WO2025125576A2 - Inhibiteurs d'expression et/ou de fonction - Google Patents

Inhibiteurs d'expression et/ou de fonction Download PDF

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Publication number
WO2025125576A2
WO2025125576A2 PCT/EP2024/086234 EP2024086234W WO2025125576A2 WO 2025125576 A2 WO2025125576 A2 WO 2025125576A2 EP 2024086234 W EP2024086234 W EP 2024086234W WO 2025125576 A2 WO2025125576 A2 WO 2025125576A2
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seq
etx
strand
nucleosides
nucleoside
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WO2025125576A3 (fr
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Natalie PURSELL
Alan VICTOR WHITMORE
Sophia MALANDRAKI-MILLER
Ines DE SANTIAGO
Gaye SAGINC GIANNOUSTAS
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E Therapeutics PLC
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E Therapeutics PLC
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Priority claimed from PCT/EP2024/052934 external-priority patent/WO2024165571A2/fr
Application filed by E Therapeutics PLC filed Critical E Therapeutics PLC
Priority to PCT/EP2024/088248 priority Critical patent/WO2025133348A1/fr
Priority to AU2024408289A priority patent/AU2024408289A1/en
Priority to IL330165A priority patent/IL330165A/en
Publication of WO2025125576A2 publication Critical patent/WO2025125576A2/fr
Publication of WO2025125576A3 publication Critical patent/WO2025125576A3/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
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    • 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
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    • 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
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    • 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
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/346—Spatial arrangement of the modifications having a combination of backbone and sugar modifications
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate

Definitions

  • the present invention provides inhibitors, such as nucleic acid compounds, such as siRNA, suitable for therapeutic use. Additionally, the present invention provides methods of making these compounds, as well as methods of using such compounds for the treatment of various diseases and condition BACKGROUND OF THE INVENTION Inhibitors, such as oligonucleoside/ oligonucleotide compounds which are inhibitors of gene expression and/or expression or function of other targets such as LNCRNAs, can have important therapeutic applications in medicine. Oligonucleotides/ oligonucleosides can be used to silence genes that are responsible for a particular disease. Gene-silencing prevents formation of a protein by inhibiting translation.
  • siRNA, antisense RNA, and micro-RNA are oligonucleoside /oligonucleotides that prevent the formation of proteins by gene-silencing.
  • a number of modified siRNA compounds in particular have been developed in the last two decades for diagnostic and therapeutic purposes, including siRNA / RNAi therapeutic agents for the treatment of various diseases including central-nervous-system diseases, inflammatory diseases, metabolic disorders, oncology, infectious diseases, and ocular diseases.
  • the present invention relates to inhibitors, such oligomers e.g. nucleic acids, e.g.
  • the SLC25A5 gene belongs to the ANT gene family, which itself belongs to the superfamily that includes genes encoding brown fat mitochondrial uncoupling proteins and mitochondrial phosphate carrier proteins. This gene is a member of the mitochondrial carrier subfamily of solute carrier protein genes. The product of this gene, adenine nucleotide translocator 2 (ANT2), functions as a major constituent of the mitochondrial permeability-transition pore complex that catalyses the exchange of mitochondrial ATP with cytosolic ADP.
  • ANT2 adenine nucleotide translocator 2
  • ANT2 maintains mitochondrial membrane potential by regulating ADP/ATP ratios in oxidative phosphorylation.
  • ANT2 facilitates uncoupling of the mitochondrial membrane when acylated by SIRT4. Though uncoupling the membrane potential typically leads to apoptosis, ANT2 was found to be antiapoptotic. As a result, it is postulated to mediate the TFIIH- dependent response to DNA damage as a component of the MMS19-XPD.
  • the invention relates to an inhibitor of expression and / or function of SLC25A5/ANT2, wherein said inhibitor is conjugated to one or more ligand moieties.
  • the invention relates to an inhibitor according to the invention, wherein said inhibitor is an siRNA oligomer.
  • the invention relates to an inhibitor of expression and / or function of SLC25A5/ANT2, wherein said inhibitor is an siRNA oligomer.
  • the invention relates to an inhibitor according to the invention, wherein said inhibitor comprises an siRNA oligomer conjugated to one or more ligand moieties.
  • the invention relates to an inhibitor according to the invention, wherein said one or more ligand moieties comprise one or more GalNAc ligands or comprise one or more GalNAc ligand derivatives. In a further aspect, the invention relates to an inhibitor according to the invention, wherein said one or more ligand moieties comprise one or more GalNAc ligand derivatives. In a further aspect, the invention relates to an inhibitor according to the invention, wherein the target of the inhibitor is SLC25A5/ANT2.
  • the invention relates to an inhibitor according to the invention, wherein the inhibitor is a nucleic acid for inhibiting expression of SLC25A5 comprising a duplex region that comprises a first strand and a second strand that is at least partially complementary to the first strand, wherein the first strand is: (i) at least partially complementary to a portion of RNA transcribed from the SLC25A5 gene, and (ii) comprises at least 17 contiguous nucleosides differing by 0 or 1 nucleosides from any one of the first strand sequences as listed in Table 2.
  • the inhibitor is a nucleic acid for inhibiting expression of SLC25A5 comprising a duplex region that comprises a first strand and a second strand that is at least partially complementary to the first strand, wherein the first strand is: (i) at least partially complementary to a portion of RNA transcribed from the SLC25A5 gene, and (ii) comprises at least 17 contiguous nucleosides differing by 0 or 1
  • the invention relates to an inhibitor according to the invention, wherein the inhibitor is a nucleic acid for inhibiting expression of SLC25A5 comprising a duplex region that comprises a first strand and a second strand that is at least partially complementary to the first strand, wherein said first strand is: (i) at least partially complementary to a portion of RNA transcribed from the SLC25A5 gene, and (ii) comprises at least 17 contiguous nucleosides differing by 0 or 1 nucleosides from any one of the first strand modified sequences as listed in Table 3.
  • the inhibitor is a nucleic acid for inhibiting expression of SLC25A5 comprising a duplex region that comprises a first strand and a second strand that is at least partially complementary to the first strand, wherein said first strand is: (i) at least partially complementary to a portion of RNA transcribed from the SLC25A5 gene, and (ii) comprises at least 17 contiguous nucleosides differing by 0 or
  • the invention relates to an inhibitor according to the invention, wherein the first strand comprises nucleosides 2-18 of any one of the sequences defined in claim 8 or 9, in particular wherein the first strand comprises nucleosides 2-18 of any one of the sequences defined in Tables 2 or 3.
  • the invention relates to an inhibitor according to the invention, wherein the second strand comprises a nucleoside sequence of at least 17 contiguous nucleosides differing by 0 or 1 nucleosides from any one of the second strand sequences as listed in Table 2, and wherein the second strand has a region of at least 85% complementarity over the 17 contiguous nucleosides to the first strand.
  • the invention relates to an inhibitor according to the invention, wherein the second strand comprises a nucleoside sequence of at least 17 contiguous nucleosides differing by 0 or 1 nucleosides from any one of the second strand modified sequences as listed in Table 4, and wherein the second strand has a region of at least 85% complementarity over the 17 contiguous nucleosides to the first strand.
  • the invention relates to an inhibitor according to the invention, wherein the first strand comprises any one of the first strand sequences as listed in Table 2.
  • the invention relates to an inhibitor according to the invention, wherein the first strand comprises any one of the first strand modified sequences as listed in Table 3.
  • the invention relates to an inhibitor according to the invention, wherein the second strand comprises any one of the second strand sequences as listed in Table 2. In a further aspect, the invention relates to an inhibitor according to the invention, wherein the second strand comprises any one of the first strand modified sequences as listed in Table 4. In a further aspect, the invention relates to an inhibitor according to the invention, wherein the first strand comprises any one of the following sequences: SEQ ID NO:304, SEQ ID NO:323, SEQ ID NO:439, SEQ ID NO:453 and SEQ ID NO:496. In a further aspect, the invention relates to an inhibitor according to the invention, wherein the first strand comprises SEQ ID NO: 304 or SEQ ID NO: 280.
  • the invention relates to an inhibitor according to the invention, wherein the first strand comprises any one of the following sequences: SEQ ID NO:856, SEQ ID NO:875, SEQ ID NO:991, SEQ ID NO:1005 and SEQ ID NO:1048.
  • the invention relates to an inhibitor according to the invention, wherein the first strand comprises SEQ ID NO: 856 or SEQ ID NO: 832.
  • the invention relates to an inhibitor according to the invention, wherein the second strand comprises any one of the following sequences: SEQ ID NO:580, SEQ ID NO:599, SEQ ID NO:715, SEQ ID NO:729 and SEQ ID NO:772.
  • the invention relates to an inhibitor according to the invention, wherein the second strand comprises SEQ ID NO:580 or SEQ ID NO: 556. In a further aspect, the invention relates to an inhibitor according to the invention, wherein the second strand comprises any one of the following sequences: SEQ ID NO:1132, SEQ ID NO:1151, SEQ ID NO:1267, SEQ ID NO:1281 and SEQ ID NO:1324. In a further aspect, the invention relates to an inhibitor according to the invention, wherein the second strand comprises SEQ ID NO:1132 or SEQ ID NO:1108.
  • the invention relates to an inhibitor according to the invention, comprising first and second strands that comprise, consist of, or consist essentially of a nucleoside sequence differing by 0 or 1 nucleosides from any one of the following combinations of first and second sequences: Unmodified first strand Unmodified second strand SEQ ID NO: 304 SEQ ID NO: 580 SEQ ID NO: 323 SEQ ID NO: 599 SEQ ID NO: 439 SEQ ID NO: 715 SEQ ID NO: 453 SEQ ID NO: 729 SEQ ID NO: 496 SEQ ID NO: 772
  • the invention relates to an inhibitor according to the invention, comprising first and second strands that comprise, consist of, or consist essentially of a nucleoside sequence differing by 0 or 1 nucleosides from any one of the following combinations of first and second sequences: Unmodified first strand Unmodified second strand SEQ ID NO: 304 SEQ ID NO: 580
  • the invention relates to an inhibitor according to the invention, comprising
  • the invention relates to an inhibitor according to the invention, wherein the first strand has a length in the range of 17 to 30 nucleosides, preferably 19 to 25 nucleosides, more preferably 19 or 23 nucleosides.
  • the invention relates to an inhibitor according to the invention, wherein the second strand has a length in the range of 17 to 30 nucleosides, preferably 19 to 25 nucleosides, more preferably 19 or 21 or 23 nucleosides.
  • the invention relates to an inhibitor according to the invention, wherein the duplex region of the nucleic acid is between 17 and 30 nucleosides in length, more preferably is 19 or 21 or 23 nucleosides in length.
  • the invention relates to an inhibitor according to the invention, wherein the region of complementarity between the first strand and the portion of RNA transcribed from the SLC25A5 gene is between 17 and 30 nucleosides in length.
  • the invention relates to an inhibitor according to the invention, wherein the nucleic acid further comprises one or more single-stranded nucleoside overhangs, optionally wherein the overhang is present on the first or second strand, preferably at the 3’ terminus of the first or second strand, and/or wherein the overhang comprises 1 to 4 nucleosides, more preferably 2 nucleosides.
  • the invention relates to an inhibitor according to the invention, wherein the nucleic acid is an siRNA oligonucleoside.
  • the invention relates to an inhibitor according to the invention, wherein the second sense strand further comprises one or more abasic nucleosides in a terminal region of the second strand, and wherein said abasic nucleoside(s) is / are connected to an adjacent nucleoside through a reversed internucleoside linkage.
  • the invention relates to an inhibitor according to the invention, wherein the second strand comprises: i) 2, or more than 2, abasic nucleosides in a terminal region of the second strand; and / or ii) 2, or more than 2, abasic nucleosides in either the 5’ or 3’ terminal region of the second strand; and / or iii) 2, or more than 2, abasic nucleosides in either the 5’ or 3’ terminal region of the second strand, wherein the abasic nucleosides are present in an overhang as herein described; and / or iv) 2, or more than 2, consecutive abasic nucleosides in a terminal region of the second strand, wherein preferably one such abasic nucleoside is a terminal nucleoside; and / or v) 2, or more than 2, consecutive abasic nucleosides in either the 5’ or 3’ terminal region of the second strand, wherein preferably one such
  • the invention relates to an inhibitor according to the invention, wherein the reversed internucleoside linkage is at a terminal region which is distal to the 5’ terminal region of the second strand, or at a terminal region which is distal to the 3’ terminal region of the second strand.
  • the invention relates to an inhibitor according to the invention, wherein the reversed internucleoside linkage is a 3’3 reversed linkage.
  • the invention relates to an inhibitor according to the invention, wherein the reversed internucleoside linkage is a 5’5 reversed linkage.
  • the invention relates to an inhibitor according to the invention, wherein the second sense strand further comprises one or more abasic nucleosides in a terminal region of the second strand, and wherein said abasic nucleoside(s) is / are connected to an adjacent nucleoside through a reversed internucleoside linkage.
  • the invention relates to an inhibitor according to the invention, wherein the second strand comprises 2 consecutive abasic nucleosides in the 5’ terminal region of the second strand, wherein one such abasic nucleoside is a terminal nucleoside at the 5’ terminal region of the second strand and the other abasic nucleoside is a penultimate nucleoside at the 5’ terminal region of the second strand, wherein: (a) said penultimate abasic nucleoside is connected to an adjacent first basic nucleoside in an adjacent 5’ near terminal region through a reversed internucleoside linkage; and (b) the reversed linkage is a 5-5’ reversed linkage; and (c) the linkage between the terminal and penultimate abasic nucleosides is 3’5’ when reading towards the terminus comprising the terminal and penultimate abasic nucleosides.
  • An inhibitor, or inhibitor for use, according to the invention wherein the reversed internucleoside linkage is at a terminal region which is distal to the 5’ terminal region of the second strand, or at a terminal region which is distal to the 3’ terminal region of the second strand.
  • the invention relates to an inhibitor according to the invention, wherein (i) the first strand and the second strand each has a length of 23 nucleosides; (ii) two phosphorothioate internucleoside linkages are respectively between three consecutive positions in said 5’ near terminal region of the second strand, wherein a first phosphorothioate internucleoside linkage is present between said adjacent first basic nucleoside of (a) and an adjacent second basic nucleoside in said 5’ near terminal region of the second strand, and a second phosphorothioate internucleoside linkage is present between said adjacent second basic nucleoside and an adjacent third basic nucleoside in said 5’ near terminal region of the second strand; (iii) two phosphorothioate internucleoside linkages are respectively between three consecutive positions in both 5’ and 3’ terminal regions of the first strand, whereby a terminal nucleoside respectively at each of the 5’ and 3’ terminal regions of said first strand
  • the invention relates to an inhibitor according to the invention, wherein the 2 consecutive inverted abasic nucleosides in the 5’ terminal region of the second strand present as the following 5’ terminal motif wherein: T represents a 2’Me ribose modification, B represents the nucleoside bases of the first two basic nucleosides in the 5' terminal region of the second strand, and Z represents the remaining 19 contiguous basic nucleosides of said second strand.
  • T represents a 2’Me ribose modification
  • B represents the nucleoside bases of the first two basic nucleosides in the 5' terminal region of the second strand
  • Z represents the remaining 19 contiguous basic nucleosides of said second strand.
  • the invention relates to an inhibitor according to the invention, wherein one or more nucleosides on the first strand and / or the second strand is / are modified, to form modified nucleosides.
  • the invention relates to an inhibitor according to the invention, wherein the modification is a modification at the 2’-OH group of the ribose sugar, optionally selected from 2'-Me or 2’-F modifications.
  • the invention relates to an inhibitor according to the invention, wherein the first strand comprises a 2’-F at any of position 14, position 2, position 6, or any combination thereof, counting from position 1 of said first strand.
  • the invention relates to an inhibitor according to the invention, wherein the second strand comprises a 2’-F modification at position 7 and / or 9, and / or 11 and / or 13, counting from position 1 of said second strand.
  • the invention relates to an inhibitor according to the invention, wherein the first and second strand each comprise 2'-Me and 2’-F modifications.
  • the invention relates to an inhibitor according to the invention, which is an siRNA, wherein the siRNA comprises at least one thermally destabilizing modification, suitably at one or more of positions 1 to 9 of the first strand counting from position 1 of the first strand, and / or at one or more of positions on the second strand aligned with positions 1 to 9 of the first strand, wherein the destabilizing modification is selected from a modified unlocked nucleic acid (UNA) and a glycol nucleic acid (GNA), preferably a glycol nucleic acid.
  • UUA modified unlocked nucleic acid
  • GNA glycol nucleic acid
  • the invention relates to an inhibitor according to the invention, wherein the siRNA comprises at least one thermally destabilizing modification at position 7 of the first strand, counting from position 1 of the first strand.
  • the invention relates to an inhibitor according to the invention, which is an siRNA, wherein the siRNA comprises 3 or more 2’-F modifications at positions 7 to 13 of the second strand, such as 4, 5, 6 or 72’-F modifications at positions 7 to 13 of the second strand, counting from position 1 of said second strand
  • the invention relates to an inhibitor according to the invention, which is an siRNA, wherein said second strand comprises at least 3, such as 4, 5 or 6, 2’-Me modifications at positions 1 to 6 of the second strand, counting from position 1 of said second strand.
  • the invention relates to an inhibitor according to the invention, which is an siRNA, wherein said first strand comprises at least 52’-Me consecutive modifications at the 3’ terminal region, preferably including the terminal nucleoside at the 3’ terminal region, or at least within 1 or 2 nucleosides from the terminal nucleoside at the 3’ terminal region.
  • the invention relates to an inhibitor according to the invention, which is an siRNA wherein said first strand comprises 72’-Me consecutive modifications at the 3’ terminal region, preferably including the terminal nucleoside at the 3’ terminal region.
  • the invention relates to an inhibitor according to the invention, wherein the siRNA oligomer further comprises one or more phosphorothioate internucleoside linkages.
  • the invention relates to an inhibitor according to the invention, wherein said one or more phosphorothioate internucleoside linkages are respectively between at least three consecutive positions in a 5’ or 3’ near terminal region of the second strand, whereby said near terminal region is preferably adjacent said terminal region wherein said one or more abasic nucleosides of said second strand is / are located as defined herein.
  • the invention relates to an inhibitor according to the invention, wherein said one or more phosphorothioate internucleoside linkages are respectively between at least three consecutive positions in a 5’ and / or 3’ terminal region of the first strand, whereby preferably a terminal position at the 5’ and / or 3’ terminal region of said first strand is attached to its adjacent position by a phosphorothioate internucleoside linkage.
  • the invention relates to an inhibitor according to the invention, wherein the oligomer is an siRNA and the second strand of the siRNA is conjugated directly or indirectly to one or more ligand moiety(s), wherein said ligand moiety is typically present at a terminal region of the second strand, preferably at the 3’ terminal region thereof.
  • the invention relates to an inhibitor according to the invention, wherein the ligand moiety comprises i) one or more GalNAc ligands; and / or ii) one or more GalNAc ligand derivatives; and / or iii) one or more GalNAc ligands and / or GalNAc ligand derivatives conjugated to said siRNA through a linker.
  • the invention relates to an inhibitor according to the invention, wherein said one or more GalNAc ligands and / or GalNAc ligand derivatives are conjugated directly or indirectly to the 5’ or 3’ terminal region of the second strand of the siRNA oligomer, preferably at the 3’ terminal region thereof.
  • the invention relates to an inhibitor according to the invention, wherein the ligand moiety comprises
  • the invention relates to an inhibitor according to the invention, comprising the structure , wherein oligonucleotide represents the contiguous nucleosides of the second strand.
  • the invention relates to an inhibitor according to the invention, comprising the structure wherein: r and s are independently an integer selected from 1 to 16; and Z is an oligonucleoside moiety.
  • the invention relates to an inhibitor according to the invention, comprising the structure , wherein oligonucleotide represents the contiguous nucleosides of the second strand.
  • the invention relates to an inhibitor according to the invention, wherein the structure is conjugated to the 3’ terminal region of the second strand.
  • the invention relates to an inhibitor according to the invention, formulated as a pharmaceutical composition with an excipient and / or carrier.
  • the invention relates to a pharmaceutical composition comprising an inhibitor according to one or more preceding aspects, in combination with a pharmaceutically acceptable excipient or carrier.
  • the invention relates to the pharmaceutical composition according to the invention, further comprising a GLP-1 agonist and/or a THR-beta agonist.
  • the invention relates to the pharmaceutical composition according to the invention, wherein the GLP-1 agonist is a GLP-1/GIP dual agonist, a GLP-1/FGF21 dual agonist, a GLP-1/GCGR dual agonist, or a GLP-1/GIP/GCGR triple agonist.
  • the invention relates to the pharmaceutical composition according to the invention, wherein the GLP-1 agonist is semaglutide.
  • the invention relates to the pharmaceutical composition according to the invention, wherein the THR-beta agonist is resmetirom.
  • the invention relates to the pharmaceutical composition according to the invention, wherein the GLP-1/GIP dual agonist is tirzepatide.
  • the invention relates to the pharmaceutical composition according to the invention, further comprising one or more of: an amylin receptor agonist (such as pramlintide), and/or a dual amylin + calcitonin receptor agonist, and/or a glucagon receptor agonist, and/or an FXR receptor agonist (such as cilofexor or obeticholic acid), and/or an FGF-21 analogue or FGF-21 receptor agonist (such as efruxifermin), and/or an FGF-19 analogue or FGF-19 receptor agonist (such as aldafermin), and/or a galectin 3 inhibitor (such as belapectin), and/or a PPAR ⁇ agonist (such as elafibrinor), and/or a PPAR ⁇ agonist (such as pioglitazone or rosiglitazone), and/or a mixed PPAR ⁇ and/or ⁇ and/or ⁇ agonist,
  • the invention relates to an inhibitor according to the invention or a pharmaceutical composition according to the invention for use in therapy.
  • the invention relates to an inhibitor according to the invention or a pharmaceutical composition according to the invention for use in the prevention and/or treatment of metabolic disease or disorder, such as a metabolic disease or disorder associated with non-alcoholic fatty liver disease (NAFLD), and/or obesity and/or a disease or disorder associated with adipogenesis and/or for use in reducing adipogenesis.
  • the invention relates to an inhibitor for use according to the invention or a pharmaceutical composition for use according to the invention, wherein the inhibitor or the pharmaceutical composition is to be used in combination with a GLP-1 agonist and/or a THR-beta agonist.
