CN116802290A

CN116802290A - Nucleic acids for inhibiting PROS1 expression in cells

Info

Publication number: CN116802290A
Application number: CN202180088780.8A
Authority: CN
Inventors: 尤特·舍佩尔; 西比勒·戴慕兹; 艾略特·莫里森; 拉贾·普林斯·艾兰纳尼; 安尼·安吉利略·谢勒
Original assignee: Universitaet Bern; Silence Therapeutics GmbH
Current assignee: Universitaet Bern; Silence Therapeutics GmbH
Priority date: 2020-11-04
Filing date: 2021-11-02
Publication date: 2023-09-22

Abstract

The present invention relates to nucleic acid products that interfere with or inhibit the expression of the PROS1 gene. The invention also relates to therapeutic uses of PROS1 inhibition for treating bleeding disorders.

Description

Nucleic acids for inhibiting PROS1 expression in cells

Technical Field

The present invention relates to nucleic acid products that interfere with or inhibit the expression of the PROS1 (protein S) gene. The invention also relates to therapeutic uses of PROS1 inhibition for treating bleeding disorders.

Background

Double-stranded RNA (dsRNA) capable of complementarily binding to expressed mRNA has been shown to block gene expression (Fire et al 1998,Nature.1998Feb 19;391 (6669): 806-11 and Elbashir et al 2001,Nature.2001May 24;411 (6836): 494-8), the mechanism of which is known as RNA interference (RNAi). Short dsrnas direct gene-specific, post-transcriptional silencing in many organisms, including vertebrates, and have become useful tools for studying gene function. RNAi is mediated by the RNA-induced silencing complex (RISC), a sequence-specific multicomponent nuclease that degrades messenger RNA homologous to the silencing trigger loaded into the RISC complex. Interfering RNAs, such as sirnas, antisense RNAs, and micrornas are oligonucleotides that prevent the formation of a protein by gene silencing, i.e., inhibiting the translation of a protein by degradation of an mRNA molecule. Gene silencing agents are becoming increasingly important for therapeutic applications in medicine.

According to Watts and Corey, in Journal of Pathology (2012; vol 226, p 365379), there are algorithms that can be used to design triggers for nucleic acid silencing, but all have serious limitations. Because the algorithm does not take into account factors such as the tertiary structure of the target mRNA or the involvement of the RNA-binding protein, various experimental methods may be required to determine an effective iRNA. Thus, the discovery of an effective nucleic acid silencing trigger with minimal off-target effects is a complex process. For drug development of these highly charged molecules, they must be able to be economically synthesized, distributed to the target tissue, enter the cells and function within acceptable toxicity limits.

Hemophilia a and hemophilia B are the most common bleeding disorders caused by deficiency of procoagulant Factor VIII (FVIII) or factor IX (FVIX), respectively (Weyand and Pipe, 2019). The severity of hemophilia is classified according to the residual endogenous factor levels (balkuransinggh and Young 2017). Patients with severe hemophilia often suffer from spontaneous bleeding in the musculoskeletal system, such as joint hematochezia. This may lead to disabilities at a young age if left untreated.

Hemostasis is tightly regulated by the interaction of procoagulant factors and anticoagulant factors to control excessive bleeding events and prevent thrombotic events. Blood clotting is activated in response to injury to the vessel wall, where FVIIa binds to the exposed tissue factor, and then the FVIIa tissue factor complex effectively activates FX. FXa and FVa then form a prothrombinase complex, which produces thrombin. Furthermore, the FVIIa-tissue factor complex activates FIX, which together with its cofactor FVIIIa activates FX. The blood coagulation efficiency is determined by the amount of FXa and thrombin, a multifunctional enzyme that cleaves fibrinogen to fibrin and activates platelets. In tissues with low tissue factor levels, such as joints and muscles, the amount of FXa produced by fva-TF is insufficient. Thus, the amplification provided by the FIXa-FVIIIa complex is critical for effective hemostasis

In contrast to coagulation factors such as FVIII and FIX, protein S is an anticoagulant because it acts as a cofactor for activating protein C and Tissue Factor Pathway Inhibitor (TFPI). TFPI alpha is a poor FXa inhibitor in the absence of protein S. Similarly, in the absence of protein S, APC is also less effective in inhibiting FVa and FVIIIa. Thus, mutations in the loss of protein S function lead to uncontrolled clotting in mice and humans. Nevertheless, the inventors have surprisingly found that reducing the expression of protein S with nucleic acids can be a useful treatment for bleeding disorders such as hemophilia.

Current hemophilia treatments involve treatment with replacement factors on demand or in prophylaxis to prevent bleeding and maintain joint health. However, replacement therapy may be affected by developing alloantibodies to FVIII and FIX. These occur in-25 to 40% of severe hemophilia patients. These patients require treatment with bypass agents and induction of immune tolerance to eradicate the inhibitors (Weyand and Pipe 2019).

Thus, there is a clear need in the art for new methods of treating bleeding disorders such as hemophilia. The present invention addresses this need.

Disclosure of Invention

One aspect of the invention is a double stranded nucleic acid for inhibiting expression of PROS1, wherein the nucleic acid comprises a first strand and a second strand, wherein the first strand sequence comprises a sequence that differs by NO more than 15 nucleotides from any of the sequences selected from the group consisting of SEQ ID NO 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 255, 19, 15, 1, 3, 5, 7, 9, 11, 13, 17, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 and 49.

One aspect relates to a double stranded nucleic acid capable of inhibiting expression of PROS1, in particular in a cell, for use as a medicament or for use in a related diagnostic or therapeutic method, wherein the nucleic acid comprises or consists of in particular a first strand and a second strand, and in particular wherein the first strand comprises a sequence sufficiently complementary to a PROS1 mRNA to mediate RNA interference.

One aspect relates to compositions comprising a nucleic acid as disclosed herein and a solvent (particularly water) and/or a delivery vehicle and/or a physiologically acceptable excipient and/or carrier and/or salt and/or diluent and/or buffer and/or preservative.

One aspect relates to a composition comprising a nucleic acid as disclosed herein and an additional therapeutic agent selected from, for example, oligonucleotides, small molecules, monoclonal antibodies, polyclonal antibodies, and peptides.

One aspect relates to a nucleic acid or composition disclosed herein for use as a medicament or in a related method.

One aspect relates to a nucleic acid as disclosed herein or a composition comprising the same for use in preventing a bleeding disorder, reducing the risk of suffering from a bleeding disorder, or treating a bleeding disorder.

One aspect relates to the use of a nucleic acid as disclosed herein or a composition comprising the same, in preventing a bleeding disorder, reducing the risk of suffering from a bleeding disorder, or treating a bleeding disorder. The bleeding disorders are in particular coagulation deficiency disorders. The coagulation-deficient condition may be a condition associated with prolonged bleeding events and/or reduced thrombin and/or deficient clot formation. Bleeding disorders in particular hemophilia, hereditary hemophilia, hemophilia a, hemophilia B, hemophilia C, von willebrand's disease, von willebrand syndrome, fibrinogen free disorder, hypofibrinogenemia, parahaemophilia, arthritic blood (AH), clotting factor deficiency, factor II, V, VII, X and/or XI hereditary deficiency, factor V and VIII combined deficiency, acquired hemophilia, acquired clotting factor deficiency and acquired bleeding disorder. More particularly, it is hemophilia, in particular hemophilia a or B, most particularly hemophilia a.

One aspect relates to a method of preventing, reducing the risk of developing, or treating a bleeding disorder, comprising administering to a subject in need of treatment a pharmaceutically effective dose or amount of a nucleic acid disclosed herein or a composition comprising the same, particularly wherein the nucleic acid or composition is administered to the subject by subcutaneous, intravenous or oral, rectal, pulmonary, intramuscular, or intraperitoneal administration.

Detailed Description

The present invention relates to a nucleic acid and compositions thereof that are double stranded and comprise sequences that are homologous and/or complementary to a portion of an expressed RNA transcript of PROS 1. These nucleic acids, or conjugates thereof, or combinations thereof, may be used to treat and prevent bleeding disorders.

One aspect of the invention is a double stranded nucleic acid for inhibiting expression of PROS1, particularly in a cell, wherein the nucleic acid comprises a first strand and a second strand, wherein the first strand sequence comprises a sequence that differs by NO more than 15 nucleotides from any of the sequences selected from the group consisting of SEQ ID NOs 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 255, 19, 15, 1, 3, 5, 7, 9, 11, 13, 17, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 and 49. These nucleic acids have the advantage of being active and/or of having little or no associated off-target effects in a variety of species associated with preclinical and clinical development. Little to no associated off-target effect means that the nucleic acid specifically inhibits the intended target without significantly inhibiting other genes or inhibiting only one or a few other genes at a therapeutically acceptable level.

In particular, the first strand sequence comprises, or consists essentially of, a sequence differing by NO more than 3 nucleotides, in particular NO more than 2 nucleotides, more in particular NO more than 1 nucleotide, most in particular NO nucleotide difference from any of the sequences selected from the group consisting of SEQ ID NOs 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 255, 19, 15, 1, 3, 5, 7, 9, 11, 13, 17, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 and 49.

In particular, the first strand sequence of the nucleic acid consists of one of the sequences selected from the group consisting of SEQ ID NO 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 255, 19, 15, 1, 3, 5, 7, 9, 11, 13, 17, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 and 49. However, the sequence may be modified by some nucleic acid modification that does not alter the nucleotide properties. For example, modification of the backbone or sugar residues of a nucleic acid does not alter the properties of the nucleotide, as the base itself remains the same as in the reference sequence.

A nucleic acid comprising a reference sequence according to the present disclosure means that the nucleic acid comprises a sequence of consecutive nucleotides in the order defined in the reference sequence.

When reference is made herein to a sequence comprising or consisting of a number of nucleotides not shown to be modified in the sequence, the reference also encompasses the same nucleotide sequence, wherein one, several, e.g. two, three, four, five, six, seven or more, including all, of the nucleotides are modified by modification, e.g. 2'-OMe, 2' -F, linked to a ligand or linker, with 3 'or 5' end modification or any other modification. It also refers to sequences in which two or more nucleotides are linked to each other by a natural phosphodiester linkage or by any other linkage such as phosphorothioate or phosphorodithioate linkages.

A double-stranded nucleic acid is a nucleic acid in which the first strand and the second strand hybridize to each other over at least part of their length, and are therefore capable of forming a double-stranded region under physiological conditions, for example in PBS at 37℃with a concentration of 1. Mu.M for each strand. The first and second strand are in particular capable of hybridizing to one another and thus form a double-stranded region over a region of at least 15 nucleotides, in particular 16, 17, 18 or 19 nucleotides. The double-stranded region comprises nucleotide base pairing between the two strands, particularly based on Watson-Crick base pairing and/or wobble base pairing (e.g., GU base pairing). All nucleotides of the two strands within the double-stranded region do not have to base pair with each other to form the double-stranded region. A number of mismatches, deletions or insertions between the nucleotide sequences of the two strands are acceptable. An overhang on either end of the first or second strand or unpaired nucleotide on either end of the double stranded nucleic acid is also possible. The double-stranded nucleic acid is particularly a stable double-stranded nucleic acid under physiological conditions, and particularly has a melting temperature (Tm) of 45 ℃ or more, 50 ℃ or more, 55 ℃ or more, 60 ℃ or more, 65 ℃ or more, 70 ℃ or more, 75 ℃ or more, 80 ℃ or more, or 85 ℃ or more, for example in PBS with a concentration of 1 μm per strand.

The first strand and the second strand are in particular capable of forming a double-stranded region (i.e. complementary to each other) over at least part of their length, in particular 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 strand and the second strand. Strands that are complementary to each other over a length means that the strands are capable of base pairing with each other over that length by Watson-Crick or wobble base pairing. Each nucleotide of this length need not necessarily be capable of base pairing with the counterpart in the other strand throughout the given length, so long as a stable double-stranded nucleotide can be formed under physiological conditions. However, in certain embodiments, it is preferred if each nucleotide of that length can base pair with the counterpart in the other strand over the given length.

A certain number of mismatches, deletions or insertions between the first strand and the target sequence, or between the first strand and the second strand, may be tolerated in the case of siRNA, even in some cases have the potential to increase the activity of RNA interference (e.g., inhibition).

The inhibitory activity of the nucleic acids of the invention depends on the formation of a double-stranded region between all or part of the first strand and a portion of the target nucleic acid. The portion of the target nucleic acid that forms a double-stranded region with the first strand, defined as starting at the first base pair formed between the first strand and the target sequence and ending at the last base pair formed between the first strand and the target sequence, inclusive, is the target nucleic acid sequence or simply the target sequence. The duplex region formed between the first strand and the second strand need not be identical to 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, at least under physiological conditions, the first strand must be able to form a double-stranded structure with both the second strand and the target sequence.

The complementarity between the first strand and the target sequence may be complete (i.e., 100% identity with no nucleotide mismatches or insertions or deletions in the first strand as compared to the target sequence).

The complementarity between the first strand and the target sequence may not be complete. Complementarity may be about 70% to about 100%. More specifically, complementarity may be at least 70%, 80%, 85%, 90% or 95% and intermediate values.

The identity between the first strand and the complement of the target sequence may be in the range of about 75% to about 100%. More specifically, complementarity may be at least 75%, 80%, 85%, 90%, or 95% and intermediate, provided that the nucleic acid is capable of reducing or inhibiting expression of PROS 1.

A nucleic acid having less than 100% complementarity between the first strand and the target sequence may be capable of reducing expression of PROS1 to the same level as a nucleic acid having complete complementarity between the first strand and the target sequence. Alternatively, it may be capable of reducing expression of PROS1 to a level of 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of the reduced level achieved by the fully complementary nucleic acid.

In one aspect, the nucleic acid of the present disclosure is a nucleic acid, wherein

(a) The first strand sequence comprises a sequence that differs from any one of the first strand sequences of table 1 by no more than 3 nucleotides, and optionally wherein the second strand sequence comprises a sequence that differs from a second strand sequence of the same row of the table by no more than 3 nucleotides;

(b) The first strand sequence comprises a sequence that differs from any one of the first strand sequences of table 1 by no more than 2 nucleotides, and optionally wherein the second strand sequence comprises a sequence that differs from a second strand sequence of the same row of the table by no more than 2 nucleotides;

(c) The first strand sequence comprises a sequence that differs from any one of the first strand sequences of table 1 by no more than 1 nucleotide, and optionally wherein the second strand sequence comprises a sequence that differs from a second strand sequence of the same row of the table by no more than 1 nucleotide;

(d) The first strand sequence comprises a sequence corresponding to nucleotides 2 to 17 from the 5 'end of any one of the first strand sequences of table 1, and optionally wherein the second strand sequence comprises a sequence corresponding to nucleotides 2 to 17 from the 5' end of the second strand sequence of the same row of the table;

(e) The first strand sequence comprises a sequence corresponding to nucleotides 2 to 18 from the 5 'end of any one of the first strand sequences of table 1, and optionally wherein the second strand sequence comprises a sequence corresponding to nucleotides 2 to 18 from the 5' end of the second strand sequence of the same row of the table;

(f) The first strand sequence comprises a sequence corresponding to nucleotides 2 to 19 from the 5 'end of any one of the first strand sequences of table 1, and optionally wherein the second strand sequence comprises a sequence corresponding to nucleotides 2 to 19 from the 5' end of the second strand sequence of the same row of the table;

(g) The first strand sequence comprises a sequence corresponding to nucleotides 2 to 19 from the 5 'end of any one of the first strand sequences of table 1, and optionally wherein the second strand sequence comprises a sequence corresponding to nucleotides 1 to 18 from the 5' end of the second strand sequence of the same row of the table;

(h) The first strand sequence comprises the sequence of any one of the first strand sequences of table 1, and optionally wherein the second strand sequence comprises the sequence of the second strand sequence of the same row of the table; or (b)

(i) The first strand sequence consists of the sequence of any one of the first strand sequences of table 1, and optionally wherein the second strand sequence consists of the sequence of the second strand sequence of the same row of the table;

wherein, table 1 is:

TABLE 1

In one aspect, the nucleic acid is a nucleic acid, wherein:

(a) The first strand sequence comprises the sequence of SEQ ID NO. 209, and optionally wherein the second strand sequence comprises the sequence of SEQ ID NO. 210;

(b) The first strand sequence comprises the sequence of SEQ ID NO. 229, and optionally wherein the second strand sequence comprises the sequence of SEQ ID NO. 230;

(c) The first strand sequence comprises the sequence of SEQ ID NO:199, and optionally wherein the second strand sequence comprises the sequence of SEQ ID NO: 200; or (b)

(d) The first strand sequence comprises the sequence of SEQ ID NO. 203, and optionally wherein the second strand sequence comprises the sequence of SEQ ID NO. 204.

In one aspect, if the most 5' nucleotide of the first strand is a nucleotide other than a or U, then that nucleotide is replaced with a or U. In particular, in the sequence, if the most 5 'nucleotide of the first strand is a nucleotide other than U, then that nucleotide is replaced by U, more particularly by U with a 5' (E) -vinyl phosphonate.

In one aspect, there is a mismatch between the first nucleotide at the 5' end of the first strand and the corresponding nucleotide in the second strand (the nucleotide with which the base pair is formed if there is no mismatch). For example, the 5' nucleotide of the first strand may be U and the corresponding nucleotide in the second strand may be any nucleotide other than a. In this case, two nucleotides cannot form classical Watson-Crick base pairs, and there is a mismatch between the two nucleotides.

When a nucleic acid of the invention does not comprise the complete sequence of the reference first strand and/or second strand sequences, e.g. as given in table 1, or one or both strands differ from the corresponding reference sequence by one, two or three nucleotides, the nucleic acid particularly retains at least 30%, more particularly at least 50%, more particularly at least 70%, more particularly at least 80%, even more particularly at least 90%, still more particularly at least 95% and most particularly 100% of the inhibition activity of the PROS1 compared to the inhibition activity of the corresponding nucleic acid comprising the complete first strand and second strand reference sequences in a comparable experiment.

In one aspect, the nucleic acid is a nucleic acid, wherein the first strand sequence comprises or in particular consists of the sequence of SEQ ID No. 209, and optionally wherein the second strand sequence comprises or in particular consists of the sequence of at least 15, in particular at least 16, more in particular at least 17, still more in particular at least 18, and most in particular all nucleotides of the sequence of SEQ ID No. 210; or wherein the first strand sequence comprises or in particular consists of the sequence of SEQ ID No. 229, and optionally wherein the second strand sequence comprises or in particular consists of the sequence of at least 15, in particular at least 16, more in particular at least 17, still more in particular at least 18, and most in particular all nucleotides of the sequence of SEQ ID No. 230; or wherein the first strand sequence comprises or in particular consists of the sequence of SEQ ID NO:199, and optionally wherein the second strand sequence comprises or in particular consists of the sequence of at least 15, in particular at least 16, more in particular at least 17, still more in particular at least 18, and most in particular all nucleotides of the sequence of SEQ ID NO: 200; or wherein the first strand sequence comprises or in particular consists of the sequence of SEQ ID NO. 203, and optionally wherein the second strand sequence comprises or in particular consists of the sequence of at least 15, in particular at least 16, more in particular at least 17, still more in particular at least 18, and most in particular all nucleotides of the sequence of SEQ ID NO. 204.

In one aspect, the nucleic acid is a double-stranded nucleic acid for inhibiting expression of PROS1, particularly in a cell, wherein the nucleic acid comprises a first nucleic acid strand and a second nucleic acid strand, wherein the first strand is capable of hybridizing under physiological conditions to a nucleic acid selected from the group consisting of sequences of SEQ ID NOs 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 20, 16, 2, 4, 6, 8, 10, 12, 14, 18, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 and 50; and is also provided with

Wherein the second strand is capable of hybridizing to the first strand under physiological conditions to form a double-stranded region.

Nucleic acids capable of hybridizing under physiological conditions are nucleic acids capable of forming base pairs, particularly Watson-Crick or wobble base pairs, between at least a portion of the opposing nucleotides in a strand so as to form at least one double-stranded region. Such double stranded nucleic acids are in particular double stranded nucleic acids which are stable under physiological conditions (e.g.in PBS at 37℃with a concentration of 1. Mu.M for each strand), which means that under such conditions the two strands remain hybridized to each other. The Tm of the double-stranded nucleotide is particularly 45℃or more, particularly 50℃or more, more particularly 55℃or more.

One aspect of the invention relates to a nucleic acid for inhibiting expression of PROS1, in particular in a cell, wherein the nucleic acid comprises a first sequence of at least 15, in particular at least 16, more in particular at least 17, still more in particular at least 18, most in particular all nucleotides differing from any of the sequences of Table 4 by no more than 3 nucleotides, in particular no more than 2 nucleotides, more in particular no more than 1 nucleotide, most in particular no more than any nucleotide, which is capable of hybridizing under physiological conditions to a target gene transcript (e.g.mRNA). In particular, the nucleic acid further comprises a second sequence of at least 15, in particular at least 16, more in particular at least 17, still more in particular at least 18, most in particular all nucleotides differing from any of the sequences of table 4 by no more than 3 nucleotides, in particular no more than 2 nucleotides, more in particular no more than 1 nucleotide, most in particular no difference from any of the nucleotides, wherein the second sequence is capable of hybridizing to the first sequence under physiological conditions, in particular wherein the nucleic acid is an siRNA capable of inhibiting expression of PROS1 via an RNAi pathway.

One aspect relates to any double-stranded nucleic acid disclosed in table 2, provided that the double-stranded nucleic acid is capable of inhibiting PROS1 expression. These nucleic acids are all siRNAs with various nucleotide modifications. Some of these are conjugates comprising GalNAc moieties that can specifically target cells, such as hepatocytes, that have GalNAc receptors.

The nucleic acids described herein may be capable of inhibiting expression of PROS1, particularly in cells. The nucleic acid may be capable of completely inhibiting PROS1 expression, resulting in a residual expression of 0% after treatment with the nucleic acid. The nucleic acid may be capable of partially inhibiting PROS1 expression. Partial inhibition refers to a 15%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more reduction in PROS1 expression, or an intermediate value, as compared to the absence of nucleic acid under comparable conditions. The level of inhibition can be measured by comparing the treated sample to an untreated sample or to a control treated sample, e.g., siRNA that does not target PROS 1. Inhibition may be measured by measuring the levels of PROS1 mRNA and/or protein or the levels of biomarkers or indicators associated with the presence or activity of protein S. It can be measured in cells that have been treated in vitro with the nucleic acids described herein. Alternatively, or in addition, inhibition may be measured in cells such as hepatocytes, or tissues such as liver tissue, or organs such as liver, or in bodily fluids such as blood, serum, stranguria, or in any other body part or bodily fluid obtained from a subject previously treated with a nucleic acid disclosed herein. In particular, inhibition of PROS1 expression is determined by comparing the levels of PROS1 mRNA measured in a cell expressing PROS1 after in vitro treatment with the double stranded RNA disclosed herein under ideal conditions (see examples of suitable concentrations and conditions) for 24 or 48 hours with the levels of PROS1 mRNA measured in control cells untreated or mock-treated or treated with control double stranded RNA under the same or at least comparable conditions.

One aspect of the invention relates to a nucleic acid, wherein the first strand and the second strand are present on a single strand of the nucleic acid, the nucleic acid being looped such that the first strand and the second strand are capable of hybridizing to each other, thereby forming a double-stranded nucleic acid having a double-stranded region.

In particular, the first strand and the second strand of the nucleic acid are separate strands. The two separate strands are in particular each 17 to 25 nucleotides long, more in particular 18 to 25 nucleotides long. The two strands may have the same or different lengths. The first strand may be 17-25 nucleotides in length, in particular it may be 18-24 nucleotides in length, it may be 18, 19, 20, 21, 22, 23 or 24 nucleotides in length. Most particularly, the first strand is 19 nucleotides long. The second strand may independently be 17-25 nucleotides in length, in particular it may be 18-24 nucleotides in length, which may be 18, 19, 20, 21, 22, 23 or 24 nucleotides in length. More particularly, the second strand is 18 or 19 or 20 nucleotides in length, and most particularly 19 nucleotides in length.

In particular, the first strand and the second strand of the nucleic acid form a double-stranded region of 17-25 nucleotides in length. More particularly, the double stranded region is 18-24 nucleotides in length. The double stranded region may be 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. In the most particular embodiment, the double stranded region is 18 or 19 nucleotides in length. A double-stranded region is defined herein as the region between and including the region from the most 5 'nucleotide of the first strand that base pairs with the nucleotide of the second strand to the most 3' nucleotide of the first strand that base pairs with the nucleotide of the second strand. . The double-stranded region may include nucleotides in either or both strands that do not base pair with nucleotides in the other strand. It may comprise one, two, three or four such nucleotides on the first strand and/or the second strand. However, in particular, the double-stranded region consists of 17-25 consecutive nucleotide base pairs. That is, it comprises, in particular, 17-25 consecutive nucleotides on both strands, all bases being paired with nucleotides in the other strand. More particularly, the double stranded region consists of 18 or 19 consecutive nucleotide base pairs, most particularly 18 consecutive nucleotide base pairs.

In each of the embodiments disclosed herein, the nucleic acid may be blunt at both ends; having a protruding end at one end and a flat end at the other end; or have protruding ends at both ends.

The nucleic acid may have an overhang at one end and a blunt end at the other end. The nucleic acid may have overhangs at both ends. The nucleic acid may be blunt at both ends. The nucleic acid may be blunt-ended at the 5 'end of the first strand and the 3' end of the second strand, or blunt-ended at the 3 'end of the first strand and the 5' end of the second strand.

The nucleic acid may include an overhang at the 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 overhangs at both the 5 'and 3' ends of the first strand. The nucleic acid may have overhangs at both the 5 'and 3' ends 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.

The overhang at the 3 'end or the 5' end of the second strand or the first strand may consist of lengths of 1, 2, 3, 4 and 5 nucleotides. Alternatively, the overhang may consist of 1 or 2 nucleotides, which may or may not be modified.

In one embodiment, the 5' end of the first strand is a single stranded overhang of one, two or three nucleotides, in particular one nucleotide.

In particular, the nucleic acid is an siRNA. siRNA is a short interfering or short silencing RNA that is capable of inhibiting the expression of a target gene through an RNA interference (RNAi) pathway. Inhibition occurs through targeted degradation of mRNA transcripts of the target gene after transcription. The siRNA forms part of RISC complex. RISC complexes specifically target RNA through sequence complementarity of the first (antisense) strand to the target sequence.

In particular, the nucleic acid is capable of inhibiting PROS1. The inhibition is mediated in particular by an RNA interference (RNAi) mechanism. In particular, the nucleic acid mediates RNA interference (i.e., it is capable of inhibiting its target) with an efficacy of at least 50% inhibition, more particularly at least 70%, more particularly at least 80%, even more particularly at least 90%, still more particularly at least 95%, most particularly 100% inhibition. In comparable experiments, inhibition efficacy is measured in particular by comparing the levels of PROS1 mRNA in cells treated with PROS 1-specific siRNA, such as hepatocytes, and in cells treated with controls. The control may be a treatment with or without a non-PROS 1 targeted siRNA. Thus, the nucleic acid or at least the first strand of the nucleic acid can in particular be integrated into the RISC complex. As a result, the nucleic acid, or at least the first strand of the nucleic acid, is thus able to direct the RISC complex to a specific target RNA; in turn, the nucleic acid, or at least the first strand of the nucleic acid, is at least partially complementary to the target RNA. The RISC complex then specifically cleaves the target RNA, resulting in inhibition of expression of the gene from which the RNA is derived.

A particularly preferred embodiment is a nucleic acid, wherein the first strand comprises or consists of SEQ ID NO. 233 and the second strand optionally comprises or consists of SEQ ID NO. 256. The nucleic acid may be further conjugated to a ligand. Even more preferred is a nucleic acid wherein the first strand comprises or consists of SEQ ID NO:233 and the second strand optionally comprises the sequence of SEQ ID NO:234 or consist thereof. Most preferred in this case is the sequence represented by SEQ ID NO:233 and SEQ ID NO:234, and a siRNA consisting of 234. One aspect of the invention is EU161.

Another particularly preferred embodiment is a nucleic acid wherein the first strand comprises SEQ ID NO:237 or consists of SEQ ID NO:257 or consist of the same. The nucleic acid may be further conjugated to a ligand. Even more preferred is a nucleic acid wherein the first strand comprises SEQ ID NO:237 or consists of SEQ ID NO:238 or consist thereof. Most preferred in this case is the sequence represented by SEQ ID NO:237 and SEQ ID NO: 238. One aspect of the invention is EU163.

Another particularly preferred embodiment is a nucleic acid wherein the first strand comprises SEQ ID NO:251 or consists of, and the second strand optionally comprises SEQ ID NO:258 or consist thereof. The nucleic acid may be further conjugated to a ligand. Even more preferred is a nucleic acid wherein the first strand comprises SEQ ID NO:251 or consists of, and the second strand optionally comprises SEQ ID NO:252 or consist thereof. Most preferred in this case is the sequence represented by SEQ ID NO:251 and SEQ ID NO: 252. One aspect of the invention is EU170.

One aspect of the invention relates to protein S inhibitors, such as siRNA, antibodies, small molecules, peptides, proteins or any other agent that reduces the level of protein S in the blood or blocks its activity, for use in the treatment of blood disorders, in particular hemophilia. In particular, protein S inhibitors are useful for inhibiting human protein S, and in particular for treating human subjects in need thereof.

Nucleic acid modification

The nucleic acids discussed herein comprise unmodified RNA and RNA that has been modified (e.g., to improve efficacy or stability). Unmodified RNA refers to molecules in which the components of the nucleic acid, i.e., sugar, base, and phosphate moieties, are identical or substantially identical to those that occur naturally (e.g., those that occur naturally in humans). The term "modified nucleotide" as used herein refers to a nucleotide in which one or more components of the nucleotide, i.e., sugar, base, and phosphate moieties, are different from those that occur naturally. The term "modified nucleotides" also refers in some cases to molecules that are not nucleotides in the strict sense of the term, as they lack essential components of nucleotides such as sugar, base or phosphate moieties or have substitutes therefor. A nucleic acid comprising such modified nucleotides is still to be understood as a nucleic acid, even if one or more of the nucleotides of the nucleic acid have been replaced by a modified nucleotide, which modified nucleotide lacks the essential components of the nucleotide or has a substitute therefor.

Modification of the nucleic acids of the invention generally provides a powerful tool to overcome potential limitations, including but not limited to the inherent in vitro and in vivo stability and bioavailability of natural RNA molecules. The nucleic acids of the invention may be modified by chemical modification. The modified nucleic acid may also minimize the possibility of inducing interferon activity in humans. The modification may further enhance functional delivery of the nucleic acid to the target cell. The modified nucleic acids of the invention may include one or more chemically modified ribonucleotides of either the first strand or the second strand or both. Ribonucleotides may include chemical modification of base, sugar or phosphate moieties. Ribonucleic acids may be modified by substitution or insertion with nucleic acids or analogs of bases.

Throughout the present specification, "identical or co-modified" refers to the same modification to any nucleotide, i.e. A, G, C or U is modified with groups such as methyl (2 '-OMe) or fluoro (2' -F). For example, 2'-F-dU, 2' -F-dA, 2'-F-dC, 2' -F-dG are all considered identical or co-modified, while 2'-OMe-rU, 2' -OMe-rA;2' -OMe-rC; the same is true for 2' -OMe-rG. In contrast, the 2'-F modification is a different modification compared to the 2' -OMe modification.

