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WO2025021034A1 - Agents oligonucléotidiques double brin et utilisations associées - Google Patents

Agents oligonucléotidiques double brin et utilisations associées Download PDF

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Publication number
WO2025021034A1
WO2025021034A1 PCT/CN2024/106419 CN2024106419W WO2025021034A1 WO 2025021034 A1 WO2025021034 A1 WO 2025021034A1 CN 2024106419 W CN2024106419 W CN 2024106419W WO 2025021034 A1 WO2025021034 A1 WO 2025021034A1
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Prior art keywords
double
sense strand
nucleotide
stranded oligonucleotide
oligonucleotide agent
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Inventor
Xiaoming ZOU
Jianyu LU
Qingfeng Li
Cuicui GAO
Qionger HE
Hongkuan YANG
Mingjie Wang
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Visirna Therapeutics Suzhou Co Ltd
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Visirna Therapeutics Suzhou Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/312—Phosphonates
    • C12N2310/3125—Methylphosphonates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/317—Chemical structure of the backbone with an inverted bond, e.g. a cap structure
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/322—2'-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate

Definitions

  • RNA interference RNA interference
  • RNA interference (RNAi) technology refers to the highly conserved, efficient and specific degradation of homologous mRNA induced by double-stranded RNA (dsRNA) in the process of evolution.
  • dsRNA double-stranded RNA
  • the dsRNA inhibits the expression of a target gene by destroying a target mRNA.
  • the RNAi technology is capable to specifically eliminate or turn off the expression of the target gene, and thus has quickly become one of the most useful research tools in exploring gene functions and therapeutic agents for the treatment of many diseases including metabolic diseases, infectious diseases and malignant tumors, among others.
  • oligonucleotide agent comprising a sense strand and an anti-sense strand, wherein:
  • the sense strand and the anti-sense strand form a double-stranded portion of 15 to 27 base pairs in length and a 5’ extension in the anti-sense strand;
  • the 5’ extension has a length of at least three nucleotides and is cleavable from the most 3’ nucleotide of the 5’ extension, and the cleaved double-stranded oligonucleotide agent is capable of silencing a target RNA or inhibiting expression of a target gene via RNA interference.
  • oligonucleotide agent comprising a structure represented by Formula (C) :
  • first fragment in the anti-sense strand and the second fragment in the sense strand form a double -stranded portion by base pairing, wherein the first fragment and the second fragment are of equal length;
  • the 5’ extension comprises at least three nucleotides in length
  • the anti-sense strand comprises a cleavage region comprising the most 5’ nucleotide (X2) of the first fragment and the two most 3’ nucleotides (Y-Z) of the 5’ extension, and the cleavage region comprises a nucleotide sequence set forth in Formula A: (3’ -5’ ) X2-Y-Z, which is cleavable between X2 and Y, and the Formula A is as defined below;
  • the sense strand has a length of 15 to 35, 15 to 23, 15 to 22, or 15 to 21 nucleotides
  • anti-sense strand has a length of 25 to 35, 26 to 35, 26 to 30, 25 to 27, or 26 to 27 nucleotides.
  • a double-stranded oligonucleotide agent of Formula (C) does not require the complementary region and the target region to form a blunt end. It is possible that the targeting region comprising a 3’ overhand, or the complementary region comprise a 5’ extension, or the complementary region and the target region to form a blunt end.
  • oligonucleotide agent comprising a structure represented by Formula (D) :
  • the first fragment in the anti-sense strand and the second fragment in the sense strand form a double-stranded portion by base pairing, wherein the first fragment and the second fragment are of equal length;
  • the 5’ extension comprises at least three nucleotides in length
  • the anti-sense strand comprises a cleavage region comprising the most 5’ nucleotide (X2) of the first fragment and the two most 3’ nucleotides (Y-Z) of the 5’ extension
  • the cleavage region comprises a nucleotide sequence set forth in Formula A: (3’ -5’ ) X2-Y-Z, which is cleavable between X2 and Y, and upon cleavage, the 5’ extension is removed from its most 3’ nucleotide (Y)
  • the Formula A is as defined below
  • the sense strand has a length of 15 to 35, 15 to 23, 15 to 22, 15 to 21, 16 to 25, 17 to 23, 18 to 23, 19 to 23, 19 to 21, 20 to 23, 20 to 21, 21 to 23, such as 17, 18, 19, 20, 21, 22, or 23 nucleotides;
  • the anti-sense strand has a length of 25 to 35, 25 to 30, 26 to 35, 26 to 30, 26 to 27, such as 25, 26, 27, 28, 29, or 30 nucleotides.
  • a double-stranded oligonucleotide agent comprising a double-stranded oligonucleotide operably linked to a blocking group, wherein the blocking group comprises M03 or M06.
  • a double-stranded oligonucleotide agent comprising a double-stranded oligonucleotide operably linked to a ligand, where the ligand comprises a chemical structure selected from the group consisting of: VSDL-01, VSDL-01A, VSDL-02, VSDL-02A, VSDL-03, VSDL-03A, VSDL-04, VSDL-04A, VSDL-05, VSDL-05A, VSDL-06, VSDL-06A, VSDL-07, VSDL-07A, VSDL-08, VSDL-08A, VSDL-09, VSDL-10, VSDL-11, VSDL-12, VSDL-13, and VSDL-14.
  • the ligand comprises a chemical structure selected from the group consisting of: VSDL-01, VSDL-01A, VSDL-02, VSDL-02A, VSDL-03, VSDL-03A, VSDL-04,
  • composition comprising a double-stranded oligonucleotide agent disclosed herein or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
  • a method of inhibiting the expression of a target gene in a subject in need thereof comprising administering to the subject a pharmaceutically effective amount of a double-stranded oligonucleotide agent disclosed herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition disclosed herein.
  • a method of treating a disease or a disorder in a subject in need thereof comprising administering to the subject a pharmaceutically effective amount of a double-stranded oligonucleotide agent disclosed herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition disclosed herein.
  • Fig. 1 shows relative AGT mRNA expression in liver for Test Compounds (ds101 and ds100) .
  • Fig. 2 shows relative SOD-1 expression in different tissues (frontal cortex, hippocampus, striatum and heart) for Test Compounds (ds110, ds111, ds112 and ds113) .
  • Fig. 3 shows relative SOD-1 expression in different tissues (prefrontal cortex and lumbar spinal cord) for Test Compounds (ds114 and ds115) .
  • Fig. 4 shows relative mTTR expression for Test Compounds (ds118 and ds119) .
  • Fig. 5 shows the results of liver homogenate processing reaction for Test Compound.
  • Fig. 6 shows plasma relative AGT levels for Test Compounds (ds101 and ds86) in NHP model.
  • references to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se.
  • description referring to “about X” includes description of “X” .
  • Numeric ranges are inclusive of the numbers defining the range.
  • the term “about” refers to the indicated value of the variable and to all values of the variable that are within the experimental error of the indicated value (e.g. within the 95%confidence interval for the mean) or within 10 percent of the indicated value, whichever is greater.
  • the terms “optional” or “optionally” means the described event or condition may or may not occur and includes both the situation where the event or condition occurs and where it does not occur.
  • “optionally modified” includes both unmodified and modified
  • nucleotide optionally modified includes nucleotide both unmodified and modified.
  • disorder refers to any disease, disorder or condition that impairs the normal functioning of a subject (e.g., human) .
  • the term “effective amount” means the amount of a pharmaceutical agent that produces some desired local or systemic therapeutic effect at a reasonable benefit/risk ratio applicable to any treatment alone or together with further doses.
  • the desired local or systemic therapeutic effect preferably relates to inhibition of the course of the disorder. This comprises slowing down the progression of the disorder and, in particular, interrupting or reversing the progress of the disorder.
  • the amount is sufficient to avoid or delay onset of the disorder.
  • An effective amount need not be curative or prevent a disorder from ever occurring.
  • an effective amount of the pharmaceutical agent described herein will depend on the condition to be treated, the severeness of the disorder, the individual parameters of the patient, including age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present) , the specific route of administration and similar factors. Accordingly, the doses administered of the pharmaceutical agent described herein may depend on various of such parameters. In the case that a reaction in a patient is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used. In certain embodiments, an effective amount of a pharmaceutical agent will depend on its therapeutic index, solubility, and the like.
  • inhibitor as used herein, is used interchangeably with “reducing, ” “silencing, ” “downregulating, ” “suppressing” and other similar terms, and includes any level of inhibition, in particular, a statistically significant or clinically significant inhibition.
  • the term “inhibiting expression” of a target gene refers to any significant level of decrease in the level of target gene expression in cells, cell populations or tissues treated with an agent of interest, compared to those not treated.
  • Gene expression level can be measured by, for example, the level of mRNA transcript of the target gene, or level of protein expressed from the target gene.
  • the inhibition of expression of a target gene results in a clinically relevant inhibition of the level of target gene expression, e.g., sufficiently inhibited to permit an effective therapeutic response.
  • the term “pharmaceutically acceptable” indicates that the substance or composition is compatible chemically and/or toxicologically, with the other ingredients comprising a formulation, and/or the subjects being treated therewith.
  • the term “pharmaceutically acceptable salt” includes salts that retain the biological effectiveness of the free acids and bases of the specified compound and that are not biologically or otherwise undesirable.
  • Contemplated pharmaceutically acceptable salt forms include, but are not limited to, mono, bis, tris, tetrakis, and so on.
  • Pharmaceutically acceptable salts are non-toxic in the amounts and concentrations at which they are administered. The preparation of such salts can facilitate the pharmacological use by altering the physical characteristics of a compound without preventing it from exerting its physiological effect. Useful alterations in physical properties include lowering the melting point to facilitate transmucosal administration and increasing the solubility to facilitate administering higher concentrations of the drug.
  • Pharmaceutically acceptable salts can include acid addition salts such as those containing sulfate, chloride, fumarate, maleate, phosphate, sulfamate, acetate, citrate, lactate, tartrate, malonate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate and quinate.
  • acid addition salts such as those containing sulfate, chloride, fumarate, maleate, phosphate, sulfamate, acetate, citrate, lactate, tartrate, malonate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate and quinate.
  • Pharmaceutically acceptable salts can be obtained from acids such as sulfuric acid, hydrochloric acid, fumaric acid, maleic acid, phosphoric acid, sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid, and quinic acid.
  • acids such as sulfuric acid, hydrochloric acid, fumaric acid, maleic acid, phosphoric acid, sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid, and quinic acid.
  • subject includes human and non-human animals.
  • Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, mouse, rat, cat, rabbit, sheep, dog, cow, chickens, amphibians, and reptiles.
  • treatment refers to managing, eliminating, reducing or ameliorating a disorder and/or a symptom associated therewith.
  • treatment of a disorder does not require that the disorder, or symptoms associated therewith be completely eliminated.
  • treatment may include “prophylactic treatment” that is applied before development of any symptom or manifestation of a disorder to reduce the possibility of occurrence or recurrence of a disorder, or reducing the possibility of relapse of a previously controlled disorder, in a subject who is not afflicted with a disorder but at risk, or who is susceptible to recurrence of the disorder, or who is at risk or susceptible to relapse of the disorder.
  • “treatment” also includes prevention of relapse or prevention stages, as well as treatment of acute or chronic signs, symptoms and/or dysfunction. Treatment can target symptoms, for example, to suppress symptoms. It can function in a short period of time, for a medium period of time, or can be a long-term treatment, such as in the case of maintenance therapy.
  • nucleotide means a base linked to a sugar, with a phosphate group covalently linked to the sugar portion and is intended to encompass both natural (i.e. unmodified) nucleotide and modified nucleotide.
  • a nucleotide is an unmodified ribonucleotide.
  • a ribonucleotide is a 3’ -ribonucleotide.
  • a ribonucleotide is a 5’ -ribonucleotide.
  • a modified or unmodified nucleotide may be further modified optionally.
  • a natural nucleotide consists of a natural base, a natural sugar moiety, and a phosphoric acid moiety.
  • a natural nucleotide refers to an adenine ribonucleotide, adenine deoxyribonucleotide, guanine ribonucleotide, guanine deoxyribonucleotide, cytosine ribonucleotide, cytosine deoxyribonucleotide, uracil ribonucleotide, thymine ribonucleotide or thymine deoxyribonucleotide.
  • “Ribonucleotide” means a nucleotide having a hydroxy at the 2'position of the sugar portion of the nucleotide. “Deoxyribonucleoside” means a nucleotide having a hydrogen at the 2'position of the sugar portion of the nucleotide.
  • a natural base of RNA includes A (adenine) , G (guanine) , C (cytosine) , U (uracil) , and T (thymine) .
  • nucleotide “G, ” “C, ” “A, ” “T, ” and “U” each refers to a natural or a modified nucleotide that contains guanine, cytosine, adenine, thymidine, and uracil as a base, respectively.
  • a nucleotide may be replaced by an analogue thereof, including natural and non-natural analogues.
  • analogues of guanosine includes 6-thioguanosine, 8-azaguanosine, 8-oxoguanosine, 2-aminopurine riboside, and the like.
  • analogues of adenosine includes cordycepin (3’ -deoxyadenosine) , N6-benzyladenosine, 2-chloroadenosine, and the like.
