WO2023212810A1

WO2023212810A1 - Methods and compositions for modulating a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (adamts13)

Info

Publication number: WO2023212810A1
Application number: PCT/CA2023/050593
Authority: WO
Inventors: Christian Kastrup; Francesca FERRARESSO; Amy Wong STRILCHUK
Original assignee: The University Of British Columbia
Priority date: 2022-05-05
Filing date: 2023-05-02
Publication date: 2023-11-09

Abstract

The present disclosure provides a lipid nanoparticle comprising an siRNA molecule against ADAMTS13, the siRNA molecule containing modified or unmodified nucleotides. Further provided is an siRNA molecule against ADAMTS13, the siRNA molecule containing modified or unmodified nucleotides and is between 15 and 35 nucleotides in length and has at least 80% sequence identity to SEQ ID NOs: 1-10.

Description

METHODS AND COMPOSITIONS FOR MODULATING A DISINTEGRIN AND METALLOPROTEINASE WITH A THROMBOSPONDIN TYPE 1 MOTIF, MEMBER 13 (ADAMTS13)

TECHNICAL FIELD

The present disclosure relates to nucleic acid for targeting ADAMTS13 and pharmaceutical formulations thereof.

BACKGROUND

A disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS- 13) is primarily synthesized by hepatic stellate cells in the liver and is secreted into blood as a constitutionally active protease. It is found in blood plasma at a concentration of approximately 1 pg/mL and has a half-life of 2-3 days. ADAMTS13 cleaves von Willebrand Factor (VWF), a multimeric glycoprotein that mediates platelet aggregation, one of the first steps in clot formation. The size of VWF multimers is the main determinant of its platelet-recruiting function; larger VWF multimers have more platelet binding sites. Upon formation of large VWF multimers during blood vessel damage or high shear stress, ADAMTS13 prevents blockage of the blood vessel by cleaving VWF to destabilizes and promote degradation of clots. Low ADAMTS13 levels can lead to formation of obstructive thrombi, such as in thrombotic thrombocytopenic purpura (TTP) and various cardiovascular diseases. TTP has only been observed in patients that express normal levels of VWF with low levels of ADAMTS13 at the same time.

Von Willebrand Disease (VWD) is a genetic bleeding disorder which affects approximately 1% of the world population. There are 3 types of VWD: Type 1 VWD is characterized by partial deficiency in VWF and is the most common, affecting -60-80% of patients; Type 2 VWD is characterized by normal levels of VWF with defective function; and Type 3 VWD is characterized by complete deficiency in VWF. In the case of Type 1 VWD, significantly lower than normal VWF levels leads to decreased interaction with platelets, resulting in an unstable clot that is easily degraded, contributing to bleeding. ADAMTS13 contribute to bleeding by cleaving large VWF multimers into smaller, less procoagulant fragments. Thus, decreasing systemic ADAMTS13 levels may have therapeutic utility in Type 1 VWD, as decreased cleavage of VWF leads to a pool of large, more procoagulant VWF multimers in circulation that can readily interact with platelets to form a stable blood clot.

Despite pathologies associated with low ADAMTS13 levels, decreasing circulating ADAMTS13 levels may have therapeutic benefit in certain scenarios, such as in Type 1 VWD. However, there are currently no ADAMTS13 inhibitors or gene therapies that decrease ADAMTS13 levels.

SUMMARY

The present disclosure in some embodiments provides a method for modifying the expression of ADAMTS13, thereby treating and/or preventing one or more disorders for which it is desirable to reduce ADAMTS13 levels.

The present disclosure in some embodiments provides a lipid nanoparticle (LNP) comprising siRNA for reducing or inhibiting the expression of ADAMTS13, thereby treating and/or preventing one or more conditions, diseases or disorders for which it is desirable to modulate ADAMTS13 levels, such as in bleeding disorders. In some examples, the inventors have discovered that lipid nanoparticles having lipid components as described herein and encapsulating siRNA targeting ADAMTS13 mRNA could achieve controlled and/or sustained reduction of ADAMTS13 levels in the blood or other bodily sites.

According to one aspect of the disclosure, there is provided a lipid nanoparticle comprising: an siRNA molecule against ADAMTS13 mRNA; an ionizable, cationic amino lipid having a pKa of between 5.5 and 7.0 and that is present at between 10 mol% and 85 mol%; a neutral, vesicleforming lipid selected from at least one of a phospholipid and a triglyceride; a sterol; and a hydrophilic polymer-lipid conjugate present at between 0.5 mol% and 5 mol%.

According to one embodiment of the disclosure, the ADAMTS13 is human.

According to another example of any aspect or embodiment herein, the siRNA molecule comprises modified or unmodified nucleotides, and wherein the modified nucleotides are methylated. According to another example of any aspect or embodiment herein, at least one strand of the duplex siRNA has a sequence that has at least 80% sequence identity to any one of SEQ ID NOs: 1 to 10 or 17-26.

In a further example of any aspect or embodiment herein, at least one strand of the duplex siRNA has a sequence that has at least 80% sequence identity to any one of SEQ ID NOs: 1 to 10 or 17- 26.

According to another example of any aspect or embodiment herein, at least one strand of the duplex siRNA has a sequence that has at least 85% sequence identity to any one of SEQ ID NOs: 1 to 10 or 17-26.

According to a further example of any aspect or embodiment herein, at least one strand of the duplex siRNA has a sequence that has at least 90% sequence identity to any one of SEQ ID NOs: 1 to 10 or 17-26.

In a further example of any aspect or embodiment herein, at least one strand of the duplex siRNA has a sequence that has at least 95% sequence identity to any one of SEQ ID NOs: 1 to 10 or 17- 26.

In a further example of any aspect or embodiment herein, the siRNA molecule is 15 to 35 nucleotides in length.

According to a further example of any aspect or embodiment herein, the siRNA molecule is 18 to 35 nucleotides in length.

According to a further example of any aspect or embodiment herein, the siRNA molecule is 20 to 30 nucleotides in length.

