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WO2025201529A1 - Composés pour synthétiser des molécules de petit arn activateur modifié, molécules de petit arn activateur modifié et leurs utilisations - Google Patents

Composés pour synthétiser des molécules de petit arn activateur modifié, molécules de petit arn activateur modifié et leurs utilisations

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Publication number
WO2025201529A1
WO2025201529A1 PCT/CN2025/085845 CN2025085845W WO2025201529A1 WO 2025201529 A1 WO2025201529 A1 WO 2025201529A1 CN 2025085845 W CN2025085845 W CN 2025085845W WO 2025201529 A1 WO2025201529 A1 WO 2025201529A1
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Prior art keywords
alkyl
alkylene
substituted
compound
modified
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English (en)
Inventor
Fulu ZHAO
Zubao GAN
Moorim KANG
Longcheng Li
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Ractigen Therapeutics
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Ractigen Therapeutics
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Publication of WO2025201529A1 publication Critical patent/WO2025201529A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • 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

Definitions

  • Oligonucleotide-based therapeutics have emerged as a promising technology due to their ability to regulate the expression of a wide range of target genes.
  • the administration of oligonucleotides to patients, organs, tissues, or cells can elicit various biochemical reactions, allowing for functions such as gene silencing, inhibition, activation, and modulation.
  • PK pharmacokinetics
  • PD pharmacodynamics
  • chemical modifications have been explored for enhancing the drug properties of oligonucleotides. These modifications can be applied to the nucleosides, backbones, bases, and terminals of the oligonucleotides.
  • the present disclosure provides a compound having a structure of Formula 1.
  • the compound can be used for synthesizing a modified saRNA to enhance gene expression modulation activities of saRNA both in vitro and in vivo.
  • a saRNA which comprises a sense strand comprising a first nucleotide sequence, and an antisense strand comprising a second nucleotide sequence.
  • the first nucleotide sequence can be 15 to 30 nucleotides in length and has at least about 70%identity to a corresponding target gene sequence.
  • the second nucleotide sequence can be 15 to 30 nucleotides in length and has at least about 70%complementarity to the corresponding target gene sequence.
  • Also provided herein is a method of modulating the expression of a target gene, the method comprising contacting a cell with the pharmaceutical composition of the present disclosure.
  • FIG. 1 shows the activity of chemically modified saRNAs (CM-saRNAs) with (E) -deuterated viny1 phosphonate (VPD) modification (referred to as “VPD-saRNAs” ) on the expression of SERPING1 mRNA in transformed human liver epithelial-2 cells (THLE-2 cells) .
  • CM-saRNAs chemically modified saRNAs
  • VPD-saRNAs -deuterated viny1 phosphonate
  • SERPING1 mRNA SERPING1 mRNA in transformed human liver epithelial-2 cells (THLE-2 cells) .
  • CM-saRNA (s) with (E) -vinyl phosphonate (VP) modification are referred to as “VP-saRNA (s) ” .
  • SERPING1 mRNA levels were quantified by two step RT-qPCR using a gene specific primer set in each of the PCR reactions. Geometric means of the mRNA levels of HPRT1 and TBP were served as an internal control. Data represents mean expression levels of SERPING1 relative to Mock treatment after normalized to HPRT1 and TBP (mean ⁇ SEM of two replicated transfection wells) .
  • FIG. 2 shows the activity of saRNAs on the expression of human UTRN (hUTRN or UTRN) mRNA in human malignant embryonic rhabdomyoma cells (RD cells) .
  • the indicated saRNAs i.e., RD-12027, RD-12028, RD-12031, RD-12033, RD-12034, RD-12299, RD-12303, RD-12304, RD-12305, RD-12313 and RD-12316
  • their corresponding newly designed saRNAs i.e., RD-13587, RD-13580, RD-13584, RD-13716, RD-13720, RD-13629, RD-13727, RD-13632, RD-13732, RD-13737 and RD-13740
  • FIGs. 4A and 4B show the UTRN mRNA levels as quantified by two step RT-qPCR using a gene specific primer set in each of the PCR reactions. TBP was amplified as an internal control. Data represents mean expression levels of UTRN mRNA relative to Mock treatment after normalized to TBP (mean ⁇ SEM of two replicated transfection wells) .
  • CML-saRNA i.e., RD-15637
  • VPL-saRNAs i.e., RD-15638 and RD-15639
  • RD cells were transfected into RD cells at the indicated concentrations (i.e., 0.024, 0.1, 0.4, 1.56, 6.25, 25, 100 and 200 nM) for 3 days.
  • Cells were transfected in absence of oligonucleotide as Mock treatments (not shown) .
  • dsCon2M8 served as a non-targeting duplex control (not shown) .
  • UTRN mRNA levels were quantified by two step RT-qPCR using a gene specific primer set in each of the PCR reactions.
  • FIGs. 9A-9E show the activity of VPDL-saRNA in inducing Utrn mRNA expression in hUTRNp (KI/KI) ⁇ mdx male mice.
  • the indicated VPDL-saRNA i.e., RD-18997
  • hUTRNp KI/KI
  • mdx male mice via SC injection for a total of five doses (50 mg/kg per dose) on day 0, 2, 4, 7 and 14.
  • the male mice were sacrificed at day 28 post first dosing.
  • FIGs. 10A-10G show the concentration and biodistribution of VPDL-saRNA across various tissues in C57BL/6J male mice.
  • the indicated VPDL-saRNA (i.e., RD-18997) was administered to C57BL/6J male mice at a single dose of 50 mg/kg via SC injection on day 0. Mice were sacrificed at 5 min, 2 hr, and on day 1, 7, 15, 30, 45, and 60 post-dosing. Two mice were sacrificed prior to dosing and served as negative controls. Plasma, heart, liver, kidney, platysma, diaphragm, and gluteus tissues were collected at the corresponding time points. FIGs. 10A-1 0G showed the concentrations of RD-18997 as quantified in these tissues via LC-MS/MS analysis.
  • the black dashed line indicated the lower limit of quantitation (LLOQ) for the LC-MS/M S assay.
  • the LLOQ was 10 ng/g for heart, liver, kidney, platysma, diaphragm, and gluteus tissue.
  • the LLOQ was 25 ng/mL for plasma.
  • Oligonucleotides offer great potential for prevention or treatment of a variety of diseases, disorders or conditions by modulating, e.g., up-regulating or down-regulating protein expression of disease-associated genes and their variants.
  • saRNAs as up-regulators have become an emerging class of therapeutic agents and under active development.
  • the newly developed compounds of the present application when used as a moiety of a saRNA, can significantly enhance gene expression modulation activities of the saRNA both in vitro and in vivo, as compared to saRNAs modified in a conventional manner of the prior art, and thus may improve bioactive and pharmacological properties (e.g., biodistribution, bioavailability, pharmacokinetics, activity, potency, etc. ) .
  • the improvement of bioactive properties may be caused by improved cell uptake, higher potency, and longer duration/half-life.
  • the improvement of pharmacological properties may also lead to lower toxicity, lower dose, less frequent administrations, and less undesired immune responses.
  • the present compounds, and thus the resultant saRNAs involve a simpler synthesis process and thus have better processability in manufacturing.
  • any numerical ranges include any numerical values in the range (including the upper and lower values) , and any sub-ranges in the range.
  • the range from 1 to 3 may include any of the numerical values 1, 2 and 3, as well as sub-ranges from 1 to 2 and from 2 to 3.
  • oligonucleotide As used herein, the terms “oligonucleotide” , “polynucleotide” or “oligo” are interchangeable and refer to polymers of nucleotides, and particularly refer to single-stranded nucleic acid molecules of DNA, RNA, or DNA/RNA hybrid, oligonucleotide strands containing regularly and irregularly alternating deoxyribosyl portions and ribosyl portions, as well as modified and naturally or unnaturally existing frameworks for such oligonucleotides, such as a phosphorodiamidate morpholino oligomer (PMO) .
  • PMO phosphorodiamidate morpholino oligomer
  • the oligonucleotide for activating target gene transcription described herein can be or may comprise a small activating nucleic acid molecule (saRNA) .
  • the term “complementary” refers to the capability of forming base pairs between two oligonucleotide strands.
  • the base pairs are generally formed through hydrogen bonds between nucleotides in the antiparallel oligonucleotide strands.
  • the bases of the complementary oligonucleotide strands can be paired in the Watson-Crick manner (such as A to T, A to U, and C to G) or in any other manner allowing the formation of a duplex (such as Hoogsteen or reverse Hoogsteen base pairing) .
  • Complementarity includes complete complementarity and incomplete complementarity.
  • “Complete complementarity” or “100%complementarity” means that each nucleotide from the first oligonucleotide strand can form a hydrogen bond with a nucleotide at a corresponding position in the second oligonucleotide strand in the double-stranded region of the double-stranded oligonucleotide molecule, with no base pair being “mispaired” .
  • Incomplete complementarity means that not all the nucleotide units of the two strands are bound with each other by hydrogen bonds.
