WO2024023267A2 - Nucleic acid compounds - Google Patents

Nucleic acid compounds Download PDF

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WO2024023267A2
WO2024023267A2 PCT/EP2023/070927 EP2023070927W WO2024023267A2 WO 2024023267 A2 WO2024023267 A2 WO 2024023267A2 EP 2023070927 W EP2023070927 W EP 2023070927W WO 2024023267 A2 WO2024023267 A2 WO 2024023267A2
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strand
nucleic acid
nucleosides
seq
nucleoside
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PCT/EP2023/070927
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French (fr)
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WO2024023267A3 (en
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Amy MCCARTHY
Graham CRAGGS
James LONGDEN
Ines DE SANTIAGO
Duncan Brown
Ahmad Ali MORTAZAVI
Viviana MANNELLA
Muthusamy Jayaraman
Alexandre DEBACKER
Adrian James MOGG
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E-Therapeutics Plc
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Publication of WO2024023267A2 publication Critical patent/WO2024023267A2/en
Publication of WO2024023267A3 publication Critical patent/WO2024023267A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/322—2'-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01038—Beta-N-acetylglucosaminylglycopeptide beta-1,4-galactosyltransferase (2.4.1.38)

Definitions

  • 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, for use in the treatment and / or prevention of disease.
  • a nucleic acid for inhibiting expression of B4GALT1 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 B4GALT1 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 B4GALT1 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 B4GALT1 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 above second aspect of the present invention, wherein the first strand comprises any one of the first strand modified sequences as listed in Table 3.
  • a nucleic acid according to the above first aspect of the present invention wherein the first strand comprises any one of the following sequences: SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41.
  • a nucleic acid according to the above second aspect of the present invention wherein the first strand comprises any one of the following sequences: SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81.
  • a nucleic acid according to the above first aspect of the present invention wherein the second strand comprises any one of the following sequences: SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61.
  • a nucleic acid according to the above second aspect of the present invention wherein the second strand comprises any one of the following sequences: SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101.
  • a nucleic acid comprising first and second strands that comprise, consist of, or consist essentially of a nucleoside sequence differing by 0 or 1 nucleosides from any one of the following first and second sequences:
  • a nucleic acid comprising first and second strands that comprise, consist of, or consist essentially of a nucleoside sequence differing by 0 or 1 nucleosides from any one of the following first and second sequences: [0022] A nucleic acid comprising first and second strands that comprise, consist of, or consist essentially of a nucleoside sequence differing by 0 or 1 nucleosides from any one of the following first and second sequences:
  • a nucleic acid comprising first and second strands that comprise, consist of, or consist essentially of a nucleoside sequence differing by 0 or 1 nucleosides from any one of the following first and second sequences:
  • a nucleic acid comprising first and second strands that comprise, consist of, or consist essentially of a nucleoside sequence differing by 0 or 1 nucleosides from any one of the following first and second sequences:
  • a nucleic acid comprising first and second strands that comprise, consist of, or consist essentially of a nucleoside sequence differing by 0 or 1 nucleosides from any one of the following first and second sequences:
  • a nucleic acid comprising first and second strands that comprise, consist of, or consist essentially of a nucleoside sequence differing by 0 or 1 nucleosides from any one of the following first and second sequences:
  • a nucleic acid comprising first and second strands that comprise, consist of, or consist essentially of a nucleoside sequence differing by 0 or 1 nucleosides from any one of the following first and second sequences:
  • a nucleic acid comprising first and second strands that comprise, consist of, or consist essentially of a nucleoside sequence differing by 0 or 1 nucleosides from any one of the following first and second sequences:
  • a nucleic acid comprising first and second strands that comprise, consist of, or consist essentially of a nucleoside sequence differing by 0 or 1 nucleosides from any one of the following first and second sequences:
  • a conjugate for inhibiting expression of B4GALT1 target gene in a cell comprising a nucleic acid as disclosed herein and one or more ligand moieties.
  • 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 diabetes.
  • a nucleic acid or pharmaceutical composition for use in prevention or treatment of cardiovascular disease.
  • Figure 1 Linker and ligand portions of constructs suitable for use according to the present invention including tether la. While Figure 1 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 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 occurring as a result of hydrolytic displacement, such as an OH substituent.
  • tether la 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 la 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 a substituent occurring as a result of hydrolytic displacement, such as an OH substituent, or (c) tether la 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 lb. While Figure 2 depicts the linker to be conjugated to an oligonucleotide, it is to be understood that the present invention also encompasses conjugates of the same linker with an oligonucleoside as disclosed herein.
  • tether lb 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 lb 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 a substituent occurring as a result of hydrolytic displacement, such as an OH substituent, or (c) tether lb 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. While 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.
  • 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.
  • Figure 8 Duplex construct according to Table 5.
  • Figure 9 Results of an RNAi molecule screen for inhibition of B4GALT1 mRNA expression in human Huh7 cells. Each bar represents mean relative expression of B4GALT1 mRNA compared to untreated wells after treatment with an individual siRNA construct at 5 nM.
  • Figure 10 Results of an RNAi molecule screen for inhibition of B4GALT1 mRNA expression in human Huh7 cells. Each bar represents mean relative expression of B4GALT1 mRNA compared to untreated wells after treatment with an individual siRNA construct at 0.1 nM.
  • Figure 11 Results of dose-response experiments for inhibition of B4GALT1 mRNA expression in human Huh7 cells. Points represent mean relative expression of B4GALT1 mRNA compared to untreated wells after treatment with siRNA construct at the indicated concentrations on the x-axis. Error bars represent standard deviation of the mean. 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 12 Time course inhibition of B4GALT1 expression in mice.
  • FIG. 13 Inhibition of B4GALT1 expression by ETXM1200 (ETXS2400 & ETXS2399), ETXM1201 (ETXS2402 & ETXS2401), ETXM1217 (ETXS2434 & ETXS2401), ETXM1764 (ETXS3528 & ETXS2401), ETXM1765 (ETXS3530 & ETXS2401), ETXM1766 (ETXS3532 & ETXS2401), ETXM 1767 (ETXS3534 & ETXS2401), ETXM 1768 (ETXS3536 & ETXS2401), ETXM1769 (ETXS3538 & ETXS2401) and ETXM1770 (ETXS3540 & ETXS2401).
  • ETXM1200 ETXS2400 & ETXS2399
  • ETXM1201 ETXS2402 & ETXS2401
  • ETXM1217 ETXS2434
  • FIG. 14 Inhibition of ZPI expression by ETXM1203 (ETXS2406 & ETXS2405), ETXM1204 (ETXS2408 & ETXS2407), ETXM1218 (ETXS2436 & ETXS2407), ETXM1772 (ETXS3544 & ETXS2407), ETXM1773 (ETXS3546 & ETXS2407), ETXM1774 (ETXS3548 & ETXS2407), ETXM 1775 (ETXS3550 & ETXS2407), ETXM 1776 (ETXS3552 & ETXS2407), ETXM1777 (ETXS3554 & ETXS2407) and ETXM1778 (ETXS3556 & ETXS2407).
  • ETXM1203 ETXS2406 & ETXS2405
  • ETXM1204 ETXS2408 & ETXS2407
  • ETXM1218 ETXS2436 & ET
  • Figure 15 Selection of active GalNAc-siRNAs with EC50 values less than lOOnM. Dose-response in B4GALT1 gene knockdown in primary mouse hepatocytes was measured after 48hr incubation with GalNAc-siRNAs targeting mouse B4GALT1 at 10 serial dilutions from lOOOnM . ECso values were determined by fitting data to a 4-parameter sigmoidal doseresponse (variable slope) equation using GraphPad Prism. 4 active GalNAc-siRNAs, ETXM619, ETXM624, ETXM628 and ETXM633, were selected for in vivo pharmacology.
  • Figure 16 Summary of B4GALT1 mRNA knockdown effects of multiple dosing of GalNAc-siRNAs, ETXM619, ETXM624, ETXM628 and ETXM633 (lOmg/kg) in mouse liver tissues.
  • FIG. 17 Effect of B4GALT1 mRNA knockdown in plasma LDL-c, glucose and fibrinogen levels.
  • Plasma samples were collected on day 14 after three dosings of ETXMs (lOmg/kg, s.c.) on day 0, day 3 and day 7.
  • ETXMs lOmg/kg, s.c.
  • the ETXM treated group shows significantly reduced levels of LDL-c, glucose and fibrinogen in normal C57BL/6 mice.
  • Data presented here are Meani SD.
  • the term "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” 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.
  • a double stranded nucleic acid e.g. siRNA agent of the invention includes a nucleoside mismatch in the sense strand.
  • the nucleoside mismatch is, for example, within 5, 4, 3, 2, or 1 nucleosides from the 3 '-end of the nucleic acid e.g. siRNA.
  • the nucleoside mismatch is, for example, in the 3'- terminal nucleoside of the nucleic acid e.g. siRNA.
  • a "target sequence” (which may also be called a target RNA or a target mRNA) refers to a contiguous portion of the nucleoside sequence of an mRNA molecule formed during the transcription of a gene, including mRNA that is a product of RNA processing of a primary transcription product.
  • 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,
  • 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.
  • RNA interference agent refers to an agent that contains RNA, and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. siRNA directs the sequence-specific degradation of mRNA through RNA interference (RNAi).
  • RISC RNA-induced silencing complex
  • 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.
  • each strand of the nucleic acid e.g. a dsiRNA molecule
  • 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 intemucleoside 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.
  • the two strands forming the duplex structure may be different portions of one larger molecule, or they may be separate molecules e.g. RNA molecules.
  • 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.
  • Bosset 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.
  • first 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 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.
  • Such 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.
  • non- Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogstein base pairing.
  • nucleic acid eg dsiRNA
  • 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 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.
  • 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:2-21 or 102-201.
  • 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 B4GALTlgene. In certain embodiments, 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 B4GALTlmRNA. 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 B4GALTlmRNA.
  • the first strand of the nucleic acid according to the invention is partially or fully complementary to a contiguous portion of 17, 18 or 19 nucleosides of any one of the sequences as listed in Table 1, i.e., any one of SEQ ID NOs: 2-21 or 102-201.
  • the first (antisense) strand of the nucleic acid according to the invention is partially complementary to a contiguous portion of the B4GALTlmRNA 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 B4GALTlmRNA.
  • 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: 2-21 or 102-201.
  • 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: 2-21 or 102-201.
  • 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.
  • treating refers to a beneficial or desired result including, but not limited to, alleviation or amelioration of one or more symptoms associated with gene expression.
  • Treatment can also mean prolonging survival as compared to expected survival in the absence of treatment. Treatment can include prevention of development of comorbidities, e.g., reduced liver damage in a subject with a hepatic infection.
  • “Therapeutically effective amount,” as used herein, is intended to include the amount of a nucleic acid e.g. an 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).
  • a nucleic acid e.g. an 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).
  • phrases "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.
  • 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.
  • the term "at least" prior to a number or series of numbers is understood to include the number adjacent to the term “at least”, and all subsequent numbers or integers that could logically be included, as clear from context.
  • the number of 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.
  • nucleoside overhang As used herein, "no more than” or “less than” is understood as the value adjacent to the phrase and logical lower values or integers, as logical from context, to zero. For example, a duplex with an overhang of "no more than 2 nucleosides" has a 2, 1, or 0 nucleoside overhang. When “no more than” is present before a series of numbers or a range, it is understood that “no more than” can modify each of the numbers in the series or range.
  • the terminal region of a strand is the last 5 nucleosides from the 5’ or the 3’ end.
  • nucleic acids 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):
  • 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;
  • abasic nucleoside 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
  • abasic nucleoside 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 the 5’ or 3’ terminal region of the second strand; and / or a reversed internucleoside linkage connects at least one abasic nucleoside to an adjacent basic nucleoside in a terminal region of the second strand; and / or a reversed internucleoside linkage connects at least one abasic nucleoside to an adjacent basic nucleoside in either the 5’ or 3’ terminal region of the second strand; and /or an abasic nucleoside as the penultimate nucleoside which is connected via the reversed linkage to the nucleoside which is not the terminal nucleoside (called the antepenultimate nucleoside herein); and/or abasic nucleosides as the 2
  • the reversed linkage is a 5-5’ reversed linkage and the linkage between the terminal and penultimate abasic nucleosides is 3’5’ when reading towards the terminus comprising the terminal and penultimate abasic nucleosides; or
  • the reversed linkage is a 3-3’ reversed linkage and the linkage between the terminal and penultimate abasic nucleosides is 5’3’ when reading towards the terminus comprising the terminal and penultimate abasic nucleosides.
  • abasic nucleoside at the terminus of the second strand.
  • abasic nucleosides in the terminal region of the second strand, preferably at the terminal and penultimate positions.
  • abasic nucleosides are consecutive, for example all abasic nucleosides may be consecutive.
  • 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 moi eties 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 intemucleoside linkage is located between the penultimate (abasic) nucleoside and the antepenultimate nucleoside.
  • 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 intemucleoside 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 pen
  • 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 intemucleoside 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 intemucleoside 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 intemucleoside 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 intemucleoside 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’
  • 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)
  • the 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 intemucleoside 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.
  • 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, it will also be appreciated that with the presence of one or more additional reversed linkages and / or nucleosides having an inverted orientation, then the overall nucleic acid may have 3’ - 5’ end structures corresponding to the conventionally positioned 5’ / 3’ ends.
  • the nucleic acid may have a 3 ’-3’ reversed linkage, and the terminal sugar moiety may comprise a 5’ OH rather than a 5’ phosphate group at the 5’ position of that terminal sugar.
  • the 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
  • 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 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 C1-4 alkyl (preferably H),
  • Z represents the remaining nucleosides of said second strand, more preferably the following 5’ terminal motif wherein:
  • 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 eg RNA which is distal to a ligand moiety, such as a GalNAc containing portion, of the molecule.
  • a ligand moiety such as a GalNAc containing portion
  • GalNAc-siRNA constructs with a 5 ’-GalNAc on the sense strand can have a reversed linkage on the opposite end of the sense strand.
  • GalNAc-siRNA constructs with a 3’-GalNAc on the sense strand can have a reversed linkage on the opposite end of the sense strand.
  • the 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 (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 C1-4 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 wherein:
  • 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.
  • 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 B4GALT1 gene is between 17 and 30 nucleosides in length.
  • the nucleic acid e.g. an RNA of the invention e.g., a dsiRNA
  • the nucleic acid does not comprise further modifications, e.g., chemical modifications or conjugations known in the art and described herein.
  • the nucleic acid e.g. RNA of the invention e.g., a dsiRNA
  • nucleosides are modified.
  • 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,
  • 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 intemucleoside 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'.
  • Various salts, mixed salts and free acid forms are also included.
  • Modified nucleic acids e.g. RNAs can also contain one or more substituted sugar moieties.
  • the nucleic acids e.g. siRNAs, e.g., dsiRNAs, featured herein can include one of the following at the 2'-position: OH; F; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; 0-, S- or N- alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted. 2’ O- methyl and 2’ -F are preferred modifications.
  • the nucleic acid comprises at least one modified 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' - O-alkyl- modified nucleoside, 2'-hydroxly-modified nucleoside, a 2'- methoxyethyl modified nucleo
  • 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.
  • One preferred modification is a modification at the 2’ -OH group of the ribose sugar, optionally selected from 2'-Me or 2’-F modifications.
  • Preferred nucleic acid comprise one or more nucleosides on the first strand and / or the second strand which are modified, to form modified nucleosides, as follows:
  • a nucleic acid wherein the modification is a modification at the 2’ -OH group of the ribose sugar, optionally selected from 2'-Me or 2’-F modifications.
  • a nucleic acid wherein the first strand comprises a 2’-F modification at any of position 2, position 6, position 14, or any combination thereof, counting from position 1 of said first strand.
  • a nucleic acid wherein the second strand comprises a 2’-F modification at any of position 7, position 9, position 11, or any combination thereof, counting from position 1 of said second strand.
  • a nucleic acid wherein the first and second strand each comprise 2'-Me and 2’-F modifications.
  • a nucleic which comprises at least one thermally destabilizing modification, suitably at one or more of positions 1 to 9 of the first strand counting from position 1 of the first strand, and / or at one or more of positions on the second strand aligned with positions 1 to 9 of the first strand, wherein the destabilizing modification is selected from a modified unlocked nucleic acid (UNA) and a glycol nucleic acid (GNA), preferably a glycol nucleic acid, more preferably an (S)-glycol nucleic acid.
  • UUA modified unlocked nucleic acid
  • GNA glycol nucleic acid
  • a nucleic acid which is an siRNA oligonucleoside, wherein said first strand comprises
  • a nucleic acid which is an siRNA oligonucleoside wherein each of the first and second strands comprises an alternating modification pattern, preferably a fully alternating modification pattern along the entire length of each of the first and second strands, wherein the nucleosides of the first strand are modified by (i) 2’Me modifications on the odd numbered nucleosides counting from position 1 of the first strand, and (ii) 2’F modifications on the even numbered nucleosides counting from position 1 of the first strand, and nucleosides of the second strand are modified by (i) 2’F modifications on the odd numbered nucleosides counting from position 1 of the second strand, and (ii) 2’Me modifications on the even numbered nucleosides counting from position 1 of the second strand.
  • Such fully alternating modification patterns are present in a blunt ended oligonucleoside, wherein each of the first and second strands are 19 nucleosides in length.
  • 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 moi eties of the backbone, and reading in a direction away from that end of the molecule.
  • 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. As hereinbefore described, there will be a region of complementarity between the sense and antisense strands, and in this way the antisense strand will also have a region of complementarity to the target nucleic acid sequence as referred to above.
  • the nucleic acid e.g. siRNA agent further comprises at least one phosphorothioate or methylphosphonate internucleoside linkage.
  • the phosphorothioate or methylphosphonate intemucleoside 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 intemucleoside 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 intemucleoside 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 intemucleoside 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 intemucleoside 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 intemucleoside 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 intemucleoside linkage.
  • the nucleic acid strand may be an RNA comprising a phosphorothioate intemucleoside 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 moi eties 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.
  • the same nucleic acid may also comprise a 2’ F modification at positions 7, 9 and 11 of the second strand.
  • a nucleic acid wherein modified nucleosides of said second strand comprise a modification pattern according to any one of the following (5’ -3 ’):
  • a nucleic acid wherein modified nucleosides of said second strand comprise a modification pattern according to any one of the following (5’-3’):
  • a nucleic acid wherein modified nucleosides of said second strand comprise a modification pattern according to any one of the following (5’-3’): ia - ia - Me - Me - Me - Me - Me - Me - F- F- F- F- F- F- Me - Me - Me - Me - Me - Me - Me - Me - Me - Me -
  • ia represents an inverted abasic nucleoside
  • ia represents an inverted abasic nucleoside
  • a nucleic acid wherein modified nucleosides of said second strand comprise a modification pattern according to any one of the following (5’-3’): ia - ia - Me(s)Me(s)Me - Me - Me - Me - F- F- F- F- F- Me - Me - Me - Me - Me - Me - Me - F - Me - Me, or ia - ia - Me(s)Me(s)Me - Me - Me - F - F - Me - F- F- F- F- F- F- F- F- F- Me - Me - Me - Me - Me - Me - Me - Me - Me - Me, or ia - ia - Me(s)Me(s)Me - Me - Me - F - F - Me - Me - Me - Me - Me - Me - Me, or ia
  • (s) is a phosphorothioate internucleoside linkage
  • ia represents an inverted abasic nucleoside
  • modified nucleosides comprise any one of the following modification patterns:
  • modified nucleosides comprise any one of the following modification patterns:
  • Modification pattern 1 Second strand (5’ -3 ’): Me(s)Me(s)Me - Me - Me - Me - F - F - F - F - F
  • modified nucleosides comprise any one of the following modification patterns:
  • modified nucleosides comprise any one of the following modification patterns:
  • Modification pattern 1 Second strand (5’-3’): ia - ia - Me - Me - Me - Me - Me - Me - F - F
  • modified nucleosides comprise any one of the following modification patterns:
  • ia represents an inverted abasic nucleoside
  • ia - ia when the inverted abasic nucleosides as represented by ia - ia are present at the 3’ terminus of the second strand, said inverted abasic nucleosides are present in a 2 nucleoside overhang.
  • modified nucleosides comprise any one of the following modification patterns:
  • (s) is a phosphorothioate internucleoside linkage, ia represents an inverted abasic nucleoside.
  • modified nucleosides comprise any one of the following modification patterns:
  • (s) is a phosphorothioate internucleoside linkage
  • ia represents an inverted abasic nucleoside
  • when the inverted abasic nucleosides as represented by ia - ia are present at the 3’ terminus of the second strand, said in
  • nucleic acid wherein the modified nucleosides comprise the following modification pattern:
  • Modification pattern 5 Second strand (5’-3 ’): ia - ia - Me(s)Me(s)Me - Me - Me - Me - Me - Me
  • a nucleic acid wherein the first strand comprises a 2’ sugar modification pattern wherein said modifications are selected at least from 2’Me and 2’F sugar modifications, provided that the overall number of 2’F sugar modifications in the first strand does not consist of four, or six, 2’F modifications.
  • a nucleic acid wherein the first strand comprises a 2’ sugar modification pattern wherein said modifications are selected at least from 2’Me and 2’F sugar modifications, wherein the overall number of 2’F sugar modifications in the first strand consists of three, five or seven 2’F modifications.
  • a nucleic acid wherein the first strand comprises a 2’ sugar modification pattern wherein said modifications are selected at least from 2’Me and 2’F sugar modifications, wherein the overall number of 2’F sugar modifications in the first strand consists of three 2’F modifications.
  • a nucleic acid wherein the first strand comprises a 2’ sugar modification pattern wherein said modifications are selected at least from 2’Me and 2’F sugar modifications, wherein the overall number of 2’F sugar modifications in the first strand consists of five 2’F modifications.
  • a nucleic acid wherein the first strand comprises a 2’ sugar modification pattern as follows (5 ’-3’):
  • a nucleic acid wherein the first strand comprises a 2’ sugar modification pattern as follows (5 ’-3’):
  • a nucleic acid wherein the first strand comprises a 2’ sugar modification pattern as follows (5 ’-3’):
  • a nucleic acid wherein the first strand comprises a 2’ sugar modification pattern as follows (5 ’-3’):
  • a nucleic acid wherein the first strand comprises a 2’ sugar modification pattern wherein said modifications are selected at least from 2’Me and 2’F sugar modifications, wherein the overall number of 2’F sugar modifications in the first strand consists of seven 2’F modifications.
  • a nucleic acid wherein the first strand comprises a 2’ sugar modification pattern as follows (5 ’-3’):
  • X 2 , X 3 and X 4 are selected from 2’Me and 2’F sugar modifications, provided that for X 2 , X 3 and X 4 at least one is a 2’F sugar modification, and the other two sugar modifications are 2’Me sugar modifications.
  • a nucleic acid wherein the first strand comprises a 2’ sugar modification pattern as follows (5 ’-3’):
  • a nucleic acid wherein the first strand comprises a 2’ sugar modification pattern as follows (5 ’-3’):
  • a nucleic acid wherein the first strand comprises a 2’ sugar modification pattern as follows (5 ’-3’):
  • a nucleic acid wherein the first strand comprises a 2’ sugar modification pattern as follows (5 ’-3’):
  • a nucleic acid wherein the first strand comprises a 2’ sugar modification pattern as follows (5 ’-3’):
  • a nucleic acid wherein the second strand comprises a 2’ sugar modification pattern as follows (5 ’-3’):
  • a nucleic acid wherein the second strand comprises a 2’ sugar modification pattern as follows (5 ’-3’):
  • the first strand comprises a 2’ sugar modification pattern wherein said modifications are selected at least from 2’Me and 2’F sugar modifications, provided that the overall number of 2’F sugar modifications in the first strand does not consist of four, or six, 2’F modifications.
  • a nucleic acid wherein the second strand comprises a 2’ sugar modification pattern as follows (5 ’-3’):
  • the first strand comprises a 2’ sugar modification pattern wherein said modifications are selected at least from 2’Me and 2’F sugar modifications, wherein the overall number of 2’F sugar modifications in the first strand consists of three, five or seven 2’F modifications.
  • a nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar modification pattern as follows (5’-3 ’):
  • nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
  • a nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar modification pattern as follows (5’-3 ’):
  • nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
  • a nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar modification pattern as follows (5’-3 ’):
  • nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
  • a nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar modification pattern as follows (5’-3 ’):
  • nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
  • a nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar modification pattern as follows (5’-3 ’):
  • nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
  • a nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar modification pattern as follows (5’-3 ’):
  • nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
  • a nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar modification pattern as follows (5’-3’):
  • nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
  • a nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar modification pattern as follows (5’-3 ’):
  • nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
  • a nucleic acid wherein the second strand comprises a 2’ sugar, and abasic modification pattern as follows (5’-3 ’): ia-ia-(Me)8 - (F) 3 - (Me) 10 wherein ia represents an inverted abasic nucleoside.
  • a nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar, and abasic modification pattern as follows (5’-3 ’): ia-ia-(Me)8 - (F) 3 - (Me)10, wherein ia represents an inverted abasic nucleoside; and wherein nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
  • a nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar, and abasic modification pattern as follows (5’-3’): ia-ia-(Me)8 - (F) 3 - (Me) 10 , wherein ia represents an inverted abasic nucleoside; and wherein nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
  • a nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar, and abasic modification pattern as follows (5’-3’): ia-ia-(Me)8 - (F) 3 - (Me) 10 , wherein ia represents an inverted abasic nucleoside; and wherein nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
  • a nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar, and abasic modification pattern as follows (5’-3 ’): ia-ia-(Me)8 - (F) 3 - (Me) 10 , wherein ia represents an inverted abasic nucleoside; and wherein nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
  • a nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar, and abasic modification pattern as follows (5’-3 ’): ia-ia-(Me)8 - (F) 3 - (Me) 10 , wherein ia represents an inverted abasic nucleoside; and wherein nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
  • a nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar, and abasic modification pattern as follows (5’-3 ’): ia-ia-(Me)8 - (F) 3 - (Me) 10 , wherein ia represents an inverted abasic nucleoside; and wherein nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
  • a nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar, and abasic modification pattern as follows (5’-3 ’): ia-ia-(Me)8 - (F) 3 - (Me) 10 , wherein ia represents an inverted abasic nucleoside; and wherein nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
  • a nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar, and abasic modification pattern as follows (5’-3 ’): ia-ia-(Me)8 - (F) 3 - (Me) 10 , wherein ia represents an inverted abasic nucleoside; and wherein nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
  • a nucleic acid wherein the second strand comprises a 2’ sugar modification pattern as follows (5 ’-3’): ia-ia-Me(s)Me(s) (Me) 6 - (F) 3 - (Me) 10 , wherein ia represents an inverted abasic nucleoside, and (s) represents a phosphorothioate linkage.
  • a nucleic acid wherein the second strand comprises a 2’ sugar modification pattern as follows (5 ’-3’): ia-ia-Me(s)Me(s) (Me) 6 - (F) 3 - (Me) 10, wherein ia represents an inverted abasic nucleoside, and (s) represents a phosphorothioate linkage; and wherein the first strand comprises a 2’ sugar modification pattern wherein said modifications are selected at least from 2’Me and 2’F sugar modifications, provided that the overall number of 2’F sugar modifications in the first strand does not consist of four, or six, 2’F modifications.
  • a nucleic acid wherein the second strand comprises a 2’ sugar modification pattern as follows (5 ’-3’): ia-ia-Me(s)Me(s) (Me) 6 - (F)3 - (Me) io, wherein ia represents an inverted abasic nucleoside, and (s) represents a phosphorothioate linkage; and wherein the first strand comprises a 2’ sugar modification pattern wherein said modifications are selected at least from 2’Me and 2’F sugar modifications, wherein the overall number of 2’F sugar modifications in the first strand consists of three, five or seven 2’F modifications.
  • a nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar, and abasic modification pattern as follows (5’-3 ’): ia-ia-Me(s)Me(s) (Me) 6 - (F) 3 - (Me) 10 , wherein ia represents an inverted abasic nucleoside, and (s) represents a phosphorothioate linkage, and wherein nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
  • a nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar, and abasic modification pattern as follows (5’-3’): ia-ia-Me(s)Me(s) (Me) 6 - (F) 3 - (Me) 10 , wherein ia represents an inverted abasic nucleoside, and (s) represents a phosphorothioate linkage, and wherein nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
  • a nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar, and abasic modification pattern as follows (5’-3 ’): ia-ia-Me(s)Me(s) (Me) 6 - (F) 3 - (Me) 10 , wherein ia represents an inverted abasic nucleoside, and (s) represents a phosphorothioate linkage, and wherein nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
  • a nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar, and abasic modification pattern as follows (5’-3 ’): ia-ia-Me(s)Me(s) (Me) 6 - (F) 3 - (Me) 10 , wherein ia represents an inverted abasic nucleoside, and (s) represents a phosphorothioate linkage, and wherein nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
  • a nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar, and abasic modification pattern as follows (5’-3 ’): ia-ia-Me(s)Me(s) (Me) 6 - (F) 3 - (Me) 10 , wherein ia represents an inverted abasic nucleoside, and (s) represents a phosphorothioate linkage, and wherein nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
  • a nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar, and abasic modification pattern as follows (5’-3 ’): ia-ia-Me(s)Me(s) (Me) 6 - (F) 3 - (Me) 10 , wherein ia represents an inverted abasic nucleoside, and (s) represents a phosphorothioate linkage, and wherein nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
  • a nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar, and abasic modification pattern as follows (5’-3 ’): ia-ia-Me(s)Me(s) (Me) 6 - (F) 3 - (Me) 10 , wherein ia represents an inverted abasic nucleoside, and (s) represents a phosphorothioate linkage, and wherein nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
  • a nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar, and abasic modification pattern as follows (5’-3 ’): ia-ia-Me(s)Me(s) (Me) 6 - (F) 3 - (Me) 10 , wherein ia represents an inverted abasic nucleoside, and (s) represents a phosphorothioate linkage, and wherein nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
  • 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 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 eg 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 sense 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 SEQ ID NO:302 or SEQ ID NO:305, preferably wherein the linker is conjugated to the 3' terminal region of the second strand, i.e., to the 3' terminal region of SEQ ID NO:302 or SEQ ID NO:305, 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 SEQ ID NO:302 or SEQ ID NO:305, 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 SEQ ID NO:302 or SEQ ID NO:305, preferably wherein the linker is conjugated to the 3' terminal region of the second strand, i.e., to the 3' terminal region of SEQ ID NO:302 or SEQ ID NO:305, 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 SEQ ID NO:302 or SEQ ID NO:305, preferably wherein the linker is conjugated to the 3' terminal region of the second strand, i.e., to the 3' terminal region of SEQ ID NO:302 or SEQ ID NO:305, 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 SEQ ID NO:611 or SEQ ID NO:613, preferably wherein the linker is conjugated to the 3' terminal region of the second strand, i.e., to the 3' terminal region of SEQ ID NO:611 or SEQ ID NO:613, 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 SEQ ID NO:611 or SEQ ID NO:613, 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 SEQ ID NO:611 or SEQ ID NO:613, preferably wherein the linker is conjugated to the 3' terminal region of the second strand, i.e., to the 3' terminal region of SEQ ID NO:611 or SEQ ID NO:613, 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 SEQ ID NO:611 or SEQ ID NO:613, 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 SEQ ID NO:611 or SEQ ID NO:613, preferably wherein the linker is conjugated to the 3' terminal region of the second strand, i.e., to the 3' terminal region of SEQ ID NO:611 or SEQ ID NO:613, 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 first strand comprising or consisting of SEQ ID NO:503 and a modified second strand comprising or consisting of SEQ ID NO:611, preferably wherein the linker is conjugated to the 3' terminal region of the second strand, i.e., to the 3' terminal region of SEQ ID NO:611, 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 SEQ ID NO:611, 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 first strand comprising or consisting of SEQ ID NO: 503 and a modified second strand comprising or consisting of SEQ ID NO:611, preferably wherein the linker is conjugated to the 3' terminal region of the second strand, i.e., to the 3' terminal region of SEQ ID NO:611, 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 first strand comprising or consisting of SEQ ID NO: 503 and a modified second strand comprising or consisting of SEQ ID NO:611, preferably wherein the linker is conjugated to the 3' terminal region of the second strand, i.e., to the 3' terminal region of SEQ ID NO:611, 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 first strand comprising or consisting of SEQ ID NO:505 and a modified second strand comprising or consisting of SEQ ID NO:613, preferably wherein the linker is conjugated to the 3' terminal region of the second strand, i.e., to the 3' terminal region of SEQ ID NO:613, 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 SEQ ID NO:613, 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 first strand comprising or consisting of SEQ ID NO: 505 and a modified second strand comprising or consisting of SEQ ID NO:613, preferably wherein the linker is conjugated to the 3' terminal region of the second strand, i.e., to the 3' terminal region of SEQ ID NO:613, 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 first strand comprising or consisting of SEQ ID NO: 505 and a modified second strand comprising or consisting of SEQ ID NO:613, preferably wherein the linker is conjugated to the 3' terminal region of the second strand, i.e., to the 3' terminal region of SEQ ID NO:613, 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 first strand comprising or consisting of SEQ ID NO: 503 and a modified second strand comprising or consisting of SEQ ID NO:611, wherein the second strand has the following structure wherein:
  • T represents a 2’Me ribose modification
  • B represents the nucleoside bases of the first two basic nucleosides in the 5' terminal region of SEQ ID NO:611, and
  • Z represents the remaining 19 contiguous basic nucleosides of SEQ ID NO:611.
  • 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 first strand comprising or consisting of SEQ ID NO: 503 and a modified second strand comprising or consisting of SEQ ID NO:611, wherein the second strand has the following structure wherein:
  • T represents a 2’Me ribose modification
  • B represents the nucleoside bases of the first two basic nucleosides in the 5' terminal region of SEQ ID NO:611, and
  • Z represents the remaining 19 contiguous basic nucleosides of SEQ ID NO:611.
  • 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 first strand comprising or consisting of SEQ ID NO: 505 and a modified second strand comprising or consisting of SEQ ID NO:613, wherein the second strand has the following structure wherein:
  • T represents a 2’Me ribose modification
  • B represents the nucleoside bases of the first two basic nucleosides in the 5' terminal region of SEQ ID NO:613, and
  • Z represents the remaining 19 contiguous basic nucleosides of SEQ ID NO:613.
  • 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 first strand comprising or consisting of SEQ ID NO: 505 and a modified second strand comprising or consisting of SEQ ID NO:613, wherein the second strand has the following structure wherein:
  • T represents a 2’Me ribose modification
  • B represents the nucleoside bases of the first two basic nucleosides in the 5' terminal region of SEQ ID NO:613, and
  • Z represents the remaining 19 contiguous basic nucleosides of SEQ ID NO:613.
  • the invention provides a cell containing a nucleic acid, such as inhibitory RNA [RNAi] as described herein.
  • 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 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.
  • Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com 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
  • 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.
  • 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. 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. In some embodiments, the nucleic acid e.g. siRNA agent is administered at a dose of about 10 mg/kg to about 30 mg/kg. In certain embodiments, the nucleic acid e.g. siRNA agent is administered at a dose selected from about 0.5 mg/kg 1 mg/kg, 1.5 mg/kg, 3 mg/kg, 5 mg/kg, 10 mg/kg, and 30 mg/kg. In certain embodiments, the nucleic acid e.g.
  • the nucleic acid e.g. siRNA agent is administered about once per week, once per month, once every other two months, or once a quarter (i.e., once every three months) at a dose of about 0.1 mg/kg to about 5.0 mg/kg.
  • the nucleic acid e.g. siRNA agent is administered to the subject once a week.
  • the nucleic acid e.g. siRNA agent is administered to the subject once a month.
  • the nucleic acid e.g. siRNA agent is administered once per quarter (i.e. , every three months).
  • the treatments can be administered on a less frequent basis. For example, after administration weekly or biweekly for three months, administration can be repeated once per month, for six months, or a year; or longer.
  • the pharmaceutical composition can be administered once daily, or administered as two, three, or more sub-doses at appropriate intervals throughout the day or even using continuous infusion or delivery through a controlled release formulation.
  • 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.
  • 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. In certain preferred embodiments, the compositions are administered by intravenous infusion or injection. In certain embodiments, 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).
  • the present invention also provides methods of inhibiting expression of B4GALT1 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 B4GALT1 gene in the cell, thereby inhibiting expression of the B4GALT1 gene in the cell.
  • a nucleic acid “for inhibiting the expression of B4GALT1” is a nucleic acid that is capable of inhibiting B4GALT1 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.
  • inhibitor As used herein, is used interchangeably with “reducing,” “silencing,” “downregulating”, “suppressing”, and other similar terms, and includes any level of inhibition.
  • expression of B4GALT1 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 B4GALT1 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 B4GALT1 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 B4GALT1 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 B4GALT1 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 B4GALT1 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 B4GALT1 gene with an IC50 value lower than 100 pM.
  • Inhibition of expression of the B4GALT1 gene may be quantified by the following method:
  • Huh7 cells human hepatocyte-derived cell line, obtained from JCRB Cell Bank
  • DMEM Dulbecco’s Modified Eagle Medium
  • FBS FBS
  • Cells may then be transfected with siRNA duplexes targeting B4GALT1 mRNA or a negative control siRNA (siRNA-control; sense strand 5’- UUCUCCGAACGUGUCACGUTT-3’ (SEQ ID NO:934), antisense strand 5’- ACGUGACACGUUCGGAGAATT-3’ (SEQ ID NO:933)) using 10x3-fold serial dilutions over a final duplex concentration range of 20 nM to 1 pM.
  • siRNA duplexes targeting B4GALT1 mRNA or a negative control siRNA (siRNA-control; sense strand 5’- UUCUCCGAACGUGUCACGUTT-3’ (SEQ ID NO:934), antisense strand 5’- ACGUGACACGUUCGGAGAATT-3’ (S
  • 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% CO2 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).
  • Realtime quantitative PCR may be performed on an ABI Prism 7900HT or ABI QuantStudio 7 with primers specific for human B4GALT1 (Hs00155245_ml) and human GAPDH (Hs02786624_gl) 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 B4GALT1 expression may be calculated from mean Ct values using the comparative Ct (AACt) method, normalised to GAPDH and relative to untreated cells.
  • Maximum percent inhibition of B4GALT1 expression and IC50 values may be calculated using a four parameter (variable slope) model using GraphPad Prism 9.
  • the inhibitory potential of a nucleic acid of the invention may be quantified without prior transfection of a target cell with said nucleic acid.
  • the nucleic acid of the invention when cells are incubated with a nucleic acid of the invention, inhibits expression of the B4GALT1 gene with an EC50 value lower than 1000 nM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200 nM, or 100 nM, 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 cells are incubated with a nucleic acid of the invention, the nucleic acid of the invention inhibits expression of the B4GALT1 gene with an EC50 value lower than 1000 nM. In a more preferred embodiment, when cells are incubated with a nucleic acid of the invention, the nucleic acid of the invention inhibits expression of the B4GALT1 gene with an EC50 value lower than 500 nM. In an even more preferred embodiment, when cells are incubated with a nucleic acid of the invention, the nucleic acid of the invention inhibits expression of the B4GALT1 gene with an EC50 value lower than 200 nM. In a most preferred embodiment, when cells are incubated with a nucleic acid of the invention, the nucleic acid of the invention inhibits expression of the B4GALT1 gene with an EC50 value lower than 100 nM.
  • Inhibition of expression of the B4GALT1 gene in the presence of free nucleic acids may be quantified using the following method:
  • PMHs Primary C57BL/6 mouse hepatocytes
  • DMEM Gibco-11995-092
  • FBS Penicillin/Streptomycin
  • HEPES HEPES
  • L-glutamine L-glutamine
  • Cells may be cultured at 37°C in an atmosphere with 5% CO2 in a humidified incubator.
  • PMHs may be seeded at a density of 36,000 cells/well in regular 96-well tissue culture plates.
  • Dose response analysis in PMHs may be done by direct incubation of cells in a gymnotic free uptake setting with final GalNAc-siRNA concentrations of 1000, 500, 250, 125, 62.5, 31.3, 15.6, 7.8, 3.9, 1.95 nM.
  • cells may be incubated without GalNAc-siRNA.
  • After 48hr incubation, cells may be harvested for RNA extraction. Total RNA may be extracted using RNeasy Kit following the manufacturer’s instructions (Qiagen, Shanghai, China).
  • real-time quantitative PCR may be performed using an ABI Prism 7900HT to detect the relative abundance of B4GALT1 mRNA normalized to the housekeeping gene GAPDH.
  • the expression of the target gene in each test sample may be determined by relative quantitation using the comparative Ct (AACt) method. This method measures the Ct differences (ACt) between target gene and housekeeping gene.
  • AACt comparative Ct
  • ACt Ct differences between target gene and housekeeping gene.
  • inhibition of expression of the B4GALT1 gene may be characterized by a reduction of mean relative expression of the B4GALT1 gene.
  • the mean relative expression of B4GALT1 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 B4GALT1 is below 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3 or 0.2, preferably when determined by qPCR, more preferably by reverse transcriptase (RT)-qPCR, as described herein.
  • Mean relative expression of the B4GALT1 gene may be quantified by the following method:
  • Huh7 cells human hepatocyte-derived cell line, obtained from JCRB Cell Bank
  • DMEM Dulbecco’s Modified Eagle Medium
  • FBS FBS
  • Cells may be transfected with siRNA duplexes targeting B4GALT1 mRNA or a negative control siRNA (siRNA-control; sense strand 5’- UUCUCCGAACGUGUCACGUTT-3’ (SEQ ID NO:934), antisense strand 5’- ACGUGACACGUUCGGAGAATT-3’ (SEQ ID NO:933)) at a final duplex concentration of 5 nM and 0.1 nM.
  • siRNA duplexes targeting B4GALT1 mRNA or a negative control siRNA (siRNA-control; sense strand 5’- UUCUCCGAACGUGUCACGUTT-3’ (SEQ ID NO:934), antisense strand 5’- ACGUGACACGUUCGGAGAATT-3’ (SEQ ID NO:933)
  • 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% CO2 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).
  • Realtime quantitative PCR may be performed on an ABI Prism 7900HT or ABI QuantStudio 7 with primers specific for human B4GALT1 (Hs00155245_ml) and human GAPDH (Hs02786624_gl) using a TaqMan Gene Expression Assay Kit (ThermoFisher Scientific). qPCR may be performed in duplicate on cDNA derived from each well and the mean Ct calculated. Relative B4GALT1 expression may be calculated from mean Ct values using the comparative Ct (AACt) method, normalised to GAPDH and relative to untreated cells.
  • AACt comparative Ct
  • Inhibition of the expression of B4GALT1 gene may be manifested by a reduction of the amount of mRNA of the target B4GALT1 gene in comparison to a suitable control.
  • inhibition of the expression of B4GALT1 gene may be assessed in terms of a reduction of a parameter that is functionally linked to gene expression, e.g , protein expression or signaling pathways.
  • the present invention also provides methods of using nucleic acid e.g. an siRNA of the invention or a composition containing nucleic acid e.g. an siRNA of the invention to reduce or inhibit B4GALT1 gene expression in a cell.
  • the methods include contacting the cell with a nucleic acid e.g. dsiRNA of the invention and maintaining the cell for a time sufficient to obtain degradation of the mRNA transcript of B4GALT1, thereby inhibiting expression of the B4GALT1 gene in the cell. Reduction in gene expression can be assessed by any methods known in the art.
  • the cell may be contacted in vitro or in vivo, i.e., the cell may be within a subject.
  • a cell suitable for treatment using the methods of the invention may be any cell that expresses a gene of interest associated with diabetes or cardiovascular disease.
  • the in vivo methods of the invention may include administering to a subject a composition containing a nucleic acid of the invention e.g. an 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 B4GALT1 gene of the mammal to be treated.
  • a nucleic acid of the invention e.g. an siRNA
  • the nucleic acid e.g. siRNA includes a nucleoside sequence that is complementary to at least a part of an RNA transcript of B4GALT1 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 B4GALT1 gene, in a therapeutically effective amount e.g. a nucleic acid such as an siRNA targeting B4GALT1 or a pharmaceutical composition comprising the nucleic acid targeting B4GALT1.
  • a nucleic acid such as an siRNA of the invention
  • a pharmaceutical composition comprising the nucleic acid targeting B4GALT1.
  • the disease to be treated is diabetes or a cardiovascular disease.
  • diabetes refers to group of metabolic diseases in which a subject has high blood sugar, either because the body does not produce enough insulin, or because cells do not respond to the insulin that is produced.
  • Type 1 diabetes results from the body's failure to produce insulin, and presently requires the person to inject insulin.
  • IDDM insulin-dependent diabetes mellitus
  • Type 2 diabetes T2D results from insulin resistance, a condition in which cells fail to use insulin properly, sometimes combined with an absolute insulin deficiency.
  • NIDDM non-insulin-dependent diabetes mellitus
  • GD Gestational diabetes
  • the nucleic acid according to the invention or a pharmaceutical composition comprising said nucleic acid is used for the treatment of diabetes, preferably type 2 diabetes (T2D).
  • diabetes preferably type 2 diabetes (T2D).
  • the nucleic acid according to the invention pharmaceutical composition comprising said nucleic acid is used for the treatment of diabetes, preferably type 2 diabetes (T2D), wherein the treatment results in a reduction in LDL-cholesterol (LDL- c) levels in the blood.
  • the nucleic acid according to the invention pharmaceutical composition comprising said nucleic acid is used for the treatment of diabetes, preferably type 2 diabetes (T2D), wherein the treatment results in a reduction in fasting glcuose levels in the blood.
  • the nucleic acid according to the invention pharmaceutical composition comprising said nucleic acid is used for the treatment of diabetes, preferably type 2 diabetes (T2D), wherein the treatment results in a reduction in fibrinogen levels in the blood.
  • diabetes preferably type 2 diabetes (T2D)
  • T2D type 2 diabetes
  • cardiovascular disease refers to any condition, disorder or disease state associated with, resulting from or causing a structural or functional abnormality of the heart, or of the blood vessels supplying the heart, that impairs its normal functioning.
  • Cardiovascular disease may comprise coronary artery disease, atherosclerosis, myocardial infarction, arteriosclerosis, hypertension, angina, deep vein thrombosis, stroke, congestive heart failure or arrhythmia.
  • the cardiovascular disease is coronary artery disease.
  • the nucleic acid according to the invention pharmaceutical composition comprising said nucleic acid is used for the treatment of cardiovascular disease, preferably coronary artery disease.
  • the nucleic acid according to the invention pharmaceutical composition comprising said nucleic acid is used for the treatment of cardiovascular disease, preferably coronary artery disease, wherein the treatment results in a reduction in LDL- cholesterol (LDL-c) levels in the blood.
  • cardiovascular disease preferably coronary artery disease
  • LDL-c LDL- cholesterol
  • the nucleic acid according to the invention pharmaceutical composition comprising said nucleic acid is used for the treatment of cardiovascular disease, preferably coronary artery disease, wherein the treatment results in a reduction in fibrinogen levels in the blood.
  • An 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 B4GALT1 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 B4GALT1 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 treat disease related to diabetes or cardiovascular disease.
  • nucleic acid e.g. siRNA
  • 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 B4GALT1 target gene , e.g., in a cell or tissue of the patient by at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or below the level of detection of the assay method used. In certain embodiments, administration results in clinical stabilization or preferably clinically relevant reduction of at least one sign or symptom of a B4GALT1 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 B4GALT1 as defined in any of the sentences hereinafter.
  • R 1 at each occurrence is independently selected from the group consisting of hydrogen, methyl and ethyl;
  • X 1 and X2 at each occurrence are independently selected from the group consisting of methylene, oxygen and sulfur;
  • m is an integer of from 1 to 6;
  • n is an integer of from 1 to 10;
  • q, r, s, t, v are independently integers from 0 to 4, with the proviso that:
  • Z is an oligonucleoside moiety.
  • a compound according to any of Sentences 1 to 17, wherein m 3.
  • a compound according to any of Sentences 1 to 18, wherein n 6.
  • Zi, Z2, Z3, Z4 are independently at each occurrence oxygen or sulfur; and one the bonds between P and Z2, and P and Z3 is a single bond and the other bond is a double bond.
  • RNA compound comprises an RNA duplex comprising first and second strands, wherein the first strand is at least partially complementary to an RNA sequence of a target gene, and the second strand is at least partially complementary to said first strand, and wherein each of the first and second strands have 5’ and 3’ ends.
  • a composition comprising a compound of Formula (II) as defined in Sentence 27, and a compound of Formula (III) as defined in Sentence 28, optionally dependent on Sentence 29.
  • 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.
  • 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.
  • 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 intemucleoside 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 any of Sentences 1 to 29, or 32 to 34, or 37 to 40, wherein said ligand moiety as depicted in Formula (I) in Sentence 1 comprises one or more ligands.
  • a compound according to Sentence 41, wherein said ligand moiety as depicted in Formula (I) in Sentence 1 comprises one or more carbohydrate ligands.
  • a compound according to Sentence 42, wherein said one or more carbohydrates can be a monosaccharide, di saccharide, tri saccharide, tetrasaccharide, oligosaccharide or polysaccharide.
  • a compound according to Sentence 43 wherein said one or more carbohydrates comprise one or more galactose moieties, one or more lactose moieties, one or more N- AcetylGalactosamine moieties, and / or one or more mannose moieties.
  • 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 any of Formulae (Via), (VIb) or (Vic), preferably Formula (Via):
  • Ai is hydrogen, or a suitable hydroxy protecting group; a is an integer of 2 or 3; and b is an integer of 2 to 5; or
  • Ai is hydrogen, or a suitable hydroxy protecting group; a is an integer of 2 or 3; and c and d are independently integers of 1 to 6; or
  • Ai is hydrogen, or a suitable hydroxy protecting group; a is an integer of 2 or 3; and e is an integer of 2 to 10.
  • a compound according to Sentences 46 to 48, wherein said moiety: as depicted in Formula (I) in Sentence 1 is Formula (VII): wherein:
  • Ai is hydrogen; a is an integer of 2 or 3.
  • a compound according to Sentence 49 or 50, wherein a 2.
  • a compound according to Sentence 49 or 50, wherein a 3.
  • a compound according to Sentence 49, wherein b 3.
  • 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.
  • Formula (XI) 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.
  • a composition according to Sentence 62 wherein said compound of Formula (XI) as defined in Sentence 60 is present in an amount in the range of 10 to 15% by weight of said composition.
  • R 1 at each occurrence is independently selected from the group consisting of hydrogen, methyl and ethyl;
  • X 1 and X2 at each occurrence are independently selected from the group consisting of methylene, oxygen and sulfur;
  • m is an integer of from 1 to 6;
  • n is an integer of from 1 to 10;
  • q, r, s, t, v are independently integers from 0 to 4, with the proviso that:
  • Z is an oligonucleoside moiety; and where appropriate carrying out deprotection of the ligand and / or annealing of a second strand for the oligonucleoside moiety.
  • R 1 at each occurrence is independently selected from the group consisting of hydrogen, methyl and ethyl;
  • X 1 and X2 at each occurrence are independently selected from the group consisting of methylene, oxygen and sulfur;
  • q, r, s, t, v are independently integers from 0 to 4, with the proviso that:
  • Z is an oligonucleoside moiety.