According to a further example of any aspect or embodiment herein, the siRNA molecule is a conjugate molecule. For example, the conjugate molecule may comprise a sugar group. In one embodiment, the sugar group comprises GalNAc. According to a further aspect, the disclosure provides an siRNA molecule that has at least 80%, 85%, 90%, 95% or 97% sequence identity to any one of SEQ ID NOs: 1-10 or 17-26.

According to a further aspect, the disclosure provides an siRNA molecule that has at least 70%, 75%, 80%, 85%, 90%, 95% or 97% sequence identity to any one of SEQ ID NOs: 17-26.

In some embodiments, there is provided a composition comprising a duplex or single-stranded siRNA molecule as described herein, and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, but are not limited to, cationic lipids, cationic peptides, cationic polymers or dendrimers, lipid nanoparticles, micelles, nanoplexes, nanocapsules, nanogels, etc.

According to a further aspect, the disclosure provides a pharmaceutical composition comprising the siRNA molecule or the lipid nanoparticle as described in any aspect or embodiment herein and wherein the pharmaceutical composition comprises a pharmaceutically acceptable salt and/or excipient.

A further embodiment includes use of the pharmaceutical composition or lipid nanoparticle described above to treat a disorder by modulating levels of ADAMTS13 in the blood or other bodily sites in a patient in need of such treatment thereof. In some embodiments, the disorder is a bleeding disorder.

Another embodiment includes use of the pharmaceutical composition or lipid nanoparticle described above in the manufacture of a medicament to treat a disorder by modulating levels of ADAMTS13 in the blood or other bodily sites. In some embodiments, the disorder is a bleeding disorder.

Yet further, there is provided a method of treating a patient having a disorder resulting by modulating levels of ADAMTS13 in the blood or other bodily sites comprising administering the pharmaceutical composition or lipid nanoparticle described above to a patient in need of such treatment thereof.

In another embodiment, there is provided an siRNA for targeting mammalian AD AMTS 13. In some embodiments, the siRNA decreases bleeding in bleeding disorders. According to a further example of any aspect or embodiment herein, the pharmaceutical composition is for inhibiting the expression of ADAMTS13, thereby treating and/or preventing one or more blood coagulation disorders.

According to a further example of any aspect or embodiment herein, there is provided a use of the pharmaceutical composition in the manufacture of a medicament to treat a blood coagulation disorder in a patient in need of such treatment thereof.

According to a further example of any aspect or embodiment herein, there is provided a method of treating a patient having a blood coagulation disorder, the method comprising: administering the pharmaceutical composition as described in any aspect of embodiment herein to a patient in need of such treatment thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows human ADAMTS13 mRNA relative to control (%) LNP duplex siRNA sequences, hs.Ri . AD AMTS 13.13.1 (hs.13.1 ) (duplex siRNA of SEQ ID Nos 1 and 2), hs.Ri . AD AMTS 13.13.9 (hs.13.9) (duplex siRNA of SEQ ID Nos 3 and 4), hs.Ri.ADAMTS13.13.6 (hs.13.6) (duplex siRNA of SEQ ID Nos 5 and 6) hs.Ri.ADAMTS13.13.10 (hs.13.10) (duplex siRNA of SEQ ID Nos 7 and 8), and hs.Ri.ADAMTS13.13.7 (hs.13.7) (duplex siRNA of SEQ ID Nos 9 and 10) (Table 1) after addition to HUH7 cells in vitro, and compared to cells treated with empty LNP as controls.

Figure 2A shows hepatic ADAMTS13 mRNA relative to control (%) for a luciferase siRNA control (siLuc), and for duplex siRNA targeting ADAMTS13 corresponding to ms. AD AMTS 13.1 (ms.l) (duplex siRNA of SEQ ID Nos 11 and 12), ms.ADAMTS13.2 (ms.2) (duplex siRNA of SEQ ID Nos 13 and 14), ms.ADAMTS13.3 (ms.3) (duplex siRNA of SEQ ID Nos 15 and 16) (Table 2) one week post injection.

Figure 2B shows hepatic ADAMTS13 mRNA relative to control (%) for a luciferase siRNA control (siLuc), and siRNA targeting ADAMTS13 corresponding to ms. AD AMTS 13.3 (si Al 3) (duplex siRNA of SEQ ID Nos 15 and 16) (Table 2) one week post injection. Figure 2C is a western blot detecting ADAMTS13 in blood plasma from mice treated with siRNA targeting ADAMTS13 corresponding to ms. AD AMTS 13.3 (si Al 3) (duplex siRNA of SEQ ID Nos 15 and 16) (Table 2) one week post-injection.

Figure 2D shows levels of ADAMTS13 (Al 3) and the control Peptidylprolyl Isomerase A (PPIA) mRNA in hepatic stellate cells isolated from livers of mice treated with control siRNA targeting luciferase (siLuc) and siRNA targeting ADAMTS13 corresponding to ms.ADAMTS13.3 (siAl 3) (duplex siRNA of SEQ ID Nos 15 and 16) (Table 2) one week post-injection.

Figure 2E shows activity of ADAMTS13 in blood plasma, represented as relative fluorescence unit (RFU), from mice treated with control siRNA targeting luciferase (siLuc) and siRNA targeting ADAMTS13 corresponding to ms.ADAMTS13.3 (siAl 3) (duplex siRNA of SEQ ID Nos 15 and 16) (Table 2) one week post-injection.

DETAILED DESCRIPTION

Throughout this specification, unless the context requires otherwise, the words “comprise”, “comprising” and the like, are to be construed in an inclusive sense as opposed to an exclusive sense, that is to say, in the sense of “including, but not limited to”.

One embodiment of the disclosure provides a lipid nanoparticle comprising an siRNA sequence to reduce the expression of ADAMTS13. In some embodiments, mRNA encoding ADAMTS13 is targeted by the siRNA sequence and thereby reduces or prevents the assembly of the ADAMTS13 protein by the liver. In some embodiments, this in turn reduces secretion of ADAMTS13 into the blood.

