Nucleic acids comprising locked nucleic acids (LNA) for inhibiting gene expression in a cell

Abstract

The invention relates to double stranded nucleic acids of formula (I), in particular, conjugated double stranded nucleic acids comprising at least one or two LNA (= locked nucleic acid) modification(s) in one or two defined positions of the sense strand. Such (conjugated) nucleic acids can be used to inhibit expression of a target gene. The invention further relates to compositions comprising said (conjugated) nucleic acids for use in the prophylaxis or treatment of a disease.

Description

[0001] Nucleic acids comprising locked nucleic acids (LNA) for inhibiting gene expression in a cell

[0002] Field of the invention

[0003] The invention relates to double stranded nucleic acids of formula (I), in particular, conjugated double stranded nucleic acids comprising at least one or two LNA (= Locked Nucleic Acid) modification(s) in one or two defined positions of the sense strand. Such (conjugated) nucleic acids can be used to inhibit expression of a target gene. The invention further relates to compositions comprising said (conjugated) nucleic acids for use in the prophylaxis or treatment of a disease.

[0004] Background

[0005] 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. Such gene silencing agents are becoming increasingly important for therapeutic applications in medicine. For the pharmaceutical development of such 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.

[0006] One of the key requirements for the use of inhibitory nucleic acids in medical applications is metabolic stability of the nucleic acids and any ligand they are conjugated to. This is mostly achieved through specific chemical modifications. One commonly used modification is the replacement of metabolically, relatively labile phosphodiester (PO) linkages with more stable phosphorothioate (PS) linkages. The downside of using phosphorothioate (PS) linkages however is that they are in general not stereo defined in solid phase synthesis, in which case each phosphorothioate (PS) modification can be present in the S or R form in the final molecule. The use of such linkages in nucleic acids or ligands leads to a mix of stereo chemically different molecules in the final product (for example, several siRNAs will have a phosphorothioate in the Spconfiguration at a given position, and others will have a phosphorothioate in the Rpconfiguration). With each additional phosphorothioate (PS) that is not stereo defined in a product, the number of possible stereochemical forms of the resulting product are multiplied by two. This in turn leads to higher complexity of compositions comprising such products, as they in fact comprise a mix of products with different stereochemical conformations. This increased complexity can have negative effects on downstream processes, such as purification of the drug product, and creates uncertainty in clinical development, as it is not certain whether each of the forms of the product is as potent as the others. Further, the increase of the numbers of phosphorothioates (PS) within a nucleic acid has been described to correlate with certain unintended effects, such as increased protein binding and toxicity. It is therefore beneficial to reduce the number of stereogenic phospohorothioate linkages in nucleic acid products.

[0007] Studies have previously shown that 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 stabilization 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 21A Locked Nucleic Acid is a nucleotide where the ribose is “locked” by a methylene bridge between 2' and 4' carbon atoms of the sugar.131This non-natural modification aggravates enzymatic digestion. Additionally, LNA modifications in oligonucleotides have shown increased thermal stability.141For siRNA design, it is crucial that the siRNA duplex comprises a thermodynamic asymmetry.1561Functional 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.15 61Asymmetry 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. Several approaches have shown that two or three LNA modifications are generally accepted at the 3' terminus of the antisense strand, but in some cases a trade-off of decreased on-target activity had to be made.11 J’81These examples showed that at least two contiguous LNA modifications are needed to achieve the intended effects of improved metabolic stability. However, having at least two contiguous LNA modifications can have negative effects on knockdown efficacy as shown in the examples above.

[0008] There is a clear need in the industry for more potent and stable inhibitory nucleic acids, such as siRNAs. The present invention provides an inhibitory nucleic acid showing increased and / or longer lasting knockdown activity, while stability is improved.

[0009] Summary of the invention

[0010] One aspect of the invention is a 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

[0011] 5' Xa- Yf - Y2- Y3- Y4- Y5- Z - Y-5 - Y-4 - Y-3 - Y-2 - Yu- Xb3'

[0012] Second (sense or passenger) strand

[0013] 3' Xa'- Yf'- Y2' - Y3'- Y4' - Y5'- Z' - Y-5 - Y.4' - Y.3'- Y-2' - Yu'- Xb' 5'

[0014] (I), wherein: each of Xa, Xa1, Xb and Xb1independently represents an overhang nucleotide sequence comprising 0-15, 0-10, or 0-6 nucleotides, each of Yf and Yf' independently represents a nucleotide, wherein 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 Yuand Yu' independently represents a nucleotide, wherein Yuand Yu' represent the ultimate position of the double stranded region between the first and the second strands, wherein Yuand Yu' are opposite each other in their respective positions in the first and second strands and may be complementary to each other, each of Y2to Ys and Y2' to Ys' independently represents a nucleotide within the double stranded region, wherein Y2to Ys and Y2' 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.2to Y.s and Y.2' to Y-s' independently represents a nucleotide within the double stranded region, wherein Y.2to Y-s and Y.2' to Y-s'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 a) Y-2' is an LNA modified nucleotide, b) Y2' is an LNA modified nucleotide, or c) Y-2' and Y2' each are LNA modified nucleotides.

[0015] One aspect of the invention is 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.

[0016] 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 the nucleic acid of formula (I), or the conjugated nucleic acid, or the composition according to the invention, to said subject.

[0017] One aspect of the invention is the nucleic acid of formula (I), or the conjugated nucleic acid, or the composition of the invention for use in the prophylaxis or treatment of a disease, disorder or syndrome.

[0018] One aspect of the invention is the use of the nucleic acid of formula (I), or the conjugated nucleic acid, or the composition of the invention in the preparation of a medicament for prophylaxis or treatment of a disease, disorder or syndrome.

[0019] Detailed description of the invention

[0020] The present invention relates to a double stranded nucleic acid of formula (I), preferably 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 in one or two defined position(s) of the sense strand, and, optionally, may further comprise other modifications. The nucleic acid of formula (I), or the conjugate or 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.

[0021] A first aspect of the invention is a double stranded nucleic acid for inhibiting expression of a target gene, comprising a first strand and a second strand, represented by formula (I):

[0022] First (antisense) strand

[0023] 5' Xa- Yf - Y2- Y3- Y4- Y5- Z - Y-5 - Y-4 - Y-3 - Y-2 - Yu- Xb3'

[0024] Second (sense) strand

[0025] 3' Xa'- Yf'- Y2' - Y3'- Y4' - Y5'- Z' - Y-5 - Y.4' - Y.3'- Y-2' - Yu'- Xb' 5'

[0026] (I), wherein: each of Xa, Xa1, Xb and Xb1independently represents an overhang nucleotide sequence comprising 0-10, or 0-6 nucleotides, each of Yf and Yf' independently represents a nucleotide, wherein 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 Yuand Yu' independently represents a nucleotide, wherein Yuand Yu' represent the ultimate position of the double stranded region between the first and the second strands, wherein Yuand Yu' 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 Ysand Y2' to Y5' independently represents a nucleotide within the double stranded region, wherein Y2 to Ys and Y2' 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' to Y.s' independently represents a nucleotide within the double stranded region, wherein Y-2 to Y-s and Y-2' to Y-s'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 a) Y-2' is an LNA modified nucleotide, b) Y2' is an LNA modified nucleotide, or c) Y-2' and Y2' each are LNA modified nucleotides.

[0027] 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.

[0028] In one embodiment of the double stranded nucleic acid of formula (I), each of Xa, Xa1, Xb and Xb1independently represents an overhang nucleotide sequence comprising 0-15, 0-10, 0-6, 1- 9, 1-8, 1-6, or 1-3 nucleotides.

[0029] In one preferred embodiment of the double stranded nucleic acid of formula (I), each of Xa, Xa', Xb and Xb' comprises 0 nucleotides.

[0030] In one embodiment of the double stranded nucleic acid of formula (I), 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.

[0031] In one embodiment of the double stranded nucleic acid of formula (I) of the invention, Y-2' is an LNA modified nucleotide, or Y2' is an LNA modified nucleotide, and the second strand comprises no additional LNA modified nucleotide. In one embodiment of the double stranded nucleic acid of formula (I) of the invention, Y-2' and Y2' each are LNA modified nucleotides, and the second strand comprises no additional LNA modified nucleotide.

[0032] In one embodiment, the first strand of the double stranded nucleic acid of formula (I) of the invention comprises no LNA modified nucleotide.

[0033] In a further embodiment, the second strand of the double stranded nucleic acid of formula (I) of the invention comprises one phosphorothioate linkage between Y2' and Yf'.

[0034] In a further embodiment, the second strand of the double stranded nucleic acid of formula (I) of the invention comprises at least one phosphorothioate linkage between Y2' and Yf'.

[0035] In a further embodiment, the second strand of the double stranded nucleic acid of formula (I) of the invention comprises two phosphorothioate linkages between Y2'and Y / and between Y2' and Y3'.

[0036] In a further embodiment, the second strand of the double stranded nucleic acid of formula (I) of the invention comprises at least two phosphorothioate linkages between Y2' and Yf' and between Y2' and Y3'.

[0037] In a further embodiment, the first strand of the double stranded nucleic acid of formula (I) of the invention further comprises a) at least one phosphorothioate linkage between Yf and Y2, or b) at least two phosphorothioate linkages between Yf and Y2 and between Y2 and Y3.

[0038] In a further embodiment, the first strand of the double stranded nucleic acid of formula (I) of the invention further comprises a) one phosphorothioate linkage between Yf and Y2, or b) two phosphorothioate linkages between Yf and Y2 and between Y2 and Y3.

[0039] In a further embodiment, the first strand of the double stranded nucleic acid of formula (I) of the invention comprises a (E)-vinylphosphonate nucleotide at a) position Yf, when Xa = 0; or b) the position of the first nucleotide Xacounted from the 5’-end of the first strand, when Xa= 1-6. The (E)-vinylphosphonate nucleotide may be a DNA or RNA nucleotide. Preferably, the 5’ (E)- vinylphosphonate nucleotide is an RNA nucleotide, more preferably a (vp)-ll.

[0040] In one embodiment, if Yf of the first strand is a nucleotide other than A or II, this nucleotide is replaced by an A or II. Preferably, if Yf of the first strand is a nucleotide other than II, this nucleotide is replaced by II, and more preferably by II with a 5’ vinylphosphonate (= (vp)-ll).

[0041] In a further embodiment, the second strand of the double stranded nucleic acid of formula (I) of the invention is modified with 2' fluoro modifications at those second strand positions that are opposite to positions 11 , 12 and 13 of the first strand counting from Yf, optionally wherein the remaining nucleotides of the second strand are modified with 2' O-methyl modifications, and optionally wherein the first strand is modified with alternating 2' O-methyl modifications and 2' fluoro modifications.

[0042] In a further embodiment, the second strand of the double stranded nucleic acid of formula (I) of the invention comprises at least one inverted nucleotide. Preferably, the inverted nucleotide is at position Yu' directly adjacent to the LNA modified nucleotide and, optionally, the nucleic acid of formula (I) further comprises a phosphorothioate linkage between Yu' and Y-2' and / or between Y-2' and Y-3'.

[0043] In a further embodiment, the second strand of the double stranded nucleic acid of formula (I) of the invention comprises one inverted nucleotide. Preferably, the inverted nucleotide is at position Yu' directly adjacent to the LNA modified nucleotide and, optionally, the nucleic acid of formula (I) further comprises one phosphorothioate linkage between Yu' and Y-2' and / or one phosphorothioate linkage between Y_2' and Y_3'.

[0044] In another embodiment, the second strand of the double stranded nucleic acid of formula (I) of the invention comprises at least one inverted nucleotide, which is preferably at position Yf' directly adjacent to the LNA modified nucleotide and, optionally, the nucleic acid of formula (I) further comprises a phosphorothioate linkage between Y and Y2' and / or between Y2' and Y3'.

[0045] In another embodiment, the second strand of the double stranded nucleic acid of formula (I) of the invention comprises one inverted nucleotide, which is preferably at position Yf' directly adjacent to the LNA modified nucleotide and, optionally, the nucleic acid of formula (I) further comprises one phosphorothioate linkage between Yf’ and Y2' and / or one phosphorothioate linkage between Y2' and Y3'.

[0046] 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. Likewise, 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.

[0047] In another embodiment of the invention, 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.

[0048] In another embodiment of the invention, 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.

[0049] In one embodiment of the invention, 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. In another embodiment of the invention, the inverted nucleotide is a 2 - deoxynucleotide.

[0050] When a given nucleotide of the second strand is an inverted nucleotide, the inverted nucleotide is preferably an unmodified nucleotide in that it does not comprise any further modifications compared to the natural nucleotide counterpart.

[0051] When the 3’ terminal nucleotide of the second strand, preferably at position Y is an inverted nucleotide, preferably an inverted RNA nucleotide, the inverted nucleotide or inverted RNA nucleotide is preferably an unmodified nucleotide. Specifically, the inverted RNA nucleotide is preferably a 2’-OH nucleotide. Preferably, in this embodiment when the 3’ terminal nucleotide of the second strand is an inverted nucleotide or inverted RNA nucleotide, the nucleic acid of formula (I) is blunt ended at least at the end that comprises the 5’ end of the first strand.