  • Formula (Xlllb) 75 A process according to Sentences 69, as dependent on Sentences 70 to 73, wherein: compound of Formula (XIV) is either Formula (XlVa) or Formula (XlVb):
  • Formula (XlVb) and compound of Formula (XV) is either Formula (XVa) or Formula (XlVb):
  • Formula (XVb) 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 (i) said RNA duplex is attached at the 5’ end of its second strand to the adjacent phosphate in Formula (XVa), or (ii) said RNA duplex is attached at the 3’ end of its second strand to the adjacent phosphate in Formula (XVb).
  • R 1 at each occurrence is independently selected from the group consisting of hydrogen, methyl and ethyl;
  • X 1 and X2 at each occurrence are independently selected from the group consisting of methylene, oxygen and sulfur;
  • q, r, s, t, v are independently integers from 0 to 4, with the proviso that:
  • Z is an oligonucleoside moiety.
  • R 1 at each occurrence is independently selected from the group consisting of hydrogen, methyl and ethyl; m is an integer of from 1 to 6; n is an integer of from 1 to 10.
  • a compound of Formula (XIV) Formula (XIV) wherein: R 1 is selected from the group consisting of hydrogen, methyl and ethyl;
  • X2 is selected from the group consisting of methylene, oxygen and sulfur; s, t, v are independently integers from 0 to 4, with the proviso that s, t and v cannot all be 0 at the same time.
  • 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.
  • a compound according to Sentence 77 for the preparation of a compound according to any of Sentences 20, 25, 27, 29, 54, 56, and / or a composition according to any of Sentences 30, 31, 57, 58.
  • Use of a compound according to Sentence 78 for the preparation of a compound according to any of Sentences 20, 25, 28, 29, 55, 56, and / or a composition according to any of Sentences 30, 31, 57, 58.
  • Use of a compound according to Sentence 79 for the preparation of a compound according to any of Sentences 21, 26, 32, 34, 59, 61, and / or a composition according to any of Sentences 35, 36, 62, 63.
  • a compound according to Sentence 80 for the preparation of a compound according to any of Sentences 21, 26, 33, 34, 60, 61, and / or a composition according to any of Sentences 35, 36, 62, 63.
  • Use of a compound according to Sentence 88 for the preparation of a compound according to any of Sentences 20, 25, 27 to 29, 54 to 56, and / or a composition according to any of Sentences 30, 31, 57, 58.
  • Use of a compound according to Sentence 89 for the preparation of a compound according to any of Sentences 21, 26, 32 to 34, 59 to 61, and / or a composition according to any of Sentences 35, 36, 62, 63.
  • 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.
  • an oligonucleoside moiety as represented by Z in any of the following clauses can comprise a nucleic acid for inhibiting expression of B4GALT1 as defined in any of the sentences hereinafter.
  • Z is an oligonucleoside moiety.
  • Zi, Z2, Z3, Z4 are independently at each occurrence oxygen or sulfur; and one the bonds between P and Z2, and P and Z3 is a single bond and the other bond is a double bond.
  • oligonucleoside is an RNA compound capable of modulating, preferably inhibiting, expression of a target gene.
  • RNA compound comprises an RNA duplex comprising first and second strands, wherein the first strand is at least partially complementary to an RNA sequence of a target gene, and the second strand is at least partially complementary to said first strand, and wherein each of the first and second strands have 5’ and 3’ ends.
  • Formula (III) A compound as defined in any of Clauses 1 to 15, 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.
  • a compound according to Clause 16 wherein the modifications are chosen from 2’-O- methyl, 2’ -deoxy -fluoro, and 2’-deoxy.
  • the oligonucleoside further comprises one or more degradation protective moieties at one or more ends.
  • a compound according to Clause 21, wherein said one or more carbohydrates can be a monosaccharide, di saccharide, tri saccharide, tetrasaccharide, oligosaccharide or polysaccharide.
  • a compound according to Clauses 20 to 27, wherein said moiety: as depicted in Formula (I) in Clause 1 is any of Formulae (IV), (V) or (VI), preferably Formula (IV):
  • Ai is hydrogen, or a suitable hydroxy protecting group; a is an integer of 2 or 3; and b is an integer of 2 to 5; or
  • Ai is hydrogen, or a suitable hydroxy protecting group; a is an integer of 2 or 3; and c and d are independently integers of 1 to 6; or
  • Ai is hydrogen, or a suitable hydroxy protecting group; a is an integer of 2 or 3; and e is an integer of 2 to 10.
  • Ai is hydrogen; a is an integer of 2 or 3.
  • Formula (IX) 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.
  • a compound according to Clause 35, wherein the modifications are chosen from 2’-O- methyl, 2’ -deoxy -fluoro, and 2’-deoxy.
  • a compound according to Clause 37 wherein said one or more degradation protective moieties are not present at the end of the 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.
  • 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.
  • 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.
  • Z is an oligonucleoside moiety; and where appropriate carrying out deprotection of the ligand and / or annealing of a second strand for the oligonucleoside.
  • Formula (Xia) 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.
  • Formula (Xia) 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.
  • Z is an oligonucleoside moiety.
  • a pharmaceutical composition comprising of a compound according to any of Clauses 1 to 40, together with a pharmaceutically acceptable carrier, diluent or excipient.
  • TLC Thin layer chromatography
  • fluorescence indicator 254 nm from Macherey -Nagel.
  • Compounds were visualized under UV light (254 nm), or after spraying with the 5% H2SO4 in methanol (MeOH) or ninhydrin reagent according to Stahl (from Sigma-Aldrich), followed by heating.
  • Flash chromatography was performed with a Biotage Isol era One flash chromatography instrument equipped with a dual variable UV wavelength detector (200-400 nm) using Biotage Sfar Silica 10, 25, 50 or 100 g columns (Uppsala, Sweden).
  • HPLC/ESI-MS was performed on a Dionex UltiMate 3000 RS UHPLC system and Thermo Scientific MSQ Plus Mass spectrometer using an Acquity UPLC Protein BEH C4 column from Waters (300A, 1.7 pm, 2.1 x 100 mm) at 60 °C.
  • the solvent system consisted of solvent A with H2O containing 0.1% formic acid and solvent B with acetonitrile (ACN) containing 0.1% formic acid. A gradient from 5-100% of B over 15 min with a flow rate of 0.4 mL/min was employed.
  • Detector and conditions Corona ultra-charged aerosol detection (from esa). Nebulizer Temp.: 25 °C. N2 pressure: 35.1 psi. Filter: Corona.
  • A,A,A',M-tetramethyl-O-(lH-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 NN- 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 1- hydroxybenzotriazole hydrate
  • TriGalNAc (12) Triantennary GalNAc compound 10 (0.35 g, 0.24 mmol, 1.0 eq.) and compound 11 (0.11 g, 0.31 mmol, 1.5 eq.) were dissolved in DCM (5 mL) under argon and triethylamine (0.1 mL, 0.61 mmol, 3.0 eq.) was added. The reaction was stirred at room temperature overnight. The solvent was removed under reduced pressure, the residue was dissolved in EtOAc (100 mL) and washed with water (100 mL). The organic layer was separated and dried over Na2SO4.
  • Tether 1 conjugation of Tether 1 to a siRNA strand: Monofluoro cyclooctyne (MFCO) conjugation at 5’-or 3’-end
  • reaction mixture was diluted 15-fold with water, filtered through a 1.2 pm filter from Sartorius and then purified by reserve phase (RP HPLC) on an Akta Pure instrument (GE Healthcare).
  • RP HPLC purification was performed using a XBridge C18 Prep 19 x 50 mm column from Waters.
  • Buffer A was 100 mM tri ethyl ammonium acetate pH 7 and buffer B contained 95% acetonitrile in buffer A.
  • a flow rate of 10 mL/min and a temperature of 60°C were employed.
  • UV traces at 280 nm were recorded.
  • a gradient of 0-100% B within 60 column volumes was employed.
  • conjugates were desalted by size exclusion chromatography using Sephadex G25 Fine resin (GE Healthcare) on an Akta Pure (GE Healthcare) instrument to yield the conjugated oligonucleotides in an isolated yield of 50-70%.
  • the two complementary strands were annealed by combining equimolar aqueous solutions of both strands. The mixtures were placed into a water bath at 70°C for 5 minutes and subsequently allowed to cool to ambient temperature within 2 h. The duplexes were lyophilized for 2 days and stored at -20°C.
  • duplexes were analyzed by analytical SEC HPLC on SuperdexTM 75 Increase 5/150 GL column 5 x 153-158 mm (Cytiva) on a Dionex Ultimate 3000 (Thermo Fisher Scientific) HPLC system.
  • Mobile phase consisted of lx PBS containing 10% acetonitrile.
  • An isocratic gradient was run in 10 min at a flow rate of 1.5 mL/min at room temperature. UV traces at 260 and 280 nm were recorded.
  • Water (LC-MS grade) was purchased from Sigma- Aldrich and Phosphate-buffered saline (PBS; lOx, pH 7.4) was purchased from GIBCO (Thermo Fisher Scientific).
  • TLC Thin layer chromatography
  • silica-coated aluminium plates with fluorescence indicator 254 nm from Macherey -Nagel. Compounds were visualized under UV light (254 nm), or after spraying with the 5% H2SO4 in methanol (MeOH) or ninhydrin reagent according to Stahl (from Sigma-Aldrich), followed by heating.
  • Flash chromatography was performed with a Biotage Isol era One flash chromatography instrument equipped with a dual variable UV wavelength detector (200-400 nm) using Biotage Sfar Silica 10, 25, 50 or 100 g columns (Uppsala, Sweden).
  • HPLC/ESI-MS was performed on a Dionex UltiMate 3000 RS UHPLC system and Thermo Scientific MSQ Plus Mass spectrometer using an Acquity UPLC Protein BEH C4 column from Waters (300A, 1.7 ⁇ m, 2.1 x 100 mm) at 60 °C.
  • the solvent system consisted of solvent A with H2O containing 0.1% formic acid and solvent B with acetonitrile (ACN) containing 0.1% formic acid. A gradient from 5-100% of B over 15 min with a flow rate of 0.4 mL/min was employed.
  • Detector and conditions Corona ultra-charged aerosol detection (from esa). Nebulizer Temp.: 25 °C. N2 pressure: 35.1 psi. Filter: Corona.
  • A,A,A',A'-tetramethyl-O-(lH-benzotriazol-l- yl)uronium hexafluorophosphate (HBTU) (2.78 g, 7.44 mmol, 5.0 eq.), 1- hydroxybenzotriazole hydrate (HOBt) (1.05 g, 7.44 mmol, 5.0 eq.) and 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 diisopropylethylamine
  • TriGalNAc Triantennary GalNAc compound 14 (0.31 g, 0.15 mmol, 1.0 eq.) was dissolved in EtOAc (15 mL) and Pd/C (40 mg) was added. The reaction mixture was degassed by using vacuum/argon cycles (3x) and hydrogenated under balloon pressure overnight. The completion of the reaction was monitored by mass spectrometry and the resulting mixture was filtered through a thin pad of celite. The solvent was removed under reduced pressure and the resulting residue was dried under high vacuum overnight. The residue was used for conjugations to oligonucleosides without further purification (0.28 g, quantitative yield). MS: calculated for C81H131N7O42, 1874.9. Found 1875.3.
  • Tether 2 Conjugation of Tether 2 to a siRNA strand: TriGalNAc tether 2 (GalNAc-T2) conjugation at 5’-end or 3’-end
  • 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.
  • conjugates were desalted by size exclusion chromatography using Sephadex G25 Fine resin (GE Healthcare) on an Akta Pure (GE Healthcare) instrument to yield the conjugated oligonucleotides in an isolated yield of 60-80%.
  • the conjugates were characterized by HPLC-MS analysis with a 2.1 x 50 mm XBridge C18 column (Waters) on a Dionex Ultimate 3000 (Thermo Fisher Scientific) HPLC system equipped with a Compact ESLQq-TOF mass spectrometer (Bruker Daltonics).
  • Buffer A was 16.3 mM triethylamine, 100 mM HFIP in 1% MeOH in H2O and buffer B contained 95% MeOH in buffer A.
  • a flow rate of 250 ⁇ L/min and a temperature of 60°C were employed.
  • UV traces at 260 and 280 nm were recorded.
  • a gradient of 1-100% B within 31 min was employed.
  • the two complementary strands were annealed by combining equimolar aqueous solutions of both strands. The mixtures were placed into a water bath at 70°C for 5 minutes and subsequently allowed to cool to ambient temperature within 2 h. The duplexes were lyophilized for 2 days and stored at -20°C.
  • duplexes were analyzed by analytical SEC HPLC on SuperdexTM 75 Increase 5/150 GL column 5 x 153-158 mm (Cytiva) on a Dionex Ultimate 3000 (Thermo Fisher Scientific) HPLC system.
  • Mobile phase consisted of lx PBS containing 10% acetonitrile.
  • An isocratic gradient was run in 10 min at a flow rate of 1.5 mL/min at room temperature. UV traces at 260 and 280 nm were recorded.
  • Water (LC-MS grade) was purchased from Sigma- Aldrich and Phosphate-buffered saline (PBS; 10x, pH 7.4) was purchased from GIBCO (Thermo Fisher Scientific).
  • TriGalNAc Tether2 Conjugation of Tether 2 to a siRNA strand: TriGalNAc tether 2 (GalNAc-T2) conjugation at 5’-end or 3’-end
  • 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)- ur
  • 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)
  • 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 NH4OH:EtOH solution 4: 1 (v/v) for 20 hours at 45°C (TCI).
  • TCI 45°C
  • the solid support was then filtered off, the filter was thoroughly washed with H2O 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 IM sodium acetate.
  • Example 7 Solid phase synthesis method: scale >5 ⁇ mol
  • Syntheses of siRNA sense and antisense strands were performed on a MerMadel2 synthesiser with commercially available solid supports made of controlled pore glass with universal linker (Universal CPG, with a loading of 40 pmol/g; LGC Biosearch or Glen Research) at 5 ⁇ mol scale.
  • Sense strand destined to 3' conjugation were sytnthesised at 12 pmol on 3'-PT-Amino-Modifier C6 CPG 500 A solid support with a loading of 86 pmol/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)- ur
  • 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)
  • oligonucleotides were cleaved from the solid support using a NH4OH:EtOH solution 4: 1 (v/v) for 20 hours at 45°C (TCI). The solid support was then filtered off, the filter was thoroughly washed with H2O and the volume of the combined solution was reduced by evaporation under reduced pressure. [00390] Oligonucleotide were treated to form the sodium salt by EtOH precipitation from IM sodium acetate.
  • the single strand oligonucleotides were purified by IP-RP HPLC on Xbridge BEH C18 5 pm, 130 A, 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.
  • Sense strands were conjugated as per protocol provided in any of Examples 1, 3, 5.
  • Sense and Antisense strands were then annealed in water to form the final duplex siRNA and duplex purity were assessed by size exclusion chromatography.
  • siRNA oligonucleosides according to the present invention target B4GALT1.
  • DNA sequence of the B4GALT1 target is as follows (SEQ ID NO: 1):

Abstract

The present invention provides novel nucleic acid compound 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.

Description

NUCLEIC ACID COMPOUNDS
FIELD
[0001] 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.
BACKGROUND OF THE INVENTION
[0002] 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.
[0003] A number of modified siRNA compounds in particular have been developed in the last two decades for diagnostic and therapeutic purposes, including siRNA / RNAi therapeutic agents for the treatment of various diseases including central-nervous-system diseases, inflammatory diseases, metabolic disorders, oncology, infectious diseases, and ocular diseases.
[0004] The present invention relates to nucleic acid compounds, for use in the treatment and / or prevention of disease.
STATEMENTS OF INVENTION
[0005] According to a first aspect of the present invention, there is provided a nucleic acid for inhibiting expression of B4GALT1, 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 B4GALT1 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.
[0006] According to a second aspect of the present invention, there is provided a nucleic acid for inhibiting expression of B4GALT1, 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 B4GALT1 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.
[0007] 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.
[0008] 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.
[0009] 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.
[0010] 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.
[0011] 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. [0012] 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.
[0013] A nucleic acid according to the above second aspect of the present invention, wherein the first strand comprises any one of the first strand modified sequences as listed in Table 3.
[0014] A nucleic acid according to the above first aspect of the present invention, wherein the second strand comprises any one of the second strand sequences as listed in Table 2.
[0015] A nucleic acid according to the above second aspect of the present invention, wherein the second strand comprises any one of the second strand modified sequences as listed in Table 4.
[0016] A nucleic acid according to the above first aspect of the present invention, wherein the first strand comprises any one of the following sequences: SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41.
[0017] A nucleic acid according to the above second aspect of the present invention, wherein the first strand comprises any one of the following sequences: SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81.
[0018] A nucleic acid according to the above first aspect of the present invention, wherein the second strand comprises any one of the following sequences: SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61.
[0019] A nucleic acid according to the above second aspect of the present invention, wherein the second strand comprises any one of the following sequences: SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101.
[0020] A nucleic acid comprising first and second strands that comprise, consist of, or consist essentially of a nucleoside sequence differing by 0 or 1 nucleosides from any one of the following first and second sequences:
Figure imgf000005_0001
[0021] A nucleic acid comprising first and second strands that comprise, consist of, or consist essentially of a nucleoside sequence differing by 0 or 1 nucleosides from any one of the following first and second sequences:
Figure imgf000005_0002
[0022] A nucleic acid comprising first and second strands that comprise, consist of, or consist essentially of a nucleoside sequence differing by 0 or 1 nucleosides from any one of the following first and second sequences:
Figure imgf000006_0001
[0023] A nucleic acid comprising first and second strands that comprise, consist of, or consist essentially of a nucleoside sequence differing by 0 or 1 nucleosides from any one of the following first and second sequences:
Figure imgf000006_0002
Figure imgf000007_0001
[0024] A nucleic acid comprising first and second strands that comprise, consist of, or consist essentially of a nucleoside sequence differing by 0 or 1 nucleosides from any one of the following first and second sequences:
Figure imgf000007_0002
Figure imgf000008_0001
[0025] A nucleic acid comprising first and second strands that comprise, consist of, or consist essentially of a nucleoside sequence differing by 0 or 1 nucleosides from any one of the following first and second sequences:
Figure imgf000008_0002
Figure imgf000009_0001
Figure imgf000010_0001
[0026] A nucleic acid comprising first and second strands that comprise, consist of, or consist essentially of a nucleoside sequence differing by 0 or 1 nucleosides from any one of the following first and second sequences:
Figure imgf000010_0002
[0027] A nucleic acid comprising first and second strands that comprise, consist of, or consist essentially of a nucleoside sequence differing by 0 or 1 nucleosides from any one of the following first and second sequences:
Figure imgf000010_0003
Figure imgf000011_0001
[0028] A nucleic acid comprising first and second strands that comprise, consist of, or consist essentially of a nucleoside sequence differing by 0 or 1 nucleosides from any one of the following first and second sequences:
Figure imgf000011_0002
[0029] A nucleic acid comprising first and second strands that comprise, consist of, or consist essentially of a nucleoside sequence differing by 0 or 1 nucleosides from any one of the following first and second sequences:
Figure imgf000011_0003
[0030] A conjugate for inhibiting expression of B4GALT1 target gene in a cell, said conjugate comprising a nucleic acid as disclosed herein and one or more ligand moieties.
[0031] A pharmaceutical composition comprising a nucleic acid as disclosed herein, in combination with a pharmaceutically acceptable excipient or carrier.
[0032] A nucleic acid or pharmaceutical composition, for use in therapy.
[0033] A nucleic acid or pharmaceutical composition, for use in prevention or treatment of diabetes. [0034] A nucleic acid or pharmaceutical composition, for use in prevention or treatment of cardiovascular disease.
FIGURES
[0035] Figure 1: Linker and ligand portions of constructs suitable for use according to the present invention including tether la. While Figure 1 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.
It should also be understood that while 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 occurring as a result of hydrolytic displacement, such as an OH substituent. In this way, (a) tether la 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 la 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 a substituent occurring as a result of hydrolytic displacement, such as an OH substituent, or (c) tether la constructs can comprise a mixture of molecules as defined in (a) and/or (b).
[0036] Figure 2: Linker and ligand portions of constructs suitable for use according to the present invention including tether lb. While Figure 2 depicts the linker to be conjugated to an oligonucleotide, it is to be understood that the present invention also encompasses conjugates of the same linker with an oligonucleoside as disclosed herein.
The comments made in relation to Figure 1 and the possible replacement of the F substituent as shown in Figure 1 on the cyclo-octyl ring replaced by a substituent occurring as a result of hydrolytic displacement, such as an OH substituent, apply equally to tether lb constructs. In this way, (a) tether lb 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 lb 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 a substituent occurring as a result of hydrolytic displacement, such as an OH substituent, or (c) tether lb constructs can comprise a mixture of molecules as defined in (a) and/or (b).
[0037] Figure 3: Linker and ligand portions of constructs suitable for use according to the present invention including tether 2a. While 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.
[0038] 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.
[0039] Figure 5: Formulae described in Sentences 1-101 disclosed herein.
[0040] Figure 6: Formulae described in Clauses 1-56 disclosed herein
[0041] Figures 7a and 7b: Inverted abasic constructs that can be used with nucleic acid sequences according to the present invention as described herein. For Figure 7a, a galnac linker is attached to the 5’ end region of the sense strand in use (not depicted in Figure 7a). For Figure 7b, 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.
[0042] Figure 8: Duplex construct according to Table 5.
[0043] Figure 9: Results of an RNAi molecule screen for inhibition of B4GALT1 mRNA expression in human Huh7 cells. Each bar represents mean relative expression of B4GALT1 mRNA compared to untreated wells after treatment with an individual siRNA construct at 5 nM.
[0044] Figure 10: Results of an RNAi molecule screen for inhibition of B4GALT1 mRNA expression in human Huh7 cells. Each bar represents mean relative expression of B4GALT1 mRNA compared to untreated wells after treatment with an individual siRNA construct at 0.1 nM.
[0045] Figure 11: Results of dose-response experiments for inhibition of B4GALT1 mRNA expression in human Huh7 cells. Points represent mean relative expression of B4GALT1 mRNA compared to untreated wells after treatment with siRNA construct at the indicated concentrations on the x-axis. Error bars represent standard deviation of the mean. 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.
[0046] Figure 12: Time course inhibition of B4GALT1 expression in mice.
[0047] Figure 13: Inhibition of B4GALT1 expression by ETXM1200 (ETXS2400 & ETXS2399), ETXM1201 (ETXS2402 & ETXS2401), ETXM1217 (ETXS2434 & ETXS2401), ETXM1764 (ETXS3528 & ETXS2401), ETXM1765 (ETXS3530 & ETXS2401), ETXM1766 (ETXS3532 & ETXS2401), ETXM 1767 (ETXS3534 & ETXS2401), ETXM 1768 (ETXS3536 & ETXS2401), ETXM1769 (ETXS3538 & ETXS2401) and ETXM1770 (ETXS3540 & ETXS2401).
[0048] Figure 14: Inhibition of ZPI expression by ETXM1203 (ETXS2406 & ETXS2405), ETXM1204 (ETXS2408 & ETXS2407), ETXM1218 (ETXS2436 & ETXS2407), ETXM1772 (ETXS3544 & ETXS2407), ETXM1773 (ETXS3546 & ETXS2407), ETXM1774 (ETXS3548 & ETXS2407), ETXM 1775 (ETXS3550 & ETXS2407), ETXM 1776 (ETXS3552 & ETXS2407), ETXM1777 (ETXS3554 & ETXS2407) and ETXM1778 (ETXS3556 & ETXS2407). [0049] Figure 15: Selection of active GalNAc-siRNAs with EC50 values less than lOOnM. Dose-response in B4GALT1 gene knockdown in primary mouse hepatocytes was measured after 48hr incubation with GalNAc-siRNAs targeting mouse B4GALT1 at 10 serial dilutions from lOOOnM . ECso values were determined by fitting data to a 4-parameter sigmoidal doseresponse (variable slope) equation using GraphPad Prism. 4 active GalNAc-siRNAs, ETXM619, ETXM624, ETXM628 and ETXM633, were selected for in vivo pharmacology.
[0050] Figure 16: Summary of B4GALT1 mRNA knockdown effects of multiple dosing of GalNAc-siRNAs, ETXM619, ETXM624, ETXM628 and ETXM633 (lOmg/kg) in mouse liver tissues. The y-axis values are the relative mRNA expression to the non-treated group (n=5). Each data point represents the relative mRNA expression as Mean ± SD from n=3 experiment. Red arrows on the top of the graph indicate the days test articles were administered.
[0051] Figure 17: Effect of B4GALT1 mRNA knockdown in plasma LDL-c, glucose and fibrinogen levels. Plasma samples were collected on day 14 after three dosings of ETXMs (lOmg/kg, s.c.) on day 0, day 3 and day 7. Compared to the non-treated group ( n=5), the ETXM treated group (n=12) shows significantly reduced levels of LDL-c, glucose and fibrinogen in normal C57BL/6 mice. Data presented here are Meani SD.
DEFINITIONS
[0052] The “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. As used herein, the term "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. In some embodiments, a double stranded nucleic acid e.g. an siRNA agent of the invention includes a nucleoside mismatch in the antisense strand.
[0053] 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. [0054] In the context of molecule comprising a nucleic acid provided with a ligand moiety, optionally also with a linker moiety, the nucleic acid of the invention may be referred to as an oligonucleoside or an oligonucleoside moiety.
[0055] 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.
[0056] It is preferred herein that the nucleic acid according to the invention is a double stranded oligonucleoside comprising one or more phosphorothioate backbone bonds between nucleosides. 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.
[0057] In some embodiments, a double stranded nucleic acid e.g. siRNA agent of the invention includes a nucleoside mismatch in the sense strand. In some embodiments, 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.
[0058] In another embodiment, the nucleoside mismatch is, for example, in the 3'- terminal nucleoside of the nucleic acid e.g. siRNA.
[0059] A "target sequence" (which may also be called a target RNA or a target mRNA) refers to a contiguous portion of the nucleoside sequence of an mRNA molecule formed during the transcription of a gene, including mRNA that is a product of RNA processing of a primary transcription product. [0060] The target sequence may be from about 10-35 nucleosides in length, e.g., about 15-30 nucleosides in length. For example, 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. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.
[0061] The term "ribonucleoside" or "nucleoside" can also refer to a modified nucleoside, as further detailed below.
[0062] A nucleic acid can be a DNA or an RNA, and can comprise modified nucleosides. RNA is a preferred nucleic acid.
[0063] The terms "iRNA", “siRNA”, "RNAi agent," and "iRNA agent," "RNA interference agent" as used interchangeably herein, refer to an agent that contains RNA, and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. siRNA directs the sequence-specific degradation of mRNA through RNA interference (RNAi).
[0064] 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.
[0065] The majority of nucleosides of each strand of the nucleic acid, e.g. a dsiRNA molecule, 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. In addition, as used in this specification, an "siRNA" may include ribonucleosides with chemical modifications.
[0066] The term "modified nucleoside" refers to a nucleoside having, independently, a modified sugar moiety, a modified internucleoside linkage, or modified nucleobase, or any combination thereof. Thus, the term modified nucleoside encompasses substitutions, additions or removal of, e.g., a functional group or atom, to intemucleoside 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.
[0067] The two strands forming the duplex structure may be different portions of one larger molecule, or they may be separate molecules e.g. RNA molecules.
[0068] The term "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. Furthermore, the nucleoside(s) of an overhang can be present on the 5'-end, 3'-end, or both ends of either an antisense or sense strand.
[0069] In certain embodiments, 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.
[0070] "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.
[0071] Unless otherwise indicated, 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).
[0072] 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. Such sequences can be referred to as "fully complementary" with respect to each other herein. However, where a 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. For example, 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".
[0073] "Complementary" sequences, as used herein, 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.
[0074] 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 eg dsiRNA, or between the antisense strand of a double stranded nucleic acid e.g. siRNA agent and a target sequence.
[0075] Within the present invention, the second strand of the nucleic acid according to the invention, in particular a dsiRNA for inhibiting B4GALT1, is at least partially complementary to the first strand of said nucleic acid. In certain embodiments, 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.
[0076] 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. [0077] Alternatively, 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.
[0078] 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, wherein at least 14, 15, 16, 17, 18 or all 19 base pairs are complementary base pairs, in particular Watson-Crick 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, wherein at least 16, 17, 18, 19, 20 or all 21 base pairs are complementary base pairs, in particular Watson-Crick base pairs.
[0079] As used herein, 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). In certain embodiments, the contiguous portion of the mRNA is a sequence as listed in Table 1, i.e., any one of SEQ ID NOs:2-21 or 102-201. For example, 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.
[0080] Accordingly, in some preferred embodiments, the antisense oligonucleosides as disclosed herein are fully complementary to the target gene sequence.
[0081] In other embodiments, 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.
[0082] In certain embodiments, 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 B4GALTlgene. In certain embodiments, 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 B4GALTlmRNA. 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 B4GALTlmRNA. 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 or 19 nucleosides of any one of the sequences as listed in Table 1, i.e., any one of SEQ ID NOs: 2-21 or 102-201.
[0083] In certain embodiments, the first (antisense) strand of the nucleic acid according to the invention is partially complementary to a contiguous portion of the B4GALTlmRNA 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 B4GALTlmRNA. In certain embodiments, 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: 2-21 or 102-201. In certain embodiments, 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: 2-21 or 102-201.
[0084] In some embodiments, 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.
[0085] In some embodiments, 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.
[0086] As used herein, 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. In certain preferred embodiments, the subject is a human.
[0087] 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. Treatment can include prevention of development of comorbidities, e.g., reduced liver damage in a subject with a hepatic infection.
[0088] "Therapeutically effective amount," as used herein, is intended to include the amount of a nucleic acid e.g. an 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).
[0089] The phrase "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.
[0090] The phrase "pharmaceutically-acceptable carrier" as used herein 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.
[0091] Where 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.
[0092] The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article.
[0093] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to". [0094] The term "or" is used herein to mean, and is used interchangeably with, the term "and/or," unless context clearly indicates otherwise. For example, "sense strand or antisense strand" is understood as "sense strand or antisense strand or sense strand and antisense strand."
[0095] 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%. When about is present before a series of numbers or a range, it is understood that "about" can modify each of the numbers in the series or range.
[0096] The term "at least" prior to a number or series of numbers is understood to include the number adjacent to the term "at least", and all subsequent numbers or integers that could logically be included, as clear from context. For example, the number of nucleosides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleosides of a 21 nucleoside nucleic acid molecule" means that 18, 19, 20, or 21 nucleosides have the indicated property. When at least is present before a series of numbers or a range, it is understood that "at least" can modify each of the numbers in the series or range.
[0097] As used herein, "no more than" or "less than" is understood as the value adjacent to the phrase and logical lower values or integers, as logical from context, to zero. For example, a duplex with an overhang of "no more than 2 nucleosides" has a 2, 1, or 0 nucleoside overhang. When "no more than" is present before a series of numbers or a range, it is understood that "no more than" can modify each of the numbers in the series or range.
[0098] The terminal region of a strand is the last 5 nucleosides from the 5’ or the 3’ end.
[0099] Various embodiments of the invention can be combined as determined appropriate by one of skill in the art.
Abasic Nucleosides
[00100] 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.
Typically, there will be a hydrogen at position 1 of the sugar moiety of the abasic nucleosides present in a nucleic acid according to the present invention. [00101] 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.
[00102] 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 the 5’ or 3’ terminal region of the second strand; and / or a reversed internucleoside linkage connects at least one abasic nucleoside to an adjacent basic nucleoside in a terminal region of the second strand; and / or a reversed internucleoside linkage connects at least one abasic nucleoside to an adjacent basic nucleoside in either the 5’ or 3’ terminal region of the second strand; and /or an abasic nucleoside as the penultimate nucleoside which is connected via the reversed linkage to the nucleoside which is not the terminal nucleoside (called the antepenultimate nucleoside herein); and/or abasic nucleosides as the 2 terminal nucleosides connected via a 5 ’-3’ linkage when reading the strand in the direction towards the terminus comprising the terminal nucleosides; abasic nucleosides as the 2 terminal nucleosides connected via a 3 ’-5’ linkage when reading the strand in the direction towards the terminus comprising the terminal nucleosides; abasic nucleosides as the terminal 2 positions, wherein the penultimate nucleoside is connected via the reversed linkage to the antepenultimate nucleoside, and wherein the reversed linkage is a 5-5’ reversed linkage or a 3’-3’ reversed linkage; abasic nucleosides as the terminal 2 positions, wherein the penultimate nucleoside is connected via the reversed linkage to the antepenultimate nucleoside, and wherein either
(1) the reversed linkage is a 5-5’ reversed linkage and the linkage between the terminal and penultimate abasic nucleosides is 3’5’ when reading towards the terminus comprising the terminal and penultimate abasic nucleosides; or
(2) the reversed linkage is a 3-3’ reversed linkage and the linkage between the terminal and penultimate abasic nucleosides is 5’3’ when reading towards the terminus comprising the terminal and penultimate abasic nucleosides.
[00103] Preferably there is an abasic nucleoside at the terminus of the second strand.
[00104] Preferably there are 2 or at least 2 abasic nucleosides in the terminal region of the second strand, preferably at the terminal and penultimate positions.
[00105] Preferably 2 or more abasic nucleosides are consecutive, for example all abasic nucleosides may be consecutive. For example, the terminal 1 or terminal 2 or terminal 3 or terminal 4 nucleosides may be abasic nucleosides.
[00106] 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.
[00107] A reversed linkage (which may also be referred to as an inverted linkage, which is also seen in the art), comprises either a 5’-5’, a 3’3’, a 3’-2’ or a 2’-3’ phosphodiester linkage between the adjacent sugar moi eties of the nucleosides.
[00108] 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.
[00109] A preferred embodiment comprises 2 abasic nucleosides at the terminal and penultimate positions of the second strand, and wherein the reversed intemucleoside linkage is located between the penultimate (abasic) nucleoside and the antepenultimate nucleoside. [00110] Preferably there are 2 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 intemucleoside 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).
[00111] Preferably 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.
[00112] Typically 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. More typically, (i) the first strand and the second strand each has a length of 23 nucleosides; (ii) two phosphorothioate internucleoside linkages are respectively between three consecutive positions in said 5’ near terminal region of the second strand, wherein a first phosphorothioate internucleoside linkage is present between said adjacent first basic nucleoside of (a) and an adjacent second basic nucleoside in said 5’ near terminal region of the second strand, and a second phosphorothioate internucleoside linkage is present between said adjacent second basic nucleoside and an adjacent third basic nucleoside in said 5’ near terminal region of the second strand; (iii) two phosphorothioate intemucleoside 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 penultimate nucleoside by a phosphorothioate intemucleoside linkage, and each first 5’ and 3’ penultimate nucleoside is attached to a respective 5’ and 3’ adjacent antepenultimate nucleoside by a phosphorothioate intemucleoside linkage; and (iv) the second strand of the nucleic acid is conjugated directly or indirectly to one or more ligand moi eties at the 3’ terminal region of the second strand. [00113] Alternatively 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 intemucleoside 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. More typically, (i) the first strand and the second strand each has a length of 23 nucleosides; (ii) two phosphorothioate internucleoside linkages are respectively between three consecutive positions in said 3’ near terminal region of the second strand, wherein a first phosphorothioate intemucleoside 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 intemucleoside 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; (iii) two phosphorothioate intemucleoside 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 penultimate nucleoside by a phosphorothioate intemucleoside linkage, and each first 5’ and 3’ penultimate nucleoside is attached to a respective 5’ and 3’ adjacent antepenultimate nucleoside by a phosphorothioate intemucleoside linkage; and (iv) the second strand of the nucleic acid is conjugated directly or indirectly to one or more ligand moi eties at the 5’ terminal region of the second strand.
[00114] Examples of the structures are as follows (where the specific 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)
Figure imgf000028_0002
B Illustrating a 5’ -5’ reversed bond (and also showing the 3’-5’ direction of the last phosphodiester bond between the two abasic molecules reading towards the terminus of the molecule)
Figure imgf000028_0001
[00115] The 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 intemucleoside 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. [00116] 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. Whilst certain structures drawn and referenced herein are represented using conventional 5’ - 3’ direction (with reference to the numbering of ring atoms on the nucleoside sugars), it will be appreciated that the presence of a terminal nucleoside having a change of orientation and a proximal 3 ’-3’ reversed linkage, will result in a nucleic acid having an overall 5’- 5’ end structure (i.e. the conventional 3’ end nucleoside becomes a 5’ end nucleoside). Alternatively, it will be appreciated that the presence of a terminal nucleoside having a change of orientation and a proximal 5 ’-5’ reversed linkage will result in a nucleic acid with an overall 3’- 3’ end structure.
[00117] 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. Alternatively, 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. While a skilled addressee will appreciate that inverted orientations as described above can result in nucleic acid molecules having overall 3’ - 3’ or 5’- 5’ end structures as described herein, it will also be appreciated that with the presence of one or more additional reversed linkages and / or nucleosides having an inverted orientation, then the overall nucleic acid may have 3’ - 5’ end structures corresponding to the conventionally positioned 5’ / 3’ ends.
[00118] In one aspect 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.
[00119] A skilled person would therefore clearly understand that 5’- 5’, 3 ’-3’ and 3’ - 5’ (reading in the direction of that terminus) end variants of the more conventional 5’- 3’ structures (with reference to the numbering of ring atoms on the end nucleoside sugars) drawn herein are included in the scope of the disclosure, where a reversed linkage or linkages is / are present. [00120] In the situation of eg a reversed intemucleoside linkage and / or one or more nucleosides having an inverted orientation creating an inverted end, and where the relative position of a linkage (eg 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. It is possible to tell from these internal bonds and / or nucleoside orientation which ends of the nucleic acid would constitute the conventional 5’ and 3’ ends (with reference to the numbering of ring atoms on the end nucleoside sugars) of the molecule absent the reversed linkage.
[00121] In some embodiments, 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
Figure imgf000030_0001
wherein:
B represents a nucleoside base,
T represent H, OH or a 2’ ribose modification,
Z represents the remaining nucleosides of said second strand. [00122] In some embodiments, 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
Figure imgf000031_0001
wherein:
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 C1-4 alkyl (preferably H),
Z represents the remaining nucleosides of said second strand, more preferably the following 5’ terminal motif
Figure imgf000032_0001
wherein:
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.
[00123] The reversed bond is preferably located at the end of the nucleic acid eg RNA which is distal to a ligand moiety, such as a GalNAc containing portion, of the molecule.
[00124] GalNAc-siRNA constructs with a 5 ’-GalNAc on the sense strand can have a reversed linkage on the opposite end of the sense strand. [00125] GalNAc-siRNA constructs with a 3’-GalNAc on the sense strand can have a reversed linkage on the opposite end of the sense strand.
[00126] In a preferred embodiment, 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
Figure imgf000033_0001
wherein:
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 C1-4 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
Figure imgf000034_0001
wherein:
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
[00127] 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.
[00128] Typically the duplex region of the nucleic acid is between 17 and 30 nucleosides in length, more preferably is 19 or 21 nucleosides in length. Similarly, the region of complementarity between the first strand and the portion of RNA transcribed from the B4GALT1 gene is between 17 and 30 nucleosides in length. Nucleic Acid Modifications
[00129] In certain embodiments, the nucleic acid e.g. an RNA of the invention e.g., a dsiRNA, does not comprise further modifications, e.g., chemical modifications or conjugations known in the art and described herein.
[00130] In other preferred embodiments, the nucleic acid e.g. RNA of the invention, e.g., a dsiRNA, is further chemically modified to enhance stability or other beneficial characteristics.
[00131] In certain embodiments of the invention, substantially all of the nucleosides are modified.
[00132] 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.
[00133] 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.
[00134] Specific examples of 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. For the purposes of this specification, and as sometimes referenced in the art, modified nucleic acids e.g. RNAs that do not have a phosphorus atom in their intemucleoside backbone can also be considered to be oligonucleosides. In some embodiments, a modified nucleic acid e.g. an siRNA will have a phosphorus atom in its internucleoside backbone.
[00135] Modified nucleic acid e.g. RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3 '-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5'-linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 5'-3' or 5'-2'. Various salts, mixed salts and free acid forms are also included.
[00136] 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; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; 0-, S- or N- alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted. 2’ O- methyl and 2’ -F are preferred modifications.
[00137] In certain preferred embodiments, the nucleic acid comprises at least one modified nucleoside.
[00138] The nucleic acid of the invention may comprise one or more modified nucleosides on the first strand and/or the second strand.
[00139] In some embodiments, substantially all of the nucleosides of the sense strand and all of the nucleosides of the antisense strand comprise a modification.
[00140] In some embodiments, all of the nucleosides of the sense strand and substantially all of the nucleosides of the antisense strand comprise a modification.
[00141] In some embodiments, all of the nucleosides of the sense strand and all of the nucleosides of the antisense strand comprise a modification.
[00142] In one embodiment, 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' - O-alkyl- modified nucleoside, 2'-hydroxly-modified nucleoside, a 2'- methoxyethyl modified nucleoside, a 2'-O-alkyl-modified nucleoside, a morpholino nucleoside, a phosphoramidate, a non-natural base comprising nucleoside, a tetrahydropyran modified nucleoside, a 1 ,5- anhydrohexitol modified nucleoside, a cyclohexenyl modified nucleoside, a nucleoside comprising a phosphorothioate group, a nucleoside comprising a methylphosphonate group, a nucleoside comprising a 5 '-phosphate, and a nucleoside comprising a 5 '-phosphate mimic. In another embodiment, the modified nucleosides comprise a short sequence of 3 '-terminal deoxy -thymine nucleosides (dT).
[00143] 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. In another embodiment, the modifications on the nucleosides are 2'-O-methyl (“2'-Me”) or 2'-fluoro modifications.
[00144] One preferred modification is a modification at the 2’ -OH group of the ribose sugar, optionally selected from 2'-Me or 2’-F modifications.
[00145] Preferred nucleic acid comprise one or more nucleosides on the first strand and / or the second strand which are modified, to form modified nucleosides, as follows:
[00146] A nucleic acid wherein the modification is a modification at the 2’ -OH group of the ribose sugar, optionally selected from 2'-Me or 2’-F modifications.
[00147] A nucleic acid wherein the first strand comprises a 2’-F modification at any of position 2, position 6, position 14, or any combination thereof, counting from position 1 of said first strand.
[00148] A nucleic acid wherein the second strand comprises a 2’-F modification at any of position 7, position 9, position 11, or any combination thereof, counting from position 1 of said second strand.
[00149] A nucleic acid wherein the first and second strand each comprise 2'-Me and 2’-F modifications.
[00150] A nucleic which comprises at least one thermally destabilizing modification, suitably at one or more of positions 1 to 9 of the first strand counting from position 1 of the first strand, and / or at one or more of positions on the second strand aligned with positions 1 to 9 of the first strand, wherein the destabilizing modification is selected from a modified unlocked nucleic acid (UNA) and a glycol nucleic acid (GNA), preferably a glycol nucleic acid, more preferably an (S)-glycol nucleic acid.
[00151] A nucleic acid which comprises at least one thermally destabilizing modification at position 7 of the first strand, counting from position 1 of the first strand. [00152] A nucleic acid which is an siRNA oligonucleoside, wherein the siRNA oligonucleoside comprises 3 or more 2’-F modifications at positions 6 to 12 of the second strand, such as 4, 5,
6 or 7 2’-F modifications at positions 6 to 12 of the second strand, counting from position 1 of said second strand.
[00153] A nucleic acid which is an siRNA oligonucleoside, wherein said second strand comprises at least 3, such as 4, 5 or 6, 2’-Me modifications at positions 1 to 6 of the second strand, counting from position 1 of said second strand.
[00154] A nucleic acid which is an siRNA oligonucleoside, wherein said first strand comprises at least 5 2’ -Me consecutive modifications at the 3’ terminal region, preferably including the terminal nucleoside at the 3’ terminal region, or at least within 1 or 2 nucleosides from the terminal nucleoside at the 3’ terminal region.
[00155] A nucleic acid which is an siRNA oligonucleoside, wherein said first strand comprises
7 2’ -Me consecutive modifications at the 3’ terminal region, preferably including the terminal nucleoside at the 3’ terminal region.
[00156] A nucleic acid which is an siRNA oligonucleoside, wherein each of the first and second strands comprises an alternating modification pattern, preferably a fully alternating modification pattern along the entire length of each of the first and second strands, wherein the nucleosides of the first strand are modified by (i) 2’Me modifications on the odd numbered nucleosides counting from position 1 of the first strand, and (ii) 2’F modifications on the even numbered nucleosides counting from position 1 of the first strand, and nucleosides of the second strand are modified by (i) 2’F modifications on the odd numbered nucleosides counting from position 1 of the second strand, and (ii) 2’Me modifications on the even numbered nucleosides counting from position 1 of the second strand. Typically such fully alternating modification patterns are present in a blunt ended oligonucleoside, wherein each of the first and second strands are 19 nucleosides in length.
[00157] 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 moi eties of the backbone, and reading in a direction away from that end of the molecule.
[00158] 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. Typically, 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.
[00159] As used herein, “position 1 of the antisense strand” is the 5’ most nucleoside (not including abasic nucleosides) at the conventional 5’ end of the antisense strand. As hereinbefore described, there will be a region of complementarity between the sense and antisense strands, and in this way the antisense strand will also have a region of complementarity to the target nucleic acid sequence as referred to above.
[00160] In certain embodiments, the nucleic acid e.g. siRNA agent further comprises at least one phosphorothioate or methylphosphonate internucleoside linkage. For example the phosphorothioate or methylphosphonate intemucleoside 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.
[00161] In certain embodiments, the phosphorothioate or methylphosphonate intemucleoside 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.
[00162] In certain embodiments, a phosphorothioate or a methylphosphonate intemucleoside 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.
[00163] Any nucleic acid may comprise one or more phosphorothioate (PS) modifications within the nucleic acid, such as at least two PS intemucleoside bonds at the ends of a strand.
[00164] At least one of the oligoribonucleoside strands preferably comprises at least two consecutive phosphorothioate modifications in the last 3 nucleosides of the oligonucleoside.
[00165] The invention therefore also relates to: A nucleic acid disclosed herein which comprises phosphorothioate intemucleoside 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. [00166] A nucleic acid disclosed herein which comprises phosphorothioate intemucleoside 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 intemucleoside linkage.
[00167] The nucleic acid strand may be an RNA comprising a phosphorothioate intemucleoside linkage between the three nucleosides contiguous with 2 terminally located abasic nucleosides.
[00168] 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 moi eties 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.
Further preferred, the same nucleic acid may also comprise a 2’ F modification at positions 7, 9 and 11 of the second strand.
[00169] Preferred modifications are as follows.
[00170] A nucleic acid wherein modified nucleosides of said second strand comprise a modification pattern according to any one of the following (5’ -3 ’):
Me - Me - Me - Me - Me - Me - F - F - F - F - F - Me - Me - Me - Me - Me - Me - Me - F - Me - Me, or
Me - Me - Me - Me - Me - F - F - Me - F - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me, or
Me - Me - Me - Me - Me - Me -F- Me - F - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me, or
Me - Me - Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me, or
Me - Me - Me - Me - Me - Me - F - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me
- Me - Me. [00171] A nucleic acid wherein modified nucleosides of said second strand comprise a modification pattern according to any one of the following (5’-3’):
Me(s)Me(s)Me - Me - Me - Me -F-F-F-F-F- Me - Me - Me - Me - Me - Me - Me - F - Me - Me, or
Me(s)Me(s)Me - Me - Me - F - F - Me -F-F-F-F- Me - Me - Me - Me - Me - Me - Me - Me - Me, or
Me(s)Me(s)Me - Me - Me - Me -F- Me - F- F- F- F- Me - Me - Me - Me - Me - Me - Me - Me - Me, or
Me(s)Me(s)Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me, or
Me(s)Me(s)Me - Me - Me - Me - F - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me, or
Me - Me - Me - Me - Me - Me - F- F- F- F- F- Me - Me - Me - Me - Me - Me - Me -
F(s)Me(s)Me, or
Me - Me - Me - Me - Me - F - F - Me - F- F- F- F- Me - Me - Me - Me - Me - Me - Me(s)Me(s)Me, or
Me - Me - Me - Me - Me - Me -F- Me - F- F- F- F- Me - Me - Me - Me - Me - Me - Me(s)Me(s)Me, or
Me - Me - Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me(s)Me(s)Me, or
Me - Me - Me - Me - Me - Me - F - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me(s)Me(s)Me, wherein (s) is a phosphorothioate intemucleoside linkage.
[00172] A nucleic acid wherein modified nucleosides of said second strand comprise a modification pattern according to any one of the following (5’-3’): ia - ia - Me - Me - Me - Me - Me - Me - F- F- F- F- F- Me - Me - Me - Me - Me - Me -
Me - F - Me - Me, or ia - ia - Me - Me - Me - Me - Me - F - F - Me - F- F- F- F- Me - Me - Me - Me - Me - Me -
Me - Me - Me, or ia - ia - Me - Me - Me - Me - Me - Me -F- Me -F-F-F-F- Me - Me - Me - Me - Me - Me
- Me - Me - Me, or ia - ia - Me - Me - Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me
- Me - Me - Me - Me, or ia - ia - Me - Me - Me - Me - Me - Me - F - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me, or
Me - Me - Me - Me - Me - Me -F-F-F-F-F- Me - Me - Me - Me - Me - Me - Me - F -
Me - Me - ia - ia, or
Me - Me - Me - Me - Me - F - F - Me - F- F- F- F- Me - Me - Me - Me - Me - Me - Me -
Me - Me - ia - ia, or
Me - Me - Me - Me - Me - Me -F- Me - F- F- F- F- Me - Me - Me - Me - Me - Me - Me -
Me - Me - ia - ia, or
Me - Me - Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me - ia - ia, or
Me - Me - Me - Me - Me - Me - F - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me
- Me - Me- ia - ia, wherein ia represents an inverted abasic nucleoside, and when the inverted abasic nucleosides as represented by ia - ia are present at the 3’ terminus of the second strand, said inverted abasic nucleosides are present in a 2 nucleoside overhang.
[00173] A nucleic acid wherein modified nucleosides of said second strand comprise a modification pattern according to any one of the following (5’-3’): ia - ia - Me(s)Me(s)Me - Me - Me - Me - F- F- F- F- F- Me - Me - Me - Me - Me - Me - Me - F - Me - Me, or ia - ia - Me(s)Me(s)Me - Me - Me - F - F - Me - F- F- F- F- Me - Me - Me - Me - Me - Me
- Me - Me - Me, or ia - ia - Me(s)Me(s)Me - Me - Me - Me -F- Me -F-F-F-F- Me - Me - Me - Me - Me - Me - Me - Me - Me, or ia - ia - Me(s)Me(s)Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me
- Me - Me - Me - Me, or ia - ia - Me(s)Me(s)Me - Me - Me - Me - F - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me, or
Me - Me - Me - Me - Me - Me -F-F-F-F-F- Me - Me - Me - Me - Me - Me - Me - F(s)Me(s)Me - ia - ia, or
Me - Me - Me - Me - Me - F - F - Me - F- F- F- F- Me - Me - Me - Me - Me - Me - Me(s)Me(s)Me - ia - ia, or
Me - Me - Me - Me - Me - Me -F- Me - F- F- F- F- Me - Me - Me - Me - Me - Me - Me(s)Me(s)Me - ia - ia, or
Me - Me - Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me(s)Me(s)Me - ia - ia, or
Me - Me - Me - Me - Me - Me - F - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me(s)Me(s)Me - ia - ia, wherein:
(s) is a phosphorothioate internucleoside linkage, ia represents an inverted abasic nucleoside, and when the inverted abasic nucleosides as represented by ia - ia are present at the 3’ terminus of the second strand, said inverted abasic nucleosides are present in a 2 nucleoside overhang.
