EP4176061A1 - Acides nucléiques contenant des nucléotides abasiques - Google Patents

Acides nucléiques contenant des nucléotides abasiques

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Publication number
EP4176061A1
EP4176061A1 EP22704882.4A EP22704882A EP4176061A1 EP 4176061 A1 EP4176061 A1 EP 4176061A1 EP 22704882 A EP22704882 A EP 22704882A EP 4176061 A1 EP4176061 A1 EP 4176061A1
Authority
EP
European Patent Office
Prior art keywords
strand
nucleic acid
nucleotide
nucleotides
acid according
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP22704882.4A
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German (de)
English (en)
Inventor
Ahmad Ali MORTAZAVI
Viviana MANNELLA
Muthusamy Jayaraman
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
E Therapeutics PLC
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E Therapeutics PLC
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Filing date
Publication date
Application filed by E Therapeutics PLC filed Critical E Therapeutics PLC
Publication of EP4176061A1 publication Critical patent/EP4176061A1/fr
Pending legal-status Critical Current

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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
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
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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
    • 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
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • 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 N.A.
    • 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
    • C—CHEMISTRY; METALLURGY
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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
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/332—Abasic residue
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/343—Spatial arrangement of the modifications having patterns, e.g. ==--==--==--
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/344—Position-specific modifications, e.g. on every purine, at the 3'-end
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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
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    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
    • C12Y101/03015—(S)-2-Hydroxy-acid oxidase (1.1.3.15)

Definitions

  • the present invention provides novel oligonucleotide compounds, which are nucleic acid compounds, 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 conditions.
  • Oligonucleotide compounds have important therapeutic applications in medicine. Oligonucleotides can be used to silence genes that are responsible for a particular disease. Gene-silencing prevents formation of a protein by inhibiting translation. Importantly, genesilencing agents are a promising alternative to traditional small, organic compounds that inhibit the function of the protein linked to the disease. siRNA, antisense RNA, and micro- RNA are oligonucleotides that prevent the formation of proteins by gene-silencing.
  • 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 such oligonucleotide compounds, which are nucleic acid compounds, for use in the treatment and / or prevention of disease.
  • a nucleic acid for inhibiting expression of a target gene in a cell, comprising at least one duplex region that comprises at least a portion of a first strand and at least a portion of a second strand that is at least partially complementary to the first strand, wherein said first strand is at least partially complementary to at least a portion of RNA transcribed from said target gene to be inhibited, wherein the second strand comprises one or more abasic nucleotides in a terminal region of the second strand, and wherein said abasic nucleotide(s) is / are connected to an adjacent nucleotide through a reversed internucleotide linkage.
  • a conjugate for inhibiting expression of 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.
  • a pharmaceutical composition comprising a nucleic acid as disclosed herein or a conjugate as disclosed herein and a physiologically acceptable excipient.
  • FIG. 3 shows analysis of hsTTR mRNA expression levels in a total of 45 human-derived cancer cell lysates and lysates of primary human hepatocytes (PHHs). mRNA expression levels are shown in relative light units [RLUs],
  • FIGs. 4A-B shows the results from the dose-response analysis of hsTTR targeting GalNAc-siRNAs in HepG2 cells in Example 1.
  • FIG 16. Single dose mouse pharmacology of ETX005. HAO1 mRNA expression is shown relative to the saline control group. Each point represents the mean and standard deviation of 3 mice.
  • FIG 20 Single dose mouse pharmacology of ETX014. C5 mRNA expression is shown relative to the saline control group. Each point represents the mean and standard deviation of 3 mice.
  • FIG 21 Single dose mouse pharmacology of ETX0014. Serum C5 concentration is shown relative to the saline control group. Each point represents the mean and standard deviation of 3 mice.
  • FIG 22 Single dose mouse pharmacology of ETX015. C5 mRNA expression is shown relative to the saline control group. Each point represents the mean and standard deviation of 3 mice.
  • FIG. 26 Single dose NHP pharmacology of ETX019. Serum TTR concentration is shown relative to day 1 of the study and also pre-dose.. Each point represents the mean and standard deviation of 3 animals. Time points up to 84 days are shown.
  • FIG. 28a Single dose NHP pharmacology of ETX023. Serum TTR concentration is shown relative to day 1 of the study and also pre-dose. Each point represents the mean and standard deviation of 3 animals. Time points up to 84 days are shown.
  • ETX014 as a product includes molecules based on the linker and ligand portions as specifically depicted in FIG 30 attached to an oligonucleotide moiety as also depicted herein
  • this ETX014 product may alternatively further comprise, or consist essentially of, molecules wherein the linker and ligand portions are essentially as depicted in FIG 30 attached to an oligonucleotide moiety but having the F substituent as shown in FIG 30 on the cyclo-octyl ring replaced by a substituent occurring as a result of hydrolytic displacement, such as an OH substituent.
  • ETX001 can consist essentially of molecules having linker and ligand portions specifically as depicted in FIG 31, with a F substituent on the cyclo-octyl ring; or (b) ETX001 can consist essentially of molecules having linker and ligand portions essentially as depicted in FIG 31 but having the F substituent as shown in FIG 31 on the cyclo-octyl ring replaced by a substituent occurring as a result of hydrolytic displacement, such as an OH substituent, or (c) ETX001 can comprise a mixture of molecules as defined in (a) and/or (b).
  • ETX010 can consist essentially of molecules having linker and ligand portions specifically as depicted in FIG 31, with a F substituent on the cyclo-octyl ring; or (b) ETX010 can consist essentially of molecules having linker and ligand portions essentially as depicted in FIG 31 but having the F substituent as shown in FIG 31 on the cyclo-octyl ring replaced by a substituent occurring as a result of hydrolytic displacement, such as an OH substituent, or (c) ETX010 can comprise a mixture of molecules as defined in (a) and/or (b).
  • FIG 36 Creatinine (CREA) concentration from animals treated with a single 1 mg/kg dose of ETX023. Each point represents the mean and standard deviation of 3 animals. The dotted lines show the range of values considered normal for this species (Park et al. 2016 Reference values of clinical pathology parameter in cynomolgus monkeys used in preclinical studies. Lab Anim Res 32:79-86.)
  • FIG. 43 shows the detail of the formulae described in Sentences 1-101 disclosed herein.
  • Figure 45b shows the underlying nucleotide sequences for the sense (SS) and antisense (AS) strands of constructs ETX005, ETX006 as described herein.
  • SS sense
  • AS antisense
  • iaia as shown at the 3’ end region of the sense strand in Figure 46a represents (i) two abasic nucleotides provided as the penultimate and terminal nucleotides at the 3’ end region of the sense strand, (ii) wherein a 3 ’-3’ reversed linkage is provided between the antepenultimate nucleotide (namely A at position 21 of the sense strand, wherein position 1 is the terminal 5’ nucleotide of the sense strand, namely terminal A at the 5 ’end region of the sense strand) and the adjacent penultimate abasic residue of the sense strand, and (iii) the linkage between the terminal and penultimate abasic nucleotides is 5’-3’ when reading towards the 3’ end region comprising the terminal and penultimate abasic nucleotides.
  • the present invention provides nucleic acids such as inhibitory RNA molecules (which may be referred to as iRNA), and compositions containing the same which can affect expression of a target gene.
  • the gene may be within a cell, e.g. a cell within a subject, such as a human.
  • the nucleic acids can be used to prevent and/or treat medical conditions associated with the expression of a target gene.
  • Inhibitory RNA (iRNA) is the preferred nucleic acid herein.
  • a double stranded nucleic acid e.g. RNAi agent of the invention includes a nucleotide mismatch in the sense strand.
  • the nucleotide mismatch is, for example, within 5, 4, 3, 2, or 1 nucleotides from the 3 '-end of the nucleic acid e.g. iRNA.
  • the nucleotide mismatch is, for example, in the 3'- terminal nucleotide of the nucleic acid e.g. iRNA.
  • ribonucleotide or “nucleotide” can also refer to a modified nucleotide, as further detailed below.
  • RNAi agent refers to an agent that contains RNA, and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway.
  • RISC RNA-induced silencing complex
  • iRNA directs the sequence-specific degradation of mRNA through RNA interference (RNAi).
  • nucleotide overhang refers to at least one unpaired nucleotide that extends from the duplex structure of a double stranded nucleic acid.
  • a ds nucleic acid can comprise an overhang of at least one nucleotide; alternatively the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more.
  • a nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside.
  • the overhang(s) can be on the sense strand, the antisense strand, or any combination thereof.
  • the nucleotide(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 nucleotide, 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 nucleotide, overhang at the 3'-end or the 5'-end.
  • the term "complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide 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 can also include, or be formed entirely from, non- Watson-Crick base pairs or base pairs formed from non-natural and modified nucleotides, in so far as the above requirements with respect to their ability to hybridize are fulfilled.
  • non- Watson- Crick base pairs include, but are not limited to, G:U Wobble or Hoogstein base pairing.
  • a nucleic acid or polynucleotide that is "substantially complementary” to at least part of a messenger RNA refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g. , an mRNA encoding a gene).
  • mRNA messenger RNA
  • a polynucleotide is complementary to at least a part of an mRNA of a gene of interest if the sequence is substantially complementary to a non- interrupted portion of an mRNA encoding that gene.
  • the antisense polynucleotides disclosed herein are substantially complementary to a target RNA sequence and comprise a contiguous nucleotide 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.
  • a nucleic acid e.g. an iRNA of the invention includes a sense strand that is substantially complementary to an antisense polynucleotide which, in turn, is complementary to a target gene sequence and comprises a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to the equivalent region of the nucleotide 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 iRNA of the invention includes an antisense strand that is substantially complementary to the target sequence and comprises a contiguous nucleotide 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.
  • treating refers 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. Treatment can include prevention of development of comorbidities, e.g. , reduced liver damage in a subject with a hepatic infection.
  • “Therapeutically effective amount,” as used herein, is intended to include the amount of a nucleic acid e.g. an iRNA 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 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.
  • 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.
  • sense strand or antisense strand is understood as “sense strand or antisense strand or sense strand and antisense strand.”
  • the term "at least" prior to a number or series of numbers is understood to include the number adjacent to the term “at least”, and all subsequent numbers or integers that could logically be included, as clear from context.
  • the number of nucleotides in a nucleic acid molecule must be an integer.
  • "at least 18 nucleotides of a 21 nucleotide nucleic acid molecule” means that 18, 19, 20, or 21 nucleotides have the indicated property.
  • nucleotide overhang As used herein, "no more than” or “less than” is understood as the value adjacent to the phrase and logical lower values or integers, as logical from context, to zero. For example, a duplex with an overhang of "no more than 2 nucleotides” has a 2, 1, or 0 nucleotide overhang. When “no more than” is present before a series of numbers or a range, it is understood that “no more than” can modify each of the numbers in the series or range.
  • the terminal region of a strand is the last 5 nucleotides from the 5’ or the 3’ end.
  • abasic nucleotides there are 1, e.g. 2, e.g. 3, e.g. 4 or more abasic nucleotides present in the nucleic acid.
  • Abasic nucleotides are modified nucleotides because they lack the base normally seen at position 1 of the sugar moiety. Typically, there will be a hydrogen at position 1 of the sugar moiety of the abasic nucleotides present in a nucleic acid according to the present invention.
  • the abasic nucleotides are in the terminal region of the second strand, preferably located within the terminal 5 nucleotides of the end of the strand.
  • the terminal region may be the terminal 5 nucleotides, which includes abasic nucleotides.
  • the second strand may comprise, as preferred features (which are all specifically contemplated in combination unless mutually exclusive):
  • abasic nucleotides in either the 5’ or 3’ terminal region of the second strand, wherein the abasic nucleotides are present in an overhang as herein described;
  • the reversed linkage is a 5-5’ reversed linkage and the linkage between the terminal and penultimate abasic nucleotides is 3’5’ when reading towards the terminus comprising the terminal and penultimate abasic nucleotides; or
  • the reversed linkage is a 3-3’ reversed linkage and the linkage between the terminal and penultimate abasic nucleotides is 5’3’ when reading towards the terminus comprising the terminal and penultimate abasic nucleotides.