  • Examples of analogues of cytidine includes gemcitabine (2’ , 2’ -difluoro-2’ -deoxycytidine) , cytarabine (1- ⁇ -D-arabinofuranosylcytosine) , decitabine (5-aza-2’ -deoxycytidine) , and the like.
  • Examples of analogues of uridine includes 5-fluorouridine, pseudo-uridine, 5-bromo-uridine, 4-thiouridine, 5-azido-uridine and the like.
  • RNA ribonucleic acid
  • RNA is a carrier of genetic information that exists in cells and some viruses and viroids.
  • RNA consists of ribonucleotides linked by internucleotide linkages forming a strand, including single-stranded RNA and double-stranded RNA. Natural internuleotide linkage is phosphodiester bond.
  • modified nucleotide refers to a nucleotide having at least a modified base, a modified sugar or a modified phosphate group that can form a modified internucleotide linkage.
  • a modified nucleotide may comprise one, two, three or more modifications.
  • a nucleotide may comprise one modification.
  • a nucleotide may comprise two modifications.
  • a nucleotide may comprise three modifications.
  • modified bases include, but not limited to, hypoxanthine (I) , xanthine, 7-methylguanine, 5, 6-dihydrouracil, 5-methylcytosine (m5C) , 5-hydroxymethoylcytosine, N6-methyladenosine (m6A) , 3-methyluridine (m3U) , 5-methyluridine (m5U) , pseudouridine, 2-thiouridine (s2U) and 5-propyluridine (5-pU) .
  • modified sugars include, but not limited to, 2’ -sugar modified, 3’ -sugar modified and 5’ -sugar modified, such as 2’ -OMe (2’ -O-methyl) modification, 2’ -F (2’ -deoxy-2’ -fluoro) modification, 2’ -O-MOE (2’ -O-methoxyethyl) modification, 2’ -deoxy (2’ -d) modification, 5’-morpholine (5’ -Mo) modification, unlocked nucleic acid (UNA) modification, glycol nucleic acid (GNA) modification, locked nucleic acid (LNA) modification, tricyclo-DNA (tcDNA) modification, (S) -constrained ethyl bridged nucleic acid ( (S) -cEt-BNA) modification, 5’ - (E) -vinylphosphate (VP) modificatio, 2’ -O-C16 modification, 2’ -C16 modification, conjugating with invert
  • modified internucleotide linkages include, but not limited to, methylphosphonate (MP) , methoxypropyl methylphosphonate (MOP) , phosphorothioate (PS) , phosphorodithioate (PS2) , phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate, phosphoramidate, -O-P (O) (OR) -O-, -O-P (S) (OR) -O-, -O-P (S) (SR) -O-, -S-P (O) (OR) -O-, -O-P (O) (OR) -S-, -S-P (O) (OR) -S-, -S-P (O) (OR) -S-, -O-P (S) (OR) -S-, -O-P (S) (OR) -S-, -
  • the modified internucleotide linkage does not contain phosphorous but rather contains peptide bonds, for example in a peptide nucleic acid (PNA) , or linking groups including carbamate, amides, and linear and cyclic hydrocarbon groups.
  • PNA peptide nucleic acid
  • the modified internucleotide linkage is a phosphothioate linkage.
  • small interfering RNA e.g., mRNA, e.g., a transcript of a gene that encodes a protein.
  • mRNA e.g., a transcript of a gene that encodes a protein.
  • mRNA to be silenced e.g., a transcript of a gene that encodes a protein.
  • mRNA to be silenced e.g., a transcript of a gene that encodes a protein.
  • mRNA to be silenced e.g., a transcript of a gene that encodes a protein.
  • mRNA e.g., a transcript of a gene that encodes a protein.
  • target gene e.g., a gene that encodes a protein.
  • the RNA to be silenced is an endogenous gene or a pathogen gene.
  • RNAs other than mRNA e.g., tRNAs, and viral RNAs, can also be targeted.
  • an siRNA comprises a double-stranded region of less than 60, 50, 40 or 30 complementary base pairs; preferably, an siRNA comprises a double-stranded region of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 complementary base pairs.
  • the sense and/or antisense strands of the siRNA independently have a length of 15-35 nucleotides, forming a complementary double-stranded region with a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 base pairs.
  • the sense strand of the siRNA has a length of 15-35 nucleotides
  • the antisense strand of the siRNA has a length of 25-35 nucleotides, forming a complementary double-stranded region with a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 base pairs.
  • the sense strand of the siRNA has a length of 17-23 nucleotides
  • the antisense strand of the siRNA has a length of 25-30 nucleotides.
  • the sense strand of the siRNA has a length of 21-23 nucleotides
  • the antisense strand of the siRNA has a length of 26-30 nucleotides.
  • the sense and antisense strands of the siRNA are fully complementary with a length of 15-30 base pairs.
  • the sense and antisense strands of the siRNA are fully complementary with a length of 17, 18, 19, 20, 21, 22, or 23 base pairs.
  • the siRNA (e.g., the anti-sense strand) is sufficiently complementary to the target RNA such that the siRNA silences the target RNA, for example inhibits production of protein encoded by the target RNA.
  • the term “sufficiently complementary” is used to indicate a sufficient degree of complementarity such that stable and specific binding occurs between an siRNA, in particular its anti-sense strand, and a target RNA molecule. Such degree of complementarity is sufficient to avoid non-specific binding of the siRNA to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of assays or therapeutic treatment, or in the case of in vitro assays, under conditions in which the assays are performed.
  • the non-target sequences typically differ by at least 4 nucleotides or more. In some embodiments, the non-target sequences differ by at least 8 nucleotides or more. Alternatively, or additionally, such degree of complementarity is sufficient to allow the siRNA to silence target RNA, for example to reduce production of protein encoded by the target mRNA.
  • complementarity is measured by the percentage of bases in each strand that can form hydrogen bonds with one another, as dictated by established base-pairing rules.
  • the oligonucleotide sequences do not need to be entirely complementary (i.e., perfectly complementary) to their corresponding nucleic acid sequences.
  • a first nucleotide sequence may be deemed complementary to a second nucleotide sequence if it exhibits a certain level of sequence complementarity, such as at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.
  • 18 out of 20 nucleobases of a first nucleotide sequence align with a corresponding region of a second nucleotide sequence, achieving 90 percent complementarity.
  • the non-complementary nucleobases are also referred to as mismatches and might be grouped or interspersed among the complementary bases and do not need to be adjacent to each other or to the complementary nucleobases.
  • mismatch includes, but is not limited to:
  • mismatches include wobble base pair and Hoogstein base pair.
  • exactly complementary is intended to mean that a first nucleotide sequence and a second nucleotide sequence form a hybrid made exclusively of Watson-Crick base pairs in the region of exact complementarity.
  • a “sufficiently complementary” oligonucleotide can include an internal region (e.g., of at least 7, 8, 9, or 10 nucleotides) that is exactly complementary to a target RNA.
  • oligonucleotide refers to a nucleic acid molecule (RNA or DNA) for example of length less than 100, 200, 300, or 400 nucleotides.
  • “monomer” refers to a class of compounds that can be assembled into a ribonucleic acid chain and can perform certain functions.
  • “monomer” includes, but is not limited to, natural nucleotides, unnatural nucleotides (such as modified nucleotides, nucleotide analogs, abasic deoxyribonucleotides, GNA, LNA, etc. ) , blocking groups, M03 as disclosed herein and M06 as disclosed herein.
  • linkage refers to the connection between the residues of two monomers (such as nucleotides) , such as nucleotide residues, via a single bond or a linking group (such as through a phosphodiester linkage, phosphorothioate linkage, or phosphordithioate linkage) .
  • the linkage refers to the connection between the residues of two monomers (such as nucleotides) via an internucleotide linkage, such as phosphodiester linkage, phosphorothioate linkage, or phosphordithioate linkage.
  • nucleotide linkage refers the linkage (e.g., a bond or a linking group) between two moieties of the oligonucleotide disclosed herein such as between two monomers, including linkage between to nucleotides, between one nucleotide and one ligand, between one nucleotide and one blocking group and between one nucleotide and an abasic nucleotide of the oligonucleotide disclosed herein.
  • alternating motif or “alternative pattern” as used herein refers to a motif having one or more modifications, each modification occurring on alternating nucleotides of one strand.
  • the alternating nucleotide may refer to one per every other nucleotide or one per every three nucleotides, or a similar pattern.
  • the alternating motif can be “ABABABABABAB...” , “AABBAABBAABB...” , “AABAABAABAAB...” , “AAABAAABAAAB...” , “AAABBBAAABBB...” , or “ABCABCABCABC...” . etc.
  • the type of modifications contained in the alternating motif may be the same or different.
  • the alternating pattern i.e., modifications on every other nucleotide, may be the same, but each of the sense strand or antisense strand can be selected from several possibilities of modifications within the alternating motif such as “ABABA...” , “ACACAC...” , “BDBDBD...” or “CDCDCD...” , etc.
  • conjugate refers to residues of two molecules (e.g., two nucleotides) such as residues of two nucleotides are linked via a bond (e.g., a single bond) or a linking group (e.g., phosphodiester, phosphorothioate or phosphorodithioate) .
  • conjuggate as a noun refers to a compound or complex formed by the covalent connection between various chemical moieties.
  • double-stranded RNA conjugate represents a compound or complex formed by the covalent connection of one or more chemical moieties (such as conjugate groups, ligand groups, or delivery systems) to double- stranded RNA.
  • a double-stranded RNA conjugate disclosed herein may also referred to as “conjugate” for brevity, which should be understood as the full term “double-stranded RNA conjugate” based on the context.
  • the delivery system, ligand group, or conjugate group can be attached to any available position of any nucleotide of the oligonucleotide agent, including the phosphate group, the sugar ring (including covalent connection of the delivery system, ligand group, or conjugate group to the atom at the 3’ or 5” position of the sugar ring of a nucleotide through a phosphodiester linkage) , the 2’ -hydroxyl group, the 5’ -hydroxyl group, and the base.
  • the ligand group or delivery system can be attached to the 3’ -position of a nucleotide, and unless otherwise specified, the nucleotides are connected by 3’ -5’ phosphodiester linkages.
  • the delivery system, ligand group, or conjugate group can also be attached to the 2’ -position of a nucleotide, and unless otherwise specified, the nucleotides are connected by 2’ -5’ phosphodiester bonds.
  • the double-stranded oligonucleotide agent disclosed herein may optionally be conjugated to one or more ligands.
  • the ligand can be attached to the sense strand, antisense strand or both strands, at the 3’ -end, 5’ -end or both ends.
  • the ligand may be conjugated to the sense strand, in particular, the 3’ -end of the sense strand.
  • the ligand is conjugated to the anti-sense strand, in particular, the 5’ -end of the anti-sense strand.
  • moieties are ligands, which are coupled, preferably covalently, either directly or indirectly via an intervening tether.
  • a ligand alters the distribution, targeting or lifetime of the molecule into which it is incorporated.
  • a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, receptor e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand.
  • Ligands providing enhanced affinity for a selected target are also termed targeting ligands.
  • Some ligands can have endosomolytic properties.
  • the endosomolytic ligands promote the lysis of the endosome and/or transport of the composition disclosed herein, or its components, from the endosome to the cytoplasm of the cell.
  • Ligands can improve transport, hybridization, and specificity properties and may also improve nuclease resistance of the resultant natural or modified oligoribonucleotide, or a polymeric molecule comprising any combination of monomers described herein and/or natural or modified ribonucleotides.
  • Ligands in general can include therapeutic modifiers, e.g., for enhancing uptake; diagnostic compounds or reporter groups e.g., for monitoring distribution; cross-linking agents; and nuclease-resistance conferring moieties.
  • General examples include lipids, steroids, vitamins, sugars, proteins, peptides, polyamines, and peptide mimics.
  • Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell.
  • ligands include dyes, intercalating agents (e.g., acridines) , cross-linkers (e.g., psoralene, mitomycin C) , porphyrins (e.g., TPPC4, texaphyrin, Sapphyrin) , polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine) , artificial endonucleases or a chelator (e.g., EDTA) , lipophilic molecules, e.g, cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1, 3-bis-O- (hexadecyl) glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1, 3-propanediol, heptadecyl group,
  • Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell.
  • the ligand can be a substance, e.g., a drug, which can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments.
  • the ligand can increase the uptake of the oligonucleotide into the cell by activating an inflammatory response, for example.
  • exemplary ligands that would have such an effect include tumor necrosis factor alpha (TNFalpha) , interleukin-1 beta, or gamma interferon.
  • TNFalpha tumor necrosis factor alpha
  • interleukin-1 beta interleukin-1 beta
  • gamma interferon e.g., interleukin-1 beta
  • the ligand is a lipid or lipid-based molecule.
  • a lipid-based ligand can be used to modulate, e.g., control the binding of the conjugate to a target tissue.
  • the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell.
  • the ligand is a cell-permeation agent, preferably a helical cell-permeation agent.
  • the ligand can be a peptide or peptidomimetic.
  • a targeting peptide can be an amphipathic ⁇ -helical peptide.
  • the targeting ligand can be any ligand that is capable of targeting a specific receptor. Examples are: folate, GalNAc, galactose, mannose, mannose-6P, clusters of sugars such as GalNAc cluster, mannose cluster, galactose cluster, or an apatamer. A cluster is a combination of two or more sugar units.