[0052] When the 5’ terminal nucleotide of the second strand, preferably at position Yu’ is an inverted nucleotide, preferably an inverted RNA nucleotide, the inverted nucleotide or inverted RNA nucleotide is preferably an unmodified nucleotide. Specifically, the inverted RNA nucleotide is preferably a 2’-OH nucleotide. Preferably, in this embodiment when the 5’ terminal nucleotide of the second strand is an inverted nucleotide or inverted RNA nucleotide, the nucleic acid of formula (I) is blunt ended at least at the end that comprises the 3’ end of the first strand. In one embodiment, the nucleic acid 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'-OMe, 2'-F, a ligand, a linker, a 3' end or 5' end modification or of any other modification well known to those with skill in the art.

[0053] In one embodiment, the nucleic acid of the invention, particularly the conjugated nucleic acid of the invention, is a siRNA, preferably wherein the nucleic acid mediates RNA interference.

[0054] In one embodiment, the invention relates to a nucleic acid of formula (I), wherein i) the first strand of the nucleic acid has a length of between 14 and 30 nucleotides; and / or ii) the second strand of the nucleic acid has a length of between 14 and 30 nucleotides.

[0055] In one embodiment, 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.

[0056] In one embodiment, the invention relates to a nucleic acid of formula (I), wherein i) the first strand of the nucleic acid has a length of between 19 and 25 nucleotides; and / or ii) the second strand of the nucleic acid has a length of between 19 and 25 nucleotides.

[0057] In one embodiment, 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.

[0058] In one embodiment, 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.

[0059] In one embodiment, the invention relates to a nucleic acid of formula (I), wherein i) the first 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 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

[0060] (iii) the double stranded region between the first and the second strands is blunt ended at both ends of the double stranded region. In one embodiment, 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.

[0061] In one embodiment, 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

[0062] (iii) 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.

[0063] In one embodiment, 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.

[0064] In one embodiment, 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

[0065] (iii) 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.

[0066] In one embodiment, the invention relates to a nucleic acid of formula (I), wherein the first strand and the second strand 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.

[0067] In one embodiment, 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.

[0068] In a preferred embodiment, 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.

[0069] In one embodiment, 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.

[0070] In a preferred embodiment, 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.

[0071] In one embodiment, 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.

[0072] In a preferred embodiment, 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.

[0073] In one embodiment, 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.

[0074] In a preferred embodiment, 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.

[0075] A double-stranded nucleic acid 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. As shown in formula (I), the duplex region between the first and the second strand begins at Yf and Y and ends at Yuand Yu', respectively on the first and second strands. This duplex region comprises nucleotide base pairings 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. A certain number of mismatches, deletions or insertions between the nucleotide sequences of the two strands are acceptable. Overhangs on either end of the first or second strand or unpaired nucleotides at either end of the double-stranded nucleic acid are also possible. The overhangs of formula (I) are represented as Xa, Xa', Xb and Xb', respectively, on the first and second strands at the 5’ and 3’ ends. 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 ormore, 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.

[0076] The first strand and the second strand 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.

[0077] 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 according to the present invention and even have the potential in certain cases to increase RNA interference (e.g., inhibition) activity.

[0078] The inhibition (or “knockdown”) activity of the nucleic acids according to 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.

[0079] 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).

[0080] The complementarity between the first strand and the complementary sequence of the target sequence may range from about 75% to about 100%. More specifically, the complementarity may be at least 75%, 80%, 85%, 90% or 95% and intermediate values, provided a nucleic acid is capable of reducing or inhibiting the expression of a desired target gene.

[0081] A nucleic acid having less than 100% complementarity between the first strand and the target sequence may be able to reduce the expression of a desired target gene to the same level as a nucleic acid having perfect complementarity between the first strand and target sequence. Alternatively, it may be able to reduce expression of a desired target gene to a level that is 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of the level of reduction achieved by the nucleic acid with perfect complementarity.

[0082] One embodiment 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. Herein the duplex region is formed between and including Yf to Yuand Y to Yu' of the first strand and the second strand, respectively, of formula (I). Preferably, 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.

[0083] In certain embodiments, 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 Yuand Yu'. 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.

[0084] In each of the embodiments disclosed herein, the nucleic acid 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.

[0085] The nucleic acid may have an overhang at one end and a blunt end at the other end. The nucleic acid may have an overhang at both ends. The nucleic acid may be blunt ended at both ends. The nucleic acid 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.

[0086] The nucleic acid may comprise an overhang at a 3' or 5' end. The nucleic acid may have a 3' overhang on the first strand. The nucleic acid may have a 3' overhang on the second strand. The nucleic acid may have a 5' overhang on the first strand. The nucleic acid may have a 5' overhang on the second strand. The nucleic acid may have an overhang at both the 5' end and 3' end of the first strand. The nucleic acid may have an overhang at both the 5' end and 3' end of the second strand. The nucleic acid may have a 5' overhang on the first strand and a 3' overhang on the second strand. The nucleic acid may have a 3' overhang on the first strand and a 5' overhang on the second strand. The nucleic acid may have a 3' overhang on the first strand and a 3' overhang on the second strand. The nucleic acid may have a 5' overhang on the first strand and a 5' overhang on the second strand.

[0087] An overhang at the 3’ end or 5’ end of the second strand or the first strand may consist of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 nucleotides in length. Optionally, an overhang may consist of 1 , 2 or 3 nucleotides, which may or may not be modified.

[0088] In one embodiment, the 5’ end of the first strand is a single-stranded overhang of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 nucleotides, preferably of 1 , 2 or 3 nucleotides.

[0089] A double stranded nucleic acid of the present invention according to formula (I), particularly a conjugated nucleic acid, may be a nucleic acid, wherein the first strand and / or the second strand comprise further phosphorothioate linkages.

[0090] Optionally all remaining linkages between nucleotides of the first and / or of the second strand are phosphodiester linkages

[0091] Nucleic acid modifications

[0092] Nucleic acids of formula (I) of the invention 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. The term “modified nucleotide” as used herein 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. The term “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.

[0093] Modifications of the nucleic acid of formula (I) 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 of formula (I) 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. Preferably, the modified nucleic acids of formula (I) 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.

[0094] Throughout the description of the invention, “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’-OMe) or a fluoro group (2’-F). For example, 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'-OMe- rC; 2'-OMe-rG. In contrast, a 2’-F modification is a different modification compared to a 2’-OMe modification.

[0095] Preferably, at least one nucleotide of the first and / or second strand of the nucleic acid of formula (I) is a modified nucleotide, preferably a non-naturally occurring nucleotide such as preferably a 2’-F modified nucleotide.

[0096] 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.

[0097] Examples of “oxy”-2' hydroxyl group modifications include alkoxy or aryloxy (OR, e.g., R=H, alkyl (such as methyl), cycloalkyl, aryl, aralkyl, heteroaryl or sugar); polyethyleneglycols (PEG), O(CH2CH2O)nCH2CH2OR; O-AMINE (AMINE=NH2, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, or polyamino) and aminoalkoxy, O(CH2)nAMINE, (e.g., AMINE=NH2, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, or polyamino).

[0098] “Deoxy” modifications include hydrogen, halogen, amino (e.g., NH2, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); NH(CH2CH2NH)nCH2CH2-AMINE (AMINE=NH2, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino), — NHC(O)R (R=alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted with e.g., an amino functionality. Other substituents of certain embodiments include 2'-methoxyethyl, 2'- OCH3, 2'-O-allyl, 2'-C-allyl, and 2'-fluoro.

[0099] The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, 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.

[0100] The 2' modifications may be used in combination with one or more phosphate internucleotide linker modifications (e.g., phosphorothioate or phosphorodithioate).

[0101] One or more nucleotides of a nucleic acid of formula (I) of the present invention may be modified. The nucleic acid of formula (I) 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 (Yu) may be a modified nucleotide. The 3’- terminal nucleotide of the duplex region in the second strand (Yf ) may be a modified nucleotide. The 5’-terminal nucleotide of the duplex region in the first strand (Yf) may be a modified nucleotide. The 5’-terminal nucleotide of the duplex region in the second strand (Yu') may be a modified nucleotide.

[0102] 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. In each case the 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.

[0103] 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’-0Me) 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 bisdecylamide group.

[0104] 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-

[0105] 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.

[0106] 6-methyluridine), propyne, quesosine, 2-thiouridine, 4-thiouridine, wybutosine, wybutoxosine,

[0107] 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, 5'-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, beta-D- galactosylqueosine, 1 -methyladenosine, 1-methylinosine, 2,2-dimethylguanosine,

[0108] 3-methylcytidine, 2-methyladenosine, 2-methylguanosine, N6-methyladenosine,

[0109] 7-methylguanosine, 5-methoxyaminomethyl-2- thiouridine, 5-methylaminomethyluridine,

[0110] 5-methylcarbonylmethyluridine, 5-methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio-N6- isopentenyladenosine, beta-D-mannosylqueosine, uridine-5-oxyacetic acid and 2-thiocytidine.

[0111] Many of the modifications described herein and that occur within a nucleic acid of formula (I) 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 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.

[0112] 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.

[0113] 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.

[0114] Modified nucleic acids of formula (I), as used herein, 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);

[0115] (i) alteration, e.g., replacement, of a constituent of the ribose sugar, e.g., of the 2' hydroxyl on the ribose sugar;

[0116] (ii) replacement of the phosphate moiety with “dephospho” linkers;

[0117] (iii) modification or replacement of a naturally occurring base;

[0118] (iv) replacement or modification of the ribose-phosphate backbone; and

[0119] (v) 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.

[0120] The terms “replacement”, “modification” and “alteration” indicate a difference from a naturally occurring molecule.

[0121] Specific modifications are discussed in more detail below.

[0122] The use of a chemical modification pattern of the nucleic acids confers nuclease stability in serum and makes for example a subcutaneous application route feasible. 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.

[0123] Some representative modified nucleic acid sequences of formula (I) of the present invention are shown in the examples. These examples are meant to be representative only and not limiting to the scope of the invention.

[0124] In one embodiment, 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 Yuat the 3’ end of the duplex region of the first strand. The first common modification is preferably 2’-F.

[0125] In one embodiment, 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.

[0126] In one embodiment, 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. Preferably, 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’-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 modification is preferably 2’-OMe.

[0127] In the context of this disclosure, the size or volume of a substituent, such as a 2’ ribose modification, is preferably measured as the van der Waals volume.

[0128] In one embodiment, 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. 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. In addition, or alternatively, the odd-numbered nucleotides of the first strand are modified with a second modification. Preferably, 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.

[0129] 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 of a duplex.

[0130] In one embodiment, 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. In addition, or alternatively, the odd-numbered nucleotides of the first strand are modified with a second modification. In addition, or alternatively, 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’-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’-OMe 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.

[0131] In one embodiment of the 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. Preferably, 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. The positions are counted starting from Y at the 3’ end of the second strand towards Yu’ at the 5’ end, wherein Y represents nucleotide number 1 , Y2’ represents nucleotide number 2 and so on. 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. In addition, or alternatively, 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’-OMe 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.

[0132] In one embodiment of the nucleic acid of formula (I), 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.

[0133] In one embodiment of the nucleic acid of formula (I), 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’-OMe. In one embodiment in this embodiment, the 3’ terminal nucleotide of the second strand, preferably at position Y , is an inverted nucleotide, preferably an inverted RNA nucleotide. In another embodiment in this embodiment, the 5’ terminal nucleotide of the second strand, preferably at position Yu', is an inverted nucleotide, preferably an inverted RNA nucleotide.

[0134] 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. In one embodiment, “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.

[0135] 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’-OMe 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’-OMe modification (in other words, they are not modified or are modified with a modification other than 2’-OMe). In one embodiment, the nucleotide on the second strand which corresponds to position 13 of the duplex region of the first strand (wherein Yf is position 1 , Y2 is position 2 and so on), is the nucleotide that forms a base pair with position 13 of the first strand.

[0136] In one embodiment, the nucleotide on the second strand which corresponds to position 11 of the duplex region of the first strand (wherein Yf is position 1 , Y2 is position 2 and so on), is the nucleotide that forms a base pair with position 11 of the first strand.

[0137] In one embodiment, the nucleotide on the second strand which corresponds to position 12 of the duplex region of the first strand (wherein Yf is position 1 , Y2 is position 2 and so on), is the nucleotide that forms a base pair with position 12 of the first strand.

[0138] For example, in a 19-mer nucleic acid which is double-stranded and blunt ended, 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.

[0139] In one embodiment, in the case of a partially complementary first and 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.

[0140] 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’-OMe 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.

[0141] 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’-OMe modification.

[0142] 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 modified with a 2'-F modification.

[0143] 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.

[0144] 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’-OMe, other 2’ sugar modifications, in particular a 2’-H modification resulting in a DNA nucleotide.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] 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. Preferably the nucleotides that are not modified with 2’- OMe are modified with fluoro at the 2’ position (2’-F modification).

[0149] In certain embodiments, 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. Preferably these nucleotides are modified with a 2’-F modification where the modification is not a 2’-OMe modification.

[0150] In one embodiment 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. Preferably 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. Preferably 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. Y ), and the remaining modifications are naturally occurring modifications, preferably 2’-OMe. 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.