  • abasic nucleotide at the terminus of the second strand.
  • abasic nucleotides in the terminal region of the second strand, preferably at the terminal and penultimate positions.
  • abasic nucleotides are consecutive, for example all abasic nucleotides may be consecutive.
  • terminal 1 or terminal 2 or terminal 3 or terminal 4 nucelotides may be abasic nucleotides.
  • An abasic nucleotide may also be linked to an adjacent nucleotide through a 5 ’-3’ phosphodiester linkage or reversed linkage unless there is only 1 abasic nucleotide at the terminus, in which case it will have a reversed linkage to the adjacent nucleotide.
  • 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 moi eties of the nucleotides.
  • Abasic nucleotides which are not terminal will have 2 phosphodiester bonds, one with each adjacent nucleotide, 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 nucleotides at the terminal and penultimate positions of the second strand, and wherein the reversed internucleotide linkage is located between the penultimate (abasic) nucleotide and the antepenultimate nucleotide.
  • abasic nucleotides at the terminal and penultimate positions of the second strand and the penultimate nucleotide is linked to the antepenultimate nucleotide through a reversed internucleotide linkage and is linked to the terminal nucleotide through a 5 ’-3’ or 3 ’-5’ phosphodiester linkage (reading in the direction of the terminus of the molecule).
  • the reversed internucleotide linkage is a 3’3 reversed linkage.
  • the reversed internucleotide linkage is at a terminal region which is distal to the 5’ terminal phosphate of the second strand.
  • the reversed internucleotide linkage is a 5’5 reversed linkage.
  • the reversed internucleotide linkage is at a terminal region which is distal to the 3’ terminal hydroxide of the second strand.
  • RNA nucleotides shown are not limiting and could be any RNA nucleotide:
  • a A 3’-3’ reversed bond (and also showing the 5’-3 direction of the last phosphodiester bond between the two abasic molecules reading towards the terminus of the molecule)
  • the abasic nucleotide or abasic nucleotides present in the nucleic acid are provided in the presence of a reversed internucleotide linkage or linkages, namely a 5 ’-5’ or a 3 ’-3’ reversed internucleotide linkage.
  • a reversed linkage occurs as a result of a change of orientation of an adjacent nucleotide sugar, such that the sugar will have a 3’ - 5’ orientation as opposed to the conventional 5’ - 3’ orientation (with reference to the numbering of ring atoms on the nucleotide sugars).
  • the abasic nucleotide or nucleotides as present in the nucleic acids of the invention preferably include such inverted nucleotide sugars.
  • the proximal 3’-3’ or 5’ -5’ reversed linkage as herein described may comprise the reversed linkage being directly adjacent / attached to a terminal nucleotide having an inverted orientation, such as a single terminal nucleotide having an inverted orientation.
  • the proximal 3 ’-3’ or 5 ’-5’ reversed linkage as herein described may comprise the reversed linkage being adjacent 2, or more than 2, nucleotides having an inverted orientation, such as 2, or more than 2, terminal region nucleotides having an inverted orientation, such as the terminal and penultimate nucleotides. In this way, the reversed linkage may be attached to a penultimate nucleotide having an inverted orientation.
  • nucleic acid molecules having overall 3’ - 3’ or 5’- 5’ end structures as described herein, it will also be appreciated that with the presence of one or more additional reversed linkages and / or nucleotides having an inverted orientation, then 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 majority of the molecule will comprise conventional internucleotide 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 nucleotide sugars), which can be used to determine the conventional 5’ and 3’ ends that would be found absent the inverted end configuration.
  • the reversed bond is preferably located at the end of the nucleic acid eg RNA which is distal to a ligand moiety, such as a GalNAc containing portion, of the molecule.
  • a ligand moiety such as a GalNAc containing portion
  • GalNAc-siRNA constructs with a 5 ’-GalNAc on the sense strand can have a reversed linkage on the opposite end of the sense strand.
  • GalNAc-siRNA constructs with a 3 ’-GalNAc on the sense strand can have a reversed linkage on the opposite end of the sense strand.
  • the first strand of the nucleic acid has a length in the range of 15 to 30 nucleotides, preferably 19 to 25 nucleotides, more preferably 23 or 25; and / or ii) the second strand of the nucleic acid has a length in the range of 15 to 30 nucleotides, preferably 19 to 25 nucleotides, more preferably 23.
  • the duplex structure of the nucleic acid e.g. an iRNA is about 15 to 30 base pairs 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,
  • 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 nucleotides 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, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in
  • 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 nucleotides in length.
  • the region of complementarity between the antisense strand and the target is 19 to 21 nucleotides in length, for example, the region of complementarity is 21 nucleotides in length.
  • each strand is no more than 30 nucleotides in length.
  • a nucleic acid e.g. a dsRNA as described herein can further include one or more singlestranded nucleotide overhangs e.g., 1-4, 2-4, 1-3, 2-3, 1, 2, 3, or 4 nucleotides.
  • a nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside.
  • the overhang(s) can be on the sense strand, the antisense strand, or any combination thereof.
  • the nucleotide(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 nucleotide, e.g. , at least one strand comprises a 3' overhang of at least 2 nucleotides.
  • the overhang is suitably on the antisense/ guide strand and/or the sense / passenger strand.
  • the nucleic acid e.g. an RNA of the invention e.g., a dsRNA
  • the nucleic acid does not comprise further modifications (beyond the required abasic modifications), e.g., chemical modifications or conjugations known in the art and described herein.
  • the nucleic acid e.g. RNA of the invention e.g., a dsRNA
  • 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. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is hereby incorporated herein by reference.
  • Modifications include, for example, end modifications, e.g., 5'-end modifications (phosphorylation, conjugation, inverted linkages) or 3 '-end modifications (conjugation, DNA nucleotides within an RNA, or RNA nucleotides 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. , at the 2'-position or 4'- position) or replacement of the sugar; or backbone modifications, including modification or replacement of the phosphodiester linkages.
  • end modifications e.g., 5'-end modifications (phosphorylation, conjugation, inverted linkages) or 3 '-end modifications (conjugation, DNA nucleotides within an RNA, or RNA nucleotides within a DNA, inverted linkages, etc.
  • base modifications e.g., replacement with stabilizing bases, destabilizing bases,
  • nucleic acids such as iRNA 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 iRNA 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'.
  • Various salts, mixed salts and free acid forms are also included.
  • Modified nucleic acids e.g. RNAs can also contain one or more substituted sugar moieties.
  • the nucleic acids e.g. iRNAs, e.g., dsRNAs, featured herein can include one of the following at the 2'-position: OH; F; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; 0-, 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 nucleotide.
  • the nucleic acid of the invention may comprise one or more modified nucleotides on the first strand and/or the second strand.
  • substantially all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a modification.
  • all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand comprise a modification.
  • all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a modification.
  • At least one of the modified nucleotides is selected from the group consisting of a deoxy- nucleotide, a 3 '-terminal deoxy-thymine (dT) nucleotide, a 2'-0- methyl modified nucleotide (also called herein 2’-Me, where Me is a methoxy) , a 2'-fluoro modified nucleotide, a 2'-deoxy- modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2' -amino- modified nucleotide, a 2'- O-allyl- modified nucleotide, 2' -C-alkyl- modified nucleotide, 2'-hydroxly-modified nucleotide, a 2'
  • Modifications on the nucleotides may preferably be selected from the group including, but not limited to, LNA, HNA, CeNA, 2 -methoxyethyl, 2'-0-alkyl, 2 -0-allyl, 2'-C- allyl, 2'- fluoro, 2'-deoxy, 2'- hydroxyl, and combinations thereof.
  • the modifications on the nucleotides are 2 -0-methyl (“2-Me”) or 2'-fluoro modifications.
  • Preferred nucleic acid comprise one or more nucleotides on the first strand and / or the second strand which are modified, to form modified nucleotides, as follows:
  • a nucleic acid wherein the modification is a modification at the 2’ -OH group of the ribose sugar, optionally selected from 2'-Me or 2’-F modifications.
  • a nucleic acid 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.
  • a nucleic acid wherein the second strand comprises a 2’-F modification at position 7 and 9 and 11 counting from position 1 of said second strand.
  • a nucleic acid wherein the first and second strand each comprise 2'-Me and 2’-F modifications.
  • IMUNA modified unlocked nucleic acid
  • GNA glycol nucleic acid
  • the nucleic acid may be a double stranded molecule, preferably double stranded RNA, which has a melting temperature in the range of about 40 to 80°C.
  • the nucleic acid may comprise at least one thermally destabilizing modification at position 7 of the first strand.
  • a nucleic acid wherein the nucleic acid comprises 3 or more 2’-F modifications at positions 7 to 13 of the second strand, such as 4, 5, 6 or 7 2’-F modifications at positions 7 to 13 of the second strand, counting from position 1 of said second strand.
  • a nucleic acid 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.
  • a nucleic acid wherein said first strand comprises at least 5 2’ -Me consecutive modifications at the 3’ terminal region, preferably including the terminal nucleotide at the 3’ terminal region, or at least within 1 or 2 nucleotides from the terminal nucleotide at the 3’ terminal region.
  • a nucleic acid wherein said first strand comprises 7 2’-Me consecutive modifications at the 3’ terminal region, preferably including the terminal nucleotide at the 3’ terminal region.
  • Preferred modification patterns include:
  • a nucleic acid wherein the second strand includes the following modification pattern:
  • N represents a nucleotide with a first modification
  • N A represents a nucleotide with a second modification different to the first modification of N
  • N B represents a nucleotide with a third modification different to the first modification of N, but either the same or different to the second modification of N A ; and wherein said pattern has a 5’ to 3’ directionality along the second strand.
  • a nucleic acid wherein the second strand includes the following modification pattern:
  • a nucleic acid wherein the second strand includes the following modification pattern:
  • a nucleic acid wherein the second strand includes the following modification pattern:
  • a nucleic acid wherein the second strand includes the following modification pattern:
  • a nucleic acid wherein the second strand includes the following modification pattern:
  • N c and N D which may be the same or different, respectively denote a plurality of 5’ and 3’ terminal region chemically modified nucleotides, wherein at least N c comprises at least two differently modified nucleotides.
  • a nucleic acid wherein the second strand includes the following modification pattern:
  • a nucleic acid wherein the second strand includes the following modification pattern:
  • a nucleic acid wherein the second strand includes the following modification pattern:
  • a nucleic acid wherein the second strand includes the following modification pattern:
  • a nucleic acid wherein the first strand includes the following modification pattern:
  • M represents a nucleotide with a first modification and wherein typically (M) 3 -s are substantially aligned with (N) 3 -5 in said second strand;
  • M A represents a nucleotide with a second modification different to the first modification of M
  • M B represents a nucleotide with a third modification different to the first modification of M, but either the same or different to the second modification of M A .
  • a nucleic acid, wherein the first strand includes the following modification pattern:
  • a nucleic acid, wherein the first strand includes the following modification pattern:
  • a nucleic acid wherein the first strand includes the following modification pattern:
  • a nucleic acid wherein the first strand includes the following modification pattern:
  • a nucleic acid, wherein the first strand includes the following modification pattern:
  • a nucleic acid wherein the first strand includes the following modification pattern:
  • a nucleic acid wherein the first strand includes the following modification pattern:
  • position 1 of the sense strand is the 5’ most nucleotide (not including abasic nucleotides) at the conventional 5’ end of the sense strand.
  • the nucleotide at this position 1 of the sense strand will be equivalent to the 5’ nucleotide of the selected target nucleic acid sequence, and more generally the sense strand will have equivalent nucleotides 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.
