ 3' _3' - 倒的碱基部分 （ 3' -3' -inverted nucleotide moiety )、 3' _3' -倒脱碱基部分） ( 3' -3' -inverted abasic moiety ) 3' _2 ' -倒置核苷酸部分 ( 3' -2' -inverted nucleotide moiety )、 3' _2' -倒置脱碱基部分的磷酸盐、 1, 4-丁二醇； 3' -磷酸酯 ( 1, 4- butanediol phosphate )^ hexylphosphate 氨基己基磷酸酉旨 ( aminohexyl phosphate )^ 3 ' -磷酸、 3' - 硫代磷酸酯、 二硫代磷酸酯 （ph0Sph0r0dithi0ate )、 或桥接或非桥接的甲基膦酸酯基团 sti l lben 禾口芘 ( bridging or non-bridging methylphosphonate moiety sti l lben and
3' -帽的实施例包括， 但不限于甘油酯、 倒置的脱氧脱碱基残基（部分）、 4'， 5' -亚甲基 核苷 酸 （ 4' , 5' -methylene nucleotide ) 、 1_ ( β _D_ 赤呋喃 糖 ） 核苷 酸
Figure imgf000010_0001
4' , 5' -methylene nucleotide；
1- (beta-D-erythrofuranosyl) nucleotide )、 4 ' -硫代核苷酸碳环核苷酸 ( 4' -thio nucleotide, carbocycl ic nucleotide ) ^ 4'-硫代核苷酸 ( 4， -thio nucleotide ) ^ 5' _氨基院 基磷酸酯（5' -amino-alkyl phosphate )^ 1, 3_二氨基 _2_丙基磷酸酯（1, 3-diamino-2-propyl phosphate ) 3—氛基丙基憐酸酉旨 ( 3-aminopropyl phosphate ) 6—氛基己基憐酸酉旨 ( 6-aminohexyl phosphate )、 1, 2-氨基十二烷基磷酸羟丙基磷酸酯 ( 1, 2-aminododecyl phosphate ) 1, 5-anhydrohexitol 核苷酸、 L -核苷酸 ( L-nucleotide ) α _核苷酸 ( alpha-nucleotide ) 修饰的核苷酸 ( modified base nucleotide )、 硫代磷酸酉旨 ( phosphorodithioate ) 苏式-呋喃戊糖基核苷酸 ( threo-pentofuranosyl nucleotide )^ 无环的 3'， 4' -塞科核苷酸 （acycl ic 3' , 4' -seco nucleotide ) 3, 4- 二羟基核苷酸 ( 3, 4-dihydroxybutyl nucleotide )^ 3, 5-dihydroxypentyl 核苷酸、 5' _5'反相核苷酸部 分 ( 5' -5' -inverted nucleotide moiety) 5' _5 ' -倒置脱碱基部分 ( 5' -5' -inverted abasic moiety) 5， _磷酰胺 ( 5' -phosphoramidate ) 5，-硫代磷酸酷 ( 5' -phosphorothioate ) 1, 4- 丁二醇磷酸酯 ( 1, 4-butanediol phosphate )^ 5' -氨基 ( 5' _amino)、 桥接和 /或 5' -氨基磷 酸酉旨 ( bridging and/or non-bridging 5' -phosphoramidate ) ¾代憐酸酉旨禾口 /非桥或憐酸酉旨 (phosphorothioate and/or phosphorodithioate)、 非桥接或桥接甲基膦酸二甲酯和 5' -巯 基 (bridging or non-bridging methylphosphonate and 5' -mercapto moieties )。 (有关 详细信息， 参照 Beaucage and Lyer, Tetrahedron 49 : 1925, 1993) and sti l lben 和芘 ( pyrene)。
"非对称发夹"是线性 iNA分子有一个含有环型区段， 可以由核苷酸或非核苷酸构成的反 义链， 和一个正义链， 其中， 正义链含有比反义链更少的核苷酸， 但是足以互补于反义链区 段， 形成一个含有环型结构的双链。
本文所用的 "非对称双链"是指有两个单独的链， 包含有正义链和反义链的 iNA分子。 其 中， 正义链含有比反义链更少的核苷酸， 但是足以互补于反义链区段， 形成一个双链。
iNA可以通过化学合成或生物生产。 化学合成的方法， 可以在 Nucleic Acids Research, 624-627, 1999 and Nucleic Acids Research, 3547-3553， 1955 分别找到。 分子克隆技术 可以用于生物生产。
术语小干扰 RNA ( siRNA)有时也被称为短干扰 RNA或沉默 RNA， 是用来指双链 RNA分子具 有 16-29个核苷酸的长度， 可以发挥多种生物学作用。 最值得注意的是， siRNA通过 RNA干 扰途径， 能干扰一个特定的基因表达。 除此外， siRNA的作用在 RNAi相关功能， 例如， 抗病 毒机制或染色质结构的形成， 这些复杂的途径现在才阐明。
术语 RNAa指的双链 RNA激活基因表达的现象。这种现象被称为 "小 RNA介导的基因激活" 或 RNAa。 双链 RNA靶向性基因的启动子， 诱导强有力的基因转录。 最近， RNAa已经在几个其 他哺乳动物， 包括非人灵长类、 小鼠、 大鼠被证明。
本文所用术语 "RNA干扰是指的依赖 RNA的基因沉默的过程， 是在细胞中由双链 RNA诱导 的沉默复合物， 在那里它们与 RISC的催化单位 ARG0NAUTE结合。 当双链 RNA或 RNA-类似 iNA 是外生的 （来自感染了病毒的 RNA基因组或从转染的 iNA或 siRNA), RNA或 iNA被直接导入 到细胞质， 由 Dicer酶切割成短的片段 （siRNA)。 dsRNA可以是内源性的 （源于细胞） 如前 的 microRNA从 RNA编码基因的基因组表达。基因的初级转录物首先被处理， 以在细胞核中形 成的特征的茎-环结构的 pre-miRNA， 然后将其导出到细胞质中， 由 Dicer切割。 因此， 有两 个 dsRNA的途径， 外源性和内源性。 RNA-诱导的沉默复合物 （RISC) 的活性成分， 核酸内切 酶被称为 Argonaute蛋白， 解开与它们结合的双链 siRNA, 降解与 siRNA反义链互补的 RNA。
双链 RNA， 被 Dicer切割后， 只有导向链（反义链）结合 Argonaute蛋白和引起基因沉默。 正义链在 RISC激活后被降解。
方案实施 1
分段式结构的 iNA包括针对两个不同的 HBV RNA位点的反义链。 在连续的链中间有几个 没有互补的核苷酸， 例如：
iNA序列 231号 （iNA ID #2 and #8)：
正义链： GGGUUUUUCUUGUUGACAAdTdTuauaCCGUGUGCACUUCGCUUCAdTdT
反义链 1 : UUGUCAACAAGAAAAACCCCG
反义链 2 : UGAAGCGAAGUGCACACGGUC
iNA ID #231A (按照 iNA ID #2 and #8 iNA设计的分段式 iNA) :
Sense strand： CCGUGUGCACUUCGCUUCAdTdTuauaGGGUUUUUCUUGUUGACAAdTdT
Anti-sense strand 1 : UUGUCAACAAGAAAAACCCCG
Anti-sense strand 2 : UGAAGCGAAGUGCACACGGUC iNA ID #232 (按照 iNA ID #2和 #5iNA设计的分段式 iNA)
正义链： GGGUUUUUCUUGUUGACAAdTdTuauaGGAUGUGUCUGCGGCGUUUdTdT
Ant i-sense strand 1 : UUGUCAACAAGAAAAACCCCG
Ant i-sense strand 2 : AAACGCCGCAGACACAUCCAG
iNA ID #233 (按照 iNA ID #2和 #6iNA设计的分段式）
正义链： GGGUUUUUCUUGUUGACAAdTdTuauaUCUUGUUGGUUCUUCUGGAdTdT
反义链 1 : UUGUCAACAAGAAAAACCCCG
反义链 2 : UCCAGAAGAACCAACAAGAAG
iNA ID #234 (按照 iNA ID #2和 #7 iNA设计的分段式 iNA)
正义链： GGGUUUUUCUUGUUGACAAdTdTuauaCGGGGCGCACCUCUCUUUAdTdT
反义链 1 : UUGUCAACAAGAAAAACCCCG
反义链 2 : UAAAGAGAGGUGCGCCCCGUG