[0151] In one embodiment of the nucleic acid of formula (I), each of the nucleotides of the first strand and of the second strand is a modified nucleotide.

[0152] The term “odd numbered” as described herein means a number not divisible by two. Examples of odd numbers are 1 , 3, 5, 7, 9, 11 and so on. The term “even numbered” as described herein 1 means a number which is evenly divisible by two. Examples of even numbers are 2, 4, 6, 8, 10, 12, 14 and so on.

[0153] Unless specifically stated otherwise, herein the 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.

[0154] 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.

[0155] 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).

[0156] One or more of the odd-numbered nucleotides of the second strand (wherein the nucleotides are numbered contiguously starting with nucleotide number 1 (Yf ) at the 3’ end 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.

[0157] 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.

[0158] In some embodiments of the nucleic acid of formula (I) of the invention, 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.

[0159] 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.

[0160] In certain embodiments, 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 of 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.

[0161] 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.

[0162] 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.

[0163] The nucleic acid of formula (I) 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 . 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, 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 Xa, Xband Xa', Xb' if these are present.

[0164] 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.

[0165] 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.

[0166] 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.

[0167] Clearly, if the 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 Xa, Xband Xa', Xb' respectively.

[0168] 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. In other words, 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. Another option is to have dissimilar modifications in each of the strands. 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.

[0169] The modification and / or modifications may each and individually be selected from the group consisting of 3' terminal deoxy thymine, 2'-OMe, 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 a locked nucleotide, an abasic nucleotide or a non-natural base comprising nucleotide.

[0170] At least one modification may be 2'-OMe 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.

[0171] 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. Preferably, the nucleic acid 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 comprises an inverted nucleotide at the 3’ end of the second strand. Most preferably, the nucleic acid comprises an inverted RNA nucleotide at the 3’ end of the second strand and this nucleotide is preferably an inverted A (= inverted adenosine). 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 of formula (I) 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. A nucleic acid with such a nucleotide is stable and easy to synthesize. 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. Specifically, the inverted RNA nucleotide is preferably a 2’-OH nucleotide.

[0172] 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 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 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.

[0173] Some representative modified nucleic acid sequences of formula (I) of the present invention are shown in the examples. These examples are meant to be representative and not limiting.

[0174] In certain preferred embodiments, 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'-OMe modification and 2'-F modification. The nucleic acid may comprise a modification that is 2'-OMe that may be a first modification, and a second modification that is 2'-F. The nucleic acid 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.

[0175] In one embodiment of the nucleic acid of formula (I), 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:

[0176] (i) at least one or both of the nucleotides 2 and 14 of the first strand is / are modified by a first modification; and / or

[0177] (ii) at least one, several, or all the even-numbered nucleotides of the first strand is / are modified by a first modification; and / or

[0178] (iii) at least one, several, or all the odd-numbered nucleotides of the first strand is / are modified by a second modification; and / or wherein if the second strand comprises at least one modified nucleotide:

[0179] (iv) at least one, several, or all the nucleotides of the second strand in a position corresponding to an even-numbered nucleotide of the first strand is / are modified by a third modification; and / or

[0180] (v) at least one, several, or all the nucleotides of the second strand in a position corresponding to an odd-numbered nucleotide of the first strand is / are modified by a fourth modification; and / or

[0181] (vi) 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; and / or

[0182] (vii) at least one, several, or all the nucleotides of the second strand in a position other than the 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; wherein the 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:

[0183] (a) the first modification is preferably different from the second and from the third modification;

[0184] (b) the first modification is preferably the same as the fourth modification;

[0185] (c) the second and the third modification are preferably the same modification;

[0186] (d) the first modification is preferably a 2’-F modification;

[0187] (e) the second modification is preferably a 2’-OMe modification;

[0188] (f) the third modification is preferably a 2’-OMe modification; and / or

[0189] (g) the fourth modification is preferably a 2’-F modification; and wherein optionally the nucleic acid is conjugated to a heterologous moiety.

[0190] One aspect of the invention is the conjugated nucleic acid of formula (I) wherein the further modifications on the first and / or second strands are non-naturally occurring nucleotides such as a 2’-F modified nucleotide.

[0191] 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. For example, 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). 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. Alternatively, the linker can connect to or replace the terminal atom of a nucleotide surrogate (e.g., PNAs). These spacers or linkers can include e.g., — (CH2)n — , — (CH2)nN — , — (CH2)nO — , — (CH2)nS — , — (CH2CH2O)nCH2CH2O — (e.g., n=3 or 6), abasic sugars, amide, carboxy, amine, oxyamine, oxyimine, thioether, disulfide, thiourea, sulfonamide, or morpholino, or biotin and fluorescein reagents. The 3' end can be an — OH group.

[0192] Other examples of 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, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles).

[0193] 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.

[0194] 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: 5'- monophosphate ((HO)2(O)P— 0-5'); 5'-diphosphate ((HO)2(O)P— O— P(HO)(O)— 0-5'); 5'- triphosphate ((HO)2(O)P— O— (HO)(O)P— O— P(HO)(O)— O-51); 5'-guanosine cap (7- methylated or non-methylated) (7m-G-O-5'-(HO)(O)P— O— (HO)(O)P— O— P(HO)(O)— 0-5'); 5'-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N — 0-5'- (HO)(O)P — O — (HO)(O)P — O — P(HO)(O) — 0-5'); 5'-monothiophosphate (phosphorothioate; (HO)2(S)P — 0-5'); 5'-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P — 0-5'), 5'- phosphorothiolate ((HO)2(O)P — S-5'); any additional combination of oxygen / sulfur replaced monophosphate, diphosphate and triphosphates (e.g., 5'-alpha-thiotriphosphate, 5'-gamma- thiotriphosphate, etc.), 5'-phosphoramidates ((HO)2(O)P — NH-5', (HO)(NH2)(O)P — 0-5'), 5'- alkylphosphates (alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g., RP(OH)(O) — 0-5'- (wherein R is an alkyl), (OH)2(O)P-5'-CH2-), 5' (E)-vinylphosphonate, 5’(Z)-vinylphosphonate, 5’cyclopropyl phosphonate, 5'-alkyletherphosphonates (alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g., RP(OH)(O) — 0-5'-) (wherein R is an alkylether)), 5’ phosphate consisting of carbocyclic ribonucleotides with 5’ alkylphosphates (alkyl=methyl, ethyl, isopropyl, etc., e.g. RP((0H)(0)-0-5’- (wherein R is an alkyl), (OH)2(O)P-5'-CH2-), and carbocyclic ribonucleotides with phosphonates, 5’cyclopropyl phosphonate, 5’(Z)- vinylphosphonate, 5’(E)-vinylphosphonate, 5’alkyletherphosphonates

[0195] (alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc.; e.g., RP(0H)(0)-0-5’-) (wherein R is an alkylether)).

[0196] Certain 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-aminopropyl phosphate; 6-aminohexyl phosphate; 12-aminododecyl phosphate; hydroxypropyl phosphate; 1 ,5-anhydrohexitol nucleotide; alpha-nucleotide; threo-pentofuranosyl nucleotide; acyclic 3', 4'- seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5'-5'-inverted abasic moiety; 1 ,4-butanediol phosphate; 5'-amino; and bridging or non-bridging methylphosphonate and 5'-mercapto moieties.

[0197] In each sequence described herein, a C-terminal “-OH” moiety may be substituted for a C- terminal “-NH2” moiety, and vice-versa.

[0198] The invention also provides a nucleic acid 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. Preferably, the terminal 5’ (E)- vinylphosphonate (“vp”) nucleotide is a uridine (“vp-ll”).

[0199] The first strand of the nucleic acid may comprise formula (I):

[0200] (vp)-N(po)[N(po)]n- (I) where ‘(vp)-’ is the 5’ (E)-vinylphosphonate, ‘N’ is a nucleotide, ‘po’ is a phosphodiester linkage, and 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).

[0201] Preferably, the terminal 5’ (E)-vinylphosphonate nucleotide is an RNA nucleotide, preferably a (vp)-U.

[0202] 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:

[0203] Nucleotide with a phosphate at the 5’-end of the ribose

[0204] Nucleotide with a terminal 5’ (E)-vinylphosphonate at the 5’-end of the ribose.

[0205] In one embodiment, the first strand 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.

[0206] In one embodiment, 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.

[0207] In one embodiment, the first strand and / or the second strand of the nucleic acid comprises more than one phosphorothioate and / or more than one phosphorodithioate linkage.

[0208] In one embodiment, 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 can further comprise phosphorothioate or phosphorodithioate linkages between the terminal two or three 3’ nucleotides of the duplex region.

[0209] In one embodiment, 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.

[0210] In one embodiment, 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. Optionally, each or either end of the first strand may comprise one or two or three phosphorothioate or phosphorodithioate modified nucleotides (internucleotide linkage). Optionally, each or either end of the second strand may comprise one or two or three phosphorothioate or phosphorodithioate modified nucleotides (internucleotide linkage).

[0211] In one embodiment, 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. Preferably, the nucleic acid 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.

[0212] In one embodiment, 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 nucleotides at the 5’ end of the duplex region of the first strand.

[0213] In one embodiment, the nucleic acid of formula (I) comprises a phosphorothioate linkage between the terminal three 5’ nucleotides of the duplex region of the first strand.

[0214] In one embodiment, the nucleic acid of formula (I): has two phosphorothioate linkages between the terminal three 5’ nucleotides of the first strand, or between the terminal three 5’ nucleotides of the duplex region of the first strand and may comprise one or more further phosphorothioate linkages;

[0215] (i) is conjugated to a triantennary ligand either on the 3’ end nucleotide or on the 5’ end nucleotide of the second strand;

[0216] (ii) has two phosphorothioate linkages between the terminal three or has one phosphorothioate linkage between the terminal two nucleotides of the second strand at the non-conjugated end and may comprise further phosphorothioate linkages, particularly one, two or three phosphorothioate linkages; and

[0217] (iii) optionally all remaining linkages between nucleotides of the first and / or of the second strand are phosphodiester linkages.

[0218] In one embodiment, the nucleic acid of formula (I): has two phosphorothioate linkages between the terminal three 3’ nucleotides of the first strand, or between the terminal three 3’ nucleotides of the duplex region of the first strand and may comprise one or more further phosphorothioate linkages;

[0219] (i) is conjugated to a triantennary ligand either on the 3’ end nucleotide or on the 5’ end nucleotide of the second strand;

[0220] (ii) has two phosphorothioate linkages between the terminal three or has one phosphorothioate linkage between the terminal two nucleotides of the second strand at the non-conjugated end and may comprise further phosphorothioate linkages, particularly one, two or three phosphorothioate linkages; and

[0221] (iii) optionally all remaining linkages between nucleotides of the first and / or of the second strand are phosphodiester linkages.

[0222] In one embodiment, the nucleic acid of formula (I):

[0223] (i) has a terminal 5’ (E)-vinylphosphonate nucleotide at the 5’ end of the first strand, preferably the 5’ end of the duplex region of the first strand; (ii) has two phosphorothioate linkages between the terminal three 3’ nucleotides on the second strand, preferably the 3’ end of the duplex region of the second strand,

[0224] (iii) has one or more phosphorothioate linkage(s) on the first and / or second strand, and

[0225] (iv) optionally all remaining linkages between nucleotides of the first and / or of the second strand are phosphodiester linkages.

[0226] The use of 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.

[0227] In one embodiment, the nucleic acid comprises one phosphorodithioate linkage between the two terminal nucleotides at the 3’ end of the duplex region of the second strand and one 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.

[0228] Preferably, 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.

[0229] Preferably, 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.

[0230] In one embodiment, 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.

[0231] In one embodiment, all the linkages of the nucleic acid 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.

[0232] Other phosphate linkage modifications are possible.

[0233] 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.

[0234] The phosphate groups can also individually be replaced by non-phosphorus containing connectors.

[0235] Examples of 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. In certain embodiments, replacements may include the methylenecarbonylamino and methylenemethylimino groups.

[0236] The phosphate linker and ribose sugar may be replaced by nuclease resistant nucleotides. Examples include the morpholino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates. In certain embodiments, PNA surrogates may be used.

[0237] In one embodiment, the nucleic acid of the invention 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:

[0238] (i) a modified nucleotide;

[0239] (ii) 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 with a given SEQ ID No., preferably a 2’-F modified nucleotide; (iii) 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 with a given SEQ ID No. are 2’-0Me modified nucleotides;

[0240] (iv) 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 with a given SEQ ID No. are 2’-F modified nucleotides;

[0241] (v) the second strand nucleotide corresponding to position 11 and / or 13 or 11-13 of the first strand with a given SEQ ID No. 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;

[0242] (vi) an inverted nucleotide, preferably a 3’-3’ linkage at the 3’ end of the duplex region of the second strand with a given SEQ ID No.;

[0243] (vii) one or more phosphorothioate linkages;

[0244] (viii) one or more phosphorodithioate linkages; and / or

[0245] (ix) the duplex region of the first strand with a given SEQ ID No. 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.

[0246] All modifications and modification patterns described herein of the first and second strand of the nucleic acid of the invention can be combined with all other embodiments of the invention disclosed herein.

[0247] Heterologous moieties

[0248] The nucleic acids 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.