  • the targeting ligands also include integrin receptor ligands, Chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL and HDL ligands.
  • the ligands can also be based on nucleic acid, e.g., an aptamer.
  • the aptamer can be unmodified or have any combination of modifications disclosed herein.
  • the ligands can all have same properties, all have different properties or some ligands have the same properties while others have different properties.
  • a ligand can have targeting properties, have endosomolytic activity or have PK modulating properties. In some embodiments, all the ligands have different properties.
  • Ligands can be coupled to the oligonucleotides at various places, for example, 3’ -end, 5’ -end, and/or at an internal position.
  • the ligand is attached to the oligonucleotides via an intervening tether.
  • the ligand or tethered ligand may be present on a monomer when said monomer is incorporated into the growing strand.
  • the ligand may be incorporated via coupling to a “precursor” monomer after said “precursor” monomer has been incorporated into the growing strand.
  • a monomer having, e.g., an amino-terminated tether (i.e., having no associated ligand) may be incorporated into a growing oligonucleotide strand.
  • a ligand having an electrophilic group e.g., a pentafluorophenyl ester or aldehyde group, can subsequently be attached to the precursor monomer by coupling the electrophilic group of the ligand with the terminal nucleophilic group of the precursor monomer’s tether.
  • a monomer having a chemical group suitable for taking part in Click Chemistry reaction may be incorporated e.g., an azide or alkyne terminated tether/linker.
  • a ligand having complementary chemical group e.g., an alkyne or azide can be attached to the precursor monomer by coupling the alkyne and the azide together.
  • ligands can be attached to one or both strands.
  • a double-stranded iRNA agent contains a ligand conjugated to the sense strand.
  • a double-stranded iRNA agent contains a ligand conjugated to the anti-sense strand.
  • ligand can be conjugated to bases, sugars, or internucleotide linkages of nucleic acid molecules. Conjugation to purine bases or derivatives thereof can occur at any position including, endocyclic and exocyclic atoms. In some embodiments, the 2-, 6-, 7-, or 8-positions of a purine base are attached to a conjugate moiety. Conjugation to pyrimidine bases or derivatives thereof can also occur at any position. In some embodiments, the 2-, 5-, and 6-positions of a pyrimidine base can be substituted with a conjugate moiety. Conjugation to sugars of nucleosides can occur at any carbon atom.
  • Example carbon atoms of a sugar moiety that can be attached to a conjugate moiety include the 2’ , 3’ , and 5’ carbon atoms.
  • the 1’ position can also be attached to a conjugate moiety, such as in an abasic residue.
  • Internucleotide linkages can also bear conjugate moieties.
  • phosphorus-containing linkages e.g., phosphodiester, phosphorothioate, phosphorodithiotate, phosphoroamidate, and the like
  • the conjugate moiety can be attached directly to the phosphorus atom or to an O, N, or S atom bound to the phosphorus atom.
  • amine-or amide-containing internucleotide linkages e.g., PNA
  • the conjugate moiety can be attached to the nitrogen atom of the amine or amide or to an adjacent carbon atom.
  • ligand in the field of RNA interference may be used, although the ligand is typically a carbohydrate e.g., monosaccharide (such as GalNAc) , disaccharide, trisaccharide, tetrasaccharide, polysaccharide.
  • monosaccharide such as GalNAc
  • Linkers that conjugate the ligand to the nucleic acid include those discussed above.
  • the ligand can be one or more GalNAc (N-acetylglucosamine) derivatives attached through a bivalent or trivalent branched linker.
  • GalNAc N-acetylglucosamine
  • the ligand is conjugated to the oligonucleotide (e.g., the 5’ end of the anti-sense strand) via an internucleotide linkage, and the internucleotide linkage is optionally modified as described above.
  • the ligand is a GalNAc ligand, a lipophilic ligand or other ligands targeting a receptor that could facilitate the endocytosis of siRNA conjugate, such as TfR targeting ligands, LDL-R targeting ligands, or integrin targeting ligands.
  • the double-stranded oligonucleotide agent disclosed herein may optionally be conjugated to one or more blocking groups.
  • the blocking group can be attached to the sense strand, antisense strand or both strands, at the 3’ -end, 5’ -end or both ends.
  • the blocking group is conjugated to the anti-sense strand, in particular, the 5’ -end of the anti-sense strand.
  • the blocking group is conjugated to the oligonucleotide (e.g., the 5’ end of the anti-sense strand) via an internucleotide linkage, and the internucleotide linkage is optionally modified as described above.
  • the blocking group is linked to the double-stranded oligonucleotide agent with a phosphorothioate.
  • the blocking group is linked to the double-stranded oligonucleotide agent with a phosphodiester.
  • a “blocking group” refers to a group may be conjugated to an oligonucleotide disclosed herein, e.g., at the 5’ end of the anti-sense strand, which may reduce or inhibit exonulcease catabolism.
  • the blocking group may reduce or inhibit the RNA interference effect of the oligonucleotide.
  • the blocking group is cleaved from the oligonucleotide before providing the RNA interference effect.
  • blocking groups include but not limited to an abasic residue, an inverted abasic residue, M03 and M06.
  • the present disclosure provides a double-stranded oligonucleotide agent comprising a sense strand and an anti-sense strand.
  • the sense strand and the anti-sense strand form a double-stranded portion and a 5’ extension in the anti-sense strand.
  • the 5’ extension has a length of at least three nucleotides.
  • 5’ extension in the anti-sense strand can be designed such that it can be cleaved at a specific cleavage site.
  • the double-stranded oligonucleotide agent provided herein is particularly advantageous in that the 5’ extension is cleavable from the most 3’ nucleotide of the 5’ extension.
  • the double-stranded oligonucleotide agent reaches the target tissue, it can be converted into the cleaved product .
  • Such a cleaved double-stranded oligonucleotide agent is found to be capable of inhibiting expression of a target gene via RNA interference.
  • RNA interference refers to the ability to silence, in a sequence specific manner, a target RNA, by a double-stranded short interfering RNA (siRNA) .
  • the first step in RNA interference (RNAi) involves activating the RNA-induced silencing complex (RISC) , which necessitates the degradation of the sense strand of the double-stranded RNA (dsRNA) duplex.
  • RISC RNA-induced silencing complex
  • Ago2 Argonaute 2
  • the RISC is activated by the antisense strand (Rand et al. (2005) Cell 123, 621) .
  • the double-stranded oligonucleotide agents provided herein have enhanced activity in inhibition of the target gene expression than a reference siRNA having otherwise comparable anti-sense sequence except for the 5’ extension or the cleavage region provided herein.
  • the double-stranded oligonucleotide agents provided herein comprises, essentially consists of or consists of ribonucleic acids (RNA) .
  • the sense stand of the double-stranded oligonucleotide agents provided herein comprises, essentially consists of or consists of RNA.
  • the anti-sense stand of the double-stranded oligonucleotide agents provided herein comprises, essentially consists of or consists of RNA.
  • the double-stranded oligonucleotide agents provided herein can be converted into an siRNA, optionally after delivery to a target tissue or a target cell.
  • the double-stranded oligonucleotide agents provided herein is can be converted into an siRNA by an enzyme, or optionally by RNAase III, or optionally by dicer.
  • the cleaved double-stranded oligonucleotide agents provided herein is an siRNA.
  • the double-stranded portion is formed by base pairing between a first fragment in the anti-sense strand and a second fragment in the sense strand, wherein the first fragment and the second fragment are of equal length.
  • the base pairing between the first fragment and the second fragment can include base pairs in a Watson-Crick manner and/or in any other manner that allows the formation of a stable double strand.
  • Watson-Crick base pairing adenine (A) is paired with thymine (T) in DNA and with uracil (U) in RNA; Guanine (G) is paired with cytosine (C) .
  • Base pairs can also be formed by non-Watson-Crick base pairs, which include, but not limited to, G-U wobble base pair and Hoogstein base pair.
  • a nucleotide containing hypoxanthine as its base may be paired with a nucleotide base containing adenine, cytosine, or uracil.
  • a nucleotide containing uracil, guanine, or adenine may be replaced by a nucleotide containing, for example, inosine (as used herein, “I” may indicate a hypoxanthine base, inosine, or an inosine-containing nucleotide depending on its context) . This substitution is referred to “I modification” .
  • adenine and cytosine anywhere in the oligonucleotide can be replaced by guanine and uracil, respectively, to form a G-U wobble base pair with the target mRNA.
  • the base pairs formed in the double-stranded portion can comprise or consist essentially of Watson-Crick base pairing, and/or non-Watson-Crick base pairs such as G-U wobble base pair and Hoogstein base pair, or a combination thereof.
  • the first fragment in the sense strand and the second fragment in the anti-sense strand are of equal length, and are complementary to form the double-stranded portion. In some embodiments, the first fragment and the second fragment are at least 80%, 85%, 90%, 95%, or 100%complementary.
  • the double-stranded portion is of 15 to 27 nucleotide pairs in length. In certain embodiments, the double-stranded portion has a length of 16 to 27 nucleotide pairs, 16 to 26 nucleotide pairs, 16 to 25 nucleotide pairs, 16 to 24 nucleotide pairs, 16 to 23 nucleotide pairs, 16 to 22 nucleotide pairs, 16 to 21 nucleotide pairs, 16 to 20 nucleotide pairs, 17 to 23 nucleotide pairs, 17 to 22 nucleotide pairs, 17 to 21 nucleotide pairs, 17 to 20 nucleotide pairs, 18 to 23 nucleotide pairs, 18 to 22 nucleotide pairs, 18 to 21 nucleotide pairs, 18 to 20 nucleotide pairs, 19 to 23 nucleotide pairs, 19 to 22 nucleotide pairs, 19 to 21 nucleotide pairs.
  • the double-stranded portion has a length of 16 nucleotide pairs, 17 nucleotide pairs, 18 nucleotide pairs, 19 nucleotide pairs, 20 nucleotide pairs, 21 nucleotide pairs, 22 nucleotide pairs, or 23 nucleotide pairs.
  • the sense strand of the double-stranded oligonucleotide agent has a length of 15 to 35 nucleotides (nt) , 16 to 35, 16 to 30, 16 to 27, 16 to 26, 16 to 25, 16 to 24, 16 to 23, 16 to 22, 16 to 21, 17 to 35, 17 to 30, 17 to 25, 17 to 23, 17 to 22, 17 to 21, 18 to 35, 18 to 30, 18 to 25, 18 to 23, 18 to 22, 18 to 21, 19 to 35, 19 to 30, 19 to 25, 19 to 23, 19 to 22, 19 to 21, 20 to 35, 20 to 30, 20 to 25, 20 to 23, 21 to 35, 21 to 30, 21 to 25, 21 to 23, such as 35, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, or 15 nt.
  • nt nucleotides
  • the sense strand of the double-stranded oligonucleotide agent has a length of 15 to 23, 15 to 22, or 15 to 21 nt.
  • the 5’ extension has a length of at least 3, 4, 5, 6, 7 or more nucleotides.
  • the double-stranded oligonucleotide agent further comprises a 3’ extension in the anti-sense strand.
  • the 3’ extension has a length of at least 1, 2, or more nucleotides.
  • the 3’ extension can be an overhang having a length of at least 1, 2, 3, 4, or 5 nucleotides.
  • An extension (e.g., 3’ extension) can be 1-6 nucleotides in length, for instance 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5 nucleotides in length, 1-4 nucleotides in length, 2-4 nucleotides in length, 1-3 nucleotides in length, 2-3 nucleotides in length, or 1-2 nucleotides in length.
  • the extensions e.g., 5’ extension or 3’ extension
  • the nucleotides in the extension region can each independently be a modified or unmodified nucleotide including, but not limited to 2’ -sugar modification, such as, 2’ -F, or 2’ -OMe.
  • the 5’ -or 3’ -extensions at the sense strand, antisense strand or both strands may be phosphorylated.
  • the extension region contains two nucleotides having a phosphorothioate linkage between the two nucleotides, where the two nucleotides can be the same or different. In one embodiment, this 3’ -extension is present in the antisense strand.
  • the anti-sense strand of the double-stranded oligonucleotide agent has a length of at least 25 to 35, 25 to 34, 25 to 33, 25 to 32, 25 to 31, 25 to 30, 25 to 29, 25 to 28, 25 to 27, 25 to 26, 26 to 35, 26 to 34, 26 to 33, 26 to 32, 26 to 31, 26 to 30, 26 to 29, 26 to 28, 26 to 27 nt. In certain embodiments, the anti-sense strand of the double-stranded oligonucleotide agent has a length of 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, 30 nt.
  • the first fragment comprises a targeting region which is sufficiently complementary to part of a target RNA, optionally a mRNA encoding the target gene.
  • the targeting region disclosed herein is at least 90%, at least 95%, or 100%complementary to the part of the target RNA.
  • the targeting region binds to the target RNA and forms a duplex made exclusively of Watson-Crick base pairs in the region of complementarity.
  • the target region can include an internal region (e.g., of at least 10 nucleotides) that is 100%complementary to a target RNA.
  • the internal region 100%complementary to a target RNA can be the seed region which is from position 2 to position 8 from the 5’ end of the targeting region in the anti-sense strand.
  • the target region is 100%complementary to a target RNA, e.g., the target RNA and the dsRNA duplex agent anneal, for example to form a hybrid made exclusively of Watson-Crick base pairs in the region of exact complementarity.