  • a preferred nucleic acid is a double stranded RNA comprising 2 adjacent abasic nucleotides at the 5’ terminus of the second strand and a ligand moiety comprising one or more GalNAc ligand moi eties at the opposite 3’ end of the second strand.
  • 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.
  • the same nucleic acid may also comprise a 2’ F modification at positions 7, 9 and 11 of the second strand.
  • the invention provides a pharmaceutical composition for inhibiting expression of a target gene, the composition comprising a nucleic acid as disclosed herein.
  • the pharmaceutically acceptable composition may comprise an excipient and or carrier.
  • a repeat-dose regimen may include administration of a therapeutic amount of a nucleic acid e.g. iRNA on a regular basis, such as every other day or once a year.
  • the nucleic acid e.g. iRNA is administered about once per month to about once per quarter (i.e., about once every three months).
  • the present invention also provides methods of inhibiting expression of a gene in a cell.
  • the methods include contacting a cell with an nucleic acid of the invention e.g. RNAi agent, such as double stranded RNAi agent, in an amount effective to inhibit expression of the gene in the cell, thereby inhibiting expression of the gene in the cell.
  • an nucleic acid of the invention e.g. RNAi agent, such as double stranded RNAi agent
  • RNAi agent e.g. a double stranded RNAi agent
  • Contacting a cell in vivo with nucleic acid e.g. iRNA includes contacting a cell or group of cells within a subject, e.g., a human subject, with the nucleic acid e.g. iRNA. 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 RNAi agent to a site of interest.
  • expression of a gene 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.
  • the methods include a clinically relevant inhibition of expression of a target gene e.g. as demonstrated by a clinically relevant outcome after treatment of a subject with an agent to reduce the expression of the gene [00237] Inhibition of the expression of a gene may be manifested by a reduction of the amount of mRNA of the target gene of interest in comparison to a suitable control.
  • the present invention also provides methods of using nucleic acid e.g. an iRNA of the invention or a composition containing nucleic acid e.g. an iRNA of the invention to reduce or inhibit gene expression in a cell.
  • the methods include contacting the cell with a nucleic acid e.g. dsRNA 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 can be assessed by any methods known in the art.
  • 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 disease.
  • 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 iRNA, where the nucleic acid e.g. iRNA includes a nucleotide sequence that is complementary to at least a part of an RNA transcript of the gene of the mammal to be treated.
  • a nucleic acid of the invention e.g. an iRNA
  • the nucleic acid e.g. iRNA includes a nucleotide sequence that is complementary to at least a part of an RNA transcript of the 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 iRNA of the invention to a subject, e.g., a subject that would benefit from a reduction or inhibition of the expression of a gene, in a therapeutically effective amount e.g. a nucleic acid such as an iRNA targeting a gene or a pharmaceutical composition comprising the nucleic acid targeting a gene.
  • 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. iRNA, such as about 0.01 mg/kg to about 200 mg/kg.
  • iRNA nucleic acid
  • the nucleic acid e.g. iRNA 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 iRNA 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.
  • Xi and X2 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:
  • 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, and wherein said RNA duplex is attached at the 3’ end of its second strand to the adjacent phosphate.
  • 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.
  • oligonucleotide comprises an RNA duplex which further comprises one or more riboses modified at the 2’ position, preferably a plurality of riboses modified at the 2’ position.
  • a compound according to Sentence 39 wherein said one or more degradation protective moieties are not present at the end of the oligonucleotide strand that carries the ligand moieties, and / or wherein said one or more degradation protective moieties is selected from phosphorothioate intemucleotide linkages, phosphorodithioate internucleotide linkages and inverted abasic nucleotides, wherein said inverted abasic nucleotides are present at the distal end of the strand that carries the ligand moieties.
  • a compound according to any of Sentences 1 to 29, or 32 to 34, or 37 to 40, wherein said ligand moiety as depicted in Formula (I) in Sentence 1 comprises one or more ligands.
  • a compound according to Sentence 41, wherein said ligand moiety as depicted in Formula (I) in Sentence 1 comprises one or more carbohydrate ligands.
  • 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 45 which comprises two or three N- AcetylGalactosamine moieties.
  • a compound according to Sentence 47 wherein said one or more ligands are attached as a biantennary or triantennary branched configuration.
  • Moiety Moiety as depicted in Formula (I) in Sentence 1 is any of Formulae (Via), (VIb) or (Vic), preferably Formula (Via):
  • Ai 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
  • Ai 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
  • Ai 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.
  • a compound according to Sentences 46 to 48, wherein said moiety: as depicted in Formula (I) in Sentence 1 is Formula (VII):
  • Ai is hydrogen; a is an integer of 2 or 3.
  • 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, and wherein said RNA duplex is attached at the 5’ end of its second strand to the adjacent phosphate.
  • a composition comprising a compound of Formula (VIII) as defined in Sentence 54, and a compound of Formula (IX) as defined in Sentence 55, optionally dependent on Sentence 56.
  • a composition according to Sentence 62 wherein said compound of Formula (XI) as defined in Sentence 60 is present in an amount in the range of 10 to 15% by weight of said composition.
  • Ri at each occurrence is independently selected from the group consisting of hydrogen, methyl and ethyl;
  • Xi and X2 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:
  • Z is an oligonucleotide moiety; and where appropriate carrying out deprotection of the ligand and / or annealing of a second strand for the oligonucleotide moiety.
  • Sentence 68 wherein a compound of Formula (XII) is prepared by reacting compounds of Formulae (XIV) and (XV):
  • Ri at each occurrence is independently selected from the group consisting of hydrogen, methyl and ethyl;
  • Xi and X2 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:
  • Z is an oligonucleotide moiety.
  • oligonucleotide 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, and wherein said RNA duplex is attached at the 5’ end of its second strand to the adjacent phosphate.
  • oligonucleotide 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, and wherein said RNA duplex is attached at the 5’ end of its second strand to the adjacent phosphate.
  • oligonucleotide 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, and wherein said RNA duplex is attached at the 3’ end of its second strand to the adjacent phosphate.
  • oligonucleotide 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, and wherein said RNA duplex is attached at the 3’ end of its second strand to the adjacent phosphate.
  • Formula (XlVb) and compound of Formula (XV) is either Formula (XVa) or Formula (XlVb):
  • Formula (XVb) wherein the oligonucleotide 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, and wherein (i) said RNA duplex is attached at the 5’ end of its second strand to the adjacent phosphate in Formula (XVa), or (ii) said RNA duplex is attached at the 3’ end of its second strand to the adjacent phosphate in Formula (XVb).
  • Ri at each occurrence is independently selected from the group consisting of hydrogen, methyl and ethyl;
  • Xi and X2 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:
  • Z is an oligonucleotide moiety.
  • Ri at each occurrence is independently selected from the group consisting of hydrogen, methyl and ethyl; m is an integer of from 1 to 6; n is an integer of from 1 to 10.
  • Ri is selected from the group consisting of hydrogen, methyl and ethyl
  • X2 is selected from the group consisting of methylene, oxygen and sulfur; s, t, v are independently integers from 0 to 4, with the proviso that s, t and v cannot all be 0 at the same time.
  • R1 at each occurrence is independently selected from the group consisting of hydrogen, methyl and ethyl;
  • XI is selected from the group consisting of methylene, oxygen and sulfur; q and r are independently integers from 0 to 4, with the proviso that q and r cannot both be 0 at the same time;
  • Z is an oligonucleotide moiety.
  • a pharmaceutical composition comprising of a compound according to any of Sentences 1 to 29, 32 to 34, 37 to 56, 59 to 61, and 64 to 67, and / or a composition according to any of Sentences 30, 31, 35, 36, 57, 58, 62 and 63, together with a pharmaceutically acceptable carrier, diluent or excipient.
  • a compound comprising the following structure: Formula (I) wherein: r and s are independently an integer selected from 1 to 16; and
  • Z is an oligonucleotide moiety.
  • Zi, Z2, Z3, Z4 are independently at each occurrence oxygen or sulfur; and one the bonds between P and Z2, and P and Z 3 is a single bond and the other bond is a double bond.
  • oligonucleotide is an RNA compound capable of modulating, preferably inhibiting, expression of a target gene.
  • 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.
  • oligonucleotide comprises an RNA duplex which further comprises one or more riboses modified at the 2’ position, preferably a plurality of riboses modified at the 2’ position.
  • a compound according to Clause 21, wherein said one or more carbohydrates can be a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide or polysaccharide.
  • Ai 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
  • Ai 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
  • Ai 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.
  • Ai is hydrogen; a is an integer of 2 or 3. 30.
  • a compound according to Clause 28 or 29, wherein a 2.
  • Formula (IX) A compound according to Clause 33 or 34, wherein the oligonucleotide comprises an RNA duplex which further comprises one or more riboses modified at the 2’ position, preferably a plurality of riboses modified at the 2’ position.
  • a compound according to Clause 35, wherein the modifications are chosen from 2’-O- methyl, 2’-deoxy-fluoro, and 2’-deoxy.
  • a compound according to Clause 37 wherein said one or more degradation protective moieties are not present at the end of the oligonucleotide strand that carries the linker / ligand moieties, and / or wherein said one or more degradation protective moieties is selected from phosphorothioate internucleotide linkages, phosphorodithioate intemucleotide linkages and inverted abasic nucleotides, wherein said inverted abasic nucleotides are present at the distal end of the same strand to the end that carries the linker / ligand moieties.
  • the oligonucleotide 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, and wherein said RNA duplex is attached at the 5’ end of its second strand to the adjacent phosphate.
  • 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, and wherein said RNA duplex is attached at the 3’ end of its second strand to the adjacent phosphate.
  • Z is an oligonucleotide moiety; and where appropriate carrying out deprotection of the ligand and / or annealing of a second strand for the oligonucleotide.
  • Formula (Xa) and compound of Formula (XI) is Formula (Xia):
  • oligonucleotide 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, and wherein said RNA duplex is attached at the 5’ end of its second strand to the adjacent phosphate.
  • oligonucleotide 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, and wherein said RNA duplex is attached at the 3’ end of its second strand to the adjacent phosphate.
  • Z is an oligonucleotide moiety.
  • a pharmaceutical composition comprising of a compound according to any of Clauses 1 to 40, together with a pharmaceutically acceptable carrier, diluent or excipient.
  • references to (invabasic)(invabasic) refers to nucleotides in an overall polynucleotide which are the terminal 2 nucleotides which have sugar moieties that are (i) abasic, and (ii) in an inverted configuration, whereby the bond between the penultimate nucleotide and the antepenultimate nucleotide has a reversed linkage, namely either a 5-5 or a 3-3 linkage. Again, this similarly applies to all other references to (invabasic)(invabasic) herein.
  • linker portions as shown in FIG 30 / FIG 31 which can be present in any of products ETX001, ETX005, ETX010, ETX014, ETX019, ETX023 according to the present invention, that while these products can include molecules based on the linker and ligand portions as specifically depicted in FIG 30 / FIG 31 attached to an oligonucleotide moiety as also depicted herein, these products may alternatively further comprise, or consist essentially of, molecules wherein the linker and ligand portions are essentially as depicted in FIG 30 / FIG 31 attached to an oligonucleotide moiety but having the F substituent as shown in FIG 30 / FIG 31 on the cyclo-octyl ring replaced by a substituent occurring as a result of hydrolytic displacement, such as an OH substituent.
  • these products can consist essentially of molecules having linker and ligand portions specifically as depicted in FIG 30 / FIG 31, with a F substituent on the cyclo-octyl ring; or (b) these products can consist essentially of molecules having linker and ligand portions essentially as depicted in FIG 30 / FIG 31 but having the F substituent as shown in FIG 30 / FIG 31 on the cyclo-octyl ring replaced by a substituent occurring as a result of hydrolytic displacement, such as an OH substituent, or (c) these products can comprise a mixture of molecules as defined in (a) or (b).