iNA ID #235 (按照 iNA ID #2和 #9 iNA设计的分段式 iNA)
正义链： GGGUUUUUCUUGUUGACAAdTdTuauaGGCAGGUCCCCUAGAAGAAdTdT
反义链 1 : UUGUCAACAAGAAAAACCCCG
反义链 2 : UUCUUCUAGGGGACCUGCCUC
iNA ID #236 (按照 iNA ID #2和 #11 iNA设计的分段式 iNA)
正义链： GGGUUUUUCUUGUUGACAAdTdTuauaGCAUGGAGACCACCGUGAAdTdT
反义链 1 : UUGUCAACAAGAAAAACCCCG
反义链 2 : UUCACGGUGGUCUCCAUGCGA
iNA ID #237 (按照 iNA ID #5和 #8 iNA设计的分段式 iNA
正义链： GGAUGUGUCUGCGGCGUUUdTdTuauaCCGUGUGCACUUCGCUUCAdTdT
反义链 1 : AAACGCCGCAGACACAUCCAG
反义链 2 : UGAAGCGAAGUGCACACGGUC
iNA ID #238 (按照 iNA ID #6和 #8 iNA设计的分段式 iNA)
正义链： UCUUGUUGGUUCUUCUGGAdTdTuauaCCGUGUGCACUUCGCUUCAdTdT
反义链 1： UCCAGAAGAACCAACAAGAAG
反义链 2 : UGAAGCGAAGUGCACACGGUC
iNA ID #239 (按照 iNA ID #7和 #8 iNA设计的分段式 iNA)
正义链： CGGGGCGCACCUCUCUUUAdTdTuauaCCGUGUGCACUUCGCUUCAdTdT
反义链 1 : UAAAGAGAGGUGCGCCCCGUG
反义链 2 : UGAAGCGAAGUGCACACGGUC
iNA ID #240 (按照 iNA ID #9和 #8 iNA设计的分段式 iNA) :
正义链： GGCAGGUCCCCUAGAAGAAdTdTuauaCCGUGUGCACUUCGCUUCAdTdT
反义链 1 : UUCUUCUAGGGGACCUGCCUC
反义链 2 : UGAAGCGAAGUGCACACGGUC
iNA ID #241 (按照 iNA ID #11和 #8 iNA设计的分段式 iNA) :
正义链： GCAUGGAGACCACCGUGAAdTdTuauaCCGUGUGCACUUCGCUUCAdTdT
反义链 1 : UUCACGGUGGUCUCCAUGCGA
反义链 2 : UGAAGCGAAGUGCACACGGUC
实施例 2
对于环形或类似环形的结构， 每个 iNA是如下所示。 一条链包括两段多核苷酸， 方向相 反 （一段 5'到 3'另一段 3'至 5' )， 其中 5' -3'方向与靶 RNA的序列相同顺序。 在溶液中， 将两个端部的多核苷酸杂交在一起， 以形成一个环形结构。另一条链将与环形的单链区域互 补， 也和靶 RNA互补。 这样的序列的例子， 如下所示：
iNA ID #242 (按 iNA ID #2设计的类环形 HBV iNA)： 正义链： GGGCCCGGGUUUUUCUUGUUGACAAUUCCCGGG (划线的部分是 3' ~5' 方向） 反义链： UUGUCAACAAGAAAAACCCCG
iNA ID #243 (按 iNA ID #5设计的类环形 HBV iNA)：
正义链： GGGCCCGGAUGUGUCUGCGGCGUUUUUCCCGGG (划线的部分是 3' -5' 方向） 反义链： AAACGCCGCAGACACAUCCAG
iNA ID #244 (按 iNA ID #6设计的类环形 HBV ΪΝΑ6)：
正义链： GGGCCCUCUUGUUGGUUCUUCUGGAUUCCCGGG (划线的部分是 3' -5' 方向） 反义链： UCCAGAAGAACCAACAAGAAG
iNA ID #245 (按 iNA ID #7设计的类环形 HBV)：
正义链： GGGCCCCGGGGCGCACCUCUCUUUUUCCCGGG (划线的部分是 3' ~5' 方向）
反义链： UAAAGAGAGGUGCGCCCCGUG
iNA ID #246 (按 iNA ID #8设计的类环形 HBV iNA)：
正义链： GGGCCCCCGUGUGCACUUCGCUUCAUUCCCGGG (划线的部分是 3' -5' 方向）.
反义链： UGAAGCGAAGUGCACACGGUC
iNA ID #247 (按 iNA ID #9设计的类环形 HBV iNA)：
正义链： GGGCCCGGCAGGUCCCCUAGAAGAAUUCCCGGG (划线的部分是 3' -5' 方向） 反义链： UUCUUCUAGGGGACCUGCCUC
iNA ID #248 (按 iNA ID #11设计的类环形 HBV iNA)：
正义链： GGGCCCGCAUGGAGACCACCGUGAAUUCCCGGG (划线的部分是 3' -5' 方向） 反义链： UUCACGGUGGUCUCCAUGCGA
实施方案 3
化学修饰：
iNA ID #249 (按 iNA ID #2和 # 8设计的类环形 HBV iNA) 反义链 1 : UUGUCAACAAGAAAAACCCCG
反义链 2 : UGAAGCGAAGUGCACACGGUC
( "m"在前的碱基指的是 2 ' 甲氧基 （2 ' -O-Methyl ) RNA碱基. " d" 在前的碱基指 的是 DNA碱基）
iNA ID #250 (按照 iNA ID #2和 #8 iNA设计的分段式 ）： 反义链 1： UUGUmCAAmCAAGAAAAACCCCG
反义链 2 : UGAAGCGAAGUGCACACGGUC
( "m"在前的碱基指的是 2 ' 甲氧基 （2 ' -O-Methyl ) RNA碱基. " d" 在前的碱基指 的是 DNA碱基）
iNA ID #250A (按照 iNA ID #2和 #8 iNA设计的分段式）： 反义链 1： UUGUmCAAmCAAGAAAAACCCCG
反义链 2 : UGAAGCGAAGUGCACACGGUC
( "m"在前的碱基指的是 2 ' 甲氧基 （2 ' -O-Methyl ) RNA碱基. " d" 在前的碱基指 的是 DNA碱基）
iNA ID #251 (按照 iNA ID #2和 #8 iNA设计的分段式）： 反义链 1： UUGUmCAAmCAAGAAAAACCCCG
反义链 2 : UGAAGCGAAGUGmCAmCACGGUC
( "m"在前的碱基指的是 2 ' 甲氧基 （2 ' -O-Methyl ) RNA碱基. " d" 在前的碱基指 的是 DNA碱基） iNA ID #252 (按照 iNA ID #2和 #8 iNA设计的分段式）： 反义链 1 : UUGUCAACAAGAAAAACCCCG
反义链 2 : UGAAGCGAAGUGmCAmCACGGUC
( "m"在前的碱基 指的是 2 ' 甲氧基 （2 ' -O-Methyl ) RNA碱基. " d" 在前的碱基 is DNA碱基）
iNA ID #253 (按照 iNA ID #2 iNA设计的分段式）：
正义链： mGGGUUUUUCUUGUUGAmCAAdTdT
反义链： UUGUmCAAmCAAGAAAAACCCCG
( "m"在前的碱基指的是 2 ' 甲氧基 （2 ' -O-Methyl ) RNA碱基. " d" 在前的碱基指 的是 DNA碱基）
iNA ID #254 (按照 iNA ID#2 iNA设计的分段式）：
正义链： mGGGUUUUUCUUGUUGAmCAAdTdT
反义链： UUGUCAACAAGAAAAACCCCG
( "m"在前的碱基指的是 2 ' 甲氧基 （2 ' -O-Methyl ) RNA碱基. " d" 在前的碱基指 的是 DNA碱基）
iNA ID #255 (按照 iNA ID #8 iNA设计的分段式）：
正义链： CCGmUGUGmCACUUCGCUUmCAdTdT
反义链： UGAAGCGAAGUGmCAmCACGGUC
( "m"在前的碱基指的是 2 ' 甲氧基 （2 ' -O-Methyl ) RNA碱基. " d" 在前的碱基指 的是 DNA碱基）
iNA ID #256 (按照 iNA ID #8 iNA设计的分段式）：
正义链： CCGmUGUGmCACUUCGCUUmCAdTdT
反义链： UGAAGCGAAGUGCACACGGUC
( "m"在前的碱基指的是 2 ' 甲氧基 （2 ' -O-Methyl ) RNA碱基. " d" 在前的碱基指 的是 DNA碱基）
实施方案 4
对于类似的茎-环结构，每个 iNA是一条链的多核苷酸可以形成-个环形， 由 5' -末端核 苷酸链和 3' -末端核苷酸由 Watson-Crick的原则而结合的双链结构 另一个链将被互补到 环的单链区段以形成双链 iNA。 如下所示：
iNA ID #257 (按照 iNA ID #2 iNA设计的茎一环型 iNA)：
正义链： GGGCCCGGGUUUUUCUUGUUGACAAUUGGGCCC_