In particular, at least one nucleotide of the first and/or second strand of the nucleic acid is a modified nucleotide, in particular a non-naturally occurring nucleotide, such as in particular a 2' -F modified nucleotide.

The modified nucleotide may be a glycosyl modified nucleotide. The 2' hydroxyl (OH) group may be modified or replaced with a number of different "oxy" or "deoxy" substituents.

Examples of "oxy" -2' hydroxyl modifications include alkoxy OR aryloxy (OR, e.g., r=h, alkyl (e.g., methyl), cycloalkyl, aryl, aralkyl, heteroaryl, OR sugar); polyethylene glycol (PEG), O (CH) ₂ CH ₂ O) _n CH ₂ CH ₂ OR; "locked" nucleic acids (LNA) in which the 2' hydroxyl group is 4' to the same ribose, e.g.via a methylene bridge 'Carbon linkage; o-(Alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino or diheteroarylamino, ethylenediamine or polyamino) and aminoalkoxy,/->(e.g.)>Alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino or diheteroarylamino, ethylenediamine or polyamino).

"deoxidizing" modifications include hydrogen, halogen, amino (e.g., NH) ₂ An alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); (Alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino), -NHC (O) R (r=alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar), cyano; a mercapto group; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl, and alkynyl groups, which may be optionally substituted with, for example, amino functional groups. Other substituents of certain embodiments include 2 '-methoxyethyl, 2' -OCH ₃ 2' -O-allyl, 2' -C-allyl and 2' -fluoro.

The glycosyl group may also contain one or more carbons having a stereochemical configuration opposite to that of the corresponding carbon in ribose. Thus, the modified nucleotide may contain a sugar, such as arabinose.

Modified nucleotides may also contain "abasic" sugars, which lack nucleobases at C-1'. These abasic sugars may further contain modifications on one or more of the constituent sugar atoms.

The 2' modification may be used in combination with one or more phosphate internucleoside modifications (e.g., phosphorothioate or phosphorodithioate).

One or more nucleotides of the nucleic acids of the invention may be modified. The nucleic acid may comprise at least one modified nucleotide. The modified nucleotide may be in the first strand. The modified nucleotide may be in the second strand. The modified nucleotide may be in a double stranded region. The modified nucleotide may be outside the double-stranded region, i.e., in the single-stranded region. The modified nucleotide may be on the first strand and may be outside the double-stranded region. The modified nucleotide may be on the second strand and may be outside the double stranded 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' end nucleotide of the first strand may be a modified nucleotide. The 5' end nucleotide of the second strand may be a modified nucleotide.

The nucleic acid of the invention may have 1 modified nucleotide, or the nucleic acid of the invention may have about 2-4 modified nucleotides, or the 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 the modified nucleotide retains at least 50% of its activity compared to the same nucleic acid without the modified nucleotide, and vice versa. The nucleic acid may retain 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of its activity and intermediate values compared to the same nucleic acid without the modified nucleotide, or may have more than 100% of the activity of the same nucleic acid without the modified nucleotide.

The modified nucleotide may be a purine or pyrimidine. At least half of the purines may be modified. At least half of the pyrimidines may be modified. All purines can be modified. All pyrimidines may be modified. The modified nucleotide may be selected from the group consisting of a 3 'deoxythymine (dT) nucleotide, a 2' -O-methyl (2 '-OMe) modified nucleotide, a 2' deoxymodified nucleotide, a locked 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, an phosphoramidate, a non-natural base including a nucleotide, a nucleotide including a 5' -phosphorothioate group, a nucleotide including a 5 '-phosphate or a 5' -phosphate mimetic, and a terminal nucleotide attached to a cholesteryl derivative or a didecyl dodecanoamide group.

The nucleic acid may comprise a nucleotide comprising a modified base, wherein the base is selected from the group consisting of 2-aminoadenosine, 2, 6-diaminopurine, inosine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4, 6-trimethoxybenzene, 3-methyluracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidine (e.g., 5-methylcytidine), 5-alkyluridine (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine), 6-azapyrimidine, 6-alkylpyrimidine (e.g., 6-methyluridine), propyne, braided glycoside, 2-thiouridine, 4-thiouridine, huai Dinggan, huai Dingyang nucleoside, 4-acetylcytidine, 5- (carboxymethyl) uridine, 5' -carboxymethyl-2-thiouridine, 5-carboxymethyl aminomethyluridine, beta-D-galactosyl uridine, 1-methyladenosine, 1-methyl inosine, 2-dimethyl guanosine, 3-methylcytidine, 2-methyl adenosine, 2-methyl guanosine, N-methyl guanosine, 6-methyl guanosine, 5-methoxymethyl-5-oxo-guanosine, 5-methyl-2-thiouridine, 5-oxo-methylguanosine, 5-D-methylguanosine, 5-methyl-2-thiouridine, 5-D-methylguanosine, uridine-5-oxyacetic acid and 2-thiocytidine.

Many of the modifications described herein and occurring within a nucleic acid will be repeated within a polynucleotide molecule, such as modification of a base or phosphate moiety, or non-linking O of a phosphate moiety. In some cases, the modification will occur at all possible sites/nucleotides in the polynucleotide, but in many cases it will not occur. Modification may occur only at the 3 'or 5' end position, may occur only in the terminal region, for example at a position on the terminal nucleotide or in the last 2, 3, 4, 5 or 10 nucleotides of the strand. Modification may occur in the double-stranded region, the single-stranded region, or both. The modification may occur only in the double-stranded region of the nucleic acid of the invention, or may occur only in the single-stranded region of the nucleic acid of the invention. Phosphorothioate or phosphorodithioate modifications at non-linked O positions may occur only at one or both ends, may occur only in the terminal region, for example, at a position on a terminal nucleotide or in the last 2, 3, 4 or 5 nucleotides of a strand, or may occur in a duplex and/or in a single stranded region, particularly at the ends. The 5 'and/or 3' ends may be phosphorylated.

The stability of the nucleic acids of the invention may be increased by including specific bases at the overhangs, or by including modified nucleotides at single stranded overhangs, such as at the 5 'or 3' overhangs, or both. Purine nucleotides may be included in the overhangs. All or some of the bases in the 3 'or 5' overhangs may be modified. Modifications may include modifications on the 2' oh group using ribose, the use of deoxyribonucleotides instead of ribonucleotides, and modifications on the phosphate group, such as phosphorothioate or phosphorodithioate modifications. The overhangs need not be homologous to the target sequence.

The nuclease can hydrolyze the nucleic acid phosphodiester bond. However, chemical modifications to nucleic acids may impart improved properties and may make oligoribonucleotides more stable to nucleases.

As used herein, a modified nucleic acid may comprise one or more of the following:

(i) Changes, such as substitution of one or two non-linked phosphate oxygens and/or one or more linked phosphate oxygens (meaning linked, even at the 5 'and 3' ends of the nucleic acids of the invention);

(ii) Alterations such as substitution of ribose moiety, e.g., 2' hydroxyl on ribose;

(iii) Replacing the phosphate moiety with a "dephosphorylation" linker;

(iv) Modification or substitution of naturally occurring bases;

(v) Substitution or modification of the ribose-phosphate backbone; and

(vi) Modification of the 3 'or 5' end of the first and/or second strand, e.g., removal, modification or substitution of terminal phosphate groups, or conjugation of a moiety, e.g., a fluorescently labeled moiety, to the 3 'or 5' end of one or both strands.

The terms substitution, modification and alteration mean a difference from a naturally occurring molecule.

Specific modifications are discussed in more detail below.

The nucleic acid may comprise one or more modified nucleotides on the second and/or first strand. Alternate nucleotides may be modified to form modified nucleotides.

Alternating as described herein means that it occurs one after the other in a conventional manner. In other words, alternating means that it occurs repeatedly in sequence. For example, if one nucleotide is modified, the next consecutive nucleotide is not modified, and the next consecutive nucleotide is modified again, and so on. One nucleotide may be modified with a first modification, the next consecutive nucleotide may be modified with a second modification, and subsequent consecutive nucleotides may be modified with the first modification, and so on, wherein the first modification and the second modification are different.

Some representative modified nucleic acid sequences of the invention are shown in the examples. These examples are representative and not limiting.

In one aspect of the nucleic acid, at least positions 2 and 14 of the first strand are modified, in particular by a first common modification, the nucleotides being numbered consecutively starting from nucleotide number 1 at the 5' end of the first strand. The first modification is in particular 2' -F.

In one aspect, at least one, several or in particular all even numbered nucleotides of the first strand are modified, in particular by a first common modification, which nucleotides are consecutively numbered starting from nucleotide number 1 at the 5' end of the first strand. The first modification is in particular 2' -F.

In one aspect, at least one, several or in particular all odd numbered nucleotides of the first strand are modified, which nucleotides are numbered consecutively starting with nucleotide number 1 at the 5' end of the first strand. In particular, they are modified by a second modification. If the nucleic acid also comprises a first modification, for example of nucleotides 2 and 14 or of all even numbered nucleotides of the first strand, the second modification differs in particular from the first modification. The first modification is in particular any 2' -ribose modification, or Locked Nucleic Acid (LNA), or Unlocked Nucleic Acid (UNA), or 2' -fluoroarabi nucleic acid (FANA) modification of the same size or smaller volume than the 2' -OH group. The 2 '-ribose modification of the same size or smaller volume than the 2' -OH group may be, for example, 2'-F, 2' -H, 2 '-halogen, or 2' -NH ₂ . The second modification is in particular any 2 'ribose modification which is bulky more than the 2' -OH group. The 2' ribose modification which is bulky more than the 2' -OH group may be, for example, 2' -OMe, 2' -O-MOE (2 ' -O-methoxyethyl), 2' -O-allyl or 2' -O-alkyl, provided that under comparable conditions the nucleic acid is capable of reducing expression of the target gene to at least the same extent as the same nucleic acid without the modification. The first modification is in particular 2'-F and/or the second modification is in particular 2' -OMe.

In the context of the present disclosure, the size or volume of the substituents such as 2' ribose modification is measured, in particular, in van der waals volumes.

In one aspect, at least one, several or in particular all nucleotides of the second strand corresponding to even numbered nucleotides of the first strand are modified, in particular by a third modification. In particular, in the same nucleic acid, the 2 nd and 14 th nucleotides or all even numbered nucleotides of the first strand are modified by the first modification. In addition, or alternatively, the odd nucleotides of the first strand are modified by a second modification. In particular, 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 in particular any 2' -ribose modification, or Locked Nucleic Acid (LNA), or Unlocked Nucleic Acid (UNA), or 2' -fluoroarabi nucleic acid (FANA) modification of the same size or smaller volume than the 2' -OH group. And 2' -OH group size The same or smaller 2' -ribose modification may be, for example, 2' -F, 2' -H, 2' -halogen, or 2' -NH ₂ . The second and/or third modification is in particular any 2 'ribose modification that is bulky larger than the 2' -OH group. The 2' ribose modification which is bulky more than the 2' -OH group may be, for example, 2' -OMe, 2' -O-MOE (2 ' -O-methoxyethyl), 2' -O-allyl or 2' -O-alkyl, provided that under comparable conditions the nucleic acid is capable of reducing expression of the target gene to at least the same extent as the same nucleic acid without the modification. The first modification is in particular 2'-F and/or the second and/or third modification is in particular 2' -OMe. Nucleotides on the first strand are numbered consecutively starting from nucleotide number 1 at the 5' end of the first strand.

The nucleotides in the second strand that correspond to, for example, the even-numbered nucleotides of the first strand are nucleotides of the second strand that base pair with the even-numbered nucleotides of the first strand.

In one aspect, at least one, several or in particular all nucleotides of the second strand corresponding to odd numbered nucleotides of the first strand are modified, in particular by a fourth modification. In particular, in the same nucleic acid, the 2 nd and 14 th nucleotides or all even numbered nucleotides of the first strand are modified by the first modification. In addition, or alternatively, the odd nucleotides of the first strand are modified by a second modification. In addition, or alternatively, all nucleotides of the second strand in positions corresponding to the even-numbered nucleotides of the first strand are third modified. The fourth modification is in particular different from the second modification, and in particular different from the third modification, and the fourth modification is in particular identical to the first modification. The first and/or fourth modification is in particular any 2' -ribose modification, or Locked Nucleic Acid (LNA), or Unlocked Nucleic Acid (UNA), or 2' -fluoroarabinoic acid (FANA) modification of the same size or smaller volume than the 2' -OH group. The 2 '-ribose modification of the same size or smaller volume than the 2' -OH group may be, for example, 2'-F, 2' -H, 2 '-halogen, or 2' -NH2. The second and/or third modification is in particular any 2 'ribose modification that is bulky larger than the 2' -OH group. The 2' ribose modification which is bulky more than the 2' -OH group may be, for example, 2' -OMe, 2' -O-MOE (2 ' -O-methoxyethyl), 2' -O-allyl or 2' -O-alkyl, provided that under comparable conditions the nucleic acid is capable of reducing expression of the target gene to at least the same extent as the same nucleic acid without the modification. The first and/or fourth modification is in particular a 2'-OMe modification, and/or the second and/or third modification is in particular a 2' -F modification. Nucleotides on the first strand are numbered consecutively starting from nucleotide number 1 at the 5' end of the first strand.

In one aspect of the nucleic acid, the nucleotide of the second strand corresponding to the 11 th nucleotide or 13 th nucleotide or 11 and 13 th nucleotides or 11-13 th nucleotides of the first strand is modified by a fourth modification. In particular, all nucleotides of the second strand except for the 11 th nucleotide or 13 th nucleotide or 11 and 13 th nucleotides or 11-13 th nucleotide positions of the first strand are modified by the third modification. In particular, in the same nucleic acid, the 2 nd and 14 th nucleotides or all even numbered nucleotides of the first strand are modified by the first modification. In addition, or alternatively, the odd nucleotides of the first strand are modified by a second modification. The fourth modification is in particular different from the second modification, and in particular different from the third modification, and the fourth modification is in particular identical to the first modification. The first and/or fourth modification is in particular any 2' -ribose modification, or Locked Nucleic Acid (LNA), or Unlocked Nucleic Acid (UNA), or 2' -fluoroarabinoic acid (FANA) modification of the same size or smaller volume than the 2' -OH group. The 2 '-ribose modification of the same size or smaller volume than the 2' -OH group may be, for example, 2'-F, 2' -H, 2 '-halogen, or 2' -NH2. The second and/or third modification is in particular any 2 'ribose modification that is bulky larger than the 2' -OH group. The 2' ribose modification which is bulky more than the 2' -OH group may be, for example, 2' -OMe, 2' -O-MOE (2 ' -O-methoxyethyl), 2' -O-allyl or 2' -O-alkyl, provided that under comparable conditions the nucleic acid is capable of reducing expression of the target gene to at least the same extent as the same nucleic acid without the modification. The first and/or fourth modification is in particular a 2'-OMe modification, and/or the second and/or third modification is in particular a 2' -F modification. Nucleotides on the first strand are numbered consecutively starting from nucleotide number 1 at the 5' end of the first strand.

In one aspect of the nucleic acid, all even-numbered nucleotides of the first strand are modified by a first modification, all odd-numbered nucleotides of the first strand are modified by a second modification, all nucleotides of the second strand at positions corresponding to the even-numbered nucleotides of the first strand are modified by a third modification, all nucleotides of the second strand at positions corresponding to the odd-numbered nucleotides of the first strand are modified by a fourth modification, wherein the first and/or fourth modification is 2'-F and/or the second and/or third modification is 2' -OMe.

In one aspect of the nucleic acid, all even numbered nucleotides of the first strand are modified by a first modification, all odd numbered nucleotides of the first strand are modified by a second modification, all nucleotides of the second strand corresponding to positions 11-13 of the first strand are modified by a fourth modification, all nucleotides of the second strand except for nucleotides corresponding to positions 11-13 of the first strand are modified by a third modification, wherein the first and fourth modifications are 2'-F and the second and third modifications are 2' -OMe. In one embodiment of this aspect, the 3 'end nucleotide of the second strand is an inverted RNA nucleotide (i.e., the nucleotide is attached to the 3' end of the strand through its 3 'carbon rather than, typically, through its 5' carbon). When the 3' end nucleotide of the second strand is an inverted RNA nucleotide, the inverted RNA nucleotide is particularly an unmodified nucleotide, since it does not include any modification compared to the natural nucleotide counterpart. Specifically, the inverted RNA nucleotide is particularly a 2' -OH nucleotide. In particular, in this aspect, when the 3 'terminal nucleotide of the second strand is an inverted RNA nucleotide, the nucleic acid is blunt-ended at least at the end comprising the 5' end of the first strand.

An aspect of the invention is a nucleic acid disclosed herein for inhibiting expression of the PROS1 gene, particularly in a cell, wherein the first strand comprises modified nucleotides or unmodified nucleotides at a plurality of positions to facilitate RISC processing of the nucleic acid.

In one aspect, "RISC-facilitated" means that the nucleic acid can be processed by RISC, e.g., any modification present will allow the nucleic acid to be processed by RISC, particularly in favor of RISC, suitably such that siRNA activity can occur.

One aspect is a nucleic acid as disclosed herein wherein the nucleotides at positions 2 and 14 from the 5 'end of the first strand are not modified by a 2' OMe modification, whereas the nucleotides at positions 11 or 13 or 11 and 13 or 11, 12 and 13 on the second strand corresponding to the first strand are not modified by a 2'-OMe modification (in other words, they are naturally occurring nucleotides or are modified by a modification other than 2' -OMe).

In one aspect, the nucleotide on the second strand that corresponds to position 13 of the first strand is a nucleotide that forms a base pair with position 13 (from the 5' end) of the first strand.

In one aspect, the nucleotide on the second strand that corresponds to position 11 of the first strand is a nucleotide that forms a base pair with position 11 (from the 5' end) of the first strand.

In one aspect, the nucleotide on the second strand that corresponds to position 12 of the first strand is a nucleotide that forms a base pair with position 12 (from the 5' end) of the first strand.

For example, in a double-stranded and blunt-ended 19-mer nucleic acid, position 13 of the first strand (from the 5 'end) will pair with position 7 of the second strand (from the 5' end). The 11 th (from the 5 'end) of the first strand will pair with the 9 th (from the 5' end) of the second strand. This nomenclature may be applied to other positions of the second strand.

In one aspect, in the case of partially complementary first and second strands, if the position on the second strand is a position where there is a mismatch, the nucleotides on the second strand that "correspond" to the position on the first strand may not necessarily form base pairs, but the nomenclature still applies.

One aspect is a nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand are not modified by a 2' -OMe modification, and the nucleotides on the second strand corresponding to positions 11, or 13, or 11 and 13, or 11-13 of the first strand are modified by a 2' -F modification.

One aspect is a nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand are modified by a 2' -F modification, and the nucleotides at positions 11, or 13, or 11 and 13, or 11-13 on the second strand corresponding to the first strand are not modified by a 2' -OMe modification.

One aspect is a nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand are modified by a 2' -F modification, and the nucleotides at positions 11, or 13, or 11 and 13, or 11-13 on the second strand corresponding to the first strand are modified by a 2' -F modification.

One aspect is a nucleic acid disclosed herein, wherein greater than 50% of the nucleotides of the first and/or second strand comprise a 2'-OMe modification, e.g., greater than 55%, 60%, 65%, 70%, 75%, 80%, or 85%, or more of the nucleotides of the first and/or second strand comprise a 2' -OMe modification.

One aspect is a nucleic acid disclosed herein, wherein greater than 50% of the nucleotides of the first and/or second strand comprise naturally occurring RNA modifications, e.g., wherein greater than 55%, 60%, 65%, 70%, 75%, 80%, or 85% or more of the first and/or second strand comprise such modifications. Suitable naturally occurring modifications include, as well as 2' -OMe, other 2' sugar modifications, particularly 2' -H modifications made at DNA nucleotides.

One aspect is a nucleic acid disclosed herein comprising no more than 20%, such as no more than 15%, such as no more than 10%, of 2 'modified nucleotides having a modification on the first and/or second strand that is not a 2' -OMe modification.

One aspect is a nucleic acid as disclosed herein, wherein the number of nucleotides in the first and/or second strand having a 2 '-modification that is not a 2' -OMe modification is not more than 7, more particularly not more than 5, and most particularly not more than 3.

One aspect is a nucleic acid disclosed herein that includes no more than 20% (e.g., no more than 15% or no more than 10%) of 2' -F modifications on the first and/or second strand.

An aspect is a nucleic acid as disclosed herein, wherein the number of nucleotides in the first and/or second strand having a 2' -F modification is no more than 7, more particularly no more than 5, and most particularly no more than 3.

One aspect is a nucleic acid as disclosed herein, wherein all nucleotides are modified by a 2'-OMe modification except for the nucleotides at positions 2 and 14 of the 5' end of the first strand and at positions 11, or 13, or 11 and 13, or 11-13 of the second strand corresponding to the first strand. In particular, nucleotides not modified by 2' -OMe are modified by fluorine at the 2' -position (2 ' -F modification).

In particular, all nucleotides of a nucleic acid are modified at the 2' position of the sugar. In particular, these nucleotides are modified by 2'-F, wherein the modification is not a 2' -OMe modification.

In one aspect, the nucleic acid is modified alternately by a 2' -OMe modification and a 2-F modification on the first strand, and the 2 nd and 14 th positions (starting from the 5' end) are modified by a 2' -F. In particular, the second strand is modified with 2' -F at the nucleotides on the second strand corresponding to positions 11, or 13, or 11 and 13, or 11-13 of the first strand. In particular, the second strand is modified at positions 11-13 counted from the 3' -end of the first position of the complementary (double stranded) region by a 2' -F modification, and the remaining modifications are naturally occurring modifications, in particular 2' -OMe. At least in this case, the complementary region starts at a first position of a second strand having the corresponding nucleotide in the first strand, regardless of whether the two nucleotides are capable of base pairing with each other.

In one aspect of the nucleic acid, each nucleotide of the first strand and the second strand is a modified nucleotide.

Unless specifically stated otherwise, nucleotides of the first strand herein are numbered consecutively starting from nucleotide number 1 at the 5' end of the first strand. The nucleotides of the second strand are numbered consecutively starting from nucleotide number 1 at the 3' end of the second strand.

An "odd-numbered" nucleotide is a nucleotide that is odd-numbered in the strand, wherein the nucleotides are numbered consecutively starting from the designated end of the strand, or from the 5' end if the end at which the nucleotides begin to be numbered is not indicated. "even-numbered" nucleotides are those that are used in the strand with an even number, wherein the nucleotides are consecutively numbered starting from the designated end of the strand, or from the 5' end if the end at which the nucleotides begin to be numbered is not indicated.

One or more nucleotides on the first and/or second strand may be modified to form modified nucleotides. One or more odd numbered nucleotides of the first strand may be modified. One or more 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-numbered 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.

In the nucleic acid of the present invention, a plurality of odd-numbered nucleotides in the first strand may be modified. The plurality of even numbered nucleotides in the first strand may be modified by a second modification. The first strand may include adjacent nucleotides modified by the common modification. The first strand may also include adjacent nucleotides that are modified by a second, different modification (i.e., the first strand may include nucleotides that are adjacent to each other and that are modified by a first modification and other nucleotides that are adjacent to each other and that are modified by a second modification that is different from the first modification).

One or more odd-numbered nucleotides of the second strand (wherein the nucleotides are numbered consecutively from nucleotide number 1 at the 3 'end of the second strand) may be modified by a different modification than the odd-numbered nucleotides of the first strand (wherein the nucleotides are numbered consecutively from nucleotide number 1 at the 5' end of the first strand), and/or one or more even-numbered nucleotides of the second strand may be modified by the same modification as 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. The plurality of odd numbered nucleotides of the second strand may be modified by a common modification and/or the plurality of even numbered nucleotides may be modified by the same modification present on the odd numbered nucleotides of the first strand. The plurality of odd-numbered nucleotides of the second strand may be modified by a different modification than the modification of the odd-numbered nucleotides of the first strand.

The second strand may include adjacent nucleotides that are modified by a common modification, which may be a different modification than the modification of the odd numbered nucleotides of the first strand.

In the nucleic acid of the present invention, each odd-numbered nucleotide in the first strand and each even-numbered nucleotide in the second strand may be modified by a common modification, and each even-numbered nucleotide in the first strand may be modified by a different modification, and each odd-numbered nucleotide in the second strand may be modified by a different modification.

The nucleic acids of the invention may have modified nucleotides of the first strand displaced by at least one nucleotide relative to unmodified or differently modified nucleotides of the second strand.

One or more or each odd numbered nucleotide may be modified in the first strand and one or more or each even numbered nucleotide may be modified in the second strand. One or more or each alternative nucleotide on either or both strands may be modified by a second modification. One or more or each even-numbered nucleotide may be modified in the first strand and one or more or each even-numbered nucleotide may be modified in the second strand. One or more or each alternative nucleotide on either or both strands may be modified by a second modification. One or more or each odd numbered nucleotide may be modified in the first strand and one or more odd numbered nucleotide may be modified in the second strand by common modification. One or more or each alternative nucleotide on either or both strands may be modified by a second modification. One or more or each even numbered nucleotide may be modified in the first strand and one or more or each odd numbered nucleotide may be modified in the second strand by common modification. One or more or each alternative nucleotide on either or both strands may be modified by a second modification.

The nucleic acids of the invention may comprise single-stranded or double-stranded constructs comprising at least two regions of alternating modification in one or both strands. These alternating regions may comprise up to about 12 nucleotides, but in particular comprise from about 3 to about 10 nucleotides. The region of alternating nucleotides may be located at the end of one or both strands of the nucleic acid of the invention. The nucleic acid may comprise alternating nucleotides of 4 to about 10 nucleotides at each end (3 'and 5'), and these regions may be separated by about 5 to about 12 consecutive unmodified or different or co-modified nucleotides.

The odd numbered nucleotides of the first strand may be modified and the even numbered nucleotides may be modified by a second modification. The second strand may include adjacent nucleotides that are modified by 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 by the second modification. One or more nucleotides having the second modification may be adjacent to each other and to nucleotides having the same modification as the modification of the odd numbered nucleotides of the first strand. The first strand may also include phosphorothioate linkages between the two nucleotides at the 3' and 5' ends or phosphorodithioate linkages between the two nucleotides at the 3' end. The second strand may include phosphorothioate or phosphorodithioate linkages between two nucleotides at the 5' end. The second strand may also be conjugated to a ligand at the 5' end.

The nucleic acids of the invention may include a first strand comprising adjacent nucleotides that are modified by common modification. One or more such nucleotides may be adjacent to one or more nucleotides that may be modified by the second modification. One or more nucleotides having the second modification may be contiguous. The second strand may include adjacent nucleotides that are modified by a common modification, which may be identical to 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 by the second modification. One or more nucleotides having the second modification may be contiguous. The first strand may also include phosphorothioate linkages between the two nucleotides at the 3' and 5' ends or phosphorodithioate linkages between the two nucleotides at the 3' end. The second strand may include phosphorothioate or phosphorodithioate linkages between two nucleotides at the 3' end. The second strand may also be conjugated to a ligand at the 5' end.

The nucleotides numbered 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 and 25 from 5 'to 3' on the first strand and from 3 'to 5' on the second strand may be modified by modification on the first strand. The nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 may be modified by a second modification on the first strand. The nucleotides of numbers 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 may be modified by modification on the second strand. The nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 may be modified by a second modification on the second strand. For the nucleic acids of the invention, the nucleotides are numbered 5 'to 3' on the first strand and 3 'to 5' on the second strand.

The nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 may be modified by modification on the first strand. The nucleotides of numbers 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 may be modified by a second modification on the first strand. The nucleotides of numbers 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 may be modified by modification on the second strand. The nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 may be modified by a second modification on the second strand.

Obviously, if the length of the first and/or second strand is shorter than 25 nucleotides, e.g. 19 nucleotides in length, no nucleotides numbered 20, 21, 22, 23, 24 and 25 are to be modified. The skilled person will therefore understand that the above description applies to shorter chains.

One or more modified nucleotides on the first strand may be paired with a modified nucleotide on the second strand that has a common modification. One or more modified nucleotides on the first strand may be paired with a modified nucleotide 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, alternating nucleotides may be aligned on both strands, e.g., all modifications in alternating regions of the second strand are paired with the same modification in the first strand, or modifications may be offset by one nucleotide, where common modifications in alternating regions of one strand are paired with dissimilar modifications (i.e., second or further modifications) in the other strand. Another option is to have different modifications in each strand.

The modification on the first strand may be shifted by one nucleotide relative to the modified nucleotide on the second strand such that the commonly modified nucleotides are not paired with each other.

The modification and/or modifications may each and individually be selected from the group consisting of 3 'deoxythymine, 2' -OMe, 2 'deoxymodification, 2' amino modification, 2 'alkyl modification, morpholino modification, phosphoramidate modification, 5' -phosphorothioate group modification, 5 'phosphate or 5' phosphate mimetic modification and cholesterol derivative or didecyl dodecanoamide group modification, and/or the modified nucleotide may be any of a locked nucleotide, an abasic nucleotide, or a non-natural base including a nucleotide.

At least one modification may be 2'-OMe and/or at least one modification may be 2' -F. Further modifications described herein may be present on the first and/or second strand.

The nucleic acids of the invention may comprise inverted RNA nucleotides at one or several strand ends. Such inverted nucleotides provide stability to the nucleic acid. In particular, the nucleic acid comprises at least one inverted nucleotide at the 3 'end of the first and/or second strand and/or the 5' end of the second strand. More particularly, the nucleic acid comprises an inverted nucleotide at the 3' end of the second strand. Most particularly, the nucleic acid comprises a reverse RNA nucleotide at the 3' end of the second strand, in particular a reverse A. An inverted nucleotide is a nucleotide that is attached to the 3 'end of a nucleic acid through its 3' carbon rather than the usual 5 'carbon, or is attached to the 5' end of a nucleic acid through its 5 'carbon rather than the usual 3' carbon. The inverted nucleotide is present in particular at the end of a strand, not as an overhang, but opposite the corresponding nucleotide in the other strand. Thus, the nucleic acid is at the end comprising the inverted RNA nucleotide, particularly at the blunt end. The inverted RNA nucleotide present at the end of the strand refers in particular to the inverted RNA nucleotide that is the last nucleotide at the end of the strand. Nucleic acids having such nucleotides are stable and easy to synthesize. Reverse RNA nucleotides are particularly unmodified nucleotides because they do not include any modification compared to the natural nucleotide counterpart. Specifically, the inverted RNA nucleotide is particularly a 2' -OH nucleotide.

The nucleic acids of the invention may include one or more nucleotides modified at the 2 '-position with 2' -H, thus having DNA nucleotides in the nucleic acid. The nucleic acid of the invention may comprise DNA nucleotides at positions 2 and/or 14 of the first strand counted from the 5' end of the first strand. The nucleic acid may comprise a DNA nucleotide on the second strand corresponding to positions 11, or 13, or 11 and 13, or 11-13 of the first strand.

In one aspect, there is no more than one DNA nucleotide in each nucleic acid of the invention.

The nucleic acids of the invention may comprise one or more LNA nucleotides. The nucleic acids of the invention may comprise LNA nucleotides at positions 2 and/or 14 counted starting from the 5' end of the first strand. The nucleic acid may comprise an LNA at position 11, or 13, or 11 and 13, or 11-13 on the second strand corresponding to the first strand.