  • the invention relates to an inhibitor for use according to the invention or a pharmaceutical composition for use according to the invention, wherein the GLP-1 agonist is a GLP-1/GIP dual agonist, a GLP-1/FGF21 dual agonist, a GLP-1/GCGR dual agonist, or a GLP-1/GIP/GCGR triple agonist.
  • the invention relates to an inhibitor for use according to the invention or a pharmaceutical composition for use according to the invention, wherein the GLP-1 agonist is semaglutide.
  • the invention relates to an inhibitor for use according to the invention or a pharmaceutical composition for use according to the invention, wherein the THR-beta agonist is resmetirom.
  • the invention relates to an inhibitor for use according to the invention or a pharmaceutical composition for use according to the invention, wherein the GLP-1/GIP dual agonist is tirzepatide.
  • the invention relates to an inhibitor for use according to the invention or a pharmaceutical composition for use according to the invention, wherein the inhibitor or the pharmaceutical composition is to be used in combination with one or more of: an amylin receptor agonist (such as pramlintide), and/or a dual amylin + calcitonin receptor agonist, and/or a glucagon receptor agonist, and/or an FXR receptor agonist (such as cilofexor or obeticholic acid), and/or an FGF-21 analogue or FGF-21 receptor agonist (such as efruxifermin), and/or an FGF-19 analogue or FGF-19 receptor agonist (such as aldafermin), and/or a galectin 3 inhibitor (such as belapectin), and/
  • the invention relates to the use of SLC25A5/ANT2 as a target for identifying one or more therapeutic agents for the treatment or prevention of a metabolic disease or disorder, such as a metabolic disease or disorder associated with non-alcoholic fatty liver disease (NAFLD), and/or obesity and/or a disease or disorder associated with adipogenesis and/or for reducing adipogenesis.
  • a metabolic disease or disorder such as a metabolic disease or disorder associated with non-alcoholic fatty liver disease (NAFLD), and/or obesity and/or a disease or disorder associated with adipogenesis and/or for reducing adipogenesis.
  • NAFLD non-alcoholic fatty liver disease
  • the invention relates to a method of treating or preventing metabolic disease or disorder, such as a metabolic disease or disorder associated with non-alcoholic fatty liver disease (NAFLD), and/or obesity and/or a disease or disorder associated with adipogenesis and/or a method of reducing adipogenesis, which comprises administering to a patient an inhibitor of expression and/or function of SLC25A5/ANT2, such as an inhibitor according to the invention.
  • the invention relates to a method according to the invention, wherein the inhibitor of expression and/or function of SLC25A5/ANT2 is administered together with a GLP-1 agonist and/or a THR-beta agonist.
  • the invention relates to a method according to the invention, wherein the GLP-1 agonist is a GLP-1/GIP dual agonist, a GLP-1/FGF21 dual agonist, a GLP-1/GCGR dual agonist, or a GLP- 1/GIP/GCGR triple agonist.
  • the invention relates to a method according to the invention, wherein the GLP-1 agonist is semaglutide.
  • the invention relates to a method according to the invention, wherein the GLP-1/GIP dual agonist is tirzepatide.
  • the invention relates to a method according to the invention, wherein the THR-beta agonist is resmetirom.
  • the invention relates to a method according to the invention, wherein the inhibitor of expression and/or function of SLC25A5/ANT2 is administered together with one or more of: an amylin receptor agonist (such as pramlintide), and/or a dual amylin + calcitonin receptor agonist, and/or a glucagon receptor agonist, and/or an FXR receptor agonist (such as cilofexor or obeticholic acid), and/or an FGF-21 analogue or FGF-21 receptor agonist (such as efruxifermin), and/or an FGF-19 analogue or FGF-19 receptor agonist (such as aldafermin), and/or a galectin 3 inhibitor (such as belapectin), and/or a PPAR ⁇ agonist (such as elafibrinor), and/or a PPAR ⁇ agonist (such as pioglitazone or rosiglitazone), and/or
  • the invention relates to the use of an inhibitor according to the invention or a pharmaceutical composition according to the invention, in the preparation of a medicament for the treatment of a metabolic disease or disorder, such as a metabolic disease or disorder associated with non- alcoholic fatty liver disease (NAFLD), and/or obesity and/or a disease or disorder associated with adipogenesis and/or for reducing adipogenesis
  • a metabolic disease or disorder such as a metabolic disease or disorder associated with non- alcoholic fatty liver disease (NAFLD), and/or obesity and/or a disease or disorder associated with adipogenesis.
  • NAFLD non-alcoholic fatty liver disease
  • the invention relates to SLC25A5/ANT2 for use as a biomarker of a metabolic disease or disorder, such as a metabolic disease or disorder associated with non-alcoholic fatty liver disease (NAFLD), and/or obesity and/or a disease or disorder associated with adipogenesis.
  • the invention relates to SLC25A5/ANT2 for use in an in vivo method of predicting susceptibility to prevention and/or treatment of metabolic disease or disorder, such as a metabolic disease or disorder associated with non-alcoholic fatty liver disease (NAFLD), and/or obesity and/or a disease or disorder associated with adipogenesis, typically by monitoring the sequence and/ or level of expression and / or function of SLC25A5/ANT2 in a sample obtained from a patient.
  • metabolic disease or disorder such as a metabolic disease or disorder associated with non-alcoholic fatty liver disease (NAFLD), and/or obesity and/or a disease or disorder associated with adipogenesis
  • the invention in another aspect, relates to a method of predicting susceptibility to a metabolic disease or disorder, such as a metabolic disease or disorder associated with non-alcoholic fatty liver disease (NAFLD), and/or obesity and/or a disease or disorder associated with adipogenesis, in a patient, said method comprising: (a) obtaining a sample from the patient, (b) detecting the sequence and / or expression and / or function of SLC25A5/ANT2 in said sample obtained from the patient, (c) predicting susceptibility to a disease related to a metabolic disease or disorder, such as a metabolic disease or disorder associated with non-alcoholic fatty liver disease (NAFLD) and/or obesity and/or a disease or disorder associated with adipogenesis, based on the sequence and / or expression and / or function of SLC25A5/ANT2 in said sample obtained from the patient, (d) preferably administering to the diagnosed patient an effective amount of an inhibitor of SLC25A5/ANT2.
  • a metabolic disease or disorder such
  • FIGURES Figure 1a An exemplary linear configuration for a conjugate.
  • Figure 1b An exemplary branched configuration for a conjugate.
  • Figure 2 Linker and ligand portions of constructs suitable for use according to the present invention including tether 1a. While Figure 2 depicts the linker to be conjugated to an oligonucleotide, it is to be understood that the present invention also encompasses conjugates of the same linker with an oligonucleoside as disclosed herein.
  • Figure 2 depicts as a product molecules based on the linker and ligand portions as specifically depicted in Figure 2 attached to an oligonucleoside moiety as also depicted herein
  • this product may alternatively further comprise, or consist essentially of, molecules wherein the linker and ligand portions are essentially as depicted in Figure 2 attached to an oligonucleoside moiety but having the F substituent as shown in Figure 2 on the cyclo-octyl ring replaced by a substituent, which could occur as a result of hydrolytic displacement, such as an OH substituent, or the OH substituent could be synthesized as a linker in its own right.
  • tether 1a constructs can consist essentially of molecules having linker and ligand portions specifically as depicted in Figure 2, with a F substituent on the cyclo-octyl ring; or (b) tether 1a constructs can consist essentially of molecules having linker and ligand portions essentially as depicted in Figure 2 but having the F substituent as shown in Figure 2 on the cyclo- octyl ring replaced by an OH substituent, or (c) tether 1a constructs can comprise a mixture of molecules as defined in (a) and/or (b).
  • Figure 3 Linker and ligand portions of constructs suitable for use according to the present invention including tether 1b.
  • Figure 3 depicts the linker to be conjugated to an oligonucleotide
  • the present invention also encompasses conjugates of the same linker with an oligonucleoside as disclosed herein.
  • tether 1b constructs can consist essentially of molecules having linker and ligand portions specifically as depicted in Figure 3, with a F substituent on the cyclo-octyl ring; or (b) tether 1b constructs can consist essentially of molecules having linker and ligand portions essentially as depicted in Figure 3 but having the F substituent as shown in Figure 3 on the cyclo- octyl ring replaced by an OH substituent, or (c) tether 1b constructs can comprise a mixture of molecules as defined in (a) and/or (b).
  • Figure 4 Linker and ligand portions of constructs suitable for use according to the present invention including tether 2a.
  • Figure 4 depicts the linker to be conjugated to an oligonucleotide, it is to be understood that the present invention also encompasses conjugates of the same linker with an oligonucleoside as disclosed herein.
  • Figure 5 Linker and ligand portions of constructs suitable for use according to the present invention including tether 2b. While Figure 5 depicts the linker to be conjugated to an oligonucleotide, it is to be understood that the present invention also encompasses conjugates of the same linker with an oligonucleoside as disclosed herein.
  • Figure 6 Formulae described in Sentences 1-101 disclosed herein.
  • Figure 7 Formulae described in Clauses 1-56 disclosed herein.
  • Figures 8a and 8b Inverted abasic constructs that can be used with nucleic acid sequences according to the present invention as described herein.
  • a GalNAc linker is attached to the 5’ end region of the sense strand in use (not depicted in Figure 8a).
  • a GalNAc linker is attached to the 3’ end region of the sense strand in use (not depicted in Figure 8b).
  • iaia as shown at the 3’ end region of the sense strand in Figure 8a represents (i) two abasic nucleosides provided as the penultimate and terminal nucleosides at the 3’ end region of the sense strand, (ii) wherein a 3’-3’ reversed linkage is provided between the antepenultimate nucleoside (namely at position 21 of the sense strand, wherein position 1 is the terminal 5’ nucleoside of the sense strand) and the adjacent penultimate abasic residue of the sense strand, and (iii) the linkage between the terminal and penultimate abasic nucleosides is 5’-3’ when reading towards the 3’ end region comprising the terminal and penultimate abasic nucleosides.
  • iaia as shown at the 5’ end region of the sense strand in Figure 8b represents (i) two abasic nucleosides provided as the penultimate and terminal nucleosides at the 5’ end region of the sense strand, (ii) wherein a 5’-5’ reversed linkage is provided between the antepenultimate nucleoside (namely at position 1 of the sense strand, not including the iaia motif at the 5’ end region of the sense strand in the nucleoside position numbering on the sense strand) and the adjacent penultimate abasic residue of the sense strand, and (iii) the linkage between the terminal and penultimate abasic nucleosides is 3’-5’ when reading towards the 5’ end region comprising the terminal and penultimate abasic nucleosides.
  • Figures 9a and 9b Duplex constructs according to Table 5.
  • Figures 10 Summary of mRNA and protein knockdown effects of a single dose of GalNAc-siRNAs, ETX- M00001397, ETX-M00001570, ETX-M00001378, ETX-M00001513, and ETX-M00001527 (1 mg/kg or 3 mg/kg) in mouse liver tissues.
  • a negative control a non-target specific siRNA was used.
  • FIG 11 Liver samples stained with H&E were given a score for NAFLD Activity Score (NAS) using the clinical criteria outlined by Kleiner et al. (2005).
  • Total NAS represents the sum of scores for steatosis, inflammation, and ballooning, and ranges from 0-8.
  • NAS score was determined by Gubra Histopathological Objective Scoring Technology (GHOST) deep learning app developed by Gubra using the VIS software (Visiopharm, Denmark) for a more accurate and objective method for staging disease in DIO-NASH mouse models. Results are presented as change in NAS score (improvement or worsening) at study termination compared to the pre-dose biopsy. Also shown is the percentage of animals with at least a 1- or 2-point improvement in NAS.
  • GHOST Gubra Histopathological Objective Scoring Technology
  • TIMP-1 and PIIINP are non-invasive blood biomarkers for NAFLD/NASH that predict hepatic fibrosis.
  • TIMP-1 was measured in plasma collected in EDTA tubes using a commercial ELISA kit (R&D Systems).
  • PIIINP was measured in plasma collected in EDTA tubes using a commercial ELISA kit (Cusabio).
  • TIMP-1 and PIIINP levels were increased in DIO-NASH mice (vehicle sc, siCtrl, vehicle PO).
  • FIG. 15 Overview of Human Hydrodynamic Injection (HDI) Mouse study.
  • Figure 16 Human SLC25A5 mRNA knockdown in Hydrodynamic Injection (HDI) mouse model. Mice received siRNA (3 mg/kg) subcutaneously on Day -6, followed by HDI of human SLC25A5 plasmid on Day 0. qPCR on Day 1 confirmed reduced human SLC25A5 mRNA expression, with several siRNAs including ETX-M00001378 (ETX-M00001751) and ETX-M00001354 (ETX-M00002666) showing the most significant knockdown relative to controls.
  • ETX-M00001378 ETX-M00001751
  • ETX-M00001354 ETX-M00002666
  • ALT Alanine Transaminase
  • AST Aspartate Transaminase
  • ALP Alkaline Phosphatase
  • GGT Gamma-Glutamyltransferase. Dashed lines indicate the reference range for normal values.
  • DETAILED DESCRIPTION The present invention, inter alia, provides inhibitors, for example oligomers such as nucleic acids, such as inhibitory RNA molecules (which may be referred to as iRNA or siRNA), and compositions containing the same which can affect expression of a target, for example by binding to mRNA transcribed from a gene.
  • the target may be within a cell, e.g.
  • the inhibitors can be used to prevent and/or treat medical conditions associated with the e.g. the expression of a target gene.
  • the present invention identifies inhibitors of expression and/or function of SLC25A5/ANT2 as useful in the prevention and/or treatment of a metabolic disease or disorder, such as a metabolic disease or disorder associated with non-alcoholic fatty liver disease (NAFLD) and/or obesity and/or a disease or disorder associated with adipogenesis.
  • ADP/ATP translocase 2 (ANT2) is a protein that in humans is encoded by the SLC25A5 gene on the X chromosome. This protein functions as an antiporter for ADP/ATP exchange between the mitochondrial matrix and cytoplasm.
  • the present invention relates to an inhibitor of expression and/or function of SLC25A5/ANT2. Accordingly, in certain embodiments, the invention relates to an inhibitor of expression of the SLC25A5 gene, such as an siRNA that targets an mRNA transcribed from the SLC25A5 gene. In certain embodiments, the invention relates to an inhibitor of function of the gene product ANT2. Both options are encompassed when it referred herein to an inhibitor of SLC25A5/ANT2 or an inhibitor of the invention. In humans, ANT2 is encoded by the SLC25A5 gene (SEQ ID NO:1381).
  • the approach takes advantage of information that is usually ignored in standard gene set analyses – the known and predicted interactions between genes (and proteins) and the inclusion of other genes in the same or related pathways.
  • GWAS Genome Wide Association Studies
  • NAFLD Non-Alcoholic Fatty Liver Disease
  • the inhibition disclosed herein may be of the gene SLC25A5 or protein ANT2 resulting from expression of the SLC25A5 gene and reference to SLC25A5/ANT2 hereby explicitly incorporates a reference to inhibition of the expression or function of the gene and, separately, of the protein product.
  • first strand also called the antisense strand or guide strand herein and which can be used interchangeably herein, refers to the nucleic acid strand, e.g. the strand of an siRNA, e.g. a dsiRNA, which includes a region that is substantially complementary to a target sequence, e.g. to an mRNA.
  • region of complementarity refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence. Where the region of complementarity is not fully complementary to the target sequence, the mismatches can be in the internal or terminal regions of the molecule.
  • a double stranded nucleic acid e.g. an siRNA agent of the invention includes a nucleotide mismatch in the antisense strand.
  • the “second strand” also called the sense strand or passenger strand herein, and which can be used interchangeably herein, refers to the strand of a nucleic acid e.g. siRNA that includes a region that is substantially complementary to a region of the antisense strand as that term is defined herein.
  • nucleic acid of the invention may be referred to as an oligonucleotide moiety or oligonucleoside moiety
  • Oligonucleotides are short nucleic acid polymers. Whilst oligonucleotides contain phosphodiester bonds between the nucleoside component thereof (base plus sugar), the present invention is not limited to oligonucleotides always joined by such a phosphodiester bond between adjacent nucleosides, and other oligomers of nucleosides joined by bonds which are bonds other than a phosphate bond are contemplated.
  • a bond between nucleotides may be a phosphorothioate bond. Therefore, the term “oligonucleoside” herein covers both oligonucleotides and other oligomers of nucleosides.
  • An oligonucleoside which is a nucleic acid having at least a portion which is an oligonucleotide is preferred according to the present invention.
  • An oligonucleoside having one or more, or a majority of, phosphodiester backbone bonds between nucleosides is also preferred according to the present invention.
  • an oligonucleoside having one or more, or a majority of, phosphodiester backbone bonds between nucleosides, and also having one or more phosphorothioate backbone bonds between nucleosides (typically in a terminal region of the first and / or second strands) is also preferred according to the present invention. It is preferred herein that the nucleic acid according to the invention is a double stranded oligonucleoside comprising one or more phosphorothioate backbone bonds between nucleosides.
  • a double stranded nucleic acid e.g. siRNA agent of the invention includes a nucleoside mismatch in the sense strand.
  • the nucleoside mismatch is, for example, within 5, 4, 3, 2, or 1 nucleosides from the 3 '-end of the nucleic acid e.g. siRNA.
  • the nucleoside mismatch is, for example, in the 3'- terminal nucleoside of the nucleic acid e.g. siRNA.
  • a "target sequence” (which may be called a target RNA or a target mRNA) refers to a contiguous portion of the nucleoside sequence of an mRNA molecule formed during the transcription of a gene, including mRNA that is a product of RNA processing of a primary transcription product, or can be a contiguous portion of the nucleotide sequence of any RNA molecule such as a LNCRNA which it is desired to inhibit.
  • the target sequence may be from about 10-35 nucleosides in length, e.g., about 15-30 nucleosides in length.
  • the target sequence can be from about 15-30 nucleosides, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18- 28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20- 21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleosides in length.
  • ribonucleoside or “nucleoside” can also refer to a modified nucleoside as further detailed below.
  • a nucleic acid can be a DNA or an RNA, and can comprise modified nucleosides.
  • RNA is a preferred nucleic acid.
  • iRNA RNA-induced silencing complex
  • RISC RNA-induced silencing complex
  • siRNA directs the sequence- specific degradation of mRNA through RNA interference (RNAi).
  • RNAi RNA interference
  • a double stranded RNA is referred to herein as a “double stranded siRNA (dsiRNA) agent", “double stranded siRNA (dsiRNA) molecule”, “double stranded RNA (dsRNA) agent”, “double stranded RNA (dsRNA) molecule”, “dsiRNA agent”, “dsiRNA molecule”, or “dsiRNA”, which refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary nucleic acid strands, referred to as having "sense” and “antisense” orientations with respect to a target RNA.
  • nucleosides of each strand of the nucleic acid are preferably ribonucleosides, but in that case each or both strands can also include one or more non- ribonucleosides, e.g., a deoxyribonucleoside or a modified ribonucleoside.
  • an "siRNA” may include ribonucleosides with chemical modifications.
  • modified nucleoside refers to a nucleoside having, independently, a modified sugar moiety, a modified internucleoside linkage, or modified nucleobase, or any combination thereof.
  • modified nucleoside encompasses substitutions, additions or removal of, e.g., a functional group or atom, to internucleoside linkages, sugar moieties, or nucleobases. Any such modifications, as used in a siRNA type molecule, are encompassed by "iRNA” or “RNAi agent” or “siRNA” or “siRNA agent” for the purposes of this specification and claims.
  • iRNA or “RNAi agent” or “siRNA” or “siRNA agent” for the purposes of this specification and claims.
  • the duplex region of a nucleic acid of the invention e.g.
  • a dsRNA may range from about 9 to 40 base pairs in length such as 9 to 36 base pairs in length, e.g., about 15- 30 base pairs in length, for example, about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, such as about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18- 27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21- 30, 21-29, 21-28,
  • nucleoside overhang refers to at least one unpaired nucleoside that extends from the duplex structure of a double stranded nucleic acid.
  • a ds nucleic acid can comprise an overhang of at least one nucleoside; alternatively, the overhang can comprise at least two nucleosides, at least three nucleosides, at least four nucleosides, at least five nucleosides, or more.
  • a nucleoside overhang can comprise or consist of a nucleoside analog, including a deoxynucleoside.
  • the overhang(s) can be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the /nucleoside(s) of an overhang can be present on the 5'-end, 3'-end, or both ends of either an antisense or sense strand.
  • the antisense strand has a 1-10 nucleoside, e.g., 0-3, 1-3, 2-4, 2-5, 4-10, 5-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleoside overhang at the 3'-end or the 5'-end.
  • Bosset or Blunt end means that there are no unpaired nucleoside at that end of the double stranded nucleic acid, i.e., no nucleoside overhang.
  • the nucleic acids of the invention include those with no nucleoside overhang at one end or with no nucleoside overhangs at either end.
  • the term "complementary,” when used to describe a first nucleoside sequence in relation to a second nucleoside sequence, refers to the ability of an oligonucleoside comprising the first nucleoside sequence to hybridize and form a duplex structure under certain conditions with an oligonucleoside or polynucleoside comprising the second nucleoside sequence, as will be understood by the skilled person.
  • Such conditions can, for example, be stringent conditions, where stringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C for 12-16 hours followed by washing (see, e.g., "Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Complementary sequences within nucleic acid e.g.
  • a dsiRNA as described herein, include base-pairing of the oligonucleoside or polynucleoside comprising a first nucleoside sequence to an oligonucleoside or polynucleoside comprising a second nucleoside sequence over the entire length of one or both nucleoside sequences.
  • sequences can be referred to as "fully complementary” with respect to each other herein.
  • first sequence is referred to as “substantially complementary” or “partially complementary”with respect to a second sequence herein
  • the two sequences can be fully complementary, or they can form one or more mismatched base pairs, such as 2, 4, or 5 mismatched base pairs, but preferably not more than 5, while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., inhibition of gene expression via a RISC pathway.
  • Overhangs shall not be regarded as mismatches with regard to the determination of complementarity.
  • a nucleic acid e.g.
  • dsRNA comprising one oligonucleoside 17 nucleosides in length and another oligonucleoside 19 nucleosides in length, wherein the longer oligonucleoside comprises a sequence of 17 nucleosides that is fully complementary to the shorter oligonucleoside, can yet be referred to as "fully complementary”.