[0249] Any given nucleic acid of formula (I) may be conjugated to a plurality of heterologous moieties, which may be the same or different.

[0250] 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.

[0251] For example, 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.

[0252] 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.

[0253] Alternatively, 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.

[0254] In this context, 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.

[0255] Thus, 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.

[0256] 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. Thus, 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. In other embodiments, 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. Asialoglycoprotein receptor complex (ASG P)

[0257] One example of 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.

[0258] The ASGP-R complex recognizes specifically terminal p-galactosyl subunits of glycosylated proteins or other oligosaccharides191and 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 moiety1101.

[0259] The ASGP-R complex is a mediator for an active uptake of terminal p-galactosyl containing glycoproteins to the cell’s endosomes. Thus, the ASGPR is highly suitable for targeted delivery of drug candidates conjugated to such ligands like, e.g., nucleic acids into receptor-expressing cells

[0011] .

[0260] More generally, the ligand can comprise a saccharide that is selected to have an affinity for at least one type of receptor on a target cell. In particular, the receptor is on the surface of a mammalian liver cell, for example, the hepatic asialoglycoprotein receptor complex described before (ASGP-R).

[0261] 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.

[0262] 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 aspects. 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.

[0263] In one aspect, the invention relates to the conjugated nucleic acid, wherein the nucleic acid is conjugated to a heterologous moiety.

[0264] In one embodiment, 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.

[0265] In one embodiment the conjugated nucleic acid is conjugated to a heterologous moiety comprising a compound of formula (II):

[0266] [S-X1-P-X2]3-A-X3- (II) wherein:

[0267] S represents a functional component, e.g. a ligand, such as a saccharide, preferably wherein the saccharide is N-acetyl galactosamine;

[0268] X1represents C3-C6 alkylene or (-CH2-CH2-O)m(-CH2)2- wherein m is 1 , 2, or 3;

[0269] P is a phosphate or modified phosphate, preferably a thiophosphate;

[0270] X2is alkylene or an alkylene ether of the formula (-CH2)n-O-CH2- where n = 1- 6;

[0271] A is a branching unit;

[0272] X3represents a bridging unit; wherein a nucleic acid as defined herein is conjugated to X3via a phosphate or modified phosphate, preferably a thiophosphate.

[0273] In formula (II), 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.

[0274] The branching unit A may have a structure selected from: wherein each Ai independently represents O, S, C=O or NH; and each n independently represents an integer from 1 to 20.

[0275] The branching unit may have a structure selected from: wherein each Ai independently represents O, S, C=O or NH; and each n independently represents an integer from 1 to 20.

[0276] The branching unit may have a structure selected from: wherein Ai is O, S, C=O or NH; and each n independently represents an integer from 1 to 20.

[0277] The branching unit may have the structure:

[0278] The branching unit may have the structure: The branching unit may have the structure:

[0279] Alternatively, the branching unit A may have a structure selected from: wherein:

[0280] R1is hydrogen or C1-C10 alkylene; and R2is C1-C10 alkylene.

[0281] Optionally, the branching unit consists of only a carbon atom.

[0282] The “X3” portion is a bridging unit. The bridging unit is linear and is covalently bound to the branching unit and the nucleic acid.

[0283] X3may 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(O)-Ci-C4 alkylene-, -Ci- 04 alkylene-C(O)NH-Ci-C4 alkylene-, -C1-C4 alkylene-SC(O)-Ci-C4 alkylene-, -C1-C4 alkylene- C(0)S-Ci-C4 alkylene-, -C1-C4 alkylene-OC(O)-Ci-C4 alkylene-, -C1-C4 alkylene-C(O)O-Ci-C4 alkylene-, and -Ci-Ce alkylene-S-S-Ci-Ce alkylene-.

[0284] X3may be an alkylene ether of formula -(C1-C20 alkylene)-0-(Ci-C2o alkylene)-. X3may be an alkylene ether of formula -(C1-C20 alkylene)-0-(C4-C2o alkylene)-, wherein said (C4-C20 alkylene) is linked to Z. X3may 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. In one embodiment, the nucleic acid is conjugated to a heterologous moiety of formula (III):

[0285] [S-X1-P-X2]3-A-X3- (III) wherein:

[0286] S represents a functional component, e.g. a ligand, such as a saccharide, preferably GalNAc;

[0287] X1represents C3-Ce 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;

[0288] X2is Ci-Cs alkylene;

[0289] A is a branching unit selected from:

[0290] X3is a bridging unit; wherein a nucleic acid according to the present invention is conjugated to X3via a phosphate or a modified phosphate, preferably a thiophosphate.

[0291] The branching unit A may have the structure:

[0292] The branching unit A may have the structure: wherein X3is attached to the nitrogen atom.

[0293] X3may be C1-C20 alkylene. Preferably, X3is selected from the group consisting of -C3H6-, -C4H8-, -CeH 12- and -CsHie-, especially -C4H8-, -C6HI2- and -CsHie-.

[0294] In one embodiment, the nucleic acid is conjugated to a ligand comprising a compound of formula (IV):

[0295] [S-X1-P-X2]3-A-X3- (IV) wherein:

[0296] S represents a functional component, e.g., a ligand, such as a saccharide, preferably GalNAc;

[0297] X1represents 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;

[0298] X2is an alkylene ether of formula -C3H6-O-CH2-; A is a branching unit;

[0299] X3is an alkylene ether of formula selected from the group consisting of -CH2-O-CH2-,

[0300] - CH2-O-C2H4-, -CH2-O-C3H6-, -CH2-O-C4H8-, -CH2-O-C5H10-, -CH2-O-C6Hi2-, -CH2-O-, C7H14-, and -CH2-O-C8H16-, wherein in each case the -CH2- group is linked to A, and wherein X3is conjugated to a nucleic acid according to the present invention by a phosphate or modified phosphate, preferably a thiophosphate.

[0301] The branching unit may comprise carbon. Preferably, the branching unit is a carbon.

[0302] X3may be selected from the group consisting of -CH2-O-C4H8-, -CH2-O-C5H10-, -CH2-O-C6H12-, -CH2-O-C7H14-, and -CH2-O-C8H16-. Preferably, X3is selected from the group consisting of -CH2-O-C4H8-, -CH2-O-C6H12- and -CH2-O-C8H16.

[0303] X1may be (-CH2-CH2-O)(-CH2)2-. X1may be (-CH2-CH2-O)2(-CH2)2-. X1may be (-CH2-CH2- O)S(-CH2)2-. Preferably, X1is (-CH2-CH2-O)2(-CH2)2-. Alternatively, X1represents C3-C6 alkylene. X1may be propylene. X1may be butylene. X1may be pentylene. X1may be hexylene. Preferably the alkyl is a linear alkylene. In particular, X1may be butylene.

[0304] X2represents an alkylene ether of formula -C3H6-O-CH2- i.e. C3 alkoxy methylene, or - CH2CH2CH2OCH2-.

[0305] For any of the above embodiments, when P represents a modified phosphate group, P can be represented by: wherein Y1and Y2each independently represent =0, =S, -O -OH, -SH, -BH3, -OCH2CO2, - OCH2CO2RX, -0CH2C(S)0RX, and -ORX, wherein Rxrepresents Ci-Ce alkyl and wherein H indicates attachment to the remainder of the compound.

[0306] By modified phosphate it is meant a phosphate group wherein one or more of the non-linking oxygens is replaced. Examples of 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).

[0307] 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.

[0308] For example, Y1may represent -OH and Y2may represent =0 or =S; or Y1may represent -O' and Y2may represent =0 or =S;

[0309] Y1may represent =0 and Y2may represent -CH3, -SH, -ORX, or -BH3 Y1may represent =S and Y2may represent -CH3, ORXor -SH.

[0310] It will be understood by the skilled person that in certain instances there will be delocalisation between Y1and Y2.

[0311] Preferably, the modified phosphate group is a thiophosphate group. Thiophosphate groups include bithiophosphate (i.e., where Y1represents =S and Y2represents -S') and monothiophosphate (i.e., where Y1represents -O' and Y2represents =S, or where Y1represents =0 and Y2represents -S'). Preferably, P is a monothiophosphate. The inventors have found that conjugates having thiophosphate groups in replacement of phosphate groups have improved potency and duration of action in vivo. P may also be an ethylphosphate (i.e. where Y1represents =0 and Y2represents OCH2CH3).

[0312] The ligand, e.g., saccharide, may be selected to have an affinity for at least one type of receptor on a target cell. In particular, the receptor is on the surface of a mammalian liver cell, for example, the hepatic asialoglycoprotein receptor complex (ASGP-R).

[0313] For any of the above or below embodiments, the saccharide may be selected from N-acetyl with one or more of galactosamine, mannose, galactose, glucose, glucosamine and fructose. Typically, a ligand to be used in the present invention may include N-acetyl galactosamine (GalNAc). Preferably 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. In certain embodiments, 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. Preferably, the compounds of the invention comprise the p-form, 2-(Acetylamino)-2-deoxy-p-D-galactopyranose.

[0314] 2-(Acetylamino)-2-deoxy-p-D-galactopyranose

[0315] 2-(Acetylamino)-2-deoxy-a-D-galactopyranose

[0316] In one embodiment, the nucleic acid 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: wherein Z is any nucleic acid as defined herein.

[0317] In certain embodiments, 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.

[0318] In certain embodiments, the heterologous moiety (“triantennary ligand”) is conjugated to the 3’ position of the ribose of the terminal nucleotide of the second (sense) strand of Z.

[0319] In other embodiments, the heterologous moiety (“triantennary ligand”) is conjugated to the 5’ position of the ribose of the terminal nucleotide of the second (sense) strand of Z.

[0320] In other embodiments, the heterologous moiety (“triantennary ligand”) is conjugated to the 3’ position of the ribose of the terminal nucleotide of the first (antisense) strand of Z.

[0321] Preferably, the nucleic acid is a conjugated nucleic acid, wherein the nucleic acid is conjugated to a triantennary ligand with one of the following structures: wherein Z is any nucleic acid as defined herein.

[0322] In a preferred embodiment, 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.

[0323] Preferably, the triantennary ligand” is conjugated to the 5’ position of the ribose of the terminal nucleotide of the second (sense) strand of Z.

[0324] CD206 mannose

[0325] Another example of a ligand or a targeting agent for targeting the nucleic acid of formula (I) as described herein to CD206 receptor expressing cells is the chemically modified mannose ligand linker (CMM) moiety, which has high affinity to the CD206 receptor and which has the following structure: llehara et al. (Nucleic Acids Research, Volume 50, Issue 9, 20 May 2022, Pages 4840-4859) describe methods to conjugate the CMM moiety to siRNAs.

[0326] Ye et al. (Molecular Therapy Vol. 31 No. 1 , doi.org / 10.1016 / j.ymthe.2022.09.009) describe a hexavalent mannose ligand of structure S24 that targets CD206 receptor:

[0327] Glucagon Like Peptide 1 Receptor (GLP1 R)

[0328] Another example of a ligand or targeting agent for targeting the nucleic acid of formula (I) as described herein to the Glucagon Like Peptide 1 Receptor (GLP1 R) is the GLPR1 peptide agonist described by Ammala et al. (ScienceAdvances, Vol.4 No. 10, 17 Oct 2018), particularly the eGLP1 ligand, described in Fig. S1 of Ammala et al.:

[0329] NH2 - H(Aib)EGT5 FTSDV10 SSYLE15 EQAAK20 EFIAW25 LVKGG30 PSSGA35 PPPSC40- NH2, wherein “Aib” represents

[0330] 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

[0331] Examples of 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).

[0332] Folic Acid

[0333] Examples of 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.

[0334] A heterologous moiety of formula (II), (III) or (IV) or any one of the triantennary ligands or targeting agents 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 of the nucleic acid of formula

[0335] (I). The nucleic acid of formula (I) can comprise more than one heterologous moiety of formula

[0336] (II), (III) or (IV) or any one of the triantennary ligands or targeting agents disclosed herein. However, 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. Preferably in that case, 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.

[0337] Preferably, 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.

[0338] A nucleic acid with a single heterologous moiety (e.g., of formula (II), (III) or (IV) or any one of the triantennary ligands or targeting agents 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. Preferably therefore, a single heterologous moiety (e.g., of any of formulae (II), (III) or (IV) or any one of the triantennary ligands or targeting agents disclosed herein) is covalently attached to (conjugated with) the 5’ end of the second strand of the nucleic acid. In one embodiment the first strand of the nucleic acid comprises a compound of formula (V): wherein b is 0 or 1 ; and wherein the second strand is a compound of formula (VI):

[0339] (VI); wherein: c and d are independently 0 or 1;

[0340] Zi and Z2 are respectively the first and second strand of the nucleic acid;

[0341] Y is independently O or S; n is independently 0, 1 , 2 or 3; and

[0342] 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 2 or 3.

