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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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
  • A61K31/713—Double-stranded nucleic acids or oligonucleotides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
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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
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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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  • C12N2320/00—Applications; Uses
  • C12N2320/10—Applications; Uses in screening processes
  • C12N2320/11—Applications; Uses in screening processes for the determination of target sites, i.e. of active nucleic acids

Definitions

• NUCLEIC ACID COMPOUNDS FIELD The present invention provides novel 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.
• Nucleic acid compounds have important therapeutic applications in medicine. Nucleic acids can be used to silence genes that are responsible for a particular disease. Gene-silencing prevents formation of a protein by inhibiting translation. Importantly, gene-silencing 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 / oligonucleosides 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 nucleic acid compounds that inhibit the expression of the gene ZPI, for use in the treatment and / or prevention of disease.
• a nucleic acid for inhibiting expression of ZPI comprising a duplex region that comprises a first strand and a second strand that is at least partially complementary to the first strand, wherein said first strand is: (i) at least partially complementary to a portion of RNA transcribed from the ZPI gene, and (ii) comprises at least 17 contiguous nucleosides differing by 0 or 1 nucleosides from any one of the first strand sequences as listed in Table 2.
• a nucleic acid for inhibiting expression of ZPI comprising a duplex region that comprises a first strand and a second strand that is at least partially complementary to the first strand, wherein said first strand is: (i) at least partially complementary to a portion of RNA transcribed from the ZPI gene, and (ii) comprises at least 17 contiguous nucleosides differing by 0 or 1 nucleosides from any one of the first strand modified sequences as listed in Table 3.
• a nucleic acid as described herein, wherein the first strand comprises nucleosides 2-18 of any one of the sequences according to the above first and second aspects of the present invention.
• a nucleic acid according to the above first aspect of the present invention wherein the second strand comprises a nucleoside sequence of at least 17 contiguous nucleosides differing by 0 or 1 nucleosides from any one of the second strand sequences as listed in Table 2, and wherein the second strand has a region of at least 85% complementarity over the 17 contiguous nucleosides to the first strand.
• a nucleic acid according to the above first aspect of the present invention wherein the second strand comprises a nucleoside sequence of at least 17 contiguous nucleosides differing by 0 or 1 nucleosides from any one of the second strand sequences as listed in Table 2, and wherein the duplex region comprises at least 14, 15, 16 or 17 complementary base pairs.
• a nucleic acid according to the above second aspect of the present invention wherein the second strand comprises a nucleoside sequence of at least 17 contiguous nucleosides differing by 0 or 1 nucleosides from any one of the second strand modified sequences as listed in Table 4, and wherein the second strand has a region of at least 85% complementarity over the 17 contiguous nucleosides to the first strand.
• a nucleic acid according to the above second aspect of the present invention wherein the second strand comprises a nucleoside sequence of at least 17 contiguous nucleosides differing by 0 or 1 nucleosides from any one of the second strand modified sequences as listed in Table 4, and wherein the duplex region comprises at least 14, 15, 16 or 17 complementary base pairs.
• a nucleic acid according to the above first aspect of the present invention wherein the first strand comprises any one of the first strand sequences as listed in Table 2.
• a nucleic acid according to the invention, wherein the first strand comprises SEQ ID NO: 148 or SEQ ID NO: 145.
• a pharmaceutical composition comprising a nucleic acid as disclosed herein, in combination with a pharmaceutically acceptable excipient or carrier.
• a nucleic acid or pharmaceutical composition for use in therapy.
• a nucleic acid or pharmaceutical composition for use in prevention or treatment of a disease related to a disorder of haemostasis, such as haemophilia.
• a nucleic acid or pharmaceutical composition for use in prevention or treatment of Von Willebrand disease.
• a nucleic acid or pharmaceutical composition for use in prevention or treatment of Factor X Deficiency.
• FIGURES Figure 1 Linker and ligand portions of constructs suitable for use according to the present invention including tether 1a.
• Figure 1 depicts the linker to be conjugated to an oligonucleotide
• the present invention also encompasses conjugates of the same linker with an oligonucleoside as disclosed herein.
• Figure 1 depicts as a product molecules based on the linker and ligand portions as specifically depicted in Figure 1 attached to an oligonucleoside moiety as also depicted herein, this product may alternatively further comprise, or consist essentially of, molecules wherein the linker and ligand portions are essentially as depicted in Figure 1 attached to an oligonucleoside moiety but having the F substituent as shown in Figure 1 on the cyclo-octyl ring replaced by a substituent, which could occur as a result of hydrolytic displacement, such as an OH substituent, or the OH substituent could be synthesized as a linker in its own right.
• tether 1a constructs can consist essentially of molecules having linker and ligand portions specifically as depicted in Figure 1, with a F substituent on the cyclo-octyl ring; or (b) tether 1a constructs can consist essentially of molecules having linker and ligand portions essentially as depicted in Figure 1 but having the F substituent as shown in Figure 1 on the cyclo- octyl ring replaced by an OH substituent, or (c) tether 1a constructs can comprise a mixture of molecules as defined in (a) and/or (b).
• Figure 2 Linker and ligand portions of constructs suitable for use according to the present invention including tether 1b.
• Figure 2 depicts the linker to be conjugated to an oligonucleotide
• the present invention also encompasses conjugates of the same linker with an oligonucleoside as disclosed herein.
• tether 1b constructs can consist essentially of molecules having linker and ligand portions specifically as depicted in Figure 2, with a F substituent on the cyclo-octyl ring; or (b) tether 1b constructs can consist essentially of molecules having linker and ligand portions essentially as depicted in Figure 2 but having the F substituent as shown in Figure 2 on the cyclo- octyl ring replaced by an OH substituent, or (c) tether 1b constructs can comprise a mixture of molecules as defined in (a) and/or (b).
• Figure 3 Linker and ligand portions of constructs suitable for use according to the present invention including tether 2a.
• Figure 3 depicts the linker to be conjugated to an oligonucleotide, it is to be understood that the present invention also encompasses conjugates of the same linker with an oligonucleoside as disclosed herein.
• Figure 4 Linker and ligand portions of constructs suitable for use according to the present invention including tether 2b. While Figure 4 depicts the linker to be conjugated to an oligonucleotide, it is to be understood that the present invention also encompasses conjugates of the same linker with an oligonucleoside as disclosed herein.
• Figure 5 Formulae described in Sentences 1-101 disclosed herein.
• Figure 6 Formulae described in Clauses 1-56 disclosed herein
• Figures 7a and 7b Inverted abasic constructs that can be used with nucleic acid sequences according to the present invention as described herein.
• a GalNAc linker is attached to the 5’ end region of the sense strand in use (not depicted in Figure 7a).
• a GalNAc linker is attached to the 3’ end region of the sense strand in use (not depicted in Figure 7b).
• iaia as shown at the 3’ end region of the sense strand in Figure 7a represents (i) two abasic nucleosides provided as the penultimate and terminal nucleosides at the 3’ end region of the sense strand, (ii) wherein a 3’-3’ reversed linkage is provided between the antepenultimate nucleoside (namely at position 21 of the sense strand, wherein position 1 is the terminal 5’ nucleoside of the sense strand) and the adjacent penultimate abasic residue of the sense strand, and (iii) the linkage between the terminal and penultimate abasic nucleosides is 5’-3’ when reading towards the 3’ end region comprising the terminal and penultimate abasic nucleosides.
• iaia as shown at the 5’ end region of the sense strand in Figure 7b represents (i) two abasic nucleosides provided as the penultimate and terminal nucleosides at the 5’ end region of the sense strand, (ii) wherein a 5’-5’ reversed linkage is provided between the antepenultimate nucleoside (namely at position 1 of the sense strand, not including the iaia motif at the 5’ end region of the sense strand in the nucleoside position numbering on the sense strand) and the adjacent penultimate abasic residue of the sense strand, and (iii) the linkage between the terminal and penultimate abasic nucleosides is 3’-5’ when reading towards the 5’ end region comprising the terminal and penultimate abasic nucleosides.
• Figures 8a and 8b Duplex constructs according to Table 5.
• Figure 9 The correlation between predicted and experimentally determined siRNA efficacy values i.e. maximum RNA knockdown where 1 represents maximum knockdown and 0 is no reduction in mRNA levels. Data displayed are for the test dataset in the best performing siRNAdesignR model.
• Figure 10 Performance metrics for the best performing siRNAdesignR model in the test data set and also the validation dataset. In both cases the model scored above 0.5 in the Precision@20 metric, and the best performing siRNA (experimentally determined) was in the top 20 predictions of the model (nSiRNAsForBest).
• Figure 11 siRNAdesignR ranking for 276 siRNAs.
• Figure 12 A dose response curve of SLC25A5 mRNA knockdown following 24 hour exposure to siRNA. Cells were tested in triplicate repeats on two separate days (replicates 1 and 2). Data are mean +/- standard deviation with knockdown normalised to untreated wells.
• Figure 13 Change in SLC25A5 mRNA knockdown over 28 days following one subcutaneous dose of siRNA at day 0. As mRNA measurements are taken from liver tissue measurements are taken from different mice at each time point. Data are mean +/- standard deviation from 16 mice per timepoint per dose, normalised to saline control.
• Figure 14 Change in SLC25A5 protein expression over 28 days following one subcutaneous dose of siRNA at day 0. As mRNA measurements are taken from liver tissue measurements are taken from different mice at each time point. Data are mean +/- standard deviation from 16 mice per timepoint per dose, normalized to saline control.
• Figure 15 Results of dose-response experiments for inhibition of B4GALT1 mRNA expression in human Huh7 cells. Data are mean +/- standard deviation from triplicate repeats with knockdown normalized to untreated wells. Dotted curves represent 95% confidence intervals. Dotted lines and shaded areas represent the mean relative expression +/- standard deviation from untreated wells on the same plate.
• Figure 16 Results of time course experiments for inhibition of B4GALT1 mRNA expression in C57BL/6 mice. Data are mean +/- standard deviation from 12 mice per timepoint per dose, normalised mice prior to treatment with siRNA construct.
• Figure 17 Results of an RNAi molecule screen targeting the expression of B4GALT1 in human Huh7 cells. Data are mean +/- standard deviation from triplicate repeats with knockdown at 3 nM normalised to untreated cells. ETX-M00001217 was included as a non-targeting control and ETX-M00001850 was the lead sequence from the original screen included for comparison.
• Figure 18 Overview of the hydrodynamic injection (HDI) study in mice.
• HDI hydrodynamic injection
• Figure 19 Results of an siRNA molecule screen targeting the expression of human ZPI following hydrodynamic injection in BALB/c mice.
• Non-na ⁇ ve cynomolgus macaques received a single 3 mg/kg subcutaneous dose of ETX siRNA on day 0.
• Plasma samples were collected on days -7, -3, 0, 7, 14, 28, 42, 56, 70, and 84.
• Liver biopsies were collected on days -7, 28, 56, and 84.
• ALT Alanine Transaminase
• AST Aspartate Transaminase
• ALP Alkaline Phosphatase
• GGT Gamma- Glutamyltransferase.
• the grey areas indicate the reference range of normal values for each marker.
• Figure: 23 Overview of studies in von Willebrand Factor (vwf) knockout (KO) mice.
• Figure 24 Ex vivo rotational thromboelastometry (ROTEM) analysis of vwf KO mice treated with ETX- M00001185. Vwf KO mice received two 10 mg/kg subcutaneous doses of ETX siRNA on days -10 and -3. Blood samples were collected on day 0 for ROTEM analysis.
• ROTEM Ex vivo rotational thromboelastometry
• FIG. 25 In vivo tail vein transection (TVT) testing in vwf KO mice treated with ETX-M00001185. Vwf KO mice received two 10 mg/kg subcutaneous doses of ETX siRNA on days -10 and -3. On day 0 a transection was made to the tail vein of each mouse. The amount of blood loss over twenty minutes was recorded. Treatment resulted in statistically significant reductions blood loss. Data is represented as Mean +/- SEM.
• first strand also called the antisense strand or guide strand herein and which can be used interchangeably herein, refers to the nucleic acid strand, e.g. the strand of an siRNA, e.g. a dsiRNA, which includes a region that is substantially complementary to a target sequence, e.g. to an mRNA.
• region of complementarity refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence. Where the region of complementarity is not fully complementary to the target sequence, the mismatches can typically be in the internal or terminal regions of the molecule.
• a double stranded nucleic acid e.g. an siRNA agent of the invention includes a nucleoside mismatch in the antisense strand.
• the “second strand” also called the sense strand or passenger strand herein, and which can be used interchangeably herein, refers to the strand of a nucleic acid e.g. siRNA that includes a region that is substantially complementary to a region of the antisense strand as that term is defined herein.
• the nucleic acid of the invention may be referred to as an oligonucleoside or an oligonucleoside moiety.
• Oligonucleotides are short nucleic acid polymers. Whilst oligonucleotides contain phosphodiester bonds between the nucleoside component thereof (base plus sugar), the present invention is not limited to oligonucleotides always joined by such a phosphodiester bond between adjacent nucleosides, and other oligomers of nucleosides joined by bonds which are bonds other than a phosphodiester bond are contemplated. For example, a bond between nucleosides may be a phosphorothioate bond. Therefore, the term “oligonucleoside” as used herein covers both oligonucleotides and other oligomers of nucleosides.
• An oligonucleoside which is a nucleic acid having at least a portion which is an oligonucleotide is preferred according to the present invention.
• An oligonucleoside having one or more, or a majority of, phosphodiester backbone bonds between nucleosides is also preferred according to the present invention.
