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WO2025163186A1 - Acides nucléiques conjugués et acides nucléiques comprenant des nucléosides verrouillés et des nucléotides inversés pour inhiber l'expression génique dans une cellule - Google Patents

Acides nucléiques conjugués et acides nucléiques comprenant des nucléosides verrouillés et des nucléotides inversés pour inhiber l'expression génique dans une cellule

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Publication number
WO2025163186A1
WO2025163186A1 PCT/EP2025/052612 EP2025052612W WO2025163186A1 WO 2025163186 A1 WO2025163186 A1 WO 2025163186A1 EP 2025052612 W EP2025052612 W EP 2025052612W WO 2025163186 A1 WO2025163186 A1 WO 2025163186A1
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Prior art keywords
strand
nucleic acid
nucleotide
nucleotides
modification
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2025/052612
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English (en)
Inventor
Sophie SCHÖLLKOPF
Nina WAS
Lucas Bethge
Judith HAUPTMANN
Eliot MORRISON
Marie WIKSTRÖM LINDHOLM
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Silence Therapeutics GmbH
Original Assignee
Silence Therapeutics GmbH
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Publication date
Application filed by Silence Therapeutics GmbH filed Critical Silence Therapeutics GmbH
Publication of WO2025163186A1 publication Critical patent/WO2025163186A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/322—2'-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/344—Position-specific modifications, e.g. on every purine, at the 3'-end
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/346—Spatial arrangement of the modifications having a combination of backbone and sugar modifications
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate

Definitions

  • Conjugated nucleic acids and nucleic acids comprising locked nucleosides and inverted nucleotides for inhibiting gene expression in a cell
  • the invention relates to novel nucleic acids of formula (I), in particular, nucleic acids, and preferably conjugated nucleic acids, comprising at least one LNA modification and optionally further comprising inverted nucleotides and/or phosphorothioate linkages.
  • Such (conjugated) nucleic acids of formula (I) can be used to inhibit expression of a target gene.
  • the invention further relates to compositions comprising said (conjugated) nucleic acids of formula (I) for use in the prophylaxis or treatment of a disease.
  • Inhibitory nucleic acids such as siRNAs are short nucleic acids that inhibit the formation of proteins by causing targeted degradation of the mRNA molecules that encode proteins.
  • gene silencing agents are becoming increasingly important for therapeutic applications in medicine.
  • nucleic acids it is among other things necessary that they can be synthesized economically, are metabolically stable, are specifically targeted to a tissue, and are able to enter cells and function within acceptable limits of toxicity.
  • inverted ribonucleotides can be used for end stabilization of siRNA conjugates (e.g., WO2018185239A1). Inverted ribonucleotides can be used at the siRNA termini and do not require additional stabilisation of the respective terminus by phosphorothioate linkages. It has also been shown that oligonucleotides with Locked Nucleic Acids (LNA) have increased metabolic stability in siRNA and antisense applications. 11 21 A Locked Nucleic Acid is a nucleotide where the ribose is “locked” by a methylene bridge between 2' and 4' carbon atoms of the sugar. 131 This non-natural modification aggravates enzymatic digestion.
  • LNA Locked Nucleic Acid
  • siRNA duplex comprises a thermodynamic asymmetry.
  • 1561 Functional siRNAs are characterized by a lower internal stability at the 5' end of the first strand compared to the internal stability at the 5' end of the second strand. This facilitates loading of the correct (first) strand into RISC. 1561 Asymmetry can also be introduced by increasing the thermal stability of the 3' terminus of the first strand, e.g. by introduction of one or more LNA modifications.
  • the present invention provides an inhibitory nucleic acid showing increased knock down activity, while stability is improved.
  • One aspect of the invention is a (conjugated) double stranded nucleic acid for inhibiting expression of a target gene, comprising a first strand and a second strand, represented by formula (I): First (antisense or guide) strand
  • each of X a , X a ', Xb and Xb' independently represents an overhang nucleotide sequence comprising 0-15, 0-10, or 0-6 nucleotides
  • each of Yf and Yf' independently represents a nucleotide
  • Yf and Yf' represent the first position of a double stranded region between the first and the second strands, wherein Yf and Y are opposite each other in their respective positions in the first and second strands and may be complementary to each other
  • each of Y u and Y u ' independently represents a nucleotide, wherein Y u and Y u ' represent the ultimate position of the double stranded region between the first and the second strands, wherein Y u and Y u ' are opposite each other in their respective positions in the first and second strands and may be complementary to each other, each of Y2 to Y5 and Y2' to
  • One aspect of the invention is a composition
  • a composition comprising a nucleic acid of formula (I) of the invention and a solvent and/or a delivery vehicle and/or a physiologically acceptable excipient and/or a carrier and/or a salt and/or a diluent and/or a buffer and/or a preservative and/or a further therapeutic agent selected from the group comprising an oligonucleotide, a small molecule, a monoclonal antibody, a polyclonal antibody and a peptide.
  • One aspect of the invention is a method for the prophylaxis or treatment of a disease or disorder in a subject in need thereof, comprising administering a (conjugated) nucleic acid of formula (I) of the invention, to said subject.
  • One aspect of the invention is the (conjugated) nucleic acid of formula (I) of the invention for use in the prophylaxis or treatment of a disease, disorder or syndrome.
  • One aspect of the invention is the use of the (conjugated) nucleic acid of formula (I) or the composition of the invention in the preparation of a medicament for the prophylaxis or treatment of a disease, disorder or syndrome.
  • the present invention relates to a (conjugated) double stranded nucleic acid of formula (I) for inhibiting expression of a target gene, and compositions thereof.
  • the nucleic acid of formula (I), or the conjugate or composition of the invention comprise at least one LNA modification and may further comprise other modifications.
  • the nucleic acid of formula (I), or the conjugate or the composition of the invention may be used in the prophylaxis and treatment of a variety of diseases, disorders and syndromes in which reduced expression of a desired target gene product is desirable.
  • a first aspect of the invention is a (conjugated) double stranded nucleic acid of formula (I) for inhibiting expression of a target gene, comprising a first strand and a second strand, represented by formula (I): First strand
  • each of X a , X a ', Xb and Xb' independently represents an overhang nucleotide sequence comprising 0-15 or 0-10 nucleotides
  • each of Yf and Yf' independently represents a nucleotide
  • Yf and Yf' represent the first position of a double stranded region between the first and the second strands
  • Yf and Yf' are opposite each other in their respective positions in the first and second strands and may be complementary to each other
  • each of Y u and Y u ' independently represents a nucleotide
  • Y u and Y u ' represent the ultimate position of the double stranded region between the first and the second strands
  • Y u and Y u ’ are opposite each other in their respective positions in the first and second strands and may be complementary to each other
  • each of Z and Z' represents a nucleotide sequence comprising 0-25 nucleotides within the double stranded region between the first and the second strands, wherein Z and Z' may be at least partially complementary to each other, wherein the first strand comprises one LNA modified nucleotide, preferably Y-2 comprises an LNA modified nucleotide, and wherein the second strand comprises none, one or two LNA modifications.
  • One embodiment of the first aspect of the invention concerns a (conjugated) double stranded nucleic acid of formula (I) for inhibiting expression of a target gene, comprising a first strand and a second strand, represented by formula (I): First (antisense or guide) strand
  • each of X a , X a ', Xb and Xb' independently represents an overhang nucleotide sequence comprising 0-15, 0-10, or 0-6 nucleotides
  • each of Yf and Yf' independently represents a nucleotide
  • Yf and Yf' represent the first position of a double stranded region between the first and the second strands, wherein Yf and Y are opposite each other in their respective positions in the first and second strands and may be complementary to each other
  • each of Y u and Y u ' independently represents a nucleotide, wherein Y u and Y u ' represent the ultimate position of the double stranded region between the first and the second strands, wherein Y u and Y u ' are opposite each other in their respective positions in the first and second strands and may be complementary to each other
  • each of Y 2 to Ys and Y 2 ' independently represents an
  • Y 2 , Y 3 , Y 4 , Ys, Y 2 ', Y 3 ', Y 4 ', and Ys independently represents a nucleotide within the double stranded region, wherein Y 2 to Ys and Y 2 ' to Ys' are opposite each other in their respective positions in the first and second strands and may be complementary to each other, each of Y. 2 to Y-s and Y-2 1 to Y-s' (i.e. Y. 2 , Y. 3 , Y. 4 , Y-s, Y-2 1 , Y. 3 ', Y.
  • each of Z and Z' represents a nucleotide sequence comprising 0-25 nucleotides within the double stranded region between the first and the second strands, wherein Z and Z' may be at least partially complementary to each other, preferably comprising 0-15 nucleotides within the double stranded region between the first and the second strands, wherein the first strand comprises one LNA modified nucleotide, preferably at position Y-2, wherein the second strand comprises none, one or two LNA modifications, wherein the first and/or the second strands independently comprise at least one phosphorothioate linkage, and wherein the first and/or the second strands may each comprise further modifications.
  • (conjugated) double stranded nucleic acid of formula (I) as used herein means a double stranded nucleic acid of formula (I), preferably a conjugated double stranded nucleic acid of formula (I).
  • First and second strands that “may be complementary to each other” as described herein are complementary (100% complementarity) or at least partially complementary (less than 100% complementarity) to each other.
  • each of X a , Xa', Xb and Xb' independently represents an overhang nucleotide sequence comprising 0-15, 0-10, or 0-6, 1-9, 1-8, 1-6, or 1-3 nucleotides.
  • each of X a , X a ', Xb and Xb' comprises 0 nucleotide.
  • each of Z and Z' independently represents a nucleotide sequence comprising 4-20, preferably 7-15, more preferably 8-13 nucleotides within the double stranded region between the first and the second strands, wherein Z and Z' may be at least partially complementary to each other, preferably Z and Z' are complementary.
  • At least one of Y 5 ',Y 4 ', Y 3 ',Y 2 ' and/or Y- 5 ',Y- 4 ', Y. 3 ',Y- 2 ' comprises an LNA modified nucleotide.
  • one of Y5',Y4',Y3', Y2' and/or Y-s', Y-4 1 , Y-3', Y-2' comprises an LNA modified nucleotide.
  • Y-2' comprises an LNA modified nucleotide.
  • Y2' and Y-2' each comprise an LNA modified nucleotide.
  • Y-2 is an LNA modified nucleotide and at least one phosphorothioate linkage is present between Y-2 and Yu.
  • the nucleic acid of formula (I) of the invention may comprise one or several, such as two, three, four, five, six, seven or more, including all, nucleotides that are modified by modifications or conjugations, such as, e.g., 2'-0Me, 2'-F, a ligand, a linker, a 3’ end or 5’ end modification or of any other modification well known to those if skill in the art.
  • modifications or conjugations such as, e.g., 2'-0Me, 2'-F, a ligand, a linker, a 3’ end or 5’ end modification or of any other modification well known to those if skill in the art.
  • the (conjugated) nucleic acid of formula (I) of the invention is an siRNA, preferably wherein the nucleic acid mediates RNA interference.
  • a double-stranded nucleic acid of formula (I) as defined herein is a nucleic acid in which the first strand and the second strand hybridise to each other over at least part of their lengths and are therefore capable of forming a duplex region under physiological conditions, such as in PBS at 37°C at a concentration of 1 pM of each strand.
  • the first and second strands are preferably able to hybridise to each other and therefore form a duplex region over a region of at least 15 nucleotides, preferably 16, 17, 18 or 19 nucleotides or more.
  • the duplex region between the first and the second strand begins at Yf and Y and ends at Y u and Y u ', respectively on the first and second strands.
  • This duplex region comprises nucleotide base parings between the two strands, preferably based on Watson-Crick base pairing and/or wobble base pairing (such as Gil base pairing). All the nucleotides of the two strands within a duplex region do not have to base pair to each other to form a duplex region.
  • the double-stranded nucleic acid is preferably a stable double-stranded nucleic acid under physiological conditions, and preferably has a melting temperature (Tm) 45°C or more, preferably 50°C or more, preferably 60°C or more, and more preferably between 75- 85°C for example in PBS at a concentration of 1 pM of each strand.
  • Tm melting temperature
  • the first strand and the second strand of the nucleic acid of formula (I) are preferably capable of forming a duplex region (i.e. , are complementary to each other) over i) at least a portion of their lengths, preferably over at least 10 nucleotides of both of their lengths, preferably over at least 15 nucleotides of both of their lengths, ii) over the entire length of the first strand, iii) over the entire length of the second strand or iv) over the entire length of both the first and the second strand.
  • Strands being complementary to each other over a certain length means that the strands are able to base pair to each other, either via Watson-Crick or wobble base pairing, over that length.
  • Each nucleotide of the length does not necessarily have to be able to base pair with its counterpart in the other strand over the entire given length as long as a stable double-stranded nucleotide under physiological conditions can be formed. It is however, preferred, in certain embodiments, if each nucleotide of the length can base pair with its counterpart in the other strand over the entire given length.
  • a certain number of mismatches, deletions or insertions between the first strand and the target sequence, or between the first strand and the second strand can be tolerated in the context of the nucleic acids of formula (I) of the present invention and even have the potential in certain cases to increase RNA interference (e.g., inhibition) activity.
  • the inhibition (or “knockdown”) activity of the nucleic acids of formula (I) of the present invention relies on the formation of a duplex region between all or a portion of the first strand and a portion of a target nucleic acid.
  • the portion of the target nucleic acid that forms a duplex region with the first strand defined as beginning with the first base pair formed between the first strand and the target sequence and ending with the last base pair formed between the first strand and the target sequence, inclusive, is the target nucleic acid sequence or simply, target sequence.
  • the duplex region formed between the first strand and the second strand need not be the same as the duplex region formed between the first strand and the target sequence. That is, the second strand may have a sequence different from the target sequence; however, the first strand must be able to form a duplex structure with both the second strand and the target sequence, at least under physiological conditions.
  • the complementarity between the first strand and the target sequence may be perfect (i.e. , 100% identity with no nucleotide mismatches or insertions or deletions in the first strand as compared to the target sequence).
  • the complementarity between the first strand and the complementary sequence of the target sequence may range from about 75% to less thanl 00%. More specifically, the complementarity may be at least 75%, 80%, 85%, 90% or 95% and intermediate values, provided a nucleic acid of formula (I) is capable of reducing or inhibiting the expression of a desired target gene.
  • One embodiment of the first aspect of the present invention relates to a nucleic acid, wherein the first strand and the second strand are present on a single strand of a nucleic acid that loops around so that the first strand and the second strand are able to hybridise to each other and to thereby form a double-stranded nucleic acid with a duplex region.
  • the duplex region is formed between and including Yf to Y u and Y to Y u ' of the first strand and the second strand, respectively, of formula (I).
  • the first strand and the second strand of the nucleic acid are separate strands.
  • the two separate strands are each 10-35 nucleotides in length, preferably 17-25 nucleotides in length, more preferably 18-25 nucleotides in length.
  • the two strands may be of the same or different lengths.
  • the first strand may be 17-25 nucleotides in length, preferably it may be 18-24 nucleotides in length, it may be 18, 19, 20, 21 , 22, 23 or 24 nucleotides in length. Most preferably, the first strand is 19 nucleotides in length.
  • the second strand may independently be 17-25 nucleotides in length, preferably it may be 18-24 nucleotides in length, it may be 18, 19, 20, 21 , 22, 23 or 24 nucleotides in length. More preferably, the second strand is 18 or 19 or 20 nucleotides in length, and most preferably it is 19 nucleotides in length.
  • the first strand and the second strand of the nucleic acid form a duplex region of 10-35 nucleotides in length, preferably 17-25 nucleotides in length. More preferably, the duplex region is 18-24 nucleotides in length.
  • the duplex region may be 10, 11 , 12, 13, 14,15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or 25 nucleotides in length, preferably 17, 18, 19, 20, 21 , 22, 23, 24 or 25 nucleotides in length. In the most preferred embodiment, the duplex region is 18 or 19 nucleotides in length.
  • the duplex region is defined here as the region between and including Yf and Y? to Y u and Y u '.
  • the duplex region may comprise nucleotides in either or both strands that are not base-paired to a nucleotide in the other strand. It may comprise one, two, three or four such nucleotides on the first strand and/or on the second strand. However, preferably, the duplex region consists of 17-25 consecutive nucleotide base pairs. That is to say that it preferably comprises 17-25 consecutive nucleotides on both of the strands that all bases pair to a nucleotide in the other strand. More preferably, the duplex region consists of 18 or 19 consecutive nucleotide base pairs, most preferably 18.
  • nucleic acid of formula (I) may be blunt ended at both ends; have an overhang at one end and a blunt end at the other end; or have an overhang at both ends.
  • the nucleic acid of formula (I) may have an overhang at one end and a blunt end at the other end.
  • the nucleic acid of formula (I) may have an overhang at both ends.
  • the nucleic acid of formula (I) may be blunt ended at both ends.
  • the nucleic acid of formula (I) may be blunt ended at the end with the 5' end of the first strand and the 3' end of the second strand or at the 3’ end of the first strand and the 5' end of the second strand.
  • the nucleic acid of formula (I) may comprise an overhang at a 3' or 5' end.
  • the nucleic acid of formula (I) may have a 3' overhang on the first strand.
  • the nucleic acid of formula (I) may have a 3' overhang on the second strand.
  • the nucleic acid of formula (I) may have a 5' overhang on the first strand.
  • the nucleic acid of formula (I) may have a 5' overhang on the second strand.
  • the nucleic acid of formula (I) may have an overhang at both the 5' end and 3' end of the first strand.
  • the nucleic acid of formula (I) may have an overhang at both the 5' end and 3' end of the second strand.
  • An overhang at the 3’ end or 5’ end of the second strand or the first strand of the nucleic acid of formula (I) may consist of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 12, 13, 14, or15 nucleotides in length.
  • an overhang may consist of 1 , 2 or 3 nucleotides, which may or may not be modified.
  • the 5’ end of the first strand of the nucleic acid of formula (I) is a singlestranded overhang of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15nucleotides, preferably of 1 , 2 or 3 nucleotides.
  • a (conjugated) double stranded nucleic acid of the present invention according to formula (I), may be a nucleic acid, wherein the first strand and/or the second strand comprise further phosphorothioate linkages.
  • the nucleic acid of formula (I) of the invention further comprises a (E)-vinylphosphonate nucleotide, preferably at position Y f .
  • the 5’ (E)- vinylphosphonate nucleotide maybe a DNA or RNA nucleotide.
  • the 5’ (E)- vinylphosphonate nucleotide is an RNA nucleotide, more preferably a (vp)-ll.
  • Y-2 and Y u further comprises: a) one phosphorothioate linkage between Yf and Y2, and/or b) an (E)-vinylphosphonate nucleotide, preferably at position Yf, preferably wherein the
  • (E)- vinylphosphonate is an RNA nucleotide, preferably a uridine.
  • the first strand of the nucleic acid of formula (I) comprises a phosphorothioate linkage between Y-2 and Y u and further comprises one phosphorothioate linkage between Yf and Y2 and one phosphorothioate linkage between Y2 and Y3.
  • the first strand of the nucleic acid of formula (I) comprises a phosphorothioate linkage between Y-2 and Y u and further comprises one phosphorothioate linkage between Yf and Y2 and one phosphorothioate linkage between Y2 and Y3, and an (E)- vinylphosphonate nucleotide, preferably at position Yf, preferably wherein the (E)- vinylphosphonate is an RNA nucleotide, preferably a uridine.
  • the (E)-vinylphosphonate nucleotide maybe a DNA or RNA nucleotide.
  • the 5’ (E)- vinylphosphonate nucleotide is an RNA nucleotide, more preferably a (vp)-ll.
  • the first strand of the nucleic acid of formula (I) is a nucleotide other than A or II
  • this nucleotide is replaced by an A or II.
  • this nucleotide is replaced by II, and more preferably by II with a 5’ vinylphosphonate.
  • one of Ys‘, YT, 3 , Y 2 ‘ and/or Y.s‘, Y./, Y.3 1 , -2 comprises an LNA modification, preferably Y 2 ‘ and/or Y-2 1 , preferably Y2 1 .
  • the (conjugated) double stranded nucleic acid of formula (I) of the invention comprises a second strand, wherein the second strand comprises at least one phosphorothioate linkage, wherein the at least one phosphorothioate linkage is between Yf and Y2’, or between Y2’ and Y3’ of formula (I).
  • the second strand of the nucleic acid of formula (I) may comprise two phosphorothioate linkages, one between Y and Y2’ and a further one between Y2’ and Y3’.
  • the second strand of the nucleic acid of formula (I) comprises: a) one phosphorothioate linkage between Y and Y2’, or b) one phosphorothioate linkage between Y2’ and Y3’.
  • the second strand of formula (I) comprises a phosphorothioate linkage between Y f ’ and Y 2 ’ and preferably also between Y 2 ’ and Y 3 ’.
  • the second strand of the nucleic acid of formula (I) comprises one phosphorothioate linkage between Yf’ and Y 2 ’ and one phosphorothioate linkage between Y 2 ’ and Y 3 ’.
  • the second strand of the nucleic acid of formula (I) comprises a phosphorothioate linkage between Yf’ and Y 2 ’ and between Y 2 ’ and Y 3 ’.
  • the second strand of the nucleic acid of formula (I) comprises a phosphorothioate linkage between Y 2 ’ and Y 3 ’, preferably further at least one LNA modified nucleotide, and at least one inverted nucleotide.
  • the at least one LNA modified nucleotide is at position Y 2 ' and the at least one inverted nucleotide is at position Y f ’.
  • the second strand of the nucleic acid of formula (I) comprises a) a phosphorothioate linkage between Yf’ and Y 2 ’ and preferably also between Y 2 ’ and
  • the second strand of the nucleic acid of formula (I) comprises a) one phosphorothioate linkage between Yf’ and Y 2 ’, and/or b) (1) at least one LNA modified nucleotide, preferably at position Y 2 ';
  • the second strand of the nucleic acid of formula (I) comprises at least one LNA modified nucleotide and at least one inverted nucleotide and at least one phosphorothioate linkage.
  • the second strand of the nucleic acid of formula (I) comprises at least one LNA modified nucleotide at position Y 2 ' and at least one inverted nucleotide and at least one phosphorothioate linkage.
  • the second strand of the nucleic acid of formula (I) comprises at least one LNA modified nucleotide at position Y 2 ' and at least one inverted nucleotide at position Yf and at least one phosphorothioate linkage.
  • the second strand of the nucleic acid of formula (I) comprises at least one LNA modified nucleotide at position Y 2 ' and at least one inverted nucleotide at position Yf and at least one phosphorothioate linkage between Y 2 ' and Y3'.
  • the second strand of the nucleic acid of formula (I) comprises a) one phosphorothioate linkage between Y and Y 2 ’ and one phosphorothioate linkage between Y 2 ’ and Y3’, or b) (1) at least one LNA modified nucleotide, preferably at position Y 2 ';
  • the second strand of the nucleic acid of formula (I) comprises one phosphorothioate linkage between Y and Y 2 ’ and one phosphorothioate linkage between Y 2 ’ and Y3’, and at least one LNA modified nucleotide.
  • the second strand of the nucleic acid of formula (I) comprises one phosphorothioate linkage between Y and Y 2 ’ and one phosphorothioate linkage between Y 2 ’ and Y3’, and at least one LNA modified nucleotide at position Y 2 '.
  • all remaining linkages between nucleotides of the first and/or of the second strand of the nucleic acid of formula (I) are phosphodiester linkages.
  • the phosphorothioate linkages and (E)-vinylphosphonate nucleotide are used for improving the stabilization of the nucleic acid, therefore, these are considered to be representative examples and not limiting.
  • the phosphorothioate linkages and (E)-vinylphosphonate nucleotides may be combined or exchanged by any other tool known in the art. Some examples are (Z)-vinylphosphonate, 5'alkyletherphosphonates, 5'-cyclopropane phosphonate.
  • the (conjugated) double stranded nucleic acid further comprises at least one inverted nucleotide in the first and/or second strand of the nucleic acid of formula (I).
  • at least one of Yf’, Y u , and/or Y u ’ comprises the inverted nucleotide.
  • Y comprises the inverted nucleotide.
  • the inverted nucleotide at any of positions Yf’, Y u , and/or Y u ’ is directly adjacent to an LNA modified nucleotide, preferably, the LNA is attached through a phosphorothioate linkage to the next adjacent nucleotide, which does not comprise the inverted nucleotide.
  • the (conjugated) double stranded nucleic acid of formula (I) of the invention comprises one or more of the following modifications: a) Y2 1 is an LNA modified nucleotide and Yf’ is an inverted nucleotide, b) Y-2 1 is an LNA modified nucleotide and Y u ’ is an inverted nucleotide, or c) Y-2 is an LNA modified nucleotide and Y u is an inverted nucleotide.
  • the (conjugated) double stranded nucleic acid may comprise the modifications of a), b) or c), or any combination thereof, such as, a) and b), a) and c), or b) and c).