  • the dsRNA agent of the invention specifically discriminates a single-nucleotide difference. In this case, the dsRNA agent only mediates RNAi if exact complementarity is found in the region (e.g., within 7 nucleotides of) the single-nucleotide difference.
  • the anti-sense strand of the double-stranded oligonucleotide agent comprises a cleavage region that allows specific cleavage from the most 3’ nucleotide of the 5’ extension.
  • a double-stranded oligonucleotide agent disclosed herein undergoes cleavage mediated by an enzyme (e.g., an endonuclease) before mediates RNAi.
  • the cleavage is mediated by a ribonuclease (RNase) .
  • the cleavage is mediated by an endonuclease.
  • the cleavage is mediated by a RNAase III.
  • RNase III represents a class of endoribonucleases producing small RNAs for RNA silencing pathways.
  • the RNAase III is dicer.
  • the cleavage is a specific cleavage.
  • the term “specific” or “specifically” with respect to cleavage of an oligonucleotide agent refers to controlled or selective cleavage at a specific position, i.e., between two desired nucleotides of the oligonucleotide strand undergoing specific cleavage.
  • the products of a specific cleavage may comprise a plurality of cleavage products, wherein the desired cleavage product (i.e., the product obtained by cleavage at the specific or desired position) is the most abundant cleavage product.
  • the desired cleavage product counts for at least 30%, 40%, 50%, 60%, 70%, 80%or 90%of all cleavage products (by molar or weight) .
  • the amount of desired cleavage product (by molar or weight) is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold of any other cleavage products. While not wishing to be bound by theory, a specific cleavage is based on the recognition of a certain sequence, features, motifs or a combination thereof of the oligonucleotide agent undergoing cleavage by the enzyme mediating the cleavage.
  • the cleavage region comprises a nucleotide sequence set forth in Formula A: (3’ -5’ ) X2-Y-Z, which is cleavable between X2 and Y, wherein X2 is the most 5’ nucleotide of the first fragment and Y-Z are the two most 3’ nucleotides of the 5’ extension.
  • the cleavage region further comprises a fourth nucleotide (N1) which is the third nucleotide from the 3’ end of the 5’ extension, and the cleavage region comprises a nucleotide sequence set forth in Formula B: (3’ -5’ ) X2-Y-Z-N1, which is cleavable between X2 and Y.
  • the cleavage region further comprises a third fragment (N) , the third fragment comprises at least one nucleotide, wherein N1 is the most 3’ nucleotide of the third fragment, and the cleavage region comprises a nucleotide sequence set forth in Formula B’ : (3’ -5’) X2-Y-Z-N, which is cleavable between X2 and Y, the length of N is 1-10 nucleotides, preferably the length of N is 1-5 nucleotides, further preferably the length of N is 1 nucleotides.
  • the enzymatic cleavage can be determined using target tissue (e.g., liver, eye, lung, kidney, brain, spin cord, muscle, fat, etc. ) homogenate processing reaction assay, see for example the methods described in Example 4.6, or using Dicer Endonuclease Treatment assay.
  • target tissue e.g., liver, eye, lung, kidney, brain, spin cord, muscle, fat, etc.
  • Dicer Endonuclease Treatment assay see for example the methods described in Example 4.6, or using Dicer Endonuclease Treatment assay.
  • Z is selected from G or A or a natural or non-natural analogue thereof. In certain embodiments, Z is selected from G or a natural or non-natural analogue thereof.
  • X2 is selected from A or U, or a natural or non-natural analogue thereof.
  • a double-stranded oligonucleotide agent disclosed herein is capable of being specifically cleaved between X2 and Y, to remove the 5’ extension, thereby generating a cleaved double-stranded oligonucleotide agent.
  • the cleaved double-stranded oligonucleotide is believed to be capable of mediating RNAi.
  • the cleaved double-stranded oligonucleotide agent has a blunt end at the 5’ end of the anti-sense strand, where the first base pair at the 5’ end the anti-sense strand composed of X2 and its complementary nucleotide in the sense strand.
  • such first base pair at the 5’ end of the anti-sense strand is an AU base pair.
  • the Formula A has the nucleotide sequence from 3’ -5’s elected from the group consisting of: UUG, UAG, AUG, AAG, UUA, UAA, AUA, AAA, UCG, UGG, ACG, AGG, UCA, UGA, ACA, and AGA, or a natural or non-natural analogue thereof.
  • the nucleotide Y is selected from A or U, or a natural or non-natural analogue thereof.
  • the Formula A has the nucleotide sequence from 3’ -5’s elected from the group consisting of: UUG, UAG, AUG, AAG, UUA, UAA, AUA, and AAA.
  • At least one nucleotide in the cleavage region is a modified nucleotide, preferably all nucleotides in the cleavage region are modified nucleotides.
  • the modified nucleotide has a modified base, a modified sugar or a modified internucleotide linkage.
  • the modified nucleotide has a modified base, and optionally the modified base comprises hypoxanthine (I) , xanthine, 7-methylguanine, 5, 6-dihydrouracil, 5-methylcytosine (m5C) , 5-hydroxymethoylcytosine, N6-methyladenosine (m6A) , 3-methyluridine (m3U) , 5-methyluridine (m5U) , pseudouridine, 2-thiouridine (s2U) or 5-propyluridine (5-pU) .
  • the modified nucleotide has modified sugar, and optionally the modified sugar comprises 2’ -sugar modification, 3’ -sugar modification and 5’ -sugar modification, such as 2’ -OMe (2’ -O-methyl) modification, 2’ -F (2’ -deoxy-2’ -fluoro) modification, 2’ -O-MOE (2’-O-methoxyethyl) modification, 2’ -deoxy (2’ -d) modification, 5’ -morpholine (5’ -Mo) modification, unlocked nucleic acid (UNA) modification, glycol nucleic acid (GNA) modification, locked nucleic acid (LNA) modification, tricyclo-DNA (tcDNA) modification, (S) -constrained ethyl bridged nucleic acid ( (S) -cEt-BNA) modification, 5’ - (E) -vinylphosphate (VP) modificatio, 2’-O-C16 modification, conjugating with 2’ -
  • the modified nucleotide has modified internucleotide linkage, and optionally the modified internucleotide linkage comprises methylphosphonate (MP) , methoxypropyl methylphosphonate (MOP) , phosphorothioate (PS) , phosphorodithioate (PS2) , phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate, phosphoramidate and PNA.
  • MP methylphosphonate
  • MOP methoxypropyl methylphosphonate
  • PS phosphorothioate
  • PS2 phosphorodithioate
  • phosphoroselenoate phosphorodiselenoate
  • phosphoroanilothioate phosphoraniladate
  • PNA phosphoramidate
  • At least one nucleotide in the cleavage region is 2’ -OMe modified or 2’ -F modified. In certain embodiments, all nucleotides in the cleavage region are modified nucleotides selected from 2’ -OMe modified and 2’ -F modified. In certain embodiments, at least one nucleotide in the cleavage region is 2’ -F modified. In certain embodiments, no more than two nucleotides in the cleavage region are 2’ -F modified.
  • Z in the Formula A or Formula B is 2’ -F modified, and optionally X2 in the Formula A or Formula B is 2’ -F modified.
  • N1 in the Formula A or Formula B is 2’ -F modified.
  • both X2 and Y in the Formula A or Formula B are 2’ -OMe modified, and both Z and N1 in the Formula A or Formula B are 2’ -F modified.
  • all of X2, Y and Z in the Formula A or Formula B are 2’ -OMe modified, and N1 in the Formula A or Formula B is 2’ -F modified.
  • At least one of the internucleotide linkages between the nucleotides in the cleavage region is not phosphorothioate linkage. In certain embodiments, the internucleotide linkage between X2 and Y is not phosphorothioate linkage. In certain embodiments, the internucleotide linkage between Y and Z is not phosphorothioate linkage. In certain embodiments, none of the internucleotide linkages between the nucleotides in the cleavage region is phosphorothioate linkage. In certain embodiments, at least one of the internucleotide linkages between the nucleotides in the cleavage region is phosphodiester linkage.
  • the internucleotide linkage between X2 and Y is phosphodiester linkage. In certain embodiments, the internucleotide linkage between Y and Z is phosphodiester linkage. In certain embodiments, each of the internucleotide linkages between the nucleotides in the cleavage region is phosphodiester linkage.
  • the double-stranded oligonucleotide agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.
  • the double-stranded oligonucleotide agent provided herein comprises at least one phosphorothioate or methylphosphonate internucleotide linkage at position 1 and/or 2 of the first fragment (counting from its 5’ -end) . In certain embodiments, the double-stranded oligonucleotide agent provided herein comprises at least one phosphorothioate or methylphosphonate internucleotide linkage at position 1 and/or 2 of the first fragment (counting from its 3’ -end) .
  • the double-stranded oligonucleotide agent provided herein comprises phosphorothioate or methylphosphonate internucleotide linkage between the first fragment (e.g. the nucleotide at position 1 counting from its 5’ -end) and the X2 of Formula A or Formula B.
  • the double-stranded oligonucleotide agent provided herein comprises at least one phosphorothioate or methylphosphonate internucleotide linkage at positions 1 to 8, or at positions 1 to 6, or at position 1 and/or 2 of the second fragment (counting from its 5’ -end) . In certain embodiments, the double-stranded oligonucleotide agent provided herein comprises at least one phosphorothioate or methylphosphonate internucleotide linkage at positions 1 to 8, or at positions 1 to 6, or at position 1 and/or 2 of the second fragment (counting from its 5’ -end) .
  • each nucleotide of the first fragment of the anti-sense strand is modified. In certain embodiments, each nucleotide of the anti-sense strand is modified. In certain embodiments, each nucleotide of the second fragment of the sense strand is modified. In certain embodiments, each nucleotide of the sense strand is modified.
  • each nucleotide of the anti-sense strand is either 2’ -F modified or 2’-OMe modified; and/or each nucleotide of the sense strand is either 2’ -F modified or 2’ -OMe modified.
  • the sense strand further comprises a) a motif of three consecutive 2’-F modified nucleotides, optionally the motif occurs at positions 9, 10 and 11 counting from 5’ end nucleotide of the sense strand; and/or b) 2’ -F modified nucleotide at position 7 and/or 18 counting from 5’ end nucleotide of the sense strand.
  • the sense strand further comprises an alternating motif from position 9 to 13 counting from 5’ end, optionally 2’ -F modification and 2’ -OMe modification occur on alternating nucleotides in the motif.
  • the first fragment of the anti-sense strand and/or the second fragment of the sense strand further comprises an alternating motif where modification occurs on alternating nucleotides in the motif, optionally 2’ -F modification occurs on alternating nucleotides in the motif and/or 2’ -OMe modification occurs on alternating nucleotides in the motif.
  • the first fragment of the anti-sense strand and/or the second fragment of the sense strand comprises modification selected from the group consisting of 2’ -OMe (2’-O-methyl) modification, 2’ -F (2’ -deoxy-2’ -fluoro) modification, 2’ -O-MOE (2’ -O- methoxyethyl) modification, 2’ -deoxy (2’ -d) modification, 5’ -morpholine (5’ -Mo) modification, unlocked nucleic acid (UNA) modification, glycol nucleic acid (GNA) modification, locked nucleic acid (LNA) modification, tricyclo-DNA (tcDNA) modification, (S) -constrained ethyl bridged nucleic acid ( (S) -cEt-BNA) modification, 5’ - (E) -vinylphosphate (VP) modificatio, 2’ -O-C16 modification, conjugating with invert abasic nucleotide (invAB
  • the double-stranded oligonucleotide agent disclosed herein may optionally be linked to one or more blocking groups.
  • the blocking group can be attached to the sense strand, antisense strand or both strands, at the 3’ -end, 5’ -end or both ends.
  • the blocking group is linked to the anti-sense strand, in particular, the 5’ -end of the anti-sense strand.
  • the blocking group is linked to the oligonucleotide (e.g., the 5’ end of the anti-sense strand) via an internucleotide linkage, and the internucleotide linkage is optionally modified as described above.
  • the blocking group is linked to the double-stranded oligonucleotide agent with a phosphorothioate.
  • the blocking group is linked to the double-stranded oligonucleotide agent with a phosphodiester.
  • nucleotide linked e.g., linked at 5’ end or 3’ end
  • an inverted abasic deoxyribonucleotide has a structure of
  • a nucleotide linked (e.g., linked at 5’ end or 3’ end) with M03 has a structure of
  • the conjugation with M03 is by the reaction of a nucleotide and
  • a nucleotide linked (e.g., linked at 5’ end or 3’ end) with M06 has a structure of
  • the linkage to M06 is by the reaction of a nucleotide and
  • a method of reducing or inhibiting the exonulcease catabolism of an oligonucleotide e.g., a double-stranded oligonucleotide such as a siRNA
  • conjugating or linking a blocking group to the oligonucleotide.
  • the blocking group is an abasic residue, an inverted abasic residue, M03 or M06.
  • the blocking group is M03 or M06.
  • the blocking group (e.g., M03 or M06) is linked to an anti-sense strand of the oligonucleotide, in particular, the 5’ end or 3’ end of the anti-sense strand.
  • the blocking group (e.g., M03 or M06) is linked to the 5’ end of the anti-sense strand. In some embodiments, the blocking group may reduce or inhibit the RNA interference effect of the oligonucleotide.