  • "Complementary" sequences can also include, or be formed entirely from, non-Watson- Crick base pairs or base pairs formed from non-natural and modified nucleosides, in so far as the above requirements with respect to their ability to hybridize are fulfilled.
  • Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogstein base pairing.
  • the terms “complementary,” “fully complementary” and “substantially/partially complementary” herein can be used with respect to the base matching between the sense strand and the antisense strand of a nucleic acid e.g. dsiRNA, or between the antisense strand of a double stranded nucleic acid e.g. siRNA agent and a target sequence.
  • the second strand of the nucleic acid according to the invention in particular a dsiRNA for inhibiting SLC25A5
  • a first and second strand of a nucleic acid according to the invention are partially complementary if they form a duplex region having a length of at least 17 base pairs and comprising not more than 1, 2, 3, 4, or 5 mismatched base pairs. In certain embodiments, a first and second strand of the nucleic acid according to the invention are partially complementary if they form a duplex region having a length of 19 base pairs and comprising not more than 1, 2, 3, 4, or 5 mismatched base pairs. In certain embodiments, a first and second strand of the nucleic acid according to the invention are partially complementary if they form a duplex region having a length of 21 base pairs comprising not more than 1, 2, 3, 4, or 5 mismatched base pairs.
  • a first and second strand of the nucleic acid according to the invention are partially complementary if they form a duplex region having a length of at least 17 base pairs, wherein at least 14, 15, 16 or 17 of said base pairs are complementary base pairs, in particular Watson-Crick base pairs.
  • a first and second strand of the nucleic acid according to the invention are partially complementary if they form a duplex region having a length of 19 base pairs, wherein at least 14, 15, 16, 17, 18 or all 19 base pairs are complementary base pairs, in particular Watson-Crick base pairs.
  • a first and second strand of the nucleic acid according to the invention are partially complementary if they form a duplex region having a length of 21 base pairs, wherein at least 16, 17, 18, 19, 20 or all 21 base pairs are complementary base pairs, in particular Watson-Crick base pairs.
  • a nucleic acid that is "substantially complementary” or “partially complementary” to at least part of a messenger RNA (mRNA) refers to a polynucleoside that is substantially or partially complementary to a contiguous portion of the mRNA of interest (e.g., an mRNA encoding a gene).
  • the contiguous portion of the mRNA is a sequence as listed in Table 1, i.e., any one of SEQ ID NOs:1-276.
  • a polynucleoside is complementary to at least a part of an mRNA of a gene of interest if the sequence is substantially or partially complementary to a non-interrupted portion of an mRNA encoding that gene.
  • the antisense oligonucleosides as disclosed herein are fully complementary to the target gene sequence.
  • the antisense oligonucleosides disclosed herein are substantially or partially complementary to a target RNA sequence and comprise a contiguous nucleoside sequence which is at least about 80% complementary over its entire length to the equivalent region of the target RNA sequence, such as at least about 85%, 86%, 87%, 88%, 89%, about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary or 100% complementary.
  • the first (antisense) strand of a nucleic acid according to the invention is partially or fully complementary to a contiguous portion of RNA transcribed from the SLC25A5 gene.
  • the first strand of the nucleic acid according to the invention is partially or fully complementary to a contiguous portion of at least 17 nucleosides of the SLC25A5 mRNA. In certain embodiments, the first strand of the nucleic acid according to the invention is partially or fully complementary to a contiguous portion of 17, 18, 19, 20, 21, 22 or 23 nucleosides of the SLC25A5 mRNA. In certain embodiments, the first strand of the nucleic acid according to the invention is partially or fully complementary to a contiguous portion of 17, 18, 19, 20, 21, 22 or 23 nucleosides of any one of the sequences as listed in Table 1, i.e., any one of SEQ ID NOs: 1-276.
  • the first (antisense) strand of the nucleic acid according to the invention is partially complementary to a contiguous portion of the SLC25A5 mRNA if it comprises a contiguous nucleoside sequence of at least 17 nucleosides, wherein at least 14, 15, 16 or 17 nucleosides of said contiguous nucleoside sequence are complementary to a contiguous portion of the SLC25A5 mRNA.
  • the first strand of the nucleic acid according to the invention comprises a contiguous nucleoside sequence of at least 17 nucleosides, wherein at least 14, 15, 16 or 17 nucleosides of said contiguous nucleoside sequence are complementary to a contiguous portion of any one of the sequences listed in Table 1, i.e., any one of SEQ ID NOs: 1-276.
  • the first strand of the nucleic acid according to the invention comprises a contiguous nucleoside sequence of 19 nucleosides, wherein at least 14, 15, 16, 17, 18 or all 19 nucleosides of said contiguous nucleoside sequence are complementary to a contiguous portion of any one of the sequences listed in Table 1, i.e., any one of SEQ ID NOs: 1-276.
  • the first strand of the nucleic acid according to the invention comprises a contiguous nucleoside sequence of 23 nucleosides, wherein at least 18, 19, 20, 21, 22 or all 23 nucleosides of said contiguous nucleoside sequence are complementary to a contiguous portion of any one of the sequences listed in Table 1, i.e., any one of SEQ ID NOs: 1-276.
  • a nucleic acid e.g. an siRNA of the invention includes a sense strand that is substantially or partially complementary to an antisense oligonucleoside which, in turn, is complementary to a target gene sequence and comprises a contiguous nucleoside sequence.
  • the nucleoside sequence of the sense strand is typically at least about 80% complementary over its entire length to the equivalent region of the nucleoside sequence of the antisense strand, such as about 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary, or 100% complementary.
  • a nucleic acid e.g.
  • an siRNA of the invention includes an antisense strand that is substantially or partially complementary to the target sequence and comprises a contiguous nucleoside sequence which is at least 80% complementary over its entire length to the target sequence such as about 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary, or 100% complementary.
  • a "subject” is an animal, such as a mammal, including a primate (such as a human, a non- human primate, e.g., a monkey, and a chimpanzee), or a non-primate or a bird that expresses the target gene, either endogenously or heterologously, when the target gene sequence has sufficient complementarity to the nucleic acid e.g. iRNA agent to promote target knockdown.
  • the subject is a human.
  • the terms "treating” or “treatment” refer to a beneficial or desired result including, but not limited to, alleviation or amelioration of one or more symptoms associated with gene expression.
  • Treatment can also mean prolonging survival as compared to expected survival in the absence of treatment.
  • the terms “prevent” or “prevention” as used herein are defined as eliminating or reducing the likelihood of occurrence of one or more symptoms of a disease or disorder.
  • the inhibitor disclosed herein can be used to prevent the occurrence of a metabolic disease or disorder associated with non-alcoholic fatty liver disease (NAFLD), and/or obesity and/or a disease or disorder associated with adipogenesis.
  • NAFLD non-alcoholic fatty liver disease
  • Therapeutically effective amount is intended to include the amount of a nucleic acid e.g.
  • RNA that, when administered to a patient for treating a subject having disease, is sufficient to effect treatment of the disease (e.g., by diminishing, ameliorating or maintaining the existing disease or one or more symptoms of disease or its related comorbidities).
  • pharmaceutically acceptable is employed herein to refer to compounds, materials, compositions, or dosage forms which are suitable for use in contact with the tissues of human subjects and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically-acceptable carrier means a pharmaceutically- acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid 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 subject being treated.
  • a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also intended to be part of this invention.
  • the articles “a” and “an” are used herein to refer to one or to more than one (i.e.
  • sense strand or antisense strand is understood as “sense strand or antisense strand or sense strand and antisense strand.”
  • the term “about” is used herein to mean within the typical ranges of tolerances in the art. For example, “about” can be understood as about 2 standard deviations from the mean. In certain embodiments, about means +10%. In certain embodiments, about means +5%.
  • nucleobase sequence is the sequence of the bases of the nucleic acid in an oligomer.
  • TARGET A target for inhibition disclosed herein may be, without limitation, an mRNA, polypeptide, protein, or gene. These targets are a target the inhibition of which helps in the prevention and/or treatment of a metabolic disease or disorder associated with non-alcoholic fatty liver disease (NAFLD) and/or obesity and/or a disease or disorder associated with adipogenesis and/or in reducing adipogenesis.
  • NAFLD non-alcoholic fatty liver disease
  • the target for inhibition is the gene SLC25A5 or a gene product thereof, such as an mRNA transcribed from the SLC25A5 gene or the ANT2 protein, and inhibition may be effected by inhibition of expression or function of the SLC25A5/ANT2 gene or protein or both.
  • the target is an mRNA expressed from the SLC25A5 gene.
  • Exemplary target sequences on the SLC25A5 mRNA are listed below in Table 1. Following Table 1 provides oligonucleoside mRNA target sequences of SLC25A5, together with the corresponding positions in transcript ENST00000317881.9. It is to be understood that SEQ ID NO: 1 to 276 refer to human (Homo sapiens) mRNA sequences.
  • the present invention further provides methods of treatment of a subject in need thereof.
  • the treatment methods of the invention include administering a nucleic acid such as an siRNA of the invention to a subject, e.g., a subject that would benefit from a reduction or inhibition of the expression of SLC25A5 gene, in a therapeutically effective amount e.g. a nucleic acid such as an siRNA targeting SLC25A5 or a pharmaceutical composition comprising the nucleic acid targeting SLC25A5.
  • the disease to be treated is related to a metabolic disease or disorder, such as a metabolic disease or disorder associated with non- alcoholic fatty liver disease (NAFLD) and/or obesity and/or a disease or disorder associated with adipogenesis and/or adipogenesis.
  • the disease to be treated is related to a metabolic disease or disorder, such as a metabolic disease or disorder associated with non-alcoholic fatty liver disease (NAFLD) and/or obesity and/or a disease or disorder associated with adipogenesis and/or adipogenesis.
  • the inhibitor according to the invention may be used in the prevention and/or treatment of a metabolic disease or disorder.
  • the term "metabolic disease” refers to a disease or condition affecting a metabolic process in a subject and is often caused by disruption of normal metabolism.
  • the patient to be treated may be a patient that already has a metabolic disease or disorder or that is at risk of developing a metabolic disease or disorder. That is, in certain embodiments, the inhibitor of the present invention may be used in the treatment and/or management of an existing metabolic disease or disorder. Treatment and/or management of an existing metabolic disease or disorder with the inhibitor of the present invention may prevent worsening of the metabolic disease or disorder and/or reverse the metabolic disease or disorder. In some instances, treatment of an existing metabolic disease or disorder with the inhibitor of the present invention may even cure the metabolic disease or disorder.
  • the inhibitor of the present invention may be used to prevent manifestation of a metabolic disease or disorder in a patient that is at risk of developing a metabolic disease or disorder.
  • the skilled person is capable of diagnosing whether a patient has a metabolic disease or disorder or is at risk of developing a metabolic disease or disorder.
  • a metabolic disease or disorder may be diagnosed based weight gain and/or one or more blood markers, including, without limitation, blood glucose levels, blood insulin levels, blood free fatty acid levels, blood HbA1c levels, blood fibrinogen levels, blood cholesterol levels and blood triglyceride levels.
  • the skilled person is aware of threshold values of one or more blood markers that indicate the presence of a metabolic disease or disorder or the risk of developing a metabolic disease or disorder.
  • the metabolic disease is fatty liver disease, in particular non-alcoholic fatty liver disease (NAFLD).
  • fatty-liver disease refers to a disease wherein fat is excessively accumulated in the liver and can cause severe diseases such as chronic hepatitis and hepatic cirrhosis.
  • lipids, particularly neutral fat accumulate in hepatocytes to the extent that the amount exceeds the physiologically permissible range. From a biochemical point of view, a standard for judgment of fatty liver is that the weight of neutral fat is about 10% (100 mg/g wet weight) or more of the wet weight of hepatic tissue.
  • Fatty liver disease is generally detected by observation of elevated serum levels of liver-specific enzymes such as the transaminases ALT and AST, which serve as indices of hepatocyte injury, as well as by presentation of symptoms, which include fatigue and pain in the region of the liver, though definitive diagnosis often requires a biopsy and may be supported by imaging such as ultrasound and/or MRI.
  • the term “NAFLD” or “non-alcoholic fatty liver disease”, as used herein, relates to a condition occurring when fat is deposited in the liver (steatosis) not due to excessive alcohol use. It is related to insulin resistance and the metabolic syndrome.
  • the fatty liver disease is Non-Alcoholic SteatoHepatitis (NASH).
  • NASH refers to a liver disease characterized by an accumulation of fat (lipid droplets), along with inflammation and degeneration of hepatocytes. Once initiated, the disease is accompanied with a high risk of cirrhosis, a state wherein liver functions are altered that can progress to liver insufficiency. Thereafter, NASH often progresses to liver cancer.
  • the invention relates to the inhibitor according to the invention for use in reducing one or more of steatosis, lobular inflammation, and/or hepatocytic ballooning.
  • the invention relates to the inhibitor according to the invention for use in treating and/or preventing a disease or disorder associated with increased steatosis, increased lobular inflammation, and/or increased hepatocytic ballooning.
  • the invention relates to the inhibitor according to the invention for use in the treatment and/or prevention of fatty liver disease, such as NAFLD or NASH, wherein the inhibitor according to the invention results in one or more of: reduced steatosis, reduced lobular inflammation, and/or reduced hepatocytic ballooning.
  • the invention relates to the inhibitor according to the invention for use in the treatment and/or prevention of liver steatosis.
  • the invention relates to the inhibitor according to the invention for use in the treatment and/or prevention of lobular inflammation of the liver. In certain embodiments, the invention relates to the inhibitor according to the invention for use in the treatment and/or prevention of hepatocytic ballooning of the liver. In certain embodiments, the invention relates to the inhibitor according to the invention for use in the treatment or prevention of liver fibrosis in a patient. In certain embodiments, the invention relates to the inhibitor according to the invention for use in treating and/or preventing a disease or disorder associated with liver fibrosis.
  • the invention relates to the inhibitor according to the invention for use in the treatment and/or prevention of fatty liver disease, such as NAFLD or NASH, wherein the inhibitor according to the invention reduces fibrosis stage.
  • fatty liver disease such as NAFLD or NASH
  • the invention relates to the inhibitor according to the invention for use in reducing the levels of one or more of Alanine transaminase (ALT), Aspartate transaminase (AST), Tissue inhibitor of metalloproteinases-1 (TIMP-1) and/or Type III Procollagen Peptide (PIIINP) in a patient.
  • ALT Alanine transaminase
  • AST Aspartate transaminase
  • Tissue inhibitor of metalloproteinases-1 Tissue inhibitor of metalloproteinases-1
  • PIIINP Type III Procollagen Peptide
  • the invention relates to the inhibitor according to the invention for use in treating and/or preventing a disease associated with elevated levels of one or more of Alanine transaminase (ALT), Aspartate transaminase (AST), Tissue inhibitor of metalloproteinases-1 (TIMP-1) and/or Type III Procollagen Peptide (PIIINP) in a patient.
  • ALT Alanine transaminase
  • AST Aspartate transaminase
  • PIIINP Type III Procollagen Peptide
  • the invention relates to the inhibitor according to the invention for use in treating and/or preventing a metabolic disease or disorder associated with elevated levels of one or more of Alanine transaminase (ALT), Aspartate transaminase (AST), Tissue inhibitor of metalloproteinases-1 (TIMP-1) and/or Type III Procollagen Peptide (PIIINP) in a patient.
  • ALT Alanine transaminase
  • AST Aspartate transaminase
  • Tissue inhibitor of metalloproteinases-1 Tissue inhibitor of metalloproteinases-1
  • PIIINP Type III Procollagen Peptide
  • the invention relates to the inhibitor according to the invention for use in treating and/or preventing a metabolic disease or disorder, wherein the inhibitor according to the invention reduces the levels of one or more of Alanine transaminase (ALT), Aspartate transaminase (AST), Tissue inhibitor of metalloproteinases-1 (TIMP-1) and/or Type III Procollagen Peptide (PIIINP) in a patient.
  • ALT Alanine transaminase
  • AST Aspartate transaminase
  • Tissue inhibitor of metalloproteinases-1 Tissue inhibitor of metalloproteinases-1
  • PIIINP Type III Procollagen Peptide
  • the invention relates to the inhibitor according to the invention for use in treating and/or preventing a fatty liver disease, such as NAFLD or NASH, wherein the inhibitor according to the invention reduces the levels of one or more of Alanine transaminase (ALT), Aspartate transaminase (AST), Tissue inhibitor of metalloproteinases-1 (TIMP-1) and/or Type III Procollagen Peptide (PIIINP) in a patient.
  • ALT Alanine transaminase
  • AST Aspartate transaminase
  • Tissue inhibitor of metalloproteinases-1 Tissue inhibitor of metalloproteinases-1
  • PIIINP Type III Procollagen Peptide
  • the invention relates to the inhibitor according to the invention for use in reducing the ratio between liver weight and body weight.
  • the invention relates to the inhibitor according to the invention for use in treating and/or preventing a disease or disorder associated with increased liver weight. In certain embodiments, the invention relates to the inhibitor according to the invention for use in treating and/or preventing a metabolic disease or disorder associated with increased liver weight. In certain embodiments, the invention relates to the inhibitor according to the invention for use in treating and/or preventing a metabolic disease or disorder, wherein the inhibitor according to the invention reduces the ratio between liver weight and body weight in a patient. In certain embodiments, the invention relates to the inhibitor according to the invention for use in treating and/or preventing a fatty liver disease, such as NAFLD or NASH, wherein the inhibitor according to the invention reduces the ratio between liver weight and body weight in a patient.
  • a fatty liver disease such as NAFLD or NASH
  • the metabolic disease is obesity.
  • the term “obesity” as used herein refers to a condition in which the natural energy reserve, stored in the fatty tissue of animals, in particular humans and other mammals, is increased to a point where it is associated with certain health conditions or increased mortality.
  • the term “obese” as used herein is defined for an adult human as having a body mass index (BMI) greater than 30. Obesity is commonly associated with excessive body weight gain, in particular diet- induced body weight gain. "(Diet-induced) body weight gain” is defined herein as body weight gain resulting from an excessive dietary intake, including an excessive dietary intake of fat, in particular saturated fat, and optionally an excessive dietary intake of simple sugars, including sucrose and fructose.
  • an excessive dietary intake in particular of fat and optionally of simple sugars, refers to the consumption of an amount of diet, in particular of fat and optionally of simple sugars, higher than the amount necessary to meet the physiological needs and maintain the energy balance of said subject.
  • the effect of a treatment on reduction of - or prevention - of diet-induced body weight gain in a subject can be assessed by comparing body weight gain observed in a subject receiving the treatment with those observed in the same subject without treatment receiving the same diet and having the same level of physical activity.
  • a patient is at risk of developing obesity if the patient has a BMI greater than 25.
  • a patient is obese if the patient has a BMI greater than 30.
  • body mass index means the ratio of weight in kg divided by the height in metres, squared.
  • a “disease associated with adipogenesis” refers to a medical condition characterized by the abnormal proliferation and differentiation of adipocytes (fat cells) within the body, leading to an excessive accumulation of adipose tissue. This condition often results in health complications such as obesity, metabolic disorders, and associated comorbidities.
  • a “reduction of adipogenesis” refers to a medical or pharmaceutical intervention designed to decrease the formation and accumulation of adipocytes within the body. A reduction in adipogenesis may be determined and/or quantified based on the size and/or number of adipocytes in a tissue sample obtained from a patient.
  • a reduction in adipogenesis may be determined and/or quantified by gene expression analysis, measurement of adipogenic markers (i.e., by ELISA), assessment of lipid accumulation in a sample and/or measurement of triglyceride levels in cells or tissues.
  • the invention relates to an inhibitor suitable for use, or for use, in prevention and/or treatment of metabolic disease or disorder, such as a metabolic disease or disorder associated with non-alcoholic fatty liver disease (NAFLD) and/or obesity and/or a disease or disorder associated with adipogenesis and/or for use in reducing adipogenesis.
  • metabolic disease or disorder such as a metabolic disease or disorder associated with non-alcoholic fatty liver disease (NAFLD) and/or obesity and/or a disease or disorder associated with adipogenesis and/or for use in reducing adipogenesis.
  • INHIBITORS Inhibitors of the invention include nucleic acids such as siRNAs, antibodies and antigen binding fragments thereof, e.g., monoclonal antibodies, polypeptides, antibody–drug conjugates, and small molecules. Preferred are nucleic acids such as siRNA. Certain preferred features of inhibitors of the invention, where these are oligonucelosides such as siRNA, are given below.
  • the nucleic acid comprises a first strand comprising a sequence that is at least partially complementary to a portion of RNA transcribed from the SLC25A5 gene (SEQ ID NO:1381).
  • the nucleic acid comprises a first strand comprising a sequence that is at least partially complementary to an SLC25A5 mRNA.
  • the nucleic acid for inhibiting expression of the SLC25A5 gene comprises a duplex region that comprises a first strand and a second strand that is at least partially complementary to the first strand, wherein said first strand is: (i) at least partially complementary to a portion of RNA transcribed from the SLC25A5 gene, and (ii) comprises at least 17 contiguous nucleosides differing by 0 or 1 nucleosides from any one of SEQ ID NO:277-552.
  • the first strand comprises nucleosides 2-18 of any one of the sequences set forth in SEQ ID NO: 277-552.
  • the first strand comprises any one of SEQ ID NO: 277-552.
  • the second strand comprises a nucleoside sequence of at least 17 contiguous nucleosides differing by 0 or 1 nucleosides from any one of SEQ ID NO: 553-828; wherein the second strand has a region of at least 85% complementarity over the 17 contiguous nucleosides to the first strand. In certain embodiments, the second strand comprises any one of SEQ ID NO: 553-828.
  • the nucleic acid for inhibiting expression of the SLC25A5 gene comprises a duplex region that comprises a first strand and a second strand that is at least partially complementary to the first strand, wherein said first strand is: (i) at least partially complementary to a portion of RNA transcribed from the SLC25A5 gene, and (ii) comprises at least 21 contiguous nucleosides differing by 0 or 1 nucleosides from any one of SEQ ID NO: 277-552.
  • the first strand comprises nucleosides 2-22 of any one of the sequences set forth in SEQ ID NO: 277-552.
  • the second strand comprises a nucleoside sequence of at least 17 contiguous nucleosides differing by 0 or 1 nucleosides from any one of SEQ ID NO:553-828; wherein the second strand has a region of at least 85% complementarity over the 17 contiguous nucleosides to the first strand. In certain embodiments, the second strand comprises any one of SEQ ID NO: 553-828.