In particular, the nucleic acid may comprise a first modification and a second or further modification, each and independently selected from the group comprising a 2'-OMe modification and a 2' -F modification. The nucleic acid may include a 2'-OMe modification, which may be a first modification, and a second modification, which may be a 2' -F modification. The nucleic acids of the invention may also comprise phosphorothioate or phosphorodithioate modifications and/or deoxy modifications, which may be present in or between the terminal 2 or 3 nucleotides of each or either end of each or both strands.

In one aspect of the nucleic acid, 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:

(i) At least one or both of nucleotides 2 and 14 of the first strand are modified by the first modification; and/or

(ii) At least one, a few or all even numbered nucleotides of the first strand are modified by a first modification; and/or

(iii) At least one, a few or all odd numbered nucleotides of the first strand are modified by a second modification; and/or

Wherein if the second strand comprises at least one modified nucleotide:

(iv) At least one, several or all nucleotides in the second strand corresponding to the even numbered nucleotides of the first strand are modified by a third modification; and/or

(v) At least one, several or all nucleotides in the second strand corresponding to the odd-numbered nucleotides of the first strand are modified by a fourth modification; and/or

(vi) At least one, several or all nucleotides of the second strand corresponding to the 11 th nucleotide or 13 th nucleotide or 11 th and 13 th nucleotides or 11 th to 13 th nucleotides of the first strand are modified by a fourth modification; and/or

(vii) At least one, several or all nucleotides of the second strand except for the positions corresponding to nucleotide 11 or nucleotide 13 or nucleotide 11 and 13 or nucleotide 11-13 of the first strand are modified by a third modification;

Wherein the nucleotides on the first strand are numbered consecutively starting with nucleotide number 1 at the 5' end of the first strand;

wherein the modification is in particular at least one of the following:

(a) The first modification is in particular different from the second and third modifications;

(b) The first modification is in particular identical to the fourth modification;

(c) The second and third modifications are in particular identical modifications;

(d) The first modification is in particular a 2' -F modification;

(e) The second modification is in particular a 2' -OMe modification;

(f) The third modification is in particular a 2' -OMe modification; and/or

(g) The fourth modification is in particular the 2' -F modification; and

wherein optionally the nucleic acid is conjugated to a ligand.

One aspect is a double stranded nucleic acid for inhibiting expression of PROS1, particularly in a cell, wherein the nucleic acid comprises a first strand and a second strand, wherein the first strand sequence comprises a sequence of at least 15 nucleotides differing by NO more than 3 nucleotides from any of SEQ ID NO:187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 255, 19, 15, 1, 3, 5, 7, 9, 11, 13, 17, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 and 49, particularly SEQ ID NO:199, 203, 209 or 229, wherein all even numbered nucleotides of the first strand are modified by a first modification, all odd numbered nucleotides of the first strand are modified by a second modification, all nucleotides at positions of the second strand corresponding to the even numbered nucleotides of the first strand are modified by a third strand, the first strand and the odd numbered nucleotides of the first strand are modified by a fourth modification and all the fourth modification are modified by a fourth modification, 2' -and 2.

One aspect is a double stranded nucleic acid for inhibiting expression of PROS1 expression, particularly in a cell, wherein the nucleic acid comprises a first strand and a second strand, wherein the first strand sequence comprises a sequence of at least 15 nucleotides differing by NO more than 3 nucleotides from any of SEQ ID NO. 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 255, 19, 15, 1, 3, 5, 7, 9, 11, 13, 17, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 and 49, particularly SEQ ID NO. 199, 203, 209 or 229, wherein all even numbered nucleotides of the first strand are modified by a first modification, all odd numbered nucleotides of the first strand are modified by a second modification, all nucleotides of the second strand at positions corresponding to nucleotides of the first strand are modified by a fourth strand, and wherein the modified nucleotides of the second strand are modified by a fourth strand by a modification of at least 15 nucleotides and wherein the fourth strand is modified by a modification of the first strand is modified by a fourth strand and the fourth strand is modified by a modification of 2' -and 2.

The 3 'and 5' ends of the oligonucleotides may be modified. Such modifications may be at the 3 'or 5' or both ends of the molecule. They may comprise one or more atoms that modify or replace the entire terminal phosphate or phosphate group. For example, the 3 'and 5' ends of the oligonucleotides can be conjugated with other functional molecular entities, such as labeling moieties, e.g., fluorophores (e.g., pyrene, TAMRA, fluorescein, cy3 or Cy5 dyes) or protecting groups (e.g., based on sulfur, silicon, boron, or esters). The functional molecular entity may be linked to the sugar by a phosphate group and/or a linker. The terminal atom of the linker being attached to or substituted for the linking atom of the phosphate group or C-3' or C-5 of the saccharide A' O, N, S or C group. Alternatively, the linker may be attached to or replace the terminal atom of a nucleotide substitute (e.g., PNA). These spacers or linkers may comprise, for example, - (CH) ₂ ) _n -、-(CH ₂ ) _n N-、-(CH ₂ ) _n O-、-(CH ₂ ) _n S-、-(CH ₂ CH ₂ O) _n CH ₂ CH ₂ O- (e.g., n=3 or 6), abasic sugar, amide, carboxyl, amine, oxyamine, oxyimine, thioether, disulfide, thiourea, sulfonamide, or morpholino, or biotin and fluorescein reagents. The 3' end may be an-OH group.

Other examples of end modifications include dyes, intercalators (e.g., acridine), crosslinkers (e.g., psoralen, mitomycin C), porphyrins (TPPC 4, texaphyrin, sapphirin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases, EDTA, lipophilic carriers (e.g., cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1, 3-bis-O- (hexadecyl) glycerol, geranyloxyhexyl, hexadecyl glycerol, borneol, menthol, 1, 3-propanediol, heptadecyl, palmitic acid, myristic acid, O3- (oleoyl) lithocholic acid, O3- (oleoyl) cholanic acid, dimethoxytrityl, or phenoneOxazine) and peptide conjugates (e.g., antennapedia peptide, tat peptide), alkylating agents, phosphates, amino groups, sulfhydryl groups, PEG (e.g., PEG-40K), MPEG, [ MPEG ] ]2. Polyamino groups, alkyl groups, substituted alkyl groups, radiolabelled markers, enzymes, haptens (e.g. biotin), transport/absorption enhancers (e.g. aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g. imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, tetraazamacrocyclic Eu) ³⁺ A complex).

Terminal modifications can also be used to monitor the distribution, in which case the groups to be added can contain fluorophores, such as fluorescein or Alexa dyes. Terminal modifications may also be used to enhance uptake, for which useful modifications include cholesterol. Terminal modifications can also be used to crosslink the RNA agent to another moiety.

Terminal modifications may be added for a variety of reasons including modulating activity or modulating resistance to degradation. Terminal modifications for modulating activity include modification of the 5' end with a phosphate or phosphate analog. The nucleic acids of the invention may be 5 'phosphorylated on the first strand or the second strand or comprise a phosphoryl analogue at the 5' end. The 5' -phosphate modification comprises a modification compatible with RISC-mediated gene silencing. Suitable modifications include: 5' -monophosphate ((HO) ₂ (O) P-O-5 '), 5' -diphosphate ((HO) ₂ (O) P-O-P (HO) (O) -O-5 '), 5' -triphosphate ((HO) ₂ (O) P-O- (HO) (O) P-O-P (HO) (O) -O-5'); 5' -guanosine cap (7-methylated or unmethylated) (7 m-G-O-5' - (HO) (O) P-O-P (HO) (O) -O-5 '); 5' -adenosine caps (Appp), and any modified or unmodified nucleotide cap structures (N-O-5 ' - (HO) (O) P-O-P (HO) (O) -O-5 '); 5' -Monothiophosphate (phosphorothioate; (HO)) ₂ (S) P-O-5'); 5' -Monodithiophosphate (dithiophosphate; (HO) (S) P-O-5 '), 5' -thiophosphate ((HO) ₂ (O) P-S-5'); any other combination of oxygen/sulfur substituted mono-, di-, and tri-phosphates (e.g., 5' -alpha-thiophosphoric acid ester, 5' -gamma-thiophosphoric acid ester, etc.), 5' -phosphoramidates ((HO) ₂ (O)P-NH-5′、(HO)(NH ₂ ) (O) P-O-5 '), 5' -alkylphosphonates (alkyl = methyl, ethyl, isopropyl, propyl, etc., e.g., RP (OH) (O) -O-5' - (wherein R is alkyl), (OH) ₂ (O) P-5' -CH 2-), 5' -vinylphosphonate, 5' -alkyl ether phosphonate (alkyl ether = methoxymethyl (MeOCH) ₂ (-), ethoxymethyl, etc., e.g., RP (OH) (O) -O-5' -) (where R is an alkyl ether).

Some moieties may be attached to the 5' end of the first strand or the second strand. These include an abasic ribose moiety, an abasic deoxyribose moiety, a modified abasic ribose, and an abasic deoxyribose moiety including 2' -O alkyl modifications; reverse abasic ribose and abasic deoxyribose moieties and modifications thereof, C6-imino-Pi; mirror nucleotides comprising L-DNA and L-RNA; a 5' ome nucleotide; and nucleotide analogs including 4',5' -methylene nucleotides; 1- (β -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 nucleotides; an alpha-nucleotide; threo-type furanose nucleotides; acyclic-3', 4+ -nucleotide breaks; 3, 4-dihydroxybutyl nucleotide; 3, 5-dihydroxyamyl nucleotide, 5'-5' -reverse abasic moiety; 1, 4-butanediol phosphate; a 5' -amino group; and bridged or unbridged methylphosphonate and 5' -mercapto moieties.

In each of the sequences described herein, the C-terminal "-OH" moiety may be replaced by the C-terminal "-NH ₂ "partial substitution" and vice versa.

The invention also provides a nucleic acid according to any aspect of the invention described herein, wherein the first strand has a terminal 5 '(E) -vinylphosphonate nucleotide at its 5' end. The terminal 5' (E) -vinylphosphonate nucleotide is linked to the second nucleotide in the first strand, in particular by a phosphodiester bond.

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

(vp)-N( _po )[N( _po )] _n -(I)

wherein "(vp) -" is 5' (E) -vinylphosphonate, "N" is a nucleotide, "po" is a phosphodiester linkage, and N is 1 to (total number of nucleotides in the first strand-2), particularly wherein N is 1 to (total number of nucleotides in the first strand-3), more particularly wherein N is 1 to (total number of nucleotides in the first strand-4).

In particular, the terminal 5' (E) -vinylphosphonate nucleotide is an RNA nucleotide, in particular (vp) -U.

Terminal 5 '(E) -vinylphosphonate nucleotides are nucleotides in which the natural phosphate group at the 5' end has been replaced by an E-vinylphosphonate, in which the bridging 5 '-oxygen atom of the terminal nucleotide of the 5' phosphorylated chain is replaced by a methane group (-ch=) group:

5 '(E) -vinyl phosphonate is a 5' phosphate mimic. A biomimetic is a molecule that is capable of performing the same function and is very similar in structure to the original molecule being simulated. In the context of the present invention, 5 '(E) -vinyl phosphonate mimics the function of normal 5' phosphate, e.g., enabling efficient RISC loading. In addition, due to a slight change in its structure, 5 '(E) vinylphosphonates are able to stabilize the 5' nucleotide by protecting it from dephosphorylation by enzymes such as phosphatases.

In one aspect, the first strand has a terminal 5' (E) -vinylphosphonate nucleotide at its 5' end, the terminal 5' (E) -vinylphosphonate nucleotide is linked to a second nucleotide in the first strand by a phosphodiester linkage, and the first strand comprises a) more than 1 phosphodiester linkage; b) At least a phosphodiester linkage between terminal three 5 'nucleotides and/or c) at least a phosphodiester linkage between terminal four 5' nucleotides.

In one aspect, the first strand and/or the second strand of the nucleic acid comprises at least one Phosphorothioate (PS) and/or at least one phosphorodithioate (PS 2) linkage between the two nucleotides.

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

In one aspect, the first strand and/or the second strand of the nucleic acid comprises phosphorothioate or phosphorodithioate linkages between the terminal two 3 'nucleotides, or phosphorothioate or phosphorodithioate linkages between the terminal three 3' nucleotides. In particular, the bonds between other nucleotides in the first strand and/or the second strand are phosphodiester bonds.

In one aspect, the first strand and/or the second strand of the nucleic acid comprises a phosphorothioate linkage between two terminal 5 'nucleotides or a phosphorothioate linkage between three terminal 5' nucleotides.

In one aspect, the nucleic acids of the invention comprise one or more phosphorothioate or phosphorodithioate modifications at one or more ends of the first and/or second strand. Alternatively, each or either end of the first strand may comprise one or two or three phosphorothioate or phosphorodithioate modified nucleotides (internucleoside linkages). Alternatively, each or either end of the second strand may comprise one or two or three phosphorothioate or phosphorodithioate modified nucleotides (internucleoside linkages).

In one aspect, the nucleic acid comprises phosphorothioate linkages between two or three 3 'nucleotides and/or 5' nucleotides at the ends of the first and/or second strand. In particular, the nucleic acid includes phosphorothioate linkages between the terminal three 3 'nucleotides and the terminal three 5' nucleotides of the first and second strands. In particular, all remaining bonds between nucleotides of the first strand and/or the second strand are phosphodiester bonds.

In one aspect, the nucleic acid includes a phosphorodithioate linkage between each of the two, three, or four terminal nucleotides at the 3 'end of the first strand and/or includes a phosphorodithioate linkage between each of the two, three, or four terminal nucleotides at the 3' end 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 and includes a linkage other than a phosphorodithioate linkage between the two, three, or four terminal nucleotides at the 5' end of the first strand.

In one aspect, the nucleic acid includes phosphorothioate linkages between the terminal three 3 'nucleotides and the terminal three 5' nucleotides of the first strand and the second strand. In particular, all remaining bonds between nucleotides of the first strand and/or the second strand are phosphodiester bonds.

In one aspect, the nucleic acid:

(i) Having phosphorothioate linkages between the terminal three 3 'nucleotides and the terminal three 5' nucleotides of the first strand;

(ii) Conjugation to a tri-antennary ligand at the 3 'or 5' terminal nucleotide of the second strand;

(iii) Having phosphorothioate linkages between the three nucleotides at the end of the second strand opposite the end conjugated to the tri-antennary ligand; and is also provided with

(iv) Alternatively, all remaining bonds between nucleotides of the first strand and/or the second strand are phosphodiester bonds.

In one aspect, the nucleic acid:

(i) Having a terminal 5 '(E) -vinylphosphonate nucleotide at the 5' end of the first strand;

(ii) Having phosphorothioate linkages between the terminal three 3 'nucleotides of the first and second strands and between the terminal three 5' nucleotides of the second strand, or having phosphorodithioate linkages between the terminal two 3 'nucleotides of the first and second strands and between the terminal two 5' nucleotides of the second strand; and is also provided with

(iii) Alternatively, all remaining bonds between nucleotides of the first strand and/or the second strand are phosphodiester bonds.

The use of phosphorodithioate linkages in the nucleic acids of the invention reduces the variation in stereochemistry of the population of nucleic acid molecules compared to molecules comprising phosphorothioate at the same position. Phosphorothioate linkages introduce chiral centers and it is difficult to control which non-linking oxygen is substituted for sulfur. Depending on the number of phosphorodithioates and phosphorothioate linkages used in the nucleic acid molecule, the use of phosphorodithioates ensures that no chiral centers are present in the linkages and thus reduces or eliminates any changes in the population of nucleic acid molecules.

In one aspect, the nucleic acid includes a phosphorodithioate linkage between two terminal nucleotides at the 3 'end of the first strand and a phosphorodithioate linkage between two terminal nucleotides at the 3' end of the second strand and a phosphorodithioate linkage between two terminal nucleotides at the 5 'end of the second strand and includes linkages other than phosphorodithioate linkages between two, three, or four terminal nucleotides at the 5' end of the first strand. In particular, the first strand has a terminal 5 '(E) -vinylphosphonate nucleotide at its 5' end. The terminal 5' (E) -vinylphosphonate nucleotide is linked to the second nucleotide in the first strand, in particular by a phosphodiester bond. In particular, all bonds between nucleotides of the two strands are phosphodiester bonds, except for the bond between the two terminal nucleotides at the 3' end of the first strand and the bond between the two terminal nucleotides at the 3' and 5' ends of the second strand.

In one aspect, when there is no phosphorodithioate linkage between each of the three terminal 3 'nucleotides and/or between each of the three terminal 5' nucleotides of the first strand and/or between each of the three terminal 3 'nucleotides and/or between each of the three terminal 5' nucleotides of the second strand, the nucleic acid includes phosphorothioate linkages between the terminals. The absence of a phosphorodithioate linkage at the end means that the linkage between two terminal nucleotides at the end of the nucleic acid in question, or in particular between the three terminal nucleotides in question, is a linkage other than a phosphorodithioate linkage.

In one aspect, all bonds of the nucleic acid between the nucleotides of the two strands are phosphodiester bonds, except for the bond between the two terminal nucleotides of the 3' end of the first strand and the bond between the two terminal nucleotides of the 3' and 5' ends of the second strand.

Other phosphate bond modifications are also possible.

Phosphate linkers can also be modified by replacing the linking oxygen with nitrogen (bridged phosphoramidate), sulfur (bridged phosphorothioate), and carbon (bridged methylenephosphonate). The substitution may be performed at the terminal oxygen. Replacement of the non-linking oxygen with nitrogen is possible.

The phosphate group may be replaced with a linker that does not contain phosphorus.

Examples of moieties that can replace the phosphate group include siloxanes, carbonates, carboxymethyl, carbamates, amides, thioethers, ethylene oxide linkers, sulfonates, sulfonamides, thiomethylals, methylals, oximes, methyleneimino groups, methylenemethylimino groups, methylenehydrazines, methylenedimethylhydrazine, and methyleneoxymethylimino groups. In certain embodiments, the substitution may comprise a methylenecarbonylamino group and a methyleneimino group.

The phosphate linker and ribose may be replaced with nuclease resistant nucleotides. Examples include morpholino, cyclobutyl, pyrrolidine, and Peptide Nucleic Acid (PNA) nucleoside substitutes. In certain embodiments, PNA alternatives may be used.

In one aspect, a nucleic acid, particularly an siRNA, that inhibits expression of PROS1, particularly by RNAi, and particularly in a cell, comprises one or more or all of:

(i) Modified nucleotides;

(ii) Modified nucleotides other than the 2' -OMe modified nucleotides at positions 2 and 14 of the 5' end of the first strand, in particular 2' -F modified nucleotides;

(iii) Each odd numbered nucleotide of the first strand numbered from the 5 'end of the first strand is a 2' -OMe modified nucleotide;

(iv) Each even numbered nucleotide of the first strand numbered from the 5 'end of the first strand is a 2' -F modified nucleotide;

(v) The second strand nucleotides corresponding to positions 11 and/or 13 or 11-13 of the first strand are modified by a modification other than a 2'-OMe modification, in particular wherein one or two or all of these positions comprise a 2' -F modification;

(vi) Reverse nucleotides, particularly the 3' -3' bond at the 3' end of the second strand;

(vii) One or more phosphorothioate linkages;

(viii) One or more phosphorodithioate linkages; and/or

(ix) The first strand has a terminal 5' (E) -vinylphosphonate nucleotide at its 5' end, in which case the terminal 5' (E) -vinylphosphonate nucleotide is in particular uridine and is linked to the second nucleotide in the first strand, in particular via a phosphodiester bond.

All features of the nucleic acids may be combined with all other aspects of the invention disclosed herein.

Ligand

The nucleic acids of the invention may be conjugated to a ligand. Effective delivery of oligonucleotides, particularly double-stranded nucleic acids of the invention, to cells in vivo is important, and specific targeting and substantial protection from extracellular environments, particularly serum proteins, is required. One way to achieve specific targeting is to conjugate the ligand to the nucleic acid. In some embodiments, the ligand aids in targeting the nucleic acid to a target cell having a cell surface receptor that binds and internalizes the conjugated ligand. In such embodiments, a suitable ligand for the desired receptor molecule is required to be conjugated so that the conjugated molecule is taken up by the target cell by a mechanism such as a different receptor-mediated endocytic pathway or functionally similar process. In other embodiments, ligands that can mediate internalization of nucleic acids into target cells through mechanisms other than receptor-mediated endocytosis can alternatively be conjugated to the nucleic acids of the invention for cell or tissue specific targeting.

One example of a conjugate that mediates receptor-mediated endocytosis is the asialoglycoprotein receptor complex (ASGP-R), which has a high affinity for GalNAc moieties described herein. The ASGP-R complex consists of polymers of membrane ASGR1 and ASGR2 receptors in varying proportions, which are highly abundant in hepatocytes. One of the first disclosures of using a tri-antennary cluster glycoside as a conjugation ligand is in US patent 5,885,968. Conjugates having three GalNAc ligands and comprising a phosphate group are known and described in Dubber et al (bioconjug. Chem.2003Jan-Feb;14 (1): 239-46). ASGP-R complex showed 50-fold higher affinity for N-acetyl-D-galactosamine (GalNAc) than D-Gal.

ASGP-R complexes specifically recognize the terminal β -galactosyl subunit of glycosylated proteins or other oligosaccharides (Weigel, P.H.et.al., biochim.Biophys.Acta.2002Sep 19;1572 (2-3): 341-63) and can be used to deliver drugs to hepatocytes expressing receptor complexes by covalent coupling of galactose or galactosamine to the drug (Ishibashi, S.; et al, jbiol. Chem.1994nov 11;269 (45): 27803-6). Furthermore, binding affinity can be significantly increased by multivalent effects achieved by repetition of targeting moieties (Biessen EA, et al, J Med chem.1995Apr 28;38 (9): 1538-46).

ASGP-R complex is the medium for active uptake of glycoproteins containing terminal β -galactosyl groups by the cell endosome. ASGPR is therefore highly suitable for targeted delivery of drug candidates conjugated to such ligands, e.g., nucleic acids, to receptor expressing cells (Akinec et al, molgher. 2010Jul;18 (7): 1357-64).

More generally, the ligand may include a sugar, the sugar being selected to have an affinity for at least one type of receptor on the target cell. In particular, the receptor is located on the surface of mammalian hepatocytes, for example, the hepatic asialoglycoprotein receptor complex (ASGP-R) described previously.

The sugar may be selected from N-acetylgalactosamine, mannose, galactose, glucose, glucosamine and fucose. The sugar may be N-acetylgalactosamine (GalNAc).

Thus, the ligands for use in the present invention may comprise (i) one or more N-acetylgalactosamine (GalNAc) moieties and derivatives thereof, and (ii) a linker, wherein the linker conjugates the GalNAc moiety to a nucleic acid as defined in any of the preceding aspects. The linker may be of monovalent structure or of divalent or trivalent or tetravalent branching structure. Nucleotides may be modified as defined herein.

Thus, the ligand may comprise GalNAc.

In one aspect, the nucleic acid is conjugated to a ligand comprising a compound of formula (II):

[S-X ¹ -P-X ² ] ₃ -A-X ³ - (II)

Wherein:

s represents a sugar, in particular wherein the sugar is N-acetylgalactosamine;

X ¹ represents C ₃ ～C ₆ Alkylene or (-CH) ₂ -CH ₂ -O) _m (-CH ₂ ) ₂ -, wherein m is 1, 2, or 3;

p is a phosphate or modified phosphate, in particular a phosphorothioate;

X ² is alkylene or of the formula (-CH) ₂ ) _n -O-CH ₂ -an alkylene ether, wherein n = 1-6;

a is a branching unit;

X ³ represents a bridging unit;

wherein the nucleic acids according to the invention are bound to X by means of phosphates or modified phosphates, in particular phosphorothioates ³ And (3) coupling.

In formula (II), branching unit "a" branches in particular into three to accommodate three sugar ligands. The branching unit is in particular covalently linked to the ligand and the remaining tethered portion of the nucleic acid. The branching unit may comprise a branched aliphatic group comprising a group selected from the group consisting of alkyl, amide, disulfide, polyethylene glycol, ether, thioether, and hydroxyamino groups. The branching unit may comprise a group selected from alkyl groups and ether groups.

The branching unit a may have a structure selected from:

wherein each A ₁ O, S, C =o or NH; and each n independently represents an integer of 1 to 20.

The branching unit may have a structure selected from:

wherein A is ₁ O, S, C =o or NH; and each n independently represents an integer of 1 to 20.

The branching unit may have the following structure:

the branching unit may have the following structure:

alternatively, branching unit a may have a structure selected from: ,

wherein:

R ¹ is hydrogen or C1-C10 alkylene;

and R is ² Is a C1-C10 alkylene group.

Alternatively, the branching unit consists of carbon atoms only.

“X ³ Part is a bridging unit. The bridging unit is linear and is covalently bound to the branching unit and the nucleic acid.

X ³ Can be selected from-C ₁ ～C ₂₀ Alkylene-, -C ₂ ～C ₂₀ Alkenylene-, C ₁ ～C ₂₀ Alkylene) -O- (C ₁ ～C ₂₀ Alkylene-alkylene ether, -C (O) -C ₁ ～C ₂₀ Alkylene-, -C ₀ ～C ₄ Alkylene (Cy) C ₀ ～C ₄ Alkylene- (wherein Cy represents a substituted or unsubstituted 5-or 6-membered cycloalkylene, arylene, heterocyclylene or heteroarylene ring), -C ₁ ～C ₄ alkylene-NHC (O) -C ₁ ～C ₄ Alkylene-, -C ₁ ～C ₄ alkylene-C (O) NH-C ₁ ～C ₄ Alkylene-, -C ₁ ～C4 ₄ alkylene-SC (O) -C ₁ ～C ₄ Alkylene-, -C ₁ ～c ₄ alkylene-C (O) s-C ₁ ～C ₄ Alkylene-, -C ₁ ～C ₄ alkylene-OC (O) -C ₁ ～C ₄ Alkylene-, -C ₁ ～C ₄ alkylene-C (O) O-C ₁ ～C ₄ Alkylene-, and-C ₁ ～C ₆ alkylene-S-S-C ₁ ～C ₆ Alkylene-.

X ³ Can be- (C) ₁ ～C ₂₀ Alkylene) -O- (C ₁ ～C ₂₀ Alkylene) -alkylene ethers. X is X ³ Can be- (C) ₁ ～C ₂₀ Alkylene) -o- (C ₄ ～C ₂₀ Alkylene) -alkylene ethers, wherein (C ₄ ～c ₂₀ Alkylene) is attached to Z. X is X ³ Can be selected from the group consisting of-CH ₂ -O-C ₃ H ₆ -、-CH ₂ -O-C ₄ H ₈ -、-CH ₂ -O-C ₆ H ₁₂ -and-CH ₂ -O-C ₈ H ₁₆ -, in particular-CH ₂ -O-C ₄ H ₈ -、-CH ₂ -O-C ₆ H ₁₂ -and-CH ₂ -O-C ₈ H ₁₆ -a group consisting of, in each case, -CH ₂ -the group is linked to a.

In one aspect, the nucleic acid is conjugated to a ligand comprising a compound of formula (III):

[S-X ¹ -P-X ² ] ₃ -A-X ³ - (III)

wherein:

s represents a sugar, in particular GalNAc;

X ¹ represents C ₃ ～C ₆ Alkylene or (-CH) ₂ -CH ₂ -O)m(-CH ₂ ) ₂ -, wherein m is 1, 2, or 3;

p is a phosphate or modified phosphate, in particular a phosphorothioate;

X ² is C ₁ ～C ₈ An alkylene group;

a is a branching unit selected from:

X ³ is a bridging unit;

Branching unit a may have the following structure:

branching unit a may have the following structure:wherein X3 is attached to the nitrogen atom.

X ³ Can be C ₁ ～C ₂₀ An alkylene group. In particular, X ³ Selected from the group consisting of-C ₃ H ₆ -、-C ₄ H ₈ -、-C ₆ H ₁₂ -and-C ₈ H ₁₆ -, in particular-c ₄ H ₈ -、-C ₆ H ₁₂ -and-C ₈ H ₁₆ -a group of.

In one aspect, the nucleic acid is conjugated to a ligand comprising a compound of formula (IV):

[S-X ¹ -P-X ² ] ₃ -A-X ³ - (IV)

wherein:

s represents a sugar, in particular GalNAc;

P is a phosphate or modified phosphate, in particular a phosphorothioate;

X ² is of the formula-C ₃ H ₆ -O-CH ₂ -an alkylene ether;

a is a branching unit;

X ³ is selected from the group consisting of-CH ₂ -O-CH ₂ -、-CH ₂ -O-C ₂ H ₄ -、-CH ₂ -O-C ₃ H ₆ -、-CH ₂ -O-C ₄ H ₈ -、-CH ₂ -O-C ₅ H ₁₀ -、-CH ₂ -O-C ₆ H ₁₂ -、-CH ₂ -O-C ₇ H ₁₄ -, and-CH ₂ -O-C ₈ H ₁₆ -alkylene ethers of the group consisting of, in each case, -CH ₂ The group is linked to a,

and wherein X is ³ The binding to the nucleic acids of the invention is by means of phosphates or modified phosphates, in particular phosphorothioates.

The branching unit may comprise carbon. In particular, the branching unit is carbon.

X ³ Can be selected from the group consisting of-CH ₂ -O-C ₄ H ₈ -、-CH ₂ -O-C ₅ H ₁₀ -、-CH ₂ -O-C ₆ H ₁₂ -、-CH ₂ -O-C ₇ H-, and-CH ₂ -O-C ₈ H ₁₆ -a group of groups. In particular, X ³ Selected from the group consisting of-CH ₂ -O-C ₄ H ₈ -、-CH ₂ -O-C ₆ H ₁₂ -and-CH ₂ -O-C ₈ H ₁₆ A group of groups.

X ¹ Can be (-CH) ₂ -CH ₂ -O)(-CH ₂ ) ₂ -。X ¹ Can be (-CH) ₂ -CH ₂ -O) ₂ (-CH ₂ ) ₂ -。X ¹ Can be (-CH) ₂ -CH ₂ -O) ₃ (-CH ₂ ) ₂ -. In particular, X ¹ Is (-CH) ₂ -CH ₂ -O) ₂ (-CH ₂ ) ₂ -. Alternatively, X ¹ Represents C ₃ ～C ₆ An alkylene group. X is X ¹ Can be propylene. X is X ¹ May be butylene. X is X ¹ Can be a pentylene group. X is X ¹ Can be a hexylene group. In particular, alkyl is a linear alkylene. In particular, X ¹ May be butylene.

X ¹ representative-C ₃ H ₆ -O-CH ₂ -, C ₃ Alkylene ethers of alkoxymethylene groups, or-CH ₂ CH ₂ CH ₂ OCH ₂ -。

For any of the above aspects, when P represents a modified phosphate group, P may be defined byAnd (3) representing.

Wherein Y is ¹ And Y ¹ Each independently represents =o, =s, -O-, -OH, -SH, -BH ₃ 、-OCH ₂ CO ₂ 、-OCH ₂ CO ₂ Rx、-OCH ₂ C(S)OR ^x and-OR ^x Wherein R is ^x Represent C ₁ ～C ₆ Alkyl group, and whereinMeaning that it is attached to the remainder of the compound.

Modified phosphates are phosphate groups in which one or more non-linking oxygen is substituted. Examples of modified phosphate groups include phosphorothioates, phosphorodithioates, phosphoroselenates, borophosphates, hydrogen phosphates, phosphoramidates, alkyl or aryl phosphonates and phosphotriesters. Dithiophosphate has two non-linking oxygens replaced with sulfur. One, each, OR two of the non-linking oxygen groups of the phosphate groups may independently be either S, se, B, C, H, N OR (R is alkyl OR aryl).