  • oligonucleotide strand , “strand” and “oligonucleotide sequence” as used herein can be used interchangeably, referring to a generic term for short nucleotide sequences having less than 35 bases (including nucleotides in deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) ) .
  • the length of a strand can be any length from 15 to 35 nucleotides.
  • target gene can refer to nucleic acid sequences, transgenes, viral or bacterial sequences, chromosomes or extrachromosomal genes that are naturally present in organisms, and/or can be transiently or stably transfected or incorporated into cells and/or chromatins thereof.
  • the target gene can be a protein-coding gene or a non-protein-coding gene (such as a microRNA gene and a long non-coding RNA gene) .
  • the target gene generally contains a promoter sequence, and the positive regulation for the target gene can be achieved by designing a saRNA having sequence identity (also called homology) to the promoter sequence, characterized as the up-regulation of expression of the target gene.
  • Target sequence or “target site” used interchangeably refers to a sequence fragment in the sequence of a target gene, such as, a target gene promoter, which is homologous or complementary with a sense strand or an antisense strand of a saRNA.
  • the target gene can also include one or more regulatory elements where one or more saRNA are designed to have sequence identity to a regulatory element.
  • Non-limiting examples of one or more regulatory elements include: a promoter, an enhancer, a silencer, an insulator, a TATA box, a GC box, a CAAT box, a transcriptional start site, a DNA binding motif ofa transcription factor or other protein that regulates transcription, and a 5'untranslated region.
  • guide strand or “G strand” refers to a strand in a small RNA duplex that assembles with the argonaute (AGO) protein.
  • the other strand partially or completely complementary to the guide strand is called “passenger strand” or “P strand” .
  • the strand carrying the complementary sequence to the target is the antisense strand and, if properly designed, will be preferentially chosen to be the guide strand.
  • the passenger strand is the sense strand.
  • antisense strand is guide strand and sense strand is passenger strand.
  • sense strand is guide strand and antisense strand is passenger strand.
  • saRNA duplex refers to the strand having sequence homology or sequence identity with a fragment of the coding strand of the sequence of a target gene.
  • antisense strand of a saRNA in the saRNA duplex refers to the strand being sequence complementary to the sense strand. Said antisense strand may interact with a target region of the target gene to activate or up-regulate gene expression, said target region may be a segment of the coding strand of the sequence of a target gene.
  • coding strand refers to a DNA strand in the target gene which cannot be used for transcription, and the nucleotide sequence of this strand is the same as that of an RNA produced from transcription (in the RNA, T in DNA is replaced by U) .
  • the coding strand of the double-stranded DNA sequence of the target gene promoter described herein refers to a promoter sequence on the same DNA strand as the DNA coding strand of the target gene.
  • template strand refers to the other strand complementary with the coding strand in the double-stranded DNA of the target gene, i.e., the strand that, as a template, can be transcribed into RNA, and this strand is complementary with the transcribed RNA (A to U and G to C) .
  • RNA polymerase binds to the template strand, moves along the 3′ ⁇ 5′ direction of the template strand, and catalyzes the synthesis of the RNA along the 5′ ⁇ 3′ direction.
  • the template strand of the double-stranded DNA sequence of the target gene promoter described herein refers to a promoter sequence on the same DNA strand as the DNA template strand of the target gene.
  • overhang refers to non-base-paired nucleotides at the terminus (5′ or 3′) of an oligonucleotide strand, which is formed by one strand extending out of the other strand in a double-stranded oligonucleotide.
  • a single-stranded region extending out of the 3′ terminus and/or 5′ terminus of a duplex is referred to as an overhang.
  • natural overhang refers to an overhang which consists of one or more nucleotides homology to or complementary to the corresponding position on the target sequence.
  • a natural overhang on a sense strand consists of one or more nucleotides homology to the corresponding position on the mRNA target.
  • a natural overhang on an antisense strand consists of one or more nucleotides complementary to the corresponding position on the mRNA target.
  • promoter refers to a sequence which is spatially associated with a protein-coding or RNA-coding nucleic acid sequence and plays a regulatory role for the transcription of the protein-coding or RNA-coding nucleic acid sequence.
  • a eukaryotic gene promoter contains 100 to 5000 base pairs, although this length range is not intended to limit the term “promoter” as used herein.
  • the promoter sequence is generally located at the 5′ terminus of a protein-coding or RNA-coding sequence, it may also exist in exon and intron sequences.
  • gene activation or “activating gene expression” and “gene up-regulation” or “up-regulating gene expression” can be used interchangeably, and mean an increase in transcription, translation, expression or activity of a certain nucleic acid as determined by measuring the transcriptional level, mRNA level, protein level, enzymatic activity, methylation state, chromatin state or configuration, translation level or the activity or state in a cell or biological system of a gene. These activities or states can be determined directly or indirectly.
  • gene activation refers to an increase in activity associated with a nucleic acid sequence, regardless of the mechanism of such activation. For example, gene activation occurs at the transcriptional level to increase transcription into RNA and the RNA is translated into a protein, thereby increasing the expression of the protein.
  • small activating RNA As used herein, the terms “small activating RNA” , “saRNA” , and “small activating nucleic acid molecule” can be used interchangeably, and refer to a nucleic acid molecule that can up-regulate target gene expression and can be composed of a first nucleic acid fragment (sense strand) containing a nucleotide sequence having high sequence identity to the non-coding nucleic acid sequence (e.g., a promoter or an enhancer) of a target gene and a second nucleic acid fragment (antisense strand) containing a nucleotide sequence complementary with the first nucleic acid fragment, wherein the first nucleic acid fragment and the second nucleic acid fragment form a duplex.
  • the saRNA can also be comprised of a synthesized or vector-expressed single-stranded RNA molecule that can form a hairpin structure by two complementary regions within the molecule, wherein the first region contains a nucleotide sequence having sequence identity to the target region of a promoter of a gene, and the second region contains a nucleotide sequence which is complementary with the first region.
  • the length of the duplex region of the saRNA is typically about 15 to about 35, about 16 to about 32, about 17 to about 30, about 18 to about 28, about 19 to about 26, about 20 to about 24, and about 21 to about 22 base pairs, and typically about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22 or about 23 base pairs.
  • RNA small activating RNA
  • nucleic acid molecule also contain nucleic acids other than the ribonucleotide, including, but not limited to, modified nucleotides or analogues.
  • oligonucleotide agent refers to an oligonucleotide-containing substance which at least comprises or consists of one or more saRNA of the invention and has the activity of modulating target gene expression or enhance the effect of the saRNA, and may further comprise other oligonucleotide moieties/components (such as ASO) or non-oligonucleotide moieties/components conjugated, combined or mixed with the saRNA (s) , such as a lipid, a cell-penetrating peptide, a polyethylene glycol, an alkaloid, a tryptamine, a benzimidazole, a quinolone, an amino acid, a cholesterol, a glucose, a N-acetylgalactosamine, and any combinations thereof.
  • saRNA such as a lipid, a cell-penetrating peptide, a polyethylene glycol, an alkaloid, a tryptamine, a
  • the oligonucleotide agent comprises an RNA (such as the saRNA of the invention) , a DNA, a bridged nucleic acid (BNA) , a locked nucleic acid (LNA) , a glycol nucleic acids (GNA) or a peptide nucleic acid (PNA) .
  • RNA such as the saRNA of the invention
  • BNA bridged nucleic acid
  • LNA locked nucleic acid
  • GNA glycol nucleic acids
  • PNA peptide nucleic acid
  • prevent refers to the slowing of the progression of a disease, disorder or condition from an existing state to a more deleterious state.
  • treat refers to preventing, ameliorating, reverting, curing and/or delaying a disease, disorder or condition.
  • administration refers to ordinary enteral administration, topical administration, and especially, injection administration, and may include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal, and intrastemal injection and infusion.
  • the term “pharmaceutical composition” refers to an active agent, optionally formulated together with one or more pharmaceutically acceptable carriers and other additives.
  • the active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population.
  • compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: 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; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.
  • oral administration for example, drenches (aqueous or non-aqueous solutions
  • the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, 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.
  • a pharmaceutically acceptable material, composition or vehicle such as a liquid or solid filler, diluent, excipient, 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.
  • 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: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ring
  • the term “subject” , “test subject” and related terms, as used herein, refer to any organism to which a provided compound or composition is administered in accordance with the present invention e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans; fishes; birds; insects; worms; etc. ) and plants. In some embodiments, a subject may be suffering from, and/or susceptible to a disease, disorder, and/or condition. In some embodiments, a subject is a human being or other mammal. In some embodiments, a subject can be male or female.
  • the animal is a vertebrate such as a primate, rodent, domestic animal or game animal.
  • primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., Rhesus.
  • Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters.
  • domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon.
  • the subject is a mammal, e.g., a primate, e.g., a human.
  • the mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples.
  • a mammal other than a human can be advantageously used as subjects that represent animal models of disorders associated with autoimmune disease or inflammation.