  • the second strand comprises a nucleoside sequence of at least 19 contiguous nucleosides differing by 0 or 1 nucleosides from any one of SEQ ID NO: 553-828; wherein the second strand has a region of at least 85% complementarity over the 19 contiguous nucleosides to the first strand.
  • the second strand comprises a nucleoside sequence of at least 21 contiguous nucleosides differing by 0 or 1 nucleosides from any one of SEQ ID NO: 553-828; wherein the second strand has a region of at least 85% complementarity over the 21 contiguous nucleosides to the first strand.
  • the nucleic acid comprises a first strand that comprises, consists of, or consists essentially of a nucleoside sequence differing by 0 or 1 nucleosides from any one of SEQ ID NO: 277-552; and a second strand that comprises, consists of, or consists essentially of a nucleoside sequence differing by 0 or 1 nucleosides from any one of SEQ ID NO: 553-828.
  • the duplex region is formed between a first (antisense) strand and a complementary second (sense) strand. Exemplary pairs of complementary antisense and sense strands are listed in Table 2 below.
  • Table 2 provides the unmodified first (antisense) and corresponding unmodified second (sense) strand sequences for siRNA oligonucleosides according to the present invention, together with the corresponding positions in the overall gene sequence of SEQ ID NO:1381 as follows.
  • SEQ ID Antisense Strand Base Sequence SEQ ID Sense Strand Base Sequence Corresponding NO (AS) 5’ ⁇ 3’ NO (SS) 5’ ⁇ 3’ positions on (Shown as an Unmodified (Shown as an Unmodified ENST00000317 Nucleotide Sequence) Nucleotide Sequence) 881.9 SEQ ID UUAAGAGAAAACUGGUCAG SEQ ID AUCUGACCAGUUUUCU 1107-1129 NO: 304 AUGA NO: 580 CUUAA SEQ ID UAUCAGGAGUGACUGAAAU SEQ ID UUAUUUCAGUCACUCC 1170-1192 NO: 323 AAAA NO: 599 UGAUA SEQ ID UCCCUUUGCAGUGUCAUAG SEQ ID AUCUAUGACACUGCAA 650-672 NO: 439 AUAC NO: 715 AGGGA SEQ ID AUCAGAUUUGUAGAUCUUA SEQ ID GUUAAGAUCUACAAAU 554-576 NO: 453 ACCA
  • the first strand comprises nucleosides 2-18 of any one of the sequences set forth in SEQ ID NO: 829-1104.
  • the nucleic acid for inhibiting expression of the SLC25A5 gene comprises a duplex region that comprises a first strand and a second strand that is at least partially complementary to the first strand, wherein said first strand is: (i) at least partially complementary to a portion of RNA transcribed from the SLC25A5 gene, and (ii) comprises at least 21 contiguous nucleosides differing by 0 or 1 nucleosides from any one of SEQ ID NO: 829-1104.
  • the first strand comprises nucleosides 2-22 of any one of the sequences set forth in SEQ ID NO: 829-1104. In certain embodiments, the first strand comprises any one of SEQ ID NO: 829-1104.
  • the modification pattern of the nucleic acids as set forth in SEQ ID NO: 829-1104 is summarized in Table 3 below: Table 3 provides the modified first (antisense) sequences, together with the corresponding unmodified first (antisense) sequences for siRNA oligonucleosides according to the present invention as follows.
  • the second strand comprises a nucleoside sequence of at least 19 contiguous nucleosides differing by 0 or 1 nucleosides from any one of SEQ ID NO: 1105-1380; wherein the second strand has a region of at least 85% complementarity over the 19 contiguous nucleosides to the first strand.
  • the second strand comprises a nucleoside sequence of at least 21 contiguous nucleosides differing by 0 or 1 nucleosides from any one of SEQ ID NO: 1105-1380; wherein the second strand has a region of at least 85% complementarity over the 21 contiguous nucleosides to the first strand.
  • the second strand comprises any one of SEQ ID NO: 1105-1380.
  • the modification pattern of the nucleic acids as set forth in SEQ ID NO: 1105-1380 is summarized in Table 4 below. Table 4 provides the modified second (sense) sequences, together with the corresponding unmodified second (sense) sequences for siRNA oligonucleosides according to the present invention as follows.
  • the letter “s” is used as abbreviation for a phosphorothioate linkage between two consecutive (modified) nucleosides.
  • the abbreviation “AmsAm” is used for two consecutive 2'-O-methyl- adenosine nucleosides that are linked via a 3’5’ phosphorothioate linkage.
  • No abbreviation is used for nucleosides that are linked via a standard 3’5’ phosphodiester linkage.
  • the abbreviation “AmAm” is used for two consecutive 2'-O-methyl-adenosine nucleosides that are linked via a 3’5’ phosphodiester linkage.
  • the nucleic acid comprises a first strand that comprises, consists of, or consists essentially of a (modified) nucleoside sequence differing by 0 or 1 nucleosides from any one of SEQ ID NO: 829-1104; and a second strand that comprises, consists of, or consists essentially of a (modified) nucleoside sequence differing by 0 or 1 nucleosides from any one of SEQ ID NO:1105-1380.
  • the invention relates to a nucleic acid comprising first and second strands that comprise, consist of, or consist essentially of a nucleoside sequence differing by 0 or 1 nucleosides from any one of the following first and second sequences: Duplex ID Modified first strand Modified second strand ETX-S00003198 ETX-S00003197 ETX-M00001378 (SEQ ID NO: 856) (SEQ ID NO: 1132) ETX-S00003274 ETX-S00003273 ETX-M00001397 (SEQ ID NO: 875) (SEQ ID NO: 1151) ETX-S00003738 ETX-S00003737 ETX-M00001513 (SEQ ID NO: 991) (SEQ ID NO: 1267) ETX-S00003794 ETX-S00003793 ETX-M00001527 (SEQ ID NO: 1005) (SEQ ID NO: 1281) ETX-S00003966 ETX-S00003965 ETX-M00001570
  • ABASIC NUCLEOTIDES there are 1, e.g.2, e.g.3, e.g.4 or more abasic nucleosides present in nucleic acids according to the invention.
  • Abasic nucleosides are modified nucleosides because they lack the base normally seen at position 1 of the sugar moiety.
  • the abasic nucleosides are in the terminal region of the second strand, preferably located within the terminal 5 nucleosides of the end of the strand.
  • the terminal region may be the terminal 5 nucleosides, which includes abasic nucleosides.
  • the second strand may comprise, as preferred features (which are all specifically contemplated in combination unless mutually exclusive): 2, or more than 2, abasic nucleosides in a terminal region of the second strand; and/or 2, or more than 2, abasic nucleosides in either the 5’ or 3’ terminal region of the second strand; and/or 2, or more than 2, abasic nucleosides in either the 5’ or 3’ terminal region of the second strand, wherein the abasic nucleosides are present in an overhang as herein described; and/or 2, or more than 2, consecutive abasic nucleosides in a terminal region of the second strand, wherein preferably one such abasic nucleosides is a terminal nucleosides; and/or 2, or more than 2, consecutive abasic nucleosides in either the 5’ or 3’ terminal region of the second
  • abasic nucleoside at the terminus of the second strand.
  • the terminal 1 or terminal 2 or terminal 3 or terminal 4 nucelotides may be abasic nucleosides.
  • An abasic nucleoside may also be linked to an adjacent nucleoside through a 5’-3’ phosphodiester linkage or reversed linkage unless there is only 1 abasic nucleoside at the terminus, in which case it will have a reversed linkage to the adjacent nucleoside.
  • a reversed linkage (which may also be referred to as an inverted linkage, which is also seen in the art), comprises either a 5’-5’, a 3-’3’, a 3’-2’ or a 2’-3’ phosphodiester linkage between the adjacent sugar moieties of the nucleosides.
  • Abasic nucleosides which are not terminal will have 2 phosphodiester bonds, one with each adjacent nucleoside, and these may be a reversed linkage or may be a 5’-3 phosphodiester bond or may be one of each.
  • a preferred embodiment comprises 2 abasic nucleosides at the terminal and penultimate positions of the second strand, and wherein the reversed internucleoside linkage is located between the penultimate (abasic) nucleoside and the antepenultimate nucleoside.
  • abasic nucleosides at the terminal and penultimate positions of the second strand and the penultimate nucleoside is linked to the antepenultimate nucleoside through a reversed internucleoside linkage and is linked to the terminal nucleoside through a 5’-3’ or 3’-5’ phosphodiester linkage (reading in the direction of the terminus of the molecule).
  • a nucleic acid according to the present invention comprises one or more abasic nucleosides, optionally wherein the one or more abasic nucleosides are in a terminal region of the second strand, and/or wherein at least one abasic nucleoside is linked to an adjacent basic nucleoside through a reversed internucleoside linkage.
  • the reversed internucleoside linkage is a 3’-3’ reversed linkage.
  • the reversed internucleoside linkage is at a terminal region which is distal to the 5’ terminal phosphate of the second strand.
  • the reversed internucleoside linkage is a 5’-5’ reversed linkage.
  • the reversed internucleoside linkage is at a terminal region which is distal to the 3’ terminal hydroxide of the second strand.
  • the second strand comprises 2 consecutive abasic nucleosides in the 5’ terminal region of the second strand, wherein one such abasic nucleoside is a terminal nucleoside at the 5’ terminal region of the second strand and the other abasic nucleoside is a penultimate nucleoside at the 5’ terminal region of the second strand, wherein: (a) said penultimate abasic nucleoside is connected to an adjacent first basic nucleoside in an adjacent 5’ near terminal region through a reversed internucleoside linkage; and (b) the reversed linkage is a 5-5’ reversed linkage; and (c) the linkage between the terminal and penultimate abasic nucleosides is 3’5’ when reading towards the terminus comprising the terminal and penultimate abasic nucleoside
  • the first strand and the second strand each has a length of 23 nucleosides;
  • two phosphorothioate internucleoside linkages are respectively between three consecutive positions in said 5’ near terminal region of the second strand, wherein a first phosphorothioate internucleoside linkage is present between said adjacent first basic nucleoside of (a) and an adjacent second basic nucleoside in said 5’ near terminal region of the second strand, and a second phosphorothioate internucleoside linkage is present between said adjacent second basic nucleoside and an adjacent third basic nucleoside in said 5’ near terminal region of the second strand;
  • two phosphorothioate internucleoside linkages are respectively between three consecutive positions in both 5’ and 3’ terminal regions of the first strand, whereby a terminal nucleoside respectively at each of the 5’ and 3’ terminal regions of said first strand is each attached to a respective 5’ and 3’ adjacent penul
  • the second strand comprises 2 consecutive abasic nucleosides preferably in an overhang in the 3’ terminal region of the second strand, wherein one such abasic nucleoside is a terminal nucleoside at the 3’ terminal region of the second strand and the other abasic nucleoside is a penultimate nucleoside at the 3’ terminal region of the second strand, wherein: (a) said penultimate abasic nucleoside is connected to an adjacent first basic nucleoside in an adjacent 3’ near terminal region through a reversed internucleoside linkage; and (b) the reversed linkage is a 3-3’ reversed linkage; and (c) the linkage between the terminal and penultimate abasic nucleosides is 5’-3’ when reading towards the terminus comprising the terminal and penultimate abasic nucleosides.
  • nucleic acid molecules having overall 3’ - 3’ or 5’- 5’ end structures as described herein
  • the overall nucleic acid may have 3’ - 5’ end structures corresponding to the conventionally positioned 5’ / 3’ ends.
  • the nucleic acid may have a 3’-3’ reversed linkage, and the terminal sugar moiety may comprise a 5’ OH rather than a 5’ phosphate group at the 5’ position of that terminal sugar.
  • the 5’ or 3’ end is the conventional 5’ or 3’ end which would have existed had a reversed linkage not been in place, and wherein the conventional 5’ or 3’ end is determined by consideration of the directionality of the majority of the internal nucleoside linkages and / or nucleoside orientation within the nucleic acid. It is possible to tell from these internal bonds and / or nucleoside orientation which ends of the nucleic acid would constitute the conventional 5’ and 3’ ends (with reference to the numbering of ring atoms on the end nucleoside sugars) of the molecule absent the reversed linkage.
  • the “5’” end indicated in the diagram below which is the conventional 5’ end, can in fact comprise a 3’ OH in view of the inverted nucleoside at the terminal position.
  • the majority of the molecule will comprise conventional internucleoside linkages that run from the 3’ OH of the sugar to the 5’ phosphate of the next sugar, when reading in the standard 5’ [PO4] to 3’ [OH] direction of a nucleic acid molecule (with reference to the numbering of ring atoms on the nucleoside sugars), which can be used to determine the conventional 5’ and 3’ ends that would be found absent the inverted end configuration.
  • a 5’ A-A-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me 3’
  • the reversed bond is preferably located at the end of the nucleic acid e.g. RNA which is distal to a ligand moiety, such as a GalNAc containing portion, of the molecule.
  • GalNAc-siRNA constructs with a 5’-GalNAc on the sense strand can have a reversed linkage on the opposite end of the sense strand.
  • B represents a nucleoside base
  • T represent H, OH or a 2’ ribose modification (preferably a 2’ ribose modification, more preferably a 2’Me or 2’F ribose modification)
  • V represent O or S (preferably O)
  • R represent H or C 1-4 alkyl (preferably H)
  • Z comprises 11 to 26 contiguous nucleosides, preferably 15 to 21 contiguous nucleosides, and more preferably 19 contiguous nucleosides, more preferably the following 5’ terminal motif
  • B represents a nucleoside base
  • T represents a 2’ ribose modification (preferably a 2’Me or 2’F ribose modification)
  • Z comprises 19 contiguous nucleosides.
  • NUCLEIC ACID LENGTHS In one aspect the i) the first strand of the nucleic acid has a length in the range of 17 to 30 nucleosides, preferably 19 to 25 nucleosides, more preferably 19 or 23 nucleosides; and / or ii) the second strand of the nucleic acid has a length in the range of 17 to 30 nucleosides, preferably 19 to 25 nucleosides, more preferably 19 or 21 nucleosides.
  • the duplex region of the nucleic acid is between 17 and 30 nucleosides in length, more preferably is 19 or 21 nucleosides in length.
  • the region of complementarity between the first strand and the portion of RNA transcribed from the SLC25A5 gene is between 17 and 30 nucleosides in length.
  • the duplex structure of the nucleic acid e.g.
  • the region of complementarity of an antisense sequence to a target sequence and/or the region of complementarity of an antisense sequence to a sense sequence is about 15 to 30 nucleosides in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20- 24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-26, 21-25, 21- 24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-
  • the region of complementarity of an antisense sequence to a target sequence and/or the region of complementarity of an antisense sequence to a sense sequence is at least 17 nucleosides in length.
  • the region of complementarity between the antisense strand and the target is 19 to 21 nucleosides in length, for example, the region of complementarity is 21 nucleosides in length.
  • each strand is no more than 30 nucleosides in length.
  • the duplex structure of the nucleic acid e.g. an siRNA is 19 or 21 base pairs in length.
  • the duplex may have one of the following structures: e.g., ETX-M00001351 – ETX-M00001626
  • a nucleic acid e.g. a dsRNA as described herein can further include one or more single-stranded nucleoside overhangs e.g., 1-4, 2-4, 1-3, 2-3, 1, 2, 3, or 4 nucleosides.
  • a nucleoside overhang can comprise or consist of a nucleoside/nucleoside analog, including a deoxynucleoside/nucleoside.
  • the overhang(s) can be on the sense strand, the antisense strand, or any combination thereof.
  • nucleoside(s) of an overhang can be present on the 5'-end, 3'- end, or both ends of an antisense or sense strand of a nucleic acid e.g. a dsRNA.
  • at least one strand comprises a 3' overhang of at least 1 nucleoside, e.g., at least one strand comprises a 3' overhang of at least 2 nucleosides.
  • the overhang is suitably on the antisense/ guide strand and/or the sense / passenger strand.
  • NUCLEIC ACID MODIFICATIONS In certain embodiments, the nucleic acid e.g.
  • an RNA of the invention e.g., a dsiRNA
  • the nucleic acid e.g. RNA of the invention, e.g., a dsiRNA
  • substantially all of the nucleosides are modified.
  • the nucleic acids featured in the invention can be synthesized or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, S.L. et al.
  • Modifications include, for example, end modifications, e.g., 5'-end modifications (phosphorylation, conjugation, inverted linkages) or 3 '-end modifications (conjugation, DNA nucleosides within an RNA, or RNA nucleosides within a DNA, inverted linkages, etc.); base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, conjugated bases; sugar modifications (e.g.
  • nucleic acids such as siRNA compounds useful in the embodiments described herein include, but are not limited to RNAs containing modified backbones or no natural internucleoside linkages.
  • Nucleic acids such as RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone.
  • modified nucleic acids e.g. RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.
  • a modified nucleic acid e.g. an siRNA will have a phosphorus atom in its internucleoside backbone.
  • Modified nucleic acid e.g. RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5'-linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 5'-3' or 5'-2'
  • Modified nucleic acids e.g. RNAs can also contain one or more substituted sugar moieties.
  • the nucleic acids e.g. siRNAs, e.g., dsiRNAs, featured herein can include one of the following at the 2'-position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted. 2’ O- methyl and 2’-F are preferred modifications.
  • the nucleic acid comprises at least one modified nucleoside.
  • the nucleic acid of the invention may comprise one or more modified nucleosides on the first strand and/or the second strand.
  • substantially all of the nucleosides of the sense strand and all of the nucleosides of the antisense strand comprise a modification.
  • all of the nucleosides of the sense strand and substantially all of the nucleosides of the antisense strand comprise a modification.
  • all of the nucleosides of the sense strand and all of the nucleosides of the antisense strand comprise a modification.
  • At least one of the modified nucleosides is selected from the group consisting of a deoxy- nucleoside, a 3 '-terminal deoxy-thymine (dT) nucleoside, a 2'-O-methyl modified nucleoside (also called herein 2’-Me, where Me is a methoxy) , a 2'-fluoro modified nucleoside, a 2'-deoxy- modified nucleoside, a locked nucleoside, an unlocked nucleoside, a conformationally restricted nucleoside, a constrained ethyl nucleoside, an abasic nucleoside, a 2' -amino- modified nucleoside, a 2'-O-allyl- modified nucleoside, 2' -C-alkyl- modified nucleoside, 2'-hydroxly-modified nucleoside, a 2'- methoxyethyl modified nucleoside, a 2'-O-
  • the modified nucleosides comprise a short sequence of 3 '-terminal deoxy-thymine nucleosides (dT).
  • Modifications on the nucleosides may preferably be selected from the group including, but not limited to, LNA, HNA, CeNA, 2 -methoxyethyl, 2'-O-alkyl, 2-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxyl, and combinations thereof.
  • the modifications on the nucleosides are 2 -O-methyl (“2- Me”) or 2'-fluoro modifications.
  • the nucleic acid e.g. RNAi agent further comprises at least one phosphorothioate or methylphosphonate internucleoside linkage.
  • the phosphorothioate or methylphosphonate internucleoside linkage can be at the 3 '-terminus or in the terminal region of one strand, i.e. , the sense strand or the antisense strand; or at the ends of both strands, the sense strand and the antisense strand.
  • the phosphorothioate or methylphosphonate internucleoside linkage is at the 5 'terminus or in the terminal region of one strand, i.e. , the sense strand or the antisense strand; or at the ends of both strands, the sense strand and the antisense strand.
  • a phosphorothioate or a methylphosphonate internucleoside linkage is at both the 5'- and 3 '-terminus or in the terminal region of one strand, i.e. , the sense strand or the antisense strand; or at the ends of both strands, the sense strand and the antisense strand.
  • Any nucleic acid may comprise one or more phosphorothioate (PS) modifications within the nucleic acid, such as at least two PS internucleoside bonds at the ends of a strand.
  • PS phosphorothioate
  • At least one of the oligoribonucleoside strands preferably comprises at least two consecutive phosphorothioate modifications in the last 3 nucleosides of the oligonucleoside.
  • the invention therefore also relates to: A nucleic acid disclosed herein which comprises phosphorothioate internucleoside linkages respectively between at least two or three consecutive positions, such as in a 5’ and/or 3’ terminal region and/or near terminal region of the second strand, whereby said near terminal region is preferably adjacent said terminal region wherein said one or more abasic nucleosides of said second strand is / are located.
  • a nucleic acid disclosed herein which comprises phosphorothioate internucleoside linkages respectively between at least two or three consecutive positions in a 5’ and / or 3’ terminal region of the first strand, whereby preferably the terminal position at the 5’ and / or 3’ terminal region of said first strand is attached to its adjacent position by a phosphorothioate internucleoside linkage.
  • the nucleic acid strand may be an RNA comprising a phosphorothioate internucleoside linkage between the three nucleosides contiguous with 2 terminally located abasic nucleosides.
  • a preferred nucleic acid is a double stranded RNA comprising 2 adjacent abasic nucleosides at the 5’ terminus of the second strand and a ligand moiety comprising one or more GalNAc ligand moieties at the opposite 3’ end of the second strand. Further preferred, the same nucleic acid may also comprise a phosphorothioate bond between nucelotides at positions 3-4 and 4-5 of the second strand, reading from the position 1 of the second strand.
  • Position 1 of the first or the second strand is the nucleoside which is the closest to the end of the nucleic acid (ignoring any abasic nucleosides) and that is joined to an adjacent nucleoside (at Position 2) via a 3’ to 5’ internal bond, with reference to the bonds between the sugar moieties of the backbone, and reading in a direction away from that end of the molecule. It can therefore be seen that “position 1 of the sense strand” is the 5’ most nucleoside (not including abasic nucleosides) at the conventional 5’ end of the sense strand.
  • the nucleoside at this position 1 of the sense strand will be equivalent to the 5’ nucleoside of the selected target nucleic acid sequence, and more generally the sense strand will have equivalent nucleosides to those of the target nucleic acid sequence starting from this position 1 of the sense strand, whilst also allowing for acceptable mismatches between the sequences.
  • “position 1 of the antisense strand” is the 5’ most nucleoside (not including abasic nucleosides) at the conventional 5’ end of the antisense strand.