[00174] A nucleic acid wherein modified nucleosides comprise any one of the following modification patterns:
Modification pattern 1 : Second strand (5’-3’): Me - Me - Me - Me - Me - Me -F-F-F-F- F - Me - Me - Me - Me - Me - Me - Me - F - Me - Me, First strand (5’-3’): Me - F - Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me
Or Modification pattern 2: Second strand (5’-3’): Me - Me - Me - Me - Me - F - F - Me - F
- F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me, First strand (5’-3’): Me - F - Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me
Or Modification pattern 3: Second strand (5’-3’): Me - Me - Me - Me - Me - Me -F- Me - F
- F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me, First strand (5’-3’): Me - F - Me
- F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me
Or Modification pattern 4: Second strand (5’-3’): Me - Me - Me - Me - Me - Me - F - Me - F - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me, First strand (5’-3’): Me - F - Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me
- Me - Me
Or Modification pattern 5: Second strand (5’-3’): Me - Me - Me - Me - Me - Me - Me - Me
- F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me, First strand (5’-3’): Me - F
- Me - Me - Me - F - Me - Me - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me
Or Modification pattern 6: Second strand (5’-3’): Me - Me - Me - Me - Me - Me - F - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me, First strand (5’-3’): Me - F - Me - Me - Me - F - Me - Me - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me
- Me - Me.
[00175] A nucleic acid wherein modified nucleosides comprise any one of the following modification patterns:
Modification pattern 1 : Second strand (5’ -3 ’): Me(s)Me(s)Me - Me - Me - Me - F - F - F - F
- F - Me - Me - Me - Me - Me - Me - Me - F - Me - Me, First strand (5’-3’): Me(s)F(s)Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me -
Me(s)Me(s)Me
Or Modification pattern 2: Second strand (5’-3 ’): Me(s)Me(s)Me - Me - Me - F - F - Me - F
- F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me, First strand (5’-3’): Me(s)F(s)Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me(s)Me(s)Me
Or Modification pattern 3: Second strand (5’-3 ’): Me(s)Me(s)Me - Me - Me - Me -F- Me - F
- F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me, First strand (5’-3’): Me(s)F(s)Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me(s)Me(s)Me
Or Modification pattern 4: Second strand (5’-3 ’): Me(s)Me(s)Me - Me - Me - Me - F - Me - F - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me, First strand (5’-3’): Me(s)F(s)Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me(s)Me(s)Me
Or Modification pattern 5: Second strand (5’-3 ’): Me(s)Me(s)Me - Me - Me - Me - Me - Me
- F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me, First strand (5’-3’): Me(s)F(s)Me - Me - Me - F - Me - Me - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me -
Me - Me(s)Me(s)Me
Or Modification pattern 6: Second strand (5’-3 ’): Me(s)Me(s)Me - Me - Me - Me - F - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me, First strand (5’-3’): Me(s)F(s)Me - Me - Me - F - Me - Me - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me -
Me - Me(s)Me(s)Me wherein (s) is a phosphorothioate intemucleoside linkage.
[00176] A nucleic acid wherein modified nucleosides comprise any one of the following modification patterns:
Modification pattern 1 : Second strand (5’-3’): Me - Me - Me - Me - Me - Me - F - F - F - F
- F - Me - Me - Me - Me - Me - Me - Me - F(s)Me(s)Me, First strand (5’-3’): Me(s)F(s)Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me -
Me(s)Me(s)Me
Or Modification pattern 2: Second strand (5’-3’): Me - Me - Me - Me - Me - F - F - Me - F
- F - F - F - Me - Me - Me - Me - Me - Me - Me(s)Me(s)Me, First strand (5’-3’): Me(s)F(s)Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me -
Me - Me(s)Me(s)Me
Or Modification pattern 3: Second strand (5’-3’): Me - Me - Me - Me - Me - Me -F- Me - F
- F - F - F - Me - Me - Me - Me - Me - Me - Me(s)Me(s)Me, First strand (5 ’-3’): Me(s)F(s)Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me -
Me - Me(s)Me(s)Me Or Modification pattern 4: Second strand (5’-3’): Me - Me - Me - Me - Me - Me - F - Me - F - F - F - F - Me - Me - Me - Me - Me - Me - Me(s)Me(s)Me, First strand (5’-3’): Me(s)F(s)Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me(s)Me(s)Me
Or Modification pattern 5: Second strand (5 ’-3’): Me - Me - Me - Me - Me - Me - Me - Me
- F - F - F - Me - Me - Me - Me - Me - Me - Me - Me(s)Me(s)Me, First strand (5’-3’): Me(s)F(s)Me - Me - Me - F - Me - Me - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me -
Me - Me(s)Me(s)Me
Or Modification pattern 6: Second strand (5’-3’): Me - Me - Me - Me - Me - Me - F - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me(s)Me(s)Me, First strand (5 ’-3’): Me(s)F(s)Me - Me - Me - F - Me - Me - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me -
Me - Me(s)Me(s)Me wherein (s) is a phosphorothioate intemucleoside linkage.
[00177] A nucleic acid wherein modified nucleosides comprise any one of the following modification patterns:
Modification pattern 1 : Second strand (5’-3’): ia - ia - Me - Me - Me - Me - Me - Me - F - F
- F - F - F - Me - Me - Me - Me - Me - Me - Me - F - Me - Me, First strand (5’-3’): Me - F - Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me -
Me - Me
Or Modification pattern 2: Second strand (5’-3 ’): ia - ia - Me - Me - Me - Me - Me - F - F - Me - F - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me, First strand (5’-3’): Me - F - Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me
- Me - Me
Or Modification pattern 3: Second strand (5’-3’): ia - ia - Me - Me - Me - Me - Me - Me -F- Me - F - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me, First strand (5’-3’): Me - F - Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me
- Me - Me
Or Modification pattern 4: Second strand (5 ’-3’): ia - ia - Me - Me - Me - Me - Me - Me - F
- Me - F - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me, First strand (5’-3’): Me - F - Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me
- Me - Me - Me
Or Modification pattern 5: Second strand (5’-3 ’): ia - ia - Me - Me - Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me, First strand (5’- 3 ’): Me - F - Me - Me - Me - F - Me - Me - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me
Or Modification pattern 6: Second strand (5’-3’): ia - ia - Me - Me - Me - Me - Me - Me - F
- Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me, First strand (5’-3’): Me - F - Me - Me - Me - F - Me - Me - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me -
Me - Me - Me - Me, wherein ia represents an inverted abasic nucleoside.
[00178] A nucleic acid wherein modified nucleosides comprise any one of the following modification patterns:
Modification pattern 1 : Second strand (5’-3’): Me - Me - Me - Me - Me - Me - F - F - F - F - F - Me - Me - Me - Me - Me - Me - Me - F - Me - Me - ia - ia, First strand (5’-3’): Me - F - Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me -
Me - Me
Or Modification pattern 2: Second strand (5’-3’): Me - Me - Me - Me - Me - F - F - Me - F - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - ia - ia, First strand (5’-3’): Me - F - Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me
- Me - Me
Or Modification pattern 3: Second strand (5’-3’): Me - Me - Me - Me - Me - Me -F- Me - F
- F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - ia - ia, First strand (5’-3’): Me
- F - Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me -
Me - Me - Me
Or Modification pattern 4: Second strand (5’-3’): Me - Me - Me - Me - Me - Me - F - Me - F - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - ia - ia, First strand (5’-3’): Me - F - Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me - F - Me - F - Me - Me - Me -
Me - Me - Me - Me Or Modification pattern 5: Second strand (5’-3’): Me - Me - Me - Me - Me - Me - Me - Me
- F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me - ia - ia, First strand (5’-3’): Me - F - Me - Me - Me - F - Me - Me - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me
Or Modification pattern 6: Second strand (5’-3’): Me - Me - Me - Me - Me - Me - F - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me - ia - ia,- First strand (5’-3’): Me - F - Me - Me - Me - F - Me - Me - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me -
Me - Me - Me - Me, wherein ia represents an inverted abasic nucleoside, and when the inverted abasic nucleosides as represented by ia - ia are present at the 3’ terminus of the second strand, said inverted abasic nucleosides are present in a 2 nucleoside overhang.
[00179] A nucleic acid wherein modified nucleosides comprise any one of the following modification patterns:
Modification pattern 1 : Second strand (5’ -3 ’): ia - ia - Me(s)Me(s)Me - Me - Me - Me - F - F - F - F - F - Me - Me - Me - Me - Me - Me - Me - F - Me - Me, First strand (5’-3’): Me(s)F(s)Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me(s)Me(s)Me
Or Modification pattern 2: Second strand (5’-3 ’): ia - ia - Me(s)Me(s)Me - Me - Me - F - F
- Me - F - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me, First strand (5’-3’): Me(s)F(s)Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me(s)Me(s)Me
Or Modification pattern 3: Second strand (5’-3 ’): ia - ia - Me(s)Me(s)Me - Me - Me - Me - F- Me - F - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me, First strand (5’-3’): Me(s)F(s)Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me(s)Me(s)Me
Or Modification pattern 4: Second strand (5’-3 ’): ia - ia - Me(s)Me(s)Me - Me - Me - Me -
F - Me - F - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me, First strand (5’-3’): Me(s)F(s)Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me(s)Me(s)Me Or Modification pattern 5: Second strand (5’-3 ’): ia - ia - Me(s)Me(s)Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me, First strand (5’- 3’): Me(s)F(s)Me - Me - Me - F - Me - Me - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me(s)Me(s)Me
Or Modification pattern 6: Second strand (5’-3 ’): ia - ia - Me(s)Me(s)Me - Me - Me - Me - F - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me, First strand (5’-3’): Me(s)F(s)Me - Me - Me - F - Me - Me - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me(s)Me(s)Me wherein:
(s) is a phosphorothioate internucleoside linkage, ia represents an inverted abasic nucleoside.
[00180] A nucleic acid wherein modified nucleosides comprise any one of the following modification patterns:
Modification pattern 1 : Second strand (5’-3’): Me - Me - Me - Me - Me - Me - F - F - F - F
- F - Me - Me - Me - Me - Me - Me - Me - F(s)Me(s)Me - ia - ia, First strand (5’-3’): Me(s)F(s)Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me(s)Me(s)Me
Or Modification pattern 2: Second strand (5’-3’): Me - Me - Me - Me - Me - F - F - Me - F
- F - F - F - Me - Me - Me - Me - Me - Me - Me(s)Me(s)Me - ia - ia, First strand (5’-3’): Me(s)F(s)Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me(s)Me(s)Me
Or Modification pattern 3: Second strand (5’-3’): Me - Me - Me - Me - Me - Me -F- Me - F
- F - F - F - Me - Me - Me - Me - Me - Me - Me(s)Me(s)Me - ia - ia, First strand (5’-3’): Me(s)F(s)Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me(s)Me(s)Me
Or Modification pattern 4: Second strand (5’-3’): Me - Me - Me - Me - Me - Me - F - Me - F - F - F - F - Me - Me - Me - Me - Me - Me - Me(s)Me(s)Me - ia - ia, First strand (5’-3’): Me(s)F(s)Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me(s)Me(s)Me
Or Modification pattern 5: Second strand (5’-3’): Me - Me - Me - Me - Me - Me - Me - Me
- F - F - F - Me - Me - Me - Me - Me - Me - Me - Me(s)Me(s)Me - ia - ia, First strand (5’- 3’): Me(s)F(s)Me - Me - Me - F - Me - Me - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me(s)Me(s)Me
Or Modification pattern 6: Second strand (5’-3’): Me - Me - Me - Me - Me - Me - F - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me(s)Me(s)Me - ia - ia, First strand (5’-3’): Me(s)F(s)Me - Me - Me - F - Me - Me - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me(s)Me(s)Me wherein: (s) is a phosphorothioate internucleoside linkage, ia represents an inverted abasic nucleoside, and when the inverted abasic nucleosides as represented by ia - ia are present at the 3’ terminus of the second strand, said inverted abasic nucleosides are present in a 2 nucleoside overhang.
[00181] Particularly preferred is a nucleic acid wherein the modified nucleosides comprise the following modification pattern:
Modification pattern 5: Second strand (5’-3 ’): ia - ia - Me(s)Me(s)Me - Me - Me - Me - Me
- Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me, First strand (5’-3’): Me(s)F(s)Me - Me - Me - F - Me - Me - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me -
Me - Me(s)Me(s)Me wherein: (s) is a phosphorothioate internucleoside linkage, ia represents an inverted abasic nucleoside.
[00182] A nucleic acid wherein the first strand comprises a 2’ sugar modification pattern wherein said modifications are selected at least from 2’Me and 2’F sugar modifications, provided that the overall number of 2’F sugar modifications in the first strand does not consist of four, or six, 2’F modifications.
[00183] A nucleic acid wherein the first strand comprises a 2’ sugar modification pattern wherein said modifications are selected at least from 2’Me and 2’F sugar modifications, wherein the overall number of 2’F sugar modifications in the first strand consists of three, five or seven 2’F modifications.
[00184] A nucleic acid wherein the first strand comprises a 2’ sugar modification pattern wherein said modifications are selected at least from 2’Me and 2’F sugar modifications, wherein the overall number of 2’F sugar modifications in the first strand consists of three 2’F modifications. [00185] A nucleic acid wherein the first strand comprises a 2’ sugar modification pattern wherein said modifications are selected at least from 2’Me and 2’F sugar modifications, wherein the overall number of 2’F sugar modifications in the first strand consists of five 2’F modifications.
[00186] A nucleic acid wherein the first strand comprises a 2’ sugar modification pattern as follows (5 ’-3’):
Me - F - Me - X2 - Me - F - (Me)7 - (F - Me)2 - X3 - Me - X4 - (Me)3 wherein X2, X3 and X4 are selected from 2’Me and 2’F sugar modifications, provided that for X2, X3 and X4 at least one is a 2’F sugar modification, and the other two sugar modifications are 2’Me sugar modifications.
[00187] A nucleic acid wherein the first strand comprises a 2’ sugar modification pattern as follows (5 ’-3’):
Me - F - Me - X2 - Me - F - (Me)7 - (F - Me)2 - X3 - Me - X4 - (Me)3 wherein X2 is a 2’F sugar modification, and X3 and X4 are 2’Me sugar modifications.
[00188] A nucleic acid wherein the first strand comprises a 2’ sugar modification pattern as follows (5 ’-3’):
Me - F - Me - X2 - Me - F - (Me)7 - (F - Me)2 - X3 - Me - X4 - (Me)3 wherein X3 is a 2’F sugar modification, and X2 and X4 are 2’Me sugar modifications.
[00189] A nucleic acid wherein the first strand comprises a 2’ sugar modification pattern as follows (5 ’-3’):
Me - F - Me - X2 - Me - F - (Me)7 - (F - Me)2 - X3 - Me - X4 - (Me)3 wherein X4 is a 2’F sugar modification, and X2 and X3 are 2’Me sugar modifications.
[00190] A nucleic acid wherein the first strand comprises a 2’ sugar modification pattern wherein said modifications are selected at least from 2’Me and 2’F sugar modifications, wherein the overall number of 2’F sugar modifications in the first strand consists of seven 2’F modifications. [00191] A nucleic acid wherein the first strand comprises a 2’ sugar modification pattern as follows (5 ’-3’):
Me - F - Me - X2 - Me - F - Me - (F)2 - (Me)4 - (F - Me)2 - X3 - Me - X4 - (Me)3 wherein X2, X3 and X4 are selected from 2’Me and 2’F sugar modifications, provided that for X2, X3 and X4 at least one is a 2’F sugar modification, and the other two sugar modifications are 2’Me sugar modifications.
[00192] A nucleic acid wherein the first strand comprises a 2’ sugar modification pattern as follows (5 ’-3’):
Me - F - Me - X2 - Me - F - Me - (F)2 - (Me)4 - (F - Me)2 - X3 - Me - X4 - (Me)3 wherein X2 is a 2’F sugar modification, and X3 and X4 are 2’Me sugar modifications.
[00193] A nucleic acid wherein the first strand comprises a 2’ sugar modification pattern as follows (5 ’-3’):
Me - F - Me - X2 - Me - F - Me - (F)2 - (Me)4 - (F - Me)2 - X3 - Me - X4 - (Me)3 wherein X3 is a 2’F sugar modification, and X2 and X4 are 2’Me sugar modifications.
[00194] A nucleic acid wherein the first strand comprises a 2’ sugar modification pattern as follows (5 ’-3’):
Me - F - Me - X2 - Me - F - Me - (F)2 - (Me)4 - (F - Me)2 - X3 - Me - X4 - (Me)3 wherein X4 is a 2’F sugar modification, and X2 and X3 are 2’Me sugar modifications.
[00195] A nucleic acid wherein the first strand comprises a 2’ sugar modification pattern as follows (5 ’-3’):
Me - F - (Me)3 - X1 - (Me)7 - F - Me - F - (Me)7 wherein X1 is a thermally destabilising modification.
[00196] A nucleic acid wherein the first strand comprises a 2’ sugar modification pattern as follows (5 ’-3’):
Me - F - (Me)3 - X1 - Me - (F)2 - (Me)4 - F - Me - F - (Me)7 wherein X1 is a thermally destabilising modification.
[00197] A nucleic acid wherein the second strand comprises a 2’ sugar modification pattern as follows (5 ’-3’):
(Me)8 - (F)3 - (Me)10.
[00198] A nucleic acid wherein the second strand comprises a 2’ sugar modification pattern as follows (5 ’-3’):
(Me)8 - (F)3 - (Me)10, and wherein the first strand comprises a 2’ sugar modification pattern wherein said modifications are selected at least from 2’Me and 2’F sugar modifications, provided that the overall number of 2’F sugar modifications in the first strand does not consist of four, or six, 2’F modifications.
[00199] A nucleic acid wherein the second strand comprises a 2’ sugar modification pattern as follows (5 ’-3’):
(Me)8 - (F)3 - (Me)10, and wherein the first strand comprises a 2’ sugar modification pattern wherein said modifications are selected at least from 2’Me and 2’F sugar modifications, wherein the overall number of 2’F sugar modifications in the first strand consists of three, five or seven 2’F modifications.
[00200] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar modification pattern as follows (5’-3 ’):
(Me)8 - (F)3 - (Me)10, and wherein nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
Me - F - (Me)3 - X1 - (Me)7 - F - Me - F - (Me)7, wherein X1 is a thermally destabilising modification. [00201] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar modification pattern as follows (5’-3 ’):
(Me)8 - (F)3 - (Me)10, and wherein nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
(Me - F)3 - (Me)7 - F - Me - F - (Me)7.
[00202] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar modification pattern as follows (5’-3 ’):
(Me)8 - (F)3 - (Me)10, and wherein nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
Me - F - (Me)3 - F - (Me)7 - (F - Me)2 - F - (Me)5.
[00203] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar modification pattern as follows (5’-3 ’):
(Me)8 - (F)3 - (Me)10, and wherein nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
Me - F - (Me)3 - F - (Me)7 - F - Me - F - (Me)3 - F - (Me)3. [00204] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar modification pattern as follows (5’-3 ’):
(Me)8 - (F)3 - (Me)10, and wherein nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
Me - F - (Me)8 - X1 - Me - (F)2 - (Me)4 - F - Me - F - (Me)7, wherein X1 is a thermally destabilising modification.
[00205] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar modification pattern as follows (5’-3 ’):
(Me)8 - (F)3 - (Me)10, and wherein nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
(Me - F)3 - Me - (F)2 - (Me)4 - (F - Me)2 - (Me)6.
[00206] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar modification pattern as follows (5’-3’):
(Me)8 - (F)3 - (Me)10, and wherein nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
Me - F - (Me)3 - F - Me - (F)2 - (Me)4 - (F - Me)2 - F - (Me)5. [00207] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar modification pattern as follows (5’-3 ’):
(Me)8 - (F)3 - (Me)10, and wherein nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
Me - F - (Me)3 - F - Me - (F)2 - (Me)4 - (F - Me)2 - (Me)2 - F - (Me)3.
[00208] A nucleic acid wherein the second strand comprises a 2’ sugar, and abasic modification pattern as follows (5’-3 ’): ia-ia-(Me)8 - (F)3 - (Me)10 wherein ia represents an inverted abasic nucleoside.
[00209] A nucleic acid wherein the second strand comprises a 2’ sugar, and abasic modification pattern as follows (5’-3 ’): ia-ia-(Me)8 - (F)3 - (Me)10, wherein ia represents an inverted abasic nucleoside; and wherein the first strand comprises a 2’ sugar modification pattern wherein said modifications are selected at least from 2’Me and 2’F sugar modifications, provided that the overall number of 2’F sugar modifications in the first strand does not consist of four, or six, 2’F modifications.
[00210] A nucleic acid wherein the second strand comprises a 2’ sugar, and abasic modification pattern as follows (5’-3 ’): ia-ia-(Me)8 - (F)3 - (Me)10, wherein ia represents an inverted abasic nucleoside; and wherein the first strand comprises a 2’ sugar modification pattern wherein said modifications are selected at least from 2’Me and 2’F sugar modifications, wherein the overall number of 2’F sugar modifications in the first strand consists of three, five or seven 2’F modifications. [00211] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar, and abasic modification pattern as follows (5’-3 ’): ia-ia-(Me)8 - (F)3 - (Me)10, wherein ia represents an inverted abasic nucleoside; and wherein nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
Me - F - (Me)8 - X1 - (Me)7 - F - Me - F - (Me)7, wherein X1 is a thermally destabilising modification.
[00212] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar, and abasic modification pattern as follows (5’-3’): ia-ia-(Me)8 - (F)3 - (Me)10, wherein ia represents an inverted abasic nucleoside; and wherein nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
(Me - F)3 - (Me)7 - F - Me - F - (Me)7.
[00213] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar, and abasic modification pattern as follows (5’-3’): ia-ia-(Me)8 - (F)3 - (Me)10, wherein ia represents an inverted abasic nucleoside; and wherein nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
Me - F - (Me)3 - F - (Me)7 - (F - Me)2 - F - (Me)5. [00214] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar, and abasic modification pattern as follows (5’-3 ’): ia-ia-(Me)8 - (F)3 - (Me)10, wherein ia represents an inverted abasic nucleoside; and wherein nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
Me - F - (Me)3 - F - (Me)7 - F - Me - F - (Me)3 - F - (Me)3.
[00215] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar, and abasic modification pattern as follows (5’-3 ’): ia-ia-(Me)8 - (F)3 - (Me)10, wherein ia represents an inverted abasic nucleoside; and wherein nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
Me - F - (Me)3 - X1 - Me - (F)2 - (Me)4 - F - Me - F - (Me)7, wherein X1 is a thermally destabilising modification.
[00216] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar, and abasic modification pattern as follows (5’-3 ’): ia-ia-(Me)8 - (F)3 - (Me)10, wherein ia represents an inverted abasic nucleoside; and wherein nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
(Me - F)3 - Me - (F)2 - (Me)4 - (F - Me)2 - (Me)6. [00217] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar, and abasic modification pattern as follows (5’-3 ’): ia-ia-(Me)8 - (F)3 - (Me)10, wherein ia represents an inverted abasic nucleoside; and wherein nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
Me - F - (Me)3 - F - Me - (F)2 - (Me)4 - (F - Me)2 - F - (Me)5.
[00218] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar, and abasic modification pattern as follows (5’-3 ’): ia-ia-(Me)8 - (F)3 - (Me)10, wherein ia represents an inverted abasic nucleoside; and wherein nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
Me - F - (Me)3 - F - Me - (F)2 - (Me)4 - (F - Me)2 - (Me)2 - F - (Me)3.
[00219] A nucleic acid wherein the second strand comprises a 2’ sugar modification pattern as follows (5 ’-3’): ia-ia-Me(s)Me(s) (Me)6 - (F)3 - (Me)10, wherein ia represents an inverted abasic nucleoside, and (s) represents a phosphorothioate linkage.
[00220] A nucleic acid wherein the second strand comprises a 2’ sugar modification pattern as follows (5 ’-3’): ia-ia-Me(s)Me(s) (Me)6 - (F)3 - (Me) 10, wherein ia represents an inverted abasic nucleoside, and (s) represents a phosphorothioate linkage; and wherein the first strand comprises a 2’ sugar modification pattern wherein said modifications are selected at least from 2’Me and 2’F sugar modifications, provided that the overall number of 2’F sugar modifications in the first strand does not consist of four, or six, 2’F modifications.
[00221] A nucleic acid wherein the second strand comprises a 2’ sugar modification pattern as follows (5 ’-3’): ia-ia-Me(s)Me(s) (Me)6 - (F)3 - (Me) io, wherein ia represents an inverted abasic nucleoside, and (s) represents a phosphorothioate linkage; and wherein the first strand comprises a 2’ sugar modification pattern wherein said modifications are selected at least from 2’Me and 2’F sugar modifications, wherein the overall number of 2’F sugar modifications in the first strand consists of three, five or seven 2’F modifications.
[00222] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar, and abasic modification pattern as follows (5’-3 ’): ia-ia-Me(s)Me(s) (Me)6 - (F)3 - (Me)10, wherein ia represents an inverted abasic nucleoside, and (s) represents a phosphorothioate linkage, and wherein nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
Me(s)F(s)(Me)3 - X1 - (Me)7 - F - Me - F - (Me)8(s)Me(s)Me, wherein X1 is a thermally destabilising modification.
[00223] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar, and abasic modification pattern as follows (5’-3’): ia-ia-Me(s)Me(s) (Me)6 - (F)3 - (Me)10, wherein ia represents an inverted abasic nucleoside, and (s) represents a phosphorothioate linkage, and wherein nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
Me(s)F(s)Me - F - Me - F - (Me)7 - F - Me - F - (Me)5(s)Me(s)Me.
[00224] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar, and abasic modification pattern as follows (5’-3 ’): ia-ia-Me(s)Me(s) (Me)6 - (F)3 - (Me)10, wherein ia represents an inverted abasic nucleoside, and (s) represents a phosphorothioate linkage, and wherein nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
Me(s)F(s)(Me)3 - F - (Me)7 - (F - Me)2 - F - (Me)3(s)Me(s)Me.
[00225] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar, and abasic modification pattern as follows (5’-3 ’): ia-ia-Me(s)Me(s) (Me)6 - (F)3 - (Me)10, wherein ia represents an inverted abasic nucleoside, and (s) represents a phosphorothioate linkage, and wherein nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
Me(s)F(s)(Me)3 - F - (Me)7 - F - Me - F - (Me)3 - F - Me(s)Me(s)Me.
[00226] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar, and abasic modification pattern as follows (5’-3 ’): ia-ia-Me(s)Me(s) (Me)6 - (F)3 - (Me)10, wherein ia represents an inverted abasic nucleoside, and (s) represents a phosphorothioate linkage, and wherein nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
Me(s)F(s)(Me)3 - X1 - Me - (F)2 - (Me)i - F - Me - F - (Me)8(s)Me(s)Me, wherein X1 is a thermally destabilising modification.
[00227] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar, and abasic modification pattern as follows (5’-3 ’): ia-ia-Me(s)Me(s) (Me)6 - (F)3 - (Me)10, wherein ia represents an inverted abasic nucleoside, and (s) represents a phosphorothioate linkage, and wherein nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
Me(s)F(s)Me - F - Me - F - Me - (F)2 - (Me)4 - (F - Me)2 - (Me)4(s)Me(s)Me.
[00228] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar, and abasic modification pattern as follows (5’-3 ’): ia-ia-Me(s)Me(s) (Me)6 - (F)3 - (Me)10, wherein ia represents an inverted abasic nucleoside, and (s) represents a phosphorothioate linkage, and wherein nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
Me(s)F(s)(Me)3 - F - Me - (F)2 - (Me)4 - (F - Me)2 - F - (Me)3(s)Me(s)Me.
[00229] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein said first and second strands form a duplex region of at least 17 nucleosides in length, and wherein nucleosides of said second strand comprise a 2’ sugar, and abasic modification pattern as follows (5’-3 ’): ia-ia-Me(s)Me(s) (Me)6 - (F)3 - (Me)10, wherein ia represents an inverted abasic nucleoside, and (s) represents a phosphorothioate linkage, and wherein nucleosides of said first strand comprise a 2’ sugar modification pattern as follows (5’-3’):
Me(s)F(s)(Me)3 - F - Me - (F)2 - (Me)4 - (F - Me)2 - (Me)2 - F - Me(s)Me(s)Me.
[00230] Preferred modifications are as follows:
Modification pattern 1 :
Second strand (5’-3’): ia - ia - Me - Me - Me - Me - Me - Me - Me - Me - F - F - F - Me
- Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me - F - Me - Me - Me - X1 - Me - Me - Me - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me, wherein X1 is a thermally destabilising modification;
Or Modification pattern 2:
Second strand (5’-3’): ia - ia - Me - Me - Me - Me - Me - Me - Me - Me - F - F - F - Me
- Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me - F - Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me;
Or Modification pattern 3 :
Second strand (5’-3’): ia - ia - Me - Me - Me - Me - Me - Me - Me - Me - F - F - F - Me
- Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me - F - Me - Me - Me - F - Me - Me - Me - Me - Me - Me - Me - F - Me - F - Me - F - Me - Me - Me - Me - Me;
Or Modification pattern 4: Second strand (5’-3’): ia - ia - Me - Me - Me - Me - Me - Me - Me - Me - F - F - F - Me
- Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me - F - Me - Me - Me - F - Me - Me - Me - Me - Me - Me - Me - F
- Me - F - Me - Me - Me - F - Me - Me - Me;
Or Modification pattern 5:
Second strand (5’-3’): ia - ia - Me - Me - Me - Me - Me - Me - Me - Me - F - F - F - Me
- Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me - F - Me - Me - Me - X1 - Me - F - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me, wherein X1 is a thermally destabilising modification;
Or Modification pattern 6:
Second strand (5’-3’): ia - ia - Me - Me - Me - Me - Me - Me - Me - Me - F - F - F - Me
- Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me - F - Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me
- F - Me - Me - Me - Me - Me - Me - Me;
Or Modification pattern 7:
Second strand (5’-3’): ia - ia - Me - Me - Me - Me - Me - Me - Me - Me - F - F - F - Me
- Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me - F - Me - Me - Me - F - Me - F - F - Me- Me - Me - Me - F - Me - F - Me - F - Me - Me - Me - Me - Me;
Or Modification pattern 8:
Second strand (5’-3’): ia - ia - Me - Me - Me - Me - Me - Me - Me - Me - F - F - F - Me
- Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me - F - Me - Me - Me - F - Me - F - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - F - Me - Me - Me wherein ia represents an inverted abasic nucleoside. [00231] Further preferred modifications are as follows:
Modification pattern 1 :
Second strand (5 ’-3’): ia - ia - Me(s)Me(s)Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me(s)F(s)Me - Me - Me - X1 - Me - Me - Me - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me(s)Me(s)Me, wherein X1 is a thermally destabilising modification;
Or Modification pattern 2:
Second strand (5 ’-3’): ia - ia - Me(s)Me(s)Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me(s)F(s)Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me - F
- Me - F - Me - Me - Me - Me - Me(s)Me(s)Me;
Or Modification pattern 3 :
Second strand (5 ’-3’): ia - ia - Me(s)Me(s)Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me(s)F(s)Me - Me - Me - F - Me - Me - Me - Me - Me - Me - Me - F - Me - F - Me - F - Me - Me - Me(s)Me(s)Me;
Or Modification pattern 4:
Second strand (5 ’-3’): ia - ia - Me(s)Me(s)Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me(s)F(s)Me - Me - Me - F - Me - Me - Me - Me - Me - Me - Me - F
- Me - F - Me - Me - Me - F - Me(s)Me(s)Me;
Or Modification pattern 5:
Second strand (5 ’-3’): ia - ia - Me(s)Me(s)Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me, First strand (5’-3’): Me(s)F(s)Me - Me - Me - X1 - Me - F - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me(s)Me(s)Me, wherein X1 is a thermally destabilising modification;
Or Modification pattern 6:
Second strand (5 ’-3’): ia - ia - Me(s)Me(s)Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me(s)F(s)Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me(s)Me(s)Me;
Or Modification pattern 7:
Second strand (5 ’-3’): ia - ia - Me(s)Me(s)Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me(s)F(s)Me - Me - Me - F - Me - F - F - Me- Me - Me - Me - F - Me - F - Me - F - Me - Me - Me(s)Me(s)Me;
Or Modification pattern 8:
Second strand (5 ’-3’): ia - ia - Me(s)Me(s)Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me(s)F(s)Me - Me - Me - F - Me - F - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - F - Me(s)Me(s)Me; wherein (s) is a phosphorothioate intemucleoside linkage and ia represents an inverted abasic nucleoside.
Conjugation
[00232] Another modification of the nucleic acid e.g. 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.
[00233] In some embodiments, 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. The term "linker" or "linking group" means an organic moiety that connects two parts of a compound, e.g., covalently attaches two parts of a compound.
[00234] The ligand can be attached to the 3' or 5’ end of the sense strand.
[00235] The ligand is preferably conjugated to 3’ end of the sense strand of the nucleic acid e.g. an siRNA agent.
[00236] 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.
[00237] In one aspect 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.
[00238] In certain embodiments, the ligand moiety comprises a GalNAc or GalNAc derivative attached to the nucleic acid eg dsiRNA through a linker.
[00239] Therefore 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.
[00240] Said GalNAc ligand may be conjugated directly or indirectly to the 5’ or 3’ terminal region of the sense strand of the nucleic acid, preferably at the 3’ terminal region thereof.
[00241] GalNAc ligands are well known in the art and described in, inter alia, EP3775207A1.
[00242] In some embodiments, 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. Preferably, 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. [00243] In some embodiments, 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. Preferably, 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.
[00244] In some embodiments, 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. Preferably, 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.
[00245] In some embodiments, 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 SEQ ID NO:302 or SEQ ID NO:305, preferably wherein the linker is conjugated to the 3' terminal region of the second strand, i.e., to the 3' terminal region of SEQ ID NO:302 or SEQ ID NO:305, via a phosphodiester bond.
[00246] In some embodiments, 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 SEQ ID NO:302 or SEQ ID NO:305, preferably wherein the linker is conjugated to the 3' terminal region of the second strand, i.e., to the 3' terminal region of SEQ ID NO:302 or SEQ ID NO:305, via a phosphodiester bond.
[00247] In some embodiments, 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 SEQ ID NO:302 or SEQ ID NO:305, preferably wherein the linker is conjugated to the 3' terminal region of the second strand, i.e., to the 3' terminal region of SEQ ID NO:302 or SEQ ID NO:305, via a phosphodiester bond. [00248] In some embodiments, 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 SEQ ID NO:611 or SEQ ID NO:613, preferably wherein the linker is conjugated to the 3' terminal region of the second strand, i.e., to the 3' terminal region of SEQ ID NO:611 or SEQ ID NO:613, via a phosphodiester bond.
[00249] In some embodiments, 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 SEQ ID NO:611 or SEQ ID NO:613, preferably wherein the linker is conjugated to the 3' terminal region of the second strand, i.e., to the 3' terminal region of SEQ ID NO:611 or SEQ ID NO:613, via a phosphodiester bond.
[00250] In some embodiments, 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 SEQ ID NO:611 or SEQ ID NO:613, preferably wherein the linker is conjugated to the 3' terminal region of the second strand, i.e., to the 3' terminal region of SEQ ID NO:611 or SEQ ID NO:613, via a phosphodiester bond.
[00251] In some embodiments, 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 first strand comprising or consisting of SEQ ID NO:503 and a modified second strand comprising or consisting of SEQ ID NO:611, preferably wherein the linker is conjugated to the 3' terminal region of the second strand, i.e., to the 3' terminal region of SEQ ID NO:611, via a phosphodiester bond.
[00252] In some embodiments, 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 first strand comprising or consisting of SEQ ID NO: 503 and a modified second strand comprising or consisting of SEQ ID NO:611, preferably wherein the linker is conjugated to the 3' terminal region of the second strand, i.e., to the 3' terminal region of SEQ ID NO:611, via a phosphodiester bond. [00253] In some embodiments, 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 first strand comprising or consisting of SEQ ID NO: 503 and a modified second strand comprising or consisting of SEQ ID NO:611, preferably wherein the linker is conjugated to the 3' terminal region of the second strand, i.e., to the 3' terminal region of SEQ ID NO:611, via a phosphodiester bond.
[00254] In some embodiments, 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 first strand comprising or consisting of SEQ ID NO:505 and a modified second strand comprising or consisting of SEQ ID NO:613, preferably wherein the linker is conjugated to the 3' terminal region of the second strand, i.e., to the 3' terminal region of SEQ ID NO:613, via a phosphodiester bond.
[00255] In some embodiments, 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 first strand comprising or consisting of SEQ ID NO: 505 and a modified second strand comprising or consisting of SEQ ID NO:613, preferably wherein the linker is conjugated to the 3' terminal region of the second strand, i.e., to the 3' terminal region of SEQ ID NO:613, via a phosphodiester bond.
[00256] In some embodiments, 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 first strand comprising or consisting of SEQ ID NO: 505 and a modified second strand comprising or consisting of SEQ ID NO:613, preferably wherein the linker is conjugated to the 3' terminal region of the second strand, i.e., to the 3' terminal region of SEQ ID NO:613, via a phosphodiester bond.
[00257] In some embodiments, 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 first strand comprising or consisting of SEQ ID NO: 503 and a modified second strand comprising or consisting of SEQ ID NO:611, wherein the second strand has the following structure
Figure imgf000071_0001
wherein:
T represents a 2’Me ribose modification,
B represents the nucleoside bases of the first two basic nucleosides in the 5' terminal region of SEQ ID NO:611, and
Z represents the remaining 19 contiguous basic nucleosides of SEQ ID NO:611.
[00258] In some embodiments, 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 first strand comprising or consisting of SEQ ID NO: 503 and a modified second strand comprising or consisting of SEQ ID NO:611, wherein the second strand has the following structure
Figure imgf000072_0001
wherein:
T represents a 2’Me ribose modification,
B represents the nucleoside bases of the first two basic nucleosides in the 5' terminal region of SEQ ID NO:611, and
Z represents the remaining 19 contiguous basic nucleosides of SEQ ID NO:611.
[00259] In some embodiments, 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 first strand comprising or consisting of SEQ ID NO: 505 and a modified second strand comprising or consisting of SEQ ID NO:613, wherein the second strand has the following structure
Figure imgf000073_0001
wherein:
T represents a 2’Me ribose modification,
B represents the nucleoside bases of the first two basic nucleosides in the 5' terminal region of SEQ ID NO:613, and
Z represents the remaining 19 contiguous basic nucleosides of SEQ ID NO:613.
[00260] In some embodiments, 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 first strand comprising or consisting of SEQ ID NO: 505 and a modified second strand comprising or consisting of SEQ ID NO:613, wherein the second strand has the following structure
Figure imgf000074_0001
wherein:
T represents a 2’Me ribose modification,
B represents the nucleoside bases of the first two basic nucleosides in the 5' terminal region of SEQ ID NO:613, and
Z represents the remaining 19 contiguous basic nucleosides of SEQ ID NO:613.
Vector And Cell
[00261] In one aspect, the invention provides a cell containing a nucleic acid, such as inhibitory RNA [RNAi] as described herein.
[00262] In one aspect, the invention provides a cell comprising a vector as described herein.
Pharmaceutically Acceptable Compositions
[00263] 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. [00264] The pharmaceutically acceptable composition may comprise an excipient and or carrier.
[00265] Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com 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 laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen- free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or poly anhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; and (22) other nontoxic compatible substances employed in pharmaceutical formulations.
[00266] 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).
[00267] Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids 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.
[00268] 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.
[00269] In one embodiment, the nucleic acid or composition is administered in an unbuffered solution. In certain embodiments, the unbuffered solution is saline or water. In other embodiments, the nucleic acid e.g. siRNA agent is administered in a buffered solution. In such embodiments, the buffer solution can comprise acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. For example, the buffer solution can be phosphate buffered saline (PBS).
Dosages
[00270] The pharmaceutical compositions of the invention may be administered in dosages sufficient to inhibit expression of a gene. In general, 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. Typically, 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.
[00271] A repeat-dose regimen may include administration of a therapeutic amount of a nucleic acid e.g. siRNA on a regular basis, such as every other day or once a year. In certain embodiments, the nucleic acid e.g. siRNA is administered about once per month to about once per quarter (i.e., about once every three months).
[00272] In various embodiments, 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. In some embodiments, the nucleic acid e.g. siRNA agent is administered at a dose of about 10 mg/kg to about 30 mg/kg. In certain embodiments, the nucleic acid e.g. siRNA agent is administered at a dose selected from about 0.5 mg/kg 1 mg/kg, 1.5 mg/kg, 3 mg/kg, 5 mg/kg, 10 mg/kg, and 30 mg/kg. In certain embodiments, the nucleic acid e.g. agent is administered about once per week, once per month, once every other two months, or once a quarter (i.e., once every three months) at a dose of about 0.1 mg/kg to about 5.0 mg/kg. In certain embodiments, the nucleic acid e.g. siRNA agent is administered to the subject once a week. In certain embodiments, the nucleic acid e.g. siRNA agent is administered to the subject once a month. In certain embodiments, the nucleic acid e.g. siRNA agent is administered once per quarter (i.e. , every three months).
[00273] 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.
[00274] 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. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.
[00275] In other embodiments, 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. In some embodiments of the invention, a single dose of the pharmaceutical compositions of the invention is administered once per week. In other embodiments of the invention, a single dose of the pharmaceutical compositions of the invention is administered bimonthly. In certain embodiments, the siRNA is administered about once per month to about once per quarter (i.e. , about once every three months), or even every 6 months or 12 months.
[00276] 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.
[00277] 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. In certain preferred embodiments, the compositions are administered by intravenous infusion or injection. In certain embodiments, the compositions are administered by subcutaneous injection.
[00278] In one embodiment, the nucleic acid e.g. agent is administered to the subject subcutaneously.
[00279] 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 B4GALT1 Gene Expression
[00280] The present invention also provides methods of inhibiting expression of B4GALT1 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 B4GALT1 gene in the cell, thereby inhibiting expression of the B4GALT1 gene in the cell. It is to be noted that a nucleic acid “for inhibiting the expression of B4GALT1” is a nucleic acid that is capable of inhibiting B4GALT1 expression, preferably as described herein below.
[00281] 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.
Furthermore, contacting a cell may be accomplished via a targeting ligand moiety, including any ligand moiety described herein or known in the art. In preferred embodiments, 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.
[00282] The term "inhibiting," as used herein, is used interchangeably with "reducing," "silencing," "downregulating", "suppressing", and other similar terms, and includes any level of inhibition.
[00283] In some embodiments of the methods of the invention, expression of B4GALT1 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. In certain embodiments, the methods include a clinically relevant inhibition of expression of B4GALT1 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.
[00284] In some embodiments, when transfected into the cells, the nucleic acid of the invention inhibits expression of the B4GALT1 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.
[00285] In a preferred embodiment, when transfected into the cells, the nucleic acid of the invention inhibits expression of the B4GALT1 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 B4GALT1 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 B4GALT1 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 B4GALT1 gene with an IC50 value lower than 100 pM.
[00286] Inhibition of expression of the B4GALT1 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 an atmosphere of 5% CO2. Cells may then be transfected with siRNA duplexes targeting B4GALT1 mRNA or a negative control siRNA (siRNA-control; sense strand 5’- UUCUCCGAACGUGUCACGUTT-3’ (SEQ ID NO:934), antisense strand 5’- ACGUGACACGUUCGGAGAATT-3’ (SEQ ID NO:933)) 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 μL Opti-MEM (ThermoFisher) plus 0.3 μL Lipofectamine RNAiMAX (ThermoFisher) to 10 μL of each siRNA duplex. The mixture may be incubated at room temperature for 15 minutes before being added to 100 μL of complete growth medium containing 20,000 Huh7 cells. Cells may be incubated for 24 hours at 37°C/5% CO2 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). Realtime quantitative PCR (qPCR) may be performed on an ABI Prism 7900HT or ABI QuantStudio 7 with primers specific for human B4GALT1 (Hs00155245_ml) and human GAPDH (Hs02786624_gl) using a TaqMan Gene Expression Assay Kit (ThermoFisher Scientific).
[00287] qPCR may be performed in duplicate on cDNA derived from each well and the mean cycle threshold (Ct) calculated. Relative B4GALT1 expression may be calculated from mean Ct values using the comparative Ct (AACt) method, normalised to GAPDH and relative to untreated cells. Maximum percent inhibition of B4GALT1 expression and IC50 values may be calculated using a four parameter (variable slope) model using GraphPad Prism 9.
[00288] Alternatively or in addition, the inhibitory potential of a nucleic acid of the invention may be quantified without prior transfection of a target cell with said nucleic acid.
[00289] Thus, in some embodiments, when cells are incubated with a nucleic acid of the invention, the nucleic acid of the invention inhibits expression of the B4GALT1 gene with an EC50 value lower than 1000 nM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200 nM, or 100 nM, preferably when determined by qPCR, more preferably by reverse transcriptase (RT)-qPCR, as described herein.
[00290] In a preferred embodiment, when cells are incubated with a nucleic acid of the invention, the nucleic acid of the invention inhibits expression of the B4GALT1 gene with an EC50 value lower than 1000 nM. In a more preferred embodiment, when cells are incubated with a nucleic acid of the invention, the nucleic acid of the invention inhibits expression of the B4GALT1 gene with an EC50 value lower than 500 nM. In an even more preferred embodiment, when cells are incubated with a nucleic acid of the invention, the nucleic acid of the invention inhibits expression of the B4GALT1 gene with an EC50 value lower than 200 nM. In a most preferred embodiment, when cells are incubated with a nucleic acid of the invention, the nucleic acid of the invention inhibits expression of the B4GALT1 gene with an EC50 value lower than 100 nM.
[00291] Inhibition of expression of the B4GALT1 gene in the presence of free nucleic acids may be quantified using the following method:
[00292] Primary C57BL/6 mouse hepatocytes (PMHs) may be isolated fresh by two-step collagenase liver perfusion. Cells may be maintained in DMEM (Gibco-11995-092) supplemented with FBS, Penicillin/Streptomycin, HEPES and L-glutamine. Cells may be cultured at 37°C in an atmosphere with 5% CO2 in a humidified incubator. Within 2 hours post isolation, PMHs may be seeded at a density of 36,000 cells/well in regular 96-well tissue culture plates. Dose response analysis in PMHs may be done by direct incubation of cells in a gymnotic free uptake setting with final GalNAc-siRNA concentrations of 1000, 500, 250, 125, 62.5, 31.3, 15.6, 7.8, 3.9, 1.95 nM. In control wells, cells may be incubated without GalNAc-siRNA. After 48hr incubation, cells may be harvested for RNA extraction. Total RNA may be extracted using RNeasy Kit following the manufacturer’s instructions (Qiagen, Shanghai, China). After reverse transcription, real-time quantitative PCR may be performed using an ABI Prism 7900HT to detect the relative abundance of B4GALT1 mRNA normalized to the housekeeping gene GAPDH. The expression of the target gene in each test sample may be determined by relative quantitation using the comparative Ct (AACt) method. This method measures the Ct differences (ACt) between target gene and housekeeping gene. The formula is as follows: ACt = average Ct of B4GALT1 -average Ct of GAPDH, AACt=ACt (sample) - average ACt (untreated control), relative expression of target gene mRNA = 2'AACt.
[00293] Alternatively or in addition, inhibition of expression of the B4GALT1 gene may be characterized by a reduction of mean relative expression of the B4GALT1 gene.
[00294] In some embodiments, when cells are transfected with 0. 1 nM of the nucleic acid of the invention, the mean relative expression of B4GALT1 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.
[00295] In some embodiments, when cells are transfected with 5 nM of the nucleic acid of the invention, the mean relative expression of B4GALT1 is below 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3 or 0.2, preferably when determined by qPCR, more preferably by reverse transcriptase (RT)-qPCR, as described herein.
[00296] Mean relative expression of the B4GALT1 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% CO2. Cells may be transfected with siRNA duplexes targeting B4GALT1 mRNA or a negative control siRNA (siRNA-control; sense strand 5’- UUCUCCGAACGUGUCACGUTT-3’ (SEQ ID NO:934), antisense strand 5’- ACGUGACACGUUCGGAGAATT-3’ (SEQ ID NO:933)) at a final duplex concentration of 5 nM and 0.1 nM. Transfection may be carried out by adding 9.7 μL Opti-MEM (ThermoFisher) plus 0.3 μL Lipofectamine RNAiMAX (ThermoFisher) to 10 μL of each siRNA duplex. The mixture may be incubated at room temperature for 15 minutes before being added to 100 μL of complete growth medium containing 20,000 Huh7 cells. Cells may be incubated for 24 hours at 37°C/5% CO2 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). Realtime quantitative PCR (qPCR) may be performed on an ABI Prism 7900HT or ABI QuantStudio 7 with primers specific for human B4GALT1 (Hs00155245_ml) and human GAPDH (Hs02786624_gl) using a TaqMan Gene Expression Assay Kit (ThermoFisher Scientific). qPCR may be performed in duplicate on cDNA derived from each well and the mean Ct calculated. Relative B4GALT1 expression may be calculated from mean Ct values using the comparative Ct (AACt) method, normalised to GAPDH and relative to untreated cells.
[00297] Inhibition of the expression of B4GALT1 gene may be manifested by a reduction of the amount of mRNA of the target B4GALT1 gene in comparison to a suitable control.
[00298] In other embodiments, inhibition of the expression of B4GALT1 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 B4GALT1 Gene Expression
[00299] 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 B4GALT1 gene expression in a cell. The methods include contacting the cell with a nucleic acid e.g. dsiRNA of the invention and maintaining the cell for a time sufficient to obtain degradation of the mRNA transcript of B4GALT1, thereby inhibiting expression of the B4GALT1 gene in the cell. Reduction in gene expression can be assessed by any methods known in the art.
[00300] In the methods of the invention the cell may be contacted in vitro or in vivo, i.e., the cell may be within a subject. [00301] A cell suitable for treatment using the methods of the invention may be any cell that expresses a gene of interest associated with diabetes or cardiovascular disease.
[00302] 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 B4GALT1 gene of the mammal to be treated.
[00303] 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 B4GALT1 gene, in a therapeutically effective amount e.g. a nucleic acid such as an siRNA targeting B4GALT1 or a pharmaceutical composition comprising the nucleic acid targeting B4GALT1. The disease to be treated is diabetes or a cardiovascular disease.
[00304] According to the invention, the term “diabetes” as used herein, refers to group of metabolic diseases in which a subject has high blood sugar, either because the body does not produce enough insulin, or because cells do not respond to the insulin that is produced. There are three main types of diabetes: (1) Type 1 diabetes (T1D): results from the body's failure to produce insulin, and presently requires the person to inject insulin. (Also referred to as insulin-dependent diabetes mellitus, IDDM for short, and juvenile diabetes.) (2) Type 2 diabetes T2D): results from insulin resistance, a condition in which cells fail to use insulin properly, sometimes combined with an absolute insulin deficiency. (Formerly referred to as non-insulin-dependent diabetes mellitus, NIDDM for short, and adult-onset diabetes.) (3) Gestational diabetes (GD): is when pregnant women, who have never had diabetes before, have a high blood glucose level during pregnancy. It may precede development of T2D.
[00305] In certain embodiments, the nucleic acid according to the invention or a pharmaceutical composition comprising said nucleic acid is used for the treatment of diabetes, preferably type 2 diabetes (T2D).