One embodiment of the disclosure provides siRNA sequences to reduce the expression of coagulation factors to alter clotting. In one embodiment, the coagulation factor is AD AMTS 13. The siRNA may be a duplex siRNA. In such embodiment, the siRNA comprises a sense strand and an antisense strand, each nucleotide of the siRNA being a modified or unmodified nucleotide, and the sense and antisense strands having at least partial complementarity. In another embodiment, the siRNA is single-stranded. Further non-limiting examples of the disclosure are described in more detail hereinafter. siRNA

The expression “siRNA molecule against ADAMTS13” as used herein includes a single-stranded RNA (e.g., mature miRNA) or double-stranded RNA (i.e., duplex RNA such as siRNA, aiRNA, or pre-miRNA) that is capable of reducing or inhibiting the expression of ADAMTS13 such as by mediating the degradation or inhibiting the translation of an mRNA that is complementary to the siRNA sequence as measured in vitro or in vivo. The siRNA may have substantial or complete identity to the gene that encodes ADAMTS13 or sequence, or may comprise a region of mismatch (i.e., a mismatch motif). The sequence of the siRNA can correspond to the full-length target sequence, or a subsequence thereof.

The expression “siRNA molecule against ADAMTS13 mRNA” as used herein includes a doublestranded RNA (i.e., duplex RNA such as siRNA, aiRNA, or pre-miRNA) that reduces or inhibits the expression of ADAMTS13 such as by mediating the degradation or inhibiting the translation of an mRNA that is complementary to the siRNA sequence as measured in vitro or in vivo. The siRNA may have substantial or complete identity to the gene that encodes ADAMTS13 or sequence, or may comprise a region of mismatch (i.e., a mismatch motif). The sequence of the siRNA can correspond to the full-length target sequence, or a subsequence thereof.

In some embodiments, the siRNA is 15 to 40 or 20 to 35 nucleotides in length. Since the siRNA is double-stranded, the nucleotide length corresponds to the length of the shorter of an antisense or sense strand.

The siRNA described herein may comprise a “mismatch motif’ or “mismatch region”, which refers to a portion of the siRNA sequence that does not have 100% complementarity to its target sequence. An siRNA may have at least one, two, three, four, five, six, or more mismatch regions. The mismatch regions may be contiguous or may be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more nucleotides. The mismatch motifs or regions may comprise a single nucleotide or may comprise two, three, four, five, or more nucleotides.

In some embodiments, the siRNA reduces or inhibits expression of ADAMTS13 as measured in vitro or in vivo. Inhibition or reduction of expression of ADAMTS13 is achieved when reduction of mRNA obtained with an siRNA relative to a relevant control (e.g., buffer or an empty lipid nanoparticle) is about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or 0%. Suitable assays for measuring expression of a target gene or target sequence include, e.g., examination of protein or RNA levels using techniques known to those of skill in the art such as quantitative PCR (qPCR), western blots, dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, as well as phenotypic assays known to those of skill in the art. The reduction in expression and activity in vitro may be measured using an assay as described in the Example section.

The expression “inhibiting or reducing expression of ADAMTS13”, includes inhibition or reduction of ADAMTS13 expression that is achieved when the value obtained with an interfering RNA relative to a relevant control is about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or 0% using any one of the assays set forth above. Either mRNA or protein levels may be assayed in certain embodiments.

The nucleotides of the siRNA may be modified. Examples of modifications include, but are not limited to, 2'-O-alkyl modifications such as 2'-0-Me modifications and 2'-halogen modifications such as 2'-fluoro modifications.

The siRNA may have sequence identity to any one of the nucleotide sequences set forth in Table 1, Table 2 and Table 3 below. More typically, the siRNA has sequence identity to the human nucleotide sequences set forth in Table 1 or Table 3. The expression “sequence identity” when referring to two nucleic acids herein, refers to two sequences or subsequences that are the same or have a specified percentage of nucleotides that are the same, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a known comparison algorithm or by manual alignment and visual inspection.

For determining sequence identity, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. The sequence identity is typically measured by BLAST, which is well- known to those of skill in the art.

In one embodiment, the siRNA has at least 30% to 100% sequence identity to any one of SEQ ID NOs: 1-26 in Table 1, Table 2 and Table 3 below. For example, the siRNA may have at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% sequence identity to any one of SEQ ID Nos: 1-26. In one embodiment, a strand of the siRNA consists essentially of any one of SEQ ID NOs: 1-26 meaning that the strand differs by no more than 4 nucleotides but excluding modifications of the nucleotides, such as methylation or a halogen modification (described below).

In a further embodiment, the siRNA or a strand of a duplex siRNA differs by no more than 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14 or 15 nucleotides as set forth in SEQ ID NOs: 1-26. In one embodiment, the siRNA differs by no more than 10 nucleotides or no more than 5 nucleotides. In in one embodiment, this excludes differences due to modifications of a given nucleotide, such as methylation or a halogen modification (described below).

In another embodiment the present disclosure provides one or more exemplary siRNA sequences or duplexes thereof selected from SEQ ID NOs: 1-10 or SEQ ID NOs: 17-26 (human sequences) to inhibit or reduce the expression of AD AMTS 13.

In one embodiment, the siRNA has at least 30% to 100% sequence identity to any one of SEQ ID NOs: 1-10 in Table 1 or SEQ ID NOs 17-26 in Table 3 below. For example, the siRNA may have at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% sequence identity to any one of SEQ ID Nos: 1-10 in Table 1 or SEQ ID NOs 17-26 in Table 3 below. In one embodiment, the siRNA consists essentially of any one of SEQ ID NOs: 1-10 in Table 1 or SEQ ID NOs: 17-26 in Table 3 below, meaning that it differs by no more than 4 nucleotides excluding modifications of the nucleotides, such as methylation or a halogen modification (described below). It should be appreciated that the sequence identity herein need not require an exact match of two nucleotides. To illustrate, a given nucleotide can be methylated and will be considered to have identity to an unmethylated nucleotide.

In a further embodiment, the siRNA or a strand of a duplex siRNA differs by no more than 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14 or 15 nucleotides as set forth in SEQ ID NOs: 1-10 in Table 1 and SEQ ID NOs: 17-26 in Table 3 below. In one embodiment, the siRNA differs by no more than 10 nucleotides or no more than 8, 7, 6 or 5 nucleotides from the sequences in Table 1 and Table 3 below. In in one embodiment, this excludes differences due to modifications of a given nucleotide, such as methylation or a halogen modification (described below).