  • a method and composition described herein can be used to treat domesticated animals and/or pets.
  • SERPING1 refers to a human gene.
  • SERPING1 mRNA refers to a message RNA (mRNA) generated from the expression of SERPING1 gene, or the transcription of SERPING1 gene.
  • HAE refers to Hereditary Angioedema.
  • UTRN As used herein, the upper cased “UTRN” or “UTRN gene” refers to a human gene. As used herein, the term “UTRN mRNA” refers to a mRNA generated from the expression of UTRN gene, or the transcription of UTRN gene. “Utrn” or “Utrn gene” refers to a mouse gene. As used herein, the term “Utrn mRNA” refers to a mRNA generated from the expression of Utrn gene, or the transcription of Utrn gene. As used herein, DMD refers to Duchenne Muscular Dystrophy. Compounds
  • R b is independently selected from a group consisting of-OH, -O-R c , -SH, - (C 1 -C 22 ) alkyl, halogenated- (C 1 -C 22 ) alkyl, - (C 2 -C 22 ) alkenyl, - (C 3 -C 22 ) cycloalkyl, - (C 3 -C 22 ) cycloalkenyl, - (C 1 -C 22 ) alkylene- (C 3 -C 22 ) cycloalkyl, - (C 1 -C 22 ) alkylene-R c , - (C 1 -C 22 ) alkylene-O-R c , - (C 1 -C 22 ) alkylene-COOR c , -C (O) O-R c , -O- (C 1 -C 22 ) alkyl, -S- (C 1 -C 22 ) alky
  • R a is selected from a group consisting of H, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, or unsubstituted or substituted nucleobase.
  • R 1 is not deuterated or at least one hydrogen, if present, of R 1 is substituted with deuterium.
  • at least two hydrogens of R 1 are deuterated.
  • at least three hydrogens of R 1 are deuterated.
  • at least four hydrogens of R 1 are deuterated.
  • at least five hydrogens of R 1 are deuterated.
  • all hydrogens of R 1 are deuterated.
  • R 2 is selected from a group consisting of H, C 1 -C 12 alkyl, (C 1 -C 12 ) alkylcarbonyl, (C 1 -C 12 ) alkoxycarbonyl, (C 6 -C 16 ) aryl, (C 6 -C 16 ) arylcarbonyl, (C 6 -C 16 ) aryloxycarbonyl, and PR e R f .
  • the vinyl group is in either E or Z configuration. In an embodiment of the present application, the vinyl group is in E configuration. In another embodiment of the present application, the vinyl group is in Z configuration.
  • the hydrogens bonded directly to the vinyl group are not deuterated or at least one hydrogen is substituted with deuterium. In an embodiment, neither of the hydrogens bonded directly to the vinyl group is deuterated.
  • R 3 and R 4 are the same.
  • R 3 and R 4 are different.
  • the formula 1 has the following configuration:
  • m is 0 and A is O.
  • the compound has the structure of In this case, we number the atoms based on carbons to facilitate illustration. Accordingly, -R 1 is at position 2, and it may be shortened as 2'-R 1 . Further, the vinyl begins at position 5, and thus it can be shortened as 5'- (E) -vinyl.
  • formula 1 may have stereochemistry of Alternatively, as mentioned above, stereoisomers, such as the enantiomer, diastereoisomers of the above configuration are possible in the present application.
  • R a is an optionally substituted aryl.
  • the aryl is phenyl.
  • the phenyl is naphthyl.
  • the aryl is tetrahydronaphthyl. In some embodiments, the aryl is substituted.
  • R a is an optionally substituted heteroaryl.
  • the heteroaryl is selected from a group consisting of thienyl, pyridinyl, furyl, oxazolyl, oxadiazolyl, pyrrolyl, imidazolyl, triazolyl, thiodiazolyl, pyrazolyl, isoxazolyl, thiadiazolyl, pyranyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, thiazolyl benzothienyl, benzoxadiazolyl, benzofuranyl, benzimidazolyl, benzotriazolyl, cinnolinyl, indazolyl, indolyl, isoquinolinyl, isothiazolyl, naphthyridinyl, purinyl, thienopyridinyl, pyrido [2, 3-d]
  • the heteroaryl is substituted.
  • R a is an optionally substituted nucleobase selected from a group consisting of thymine (T) , cytosine (C) , guanine (G) , adenine (A) , uracil (U) , and an analogue or derivative thereof.
  • the nucleobase is substituted with -C 1-4 alkyl, such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl or tert-butyl.
  • the nucleobase is substituted with -C 1-6 alkylphenyl, such as methylphenyl (i.e., benzyl) , ethylphenyl, or the like.
  • the substitution can be performed on any position of the nucleobase, including any ring of the nucleobase, the amino group of the nucleobase, or even the carbonyl of the nucleobase.
  • R a is not deuterated. In some embodiments, at least one hydrogen in R a is substituted with deuterium.
  • R 2 is PR e R f .
  • each of R e and R f is independently selected from N (C 1 -C 12 alkyl) (C 1 -C 12 alkyl) or O- (C 1 -C 12 alkyl) .
  • the alkyl of each of R e and R f is unsubstituted.
  • substitution can be performed at any position of C 1 -C 12 alkyl.
  • the alky groups in each of N (C 1 -C 12 alkyl) (C 1 -C 12 alkyl) and O- (C 1 -C 12 alkyl) are the same. In some embodiments, the alky groups in each of N (C 1 -C 12 alkyl) (C 1 -C 12 alkyl) and O- (C 1 -C 12 alkyl) are different. In some embodiments, all the alky groups in N (C 1 -C 12 alkyl) (C 1 -C 12 alkyl) and O- (C 1 -C 12 alkyl) are the same.
  • the alky groups in N (C 1 -C 12 alkyl) (C 1 -C 12 alkyl) are the same, but different from the alkyl group in O- (C 1 -C 12 alkyl) .
  • none of the alky groups in N (C 1 -C 12 alkyl) (C 1 -C 12 alkyl) and O- (C 1 -C 12 alkyl) is the same with each other.
  • R e and R f are independently selected from N (C 1 -C 6 alkyl) (C 1 -C 6 alkyl) or O- (C 1 -C 6 alkyl) .
  • substitution can be performed at any position of C 1 -C 6 alkyl.
  • the alky groups in each of N (C 1 -C 6 alkyl) (C 1 -C 6 alkyl) and O- (C 1 -C 6 alkyl) are the same.
  • the alky groups in each ofN (C 1 -C 6 alkyl) (C 1 -C 6 alkyl) and O- (C 1 -C 6 alkyl) are different.
  • all the alky groups in N (C 1 -C 12 alkyl) (C 1 -C 6 alkyl) and O- (C 1 -C 6 alkyl) are the same.
  • the alky groups in N (C 1 -C 6 alkyl) (C 1 -C 6 alkyl) are the same, but different from the alkyl group in O- (C 1 -C 6 alkyl) . In some embodiments, none of the alky groups in N (C 1 -C 6 alkyl) (C 1 -C 6 alkyl) and O- (C 1 -C 6 alkyl) is the same with each other.
  • the C 1 -C 6 alkyl groups of the N (C 1 -C 6 alkyl) (C 1 -C 6 alkyl) and the O- (C 1 -C 6 alkyl) of R e and R f are independently selected from methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl and tert-butyl, and the C 1 -C 6 alky groups in each of R e and R f are the same. In other embodiments, the C 1 -C 6 alky groups in each of R e and R f are different.
  • one of R e and R f is N (C 1 -C 6 alkyl) (C 1 -C 6 alkyl) , and the other of R e and R f is O- (C 1 -C 6 alkyl) .
  • the vinyl group is in E configuration. In some embodiments, the vinyl group is in Z configuration. In some embodiments, the carbon-carbon double bond of the vinyl group is not deuterated. In some embodiments, one hydrogen directly bonded to the carbon-carbon double bond of the vinyl group is substituted with deuterium. As a result, the compound has a structure of with R a , R 1 , R 2 , R 3 and R 4 , and the stereochemistry as defined in the present disclosure; or in other embodiments, a structure of with R a , R 1 , R 2 , R 3 and R 4 , and the stereochemistry as defined in the present disclosure. In some embodiments, both of the hydrogens of the vinyl group are deuterated, and thus the compound has a structure of with R a , R 1 , R 2 , R 3 and R 4 , and the stereochemistry as defined in the present disclosure.
  • R 3 and R 4 are independently C 1 -C 6 alkyl.
  • the vinyl group of A1-A78 is in E configuration.
  • saRNAs as up-regulators have become an emerging class of therapeutic agents and are under active development.
  • a modified saRNA which comprises or is at least modified with the compound of the present application is provided. With such chemical modification, gene expression modulation activities of saRNA both in vitro and in vivo are enhanced.
  • a modified saRNA comprising a sense strand comprising a first nucleotide sequence, and an antisense strand comprising a second nucleotide sequence.
  • the first nucleotide sequence is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%homology to a target gene sequence.