[00306] In certain embodiments, the nucleic acid according to the invention pharmaceutical composition comprising said nucleic acid is used for the treatment of diabetes, preferably type 2 diabetes (T2D), wherein the treatment results in a reduction in LDL-cholesterol (LDL- c) levels in the blood. [00307] In certain embodiments, the nucleic acid according to the invention pharmaceutical composition comprising said nucleic acid is used for the treatment of diabetes, preferably type 2 diabetes (T2D), wherein the treatment results in a reduction in fasting glcuose levels in the blood.
[00308] In certain embodiments, the nucleic acid according to the invention pharmaceutical composition comprising said nucleic acid is used for the treatment of diabetes, preferably type 2 diabetes (T2D), wherein the treatment results in a reduction in fibrinogen levels in the blood.
[00309] The term "cardiovascular disease" as used herein refers to any condition, disorder or disease state associated with, resulting from or causing a structural or functional abnormality of the heart, or of the blood vessels supplying the heart, that impairs its normal functioning. Cardiovascular disease may comprise coronary artery disease, atherosclerosis, myocardial infarction, arteriosclerosis, hypertension, angina, deep vein thrombosis, stroke, congestive heart failure or arrhythmia. In a preferred embodiment, the cardiovascular disease is coronary artery disease. In certain embodiments, the nucleic acid according to the invention pharmaceutical composition comprising said nucleic acid is used for the treatment of cardiovascular disease, preferably coronary artery disease.
[00310] In certain embodiments, the nucleic acid according to the invention pharmaceutical composition comprising said nucleic acid is used for the treatment of cardiovascular disease, preferably coronary artery disease, wherein the treatment results in a reduction in LDL- cholesterol (LDL-c) levels in the blood.
[00311] In certain embodiments, the nucleic acid according to the invention pharmaceutical composition comprising said nucleic acid is used for the treatment of cardiovascular disease, preferably coronary artery disease, wherein the treatment results in a reduction in fibrinogen levels in the blood.
[00312] An 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. In one embodiment, the buffer solution is phosphate buffered saline (PBS). The pH and osmolarity of the buffer solution can be adjusted such that it is suitable for administering to a subject. [00313] Alternatively, a nucleic acid e.g. siRNA of the invention may be administered as a pharmaceutical composition, such as a dsiRNA liposomal formulation.
[00314] In one embodiment, the method includes administering a composition featured herein such that expression of B4GALT1 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. In one embodiment, expression of B4GALT1 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.
[00315] 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 treat disease related to diabetes or cardiovascular disease.
[00316] 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 B4GALT1 target gene , e.g., in a cell or tissue of the patient by at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or below the level of detection of the assay method used. In certain embodiments, administration results in clinical stabilization or preferably clinically relevant reduction of at least one sign or symptom of a B4GALT1 gene- associated disorder.
[00317] Alternatively, 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. In certain embodiments, after an initial treatment regimen, 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. In certain embodiments, the nucleic acid is administered about once per month to about once per quarter (i.e. , about once every three months).
[00318] In one aspect 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. Specifically, an oligonucleoside moiety as represented by Z in any of the following sentences can comprise a nucleic acid for inhibiting expression of B4GALT1 as defined in any of the sentences hereinafter.
1. A compound comprising the following structure:
Figure imgf000086_0001
Formula (I) wherein: R1 at each occurrence is independently selected from the group consisting of hydrogen, methyl and ethyl;
R2 is selected from the group consisting of hydrogen, hydroxy, -OC1-3alkyl, -C(=O)OC1-3alkyl, halo and nitro; X1 and X2 at each occurrence are independently selected from the group consisting of methylene, oxygen and sulfur; m is an integer of from 1 to 6; n is an integer of from 1 to 10; q, r, s, t, v are independently integers from 0 to 4, with the proviso that:
(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.
2. A compound according to Sentence 1, wherein R1 is hydrogen at each occurrence.
3. A compound according to Sentence 1, wherein R1 is methyl.
4. A compound according to Sentence 1, wherein R1 is ethyl.
5. A compound according to any of Sentences 1 to 4, wherein R2 is hydroxy. A compound according to any of Sentences 1 to 4, wherein R2 is halo. A compound according to Sentence 6, wherein R2 is fluoro. A compound according to Sentence 6, wherein R2 is chloro. A compound according to Sentence 6, wherein R2 is bromo. A compound according to Sentence 6, wherein R2 is iodo. A compound according to Sentence 6, wherein R2 is nitro. A compound according to any of Sentences 1 to 11, wherein X1 is methylene. A compound according to any of Sentences 1 to 11, wherein X1 is oxygen. A compound according to any of Sentences 1 to 11, wherein X1 is sulfur. A compound according to any of Sentences 1 to 14, wherein X2 is methylene. A compound according to any of Sentences 1 to 15, wherein X2 is oxygen. A compound according to any of Sentences 1 to 16, wherein X2 is sulfur. A compound according to any of Sentences 1 to 17, wherein m = 3. A compound according to any of Sentences 1 to 18, wherein n = 6. A compound according to Sentences 13 and 15, wherein X1 is oxygen and X2 is methylene, and preferably wherein: q = 1, r = 2, s = 1, t = 1, v = 1. A compound according to Sentences 12 and 15, wherein both X1 and X2 are methylene, and preferably wherein: q = 1, r = 3, s = 1, t = 1, v = 1.
22. A compound according to any of Sentences 1 to 21, wherein Z is:
Figure imgf000088_0001
wherein:
Zi, Z2, Z3, Z4 are independently at each occurrence oxygen or sulfur; and one the bonds between P and Z2, and P and Z3 is a single bond and the other bond is a double bond.
23. A compound according to Sentence 22, wherein said oligonucleoside is an RNA compound capable of modulating, preferably inhibiting, expression of a target gene.
24. A compound according to Sentence 23, wherein 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.
25. A compound according to Sentence 24, wherein the RNA compound is attached at the 5’ end of its second strand to the adjacent phosphate.
26. A compound according to Sentence 24, wherein the RNA compound is attached at the 3’ end of its second strand to the adjacent phosphate. 27. A compound of Formula (II):
Figure imgf000089_0001
Formula (II)
28. A compound of Formula (III):
Figure imgf000089_0002
Formula (III)
29. A compound according to Sentence 27 or 28, 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.
30. 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 composition according to Sentence 30, wherein said compound of Formula (III) as defined in Sentence 28 is present in an amount in the range of 10 to 15% by weight of said composition.
32. A compound of Formula (IV):
Figure imgf000089_0003
Formula (IV) 33. A compound of Formula (V):
Figure imgf000090_0001
Formula (V)
34. 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.
35. 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. A compound according to any of Sentences 1 to 29, or 32 to 34, or 37 to 38, wherein the oligonucleoside further comprises one or more degradation protective moieties at one or more ends.
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 intemucleoside linkages and inverted abasic nucleosides, wherein said inverted abasic nucleosides are present at the distal end of the strand that carries the ligand moieties.
41. A compound according to any of Sentences 1 to 29, or 32 to 34, or 37 to 40, wherein said ligand moiety as depicted in Formula (I) in Sentence 1 comprises one or more ligands.
42. A compound according to Sentence 41, wherein said ligand moiety as depicted in Formula (I) in Sentence 1 comprises one or more carbohydrate ligands.
43. A compound according to Sentence 42, wherein said one or more carbohydrates can be a monosaccharide, di saccharide, tri saccharide, 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.
45. A compound according to Sentence 44, wherein said one or more carbohydrates comprise one or more N-Acetyl-Galactosamine moieties.
46. A compound according to Sentence 45, which comprises two or three N- AcetylGalactosamine moieties.
47. A compound according to any of Sentences 41 to 46, wherein said one or more ligands are attached in a linear configuration, or in a branched configuration.
48. A compound according to Sentence 47, wherein said one or more ligands are attached as a biantennary or triantennary branched configuration.
49. A compound according to Sentences 46 to 48, wherein said moiety:
Figure imgf000091_0001
as depicted in Formula (I) in Sentence 1 is any of Formulae (Via), (VIb) or (Vic), preferably Formula (Via):
Figure imgf000092_0001
Formula (Via) wherein:
Ai is hydrogen, or a suitable hydroxy protecting group; a is an integer of 2 or 3; and b is an integer of 2 to 5; or
Figure imgf000092_0002
Formula (VIb) wherein:
Ai is hydrogen, or a suitable hydroxy protecting group; a is an integer of 2 or 3; and c and d are independently integers of 1 to 6; or
Figure imgf000093_0001
wherein:
Ai is hydrogen, or a suitable hydroxy protecting group; a is an integer of 2 or 3; and e is an integer of 2 to 10.
50. A compound according to Sentences 46 to 48, wherein said moiety:
Figure imgf000093_0002
as depicted in Formula (I) in Sentence 1 is Formula (VII):
Figure imgf000093_0003
wherein:
Ai is hydrogen; a is an integer of 2 or 3. A compound according to Sentence 49 or 50, wherein a = 2. A compound according to Sentence 49 or 50, wherein a = 3. A compound according to Sentence 49, wherein b = 3. A compound of Formula (VIII):
Figure imgf000094_0001
Formula (VIII) A compound of F ormula (IX) :
Figure imgf000094_0002
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 composition according to Sentence 57, wherein said compound of Formula (IX) as defined in Sentence 55 is present in an amount in the range of 10 to 15% by weight of said composition. A compound of Formula (X):
Figure imgf000095_0001
Formula (X) A compound of Formula (XI):
Figure imgf000095_0002
Formula (XI) 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. A composition according to Sentence 62, wherein said compound of Formula (XI) as defined in Sentence 60 is present in an amount in the range of 10 to 15% by weight of said composition. 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. A compound according to Sentence 64, wherein the modifications are chosen from 2’-O- methyl, 2’ -deoxy -fluoro, and 2’-deoxy. A compound according to any of Sentences 54 to 65, wherein the oligonucleoside further comprises one or more degradation protective moieties at one or more ends. A compound according to Sentence 66, wherein said one or more degradation protective moieties are not present at the end of the oligonucleoside strand that carries the ligand moieties, and / or wherein said one or more degradation protective moieties is selected from phosphorothioate internucleoside linkages, phosphorodithioate intemucleoside linkages and inverted abasic nucleosides, wherein said inverted abasic nucleosides are present at the distal end of the strand that carries the ligand moieties, as shown in any of Formulae (VIII), (IX), (X) or (XI) in any of Sentences 54, 55, 59 or 60. A process of preparing 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, 63, which comprises reacting compounds of Formulae (XII) and (XIII):
Figure imgf000096_0001
Formula (XII)
Figure imgf000097_0001
Formula (XIII) herein: R1 at each occurrence is independently selected from the group consisting of hydrogen, methyl and ethyl;
R2 is selected from the group consisting of hydrogen, hydroxy, -OC1-3alkyl, -C(=O)OC1-3alkyl, halo and nitro; X1 and X2 at each occurrence are independently selected from the group consisting of methylene, oxygen and sulfur; m is an integer of from 1 to 6; n is an integer of from 1 to 10; q, r, s, t, v are independently integers from 0 to 4, with the proviso that:
(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; and where appropriate carrying out deprotection of the ligand and / or annealing of a second strand for the oligonucleoside moiety.
69. A process according to Sentence 68, wherein a compound of Formula (XII) is prepared by reacting compounds of Formulae (XIV) and (XV):
Figure imgf000097_0002
Formula (XIV)
Figure imgf000098_0001
Formula (XV) R1 at each occurrence is independently selected from the group consisting of hydrogen, methyl and ethyl;
R2 is selected from the group consisting of hydrogen, hydroxy, -OCi-salkyl, -C(=O)OC1-3alkyl, halo and nitro; X1 and X2 at each occurrence are independently selected from the group consisting of methylene, oxygen and sulfur; q, r, s, t, v are independently integers from 0 to 4, with the proviso that:
(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.
70. A process according to Sentence 68, to prepare a compound according to any of Sentences
20, 25, 27, 29, 54, 56, and / or a composition according to any of Sentences 30, 31, 57, 58, wherein: compound of Formula (XII) is Formula (Xlla):
Figure imgf000098_0002
Formula (Xlla) and compound of Formula (XIII) is Formula (Xllla):
Figure imgf000099_0003
Formula (Xllla) 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.
71. A process according to Sentence 68, to prepare a compound according to any of Sentences 20, 25, 28, 29, 55, 56, and / or a composition according to any of Sentences 30, 31, 57, 58, wherein: compound of Formula (XII) is Formula (Xllb):
Figure imgf000099_0001
Formula (Xllb) and compound of Formula (XIII) is Formula (Xllla):
Figure imgf000099_0002
Formula (Xllla) 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.
72. A process according to Sentence 68, to prepare a compound according to any of Sentences 21, 26, 32, 34, 59, 61, and / or a composition according to any of Sentences 35, 36, 62, 63, wherein: compound of Formula (XII) is Formula (XIIc):
Figure imgf000100_0001
Formula (XIIc) and compound of Formula (XIII) is Formula (Xllla):
Figure imgf000100_0002
Formula (Xllla) 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.
73. A process according to Sentence 68, to prepare a compound according to any of Sentences 21, 26, 33, 34, 60, 61, and / or a composition according to any of Sentences 35, 36, 62, 63, wherein: compound of Formula (XII) is Formula (Xlld):
Figure imgf000101_0001
Formula (Xlld) and compound of Formula (XIII) is Formula (Xllla):
Figure imgf000101_0002
Formula (Xllla) 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.
74. A process according to any of Sentences 70 to 73, wherein: compound of Formula (Xllla) is Formula (Xlllb):
Figure imgf000101_0003
Formula (Xlllb) 75. A process according to Sentences 69, as dependent on Sentences 70 to 73, wherein: compound of Formula (XIV) is either Formula (XlVa) or Formula (XlVb):
Figure imgf000102_0001
Formula (XI Va)
Figure imgf000102_0002
Formula (XlVb) and compound of Formula (XV) is either Formula (XVa) or Formula (XlVb):
Figure imgf000102_0003
Formula (XVa)
Figure imgf000102_0004
Formula (XVb) 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 (i) said RNA duplex is attached at the 5’ end of its second strand to the adjacent phosphate in Formula (XVa), or (ii) said RNA duplex is attached at the 3’ end of its second strand to the adjacent phosphate in Formula (XVb).
76. A compound of Formula (XII):
Figure imgf000103_0001
Formula (XII) wherein: R1 at each occurrence is independently selected from the group consisting of hydrogen, methyl and ethyl;
R2 is selected from the group consisting of hydrogen, hydroxy, -OC1-3alkyl, -C(=O)OC1-3alkyl, halo and nitro; X1 and X2 at each occurrence are independently selected from the group consisting of methylene, oxygen and sulfur; q, r, s, t, v are independently integers from 0 to 4, with the proviso that:
(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.
77. A compound of Formula (Xlla):
Figure imgf000103_0002
Formula (Xlla) A compound of Formula (Xllb):
Figure imgf000104_0001
Formula (Xllb) A compound of Formula (XIIc):
Figure imgf000104_0002
Formula (XIIc) A compound of Formula (Xlld):
Figure imgf000104_0003
Formula (Xlld) A compound of Formula (XIII):
Figure imgf000104_0004
Formula (XIII) wherein: R1 at each occurrence is independently selected from the group consisting of hydrogen, methyl and ethyl; m is an integer of from 1 to 6; n is an integer of from 1 to 10.
82. A compound of Formula (Xllla):
Figure imgf000105_0001
Formula (Xllla)
83. A compound of Formula (Xlllb):
Figure imgf000105_0003
Formula (Xlllb)
84. A compound of Formula (XIV):
Figure imgf000105_0002
Formula (XIV) wherein: R1 is selected from the group consisting of hydrogen, methyl and ethyl;
R2 is selected from the group consisting of hydrogen, hydroxy, -OC1-3alkyl, -C(=O)OC1-3alkyl, halo and nitro;
X2 is selected from the group consisting of methylene, oxygen and sulfur; s, t, v are independently integers from 0 to 4, with the proviso that s, t and v cannot all be 0 at the same time.
85. A compound of Formula (XlVa):
Figure imgf000106_0001
Formula (XI Va)
86. A compound of Formula (XlVb):
Figure imgf000106_0002
Formula (XI Vb)
87. A compound of Formula (XV):
Figure imgf000106_0003
Formula (XV) wherein: R1 at each occurrence is independently selected from the group consisting of hydrogen, methyl and ethyl; X1 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):
Figure imgf000107_0001
Formula (XVa)
89. A compound of Formula (XVb):
Figure imgf000107_0002
Formula (XVb)
90. Use of a compound according to any of Sentences 76, 81 to 84, 87, for the preparation 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.
91. Use of a compound according to Sentence 85, for the preparation 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, wherein R2 = F. Use of a compound according to Sentence 86, for the preparation 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, wherein R2 = OH. Use of a compound according to Sentence 77, for the preparation of a compound according to any of Sentences 20, 25, 27, 29, 54, 56, and / or a composition according to any of Sentences 30, 31, 57, 58. Use of a compound according to Sentence 78, for the preparation of a compound according to any of Sentences 20, 25, 28, 29, 55, 56, and / or a composition according to any of Sentences 30, 31, 57, 58. Use of a compound according to Sentence 79, for the preparation of a compound according to any of Sentences 21, 26, 32, 34, 59, 61, and / or a composition according to any of Sentences 35, 36, 62, 63. Use of a compound according to Sentence 80, for the preparation of a compound according to any of Sentences 21, 26, 33, 34, 60, 61, and / or a composition according to any of Sentences 35, 36, 62, 63. Use of a compound according to Sentence 88, for the preparation of a compound according to any of Sentences 20, 25, 27 to 29, 54 to 56, and / or a composition according to any of Sentences 30, 31, 57, 58. Use of a compound according to Sentence 89, for the preparation of a compound according to any of Sentences 21, 26, 32 to 34, 59 to 61, and / or a composition according to any of Sentences 35, 36, 62, 63. A compound or composition obtained, or obtainable by a process according to any of Sentences 68 to 75. 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. 101. 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, for use in therapy.
[00319] In another aspect the present invention may be applied in the compounds, processes, compositions or uses of the following Clauses numbered 1-56 wherein reference to any Formula in the Clauses refers only to those Formulas that are defined within Clause 1-56. These formulae are reproduced in Figure 6. Specifically, an oligonucleoside moiety as represented by Z in any of the following clauses can comprise a nucleic acid for inhibiting expression of B4GALT1 as defined in any of the sentences hereinafter.
1. A compound comprising the following structure:
Figure imgf000109_0001
Formula (I) wherein: r and s are independently an integer selected from 1 to 16; and
Z is an oligonucleoside moiety.
2. A compound according to Clause 1, wherein s is an integer selected from 4 to 12.
3. A compound according to Clause 2, wherein s is 6.
4. A compound according to any of Clauses 1 to 3, wherein r is an integer selected from 4 to 14.
5. A compound according to Clause 4, wherein r is 6.
6. A compound according to Clause 4, wherein r is 12.
7. A compound according to Clause 5, which is dependent on Clause 3.
8. A compound according to Clause 6, which is dependent on Clause 3.
9. A compound according to any of Clauses 1 to 8, wherein Z is:
Figure imgf000110_0001
wherein:
Zi, Z2, Z3, Z4 are independently at each occurrence oxygen or sulfur; and one the bonds between P and Z2, and P and Z3 is a single bond and the other bond is a double bond.
10. A compound according to any of Clauses 1 to 9, wherein said oligonucleoside is an RNA compound capable of modulating, preferably inhibiting, expression of a target gene.
11. A compound according to any of Clause 10, wherein 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.
12. A compound according to Clause 11, preferably also dependent on Clauses 3 and 6, wherein the RNA compound is attached at the 5’ end of its second strand to the adjacent phosphate.
13. A compound according to Clause 11, preferably also dependent on Clauses 3 and 5, wherein the RNA compound is attached at the 3’ end of its second strand to the adjacent phosphate.
14. A compound of Formula (II), preferably dependent on Clause 12:
Figure imgf000110_0002
Formula (II)
15. A compound of Formula (III), preferably dependent on Clause 13:
Figure imgf000111_0001
Formula (III) A compound as defined in any of Clauses 1 to 15, 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. A compound according to Clause 16, wherein the modifications are chosen from 2’-O- methyl, 2’ -deoxy -fluoro, and 2’-deoxy. A compound according to any of Clauses 1 to 17, wherein the oligonucleoside further comprises one or more degradation protective moieties at one or more ends. A compound according to Clause 18, 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. A compound according to any of Clauses 1 to 19, wherein said ligand moiety as depicted in Formula (I) in Clause 1 comprises one or more ligands. A compound according to Clause 20, wherein said ligand moiety as depicted in Formula (I) in Clause 1 comprises one or more carbohydrate ligands. A compound according to Clause 21, wherein said one or more carbohydrates can be a monosaccharide, di saccharide, tri saccharide, tetrasaccharide, oligosaccharide or polysaccharide. A compound according to Clause 22, 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. 24. A compound according to Clause 23, wherein said one or more carbohydrates comprise one or more N-Acetyl-Galactosamine moieties.
25. A compound according to Clause 24, which comprises two or three N- AcetylGalactosamine moieties.
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 26, wherein said one or more ligands are attached as a biantennary or triantennary branched configuration.
28. A compound according to Clauses 20 to 27, wherein said moiety:
Figure imgf000112_0001
as depicted in Formula (I) in Clause 1 is any of Formulae (IV), (V) or (VI), preferably Formula (IV):
Figure imgf000112_0002
Formula (IV) wherein:
Ai is hydrogen, or a suitable hydroxy protecting group; a is an integer of 2 or 3; and b is an integer of 2 to 5; or
Figure imgf000113_0001
Formula (V) wherein:
Ai is hydrogen, or a suitable hydroxy protecting group; a is an integer of 2 or 3; and c and d are independently integers of 1 to 6; or
Figure imgf000113_0002
Formula (VI) wherein:
Ai is hydrogen, or a suitable hydroxy protecting group; a is an integer of 2 or 3; and e is an integer of 2 to 10.
29. A compound according to any of Clauses 1 to 28, wherein said moiety:
Figure imgf000114_0001
as depicted in Formula (I) in Clause 1 is Formula (VII):
Figure imgf000114_0002
Formula (VII) wherein:
Ai is hydrogen; a is an integer of 2 or 3.
30. A compound according to Clause 28 or 29, wherein a = 2.
31. A compound according to Clause 28 or 29, wherein a = 3.
32. A compound according to Clause 28, wherein b = 3.
33. A compound of Formula (VIII):
Figure imgf000114_0003
Formula (VIII)
34. A compound of Formula (IX):
Figure imgf000115_0001
Formula (IX) 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. A compound according to Clause 35, wherein the modifications are chosen from 2’-O- methyl, 2’ -deoxy -fluoro, and 2’-deoxy. A compound according to any of Clauses 33 to 36, wherein the oligonucleoside further comprises one or more degradation protective moieties at one or more ends. 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. A compound according to Clause 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 5’ end of its second strand to the adjacent phosphate. A compound according to Clause 34, 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.
41. A process of preparing a compound according to any of Clauses 1 to 40, which comprises reacting compounds of Formulae (X) and (XI):
Figure imgf000116_0001
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. A process according to Clause 41, to prepare a compound according to any of Clauses 6, 8 to 14, 16 to 33, and 35 to 40, wherein: compound of Formula (X) is Formula (Xa):
Figure imgf000116_0002
Formula (Xa) and compound of Formula (XI) is Formula (Xia):
Figure imgf000117_0001
Formula (Xia) 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.
43. A process according to Clause 41, to prepare a compound according to any of Clauses 5, 7, 9 to 13, 15 to 32, and 34 to 40, wherein: compound of Formula (X) is Formula (Xb):
Figure imgf000117_0002
Formula (Xb) and compound of Formula (XI) is Formula (Xia):
Figure imgf000117_0003
Formula (Xia) 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.
44. A process according to Clauses 42 or 43, wherein: compound of Formula (Xia) is Formula (Xlb):
Figure imgf000118_0001
Formula (Xlb)
45. A compound of Formula (X):
Figure imgf000118_0002
Formula (X) wherein: r is independently an integer selected from 1 to 16; and Z is an oligonucleoside moiety.
46. A compound of Formula (Xa):
Figure imgf000119_0001
Formula (Xa)
47. A compound of Formula (Xb):
Figure imgf000119_0002
Formula (Xb)
48. A compound of Formula (XI):
Figure imgf000119_0003
Formula (XI) wherein: s is independently an integer selected from 1 to 16; and
Z is an oligonucleoside moiety.
49. A compound of Formula (Xia):
Figure imgf000119_0004
Formula (Xia)
50. A compound of Formula (Xlb):
Figure imgf000120_0001
Formula (Xlb)
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.
56. A compound according to any of Clauses 1 to 40, for use in therapy.
EXAMPLES
[00320] The invention will be more fully understood by reference to the following examples.
They should not, however, be construed as limiting the scope of the invention. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended Clauses. Example 1: Synthesis of tether 1
General Experimental conditions:
[00321] Thin layer chromatography (TLC) was performed on silica-coated aluminium plates with fluorescence indicator 254 nm from Macherey -Nagel. Compounds were visualized under UV light (254 nm), or after spraying with the 5% H2SO4 in methanol (MeOH) or ninhydrin reagent according to Stahl (from Sigma-Aldrich), followed by heating. Flash chromatography was performed with a Biotage Isol era One flash chromatography instrument equipped with a dual variable UV wavelength detector (200-400 nm) using Biotage Sfar Silica 10, 25, 50 or 100 g columns (Uppsala, Sweden).
[00322] All moisture-sensitive reactions were carried out under anhydrous conditions using dry glassware, anhydrous solvents, and argon atmosphere. All commercially available reagents were purchased from Sigma-Aldrich and solvents from Carl Roth GmbH + Co. KG. D- Galactosamine pentaacetate was purchased from AK scientific.
[00323] HPLC/ESI-MS was performed on a Dionex UltiMate 3000 RS UHPLC system and Thermo Scientific MSQ Plus Mass spectrometer using an Acquity UPLC Protein BEH C4 column from Waters (300A, 1.7 pm, 2.1 x 100 mm) at 60 °C. The solvent system consisted of solvent A with H2O containing 0.1% formic acid and solvent B with acetonitrile (ACN) containing 0.1% formic acid. A gradient from 5-100% of B over 15 min with a flow rate of 0.4 mL/min was employed. Detector and conditions: Corona ultra-charged aerosol detection (from esa). Nebulizer Temp.: 25 °C. N2 pressure: 35.1 psi. Filter: Corona.
[00324] 1 H and 13C NMR spectra were recorded at room temperature on a Varian spectrometer at 500 MHz ('H NMR) and 125 MHz (13C NMR). Chemical shifts are given in ppm referenced to the solvent residual peak (CDCI3 - 'H NMR: 5 at 7.26 ppm and 13C NMR 5 at 77.2 ppm; DMSO4 - 1H NMR: 5 at 2.50 ppm and 13C NMR 5 at 39.5 ppm). Coupling constants are given in Hertz. Signal splitting patterns are described as singlet (s), doublet (d), triplet (t) or multiplet (m).
[00325] Synthesis route for the conjugate building block TriGalNAc Tether 1 :
Figure imgf000122_0001
[00326] 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. NaHCO3 (100 mL) and water (100 mL). The organic layer was separated, dried over Na2SO4 and concentrated to afford the title compound as yellow oil, which was purified by flash chromatography (gradient elution: 0-10% MeOH in DCM in 10 CV). The product was obtained as colourless oil (2.5 g, 98%, rf= 0.45 (2% MeOH in DCM)).
Figure imgf000122_0002
[00327] Preparation of compound 4: Compound 2 (2.30 g, 6.98 mmol, 1.0 eq.) and azido- PEG3-OH (1.83 g, 10.5 mmol, 1.5 eq.) were dissolved in anhydrous DCM (40 mL) under argon and molecular sieves 3 A (5 g) were added to the solution. The mixture was stirred at room temperature for 1 h. TMSOTf (0.77 g, 3.49 mmol, 0.5 eq.) was then added to the mixture and the reaction was stirred overnight. The molecular sieves were filtered, the filtrate was diluted with DCM (100 mL) and washed with cold saturated aq. NaHCCh (100 mL) and water (100 mL). The organic layer was separated, dried over Na2SO4 and the solvent was removed under reduced pressure. The crude material was purified by flash chromatography (gradient elution: 0-3% MeOH in DCM in 10 CV) to afford the title product as light yellow oil (3.10 g, 88%, rf = 0.25 (2% MeOH in DCM)). MS: calculated for C20H32N4O11, 504.21. Found 505.4. XH NMR (500 MHz, CDCI3) 5 6.21-6.14 (m, 1H), 5.30 (dd, J= 3.4, 1.1 Hz, 1H), 5.04 (dd, J= 11.2, 3.4 Hz,lH), 4.76 (d, J= 8.6 Hz, 1H), 4.23-4.08 (m, 3H), 3.91-3.80 (m, 3H), 3.74-3.59 (m, 9H), 3.49-3.41 (m, 2H), 2.14 (s, 3H), 2.02 (s, 3H), 1.97 (d, J= 4.2 Hz, 6H). 13C NMR (125 MHz, CDCI3) 5 170.6 (C), 170.5 (C), 170.4 (C), 170.3 (C), 102.1 (CH), 71.6 (CH), 70.8 (CH), 70.6 (CH), 70.5 (CH), 70.3 (CH2), 69.7 (CH2), 68.5 (CH2), 66.6 (CH2), 61.5 (CH2), 23.1 (CH3), 20.7 (3xCH3).
Figure imgf000123_0001
[00328] Preparation of compound 5: Compound 4 (1.00 g, 1.98 mmol, 1.0 eq.) was dissolved in a mixture of ethyl acetate (EtOAc) and MeOH (30 mL 1 : 1 v/v) and Pd/C (100 mg) was added. The reaction mixture was degassed using vacuum/argon cycles (3x) and hydrogenated under balloon pressure overnight. The reaction mixture was filtered through celite and washed with EtOAc (30 mL). The solvent was removed under reduced pressure to afford the title compound as colourless oil (0.95 g, quantitative yield, rf = 0.25 (10% MeOH in DCM)). The compound was used without further purification. MS: calculated for C20H34N2O11, 478.2. Found 479.4.
Figure imgf000123_0002
[00329] Preparation of compound 7: Tris{[2-(tert-butoxycarbonyl)ethoxy]methyl}-methylamine 6 (3.37 g, 6.67 mmol, 1.0 eq.) was dissolved in a mixture of DCM/water (40 mL 1 : 1 v/v) and Na2CO3 (0.18 g, 1.7 mmol, 0.25 eq.) was added while stirring vigorously. Benzyl chloroformate (2.94 mL, 20.7 mmol, 3.10 eq.) was added dropwise to the previous mixture and the reaction was stirred at room temperature for 24 h. The reaction mixture was diluted with CH2Q2 (100 mL) and washed with water (100 mL). The organic layer was separated and dried over Na2SO4. The solvent was removed under reduced pressure and the resulting crude material was purified by flash chromatography (gradient elution: 0-10% EtOAc in cyclohexane in 12 CV) to afford the title compound as pale yellowish oil (3.9 g, 91%, rf = 0.56 (10% EtOAc in cyclohexane)). MS: calculated for C33Hs3NOn, 639.3. Found 640.9. 'H NMR (500 MHz, DMSO-d6) 5 7.38-7.26 (m, 5H), 4.97 (s, 2H), 3.54 (t, 6H), 3.50 (s, 6H), 2.38 (t, 6H), 1.39 (s, 27H). 13C NMR (125 MHz, DMSO-d6 ) 5 170.3 (3xC), 154.5 (C), 137.1 (C), 128.2 (2xCH), 127.7 (CH), 127.6 (2xCH), 79.7 (3xC), 68.4 (3xCH2), 66.8 (3xCH2), 64.9 (C), 58.7 (CH2), 35.8 (3xCH2), 27.7 (9xCH3).
Figure imgf000124_0001
[00330] Preparation of compound 8: Cbz-NH-tris-Boc-ester 7 (0.20 g, 0.39 mmol, 1.0 eq.) was dissolved in CH2Q2 (1 mL) under argon, trifluoroacetic acid (TFA, 1 mL) was added and the reaction was stirred at room temperature for 1 h. The solvent was removed under reduced pressure, the residue was co-evaporated 3 times with toluene (5 mL) and dried under high vacuum to get the compound as its TFA salt (0.183 g, 98%). The compound was used without further purification. MS: calculated for C21H29NO11, 471.6. Found 472.4.
Figure imgf000124_0002
[00331] Preparation of compound 9: CbzNH-tris-COOH 8 (0.72 g, 1.49 mmol, 1.0 eq.) and GalNAc-PEG3-NH2 5 (3.56 g, 7.44 mmol, 5.0 eq.) were dissolved in N,N- dimethylformamide (DMF) (25 mL). Then A,A,A',M-tetramethyl-O-(lH-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 NN- 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. NaHCO3 (100 mL). The organic layer was dried over NafeSO4.the solvent evaporated and the crude material was purified by flash chromatography (gradient elution: 0-5% MeOH in DCM in 14 CV). The product was obtained as pale yellowish oil (1.2 g, 43%, rf = 0.20 (5% MeOH in DCM)). MS: calculated for C81H125N7O41, 1852.9. Found 1854.7. *HNMR (500 MHz, DMSO-d6) 5 7.90- 7.80 (m, 10H), 7.65-7.62 (m, 4H), 7.47-7.43 (m, 3H), 7.38-7.32 (m, 8H), 5.24-5.22 (m, 3H), 5.02-4.97 (m, 4H), 4.60-4.57 (m, 3 H), 4.07-3.90 (m 10H), 3.67-3.36 (m, 70H), 3.23-3.07 (m, 25H), 2.18 (s, 1 OH), 2.00 (s, 13H), 1.89 (s, 11H), 1.80-1.78 (m, 17H). 13C NMR (125 MHz, DMSO-d6) 5 170.1 (C), 169.8 (C), 169.7 (C), 169.4 (C), 169.2 (C), 169.1 (C), 142.7 (C), 126.3 (CH), 123.9 (CH), 118.7 (CH), 109.7 (CH), 100.8 (CH), 70.5 (CH), 69.8 (CH), 69.6 (CH), 69.5 (CH), 69.3 (CH2), 69.0 (CH2), 68.2 (CH2), 67.2 (CH2), 66.7 (CH2), 61.4 (CH2), 22.6 (CH2), 22.4 (3xCH3), 20.7 (9xCH3).
Figure imgf000125_0002
[00332] Preparation of compound 10: 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 C73H119N7O39, 1718.8. Found 1719.3.
Figure imgf000125_0001
[00333] Preparation of compound 11: Commercially available suberic acid bis(A- hydroxysuccinimide ester) (3.67 g, 9.9 mmol, 1.0 eq.) was dissolved in DMF (5 mL) and triethylamine (1.2 mL) was added. To this solution was added dropwise a solution of 3- azido-1 -propylamine (1.0 g, 9.9 mmol, 1.0 eq.) in DMF (5 mL). The reaction was stirred at room temperature for 3 h. The reaction mixture was diluted with EtOAc (100 mL) and washed with water (50 mL). The organic layer was separated, dried over ISfeSCU and the solvent was removed under reduced pressure. The crude material was purified by flash chromatography (gradient elution: 0-5% MeOH in DCM in 16 CV). The product was obtained as white solid (1.54 g, 43%, rf = 0.71 (5% MeOH in DCM)). MS: calculated for C15H23N5O5, 353.4. Found 354.3.
Figure imgf000126_0002
[00334] Preparation of TriGalNAc (12): Triantennary GalNAc compound 10 (0.35 g, 0.24 mmol, 1.0 eq.) and compound 11 (0.11 g, 0.31 mmol, 1.5 eq.) were dissolved in DCM (5 mL) under argon and triethylamine (0.1 mL, 0.61 mmol, 3.0 eq.) was added. The reaction was stirred at room temperature overnight. The solvent was removed under reduced pressure, the residue was dissolved in EtOAc (100 mL) and washed with water (100 mL). The organic layer was separated and dried over Na2SO4. The solvent was evaporated and the resulting crude material was purified by flash chromatography (elution gradient: 0-10% MeOH in DCM in 20 CV) to afford the title compound as white fluffy solid (0.27 g, 67%, rf = 0.5 (10% MeOH in DCM)). MS: calculated for C84H137N11O41, 1957.1. Found 1959.6.
Conjugation of Tether 1 to a siRNA strand: Monofluoro cyclooctyne (MFCO) conjugation at 5’-or 3’-end
5‘-end MFCO conjugation
Figure imgf000126_0001
3‘-end MFCO conjugation
Figure imgf000127_0001
[00335] General conditions for MFCO conjugation: Amine-modified single strand was dissolved at 700 OD/mL in 50 mM carbonate/bicarbonate buffer pH 9.6/dimethyl sulfoxide (DMSO) 4:6 (v/v) and to this solution was added one molar equivalent of a 35 mM solution of MFCO-C6-NHS ester (Berry& Associates, Cat. # LK 4300) in DMF. 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 pm filter from Sartorius and then purified by reserve phase (RP HPLC) on an Akta Pure instrument (GE Healthcare).
[00336] 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.
[00337] 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%.
5’-GalNAc-Tl conjugates
Figure imgf000127_0002
3’-GalNAc-Tl conjugates
Figure imgf000128_0001
[00338] General procedure for TriGalNAc conjugation: MFCO-modified single strand was dissolved at 2000 OD/mL in water and to this solution was added one equivalent solution of compound 12 (10 mM) in DMF. The reaction was carried out at room temperature and after 3 h 0.7 molar equivalent of the compound 12 solution was added. The reaction was allowed to proceed overnight and completion was monitored by LCMS. The conjugate was diluted 15-fold in water, filtered through a 1.2 pm filter from Sartorius and then purified by RP HPLC on an Akta Pure instrument (GE Healthcare).
[00339] RP HPLC purification was performed using a XBridge C18 Prep 19 x 50 mm column from Waters. Buffer A was 100 mM tri ethyl ammonium 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.
[00340] 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 (9-acetates were removed by adding 20% aqueous ammonia. Quantitative removal of these protecting groups was verified by LC-MS.
[00341] The conjugates were desalted by size exclusion chromatography using Sephadex G25 Fine resin (GE Healthcare) on an Akta Pure (GE Healthcare) instrument to yield the conjugated oligonucleotides in an isolated yield of 50-70%.
[00342] The following schemes further set out the routes of synthesis: Scheme 1
Figure imgf000129_0001
Scheme 2:
Figure imgf000130_0001
Scheme 3:
5' end MFCO conjugation
Figure imgf000131_0001
Scheme 4:
Figure imgf000132_0001
Scheme 5:
Figure imgf000133_0001
Example 2: Duplex Annealing
[00343] 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.
[00344] The duplexes were analyzed by analytical SEC HPLC on Superdex™ 75 Increase 5/150 GL column 5 x 153-158 mm (Cytiva) on a Dionex Ultimate 3000 (Thermo Fisher Scientific) HPLC system. Mobile phase consisted of lx PBS containing 10% acetonitrile. An isocratic gradient was run in 10 min at a flow rate of 1.5 mL/min at room temperature. UV traces at 260 and 280 nm were recorded. Water (LC-MS grade) was purchased from Sigma- Aldrich and Phosphate-buffered saline (PBS; lOx, pH 7.4) was purchased from GIBCO (Thermo Fisher Scientific).
Example 3: Synthesis of tether 2
General Experimental conditions:
[00345] Thin layer chromatography (TLC) was performed on silica-coated aluminium plates with fluorescence indicator 254 nm from Macherey -Nagel. Compounds were visualized under UV light (254 nm), or after spraying with the 5% H2SO4 in methanol (MeOH) or ninhydrin reagent according to Stahl (from Sigma-Aldrich), followed by heating. Flash chromatography was performed with a Biotage Isol era One flash chromatography instrument equipped with a dual variable UV wavelength detector (200-400 nm) using Biotage Sfar Silica 10, 25, 50 or 100 g columns (Uppsala, Sweden).
[00346] All moisture-sensitive reactions were carried out under anhydrous conditions using dry glassware, anhydrous solvents, and argon atmosphere. All commercially available reagents were purchased from Sigma-Aldrich and solvents from Carl Roth GmbH + Co. KG. D- Galactosamine pentaacetate was purchased from AK scientific.
[00347] HPLC/ESI-MS was performed on a Dionex UltiMate 3000 RS UHPLC system and Thermo Scientific MSQ Plus Mass spectrometer using an Acquity UPLC Protein BEH C4 column from Waters (300A, 1.7 μm, 2.1 x 100 mm) at 60 °C. The solvent system consisted of solvent A with H2O containing 0.1% formic acid and solvent B with acetonitrile (ACN) containing 0.1% formic acid. A gradient from 5-100% of B over 15 min with a flow rate of 0.4 mL/min was employed. Detector and conditions: Corona ultra-charged aerosol detection (from esa). Nebulizer Temp.: 25 °C. N2 pressure: 35.1 psi. Filter: Corona.
[00348] JH and 13C NMR spectra were recorded at room temperature on a Varian spectrometer at 500 MHz ('H NMR) and 125 MHz (13C NMR). Chemical shifts are given in ppm referenced to the solvent residual peak (CDCI3 - 'H NMR: 5 at 7.26 ppm and 13C NMR 5 at 77.2 ppm; DMSO4 - 1HNMR: 5 at 2.50 ppm and 13C NMR 5 at 39.5 ppm). Coupling constants are given in Hertz. Signal splitting patterns are described as singlet (s), doublet (d), triplet (t) or multiplet (m).
Synthesis route for the conjugate building block TriGalNAc Tetherl:
Figure imgf000135_0001
[00349] 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. NaHCCL (100 mL) and water (100 mL). The organic layer was separated, dried over Na2SO4, and concentrated to afford the title compound as yellow oil, which was purified by flash chromatography (gradient elution: 0-10% MeOH in DCM in 10 CV). The product was obtained as colourless oil (2.5 g, 98%, rf= 0.45 (2% MeOH in DCM)).
Figure imgf000135_0002
[00350] Preparation of compound 4: Compound 2 (2.30 g, 6.98 mmol, 1.0 eq.) and azido- PEG3-OH (1.83 g, 10.5 mmol, 1.5 eq.) were dissolved in anhydrous DCM (40 mL) under argon and molecular sieves 3 A (5 g) were added to the solution. The mixture was stirred at room temperature for 1 h. TMSOTf (0.77 g, 3.49 mmol, 0.5 eq.) was then added to the mixture and the reaction was stirred overnight. The molecular sieves were filtered, the filtrate was diluted with DCM (100 mL) and washed with cold saturated aq. NaHCO3 (100 mL) and water (100 mL). The organic layer was separated, dried over Na2SO4 and the solvent was removed under reduced pressure. The crude material was purified by flash chromatography (gradient elution: 0-3% MeOH in DCM in 10 CV) to afford the title product as light-yellow oil (3.10 g, 88%, rf = 0.25 (2% MeOH in DCM)). MS: calculated for C20H32N4O11, 504.21. Found 505.4. 1H NMR (500 MHz, CDCI3) 5 6.21-6.14 (m, 1H), 5.30 (dd, J= 3.4, 1.1 Hz, 1H), 5.04 (dd, J= 11.2, 3.4 Hz,lH), 4.76 (d, J= 8.6 Hz, 1H), 4.23-4.08 (m, 3H), 3.91-3.80 (m, 3H), 3.74-3.59 (m, 9H), 3.49-3.41 (m, 2H), 2.14 (s, 3H), 2.02 (s, 3H), 1.97 (d, J= 4.2 Hz, 6H). 13C NMR (125 MHz, CDCI3) 5 170.6 (C), 170.5 (C), 170.4 (C), 170.3 (C), 102.1 (CH), 71.6 (CH), 70.8 (CH), 70.6 (CH), 70.5 (CH), 70.3 (CH2), 69.7 (CH2), 68.5 (CH2), 66.6 (CH2), 61.5 (CH2), 23.1 (CH3), 20.7 (3xCH3).
Figure imgf000136_0001
[00351] Preparation of compound 5: Compound 4 (1.00 g, 1.98 mmol, 1.0 eq.) was dissolved in a mixture of ethyl acetate (EtOAc) and MeOH (30 mL 1 : 1 v/v) and Pd/C (100 mg) was added. The reaction mixture was degassed using vacuum/argon cycles (3x) and hydrogenated under balloon pressure overnight. The reaction mixture was filtered through celite and washed with EtOAc (30 mL). The solvent was removed under reduced pressure to afford the title compound as colourless oil (0.95 g, quantitative yield, rf = 0.25 (10% MeOH in DCM)). The compound was used without further purification. MS: calculated for C20H34N2O11, 478.2. Found 479.4.
Figure imgf000137_0001
[00352] Preparation of compound 7: Tris{[2-(tert-butoxycarbonyl)ethoxy]methyl}-methylamine 6 (3.37 g, 6.67 mmol, 1.0 eq.) was dissolved in a mixture of DCM/water (40 mL 1:1 v/v) and Na2CO3 (0.18 g, 1.7 mmol, 0.25 eq.) was added while stirring vigorously. Benzyl chloroformate (2.94 mL, 20.7 mmol, 3.10 eq.) was added dropwise to the previous mixture and the reaction was stirred at room temperature for 24 h. The reaction mixture was diluted with CH2Cl2 (100 mL) and washed with water (100 mL). The organic layer was separated and dried over Na2SO4. The solvent was removed under reduced pressure and the resulting crude material was purified by flash chromatography (gradient elution: 0-10% EtOAc in cyclohexane in 12 CV) to afford the title compound as pale yellowish oil (3.9 g, 91%, rf = 0.56 (10% EtOAc in cyclohexane)). MS: calculated for C33H53NO11, 639.3. Found 640.9.1H NMR (500 MHz, DMSO-d6) ^ 7.38-7.26 (m, 5H), 4.97 (s, 2H), 3.54 (t, 6H), 3.50 (s, 6H), 2.38 (t, 6H), 1.39 (s, 27H).13C NMR (125 MHz, DMSO-d6) ^ 170.3 (3xC), 154.5 (C), 137.1 (C), 128.2 (2xCH), 127.7 (CH), 127.6 (2xCH), 79.7 (3xC), 68.4 (3xCH2), 66.8 (3xCH2), 64.9 (C), 58.7 (CH2), 35.8 (3xCH2), 27.7 (9xCH3).
Figure imgf000137_0002
[00353] Preparation of compound 8: Cbz-NH-tris-Boc-ester 7 (0.20 g, 0.39 mmol, 1.0 eq.) was dissolved in CH2Cl2 (1 mL) under argon, trifluoroacetic acid (TFA, 1 mL) was added and the reaction was stirred at room temperature for 1 h. The solvent was removed under reduced pressure, the residue was co-evaporated 3 times with toluene (5 mL) and dried under high vacuum to get the compound as its TFA salt (0.183 g, 98%). The compound was used without further purification. MS: calculated for C21H29NO11, 471.6. Found 472.4.
Figure imgf000138_0001
[00354] Preparation of compound 9: CbzNH-tris-COOH 8 (0.72 g, 1.49 mmol, 1.0 eq.) and GalNAc-PEG3-NH2 5 (3.56 g, 7.44 mmol, 5.0 eq.) were dissolved in N,N- dimethylformamide (DMF) (25 mL). Then A,A,A',A'-tetramethyl-O-(lH-benzotriazol-l- yl)uronium hexafluorophosphate (HBTU) (2.78 g, 7.44 mmol, 5.0 eq.), 1- hydroxybenzotriazole hydrate (HOBt) (1.05 g, 7.44 mmol, 5.0 eq.) and 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. NaHCCL (100 mL). The organic layer was dried over Na2SO4,the solvent evaporated and the crude material was purified by flash chromatography (gradient elution: 0-5% MeOH in DCM in 14 CV). The product was obtained as pale yellowish oil (1.2 g, 43%, rf = 0.20 (5% MeOH in DCM)). MS: calculated for C81H125N7O41, 1852.9. Found 1854.7. 'H NMR (500 MHz, DMSO-d6) 5 7.90- 7.80 (m, 10H), 7.65-7.62 (m, 4H), 7.47-7.43 (m, 3H), 7.38-7.32 (m, 8H), 5.24-5.22 (m, 3H), 5.02-4.97 (m, 4H), 4.60-4.57 (m, 3 H), 4.07-3.90 (m 10H), 3.67-3.36 (m, 70H), 3.23-3.07 (m, 25H), 2.18 (s, 1 OH), 2.00 (s, 13H), 1.89 (s, 11H), 1.80-1.78 (m, 17H). 13C NMR (125 MHz, DMSO-d6) 5 170.1 (C), 169.8 (C), 169.7 (C), 169.4 (C), 169.2 (C), 169.1 (C), 142.7 (C), 126.3 (CH), 123.9 (CH), 118.7 (CH), 109.7 (CH), 100.8 (CH), 70.5 (CH), 69.8 (CH), 69.6 (CH), 69.5 (CH), 69.3 (CH2), 69.0 (CH2), 68.2 (CH2), 67.2 (CH2), 66.7 (CH2), 61.4 (CH2), 22.6 (CH2), 22.4 (3xCH3), 20.7 (9xCH3).
Figure imgf000139_0002
[00355] Preparation of compound 10: 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 C73H119N7O39, 1718.8. Found 1719.3.
Figure imgf000139_0001
[00356] Preparation of compound 14: 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 Na2SO4. The solvent was evaporated, and the crude material was purified by flash chromatography (gradient elution: 0-5% MeOH in DCM in 20 CV). The product was obtained as white fluffy solid (0.25 g, 48%, rf = 0.4 (10% MeOH in DCM)). MS: calculated for C88H137N7O42, 1965.1. Found 1965.6.
Figure imgf000140_0002
Figure imgf000140_0003
[00357] Preparation of TriGalNAc (15): Triantennary GalNAc compound 14 (0.31 g, 0.15 mmol, 1.0 eq.) was dissolved in EtOAc (15 mL) and Pd/C (40 mg) was added. The reaction mixture was degassed by using vacuum/argon cycles (3x) and hydrogenated under balloon pressure overnight. The completion of the reaction was monitored by mass spectrometry and the resulting mixture was filtered through a thin pad of celite. The solvent was removed under reduced pressure and the resulting residue was dried under high vacuum overnight. The residue was used for conjugations to oligonucleosides without further purification (0.28 g, quantitative yield). MS: calculated for C81H131N7O42, 1874.9. Found 1875.3.
Conjugation of Tether 2 to a siRNA strand: TriGalNAc tether 2 (GalNAc-T2) conjugation at 5’-end or 3’-end
5’-GalNAc-T2 conjugates
Figure imgf000140_0001
[00358] Preparation of TriGalNAc tether 2 NHS ester: To a solution of carboxylic acid tether 2 (compound 15, 227 mg, 121 pmol) in DMF (2.1 mL), N-hydroxysuccinimide (NHS) (15.3 mg, 133 pmol) and N,N'-diisopropylcarbodiimide (DIC) (19.7 μL, 127 pmol) were added. The solution was stirred at room temperature for 18 h and used without purification for the subsequent conjugation reactions.
[00359] General procedure for triGalNAc tether 2 conjugation: Amine-modified single strand was dissolved at 700 OD/mL in 50 mM carbonate/bicarbonate buffer pH 9.6/DMSO 4:6 (v/v) and to this solution was added one molar equivalent of Tether 2 NHS ester (57 mM) solution in DMF. The reaction was carried out at room temperature and after 1 h another molar equivalent of the NHS ester solution was added. The reaction was allowed to proceed for one more hour and reaction progress was monitored by LCMS. At least two molar equivalent excess of the NHS ester reagent relative to the amino modified oligonucleoside were needed to achieve quantitative consumption of the starting material. The reaction mixture was diluted 15-fold with water, filtered once through 1.2 μm filter from Sartorius and then purified by reserve phase (RP HPLC) on an Akta Pure (GE Healthcare) instrument.