• An oligonucleoside having one or more, or a majority of, phosphodiester backbone bonds between nucleosides, and also having one or more phosphorothioate backbone bonds between nucleosides (typically in a terminal region of the first and / or second strands) is also preferred according to the present invention.
• the nucleic acid according to the invention is a double stranded oligonucleoside comprising one or more phosphorothioate backbone bonds between nucleosides. Accordingly, in all instances in which the present application refers to an oligonucleotide, particularly in the chemical structures disclosed herein, the oligonucleotide may equally be an oligonucleoside as defined herein. Similarly, in all instances in which the present application refers to an oligonucleoside, particularly in the chemical structures disclosed herein, the oligonucleoside may specifically be an oligonucleotide as defined herein. In some embodiments, a double stranded nucleic acid e.g.
• siRNA agent of the invention includes a nucleoside mismatch in the sense strand.
• the nucleoside mismatch is, for example, within 5, 4, 3, 2, or 1 nucleosides from the 3 '-end of the nucleic acid e.g. siRNA.
• the nucleoside mismatch is, for example, in the 3'- terminal nucleoside of the nucleic acid e.g. siRNA.
• a “target sequence” refers to a contiguous portion of the nucleoside sequence of an mRNA molecule formed during the transcription of a gene, including mRNA that is a product of RNA processing of a primary transcription product, or can be a contiguous portion of the nucleoside sequence of any RNA molecule such as a LNCRNA which it is desired to inhibit.
• the target sequence may be from about 10-35 nucleosides in length, e.g., about 15-30 nucleosides in length.
• the target sequence can be from about 15-30 nucleosides, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18- 28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20- 21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleosides in length.
• ribonucleoside or “nucleoside” can also refer to a modified nucleoside, as further detailed below.
• a nucleic acid can be a DNA or an RNA, and can comprise modified nucleosides.
• RNA is a preferred nucleic acid.
• iRNA RNA-induced silencing complex
• RISC RNA-induced silencing complex
• siRNA directs the sequence- specific degradation of mRNA through RNA interference (RNAi).
• RNAi RNA interference
• a double stranded RNA is referred to herein as a “double stranded siRNA (dsiRNA) agent", “double stranded siRNA (dsiRNA) molecule”, “double stranded RNA (dsRNA) agent”, “double stranded RNA (dsRNA) molecule”, “dsiRNA agent”, “dsiRNA molecule”, or “dsiRNA”, which refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary nucleic acid strands, referred to as having "sense” and “antisense” orientations with respect to a target RNA.
• nucleosides of each strand of the nucleic acid are preferably ribonucleosides, but in that case each or both strands can also include one or more non-ribonucleosides, e.g., a deoxyribonucleoside or a modified nucleoside.
• an "siRNA” may include ribonucleosides with chemical modifications.
• modified nucleoside refers to a nucleoside having, independently, a modified sugar moiety, a modified internucleoside linkage, or modified nucleobase, or any combination thereof.
• modified nucleoside encompasses substitutions, additions or removal of, e.g., a functional group or atom, to internucleoside linkages, sugar moieties, or nucleobases. Any such modifications, as used in an siRNA type molecule, are encompassed by "iRNA” or “RNAi agent” or “siRNA” or “siRNA agent” for the purposes of this specification and claims.
• iRNA or “RNAi agent” or “siRNA” or “siRNA agent” for the purposes of this specification and claims.
• the duplex region of a nucleic acid of the invention e.g.
• a dsRNA may range from about 9 to 40 base pairs in length such as 9 to 36 base pairs in length, e.g., about 15- 30 base pairs in length, for example, about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, such as about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18- 27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21- 30, 21-29, 21-28,
• nucleoside overhang refers to at least one unpaired nucleoside that extends from the duplex structure of a nucleic acid according to the present invention.
• a nucleic acid according to the present invention can comprise an overhang of at least one nucleoside; alternatively the overhang can comprise at least two nucleosides, at least three nucleosides, at least four nucleosides, at least five nucleosides or more.
• a nucleoside overhang can comprise or consist of a nucleoside/nucleoside analog, including a deoxynucleoside.
• the overhang(s) can be on the sense strand, the antisense strand, or any combination thereof.
• the nucleoside(s) of an overhang can be present on the 5'-end, 3'-end, or both ends of either an antisense or sense strand.
• the antisense strand has a 1-10 nucleoside, e.g., 0-3, 1-3, 2-4, 2-5, 4-10, 5-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleoside overhang at the 3'-end or the 5'-end.
• “Blunt” or “blunt end” means that there are no unpaired nucleosides at that end of the double stranded nucleic acid, i.e., no nucleoside overhang.
• the nucleic acids of the invention include those with no nucleoside overhang at one end or with no nucleoside overhangs at either end.
• the term "complementary,” when used to describe a first nucleoside sequence in relation to a second nucleoside sequence refers to the ability of an oligonucleoside comprising the first nucleoside sequence to hybridize and form a duplex structure under certain conditions with an oligonucleoside comprising the second nucleoside sequence, as will be understood by the skilled person.
• Such conditions can, for example, be stringent conditions, where stringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C for 12-16 hours followed by washing (see, e.g., "Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press).
• Complementary sequences within nucleic acid e.g. a dsiRNA, as described herein include base-pairing of the oligonucleoside comprising a first nucleoside sequence to an oligonucleoside comprising a second nucleoside sequence over the entire length of one or both nucleoside sequences.
• sequences can be referred to as “fully complementary” with respect to each other herein.
• first sequence is referred to as “substantially complementary” or “partially complementary” with respect to a second sequence herein
• the two sequences can be fully complementary, or they can form one or more mismatched base pairs, such as 2, 4, or 5 mismatched base pairs, but preferably not more than 5 , while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., inhibition of gene expression via a RISC pathway. Overhangs shall not be regarded as mismatches with regard to the determination of complementarity.
• a nucleic acid e.g.
• dsiRNA comprising one oligonucleoside 17 nucleosides in length and another oligonucleoside 19 nucleosides in length, wherein the longer oligonucleoside comprises a sequence of 17 nucleosides that is fully complementary to the shorter oligonucleoside, can yet be referred to as "fully complementary”.
• "Complementary" sequences can also include, or be formed entirely from, non- Watson- Crick base pairs or base pairs formed from non-natural and modified nucleosides, in so far as the above requirements with respect to their ability to hybridize are fulfilled.
• Such non- Watson- Crick base pairs include, but are not limited to, G:U Wobble or Hoogstein base pairing.
• the terms “complementary,” “fully complementary” and “substantially/partially complementary” herein can be used with respect to the base matching between the sense strand and the antisense strand of a nucleic acid e.g., dsiRNA, or between the antisense strand of a double stranded nucleic acid e.g. siRNA agent and a target sequence.
• the second strand of the nucleic acid according to the invention in particular a dsiRNA for inhibiting expression of ZPI, is at least partially complementary to the first strand of said nucleic acid.
• a first and second strand of a nucleic acid according to the invention are partially complementary if they form a duplex region having a length of at least 17 base pairs and comprising not more than 1, 2, 3, 4, or 5 mismatched base pairs. In certain embodiments, a first and second strand of the nucleic acid according to the invention are partially complementary if they form a duplex region having a length of 19 base pairs and comprising not more than 1, 2, 3, 4, or 5 mismatched base pairs. In certain embodiments, a first and second strand of the nucleic acid according to the invention are partially complementary if they form a duplex region having a length of 21 base pairs comprising not more than 1, 2, 3, 4, or 5 mismatched base pairs.
• a first and second strand of the nucleic acid according to the invention are partially complementary if they form a duplex region having a length of at least 17 base pairs, wherein at least 14, 15, 16 or 17 of said base pairs are complementary base pairs, in particular Watson-Crick base pairs.
• a first and second strand of the nucleic acid according to the invention are partially complementary if they form a duplex region having a length of 19 base pairs, wherein at least 14, 15, 16, 17, 18 or all 19 base pairs are complementary base pairs, in particular Watson-Crick base pairs.
• a first and second strand of the nucleic acid according to the invention are partially complementary if they form a duplex region having a length of 21 base pairs, wherein at least 16, 17, 18, 19, 20 or all 21 base pairs are complementary base pairs, in particular Watson-Crick base pairs.
• a nucleic acid that is "substantially complementary” or “partially complementary” to at least part of a messenger RNA (mRNA) refers to a nucleic acid that is substantially or partially complementary to a contiguous portion of the mRNA of interest (e.g., an mRNA encoding a gene).
• the contiguous portion of the mRNA is a sequence as listed in Table 1, i.e., any one of SEQ ID NOs:1-47.
• a nucleic acid is complementary to at least a part of an mRNA of a gene of interest if the sequence is substantially or partially complementary to a non-interrupted portion of an mRNA encoding that gene.
• the antisense oligonucleosides as disclosed herein are fully complementary to the target gene sequence.
• the antisense oligonucleosides disclosed herein are substantially or partially complementary to a target RNA sequence and comprise a contiguous nucleoside sequence which is at least about 80% complementary over its entire length to the equivalent region of the target RNA sequence, such as at least about 85%, 86%, 87%, 88%, 89%, about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary or 100% complementary.
• the first (antisense) strand of a nucleic acid according to the invention is partially or fully complementary to a contiguous portion of RNA transcribed from the ZPI gene.
• the first strand of the nucleic acid according to the invention is partially or fully complementary to a contiguous portion of at least 17 nucleosides of the ZPI mRNA. In certain embodiments, the first strand of the nucleic acid according to the invention is partially or fully complementary to a contiguous portion of 17, 18, 19, 20, 21, 22 or 23 nucleosides of the ZPI mRNA. In certain embodiments, the first strand of the nucleic acid according to the invention is partially or fully complementary to a contiguous portion of 17, 18, 19, 20, 21, 22 or 23 nucleosides of any one of the sequences as listed in Table 1, i.e., any one of SEQ ID NOs:1-47.
• the first (antisense) strand of the nucleic acid according to the invention is partially complementary to a contiguous portion of the ZPI mRNA if it comprises a contiguous nucleoside sequence of at least 17 nucleosides, wherein at least 14, 15, 16 or 17 nucleosides of said contiguous nucleoside sequence are complementary to a contiguous portion of the ZPI mRNA.
• the first strand of the nucleic acid according to the invention comprises a contiguous nucleoside sequence of at least 17 nucleosides, wherein at least 14, 15, 16 or 17 nucleosides of said contiguous nucleoside sequence are complementary to a contiguous portion of any one of the sequences listed in Table 1, i.e., any one of SEQ ID NOs: 1-47.
• the first strand of the nucleic acid according to the invention comprises a contiguous nucleoside sequence of 19 nucleosides, wherein at least 14, 15, 16, 17, 18 or all 19 nucleosides of said contiguous nucleoside sequence are complementary to a contiguous portion of any one of the sequences listed in Table 1, i.e., any one of SEQ ID NOs: 1-47.
• the first strand of the nucleic acid according to the invention comprises a contiguous nucleoside sequence of 23 nucleosides, wherein at least 18, 19, 20, 21, 22 or all 23 nucleosides of said contiguous nucleoside sequence are complementary to a contiguous portion of any one of the sequences listed in Table 1, i.e., any one of SEQ ID NOs: 1-47.
• a nucleic acid e.g. an siRNA of the invention includes a sense strand that is substantially or partially complementary to an antisense oligonucleoside which, in turn, is complementary to a target gene sequence and comprises a contiguous nucleoside sequence.
• the nucleoside sequence of the sense strand is typically at least about 80% complementary over its entire length to the equivalent region of the nucleoside sequence of the antisense strand, such as about 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary, or 100% complementary.
• a nucleic acid e.g.
• an siRNA of the invention includes an antisense strand that is substantially or partially complementary to the target sequence and comprises a contiguous nucleoside sequence which is at least 80% complementary over its entire length to the target sequence such as about 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary, or 100% complementary.
• a "subject” is an animal, such as a mammal, including a primate (such as a human, a non- human primate, e.g., a monkey, and a chimpanzee), or a non-primate or a bird that expresses the target gene, either endogenously or heterologously, when the target gene sequence has sufficient complementarity to the nucleic acid e.g. siRNA agent to promote target knockdown.
• the subject is a human.
• the terms "treating” or “treatment” refer to a beneficial or desired result including, but not limited to, alleviation or amelioration of one or more symptoms associated with gene expression.
• Treatment can also mean prolonging survival as compared to expected survival in the absence of treatment.
• the terms “prevent” or “prevention” as used herein are defined as eliminating or reducing the likelihood of occurrence of one or more symptoms of a disease or disorder.
• the inhibitor disclosed herein can be used to prevent the occurrence of a disease related to a disorder of haemostasis, such as haemophilia.
• the inhibitor disclosed herein can be used to prevent the occurrence of Von Willebrand disease.
• the inhibitor disclosed herein can be used to prevent the occurrence of Factor X Deficiency.
• “Therapeutically effective amount,” as used herein, is intended to include the amount of a nucleic acid e.g.
• siRNA that, when administered to a patient for treating a subject having disease, is sufficient to effect treatment of the disease (e.g., by diminishing, ameliorating or maintaining the existing disease or one or more symptoms of disease or its related comorbidities).
• pharmaceutically acceptable is employed herein to refer to compounds, materials, compositions, or dosage forms which are suitable for use in contact with the tissues of human subjects and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
• pharmaceutically-acceptable carrier means a pharmaceutically- acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
• Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject being treated.
• a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also intended to be part of this invention.
• the articles “a” and “an” are used herein to refer to one or to more than one (i.e.
• sense strand or antisense strand is understood as “sense strand or antisense strand or sense strand and antisense strand.”