In another embodiment the present disclosure provides one or more siRNA sequences or duplexes thereof selected from SEQ ID NOs: 1-10 (Table 1) and SEQ ID NOs: 17-26 (Table 3) to inhibit or reduce the expression of AD AMTS 13.

In another embodiment, the present disclosure provides one or more siRNA sequences or duplexes thereof selected from SEQ ID NOs: 1-10 (Table 1) to inhibit or reduce the expression of ADAMTS13 and the siRNA or a strand of a duplex siRNA differs by no more than 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14 or 15 nucleotides and/or 0 to 50% or 10 to 40% of the nucleotides have 2'- O-alkyl modifications such as 2'-O-Me modifications and/or 2'-halogen modifications.

Without being limiting, the siRNA sequences may exhibit a modification pattern similar to that set forth in Table 2 or Table 3 below.

Table 1. Base composition of duplex siRNA sequence targeting human ADAMTS13 mRNA.

Table 2. Base modification of duplex siRNA sequences targeting murine ADAMTS13 mRNA. “r” designates unmodified base, “m” designates 2’O-methylated base

Table 3: Human siRNA sequences with modifications, “r” designates unmodified base, “m” designates 2’O-methylated base

It should be appreciated that an siRNA having a sequence similar to those set forth in the sequence listings may optionally be conjugated with another moiety, such as but not limited to a ligand, as described below.

Within an siRNA, the antisense strand and the sense strand may be designed such that when they form a duplex due to complementarity of base-pairs, they can anneal with no overhangs and thus form blunt ends at both ends of the duplex, or with an overhang at one or more of the 3' end of the sense strand, the 3' end the antisense strand, the 5' end of the sense strand, and the 5' end of the antisense strand. In some embodiments, there are no 5' overhangs and there is no 3' antisense overhang, but there is a 3' sense overhang. In other aspects, there are no 5' overhangs, but there are a 3' antisense overhang and a 3' sense overhang.

When overhangs are present, they may, for example, be 1 to 6 nucleotides long. In some aspects, the overhang is a dinucleotide. By way of a non-limiting example, in one aspect, there is a 3' sense overhang that is dTdT, and there are no overhangs on the antisense strand and no 5' sense overhang. By way of another non-limiting example, in another aspect, there are a 3' sense overhang that is dTdT and a 3' antisense overhang that also is dTdT, but there are no 5’ overhangs on either the antisense strand or the sense strand. By way of another non-limiting example, in one aspect, there is a 3' sense overhang that is dTdT, and a 3' dinucleotide antisense overhang that is complementary to two nucleotides on the target molecule adjacent to the region of the target molecule to which the region of the antisense strand within the duplex is complementary. In this aspect, there are no 5’ overhangs on either the antisense strand or the sense strand. When an overhang is present, the nucleotides within it are included in the aforementioned range of 18 to 30 nucleotides for each strand.

In some aspects, the siRNA are covalently bound to one or more other molecules to form a conjugate. In some aspects, the conjugates are selected because they facilitate delivery of the siRNA to an organism or into cells. An siRNA may be bound to a conjugate at, for example, the 5' end of the antisense strand, the 3' end of the antisense strand, the 5' end of the sense strand, the 3' end of the sense strand, or to a nucleotide at a position that is not at the 3' end or 5' end of either strand.

Examples of conjugates include but are not limited to one or more of an antibody, a peptide, an amino acid, an aptamer, a phosphate group, a cholesterol moiety, a lipid, a cell- penetrating peptide polymer, and a sugar group, which includes a sugar monomer, an oligosaccharide and modifications thereof. In one aspect, the conjugate is N- Acetylgalactosamine (GalNAc). Lipid nanoparticles

In one embodiment, the disclosure provides a nucleic acid against ADAMTS13 mRNA that is encapsulated within a lipid nanoparticle. In one embodiment, the nucleic acid is for inhibiting or reducing expression of ADAMTS13.

It will be understood that the invention is not limited by the location or the nature of the incorporation of the nucleic acid within the lipid nanoparticle. That is, the term “encapsulated” is not meant to be limited to any specific interaction between the nucleic acid and the lipid nanoparticle. The nucleic acid may be incorporated in the aqueous portion, within any lipid layer or both.

The lipid nanoparticle (LNP) described herein may comprise an ionizable lipid that may associate or complex with the nucleic acid. The term “ionizable lipid” refers to any of a number of lipid species that carry a net positive charge at a selected pH below its pKa. In some embodiments, the cationic lipid has a head group comprising an amino group. In some embodiments, the cationic lipids comprise a protonatable tertiary amine (e.g., pH titratable) head group, C16 to Cl 8 alkyl chains, ether linkages between the head group and alkyl chains, and 0 to 3 double bonds.

In certain embodiments, the cationic lipid content is from 20 mol% to 70 mol% or 30 mol% to 55 mol% or 35 mol% to 55 mol% of total lipid present in the lipid nanoparticle.

The lipid nanoparticle (LNP) described herein may comprise a helper lipid in addition to the ionizable lipid. In the context of the present disclosure, the term “helper lipid” includes any vesicle-forming lipid (e.g., bilayer-forming lipid) that may be selected from a phosphatidylcholine lipid, sphingomyelin, or mixtures thereof. In some embodiments, the helper lipid is selected from sphingomyelin, distearoylphosphatidylcholine (DSPC), di oleoylphosphatidylcholine (DOPC), 1- palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and dipalmitoyl-phosphatidylcholine (DPPC). In certain embodiments, the helper lipid is DOPC, DSPC or sphingomyelin. In one embodiment, the helper lipid is DSPC. The helper lipid content may include mixtures of two or more different types of different helper lipids. For example, in certain embodiments, the phosphatidylcholine content is from 20 mol% to 60 mol% or 25 mol% to 60 mol% or 30 mol% to 60 mol% or 35 mol% to 60 mol% or 40 mol% to 60 mol% of total lipid present in the lipid nanoparticle. The phosphatidylcholine lipid content is determined based on the total amount of lipid in the lipid nanoparticle, including the sterol.