[00360] 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.
[00361] Fractions containing full-length conjugated oligonucleosides were pooled together, precipitated in the freezer with 3 M NaOAc, pH 5.2 and 85% ethanol and then dissolved at 1000 OD/mL in water. The O-acetates were removed with 20% ammonium hydroxide in water until completion (monitored by LC-MS).
[00362] The conjugates were desalted by size exclusion chromatography using Sephadex G25 Fine resin (GE Healthcare) on an Akta Pure (GE Healthcare) instrument to yield the conjugated oligonucleotides in an isolated yield of 60-80%.
[00363] The conjugates were characterized by HPLC-MS analysis with a 2.1 x 50 mm XBridge C18 column (Waters) on a Dionex Ultimate 3000 (Thermo Fisher Scientific) HPLC system equipped with a Compact ESLQq-TOF mass spectrometer (Bruker Daltonics). Buffer A was 16.3 mM triethylamine, 100 mM HFIP in 1% MeOH in H2O and buffer B contained 95% MeOH in buffer A. A flow rate of 250 μL/min and a temperature of 60°C were employed. UV traces at 260 and 280 nm were recorded. A gradient of 1-100% B within 31 min was employed.
[00364] The following schemes further set out the routes of synthesis:
Scheme 6
Figure imgf000143_0001
Scheme 7:
Figure imgf000144_0001
Scheme 8:
Figure imgf000145_0001
Scheme 9:
Figure imgf000146_0001
Example 4: Duplex Annealing
[00365] 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.
[00366] The duplexes were analyzed by analytical SEC HPLC on Superdex™ 75 Increase 5/150 GL column 5 x 153-158 mm (Cytiva) on a Dionex Ultimate 3000 (Thermo Fisher Scientific) HPLC system. Mobile phase consisted of lx PBS containing 10% acetonitrile. An isocratic gradient was run in 10 min at a flow rate of 1.5 mL/min at room temperature. UV traces at 260 and 280 nm were recorded. Water (LC-MS grade) was purchased from Sigma- Aldrich and Phosphate-buffered saline (PBS; 10x, pH 7.4) was purchased from GIBCO (Thermo Fisher Scientific).
Example 5: Alternative synthesis route for the conjugate building block TriGalNAc Tether2:
Figure imgf000147_0001
Figure imgf000148_0001
Conjugation of Tether 2 to a siRNA strand: TriGalNAc tether 2 (GalNAc-T2) conjugation at 5’-end or 3’-end
Conjugation conditions
Figure imgf000149_0001
[00367] Pre-activation: To a solution of compound 15 (16 umol, 4 eq.) in DMF (160 μL) was added TFA-O-PFP (15 l, 21 eq.) followed by DIPEA (23 pl, 32 eq.) at 25°C. The tube was shaken for 2 h at 25°C. The reaction was quenched with H2O (10 μL).
[00368] Coupling: The resulting mixture was diluted with DMF (400 pl), followed by addition of oligo-amine solution (4.0 pmol in 10 x PBS, pH 7.4, 500 μL; final oligo concentration in organic and aqueous solution: 4 pmol/ml = 4 mM). The tube was shaken at 25°C for 16 h and the reaction was analysed by LCMS. The resulting mixture was treated with 28% NH4OH (4.5 ml) and shaken for 2 h at 25°C. The mixture was analysed by LCMS, concentrated, and purified by IP-RP HPLC to produce the oligonucleotides conjugated to tether 2 GalNAc.
5’-GalNAc-T2 conjugates
Figure imgf000149_0002
3’-GalNAc-T2 conjugates
Figure imgf000150_0001
Example 6: Solid phase synthesis method: scale <1μmol
[00369] Syntheses of siRNA sense and antisense strands were performed on a MerMadel92X synthesiser with commercially available solid supports made of controlled pore glass with universal linker (Universal CPG, with a loading of 40 pmol/g; LGC Biosearch or Glen Research).
[00370] RNA phosphoramidites were purchased from ChemGenes or Hongene.
[00371] 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'-O-methyl-3'-[(2-cyanoethyl)- (N,N-diisopropyl)]-phosphoramidite.
[00372] 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-diisopropyl)]-phosphoramidite.
[00373] 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). Iodine at 0.02M in acetonitrile/Pyridine/H2O (DNAchem) was used as oxidizing reagent. Thiolation for phosphorothioate linkages was performed with 0.2 M PADS (TCI) in acetonitrile/pyridine 1 : 1 v/v. 5-Ethyl thiotetrazole (ETT), 0.25M mM in acetonitrile was used as activator solution. [00374] 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).
[00375] At each cycle, the DMT was removed by deblock solution, 3% TCA in DCM (DNAchem).
[00376] The coupling time was 180 seconds. The oxidizer contact time was set to 80 seconds and thiolation time was 2*100 seconds.
[00377] At the end of the synthesis, the oligonucleotides were cleaved from the solid support using a NH4OH:EtOH solution 4: 1 (v/v) for 20 hours at 45°C (TCI). The solid support was then filtered off, the filter was thoroughly washed with H2O and the volume of the combined solution was reduced by evaporation under reduced pressure.
[00378] 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 IM sodium acetate.
[00379] The single strands identity were assessed by MS ESI- and then, were annealed in water to form the final duplex siRNA and duplex purity were assessed by size exclusion chromatography.
Example 7: Solid phase synthesis method: scale >5 μ mol
[00380] Syntheses of siRNA sense and antisense strands were performed on a MerMadel2 synthesiser with commercially available solid supports made of controlled pore glass with universal linker (Universal CPG, with a loading of 40 pmol/g; LGC Biosearch or Glen Research) at 5 μmol scale. Sense strand destined to 3' conjugation were sytnthesised at 12 pmol on 3'-PT-Amino-Modifier C6 CPG 500 A solid support with a loading of 86 pmol/g (LGC).
[00381] RNA phosphoramidites were purchased from ChemGenes or Hongene.
[00382] 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'-O-methyl-3'-[(2-cyanoethyl)- (N,N-diisopropyl)]-phosphoramidite.
[00383] 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-diisopropyl)]-phosphoramidite.
[00384] 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).
[00385] 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). Iodine at 0.02M in acetonitrile/Pyridine/H2O (DNAchem) was used as oxidizing reagent. Thiolation for phosphorothioate linkages was performed with 0.2 M PADS (TCI) in acetonitrile/pyridine 1 : 1 v/v. 5-Ethyl thiotetrazole (ETT), 0.25M mM in acetonitrile was used as activator solution.
[00386] At each cycle, the DMT was removed by deblock solution, 3% TCA in DCM (DNAchem).
[00387] For strands synthesised on universal CPG the coupling was performed with 8 eq. of amidite for 130 seconds. The oxidation time was 47 seconds, the thiolation time was 210 seconds.
[00388] For strands synthesised on 3'-PT-Amino-Modifier C6 CPG 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
[00389] At the end of the synthesis, the oligonucleotides were cleaved from the solid support using a NH4OH:EtOH solution 4: 1 (v/v) for 20 hours at 45°C (TCI). The solid support was then filtered off, the filter was thoroughly washed with H2O and the volume of the combined solution was reduced by evaporation under reduced pressure. [00390] Oligonucleotide were treated to form the sodium salt by EtOH precipitation from IM sodium acetate.
[00391] The single strand oligonucleotides were purified by IP-RP HPLC on Xbridge BEH C18 5 pm, 130 A, 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.
[00392] The single strands purity and identity were assessed by UPLC/MS ESI- on Xbridge BEH C18 2.5 pm, 3x50 mm (Waters) column with an increasing gradient of B in A. Mobile phase A: 100 mM HFIP, 5 mM TEA in water; mobile phase B: 20% mobile phase A: 80% Acetonitrile (v/v).
[00393] Sense strands were conjugated as per protocol provided in any of Examples 1, 3, 5.
[00394] Sense and Antisense strands were then annealed in water to form the final duplex siRNA and duplex purity were assessed by size exclusion chromatography.
Example 8: Nucleic acid sequences:
[00395] siRNA oligonucleosides according to the present invention target B4GALT1. The full
DNA sequence of the B4GALT1 target is as follows (SEQ ID NO: 1):
GAGGCATGAAGAAATAATTGTGCATGACTGAGGACTTTCCAGACCTCCCCTTTCCTTCCACCAGTTACT
TACTAATCTCAGAATCCACCCCCCAAAATTTTTCTGATAAAAACACTACCTTAAAGCCAGCCCAGGGA
GACTTGAGCCAGCCCAGGGAGACCTAAAGTCACCACAGGGAGATTTCAGCTGGACTCTTCTATCTCCT
TGTTGGCCTACCTGCAGTACAAAGCTTTTCTTTTCTCAAAAACCAGGTGTCACAGTATTGGTTTCTAGA
ACATTGGGCAGTGAGTGCTTTTGCGCTTTGGTCGGTAACACCTGGATCTGATTTAGACAATACTTTGGA
CCTGAAGTCTTAATTAGTTGAACTTTTGGGGGATTTTAAGAAGACACTAATGTATTTTACCTGTGAGAA
GAATCTAAATAATCTGTGGCCATTGGGCAAACTACTGTGGAATAAAGGTGCCTGACAATTCTTTGTCC
CTCCTCCCATCAAGAGGTGGAGTCAGCCAGGTGAAATGGCTCATGCTGGTAATCTCAGCACTTTGGGA
GGCCAAAGCAGGAAGACTGCGTGAGCTCAGGAGTTCGAGACTAGCCTGAGCAATATCGCAACATCTC
ATCTCTACTAAAAATTTTAAAATTAGCTGGACGTGGAGGCGCATCCCGGTAGTCCCAGCTACTCGGGA
GGCTGAGGCAGGAGAATCACTTGAGCCCAGGAGTTTGACGTTATAGTGACCTATGATCACACCACTGC
ACTACAGGCTGGTTGATAAAGGAAGATCCTGTCTAAAAAAAAAAGTAAAAACAAGAGGCCGAGCCAG
TTTTATTCCCCTTGAATCTGGCCTGCCCTATAAACTTGTTTTAAGCAAAAGAATGCTTTAGAAGTGATG
CTAAGGCTGGGCTTTCAGGGATCTCCATCTTCTGTATTTTTGAAATGCTCCTTTTTGGAATGCTTCCTCT
AGTTTGTGAGGAAACCCAAGCAGCCACATGGAGAGTCCTTTGTGGAGAGATCCAAGTGGAGAATGAA
GGCCCCATGACCCAACCCATTCTGAGTTTCCAGCCCCCAGACAGCCCCAACTGCCATTCACATGAGTG
AAGCCATTTTGGAACTTCCAACTGTGCCAGTGCCTCAGCTGACACCATGTGAGGCAAAGCTGCCCAGC
CAACTGCAAAACTGCGAGAAATTGTTGCTTCAAAACAGTAAGTTTTGGGGTAGGTGTTACGCTGCAAT
AGATGACTGAAATAACTGTCTACCATGTGCCGGGCACTATTTGATGCCCTTCTGATCCATGAGGGTAA
AAACAGAAATGTAACCTGGCAGGTGCAGAAGAGGGCGCCATAGGAGGGCAGAGGAAGGCCAGCTGC
AGGGAGAAGCAGGGAGCTGGTGATTCTGGGCAGATGAGCACATGGATGGGCCAACGGCCAAGCCCCC
ATGCCAGCTTTTGGCCAATCAGCACTGCAACTTCCTCCTGCATTTGTCTCGCCGGATGGGATTAATTTT
TCACCTGACGAAGTAGAGAGTGGAAAAGAGCTGGAGACAGTGGGGAGAAAGGTTGCCTGGGTCTGTC
TCACTAGCACCAGTTAATGTCTGGACTGCTGGACAATGTTGTCCCAAAGGTTTCTGGGCCATCTGTATT
ATTTGTAATTGACTGCTTCTAGGTGCCTGTGGATCAGGGGCAGCTGAGACTAGTGCTCAGGCCTCAGT
GGACTCTGCAAGTTCCTGAGGGATAGGCAATCAGCAAGTGTTGTTCCTTTTCCTCGATTTCTGGCCACG TGTGTCCTGGGACAGGTCTGTGATTCTTAATAACCCCCGCAGTCCTGTCTCCTGGCTATCATCTATACC
AATGGAAGACACATCCCCATTTCCCCCTCCACTTAATTTTCAGTTGCAGGACTAATCTGACCCACCCTC
ACTCATTGGCCAGGCCGACTTTACCCCTAGACACAGGATGCTGGGGTCAGCTTCACCTTTACCAACTCC
TTGGAGAACTCCACTTTACGTTCTAAACTAAGTTAGCAATAATTTTTCCCTTCTCTCCTTCCCACATCAT
TAAGATGATCACAGTATTTAAAAAGTATTTTAACAAATATCGGCCGGGCACGGTGGCTCACAACTGTA
ATCCCAGCACTTTGGGAGGCCGAGGCAGGCAGATCACGAGGTCAAAAGATTGAGACCATTCTGGATA
ACACGGTGAAACCCCATCTCTACTAAAAATACAAACAAATTAGCCGGGCATGGTGGCAGGCACCTGT
AGTCCCAGCTACTTGGGAGGCTGAGGCAGGAGAATGGCGTGAACCCAGGAGGCAGAGCTTGCAGTGA
GCCAAGATCACGCCACTGCACTCCAGCCTGGGTGACAGAGTGAGACTCCGTCTCAAAAAAAAAAAAA
AAAAAATCTAGGGGCTGAAGATACAGTAGTGAACAAGAGAGAAATTTCCTGTTCTCATGAAGCTGATT
TTCTAATGAGGGAGGCAAGACAACAGAAAATAAATGCATAATGTTGGGTAGTTGATATCCACTCTGAA
AAAAATCAAGCAGGTTAAGGCCGGGCGCGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCG
AGGCGGGCGGATCACCTGAGGTCAGGAGTTTGAAACCAGCCTGGCCAACATGGTGAAATCCGTCTCTA
CTAAAAACACAAAAAATTAGCCCGGCGTGATGGCAGGCACCTGTAATCCCAGCTACTCAAGAGGCTG
AGGCAGGAGAATCGCTTGAACCCGGGAGGCAGAGGTTGCAGTGAGGTGAGATTGCACCATTGCACTC
CAACCTGGGGGACAAAAGCAAGACTTTGTCTCAAAAAAAAAAAAAAAAAAATTCAAGCAGTTTAAGC
AGATTTGGGCAGGAGGCCATTCTGCATAAGGTAGTCTGAAAAGGTCTCTGTCATAAGGTGACATTTAA
GAGACCTGAATTGAATGAAATATTGGGGACAAGTGTTTCAGGCTAAATGAACAGCAAGTACAAAGGC
CCTGAGGCAGGAAGAAATATGGCAAGTTCAAGGAATAGCTATCAGGCTAGTGTGGCTAAGGCAGGTC
CAGCATGGTAGAGTGACAGATGTGGGTGGGGAGGGAAATAGGAACCAGATTGAACAGGGTTTCTGTG
GATTTGGTTCTGAACAAAATGGCATGATCTGATTTATGCTTACAAAGATTTCCTGGATGCTCTGTGGAA
AACAGACTAGGGAGGAGGAATGGAGGAGGTGGAAGCAGGGTGACCAATTAGTAGCTGCCATATAACC
CAGGGCAAGATGATGGTGGCTTGATCGAGGATGGTTACATCAGAGTTGGCTGGTGGTGAATTTTGATG
TTTTGAAGGTAGCACTGACAAAGCTGGCTGAGGGCTTGCAAATGGCATTGAGAGCAAGAGAAGCACA
TCAAGGACACCTTTTAGATTCTGGGGAACTGAATAAACAATAGTATCACTCTCTGAGGGAGGTAAGAA
CGGGAGTGGTGTGTAAGGAGTAGGTTGCTGAGGGCAAGATGTATTGTGTTTGAGATGCCAGTAAAATA
AGCAGTTGAATCTGGAGGTCAGGGAAGAGATCTGGGCTGGAGACAAATCAGTGATCAGCATTTGGAT
ATTATAAATCATTCCGAGGCAGTAAGTGTAGACACAAAAGAACATCATGGACTATGGCTGGGGCCTTC
AGCAACTGGGGAAGAAGTCCAGAGAGGAGACAGAAATGGCCAGTGAAGTGAGGAAGATCAGAAGGA
CCTGGTGTCCAGGAAGTCAAGTGAGGAAAGTTGATTCTGTATGATCACAACCAAAGTGTCAACTCATA
AGCCTTATTTTCTCATCTGTGAAATGGACACCGTAACACCACCTACTTCATGGCAGATAGTACTGGCAC
ACAGCAAACTCTCAAAATAAGGTAGCTACTGTTATTCCCTGATGGTTGGCTGCCAGAGCCCTCAACTT
CCCTATCCACATTACTGACAGCACCTCCATGAGTCTTTCTCTGGGGTGAGGTGTCTCTGCTCACTCAGG
GCCTGAGGCCTCTGGGTCAAATCGAGGTCAAGTGGCTTCAGTGCCTAAGTCTCTCACCCACACAGCCT
TCAGCCCTTACTTGCAAATCAACAAAGGGTAAACCTGTAGAAAACATGGGTTTCGGAGCCAGAATTCT
GCCTCTTGCCAGCTGTGGGCTCTTAGGAAAGTTTCTTAATCTGCCGGGGCCCCACTCTACGACATGGGG
AGAACTGCTACTTCATGGGACAGTGGGTAGCCCAGTGTAGACTGTAACGCCGGCTGATCTCCTGCACG
CTGGCCTGGGAGTTAGAGGCTTCTTGCTGCTCTCCTCTTCAAAGTATACAGGACTCCCGCCACACACAC
ATCTGGAACCAAGCTGGTCTGAGAGCCCCTTATAGCCCAGGCTACCTGATGGGGAGGCACAGAAGTG
GCAACCCGTCCACTTTCTTTGCCGCAGGACCCCCCGTTAAGCAGCGGGGTCCAGCCGGGCTGAGTTAG
GGAGGGGGTTTCGAACGTGCCACTCCTCGCCCGGCGTCGAAGCCCGTTTCCTGGGTAACCTTTTTCTGC
CTCTCTTCCTAGCCCACCAAGGCCCACTGGCCAGAACGCCGCCGCGGCCCCAAACCACTCCAGATAAC
CACCCGCCAGCTGTCCTCTCCGTTCTCTCCGCCGCCGCGCTGCAGGCCCAGGCTCGCACCCGAGTCCCT
TCGCACCCCAGGAAGTGGCGCGGCCTGTCGAGGGCAGCGTGGAGGAGGAAGAGGAGGCGCGGCTCA
ACGCGACCGAAGCTCCGCCGCAAAGGCTCGGGAGGAAGAGGGCGGTGCGCGGCCAAGCGTCGGAGCT
GCAGTCATACTCCGGGGACCCCACGACGGCGCCCCGCCCGCTGCCCACCCTCCCGAGGCCCCGCCCAG
CGCGCCCATCCCGCCACGGGCTGCCCCGCCTTCCCGCCCTCGTCCAGAAAACCCCGCGCCCGGCCCCG
CCCCCGCCTTCGCCGGGGCCCCGCCCCTCCCCTCTCCGCCGGCGCCTCGGGCGGCTTCTCGCCGCTCCC
AGGTCTGGCTGGCTGGAGGAGTCTCAGCTCTCAGCCGCTCGCCCGCCCCCGCTCCGGGCCCTCCCCTA
GTCGCCGCTGTGGGGCAGCGCCTGGCGGGCGGCCCGCGGGCGGGTCGCCTCCCCTCCTGTAGCCCACA
CCCTTCTTAAAGCGGCGGCGGGAAGATGAGGCTTCGGGAGCCGCTCCTGAGCGGCAGCGCCGCGATG
CCAGGCGCGTCCCTACAGCGGGCCTGCCGCCTGCTCGTGGCCGTCTGCGCTCTGCACCTTGGCGTCACC
CTCGTTTACTACCTGGCTGGCCGCGACCTGAGCCGCCTGCCCCAACTGGTCGGAGTCTCCACACCGCTG
CAGGGCGGCTCGAACAGTGCCGCCGCCATCGGGCAGTCCTCCGGGGAGCTCCGGACCGGAGGGGCCC
GGCCGCCGCCTCCTCTAGGCGCCTCCTCCCAGCCGCGCCCGGGTGGCGACTCCAGCCCAGTCGTGGAT
TCTGGCCCTGGCCCCGCTAGCAACTTGACCTCGGTCCCAGTGCCCCACACCACCGCACTGTCGCTGCCC
GCCTGCCCTGAGGAGTCCCCGCTGCTTGGTAAGGACTCGGGTCGGCGCCAGTCGGAGGATTGGGACCC
CCCCGGATTTCCCCGACAGGGTCCCCCAGACATTCCCTCAGGCTGGCTCTTCTACGACAGCCAGCCTCC
CTCTTCTGGATCAGAGTTTTAAATCCCAGACAGAGGCTTGGGACTGGATGGGAGAGAAGGTTTGCGAG
GTGGGTCCCTGGGGAGTCCTGTTGGAGGCGTGGGGCCGGGACCGCACAGGGAAGTCCCGAGGCCCCT
CTAGCCCCAGAACCAGAGAAGGCCTTGGAGACTTCCCTGCTGTGGCCCGAGGCTCAGGAAGTTTTGGA
GTTTGGGTCTGCTTAGGGCTTCGAGCAGCCTTGCACTGAGAACTCTGGTAGGGACCTCGAGTAATCCA CTCCCTTTTGGGGACTGACGTGAGGCTCCCGGTGGGGAAGGAGACTGACCTCTCGGTTCACGTGTCTT
GCCATAGAGCCACTCTCCTGAGTGGGTTTTTCTCCTGATCGTTTGGGCCAAGTGACTTCTCTCTGAACC
TCATATTTCTCTTCTGGGATAATAAATGGTCACCCTTTCAAGGGGTTGTTTTGGAAGATATTGTGAACA
ATGGTAAATAAGGGCTTAATTAATGAGGGTAAGCCCTCAGTAAATTGTCACTGTGTGTTCATTTCTTCC
TCTGTGTGGATCGTGACCGAGAGCCCTTCCCCCTAGCCTCCTCCTGGTATGGGTACCCAAAACCTAGGT
GAGCAGGGATCTCTCCCAGGGGCAGAGAGCTTGTGTACTCTGGGTGTTAGAGGGCTAAAATATAACCA
GTCAACACCACGTTGCCCATTTCTGGTACTTCCGGTAGCAGCCTGAGTCTCAATTATCTTGCCCAGATG
ATCTGAACTCTGACCTCTAGCCTGTTTCAGCATAGGCAGAGAGCTTGAGTAGGTGAGTTTGCATTCCTC
ATAGCAGCTGGCTGAGCCTAGTCTGGACTTCTCTTTGACCTGTAACCTACAGGCCCACAGGCCCAAGG
CAACCACAGGTTGCTTCCAGGGTTACCACACAGGTGGTTTCTCATTTCTAATGCTAGGTTTTAGATAAT
TGTTGTAAGTGAGGGGCCCTGGCAGGCAGGATGACATCCTGCCAATAGGAGTTTTCTGTCACTTTCCC
ACAGAGCCCTGGCTACTACATACTCTTGCTCAATTTCGCCAGTAATTGCGTCAATGTGTTCATATCAAG
TTTGGGAAGAACATCTTGGAATTGGTCAGACGTGAACTGTGGTAATAATGGGGGCTTGTTTTTTTAAG
CAGATAATTAAATTCCTTTGCATTTGATGATTATTCTGGGAAGCAGACTAGTCCCATAAAATGAAATG
GACTCTGCCTTGCTGCTAAGTGTCTGACTTGAGACATGCTATCGAGTTTCTCAAAATCTCTTCCTTGTGT
AAAATGTGGTTGTCGATGATTACCTTACAGGGGTTTTTTTAAGACTAAATGAGATCGTGTACATTAAAT
ACAGGCACTCAGGCTGGGCATGGTGGCTCACGCCTGTAATCCTAGCACTTTGGGAGGCTGAGGGGAGT
GGATCACTTGAGGTTAGGAGTTTGAGACCAGCCTGGCCAATATGGTGAAACACCATCCCATCTCTACA
AAAATACAAAAAAGTTAGCCAGGGGTGGTGGCATCGCAGCTACTCAGGAGGCCGAGGCAGGAGAATT
GCTTGAACCTGGGAGGCAGAGGTTGCAGTGAGTCAAGATTGTGCCAGTACACTCCAGCCTGGGCGAC
GAAGCAAGACTGTCTAAAAAAAAAAAAAAAAAAAAAAATACGGGCACTCAATACACCGTATAATAAT
AATATAGTAATAATATTTGCTTAGGATCTTTAAAAAGTTTCATTTTTTCAGACTCCCACAGAAATGGCT
CTGCACAGCAGAGTGAAGGGGGAGAGAGACTGAGTCTCCAGGCCAGAAAAAGGCCAGGTTTTTTGCT
TTTGTTTTTAGTTGTTGCCTGGATATTGCACAGAAAGAAAAAATAATTAGCAAGTTAAACAAAAGTAC
CGCAAAGTTGATTACATTGGTATTTGAGTATCACATCTTCTCTCAGAAGCGTAAGAGACAAGGTCGTG
ACCATACCTCTGCTTAGTTTTGTTTTGTAATGGTGTTGCTAGTGATCGGCTTGTCACCAGTTACTGGTGT
TTCTAAATGGACTATAATTGGCTACTTGAAAGGACTTCCTGAGAAAGAACATTTTGGAGGACGAGGAG
AGAGTGCCTTCTCTATTTTGGCTGCTTTCATGTGACATGCAAGAGACCATGACGTTTAGGCTGCTGCTG
AGGCAGCCCCAGAAATGGGGGCCGAGAGGTCTTTTCTTCATTTTAATAGGGTCTGTAGGTTTGGGTGG
TTAGGTACAGTTCTCAGAATGGAGGTTCCTGGCTATGAGGCCTTGAGAAAGCTGAAAGTCTCCTTGGG
AGTGTGTGGGTGGGGGGAGTCGAGCCCATCTGTTCATGGGCAGGTGTCAGCCAAAGCCCTTGCGGGTG
GTTTTGAGGTTGGTGGGAGAAAGCATCCGTGGGGTTTAGAGTTGTGGCCTTTTCACTACTTGCAGTTCC
TTTCCCCGACTTGGCTTTACTTTCTGGTGTCCAGGGGTCTGGGCCAGATGCTGAGATTCCTCTCAGCTG
ACAGGTGTGGGTTATGGGCAAACCCTTCCCTGGAGGACATAAGGCACCGGATTGGACTGCTGATGGGT
TGCTGTTGGAGTTGTCAGGGCCTTGGAATAGTCTTCAGATAGACTTGGGTTAGTGTGACCTGGGGCAG
GCTGCAGGTTTGGAGCCATAGTACCCCCCGCCCCCACACCGGGCACCCTGCTCTGGGCTAATGTGAGG
CTTGCAGGAGTGAGTGATGCAGTGGGAAGGGGGGCCTTTCCTGAGGATTCTACAGCTTTCTCCAGGGA
ATCCTCCCAGGTAGTTTAGGCCTGCAGGTGCTATGCTATCCTTCTTTCCTAACCCTGTCTCAGGTCCTCA
GCGGGGCCATGCGGCATCCACTTATAACCCTGCAGCGAGGCCCTCTTTTCTGGCCACCTGGGTGTTTGC
CTGCTGAGATGGGAGGAACAGTGGCCTTGGGCTTCTTCCCCCGTCATGTTTATCTCTGCTCAGATTGGG
CAGCAGCTCAATGGGACTTGACCAGCTGTGGCACTGCCAGTCTGAAGATGAGTAGGGTGATGGGGGG
AGGTGGGCAGTACCTGAAGCTGAACTGGTGAGAGAGGCAGGCTGGCCTGGGGGCTCAGCTGGGGCCT
GGGATGGTTGGTACAGTCCCCTCAGGGGGGTAGGGGAGTGAGTGTTAGACTGCTTAAGCCTCAGAGG
CCGCTCTTGCCCACCTATGCTTTGAGGAGATCCTCTTCATTTGTTCAAAGGGAAGACTCTGATCTAGAG
ATGGGCACTTGGACCAGCAAACAGCAGCTACAGGTAGCCAGGGCACCCGAGGAGCACTTGCTCATGA
GCCGGTTTCCCTGGTTTTTATGGGGGCTGTTGCTGAGCGTCTGCCAGGGTTTGTGTCCTAGCACTTGCT
GGTCTTTGCTGGGCTCTCAGCTCTCAGGTGTTTCTCTACCAGCACGTTTCCCCCTCCCTCATATGCACAC
ATGTGGACACAAGCAGGCTGCCCAGGACAGAGTGTACTTTGAGGCTTGGGAAAGGACTCTCTCTCGCC
CTTTTGGGGATGAGCCTTGGAACCTCATCACCTTCCGGCTTGGGGTGGAGCTTCATCCTGGGGGTTGAA
GCTTTAGGCTCAGATAACTAGTCTTGTAAGCCAGTTTTGTCCTGTTGTTTTTTTCGTGGAAAATAATGT
ATTGACGTATACACAGACATTCTTTGTCTAACAGTCTGAGATTGAGAAATACCCTCCATGACTATTTGG
TTTGCTTTCATGGTGAAACTTGGTCGCTTTCTTAGACACAGCCTATGGCAATAAGAGTGATCCCTGGCT
GCTGTAATTCATTCCAGACTTTGAGCAAACACAAGGCACCGCCTCCACCTGCAGTGGAGCCTCTGATG
AACCAAATGGAAACTCCTTGGGGAATGGGGAGTAAGAGCCAAATGTGGGATTGGACTTAAACTGCAG
CTTCTTAGAACTGTAGCATTCCACGATGGGATTGTCTAGTGCTCTTCCTGGAGGTTACTATTCAATAGT
TGGCTAGTGCACAGGTTCAGGGGTGACCTGATATGCCCTAGCGTTTCAGAAGATCCCTGCAAGGTGTG
TCTTTTGGTCCATCTGAAGGGTCTTGTATGGTGATCTTGTATGGATATCCGTGACGGCTAAGGCATCTG
ATAACTTCATTCCTTCAGTTCCAGCAGTGTTCCTGTATTATGCTGGGCACTAGAGCTACAAAGAAGAA
AACAAAGTGCCTCCTCTTCAGGAACTCTTAATTTAGGCAGGGGAGGCATAATTGAACAGTGCTGAGGT
CATCTAGGGGAACCAAAGTGTGTATTTATCCCCTTCCCTATCACTCCCCTCCCTCCTTCATTTCTTCCTT
TCTTCTTTCAGAAACTCCAAGTTCATATCAAAATTCTCCAGCCCTGGTTTTATTTGGTTGTGTGAAAATT
TTCCTCTAATTTCTGAAGCTATGCATTAGTTCTGCTGAGTAATCTTTAACTTGCTGCTTTATAATGATTA TAATGAGATATCACTGGGTATTATGGTCTTTGGGTAGCAGCAGGGTAGGGATTTCCAGGCTGGGACTA
AGCTAATTTATGGGTTGGGAATTATGGGGCAGTTAATAGCAAGGCAGTCCAAGCTTTCCACAGATTCC
ACCCTAGGGACCATCCAGACTTAAGGAACAGGGCCGGCAGGCTCATCCCCTTTGCACTCAGCTGGGCT
ATGGGTGTGTGTTTGTGAAAGAGGTTTATTCAGTAGTCATACCTGCTGATTTCCCTGCTATCTGTTTAC
CCAGTGCCTCCTGTACCTTGTTTCTTACTCTTTGTTCTCTGCTCTTACTATGAAGAAGCAGAGACTGGA
ATTCTGCTTGAACCCACATCTACCTGGAAATTCCAGTTTTTCTTGTCCAGTGGAGCAGCAATCCAGTTG
TTTTAGGACAAATGGTCTGCCCTTGAAGCTTAAATCCTTTGAGGGCCTGGCATGGTGACAGTTTTACAT
TTGGCTTTGGTATAGACTGGTGTGGTCCCTGGGCAGTGAGGTCACTGTAAGGCCAGCCAGCCAGACCC
TGGCTCCTAGGGGAATTAACAAGGCATGGGATTAGACTCACAGGGTCCCTCCTGTCCCTAAACTTGGT
AGGGGTTCCTGGGAGCCAGACTGCGATTAAGATTGTAGAGACCTGAGACCTGAGTTGTAGGGGCCTCT
GTGTTGATCTGGGCCATTGCCGGGTGAGCTGAGGCGGTCACTAGCTCAAGGAGTGATCTCAGGATATT
GTTCTGTAAGTCAGAGACCTCCAGGTTGGAGAGTGGGGCTTGGGGGTGGGGGACAGGGTTTAGTGGG
GAGCTGGTTCTGGGTGAATGTGGCCTAAAGGGATTTGTCCTTAGAAGACAGAGGGGTGAGTCACACAC
TCAGTGCTTCAGGTTCCACTTTGCGGCTTGGCCTCAGCCCGCCCCTTCCCTGCACAAATGAAGGCCAGG
GGCTATATAATTGGCTGTTGCTGAATTCTTTGGCAGTGATTTTAAAGTCTGGTCTGGGTGTGTTATGTA
GCTGCTTCTCTATCCACTCCCCACACCCGCTGCTTCTCCAGAGCCCCTCACAAAGCCCAGGCAGAGAG
AGAGAGAGAGAGAGAGAGAATGACTTGCCTCACAGAGATGTTGGGGATAGGGATAGGGGTATGGGTC
TTTGCTTTTGCCTTTTGAGGGGGGATAATCTCTTCCTTCATTTTAAAAGTAAAAAGTAATGCAGGCTCA
TTGAAAATAATTTGAAAAGTTGAAAGAGATATAAAAGCACACCCAAATTCCTATCACCCAAAAGAAA
CATACCGGCATATTTCCTACTAGTCTTTTTCATGTTTAAGAATATAGCTGATATATTTTTTTTTCTTTTTC
TTTTTGAGACAGGGTTTTTGCTCTGTCACCCAGGCTGGAGTGCAGTGATCACGGCTCACTGCAGCCTCG
ACCTCTCGGGCTAAGCGATTCTCCCACTTCAGTCTCCCGAGTTGCTGGGACCACAGGTGCACACCGCC
ATGCCTGACTAATTTTTGTATTTTTTGTAGAGATGGGGTTTTGCCATGTTGCCTAGGCTGGTCTCGAACT
CCAGAGCTCAAGTGATTCACCTGCCTTGGCCTCCCAAAGCGCTGGGATTATAGGTGTCAGTCACCACA
CCCAGTGTTATAGCTGTTGTCTTTATAGATGAACAGATAGATTGACATAGATTCATGTAGATAGCCTGG
TGTTCAGCATTTTTCATTTAAGATTCTGTCACAGACTTGACCCTATACCTTTAAAAATCACAAAGGCAG
TATCATAGTCTGTCAGCTGAATATGCCATAACTTAAAAAAATCATTCAACTGTTGCTGAACACACACA
TATACATATATAGTTTTTGTTTTTTCTTAGTGATGTAGTGATGCTTGTGCAGAAAGCTTTATGTACTTTT
TGGATGGTTTCTGTAGGAGAGCTTTCTAAAAAAGGAAAAAAAGTGTTGAATGTTTTTTGAGAAGGGCT
AGATTTTCAAGCCAGTCTTACAAAAGGATAGACTCATTGGAAATTCCAGATTTGCTTAGTGCTGGCAG
ATGAGTATCACTTATTGCTGAACAATGTGTCTAGAATTCTGATTAAAAAAGAAACTAGGTCCAGGAAG
TGCCTGGGGGCAGGGGCAAAGGGCCAGGCTGCAGGATAGGCTCTTAGGATCTGGCTGAGCAGAAATC
TGCTGTGAACAGAATCGGTGGGGGTGATGCTTTCTCAGTAACTTCTCCATTTGTTTCTTTAGCAGCTAA
GTCCCTGTGCTGGACTTCTGTGGACTACTGTGGCTCTGGGGCTGTGGTTGTGGGTGAACAACAGCTAG
CTAAACCAGTGCTGTTGACATCATTGAGATGTGACGCACAGGAAGGTGGGAGCAAGCTTGCAAATCA
GATTCTGAAACATATAGCACAGCTCTCCCACCTCCAGGTGGTCCTGAGATCTAGGGAGGAGCCATAGT
GAGAAACTTTAGGTTTCTAGGAATTCTCTTAGGGAGAAGCTCTCTTAGGGAGAGGCAGAACCTGGTTC
TCAGTTGGGGCTGATTCAGGTGGGTTAGATCAATAAAGCCTCAGGCCAGTGTGCCAGGCTATTCCCAA
GGAGTATACTTTGAAGTTACTCCCTTTAGAATGTCCTCAGTGGAGATAAATTCTCTCTGAGGAGCAGTT
TTGTCTGCCGGGGTCATTTGGCACAAAGCCTGGAGTGCTAGGGCGAGGTTGCACTGAGGGAAGGGGC
AGGATTATGTCAGCAGTGTGACGGATACAGTGTGAGGTCAGGCTCCTTCCTGCCCCACCACGGGGGCC
TAGAGGTCATGGGGAGGGTCCCTGGCAGGGGATTCAATCATTGCTTGGCCCCATGACAGAGTATATTC
TAAAAATGCCTTAAGTTTTTTTCTTTCAAAGTTTCTTCCTGTTTTGCATAATGGCCTTTTGCCTTTGACA
TCCTGAAACCGCAGAGCTGTCATTGGTGTTGCAGGACACTGCCAGCTTGAAAAAAATCAACAACAAA
AAAAGAAACAGGAAAGGATGTGGAGTTCAGGGTGCGGCCTAGGGAAGCTGGTATTTGCGTTATGGGA
TTGTGGGGATGTGGTATTAAGGTGTTGGGTAGCGCCTGACATTTAGAGGAGTACTCTGGGCAGAGTCC
CTGCCTGCCCAAGAATAGGTAGAATTGAGTCTTCACACCAAAGTCAGGAGAGACCCCCTCCCCCCAGG
AAGAGAATGAACAGGGACTCATTTCCTCATTCAGCAAACTTTTATTGGTAACTACACTATATGAAGTG
TGAGAGATAGACATGAACAAGAGAGGCCCCCACTCTTGGGCAGTCCCTTAGTAGTAGTAGATAGACTC
TGGCAATATGGTGTGGTCAGAGAGAGGAAGCCTGGGTGCTTTGAGGGTACTGAGGAGGTGCAGGGAG
CCAAATGGGTGGTCTGGGCCAGGGCCAGAGTCAGAATGAAGGACCTCTCTTCCAGACGTTGATTTTAG
CATCTCTGTCTCTCAGTATGTTTGAACAGTCTCCCTTATTGGAAGGGCAGGAGTCTACTGCTAAAAGTA
ACCTGCGATTTCCTCTACTTGCTGTCATGTGGAAAGAATACTAAAGCTGAAATTCCAAAAGTTGCACA
CCTTTACCAGCAGGGCAGGAGAGGAAAGGAAATGGAGGCAGAGTGAGCTGAAGATGATAAAAGAAA
GAGAAGGTGGTGCAGTTTGGACTGTTATGGACAGAGGAAGTCTGAGGGTAGCTGGACTGAGGGATCA
AAGGGAGGCAGTTGAAAGGGAAGAGAGCTGCAGAGAGGGATTTCTTGGTCTGCAGAGGGTAGGAGC
AAGCCTTGAAGGCTGCTGGAGTGAGGATTCCGAGCCCTGGTCTTTATTCTTTTTCTAATTCATTACATC
ATTTTAGGCAAGTCCTAACTCCTTTGGTCTCTGTTGTCTTTCTGAAATTTGAGTGGGCTGGGCCTGCTG
GTCTTTAGCCTCTGTCTTTCTCTACCTCCTAGATTCCAGTTTGGCGAGTGGGGGGGAAAACCTGGTTGT
ATATGCAACGTGAAAGGCCTCTGGAATTCCTTTTGAAGCTCACTACCCATGAGGCTTCTGCTAAGGATT
TCATCATGTCTGTCTAAGCAGACATAAAAATTTTAGCAGGTGGATGACCCGTAGAAATGGCACAAGGA
ATGTTTCTTTCTGTCACACTGTGGTATTTGATTTAAGAAAGTTGTTATCCTCTCTGTGCCTCAGTGTTCT CACTTGTAAAATGGCAATAACAGTATCCACCTCATAGATGTTATGAAATACAGGTAGTAGCCACGAAA
GGGCTTAAAACAGTGCCTAACACAGAATAAGTTGTGAATATATGTTATTTATTATTGGTAGTATAATG
CTTATTTGTGAAGATTTTGGCTTTTGCTTTATAGGACCTTTTTTTTTTTTAGTTGAAAATACAATGTTAC
CATGTTAAATGTTAAAAAAAATTCTACTTACCATTGTAACAGAACATGCTCCCACTTCTGTAACAGAG
CTTGCTATTACTTTTCAAATGCATACATATTCCAATGCATATATTCCAATGCAGTTGTAGAGTGAAACT
GTTTGCATGCAGCCATTTTTATCCAACATTATCTTATAAAATGTTATGTTGTTTATGATTATCCTAATTA
TCTTTTGTTGCTGTCTAGTATCCTTATAGATATTCCATTAGCATACACTATTCCAGGTTTCACTATCGTC
GATAATCTAGATATGAACATTTTTGTAGTGTGTAGCTCTTTGCTTCAGTTGAATTACTTTCCTGGGATA
AATTCCTGGGGAAGAATTTCTAGGCCAGAGGATATGGTCATCTTGACAATACTGATTCACATTGCTGC
ATTGCTTTCCAAGAGGTTTGGAATCATTCACAGGTTCTAAATTGGAAAATCCTGGCTTTTGAAGTATGT
GGATTCTAAGGGCGATTTGGATCTAGCTGGAGCCTCACACTGACACTTCCAGCCAGTGTGTGTGTGTG
TGTGTGTGTGTGTGTGTGTGTGTGTAGTTCCCTATGCTGGACACCGTGTGTGTGTGTGTGTGTGTGTGT
GTGTGTGTGTGTGTAGTTCCCTATGCTGGACACCATGTGGCCTTTCTGGACATTAGGGTTTTCCTGTGA
TTGCCTCAGAGCAGTTCCTGTTGAATTCACTCTGTGTCCACAAAAGGAGCCTTACTGTGGCTCTTTCAA
CACCCACCTACCTTTGCCAAGTTGGTTTACAGAAAGTAAGAACATTCTTTCCTTCTTCCTTGATATGTG
GCGCTAAACCTATAGCATGGGGCAGGCTCTGGCTTTAAAAACCTGACTTAAAAATAATGGTGTTGATC
AAAAAGTTTGTGGATCAGTTTTTGGAAACACTGCATGTAGCCATCCATAGAAACTTATATTCTGTTGGG
CTAGCCTGGGCGCCTGATCATTTAACTCATGTGGATGAACTTCTATGTAATAGCCCTGGTGTATGGGAT
CCAGAAACAGGGCCCTAATGAAGAAAGGCTTTTAAATTATGTTGGATAAAAATAAGTTGTTACAATAG
CCCAAAGTCTGCAAATATGAATTGCCAGTTCTGTCCTTGTAGTCATCCACCATGTGCCTGCATCTTTTG
TAGACTCTTGTAGATTCAGAAGCCCACTGAATTGCATAAATGATGGAATGATTTTAGACTTAGTGATTT
CAGTGACTAAAAGTTTACAGATCCTGGCCGGGCACAGTGGCTCACACCCGTATTCCCAGCACTTTGGG
AGGCCGAGGTGGGTGGATCACCTGAGGTCAGGAGTTTGAGACCAGCCTGGCCAACATGGTGAAACCT
TGTCTCTACTAAAAATACAAAAATTAGCCGGGTGTGGTGGCATGCACCTGTTGTCCCAGCTACTTGGG
AGGCTGAGGTGGGAGAATGGCTTGAACCTGGGAGGCGGAGGTTGCAGTGAGCCCACATCAGGCCACT
GCACTCCAGCCTGGGTGACAGAGTGAGACTCTGTCTCCACCTCCCCCGCCCCCCGAAAAAAAAAAAAG
TTTACAGATCCAGCAGATGGGGCATATTCAATTTGTGACAGCCACTCCCTTCACCTTATAGCTATGTCA
TATGTCTTCTTCTCCTTTGACTGCATTCTGCAGCAGTCAGTTGTGACTTAATATGGCACTCTGGGCCCAC
TGAATTAGGTCAGAGCTGCTAGTAGTATATTGTTCCTAGAGACCTAGGGCAAGATTTTCTTACTACATA
AAATGAGGGAGATAATTTCTTACCTCAAGATGTTGGTAAGAGGAGTGAATGAGGTTAGTTATATGGTA
ATATCAGTACTCTGAATGTCTTTTGATCAATGCCTAACTCATCTTCTTGGGCACAAAAGGCATACAGTC
AGCACCCTTAGGCCACATATAAAATTCCTCCAAATGCAGGTTTTCATCTGCCTTGGGGCAGAGTCAAG
AGAAAGAAGAGGAAGAGGCGTGAGGCTCTGACCACAACTTAGGGACAGAATATAGCCCAAAGCGAG
TACCCCAGGCCACAAGGAGAAGGCCGCTATCTTGTTGAATCCACAGCACTGGAAACTTGGAGTGTGTG
TTCCCCTGTGTCAGTTACACTGGAATTTTATGGCTGCTCACATTCTTCCCTTCAGGTGGACGTTGTTCAT
CAGTATCCTGGGCAAGAGGCCATCATAAACCACAGACAGCTGAGTGATTAGGAAGAGGAGCTGAAGA
GGGAGCATTAGATGTTTGATTGAGTCTTAGGTGAGAAAGTATATCATTAAAACAAAAAGATAGATGTA
GGCGGGCTCAGTCTTGTGTGCCTGGTGTGTTGGTAGAAAAACTAAAGCACAAGCCTGTAGATAACCTG
CTTTATTCTACCTCGGGGCTGGTGTTGGAATCCAGGATGCCAGACCCTAAAGTCCAGCTCTCTTTCCAA
CCTACTGAATAATCCGAGAGAAATCATGTTCTCTCTCTGGGCCTCAGTTTGCCCATGTATAAAATGAGA
TGAAGGATTGGCTGGGATGCTCTCCAGAGTCTCTTCCTGCCTGGAGTTCTGACGTAGCCATGTACTCCT
GCTCAGCATCGCTAAATGGCTTTGTGGTAGGACCATTGAGTGCTGCCTCCATTAGGGCCAGCTATGTA
ATGCTGGGGTGGCTGTCACTGGGCCCTAAGAGCCAGGATTGGTCTTACTGGAGAAATCCACATCCACC
TAAACTTAAGACCCAGGGGTGTCCAATCTTTTGGCTTCCCCAGGCCACACTGGAAGAAGAATTGTCTT
GGACCGCATATAAAATACACTAATTATAGCCGATGAGGTTAAAAAAAAAAAACTCAATATTTTAAGA