  • the (conjugated) double stranded nucleic acid of formula (I) of the invention comprises one or more of the following modifications: a) Y2 1 is an LNA modified nucleotide and Yf’ is an inverted nucleotide, and b) Y-2 1 is an LNA modified nucleotide.
  • the (conjugated) double stranded nucleic acid of formula (I) of the invention comprises one or more of the following modifications: a) Y2 1 is an LNA modified nucleotide and Yf’ is an inverted nucleotide, and b) Y u ’ is an inverted nucleotide.
  • the (conjugated) double stranded nucleic acid of formula (I) of the invention comprises one or more of the following modifications: a) Y2 1 is an LNA modified nucleotide and Yf’ is an inverted nucleotide, and b) Y-2 1 is an LNA modified nucleotide and Y u ’ is an inverted nucleotide.
  • the (conjugated) double stranded nucleic acid of formula (I) of the invention comprises one or more of the following modifications: a) Y2 1 is an LNA modified nucleotide and Yf’ is an inverted nucleotide, and b) Y-2 is an LNA modified nucleotide.
  • the (conjugated) double stranded nucleic acid of formula (I) of the invention comprises one or more of the following modifications: a) Y2 1 is an LNA modified nucleotide and Yf’ is an inverted nucleotide, and b) Y u is an inverted nucleotide.
  • the (conjugated) double stranded nucleic acid of formula (I) of the invention comprises one or more of the following modifications: a) Y2 1 is an LNA modified nucleotide and Yf’ is an inverted nucleotide, and b) Y-2 is an LNA modified nucleotide and Y u is an inverted nucleotide.
  • the LNA modified nucleotide of a) is attached through a phosphorothioate linkage to Y3’
  • the LNA modified nucleotide of b) is attached through a phosphorothioate linkage to Y-3’
  • the LNA modified nucleotide of c) is attached through a phosphorothioate linkage to Y-3.
  • Y-2 is an LNA modified nucleotide and a phosphorothioate linkage is present between Y-2 and Y u , preferably further phosphorothioate linkages are present between Yf and Y2, and preferably also between Y2 and Y3.
  • a phosphorothioate linkage is present between Yf’ and Y2’ and preferably also between Y2’ and Y3’.
  • Y-2 is an LNA modified nucleotide and an (E)-vinylphosphonate nucleotide is present, preferably at position Yf, preferably wherein the (E)-vinylphosphonate is an RNA nucleotide, preferably a uridine.
  • a phosphorothioate linkage is present between Yf’ and Y2’ and preferably also between Y2’ and Y 3 ’.
  • Y-2 in the first strand of the nucleic acid of formula (I) is an LNA modified nucleotide and a phosphorothioate linkage is present between Y-2 and Y u , preferably further phosphorothioate linkages are present between Yf and Y2, and preferably also between Y2 and Y 3 .
  • Y 2 ‘ is an LNA modified nucleotide and Y is an inverted nucleotide and one phosphorothioate linkage is present between Y2 and Y 3 .
  • Y-2 in the first strand of the nucleic acid of formula (I) Y-2 is an LNA modified nucleotide and an (E)-vinylphosphonate nucleotide is present, preferably at position Yf, preferably wherein the (E)-vinylphosphonate is an RNA nucleotide, preferably a uridine.
  • the second strand of the nucleic acid of formula (I) 2 is an LNA modified nucleotide and Y is an inverted nucleotide and one phosphorothioate linkage is present between Y2 and Y 3 .
  • siRNA conjugates with combined LNA and inverted ribonucleotides increased reduction of target mRNA levels in vitro and in vivo as compared to siRNA conjugates without LNA and/or inverted ribonucleotide modifications.
  • siRNAs with an LNA modification at position Y-2 of the first strand and/or Y2' of the second strand and one phosphorothioate internucleotide linkage between Y-2 and Y u , and between Y2’ and Ya’ show improved or at least comparable stability over siRNA molecules with the same LNA modifications but with two phosphorothioate internucleotide linkages between the three terminal nucleotides each at the 3' end of the first and second strand.
  • An inverted nucleotide refers to a nucleotide which is linked to the adjoining nucleotide through its 3’ OH group of the ribose moiety to form a 3'-3' phosphodiester bond, rather than through its 5’ OH group, as would normally be the case.
  • an inverted nucleotide refers to a nucleotide that is linked to the adjoining nucleotide through its 5' OH group of the ribose moiety to form a 5'-5' phosphodiester bond, rather than through it 3' OH group, as would normally be the case.
  • the inverted nucleotide is linked to the adjoining nucleotide through its 3’ OH group of the ribose moiety to form a 3’-3’ phosphorothioate bond.
  • the inverted nucleotide is linked to the adjoining nucleotide through its 5’ OH group of the ribose moiety to form a 5’-5’ phosphorothioate bond.
  • the inverted nucleotide is linked to the adjoining nucleotide through its 5’ OH group of the ribose moiety to form a 5’-5’ phosphorothioate bond.
  • the inverted nucleotide is a ribonucleotide, preferably wherein the ribonucleotide is a purine, preferably wherein the purine is an adenine or guanine, preferably an adenine.
  • the inverted nucleotide is a 2'- deoxynucleotide.
  • Nucleic acids of the invention of formula (I) include unmodified RNA as well as RNA which has been modified, e.g., to improve efficacy or stability.
  • Unmodified RNA refers to a molecule in which the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are the same or essentially the same as those which occur in nature, for example as occur naturally in the human body.
  • modified nucleotide refers to a nucleotide in which one or more of the components of the nucleotide, namely the sugar, base, and phosphate moiety, is/are different from those which occur in nature.
  • modified nucleotide also refers in certain cases to molecules that are not nucleotides in the strict sense of the term because they lack, or have a substitute of, an essential component of a nucleotide, such as the sugar, base or phosphate moiety.
  • a nucleic acid comprising such modified nucleotides is still to be understood as being a nucleic acid, even if one or more of the nucleotides of the nucleic acid has been replaced by a modified nucleotide that lacks, or has a substitution of, an essential component of a nucleotide.
  • Modifications of the nucleic acid of the present invention generally provide a powerful tool in overcoming potential limitations including, but not limited to, in vitro and in vivo stability and bioavailability inherent to native RNA molecules.
  • the nucleic acids according to the invention may be modified by chemical modifications. Modified nucleic acids can also minimise the possibility of inducing interferon activity in humans. Modifications can further enhance the functional delivery of a nucleic acid to a target cell.
  • the modified nucleic acids of the present invention may comprise one or more chemically modified ribonucleotides of either or both of the first strand or the second strand.
  • a ribonucleotide may comprise a chemical modification of the base, sugar or phosphate moieties.
  • the ribonucleic acid may be modified by substitution with or insertion of analogues of nucleic acids or bases.
  • “same or common modification” means the same modification to any nucleotide, be that A, G, C or II modified with a group such as a methyl group (2’-0Me) or a fluoro group (2’-F).
  • 2'-F-dll, 2'-F-dA, 2'-F-dC, 2'-F-dG are all considered to be the same or common modification, as are 2'-OMe-rll, 2'-OMe-rA; 2'-0Me- rC; 2'-OMe-rG.
  • a 2’-F modification is a different modification compared to a 2’-0Me modification.
  • At least one nucleotide of the first and/or second strand of the nucleic acid is a modified nucleotide, preferably a non-naturally occurring nucleotide such as preferably a 2’-F modified nucleotide.
  • a modified nucleotide can be a nucleotide with a modification of the sugar group.
  • the 2' hydroxyl group (OH) can be modified or replaced with a number of different “oxy” or “deoxy” substituents.
  • Other substituents of certain embodiments include 2'
  • the sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose.
  • a modified nucleotide may contain a sugar such as arabinose.
  • Modified nucleotides can also include “abasic” sugars, which lack a nucleobase at C - T. These abasic sugars can further contain modifications at one or more of the constituent sugar atoms.
  • the 2' modifications may be used in combination with one or more phosphate internucleotide linker modifications (e.g., phosphorothioate or phosphorodithioate).
  • phosphate internucleotide linker modifications e.g., phosphorothioate or phosphorodithioate.
  • nucleic acid of formula (I) of the present invention may be modified.
  • the nucleic acid may comprise at least one modified nucleotide.
  • the modified nucleotide may be in the first strand.
  • the modified nucleotide may be in the second strand.
  • the modified nucleotide may be in the duplex region.
  • the modified nucleotide may be outside the duplex region, i.e. , in a single-stranded region.
  • the modified nucleotide may be on the first strand and may be outside the duplex region.
  • the modified nucleotide may be on the second strand and may be outside the duplex region.
  • the 3’-terminal nucleotide of the first strand may be a modified nucleotide.
  • the 3’-terminal nucleotide of the second strand may be a modified nucleotide.
  • the 5’-terminal nucleotide of the first strand may be a modified nucleotide.
  • the 5’- terminal nucleotide of the second strand may be a modified nucleotide.
  • the 3’-terminal nucleotide of the duplex region in the first strand (Y u ) may be a modified nucleotide.
  • the 3’- terminal nucleotide of the duplex region in the second strand (Y ) may be a modified nucleotide.
  • the 5’-terminal nucleotide of the duplex region in the first strand may be a modified nucleotide.
  • the 5’-terminal nucleotide of the duplex region in the second strand may be a modified nucleotide.
  • a nucleic acid of formula (I) of the invention may have 1 modified nucleotide or a nucleic acid of formula (I) of the invention may have about 2-4 modified nucleotides, or a nucleic acid may have about 4-6 modified nucleotides, about 6-8 modified nucleotides, about 8-10 modified nucleotides, about 10-12 modified nucleotides, about 12-14 modified nucleotides, about 14-16 modified nucleotides about 16-18 modified nucleotides, about 18-20 modified nucleotides, about 20-22 modified nucleotides, about 22-24 modified nucleotides, about 24-26 modified nucleotides or about 26- 28 modified nucleotides.
  • nucleic acid comprising said modified nucleotides retains at least 50% of its activity as compared to the same nucleic acid but without said modified nucleotides or vice versa.
  • the nucleic acid may retain 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% and intermediate values of its activity as compared to the same nucleic acid but without said modified nucleotides or may have more than 100% of the activity of the same nucleic acid without said modified nucleotides.
  • the modified nucleotide may be a purine or a pyrimidine. At least half of the purines may be modified. At least half of the pyrimidines may be modified. All of the purines may be modified. All of the pyrimidines may be modified.
  • the modified nucleotides may be selected from the group consisting of a 3' terminal deoxy thymine (dT) nucleotide, a 2'-O-methyl (2’-OMe) modified nucleotide, a 2’ modified nucleotide, a 2' deoxy modified nucleotide, an abasic nucleotide, a 2' amino modified nucleotide, a 2' alkyl modified nucleotide, a 2’-deoxy-2’-fluoro (2’-F) modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a nucleotide comprising a 5'-phosphorothioate group, a nucleotide comprising a 5' phosphate or 5' phosphate mimic and a terminal nucleotide linked to a cholesteryl derivative or a dodecanoic acid bisdecy
  • the nucleic acid of formula (I) may comprise a nucleotide comprising a modified base, wherein the base is selected from 2-aminoadenosine, 2, 6-diaminopurine, inosine, pyridin-4-one, pyridin- 2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidine (e.g., 5-methylcytidine), 5-alkyluridine (e.g., ribothymidine), 5- halouridine (e.g., 5-bromouridine), 6-azapyrimidine, 6-alkylpyrimidine (e.g.
  • 6-methyluridine 6-methyluridine
  • propyne quesosine, 2-thiouridine, 4-thiouridine, wybutosine, wybutoxosine,4-acetylcytidine,5-(carboxyhydroxymethyl)uridine, 5'carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, beta-D- galactosylqueosine, 1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine, 3- methylcytidine, 2-methyladenosine, 2-methylguanosine, N6-methyladenosine, 7- methylguanosine, 5-methoxyaminomethyl-2- thiouridine, 5-methylaminomethyluridine, 5- methylcarbonylmethyluridine, 5-methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio-N6- isopentenyladenosine, beta-D-mannosylque
  • modifications described herein and that occur within a nucleic acid will be repeated within a polynucleotide molecule, such as a modification of a base, or a phosphate moiety, or a non-linking O of a phosphate moiety. In some cases, the modification will occur at all of the possible positions/nucleotides in the polynucleotide but in many cases it will not.
  • a modification may only occur at a 3' or 5' terminal position, may only occur in a terminal region, such as at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand.
  • a modification may occur in a double-strand region, a single-strand region, or in both.
  • a modification may occur only in the double-strand region of a nucleic acid of formula (I) of the invention or may only occur in a single-strand region of a nucleic acid of the invention.
  • a phosphorothioate or phosphorodithioate modification at a non-linking O position may only occur at one or both termini, may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4 or 5 nucleotides of a strand, or may occur in duplex and/or in single-strand regions, particularly at termini.
  • the 5' end and/or 3’ end may be phosphorylated.
  • Stability of a nucleic acid of formula (I) of the invention may be increased by including particular bases in overhangs, or by including modified nucleotides, in single-strand overhangs, e.g., in a 5' or 3' overhang, or in both.
  • Purine nucleotides may be included in overhangs. All or some of the bases in a 3' or 5' overhang may be modified. Modifications can include the use of modifications at the 2' OH group of the ribose sugar, the use of deoxyribonucleotides, instead of ribonucleotides, and modifications in the phosphate group, such as phosphorothioate or phosphorodithioate modifications. Overhangs need not be homologous with the target sequence.
  • Nucleases can hydrolyse nucleic acid phosphodiester bonds. However, chemical modifications to nucleic acids can confer improved properties, and can render oligoribonucleotides more stable to nucleases.
  • Modified nucleic acids can include one or more of:
  • alteration e.g., replacement, of one or both of the non-linking phosphate oxygens and/or of one or more of the linking phosphate oxygens (referred to as linking even if at the 5' and 3' terminus of the nucleic acid of the invention);
  • modification of the 3' end or 5' end of the first strand and/or the second strand e.g., removal, modification or replacement of a terminal phosphate group or conjugation of a moiety, e.g., a fluorescently labelled moiety, to either the 3' or 5' end of one or both strands.
  • replacement means a difference from a naturally occurring molecule.
  • the nucleic acid of formula (I) may comprise one or more nucleotides on the second and/or first strands that are modified. Alternating nucleotides may be modified, to form modified nucleotides. “Alternating” as described herein means to occur one after another in a regular way. In other words, alternating means to occur in turn repeatedly. For example, if one nucleotide is modified, the next contiguous nucleotide is not modified and the following contiguous nucleotide is modified and so on. One nucleotide may be modified with a first modification, the next contiguous nucleotide may be modified with a second modification and the following contiguous nucleotide is modified with the first modification and so on, where the first and second modifications are different.
  • the nucleic acid of formula (I) of the invention comprises a duplex region with at least 15 nucleotides, wherein at least nucleotides 2 and 14 of the first strand are modified, preferably by a first common modification, the nucleotides being numbered consecutively starting with nucleotide number 1 at the 5’ end of the duplex region of the first strand, herein represented by Yf (“f” for first). Accordingly, for example, nucleotide Yf is nucleotide number 1 , Y2 is nucleotide 2, and so on, counting towards the Y u at the 3’ end of the duplex region of the first strand.
  • the first common modification is preferably 2’-F.
  • At least one, several or preferably all the even-numbered nucleotides of the first strand are modified, preferably by a first common modification, the nucleotides being numbered consecutively starting with nucleotide number 1 (Yf) at the 5’ end of the duplex region in the first strand.
  • the first common modification is preferably 2’-F.
  • At least one, several or preferably all the odd-numbered nucleotides of the first strand are modified, the nucleotides being numbered consecutively starting with nucleotide number 1 (Yf) at the 5’ end of the duplex region of the first strand.
  • they are modified by a second modification. This second modification is preferably different from the first modification if the nucleic acid also comprises a first modification, for example of nucleotides 2 and 14 or of all the even-numbered nucleotides of the first strand.
  • the first modification is preferably any 2’ ribose modification that is of the same size or smaller in volume than a 2’-OH group, or an unlocked nucleic acid (UNA), or a 2'-Fluoroarabino Nucleic Acid (FANA) modification.
  • a 2’ ribose modification that is of the same size or smaller in volume than a 2’- OH group can for example be a 2’-F, 2’-H, 2’-halo, or 2’-NH2.
  • the second modification is preferably any 2’ ribose modification that is larger in volume than a 2’-OH group.
  • a 2’ ribose modification that is larger in volume than a 2’-OH group can for example be a 2’-0Me, 2’-O- MOE (2’-O-methoxyethyl), 2’-O-allyl or 2’-O-alkyl, with the proviso that the nucleic acid is capable of reducing the expression of the target gene to at least the same extent as the same nucleic acid without the modification(s) under comparable conditions.
  • the first modification is preferably 2’-F and/or the second modification is preferably 2’-0Me.
  • the size or volume of a substituent is preferably measured as the van der Waals volume.
  • At least one, several or preferably all the nucleotides of the second strand in a position corresponding to an even-numbered nucleotide of the first strand are modified, optionally by a third modification.
  • a third modification Preferably in the same nucleic acid nucleotides 2 and 14 or all the even numbered nucleotides of the first strand are modified with a first modification.
  • the odd-numbered nucleotides of the first strand are modified with a second modification.
  • the third modification is different from the first modification and/or the third modification is the same as the second modification.
  • the first modification is preferably any 2’ ribose modification that is of the same size or smaller in volume than a 2’-OH group, or an unlocked nucleic acid (UNA), or a 2'-Fluoroarabino Nucleic Acid (FANA) modification.
  • a 2’ ribose modification that is of the same size or smaller in volume than a 2’- OH group can for example be a 2’-F, 2’-H, 2’-halo, or 2’-NH2.
  • the second and/or third modification is preferably any 2’ ribose modification that is larger in volume than a 2’-OH group.
  • a 2’ ribose modification that is larger in volume than a 2’-OH group can for example be a 2’- OMe, 2’-O-MOE (2’-O-methoxyethyl), 2’-O-allyl or 2’-O-alkyl, with the proviso that the nucleic acid is capable of reducing the expression of the target gene to at least the same extent as the same nucleic acid without the modification(s) under comparable conditions.
  • the first modification is preferably 2’-F and/or the second and/or third modification is/are preferably 2’- OMe.
  • the nucleotides on the first strand are numbered consecutively starting with nucleotide number 1 (Yf) at the 5’ end of the duplex region of the first strand.
  • a nucleotide of the second strand that is in a position corresponding, for example, to an even- numbered nucleotide of the first strand is a nucleotide of the second strand that is base-paired to an even-numbered nucleotide of the first strand, i.e., nucleotides opposite each other respectively in the first and second strand.
  • At least one, several or preferably all the nucleotides of the second strand in a position corresponding to an odd-numbered nucleotide of the first strand are modified, optionally by a fourth modification.
  • Preferably in the same nucleic acid nucleotides 2 and 14 or all the even numbered nucleotides of the first strand are modified with a first modification.
  • the odd-numbered nucleotides of the first strand are modified with a second modification.
  • all the nucleotides of the second strand in a position corresponding to an even-numbered nucleotide of the first strand are modified with a third modification.
  • the fourth modification is preferably different from the second modification and preferably different from the third modification and the fourth modification is preferably the same as the first modification.
  • the first and/or fourth modification is preferably any 2’ ribose modification that is of the same size or smaller in volume than a 2’-OH group, or an unlocked nucleic acid (UNA), or a 2'-Fluoroarabino Nucleic Acid (FANA) modification.
  • a 2’ ribose modification that is of the same size or smaller in volume than a 2’-OH group can for example be a 2’-F, 2’-H, 2’-halo, or 2’-NH2.
  • the second and/or third modification is preferably any 2’ ribose modification that is larger in volume than a 2’-OH group.
  • a 2’ ribose modification that is larger in volume than a 2’-OH group can for example be a 2’-0Me, 2’-0-M0E (2’-O- methoxyethyl), 2’-O-allyl or 2’-O-alkyl, with the proviso that the nucleic acid is capable of reducing the expression of the target gene to at least the same extent as the same nucleic acid without the modification(s) under comparable conditions.
  • the first and/or the fourth modification is/are preferably a 2’-0Me modification and/or the second and/or third modification is/are preferably a 2’-F modification.
  • the nucleotides on the first strand are numbered consecutively starting with nucleotide number 1 (Yf) at the 5’ end of the duplex region of the first strand.
  • nucleic acid of formula (I) wherein the duplex region of the second strand comprises at least 15 nucleotides, the nucleotide/nucleotides of the second strand in a position corresponding to nucleotide 11 or nucleotide 13 or nucleotides 11 and 13 or nucleotides 11- 13 of the first strand is/are modified by a fourth modification.
  • all the nucleotides of the second strand other than the nucleotide/nucleotides in a position corresponding to nucleotide 11 or nucleotide 13 or nucleotides 11 and 13 or nucleotides 11-13 of the first strand is/are modified by a third modification.
  • Y represents nucleotide number 1
  • Y2’ represent nucleotide number 2 and so on.
  • Y represents nucleotide number 1
  • Y2’ represent nucleotide number 2 and so on.
  • the even numbered nucleotides of the first strand are modified with a first modification.
  • the odd-numbered nucleotides of the first strand are modified with a second modification.
  • the fourth modification is preferably different from the second modification and preferably different from the third modification and the fourth modification is preferably the same as the first modification.
  • the first and/or fourth modification is preferably any 2’ ribose modification that is of the same size or smaller in volume than a 2’- OH group, or an unlocked nucleic acid (UNA), or a 2'-Fluoroarabino Nucleic Acid (FANA) modification.
  • a 2’ ribose modification that is of the same size or smaller in volume than a 2’- OH group can for example be a 2’-F, 2’-H, 2’-halo, or 2’-NH2.
  • the second and/or third modification is preferably any 2’ ribose modification that is larger in volume than a 2’-OH group.
  • a 2’ ribose modification that is larger in volume than a 2’-OH group can for example be a 2’- OMe, 2’-O-MOE (2’-O-methoxyethyl), 2’-O-allyl or 2’-O-alkyl, with the proviso that the nucleic acid is capable of reducing the expression of the target gene to at least the same extent as the same nucleic acid without the modification(s) under comparable conditions.
  • the first and/or the fourth modification is/are preferably a 2’-0Me modification and/or the second and/or third modification is/are preferably a 2’-F modification.
  • the nucleotides on the first strand are numbered consecutively starting with nucleotide number 1 (Yf) at the 5’ end of the first strand.
  • all the even-numbered nucleotides of the first strand are modified by a first modification
  • all the odd-numbered nucleotides of the first strand are modified by a second modification
  • all the nucleotides of the second strand in a position corresponding to an even-numbered nucleotide of the first strand are modified by a third modification
  • all the nucleotides of the second strand in a position corresponding to an odd-numbered nucleotide of the first strand are modified by a fourth modification, wherein the first and/or fourth modification is/are 2’-F and/or the second and/or third modification is/are 2’- OMe.
  • all the even-numbered nucleotides of the first strand are modified by a first modification
  • all the odd-numbered nucleotides of the first strand are modified by a second modification
  • all the nucleotides of the second strand in positions corresponding to nucleotides 11-13 of the first strand are modified by a fourth modification
  • all the nucleotides of the second strand other than the nucleotides corresponding to nucleotides 11-13 of the first strand are modified by a third modification, wherein the first and fourth modification are 2’-F and the second and third modification are 2’-0Me.
  • the 3’ terminal nucleotide of the second strand is an inverted RNA nucleotide (i.e. , the nucleotide is linked to the 3’ end of the strand through its 3’ carbon, rather than through its 5’ carbon as would normally be the case).
  • the inverted RNA nucleotide is preferably an unmodified nucleotide in the sense that it does not comprise any modifications compared to the natural nucleotide counterpart.
  • the inverted RNA nucleotide is preferably a 2’-OH nucleotide.
  • the nucleic acid is blunt ended at least at the end that comprises the 5’ end of the first strand.
  • One embodiment of the present invention is a nucleic acid of formula (I) as disclosed herein for inhibiting expression of a target gene, preferably in a cell, wherein said first strand includes modified nucleotides or unmodified nucleotides at a plurality of positions in order to facilitate processing of the nucleic acid by RISC.
  • “facilitate processing by RISC” means that the nucleic acid can be processed by RISC, for example any modification present will permit the nucleic acid to be processed by RISC and preferably, will be beneficial to processing by RISC, suitably such that siRNA activity can take place.
  • nucleic acid of formula (I) as disclosed herein wherein the nucleotides at positions 2 and 14 from the 5’ end of the duplex region of the first strand are not modified with a 2’-0Me modification, and the nucleotide/nucleotides on the second strand which corresponds to position 11 or position 13 or positions 11 and 13 or positions 11 , 12 and 13 of the first strand is/are not modified with a 2’-0Me modification (in other words, they are not modified or are modified with a modification other than 2’-0Me).