  • a blocking group in reducing or inhibiting the exonulcease catabolism of an oligonucleotide (e.g., a double-stranded oligonucleotide such as a siRNA) , wherein the blocking group is conjugated or linked to the oligonucleotide, in particular, the 5’ end or 3’ end of the anti-sense strand.
  • the blocking group is an abasic residue, an inverted abasic residue, M03 or M06.
  • the blocking group is M03 or M06.
  • the blocking group is M03 or M06.
  • the blocking group (e.g., M03 or M06) is linked to the 5’ end of the anti-sense strand. In some embodiments, the blocking group may reduce or inhibit the RNA interference effect of the oligonucleotide.
  • the double-stranded oligonucleotide agent provided herein comprises a structure represented by Formula (C) :
  • first fragment in the anti-sense strand and the second fragment in the sense strand form a double -stranded portion by base pairing, wherein the first fragment and the second fragment are of equal length;
  • the 5’ extension comprises at least three nucleotides in length
  • the anti-sense strand comprises a cleavage region comprising the most 5’ nucleotide (X2) of the first fragment and the two most 3’ nucleotides (Y-Z) of the 5’ extension, and the cleavage region comprises a nucleotide sequence set forth in Formula
  • the sense strand has a length of 15 to 35, 15 to 23, 15 to 22, or 15 to 21 nucleotides
  • anti-sense strand has a length of 25 to 35, 26 to 35, 26 to 30, 25 to 27, or 26 to 27 nucleotides.
  • the sense strand of the double-stranded oligonucleotide agent of Formula (C) has a length of 17 to 23, 17 to 22, or 17 to 21 nucleotides; and the anti-sense strand of the double-stranded oligonucleotide agent of Formula (C) has a length of 20-26, 20 to 25, 20 to 24 nucleotides.
  • the sense strand of the double-stranded oligonucleotide agent of Formula (C) and the anti-sense strand of the double-stranded oligonucleotide agent of Formula (C) have a length of a) 17nt and 20nt respectively; b) 18nt and 21nt, respectively; c) 19nt and 22nt, respectively; d) 20nt and 23nt, respectively; or e) 21nt and 24nt, respectively.
  • the double-stranded oligonucleotide agent disclosed herein may optionally be linked to one or more blocking groups.
  • Any suitable blocking group provided in the present disclosure can be used.
  • the blocking group can be attached to the sense strand, antisense strand or both strands, at the 3’ -end, 5’ -end or both ends.
  • the blocking group is linked to the anti-sense strand, in particular, the 5’ -end of the anti-sense strand.
  • the blocking group is linked to the 5’ end of the 5’ extension.
  • oligonucleotide agent comprising a structure represented by Formula (D) :
  • the first fragment in the anti-sense strand and the second fragment in the sense strand form a double-stranded portion by base pairing, wherein the first fragment and the second fragment are of equal length;
  • the 5’ extension comprises at least three nucleotides in length
  • the anti-sense strand comprises a cleavage region comprising the most 5’ nucleotide (X2) of the first fragment and the two most 3’ nucleotides (Y-Z) of the 5’ extension
  • the cleavage region comprises a nucleotide sequence set forth in Formula A: (3’ -5’ ) X2-Y-Z, which is cleavable between X2 and Y, and upon cleavage, the 5’ extension is removed from its most 3’ nucleotide (Y)
  • the Formula A is as defined herein,
  • the sense strand has a length of 15 to 35, 15 to 23, 15 to 22, 15 to 21, 16 to 25, 17 to 23, 18 to 23, 19 to 23, 19 to 21, 20 to 23, 20 to 21, 21 to 23, such as 17, 18, 19, 20, 21, 22, or 23 nucleotides;
  • the anti-sense strand has a length of 25 to 35, 25 to 30, 26 to 35, 26 to 30, 26 to 27, such as 25, 26, 27, 28, 29, or 30 nucleotides.
  • the sense strand of the double-stranded oligonucleotide agent of Formula (D) has a length of 17 to 23, 17 to 22, or 17 to 21 nucleotides; and wherein the anti-sense strand of the double-stranded oligonucleotide agent of Formula (D) has a length of 22-28, 22 to 27, or 22 to 26 nucleotides.
  • the sense strand of the double-stranded oligonucleotide agent of Formula (D) and the anti-sense strand of the double-stranded oligonucleotide agent of Formula (D) have a length of a) 17nt and 22nt respectively; b) 18nt and 23nt, respectively; c) 19nt and 24nt, respectively; d) 20nt and 25nt, respectively; or e) 21nt and 26nt, respectively.
  • the double-stranded oligonucleotide agent disclosed herein may optionally be linked to one or more blocking groups.
  • Any suitable blocking group provided in the present disclosure can be used.
  • the blocking group can be attached to the sense strand, antisense strand or both strands, at the 3’ -end, 5’ -end or both ends.
  • the blocking group is linked to the anti-sense strand, in particular, the 5’ -end of the anti-sense strand.
  • the blocking group is linked to the 5’ end of the 5’ extension.
  • the sense strand of the oligonucleotide agent disclosed herein comprises at least 19, 20, 21 or 22 consecutive nucleotides of any one of SEQ ID NOs: 1-97. In some embodiments, the sense strand of the oligonucleotide agent provided herein is any one of SEQ ID NOs: 1-97.
  • the anti-sense strand of the oligonucleotide agent disclosed herein comprises at least 19, 20, 21 or 22 consecutive nucleotides of any one of SEQ ID NOs: 98-194. In some embodiments, the anti-sense strand of the oligonucleotide agent provided herein is any one of SEQ ID NOs: 98-194.
  • the anti-sense strand of the oligonucleotide agent disclosed herein comprises a pair of sense strand or anti-sense strand as set forth in Table 1.
  • the sense strand of the oligonucleotide agent provided herein is any one of SEQ ID NOs: 1-97
  • the anti-sense strand of the oligonucleotide agent provided herein is any one of SEQ ID NOs: 98-194
  • At least one of the internucleotide linkages between the nucleotides in the cleavage region is phosphodiester linkage
  • the internucleotide linkage between Y and Z is phosphodiester linkage
  • each of the internucleotide linkages between the nucleotides in the cleavage region is phosphodiester linkage.
  • the sense strand of the oligonucleotide agent provided herein is any one of SEQ ID NOs: 1-97
  • the anti-sense strand of the oligonucleotide agent provided herein is any one of SEQ ID NOs: 98-194
  • At least one nucleotide in the cleavage region is 2’ -OMe modified or 2’ -F modified;
  • each nucleotide in the cleavage region is either 2’ -OMe modified or 2’ -F modified;
  • At least one nucleotide in the cleavage region is 2’ -F modified
  • N1 in the Formula A or Formula B is 2’ -F modified;
  • both X2 and Y in the Formula A or Formula B are 2’ -OMe modified, and both Z and N1 in the Formula A or Formula B are 2’ -F modified; and/or
  • the oligonucleotide agent disclosed herein is any one of ds1-ds121.
  • the double-stranded oligonucleotide agent disclosed herein may optionally be linked to one or more blocking groups. Any suitable blocking group provided in the present disclosure can be used.
  • the blocking group can be attached to the sense strand, antisense strand or both strands, at the 3’ -end, 5’ -end or both ends.
  • the blocking group is linked to the anti-sense strand, in particular, the 5’ -end of the anti-sense strand.
  • the cleaved double-stranded oligonucleotide agent comprises a 3’ extension on the anti-sense strand.
  • Each nucleotide of the cleaved double-stranded oligonucleotide agent may be independently modified or unmodified as describe above. In some embodiments, all nucleotides of the cleaved double-stranded oligonucleotide agent are independently modified. In some embodiments, all nucleotides of the cleaved double-stranded oligonucleotide agent are independently modified with 2’ -F or 2’ -OMe.
  • Each internucleotide linkage of the cleaved double-stranded oligonucleotide agent may independently modified or unmodified as describe above.
  • the cleaved double-stranded oligonucleotide agent comprises at least one modified internucleotide linkage.
  • the modified internucleotide linkage is phosphorothioate (PS) or phosphorodithioate (PS2) .
  • the cleaved double-stranded oligonucleotide agent comprises modifications of an alternating pattern as described above.
  • the cleaved double-stranded oligonucleotide agent comprises phosphorothioate or methylphosphonate internucleotide linkage.
  • the phosphorothioate or methylphosphonate internucleotide linkage modification may occur on any nucleotide of the sense strand or antisense strand or both in any position of the strand.
  • the internucleotide linkage modification may occur on every nucleotide on the sense strand and/or antisense strand; each internucleotide linkage modification may occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand comprises both internucleotide linkage modifications in an alternating pattern.
  • the alternating pattern of the internucleotide linkage modification on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the internucleotide linkage modification on the sense strand may have a shift relative to the alternating pattern of the internucleotide linkage modification on the antisense strand.
  • the cleaved double-stranded oligonucleotide agent comprises the phosphorothioate or methylphosphonate internucleotide linkage modification in the extension (e.g., 3’extension) region.
  • the extension (e.g., 3’ extension) region comprises two nucleotides having a phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides.
  • Internucleotide linkage modifications also may be made to link the extension nucleotides with the terminal paired nucleotides within duplex region.
  • extension nucleotides may be linked through phosphorothioate or methylphosphonate internucleotide linkage, and optionally, there may be additional phosphorothioate or methylphosphonate internucleotide linkages linking the extension nucleotide with a paired nucleotide that is next to the extension nucleotide.
  • these terminal three nucleotides may be at the 3 ‘-end of the antisense strand.
  • the sense strand of the cleaved double-stranded oligonucleotide agent comprises 1-10 blocks of two to ten phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said sense strand is paired with an antisense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphophonate or phosphate linkage.
  • the antisense strand of the cleaved double-stranded oligonucleotide agent comprises two blocks of two phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphophonate or phosphate linkage.
  • the cleaved double-stranded oligonucleotide agent further comprises one to five phosphorothioate or methylphosphonate internucleotide linkage modification (s) within position 1-5 and one to five phosphorothioate or methylphosphonate internucleotide linkage modification (s) within position 18-23 of the sense strand (counting from the 5’ -end) , and one to five phosphorothioate or methylphosphonate internucleotide linkage modification at positions 1 and 2 and one to five within positions 18-23 of the antisense strand (counting from the 5’ -end) .
  • the cleaved double-stranded oligonucleotide agent comprises the pattern of the alternating motif of 2’ -OMe modification and 2’ -F modification on the sense strand initially has a shift relative to the pattern of the alternating motif of 2’ -OMe modification and 2’ -F modification on the antisense strand initially, i.e., the 2’ -OMe modified nucleotide on the sense strand base pairs with a 2’ -F modified nucleotide on the antisense strand and vice versa.
  • the 1 position of the sense strand may start with the 2’ -F modification
  • the 1 position of the antisense strand may start with the 2’ -OMe modification.
  • the introduction of one or more motifs of three identical modifications on three consecutive nucleotides to the sense strand and/or antisense strand interrupts the initial modification pattern present in the sense strand and/or antisense strand.
  • This interruption of the modification pattern of the sense and/or antisense strand by introducing one or more motifs of three identical modifications on three consecutive nucleotides to the sense and/or antisense strand surprisingly enhances the gene silencing activity to the target gene.
  • the double-stranded oligonucleotide agent disclosed herein may optionally be conjugated to one or more ligands.
  • the ligand can be attached to the sense strand, antisense strand or both strands, at the 3’ -end, 5’ -end or both ends or at any available nucleotides (e.g., at the sugar ring) .
  • the ligand may be conjugated to the sense strand, in particular, the 3’ -end of the sense strand.
  • the ligand is conjugated to the anti-sense strand, in particular, the 5’ -end of the anti-sense strand.
  • a nucleotide e.g., of the sense stand or anti-sense strand
  • a nucleotide conjugate to a ligand is also encompassed in the range of a “ligand” .
  • the ligand is a GalNAc ligand, a lipophilic ligand or other ligands targeting a receptor that could facilitate the endocytosis of siRNA conjugate, such as TfR targeting ligands, LDL-R targeting ligands, or integrin targeting ligands.
  • the ligand is conjugated to the oligonucleotide (e.g., the 5’ end of the anti-sense strand) via an internucleotide linkage, and the internucleotide linkage is optionally modified as described above.
  • the ligand is linked to the double-stranded oligonucleotide agent with a phosphorothioate.
  • the ligand is L96, having a structure of
  • the ligand is hexadecyloxy.
  • a C16 ligand may conjugate to a uridine nucleotide at 2’ position, which is abbreviated as “C16U” herein and has a structure of
  • the ligand is VSDL-01. In some embodiments, the ligand is VSDL-01A. In some embodiments, the ligand is VSDL-02. In some embodiments, the ligand is VSDL-02A. In some embodiments, the ligand is VSDL-03. In some embodiments, the ligand is VSDL-03A. In some embodiments, the ligand is VSDL-04. In some embodiments, the ligand is VSDL-04A. In some embodiments, the ligand is VSDL-05. In some embodiments, the ligand is VSDL-05A. In some embodiments, the ligand is VSDL-06. In some embodiments, the ligand is VSDL-06A.