• the term “about” is used herein to mean within the typical ranges of tolerances in the art. For example, “about” can be understood as about 2 standard deviations from the mean. In certain embodiments, about means +10%. In certain embodiments, about means +5%.
• nucleosides in a nucleic acid molecule must be an integer.
• at least 18 nucleosides of a 21 nucleoside nucleic acid molecule means that 18, 19, 20, or 21 nucleosides have the indicated property.
• nucleobase sequence is the sequence of the bases of the nucleic acid in an oligomer.
• Positions of the first and second strands, such as ‘position 1’ are counted from the 5’ end of the relevant strand, excluding any inverted abasic nucleosides.
• Various embodiments of the invention can be combined as determined appropriate by one of skill in the art.
• Abasic Nucleosides In certain embodiments, there are 1, e.g.2, e.g.3, e.g.4 or more abasic nucleosides present in nucleic acids according to the present invention.
• Abasic nucleosides are modified nucleosides because they lack the base normally seen at position 1 of the sugar moiety.
• the abasic nucleosides are in the terminal region of the second strand, preferably located within the terminal 5 nucleosides of the end of the strand.
• the terminal region may be the terminal 5 nucleosides, which includes abasic nucleosides.
• the second strand may comprise, as preferred features (which are all specifically contemplated in combination unless mutually exclusive): 2, or more than 2, abasic nucleosides in a terminal region of the second strand; and / or 2, or more than 2, abasic nucleosides in either the 5’ or 3’ terminal region of the second strand; and / or 2, or more than 2, abasic nucleosides in either the 5’ or 3’ terminal region of the second strand, wherein the abasic nucleosides are present in an overhang as herein described; and/or 2, or more than 2, consecutive abasic nucleosides in a terminal region of the second strand, wherein preferably one such abasic nucleoside is a terminal nucleoside; and / or 2, or more than 2, consecutive abasic nucleosides in either the 5’ or 3’ terminal region of the second strand, wherein preferably one such abasic nucleoside is a terminal nucleoside in either
• abasic nucleoside at the terminus of the second strand.
• the terminal 1 or terminal 2 or terminal 3 or terminal 4 nucleosides may be abasic nucleosides.
• An abasic nucleoside may also be linked to an adjacent nucleoside through a 5’-3’ phosphodiester linkage or reversed linkage unless there is only 1 abasic nucleoside at the terminus, in which case it will have a reversed linkage to the adjacent nucleoside.
• a reversed linkage (which may also be referred to as an inverted linkage, which is also seen in the art), comprises either a 5’-5’, a 3’3’, a 3’-2’ or a 2’-3’ phosphodiester linkage between the adjacent sugar moieties of the nucleosides.
• Abasic nucleosides which are not terminal will have 2 phosphodiester bonds, one with each adjacent nucleoside, and these may be a reversed linkage or may be a 5’-3 phosphodiester bond or may be one of each.
• a preferred embodiment comprises 2 abasic nucleosides at the terminal and penultimate positions of the second strand, and wherein the reversed internucleoside linkage is located between the penultimate (abasic) nucleoside and the antepenultimate nucleoside.
• abasic nucleosides at the terminal and penultimate positions of the second strand and the penultimate nucleoside is linked to the antepenultimate nucleoside through a reversed internucleoside linkage and is linked to the terminal nucleoside through a 5’-3’ or 3’-5’ phosphodiester linkage (reading in the direction of the terminus of the molecule).
• a nucleic acid according to the present invention comprises one or more abasic nucleosides, optionally wherein the one or more abasic nucleosides are in a terminal region of the second strand, and/or wherein at least one abasic nucleoside is linked to an adjacent basic nucleoside through a reversed internucleoside linkage.
• the second strand comprises 2 consecutive abasic nucleosides in the 5’ terminal region of the second strand, wherein one such abasic nucleoside is a terminal nucleoside at the 5’ terminal region of the second strand and the other abasic nucleoside is a penultimate nucleoside at the 5’ terminal region of the second strand, wherein: (a) said penultimate abasic nucleoside is connected to an adjacent first basic nucleoside in an adjacent 5’ near terminal region through a reversed internucleoside linkage; and (b) the reversed linkage is a 5-5’ reversed linkage; and (c) the linkage between the terminal and penultimate abasic nucleosides is 3’5’ when reading towards the terminus comprising the terminal and penultimate abasic nucleosides.
• the first strand and the second strand each has a length of 23 nucleosides;
• two phosphorothioate internucleoside linkages are respectively between three consecutive positions in said 5’ near terminal region of the second strand, wherein a first phosphorothioate internucleoside linkage is present between said adjacent first basic nucleoside of (a) and an adjacent second basic nucleoside in said 5’ near terminal region of the second strand, and a second phosphorothioate internucleoside linkage is present between said adjacent second basic nucleoside and an adjacent third basic nucleoside in said 5’ near terminal region of the second strand;
• two phosphorothioate internucleoside linkages are respectively between three consecutive positions in both 5’ and 3’ terminal regions of the first strand, whereby a terminal nucleoside respectively at each of the 5’ and 3’ terminal regions of said first strand is each attached to a respective 5’ and 3’ adjacent penul
• the second strand comprises 2 consecutive abasic nucleosides preferably in an overhang in the 3’ terminal region of the second strand, wherein one such abasic nucleoside is a terminal nucleoside at the 3’ terminal region of the second strand and the other abasic nucleoside is a penultimate nucleoside at the 3’ terminal region of the second strand, wherein: (a) said penultimate abasic nucleoside is connected to an adjacent first basic nucleoside in an adjacent 3’ near terminal region through a reversed internucleoside linkage; and (b) the reversed linkage is a 3-3’ reversed linkage; and (c) the linkage between the terminal and penultimate abasic nucleosides is 5’-3’ when reading towards the terminus comprising the terminal and penultimate abasic nucleosides.
• the first strand and the second strand each has a length of 23 nucleosides;
• two phosphorothioate internucleoside linkages are respectively between three consecutive positions in said 3’ near terminal region of the second strand, wherein a first phosphorothioate internucleoside linkage is present between said adjacent first basic nucleoside of (a) and an adjacent second basic nucleoside in said 3’ near terminal region of the second strand, and a second phosphorothioate internucleoside linkage is present between said adjacent second basic nucleoside and an adjacent third basic nucleoside in said 3’ near terminal region of the second strand;
• two phosphorothioate internucleoside linkages are respectively between three consecutive positions in both 5’ and 3’ terminal regions of the first strand, whereby a terminal nucleoside respectively at each of the 5’ and 3’ terminal regions of said first strand is each attached to a respective 5’ and 3’ adjacent penul
• RNA nucleosides shown are not limiting and could be any RNA nucleoside
• 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
• abasic nucleoside or abasic nucleosides present in the nucleic acid are provided in the presence of a reversed internucleoside linkage or linkages, namely a 5’-5’ or a 3’-3’ reversed internucleoside linkage.
• a reversed linkage occurs as a result of a change of orientation of an adjacent nucleoside 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 nucleoside sugars).
• the abasic nucleoside or nucleosides as present in the nucleic acids of the invention preferably include such inverted nucleoside sugars. In the case of a terminal nucleoside having an inverted orientation, then this will result in an “inverted” end configuration for the overall nucleic acid.
• the proximal 3’-3’ or 5’-5’ reversed linkage as herein described may comprise the reversed linkage being directly adjacent / attached to a terminal nucleoside having an inverted orientation, such as a single terminal nucleoside 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, nucleosides having an inverted orientation, such as 2, or more than 2, terminal region nucleosides having an inverted orientation, such as the terminal and penultimate nucleosides. In this way, the reversed linkage may be attached to a penultimate nucleoside having an inverted orientation.
• nucleic acid molecules having overall 3’ - 3’ or 5’- 5’ end structures as described herein
• the overall nucleic acid may have 3’ - 5’ end structures corresponding to the conventionally positioned 5’ / 3’ ends.
• the nucleic acid may have a 3’-3’ reversed linkage, and the terminal sugar moiety may comprise a 5’ OH rather than a 5’ phosphate group at the 5’ position of that terminal sugar.
• a reversed internucleoside linkage and / or one or more nucleosides having an inverted orientation creating an inverted end and where the relative position of a linkage (e.g., to a linker) or the location of an internal feature (such as a modified nucleoside) is defined relative to the 5’ or 3’ end of the nucleic acid, then the 5’ or 3’ end is the conventional 5’ or 3’ end which would have existed had a reversed linkage not been in place, and wherein the conventional 5’ or 3’ end is determined by consideration of the directionality of the majority of the internal nucleoside linkages and / or nucleoside orientation within the nucleic acid.
• the majority of the molecule will comprise conventional internucleoside linkages that run from the 3’ OH of the sugar to the 5’ phosphate of the next sugar, when reading in the standard 5’ [PO 4 ] to 3’ [OH] direction of a nucleic acid molecule (with reference to the numbering of ring atoms on the nucleoside sugars), which can be used to determine the conventional 5’ and 3’ ends that would be found absent the inverted end configuration.
• the second (sense) strand of the nucleic acid according to the invention comprises 2 consecutive abasic nucleosides in the 5’ terminal region as shown in the following 5’ terminal motif wherein: B represents a nucleoside base, T represent H, OH or a 2’ ribose modification, Z represents the remaining nucleosides of said second strand.
• the second (sense) strand of the nucleic acid according to the invention comprises 2 consecutive abasic nucleosides in the 5’ terminal region as shown in the following 5’ terminal motif
• B represents a nucleoside base
• T represents H, OH or a 2’ ribose modification (preferably a 2’ ribose modification, more preferably a 2’Me or 2’F ribose modification)
• V represents O or S (preferably O)
• R represents H or C 1-4 alkyl (preferably H)
• Z represents the remaining nucleosides of said second strand, more preferably the following 5’ terminal motif
• B represents a nucleoside base
• T represents a 2’ ribose modification (preferably a 2’Me or 2’F ribose modification)
• Z represents the remaining nucleosides of said second strand.
• the reversed bond is preferably located at the end of the nucleic acid e.g., RNA which is distal to a ligand moiety, such as a GalNAc containing portion, of the molecule.
• GalNAc-siRNA constructs with a 5’-GalNAc on the sense strand can have a reversed linkage on the opposite end of the sense strand.
• 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 second (sense) strand of the nucleic acid according to the invention comprises 2 consecutive abasic nucleosides in the 5’ terminal region as shown in the following 5’ terminal motif
• B represents a nucleoside base
• T represent H, OH or a 2’ ribose modification (preferably a 2’ ribose modification, more preferably a 2’Me or 2’F ribose modification)
• V represent O or S (preferably O)
• R represent H or C 1-4 alkyl (preferably H)
• Z comprises 11 to 26 contiguous nucleosides, preferably 15 to 21 contiguous nucleosides, and more preferably 19 contiguous nucleosides, more preferably the following 5’ terminal motif
• B represents a nucleoside base
• T represents a 2’ ribose modification (preferably a 2’Me or 2’F ribose modification)
• Z comprises 19 contiguous nucleosides.
• Nucleic Acid Lengths In one aspect the i) the first strand of the nucleic acid has a length in the range of 17 to 30 nucleosides, preferably 19 to 25 nucleosides, more preferably 19 or 23 nucleosides; and / or ii) the second strand of the nucleic acid has a length in the range of 17 to 30 nucleosides, preferably 19 to 25 nucleosides, more preferably 19 or 21 nucleosides.
• the duplex region of the nucleic acid is between 17 and 30 nucleosides in length, more preferably is 19 or 21 nucleosides in length.
• the region of complementarity between the first strand and the portion of RNA transcribed from the ZPI gene is between 17 and 30 nucleosides in length.
• RNA of the invention e.g., a dsiRNA
• RNA of the invention is further chemically modified to enhance stability or other beneficial characteristics.
• substantially all of the nucleosides are modified.
• the nucleic acids featured in the invention can be synthesized or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry,” Beaucage, S.L. et al. (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 nucleosides within an RNA, or RNA nucleosides within a DNA, inverted linkages, etc.); base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, conjugated bases; sugar modifications (e.g., 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 nucleosides within an RNA, or RNA nucleosides within a DNA, inverted linkages, etc.
• base modifications e.g., replacement with stabilizing bases, destabilizing bases, or
• nucleic acids such as siRNA compounds useful in the embodiments described herein include, but are not limited to RNAs containing modified backbones or no natural internucleoside linkages.
• Nucleic acids such as RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone.
• modified nucleic acids e.g., RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.
• a modified nucleic acid e.g., an siRNA will have a phosphorus atom in its internucleoside backbone.
• Modified nucleic acid e.g. RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5'-linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 5'-3' or 5'-2'.
• Modified nucleic acids e.g. RNAs can also contain one or more substituted sugar moieties.
• the nucleic acids e.g. siRNAs, e.g., dsiRNAs, featured herein can include one of the following at the 2'-position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted. 2’ O- methyl and 2’ -F are preferred modifications.
• the nucleic acid comprises at least one modified nucleoside.
• the nucleic acid of the invention may comprise one or more modified nucleosides on the first strand and/or the second strand.
• substantially all of the nucleosides of the sense strand and all of the nucleosides of the antisense strand comprise a modification.
• all of the nucleosides of the sense strand and substantially all of the nucleosides of the antisense strand comprise a modification.
• all of the nucleosides of the sense strand and all of the nucleosides of the antisense strand comprise a modification.