  • GalNAc-siRNAs targeting either hsHAOl, hsC5 or hsTTR mRNA were synthesized and QC-ed. The entire set of siRNAs (except siRNAs targeting HAO1) was first studied in a dose-response setup in HepG2 cells by transfection using RNAiMAX, followed by a doseresponse analysis in a gymnotic free uptake setup in primary human hepatocytes.
  • the aim of this set of experiments was to analyze the in vitro activity of different GalNAc-ligands in the context of siRNAs targeting three different on-targets, namely hsHAO 1 , hsC5 or hsTTR mRNA.
  • HepG2 cells were supplied by American Tissue Culture Collection (ATCC) (HB-8065, Lot # 63176294) and cultured in ATCC- formulated Eagle's Minimum Essential Medium supplemented to contain 10 % fetal calf serum (FCS).
  • ATCC American Tissue Culture Collection
  • FCS fetal calf serum
  • Primary human hepatocytes (PHHs) were sourced from Primacyt (Schwerin, Germany) sf-4589539 (Lot#: CyHufl9009HEc). Cells are derived from a malignant glioblastoma tumor by explant technique. All cells used in this study were cultured at 37°C in an atmosphere with 5% CO2 in a humidified incubator.
  • Control wells were transfected into HepG2 cells or directly incubated with primary human hepatocytes at the highest test siRNA concentrations studied on the corresponding plate. All control siRNAs included in the different project phases next to mock treatment of cells are summarized and listed in Table 2. For each siRNA and control, at least four wells were transfected/directly incubated in parallel, and individual data points were collected from each well.
  • branched DNA (bDNA) assay was performed according to manufacturer’ s instructions. Luminescence was read using a 1420 Luminescence Counter (WALLAC VICTOR Light, Perkin Elmer, Rodgau-Jugesheim, Germany) following 30 minutes incubation in the presence of substrate in the dark. For each well, the on-target mRNA levels were normalized to the hsGAPDH mRNA levels. The activity of any siRNA was expressed as percent on-target mRNA concentration (normalized to hsGAPDH mRNA) in treated cells, relative to the mean on- target mRNA concentration (normalized to hsGAPDH mRNA) across control wells.
  • bDNA probe sets were designed and synthesised specific for Homo sapiens GAPDH, AHSA1, hsHAOl, hsC5 and hsTTR.
  • bDNA probe sets were initially tested by bDNA analysis according to manufacturer’s instructions, with evaluation of levels of mRNAs of interest in two different lysate amounts, namely lOpl and 50pl, of the following human and monkey cancer cell lines next to primary human hepatocytes: SJSA-1, TF1, NCI-H1650, Y-79, Kasumi-1, EAhy926, Caki-1, Colo205, RPTEC, A253, HeLaS3, Hep3B, BxPC3, DU145, THP-1, NCI-H460, IGR37, LS174T, Be(2)-C, SW 1573, NCI-H358, TC71, 22Rvl, BT474, HeLa, KBwt, Panc-1, U
  • Fig. 1 to Fig. 3 show mRNA expression data for the three on-targets of interest, namely hsC5, hsHAOl and hsTTR, in lysates of a diverse set of human cancer cell lines plus primary human hepatocytes. Cell numbers per lysate volume are identical with each cell line tested, this is necessary to allow comparisons of expression levels amongst different cell types.
  • Fig. 1 shows hsC5 mRNA expression data for all cell types tested.
  • mRNA expression levels for all three on-targets of interest are high enough in primary human hepatocytes (PHHs).
  • HepG2 cells could be used to screen GalNAc-siRNAs targeting hsC5 and hsTTR mRNAs, in contrast, no cancer cell line could be identified which would be suitable to test siRNAs specific for hsHAOl mRNA.
  • Table 3 Target, incubation time, external ID, IC20/IC50/IC80 values and maximal inhibition of hsTTR targeting siRNAs in HepG2 cells. The listing is ordered according to external ID, with 4h of incubation listed on top and 24h of incubation on the bottom.
  • hsTTR GalNAc-siRNAs have been identified that silence the on-target mRNA >95% with IC50 values in the low double-digit pM range.
  • the second target of interest was tested in an identical dose-response setup (with minimally different final siRNA test concentrations, however) by transfection of HepG2 cells using RNAiMAX with GalNAc-siRNAs sharing identical linger/position/GalNAc-ligand variations as with hsTTR siRNAs, but sequences specific for the on-target hsC5 mRNA.
  • Table 4 Target, incubation time, external ID, IC20/IC50/IC80 values and maximal inhibition of hsC5 targeting siRNAs in HepG2 cells. The listing is ordered according to external ID, with 4h of incubation listed on top and 24h of incubation on the bottom.
  • the dose-response analysis of the two GalNAc-siRNA sets in human cancer cell line HepG2 should demonstrate (and ensure) that all new GalNAc-/linker/position/cap variants are indeed substrates for efficient binding to AGO2 and loading into RISC, and in addition, able to function in RNAi-mediated cleavage of target mRNA.
  • dose-response analysis experiments should be done in primary human hepatocytes by gymnotic, free uptake setup.
  • Hepatocytes do exclusively express the Asialoglycoprotein receptor (ASGR1) to high levels, and this receptor generally is used by the liver to remove target glycoproteins from circulation.
  • ASGR1 Asialoglycoprotein receptor
  • oligonucleotides e.g. siRNAs or ASOs
  • GalNAc-ligands conjugated to GalNAc-ligands are recognized by this high turnover receptor and efficiently taken up into the cytoplasm via clathrin-coated vesicles and trafficking to endosomal compartments. Endosomal escape is thought to be the rate-limiting step for oligonucleotide delivery.
  • FIG. 6 shows the absolute mRNA expression data for all three on-targets of interest - hsTTR, hsC5 and hsHAOl - in the primary human hepatocyte batches BHuf 16087, CHF2101 and CyHufl9009. mRNA expression levels of hsGAPDH and hsAHSAl are shown in Figure 7.
  • Table 5 Target, incubation time, external ID, IC20/IC50/IC80 values and maximal inhibition of hsHAOl targeting GalNAc-siRNAs in primary human hepatocytes (PHHs). The listing is organized according to external ID, with 4h and 72h incubation listed on top and bottom, respectively.
  • the second target of interest, hsC5 mRNA was tested in an identical dose-response setup by gymnotic, free uptake in PHHs with GalNAc-siRNAs sharing identical linker/position/GalNAc-ligand variations as with hsTTR and hsHAOl tested in the assays before, but sequences specific for the on-target hsC5 mRNA. Sequences for the GalNAc- siRNAs targeting hsC5 and all sequences and information about control siRNAs are listed in Table 1 and Table 2, respectively. The experiment ended after 4h and 72h direct incubation of PHHs. Table 6 lists activity data for all hsC5 targeting GalNAc-siRNAs studied.
  • Table 6 Target, incubation time, external ID, IC20/IC50/IC80 values and maximal inhibition of hsC5 targeting GalNAc-siRNAs in PHHs. The listing is organized according to external ID, with 4h and 72h incubation listed on top and bottom, respectively.
  • hsTTR mRNA The last target of interest, hsTTR mRNA, was again tested in a gymnotic, free uptake in PHHs in an identical dose-response setup as for the targets hsHAOl and hsC5, with the only difference being that specific siRNA sequences for the on-target hsTTR mRNA was used (see Table 1).
  • Table 7 Target, incubation time, external ID, IC20/IC50/IC80 values and maximal inhibition of hsTTR targeting GalNAc-siRNAs in primary human hepatocytes (PHHs). The listing is organized according to external ID.
  • Results are also shown in Figures 10A-B.
  • Gymnotic, free uptake of GalNAc-siRNAs targeting hsTTR did lead to significant on- target silencing within 72h, ranging between 46 to 82.5% maximal inhibition.
  • hsTTR GalNAc- siRNAs were identified that silence the on-target mRNA with IC50 values in the low doubledigit nM range.
  • siRNA sets specific for each target were composed of siRNAs with different linker/cap/modification/GalNAc-ligand chemistries in the context of two different antisense strands each.
  • GalNAc-siRNAs from Table 1 were identified that showed a high overall potency and low IC50 value.
  • TLC Thin layer chromatography
  • fluorescence indicator 254 nm from Macherey -Nagel.
  • Compounds were visualized under UV light (254 nm), or after spraying with the 5% H2SO4 in methanol (MeOH) or ninhydrin reagent according to Stahl (from Sigma-Aldrich), followed by heating.
  • 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 Sfar Silica 10, 25, 50 or 100 g columns (Uppsala, Sweden).
  • 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. N2 pressure: 35.1 psi. Filter: Corona.
  • A,A,A',A'-tetramethyl-O-(lH-benzotriazol-l-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 A,A-diisopropylethylamine (DIPEA) (2.07 mL, 11.9 mmol, 8.0 eq.) were added to the solution and the reaction was stirred for 72 h.
  • DIPEA 1,1-diisopropylethylamine
  • TriGalNAc (12) Triantennary GalNAc compound 10 (0.35 g, 0.24 mmol, 1.0 eq.) and compound 11 (0.11 g, 0.31 mmol, 1.5 eq.) were dissolved in DCM (5 mL) under argon and triethylamine (0.1 mL, 0.61 mmol, 3.0 eq.) was added. The reaction was stirred at room temperature overnight. The solvent was removed under reduced pressure, the residue was dissolved in EtOAc (100 mL) and washed with water (100 mL). The organic layer was separated and dried over Na2SO4.
  • RNA nucleotides a, c, g, u: 2’-O-Me RNA nucleotides dT DNA nucleotides s: Phosphorothioate invabasic: 1, 2-dideoxyribose
  • Oligonucleotides were synthesized on solid phase according to the phosphoramidite approach. Depending on the scale either a Mermade 12 (BioAutomation Corporation) or an AKTA Oligopilot (GE Healthcare) was used.
  • 2' -O-Methyl phosphoramidites include: 5'-(4,4'-dimethoxytrityl)-A-benzoyl-adenosine 2'-O-methyl-3'- [(2-cyanoethyl)-(A,A-diisopropyl)]-phosphoramidite, 5'-(4,4'-dimethoxytrityl)- A-benzoyl-cytidine 2'-O-methyl-3'- [(2-cyanoethyl)-(A,A-diisopropyl)]-phosphoramidite, 5'- (4,4'-dimethoxytrityl)-A-dimethylformamidine-guanosine 2'-O-methyl-3'-[(2-cyanoethyl)-(A,A- diisopropyl)]-phosphoramidite, 5'-(4,4'-dimethoxytrityl)-uridine 2'-
  • 2’ -F phosphoramidites include: 5'-dimethoxytrityl-A-benzoyl-deoxyadenosine 2'- fluoro-3'-[(2-cyanoethyl)-(A,A-diisopropyl)]-phosphoramidite, 5'-dimethoxytrityl-A-acetyl- deoxycytidine 2'-fluoro-3'-[(2-cyanoethyl)-(A,A-diisopropyl)]-phosphoramidite, 5'- dimethoxytrityl-A-isobutyryl-deoxyguanosine 2'-fluoro-3'- [(2-cyanoethyl)-(A,A-diisopropyl)]- phosphoramidite and 5'-dimethoxytrityl-deoxyuridine 2'-fluoro-3'-[(2-cyanoethyl)-(A,A-di
  • the invabasic modification was introduced using 5-O-dimethoxytrityl-l,2- dideoxyribose-3-[(2-cyanoethyl)-(A,A-diisopropyl)]-phosphoramidite (ChemGenes Cat. # ANP- 1422).
  • the oligonucleotides were cleaved from the solid support using a 1 : 1 volume solution of 28-30% ammonium hydroxide (Sigma-Aldrich, Cat. #221228) and 40% aqueous methylamine (Sigma- Aldrich, Cat. #8220911000) for 16 hours at 6°C.