  • the first nucleotide sequence is 15 to 30 nucleotides in length.
  • the first nucleotide sequence is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 nucleotides in length.
  • the first nucleotide sequence comprises at least one modified nucleotide having at least one of the following modifications: a) modification of a phosphodiester bond connecting nucleotides in the nucleotide sequence; b) modification of 2′-OH of a ribose in the nucleotide sequence; and c) modification of a base in the nucleotide sequence.
  • the first nucleotide sequence comprises at least one modified nucleotide which is modified with 2' -F nucleotide, 2' -OCH 2 CH 2 OCH 3 nucleotide, 2' -OCD 3 nucleotide, 5' - (E) -vinyl phosphonate, and/or 5' - (E) -deuterated vinyl phosphonate.
  • at least one modified nucleotide is modified with a 2' -OCD3 nucleotide.
  • at least one modified nucleotide is modified with a 5' - (E) -vinyl phosphonate.
  • At least one modified nucleotide is modified with a 5' - (E) -deuterated vinyl phosphonate.
  • at least one modified nucleotide is modified with a 2' -F nucleotide.
  • at least one modified nucleotide is modified with a 2' -OCH 2 CH 2 OCH 3 nucleotide.
  • the deuteration is performed on more than one position of the nucleotide.
  • the modified nucleotide is modified with both 2' -F and 5' - (E) -deuterated vinyl phosphonate.
  • the second nucleotide sequence is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%homology to a target gene sequence. In some embodiments, the second nucleotide sequence is 15 to 30 nucleotides in length. For example, the second nucleotide sequence is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 nucleotides in length.
  • the second nucleotide sequence comprises at least one modified nucleotide having at least one of the following modifications: a) modification of a phosphodiester bond connecting nucleotides in the nucleotide sequence; b) modification of 2′-OH of a ribose in the nucleotide sequence; and c) modification of a base in the nucleotide sequence.
  • the second nucleotide sequence comprises at least one modified nucleotide which is modified with 2' -F nucleotide, 2' -OCH 2 CH 2 OCH 3 nucleotide, 2' -OCD 3 nucleotide, 5' - (E) -vinyl phosphonate, and/or 5' - (E) -deuterated vinyl phosphonate.
  • at least one modified nucleotide is modified with a 2' -OCD3 nucleotide.
  • at least one modified nucleotide is modified with a 5' - (E) -vinyl phosphonate.
  • At least one modified nucleotide is modified with a 5' - (E) -deuterated vinyl phosphonate.
  • at least one modified nucleotide is modified with a 2' -F nucleotide.
  • at least one modified nucleotide is modified with a 2' -OCH 2 CH 2 OCH 3 nucleotide.
  • the deuteration is performed on more than one position of the nucleotide.
  • the modified nucleotide is modified with both 2' -F and 5' - (E) -deuterated vinyl phosphonate.
  • each of the sense strand and the antisense strand independently comprises a phosphodiester (PO) internucleoside linkage, a phosphorothioate (PS) internucleoside linkage, a mesyl phosphoramidate intemucleoside linkage (Ms) , a phosphorodithioate intemucleoside linkage, a PS-mimic internucleoside linkage, a phorothioate internucleoside linkage, a mesyl phosphoroamidate internucleoside linkage, a 5' stabilizing end cap, a phosphorylation blocker, galactosamine or a combination of two or more thereof.
  • PO phosphodiester
  • PS phosphorothioate
  • Ms mesyl phosphoramidate intemucleoside linkage
  • Ms mesyl phosphoramidate intemucleoside linkage
  • the linkage can be incorporated between any two nucleotides of the sense strand or the antisense strand.
  • the linkage can be incorporated between positions 1 and 2, between positions 2 and 3, between positions 3 and 4, between positions 4 and 5, between 5 and 6, between 6 and 7, between 7 and 8, between 8 and 9, between 9 and 10, between 11 and 12, between 12 and 13, between 13 and 14, between 14 and 15, between 15 and 16, between 16 and 17, between 17 and 18, between 18 and 19, between 19 and 20, between 20 and 21, between 21 and 22, between 22 and 23, or between 24 and 25 from 5' -end of the first or second nucleotide sequences.
  • the first or second nucleotide sequences comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 the above linkages, caps or blocks.
  • the first nucleotide sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%identity to a nucleotide sequence selected from any one of SEQ ID NOs: 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 69, 72, 84, 86, 88, and 90.
  • the second nucleotide sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%identity to a nucleotide sequence selected from any one of SEQ ID NOs: 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 70, 73, 85, 87, 89, and 91.
  • the saRNA comprises a sense strand and an antisense strand independently selected from any of the sequences listed in Table 1.
  • the first nucleotide sequence comprises a modified nucleotide sequence selected from any one of SEQ ID NOs: 1, 3, 4, 5, 7, 8, 9, 11, 12, 13, 17, 18, 63, 65, 67, 71, 74, 78, 79, and 81.
  • the second nucleotide sequence comprises a modified nucleotide sequence selected from any one of SEQ ID NOs: 2, 6, 10, 14, 15, 16, 64, 66, 68, 75, 76, 77, and 80.
  • the modified saRNA comprises a duplex selected from the sequences listed in Table 1.
  • the target gene is UTRN gene. In some embodiments, the target gene is SERPING1 gene.
  • the first nucleotide sequence comprises at least one modified nucleotide independently selected or derived from a compound of the present disclosure, and R2 is a linking bond.
  • at least one modified nucleotide is at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 from the 5' end of the first nucleotide sequence, ifthe positions are present.
  • the at least one modified nucleotide is at least at position 1 from the 5' end of the first nucleotide sequence.
  • the second nucleotide sequence comprises at least one modified nucleotide independently selected or derived from a compound of the present disclosure, and R2 is a linking bond.
  • at least one modified nucleotide is at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 from the 5' end of the second nucleotide sequence, if the positions are present.
  • the at least one modified nucleotide is at least at position 1 frorn the 5' end of the second nucleotide sequence.
  • the modified nucleotide is selected from a group consisting of the following: wherein the wave line means the site linked with another nucleotide, and the vinyl is in E configuration. According to embodiments of the present application, such modified nucleotide is at position 1 from the 5'end of the first or second nucleotide sequence.
  • the modified nucleotide is B1. In a particular embodiment, the modified nucleotide is B7.
  • At least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%or 100%of the rest nucleotides in the first or second nucleotide sequence are chemically modified with any of 2'-O-methyl (2’-OMe) or 2′-fluoro (2'-F) or 2’-O-methoxyethyl (2'-MOE) , or their combinations thereof.
  • at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%or 100%of the rest nucleotides in the first or second nucleotide sequence are chemically modified with 2’-MOE.
  • At least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%or 100%of the rest nucleotides in the first or second nucleotide sequence are chemically modified with 2’-OMe. In some embodiments, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%or 100%of the rest nucleotides in the first or second nucleotide sequence are chemically modified with 2’-F.
  • At least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%or 100%of the rest nucleotides in the first or second nucleotide sequence are chemically modified with two of2’-MOE, 2’-OMe and 2’-F. In some embodiments, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%or 100%of the rest nucleotides in the first or second nucleotide sequence are chemically modified with 2’-MOE, 2'-OMe and 2'-F.
  • the 2'-OMe modified nucleotide and 2’-F modified nucleotide are both present, at least part of the 2'-OMe modified nucleotides and the 2’-F modified nucleotides occur alternatively from the 5'end of the first nucleotide sequence.
  • the 2'-OMe modified nucleotides are at least at positions 3, 5, 7, 9, 10, 11, 13, 15, 17, 18, 19, 20, 21, 22, or 23 from the 5'end of the first or second nucleotide sequence, ifthe positions are present, and the 2’-F modified nucleotides are at some of the rest positions of the first or second nucleotide sequence.
  • the 2’-F modified nucleotides are at least at positions 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 22 from the 5’ end of the first or second nucleotide sequence, ifthe positions are present, and the 2’-OMe modified nucleotides are at some of the rest positions of the first or second nucleotide sequence.
  • the ratio of the number of the 2'-OMe modified nucleotides to the 2’-F modified nucleotides is in a range from 10: 1 to 1: 10.
  • such ratio can be about 10: 1, about 9: 1, about 8: 1, about 7: 1, about 6: 1, about 5: 1, about 4: 1, about 3: 1, about 2: 1, about 1: 1, about 1: 2, about 1: 3, about 1: 4, about 1: 5, about 1: 6, about 1: 7, about 1: 8, about 1: 9, or about 1: 10, or in a range having any two of the above values as endpoints.
  • the above modified saRNA only one of the first and second nucleotide sequences involves the above deuteration, and the other nucleotide sequence does not comprise deuteration.
  • the sense strand comprising the first nucleotide sequence comprises deuteration modification
  • the antisense strand comprising the second nucleotide sequence does not comprise deuteration modification.
  • the antisense strand comprising the second nucleotide sequence comprises deuteration modification
  • the sense strand comprising the first nucleotide sequence does not comprise deuteration modification.