  • a particularly preferred modification that can be used with sequences according to the present invention can be: Modification 6: First strand modification: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmsNmsNm (5’ to 3’) Second strand modification: iaiaNmsNmsNmNmNmNmNmNmNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNm (5’ to 3’) wherein in each of the above modifications: ia represents an inverted abasic nucleoside; Nm represents a 2’Me ribose modified nucle
  • RNA e.g. an siRNA of the invention involves linking the nucleic acid e.g. the siRNA to one or more ligand moieties e.g. to enhance the activity, cellular distribution, or cellular uptake of the nucleic acid e.g. siRNA e.g., into a cell.
  • the inhibitor according to the invention is conjugated to a ligand moiety that enables and/or facilitates targeting of hepatocytes.
  • targeting of hepatocytes is achieved using N-acetylgalactosamine (GalNAc) conjugates as described in more detail herein below.
  • GalNAc N-acetylgalactosamine
  • the inhibitor according to the invention is an siRNA-GalNAc conjugate.
  • the ligand moiety described can be attached to a nucleic acid e.g. an siRNA oligonucleoside, via a linker that can be cleavable or non-cleavable.
  • linker or “linking group” means an organic moiety that connects two parts of a compound, e.g., covalently attaches two parts of a compound.
  • the ligand can be attached to the 3' or 5’ end of the sense strand.
  • the ligand is preferably conjugated to 3’ end of the sense strand of the nucleic acid e.g. an siRNA agent.
  • the invention therefore relates in a further aspect to a conjugate for inhibiting expression of a target e.g. a target gene, in a cell, said conjugate comprising a nucleic acid portion and one or more ligand moieties, said nucleic acid portion comprising a nucleic acid as disclosed herein.
  • the second strand of the nucleic acid is conjugated directly or indirectly (e.g. via a linker) to the one or more ligand moiety(s), wherein said ligand moiety is typically present at a terminal region of the second strand, preferably at the 3’ terminal region thereof.
  • the ligand moiety comprises a GalNAc or GalNAc derivative attached to the nucleic acid e.g.
  • the invention relates to a conjugate wherein the ligand moiety comprises i) one or more GalNAc ligands; and/or ii) one or more GalNAc ligand derivatives; and/or iii) one or more GalNAc ligands conjugated to said nucleic acid through a linker.
  • Said GalNAc ligand may be conjugated directly or indirectly to the 5’ or 3’ terminal region of the second strand of the nucleic acid, preferably at the 3’ terminal region thereof.
  • GalNAc ligands are well known in the art and described in, inter alia, EP3775207A1.
  • the ligand moiety comprises one or more ligands.
  • the ligand moiety comprises one or more carbohydrate ligands.
  • the one or more carbohydrates can be a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide and / or polysaccharide.
  • the one or more carbohydrates comprise one or more galactose moieties, one or more lactose moieties, one or more N-AcetylGalactosamine moieties, and / or one or more mannose moieties.
  • the one or more carbohydrates comprise one or more N-Acetyl-Galactosamine moieties.
  • the compounds as described anywhere herein comprise two or three N- AcetylGalactosamine moieties.
  • the one or more ligands are attached in a linear configuration, or in a branched configuration, for example each configuration being respectively attached to a branch point in an overall linker.
  • Exemplary linear configurations and Exemplary branched configurations are shown in Figures 1a and 1b: In Fig 1a, (linear), (a) and / or (b) can typically represent connecting bonds or groups, such as phosphate or phosphorothioate groups.
  • Fig 1b (branched), in some embodiments, the one or more ligands are attached as a biantennary or triantennary branched configuration.
  • Linker Exemplary compounds of the invention comprise a ‘linker moiety’, such as that as depicted in Formula (I), that is part of an overall ‘linker’.
  • R 1 at each occurrence is independently selected from the group consisting of hydrogen, methyl and ethyl
  • X 1 and X 2 at each occurrence are independently selected from the group consisting of methylene, oxygen and sulfur
  • m is an integer of from 1 to 6
  • n is an integer of from 1 to 10
  • q, r, s, t, v are independently integers from 0 to 4, with the proviso that: (i) q and r cannot both be 0 at the same time; and (ii) s, t and v cannot all be 0 at the same time
  • Z is an oligonucleoside moiety.
  • exemplary compounds of the invention comprise an overall linker that is located between the oligonucleoside moiety and the ligand moiety of these compounds.
  • the overall linker thereby ‘links’ the oligonucleoside moiety and the ligand moiety to each other.
  • the overall linker is often notionally envisaged as comprising one or more linker building blocks. For example, there is a linker portion that is depicted as the ‘linker moiety’ as represented in Formula (I) positioned adjacent the ligand moiety and attaching the ligand moiety, typically via a branch point, directly or indirectly to the oligonucleoside moiety.
  • the linker moiety as depicted in Formula (I) can also often be referred to as the ‘ligand arm or arms’ of the overall linker.
  • Such ‘ligand arms’ and / or ‘linker moieties’ and / or ‘tether moieties’ can be envisaged by reference to the linear and / or branched configurations as set out above.
  • Tether moiety of Formula I comprises the group of atoms between Z, namely the oligonucleoside moiety, and the linker moiety.
  • R 1 is hydrogen at each occurrence.
  • R 1 is methyl.
  • R 1 is ethyl.
  • R 2 is hydroxy.
  • both X 1 and X 2 are methylene.
  • exemplary compounds of the invention comprise the following structure: Formula (IV)
  • exemplary compounds of the invention comprise the following structure: Formula (II)
  • Alternative tether moieties During the synthesis of compounds of the present invention, alternative tether moiety structures may arise.
  • alternative tether moieties have a change of one or more atoms in the tether moiety of the overall linker compared to tether moieties described anywhere herein.
  • the alternative tether moiety is a compound of Formula (I) as described anywhere herein, wherein R 2 is hydroxy.
  • compounds of the invention comprise the following structure: Formula (V)
  • compounds of the invention comprise the following structure: Formula (III) Linker moiety
  • the ‘linker moiety’ as depicted in Formula (I) comprises the group of atoms located between the tether moiety as described anywhere herein, and the ligand moiety as described anywhere herein.
  • Formula (VIa) as depicted in Formula (I) as described anywhere herein is any of Formulae (VIa), (VIb) or (VIc), preferably Formula (VIa): Formula (VIa) wherein: A I is hydrogen, or a suitable hydroxy protecting group; a is an integer of 2 or 3; and b is an integer of 2 to 5; or Formula (VIb) wherein: A I is hydrogen, or a suitable hydroxy protecting group; a is an integer of 2 or 3; and c and d are independently integers of 1 to 6; or Formula (VIc) wherein: A I is hydrogen, or a suitable hydroxy protecting group; a is an integer of 2 or 3; and e is an integer of 2 to 10.
  • the moiety: as depicted in Formula (I) is Formula (VIa): Formula (VIa) wherein: A I is hydrogen, or a suitable hydroxy protecting group; a is 3; and b is an integer of 3.
  • the moiety: as depicted in Formula (I) as described anywhere herein is Formula (VII): Formula (VII) wherein: A I is hydrogen; a is an integer of 2 or 3, preferably 3.
  • Other exemplary compounds of the invention comprise a ‘linker moiety’, as depicted in Formula (I*), that is part of an overall ‘linker’.
  • Formula I* Where: r and s are independently an integer selected from 1 to 16; and Z is an oligonucleoside moiety.
  • exemplary compounds of the invention comprise an overall linker that is located between the oligonucleoside moiety and the ligand moiety of these compounds.
  • the overall linker thereby ‘links’ the oligonucleoside moiety and the ligand moiety to each other.
  • the overall linker is often notionally envisaged as comprising one or more linker building blocks. For example, there is a linker portion that is depicted as the ‘linker moiety’ as represented in Formula (I*) positioned adjacent the ligand moiety and attaching the ligand moiety, typically via a branch point, directly or indirectly to the oligonucleoside moiety.
  • tether moiety is that portion of the overall linker which comprises the group of atoms between Z, namely the oligonucleoside moiety, and the linker moiety as depicted in Formula (I).
  • Tether moiety In relation to Formula (I*), the ‘tether moiety’ comprises the group of atoms between Z, namely the oligonucleoside moiety, and the linker moiety.
  • s is an integer selected from 4 to 12.
  • s is 6.
  • r is an integer selected from 4 to 14.
  • r is 6.
  • r is 12.
  • the moiety: as depicted in Formula (I) as described anywhere herein is Formula (VII*): Formula (VII*) wherein: A I is hydrogen; a is an integer of 2 or 3.
  • a 2.
  • a 3.
  • b 3.
  • the GalNAc ligand is comprised in any one of the linkers shown in Figures 2 to 5 or Figure 6 (Formula XI), wherein the "oligonucleotide" may be any nucleic acid disclosed herein.
  • the "oligonucleotide” may comprise other bonds than a phosphodiester bond, such as one or more phosphorothioate bonds.
  • the nucleic acid according to the invention is a double stranded oligonucleoside as defined herein and the linker is conjugated to the second strand, more preferably to the 3' terminal region of the second strand, via a phosphodiester bond.
  • the GalNAc ligand is comprised in the linker shown in Figure 4, wherein the "oligonucleotide" may be any nucleic acid disclosed herein.
  • the "oligonucleotide” may comprise other bonds than a phosphodiester bond, such as one or more phosphorothioate bonds.
  • the nucleic acid according to the invention is a double stranded oligonucleoside as defined herein and the linker is conjugated to the second strand, more preferably to the 3' terminal region of the second strand, via a phosphodiester bond.
  • the GalNAc ligand is comprised in the linker shown in Figure 5 (Formula XI), wherein the "oligonucleotide” represents a nucleic acid according to the invention, wherein the nucleic acid according to the invention comprises a modified or unmodified second strand comprising or consisting of any one of SEQ ID NO:580, SEQ ID NO:599, SEQ ID NO:715, SEQ ID NO:729 and SEQ ID NO:772, preferably wherein the linker is conjugated to the 3' terminal region of the second strand, i.e., to the 3' terminal region of any one SEQ ID NO:580, SEQ ID NO:599, SEQ ID NO:715, SEQ ID NO:729 and SEQ ID NO:772, via a phosphodiester bond.
  • the linker is conjugated to the 3' terminal region of the second strand, i.e., to the 3' terminal region of any one SEQ ID NO:580, SEQ ID NO:599,
  • the GalNAc ligand is comprised in the linker shown in Figure 4, wherein the "oligonucleotide” represents a nucleic acid according to the invention, wherein the nucleic acid according to the invention comprises a modified second strand comprising or consisting of any one of SEQ ID NO:1105 to SEQ ID NO:1380, preferably wherein the linker is conjugated to the 3' terminal region of the second strand, i.e., to the 3' terminal region of any one of SEQ ID NO:1105 to SEQ ID NO:1380, via a phosphodiester bond.
  • the GalNAc ligand is comprised in the linker shown in Figure 6 (Formula XI), wherein the "oligonucleotide” represents a nucleic acid according to the invention, wherein the nucleic acid according to the invention comprises a modified second strand comprising or consisting of any one of SEQ ID NO:1105 to SEQ ID NO:1380, preferably wherein the linker is conjugated to the 3' terminal region of the second strand, i.e., to the 3' terminal region of any one of SEQ ID NO:1105 to SEQ ID NO:1380, via a phosphodiester bond.
  • the GalNAc ligand is comprised in any one of the linkers shown in Figures 2 to 5 or Figure 6 (Formula XI), wherein the "oligonucleotide" represents a nucleic acid according to the invention, wherein the nucleic acid according to the invention comprises a modified second strand comprising or consisting of any one of SEQ ID NO:1132, SEQ ID NO:1151, SEQ ID NO:1267, SEQ ID NO:1281 and SEQ ID NO:1324, preferably wherein the linker is conjugated to the 3' terminal region of the second strand, i.e., to the 3' terminal region of any one of SEQ ID NO:1132, SEQ ID NO:1151, SEQ ID NO:1267, SEQ ID NO:1281 and SEQ ID NO:1324, via a phosphodiester bond.
  • the linker is conjugated to the 3' terminal region of the second strand, i.e., to the 3' terminal region of any one of SEQ ID NO:1132, SEQ
  • the GalNAc ligand is comprised in the linker shown in Figure 4, wherein the "oligonucleotide” represents a nucleic acid according to the invention, wherein the nucleic acid according to the invention comprises a modified second strand comprising or consisting of any one of SEQ ID NO:1132, SEQ ID NO:1151, SEQ ID NO:1267, SEQ ID NO:1281 and SEQ ID NO:1324, preferably wherein the linker is conjugated to the 3' terminal region of the second strand, i.e., to the 3' terminal region of any one of SEQ ID NO:1132, SEQ ID NO:1151, SEQ ID NO:1267, SEQ ID NO:1281 and SEQ ID NO:1324, via a phosphodiester bond.
  • the GalNAc ligand is comprised in the linker shown in Figure 6 (Formula XI), wherein the "oligonucleotide” represents a nucleic acid according to the invention, wherein the nucleic acid according to the invention comprises a modified second strand comprising or consisting of any one of SEQ ID NO:1132, SEQ ID NO:1151, SEQ ID NO:1267, SEQ ID NO:1281 and SEQ ID NO:1324, preferably wherein the linker is conjugated to the 3' terminal region of the second strand, i.e., to the 3' terminal region of any one of SEQ ID NO:1132, SEQ ID NO:1151, SEQ ID NO:1267, SEQ ID NO:1281 and SEQ ID NO:1324, via a phosphodiester bond.
  • the linker is conjugated to the 3' terminal region of the second strand, i.e., to the 3' terminal region of any one of SEQ ID NO:1132, SEQ ID NO:1151, SEQ ID NO
  • the GalNAc ligand is comprised in the linker shown in Figures 2 to 5 or Figure 6 (Formula XI), wherein the "oligonucleotide” represents a nucleic acid according to the invention, wherein the nucleic acid according to the invention comprises a modified second strand comprising or consisting of any one of SEQ ID NO:1105 to SEQ ID NO:1380, preferably any one of SEQ ID NO:1132, SEQ ID NO:1151, SEQ ID NO:1267, SEQ ID NO:1281 and SEQ ID NO:1324, wherein the second strand has the following structure
  • T represents a 2’Me ribose modification
  • B represents the nucleoside bases of the first two basic nucleosides in the 5' terminal region of any one of SEQ ID NO:1105 to SEQ ID NO:1380, preferably any one of SEQ ID NO:1132, SEQ ID NO:1151, SEQ ID NO:1267, SEQ ID NO:1281 and SEQ ID NO:1324
  • Z represents the remaining 19 contiguous basic nucleosides of any one of SEQ ID NO:1105 to SEQ ID NO:1380, preferably any one of SEQ ID NO:1132, SEQ ID NO:1151, SEQ ID NO:1267, SEQ ID NO:1281 and SEQ ID NO:1324, respectively.
  • the GalNAc ligand is comprised in the linker shown in Figure 6 (Formula XI), wherein the "oligonucleotide” represents a nucleic acid according to the invention, wherein the nucleic acid according to the invention comprises a modified second strand comprising or consisting of any one of SEQ ID NO:1105 to SEQ ID NO:1380, preferably any one of SEQ ID NO:1132, SEQ ID NO:1151, SEQ ID NO:1267, SEQ ID NO:1281 and SEQ ID NO:1324, wherein the second strand has the following structure
  • T represents a 2’Me ribose modification
  • B represents the nucleoside bases of the first two basic nucleosides in the 5' terminal region of any one of SEQ ID NO:1105 to SEQ ID NO:1380, preferably any one of SEQ ID NO:1132, SEQ ID NO:1151, SEQ ID NO:1267, SEQ ID NO:1281 and SEQ ID NO:1324
  • Z represents the remaining 19 contiguous basic nucleosides of any one of SEQ ID NO:1105 to SEQ ID NO:1380, preferably any one of SEQ ID NO:1132, SEQ ID NO:1151, SEQ ID NO:1267, SEQ ID NO:1281 and SEQ ID NO:1324, respectively.
  • the GalNAc ligand is comprised in the linker shown in Figure 4, wherein the "oligonucleotide” represents a nucleic acid according to the invention, wherein the nucleic acid according to the invention comprises a modified second strand comprising or consisting of any one of SEQ ID NO:1105 to SEQ ID NO:1380, preferably any one of SEQ ID NO:1132, SEQ ID NO:1151, SEQ ID NO:1267, SEQ ID NO:1281 and SEQ ID NO:1324, wherein the second strand has the following structure
  • T represents a 2’Me ribose modification
  • B represents the nucleoside bases of the first two basic nucleosides in the 5' terminal region of any one of SEQ ID NO:1105 to SEQ ID NO:1380, preferably any one of SEQ ID NO:1132, SEQ ID NO:1151, SEQ ID NO:1267, SEQ ID NO:1281 and SEQ ID NO:1324
  • Z represents the remaining 19 contiguous basic nucleosides of any one of SEQ ID NO:1105 to SEQ ID NO:1380, preferably any one of SEQ ID NO:1132, SEQ ID NO:1151, SEQ ID NO:1267, SEQ ID NO:1281 and SEQ ID NO:1324, respectively.
  • the invention provides a cell containing a nucleic acid, such as inhibitory RNA [RNAi] as described herein.
  • a cell comprising a vector as described herein.
  • the invention provides a vector comprising an oligonucleotide inhibitor, e.g.an iRNA e.g. siRNA.
  • an oligonucleotide inhibitor e.g.an iRNA e.g. siRNA.
  • PHARMACEUTICALLY ACCEPTABLE COMPOSITIONS the invention provides a pharmaceutical composition for inhibiting expression of a target gene, the composition comprising an inhibitor such as an oligomer such as a nucleic acid as disclosed herein.
  • the pharmaceutically acceptable composition may comprise an excipient and or carrier.
  • 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 stearate, 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 laur
  • Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g. , magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g.
  • compositions of the present invention can also be used to formulate the compositions of the present invention.
  • suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.
  • Formulations for topical administration of nucleic acids can include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases.
  • the solutions can also contain buffers, diluents and other suitable additives.
  • Pharmaceutically acceptable organic or inorganic excipients suitable for non- parenteral administration which do not deleteriously react with nucleic acids can be used.
  • the nucleic acid or composition is administered in an unbuffered solution.
  • the unbuffered solution is saline or water.
  • the nucleic acid e.g. RNAi agent is administered in a buffered solution.
  • the buffer solution can comprise acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof.
  • the buffer solution can be phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the pharmaceutical compositions of the invention may be administered in dosages sufficient to inhibit expression of a gene or modify the expression or function of a target.
  • a suitable dose of a nucleic acid e.g. an siRNA of the invention will be in the range of about 0.001 to about 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of about 1 to 50 mg per kilogram body weight per day.
  • a suitable dose of a nucleic acid e.g.
  • an siRNA of the invention will be in the range of about 0.1 mg/kg to about 5.0 mg/kg, e.g., about 0.3 mg/kg and about 3.0 mg/kg.
  • a repeat-dose regimen may include administration of a therapeutic amount of a nucleic acid e.g. siRNA on a regular basis, such as every other day or once a year.
  • the nucleic acid e.g. siRNA is administered about once per month to about once per quarter (i.e., about once every three months).
  • the nucleic acid e.g. siRNA agent is administered at a dose of about 0.01 mg/kg to about 10 mg/kg or about 0.5 mg/kg to about 50 mg/kg.
  • the nucleic acid e.g. siRNA agent is administered at a dose of about 10 mg/kg to about 30 mg/kg. In certain embodiments, the nucleic acid e.g. siRNA agent is administered at a dose selected from about 0.5 mg/kg 1 mg/kg, 1.5 mg/kg, 3 mg/kg, 5 mg/kg, 10 mg/kg, and 30 mg/kg. In certain embodiments, the nucleic acid e.g. siRNA agent is administered about once per week, once per month, once every other two months, or once a quarter (i.e., once every three months) at a dose of about 0.1 mg/kg to about 5.0 mg/kg. In certain embodiments, the nucleic acid e.g.
  • siRNA agent is administered to the subject once a week.
  • the nucleic acid e.g. siRNA agent is administered to the subject once a month.
  • the nucleic acid e.g. siRNA agent is administered once per quarter (i.e., every three months).
  • the treatments can be administered on a less frequent basis. For example, after administration weekly or biweekly for three months, administration can be repeated once per month, for six months, or a year; or longer.
  • the pharmaceutical composition can be administered once daily, or administered as two, three, or more sub-doses at appropriate intervals throughout the day or even using continuous infusion or delivery through a controlled release formulation. In that case, the nucleic acid e.g.
  • siRNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage.
  • the dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the nucleic acid e.g. siRNA over a several day period. Sustained release formulations are well known in the art and are particularly useful for delivery of agents at a particular site, such as could be used with the agents of the present invention.
  • the dosage unit contains a corresponding multiple of the daily dose.
  • a single dose of the pharmaceutical compositions can be long lasting, such that subsequent doses are administered at not more than 3, 4, or 5 day intervals, or at not more than 1, 2, 3, or 4 week intervals.
  • a single dose of the pharmaceutical compositions of the invention is administered once per week. In other embodiments of the invention, a single dose of the pharmaceutical compositions of the invention is administered bimonthly. In certain embodiments, the siRNA is administered about once per month to about once per quarter (i.e., about once every three months), or even every 6 months or 12 months.
  • Estimates of effective dosages and in vivo half-lives for the individual nucleic acid e.g. siRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as known in the art.
  • the pharmaceutical compositions of the present invention can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated.
  • Administration can be topical (e.g., by a transdermal patch), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral.
  • Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subdermal, e.g., via an implanted device; or intracranial, e.g., by intraparenchymal, intrathecal or intraventricular administration.
  • the compositions are administered by intravenous infusion or injection.
  • the compositions are administered by subcutaneous injection.
  • the nucleic acid e.g. siRNA agent is administered to the subject subcutaneously.
  • the inhibitor e.g. nucleic acid e.g. siRNA can be delivered in a manner to target a particular tissue (e.g. in particular liver cells).
  • a particular tissue e.g. in particular liver cells.
  • METHODS FOR INHIBITING GENE EXPRESSION OR INHIBITION OF TARGET EXPRESSION OR FUNCTION IN VITRO The present invention also provides methods of inhibiting expression of SLC25A5 gene in a cell. The methods include contacting a cell with a nucleic acid of the invention e.g.
  • a nucleic acid “for inhibiting the expression of SLC25A5” is a nucleic acid that is capable of inhibiting SLC25A5 expression, preferably as described herein below.