• At least one of the modified nucleosides is selected from the group consisting of a deoxy- nucleoside, a 3 '-terminal deoxy-thymine (dT) nucleoside, a 2'-O-methyl modified nucleoside (also called herein 2’-Me, where Me is a methoxy) , a 2'-fluoro modified nucleoside, a 2'-deoxy- modified nucleoside, a locked nucleoside, an unlocked nucleoside, a conformationally restricted nucleoside, a constrained ethyl nucleoside, an abasic nucleoside, a 2' -amino- modified nucleoside, a 2'- O-allyl- modified nucleoside, 2' -C-alkyl- modified nucleoside, 2'-hydroxly-modified nucleoside, a 2'- methoxyethyl modified nucleoside, a 2'-O-
• the modified nucleosides comprise a short sequence of 3 '-terminal deoxy-thymine nucleosides (dT).
• Modifications on the nucleosides may preferably be selected from the group including, but not limited to, LNA, HNA, CeNA, 2 -methoxyethyl, 2'-O-alkyl, 2 -O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'- hydroxyl, and combinations thereof.
• the modifications on the nucleosides are 2-O-methyl (“2-Me”) or 2'-fluoro modifications.
• the nucleic acid e.g., siRNA agent further comprises at least one phosphorothioate or methylphosphonate internucleoside linkage.
• the phosphorothioate or methylphosphonate internucleoside linkage can be at the 3 '-terminus or in the terminal region of one strand, i.e., the sense strand or the antisense strand; or at the ends of both strands, the sense strand and the antisense strand.
• the phosphorothioate or methylphosphonate internucleoside linkage is at the 5 'terminus or in the terminal region of one strand, i.e., the sense strand or the antisense strand; or at the ends of both strands, the sense strand and the antisense strand.
• a phosphorothioate or a methylphosphonate internucleoside linkage is at both the 5'- and 3 '-terminus or in the terminal region of one strand, i.e., the sense strand or the antisense strand; or at the ends of both strands, the sense strand and the antisense strand.
• Any nucleic acid may comprise one or more phosphorothioate (PS) modifications within the nucleic acid, such as at least two PS internucleoside bonds at the ends of a strand.
• PS phosphorothioate
• At least one of the oligoribonucleoside strands preferably comprises at least two consecutive phosphorothioate modifications in the last 3 nucleosides of the oligonucleoside.
• the invention therefore also relates to: A nucleic acid disclosed herein which comprises phosphorothioate internucleoside linkages respectively between at least two or three consecutive positions, such as in a 5’ and/or 3’ terminal region and/or near terminal region of the second strand, whereby said near terminal region is preferably adjacent said terminal region wherein said one or more abasic nucleosides of said second strand is / are located.
• a nucleic acid disclosed herein which comprises phosphorothioate internucleoside linkages respectively between at least two or three consecutive positions in a 5’ and / or 3’ terminal region of the first strand, whereby preferably the terminal position at the 5’ and / or 3’ terminal region of said first strand is attached to its adjacent position by a phosphorothioate internucleoside linkage.
• the nucleic acid strand may be an RNA comprising a phosphorothioate internucleoside linkage between the three nucleosides contiguous with 2 terminally located abasic nucleosides.
• a preferred nucleic acid is a double stranded RNA comprising 2 adjacent abasic nucleosides at the 5’ terminus of the second strand and a ligand moiety comprising one or more GalNAc ligand moieties at the opposite 3’ end of the second strand. Further preferred, the same nucleic acid may also comprise a phosphorothioate bond between nucelotides at positions 3-4 and 4-5 of the second strand, reading from the position 1 of the second strand.
• Position 1 of the first or the second strand is the nucleoside which is the closest to the end of the nucleic acid (ignoring any abasic nucleosides) and that is joined to an adjacent nucleoside (at Position 2) via a 3’ to 5’ internal bond, with reference to the bonds between the sugar moieties of the backbone, and reading in a direction away from that end of the molecule. It can therefore be seen that “position 1 of the sense strand” is the 5’ most nucleoside (not including abasic nucleosides) at the conventional 5’ end of the sense strand.
• the nucleoside at this position 1 of the sense strand will be equivalent to the 5’ nucleoside of the selected target nucleic acid sequence, and more generally the sense strand will have equivalent nucleosides to those of the target nucleic acid sequence starting from this position 1 of the sense strand, whilst also allowing for acceptable mismatches between the sequences.
• “position 1 of the antisense strand” is the 5’ most nucleoside (not including abasic nucleosides) at the conventional 5’ end of the antisense strand.
• the sequences according to the present invention have the following modification pattern: Modification 13: First strand modification: NmsNfsNmNmNmNfNmNfNfNmNmNmNmNmNfNmNmNmNfNmNmNmNfNmsNmsNm (5’ to 3’) Second strand modification: iaiaNmsNmsNmNmNmNmNmNmNmNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmN
• the 5’VP modification is preferably on the antisense strand, but can be on the sense strand as well, or instead.
• the 5’VP modification is a 5’-(E)-vinylphosphonate (5’-(E)-VP) modification.
• a 5'-VP modification is a stable phosphate mimic added at the 5' end of an oligonucleoside. It is a modification in which the 5' carbon forms a double bond to a 6' carbon linked to the phosphorus.
• Haraszti et al 2017 Haraszti et al., 5 ⁇ -Vinylphosphonate improves tissue accumulation and efficacy of conjugated siRNAs in vivo.
• RNA e.g., an siRNA of the invention involves linking the nucleic acid e.g., the siRNA to one or more ligand moieties e.g. to enhance the activity, cellular distribution, or cellular uptake of the nucleic acid e.g. siRNA e.g. into a cell.
• the inhibitor according to the invention is conjugated to a ligand moiety that enables and/or facilitates targeting of hepatocytes.
• the inhibitor according to the invention is an siRNA-GalNAc conjugate.
• the ligand moiety described can be attached to a nucleic acid e.g., an siRNA oligonucleoside, via a linker that can be cleavable or non-cleavable.
• linker or “linking group” means an organic moiety that connects two parts of a compound, e.g., covalently attaches two parts of a compound.
• the ligand can be attached to the 3' or 5’ end of the sense strand.
• the ligand is preferably conjugated to 3’ end of the sense strand of the nucleic acid e.g., an siRNA agent.
• the invention therefore relates in a further aspect to a conjugate for inhibiting expression of a target gene in a cell, said conjugate comprising a nucleic acid portion and one or more ligand moieties, said nucleic acid portion comprising a nucleic acid as disclosed herein.
• the second strand of the nucleic acid is conjugated directly or indirectly (e.g., via a linker) to the one or more ligand moiety(s), wherein said ligand moiety is typically present at a terminal region of the second strand, preferably at the 3’ terminal region thereof.
• the ligand moiety comprises a GalNAc or GalNAc derivative attached to the nucleic acid e.g., dsiRNA through a linker.
• the invention relates to a conjugate wherein the ligand moiety comprises: i) one or more GalNAc ligands; and/or ii) one or more GalNAc ligand derivatives; and/or iii) one or more GalNAc ligands conjugated to said nucleic acid through a linker.
• Said GalNAc ligand may be conjugated directly or indirectly to the 5’ or 3’ terminal region of the second strand of the nucleic acid, preferably at the 3’ terminal region thereof.
• GalNAc ligands are well known in the art and described in, inter alia, EP3775207A1.
• the GalNAc ligand is comprised in any one of the linkers shown in Figures 1 to 4 or Figure 5 (Formula XI), wherein the "oligonucleotide” may be any nucleic acid disclosed herein. Accordingly, the "oligonucleotide” may comprise other bonds than a phosphodiester bond, such as one or more phosphorothioate bonds.
• the nucleic acid according to the invention is a double stranded oligonucleoside as defined herein and the linker is conjugated to the second strand, more preferably to the 3' terminal region of the second strand, via a phosphodiester bond.
• the GalNAc ligand is comprised in the linker shown in Figure 3, wherein the "oligonucleotide” may be any nucleic acid disclosed herein. Accordingly, the "oligonucleotide” may comprise other bonds than a phosphodiester bond, such as one or more phosphorothioate bonds.
• the nucleic acid according to the invention is a double stranded oligonucleoside as defined herein and the linker is conjugated to the second strand, more preferably to the 3' terminal region of the second strand, via a phosphodiester bond.
• the GalNAc ligand is comprised in the linker shown in Figure 5 (Formula XI), wherein the "oligonucleotide” may be any nucleic acid disclosed herein. Accordingly, the "oligonucleotide” may comprise other bonds than a phosphodiester bond, such as one or more phosphorothioate bonds.
• the nucleic acid according to the invention is a double stranded oligonucleoside as defined herein and the linker is conjugated to the second strand, more preferably to the 3' terminal region of the second strand, via a phosphodiester bond.
• the GalNAc ligand is comprised in any one of the linkers shown in Figures 1 to 4 or Figure 5 (Formula XI), wherein the "oligonucleotide" represents a nucleic acid according to the invention, wherein the nucleic acid according to the invention comprises a modified or unmodified second strand comprising or consisting of any one of SEQ ID NO:95 to SEQ ID NO:141, preferably wherein the linker is conjugated to the 3' terminal region of the second strand, i.e., to the 3' terminal region of any one of SEQ ID NO:95 to SEQ ID NO:141, via a phosphodiester bond.
• the linker is conjugated to the 3' terminal region of the second strand, i.e., to the 3' terminal region of any one of SEQ ID NO:95 to SEQ ID NO:141, via a phosphodiester bond.
• the GalNAc ligand is comprised in the linker shown in Figure 3, wherein the "oligonucleotide” represents a nucleic acid according to the invention, wherein the nucleic acid according to the invention comprises a modified or unmodified second strand comprising or consisting of any one of SEQ ID NO:95 to SEQ ID NO:141, preferably wherein the linker is conjugated to the 3' terminal region of the second strand, i.e., to the 3' terminal region of any one of SEQ ID NO:95 to SEQ ID NO:141, via a phosphodiester bond.
• the GalNAc ligand is comprised in the linker shown in Figure 5 (Formula XI), wherein the "oligonucleotide” represents a nucleic acid according to the invention, wherein the nucleic acid according to the invention comprises a modified or unmodified second strand comprising or consisting of any one of SEQ ID NO:95 to SEQ ID NO:141, preferably wherein the linker is conjugated to the 3' terminal region of the second strand, i.e., to the 3' terminal region of any one SEQ ID NO:95 to SEQ ID NO:141, via a phosphodiester bond.
• the GalNAc ligand is comprised in any one of the linkers shown in Figures 1 to 4 or Figure 5 (Formula XI), wherein the "oligonucleotide" represents a nucleic acid according to the invention, wherein the nucleic acid according to the invention comprises a modified second strand comprising or consisting of any one of SEQ ID NO:189 to SEQ ID NO:235, preferably wherein the linker is conjugated to the 3' terminal region of the second strand, i.e., to the 3' terminal region of any one of SEQ ID NO:189 to SEQ ID NO:235, via a phosphodiester bond.
• the GalNAc ligand is comprised in the linker shown in Figure 3, wherein the "oligonucleotide” represents a nucleic acid according to the invention, wherein the nucleic acid according to the invention comprises a modified second strand comprising or consisting of any one of SEQ ID NO:189 to SEQ ID NO:235, preferably wherein the linker is conjugated to the 3' terminal region of the second strand, i.e., to the 3' terminal region of any one of SEQ ID NO:189 to SEQ ID NO:235, via a phosphodiester bond.
• the GalNAc ligand is comprised in the linker shown in Figure 5 (Formula XI), wherein the "oligonucleotide” represents a nucleic acid according to the invention, wherein the nucleic acid according to the invention comprises a modified second strand comprising or consisting of any one of SEQ ID NO:189 to SEQ ID NO:235, preferably wherein the linker is conjugated to the 3' terminal region of the second strand, i.e., to the 3' terminal region of any one of SEQ ID NO:189 to SEQ ID NO:235, via a phosphodiester bond.
• the GalNAc ligand is comprised in the linker shown in Figures 1 to 4 or Figure 5 (Formula XI), wherein the "oligonucleotide” represents a nucleic acid according to the invention, wherein the nucleic acid according to the invention comprises a modified second strand comprising or consisting of any one of SEQ ID NO:189 to SEQ ID NO:235, preferably SEQ ID NO: 195 or SEQ ID NO: 192, wherein the second strand has the following structure
• T represents a 2’Me ribose modification
• B represents the nucleoside bases of the first two basic nucleosides in the 5’ terminal region of any one of SEQ ID NO:189 to SEQ ID NO:235, preferably SEQ ID NO: 195 or SEQ ID NO: 192
• Z represents the remaining 19 contiguous basic nucleosides of any one of SEQ ID NO:189 to SEQ ID NO:235, preferably SEQ ID NO: 195 or SEQ ID NO: 192.
• the GalNAc ligand is comprised in the linker shown in Figure 5 (Formula XI), wherein the "oligonucleotide” represents a nucleic acid according to the invention, wherein the nucleic acid according to the invention comprises a modified second strand comprising or consisting of any one of SEQ ID NO:189 to SEQ ID NO:235, preferably SEQ ID NO: 195 or SEQ ID NO: 192, wherein the second strand has the following structure
• T represents a 2’Me ribose modification
• B represents the nucleoside bases of the first two basic nucleosides in the 5' terminal region of any one of SEQ ID NO:189 to SEQ ID NO:235
• Z represents the remaining 19 contiguous basic nucleosides of any one of SEQ ID NO:189 to SEQ ID NO:235.