  • the solid support was then filtered off, the filter was thoroughly washed with H2O and the volume of the combined solution was reduced by evaporation under reduced pressure.
  • the pH of the resulting solution was adjusted to pH 7 with 10% AcOH (Sigma- Aldrich, Cat. # A6283).
  • the crude materials were purified either by reversed phase (RP) HPLC or anion exchange (AEX) HPLC.
  • RP HPLC purification was performed using a XBridge C18 Prep 19 x 50 mm column (Waters) on an AKTA Pure instrument (GE Healthcare).
  • Buffer A was 100 mM tri ethylammonium acetate (TEAAc, Biosolve) 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% B to 100% B within 120 column volumes was employed. Appropriate fractions were pooled and precipitated in the freezer with 3 M sodium acetate (NaOAc) (Sigma- Aldrich), pH 5.2 and 85% ethanol (VWR).
  • Pellets were isolated by centrifugation, redissolved in water (50 mL), treated with 5 M NaCl (5 mL) and desalted by Size exclusion HPLC on an Akta Pure instrument using a 50 x 165mm ECO column (YMC, Dinslaken, Germany) filled with Sephadex G25-Fine resin (GE Healthcare).
  • AEX HPLC purification was performed using a TSK gel SuperQ-5PW 20 x 200 mm (BISCHOFF Chromatography) on an AKTA Pure instrument (GE Healthcare).
  • Buffer A was 20 mM sodium phosphate (Sigma-Aldrich) pH 7.8 and buffer B was the same as buffer A with the addition of 1.4 M sodium bromide (Sigma- Aldrich).
  • 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 10% B to 100% B within 27 column volumes was employed.
  • Appropriate fractions were pooled and precipitated in the freezer with 3 M NaOAc, pH 5.2 and 85% ethanol.
  • Pellets were isolated by centrifugation, redissolved in water (50 mL), treated with 5 M NaCl (5 mL) and desalted by size exclusion chromatography.
  • reaction mixture was diluted 15-fold with water, filtered through a 1.2 pm filter from Sartorius and then purified by reserve phase (RP HPLC) on an Akta Pure instrument (GE Healthcare).
  • SUBSTITUTE SHEET (RULE 26) 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.
  • RP HPLC purification was performed using a XBridge C18 Prep 19 x 50 mm column from Waters.
  • Buffer A was 100 mM tri ethylammonium 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.
  • conjugates were desalted by size exclusion chromatography using Sephadex G25 Fine resin (GE Healthcare) on an Akta Pure (GE Healthcare) instrument to yield the conjugated nucleotide in an isolated yield of 50-70%.
  • the two complementary strands were annealed by combining equimolar aqueous solutions of both strands. The mixtures were placed into a water bath at 70°C for 5 minutes and subsequently allowed to cool to ambient temperature within 2 h. The duplexes were lyophilized for 2 days and stored at -20°C.
  • duplexes were analyzed by analytical SEC HPLC on SuperdexTM 75 Increase 5/150 GL column 5 x 153-158 mm (Cytiva) on a Dionex Ultimate 3000 (Thermo Fisher Scientific) HPLC system.
  • Mobile phase consisted of lx PBS containing 10% acetonitrile.
  • An isocratic gradient was run in 10 min at a flow rate of 1.5 mL/min at room temperature. UV traces at 260 and 280 nm were recorded.
  • Water (LC-MS grade) was purchased from Sigma-Aldrich and Phosphate-buffered saline (PBS; lOx, pH 7.4) was purchased from GIBCO (Thermo Fisher Scientific).
  • GalNAc conjugates prepared are compiled in the table below. These were directed against 3 different target genes. siRNA coding along with the corresponding single strands, sequence information as well as purity for the duplexes is captured.
  • GalNAc-siRNAs targeting either hsHAOl, hsC5 or hsTTR mRNA were synthesized and QC-ed. The entire set of siRNAs (except siRNAs targeting HA01) was first studied in a dose-response setup in HepG2 cells by transfection using RNAiMAX, followed by a doseresponse analysis in a gymnotic free uptake setup in primary human hepatocytes.
  • the aim of this set of experiments was to analyze the in vitro activity of different GalNAc-ligands in the context of siRNAs targeting three different on-targets, namely hsHAOl, hsC5 or hsTTR mRNA.
  • Work packages of this study included (i) assay development to design, synthesize and test bDNA probe sets specific for each and every individual on-target of interest, (ii) to identify a cell line suitable for subsequent screening experiments, (iii) dose-response analysis of potentially all siRNAs (by transfection) in one or more human cancer cell lines, and (iv) dose-response analysis of siRNAs in primary human hepatocytes in a gymnotic, free uptake setting. In both settings, IC50 values and maximal inhibition values should be calculated followed by ranking of the siRNA study set according to their potency.
  • Oligonucleotide synthesis [00345] Standard solid-phase synthesis methods were used to chemically synthesize siRNAs of interest (see Table 12) as well as controls (see Table 13).
  • HepG2 cells were supplied by American Tissue Culture Collection (ATCC) (HB-8065, Lot #: 63176294) and cultured in ATCC-formulated Eagle's Minimum Essential Medium supplemented to contain 10 % fetal calf serum (FCS).
  • ATCC American Tissue Culture Collection
  • FCS fetal calf serum
  • Primary human hepatocytes (PHHs) were sourced from Primacyt (Schwerin, Germany) (Lot#: CyHufl 9009HEc). Cells are derived from a malignant glioblastoma tumor by explant technique. All cells used in this study were cultured at 37°C in an atmosphere with 5% CO2 in a humidified incubator.
  • Control wells were transfected into HepG2 cells or directly incubated with primary human hepatocytes at the highest test siRNA concentrations studied on the corresponding plate. All control siRNAs included in the different project phases next to mock treatment of cells are summarized and listed in Table 13. For each siRNA and control, at least four wells were transfected/directly incubated in parallel, and individual data points were collected from each well.
  • branched DNA (bDNA) assay was performed according to manufacturer’ s instructions. Luminescence was read using a 1420 Luminescence Counter (WALLAC VICTOR Light, Perkin Elmer, Rodgau-Jugesheim, Germany) following 30 minutes incubation in the presence of substrate in the dark. For each well, the on-target mRNA levels were normalized to the hsGAPDH mRNA levels. The activity of any siRNA was expressed as percent on-target mRNA concentration (normalized to hsGAPDH mRNA) in treated cells, relative to the mean on-target mRNA concentration (normalized to hsGAPDH mRNA) across control wells.
  • bDNA probe sets were designed and synthesised specific for Homo sapiens GAPDH, AHSA1, hsHAOl, hsC5 and hsTTR.
  • bDNA probe sets were initially tested by bDNA analysis according to manufacturer’s instructions, with evaluation of levels of mRNAs of interest in two different lysate amounts, namely lOpl and 50pl, of the following human and monkey cancer cell lines next to primary human hepatocytes: SJSA-1, TF1, NCLH1650, Y-79, Kasumi-1, EAhy926, Caki-1, Colo205, RPTEC, A253, HeLaS3, Hep3B, BxPC3, DU145, THP-1, NCI-H460, IGR37, LS174T, Be(2)-C, SW 1573, NCLH358, TC71, 22Rvl, BT474, HeLa, KBwt, Panc-1, U87MG
  • Fig. 1 to Fig. 3 show mRNA expression data for the three on-targets of interest, namely hsC5, hsHAOl and hsTTR, in lysates of a diverse set of human cancer cell lines plus primary human hepatocytes. Cell numbers per lysate volume are identical with each cell line tested, this is necessary to allow comparisons of expression levels amongst different cell types.
  • Fig. 1 shows hsC5 mRNA expression data for all cell types tested.
  • the identical type of cells were also screened for expression of hsHAOl mRNA, results are shown in bar diagrams as part of Fig. 2.
  • mRNA expression levels for all three on-targets of interest are high enough in primary human hepatocytes (PHHs).
  • HepG2 cells could be used to screen GalNAc-siRNAs targeting hsC5 and hsTTR mRNAs, in contrast, no cancer cell line could be identified which would be suitable to test siRNAs specific for hsHAOl mRNA.
  • HepG2 cells were transfected with the entire set of hsTTR targeting GalNAc-siRNAs (see Table 12) in a dose-response setup using RNAiMAX. The highest final siRNA test concentration was 24nM, going down in ninecells. Table 14 lists activity data for all hsTTR targeting GalNAC-siRNAs studied.
  • Table 14 Target, incubation time, external ID, IC20/IC50/IC80 values and maximal inhibition of hsTTR targeting siRNAs in HepG2 cells. The listing is ordered according to external ID, with 4h of incubation listed on top and 24h of incubation on the bottom.
  • hsTTR GalNAc-siRNAs have been identified that silence the on-target mRNA >95% with IC50 values in the low double-digit pM range.
  • hsC5 mRNA The second target of interest, hsC5 mRNA, was tested in an identical dose-response setup (with minimally different final siRNA test concentrations, however) by transfection of HepG2 cells using RNAiMAX with GalNAc-siRNAs sharing identical linger/position/GalNAc-ligand variations as with hsTTR siRNAs, but sequences specific for the on-target hsC5 mRNA.
  • Table 15 Target, incubation time, external ID, IC20/IC50/IC80 values and maximal inhibition of hsC5 targeting siRNAs in HepG2 cells. The listing is ordered according to external ID, with 4h of incubation listed on top and 24h of incubation on the bottom.
  • Hepatocytes do exclusively express the Asialoglycoprotein receptor (ASGR1) to high levels, and this receptor generally is used by the liver to remove target glycoproteins from circulation.
  • ASGR1 Asialoglycoprotein receptor
  • oligonucleotides e.g. siRNAs or ASOs
  • GalNAc-ligands conjugated to GalNAc-ligands are recognized by this high turnover receptor and efficiently taken up into the cytoplasm via clathrin-coated vesicles and trafficking to endosomal compartments. Endosomal escape is thought to be the ratelimiting step for oligonucleotide delivery.
  • FIG. 6 shows the absolute mRNA expression data for all three on-targets of interest - hsTTR, hsC5 and hsHAOl - in the primary human hepatocyte batches BHufl6087, CHF2101 and CyHufl9009. mRNA expression levels of hsGAPDH and hsAHSAl are shown in Figure 7.
  • Table 16 Target, incubation time, external ID, IC20/IC50/IC80 values and maximal inhibition ofhsHAOl targeting GalNAc-siRNAs in primary human hepatocytes (PHHs). The listing is organized according to external ID, with 4h and 72h incubation listed on top and bottom, respectively.
  • Table 17 Target, incubation time, external ID, IC20/IC50/IC80 values and maximal inhibition of hsC5 targeting GalNAc-siRNAs in PHHs. The listing is organized according to external ID, with 4h and 72h incubation listed on top and bottom, respectively.
  • Table 18 Target, incubation time, external ID, IC20/IC50/IC80 values and maximal inhibition ofhsTTR targeting GalNAc-siRNAs in primary human hepatocytes (PHHs). The listing is organized according to external ID.
  • siRNA sets specific for each target were composed of siRNAs with different linker/cap/modification/GalNAc-ligand chemistries in the context of two different antisense strands each.
  • GalNAc-siRNAs from Table 12 were identified that showed a high overall potency and low IC50 value.
  • 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 (300A, 1.7 pm, 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. N2 pressure: 35.1 psi. Filter: Corona.
  • 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
  • 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. The completion of the reaction was monitored by mass spectrometry and the resulting mixture was filtered through a thin pad of celite. The solvent was removed under reduced pressure and the resulting residue was dried under high vacuum over night. The residue was used for conjugations to oligonucleotides without further purification (0.28 g, quantitative yield). MS: calculated for C81H131N7O42, 1874.9. Found 1875.3. ix) Oligonucleotide Synthesis
  • RNA nucleotides a, c, g, u: 2 ’-O-Me RNA nucleotides dT DNA nucleotides s: Phosphorothioate invabasic: 1, 2-dideoxyribose
  • Oligonucleotides were synthesized on solid phase according to the phosphoramidite approach. Depending on the scale either a Mermade 12 (BioAutomation Corporation) or an KTA Oligopilot (GE Healthcare) was used.