[0343] Preferably, Li in formulae (V) and (VI) is of formula (VII): wherein:

[0344] L is selected from the group comprising, or preferably consisting of:

[0345] -(CH2)r-C(O)-, wherein r = 2-12;

[0346] -(CH2-CH2-O)S-CH2-C(O)-, wherein s = 1-5;

[0347] -(CH2)t-CO-NH-(CH2)t-NH-C(O)-, wherein t is independently 1-5;

[0348] -(CH2)U-CO-NH-(CH2)U-C(O)-, wherein u is independently 1-5; and

[0349] -(CH2)V-NH-C(O)-, wherein v is 2-12; and wherein the terminal C(O), if present, is attached to X of formula (VII), or if X is absent, to Wi of formula (VII), or if Wi is absent, to V of formula (VII);

[0350] Wi, W3and W5 are individually absent or selected from the group comprising, or preferably consisting of:

[0351] -(CH2)r-, wherein r = 1-7;

[0352] -(CH2)S-O-(CH2)S-, wherein s is independently 0-5;

[0353] -(CH2)t-S-(CH2)t-, wherein t is independently 0-5;

[0354] X is absent or is selected from the group comprising, or preferably consisting of: NH, NCH3or NC2H5;

[0355] V is selected from the group comprising, or preferably consisting of:

[0356] wherein B, if present, is a modified or natural nucleobase.

[0357] In one embodiment, the first strand comprises 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:

[0358] Zi and Z2 are respectively the first and second strand of the nucleic acid;

[0359] Y is independently O or S;

[0360] R1 is H or methyl; n is independently preferably 0, 1 , 2 or 3; and

[0361] 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:

[0362] -(CH2)r-C(O)-, wherein r = 2-12;

[0363] -(CH2-CH2-O)S-CH2-C(O)-, wherein s = 1-5;

[0364] -(CH2)t-CO-NH-(CH2)t-NH-C(O)-, wherein t is independently 1-5;

[0365] -(CH2)U-CO-NH-(CH2)U-C(O)-, wherein u is independently 1-5; and -(CH2)V-NH-C(O)-, wherein v is 2-12; and wherein the terminal C(O), if present, is attached to the NH group (of the linker, not of the targeting ligand); and wherein b + c + d is preferably 2 or 3. In one embodiment, the first strand of the nucleic acid comprises a compound of formula (X): wherein b is preferably 0 or 1 ; and the second strand is a compound of formula (XI):

[0366] (XI); wherein: c and d are independently preferably 0 or 1 ;

[0367] Z1 and Z2 are respectively the first and second RNA strand of the nucleic acid; Y is independently O or S; n is independently preferably 0, 1 , 2 or 3; and

[0368] 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:

[0369] , H

[0370] -- .GalNAc

[0371] ' F L and the terminal OH group is absent such that the following moiety is formed: wherein:

[0372] F is a saturated branched or unbranched (such as unbranched) Ci-3alkyl (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;

[0373] L is the same or different in formulae (X) and (XI) and is selected from the group comprising, or preferably consisting of:

[0374] -(CH2)r-C(O)-, wherein r = 2-12;

[0375] -(CH2-CH2-O)S-CH2-C(O)-, wherein s = 1-5;

[0376] -(CH2)t-CO-NH-(CH2)t-NH-C(O)-, wherein t is independently 1-5;

[0377] -(CH2)U-CO-NH-(CH2)U-C(O)-, wherein u is independently 1-5; and -(CH2)V-NH-C(O)-, wherein v is 2-12; and wherein the terminal C(O), if present, is attached to the NH group (of the linker, not of the targeting ligand); and wherein b + c + d is preferably 2 or 3.

[0378] In one embodiment, 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). Preferably, 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. Most preferably, b is 0, c is 1 and d is 1.

[0379] In one embodiment, Y is O in any of the nucleic acids of formulae (V) and (VI) or (VIII) and

[0380] (IX) or

[0381] (X) and (XI). In another embodiment, Y is S. In a preferred embodiment, Y is independently selected from O or S in the different positions in the formulae.

[0382] In one embodiment, R1 is H or methyl in any of the nucleic acids of formulae (VIII) and (IX). In one embodiment, R1 is H. In another embodiment, R1 is methyl.

[0383] In one embodiment, 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). Preferably, n is 0.

[0384] Examples of F moieties in any of the nucleic acids of formulae (X) and (XI) include (CH2)I-6 e.g.

[0385] (CH2)I-4e.g. CH2, (CH2)4, (CH2)5or (CH2)6, or CH2O(CH2)2.3, e.g. CH2O(CH2)CH3.

[0386] In one embodiment, L2 in formulae (X) and (XI) is:

[0387] In one embodiment, L2 is:

[0388] In one embodiment, n is 0 and L2 is: and the terminal OH group is absent such that the following moiety is formed: wherein Y is O or S.

[0389] In one embodiment, 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:

[0390] -(CH2)r-C(O)-, wherein r = 2-12;

[0391] -(CH2-CH2-O)S-CH2-C(O)-, wherein s = 1-5;

[0392] -(CH2)t-CO-NH-(CH2)t-NH-C(O)-, wherein t is independently 1-5;

[0393] -(CH2)U-CO-NH-(CH2)U-C(O)-, wherein u is independently 1-5; and -(CH2)V-NH-C(O)-, wherein v is 2-12; wherein the terminal C(0) is attached to the NH group.

[0394] Preferably, L is -(CH2)r-C(0)-, wherein r = 2-12, more preferably r = 2-6 even more preferably, r = 4 or 6 e.g. 4.

[0395] Preferably, L is:

[0396] Within the moiety bracketed by b, c and d, L2in the nucleic acids of formulae (X) and (XI) is typically the same. Between moieties bracketed by b, c and d, L2may be the same or different. In an embodiment, L2in the moiety bracketed by c is the same as the L2in the moiety bracketed by d. In an embodiment, L2in the moiety bracketed by c is not the same as L2in the moiety bracketed by d. In an embodiment, the L2in the moieties bracketed by b, c and d is the same, for example when the linker moiety is a serinol-derived linker moiety.

[0397] 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. In an embodiment of the invention, the serinol-GalNAc moiety (SerGN) has the following stereochemistry:

[0398] (S)-Serinol building blocks i.e., is based on an (S)-serinol-amidite or (S)-serinol succinate solid supported building block derived from L-serine isomer.

[0399] In an embodiment, the first strand of the nucleic acid comprises 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,

[0400] Zi and Z2 are respectively the first and second strand of the nucleic acid,

[0401] Y is S,

[0402] R1 is H, and

[0403] L is -(CH2)4-C(O)-, wherein the terminal C(O) of L is attached to the N atom of the linker (i.e. , not a possible N atom of a targeting ligand).

[0404] In another embodiment, the first strand of the nucleic acid comprises 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,

[0405] Z1 and Z2are respectively the first and second strand of the nucleic acid,

[0406] Y is S,

[0407] Li is of formula (VII), wherein:

[0408] W1 is -CH2-O-(CH2)3-,

[0409] W3is -CH2-,

[0410] W5 is absent,

[0411] V is CH,

[0412] X is NH, and

[0413] L is -(CH2)4-C(O)- wherein the terminal C(O) of L is attached to the N atom of X in formula (VII).

[0414] In another aspect, the first strand of the nucleic acid comprises 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,

[0415] Z1 and Z2are respectively the first and second strand of the nucleic acid,

[0416] Y is S,

[0417] Li is of formula (VII), wherein:

[0418] W1, W3and 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).

[0419] In one embodiment, the nucleic acid is conjugated to a triantennary ligand with the following structure: wherein the nucleic acid 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.

[0420] In one embodiment of the nucleic acid, the cells that are targeted by the nucleic acid with a ligand are hepatocytes.

[0421] In any one of the above ligands where GalNAc is present, the GalNAc may be substituted for any other targeting ligand, such as those mentioned herein, in particular mannose, galactose, glucose, glucosamine and fucose.

[0422] In one embodiment, the nucleic acid is conjugated to a heterologous moiety that comprises a lipid, and more preferably, a cholesterol.

[0423] 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 a 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. Preferably, 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.

[0424] Compositions, uses and methods

[0425] The present invention also provides compositions comprising a 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. For example, 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.

[0426] Compositions disclosed herein are particularly pharmaceutical compositions. Such compositions are suitable for administration to a subject.

[0427] In one aspect, the composition comprises 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. 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 described herein.

[0428] 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.

[0429] 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.

[0430] 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 the invention, or a salt thereof, in a pharmaceutically acceptable carrier.

[0431] 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 administered 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.

[0432] In one aspect, the composition comprises a nucleic acid of formula (I) or 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.

[0433] In one aspect, 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. Preferably, the further therapeutic agent is an agent that targets, preferably inhibits the expression or the activity, of a desired target gene. Preferably, 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.

[0434] In some embodiments, 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, timolol; a vasodilator, such as for example a direct vasodilator, such as hydrazaline and minoxidil; a calcium channel blocker (also referred to as calcium channel antagonists), such as for example amlodipine, diltiazem, verapamil, nifedipine, nisoldipine, felodipine, nimodipine, isradipine, levamlodipine, clevidipine, nicardipine; an aldosterone antagonist, such as for example spironolactone, eplerenone, finerenone; an alpha2-agonist, such as for example clonidine, methydopa, tizanidine, guanfacine, lofexidine; a renin inhibitor, such as for example aliskiren; an alpha-blocker, such as for example doxazosin, prazosin, terazosin; a central acting sympatholytic, such as for example methyldopa, clonidine, guanabenz, or guanfacine; a peripheral acting sympatholytic, a selective D1 receptor partial agonist, such as for example clozapine, fenoldopam; a nonselective alpha-adrenergic antagonist such as for example phenoxybenzamine, a synthetic, and steroidal antimineralocorticoid agent, such as for example spironolactone, eplerenone, canrenone, finereonone, mexrenone; an angiotensin receptor-neprilysin inhibitors (ARNi), such as for example sacubitril, sacubitril / valsartan; or an endothelin receptor antagonist (ERA), such as for example sitaxentan, ambrisentan, atrasentan, BQ-123, zibotentan, bosentan, macitentan, and tezosentan; vasopressin inhibitors, such as for example tolvaptan, conivaptan, lixivaptan, mozavaptan, stavaptan, relcovaptan; nitrates; potassium channel openers; such as for example minoxidil, nicorandil, pinacidil, levcromakalim; imidazolines, such as for example clonidine, moxonidin, oxymetazolin; a combination of any of the foregoing; and a hypertension therapeutic agent formulated as a combination of agents.

[0435] Preferably, the further therapeutic agent comprises an antagonist of the desired target gene.

[0436] In certain embodiments, two or more nucleic acids of the invention of formula (I) with different sequences may be administered simultaneously or sequentially.

[0437] In another aspect, 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.

[0438] In another aspect, the present invention provides a pharmaceutical composition comprising a nucleic acid of formula (I) of the invention and at least one pharmaceutically acceptable carrier. Dosage levels for the therapeutic agents and compositions of the invention can be determined by those skilled in the art by experimentation. In one aspect, 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. Alternatively, 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. Alternatively, 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.

[0439] A dose unit of these nucleic acids of formula (I) 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 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. In certain prophylactic applications, 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. In certain therapeutic applications, 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.

[0440] Actual dosage levels of a nucleic acid of formula (I) of the invention alone or in combination with one or more other active ingredients in the pharmaceutical 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.

[0441] The pharmaceutical composition of the invention may be a sterile injectable aqueous suspension or solution, or in a lyophilized form.

[0442] The pharmaceutical composition of the invention can be in unit dosage form. In such 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.

[0443] 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. On this basis, “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.

[0444] Pharmaceutical 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. Examples of 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.

[0445] Pharmaceutical compositions of the invention are typically sterile and stable under the conditions of manufacture and storage. The composition of the invention 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. In certain embodiments, 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.

[0446] One aspect of the invention is a nucleic acid of formula (I) or a composition, preferably a pharmaceutical composition, disclosed herein for use as a therapeutic agent. The nucleic acid of formula (I) or composition, preferably a pharmaceutical composition, is preferably for use in the prophylaxis or treatment of a disease, disorder or syndrome.

[0447] 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. One aspect of the invention is a nucleic acid of formula (I), preferably a conjugated nucleic acid of formula (I), or a composition as disclosed herein for use as a therapeutic agent.

[0448] One aspect of the invention is a nucleic acid of formula (I), preferably a conjugated nucleic acid of formula (I), or a composition, preferably a pharmaceutical composition, as disclosed herein for use in the prophylaxis or treatment of a disease, disorder or syndrome.

[0449] One aspect of the invention is the use of a nucleic acid of formula (I), preferably a conjugated nucleic acid of formula (I), 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.

[0450] One aspect of the invention is the use of a nucleic acid of formula (I), preferably a conjugated 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.

[0451] Nucleic acids of formula (I), preferably a conjugated nucleic acid 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 formula (I), preferably a conjugated nucleic acid of formula (I), of the invention.

[0452] 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 a conjugated nucleic acid of formula (I), or a pharmaceutical composition of the invention.

[0453] 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. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through experimentation, namely by monitoring a subject's response to administration of a compound and adjusting the dosage accordingly. See, e.g., Remington: The Science and Practice of Pharmacy 21st Ed., Univ, of Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins, Philadelphia, PA, 2005.

[0454] In certain embodiments, nucleic acids of formula (I) and pharmaceutical compositions of the invention may be used to treat or prevent a disease, disorder or syndrome.

[0455] In certain embodiments, 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), preferably a conjugated nucleic acid of formula (I), as disclosed herein.