• the GalNAc ligand is comprised in the linker shown in Figure 3, wherein the "oligonucleotide” represents a nucleic acid according to the invention, wherein the nucleic acid according to the invention comprises a modified second strand comprising or consisting of any one of SEQ ID NO:189 to SEQ ID NO:235, preferably SEQ ID NO: 195 or SEQ ID NO: 192, wherein the second strand has the following structure
• T represents a 2’Me ribose modification
• B represents the nucleoside bases of the first two basic nucleosides in the 5' terminal region of any one of SEQ ID NO:189 to SEQ ID NO:235, preferably SEQ ID NO: 195 or SEQ ID NO: 192
• Z represents the remaining 19 contiguous basic nucleosides of any one of SEQ ID NO:189 to SEQ ID NO:235, preferably SEQ ID NO: 195 or SEQ ID NO: 192.
• Vector And Cell provides a cell containing a nucleic acid, such as inhibitory RNA [RNAi] as described herein.
• the invention provides a cell comprising a vector as described herein.
• the invention provides a vector comprising an oligonucleoside inhibitor, e.g.an iRNA e.g. siRNA.
• an oligonucleoside inhibitor e.g.an iRNA e.g. siRNA.
• Pharmaceutically Acceptable Compositions in one aspect, 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.
• materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laur
• Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc).
• binding agents e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropy
• compositions of the present invention can also be used to formulate the compositions of the present invention.
• suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.
• Formulations for topical administration of nucleic acids can include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases.
• the solutions can also contain buffers, diluents and other suitable additives.
• Pharmaceutically acceptable organic or inorganic excipients suitable for non- parenteral administration which do not deleteriously react with nucleic acids can be used.
• the nucleic acid or composition is administered in an unbuffered solution.
• the unbuffered solution is saline or water.
• the nucleic acid e.g. siRNA agent is administered in a buffered solution.
• the buffer solution can comprise acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof.
• the buffer solution can be phosphate buffered saline (PBS).
• compositions of the invention may be administered in dosages sufficient to inhibit expression of a gene.
• a suitable dose of a nucleic acid e.g. an siRNA of the invention will be in the range of about 0.001 to about 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of about 1 to 50 mg per kilogram body weight per day.
• a suitable dose of a nucleic acid e.g. an siRNA of the invention will be in the range of about 0.1 mg/kg to about 5.0 mg/kg, e.g., about 0.3 mg/kg and about 3.0 mg/kg.
• a repeat-dose regimen may include administration of a therapeutic amount of a nucleic acid e.g.
• the nucleic acid e.g. siRNA on a regular basis, such as every other day or once a year.
• the nucleic acid e.g. siRNA is administered about once per month to about once per quarter (i.e., about once every three months).
• the nucleic acid e.g. siRNA agent is administered at a dose of about 0.01 mg/kg to about 10 mg/kg or about 0.5 mg/kg to about 50 mg/kg.
• the nucleic acid e.g. siRNA agent is administered at a dose of about 10 mg/kg to about 30 mg/kg.
• siRNA agent is administered at a dose selected from about 0.5 mg/kg 1 mg/kg, 1.5 mg/kg, 3 mg/kg, 5 mg/kg, 10 mg/kg, and 30 mg/kg.
• the nucleic acid e.g. agent is administered about once per week, once per month, once every other two months, or once a quarter (i.e., once every three months) at a dose of about 0.1 mg/kg to about 5.0 mg/kg.
• the nucleic acid e.g. siRNA agent is administered to the subject once a week.
• the nucleic acid e.g. siRNA agent is administered to the subject once a month.
• siRNA agent is administered once per quarter (i.e., every three months). After an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after administration weekly or biweekly for three months, administration can be repeated once per month, for six months, or a year; or longer.
• the pharmaceutical composition can be administered once daily, or administered as two, three, or more sub-doses at appropriate intervals throughout the day or even using continuous infusion or delivery through a controlled release formulation. In that case, the nucleic acid e.g. siRNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage.
• the dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the nucleic acid e.g. siRNA over a several day period.
• Sustained release formulations are well known in the art and are particularly useful for delivery of agents at a particular site, such as could be used with the agents of the present invention.
• the dosage unit contains a corresponding multiple of the daily dose.
• a single dose of the pharmaceutical compositions can be long lasting, such that subsequent doses are administered at not more than 3, 4, or 5 day intervals, or at not more than 1, 2, 3, or 4 week intervals.
• a single dose of the pharmaceutical compositions of the invention is administered once per week.
• a single dose of the pharmaceutical compositions of the invention is administered bimonthly.
• the siRNA is administered about once per month to about once per quarter (i.e., about once every three months), or even every 6 months or 12 months.
• Estimates of effective dosages and in vivo half-lives for the individual nucleic acid e.g. siRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as known in the art.
• the pharmaceutical compositions of the present invention can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated.
• Administration can be topical (e.g., by a transdermal patch), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral.
• Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subdermal, e.g. via an implanted device; or intracranial, e.g., by intraparenchymal, intrathecal or intraventricular administration.
• the compositions are administered by intravenous infusion or injection.
• the compositions are administered by subcutaneous injection.
• the nucleic acid e.g. agent is administered to the subject subcutaneously.
• the nucleic acid e.g. siRNA can be delivered in a manner to target a particular tissue (e.g. in particular liver cells).
• Methods For Inhibiting ZPI Gene Expression The present invention also provides methods of inhibiting expression of ZPI gene in a cell. The methods include contacting a cell with a nucleic acid of the invention e.g. siRNA agent, such as double stranded siRNA agent, in an amount effective to inhibit expression of the ZPI gene in the cell, thereby inhibiting expression of the ZPI gene in the cell.
• a nucleic acid “for inhibiting the expression of ZPI” is a nucleic acid that is capable of inhibiting ZPI expression, preferably as described herein below.
• Contacting of a cell with the nucleic acid e.g. an siRNA, such as a double stranded siRNA agent may be done in vitro or in vivo.
• Contacting a cell in vivo with nucleic acid e.g. includes contacting a cell or group of cells within a subject, e.g., a human subject, with the nucleic acid e.g. siRNA. Combinations of in vitro and in vivo methods of contacting a cell are also possible.
• Contacting a cell may be direct or indirect, as discussed above.
• contacting a cell may be accomplished via a targeting ligand moiety, including any ligand moiety described herein or known in the art.
• the targeting ligand moiety is a carbohydrate moiety, e.g. a GalNAc3 ligand, or any other ligand moiety that directs the siRNA agent to a site of interest.
• the term "inhibiting,” as used herein, is used interchangeably with “reducing,” “silencing,” “downregulating”, “suppressing”, and other similar terms, and includes any level of inhibition.
• expression of ZPI 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, preferably when determined by qPCR as described herein and/or when the siRNA is introduced into the target cell by transfection.
• the methods include a clinically relevant inhibition of expression of ZPI 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.
• the nucleic acid of the invention when transfected into the cells, inhibits expression of the ZPI gene with an IC50 value lower than 2500 pM, 2400 pM, 2300 pM, 2200 pM, 2100 pM, 2000 pM, 1900 pM, 1800 pM, 1700 pM, 1600 pM, 1500 pM, 1400 pM, 1300 pM, 1200 pM, 1100 pM, 1000 pM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM or 100 pM, preferably when determined by qPCR, more preferably by reverse transcriptase (RT)-qPCR, as described herein.
• RT reverse transcriptase
• the nucleic acid of the invention when transfected into the cells, inhibits expression of the ZPI gene with an IC50 value lower than 2500 pM. In a more preferred embodiment, when transfected into the cells, the nucleic acid of the invention inhibits expression of the ZPI gene with an IC50 value lower than 1000 pM. In an even more preferred embodiment, when transfected into the cells, the nucleic acid of the invention inhibits expression of the ZPI gene with an IC50 value lower than 500 pM. In a most preferred embodiment, when transfected into the cells, the nucleic acid of the invention inhibits expression of the ZPI gene with an IC50 value lower than 100 pM.
• Huh7 cells human hepatocyte-derived cell line, obtained from JCRB Cell Bank
• DMEM Modified Eagle Medium
• siRNA duplexes targeting ZPI mRNA or a negative control siRNA (siRNA-control; sense strand 5’-UUCUCCGAACGUGUCACGUTT-3’ (SEQ ID NO:237), antisense strand 5’-ACGUGACACGUUCGGAGAATT-3’ (SEQ ID NO:238)) using 10x3-fold serial dilutions over a final duplex concentration range of 20 nM to 1 pM.
• Transfection may be carried out by adding 9.7 ⁇ L Opti-MEM (ThermoFisher) plus 0.3 ⁇ L Lipofectamine RNAiMAX (ThermoFisher) to 10 ⁇ L of each siRNA duplex.
• the mixture may be incubated at room temperature for 15 minutes before being added to 100 ⁇ L of complete growth medium containing 20,000 Huh7 cells. Cells may be incubated for 24 hours at 37 ⁇ C/5% CO 2 prior to total RNA purification using a RNeasy 96 Kit (Qiagen). Each duplex may be tested by transfection in duplicate wells in a single experiment. cDNA synthesis may be performed using FastQuant RT (with gDNase) Kit (Tiangen).
• Real-time quantitative PCR may be performed on an ABI Prism 7900HT or ABI QuantStudio 7 with primers specific for human ZPI (Hs01547819_m1) and human GAPDH (Hs02786624_g1) using a TaqMan Gene Expression Assay Kit (ThermoFisher Scientific).
• qPCR may be performed in duplicate on cDNA derived from each well and the mean cycle threshold (Ct) calculated.
• Relative ZPI expression may be calculated from mean Ct values using the comparative Ct ( ⁇ Ct) method, normalised to GAPDH and relative to untreated cells.
• Maximum percent inhibition of ZPI expression and IC50 values may be calculated using a four parameter (variable slope) model using GraphPad Prism 9.
• inhibition of expression of the ZPI gene may be characterized by a reduction of mean relative expression of the ZPI gene.
• the mean relative expression of ZPI is below 1, 0.9, 0.8, 0.7, 0.6, 0.5, or 0.4, preferably when determined by qPCR, more preferably by reverse transcriptase (RT)-qPCR, as described herein.
• the mean relative expression of ZPI is below 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2 or 0.1, preferably when determined by qPCR, more preferably by reverse transcriptase (RT)-qPCR, as described herein.
• Mean relative expression of the ZPI gene may be quantified by the following method: Huh7 cells (human hepatocyte-derived cell line, obtained from JCRB Cell Bank) may be maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS at 37 ⁇ C in at atmosphere of 5% CO 2 .
• DMEM Modified Eagle Medium
• Cells may be transfected with siRNA duplexes targeting ZPI mRNA or a negative control siRNA (siRNA-control; sense strand 5’-UUCUCCGAACGUGUCACGUTT-3’ (SEQ ID NO:237), antisense strand 5’-ACGUGACACGUUCGGAGAATT-3’(SEQ ID NO:238)) at a final duplex concentration of 3 nM.
• Transfection may be carried out by adding 9.7 ⁇ L Opti-MEM (ThermoFisher) plus 0.3 ⁇ L Lipofectamine RNAiMAX (ThermoFisher) to 10 ⁇ L of each siRNA duplex.
• the mixture may be incubated at room temperature for 15 minutes before being added to 100 ⁇ L of complete growth medium containing 20,000 Huh7 cells. Cells may be incubated for 24 hours at 37 ⁇ C/5% CO 2 prior to total RNA purification using a RNeasy 96 Kit (Qiagen). Each duplex may be tested by transfection in duplicate wells in two independent experiments. cDNA synthesis may be performed using FastQuant RT (with gDNase) Kit (Tiangen).
• Real-time quantitative PCR may be performed on an ABI Prism 7900HT or ABI QuantStudio 7 with primers specific for human ZPI (Hs01547819_m1) and human GAPDH (Hs02786624_g1) using FastStart Universal Probe Master Kit (Roche).
• qPCR may be performed in duplicate on cDNA derived from each well and the mean Ct calculated.
• Relative ZPI expression may be calculated from mean Ct values using the comparative Ct ( ⁇ Ct) method, normalised to GAPDH and relative to untreated cells. Inhibition of the expression of ZPI gene may be manifested by a reduction of the amount of mRNA of the target ZPI gene in comparison to a suitable control.
• inhibition of the expression of ZPI gene may be assessed in terms of a reduction of a parameter that is functionally linked to gene expression, e.g, protein expression or signaling pathways.
• Methods Of Treating Or Preventing Diseases Associated With ZPI Gene Expression The present invention also provides methods of using nucleic acid e.g. an siRNA of the invention or a composition containing nucleic acid e.g. an siRNA of the invention to reduce or inhibit ZPI gene expression in a cell or reduce expression or function of a target. The methods include contacting the cell with a nucleic acid e.g.
• the cell may be contacted in vitro or in vivo, i.e., the cell may be within a subject.
• a cell suitable for treatment using the methods of the invention may be any cell that expresses a gene of interest associated with a disease related to a disorder of haemostasis, such as haemophilia.
• a cell suitable for treatment using the methods of the invention may be any cell that expresses a gene of interest associated with Von Willebrand disease.
• a cell suitable for treatment using the methods of the invention may be any cell that expresses a gene of interest associated with Factor X Deficiency.
• the in vivo methods of the invention may include administering to a subject a composition containing a nucleic acid of the invention e.g. an siRNA, where the nucleic acid e.g. siRNA includes a nucleoside sequence that is complementary to at least a part of an RNA transcript of ZPI gene of the mammal to be treated.
• the present invention further provides methods of treatment of a subject in need thereof.
• the treatment methods of the invention include administering a nucleic acid such as an siRNA of the invention to a subject, e.g., a subject that would benefit from a reduction or inhibition of the expression of ZPI gene, in a therapeutically effective amount e.g. a nucleic acid such as an siRNA targeting ZPI or a pharmaceutical composition comprising the nucleic acid targeting ZPI.