反义链： UUGUCAACAAGAAAAACCCCG
iNA ID #258 (按照 iNA ID #5 iNA设计的茎一环型 iNA)：
正义链： GGGCCCGGAUGUGUCUGCGGCGUUUUUGGGCCC_
反义链： AAACGCCGCAGACACAUCCAG
iNA ID #259 (按照 iNA ID #6 iNA设计的茎一环型 iNA
正义链： GGGCCCUCUUGUUGGUUCUUCUGGAUUGGGCCC_
反义链： UCCAGAAGAACCAACAAGAAG
iNA ID #260 (按照 iNA ID #7 iNA设计的茎一环型 iNA)：
正义链： GGGCCCCGGGGCGCACCUCUCUUUUUGGGCCC_
反义链： UAAAGAGAGGUGCGCCCCGUG
iNA ID #261 (按照 iNA ID #8 iNA设计的茎一环型 iNA)：
正义链： GGGCCCCCGUGUGCACUUCGCUUCAUUGGGCCC_
反义链： UGAAGCGAAGUGCACACGGUC
iNA ID #262 (按照 iNA ID #9 iNA设计的茎一环型 iNA)： 正义链： GGGCCCGGCAGGUCCCCUAGAAGAAUUGGGCCC
反义链： UUCUUCUAGGGGACCUGCCUC
iNA ID #263 (按照 iNA ID #11 iNA设计的茎一环型 iNA)：
正义链： GGGCCCGCAUGGAGACCACCGUGAAUUGGGCCC
反义链： UUCACGGUGGUCUCCAUGCGA
iNA ID #264 (按照 iNA ID #2 iNA设计的茎一环型 iNA)：
正义链： GGGCCCmGGGUUUUUCUUGUUGAmCAAUUGGGCCC_
反义链： UUGUCAACAAGAAAAACCCCG
( "m"在前的碱基指的 2， 甲氧基 （2 ' -0-Methyl ) RNA碱基。 " d"在前的碱基指的是 DNA碱基）。
iNA ID #265 (按照 iNA ID #8 iNA设计的茎一环型 iNA )：
正义链： GGGCCC mCCGmUGUGmCACUUCGCUUmCAUUGGGCCC_
反义链： UGAAGCGAAGUGCACACGGUC
( "m"在前的碱基指的是 2 ' 甲氧基 （2 ' -0-Methyl ) RNA碱基。 " d"在前的碱基指的 是 DNA碱基）。
iNA ID #266 (按照 iNA ID #8 iNA设计的茎一环型 iNA )：
正义链： GGGCCC mCCGmUGUGmCACUUCGCUUmCAUUGGGCCC_
反义链： UGAAGCGAAGUGmCAmCACGGUC
( "m"在前的碱基指的是 2 ' 甲氧基 （2 ' -0-Methyl ) RNA碱基。 " d"在前的碱基指的 是 DNA碱基）。
方案实施 5 : 更多 iNAs
iNA ID #267 (按照 iNA ID #8 HBV iNA)：
正义链： [mC] CG [mU] GUG [mC] ACUUCGCUU [mC] A [dT] [dT]
反义链： UGAAGCGAAGUG [mC] A [mC] ACGGUC
( "m"在前的碱基指的是 2 ' 甲氧基 （2 ' -0-Methyl ) RNA碱基。 " d"在前的碱基指的 是 DNA碱基）。
iNA ID #268 (按照 iNA ID #2 HBV iNA)：
正义链： [mG] GGUUUUUCUUGUUGA [mC] AA [dT] [dT]
反义链： UUGUCAACAAGAAAAACCCCG
( "m"在前的碱基指的是 2 ' 甲氧基 （2 ' -0-Methyl ) RNA碱基。 " d"在前的碱基指的 是 DNA碱基）。
iNA ID #269 (按照 iNA ID#2禾卩 #8 HBV iNA )：
正 义 链 ： [mG] GGUUUUUCUUGUUGA [mC] AA [dT] [dT] UAUA [mC] CG [mU] GUG [mC] ACUUCGCUU [mC] A [dT] [dT] 反义链 1 : UUGUCAACAAGAAAAACCCCG
反义链 2 : UGAAGCGAAGUG [mC] A [mC] ACGGUC
( "m"在前的碱基指的是 2 ' 甲氧基 （2 ' -0-Methyl ) RNA碱基。 " d"在前的碱基指的 是 DNA碱基）。
iNA ID #270 (按照 iNA ID#2禾卩 #8 HBV iNA)：
正 义 链 ： [mC] CG [mU] GUG [mC] ACUUCGCUU [mC] A [dT] [dT] UAUA [mG] GGUUUUUCUUGUUGA [mC] AA [dT] [dT] 反义链 1 : UUGUCAACAAGAAAAACCCCG
反义链 2 : UGAAGCGAAGUG [mC] A [mC] ACGGUC
( "m"在前的碱基指的是 2 ' 甲氧基 （2 ' -0-Methyl ) RNA碱基。 " d在前的碱基指的是 DNA碱基）。 iNA ID #271 (按照 iNA ID#2和 #8 HBV iNA)：
正 义 链 ： [mG] GGUUUUUCUUGUUGA [mC] AA [dT] [dT] NNNNN [mC] CG [mU] GUG [mC] ACUUCGCUU [mC] A [dT] [dT] 反义链 1 : UUGUCAACAAGAAAAACCCCG
反义链 2 : UGAAGCGAAGUG [mC] A [mC] ACGGUC
( "m"在前的碱基指的是 2， 甲氧基 （2' -O-Methyl ) RNA碱基。 " d" 在前的碱基指 的是 DNA碱基。 画有线的 N区域指的是在正义区域的 nt数目可以不等 （1-1000)， 也可是化 学修饰的 RNA或 DNA碱基或不同的连接健）。
iNA ID #272 (按照 iNA ID#2禾卩 #8 HBV iNA)：
正 义 链 ： [mC] CG [mU] GUG [mC] ACUUCGCUU [mC] A [dT] [dT] NNNN [mG] GGUUUUUCUUGUUGA [mC] AA [dT] [dT] 反义链 1 : UUGUCAACAAGAAAAACCCCG
反义链 2： UGAAGCGAAGUG [mC] A [mC] ACGGUC
( "m"在前的碱基指的是 2' 甲氧基 （2' -O-Methyl ) RNA碱基。 "d"在前的碱基指的 是 DNA碱基。 画有线的 N区域指的是在正义区域的 nt数目可以不等 （1-1000)， 也可是化学 修饰的 RNA或 DNA碱基， 或不同的连接健）。
方案实施例 6
siRNA制剂配方.
D0TMA溶在乙醇中， 与 siRNA混合产生水不溶性的沉淀物， 分离和干燥沉淀物后， 将沉淀 物溶解在氯仿或类似溶剂中， 并进一步与其他脂质在氯仿混合， 如 （WO/2010/135207) 所述 工艺。 除去有机溶剂后， 干燥制剂与 9%蔗糖水水合， 即可动物给药。
方案实施例 7
实验方法
细胞转染： HepG2. 2. 15细胞接种于 96孔板中， 在 C02中 （5 %C02)， 37°C培养箱中培养 过夜。 第二天早晨细胞覆盖到 40%的孔板面积。 用 lO L DMEM培养液稀释 0. 5 L siRNA ( Ι μ Μ储备溶液）。 用 10 μ L的 DMEM培养液稀释 0. 4 μ L RFect (百传生物科技）， 并在室 温下保持 5分钟。 混合稀释的 siRNA和稀释的 RFect, 涡旋震荡 10秒， 并保持在室温下 20 分钟。 加 20 L转染复合体于含 80 LDMEM培养液的孔中。 在 C02中 （5 %C02)， 37°C培养箱 中继续培养。
在 HepG2. 2. 15 细胞上筛选高效 HBV iNA 的例子。 将 iNA用 RFect 转染 iNAs ( 5nM)至 HepG2. 2. 15细胞。 两天后， 收集并破碎细胞， 用实时定量 RT-PCR方法分析基因表达的变化， 如图 2所示。
在 ftepG2. 2. 15细胞上筛选高效 HBV iNA的例子。 用 RFect转染能作用于 HBV RNA两个不 同位点的 iNAs ( 5nM) 至 HepG2. 2. 15细胞。 两天后， 收集并破碎细胞， 用实时定量 RT-PCR 方法分析基因表达的变化， 其结果如图 3所示。
实施例 8
体内实验动物的研究中使用的所有程序是机构动物管理和使用委员会（IACUC)批准的， 并按照当地， 州和联邦法规进行。 HBV转基因小鼠， 通过尾静脉注射 0. 2毫升注射 siRNA 的配方。 收获的组织和血液用于分析基因表达的变化。
如图 4 : iNA抑制 HBV在 HBV转基因动物体内的复制的例子。 乙肝病毒转基因小鼠一次 静脉注射传输系统包裹的抗病毒 iNA (siRNA) , 特异性地抑制 HBV病毒基因在肝细胞内的复 制， 而对照组的 ApoB siRNA对 HBV的复制没有影响。