  • Contacting of a cell with the nucleic acid e.g. an siRNA, such as a double stranded siRNA agent may be done in vitro or in vivo. Contacting a cell in vivo with nucleic acid e.g.
  • contacting a cell or group of cells within a subject e.g., a human subject
  • the nucleic acid e.g. siRNA e.g. siRNA
  • Combinations of in vitro and in vivo methods of contacting a cell are also possible.
  • Contacting a cell may be direct or indirect, as discussed above.
  • contacting a cell may be accomplished via a targeting ligand moiety, including any ligand moiety described herein or known in the art.
  • the targeting ligand moiety is a carbohydrate moiety, e.g. a GalNAc3 ligand, or any other ligand moiety that directs the siRNA agent to a site of interest.
  • inhibitor as used herein, is used interchangeably with “reducing,” “silencing,” “downregulating”, “suppressing”, and other similar terms, and includes any level of inhibition.
  • expression or activity of a gene or an inhibition target is inhibited by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or to below the level of detection of the assay, preferably when determined by qPCR as described herein and/or when the siRNA is introduced into the target cell by transfection.
  • the methods include a clinically relevant inhibition of expression of a target gene e.g.
  • the nucleic acid of the invention when transfected into the cells, inhibits expression of the SLC25A5 gene with an IC50 value below a defined threshold value.
  • the threshold value may be 2500 pM, 2000 pM, 1900 pM, 1800 pM, 1700 pM, 1600 pM, 1500 pM, 1400 pM, 1300 pM, 1200 pM, 1100 pM, 1000 pM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM or 100 pM, preferably when determined by qPCR, more preferably by reverse transcriptase (RT)- qPCR, as described herein.
  • RT reverse transcriptase
  • the nucleic acid of the invention when transfected into the cells, inhibits expression of the SLC25A5 gene with an IC50 value lower than 2500 pM. In a more preferred embodiment, when transfected into the cells, the nucleic acid of the invention inhibits expression of the SLC25A5 gene with an IC50 value lower than 1000 pM. In an even more preferred embodiment, when transfected into the cells, the nucleic acid of the invention inhibits expression of the SLC25A5 gene with an IC50 value lower than 500 pM. In a most preferred embodiment, when transfected into the cells, the nucleic acid of the invention inhibits expression of the SLC25A5 gene with an IC50 value lower than 100 pM.
  • Huh7 cells human hepatocyte-derived cell line, obtained from JCRB Cell Bank
  • DMEM Modified Eagle Medium
  • siRNA duplexes targeting SLC25A5 mRNA or a negative control siRNA (siRNA-control; sense strand 5’- GCCTGTACCAAGGCTTTAA -3’ (SEQ ID NO: 1383), antisense strand 5’- TTAAAGCCTTGGTACAGGC -3’ (SEQ ID NO: 1382)) using six 10-fold serial dilutions over a final duplex concentration range of 3 nM to 0.03 pM.
  • Transfection may be carried out by adding 9.7 ⁇ L Opti- MEM (ThermoFisher) plus 0.3 ⁇ L Lipofectamine RNAiMAX (ThermoFisher) to 10 ⁇ L of each siRNA duplex.
  • the mixture may be incubated at room temperature for 15 minutes before being added to 100 ⁇ L of complete growth medium containing 20,000 Huh7 cells. Cells may be incubated for 24 hours at 37 ⁇ C/5% CO 2 prior to total RNA purification using a RNeasy 96 Kit (Qiagen). Each duplex may be tested by transfection in duplicate wells in a single experiment. cDNA synthesis may be performed using FastKing RT (with gDNase) Kit (Tiangen).
  • Real-time quantitative PCR may be performed on an ABI Prism 7900HT or ABI QuantStudio 7 with primers specific for human SLC25A5 (forward: ACTGACATCATGTACACAGGCAC (SEQ ID NO:1384) , reverse: ACCCATGCCTCTGAGAACATT (SEQ ID NO:1385) ) and human GAPDH (forward: GAAGGTGAAGGTCGGAGTC (SEQ ID NO:1386) , reverse: GAAGATGGTGATGGGATTTC (SEQ ID NO:1387) ) using a SensiFAST SYBR Hi-ROX kit (Meridian). qPCR may be performed in duplicate on cDNA derived from each well and the mean cycle threshold (Ct) calculated.
  • Ct mean cycle threshold
  • Relative SLC25A5 expression may be calculated from mean Ct values using the comparative Ct ( ⁇ Ct) method, normalised to GAPDH and relative to untreated cells. Maximum percent inhibition of SLC25A5 expression and IC50 values may be calculated using a four parameter (variable slope) model using GraphPad Prism 9.
  • the nucleic acid of the invention inhibits expression of the SLC25A5 gene with an pEC50 value lower than 5, 6, 7, 8, 9 or 10, preferably when determined by qPCR, more preferably by reverse transcriptase (RT)-qPCR, as described herein.
  • Huh7 cells human hepatocyte-derived cell line, obtained from JCRB Cell Bank
  • DMEM Modified Eagle Medium
  • Cells may be transfected with siRNA duplexes targeting either SLC25A5 mRNA or a negative control siRNA (siRNA-control; sense strand 5’- GCCTGTACCAAGGCTTTAA -3’ (SEQ ID NO:1383), antisense strand 5’- TTAAAGCCTTGGTACAGGC -3’ (SEQ ID NO:1382)) in a 6-point, log dose response curve to give final in assay concentrations of 3nM to 0.03pM.
  • Transfection may be carried out by diluting Lipofectamine RNAiMAX (ThermoFisher) in Opti-MEM (ThermoFisher) medium at a ratio of 48.5:1.5.
  • This solution may be added to an equal volume of siRNA, diluted to the required concentration in phosphate buffered saline.
  • the lipofectamine RNAiMAX and siRNA mixture may be incubated at room temperature for 15 minutes before 20 ⁇ L may be added to wells of a 96 well plate.
  • Huh7 cells may be dissociated from flasks using trypsin and resuspended at a density of 200,000 cells/mL. 100 ⁇ L of Huh7 cell suspension may be added to each well of the siRNA-containing 96-well plates. Cells may be incubated for 24 hours at 37 ⁇ C, 5% CO 2 ,95% humidity. Each siRNA may be tested in triplicate wells and on two separate days for a total of six replicates.
  • Intracellular RNA may be isolated using an Rneasy kit (Qiagen) according to the manufacturer’s instructions.
  • cDNA synthesis may be performed using a FastKing RT kit, with gDNase (Tiangen).
  • Target cDNA may be the quantified by qPCR on an ABI Prism 7900HT or ABI QuantStudio 7 with primers specific for human SLC25A5 (forward: ACTGACATCATGTACACAGGCAC (SEQ ID NO:1384) , reverse: ACCCATGCCTCTGAGAACATT (SEQ ID NO:1385) ) and human GAPDH (forward: GAAGGTGAAGGTCGGAGTC (SEQ ID NO:1386) , reverse: GAAGATGGTGATGGGATTTC (SEQ ID NO:1387) ) using a SensiFAST SYBR Hi-ROX kit (Meridian).
  • qPCR may be performed in duplicate on cDNA derived from each well and the mean Ct calculated.
  • Relative SLC25A5 expression may be calculated from mean Ct values using the comparative Ct ( ⁇ Ct) method, normalised to GAPDH and relative to untreated cells.
  • Maximum percent inhibition of SLC25A5 expression and pEC50 values (-log 10 of the EC50) may be calculated using a four parameter (variable slope) model using NumPy (Python).
  • inhibition of expression of the SLC25A5 gene may be characterized by a reduction of mean relative expression of the SLC25A5 gene.
  • the mean relative expression of SLC25A5 when cells are transfected with 0.1 nM of the nucleic acid of the invention, the mean relative expression of SLC25A5 is below 1, 0.9, 0.8, 0.7, 0.6, 0.5, or 0.4, preferably when determined by qPCR, more preferably by reverse transcriptase (RT)-qPCR, as described herein. In some embodiments, when cells are transfected with 5 nM of the nucleic acid of the invention, the mean relative expression of SLC25A5 is below 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4 or 0.3, preferably when determined by qPCR, more preferably by reverse transcriptase (RT)-qPCR, as described herein.
  • RT reverse transcriptase
  • Mean relative expression of the SLC25A5 gene may be quantified by the following method: Huh7 cells (human hepatocyte-derived cell line, obtained from JCRB Cell Bank) may be maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS at 37 ⁇ C in at atmosphere of 5% CO 2 .
  • DMEM Modified Eagle Medium
  • Real-time quantitative PCR may be performed on an ABI Prism 7900HT or ABI QuantStudio 7 with primers specific for human SLC25A5 (forward: ACTGACATCATGTACACAGGCAC (SEQ ID NO:1384) , reverse: ACCCATGCCTCTGAGAACATT (SEQ ID NO:1385) ) and human GAPDH (forward: GAAGGTGAAGGTCGGAGTC (SEQ ID NO:1386) , reverse: GAAGATGGTGATGGGATTTC (SEQ ID NO:1387) ) using a SensiFAST SYBR Hi-ROX kit (Meridian) .
  • qPCR may be performed in duplicate on cDNA derived from each well and the mean Ct calculated.
  • Relative SLC25A5 expression may be calculated from mean Ct values using the comparative Ct ( ⁇ Ct) method, normalised to GAPDH and relative to untreated cells. Inhibition of the expression of SLC25A5 gene may be manifested by a reduction of the amount of mRNA of the target SLC25A5 gene in comparison to a suitable control. In other embodiments, inhibition of the expression of SLC25A5 gene may be assessed in terms of a reduction of a parameter that is functionally linked to gene expression, e.g , protein expression or signaling pathways.
  • the present invention also provides methods of using nucleic acid e.g. an siRNA of the invention or a composition containing nucleic acid e.g. an siRNA of the invention to reduce or inhibit gene expression in a cell or reduce expression or function of a target.
  • the methods include contacting the cell with a nucleic acid e.g. dsiRNA of the invention and maintaining the cell for a time sufficient to obtain degradation of the mRNA transcript of a gene, thereby inhibiting expression of the gene in the cell. Reduction in gene expression or function of a target can be assessed by any methods known in the art.
  • the gene is SLC25A5.
  • the cell may be contacted in vitro or in vivo, i.e., the cell may be within a subject.
  • a cell suitable for treatment using the methods of the invention may be any cell that expresses a gene of interest associated with a metabolic disease or disorder, such as a metabolic disease or disorder associated with non-alcoholic fatty liver disease (NAFLD) and/or obesity and/or a disease or disorder associated with adipogenesis and/or adipogenesis.
  • the in vivo methods of the invention may include administering to a subject a composition containing a nucleic acid of the invention e.g. an siRNA, where the nucleic acid e.g.
  • siRNA includes a nucleoside sequence that is complementary to at least a part of an RNA transcript of SLC25A5 gene of the mammal to be treated.
  • the present invention further provides methods of treatment of a subject in need thereof.
  • the treatment methods of the invention include administering a nucleic acid such as an siRNA of the invention to a subject, e.g., a subject that would benefit from a reduction or inhibition of the expression of a gene and/or expression and/or function of a target, in a therapeutically effective amount e.g. a nucleic acid such as an siRNA targeting a gene or a pharmaceutical composition comprising the nucleic acid targeting a gene.
  • a nucleic acid e.g.
  • siRNA of the invention may be administered as a "free” nucleic acid or “free” siRNA, administered in the absence of a pharmaceutical composition.
  • the naked nucleic acid may be in a suitable buffer solution.
  • the buffer solution may comprise acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof.
  • the buffer solution is phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the pH and osmolarity of the buffer solution can be adjusted such that it is suitable for administering to a subject.
  • a nucleic acid e.g. siRNA of the invention may be administered as a pharmaceutical composition, such as a dsiRNA liposomal formulation.
  • the method includes administering a composition featured herein such that expression of the target gene is decreased, such as for about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18, 24 hours, 28, 32, or about 36 hours.
  • expression of the target gene is decreased for an extended duration, e.g., at least about two, three, four days or more, e.g., about one week, two weeks, three weeks, or four weeks or longer, e.g., about 1 month, 2 months, or 3 months.
  • Subjects can be administered a therapeutic amount of nucleic acid e.g. siRNA, such as about 0.01 mg/kg to about 200 mg/kg.
  • the nucleic acid e.g.
  • siRNA can be administered by intravenous infusion over a period of time, on a regular basis. In certain embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. Administration of the siRNA can reduce gene product levels of a target gene , e.g., in a cell or tissue of the patient by at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or below the level of detection of the assay method used. In certain embodiments, administration results in clinical stabilization or preferably clinically relevant reduction of at least one sign or symptom of a gene-associated disorder.
  • the nucleic acid e.g.
  • siRNA can be administered subcutaneously, i.e., by subcutaneous injection.
  • One or more injections may be used to deliver the desired daily dose of nucleic acid e.g. s iRNA to a subject.
  • the injections may be repeated over a period of time.
  • the administration may be repeated on a regular basis.
  • the treatments can be administered on a less frequent basis.
  • a repeat-dose regimen may include administration of a therapeutic amount of nucleic acid on a regular basis, such as every other day or to once a year.
  • the nucleic acid is administered about once per month to about once per quarter (i.e., about once every three months).
  • the inhibitor of the present invention may be combined with other therapeutic agents for use in therapy, in particular for use in treatment of any of the metabolic diseases or disorders disclosed herein.
  • the inhibitor of the present invention such as any of the siRNA molecules disclosed herein, may be combined with a GLP-1 agonist, including, without limitation, GLP-1/GIP dual agonists, GLP-1/FGF21 dual agonists, GLP-1/GCGR dual agonists and GLP-1/GIP/GCGR triple agonists, and/or a THR-beta agonist. That is, in certain embodiments, the inhibitor of the invention, in particular the siRNA molecules disclosed herein, may be combined with a GLP-1 agonist for the treatment of metabolic diseases or disorders.
  • the inhibitor of the invention in particular the siRNA molecules disclosed herein, may be combined with a GLP-1 agonist for the treatment of fatty liver diseases, in particular NAFLD and/or NASH.
  • GLP-1 agonist refers to a compound, which fully or partially activates the human GLP-1 receptor. The term is thus equal to the term “GLP-1 receptor agonist” used in other documents.
  • GLP-1 agonist as well as the specific GLP-1 agonists described herein also encompass salt forms thereof. It follows that the GLP-1 agonist should display “GLP-1 activity” which refers to the ability of the compound, i.e.
  • the “GLP-1 agonist” binds to a GLP-1 receptor, e.g., with an affinity constant (K D ) or activate the receptor with a potency (EC 5 o) of below 1 mM, e.g. below 100 nM as measured by methods known in the art (see e.g. WO 98/08871) and exhibits insulinotropic activity, where insulinotropic activity may be measured in vivo or in vitro assays known to those of ordinary skill in the art.
  • K D affinity constant
  • EC 5 o potency
  • the GLP-1 agonist may be administered to an animal with increased blood glucose (e.g. obtained using an Intravenous Glucose Tolerance Test (IVGTT).
  • IVGTT Intravenous Glucose Tolerance Test
  • a person skilled in the art will be able to determine a suitable glucose dosage and a suitable blood sampling regime, e.g. depending on the species of the animal, for the IVGTT) and measure the plasma insulin concentration overtime. Suitable assays have been described in such as WO 2015/155151.
  • the term half maximal effective concentration (EC 5 o) generally refers to the concentration which induces a response halfway between the baseline and maximum, by reference to the dose response curve. EC 5 o is used as a measure of the potency of a compound and represents the concentration where 50% of its maximal effect is observed.
  • the in vitro potency of the GLP-1 agonist may be determined as described in WO 2015/155151 , example 29 without Human Serum Albumin (HSA), and the EC 5 o determined. The lower the EC 5 o value, the better the potency.
  • the potency (EC50) as determined (without HSA) is 5-1000 pM, such as 10-750 pM, 10-500 pM or 10-200 pM. In one embodiment the EC50 (without HSA) is at most 500 pM, such as at most 300 pM, such as at most 200 pM.
  • the EC50 (without HSA) is comparable to human GLP-1 (7-37). In one embodiment the EC50 (without HSA) is at most 50 pM. In a further such embodiment the EC50 is at most 40 pM, such as at most 30 pM such as at most 20 pM, such as at most 10 pM. In one embodiment the EC50 is around 10 pM.
  • the binding of the GLP-1 agonist to albumin may be measured using the in vitro potency assay of Example 29 of WO 2015/155151 including HSA. An increase of the in vitro potency, EC 5 o value, in the presence of serum albumin reflects the affinity to serum albumin.
  • the potency (EC50) as determined (with 1 % HSA) is 5-1000 pM, such as 100-750 pM, 200-500 pM or 100-400 pM. In one embodiment the EC50 (with 1 % HSA) is at most 750 pM, such as at most 500 pM, such as at most 400 pM, such as at most 300 or such as at most 250 pM.
  • the fold variation in relation to a known GLP-1 receptor agonist may be calculated as EC50(test analogue)/EC50(known analogue), and if this ratio is such as 0.5- 1.5, or 0.8-1.2 the potencies are considered to be equivalent.
  • the potency, EC50 (without HSA), is equivalent to the potency of liraglutide. In one embodiment the potency, EC50 (without HSA), is equivalent to the potency of semaglutide.
  • the GLP-1 agonist is a GLP-1 analogue, optionally comprising “one substituent”.
  • analogue as used herein referring to a GLP-1 peptide (hereafter “peptide”) means a peptide wherein at least one amino acid residue of the peptide has been substituted with another amino acid residue and/or wherein at least one amino acid residue has been deleted from the peptide and/or wherein at least one amino acid residue has been added to the peptide and/or wherein at least one amino acid residue of the peptide has been modified. Such addition or deletion of amino acid residues may take place at the N- terminal of the peptide and/or at the C-terminal of the peptide.
  • GLP-1 analogue or “analogue of GLP-1” as used herein refers to a peptide, or a compound, which is a variant of the human Glucagon-Like Peptide-1 (GLP-1 (7-37)).
  • GLP-1 (7-37) has the sequence HAEGTFTSDV SSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 1388).
  • variant refers to a compound which comprises one or more amino acid substitutions, deletions, additions and/or insertions.
  • the GLP-1 agonist exhibits at least 60%, 65%, 70%, 80% or 90% sequence identity to GLP-1 (7-37) over the entire length of GLP-1 (7-37).
  • the term GLP-1 agonist is meant to encompass the GLP-1 agonist and any pharmaceutically acceptable salt, amide, or ester thereof.
  • the composition comprises the GLP-1 agonist or a pharmaceutically acceptable salt, amide, or ester thereof.
  • the composition comprises the GLP-1 agonist and one or more pharmaceutically acceptable counter ions.
  • the inhibitor of the present invention in particular the siRNA molecules of the present invention, may be administered with a GLP-1 agonist selected from one or more of the GLP-1 agonists disclosed in W093/19175, W096/29342, WO98/08871, WO99/43707, WO99/43706, W099/43341 , WO99/43708, W02005/027978, W02005/058954, W02005/058958, W02006/005667, W02006/037810, W02006/037811 , W02006/097537, W02006/097538, W02008/023050, W02009/030738, W02009/030771, W02009/030774 and WO2021/219710, which are incorporated herein by reference in their entirety.
  • a GLP-1 agonist selected from one or more of the GLP-1 agonists disclosed in W093/19175, W096/29342, WO98/08871, WO99/4
  • the inhibitor of the present invention in particular the siRNA molecules of the present invention, may be administered with a GLP-1 agonist selected from the group consisting of: Dulaglutide (Trulicity®), Exenatide (Byetta®), Exenatide extended-release (Bydureon®), Liraglutide (Victoza®), Lixisenatide (Adlyxin®), Semaglutide injection (Ozempic®), and Semaglutide tablets (Rybelsus®).
  • a GLP-1 agonist selected from the group consisting of: Dulaglutide (Trulicity®), Exenatide (Byetta®), Exenatide extended-release (Bydureon®), Liraglutide (Victoza®), Lixisenatide (Adlyxin®), Semaglutide injection (Ozempic®), and Semaglutide tablets (Rybelsus®).
  • the GLP-1 agonist is semaglutide having a formula of N- epsilon26-[2-(2- ⁇ 2-[2-(2- ⁇ 2-[(S)-4-carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamino]ethoxy ⁇ ethoxy) acetylamino]ethoxy ⁇ ethoxy)acetyl] [Aib8,Arg34]GLP-1 (7-37).
  • GLP-1 agonist as used herein also encompasses dual or triple agonists that can activate more than one receptor. These dual or triple agonists may be molecules that have the ability to activate the GLP- 1 receptor and at least one further receptor.
  • the dual or triple agonist may be a chimeric molecule comprising a first portion that activates the GLP-1 receptor and further portions that activate further receptors.
  • the GLP-1 agonist is a dual or triple agonist that, in addition to the GLP-1 receptor, further activates one or more of: a glucose-dependent insulinotropic polypeptide (GIP) receptor, a Fibroblast growth factor 21 (FGF21) receptor, and a glucagon receptor (GCGR).
  • GIP glucose-dependent insulinotropic polypeptide
  • FGF21 Fibroblast growth factor 21
  • GCGR glucagon receptor
  • the inhibitor of the invention in particular the siRNA molecules disclosed herein, may be combined with a GLP-1/GIP dual agonist for the treatment of metabolic diseases or disorders.
  • the inhibitor of the invention in particular the siRNA molecules disclosed herein, may be combined with a GLP-1/GIP dual agonist for the treatment of fatty liver diseases, in particular NAFLD and/or NASH.
  • GLP-1/GIP dual agonist refers to a substance or ligand that can activate the GLP-1 receptor and the glucose-dependent insulinotropic polypeptide (GIP) receptor.
  • GLP-1/GIP receptor co-agonists and their potential medical uses are described in several patent applications such as WO 2010/011439, WO 2013/164483, WO 2014/192284, WO 2015/067715, WO 2015/022420, WO 2015/086728, WO 2015/086729, WO 2016/111971, WO 2020/023386, US 9745360, US 2014/162945, US 2014/0357552, WO 2021/150673, WO 2021/260530, WO 2022/018185, and WO 2022/079639, which are fully incorporated herein by reference.
  • the inhibitor of the invention in particular the siRNA molecules disclosed herein, may be combined with the GLP-1/GIP dual agonist tirzepatide (Mounjaro®) for the treatment of metabolic diseases or disorders.