  • the corresponding nucleotide sequence can be chemically modified with any of 2'-O-methyl (2’-OMe) or 2′-fluoro (2’-F) or 2’-O-methoxyethyl (2’-MOE) , as described above.
  • Conjugate agent and delivery moieties
  • the current disclosure also provides an oligonucleotide agent comprising the oligonucleotide and at least one delivery moiety.
  • saRNAs disclosed in the present application are covalently attached to one or more conjugate moieties.
  • delivery moieties modify one or more properties of the attached oligonucleotide, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, cellular distribution, cellular uptake, charge and clearance.
  • delivery moieties impart a new property on the attached oligonucleotide, e.g., fluorophores or reporter groups that enable detection of the oligonucleotide.
  • lipid fatty acid, WO2024002046A1
  • cholesterol moiety Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556
  • cholic acid Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053-1060
  • thioether e.g., hexyl-S-tritylthiol
  • Acids Res., 1990, 18, 3777-3783) a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides &Nucleotides, 1995, 14, 969-973) , or adamantane acetic acid, a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237) , an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp.
  • the saRNA of the present application relates to the sense strand or the antisense strand of the double-stranded oligonucleotide that is conjugated to one or more delivery moieties selected from: lipid, intercalators, reporter molecules, polyamines, polyamides, peptides, carbohydrates, vitamin moieties, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins, fluorophores, and dyes.
  • delivery moieties selected from: lipid, intercalators, reporter molecules, polyamines, polyamides, peptides, carbohydrates, vitamin moieties, polyethylene glycols, thioethers, polyethers, cholesterols, thiochol
  • a delivery moiety comprises an active drug substance, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fen-bufen, ketoprofen, (S) - (+) -pranoprofen, carprofen, dansylsarcosine, 2, 3, 5-triiodobenzoic acid, fingolimod, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indo-methicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.
  • an active drug substance for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fen-bufen, ketoprofen, (S) - (+) -pranoprofen, car
  • the saRNA of the present application is conjugated to one or more delivery moieties selected from: an oligonucleotide, a lipid, a fatty acid, a fluorophore, a ligand, a saccharide, a peptide, and an antibody.
  • the sense strand or the antisense strand of the oligonucleotide agent is conjugated to one or more delivery moieties selected from a single-stranded oligonucleotide, a cell-penetrating peptide, polyethylene glycol, an alkaloid, a tryptamine, a benzimidazole, a quinolone, an amino acid, a cholesterol, glucose and N-acetylgalactosamine.
  • the saRNA is conjugated to a lipid selected from C 4-30 fatty acid.
  • the delivery moiety is a lipid/fatty acid having a saturated or unsaturated, linear or branched C 2-16 carbon chain.
  • said delivery moiety is fatty acid comprising a carbon chain length of 16, 18 or 22 carbon atoms.
  • the delivery moiety is selected from lipophilic moieties as described in WO2024002046A1.
  • the double-stranded oligonucleotide may comprise 1, 2, 3, 4, or even more oligonucleotides separately conjugated to 1, 2, 3, 4 or even more of the delivery moieties via 1, 2, 3, 4 or even more linking moieties.
  • the sense strand or the antisense strand of the double-stranded oligonucleotide is conjugated to lipid compound C5x5, as shown below:
  • the conjugation group derived from C5x5 has a structure as shown below: wherein the asterisk represents the site where the conjugation group is attached to the strand, either directly or via a linking moiety.
  • C5x5 conjugated to the 5′end of sense strand and/or antisense strand of the double-stranded oligonucleotide In certain embodiments, the 5′end of the sense strand and/or antisense strand of the double-stranded oligonucleotide conjugated to one or more C5x5. In some embodiments, the 5′end of the sense strand of the saRNA conjugated to a C5x5. In certain embodiments, the conjugation group conjugated to the saRNA is derived from C5x5. In certain embodiments, the saRNA attached with a conjugation group has a structure as shown below:
  • the delivery moieties can be synthesized via procedures known in the art, for example WO2024002046A 1 is fully incorporated herein for synthetic process of compound C5x5.
  • saRNAs disclosed in the present application are covalently attached to one or more conjugate moieties through linking moieties.
  • the linking moieties when present, can be selected from the group consisting of-O-, -S-, -C (O) -, -NH-, -N ( (C 1 -C 12 ) alkyl) -, -N ( (C 1 -C 12 ) alkyl) -C (O) -O-, -O-C (O) -, -C (O) -O-, -O-C (O) -O-, -C (O) -NH-, -OP (O) 2 O-, -P (O) (O-) O-, -OP (O) O-, -OP (O) (S) O-, -O-S (O) 2 -O-, -S (O) 2 -O-, -S (O) -O-, -O-O-,
  • the saRNA conjugated to one or more delivery moieties disclosed in the embodiments e.g., C5x5.
  • the saRNA is directly contacted, transferred, delivered or administrated to a cell or a patient.
  • the modified saRNA further comprises one or more moieties or components conjugated or combined with the saRNA.
  • the one or more moieties or components is independently selected from a group consisting of a lipid, a fatty acid (such as fatty acid comprising a carbon chain with 4-30, 12-24, or 16-22 carbon atoms) , a fluorophore, a ligand, a saccharide, a peptide, and an antibody.
  • the one or more moieties or components is independently selected from a lipid, a cell-penetrating peptide, a polyethylene glycol, an alkaloid, a tryptamine, a benzimidazole, a quinolone, an amino acid, a cholesterol, a glucose, a N-acetylgalactosamine, and any combinations thereof.
  • the one or more moieties or components is independently selected from S9, tC2, tC2x6, C5x5, and any combinations thereof, wherein represents a support material.
  • compositions comprising the saRNA molecule of the present disclosure.
  • the pharmaceutical composition may comprise one or more additional ingredients, such as pharmaceutically acceptable carrier, excipient, solvent, diluent, stabilizer, dispersant, buffer, compatibilizer, preservative agent and combinations thereof.
  • Another embodiment of the present disclosure provides a method of modulating the expression of a target gene in vitro or in vivo, comprising the step of administrating the pharmaceutical composition to a subject, or contacting the pharmaceutical composition with cells of the subject.
  • the saRNA molecule and pharmaceutical composition can be applied in various organs, tissues and cells, such as liver, lung, kidney, intestine, pancreas, cholecyst, muscle, bone, central nervous system (CNS) , and modulate the expression of one or more target genes in the cell thereof.
  • the saRNA molecule and pharmaceutical composition can increase the expression of the target gene.
  • the subject is a mammal, such as a non-human primate or a human.
  • the subject is a rodent, such as a rat or a mouse.
  • the subject is a non-human primate.
  • the target gene is associated with a disease or disorder.
  • the target gene is associated with a disease or disorder in the central nervous system, eye, spleen, muscle, heart, kidney, or liver.
  • the cell is a mammalian cell.
  • the mammalian cell is a mouse cell.
  • the mammalian cell is a rat cell.
  • the mammalian cell is a non-human primate cell.
  • the mammalian cell is a human cell.
  • the target gene is associated with a disease or disorder.
  • the target gene is associated with a disease or disorder in the central nervous system, eye, spleen, muscle, heart, kidney, or liver. Particular embodiments
  • Embodiment 2 relates to the compound of embodiment 1, wherein the formula 1 has the following configuration:
  • Embodiment 3 relates to the compound of either of embodiment 1 or embodiment 2, wherein m is 0 and A is O.
  • Embodiment 5 relates to the compound of any of embodiments 1-3, wherein R a is an optionally substituted heteroaryl selected from a group consisting ofthienyl, pyridinyl, furyl, oxazolyl, oxadiazolyl, pyrrolyl, imidazolyl, triazolyl, thiodiazolyl, pyrazolyl, isoxazolyl, thiadiazolyl, pyranyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, thiazolyl benzothienyl, benzoxadiazolyl, benzofuranyl, benzimidazolyl, benzotriazolyl, cinnolinyl, indazolyl, indolyl, isoquinolinyl, isothiazolyl, naphthyridinyl, purinyl, thienopyridinyl, pyrido [
  • Embodiment 7 relates to the compound of any of embodiments 1-6, wherein R1 is selected from a group consisting of halogen and linear or branched OC 1 -C 6 alkyl which is optionally substituted.
  • Embodiment 8 relates to the compound of embodiment 7, wherein at least one hydrogen of the linear or branched OC 1 -C 6 alkyl which is optionally substituted is substituted with deuterium.
  • Embodiment 10 relates to the compound of embodiment 7, wherein R 1 is -CH 3 .
  • Embodiment 11 relates to the compound of either of embodiment 8 or 9, wherein R 1 is -CH 2 D, -CHD 2 or -CD 3 .
  • Embodiment 14 relates to the compound of embodiment 13, wherein the C 1 -C 6 alkyl groups of the N (C 1 -C 6 alkyl) (C 1 -C 6 alkyl) and the O- (C 1 -C 6 alkyl) of R e and R f are independently selected from methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl and tert-butyl, and wherein the C 1 -C 6 alky groups in each of R e and R f are the same or different.