HBVl ( iNA编号 267)、 HBV2 ( iNA编号 269)、 和 AP0-B的 iNA静脉给药降低 HBV转基因小鼠 肝内 HBV DNA的例子。 HBV转基因小鼠经尾静脉每周一次注射 iNA共三次。 阿德福韦酯（ADV) 组的 HBV转基因小鼠每天一次口服给药，连续 14天（ lOmg/kg/天）。在最后一次给药三天后取 组织和血。 A) Southern印迹杂交分析转基因小鼠肝内 HBV DNA的变化； B) PCR分析转基因 小鼠肝内 HBV DNA的变化。 P〈0. 001 (使用单向方差分析和 Bonferroni法的多重比较 检验） ，结果如图 5。
HBV1 ( iNA编号 267)、 HBV2 ( iNA编号 269)、 AP0-B的 iNA静脉给药降低 HBV转基因小 鼠血浆 HBeAg的例子。 HBV转基因小鼠经尾静脉每周一次注射 iNA共三次。阿德福韦酯（ADV) 组的 HBV转基因小鼠每天一次阿德福韦酯（Adefovir dipivoxi ) 1口服给药 14天（10mg/kg/ 天）。 在最后一次给药三天后取材。 A) 给物前， B) 给物 17天后。 P 〈0. 001 (使用单 向方差分析和 Bonferroni法的多重比较检验）， 结果如图 6。
HBV1 ( iNA编号 267)、 HBV2 ( iNA编号 269)、 和 AP0-B的 iNA静脉给药降低 HBV转基因 小鼠血浆 HBsAg的例子。 HBV转基因小鼠经尾静脉每周一次注射纳米胶粒传输系统包裹的 iNA 共三次。 ADV组的 HBV转基因小鼠每天一次阿德福韦酯口服给药 14天 （10mg/kg/天）。 在最 后一次给药三天后取材。 P〈0. 001 (使用单向方差分析和 Bonferroni法的多重比较检 验）。 结果如图 7。
HBV1 ( iNA编号 267)、 HBV2 ( iNA编号 269)、 和 AP0-B的 iNA静脉给药降低 HBV转基 因小鼠肝内 HBV RNA的例子。 HBV转基因小鼠经尾静脉每周一次注射 iNA共三次。 ADV组的 HBV转基因小鼠每天一次阿德福韦酯口服给药 14天 （10mg/kg/天）。 在最后一次给药三天后 取材。 P〈0. 001 (使用单向方差分析和 Bonferroni法的多重比较检验）。 结果如图 8。
HBV1 ( iNA编号 267)、 HBV2 ( iNA编号 269)、 和 AP0-B的 iNA静脉给 HBV转基因小鼠药 后血浆化学分析的例子。 HBV转基因小鼠经尾静脉每周一次注射 iNA共三次。 ADV组的 HBV转 基因小鼠每天一次阿德福韦酯口服给药 14天（10mg/kg/天）。在最后一次给药三天后取材。 *， P 〈0. 05 (使用单向方差分析和 Bonferroni法的多重比较检验）。 结果如图 9。
实施例 9:
实施例 7、 8的 SiRNA乙肝病毒抑制结果的检验方法：
mRNA 的分离： 转染后两天， 将细胞用 100 PBS 洗一次， 然后加入 100 μ L
( Turbocapture试剂盒， Qiagen公司制）裂解缓冲液。 溶解的细胞产物（80 L)转移到一 个 96孔的 mRNA的捕捉板， 在室温下孵育 1小时。 对于小鼠组织， 给药两天后， 小鼠采集小 鼠肝脏组织。 用 Polytron ( Turbocapture试剂盒， Qiagen公司制）在裂解缓冲液匀浆。 然 后转移 80 μ L到一个 96孔的 mRNA的捕捉板， 在室温下孵育 1小时。 用 100 μ L洗涤缓冲液 洗涤三次， 然后 80 的洗脱缓冲液加入到各孔中， 在 65°C下温育 5分钟。 洗脱溶液 （含 有 mRNA的） 被转移到一个新的 96孔清晰板。
实时 RT-PCR: 3 L分离的 mRNA用来实时 RT-PCR。 RT-PCR方法采用 SYBR Green—步实 时 RT-PCR试剂盒 ( SensiMix一步 SYBR Green试剂盒， BI0LINE)。 混合 11 μ L master mix (含逆转录酶）， 1 的正向和反向引物 （6 μ Μ)， 0. 3 L 50X的 SYBR Green和 2. 7 μ L 水。反转录反应的温度在 42° C， 30分钟后， 之后 95° C， 15分钟用于激活 Tag聚合酶； PCR 循环的温度和时间是 95° C， 15秒， 60° C， 30秒， 72° C， 20秒。 用 A A CT方法分析基 因表达的变化。
Southern印迹杂交分析肝 HBV DNA : 肝组织匀浆于裂解液。 肝组织 （约 0. 1克） 在含有 裂解缓冲液 （ ImM的 EDTA， 10mM的 Tris， lOmM NaCl中， 0. 5 %SDS， 蛋白酶 K)用研磨杵匀 浆。 为了提取 DNA， 将匀浆在 55°C下孵育 12小时， 然后， 加入等体积的苯酚 （Phenol)。 将 样品混合， 并以 12, 000 xg离心 10分钟。， 然后将氯仿加入上清液， 并再次离心。 然后用 NaCl和乙醇沉淀出 DNA。将沉淀出的 DNA沉淀溶于含有核糖核酸酶 A的 TE缓冲液（pH8. 0) . 一定数量的 DNA (通常含有 40微克 DNA)， 用 Hindi I I消化酶 (New England Biolabs公司， MA) 在 37° C消化 3小时已被证明 HBV基因序列是不含 Hindl l l内切位点的。 消化的 DNA, 再萃取， 并通过 1 %TAE琼脂糖凝胶电泳分离。 DNA然后被转移到 BioDyne B带正电荷的尼 龙膜。 DNA经 UV照射后固定于膜上。 杂交使用 [32P] CTP-标记的， 用 Haelll消化的被克隆到 pBluescript质粒的 HBV基因组作探针。 杂交在含有 10 %PEG- 8000， 0. 05M磷酸钠， 0. 33 毫克 /毫升鲑鱼精子 DNA， 7 %的 SDS 的溶液中在 60 °C下过夜进行。 使用磷成像方法 (Optiquant ) 测定放射性信号并测定放射性条带的密度。
病毒 DNA相对于宿主小鼠 DNA的量是用病毒 DNA条带相对于转基因 DNA条带的比值来确 定。 计算是根据每一宿主小鼠的细胞含有 1. 3份的 HBV转基因进行的。
半定量 PCR分析肝 HBV DNA : PCR方法： 95°C下 2分钟， 然后 40个循环：在 95°C下 10 秒， 在 60°C下 30秒。 使用标准曲线的方法测定。
血浆 HBeAg和 HBsAg分析： 用 ELISA方法按照生产商的说明 （国际免疫诊断， 福斯特 市， CA) 测定。
工业实用性
此申请提供了一种方法由一种或多种类型的 siRNA或 iNA， 通过抑制 HBV基因组的一个 或多个位点治疗乙型肝炎病毒性感染。
表 1 HBV病毒的 iNA (siRNA) 靶位点
siRNA 起始 点 （ 参 照
序号 AF100309的顺序） 正义链 反义链
1 185 GACCCCUGCUCGUGUUACAdTdT UGUAACACGAGCAGGGGUCCU
2 208 GGGUUUUUCUUGUUGACAAdTdT UUGUCAACAAGAAAAACCCCG
3 247 AGUCUAGACUCGUGGUGGAdTdT UCCACCACGAGUCUAGACUCU
4 257 CGUGGUGGACUUCUCUCAAdTdT UUGAGAGAAGUCCACCACGAG
5 377 GGAUGUGUCUGCGGCGUUUdTdT AAACGCCGCAGACACAUCCAG
6 434 UCUUGUUGGUUCUUCUGGAdTdT UCCAGAAGAACCAACAAGAAG
7 1522 CGGGGCGCACCUCUCUUUAdTdT UAAAGAGAGGUGCGCCCCGUG
8 1577 CCGUGUGCACUUCGCUUCAdTdT UGAAGCGAAGUGCACACGGUC
9 2359 GGCAGGUCCCCUAGAAGAAdTdT UUCUUCUAGGGGACCUGCCUC
10 2368 CCUAGAAGAAGAACUCCCUdTdT AGGGAGUUCUUCUUCUAGGGG
11 1608 GCAUGGAGACCACCGUGAAdTdT UUCACGGUGGUCUCCAUGCGA
12 179 UCCUAGGACCCCUGCUCGUUU ACGAGCAGGGGUCCUAGGAUU
13 180 CCUAGGACCCCUGCUCGUGUU CACGAGCAGGGGUCCUAGGUU
14 181 CUAGGACCCCUGCUCGUGUUU ACACGAGCAGGGGUCCUAGUU
15 182 UAGGACCCCUGCUCGUGUUUU AACACGAGCAGGGGUCCUAUU
16 183 AGGACCCCUGCUCGUGUUAUU UAACACGAGCAGGGGUCCUUU
17 184 GGACCCCUGCUCGUGUUACUU GUAACACGAGCAGGGGUCCUU
18 186 ACCCCUGCUCGUGUUACAGUU CUGUAACACGAGCAGGGGUUU
19 187 CCCCUGCUCGUGUUACAGGUU CCUGUAACACGAGCAGGGGUU