Phosphate esters can also be modified by replacing the linking oxygen with nitrogen (bridged phosphoramidate), sulfur (bridged phosphorothioate) and carbon (bridged methylenephosphonate). The substitution may be performed at the terminal oxygen. Replacement of the non-linking oxygen with nitrogen is possible.

For example, Y ¹ Can be represented by-OH, Y ² May represent =o or =s; or (b)

Y ¹ Can be represented by-O-, Y ² Can represent either =o or =s;

Y ¹ can be expressed as =o, Y ² Can represent-CH ₃ 、-SH、-OR ^x or-BH ₃

Y ¹ Can be expressed as =s, Y ² Can represent-CH ₃ 、OR ^x or-SH.

Those skilled in the art will appreciate that in some cases Y ¹ And Y ² There will be a delocalization between them.

In particular, the modified phosphate group is a phosphorothioate group. The phosphorothioate group comprises a phosphorodithioate (i.e., wherein Y ¹ Represent =s and Y ² representation-S ^- ) And a monothiophosphate (i.e., wherein Y ¹ Representation of-O ^- And Y is ² Representation = -S ^- Or wherein Y ¹ Represents =o and Y ² representation-S ^- ). In particular, P is a monothiophosphate. The inventors have found that conjugates with phosphorothioate groups replacing phosphate groups have improved in vivo efficacy and duration of action.

P may also be ethyl phosphate (i.e., wherein Y ¹ Represents =o and Y ² Represents OCH ₂ CH ₃ )。

A sugar having affinity for at least one type of receptor on the target cell may be selected. In particular, the receptor is located on the surface of mammalian hepatocytes, for example, the hepatic asialoglycoprotein receptor complex (ASGP-R).

For any of the above or below aspects, the sugar may be selected from one or more of galactosamine with N-acetyl group, mannose, galactose, glucose, glucosamine and fructose. Typically, the ligand used in the present invention may comprise N-acetylgalactosamine (GalNAc). In particular, the compounds of the invention may have 3 ligands, which in particular each comprise N-acetylgalactosamine.

"GalNAc" refers to 2- (acetamido) -2-deoxy-D-galactopyranose, which is commonly referred to in the literature as N-acetylgalactosamine. References to "GalNAc" or "N-acetylgalactosamine" include the β form: 2- (acetamido) -2-deoxy- β -D-galactopyranose and α:2- (acetamido) -2-deoxy-alpha-D-galactopyranose. In certain embodiments, form β:2- (acetamido) -2-deoxy- β -D-galactopyranose and α:2- (acetamido) -2-deoxy-alpha-D-galactopyranose may be used interchangeably. In particular, the compounds of the present invention include beta-form, 2- (acetamido) -2-deoxy-beta-D-galactopyranose.

2- (acetamido) -2-deoxy-D-galactopyranose

2- (acetamido) -2-deoxy-beta-D-galactopyranose2- (acetamido) -2-deoxy-alpha-D-galactopyranose in one aspect, the nucleic acid is a conjugated nucleic acid, wherein the nucleic acid is conjugated to a tri-antennary ligand having one of the following structures: />

Wherein Z is any nucleic acid defined herein.

In particular, the nucleic acid is a conjugated nucleic acid, wherein the nucleic acid is conjugated to a triple antenna ligand having the structure:

wherein Z is any nucleic acid defined herein.

The ligands of formula (II), (III) or (IV) or any one of the tri-antennary ligands disclosed herein may be attached at the 3' end of the first (antisense) strand and/or at any 3' and/or 5' end of the second (sense) strand. The nucleic acid may comprise more than one ligand of formula (II), (III) or (IV) or any of the tri-antennary ligands disclosed herein. However, a single ligand of formula (II), (III) or (IV) or any of the tri-antennary ligands disclosed herein is preferred, as a single such ligand is sufficient to target the nucleic acid effectively to the target cell. In particular in this case, at least the last two, in particular at least the last three, more in particular at least the last four nucleotides at the end of the nucleic acid to which the ligand is attached are linked by phosphodiester bonds.

In particular, the 5' end of the first (antisense) strand is not linked to any of the ligands of formulae (II), (III) or (IV) or the tri-antennary ligands disclosed herein, as the ligand at that position may potentially interfere with the biological activity of the nucleic acid.

A nucleic acid having a single ligand of formula (II), (III) or (IV) at the 5 'end of the strand or any of the tri-antennary ligands disclosed herein is more readily synthesized and therefore less expensive than the same nucleic acid having the same ligand at the 3' end. Thus, in particular, any of the single ligands of formula (II), (III) or (IV) disclosed herein, or any of the tri-antennary ligands, is covalently linked (conjugated) to the 5' end of the second strand of the nucleic acid.

In one aspect, the first strand of the nucleic acid is a compound of formula (V):

wherein b is in particular 0 or 1; and

the second chain is a compound of formula (VI):

wherein:

c and d are independently especially 0 or 1;

Z ₁ and Z ₂ First and second strands of nucleic acid, respectively;

y is independently O or S;

n is independently 0, 1, 2 or 3; and

L ₁ is a linker to which the ligand is attached, wherein L ₁ In formulae (V) and (VI) are identical or different and when L ₁ When present more than once in formulae (V) and (VI), are identical or different in formulae (V) and (VI), wherein L ₁ In particular of formula (VII);

And wherein b+c+d is in particular 2 or 3.

In particular, L in the formulae (V) and (VI) ₁ Having the formula (VII):

wherein:

l is selected from the group comprising, or in particular consisting of:

-(CH ₂ ) _r -C (O) -, wherein r = 2-12;

-(CH ₂ -CH ₂ -O) _s -CH ₂ -C (O) -, wherein s=1-5；

-(CH ₂ ) _t -CO-NH-(CH ₂ ) _t -NH-C (O) -, wherein t is independently 1-5;

-(CH ₂ ) _u -CO-NH-(CH ₂ ) _u -C (O) -, wherein u is independently 1-5; and

-(CH ₂ ) _v -NH-C (O) -, wherein v is 2-12; and

wherein if terminal C (O) is present, it is linked to X of formula (VII) or if X is not present, it is linked to W of formula (VII) ₁ Or if W ₁ If not, it is linked to V of formula (VII);

W ₁ 、W ₃ and W is ₅ Each absent or selected from the group comprising, or in particular consisting of:

-(CH ₂ ) _r -, wherein r=1-7;

-(CH ₂ ) _s -O-(CH ₂ ) _s -wherein s is independently 0-5;

-(CH ₂ ) _t -S-(CH ₂ ) _t -, wherein t is independently 0 to 5;

x is absent or selected from the group consisting of: NH, NCH ₃ Or NC (numerical control) ₂ H ₅ Or in particular a group consisting thereof;

v is selected from the group consisting of: CH. N is,Or in particular a group consisting thereof; wherein B, if present, is a modified or natural nucleobase.

In one aspect, the first chain is a compound of formula (VIII)

Wherein b is in particular 0 or 1; and

the second chain is a compound of formula (IX):

wherein c and d are independently especially 0 or 1;

wherein:

Z ₁ and Z ₂ First and second strands of nucleic acid, respectively;

Y is independently O or S;

R ₁ is H or methyl;

n is independently especially 0, 1, 2 or 3; and

l is the same or different in formulae (VIII) and (IX), and when L is present more than once in the same formula, is the same or different in formulae (VIII) and (IX) and is selected from the group comprising, or in particular consisting of:

-(CH ₂ ) _r -C (O) -, wherein r = 2-12;

-(CH ₂ -CH ₂ -O) _s -CH ₂ -C (O) -, wherein s = 1-5;

-(CH ₂ ) _t -CO-NH-(CH ₂ ) _t -NH-C (O) -, wherein t is independently 1-5;

-(CH ₂ ) _u -CO-NH-(CH ₂ ) _u -C (O) -, wherein u is independently 1-5; and

-(CH ₂ ) _v -NH-C (O) -, wherein v is 2-12; and

wherein if terminal C (O) is present, it is attached to the NH group (of the linker, not of the targeting ligand);

and wherein b+c+d is in particular 2 or 3.

In one aspect, the first strand of the nucleic acid is a compound of formula (X):

wherein b is in particular 0 or 1; and

the second chain is a compound of formula (XI):

wherein:

c and d are independently especially 0 or 1;

Z ₁ and Z ₂ First and second RNA strands of a nucleic acid, respectively;

y is independently O or S;

n is independently especially 0, 1, 2 or 3; and

L ₂ identical or different in formulae (X) and (XI), identical or different in the moiety bracketed by b, c and d, and selected from the group consisting of:

or in particular a group consisting of them, or

n is 0 and L ₂ The method comprises the following steps:

and terminal OH groups are absent, thereby forming A portion;

wherein:

f is a saturated branched or unbranched (e.g. unbranched) C _1-8 Alkyl (e.g. C _1-6 Alkyl) chains in which one of the carbon atoms is optionally replaced by an oxygen atom, provided that the oxygen atom is separated from another heteroatom (e.g., O or N atom) by at least 2 carbon atoms;

l is identical or different in formulae (X) and (XI) and is selected from the group comprising or in particular consisting of:

-(CH ₂ ) _r -C (O) -, wherein r = 2-12;

-(CH ₂ -CH ₂ -O) _s -CH ₂ -C (O) -, wherein s = 1-5;

-(CH ₂ ) _t -CO-NH-(CH ₂ ) _t -NH-C (O) -, wherein t is independently 1-5;

-(CH ₂ ) _u -CO-NH-(CH ₂ ) _u -C (O) -, wherein u is independently 1-5; and

-(CH ₂ ) _v -NH-C (O) -, wherein v is 2-12; and

and wherein b+c+d is in particular 2 or 3.

In one aspect, b is 0, c is 1, d is 1; b is 1, c is 0 and d is 1; b is 1, c is 1 and d is 0; or in any of the nucleic acids of the formulae (V) and (VI) or (VIII) and (IX) or (X) and (XI), b is 1, c is 1 and d is 1. In particular, 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 1. Most particularly, b is 0, c is 1 and d is 1.

In one aspect, in any one of the nucleic acids of formulas (V) and (VI) or (VIII) and (IX) or (X) and (XI), Y is O. In another aspect, Y is S. In a particular aspect, Y is independently selected from O or S at different positions in the formula.

In one aspect, in any nucleic acid of formulas (VIII) and (IX), R ₁ Is H or methyl. In one aspect, R ₁ H. In another aspect, R ₁ Is methyl.

In one aspect, in any one of the nucleic acids of formulas (V) and (VI) or (VIII) and (IX) or (X) and (XI), n is 0, 1, 2 or 3. In particular, n is 0.

Examples of the F moiety in any of the nucleic acids of formulas (X) and (XI) include (CH) ₂ ) _1-6 For example (CH) ₂ ) _1-4 For example CH ₂ 、(CH ₂ ) ₄ 、(CH ₂ ) ₅ Or (CH) ₂ ) ₆ Or CH ₂ O(CH ₂ ) _2-3 For example CH ₂ O(CH ₂ )CH ₃ 。

In one aspect, L in formulas (X) and (XI) ₂ The method comprises the following steps:

in one aspect, L ₂ The method comprises the following steps:

in one aspect, n is 0 and L ₂ The method comprises the following steps:

and terminal OH groups are absent, thereby forming the following moiety:wherein Y is O or S.

In one aspect, L in the nucleic acid of formulae (V) and (VI) or (VIII) and (IX) or (X) and (X1) is selected from the group consisting of:

-(CH ₂ ) _r -C (O) -, wherein r = 2-12;

-(CH ₂ -CH ₂ -O) _s -CH ₂ -C (O) -, wherein s = 1-5;

-(CH ₂ ) _t -CO-NH-(CH ₂ ) _t -NH-C (O) -, wherein t is independently 1-5;

-(CH ₂ ) _u -CO-NH-(CH ₂ ) _u -C (O) -, wherein u is independently 1-5; and

-(CH ₂ ) _v -NH-C (O) -, wherein v is 2-12; or in particular a group consisting thereof;

wherein terminal C (O) is linked to an NH group.

In particular, L is- (CH) ₂ ) _r -C (O) -, wherein r=2-12, more particularly r=2-6, even more particularly r=4 or 6, for example 4.

In particular, L is:

in the parts enclosed by b, c and d, L in the nucleic acids of the formulae (X) and (XI) ₂ Are generally identical. Between the parts bracketed by b, c and d, L ₂ May be the same or different. In one embodiment, L in the parts bracketed by c ₂ With L in the part bracketed by d ₂ The same applies. In one embodiment, L in the parts bracketed by c ₂ With L in the part bracketed by d ₂ Different. In one embodiment, L in the section bracketed by b, c and d ₂ As for example when the linker moiety is a serine alcohol derived linker moiety.

The serinol-derived linker moiety may be based on any stereochemical serinol, i.e. derived from the L-serine isomer, the D-serine isomer, racemic serine or other combinations of isomers. In a preferred aspect of the invention, the serinol-GalNAc moiety (SerGN) has the following stereochemistry:

i.e. solid-supported structural units derived from the L-serine isomer based on (S) -serinamide or (S) -serinesuccinate.

In a particular aspect, the first strand of the nucleic acid is a compound of formula (VIII) and the second strand of the nucleic acid is a compound of formula (IX), wherein:

b is 0;

c and d are 1 and are each independently selected from the group consisting of,

n is 0, and the number of the n is 0,

Z ₁ and Z ₂ First and second strands of nucleic acid, respectively,

y is S, and the Y is S,

R ₁ is H, and

l is- (CH) ₂ ) ₄ -C (O) -, wherein the terminal C (O) of L is linked to the N atom of the linker (i.e. not to a possible N atom of the targeting ligand)。

In another particular aspect, the first strand of the nucleic acid is a compound of formula (V), and the second strand of the nucleic acid is a compound of formula (VI), wherein:

b is 0 and is preferably selected from the group consisting of,

c and d are 1 and are each independently selected from the group consisting of,

n is 0, and the number of the n is 0,

Z ₁ and Z ₂ First and second strands of nucleic acid, respectively,

y is S, and the Y is S,

L ₁ is of formula (VII), wherein:

W ₁ is-CH ₂ -O-(CH ₃ ) ₃ -，

W ₃ is-CH ₃ -，

W ₅ In order to be absent from the device,

v is CH, and the V is CH,

x is NH, and

l is- (CH) ₂ ) ₄ -C (O) -, wherein the terminal C (O) of L is linked to the N atom of X in formula (VII).

b is 0 and is preferably selected from the group consisting of,

c and d are 1 and are each independently selected from the group consisting of,

n is 0, and the number of the n is 0,

Z ₁ and Z ₂ First and second strands of nucleic acid, respectively,

y is S, and the Y is S,

L ₁ is of formula (VII), wherein:

W ₁ 、W ₃ and W is ₅ There is no time for the existence of the non-woven fabric,

v is

X is absent, and

l is- (CH) ₂ ) ₄ -C(O)-NH-(CH ₂ ) ₅ -C (O) -, wherein the terminal C (O) of L is linked to the N atom of V in formula (VII).

In one aspect, the nucleic acid is conjugated to a tri-antennary ligand having the structure:

wherein the nucleic acid is conjugated to the last nucleotide at the 5' end of the second strand via the phosphate group of ligand a); b) The last nucleotide to the 3' end of the second strand; or c) the last nucleotide at the 3' end of the first strand.

In one aspect of the nucleic acid, the cell targeted by the nucleic acid having the ligand is a hepatocyte.

In any of the above ligands in which GalNAc is present, galNAc may be substituted with any other targeting ligand, such as those mentioned herein, in particular mannose, galactose, glucose, glucosamine and fucose.

In one aspect, the nucleic acid is conjugated to a ligand comprising a lipid, more particularly, to a ligand comprising cholesterol.

Compositions, uses and methods

The invention also provides compositions comprising the nucleic acids of the invention. The nucleic acids and compositions may be used as pharmaceuticals or diagnostic agents, alone or in combination with other agents. For example, one or more nucleic acids of the invention may be combined with a delivery vehicle (e.g., liposome) and/or excipient (e.g., carrier, diluent). Other agents such as preservatives and stabilizers may also be added. Pharmaceutically acceptable salts or solvates of any of the nucleic acids of the invention are also within the scope of the invention. Methods of delivering nucleic acids are known in the art and are within the knowledge of those skilled in the art.

The compositions disclosed herein are particularly pharmaceutical compositions. Such compositions are suitable for administration to a subject.

In one aspect, the composition comprises a nucleic acid disclosed herein, or a pharmaceutically acceptable salt or solvate thereof, and a solvent (particularly water) and/or a delivery vehicle and/or a physiologically acceptable excipient and/or carrier and/or salt and/or diluent and/or buffer and/or 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. The subcutaneous or transdermal modes of administration may be particularly suitable for the compounds described herein.

The therapeutically effective amount of the nucleic acids of the invention will depend on the route of administration, the type of mammal being treated and the physical characteristics of the particular mammal being considered. These factors and their relationship to determining the amount are well known to those skilled in the medical arts. The amount and method of administration may be adjusted to achieve optimal efficacy and may depend on factors well known to those skilled in the medical arts such as body weight, diet, concurrent administration, and other factors. The dosage size and regimen that is most suitable for human use can be guided by the results obtained in the present invention and can be confirmed in appropriately designed clinical trials.

The effective dose and treatment regimen can be determined by conventional means, starting with a low dose in the experimental animal, then increasing the dose while monitoring the effect, and also systematically varying the dose regimen. The clinician may consider a number of factors when determining the optimal dose for a given subject. These considerations are known to those skilled in the art.

The nucleic acids of the invention, or salts thereof, may be formulated to prepare pharmaceutical compositions for storage or administration, which generally comprise a therapeutically effective amount of the nucleic acids of the invention, or salts thereof, in a pharmaceutically acceptable carrier.

The nucleic acids or conjugated nucleic acids of the invention may also be administered in combination with other therapeutic compounds, alone or simultaneously, for example as a combined unit dose. The invention also encompasses compositions comprising one or more nucleic acids according to the invention in physiologically/pharmaceutically acceptable excipients such as stabilizers, preservatives, diluents, buffers, and the like.

In one aspect, the composition comprises a nucleic acid as disclosed herein and an additional therapeutic agent comprising a group of oligonucleotides, small molecules, monoclonal antibodies, polyclonal antibodies, peptides, and proteins. If the additional therapeutic agent is a protein, it is in particular FVIII and/or FIX.

In certain embodiments, two or more nucleic acids of the invention having different sequences may be administered simultaneously or sequentially.

In another aspect, the invention provides a composition, e.g., a pharmaceutical composition, comprising one nucleic acid or a combination of different nucleic acids of the invention and at least one pharmaceutically acceptable carrier.

Dosage levels of the medicaments and compositions of the present invention can be determined experimentally by those skilled in the art. In one aspect, a unit dose may contain from about 0.01mg/kg to about 100mg/kg of body weight of nucleic acid or conjugated nucleic acid. Alternatively, the dosage may be 10mg/kg to 25mg/kg body weight, or 1mg/kg to 10mg/kg body weight, or 0.05mg/kg to 5mg/kg body weight, or 0.1mg/kg to 1mg/kg body weight, or 0.1mg/kg to 0.5mg/kg body weight, or 0.5mg/kg to 1mg/kg body weight. Alternatively, the dose may be about 0.5mg/kg to about 10mg/kg body weight, or about 0.6mg/kg to about 8mg/kg body weight, or about 0.7mg/kg to about 7mg/kg body weight, or about 0.8mg/kg to about 6mg/kg body weight, or about 0.9mg/kg to about 5.5mg/kg body weight, or about 1mg/kg to about 5mg/kg body weight, or about 2mg/kg to about 5mg/kg body weight, or about 3mg/kg to about 5mg/kg body weight, or about 1mg/kg body weight, or about 3mg/kg body weight, or about 5mg/kg body weight, wherein "about" is a deviation from the specified value of at most 30%, particularly at most 20%, more particularly at most 10%, still more particularly at most 5%, most particularly 0%. The dose level may also be calculated by other parameters such as body surface area.

The dosage and frequency of administration may vary depending on whether the treatment is therapeutic or prophylactic (e.g., prophylactic), and may be adjusted during the course of treatment. In certain prophylactic applications, relatively low doses are administered at relatively infrequent intervals over a relatively long period of time. Some subjects may continue to receive treatment throughout their lives. In certain therapeutic applications, relatively high doses at relatively short intervals are sometimes required until the progression of the disease is reduced or until the patient shows a partial or complete improvement in the symptoms of the disease. Thereafter, the patient may be switched to an appropriate prophylactic dosing regimen.

The actual dosage level of the nucleic acids of the invention in the pharmaceutical compositions of the invention, alone or in combination with one or more other active ingredients, may be varied in order to obtain an amount of active ingredient effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration without causing adverse side effects to the subject or patient. The selected dosage level will depend on a variety of factors, such as pharmacokinetic factors, including the activity of the particular nucleic acid or composition being used, the route of administration, the time of administration, the rate of excretion of the particular nucleic acid being used, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular composition being used, the age, sex, weight, condition, general health and prior medical history of the subject or patient being treated, and like factors well known in the medical arts.

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

The pharmaceutical composition may be in unit dosage form. In this form, the composition is divided into unit doses containing appropriate amounts of the active component. The unit dosage form may be a packaged formulation containing discrete amounts of the formulation, such as packaged tablets, capsules, and powders in vials or ampoules. The unit dosage form may also be a capsule, cachet, or tablet itself, or it may be the appropriate number of any of these packaged forms. It may be provided in a single dose injectable form, for example in the form of a pen. The compositions may be formulated for any suitable route and means of administration.

The pharmaceutical compositions and medicaments of the invention may be administered to a mammalian subject in a pharmaceutically effective dose. The mammal may be selected from humans, non-human primates, apes or primordial monkeys, dogs, cats, horses, cows, pigs, goats, sheep, mice, rats, hamsters, hedgehog and guinea pigs, or other related species. On the basis of this, the term "PROS1" as used herein refers to a nucleic acid or protein in any of the above-mentioned species, if naturally or artificially expressed therein, but in particular the expression refers to a human nucleic acid or protein.

The pharmaceutical compositions of the present invention may be administered alone or in combination with one or more other therapeutic or diagnostic agents. Combination therapy may comprise a combination of a nucleic acid of the invention 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, therapeutically active small molecules or polypeptides, single chain antibodies, classical antibodies or fragments thereof, or nucleic acid molecules that modulate gene expression of one or more other genes, and similar modulating therapeutics that may supplement or otherwise benefit therapeutic or prophylactic regimens.

Pharmaceutical compositions are generally sterile and stable under the conditions of manufacture and storage. The composition may be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentrations. The carrier may be a solvent or dispersion medium containing, for example, water, alcohols such as ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), or any suitable mixture. For example, proper fluidity can 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 dispersions, and by the use of surfactants according to formulation chemistry well known in the art. In certain embodiments, isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride may be desirable in the composition. Prolonged absorption of the injectable compositions can be brought about by the inclusion in the composition of agents which delay absorption, for example, monostearates and gelatins.

One aspect of the invention is a nucleic acid or composition as disclosed herein for use as a medicament. The nucleic acid or composition is particularly useful for preventing a bleeding disorder, reducing the risk of developing a bleeding disorder, or treating a bleeding disorder.

The present invention provides nucleic acids, alone or in combination with one or more additional therapeutic agents, in pharmaceutical compositions, for treating or preventing conditions, diseases and disorders responsive to inhibition of PROS1 expression.

One aspect of the invention is the use of a nucleic acid or composition disclosed herein for preventing a bleeding disorder, reducing the risk of developing a bleeding disorder, or treating a bleeding disorder.

One aspect of the invention is the use of a nucleic acid or composition disclosed herein in a method of inhibiting expression of PROS1 in a cell, preferably in vitro.

One aspect of the invention is a method of inhibiting expression of PROS1 in a cell, preferably in vitro, comprising the step of administering a nucleic acid or composition disclosed herein to the cell, preferably in vitro.

The nucleic acids and pharmaceutical compositions of the invention can be used to treat a variety of conditions, disorders or diseases. Treatment with the nucleic acids of the invention leads in particular to the consumption of protein S in vivo, in particular in the liver and/or blood. Thus, the nucleic acids of the invention and compositions comprising them will be useful in methods of treating a variety of pathological conditions in which inhibition of protein S expression may be beneficial, such as, inter alia, bleeding disorders. The present invention provides a method for treating a bleeding disorder comprising the step of administering to a subject in need thereof a therapeutically effective amount of a nucleic acid of the invention.

Accordingly, the present invention provides a method of treating or preventing a bleeding disorder, the method comprising the step of administering to a subject (e.g., patient) in need thereof a therapeutically effective amount of a nucleic acid of the invention or a pharmaceutical composition comprising a nucleic acid of the invention.

The most desirable therapeutically effective amount is an amount that will result in the desired efficacy of the particular treatment selected by one of skill in the art for a given subject in need thereof. The amount will vary depending upon a variety of factors understood by the skilled artisan, 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 dose, and type of drug treatment), the nature of the one or more pharmaceutically acceptable carriers in the formulation, and the route of administration. Those skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount by experimentation, i.e., by monitoring the subject's response to administration of a compound and adjusting the dosage accordingly. See, e.g., remington: the Science and Practice of Pharmacy st Ed., univ.of Sciences In Philadelphia (USIP), lippincottWilliams & Wilkins, philadelphia, pa., 2005.

In certain embodiments, the nucleic acids and pharmaceutical compositions of the invention are useful for treating or preventing bleeding disorders.

In certain embodiments, the present invention provides a method for treating a bleeding disorder in a mammalian subject, e.g., a human, comprising the step of administering to a subject in need thereof a therapeutically effective amount of a nucleic acid as disclosed herein.

Administration of a "therapeutically effective dose" of a nucleic acid of the invention may result in a decrease in severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or prevention of injury or disability due to disease distress.

The nucleic acids of the invention may be useful in the treatment or diagnosis of bleeding disorders that may be diagnosed or treated using the methods described herein. Treatment and diagnosis of other bleeding disorders are also considered to fall within the scope of the present invention.

One aspect of the invention is a method of preventing, reducing the risk of developing, or treating a bleeding disorder, comprising administering to an individual in need of treatment a pharmaceutically effective dose or amount of a nucleic acid or composition disclosed herein, particularly wherein the nucleic acid or composition is administered to a subject subcutaneously, intravenously, or by oral, rectal, pulmonary, intramuscular, or intraperitoneal administration. In particular, it is administered subcutaneously.

In certain embodiments, the bleeding disorder is a coagulation-deficient disorder. The coagulation-deficient condition may be a condition associated with prolonged bleeding events and/or reduced thrombin and/or deficient clot formation. Bleeding disorders in particular hemophilia, hereditary hemophilia, hemophilia a, hemophilia B, hemophilia C, von willebrand's disease, von willebrand syndrome, fibrinogen free disorder, hypofibrinogenemia, parahaemophilia, arthritic blood (AH), clotting factor deficiency, factor II, V, VII, X and/or XI hereditary deficiency, factor V and VIII combined deficiency, acquired hemophilia, acquired clotting factor deficiency and acquired bleeding disorder. More particularly, it is hemophilia or joint effusion (AH). More particularly, it is hemophilia, in particular hemophilia a or B, most particularly hemophilia a. Alternatively, it is joint effusion. For the use of the pharmaceutical composition of the invention, each such disease, condition, disorder or symptom is considered a separate embodiment.

In one embodiment, the nucleic acid or composition of the invention is used or used in a method of treatment to:

a) Increasing blood coagulation; and/or

b) Reducing bleeding.

In particular, use of a nucleic acid or composition disclosed herein increases blood clotting in blood of a subject treated with the nucleic acid or composition to a corresponding level expected in a healthy subject. Alternatively, it increases coagulation in the blood of a subject treated with the nucleic acid or composition such that the difference between coagulation in the blood of the subject prior to treatment and the corresponding level expected in a healthy subject is at least temporarily reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%.

In particular, use of a nucleic acid or composition disclosed herein reduces bleeding in a subject treated with the nucleic acid or composition to a corresponding level expected in a healthy subject. Alternatively, it reduces bleeding in a subject treated with the nucleic acid or composition such that the difference between the pre-treatment subject's bleeding and the corresponding level expected in a healthy subject is reduced at least temporarily by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%.

Obviously, an appropriate dosage regimen of the nucleic acid or composition is necessary to achieve these results. The skilled person will be able to determine the dosage regimen necessary to achieve these results.

The nucleic acids or compositions disclosed herein can be used in a treatment regimen comprising once or twice a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, once every twelve weeks, once every three months, once every four months, once every five months, once every six months, or in a regimen having a different dosing frequency, such as a combination of the foregoing intervals. The nucleic acid or composition may be used subcutaneously, intravenously or using any other route of administration, such as oral, rectal, pulmonary, intramuscular or intraperitoneal. In particular, it is intended for subcutaneous use.

Exemplary treatment regimens are administered once every two weeks, once every three weeks, once every four weeks, once every month, once every two or three months, or once every three, four, five, or six or more months. The dosage can be selected and readjusted as desired by the skilled healthcare professional to maximize the therapeutic benefit to a particular subject, such as a patient. Nucleic acids will typically be administered on a number of occasions. The interval between single doses may be, for example, 2-5 days, weekly, biweekly, monthly, bi-or tri-monthly, tetra-or five-month, six-month, or yearly. The interval between administrations may also be irregular based on the level of the nucleic acid target gene product, e.g., in the blood or liver of a subject or patient.

In cells and/or subjects treated with or receiving a nucleic acid or composition disclosed herein, PROS1 expression can be inhibited by 15% to 100% but at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% or an intermediate value as compared to untreated cells and/or subjects. The inhibition level may treat bleeding disorders or may be used to further study the functional and physiological effects of the PROS1 gene product. The level of inhibition is preferably measured in the liver or in the blood or in the kidney, preferably in the blood, of a subject treated with the nucleic acid or composition.

One aspect is the use of a nucleic acid or composition disclosed herein in the manufacture of a medicament for treating a bleeding disorder, such as those listed above, or other pathology requiring inhibition of PROS1 expression. The medicament is a pharmaceutical composition.

Each of the nucleic acids of the invention, and pharmaceutically acceptable salts and solvates thereof, constitute a separate embodiment of the invention.

The invention also encompasses methods of treating or preventing bleeding disorders, such as those listed above, comprising administering to an individual in need of treatment a composition comprising a nucleic acid or composition described herein (to ameliorate such a condition). The nucleic acid or composition may be administered in a regimen comprising twice weekly, once weekly, biweekly, tricyclically, four weeks, five weeks, six weeks, seven weeks, or eight to twelve weeks or more of treatment or in a combination of regimens having different dosing frequencies, such as the foregoing intervals. The nucleic acid or bound nucleic acid may be used subcutaneously or intravenously or other route of administration, such as orally, rectally or intraperitoneally.

The nucleic acids of the invention may be administered by any suitable route of administration known in the art, including but not limited to aerosol, enteral, nasal, ocular, oral, parenteral, rectal, vaginal, or transdermal (e.g., topical administration of creams, gels or ointments, or by transdermal patches). "parenteral administration" is typically associated with injection at or in communication with the intended site of action and includes intraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or tracheal administration.

The use of a pattern of chemical modification of the nucleic acid confers nuclease stability in serum and makes, for example, a subcutaneous route of administration viable.