  • the nucleotide on the second strand which corresponds to position 13 of the duplex region of the first strand is the nucleotide that forms a base pair with position 13 of the first strand.
  • the nucleotide on the second strand which corresponds to position 11 of the duplex region of the first strand is the nucleotide that forms a base pair with position 11 of the first strand.
  • the nucleotide on the second strand which corresponds to position 12 of the duplex region of the first strand is the nucleotide that forms a base pair with position 12 of the first strand.
  • position 13 (from the 5’ end) of the duplex region of the first strand would pair with position 7 (from the 5’ end) of the duplex region of the second strand.
  • Position 11 (from the 5’ end) of the duplex region of the first strand would pair with position 9 (from the 5’ end) of the duplex region of the second strand. This nomenclature may be applied to other positions of the second strand.
  • the nucleotide on the second strand that “corresponds to” a position on the first strand may not necessarily form a base pair if that position is the position in which there is a mismatch, but the principle of the nomenclature still applies.
  • One embodiment is a nucleic acid of formula (I) as disclosed herein, wherein the nucleotides at positions 2 and 14 from the 5’ end of the duplex region of the first strand are not modified with a 2’-0Me modification, and the nucleotides on the second strand which correspond to position 11 , or 13, or 11 and 13, or 11-13 of the duplex region of the first strand are modified with a 2'-F modification.
  • One embodiment is a nucleic acid of formula (I) as disclosed herein, wherein the nucleotides at positions 2 and 14 from the 5’ end of duplex region of the first strand are modified with a 2'-F modification, and the nucleotides on the second strand which correspond to position 11 , or 13, or 11 and 13, or 11-13 of the first strand are not modified with a 2’-0Me modification.
  • One embodiment is a nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 from the 5’ end of duplex region of the first strand are modified with a 2'-F modification, and the nucleotides on the second strand which correspond to position 11 , or 13, or 11 and 13, or 11-13 of the first strand are modified with a 2'-F modification.
  • One embodiment is a nucleic acid of formula (I) as disclosed herein wherein greater than 50% of the nucleotides of the first and/or second strand comprise a 2’-OMe modification, such as greater than 55%, 60%, 65%, 70%, 75%, 80%, or 85%, or more, of the first and/or second strand comprise a 2’-OMe modification, preferably measured as a percentage of the total nucleotides of both the first and second strands.
  • a 2’-OMe modification such as greater than 55%, 60%, 65%, 70%, 75%, 80%, or 85%, or more
  • One embodiment is a nucleic acid of formula (I) as disclosed herein wherein greater than 50% of the nucleotides of the first and/or second strand comprise a naturally occurring RNA modification, such as wherein greater than 55%, 60%, 65%, 70%, 75%, 80%, or 85% or more of the first and/or second strands comprise such a modification, preferably measured as a percentage of the total nucleotides of both the first and second strands.
  • Suitable naturally occurring modifications include, as well as 2’-0Me, other 2’ sugar modifications, in particular a 2’-H modification resulting in a DNA nucleotide.
  • One embodiment is a nucleic acid of formula (I) as disclosed herein comprising no more than 20%, such as no more than 15% such as no more than 10%, of nucleotides which have 2' modifications that are not 2’-OMe modifications on the first and/or second strand, preferably as a percentage of the total nucleotides of both the first and second strands.
  • One embodiment is a nucleic acid of formula (I) as disclosed herein, wherein the number of nucleotides in the first and/or second strand with a 2’-modification that is not a 2’-OMe modification is no more than 7, more preferably no more than 5, and most preferably no more than 3.
  • One embodiment is a nucleic acid of formula (I) as disclosed herein comprising no more than 20%, (such as no more than 15% or no more than 10%) of 2’-F modifications on the first and/or second strand, preferably as a percentage of the total nucleotides of both strands.
  • One embodiment is a nucleic acid of formula (I) as disclosed herein, wherein the number of nucleotides in the first and/or second strand with a 2’-F modification is no more than 7, more preferably no more than 5, and most preferably no more than 3.
  • One embodiment is a nucleic acid of formula (I) as disclosed herein, wherein all nucleotides are modified with a 2’-OMe modification except positions 2 and 14 from the 5’ end of the duplex region of the first strand and the nucleotides on the second strand which correspond to position 11 , or 13, or 11 and 13, or 11-13 of the first strand.
  • the nucleotides that are not modified with 2’-OMe are modified with fluoro at the 2’ position (2’-F modification).
  • a preferred embodiment is a nucleic acid of formula (I) as disclosed herein wherein all nucleotides of the nucleic acid are modified at the 2’ position of the sugar.
  • these nucleotides are modified with a 2’-F modification where the modification is not a 2’-OMe modification.
  • the nucleic acid of formula (I) is modified in the duplex region on the first strand with alternating 2’-OMe modifications and 2-F modifications, and positions 2 and 14 (starting from the 5’ end) are modified with 2’-F.
  • the second strand is modified with 2’-F modifications at nucleotides on the second strand which correspond to position 11 , or 13, or 11 and 13, or 11- 13 of the duplex region of the first strand.
  • the second strand is modified with 2’-F modifications at positions 11-13 counting from the 3’ end starting at the first position of the complementary (double-stranded) region (or duplex region, i.e. Yf’), and the remaining modifications are naturally occurring modifications, preferably 2’-0Me.
  • the complementary region at least in this case starts at the first position of the second strand (Y ) that has a corresponding nucleotide in the first strand (Yf), regardless of whether the two nucleotides are able to base pair to each other.
  • each of the nucleotides of the first strand and of the second strand is a modified nucleotide.
  • odd numbered means a number not divisible by two. Examples of odd numbers are 1 , 3, 5, 7, 9, 11 and so on.
  • even numbered means a number which is evenly divisible by two. Examples of even numbers are 2, 4, 6, 8, 10, 12, 14 and so on.
  • nucleotides of the first strand are numbered contiguously starting with nucleotide number 1 (Yf) at the 5’ end of the duplex region of the first strand.
  • Nucleotides of the second strand are numbered contiguously starting with nucleotide number 1 (Yf ) at the 3’ end of the duplex region of the second strand.
  • One or more nucleotides on the first and/or second strand may be modified, to form modified nucleotides.
  • One or more of the odd-numbered nucleotides of the first strand may be modified.
  • One or more of the even-numbered nucleotides of the first strand may be modified by at least a second modification, wherein the at least second modification is different from the modification on the one or more odd nucleotides. At least one of the one or more modified even numbered-nucleotides may be adjacent to at least one of the one or more modified odd- numbered nucleotides.
  • a plurality of odd-numbered nucleotides in the first strand may be modified in the nucleic acid of the invention.
  • a plurality of even-numbered nucleotides in the first strand may be modified by a second modification.
  • the first strand may comprise adjacent nucleotides that are modified by a common modification.
  • the first strand may also comprise adjacent nucleotides that are modified by a second different modification (i.e., the first strand may comprise nucleotides that are adjacent to each other and modified by a first modification as well as other nucleotides that are adjacent to each other and modified by a second modification that is different to the first modification).
  • One or more of the odd-numbered nucleotides of the second strand may be modified by a modification that is different to the modification of the odd- numbered nucleotides on the first strand (wherein the nucleotides are numbered contiguously starting with nucleotide number 1 (Yf) at the 5’ end of the first strand) and/or one or more of the even-numbered nucleotides of the second strand may be modified by the same modification of the odd-numbered nucleotides of the first strand.
  • At least one of the one or more modified even-numbered nucleotides of the second strand may be adjacent to the one or more modified odd-numbered nucleotides.
  • a plurality of odd-numbered nucleotides of the second strand may be modified by a common modification and/or a plurality of even-numbered nucleotides may be modified by the same modification that is present on the first strand odd- numbered nucleotides.
  • a plurality of odd-numbered nucleotides on the second strand may be modified by a modification that is different from the modification of the first strand odd- numbered nucleotides.
  • the second strand may comprise adjacent nucleotides that are modified by a common modification, which may be a modification that is different from the modification of the odd- numbered nucleotides of the first strand.
  • each of the odd- numbered nucleotides in the first strand and each of the even-numbered nucleotides in the second strand may be modified with a common modification and, each of the even-numbered nucleotides may be modified in the first strand with a different modification and each of the odd-numbered nucleotides may be modified in the second strand with the different modification.
  • the nucleic acid of formula (I) of the invention may have the modified nucleotides of the first strand shifted by at least one nucleotide relative to the unmodified or differently modified nucleotides of the second strand.
  • one or more or each of the odd numbered-nucleotides may be modified in the first strand and one or more or each of the even-numbered nucleotides may be modified in the second strand.
  • One or more or each of the alternating nucleotides on either or both strands may be modified by a second modification.
  • One or more or each of the even- numbered nucleotides may be modified in the first strand and one or more or each of the even- numbered nucleotides may be modified in the second strand.
  • One or more or each of the alternating nucleotides on either or both strands may be modified by a second modification.
  • One or more or each of the odd-numbered nucleotides may be modified in the first strand and one or more of the odd-numbered nucleotides may be modified in the second strand by a common modification.
  • One or more or each of the alternating nucleotides on either or both strands may be modified by a second modification.
  • One or more or each of the even-numbered nucleotides may be modified in the first strand and one or more or each of the odd-numbered nucleotides may be modified in the second strand by a common modification.
  • One or more or each of the alternating nucleotides on either or both strands may be modified by a second modification.
  • the nucleic acid of formula (I) of the invention may comprise single- or double-stranded constructs that comprise at least two regions of alternating modifications in one or both of the strands. These alternating regions can comprise up to about 12 nucleotides but preferably comprise from about 3 to about 10 nucleotides. The regions of alternating nucleotides may be located at the termini of one or both strands of the nucleic acid of the invention.
  • the nucleic acid may comprise from 4 to about 10 nucleotides of alternating nucleotides at each of the termini (3' and 5') and these regions may be separated by from about 5 to about 12 contiguous unmodified or differently or commonly modified nucleotides.
  • the odd numbered nucleotides of the first strand may be modified and the even numbered nucleotides may be modified with a second modification.
  • the second strand may comprise adjacent nucleotides that are modified with a common modification, which may be the same as the modification of the odd-numbered nucleotides of the first strand.
  • One or more nucleotides of the second strand may also be modified with the second modification.
  • One or more nucleotides with the second modification may be adjacent to each other and to nucleotides having a modification that is the same as the modification of the odd-numbered nucleotides of the first strand.
  • the first strand may also comprise phosphorothioate linkages between the two nucleotides at the 3’ end and at the 5’ end or a phosphorodithioate linkage between the two nucleotides at the 3’ end.
  • the second strand may comprise a phosphorothioate or phosphorodithioate linkage between the two nucleotides at the 5’ end.
  • the second strand may also be conjugated to a ligand at the 5’ end.
  • the nucleic acid of the invention may comprise a first strand comprising adjacent nucleotides that are modified with a common modification.
  • One or more such nucleotides may be adjacent to one or more nucleotides which may be modified with a second modification.
  • One or more nucleotides with the second modification may be adjacent.
  • the second strand may comprise adjacent nucleotides that are modified with a common modification, which may be the same as one of the modifications of one or more nucleotides of the first strand.
  • One or more nucleotides of the second strand may also be modified with the second modification.
  • One or more nucleotides with the second modification may be adjacent.
  • the first strand may also comprise phosphorothioate linkages between the two nucleotides at the 3’ end and at the 5’ end or a phosphorodithioate linkage between the two nucleotides at the 3’ end.
  • the second strand may comprise a phosphorothioate or phosphorodithioate linkage between the two nucleotides at the 3’ end.
  • the second strand may also be conjugated to a ligand at the 5’ end.
  • the nucleotides numbered from 5' to 3' on the first strand and 3' to 5' on the second strand, 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23 and 25 may be modified by a modification on the first strand, wherein “1” corresponds to Yf or Y .
  • the nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 may be modified by a second modification on the first strand, wherein “2” corresponds to Y2.
  • the nucleotides numbered 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23 may be modified by a modification on the second strand, wherein “1” corresponds to Y .
  • nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 may be modified by a second modification on the second strand, wherein “2” corresponds to Y2’.
  • Nucleotides are numbered for the sake of the nucleic acid of the present invention from 5' to 3' on the first strand and 3' to 5' on the second strand. The modifications may continue and extend in the same pattern over the nucleotides of X a , X b and X a ', X b ' if these are present.
  • the nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 may be modified by a modification on the first strand.
  • the nucleotides numbered 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23 may be modified by a second modification on the first strand.
  • the nucleotides numbered 1 ,3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23 may be modified by a modification on the second strand.
  • the nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 may be modified by a second modification on the second strand.
  • first and/or the second strand are shorter than 25 nucleotides in length, such as 19 nucleotides in length, there are no nucleotides numbered 20, 21 , 22, 23, 24 and 25 to be modified.
  • the skilled person understands the description above to apply to shorter strands, accordingly.
  • the disclosed modifications may be at the mentioned positions within the duplex region of the first and second strands, however, if any overhangs are present, the overhangs may also comprise any of the disclosed modifications.
  • the skilled person understands how to extend a certain modification pattern such as “odd numbered” and “even numbered” modification pattern, to a present overhang, i.e.
  • nucleotides outside the duplex region herein represented by X a , X b and X a ', X b ' respectively.
  • One or more modified nucleotides on the first strand may be paired with modified nucleotides on the second strand having a common modification.
  • One or more modified nucleotides on the first strand may be paired with modified nucleotides on the second strand having a different modification.
  • One or more modified nucleotides on the first strand may be paired with unmodified nucleotides on the second strand.
  • One or more modified nucleotides on the second strand may be paired with unmodified nucleotides on the first strand.
  • the alternating nucleotides can be aligned on the two strands such as, for example, all the modifications in the alternating regions of the second strand are paired with identical modifications in the first strand or alternatively the modifications can be offset by one nucleotide with the common modifications in the alternating regions of one strand pairing with dissimilar modifications (i.e., a second or further modification) in the other strand.
  • dissimilar modifications i.e., a second or further modification
  • the modifications on the first strand may be shifted by one nucleotide relative to the modified nucleotides on the second strand, such that common modified nucleotides are not paired with each other.
  • the modification and/or modifications may each and individually be selected from the group consisting of 3' terminal deoxy thymine, 2'-0Me, a 2' deoxy modification, a 2' amino modification, a 2' alkyl modification, a morpholino modification, a phosphoramidate modification, 5'-phosphorothioate group modification, a 5' phosphate or 5' phosphate mimic modification and a cholesteryl derivative or a dodecanoic acid bisdecylamide group modification and/or the modified nucleotide may be any one of an abasic nucleotide or a nonnatural base comprising nucleotide.
  • At least one modification may be 2'-0Me and/or at least one modification may be 2'-F. Further modifications as described herein may be present on the first and/or second strand.
  • the nucleic acid of formula (I) of the invention may comprise an inverted RNA nucleotide at one or several of the strand ends.
  • Such inverted nucleotides provide stability to the nucleic acid.
  • the nucleic acid of formula (I) comprises at least an inverted nucleotide at the 3’ end of the duplex region of the first and/or the second strand and/or at the 5’ end of the second strand. More preferably, the nucleic acid of formula (I) comprises an inverted nucleotide at the 3’ end of the second strand.
  • the nucleic acid of formula (I) comprises an inverted RNA nucleotide at the 3’ end of the second strand and this nucleotide is preferably an inverted A.
  • the inverted nucleotide is preferably present at an end of a strand not as an overhang but opposite a corresponding nucleotide in the other strand, i.e. within the duplex region. Accordingly, the nucleic acid is preferably blunt ended at the end that comprises the inverted RNA nucleotide.
  • An inverted RNA nucleotide being present at the end of a strand preferably means that the last nucleotide at this end of the strand is the inverted RNA nucleotide.
  • the inverted RNA nucleotide is preferably an unmodified nucleotide in the sense that it does not comprise any modifications compared to the natural nucleotide counterpart.
  • the inverted RNA nucleotide is preferably a 2’-OH nucleotide.
  • Nucleic acids of formula (I) of the invention may comprise one or more nucleotides modified at the 2’ position with a 2’-H, and therefore having a DNA nucleotide within the nucleic acid.
  • Nucleic acids of formula (I) of the invention may comprise DNA nucleotides at positions 2 and/or 14 of the first strand counting from the 5’ end of the first strand within the duplex region (i.e. counting from Yf).
  • Nucleic acids of formula (I) may comprise DNA nucleotides on the second strand which correspond to position 11 , or 13, or 11 and 13, or 11-13 of the first strand.
  • the nucleic acid of formula (I) may comprise a first modification and a second or further modification which are each and individually selected from the group comprising 2'-0Me modification and 2'-F modification.
  • the nucleic acid of formula (I) may comprise a modification that is 2'-0Me that may be a first modification, and a second modification that is 2'-F.
  • the nucleic acid of formula (I) of the invention may also include a phosphorothioate or phosphorodithioate modification and/or a deoxy modification which may be present in or between the terminal 2 or 3 nucleotides of each or any end of each or both strands.
  • At least one nucleotide of the first and/or second strand is a modified nucleotide, wherein if the first strand comprises at least one modified nucleotide:
  • At least one, several, or all the nucleotides of the second strand in a position corresponding to nucleotide 11 or nucleotide 13 or nucleotides 11 and 13 or nucleotides 11-13 of the first strand is/are modified by a fourth modification;
  • nucleotides on the first strand are numbered consecutively starting with nucleotide number 1 (Yf) at the 5’ end of the first strand; wherein the modifications are preferably at least one of the following:
  • the first modification is preferably different from the second and from the third modification
  • the first modification is preferably the same as the fourth modification
  • the first modification is preferably a 2’-F modification
  • the second modification is preferably a 2’-0Me modification
  • the third modification is preferably a 2’-0Me modification.
  • the fourth modification is preferably a 2’-F modification; and wherein optionally the nucleic acid is conjugated to a heterologous moiety.
  • One embodiment of the invention is the nucleic acid of formula (I), particularly the conjugated nucleic acid as defined herein, wherein the further modifications on the first and/or second strands are non-naturally occurring nucleotides such as a 2’-F modified nucleotide.
  • One embodiment of the invention is the nucleic acid of formula (I), particularly the conjugated nucleic acid as defined herein, wherein i) the first strand of the nucleic acid has a length in the range of 15-30 nucleotides, preferably 19-25 nucleotides; and/or ii) the second strand of the nucleic acid has a length in the range of 15-30 nucleotides, preferably 19-25 nucleotides.
  • One embodiment of the invention is the nucleic acid of formula (I), particularly the conjugated nucleic acid as defined herein, wherein i) the first strand of the nucleic acid has a length in the range of 14-30 nucleotides, preferably 19-25 nucleotides; and/or ii) the second strand of the nucleic acid has a length in the range of 14-30 nucleotides, preferably 19-25 nucleotides.
  • the invention relates to a nucleic acid of formula (I), wherein i) the first strand of the nucleic acid has a length of between 16 and 28 nucleotides; and/or ii) the second strand of the nucleic acid has a length of between 18 and 23 nucleotides.
  • One embodiment of the invention is the nucleic acid of formula (I), particularly the conjugated nucleic acid as defined herein, wherein i) the first strand of the nucleic acid has a length in the range of 19-25 nucleotides; and/or ii) the second strand of the nucleic acid has a length in the range of 19-25 nucleotides.
  • the invention relates to a nucleic acid of formula (I), wherein i) the first strand of the nucleic acid has a length of between 17 and 25 nucleotides; and ii) the second strand of the nucleic acid has a length of between 18 and 23 nucleotides.
  • the invention relates to a nucleic acid of formula (I), wherein the first strand and the second of the nucleic acid each has a length of 19 nucleotides.
  • the invention relates to a nucleic acid of formula (I), wherein i) the first strand of the nucleic acid has a length of between of between 14 and 30, preferably between 16 and 28, more preferably between 19 and 23 nucleotides; and ii) the second strand of the nucleic acid has a length of between 14 and 30, preferably between 16 and 28, more preferably between 19 and 23 nucleotides; and
  • the double stranded region between the first and the second strands is blunt ended at both ends of the double stranded region.
  • the invention relates to a nucleic acid of formula (I), wherein the first strand and the second of the nucleic acid each has a length of 19 nucleotides and wherein the double stranded region between the first and the second strands is blunt ended at both ends of the double stranded region.
  • the invention relates to a nucleic acid of formula (I), wherein i) the first strand of the nucleic acid has a length of between of between 14 and 30, preferably between 16 and 28, more preferably between 19 and 23 nucleotides; and ii) the second strand of the nucleic acid has a length of between 14 and 30, preferably between 16 and 28, more preferably between 19 and 23 nucleotides; and
  • the double stranded region between the first and the second strands is blunt ended at the end that comprises the 3’ end of the first strand end of the double stranded region.
  • the invention relates to a nucleic acid of formula (I), wherein the first strand and the second of the nucleic acid each has a length of 19 nucleotides and wherein the double stranded region between the first and the second strands is blunt ended at the end that comprises the 3’ end of the first strand end of the double stranded region.
  • the invention relates to a nucleic acid of formula (I), wherein i) the first strand of the nucleic acid has a length of between of between 14 and 30, preferably between 16 and 28, more preferably between 19 and 23 nucleotides; and ii) the second strand of the nucleic acid has a length of between 14 and 30, preferably between 16 and 28, more preferably between 19 and 23 nucleotides; and
  • the double stranded region between the first and the second strands is blunt ended at the end that comprises the 5’ end of the first strand end of the double stranded region.
  • the invention relates to a nucleic acid of formula (I), wherein the first strand and the second of the nucleic acid each has a length of 19 nucleotides and wherein the double stranded region between the first and the second strands is blunt ended at the end that comprises the 5’ end of the first strand end of the double stranded region.
  • the invention relates to a nucleic acid of formula (I), wherein the doublestranded region between the first and the second strands has a length in the range of 14-30, preferably of 16-28 nucleotides, more preferably of 18-25, most preferably of 19-23 nucleotides.
  • the invention relates to a nucleic acid of formula (I), wherein the double-stranded region between the first and the second strands has a length of 19 nucleotides.
  • the invention relates to a nucleic acid of formula (I), wherein the double stranded region between the first and the second strands has a length in the range of 14-30, preferably of 16-28 nucleotides, more preferably of 18-25, most preferably of 19-23 nucleotides; and wherein the nucleic acid of formula (I) is blunt ended at both ends.
  • the invention relates to a nucleic acid of formula (I), wherein the double-stranded region between the first and the second strands has a length of 19 nucleotides and wherein the nucleic acid of formula (I) is blunt ended at both ends.
  • the invention relates to a nucleic acid of formula (I), wherein the double stranded region between the first and the second strands has a length in the range of 14-30, preferably of 16-28 nucleotides, more preferably of 18-25, most preferably of 19-23 nucleotides; and wherein the nucleic acid of formula (I) is blunt ended at the end that comprises the 3’ end of the first strand end of the double stranded region.
  • the invention relates to a nucleic acid of formula (I), wherein the double-stranded region between the first and the second strands has a length of 19 nucleotides and wherein the nucleic acid of formula (I) is blunt ended at the end that comprises the 3’ end of the first strand end of the double stranded region.
  • the invention relates to a nucleic acid of formula (I), wherein the double stranded region between the first and the second strands has a length in the range of 14-30, preferably of 16-28 nucleotides, more preferably of 18-25, most preferably of 19-23 nucleotides; and wherein the nucleic acid of formula (I) is blunt ended at the end that comprises the 5’ end of the first strand end of the double stranded region.
  • the invention relates to a nucleic acid of formula (I), wherein the double-stranded region between the first and the second strands has a length of 19 nucleotides and wherein the nucleic acid of formula (I) is blunt ended at the end that comprises the 5’ end of the first strand end of the double stranded region.
  • the 3' and 5' ends of an oligonucleotide can be modified. Such modifications can be at the 3' end or the 5' end or both ends of the molecule. They can include modification or replacement of an entire terminal phosphate or of one or more of the atoms of the phosphate group.
  • the 3' and 5' ends of an oligonucleotide can be conjugated to other functional molecular entities such as labelling moieties, e.g., fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) or protecting groups (based, e.g., on sulfur, silicon, boron or ester).
  • labelling moieties e.g., fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) or protecting groups (based, e.g., on sulfur, silicon, boron or ester).
  • the functional molecular entities can be attached to the sugar through a phosphate group and/or a linker.
  • the terminal atom of the linker can connect to or replace the linking atom of the phosphate group or the C-3' or C-5' O, N, S or C group of the sugar.
  • the linker can connect to or replace the terminal atom of a nucleotide surrogate (e.g., PNAs).
  • the 3' end can be an — OH group.
  • terminal modifications include dyes, intercalating agents (e.g., acridines), cross-linkers (e.g., psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases, EDTA, lipophilic carriers (e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1 ,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1 ,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid
  • Terminal modifications can also be useful for monitoring distribution, and in such cases the groups to be added may include fluorophores, e.g., fluorescein or an Alexa dye. Terminal modifications can also be useful for enhancing uptake, useful modifications for this include cholesterol. Terminal modifications can also be useful for cross-linking an RNA agent to another moiety.