GAGTTCATGAATTTGTGTTGAGCTGCATTCAAAGCCATCCTGGCCGCATGTGGCCCATGGGCCATCGG
TTGGACATGCTTGCTTTAGACCTCCCAGCAATTCTAGTCTCTAAACAGGAAATCAAAAGTCAAGATGA
ATAGATAAGTTGGTCAGTGTGAAAAAGTAATTGGTGGGAGCCACTGTAGATGCAGGGTTCTAGGCTCC
ATCAACAACCACCTACATCACTGAACGAAAGATAATGCTTGTTCAGCACTTATTACATGCCAACCATG
GTAAAAATACTTCAGATGCATTGTTTTCATGAACTCTCACAGCAGCTCTTTTTCTTGCCTAAATGCCCC
GTTAGAACCTCCAGTACAATGTTAAATAGATATGCTAAGAGACAACATATGTGTCTTGTTAGGGGGAA
AATATCCAGTCTTTGACTATTAAGAATGGTGTTAGCAGTGGGTTTTTCCTAGGTGCCCTTTATCAGGTT
GAGGAAGTTCCTTTCTATTCCTGGTTTGTTGAGTATTTTTATCATGAAAAGGTGATGGGTTTTGTCAAA
TGCTTTTCTGTGTCTGTTGAGATGATCATGTTTTTTTGTCATTTATTCTATTGATATGGTATATTATACAT
TGATTTTTCAGATATTAATCTTGCATACCTGGGATAAATCCCACTTGGTCATGGTGTATAATTCTTTTTA
TTTGTTGCTGGATTGAGTTTGCTAGTATTTTGTTGATTTGTATTCATAACAGATAGTGGTCTGTAGTCTT
TCCCTCCCTCCCTCCCTCCCTCCCTCCCTCCCTTCCTTCCTTCCTCTCTCTCTCTCTCTCTCCCCTCCCCTC
CCTTCTTTTCCCCTCCTCTCCCCTCCCCTTCCCTTTCTTCTCTTTCATAGTTGTTTACCACTGTCAGAAAA
GGTCTGTTCGTTTTCTTTCGTCGTGAGATCTTTGTTTGGTTTTGGTATCAGGGTAATACTGCCTCAAAAA
ATGAGTAGGGAAGTGTTCCTTCCTCTTCTGTATTTTGAGAGAGTTTGTGGTCGGTTTTTATTAATTCTTC
TTTAAATATCTGGTAGCGTTCACCAGTAAAGCCATCTGGGCCTGATGTTTTCTTTGTGGAAAACTTTTT
GATTCCTAATTCAGTTTCTGGTTATAGGTCTATTCAGACCTTCTATTTTTTCTTAAGTCAGTTTTGATAG TTTGTGTCTTCCAAGGAGTTTGCTTCATCTAAGTCATCTAATTTGTTGGCATACATTTCATAGTGATTCC
TTATGATCCTTTTTATTTCCGTTAAAGTTGGTGTAGGGATAGTCCCTCTTTCATTACTGATTATAATAAT
TTGAATTTTCTTTTTTTCTTAGTCTTGCCAAAAGCTTGTCATTTTTATTGATCTTTTCAGAGGACCAACTT
TGAGTTCATTATTTGTTCTCTTTGTTCTTATTTTTCTGCTTCATTAACTTCTCTAATCTTTATTCTTTCATT
CTGCTTGCTTTTGGTTAAGTTTGCTTTTTCTGGTGTCTTAAGGTAGAAGGTTAGGTTACTGATTTGAGAT
TTAAAGATCATGCTCTTTAAACGTTTTGATAGATACTGTCAGTTTGCCCTCTGGCTTTTTCTCATTAACA
GTGTATAGGAGTGCTTATTCCTCACACTCATACCAGCCCTGGGTGTTACTAACCTTTATATATTTGCCA
GTATCATATTCAGACATAGTATCTTGTTTTAATATGTTTCTCTGATTACTGATGAAGTTAAGCAAATTTT
CACGTGTTTATTGGCCATCTGTCTTTCTTTTTTCATCCTTTCTTTCAAGATGGGAGTCTTTGCCATGTTGC
CCAGGCTGGACTCGAACTCCTGGGCTCAAATGATCTTCCTGCCTCAGCCTCCTGAGTAGCTGGGACTAT
AGGCGTGAGCCACCATGGCTGGCTTGCCCATTTGTATTTCTTATGTGAGTATTTTTTCTTTTTTTTTGAA
GTGGAGTCTCACTCCATCCCCCAGAGTGGAGTGCAGTTGTCCGATCTTGGCTCACTGCAACCACCGCCT
CCCAGGTTCAAGTGATTCTCACACCTTAGCCTCCCAAGTATCTGGGACTATAGGTGTGTGCCACCACAC
CTGGCTAATATTTGTATTTTTAGCAGAGATGGGGTTTCACCATGTTGGCCAGGCTGGTTTCAAACTGGC
CTCAAGTGATTCACCTGCCTCGGCCTCCCAAAGTGCTGGGATTACAGGTGTGAGCCACTGTGCCCAGC
TGACTTTTTTTTTCTTTTTTTTAACCCTTTTTTTTTTTTACCCTTTTTTTGGCCCATTTTTTTTTACCCTTTT
TCTTTTAACCCATTTTTCTATTAGTTTTAAAAATATGTTTGCAGGAGCTTTTTATATTGTGGATTTTTCTT
GTTTATTACATATCATTTGTAAATATGGTCTCTCCATCTGTCACTCTTCTTTATCTCTGGTTTCTTTAGCT
A TTTTGTTTATGGAGAAGGTATGGGTGTAATGGTTCTTATTGGCTTTCATGTGTTCTGACTCGCTATGAGTCGTTGTGAATGGTTGTCAATGGTTTGAGTTGGTATAAATTGCTTTCAGTGGCTTTCAATCGTTGTCA
AACCTCTGCCTCCTGGGTTCAAGCGATTCTCCTGCCTCAGCTTCCCGAGAAGCTGTGATTACAGGCACC
CGCCACCACACCCAGCTAATTTTTGTGTTTTAGTAGAGACGGGGTTTCACTATGTAGGTCAAGCTGATC
TCAAACTCCTGATCTCAAATGATCCTCCCAAAGTGCTGGGGTTACAGGCGTGAGCCACTGCACTCGGC
CAGAAGTTTTGAATTTTTATGTGTTTAAATCTATGTTTTCCTTTATGACTTCAGGTTGCTTTCATACTTA
AGCAGGTCTTCACCATCCCAAAATGATAAAATTTTTCTCCTGAGTTTTCTTCTAAGTTGGTTCTTTAGA
AGCCACCAACTTGGCTTCGACAGCAAAAGATGAACAGAATTTCTGTTCAACTCTCATGCTGCAAGAAG
CTTTATGTAATACTCCAGGGACCCTTTAAGGTCCCAGAGTTTTCCTCCAAATCTATCAGTGATTCTAGT
GGCTAAGAGTAGAAATGTGAAAATTTAGCCATGTGTGCTGATAGAGCTGTAGTAATTTGTAAGCTCTG
AAGTTCTAAGGAGTCAGGGGAGAAGGGAAAGTAACATTTATTGAACATCTATTAGCTCAATAAGAAC
ATGCGATAAGTATGTATATGTATTATTTCACTTACATCTGAAAGGAAGGCATAATTATCCCCACTCCTT
AGAGAAGGAAATTGGAGCTGGCTACATTTAAAGTAGTCCTGACACCAGAGAGATATTGCCAGGAGTA
CTTGGCTGGCTGAGTGCCCAGATGGCCCATAGGAGTAGTGGGCCCTCCACAGTCCAAGGTCTGGTTCT
AGGTGGAGAGAGAAGGATGTGCTCGTAGTCAGCACCGCAGCTCCAGAAAATCTGCTGGGGCTCCAAA
ACTGATTAGAGGGGCAGCTGACTCAGTAATAAAACTCCCAGGAGACTTACTTACATACTGGAATGCAA
AGTTGCAGCTTTACTGGGAAGATTAGAACTGTTATTGAGTAGCTTAGAAATCTCTGGCTGAATTCACTG
CAAGGGAAGCCGCAGGATAAGCTAACTGCTGGTGAGTCAGCAGTCAGAGCAGGGAAGTGAATTTAAC
ATTAGATGGGTCAGTCTCTCGTGGCTGATGAATTCATCCCCACAATACTGTACACCTGCCTTAGGGACC
TTTGTCTGGACTAGGGGTTGGGGTCCCCCTCCTTTGTACAGCCCTGGAAGGACACATCCAGCTCCATCC
GCCATCTCTCCCTTACTTATTTCCTTCCTTCCTTCCTTCTTTCCATCCAGCCATCAAGCTTCCTTTCATGG
CCAATAATCATCATTGGGGTCTACTCATGGACTCTCTTGCCTCATGTATTTGTTTTATTTTGTCCTCATT
CCCACTTCTATTTCCCAGGTATATCACAGGCAACTATTCTAACGTATTTATAGTTTGTGTATCTGTTTTT
GCTCTTGCCAAAATGGAAGCCACTGCTTTATACATAGATGTATTCTTAACTTTAAAAAAAATTTTTTTA
GATTAACCTACAATAAAATTGGCTTTTTGGCATATAGTCTATAAATTTTAACACATACATATTTTTGTG
TATCTACCACCACAATCAGGATACAGAACAGTTCCATCACCCCAAAAAAATCCCTCTTGTAGTCACAT
TCTCCTCCCACCCTTAATCCCAGGCAACCACTGATCTATTCTTCATTACTATTGTTTTGTCTTTTTGAGG
ATGTCACATAAATGGAGTCACACAGTATATATACATTTTTTTAAACATATGTAAATGGCATTTTATAGC
TCATTTTGATTATATGTTTTTCATCCAGTTCTGTTTTTTTTTTTTATTTTTAAAAAGTTTGACATAACTTC
AGACTTACAGAAAAGTTGTTAGACTAATACAAAGAATTCCTGGATATCCTTTGGAGTCCCTAAATGTT
AACATTTTACTATATTTACTTTTTCCTTCTCTCTCTCTCTCTCTCTCGCTCTGTGTGTGTGTGTGTGTGTG
TGTGTGTGTGTGTATCTACCTGTAGATAGATAGATATTAATATAATTTTAGATAGATGTATCTAGATCT CTCTCTCTCATATATATGTGTGTGTGTATATATCTATATCTATATCTATATATATCTCCTTTTACCCTTAA ATATTCAGTGTATATTTCCTAACAACAAGGTGATTTAAAAATATATATATAAACATAGTATAATTAAC
AATCAGGACATCAACATTGAAACATTTCTGCTATGTCATCTACAGGCCTTAGGAAGACTTTGTCAGGT
GCCCCAATAATAGCCTTGATGGTAGAAGAAAACCATGTGTTGTATTCAGTTGTCATGTCTCTTAGTGTC
TTGTAATCTGAAATAATTCCCAAGCCCTTTGGATTTCATGACAGTGACATTGTTGAAGAGTACAGGCC
AGTTATTTTGTAGAAGGTCTCTCAGTTTAGGTCTGTCTGATGTTTCCTCCTGATCAGATTCAGGTTATTC
ACTTTTGACAGGAATACCACTGAAATGATGCTGAGTTCTTCTCAGTGTAACGAGATCTAGAGACACAC
ACTGTCAGTTTGTTCCTTATTGGCAGTGTGAACCTTGAGGATTTCATTGTAGTGGCATTTGGCATTACT
CCATTATAGTTACTATTTTACCATTTTAAATTAAAACTATCTGGCCGGGCGTAGTAGCTCATGTCTGTA
ATCCCAGCACTTTAGGAGGCTGAGGCGGGCAAATTGCTTGAGGTCAGAAGTTTGAAACCATCCTAGCC AACATAACATGGTGAAACGCCATCTCTATAAAAAATACAAAAAATTAGCCTGGCGTGGTGGCGCATTT GTAGTTCCAGCTACTCAGGAGGCTGAGGCACAAGGCTTGCTTGAGCCTGGGAGGCGGAGGTTGCAGT GAGCTGAAATCACGCCACTGCACTCTAGCCAGGGTGACAGAGTGAGACTCTGTCTCAAAAAAAAAAA
GTAAATAAATAAAAAAATTTTTTAAGTATCTTATGGGCATATACTTGTCCTGTTACTCCTCAAACTTTC
ATCCACTTTTTTTTTTTTAAATTTTTTTTCTTACCTTTCATCGTTTTCTTGATATCCACTGGGTTTTAGCAT
CTACAAATGATTCTTGCCTGAATCAGTTATTATGGTAGTTGATGGTTTTCTAATTCCATTATTCCTTCTA
TGTTTGTTAATTTTGGCATTCTTCTATAAGGAAGAGCTTACCCTTTTTCCCTATTAATTAATTCATATAT
TAATGCAGACCTATGCATTCTTACTTCATTAAATCATAATCCTTTACTATCATTATGTATTCTGATGTTC
AGACTATCCCAGATTTAGCCAATAAGATCCCCTTCAGGGGAATGGTCTTTGGGATTCCTCTTTAGAGGT
TCCTGGTTCCTGTTTTCTTTTGACATATCCTATTACTCTTTGAGCATTTTTTTTTTTTTTTTTACTTTTAGG
CACAGCAAGAAGTTCCATGGTCCTCTTGTTCTTTCCCCAACTCAGCCCTAGAGTCAGTCACTTCTCCAA
TGAGCTCTAGTTCCTTTTAGTAGAGAATCATAATTAGAAAACAAGAATCAGTGCCAAGTGTGCACCTT
TGTTTTTAAGGTCCATCCACGTTGCCGTGTATATGTCCAGCATGTTGATTCTAACTGCTGAATAATACC
TCATGATTGTCATCCATCCCAGTGTTTCTTTTTCCCTTCTGTAATGAGGGACTCCTGGACTGCCTCCAGC
ATTACCTTCACAAATATTGCTGTGAGGAAAATCCTTAAACGTTTCCTTTATGGGCAACGTGTGAGCATG
TTTATGTTGATTCAGGGGTGCCAGACACAGCTCCAGAATGGCTGCCTCAGTTTACATTTCCACCAGCAG
AGCATGACAGGCTCTGTGTCTCCGTGAATAATCAGCATTAACCAGCTTCCTATTTTTTGCCAAACTAAT
AGATGTGCTAGGATAACTCTTTGTTTTAACTTGTTTTTCTCTGATTACCAATGAGCTGGAGCATTTCTTC
ATATGCCTGATGGTCTTTGGGATTCCTCTTAGGTAAATTGCTTATTCATTATAATCCTTTGCCTGTTTTT
CACTGGAGTTCTTATATTTTTCTTGAAGATATGCAGGAATTCCTTATACATCCTAGATATTAATCCCTTC
CTGGTCTCAGACATTGCAGATATCTTCTGAATCTGTTATTTACTTATTTATTTACAATTTTTTTTTTAAG
AGTTGGGGTTTTGCTCTGTCACCCAGACTGGAGTGCAGTGGTATGATCATGACTCATTGTGGCCTCGCA
ATCCTGGGCTTAAGCGATCCTCCCACCTCAGCCTCCTGAGTAGTTGGGACTACAGGTATGCACCACCA
GACTTGGCTAATTTTATTTTATTTTTTAGAGATGGAAGTCTTAATATGTTGCTCAGGCCAATCTTGAACT
CCTGGCCTCAAGCAATCTTTCCACCTCAGCCTCCTGCATCTATTATATATATGTTCACTTTGCTCATGCT
GTATTTTGTTGCAACATAAAACTATTTTTCCCATTGTTTTGTGCAGTCTCTCACCAGCACTCTTCTTTTT
CTGTAACTGTGTTAATGCCCTTTGTTCTTCCATATGTTAGGTATGCTGGTATAGTTGAACTCTGCTGACT
CTCCTCAGTAAACAGTCTCTTTTTATGACACCTTATCCTCTACTGAATTCTCTCTATCAAGAATGACTTG
GCCGGGCATGGGGGCTCATGCCTGTAATCCCAGCATTCTGGGAGGCCGAGGTGGGCAGATCACCCGA
GGTCAGAAGTTCAAGACCAGCCCGGCCAACACGGTGAAACCCTGTCTCTATGAAAATACAAAAATCA
GCTGGGCGTGGTGGCAGGTGCCTGTAATCCCAGCTACTTGGGAGGCTGAGGCGGGAGAATCACTTGA
ACCTGAGGGGGAGGTTGCAGTAAGCCGGGATGGCACATTGCACTCCAGACTGGGTGATGGAGAAACT
CCATCTCAGGGGGAAAAAAAAAAAAAAAAAAAGAATGACTTGTCTTCCTCTTAGAGTGTGAGGTCTA
CATACAAATATTATTCTTGTATTCAGCAAATGTATGTCATAGGCCTAGTGTGTGTTAGGAACTGTGCTG
TCACCAACAAAGTTTAGAGAGGTTATAAAACTTGACTGTAGCTTTTTAGAGGTGGAGGAGTGATTTGA
AACCTAGGCTGTAATTCCTTCCTCCTGTGATTCCTTCCTACTGTGTTGCCTTCCCTTGAAAATTGCATTT
GGGGGCCAGGTGTGGTGGCTCTCGCCTGTAATCCCAGCACTTTGGGAGGCTGAGGCGGGTGGATCACC
TGAGGTCAGGAGTTCAAGACCAGCCTGGCCAACATGGCGAAACCCCGTCTTTACTAAAAATACAAAA
ATTAGCTGGATGTGGTGTGTGGTGACATGCACCTATATTCCCAGGTACTCAGTAGGCTGAGGCAAGAG
AATCACTTGAACCCAGGAGGCAGAGGCTGCAGTGAGCTGAAATTGCACCACTGCACTCCAGCCTGAGT
GACAGAGTGAGACTCTGTCTCAAAAAAAAAAAAAAGAAAAGAAAGAAAATTGCATTTAGTTCCTGTA
GACTGTGTGTCAAATGTCTAAATCTCTTCTAACAAATGGCCTAAGGAGGTGCAAAGCGAAGCATCCTC
ACCAGCATCCTGACTTGGCAGTGAGGCATGGGACCCTGGAGGGAGTAGTGGTAAGTGTGACTCTGGA
ATTCTTCCTGGGCTACTTGTCAGTGACTGGCTCCAGATTGAGAGGAGAGCCCAGAGGACACAGGTGGC
TGCCCCAGCCTGGAGGTGAAAGTCTTAAAATAAAATGCCAGATGCCTAGACCATTCTAAACCTTTCTG
AGAAGCTGAAATCATCCCTTCTGGAAGCGCTCTAGTTCTAAAAGGACAGATATACAGCAAGATCTTCC
TGGGGCTAATATGGAGTTTATAGGCAAGTAGGCCTCAGAACCTTTCCCTGGTAGTGATATCTGTGGGC
AGGCACAGTTTCCACACTTTCCAGAAATTCCAGCGGAAGGAGTGAGAAGGAGGAATCTGCCCTTGAGT
GAGGACCAAAGAAAGCAGAAATTCCTCTTGGGAATTTTTCCTCCAGAGACCAAACACTACTTGGGAGC
TTGTTTACTGGGCTTTAAAAGCTTGTGACCCCCAGTCACTCTTTCTTGACCCCAAGGCTTTGCATTTCTG
TGGCTTCCCCACTGGACAGAAGTGGAACTGTCATGCTGCCTGTTCTGGGGTCTCCCAGAGGTTTCCCCA
TGTCCTCTCCTTGCTTCTACTGCCCCACAGAATTGGGGATCTGTGACCACATATGGTATAGAATTAATG
CTTGAGAATGGTTTAGTTCAGTGATGTCAAATAAGATTCACTTTTATGCCACCTCCATCAGTTGAAGGC
CCCCCTGGCCCCTAAATTGGAAAAGATTCTGAGACAGAATCCCCGTGGGTACAGCGCAGGGACAGTA
AAGGCACGTGTGCTGTGATTTGCTATCCACTGTGTGGATGCATCCAGGAATATCAGAACCCTGGAAGA
TTATTTAAGGGGAAGTTAGGACAGCTTTTTTGCCAATCCAAGGGTGTTCTTGAGGAAGTCTGTCTTCCT
GTATGGCCTTCAGTTTCTTTCCTGTGTAACCATGGGGCCAACACATAATTCCCACAGCTCTATTGGCCC
TTGTCTGCCAGGATTCTCTAGGGTCTGATTCGAGGTGGATCCTGGCCCTTTGAGGTGGCAGAATCTGAT
CATGGTGCTGTTTCCTTAGATTTAGGCCTTGATACCCTTGGCGAGAGCATCCTGGGCTGAGTGACCACC
TGAGGTTTTTCTGGTGATTTTGTGACCCATGTAAAACTTTGAGCTTTGGGATTATTCTCTCAAGGAAAT
AGTGACATTTGGTGAAGAGCCTGTTTGGTGTGGCTATGTGAGGCTTAGCCAAGAAAATGCACCATTTT
TATTAGGAGGTTAGGCCATCCGTTGCCACAAAGTGTCAGATGCTAGGCCTAGAGCCTGGAGAAAACTT
ATTTTAAAATTGATGGGGTGCTGGAGGGGTTGGGGGGTGGTGGCTGTAGCTCATGAATCAGGTGCTAA
ACCTAGAAACAAAAGGCCTCATGTGGCAGACTGTTTCTGAGCACAGATGAATGGATGAGCAACTGGC GCAACTTTGCCCAGTTGGTCCAGCTTCCCACTTGGCCACCTAGGCTTGCTGTGAAGACCTCGTCTGGCA
GAAATGAGAGTGTTTTTGCCCCATCTTGATCTTAACTGTAATTTAAGACTAAAATCTTAGATTCTAAAA
CATCAAAGGCAAGATGGCTCCCAGCTCTGTGAGCTCAGCTTCTCACCTCTTAGTTGAACAAGTGCAGT
GTGGGTCAATACATGATTGCTGCTCTTGCTGCCAGGAACTGTCCCAGCATAGAAAGGAATGGGACACA
ATCCCTGCCGTCAAGATTCTAAGGGAGGAAGCAGGCAGGTCGACTGGTGCCTCATCTCTGCAGGGCTC
CAGCCAAGGTTTGTGAAGGATTTTGCAGGCATATGGAGTGGGGACTGATTGATCCCGAGAGGGGACT
GGGGAAAGCTCTGAAGAGGGGATGACATTTGGTTTGAACTCCAAAAAATGGTTGCTTTACCTGTTTCC
TGAAGTTTTTGAGGTGGCTTATAAGAACATATACCATAAAAAGGACCAATATAAATTTAAAATCAGAA
AAAGAGAAAATGGGCTGGGCATGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAGGCCAAGGTGG
GTGGATCGTGAGGTCAGGAGATCGAGACCATCCTGCCTGGCCAACATGGTGAAACCCCGGCTCTACTA
AAAATACAAAAAATTAGCTGGGTGTGGTGGCACATGCCTGTAGTCCCACCTACTTGGGAGGCTGAGGC
AGGAGAATCGCTTGAAACCTGGGAGGCGGAGGTTGCAGTGAGCTGAGATCGCACCACTGCACTCCAG
CCTGGGCGACAGAGTGAGACTCCTCCTCAAAAATAAATAAATAAAGAGAAAATGGAACTTAGAAAAT
TAAGAGGAAGAGTGAAAAGGTAGATATTTAGTCAGGCACAGTGGCTCATGCCTGTAATCCCAACACTT
TGGGAGGCCAAGACAGGAAAATCTCTTGAGACCAGGAGCTTGAGACTTGCCTGGCAACATCTCAGGT
GAGACCTTATCTCTACAAAAAATTTAAAAATTAGCTGAGCTGTGTGGCTCGTGACTGTGATCCCAGCT
ACTCAGGAGGCCGAGACCACAGCCCAGGAGGATCGCTTGGGCCCAGCAGTTTGAGGCTGCAGTGAGC
TGGCACCACTGCAATTCAGCCTGGGCTACAGAGCAAGACCCAGTTTAAAAAAAAAAAAAAAGATATT
CAAACCATGGGTCCCAACGTAGTTATTATATTTGACCATTTGCAAAAGCTGAAAGCAAAACATGTTAC
ACATTTTCAGAGAGGAAAATACACAGTAGTTCCTGAGTGTAAGTTGTTTTTCTTGACCTCATTCTTAAA
TTGCTTCATGAGGGTGGGAGGGAAGTGGTAGTTAATAAGTGAACCTGTAAACCAGCGTTTCTCAAAAT
GTAGTCCAGGGAATTGCATCAAAATTGCAGTTACCTACAGTGCTTGTTAAAATGCAGATTCCTGGGCC
CCTGCCCCAGGCTTATCAAATCAATCTGGTGAGTAGGACTCAAGAACCTGTAAATTCACATACTTCTG
CAGATGATTCTTCTTGCACTGCACAGCATGAAAGCCTCTGCAATAGACAGAAAGCTACCAGCATTGCG
AAAGCAACTTGAGTGCTTGGCCTTTGAAGGTTGAGTGGGACTTTAATGAGGGAGAGAGTAAGGCATG
AGAAATGGCAGTTCCACTGAGGTCAGTCAGTGGTTCATTGCTGACGAAGTCACTTTTAAGTCATGTTTT
AGAAGAACTACCAAGTGTGGCAGGTCAGGCATGTGGCAGGACTGTTTCTGAGCACAGATGAATGGAT
GAGCACCTGGCCCCACTGTGCCCAGTTGGTCTAGCTTCCCACTTGGCCACCTACGGTCTGCTGTGTGGA
CCTTGTCTGGCAGTCTCCTTTAATTTATTTTTTATTATTTTTTTCTTTTTGAGATGGAGTCTTGCTTTGTT
GCCCAGGCTAGAGTGCAGTGGCATGATCTCGGCTCACTGCAGCCTCCACTTCCCAGGTTCCAGCGATT
CTCCTGCCTCAGCCTCCCAGGTAGCTGGGATCACAGGCAAGTGCCACCACGCCCAGCTAATTTTTGTAT
TTTTAATAGAGACATGGTTTTACCATGTTGGCCAGGCTGGTCTCGAACTCCTGACCTCAGGTGATCCAC
CCATCTCAGCCTCCCAAAATGCTGGAATTACAGGTGTGAGCCACCGCACCTGGCCTATTTTTTTTCAGC
AAATTCTTTGTTTTTCTCTCTGTTCCCAAATGCAGGGTACTGAGACCACAGATGTATTCTGTTTCCTGTT
GAAAAAATGTTTCTCACTTAGCTGGGTGTGGTAGCATGCACTGCAGTCCCACGGGAGGCTGAGGCGAG
AGGATTGCTTGAGCCCAGGAGTTCGATAATCATGCCATTGCACTCTGGTCTGGGTAACAGAGCGAGAA
ACTGTCTCTTAAAAAAAAGAAAAAGAAAAAGAGGTCCTAGGGAAAGAAACAAATAGTGGCTTGGATG
GTGAGTTGGTGGAAAGAACAGTGGGTGTTGGGGGTGTTGAACTTGTGTTTGTGTGTGGTGTACCCAAG
ACATATCATGTCAGCATTAAGAATAGACTATTCCTGTTTTCTGGTCACTGAGTTGTATGTTTTGACATC
CTTATTTTGGAAGATACTTCCTTACTAGGAATGGGATAGGGAGGGGGTCACCTTTCCCATCTGTGGGTC
ATATTTTAAAATATTTATTGTTCAAGTTTAAAGATATAACCAAAGGTATAAAGAAAAATACCACAAAC
ATCTGATTTAAGAAACAAACCAGCCGAGCGCGGTGGCTCGTGCCTGTAATCCCAGCACTGTGGGAGGC
CGAGGCAGGCAGATCATGAGGTCAAGAGATCGAGACCATCCTGGCCAACATGGTGAAACCCCGTCTC
TACTGAAAATACAAAAATTAACTGGTCATGGTGGTGTGTGCCTGTAGTCCCAGCTACTCGGGAGGCTG
TGGCAGGAGAATCGCTTGAACCCAGGAGGCGGAGGTTGTAGTGAGCCAAGATTGTGCCACTGCATTCT
AGCCTGGCGACAGAGTGAGACTCCGTCTCAAAAAGAAAAAAAAAAGAAAGAAATCATTTCCTACACC
TTCGAAGCCTTCATGAGTTAGATTTTGAAACAGTGCAAAATGCTTCACGTGAGAATCGAGAGTCCCTT
CTGGTGGCTCTCCATCCCCTGCTCTTCTGTCAGGTTTTCTTGTAGGTTTATGGAAACCTTTGTTACTTGT
GCAGGTGGCAGAGAAGCAGAGAGGATAGCTGCGCGCCACCCACACAGCTAGGATTTATTGGCGTACT
CCCACGTGCATGGCAGCCAAGTGGACACAACTCTGTGATGAATCCTCCCAAGAGAACTGAGGGGCCCT
GATGGAGGAGCTGCTTCTTTGCAAAGCTTTCCTTGACTCTCTTCCTGTCCCCTAGTTGATTCCCCTTCTG
TGCTAGTTTTAGCTTATTGTTTGTTACCTGTCACACTTAGCAGTACTGTTGGCTTTGCTGGTCTCCTTGA
CTACTGGGGGTAAAGACCTTTTGTTGTTGTTGTTGAGACAGAGTCTTGCTCTGTCGCCCAGGCTGGAGT
GCAATGGCGTGATTTCGGCTCACTGCAACCTTCACCTCCCAGGTTCAAGAGATTCTCCTGCCTCAGCCT
CCTAAGTAGCTGGGATTACAGCTACACCACACCCGGTTAATTTTTGTATTTTTAATAGAGATGGGGTTT
AGTAGAGATGGGGTTTCACCATGTTGGCCAGGCTGGTCTCAAGCCCCTGACCTCAAGGTGACCTGCCT
GTCTCAGCCTCCCAAAGTGCTGGGATTACAGACATGAGCCACCATGCCCAGCCTCAAAGACCTCTTCT
TTACTTGCTCACCCTGCCGCCCACTCCCCTACCAACCCCTGCATGCCCTATACCACCTGGCACATGATA
CATACTAACTGGGTACATGTTTGAATATGAATGGATGTGGTGCTGTGAATGCTTAGGGGAAGTGGGTG
AAATGCTTAAGAACCAACCTTGAGTGGTCTGGGAAGGCTTCCTGGGAGGGTGGTGTTTGAGCTAAGGC
CAGGCAGCTGTTAGATTTGTTAGACTGAAGCCCTTGCAGACTTAGAGAGCTTGTGCTCTTCCCAGAAT
GACGGGTGAGCCACGTACAGTAAATGGTGCTTCTCATTTCTAGCCCAAGGGGCCTCAAGGGGCACCGT GATTTCACGAGAATGCTGCAAGCAAATCTTTTCTCAAGCTGGGGAATTTGGTGGTAATGCCTGGCTCA
GCTTGCGGTGCGCACCTGGCCTTTGGAAGATTGGTACAGAGAGAAGCGGCCCATCCACATGAGCCTGT
GGAACAGCACTGGTGGGGGAGCTGATTTGTGAAGAGGGGCTGTGCAGTGTACTGTCAGGTCTGAGAC
CCAGGAAGAAATTCCAGTATCCCAGCTCTCAGAATCACAGAGTTCTAGGCACTGCCTAGTTCCACGTG
TTCCCAAATGTTTCCTGAATACTTGGATTTCCTGTCCAGAGAATTTTCAAAACAAACTTAGAGGCCTGA
CCCATGGCTGCCAAGGAAGGATTTTTTTTTTAAATTAAATTTTAAAAATCAGTCCAGCATGAAAATCTA
TGATGATTTCATAAGAGAAAGGACATTTTAATATTCAAAGAGTAAGAAGCACTTAATCTTGGAAGAAA
GGGCATTCCTATACTTTGATTACCTTTAGTTTAATTAAAAAACACCTACATGGTCTTTACTTCTGTGATT
TCATTCCTGGGCTAGTGAAACATTGTCACAATAAAGCATCAGGCCAACGCTTCTTTCGACCCACTGGC
CAATCAGTTGACAAACAGTGACTAGATGTTTCAGCCTATTTTGCTGAGGCTAAAGGATTGAACTAGTG
CTTCAGCCAGCATGAAAACCAGTCAGGAGTCCGTGCTGGTGTTGGCTTAGATTAGCAGGGCCTTTGAT
GGAGGGGCATGTATGTGTTTGGGTTTGCTGTGCCAGGCAGGGGAGCAGTGGAATTTGTCTGAATTGAG
CTCACACATTGAAGTTATTGAGCGACTTACATGCAAGGCCATGACCTGGACTCCCAGCCGAGAGGCCC
ACGTGGCGGGGCTTGAGCTGGGGGAGCCGAGGACAGCTTACATCTGCTCATCTGCTTACGTAACCCTG
CCTCCCAGCTTCCAGAGCCAAGAAAACACACAAGCCAGCCCAGCGGGGCCGAGAGCCTGTGGTAGCA
CACGCCATGCGCCGCACAGCAAGGGCGCCTTGGCTCGGCTTGAGGCCTGTCATGAAGCCCTCAGCCCT
CTGCCTCCTCCCAGAGCTTCTCCCCACCACCCCAGGCAGTGGCTCTGAAACCTGGTCGCAGGTCTGCAT
GATTCTGAACAGAGGTAGTCGTTGCCTTCCTGGAGTCTGAGCTCTCTGGAGTTTCTCACTGGGACAGA
GCCAGGTGTGTAGCAGAGCATGGTCCCTGCAGTATGGCAGGAGGTGTGCAGGGCATTCAGGAGGCCT
CCTGGCTGGCACTCGACCCAATTAGTCATTCAACGCCAGGTCTGGGGCTGCTGTCTGTTGTCTCAAAGG
TGTGAGCTGCAAGATCCTTAGAGTTGTGGAGAAAAAATTGCCAGATTGGCAAGAAGGGCAGGATTGG
GGGTCAAGGTGTCTCAGTGTGTTGGAAGCATGATGGGGGTTGTGCAAGGGGCACAGCGAGTTCAGAA
GGGAGCAGGAGAGTGAGAAGAGGCTGTTCAGTGATAAAGCTCTGCACAGAGCCATTGGAGGAGCAAG
CTCCTTGACCATCCTTAAACCAGGGTAATTTTCATTTAGGTTCTGCCACACGCTCAGCAGGGAACTCCT
GGAAGGCAGGATTTGTCTTGTCCATCCTCCCTCCCTACCTCAACCCACTCCTCCTTGGGCTGGCACACA
GTAGGTACCCAGAAAGTATCAATTGAAACAAATTGAAAGTGGTCTTGATACATATCACAGGGCAAGTT
TGCAGTTAACAGACATTTCAGAGTAAAGACTCTCTGGCTTGGTGCTCGATCGGCTTCTGTGGGTTGTCA
GCATGCTGTGGACAGCCCCGGCATGGGAGCGAGTGGGCGTGTGTGTGTGTGTATGTGAGGGTGAGAG
AGCGTTAGTGTGTGTGTTGGGGTTGGGGAGAGAGGAGGGGGAATAGAAGATGGACCACCCGGGTATC
AGCTTCTGCCCTGGGGAGATGGTGGTGTCAGTTGCTGAGGGAATCCTGAGAAGCAGGTCTGGCTGTAG
GTGGTGATGGTGGTGGGGTTGCATGAGAATCCATTTGGGGCAGGTTGAATTTGAGGTGCCCATGACAT
ATGGCTAGCCATGTTCTGTTGGCTGTGAGGTCAGGAGAGAGACATGAGATGGAAACAGAGGTTTGGG
AACTGTCATGTGCTTAAACCAAAGACCTGGGTATAGGGAGAGTGAGAAGAGAAGGGGGCAAAGATGG
ACATCCAAGAAAGAAGCTGAGAAAGCCTAGGAATTTGAGGTAAGAGGAGACGTAGGTAAATGTGACG
CTTGGTGATCAAGGCTTCTTTCCACCTCTCCTATGCTGGACACTCACGTCTCCTGTCTGCTTGGAAATTC
ATGCTGAGGGCAGGGAAGGTGGGAGCAAGGATTTGTCTAAAGATCTTGCTTTGGATCCCTGCACTCCT
CCTGGTTTACCAAGTGTCACTGGACACGTCAGGGCGTTCTGAGACCTTAGAGAGCATCCAGTCCTGTC
CCTGCAGTTTACAAATGAGGAAACCAGTACCCTGAGAGTGGCTGTACTATCCACTCTCAGGATACCAA
AGATCATCTGGAAAGTCACTGGTGGAGCTGGACCGGGGCCCAGGCATCTCTTCTCCTGTCCGGGGCTC
TTGACTTCAGGACCACCTTTCTGAAACCCATGATGGGGCAACACCAGGACACTTTCCAGCCTGCAGGT
GTCTGTCCCGCGGAAGCGAGCCAGGCCACATGTGAATTCCTGTTTTCTGGGTGGGTTTCAGAAGGTAC
GAGCAAGTCGGCAGGGTGACAGCCCAGGTGCTTCTTGGGTTCCCCAAAACGCGGTTATGTTTAGCAGC
ATCCTCAGAACCAAAGGTGGGGTGGGGGCTGCAGATGTTGTGGGGGCCCTCTGAAGTGAAAAGAGCC
CTGTGACAGATCTTTTCTTCATGTTTTTCACAAGTTCACTGTGCAGCAGGGCCCCCCCAGTAGCCTTTG
CCCAGGGTTGGGTGTTGGGCAGCCCAGGCCTGGCTGACCTTGTGGGGAAGGGTGTGAATGGTGGGAA
TCCCCGAGGGCCCTCTTTGCCCGAAAGCCCTAAGCCTTGACATCAGATGCCCATCAGATGGTCCATCG
GAGCCCTACTACCCAGCTTGCCCAGTGAGAATCATCTGGGCTCCTTGTTAGGTAGCCATTTAGGTCCTT
CCCAAAATCCACAGACTCTCTAAGGGAAGGGCCCGAGATGCTGTACTTGTACTAACTTCCTCAAGCAA
TTCTTGTGATAGGTTTGGGAAAAACTTGTCCAGGGTGACCACTGACTGAGTCCTGGTCTTCTCTGAAGA
GCACAGTGCCTGCTCACTTTAGGGCACCCTGGGAGGTGGGAGCTGGCTCAGCAGGCAGTCTTATAAGG
GACTGAGCTTCAAGGCCTCTGTCCCTCCAGGAGGGAGGTGCATGACCAGAGAGGGAGGCCTGAGGAT
CTTCTTCCCTGCCCCAGAGGGTCTGCTGCCTGAGCTCTGTGATAGCGCAGAGAGTAAAAGGATCAAGC
TTGATTGAGGCCTATCTCTCAATGCGAAAGTTTGCTAGTTAAGAGGAGAGTGGGAAGGGCATTTCTGG
CAAAGAGAAAAGTGTGGACAGGCATGGCTTAAGGGATGGGGAGGGAGACAGACAGAGCTGAGGGTG
AAGGGCCTTTTGCTCAGCTGTGGGCCTTGGCCTTCCCTTGTGCAGGGACACACAGCCTTAGAGCCACT
GGAGGTTTTAGTGGGAAAGTAATATGGTCGGGGCTGTATCTCAGAAGAAAACAAACTAATGGGAACA
GGTCCTGTGATGGTGGACCTGGGTCAGCTACGGAGGGAGGGAAGATGTGAGATGTGTACTGGGGAAG
GGGGTGGAAGTGGCAGCTATCTGGTGAGAGGAAGCAGGCCCACAGCTTTTTTTCTCAAGCTGTTGAAT
TCAGAAGGGCGAGTGATTCCGGGAGTAGGGGGTGCTTGGAGAGCCACGCGTTATTGATAAACAGGGC
AGGCTGAAGCCTGCTCACTGGCCCTGGGCGGGTTCTCACCAGCATGTTTCAGGTTTTGATCTGTGCTTG
TGGTTGGTGTTCCTACCTGTTCTCTAGGTTCCTTCCTTTGTTCTTGTGGCTCATTTGCTTCACAGGTGAA
GCTGGTTACACTAGAGTAACAGTTCCCAAAGTGTGTTCCCTGGAAAAATGGTTCTGTAGCCAAATAAG CTTGGGAAATGGTGGGTTAAATATAACGAAGGGGGTTTTTCGACTGCACAACTTCTCAGAGCCTTTGG
TGTGTGTCGTGACTTTGCAGAAGCAGGATTTAATACGCAGCATTCCCGTTCTTATTTGACCACGAGACA
TGTTTTTCCATTAAGCATCTTGCTGGGTCTGATGTTTTCTGGAACCCATTTTGAGGCGGTCTGGTCTGCA
GAGAGTATGGGGAGCCTGGGTTCAAGCCTTGGCTCTTGACTCTCAGCAGAGCCTTGATTCCCTGTGTTG
CCTGGACTGCACCACGTGTACCACATACCCGGTATGTGACGTTTTCCTCATCCCTCTTCCCACCTGCCG
TTACCTCACAATCCACAATCTGCACCTCATCCATTTTTCTTCTGAGGCAAGCACTCTCTTACTAACTTAC
TTATCTCATCTGCATCCATGTTCTTCTAGGCCAGAAACTTGGGAGTCATCCCTCCCTCTTTGTTACTTCT
TCTTCCTCTTTGTTACTTTATCCCCTCTGTTACTAAACATTCTTCTGTGTTTCCAGCTATTTCTTTTATTTT
CCCTCGGTCTCCTTTGGGGTTTCTTTGCCTCCATCTCTCCCAGACCTTGGTTCACCTTCCATCGAGTCCC
TTCCTGGGACATGGGCACTCATGCCACTCCTGCTACCTTCCACTTCGAAGCTAACTCCCTCCACACTGA
CGTCCCCAACATGCATGCATACACACACACACACACACACACACATACACACACACACACACACACTT
CCCCAGTTAGGCTAGAATCAGAGAGATGATGTCAGCCATTTGTCCAAGGCCACGCAGCTGGGAGGTCA
CAGAGCTAAGTCTCAACCTCAGGGGTTTTGAGAAATTGCCTTCTCATCCGTGATCACTGATTTCTACAA
CAGCCTGTCAGGAAGTCTGGGTAGAAATTACTTCCATTTTACAGTGGAGTCAGAGCGGGGAGGGTCCT
GGGCAGGCGAGTGCTTCACAGAGTGACCAACCATCTAGGTTTGCCCCACACTGAAGGGGGTTTCTGGG
GATGGTTGGTCACCCTAATGCTGGATGTGGTGCCTGATGCTGGGCAGGAGGGCCCTCTCCGTGGCCAC
GTTGCCTCCCAGGAGGAGACATTTCCTCTGCAGCTGCAGCTGCAGCCTGGCCATCTGATGCAGCCTGT
GGAGCGGTGGCGAGTCCTGTGGCCTGCTAACTTCTCCCTCCCTCCACCTCTCTAGTGGGCCCCATGCTG
ATTGAGTTTAACATGCCTGTGGACCTGGAGCTCGTGGCAAAGCAGAACCCAAATGTGAAGATGGGCG
GCCGCTATGCCCCCAGGGACTGCGTCTCTCCTCACAAGGTGGCCATCATCATTCCATTCCGCAACCGGC
AGGAGCACCTCAAGTACTGGCTATATTATTTGCACCCAGTCCTGCAGCGCCAGCAGCTGGACTATGGC
ATCTATGTTATCAACCAGGTGAGGCCTGGGAAGGTGGAATGAGAGAGGGTGTGTGTGCATGCAGATG
TGTATCAGATGTGTGTGTAATGAGGGCAGGGGAAGGGGAGTGATTTCACAGACACCTGGCACTTACA
GCGAGGAACCAGCCCCCCAGCCACCACCAGTGCAGATGAGGTAAACGCCAAACAGTGTGCTTGCCTA
TTGCTGTCAACTCTATAGCCAAGGGAAATGCTGGAGTGTTTTCGTTGTTCTGTTTTTGTTTTCTGGAAGT
AGCCTTCCAGCAAGATTGGGAAAAAAGACAACCCTAATTATTCCAAAGTACACACTGATTATTCCCTG
GCTTTGTGTAGCTGTGTATTTTCCTTTTAAAAATAAAACCACCATTTAGATGTCAGACTTTTAGGTAAC
TTCAAAGTTTATCCAGTCAGTCAGAGCGTGTCTCCTGGGGCACCTGGAGACAGTGCCCTTAGTTCAGG
TCACATGCCTACATGCCAGCCCCTGGTGAAATATCTGGAGAAGTCTGATTCGTGGGCCATCTGAGAGT
TATGTGGACTGGGCCGAGTCTGAGAAAAAGTTTCTCACTGCTCGTCTGATCCATATGTGTTGGGCTTTA
GCCCTGCTTAGGAAAGTAATGCTAAGGATAGGTCAACTTTCATCACCATGGCATGGAGAATCAGATTG
ATCTAAGAGGCATCTTTATTGAAATAAATTTTTCAGTTTATTTGAGGAGCATTATTTTCCCAAGAGTAT
AACTTTGATATTTCAAGATTACCCCTAACACTTAAATTCATGTTTTTAGACTATAACCTCCTAGGTGCA
ATGACACATCTAACTTATCTAAGCACCCAGTTTCATTGAAATTCATTTGAAGAGTCTGAGTACGCCCAT
TTCTACAAGGCCCAATGTCCATTTCATTTCGAGATAAACTCTGCTTTAGGTAGGAGGATTGTTGGCAGT
TTACGGCTTCCATCAAGGTCAAGGAACTCTGTGCACCTTCCCTATGACCCCAGGGGAAGCACTCGAGG
ACTGCTGTGGCATTGTGCTGCATCACTTGCTGCAGGGAGATTCTGAAGAAGTGTAAGGTCTCAGTCCT
GCCCTGTCCCGAAGCCTCCAACCCACTTCTGGCAAGTGGGACCTTCCCAGGGAACAATTTGTTAACAG
ACCCAAATATCCTGTGATTGGATGGTGGCTGCCAAATGCTTTGGAAGCTCAGAGGAAGGAGAGAGAG
CAATGGCTTGGAAGAACCAGGATATAAACTAGGTTCTAAAGTCTGCAGGGAGATGGGCTTCTCAGCTG
GGGCCAGTGAGCAGGGACCTTAAGGCAGAAAGGAGCCTTGCATGTTCCTGGAAATTGAGATGCCCAC
TGGGGTAGGAAAGCACCAGAAGCTCTGGGACCAGGTGTCAGAGTTAAGCCTGTGAGGCAGGAGAGAG
CAGAACAAGCCCTGTTACAAGGAAACTGAAGCAGGAGAGCAGGTGGTGGGCAAACCCCTTGAGGCTG
TTTGAATTCTTCGGCCAAGTGAGGTACAGACCAGGGCCCTATGAACACCTGCAAGCAAGACAGCCACG
CAGTTGTGGGTCACCTTGGAAGAATATTGGAGAATGCAAGAGAGAACAGGTAAATGTCCTGCAAAAT
GCGGGTCACTTTAACCCAACACATATTCATTTAAGAAAAGCTCTGTGATTGAGAAACATTTGTCTGAT
GCCAGTTAGCACATACCAATGACGGCAAGATTCAGGAGCCTGTTATTAAAGCAGTGGCAGCGAGCAC
CTGGAAGAGGCGGCCACCATCACCAGGAGCCAGCAGGGATGACTAATAAGCCGTGCCAGCTGCATCT
CGTTTCTCTCTTGACAGTTGCTATGCCAGTAGATGAGGGATGTACTGTGGATACAATGCTGTCATATCT
TATTCAGCAGGGCATCTGATAGCATCCCACAAATCTGCCTGAGTAGAAGACAGACAGCTGTGGTCTGG
GTGCCATATAGGTAGGTTAAAATATATATTTGGGCCTAGGCGCAGTGGCTCATGCCTGTAATCCCAGC
ACTTTGGGAGGCCAAGGCAGGCGGATCACTTGAAGTCAGGAGTTCAAGACCAGCCTGGCCAACATGG
CGAAACCCCGTCTCTACTAAAAATACAAAAATTAGCTGGACATAGTGGTGGGCGGCTGTAATCCCAGC
TACTCGGGAGGCTGAGGCAGGAGAATCTCTTGAACCCAGGAGGCAGAGGTTGCAGTGAGCCGAGATC
ATGCCACTGCACTCCAGCCTGGGCAACAGAGTGAGACTCTGTCTCAAAAAAATAAAATAAATAAATA
AATAAATAAAATATATACTTGGGTAAAGAGGATAAAAGAGTTAGCGATGATGCTGAATTTTTGAACTG
AGGTGGCTGTTTTCAAGGAAGACTGGAGGGTGGGATGCTACGTCTAGATATGTTGCAGTTTAGGTGAA
TGTGAGACTTCCCTGTTTTGAAGTCAAATATTGGACCAGTAAAATCTAGCCATCAGCTTAAATTCCTAT
GATACAATTTACATACTCCCCAGGCTCAACACAGTAGATTTCTGAATGTCCTCTGCCAGCTACATGCTC
CTGCCCACCTCAATCCGAGTAGATGGAACAACTAACCAAGCCAGCTCAGACCGGTGGCACAGCTGTGC
TGGCTAACACTGGGCACCACCTAAGAGAGTGCTTCTCCAAAAGTGTGCTTCCCCAAATGGAGCGAAAT
ACGCTTGAGGAATGTTGGGTTGAACCATGTAAAGCAGGTCTCATTCCCGCAGAGCCTTTGGTACCCCG GTGTACACTGTAACCCCAGAAGTGTTTCCTGAGCTTGCCTGACGAGACAACTTTTCCAAGAACCGTCTC
AAGTGATGAGTGTTTTGTGAGTCACACTTTGGGGAAAGCGGGCCTAAGTTAGCATCTCCTCCCAGCTG
CCTCCCTGCTTTCCCTGGAACACTAGGAACTGCCCGTCCTCCCTCCCTCCCTCCTCTTCCCACTTCACAA
CTTAGCATCAGGAATATTTTAGTTTTGGTTTTTCAAACATATATACCTCCTTTTTTCTTATCTTGTCAAT
ATCATCTTTTTTTTTTCTTTGCTTTTCCTCATACTTTTTTTTCTCTTCATCCTTTCCTTCTCCAAGGGTTAA
CTTTCCACCTTAGGAGAATCTTTTCTGCTTTTTCTCCCACTTCCCCAGCTACTCTCTTATCATCTGCTCCA
ATCTCACCCTAATTGATCATTTTGGGAAAATATGGTCAGAGTCCAGATAACTAAGTTGAGAAATGCTT
AAACTCTGCCATACCTTTCCAGTAAAGAATATTACCTAATAAATAATAAAATGGTAATGGGAAACCTG
AACCCTGAAAAAAAAGAGGTGGAAGGAGAAACATTTGGAGCACATCCTGTCTACAAATTAGGAACTG
CCTGTGTTATCTGTTTTATGGTTATATTCTAGAAGAAGAAAGGGATTTTGTAGCACCTGGTTTTGACCT
TTCTGCACTGTTTGTTGAGCAAATAAACCTTATGGGCTGTTAGCCCTCTTTATAGCCTCTCAGCTTATCC
CTGGCCCAGACACCCTGCTGTCATTTTGACTTTTCATTCCCACACACACATACACATGCACACACATGT
ACACACACACACATACCATTTAAGATTAGACAGAAGTAATGCTCAAAATGGAGTGGCTTCTGAGACAT
TTAGTCCAAGGGTTCCCAAACAGGCTTTTCAGTATCAGATTTCTTTCTGCCCCATTGAAATGCTACACA
ACCTTCCGCTTACAGCAGGTCACAAGGGTTTCATTCTACTTGAAGTAGGGGCCATGTCCCATTTCCACT
TCCTTGGCTTCCCATTCAGTCACTGCTAGGATTTGCCTAGACCCCTGAGGCCAGACAATGTAGAAACTT
CTGCTCCATGTCACAGGTGAGGAAACAGGCTCAGAGAGGGACAGGCTCCGAAAGTCACATAGACAAC
AGTAGGGCTGCGGCTCAAACCCCAGCGTCTGACTCCAGGTTTAGTGCCTTCTCAGGGCATCAGTGACA
CTCCTCATGGCCAGGGTGCCCCCAGTGTTGCTCACAGTCTGGTATCCAGGGCTGAGAGTGTGCTGTGT
GCTCAGACTGCCTGGGTTCAGTCCTGGCACTGCCACTTTACAGTCAGTGACCTCAGGCAGGTTACTTAA
GCTCTGCAGGCCTCAGTTTCCTCCTTGGTGGGGAGGGTTATGAGGCATCCTTCTCATGGTAAACCTTCA
GTAAATACCAGCCGTTACTAGGAGGGTCCACTCCTGCCTCTCCACTCTCCATTCATCCTGCCTGTTTCC
TCTGCCTGCTTCCTCTGCCTGCTTCTGTGGTGGTGAATTCTTCATGGCTCCCACCGCCTCCTGCTGCACC
CCCACTCAGGGCCCGCATCAGGACCCTTCCTCCTATTGGTTTGAACTCCTTGGAGTCAGAGGGTAATG
GATAGTGGAGTGAGCCAGGTGGCAGAATCTCAGAGGCCATCCCGGGCCTATAAGCCTCTTCAAAATA
GGGCCACGTATCAAGCTTTACACACAGGAGTGAACTTTCACAAGTTGTTATGACTCATACTCTGTCTAT
AGTAAGCTGTTAACCACTCCCATTTGGCTTATGCCTCTGTAATTATTGTACTAACTTATATCTTAAAAT
AAGGATATTGAAGGAATGAGCCGGGAGAGGCTTTCCTGGTTGAGATATAGAAGAACAAGAGTTGCTC
TTTTTCCTTAAGGTCTCTCCTCCCACCCCTGACCTTAGCTCACCAGCATGGGAGAATACTATTTGACTC
CTTGTACTCTGAGACGTGGATTTCAAGATATAGCATTCCAACTTCAACGGCAGCAAGAAAAGAAGCAA
CAGAAGGAGAAGACATCATAGCAAACAGGGATGCATGCTGCATTTCCTAATACTCAAACCCGGAAAC
GAGACTTCACTCAAGGTGAAGGGAGGGCAGGTCACCACCTGGTAGCACTAGCCCTAAATTAAGGAAT
GCAGAATGTTTGTGGGATTGCCCATCATAAAAATTACAAAATGAGTAAGGAATGCAGGCACAGCTGG
CCAGGTGGGTTTGTCACAACCATGGCAGCCCTTTGCCCCACAGCCAGTACACAGAACTGGTCTCTCCA
ATTCCGATTGCATATCTTCTGGCACCTCTGTTCCTCTCCCTCAGCTGCCCAGGATTTTTCTGGTTCTGAC
CATGTTACTTCCTCTTTTAAACCTGTTAGCATTTCACGACTGCCTACAGGCAACGGTCTAAATGGTCGG
AAGGCCCAAGCTTAGCATCCGAGACCCTGACCTACCTCCAGCCACTTCCTCCTCCTCTCCACTTCACTG
GACTCCCCATCTCCACCCAGACACCTCTGTTCTCCCCTCTGTGTGCCTTTGCTTATGCTGTCCCCTGTGT
TCCTAGTGTGTCTCTGGCTATCTTTTAAGCTTCCCTCCCCAACCTCATTAGTTCTGTGGAGCCCCTGGAA
TAGAGCTGACTTCTCCTTCCCTGCTGCTCCCAGGCTGCTCAGAACTTTCTGGAAAGGGATGATTATCTG
AGTTCCAGCCTCACCCCAGCCCCCGGACTCTGAGTCCCTCATGTCTGCCTCCCTTCTTTCTCTCTGACCA
CACAGCTGGTACATAGTCAGTACAGACGCAGTCAGTGAGTGGAGCACGGGGCTTCTCTCCAGGATTCC