Solutions or suspensions for intradermal or subcutaneous application typically contain one or more of the following: sterile diluents, such as water for injection, saline solutions, non-volatile oils, polyethylene glycols, glycerol, propylene glycol or other synthetic solvents; antimicrobial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediamine tetraacetic acid; buffers such as acetate, citrate or phosphate; and/or tonicity adjusting agents such as sodium chloride or dextrose. The pH may be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide, or with a buffer having citrate, phosphate, acetate, etc. These formulations may be packaged in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Sterile injectable solutions may be prepared by incorporating the nucleic acids in the required amount in the appropriate solvent with one or a combination of the ingredients described above as required followed by sterile microfiltration. Dispersions can be prepared by incorporating the active compound into a sterile vehicle which contains the dispersing 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) which yield a powder of the active ingredient plus any additional desired ingredient from a sterile-filtered solution thereof.

When a therapeutically effective amount of a nucleic acid of the invention is administered, for example, by intravenous injection, cutaneous injection, or subcutaneous injection, the nucleic acid will take the form of a pyrogen-free, parenterally acceptable aqueous solution. Methods of preparing parenterally acceptable solutions are within the skill of the art in view of suitable pH, isotonicity, stability, and the like. Preferred pharmaceutical compositions for intravenous, cutaneous or subcutaneous injection contain, in addition to the nucleic acid, an isotonic vehicle, for example, sodium chloride injection, ringer's injection, dextrose and sodium chloride injection, lactated ringer's injection, or other vehicle known in the art. The pharmaceutical compositions of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives well known to those skilled in the art.

The amount of nucleic acid that can be combined with the carrier material to produce a single dosage form will vary depending on various factors including the subject to be treated and the particular mode of administration. In general, it will be the amount of the composition that produces the appropriate therapeutic effect in the particular situation. Typically, in one hundred percent, the amount ranges from about 0.01% to about 99% nucleic acid, from about 0.1% to about 70%, or from about 1% to about 30% nucleic acid in combination with a pharmaceutically acceptable carrier.

The nucleic acids may be prepared with carriers that protect the compounds from rapid release, such as controlled release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used. Many methods for preparing such formulations have been patented or are 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.

The dosage regimen can be adjusted to provide the best desired response (e.g., therapeutic response). For example, a dose may be administered, several divided doses may be administered over a period of time, or the dose may be proportionally reduced or increased depending on the particular circumstances, as indicated by the particular circumstances of the therapeutic situation. The ease of formulating parenteral compositions in dosage unit form for administration and uniformity of dosage is particularly advantageous when administered to a subject or patient. As used herein, dosage unit form refers to physically discrete units suitable as unitary dosages for the subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect. The specification of the dosage unit forms of the invention will depend on the particular nature of the active compound and the particular therapeutic effect to be achieved, as well as the treatment and sensitivity of any individual patient.

The nucleic acids or compositions of the invention may be produced using methods conventional in the art, including chemical synthesis, such as solid phase chemical synthesis.

The nucleic acids 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, the nucleic acids of the invention may be administered with a needleless hypodermic device. Examples of well known implants and modules that may be used in the present invention are in the art, including, for example, implantable micro infusion pumps for controlled rate delivery; a device for transdermal application; an infusion pump for delivery at a precise infusion rate; variable flow implantable infusion devices for continuous drug delivery; an osmotic drug delivery system. These and other such implants, administration systems and modules are known to those skilled in the art.

In certain embodiments, the nucleic acids or compositions of the invention may be formulated to ensure a desired distribution in vivo. In order to target therapeutic compounds or compositions of the invention to specific in vivo locations, they may be formulated, for example, in liposomes that may include one or more moieties that selectively translocate into specific cells or organs, thus enhancing targeted drug delivery.

The invention is characterized by high specificity at the level of molecular and tissue-directed delivery. The nucleic acid sequences of the invention are highly specific for their targets, meaning that they do not inhibit the expression of genes they are not designed to target, or only minimally inhibit the expression of genes they are not designed to target, and/or only inhibit the expression of low numbers of genes they are not designed to target. Further levels of specificity are achieved when the nucleic acid is linked to a ligand that is specifically recognized and internalized by a particular cell type. This is the case, for example, when the nucleic acid is linked to a ligand comprising a GalNAc moiety which is specifically recognized and internalized by the hepatocyte. This results in the nucleic acid inhibiting expression of its target only in cells targeted by the ligand to which the nucleic acid is linked. These two levels of specificity potentially confer a better safety profile than currently available treatments. In certain embodiments, the invention thus provides a nucleic acid of the invention linked to a ligand comprising one or more GalNAc moieties, or comprising one or more other moieties, which confers cell-type or tissue-specific internalization of the nucleic acid, thereby conferring additional specificity for target gene knockdown by RNA interference.

Nucleic acids as described herein may be formulated with lipids in the form of liposomes. Such formulations may be described in the art as liposome complexes (lipoplex). Compositions with lipids/liposomes can be used to aid in the delivery of the nucleic acids of the invention to target cells. The lipid delivery systems described herein may be used as alternatives to conjugated ligands. The modifications described herein may be present when the nucleic acids of the invention are used with lipid delivery systems or with ligand conjugate delivery systems.

Such lipid complexes may include lipid compositions comprising:

i) A cationic lipid, or a pharmaceutically acceptable salt thereof;

ii) a steroid;

iii) Phosphatidylethanolamine phospholipids; and/or

iv) pegylated lipids.

The cationic lipid may be an amino cationic lipid.

The cationic lipid component may be present in an amount of about 55 mole% to about 65 mole% of the total lipid content of the composition. In particular, the cationic lipid component is about 59 mole% of the total lipid content of the composition.

The composition may further comprise a steroid. The steroid may be cholesterol. The steroid may be present in an amount of about 26 mole% to about 35 mole% of the total lipid content of the lipid composition. More particularly, the steroid may be present in an amount of about 30 mole% of the total lipid content of the lipid composition.

Phosphatidylethanolamine phospholipids may be selected from the group consisting of 1, 2-di-phytyl-sn-glycero-3-phosphate ethanolamine (DPPE), 1, 2-di-oleoyl-sn-glycero-3-phosphate ethanolamine (DOPE), 1, 2-di-stearoyl-sn-glycero-3-phosphate ethanolamine (DSPE), 1, 2-di-lauroyl-sn-glycero-3-phosphate ethanolamine (DLPE), 1, 2-di-myristoyl-sn-glycero-3-phosphate ethanolamine (DMPE), 1, 2-di-palmitoyl-sn-glycero-3-phosphate ethanolamine (DPPE), 1, 2-di-oleoyl-sn-glycero-3-phosphate ethanolamine (dlospe), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate ethanolamine (POPE), 1, 2-di-erucyl-sn-glycero-3-phosphate ethanolamine (DSPE), 1, 2-di-squaloyl-sn-glycero-3-phosphate ethanolamine (DSPE), and 1-oleoyl-sn-glycero-3-phosphate ethanolamine (SLPE). The amount of phospholipid may be about 10 mole% of the total lipid content of the composition.

The pegylated lipid may be selected from the group consisting of 1, 2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol (DMG-PEG) and C16-ceramide-PEG. The amount of pegylated lipid may be about 1 to 5 mole% of the total lipid content of the composition.

The cationic lipid component may be present in the composition in an amount of about 55 mole% to about 65 mole% of the total lipid content of the lipid composition, particularly about 59 mole% of the total lipid content of the lipid composition.

The composition may have a ratio 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 a molar ratio of components i) i:i) i:ii) i:v) of 65:24:10:1.

Neutral liposome compositions can be formed, for example, from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). The anionic liposome composition may be formed from dimyristoyl phosphatidylglycerol, while the anionic fusogenic liposome may be formed predominantly from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposome composition can be formed from Phosphatidylcholine (PC), such as soybean PC and egg PC. Another type is formed by a mixture of phospholipids and/or phosphatidylcholine and/or cholesterol.

Positively charged synthetic cationic lipids, N- [1- (2, 3-dioleoyloxy) propyl ] -N, N-trimethylammonium chloride (DOTMA), can be used to form small liposomes that spontaneously interact with nucleic acids to form lipid-nucleic acid complexes capable of fusing with negatively charged lipids of cell membranes of tissue culture cells. DOTMA analogs can also be used to form liposomes.

Derivatives and analogs of the lipids described herein can also be used to form liposomes.

Liposomes containing nucleic acids can be prepared by a variety of methods. In one example, the lipid component of the liposome is dissolved in a detergent such that the lipid component forms micelles. For example, the lipid component may be an ampholytic cationic lipid or a lipid conjugate. The detergent may have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholate, CHAPS, octyl glucoside, deoxycholate, and lauroyl sarcosine. Then, the nucleic acid preparation is added to the micelle containing the lipid component. Cationic groups on the lipids interact with the nucleic acids and condense around the nucleic acids to form liposomes. After coagulation, the detergent is removed, for example by dialysis, to obtain a liposome preparation of the nucleic acid.

If desired, carrier compounds which aid in the coagulation can be added during the coagulation reaction, for example by controlled addition. For example, the carrier compound may be a polymer other than a nucleic acid (e.g., spermine or spermidine). The pH may also be adjusted to promote coagulation.

The nucleic acid preparation of the present invention may contain a surfactant. In one embodiment, the nucleic acid is formulated as an emulsion comprising a surfactant.

The non-ionized surfactant is a nonionic surfactant. Examples include nonionic esters such as ethylene glycol esters, propylene glycol esters, glycerol esters, and the like, nonionic alkanolamides, and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers.

Surfactants that carry a negative charge when dissolved or dispersed in water are anionic surfactants. Examples include carboxylates such as soaps, acyl lactates, acyl amides of amino acids, sulfates such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonate, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates.

Surfactants that carry a positive charge when dissolved or dispersed in water are cationic surfactants. Examples include quaternary ammonium salts and ethoxylated amines.

Surfactants having the ability to carry a positive or negative charge are amphoteric surfactants. Examples include acrylic acid derivatives, substituted alkylamides, N-alkyl betaines and phospholipids.

"micelle" is defined herein as a specific type of molecular assembly in which amphiphilic molecules are arranged in a spherical structure such that all hydrophobic portions of the molecule are directed inward, leaving hydrophilic portions in contact with the surrounding water. If the environment is hydrophobic, the opposite arrangement exists. Micelles may be formed by mixing an aqueous solution of nucleic acid, an alkali metal alkyl sulfate, and at least one micelle forming compound.

Exemplary micelle-forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, oleic acid monoglyceride, monooleate, monolaurate, borage oil, evening primrose oil, menthol, trihydroxy oxo-cholesteryl glycine and pharmaceutically acceptable salts thereof, glycerol, polyglycerol, lysine, polylysine, triolein, polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethers and analogues thereof, chenodeoxycholate, deoxycholate, and mixtures thereof.

Phenol and/or m-cresol may be added to the mixed micelle composition to act as a stabilizer and preservative. Isotonic agents such as glycerol may be added.

The nucleic acid formulation may be incorporated into particles, such as microparticles. Microparticles may be prepared by spray drying, lyophilization, evaporation, fluid bed drying, vacuum drying, or a combination of these methods.

Definition of the definition

As used herein, the terms "inhibit," "down-regulate," or "reduce" with respect to gene expression refer to the reduction of gene expression, or the level of an RNA molecule or equivalent RNA molecule (e.g., mRNA) encoding one or more proteins or protein subunits, or the activity of one or more proteins or protein subunits, below that observed in the absence of a nucleic acid or conjugated nucleic acid of the invention, or as compared to that obtained with an siRNA molecule having no known homology to a human transcript (referred to herein as a non-silencing control). Such controls can be conjugated and modified in a similar manner to the molecules of the invention and delivered into the target cells by the same route. Expression after treatment with a nucleic acid of the invention can be reduced to 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 5% or 0% or intermediate values, or less than that observed in the absence of the nucleic acid or conjugated nucleic acid. Expression can be measured in the cell to which the nucleic acid is applied. Alternatively, the level may be measured in a different set of cells or in a tissue or organ or in a body fluid such as blood or plasma, especially if the nucleic acid is administered to the subject. The inhibition levels are in particular measured under selected conditions, since 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 can be measured, for example, after 24 hours or 48 hours of treatment with nucleic acids at a concentration of 0.038nM to 10. Mu.M, in particular 1nM, 10nM or 100 nM. These conditions may be different for different nucleic acid sequences or for different types of nucleic acids, for example for unmodified or conjugated to a ligand or unconjugated nucleic acid. Examples of suitable conditions for determining the level of inhibition are described in the examples.

Nucleic acid refers to a nucleic acid comprising two strands of nucleotides, which is capable of interfering 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 chain, may also be a guide chain; and a second chain, which may also be a passenger chain. 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.

Nucleic acids may include ribonucleotides, modified ribonucleotides, deoxyribonucleotides, or nucleotide analogs that are non-nucleotides capable of mimicking a nucleotide, such that they can be "paired" with a corresponding base on a target sequence or complementary strand. The nucleic acid may further comprise a double-stranded nucleic acid portion or double-stranded region formed by all or a portion of the first strand (also referred to in the art as a guide strand) and all or a portion of the second strand (also referred to in the art as a passenger strand). A double-stranded region is defined as starting at 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.

Double-stranded region refers to a region in two complementary or substantially complementary oligonucleotides that form base pairs with each other by Watson-Crick base pairing or any other means that allows a duplex to form between complementary or substantially complementary oligonucleotide strands. For example, an oligonucleotide strand having 21 nucleotide units may base pair with another oligonucleotide having 21 nucleotide units, but only 19 nucleotides on each strand are complementary or substantially complementary, such that a "double stranded region" consists of 19 base pairs. The remaining base pairs may exist as 5 'and 3' overhangs, or as single stranded regions. Further, within the double-stranded region, 100% complementarity is not required; substantial complementarity is permitted within the double-stranded region. Substantial complementarity refers to complementarity between strands such that they are capable of annealing under biological conditions. Techniques for empirically determining whether two strands are capable of annealing under biological conditions are well known in the art. Alternatively, both chains may be synthesized and added together under biological conditions to determine whether they anneal to each other. The first strand and a portion of the second strand forming at least one duplex region may be fully complementary to each other and at least partially complementary. Depending on the length of the nucleic acid, a perfect match in base complementarity between the first strand and the second strand is not necessarily required. However, the first strand and the second strand must be capable of hybridizing under physiological conditions.

As used herein, the term "unpaired nucleotide analog" refers to a nucleotide analog comprising a non-base pairing moiety, including but not limited to: 6-deaminoadenosine (gourmet), 4-Me-indole, 3-nitropyrrole, 5-nitroindole, ds, pa, N3-Me ribo U, N3-Me riboT, N3-MedC, N3-Me-dT, N1-Me-dG, N1-Me-dA, N3-ethyl-dC, and N3-MedC. In some embodiments, the non-base pairing nucleotide analog is a ribonucleotide. In other embodiments, it is a deoxyribonucleotide.

As used herein, the term "terminal functional group" includes, but is not limited to, halogen, alcohol, amine, carboxylic acid, ester, amide, aldehyde, ketone, and ether groups.

As used herein, an "overhang" has its normal and customary meaning in the art, i.e., a single-stranded portion of a nucleic acid that extends beyond the terminal nucleotides of the complementary strand in a double-stranded nucleic acid. The term "blunt end" encompasses double-stranded nucleic acids whereby the two strands terminate at the same position, whether or not the terminal nucleotides base pair. The terminal nucleotides of the first strand and the second strand at the blunt end may be base paired. The terminal nucleotides of the first strand and the second strand at the blunt end may not be paired. The two nucleotides of the first strand and the second strand at the blunt end may be base paired. The two nucleotides of the first strand and the second strand at the end of the blunt end may not be paired.

The term "serinol-derived linker moiety" refers to a linker moiety comprising the structure:

the O atom of the structure is typically attached to the RNA strand and the N atom is typically attached to the targeting ligand.

"protein S" in the context of the present invention relates to the human "vitamin K-dependent protein S" encoded by the gene PROS1 (NCBI gene ID: 5627) (UniProt ID P07225).

In the context of the present specification, the term "hemophilia" relates to a condition in which the body's ability to produce blood clots is impaired. The term "hemophilia" encompasses conditions or disorders which are hereditary hemophilia, hemophilia a or B or C, acquired hemophilia, fibrinogen free, hypofibrinogen, paramemophilia, arthrosis (AH), genetic defects of factors II, V, VII, X and/or XI, combined defects of factors V and VIII, von willebrand's disease, von willebrand's syndrome, acquired coagulation factor defects.

The terms "patient," "subject," and "individual" are used interchangeably and refer to a human or non-human animal. These terms include mammals, such as humans, primates, livestock animals (e.g., cows, pigs), companion animals (e.g., dogs, cats), and rodents (e.g., mice and rats).

As used herein, "treatment" or "treatment" and grammatical variants thereof refer to methods for achieving beneficial or desired clinical results. The term may refer to slowing the rate of onset or progression of a condition, disorder or disease, reducing or alleviating symptoms associated therewith, producing complete or partial regression of the condition, or some combination of any of the foregoing. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, reduced or lessened symptoms, reduced extent of disease, stabilization of disease state (i.e., not worsening), delayed or slowed progression of disease, improved or slowed progression of disease state, and remission (whether partial or total). "treatment" may also refer to prolonged survival relative to the expected survival time when untreated. Thus, a subject (e.g., a human) in need of treatment may be a subject who has been afflicted with the disease or condition in question. The term "treatment" encompasses inhibition or reduction of an increase in the severity of a pathological state or symptom relative to the absence of treatment, and is not necessarily meant to imply a complete cessation of the associated disease, disorder, or condition.

As used herein, the term "preventing" and grammatical variants thereof refers to methods for preventing the development of a condition, disease, or disorder, or altering the pathological condition of a condition, disease, or disorder. Accordingly, "prevention" may refer to preventive or preventative measures. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, prevention or alleviation of the symptoms, progression or development of a disease, whether detectable or undetectable. Thus, a subject (e.g., a human) in need of prevention may be a subject that has not been afflicted with the disease or condition in question. The term "preventing" encompasses slowing the onset of the disease relative to the absence of treatment, and is not necessarily meant to imply permanent prevention of the disease, disorder or condition in question. Thus, in certain contexts, "preventing" or "prevention" of a condition may refer to reducing the risk of developing the condition, or preventing or delaying the development of symptoms associated with the condition.

As used herein, an "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, e.g., prevents or treats a condition of interest or beneficially alleviates symptoms associated with the condition.

As used herein, the term "pharmaceutically acceptable salt" refers to a salt that is not harmful to the patient or subject to whom the salt in question is administered. It may be, for example, a salt selected from acid addition salts and basic salts. Examples of acid addition salts include chloride salts, citrate salts, and acetate salts. Examples of the basic salt include a salt in which the cation is selected from alkali metal cations such as sodium or potassium ionsAlkaline earth metal cations such as calcium or magnesium ions, and substituted ammonium ions such as type N (R ¹ )(R ² )(R ³ )(R ⁴ ) ⁺ Wherein R is ¹ 、R ² 、R ³ And R is ⁴ Typically independently hydrogen, optionally substituted C _1-6 Alkyl or optionally substituted C _2-6 Alkenyl groups. Correlation C _1-6 Examples of alkyl groups include methyl, ethyl, 1-propyl and 2-propyl. C possibly related _2-6 Examples of alkenyl groups include vinyl, 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 newer versions thereof), "Encyclopaedia of Pharmaceutical Technology",3rd edition,James Swarbrick (Ed.), informa Healthcare USA (inc.), NY, USA,2007, and j.pharm.sci.66:2 (1977). The "pharmaceutically acceptable salts" retain the desired biological activity of the parent compound in nature without imparting any undesirable effects relative to the compound. Examples of pharmaceutically acceptable salts include acid addition salts and base addition salts. The acid addition salts include salts derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphorous acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, and the like, or from non-toxic organic acids such as aliphatic monocarboxylic and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic 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, and salts derived from non-toxic organic amines such as N, N' -dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and the like.

The term "pharmaceutically acceptable carrier" encompasses any standard pharmaceutical carrier. Pharmaceutically acceptable carriers for therapeutic use are well known in the pharmaceutical arts and are described, for example, in Remington's Pharmaceutical Sciences, mack Publishing co. (a.r. gennaro dit.1985). For example, sterile saline at slightly acidic or physiological pH as well as phosphate buffered saline may be used. Exemplary pH buffers include phosphate, citrate, acetate, TRIS (hydroxymethyl) aminomethane (TRIS), N-TRIS (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS), ammonium bicarbonate, diethanolamine, histidine (which is a preferred buffer), arginine, lysine, or acetate or mixtures thereof. The term further encompasses any of the agents listed in the united states pharmacopeia for use in animals, including humans. "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 the route of administration selected, the nucleic acid may be coated in one or more materials that are expected to protect the compound from acids to which the nucleic acid may be exposed and other natural inactivating conditions when administered to a subject by a particular route of administration.

In the context of the present invention, the term "solvate" refers to a complex of defined stoichiometry formed between a solute (in this case, a nucleic acid compound according to the invention or a pharmaceutically acceptable salt thereof) and a solvent. The solvent in this regard may be, for example, water or another pharmaceutically acceptable, typically small molecule, organic species such as, but not limited to, acetic acid or lactic acid. When the solvent in question is water, such solvates are generally referred to as hydrates.

The invention will now be described with reference to the following non-limiting drawings and examples.

Drawings

FIG. 1 shows possible synthetic pathways for DMT-serinol (GalNAc) -CEP and CPG.

FIG. 2 shows inhibition of PROS1 mRNA levels in human cells by transfection of different PROS1 siRNAs.

FIG. 3 shows a dose response test for decreasing PROS1 mRNA levels in human cells by transfection of PROS1 siRNA.

Fig. 4 shows inhibition of PROS1 target gene expression in primary mouse hepatocytes by receptor-mediated uptake of PROS1 siRNA conjugates.

Fig. 5 shows inhibition of PROS1 target gene expression in primary human hepatocytes by receptor-mediated uptake of PROS1 siRNA conjugates.

FIG. 6 shows that loss of X-enzyme activity rescues PROS1 ^-/- And (3) a mouse. A, schematic model of thrombin generation under hemophilia conditions. One of the major coagulation complexes is the intrinsic decaase (X-ase) complex. The decases include activated FIX (FIXa) as protease, activated FVIII (FVIIIa) as cofactor and Factor X (FX) as substrate. Although Tissue Factor (TF) production or exposure at the site of injury is the major event in initiating coagulation through an exogenous pathway, endogenous pathway decases are important because the amount of active TF available in vivo is limited and TFPI is present, which inhibits TF/activated factor VII (FVIIa) complex when complexed with activated FX (FXa) (fig. 6A). Thus, sustained thrombin generation was dependent on FIX and FVIII activation (fig. 6A). This process is amplified because FVIII is activated by FXa and thrombin, FIX is activated by FVIIa and activated factor XI (FXIa), the latter factor previously activated by thrombin. Thus, FVIII and FIX activation gradually increases as FXa and thrombin B are formed, an experimental approach to enhance thrombin generation in severe hemophilia a and B by targeting PROS 1. C-D, mouse model validation and DIC hematological parameter evaluation in hemophilia adult mice with and without PROS1 deficiency: F8F 8 ^-/- PROS1 ^+/+ 、F8 ^-/- PROS1 ^+/- And F8 ^-/- PROS1 ^-/- (C) And F9 ^-/- PROS1 ^+/+ 、F9 ^-/- PROS1 ^+/- And F9 ^-/- PROS1 ^-/- PS (protein S; antigenic), FVIII (thrombotic activity) or FIX (thrombotic activity) plasma levels (n=5/group) in adult mice (D); platelets (n=7/group), fibrinogen (n=8/group), PT (n=6/group), and TAT (n=6/group) in hemophilia a group (C); and platelets (n=5/group), fibrinogen (n=4/group), PT (n=4/group), and TAT (n=4/group) in hemophilia B group (D). E-F, single intravenous infusion of 2U/g recombinant FVIIIF8 after 24 hours ^-/- PROS1 ^-/- Macroscopic images of the lungs of mice (E), and corresponding microscopic evaluation of fibrin clots in lung sections (F). G, at F8 ^-/- PROS1 ^+/+ And F8 ^-/- PROS1 ^-/- Recombinant FVIII>0.3u/g +.5 intravenous injections>After hours (injection time point: 1 hour before catheterization, 1 hour, 4 hours, 8 hours and 16 hours after catheterization) (n=3) (G, white and black bars), and at F8 ^-/- PROS1 ^-/- (n=3) (G, dashed line) plasma levels of fibrinogen and TAT 24 hours after one intravenous injection, and representative immunohistochemical analysis can be performed at repeated intravenous injections of 0.3 u/G->(H) After 24 hours, and one intravenous injection of 0.3U/g +.>(i) After F8 ^-/- PROS1 ^-/- Fibrin clots were detected in lung and liver sections. All data are expressed as mean ± standard error (s.e.m.); ns, not significant; * P < 0.05; p is less than 0.005.

Fig. 7 shows a mouse model of thrombosis. A-C, at F8 ^+/+ PROS1 ^+/+ 、F8 ^-/- PROSl ^+/+ 、F8 ^-/- PROS1 ^+/- And F8 ^-/- PROS1 ^-/- TF-induced venous thromboembolism (n=10/genotype) in mice. Anesthetized mice were injected intravenously via the inferior vena cava with varying doses of recombinant TF (Innovin): 1/2 dilution in A (. About.4.3 nM TF) and 1/4 dilution in B-C (. About.2.1 nM TF). In (A), a group F8 ^+/+ PROS1 ^+/+ Mice were injected with low molecular weight heparin (enoxaparin 60 μg/g subcutaneous injection (s.c.)). The time to respiratory arrest onset was recorded for at least 2 minutes. The experiment was terminated at 20 minutes. Kaplan-Meier survival curve (a-B). C, at respirationAt 2 minutes after cessation of onset or at the completion of the 20 minute observation period, the lungs were excised and examined for fibrin clots (immunostaining for insoluble fibrin, mAb clone 102-10). D, at F8 ^+/+ PROS1 ^+/+ 、F8 ^-/- PROS1 ^+/+ And F8 ^-/- PROS1 ^-/- Thrombosis in mesenteric artery under FeCl3 injury recorded by in vivo microscopy in mice, representative experiment (n=3/genotype). D, at F8 ^+/+ PROS1 ^+/+ 、F8 ^-/- PROS1 ^+/+ And F8 ^-/- PROS1 ^-/- FeCl recorded by in vivo microscopy in mice ₃ Thrombosis in mesenteric arteries under injury, representative experiment (n=3/genotype).

Fig. 8 shows a tail bleeding model. Blood was collected in fresh saline tubing after tail transection for 30 minutes (a) and 10 minutes (B) at 2mm (a) and 4mm (B); total blood loss (μl) was then measured. F8F 8 ^+/- PROS1 ^+/+ And F8 ^+/+ PROS1 ^+/+ Mice (white bars) were used as controls (all groups n=5 in a, all groups n=6 in a). C, anti-human Ps antibodies altered tail bleeding after 4 mm transection.

Fig. 9 shows an acute arthrodesis model. A, at F8 ^-/- PROS1 ^+/+ 、F8 ^-/- PROS1 ^+/- 、F8 ^-/- PROS1 ^-/- And F8 ^+/+ PROS1 ^+/+ Differences in knee diameters in mice 72 hours post-injury and pre-injury. B, at F8 ^+/+ PROS1 ^+/+ 、F8 ^-/- PROS1 ^+/+ And F8 ^-/- PROS1 ^-/- Microscopic evaluation in mice (Masson trichrome staining and immunostaining of insoluble fibrin) represents intact and injured knee joint cavities for 72 hours. C, in vivo (in vivo) mPS silencing using specific siRNA: f8 treated with single intraperitoneal (i.p.) infusion of mPS siRNA or control siRNA ^-/- PROS1 ^+/- And F8 ^-/- PROS1 ^+/+ Mice were evaluated for joint diameter 72 hours after injury. D, F8 previously treated with mPS siRNA or Ctrl siRNA ^-/- PROS1 ^+/+ In mice, microscopic evaluation (Masson trichromatic staining) of the knee joint cavity after 72 hours of representative leg injuryAnd (3) inner part. The measured values are expressed as mean ± standard error. * P is less than 0.05; * P < 0.005; * P < 0.0005; * P < 0.0001.

Fig. 10 shows that both PS and TFPI are expressed in mouse membranes. A, for F8 from previous treatment with Ctrl-siRNA or mPS-siRNA ^-/- PROS1 ^+/+ The mice were immunostained for Ps and TFPI in the knee joint cavity of the injured knee. Arrow points to synovial tissue, arrow points to vascular structure, and both PS and TFPI are positive. The boxes in the upper graph (scale: 200 μm) show the enlarged areas in the lower graph (scale: 50 μm). B, for F8 from ^-/- PROS1 ^+/+ And F8 ^-/- PROSl ^-/- Mice were immunostained for TFPI in the knee joint cavity of the intact knee. C-E Western blot analysis of conditioned medium from primary mouse fibroblast-like synoviocytes (FLS) cultures using anti-PS (C) and anti-TFPI (d) antibodies. Platelet Free Plasma (PFP), platelet Protein Lysate (PLT), mouse PS (mPS) was used as positive control (c). Expression of TFPI isoforms was determined by comparing the molecular weights of deglycosylated TFPI and fully glycosylated TFPI. Mouse placenta was used as a positive control for TFPI alpha. E-F, western blot analysis of total protein lysates isolated from FLS after 24 hours of incubation with anti-PS (F) and anti-TFPI (E) antibodies to thrombin (Thr, +) or solvent (vehicle, -). Human recombinant TFPI full length was used as a positive control for TFPI alpha (hrTFPI). Blots represent three independent experiments.

Fig. 11 shows PS and TFPI in human synovium. A, PS and TFPI are expressed in synovial tissue of HA (on demand and prophylaxis), HB (on demand) or Osteoarthritis (OA) patients. The arrow points to the synovial lining layer and the arrow points to the vascular structure in the lining layer, positive for both PS and TFPI. Scale bar: 50 μm. B, western blot analysis was performed on conditioned medium of primary human FLS (hFLS) cultures from healthy individuals and OA patients, using anti-TFPI antibodies before and after deglycosylation. Human platelet lysate (hPLT) was used as a positive control for TFPI alpha. Blots represent three independent experiments.

FIG. 12 shows thrombin generation and fibrin network in hemophilia A, TF- (1 pM) induced from F8 ^-/- PROS1 ^+/+ And F8 ^-/- PROS1 ^-/- Thrombin generation in PRP in mice, shows TFPI-dependent PS activity. B from F8 ^-/- PROS1 ^+/+ And F8 ^-/- PROS1 ^-/- APC-dependent PS activity in PRP and PFP in mice. C, from F8 ^+/+ PROS1 ^+/+ 、F8 ^-/- PROS1 ^+/+ And F8 ^-/- PROS1 ^-/- And from F9 ^+/+ PROS1 ^+/+ 、F9 ^-/- PROS1 ^+/+ And F9 ^-/- PROS1 ^-/- Representative scanning electron microscope images of fibrin structures. D-G, thrombin generation initiated by low TF concentration (1 pM) in PFP (D-E) and PRP (F-G) from severe HA patients (FVIII < 1%) without (D, F) and with high titer inhibitors (E, G). The measured values are expressed as mean ± standard error. * P < 0.005; * P < 0.0005.