  • the ligand is VSDL-07. In some embodiments, the ligand is VSDL-07A. In some embodiments, the ligand is VSDL-08. In some embodiments, the ligand is VSDL-08A. In some embodiments, the ligand is VSDL-09. In some embodiments, the ligand is VSDL-10. In some embodiments, the ligand is VSDL-11. In some embodiments, the ligand is VSDL-12. In some embodiments, the ligand is VSDL-13. In some embodiments, the ligand is VSDL-14.
  • the double-stranded oligonucleotide agent provided herein is capable of inhibiting expression of a target gene.
  • the target gene is selected from the group consisting of AGT, complement factor B, DGAT2, DUX, ANGPTL8, APOC3, F12, INHBE, PNPLA3, Serpinc1, APP, SOD1, TMPRSS6, KHK, PCSK9, VEGFA, ANGPTL3, ANGPTL4, C3, C5, TTR, IGF-1R, VEGFR, ANG2, GIPR, GPR75, ActRII, NUDT21, PLN, HSD17b13, CNOT6L, PTP1B, CFHR, ATX, CIDEB, mARC1, TSHR, CB1 and LPA.
  • the targeting region contained in the first fragment provided herein is sufficiently complementary to a part of an mRNA encoding the target gene. In some embodiments, the targeting region is at least 80%, 85%, 90%or 95%complementary to a part of an mRNA encoding the target gene. In some embodiments, the targeting region is 100%complementary (perfect complementary) to a part of an mRNA encoding the target gene.
  • a double-stranded oligonucleotide agent comprising a double-stranded oligonucleotide operably linked to a blocking group, wherein the blocking group comprises M03 or M06.
  • the double-strand oligonucleotide can be any suitable siRNA or RNAi agent.
  • a double-stranded oligonucleotide agent comprising a double-stranded oligonucleotide operably linked to a ligand, where the ligand comprises a chemical structure selected from the group consisting of: VSDL-01, VSDL-01A, VSDL-02, VSDL-02A, VSDL-03, VSDL-03A, VSDL-04, VSDL-04A, VSDL-05, VSDL-05A, VSDL-06, VSDL-06A, VSDL-07, VSDL-07A, VSDL-08, VSDL-08A, VSDL-09, VSDL-10, VSDL-11, VSDL-12, VSDL-13, and VSDL-14.
  • the double-strand oligonucleotide can be any suitable siRNA or RNAi agent.
  • the double-stranded oligonucleotide agent operably linked to a blocking group or a ligand comprising a sense strand and an anti-sense strand. In some embodiments, the double-stranded oligonucleotide agent operably linked to a blocking group or a ligand comprising a blocking group conjugated to the 5’ end of the anti-sense strand. In some embodiments, the double-stranded oligonucleotide agent operably linked to a blocking group or a ligand comprising a ligand conjugated to the 5’ end of the anti-sense strand, 5’ end of the sense strand or 3’ end of the sense strand.
  • the double-stranded oligonucleotide agent operably linked to a blocking group or a ligand is operably linked to the blocking group at one end, and is operably linked to the ligand at another end. In some embodiments, the double-stranded oligonucleotide agent operably linked to a blocking group or a ligand is capable of inhibiting expression of a target gene via RNA interference.
  • the double-stranded oligonucleotide agent operably linked to a blocking group or a ligand comprises a sense strand and an anti-sense strand, wherein each strand has 14 to 30 nucleotides.
  • the anti-sense strand of the double-stranded oligonucleotide agent operably linked to a blocking group or a ligand is sufficiently complementary to part of a target RNA or an mRNA encoding a target gene, and the sense strand of the double-stranded oligonucleotide agent operably linked to a blocking group or a ligand is sufficiently complementary to the targeting region of the anti-sense strand.
  • the blocking group is conjugated to the 5’ end of the anti-sense strand.
  • the ligand is conjugated to the 5’ end of the anti-sense strand, 5’ end of the sense strand or 3’ end of the sense strand.
  • the anti-sense strand of the double-stranded oligonucleotide agent operably linked to a blocking group or a ligand is operably linked to the blocking group at one end, and is operably linked to the ligand at another end, and/or
  • the anti-sense strand of the double-stranded oligonucleotide agent operably linked to a blocking group or a ligand capable of silencing a target RNA or inhibiting expression of a target gene via RNA interference.
  • dsRNA double-stranded RNA
  • Formula (I) a structure represented by Formula (I) :
  • the sense strand consists of region A1, region A2 and X1 (from 5’ to 3’ ) ,
  • the anti-sense strand consists of region N, Z, Y, X2, region B2 and region B1 (from 5’ to 3’ ) , the region A1 is 0-15 nucleotides,
  • the region B1 is 0-15 nucleotides
  • region A2 and region B2 has the same length and is at least 1 nucleotide
  • region A2 and region B2 comprise a complementary double-stranded region
  • each of X1, X2, Y and Z is independently 1 nucleotide, wherein X1 and X2 can form a hydrogen bond,
  • the region N is at least 1 nucleotide
  • the total length of the sense strand is 17-23 nt
  • the total length of the anti-sense strand is at least 26 nt
  • the dsRNA comprises no more than 23 complementary base pairs
  • each of the nucleotides of the dsRNA is independently optionally modified.
  • the sense strand consists of region A1, region A2 and X1 (from 5’ to 3’ )
  • the anti-sense strand consists of region N, Z, Y, X2, region B2 and region B1 (from 5’ to 3’)
  • the region A1 is 1-6 nucleotides
  • the region B1 is 0-6 nucleotides
  • the region A2 and region B2 has the same length and is at least 1 nucleotide
  • the region A2 and region B2 comprise a complementary double-stranded region
  • each of X1, X2, Y and Z is independently 1 nucleotide, wherein X1 and X2 are complementary
  • the region N is at least 1 nucleotide
  • the total length of the sense strand is 17-23 nt
  • the total length of the anti-sense strand is at least 26 nt
  • the dsRNA comprises no more than 23 complementary base pairs
  • each of the nucleotides of the dsRNA is
  • each of X2 and Y is independently A, dA, U or dU, and Z is G or dG.
  • each of X2 and Y is independently A, dA, U or dU, Z is G or dG, and each of the nucleotides of the dsRNA is independently modified.
  • X2 is A or U, and Z is G.
  • X2 is A or U
  • Z is G
  • each of the nucleotides of the dsRNA is independently modified.
  • Y is A or U.
  • Y is A or U, and each of the nucleotides of the dsRNA is independently modified.
  • X2 is A or U and Y is A or U.
  • X2 is A or U
  • Y is A or U
  • each of the nucleotides of the dsRNA is independently modified.
  • Y is A.
  • Y is A, and each of the nucleotides of the dsRNA is independently modified.
  • X1 is A or U.
  • X1 is A, U or I.
  • X1 is A, U, G or I.
  • X1 is A, U or I, and each of the nucleotides of the dsRNA is independently modified.
  • At least 1 nucleotide of Y, Z and region N is modified.
  • the region N consists of 1 nucleotide and group X
  • group X is nucleotide, blocking group or a combination of nucleotide and blocking group
  • the blocking group is a structure resisting nuclease decomposition
  • the blocking group is linked to the region N at 5’ end, and the blocking group is independently optionally modified.
  • the blocking group is selected from inverted abasic deoxyribonucleotide or RX, wherein RX is
  • the region N consists of 1 nucleotide and 1 blocking group.
  • the 1 nucleotide and 1 blocking group of the region N are linked via phosphorothioate linkage.
  • the region N consists of 2 nucleotides and 1 blocking group.
  • the 2 nucleotides and 1 blocking group of the region N are linked via phosphorothioate linkage.
  • the region N consists of 1 nucleotide and 2 blocking groups.
  • the 1 nucleotide and 2 blocking groups of the region N are linked via phosphorothioate linkage.
  • the region N consists of 1 nucleotide and 2 blocking groups.
  • the 1 nucleotide and 2 blocking groups of the region N are linked via a linkage, wherein the linkage is via a single bond.
  • the 1 nucleotide and 2 blocking groups of the region N are linked via a linkage, wherein the linkage is via a phosphodiester, phosphorothioate or phosphorodithioate linkage.
  • all nucleotides of the dsRNA are independently modified.
  • the nucleotide modification comprises 1, 2 or more selected from the group consisting of base modifications, sugar ring modifications, phosphate skeleton modifications, and end modifications.
  • Base modifications include substitutions with a stabilized base, a destabilized base, or a base paired with extended partner libraries; base removal (an abasic nucleotide) , or a conjugated base.
  • Sugar modifications include modifications at the 2’ position, 3’ position, or 4’ position or sugar substitutions.
  • Phosphate skeleton modifications comprise modifications or replacement of a phosphodiester linkage.
  • End modifications comprise a 5’ end modifications (phosphorylation, conjugation, inverted linkage, etc. ) and 3’ end modifications (conjugation, DNA nucleotide, inverted linkage, etc. ) .
  • the nucleotide modification comprises 1, 2 or more selected from the group consisting of 2’ -OMe modification, 2’ -F modification, 2’ -deoxy modification, VP modification, 5’ -MP modification, PS modification, PS2 modification, MP modification, MOP modification, invAB modification, LNA modification, UNA modification and a modification enhancing the affinity of dsRNA and ARGO protein.
  • the modification enhancing the affinity of dsRNA and ARGO protein includes replacement of a nucleotide with
  • the nucleotide modification comprises 1, 2 or more selected from the group consisting of 2’ -OMe, 2’ -F, 2’ -deoxy, VP, 5’ -MP, PS, PS2, MP, MOP, invAB and a modification enhancing the affinity of dsRNA and ARGO protein.
  • the modification enhancing the affinity of dsRNA and ARGO protein is on X2.
  • the region A1 and the region B1 comprise a complementary double-strand region.
  • the region A1 is 1 nucleotide, and the region B1 is 3 nucleotides.
  • the length of region A2 or B2 is 20 nt.
  • the region A1 is 0 nucleotide, and the region B1 is 2 nucleotides.
  • the total length of the sense strand is 17-21 nt. In some embodiments, the total length of the sense strand is 18-21 nt.
  • the total length of the anti-sense strand is 26-35 nt. In some embodiments, the total length of the anti-sense strand is 26-32 nt. In some embodiments, the total length of the anti-sense strand is 26-28 nt. In some embodiments, the total length of the anti-sense strand is 26-27 nt. In some embodiments, the total length of the anti-sense strand is 26 nt, 27 nt, 28 nt, 29 nt or 30 nt.
  • the region A1 is 1-4 nucleotides.
  • the total length of the anti-sense strand is 26-27 nt, and the region A1 is 1-4 nucleotides.
  • the region N comprises inverted abasic deoxyribonucleotide (invAB) .
  • the total length of the anti-sense strand is 26-27 nt
  • the region A1 is 1-4 nucleotides
  • the region N comprises inverted abasic deoxyribonucleotide (invAB) .
  • Z and the region N is linked via a phosphorothioate linkage.
  • X2 and the region B2 is linked via a phosphorothioate linkage.
  • Z and the region N is linked via a phosphorothioate linkage
  • X2 and the region B2 is linked via a phosphorothioate linkage
  • the total length of the anti-sense strand is 26-27 nt
  • the region A1 is 1-4 nucleotides
  • Z and the region N is linked via a phosphorothioate linkage.
  • the total length of the anti-sense strand is 26-27 nt
  • the region A1 is 1-4 nucleotides
  • X2 and the region B2 is linked via a phosphorothioate linkage.
  • the total length of the anti-sense strand is 26-27 nt
  • the region A1 is 1-4 nucleotides
  • Z and the region N is linked via a phosphorothioate linkage
  • X2 and the region B2 is linked via a phosphorothioate linkage.
  • the total length of the anti-sense strand is 26-27 nt
  • the region A1 is 1-4 nucleotides
  • the region N comprises inverted abasic deoxyribonucleotide (invAB)
  • Z and the region N is linked via a phosphorothioate linkage.
  • the total length of the anti-sense strand is 26-27 nt
  • the region A1 is 1-4 nucleotides
  • the region N comprises inverted abasic deoxyribonucleotide (invAB)
  • X2 and the region B2 is linked via a phosphorothioate linkage.
  • the total length of the anti-sense strand is 26-27 nt
  • the region A1 is 1-4 nucleotides
  • the region N comprises inverted abasic deoxyribonucleotide (invAB)
  • Z and the region N is linked via a phosphorothioate linkage
  • X2 and the region B2 is linked via a phosphorothioate linkage.
  • the total length of the sense strand is 17-21 nt
  • the total length of the anti-sense strand is 26-27 nt
  • the region A1 is 1-4 nucleotides.
  • the total length of the sense strand is 17-21 nt, and the region N comprises inverted abasic deoxyribonucleotide (invAB) .
  • invAB inverted abasic deoxyribonucleotide
  • the total length of the sense strand is 17-21 nt
  • the region A1 is 1-4 nucleotides
  • the region N comprises inverted abasic deoxyribonucleotide (invAB) .
  • the total length of the anti-sense strand is 26-27 nt
  • the region A1 is 1-4 nucleotides
  • position 1 counting from its 5’ end of the region N is replaced with RX, wherein the RX is and Z and the region N is linked via a phosphorothioate linkage.
  • the total length of the anti-sense strand is 26-27 nt
  • the region A1 is 1-4 nucleotides
  • position 1 counting from its 5’ end of the region N is replaced with RX
  • the RX is and X2 and the region B2 is linked via a phosphorothioate linkage.