[0456] Administration of a "therapeutically effective dosage" of a nucleic acid 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.

[0457] Nucleic acids 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. Treatment and diagnosis of other diseases, disorders or syndromes are also considered to fall within the scope of the present invention.

[0458] 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 a nucleic acid or a composition disclosed herein to an individual in need of treatment, preferably wherein the nucleic acid or composition is administered to the subject subcutaneously, intravenously or by oral, rectal, pulmonary, intramuscular or intraperitoneal administration. Preferably, it is administered subcutaneously.

[0459] 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.

[0460] A nucleic acid of formula (I), preferably a conjugated nucleic acid of formula (I), or 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), preferably a conjugated 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.

[0461] In cells and / or subjects treated with or receiving a conjugated nucleic acid or composition as disclosed herein, 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 may 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 of the invention.

[0462] One aspect is the use of a nucleic acid of formula (I), preferably a conjugated nucleic acid of formula (I), or 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.

[0463] Each of the nucleic acids of formula (I) of the invention and pharmaceutically acceptable salts and solvates thereof constitutes an individual embodiment of the invention. Also included in the invention is a method of prophylaxis or treatment of a disease, disorder or syndrome, such as those listed above, comprising administration of a composition comprising a nucleic acid of formula (I), preferably a conjugated nucleic acid of formula (I), or composition as described herein, to an individual in need thereof. The nucleic acid 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 may be for use subcutaneously or intravenously or other application routes such as oral, rectal or intraperitoneal.

[0464] The nucleic acid of formula (I) or compositions 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). "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.

[0465] 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. 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. Such preparations may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0466] 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. In the case of sterile powders for the preparation of sterile injectable solutions, 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.

[0467] 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.

[0468] The amount of 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.

[0469] 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. 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.

[0470] 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. As used herein, 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.

[0471] The nucleic acid or composition of the present invention can be produced using routine methods in the art including chemical synthesis, such as solid phase chemical synthesis.

[0472] Nucleic acids of formula (I), preferably a conjugated nucleic acid 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. For example, in one embodiment, a nucleic acid of the invention may be administered with a needleless hypodermic injection device. Examples of well-known 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.

[0473] In certain embodiments, the nucleic acid or composition of the invention may be formulated to ensure a desired distribution in vivo. To target 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.

[0474] The present 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 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. This leads to the nucleic acid inhibiting the expression of their target only in the cells that are targeted by the ligand to which they are linked. These two levels of specificity potentially confer a better safety profile than the currently available treatments. In certain embodiments, 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 internalisation of the nucleic acid thereby conferring additional specificity of target gene knockdown by RNA interference.

[0475] The nucleic acid 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 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 the invention with a lipid delivery system or with a ligand conjugate delivery system.

[0476] 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.

[0477] The cationic lipid may be an amino cationic lipid.

[0478] The cationic lipid may have the formula (XII): or a pharmaceutically acceptable salt thereof, wherein:

[0479] X represents O, S or NH;

[0480] R1and R2each 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, R3and R4each independently represent hydrogen, methyl, ethyl, a mono- or polyamine moiety, or R3and R4together form a heterocyclyl ring; when X represents O, R3and R4each independently represent hydrogen, methyl, ethyl, a mono- or polyamine moiety, or R3and R4together form a heterocyclyl ring, or R3represents hydrogen and R4represents C(NH)(NH2). The cationic lipid may have the formula (XIII): or a pharmaceutically acceptable salt thereof.

[0481] The cationic lipid may have the formula (XIV): or a pharmaceutically acceptable salt thereof.

[0482] 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. In particular, the cationic lipid component is about 59 mol% of the overall lipid content of the composition.

[0483] The 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), 1 ,2-Dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1 ,2-Dimyristoyl-sn-glycero-3- phosphoethanolamine (DMPE), 1 ,2-Dipalmitoyl-sn- glycero-3-phosphoethanolamine (DPPE), 1 ,2-Dilinoleoyl-sn-glycero-3-phosphoethanolamine (DLoPE), 1-Palmitoyl-2-oleoyl-sn-glycero- 3-phosphoethanolamine (POPE), 1 ,2-Dierucoyl-sn- glycero-3-phosphoethanolamine (DEPE), 1 ,2-Disqualeoyl-sn-glycero-3-phosphoethanolamine (DSQPE) and 1-Stearoyl-2-linoleoyl-sn- glycero-3-phosphoethanolamine (SLPE). The content of the phospholipid may be about 10 mol% of the overall lipid content of the composition.

[0484] 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.

[0485] 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. a phosphatidylethanolamine phospholipid having the structure DPhyPE and a PEGylated lipid having the structure mPHG ’UUU :)\!G

[0486] 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.

[0487] A positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride (DOTMA) 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.

[0488] Derivatives and analogues of lipids described herein may also be used to form liposomes.

[0489] A liposome containing a nucleic acid can be prepared by a variety of methods. In one example, the lipid component of a liposome is dissolved in a detergent so that micelles are formed with the lipid component. For example, 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. After condensation, the detergent is removed, e.g., by dialysis, to yield a liposomal preparation of nucleic acid.

[0490] If necessary, a carrier compound that assists in condensation can be added during the condensation reaction, e.g., by controlled addition. For example, 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.

[0491] Nucleic acid formulations of the present invention may include a surfactant. In one embodiment, the nucleic acid is formulated as an emulsion that includes a surfactant.

[0492] A surfactant that is not ionized is a non-ionic surfactant. Examples include 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.

[0493] A surfactant that carries a negative charge when dissolved or dispersed in water is an 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.

[0494] 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.

[0495] A surfactant that has the ability to carry either a positive or negative charge is an amphoteric surfactant. Examples include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

[0496] "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.

[0497] 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.

[0498] 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.

[0499] 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.

[0500] Definitions

[0501] As used herein, 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.

[0502] By nucleic acid 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.

[0503] 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.

[0504] By 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. For example, 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 —

[0505] 3' 5’ wherein the enclosed area in this schematic represents the double strand or duplex region. In this specific example, the first nucleotide on the 5’ end of the first strand corresponds to Yf and the last nucleotide within the enclosed area on the 3’ terminal end of the first strand corresponds to Yu, while the last two nucleotides (two boxes) outside the enclosed area at the 3’ end of the first strand correspond to Xb. In the same manner, the first nucleotide on the 3’ end of the second strand corresponds to Yf and the last nucleotide within the enclosed area on the 5’ terminal end of the second strand corresponds to Yu. Any nucleotides outside of the enclosed area, if present, correspond respectively to Xa, Xb and Xa’ and Xb’ on the first and second strand. In this disclosure, 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.

[0506] Further, within the duplex region, 100% complementarity is not required; substantial complementarity is allowable within a duplex region. Substantial 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.

[0507] As used herein, the terms “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. In some embodiments the non-base pairing nucleotide analogue is a ribonucleotide. In other embodiments it is a deoxyribonucleotide.

[0508] As used herein, the term, “terminal functional group” includes without limitation a halogen, alcohol, amine, carboxylic, ester, amide, aldehyde, ketone, and ether groups.

[0509] 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:

[0510] An O atom of said structure typically links to an RNA strand and the N atom typically links to the targeting ligand.

[0511] The terms “patient,” “subject,” and “individual” may be used interchangeably and refer to either a human or a non-human animal. These terms include mammals such as humans, primates, livestock animals (e.g., bovines, porcines), companion animals (e.g., canines, felines) and rodents (e.g., mice and rats).

[0512] As used herein, “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. For the purposes of this invention, 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) in need of treatment may thus be a subject already afflicted with the disease or disorder in question. The term “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.

[0513] As used herein, the terms "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. For the purposes of this invention, 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) in need of prophylaxis may thus be a subject not yet afflicted with the disease or disorder in question. The term “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. Thus “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.

[0514] As used herein, 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.

[0515] As used herein, 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(R1)(R2)(R3)(R4)+, wherein R1, R2, R3and R4independently 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. Examples of 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. Examples of pharmaceutically acceptable salts include acid addition salts and base addition salts. 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. 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.

[0516] The term "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). For example, 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. The term further encompasses any agents listed in the US Pharmacopeia for use in animals, including humans. 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. In certain embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on selected route of administration, 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.

[0517] The term “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. When the solvent in question is water, such a solvate is normally referred to as a hydrate.

[0518] The invention will now be described with reference to the following non-limiting Figures and Examples.

[0519] Brief Description of the Figures

[0520] Figure 1 - 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 Y of the second strand.

[0521] Figure 2 - GalNAc-siRNA conjugates with LNA modifications at positions Y2’ and Y of the second strand require one phosphorothioate internucleotide linkage to be stable in acidic rat liver tritosome lysate.

[0522] Figure 3 - in vitro study showing that modifying the first strand at Y-2 and the second strand at Y2’ with one LNA is tolerated in a sequence specific manner. Two LNA modifications per strand lead to a reduced mRNA knockdown. Figure 4 - 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).

[0523] Figure 5- in vitro study showing LNA modifications in combination with inverted ribonucleotides decrease target mRNA knockdown.

[0524] Figure 6 - in vitro study showing that activity of 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.

[0525] Figure 7 - 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.

[0526] Figure 8 - 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.

[0527] Figure 9 - 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.

[0528] Figure 10 - in vitro study showing that 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.

[0529] Figures 11a, 11 b, 11c - 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

[0530] Figures 12a, 12b, 12c - 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

[0531] Figure 13 - In vitro activity of GalNAc siRNA conjugates with LNA modifications at the 5'-end of the second strand.

[0532] Figure 14 - T ritosome stability of GalNAc siRNA conjugates with LNA modifications at positions 1 or 2 of the second strand 5'-end.

[0533] Figure 15 - 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.

[0534] Figure 16 - T ritosome 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.

[0535] Figure 17 - In vitro activity of GalNAc siRNA conjugates with different combinations of LNA- containing end modifications.

[0536] Figure 18 - Improved tritosome stability of GalNAc-siRNA conjugates targeting Ttr with LNA at penultimate position 18 of the second strand.

[0537] Figure 19 - Improved tritosome stability of GalNAc-siRNA conjugates targeting At3 with LNA at penultimate position 18 of the second strand.

[0538] Figure 20 - Plasma TTR levels after treatment with GalNAc-siRNA conjugates with and without LNA at penultimate position 18 of the second strand.

[0539] Figure 21 - Target mRNA levels in mouse liver after treatment with GalNAc siRNA conjugates with and without LNA at penultimate position 18 of the second strand. Figure 22 - In vitro activity of GalNAc siRNA conjugates targeting Cfb with LNA at penultimate position 18 of the second strand.

[0540] Figure 23 - Plasma Cfb levels after treatment with GalNAc siRNA conjugates with and without LNA at penultimate position 18 of the second strand.

[0541] Figure 24 - Plasma At3 levels after treatment with GalNAc siRNA conjugates with different combinations of LNA-containing end modifications.

[0542] Examples

[0543] 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.

[0544] Building block synthesis

[0545] Synthesis of the phosphoramidite derivatives of ST41 (ST41-phos), ST43 (ST43-phos) as well as ST23 (ST23-phos) and their precursor compounds can be performed as described in WO2017 / 174657:

[0546] ST41-phos:

[0547] ST43-phos:

[0548] ST23-phos:

[0549] ST23-phos Synthesis of Oligonucleotides

[0550] 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.

[0551] 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 I2 in pyridine / H2O). 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.

[0552] All 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. Upon completion of the TBDMS cleavage reaction and cooling to RT the crude product was precipitated by addition of 20 mL / PrOH (- 20°C) and 0.2 mL 2 M NaAc and harvested by centrifugation and decantation. The residual pellet was then reconstituted to a concentration of ~10 mg / mL in water. Crude products were further purified by ion exchange chromatography (SourceQ. 7.5 mL. GE Healthcare) on an AKTA Pure HPLC System using a sodium chloride gradient (A: 25mM Tris, 10% ACN, 90% H2O, pH 7.5 B: 25mM Tris, 2M NaCI, 10% ACN, 90% H2O, pH 7.5). Product containing fractions were pooled, desalted on a size exclusion column (Zetadex, EMP Biotech) and lyophilised.

[0553] To prove their purity all final single stranded products were analyzed by AEX-HPLC (DNA Pac PA200 4.0x250mm & DNAPac PA200 Guard 4x50 mm at 80 °C, using a gradient of 25-70% B (10% MeCN, 20 mM TRIS, 0.4 M LiCIO4in water, pH =7.4) in A (10% MeCN, 20 mM TRIS in water) over 10 min, flow rate 1 mL / min). Purity is given in %FLP (% full length product) which is the percentage of the UV-area under the assigned product signal in the UV-trace of the AEX- HPLC analysis of the final product. Identity of the respective single stranded products was confirmed by UPLC-MS analysis (Acquity LIPLC BEH C182.1x50mm, 1.7 pm Acquity BEH C18 1.7 pM Vanguard Pre-Col at 60°C, using a gradient of 10-22% B (100 mM HFIP, 15 mM TEA in MeOH) in A (100 mM HFIP, 15 mM TEA, 5% MeOH in water) over 5 min, flow rate 0.3 mL / min).