In one embodiment, the LNP comprises a sterol, a hydrophilic polymer-lipid conjugate or both.

Examples of sterols include cholesterol, or a cholesterol derivative, such as cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2 '-hydroxy ethyl ether, cholesteryl-4'- hydroxybutyl ether, beta-sitosterol, fucosterol and the like. In one embodiment, the sterol is present at from 15 mol% to 65 mol%, 18 mol% to 50 mol%, 20 mol% to 50 mol%, 25 mol% to 50 mol% or 30 mol% to 50 mol% based on the total lipid present in the lipid nanoparticle. In another embodiment, the sterol is cholesterol and is present at from 15 mol% to 65 mol%, 18 mol% to 50 mol%, 20 mol% to 50 mol%, 25 mol% to 50 mol% or 30 mol% to 50 mol% based on the total lipid and sterol present in the lipid nanoparticle.

In one embodiment, the hydrophilic-polymer lipid conjugate includes (i) a vesicle- forming lipid having a polar head group, and (ii) covalently attached to the head group, a polymer chain that is hydrophilic. Example of hydrophilic polymers include polyethyleneglycol (PEG), polyvinylpyrrolidone, polyvinylmethylether, polyhydroxypropyl methacrylate, polyhydroxypropylmethacrylamide, polyhyd. -oxy ethyl acrylate, polymethacrylamide, polydimethylacrylamide, polymethyloxazoline, polyethyloxazoline, polyhydroxyethyloxazoline, polyhydroxypropyloxazoline, polysarcosine and polyaspartamide. In one embodiment, the hydrophilic-polymer lipid conjugate is a PEG-lipid conjugate.

The hydrophilic polymer lipid conjugate may be present in the nanoparticle at 0 mol% to 5 mol%, or at 0.5 mol% to 3 mol%, or at 0.5 mol% to 2.5 mol% or at 0.5 mol% to 2.0 mol% or at 0.5 mol% to 1.8 mol% of total lipid. In another embodiment, the PEG-lipid conjugate is present in the nanoparticle at 0 mol% to 5 mol%, or at 0.5 mol% to 3 mol% or at 0.5 mol% to 2.5 mol% or at 0.5 mol% to 2.0 mol% or at 0.5 mol% to 1.8 mol% of total lipid. In certain embodiments, the PEG- lipid conjugate may be present in the nanoparticle at 0 mol% to 5 mol%, or at 0 mol% to 3 mol%, or at 0 mol% to 2.5 mol% or at 0 mol% to 2.0 mol% or at 0 mol% to 1.8 mol% of total lipid.

Methods to treat or prevent blood coagulation disorders

In another aspect, the present disclosure provides methods of treating a subject having any disorder or condition that would benefit from a reduction in ADAMTS13 expression.

This includes a “bleeding disorder”, which as used herein includes any condition, of any severity, that results in abnormal amounts of bleeding in a subject, such as but not limited to a blood clotting disorder. The bleeding disorder includes but is not limited to hemophilia A and B, von Willebrand Disease (VWD), platelet disorders, menorrhagia and other rare bleeding disorders or conditions. The methods include administering to the subject a therapeutically effective amount of the siRNA, optionally encapsulated in a lipid nanoparticle, thereby treating the subject or providing a prophylactic effect.

As used herein, the term “subject” includes any human or non-human mammalian subject that would benefit from a reduction in ADAMTS13 expression relative to lack of treatment thereof. This includes a prophylactic benefit in some embodiments. In some embodiments, the subject is a human.

In one embodiment, the disclosure provides methods of preventing at least one symptom, e.g., bleeding, in a subject having a bleeding disorder that would benefit from reduction in ADAMTS13 expression. The methods include administering to the subject a therapeutically effective amount of the siRNA, thereby preventing at least one symptom in the subject having a disorder that would benefit from reduction in ADAMTS13 expression.

In one embodiment, the administration of the siRNA to the subject causes a decrease in bleeding, and/or a decrease in ADAMTS13 expression and/or accumulation.

In another embodiment the present disclosure provides a method of treating a patient by modulating coagulation, the method comprising: administering siRNA to a subject in need thereof to inhibit the expression of AD AMTS 13. ADAMTS13 expression or activity can be assessed as set forth in the Example section herein.

Further methods for assessing knockdown, inhibition and/or reduction in ADAMTS13 expression include quantifying hepatic ADAMTS13 mRNA level, plasma ADAMTS13 protein concentration, and/or ADAMTS13 activity. Inhibition of expression of a target gene or target sequence is achieved when the value obtained with an siRNA relative to a relevant control is about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%.

In another embodiment, the siRNA is used to treat a cell in vitro or in vivo. The cell may be within a subject, such as a mammalian subject, for example a human subject suffering from a bleeding disorder. One embodiment of the disclosure provides a method to knock-down ADAMTS13 using siRNA delivered to hepatic stellate cells.

Pharmaceutical formulations

In some embodiments, the siRNA or lipid nanoparticle comprising a nucleic acid reducing expression ADAMTS13 is part of a pharmaceutical composition and is administered to treat and/or prevent a disease condition. The treatment may provide a prophylactic (preventive), ameliorative or a therapeutic benefit to treat a bleeding disorder. The pharmaceutical composition will be administered at any suitable dosage.

In one embodiment, the pharmaceutical composition is administered parenterally, i.e., intraarterially, intravenously, subcutaneously or intramuscularly. In another embodiment, the pharmaceutical compositions are administered intranasally, intravitreally, subretinally, intrathecally or via other local routes.

The pharmaceutical composition comprises pharmaceutically acceptable salts and/or excipients. Used herein, the term "pharmaceutically acceptable salt" refers to pharmaceutically acceptable salts derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, and tetraalkylammonium, and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, and oxalate. Suitable salts include those described in P. Heinrich Stahl, Camille G. Wermuth (Eds.), Handbook of Pharmaceutical Salts Properties, Selection, and Use; 2002.

As used herein, the term "excipient" means the substances used to formulate active pharmaceutical ingredients (API) into pharmaceutical formulations. Non-limiting examples include mannitol, Captisol®, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, sodium crosscarmellose, glucose, gelatin, sucrose, magnesium carbonate, and the like. Acceptable excipients are non-toxic and may be any solid, liquid, semi-solid excipient that is generally available to one of skill in the art.