20 188 CCCUGCUCGUGUUACAGGCUU GCCUGUAACACGAGCAGGGUU
21 189 CCUGCUCGUGUUACAGGCGUU CGCCUGUAACACGAGCAGGUU
22 190 CUGCUCGUGUUACAGGCGGUU CCGCCUGUAACACGAGCAGUU
23 191 UGCUCGUGUUACAGGCGGGUU CCCGCCUGUAACACGAGCAUU
24 192 GCUCGUGUUACAGGCGGGGUU CCCCGCCUGUAACACGAGCUU
25 193 CUCGUGUUACAGGCGGGGUUU ACCCCGCCUGUAACACGAGUU
26 194 UCGUGUUACAGGCGGGGUUUU AACCCCGCCUGUAACACGAUU
27 195 CGUGUUACAGGCGGGGUUUUU AAACCCCGCCUGUAACACGUU
28 196 GUGUUACAGGCGGGGUUUUUU AAAACCCCGCCUGUAACACUU
29 197 UGUUACAGGCGGGGUUUUUUU AAAAACCCCGCCUGUAACAUU
30 198 GUUACAGGCGGGGUUUUUCUU GAAAAACCCCGCCUGUAACUU
31 199 UUACAGGCGGGGUUUUUCUUU AGAAAAACCCCGCCUGUAAUU
32 200 UACAGGCGGGGUUUUUCUUUU AAGAAAAACCCCGCCUGUAUU
33 201 ACAGGCGGGGUUUUUCUUGUU CAAGAAAAACCCCGCCUGUUU 聽vvvvvvvv謹〕聽 §v n圈圈
vvvvvvvvv讓聽〕 isv圈圈 nnnn
vvvvvvvvv聽聽誠 §。 nn nn圈
§vvvvvvvvv麵謹謹誠 §02 nn圈 9
§vvvvvvvvv vvvvv謹誠nnnnn圈
vvvvvvvvv vvvvvv謹誠。。nnnnnn圈
vv §v謹ivvvvvvvvvvnnn圈 nnnn
。薩謹麵謹vv vvvvvvvv nnnnnn
聽vvvv聽謹〕v〕v nnnn n n n
〕vvvv謹謹〕v〕v圈 nnn nn n n
vvvv聽謹謹〕v〕 nnnnnn n
vvvvv。謹謹〕v〕 g nnnnn nnn
vvvv§〕 V。謹謹〕v〕 i nnnn圈 n n
聽 vvvvvv。謹謹〕v〕 02nnn圈 n n9
ivvvv誠〕 V。謹謹〕vv nnn nn nn
〕vvvvv誠〕 V。謹謹〕vv圈 nn圈 n
vvvvv。謹謹〕v nnnn nnnnnn
聽 vvvvvv〕誠〕 V。謹謹〕v nn圈 n n
vvvvv謹vvv。謹謹〕n nnnn
vvvvv薩麵〕v。謹謹〕 92 nnn nnnn n9
vvvvv謹 v vvv。謹謹 nnn圈nn
麵〕〕 vvvv謹 vv麵〕vnn圈 nn
vvvv謹 vvvvvv。謹。 092 nnn圈 nnnn
vvvv謹 WW vvv。謹 nnn圈圈nn
麵〕〕 svvv謹 VVVV3誠vv§ nn nnnnnnn
vvv謹 vvvvv誠vv§ nnn圈 nnnnnn
i誠vvvi3 n nnnn
聽 VVV謹 vvvvv聽 v誠vvv nnn nnnn
Figure imgf000019_0001
聽 VVV謹 vvvvv聽誠vv 992n nnnn
vvv謹 vvvvv聽。誠vv nnn nnnn
vv謹 vvvvv聽 v誠vv nnnn nnnnn
謹謹。。vvv§v。謹vv〕〕讓讓〕〕vv nnn
vvvv§vvv麵聽〕§§謹§nn n
聽§§§麵〕。v§§v ΐΐ n nnnnnn
§§§麵〕。。薩麵〕v§§v圈 nnnn
麵。。§§麵〕。。薩 V vv§§圈nnnn
聽。§§麵〕。。薩 V
聽 §§麵〕。。薩 vv〕vv§§圈 nnnn
麵〕。§麵〕。。薩 vvvvv§§圈 nnnnn
§§麵〕。。薩 vvv誠〕vv§§ S n nn nnn
聽 §麵§薩 vvvv誠〕vi§圈 nn
麵〕。麵〕。。薩 vvvvv誠〕vv§ n圈 nnnn
§vv§vvvvvv謹誠〕vv§ nnn nn nnn 聽麵〕。。薩 vvvvvv謹誠〕vi圈nn 麵〕。。薩 vvvvvvv聽謹謹誠〕v nnn
§vvvvvvvv聽謹謹誠〕v nn n nn
聽vvvvvvvvv聽謹謹誠〕vn nn nn
聽薩 ννννννννν聽謹謹誠〕v nn
薩 νννννννννν〕聽謹謹誠〕v ηη圈 nn
聽ννννννννννν 3聽謹謹誠〕v η圈n
ννννννννννν誠〕聽謹謹誠〕v圈 nn n
vvvvvvvvvvvv誠〕聽謹謹誠〕 nn圈 n
聽 ννννννννννν讓誠〕聽謹謹誠〕 n
ννννννννννν聽 §聽謹謹誠 ηη
ννννννννννi§〕聽謹。ηηnnn n 9
νννννννννν〕 §〕聽謹謹 ηηη圈nnn
ηηννννννννί3νi§〕聽謹。ηη nnnn n
vvvvvvvvv謹vvi§〕聽。nnnnn
〕 vvv§vvv§vvv〕〕讓聽：nnnnnnn - vv§薩 V謹 vvvv§§vvnnnnnnnnn
§薩 V謹謹。。§§vvvvvv§nnnn
聽。〕謹 §§〕謹 vvv§vv§§v nn
§〕§〕謹聽。§ vv§vvv§v§11n
Figure imgf000020_0001
。§§薩§聽v 。画〕〕〕v§vv〕§〕〕n nn
v§vv§vv謹 vvvvv。§聽vnnn nn
§vv§vv。vvvvv。§聽vnn圈n nn
薩 vv§vvvvvvv。§聽vnn圈n nn
vv§vv謹聽vvvvvv。§聽nnnn n
§§聽 §isvv麵讓〕〕§vvv§vnnnnn
薩 §iv聽 v誠§ n
聽〕viv§vvv麵〕〕誠麵。§ nnn
。謹 §聽 ivvv謹 vvvvvivv§vv∞οΐnnn
讓〕：聽 ivvv6nn圈 -
。§聽 ivvv謹n圈
§聽 ivvvvvvvvvvivvvnnnn圈nnn
薩聽 vvvvvv§vv§麵聽讓〕 vvv n圈 n
麵〕〕：麵〕 V謹。〕vvvvivv§ 3圈 nn nnnn - vv:麵〕 V謹。〕麵謹 vvvvivv§ΐnnnn -
§：麵〕 v謹。〕vv讓vvvvivvvi3圈 nn nn - 謹 vvi§vvvv§i§讓 vn nnn
v:麵讓 §ivnnn - i§vvi謹〕謹vvv§vv:麵讓 §vn ηn - ν謹〕§ννν。麵〕 ν vvv麵 vv誠vvηηηηn n
ν§§麵 §〕誠vv§§麵〕〕vηη n n
§vvv§isvvvi3vn
ν§νννν§§v§v§v sηηηnnnn 聽。〕。謹〕〕〕〕§ν〕〕§§§§§〕 nη圈 57 UCGCUUGUCCGUUU UUCGUUGCUGCUGCU 21AAAAAAAAAAAAA
7056 GUCGCUUGUCCGUUU UCGUUGCUGCUGCU 121AAAAAAAAAAAAA
GGUUGUGUCGCUUGUUGCUGCUCUUUCCCUAAAAAA AAAAAA
GGGUUGUGUCGCUUG UGCUGCUCUUUCCCUGAAAAAAAAAAA
GGUGGGG面画 GGG UUCCUUCUUCCCCCCAAAAAAAAAAA
086 GGGUGGUUGUGUCUGC UGUUCUCCUCCCCC 2AAAAAAAAAAAA
039 CUUGUUCCCUCCCUCG UUGGUGGGUGCUGUU 2AAAAAAAAAAA
δ903 GGCUUGCCCGiG UUUUCGGGUCUGUCCU 1AAAAAAAAAAAA
CCUCCGCUGUGCCUUGGUU CCGGCCGCUUGGGGUUAAAAAAA
870 GCCUCCGCUGUGCCUUGUU CGGCCGCUUGGGGCUU 1AAAAAAA
GCCUCCGCUGUGCCUUUUGGCCGCUUGGGGCUUU AAA AAAAA
60GCCUCCGCUGUGCCUUUGGCCGCUUGGGGCUUUU 1 AAAA AAAA
CGCCUCCGCUGUGCCUU GGCCGCUUGGGGCUUGUUAAAAAAA
UCGCCUCCGCUGUGCUU GCCGCUUGGGGCUUGUUAAAAAAAA
UUCGCCUCCGCUGUGUU CCGCUUGGGGCUUGUUAAAAAAAAA
CUCUGUUCGCCUCCGC UUGGGGCUUGCGUGGAAAAAAAAAAA
GUUCUGUCCUCUGUUCG UGCGUGGCUGCUAAAAAAAAAAAAA
sUCUCUGUUCUGUCCUCU UGGCUGCUGGUG AAAAAAAAAAAAA
GGUGUGUUCCCGCCCUG UGGUGCUGGUGCCCCAAAAAAAAAA
5 GGGGGGGGUUGGU面G UUCCUCCUCCUCCCCC 12AAA AAAAAA
GGGGGGGGGUUGGUI UCCUCCUCCUCCCCCAAAJAAAAAAAAA
50 GCUGUGUG画UGGUGGCUCUUCCCGUCUU 1A AAA AAAAAAAA
GCUGUGUGUUUUGGU UCUICCCGUCUUUG AAAAAAAJAAAAAA