  • fluorophores e.g., fluorescein or an Alexa dye.
  • Terminal modifications can also be useful for enhancing uptake, useful modifications for this include cholesterol. Terminal modifications can also be useful for cross-linking an RNA agent to another moiety.
  • Terminal modifications can be added for a number of reasons, including to modulate activity or to modulate resistance to degradation.
  • Terminal modifications useful for modulating activity include modification of the 5' end with phosphate or phosphate analogues.
  • Nucleic acids of the invention, on the first or second strand, may be 5' phosphorylated or include a phosphoryl analogue at the 5' prime terminus.
  • 5'-phosphate modifications include those which are compatible with RISC mediated gene silencing. Suitable modifications include:
  • alkylether methoxymethyl (MeOCH2-), ethoxymethyl, etc.; e.g., RP(0H)(0)-0-5’-) (wherein R is an alkylether)).
  • moieties may be linked to the 5' terminus of the first strand or the second strand. These include abasic ribose moiety, abasic deoxyribose moiety, modifications abasic ribose and abasic deoxyribose moieties including 2'-0 alkyl modifications; inverted abasic ribose and abasic deoxyribose moieties and modifications thereof, C6-imino-Pi; a mirror nucleotide including L-DNA and L-RNA; 5'0Me nucleotide; and nucleotide analogues including 4', 5'- methylene nucleotide; 1-(P-D-erythrofuranosyl)nucleotide; 4 '-thio nucleotide, carbocyclic nucleotide; 5'-amino-alkyl phosphate; 1 ,3-diamino-2-propyl phosphate, 3-
  • a C-terminal “-0H” moiety may be substituted for a C- terminal “-NH2” moiety, and vice-versa.
  • the invention also provides a nucleic acid of formula (I) according to any embodiment of the invention described herein, wherein the first strand has a terminal 5’ (E)-vinylphosphonate nucleotide at its 5’ end.
  • This terminal 5’ (E)-vinylphosphonate nucleotide is preferably linked to the second nucleotide in the first strand by a phosphodiester linkage.
  • the terminal 5’ (E)-vinylphosphonate (“vp”) nucleotide is a uridine (“vp-ll”).
  • the first strand of the nucleic acid may comprise formula (I):
  • n is from 1 to (the total number of nucleotides in the first strand - 2), preferably wherein n is from 1 to (the total number of nucleotides in the first strand -3), more preferably wherein n is from 1 to (the total number of nucleotides in the first strand -4).
  • the terminal 5’ (E)-vinylphosphonate nucleotide is an RNA nucleotide, preferably a (vp)-U.
  • a terminal 5’ (E)-vinylphosphonate nucleotide is a nucleotide wherein the phosphate group at the 5’-end of the ribose has been replaced with a E-vinylphosphonate group:
  • the first strand of the nucleic acid of formula (I) has a terminal 5’ (E)- vinylphosphonate nucleotide at its 5’ end, preferably at the 5’ end of the duplex region, the terminal 5’ (E)-vinylphosphonate nucleotide is linked to the second nucleotide in the first strand by a phosphodiester linkage and the first strand comprises a) more than 1 phosphodiester linkage; b) phosphodiester linkages between at least the terminal three 5’ nucleotides and/or c) phosphodiester linkages between at least the terminal four 5’ nucleotides.
  • the first strand and/or the second strand of the nucleic acid of formula (I) comprises at least one phosphorothioate (ps) and/or at least one phosphorodithioate (ps2) linkage between two nucleotides.
  • the first strand and/or the second strand of the nucleic acid comprises more than one phosphorothioate and/or more than one phosphorodithioate linkage.
  • the first strand and/or the second strand of the nucleic acid of formula (I) comprises a phosphorothioate or phosphorodithioate linkage between the terminal two 3’ nucleotides of the duplex region.
  • the second strand of the nucleic acid of formula (I) can further comprise phosphorothioate or phosphorodithioate linkages between the terminal two or three 3’ nucleotides of the duplex region.
  • the first strand and/or the second strand of the nucleic acid of formula (I) comprises a phosphorothioate linkage between the terminal two 5’ nucleotides or a phosphorothioate linkages between the terminal three 5’ nucleotides, preferably the nucleic acid comprises a phosphorothioate linkage between the terminal two 5’ nucleotides of the duplex region or a phosphorothioate linkages between the terminal three 5’ nucleotides of the duplex region.
  • the nucleic acid of formula (I) of the present invention comprises one or more phosphorothioate or phosphorodithioate modifications on one or more of the terminal ends of the first and/or the second strand, preferably of the terminal ends of the duplex region of the first and/or the second strand.
  • each or either end of the first strand may comprise one or two or three phosphorothioate or phosphorodithioate modified nucleotides (internucleotide linkage).
  • each or either end of the second strand may comprise one or two or three phosphorothioate or phosphorodithioate modified nucleotides (internucleotide linkage).
  • the nucleic acid of formula (I) comprises a phosphorothioate linkage between the terminal two or three 3’ nucleotides of the duplex region of the second strand and/or 5’ nucleotides of the duplex region of the first and/or the second strand, preferably the terminal two or three 3’ nucleotides of the duplex region of the second strand and/or 5’ nucleotides of the duplex region of the first and/or the second strands.
  • the nucleic acid of formula (I) comprises a phosphorothioate linkage between each of the terminal three 5’ nucleotides of the duplex region of the first strand and the terminal three 3’ nucleotides of the duplex region of the second strand.
  • the nucleic acid of formula (I) comprises a phosphorodithioate linkage between each of the two, three or four terminal nucleotides at the 3’ end of the first strand, preferably the terminal nucleotides at the 3’ end of the duplex region of the first strand, and/or comprises a phosphorodithioate linkage between each of the two, three or four terminal nucleotides at the 3’ end of the second strand, preferably the terminal nucleotides at the 3’ end of duplex region of the second strand, and/or a phosphorodithioate linkage between each of the two, three or four terminal nucleotides at the 5’ end of the second strand, preferably the terminal nucleotides at the 5’ end of the duplex region of the second strand and comprises a linkage other than a phosphorodithioate linkage between the two, three or four terminal nucleotides at the 5’ end of the first strand, preferably the terminal nucle
  • the nucleic acid of formula (I) comprises a phosphorothioate linkage between the terminal three 5’ nucleotides of the duplex region of the first strand.
  • (i) has a phosphorothioate linkage between the terminal three 5’ nucleotides of the first strand, or the terminal three 5’ nucleotides of the duplex region of the first strand and may comprise further phosphorothioate linkages;
  • (ii) is conjugated to a triantennary ligand either on the 3’ end nucleotide or on the 5’ end nucleotide of the second strand;
  • (iii) has a phosphorothioate linkage between the terminal three or two nucleotides of the second strand at the end opposite to the one conjugated to the triantennary ligand and may comprise further phosphorothioate linkages;
  • a phosphorodithioate linkage in the nucleic acid of formula (I) of the invention reduces the variation in the stereochemistry of a population of nucleic acid molecules compared to molecules comprising a phosphorothioate in that same position.
  • Phosphorothioate linkages introduce chiral centres and it is difficult to control which non-linking oxygen is substituted for sulphur.
  • the use of a phosphorodithioate ensures that no chiral centre exists in that linkage and thus reduces or eliminates any variation in the population of nucleic acid molecules, depending on the number of phosphorodithioate and phosphorothioate linkages used in the nucleic acid molecule.
  • the nucleic acid of formula (I) comprises a phosphorodithioate linkage between the two terminal nucleotides at the 3’ end of the duplex region of the second strand and a phosphorodithioate linkage between the two terminal nucleotides at the 5’ end of the duplex region of the second strand and comprises a linkage other than a phosphorodithioate linkage between the two, three or four terminal nucleotides at the 5’ end of the duplex region of the first strand.
  • the first strand has a terminal 5’ (E)-vinylphosphonate nucleotide at its 5’ end of the duplex region.
  • This terminal 5’ (E)-vinylphosphonate nucleotide is preferably linked to the second nucleotide in the first strand by a phosphodiester linkage.
  • all the linkages between the nucleotides of both strands other than the linkage between the two terminal nucleotides at the 3’ end of the duplex region of the first strand and the linkages between the two terminal nucleotides at the 3’ end and at the 5’ end of the duplex region of the second strand are phosphodiester linkages.
  • the nucleic acid of formula (I) comprises a phosphorothioate linkage between each of the three terminal 5’ nucleotides on the first strand, preferably of the terminal 5’ nucleotides of the duplex region on the first strand, and/or between each of the three terminal 3’ nucleotides of the duplex region of the second strand and/or between each of the three terminal 5’ nucleotides of the duplex region of the second strand when there is no phosphorodithioate linkage present at that end, preferably of the terminal 3’ and/or 5’ nucleotides of the duplex region on the second strand.
  • No phosphorodithioate linkage being present at an end means that the linkage between the two terminal nucleotides, or preferably between the three terminal nucleotides of the nucleic acid end in question are linkages other than phosphorodithioate linkages.
  • all the linkages of the nucleic acid of formula (I) between the nucleotides of both strands other than the linkage between the two terminal nucleotides at the 3’ end of the first strand and the linkages between the two terminal nucleotides at the 3’ end and at the 5’ end of the second strand are phosphodiester linkages.
  • the phosphate linker can also be modified by replacement of a linking oxygen with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at a terminal oxygen. Replacement of the non-linking oxygens with nitrogen is possible.
  • the phosphate groups can also individually be replaced by non-phosphorus containing connectors.
  • moieties which can replace the phosphate group include siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.
  • replacements may include the methylenecarbonylamino and methylenemethylimino groups.
  • the phosphate linker and ribose sugar may be replaced by nuclease resistant nucleotides.
  • nuclease resistant nucleotides examples include the morpholino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates.
  • PNA surrogates may be used.
  • the nucleic acid of formula (I), which is preferably an siRNA that inhibits expression of a desired target gene, preferably via RNAi, and preferably in a cell, comprises one or more or all of:
  • a modified nucleotide other than a 2’-OMe modified nucleotide at positions 2 and 14 from the 5’ end of the duplex region of the first strand preferably a 2’-F modified nucleotide
  • each of the odd-numbered nucleotides of the first strand as numbered starting from one at the 5’ end of the duplex region of the first strand are 2’-OMe modified nucleotides;
  • each of the even-numbered nucleotides of the first strand as numbered starting from one at the 5’ end of the duplex region of the first strand are 2’-F modified nucleotides;
  • the second strand nucleotide corresponding to position 11 and/or 13 or 11-13 of the first strand is modified by a modification other than a 2’-0Me modification, preferably wherein one or both or all of these positions comprise a 2’-F modification;
  • the duplex region of the first strand has a terminal 5’ (E)- vinylphosphonate nucleotide at its 5’ end, in which case the terminal 5’ (E)-vinylphosphonate nucleotide is preferably a uridine and is preferably linked to the second nucleotide in the first strand by a phosphodiester linkage.
  • the nucleic acids of formula (I) of the invention may be conjugated to a heterologous moiety.
  • a heterologous moiety is any moiety which is not a nucleic acid molecule capable of inhibiting expression of a desired target gene.
  • a heterologous moiety may be, or may comprise, a peptide (or polypeptide), a saccharide (or polysaccharide), a lipid, a different nucleic acid, or any other suitable molecule.
  • Any given nucleic acid of formula (I) may be conjugated to a plurality of heterologous moieties, which may be the same or different.
  • An individual heterologous moiety may itself comprise one or more functional moieties (such as targeting agents as described in more detail below), each optionally covalently associated to the nucleic acid via a linker.
  • the heterologous moiety may comprise a targeting agent.
  • the targeting agent targets the single-stranded or a double-stranded nucleic acid of formula (I), particularly the single-stranded or the double-stranded RNA, to a target cell.
  • a heterologous moiety, or the functional component thereof may serve for example to modulate bioavailability or pharmacokinetics. For example, it may increase half-life in vivo.
  • a heterologous moiety (or the functional component thereof) may comprise a targeting agent. Efficient delivery of oligonucleotides, in particular double-stranded nucleic acids of the invention, to cells in vivo is important and requires specific targeting and substantial protection from the extracellular environment, particularly serum proteins.
  • One method of achieving specific targeting is to conjugate a targeting agent to the nucleic acid, wherein the targeting agent helps in targeting the nucleic acid to a target cell which has a cell surface receptor that binds to the targeting agent.
  • the term “receptor” is used to include any molecule on the surface of a target cell capable of binding to the targeting agent and should not be taken to imply any particular function for the cell surface receptor.
  • the targeting agent may be regarded as a “ligand” for the cell surface receptor.
  • the terms “targeting agent” and “ligand” may be used interchangeably. Again, this terminology should not be taken to imply any particular function for the targeting agent or the cell surface receptor, or any particular relationship between the two molecules other than the ability of one to bind to the other.
  • the targeting agent may be any moiety having affinity for the chosen receptor. It may, for example, be an affinity protein (such as an antibody or a fragment thereof having affinity for the chosen receptor), an aptamer, or any other suitable moiety. In some embodiments, the targeting agent may be a physiological ligand for the receptor.
  • Binding between the targeting agent and the receptor may promote uptake of the conjugated nucleic acid by the target cell, e.g., via internalization of the receptor, or any other suitable mechanism.
  • appropriate ligands for the desired receptor molecules may be used as targeting agents in order for the conjugated nucleic acids to be taken up by the target cells by mechanisms such as different receptor-mediated endocytosis pathways or functionally analogous processes.
  • a ligand which can mediate internalization of the nucleic acid into a target cell by mechanisms other than receptor mediated endocytosis may alternatively be conjugated to a nucleic acid of the invention for cell or tissue specific targeting.
  • ASGP Asialoglycoprotein receptor complex
  • a ligand that mediates receptor mediated endocytosis is the GalNAc moiety described herein, which has high affinity to the asialoglycoprotein receptor complex (ASGP- R).
  • the ASGP-R complex is composed of varying ratios of multimers of membrane ASGR1 and ASGR2 receptors, which are highly abundant on hepatocytes.
  • One of the first disclosures of the use of triantennary cluster glycosides as conjugated ligands was in US patent number US 5,885,968.
  • Conjugates having three GalNAc ligands and comprising phosphate groups are known and are described in Dubber et al. (Bioconjug. Chem. 2003 Jan-Feb; 14(1):239-46.).
  • the ASGP-R complex shows a 50-fold higher affinity for N-Acetyl-D-Galactosamine (GalNAc) than D-Gal.
  • the ASGP-R complex recognizes specifically terminal p-galactosyl subunits of glycosylated proteins or other oligosaccharides 191 and can be used for delivering a drug to the liver’s hepatocytes expressing the receptor complex by covalent coupling of galactose or galactosamine to the drug substance (Ishibashi.S. ; et. al., J Biol. Chem. 1994 Nov 11 ;269(45):27803-6). Furthermore, the binding affinity can be significantly increased by the multi-valency effect, which is achieved by the repetition of the targeting moiety 1101 .
  • the ASGP-R complex is a mediator for an active uptake of terminal p-galactosyl containing glycoproteins to the cell’s endosomes.
  • the ASGPR is highly suitable for targeted delivery of drug candidates conjugated to such ligands like, e.g., nucleic acids into receptor-expressing cells [11] .
  • the ligand can comprise a saccharide that is selected to have an affinity for at least one type of receptor on a target cell.
  • the receptor is on the surface of a mammalian liver cell, for example, the hepatic asialoglycoprotein receptor complex described before (ASGP-R).
  • the saccharide may be selected from N-acetyl galactosamine, mannose, galactose, glucose, glucosamine and fucose.
  • the saccharide may be N-acetyl galactosamine (GalNAc).
  • the heterologous moiety may comprise a plurality of such saccharides, e.g., two or especially three such saccharides, e.g. three GalNAc groups.
  • a heterologous moiety may therefore comprise (i) one or more functional components, and (ii) a linker, wherein the linker conjugates the functional components to a nucleic acid as defined in any preceding embodiments.
  • the linker may be a monovalent structure or bivalent or trivalent or tetravalent branched structure.
  • the nucleotides may be modified as defined herein.
  • the functional components may therefore be ligands (or targeting agents). Where multiple functional components are present, they may be the same or different. Where the functional components are ligands, they may be saccharides, and may therefore be (or comprise) GalNAc.
  • the invention relates to the conjugated nucleic acid, wherein the nucleic acid of formula (I) is conjugated to a heterologous moiety.
  • the conjugated nucleic acid is conjugated to a heterologous moiety, wherein the heterologous moiety comprises (i) one or more N-acetyl galactosamine (GalNAc) moieties or derivatives thereof, and (ii) a linker, wherein the linker conjugates the at least one GalNAc moiety or derivative thereof to the nucleic acid.
  • the conjugated nucleic acid is conjugated to a heterologous moiety comprising a compound of formula (II):
  • S represents a functional component, e.g. a ligand, such as a saccharide, preferably wherein the saccharide is N-acetyl galactosamine;
  • X 1 represents C3-C6 alkylene or (-CH2-CH2-O) m (-CH2)2- wherein m is 1 , 2, or 3;
  • P is a phosphate or modified phosphate, preferably a thiophosphate
  • A is a branching unit
  • X 3 represents a bridging unit; wherein a nucleic acid as defined in any of the preceding claims is conjugated to X 3 via a phosphate or modified phosphate, preferably a thiophosphate.
  • the branching unit “A” preferably branches into three in order to accommodate three saccharide ligands.
  • the branching unit is preferably covalently attached to the remaining tethered portions of the ligand and the nucleic acid.
  • the branching unit may comprise a branched aliphatic group comprising groups selected from alkyl, amide, disulphide, polyethylene glycol, ether, thioether and hydroxyamino groups.
  • the branching unit may comprise groups selected from alkyl and ether groups.
  • the branching unit may have the structure:
  • the branching unit may have the structure:
  • the branching unit may have the structure:
  • branching unit A may have a structure selected from: wherein:
  • R 1 is hydrogen or 01-010 alkylene; and R 2 is 01-010 alkylene.
  • the branching unit consists of only a carbon atom.
  • the “X 3 ” portion is a bridging unit.
  • the bridging unit is linear and is covalently bound to the branching unit and the nucleic acid.
  • X 3 may be selected from -C1-C20 alkylene-, -C2-C20 alkenylene-, an alkylene ether of formula - (C1-C20 alkylene)-0-(Ci-C2o alkylene)-, -C(0)-Ci-C2o alkylene-, -C0-C4 alkylene(Cy)Co-C4 alkylene- wherein Cy represents a substituted or unsubstituted 5 or 6 membered cycloalkylene, arylene, heterocyclylene or heteroarylene ring, -C1-C4 alkylene-NHC(0)-Ci-C4 alkylene-, -Ci- C4 alkylene-C(0)NH-Ci-C4 alkylene-, -C1-C4 alkylene-SC(0)-Ci-C4 alkylene-, -C1-C4 alkylene- C(0)S-Ci-C4 alkylene-, -C1-C4 alky
  • X 3 may be an alkylene ether of formula -(C1-C20 alkylene)-0-(Ci-C2o alkylene)-.
  • X 3 may be an alkylene ether of formula -(C1-C20 alkylene)-0-(C4-C2o alkylene)-, wherein said (C4-C20 alkylene) is linked to Z.
  • X 3 may be selected from the group consisting of -CH2-O-C3H6-, -CH2- O-C4H8-, -CH2-O-C6H12- and -CH2-O-C8H16-, especially -CH2-O-C4H8-, -CH2-O-C6H12- and -CH2-O-C8H16-, wherein in each case the -CH2- group is linked to A.
  • nucleic acid of formula (I) is conjugated to a heterologous moiety of formula (HI):
  • S represents a functional component, e.g. a ligand, such as a saccharide, preferably
  • X 1 represents C3-C6 alkylene or (-CH2-CH2-O) m (-CH2)2- wherein m is 1 , 2, or 3;
  • P is a phosphate or modified phosphate, preferably a thiophosphate
  • X 2 is C1- Cs alkylene
  • A is a branching unit selected from:
  • X 3 is a bridging unit; wherein a nucleic acid according to the present invention is conjugated to X 3 via a phosphate or a modified phosphate, preferably a thiophosphate.
  • the branching unit A may have the structure:
  • the branching unit A may have the structure: , wherein X 3 is attached to the nitrogen atom.
  • X 3 may be C1-C20 alkylene.
  • X 3 is selected from the group consisting of -C3H6-, - C4H 8 -, -C6Hi2- and -CsHie-, especially -C4H 8 -, -C6HI 2 - and -CsHie-.
  • nucleic acid of formula (I) is conjugated to a ligand comprising a compound of formula (IV):
  • S represents a functional component, e.g., a ligand, such as a saccharide, preferably GalNAc;
  • X 1 represents C3-C6 alkylene or (-CH2-CH2-O) m (-CH2)2- wherein m is 1 , 2, or 3;
  • P is a phosphate or modified phosphate, preferably a thiophosphate
  • X 2 is an alkylene ether of formula -C3H6-O-CH2-;
  • A is a branching unit
  • X 3 is an alkylene ether of formula selected from the group consisting of -CH2-O-CH2-, - CH2-O-C2H4-, -CH2-O-C3H6-, -CH2-O-C4H8-, -CH2-O-C5H10-, -CH2-O-C6H12-, -CH2-O-C7H14-, and -CH2-O-C8H16-, wherein in each case the -CH2- group is linked to A, and wherein X 3 is conjugated to a nucleic acid of formula (I) according to the present invention by a phosphate or modified phosphate, preferably a thiophosphate.
  • the branching unit may comprise carbon.
  • the branching unit is a carbon.
  • X 3 may be selected from the group consisting of -CH2-O-C4H8-, -CH2-O-C5H10-, -CH2-O-C6H12-, -CH2-O-C7H14-, and -CH2-O-C8H16-.
  • X 3 is selected from the group consisting of -CH2-O-C4H8-, -CH2-O-C6H12- and -CH2-O-C8H16.
  • X 1 may be (-CH 2 -CH 2 -O)(-CH 2 )2-.
  • X 1 may be (-CH2-CH 2 -O)2(-CH 2 )2-.
  • X 1 may be (-CH2-CH 2 -O)3(-CH 2 )2-.
  • X 1 is (-CH2-CH2-O)2(-CH2)2-.
  • X 1 represents C3-C6 alkylene.
  • X 1 may be propylene.
  • X 1 may be butylene.
  • X 1 may be pentylene.
  • X 1 may be hexylene.
  • the alkyl is a linear alkylene. In particular, X 1 may be butylene.
  • X 2 represents an alkylene ether of formula -C3H6-O-CH2- i.e. C3 alkoxy methylene, or - CH2CH2CH2OCH2-.
  • modified phosphate it is meant a phosphate group wherein one or more of the non-linking oxygens is replaced.
  • modified phosphate groups include phosphorothioate, phosphorodithioates, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters.
  • Phosphorodithioates have both non-linking oxygens replaced by sulphur.
  • One, each or both non-linking oxygens in the phosphate group can be independently any one of S, Se, B, C, H, N, or OR (R is alkyl or aryl).
  • the phosphate can also be modified by replacement of a linking oxygen with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates).
  • the replacement can occur at a terminal oxygen. Replacement of the non-linking oxygens with nitrogen is possible.
  • the modified phosphate group is a thiophosphate group.
  • P is a monothiophosphate.
  • the ligand e.g., saccharide
  • the ligand may be selected to have an affinity for at least one type of receptor on a target cell.
  • the receptor is on the surface of a mammalian liver cell, for example, the hepatic asialoglycoprotein receptor complex (ASGP-R).
  • ASGP-R hepatic asialoglycoprotein receptor complex
  • the saccharide may be selected from N-acetyl with one or more of galactosamine, mannose, galactose, glucose, glucosamine and fructose.
  • a ligand to be used in the present invention may include N-acetyl galactosamine (GalNAc).
  • the compounds of the invention may have 3 ligands, which will each preferably include N-acetyl galactosamine.
  • GalNAc refers to 2-(Acetylamino)-2-deoxy-D-galactopyranose, commonly referred to in the literature as N-acetyl galactosamine.
  • Reference to “GalNAc” or “N-acetyl galactosamine” includes both the p- form: 2-(Acetylamino)-2-deoxy-p -D-galactopyranose and the a-form: 2- (Acetylamino)-2-deoxy-a-D- galactopyranose.
  • both the p-form: 2- (Acetylamino)-2-deoxy-p-D-galactopyranose and a-form: 2-(Acetylamino)-2-deoxy-a-D- galactopyranose may be used interchangeably.
  • the compounds of the invention comprise the p-form, 2-(Acetylamino)-2-deoxy-p-D-galactopyranose.
  • the nucleic acid of formula (I) is a conjugated nucleic acid, wherein the nucleic acid is conjugated to a heterologous moiety with one of the following structures, which may be referred to as “triantennary ligands” for ease of reference:
  • Z is any nucleic acid as defined herein.