  • the inhibitor of the invention, in particular the siRNA molecules disclosed herein may be combined with the GLP-1/GIP dual agonist tirzepatide (Mounjaro®) for the treatment of fatty liver diseases, in particular NAFLD and/or NASH.
  • “tirzepatide” means a GLP-1/GIP dual agonist peptide as described in U.S. Pat. No. 9,474,780 and described by CAS Registry Number: 2023788-19-2.
  • Tirzepatide is described in Example 1 of U.S. Pat. No.9,474,780, with the following sequence: YX 1 EGTFTSDYSIX 2 LDKIAQKAFVQWLMGGPSSGAPPPS (SEQ ID NO: 1389) wherein X 1 is ⁇ -amino isobutyric acid (Aib); X 2 is Aib; K at position 20 is chemically modified through conjugation to the epsilon-amino group of the K side-chain with (2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl) 2 - ( ⁇ Glu) 1 -CO—(CH 2 ) 18 —CO 2 H; and the C-terminal amino acid is amidated as a C-terminal primary amide.
  • X 1 is ⁇ -amino isobutyric acid (Aib)
  • X 2 is Aib
  • K at position 20 is chemically modified through conjugation to the epsilon
  • the inhibitor of the invention in particular the siRNA molecules disclosed herein, may be combined with a GLP-1/ FGF21 dual agonist for the treatment of metabolic diseases or disorders.
  • the inhibitor of the invention, in particular the siRNA molecules disclosed herein may be combined with a GLP-1/FGF21 dual agonist for the treatment of fatty liver diseases, in particular NAFLD and/or NASH.
  • GLP-1/FGF21 dual agonist refers to a substance or ligand that can activate the GLP-1 receptor and the Fibroblast growth factor 21 (FGF21) receptor.
  • the GLP-1/FGF21 dual agonist is a chimeric molecule comprising a GLP-1 agonist as defined herein above and the molecule FGF21 or a functionally active variant or analogue thereof.
  • the GLP-1/FGF21 dual agonist may further comprise an antibody Fc region.
  • GLP-1/ FGF21 receptor co-agonists and their potential medical uses are described in several patent applications such as WO 2010/142665, WO2014/037373, WO 2018/115401, WO 2018/166461, WO 2019/243557, and WO 2022/002408, which are fully incorporated herein by reference.
  • the inhibitor of the invention in particular the siRNA molecules disclosed herein, may be combined with a GLP-1/ GCGR dual agonist for the treatment of metabolic diseases or disorders.
  • the inhibitor of the invention in particular the siRNA molecules disclosed herein, may be combined with a GLP-1/GCGR dual agonist for the treatment of fatty liver diseases, in particular NAFLD and/or NASH.
  • GLP-1/ GCGR dual agonist refers to a substance or ligand that can activate the GLP-1 receptor and the glucagon receptor (GCGR).
  • GLP-1/GCGR receptor co-agonists and their potential medical uses are described in several patent applications such as, WO 2008/101017, WO 2009/155258, WO 2011/075393, WO 2011/160630, WO 2014/056872, WO 2014/091316, WO 2015/086733, WO 2017/181452, WO 2018/100174, WO 2019/030268, WO 2019/060660 and WO 2023/006923, which are fully incorporated herein by reference.
  • the inhibitor of the invention in particular the siRNA molecules disclosed herein, may be combined with the GLP-1/ GCGR dual agonist Survodutide (BI 456906) for the treatment of metabolic diseases or disorders.
  • the inhibitor of the invention in particular the siRNA molecules disclosed herein, may be combined with the GLP-1/ GCGR dual agonist Survodutide (BI 456906) for the treatment of fatty liver diseases, in particular NAFLD and/or NASH.
  • the inhibitor of the invention in particular the siRNA molecules disclosed herein, may be combined with a GLP-1/GIP/GCGR triple agonist for the treatment of metabolic diseases or disorders.
  • the inhibitor of the invention, in particular the siRNA molecules disclosed herein may be combined with a GLP-1/GIP/GCGR dual agonist for the treatment of fatty liver diseases, in particular NAFLD and/or NASH.
  • GLP-1/GIP/GCGR triple agonist refers to a substance or ligand that can activate the GLP-1 receptor, the GIP receptor and the glucagon receptor (GCGR).
  • GLP-1/GIP/GCGR receptor co-agonists and their potential medical uses are described in several patent applications such as, WO 2014/096150, WO 2015/067716, WO 2019/125292, WO 2022/090447, and WO 2022/268029, which are fully incorporated herein by reference.
  • the inhibitor of the invention in particular the siRNA molecules disclosed herein, may be combined with Retatrutid (LY-3437943) for the treatment of metabolic diseases or disorders.
  • the THR-beta agonist is KB-2115 (eprotirome) or any of the compounds disclosed in WO 2007/110226 or WO 2009/077147, which are incorporated herein by reference in their entirety.
  • the person skilled in the art is aware of ways to formulate THR-beta agonists for any suitable route of administration.
  • the inhibitor of the invention in particular the siRNA molecules disclosed herein, may be combined with a GLP-1 agonist, such as any one of the GLP-1 agonists disclosed herein above, including dual or triple agonists, and a THR-beta agonist, such as any one of the THR-beta agonists disclosed herein above, for the treatment of fatty liver diseases, in particular NAFLD and/or NASH.
  • a GLP-1 agonist such as any one of the GLP-1 agonists disclosed herein above, including dual or triple agonists
  • a THR-beta agonist such as any one of the THR-beta agonists disclosed herein above
  • the inhibitor of the invention in particular the siRNA molecules disclosed herein, may alternatively, or in addition, be combined and/or co-administered with one or more of: an amylin receptor agonist (such as pramlintide), and/or a dual amylin + calcitonin receptor agonist, and/or a glucagon receptor agonist, and/or an FXR receptor agonist (such as cilofexor or obeticholic acid), and/or an FGF-21 analogue or FGF-21 receptor agonist (such as efruxifermin), and/or an FGF-19 analogue or FGF-19 receptor agonist (such as aldafermin), and/or a galectin 3 inhibitor (such as belapectin), and/or a PPAR ⁇ agonist (such as elafibrinor), and/or a PPAR ⁇ agonist (such as pioglitazone or rosiglitazone), and/or a mixed PP
  • the inhibitor of the invention in particular the siRNA molecules disclosed herein, may be used for the treatment of any of the metabolic diseases or disorders disclosed herein, more preferably for the treatment of NAFLD, in combination with any of the molecules disclosed in Batchuluun et al. (Nat Rev Drug Discov, 2022, 21(4):283-305.
  • a citrate/isocitrate carrier (CIC) inhibitor such as Benzenetricarboxylate, CPTI-1 or CPTI-2
  • an ATP-citrate lyase (ACLY) inhibitor such as Bempedoic acid, Hydroxycitrate, BMS-303141, Emodin derivates, Furan carboxylate derivates, MEDICA 16, SB- 204990, or NDI-091143
  • an acetyl-CoA carboxylase (ACC) inhibitor such as Firsocostat, PF-05221304, PF-05175157, MK-4074, A-908292
  • Carboxamide derivative-1k CP-640186, Monocyclic derivate-1q, ND-654, ND-646, Olefin derivate-2e, (S)-9c, Soraphen A, TOFA, or WZ66), and/
  • the inhibitor according to the invention in particular the siRNA according to the invention, may be co-administered with a GLP-1 agonist, including GLP-1/GIP dual agonists, GLP- 1/FGF21 dual agonists, GLP-1/GCGR dual agonists and GLP-1/GIP/GCGR triple agonists, and/or a THR- beta agonist, wherein the inhibitor according to the invention may be administered prior to, after, or at the same time as the GLP-1 agonist and/or the THR-beta agonist.
  • a GLP-1 agonist including GLP-1/GIP dual agonists, GLP- 1/FGF21 dual agonists, GLP-1/GCGR dual agonists and GLP-1/GIP/GCGR triple agonists, and/or a THR- beta agonist
  • a GLP-1 agonist including GLP-1/GIP dual agonists, GLP- 1/FGF21 dual agonists, GLP-1/GCGR dual agonists and GLP
  • the inhibitor according to the invention and the at least one more additional therapeutic agent may be delivered orally, subcutaneously, intravenously, intranasally, transdermally, intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally, or intraocularly.
  • the inhibitor according to the invention may be administered intravenously (IV) and/or subcutaneously, and the GLP-1 agonist may be administered subcutaneously.
  • the present invention may be applied in the compounds, processes, compositions or uses of the following Sentences numbered 1-101 wherein reference to any Formula in the Sentences 1-101 refers only to those Formulas that are defined within Sentences 1-101. These formulae are reproduced in Figure 6.
  • a compound according to any of Sentences 1 to 11, wherein X 1 is methylene.
  • a compound according to any of Sentences 1 to 11, wherein X 1 is oxygen.
  • a compound according to any of Sentences 1 to 11, wherein X 1 is sulfur.
  • a compound according to any of Sentences 1 to 14, wherein X 2 is methylene.
  • a compound according to any of Sentences 1 to 15, wherein X 2 is oxygen.
  • a compound according to any of Sentences 1 to 16, wherein X 2 is sulfur.
  • a compound according to any of Sentences 1 to 17, wherein m 3.
  • a compound according to any of Sentences 1 to 18, wherein n 6.
  • Z is: wherein: Z 1 , Z 2 , Z 3 , Z 4 are independently at each occurrence oxygen or sulfur; and one the bonds between P and Z 2 , and P and Z 3 is a single bond and the other bond is a double bond.
  • said RNA compound comprises an RNA duplex comprising first and second strands, wherein the first strand is at least partially complementary to an RNA sequence of a target gene, and the second strand is at least partially complementary to said first strand, and wherein each of the first and second strands have 5’ and 3’ ends.
  • a composition comprising a compound of Formula (II) as defined in Sentence 27, and a compound of Formula (III) as defined in Sentence 28, optionally dependent on Sentence 29.
  • a composition comprising a compound of Formula (IV) as defined in Sentence 32, and a compound of Formula (V) as defined in Sentence 33, optionally dependent on Sentence 34.
  • a composition according to Sentence 35 wherein said compound of Formula (V) as defined in Sentence 33 is present in an amount in the range of 10 to 15% by weight of said composition.
  • a compound according to Sentence 37, wherein the modifications are chosen from 2’-O-methyl, 2’-deoxy-fluoro, and 2’-deoxy.
  • a compound according to Sentence 42, wherein said one or more carbohydrates can be a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide or polysaccharide.
  • a compound according to Sentence 43 wherein said one or more carbohydrates comprise one or more galactose moieties, one or more lactose moieties, one or more N-AcetylGalactosamine moieties, and / or one or more mannose moieties.
  • said one or more carbohydrates comprise one or more N-Acetyl-Galactosamine moieties.
  • a compound according to Sentence 45 which comprises two or three N-AcetylGalactosamine moieties.
  • a compound according to Sentence 66 wherein said one or more degradation protective moieties are not present at the end of the oligonucleoside strand that carries the ligand moieties, and / or wherein said one or more degradation protective moieties is selected from phosphorothioate internucleoside linkages, phosphorodithioate internucleoside linkages and inverted abasic nucleosides, wherein said inverted abasic nucleosides are present at the distal end of the strand that carries the ligand moieties, as shown in any of Formulae (VIII), (IX), (X) or (XI) in any of Sentences 54, 55, 59 or 60.
  • Formula (XII) Formula (XIII) herein: R 1 at each occurrence is independently selected from the group consisting of hydrogen, methyl and ethyl; R 2 is selected from the group consisting of hydrogen, hydroxy, -OC 1-3 alkyl, -C( O)OC 1-3 alkyl, halo and nitro;
  • X 1 and X 2 at each occurrence are independently selected from the group consisting of methylene, oxygen and sulfur;
  • m is an integer of from 1 to 6;
  • n is an integer of from 1 to 10;
  • q, r, s, t, v are independently integers from 0 to
  • Formula (XIV) Formula (XV) R 1 at each occurrence is independently selected from the group consisting of hydrogen, methyl and ethyl;
  • X 1 and X 2 at each occurrence are independently selected from the group consisting of methylene, oxygen and sulfur;
  • q, r, s, t, v are independently integers from 0 to 4, with the proviso that: (i) q and r cannot both be 0 at the same time; and (ii) s, t and v cannot all be 0 at the same time;
  • Z is an oligonucleoside moiety.
  • a compound according to Sentence 80 for the preparation of a compound according to any of Sentences 21, 26, 33, 34, 60, 61, and / or a composition according to any of Sentences 35, 36, 62, 63. 97.
  • Use of a compound according to Sentence 88 for the preparation of a compound according to any of Sentences 20, 25, 27 to 29, 54 to 56, and / or a composition according to any of Sentences 30, 31, 57, 58. 98.
  • Use of a compound according to Sentence 89 for the preparation of a compound according to any of Sentences 21, 26, 32 to 34, 59 to 61, and / or a composition according to any of Sentences 35, 36, 62, 63. 99.
  • HepNet a proprietary computational platform for the identification and validation of drug targets in hepatocytes.
  • Two independent approaches were used for the identification of SLC25A5/ANT2, the first utilised omics data in the form of Genome Wide Association Studies (GWAS) metanalyses to identify targets for Non- Alcoholic Fatty Liver Disease (NAFLD) from experimental data.
  • the second approach utilised the HepNet knowledge graph, a semantic network describing interactions between entities such as genes, drugs, diseases and/or biological processes in the context of hepatocytes.
  • GWAS-based approach metanalyses were QC'ed for sample size, target population, and quality of statistical analysis.
  • Protein-protein interaction networks were then built around the selected processes and the nodes scored for their importance to the network using a proprietary algorithm (algorithm C).
  • algorithm C The highest scoring nodes from algorithm C were manually assessed by an internal team to identify targets of interest for the treatment of NAFLD metabolic diseases.
  • algorithm C machine learning algorithms were used to predict interactions between genes and disease based upon the structure of the knowledge graph, a task commonly referred to as ‘link prediction’. This generated a set of genes that could be used for network-based target identification using proprietary algorithms A-C as described above.
  • the networks and other outputs from these algorithms were different from the ones generated by the GWAS-based analysis and were assessed by a different internal team.
  • ANT2 was surprisingly identified as a drug target for NAFLD among various other targets that have been previously associated with metabolic disorders, such NAFLD (APOA5, HMDH, APOC3, NR1H3, MTP, PCSK9, SOAT1 and ABCA1).
  • the reaction was stirred at room temperature for 3 h.
  • the reaction mixture was diluted with DCM (50 mL) and washed with cold saturated aq. NaHCO 3 (100 mL) and water (100 mL).
  • the organic layer was separated, dried over Na 2 SO 4 and concentrated to afford the title compound as yellow oil, which was purified by flash chromatography (gradient elution: 0-10% MeOH in DCM in 10 CV).
  • the reaction was carried out at room temperature and after 1 h another molar equivalent of the MFCO solution was added. The reaction was allowed to proceed for an additional hour and was monitored by LC/MS. At least two molar equivalent excess of the MFCO NHS ester reagent relative to the amino modified oligonucleotide were needed to achieve quantitative consumption of the starting material.
  • the reaction mixture was diluted 15-fold with water, filtered through a 1.2 ⁇ m filter from Sartorius and then purified by reserve phase (RP HPLC) on an ⁇ kta Pure instrument (GE Healthcare). Purification was performed using a XBridge C18 Prep 19 x 50 mm column from Waters.
  • Buffer A was 100 mM TEAAc pH 7 and buffer B contained 95% acetonitrile in buffer A.
  • a flow rate of 10 mL/min and a temperature of 60°C were employed. UV traces at 280 nm were recorded. A gradient of 0-100% B within 60 column volumes was employed.
  • Fractions containing full length conjugated oligonucleotide were pooled, precipitated in the freezer with 3 M NaOAc, pH 5.2 and 85% ethanol and the collected pellet was dissolved in water. Samples were desalted by size exclusion chromatography and concentrated using a speed-vac concentrator to yield the conjugated oligonucleotide in an isolated yield of 40–80%.
  • RP HPLC purification was performed using a XBridge C18 Prep 19 x 50 mm column from Waters.
  • Buffer A was 100 mM triethylammonium acetate pH 7 and buffer B contained 95% acetonitrile in buffer A.
  • a flow rate of 10 mL/min and a temperature of 60°C were employed.
  • UV traces at 280 nm were recorded.
  • a gradient of 0-100% B within 60 column volumes was employed.
  • Fractions containing full-length conjugated oligonucleotide were pooled, precipitated in the freezer with 3 M NaOAc, pH 5.2 and 85% ethanol and the collected pellet was dissolved in water to give an oligonucleotide solution of about 1000 OD/mL.
  • Flash chromatography was performed with a Biotage Isolera One flash chromatography instrument equipped with a dual variable UV wavelength detector (200-400 nm) using Biotage Sfär Silica 10, 25, 50 or 100 g columns (Uppsala, Sweden). All moisture-sensitive reactions were carried out under anhydrous conditions using dry glassware, anhydrous solvents, and argon atmosphere. All commercially available reagents were purchased from Sigma-Aldrich and solvents from Carl Roth GmbH + Co. KG. D-Galactosamine pentaacetate was purchased from AK scientific.
  • HPLC/ESI-MS was performed on a Dionex UltiMate 3000 RS UHPLC system and Thermo Scientific MSQ Plus Mass spectrometer using an Acquity UPLC Protein BEH C4 column from Waters (300 ⁇ , 1.7 ⁇ m, 2.1 x 100 mm) at 60 °C.
  • the solvent system consisted of solvent A with H2O containing 0.1% formic acid and solvent B with acetonitrile (ACN) containing 0.1% formic acid. A gradient from 5-100% of B over 15 min with a flow rate of 0.4 mL/min was employed.
  • Detector and conditions Corona ultra-charged aerosol detection (from esa). Nebulizer Temp.: 25 °C.
  • TriGalNAc _Tether2 Preparation of compound 2: D-Galactosamine pentaacetate (3.00 g, 7.71 mmol, 1.0 eq.) was dissolved in anhydrous dichloromethane (DCM) (30 mL) under argon and trimethylsilyl trifluoromethanesulfonate (TMSOTf, 4.28 g, 19.27 mmol, 2.5 eq.) was added. The reaction was stirred at room temperature for 3 h. The reaction mixture was diluted with DCM (50 mL) and washed with cold saturated aq. NaHCO 3 (100 mL) and water (100 mL).
  • DCM dichloromethane
  • TMSOTf trimethylsilyl trifluoromethanesulfonate
  • N,N,N′,N′- tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU) (2.78 g, 7.44 mmol, 5.0 eq.), 1-hydroxybenzotriazole hydrate (HOBt) (1.05 g, 7.44 mmol, 5.0 eq.) and N,N-diisopropylethylamine (DIPEA) (2.07 mL, 11.9 mmol, 8.0 eq.) were added to the solution and the reaction was stirred for 72 h. The solvent was removed under reduced pressure, the residue was dissolved in DCM (100 mL) and washed with saturated aq.
  • DIPEA N,N,N′,N′- tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate
  • Triantennary GalNAc compound 10 (0.45 g, 0.26 mmol, 1.0 eq.), HBTU (0.19 g, 0.53 mmol, 2.0 eq.) and DIPEA (0.23 mL, 1.3 mmol, 5.0 eq.) were dissolved in DCM (10 mL) under argon. To this mixture, it was added dropwise a solution of compound 13 (0.14 g, 0.53 mmol, 2.0 eq.) in DCM (5 mL). The reaction was stirred at room temperature overnight. The solvent was removed, and the residue was dissolved in EtOAc (50 mL), washed with water (50 mL) and dried over Na 2 SO 4 .
  • TriGalNAc Triantennary GalNAc compound 14 (0.31 g, 0.15 mmol, 1.0 eq.) was dissolved in EtOAc (15 mL) and Pd/C (40 mg) was added. The reaction mixture was degassed by using vacuum/argon cycles (3x) and hydrogenated under balloon pressure overnight.
  • TriGalNAc tether 2 (GalNAc-T2) conjugation at 5’-end or 3’- end 5’-GalNAc-T2 conjugates
  • TriGalNAc tether 2 NHS ester To a solution of carboxylic acid tether 2 (compound 15, 227 mg, 121 ⁇ mol) in DMF (2.1 mL), N-hydroxysuccinimide (NHS) (15.3 mg, 133 ⁇ mol) and N,N′- diisopropylcarbodiimide (DIC) (19.7 ⁇ L, 127 ⁇ mol) were added. The solution was stirred at room temperature for 18 h and used without purification for the subsequent conjugation reactions.
  • NHS N-hydroxysuccinimide
  • DIC N,N′- diisopropylcarbodiimide
  • the reaction mixture was diluted 15-fold with water, filtered once through 1.2 ⁇ m filter from Sartorius and then purified by reserve phase (RP HPLC) on an ⁇ kta Pure (GE Healthcare) instrument.
  • the purification was performed using a XBridge C18 Prep 19 x 50 mm column from Waters.
  • Buffer A was 100 mM TEAA pH 7 and buffer B contained 95% acetonitrile in buffer A.
  • a flow rate of 10 mL/min and a temperature of 60°C were employed.
  • UV traces at 280 nm were recorded.
  • a gradient of 0–100% B within 60 column volumes was employed.
  • Tether 2 Conjugation of Tether 2 to a siRNA strand: TriGalNAc tether 2 (GalNAc-T2) conjugation at 5’-end or 3’- end Conjugation conditions
  • Pre-activation To a solution of compound 15 (16 umol, 4 eq.) in DMF (160 ⁇ L) was added TFA-O-PFP (15 ⁇ l, 21 eq.) followed by DIPEA (23 ⁇ l, 32 eq.) at 25°C. The tube was shaken for 2 h at 25°C. The reaction was quenched with H2O (10 ⁇ L).
  • Example 7 Solid phase synthesis method: scale ⁇ 1 ⁇ mol Syntheses of siRNA sense and antisense strands were performed on a MerMade192X synthesiser with commercially available solid supports made of controlled pore glass with universal linker (Universal CPG, with a loading of 40 ⁇ mol/g; LGC Biosearch or Glen Research). RNA phosphoramidites were purchased from ChemGenes or Hongene.