709 CGCUGUGUG画UGUUCCCGUCUUUG 1AAAA AAA AAAAAAAAA
708 UCGCUGUGUG画UG画CCCGUCUUUGG 1AAAA AA AAAAAAA
706 CUUCGCUGUGUGUU面CCCGUCUUUGGU 1AAAA AAAAAAAAA
CGCCGCCUUGGGCUCU UUGCCUCGGUCGGUCGUUAAAAAAAA
CGCUGUCCGCCGCC UCGGUCGUUGCUUGCUGAAAAAAAAAAA
GGCUUUCGCUGUCCG UUGCUUGCUGGUCCAAAAAAAAAAAAA
606 GCUGGGCCCCGUGCG UUCCGGUGGUCUCCUGCG 1AAAAAAAAA
CCUUCGCUUCCCUCUGCUU GCGGGUGGCGGUGUUAAAAAAAA
0 GCCUUCGCUUCCCUCUGUU CGGGUGGCGGUGCUU 14AAAAAAAA
58 UGCCUUCGCUUCCCUCUUUGGGUGGCGGUGCUU 12AA AAAAAAA
GUGCCUUCGCUUCCCUCUU GGGUGGCGGUGCCUUAAAAAAAA
580 UGUGCCUUCGCUUCCCUUUGGUGGCGGUGCCUU 1AA AAAAAAA
GUGUGCCUUCGCUUCCCUU GGUGGCGGUGCCCUUAAAAAAAA
CGUGUGCCUUCGCUUCCUU GUGGCGGUGCCCGUUAAAAAAAA
;57 CGCCCUCUCUUUCGCGGUU CCGCGUGGGGUGCGUU i 12AAAAAAA
56 GCGCCCUCUCUUUCGCGUU CGCGUGGGGUGCGCUU 12AAAAAAA
355 GGCGCCCUCUCUUUCGCUU GCGUGGGGUGCGCCUU 1212AAAAAAA
GGGCGCCCUCUCUUUCGUU CGUGGGGUGCGCCCUUAAAAAAA
3053 GGGGCGCCCUCUCUUUCUU GUGGGGUGCGCCCCUU 112AAAAAAA
CGGGGCGCCCUCUCUUUUUGGGGUGCGCCCCGUUU AA AAAAA
50 CCGGGGCGCCCUCUCUUUUGGGGUGCGCCCCGUGUU 12AA AAAA
7 CCCGGGGCGCCCUCUCUUUGGGGUGCGCCCCGUGGUU 12AA AAA UUUU UUUUU 760 GGGC面UIUUUGCUUUUUUUGUCUCCUG 2122AA AAJAAA AAAAAA
GGGCUUUUUUG IU面画 UGUCUCCUGU AAAAAAAAAAJAAA
567CGGGCUUUUUUUUUUUGUCUCCUGUUU 2121 AAAAAAAAAAA AAAA
CGGGCUUUUU画UUUUGUCUCCUGUUUU 2 AAAAAAAAAAA AA
357 GUUGUGUG面 GCUUUUUGCUUCUCUUCC 2121AAAAA AAAAAAAA
570 UGUUGUGUG面 GCUU UUGCUUCUCUUCCU 2122AAAAAAAAAAAA
565 GCUUGUUGUGUG面 G UCUCUUCCUGUCCU 2AAAAAAAAAAAA
056 GGCUUGUUGUGUG面CUCUUCCUGUCCUC 2124AAAAA AAAAAAA
0956 GGGGCUUGUUGUGUG UCUUCCUGUCCUCCUG 221AAAAAAAAAAA
08558 GCGGGGCUUGUUGUGUCCUGUCCUCCUGC 22AAAAAA AAAAAAA
07505 GUCCUUGCUUUUCCU UGGU面GCGGUCG 22AAAAAAAA AAAAA
0689 GCUUUUUCUUCUCUGUCC UCGUGGIGCCC 224AAAAAAAAAAJAAA
05 GCGUCGCGGUCUCUC UUGGUCUUCUGCGCGCGG 2AAAAAAAAA
CM390 CGCGCGGGUCUCUCGUUGGCCUUCGUCUGCGG 22AAAAAA AAAA
03379CUCCCUCGCCUCGCGUU UCUGCGGGCGGGGGUUUU 22 AAAAAAA
0378 GCUCCCUCGCCUCGCGUU CUGCGGGCGGGGGUUCUU 222AAAAAA
0377GCUCCCUCGCCUCGCUU UGCGGGCGGGGGUUCUUU 212 AAAAAAA
00375 GGCUCCCUCGCCUCGUU CGGGCGGGGGUUCUUCUU 22AAAAAAA
37GGCUCCCUCGCCUCUU GGGCGGGGGUUCUUCUUU 24 AAAAAAAA
373GGCUCCCUCGCCUUUGGCGGGGGUUCUUCUUUU 2 AAAAAA AAA
37 GGGCUCCCUCGCCUU GGCGGGGGUUCUUCUUCUU 22AAAAAAAA
37GGGCUCCCUCGCUU GCGGGGGUUCUUCUUCUUU 21 AAAAAAAAA
370 UGGGCUCCCUCGUU CGGGGGUUCUUCUUCUUU 2AAAAAAAAAA
369 CUGGGCUCCCUCUU GGGGGUUCUUCUUCUGUU 2AAAAAAAAAA
367 CCCUGGGCUCCCUU GGGGUUCUUCUUCUGGGUU 2AAAAAAAAA
366 CCCCUGGGCUCCUU GGGUUCUUCUUCUGGGGUU 2AAAAAAAAA
365 UCCCCUGGGCUCUU GGUUCUUCUUCUGGGGUU 2AAAAAAAAAA
9036 GUCCCCUGGGCUUUGUUCUUCUUCUGGGGCUU 124AAAAAAA AAA
363 GGUCCCCUGGGCUU GUUCUUCUUCUGGGGCCUU 2AAAAAAAAA
36GGUCCCCUGGGUU UUCUUCUUCUGGGGCCUUU 22 AAAAAAAAAA
36 CGGUCCCCUGGGUU UCUUCUUCUGGGGCCUGUU 21AAAAAAAAA
360 GCGGUCCCCUGGGUU CUUCUUCUGGGGCCUGCUU 2AAAAAAAA
358GGCGGUCCCCUGGUU UCUUCUGGGGCCUGCCUUU 2 AAAAAAAA
3 GUUGUUGCGGGGCGG UGCCUCUUCGUC面 CCG 242AAAAAAAAA
338 UCUGUUGUUGCGGGG UCUUCGUC面 CCGUGU 2AAAAAAAAAA
336CUCUGUUGUUGCGG UUCGUC面 CCGUGUUU 2 AAAAAAAAAAA
33 GGCUCUGUUGUUGCG UCICCGUGUUUCCGG 22AAAAAAJAAAAAA
80330 CCGGCUCUGUUGUUG ICCGUGUUUCCGG 12AAAAAAJAAAAAAAA
89 CUGCUUGCCCCUG UUUGGUGGUCUUUGCGG 22AAAAAAAAAAAA
88 CCUGCUUGCCCCU UUGGUGGUCUUUGCGGG 22AAAAAAAAAAAA
33 GGGCUGUUCUUGU UUCGCGUUUCUCUCCC 22AAAAAAAAAAAAA
3 GGGGCUGUUCUUGU UCGCGUUUCUCUCCC 222AAAAAAAAAAAAA
GCCUUUGUGGUUUCC UGCCCUGUUGUCUGAAAAAAAAAAAAA
GGGCIUCGCCU UUGUCUGUUUUUUGCCCUAJAAAAAAAAAAAA
70 CGUIUUGGGCIU UUUUUGCCCUU面 CGUU 21JAAAAJAAAAAAAA
7 UUUU UUUUUUUUU
CCCUUUCUCCCCUCUGG UUGGGUGGGIGGGGAAAAAAAAJAAA
GUUGGCCCUGCUUCGC UUUGUGCGGGUCCCUGAAAAAAAAAA GUUGGCCCUGCUUCG UUGUGCGGGUCCCUG AAAAAAAAAAAA
GGUCUUCCCCUCGG UUUCGGGUUUGGGCCAAAAAAAAAAAAA
GGGGGUUGGUCUUCCCC UUUGGGCCCCUCCCUAAAAAAAAAA
GGCGUCUCGCUGGUGCUGUGUCUUGUUCCCAAAAAAAA AAAA
GCGGGGUUUUiG UUUUiUCCCCGCCUAAAAAAAAAAAA
GGCGGGGUUUUUUG UUUiUCCCCGCCUUAAAAAAAAAAAA
GUUGUGUUGUCUUCU UUGUCCUCUCUCU AAAAAAAAAAAAAA GGUUGUGUUGUCUUUGUCCUCUCUCUGG AAAAAAA AAAAAAA
CGGUUGUGUUGUCUU UGUCCUCUCUCUGGAAAAAAAAAAAAA
CCUGUUC面UUUUGCUUUUGUCUUGGGAAA AA AAAAAAAAA GUUUUUCCCUGUUCU UCUUGGGUUCUG AAAAAAAAAAAAAAA