• the disease to be treated is a disease related to a disorder of haemostasis, such as haemophilia.
• the disease related to a disorder of haemostasis is Von Willebrand disease.
• the disease related to a disorder of haemostasis is Factor X Deficiency.
• the nucleic acid according to the invention may be used in the prevention and/or treatment of a disease related to a disorder of haemostasis, such as haemophilia.
• haemophilia refers to a group of disease states broadly characterized by reduced blood clotting or coagulation. Haemophilia may refer to Type A, Type B, or Type C haemophilia, or to the composite of all three diseases types.
• the nucleic acid according to the invention may be used in the prevention and/or treatment of Von Willebrand disease.
• the patient to be treated may be a patient that already has a disease related to a disorder of haemostasis or that is at risk of developing a disease related to a disorder of haemostasis. That is, in certain embodiments, the nucleic acid of the present invention may be used in the treatment and/or management of an existing disease related to a disorder of haemostasis. Treatment and/or management of an existing disease related to a disorder of haemostasis with the nucleic acid of the present invention may prevent worsening of the disease related to a disorder of haemostasis and/or reverse the disease related to a disorder of haemostasis.
• treatment of an existing disease related to a disorder of haemostasis with the nucleic acid of the present invention may even cure the disease related to a disorder of haemostasis.
• the nucleic acid of the present invention may be used to prevent manifestation of a disease related to a disorder of haemostasis in a patient that is at risk of developing a disease related to a disorder of haemostasis.
• the skilled person is capable of diagnosing whether a patient has a disease related to a disorder of haemostasis or is at risk of developing a disease related to a disorder of haemostasis.
• diagnosing a disease related to a disorder of haemostasis may involve clotting factor tests as known in the art.
• Haemophilia, or hemophilia is a mostly inherited genetic disorder that impairs the body's ability to make blood clots, a process needed to stop bleeding. This results in subjects bleeding for a longer time after an injury, easy bruising, and an increased risk of bleeding inside joints or the brain.
• Subjects with a mild case of the disease may have symptoms only after an accident or during surgery. Bleeding into a joint, also referred to as haemarthrosis, can result in permanent damage while bleeding in the brain can result in long term headaches, seizures, or a decreased level of consciousness.
• haemophilia A which occurs due to low amounts of clotting factor VIII
• haemophilia B which occurs due to low levels of clotting factor IX. They are typically inherited from one's parents through an X chromosome carrying a nonfunctional gene. Rarely a new mutation may occur during early development or haemophilia may develop later in life due to antibodies forming against a clotting factor.
• haemophilia C which occurs due to low levels of factor XI
• Von Willebrand disease which occurs due to low levels of a substance called von Willebrand factor
• parahaemophilia which occurs due to low levels of factor V.
• the nucleic acid of the present invention is suitable for treatment, or for treatment of haemophilia A, B and/or C. In certain embodiments, the nucleic acid of the present invention is suitable for treatment, or for treatment of haemophilia A and/or B.
• the nucleic acid of the present invention is suitable for treatment, or for treatment of acquired haemophilia. In certain embodiments, the nucleic acid of the present invention is suitable for treatment, or for treatment of Willebrand disease. In certain embodiments, the nucleic acid of the present invention is suitable for treatment, or for treatment of parahaemophilia.
• treatment with the nucleic acid of the invention results in a boost of clotting factor levels such that bleeding can be reduced or prevented.
• treatment with the nucleic acid of the invention reduces or prevents bleeding episodes in a subject suffering from haemophilia.
• treatment with the nucleic acid of the invention reduces or prevents bleeding into a joint of a subject suffering from haemophilia. In certain embodiments, treatment with the nucleic acid of the invention reduces or prevents bleeding into a muscle or into the brain of a subject suffering from haemophilia.
• treatment of a subject, preferably a subject having a disorder of haemostasis, such as haemophilia, with the nucleic acid of the invention may result in one or more of more of the following:
• treatment of a subject, preferably a subject having a disorder of haemostasis, such as haemophilia, with the nucleic acid of the invention results in one or more of the following: reduced bone marrow hyperplasia, reduced osteoarthritis, reduced chondrocyte degeneration/necrosis, reduced haemorrhage, reduced haemosiderin deposition, reduced occurrence of haematoma, reduced osteoclastogenic bone resorption, reduced osteolysis, reduced periostitis, reduced sub-chondral bone sclerosis, reduced tendon degeneration, reduced tendonitis, and/or reduced tenosynovitis.
• the invention relates to a nucleic acid suitable for use, or for use, in treatment of haemophilia, wherein the treatment of haemophilia is characterized by reduced bleeding and one or more of: reduced bone marrow hyperplasia, reduced osteoarthritis, reduced chondrocyte degeneration/necrosis, reduced haemorrhage, reduced haemosiderin deposition, reduced haematoma, reduced osteoclastogenic bone resorption, reduced osteolysis, reduced periostitis, reduced sub-chondral bone sclerosis, reduced tendon degeneration, reduced tendonitis, and/or reduced tenosynovitis.
• reduced bone marrow hyperplasia reduced osteoarthritis
• reduced chondrocyte degeneration/necrosis reduced haemorrhage
• reduced haemosiderin deposition reduced haematoma
• reduced osteoclastogenic bone resorption reduced osteolysis
• reduced periostitis reduced sub-chondral bone sclerosis
• the disease or disorder can be Von Willebrand disease.
• a nucleic acid e.g. siRNA of the invention may be administered as a “free” nucleic acid or “free” siRNA, administered in the absence of a pharmaceutical composition.
• the naked nucleic acid may be in a suitable buffer solution.
• the buffer solution may comprise acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof.
• the buffer solution is phosphate buffered saline (PBS).
• PBS phosphate buffered saline
• the pH and osmolarity of the buffer solution can be adjusted such that it is suitable for administering to a subject.
• a nucleic acid e.g. siRNA of the invention may be administered as a pharmaceutical composition, such as a dsiRNA liposomal formulation.
• the method includes administering a composition featured herein such that expression of ZPI 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 ZPI target gene is decreased for an extended duration, e.g., at least about two, three, four days or more, e.g., about one week, two weeks, three weeks, or four weeks or longer, e.g., about 1 month, 2 months, or 3 months.
• Subjects can be administered a therapeutic amount of nucleic acid e.g.
• siRNA such as about 0.01 mg/kg to about 200 mg/kg, so as to prevent and/or treat a disease related to a disorder of haemostasis, such as haemophilia.
• the nucleic acid e.g. siRNA can be administered by intravenous infusion over a period of time, on a regular basis. In certain embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis.
• Administration of the siRNA can reduce gene product levels of ZPI 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.
• administration results in clinical stabilization or preferably clinically relevant reduction of at least one sign or symptom of a ZPI gene-associated disorder.
• the nucleic acid e.g. siRNA can be administered subcutaneously, i.e., by subcutaneous injection. One or more injections may be used to deliver the desired daily dose of nucleic acid e.g., siRNA to a subject.
• the injections may be repeated over a period of time.
• the administration may be repeated on a regular basis.
• the treatments can be administered on a less frequent basis.
• a repeat-dose regimen may include administration of a therapeutic amount of nucleic acid on a regular basis, such as every other day or to once a year.
• the nucleic acid is administered about once per month to about once per quarter (i.e., about once every three months).
• the present invention may be applied in the compounds, processes, compositions or uses of the following Sentences numbered 1-101 wherein reference to any Formula in the Sentences 1-101 refers only to those Formulas that are defined within Sentences 1-101. These formulae are reproduced in Figure 5.
• an oligonucleoside moiety as represented by Z in any of the following sentences can comprise a nucleic acid for inhibiting expression of ZPI as defined in any of the claims hereinafter.
• a compound according to Sentence 1, wherein R 1 is hydrogen at each occurrence.
• a compound according to Sentence 1, wherein R 1 is methyl.
• a compound according to Sentence 1, wherein R 1 is ethyl.
• a compound according to any of Sentences 1 to 4, wherein R 2 is hydroxy.
• a compound according to any of Sentences 1 to 4, wherein R 2 is halo.
• a compound according to Sentence 6, wherein R 2 is chloro.
• a compound according to Sentence 6, wherein R 2 is bromo.
• a compound according to Sentence 6, wherein R 2 is iodo.
• a compound according to Sentence 6, wherein R 2 is nitro. 12.
• said RNA compound comprises an RNA duplex comprising first and second strands, wherein the first strand is at least partially complementary to an RNA sequence of a target gene, and the second strand is at least partially complementary to said first strand, and wherein each of the first and second strands have 5’ and 3’ ends.
• the oligonucleoside 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.
• a composition comprising a compound of Formula (II) as defined in Sentence 27, and a compound of Formula (III) as defined in Sentence 28, optionally dependent on Sentence 29. 31.
• a compound according to Sentence 32 or 33 wherein the oligonucleoside 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.
• 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. 36.
• a composition according to Sentence 35 wherein said compound of Formula (V) as defined in Sentence 33 is present in an amount in the range of 10 to 15% by weight of said composition.
• 37 A compound as defined in any of Sentences 1 to 29, or 32 to 34, wherein the oligonucleoside 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.
• 38. A compound according to Sentence 37, wherein the modifications are chosen from 2’-O-methyl, 2’-deoxy-fluoro, and 2’-deoxy. 39.
• 40. A compound according to Sentence 39, wherein said one or more degradation protective moieties are not present at the end of the oligonucleoside strand that carries the ligand moieties, and / or wherein said one or more degradation protective moieties is selected from phosphorothioate internucleoside linkages, phosphorodithioate internucleoside linkages and inverted abasic nucleosides, wherein said inverted abasic nucleosides are present at the distal end of the strand that carries the ligand moieties.
• a compound according to Sentence 42, wherein said one or more carbohydrates can be a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide or polysaccharide. 44.
• a compound according to Sentence 43 wherein said one or more carbohydrates comprise one or more galactose moieties, one or more lactose moieties, one or more N-AcetylGalactosamine moieties, and / or one or more mannose moieties.
• said one or more carbohydrates comprise one or more N-Acetyl-Galactosamine moieties.
• a compound according to Sentence 45 which comprises two or three N-AcetylGalactosamine moieties.
• a compound according to Sentence 47 wherein said one or more ligands are attached as a biantennary or triantennary branched configuration.
• a compound according to Sentences 46 to 48, wherein said moiety: as depicted in Formula (I) in Sentence 1 is Formula (VII): Formula (VII) wherein: A I is hydrogen; a is an integer of 2 or 3. 51.
• a compound according to Sentence 49 or 50, wherein a 2.
• a compound according to Sentence 49 or 50, wherein a 3. 53.
• a compound according to Sentence 49, wherein b 3. 54.
• a compound of Formula (IX) A compound according to Sentence 54 or 55, wherein the oligonucleoside 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.
• 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 compound according to Sentence 59 or 60 wherein the oligonucleoside 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.
• a composition comprising a compound of Formula (X) as defined in Sentence 59, and a compound of Formula (XI) as defined in Sentence 60, optionally dependent on Sentence 61.
• 64. A compound as defined in any of Sentences 54 to 63, wherein the oligonucleoside 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.
• 65. A compound according to Sentence 64, wherein the modifications are chosen from 2’-O-methyl, 2’-deoxy-fluoro, and 2’-deoxy. 66.
• Formula (XIII) herein: R 1 at each occurrence is independently selected from the group consisting of hydrogen, methyl and ethyl; R 2 is selected from the group consisting of hydrogen, hydroxy, -OC 1-3 alkyl, -C( O)OC 1-3 alkyl, halo and nitro;
• X 1 and X 2 at each occurrence are independently selected from the group consisting of methylene, oxygen and sulfur;
• m is an integer of from 1 to 6;
• n is an integer of from 1 to 10;
• q, r, s, t, v are independently integers from 0 to 4, with the proviso
• Formula (XV) R 1 at each occurrence is independently selected from the group consisting of hydrogen, methyl and ethyl;
• X 1 and X 2 at each occurrence are independently selected from the group consisting of methylene, oxygen and sulfur;
• q, r, s, t, v are independently integers from 0 to 4, with the proviso that: (i) q and r cannot both be 0 at the same time; and (ii) s, t and v cannot all be 0 at the same time;
• Z is an oligonucleoside moiety.
• a compound of Formula (XIVa) Formula (XIVa)
• a compound of Formula (XIVb) Formula (XIVb) 87.
• a compound of Formula (XV) Formula (XV) wherein: R 1 at each occurrence is independently selected from the group consisting of hydrogen, methyl and ethyl; X 1 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 oligonucleoside moiety. 88.
• a compound of Formula (XVa) Formula (XVa)
• a compound of Formula (XVb) Formula (XVb) 90.
• Sentence 88 for the preparation of a compound according to any of Sentences 20, 25, 27 to 29, 54 to 56, and / or a composition according to any of Sentences 30, 31, 57, 58. 98.
• 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 pharmaceutically acceptable carrier diluent or excipient.
• an oligonucleoside moiety as represented by Z in any of the following clauses can comprise a nucleic acid for inhibiting expression of ZPI as defined in any of the claims hereinafter.
• 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 oligonucleoside moiety.
• r and s are independently an integer selected from 1 to 16; and Z is an oligonucleoside moiety.
• s is an integer selected from 4 to 12. 3.
• said RNA compound comprises an RNA duplex comprising first and second strands, wherein the first strand is at least partially complementary to an RNA sequence of a target gene, and the second strand is at least partially complementary to said first strand, and wherein each of the first and second strands have 5’ and 3’ ends.