  • the nucleic acid Z is conjugated to the triantennary ligand via the phosphate or thiophosphate group which links the triantennary ligand to the 3’ or 5’ position of the sugar, particularly to the 3’ or 5’ position of the ribose, of the terminal nucleotide of said nucleic acid Z.
  • heterologous moiety (“triantennary ligand”) is conjugated to the 3' position of the ribose of the terminal nucleotide of the second (sense) strand of Z.
  • heterologous moiety (“triantennary ligand”) is conjugated to the 5' position of the ribose of the terminal nucleotide of the second (sense) strand of Z.
  • heterologous moiety (“triantennary ligand”) is conjugated to the 3' position of the ribose of the terminal nucleotide of the first (antisense) strand of Z.
  • the nucleic acid is a conjugated nucleic acid, wherein the nucleic acid is conjugated to a triantennary ligand with one of the following structures:
  • Z is any nucleic acid as defined herein.
  • the nucleic acid Z is conjugated to the triantennary ligand via the phosphate or thiophosphate group which links the triantennary ligand to the 3’ or 5’ position of the ribose of the terminal nucleotide of said nucleic acid Z.
  • the triantennary ligand is conjugated to the 5' position of the ribose of the terminal nucleotide of the second (sense) strand of Z.
  • a heterologous moiety of formula (II), (III) or (IV) or any one of the triantennary ligands disclosed herein can be attached at the 3’-end of the first (antisense) strand and/or at any of the 3’ and/or 5’ end of the second (sense) strand.
  • the nucleic acid can comprise more than one heterologous moiety of formula (II), (III) or (IV) or any one of the triantennary ligands disclosed herein.
  • a single heterologous moiety of formula (II), (III) or (IV) or any one of the triantennary ligands disclosed herein is preferred because a single such moiety is sufficient for efficient targeting of the nucleic acid to the target cells.
  • at least the last two, preferably at least the last three and more preferably at least the last four nucleotides at the end of the nucleic acid to which the ligand is attached are linked by a phosphodiester linkage.
  • the 5’-end of the first (antisense) strand is not attached to a heterologous moiety, since attachment at this position can potentially interfere with the biological activity of the nucleic acid.
  • a nucleic acid with a single heterologous moiety (e.g., of formula (II), (III) or (IV) or any one of the triantennary ligands disclosed herein) at the 5’ end of a strand is easier and therefore cheaper to synthesise than the same nucleic acid with the same group at the 3’ end.
  • a single heterologous moiety e.g., of any of formulae (II), (III) or (IV) or any one of the triantennary ligands disclosed herein
  • the first strand of the nucleic acid is a compound of formula (V): wherein b is preferably 0 or 1 ; and the s wherein: c and d are independently preferably 0 or 1 ;
  • Zi and Z2 are respectively the first and second strand of the nucleic acid
  • Li is a linker to which a ligand is attached, wherein Li is the same or different in formulae (V) and (VI), and is the same or different within formulae (V) and (VI) when Li is present more than once within the same formula, wherein Li is preferably of formula (VII); and wherein b + c + d is preferably 2 or 3.
  • Li in formulae (V) and (VI) is of formula (VII): wherein: L is selected from the group comprising, or preferably consisting of:
  • Wi, W3 and W5 are individually absent or selected from the group comprising, or preferably consisting of:
  • X is absent or is selected from the group comprising, or preferably consisting of: NH, NCH3 or NC 2 H 5 ;
  • V is selected from the group comprising, or preferably consisting of: wherein B, if present, is a modified or natural nucleobase
  • the first strand is a compound of formula (VIII) wherein b is preferably 0 or 1; and the second strand is a compound of formula (IX): wherein c and d are independently preferably 0 or 1 ; wherein:
  • Zi and Z2 are respectively the first and second strand of the nucleic acid
  • Y is independently O or S
  • R1 is H or methyl; n is independently preferably 0, 1 , 2 or 3; and
  • L is the same or different in formulae (VIII) and (IX), and is the same or different within formulae (VIII) and (IX) when L is present more than once within the same formula, and is selected from the group comprising, or preferably consisting of:
  • the first strand of the nucleic acid is a compound of formula (X): wherein b is preferably 0 or 1 ; and the second strand is a compound of formula (XI): wherein: c and d are independently preferably 0 or 1 ;
  • Z1 and Z2 are respectively the first and second RNA strand of the nucleic acid
  • L2 is the same or different in formulae (X) and (XI) and is the same or different in moieties bracketed by b, c and d, and is selected from the group comprising, or preferably consisting of: n is 0 and L2 is:
  • F is a saturated branched or unbranched (such as unbranched) Ci-salkyl (e.g., Ci-ealkyl) chain wherein one of the carbon atoms is optionally replaced with an oxygen atom provided that said oxygen atom is separated from another heteroatom (e.g., an O or N atom) by at least 2 carbon atoms;
  • L is the same or different in formulae (X) and (XI) and is selected from the group comprising, or preferably consisting of:
  • b is 0, c is 1 and d is 1 ; b is 1 , c is 0 and d is 1 ; b is 1 , c is 1 and d is 0; or b is 1 , c is 1 and d is 1 in any of the nucleic acids of formulae (V) and (VI) or (VIII) and (IX) or (X) and (XI).
  • b is 0, c is 1 and d is 1 ; b is 1 , c is 0 and d is 1 ; or b is 1 , c is 1 and d is 1.
  • b is 0, c is 1 and d is 1.
  • Y is O in any of the nucleic acids of formulae (V) and (VI) or (VIII) and (IX) or (X) and (XI).
  • Y is S.
  • Y is independently selected from O or S in the different positions in the formulae.
  • Ri is H or methyl in any of the nucleic acids of formulae (VIII) and (IX). In one embodiment, Ri is H. In another embodiment, Ri is methyl.
  • n is 0, 1 , 2 or 3 in any of the nucleic acids of formulae (V) and (VI) or (VIII) and (IX) or (X) and (XI).
  • n is 0.
  • F moieties in any of the nucleic acids of formulae (X) and (XI) include (CH2)I-6 e.g. (CH 2 )I- 4 e.g. CH 2 , (CH 2 ) 4 , (CH 2 ) 5 or (CH 2 ) 6 , or CH 2 O(CH 2 ) 2.3 , e.g. CH 2 O(CH 2 )CH 3 .
  • L 2 in formulae (X) and (XI) is:
  • L 2 is:
  • n 0 and L 2 is: and the terminal OH group is absent such that the following moiety is formed:
  • GalNAc wherein Y is O or S.
  • L in the nucleic acids of formulae (V) and (VI) or (VIII) and (IX) or (X) and (XI), is selected from the group comprising, or preferably consisting of:
  • L is:
  • L 2 in the nucleic acids of formulae (X) and (XI) is typically the same. Between moieties bracketed by b, c and d, L 2 may be the same or different. In an embodiment, L 2 in the moiety bracketed by c is the same as the L 2 in the moiety bracketed by d. In an embodiment, L 2 in the moiety bracketed by c is not the same as L 2 in the moiety bracketed by d. In an embodiment, the L 2 in the moieties bracketed by b, c and d is the same, for example when the linker moiety is a serinol-derived linker moiety.
  • Serinol derived linker moieties may be based on serinol in any stereochemistry i.e. derived from L-serine isomer, D-serine isomer, a racemic serine or other combination of isomers.
  • the serinol-GalNAc moiety (SerGN) has the following stereochemistry:
  • (S)-Serinol building blocks Serlnol derived linker moieties i.e. , is based on an (S)-serinol-amidite or (S)-serinol succinate solid supported building block derived from L-serine isomer.
  • the first strand of the nucleic acid is a compound of formula (VIII) and the second strand of the nucleic acid is a compound of formula (IX), wherein: b is 0; c and d are 1, n is 0,
  • Zi and Z2 are respectively the first and second strand of the nucleic acid, Y is S,
  • R1 is H
  • L is -(CH 2 ) 4 -C(O)-, wherein the terminal C(O) of L is attached to the N atom of the linker (ie not a possible N atom of a targeting ligand).
  • the first strand of the nucleic acid is a compound of formula (V) and the second strand of the nucleic acid is a compound of formula (VI), wherein: b is 0, c and d are 1, n is 0,
  • Z1 and Z 2 are respectively the first and second strand of the nucleic acid, Y is S,
  • Li is of formula (VII), wherein:
  • W1 is -CH 2 -O-(CH 2 ) 3 -
  • W 3 is -CH 2 -
  • W5 is absent, V is CH,
  • X is NH
  • L is -(CH 2 )4-C(O)- wherein the terminal C(O) of L is attached to the N atom of X in formula (VII).
  • the first strand of the nucleic acid is a compound of formula (V) and the second strand of the nucleic acid is a compound of formula (VI), wherein: b is 0, c and d are 1, n is 0,
  • Zi and Z2 are respectively the first and second strand of the nucleic acid, Y is S,
  • Li is of formula (VII), wherein:
  • W1, W3 and W5 are absent
  • L is -(CH2)4-C(O)-NH-(CH2)S-C(O)-, wherein the terminal C(O) of L is attached to the N atom of V in formula (VII).
  • the nucleic acid is conjugated to a triantennary ligand with the following structure: wherein the nucleic acid of formula (I) is conjugated to the triantennary ligand via the phosphate group of the ligand to the a) 3' position of the ribose of the terminal nucleotide of the second (sense) strand of Z, or b) 5' position of the ribose of the terminal nucleotide of the second (sense) strand of Z, or c) 3' position of the ribose of the terminal nucleotide of the first (antisense) strand of Z.
  • the cells that are targeted by the nucleic acid with a ligand are hepatocytes.
  • the GalNAc may be substituted for any other targeting ligand, such as those mentioned herein, in particular mannose, galactose, glucose, glucosamine and fucose.
  • the nucleic acid is of formula (I) conjugated to a heterologous moiety that comprises a lipid, and more preferably, a cholesterol.
  • CD206 receptor (a mannose receptor)
  • CMM mannose ligand linker
  • GLP1 R Glucagon Like Peptide 1 Receptor
  • Knerr et al. J. Am. Chem. Soc., 143, 9, (2021), 3416-3429) discloses a number of GLPR1 peptide agonists that can be used, particularly for the extrahepatic delivery of ASOs, and methods for their synthesis.
  • Transferrin 1 Receptor TfR1
  • ligands or targeting agents for targeting the nucleic acid of formula (I) as described herein to the TfR1 receptor are described in Sugo et al. (Journal of Controlled Release 237, (2016), 1-13).
  • ligands or targeting agents for targeting the nucleic acid as described herein to the folic acid receptor are described in Dohmen et. al. Particularly, folic acid targeted PEG ligands that are conjugated to siRNA (e.g., FolA-PEG24-siRNA, Figure 1) and their synthesis are described.
  • siRNA e.g., FolA-PEG24-siRNA, Figure 1
  • the nucleic acids described herein may be capable of inhibiting the expression of a desired target gene. Inhibition may be complete, i.e., 0% remaining expression. Inhibition of the desired target gene expression may be partial, i.e., it may be 15%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more, or intermediate values of inhibition of the level of the desired target gene expression in the absence of a nucleic acid of the invention. The level of inhibition may be measured by comparing a treated sample with an untreated sample or with a sample treated with a control such as for example an siRNA that does not target the desired target gene.
  • a control such as for example an siRNA that does not target the desired target gene.
  • Inhibition may be measured by measuring the desired target gene mRNA and/or protein levels or levels of a biomarker or indicator that correlates with the desired target gene presence or activity. It may be measured in cells that may have been treated in vitro with a nucleic acid described herein. Alternatively, or in addition, inhibition may be measured in cells, such as hepatocytes, or tissue, such as liver tissue, or an organ, such as the liver, or in a body fluid such as blood, serum, lymph or any other body part or fluid that has been taken from a subject previously treated with a nucleic acid disclosed herein.
  • inhibition of the desired target gene expression is determined by comparing the desired target gene mRNA level measured in target gene-expressing cells after 24 or 48 hours of in vitro treatment with a double-stranded RNA disclosed herein under ideal conditions (see the examples for appropriate concentrations and conditions) to the desired target gene mRNA level measured in control cells that were untreated or mock treated or treated with a control double-stranded RNA under the same conditions.
  • compositions uses and methods
  • the present invention also provides compositions comprising the nucleic acid of formula (I) of the invention.
  • the nucleic acids of formula (I) and compositions may be used as therapeutic or diagnostic agents, alone or in combination with other agents.
  • one or more nucleic acid(s) of formula (I) of the invention can be combined with a delivery vehicle (e.g., liposomes) and/or excipients, such as carriers, diluents. Other agents such as preservatives and stabilizers can also be added.
  • Pharmaceutically acceptable salts or solvates of any of the nucleic acids of formula (I) of the invention are likewise within the scope of the present invention.
  • Methods for the delivery of nucleic acids are known in the art and within the knowledge of the person skilled in the art.
  • Compositions disclosed herein are particularly pharmaceutical compositions. Such compositions are suitable for administration to a subject.
  • the composition comprises nucleic acid of formula (I) or a conjugated nucleic acid of formula (I) disclosed herein, or a pharmaceutically acceptable salt or solvate thereof, and a solvent (preferably water) and/or a delivery vehicle and/or a physiologically acceptable excipient and/or a carrier and/or a salt and/or a diluent and/or a buffer and/or a preservative.
  • a solvent preferably water
  • a delivery vehicle preferably water
  • a physiologically acceptable excipient and/or a carrier and/or a salt and/or a diluent and/or a buffer and/or a preservative.
  • Pharmaceutically acceptable carriers or diluents include those used in formulations suitable for oral, rectal, nasal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, and transdermal) administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy.
  • Subcutaneous or transdermal modes of administration may be particularly suitable for the compounds of formula (I) described herein.
  • the prophylactically or therapeutically effective amount of a nucleic acid of formula (I) of the present invention will depend on the route of administration, the type of mammal being treated, and the physical characteristics of the specific mammal under consideration. These factors and their relationship to determining this amount are well known to skilled practitioners in the medical arts. This amount and the method of administration can be tailored to achieve optimal efficacy, and may depend on such factors as weight, diet, concurrent medication and other factors, well known to those skilled in the medical arts. The dosage sizes and dosing regimen most appropriate for human use may be guided by the results obtained by the present invention and may be confirmed in properly designed clinical trials.
  • An effective dosage and treatment protocol may be determined by conventional means, starting with a low dose in laboratory animals and then increasing the dosage while monitoring the effects, and systematically varying the dosage regimen as well. Numerous factors may be taken into consideration by a clinician when determining an optimal dosage for a given subject. Such considerations are known to the skilled person.
  • Nucleic acids of formula (I) of the present invention, or salts thereof may be formulated as pharmaceutical compositions prepared for storage or administration, which typically comprise a prophylactically or therapeutically effective amount of a nucleic acid of formula (I) of the invention, or a salt thereof, in a pharmaceutically acceptable carrier.
  • the nucleic acid of formula (I) or conjugated nucleic acid of the present invention can also be administered in combination with other therapeutic compounds, either administrated separately or simultaneously, e.g., as a combined unit dose.
  • the invention also includes a composition comprising one or more nucleic acids of formula (I) according to the present invention in a physiologically/pharmaceutically acceptable excipient, such as a stabilizer, preservative, diluent, buffer, and the like.
  • the composition comprises a conjugated nucleic acid disclosed herein and a solvent and/or a delivery vehicle and/or a physiologically acceptable excipient and/or a carrier and/or a salt and/or a diluent and/or a buffer and/or a preservative and/or a further therapeutic agent selected from the group comprising an oligonucleotide, a small molecule, a monoclonal antibody, a polyclonal antibody and a peptide.
  • the composition comprises a nucleic acid of formula (I) disclosed herein and a further therapeutic agent selected from the group comprising an oligonucleotide, a small molecule, a monoclonal antibody, a polyclonal antibody and a peptide.
  • the further therapeutic agent is an agent that targets, preferably inhibits the expression or the activity, of a desired target gene.
  • the further therapeutic agent is one of the following: a) a peptide that inhibits the expression or activity of a desired target gene, b) an antibody that specifically binds under physiological conditions to the desired target gene, or one of their subunits or proteolytic cleavage products.
  • the further therapeutic agent is selected from the group consisting of of a diuretic, such as for example, thiazide, thiazide-like, loop and potassium sparing diuretics; an angiotensin converting enzyme (ACE) inhibitor, such as for example benazepril, captopril, enalapril, fosinopril, lisinopril; an angiotensin II receptor antagonist, a beta-blocker, such as for example acebutolol, alprenolol, atenolol, betaxolol, bisoprolol, bunolol, carteolol, carvedilol, celiprolol, esmolol, labetalol, levobunolol, metipranolol, metoprolol, nadolol, oxpreolol, pindolol, propranolol, sotalol,
  • the further therapeutic agent comprises an antagonist of the desired target gene.
  • two or more nucleic acids of formula (I) of the invention with different sequences may be administered simultaneously or sequentially.
  • the present invention provides a composition, e.g., a pharmaceutical composition, comprising one or a combination of different nucleic acids of formula (I) of the invention and at least one pharmaceutically acceptable carrier.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a nucleic acid of formula (I) of the invention and at least one pharmaceutically acceptable carrier.
  • a unit dose may contain between about 0.01 mg/kg and about 100 mg/kg body weight of nucleic acid or conjugated nucleic acid.
  • the dose can be from 10 mg/kg to 25 mg/kg body weight, or 1 mg/kg to 10 mg/kg body weight, or 0.05 mg/kg to 5 mg/kg body weight, or 0.1 mg/kg to 5 mg/kg body weight, or 0.1 mg/kg to1 mg/kg body weight, or 0.1 mg/kg to 0.5 mg/kg body weight, or 0.5 mg/kg to 1 mg/kg body weight.
  • the dose can be from about 0.5 mg/kg to about 10 mg/kg body weight, or about 0.6 mg/kg to about 8 mg/kg body weight, or about 0.7 mg/kg to about 7 mg/kg body weight, or about 0.8 mg/kg to about 6 mg/kg body weight, or about 0.9 mg/kg to about 5.5 mg/kg body weight, or about 1 mg/kg to about 5 mg/kg body weight, or about 1 mg/kg body weight, or about 3 mg/kg body weight, or about 5 mg/kg body weight, wherein “about” is a deviation of up to 30%, preferably up to 20%, more preferably up to 10%, yet more preferably up to 5% and most preferably 0% from the indicated value. Dosage levels may also be calculated via other parameters such as, e.g., body surface area.
  • a dose unit of these nucleic acids preferably comprises about 1 mg/kg to about 5 mg/kg body weight, or about 1 mg/kg to about 3 mg/kg body weight, or about 1 mg/kg body weight, or about 3 mg/kg body weight, or about 5 mg/kg body weight.
  • the target gene mRNA level in the liver and/or the target gene protein level in the plasma or blood of a subject treated by a dose unit of the nucleic acid is preferably decreased at the time point of maximum effect by at least 30%, at least 40%, at least 50%, at least 60% or at least 70% as compared to a control that was not treatment with the nucleic acid or treated with a control nucleic acid under comparable conditions.
  • the dosage and frequency of administration may vary depending on whether the treatment is therapeutic or prophylactic (e.g., preventative), and may be adjusted during the course of treatment.
  • a relatively low dosage is administered at relatively infrequent intervals over a relatively long period of time. Some subjects may continue to receive treatment over their lifetime.
  • a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patient may be switched to a suitable prophylactic dosing regimen.
  • compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without causing deleterious side effects to the subject or patient.
  • a selected dosage level will depend upon a variety of factors, such as pharmacokinetic factors, including the activity of the particular nucleic acid or composition employed, the route of administration, the time of administration, the rate of excretion of the particular nucleic acid being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the subject or patient being treated, and similar factors well known in the medical arts.
  • factors such as pharmacokinetic factors, including the activity of the particular nucleic acid or composition employed, the route of administration, the time of administration, the rate of excretion of the particular nucleic acid being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the subject or patient being treated, and similar factors well known in the medical arts.
  • the pharmaceutical composition may be a sterile injectable aqueous suspension or solution, or in a lyophilized form.
  • the pharmaceutical compositions can be in unit dosage form.
  • the composition is divided into unit doses containing appropriate quantities of the active component.
  • the unit dosage form can be a packaged preparation, the package containing discrete quantities of the preparations, for example, packeted tablets, capsules, and powders in vials or ampoules.
  • the unit dosage form can also be a capsule, cachet, or tablet itself, or it can be the appropriate number of any of these packaged forms. It may be provided in single dose injectable form, for example in the form of a pen.
  • Compositions may be formulated for any suitable route and means of administration.
  • the nucleic acid of formula (I) and pharmaceutical compositions of the present invention may be administered to a mammalian subject in a pharmaceutically effective dose.
  • the mammal may be selected from a human, a non-human primate, a simian or prosimian, a dog, a cat, a horse, cattle, a pig, a goat, a sheep, a mouse, a rat, a hamster, a hedgehog and a guinea pig, or other species of relevance.
  • target gene or “desired target gene” as used herein denotes nucleic acid or protein in any of the above-mentioned species, if expressed therein naturally or artificially, but preferably this wording denotes human nucleic acids or proteins.
  • compositions of the invention may be administered alone or in combination with one or more other therapeutic or diagnostic agents.
  • a combination therapy may include a nucleic acid of the present invention combined with at least one other therapeutic agent selected based on the particular patient, disease or condition to be treated.
  • other such agents include, inter alia, a therapeutically active small molecule or polypeptide, a single chain antibody, a classical antibody or fragment thereof, or a nucleic acid molecule which modulates gene expression of one or more additional genes, and similar modulating therapeutics which may complement or otherwise be beneficial in a therapeutic or prophylactic treatment regimen.
  • compositions of the invention are typically sterile and stable under the conditions of manufacture and storage.
  • the composition may be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.
  • the carrier may be a solvent or dispersion medium containing, for example, water, alcohol such as ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), or any suitable mixtures.
  • the proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by use of surfactants according to formulation chemistry well known in the art.
  • isotonic agents e.g., sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride may be desirable in the composition.
  • Prolonged absorption of injectable compositions may be brought about by including in the composition an agent that delays absorption for example, monostearate salts and gelatine.
  • nucleic acid of formula (I) or a composition, preferably a pharmaceutical composition, disclosed herein for use as a therapeutic agent is preferably for use in the prophylaxis or treatment of a disease, disorder or syndrome.
  • the present invention provides a nucleic acid of formula (I) for use, alone or in combination with one or more additional therapeutic agents in a pharmaceutical composition, for treatment or prophylaxis of conditions, diseases and disorders responsive to inhibition of a desired target gene expression.
  • nucleic acid of formula (I) preferably a conjugated nucleic acid, or a composition, preferably a pharmaceutical composition, as disclosed herein for use as a therapeutic agent.
  • nucleic acid of formula (I) preferably a conjugated nucleic acid, or a composition, preferably a pharmaceutical composition, as disclosed herein for use in the prophylaxis or treatment of a disease, disorder or syndrome.
  • nucleic acid of formula (I) preferably the use of a conjugated nucleic acid, or composition, preferably a pharmaceutical composition, as disclosed herein in the preparation of a medicament for prophylaxis or treatment of a disease, disorder or syndrome.
  • nucleic acid of formula (I) or a composition, preferably a pharmaceutical composition, as disclosed herein in the prophylaxis or treatment of a disease, disorder or syndrome.
  • Nucleic acids of formula (I), and pharmaceutical compositions, preferably pharmaceutical compositions, of the invention may be used in the treatment of a variety of conditions, disorders or diseases. Treatment with a nucleic acid of formula (I) of the invention preferably leads to in vivo depletion of the selected target gene, preferably in the liver and/or in blood. As such, nucleic acids of the invention, and compositions comprising them, will be useful in methods for treating a variety of pathological disorders in which inhibiting the expression of the target gene may be beneficial.
  • the present invention provides methods for treating a disease, disorder or syndrome comprising the step of administering to a subject in need thereof a prophylactically or therapeutically effective amount of a nucleic acid of the invention.
  • the invention thus provides methods of prophylaxis or treatment of a disease, disorder or syndrome, the method comprising the step of administering to a subject (e.g., a patient) in need thereof a therapeutically effective amount of a nucleic acid of formula (I), preferably of a conjugated nucleic acid, or pharmaceutical composition comprising a nucleic acid of formula (I), preferably a conjugated nucleic acid, of the invention.
  • a subject e.g., a patient
  • a therapeutically effective amount of a nucleic acid of formula (I) preferably of a conjugated nucleic acid
  • pharmaceutical composition comprising a nucleic acid of formula (I), preferably a conjugated nucleic acid, of the invention.
  • the most desirable therapeutically effective amount is an amount that will produce a desired efficacy of a particular treatment selected by one of skill in the art for a given subject in need thereof. This amount will vary depending upon a variety of factors understood by the skilled worker, including but not limited to the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration.