Claims

权利要求书 、 用来抑制乙肝病毒的复制或繁殖的 iNA， 包括一个或多个正义链， 和一个或多个反义链， 其中的一个或多个的多核苷酸链与 HBV RNA的一个或多个区域互补。
、 如权利要求 1中所述的 iNA， 其特征在于， 所述的 iNA为连续双链或间断性双链结构， 该 连续双链或间断性双链结构为直链、 环形、 类环形、 发卡型、 茎一环、 单向、 或双向形的 结构。
、 如权利要求 2中所述的 iNA, 其特征在于， 间断性双链 iNAs, 具有两条或多条多核苷酸， 有一个或多个缺口或切口， 在缺口或切口之间形成一个或多个互补的双链区。
、 如权利要求 2中所述的 iNA, 其特征在于， 双链 iNA长 10〜200个核苷酸， 或为 15〜50 个核苷酸， 或是 19- 29个核苷酸。
、 如权利要求 2中所述的 iNA, 其特征在于， 双链的互补区域的多核苷酸链不少于 10个核 苷酸， 或是 19- 29个核苷酸。
、 如权利要求 2中所述的 iNA， 其特征在于， 至少 50%， 70% , 75% , 80% , 85% , 90% , 95% , 96% , 97 % , 98 % iNA , 99% , 或 100%的一个链中的核苷酸的任何双链区域互补 于另一条链。
、 如权利要求 2中所述的 iNA, 其特征在于， 双链 iNA或 siRNA具有粘端或钝端， 其中， 粘 端位于一条或两条链上， 末端突出碱基长 1-5个核苷。
、 如权利要求 1中所述的 iNA， 其特征在于， 包含一个长的正义链和两个短的反义链， 反义 链与 HBV RNA的两个不同位置互补。
、 如权利要求 1中所述的 iNA, 其特征在于， 包含一个长的反义链， 反义链包含与 HBV RNA 的两个不同位置互补的反义片段； 和两个短的正义链， 两个正义链的序列与 HBV RNA两 个不同位置的序列一样或接近。
0、 如权利要求 8或 9所述的 iNA， 其特征在于： 长链的长度为 15至 80个核苷酸， 或为 19 50个核苷酸； 两个短链每条长 10至 40个核苷酸， 或为 19至 27个核苷酸。 替换页 （细则第 26条) 1、 如权利要求 1所述的 iNA，其特征在于： 具有下述结构 1-31中的任一结构:
Figure imgf000025_0001
替换页 （细则第 26条) 21mer 2Imer
结构 14 JL
45mer 结构 15
Figure imgf000026_0001
21mer 21mer
结构 16 ΤΤΠ
45mer
9mer to 30mer 9merto 30ffn ier
Figure imgf000026_0002
0至靶 mRNA全长 9nw to 30mer 0至耙 mRNA全长 短链
结构 18
长链
Figure imgf000026_0003
两个或两个以上短链 两个或两个以上短链
0至靶 mRNA全长 9mer to 30mer 0至靶 mRNA全长 9merto 30mer, , 0至耙 mRNA全长
< >
短链
结构 19
长链
26mer至耙 mRNA全长
替换页 （细则第 26条) 结构
Figure imgf000027_0001
结构 20A
Figure imgf000027_0002
未 K对的核苷酸区段 未 对的核苷酸区段
Figure imgf000027_0003
未 S对的核苷酸区段
替换页 （细则第 26条) 结构 21
Figure imgf000028_0001
Figure imgf000028_0002
替换页 （细则第 26条) 结构 24
Figure imgf000029_0001
替换页 （细则第 26条)
Figure imgf000030_0001
正义区段 反义区段
反义区段 3† ° 正义区段
缺口
替换页 （细则第 26条)
Figure imgf000031_0001
结构 30
Figure imgf000031_0002
替换页 （细则第 26条)
Figure imgf000032_0001
12. 如权利要求 1中所述的 iNA, 其特征在于， 包含 iNA ID No. 1-272中的任意一种或其 组合， 或 iNA ID NO. 1-11 和 231-272中的任意一种或其组合。
13. 如权利要求 1-12中任一权利要求中所述的 iNA， 其特征在于： 其中的一个或多个核糖核 酸用化学修饰的或脱氧核糖核酸取代。
14. 如权利要求.1-12中任一权利要求中所述的 iNA, 其特征在于： 包含有硫代磷酸酯或 2' - 修饰的核苷酸。
15.如权利要求 1-14任一权利要求中所述的 iNA的用途， 其用于在哺乳细胞中抑制 HBV基因 表达和复制。
16. 一个药物配方， 包含权利要求 1至 14中任一权利要求中的的 iNA和药物用载体。
替换页 （细则第 26条)
PCT/CN2013/090055 2012-12-21 2013-12-20 治疗乙型病毒性肝炎的rna干扰制剂 WO2014094645A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201210564083.8A CN103333890B (zh) 2012-12-21 2012-12-21 治疗乙型病毒性肝炎的rna干扰制剂
CN201210564083.8 2012-12-21