• a compound according to Clause 16 wherein the modifications are chosen from 2’-O-methyl, 2’-deoxy-fluoro, and 2’-deoxy.
• said one or more carbohydrates can be a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide or polysaccharide. 23.
• 26. A compound according to any of the preceding Clauses, wherein said one or more ligands are attached in a linear configuration, or in a branched configuration. 27.
• a compound according to Clause 33 or 34, wherein the oligonucleoside 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.
• 36. A compound according to Clause 35, wherein the modifications are chosen from 2’-O-methyl, 2’-deoxy-fluoro, and 2’-deoxy.
• 37. A compound according to any of Clauses 33 to 36, wherein the oligonucleoside further comprises one or more degradation protective moieties at one or more ends. 38.
• a compound according to Clause 37 wherein said one or more degradation protective moieties are not present at the end of the oligonucleoside strand that carries the linker / ligand moieties, and / or wherein said one or more degradation protective moieties is selected from phosphorothioate internucleoside linkages, phosphorodithioate internucleoside linkages and inverted abasic nucleosides, wherein said inverted abasic nucleosides are present at the distal end of the same strand to the end that carries the linker / ligand moieties. 39.
• the oligonucleoside 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. 40.
• the oligonucleoside 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.
• a process of preparing a compound according to any of Clauses 1 to 40 which comprises reacting compounds of Formulae (X) and (XI): Formula (XI) wherein: r and s are independently an integer selected from 1 to 16; and Z is an oligonucleoside moiety; and where appropriate carrying out deprotection of the ligand and / or annealing of a second strand for the oligonucleoside. 42.
• Formula (XIb) 51 Use of a compound according to any of Clauses 45 and 48 to 50, for the preparation of a compound according to any of Clauses 1 to 40. 52. Use of a compound according to Clause 46, for the preparation of a compound according to any of Clauses 6, 8 to 14, 16 to 33, and 35 to 40. 53. Use of a compound according to Clause 47, for the preparation of a compound according to any of Clauses 5, 7, 9 to 13, 15 to 32, and 34 to 40. 54. A compound or composition obtained, or obtainable by a process according to any of Clauses 41 to 44. 55. A pharmaceutical composition comprising of a compound according to any of Clauses 1 to 40, together with a pharmaceutically acceptable carrier, diluent or excipient.
• D-Galactosamine pentaacetate was purchased from AK scientific.
• HPLC/ESI-MS was performed on a Dionex UltiMate 3000 RS UHPLC system and Thermo Scientific MSQ Plus Mass spectrometer using an Acquity UPLC Protein BEH C4 column from Waters (300 ⁇ , 1.7 ⁇ m, 2.1 x 100 mm) at 60 °C.
• the solvent system consisted of solvent A with H 2 O containing 0.1% formic acid and solvent B with acetonitrile (ACN) containing 0.1% formic acid.
• ACN acetonitrile
• 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).
• TriGalNAc _Tether1 Preparation of compound 2: D-Galactosamine pentaacetate (3.00 g, 7.71 mmol, 1.0 eq.) was dissolved in anhydrous dichloromethane (DCM) (30 mL) under argon and trimethylsilyl trifluoromethanesulfonate (TMSOTf, 4.28 g, 19.27 mmol, 2.5 eq.) was added. The reaction was stirred at room temperature for 3 h. The reaction mixture was diluted with DCM (50 mL) and washed with cold saturated aq.
• DCM dichloromethane
• TMSOTf trimethylsilyl trifluoromethanesulfonate
• N,N,N′,N′- tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU) (2.78 g, 7.44 mmol, 5.0 eq.), 1-hydroxybenzotriazole hydrate (HOBt) (1.05 g, 7.44 mmol, 5.0 eq.) and N,N-diisopropylethylamine (DIPEA) (2.07 mL, 11.9 mmol, 8.0 eq.) were added to the solution and the reaction was stirred for 72 h. The solvent was removed under reduced pressure, the residue was dissolved in DCM (100 mL) and washed with saturated aq.
• DIPEA N,N,N′,N′- tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate
• Triantennary GalNAc compound 9 (0.27 g, 0.14 mmol, 1.0 eq.) was dissolved in MeOH (15 mL), 3 drops of acetic acid (AcOH) and Pd/C (30 mg) was added. The reaction mixture was degassed using vacuum/argon cycles (3x) and hydrogenated under balloon pressure overnight. The completion of the reaction was followed by mass spectrometry and the resulting mixture was filtered through a thin pad of celite. The solvent was evaporated and the residue obtained was dried under high vacuum and used for the next step without further purification. The product was obtained as pale yellowish oil (0.24 g, quantitative yield). MS: calculated for C 73 H 119 N 7 O 39 , 1718.8.
• 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 carried out at room temperature and after 1 h another molar equivalent of the MFCO solution was added. The reaction was allowed to proceed for an additional hour and was monitored by LC/MS. At least two molar equivalent excess of the MFCO NHS ester reagent relative to the amino modified oligonucleotide were needed to achieve quantitative consumption of the starting material.
• the reaction mixture was diluted 15-fold with water, filtered through a 1.2 ⁇ m filter from Sartorius and then purified by reserve phase (RP HPLC) on an ⁇ kta Pure instrument (GE Healthcare). Purification was performed using a XBridge C18 Prep 19 x 50 mm column from Waters.
• Buffer A was 100 mM TEAAc pH 7 and buffer B contained 95% acetonitrile in buffer A.
• a flow rate of 10 mL/min and a temperature of 60°C were employed. UV traces at 280 nm were recorded. A gradient of 0-100% B within 60 column volumes was employed.
• Fractions containing full length conjugated oligonucleotide were pooled, precipitated in the freezer with 3 M NaOAc, pH 5.2 and 85% ethanol and the collected pellet was dissolved in water. Samples were desalted by size exclusion chromatography and concentrated using a speed-vac concentrator to yield the conjugated oligonucleotide in an isolated yield of 40–80%.
• RP HPLC purification was performed using a XBridge C18 Prep 19 x 50 mm column from Waters.
• Buffer A was 100 mM triethylammonium acetate pH 7 and buffer B contained 95% acetonitrile in buffer A.
• a flow rate of 10 mL/min and a temperature of 60°C were employed.
• UV traces at 280 nm were recorded.
• a gradient of 0-100% B within 60 column volumes was employed.
• Fractions containing full-length conjugated oligonucleotide were pooled, precipitated in the freezer with 3 M NaOAc, pH 5.2 and 85% ethanol and the collected pellet was dissolved in water to give an oligonucleotide solution of about 1000 OD/mL.
• the O-acetates were removed by adding 20% aqueous ammonia. Quantitative removal of these protecting groups was verified by LC-MS.
• the conjugates were desalted by size exclusion chromatography using Sephadex G25 Fine resin (GE Healthcare) on an ⁇ kta Pure (GE Healthcare) instrument to yield the conjugated oligonucleotides in an isolated yield of 50–70%.
• the following schemes further set out the routes of synthesis:
• Example 2 Duplex Annealing To generate the desired siRNA duplex, 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. The 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 1x PBS containing 10% acetonitrile.
• D-Galactosamine pentaacetate was purchased from AK scientific.
• HPLC/ESI-MS was performed on a Dionex UltiMate 3000 RS UHPLC system and Thermo Scientific MSQ Plus Mass spectrometer using an Acquity UPLC Protein BEH C4 column from Waters (300 ⁇ , 1.7 ⁇ m, 2.1 x 100 mm) at 60 °C.
• the solvent system consisted of solvent A with H 2 O containing 0.1% formic acid and solvent B with acetonitrile (ACN) containing 0.1% formic acid.
• ACN acetonitrile
• 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).
• TriGalNAc _Tether2 Preparation of compound 2: D-Galactosamine pentaacetate (3.00 g, 7.71 mmol, 1.0 eq.) was dissolved in anhydrous dichloromethane (DCM) (30 mL) under argon and trimethylsilyl trifluoromethanesulfonate (TMSOTf, 4.28 g, 19.27 mmol, 2.5 eq.) was added. The reaction was stirred at room temperature for 3 h. The reaction mixture was diluted with DCM (50 mL) and washed with cold saturated aq.
• DCM dichloromethane
• TMSOTf trimethylsilyl trifluoromethanesulfonate
• N,N,N′,N′- tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU) (2.78 g, 7.44 mmol, 5.0 eq.), 1-hydroxybenzotriazole hydrate (HOBt) (1.05 g, 7.44 mmol, 5.0 eq.) and N,N-diisopropylethylamine (DIPEA) (2.07 mL, 11.9 mmol, 8.0 eq.) were added to the solution and the reaction was stirred for 72 h. The solvent was removed under reduced pressure, the residue was dissolved in DCM (100 mL) and washed with saturated aq.
• DIPEA N,N,N′,N′- tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate
• Triantennary GalNAc compound 10 (0.45 g, 0.26 mmol, 1.0 eq.), HBTU (0.19 g, 0.53 mmol, 2.0 eq.) and DIPEA (0.23 mL, 1.3 mmol, 5.0 eq.) were dissolved in DCM (10 mL) under argon. To this mixture, it was added dropwise a solution of compound 13 (0.14 g, 0.53 mmol, 2.0 eq.) in DCM (5 mL). The reaction was stirred at room temperature overnight. The solvent was removed, and the residue was dissolved in EtOAc (50 mL), washed with water (50 mL) and dried over Na 2 SO 4 .
• TriGalNAc Triantennary GalNAc compound 14 (0.31 g, 0.15 mmol, 1.0 eq.) was dissolved in EtOAc (15 mL) and Pd/C (40 mg) was added. The reaction mixture was degassed by using vacuum/argon cycles (3x) and hydrogenated under balloon pressure overnight.
• TriGalNAc tether 2 (GalNAc-T2) conjugation at 5’-end or 3’-end 5’-GalNAc-T2 conjugates
• TriGalNAc tether 2 NHS ester To a solution of carboxylic acid tether 2 (compound 15, 227 mg, 121 ⁇ mol) in DMF (2.1 mL), N-hydroxysuccinimide (NHS) (15.3 mg, 133 ⁇ mol) and N,N′- diisopropylcarbodiimide (DIC) (19.7 ⁇ L, 127 ⁇ mol) were added. The solution was stirred at room temperature for 18 h and used without purification for the subsequent conjugation reactions.
• NHS N-hydroxysuccinimide
• DIC N,N′- diisopropylcarbodiimide
• the reaction mixture was diluted 15-fold with water, filtered once through 1.2 ⁇ m filter from Sartorius and then purified by reserve phase (RP HPLC) on an ⁇ kta Pure (GE Healthcare) instrument.
• the purification was performed using a XBridge C18 Prep 19 x 50 mm column from Waters.
• Buffer A was 100 mM TEAA pH 7 and buffer B contained 95% acetonitrile in buffer A.
• a flow rate of 10 mL/min and a temperature of 60°C were employed.
• UV traces at 280 nm were recorded.
• a gradient of 0–100% B within 60 column volumes was employed.
• Example 4 Duplex Annealing To generate the desired siRNA duplex, 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. The 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 1x PBS containing 10% acetonitrile.
• Tether 2 Conjugation of Tether 2 to a siRNA strand: TriGalNAc tether 2 (GalNAc-T2) conjugation at 5’-end or 3’-end Conjugation conditions
• Pre-activation To a solution of compound 15 (16 umol, 4 eq.) in DMF (160 ⁇ L) was added TFA-O-PFP (15 ⁇ l, 21 eq.) followed by DIPEA (23 ⁇ l, 32 eq.) at 25°C. The tube was shaken for 2 h at 25°C. The reaction was quenched with H 2 O (10 ⁇ L).
• the 2'-O-Methyl phosphoramidites used were the following: 5'-(4,4'-dimethoxytrityl)-N-benzoyl- adenosine 2'-O-methyl-3'- [(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-(4,4'-dimethoxytrityl)- N-acetyl-cytidine 2'-O-methyl-3'- [(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-(4,4'- dimethoxytrityl)-N-isobutyryl-guanosine 2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]- phosphoramidite, 5'-(4,4'-dimethoxytrityl)-uridine 2'
• the 2’-F phosphoramidites used were the following: 5'-dimethoxytrityl-N-benzoyl-deoxyadenosine 2'- fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-dimethoxytrityl-N-acetyl-deoxycytidine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-dimethoxytrityl-N-isobutyryl- deoxyguanosine 2'-fluoro-3'- [(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite and 5'-dimethoxytrityl- deoxyuridine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-
• Inverted abasic phosphoramidite, 3-O-Dimethoxytrityl-2-deoxyribose-5-[(2-cyanoethyl)-(N, N- diisopropyl)]-phosphoramidite were purchased from Chemgenes (ANP-1422) or Hongene (OP-040).
• the DMT was removed by deblock solution, 3% TCA in DCM (DNAchem).
• the coupling time was 180 seconds.
• the oxidizer contact time was set to 80 seconds and thiolation time was 2*100 seconds.
• the oligonucleotides were cleaved from the solid support using a NH 4 OH:EtOH solution 4:1 (v/v) for 20 hours at 45°C (TCI).
• TCI 45°C
• the solid support was then filtered off, the filter was thoroughly washed with H 2 O and the volume of the combined solution was reduced by evaporation under reduced pressure.
• Oligonucleotide were treated to form the sodium salt by ultracentrifugation using Amicon Ultra-2 Centrifugal Filter Unit; PBS buffer (10x, Teknova, pH 7.4, Sterile) or by EtOH precipitation from 1M sodium acetate.