  • the total length of the anti-sense strand is 26-27 nt
  • the region A1 is 1-4 nucleotides
  • position 1 counting from its 5’ end of the region N is replaced with RX, wherein the RX is Z and the region N is linked via a phosphorothioate linkage
  • X2 and the region B2 is linked via a phosphorothioate linkage.
  • the total length of the sense strand is 17-21 nt
  • the region A1 is 1-4 nucleotides
  • position 1 counting from its 5’ end of the region N is replaced with RX, wherein the RX is
  • the total length of the sense strand is 17-21 nt
  • the total length of the anti-sense strand is 26-27 nt
  • the region A1 is 1-4 nucleotides
  • position 1 counting from its 5’end of the region N is replaced with RX, wherein the RX is
  • the total length of the sense strand is 17-21 nt, and Z and the region N is linked via a phosphorothioate linkage.
  • the total length of the sense strand is 17-21 nt, and X2 and the region B2 is linked via a phosphorothioate linkage.
  • the total length of the sense strand is 17-21 nt, Z and the region N is linked via a phosphorothioate linkage, and X2 and the region B2 is linked via a phosphorothioate linkage.
  • the total length of the sense strand is 17-21 nt
  • the total length of the anti-sense strand is 26-27 nt
  • the region A1 is 1-4 nucleotides
  • Z and the region N is linked via a phosphorothioate linkage.
  • the total length of the sense strand is 17-21 nt
  • the total length of the anti-sense strand is 26-27 nt
  • the region A1 is 1-4 nucleotides
  • X2 and the region B2 is linked via a phosphorothioate linkage.
  • the total length of the sense strand is 17-21 nt
  • the total length of the anti-sense strand is 26-27 nt
  • the region A1 is 1-4 nucleotides
  • Z and the region N is linked via a phosphorothioate linkage
  • X2 and the region B2 is linked via a phosphorothioate linkage.
  • the total length of the sense strand is 17-21 nt
  • the total length of the anti-sense strand is 26-27 nt
  • the region A1 is 1-4 nucleotides
  • the region N comprises inverted abasic deoxyribonucleotide (invAB)
  • Z and the region N is linked via a phosphorothioate linkage.
  • the total length of the sense strand is 17-21 nt
  • the total length of the anti-sense strand is 26-27 nt
  • the region A1 is 1-4 nucleotides
  • the region N comprises inverted abasic deoxyribonucleotide (invAB)
  • X2 and the region B2 is linked via a phosphorothioate linkage.
  • the total length of the sense strand is 17-21 nt
  • the total length of the anti-sense strand is 26-27 nt
  • the region A1 is 1-4 nucleotides
  • the region N comprises inverted abasic deoxyribonucleotide (invAB)
  • Z and the region N is linked via a phosphorothioate linkage
  • X2 and the region B2 is linked via a phosphorothioate linkage.
  • positions 1 and 2 and positions 2 and 3 counting from its 5’ end and 3’end of the sense strand and the anti-sense strand are independently optionally linked via a phosphorothioate linkage.
  • positions 1 and 2 and positions 2 and 3 counting from its 5’ end and 3’end of the sense strand and the anti-sense strand are linked via a phosphorothioate linkage.
  • a dsRNA conjugate comprising a dsRNA disclosed herein and a delivery system, wherein the delivery system is capable to deliver the dsRNA to a target RNA and thereby provide RNA interference effect, the delivery system is conjugated with the dsRNA, and the dsRNA conjugate comprises the delivery system is 1 or more.
  • a delivery system is selected from the group consisting of a liposome, lipid nanoparticle (LNP) , lipid complex, lipid polymeric complex (LPP) , mannose delivery system, N-acetylgalactosamine (GalNAc) conjugated delivery system, Apelin receptor targeting delivery system, integrin receptor targeting delivery system, peptide, antibody and any combination thereof.
  • the delivery system is conjugated at the 3’ end of the sense strand.
  • the delivery system is conjugated at the 5’ end of the sense strand.
  • the delivery system is conjugated at the 3’ end of the anti-sense strand.
  • the delivery system is conjugated at the 5’ end of the anti-sense strand.
  • the delivery system is conjugated at a nucleotide which is not at an end of the dsRNA.
  • composition comprising a dsRNA disclosed herein and a pharmaceutically acceptable carrier or excipient.
  • dsRNA, dsRNA conjugate or composition disclosed herein in the manufacturing of a medicament for treatment of a disease or disorder associated with RNA interference of a gene expression.
  • the gene is selected from the group consisting of AGT, complement factor B, DGAT2, DUX, ANGPTL8, APOC3, F12, INHBE, PNPLA3, Serpinc1, APP, SOD1, TMPRSS6, KHK, PCSK9, VEGFA, ANGPTL3, ANGPTL4, C3, C5, TTR, IGF-1R, VEGFR, ANG2, GIPR, GPR75, ActRII, NUDT21, PLN, HSD17b13, CNOT6L, PTP1B, CFHR, ATX, CIDEB, mARC1, TSHR, CB1 and LPA.
  • the gene is selected from the group consisting of AGT, complement factor B, DGAT2, SOD1, KHK, APP, DUX, ANGPTL3, ANGPTL4 and ANGPTL8.
  • dsRNA, dsRNA conjugate or composition disclosed herein for use of treating a disease or disorder associated with RNA interference of a gene expression.
  • Embodiment I-1 A double-stranded RNA (dsRNA) , comprising a sense strand and an anti-sense strand, of a structure represented by Formula (I) :
  • the sense strand consists of region A1, region A2 and X1 (from 5’ to 3’ ) ,
  • the anti-sense strand consists of region N, Z, Y, X2, region B2 and region B1 (from 5’ to 3’ ) ,
  • the region A1 is 0-15 nucleotides
  • the region B1 is 0-15 nucleotides
  • region A2 and region B2 are of equal length which is at least 1 nucleotide
  • region A2 and region B2 comprise a double-stranded region that forms reverse complements, each of X1, X2, Y and Z is independently 1 nucleotide, wherein X1 and X2 can associate with a hydrogen bond,
  • the region N is at least 1 nucleotide
  • the total length of the sense strand is 17-23 nt
  • the total length of the anti-sense strand is at least 26 nt
  • the dsRNA comprises no more than 23 complementary base pairs
  • each of the nucleotides of the dsRNA is independently optionally modified.
  • Embodiment I-2 The dsRNA of Embodiment I-1, wherein X2 is A or U, and Z is G.
  • Embodiment I-3 The dsRNA of Embodiment I-2, wherein Y is A or U.
  • Embodiment I-4 The dsRNA of Embodiment I-3, wherein Y is A.
  • Embodiment I-5 The dsRNA of any one of Embodiments I-1 to I-4, wherein the region N consists of 1 nucleotide and group X, wherein the group X is nucleotide, blocking group or a combination of nucleotide and blocking group, wherein the blocking group is a structure capable of resisting nuclease degradation, the blocking group is linked to the region N at 5’ end, and the blocking group is independently optionally modified.
  • Embodiment I-6 The dsRNA of Embodiment I-5, wherein the blocking group is selected from inverted abasic deoxyribonucleotide or RX, wherein RX is
  • Embodiment I-7 The dsRNA of Embodiment I-6, wherein the region N consists of 1 nucleotide and 1 blocking group.
  • Embodiment I-8 The dsRNA of Embodiment I-7, wherein the 1 nucleotide and 1 blocking group of the region N are linked via phosphorothioate linkage.
  • Embodiment I-9 The dsRNA of any one of Embodiments I-1 to I-8, wherein all nucleotides of the dsRNA are independently modified.
  • Embodiment I-10 The dsRNA of any one of Embodiments I-1 to I-8, wherein the nucleotide modification comprises 1, 2 or more selected from the group consisting of 2’ -OMe, 2’-F, 2’ -deoxy, VP, 5’ -MP, PS, PS2, MP, MOP, invAB and a modification enhancing the affinity of dsRNA and ARGO protein.
  • Embodiment I-11 The dsRNA of Embodiment I-10, wherein the modification enhancing the affinity of dsRNA and ARGO protein is on X2.
  • Embodiment I-12 The dsRNA of any one of Embodiments I-1 to I-11, wherein the total length of the sense strand is 17-21 nt; further, the total length of the sense strand is 18-21 nt.
  • Embodiment I-13 The dsRNA of any one of Embodiments I-1 to I-11, wherein the total length of the anti-sense strand is 26-35 nt; further, the total length of the anti-sense strand is 26-27 nt.
  • Embodiment I-14 The dsRNA of any one of Embodiments I-1 to I-11, wherein the region A1 and the region B1 comprise a complementary double-strand region.
  • Embodiment I-15 The dsRNA of Embodiment I-14, wherein the region A1 is 1 nucleotide, and the region B1 is 3 nucleotides.
  • Embodiment I-16 The dsRNA of Embodiment I-15, wherein the length of region A2 or B2 is 20 nt.
  • Embodiment I-17 The dsRNA of any one of Embodiments I-1 to I-11, wherein the region A1 is 0 nucleotide, and the region B1 is 2 nucleotides.
  • Embodiment I-18 Use of the dsRNA of any one of Embodiments I-1 to I-17 or a pharmaceutical composition thereof in the manufacturing of a medicament for treatment of a disease or disorder associated with RNA interference of a gene expression; further, the gene is selected from the group consisting of AGT, complement factor B, DGAT2, DUX, ANGPTL8, APOC3, F12, INHBE, PNPLA3, Serpinc1, APP, SOD1, TMPRSS6, KHK, PCSK9, VEGFA, ANGPTL3, ANGPTL4, C3, C5, TTR, IGF-1R, VEGFR, ANG2, GIPR, GPR75, ActRII, NUDT21, PLN, HSD17b13 and LPA.
  • a method of modulating the expression of a target gene in a subject in need thereof comprising administering to the subject a pharmaceutically effective amount of a double-stranded oligonucleotide agent disclosed herein or a pharmaceutical composition disclosed herein.
  • a method of inhibiting the expression of a target gene in a subject in need thereof comprising administering to the subject a pharmaceutically effective amount of a double-stranded oligonucleotide agent disclosed herein or a pharmaceutical composition disclosed herein.
  • a method of treating a disease or disorder in a subject in need thereof, by modulating (e.g., inhibiting) the expression of a target gene for example comprising administering to the subject a pharmaceutically effective amount of a double-stranded oligonucleotide agent disclosed herein or a pharmaceutical composition disclosed herein.
  • a method of treating a disease or disorder in a subject in need thereof comprising administering to the subject a pharmaceutically effective amount of a double-stranded oligonucleotide agent disclosed herein or a pharmaceutical composition disclosed herein.
  • the disease or disorder is associated with the target gene. In some embodiments the disease or disorder is associated with the overexpression or activation of the target gene.
  • the target gene includes, but not limited to, AGT, CFB, DGAT2, DUX, ANGPTL8, APOC3, F12, INHBE, PNPLA3, Serpinc1, APP, SOD1, TMPRSS6, KHK, PCSK9, VEGFA, ANGPTL3, ANGPTL4, C3, C5, TTR, IGF-1R, VEGFR, ANG2, GIPR, GPR75, ActRII, NUDT21, PLN, HSD17b13, CNOT6L, PTP1B, CFHR, ATX, CIDEB, mARC1, TSHR, CB1 and LPA.
  • the double-stranded oligonucleotide agent disclosed herein or a pharmaceutical composition disclosed herein can be administered to a subject by a variety of routes, depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal) , oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or intraventricular administration.
  • the route and site of administration may be chosen to enhance targeting.
  • intramuscular injection into the muscles of interest would be a logical choice.
  • Lung cells might be targeted by administering the oligonucleotide in aerosol form.
  • the defined amount can be an amount effective to treat or prevent a disease or disorder, e.g., a disease or disorder associated with the target RNA.
  • the unit dose for example, can be administered by injection (e.g., intravenous, subcutaneous or intramuscular) , an inhaled dose, or a topical application.
  • the unit dose is administered less frequently than once a day, e.g., less than every 2, 4, 8 or 30 days.
  • the unit dose is not administered with a frequency (e.g., not a regular frequency) .
  • the unit dose may be administered a single time.
  • the effective dose is administered with other traditional therapeutic modalities.
  • the effective dose can be administered in a single dose or in two or more doses, as desired or considered appropriate under the specific circumstances. If desired to facilitate repeated or frequent infusions, implantation of a delivery device, e.g., a pump, semi-permanent stent (e.g., intravenous, intraperitoneal, intracisternal or intracapsular) , or reservoir may be advisable.
  • compositions comprise the oligonucleotide agent (e.g., a pharmaceutically effective amount of the oligonucleotide agent) disclosed herein, taken alone or formulated together with one or more pharmaceutically acceptable carriers (additives) , excipient and/or diluents.
  • compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions) , tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; (3) topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8) nasally. Delivery using subcutaneous or intravenous methods can be particularly advantageous.
  • the pharmaceutically-acceptable carrier can be a composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid) , or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
  • manufacturing aid e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid
  • solvent encapsulating material involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium state, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (1
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.
  • a formulation of the present invention comprises an excipient selected from the group consisting of cyclodextrins, celluloses, liposomes, micelle forming agents, e.g., bile acids, and polymeric carriers, e.g., polyesters and polyanhydrides; and an oligonucleotide disclosed herein.