The examples are intended to illustrate preparations and properties of the invention but are in no way intended to limit the scope of the invention.

EXAMPLES

Materials and methods siRNA-LNP preparation and analysis

2’-O-methylated siRNA targeting murine ADAMTS13, siRNA targeting human ADAMTS13, and a negative control siRNA targeting Luciferase (siLuc) were obtained commercially (Integrated DNA Technologies (IDT), Coralville, USA). siRNAs were encapsulated in LNPs as previously described. Briefly, siRNAs were dissolved in sodium acetate (pH 4) and combined with a lipid solution at an amine-to-phosphate (N/P) ratio of 3. The lipid formulations consisted of DSPC, cholesterol and PEG-DMG at a 10:38.5: 1.5 % molar ratio, with 50% ALC-0315 (DC Chemicals, Shanghai, China). The LNPs were dialysed against phosphate buffered saline (PBS) at pH 7.4 in 500-fold excess. For quality control, cholesterol content was measured using the Cholesterol E Assay Kit (Wako Chemicals, Mountain View, CA). To determine siRNA concentration and entrapment a RiboGreen assay (Quant-IT Ribogreen RNA Assay Kit, ThermoFisher) was used. Encapsulation efficiency was measured by comparing Ribogreen signal in LNP samples with or without Triton X-100 detergent. Particle size was measured using a Malvern-Zetasizer Nano). The resulting LNPs were diluted to a final concentration of 0.1 mg siRNA per mL in PBS prior to intravenous (IV) injection. Mice

Procedures performed at each institution were approved by the local Animal Care Committee. Wild-type (WT) C57BL/6J mice (stock #000664; The Jackson Laboratory) age 8-14 weeks were used unless otherwise indicated.

LNP-siRNA injection

Mice were injected with 1 mg siRNA per kg body weight (mg/kg). One week after administration, liver tissue and blood were collected to measure target mRNA and protein levels, respectively, and compared to siLuc-treated mice.

Tissue collection

All blood samples were collected via cardiac puncture and liver tissues were surgically excised following euthanasia by chest cavity opening. Plasma samples were separated from whole blood by spinning at 2000 x g for 10 minutes, after blood collection into a syringe containing sodium citrate (0.32 % final).

Isolation of HSC from liver

The caudal vena cava was cannulated and the livers were perfused with perfusion buffer (50 mM EGTA, 1 M Glucose, and 1% Penicillin-Streptomycin in Hank’s Balanced Salt Solution (HBSS, without calcium and magnesium). Livers were then perfused with HBSS containing 1.0 M calcium chloride, 1.0 M glucose, 1% Penicillin-Streptomycin, and 5.0 mg Pronase (Roche, Indianapolis, IN). Finally, livers were perfused with HBSS containing 1.0 M calcium chloride, 1.0 M glucose, 1% Penicillin-Streptomycin, and 5.0 mg Collagenase (Sigma-Aldrich). The digested livers were transferred to cold media (DMEM, 1% Pen/Strep, 10% heat-inactivated FBS) and mechanically dissociated, and DNAse solution (16 pg/mL DNAse final concentration in sterile saline) was added. The solutions were centrifuged 3 times at 50 x g for 2 min, and the hepatocyte pellet was discarded. The collected supernatants were centrifuged at 1000 x g for 6 min to collect non- parenchymal cells. Hepatic stellate cells were separated from the non-parenchymal cells using an 8% Histodenz (Sigma-Aldrich) gradient, as described previously. The hepatic stellate cells were collected from the interface and diluted to 50 mL in HBSS, then centrifuged at 1000 x g for 8 minutes. The resulting hepatic stellate cells were plated in 12-well tissue culture plates (Corning) in DMEM containing 10% FBS and 1% Penicillin-Streptomycin for 2 hours at 37 C. Following incubation, the adherent hepatic stellate cells were gently rinsed in HBSS, then lysed in TRI Reagent, and mRNA was isolated using DirectZol mRNA MiniPrep spin columns (Zymo Research, Irvine CA). mRNA quantification

Livers were homogenized in Trizol (ThermoFisher, Waltham, USA) and DNA and RNA were isolated by precipitating them in phenol-chloroform. DNA was digested by incubating samples with TURBO DNase and 10-times TURBO DNase Buffer (ThermoFisher). DNAse was then removed by repeating the precipitation of RNA in Trizol-phenol-chloroform. Reverse transcription was performed using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, USA). Quantitative PCR, was performed using the SYBR Green Master Mix (ThermoFisher), and DNA primers against ADAMTS13 (F:5’-GTGCTCACTAATCTCAATATC-3’, R:5’- AAGGATGAGGTGATGTTG-3’). PCR was run for 55 cycles, ADAMTS13 expression was quantified using the AACt method, relative to the expression of the housekeeping gene Ppia (F:5’- GCGTCTCCTTCGAGCTGTT-3’, R:5’-TGTAAAGTCACCACCCTGGC-3’). All primers were synthesized by IDT. Data was collected and analyzed using the 7500 Software v 2.3.

Protein quantification

Plasma samples for the blots visualizing ADAMTS13 samples were not reduced. Samples were heated and separated on 4-15 % acrylamide gradient gels (Bio-Rad). Following electrophoresis, the samples were transferred to a PVDF membrane and blocked with protein-free Blot Blocking Buffer (Azure, VWR, Radnor, USA). The membranes were treated with a primary antibody against ADAMTS13 (1 : 1000; NB110-82382, Novus Biologicals, Littleton, USA), washed, and treated with HRP-conjugated secondary antibody (Goat Anti-Rabbit: 1 :2000, Cell Signaling Inc.; Donkey Anti-Goat: 1/15,000, ab7125, Abeam; or Goat Anti-Mouse IgG: 1 : 15,000, ab97040, Abeam). Bands were detected using ECL substrates (Bio-Rad) and imaged on a Sapphire Biomolecular Imager (Azure Biosystem). Protein quantification of the bands was performed using ImageJ software. Relative intensity was determined using IgG as the control. Quantifying AD AMTS 13 activity in blood plasma

Mouse plasma samples (60 pL) were diluted to 100 pL with a buffer (5 mmol/L Bis-Tris, 25 mmol/L CaCh, 0.005% Tween-20 at pH 6) on a 96 well-plate. Next, 100 pL of 4 pmol/L of FRETS-VWF73 (Peptide Institute Inc.) was added to the diluted plasma and incubated for 20 min at 37 °C before measuring fluorescence (emission wavelength of 460 nm and excitation of 330 nm) at 30 °C (Tecan microplate reader). Readings were recorded every 20 sec for 40 min. As a negative control, the activity of ADAMTS13 was inhibited by chelating the Ca²⁺ and Zn²⁺ cofactors using EDTA. To account for differences in hemolysis, the baseline value was subtracted from each sample curve, then the data from each plasma sample was normalized by subtracting the EDTA-treated plasma (negative control) curve.