  • 2'-O-Methyl phosphoramidites include: 5'-(4,4'-dimethoxytrityl)-N-benzoyl-adenosine 2'-O-methyl-3'- [(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-(4,4'- dimethoxytrityl)-N-benzoyl-cytidine 2'-O-methyl-3'- [(2-cyanoethyl)-(N,N-diisopropyl)]- phosphoramidite, 5'-(4,4'-dimethoxytrityl)-N-dimethylformamidine-guanosine 2'-O-methyl- 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-(4,4'-dimethoxytrityl)-uridine 2
  • 2’-F phosphoramidites include: 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-
  • the oligonucleotides were cleaved from the solid support using a 1 : 1 volume solution of 28-30% ammonium hydroxide (Sigma-Aldrich, Cat. #221228) and 40% aqueous methylamine (Sigma-Aldrich, Cat. #8220911000) for 16 hours at 6°C.
  • the solid support was then filtered off, the filter was thoroughly washed with H2O and the volume of the combined solution was reduced by evaporation under reduced pressure.
  • the pH of the resulting solution was adjusted to pH 7 with 10% AcOH (Sigma- Aldrich, Cat. # A6283).
  • RP HPLC purification was performed using a XBridge C18 Prep 19 x 50 mm column (Waters) on an AKTA Pure instrument (GE Healthcare).
  • Buffer A was 100 mM triethylammonium acetate (TEAAc, Biosolve) 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% B to 100% B within 120 column volumes was employed. Appropriate fractions were pooled and precipitated in the freezer with 3 M sodium acetate (NaOAc) (Sigma-Aldrich), pH 5.2 and 85% ethanol (VWR).
  • NaOAc sodium acetate
  • VWR 85% ethanol
  • Pellets were isolated by centrifugation, redissolved in water (50 mL), treated with 5 M NaCl (5 mL) and desalted by Size exclusion HPLC on an Akta Pure instrument using a 50 x 165mm ECO column (YMC, Dinslaken, Germany) filled with Sephadex G25-Fine resin (GE Healthcare).
  • AEX HPLC purification was performed using a TSK gel SuperQ-5PW 20 x 200 mm (BISCHOFF Chromatography) on an AKTA Pure instrument (GE Healthcare).
  • Buffer A was 20 mM sodium phosphate (Sigma-Aldrich) pH 7.8 and buffer B was the same as buffer A with the addition of 1.4 M sodium bromide (Sigma- Aldrich).
  • 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 10% B to 100% B within 27 column volumes was employed. Appropriate fractions were pooled and precipitated in the freezer with 3 M NaOAc, pH 5.2 and 85% ethanol.
  • Pellets were isolated by centrifugation, redissolved in water (50 mL), treated with 5 M NaCl (5 mL) and desalted by size exclusion chromatography. [00404] The MMT group was removed with 25% acetic acid in water. Once the reaction was complete the solution was neutralized and the samples were desalted by size exclusion chromatography.
  • conjugates were desalted by size exclusion chromatography using Sephadex G25 Fine resin (GE Healthcare) on an Akta Pure (GE Healthcare) instrument to yield the conjugated oligonucleotides in an isolated yield of 60-80%.
  • the conjugates were characterized by HPLC-MS analysis with a 2.1 x 50 mm XBridge C18 column (Waters) on a Dionex Ultimate 3000 (Thermo Fisher Scientific) HPLC system equipped with a Compact ESI-Qq-TOF mass spectrometer (Bruker Daltonics).
  • Buffer A was 16.3 mM triethylamine, 100 mM HFIP in 1% MeOH in H2O and buffer B contained 95% MeOH in buffer A.
  • a flow rate of 250 pL/min and a temperature of 60°C were employed.
  • UV traces at 260 and 280 nm were recorded.
  • a gradient of 1-100% B within 31 min was employed.
  • the two complementary strands were annealed by combining equimolar aqueous solutions of both strands. The mixtures were placed into a water bath at 70°C for 5 minutes and subsequently allowed to cool to ambient temperature within 2 h. The duplexes were lyophilized for 2 days and stored at -20°C.
  • duplexes were analyzed by analytical SEC HPLC on SuperdexTM 75 Increase 5/150 GL column 5 x 153-158 mm (Cytiva) on a Dionex Ultimate 3000 (Thermo Fisher Scientific) HPLC system.
  • Mobile phase consisted of lx PBS containing 10% acetonitrile.
  • An isocratic gradient was run in 10 min at a flow rate of 1.5 mL/min at room temperature. UV traces at 260 and 280 nm were recorded.
  • Water (LC-MS grade) was purchased from Sigma- Aldrich and Phosphate-buffered saline (PBS; lOx, pH 7.4) was purchased from GIBCO (Thermo Fisher Scientific).
  • GalNAc conjugates prepared are compiled in the table below. These were directed against 3 different target genes. siRNA coding along with the corresponding single strands, sequence information as well as purity for the duplexes is captured.
  • mice with an age of about 8 weeks were randomly assigned into groups of 21 mice.
  • the animals received a single subcutaneous dose of 0.3 or 3 mg/kg GalNAc-siRNA dissolved in saline (sterile 0.9% sodium chloride) or saline only as control.
  • saline sterile 0.9% sodium chloride
  • mice from each group were euthanised and serum and liver samples taken.
  • Serum was taken from a group of 5 untreated mice at day 0 to provide a baseline measurement of glycolate concentration.
  • Serum was stored at -80°C until further analysis. Liver sample (approximately 50 mg) were treated with RNAlater and stored overnight at 4°C, before being stored at -80°C.
  • Liver samples were analysed using quantitative real-time PCR for HA01 mRNA (Thermo assay ID Mm00439249_ml) and the housekeeping gene GAPDH mRNA (Thermo assay ID Mm99999915_gl).
  • the delta delta Ct method was used to calculated changes in HA01 expression normalised to GAPDH and relative to the saline control group.
  • a single 3 mg/kg dose of ETX005 inhibited HA01 mRNA expression by greater than 80% after 7 days (FIG 16). The suppression of HAO 1 expression was durable, with a single 3 mg/kg dose of ETX005 maintaining greater than 60% inhibition of HAO 1 mRNA at the end of the study on day 28. A single dose of 0.3 mg/kg ETX005 also inhibited HA01 expression when compared with the saline control group, with HAO1 expression levels reaching normal levels only at day 28 of the study.
  • HAO 1 mRNA expression is expected to cause an increase in serum glycolate levels.
  • Serum glycolate concentration was measured using LC-MS/MS (FIG 17).
  • a single 3 mg/kg dose of ETX005 caused a significant increase in serum glycolate concentration, reaching peak levels 14 days after dosing and remaining higher than baseline level (day 0) and the saline control group until the end of the study at day 28.
  • a single 0.3 mg/kg dose of ETX005 showed a smaller and more transient increase in serum glycolate concentration above the level seen in a baseline and saline control group, demonstrating that a very small dose can suppress HA01 mRNA at a magnitude sufficient to affect the concentration of a metabolic biomarker in serum.
  • FIG 16. Single dose mouse pharmacology of ETX005. HA01 mRNA expression is shown relative to the saline control group. Each point represents the mean and standard deviation of 3 mice.
  • FIG. Single dose mouse pharmacology of ETX005. Serum glycolate concentration is shown. Each point represents the mean and standard deviation of 3 mice, except for baseline glycolate concentration (day 0) which was derived from a group of 5 mice.
  • mice with an age of about 8 weeks were randomly assigned into groups of 21 mice.
  • the animals received a single subcutaneous dose of 0.3 or 3 mg/kg GalNAc-siRNA dissolved in saline (sterile 0.9% sodium chloride) or saline only as control.
  • saline sterile 0.9% sodium chloride
  • mice from each group were euthanised and serum and liver samples taken.
  • Serum was taken from a group of 5 untreated mice at day 0 to provide a baseline measurement of glycolate concentration.
  • Serum was stored at -80°C until further analysis. Liver sample (approximately 50 mg) were treated with RNAlater and stored overnight at 4°C, before being stored at -80°C.
  • Liver samples were analysed using quantitative real-time PCR for HA01 mRNA (Thermo assay ID Mm00439249_ml) and the housekeeping gene GAPDH mRNA (Thermo assay ID Mm99999915_gl).
  • the delta delta Ct method was used to calculated changes in HA01 expression normalised to GAPDH and relative to the saline control group.
  • a single 3 mg/kg dose of ETX006 inhibited HA01 mRNA expression by than 80% after 7 days (FIG 18). The suppression of HAO 1 expression was durable and continued until the end of the study, with ETX006 maintaining greater than 60% inhibition of HAO 1 mRNA at day 28.
  • a single dose of 0.3 mg/kg also inhibited HA01 expression when compared with the saline control group, with HA01 expression levels reaching normal levels only at day 28 of the study.
  • HAO 1 mRNA expression is expected to cause an increase in serum glycolate levels.
  • Serum glycolate concentration was measured using LC-MS/MS (FIG 19).
  • a single 3 mg/kg dose of ETX006 caused a significant increase in serum glycolate concentration, reaching peak levels 14 days after dosing and remaining higher than baseline levels (day 0) and the saline control group until the end of the study at day 28.
  • a single 0.3 mg/kg dose of ETX006 showed a smaller and more transient increase in serum glycolate concentration above the level seen in a baseline and saline control groups, demonstrating that a very small dose can also affect the concentration of a metabolic biomarker in serum.
  • FIG 18. Single dose mouse pharmacology of ETX006. HA01 mRNA expression is shown relative to the saline control group. Each point represents the mean and standard deviation of 3 mice.
  • FIG 19. Single dose mouse pharmacology of ETX006. Serum glycolate concentration is shown. Each point represents the mean and standard deviation of 3 mice, except for baseline glycolate concentration (day 0) which was derived from a group of 5 mice.
  • Serum was stored at -80°C until further analysis. Liver sample (approximately 50 mg) were treated with RNAlater and stored overnight at 4°C, before being stored at -80°C.
  • liver samples were analysed using quantitative real-time PCR for C5 mRNA (Thermo assay ID Mm00439275_ml) and the housekeeping gene GAPDH mRNA (Thermo assay ID Mm99999915_g1).
  • the delta delta Ct method was used to calculated changes in C5 expression normalised to GAPDH and relative to the saline control group.
  • ETX014 inhibited C5 mRNA expression in a dose-dependent manner (FIG 20) with the 3 mg/kg dose achieving greater than 90% reduction in C5 mRNA at day 14.
  • the suppression of C5 expression by ETX014 was durable, with the 3 mg/kg dose of each molecule showing clear knockdown of C5 mRNA until the end of the study at day 28.
  • C5 protein level analysis serum samples were measured using a commercially available C5 ELISA kit (Abeam ab264609). Serum C5 levels were calculated relative to the saline group means at matching timepoints.
  • Serum protein data support the mRNA analysis (FIG 21).
  • Treatment with ETX014 caused a dose-dependent decrease in serum C5 protein concentration. All doses of ETX014 reduced C5 protein levels by greater than 70%, with the 3 mg/kg dose reducing C5 levels to almost undetectable levels at day 7 of the study. Reduction of serum C5 was sustained by all doses until study termination, with even the lowest dose of 0.3 mg/kg still showing inhibition of approximately 40% at day 28.
  • FIG 20 Single dose mouse pharmacology of ETX014. C5 mRNA expression is shown relative to the saline control group. Each point represents the mean and standard deviation of 3 mice.
  • FIG 21 Single dose mouse pharmacology of ETX0014. Serum C5 concentration is shown relative to the saline control group. Each point represents the mean and standard deviation of 3 mice.