  • Embodiment 15 relates to the compound of embodiment 14, wherein one of R e and R f is N (C 1 -C 6 alkyl) (C 1 -C 6 alkyl) , and the other of R e and R f is O- (C 1 -C 6 alkyl) .
  • Embodiment 26 relates to the compound of embodiment 1, wherein the oligonucleotide is a saRNA, a siRNA, an ASO or a miRNA.
  • Embodiment 27 A modified saRNA, wherein the saRNA comprises a sense strand comprising a first nucleotide sequence, and an antisense strand comprising a second nucleotide sequence, wherein the first nucleotide sequence is 15 to 30 nucleotides in length and at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%homology to a target gene sequence; and/or wherein the second nucleotide sequence is 15 to 30 nucleotides in length and at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%complementary to the target gene sequence; and/or wherein the first nucleotide sequence or the second nucleotide sequence comprises at least
  • Embodiment 29 relates to the modified saRNA of embodiment 28, wherein the modification of a phosphodiester bond connecting nucleotides is selected from a phosphorothioate modification and boranophosphate modification; and/or the modification of 2′-OH is selected from the group consisting of 2′-fluoro modification, 2′-oxymethyl modification, 2′-oxyethylidene methoxy modification, 2′-oxymethylidene ethoxy modification, 2, 4′-dinitrophenol modification, 2′-amino modification and 2′-deoxy modification; and/or the modification of a base is selected from the group consisting of 5 ′-bromouracil modification, 5′-iodouracil modification, N-methyluracil modification, and 2, 6-diaminopurine modification.
  • Embodiment 45 relates to the oligonucleotide agent of embodiment 44, wherein the one or more moieties or components is independently selected from S9, tC2, tC2x6, C5x5, and any combinations thereof, wherein represents a support material.
  • Embodiment 46. A pharmaceutical composition comprising: a) the saRNA molecule according to any one of embodiments 27-43, or the oligonucleotide agent according to any of embodiments 44-45; and b) optionally, one or more ingredients selected from the group consisting of pharmaceutically acceptable carrier, excipient, solvent, diluent, stabilizer, dispersant, buffer, compatibilizer, preservative agent and combinations thereof.
  • Embodiment 47 Embodiment 47.
  • the crude intermediate was dissolved in dry pyridine (80 mL) , and then Isobutyryl chloride (6.5 mL, 62.6 mmol, 2.0 eq) was added to the mixture at ice bath under nitrogen atmosphere. Then the mixture was stirred at room temperature for 2 h. After that the reaction was moved to ice bath, ammonium hydroxide (7 mL) was added slowly into the mixture. Then the reaction was extracted two times with ethyl acetate, the organic phase was washed one time by brine. Then dried by anhydrous Na 2 SO 4 and concentrated under reduced pressure. The resultant residue was dissolved in 1, 4-dioxane/H 2 O (1: 1, 100 mL) .
  • the crude product (300 mg, 0.41 mmol, 1.0 eq) was dissolved in anhydrous DCM (5 mL) then DIPEA (204 ⁇ L, 1.23 mmol, 3.0 eq) , - ( (chloro (diisopropylamino) phosphanyl) oxy) propanenitrile compound 47 (274 tL, 1.23 mmol, 3.0 eq. ) were added under nitrogen atmosphere at 25°C. The reaction mixture was stirred for 1 h. The mixture was extracted two times with DCM, then washed with brine and dried with anhydrous Na 2 SO 4 .
  • Oligonucleotide conjugate with C5x5 was generated by using a conjugation group derived from the compound C5x5 by using C5x5 as terminus amidite according to the above methods of general synthesis method of oligonucleotide.
  • Exemplary structure of the lipid-conjugated oligonucleotide is C5x5-saRNA as illustrated below: C5x5-saRNA (Compound C5x5 application) .
  • the conjugation derived from the delivery enhancing compound C5x5 is linked with saRNA duplexes at the 5'-end of the sense strand (S) via a linking moiety, such as -OP (O) 2 O-, -OP (O) (S) O-or -P (O) -O-, wherein (S) is the sense strand and (AS) is the antisense strand.
  • a linking moiety such as -OP (O) 2 O-, -OP (O) (S) O-or -P (O) -O-
  • CM-saRNAs i.e., RD-17235, RD-18069 and RD-172378
  • their corresponding VP-saRNAs i.e., RD-17236, RD-18071 and RD-1 7239
  • their corresponding VPD-saRNAs i.e., RD-18057, RD-18073 and RD-18053
  • RD-17268 was transfected and served as a siRNA control to silence SERPING1 mRNA expression.
  • SERPING1 mRNA levels as quantified by two step RT-qPCR are plotted in FIG. 1 and summarized in Table 2.
  • Table 2. SERPING1 mRNA levels in THLE-2 cells Note: “-" represents not available. “/” represents not tested. SEM represents Standard Error of the Mean.
  • saRNAs were designed and tested.
  • the indicated saRNAs i.e., RD-12027, RD-12028, RD-12031, RD-12033, RD-12034, RD-12299, RD-12303, RD-12304, RD-12305, RD-12313 and RD-12316
  • their corresponding newly designed saRNA i.e., RD-13587, RD-13580, RD-13584, RD-13716, RD-13720, RD-13629, RD-13727, RD-13632, RD-13732, RD-13737 and RD-13740
  • UTRN mRNA levels as quantified by two step RT-qPCR are plotted in FIG. 2.
  • newly designed saRNAs exhibited comparable or superior activity in inducing UTRN mRNA expression compared to their parent saRNAs.
  • the results indicate that asymmetric double-stranded structure can improve the activation activity of saRNA.
  • CM-saRNAs i.e., RD-14734, RD-13869, RD-14752 and RD-13870
  • the indicated CM-saRNAs i.e., RD-14734, RD-13869, RD-14752 and RD-13870
  • RD-14734, RD-13869, RD-14752 and RD-13870 were transfected into RD cells at 25 nM for 3 days.
  • UTRN mRNA levels as quantified by two step RT-qPCR are plotted in FIG. 3.
  • CM-saRNAs induced a greater than 1.5-fold increase in UTRN mRNA expression.
  • the results indicate that saRNAs can improve their activation activity by means of chemical modifications.
  • Example 10 In vitro activity of VPD-saRNAs in inducing UTRN mRNA expression levels in RD cells
  • the indicated saRNAs i.e., RD-12027 and RD-12305
  • their corresponding CM-saRNAs i.e., RD-21728 and RD-21731
  • their corresponding VP-saRNAs i.e., RD-20359 and RD-21732
  • their corresponding VPD-saRNAs i.e., RD-18342 and RD-21733
  • concentrations i.e., 0.02, 0.07, 0.21, 0.62, 1.85, 5.56, 16.67 and 50 nM
  • VPD-saRNAs showed lowest EC 50 and highest E max values compared to other saRNA treatments. The results indicate that VPD-saRNA showed the highest potency in inducing UTRN mRNA expression, suggesting that VPD modification can improve the potency of the saRNAs in activation of target gene expression.
  • Example 11 In vitro activity of VPDL-saRNAs in inducing UTRN mRNA expression levels in RD cells
  • lipid-conjugated saRNAs were designed and tested.
  • the lipid (C5x5) conjugated to CM-saRNA (s) are referred to as “CML-saRNA (s) ”
  • conjugated to VP-saRNA (s) are referred to as “VPL-saRNA (s) ”
  • conjugated to VPD-saRNA (s) are referred to as “VPDL-saRNA (s) ” .
  • the indicated CML-saRNA (RD-15637) and two VPL-saRNAs (RD-15638 and RD-15639) were first tested in vitro to assess the UTRN mRNA expression. These compounds were transfected into RD cells at the indicated concentrations (i.e., 0.024, 0.1, 0.4, 1.56, 6.25, 25, 100 and 200 nM) for 3 days.
  • the activation activity of saRNAs on UTRN mRNA expression generated dose response curve via two step RT-qPCR are plotted in FIG. 5. EC 50 values were extrapolated to define potency in context to maximal activity for each of the tested saRNAs.
  • the areas under the curve (AUC) of UTRN mRNA expression were calculated by GraphPad Prism software as described in the Materials and Methods section.
  • the resulting EC 50 values, E max and AUC following CML-saRNA and VPL-saRNA treatments in RD cells are summarized in Table 4. As shown in FIG. 5 and Table 4, VPL-saRNAs showed lower EC 50 values compared to CML-saRNA. Table 4. EC 50 , E max and AUC values in RD cells
  • the indicated CML-saRNA i.e., RD-18037
  • its corresponding VPL-saRNA i.e., RD-18036
  • its corresponding VPDL-saRNA i.e., RD-18035
  • the indicated concentrations i.e., 0.02, 0.07, 0.21, 0.62, 1.85, 5.56, 16.67 and 50 nM
  • CM-saRNA i.e., RD-14752
  • VPL-saRNA i.e., RD-15639
  • VPDL-saRNA i.e., RD-18031
  • EC 50 values following saRNA treatment in RD cells are summarized in Table 5. As shown in FIG. 6A-6B and Table 5, VPDL-saRNAs showed the lowest EC 50 values compared to CML-saRNA and VPL-saRNA treatments. Table 5. EC 50 values in RD cells
  • VPDL-saRNA showed superior potency in activating UTRN mRNA expression, as evidenced by its lower EC 50 , suggesting that VPD modification can improve the target engagement and cellular response of the saRNA.