  • nucleic acids of formula (I) and pharmaceutical compositions of the invention may be used to treat or prevent a disease, disorder or syndrome.
  • the present invention provides methods for prophylaxis or treatment of a disease, disorder or syndrome in a mammalian subject, such as a human, the method comprising the step of administering to a subject in need thereof a prophylactically or therapeutically effective amount of a nucleic acid of formula (I) as disclosed herein.
  • Administration of a "therapeutically effective dosage" of a nucleic acid of formula (I) of the invention may result in a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction.
  • Nucleic acids of formula (I) of the invention may be beneficial in treating or diagnosing a disease, disorder or syndrome that may be diagnosed or treated using the methods described herein. T reatment and diagnosis of other diseases, disorders or syndromes are also considered to fall within the scope of the present invention.
  • One aspect of the invention is a method of prophylaxis or treatment of a disease, disorder or syndrome comprising administering a pharmaceutically effective dose or amount of a nucleic acid of formula (I) or a composition, preferably a pharmaceutical composition, disclosed herein to an individual in need of treatment, preferably wherein the nucleic acid of formula (I) or composition is administered to the subject subcutaneously, intravenously or by oral, rectal, pulmonary, intramuscular or intraperitoneal administration. Preferably, it is administered subcutaneously.
  • the disease, disorder or syndrome is typically a target gene-mediated disease, disorder or syndrome associated with aberrant activation and/or over-activation (hyper-activation) of the target gene and/or with over-expression or ectopic expression or localisation or accumulation of the target gene.
  • a nucleic acid of formula (I) or compositions, preferably pharmaceutical compositions, disclosed herein may be for use in a regimen comprising treatments once or twice weekly, every week, every two weeks, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, every eight weeks, every nine weeks, every ten weeks, every eleven weeks, every twelve weeks, every three months, every four months, every five months, every six months or in regimens with varying dosing frequency such as combinations of the before-mentioned intervals.
  • the nucleic acid of formula (I) or composition may be for use subcutaneously, intravenously or using any other application routes such as oral, rectal, pulmonary, or intraperitoneal. Preferably, it is for use subcutaneously.
  • the target gene expression may be inhibited compared to untreated cells and/or subjects by a range from 15% up to 100% but at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% or intermediate values.
  • the level of inhibition may allow treatment of a disease associated with the target gene expression or overexpression or complement over-activation, or may serve to further investigate the functions and physiological roles of the target gene products.
  • the level of inhibition is preferably measured in the liver or in the blood or in the kidneys, preferably in the blood, of the subject treated with the nucleic acid or composition.
  • nucleic acid of formula (I) or composition preferably a pharmaceutical composition, as disclosed herein in the manufacture of a medicament for treating a disease, disorder or syndromes, such as those as listed above or additional pathologies associated with elevated levels of the target gene, preferably in the blood or in the kidneys, or over activation of the complement pathway, or additional therapeutic approaches where inhibition of a selected target gene expression is desired.
  • a medicament is a pharmaceutical composition.
  • nucleic acids of formula (I) of the invention and pharmaceutically acceptable salts and solvates thereof constitutes an individual embodiment of the invention.
  • nucleic acid of formula (I) or composition may, for example, be administered in a regimen comprising treatments twice every week, once every week, every two weeks, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, or every eight to twelve or more weeks or in regimens with varying dosing frequency such as combinations of the before-mentioned intervals.
  • the nucleic acid of formula (I) or conjugated nucleic acid or pharmaceutical composition may be for use subcutaneously or intravenously or other application routes such as oral, rectal or intraperitoneal.
  • a nucleic acid of formula (I) of the invention may be administered by any appropriate administration pathway known in the art, including but not limited to aerosol, enteral, nasal, ophthalmic, oral, parenteral, rectal, vaginal, or transdermal (e.g., topical administration of a cream, gel or ointment, or by means of a transdermal patch).
  • any appropriate administration pathway known in the art including but not limited to aerosol, enteral, nasal, ophthalmic, oral, parenteral, rectal, vaginal, or transdermal (e.g., topical administration of a cream, gel or ointment, or by means of a transdermal patch).
  • Parenteral administration is typically associated with injection at or in communication with the intended site of action, including infraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal administration.
  • nucleic acids confers nuclease stability in serum and makes for example subcutaneous application route feasible.
  • Solutions or suspensions used for intradermal or subcutaneous application typically include one or more of: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and/or tonicity adjusting agents such as, e.g., sodium chloride or dextrose.
  • a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents
  • antibacterial agents such as benzyl alcohol or methyl parabens
  • antioxidants such as ascorbic acid or sodium bisulfite
  • chelating agents
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide, or buffers with citrate, phosphate, acetate and the like.
  • acids or bases such as hydrochloric acid or sodium hydroxide, or buffers with citrate, phosphate, acetate and the like.
  • Such preparations may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • Sterile injectable solutions may be prepared by incorporating a nucleic acid in the required amount in an appropriate solvent with one or a combination of ingredients described above, as required, followed by sterilization microfiltration.
  • Dispersions may be prepared by incorporating the active compound into a sterile vehicle that contains a dispersion medium and optionally other ingredients, such as those described above.
  • the methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient in addition to any additional desired ingredient from a sterile-filtered solution thereof.
  • nucleic acid of the invention When a prophylactically or therapeutically effective amount of a nucleic acid of the invention is administered by, e.g., intravenous, cutaneous or subcutaneous injection, the nucleic acid will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. Methods for preparing parenterally acceptable solutions, taking into consideration appropriate pH, isotonicity, stability, and the like, are within the skill in the art.
  • a preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection will contain, in addition to a nucleic acid, an isotonic vehicle such as sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection, or other vehicle as known in the art.
  • a pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives well known to those of skill in the art.
  • nucleic acid which can be combined with a carrier material to produce a single dosage form will vary depending on a variety of factors, including the subject being treated, and the particular mode of administration. In general, it will be an amount of the composition that produces an appropriate therapeutic effect under the particular circumstances. Generally, out of one hundred percent, this amount will range from about 0.01% to about 99% of nucleic acid, from about 0.1% to about 70%, or from about 1% to about 30% of nucleic acid in combination with a pharmaceutically acceptable carrier.
  • the nucleic acid may be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.
  • a controlled release formulation including implants, transdermal patches, and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
  • Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a dose may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the particular circumstances of the therapeutic situation, on a case-by-case basis. It is especially advantageous to formulate parenteral compositions in dosage unit forms for ease of administration and uniformity of dosage when administered to the subject or patient.
  • a dosage unit form refers to physically discrete units suitable as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce a desired therapeutic effect.
  • the specification for the dosage unit forms of the invention depends on the specific characteristics of the active compound and the particular therapeutic or prophylactic effect(s) to be achieved and the treatment and sensitivity of any individual patient.
  • nucleic acid of formula (I) or composition of the present invention can be produced using routine methods in the art including chemical synthesis, such as solid phase chemical synthesis.
  • Nucleic acids of formula (I) or compositions of the invention may be administered with one or more of a variety of medical devices known in the art.
  • a nucleic acid of the invention may be administered with a needleless hypodermic injection device.
  • implants and modules useful in the present invention are in the art, including e.g., implantable micro-infusion pumps for controlled rate delivery; devices for administering through the skin; infusion pumps for delivery at a precise infusion rate; variable flow implantable infusion devices for continuous drug delivery; and osmotic drug delivery systems. These and other such implants, delivery systems, and modules are known to those skilled in the art.
  • the nucleic acid or composition of the invention may be formulated to ensure a desired distribution in vivo.
  • a therapeutic compound or composition of the invention to a particular in vivo location, they can be formulated, for example, in liposomes which may comprise one or more moieties that are selectively transported into specific cells or organs, thus enhancing targeted drug delivery.
  • the invention is characterized by high specificity at the molecular and tissue-directed delivery level.
  • the sequences of the nucleic acids of formula (I) of the invention are highly specific for their target, meaning that they do not inhibit the expression of genes that they are not designed to target or only minimally inhibit the expression of genes that they are not designed to target and/or only inhibit the expression of a low number of genes that they are not designed to target.
  • a further level of specificity is achieved when nucleic acids of formula (I) are linked to a ligand that is specifically recognised and internalised by a particular cell type. This is for example the case when a nucleic acid is linked to a ligand comprising GalNAc moieties, which are specifically recognised and internalised by hepatocytes.
  • the present invention thus provides nucleic acids of the invention linked to a ligand comprising one or more GalNAc moieties, or comprising one or more other moieties that confer cell-type or tissue-specific internalization of the nucleic acid thereby conferring additional specificity of target gene knockdown by RNA interference.
  • the nucleic acid of formula (I) as described herein may be formulated with a lipid in the form of a liposome. Such a formulation may be described in the art as a lipoplex.
  • the composition with a lipid/liposome may be used to assist with delivery of the nucleic acid of formula (I) of the invention to the target cells.
  • the lipid delivery system herein described may be used as an alternative to a conjugated ligand.
  • the modifications herein described may be present when using the nucleic acid of formula (I) of the invention with a lipid delivery system or with a ligand conjugate delivery system.
  • Such a lipoplex may comprise a lipid composition comprising: i) a cationic lipid, or a pharmaceutically acceptable salt thereof; ii) a steroid; iii) a phosphatidylethanolamine phospholipid; and/or iv) a PEGylated lipid.
  • the cationic lipid may be an amino cationic lipid.
  • the cationic lipid may have the formula (XII): or a pharmaceutically acceptable salt thereof, wherein:
  • X represents O, S or NH
  • R 1 and R 2 each independently represents a C4-C22 linear or branched alkyl chain or a C4-C22 linear or branched alkenyl chain with one or more double bonds, wherein the alkyl or alkenyl chain optionally contains an intervening ester, amide or disulfide; when X represents S or NH, R 3 and R 4 each independently represent hydrogen, methyl, ethyl, a mono- or polyamine moiety, or R 3 and R 4 together form a heterocyclyl ring; when X represents O, R 3 and R 4 each independently represent hydrogen, methyl, ethyl, a mono- or polyamine moiety, or R 3 and R 4 together form a heterocyclyl ring, or R 3 represents hydrogen and R 4 represents C(NH)(NH2).
  • the cationic lipid may have the formula (XIII): or a pharmaceutically acceptable salt thereof.
  • the cationic lipid may have the formula (XIV): or a pharmaceutically acceptable salt thereof.
  • the content of the cationic lipid component may be from about 55 mol% to about 65 mol% of the overall lipid content of the composition.
  • the cationic lipid component is about 59 mol% of the overall lipid content of the composition.
  • compositions can further comprise a steroid.
  • the steroid may be cholesterol.
  • the content of the steroid may be from about 26 mol% to about 35 mol% of the overall lipid content of the lipid composition. More particularly, the content of steroid may be about 30 mol% of the overall lipid content of the lipid composition.
  • the phosphatidylethanolamine phospholipid may be selected from the group consisting of 1 ,2- diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE), 1 ,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE),
  • DPhyPE diphytanoyl-sn-glycero-3-phosphoethanolamine
  • DOPE 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine
  • DSPE 1,2-distearoyl-sn-glycero-3-phosphoethanolamine
  • DLPE 1.2-Dilauroyl-sn-glycero-3-phosphoethanolamine
  • DMPE 1,2-Dimyristoyl-sn-glycero-3- phosphoethanolamine
  • DPPE 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine
  • DLoPE 1.2-Dilinoleoyl-sn-glycero-3-phosphoethanolamine
  • POPE 1-Palmitoyl-2-oleoyl-sn-glycero- 3-phosphoethanolamine
  • DEPE 1,2-Dierucoyl-sn-glycero-3-phosphoethanolamine
  • DSQPE 1.2-Disqualeoyl-sn-glycero-3-phosphoethanolamine
  • SLPE 1-Stearoyl-2-linoleoyl-sn- glycero-3-phosphoethanolamine
  • the content of the phospholipid may be about 10 mol% of the overall lipid content of the composition.
  • the PEGylated lipid may be selected from the group consisting of 1 ,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol (DMG-PEG) and 016-Ceramide-PEG.
  • the content of the PEGylated lipid may be about 1 to 5 mol% of the overall lipid content of the composition.
  • the content of the cationic lipid component in the composition may be from about 55 mol% to about 65 mol% of the overall lipid content of the lipid composition, preferably about 59 mol% of the overall lipid content of the lipid composition.
  • the composition may have a molar ratio of the components of i):ii):iii):iv) selected from 55:34:10:1; 56:33:10:1; 57:32:10:1; 58:31:10:1; 59:30:10:1; 60:29:10:1; 61:28:10:1; 62:27:10:1; 63:26:10:1; 64:25:10:1; and 65:24:10:1.
  • Neutral liposome compositions may be formed from, for example, dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).
  • Anionic liposome compositions may be formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes may be formed primarily from dioleoyl phosphatidylethanolamine (DOPE).
  • Another type of liposomal composition may be formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.
  • a positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride can be used to form small liposomes that interact spontaneously with nucleic acid to form lipid-nucleic acid complexes which are capable of fusing with the negatively charged lipids of the cell membranes of tissue culture cells.
  • DOTMA analogues can also be used to form liposomes.
  • lipids described herein may also be used to form liposomes.
  • a liposome containing a nucleic acid can be prepared by a variety of methods.
  • the lipid component of a liposome is dissolved in a detergent so that micelles are formed with the lipid component.
  • the lipid component can be an amphipathic cationic lipid or lipid conjugate.
  • the detergent can have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine.
  • the nucleic acid preparation is then added to the micelles that include the lipid component.
  • the cationic groups on the lipid interact with the nucleic acid and condense around the nucleic acid to form a liposome.
  • the detergent is removed, e.g., by dialysis, to yield a liposomal preparation of nucleic acid.
  • a carrier compound that assists in condensation can be added during the condensation reaction, e.g., by controlled addition.
  • the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). pH can also be adjusted to favour condensation.
  • Nucleic acid formulations of the present invention may include a surfactant.
  • the nucleic acid is formulated as an emulsion that includes a surfactant.
  • a surfactant that is not ionized is a non-ionic surfactant.
  • non-ionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters etc., nonionic alkanolamides, and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers.
  • a surfactant that carries a negative charge when dissolved or dispersed in water is an anionic surfactant.
  • anionic surfactant examples include carboxylates, such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates.
  • carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and
  • a surfactant that carries a positive charge when dissolved or dispersed in water is a cationic surfactant.
  • examples include quaternary ammonium salts and ethoxylated amines.
  • a surfactant that has the ability to carry either a positive or negative charge is an amphoteric surfactant.
  • amphoteric surfactant examples include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.
  • Micelles are defined herein as a particular type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all the hydrophobic portions of the molecules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic.
  • a micelle may be formed by mixing an aqueous solution of the nucleic acid, an alkali metal alkyl sulphate, and at least one micelle forming compound.
  • Exemplary micelle forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleates, monolaurates, borage oil, evening of primrose oil, menthol, trihydroxy oxo cholanyl glycine and pharmaceutically acceptable salts thereof, glycerol, polyglycerol, lysine, polylysine, triolein, polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethers and analogues thereof, chenodeoxycholate, deoxycholate, and mixtures thereof. Phenol and/or m-cresol may be added to the mixed micellar composition to act as a stabiliser and preservative. An isotonic agent such as glycerine may as be added.
  • a nucleic acid preparation may be incorporated into a particle such as a microparticle.
  • Microparticles can be produced by spray-drying, lyophilisation, evaporation, fluid bed drying, vacuum drying, or a combination of these methods.
  • the terms “inhibit”, “down-regulate”, or “reduce” with respect to gene expression mean that the expression of the gene, or the level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits (e.g., mRNA), or the activity of one or more proteins or protein subunits, is reduced below that observed either in the absence of the nucleic acid or conjugated nucleic acid of the invention or as compared to that obtained with an siRNA molecule with no known homology to the human transcript (herein termed non-silencing control). Such control may be conjugated and modified in an analogous manner to the molecule of the invention and delivered into the target cell by the same route.
  • the expression after treatment with the nucleic acid of the invention may be reduced to 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 5% or 0% or to intermediate values, or less than that observed in the absence of the nucleic acid or conjugated nucleic acid.
  • the expression may be measured in the cells to which the nucleic acid is applied. Alternatively, especially if the nucleic acid is administered to a subject, the level can be measured in a different group of cells or in a tissue or an organ or in a body fluid such as blood or plasma.
  • the level of inhibition is preferably measured in conditions that have been selected because they show the greatest effect of the nucleic acid on the target mRNA level in cells treated with the nucleic acid in vitro.
  • the level of inhibition may for example be measured after 24 hours or 48 hours of treatment with a nucleic acid at a concentration of between 0.038 nM - 10 pM, preferably 0.5 nM, 1 nM, 10 nM or 100 nM.
  • These conditions may be different for different nucleic acid sequences or for different types of nucleic acids, such as for nucleic acids that are unmodified or modified or conjugated to a ligand or not. Examples of suitable conditions for determining levels of inhibition are described in the examples.
  • nucleic acid it is meant a nucleic acid comprising two strands comprising nucleotides, that is able to interfere with gene expression. Inhibition may be complete or partial and results in down regulation of gene expression in a targeted manner.
  • the nucleic acid comprises two separate polynucleotide strands; the first strand, which may also be a guide strand; and a second strand, which may also be a passenger strand.
  • the first strand and the second strand may be part of the same polynucleotide molecule that is self-complementary which 'folds' back to form a double-stranded molecule.
  • the nucleic acid may be an siRNA molecule.
  • the nucleic acid may comprise ribonucleotides, modified ribonucleotides, deoxynucleotides, deoxyribonucleotides, or nucleotide analogues non-nucleotides that are able to mimic nucleotides such that they may 'pair' with the corresponding base on the target sequence or complementary strand.
  • the nucleic acid may further comprise a double-stranded nucleic acid portion or duplex region formed by all or a portion of the first strand (also known in the art as a guide strand) and all or a portion of the second strand (also known in the art as a passenger strand).
  • the duplex region is defined as beginning with the first base pair formed between the first strand and the second strand and ending with the last base pair formed between the first strand and the second strand, inclusive.
  • duplex region it is meant the region in two complementary or substantially complementary oligonucleotides that form base pairs with one another, either by Watson-Crick base pairing or any other manner that allows for a duplex between oligonucleotide strands that are complementary or substantially complementary.
  • an oligonucleotide strand having 21 nucleotide units can base pair with another oligonucleotide of 21 nucleotide units, yet only 19 nucleotides on each strand are complementary or substantially complementary, such that the “duplex region” consists of 19 base pairs.
  • the remaining base pairs may exist as 5' and 3' overhangs, or as single-stranded regions.
  • a “double strand region” or “duplex region” therefore can be represented as in the following schematic: d bl t d i first strand second strand ' 5’ wherein the enclosed area in this schematic represents the double strand or duplex region.
  • the first nucleotide on the 5’ end of the first strand corresponds to Yf
  • the last nucleotide within the enclosed area on the 3’ terminal end of the first strand corresponds to Y u
  • the last two nucleotides (two boxes) outside the enclosed area at the 3’ end of the first strand correspond to Xb.
  • the first nucleotide on the 3’ end of the second strand corresponds to Y/ and the last nucleotide within the enclosed area on the 5’ terminal end of the second strand corresponds to Y u .
  • Any nucleotides outside of the enclosed area if present, correspond respectively to X a , Xb and Xa’ and Xb’ on the first and second strand.
  • the nucleotides are counted on the first strand starting from the 5’ end towards the 3’ end, in this specific example from 1 to 21 , wherein 1 is the terminal 5’ nucleotide on the first strand and 21 is the terminal 3’ nucleotide on the first strand.
  • the nucleotides on the second strand are counted starting from the 3’ terminal end towards the 5’ end, in this specific example from 1 to 19, wherein 1 is the terminal 3’ nucleotide on the second strand and 19 is the terminal 5’ nucleotide on the second strand.
  • ⁇ complementarity refers to complementarity between the strands such that they are capable of annealing under biological conditions. Techniques to empirically determine if two strands are capable of annealing under biological conditions are well known in the art. Alternatively, two strands can be synthesised and added together under biological conditions to determine if they anneal to one another. The portion of the first strand and second strand that forms at least one duplex region may be fully complementary and is at least partially complementary to each other. Depending on the length of a nucleic acid, a perfect match in terms of base complementarity between the first strand and the second strand is not necessarily required. However, the first and second strands must be able to hybridise under physiological conditions.
  • non-pairing nucleotide analogue means a nucleotide analogue which includes a non-base pairing moiety including but not limited to: 6 des amino adenosine (Nebularine), 4-Me-indole, 3-nitropyrrole, 5-nitroindole, Ds, Pa, N3-Me ribo II, N3-Me riboT, N3-Me dC, N3-Me-dT, N1-Me-dG, N1-Me-dA, N3-ethyl-dC, and N3-Me dC.
  • the non-base pairing nucleotide analogue is a ribonucleotide. In other embodiments it is a deoxyribonucleotide.
  • terminal functional group includes without limitation a halogen, alcohol, amine, carboxylic, ester, amide, aldehyde, ketone, and ether groups.
  • an “overhang” as used herein has its normal and customary meaning in the art, i.e. a singlestranded portion of a nucleic acid that extends beyond the terminal nucleotide of a complementary strand in a double-strand nucleic acid.
  • the term “blunt end” includes doublestranded nucleic acid whereby both strands terminate at the same position, regardless of whether the terminal nucleotide(s) are base-paired.
  • the terminal nucleotide of a first strand and a second strand at a blunt end may be base paired.
  • the terminal nucleotide of a first strand and a second strand at a blunt end may not be paired.
  • the terminal two nucleotides of a first strand and a second strand at a blunt end may be base-paired.
  • the terminal two nucleotides of a first strand and a second strand at a blunt end may not be paired.
  • the term “serinol-derived linker moiety” means the linker moiety comprises the following structure:
  • An O atom of said structure typically links to an RNA strand and the N atom typically links to the targeting ligand.
  • patient may be used interchangeably and refer to either a human or a non-human animal.
  • mammals such as humans, primates, livestock animals (e.g., bovines, porcines), companion animals (e.g., canines, felines) and rodents (e.g., mice and rats).
  • livestock animals e.g., bovines, porcines
  • companion animals e.g., canines, felines
  • rodents e.g., mice and rats.
  • treating or “treatment” and grammatical variants thereof refer to an approach for obtaining beneficial or desired clinical results.
  • the term may refer to slowing the onset or rate of development of a condition, disorder or disease, reducing or alleviating symptoms associated with it, generating a complete or partial regression of the condition, or some combination of any of the above.
  • beneficial or desired clinical results include, but are not limited to, reduction or alleviation of symptoms, diminishment of extent of disease, stabilization (i.e., not worsening) of state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Treatment can also mean prolonging survival relative to expected survival time if not receiving treatment.
  • a subject e.g., a human
  • treatment includes inhibition or reduction of an increase in severity of a pathological state or symptoms relative to the absence of treatment, and is not necessarily meant to imply complete cessation of the relevant disease, disorder or condition.
  • prophylaxis and grammatical variants thereof refer to an approach for inhibiting or preventing the development, progression, or time or rate of onset of a condition, disease or disorder, and may relate to pathology and/or symptoms.
  • beneficial or desired clinical results include, but are not limited to, prevention, inhibition or slowing of symptoms, progression or development of a disease, whether detectable or undetectable.
  • a subject e.g., a human
  • prophylaxis includes slowing the onset of disease relative to the absence of treatment and is not necessarily meant to imply permanent prevention of the relevant disease, disorder or condition.
  • prophylaxis of a condition may in certain contexts refer to reducing the risk of developing the condition, or preventing, inhibiting or delaying the development of symptoms associated with the condition. It will be understood that prophylaxis may be considered as treatment or therapy.
  • an “effective amount,” “prophylactically effective amount”, “therapeutically effective amount” or “effective dose” is an amount of a composition (e.g., a therapeutic composition or agent) that produces at least one desired therapeutic effect in a subject, such as preventing or treating a target condition or beneficially alleviating a symptom associated with the condition.
  • the term “pharmaceutically acceptable salt” refers to a salt that is not harmful to a patient or subject to which the salt in question is administered. It may be a salt chosen, e.g., among acid addition salts and basic salts. Examples of acid addition salts include chloride salts, citrate salts and acetate salts.
  • Examples of basic salts include salts wherein the cation is selected from alkali metal cations, such as sodium or potassium ions, alkaline earth metal cations, such as calcium or magnesium ions, as well as substituted ammonium ions, such as ions of the type N(R 1 )(R 2 )(R 3 )(R 4 )+, wherein R 1 , R 2 , R 3 and R 4 independently will typically designate hydrogen, optionally substituted Ci-6-alkyl groups or optionally substituted C2-6- alkenyl groups. Examples of relevant Ci-6-alkyl groups include methyl, ethyl, 1-propyl and 2- propyl groups.