  • ETX015 Targeting C5 mRNA
  • T2a inverted abasic
  • mice with an age of about 8 weeks were randomly assigned into groups of 21 mice.
  • the animals received a single subcutaneous dose of 0.3, 1, or 3 mg/kg GalNAc-siRNA dissolved in saline (sterile 0.9% sodium chloride) or saline only as control.
  • saline sterile 0.9% sodium chloride
  • mice from each group were euthanised and serum and liver samples taken.
  • Serum was stored at -80°C until further analysis. Liver sample (approximately 50 mg) were treated with RNAlater and stored overnight at 4°C, before being stored at -80°C.
  • ETX015 inhibited C5 mRNA expression in a dose-dependent manner (FIG 22) with the 3 mg/kg dose achieving greater than 85% reduction of C5 mRNA.
  • the suppression of C5 expression was durable, with the 3 mg/kg dose of each molecule showing clear knockdown of C5 mRNA until the end of the study at day 28.
  • Mice dosed with 3 mg/kg ETX015 still exhibited less than 50% of normal liver C5 mRNA levels 28 days after dosing.
  • C5 protein level analysis serum samples were measured using a commercially available C5 ELISA kit (Abeam ab264609). Serum C5 levels were calculated relative to the saline group means at matching timepoints.
  • FIG 23 Serum protein data support the mRNA analysis (FIG 23).
  • ETX015 caused a dosedependent decrease in serum C5 protein concentration.
  • the 3 mg/kg and 1 mg/kg doses of ETX015 achieved greater than 90% reduction of serum C5 protein levels.
  • the highest dose exhibited durable suppression of C5 protein expression, with a greater than 70% reduction of C5 at day 28 of the study compared to saline control.
  • FIG 22 Single dose mouse pharmacology of ETX015. C5 mRNA expression is shown relative to the saline control group. Each point represents the mean and standard deviation of 3 mice.
  • FIG 23 Single dose mouse pharmacology of ETX0014. Serum C5 concentration is shown relative to the saline control group. Each point represents the mean and standard deviation of 3 mice.
  • ETX023 pharmacology was evaluated in non-human primate (NHP) by quantifying serum transthyretin (TTR) protein levels.
  • TTR serum transthyretin
  • mice Male cynomolgus monkeys (3-5 years old, 2-3 kg) were assigned into groups of 3 animals. Animals were acclimatised for 2 weeks, and blood taken 14 days prior to dosing to provide baseline TTR concentration. A liver biopsy was performed 18 or 38 days prior to dosing to provide baseline mRNA levels. On day 0 of the study, the animals received a single subcutaneous dose of 1 mg/kg GalNAc-siRNA ETX023 dissolved in saline (sterile 0.9% sodium chloride). At day 3, day 14, day 28, day 42, day 56, day 70 and day 84 of the study, a liver biopsy was taken and RNA extracted for measurement of TTR mRNA. At day 1, day 3, day 7, day 14, day 28, day 42, day 56, day 70 and day 84 of the study, a blood sample was taken for measurement of serum TTR concentration and clinical blood chemistry analysis.
  • TTR protein concentration was measured by a commercially available ELISA kit (Abeam ab231920). TTR concentration as a fraction of day 1 was calculated for each individual animal and this was plotted as mean and standard deviation for the group of 3 animals (FIG 24).
  • a single 1 mg/kg dose of ETX023 caused a rapid and significant reduction in serum TTR concentration, reaching nadir 28 days after dosing and remaining suppressed until day 70.
  • TTR mRNA was measured by real-time quantitative PCR using a TaqMan Gene expression kit TTR (Thermo, assay ID Mf02799963_ml). GAPDH expression was also measured (Thermo, assay ID Mf04392546_gl) to provide a reference. Relative TTR expression for each animal was calculated normalised to GAPDH and relative to pre-dose levels by the AACt method. A single 1 mg/kg dose of ETX023 also caused a rapid and significant reduction in liver TTR mRNA, reaching nadir 14 days after dosing and remaining suppressed until day 84 (FIG 28b).
  • Serum was analysed within 2 hours using an automatic biochemical analyser.
  • a significant increase in ALT (alanine transaminase) and AST (aspartate transaminase) are commonly used to demonstrate liver toxicity.
  • No increase in ALT (FIG 28d) or ALT (FIG 28e) was associated with dosing of ETX023.
  • ETX024 pharmacology was evaluated in non-human primate (NHP) by quantifying serum transthyretin (TTR) protein levels.
  • TTR serum transthyretin
  • mice Male cynomolgus monkeys (3-5 years old, 2-3 kg) were assigned into groups of 3 animals. Animals were acclimatised for 2 weeks, and blood taken 14 days prior to dosing to provide baseline TTR concentration. A liver biopsy was performed 18 or 38 days prior to dosing to provide baseline mRNA levels. On day 0 of the study, the animals received a single subcutaneous dose of 1 mg/kg GalNAc-siRNA ETX024 dissolved in saline (sterile 0.9% sodium chloride). At day 3, day 14, day 28, day 42, day 56, day 70 and day 84 of the study, a liver biopsy was taken and RNA extracted for measurement of TTR mRNA. At day 1, day 3, day 7, day 14, day 28, day 42, day 56, 70 and day 84 of the study, a blood sample was taken for measurement of serum TTR concentration and clinical blood chemistry analysis.
  • TTR protein concentration was measured by a commercially available ELISA kit (Abeam ab231920). TTR concentration as a fraction of day 1 was calculated for each individual animal and this was plotted as mean and standard deviation for the group of 3 animals (FIG 25).
  • a single 1 mg/kg dose of ETX024 caused a rapid and significant reduction in serum TTR concentration, reaching nadir 28 days after dosing and remaining suppressed until day 70.
  • TTR mRNA was measured by real-time quantitative PCR using a TaqMan Gene expression kit TTR (Thermo, assay ID Mf02799963_ml). GAPDH expression was also measured (Thermo, assay ID Mf04392546_gl) to provide a reference. Relative TTR expression for each animal was calculated normalised to GAPDH and relative to pre-dose levels by the AACt method. A single 1 mg/kg dose of ETX024 caused a rapid and significant reduction in liver TTR mRNA, reaching nadir 14 days after dosing and remaining suppressed until day 84 (FIG 29b).
  • ALT alanine transaminase
  • AST aspartate transaminase
  • compounds of the invention are able to depress serum protein level of a target protein to a value below the initial (starting) concentration at day 0, over a period of up to at least about 14 days after day 0, up to at least about 21 days after day 0, up to at least about 28 days after day 0, up to at least about 35 days after day 0, up to at least about 42 days after day 0, up to at least about 49 days after day 0, up to at least about 56 days after day 0, up to at least about 63 days after day 0, up to at least about 70 days after day 0, up to at least about 77 days after day 0, or up to at least about 84 days after day 0, hereinafter referred to as the “dose duration”.
  • Day 0 as referred to herein is the day when dosing of a compound of the invention to a patient is initiated, in other words the start of the dose duration or the time post dose.
  • compounds of the invention are able to depress serum protein level of a target protein to a value of at least about 90% or below of the initial (starting) concentration at day 0, such as at least about 85% or below, at least about 80% or below, at least about 75% or below, at least about 70% or below, at least about 65% or below, at least about 60% or below, at least about 55% or below, at least about 50% or below, at least about 45% or below, at least about 40% or below, at least about 35% or below, at least about 30% or below, at least about 25% or below, at least about 20% or below, at least about 15% or below, at least about 10% or below, at least about 5% or below, of the initial (starting) concentration at day 0.
  • depression of serum protein can be maintained over a period of up to at least about 14 days after day 0, up to at least about 21 days after day 0, up to at least about 28 days after day 0, up to at least about 35 days after day 0, up to at least about 42 days after day 0, up to at least about 49 days after day 0, up to at least about 56 days after day 0, up to at least about 63 days after day 0, up to at least about 70 days after day 0, up to at least about 77 days after day 0, or up to at least about 84 days after day 0.
  • the serum protein can be depressed to a value of at least about 90% or below of the initial (starting) concentration at day 0, such as at least about 85% or below, at least about 80% or below, at least about 75% or below, at least about 70% or below, at least about 65% or below, at least about 60% or below, at least about 55% or below, at least about 50% or below, at least about 45% or below, at least about 40% or below, of the initial (starting) concentration at day 0.
  • compounds of the invention are able to achieve a maximum depression of serum protein level of a target protein to a value of at least about 50% or below of the initial (starting) concentration at day 0, such as at least about 45% or below, at least about 40% or below, at least about 35% or below, at least about 30% or below, at least about 25% or below, at least about 20% or below, at least about 15% or below, at least about 10% or below, at least about 5% or below, of the initial (starting) concentration at day 0.
  • maximum depression of serum protein occurs at about day 14 after day 0, at about day 21 after day 0, at about day 28 after day 0, at about day 35 after day 0, or at about day 42 after day 0. More typically, such maximum depression of serum protein occurs at about day 14 after day 0, at about day 21 after day 0, or at about day 28 after day 0.
  • Specific compounds of the invention can typically achieve a maximum % depression of serum protein level of a target protein and / or a % depression over a period of up to at least about 84 days as follows:
  • ETX019 can typically achieve at least 50% depression of serum protein level of a target protein, typically TTR, typically at about 7 to 21 days after day 0, in particular at about 14 days after day 0, and / or can typically maintain at least 90% depression of serum protein level of a target protein, typically TTR, over a period of up to at least about 84 days after day 0 (as hereinbefore described, “day 0” as referred to herein is the day when dosing of a compound of the invention to a patient is initiated, and as such denotes the time post dose);
  • ETX020 can typically achieve at least 30% depression of serum protein level of a target protein, typically TTR, typically at about 7 to 21 days after day 0, in particular at about 14 days after day 0, and / or can typically maintain at least 80% depression of serum protein level of a target protein, typically TTR, over a period of up to at least about 84 days after day 0 (as hereinbefore described, “day 0” as referred to herein is the day when dosing of a compound of the invention to a patient is initiated, and as such denotes the time post dose);
  • ETX023 can typically achieve at least 20% depression of serum protein level of a target protein, typically TTR, typically at about 7 to 21 days after day 0, in particular at about 14 days after day 0, and / or can typically maintain at least 50% depression of serum protein level of a target protein, typically TTR, over a period of up to at least about 84 days after day 0 (as hereinbefore described, “day 0” as referred to herein is the day when dosing of a compound of the invention to a patient is initiated, and as such denotes the time post dose);
  • ETX024 can typically achieve at least 20% depression of serum protein level of a target protein, typically TTR, typically at about 7 to 21 days after day 0, in particular at about 14 days after day 0, and / or can typically maintain at least 60% depression of serum protein level of a target protein, typically TTR, over a period of up to at least about 84 days after day 0 (as hereinbefore described, “day 0” as referred to herein is the day when dosing of a compound of the invention to a patient is initiated, and as such denotes the time post dose).
  • day 0 as referred to herein is the day when dosing of a compound of the invention to a patient is initiated, and as such denotes the time post dose).
  • the depression of serum level is determined in non-human primates by delivering a single subcutaneous dose of 1 mg/kg of the relevant active agent, eg ETX0023 or ETX0024, dissolved in saline (sterile 0.9% sodium chloride). Suitable methods are described herein. It will be appreciated that this is not limiting and other suitable methods with appropriate controls may be used.
  • the relevant active agent eg ETX0023 or ETX0024
  • saline sterile 0.9% sodium chloride
  • Example 7 ETX023 (Targeting TTR mRNA) Tla inverted abasic
  • Kidney health was monitored by assessment of urea (blood urea nitrogen, BUN) and creatinine concentration throughout the study. Both blood urea concertation (BUN) and creatinine levels remained stable and within the expected range after a single 1 mg/kg dose of ETX023 (FIG 35 and 36).