[0554] Table 1 : Single stranded oligonucleotides

[0555] Double strand formation

[0556] Individual single strands were dissolved in a concentration of 60 OD / mL in H2O, both individual oligonucleotide solutions were added together in a reaction vessel, a titration was performed for easier reaction monitoring, The first strand was added in 25% excess over the second strand as determined by UV-absorption at 260nm, The reaction mixture was heated to 80°C for 5min and then slowly cooled to RT, Double strand formation was monitored by ion pairing reverse phase HPLC (X Bridge BEH C18 2,1x50 mm 2,5pm, XP VanGuard Cartridges at 15 °C, using a gradient of 5-45% B (100 mM HFIP, 15 mM TEA in MeOH) in A (100 mM HFIP, 15 mM TEA, 5% MeOH in water) over 15 min, flow rate 0,3 mL / min), From the UV-area of the residual single strand the needed amount of the second strand was calculated and added to the reaction mixture, The reaction was heated to 80°C again and slowly cooled to RT, This procedure was repeated until less than 10% of residual single strand was detected,

[0557] Table 2: Nucleic acid conjugates Summary tables

[0558] Table 3: Summary of abbreviations Ill

[0559] The abbreviations as shown in the above abbreviation table may be used herein. The list of abbreviations may not be exhaustive and further abbreviations and their meaning may be found throughout this document.

[0560] Example 2 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 Y of the second strand.

[0561] The influence of an LNA modification at Y2’ and Y with different combinations of phosphorothioate internucleotide linkages of the second strand was analyzed using GalNAc- siRNA conjugates targeting At3 and Ttr by receptor-mediated uptake in mouse primary hepatocytes. X1335 and X1348 contain two phosphorothioate linkages at the 3' end of the second strand. X1342 and X1355 contain an LNA modification at Yf and one phosphorothioate internucleotide linkage between position Y3’ and Y2’. X1343 and X1356 contain an LNA modification at Y and two phosphorothioate internucleotide linkages at the terminal positions. X1344 (a different synthesis batch of the identical compound was called X2235, see Example 26 below), 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 Yf’ 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 1.

[0562] Examole 3

[0563] 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.

[0564] Stability of GalNAc-siRNA conjugates targeting At3 with LNA modification at Y2’ and Y with different combinations of phosphorothioate internucleotide linkages of the second strand in acidic rat liver tritosome extract was analyzed. X1335 contains two phosphorothioate internucleotide linkages at the 3' end of the second strand. X1342 contains an LNA modification at Y 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 atY2’ and Y 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. To assess stability, 5 pM GalNAc-siRNA conjugate was incubated with acidic rat liver tritosome extract (pH 5) at 37°C for 0, 24 and 96 hours. After incubation, samples were separated on 20% TBE polyacrylamide gels and visualized by ethidium bromide staining. Results are shown in Figure 2.

[0565] In vitro study showing that modifying the first strand at Y-2 and the second strand at Y2’ with one LNA is tolerated in a sequence specific manner. Two LNA modifications per strand lead to a reduced mRNA knockdown. 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 Yuas well as Yf 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 Yuas well as Yf’ 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 Yuas well as Yf’ 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.

[0566] 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 1nM 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 Figures.

[0567] Example 5

[0568] 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).

[0569] 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 Y2’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 Yuas 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 Yuas well as Y and Y2’ and an irA modification at the 3' end of the second strand.

[0570] 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 Yf of the second strand led to a further increase in stability.

[0571] To assess stability, 5 pM GalNAc-siRNA conjugate was incubated with acidic rat liver tritosome extract (pH 5) at 37°C for 0, 24 and 96 hours. After incubation, samples were separated on 20% TBE polyacrylamide gels and visualized by ethidium bromide staining. Results are shown in Figure 4.

[0572] Example 6

[0573] In vitro study showing LNA modifications in combination with inverted ribonucleotides decrease target mRNA knockdown.

[0574] The influence of an siRNA containing irA at Yf of the second strand combined with an LNA modification at Y2’ was analyzed using GalNAc-siRNA conjugates targeting At3 and Ttr by receptor-mediated uptake in mouse primary hepatocytes. X1594 and X1599 contain an irA modification at Yf 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 Y2’, 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.

[0575] 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, and Pten mRNA levels were determined by Taqman qRT-PCR. Each bar represents mean ± SD from three technical replicates. Results are shown in Figure 5. Example 7

[0576] In vitro study showing that activity of 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.

[0577] 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.

[0578] 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 siRNAs targeting At3 or 1 nM and 0.1 nM siRNAs targeting TTR directly after plating. Cells were lysed after 24 h, total RNA was extracted and Ttr, At3, Actb, and Pten mRNA levels were determined by Taqman qRT-PCR. Each bar represents mean ± SD from three technical replicates. Results are shown in Figure 6.

[0579] Example 8

[0580] Stability of GalNAc-siRNA conjugates with inverted ribonucleotides at the 3' end of the second strand in acidic rat liver tritosome extracts cannot be improved by addition of LNA modifications at Y2’ of the second strand.

[0581] Stability of GalNAc-siRNA conjugates targeting At3 containing irA at the terminal position of the second strand combined with an LNA modification at Y2’ in acidic rat liver tritosome extract was analyzed. 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 atY2’, 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.

[0582] To assess stability, 5 pM GalNAc-siRNA conjugate was incubated with acidic rat liver tritosome extract (pH 5) at 37°C for 0, 24 and 96 hours. After incubation samples were separated on 20% TBE polyacrylamide gels and visualized by ethidium bromide staining. Results are shown in Figure7.

[0583] Example 9

[0584] 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.

[0585] Stability of GalNAc-siRNA conjugates targeting At3 containing irA at the terminal position of the second strand combined with an LNA modification at Y2’ in acidic rat liver tritosome extract was analyzed. All tested molecules have a vinylphosphonate at the 5’ end of the first 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.

[0586] Surprisingly, 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.

[0587] To assess stability, 5 pM GalNAc-siRNA conjugate was incubated with acidic rat tritosome extract (pH 5) at 37°C for 0, 24 and 96 hours. After incubation, samples were separated on 20% TBE polyacrylamide gels and visualized by ethidium bromide staining. Results are shown in Figure 8.

[0588] Example 10

[0589] 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.

[0590] Stability of GalNAc-siRNA conjugates targeting At3 containing irA at the terminal position of the second strand combined with an LNA modification at Y2’ in acidic rat liver tritosome extract was analyzed. All tested molecules have a 5' vinylphosphonate in 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 Yuof 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. Surprisingly, 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. The number of phosphorothioate internucleotide linkages in both strands can therefore be reduced. This is an advantage because such molecules have fewer stereogenic centres (the phosphorothioate are stereogenic). To assess stability, 5 pM GalNAc-siRNA conjugate was incubated with acidic rat tritosome extract (pH 5) at 37°C for 0, 24 and 96 hours. After incubation samples were separated on 20% TBE polyacrylamide gels and visualized by ethidium bromide staining. Results are shown in Figure 9.

[0591] Example 11

[0592] 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.

[0593] 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 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. Addition of an irA and an LNA modification to the second strand of an siRNA targeting At3 results in a decreased mRNA knockdown, while addition of an LNA modification in the first strand results in an increase of mRNA knockdown compared to the respective siRNA without LNA and irA modifications. Surprisingly, this effect can be reversed by combining those first strand and second strand modifications. The resulting siRNA show a comparable mRNA knockdown under the tested conditions compared to the respective siRNA without LNA and irA modifications.

[0594] 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 1nM 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, and ApoB mRNA levels were determined by Taqman qRT-PCR. Each bar represents mean ± SD from three technical replicates. Results are shown in Figure 10.

[0595] Example 12

[0596] 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.

[0597] Example 12 A

[0598] 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 ofthe 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 Yuof 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.

[0599] This experiment was conducted in primary human hepatocytes. Cells were seeded at a density of 35,000 cells per 96-well and treated with 10 nM and 1 nM using siRNAs targeting At3 directly after plating. Cells were lysed after 24 h, total RNA was extracted and At3 and Actb mRNA levels were determined by Taqman qRT-PCR. Each bar represents mean ± SD from three technical replicates. Results are shown in Figure 11A.

[0600] Example 12B

[0601] 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 Yuof 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.

[0602] This experiment was conducted in primary human hepatocytes. Cells were seeded at a density of 35,000 cells per 96-well and treated with 10 nM and 1 nM using siRNAs targeting At3 directly after plating. Cells were lysed after 24 h, total RNA was extracted and At3 and Actb mRNA levels were determined by Taqman qRT-PCR. Each bar represents mean ± SD from three technical replicates. Results are shown in Figure 11 B.

[0603] Example 12C

[0604] 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 ofthe first strand. X1845 contains an LNA modification at Y2’, an irA modification at Yf 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 Yuof 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.

[0605] This experiment was conducted in primary human hepatocytes. Cells were seeded at a density of 35,000 cells per 96-well and treated with 10 nM and 1 nM using siRNAs targeting At3 directly after plating. Cells were lysed after 24 h, total RNA was extracted and At3 and Actb mRNA levels were determined by Taqman qRT-PCR. Each bar represents mean ± SD from three technical replicates. Results are shown in Figure 11C. Example 13

[0606] 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.

[0607] Example 13A

[0608] All tested molecules have a vinylphosphonate at the 5’ end of the first strand. 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 Yf 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 Yuof 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.

[0609] This experiment was conducted in primary human hepatocytes. Cells were seeded at a density of 35,000 cells per 96-well and treated with 10 nM and 1 nM using siRNAs targeting At3 or Aldh2 directly after plating. Cells were lysed after 24 h, total RNA was extracted and At3 and

[0610] Actb or Aldh2 and Ppib mRNA levels were determined by Taqman qRT-PCR. Each bar represents mean ± SD from three technical replicates. Results are shown in Figure 12A.

[0611] Example 13B

[0612] All tested molecules have a vinylphosphonate at the 5’ end 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 Yf 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 Yuof 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.

[0613] This experiment was conducted in primary human hepatocytes. Cells were seeded at a density of 35,000 cells per 96-well and treated with 10 nM and 1 nM using siRNAs targeting At3 or Aldh2 directly after plating. Cells were lysed after 24 h, total RNA was extracted and At3 and Actb or Aldh2 and Ppib mRNA levels were determined by Taqman qRT-PCR. Each bar represents mean ± SD from three technical replicates. Results are shown in Figure 12B.

[0614] Example 13C

[0615] All tested molecules have a vinylphosphonate at the 5’ end 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 Yf 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 Yuof 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.

[0616] This experiment was conducted in primary human hepatocytes. Cells were seeded at a density of 35,000 cells per 96-well and treated with 10 nM and 1 nM using siRNAs targeting At3 or Aldh2 directly after plating. Cells were lysed after 24 h, total RNA was extracted and At3 and Actb or Aldh2 and Ppib mRNA levels were determined by Taqman qRT-PCR. Each bar represents mean ± SD from three technical replicates. Results are shown in Figure 12C.

[0617] Example 14

[0618] 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.

[0619] Commercially available 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 / FhO). Phosphorothioates (PS) were introduced through coupling of phosphoramidites, phosphorodithioates (PS2) were introduced through coupling of thiophosphoramidites and subsequent sulfurization using 0.05 M ((dimethylamino-methylidene) amino)-3H-1.2.4- dithiazoline-3-thione (DDTT, Chemgenes).

[0620] Single-stranded oligonucleotides and double-stranded siRNAs and siRNA conjugates are listed in Tables 4 and 5.

[0621] Table 4: Single-stranded oligonucleotides Table 5: Double-stranded siRNAs and siRNA conjugates

[0622] Example 15

[0623] In vitro activity of GalNac siRNA conjugates with LNA near the 5' end of the second strand.

[0624] 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]3ST43.

[0625] This experiment was conducted in primary mouse hepatocytes. Cells were seeded at a density of 25,000 cells per96-well and treated with 10 nM and 1 nM using siRNAs targeting At3 directly after plating. Cells were lysed after 24 h, total RNA was extracted and At3, Actb and ApoB mRNA levels were determined by Taqman qRT-PCR. Each bar represents geometric mean ± SD from three technical replicates. Results are shown in Figure 13.

[0626] Example 16

[0627] Improved tritosome stability of GalNAc-siRNA conjugates with LNA near the 5' end of the second strand.

[0628] 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]3ST43. To assess stability, 5 pM GalNAc-siRNA conjugate was incubated with acidic rat liver tritosome extract (pH 5) at 37°C for 0, 24 and 96 hours. After incubation, samples were separated on 20% TBE polyacrylamide gels and visualized by ethidium bromide staining. Results are shown in Figure 14.

[0629] Example 17

[0630] In vitro activity of GalNAc-siRNA conjugates with LNA near the 5' end of the second strand.

[0631] X1767 (a different synthesis batch of this compound was called X2052, see Example 26 below), 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.

[0632] 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 directly after plating. Cells were lysed after 24 h, total RNA was extracted and At3, Actb and ApoB mRNA levels were determined by Taqman qRT-PCR. Each bar represents geometric mean ± SD from three technical replicates. Results are shown in Figure 15.

[0633] Example 18

[0634] Improved tritosome stability of GalNAc-siRNA conjugates with LNA near the 5' end of the second strand.

[0635] 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)]3 ST43 (ps). X1913 with the GalNAc cluster [ST23]3 ST43 was included as reference.