Publications (1)

Publication Number Publication Date
WO2014094645A1 true WO2014094645A1 (zh) 2014-06-26

Family

ID=49242092

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2013/090055 WO2014094645A1 (zh) 2012-12-21 2013-12-20 治疗乙型病毒性肝炎的rna干扰制剂

Country Status (2)

Country Link
CN (1) CN103333890B (zh)
WO (1) WO2014094645A1 (zh)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2966170A1 (en) * 2014-07-10 2016-01-13 Heinrich-Pette-Institut Leibniz-Institut für experimentelle Virologie-Stiftung bürgerlichen Rechts - HBV inactivation
EP3395363A4 (en) * 2015-11-09 2019-08-28 IDAC Theranostics, Inc. ANTIVIRAL MEDICINAL PRODUCT
US10799524B2 (en) 2017-10-20 2020-10-13 Dicerna Pharmaceuticals, Inc. Methods for treating hepatitis B infection
WO2022026387A1 (en) * 2020-07-27 2022-02-03 Aligos Therapeutics, Inc. Hbv binding oligonucleotides and methods of use
EP3873488A4 (en) * 2018-10-31 2022-08-03 The University of Sydney COMPOSITIONS AND METHODS FOR THE TREATMENT OF VIRUS INFECTION
US11517584B2 (en) 2016-08-04 2022-12-06 Arrowhead Pharmaceuticals, Inc. RNAi agents for Hepatitis B virus infection

Families Citing this family (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103333890B (zh) * 2012-12-21 2015-04-15 厦门成坤生物技术有限公司 治疗乙型病毒性肝炎的rna干扰制剂
JP6546161B2 (ja) * 2013-10-04 2019-07-17 ノバルティス アーゲー Ｂ型肝炎ウイルスを治療するための有機化合物
JOP20200092A1 (ar) 2014-11-10 2017-06-16 Alnylam Pharmaceuticals Inc تركيبات iRNA لفيروس الكبد B (HBV) وطرق لاستخدامها
CN105079021A (zh) * 2015-08-03 2015-11-25 厦门成坤生物技术有限公司 包含m10c1化合物与活性成分的组合物及其制备方法
KR20180038465A (ko) 2015-08-07 2018-04-16 애로우헤드 파마슈티컬스 인코포레이티드 Ｂ형 간염 바이러스 감염에 대한 ｒｎａｉ 치료법
JP6988481B2 (ja) * 2016-01-26 2022-01-05 日産化学株式会社 一本鎖オリゴヌクレオチド
US11324820B2 (en) 2017-04-18 2022-05-10 Alnylam Pharmaceuticals, Inc. Methods for the treatment of subjects having a hepatitis b virus (HBV) infection
JP7208911B2 (ja) * 2017-10-11 2023-01-19 日東電工株式会社 核酸分子発現の調節
MX2021001056A (es) 2018-08-13 2021-04-12 Alnylam Pharmaceuticals Inc Composiciones de agente de acido ribonucleico bicatenario (arnbc) de virus de la hepatitis b (vhb) y metodos de uso de las mismas.
CN113171371B (zh) * 2021-04-13 2022-01-25 厦门甘宝利生物医药有限公司 一种抑制乙型肝炎病毒基因表达的rna抑制剂及其应用
CA3235672A1 (en) * 2021-11-29 2023-06-01 Shanghai Argo Biopharmaceutical Co., Ltd. Compositions and methods for inhibiting expression of hepatitis b virus (hbv) protein

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030206887A1 (en) * 1992-05-14 2003-11-06 David Morrissey RNA interference mediated inhibition of hepatitis B virus (HBV) using short interfering nucleic acid (siNA)
CN101603042A (zh) * 2008-06-13 2009-12-16 厦门大学 可用于乙型肝炎病毒感染治疗的rna干扰靶点
WO2010012244A1 (zh) * 2008-08-01 2010-02-04 苏州瑞博生物技术有限公司 乙型肝炎病毒基因的小核酸干扰靶位点序列和小干扰核酸及组合物和应用
CN103333890A (zh) * 2012-12-21 2013-10-02 厦门成坤生物技术有限公司 治疗乙型病毒性肝炎的rna干扰制剂

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102191246B (zh) * 2010-10-28 2014-08-27 百奥迈科生物技术有限公司 多靶标干扰核酸分子及其应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030206887A1 (en) * 1992-05-14 2003-11-06 David Morrissey RNA interference mediated inhibition of hepatitis B virus (HBV) using short interfering nucleic acid (siNA)
CN101603042A (zh) * 2008-06-13 2009-12-16 厦门大学 可用于乙型肝炎病毒感染治疗的rna干扰靶点
WO2010012244A1 (zh) * 2008-08-01 2010-02-04 苏州瑞博生物技术有限公司 乙型肝炎病毒基因的小核酸干扰靶位点序列和小干扰核酸及组合物和应用
CN103333890A (zh) * 2012-12-21 2013-10-02 厦门成坤生物技术有限公司 治疗乙型病毒性肝炎的rna干扰制剂

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2966170A1 (en) * 2014-07-10 2016-01-13 Heinrich-Pette-Institut Leibniz-Institut für experimentelle Virologie-Stiftung bürgerlichen Rechts - HBV inactivation
EP3395363A4 (en) * 2015-11-09 2019-08-28 IDAC Theranostics, Inc. ANTIVIRAL MEDICINAL PRODUCT
US10709727B2 (en) 2015-11-09 2020-07-14 Idac Theranostics, Inc. Antiviral drugs
US11517584B2 (en) 2016-08-04 2022-12-06 Arrowhead Pharmaceuticals, Inc. RNAi agents for Hepatitis B virus infection
US11590156B2 (en) 2016-08-04 2023-02-28 Arrowhead Pharmaceuticals, Inc. RNAi agents for hepatitis B virus infection
US10799524B2 (en) 2017-10-20 2020-10-13 Dicerna Pharmaceuticals, Inc. Methods for treating hepatitis B infection
US11052105B2 (en) 2017-10-20 2021-07-06 Dicerna Pharmaceuticals, Inc. Methods for treating hepatitis B infection
US11052104B2 (en) 2017-10-20 2021-07-06 Dicerna Pharmaceuticals, Inc. Methods for treating hepatitis B infection
US11273173B2 (en) 2017-10-20 2022-03-15 Dicerna Pharmaceuticals, Inc. Methods for treating hepatitis B infection
EP3873488A4 (en) * 2018-10-31 2022-08-03 The University of Sydney COMPOSITIONS AND METHODS FOR THE TREATMENT OF VIRUS INFECTION
WO2022026387A1 (en) * 2020-07-27 2022-02-03 Aligos Therapeutics, Inc. Hbv binding oligonucleotides and methods of use

Also Published As

Publication number Publication date
CN103333890A (zh) 2013-10-02
CN103333890B (zh) 2015-04-15

Similar Documents

Publication Publication Date Title
WO2014094645A1 (zh) 治疗乙型病毒性肝炎的rna干扰制剂
US11408002B1 (en) Methods and compositions for the specific inhibition of alpha-1 antitrypsin by double-stranded RNA
JP6453275B2 (ja) 非対称性干渉ｒｎａの組成物およびその使用
CA2781896C (en) Modulation of hsp47 expression
JP5805088B2 (ja) 遺伝子発現を阻害する組成物およびその使用
CA2619876A1 (en) Chemically modified short interfering nucleic acid molecules that mediate rna interference
JP2014097072A5 (zh)
JP2014522242A (ja) イソクエン酸デヒドロゲナーゼ（ｉｄｈ１）遺伝子発現のｒｎａ干渉媒介抑制
CN117120612A (zh) 用于抑制己酮糖激酶（khk）的组合物及方法
WO2006038608A1 (ja) オリゴ二本鎖ｒｎａ及び医薬組成物
WO2012021383A2 (en) RNA INTERFERENCE MEDIATED INHIBITION OF MITOGEN-ACTIVATED PROTEIN KINASE 1 (MAPK1) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
US20150044196A1 (en) Compositions for Inhibiting Gene Expression and Uses Thereof
CN118284695A (zh) 用于治疗hbv的药物组合
CN111201319A (zh) 核酸分子表达的调节
AU2015200064A1 (en) Modulation of hsp47 expression
AU2006261653A1 (en) RNA interference mediated inhibition of histone deacetylase (HDAC) gene expression using short interfering nucleic acid (siNA)

Legal Events

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

Ref document number: 13865687

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 13865687

Country of ref document: EP

Kind code of ref document: A1