  • Example 8 ETX024 (Targeting TTR mRNA) T2a inverted abasic
  • Kidney health was monitored by assessment of urea (blood urea nitrogen, BUN) and creatinine concentration throughout the study. Both blood urea concertation (BUN) and creatinine levels remained stable and within the expected range after a single 1 mg/kg dose of ETX024 (FIG 38 and 39).
  • FIG. 40A depicts a tri-antennary GalNAc (A-acetylgalactosamine) unit.
  • FIG. 40B depicts an alternative tri-antennary GalNAc according to one embodiment of the invention, showing variance in linking groups.
  • FIG. 41A depicts tri-antennary GalNAc-conjugated siRNA according to the invention, showing variance in the linking groups.
  • FIG. 41B depicts a genera of tri-antennary GalNAc-conjugated siRNAs according to one embodiment of the invention.
  • FIG. 41C depicts a genera of bi-antennary GalNAc-conjugated siRNAs according to one embodiment of the invention, showing variance in the linking groups.
  • FIG. 41D depicts a genera of bi-antennary GalNAc-conjugated siRNAs according to another embodiment of the invention, showing variance in the linking groups.
  • FIG. 42A depicts another embodiment of the tri-antennary GalNAc-conjugated siRNA according to one embodiment of the invention.
  • FIG. 42B depicts a variant shown in Fig. 27A, having an alternative branching GalNAc conjugate.
  • FIG. 42C depicts a genera of tri-antennary GalNAc-conjugated siRNAs according to one embodiment of the invention, showing variance in the linking groups.
  • FIG. 42D depicts a genera of bi-antennary GalNAc-conjugated siRNAs according to one embodiment the invention, showing variance in the linking groups.
  • the further aspect discloses forms of ASGP-R ligand-conjugated, chemically modified RNAi agents, and methods of making and uses of such conjugated molecules.
  • the ASGP-R ligand comprises /'/-acetylgalactosamine (GalNAc).
  • the invention provides an siRNA conjugated to tri- antennary or biantennary units of GalNAc of the following formula (I):
  • n 0, 1, 2, 3, or 4.
  • the number of the ethyleneglycol units may vary independently from each other in the different branches.
  • Other embodiments my contain only two branches, as depicted in Formulae (Il-a)
  • n is chosen from 0, 1, 2, 3, or 4.
  • the number of the ethylene-glycol units may vary independently from each other in the different branches.
  • GalNAc branches can also be added, for example, 4-, 5-, 6-, 7-, 8-, 9- branched GalNAc units may be used.
  • the branched GalNAc can be chemically modified by the addition of another targeting moiety, e.g., a lipids, cholesterol, a steroid, a bile acid, targeting (poly)peptide, including polypeptides and proteins, (e.g., RGD peptide, transferrin, poly glutamate, polyaspartate, glycosylated peptide, biotin, asialoglycoprotein insulin and EGF.
  • another targeting moiety e.g., a lipids, cholesterol, a steroid, a bile acid
  • targeting (poly)peptide including polypeptides and proteins, (e.g., RGD peptide, transferrin, poly glutamate, polyaspartate, glycosylated peptide, biotin, asialoglycoprotein insulin and EGF.
  • the GalNAc units may be attached to the RNAi agent via a tether, such as the one shown in Formula (III*):
  • m is chosen from 0, 1, 2, 3, 4, or 5 and p is chosen from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, independently of m, and X is either CH2 or O.
  • Such an attachment of the GalNAc branched units via the specified tethers is preferably at a 3’ or a 5’ end of the sense strand of the RNAi agent.
  • the attachment to the 3’ of RNAi agent is through C6 amino linker as shown in Formula (V*):
  • This linker is the starting point of the synthesis as shown in Example 15.
  • the same linkers and tethers as described above can be used with alternative branched
  • the GalNAc units may be attached to the RNAi agent via a tether, such as the one shown in Formula (III*-2):
  • q is chosen from 1, 2, 3, 4, 5, 6, 7, or 8.
  • Such an attachment of the GalNAc branched units via the specified tethers preferably at a 3’ or a 5’ end of the sense strand of the double stranded RNAi agent.
  • the attachment to the 3’ of RNAi agent is as shown in Example 17.
  • the transitional linker between the tether and the 3’ end of the oligo comprises the structure of the formula (V*-a; see also Fig. 42C) or another suitable linker may be used, for example, C6 amino linker shown in Formula (V*-b):
  • Additional and/or alternative conjugation sites may include any non-terminal nucleotide, including sugar residues, phosphate groups, or nucleic acid bases.
  • the conjugated oligomeric compound (referred herein as RNA interference compound (RNAi compound)) comprises two strands, each having sequence of from 8 to 55 linked nucleotide monomer subunits (including inverted abasic (ia) nucleotide(s)) in either the antisense strand or in the sense strand.
  • the conjugated oligomeric compound strands comprise, for example, a sequence of 16 to 55, 53, 49, 40, 25, 24, 23, 21, 20, 19, 18, 17, or up to (about) 18-25, 18-23, 21-23 linked nucleotide monomer subunits.
  • RNAi agent of the invention may have a hairpin structure, having a single strand of the combined lengths of both strands as described above.
  • nucleotide as used throughout, may also refer to nucleosides (i.e., nucleotides without phosphate/phosphonothioate groups) where context so requires.)
  • the double stranded RNAi agent is blunt-ended or has an overhang at one or both ends.
  • the overhang is 1-6, 1-5, 1-4, 1-3, 2-4, 4, 3, 2 or 1 nucleotide(s) (at 3’ end or at 5’ end) of the antisense strand as well as 2-4, 3, or 2 or 1 nucleotide(s) (at 3’ end or at 5’ end) of the sense strand.
  • the RNAi agent comprises 2 nucleotide overhang at the 3’ end of the antisense strand and 2 nucleotide overhang at 3’ end of the sense strand.
  • the RNAi agents comprise 2 nucleotide overhang at the 3’ end of the antisense strand and are blunt-ended on the other end.
  • the construct is blunt-ended on both ends.
  • the RNAi agent comprises 4 nucleotide overhang in the 3’ end of the antisense strand and blunt-ended on the other end.
  • the constructs are modified with a degradation protective moiety that prevents or inhibits nuclease cleavage by using a terminal cap, one or more inverted abasic nucleotides, one or more phosphorothioate linkages, one of more deoxynucleotides (e.g., D-ribonucleotide, D-2'-deoxyribonucleotide or another modified nucleotide), or a combination thereof.
  • Such degradation protective moieties may be present at any one or all ends that are not conjugated to the ASGP-R ligand.
  • the degradation protective moiety is chosen alone or as any combination from a group consisting of 1-4, 1-3, 1-2, or 1 phosphorothioate linkages, 1-4 1-3, 1-2, or 1 deoxynucleotides, and 1-4, 1-3, 1-2, or 1 inverted abasic nucleotides.
  • the degradation protective moieties are configured as in one of the constructs 9.1, 9.2, 9.3, 10.1, 10.2, 10.3, 11.1, 11.2, 11.3, 12.1, 12.2, and 12.3, as shown in the Examples 9-18.
  • Such exemplary protective moieties’ configurations can be used in conjunction with any RNAi agents of the invention.
  • all or some riboses of the nucleotides in the sense and/or antisense strand (s) are modified. In certain embodiments, at least 50%, 60%, 70%, 80%, 90% or more (e.g., 100%) of riboses in the RNAi agent are modified. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more riboses are not modified.
  • ribose modifications include 2’ substituent groups such as 2’-O-alkyl modifications, including 2’-O-methyl, and 2’ -deoxyfluoro. Additional modifications are known in the art, including 2’-deoxy, LNA (e.g., 2'-O, 4'-C methylene bridge or 2'-O, 4'-C ethylene bridge), 2’-methoxythoxy (MOE), 2’-O-(CH2)OCH3, etc.
  • LNA e.g., 2'-O, 4'-C methylene bridge or 2'-O, 4'-C ethylene bridge
  • MOE 2’-methoxythoxy
  • a number of modifications provide a distinct pattern of modifications, for example, as shown in constructs in the Examples 9-18, or as described in US Patents Nos. 7,452,987; 7,528,188; 8,273,866; 9,150,606; and 10,266,825; all of which are incorporated by reference herein.
  • the siRNA comprises one or more thermally destabilizing nucleotides, e.g., GNA, ENA, etc., for example, at positions 11 (preferred), 12, 13 of the antisense strand and/or positions 9 and 10 (preferred) of the sense strand. .
  • thermally destabilizing nucleotides e.g., GNA, ENA, etc.
  • nucleic acid bases could be modified, for example, at the C4 position as described in US Patent No. 10,119,136.
  • the RNAi agents of the invention are directed against therapeutic targets, inhibition of which will result in prevention, alleviation, or treatment of a disease, including undesirable or pathological conditions.
  • targets include: ApoC, ApoB, ALAS1, TTR, GO, C5 (see Examples), etc.
  • targets are human, while the RNAi agent comprise an antisense strand fully or partially complementary to such a target.
  • the RNAi agents may comprise two or more chemically linked RNAi agents directed against the same or different targets.
  • Example 9 Inverted abasic chemistry with 5’-GalNAc
  • ia inverted abasic nucleotide
  • m 2’-O-methyl nucleotide
  • f 2 ’-deoxy-2’ -fluoro nucleotide
  • s phosphorothioate internucleotide linkage
  • Example 10 Inverted abasic chemistry with 3’-GalNAc

Abstract

La présente invention concerne des molécules d'acides nucléiques destinées à être utilisées dans le traitement ou la prévention de maladies.
EP22704882.4A 2021-01-30 2022-01-28 Acides nucléiques contenant des nucléotides abasiques Pending EP4176061A1 (fr)

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US8273866B2 (en) 2002-02-20 2012-09-25 Merck Sharp & Dohme Corp. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (SINA)
EP2258847B2 (fr) 2002-08-05 2020-07-01 Silence Therapeutics GmbH Autres nouvelles formes de molécules d'ARN interférant
US9150606B2 (en) 2002-11-05 2015-10-06 Isis Pharmaceuticals, Inc. Compositions comprising alternating 2'-modified nucleosides for use in gene modulation
US9150605B2 (en) 2002-11-05 2015-10-06 Isis Pharmaceuticals, Inc. Compositions comprising alternating 2′-modified nucleosides for use in gene modulation
KR100576357B1 (ko) 2003-10-21 2006-05-03 삼성전자주식회사 레졸을 함유하는 수지용액, 이를 사용하여 형성된열경화수지막 및 이를 사용하여 열경화수지막을 형성하는방법
EP2264169A1 (fr) * 2009-06-15 2010-12-22 Qiagen GmbH SiNA modifié
WO2011084193A1 (fr) * 2010-01-07 2011-07-14 Quark Pharmaceuticals, Inc. Composés oligonucléotidique comportant des extrémités sortantes non nucléotidiques
US9988627B2 (en) * 2013-10-04 2018-06-05 Novartis Ag Formats for organic compounds for use in RNA interference
KR101696704B1 (ko) * 2013-12-17 2017-01-16 주식회사 인코드젠 오프-타겟을 막기 위해 변형된 rna 간섭 유도 핵산 및 그 용도
WO2015106128A2 (fr) 2014-01-09 2015-07-16 Alnylam Pharmaceuticals, Inc. Agents d'arni modifiés
TWI788312B (zh) * 2016-11-23 2023-01-01 美商阿尼拉製藥公司 絲胺酸蛋白酶抑制因子Ａ１ ｉＲＮＡ組成物及其使用方法
SG11201912178VA (en) * 2017-09-11 2020-01-30 Arrowhead Pharmaceuticals Inc Rnai agents and compositions for inhibiting expression of apolipoprotein c-iii (apoc3)
EP3775207A1 (fr) 2018-04-05 2021-02-17 Silence Therapeutics GmbH Siarns avec vinylphosphonate à l'extrémité 5' du brin antisens

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