FIG. 13 shows a genotyping method. By hybridization F8 ^-/- PROS1 ^+/- (a-c) and F9 ^-/- PROS1 ^+/- (d-f) genotype obtained from mice. a, amplifying the PROS1 allele by multiplex PCR. Then, electrophoresis is carried out on the PCR product; according to Saller,2009, the molecular weight of the wt band (234 bp) is lower than that of the ineffective band (571 bp). b, multiplex PCR was set up to amplify the wt band (620 bp) and the null band (420 bp) of the F8 allele from genomic DNA. c, PCR products amplified from the F8 allele on the same samples as in (a) (null band: 420 bp). d, amplifying PROS1 alleles by multiplex PCR. Then, electrophoresis is carried out on the PCR product; according to Saller,2009, the molecular weight of the wt band (234 bp) is lower than that of the ineffective band (571 bp). e, multiplex PCR was set up to amplify the wt band (320 bp) and the null band (550 bp) of the F9 allele from genomic DNA. F, PCR products amplified from the F9 allele on the same samples as in (d) (null band: 550 bp).

Fig. 14 shows histology under physiological conditions. At F8 ^-/- PROS1 ^-/- And F8 ^-/- PROS1 ^+/+ F9 ^-/- PROS1 ^+/+ And F9 ^-/- PROS1 ^-/- Insoluble fibrin was immunostained on liver, lung, kidney and brain sections of mice. Scale bar: 100 μm.

FIG. 15 shows that genetic deletion of PROS1 can preventHemophilia B mice develop joint hematochezia. A, F9 ^-/- PROS1 ^+/+ 、F9 ^-/- PROS1 ^+/- 、F9 ^-/- PROS1 ^-/- And F9 ^+/+ PROS1 ^+/+ The difference between the knee diameter of the mice 72 hours after injury and before injury. B, at F9 ^+/+ PROS1 ^+/+ 、F9 ^-/- PROS1 ^+/+ And F9 ^-/- PROS1 ^-/- In mice, microscopic evaluation (Masson trichromatic staining and insoluble fibrin staining, mAb clone 102-10) represents the knee joint cavity after 72 hours of intact and leg injury. Scale bar: 500 μm. The measured values are expressed as mean ± standard error. * P < 0.0005.

Figure 16 shows the quantification of fibrin network density and fiber branching. a-b from F8 ^+/+ PROS1 ^+/+ ，F8 ^-/- PROS1 ^+/+ And F8 ^-/- PROS1 ^-/- Fibrin network in mice. c-d from F9 ^+/+ PROS1 ^+/+ ，F9 ^-/- PROS1 ^+/+ And F9 ^-/- PROS1 ^-/- Is a fibrin network of (a). Quantification of fibrin network density (a and c). Quantification of fiber branching (b and d). The measured values are expressed as mean ± standard error. * P < 0.0005.

Fig. 17 shows that different PROS1 siRNA conjugates inhibit PROS1 target gene expression in primary hepatocytes.

Fig. 18 shows inhibition of human PROS1 gene expression in primary human hepatocytes by receptor-mediated uptake of different PROS1 siRNA conjugates.

Figure 19 shows inhibition of expression of the PROS1 gene in vivo by a single administration of different PROS1 siRNA conjugates.

Figure 20 shows inhibition of PROS1 gene expression in hemophilia mice by single administration of a PROS1 siRNA conjugate.

Fig. 21 shows that treatment with PROS1 siRNA conjugate reduces knee swelling in an acute arthritic model.

Figure 22 shows a haemostatic profile of an improved hemophilia a animal model treated with a PROS1 siRNA conjugate.

FIG. 23 shows a dose-dependent decrease in protein S mRNA levels in human cells by transfection of protein S siRNA at a concentration of 1nM to 0.00001 nM.

Fig. 24 shows inhibition of PROS1 target gene expression in primary human hepatocytes by receptor-mediated uptake of PROS1 siRNA conjugates.

Fig. 25 shows inhibition of PROS1 target gene expression in primary cynomolgus monkey hepatocytes by receptor-mediated uptake of PROS1 siRNA conjugates.

Examples

Example 1 Synthesis of building blocks

The synthetic routes for DMT-serinol (GalNAc) -CEP and CPG as described below are summarized in FIG. 1. Starting material DMT-Serinol (H) (1) was prepared from commercially available L-serine according to the published procedure of the literature (Hoevelmann et al chem. Sci.,2016,7, 128-135). GalNAc (Ac) was prepared according to the method disclosed in the literature (Nair et al J.am.chem.Soc.,2014, 136 (49), pp 16958-1696, starting from commercially available peracetylated galactosamine ₃ )-C ₄ H ₈ -COOH (2). The phosphonating reagent 2-cyanoethyl-N, N-diisopropylchlorophosphazene amidinate (4) is commercially available. Synthesis of (vp) -mU-phos was performed as described by Prakash, nucleic Acids Res.2015, 43 (6), 2993-3011 and Haraszti, nucleic Acids Res.2017, 45 (13), 7581-7592. The synthesis of phosphoramidite derivatives of ST43 (ST 43-phos) and ST23 (ST 23-phos) can be carried out as described in WO 2017/174657.

DMT-serinol (GalNAc) (3)

HBTU (9.16 g,24.14 mmol) was added to GalNAc (Ac) ₃ )-C ₄ H ₈ In a stirred solution of-COOH (2) (11.4 g,25.4 mmol) and DIPEA (8.85 ml,50.8 mmol). After 2 minutes activation time, a solution of DMT-serinol (H) (1) (10 g,25.4 mmol) in acetonitrile (anhydrous) (200 ml) was added to the stirred mixture. LCMS showed good conversion after 1 hour. The reaction mixture was concentrated in vacuo. The residue was dissolved in EtOAc, followed by washing with water (2×) and brine. Na for organic layer ₂ SO ₄ Dried, filtered and concentrated under reduced pressure. By column chromatography (3% MeOH in CH) ₂ Cl ₂ +1％Et ₃ N,700g silica) further purified the residue. Combining the product-containing fractions, concentrating, andby CH ₂ Cl ₂ (2X) stripping gave 10.6g (51%) DMT-serinol (GalNAc) (3) as an off-white foam.

DMT-serinol (GalNAc) -CEP (5)

2-cyanoethyl-N, N-diisopropylchlorophosphamide (4) (5.71 ml,25.6 mmol) was slowly added to DMT-serinol (GalNAc) (3) (15.0 g,17.0 mmol), DIPEA (14.9 ml,85 mmol) and under argon atmosphere at 0deg.CThe molecular sieve was in a stirred mixture in dichloromethane (dry) (150 ml). The reaction mixture was stirred at 0 ℃ for 1 hour. TLC indicated complete conversion. The reaction mixture was filtered and concentrated in vacuo to give a thick oil. The residue was dissolved in dichloromethane and purified by flash chromatography (0-50% acetone in toluene 1% Et ₃ N,220g silica). The product containing fractions were combined and concentrated in vacuo. The resulting oil was stripped with MeCN (2×) to give 13.5g (77%) of colorless DMT-serinol (GalNAc) -CEP (5) foam.

DMT-serinol (GalNAc) -succinate (6)

DMAP (1.11 g,9.11 mmol) was added to a stirred mixture of DMT-serinol (GalNAc) (3) (7.5 g,9.11 mmol) and succinic anhydride (4.56 g,45.6 mmol) in dichloromethane (50 ml) and pyridine (50 ml) under an argon atmosphere. After stirring for 16 hours, the reaction mixture was concentrated in vacuo, the residue was taken up in EtOAc and washed with 5% citric acid (aq). The aqueous layer was extracted with EtOAc. Subsequently with saturated NaHCO ₃ The combined organic layers were washed with aqueous solution and brine, and dried over Na ₂ SO ₄ Dried, filtered and concentrated in vacuo. By flash chromatography (0-5% MeOH in CH) ₂ Cl ₂ +1％Et ₃ N,120g silica). The product containing fractions were combined and concentrated in vacuo. The residue was stripped with MeCN (3×) to give 5.9g (70%) DMT-serinol (GalNAc) -succinate (6).

DMT-serinol (GalNAc) -succinyl-xaa-CPG (7)

DMT-serinol (GalNAc) -succinate (6) (1 equivalent) and HBTU (1.1 equivalent) were dissolved in CH ₃ CN (10 ml). Diisopropylethylamine (2 eq) was added to the solution, and the mixture was spun for 2 minutes, followed by the addition of natural amino-xaa-CPG (500A, 88. Mu. Mol/g,1 eq). The suspension was gently shaken on a wrist shaker at room temperature for 16 hours, then filtered and washed with acetonitrile. The solid support was dried under reduced pressure for 2 hours. By and Ac ₂ O/2, 6-lutidine/NMI the unreacted amine on the support was capped by stirring at room temperature (2X 15 min). Washing of the support was repeated as described above. The solid was dried under vacuum to give DMT-serinol (GalNAc) -succinyl-lcaa-CPG (7) (loading: 34. Mu. Mol/g, determined by detritylation assay).

EXAMPLE 2 oligonucleotide Synthesis

The example compounds were synthesized according to the following methods and are known to those skilled in the art. The assembly of the oligonucleotide strands and the linker building blocks is carried out by solid phase synthesis using the phosphoramidite method.

Downstream cleavage, deprotection and purification follow standard procedures known in the art.

Oligonucleotide synthesis was performed on AKTA oligo lot 10 using commercially available 2'o-methyl RNA and 2' fluoro-2 ' deoxy RNA bases loaded with CPG solid support and phosphoramidite (all standard protections, chemGenes, linkTech). The synthesis of DMT- (S) -serinol (GalNAc) -succinyl-xaa-CPG (7) and DMT- (S) -serinol (GalNAc) -CEP (5) is described in example 1.

Auxiliary reagents were purchased from EMP Biotech. Anhydrous acetonitrile (< 20ppm H) using 0.1M phosphoramidite ₂ O) solution synthesis was performed using Benzylthiotetrazole (BTT) as activator (0.3M in acetonitrile). The coupling time was 10 minutes. Using Cap/OX/Cap or Cap/Thio/Cap circulation (Cap: ac) ₂ O/NMI/lutidine/acetonitrile, oxidant: 0.05M I ₂ In pyridine/H ₂ O). Phosphorothioate was introduced using a commercially available thiolating reagent, 50 mM EDITH in acetonitrile (Link Technologies). DMT cleavage was achieved by treatment with a 3% solution of dichloroacetic acid in toluene. After the completion of the program synthesis cycle, diethylamine (DEA) washing was performed. All oligonucleotides were synthesized in DMT-off mode.

Ligation of the serinol (GalNAc) moiety is achieved by using base-supported (S) -DMT-serinol (GalNAc) -succinyl-xaa-CPG (7) or (S) -DMT-serinol (GalNAc) -CEP (5). The triple antenna GalNAc cluster (ST 23/ST 43) was introduced by sequential coupling of branched trisomy imide derivatives (C6 XLT-phos) followed by coupling of GalNAc amide (ST 23-phos). The ligation of the (vp) -mU moiety is achieved using the (vp) -mU-phos in the last synthesis cycle. (vp) -mU-phos does not provide a hydroxyl group suitable for further synthesis of extension and therefore does not have a DMT-group. Thus, the coupling of (vp) -mU-phos leads to termination of synthesis.

To remove the methyl ester of the masked vinylphosphonate, the CPG carrying the fully assembled oligonucleotides was dried under reduced pressure and transferred to a 20 ml PP injection reactor for solid phase peptide synthesis equipped with a disc frit (Carl Roth GmbH). CPG was then combined with 250. Mu.L of LTMSBr and 177. Mu.L of pyridine in CH at room temperature ₂ Cl ₂ In (0.5 ml/. Mu. Mol of solid support-bound oligonucleotides) and sealing the reactor with a Luer cap. The reaction vessel was stirred slightly over 2X 15 minutes, the excess reagent was discarded and the remaining CPG was washed 2 times with 10ml of acetonitrile. Further downstream processing was not altered from any of the other example compounds.

The single strand was excised from CPG by treatment with 40% aqueous methylamine (90 min, room temperature). The resulting crude oligonucleotide was purified by ion exchange chromatography (Resource Q,6ml,GE Healthcare) using a sodium chloride gradient by AKTA pure HPLC system. The fractions containing the product were pooled, desalted on a size exclusion column (Zetadex, EMP Biotech) and lyophilized until further use.

All final single-stranded products were analyzed by AEX-HPLC to demonstrate their purity. Identity of the respective single stranded products was confirmed by LC-MS analysis.

Example 3 double strand formation

Each single strand was dissolved in H at a concentration of 60 OD/ml ₂ O. Two separate oligonucleotide solutions are added together to the reaction vessel. In order to monitor the reaction more easily, titration was performed. The first strand was added at 25% excess relative to the second strand as determined by UV absorption at 260 nmAnd (5) entering. The reaction mixture was heated to 80 ℃ for 5 minutes and then cooled slowly to room temperature. Double strand formation was monitored by ion-paired reverse phase HPLC. The desired amount of second strand was calculated from the UV area of the residual single strand and added to the reaction mixture. The reaction was again heated to 80 ℃ and slowly cooled to room temperature. This operation was repeated until less than 10% of residual single strands were detected.

Example 4 reduction of human PROS1 mRNA levels in human Hep3B cells by transfection of PROS1 siRNA

In vitro experiments showed that by transfection of any of the PROS1 siRNA molecules EU060 to EU083, the PROS1 mRNA levels in human Hep3B cells were reduced by more than 70%. Hep3B cells were seeded in 96-well plates at a density of 12000 cells per well. The following day, cells were transfected with 10 nM, 1nM or 0.1nM PROS1 siRNA or non-targeted control siRNA (EU 012) and 1 μg/ml AtuFECT. After 24 hours, the cells were lysed to extract RNA, and the mRNA levels of PROS1 and actin were determined by TaqmanqRT-PCR. The values obtained for the PROS1 mRNA were normalized to the value of housekeeping gene actin production and averaged over untreated samples (ut) for target gene expression set at 1-fold. Each bar represents the mean ± SD from three biological replicates. The siRNA duplex used in this study is listed in table 2 and further described in table 4. The results are shown in fig. 2.

Example 5 dose-dependent reduction of PROS1 mRNA levels in human cells by transfection of PROS1 siRNA

In vitro tests showed that many PROS1 siRNA molecules resulted in dose-dependent reduction of PROS1 mRNA levels in human Hep3B cells. Hep3B cells were seeded in 96-well plates at a density of 12000 cells per well. The following day, cells were transfected with 0.1nM, 0.01nM or 0.001nM PROS1 siRNA or 0.1nM non-targeted control siRNA (EU 012) and 1. Mu.g/ml AtuFECT. After 24 hours, the cells were lysed to extract RNA, and the mRNA levels of PROS1 and actin were determined by TaqmanqRT-PCR. The values obtained for the PROS1 mRNA were normalized to the value of housekeeping gene actin production and averaged over untreated samples (ut) for target gene expression set at 1-fold. Each bar represents the mean ± SD from three biological replicates. The siRNA duplex used in this study is listed in table 2 and further described in table 4. The results are shown in fig. 3.

Example 6 inhibition of primary mouse hepatocytes by receptor-mediated uptake of PROS1 siRNA conjugates PROS1 target Gene expression

This example shows a dose-dependent decrease in the levels of PROS1 mRNA in primary hepatocytes by receptor-mediated uptake of EU140 through EU 148. Primary mouse hepatocytes were seeded in 96-well plates at a density of 25000 cells per well. After attachment, they were incubated with PROS1 siRNA conjugates at 100nM, 10nM, 1nM and 0.1nM in cell culture medium, or with 100nM non-targeted control conjugate (EU 110), as shown below. The following day, the cells were lysed to extract RNA and the mRNA levels of PROS1 and ApoB were determined by Taqman qRT-PCR. The values obtained for the PROS1 mRNA were normalized to the values generated for the housekeeping gene ApoB and averaged (ut) with respect to the untreated sample set to 1-fold target gene expression. Each bar represents the mean ± sD from three biological replicates. The siRNA conjugates used in this study are listed in table 2 and further described in table 4. The results are shown in fig. 4.

Example 7 inhibition of human in primary human hepatocytes by receptor-mediated uptake of PROS1 siRNA conjugates PROS1 Gene expression

This example shows a dose-dependent decrease in human PROS1 mRNA levels in primary human hepatocytes by EU140 to 147. Primary human hepatocytes (life technologies) were seeded at a density of 35000 cells per well in 96-well plate medium and subsequently incubated with PROS1 siRNA conjugates EU140 to EU147 at concentrations of 100nM, 10nM, 1nM and 0.1nM, as shown in fig. 5, or they were incubated with 100nM of non-targeted control conjugate (EU 110). The values obtained for the PROS1 mRNA were normalized to the values generated for the housekeeping gene ApoB and averaged (ut) with respect to the untreated sample set to 1-fold target gene expression. Each bar represents the mean ± SD from three biological replicates. The siRNA conjugates used in this study are listed in table 2 and further described in table 4. The results are shown in fig. 5.

^-/- EXAMPLE 8 loss of Decaenzyme Activity rescue of PROS1 mice

PROS1 ^+/- Female and F8 ^-/- Male hybridization produced 25% F8 ^+/- PROS1 ^+/- And (5) offspring. F8F 8 ^+/- PROS1 ^+/- Female and F8 ^-/- Male co-breeding produced 25% of F8 ^-/- PROS1 ^+/- Offspring (fig. 13 a-c). By F9 ^-/- PROS1 ^+/- Similar observations were made with mice (FIGS. 13D-F). As expected, F8 ^-/- PROS1 ^-/- And F9 ^-/- PROS1 ^-/- Mice did not show FVIII and FIX plasma activity, respectively, and at F8 ^-/- PROS1 ^-/- And F9 ^-/- PROS1 ^-/- No Ps were detected in the plasma of mice (fig. 6C-D). F8 is reported ^-/- PROS1 ^+/- And F9 ^-/- PROS1 ^+/- Ps level ratio F8 in (B) ^-/- PROS1 ^+/+ And F9 ^-/- PROS1 ^+/+ Mice were 50-60% lower (FIG. 6C-D).

At F8 ^-/- PROS1 ^+/- Of 295 pups in the breeding pair, 72 (24%) were F8 ^-/- PROS1 ^+/+ 164 (56%) are F8 ^-/- PROS1 ^+/- While 59 (20%) are F8 ^-/- PROS1 ^-/- (χ2=4.8, p=0.09). Thus F8 ^-/- PROS1 ^-/- Mice were present at the expected mendelian ratio. In contrast, at F9 ^-/- PROS1 ^+/- Of 219 pups in the breeding pair, 56 (26%) were F9 ^-/- PROS1 ^+/+ 132 (60%) are F9 ^-/- PROS1 ^+/- While 31 (14%) are F9 ^-/- PROS1 ^-/- (χ2=14.95, p=0.001). This is in accordance with F9 ^-/- PROS1 ^-/- The transfer ratio distortion of the mice is compatible with F9 ^+/+ PROS1 ^+/+ Mice were mated consistently reduced in litter size (5.2±0.7 versus 9.8±1.8, n=4 mated/over 3t passages, p=0.046).

F8 ^-/- PROS1 ^-/- And F9 ^-/- PROS1 ^-/- The mice appeared completely normal. Their viability was monitored for up to 20 (n=4) and 16 months (n) =2), respectively with F8 ^-/- PROS1 ^+/+ And F9 ^-/- PROS1 ^+/+ The mice did not have any differences compared.

Since complete PROS1 deficiency in mice leads to consumable coagulopathy, we assessed F8 ^-/- PROS1 ^-/- And F9 ^-/- PROS1 ^-/- Whether the mice had DIC. DIC parameters at F8 ^-/- PROS1 ^+/+ 、F8 ^-/- PROS1 ^+/- And F8 ^-/- PROS1 ^-/- In mice (FIG. 6C), and in F9 ^-/- PROS1 ^+/+ 、F9 ^-/- PROS1 ^+/- And F9 ^-/- PROS1 ^-/- The mice (fig. 6D) were comparable. Due to lack of FVIII, in F8 ^-/- PROS1 ^+/+ (69.+ -. 2 seconds), F8 ^-/- PROS1 ^+/- (68.+ -. 3 seconds) and F8 ^-/- PROS1 ^-/- Activated partial thromboplastin time (aPTT) was equally prolonged (mean ± standard error, n=6, p=0.3 per group) in mice (63±3 seconds). At F9 ^-/- PROS1 ^+/+ 、F9 ^-/- PROS1 ^+/- And F9 ^-/- PROS1 ^-/- Comparable data were obtained in mice. In addition, at F8 ^-/- PROS1 ^-/- And F9 ^-/- PROS1 ^-/- No thrombosis or fibrin deposition was found in the brain, lung, liver and kidney of the mice (fig. 14).

Thus, loss of decase activity rescues embryonic lethality from complete PROS1 deficiency. However, the effect of rescue on loss of FIX activity is only partial. The possible explanation is that severe HB HAs a lighter condition than severe HA. Thus, PROS1 ^-/- F9 disruption in mice was less effective at rebalancing coagulation than F8 disruption.

To study recovery of F8 by FVIII infusion ^-/- PROS1 ^-/- Whether intrinsic ten enzyme activity in mice induced DIC, thrombosis and idiopathic purpura, we administered recombinant FVIII (rFVIII) intravenously. No mice died after rFVIII injection. 24 hours after a single injection of excess rFVIII, at F8 ^-/- PROS1 ^-/- Thrombus and pulmonary hemorrhage in many blood vessels were found in mice (fig. 6E-F). Coagulation 24 hours after repeated administration of normal doses of rFVIIIBlood analysis showed non-clotting Prothrombin Time (PT) (not shown), low fibrinogen and high thrombin-antithrombin (TAT) levels, compatible with overt DIC (fig. 6G). In contrast, at F8 ^-/- PROS1 ^-/- Fibrinogen and TAT levels and untreated F8 following a single injection of normal doses of rFVIII in mice ^-/- PROS1 ^-/- Mice were equivalent (fig. 6G). Although a large number of thrombi were visible in the lungs and liver (fig. 6H-I), none of these mice developed idiopathic purpura.

^-/- EXAMPLE 9 loss of Tenase Activity does not prevent TF-induced thromboembolic mortality in PROS1 mice

We have previously demonstrated that although 88% PROS1 ^+/+ Mice survived the TF-induced thromboembolic model, but only 25% of PROS1 was injected 20 minutes after low TF dose (. About.1.1 nM) ^+/- Mice survived. PROS1 when higher TF doses (4.3 nM) are used ^+/+ And PROS1 ^+/- Mice all died within 20 minutes. PROS1, however ^+/- Ratio PROS1 ^+/+ Death occurred earlier. HA and WT mice were equally sensitive to this high TF dose, with more than 85% of mice dying within 15 minutes (fig. 7A). In contrast, WT mice prevented with Low Molecular Weight Heparin (LMWH) thrombus >75% survived (FIG. 7A). Thus, HA does not protect mice from TF-induced thromboembolism compared to LMWH. Then we studied F8 in the same model ^-/- PROS1 ^+/+ ，F8 ^-/- PROS1 ^+/- And F8 ^-/- PROS1 ^-/- And (3) a mouse. After infusion of TF (-2.1 nM), 40-60% of mice die (P)>0.05 Irrespective of their PROS1 genotype (fig. 7B). However, F8 is present ^-/- PROS1 ^-/- And F8 ^-/- PROS1 ^+/- Mice earlier than F8 ^-/- PROS1 ^+/+ Trend of death of mice, F8 ^-/- PROS1 ^+/- Earlier than F8 ^-/- PROS1 ^+/+ Trend of death in mice (average death time: F8) ^-/- PROS1 ^+/+ F8 is 12+ -4 min ^-/- PROS1 ^+/- 7+ -2 min, F8 ^-/- PROS1 ^-/- For 8±3 minutes, n=4-6 per group, p=0.43). By F9 ^-/- PROS1 ^+/+ ，F9 ^-/- PROS1 ^+/- And F9 ^-/- PROS1 ^-/- Similar data were obtained for mice (data not shown).

At F8 ^-/- PROS1 ^+/+ And F8 ^-/- PROS1 ^-/- Fibrin clots were detected in the pulmonary artery of mice, which died during TF-induced thromboembolic challenge (fig. 7C). Importantly, from F8 ^-/- PROS1 ^-/- Thrombus ratio of mice of (2) derived from F8 ^-/- PROS1 ^+/+ Is (n=48 pairs 26, respectively). In addition, F8 ^-/- PROS1 ^-/- Most arteries in the lung are completely occluded, while at F8 ^-/- PROS1 ^+/+ Only a portion of the lung is occluded.

F8, which dies during TF-induced thromboembolic challenge ^-/- PROS1 ^-/- No mice developed idiopathic purpura. At F9 ^-/- PROS1 ^+/+ 、F9 ^-/- PROS1 ^+/- And F9 ^-/- PROS1 ^-/- Similar data were obtained for mice (not shown).

^-/- Example 10 missing part of FVIII protects PROS1 mice from mesenteric arteriole thrombosis Influence of

Then, we recorded thrombosis in the mesenteric arterioles, which model is sensitive to defects in the intrinsic coagulation pathway. At F8 ^+/+ PROS1 ^+/+ In mice, thrombus increased to occlusion size within 20 minutes, all injured arterioles were occluded (fig. 7D). As expected, F8 ^-/- PROS1 ^+/+ No arterioles of (C) show thrombosis, F8 ^-/- PROS1 ^-/- Mice showed partial thrombus (fig. 7D).

At F8 ^+/+ PROS1 ^+/+ Embolic events were produced during thrombus formation in mice, but at F8 ^-/- PROS1 ^+/+ No in mice. At F8 ^-/- PROS1 ^-/- In mice, multiple microembolic agents shed during part of the thrombus growth process, preventing the formation of occlusive thrombus.

Example 11 PROS1 targeting restriction but not elimination of HA miceBleeding from the tail of (2)

Bleeding phenotypes were assessed by tail transection using a mild or severe bleeding model.

In both models, F8 ^-/- PROS1 ^-/- Blood loss of F8 ^-/- PROS1 ^+/+ Mice were reduced compared (fig. 8A-B). When being attacked by mild model, F8 ^-/- PROS1 ^+/- Less bleeding than F8 in mice ^-/- PROS1 ^+/+ Mice (fig. 8A). Conversely, when exposed to severe model, F8 ^-/- PROS1 ^-/- And F8 ^-/- PROS1 ^+/- Mice exhibited comparable blood loss (fig. 8B). However, in both models, F8 ^-/- PROS1 ^-/- The bleeding of mice exceeded F8 ^+/- PROS1 ^+/+ And F8 ^+/+ PROS1 ^+/+ Mice (FIGS. 8A-B) demonstrate F8 ^-/- The missing part of PROS1 in mice corrected the bleeding phenotype.

Then, studies on how inhibition of PS activity alters F8 using PS neutralizing antibodies ^-/- PROS1 ^+/- Tail bleeding in mice. The antibody will F8 ^-/- PROS1 ^+/- Blood loss in mice (FIG. 8C) was limited to the same extent as PROS1 complete gene loss (FIG. 8B).

EXAMPLE 12 PROS1 targeting or PS inhibition fully protects HA or HB mice from acute arthritic hematopoiesis (AH)

Although bleeding may occur anywhere in hemophiliacs, most bleeding occurs at the joints. To determine whether a PROS1 deletion can prevent the formation of arthrosclerosis in hemophilia mice, we applied the AH model to F8 ^-/- PROS1 ^+/+ 、F8 ^-/- PROS1 ^+/- 、F8 ^-/- PROS1 ^-/- And F8 ^+/+ PROS1 ^+/+ And (3) a mouse. And F8 ^-/- PROS1 ^+/+ And F8 ^-/- PROS1 ^+/- F8 compared with mice ^-/- PROS1 ^-/- And F8 ^+/+ PROS1 ^+/+ Knee swelling was reduced after injury in mice (fig. 9A). F8F 8 ^-/- PROS1 ^-/- And F8 ^+/+ PROS1 ^+/+ There was no difference in knee swelling between miceDifferent (fig. 9A). At F8 ^-/- PROS1 ^+/+ Bleeding was observed in the articular cavity and synovium of (ibs=2, n=5), but in F8 ^-/- PROS1 ^-/- (ibs=0, n=5) and F8 ^+/+ PROS1 ^+/+ Mice (ibs=0, n=5) no (fig. 9B). And F8 ^-/- PROS1 ^-/- And F8 ^+/+ PROS1 ^+/+ F8 compared with mice ^-/- PROS1 ^+/+ More fibrin in the articular cavity and synovium (fig. 9B). By F9 ^-/- PROS1 ^+/+ And F9 ^-/- PROS1 ^-/- Similar data (ibs=0, n=3 and ibs=2, n=3, respectively) were obtained for mice (fig. 15A-B).

These results are obtained by the reaction at F8 ^-/- PROS1 ^+/- Subcutaneous infusion of PS neutralizing antibodies or control antibodies (knee swelling of PS neutralizing antibody group 0.43±0.07 versus control group 0.69±0.09mm, n= 9,P =0.04) was confirmed in mice (starting 1 day prior to AH induction) for 4 consecutive days. PS plasma levels were 26±6% for PS neutralizing antibody group, and 45±3% (n=5, p=0.017) for control group. In addition, PS inhibition may be achieved by treating F8 prior to AH challenge ^-/- PROS1 ^+/- And F8 ^-/- PROS1 ^+/+ Mice were alternatively achieved by intravenous injection of mouse PS (mPS) siRNA (fig. 9C-D). IBS evaluation confirmed that the mPS siRNA treated F8 compared to the control siRNA treated (1bs=2, n=3) ^-/- PROS1 ^+/+ Mice (ibs=0.5, n=3) lack intra-articular hemorrhage (fig. 9C). Importantly, PS expression was reduced by mPS siRNA in both plasma (26±3%, 84±+11% in control, n=3, p=0.006) and synovium (fig. 10A).

Example 13 expression of PS and TFPI in mouse sliding Membrane

To understand the prof 1 gene deletion and the significant intra-articular hemostasis of PS inhibition in hemophiliacs, PS and TFPI immunostaining was performed on knee sections. PS was mainly present in F8 of AH treated with control siRNA ^-/- PROS1 ^+/+ Lining of mouse synovial tissue, while in F8 of AH receiving mPS siRNA ^-/- PROS1 ^+/+ In mice, synovial staining for PS was significantly reduced (FIG. 10A). In contrast, TFPI staining was receiving mPSsiRNAIs more pronounced in synovial tissue of hemophilia mice than control siRNA treatment (fig. 10A). However, TFPI expression is at F8 ^-/- PROS1 ^+/+ And F8 ^-/- PROS1 ^-/- The mouse had a comparability in the synovial lining layer (fig. 10B).

To further demonstrate that PS is expressed by fibroblast-like synoviocytes (FLS), we were isolated from F8 ^+/+ PROS1 ^+/+ 、F8 ^-/- PROS1 ^+/+ And F8 ^-/- PROS1 ^-/- Western blots were performed on conditioned medium collected from FLS. As shown in FIG. 10C, F8 ^+/+ PROS1 ^+/+ And F8 ^-/- PROS1 ^+/+ The medium of FLS shows a band of molecular weight-75 kDa and comparable to PS and similar to that observed in plasma and platelets. As expected, in the case of F8 ^+/+ PROS1 ^-/- No staining was detected in the medium obtained with FLS (FIG. 10C).