• Example 7 Solid phase synthesis method: scale ⁇ 5 ⁇ mol Syntheses of siRNA sense and antisense strands were performed on a MerMade12 synthesiser with commercially available solid supports made of controlled pore glass with universal linker (Universal CPG, with a loading of 40 ⁇ mol/g; LGC Biosearch or Glen Research) at 5 ⁇ mol scale.
• Sense strand destined to 3' conjugation were sytnthesised at 12 ⁇ mol on 3'-PT-Amino-Modifier C6 CPG 500 ⁇ solid support with a loading of 86 ⁇ mol/g (LGC).
• RNA phosphoramidites were purchased from ChemGenes or Hongene.
• the 2'-O-Methyl phosphoramidites used were the following: 5'-(4,4'-dimethoxytrityl)-N-benzoyl- adenosine 2'-O-methyl-3'- [(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-(4,4'-dimethoxytrityl)- N-acetyl-cytidine 2'-O-methyl-3'- [(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-(4,4'- dimethoxytrityl)-N-isobutyryl-guanosine 2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]- phosphoramidite, 5'-(4,4'-dimethoxytrityl)-uridine 2'
• the 2’-F phosphoramidites used were the following: 5'-dimethoxytrityl-N-benzoyl-deoxyadenosine 2'- fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-dimethoxytrityl-N-acetyl-deoxycytidine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-dimethoxytrityl-N-isobutyryl- deoxyguanosine 2'-fluoro-3'- [(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite and 5'-dimethoxytrityl- deoxyuridine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-
• Inverted abasic phosphoramidite 3-O-Dimethoxytrityl-2-deoxyribose-5-[(2-cyanoethyl)-(N, N- diisopropyl)]-phosphoramidite were purchased from Chemgenes (ANP-1422) or Hongene (OP-040). All phosphoramidites were dissolved in anhydrous acetonitrile (Honeywell Research Chemicals) at a concentration of 0.05M, except 2’-O-methyl-uridine phosphoramidite which was dissolved in DMF/MeCN (1:4, v/v).
• the oxidation time was 47 seconds, the thiolation time was 210 seconds.
• the coupling was performed with 8 eq. of amidite for 2*150 seconds.
• the oxidation time was 47 seconds, the thiolation time was 250 seconds
• the oligonucleotides were cleaved from the solid support using a NH 4 OH:EtOH solution 4:1 (v/v) for 20 hours at 45°C (TCI).
• TCI 45°C
• the solid support was then filtered off, the filter was thoroughly washed with H 2 O and the volume of the combined solution was reduced by evaporation under reduced pressure.
• Oligonucleotide were treated to form the sodium salt by EtOH precipitation from 1M sodium acetate.
• the single strand oligonucleotides were purified by IP-RP HPLC on Xbridge BEH C18 5 ⁇ m, 130 ⁇ , 19x150 mm (Waters) column with an increasing gradient of B in A.
• Mobile phase A 240 mM HFIP, 7 mM TEA and 5% methanol in water
• mobile phase B 240 mM HFIP, 7 mM TEA in methanol.
• the single strands purity and identity were assessed by UPLC/MS ESI- on Xbridge BEH C182.5 ⁇ m, 3x50 mm (Waters) column with an increasing gradient of B in A.
• Example 8 Nucleic acid sequences siRNA oligonucleosides according to the present invention target ZPI.
• the full DNA sequence of the ZPI target gene is SEQ ID NO: 236. Following Table 1 provides oligonucleoside mRNA target sequences of ZPI, together with the corresponding positions in transcript ENST00000261994.9.
• SEQ ID NO: 1 to 47 refer to human (Homo sapiens) mRNA sequences.
• Table 1 SEQ ID NO Oligonucleoside mRNA target sequence Starting position on 5’ ⁇ 3’ ENST00000261994.9
• SEQ ID NO: 1 ACCUCUGGAAUGCUUCUGUUUCU 1412
• SEQ ID NO: 2 AGUUCAAGCUAGAUCAGAAGUAU 1146
• SEQ ID NO: 3 UUGUGGAUUACAUCUUGUUCAAA 825
• SEQ ID NO: 4 ACCUUAGUGAACUCUCAGCUACU 1227
• SEQ ID NO: 6 UACUGGAAGAAAUCUCCAAGUAU 1246
• SEQ ID NO: 7 UUCCCAAACUGUUUGAUGAGAUU 780
• SEQ ID NO: 8 CUUGUGGAUUACAUCUUGUUCAA 824
• SEQ ID NO: 9 CAGGCA
• modified second strand sequences additionally includes the Me / F modified second strand in the absence of the 5’iaia motif.
• Table 5 identifies duplexes with Duplex IDs referencing the modified antisense and sense IDs from previous Tables 3 and 4.
• the invention relates to a nucleic acid comprising first and second strands that comprise, consist of, or consist essentially of a nucleotide sequence differing by 0 or 1 nucleotides from any one of the following first and second sequences: Duplex ID Modified first strand Modified second strand ETX-M00002910 ETX-S00008818 (SEQ ID NO: 148) ETX-S00008817 (SEQ ID NO: 195) ETX-M00002907 ETX-S00008812 (SEQ ID NO: 145) ETX-S00008811 (SEQ ID NO: 192)
• the invention relates to a nucleic acid comprising first and second strands that comprise, consist of, or consist essentially of a nucleotide sequence differing by 0 or 1 nucleotides from any one of the following first and second sequences: Duplex ID Modified first strand Modified second strand ETX-M00002910 ETX-S00008818 (SEQ ID NO: 148) ETX-S000088
• siRNA duplexes targeting ZPI mRNA or a negative control siRNA (siRNA-control; sense strand 5’-UUCUCCGAACGUGUCACGUTT-3’ (SEQ ID NO:297), antisense strand 5’-ACGUGACACGUUCGGAGAATT-3’ (SEQ ID NO:298)) at a final duplex concentration of 3 nM.
• Transfection is carried out by adding 9.7 ⁇ L Opti-MEM (ThermoFisher) plus 0.3 ⁇ L Lipofectamine RNAiMAX (ThermoFisher) to 10 ⁇ L of each siRNA duplex.
• RNA purification using a RNeasy 96 Kit (Qiagen). Each duplex is tested by transfection in duplicate wells in two independent experiments.
• cDNA synthesis is performed using FastQuant RT (with gDNase) Kit (Tiangen).
• Real-time quantitative PCR (qPCR) is performed on an ABI Prism 7900HT or ABI QuantStudio 7 with primers specific for human ZPI (Hs01547819_m1) and human GAPDH (Hs02786624_g1) using FastStart Universal Probe Master Kit (Roche).
• siRNAdesignR is a computational tool developed by the inventors for the engineering, selection and optimisation of features for the prediction of siRNA efficacy.
• siRNAdesignR identified all possible siRNA target sequences in a gene (consensus regions found in all transcripts) and then filtered out sequences with undesirable characteristics (sequences that match any other human gene with 0 or 1 mismatches, sequences that are commonly mutated (in >1% of the population), sequences that encode immunostimulatory motifs or have degenerate bases and sequences in the 5’ untranslated region). For the remaining sequences, the inventors then derived over 400 features describing the nucleotide sequence of an siRNA target sequence and how these nucleotides interacted with each other and other nucleotides on the messenger RNA at a distance from the target sequence.
• siRNAdesignR predictions for efficacious antisense sequences of siRNAs targeting ZPI sequence prediction A GAAACAGAAGCAUUCCAGAGGU (SEQ ID NO:48) 0.87 A UACUUCUGAUCUAGCUUGAACU (SEQ ID NO:49) 0.86 U UUGAACAAGAUGUAAUCCACAA (SEQ ID NO:50) 0.86 A GUAGCUGAGAGUUCACUAAGGU (SEQ ID NO:51) 0.85 A CAAGAAUUAAUUUGGUUUCAGG (SEQ ID NO:52) 0.85 A UACUUGGAGAUUUCUUCCAGUA (SEQ ID NO:53) 0.84 A AUCUCAUCAAACAGUUUGGGAA (SEQ ID NO:54) 0.83 U UGAACAAGAUGUAAUCCACAAG (SEQ ID NO:55) 0.83 A AAGGUGGAGGCAAACUUGCCUG (SEQ ID NO:56) 0.82 A AAACUUCCAUGUUUCUGGUUUU (SEQ ID NO:48)
• FIG. 11 Highlighted in Figure 11 is an siRNA sequence that ranked within the top 20 predictions by siRNAdesignR for the target SLC25A5. This sequence was subsequently tested in dose response in vitro (in a Huh7 cell line), measuring the amount of mRNA knockdown (Figure 12), and in vivo (in C57BL/6 mice), measuring mRNA and protein knockdown ( Figures 13 and 14). To further validate this approach, the inventors investigated whether siRNAdesignR predictions could identify more potent siRNA molecules than those identified by in vitro screening. 100 siRNAs were originally selected for in vitro screening based upon the cross reactivity between the Homo sapiens, Macaca fascicularis and Mus musculus B4GALT1 messenger RNA sequences.
• This screen identified siRNAs that were potent in vitro ( Figure 15) and in vivo ( Figure 16). Subsequent to performing this screen efficacy predictions were generated using siRNAdesignR as described above, this identified sequences targeting B4GALT1 that were predicted to be more efficacious than the lead sequence from the original screen shown in Figures 15 and 16. Screening of the predicted sequences in an in vitro model revealed 13 sequences demonstrating similar or greater mRNA knockdown than the original screening lead (ETXM-1850, see Figure 17). Accordingly, the inventors were able to validate that siRNAdesignR is capable to predict highly potent siRNA sequences.
• Example 11 Confirmation of Lead siRNA Activity in a Human hydrodynamic injection (HDI) Mouse Model
• the transient expression of human mRNA in mice was evaluated to assess the activity of siRNAs against human mRNA sequences.
• a single subcutaneous dose of siRNAs (3 mg/kg) was administered on Day -6.
• Hydrodynamic injection (HDI) of a plasmid expressing human ZPI mRNA was performed on Day 0.
• the study was terminated, and mRNA expression was analysed on Day 1.
• saline was injected in place of an siRNA in the vehicle control group.
• An overview of the study is shown in Figure 18.
• the siRNAs tested in this study are shown in Table 7 below. Activity of all tested siRNAs was confirmed in the human HDI mouse model ( Figure 19).
• RNAi molecules are depicted in the relevant Tables herein. As can be seen from Figure 19, and Table 7 below, a particularly large effect is seen for a number of siRNAs.
• Preferred siRNAs for use in the invention are ETX-M00002910 and ETX-M00002907, and these are specifically contemplated throughout this disclosure, even when not singled out in a specific passage of this disclosure.
• an 84-day, single dose study was designed to effectively demonstrate the reduction of target mRNA and protein in healthy monkeys.
• 12 non-na ⁇ ve male cynomolgus macaques aged 4-8 years received 3 mg/kg of ETX siRNA subcutaneously on day 0.
• Liver biopsies were taken on study days -7, 28, 56 and 84 for measurement of target mRNA and protein levels.
• Blood (plasma) samples were taken on study days -7, -3, 0, 7, 14, 28, 42, 56, 70, and 84 for measurement of target protein levels.
• Plasma samples for measurement of liver enzymes (ALT, AST, ALP, and GGT) were collected on study days 0 (pre-treatment) 14, 28, 56, and 84 post-dose.
• Endpoints also included clinical observations for general health, adverse clinical signs, and injection site monitoring.
• a summary of the study design is shown in Figure 20.
• the following siRNAs were tested in this study: - ETX-M00002910 (SEQ ID NO: 54 and SEQ ID NO: 101) - ETX-M00002907 (SEQ ID NO: 51 and SEQ ID NO: 98) ZPI mRNA and ZPI protein levels were determined after a single dose of 3 mg/kg siRNA.
• Treatment of NHPs with either ETX-M00002910 or ETX-M00002907 achieved approximately 63% knockdown of ZPI mRNA and 76-86% of ZPI protein in liver and plasma after a single dose (Figure 21).
• Hepatic function measurements were performed after a single dose of 3 mg/kg siRNA. ETX-M00002910 and ETX-M00002907 were well-tolerated, with all hepatic function measurements (ALT, AST, ALP, and GGT) remaining within the normal range for NHPs as provided by the vendor [Reference ranges for ALT(U/L): 21-74.5 U/L; AST(U/L) Reference range: 26-72.8 U/L; ALP(U/L) Reference range: 79.78- 1078.88 U/L; GGT(U/L) Reference range: 36.75-170 U/L] ( Figure 22).
• Example 13 Efficacy of ETX-148 for von Willebrand Disease (vWD) This study was designed to evaluate the efficacy of ETX-148 for the treatment of vWD. Specifically, two analyses were carried out in von Willebrand Factor (vwf) knockout (KO) mice. Vwf KO mice were subcutaneously injected with two doses of ETX-148 on days -10 and -3. On day 0, the analyses were performed. A summary of the study design is shown in Figure 23.
• vwf von Willebrand Factor
• siRNA was utilized in this study: - ETX-M00001185 (Sense Strand: iaiaUACCAAGGAAAUGCCACCAUG – SEQ ID NO: 299; Anti-sense Strand: CAUGGUGGCAUUUCCUUGGUAGG – SEQ ID NO: 300).
• the efficacy of ETX-148 for vWD was determined utilizing ex vivo rotational thromboelastometry (ROTEM).
• ROTEM ex vivo rotational thromboelastometry
• ETX-148 Treatment with ETX-148 resulted in blood clots forming at a significantly fast rate in vwf KO mice, as can be seen in the decrease in clot formation time and increase in maximum clotting velocity (Figure 24).

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