  • C2-6-alkenyl groups of possible relevance include ethenyl, 1- propenyl and 2-propenyl.
  • Other examples of pharmaceutically acceptable salts are described in “Remington’s Pharmaceutical Sciences”, 17th edition, Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, PA, USA, 1985 (and more recent editions thereof), in the “Encyclopaedia of Pharmaceutical Technology”, 3rd edition, James Swarbrick (Ed.), Informa Healthcare USA (Inc.), NY, USA, 2007, and in J. Pharm. Sci. 66: 2 (1977).
  • a "pharmaceutically acceptable salt” retains qualitatively a desired biological activity of the parent compound without imparting any undesired effects relative to the compound.
  • Acid addition salts include salts derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphorous, phosphoric, sulfuric, hydrobromic, hydroiodic and the like, or from nontoxic organic acids such as aliphatic mono- and di-carboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like.
  • nontoxic inorganic acids such as hydrochloric, nitric, phosphorous, phosphoric, sulfuric, hydrobromic, hydroiodic and the like
  • nontoxic organic acids such as aliphatic mono- and di-carboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like.
  • Base addition salts include salts derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N, N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.
  • pharmaceutically acceptable carrier includes any of the standard pharmaceutical carriers.
  • Pharmaceutically acceptable carriers for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).
  • sterile saline and phosphate- buffered saline at slightly acidic or physiological pH may be used.
  • Exemplary pH buffering agents include phosphate, citrate, acetate, tris/hydroxymethyl)aminomethane (TRIS), N- Tris(hydroxymethyl)methyl-3-aminopropanesulphonic acid (TAPS), ammonium bicarbonate, diethanolamine, histidine, which is a preferred buffer, arginine, lysine, or acetate or mixtures thereof.
  • TIS tris/hydroxymethyl)aminomethane
  • TAPS N- Tris(hydroxymethyl)methyl-3-aminopropanesulphonic acid
  • ammonium bicarbonate diethanolamine
  • histidine which is a preferred buffer
  • arginine arginine
  • lysine lysine
  • a "pharmaceutically acceptable carrier” includes any and all physiologically acceptable, i.e., compatible, solvents, dispersion media, coatings, antimicrobial agents, isotonic and absorption delaying agents, and the like.
  • the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).
  • the nucleic acid may be coated in a material or materials intended to protect the compound from the action of acids and other natural inactivating conditions to which the nucleic acid may be exposed when administered to a subject by a particular route of administration.
  • solvate in the context of the present invention refers to a complex of defined stoichiometry formed between a solute (in casu, a nucleic acid compound or pharmaceutically acceptable salt thereof according to the invention) and a solvent.
  • the solvent in this connection may, for example, be water or another pharmaceutically acceptable, typically small-molecular organic species, such as, but not limited to, acetic acid or lactic acid.
  • a solvate is normally referred to as a hydrate.
  • Figure 3 GalNAc-siRNA conjugates with LNA modifications at Y-2 and Y u of the first strand require one phosphorothioate internucleotide linkage to be stable in acidic rat liver tritosome extract.
  • FIG. 5 GalNAc-siRNA conjugates with LNA modifications at positions Y2' and Yf'of the second strand require one phosphorothioate internucleotide linkage to be stable in acidic rat liver tritosome lysate.
  • Figure 7 Stability of GalNAc-siRNA conjugates with LNA at Y-2 in the first and Y2' in the second strand is improved and can be further improved by an inverted ribonucleotide (irA).
  • irA inverted ribonucleotide
  • Figure 10 Stability of GalNAc-siRNA conjugates with inverted ribonucleotides at the 3' end of the second strand in acidic rat liver tritosome lysate cannot be improved by addition of LNA modifications at Y2'of the second strand.
  • Figure 11 Stability of GalNAc-siRNA conjugates with inverted ribonucleotides at the 3' end of the second strand and a vinylphosphonate at the 5'end of the first strand in acidic rat liver tritosome extract can be improved by addition of LNA modifications at Y2'of the second strand.
  • Figure 12 Stability of GalNAc-siRNA conjugates with inverted ribonucleotides at the 3' end and LNA modification at Y2'of the second strand and a 5' vinylphosphonate at the first strand in acidic tritosome lysate can be improved by addition of LNA modifications at Y-2 of the first strand.
  • Figures 14a, 14b, 14c - GalNAc-siRNA conjugates with LNA modifications in the 3'end of the first and/or the second strand and an inverted nucleotide at the 3'end of the second strand effect reduction AT3 mRNA levels in vitro independently of the underlying modification pattern
  • Figures 15a, 15b, 15c - GalNAc siRNA conjugates with vinylphosphonate at the 5'end of the first strand and LNA modifications in the 3'end of the first and/or the second strand and an inverted ribonucleotide at the 3'end of the second strand effect reduction of ALDH2 and AT3 mRNA levels in vitro independently of the underlying modification pattern
  • Figures 18a, 18b - AT3 target mRNA reduction can be improved in vivo using GalNAc-siRNA conjugates with vinylphosphonate at the 5' end of the first strand and additionally with an LNA modification at Y-2 of the first strand, an LNA modification at Y2' and an irA modification at Yf of the second strand.
  • Figures 19A, 19B, 19C In vitro activity of siRNAs of 19, 20, 21 , 22, and 23 nt length, with and without LNA at the penultimate position of the first strand.
  • Target gene PTEN reduction is shown in Figure 19A
  • target gene PPIB reduction is shown in Figure 19B
  • target gene AT3 reduction is shown in Figure 19C.
  • Figure 20 In vitro activity of GalNAc siRNA conjugates with LNA modifications in one or both strands and phosphorodithioate internucleotide linkages at the siRNA termini.
  • Figure 21 In vitro activity of GalNAc siRNA conjugates with LNA modifications at the 5'-end of the second strand.
  • Figure 22 Tritosome stability of GalNAc siRNA conjugates with LNA modifications at positions 1 or 2 of the second strand 5'-end.
  • Figure 23 In vitro activity of GalNAc siRNA conjugates with different combinations of LNA, irA and phosphorothioate internucleotide linkages at positions 1 or 2 of the second strand 5' end.
  • Figure 24 Tritosome stability of GalNAc siRNA conjugates with different combinations of LNA, irA and phosphorothioate internucleotide linkages at positions 1 or 2 of the second strand 5' end.
  • Figure 25 In vitro activity of GalNAc siRNA conjugates with LNA at the 3'-penultimate position of the first strand and different end modifications at the 5'-end of the first strand and at the 3'-end of the second strand.
  • Figure 26 In vitro activity of GalNAc siRNA conjugates with different combinations of LNA- containing end modifications.
  • Figure 27 Improved tritosome stability of GalNAc-siRNA conjugates targeting At3 with LNA at penultimate position 18 of the second strand.
  • Example compounds were synthesised according to methods described below and methods known to the person skilled in the art. Assembly of the oligonucleotide chain and linker building blocks was performed by solid phase synthesis applying phosphoramidite methodology.
  • Oligonucleotides were synthesized on an AKTA oligopilot 10 synthesizer using standard phosphoramidite chemistry.
  • Commercially available base loaded solid supports including inverted 2’TBDMS-riboA, 2’OMe nucleotide phosphoramidites and 2’F nucleotide phosphoramidites (all standard protection) were purchased from ChemGenes, Locked Nucleic Acid (LNA) amidites were purchased from Link and used according to the manufacturers recommended procedures.
  • Ancillary reagents were purchased from EMP Biotech. Synthesis was performed using a 0.1 M solution of the phosphoramidite in dry acetonitrile and benzylthiotetrazole (BTT) was used as activator (0.3M in acetonitrile). Coupling time was 10 min. A Cap/OX/Cap or Cap/Thio/Cap cycle was applied (Cap: Ac2O/NMI/Lutidine/Acetonitrile. Oxidizer: 0.1M l 2 in pyridine/H 2 O). Phosphorothioates were introduced using 0.2M XH (0.2 M Xanthane hydride in pyridine).
  • DMT cleavage was achieved by treatment with 3% dichloroacetic acid in toluene. Upon completion of the programmed synthesis cycles a diethylamine (DEA) wash was performed. All other reagents and solvents were commercially available and used in standard reagent quality.
  • oligonucleotides were synthesized in DMT-off mode. The single strands were cleaved off the support by 40% aq. methylamine treatment (90min, RT). The resulting crude oligonucleotide was concentrated under reduced pressure to a concentration of ⁇ 10 mg/mL. Oligonucleotides containing ribose units with TBDMS protection of the 2’-OH group were further concentrated to dryness. For a 10 pmol scale synthesis 1 mL DMSO, 0.5 mL NEta and 0.75 mL NEta*3HF were added and the mixture was heated to 65°C for 2 h.
  • M 10008 and M 10014 contain an LNA modification at Y-s
  • M 10009 and M 10015 contain an LNA modification at Y.4
  • M10010 and M10016 contain an LNA modification at Y-3
  • M 10011 and M 10017 contain an LNA modification at Y-2
  • M 10012 and M 10018 contain an LNA modification at Y u .
  • siRNAs with LNA at Y-s and Y u reduced the target mRNA levels less than, whereas an siRNA with LNA at Y-2 of the first strand reduced the target mRNA levels more than the respective siRNA without LNA modification.
  • siRNAs with LNA at Y. 4 and Y-3 have similar activity as compared to the reference.
  • the cells were seeded at a density of 30,000 cells per 96-well, and simultaneously transfected with 10 nM and 1 nM siRNA and 0.3 pl RNAiMax. Cells were lysed after 24 hours (h), total RNA was extracted and ALK3, PPIB, PTEN mRNA levels were determined by Taqman qRT-PCR. Each bar represents geometric mean ⁇ SD from three technical replicates. Results are shown in Figure 1.
  • X1335 and X1348 contain two phosphorothioate linkages at the 3' end of the first strand.
  • X1336 and X1349 contain an LNA modification at Y u and one phosphorothioate internucleotide linkage between position Y-3 and Y-2.
  • X1337 and X1350 contain an LNA modification at Y u and two phosphorothioate internucleotide linkages at the terminal positions.
  • X1338 and X1351 contain an LNA modification at Y-2 and one phosphorothioate linkage between the terminal nucleotides.
  • X1339 and X1352 contain an LNA modification at Y-2 and two phosphorothioate internucleotide linkage between the terminal nucleotides.
  • X1340 and X1353 contain two LNA modifications at Y_ 2 and Y u of the first strand.
  • X1341 and X1354 contain two LNA modifications at Y-2 and Y u and two phosphorothioate internucleotide linkages at the terminal nucleotides. All tested variants of siRNAs targeting At3 show comparable mRNA knockdown under the tested conditions. siRNAs targeting Ttr reduce the target mRNA levels less than the respective siRNA without LNA modification, independent of the phosphorothioate internucleotide linkages.
  • Cells were seeded at a density of 25,000 cells per 96-well and treated with 10 nM and 1 nM using siRNAs targeting At3 or 1 nM and 0.1 nM using siRNAs targeting Ttr directly after plating.
  • Cells were lysed after 24 h, total RNA was extracted and Ttr, At3, Actb, Pten, ApoB mRNA levels were determined by Taqman qRT-PCR. Each bar represents geometric mean ⁇ SD from three technical replicates. Results are shown in Figure 2.
  • GalNAc-siRNA conjugates with LNA modifications at Y-2 and Y u of the first strand require one phosphorothioate internucleotide linkage to be stable in acidic rat liver tritosome extracts.
  • X1335 contains two phosphorothioate internucleotide linkages at the 3' end of the first strand.
  • X1336 contains an LNA modification at Y u and one phosphorothioate internucleotide linkage between Y.3 and Y. 2.
  • X1337 contains an LNA modification at Y u and two phosphorothioate internucleotide linkages at the terminal positions.
  • X1338 contains an LNA modification at Y-2 and one phosphorothioate internucleotide linkage between the terminal nucleotides.
  • X1339 contains an LNA modification at Y-2 and two phosphorothioate internucleotide linkage between the terminal nucleotides.
  • X1340 contains two LNA modifications at Y-2 and Y u of the first strand.
  • X1341 contains two LNA modifications at position Y-2 and Y u and two phosphorothioate internucleotide linkages at the terminal nucleotides.
  • GalNAc-siRNA conjugates with an LNA modification at Y u show comparable stability to the respective siRNA without LNA modification.
  • GalNAc-siRNAs modified with an LNA modification at Y-2 of the first strand with either one (X1338) or two (X1339) phosphorothioate internucleotide linkages show an improved stability in comparison to the respective siRNA without LNA modification.
  • the number of phosphorothioate internucleotide linkages can therefore be reduced while retaining stability of the siRNA conjugates.
  • GalNAc-siRNAs containing LNA modifications at Y-2 and Y u did show a comparable stability to those with LNA modifications at Y.2.
  • GalNAc siRNA conjugates with LNA modifications at Y-2 and Y u without phosphorothioate internucleotide linkages show a reduced stability in comparison to the respective siRNA without LNA modification.
  • Example 5 in vitro study showing that reduction of one phosphorothioate internucleotide linkage does not impact mRNA knockdown of siRNAs modified with LNA at Y2’ and/or Yf of the second strand.
  • X1335 and X1348 contain two phosphorothioate linkages at the 3' end of the second strand.
  • X1342 and X1355 contain an LNA modification at Y and one phosphorothioate internucleotide linkage between position Y3’ and Y2’.
  • X1343 and X1356 contain an LNA modification at Yf’ and two phosphorothioate internucleotide linkages at the terminal positions.
  • X1344 and X1357 contain an LNA modification at Y2’ and one phosphorothioate linkage between the terminal nucleotides.
  • X1345 and X1358 contain an LNA modification at Y2’ and two phosphorothioate internucleotide linkage between the terminal nucleotides.
  • X1346 and X1359 contain two LNA modifications at position Y2’ and Y of the second strand.
  • X1347 and X1360 contain two LNA modifications at position Y2’ and Y and two phosphorothioate internucleotide linkages at the terminal nucleotides. All tested variants of siRNAs show comparable mRNA knockdown under the tested conditions.
  • This experiment was conducted in primary mouse hepatocytes. Cells were seeded at a density of 25,000 cells per 96-well and treated with 10 nM and 1 nM using siRNAs targeting At3 or 1 nM and 0.1 nM using siRNAs targeting Ttr directly after plating. Cells were lysed after 24 h, total RNA was extracted and Ttr, At3, Actb, Pten, ApoB mRNA levels were determined by Taqman qRT-PCR. Each bar represents geometric mean ⁇ SD from three technical replicates. Results are shown in Figure 2.
  • GalNAc-siRNA conjugates with LNA modifications at positions Y2’ and Yf of the second strand require one phosphorothioate internucleotide linkage to be stable in acidic rat liver tritosome extracts.
  • X1335 contains two phosphorothioate internucleotide linkages at the 3' end of the second strand.
  • X1342 contains an LNA modification at Yf’ and one phosphorothioate internucleotide linkage between Y3’ and Y2’.
  • X1343 contains an LNA modification at Y and two phosphorothioate internucleotide linkages at the terminal positions.
  • X1344 contains an LNA modification at Y2’ and one phosphorothioate internucleotide linkage between the terminal nucleotides.
  • X1345 contains an LNA modification at Y2’ and two phosphorothioate internucleotide linkage between the terminal nucleotides.
  • X1346 contains two LNA modifications at Y2’ and Yf of the second strand.
  • X1347 contains two LNA modifications at position Y2’ and Yf and two phosphorothioate internucleotide linkages at the terminal nucleotides.
  • GalNAc-siRNA conjugates with an LNA modification at Y and two phosphorothioate internucleotide linkages show an improved stability in comparison to the respective siRNA without LNA modification.
  • GalNAc-siRNA conjugates with an LNA modification at Y2’ and Y without phosphorothioate internucleotide linkages show a decreased stability in comparison to the respective siRNA without LNA modification.
  • All other GalNAc-siRNAs tested show a comparable stability in comparison to the respective siRNA without LNA modification.
  • the number of phosphorothioate internucleotide linkages in the second strand can therefore be reduced without leading to unstable molecules.
  • X1335 and X1348 contain no LNA modifications and two phosphorothioate linkages at the 3' end of each strand.
  • X1604 and X1607 contain an LNA modification at Y-2 of the first and Y2’ of the second strand and one phosphorothioate internucleotide linkage between Y-2 and Y u as well as Y f ’ and Y2’.
  • X1605 and X1608 contain an LNA modification at Y-2 of the first and Y2’ of the second strand and two phosphorothioate internucleotide linkages at the terminal positions.
  • X1606 and X1609 contain two LNA modifications at Y-2 and Y u as well as Y and Y2’and two phosphorothioate linkage between the terminal nucleotides.
  • siRNAs with one LNA modification in each strand that target At3 show comparable mRNA knockdown as the respective siRNA without LNA modifications.
  • siRNA with two LNA modifications at Y-2 and Y u as well as Y f ’ and Y2’ led to a reduced mRNA knockdown. All tested siRNAs targeting Ttr reduce the target mRNA levels less than the respective siRNA without LNA modification.
  • X1335 contains no LNA modifications and two phosphorothioate linkages at the 3' end of each strand.
  • X1338 contains an LNA modification at Y-2 and one phosphorothioate internucleotide linkage between the terminal nucleotides of the first strand.
  • X1344 contains an LNA modification at Y 2 ’and one phosphorothioate internucleotide linkage between the terminal nucleotides of the second strand.
  • X1604 contains an LNA modification at Y-2 of the first and Y2’ of the second strand and one phosphorothioate internucleotide linkage between Y-2 and Y u as well as Y and Y2’.
  • X1733 contains an LNA modification at Y-2 of the first and Y2’ of the second strand and one phosphorothioate internucleotide linkage between Y-2 and Y u as well as Yf’ and Y2’ and an irA modification at the 3' end of the second strand.
  • GalNAc-siRNA conjugates with one LNA modification in the first or the second strand show an improved stability compared to the respective siRNA without LNA modification.
  • Combination of first and second strand with LNA at Y-2 and Y2’ did not further improve stability.
  • Addition of an irA modification at Y of the second strand led to a further increase in stability.
  • X1594 and X1599 contain an irA modification at Y of the second strand.
  • X1595 and X1600 contain an LNA modification at Y2’and an irA modification at the 3' end of the second strand.
  • X1596 and X1601 contain an LNA modification at Y2’, an irA modification as well as one phosphorothioate internucleotide linkage at the 3' end of the second strand.
  • X1597 and X1602 contain an LNA modification at Y 2 ’, an irA modification at the 3' end of the second strand as well as one phosphorothioate internucleotide linkage between position Y3’ and Y2’.
  • X1598 and X1603 contain an LNA modification at position Y2’, an irA modification as well as two phosphorothioate internucleotide linkages at the 3' end of the second strand. All tested variants of siRNAs targeting Ttr show comparable or slightly reduced mRNA knockdown compared to the respective siRNA without an LNA modification under the tested conditions. All tested variants of siRNAs targeting At3 show reduced mRNA knockdown compared to the respective siRNA without an LNA modification.
  • siRNAs with LNA modifications in combination with inverted ribonucleotides in the second strand can be improved by addition of vinylphosphonate to the 5’ end of the first strand.
  • the activity of an siRNA containing irA at the 3' terminal position combined with an LNA modification at Y2’ of the second strand and a vinylphosphonate at the 5’ end of the first strand was analyzed using GalNAc-siRNA conjugates targeting At3 and Ttr by receptor- mediated uptake in mouse primary hepatocytes. All tested molecules have a vinylphosphonate at the 5’ end of the first strand.
  • X1643 and X1648 contain an irA modification at the 3' end of the second strand.
  • X1644 and X1649 contain an LNA modification at Y2’ and an irA modification at the 3' end of the second strand.
  • X1645 and X1650 contain an LNA modification at Y2’ and an irA modification as well as one phosphorothioate internucleotide linkage at the 3' end of the second strand.
  • X1646 and X1651 contain an LNA modification at Y2’ and an irA modification at the 3' end of the second strand as well as one phosphorothioate internucleotide linkage between position Y3’ and Y2’.
  • X1647 and X1652 contain an LNA modification at Y2’, an irA modification as well as two phosphorothioate internucleotide linkages at the 3' end of the second strand.
  • All tested variants of siRNAs targeting At3 show a slight decrease in mRNA knockdown under the tested conditions compared to the respective siRNA without LNA modification.
  • All tested variants of siRNAs targeting Ttr show a comparable mRNA knockdown as the respective siRNA without LNA modification under the tested conditions.
  • X1594 contains an irA modification at the 3' end of the second strand.
  • X1595 contains an LNA modification at Y2’ and an irA modification at the 3' end of the second strand.
  • X1596 contains an LNA modification at Y2’, an irA modification as well as one phosphorothioate internucleotide linkage at the 3' end of the second strand.
  • X1597 contains an LNA modification at Y2’, an irA modification at the 3' end of the second strand as well as one phosphorothioate internucleotide linkage between positions Y3’ and Y2’.
  • X1598 contains an LNA modification at Y2’, an irA modification as well as two phosphorothioate internucleotide linkages at the 3' end of the second strand. All tested siRNAs show a comparable stability in comparison to the respective siRNA without LNA modification.
  • Stability of GalNAc-siRNA conjugates with inverted ribonucleotides at the 3' end of the second strand and a vinylphosphonate at the 5’ end of the first strand in acidic rat liver tritosome extract can be improved by addition of LNA modifications at Y2’ of the second strand.
  • X1643 contains an irA modification at the 3' end of the second strand.
  • X1644 contains an LNA modification at Y2’ and an irA modification at the 3' end of the second strand.
  • X1645 contains an LNA modification at Y2’ and an irA modification as well as one phosphorothioate internucleotide linkage at the 3' end of the second strand.
  • X1646 contains an LNA modification at Y2’ and an irA modification at the 3' end of the second strand as well as one phosphorothioate internucleotide linkage between position Y3’ and Y2’.
  • X1647 contains an LNA modification at Y2’ and an irA modification as well as two phosphorothioate internucleotide linkages at the 3' end of the second strand.
  • siRNAs with an LNA modification at Y2’ of the second strand and no or one phosphorothioate internucleotide linkage between the terminal nucleotides of the second strand show a comparable stability in comparison to the respective siRNA without LNA modification.
  • siRNAs with an LNA modification at Y2’ of the second strand and one phosphorothioate internucleotide linkage between Y3’ and Y2’ or two phosphorothioate internucleotide linkages at the terminal nucleotides show an improved stability at 96 h in comparison to the respective siRNA without LNA modification.
  • Stability of GalNAc-siRNA conjugates with inverted ribonucleotides at the 3' end and LNA modification at Y2’ of the second strand and a 5' vinylphosphonate at the first strand in acidic tritosome lysate can be improved by addition of LNA modifications at Y-2 of the first strand.
  • X1647 contains an LNA modification at Y2’, an irA modification as well as two phosphorothioate internucleotide linkages at the 3' end of the second strand.
  • X1698 contains an LNA modification at Y-2 and two phosphorothioate internucleotide linkages at the terminal nucleotides of the first strand.
  • X1699 contains an LNA modification at Y2’ and an irA modification as well as two phosphorothioate internucleotide linkages at the 3' end of the second strand and an LNA modification at Y- 2 and two phosphorothioate internucleotide linkages at the terminal nucleotides of the first strand.
  • X1696 contains an LNA modification at Y-2 and one phosphorothioate internucleotide linkage between nucleotides Y-2 and Y u of the first strand.
  • X1699 contains an LNA modification at Y2’ and an irA modification at Y as well as one phosphorothioate internucleotide linkage between positions Y3’ and Y2’ of the second strand and an LNA modification at Y-2 with one phosphorothioate internucleotide linkage between the terminal nucleotides of the first strand.
  • siRNAs with an LNA modification at Y-2 of the first and Y2’ of the second strand and one phosphorothioate internucleotide linkage at each 3' end shows an improved stability over the molecule with the same LNA modifications but two phosphorothioate internucleotide linkages at each 3' end.
  • Stability of GalNAc-siRNA conjugates with inverted ribonucleotides at the 3' end and LNA modification at Y2’ of the second strand and a vinylphosphonate at the 5’ end of the first strand can be improved by addition of an LNA modification at Y-2 of the first strand.
  • siRNA containing irA at the 3' terminal position combined with an LNA modification at Y2’ of the second strand and a vinylphosphonate at the 5’ end and an LNA modification at Y-2 of the first strand was analyzed using GalNAc-siRNA conjugates targeting At3 and Ttr by receptor-mediated uptake in mouse primary hepatocytes. All tested molecules have a vinylphosphonate at the 5’ end of the first strand.
  • X1560 and X1704 contain two phosphorothioate linkages at the 3' end of the first and the second strand.
  • X1643 and X1648 contain an irA modification at the 3' end of the second strand.
  • X1646 and X1651 contain an irA modification at the 3' end, an LNA modification at Y2’ and a phosphorothioate internucleotide linkage between position Y3’ and Y2’ of the second strand.