  • the 2'-O-Methyl phosphoramidites used were the following: 5'-(4,4'-dimethoxytrityl)-N-benzoyl- adenosine 2'-O-methyl-3'- [(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-(4,4'-dimethoxytrityl)- N-acetyl-cytidine 2'-O-methyl-3'- [(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-(4,4'- dimethoxytrityl)-N-isobutyryl-guanosine 2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]- phosphoramidite, 5'-(4,4'-dimethoxytrityl)-uridine 2'
  • the 2’-F phosphoramidites used were the following: 5'-dimethoxytrityl-N-benzoyl-deoxyadenosine 2'- fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-dimethoxytrityl-N-acetyl-deoxycytidine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-dimethoxytrityl-N-isobutyryl- deoxyguanosine 2'-fluoro-3'- [(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite and 5'-dimethoxytrityl- deoxyuridine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-
  • Inverted abasic phosphoramidite, 3-O-Dimethoxytrityl-2-deoxyribose-5-[(2-cyanoethyl)-(N, N- diisopropyl)]-phosphoramidite were purchased from Chemgenes (ANP-1422) or Hongene (OP-040).
  • the DMT was removed by deblock solution, 3% TCA in DCM (DNAchem).
  • the coupling time was 180 seconds.
  • the oxidizer contact time was set to 80 seconds and thiolation time was 2*100 seconds.
  • the oligonucleotides were cleaved from the solid support using a NH 4 OH:EtOH solution 4:1 (v/v) for 20 hours at 45°C (TCI).
  • TCI 45°C
  • the solid support was then filtered off, the filter was thoroughly washed with H 2 O and the volume of the combined solution was reduced by evaporation under reduced pressure.
  • Oligonucleotide were treated to form the sodium salt by ultracentrifugation using Amicon Ultra-2 Centrifugal Filter Unit; PBS buffer (10x, Teknova, pH 7.4, Sterile) or by EtOH precipitation from 1M sodium acetate.
  • Example 8 Solid phase synthesis method: scale ⁇ 5 ⁇ mol Syntheses of siRNA sense and antisense strands were performed on a MerMade12 synthesiser with commercially available solid supports made of controlled pore glass with universal linker (Universal CPG, with a loading of 40 ⁇ mol/g; LGC Biosearch or Glen Research) at 5 ⁇ mol scale.
  • Sense strand destined to 3' conjugation were sytnthesised at 12 ⁇ mol on 3'-PT-Amino-Modifier C6 CPG 500 ⁇ solid support with a loading of 86 ⁇ mol/g (LGC).
  • RNA phosphoramidites were purchased from ChemGenes or Hongene.
  • the 2'-O-Methyl phosphoramidites used were the following: 5'-(4,4'-dimethoxytrityl)-N-benzoyl- adenosine 2'-O-methyl-3'- [(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-(4,4'-dimethoxytrityl)- N-acetyl-cytidine 2'-O-methyl-3'- [(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-(4,4'- dimethoxytrityl)-N-isobutyryl-guanosine 2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]- phosphoramidite, 5'-(4,4'-dimethoxytrityl)-uridine 2'
  • the 2’-F phosphoramidites used were the following: 5'-dimethoxytrityl-N-benzoyl-deoxyadenosine 2'- fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-dimethoxytrityl-N-acetyl-deoxycytidine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-dimethoxytrityl-N-isobutyryl- deoxyguanosine 2'-fluoro-3'- [(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite and 5'-dimethoxytrityl- deoxyuridine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-
  • Inverted abasic phosphoramidite 3-O-Dimethoxytrityl-2-deoxyribose-5-[(2-cyanoethyl)-(N, N- diisopropyl)]-phosphoramidite were purchased from Chemgenes (ANP-1422) or Hongene (OP-040). All phosphoramidites were dissolved in anhydrous acetonitrile (Honeywell Research Chemicals) at a concentration of 0.05M, except 2’-O-methyl-uridine phosphoramidite which was dissolved in DMF/MeCN (1:4, v/v).
  • the oxidation time was 47 seconds, the thiolation time was 210 seconds.
  • the coupling was performed with 8 eq. of amidite for 2*150 seconds.
  • the oxidation time was 47 seconds, the thiolation time was 250 seconds
  • the oligonucleotides were cleaved from the solid support using a NH4OH:EtOH solution 4:1 (v/v) for 20 hours at 45°C (TCI).
  • TCI 45°C
  • the solid support was then filtered off, the filter was thoroughly washed with H 2 O and the volume of the combined solution was reduced by evaporation under reduced pressure.
  • Oligonucleotide were treated to form the sodium salt by EtOH precipitation from 1M sodium acetate.
  • the single strand oligonucleotides were purified by IP-RP HPLC on Xbridge BEH C18 5 ⁇ m, 130 ⁇ , 19x150 mm (Waters) column with an increasing gradient of B in A.
  • Mobile phase A 240 mM HFIP, 7 mM TEA and 5% methanol in water
  • mobile phase B 240 mM HFIP, 7 mM TEA in methanol.
  • the single strands purity and identity were assessed by UPLC/MS ESI- on Xbridge BEH C182.5 ⁇ m, 3x50 mm (Waters) column with an increasing gradient of B in A.
  • Example 9 Dose-response for Inhibition of SLC25A5 Expression in Human Huh7 Cells
  • Huh7 cells human hepatocyte-derived cell line, obtained from JCRB Cell Bank
  • DMEM Modified Eagle Medium
  • siRNA duplexes targeting either SLC25A5 mRNA or a negative control siRNA (siRNA-control; sense strand 5’- GCCTGTACCAAGGCTTTAA -3’ (SEQ ID NO:1383), antisense strand 5’- TTAAAGCCTTGGTACAGGC -3’ (SEQ ID NO:1382)) in a 6-point, log dose response curve to give final in assay concentrations of 3nM to 0.03pM.
  • Transfection was carried out by diluting Lipofectamine RNAiMAX (ThermoFisher) in Opti-MEM (ThermoFisher) medium at a ratio of 48.5:1.5.
  • This solution was added to an equal volume of siRNA, diluted to the required concentration in phosphate buffered saline.
  • the lipofectamine RNAiMAX and siRNA mixture was incubated at room temperature for 15 minutes before 20 ⁇ L was added to wells of a 96 well plate.
  • Huh7 cells were dissociated from flasks using trypsin and resuspended at a density of 200,000 cells/mL. 100 ⁇ L of Huh7 cell suspension was added to each well of the siRNA-containing 96-well plates. Cells were incubated for 24 hours at 37 ⁇ C, 5% CO 2 ,95% humidity. Each siRNA was tested in triplicate wells and on two separate days for a total of six replicates.
  • cDNA synthesis was performed using a FastKing RT kit, with gDNase (Tiangen).
  • Target cDNA was the quantified by qPCR on an ABI Prism 7900HT or ABI QuantStudio 7 with primers specific for human SLC25A5 (forward: ACTGACATCATGTACACAGGCAC (SEQ ID NO:1384) , reverse: ACCCATGCCTCTGAGAACATT (SEQ ID NO:1385) ) and human GAPDH (forward: GAAGGTGAAGGTCGGAGTC (SEQ ID NO:1386) , reverse: GAAGATGGTGATGGGATTTC (SEQ ID NO:1387) ) using a SensiFAST SYBR Hi-ROX kit (Meridian).
  • qPCR was performed in duplicate on cDNA derived from each well and the mean Ct calculated.
  • Relative SLC25A5 expression was calculated from mean Ct values using the comparative Ct ( ⁇ Ct) method, 213ormalized to GAPDH and relative to untreated cells.
  • Maximum percent inhibition of SLC25A5 expression and pEC50 values (-log 10 of the EC50) were calculated using a four parameter (variable slope) model using NumPy (Python). Results are shown in Table 6. Sequences of RNAi molecules are depicted in the relevant Tables herein. Table 6 – Results of dose-response experiments for inhibition of SLC25A5 mRNA expression in human Huh7 cells.
  • ETXM ID Minimum r1 Maximum r1 pEC50 r1 Minimum r2 Maximum r2 pEC50 r2 ETX-M00001351 0.095738517 1.012076322 9.74 0.16593971 1.135465148 9.46 ETX-M00001352 0.068977171 0.832613609 10.36 0.107726125 1.031889622 10.19 ETX-M00001353 0.076601856 0.942804039 10.09 0.105081317 0.866486243 9.79 ETX-M00001354 0.04160111 0.352837401 12.42 0.054758242 0.528360743 10.99 ETX-M00001355 0.286834824 1.105769257 9.36 0.422953861 1.267175414 9.08 ETX-M00001356 0.062109246
  • ETX-M00001403 0.272226606 1.247723091 9.26 0.332815624 1.063614207 9.1 ETX-M00001404 0.94795512 1.325845624 8.4 0.882313118 1.159683253 ? ETX-M00001405 0.216376035 1.081756406 9.38 0.353142531 1.074248491 9.08 ETX-M00001406 0.425361623 1.388509882 9.03 0.520891691 1.290040521 9.0 ETX-M00001407 0.360201247 1.028556712 9.36 0.530895788 1.15606022 8.54 ETX-M00001408 0.790249405 1.164609515 9.04 0.822216806 1.000182763 8.25 ETX-M00001409 0.384042177 1.109734635 9.07 0.588229001 0.984025099 8.11 ETX-M00001410 0.458907902 1.110918523 8.99 0.661359549 1.2032852
  • mice Thirty-two C57BL/6 male mice were allocated for each of the five GalNAc-siRNAs, ETX-M00001397, ETX- M00001570, ETX-M00001378, ETX-M00001513, and ETX-M00001527.
  • 20 mice were allocated as a control group (saline) and 16 mice were allocated for a negative control siRNA group (ETXM1198).
  • Mice were subcutaneously dosed with ETXMs (1 mg/kg or 3 mg/kg) on day 0, defined as the day mice were first dosed.
  • Four mice from saline control group were sacrificed on day 0 prior to dosing and subsequently, four mice in each treatment group at each dose level were sacrificed on day 7, 14, 21, and 28.
  • liver tissues and plasma samples were harvested for further analysis.
  • SLC25A5 mRNA knockdown in mouse liver Harvested liver samples were used to measure the SLC25A5 mRNA knockdown level by RT-qPCR.
  • each tissue was treated with RNAlater and stored at 4 °C overnight then at -80 °C until the further analysis.
  • Liver tissues were homogenized with TRIZOL for RNA extraction.
  • RNA samples, adjusted to 400 ng/ ⁇ L, were reverse transcribed to cDNA using FastKing RT Kit, manufactured by TIANGEN. After reverse transcription, real-time quantitative PCR may be performed using an ABI Prism 7900HT to detect the relative abundance of SLC25A5 mRNA normalized to the housekeeping gene GAPDH.
  • ANT2 protein knockdown in mouse liver A second sample of liver tissue was used to measure ANT2 protein knockdown by LC-MS. Forty milligrams of mouse liver with 1.5 mL of RIPA buffer were used for protein extraction.
  • 1X of protease inhibitor, 1X of phosphatase inhibitor and 1X of PMSF were included in the RIPA buffer.
  • QIAGEN Tissue Lyser was used to homogenize the liver until lysate becomes transparent. After high-speed centrifugation at 4 °C, the protein concentration in liver lysate was measured by BCA quantification kit. Three hundred microgram protein was taken from each liver lysate sample for digestion using the S- TrapTM 96-well MS sample prep kit according to the manufacturer’s instruction with one exception: DTT and IAA were used as reductant and alkylation reagents instead of TCEP and MMTS.
  • the SLC25a5 protein database from UniProt was uploaded as library search.
  • the final selection of peptides should have the following characters: 1. Elution peak without tailing; 2. Well-matched product ions; 3. Quantified peak area greater than 1e6. Consequently, the following peptides, QIFLGGVDK and EQGVLSFWR, were chosen for SLC25a5 protein quantitation.
  • Relative protein expression is calculated via time-matched normalisation of the average target peptide counts to the Vehicle Control group. Peptide counts corresponding to the reference protein (GAPDH) are not considered.
  • the results of mRNA and protein knockdown are shown in Figure 10.
  • Example 11 Pharmacological studies in Gubra-Amylin NASH (GAN) diet-induced obese (DIO) mice
  • GAN Gubra-Amylin NASH
  • DIO diet-induced obese mice
  • ETX-312 ETX-M00001378
  • ETX-M00001751 an inhibitor of SLC25A5 expression
  • NAS NAFLD Activity Score
  • a cone shaped wedge of liver tissue (approximately 50 mg) was excised from the distal portion of the lobe and fixated in 10% neutral buffered formalin (10% NBF) for histology.
  • the cut surface of the liver was instantly electrocoagulated using bipolar coagulation (ERBE VIO 100 electrosurgical unit).
  • the liver was returned to the abdominal cavity, the abdominal wall sutured and the skin closed with staplers.
  • mice received carprofen (5mg/kg) administered subcutaneously on the day of the procedure and on post-operation days 1 and 2. Animals were randomized into groups based on fibrosis stage and picrosirius red (PSR) staining (% fractional area).
  • LEAN-CHOW control saline
  • mice Body weight was recorded daily and food intake recorded once weekly for 24 hours during the study period.
  • the body composition of mice was be assessed by an EchoMRI 3-1 Body composition analyzer (EchoMRI, US) at week 12. Non-anaesthetised animals were placed in a plastic tube inside the MRI scanner for approximately 80 seconds. The body composition was expressed as fat mass, fat free mass (lean mass) and water. Water is normally excluded.
  • preterminal tail vein blood collection was conducted for non-fasting plasma Alanine transaminase (ALT), Aspartate transaminase (AST), Triglycerides (TG) and Total Cholesterol (TC) analysis.
  • ALT Alanine transaminase
  • AST Aspartate transaminase
  • TG Triglycerides
  • TC Total Cholesterol
  • Plasma samples were prepared during anesthesia with isoflurane with the abdominal cavity opened and cardiac blood drawn with a syringe into a Microvette/Vacuette of appropriate dimensions with anticoagulant and mixed by inversion 5 times. Blood was placed at 4°C until it was centrifuged at 3000 g for 10 minutes. The plasma supernatants were transferred to new tubes and immediately frozen on dry ice. The samples were stored at -70°C.
  • ALT, AST, TG, and TC assays were measured using commercial kits (Roche Diagnostics), on the Cobas c 501 autoanalyzer. Insulin was measured in plasma collected in heparinized tubes using the commercial MSD platform (Meso Scale Diagnostics). TIMP -1 was measured in plasma collected in EDTA tubes using a commercial ELISA kit (R&D Systems). PIIINP was measured in plasma collected in EDTA tubes using a commercial ELISA kit (Cusabio). After study termination, the liver was collected and weighed. Terminal adipose tissue weight was also measured. Specific liver samples and biopsies were dissected and prepared as follows.
  • the liver was divided into a left lateral lobe, medial lobe, right lateral lobe, and caudate lobe.
  • the Post-biopsy piece ( ⁇ 200 mg, less than 0.7 x 0.5 cm) was cut from the left lateral lobe, 4 mm from the prebiopsy site with an edge.
  • the tissue was collected in 10% neutral buffered formalin.
  • a liver sample collected for future analysis of mRNA expression ( ⁇ 150 mg) was dissected from the left medial lobe, collected in tubes and frozen in liquid nitrogen. The samples was stored at -70°C.
  • a second piece of liver was dissected from the center of the left lateral lobe, put in a tube and frozen in liquid nitrogen for future analysis. The samples were stored at -70°C.
  • FFPE biopsies were placed in 10% neutral buffered formalin (10% NBF) for approximately 24 hours and then transferred to 70% ethanol and stored at 4°C. The FFPE biopsies were then placed in the Histokinette to infiltrate prior to embedding in blocks. Biopsy tissues were cut at 3 ⁇ m on a microtome and the sections were mounted on Superfrost Plus slides and stored at 4 0C. Glass slides with paraffin embedded sections were deparaffinated in xylene and rehydrated in series of graded ethanol.
  • H&E staining slides are incubated in Mayer’s Hematoxylin (Dako), washed in tap water, stained in Eosin Y solution (Sigma-Aldrich), dehydrated in graded ethanol and cover slipped.
  • PSR Picrosirius red
  • Immunohistochemistry was performed using standard procedures. Briefly, after antigen retrieval and blocking of endogenous peroxidase activity, slides were incubated with primary antibody. The primary antibody was detected using a polymeric HRP-linker antibody conjugate. Next, the primary antibody was visualized with DAB as chromogen. Finally, sections were counterstained in hematoxylin and cover slipped. Slides were scanned under a 20x objective in a ScanScope AT slide scanner (Aperio).
  • Antibodies used Antigen Primary Antibody Vendor Col1a1 Goat anti-type I Collagen Southern Biotech, Cat.1310- 01 Galectin-3 (mouse) Rat anti-Galectin 3 Biolegend, Cat.125402 ⁇ -SMA Rabbit anti-alpha smooth Abcam, Cat. ab124964 muscle actin [EPR5368] Liver samples stained with H&E or PSR were given a score for NAFLD Activity Score (NAS) and fibrosis stage, respectively, using the clinical criteria outlined by Kleiner et al. (Design and validation of a histological scoring system for nonalcoholic fatty liver disease, Hepatology, 2005) shown in the table below.
  • NAS NAFLD Activity Score
  • Total NAS represents the sum of scores for steatosis, inflammation, and ballooning, and ranges from 0-8.
  • NAS and fibrosis stage was determined by Gubra Histopathological Objective Scoring Technology (GHOST) deep learning app developed by Gubra using the VIS software (Visiopharm, Denmark) for a more accurate and objective method for staging disease in DIO-NASH mouse models. The app scores were reviewed by a histopathologist blinded to the results. Terminal liver biopsy histomorphometric analysis was also conducted by immunohistochemistry (IHC) for inflammation (Gal-3), Collagen (Col1a1), and activated stellate cells (a-SMA).
  • IHC immunohistochemistry
  • NAS Scoring Feature Degree Score Steatosis (percentage of ⁇ 5% 0 hepatocytes with lipid 5-33% 1 droplets) >33-66% 2 >66% 3 Lobular inflammation No foci 0 ⁇ 2 foci/200x 1 2-4 foci/200x 2 >4 foci/200x 3 Ballooning degeneration None 0 Few 1 Many cells/prominent 2 ballooning Fibrosis stage None 0 Perisinusoidal or periportal 1 Perisinusoidal and 2 portal/periportal 3 Bridging fibrosis 4 Cirrhosis As shown in Figure 11, the inhibitor ETX-312 resulted in a significant improvement of the NAFLD activity score (NAS).
  • NAS NAFLD activity score
  • ETX-312 was greater than the effects obtained with both positive controls, i.e., the GLP-1 agonist semaglutide and the THR-beta agonist resmetirom. Surprisingly, the most significant improvement was obtained when ETX-312 was combined with semaglutide or resmetirom.
  • the impact of the inhibitor ETX-312 on the blood markers ALT, AST, TIMP-1 and PIIINP is shown in Figures 12 and 13.
  • the terminal liver weight to body weight ratio in DIO-NASH mice that received different treatments is shown in Figure 14.
  • Example 12 Confirmation of Lead siRNA Activity in a Human Hydrodynamic Injection (HDI) Mouse Model
  • the transient expression of human mRNA in mice was evaluated to assess the activity of siRNAs against human mRNA sequences.
  • a single subcutaneous dose of siRNAs (3 mg/kg) was administered on Day -6.
  • Hydrodynamic injection (HDI) of a plasmid expressing human SLC25A5 mRNA was performed on Day 0.
  • the study was terminated, and mRNA expression was analyzed on Day 1.
  • saline was injected in place of an siRNA in the vehicle control group.
  • An overview of the study is shown in Figure 15.
  • the siRNAs tested in this study are shown in Table 7 below.
  • qPCR was performed in duplicate on cDNA derived from each well and the mean Ct calculated.
  • Relative SLC25A5 expression was calculated from mean Ct values using the comparative Ct ( ⁇ Ct) method, normalized to NEO and relative to vehicle-injected controls. Three animals were excluded from the analysis due to missing data or significant variation between technical replicates (Ct difference > 0.5).
  • a statistical analysis was carried out to compare the mean mRNA expression of the treatment groups to the Vehicle Control: pairwise comparisons were made using a linear model, with coefficients estimating the average treatment effect relative to the comparator. One-tailed t-tests were performed to test if the effect was less than 0, and p-values were adjusted for multiple comparisons using the FDR method.
  • RNAi molecules are depicted in the relevant Tables herein. As can be seen from Figure 16, and Table 7 below, a particularly large effect is seen for a number of siRNAs.
  • Preferred siRNAs for use in the invention are ETX-M00001751 (antisense: SEQ ID NO: 304, sense: SEQ ID NO: 580) and ETX-M00002666 (antisense: SEQ ID NO: 280, sense: SEQ ID NO: 556), and these are specifically contemplated throughout this disclosure, even when not singled out in a specific passage of this disclosure.
  • ETX-S00003198 can equally be considered to be reference to ETX-S00009009 where allowed by context.
  • Example 13 Pharmacodynamic study of ETX-312 in non-human primates (NHPs) This study was designed to evaluate the activity of the lead siRNA and a backup siRNA in NHPs. Specifically, an 84-day, single dose study was designed to effectively demonstrate the reduction of target mRNA and protein in the livers of healthy monkeys. To this end, 12 non-na ⁇ ve male cynomolgus macaques aged 4-8 years received 3 mg/kg ETX siRNA subcutaneously on day 0.
  • liver biopsies were taken on study days -7, 28, 56 and 84 for measurement of target mRNA and protein levels.
  • Blood samples for measurement of liver enzymes were collected on study days -7, -3, 0 (pre-treatment), and 7, 14, 28, 42,56, 70 and 84 (post-treatment); however, samples collected on -7, -3, 7, 42, and 70 were not analyzed.
  • Endpoints also included clinical observations for general health, adverse clinical signs, and injection site monitoring.
  • a summary of the study design is shown in Figure 17.
  • the following siRNAs were tested in this study: - ETX-M00001378 (antisense: SEQ ID NO: 304, sense: SEQ ID NO: 580) - ETX-M00001354 (antisense: SEQ ID NO: 280, sense: SEQ ID NO: 556) Hepatic SLC25A5 mRNA and ANT2 protein levels were determined after a single dose of 3 mg/kg siRNA.
  • ETX-M00001378 ETX-M00001751
  • ETX-M00001354 ETX-M00002666

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Abstract

La présente invention concerne des inhibiteurs et des compositions contenant des inhibiteurs et leurs utilisations dans le traitement ou la prévention d'une maladie ou d'un trouble métabolique, tels qu'une maladie ou un trouble métabolique associés à la stéatose hépatique non alcoolique (NAFLD), et/ou l'obésité et/ou une maladie ou un trouble associés à l'adipogenèse.
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