Screen siRNA targeting AD AMTS 13 in human hepatoma (HUH7) cell culture

HUH7 cells were seeded at 3.5 x io⁵ cells/well one day prior to transfection. Cells were then transfected with the five different human LNP-siRNA targeting human ADAMTS13 mRNA at a dose of 3 pg/mL of siRNA, or empty LNPs as a negative control. The following day, cells were lysed, and the RNA was extracted using the PureLink RNA Mini Kit (Thermo Fisher, Waltham, MA) following the manufacturer’s protocol. AD AMTS mRNA levels was then detected and quantified as described above.

Statistical Analysis

To ensure a t-test could be used between two groups, the F-test was performed to confirm the standard deviation (SD) between groups was not statistically different. Comparisons between the mean of two groups were performed with a one-tailed unpaired parametric t-test or with Welch’s t test if the SD between groups was significantly different. For assessing ADAMTS13 activity, simple linear regression was used to determine statistical difference in the slopes. All analysis was done using GraphPad Prism (Version 9.2.0). Significance was designated at P values < 0.05.

Example 1: siRNA knock down in vitro

This example demonstrates that siRNA knocks down ADAMTS13 in human hepatocytes in vitro. Quantitative PCR as described in the Materials and Methods was used to measure human ADAMTS13 mRNA levels after administration of LNP containing siRNA sequences targeting human ADAMTS13 mRNA as set out in Table 1, namely hs.Ri.ADAMTS13.13.1 (hs.13.1) (duplex siRNA of SEQ ID Nos 1 and 2), hs.Ri.ADAMTS13.13.9 (hs.13.9) (duplex siRNA of SEQ ID Nos 3 and 4), hs.Ri.ADAMTS13.13.6 (hs.13.6) (duplex siRNA of SEQ ID Nos 5 and 6) hs.Ri.ADAMTS13.13.10 (hs.13.10) (duplex siRNA of SEQ ID Nos 7 and 8), and hs.Ri.ADAMTS13.13.7 (hs.13.7) (duplex siRNA of SEQ ID Nos 9 and 10), to human hepatocyte cells in culture and compared to cells treated with empty LNPs as negative control.

As shown in Figure 1, significant depletion of ADAMTS13 mRNA was observed in HUH7 cells after treatment with LNP containing the siRNA targeting human ADAMTS13 corresponding to hs.Ri.ADAMTS13.13.7 (hs.13.7) (duplex siRNA of SEQ ID Nos 9 and 10).

Example 2: siRNA knock down of ADAMTS13 in vivo.

This example demonstrates that siRNA knocks down ADAMTS13 mRNA in mice, resulting in depletion of circulating ADAMTS13 protein and decreased ADAMTS13 activity in vivo.

Control siRNA targeting luciferase (siLuc), or siRNA targeting mouse ADAMTS13 sequences set out in Table 2, namely ms.ADAMTS13.1 (ms.l) (duplex siRNA of SEQ ID Nos 11 and 12), ms.ADAMTS13.2 (ms.2) (duplex siRNA of SEQ ID Nos 13 and 14), and ms.ADAMTS13.3 (ms.3) (duplex siRNA of SEQ ID Nos 15 and 16), was encapsulated in lipid nanoparticles ALC- 0315 and administered to mice intravenously as described in the Material and Methods.

PCR (qPCR) as described in the Materials and Methods was used to quantify hepatic ADAMTS13 mRNA levels after administration of different siRNA sequences targeting murine ADAMTS13, respectively, to mice 1 week weeks prior to tissue collection, and compared to control siRNA targeting luciferase (siLuc). 1 The results are shown in Figure 2A. As can be seen in Figure 2A, hepatic ADAMTS13 mRNA levels significantly reduced in liver from mice administered siRNA targeting ADAMTS13 corresponding to sequences ms.ADAMTS13.3 (SEQ ID Nos 15 and 16), compared to siLuc- treated mice. siRNA targeting ADAMTS13 (siA13) was encapsulated in lipid nanoparticles ALC-0315 and administered to mice intravenously as described in the Material and Methods.

PCR (qPCR) as described in the Materials and Methods was used to quantify hepatic ADAMTS13 mRNA levels after administration of siRNA sequence targeting ADAMTS13 corresponding to ms. AD AMTS 13.3 (si Al 3) (SEQ ID Nos 15 and 16) of Table 2 to mice 1 week prior to tissue collection, and compared to control siRNA targeting luciferase (siLuc).

The results are shown in Figure 2B. As can be seen in Figure 2B, ADAMTS13 mRNA levels significantly reduced in the liver from mice administered siRNA targeting ADAMTS13 corresponding to sequences ms.ADAMTS13.3 (SEQ ID Nos 15 and 16), compared to siLuc- treated mice.

Western blot as described in the Materials and Methods was used to quantify ADAMTS13 protein levels in blood plasma after administration of siRNA sequence targeting ADAMTS13 corresponding to ms. AD AMTS 13.3 (si Al 3) (SEQ ID Nos 15 and 16) of Table 2, respectively, to mice 1 week prior to blood sampling, and compared to siLuc.

The results are shown in Figure 2C. As can be seen in Figure 2C, compared to control siLuc, ADAMTS13 protein levels were significantly reduced in the blood plasma of mice by 1 week after administration of siRNA targeting ADAMTS13 corresponding to ms.ADAMTS13.3 (siA13) (SEQ ID Nos 15 and 16) of Table 2.