TGCCCCTTTGTTTATCCCTAGTCTCAGGACTCCCTACTCCTGGTCTTCTGCCTAAATCTGTGCCTCTTGG
AAGTGAAGCCTCCGTTCCCAGTGGGGCCAGGTCCTGACCCTTGGGAACTTGCAGGATCCCTCCCTTGG
GCCTCTCCCCGAAGCTTCCAGCTCAATGCTGACCAGAGCACAGGCTGCCTGTGACAGTCCTTGGGGTG
ACCTCCCTTATCAGGAAAAATGCAGAAAACCTATTAATACCTTAGCCTTGTGATTGTTAATGGTCACA
AAACTCCTTTAGGGTCCTTTGGACTCAGCACCTTTATGGTCTCACTTTGAATTTTGAACCTCCCACCTCC
CCCCATCCCCCAGAGTAAGGCAAATGGTCTTCTGATTGTTCCTGCAGAGGGAAGGCTCCACAGGTAAG
CACACGATGGCCAGGAAGCAGAGCTGGAGCCTGCCTGAAAGGCTGTGGAGAAATGGAGGGAGGGCT
GCCCTGAGGACTCTGTCTGGCTTTGAAGTTTTCTACTGTTTCCTTTTCTTCTGTGCACTGTTTTAGGATG
ATGGGGTGATAGTTCCAGGCTGGTTGAGGATGGATTTGGAGACAGTCCTTTGTACCCTCAGTGAGCAA
GAGTATCTGTCACCCTACCTCAGCAGTTGTCTCTGTCACTGGTCCAAGCAGCTGGTTCCTACACAAGGT
CAAGATCAACTGGGGAGAAGCAGACTCCTGGGTCTATCCCATTAGTGAGGACAGCTGCCTGGGCTTAT
GGCCTCATTGGTTTGGTTTCTATCTTGATCATCTCTACCATCCCCCCATCCCGGCCTTCCATTTTCTACC
TCAGCTGTCAGTGCACAGATTGATGTGTGTGGGAACGGAGCTTGGGAGGAGTGGGGTAGGGCTGGTC
CTGTCCTGTAGCCTCCCCTTCCTTCGGGCACTTGGACCCTTTGGAGCTTGCCGGGGTGGGGAATGGGAG
TGGGAAGGCCAGGGAGTGTCTCTGCACCATCACTGTTTGAGTGTTGCCCCTTTGCTGTGTGCCCCACCT
AGTCTATGTGTGTCTCTGTTCTCTGGGGACTCAATTTGCTGGTGAATTGCTTCCATGGACATTGTTCTGG
GAAATGCCATTTTTTCTGCTCACCCATGACTCTGTGACAAGGAATGACAGCTTATTAGGAATTTGTTTT
TGCATTGGAACAGTGGTCATCAGAATGGGCCCCTTTTCCCTTGCAGCTTTGACATTTGCCTCTCTTTTCC
TCACCTCTCTCCCTTGCATCCACCCTTTTCTCTTTTTCTTCTTTTTTGTTTTCCTTCTAGCAGGGGCCTTTT
ACCTTTACTTGTTAATCCTGTTTGTAGCAAAGCAAGTGGAAGGAGGAGTTCCTCTCTGATCTGCTTCTT ATTCTCCACCTACCTTCTCTTCTGTACTTTCCGCCTCCTAGAGAGAGAGAGAGAGAGAGGAATGCCGA
CCTAACTACCGCTGCCACTGCTGCTGCCACCACCGCTGCCACCACCACCCTGGTAATGTTCACATGTCC
TCAAATCAACCCAGAGCCAGGGCCCTGCTGGTCAGGGGGAGGCTATGTAAATAATCCCATGAGTGTGC
CATCCTCAGGCCCTGGGGTCTCCTAGGCAAGACCAGGGCCTCTGTGGGCTCTCTCGGAAATGCTGAGG
TTGCTGGAAGCCAGCCCGTCATACAGGGTCTGAGAGTTTAACTTCTTTTAAATTAAACCACAGTTGAG
CTCATGCTGTGTGTGTATAAACTTTTGTATCCTGCTTTTTCCTTAAATTCTTTATCATCAGCATCTTCCC
ATGTTATTTCATAGTCTTCATCATCATCACTTTCCATACCTTCATAGTAGTTGATCGTAGAATTCCATCA
TAATTAACTTGTCTTTTCTCTCTTAGAAGTCCCTTAGGTAATGTCCAATTTTCCGTGAGTGTAAGTAATA
CCATAATGAACATCTTGGAGTCTGAAGTTTATTCTGTGTTGGTTTGTTCCACATTTAGGATCATTTTCCC
AGGCTAGATTTTCAGATGTGGGATTATGGGTTCAGATATGGTTTACACATTTTTATAGTTCTTAATACA
GATGGCCAAATTGCTTTCTGAAAGAGAAGCTTTTCTTAAGTATTTTTCTCCAACTTGTATCTTAAACAT
CCTGAACATGCTTAGCACCACTGTCTTGATATATCTGCGGAAAGCCACGTCTCCACTTTTCAGTGTGTC
GGGCCCTGGGAGAGGCAGGCATCCTGCGCTGGCTCCTTGGAGCTGGGTTTAAAATTGTCTCCTCTGGC
TGGGCGTGGTGGCTCACACCTGTAATCCCAGTACTTTGGGAGGCCGAGGTGGGCGGATCACTAGGTCA
GGAGATCGAGACCATCCTGGCTAACATGGTGAAACCCCGTCTCTACTAAAAATACAAAAAATTAGCCG
GGCGTGGTGGCGGGCACTTGAAAAGTCCCAGCTACTCGGGAGGCTGAGGCAGGAGAATGATATGAAC
CCGGGAGGCGGAGCTTGCAGTGAGCCGAGATCGCGCCACTGCACTCCAGCCTGGGCGACAGAGTGAG
ACTCCATTTTAAAAAAACAAACAAACAAAACAAAAAAACAAACAAACAAAAACTGTCTCTTCTGTGC
TCACTTCACCCAGAATCCCTGTTGGGCTCTTCAAGGAGCTCAGTTCTCTCTGAAAGCAACTTTATAGCC
TCAGTCCAGTCTGTGTTCCTGTGTGGCAGGGGTCAAGGGTATGCTCACTCTTGAGAGTGGTGTCTTTGG
TTGACCAAGAACCACTCCCATAGCCTGGTCCCTAACCCTTGAAGGCCCATCTCTCTCACTCACTGGGGT
GAAGAGTTTAAATCTCAGATCCAAGTTTTGTTGAGAGCTCTGAGCTACCATATTGCTATGGTTAACAAT
AGTTAACAATGTTAACAATGGTTAACTATGGTTAACAATAGTTAACAATGTTTAACAACTAGAGCCCA
GCTGGGTGTGGTGGCATGTGCTAACAGTCCCAGCTTCTCAAGAGGCTGAGGTGAGAAGATTGCTGGAG
TCCAGGAGCTCAAGGCCAGCCTGGGCAACATGGCGAGACCCTGTCTCCCCTGCAAAAAAACAACAAC
AACAAAAGCAAAACTAGAGCCCAACTGCTGTGAACTCATGGCTGAGTAGATATTATTAGCCCTCCACA
AACTCAGCATTTGTATAATCCCAGGCTGTTTCCAGTAATTCTCTGGGGATCATCTCCCAGCCTGTCCAC
TGTTCCAGGATCCACACTTAGGCCTATAGGAATGCCCCGTCAGAGCTTCTGCTGCCGCTGATCTGTTAC
TGTTTCATGCAACCCACTCGGCCTAGTTCCTTCCTCTTACTGTCTCAGTGGGCACAGAAAAGCATACAG
AGGGTGTTTCAGCAAACATTGCCACTGGCTGCAGACCTGCCCCCGGATCTGTCCTGTTGAGAGCTTAG
TGCTGCGTTCTTGCATGGTGGGGAGGGGTGTGGCTCTGTGATGAGCCAGGGCATGTGTATAGGAGCAA
CAGTGTCTCTCTTATCACGTAGAAGTTCTGACTCATTGCGAGTCTTGGCTTTGGGTTAATGGTTCCAGC
CATGTTGCTGCTGTGTCTTTTGGTGCAGGAGAGGCTGGGCACAGTTGGTCCCTAAGCCATTATGGATA
AGGGATGTGTCTGCTGATATACACACATGGACCTGACATCCAGGGAAGGCAGGGTGATTGGACAGAA
CAGTTCTTCCAGAAGCTGTTGGAACTTGGACAAGAGTGGCCCTTGGCTTTCTGTAGTTGGTCATCTGTC
CCCTGTTGCAATCAGGGGAAGGCCACACTTGCCTTCCTTAACCACAGTTAGGATTTTCTTGGGGATTAG
ACCAGATTCTAGCACCTGTCCTGAACCTCTCGCCCCGCCCCTACAAAGGCTGCTTGCAAGTGTAGTGC
ACATACACAGGGAGCAGGTGGGGCATGGAAGTGGAAGTGGAGCCCCTGCCTTTGGCCCTTGGGGGAG
GCACTGTCTGCTTACCCACGGTTGTTGCCTCATAGGAATCATACAACAGCTTCCTAACTGGTCTCCTTG
CCTTCAGTTGGATTGGGGCACAAATCCCTCCTTGACATATAAACCATGGTTTAAGGCTCCCTGTGGCCT
AAATAAAGATAAAGCTTAAGTATCTTAACAAGCACCTAACCCTTCTCCCCAGCCTCGGTGATTTGGCT
CATCGCTGCCTTCATGTTTCATTCTGGCTTCACTCATTCGGAATTTCTTGTAGTTCCTTGGCTGTTCTCTT
TTCCTTACCGCCTTTACAAATGCTCTCACCATGCATGCTTTTCTCTGCTCCTACAGATGCCTTCTCTCCC
AGCACCGCCTCCAGAGTCTATGTCTGGTCGATTCTGTCTGCTGTCTCCAGTCCCCATCTTGTGGCAGTC
TCTGCTCAATCATTTGGGGATTTTATATGTTTTCTGGCCTTTCTTTTGGGGGCCTGTCTTCTCCTTCTAA
AAGCAGCCAGTTGACCTAGAAGGAAGGGATAACTGTAACTCTTGTCTACCAACATAAGATTAGGCCCA
CCCTTTAAAAGCTGCGTCTTTGAAAGGGACACCTGCACCCAGCATGCTGGCTTCTCTTCACCAAGCGTG
ACTTCCTACGCATTTCACAGGCCTCCAGAGGTCCCCCTGACTCTCTTCTGCTGTGAGAAACTCTAATCA
TGTAAGCCACAGGCTAATTCCCTTGAGCCTTAAATGTTTTTAGTAATTTCCCATTCATCAGAGAAGCAG
GATTTGGGAGGAATTTTGAAGCAAACACTACAGAAGGCAGAGTCTCCAGGTAGGATATCTAAGAGAC
ATTTGGAATGGTCTGACTGTTCAAGATGGATGGGAAAGCCTCTTCCTGTAATGATAGTAGCCAACATT
TGTTGTCAGGCAGTGGGGCCCCATTTTTGAGATGGGGTCTCTGTCACCCAGGTTGGAGTGCGGTGGTG
CTGTCATGGCTCACTGCAACCTCAGCCTCCCCGGGCTGGGTCTTCTTAATTCTGAAAAACCCAGCTTTT
AAAGGGTGGACCTAATCTTATGTTGGTAGACAATGTTGTCTCATTTAATACAATGCACATGCTCTCCCC
ATAACACAAAAGAGGGAACTGAGGCCTGGAGGTGTGATGTACCCCAAGTCACATAGCTAATAAATAA
AGAAGCCAGCATTCCTGGGATTAAAAATGCATGTGTCTGTCACTGTGGTGTATTTGGTGCTTGATCAAT
GTTTACTTGAGCAAATGGAGGGGCAGAGGTACCGATGAGTGTGCTCAGTGAGGAGGGCAGGAGTGAA
GCTGGGCGTCTTCCCGCCTCTTGTGAGTGGTGGGGCTTGGTGAGCTTGCCAGGGCCTGTCTTTCTTATC
AAAGAAGGTGTGTGCCCCAGTGTTACAGCATTTCACCCAAAGCAGCCTAGAAAATGCTTGACTTTTCT
GTCATTCCGGGGAGGACACTTTCCTCCTCCACTGTTCTGCTGGCCTGGTGTACCCACGGCCCCTGATAG
ATGATAGCACCTGCTAAAGTGCACCATGCCCTTCCGTCTCACTGCATCCCACAGATGAGGCCAGGCTG
GGATGAGGGAGAAAGGGAGGGATATATAGTTCAGGTTATTTTGGAAAACTGCCTGACCAATTTTAAGT CTGGGCCGGACACTGGGGCATCTCACCACGTTGAAAGGGCCGTGGCACCCCGGGCGGTGAAAGGGGC
TGGAACCAGGTCTGCTTCTTGGGCTTCTCCTCCAGGGTGCCATTGCTCATGGGCCTTGGCTGCAGAGGT
GCTCATTCGTGGTTCCAAAATTCCAATTCCTGGGAGAGGAAAAATGCTTAGTTCAGTCTCAGTTAGGC
CTCTGCTTAGATCAAACAGCCAAGGCCAGTAGGCCCAGTCCTATGGTAGAGACATGGCCTCAAAGAGC
CCTCTGCTGCAGTTGTTGGGGAGTGTACCAAGAGAAGGGAGCATTGTCCTGGGCTGGGCAGCCCTGGG
GGTCTAGTGCATAGATGTAGAAAGGCTCTGTTGGTATACCTCCCTTTGCTTGTTGGAAAGTGCTCAACG
GGGCTGAATTGTGTTTGACAGTGTAAGTCTGGGCTGGGGTGAGGGTTGTTACAAGATTGTCAAGATGA
TTAAATGAAATGCCATTTGAAACACTTATCCATGCCTTGTGTATGGTATCCCCACCAGTGAATATTCAC
A TTGTTTATTTATTTTTATTTATTATTTAGAATGTACCCAAAGTCGATACCTTCTGCCATTCATAGTTTTTGTCCCACTAAGTGGCCATGATGTATGCTTGACAAAGCTTGTGTCGGACAACATTTCTTTTTGTTGTCTT
CACTGCAACCTCCACCTCCCGGCTTCAAGTGATTCTCCTGCCTCAGCCTCCTGAGTAGCTAGGAATCCA
GGCGCCCGCCACCACACCCAGCTAATTTTTGTATTTTTAGTAGAGACGGGCTTTCGCCATGTTGGCCAG
GCTGGTCTCAAACTCCTGACCTGAGGTGATCCACCGCCTTGGCCTTCCAAAGTGCTAGGATTACATAC
GTGAGCCACTGTGCCCGGCAATTTTTTGTGTTTTTAGTAGAGATGGGGTTTCACCATGTTGGCCAGGCT
GGTCTCGAACTCCTGACCTCAAGTGATCTGCCCGCCTCAGCCTCCCTAATGCTGGGATTACAGGTGTGA
GCCACCACGCCCAGCCTAAACTTTGAATTTCTTTGAACCCATGACTTACACAGAATTAGCTGAACGCA
GAATTCCAAATCAACTCAGCCTGTGGGACAGCCAAAAAACACAGTGTGCCTTTGGGCTCCTTCACTCA
CCACGCGGGGTTAGAAAACTTTGTCAGAGGCTTTAAAAAAGGAGCTCTTGTGTGTAAAATGTTTCCTT
GATTCTCTTTCTGGTGCCTCTCTTTCTCTAAGTGGTTTGCTTCCCCAAGTTCCCCACCTGAGTCTGGGTG
GCTGTGGCACATCTGTGCATTCTGTACGCACACAGGCAGCCTTTTGGAGTGCCAGTTTCCAGGTCTTGG
TTTTATTTATTTATTTATTTATTTTTTTGAGATGGGGGTCTCACTCTGCCGCCCAGGCTGGAGTGCAGTG
GTGCCGTCATGGCTCACTGCAACCTCAACCTCCCTGGGATCAGTTGAGCCTCCTACCTCAGCCTCCAGA
GTACTAGGGACCACCATGCCTGGCAAATTTTTGTAATTTTTTGTAGAGGCAGAGTCTCACCATGTTGCT
CAGGCTGGTCTCGAGCTCCTAGACTCAAGTGATCTGCCCACCTTGGCCTCCCAAGTGTTAGGATTACA
AGTGTGAGCCACCATGCCCAGCCCAGGTCATCTTTTGAGGGCATGGAGAGAAGACTTTGAGCATCCCA
CTTTTGAGATTGTGTACCAGTCGCAAGCCCCTATGACACACTTTTTCCCCAAAGTAGAGGGCTCTGACT
ATGTTGATCCCAAGAGAGATGGGAAAGAGCATTGAATGAGGATTCCAAAGTATTGGGCCTTAGTTCGT
TTCCTCATGTTGGTGTTGTGAAGATTCTGGTTAGGATAACAGCATGTGTGCAGGAGGCTTTGTGAACTG
CTGAGAGTGAGGCGTGGCAATGTCAGTGCTAGGTTTGTCCTTACTAACCTGGGGCCATGGGAATTGAT
AAGACCAGATTCCCAACTCTACCCCACAATGTGATCCCTGTGGTGACCCCTCACAGGGCTCTTTGGTCG
AGCTTCCCAGAAGGGATCACCATCTGCCATTGTATGTTGAACCCCATTCATTCATTCATTCATTCAGCC
AACCAGCAACTATTTGTTGAGCTCTTATTGTGTGAGAAGCAGTCTTCAAGGAACTGGGTGAATAAAAA
AAACAAAACATCCTAACCTTCATTGAGCTTACATTCTTACTGAAAGAAAACAAATAAAACATACATGT
AATCCTAGCACTTTGGGAGGCCAAGGCAGGCGGATCACTTGAGGTCAGGAATTTGAAACCAGCCTGG
CCAACGTGAAACCCATCTCTACTGAAAATTAAAAAAAAAAAAAAAAAAAAGCCGGGCATGGTGGCAC
ATGCCTGTAATCCCAGCTACTCGCGAGGCTAAGGCAGGAGAATCGCTTGAATCCTGGAGGCAGAGGTT
GCAGTGAGCCAAGATCATACCATTATACTCCAGCCTCAGTGATGAAGCAAGACTCCATCTCAAAAATA
AAAAATAAAAATAAAAATATGCATTCCCTTTGCACCAGCACACTTGGTGCCTGGGGACCTCGTGGTTG
GCACCCTGAAGCAGGTGTCCCTCTTCTGTCTTGCACACCTTGCTTCTGTCCTGGTGTGTATGGCATGGC
CTTCTGCCCTCCATGGTGAGCACTGTGAGGGCAGAGGTTGAGTTGGGTTTGCTGTATTTCTCAGGTGCC
TAGGTTTGTGCTTGACAGGTAGATGGAAGGCACACAATGTGGTCATCAAACCTCAGTCAACCATATAA
GGAAGGTAGAAGTGAAAAGTCCCATAGGTACCCAACTAATGTCACCAGTTTCCTGGATACCTTTCCTG
GAGTTTATTTATAGTGTGTATAAATAAATGATGTATGTGTTTAAATGCCTTTTTCACCTTTCCTTTTAGA
GCTGCCTCTTTTTAACAGTTCCATTCCATTGTATGGATGTACTATGATTTATTGAACCAGTTCCCTACTG
ATTATTCTGTTTTTTGCAGTCTTTTGTTATGATGAACATTCCACAGTGACAATGTTGTTCATAGTCATTC
ACACACATGCAAGTCCTTCTGCAGGATATATTTCTAGAGGGGAATTGCTGACTCAGAGGTTTTGGTAC
TCTGTGTTGATTGTAGAGTGACGGCAGAAAAGTGAGGCCCAAGAGTTTCCTAGTGACCATGTGTAGTG
GACAAGTCACCAGTCCCTGTGAGTGTTTGGCCCAAAGGCTTTAAGGCATTTGATATCACTGTTTTTGTT
TCTGCACCAGGCGGGAGACACTATATTCAATCGTGCTAAGCTCCTCAATGTTGGCTTTCAAGAAGCCTT
GAAGGACTATGACTACACCTGCTTTGTGTTTAGTGACGTGGACCTCATTCCAATGAATGACCATAATG
CGTACAGGTGTTTTTCACAGCCACGGCACATTTCCGTTGCAATGGATAAGTTTGGATTCAGGTAAGAG
ATACTCAGTCAGAATCTGTGGTAAACATGTCTCTCTCATGTGTTGACTAGGAAATGCAGTCCTGGCAG
CTCAAGAGTGCCTCTTTAAGCTCTGGAGCAGAATGCCTCCTCTGAGAAATGGGTGCTTTGTATTAGTTG
A CTGTATTTGTGTATATTATGTATAGGTATTGGATCTCTATGTATAGAATGGACCCATGGGTGATGCTTCGTGCCTTGCCTAGCTACTACCCCACGAAGCGACATAGAGAACGATAGACTATGTTTGCCCCCGCTCG
ATCTTGGCTCACCGCAACCTCTGCCTCCCGGGTTCATGCCATCCTGTCACCTCAGCCTCCTGAGTAGCT
GGGACTACAAACACTTGCCACCATGCGCAGCTAATTTTTGTATATTTTGTAGAGATGGGGTTTTGCTGT
ATTGCCCAGTCTGGTCTCGAACTCCTGAGCTCAAGCAATCCATCTGCCTTGGCCTCTCGAAGTGCTGGA
TTATAGGCATGTGGCACCATGCCTGGCCTAAGAACAGTTTTTAGCATTTGGGAGGGGCTCTCATCTTTA
AGCTCCAAATGATACTGTATTTTCTTGCTTTTTTCTTTCTCTTGCCCCACAAGTTTTGGAAAGTAAATTG
G GATTATTTATGGTATGTATCCACCGCGCGATCCTTGTAGACTTCTTAGTTTTCAAGCCCTCTAGGTGACTTAGCGCATGCTAGGCCAAGGTAGTAGCTATCCACATTTGCAGTCGCTGGCTTTCTATCGTTGTCTAT GCCTTGACTGCCTGGGCTCAATCCATCCTGCAGCCTCAGCCTCCTGAGTAGTTGGGACTACAGGCATG
AGCCAGCATGTCCAGCTAATTTTTTATTTTTAGTGGAGATGAGGTCTGGCTATGTTGCCCAAGCTGGGC
TTGAACTCTTGGGCTCAAGTGATCCTCTCACCTCAGCCTTCCAAAGCATTGGGATTACAGGTGTGAACC
ACTGCTCCCGCCCTTGGCCCTATAAGAAGGAATGTGATTCTGTTTTCCAGCAGGGCACAAACTTCTGCT
TAAATACAAAGCCCAAATTTTTCCACCAAAATGCCCCTAGTGAAGTGGCCAGCCCAGATGCCCGACTA
GCGTATTATCCAAAGCATATTGTCATTGGTGGAAAATGGCCTTATAGTCCATTGTTTTGTCTTAAAAGT
AAATATATAAATAAACTTGTATATTGTTTCCTAATTCCGTGTTTATATTAACATAAAAGTGTTTTAAATT
ACCTGTCAGTGGCCAGGTGCAGTGGCTCGTGCCTGTAATCGCAGCACTTTGGGAGGCCGAGGCGGGCA
GATCACCTGAGGTCAGGAGTTCGAGACCAGCCTGACCAGCATGGTGAAACCCTGTCTCTACTAAAAAT
ACAAAAATTAGCCAGGTGTGGTGGCAGGTGCCTGTAATCCCAGCTACTCGGGAAGCTGAGGCAGGAG
AATTGCTTGAACCCGGGAGGCAGAGGTTGCAGTGAGTTGAGATCGCGCCATTGAACTTCAACTTGGGC
AACAGAGCAAGACTCTGTCTCAGAGAAAGAAAAAAAAAAACCTATCAGTTGAATAACAAAACCCTTT
CCTTCCTTGCTTTAAGTGAATCTGAAGATCCAGGAGCTGTGCTGCAGGTACCCTCTATGTTGGGTACCC
T CTTTGTGTTTTTTCATGTGTCTTTGCTATCTTTAAGTTTATCTATTGTTTGGTAGGGATTCGAGGCATGCTACTTGTTGACGTTAGCTACGACCACGCAACGCCCCTTTGTGAATTGTTTGACGAAGTTAGCGACAGC
TGATCATGGCTCACTATAGCCTTAAACTCCCTGGCTCAAGTGATCCTCTCACCTCGGCTTTCCTAGTAG
CTGGGACCACAGGTGTGGGCCAGCACCCCTGGCTGATTTAAAAAAAAAAAAATTTTTTTTTTTAGAGA
TGTCTCACTATGTTACCCAGGCTGGTCTTGAACTCCTGGGGGCTCAAGCAATCCTCCTGCTTTGACCTC
CCAAAGTGCTGGGATGACAGGCATGAACTACTGCACCTGCTGAGATGCAACAGCTTTCTGTCAGACTC
ATTTTATTCTCATCATTTCTTCCTGTCCTCCCTTGCTGGGAGCATGAGAGCTGTGATGGGAATATAGGA
ATGTATGAAGTCCTTCTCCCAGATCAAAAATCCTAACTTCTTGTCTTAAAGGGAGGAAAATTTGAATGT
AACCTTACTTTTAGACTCTTCAGAAATCCTTCTATACCCTTCCGTCCCCGCTTTCACCCTTCCTCCCTCT
CCGTGTGTGTATCTTCTTCTCTTGAAACACACAGGTTTATACCCTGACCCCTCTTGATTCATCCCTTGAA
GCACAGTGGTGAACAAGGAAGGGGCCCGTGATGCCCTAATTCTTTGCCACAGCACCATGTTTGTTTCA
CAAGGAGCCTGGCAGGTTTGGGCTTGGGGCAGATAGGGGAGAGAAAGCAGCAGAGACAGCAAAACC
AAATCATGTCAGCTTGGCATGTACTTCCCTCTGAAATAGCTAAGAATCCATTTCTGTAAAAGCACTGAT
TATCAGAAAACCTTATTGGCCTGGCCACCTTTGGTTCAAACCCTCACATTAATAATGTGGACAGTAGTA
TGAGGTGTGCCAAAGGTGGATGACTCAGCACCTAAGTGATGACACCTAATTACGAATAGGTTCATTAA
AGCAGACCCCCTGGGGACCTTTGCTTGAGGATCCTTACAGTCAGAATTCCTGAATATATTTGAAAATA
ATAATTGCATCTTTATTTTCATATGTTCTGTATGGTTTGGCTGACTTCCCCCTCAAAGTCTGAGTTAGAG
TTTTCCTTAATTTATGTGATGGGTTTGGTCTTTTTGGATTCCAGAAAGAGCTGGGTGTGGTTTGGAGCT
GCACTCAGAGTCACACAAAACCACAGCCTTTAGAGAACCCACAGGAAGGCTTTGGGGCACGTCCTGA
TTCTTGACATTTCTCATCAGTGCTGACTTTGTATCCCTTAGGAGTTCACAATTCATAACCACTGAAATA
TTAAAATACAAAAAGTTTTGGAAGGATGAGAGCCCAGATGCTCTACTACTTGAAAATATGTTAAAACA
TAAGTTCATCATTATACATTTTGCTAAATCAGGATAAAGTCTGAAGTTTCAAAGAAGTTTTATTTTAGC
AAATTTTCAGAAACACTGCCTCAACTGTTAGGGCCAGTGTTCTAGTCAGTATGCCTTTGGAAGCATGA
AAGCTGGATTGGTCGATAGGATGGGTGTGGAAGGGGGGCTGTGACTGGGTGGGTACAGAGAGGCTCT
GAAACAATCTCAGATTCCAGGAGTTCCTGGATAAGGACTTCATGTGCGGGAACAGAGCACAGGAGAA
GCAGATTCCTGAGCCACTCAGGAAGAACTGGGCCTAGGCCTGCTCTTGTCACTGACTGGCTTTCTACAT
AACCACAGAAACAGCACTGTGTTGTAGAAAGAGGAAGATCATACTTTTTGATATCTGTGTCTAATTTA
AGGTCATCTGAGCCCTGATAGAAAAGCAAAACAGACAAAACCCTTGTAACTGCTCCCTCCCACCCCAC
CCACCATCAAAAAAGCTTTAGAGAGGCTGGACATGGTGGCTCTTGCCTGTGATCCCAGCACTTTGGGA
GGCTAAGGTGGGTGGATCACCTGAGGTCAGGAGTTCGAGACCAGCCTGACCAATATGGTGAAACCCC
ATCTGTACTAAAAATACAAAAATTAGCCAGGTGTGGTGGCACACGCCTGTAGTCCCAGCTACTTGGGA
GGCTGAGACAGGAGAATTACTTGAAAACCTGGGAGGCGGAGGTTGCAGTGAGCCGAGATCACGCCAT
TGTACTCCAGCCTGGGCTACAGAGCGAGACTCCTTCAAAAAAAAAAAAAAAAAAAGATCCGGTTTGG
TGTCTTACAACTGTAATCCCAGCACTTTGGGAGGCCGAGGCCGGTGGATCACGAGGTTAAGAGATCAA
GACCATCCTGACCAACATGGTGAAACCCTGTCTCTACTAAAAATTAGCTGGGCGTGGTGGCAGGCGCC
TGTAGTCCCAGCTCCTCAGGAGGCTGAGGCAGAAGAATCGCTTGAACCCGGGAGGCGGAAGTTGCAG
TGAGCCTAGATCGCGCCCCTGCACTCCAGCCTGGCAACAGAGCAAGACTACGTCTCAAAAAAAAAAT
AAATAAAAACTCTAGAGAAGCAAAAAGAATAACTTTAAAAGTGTTTATGTTCTCAGCAAGCTTTATTT
TGGGGATGTCAGAACTTAACTAACCACTGCTCCTTCTGTGTGTATGTTTTTCCTCCAGCCTACCTTATGT
TCAGTATTTTGGAGGTGTCTCTGCTCTAAGTAAACAACAGTTTCTAACCATCAATGGATTTCCTAATAA
TTATTGGGGCTGGGGAGGAGAAGATGATGACATTTTTAACAGGTAATGGTCATAACTTAGATATCTTT
CTCCTCTGTCAACCTTCACTTCCAGTTTTTTAACCAATGCTTGGTTGTTCCCCAAGGACTGACCCTCAGA
TGGGATGCACCCCTAGTCAGCCCACATTCTTAGGTGTGGCTTCCTACAGGTCCTGCAGGTGCTAAAAG
GGATCTGTAGGAAAATGAGTTTCTGAGATTTTTGTATTGGCCTGGAAAAATGTCAAATGGGAACCAAG
TGACGGGGCAAGTTTACTTTGACTTGCTGCATGCCGTTTTGTACTCAAGGAGTAAACCAATGTCCTTTG
TAAAAATCCCTCCTTTCATTATGGTCCCCTTTCACTGTGAAACAAGTTTCCTTGAGCAGAATCCTAACT
GTCTTCACAGAAGCTTTGTGTTATATTTTTATTTTGGAGTATTTTCACATATACAAAAGAGATACTGTA
GTATAATAAACCTTTGAGGACCTATCCAGCCCCAGCAACCATTATGGCCTGGTCAGTTCTGTCCCATCC
ACATCCTGGGGCTCTTTTTAAGCTGGTAAATCATTATGATGTGGGTTGTCATTTACAGTGGTAAAAAAC ATCTATCAGTAGCATTTGAAAGAACATTCTGCTCAGTCCTCTGGCTGTAGAGGCTTCAACCCCACCAGC
CACCGATGAGCACCTTCTCCCTCCAGGAGCCAGTCTGAGCTCATTACTGAGTTTAATATCAGAATACA
CCCTGGTGCAGCCTTTCTAAATTGCAGTACCAGTTAACAGAAGGTGTCTGTCAGAGCAACACCCAAGT
CATTCAAGTTACCATTGTGTGCAAACTTAACAGAGACCCACGTCTTCAATATAAGCCTTGAAGGAAAC
TCCAGTTTTAGTATGTAGATGGGGTATCAAGTGTGTGCACATTGAACATCTGCTGCATACAGAGCACT
GTGCCAGGCAGGCCCAGGACACTGAAAACCTGGACATAGGGTCCAGACAGAAGCAAGCCTGCTTCCA
CAGAGGCACTCCTGGGCAGACACTCTGGACTGATATGACAGTGTGCAGGGCCGACAGGATACCACAG
GTCTGAATGGTCAGAACAGCTGGGGAGGGAGGGAGCATCCGCAGGCATCTAGTCCCATGCTAACGCA
GTGGCACTAGAAGGATGGGTGGTGTGTGGAGCAACTTTCTTGAAAGATAAAGGACCTAACACTTTCTA
TGCACCACTTACTGTGTGCCAGGCAAGGCCAGGAATGTTTAAGTGGTCTGGGATCAGCCAGTTCTGCC
TCTTAACTAACTTTGCTGTCCTGCTCTCCAGGCTTTCATTTTGGTCCTCATTCCTTTTCCTTGGACCAAC
ACAGAATCCTCCACCCTGTTCTGGCTGCCTCTAGTCTTGTTCTCAGCCCTCCATTTGTTTTTTTCTGCCTT
TTCCCACATGTTCTGAAGCCCTCCATTCGTATACTACTTTCCAGAGACTTCCCCATGGCTAAAAGCATT
TTGGAAATACTGTATATTAGGCCCCTTTCAGATACTGGCAACCGTTTGTGGGATGCTCTGAGAAGGCCT
CTGTGACTTAGCCTGGCCCTTTTCAGCCCATCACCTGCCACGTCCTACCCCAGACCCTTGTCACCAGTC
CCCAGGAGCTTACGTTGCTCCCTGAGGGCACTAGGCTTGCTCTCACTTCCATGCCTTTGCCTGTGCCAT
CCTGGCTGCCCAAAATGCTATGGCAGATACCTGTTCATCCTCAACTGGGCTCTGCCTAGGCTTGCTCCA
GCAGAGGTTACAAACTCTATGCTTCTTCCTCTGTGTCTCCAACCTCATCTTCCTCTTCTCACCTCCATCC
TGGCCCTAAAGGCCCTATGTTTGAAGCATTCACACTGTATATTCTGTGGGGCACACGGCCCCAGTGTCT
GGCACATGGTAGTCAACACCACAAACCGCAGAACCAGTTGTAAAAGGACATGGAGTCGGAATGTGAG
TTTTAACCAGGGTCATGCTGGGCTGGGTTCTGGCATGATGCTGGGTTGTGGGCTGAGTGAGAACAGCA
AGGGTGATGGTGGATGGAGCAACAGTCTTGCAGCCGGGGCTCTCAGGCCAAGTGTATGGCAGCTCTGT
GATAATGACTTTCCCTTTACTCTTTGCAGATTAGTTTTTAGAGGCATGTCTATATCTCGCCCAAATGCTG
TGGTCGGGAGGTGTCGCATGATCCGCCACTCAAGAGACAAGAAAAATGAACCCAATCCTCAGAGGTG
CATTCTTTGTTTATTCATACTCCTTCCCCCTTTAGGATGAGGTAGGCTGCAGGTCCGAGGCTCTGGGCC
TAGAGGGAAATTGAGGTGGTCAGGTTACAGTGGAGAGGGAGGAGGAAGTACGTGTGATGATTTCTTC
TTAAGATTTTTGTTTTAAGACAATCTCCTTGTGCTCTTTTCCTTGTAGGTTTGACCGAATTGCACACACA
AAGGAGACAATGCTCTCTGATGGTTTGAACTCACTCACCTACCAGGTGCTGGATGTACAGAGATACCC
ATTGTATACCCAAATCACAGTGGACATCGGGACACCGAGCTAGCGTTTTGGTACACGGATAAGAGACC
TGAAATTAGCCAGGGACCTCTGCTGTGTGTCTCTGCCAATCTGCTGGGCTGGTCCCTCTCATTTTTACC
AGTCTGAGTGACAGGTCCCCTTCGCTCATCATTCAGATGGCTTTCCAGATGACCAGGACGAGTGGGAT
ATTTTGCCCCCAACTTGGCTCGGCATGTGAATTCTTAGCTCTGCAAGGTGTTTATGCCTTTGCGGGTTTC
TTGATGTGTTCGCAGTGTCACCCCAGAGTCAGAACTGTACACATCCCAAAATTTGGTGGCCGTGGAAC
ACATTCCCGGTGATAGAATTGCTAAATTGTCGTGAAATAGGTTAGAATTTTTCTTTAAATTATGGTTTT
CTTATTCGTGAAAATTCGGAGAGTGCTGCTAAAATTGGATTGGTGTGATCTTTTTGGTAGTTGTAATTT
AACAGAAAAACACAAAATTTCAACCATTCTTAATGTTACGTCCTCCCCCCACCCCCTTCTTTCAGTGGT
ATGCAACCACTGCAATCACTGTGCATATGTCTTTTCTTAGCAAAAGGATTTTAAAACTTGAGCCCTGGA
CCTTTTGTCCTATGTGTGTGGATTCCAGGGCAACTCTAGCATCAGAGCAAAAGCCTTGGGTTTCTCGCA
TTCAGTGGCCTATCTCCAGATTGTCTGATTTCTGAATGTAAAGTTGTTGTGTTTTTTTTTAAATAGTAGT
TTGTAGTATTTTAAAGAAAGAACAGATCGAGTTCTAATTATGATCTAGCTTGATTTTGTGTTGATCCAA
ATTTGCATAGCTGTTTAATGTTAAGTCATGACAATTTATTTTTCTTGGCATGCTATGTAAACTTGAATTT
CCTATGTATTTTTATTGTGGTGTTTTAAATATGGGGAGGGGTATTGAGCATTTTTTAGGGAGAAAAATA
AATATATGCTGTAGTGGCCACAAATAGGCCTATGATTTAGCTGGCAGGCCAGGTTTTCTCAAGAGCAA
AATCACCCTCTGGCCCCTTGGCAGGTAAGGCCTCCCGGTCAGCATTATCCTGCCAGACCTCGGGGAGG
ATACCTGGGAGACAGAAGCCTCTGCACCTACTGTGCAGAACTCTCCACTTCCCCAACCCTCCCCAGGT
GGGCAGGGCGGAGGGAGCCTCAGCCTCCTTAGACTGACCCCTCAGGCCCCTAGGCTGGGGGGTTGTA
AATAACAGCAGTCAGGTTGTTTACCAGCCCTTTGCACCTCCCCAGGCAGAGGGAGCCTCTGTTCTGGT
GGGGGCCACCTCCCTCAGAGGCTCTGCTAGCCACACTCCGTGGCCCACCCTTTGTTACCAGTTCTTCCT
CCTTCCTCTTTTCCCCTGCCTTTCTCATTCCTTCCTTCGTCTCCCTTTTTGTTCCTTTGCCTCTTGCCTGTC
CCCTAAAACTTGACTGTGGCACTCAGGGTCAAACAGACTATCCATTCCCCAGCATGAATGTGCCTTTTA
ATTAGTGATCTAGAAAGAAGTTCAGCCGAACCCACACCCCAACTCCCTCCCAAGAACTTCGGTGCCTA
AAGCCTCCTGTTCCACCTCAGGTTTTCACAGGTGCTCCCACCCCAGTTGAGGCTCCCACCCACAGGGCT
GTCTGTCACAAACCCACCTCTGTTGGGAGCTATTGAGCCACCTGGGATGAGATGACACAAGGCACTCC
TACCACTGAGCGCCTTTGCCAGGTCCAGCCTGGGCTCAGGTTCCAAGACTCAGCTGCCTAATCCCAGG
GTTGAGCCTTGTGCTCGTGGCGGACCCCAAACCACTGCCCTCCTGGGTACCAGCCCTCAGTGTGGAGG
CTGAGCTGGTGCCTGGCCCCAGTCTTATCTGTGCCTTTACTGCTTTGCGCATCTCAGATGCTAACTTGG
TTCTTTTTCCAGAAGCCTTTGTATTGGTTAAAAATTATTTTCCATTGCAGAAGCAGCTGGACTATGCAA
AAAGTATTTCTCTGTCAGTTCCCCACTCTATACCAAGGATATTATTAAAACTAGAAATGACTGCATTGA
GAGGGAGTTGTGGGAAATAAGAAGAATGAAAGCCTCTCTTTCTGTCCGCAGATCCTGACTTTTCCAAA
GTGCCTTAAAAGAAATCAGACAAATGCCCTGAGTGGTAACTTCTGTGTTATTTTACTCTTAAAACCAA
ACTCTACCTTTTCTTGTTGTTTTTTTTTTTTTTTTTTTTTTTTTTTTGGTTACCTTCTCATTCATGTCAAGTA
TGTGGTTCATTCTTAGAACCAAGGGAAATACTGCTCCCCCCATTTGCTGACGTAGTGCTCTCATGGGCT CACCTGGGCCCAAGGCACAGCCAGGGCACAGTTAGGCCTGGATGTTTGCCTGGTCCGTGAGATGCCGC GGGTCCTGTTTCCTTACTGGGGATTTCAGGGCTGGGGGTTCAGGGAGCATTTCCTTTTCCTGGGAGTTA TGACCGCGAAGTTGTCATGTGCCGTGCCCTTTTCTGTTTCTGTGTATCCTATTGCTGGTGACTCTGTGTG AACTGGCCTTTGGGAAAGATCAGAGAGGGCAGAGGTGGCACAGGACAGTAAAGGAGATGCTGTGCTG GCCTTCAGCCTGGACAGGGTCTCTGCTGACTGCCAGGGGCGGGGGCTCTGCATAGCCAGGATGACGGC TTTCATGTCCCAGAGACCTGTTGTGCTGTGTATTTTGATTTCCTGTGTATGCAAATGTGTGTATTTACCA TTGTGTAGGGGGCTGTGTCTGATCTTGGTGTTCAAAACAGAACTGTATTTTTGCCTTTAAAATTAAATA ATATAACGTGAATAAATGACCCTATCTTTGTAACTGCAGGTGGTTTCTGTTTGCCAGGTGTAAGGGTTG TCATGGCTGTGGGATGGGGTGGGGACAGGGTCATTCCCTGGTCTGTGACCCATACAAATACACATGCC TCCCTGGAATCAGACATTTCCCCATCTGAACTTCATTCTCTTATCTGTAAAATGGGAATAATAACACAT AGGGACTTTTTTGAGGCTTAAAAGTGACGATATATGTAAAACAATGACTAATGCCTCACAAGTACTCA CTACATAGTAGCTAGTGCCATTTCAAAGTAGAATTTTTTTCCCCTAGCAGTTCTTGGGCCACATTCTGC TATTTTCAACAGATACCAGGATCATTCAGATGTAGATCTCAGGGCCATTTGCACCAGGTGCTCACAGT GTAACTTGAAGGGAATTATCCAAAATGAGGTTTCTTGTCAGTCTCAGGAAATGTAACCATAAGCTCTA AAAGGTCTTAGTTTTTACCCAGGTGCCTCCTCCTTGGTGGCCCTGGGTCAGGCTGGTTGGATTGAATTG GCACTCCTGAAGAAGGGCTGCAGGAAACCAGTGAGCAGGAGAGCCACCCTTGGCAGGGAGCTGCAGG CCCTGCCTGCATGTCACTGCTGGAGGGATCCCTGGTGACCTCAGGCCTGTGCAAAGGTGGCCTGGGGT TCAGATCTGGCCTTCAAACAGGACAACTCTGGTCCTTTGGACAAAATGCTGCCTTAGAGGGTCTGACA AAATTAAAAACAAACAAAAAAAAACCTGTTTCTTTCCTTCTCACACACCACCACTCACAACACTTCAG TTCTGCCCCTAGATATGTAGGGATTTCTCCCCACCAACAAGCAGTTTTCTAGTGGACACTAGCTGGGTG TCCTACAGTTTAACTCAATTCTGACACTGTCTGCCTGGAGATAGCAACGGATCCCACAGGTTGAGGGC TCAGTCTCACAAGACTGCCTCCACTGCAGATGCCAGTCACAAGTAGTTGGTTGTGACCTATGCTTTACA AAAATGTTTTTTGGATACAGGGCCTTGCTGTGTCACCCAGGCTGGCCTGAAACTCCTGGGCTCACACA ATCCTCCCGCCACAACTTAGAAGTAGCTGAGCTGCAGGTTTATACCACTCACCCAGCTATAGTTGTGA CCTATACTTCTGACCAACCAGCTATAAATTGGGGTTTCTATGAGCCTCTTCTTGGGTTTAATTTGCTAG GTCAGCTTACAGAACTCAGTGTAACACTTAACATTTACTGGTCTTATTATAAGTGATATTAGAAAGGAT ACTGATGAAGAACCGGATGGAGAGATGCATAGGGCAAGGCATGGGGGAGGGGGAGAGAAGCTTCCA TGCCCTCTCCAGGGGCTCCACCCTCCAGACACCTCCACGTGTTCAGCTATCTGGAAGCTCATCTGACCC TGTCCTTCTGGTTTTTATGGAAGCTTCATCACATAGGCCTGATAGACTACATCATCGGCCATTGCCAGT CAGCTCAACCTTCAGCCCTTTTCCCCTTCCTGAAGGATGGGAGTGGGACTGAAAGTGCCAACCTTCTCA TCATGGCTTGGTCTTTCTGGTGACCAGTCCCCATCCAGGAGTTCACTGAGAATCATTTCATTAAAACAA AAGACGTTCCTATCACCCGGGAAATTCCAAGGGATTAGAAGCTCTGTCAGGAACCAGGGTCAAGCAC CAAATATTAGAACAAAAGATTCTCCTAGCATAAATATTAGAACAAAAGATTCTCCTAGCATAAATATT AGAACAAAAAATTCTCCTATTGCTCAGGAAATTATAAGAGTTTTAGGGGCTCTGTACCAGGAACCCAG CGCAGAGGCCAAATATATATATTTTATTATCTCACAGTGCCACACAGGACTTTGCAAGCTGTCAGGTCT GAGTGAGATGGAGCACACCAGTGAAAGGTTAAGTTCACCCTTTCACTGATGTGCTCCACTTCACTGAG ACACATATCCACACAGACACACAGAGACACACACATCCACCCAGACGCACGCA
[00396] Following Table 1 provides oligonucleoside mRNA target sequences of B4GALT1, together with the corresponding positions in transcript NM 001497.4. It is to be understood that SEQ ID NO: 2 to 20, 102 to 201 and 622 to 680 refer to human (Homo sapiens) mRNA sequences.
Table 1
Figure imgf000168_0001
Figure imgf000169_0001
Figure imgf000170_0001
Figure imgf000171_0001
Figure imgf000172_0001
Figure imgf000173_0001
[00397] Table 2 provides the unmodified first (antisense) and corresponding unmodified second (sense) strand sequences for siRNA oligonucleosides according to the present invention, together with the corresponding positions in the overall gene sequence of SEQ ID NO: 1 as follows.
Table 2
Figure imgf000173_0002
Figure imgf000174_0001
Figure imgf000175_0001
Figure imgf000176_0001
Figure imgf000177_0001
Figure imgf000178_0001
Figure imgf000179_0001
Figure imgf000180_0001
Figure imgf000181_0001
Figure imgf000182_0001
[00398] Table 3 provides the modified first (antisense) sequences, together with the corresponding unmodified first (antisense) sequences for siRNA oligonucleosides according to the present invention as follows.
Table 3
Figure imgf000182_0002
Figure imgf000183_0001
Figure imgf000184_0001
Figure imgf000185_0001
Figure imgf000186_0001
Figure imgf000187_0001
Figure imgf000188_0001
Figure imgf000189_0001
Figure imgf000190_0001
Figure imgf000191_0001
Figure imgf000192_0001
Figure imgf000193_0001
[00399] Table 4 provides the modified second (sense) sequences, together with the corresponding unmodified second (sense) sequences for siRNA oligonucleosides according to the present invention as follows.
Table 4
Figure imgf000193_0002
Figure imgf000194_0001
Figure imgf000195_0001
Figure imgf000196_0001
Figure imgf000197_0001
Figure imgf000198_0001
Figure imgf000199_0001
Figure imgf000200_0001
Figure imgf000201_0001
Figure imgf000202_0001
Figure imgf000203_0001
[00400] Some of the modified second strand sequences as illustrated above in Table 4 include the preferred 5’ iaia motif. However, it should also be understood that the scope of these modified second strand sequences additionally includes the Me / F modified second strand in the absence of the 5’iaia motif.
[00401] Table 5 identifies duplexes with Duplex IDs referencing the modified antisense and sense IDs from previous Tables 3 and 4.
Table 5
Figure imgf000203_0002
Figure imgf000204_0001
Figure imgf000205_0001
Figure imgf000206_0001
Figure imgf000207_0001
Figure imgf000208_0001
[00402] Definitions as provided in the above Tables:
A - adenosine
C - cytidine
G - guanosine
T - thymidine m - 2’-O-methyl f - 2’fluro s - phosphorothioate bond o - thermally destabilised nucleoside ia - inverted abasic nucleoside
Example 9: Inhibition Screen for B4GALT1 Expression in Human Huh7 Cells
[00403] Huh7 cells (human hepatocyte-derived cell line, obtained from JCRB Cell Bank) were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS at 37°C in at atmosphere of 5% CO2. Cells were transfected with siRNA duplexes targeting B4GALT1 mRNA or a negative control siRNA (siRNA-control; sense strand 5’- UUCUCCGAACGUGUCACGUTT-3 (SEQ ID NO:934)’, antisense strand 5’- ACGUGACACGUUCGGAGAATT-3’ (SEQ ID NO:933)) at a final duplex concentration of 5 nM and 0.1 nM. Transfection was carried out by adding 9.7 μL Opti-MEM (ThermoFisher) plus 0.3 μL Lipofectamine RNAiMAX (ThermoFisher) to 10 μL of each siRNA duplex. The mixture was incubated at room temperature for 15 minutes before being added to 100 μL of complete growth medium containing 20,000 Huh7 cells. Cells were incubated for 24 hours at 37°C/5% CO2 prior to total RNA purification using a RNeasy 96 Kit (Qiagen). Each duplex was tested by transfection in duplicate wells in two independent experiments.
[00404] cDNA synthesis was performed using FastQuant RT (with gDNase) Kit (Tiangen). Real-time quantitative PCR (qPCR) was performed on an ABI Prism 7900HT or ABI QuantStudio 7 with primers specific for human B4GALT1 and human GAPDH (Hs02786624_gl) using FastStart Universal Probe Master Kit (Roche).
[00405] qPCR was performed in duplicate on cDNA derived from each well and the mean Ct calculated. Relative B4GALT1 expression was calculated from mean Ct values using the comparative Ct (AACt) method, normalised to GAPDH and relative to untreated cells. Based on the results of primary screen, siRNA duplexes displaying good activity were selected for dose-response follow-up. Results are shown in Figures 9 and 10. Sequences of RNAi molecules are depicted in Table 5.