  • the reduced EC 50 highlights the potential of VPD modified saRNAs to achieve their desired biological effect at lower therapeutic doses, lower systemic exposure, and wider therapeutic windows, particularly in conditions where dose-limiting toxicity is a concern.
  • Example 12 In vivo activity of VPDL-saRNAs in inducing Utrn mRNA expression levels in human UTRN promoter KI/+ mice
  • Mouse Utrn mRNA levels as quantified in skeletal muscle (i.e., gluteus) , smooth muscle (i.e., thoracic aorta) and cardiac muscle (i.e., heart) via two-step RT-qPCR are shown in FIGs. 8A-8C and summarized in Table 6. Table 6. Mouse Utrn mRNA levels following VPL-saRNA and VPDL-saRNA treatments in male and female hUTRNp KI/+ mice Note: “-" represents not available. “/” represents not shown. "SEM”represents Standard Error of the Mean.
  • hUTRNp (KI/KI) ⁇ mdx hybrid mice were developed by crossing the hUTRNp (KI/KI) mice with the well-established mdx mice, and the indicated VPDL-saRNA (i.e., RD-18997) was administered to the male model mice via SC injection for a total of five doses (50 mg/kg per dose) on day 0, 2, 4, 7 and 14. The mice were sacrificed at day 28 post first dosing.
  • VPDL-saRNA induced a 1.34-to 2.89-fold increase in UTRN mRNA expression. These results indicate that saRNA with VPD modification and lipid-conjugated moiety can achieve promising target gene activation in skeletal muscles.
  • the PD data of the VPDL-saRNA revealed significant target engagement and biological activity in muscle tissues.
  • Example 14 Concentration and biodistribution of VPDL-saRNA across various tissues in C57BL/6J male mice.
  • the concentrations of RD-18997 in the heart, diaphragm, platysma, and gluteus were approximately 42.7%, 26.6%, 17.6%, and 20.5%of their respective maximum concentration (C max ) values.
  • the muscle tissues with the highest drug accumulation were the platysma (near the SC injection site) , followed by the heart, diaphragm, and gluteus.
  • the estimated half-life of RD-18997 in the platysma, heart, diaphragm, and gluteus was approximately 497, 848, 577, and 413 hours, or 20.7, 35.3, 24.0 and 17.2 days, respectively.
  • oligonucleotides used in the above examples were synthesized via the following general method. (1) Single-stranded synthesis
  • the single-stranded oligonucleotide was synthesized on a K&ADNA synthesizer (K&ALaborgeraete GbR, chaafheim, Germany) by a solid-phase synthesis technique.
  • the starting material was universal solid support or special solid support commercially available or synthesized as disclosure in previous context.
  • phosphoramidite monomers including various linkers and conjugates were added sequentially onto a solid support in the DNA synthesizer to generate the desired full-length oligonucleotides.
  • each cycle of amidite addition consisted of four chemical reactions including detritylation, coupling, oxidation/thiolation and capping.
  • detritylation was performed by using 3%dichloroacetic acid (DCA) in DCM for 45 seconds.
  • DCA 3%dichloroacetic acid
  • phosphoramidite coupling was conducted for 6 minutes for all amidites by 12 eq..
  • oxidation was performed by using 0.02 M iodine in THF: pyridine: water (70: 20: 10, v/v/v) for 1 minute; if phosphorothioate modification was needed, oxidation was replaced by thiolation which was carried out with 0.1 M solution of xanthane hydride in pyridine: ACN (50: 50, v/v) for 3 minutes.
  • the capping was performed by using a THF: acetic anhydride: Pyridine (80: 10: 10, v/v/v) (CAP A) and N-methylimidazole: THF (10: 90, v/v) , (CAP B) for 20 seconds.
  • the Cycles of four chemical reactions will depend on the length of oligonucleotide.
  • Deprotection I (Nucleobase Deprotection): after completion of the synthesis, the solid support was transferred to a screw-cap microcentrifuge tube. For a 1 ⁇ mol synthesis scale, 1 mL of a mixture of methylamine and ammonium hydroxide was added. The tube containing the solid support was then heated in an oven at 60°C to 65°C for 15 min and then allowed to cool to room temperature. The cleavage solution was collected and evaporated to dryness in a speedvac to provide crude single-stranded oligonucleotide.
  • Deprotection II Removal of 2’-TBDMS Group: if the crude RNA oligonucleotide, still carrying the 2’-TBDMS groups, it was dissolved in 0.1 mL of DMSO. After adding 1 mL of Triethylamine trihydrofluoride, the tube was capped, and the mixture was shaken vigorously to ensure complete dissolution. Then, it was heated in an oven at 65°C for 15 minutes. The tube was removed from the oven and cooled down to room temperature. The solution containing the completely desilylated oligonucleotide was cooled on dry ice.
  • oligonucleotides The purification of oligonucleotides was performed on an AKTA explorer 10 equipped with a Source 15Q 4.6/100 PE column using the following conditions: buffer A: (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) , B: (10 mM Tris-HCl, 1 mM EDTA, 2M NaCl, pH 7.5) , gradient: 10%B to 60%B in 25 min, flow rate: 1 mL/min.
  • buffer A (10 mM Tris-HCl, 1 mM EDTA, pH 7.5
  • B (10 mM Tris-HCl, 1 mM EDTA, 2M NaCl, pH 7.5)
  • gradient 10%B to 60%B in 25 min
  • flow rate 1 mL/min.
  • the pure oligonucleotides were collected and desalted by a HiPrep 26/10 Desalting column. (3) Annealing to form duplex
  • the sense strand and antisense strand were mixed by equal volumes at equimolar concentration in the tube. Place the tube in a heat block at 95°C for 5 min and then cool to room temperature; subsequently, lyophilized to powder.
  • Coniugate moiety
  • conjugation moieties can be synthesized via procedures known in the art, for example WO2024002046A1 is fully incorporated herein for synthetic process of tC2, tC2x6, and C5x5.
  • WO2024002046A1 is fully incorporated herein for synthetic process of tC2, tC2x6, and C5x5.
  • RD cells Human malignant embryonic rhabdomyoma cells (RD cells) (TCHu 45, Center for Excellence in Molecular Cell Science, Chinese Academy of Science, China) were cultured at 37°C with 5%CO2 in modified DMEM medium (Gibco, Thermo Fisher Scientific, Carlsbad, CA) supplemented with 10%bovine calf serum (Sigma-Aldrich) and 1%penicillin/streptomycin (Gibco) .
  • Transformed human liver epithelial-2 (THLE-2) (CBP61031, Nanjing Cobioer Biosciences CO.
  • mice were cultured at 37°C with 5%CO2 in modified BEGM medium (Gibco) supplemented with 10%bovine calf serum and 5 ng/mL epidermal growth factor (EGF) plus 70 ng/mL phosphoethanolamine.
  • modified BEGM medium Gibco
  • EGF epidermal growth factor
  • Human UTRN promoter knock-in mice (hUTRN promoter KI/+ mice or hUTRNp KI/+ mice) were created by replacing the mouse Utrn promoter ( ⁇ 12kb upstream of ATG start codon of mouse Utrn gene) with the human UTRN promoter ( ⁇ 11.5kb upstream of ATG start codon of human UTRN gene) via targeting in C57BL/6J embryonic stem cells by Cyagen Biotechnology Co., LTD (Jiangsu, China) .
  • PCR genotyping strategy for confirming mice with the human UTRNp KI involved two steps. Tail snips were gathered at postnatal day 0 (PND 0) , and each pup was identified by paw tattooing and genotyped by PCR analysis using 3 sets of specific primers. The presence of Utrn promoter deletions was confirmed using Utrn-Neo-del PCR with primers F1, 5′-AAC TC CTGCCTACAGCATAACTG-3′, and R1, 5′-ATTAGCAGGTGGGCAAAAGGTTA-3′ (one 303 bp band for homozygotes, 303 bp and 190 bp two bands for heterozygotes, one 190 bp band for wildtype allele) .
  • the hUTRNp KI status was then further verified using Utrn-hUTRNp PCR with primers F2, 5′-GCTCTCTGAATGGTTTCTCCCATA-3′, R2, 5′-GGGGAAAGTGTCCAAATTACTGAG-3′, internal control PCR primer F4, 5′-CTATCAGGGATACTCCTCTTTGCC-3′ and internal control PCR primer R4, 5′-GATACAGGAATGACAAGCTCATGGT-3′ (one 507 bp band for wild type, 507 bp and 262 bp two band for KI mutant allele) .