Hepatic stellate cells were isolated from excised livers and mRNA Materials and Methods. PCR (qPCR) as described in the Materials and Methods was used to quantify ADAMTS13 mRNA levels in isolated hepatic stellate cells after administration of siRNA targeting ADAMTS13 corresponding to ms.ADAMTS13.3 (siA13) (SEQ ID Nos 15 and 16) of Table 2 or control siRNA targeting luciferase (siLuc) to mice 1 week weeks prior to tissue collection. ADAMTS13 mRNA levels were compared to the internal housekeeping control, Peptidylprolyl Isomerase A (PPIA) mRNA levels.

The results are shown in Figure 2D. As can be seen in Figure 2D, ADAMTS13 mRNA were detected in hepatic stellate cells isolated from livers of mice treated with siRNA luciferase (siLuc), but not in hepatic stellate cells isolated from livers of mice treated with siRNA targeting ADAMTS13 corresponding to ms. AD AMTS 13.3 (si Al 3) (SEQ ID Nos 15 and 16) of Table 2. Dashed lines represent a maximum amplification cycle of 55 when performing qPCR, and data points below the dashed lines represents detectable mRNA. PPIA mRNA were detected at similar levels in hepatic stellate cells isolated from livers of mice treated with siLuc and siRNA targeting ADAMTS13 corresponding to ms.ADAMTS13.3 (siA13) (SEQ ID Nos 15 and 16) of Table 2.

ADAMTS13 activity in blood plasma was quantified after administration of siRNA targeting ADAMTS13 corresponding to ms. AD AMTS 13.3 (si Al 3) (SEQ ID Nos 15 and 16) of Table 2, respectively, to mice 1 week prior to blood sampling, and compared to siLuc.

The results are shown in Figure 2E. Blood plasma from mice treated with siRNA targeting ADAMTS13 corresponding to ms.ADAMTS13.3 (siA13) (SEQ ID Nos 15 and 16) ofTable 2 had significantly decreased ADAMTS13 activity levels, represented by relative fluorescence units (RFU), in blood plasma 1 week post-administration compared to siLuc-treated mice.

Although the invention has been described and illustrated with reference to the foregoing detailed description and examples, it will be apparent that a variety of modifications and changes may be made without departing from the invention.

Claims

CLAIMS:

1. A lipid nanoparticle comprising: an siRNA molecule against ADAMTS13 mRNA; an ionizable, cationic amino lipid having a pKa of between 5.5 and 7.0 and that is present at between 10 mol% and 85 mol%; a neutral, vesicle-forming lipid selected from at least one of a phospholipid and a triglyceride; a sterol; and a hydrophilic polymer-lipid conjugate present at between 0.5 mol% and 5 mol%.

2. The lipid nanoparticle of claim 1, wherein the ADAMTS13 is human.

3. The lipid nanoparticle of claim 1, wherein the siRNA molecule comprises modified or unmodified nucleotides, and wherein the modified nucleotides are methylated.

4. The lipid nanoparticle of any one of claims 1 to 3, wherein at least one strand of the duplex siRNA has a sequence that has at least 80% sequence identity to any one of SEQ ID NOs: 1 to 10 or 17-26.

5. The lipid nanoparticle of any one of claims 1 to 4, wherein at least one strand of the duplex siRNA has a sequence that has at least 80% sequence identity to any one of SEQ ID NOs: 1 to 10 or 17-26.

6. The lipid nanoparticle of any one of claims 1 to 4, wherein at least one strand of the duplex siRNA has a sequence that has at least 85% sequence identity to any one of SEQ ID NOs: 1 to 10 or 17-26.

7. The lipid nanoparticle of any one of claims 1 to 4, wherein at least one strand of the duplex siRNA has a sequence that has at least 90% sequence identity to any one of SEQ ID NOs: 1 to 10 or 17-26.

8. The lipid nanoparticle of any one of claims 1 to 4, wherein at least one strand of the duplex siRNA has a sequence that has at least 95% sequence identity to any one of SEQ ID NOs: 1 to 10 or 17-26.

9. The lipid nanoparticle of any one of claims 1 to 8, wherein the siRNA molecule is 15 to 35 nucleotides in length. The lipid nanoparticle of claim 9, wherein the siRNA molecule is 18 to 35 nucleotides in length. The lipid nanoparticle of claim 9, wherein the siRNA molecule is 20 to 30 nucleotides in length. The lipid nanoparticle of any one of claims 1 to 11, wherein the siRNA molecule is a conjugate molecule. The lipid nanoparticle of claim 12, wherein the conjugate molecule comprises a sugar group. The lipid nanoparticle of claim 13, wherein the sugar group comprises GalNAc. An siRNA molecule having at least 80% sequence identity to any one of SEQ ID NOs: 1- 10 or 17-26. The siRNA molecule of claim 15 having at least 85% sequence identity to any one of SEQ ID NOs: 1-10 or 17-26. The siRNA molecule of claim 15 having at least 90% sequence identity to any one of SEQ ID NOs: 1-10 or 17-26. The siRNA molecule of claim 15 having at least 95% sequence identity to any one of SEQ ID NOs: 1-10 or 17-26. A pharmaceutical composition comprising the siRNA molecule of any one of claims 15 to 18, wherein the pharmaceutical composition comprises a pharmaceutically acceptable salt and/or excipient. A pharmaceutical composition comprising the siRNA molecule of any one of claims 15 to 18, wherein the siRNA is encapsulated in a lipid nanoparticle. Use of the pharmaceutical composition of claim 19 or the lipid nanoparticle of any one of claims 1 to 14 to treat a disorder in a patient in need of such treatment thereof. Use of the pharmaceutical composition of claim 19 or the lipid nanoparticle of any one of claims 1 to 14 in the manufacture of a medicament to treat a bleeding disorder. A method of treating a patient having a bleeding disorder comprising administering the pharmaceutical composition of claim 19 or the lipid nanoparticle of any one of claims 1 to 14 to a patient in need of such treatment thereof.