Table 6. B4GALT1 relative mRNA expression
Figure imgf000209_0001
Figure imgf000210_0001
Figure imgf000211_0001
Figure imgf000212_0001
Example 10: Dose-response for Inhibition of B4GALT1 in Human Huh7 Cells [00406] Huh7 cells (human hepatocyte-derived cell line, obtained from JCRB Cell Bank) were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS at 37°C in at atmosphere of 5% CO2. Cells were transfected with siRNA duplexes targeting B4GALT1 mRNA or a negative control siRNA (siRNA-control; sense strand 5’- UUCUCCGAACGUGUCACGUTT-3’ (SEQ ID NO:934), antisense strand 5’- ACGUGACACGUUCGGAGAATT-3’ (SEQ ID NO:933)) using 10x3-fold serial dilutions over a final duplex concentration range of 20 nM to 1 pM. Transfection was carried out by adding 9.7 μL Opti-MEM (ThermoFisher) plus 0.3 μL Lipofectamine RNAiMAX (ThermoFisher) to 10 μL of each siRNA duplex. The mixture was incubated at room temperature for 15 minutes before being added to 100 μL of complete growth medium containing 20,000 Huh7 cells. Cells were incubated for 24 hours at 37°C/5% CO2 prior to total RNA purification using a RNeasy 96 Kit (Qiagen). Each duplex was tested by transfection in duplicate wells in a single experiment.
[00407] cDNA synthesis was performed using FastQuant RT (with gDNase) Kit (Tiangen). Real-time quantitative PCR (qPCR) was performed on an ABI Prism 7900HT or ABI QuantStudio 7 with primers specific for human B4GALT1 (Hs00155245_ml) and human GAPDH (Hs02786624_gl) using a TaqMan Gene Expression Assay Kit (ThermoFisher Scientific).
[00408] qPCR was performed in duplicate on cDNA derived from each well and the mean Ct calculated. Relative B4GALT1 expression was calculated from mean Ct values using the comparative Ct (AACt) method, normalised to GAPDH and relative to untreated cells. Maximum percent inhibition of B4GALT1 expression and IC50 values were calculated using a four parameter (variable slope) model using GraphPad Prism 9. Results are shown in Figure 11. Sequences of RNAi molecules are depicted in the relevant Tables herein.
Example 11: Dose-response for Inhibition of B4GALT1 in Human Huh7 Cells
[00409] Huh7 cells (human hepatocyte-derived cell line, obtained from JCRB Cell Bank) were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS at 37°C in at atmosphere of 5% CO2. Cells were transfected with siRNA duplexes designed against the target or a negative control siRNA at O.lnM and InM. Transfection was carried out by adding 9.7 μL Opti-MEM (ThermoFisher) plus 0.3 μL Lipofectamine RNAiMAX (ThermoFisher) to 10 μL of each siRNA duplex. The mixture was incubated at room temperature for 15 minutes before being added to 100 μL of complete growth medium containing 20,000 Huh7 cells. Cells were incubated for 24 hours at 37°C/5% CO2 prior to total RNA purification using a RNeasy 96 Kit (Qiagen). Each duplex was tested by transfection in duplicate wells and the experiment was repeated three time.
[00410] cDNA synthesis was performed using FastKing RT kit (with gDNase) Kit (Tiangen). Real-time quantitative PCR (qPCR) was performed on an ABI Prism 7900HT or ABI QuantStudio 7 with primers specific for human B4GALT1 (Hs00155245_ml) and human GAPDH (Hs02786624_gl) using a TaqMan Gene Expression Assay Kit (ThermoFisher Scientific).
[00411] qPCR was performed in duplicate on cDNA derived from each well and the mean Ct calculated. Relative target expression was calculated from mean Ct values using the comparative Ct (AACt) method, normalised to GAPDH and relative to untreated cells.
[00412] For the inhibition of B4GALT1, the siRNA duplexes ETXM1200, ETXM1201, ETXM1217, ETXM1764, ETXM1765, ETXM1766, ETXM1767, ETXM1768, ETXM1769 and ETXM1770 were tested (Fig.13).
[00413] In a second experiment, the siRNA duplexes ETXM1203, ETXM1204, ETXM1218, ETXM1772, ETXM1773, ETXM1774, ETXM1775, ETXM1776, ETXM1777 and ETXM1778 were tested for inhibition of B4GALTl(Fig.l4).
Example 12: B4GALT1 pharmacology study
[00414] ETX in-house computational biology analysis identified B4GALT1, encoding beta-1, 4- galactosyltransferase 1, as a gene associated with Type 2 Diabetes (T2D). Here, the inventors establish B4GALT1 as a potential therapeutic target for T2D. In-silico--designed GalNAc- siRNAs targeting mouse hepatic B4GALT1 were synthesized and tested to access the plausibility of the hypothesis that a significant knockdown of hepatic B4GALT1 mRNA lowers the plasma levels of LDL-c, fibrinogen, and fasting glucose.
[00415] In vitro dose-response assay to select potent molecules
In vitro dose-response assay measuring the gene knockdown in primary mouse hepatocytes (PMHs) was performed to test 20 GalNAc-siRNAs targeting hepatic B4GALT1. Primary C57BL/6 mouse hepatocytes (PMHs) were isolated fresh by two-step collagenase liver perfusion. Cells were maintained in DMEM (Gibco-11995-092) supplemented with FBS, Penicillin/Streptomycin, HEPES and L-glutamine. Cells were cultured at 37°C in an atmosphere with 5% CO2 in a humidified incubator. Within 2 hours post isolation, PMHs were seeded at a density of 36,000 cells/well in regular 96-well tissue culture plates. Dose response analysis in PMHs was done by direct incubation of cells in a gymnotic free uptake setting with final GalNAc-siRNA concentrations of 1000, 500, 250, 125, 62.5, 31.3, 15.6, 7.8, 3.9, 1.95 nM. In control wells, cells were incubated without GalNAc-siRNA. After 48hr incubation, cells were harvested for RNA extraction. Total RNA was extracted using RNeasy Kit following the manufacturer’s instructions (Qiagen, Shanghai, China). After reverse transcription, real-time quantitative PCR was performed using an ABI Prism 7900HT to detect the relative abundance of B4GALT1 mRNA normalized to the housekeeping gene GAPDH. The expression of the target gene in each test sample was determined by relative quantitation using the comparative Ct (AACt) method. This method measures the Ct differences (ACt) between target gene and housekeeping gene. The formula is as follows: ACt = average Ct of B4GALT1 -average Ct of GAPDH, AACt=ACt (sample) - average ACt (untreated control), relative expression of target gene mRNA = 2'AACt. Based on the results of in vitro free uptake experiment, GalNAc-siRNAs displaying good activity were selected for EC50 determination using a 10-point concentration curve (Figure 15).
[00416] In vivo pharmacology with four selected GalNAc-siRNAs
The pharmacodynamic activity of four selected B4GALT1 GalNAc-siRNAs was measured in vivo. Twelve C57BL/6 male mice were allocated for each of GalNAc-siRNAs, ETXM619, ETXM624, ETXM628 and ETXM633. 5 mice were allocated as no-treatment control group. Mice were subcutaneously dosed with ETXMs (10 mg/kg) on day 0, defined as the day mice were first dosed, day 3 and day 7. 3 mice in each treatment group were sacrificed on day 3, day 7, day 10 and day 14. Upon termination, liver tissues and plasma samples were harvested for further analysis. Day 3 samples were used to evaluate the single dose effect of ETXMs given on day 0. Day 7 samples represent the repeat dose effect of ETXMs given on day 0 and day3. Likewise, day 10 and day 14 samples represent the repeat dose effect of ETXMs given on day 0, day 3 and day 7. 5 mice allocated as the control group were sacrificed on day 14.
[00417] B4GALT1 gene knockdown in mouse liver
Harvested liver samples were used to measure the B4GALT1 mRNA knockdown level by RT-qPCR. Upon collection, each tissue was treated with RNAlater and stored at 4°C overnight then at -80°C until the further analysis. Liver tissues were homogenized with TRIZOL for RNA extraction. RNA samples, adjusted to 400 ng/μL, were reverse transcribed to cDNA using FastKing RT Kit, manufactured by TIANGEN. After gDNA removal procedure, purified cDNA samples were used for RT-qPCR. RT-qPCR method and the relative mRNA expression calculations are as described above. Figure 16 shows that all test articles exhibit > 50% gene knockdown efficiency at day 3, 7, 10 and 14.
[00418] Terminal plasma collection and measurement of plasma biomarkers using biochemical analyser
The terminal plasma samples were collected via submandibular vein after 4-5 hour fasting. Blood samples were collected in heparin sodium coated tubes then centrifuged at 7,000g at 4°C for 10 min to obtain plasma samples. The plasma samples were used for the measurements of AST, ALT, albumin, ALP, BUN, CREA, TBIL, glucose, total cholesterol, LDL-c, HDL-c, triglycerides and NEFA (free fatty acids) by a biochemical analyser.
[00419] Measurement of plasma insulin and fibrinogen levels using ELISA kits
Blood samples were collected in K2EDTA coated tubes then centrifuged at 7,000g at 4°C for 10 minutes to obtain plasma samples. The plasma insulin level was measured using Mouse Insulin ELISA kit (Mercodia, 10-1247-01) according to the manufacturer’s protocol. The fibrinogen plasma level was measured using Mouse Fibrinogen Antigen Assay kit (Innovative Research, IMSFBGKTT).
[00420] B4GALT1 gene silence effect in biomarker modulation
The means of the untreated control group (n=5) and the day-14 treatment group comprising groups administered with ETXM619, ETXM624, ETXM628 or ETXM633 subcutaneously at day 0, day 3 and day7 (n=3 per group, n=12 total) were tested for equality under the null hypothesis via a two-tailed t-test. Statistically significant differences in the means of efficacy biomarker readouts were detected with an 18.8% decrease in LDL-C (p<0.05); a 21.0% decrease in fasting glucose (p<0.05); and a 29.6% decrease in fibrinogen (p<0.01 ) (Figure 17).
[00421] The present invention is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the present disclosure.
[00422] In case of ambiguity between the sequences in this specification and the sequences in the attached sequence listing, the sequences provided herein are considered to be the correct sequences.

Claims

New PCT Patent Application based on US 63/369,619 e-therapeutics pic Voss.-Ref. : AF2698 PCT BS CLAIMS
1. A nucleic acid for inhibiting expression of B4GALT1, 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 B4GALT1 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.
2. A nucleic acid for inhibiting expression of B4GALT1, 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 B4GALT1 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.
3. A nucleic acid according to claim 1 or 2, wherein the first strand comprises nucleosides 2-18 of any one of the sequences defined in claim 1 or 2, in particular wherein the first strand comprises nucleosides 2-18 of any one of the sequences defined in Tables 2 or 3.
4. A nucleic acid according to claim 1, 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 claim 2, 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 claim 1, wherein the first strand comprises any one of the first strand sequences as listed in Table 2. A nucleic acid according to claim 2, wherein the first strand comprises any one of the first strand modified sequences as listed in Table 3. A nucleic acid according to claim 4, wherein the second strand comprises any one of the second strand sequences as listed in Table 2. A nucleic acid according to claim 5, wherein the second strand comprises any one of the second strand modified sequences as listed in Table 4. A nucleic acid according to claim 6, wherein the first strand comprises any one of the following sequences: SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41. A nucleic acid according to claim 7, wherein the first strand comprises any one of the following sequences: SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81. A nucleic acid according to claim 8, wherein the second strand comprises any one of the following sequences: SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61. A nucleic acid according to claim 9, wherein the second strand comprises any one of the following sequences: SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85,
SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90,
SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95,
SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100,
SEQ ID NO: 101. A nucleic acid according to claims 1 and 4, comprising first and second strands that comprises, consists of, or consists essentially of a nucleoside sequence differing by 0 or 1 nucleosides from any one of the following first and second sequences:
Figure imgf000220_0001
Figure imgf000221_0001
A nucleic acid according to claims 1 and 4, comprising first and second strands that comprises, consists of, or consists essentially of a nucleoside sequence differing by 0 or 1 nucleosides from any one of the following first and second sequences:
Figure imgf000221_0002
A nucleic acid according to claims 1 and 4, comprising first and second strands that comprises, consists of, or consists essentially of a nucleoside sequence differing by 0 or 1 nucleosides from any one of the following first and second sequences:
Figure imgf000221_0003
A nucleic acid according to claims 2 and 5, comprising first and second strands that comprises, consists of, or consists essentially of a nucleoside sequence differing by 0 or 1 nucleosides from any one of the following first and second sequences:
Figure imgf000221_0004
Figure imgf000222_0001
A nucleic acid according to claims 2 and 5, comprising first and second strands that comprises, consists of, or consists essentially of a nucleoside sequence differing by 0 or 1 nucleosides from any one of the following first and second sequences:
Figure imgf000222_0002
A nucleic acid according to claims 2 and 5, comprising first and second strands that comprises, consists of, or consists essentially of a nucleoside sequence differing by 0 or 1 nucleosides from any one of the following first and second sequences:
Figure imgf000222_0003
Figure imgf000223_0001
A nucleic acid according to claims 2 and 5, comprising first and second strands that comprises, consists of, or consists essentially of a nucleoside sequence differing by 0 or 1 nucleosides from any one of the following first and second sequences:
Figure imgf000223_0002
A nucleic acid according to claims 2 and 5, comprising first and second strands that comprises, consists of, or consists essentially of a nucleoside sequence differing by 0 or 1 nucleosides from any one of the following first and second sequences:
Figure imgf000223_0003
A nucleic acid according to any preceding claim, wherein the first strand has a length in the range of 17 to 30 nucleosides, preferably 19 to 25 nucleosides, more preferably 19 or 23 nucleosides. A nucleic acid according to any preceding claim, wherein the second strand has a length in the range of 17 to 30 nucleosides, preferably 19 to 25 nucleosides, more preferably 19 or 21 nucleosides. A nucleic acid according to any preceding claim, wherein the duplex region of the nucleic acid is between 17 and 30 nucleosides in length, more preferably is 19 or 21 nucleosides in length. A nucleic acid according to any preceding claim, wherein the region of complementarity between the first strand and the portion of RNA transcribed from the B4GALT1 gene is between 17 and 30 nucleosides in length. A nucleic acid according to any preceding claim, wherein the nucleic acid is either blunt ended, or comprises one or more single-stranded nucleoside overhangs, optionally wherein the overhang is present on the first or second strand, preferably at the 3’ terminus of the first or second strand, and/or wherein the overhang comprises 1 to 4 nucleosides, more preferably 2 nucleosides. A nucleic acid according to any preceding claim, wherein the nucleic acid is an siRNA oligonucleoside. A nucleic acid according to any preceding claim, wherein one or more nucleosides on the first and/or second strand are modified, to form modified nucleosides. A nucleic acid according to claim 28, wherein one or more nucleosides on the first and/or second strand comprise terminal modifications, base modifications, sugar modifications and/or backbone modifications. A nucleic acid according to claim 28, wherein one or more nucleosides on the first and/or second strand comprise sugar modifications, wherein the modification is a modification at the 2’ -OH group of the ribose sugar. A nucleic acid according to claim 30, wherein the sugar modifications comprise 2’-Me and/or 2’-F modifications. A nucleic acid according to claim 28, wherein the first strand comprises a 2’-F modification at any of position 2, position 6, position 14, or any combination thereof, counting from position 1 of said first strand. A nucleic acid according to claim 28, wherein the second strand comprises a 2’-F modification at any of position 7, position 9, position 11, or any combination thereof, counting from position 1 of said second strand. A nucleic acid according to claim 28, wherein the first and second strand each comprise 2'-Me and 2’-F modifications. A nucleic according to claim 28, wherein the nucleic acid comprises at least one thermally destabilizing modification, suitably at one or more of positions 1 to 9 of the first strand counting from position 1 of the first strand, and / or at one or more of positions on the second strand aligned with positions 1 to 9 of the first strand, wherein the destabilizing modification is selected from a modified unlocked nucleic acid (UNA) and a glycol nucleic acid (GNA), preferably a glycol nucleic acid, more preferably an (S)- glycol nucleic acid, wherein more preferably the nucleic acid comprises at least one thermally destabilizing modification at position 7 of the first strand, counting from position 1 of the first strand. A nucleic acid according to claim 28, which is an siRNA oligonucleoside, wherein the siRNA oligonucleoside comprises at least 3 2’-F modifications at positions 6 to 12 of the second strand, such as 4, 5, 6 or 7 2’-F modifications at positions 6 to 12 of the second strand, counting from position 1 of said second strand. A nucleic acid according to claim 28, which is an siRNA oligonucleoside, wherein said second strand comprises at least 3, such as 4, 5 or 6, 2’ -Me modifications at positions 1 to 6 of the second strand, counting from position 1 of said second strand. A nucleic acid according to claim 28, which is an siRNA oligonucleoside, wherein said first strand comprises at least 5 2’ -Me consecutive modifications at the 3’ terminal region, preferably including the terminal nucleoside at the 3’ terminal region, or at least within 1 or 2 nucleosides from the terminal nucleoside at the 3’ terminal region. A nucleic acid according to claim 28, which is an siRNA oligonucleoside, wherein said first strand comprises 7 2’ -Me consecutive modifications at the 3’ terminal region, preferably including the terminal nucleoside at the 3’ terminal region. A nucleic acid according to claim 28, which is an siRNA oligonucleoside, wherein each of the first and second strands comprises an alternating modification pattern, preferably a fully alternating modification pattern along the entire length of each of the first and second strands, wherein the nucleosides of the first strand are modified by (i) 2’Me modifications on the odd numbered nucleosides counting from position 1 of the first strand, and (ii) 2’F modifications on the even numbered nucleosides counting from position 1 of the first strand, and nucleosides of the second strand are modified by (i) 2’F modifications on the odd numbered nucleosides counting from position 1 of the second strand, and (ii) 2’Me modifications on the even numbered nucleosides counting from position 1 of the second strand. A nucleic acid according to claim 28, which is an siRNA oligonucleoside, wherein nucleosides of said first strand comprise a 2’ sugar modification pattern wherein said modifications are selected at least from 2’Me and 2’F sugar modifications, provided that the overall number of 2’F sugar modifications in the first strand does not consist of four, or six, 2’F modifications A nucleic acid according to claim 28, which is an siRNA oligonucleoside, wherein nucleosides of said first strand comprise a 2’ sugar modification pattern wherein said modifications are selected at least from 2’Me and 2’F sugar modifications, wherein the overall number of 2’F sugar modifications in the first strand consists of three, five or seven 2’F modifications. A nucleic acid according to any preceding claim, which further 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 intemucleoside linkage. A nucleic acid according to claim 43, wherein the second strand comprises 2 consecutive abasic nucleosides in the 5’ terminal region of the second strand, wherein one such abasic nucleoside is a terminal nucleoside at the 5’ terminal region of the second strand and the other abasic nucleoside is a penultimate nucleoside at the 5’ terminal region of the second strand, wherein:
(a) said penultimate abasic nucleoside is connected to an adjacent first basic nucleoside in an adjacent 5’ near terminal region through a reversed internucleoside linkage; and
(b) the reversed linkage is a 5-5’ reversed linkage; and
(c) the linkage between the terminal and penultimate abasic nucleosides is 3’5’ when reading towards the terminus comprising the terminal and penultimate abasic nucleosides. A nucleic acid according to claim 43, wherein 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. A nucleic acid according to claim 44, wherein
(i) the first strand and the second strand each has a length of 23 nucleosides;
(ii) two phosphorothioate internucleoside linkages are respectively between three consecutive positions in said 5’ near terminal region of the second strand, wherein a first phosphorothioate intemucleoside 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 intemucleoside linkage is present between said adjacent second basic nucleoside and an adjacent third basic nucleoside in said 5’ near terminal region of the second strand;
(iii) two phosphorothioate intemucleoside 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 penultimate nucleoside by a phosphorothioate intemucleoside linkage, and each first 5’ and 3’ penultimate nucleoside is attached to a respective 5’ and 3’ adjacent antepenultimate nucleoside by a phosphorothioate intemucleoside linkage; and
(iv) the second strand of the nucleic acid is conjugated directly or indirectly to one or more ligand moi eties at the 3’ terminal region of the second strand. A nucleic acid according to claim 45, wherein
(i) the first strand and the second strand each has a length of 23 nucleosides;
(ii) two phosphorothioate intemucleoside linkages are respectively between three consecutive positions in said 3’ near terminal region of the second strand, wherein a first phosphorothioate intemucleoside 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 intemucleoside 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;
(iii) two phosphorothioate intemucleoside 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 penultimate nucleoside by a phosphorothioate intemucleoside linkage, and each first 5’ and 3’ penultimate nucleoside is attached to a respective 5’ and 3’ adjacent antepenultimate nucleoside by a phosphorothioate intemucleoside linkage; and
(iv) the second strand of the nucleic acid is conjugated directly or indirectly to one or more ligand moi eties at the 5’ terminal region of the second strand. A nucleic acid according to any preceding claim, wherein the nucleic acid comprises one or more phosphorothioate intemucleoside linkages. A nucleic acid according to claim 48, wherein said one or more phosphorothioate intemucleoside linkages are respectively between at least three consecutive positions in a 5’ or 3’ near terminal region of the second strand, whereby said near terminal region is preferably adjacent said terminal region wherein one or more abasic nucleosides of said second strand is / are located according to at least claim 45. A nucleic acid according to claim 48 or 49, wherein said one or more phosphorothioate internucleoside linkages are respectively between at least three consecutive positions in a 5’ and / or 3’ terminal region of the first strand, whereby preferably a terminal position at the 5’ and / or 3’ terminal region of said first strand is attached to its adjacent position by a phosphorothioate internucleoside linkage. A nucleic acid according to claim 31, wherein said modified nucleosides of said second strand comprise a modification pattern according to any one of the following (5’-3 ’):
Me - Me - Me - Me - Me - Me - F - F - F - F - F - Me - Me - Me - Me - Me - Me - Me - F
- Me - Me, or
Me - Me - Me - Me - Me - F - F - Me - F - F - F - F - Me - Me - Me - Me - Me - Me - Me
- Me - Me, or
Me - Me - Me - Me - Me - Me -F- Me - F - F - F - F - Me - Me - Me - Me - Me - Me - Me
- Me - Me, or
Me - Me - Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me
- Me - Me - Me, or
Me - Me - Me - Me - Me - Me - F - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me. A nucleic acid according to claim 31, wherein said modified nucleosides of said second strand comprise a modification pattern according to any one of the following (5’-3 ’):
(Me)8 - (F)3 - (Me)10. A nucleic acid according to claim 51, wherein said modified nucleosides of said second strand comprise a modification pattern according to any one of the following (5’-3 ’):
Me(s)Me(s)Me - Me - Me - Me - F - F - F - F - F - Me - Me - Me - Me - Me - Me - Me - F - Me - Me, or Me(s)Me(s)Me - Me - Me - F - F - Me - F- F- F- F- Me - Me - Me - Me - Me - Me - Me - Me - Me, or
Me(s)Me(s)Me - Me - Me - Me -F- Me -F-F-F-F- Me - Me - Me - Me - Me - Me - Me - Me - Me, or
Me(s)Me(s)Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me, or
Me(s)Me(s)Me - Me - Me - Me - F - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me
- Me - Me - Me, or
Me - Me - Me - Me - Me - Me -F-F-F-F-F- Me - Me - Me - Me - Me - Me - Me - F(s)Me(s)Me, or
Me - Me - Me - Me - Me - F - F - Me - F- F- F- F- Me - Me - Me - Me - Me - Me - Me(s)Me(s)Me, or
Me - Me - Me - Me - Me - Me -F- Me - F- F- F- F- Me - Me - Me - Me - Me - Me - Me(s)Me(s)Me, or
Me - Me - Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me -
Me - Me(s)Me(s)Me, or
Me - Me - Me - Me - Me - Me - F - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me(s)Me(s)Me, wherein (s) is a phosphorothioate intemucleoside linkage. A nucleic acid according to claim 52, wherein said modified nucleosides of said second strand comprise a modification pattern according to any one of the following (5’-3’):
Me(s)Me(s) (Me)6 - (F)3 - (Me)10 wherein (s) is a phosphorothioate intemucleoside linkage. A nucleic acid according to claim 51, wherein said modified nucleosides of said second strand comprise a modification pattern according to any one of the following (5’-3’): ia - ia - Me - Me - Me - Me - Me - Me -F-F-F-F-F- Me - Me - Me - Me - Me - Me - Me - F - Me - Me, or ia - ia - Me - Me - Me - Me - Me - F - F - Me -F-F-F-F- Me - Me - Me - Me - Me -
Me - Me - Me - Me, or ia - ia - Me - Me - Me - Me - Me - Me -F- Me - F- F- F- F- Me - Me - Me - Me - Me - Me - Me - Me - Me, or ia - ia - Me - Me - Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me, or ia - ia - Me - Me - Me - Me - Me - Me - F - Me - F - F - F - Me - Me - Me - Me - Me -
Me - Me - Me - Me - Me, or
Me - Me - Me - Me - Me - Me - F- F- F- F- F- Me - Me - Me - Me - Me - Me - Me - F
- Me - Me - ia - ia, or
Me - Me - Me - Me - Me - F - F - Me - F- F- F- F- Me - Me - Me - Me - Me - Me - Me
- Me - Me - ia - ia, or
Me - Me - Me - Me - Me - Me -F- Me - F- F- F- F- Me - Me - Me - Me - Me - Me - Me
- Me - Me - ia - ia, or
Me - Me - Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me
- Me - Me - Me - ia - ia, or
Me - Me - Me - Me - Me - Me - F - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me- ia - ia, wherein ia represents an inverted abasic nucleoside, and when the inverted abasic nucleosides as represented by ia - ia are present at the 3’ terminus of the second strand, said inverted abasic nucleosides are present in a 2 nucleoside overhang. A nucleic acid according to claim 52, wherein said modified nucleosides of said second strand comprise a modification pattern according to any one of the following (5’-3’): ia-ia- (Me)8 - (Ff - (Me)10, wherein ia represents an inverted abasic nucleoside. A nucleic acid according to claim 51, wherein said modified nucleosides of said second strand comprise a modification pattern according to any one of the following (5’-3’): ia-ia - Me(s)Me(s)Me - Me - Me - Me -F-F-F-F-F- Me - Me - Me - Me - Me - Me - Me - F - Me - Me, or ia-ia - Me(s)Me(s)Me - Me - Me - F - F - Me -F-F-F-F- Me - Me - Me - Me - Me - Me - Me - Me - Me, or ia-ia - Me(s)Me(s)Me - Me - Me - Me -F- Me - F- F- F- F- Me - Me - Me - Me - Me - Me - Me - Me - Me, or ia-ia - Me(s)Me(s)Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me, or ia-ia - Me(s)Me(s)Me - Me - Me - Me - F - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me, or
Me - Me - Me - Me - Me - Me - F- F- F- F- F- Me - Me - Me - Me - Me - Me - Me - F(s)Me(s)Me - ia - ia, or Me - Me - Me - Me - Me - F - F - Me - F - F - F - F - Me - Me - Me - Me - Me - Me - Me(s)Me(s)Me - ia - ia, or
Me - Me - Me - Me - Me - Me -F- Me - F - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me(s)Me(s)Me - ia - ia, or
Me - Me - Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me -
Me - Me(s)Me(s)Me - ia - ia, or
Me - Me - Me - Me - Me - Me - F - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me(s)Me(s)Me - ia - ia, wherein:
(s) is a phosphorothioate internucleoside linkage, ia represents an inverted abasic nucleoside, and when the inverted abasic nucleosides as represented by ia - ia are present at the 3’ terminus of the second strand, said inverted abasic nucleosides are present in a 2 nucleoside overhang. A nucleic acid according to claim 52, wherein said modified nucleosides of said second strand comprise a modification pattern according to any one of the following (5’-3’): ia-ia-Me(s)Me(s) (Me)6 - (F)3 - (Me)10, wherein:
(s) is a phosphorothioate internucleoside linkage, and ia represents an inverted abasic nucleoside. A nucleic acid according to claim 31, wherein said modified nucleosides comprise any one of the following modification patterns:
Modification pattern 1 :
Second strand (5’-3’): Me - Me - Me - Me - Me - Me - F - F - F - F - F - Me - Me - Me - Me - Me - Me - Me - F - Me - Me, First strand (5’-3’): Me - F - Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me -
F - Me - Me - Me - Me - Me - Me - Me
Or Modification pattern 2:
Second strand (5’-3’): Me - Me - Me - Me - Me - F - F - Me - F - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me - F - Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me
Or Modification pattern 3 :
Second strand (5’-3’): Me - Me - Me - Me - Me - Me -F- Me - F - F - F - F - Me - Me -
Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me - F - Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me
Or Modification pattern 4:
Second strand (5’-3’): Me - Me - Me - Me - Me - Me - F - Me - F - F - F - F - Me - Me -
Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me - F - Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me
Or Modification pattern 5:
Second strand (5’-3’): Me - Me - Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me - F - Me - Me - Me - F - Me - Me - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me
Or Modification pattern 6:
Second strand (5’-3’): Me - Me - Me - Me - Me - Me - F - Me - F - F - F - Me - Me - Me
- Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me - F - Me - Me - Me - F - Me - Me - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me. A nucleic acid according to claim 31, wherein said modified nucleosides comprise any one of the following modification patterns:
Modification pattern 1 :
Second strand (5’-3’): Me - Me - Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me - F - Me - Me - Me - X1 - Me - Me - Me - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me, wherein X1 is a thermally destabilising modification;
Or Modification pattern 2:
Second strand (5’-3’): Me - Me - Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me - F - Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me;
Or Modification pattern 3 :
Second strand (5’-3’): Me - Me - Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me - F - Me - Me - Me - F - Me - Me - Me - Me - Me - Me - Me - F - Me - F - Me - F - Me - Me - Me - Me - Me;
Or Modification pattern 4:
Second strand (5’-3’): Me - Me - Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me - F - Me - Me - Me - F - Me - Me - Me - Me - Me - Me - Me - F - Me - F - Me - Me - Me - F - Me - Me - Me;
Or Modification pattern 5:
Second strand (5’-3’): Me - Me - Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me, First strand (5’-3’): Me - F - Me - Me - Me - X1 - Me - F - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me, wherein X1 is a thermally destabilising modification;
Or Modification pattern 6:
Second strand (5’-3’): Me - Me - Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me - F - Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me;
Or Modification pattern 7:
Second strand (5’-3’): Me - Me - Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me - F - Me - Me - Me -F - Me - F - F - Me- Me - Me - Me - F
- Me - F - Me - F - Me - Me - Me - Me - Me;
Or Modification pattern 8:
Second strand (5’-3’): Me - Me - Me - Me - Me - Me - Me - Me - F - F - F - Me -
Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me - F - Me - Me - Me - F - Me - F - F - Me - Me - Me - Me - F
- Me - F - Me - Me - Me - F - Me - Me - Me. A nucleic acid according to claim 59, wherein said modified nucleosides comprise any one of the following modification patterns:
Modification pattern 1 :
Second strand (5’-3’): Me(s)Me(s)Me - Me - Me - Me - F - F - F - F - F - Me - Me - Me -
Me - Me - Me - Me - F - Me - Me,
First strand (5’-3’): Me(s)F(s)Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me
- F - Me - Me - Me - Me - Me(s)Me(s)Me
Or Modification pattern 2: Second strand (5’-3’): Me(s)Me(s)Me - Me - Me - F - F - Me - F - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me(s)F(s)Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me
- F - Me - Me - Me - Me - Me(s)Me(s)Me
Or Modification pattern 3 :
Second strand (5 ’-3’): Me(s)Me(s)Me - Me - Me - Me -F- Me - F - F - F - F - Me - Me -
Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me(s)F(s)Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me
- F - Me - Me - Me - Me - Me(s)Me(s)Me
Or Modification pattern 4:
Second strand (5’-3’): Me(s)Me(s)Me - Me - Me - Me - F - Me - F - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me(s)F(s)Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me(s)Me(s)Me
Or Modification pattern 5:
Second strand (5 ’-3’): Me(s)Me(s)Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me(s)F(s)Me - Me - Me - F - Me - Me - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me(s)Me(s)Me
Or Modification pattern 6:
Second strand (5 ’-3’): Me(s)Me(s)Me - Me - Me - Me - F - Me - F - F - F - Me - Me -
Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me(s)F(s)Me - Me - Me - F - Me - Me - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me(s)Me(s)Me wherein (s) is a phosphorothioate intemucleoside linkage. A nucleic acid according to claim 60, wherein said modified nucleosides comprise any one of the following modification patterns: Modification pattern 1 :
Second strand (5 ’-3’): Me(s)Me(s)Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me(s)F(s)Me - Me - Me - X1 - Me - Me - Me - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me(s)Me(s)Me, wherein X1 is a thermally destabilising modification;
Or Modification pattern 2:
Second strand (5 ’-3’): Me(s)Me(s)Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me(s)F(s)Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me
- F - Me - F - Me - Me - Me - Me - Me(s)Me(s)Me;
Or Modification pattern 3 :
Second strand (5 ’-3’): Me(s)Me(s)Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me(s)F(s)Me - Me - Me - F - Me - Me - Me - Me - Me - Me - Me
- F - Me - F - Me - F - Me - Me - Me(s)Me(s)Me;
Or Modification pattern 4:
Second strand (5 ’-3’): Me(s)Me(s)Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me(s)F(s)Me - Me - Me - F - Me - Me - Me - Me - Me - Me - Me
- F - Me - F - Me - Me - Me - F - Me(s)Me(s)Me;
Or Modification pattern 5:
Second strand (5 ’-3’): Me(s)Me(s)Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me, First strand (5’-3’): Me(s)F(s)Me - Me - Me - X1 - Me - F - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me(s)Me(s)Me, wherein X1 is a thermally destabilising modification;
Or Modification pattern 6:
Second strand (5 ’-3’): Me(s)Me(s)Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me(s)F(s)Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me(s)Me(s)Me;
Or Modification pattern 7:
Second strand (5 ’-3’): Me(s)Me(s)Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me(s)F(s)Me - Me - Me - F - Me - F - F - Me- Me - Me - Me - F - Me - F - Me - F - Me - Me - Me(s)Me(s)Me;
Or Modification pattern 8: Second strand (5 ’ -3 ’): Me(s)Me(s)Me - Me - Me - Me - Me
- Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me(s)F(s)Me - Me - Me - F - Me - F - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - F - Me(s)Me(s)Me; wherein (s) is a phosphorothioate intemucleoside linkage. A nucleic acid according to claim 59, wherein said modified nucleosides comprise any one of the following modification patterns:
Modification pattern 1 :
Second strand (5’-3’): Me - Me - Me - Me - Me - Me - F - F - F - F - F - Me - Me - Me -
Me - Me - Me - Me - F(s)Me(s)Me,
First strand (5’-3’): Me(s)F(s)Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me
- F - Me - Me - Me - Me - Me(s)Me(s)Me
Or Modification pattern 2: Second strand (5’-3’): Me - Me - Me - Me - Me - F - F - Me - F - F - F - F - Me - Me - Me - Me - Me - Me - Me(s)Me(s)Me,
First strand (5’-3’): Me(s)F(s)Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me
- F - Me - Me - Me - Me - Me(s)Me(s)Me
Or Modification pattern 3 :
Second strand (5’-3’): Me - Me - Me - Me - Me - Me -F- Me - F - F - F - F - Me - Me -
Me - Me - Me - Me - Me(s)Me(s)Me,
First strand (5’-3’): Me(s)F(s)Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me
- F - Me - Me - Me - Me - Me(s)Me(s)Me
Or Modification pattern 4:
Second strand (5’-3’): Me - Me - Me - Me - Me - Me - F - Me - F - F - F - F - Me - Me -
Me - Me - Me - Me - Me(s)Me(s)Me,
First strand (5’-3’): Me(s)F(s)Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me(s)Me(s)Me
Or Modification pattern 5:
Second strand (5’-3’): Me - Me - Me - Me - Me - Me - Me - Me - F - F - F - Me - Me -
Me - Me - Me - Me - Me - Me(s)Me(s)Me,
First strand (5’-3’): Me(s)F(s)Me - Me - Me - F - Me - Me - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me(s)Me(s)Me
Or Modification pattern 6:
Second strand (5’-3’): Me - Me - Me - Me - Me - Me - F - Me - F - F - F - Me - Me - Me
- Me - Me - Me - Me - Me(s)Me(s)Me,
First strand (5’-3’): Me(s)F(s)Me - Me - Me - F - Me - Me - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me(s)Me(s)Me wherein (s) is a phosphorothioate intemucleoside linkage. A nucleic acid according to claim 59, wherein said modified nucleosides comprise any one of the following modification patterns: Modification pattern 1 :
Second strand (5’-3’): ia - ia - Me - Me - Me - Me - Me - Me -F-F-F-F-F- Me - Me - Me - Me - Me - Me - Me - F - Me - Me,
First strand (5’-3’): Me - F - Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me
Or Modification pattern 2:
Second strand (5’-3’): ia - ia - Me - Me - Me - Me - Me - F - F - Me -F-F-F-F- Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me - F - Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me
Or Modification pattern 3 :
Second strand (5’-3’): ia - ia - Me - Me - Me - Me - Me - Me -F- Me - F- F- F- F- Me
- Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me - F - Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me
Or Modification pattern 4:
Second strand (5’-3’): ia - ia - Me - Me - Me - Me - Me - Me - F - Me -F-F-F-F-
Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me - F - Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me
Or Modification pattern 5:
Second strand (5’-3’): ia - ia - Me - Me - Me - Me - Me - Me - Me - Me - F - F - F - Me
- Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me - F - Me - Me - Me - F - Me - Me - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me
Or Modification pattern 6:
Second strand (5’-3’): ia - ia - Me - Me - Me - Me - Me - Me - F - Me - F - F - F - Me -
Me - Me - Me - Me - Me - Me - Me - Me - Me First strand (5’-3’): Me - F - Me - Me - Me - F - Me - Me - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me, wherein ia represents an inverted abasic nucleoside. A nucleic acid according to claim 60, wherein said modified nucleosides comprise any one of the following modification patterns:
Modification pattern 1 :
Second strand (5’-3’): ia - ia - Me - Me - Me - Me - Me - Me - Me - Me -F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me - F - Me - Me - Me - X1 - Me - Me - Me - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me, wherein X1 is a thermally destabilising modification;
Or Modification pattern 2:
Second strand (5’-3’): ia - ia - Me - Me - Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me - F - Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me;
Or Modification pattern 3 :
Second strand (5’-3’): ia - ia - Me - Me - Me - Me - Me - Me - Me - Me -F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me - F - Me - Me - Me - F - Me - Me - Me - Me - Me - Me - Me - F - Me - F - Me - F - Me - Me - Me - Me - Me;
Or Modification pattern 4:
Second strand (5’-3’): ia - ia - Me - Me - Me - Me - Me - Me - Me - Me -F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me, First strand (5’-3’): Me - F - Me - Me - Me - F - Me - Me - Me - Me - Me - Me - Me - F - Me - F - Me - Me - Me - F - Me - Me - Me;
Or Modification pattern 5:
Second strand (5’-3’): ia - ia - Me - Me - Me - Me - Me - Me - Me - Me -F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me - F - Me - Me - Me - X1 - Me - F - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me, wherein X1 is a thermally destabilising modification;
Or Modification pattern 6:
Second strand (5’-3’): ia - ia - Me - Me - Me - Me - Me - Me - Me - Me -F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me - F - Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me;
Or Modification pattern 7:
Second strand (5’-3’): ia - ia - Me - Me - Me - Me - Me - Me - Me - Me -F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me - F - Me - Me - Me -F - Me - F - F - Me- Me - Me - Me - F
- Me - F - Me - F - Me - Me - Me - Me - Me;
Or Modification pattern 8:
Second strand (5’-3’): ia - ia - Me - Me - Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me - F - Me - Me - Me - F - Me - F - F - Me - Me - Me - Me - F
- Me - F - Me - Me - Me - F - Me - Me - Me, wherein ia represents an inverted abasic nucleoside. A nucleic acid according to claim 59, wherein said modified nucleosides comprise any one of the following modification patterns: Modification pattern 1 :
Second strand (5’-3’): Me - Me - Me - Me - Me - Me - F - F - F - F - F - Me - Me - Me - Me - Me - Me - Me - F - Me - Me - ia - ia,
First strand (5’-3’): Me - F - Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me
Or Modification pattern 2:
Second strand (5’-3’): Me - Me - Me - Me - Me - F - F - Me - F - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - ia - ia,
First strand (5’-3’): Me - F - Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me
Or Modification pattern 3 :
Second strand (5’-3’): Me - Me - Me - Me - Me - Me -F- Me - F - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - ia - ia,
First strand (5’-3’): Me - F - Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me
Or Modification pattern 4:
Second strand (5’-3’): Me - Me - Me - Me - Me - Me - F - Me - F - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - ia - ia,
First strand (5’-3’): Me - F - Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me
Or Modification pattern 5:
Second strand (5’-3’): Me - Me - Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me - ia - ia,
First strand (5’-3’): Me - F - Me - Me - Me - F - Me - Me - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me
Or Modification pattern 6:
Second strand (5’-3’): Me - Me - Me - Me - Me - Me - F - Me - F - F - F - Me - Me - Me
- Me - Me - Me - Me - Me - Me - Me - ia - ia First strand (5’-3’): Me - F - Me - Me - Me - F - Me - Me - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me, wherein ia represents an inverted abasic nucleoside, and when the inverted abasic nucleosides as represented by ia - ia are present at the 3’ terminus of the second strand, said inverted abasic nucleosides are present in a 2 nucleoside overhang. A nucleic acid according to claim 55, wherein said modified nucleosides comprise any one of the following modification patterns:
Modification pattern 1 :
Second strand (5’-3’): ia - ia - Me(s)Me(s)Me - Me - Me - Me -F-F-F-F-F- Me - Me - Me - Me - Me - Me - Me - F - Me - Me,
First strand (5’-3’): Me(s)F(s)Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me
- F - Me - Me - Me - Me - Me(s)Me(s)Me
Or Modification pattern 2:
Second strand (5’-3’): ia - ia - Me(s)Me(s)Me - Me - Me - F - F - Me -F-F-F-F- Me
- Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me(s)F(s)Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me
- F - Me - Me - Me - Me - Me(s)Me(s)Me
Or Modification pattern 3 :
Second strand (5 ’-3’): ia - ia - Me(s)Me(s)Me - Me - Me - Me -F- Me -F-F-F-F- Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me(s)F(s)Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me
- F - Me - Me - Me - Me - Me(s)Me(s)Me
Or Modification pattern 4:
Second strand (5’-3’): ia - ia - Me(s)Me(s)Me - Me - Me - Me - F - Me -F-F-F-F- Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me(s)F(s)Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me(s)Me(s)Me Or Modification pattern 5:
Second strand (5 ’-3’): ia - ia - Me(s)Me(s)Me - Me - Me - Me - Me - Me - F - F - F - Me
- Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me(s)F(s)Me - Me - Me - F - Me - Me - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me(s)Me(s)Me
Or Modification pattern 6:
Second strand (5’-3’): ia - ia - Me(s)Me(s)Me - Me - Me - Me - F - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me(s)F(s)Me - Me - Me - F - Me - Me - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me(s)Me(s)Me wherein:
(s) is a phosphorothioate internucleoside linkage, ia represents an inverted abasic nucleoside. A nucleic acid according to claim 60, wherein said modified nucleosides comprise any one of the following modification patterns:
Modification pattern 1 :
Second strand (5 ’-3’): ia - ia - Me(s)Me(s)Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me(s)F(s)Me - Me - Me - X1 - Me - Me - Me - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me(s)Me(s)Me, wherein X1 is a thermally destabilising modification;
Or Modification pattern 2:
Second strand (5 ’-3’): ia - ia - Me(s)Me(s)Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me(s)F(s)Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me
- F - Me - F - Me - Me - Me - Me - Me(s)Me(s)Me;
Or Modification pattern 3 : Second strand (5 ’-3’): ia - ia - Me(s)Me(s)Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me(s)F(s)Me - Me - Me - F - Me - Me - Me - Me - Me - Me - Me
- F - Me - F - Me - F - Me - Me - Me(s)Me(s)Me;
Or Modification pattern 4:
Second strand (5 ’-3’): ia - ia - Me(s)Me(s)Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me(s)F(s)Me - Me - Me - F - Me - Me - Me - Me - Me - Me - Me
- F - Me - F - Me - Me - Me - F - Me(s)Me(s)Me;
Or Modification pattern 5:
Second strand (5 ’-3’): ia - ia - Me(s)Me(s)Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me(s)F(s)Me - Me - Me - X1 - Me - F - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me(s)Me(s)Me, wherein X1 is a thermally destabilising modification;
Or Modification pattern 6:
Second strand (5 ’-3’): ia - ia - Me(s)Me(s)Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me(s)F(s)Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me(s)Me(s)Me;
Or Modification pattern 7:
Second strand (5 ’-3’): ia - ia - Me(s)Me(s)Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me(s)F(s)Me - Me - Me - F - Me - F - F - Me- Me - Me - Me - F - Me - F - Me - F - Me - Me - Me(s)Me(s)Me;
Or Modification pattern 8: Second strand (5 ’-3’): ia - ia - Me(s)Me(s)Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3’): Me(s)F(s)Me - Me - Me - F - Me - F - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - F - Me(s)Me(s)Me; wherein:
(s) is a phosphorothioate internucleoside linkage, ia represents an inverted abasic nucleoside. A nucleic acid according to claim 59, wherein said modified nucleosides comprise any one of the following modification patterns:
Modification pattern 1 :
Second strand (5’-3’): Me - Me - Me - Me - Me - Me - F - F - F - F - F - Me - Me - Me - Me - Me - Me - Me - F(s)Me(s)Me - ia - ia,
First strand (5’-3’): Me(s)F(s)Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me
- F - Me - Me - Me - Me - Me(s)Me(s)Me
Or Modification pattern 2:
Second strand (5’-3’): Me - Me - Me - Me - Me - F - F - Me - F - F - F - F - Me - Me -
Me - Me - Me - Me - Me(s)Me(s)Me - ia - ia,
First strand (5’-3’): Me(s)F(s)Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me
- F - Me - Me - Me - Me - Me(s)Me(s)Me
Or Modification pattern 3 :
Second strand (5’-3’): Me - Me - Me - Me - Me - Me -F- Me - F - F - F - F - Me - Me -
Me - Me - Me - Me - Me(s)Me(s)Me - ia - ia,
First strand (5’-3’): Me(s)F(s)Me - F - Me - F - Me - F - F - Me - Me - Me - Me - F - Me
- F - Me - Me - Me - Me - Me(s)Me(s)Me
Or Modification pattern 4:
Second strand (5’-3’): Me - Me - Me - Me - Me - Me - F - Me - F - F - F - F - Me - Me - Me - Me - Me - Me - Me(s)Me(s)Me - ia - ia, First strand (5’-3’): Me(s)F(s)Me - F - Me - F - Me - Me - Me - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me(s)Me(s)Me
Or Modification pattern 5:
Second strand (5’-3’): Me - Me - Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me(s)Me(s)Me - ia - ia,
First strand (5’-3’): Me(s)F(s)Me - Me - Me - F - Me - Me - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me(s)Me(s)Me
Or Modification pattern 6:
Second strand (5’-3’): Me - Me - Me - Me - Me - Me - F - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me(s)Me(s)Me - ia - ia,
First strand (5’-3’): Me(s)F(s)Me - Me - Me - F - Me - Me - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me(s)Me(s)Me wherein:
(s) is a phosphorothioate internucleoside linkage, ia represents an inverted abasic nucleoside, and when the inverted abasic nucleosides as represented by ia - ia are present at the 3’ terminus of the second strand, said inverted abasic nucleosides are present in a 2 nucleoside overhang. A nucleic acid according to any preceding claim, wherein the nucleic acid is conjugated directly or indirectly to one or more ligand moieties, optionally wherein said ligand moiety is present at a terminal region of the second strand, preferably at the 3’ terminal region thereof. A nucleic acid according to claim 70, wherein the ligand moiety comprises:
(i) one or more N-acetyl galactosamine (GalNAc) ligands, and / or
(ii) one or more N-acetyl galactosamine (GalNAc) ligand derivatives, and/or
(iii) one or more N-acetyl galactosamine (GalNAc) ligands and/or derivatives thereof, conjugated to the nucleic acid through a linker. A nucleic acid according to claim 71, wherein said one or more GalNAc ligands and / or GalNAc ligand derivatives are conjugated directly or indirectly to the 5’ or 3’ terminal region of the second strand of the nucleic acid, preferably at the 3’ terminal region thereof. A nucleic acid according to any one of claims 70 to 72, wherein the ligand moiety comprises the following structure:
Figure imgf000250_0001
A nucleic acid according to any one of claims 70 to 73, comprising the structure:
Figure imgf000250_0002
wherein: R1 at each occurrence is independently selected from the group consisting of hydrogen, methyl and ethyl;
R2 is selected from the group consisting of hydrogen, hydroxy, -OC1-3alkyl, -C(=O)OC1- 3alkyl, halo and nitro;
X1 and X2 at each occurrence are independently selected from the group consisting of methylene, oxygen and sulfur; m is an integer of from 1 to 6; n is an integer of from 1 to 10; q, r, s, t, v are independently integers from 0 to 4, with the proviso that:
(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 nucleic acid according to claim 74, comprising the structure
Figure imgf000251_0001
wherein [oligonucleotide] represents the contiguous nucleosides of the second strand. A nucleic acid according to any one of claims 70 to 73, comprising the structure:
Figure imgf000251_0002
wherein: r and s are independently an integer selected from 1 to 16; and
Z is an oligonucleoside moiety. A nucleic acid according to claim 76, comprising the structure
Figure imgf000252_0001
wherein [oligonucleotide] represents the contiguous nucleosides of the second strand. A nucleic acid according to any one of claims 70 to 77, wherein the nucleic acid comprises the modification pattern:
Second strand (5 ’-3’): ia - ia - Me(s)Me(s)Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me - Me - Me,
First strand (5’-3 ’): Me(s)F(s)Me - Me - Me - F - Me - Me - F - Me - Me - Me - Me - F - Me - F - Me - Me - Me - Me - Me(s)Me(s)Me, wherein:
(s) is a phosphorothioate internucleoside linkage, ia represents an inverted abasic nucleoside and wherein the nucleic acid is conjugated directly or indirectly to one or more ligand moieties, optionally wherein said ligand moiety is present at a terminal region of the second strand, preferably at the 3’ terminal region thereof. A nucleic acid according to claim 78, wherein the second strand comprises the structure
Figure imgf000252_0002
Figure imgf000253_0001
wherein [oligonucleotide] represents the contiguous nucleosides of the second strand, and wherein the one or more ligand moi eties are conjugated to the 3’ terminal region of the second strand via a linker. The nucleic acid according to claim 78 or 79, wherein the first and second strands comprise, consist of, or consist essentially of a nucleoside sequence differing by 0 or 1 nucleosides from any one of the following first and second sequences:
Figure imgf000253_0002
The nucleic acid according to any one of claims 78 to 80, wherein the 2 consecutive inverted abasic nucleosides in the 5’ terminal region of the second strand present as the following 5’ terminal motif
Figure imgf000254_0001
wherein:
T represents a 2’Me ribose modification,
B represents the nucleoside bases of the first two basic nucleosides in the 5' terminal region of the second strand, and
Z represents the remaining 19 contiguous basic nucleosides of said second strand. A pharmaceutical composition comprising a nucleic acid according to any preceding claim, in combination with a pharmaceutically acceptable excipient or carrier. A nucleic acid or pharmaceutical composition according to any preceding claim, for use in therapy. A nucleic acid or pharmaceutical composition according to any preceding claim, for use in prevention or treatment of diabetes. A nucleic acid or pharmaceutical composition according to any preceding claim, for use in prevention or treatment of cardiovascular disease.
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"Current protocols in nucleic acid chemistry", JOHN WILEY & SONS, INC.
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS

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