  • Mouse Rgs7 served as an internal control, using specific primers to produce a 507 bp fragment.
  • the PCR products were detected by 1%agarose gel.
  • Primers of hUTRN promoter KI mice genotyping are shown in Table 9. Human UTRNp (KI/KI) ⁇ mdx mice
  • hUTRNp KI/+ mice were further crossed with the mdx mice which carries a spontaneous mutation in the dystrophin (Dmd) gene and is the most widely used animal model of the human disease DMD [Dominic J Wells, 2018] , resulting in hUTRNp KI/KI ⁇ mdx mice.
  • Dmd dystrophin
  • the hUTRNp KI/KI ⁇ mdx mice were generated by first mating male hUTRNp KI/+mice with female Dmd KI/+ mice, resulting in male h UTRNp KI/+ ⁇ Dmd KI/Y (Y represents Y chromosome) and female hUTRNp KI/+ ⁇ Dmd KI/+ offspring. These offsprings were then further mated to produce hUTRNp KI/KI ⁇ mdx mice of both genders, which were subsequently confirmed through PCR genotyping.
  • the hUTRNp KI status was then further verified using Utrn-hUTRNp PCR with primers F2, 5’-GCTCTCTGAATGGTTTCTCCCATA-3’ and R2, 5’-GGGGAAAGTGTCCAAATTACTGAG-3’, internal control PCR primer F4, 5′-CTATCAGGGATACTCCTCTTTGCC-3′ and internal control PCR primer R4, 5′-GATACAGGAATGACAAGCTCATGGT-3′ (one 507 bp band for wild type, 507 bp and 262 bp two band for KI mutant allele) .
  • Mouse Rgs7 served as an internal control, using specific primers to produce a 507 bp fragment.
  • the PCR products were detected by 1%agarose gel. Primers of hUTRN promoter KI mice genotyping are shown in Table 9.
  • Dmd-KI-mutation PCR distinguishes WT mice (210 bp fragment) from heterozygous mice (210 and 237 bp fragments) and homozygous mice (237 bp fragment) carrying the Dmd KI mutation.
  • the PCR products were detected by 1%agarose gel.
  • Primers of hUTRNp (KI/KI) ⁇ mdx mice genotyping are shown in Table 9.
  • C57BL/6J mice ( ⁇ 4-6 weeks old) purchased from SPF Biotechnology Co., LTD (B204, Beijing, China) .
  • RNA isolation and reverse transcription-quantitative polymerase chain reaction RNA isolation and two-step RT-qPCR
  • RNA was isolated from treated cells using a RNeasy Plus Mini kit (Qiagen, Hilden, Germany) according to its manual. Animal tissues were isolated using the MagPure Total RNA Micro LQ kit (Magen, R6621, Guangzhou, China) in conjunction with the auto-pure96 machine (ALLSHENG, Hangzhou, China) . The resultant RNA ( ⁇ 1 ⁇ g) was reverse transcribed into cDNA by using a PrimeScriptTM RT reagent kit with gDNA Eraser (Takara, RR047A, Shlga, Japan) .
  • MagPure Total RNA Micro LQ kit Magen, R6621, Guangzhou, China
  • ALLSHENG auto-pure96 machine
  • the resultant cDNA was amplified in a Roche LightCycler 480 Multiwell Plate 384 (Roche, ref: 4729749001, US) using TB Premix Ex Taq TM II (Takara, RR820A, Shlga, Japan) reagents and primers specifically for amplifying target genes of interest.
  • Reaction conditions were as follows: reverse transcription reaction (stage 1) : 42°C for 5 min, 95°C for 10 sec; PCR reaction (stage 2) : 95°C for 5 sec, 60°C for 30 sec, 72°C for 10 sec, 40 cycles of amplification; and melting curve (stage 3) .
  • PCR reaction conditions are shown in Table 10 and Table 11. Primer sequences are listed in Table 9.
  • E rel 2 (CtTm-CtTs) / 2 (CtRm-CtRs) wherein CtTm was the Ct value of the target gene from the control-treated sample; CtTs was the Ct value of the target gene from the saRNA-treated sample; CtRm was the Ct value of the internal reference gene from the control-treated sample; CtRs was the Ct value of the internal reference gene from the saRNA-treated sample.
  • E rel 2 (CtTm-CtTs) / ( (2 (CtR1m-CtR1s) *2 (CtR2m-CtR2s ) ) (1/2) ) wherein CtTm was the Ct value of the target gene from the control-treated sample; CtTs was the Ct value of the target gene from the saRNA-treated sample; CtR 1 m was the Ct value of the internal reference gene 1 from the control-treated sample; CtR1s was the Ct value of the internal reference gene 1 from the saRNA-treated sample; CtR2m was the Ct value of the internal reference gene 2 from the control-treated sample; and CtR2s was the Ct value of the internal reference gene 2 from the saRNA-treated sample. Areas under
  • the Area Under the Curve (AUC) for a specific test article is derived from the curve produced by plotting the dose-response data across eight doses. This curve reflects the cumulative effect of the test article across all tested doses. Essentially, the AUC quantifies the overall response to the treatment, integrating the responses at individual doses into a single metric, such as a mean efficacy.
  • AUC agonist versus response curve from the dose-response data. Then using the ′Area under the Curve′ plugin in GraphPad Prism software, the curve can be evaluated. The value of AUC is unitless but can be used as a comparative indicator for the determination of relative response in comparison to other test articles. Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) quantitative analysis
  • the concentrations of RD-18997 in various mice tissues were quantified at the indicated time points (i.e., 0, 5 min, 2 hr, and on day 1, 7, 15, 30, 45, 60) post dosing, using LC-MS/MS analysis conducted by Accurant BioTech Inc (Zhejiang, China) .
  • Statistical analysis Statistical analysis

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Abstract

L'invention concerne un composé ayant une structure de formule 1. Le composé peut être utilisé pour synthétiser un petit ARN activateur (ARNsa) modifié pour améliorer les activités de modulation d'expression génique de l'ARNsa à la fois in vitro et in vivo. L'invention concerne également une composition pharmaceutique comprenant le composé et un procédé de modulation de l'expression d'un gène cible à l'aide de celui-ci.
PCT/CN2025/085845 2024-03-29 2025-03-28 Composés pour synthétiser des molécules de petit arn activateur modifié, molécules de petit arn activateur modifié et leurs utilisations Pending WO2025201529A1 (fr)

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WO2019193144A1 (fr) * 2018-04-05 2019-10-10 Silence Therapeutics Gmbh Siarns avec vinylphosphonate à l'extrémité 5' du brin antisens
WO2021178885A1 (fr) * 2020-03-06 2021-09-10 Aligos Therapeutics, Inc. Molécules d'acide nucléique interférent court modifiées (sina) et leurs utilisations
WO2023039076A1 (fr) * 2021-09-08 2023-03-16 Aligos Therapeutics, Inc. Molécules d'acide nucléique interférent court modifiées (sina) et leurs utilisations
WO2023122317A2 (fr) * 2021-12-23 2023-06-29 Aligos Therapeutics, Inc. MOLÉCULES D'ACIDE NUCLÉIQUE INTERFÉRENT COURT (SINA) POUR CIBLER LA β-CATÉNINE ET LEURS UTILISATIONS
WO2023138502A1 (fr) * 2022-01-20 2023-07-27 Ractigen Therapeutics Modulateurs oligonucléotidiques activant l'expression de l'utrophine
WO2024001172A1 (fr) * 2022-06-27 2024-01-04 Ractigen Therapeutics Modulateurs oligonucléotidiques activant l'expression du facteur h du complément
WO2024001170A1 (fr) * 2022-06-27 2024-01-04 Ractigen Therapeutics Petite molécule d'acide nucléique d'activation et son utilisation dans le traitement de l'oedème de quincke héréditaire

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WO2019193144A1 (fr) * 2018-04-05 2019-10-10 Silence Therapeutics Gmbh Siarns avec vinylphosphonate à l'extrémité 5' du brin antisens
WO2021178885A1 (fr) * 2020-03-06 2021-09-10 Aligos Therapeutics, Inc. Molécules d'acide nucléique interférent court modifiées (sina) et leurs utilisations
WO2023039076A1 (fr) * 2021-09-08 2023-03-16 Aligos Therapeutics, Inc. Molécules d'acide nucléique interférent court modifiées (sina) et leurs utilisations
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WO2023138502A1 (fr) * 2022-01-20 2023-07-27 Ractigen Therapeutics Modulateurs oligonucléotidiques activant l'expression de l'utrophine
WO2024001172A1 (fr) * 2022-06-27 2024-01-04 Ractigen Therapeutics Modulateurs oligonucléotidiques activant l'expression du facteur h du complément
WO2024001170A1 (fr) * 2022-06-27 2024-01-04 Ractigen Therapeutics Petite molécule d'acide nucléique d'activation et son utilisation dans le traitement de l'oedème de quincke héréditaire

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