  • a formulation renders orally bioavailable for an oligonucleotide agent disclose herein.
  • the double-stranded oligonucleotide agents can be formulated in combination with another agent, e.g., another therapeutic agent or a stabilizing agent, e.g., a protein that complexes with double-stranded oligonucleotide agent.
  • another agent e.g., another therapeutic agent or a stabilizing agent, e.g., a protein that complexes with double-stranded oligonucleotide agent.
  • Still other agents include chelators, e.g., EDTA (e.g., to remove divalent cations such as Mg 2+ ) , salts, RNAse inhibitors (e.g., a broad specificity RNAse inhibitor such as RNAsin) and so forth.
  • Methods of preparing these formulations or compositions include the step of bringing into association an oligonucleotide agent disclose herein with the carrier and, optionally, one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association an oligonucleotide disclosed herein with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
  • the absorption of the drug in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
  • An oligonucleotide agent disclose herein may be formulated for administration in any convenient way for use in human or veterinary medicine, by analogy with other pharmaceuticals.
  • nucleotide encompasses a ribonucleotide and deoxyribonucleotide.
  • G ribonucleotide
  • C ribonucleotide
  • A cytosine
  • T deoxyribonucleotide
  • U nucleotide containing guanine, cytosine, adenine, thymine and uracil, respectively.
  • dA stands for deoxyadenosine
  • dG stands for deoxyguanosine
  • dT stands for deoxythymidine
  • dU stands for deoxyuridine
  • dC stands for deoxycytidine
  • A stands for adenosine
  • G stands for guanosine
  • T stands for ribothymidine or 5-methyluridine
  • U stands for uridine
  • C stands for cytidine.
  • f when “f” is marked after A, U, C, G, and T, it strands for a nucleotide modified with 2’ -fluoro.
  • Af stands for 2’ -fluoroadenosine
  • Gf stands for 2’ -fluoroguanosine
  • Tf stands for 2’ -fluororibothymidine or 5-methyl-2’ -fluorouridine
  • Uf stands for 2’ -fluorouridine
  • Cf stands for 2’ -fluorocytidine.
  • a lowercase letter such as “a” , “u” , “c” , “g” , “t” and “i” stands for 2’ -methoxy modified A, U, C, G, T, and I, respectively.
  • a stands for 2’ -methoxyadenosine
  • g stands for 2’ -methoxyguanosine
  • t stands for 2’ -methoxyribothymidine or 5-methyl-2’ -methoxyuridine
  • u stands for 2’ -methoxyuridine
  • c stands for 2’ -methoxycytidine.
  • C16 or C16 modification refers to 2’ -O-C16 modification or to 2’ -C16 modification.
  • C16 When “C16” marked before A, U, C, G or T, it stands for a nucleotide modified with 2’ -hexadecyloxy.
  • C16U stands for
  • C16 (dU) stands for
  • GNA-G, GNA-A, GNA-C, GNA-U, and GNA-T have the following structures.
  • “*” between two nucleotides such as A, U, C, G and T stands for a phosphorothioate internucleotide linkage, i.e., the two nucleotides are linked via a phosphorothioate linkage.
  • the absence of “*” between two nucleotides such as A, U, C, G and T stands for an unmodified internucleotide linkage (i.e., phosphodiester linkage) .
  • a sequence of “5’ -AdUgCf*T-3’ ” stands for a sequence, wherein starting from the 5’ end, position 1 is adenosine, position 2 is deoxyuridine, position 3 is 2’ -methoxyguanosine, position 4 is 2’ -fluorocytidine, position 5 is ribothymidine, the position 4 and position 5 are linked via a phosphorothioate linkage and the other adjacent positions are linked via a phosphodiester linkage.
  • invAB or “invAB modification” refers to a nucleotide conjugated (e.g., conjugated at 5’ end or 3’ end) with an inverted abasic deoxyribonucleotide. For example, if a nucleotide has a structure of the corresponding invAB modified nucleotide has a structure of
  • invAb or “invAb modification” refers to replace a nucleotide with an inverted abasic deoxyribonucleotide. For example, if a nucleotide has a structure of the corresponding invAb modified nucleotide has a structure of
  • VP or “VP modification” refers to a nucleotide with (E) -vinylphosphate modification (e.g., 5’ - (E) -vinylphosphate modification) .
  • E e.g., 5’ - (E) -vinylphosphate modification
  • VP modified U, u, dU and Uf have the following structures.
  • M03 or “M03 modification” refers to a nucleotide conjugated (e.g., conjugated at 5’ end or 3’ end) with a molecule having a structure of For example, if a nucleotide has a structure of the corresponding M03 modified nucleotide has a structure of In some embodiments, the conjugation with M03 is by the reaction of a nucleotide and
  • M06 or “M06 modification” refers to a nucleotide conjugated (e.g., conjugated at 5’ end or 3’ end) with a molecule having a structure of For example, if a nucleotide has a structure of the corresponding M06 modified nucleotide has a structure of In some embodiments, the conjugation with M06 is by the reaction of a nucleotide and
  • a sequence of “5’ - (M06) *AdTgCf* (invAB) -3’ ” stands for an oligonucleotide having a structure of
  • a sequence of “5’ - (M06) *AdTgCf* [L96] -3’ ” stands for an oligonucleotide having a structure of
  • Oligonucleotide synthesis was performed on a MerMade 12 synthesizer using standard solid-phase synthesis protocols. All phosphoramidites were purchased from Hongene Biotech, including 2'-OMe-rA (Bz) , 2'-OMe-rC (Ac) , 2'-OMe-rU, 2'-OMe-rG (iBu) , and 2'-F-rA (Bz) , 2'-F-rC (Ac) , 2'-F-rU, 2'-F-rG (iBu) .
  • the crude oligonucleotide solution was concentrated by centrifugal evaporation under elevated temperature (45 °C) and reduced pressure (5.6 Torr) for 8 h to give crude oligo as a solid.
  • the resulting solid was subjected to prep-HPLC purification. The appropriate fractions were pooled and lyophilized to give the purified product.
  • the single strand oligonucleotides to be annealed were prepared with sterile RNase Free H 2 O.
  • the annealing reaction system The mixture was placed in a 95 °C water bath for 10 minutes (for ⁇ 100 nmol, 20 minutes) , then it was quickly put into a 60 °C water bath and allowed to cool, then lyophilized to give double-stranded oligonucleotides and stored at low temperature.
  • Complementary double-stranded oligonucleotides were prepared by combining equimolar single-stranded oligonucleotides solutions.
  • the expression levels of the target genes were quantified using qPCR with gene-specific primers (e.g., with commercially available Assays or primers shown below) .
  • GAPDH was measured as housekeeping gene in parallel.
  • the samples were processed in an Applied Biosystems Fast Real-Time PCR system for TaqMan cycling, cycle according to the thermal profiles below: 95°C for 10 min, then cycling at 95°C for 15 sec, 60°C for 1 min for 40 cycles.
  • the dsRNA agent disclosed herein displayed potent target gene expression inhibitory activity.
  • Crossing dsRNA agents targeting different genes, dsRNA agents with a purine at Z displayed better inhibitory activities than a pyrimidine, more preferably, guanine at Z. It can be seen that dsRNA agents with adenine or uracil at X1/X2 displayed potent inhibitory activity, while dsRNA agents with cytosine or guanine at X1/X2 displayed relatively modest activity.
  • the dsRNA agents with blocking groups e.g., M03, M06 or invAB
  • ligand e.g., VSDL-03A
  • the blocking group or ligand is cleaved together with the cleavage region of the dsRNA disclosed herein before provide RNAi effect.
  • the primary human hepatocytes (PHH) mixed with the appropriate medium, and then adjusted the cell suspension to the final cell density of 6 ⁇ 10 5 cells/mL.
  • the dsRNA was diluted and added 10 ⁇ L/well to the collagen-I coated 96-well plate, and followed by addition of cell suspension (90 ⁇ L/well) .
  • the cells were cultured at 37°C and 5%CO 2 for 48 hours.
  • the expression levels of the target genes were quantified using qPCR with gene-specific primers (e.g., with commercially available Assays or primers shown below) .
  • GAPDH was measured as housekeeping gene in parallel.
  • the samples were processed in an Applied Biosystems Fast Real-Time PCR system for TaqMan cycling, cycle according to the thermal profiles below: 95°C for 10 min, then cycling at 95°C for 15 sec, 60°C for 1 min for 40 cycles.
  • Table 7 Test Compounds knock-down activity in primary human hepatocyte free-uptake assay
  • a cleavage region with both 2’ -F and 2’ -OMe displayed potent inhibitory activity.
  • X2 and Y are 2’ -OMe modified
  • Z and N1 are 2’ -F modified.
  • internucleotide linkage modification between X2 and Y or Y and Z would reduce the inhibitory activity of the dsRNA.
  • the dsRNA agents disclosed herein with a 5’ extension on the anti-sense strand and a cleavage region displayed relatively more potent inhibitory activity against different genes and targets, in comparison with parent dsRNA agents without the 5’ extension.
  • mice (BALB/C, 6-7 weeks, female) were dosed at Day 0 with vehicle or the test siRNA (5mg/kg) .
  • injection volume (mL) mouse body weight (g) ⁇ 8%) .
  • the mass of the plasmid injected into each mouse was 10 ⁇ g.
  • All animals were sacrificed at Day 4.
  • Liver tissues from all groups were taken for target mRNA analysis via qPCR method.
  • the RNA was reverse transcribed to cDNA with III RT SuperMix for qPCR (+gDNA wiper) (Vazyme-R323) following the manual.
  • the cDNA was quantified by qPCR.
  • NEO The sequence information is as follows in the table) mRNA were detected in parallel as an internal control.
  • the dsRNA agents disclosed herein with a 5’ extension on the anti-sense strand and a cleavage region displayed significantly more potent inhibitory efficacy against target gene, in comparison with parent dsRNA agents without the 5’ extension.
  • Testing compounds were formulated at up to 20 mg/mL in 10 mM PBS (pH 7.4) and administered as 50 ⁇ L IT injections (0.9 mg/dose) by lumbar puncture. Animal tail flapping or tail tip shaking were used as the marker for successful operation. After dosing, remove the anesthesia maintenance device and put the animal back into the cage. Animals were sacrificed at Day14, and tissue samples were taken for qPCR analysis.
  • RNAs from tissue samples were extracted by Automated Nucleic Acid Extraction Systems . Transfer the sample to a fresh RNase-free tube for cDNA synthesis and qPCR.
  • the dsRNA agents disclosed herein with a 5’ extension on the anti-sense strand and a cleavage region and VSDL ligands displayed comparable inhibitory efficacy against target gene, in comparison with parent dsRNA agents with the 5’ VPu modification and C16U.
  • the dsRNA agents disclosed herein displayed more tissue-specific target knockdown compared to dsRNA agents with the 5’ VPu modification and C16U.
  • a topical antibiotic (tobramycin) was applied to both eyes twice on the day before and twice on the day after IVT injection to mice.
  • 1 ⁇ PBS or siRNA formulated in 1 ⁇ PBS was administered in both eyes by IVT injection at 3 ⁇ g per dose. All animals were sacrificed on Day 7, and whole eye were sampled for qPCR analysis.
  • the dsRNA agents disclosed herein with a 5’ extension on the anti-sense strand and a cleavage region and VSDL ligand displayed stronger inhibitory efficacy against target gene, in comparison with parent dsRNA agents with the 5’ VPu modification and C16U.
  • Results were shown in Fig. 5. After 48 h incubation, the observed products include unconsumed Test Compound (31%) , Test Compound with one nucleotide cleaved at 3’ end (15%) , Test Compound with two nucleotides cleaved at 3’ end and M06 cleaved (18%) , Test Compound with three nucleotides cleaved at 5’ end (15%+5%+10%) and other minor metabolites (5%) .
  • the only observed 5’ cleavage product is Test Compound with three nucleotides cleaved at 5’ end;
  • the observed 3’ cleavage products include Test Compound with one or two nucleotides cleaved at 3’ end; and
  • the cleavage products of Test Compound with three nucleotides cleaved at 5’ end is the major product among all cleavage products.
  • In vivo target knockdown effect of modified siRNA was evaluated in cynomolgus monkey.
  • Circulating AGT levels were quantified using an ELISA specific for human angiotensinogen (and cross-reactive with cynomolgus) , according to manufacturer's protocol (Sino Biological KIT10994) . Data were expressed as percent of baseline value and presented as mean plus/minus standard error of the mean.
  • Kidney Presence of mild basophilic granules and tubular cell vacuolation in both groups.
  • Liver mild to modest vacuolation and pigmented hepatocytes and Kupffer cells in both groups.
  • Injection Site Minimal to moderate mixed cell inflammation.

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Abstract

L'invention concerne un agent oligonucléotidique double brin et ses utilisations. L'agent oligonucléotidique double brin comprend un site de clivage spécifique pour une endonucléase, l'agent oligonucléotidique double brin clivé étant capable d'inhiber l'expression du gène cible par interférence ARN (ARNi).
PCT/CN2024/106419 2023-07-21 2024-07-19 Agents oligonucléotidiques double brin et utilisations associées Pending WO2025021034A1 (fr)

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CN202311532296 2023-11-16
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* Cited by examiner, † Cited by third party
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