We also studied F8 ^-/- PROS1 ^+/+ And F8 ^-/- PROS1 ^-/- TFPI expression in FLS conditioned medium (fig. 10D). All media showed a band of-50 kDa, similar to that observed with placenta lysate. TFPI isoform expression was studied after protein deglycosylation because fully glycosylated TFPI alpha and TFPI beta migrate at the same molecular weight. Deglycosylated TFPI from FLS medium migrates as a single band of molecular weight TFPI alpha, which has a molecular weight similar to placental TFPI (positive control of TFPI alpha) (fig. 10D). This suggests that FLS expresses TFPI alpha but not TFPI beta. Furthermore, after thrombin stimulation, F8 ^-/- PROS1 ^+/+ PS and TFPI expression increased in FLS (FIG. 10E-F).

Example 14 expression of PS and TFPI in synovial Membrane of HA or HB patients

Human HA, HB, and PS and TFPI of osteoarthritis knee synovial tissue were then analyzed (fig. 11A). On-demand strong signals for TFPI and PS were found in the synovial lining layer and the sub-lining layer of HA patients (n=7). In contrast, immunostaining for PS and TFPI was reduced in the preventative HA patients (n=5). Compared to HA-on-demand patients, HB-on-demand patients showed fewer PS and TFPI signals in the synovial lining layer and sub-lining layer (n=4). Sections from osteoarthritis patients (n=7) were similar to those from hemophiliacs, showing no intense staining of TFPI and PS. To assess which isoforms of TFPI are expressed by human FLS, western blot analysis was performed on conditioned medium of human FLS isolated from healthy subjects and osteoarthritis patients. Similar to mouse FLS, human FLS expressed tfpia but did not express tfpia (fig. 11B).

Example 15 deletion of PROS1 in HA mice results in lack of TFPI-dependent PS Activity and inhibition of APC Resistance to

In HA or HB mice lacking PROS1 or PS inhibition, the overall protection against AH can be explained, at least in part, by the lack of PS cofactor activity against APC and TFPI in the joint. However, the cause of partial hemostasis in HA mice lacking PROS1 or PS inhibition in the tail bleeding challenge model needs further investigation.

In vitro TF-initiated thrombin generation assays indicate that there is a correlation between the ability of plasma to generate thrombin and the clinical severity of hemophilia. Thus, we studied the effect of loss of PROS1 on thrombin generation in HA mouse plasma. TFPI-dependent PS activity was not assessed in platelet-free plasma (PFP), but in platelet-rich plasma (PRP), as PS activity of TFPI cofactors could not be demonstrated in mouse plasma using the thrombin generation assay. This can be explained by the absence of TFPI alpha [ and its presence in mouse platelets ] in mouse plasma.

F8 in response to 1pM TF ^-/- PROS1 ^-/- Both the thrombin peak and the Endogenous Thrombin Potential (ETP) of PRP of (2) are significantly higher than F8 ^-/- PROS1 ^+/+ (1072.+ -.160 vs 590.+ -.10 nmol/L min, n=3/group, P=0.04), indicated at F8 ^-/- PROS1 ^-/- Lack of PS TFPI-cofactor activity in PRP (fig. 12A). In agreement with previous work, F8 in the presence of 1, 2.5 or 5pM TF ^-/- PROS1 ^+/+ And F8 ^-/- PROS1 ^-/- Thrombin peaks in PFP and ETP were identical in mice (data not shown).

To evaluate F8 ^-/- PROS1 ^-/- Whether mice exhibit defective functional APCLai Xing PS Activity we performed thrombin generation tests in Ca2+ ionophore activated PRP in the absence of APC, in the presence of wild-type (WT) recombinant APC, or in the presence of mutant (L38D) recombinant mouse APC (L38 DAPC, variant with reduced PS cofactor activity (expanded)). In this assay, APC titration showed that addition of 8nMWTAPC was able to reduce ETP by 90% in the activated PRP of WT mice, while L38DAPC at the same concentration reduced ETP by only 30% (data not shown). Based on these data, thrombin generation curves (3 mice/assay) for activated PRP were recorded. Calculated APC ratio (ETP _+APCWT /ETP _+APCL38D ) Indicating F8 ^-/- PROS1 ^-/- APC resistance exists in plasma, but F8 ^-/- PROS1 ^+/+ No in plasma (0.87±0.13, 0.23±0.08, p=0.01, respectively) (fig. 12B).

In the presence of 2nM WT APC and L38D APC, also at F8 ^-/- PROS1 ^+/+ And F8 ^-/- PROS1 ^-/- APC-dependent PS activity was tested in PFP of mice (2 mice/test). Calculated APC ratio shows F8 ^-/- PROS1 ^-/- APC resistance in mice, but at F8 ^-/- PROS1 ^+/+ No in mice (1.08± 0.04,0.25 ±0.09, p=0.0003, respectively) (fig. 12B).

Example 16 improved fibrin network in PROS1 deficient HA mice

The tail bleeding mouse model is not only sensitive to platelet dysfunction, but also to clotting and fibrinolytic changes. To understand the differences in tail bleeding between genotypes studied, we studied fibrin structure using scanning electron microscopy imaging (fig. 12C). And F8 ^-/- PROS1 ^+/+ F8 compared to plasma clot ^+/+ PROS1 ^+/+ And F8 ^-/- PROS1 ^-/- The clot of plasma showed a denser, highly branched fibrin fiber network (fig. 16 a-b). In contrast, from F9 ^+/+ PROS1 ^+/+ And F9 ^-/- PROS1 ^-/- The clot of plasma did not show a ratio of F9 ^-/- PROS1 ^+/+ The plasma clot was a denser network, but a trend of increased fiber branching (fig. 16 c-d).

And from F8 ^+/+ PROS1 ^+/+ Mice or F9 ^+/+ PROS1 ^+/+ Fiber comparison from mice, from F8 ^-/- PROS1 ^-/- And F8 ^-/- PROS1 ^+/+ Mice and F9 ^-/- PROS1 ^-/- And F9 ^-/- PROS1 ^+/+ The fibrin fibers of the mice showed a larger diameter. However, F8 ^-/- PROS1 ^-/- And F9 ^-/- PROS1 ^-/- The fiber surface of the mouse is respectively matched with F8 ^-/- PROS1 ^+/+ Or F9 ^-/- PROS1 ^+/+ Mice showed less porosity, indicating F8 ^-/- PROS1 ^-/- And F9 ^-/- PROS1 ^-/- The derivative fiber may be more than F8 ^-/- PROS1 ^+/+ Or F9 ^-/- PROS1 ^+/+ The derivatized fibers have less permeability and are thus more resistant to fibrinolysis. These data, supplementing TFPI and APC cofactor activity results (fig. 12A-B), help explain why F8 ^-/- PROS1 ^-/- Tail hemorrhage with F8 ^-/- PROS1 ^+/+ Mice improved compared to, but not as much as F8 ^+/+ PROS1 ^+/+ Complete correction was as in mice.

EXAMPLE 17 inhibition of PS in plasma restores thrombin generation in HA patients

Then, we examined the effect of PS inhibition on thrombin generation in human HA plasma. ETP in PFP was increased 2-4 fold in the presence of PS neutralizing antibodies. Similar results were obtained with anti-human TFPI antibodies against the C-terminal domain, which were effective in inhibiting FXa even in the presence of FVIII inhibitors (fig. 12D-E). PS inhibition had a significant effect in PRP samples, which increased ETP more than 10-fold (1912±37 and 1872±64nm min) (fig. 12F and G, respectively). Thus, PS inhibition completely restored ETP in hemophilia plasma (in contrast to ETP in normal plasma: 1495.+ -. 2nM min). Similar results were obtained using anti-TFPI antibodies (fig. 12D-G). These data confirm the improvement in thrombin generation in PS inhibition-driven HA PFP and PRP we observed in mice in humans.

Example 18 materials and methods of examples 6-17

A mouse

F8 with C57BL/6J background obtained from Jackson laboratories ^-/- Mice (B6; 129S 4-F8) ^tm1Kaz J) and F9 ^-/- Mouse (B6.129P2-F9) ^tm1Dws /J)。PROS1 ^+/- Mice are offspring of the original colony. The Swiss federal veterinary agency approves the experiment.

TF-induced pulmonary embolism

Anesthetized 6-9 week old mice received 4.25nM (1:2 dilution) or 2.1nM (1:4 dilution) human recombinant TF (hrTF, dade Innovin, siemens) intravenously (2. Mu.L/g). After the end of the two-minute or 20-minute observation period following the onset of respiratory arrest, the lungs were collected and fixed in 4% pfa. Lung sections were stained for fibrin with hematoxylin and eosin. The extent of fibrin clot in the lungs was assessed as the number of intravascular thrombi in 10 non-overlapping regions selected at random (x 10 magnification).

Tail breaking model of HA mouse

As described, two different models of tail break were evaluated to assess bleeding phenotypes ¹⁴ . Briefly, tail ends of 8-10 week old mice were transected for 2mm (mild injury) and bleeding was venous, or for 4mm (severe injury) and bleeding was arterial and venous. Bleeding was quantified as blood loss after 30 or 10 minutes, respectively. In the severe injury model, some F8 ^-/- PROS1 ^+/- Mice received either rabbit anti-human PS-IgG (Dako) or rabbit isotype IgG (R) intravenously at a dose of 2.1mg/kg 2 minutes prior to tail transection&D Systems)。

Acute joint hematoma formation model

The joint diameters were measured at 0 and 72 hours using a digital caliper (kanagawa Sanfeng 547-301). At 72 hours, mice were sacrificed, knees were separated, fixed in 4% pfa, decalcified and embedded in paraffin. Intra-articular bleeding score (IBS) was assessed as described.

PS inhibition in vivo

Mice of 10 weeks size were continuously infused with rabbit anti-human PS-IgG (Dako Basel, switzerland) or rabbit isotype IgG (R & D Systems) at a rate of 1 mg/kg/day by subcutaneous osmotic minipump (model 2001, alzet).

Alternatively, 10 week old mice were treated with transfection reagent (Invivoffectamine 3.0,Invitrogen,Life Technologies) at a single dose of 1mg/kg of mouse specific siRNA (s 72206, life Technologies) or control siRNA (4459405,In vivo Negative Control#1Ambion,Life Technologies) as per manufacturer's instructions. The acute arthritic model was used 2.5 days after PS inhibition.

Statistical method

Values are expressed as mean ± standard error. Chi-square of the non-cognate genetic locus was used to assess isolation of mendelian alleles. Survival data in TF-induced venous thromboembolic model were plotted using the Kaplan-Meier method. The comparison curves were counted using a log rank test (Prism 6.0d; graphPad). Other data were analyzed by GraphPad Prism 6.0d t-test, one-way and two-way ANOVA assays. P values less than 0.05 are considered statistically significant.

Preparation of mouse plasma

Mice of 6-9 weeks old were anesthetized with pentobarbital (40 mg/kg) and whole blood was drawn from the inferior vena cava into 3.13% citrate (1 volume anticoagulant/9 volume blood). The centrifuge was preheated to 26 ℃, and the blood was centrifuged at 1031g for 10 minutes to obtain Platelet Rich Plasma (PRP). Alternatively, blood was centrifuged at 2400g for 10 minutes at Room Temperature (RT) to obtain platelet-poor plasma (PPP). To obtain Platelet Free Plasma (PFP), an additional centrifugation is performed at 10000g for 10 minutes.

Measurement of platelet count and coagulation parameters

Platelet counts were performed with an automated cell counter (Procyte Dx Hematology Analyzer, idex). The activity of fibrinogen, FVIII and FIX was measured on an automatic Sysmex CA-7000 coagulation analyzer (Sysmex Digitana). Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT) were measured on a coagulometer (MC 4plus, merlin Medical).

ELISA method for determining PS antigen and TAT complex of mouse

Wells from 96-well plates (Maxisorb, thermo) were coated with 50 μl of 10 μg/mL rabbit polyclonal anti-human PS (DAKO Cytomation) per well and incubated overnight at 4 ℃. After 3 washes with TBS buffer (0.05M 3- (hydroxymethyl) aminomethane, 0.15M NaCl, pH 7.5, 0.05% Tween 20), the plate was blocked with 2% TBS-BSA. Diluted plasma samples (dilution range: 1:300-1:600) were added to the wells and incubated for 2 hours at room temperature. After 3 washes, 50. Mu.L of 1. Mu.g/mL biotinylated chicken polyclonal anti-mouse protein S was added and incubated for 2 hours at room temperature. streptavidin-HRP conjugated horseradish peroxidase (Thermo) was added to amplify the signal and the plates were incubated for 1 hour. Plates were washed 3 times and 100 μl of TMB substrate (KPL) was added. The reaction was quenched by the addition of 100. Mu.L HCl (1M). Absorbance was measured at 450 nm. Standard curves were established using serial dilutions of pooled normal plasma dilutions from 14 healthy mice (7-12 weeks old, male 8, female 6). Results are expressed as a percentage relative to pooled normal plasma.

TAT levels were measured in duplicate for each plasma sample using a commercially available ELISA (Enzygnost TAT micro, siemens) according to the manufacturer's instructions.

Mouse tissue processing and sectioning, immunohistochemistry and microscopy

Untreated tissue sections (4 μm) were trichromatically stained with hematoxylin/eosin or Masson, or immunostained for insoluble fibrin PS or TFPI. The following antibodies were used: fibrin (mAb clone 102-10) was incubated at a final concentration of 15.6 μg/mL for 30 minutes at room temperature, and the secondary antibody rabbit anti-human, (Cambridge ab7155 Abcam, UK) was diluted 1:200 and incubated for 30 minutes at room temperature; PS (MAB 4976, R & D, 1:50 dilution) was incubated for 30 min at room temperature, and secondary anti-rabbit anti-rat (ab 7155 Abam) was 1:200 diluted and incubated for 30 min at room temperature; TFPI (PAHTFPI-S, hematological Technologies) was 1:200 diluted at a final concentration of 18.6. Mu.g/mL and incubated at room temperature for 30 minutes, and secondary anti-rabbit anti-sheep IgG (ab 7106, abcam) was incubated at room temperature for 30 minutes. All staining was performed with an immunostainer BOND RX (Mu Tengci Leica Biosystems, switzerland) according to the manufacturer's instructions. The entire slide was scanned using an air objective 3D HISTECH Panoramic 250Flash II with 20× (NA 0.8), 40× (NA 0.95). Image processing was performed using the Panoramic Viewer software.

In vivo administration of FVIII to mice with complete F8 Gene deletion

Mice of 6 to 9 weeks of age were anesthetized with ketamine (80 mg/kg) and xylazine (16 mg/kg). We administered 0.3U/kg recombinant FVIII intravenouslyBaxalta) to achieve 100% FVIII level at 1 hour (normal dose) or excess recombinant FVIII (2U/kg) to achieve 1 hour>200%. The normal or overdose was injected 1 hour before and 1 hour after the introduction of the jugular catheter (Mouse JVC 2Fr PU 10cm,Instech), then 4 hours, 8 hours, and 16 hours after placement of the centerline. Mice were sacrificed 24 hours after the first injection. Blood was drawn and organs were collected. FVIII, fibrinogen and thrombin-antithrombin complex (TAT) were measured as described in the examples. The lungs were isolated, fixed in 4% Paraformaldehyde (PFA) and embedded in paraffin.

FeCl in mesenteric artery ₃ Model of traumatic thrombosis

According to the reference ² With minor modifications, a model of mesenteric arterial thrombosis was studied using a living microscope. Mice were anesthetized by intraperitoneal injection of a mixture of ketamine (80 mg/kg) and xylazine (16 mg/kg). Platelets were labeled directly in vivo by injection of 100 μl of rhodamine 6G (1.0 mM). After the site of investigation was selected, a single piece of 10% FeCl was used ₃ Saturated filter paper (1M Whatman paper patch 1mm in diameter) was applied topically for 1min to cause vessel wall damage. Thrombosis was monitored in real time under a fluorescence microscope (IV-500, san diego Micron Instruments, california) equipped with an affinity corrected FITC filter set of water immersion optics (Zeiss, germany). Bright fluorescent-labeled platelets and leukocytes a 1355 μm x 965 μm field of view was observed by video-triggered stroboscopic epi illumination (Chadwick Helmuth, elmond, california). A10-fold objective Zeiss Plan-Neofluar with NA0.3 was used. All scenes were recorded on video tape using a custom low-delay silicon enhanced target camera (Dage MTI of Michigan, ind.), a timebase generator and Hi-8VCR (EVC-100 of Sony, japan). The time to occlusion of the vessel wall is measured, as determined by the cessation of blood cell flow.

Fibroblast-like synoviocyte (FLS) isolation, culture and flow cytometry

According to ³ Mouse FLS from 8-10 week old mice were isolated and cultured. After three passages, phase contrast images of cells were taken and cells were incubated with FITC conjugated rat anti-mouse CD11b antibody (M1/70,Pharmingen,BD Biosciences), PE conjugated rat anti-mouse CD90.2 antibody (30-H12, pharmingen, BD Biosciences), FITC conjugated rat anti-mouse CD106 antibody (429MVCAMA.A,Pharmingen,BD Biosciences), PE conjugated hamster anti-mouse CD54 antibody (3E2,Pharmingen,BD Biosciences) and fluorescent dye conjugated isotype control antibody for 30 minutes in the dark at 4 ℃. After the final washing and centrifugation steps, all incubated cells were analyzed on LSR II flow cytometry (BD Biosciences) and FACS Diva 7.0 software (BD Biosciences). Human FLS from healthy individuals and OA patients were purchased from Asterand, bioscience and cultured according to the manufacturing instructions.

Western blot

PS and TFPI were detected in human and mouse samples by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12% gradient SDS-PAGE, bio-Rad) under reducing conditions. Proteins were transferred onto nitrocellulose membranes (Bio-Rad) and then visualized using the following procedure: 2 μg/mL monoclonal MAB-4976 for mouse PS (R)&D system) for mouse TFPI (R&D system) 1. Mu.g/mL polyclonal AF2975. Recombinant murine PS ⁴ (30 ng), recombinant human TFPI full length (supplied by the Chamber strength of Japanese blood-change), washed platelet lysate from F8 ^-/- PROS1 ^+/+ PFP and derived from F8 in mice ^+/+ PROS1 ^+/+ Placental lysate of mice was used as PS, TFPI alpha control. After 24 hours incubation in serum-free medium (OptiMem), samples were collected from the fused mouse and human FLS conditioned medium and concentrated 40-fold using an Amicon filter (Millipore, cut-off 10 kDa). For TFPI Western blotting, samples were deglycosylated with five protein deglycosylases (PNGase F, O-glycosidase, neuraminidase, beta 1-4 galactosidase, beta-N-acetaminoglucosidase, deglycosylation kit, V4931, promega) before loading on the gelThe mixture was treated at 37℃for 12 hours. The final detection was done using horseradish peroxidase-conjugated secondary antibodies (Dako) and SuperSignal West Dura extended duration chemiluminescent substrates (Pierce), monitored with a Fuji LAS 3000IR CCD camera.

Immunohistochemistry of human knee joint synovium

Paraffin-embedded samples of synovial tissue from ten HA patients and four HB patients undergoing arthroplasty with severe knee arthropathy were collected at archives of anatomical and histological parts of the university of florens experiments and clinical department. Seven HA patients were treated as needed and five HA patients were secondary prevented. All four HB patients were treated as needed. Synovial samples from seven Osteoarthritis (OA) patients were used as controls. For immunohistochemical analysis, synovial tissue sections (5 μm thick) were dewaxed, rehydrated, boiled in sodium citrate buffer (10 mM, pH 6.0) for 10 min for antigen retrieval, followed by 3%H in methanol ₂ O ₂ Treatment was performed at room temperature for 15 minutes to block endogenous peroxidase activity. The sections were then washed in PBS and incubated with Ultra V blockers (UltraVision high volume detection System anti-multivalent antibodies, HRP, catalog number TP-125-HL, labVision) for 10 minutes at room temperature according to manufacturer's protocol. After blocking non-specific site binding, the sections were incubated with rabbit polyclonal anti-human protein S/PROS1 antibodies (1:50 dilution, catalog No. NBP1-87218,Novus Biologics) or sheep polyclonal anti-human Tissue Factor Pathway Inhibitor (TFPI) antibodies (1:500 dilution, catalog No. PAHTFPI-S, haematologic Technologies) diluted in PBS overnight at 4 ℃. For PS immunostaining, tissue sections were then incubated with biotinylated secondary antibody followed by streptavidin peroxidase (UltraVision high capacity detection System anti-multivalent antibody, HRP; labVision) according to manufacturer's protocol. For TFPI immunostaining, tissue sections were incubated with HRP-conjugated donkey anti-sheep IgG (1:1000 dilution; catalog No. ab97125; abcam) for 30 minutes. 3-amino-9-ethyl carbazole (AEC kit, catalog number TA-125-SA; labVision) was used as a chromogenic agent for the immune reaction. Finally, the synovial sections were counterstained with Mayer's hematoxylin (Bio-optics), washed and mounted on aqueous media In the blocking agent, and observed under a Leica DM 4000B microscope (Leica Microsystems). Sections not exposed to primary antibodies or incubated with isotype-matched and concentration-matched non-immune IgG (Sigma-Aldrich) served as negative controls for antibody specificity. Optical microscope images were captured with a Leica DFC310 FX 1.4 million pixel digital color camera equipped with a Leica software application suite LAS V3.8 (Leica Microsystems).

Ultrastructural investigation of fibrin clots

Fibrin clot was prepared from PFP by adding-5 nM TF (Dade Innovin, siemens) at 37 ℃. They were then fixed in 2% glutaraldehyde, dehydrated, dried and sputter coated with gold palladium for visualization using a scanning electron microscope. Semi-quantitative evaluation of network density and fiber branching was performed using STEPanizer software (www.stepanizer.com).

Calibration automatic thrombus determination in mouse specimens

Thrombin generation in PFP and PRP was determined using a Calibrated Automatic Thrombogram (CAT) method.

TFPI-dependent PS activity was assessed in PRP (150G/L) as shown below. Briefly, 10. Mu.L of mouse PRP (150G/L), 10. Mu.L of PRP reagent (Diagnostica Stago), and 30. Mu.L of buffer A (25mm Hepes,175mm NaCl,pH 7.4,5mg/mL BSA) were mixed. Thrombin generation was initiated with 10 μl of fluorogenic substrate/CaCl 2 mixture at 37 ℃. The final concentrations were as follows: 16.6% mouse plasma, 1pM hrTF, 4. Mu.M phospholipid, 16mM CaCl ₂ And 0.42mM fluorogenic substrate.

In mouse PFP and PRP, APC-dependent PS activity was assessed in CAT-based APC resistance assays. PRP (150G/L) was pre-treated with 40. Mu.M Ca ²⁺ Ionophore (A23187) was activated for 5 minutes at 37 ℃. The final concentrations were as follows: 16.6% mouse plasma, 22. Mu. M A23187, 1pM hrTF, 4. Mu.M phospholipid, 2nM (for PFP) or 8nM (for PRP) wild-type recombinant mouse APC (wt-rmAPC) or mutant recombinant mouse APC (rmAPC L38D), 16mM CaCl ₂ And 0.42mM fluorogenic substrate.

For TF titration on PFP, the following reagents were used: PPP reagent and MP reagent (Diagnostica Stago).

Use equipmentFluorsocan with dispenserThe fluorometer measures fluorescence. Fluorescence intensities were detected at wavelengths of 390nm (excitation filter) and 460nm (emission filter). Special software program->Version 3.0.0.29 (Thrombinoscope bv) is capable of calculating thrombin activity against a calibrator (Thrombinoscope bv) and displaying thrombin activity over time. All experience was repeated at 37 ℃ and measurements were typically continued for 60 minutes.

CAT determination in human samples

Written informed consent was obtained from the patient. Venous blood was drawn by venipuncture into 3.2% sodium citrate (v/v) and centrifuged at 2000g for 5 min. Platelet-poor plasma (PPP) was then centrifuged at 10000g for 10 minutes to obtain PFP. PFP was aliquoted, quick frozen and stored at-80℃until use. For PRP, blood was centrifuged at 180g×10 min. All subjects obtained informed consent for participation. According to the reference ¹³ With minor modifications, thrombin generation was assessed in human PFP and PRP. Briefly, 68. Mu.L of PFP or PRP (150G/L) was incubated with 12. Mu.L of polyclonal rabbit anti-human PS-IgG antibody (0.42 mg/mL, dako) or monoclonal antibody against TFPI or buffer A for 15 min at 37℃for (0.66. Mu.m, MW1848, sanquin). Coagulation was performed using 20. Mu.L of a 7:1 mixture of PPP low and PPP 5pm reagent (Diagnostica Stago) for PFP samples, or PRP reagent (Diagnostica stago) for PRP samples. Add 20. Mu.L CaCl ₂ And fluorogenic substrate (I-1140; bachem), thrombin generation is followed in a Fluorkanascent reader (Thermo Labsystems).

Discussion of examples 6-17

Since PS is a key regulator of thrombin generation, we believe that targeting PS may constitute a potential therapy for hemophilia.

Extensive studies in mice provide proof of concept data demonstrating that PS and TFPI play a central role in causing hemophilia mice to bleed and severe joint damage. Targeting PROS1 or inhibition of PS has the ability to improve hemophilia in mice as judged by improvement of bleeding phenotype and overall protection against the formation of joint effusion in an in vivo tail bleed assay (fig. 8A-C and 9). Because the joint exhibits very weak expression of TF and synovial cells produce large amounts of TFPI alpha and PS (fig. 10), the activity of the extrinsic pathway is greatly reduced within the joint, making the hemophilia joint prone to bleeding. In addition, both Thrombomodulin (TM) and the Endothelial Protein C Receptor (EPCR) are expressed by FLS, suggesting that in the case of AH, the TM-thrombin complex activates EPCR-bound PC to produce very potent anticoagulant APCs. Importantly, TFPI alpha expression was upregulated by thrombin (fig. 10F). Thus, AH, which typically results in significant local inflammation and joint symptoms, can last from days to weeks, also promotes the local production and secretion of various anticoagulants, i.e., APC, TFPI alpha and their mutual cofactors PS, which helps explain the pathophysiology of joint injury in hemophilia.

Observations using clinical samples from hemophiliacs are consistent with empirical training drawn from murine studies. In humans, blocking PS with or without inhibitors normalized ETP in plasma of patients with HA (fig. 12D-G). HB patients showed less TFPI and PS intra-articular expression than HA patients, consistent with current knowledge that HB patients bleed less than HA patients (fig. 11). Furthermore, patients with HA who received prophylaxis showed less TFPI and PS synovial expression than patients who received FVIII concentrate only in the case of bleeding, the so-called "on demand therapy" (fig. 11A). Finally, in agreement with what was observed in mice, human FLS secreted TFPI alpha and PS, thus enhancing the inference of human from mouse hemophilia data.

The broad findings in this report suggest that targeting PS may translate into a useful therapy for hemophilia. PS in human and mouse joints is a novel pathophysiologic contributor to joint haematoma and constitutes an attractive potential therapeutic target, especially because it has dual cofactor activity for APC and TFPI alpha within the joint. In the presence of PS, joint hematocrit increases TFPI alpha expression in the synovium. Targeting PS in mice protects them from joint hematocele. Therefore, it is believed that TFPI alpha and its cofactor PS, together with the TM-EPCR-PC pathway, produced by FLS, form an effective intra-articular anticoagulant system with important pathological effects on joint hematosis. Mouse PS-silencing RNAs that have been successfully used in hemophilia mice (fig. 9H-I and fig. 10A) are one treatment that we will develop for hemophilia patients. The advantage of silencing RNA over current factor replacement therapies is its longer half-life, reducing the frequency of injection and its potential subcutaneous route of administration.

EXAMPLE 19 PROS1 siRNA conjugates inhibit PROS1 target Gene expression in Primary hepatocytes

This example shows a dose-dependent decrease in the levels of PROS1 mRNA in primary hepatocytes by receptor-mediated uptake from EU149 to EU 160.

Primary mouse hepatocytes were seeded in 96-well plates at a density of 25000 cells per well. After attachment, they were incubated with PROS1 siRNA conjugates in cell culture medium at 100nM, 10nM, 1nM, 0.1nM and 0.01nM, as shown in FIG. 17, or with 100nM non-targeted control conjugate (EU 110). The following day, the cells were lysed to extract RNA and the mRNA levels of PROS1 and actin were determined by Taqman qRT-PCR. The values obtained for the PROS1 mRNA were normalized to the value of housekeeping gene actin production and averaged over untreated samples (ut) for target gene expression set at 1-fold. Each bar represents the mean ± SD from three biological replicates. The siRNA conjugates used in this study are listed in table 2 and further described in table 4. The results of EU149 to 153 are shown in FIG. 17A and the results of EU154 to EU160 are shown in FIG. 17B.

Example 20 inhibition of human PROS1 Gene expression in Primary human hepatocytes by receptor-mediated uptake

This example shows that human PROS1 mRNA levels in primary human hepatocytes are reduced by dose-dependent administration of EU149 to EU152, EU156, EU159 and EU160 by receptor-mediated uptake.

Primary human hepatocytes (Life Technologies) were seeded at a density of 35000 cells per well in 96-well plate medium and subsequently incubated with PROS1 siRNA conjugates of EU149 to EU152, EU156, EU159 and EU160 at concentrations of 100nM, 10nM, 1nM, 0.1nM or 0.01nM, as shown in fig. 18, or they were incubated with non-targeted control conjugates of 100nM (EU 110). The values obtained for the PROS1 mRNA were normalized to the value of housekeeping gene actin production and averaged over untreated samples (ut) for target gene expression set at 1-fold. Each bar represents the mean ± SD from three biological replicates. The siRNA conjugates used in this study are listed in table 2 and further described in table 4. The results of EU149 through 153 and EU156, EU159 and EU160 in FIG. 18A are shown in FIG. 18B.

Example 21 inhibition of PROS1 Gene expression in vivo by Single administration of PROS1 siRNA conjugates

This example shows a dose-dependent in vivo decrease in the levels of PROS1 mRNA in the liver of mice treated with EU140 to EU145, EU150 to EU152 or EU 159.

C57BL/6 mice of 9 to 12 weeks of age were treated by subcutaneous injection of 1 or 5mg of conjugate per kg body weight (EU 140 to EU145, EU150 to EU152 or EU 159) or carrier PBS, as shown in FIGS. 19A and 19B. Liver samples were collected from all mice 2 weeks after treatment and flash frozen. RNA was extracted from liver samples and the mRNA levels of PROS1 and actin were determined by Taqman qRT-PCR. The values obtained for the PROS1mRNA were normalized to the values for housekeeping gene actin production and correlated with the average of liver samples from the vector treated group (PBS) set to 1-fold target gene expression. Each bar in the scatter plot represents the median of 5-7 animals with 95% confidence intervals.

The siRNA conjugates used in this study are listed in table 2 and further described in table 4. Dose-dependent reduction of PROS1mRNA in mouse livers after treatment with PROS1 siRNA conjugate is shown in fig. 19A and 19B.

Example 22 inhibition of PROS1 Gene expression in hemophilia mice by Single administration of PROS1 siRNA conjugates Dada (Chinese character)

This example shows a decrease in the level of PROS1mRNA in the liver and a decrease in the level of PROS1 in serum of a hemophilia A mouse model treated with EU 152.