[0636] To assess stability, 5 pM GalNAc-siRNA conjugate was incubated with acidic rat liver tritosome extract (pH 5) at 37°C for 0, 24 and 96 hours. After incubation, samples were separated on 20% TBE polyacrylamide gels and visualized by ethidium bromide staining. Results are shown in Figure 16. Example 19

[0637] In vitro activity of GalNac siRNA conjugates with combined LNA modifications at different strand termini.

[0638] 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. The same activity as with the control compound is detected when LNA modifications are used without another opposing LNA (X1911 , X1344, X1604, X1733 compared to X1767; X1913 compared to X1910). In vitro activity of GalNAc siRNA conjugates with simultaneous LNA modifications at first strand position 18 and second strand position 2 is reduced when the 3' end of the second strand is modified with LNA and irA (X2124 compared to X2125; X2126 compared to X2127; X2128 compared to X2129).

[0639] 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 siRNAs targeting At3 directly after plating. Cells were lysed after 24 h, total RNA was extracted and At3, Actb and ApoB mRNA levels were determined by Taqman qRT-PCR. Each bar represents geometric mean ± SD from three technical replicates. Results are shown in Figure 17.

[0640] Example 20

[0641] Improved tritosome stability of GalNAc-siRNA conjugates with LNA at penultimate position 18 of the second strand in a blunt ended 19-mer.

[0642] X1734 and X1704 contain no LNA modifications and two phosphorothioate linkages at the 3' end of each strand. X1357 and X1957 contain an LNA modification at position 18 of the second strand and one phosphorothioate internucleotide linkage between the terminal nucleotides. X1704 and X1957 are modified with 5'-(E)-vinylphosphonate at the 5' end of the first strand. X1357 and X1957 show improved stability after 96 h incubation with rat liver tritosomes.

[0643] To assess stability, 5 pM GalNAc-siRNA conjugate was incubated with acidic rat liver tritosome extract (pH 5) at 37°C for 0, 24 and 96 hours. After incubation, samples were separated on 20% TBE polyacrylamide gels and visualized by ethidium bromide staining. Results are shown in Figure 18.

[0644] Example 21

[0645] 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.

[0646] 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.

[0647] 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.

[0648] To assess stability, 5 pM GalNAc-siRNA conjugate was incubated with acidic rat liver tritosome extract (pH 5) at 37°C for 0, 24 and 96 hours. After incubation, samples were separated on 20% TBE polyacrylamide gels and visualized by ethidium bromide staining. Results are shown in Figure19.

[0649] Example 22

[0650] GalNAc-siRNA conjugates with an LNA modification at penultimate position 18 of the second strand in a blunt ended 19-mer improved reduction of Ttr target mRNA levels in vivo in mice.

[0651] X1734 contains no LNA modifications and two phosphorothioate linkages at the 3' end of each strand. X2033 contains an LNA modification at position 18 and one phosphorothioate internucleotide linkage between the terminal nucleotides of the second strand. GalNAc-siRNA conjugates containing an LNA at position 18 of the second strand reduce the target mRNA levels more compared to the respective siRNA without LNA. TTR serum protein levels showed a comparable initial knockdown on day 8 but an elongated duration of action upon the terminal study day.

[0652] C57BL / 6 male mice (n = 4) were each subcutaneously administered 1 mg / kg of one of the siRNA GalNAc conjugates X1734 and X2033. EDTA-plasma samples were retrieved by retro- orbital bleeding at different time points before and after treatment. Plasma TTR concentrations were analyzed with a Mouse Pre-Albumin ELISA Kit (Biocat 41-PALMS-E01-AL). Results are shown in Figure 20.

[0653] Liver sections were prepared 43 days after treatment, total RNA was extracted from the tissue and liver TTR mRNA levels were quantified by AACt analysis by Multiplex TaqMan quantification against house-keeping gene expression (ApoB and PTEN). Each bar represents the geometric mean ± SD. Results are shown in Figure 21.

[0654] Example 23 Example compounds were synthesized as described in Examples 1 and 14. Single-stranded oligonucleotides and double-stranded siRNAs and siRNA conjugates are listed in Tables 1-8.

[0655] Table 6: Single stranded oligonucleotides

[0656] Table 7: Double-stranded siRNA conjugates

[0657] Table 8 is shown at the very end of the Example section.

[0658] Example 24

[0659] In vitro study showing that LNA at penultimate position 18 of the second strand does not impact mRNA knockdown.

[0660] X2193 contains two phosphorothioate linkages at the 3' end of the second strand. X2194 contains an LNA modification at position 18 and one phosphorothioate internucleotide linkage between the terminal nucleotides of the second strand.

[0661] 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 siRNAs targeting CFB directly after plating. Cells were lysed after 24 h, total RNA was extracted and CFB, and ApoB mRNA levels were determined by Taqman qRT-PCR. Each bar represents geometric mean ± SD from three technical replicates.

[0662] Results are shown in Figure 22.

[0663] Example 25

[0664] GalNAc-siRNA conjugates with an LNA modification at penultimate position 18 of the second strand improved reduction of Cfb target protein in vivo in mice.

[0665] C57BL / 6 male mice (n=4) were each subcutaneously administered 5 mg / kg of one of the siRNA GalNAc conjugates X2193 and X2194. EDTA-plasma samples were retrieved by retro-orbital bleeding at different time points before and after treatment. Plasma CFB concentrations were analyzed with a Factor B antibody (QuidelOrtho, A311) using an automated capillary electrophoresis system (JESS Simple Western System and Compass software; Protein Simple, USA). Results are shown in Figure 23.

[0666] Example 26

[0667] GalNAc-siRNA conjugates with an LNA modification at penultimate positions 2 and 18 of the second strand improved reduction of At3 target protein levels in vivo in mice. X1767 / X2052, X1911, X1344 / X2235, X1733, X1910, and X1913 contain LNA and irA modifications and phosphorothioate internucleotide linkages as detailed in Tables 1, 2, 6 and 7. X2052 and X1767 as well as X2235 and X1344 are different synthesis batches of identical sequences with identical modifications. X1910 and X1913 contain the GalNAc cluster [ST23]3 ST43, whereas all other conjugates contain the GalNAc cluster [ST23 (ps)]3 ST43 (ps).

[0668] C57BL / 6 male mice (n = 4) were each subcutaneously administered 0.5 mg / kg of one of the GalNAc siRNA conjugates X2052, X1911, X2235, X1733, X1910 and X1913. 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 24.

[0669] Table 8: First strand and second strand sequences of double-stranded siRNAs and siRNA conjugates.

[0670]

[0671]

[0672]

[0673]

[0674]

[0675]

[0676]

[0677]

[0678]

[0679]

[0680]

[0681]

[0682]

[0683]

[0684]

[0685]

[0686]

[0687] References

[0688] [1] J. Elmen, H. Thonberg, K. Ljungberg, M. Frieden, M. Westergaard, Y. Xu, B. Wahren, Z. Liang, H. 0rum, T. Koch, C. Wahlestedt, Nucleic Acids Res. 2005, 33, 439-447.

[0689] [2] J. Kurreck, E. Wyszko, C. Gillen, V. A. Erdmann, Nucleic Acids Res. 2002, 30, 1911— 1918.

[0690] [3] S. Obika, D. Nanbu, Y. Hari, K. Morio, Y. In, T. Ishida, T. Imanishi, Tetrahedron Lett. 1997, 38, 8735-8738.

[0691] [4] S. Wada, S. Obika, M.-A. Shibata, T. Yamamoto, M. Nakatani, T. Yamaoka, H. Torigoe, M. Harada-Shiba, Mol. Then - Nucleic Acids 2012, 1, e45.

[0692] [5] D. S. Schwarz, G. Hutvagner, T. Du, Z. Xu, N. Aronin, P. D. Zamore, Cell 2003, 115, 199- 208.

[0693] [6] A. Khvorova, A. Reynolds, S. D. Jayasena, Cell 2003, 115, 209-216.

[0694] [7] S. Z. Glud, J. B. Bramsen, F. Dagns-Hansen, J. Wengel, K. A. Howard, J. R. Nyengaard, J. Kjems, Oligonucleotides 2009, 19, 163-168.

[0695] [8] O. R. Mook, F. Baas, M. B. de Wissel, K. Fluiter, Mol. Cancer Then 2007, 6, 833-843.

[0696] [9] P. H. Weigel, J. H. N. Yik, Biochim. Biophys. Acta (BBA) - Gen. Subj. 2002, 1572, 341- 363.

[0697]

[0010] E. A. L. Biessen, D. M. Beuting, H. C. P. F. Roelen, G. A. van de Marel, J. H. V. Boom, T. J. C. V. Berkel, J. Med. Chem. 1995, 38, 1538-1546.

[0698]

[0011] A. Akinc, W. Querbes, S. De, J. Qin, M. Frank-Kamenetsky, K. N. Jayaprakash, M. Jayaraman, K. G. Rajeev, W. L. Cantley, J. R. Dorkin, J. S. Butler, L. Qin, T. Racie, A. Sprague, E. Fava, A. Zeigerer, M. J. Hope, M. Zerial, D. W. Sah, K. Fitzgerald, M. A. Tracy, M. Manoharan, V. Koteliansky, A. de Fougerolles, M. A. Maier, Mol. Then 2010, 18, 1357- 1364.

Claims

Claims1. A 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) strand 5' Xa - Yf - Y2- Y3- Y4- Y5- Z - Y-5 - Y-4 - Y-3 - Y-2 - Yu- Xb3' Second (sense) strand 3' Xa- Yf'- Y2' - Y3- Y4' - Y5'-Z' - Y-5 - Y-4' - Y.3'- Y-2' - Yu'- Xb' 5' (I), wherein: each of Xa, Xa', Xb and Xb' independently represents an overhang nucleotide sequence comprising 0-15 nucleotides, each of Yf and Y independently represents a nucleotide, wherein Yf and Y 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 Yuand Yu' independently represents a nucleotide, wherein Yuand Yu' represent the ultimate position of the double stranded region between the first and the second strands, wherein Yuand Yu' are opposite each other in their respective positions in the first and second strands and may be complementary to each other, each of Y2to Ysand Y2' to Ys' independently represents a nucleotide within the double stranded region, wherein Y2to Ys and Y2' 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.2to Y-s and Y.2' to Y.s' independently represents a nucleotide within the double stranded region, wherein Y.2to Y-sand Y.2' to Y-s'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 a) Y-2'is an LNA modified nucleotide, b) Y2' is an LNA modified nucleotide, or c) Y-2' and Y2' each are LNA modified nucleotides.

2. The double stranded nucleic acid of claim 1, whereina) Y-2'is an LNA modified nucleotide, or b) Y2' is an LNA modified nucleotide, and wherein the second strand comprises no additional LNA modified nucleotide.

3. The double stranded nucleic acid of claim 1, whereinY-2' and Y2' are LNA modified nucleotides and wherein the second strand comprises no additional LNA modified nucleotide.

4. The double stranded nucleic acid of any of the preceding claims, wherein the second strand comprises at least one phosphorothioate linkage between Y2' and Yf'.

5. The double stranded nucleic acid of any of the preceding claims, wherein the second strand comprises at least two phosphorothioate linkages, one between Y2'and Y / and one between Y2' and Y3'.

6. The double stranded nucleic acid of any of the preceding claims, wherein the first strand comprises a) at least one phosphorothioate linkage between Yf and Y2, or b) at least two phosphorothioate linkages, one between Yf and Y2 and one between Y2 and Y3.

7. The double stranded nucleic acid of any of the preceding claims, wherein the first strand comprises a (E)-vinylphosphonate nucleotide at a) position Yf, when Xa = 0; or b) the position of the first nucleotide Xacounted from the 5’-end of the first strand, when Xa= 1-6.

8. The double stranded nucleic acid of any of the preceding claims, wherein the second strand is modified with 2' fluoro modifications at those second strand positions that are opposite to positions 11, 12 and 13 of the first strand counting from Yf.

9. The double stranded nucleic acid of claim 8, wherein the remaining nucleotides of the second strand are modified with 2' O-methyl modifications.

10. The double stranded nucleic acid of claims 8 or 9, wherein the first strand is modified with alternating 2' O-methyl modifications and 2' fluoro modifications.

11. The double stranded nucleic acid of claim 10, wherein at least position Yf is modified with 2' O-methyl and at least position Y2 is modified with 2' fluoro.

12. The double stranded nucleic acid of any of the preceding claims, wherein the second strand comprises at least one inverted nucleotide.

13. The nucleic acid of any of the preceding claims, wherein i) the first strand of the nucleic acid has a length of between 14 and 30 nucleotides; and / or ii) the second strand of the nucleic acid has a length of between 14 and 30 nucleotides.

14. The nucleic acid of any of the preceding claims, wherein i) the first strand of the nucleic acid has a length of between 19 and 25 nucleotides; and / or ii) the second strand of the nucleic acid has a length of between 19 and 25 nucleotides.

15. The nucleic acid of any of the preceding claims, wherein the nucleic acid is conjugated to a heterologous moiety.

16. The conjugated nucleic acid of claim 15, 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.

17. A composition comprising the nucleic acid of any one of the preceding claims 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.

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