  • X1696 and X1700 contain an LNA modification at Y-2 and a phosphorothioate internucleotide linkage between the terminal nucleotides of the first strand.
  • the second strands of X1697 and X1701 contain an irA modification at the 3' end, an LNA modification at Y2’ and a phosphorothioate internucleotide linkage between position Y3’ and Y2’ while the first strand contains an LNA modification at Y-2 and a phosphorothioate internucleotide linkage between the terminal nucleotides of the 3' end.
  • All tested variants of siRNAs targeting Ttr show a comparable mRNA knockdown under the tested conditions compared to the respective siRNA without LNA and irA modifications.
  • GalNAc-siRNA conjugates with LNA modifications in the 3’ end of the first and/or the second strand and an inverted nucleotide at the 3’ end of the second strand effect reduction AT3 mRNA levels in vitro independently of the underlying modification pattern.
  • the respective GalNAc siRNA modifications were tested with three different 2’-F and 2’- OMe modification patterns.
  • X1827 contains no LNA modifications and two phosphorothioate linkages at the 3' end of each strand.
  • X1828 contains an LNA modification at Y-2 and one phosphorothioate internucleotide linkage between the terminal nucleotides of the first strand.
  • X1829 contains an LNA modification at Y2’, an irA modification at Y and one phosphorothioate internucleotide linkage between Y3’ and Y2’ of the second strand.
  • X1830 and contain an LNA modification at Y-2 of the first and Y2’ of the second strand and one phosphorothioate internucleotide linkage between Y-2 and Y u of the first strand, one phosphorothioate internucleotide linkage between position Y3’ and Y2’ of the second strand and an irA modification at the 3' end of the second strand.
  • X1831 contains an LNA modification at position Y2’ and one phosphorothioate internucleotide linkage between position Y3’ and Y2’ of the second strand.
  • X1832 contains an LNA modification at Y2’ and one phosphorothioate internucleotide linkage between position Y3’ and Y2’ of the second strand and an LNA modification at Y- 2 and one phosphorothioate internucleotide linkage between the terminal nucleotides of the first strand.
  • X1835 contains no LNA modifications and two phosphorothioate linkages at the 3' end of each strand.
  • X1836 contains an LNA modification at Y-2 and one phosphorothioate internucleotide linkage between the terminal nucleotides of the first strand.
  • X1837and contains an LNA modification at Y2’, an irA modification at Y and one phosphorothioate internucleotide linkage between position Y3’ and Y2’ of the second strand.
  • X1838 contains an LNA modification at Y-2 of the first and Y2’ of the second strand and one phosphorothioate internucleotide linkage between Y-2 and Y u of the first strand, one phosphorothioate internucleotide linkage between Y3’ and Y2’ of the second strand and an irA modification at the 3' end of the second strand.
  • X1839 contains an LNA modification at Y2’ and one phosphorothioate internucleotide linkage between position Y3’ and Y2’ of the second strand.
  • X1840 contains an LNA modification at Y2’ and one phosphorothioate internucleotide linkage between position Y3’ and Y2’ of the second strand and an LNA modification at Y-2 and one phosphorothioate internucleotide linkage between the terminal nucleotides of the first strand.
  • X1843 contains no LNA modifications and two phosphorothioate linkages at the 3' end of each strand.
  • X1844 contains an LNA modification at Y-2 and one phosphorothioate internucleotide linkage between the terminal nucleotides of the first strand.
  • X1845 contains an LNA modification at Y2’, an irA modification at Y and one phosphorothioate internucleotide linkage between Y3’ and Y2’ of the second strand.
  • X1846 contains an LNA modification at Y-2 of the first and Y2’ of the second strand and one phosphorothioate internucleotide linkage between Y-2 and Y u of the first strand, one phosphorothioate internucleotide linkage between Y3’ and Y2’ of the second strand and an irA modification at the 3' end of the second strand.
  • X1847 contains an LNA modification at Y2’ and one phosphorothioate internucleotide linkage between Y3’ and Y2’ of the second strand.
  • X1848 contains an LNA modification at Y2’ and one phosphorothioate internucleotide linkage between Y3’ and Y2’ of the second strand and an LNA modification at Y-2 and one phosphorothioate internucleotide linkage between the terminal nucleotides of the first strand.
  • GalNAc siRNA conjugates with vinylphosphonate at the 5’ end of the first strand and LNA modifications in the 3’ end of the first and/or the second strand and an inverted nucleotide at the 3’ end of the second strand reduce ALDH2 and AT3 mRNA levels in vitro independently of the underlying modification pattern.
  • the respective GalNAc siRNA modifications are tested with three different 2’-F and 2’-OMe modification patterns.
  • X1775 (At3) and X1787 (Aldh2) contain no LNA modifications and two phosphorothioate linkages at the 3' end of each strand.
  • X1776 (At3) and X1788 (Aldh2) contain an LNA modification at Y-2 and one phosphorothioate internucleotide linkage between the terminal nucleotides of the first strand.
  • X1777 (At3) and X1789 (Aldh2) contain an LNA modification at Y2’, an irA modification at Y and one phosphorothioate internucleotide linkage between Y3’ and Y2’ of the second strand.
  • X1778 (At3) and X1790 (Aldh2) contain an LNA modification at Y-2 of the first and Y2’ of the second strand and one phosphorothioate internucleotide linkage between position Y-2 and Y u of the first strand, one phosphorothioate internucleotide linkage between Y3’ and Y2’ of the second strand and an irA modification at the 3' end of the second strand.
  • X1799 (At3) and X1811 (Aldh2) contain an LNA modification at Y2’ and one phosphorothioate internucleotide linkage between position Y3’ and Y2’ of the second strand.
  • X1800 (At3) and X1812 (Aldh2) contain an LNA modification at Y2’ and one phosphorothioate internucleotide linkage between position Y3’ and Y2’ of the second strand and an LNA modification at Y-2 and one phosphorothioate internucleotide linkage between the terminal nucleotides of the first strand.
  • X1779 (At3) and X1791 (Aldh2) contain no LNA modifications and two phosphorothioate linkages at the 3' end of each strand.
  • X1780 (At3) and X1792 (Aldh2) contain an LNA modification at Y-2 and one phosphorothioate internucleotide linkage between the terminal nucleotides of the first strand.
  • X1781 (At3) and X1793 (Aldh2) contain an LNA modification at Y2’, an irA modification at Y and one phosphorothioate internucleotide linkage between position Y3’ and Y2’ of the second strand.
  • X1782 (At3) and X1794 (Aldh2) contain an LNA modification at Y-2 of the first and Y2’ of the second strand and one phosphorothioate internucleotide linkage between Y-2 and Y u of the first strand, one phosphorothioate internucleotide linkage between position Y3’ and Y2’ of the second strand and an irA modification at the 3' end of the second strand.
  • X1803 (At3) and X1815 (Aldh2) contain an LNA modification at Y2’ and one phosphorothioate internucleotide linkage between Y3’ and Y2’ of the second strand.
  • X1804 (At3) and X1816 (Aldh2) contain an LNA modification at Y2’ and one phosphorothioate internucleotide linkage between position Y3’ and Y2’ of the second strand and an LNA modification at Y-2 and one phosphorothioate internucleotide linkage between the terminal nucleotides of the first strand.
  • X1783 (At3) and X1795 (Aldh2) contain no LNA modifications and two phosphorothioate linkages at the 3' end of each strand.
  • X1784 (At3) and X1796 (Aldh2) contain an LNA modification at Y-2 and one phosphorothioate internucleotide linkage between the terminal nucleotides of the first strand.
  • X1785 (At3) and X1797 (Aldh2) contain an LNA modification at Y2’, an irA modification at Y and one phosphorothioate internucleotide linkage between position Y3’ and Y2’ of the second strand.
  • X1786 (At3) and X1798 (Aldh2) contain an LNA modification at Y-2 of the first and Y2’ of the second strand and one phosphorothioate internucleotide linkage between position Y-2 and Y u of the first strand, one phosphorothioate internucleotide linkage between Y3’ and Y2’ of the second strand and an irA modification at the 3' end of the second strand.
  • X1807 (At3) and X1819 (Aldh2) contain an LNA modification at Y2’ and one phosphorothioate internucleotide linkage between Y3’ and Y2’ of the second strand.
  • X1808 (At3) and X1820 (Aldh2) contain an LNA modification at Y2’ and one phosphorothioate internucleotide linkage between position Y3’ and Y2’ of the second strand and an LNA modification at Y-2 and one phosphorothioate internucleotide linkage between the terminal nucleotides of the first strand.
  • X1335 contains no LNA modifications and two phosphorothioate linkages at the 3' end of each strand.
  • X1730 contains an irA modification at the 3' end of the second strand.
  • X1338 contains an LNA modification at Y-2 and one phosphorothioate internucleotide linkage between the terminal nucleotides of the first strand.
  • X1733 contains an LNA modification at Y-2 of the first and Y2’ of the second strand and one phosphorothioate internucleotide linkage between position Y-2 and Y u of the first strand, one phosphorothioate internucleotide linkage between position Y3’ and Y2’ of the second strand and an irA modification at the 3' end of the second strand.
  • GalNAc-siRNA conjugates containing an LNA modification at Y-2 of the first strand reduce the target mRNA levels more compared to the respective siRNA without LNA modifications.
  • GalNAc-siRNA conjugates with an irA modification at Yf of the second strand, as well as the siRNA with an LNA modification at Y2’ and an irA modification at Yf of the second strand reduce the target mRNA levels less than the respective siRNA without LNA and irA modifications in the 3' end of the second strand. This experiment was conducted in primary human hepatocytes.
  • Cells were seeded at a density of 35,000 cells per 96-well and treated with 100 nM, 20 nM, 4 nM, 0.8 nM and 0.16 nM using siRNAs targeting At3 directly after plating. Cells were lysed after 24 h, total RNA was extracted and At3 and PPIB mRNA levels were determined by Taqman qRT- PCR. Each bar represents mean ⁇ SD from three technical replicates. Results are shown in Figure 16A.
  • X1729 contains two phosphorothioate linkages at the 3' end of the first and the second strand.
  • X1731 contains an irA modification at the 3' end of the second strand.
  • X1732 contains an LNA modification at Y2’ and an irA modification at position Y of the second strand.
  • X1696 contains an LNA modification at Y-2 and a phosphorothioate internucleotide linkage between the terminal nucleotides of the first strand.
  • the second strand of X1697 contains an irA modification at the 3' end, an LNA modification at Y2’ and a phosphorothioate internucleotide linkage between Y3’ and Y2’ while the first strand contains an LNA modification at Y-2 and a phosphorothioate internucleotide linkage between the terminal nucleotides of the 3' end.
  • GalNAc-siRNA conjugates containing an LNA at Y-2 of the first strand reduce the target mRNA levels more compared to the respective siRNA without LNA.
  • GalNAc-siRNA conjugates with an irA modification at Y of the second strand as well as the siRNA conjugates with an LNA modification at Y2’ and an irA modification at Y of the second strand reduce the target mRNA levels less than the respective siRNA conjugate without LNA and irA modifications in the 3' end of the second strand.
  • GalNAc siRNA conjugates containing an LNA at Y-2 of the first strand and an LNA modification at Y2’ and an irA modification at Y of the second strand reduce the target mRNA levels to the same extent as GalNAc-siRNA conjugates containing an LNA at Y-2 of the first strand.
  • GalNAc-siRNA conjugates with an LNA modification at Y-2 of the first strand, an LNA modification at Y2’ and an irA modification at Y of the second strand show improved reduction of AT3 target mRNA levels in vivo.
  • X1335 contains no LNA modifications and two phosphorothioate linkages at the 3' end of each strand.
  • X1338 contains an LNA modification at Y-2 and one phosphorothioate internucleotide linkage between the terminal nucleotides of the first strand.
  • X1733 contains an LNA modification at Y-2 and one phosphorothioate internucleotide linkage between the terminal nucleotides of the first strand and an LNA modification at Y2’, one phosphorothioate internucleotide linkage between positions Y2’ and Y and an irA modification at the 3' end of the second strand.
  • GalNAc-siRNA conjugates containing an LNA at Y-2 of the first strand reduce the target mRNA levels more compared to the respective siRNA without LNA.
  • GalNAc- siRNA conjugates containing an LNA modification at Y-2 of the first strand and an LNA modification at Y2’ and an irA modification at Y of the second strand reduce the target mRNA levels to a higher extent compared to GalNAc-siRNA conjugates containing an LNA at Y-2 of the first strand.
  • EDTA-plasma samples were retrieved by retro-orbital bleeding at different time points before and after treatment. Plasma AT3 concentrations were analyzed with an Antithrombin III (SERPINC1) Mouse ELISA Kit (abeam ab108800). Results are shown in Figure 17A.
  • SERPINC1 Antithrombin III
  • X1335 contains no LNA modifications and two phosphorothioate linkages at the 3' end of each strand.
  • X1338 contains an LNA modification at Y-2 and one phosphorothioate internucleotide linkage between the terminal nucleotides of the first strand.
  • X1733 contains an LNA modification at Y-2 and one phosphorothioate internucleotide linkage between the terminal nucleotides of the first strand and an LNA modification at Y2’, one phosphorothioate internucleotide linkage between positions Y2’ and Y and an irA modification at the 3' end of the second strand.
  • GalNAc-siRNA conjugates containing an LNA at Y-2 of the first strand reduce the target mRNA levels more compared to the respective siRNA without LNA.
  • GalNAc siRNA conjugates containing an LNA modification at Y-2 of the first strand and an LNA modification at Y2’ and an irA modification at Y of the second strand reduce the target mRNA levels to a higher extent compared to GalNAc-siRNA conjugates containing an LNA at Y-2 of the first strand.
  • AT3 target mRNA reduction can be improved in vivo using GalNAc-siRNA conjugates with vinylphosphonate at the 5' end of the first strand and additionally with an LNA modification at Y-2 of the first strand, an LNA modification at Y2’and an irA modification at Y of the second strand.
  • X1729 contains two phosphorothioate linkages at the 3' end of the first and the second strands.
  • X1732 contains an LNA modification at Y2’ and an irA modification at Y of the second strand.
  • X1696 contains an LNA modification at Y-2 and a phosphorothioate internucleotide linkage between the terminal nucleotides of the first strand.
  • the second strand of X1697 contains an irA modification at the 3' end, an LNA modification at Y2’ and a phosphorothioate internucleotide linkage between positions Y3’ and Y2’ while the first strand contains an LNA modification at Y-2 and a phosphorothioate internucleotide linkage between the terminal nucleotides of the 3' end.
  • GalNAc-siRNA conjugates containing an LNA at Y-2 of the first strand reduce the target mRNA levels more compared to the respective siRNAs without LNA.
  • GalNAc-siRNA conjugates with an LNA modification at Y2’ and an irA modification at Y of the second strand reduce the target mRNA levels less than the respective siRNA without LNA and irA modifications in the 3' end of the second strand.
  • GalNAc siRNA conjugates containing an LNA at Y-2 of the first strand and an LNA modification at Y2’ and an irA modification at Y of the second strand reduce the target mRNA levels to the same extent as GalNAc-siRNA conjugates containing an LNA at Y-2 of the first strand.
  • EDTA-plasma samples were retrieved by retro-orbital bleeding at different time points before and after treatment.
  • Plasma AT3 concentrations were analyzed with an Antithrombin III (SERPINC1) Mouse ELISA Kit (abeam ab108800). Due to a technical handling error, one animal of the group treated with X1696 was excluded from analysis. Results are shown in Figure 18A.
  • X1729 contains two phosphorothioate linkages at the 3' end of the first and the second strand.
  • X1732 contains an LNA modification at Y2’ and an irA modification at Y of the second strand.
  • X1696 contains an LNA modification at Y-2 and a phosphorothioate internucleotide linkage between the terminal nucleotides of the first strand.
  • the second strand of X1697 contains an irA modification at the 3' end, an LNA modification at Y2’ and a phosphorothioate internucleotide linkage between positions Y3’ and Y2’ while the first strand contains an LNA modification at Y-2 and a phosphorothioate internucleotide linkage between the terminal nucleotides of the 3' end.
  • GalNAc-siRNA conjugates containing an LNA at Y-2 of the first strand reduce the target mRNA levels more compared to the respective siRNA without LNA.
  • GalNAc-siRNA conjugates with an LNA modification at Y2’ and an irA modification at Y of the second strand reduce the target mRNA levels less than the respective siRNA without LNA and irA modifications in the 3' end of the second strand.
  • GalNAc siRNA conjugates containing an LNA at Y-2 of the first strand and an LNA modification at Y2’ and an irA modification at Y of the second strand reduce the target mRNA levels to a same extent as GalNAc- siRNA conjugates containing an LNA at position 18 of the first strand.
  • Example compounds were synthesised according to methods described in Example 1 or below and methods known to the person skilled in the art. Assembly of the oligonucleotide chain and linker building blocks was performed by solid phase synthesis applying phosphoramidite methodology.
  • nucleoside thiophosphoramidites (Hongene), were coupled using a triple couple/wash cycle over a combined period of 60 min. After coupling a Cap/OX/Cap or Cap/Thio/Cap cycle was applied (Cap: Ac2O/NMI/Lutidine/Acetonitrile, Oxidizer: 0.1 M I2 in pyridine/FLO).
  • PS Phosphorothioates
  • PS2 phosphorodithioates
  • DDTT 0.05 M ((dimethylamino-methylidene) amino)-3H-1 ,2.4-dithiazoline-3-thione
  • Single-stranded oligonucleotides and double-stranded siRNAs and siRNA conjugates are listed in Tables 4 and 5.
  • siRNAs of different lengths with and without LNA modification were conducted in Hep3B cells.
  • the cells were seeded at a density of 30,000 cells per 96-well, and simultaneously transfected with 1 nM and 0.1 nM siRNA and 0.3 pl RNAiMax. Cells were lysed after 24 h, total RNA was extracted and PTEN and PPIB mRNA levels were determined by Taqman qRT-PCR. Each bar represents mean ⁇ SD from three technical replicates. Data are shown in Figures 19A and 19B.
  • the experiment with siRNAs targeting AT3 was conducted in HepG2 cells.
  • the cells were seeded at a density of 40,000 cells per 96-well, and simultaneously transfected with 1 nM and 0.1 nM siRNA and 0.3 pl RNAiMax. Cells were lysed after 24 h, total RNA was extracted and AT3 and PPIB mRNA levels were determined by Taqman qRT-PCR. Each bar represents mean ⁇ SD from three technical replicates. Results are shown in Figure 19C.
  • X1767 is a GalNAc siRNA conjugate with two terminal phosphorothioates at the 3'-end of the first strand, the 5'-end of the second strand and the 3'-end of the second strand.
  • X2065 and X2067 contain an LNA modification at position 18 and one phosphorothioate internucleotide linkage between positions 18 and 19 of the first strand.
  • One phosphorodithioate internucleotide linkage is present at the 5'- and 3'-end of the second strand.
  • X2065 has two phosphorothioates at the 5'-end of the first strand, whereas X2067 has 5'-(E)-vinylphosphonate and phosphodiester internucleotide linkages at this end.
  • X2066 and X2068 have an LNA modification at position 18 and an inverted RNA at position 19 of the second strand.
  • a phosphorothioate internucleotide linkage is present between positions 17 and 18 of the second strand.
  • the 5'-end and 3'-end of the first strand and 5'-end of the second strand are modified as in X2065 and X2067, respectively.
  • X1910, X1912, X1913, X1921 , X1922, X1923, X1925, X1926, X1934, X1936 contain LNA and irA modifications and phosphorothioate linkages as detailed in Tables 4 and 5.
  • the siRNAs assayed here were conjugated to the GalNAc cluster [ST23]a ST43.
  • X1910, X1912, X1913, X1921 , X1922, X1923, X1925, X1926, X1934, X1936 contain LNA and irA modifications and phosphorothioate linkages as detailed in Tables 4 and 5. Best tritosome stability is observed when LNA is placed at the penultimate position of the 5' end of the second strand.
  • X1913 contains LNA at position 2 and a phosphorothioate internucleotide linkage between positions 1 and 2 of the second strand.
  • An siRNA conjugate with no phosphorothioate at this end is less stable in tritosomes (X1912).
  • the siRNA assayed here were conjugated to the GalNAc cluster [ST23]3 ST43.
  • X1767, X1911 , X1924, X1919, X1932, X1915, and X1910 contain LNA and irA modifications and phosphorothioate linkages as detailed in Tables 4 and 5.
  • the siRNAs assayed here were conjugated to the GalNAc cluster [ST23 (ps)]3 ST43 (ps).
  • X1913 with the GalNAc cluster [ST23]a ST43 was included as reference.
  • X1767, X1911 , X1924, X1919, X1932, X1915, and X1910 contain LNA and irA modifications and phosphorothioate linkages as detailed in Tables 4 and 5. Best tritosome stability is observed when LNA is placed at the penultimate position of the 5'-end of the second strand.
  • X1911 contains LNA at position 2 and a phosphorothioate linkage between the GalNAc cluster and position 1 of the second strand.
  • the siRNA is conjugated to the GalNAc cluster [ST23 (ps)]a ST43 (ps).
  • X1913 with the GalNAc cluster [ST23]3 ST43 was included as reference.
  • the tested GalNAc-siRNA conjugates contain LNA and irA modifications and phosphorothioate linkages as detailed in Tables 4 and 5.
  • In vitro activity is improved when an LNA modification is placed at position 18 of the first strand and another LNA modification is simultaneously placed at position 2 of the second strand (referring to the penultimate positions in a blunt ended 19-mer). This is the case for GalNAc clusters with and without phosphorothioate modifications and for conjugates that are additionally modified with 5'-(E)-vinylphosphonate at the first strand.
  • Tritosome stability of GalNAc-siRNA conjugates with LNA at penultimate position 18 of the second strand in a blunt ended 19-mer is improved and cannot be further improved by an LNA modification in the first strand.
  • X1335 contains no LNA modifications and two phosphorothioate linkages at the 3' end of each strand.
  • X1338 contains an LNA modification at position 18 and one phosphorothioate internucleotide linkage between the terminal nucleotides.
  • X1344 contains an LNA modification at position 18 and one phosphorothioate internucleotide linkage between the terminal nucleotides of the second strand.
  • X1345 contains an LNA modification at position 18 and two phosphorothioate internucleotide linkage between the terminal nucleotides of the second strand.
  • X1604 contains an LNA modification at position 18 of the first and the second strand and one phosphorothioate internucleotide linkage between position 18 and 19.
  • X1605 contains an LNA modification at position 18 of the first and the second strand and two phosphorothioate internucleotide linkage between position 18 and 19.
  • GalNAc-siRNA conjugates with one LNA modification in the second strand show an improved stability compared to the respective siRNA without LNA modification. Combination of first and second strand with LNA at position 18 did not further improve stability.
  • Example compounds were synthesised according to methods described herein and methods known to the person skilled in the art. Assembly of the oligonucleotide chain and linker building blocks was performed by solid phase synthesis applying phosphoramidite methodology.
  • Example compounds were synthesized as described in Examples 1 and 20. Single-stranded oligonucleotides and double-stranded siRNAs and siRNA conjugates are listed in Tables 1-7.
  • GalNAc siRNA conjugates with LNA modifications at one or more penultimate positions of the siRNA strands show improved target protein reduction in vivo.
  • the tested GalNAc siRNA conjugates contain LNA and irA modifications and phosphorothioate linkages as detailed in Tables 1-7. In vivo activity is improved when LNA modifications are placed at the penultimate position of first strand 3' end, second strand 5' end or second strand 3' end. These modifications can be combined with each other, with 5'-(E)-vinylphosphonate at the first strand, and with [ST23(ps)]3 ST41 (ps) and [ST23]a ST41 GalNAc clusters.
  • EDTA-plasma samples were retrieved by retro-orbital bleeding at day 29 after treatment.
  • Plasma AT3 concentrations were analyzed with an Antithrombin III (SERPINC1) Mouse ELISA Kit (abeam ab108800). Results are shown in Figure 28.
  • Table 7 First strand and second strand sequences of double-stranded siRNAs and siRNA conjugates.

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Abstract

L'invention concerne de nouveaux acides nucléiques de formule (I), en particulier des acides nucléiques et de préférence des acides nucléiques conjugués de formule (I), comprenant au moins une modification de LNA et, éventuellement, comprenant en outre des nucléotides inversés et/ou des liaisons phosphorothioates. De tels acides nucléiques (conjugués) de formule (I) peuvent être utilisés pour inhiber l'expression d'un gène cible. L'invention concerne en outre des compositions comprenant lesdits acides nucléiques (conjugués) de formule (I) destinés à être utilisés dans la prophylaxie ou le traitement d'une maladie.
PCT/EP2025/052612 2024-02-01 2025-01-31 Acides nucléiques conjugués et acides nucléiques comprenant des nucléosides verrouillés et des nucléotides inversés pour inhiber l'expression génique dans une cellule Pending WO2025163186A1 (fr)

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