Factor 8 knockout mice (F8) of 9 to 12 weeks of age were treated by subcutaneous injection of 3mg of EU152 or vector PBS per kg body weight ^-/- A mouse; prince et al blood (2018) 131 (12): 1360-1371) as shown in fig. 20A and 20B. Liver samples were collected from all mice 8 days after injection and flash frozen. Plasma was prepared from blood collected at the same time point. RNA was extracted from liver samples and the mRNA levels of PROS1 and actin were determined by Taqman qRT-PCR. The values obtained for the PROS1mRNA were normalized to the values for housekeeping gene actin production and correlated with the average of liver samples from the vector treated group (PBS) set to 1-fold target gene expression. The levels of PROS1 in plasma samples were measured by a specific ELISA method (Prince et al, 2018).

Each bar (a) or line (B) in the scatter plot represents the mean and standard deviation of 8-9 animals.

The siRNA conjugates used in this study are listed in table 2 and further described in table 4. The decrease in PROS1mRNA in the liver of mice after treatment with the PROS1 siRNA conjugate is shown in fig. 20A, and the decrease in plasma PROS1 level is depicted in fig. 20B.

Example 23 treatment with PROS1 siRNA conjugates to reduce knee swelling in acute arthritic models

This example shows F8 ^-/- The difference between knee diameters of mice before knee injury and after 72 hours. Joint swelling was reduced in the mice group prevented with EU 152.

Factor 8 knockout mice of 9 to 12 weeks of age were treated by subcutaneous injection of 3mg, 5mg or 10mg of EU152 or vector PBS per kg body weight (F8 ^-/- A mouse; prince et al 2018), as shown in fig. 21. Knee joint diameters were measured 5 days after injection and knee joint injuries were performed under analgesic coverage (princet al, 2018). After 72 hours, knee diameter was measured again to evaluate swelling.

Scatter plots represent median values of 7-10 animals. And (3) statistics: kruskal-Wallis test with Dunn multiplex comparison test, relative to control (PBS).

The siRNA conjugate columns used in this studyIn table 2 and further described in table 4. F8F 8 ^-/- The difference in knee diameter of the mice before knee injury and after 72 hours is shown in figure 21. Hemophilia mice treated with EU152 before injury showed a dose-dependent decrease in knee swelling compared to hemophilia animals treated with vehicle (PBS).

Example 24 treatment with PROS1 siRNA conjugates to improve hemostatic Profile of hemophilia A animal models

This example shows the results from a wild-type mouse, hemophilia a mouse model (F8 ^-/- ) Or from a hemophilia a mouse model (with PROS1 siRNA (F8) ^-/- EU 152) processing) the clotting time, clot formation time, and alpha angle of the collected whole blood sample. Clot formation was assessed by rotational thromboelastometry (ROTEM), a viscoelastic assay that allows for real-time measurement of hemostasis of total clot formation (Gorlinger et al, ann Card Anaesth (2016), 19:516-20). Clotting time and clot formation time were reduced and alpha angle was increased in hemophilia mice compared to the evaluation of these hemostatic parameters in wild type mice. Treatment of hemophilia mice with PROS1 siRNA reduced clotting time, clot formation time and increased alpha angle.

Factor 8 knockout mice (F8) of 9 to 12 weeks of age were treated by subcutaneous injection of 5mg of EU152 or vector PBS per kg body weight ^-/- A mouse; prince et al 2018), as shown in fig. 22A-C. End point blood samples were taken 7 days after treatment was completed and coagulation time, clot formation time and angle alpha were determined using ROTEM. For comparison, whole blood samples from wild-type mice were collected and analyzed by the same method.

Scatter plots represent median values for 6-11 animals. And (3) statistics: analysis of variance of Welch and T3 log transform post test of Dunnett.

The siRNA conjugates used in this study are listed in table 2 and further described in table 4. Hemophilia a mice treated with PBS (F8) from wild-type mice (WT) ^-/- PBS) or EU152 (F8) with PROS1 siRNA ^-/- Clotting times of blood samples collected from EU 152) treated hemophilia a mice are shown in fig. 22A. Clot formation times and alpha angles for blood samples collected from the same treatment group are depicted in fig. 22B and 22C, respectively.

EXAMPLE 25 reduction of human protein S mRNA levels in human Hep3B cells by transfected protein S siRNA

In vitro experiments showed a dose-dependent decrease in protein S mRNA levels in human Hep3B cells by transfection of protein S siRNA molecules (EU 199 to EU 222).

Hep3B cells were seeded in 96-well plates at a density of 12000 cells per well. The following day, cells were transfected with 0.1nM, 0.01nM or 0.001nM PROS1 siRNA or non-targeted control siRNA (EU 198) and 1. Mu.g/ml AtuFECT. After 24 hours, the cells were lysed to extract RNA and the mRNA levels of PROS1 and actin were determined by Taqman qRT-PCR. The values obtained for protein S mRNA were normalized to the values of housekeeping gene actin production and are listed in table a relative to the average value (ut) of untreated samples for target gene expression set to 1-fold. SD represents standard deviation from three biological replicates. The siRNA duplex used in this study is listed in table 2 and further described in table 4.

Table A

EXAMPLE 26 dose-dependent reduction of human by transfection of protein S siRNA at a concentration of 1nM to 0.00001nM Protein S mRNA levels in cells

In vitro experiments showed a dose-dependent decrease in protein S mRNA levels in human Hep3B cells by transfection of protein S siRNA molecules.

Hep3B cells were seeded in 96-well plates at a density of 12000 cells per well. The following day, cells were transfected with 1nM, 0.01nM, 0.001nM, 0.0001nM or 0.00001nM PROS1 siRNA or 1nM non-targeted control siRNA (EU 0198) and 1. Mu.g/ml AtuFECT. After 24 hours, the cells were lysed to extract RNA and the mRNA levels of PROS1 and actin were determined by Taqman qRT-PCR. The values obtained for protein S mRNA were normalized to the value for housekeeping gene actin production and averaged (ut) with respect to untreated samples for target gene expression set at 1-fold. Each bar represents the mean ± SD from three biological replicates. The siRNA duplex used in this study is listed in table 2 and further described in table 4. The results are shown in FIG. 23.

Example 27 inhibition of human protein S Gene expression in Primary human hepatocytes by receptor-mediated uptake

In vitro experiments showed a dose-dependent decrease in human protein S mRNA levels in primary human hepatocytes by conjugated siRNA EU161 to EU 171.

Primary human hepatocytes (Life Technologies) were seeded at a density of 35000 cells per well in 96-well plate medium and subsequently incubated with protein S siRNA conjugates EU 161-EU 171 at a concentration of 100nM, 10nM, 1nM or 0.1nM, as shown in fig. 24. The values obtained for protein S mRNA were normalized to the value for housekeeping gene actin production and averaged (ut) with respect to untreated samples for target gene expression set at 1-fold. Each bar represents the mean ± SD from three biological replicates. The siRNA conjugates used in this study are listed in table 2 and further described in table 4. The results of EU161 to 171 are shown in FIG. 24.

Example 28 inhibition of protein S Gene expression in Primary cynomolgus monkey hepatocytes by receptor-mediated uptake

In vitro experiments showed that cynomolgus monkey protein S mRNA levels in primary cynomolgus monkey hepatocytes were dose-dependently reduced by conjugated siRNA EU161 to EU 171.

Primary cynomolgus monkey hepatocytes (Life Technologies) were seeded at a density of 45000 cells per well in plate medium of 96-well plates and subsequently incubated with protein S siRNA conjugates EU161 to EU171 at a concentration of 100nM, 10nM, 1nM, or 0.1nM, as shown in fig. 25. The values obtained for protein S mRNA were normalized to the value for housekeeping gene actin production and averaged (ut) with respect to untreated samples for target gene expression set at 1-fold. Each bar represents the mean ± SD from three biological replicates. The siRNA conjugates used in this study are listed in table 2 and further described in table 4. The results of EU161 to 171 are shown in FIG. 25.

EXAMPLE 29 inhibition of protein S expression in vivo

In vivo experiments showed a decrease in protein S mRNA levels in cynomolgus monkey liver tissue collected on days 15 to 17 and 43 to 45 after treatment with EU161 alone by subcutaneous injection.

The cynomolgus monkeys (24 to 48 months of age, male and female) that were bred for the purpose were assigned to different treatment groups (2 males and 2 females per group). On day 1, one group was treated with a single dose of EU161 subcutaneous injection of 3mg per kg body weight, while control animals received a vehicle, 0.9% saline subcutaneous injection. Liver samples were collected from each animal by laparotomy and snap frozen between day 15 and 17 and day 23 and 43. RNA was extracted from liver samples and protein S and ApoB mRNA levels were determined by Taqman qRT-PCR. The values of the protein S mRNA obtained were normalized to the values generated for housekeeping gene, apo B, and relative to the average (saline) of the 0.9% saline treatment group from the same time point set to 1-fold target gene expression. Each column represents the average ± SD of 4 animals.

The siRNA conjugates used in this study are further described in tables 2 and 4. The reduction of S mRNA protein in liver after treatment with EU161 at two different time points is shown in figure 26.

Example 30 reduction of serum levels of Total protein S in vivo

In vivo experiments showed a decrease in total protein S in cynomolgus monkey serum following a single treatment with EU161 by subcutaneous injection.

The cynomolgus monkeys (24 to 48 months of age, male and female) that were bred for the purpose were assigned to different treatment groups (2 males and 2 females per group). One group was treated by subcutaneous injection with a single dose of 3mg/kg body weight EU 161. Control animals received a subcutaneous injection of vehicle, 0.9% saline. Venous blood samples were collected at weekly intervals for serum preparation beginning 2 weeks prior to treatment. Total protein S levels serum levels in these samples were measured using the human protein S ELISA kit of Abcam (ab 190808). Cynomolgus monkey serum samples were measured in triplicate and cynomolgus monkey protein S serum levels were interpolated from a human protein S standard curve. The results are expressed as median and CI is 95%.

The siRNA conjugates used in this study are further described in tables 2 and 4. The decrease in serum total protein S levels after EU161 treatment is shown in figure 27.

EXAMPLE 31 reduction of plasma levels of free protein S in vivo

In vivo experiments showed a decrease in free protein S in cynomolgus monkey plasma after a single treatment with EU161 by subcutaneous injection.

The cynomolgus monkeys (24 to 48 months of age, male and female) that were bred for the purpose were assigned to different treatment groups (2 males and 2 females per group). One group was treated by subcutaneous injection with a single dose of 3mg/kg body weight EU 161. Control animals received a subcutaneous injection of vehicle, 0.9% saline. Venous blood samples were collected into vacuum tubes containing sodium citrate as an anticoagulant to prepare platelet poor plasma 2 weeks before and about every two weeks after dosing. By using an AtellicaCoag 360 instrument (Siemens)Free PS Ag reagent measurement. The levels of free protein S in cynomolgus study samples are plotted as a percentage of 100 on the basis of the ginseng reference standard. The results are expressed as median and CI is 95%. />

The siRNA conjugates used in this study are described in tables 2 and 4. The decrease in serum free protein S levels following EU161 treatment is shown in figure 28.

Summary sheet

Total duplex table-Table 2

Table of general abbreviations-Table 3

Abbreviations as shown in the abbreviation tables above may be used herein. The list of abbreviations may not be exhaustive and further abbreviations and their meanings may be found throughout the document.

General sequence Listing-Table 4

Sequence listing

<110> university of Berney (university ä t Bern)

Sirens treatment Co., ltd (Silence Therapeutics GmbH)

<120> nucleic acid for inhibiting PROS1 expression in cells

<130> S120bEP2

<160> 258

<170> PatentIn version 3.5

<210> 1

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 1

ugcuuucauu gcuuugucc 19

<210> 2

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 2

ggacaaagca augaaagca 19

<210> 3

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 3

uuccacagac accauauuc 19

<210> 4

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 4

gaauauggug ucuguggaa 19

<210> 5

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 5

uauuccagaa gcuccuugc 19

<210> 6

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 6

gcaaggagcu ucuggaaua 19

<210> 7

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 7

uuugugucaa gguucaagg 19

<210> 8

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 8

ccuugaaccu ugacacaaa 19

<210> 9

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 9

auugacacag cuucuuagg 19

<210> 10

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 10

ccuaagaagc ugugucaau 19

<210> 11

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 11

uucuaauucu uccacagac 19

<210> 12

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 12

gucuguggaa gaauuagaa 19

<210> 13

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 13

auauccaucu ucauugcau 19

<210> 14

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 14

augcaaugaa gauggauau 19

<210> 15

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 15

uuuucaaaga ccucccugg 19

<210> 16

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 16

ccagggaggu cuuugaaaa 19

<210> 17

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 17

aguuugaauc cuuucuucc 19

<210> 18

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 18

ggaagaaagg auucaaacu 19

<210> 19

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 19

uuucauugcu uuguccaag 19

<210> 20

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 20

cuuggacaaa gcaaugaaa 19

<210> 21

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 21

cauugcuuug uccaagacg 19

<210> 22

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 22

cgucuuggac aaagcaaug 19

<210> 23

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 23

uauguuuaga aauggcuuc 19

<210> 24

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 24

gaagccauuu cuaaacaua 19

<210> 25

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 25

uguucuugca cacagcugu 19

<210> 26

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 26

acagcugugu gcaagaaca 19

<210> 27

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 27

aucuugggca aguuugaau 19

<210> 28

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 28

auucaaacuu gcccaagau 19

<210> 29

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 29

aacucuucug aucuugggc 19

<210> 30

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 30

gcccaagauc agaagaguu 19

<210> 31

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 31

uucuuccaca gacaccaua 19

<210> 32

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 32

uauggugucu guggaagaa 19

<210> 33

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 33

gucaggauaa gcauuaguu 19

<210> 34

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 34

aacuaaugcu uauccugac 19

<210> 35

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 35

acagacacca uauuccaua 19

<210> 36

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 36

uauggaauau ggugucugu 19

<210> 37

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 37

uuuggauaaa aauaauccg 19

<210> 38

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 38

cggauuauuu uuauccaaa 19

<210> 39

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 39

cucacaacuc uucugaucu 19

<210> 40

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 40

agaucagaag aguugugag 19

<210> 41

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 41

gcauucacug guguggcac 19

<210> 42

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 42

gugccacacc agugaaugc 19

<210> 43

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 43

uaggucagga uaagcauua 19

<210> 44

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 44

uaaugcuuau ccugaccua 19

<210> 45

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 45

agcacacaug uucucagag 19

<210> 46

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 46

cucugagaac augugugcu 19

<210> 47

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 47

uccacagaca ccauauucc 19

<210> 48

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 48

ggaauauggu gucugugga 19

<210> 49

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 49

ucauucacug guguggcac 19

<210> 50

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand-modification according to the Total sequence Listing at the end of the Specification

<400> 50

ucgaaguauu ccgcguacg 19

<210> 51

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 51

cguacgcgga auacuucga 19

<210> 52

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 52

ugcuuucauu gcuuugucc 19

<210> 53

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 53

ggacaaagca augaaagca 19

<210> 54

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 54

uuccacagac accauauuc 19

<210> 55

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 55

gaauauggug ucuguggaa 19

<210> 56

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 56

uauuccagaa gcuccuugc 19

<210> 57

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 57

gcaaggagcu ucuggaaua 19

<210> 58

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 58

uuugugucaa gguucaagg 19

<210> 59

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 59

ccuugaaccu ugacacaaa 19

<210> 60

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 60

auugacacag cuucuuagg 19

<210> 61

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 61

ccuaagaagc ugugucaau 19

<210> 62

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 62

uucuaauucu uccacagac 19

<210> 63

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 63

gucuguggaa gaauuagaa 19

<210> 64

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 64

auauccaucu ucauugcau 19

<210> 65

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 65

augcaaugaa gauggauau 19

<210> 66

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 66

uuuucaaaga ccucccugg 19

<210> 67

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 67

ccagggaggu cuuugaaaa 19

<210> 68

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 68

aguuugaauc cuuucuucc 19

<210> 69

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 69

ggaagaaagg auucaaacu 19

<210> 70

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 70

uuucauugcu uuguccaag 19

<210> 71

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 71

cuuggacaaa gcaaugaaa 19

<210> 72

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 72

cauugcuuug uccaagacg 19

<210> 73

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 73

cgucuuggac aaagcaaug 19

<210> 74

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 74

uauguuuaga aauggcuuc 19

<210> 75

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 75

gaagccauuu cuaaacaua 19

<210> 76

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 76

uguucuugca cacagcugu 19

<210> 77

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 77

acagcugugu gcaagaaca 19

<210> 78

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 78

aucuugggca aguuugaau 19

<210> 79

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 79

auucaaacuu gcccaagau 19

<210> 80

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 80

aacucuucug aucuugggc 19

<210> 81

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 81

gcccaagauc agaagaguu 19

<210> 82

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 82

uucuuccaca gacaccaua 19

<210> 83

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 83

uauggugucu guggaagaa 19

<210> 84

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 84

gucaggauaa gcauuaguu 19

<210> 85

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 85

aacuaaugcu uauccugac 19

<210> 86

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 86

acagacacca uauuccaua 19

<210> 87

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 87

uauggaauau ggugucugu 19

<210> 88

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 88

uuuggauaaa aauaauccg 19

<210> 89

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 89

cggauuauuu uuauccaaa 19

<210> 90

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 90

cucacaacuc uucugaucu 19

<210> 91

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 91

agaucagaag aguugugag 19

<210> 92

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 92

gcauucacug guguggcac 19

<210> 93

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 93

gugccacacc agugaaugc 19

<210> 94

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 94

uaggucagga uaagcauua 19

<210> 95

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 95

uaaugcuuau ccugaccua 19

<210> 96

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 96

agcacacaug uucucagag 19

<210> 97

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 97

cucugagaac augugugcu 19

<210> 98

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 98

uccacagaca ccauauucc 19

<210> 99

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 99

ggaauauggu gucugugga 19

<210> 100

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 100

ucgaaguauu ccgcguacg 19

<210> 101

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 101

cguacgcgga auacuucga 19

<210> 102

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 102

uuccacagac accauauuc 19

<210> 103

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 103

gaauauggug ucuguggaa 19

<210> 104

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 104

uucuaauucu uccacagac 19

<210> 105

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 105

gucuguggaa gaauuagaa 19

<210> 106

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 106

uuuucaaaga ccucccugg 19

<210> 107

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 107

ccagggaggu cuuugaaaa 19

<210> 108

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 108

uuucauugcu uuguccaag 19

<210> 109

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 109

cuuggacaaa gcaaugaaa 19

<210> 110

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 110

uguucuugca cacagcugu 19

<210> 111

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 111

acagcugugu gcaagaaca 19

<210> 112

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 112

acagacacca uauuccaua 19

<210> 113

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 113

uauggaauau ggugucugu 19

<210> 114

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 114

ucauucacug guguggcac 19

<210> 115

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 115

gugccacacc agugaaugc 19

<210> 116

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 116

agcacacaug uucucagag 19

<210> 117

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 117

cucugagaac augugugcu 19

<210> 118

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 118

uccacagaca ccauauucc 19

<210> 119

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 119

ggaauauggu gucugugga 19

<210> 120

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 120

uuccacagac accauauuc 19

<210> 121

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 121

uucuaauucu uccacagac 19

<210> 122

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 122

uuuucaaaga ccucccugg 19

<210> 123

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 123

uuucauugcu uuguccaag 19

<210> 124

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 124

ucagacacca uauuccaua 19

<210> 125

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 125

ccagggaggu cuuugaaaa 19

<210> 126

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 126

uuuucaaaga ccucccugg 19

<210> 127

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 127

ccagggaggu cuuugaaaa 19

<210> 128

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 128

uuuucaaaga ccucccugg 19

<210> 129

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 129

ccagggaggu cuuugaaaa 19

<210> 130

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 130

cuuggacaaa gcaaugaaa 19

<210> 131

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 131

uuucauugcu uuguccaag 19

<210> 132

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 132

cuuggacaaa gcaaugaaa 19

<210> 133

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 133

cuuggacaaa gcaaugaaa 19

<210> 134

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 134

cuuggacaaa gcaaugaaa 19

<210> 135

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 135

ccagggaggu cuuugaaaa 19

<210> 136

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 136

cuuggacaaa gcaaugaaa 19

<210> 137

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 137

ucgaaguauu ccgcguacg 19

<210> 138

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 138

cguacgcgga auacuucga 19

<210> 139

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 139

uuuucaaaga ccucccugg 19

<210> 140

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 140

ccagggaggu cuuugaaaa 19

<210> 141

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 141

uuauaaaagg cauucacug 19

<210> 142

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 142

cagugaaugc cuuuuauaa 19

<210> 143

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 143

uuuuguaaug uagaccuug 19

<210> 144

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 144

caaggucuac auuacaaaa 19

<210> 145

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 145

auuaauauuc acuuccaug 19

<210> 146

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 146

cauggaagug aauauuaau 19

<210> 147

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 147

uuguacuuca acaaucaca 19

<210> 148

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 148

ugugauuguu gaaguacaa 19

<210> 149

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 149

cuuuauugca caguucuuc 19

<210> 150

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 150

gaagaacugu gcaauaaag 19

<210> 151

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 151

uauuugaggg aucuuugca 19

<210> 152

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 152

ugcaaagauc ccucaaaua 19

<210> 153

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 153

auauucacuu ccaugcagc 19

<210> 154

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 154

gcugcaugga agugaauau 19

<210> 155

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 155

aguauaauua cacacaagg 19

<210> 156

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 156

ccuugugugu aauuauacu 19

<210> 157

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 157

ccuugugugu aauuauacu 19

<210> 158

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 158

aagagauggu ggucuauua 19

<210> 159

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 159

aaaugcauca caguaccag 19

<210> 160

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 160

cugguacugu gaugcauuu 19

<210> 161

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 161

gucauuuuca aagaccucc 19

<210> 162

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 162

ggaggucuuu gaaaaugac 19

<210> 163

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 163

uugaaaagag cgaagacaa 19

<210> 164

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 164

uugucuucgc ucuuuucaa 19

<210> 165

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 165

uguauguuca uucuuaagc 19

<210> 166

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 166

gcuuaagaau gaacauaca 19

<210> 167

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 167

uuaaugaguu cacuuucca 19

<210> 168

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 168

uggaaaguga acucauuaa 19

<210> 169

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 169

uuuuacagga acaguggua 19

<210> 170

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 170

uaccacuguu ccuguaaaa 19

<210> 171

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 171

cauucuuaag cugaacuuc 19

<210> 172

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 172

gaaguucagc uuaagaaug 19

<210> 173

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 173

cauuauuaua aucuaugug 19

<210> 174

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 174

cacauagauu auaauaaug 19

<210> 175

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 175

cgaauauuca aggucacau 19

<210> 176

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 176

augugaccuu gaauauucg 19

<210> 177

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 177

cacugaaugg aacaucugg 19

<210> 178

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 178

ccagauguuc cauucagug 19

<210> 179

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 179

ucuggaaugg cauugacac 19

<210> 180

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 180

gugucaaugc cauuccaga 19

<210> 181

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 181

aaguuugccu cugagacgg 19

<210> 182

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 182

ccgucucaga ggcaaacuu 19

<210> 183

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 183

uucguauaca uccaucuag 19

<210> 184

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 184

cuagauggau guauacgaa 19

<210> 185

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 185

cuuagggccu guauccgau 19

<210> 186

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 186

aucggauaca ggcccuaag 19

<210> 187

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 187

uuauaaaagg cauucacug 19

<210> 188

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 188

cagugaaugc cuuuuauaa 19

<210> 189

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 189

uuuuguaaug uagaccuug 19

<210> 190

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 190

caaggucuac auuacaaaa 19

<210> 191

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 191

auuaauauuc acuuccaug 19

<210> 192

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 192

cauggaagug aauauuaau 19

<210> 193

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 193

uuguacuuca acaaucaca 19

<210> 194

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 194

ugugauuguu gaaguacaa 19

<210> 195

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 195

cuuuauugca caguucuuc 19

<210> 196

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 196

gaagaacugu gcaauaaag 19

<210> 197

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 197

uauuugaggg aucuuugca 19

<210> 198

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 198

ugcaaagauc ccucaaaua 19

<210> 199

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 199

auauucacuu ccaugcagc 19

<210> 200

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 200

gcugcaugga agugaauau 19

<210> 201

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 201

aguauaauua cacacaagg 19

<210> 202

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 202

ccuugugugu aauuauacu 19

<210> 203

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 203

uaauagacca ccaucucuu 19

<210> 204

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 204

aagagauggu ggucuauua 19

<210> 205

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 205

aaaugcauca caguaccag 19

<210> 206

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 206

cugguacugu gaugcauuu 19

<210> 207

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 207

gucauuuuca aagaccucc 19

<210> 208

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 208

ggaggucuuu gaaaaugac 19

<210> 209

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 209

uugaaaagag cgaagacaa 19

<210> 210

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 210

uugucuucgc ucuuuucaa 19

<210> 211

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 211

uguauguuca uucuuaagc 19

<210> 212

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 212

gcuuaagaau gaacauaca 19

<210> 213

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 213

uuaaugaguu cacuuucca 19

<210> 214

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 214

uggaaaguga acucauuaa 19

<210> 215

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 215

uuuuacagga acaguggua 19

<210> 216

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 216

uaccacuguu ccuguaaaa 19

<210> 217

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 217

cauucuuaag cugaacuuc 19

<210> 218

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 218

gaaguucagc uuaagaaug 19

<210> 219

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 219

cauuauuaua aucuaugug 19

<210> 220

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 220

cacauagauu auaauaaug 19

<210> 221

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 221

cgaauauuca aggucacau 19

<210> 222

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 222

augugaccuu gaauauucg 19

<210> 223

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 223

cacugaaugg aacaucugg 19

<210> 224

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 224

ccagauguuc cauucagug 19

<210> 225

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 225

ucuggaaugg cauugacac 19

<210> 226

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 226

gugucaaugc cauuccaga 19

<210> 227

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 227

aaguuugccu cugagacgg 19

<210> 228

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 228

ccgucucaga ggcaaacuu 19

<210> 229

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 229

uucguauaca uccaucuag 19

<210> 230

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 230

cuagauggau guauacgaa 19

<210> 231

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 231

cuuagggccu guauccgau 19

<210> 232

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 232

aucggauaca ggcccuaag 19

<210> 233

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 233

auauucacuu ccaugcagc 19

<210> 234

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 234

gcugcaugga agugaauau 19

<210> 235

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 235

uuauucacuu ccaugcagc 19

<210> 236

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 236

gcugcaugga agugaauau 19

<210> 237

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 237

uaauagacca ccaucucuu 19

<210> 238

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 238

aagagauggu ggucuauua 19

<210> 239

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 239

uaauagacca ccaucucuu 19

<210> 240

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 240

aagagauggu ggucuauua 19

<210> 241

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 241

uaauagacca ccaucucuu 19

<210> 242

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 242

aagagauggu ggucuauua 19

<210> 243

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 243

uugaaaagag cgaagacaa 19

<210> 244

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 244

uugucuucgc ucuuuucaa 19

<210> 245

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 245

uugaaaagag cgaagacaa 19

<210> 246

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 246

uugucuucgc ucuuuucaa 19

<210> 247

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 247

uugaaaagag cgaagacaa 19

<210> 248

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 248

uugucuucgc ucuuuucaa 19

<210> 249

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 249

uucguauaca uccaucuag 19

<210> 250

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 250

cuagauggau guauacgaa 19

<210> 251

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 251

uucguauaca uccaucuag 19

<210> 252

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 252

cuagauggau guauacgaa 19

<210> 253

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 253

uucguauaca uccaucuag 19

<210> 254

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 254

cuagauggau guauacgaa 19

<210> 255

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA strand

<400> 255

uuauucacuu ccaugcagc 19

<210> 256

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 256

gcugcaugga agugaauau 19

<210> 257

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 257

aagagauggu ggucuauua 19

<210> 258

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<400> 258

cuagauggau guauacgaa 19

Claims

1. A double stranded nucleic acid for inhibiting expression of PROS1, wherein the nucleic acid comprises a first strand and a second strand, wherein the first strand sequence comprises a sequence of at least 15 nucleotides differing by NO more than 3 nucleotides from any of the sequences selected from the group consisting of SEQ ID NOs 199, 187, 189, 191, 193, 195, 197, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231 and 255.

2. The nucleic acid of any one of the preceding claims, wherein the first strand and the second strand form a double-stranded region of 17-25 nucleotides in length.

3. The nucleic acid of any one of the preceding claims, wherein the nucleic acid mediates RNA interference.

4. The nucleic acid according to any of the preceding claims, wherein at least one nucleotide of the first and/or second strand is a modified nucleotide, in particular a non-naturally occurring nucleotide, such as a 2' -F modified nucleotide.

5. The nucleic acid of any one of the preceding claims, wherein at least nucleotides 2 and 14 of the first strand are modified by a first modification, the nucleotides being numbered consecutively at the 5' end of the first strand starting with nucleotide number 1.

6. The nucleic acid of any one of the preceding claims, wherein the first strand has a terminal 5 '(E) -vinylphosphonate nucleotide at its 5' end.

7. The nucleic acid of any one of the preceding claims, wherein the nucleic acid comprises phosphorothioate linkages between two or three 3 'nucleotides and/or 5' nucleotides at the end of the first and/or the second strand, and in particular wherein the linkages between the remaining nucleotides are phosphodiester linkages.

8. The nucleic acid of any one of the preceding claims, comprising a phosphorodithioate linkage between each of the two, three or four terminal nucleotides at the 3 'end of the first strand and/or comprising a phosphorodithioate linkage between each of the two, three or four terminal nucleotides at the 3' end of the second strand and/or comprising a phosphorodithioate linkage between the two, three or four terminal nucleotides at the 5 'end of the second strand and comprising a linkage other than a phosphorodithioate linkage between the two, three or four terminal nucleotides at the 5' end of the first strand.

9. The nucleic acid of any one of the preceding claims, wherein the nucleic acid is conjugated to a ligand.

10. The nucleic acid of claim 10, wherein the ligand comprises (i) one or more N-acetylgalactosamine (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.

11. The nucleic acid of any one of claims 1 to 5, 7 or 9 to 10, wherein the first strand comprises SEQ ID No. 233 and the second strand optionally comprises SEQ ID No. 234.

12. The nucleic acid of any one of claims 1 to 5, 7 or 9 to 10, wherein the first strand consists of SEQ ID No. 233 and wherein the second strand consists of SEQ ID No. 234.

13. The nucleic acid of any one of claims 1 to 5, 7 or 9 to 10, wherein the first strand comprises SEQ ID No. 237 and the second strand optionally comprises SEQ ID No. 238.

14. The nucleic acid of any one of claims 1 to 5, 7 or 9 to 10, wherein the first strand consists of SEQ ID No. 237 and wherein the second strand consists of SEQ ID No. 238.

15. The nucleic acid of any one of claims 1 to 7 or 9 to 10, wherein the first strand comprises SEQ ID No. 251 and the second strand optionally comprises SEQ ID No. 252.

16. The nucleic acid of any one of claims 1 to 7 or 9 to 10, wherein the first strand consists of SEQ ID No. 251 and wherein the second strand consists of SEQ ID No. 252.

17. A composition comprising a nucleic acid according to any 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 oligonucleotides, small molecules, monoclonal antibodies, polyclonal antibodies and peptides.

18. A nucleic acid according to any one of claims 1 to 16 or a composition according to claim 17 for use as a medicament.

19. The nucleic acid of any one of claims 1-16 or the composition of claim 17 for use in preventing a bleeding disorder, reducing the risk of suffering from a bleeding disorder, or treating a bleeding disorder, in particular hemophilia a or hemophilia B.